WO1998028333A2 - USPA1 AND USPA2 ANTIGENS OF $i(MORAXELLA CATARRHALIS) - Google Patents

USPA1 AND USPA2 ANTIGENS OF $i(MORAXELLA CATARRHALIS) Download PDF

Info

Publication number
WO1998028333A2
WO1998028333A2 PCT/US1997/023930 US9723930W WO9828333A2 WO 1998028333 A2 WO1998028333 A2 WO 1998028333A2 US 9723930 W US9723930 W US 9723930W WO 9828333 A2 WO9828333 A2 WO 9828333A2
Authority
WO
WIPO (PCT)
Prior art keywords
uspa2
uspal
seq
catarrhalis
peptide
Prior art date
Application number
PCT/US1997/023930
Other languages
French (fr)
Other versions
WO1998028333A3 (en
WO1998028333A9 (en
Inventor
Eric J. Hansen
Christoph Aebi
Leslie D. Cope
Isobel Maciver
Michael J. Fiske
Ross Fredenburg
Original Assignee
The Board Of Regents, The University Of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to AT97953461T priority Critical patent/ATE497004T1/en
Priority to CA002274495A priority patent/CA2274495C/en
Priority to JP52907598A priority patent/JP2001515467A/en
Priority to DE69740108T priority patent/DE69740108D1/en
Priority to BR9714160-7A priority patent/BR9714160A/en
Priority to EP97953461A priority patent/EP0948625B1/en
Priority to AU57201/98A priority patent/AU746442B2/en
Application filed by The Board Of Regents, The University Of Texas System filed Critical The Board Of Regents, The University Of Texas System
Publication of WO1998028333A2 publication Critical patent/WO1998028333A2/en
Publication of WO1998028333A9 publication Critical patent/WO1998028333A9/en
Publication of WO1998028333A3 publication Critical patent/WO1998028333A3/en
Priority to US09/336,447 priority patent/US6310190B1/en
Priority to US09/952,267 priority patent/US6753417B2/en
Priority to US10/872,769 priority patent/US7344724B2/en
Priority to US10/872,768 priority patent/US7288646B2/en
Priority to US12/044,805 priority patent/US7576194B2/en
Priority to US12/044,818 priority patent/US7569683B2/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C07K14/212Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04Antibacterial agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1217Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates generally to the fields of microbiology, and clinical bacteriology. More particularly, it concerns sequences of the uspAl and uspAl genes which encode the proteins UspAl and UspA2. respectively, both of which encode an epitope reactive with monoclonal antibody (MAb) 17C7 and provide useful epitopes for immunodiagnosis and immunoprophylaxis.
  • MAb monoclonal antibody
  • Moraxella catarrhalis previously known as Branhamella catarrhalis or Neisseria catarrhalis, was a harmless saprophyte of the upper respiratory tract (Catlin, 1990; Berk, 1990).
  • this organism is an important human pathogen. Indeed, it has been established that this Gram- negative diplococcus is the cause of a number of human infections (Murphy, 1989).
  • M. catarrhalis is now known to be the third most common cause of both acute and chronic otitis media (Catlin, 1990; Faden et al. 1990; 1991 ; Marchant, 1990), the most common disease for which infants and children receive health care according to the 1989 Consensus Report.
  • This organism also causes acute maxillary sinusitis, generalized infections of the lower respiratory tract (Murphy and Loeb, 1989) and is an important cause of bronchopulmonary infections in patients with underlying chronic lung disease and, less frequently, of systemic infections in immunocompromised patients (Melendez and Johnson, 1990; Sarubbi et al, 1990; Schonheyder and Ejlertsen, 1989; Wright and Wallace. 1989).
  • M. catarrhalis This is particularly troublesome in that M. catarrhalis now accounts for approximately 17-20% of all otitis media infection (Murphy, 1989). In addition, M. catarrhalis is also a significant cause of sinusitis (van Cauwenberge et al, 1993) and persistent cough (Gottfarb and Brauner, 1994) in children. In the elderly, it infects patients with predisposing conditions such as chronic obstructive pulmonary disease (COPD) and other chronic cardiopulmonary conditions (Boyle et al, 1991 ; Davies and Maesen, 1988; Hager et al,
  • COPD chronic obstructive pulmonary disease
  • M. catarrhalis otitis media has been documented by means of an ELISA system using whole M. catarrhalis cells as antigen and acute and convalescent sera or middle ear fluid as the source of antibody (Leinonen et al, 1981).
  • the development of serum bactericidal antibody during M. catarrhalis infection in adults was shown to be dependent on the classical complement pathway (Chapman et al, 1985).
  • M. catarrhalis antigens that would serve as potentially important targets of the human immune response to infection (Murphy, 1989; Goldblatt et al, 1990; Murphy et al, 1990).
  • OMPs outer membrane proteins
  • LOS lipooligosaccharide
  • fimbriae fimbriae.
  • M. catarrhalis appears to be somewhat distinct from other Gram-negative bacteria in that attempts to isolate the outer membrane of this organism using detergent fractionation of cell envelopes has generally proven to be unsuccessful in that the procedures did not yield consistent results (Murphy, 1989; Murphy and Loeb, 1989).
  • preparations were found to be contaminated with cytoplasmic membranes, suggesting an unusual characteristic of the M. catarrhalis cell envelope.
  • OMPs Outer membrane proteins (OMPs) constitute major antigenic determinants of this unencapsulated organism (Bartos and Murphy, 1988) and different strains share remarkably similar OMP profiles (Bartos and Murphy, 1988; Murphy and Bartos, 1989).
  • OMPs outer membrane proteins
  • At least three different surface-exposed outer membrane antigens have been shown to be well-conserved among M. catarrhalis strains; these include the 81 kDa CopB OMP (Helminen et al, 1993b), the heat- modifiable CD OMP (Murphy et al, 1993) and the high-molecular weight UspA antigen
  • the MAb designated 17C7, was described as binding to UspA, a very high molecular weight protein that migrated with an apparent molecular weight (in SDS-PAGE) of at least 250 kDa (Helminen et al, 1994; Klingman and Murphy, 1994).
  • MAb 17C7 enhanced pulmonary clearance of M. catarrhalis from the lungs of mice when used in passive immunization studies and, in colony blot radioimmunoassay analysis, bound to every isolate of M. catarrhalis examined. This same MAb also reacted, although less intensely, with another antigen band of approximately 100 kDa, as described in U.S. Patent No. 5,552,146 (incorporated herein by reference).
  • a recombinant bacteriophage that contained a fragment of M. catarrhalis chromosomal DNA that expressed a protein product that bound MAb 17C7 was also identified and migrated at a rate similar or indistinguishable from that of the native UspA antigen from M. catarrhalis (Helminen et al. , 1994).
  • epitopic core sequences of UspAl and UspA2 which can serve as the basis for the preparation of therapeutic or prophylactic compositions or vaccines which comprise peptides of 7, 10, 20, 30, 40, 50 or even 60 amino acids in length that elicit an antigenic reaction and a pharmaceutically acceptable buffer or diluent.
  • These peptides may be coupled to a carrier, adjuvant, another peptide or other molecule such that an effective antigenic response to M. catarrhalis is retained or even enhanced.
  • these peptides may act as carriers themselves when coupled to another peptide or other molecule that elicits an antigenic response to M. catarrhalis or another pathogen.
  • UspA2 can serve as a carrier for an oligosaccharide.
  • the epitopic core sequences of UspAl and UspA2 comprise one or more isolated peptides of 7, 10, 20, 30, 40, 50 or even 60 amino acids in length having the amino acid sequence AQQQDQH (SEQ ID NO: 17).
  • nucleic acids uspAl and uspA2, which encode the UspAl and the UspA2 antigens, respectively, as well as the amino acid sequences of the UspAl and UspA2 antigens of the M. catarrhalis isolates 035E, TTA24, TTA37, and 046E. It is envisioned that nucleic acid segments and fragments of the genes uspAl and uspA2 and the UspAl and UspA2 antigens will be of value in the preparation and use of therapeutic or prophylactic compositions or vaccines for treating, inhibiting or even preventing M. catarrhalis infections.
  • a method for inducing an immune response in a mammal comprising the step of providing to the mammal an antigenic composition that comprises an isolated peptide of about 20 to about 60 amino acids that contains the identified epitopic core sequence and a pharmaceutically acceptable buffer or diluent.
  • a method for diagnosing M. catarrhalis infection which comprises the step of determining the presence, in a sample, of an M. catarrhalis amino acid sequence corresponding to residues of the epitopic core sequences of either the UspAl or UspA2 antigen.
  • This method may comprise PCR TM detection of the nucleotide sequences or alternatively an immunologic reactivity of an antibody to either a UspA 1 or UspA2 antigen.
  • a method for treating an individual having an M. catarrhalis infection which comprises providing to the individual an isolated peptide of about 20 to about 60 amino acids that comprises at least about 10 consecutive residues of the amino acid sequence identified as an epitopic core sequence of UspAl or UspA2.
  • a method for preventing or limiting an M. catarrhalis infection comprises providing to a subject an antibody that reacts immunologically with the identified epitopic core region of either UspAl or UspA2 of M. catarrhalis.
  • a method for screening a peptide for reactivity with an antibody that binds immunologically to UspAl, UspA2 or both which comprises the steps of providing the peptide and contacting the peptide with the antibody and then
  • SUBSTITUTE SHEET RULE 25 determining the binding of the antibody to the peptide.
  • This method may comprise an immunoassay such as a western blot, an ELISA, an RIA or an immunoaffinity separation.
  • a method for screening a UspAl or UspA2 peptide for its ability to induce a protective immune response against M. catarrhalis by providing the peptide, administering it in a suitable form to an experimental animal, challenging the animal with M. catarrhalis and then assaying for an M. catarrhalis infection in the animal.
  • the animal used will be a mouse that is challenged by a pulmonary exposure to M. catarrhalis and that the assaying comprises assessing the degree of pulmonary clearance by the mouse.
  • FIG. 1 Southern blot analysis of PvwII-digested chromosomal DNA from strains of M. catarrhalis using a probe from the uspAl gene. Bacterial strain designations are at the top; kilobase (kb) position markers are on the left.
  • FIG. 2A Proteins present in whole cell lysates of the wild-type strain 035E and the isogenic uspAl mutant strain. These proteins were resolved by SDS-PAGE and stained with Coomassie blue.
  • the left lane (WT) contains the wild-type strain and right lane (MUT) contains
  • SUBSTITUTE SHEET (RULE 25) the mutant.
  • the arrows indicate the protein, approximately 120 kDa in size, that is present in the wild-type and missing in the mutant. Kilodalton position markers are on the left.
  • FIG.2B Western blot analysis of whole cell lysates of the wild-type strain 035E and the isogenic uspAl mutant strain. These proteins were resolved by SDS-PAGE and probed with MAb 17C7 in western blot analysis.
  • the left lane (WT) contains the wild-type strain and the right lane (MUT) contains the mutant. Kilodalton position markers are on the left. It can been seen that both strains possess the very high molecular weight band reactive with MAb 17C7 whereas only the wild-type strain also has a band of approximately 120 kDa that binds this MAb.
  • FIG. 2C Western blot analysis of whole cell lysate (WCL) and EDTA-extracted outer membrane vesicles (OMV) from the wild-type strain O35E (WT) and the isogenic uspAl mutant (MUT) using MAb 17C7.
  • Samples were either heated at 37°C for 15 minutes (H) or at 100°C for 5 minutes (B) prior to SDS-PAGE.
  • Molecular weight position markers (in kilodaltons) are indicated on the left.
  • the open arrow indicates the position of the very high molecular weight form of the MAb 17C7-reactive antigen;
  • the closed arrow indicates the position of the approximately 120 kDa protein;
  • the open circle indicates the position of the approximately 70-80 kDa protein.
  • FIG. 3 Southern blot analysis of chromosomal DNA from the wild-type M. catarrhalis strain 035E and the isogenic uspAl mutant. Chromosomal DNA was digested with Pvu and probed with a 0.6 kb BgHl-Pvull fragment from the uspAl gene. The wild-type strain is listed as O35E at the top of this figure and the mutant strain is listed as O35E-uspAl " . Kilobase position markers are present on the left side.
  • FIG. 4 Western blot reactivity of proteins in M. catarrhalis strain 035E outer membrane vesicles (labeled 035E OMV) and the MF-4-1 GST fusion protein (labeled GST fusion protein) with MAb 17C7.
  • FIG. 5 PCRTM products obtained by the use of the T3 and P 10 primers (middle lane - 0.9 kb product) and the T7 and P9 primers (right lane - 1.7 kb product) when used in a PCRTM
  • FIG. 6A-6C SDS-PAGE and westerns of purified proteins.
  • FIG. 6A Coomassie blue stained gel of purified UspA2 (lane 2).
  • FIG. 6B Coomassie blue stained gel of purified UspAl prepared without heating of sample (lane 4), heated for 3 min at 100°C (lane 5), heated for 5 min at 100°C (lane 6), and heated for 10 min at 100°C (lane 7).
  • FIG. 6C Western of the purified UspA2 (lane 9) and purified UspAl (lane 10) probed with the 17C7 MAb. Both proteins were heated 10 min.
  • the molecular size markers in lanes 1, 3, and 8 are as indicated in kilodaltons.
  • FIG. 7 Interaction of purified UspAl and UspA2 with HEp-2 cells as determined by ELISA.
  • HEp-2 cell monolayers cultured in 96-well plate were incubated with serially diluted UspAl or UspA2.
  • 035E bacterial strain was used as the positive control.
  • the bacteria were diluted analogous to the proteins beginning with a suspension with an A 550 of 1.0.
  • the bound proteins or attached bacteria were detected with a 1 :1 mixed antisera to UspAl and UspA2 as described in the methods.
  • FIG. 8 Interaction with fibronectin and vitronectin determined by dot blot.
  • the bound vitronectin was detected with rabbit polyclonal antibodies, the protein bound to the fibronectin was detected with pooled sera made against the UspAl and UspA2.
  • FIG. 9 The levels of antibodies to the protein UspAl, UspA2 and M. catarrhalis O35E strain in normal human sera.
  • Data are the log i0 transformed end-point titers of the IgG (FIGs. 9A-9C) and IgA (FIGs. 9D-9F) antibodies determined by ELISA.
  • the individual titers were plotted according to age group and the geometric mean titer for each age group linked by a solid line. Sera for the 2-18 month old children were consecutive samples from a group of ten children.
  • FIG. 10 Subclass distribution of IgG antibodies to UspAl and UspA2 in normal human sera.
  • FIG. 10A shows titers toward UspAl and
  • FIG. 10B shows titers to UspA2.
  • FIG. 11 Relationship of serum IgG titers to UspAl (FIG. 1 1A) and UspA2 (FIG. 1 IB) with the bactericidal liter against the 035E strain determined by logistic regression (p ⁇ 0.05).
  • the solid line indicates the linear relationship between the IgG titer and bactericidal titer.
  • Broken lines represent the 95 % confidence intervals of the linear fit.
  • FIG. 12 Schematic drawing showing the relative positions of decapeptides 10-24 within the region of UspAl and UspA2 which binds to MAb 17C7.
  • FIG. 13 Western dot blot analysis demonstrating reactivity of decapeptides 10-24 with MAb 17C7.
  • FIG. 14 Partial restriction enzyme map of the uspAl (FIG. 14A) and uspA2 (FIG. 14B) genes from M. catarrhalis strain 035E and the mutated versions of these genes.
  • the shaded boxes indicate the open reading frame of each gene.
  • Relevant restriction sites are indicated.
  • PCRTM primer sites P1-P6 are indicated by arrows.
  • the DNA fragments containing the partial uspAl and uspA2 open reading frames that were derived from M. catarrhalis strain O35E chromosomal DNA by PCRTM and cloned into pBluescriptll SK+ are indicated by black bars.
  • Dotted lines connect corresponding restriction sites on these DNA inserts and the chromosome. Open bars indicate the location of the kanamycin or chloramphenicol cassettes, respectively.
  • the DNA probes specific for uspAl or uspA2 are indicated by the appropriate cross-hatched bars and were amplified by PCRTM from M. catarrhalis strain 035E chromosomal DNA by the use of the oligonucleotide primer pairs
  • FIG. 15. Detection of the UspAl and UspA2 proteins in wild-type and mutant strains of M catarrhalis O35E. Proteins present in EDTA-extracted outer membrane vesicles from the wild-type strain (lane 1), the uspAl mutant strain 035E.1 (lane 2), the uspA2 mutant strain
  • FIG. 15 A the closed arrow indicates the very high molecular weight form of the UspA antigen which is comprised of both UspAl and UspA2.
  • FIG. 15B the bracket on the left indicates the very high molecular weight forms of the UspAl and UspA2 proteins that bind MAb 17C7.
  • the open arrow indicates the 120 kDa, putative monomeric form of UspAl .
  • the closed arrow indicates the 85 kDa, putative monomeric form of UspA2.
  • Molecular weight position markers in kilodaltons
  • FIG. 16 Comparison of the rate and extent of growth of the wild-type and mutant strains of M. catarrhalis.
  • the wild-type strain 035E (closed squares), the uspAl mutant O35E.1 (open squares), the uspA2 mutant O35E.2 (closed circles), and the uspAl uspA2 double mutant 035E.12 (open circles) of M. catarrhalis O35E from overnight broth cultures were diluted to a density of 35 Klett units in BHI broth and subsequently allowed to grow at 37° with shaking. Growth was followed by means of turbidity measurements.
  • FIG. 17 Susceptibility of wild-type and mutant strains of M. catarrhalis to killing by normal human serum.
  • Cells of the wild-type parent strain 035E (diamonds), uspAl mutant O35E.1 (triangles), uspA2 mutant O35E.2 (circles), and uspAl uspA2 double mutant 035E.12 (squares) from logarithmic-phase BHI broth cultures were incubated in the presence of 10% (v/v) normal human serum (closed symbols) or heat-inactivated normal human serum (open symbols). Data are presented as the percentage of the original inoculum remaining at each time point.
  • the present invention relates to the identification of epitopes useful for developing potential vaccines against M. catarrhalis.
  • isolation of the protein which contained the epitope yielded unexpected results.
  • MAb 17C7 recognized a single epitope, but the characteristics of the protein associated with the epitope suggested the existence
  • the present invention provides insights into the antigenic structure of the UspA protein based on the analysis of the sequences of the UspAl and UspA2 proteins which comprise the protein. Characterization of the epitopic region of the molecule that is targeted by the MAb 17C7 permits the development of agents that will be useful in protecting against M. catarrhalis infections, e.g., in the preparation of prophylactic reagents. Particular embodiments relate to the amino acid and nucleic acids corresponding to the UspAl and UspA2 proteins, peptides and antigenic compositions derived therefrom, and methods for the diagnosis and treatment of M. catarrhalis disease.
  • the present invention provides for the identification of the proteins UspAl and UspA2 from M. catarrhalis strain 035E.
  • the UspAl protein comprises about 831 amino acid residues and has a predicted mass of about 88,271 daltons (SEQ ID NO:
  • the UspA2 protein comprises about 576 residues and has a predicted mass of about
  • UspA2 is not a truncated or processed form of UspAl .
  • the present invention has identified the specific epitope to which MAb 17C7 binds.
  • a common peptide sequence designated as the "3Q" peptide, found between amino acid residues 480-502 and 582-604 of the UspAl protein (SEQ ID NO:l) and
  • SUBSTTTUTE SHEET (RULE 25) residues 355-377 of the UspA2 protein (SEQ ID NO:3) of M. catarrhalis strain 035E, encompasses the region which appears to be recognized by MAb 17C7. (Note that numbering of the amino acid residues is based upon strain 035E as provided in SEQ ID NO:3.) It is envisioned that this region plays an important role in the biology of the pathogen and, from this information, one will deduce amino acids residues that are critical in MAb 17C7 antibody binding. It also is envisioned that, based upon this information, one will be able to design epitopic regions that have either a higher or lower affinity for the MAb 17C7 or other antibodies. Further embodiments of the present invention are discussed below.
  • the present invention provides DNA segments, vectors and the like comprising at least one isolated gene, DNA segment or coding region that encodes a M. catarrhalis UspAl or UspA2 protein, polypeptide, domain, peptide or any fusion protein thereof.
  • a M. catarrhalis uspAl gene comprising about 2493 base pairs (bp) (SEQ ID NO:
  • strain O35E about 3381 bp (SEQ ID NO:6) of strain O46E, about 3538 bp (SEQ ID NO: 10) of strain TTA24, or about 3292 bp (SEQ ID NO: 14) of strain TTA37.
  • strain 046E about 2673 bp (SEQ ID NO:12), or about 4228 bp (SEQ ID NO:16) of strain TTA37.
  • the uspAl and uspA2 genes will be useful in the preparation of proteins, antibodies, screening assays for potential candidate drugs and the like to treat or inhibit, or even prevent, M. catarrhalis infections.
  • the present invention also provides for the use of the UspAl or UspA2 proteins or peptides as immunogenic carriers of other agents which are useful for the treatment, inhibition or even prevention of other bacterial, viral or parasitic infections. It is envisioned that either the UspAl or UspA2 antigen, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids such that a bispecific or multivalent composition or vaccine which is useful for the treatment, inhibition or even prevention of infection by M. catarrhalis and another pathogen(s) is prepared.
  • compositions will be familiar to those of skill in the art and, for example, similar to those used to prepare conjugates to keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyannin
  • BSA bovine serum albumin
  • the present invention in one embodiment, encompasses the two new protein sequences, UspAl and UspA2, and the peptide sequence AQQQDQH (SEQ ID NO: 17) identified as the target epitope of MAb 17C7.
  • the amino acid sequences of the UspAl and UspA2 proteins from four strains of M. catarrhalis indicated that each protein contained at least one copy of the peptide YELAQQQDQH (SEQ ID NO: 18) which binds Mab 17C7 or, in one instance, a peptide nearly identical and having the amino acid sequence YDLAQQQDQH (SEQ ID NO:19).
  • the peptide (YELAQQQDQH, SEQ ID NO: 18) occurs twice in UspAl from strain O35E at residues 486-495 and 588-597 (SEQ ID NOT) and once in UspA2 from strain O35E at residues 358-367 (SEQ ID NO:3). It occurs once in UspAl from strain TTA24 at residues 497- 506 (SEQ ID NO:9) and twice in UspA2 from strain TTA24 at residues 225-234 and 413-422 (SEQ ID NOT 1).
  • the peptide YDLAQQQDQH (SEQ ID NO: 19) occurs once in UspAl from strain 046E at residues 448-457 (SEQ ID NO:5) whereas the peptide YELAQQQDQH (SEQ ID NO: 18) occurs once in this same protein at residues 649-658 (SEQ ID NO:5).
  • YELAQQQDQH (SEQ ID NO: 18) occurs once in UspA2 from strain O46E at residues 416-425 (SEQ ID NO:7).
  • the peptide YELAQQQDQH (SEQ ID NO: 18) occurs twice in UspAl from strain TTA37 at residues 478-487 and 630-639 (SEQ ID NO: 13) and twice in UspA2 from strain TTA37 at residues 522-531 and 681-690 (SEQ ID NO: 15).
  • hybrid molecules containing portions from one UspA protein for example the UspAl protein, fused with portions of the other UspA protein, in this example the UspA2 protein, or fused with other proteins which are useful for identification, such as kanamycin-resistance, or other purposes in the screening of potential vaccines or further characterization of the UspAl and UspA2 proteins.
  • a fusion could be generated with sequences from three, four or even five peptide regions represented in a
  • UspAl and UspA2 proteins may be advantageously cleaved into fragments for use in further structural or functional analysis, or in the generation of reagents such as UspA-related polypeptides and UspA-specific antibodies.
  • This can be accomplished by treating purified or unpurified UspAl and/or UspA2 with a peptidase such as endoproteinase glu-C (Boehringer, Indianapolis, IN).
  • Treatment with CNBr is another method by which UspAl and/or UspA2 fragments may be produced from their natural respective proteins.
  • SUBSTITUTE SHEET RULE 25 be conferred upon a polypeptide which result in increased activity or stability.
  • amino acid substitutions in certain polypeptides may be utilized to provide residues which may then be linked to other molecules to provide peptide-molecule conjugates which retain enough antigenicity of the starting peptide to be useful for other purposes.
  • a selected UspAl or UspA2 peptide bound to a solid support might be constructed which would have particular advantages in diagnostic embodiments.
  • hydropathic index of amino acids in conferring interactive biological function on a protein has been discussed generally by Kyte & Doolittle (1982), wherein it is found that certain amino acids may be substituted for other amino acids having a similar hydropathic index or core and still retain a similar biological activity.
  • amino acids are assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with substrate molecules.
  • Preferred substitutions which result in an antigenically equivalent peptide or protein will generally involve amino acids having index scores within ⁇ 2 units of one another, and more preferably within ⁇ 1 unit, and even more preferably, within ⁇ 0.5 units.
  • isoleucine which has a hydropathic index of +4.5
  • an amino acid such as valine (+ 4.2) or leucine (+ 3.8).
  • lysine (- 3.9) will preferably be substituted for arginine (-4.5), and so on.
  • substitution of like amino acids may also be made on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments.
  • U.S. Patent 4,554,101 incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with an important biological property of the protein.
  • each amino acid has also been assigned a hydrophilicity value. These values are detailed below in Table III.
  • one amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of R-group substituents, for example, in terms of size, electrophilic character, charge, and the like.
  • preferred substitutions which take various of the foregoing characteristics into
  • SUBSTITUTE SHEET RULE 25 consideration will be known to those of skill in the art and include, for example, the following combinations: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • peptides derived from these polypeptides are contemplated.
  • such peptides may comprise about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 consecutive residues.
  • a peptide that comprises 6 consecutive amino acid residues may comprise residues 1 to 6, 2 to 7, 3 to 8 and so on of the UspAl or UspA2 protein.
  • Such peptides may be represented by the formula
  • Syntheses of peptides are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of a commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptides synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
  • peptides may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity.
  • agents including buffers such as Tris or phosphate buffers to maintain a pH of 7.0 to 7.5.
  • agents which will inhibit microbial growth such as sodium azide or
  • SUBSTTTUTE SHEET (RULE 25) Merthiolate.
  • the solutions For extended storage in an aqueous state it will be desirable to store the solutions at 4°C, or more preferably, frozen.
  • the peptide(s) may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled, deionized) or buffer prior to use.
  • an epitopic core sequence is a relatively short stretch of amino acids that is structurally “complementary” to, and therefore will bind to, binding sites on antibodies or T-cell receptors. It will be understood that, in the context of the present disclosure, the term “complementary” refers to amino acids or peptides that exhibit an attractive force towards each other.
  • certain epitopic core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the corresponding UspA antigen to the corresponding UspA- directed antisera.
  • the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences.
  • the smallest useful core sequence anticipated by the present disclosure would be on the order of about 6 amino acids in length.
  • this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.
  • the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
  • the present invention also encompasses nucleic acids encoding the UspAl (SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO: 10 and SEQ ID NO: 14) and UspA2 (SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 12 and SEQ ID NO: 16) proteins from the exemplary M. catarrhalis strains 035E, 046E, TTA24 and TTA37, respectively. Because of the degeneracy of the genetic code, many other nucleic acids also may encode a given UspAl or UspA2 protein.
  • codons For example, four different three-base codons encode the amino acids alanine, glycine, proline, threonine and valine, while six different codons encode arginine, leucine and serine. Only methionine and tryptophan are encoded by a single codon. Table I provides a list of amino acids and their corresponding codons for use in such embodiments. In order to generate any nucleic acid encoding UspAl or UspA2, one need only refer to the codon table provided herein. Substitution of the natural codon with any codon encoding the same amino acid will result in a distinct nucleic acid that encodes UspAl or UspA2. As a practical matter, this can be accomplished by site-directed mutagenesis of an existing uspAl or uspA2 gene or de novo chemical synthesis of one or more nucleic acids.
  • substitutional mutants generated by site-directed changes in the nucleic acid sequence that are designed to alter one or more codons of a given polypeptide or epitope may provide a more convenient way of generating large numbers of mutants in a rapid fashion.
  • the nucleic acids of the present invention provide for a simple way to generate fragments (e.g., truncations) of UspAl or UspA2, UspAl-UspA2 fusion molecules (discussed above) and UspAl or UspA2 fusions with other molecules.
  • fragments e.g., truncations
  • UspAl or UspA2 fusion molecules discussed above
  • UspAl or UspA2 fusions with other molecules e.g., utilization of restriction enzymes and nuclease in the uspAl or uspA2 gene permits one to manipulate the structure of these genes, and the resulting gene products.
  • nucleic acid sequence information provided by the present disclosure also allows for the preparation of relatively short DNA (or RNA) sequences that have the ability to specifically hybridize to gene sequences of the selected uspAl or uspA2 gene.
  • nucleic acid probes of an appropriate length are prepared based on a consideration of the coding sequence of the uspAl or uspA2 gene, or flanking regions near the uspAl or uspA2 gene, such as regions downstream and upstream in the M. catarrhalis chromosome. The ability of such
  • nucleic acid probes to specifically hybridize to either uspAl or uspA2 gene sequences lends them particular utility in a variety of embodiments.
  • the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample.
  • these oligonucleotides can be inserted, in frame, into expression constructs for the purpose of screening the corresponding peptides for reactivity with existing antibodies or for the ability to generate diagnostic or therapeutic reagents.
  • the preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 10 to 20, or so, nucleotide stretch of the sequence, although sequences of 30 to 60 or so nucleotides are also envisioned to be useful.
  • a size of at least 9 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective.
  • molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of the specific hybrid molecules obtained.
  • nucleic acid molecules having either uspAl or uspA2 gene-complementary stretches of 15 to 20 nucleotides, or even longer, such as 30 to 60, where desired.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant production.
  • probes that would be useful may be derived from any portion of the sequences of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16. Therefore, probes are specifically contemplated that comprise nucleotides 1 to 9, or 2 to 10, or 3 to 11 and so forth up to a probe comprising the last 9 nucleotides of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or
  • each probe would comprise at least about 9 linear nucleotides of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16., designated by the formula "n to n + 8," where n is an integer from 1 to the number of nucleotides in the sequence. Longer probes that hybridize to the uspAl or uspA2 gene under low, medium, medium-high and high stringency conditions are
  • SUBSTITUTE 5HEET RULE 25 also contemplated, including those that comprise the entire nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO: 12 or SEQ ID NO.T4 or SEQ ID NO: 16. This hypothetical may be repeated for probes having lengths of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 and greater bases.
  • the probes of the present invention will find particular utility as the basis for diagnostic hybridization assays for detecting UspAl or UspA2 DNA in clinical samples.
  • Exemplary clinical samples that can be used in the diagnosis of infections are thus any samples which could possibly include Moraxella nucleic acid, including middle ear fluid, sputum, mucus, bronchoalveolar fluid, amniotic fluid or the like.
  • hybridization techniques and systems are known which can be used in connection with the hybridization aspects of the invention, including diagnostic assays such as those described in Falkow et al, U.S. Patent 4,358,535.
  • diagnostic assays such as those described in Falkow et al, U.S. Patent 4,358,535.
  • relatively stringent conditions for applications requiring a high degree of selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, for example, one will select relatively low salt and/or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C to 70°C. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
  • mutant clone colonies growing on solid media which contain variants of the UspAl and/or UspA2 sequence could be identified on duplicate filters using hybridization conditions and methods, such as those used in colony blot assays, to obtain hybridization only between probes containing sequence variants and nucleic acid sequence variants contained in specific colonies.
  • small hybridization probes containing short variant sequences of either the uspAl or uspA2 gene may be utilized to identify those clones growing on solid media which contain sequence variants of the entire uspAl or uspA2 gene. These clones can then be grown to obtain desired quantities of the variant UspAl or UspA2 nucleic acid sequences or the corresponding UspA antigen.
  • nucleic acid sequences of the present invention are used in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
  • colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with pathogen nucleic acid-containing samples.
  • the hybridization probes described herein will be useful both as reagents in solution hybridizations as well as in embodiments employing a solid phase.
  • the test DNA (or RNA) from suspected clinical samples such as exudates, body fluids (e.g., amniotic fluid, middle ear effusion, bronchoalveolar lavage fluid) or even tissues, is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
  • SUBSTTTUTE SHEET (RULE 25) nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
  • the nucleic acid sequences which encode for the UspAl and/or UspA2 epitopes, or their variants, may be useful in conjunction with PCRTM methodology to detect M. catarrhalis.
  • PCRTM technology as set out, e.g. , in U.S. Patent 4,603,102, one may utilize various portions of either the uspAl or uspA2 sequence as oligonucleotide probes for the PCRTM amplification of a defined portion of a uspAl or uspA2 nucleic acid in a sample.
  • the amplified portion of the uspAl or uspA2 sequence may then be detected by hybridization with a hybridization probe containing a complementary sequence. In this manner, extremely small concentrations of M. catarrhalis nucleic acid may detected in a sample utilizing uspAl or uspA2 sequences.
  • the expression cassette contains a UspAl and/or UspA2-encoding nucleic acid under transcriptional control of a promoter.
  • a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoters most commonly used in prokaryotic recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al, 1977; Goeddel et al, 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1980; EPO Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors
  • the appropriate expression cassette can be inserted into a commercially available expression vector by standard subcloning techniques.
  • E. coli vectors pUC or pBluescriptTM may be used according to the present invention to produce recombinant UspAl and/or UspA2 polypeptide in vitro.
  • the manipulation of these vectors is well known in the art.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli vectors pUC or pBluescriptTM may be used according to the present invention to produce recombinant UspAl and/or UspA2 polypeptide in vitro.
  • the manipulation of these vectors is well known in the art.
  • coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar et al, 1977).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as a transforming vector in connection with these hosts.
  • the phage lambda GEM -1 1 may be utilized in making recombinant phage vector which can be used to transform host cells, such as E. coli LE392.
  • the UspA antigen is expressed as a fusion protein by using the pGEX4T-2 protein fusion system (Pharmacia LKB, Piscataway, NJ), allowing characterization of the UspA antigen as comprising both the UspAl and UspA2 proteins.
  • fusion protein expression systems are the glutathione S-transferase system (Pharmacia,
  • fusion systems produce recombinant protein bearing only a small number of additional amino acids, which are unlikely to affect the functional capacity of the recombinant protein.
  • FLAG system and 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the protein to its native conformation.
  • Other fusion systems produce proteins where it is desirable to excise the fusion partner from the desired protein.
  • the fusion partner is linked to the
  • SUBSTITUTE SHEET (RULE 25) recombinant protein by a peptide sequence containing a specific recognition sequence for a protease.
  • suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA).
  • E. coli is a preferred prokaryotic host.
  • E. coli strain RR1 is particularly useful.
  • Other microbial strains which may be used include E. coli strains such as E. coli LE392, E. coli B, and E. coli X 1776 (ATCC No. 31537).
  • the aforementioned strains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may be used. These examples are, of course, intended to be illustrative rather than limiting.
  • Recombinant bacterial cells for example E. coli
  • suitable media for example LB
  • the cells are collected by centrifugation and washed to remove residual media.
  • the bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a selective speed.
  • the recombinant protein is expressed in the inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g. 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents such as ⁇ - mercaptoethanol or DTT (dithiothreitol).
  • urea e.g. 8M
  • chaotropic agents such as guanidine hydrochloride
  • reducing agents such as ⁇ - mercaptoethanol or DTT (dithiothreitol).
  • polypeptide may be advantageous to incubate the polypeptide for several hours under conditions suitable for the protein to undergo a refolding process into a conformation which more closely resembles that of the native protein.
  • conditions generally include low protein concentrations less than 500 ⁇ g/ml, low levels of reducing agent, concentrations of urea
  • SUBSTITUTE SHEET RULE 25 less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulfide bonds within the protein molecule.
  • the refolding process can be monitored, for example, by SDS-PAGE or with antibodies which are specific for the native molecule (which can be obtained from animals vaccinated with the native molecule isolated from bacteria).
  • the protein can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns.
  • the expression construct may comprise a virus or engineered construct derived from a viral genome.
  • viruses The ability of certain viruses to enter cells via receptor-mediated endocytosis and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).
  • the first viruses used as vectors were DNA viruses including the papovaviruses (simian virus 40 (SV40), bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986) and adeno-associated viruses.
  • Retroviruses also are attractive gene transfer vehicles (Nicolas and Rubenstein, 1988; Temin, 1986) as are vaccina virus (Ridgeway, 1988) adeno-associated virus (Ridgeway, 1988) and herpes simplex virus (HSV) (Glorioso et al, 1995).
  • Such vectors may be used to (i) transform cell lines in vitro for the purpose of expressing proteins of interest or (ii) to transform cells in vitro or in vivo to provide therapeutic polypeptides in a gene therapy scenario.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • SUBSTITUTE SHEET (RULE 25) At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a
  • TATA box such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
  • the particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • Preferred promoters include those derived from HSV, including the ⁇ 4 promoter.
  • Another preferred embodiment is the tetracycline controlled promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • Table IV lists several promoters which may be employed, in the context of the present invention, to regulate the expression of a transgene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of transgene expression but, merely, to be exemplary thereof.
  • Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Table V lists several enhancers, of course, this list is not meant to be limiting but exemplary.
  • Base EPDB could also be used to drive expression of a transgene.
  • Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • Host cells include eukaryotic microbes, such as yeast cultures may also be used.
  • Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • the plasmid YRp7 for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid already contains the
  • SUBSTITUTE SHEET (RULE 25) trp ⁇ gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977).
  • the presence of the trp ⁇ lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Suitable promoting sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (Hitzeman et al, 1980) or other glycolytic enzymes (Hess et al, 1968; Holland et al, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • 3- phosphoglycerate kinase Hitzeman et al, 1980
  • other glycolytic enzymes Hess et al, 1968; Holland et al, 1978
  • enolase glyceraldehyde-3-phosphate dehydrogena
  • the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • Other promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973).
  • useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • Antibodies to UspAl or UspA2 peptides or polypeptides may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., purified or partially purified protein, synthetic protein or fragments thereof, as discussed in the section on vaccines.
  • Animals to be immunized are mammals such as cats, dogs and horses, although there is no limitation other than that the subject be capable of mounting an immune response of some kind.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is possible. The use of rats may provide certain advantages, but mice are preferred, with the BALB/c mouse being most preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol.
  • B cells B lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer removed.
  • Spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5
  • the antibody-producing B cells from the immunized animal are then fused with cells of an immortal myeloma cell line, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells, called "hybridomas.”
  • SUBSTITUTE SHEET (RULE 25) Any one of a number of myeloma cells may be used and these are known to those of skill in the art.
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
  • NS-1 myeloma cell line (also termed
  • mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described by Kohler & Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate.
  • Fusion procedures usually produce viable hybrids at low frequencies, about 1 10 " to 1 10 . This does not pose a problem, however, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culture in a selective medium.
  • the selective medium generally is one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • SUBSTTTUTE SHEET (RULE 25)
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma can be injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • Monoclonal antibodies of the present invention also include anti-idiotypic antibodies produced by methods well-known in the art.
  • Monoclonal antibodies according to the present invention also may be monoclonal heteroconjugates, i.e., hybrids of two or more antibody molecules.
  • monoclonal antibodies according to the invention are chimeric monoclonal antibodies.
  • the chimeric monoclonal antibody is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region
  • SUBSTITUTE SHEET (RULE 25) sequences from a mouse antibody producing cell and the constant-region exons from a human antibody gene.
  • the antibody encoded by such a recombinant gene is a mouse-human chimera. Its antibody specificity is determined by the variable region derived from mouse sequences. Its isotype, which is determined by the constant region, is derived from human DNA.
  • the monoclonal antibody according to the present invention is a
  • “humanized” monoclonal antibody produced by techniques well-known in the art. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse Ig into a human V-domain, followed by the replacement of some human residues in the framework regions of their murine counterparts.
  • “Humanized” monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and therapeutic methods for treating Moraxella infections.
  • the monoclonal antibodies and fragments thereof according to this invention can be multiplied according to in vitro and in vivo methods well-known in the art.
  • Multiplication in vitro is carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth-sustaining supplements e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • feeder cells such as normal mouse peritoneal exudate cells, spleen
  • Monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo.
  • Cell clones are injected into mammals which are histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to injection.
  • fragments of the monoclonal antibody of the invention can be obtained from monoclonal antibodies produced as described above, by methods which include digestion with enzymes such as pepsin or papain and/or cleavage of
  • SUBSTITUTE SHEET (RULE 25) disulfide bonds by chemical reduction.
  • monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or they may be produced manually using techniques well known in the art.
  • the monoclonal conjugates of the present invention are prepared by methods known in the art. e.g., by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as 3 H, l25 I, I31 1 32 P, 35 S, 14 C, Cr, Cl, 3 Co, Co, Fe, Se, Eu, and mTc, are other useful labels which can be conjugated to antibodies.
  • a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate.
  • Conjugates with metal chelates are
  • Radio-labeled monoclonal antibodies of the present invention are produced according to well-known methods in the art.
  • monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • Monoclonal antibodies according to the invention may be labeled with technetium- m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.
  • a reducing agent such as SNC1 2
  • a buffer solution such as sodium-potassium phthalate solution
  • the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and western blot methods, as well as other procedures which may utilize antibodies specific to CopB epitopes. While ELISAs are preferred, it will be readily appreciated that such assays include RIAs and other non-enzyme linked antibody binding assays or procedures. Additionally, it is proposed that monoclonal antibodies specific to the particular UspA epitope may be utilized in other useful applications. For example, their use in immunoabsorbent protocols may be useful in purifying native or recombinant UspA proteins or variants thereof.
  • UspAl and UspA2 mutant peptides may be screened, in immunoassay format, for reactivity against UspAl- or UspA2-specific antibodies, such as MAb 17C7. In this way, a mutational analysis of various epitopes may be performed. Results from such analyses may then be used to determine which additional UspAl or UspA2 epitopes may be recognized by antibodies and useful in the preparation of potential vaccines for Moraxella.
  • Diagnostic immunoassays include direct culturing of bodily fluids, either in liquid culture or on a solid support such as nutrient agar.
  • a typical assay involves collecting a sample of bodily fluid from a patient and placing the sample in conditions optimum for growth of the pathogen. The determination can then be made as to whether the microbe exists in the sample.
  • Immunoassays encompassed by the present invention include, but are not limited to those described in U.S. Patent No. 4,367,1 10 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.
  • Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELI S As) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.
  • ELI S As enzyme linked immunosorbent assays
  • RIAs radioimmunoassays
  • the anti-UspA antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the desired antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen, that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a
  • SUBSTITUTE SHEET (RULE 25) third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the UspA antigen are immobilized onto the well surface and then contacted with the anti-UspA antibodies. After binding and appropriate washing, the bound immune complexes are detected. Where the initial antigen specific antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antigen specific antibody, with the second antibody being linked to a detectable label.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the primary antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed.
  • This system may provide for signal amplification if desired.
  • Competition ELISAs are also possible in which test samples compete for binding with known amounts of labeled antigens or antibodies.
  • the amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells.
  • Antigen or antibodies may also be linked to a solid support, such as in the form of beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody.
  • the presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
  • SUBSTITUTE SHEET (RULE 25) In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin
  • BSA casein and solutions of milk powder.
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • the layered antisera is then allowed to incubate for from 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C. Following incubation, the antisera- contacted surface is washed so as to remove non-immunocomplexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first.
  • the second antibody will preferably be an antibody having specificity in general for human IgG.
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
  • SUBSTITUTE SHEET (RULE 25) After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid [ABTS] and H 2 0 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. Alternatively, the label may be a chemilluminescent one. The use of such labels is described in U.S. Patent Nos. 5,310,687, 5,238,808 and 5,221,605.
  • UspAl or UspA2 polypeptides or UspAl- or UspA2-derived peptides may be used as vaccine formulations to generate protective anti- catarrhalis antibody responses in vivo.
  • protective it is only meant that the immune system of a treated individual is capable of generating a response that reduces, to any extent, the clinical impact of the bacterial infection. This may range from a minimal decrease in bacterial burden to outright prevention of infection. Ideally, the treated subject will not exhibit the more serious clinical manifestations of M. catarrhalis infection.
  • immunoprophylaxis involves the administration, to a subject at risk, of a vaccine composition.
  • the vaccine composition will contain a UspAl and/or UspA2 polypeptide or immunogenic derivative thereof in a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier diluent or excipient.
  • SUBSTITUTE 5HEET (RULE 25)
  • the stability and immunogenicity of UspAl and UspA2 antigens may vary and, therefore, it may be desirable to couple the antigen to a carrier molecule.
  • exemplary carriers are KLH, BSA, human serum albumin, myoglobin, ⁇ -galactosidase, penicillinase, CRM 197 and bacterial toxoids, such as diphtheria toxoid and tetanus toxoid.
  • Synthetic carriers such as multi-poly-DL-alanyl-poly-L-lysine and poly-L- lysine also are contemplated. Coupling generally is accomplished through amino or carboxyl- terminal residues of the antigen, thereby affording the peptide or polypeptide the greatest chance of assuming a relatively "native" conformation following coupling.
  • UspA2 antigen such that the UspAl or UspA2 antigen acts as the carrier molecule.
  • agents which protect against other pathogenic organisms such as bacteria, viruses or parasites, could be coupled to either a UspAl or UspA2 antigen to produce a multivalent vaccine or pharmaceutical composition which would be useful for the treatment or inhibition of both M. catarrhalis infection and other pathogenic infections.
  • either UspAl or UspA2 proteins or peptides could serve as immunogenic carriers for other vaccine components, for example, saccharides of pneumococcus, menigococcus or hemophylus influenza and could even be covalently coupled to these other components.
  • adjuvants which are known to stimulate the appropriate portion of the immune system of the vaccinated animal. Suitable adjuvants for the vaccination of subjects
  • StimulonTM QS-21 ; Aquila Biopharmaceuticals Inc., Wooster, MA
  • mineral gels such as aluminum hydroxide, aluminum phosphate, calcium phosphate and alum
  • surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyl- dioctadecylammonium bromide, N,N-dioctadecyl-N,N'-bis(2-hydroxyethyl)-propanediamine, methoxyhexadecylglycerol and pluronic polyols
  • polyanions such as pyran, dextran sulfate,
  • SUBSTITUTE SHEET polyacrylic acid and carbopol, peptides and amino acids such as muramyl dipeptide, dimethylglycine, tuftsin and trehalose dimycolate.
  • Agents include synthetic polymers of sugars (Carbopol), emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution , of a perfluorocarbon (Fluosol-DA) also may be employed.
  • vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601 ,903; 4,599,231; 4,599,230; 4,596,792; and 4.578,770, all incorporated herein by reference.
  • such vaccines are prepared as injectables.
  • Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
  • the vaccine preparations of the present invention also can be administered following incorporation into non-toxic carriers such as liposomes or other microcarrier substances, or after conjugation to polysaccharides, proteins or polymers or in combination with Quil-A to form "iscoms" (immunostimulating complexes). These complexes can serve to reduce the toxicity of the antigen, delay its clearance from the host and improve the immune response by acting as an adjuvant.
  • suitable adjuvants for use this embodiment of the present invention include INF, IL-2, IL-4, IL-8, IL-12 and other immunostimulatory compounds.
  • conjugates comprising the immunogen together with an integral membrane protein of prokaryotic origin, such as TraT may prove advantageous.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient
  • SUBSTTTUTE SHEET (RULE 25) in the range of 0.5% to 10%, preferably 1 -2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate. sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • the peptides may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as. for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
  • SUBSTITUTE SHEET (RULE 25) preferably one or more, usually at least about three vaccinations.
  • the vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies.
  • the course of the immunization may be followed by assays for antibodies for the supernatant antigens.
  • the assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.
  • Passive Immunotherapy Passive immunity is defined, for the purposes of this application, as the transfer to an organism of an immune response effector that was generated in another organism.
  • the classic example of establishing passive immunity is to transfer antibodies produced in one organism into a second, immunologically compatible animal.
  • immunologically compatible it is meant that the antibody can perform at least some of its immune functions in the new host animal.
  • other effectors such as certain kinds of lymphocytes, including cytotoxic and helper T cells, NK cells and other immune effector cells.
  • the present invention contemplates both of these approaches.
  • Antibodies, antisera and immune effector cells are raised using standard vaccination regimes in appropriate animals, as discussed above.
  • the primary animal is vaccinated with at least a microbe preparation or one bacterial product or by-product according to the present invention, with or without an adjuvant, to generate an immune response.
  • the immune response may be monitored, for example, by measurement of the levels of antibodies produced, using standard ELISA methods.
  • immune effector cells can be collected on a regular basis, usually from blood draws.
  • the antibody fraction can be purified from the blood by standard means, e.g., by protein A or protein G chromatography.
  • monoclonal antibody-producing hybridomas are prepared by standard means (Coligan et al, 1991). Monoclonal antibodies are then prepared from the hybridoma cells by standard means. If the primary host's monoclonal antibodies are not
  • SUBSTTTUTE SHEET (RULE 25) compatible with the animal to be treated, it is possible that genetic engineering of the cells can be employed to modify the antibody to be tolerated by the animal to be treated.
  • murine antibodies for example, may be “humanized” in this fashion.
  • Antibodies, antisera or immune effector cells are injected into hosts to provide passive immunity against microbial infestation.
  • an antibody composition is prepared by mixing, preferably homogeneously mixing, at least one antibody with at least one pharmaceutically or veterinarally acceptable carrier, diluent, or excipient using standard methods of pharmaceutical or veterinary preparation.
  • the amount of antibody required to produce a single dosage form will vary depending upon the microbial species being vaccinated against, the individual to be treated and the particular mode of administration.
  • the specific dose level for any particular individual will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the individual, time of administration, route of administration, rate of excretion, drug combination and the severity of the microbial infestation.
  • the antibody composition may be administered intravenously, subcutaneously, intranasally, orally, intramuscularly, vaginally, rectally, topically or via any other desired route. Repeated dosings may be necessary and will vary, for example, depending on the clinical setting, the particular microbe, the condition of the patient and the use of other therapies.
  • the invention also relates to a vaccine comprising a nucleic acid molecule encoding a
  • a vaccine is referred to herein as a nucleic acid vaccine or DNA vaccine and is useful for the genetic immunization of vertebrates.
  • genetic immunization refers to inoculation of a vertebrate, particularly a mammal such as a mouse or human, with a nucleic acid vaccine directed against a pathogenic agent, particularly M. catarrhalis, resulting in protection of the vertebrate against M.
  • nucleic acid vaccine or “DNA vaccine” as used herein, is a nucleic acid construct comprising a nucleic acid molecule encoding UspAl , UspA2 or an immunogenic epitope comprising SEQ ID NO: 17.
  • the nucleic acid construct can also include transcriptional promoter elements, enhancer elements, splicing signals, termination and polyadenylation signals, and other nucleic acid sequences.
  • the nucleic acid vaccine can be produced by standard methods. For example, using known methods, a nucleic acid (e.g., DNA) encoding UspAl or UspA2 can be inserted into an expression vector to construct a nucleic acid vaccine (see Maniatis et al, 1989). The individual vertebrate is inoculated with the nucleic acid vaccine (i.e., the nucleic acid vaccine is administered), using standard methods.
  • a nucleic acid e.g., DNA
  • UspAl or UspA2 can be inserted into an expression vector to construct a nucleic acid vaccine (see Maniatis et al, 1989).
  • the individual vertebrate is inoculated with the nucleic acid vaccine (i.e., the nucleic acid vaccine is administered), using standard methods.
  • the vertebrate can be inoculated subcutaneously, intravenously, intraperitoneally, intradermally, intramuscularly, topically, orally, rectally, nasally, buccally, vaginally, by inhalation spray, or via an implanted reservoir in dosage formulations containing conventional non-toxic, physiologically acceptable carriers or vehicles.
  • the vertebrate is inoculated with the nucleic acid vaccine through the use of a particle acceleration instrument (a "gene gun").
  • a particle acceleration instrument a "gene gun”
  • the form in which it is administered e.g., capsule, tablet, solution, emulsion
  • nose drops, inhalants or suppositories can be used.
  • the nucleic acid vaccine can be administered in conjunction with any suitable adjuvant.
  • the adjuvant is administered in a sufficient amount, which is that amount that is sufficient to generate an enhanced immune response to the nucleic acid vaccine.
  • the adjuvant can be administered prior to (e.g., 1 or more days before) inoculation with the nucleic acid vaccine; concurrently with (e.g., within 24 hours of) inoculation with the nucleic acid vaccine; contemporaneously (simultaneously) with the nucleic acid vaccine (e.g., the adjuvant is mixed with the nucleic acid vaccine, and the mixture is administered to the vertebrate); or after (e.g., 1 or more days after) inoculation with the nucleic acid vaccine.
  • the adjuvant can also be administered at more than one time (e.g., prior to inoculation with the nucleic acid vaccine and also after inoculation with the nucleic acid vaccine).
  • the term "in conjunction with” encompasses any time period, including those specifically described herein and combinations of the time periods specifically described herein, during which the adjuvant can be administered so as to generate an enhanced immune response to the nucleic acid vaccine
  • nucleic acid vaccine e.g., an increased antibody titer to the antigen encoded by the nucleic acid vaccine, or an
  • SUBSTTTUTE SHEET (RULE 25) increased antibody titer to M. catarrhalis).
  • the adjuvant and the nucleic acid vaccine can be administered at approximately the same location on the vertebrate; for example, both the adjuvant and the nucleic acid vaccine are administered at a marked site on a limb of the vertebrate.
  • the nucleic acid construct is co-administered with a transfection-facilitating agent.
  • the transfection-facilitating agent is dioctylglycylspermine (DOGS) (as exemplified in published PCT application publication no. WO 96/21356 and incorporated herein by reference).
  • DOGS dioctylglycylspermine
  • the transfection- facilitating agent is bupivicaine (as exemplified in U.S. Patent 5,593,972 and incorporated herein by reference).
  • Murine short-term pulmonary clearance models have now been developed (Unhanand et al, 1992; Verghese et al, 1990) which permit an evaluation of the interaction of M. catarrhalis with the lower respiratory tract as well as assessment of pathologic changes in the lungs.
  • This model reproducibly delivers an inoculum of bacteria to a localized peripheral segment of the murine lung. Bacteria multiply within the lung, but are eventually cleared as a result of (i) resident defense mechanisms, (ii) the development of an inflammatory response, and/or (iii) the development of a specific immune response.
  • serum IgG antibody can enter the alveolar spaces in the absence of an inflammatory response and enhance pulmonary clearance of nontypable H. influenzae (McGehee et al, 1989), a pathogen with a host range and disease spectrum nearly identical to those of M. catarrhalis.
  • the present invention provides methods for identifying new M. catarrhalis inhibitory compounds, which may be termed as "candidate substances," by screening for immunogenic activity with peptides that include one or more mutations to the identified immunogenic epitopic region. It is contemplated that such screening techniques will prove useful in the general identification of any compound that will serve the purpose of inhibiting, or even killing, M. catarrhalis, and in preferred embodiments, will provide candidate vaccine compounds.
  • useful compounds in this regard will in no way be limited to proteinaceous or peptidyl compounds.
  • the most useful pharmacological compounds for identification through application of the screening assays will be non-peptidyl in nature and, e.g. , which will serve to inhibit bacterial protein transcription through a tight binding or other chemical interaction.
  • Candidate substances may be obtained from libraries of synthetic chemicals, or from natural samples, such as rain forest and marine samples.
  • M. catarrhalis inhibitor To identify a M. catarrhalis inhibitor, one would simply conduct parallel or otherwise comparatively controlled immunoassays and identify a compound that inhibits the phenotype of M. catarrhalis. Those of skill in the art are familiar with the use of immunoassays for competitive screenings (for example refer to Sambrook et al. 1989).
  • a candidate substance Once a candidate substance is identified, one would measure the ability of the candidate substance to inhibit M. catarrhalis in the presence of the candidate substance. In general, one will desire to measure or otherwise determine the activity of M. catarrhalis in the absence of the added candidate substance relative to the activity in the presence of the candidate substance in order to assess the relative inhibitory capability of the candidate substance.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence
  • SUBSTTTUTE SHEET RULE 25 variants incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art.
  • the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
  • This primer is then annealed with the single- stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA polymerizing enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydrox ylamine, to obtain sequence variants.
  • Second Generation Inhibitors In addition to the inhibitory compounds initially identified, the inventor also contemplates that other sterically similar compounds may be formulated to mimic the key portions of the structure of the inhibitors. Such compounds, which may include peptidomimetics of peptide inhibitors, may be used in the same manner as the initial inhibitors.
  • Certain mimetics that mimic elements of protein secondary structure are designed using the rationale that the peptide backbone of proteins exists chiefly to orientate amino acid side chains in such a way as to facilitate molecular interactions.
  • a peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
  • ⁇ -turn structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
  • Nucleic acid sequence used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989).
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA.
  • RNA is used, it may be desired to convert the RNA to a cDNA.
  • primers that selectively hybridize to nucleic acids corresponding to UspAl or UspA2 protein or a mutant thereof are contacted with the isolated nucleic acid under conditions that permit selective hybridization.
  • the term "primer”, as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template- dependent process.
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.
  • the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
  • the amplification product is detected.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).
  • PCRTM polymerase chain reaction
  • SUBSTTTUTE SHEET RULE 25 Briefly, in PCRTM, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a DNA polymerase e.g., Taq polymerase
  • a reverse transcriptase PCRTM (RT-PCRTM) amplification procedure may be performed in order to quantify the amount of mRNA amplified or to prepare cDNA from the desired mRNA.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed December 21 , 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT US87/00880, incorporated herein by reference, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • SUBST E 25 An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]- triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • Target specific sequences can also be detected using a cyclic probe reaction (CPR).
  • CPR a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • primers are used in a PCRTM-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • the target sequence After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR 3SR
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • an RNA polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • ssDNA target single-stranded DNA
  • This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include “RACE” and “one-sided PCR” (Frohman, 1990, incorporated by reference).
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used in the amplification step of the present invention.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al, 1989.
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.
  • Amplification products must be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled, nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non- covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
  • kits This generally will comprise preselected primers for specific markers. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker primer pair.
  • Preferred pairs of primers for amplifying nucleic acids are selected to amplify the sequences specified in SEQ ID NO: 1
  • nucleic acid fragments are prepared that include a contiguous stretch of nucleotides identical to for example about 15, 20, 25, 30, 35, etc. ; 48, 49, 50, 51, etc. ; 75, 76, 77, 78, 79, 80 etc. ; 100, 101, 102, 103 etc. ; 1 18, 1 19, 120, 121 etc.; 127, 128, 129, 130, 131, etc.; 316, 317, 318, 319, etc.
  • Similar fragments may be prepared which are identical or complimentary to, for example, SEQ ID NO: 1 such that the fragments do not hybridize to, for example, SEQ ID NO:3.
  • kits will comprise hybridization probes specific for UspAl or UspA2 proteins chosen from a group including nucleic acids corresponding to the sequences specified in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16 or to intermediate lengths of the sequences specified.
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker hybridization probe.
  • one method of screening for genetic variation is based on RNase cleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes.
  • mismatch is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single and multiple base point mutations.
  • U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as +.
  • RNase mismatch cleavage assays including those performed according to U.S. Patent No. 4,946,773, require the use of radiolabeled RNA probes.
  • Myers and Maniatis in U.S. Patent No. 4,946,773 describe the detection of base pair mismatches using RNase A.
  • Other investigators have described the use of E. coli enzyme, RNase I, in mismatch assays. Because it has broader cleavage specificity than RNase A, RNase I would be a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches.
  • the use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufficiently high.
  • the RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution.
  • the assay relies on being able to easily generate high specific activity radiolabeled RNA probes complementary to the mRNA of interest by in vitro transcription.
  • the templates for in vitro transcription were recombinant plasmids containing bacteriophage promoters.
  • the probes are mixed with total cellular RNA samples to permit hybridization to their complementary targets, then the mixture is treated with RNase to degrade excess unhybridized probe.
  • the RNase used is specific for single- stranded RNA, so that hybridized double-stranded probe is protected from degradation. After inactivation and removal of the RNase, the protected probe (which is proportional in amount to the amount of target mRNA that was present) is recovered and analyzed on a polyacrylamide gel.
  • the RNase Protection assay was adapted for detection of single base mutations.
  • radiolabeled RNA probes transcribed in vitro from wild type sequences are hybridized to complementary target regions derived from test samples.
  • the test target generally comprises DNA (either genomic DNA or DNA amplified by cloning in plasmids or by PCRTM), although RNA targets (endogenous mRNA) have occasionally been used. If single nucleotide (or greater) sequence differences occur between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position ("mismatch”) can be recognized and cleaved in some cases by single-strand specific
  • M. catarrhalis strains FR3227 and FR2336 were obtained from Richard Wallace, University of Texas Health Center, Tyler, TX.
  • M. catarrhalis strain B6 was obtained from Elliot Juni, University of Michigan, Ann Arbor, MI.
  • M. catarrhalis strain TTA1 was obtained from Steven Berk, East Tennessee State University, Johnson City, TN.
  • M. catarrhalis strain 25240 was obtained from the American Type Culture Collection, Rockville, MD. M. catarrhalis strains were routinely cultured in Brain Heart Infusion (BHI) broth (Difco Laboratories, Detroit, MI) at 37°C or on BHI agar plates in an atmosphere of 95% air-5% C0 2 . Escherichia coli strains LE392 and XLl-Blue MRF' (Stratagene, La Jolla, CA) were grown on Lubria-Bertani medium (Maniatis et al, 1982) supplemented with maltose
  • BHI Brain Heart Infusion
  • Escherichia coli strains LE392 and XLl-Blue MRF' (Stratagene, La Jolla, CA) were grown on Lubria-Bertani medium (Maniatis et al, 1982) supplemented with maltose
  • MAb 17C7 is a murine IgG antibody reactive with the
  • MAbs specific for UspA material i.e., 16A7, 17B1 , and 5C12 were produced for this study by fusing spleen cells from mice immunized with outer membrane vesicles from
  • Plasmid and bacteriophage cloning vectors utilized in this work and the recombinant derivatives of these vectors are listed in Table VI.
  • Standard recombinant DNA techniques including plasmid isolation, restriction enzyme digestions, DNA modifications, ligation reactions and transformation of E. coli are familiar to those of skill in the art and were performed as previously described
  • PCRTM Polymerase Chain Reaction
  • Nucleotide sequence analysis Nucleotide sequence analysis of DNA fragments in recombinant plasmids, in bacteriophage, or derived by PCRTM was performed using an Applied Biosystems Model 373 A automated DNA sequencer (Applied Biosystems, Foster City, CA). DNA sequence information was analyzed using the Intelligenetics suite package and programs from the University of Wisconsin Genetics Computer Group software analysis package
  • Lysates were generated from E. coli cells infected with recombinant bacteriophage by using the plate lysis method as described (Helminen et al, 1994).
  • MAb-based screening of plaques formed by recombinant ZAP Express bacteriophage on E. coli XL 1 -Blue MRF' cells was performed according to the manufacturer's instructions (Stratagene, La Jolla, CA). Briefly, nitrocellulose filters soaked in 10 mM IPTG were applied to the surface of agar plates five hours after bacteriophage infection of the bacterial lawn.
  • nitrocellulose pads were removed, washed with PBS containing 0.5% (v/v) Tween 20 and 5% (w/v) skim milk (PBS-T) and incubated with hybridoma culture supernatant containing the MAb for 4 hours at room
  • M. catarrhalis protein antigens Characterization of M. catarrhalis protein antigens. Outer membrane vesicles were prepared from BHI broth-grown M. catarrhalis cells by the EDTA-buffer method (Murphy and Loeb, 1989). Proteins present in these vesicles were resolved by sodium dodecyl sulfate
  • SDS-polyacrylamide gel electrophoresis PAGE
  • SDS-PAGE-resolved proteins were electrophoretically transferred to nitrocellulose and western blot analysis was performed as described using MAb 17C7 as the primary antibody (Kimura et al, 1985).
  • MAb 17C7 MAb 17C7 as the primary antibody
  • the nucleotide sequence of the M. catarrhalis 035E uspAl gene and the deduced amino acid sequence of the UspAl protein are provided in SEQ ID NO:2 and SEQ ID NOT, respectively.
  • the predicted protein product of the uspAl ORF had a pi or 4.7, was highly hydrophilic, and was characterized by extensively repeated motifs.
  • the first motif consists of the consensus sequence NXAXXYSXIGGGXN (SEQ ID NO:24), which is extensively repeated between amino acid residues 80 and 170.
  • the second region from amino acid residues 320 to 460, contains a long sequence which is repeated three times in its entirety, but which also contains smaller units which are repeated several times themselves. This "repeat within a repeat" arrangement is also true of the third region, which extends from amino acid residues 460 to 600 .
  • This last motif consists of many repeats of the small motif QADI (SEQ ID NO:25) and two large repeats which contain the QADI (SEQ ID NO:25) motif within themselves.
  • SUBSTITUTE SHEET (RULE 25) a putative adhesin of H. influenzae Rd (GenBank accession number U32792) (Fleischmann et al, 1995), the ⁇ ia adhesin from nontypable H. influenzae (GenBank accession number U38617) (Barenkamp and St. Geme III, 1996), and the YadA invasin of Yersinia enter ocolitica (Skurnik and Wolf-Watz, 1989) (SwissProt:P31489).
  • MAb 17C7 binds to a very high molecular weight proteinaceous material of M. catarrhalis, designated UspA, that migrates with an apparent molecular weight (in SDS-PAGE) of at least 250 kDa.
  • This same MAb also reacts with another antigen band of approximately 100 kDa, as described in U.S. Patent No. 5,552,146 and incorporated herein by reference, and it is bound by a phage lysate from E. coli infected by a recombinant bacteriophage that contained a fragment of M. catarrhalis chromosomal DNA.
  • the M. catarrhalis proteinaceous material in the phage lysate that binds this MAb migrates at a rate similar or indistinguishable from that of the native UspA material (Helminen et al, 1994).
  • SUBSTTTUTE SHEET (RULE 25) Southern blot analysis of chromosomal DNA from several M. catarrhalis strains, using a
  • Proteinaceous Material Three tenths (0.3) mg of purified very high molecular weight UspA proteinaceous material (at the time of the purification this material was thought to be a single protein) was precipitated with 90% ethanol and the pellet was resuspended in 100 ml of 88% formic acid containing 12M urea. Following resuspension, 100 ml of 88% formic acid containing 2M CNBr was added and the mixture was incubated in the dark overnight at room temperature. One ml (2.0 mg) of purified UspA material was added directly to a vial containing 25 mg of either trypsin or chymotrypsin. The reaction mixtures were incubated for -48 hours, at 37°C. One ml (2.0 mg) of purified UspA material was added directly to a vial containing 15 mg of endoproteinase Lys-C. The reaction mixtures were incubated for about 48 hours at 37°C.
  • the cleavage reaction mixtures were clarified by centrifugation in an Eppendorf M centrifuge at 12,000 rpm for 5 minutes.
  • the clarified supernatant was loaded directly onto a Vydac C4 HPLC column using a mobile phase of 0.1% (v/v) aqueous trifiuoroacetic acid (Solvent A) and acetonitrile:H 2 0:trifluoroacetic acid, 80:20:0.1 (v/v/v) (Solvent B) at a flow rate of 1.0 ml/min.
  • the reaction mixtures were washed onto the column with 100% Solvent A followed by elution of cleavage fragments using a 30 minutes linear gradient (0-100%) of Solvent B. Fractions were collected manually, dried overnight in a Speed- Vac and resuspended
  • N-terminal amino acid sequences of these fragments then were determined using an Applied Biosystems Model 477A PTH Analyzer (Applied Biosystems, Foster City, CA, U.S.A.). A summary of these sequences is given in Table VII. About half of the sequences were found to match the sequence deduced from the uspAl gene, while the other half did not.
  • the high molecular weight UspA protein may comprise either a multimer of more than one distinct protein or distinct multimers of two different proteins.
  • the ambiguous residue is likely to be a serine; in SEQ ID NO:33, position 13 is likely to be aspartic acid, position 14 is likely to be glycine and position 15 is likely to be arginine; in SEQ ID NO:35 both positions 13 and 19 are likely to be serines; in SEQ ID NO:49 the ambiguous residue is likely to be an asparagine; and in SEQ ID NO:50 position 4 is likely to be serine and position 8 is likely to be threonine.
  • Peptide 1 KALESNVEEGLLDLSGR (SEQ ID NO:55) Peptide 2 ALESNVEEGLLELSGRTIDQR (SEQ ID NO:56) Peptide 3 NQAHIANNINXIYELAQQQDQK (SEQ ID NO:57) Peptide 4 NQADIAQNQTDIQDLAAYNELQ (SEQ ID NO:58) Peptide 5 ATHDYNERQTEA (SEQ ID NO:59) Peptide ⁇ KASSENTQNIAK (SEQ ID NO:60)
  • mutant uspAl construct Preparation of mutant uspAl construct.
  • the nucleotide sequence of the cloned uspAl gene was used to construct an isogenic uspAl mutant.
  • Oligonucleotide primers (if ⁇ mHI-ended P 1 and PI 6 in Table IX) were used to amplify a truncated version of the uspAl ORF from M. catarrhalis strain O35E chromosomal DNA; this PCRTM product was cloned into the BamUl site of the
  • EDTA-extracted outer membrane vesicles of both the wild-type strain (FIG. 2C, lanes 5 and 7) and mutant strain (FIG. 2C, lanes 6 and 8) possessed a protein of approximately 70-80 kDa that was reactive with MAb 17C7.
  • This approximately 70- 80 kDa band likely represents one form, perhaps the monomeric form, of the product of a second gene encoding the MAb 17C7-reactive epitope.
  • fusion proteins The epitope which binds MAb 17C7 was localized by using the nucleotide sequence of the uspAl gene described above to construct fusion proteins.
  • fusion proteins containing five peptides spanning the UspAl protein were constructed by using the pGEX4T-2 protein fusion system (Pharmacia LKB).
  • the oligonucleotide primers used in PCRTM to amplify the desired nucleotide sequences from M. catarrhalis strain 035E chromosomal DNA are listed in Table IX.
  • FIG. 4 depicts the western blot reactivity of MAb 17C7 with the MF-4-1 fusion protein.
  • These two fusion proteins had in common only a 23-residue region NNINNIYELAQQQDQHSSDIKTL (SEQ ID NO:65), suggesting that this 23-residue region, designated as the "3Q" peptide, contains the epitope that binds MAb 17C7.
  • PCRTM primers used for the production of usp A 1 gene fragments for use in the construction of fusion proteins and mutagenesis and the reactivity of the resulting fusion protein with MAb 17C7
  • a ligation-basedPCRTM system was used to verify this finding. Chromosomal DNA from the mutant strain was digested to completion with Pvull and was resolved by agarose gel electrophoresis. Fragments ranging in size from 2-3 kb were excised from the agarose, blunt- ended, and ligated into the Ec RV site in pBluescript II SK+ This ligation reaction mixture was precipitated and used in a PCRTM amplification reaction. Each PCRTM reaction contained either the T3 or T7 primer derived from the DNA encoding the 3Q peptide. This approach yielded a 1.7
  • Nucleotide sequence analysis of these two PCRTM products revealed two incomplete ORFs which, when joined at the region encoding the 3Q peptide, formed a 1,728-bp ORF encoding a protein with a calculated molecular weight of 62,483 daltons (SEQ ID NO:3).
  • the amino acid sequence of this protein had 43% identity with that of UspAl .
  • Closer examination revealed that a region extending from amino acids 278-41 1 in this second protein, designated UspA2, was nearly identical to the region in UspAl between amino acids 505-638 (SEQ ID NOT). Furthermore, these two regions both contain the 23-mer (the 3Q peptide) that likely contains the epitope that binds MAb 17C7.
  • Oligonucleotide primers PI and P2 were used to amplify a 2.5-2.6 kb fragment from M. catarrhalis strain O35E chromosomal DNA. Nucleotide sequence analysis of this PCRTM product was used to confirm the nucleotide sequence of the uspA2 ORF determined from the ligation-based PCRTM study. These results proved that M. catarrhalis strain 035E contains two different ORFs (i.e., uspAl and uspAT) which encode the same peptide (i.e., the 3Q peptide) which likely binds MAb 17C7. This 3Q peptide appears twice in UspAl and once in UspA2
  • nucleotide sequences of the two DNA segments encoding these 3Q peptides in uspAl are nearly identical, with three nucleotides being different. These nucleotide differences did not cause a change in the amino acid sequence.
  • the nucleotide sequence of the DNA segment encoding the 3Q peptide in uspA2 is identical to the DNA encoding the first 3Q peptide in UspAl .
  • lane 7 the three dominant MAb 17C7-reactive bands present in M. catarrhalis strain O35E outer membrane vesicles have apparent molecular weights of greater than
  • a new M. catarrhalis strain 035E genomic library was constructed in the bacteriophage vector ZAP Express (Stratagene, La Jolla, CA). Chromosomal DNA from this strain was partially digested with Sau2>A ⁇ and 4-9 kb DNA fragments were ligated into the vector arms according to the instructions obtained from the manufacturer.
  • This library was amplified in E. coli MRF'. An aliquot of this library was diluted and plated and the resultant plaques were screened for reactivity with MAb 17C7. Approximately 24 plaques which bound this MAb were detected; the responsible recombinant bacteriophage were purified by the single plaque isolation method, and the DNA insert from one of these bacteriophage was subjected to nucleotide sequence analysis.
  • Nucleotide sequence of the 2.6 kb DNA fragment present in this recombinant bacteriophage revealed that, on one end, it contained an incomplete ORF that encoded the 3Q peptide. Until its truncation by the vector cloning site, the sequence of this incomplete ORF was identical or nearly identical to that of the uspA2 ORF derived from the ligation-based PCRTM study described immediately above, providing further evidence that two genes which share a common epitope encode the UspA antigen.
  • TTA24 and O35E isolates were as previously described in Example I.
  • Additional isolates were obtained from the University of Rochester and the American Type Culture Collection (ATCC). The bacteria were routinely passaged on Mueller-Hinton agar (Difco, Detroit, Ml) incubated at 35°C with 5% carbon dioxide. The bacteria used for the purification of the protein were grown in sterile broth containing 10 g casamino acids (Difco, Detroit, Ml) and 15 g yeast extract (BBL, Cockeysville, MD) per liter. The isolates were stored at -70°C in Mueller-Hinton broth containing 40% glycerol.
  • ATCC American Type Culture Collection
  • SUBSTTTU EET RULE 25 Purification of UspA2.
  • Bacterial cells (-400 g wet wt. of M. catarrhalis 035E) were washed twice with 2 liters of pH 6.0, 0.03 M sodium phosphate (NaPO 4 ) containing 1.0% Triton ® X-100 (TX-100) (J.T. Baker Inc., Philipsburg, NJ) (pH 6.0) by stirring at room temperature for 60 min. Cells containing the UspA2 protein were pelleted by centrifugation at 13,700 x g for 30 min at 4°C.
  • Tris(hydroxymethyl)aminomethane-HCl Tris(hydroxymethyl)aminomethane-HCl
  • TX-100 Tris(hydroxymethyl)aminomethane-HCl
  • Cells were pelleted by centrifugation at 13,700 x g for 30 min at 4°C.
  • the supernatant, containing the UspA2 protein was collected and further clarified by sequential microfiltration through a 0.8 ⁇ m membrane (CN.8, Nalge, Rochester, NY) then a 0.45 ⁇ m membrane (cellulose acetate, low protein binding, Corning,
  • the entire filtered crude extract preparation was loaded onto a 50 x 217 mm (-200 ml) TMAE column [650(S), 0.025-0.4 mm, EM Separations, Gibbstown, NJ] equilibrated with pH 8.0, 0.03 M Tris-HCl buffer containing 0.1% TX-100 (THT).
  • the column was washed with 400 ml of equilibration buffer followed by 600 ml of 0.25 M NaCl in 0.03 M THT.
  • UspA2 was subsequently eluted with 800 ml of 1.0 M NaCl in 0.03 M THT. Fractions were screened for UspA2 by SDS-PAGE and pooled.
  • the buffer exchanged material was subsequently loaded onto a 50 x 217 mm (-425 ml) ceramic hydroxyapatite column (Type I, 40 ⁇ m, Bio-Rad) equilibrated with 10 mM PT.
  • the column was washed with 450 ml of the equilibration buffer followed by 900 ml of pH 7.0,
  • the UspAl enriched fractions collected during four separate purifications of UspA2 were pooled.
  • the combined UspAl pools were concentrated approximately threefold by ultrafiltration using an Amicon stirred cell with a YM-100 membrane under nitrogen pressure.
  • the UspAl concentrate was split into two 175 ml aliquots and the buffer exchanged by passage over a 50 x 280 mm (-550 ml) Sephadex G-25 column equilibrated with 10 mM PT.
  • the buffer exchanged material was subsequently loaded onto a 50 x 217 mm (-425 ml) ceramic hydroxyapatite column (Bio-Rad) equilibrated with 10 mM PT.
  • the column was washed with 450 ml of the equilibration buffer followed by 900 ml of pH
  • SDS-PAGE and Western blot Analysis were carried out as described by Laemmli (1970) using 4 to 20% (w/v) gradient acrylamide gels (Integrated Separation Systems).
  • PVDF polyvinylidene difluoride
  • Protein concentrations were estimated by the BCA assay (Pierce, Rockford, IL), using bovine serum albumin as the standard.
  • reaction mixture was incubated for 48 h at 37°C.
  • Tricine buffer system (Schagger and von Jagow, 1987). The fractions containing a single peptide band were submitted directly for N-terminal sequence analysis. Fractions displaying multiple peptide bands in SDS-PAGE were electrophoretically transferred onto a PVDF membrane as described above. The membrane was stained with Coomassie Brilliant Blue R-
  • the column was calibrated using the HMW Calibration Kit (Pharmacia) which contains aldolase with a size of 158,000, catalase with a size of 232,000; ferritin with a size of 440,000; thyroglobulin with a size of 669,000; and blue dextran with sizes between 2000 and 2,000,000.
  • HMW Calibration Kit Pharmacia
  • PTH phenylthiohydantoin
  • Each dose of vaccine contained 5 ⁇ g of purified protein, 100 ⁇ g of aluminum phosphate and 50 ⁇ g of MPL resuspended in a 200 ⁇ l volume.
  • Control mice were injected with 5 ⁇ g of CRM 197 with the same adjuvants. Serum samples were collected before the first vaccination and two weeks after
  • the 17C7 MAb was secreted by a hybridoma (ATCC HB 11093).
  • MAbs 13-1, 29-31, 45-2, and 6-3 were prepared as previously described (Chen et al, 1995).
  • Murine model of M. catarrhalis pulmonary clearance This model was performed as described previously (Chen et al, 1995).
  • Enzyme linked immunosorbent assay Two different ELISA procedures were used. One was used to examine the reactivity of sera to whole bacterial cells and the other the reactivity to the purified proteins.
  • the bacteria were grown overnight on Mueller-Hinton agar and swabbed off the plate into PBS.
  • the turbidity of the cells was adjusted to 0.10 at 600 nm and 100 ⁇ l added to the wells of a 96 well Nunc F Immunoplate (Nunc, Roskilde, Denmark).
  • the cells were dried overnight at 37°C, sealed with a mylar plate sealer and stored at 4°C until needed.
  • the residual protein binding sites were blocked by adding 5% non-fat dry milk in PBS with 0.1% Tween 20 (Bovine Lacto Transfer Technique Optimizer [BLOTTO]) and incubating 37°C for one hour.
  • the blocking solution was then removed and 100 ⁇ l of sera serially diluted in the wells with blotto.
  • the sera were allowed to incubate for 1 h at 37°C.
  • the plate wells were soaked with 300 ml PBS containing 0.1% Tween 20 for 30 seconds and washed 3 times for 5 seconds with a Skatron plate washer and then incubated 1 hr at 37°C with goat anti-mouse IgG conjugated to alkaline phosphatase (BioSource) diluted 1 : 1000 in blotto. After washing, the plates were developed at room temperature with 100 ⁇ l per well of 1 mg/ml p-nitrophenyl phosphate dissolved in diethanolamine buffer.
  • the proteins were diluted to a concentration of 5 ⁇ g/ml in a 50 mM sodium carbonate buffer (pH 9.8) containing 0.02% sodium azide (Sigma Chemical Co.). One hundred microliters were added to each well of a 96 well
  • Complement-dependent bactericidal assay 20 ⁇ l of the bacterial suspension containing approximately 1200 cfu bacteria in PBS supplemented with 0.1 mM
  • CaCl 2 MgCl 2 and 0.1% gelatin (PCMG) were mixed with 20 ⁇ l of serum diluted in PCMG and incubated for 30 min at 4°C.
  • Complement prepared as previously described (Chen et al, 1996), was added to a concentration of 20%, mixed, and incubated 30 min at 35°C.
  • the assay was stopped by diluting with 200 ⁇ l of cold, 4°C, PCMG. 50 ⁇ l of this suspension was spread onto Mueller-Hinton plates. Relative killing was calculated as the percent reduction in cfu in the sample relative to that in a sample in which heat inactivated complement replaced active complement.
  • the membrane was incubated with the MAb 17C7 diluted in blotto for 2 h at room temperature and then with goat anti-mouse immunoglobulin conjugated to alkaline phosphatase (BIO-RAD Lab. Hercules, Calif.) (1 :2,000 in PBS with 5% dry milk, 2 h, room temperature). The membrane was finally developed with a substrate solution containing
  • HEp-2 cells Interaction with HEp-2 cells by the purified protein.
  • a 96 well cell culture plate (Costar Corp., Cambridge, Mass.) was seeded with 5 x 10 HEp-2 cells in 0.2 ml RPMI containing 10% fetal calf serum and the plate incubated overnight in a 37°C incubator containing 5% C0 2 .
  • Purified UspAl or UspA2 (1 to 1,000 ng) in blotto was added and incubated at 37°C for 2 h.
  • the plate was washed with PBS, and incubated with the 1 : 1 mixed mouse antisera to either UspAl or UspA2 (1 : 1000 dilution in PBS containing 5% dry milk), the plate was washed and incubated with rabbit anti-mouse IgG conjugated to horseradish peroxidase (1 :5,000 in PBS containing 5% dry milk) (Brookwood Biomedical, Birmingham,
  • FIG. 7 plots the values for three fold dilutions of the bacterial suspension.
  • the inventors developed a large-scale, high yield process for extracting and purifying UspA2 from a pellet of M. catarrhalis cells.
  • the method consisted of three critical steps. First the UspA2 protein was extracted from the bacteria with pH 8.0, 0.03 M THT. Second, the cell extract was applied to a TMAE column and the UspA2 protein eluted with NaCl. Finally, the enriched fractions from the TMAE chromatography were applied to a ceramic hydroxyapatite column and the UspA2 eluted with a linear NaPO 4 gradient. A yield of 250 mg of purified UspA2 was typically obtained from -400 g wet weight of M. catarrhalis O35E strain cells. A single band was seen for the UspA2 in SDS-PAGE gels
  • SUBSTTTUTE SHEET RULE 26 range: 5,000-5,000,000) calibrated with molecular weight standards.
  • Purified UspAl exhibited a native molecular size of 1,150,000 and UspA2 a molecular size of 830,000. These sizes, however, may be affected by the presence of TX-100.
  • Asterisk (*) indicates match with UspAl . Without asterisk indicates matches with nucleotide derived amino acid sequence of UspA2.
  • ELISA titers are for total IgG and IgM antibodies for sera pooled from ten mice. c
  • UspA2 proteins were generated in mice.
  • the titers of antigen specific antibodies (IgG and IgM) as well as the cross-reactive antibodies in these sera were determined by an ELISA assay using each of the purified proteins (Table XIII). Both proteins elicited antibody titers that were greater against themselves than against the heterologous protein.
  • the reactivities of both the MAbs (Table XII) as well as the polyclonal antibodies indicate that the proteins possessed both shared and non-shared B-cell epitopes.
  • Antibody reactivity to whole bacterial cells and bactericidal activity Antisera to the
  • TTA24 14,341 7,770 800 800 a Titer determined for pool of sera from ten mice. The titer of the sera drawn before the first immunization was less than 50 for all isolates. b Bactericidal titers were determined as the inverse of the highest serum dilution killing greater than 50% of the bacteria. The titers for the sera from mice immunized contemporaneously with CRM I97 were less than 100.
  • CRM 197 0 - a Challenge method described in text. Numbers are the percentage of bacteria cleared from the immunized mice compared to control mice which were immunized with CRM, 97 .
  • the purified proteins appear to be homopolymers of their respective subunits held together by strong non-covalent forces. This is indicated by the fact that UspA2 lacks any cysteines and treatment of both proteins with reducing agents did not alter their mobilities in SDS-PAGE. Both gene sequences possess leucine zipper motifs that might mediate coil-coil interactions (O'Shea et al, 1991). Even so, it was surprising that the non-covalent bonds of both proteins were not only strong enough to resist dissociation by the conditions normally used to prepare samples for SDS-PAGE, but also high concentrations of chaotropic agents such as urea (Klingman and Murphy, 1994) and guanidine HC1.
  • catarrhalis surface inhibits the formation of the membrane attack complex, rendering the bacteria resistant to the complement dependent killing activity of the sera.
  • They have also described two types of human isolates: one that binds vitronectin and is resistant to the lytic activity of the serum and the other that does not bind vitronectin and is serum sensitive (Hoi et al, 1993). It must be noted, however, that vitronectin, like all the extracellular matrix proteins, has many forms and serves multiple functions in the host (Preissner, 1991 ; Seiffert, 1997).
  • the interaction of both UspAl and UspA2 with the extracellular matrix proteins fibronectin and vitronectin may serve the bacterium in ways beyond subverting host defenses or as receptors for bacterial adhesion.
  • mice immunized with either UspAl or UspA2 developed high antibody titers toward the homologous and heterologous bacterial isolates. Further, the sera from these mice had complement dependent bactericidal activity toward all the isolates tested. In addition, immunized mice exhibited enhanced pulmonary clearance of the homologous isolate and heterologous isolates. It is important to note that antibodies elicited by the proteins were partially cross-reactive. This was expected since both react with the 17C7 MAb and share amino acid sequence.
  • EXAMPLE V The Level and Bactericidal Capacity of Child and Adult Human Antibodies Directed against the Proteins UspAl and UspA2
  • M. catarrhalis strains 035E and TTA24 were as described in Example I.
  • the membrane was washed again with PBS, and then 10 mM Tris buffer (pH 8.0) containing 1 M sodium chloride to remove non-specific proteins.
  • the bound antibodies were eluted by incubation in 5 ml of 100 mM glycine (pH 2.5) for 2 min with shaking.
  • One ml of Tris-HCl (1M, pH 8.0) was immediately added to the eluate to neutralize the pH.
  • the eluted antibodies were dialyzed against PBS and stored at -20°C.
  • Enzyme-linked immunosorbent assay ELISA.
  • Antibody titers to the 035E and other M. catarrhalis strains were determined by a whole-cell ELISA as previously described using biotin-labeled rabbit anti-human IgG or IgA antibodies (Brookwood Biomedical. Birmingham. Alabama) (Chen et al, 1996).
  • Antibody titers to UspAl and UspA2 were determined by a similar method except that the plates were coated with 0.1 ⁇ g of purified protein in 100 ⁇ g of
  • the IgG subclass antibodies to UspAl or UspA2 were determined using sheep anti-human IgG subclass antibodies conjugated to alkaline phosphatase (The Binding Site Ltd., San Diego, Calif).
  • the antibody end point titer was defined as the highest serum dilution giving an A 415 greater than three times that of the control.
  • the control wells received all treatments except human sera and usually had absorbance values ranging from 0.03 to 0.06.
  • the specificity of biotin-labeled rabbit anti-human IgG and IgA antibodies was determined against purified human IgG, IgM and IgA (Pierce, Rockford, IL) by ELISA. No cross-reactivity was found.
  • the assay sensitivity determined by testing against purified human antibodies of appropriate isotype in an ELISA was 15 and 60 ng/ml in the IgG and IgA assays, respectively.
  • the specificity of the human IgG subclass antibody assays was confirmed in ELISA against purified human myeloma IgG subclass proteins (ICN Biomedicals, Inc., Irvine, CA), and the assay sensitivity was 15 ng/ml in the IgGl , IgG3 and IgG4 assays, and 120 ng/ml in the IgG2 assay. Two control sera were included to control for assay to assay variation.
  • the level of IgA antibodies to UspAl , UspA2 and 035E bacterial cells were age dependent (FIG. 9).
  • a serum IgA titer against the UspAl and UspA2 was detected in all twenty-six adults and children of 18-36 months of age. For children less than 18 months of age, the proportion exhibiting antigen specific IgA titers increased with age.
  • IgG subclass titers to the UspAl and UspA2 antigens were determined on sera from ten adult sera and thirty-five children's sera. The subclass distribution was found to be age-dependent. The most prominent antibodies to the UspAl and UspA2 antigens were of the IgGl and IgG3 subclasses, which were detected in almost all sera. The IgG2 and IgG4 titers were either undetectable or extremely low. Therefore, only data on IgGl and IgG3 subclasses are reported (FIG. 10).
  • the IgG3 titers against UspAl or UspA2 in the adult sera were significantly higher than the IgGl titers (p ⁇ 0.05).
  • the same subclass profile was seen in the sera from the 2 month old children, although the difference between IgGl and IgG3 titers did not reach statistical significance, probably because of the smaller sample size.
  • Sera from children between 4 and 36 months of age all had a similar subclass profile which was different from that of the adults and 2 month old children.
  • the IgGl titers in children's sera were either higher than or equivalent to the IgG3 titers.
  • the mean IgGl titer to either UspAl or UspA2 was significantly higher than
  • the bactericidal titers of seventeen sera representing different age groups were determined (Table XVI). All the adult sera and three out of five sera from the two month old children which had high IgG titers to the UspA proteins had strong bactericidal activity. Sera from 6 month old children had the least bactericidal activity. All five sera from this age group had a marginal bactericidal titer of 50, the lowest dilution assayed. The bactericidal activity of the sera from 18 to 36 month old children was highly variable with titers ranging from less than 50 to 500. There was a significant linear relationship between the bactericidal titers and the IgG antibody titers against both UspAl and UspA2 by logistic regression analysis (p ⁇ 0.0 ⁇ ) (FIG. 11).
  • Bactericidal titer was determined as the highest serum dilution resulting in killing of 50% or more of the bacteria relative to the control. Control bacteria were incubated with test serum and heat inactivated complement serum.
  • IgG titers against the UspAl and UspA2 proteins were end point titers determined with a starting serum dilution of 1 :500.
  • the bactericidal titers of the absorbed sera were determined and compared with those seen before absorption (Table XVIII). Absorption with either UspAl or UspA2 resulted in complete loss of bactericidal activity ( ⁇ 50) for all six sera when assayed against the 035E strain, the strain from which the purified proteins were made (Table XVIII). The bactericidal activity of the absorbed sera was also reduced by at least three fold when assayed against the a heterologous strain 1230-359. Absorption using UspAl resulted in greater reduction of the bactericidal titer against the heterologous strain in 3 out of 6 samples compared to abso ⁇ tions using UspA2 (Table XVIII). This result was consistent with the difference in the reductions of
  • a Sera were the same as those described in Table XVII.
  • Bactericidal titer The bactericidal activity was measured against the 035E or 1230-359 strains with 3-fold diluted sera starting at 1:50. The highest serum dilution resulting in 50% or greater killing was determined as the bactericidal titer.
  • the purified UspAl and UspA2 proteins used for abso ⁇ tion were made from the 035 E strain.
  • Affinity purified antibodies to UspAl and UspA2 To confirm their cross-reactivity and bactericidal activity, antibodies to UspAl or UspA2 from adult plasma were isolated by an affinity purification procedure. The purified antibodies reacted specifically with the UspAl and the UspA2 proteins but not with non-UspA proteins in the 035E lysates in a western blot assay. The purified antibodies to one protein also reacted to the other with almost equivalent titer in ELISA (Table XX). Both antibody preparations exhibited reactivity with five M. catarrhalis strains in the whole-cell ELISA and bactericidal assay (Table XXI). The bactericidal titers against all five M. catarrhalis strains ranged between 400 and 800, which was equivalent to
  • the antibodies were purified from plasma pooled from two healthy adults by immune elution using purified UspAl or UspA2 from the 035E strain immobilized on nitrocellulose membrane.
  • b ELISA end point titers are the highest antibody dilutions giving an A 4] 5 greater than three times the background.
  • UspAl or UspA2 from a single isolate exhibited killing against multiple strains. This result indicates that humans developed bactericidal antibodies toward the conserved epitopes of UspA proteins in response to natural infections.
  • the IgG antibodies were primarily of the IgGl and IgG3 subclasses with IgG3 being higher. This is consistent with previous reports that the IgG3 subclass is a major constituent of the immune response to M. catarrhalis in adults and children greater than 4
  • IgG3 constitutes only a minor component of the total immunoglobulin in serum.
  • IgG3 antibody has the highest affinity to interact with Clq, the initial step in the classic complement pathway leading to elimination of the bacterium by both complement-dependent killing and opsono-phagocytosis (Roitt et al, 1985). Since
  • IgG3 antibody is efficiently transferred across the placenta, it may also confer protective immunity to infants.
  • the data from this study indicate that IgG3 antibody to the UspA proteins is an important component of the immune response to natural infection and has in vitro biological activity.
  • UspA2 as a pneumococcal saccharide carrier.
  • Vaccine group consists of 5 Swiss- Webster mice. Each group immunized at wk 0 and wk 3 and serum collected at wk 6.
  • SUBSTTTUTE SHEET (RULE 25) Vaccine composed of 1 ⁇ g Pneumo Type 7F and 1 ⁇ g UspA2 adjuvanted with aluminum phosphate.
  • BC 50 titer is highest serum dilution at which >50% of bacteria were killed as compared to serum from wk 0 mice.
  • the most concentrated serum tested was a 1 TOO dilution.
  • UspA2 as an Haemophilus b Oligosaccharide Carrier.
  • HbO Haemophilus influenzae type b oligosaccharide
  • the immunogenicity of the conjugate was examined by immunizing Swiss- Webster mice.
  • the mice were immunized twice on wk 0 and wk 4 with 1 ⁇ g of carbohydrate. No adjuvant was used with the conjugate, but was used with UspA2.
  • the sera were pooled and titered.
  • the reactivity toward HbPS by the radioantigen binding assay (RABA) was similar to that seen when HbO is conjugated to CRM 197 (Table XXV).
  • RABA radioantigen binding assay
  • the whole cell titer toward the homologous M. catarrhalis isolate (035E) was similar to that seen for non-conjugated USpA2 (Table XXVI), as were the bactericidal titers (Table XXVII).
  • RABA radioantigen binding assay
  • CRM 7 to Haemophilus b polysaccharide by Radioantigen Binding Assay (RABA)
  • the 035E.2 and 035E.12 expressed a smaller truncated form UspA2 (tUspA2) that reacts with antibodies prepared by immunizing mice with purified UspA2.
  • the tUspA2 could be detected in a western blot of bacterial lysates using either polyclonal anti-UspA2 sera or the MAb 13-1.
  • the size of the smaller form was consistent with the gene truncation used for the construction of the two mutants.
  • This bactericidal capacity was tested by mixing the non-immune mouse sera, a 1 :5 dilution of human complement and a suspension of bacteria (Approx. 1000 cfu) in the wells of a microtiter plate.
  • the mouse sera were tested at both a 1 :50 and 1 TOO dilution.
  • the number of surviving bacteria was then determined by spreading a dilution of this bacterial suspension on agar growth medium. The killing was considered significant when fewer than 50% viable bacteria as cfu's were recovered relative to the samples without mouse sera. Killing by the non-immune sera was seen only for the mutants lacking a "complete" UspA2 (Table XXVIII).
  • overlapping synthetic decapeptides as shown in Table XXIX and FIG. 12, that were N-terminally bound to a membrane composed of derivatized cellulose were obtained from Research Genetics Inc. (Huntsville. AL). After five washes with PBS-Tween containing 5% (w/v) non-fat dry milk, the membrane was subsequently incubated with MAb 17C7 (in the form of hybridoma culture supernatant) overnight at 4°C. Following three washes with PBS- Tween, the membrane was incubated overnight at 4°C with gentle rocking with 10 cpm of radioiodinated (specific activity 2 x 10 cpm/ ⁇ g protein), affinity-purified goat anti-mouse immunoglobulin. The membrane was then washed as before and exposed to X-ray film (Fuji RX safety film, Fuji Industries, Tokyo, Japan).
  • peptide 12 shows no binding and binding by peptides 15, 16, 19, 22, 23 is probably non-specific.
  • a comparison of peptides 12, 13, and 14 yields the conclusion that the 7-mer AQQQDQH (SEQ ID NO: 17) is an essential epitope for MAb 17C7 to bind to
  • M. catarrhalis strains were routinely grown at 37°C on Brain-Heart Infusion (BHI) agar plates (Difco Laboratories, Detroit, MI) in an atmosphere of 95% air-5% C0 2 supplemented, when necessary, with kanamycin (20 ⁇ g/ml) (Sigma Chemicals Co., St. Louis, MO) or chloramphenicol (0.5 ⁇ g/ml) (Sigma), or in BHI broth.
  • BHI Brain-Heart Infusion
  • Escherichia coli strains were cultured on Luria-Bertani (LB) agar plates (Maniatis et al, 1982) supplemented, when necessary, with ampicillin (100 ⁇ g/ml), kanamycin (30 ⁇ g/ml), or chloramphenicol (30 ⁇ g/ml).
  • 035E.1 Isogenic mutant of 035 E Aebi e/ ⁇ /., 1997 with a kan cartridge in the uspAl structural gene
  • 035E.2 Isogenic mutant of 035E Aebi e/ ⁇ /., 1997 with a kan cartridge in the uspA2 structural gene
  • 035E.12 Isogenic mutant of 035E This study with a kan cartridge in the uspA2 structural gene and a cat cartridge in the uspAl structural gene
  • MAb 17C7 Monoclonal antibodies (MAbs).
  • MAb 17C7 a murine IgG antibody that reacts with a conserved epitope of both UspAl and UspA2 from M. catarrhalis strain 035E, as described in earlier examples herein, was used for immunologic detection of these proteins.
  • MAb 17C7 was used in the form of hybridoma culture supernatant fluid in western blot analysis and in the indirect antibody-accessibility assay.
  • MAb 3F12 an IgG MAb specific for the major outer membrane protein of Haemophilus ducreyi (Klesney-Tait et al. , 1997), was used as a negative control in the indirect antibody-accessibility assay.
  • Chromosomal DNA of M. catarrhalis strain 035E was used as the template in a polymerase chain reaction (PCRTM) system together with oligonucleotide primers derived from either just after the start of the strain 035E uspAl open reading frame (i.e., PI in FIG. 14) or just after the end of this open reading frame (i.e., P2 in
  • FIG. 14 These primers were designed to contain a BamUl restriction site at their 5'-end. The sequence of these primers was:
  • PCRTM products were extracted from 0.7% agarose gel slices using the Qiaex Gel Extraction Kit (Qiagen, Inc., Chadsworth, CA) and digested with BamUl (New England Biolabs, Inc., Beverly, MA) for subsequent ligation into the BamUl site of pBluescript II SK+ (Stratagene, La Jolla, CA). Ligation reactions were performed with overnight incubation at
  • the cat cartridge was subsequently ligated into Bglll restriction sites located in the mid-portion of cloned segment from the uspAl gene and, after transformation of
  • Serum bactericidal assay Serum bactericidal assay.
  • Complement-sufficient normal adult human serum was prepared by standard methods. Complement inactivation was achieved by heating the serum for 30 min at 56°C.
  • a M. catarrhalis broth culture in early logarithmic phase was diluted in
  • Veronal-buffered saline containing 0.10% (w/v) gelatin (GVBS) to a concentration of 1-2 x 10 3 cfu/ml, and 20 ⁇ l portions were added to 20 ⁇ l of native or heat-inactivated normal human serum together with 160 ⁇ l of Veronal-buffered saline containing 5 mM MgCl 2 and 1.5 mM CaCl 2 .
  • This mixture was incubated at 37°C in a stationary water bath. At time 0 and at 15 and 30 min, 10 ⁇ l aliquots were removed, suspended in 75 ⁇ l of BHI broth and spread onto prewarmed BHI agar plates.
  • Adherence assay A method used to measure adherence of Haemophilus influenzae to Chang conjunctival cells in vitro (St. Geme III and Falkow, 1990) was adapted for use with M. catarrhalis. Briefly, 2-3 x 10 HEp-2 cells (ATCC CCL 23) or Chang conjunctival cells
  • SUBSTTTUTE SHEET (RULE 26) saline (PBS) or PBS containing 0.15% (w/v) gelatin (PBS-G). The bacterial cells were centrifuged again and this final pellet was gently resuspended in 6-8 ml of PBS or PBS-G.
  • Portions (25 ⁇ l) of this suspension (10 CFU) were inoculated into the wells of a 24- well tissue culture plate containing monolayers of HEp-2 or Chang cells. These tissue culture plates were centrifuged for 5 min at 165 x g and then incubated for 30 min at 37°C. Non- adherent bacteria were removed by rinsing the wells gently five times with PBS or PBS-G, and the epithelial cells were then released from the plastic support by adding 200 ⁇ l of PBS containing 0.05% trypsin and 0.02% EDTA. This cell suspension was serially diluted in PBS or PBS-G and spread onto BHI plates to determine the number of viable M. catarrhalis present.
  • PCRTM PCRTM product was used to electroporate the kanamycin-resistant uspA2 strain 035E.2 and yielded the chloramphenicol - and kanamycin-resistant transformant 035E.12, a putative uspAl uspA2 double mutant.
  • the uspA 1 -specific DNA probe was obtained by PCRTM-based amplification of M. catarrhalis strain 035E chromosomal DNA using the primers P3 and P4 (FIG. 14A). A 500-bp DNA fragment was amplified from 035E chromosomal DNA by PCRTM with the primers P5 and P6 (FIG. 14B). Use of these two gene-specific probes together with the kan and cat cartridges in Southern blot analysis confirmed that strain 035E.12 was a uspAl uspA2 double mutant.
  • MAb 17C7-reactive antigen expressed by this uspAl mutant appeared to be equivalent to that expressed by the wild-type strain.
  • the uspA2 mutant 035E.2 (FIG. 15B, lane 3) expressed the
  • the uspA2 mutant had relatively little very high molecular weight antigen reactive with MAb 17C7.
  • the uspAl uspA2 double mutant 035E.12 (FIG. 15B, lane 4) expressed no detectable MAb 17C7- reactive antigens.
  • SUBSTTTUTE SHEET (RULE 26) Binding of MAb 17C7 to whole cells of the wild-type and mutant strains.
  • the indirect antibody-accessibility assay was used to determine whether both UspAl and UspA2 are exposed on the surface of M. catarrhalis and accessible to antibody.
  • MAb 3F12 a murine IgG antibody specific for a ⁇ . ducreyi outer membrane protein (Klesney-Tait et al, 1997), was included as a negative control.
  • PBS was used for washing of the monolayers and for serial dilutions of adherent M. catarrhalis.
  • c PBS-G was used for washing of the monolayers and for serial dilutions of adherent M. catarrhalis.
  • d P value when compared to the wild-type strain O35E using the two-tailed Student t- test.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically,
  • Haemophilus ducreyi consists of two OmpA homologs," J. Bacteriol, 179:1764-1773,
  • St.Geme III and Falkow "Haemophilus influenzae adheres to and enters cultured human epithelial cells," Infect. Immun. , 58:4036-4044, 1990.
  • St.Geme III, Cutter, Barenkamp "Characterization of the genetic locus encoding Haemophilus influenzae type b surface fibrils," J. Bacteriol, 178:6281-6287, 1996. Stinchcomb et al. , Nature, 282:39, 1979.
  • Moraxella (Branhamella) catarrhalis is mediated by a high-molecular-weight outer membrane protein (HMW-OMP)," Abstracts General Meeting Amer. Soc. Microbiol,

Abstract

The present invention discloses the existence of two novel proteins UspA1 and UspA2, and their respective genes uspA1 and uspA2. Each protein encompasses a region that is conserved between the two proteins and comprises an epitope that is recognized by MAb 17C7. One or more than one of these species may aggregate to form the very high molecular weight form (i.e. greater than 200 kDa) of the UspA antigen. Compositions and both diagnostic and therapeutic methods for the treatment and study of M. catarrhalis are disclosed.

Description

DESCRIPTION
JSPA1 AND USPA2 ANTIGENS OF MORAXELLA CA TARRHALIS
BACKGROUND OF THE INVENTION
I. Field of the Invention
The present invention relates generally to the fields of microbiology, and clinical bacteriology. More particularly, it concerns sequences of the uspAl and uspAl genes which encode the proteins UspAl and UspA2. respectively, both of which encode an epitope reactive with monoclonal antibody (MAb) 17C7 and provide useful epitopes for immunodiagnosis and immunoprophylaxis.
II. Description of Related Art
It was previously thought that Moraxella catarrhalis, previously known as Branhamella catarrhalis or Neisseria catarrhalis, was a harmless saprophyte of the upper respiratory tract (Catlin, 1990; Berk, 1990). However, during the previous decade, it has been determined that this organism is an important human pathogen. Indeed, it has been established that this Gram- negative diplococcus is the cause of a number of human infections (Murphy, 1989). M. catarrhalis is now known to be the third most common cause of both acute and chronic otitis media (Catlin, 1990; Faden et al. 1990; 1991 ; Marchant, 1990), the most common disease for which infants and children receive health care according to the 1989 Consensus Report. This organism also causes acute maxillary sinusitis, generalized infections of the lower respiratory tract (Murphy and Loeb, 1989) and is an important cause of bronchopulmonary infections in patients with underlying chronic lung disease and, less frequently, of systemic infections in immunocompromised patients (Melendez and Johnson, 1990; Sarubbi et al, 1990; Schonheyder and Ejlertsen, 1989; Wright and Wallace. 1989).
The 1989 Consensus Report further concluded that prevention of otitis media is an important health care goal due to both its occurrence in infants and children, as well as certain populations of all age groups. In fact, the total financial burden of otitis media has been estimated to be at least $2.5 billion annually. Vaccines were identified as the most desired approach to prevent this disease for a number of reasons. For example, it was estimated that if vaccines could reduce the incidence of otitis media by 30%, then the annual health care savings would be at least $400 million. However, while some progress has been made in the development of vaccines for 2 of the 3 common otitis media pathogens, Streptococcus pneumoniae and Haemophilus influenzae, there is no indication that similar progress has been made with respect to M. catarrhalis. This is particularly troublesome in that M. catarrhalis now accounts for approximately 17-20% of all otitis media infection (Murphy, 1989). In addition, M. catarrhalis is also a significant cause of sinusitis (van Cauwenberge et al, 1993) and persistent cough (Gottfarb and Brauner, 1994) in children. In the elderly, it infects patients with predisposing conditions such as chronic obstructive pulmonary disease (COPD) and other chronic cardiopulmonary conditions (Boyle et al, 1991 ; Davies and Maesen, 1988; Hager et al,
1987).
Despite its recognized virulence potential, little is known about the mechanisms employed by M. catarrhalis in the production of disease or about host factors governing immunity to this pathogen. An antibody response to M. catarrhalis otitis media has been documented by means of an ELISA system using whole M. catarrhalis cells as antigen and acute and convalescent sera or middle ear fluid as the source of antibody (Leinonen et al, 1981). The development of serum bactericidal antibody during M. catarrhalis infection in adults was shown to be dependent on the classical complement pathway (Chapman et al, 1985). And more recently, it was reported that young children with M. catarrhalis otitis media develop an antibody response in the middle ear but fail to develop a systemic antibody response in a uniform manner (Faden et al, 1992).
Previous attempts have been made to identify and characterize M. catarrhalis antigens that would serve as potentially important targets of the human immune response to infection (Murphy, 1989; Goldblatt et al, 1990; Murphy et al, 1990). Generally speaking, the surface of M. catarrhalis is composed of outer membrane proteins (OMPs), lipooligosaccharide (LOS) and fimbriae. M. catarrhalis appears to be somewhat distinct from other Gram-negative bacteria in that attempts to isolate the outer membrane of this organism using detergent fractionation of cell envelopes has generally proven to be unsuccessful in that the procedures did not yield consistent results (Murphy, 1989; Murphy and Loeb, 1989). Moreover, preparations were found to be contaminated with cytoplasmic membranes, suggesting an unusual characteristic of the M. catarrhalis cell envelope.
SUBSTTTUTE SHEET (RULE 25) Passive immunization with polyclonal antisera raised against outer membrane vesicles of the M. catarrhalis strain 035E was also found to protect against pulmonary challenge by the heterologous M. catarrhalis strain TTA24. In addition, active immunization with M. catarrhalis outer membrane vesicles resulted in enhanced clearance of this organism from the lungs after challenge. The positive effect of immunization in pulmonary clearance indicates that antibodies play a major role in immunoprotection from this pathogen. In addition, the protection observed against pulmonary challenge with a heterologous M. catarrhalis strain demonstrates that one or more conserved surface antigens are targets for antibodies which function to enhance clearance of M. catarrhalis from the lungs.
Outer membrane proteins (OMPs) constitute major antigenic determinants of this unencapsulated organism (Bartos and Murphy, 1988) and different strains share remarkably similar OMP profiles (Bartos and Murphy, 1988; Murphy and Bartos, 1989). At least three different surface-exposed outer membrane antigens have been shown to be well-conserved among M. catarrhalis strains; these include the 81 kDa CopB OMP (Helminen et al, 1993b), the heat- modifiable CD OMP (Murphy et al, 1993) and the high-molecular weight UspA antigen
(Helminen et al, 1994). Of these three antigens, both the CopB protein and UspA antigen have been shown to bind antibodies which exert biological activity against M. catarrhalis in an animal model (Helminen et al, 1994; Murphy et al, 1993).
The MAb, designated 17C7, was described as binding to UspA, a very high molecular weight protein that migrated with an apparent molecular weight (in SDS-PAGE) of at least 250 kDa (Helminen et al, 1994; Klingman and Murphy, 1994). MAb 17C7 enhanced pulmonary clearance of M. catarrhalis from the lungs of mice when used in passive immunization studies and, in colony blot radioimmunoassay analysis, bound to every isolate of M. catarrhalis examined. This same MAb also reacted, although less intensely, with another antigen band of approximately 100 kDa, as described in U.S. Patent No. 5,552,146 (incorporated herein by reference). A recombinant bacteriophage that contained a fragment of M. catarrhalis chromosomal DNA that expressed a protein product that bound MAb 17C7 was also identified and migrated at a rate similar or indistinguishable from that of the native UspA antigen from M. catarrhalis (Helminen et al. , 1994).
SUBSTTTUTE SHEET (RULE 26) With the rising importance of this pathogen in respiratory tract infections, identification of the surface components of this bacterium involved in virulence expression and immunity is becoming more important. To date, there are no vaccines available, against any other OMP, LOS or fimbriae, that induce protective antibodies against M. catarrhalis. Thus, it is clear that there remains a need to identify and characterize useful antigens and which can be employed in the preparation of immunoprophylactic reagents. Additionally, once such an antigen or antigens is identified, there is a need for providing methods and compositions which will allow the preparation of vaccines and in quantities that will allow their use on a wide scale basis in prophylactic protocols.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide new UspAl and UspA2 proteins and genes coding therefor. It also is an object of the present invention to provide methods of using these new proteins, for example, in the preparation of agents for the treatment and inhibition of M. catarrhalis infection. It also is contemplated that through the use of other technologies such as antibody treatment and immunoprophylaxis that one can inhibit or even prevent M. catarrhalis infections.
In satisfying these goals, there are provided epitopic core sequences of UspAl and UspA2 which can serve as the basis for the preparation of therapeutic or prophylactic compositions or vaccines which comprise peptides of 7, 10, 20, 30, 40, 50 or even 60 amino acids in length that elicit an antigenic reaction and a pharmaceutically acceptable buffer or diluent. These peptides may be coupled to a carrier, adjuvant, another peptide or other molecule such that an effective antigenic response to M. catarrhalis is retained or even enhanced. Alternatively, these peptides may act as carriers themselves when coupled to another peptide or other molecule that elicits an antigenic response to M. catarrhalis or another pathogen. For example, UspA2 can serve as a carrier for an oligosaccharide.
In one embodiment, the epitopic core sequences of UspAl and UspA2 comprise one or more isolated peptides of 7, 10, 20, 30, 40, 50 or even 60 amino acids in length having the amino acid sequence AQQQDQH (SEQ ID NO: 17).
SUBSTITUTE SHEET RULE 25) In another embodiment, there are provided nucleic acids, uspAl and uspA2, which encode the UspAl and the UspA2 antigens, respectively, as well as the amino acid sequences of the UspAl and UspA2 antigens of the M. catarrhalis isolates 035E, TTA24, TTA37, and 046E. It is envisioned that nucleic acid segments and fragments of the genes uspAl and uspA2 and the UspAl and UspA2 antigens will be of value in the preparation and use of therapeutic or prophylactic compositions or vaccines for treating, inhibiting or even preventing M. catarrhalis infections.
In another embodiment, there is provided a method for inducing an immune response in a mammal comprising the step of providing to the mammal an antigenic composition that comprises an isolated peptide of about 20 to about 60 amino acids that contains the identified epitopic core sequence and a pharmaceutically acceptable buffer or diluent.
In another embodiment, there is provided a method for diagnosing M. catarrhalis infection which comprises the step of determining the presence, in a sample, of an M. catarrhalis amino acid sequence corresponding to residues of the epitopic core sequences of either the UspAl or UspA2 antigen. This method may comprise PCR ™ detection of the nucleotide sequences or alternatively an immunologic reactivity of an antibody to either a UspA 1 or UspA2 antigen.
In a further embodiment, there is provided a method for treating an individual having an M. catarrhalis infection which comprises providing to the individual an isolated peptide of about 20 to about 60 amino acids that comprises at least about 10 consecutive residues of the amino acid sequence identified as an epitopic core sequence of UspAl or UspA2.
In a still further embodiment, there is provided a method for preventing or limiting an M. catarrhalis infection that comprises providing to a subject an antibody that reacts immunologically with the identified epitopic core region of either UspAl or UspA2 of M. catarrhalis.
In another embodiment, there is provided a method for screening a peptide for reactivity with an antibody that binds immunologically to UspAl, UspA2 or both which comprises the steps of providing the peptide and contacting the peptide with the antibody and then
SUBSTITUTE SHEET RULE 25 determining the binding of the antibody to the peptide. This method may comprise an immunoassay such as a western blot, an ELISA, an RIA or an immunoaffinity separation.
In a still further embodiment, there is provided a method for screening a UspAl or UspA2 peptide for its ability to induce a protective immune response against M. catarrhalis by providing the peptide, administering it in a suitable form to an experimental animal, challenging the animal with M. catarrhalis and then assaying for an M. catarrhalis infection in the animal. It is envisioned that the animal used will be a mouse that is challenged by a pulmonary exposure to M. catarrhalis and that the assaying comprises assessing the degree of pulmonary clearance by the mouse.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1. Southern blot analysis of PvwII-digested chromosomal DNA from strains of M. catarrhalis using a probe from the uspAl gene. Bacterial strain designations are at the top; kilobase (kb) position markers are on the left.
FIG. 2A. Proteins present in whole cell lysates of the wild-type strain 035E and the isogenic uspAl mutant strain. These proteins were resolved by SDS-PAGE and stained with Coomassie blue. The left lane (WT) contains the wild-type strain and right lane (MUT) contains
SUBSTITUTE SHEET (RULE 25) the mutant. The arrows indicate the protein, approximately 120 kDa in size, that is present in the wild-type and missing in the mutant. Kilodalton position markers are on the left.
FIG.2B. Western blot analysis of whole cell lysates of the wild-type strain 035E and the isogenic uspAl mutant strain. These proteins were resolved by SDS-PAGE and probed with MAb 17C7 in western blot analysis. The left lane (WT) contains the wild-type strain and the right lane (MUT) contains the mutant. Kilodalton position markers are on the left. It can been seen that both strains possess the very high molecular weight band reactive with MAb 17C7 whereas only the wild-type strain also has a band of approximately 120 kDa that binds this MAb.
FIG. 2C. Western blot analysis of whole cell lysate (WCL) and EDTA-extracted outer membrane vesicles (OMV) from the wild-type strain O35E (WT) and the isogenic uspAl mutant (MUT) using MAb 17C7. Samples were either heated at 37°C for 15 minutes (H) or at 100°C for 5 minutes (B) prior to SDS-PAGE. Molecular weight position markers (in kilodaltons) are indicated on the left. The open arrow indicates the position of the very high molecular weight form of the MAb 17C7-reactive antigen; the closed arrow indicates the position of the approximately 120 kDa protein; the open circle indicates the position of the approximately 70-80 kDa protein.
FIG. 3. Southern blot analysis of chromosomal DNA from the wild-type M. catarrhalis strain 035E and the isogenic uspAl mutant. Chromosomal DNA was digested with Pvu and probed with a 0.6 kb BgHl-Pvull fragment from the uspAl gene. The wild-type strain is listed as O35E at the top of this figure and the mutant strain is listed as O35E-uspAl". Kilobase position markers are present on the left side.
FIG. 4. Western blot reactivity of proteins in M. catarrhalis strain 035E outer membrane vesicles (labeled 035E OMV) and the MF-4-1 GST fusion protein (labeled GST fusion protein) with MAb 17C7.
FIG. 5. PCR™ products obtained by the use of the T3 and P 10 primers (middle lane - 0.9 kb product) and the T7 and P9 primers (right lane - 1.7 kb product) when used in a PCR™
SUBSTITUTE SHEET RULE 25 amplification with chromosomal DNA from the uspAl mutant. A kb ladder is present in the first lane; several kb position markers are listed on the left side of this figure.
FIG. 6A-6C. SDS-PAGE and westerns of purified proteins. FIG. 6A. Coomassie blue stained gel of purified UspA2 (lane 2). FIG. 6B. Coomassie blue stained gel of purified UspAl prepared without heating of sample (lane 4), heated for 3 min at 100°C (lane 5), heated for 5 min at 100°C (lane 6), and heated for 10 min at 100°C (lane 7). FIG. 6C. Western of the purified UspA2 (lane 9) and purified UspAl (lane 10) probed with the 17C7 MAb. Both proteins were heated 10 min. The molecular size markers in lanes 1, 3, and 8 are as indicated in kilodaltons.
FIG. 7. Interaction of purified UspAl and UspA2 with HEp-2 cells as determined by ELISA. HEp-2 cell monolayers cultured in 96-well plate were incubated with serially diluted UspAl or UspA2. 035E bacterial strain was used as the positive control. The bacteria were diluted analogous to the proteins beginning with a suspension with an A550 of 1.0. The bound proteins or attached bacteria were detected with a 1 :1 mixed antisera to UspAl and UspA2 as described in the methods.
FIG. 8. Interaction with fibronectin and vitronectin determined by dot blot. The bound vitronectin was detected with rabbit polyclonal antibodies, the protein bound to the fibronectin was detected with pooled sera made against the UspAl and UspA2.
FIG. 9. The levels of antibodies to the protein UspAl, UspA2 and M. catarrhalis O35E strain in normal human sera. Data are the logi0 transformed end-point titers of the IgG (FIGs. 9A-9C) and IgA (FIGs. 9D-9F) antibodies determined by ELISA. The individual titers were plotted according to age group and the geometric mean titer for each age group linked by a solid line. Sera for the 2-18 month old children were consecutive samples from a group of ten children.
FIG. 10. Subclass distribution of IgG antibodies to UspAl and UspA2 in normal human sera. FIG. 10A shows titers toward UspAl and FIG. 10B shows titers to UspA2.
SUBSTTTUTE SHEET RULE 25) FIG. 11. Relationship of serum IgG titers to UspAl (FIG. 1 1A) and UspA2 (FIG. 1 IB) with the bactericidal liter against the 035E strain determined by logistic regression (p<0.05). The solid line indicates the linear relationship between the IgG titer and bactericidal titer. Broken lines represent the 95 % confidence intervals of the linear fit.
FIG. 12. Schematic drawing showing the relative positions of decapeptides 10-24 within the region of UspAl and UspA2 which binds to MAb 17C7.
FIG. 13. Western dot blot analysis demonstrating reactivity of decapeptides 10-24 with MAb 17C7.
FIG. 14. Partial restriction enzyme map of the uspAl (FIG. 14A) and uspA2 (FIG. 14B) genes from M. catarrhalis strain 035E and the mutated versions of these genes. The shaded boxes indicate the open reading frame of each gene. Relevant restriction sites are indicated. PCR™ primer sites (P1-P6) are indicated by arrows. The DNA fragments containing the partial uspAl and uspA2 open reading frames that were derived from M. catarrhalis strain O35E chromosomal DNA by PCR™ and cloned into pBluescriptll SK+ are indicated by black bars.
Dotted lines connect corresponding restriction sites on these DNA inserts and the chromosome. Open bars indicate the location of the kanamycin or chloramphenicol cassettes, respectively. The DNA probes specific for uspAl or uspA2 are indicated by the appropriate cross-hatched bars and were amplified by PCR™ from M. catarrhalis strain 035E chromosomal DNA by the use of the oligonucleotide primer pairs
P3 (5'-GACGCTCAACAGCACTAATACG-3') (SEQ ID NO:20)/P4
(5'-CCAAGCTGATATCACTACC-3') (SEQ ID NO:21) and
P5 (5'-TCAATGCCTTTGATGGTC-3') (SEQ ID NO:22)/P6
(5'-TGTATGCCGCTACTCGCAGCT-3') (SEQ ID NO:23), respectively.
FIG. 15. Detection of the UspAl and UspA2 proteins in wild-type and mutant strains of M catarrhalis O35E. Proteins present in EDTA-extracted outer membrane vesicles from the wild-type strain (lane 1), the uspAl mutant strain 035E.1 (lane 2), the uspA2 mutant strain
035E.2 (lane 3), and the isogerύcuspAl uspA2 double mutant strain 035E.12 (lane 4) were resolved by SDS-PAGE, and either stained with Coomassie blue (FIG. 15A) or transferred to nitrocellulose and probed with MAb 17C7 followed by radioiodinated goat anti-mouse
SUBSTTTUTE SHEET RULE 25) immunoglobulin in western blot analysis. In FIG. 15 A, the closed arrow indicates the very high molecular weight form of the UspA antigen which is comprised of both UspAl and UspA2. In FIG. 15B, the bracket on the left indicates the very high molecular weight forms of the UspAl and UspA2 proteins that bind MAb 17C7. The open arrow indicates the 120 kDa, putative monomeric form of UspAl . The closed arrow indicates the 85 kDa, putative monomeric form of UspA2. Molecular weight position markers (in kilodaltons) are present on the left.
FIG. 16. Comparison of the rate and extent of growth of the wild-type and mutant strains of M. catarrhalis. The wild-type strain 035E (closed squares), the uspAl mutant O35E.1 (open squares), the uspA2 mutant O35E.2 (closed circles), and the uspAl uspA2 double mutant 035E.12 (open circles) of M. catarrhalis O35E from overnight broth cultures were diluted to a density of 35 Klett units in BHI broth and subsequently allowed to grow at 37° with shaking. Growth was followed by means of turbidity measurements.
FIG. 17. Susceptibility of wild-type and mutant strains of M. catarrhalis to killing by normal human serum. Cells of the wild-type parent strain 035E (diamonds), uspAl mutant O35E.1 (triangles), uspA2 mutant O35E.2 (circles), and uspAl uspA2 double mutant 035E.12 (squares) from logarithmic-phase BHI broth cultures were incubated in the presence of 10% (v/v) normal human serum (closed symbols) or heat-inactivated normal human serum (open symbols). Data are presented as the percentage of the original inoculum remaining at each time point.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention relates to the identification of epitopes useful for developing potential vaccines against M. catarrhalis. Early work was directed at determining the molecular nature of the UspA antigen and characterize the epitope which is recognized by the MAb 17C7. Preliminary work indicated that MAb 17C7 recognizes a single antigenic epitope and it was believed that this epitope was encoded by a single gene. However, isolation of the protein which contained the epitope yielded unexpected results. MAb 17C7 recognized a single epitope, but the characteristics of the protein associated with the epitope suggested the existence
SUBSTTTUTE SHEET RULE 25 of not one but two separate proteins. Further careful analyses led to a surprising discovery. A single epitope of the UspA antigen is recognized by the MAb 17C7, but this epitope is present in two different proteins, UspAl and UspA2, which are encoded by two different genes uspAl and uspA2, respectively, and only have 43% identity to each other. The present invention provides the nucleotide sequences of the genes uspAl and uspA2, their respective protein products, UspAl and UspA2, and the shared epitope recognized by MAb 17C7.
In addition, the present invention provides insights into the antigenic structure of the UspA protein based on the analysis of the sequences of the UspAl and UspA2 proteins which comprise the protein. Characterization of the epitopic region of the molecule that is targeted by the MAb 17C7 permits the development of agents that will be useful in protecting against M. catarrhalis infections, e.g., in the preparation of prophylactic reagents. Particular embodiments relate to the amino acid and nucleic acids corresponding to the UspAl and UspA2 proteins, peptides and antigenic compositions derived therefrom, and methods for the diagnosis and treatment of M. catarrhalis disease.
As stated previously, M. catarrhalis infections present a serious health challenge, especially to the young. Thus, there is a clear need to develop compositions and methods that will aid in the treatment and diagnosis of this disease. The present invention, by virtue of new information regarding the structure of the UspA antigen of M. catarrhalis, and discovery of the two new and distinct proteins UspAl and UspA2 provides such improved compositions and methods. UspAl and UspA2 represent important antigenic determinants, as the MAb 17C7 has been shown to protect experimental animals, as measured in a pulmonary clearance model, when provided in passive immunizations.
In a first embodiment, the present invention provides for the identification of the proteins UspAl and UspA2 from M. catarrhalis strain 035E. The UspAl protein comprises about 831 amino acid residues and has a predicted mass of about 88,271 daltons (SEQ ID
NO: l). The UspA2 protein comprises about 576 residues and has a predicted mass of about
62,483 daltons (SEQ ID NO:3). UspA2 is not a truncated or processed form of UspAl .
In a second embodiment, the present invention has identified the specific epitope to which MAb 17C7 binds. A common peptide sequence, designated as the "3Q" peptide, found between amino acid residues 480-502 and 582-604 of the UspAl protein (SEQ ID NO:l) and
SUBSTTTUTE SHEET (RULE 25) residues 355-377 of the UspA2 protein (SEQ ID NO:3) of M. catarrhalis strain 035E, encompasses the region which appears to be recognized by MAb 17C7. (Note that numbering of the amino acid residues is based upon strain 035E as provided in SEQ ID NO:3.) It is envisioned that this region plays an important role in the biology of the pathogen and, from this information, one will deduce amino acids residues that are critical in MAb 17C7 antibody binding. It also is envisioned that, based upon this information, one will be able to design epitopic regions that have either a higher or lower affinity for the MAb 17C7 or other antibodies. Further embodiments of the present invention are discussed below.
In another preferred embodiment, the present invention provides DNA segments, vectors and the like comprising at least one isolated gene, DNA segment or coding region that encodes a M. catarrhalis UspAl or UspA2 protein, polypeptide, domain, peptide or any fusion protein thereof. Herein are provided at least an isolated gene, DNA segment or coding region that encodes a M. catarrhalis uspAl gene comprising about 2493 base pairs (bp) (SEQ ID
NO:2) of strain O35E, about 3381 bp (SEQ ID NO:6) of strain O46E, about 3538 bp (SEQ ID NO: 10) of strain TTA24, or about 3292 bp (SEQ ID NO: 14) of strain TTA37. Further provided are at least an isolated gene, DNA segment or coding region that encodes a M. catarrhalis uspA2 gene comprising about 1728 bp (SEQ ID NO:4) of strain O35E, about 3295 bp (SEQ ID
NO:8) of strain 046E, about 2673 bp (SEQ ID NO:12), or about 4228 bp (SEQ ID NO:16) of strain TTA37. It is envisioned that the uspAl and uspA2 genes will be useful in the preparation of proteins, antibodies, screening assays for potential candidate drugs and the like to treat or inhibit, or even prevent, M. catarrhalis infections.
The present invention also provides for the use of the UspAl or UspA2 proteins or peptides as immunogenic carriers of other agents which are useful for the treatment, inhibition or even prevention of other bacterial, viral or parasitic infections. It is envisioned that either the UspAl or UspA2 antigen, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids such that a bispecific or multivalent composition or vaccine which is useful for the treatment, inhibition or even prevention of infection by M. catarrhalis and another pathogen(s) is prepared. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and, for example, similar to those used to prepare conjugates to keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
SUBSTTTUTE SHEET RULE 25) It is important to note that screening methods for diagnosis and prophylaxis are readily available, as set forth below. Thus, the ability to (i) test peptides, mutant peptides and antibodies for their reactivity with each other and (ii) test peptides and antibodies for the ability to prevent infections in vivo, provide powerful tools to develop clinically important reagents.
1.0 UspA Proteins, Peptides and Polypeptides
The present invention, in one embodiment, encompasses the two new protein sequences, UspAl and UspA2, and the peptide sequence AQQQDQH (SEQ ID NO: 17) identified as the target epitope of MAb 17C7. In addition, inspection of the amino acid sequences of the UspAl and UspA2 proteins from four strains of M. catarrhalis indicated that each protein contained at least one copy of the peptide YELAQQQDQH (SEQ ID NO: 18) which binds Mab 17C7 or, in one instance, a peptide nearly identical and having the amino acid sequence YDLAQQQDQH (SEQ ID NO:19).
The peptide (YELAQQQDQH, SEQ ID NO: 18) occurs twice in UspAl from strain O35E at residues 486-495 and 588-597 (SEQ ID NOT) and once in UspA2 from strain O35E at residues 358-367 (SEQ ID NO:3). It occurs once in UspAl from strain TTA24 at residues 497- 506 (SEQ ID NO:9) and twice in UspA2 from strain TTA24 at residues 225-234 and 413-422 (SEQ ID NOT 1). The peptide YDLAQQQDQH (SEQ ID NO: 19) occurs once in UspAl from strain 046E at residues 448-457 (SEQ ID NO:5) whereas the peptide YELAQQQDQH (SEQ ID NO: 18) occurs once in this same protein at residues 649-658 (SEQ ID NO:5). The peptide
YELAQQQDQH (SEQ ID NO: 18) occurs once in UspA2 from strain O46E at residues 416-425 (SEQ ID NO:7). The peptide YELAQQQDQH (SEQ ID NO: 18) occurs twice in UspAl from strain TTA37 at residues 478-487 and 630-639 (SEQ ID NO: 13) and twice in UspA2 from strain TTA37 at residues 522-531 and 681-690 (SEQ ID NO: 15).
Also encompassed in the present invention are hybrid molecules containing portions from one UspA protein, for example the UspAl protein, fused with portions of the other UspA protein, in this example the UspA2 protein, or fused with other proteins which are useful for identification, such as kanamycin-resistance, or other purposes in the screening of potential vaccines or further characterization of the UspAl and UspA2 proteins. For example, one may fuse residues 1-350 of any UspAl with residues 351-576 of any UspA2. Alternatively, a fusion could be generated with sequences from three, four or even five peptide regions represented in a
SUBSTITUTE SHEET RULE 25) single UspA antigen. Also encompassed are fragments of the disclosed UspAl and UspA2 molecules, as well as insertion, deletion or replacement mutants in which non-UspA sequences are introduced, UspA sequences are removed, or UspA sequences are replaced with non-UspA sequences, respectively.
UspAl and UspA2 proteins, according to the present invention, may be advantageously cleaved into fragments for use in further structural or functional analysis, or in the generation of reagents such as UspA-related polypeptides and UspA-specific antibodies. This can be accomplished by treating purified or unpurified UspAl and/or UspA2 with a peptidase such as endoproteinase glu-C (Boehringer, Indianapolis, IN). Treatment with CNBr is another method by which UspAl and/or UspA2 fragments may be produced from their natural respective proteins.
Recombinant techniques also can be used to produce specific fragments of UspAl or UspA2.
More subtle modifications and changes may be made in the structure of the encoded UspAl or UspA2 polypeptides of the present invention and still obtain a molecule that encodes a protein or peptide with characteristics of the natural UspA antigen. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table:
SUBSTITUTE SHEET RULE 25 TABLE I
Amino acid names and Codons abbreviations
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC uuu
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine He I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gin Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Tip w UGG
Tyrosine Tyr Y UAC UAU
It is known that certain amino acids may be substituted for other amino acids in a protein structure in order to modify or improve its antigenic or immunogenic activity (see, e.g., Kyte & Doolittle, 1982; Hopp, U.S. patent 4,554,101 , incorporated herein by reference). For example, through the substitution of alternative amino acids, small conformational changes may
SUBSTITUTE SHEET RULE 25 be conferred upon a polypeptide which result in increased activity or stability. Alternatively, amino acid substitutions in certain polypeptides may be utilized to provide residues which may then be linked to other molecules to provide peptide-molecule conjugates which retain enough antigenicity of the starting peptide to be useful for other purposes. For example, a selected UspAl or UspA2 peptide bound to a solid support might be constructed which would have particular advantages in diagnostic embodiments.
The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein has been discussed generally by Kyte & Doolittle (1982), wherein it is found that certain amino acids may be substituted for other amino acids having a similar hydropathic index or core and still retain a similar biological activity. As displayed in Table II below, amino acids are assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with substrate molecules. Preferred substitutions which result in an antigenically equivalent peptide or protein will generally involve amino acids having index scores within ±2 units of one another, and more preferably within ±1 unit, and even more preferably, within ±0.5 units.
TABLE II
Amino Acid Hydropathic Index
Isoleucine 4.5
Valine 4.2
Leucine 3.8
Phenylalanine 2.8
Cysteine/cystine 2.5
Methionine 1.9
Alanine 1.8
Glycine -0.4
Threonine -0.7
SUBSTHUTE SHEET RULE 25 Table II (Continued)
Amino Acid Hydropathic Index
Tryptophan -0.9
Serine -0.8
Tyrosine -1.3
Proline -1.6
Histidine -3.2
Glutamic Acid -3.5
Glutamine -3.5
Aspartic Acid -3.5
Asparagine -3.5
Lysine -3.9
Arginine -4.5
Thus, for example, isoleucine, which has a hydropathic index of +4.5, will preferably be exchanged with an amino acid such as valine (+ 4.2) or leucine (+ 3.8). Alternatively, at the other end of the scale, lysine (- 3.9) will preferably be substituted for arginine (-4.5), and so on.
Substitution of like amino acids may also be made on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with an important biological property of the protein.
As detailed in U.S. Patent 4,554,101, each amino acid has also been assigned a hydrophilicity value. These values are detailed below in Table III.
SUBSTITUTE SHEET RULE 25) TABLE III
Amino Acid Hydrophilic Index arginine +3.0 lysine +3.0 aspartate +3.0 + 1 glutamate +3.0 + 1 serine +0.3 asparagine +0.2 glutamine +0.2 glycine 0 threonine -0.4 alanine -0.5 histidine -0.5 proline -0.5 ± 1 cysteine -1.0 methionine -1.3 valine -1.5 leucine -1.8 isoleucine -1.8 tyrosine -2.3 phenylalanine -2.5 tryptophan -3.4
It is understood that one amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
Accordingly, these amino acid substitutions are generally based on the relative similarity of R-group substituents, for example, in terms of size, electrophilic character, charge, and the like. In general, preferred substitutions which take various of the foregoing characteristics into
SUBSTITUTE SHEET RULE 25) consideration will be known to those of skill in the art and include, for example, the following combinations: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In addition, peptides derived from these polypeptides, including peptides of at least about 6 consecutive amino acids from these sequences, are contemplated. Alternatively, such peptides may comprise about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 consecutive residues. For example, a peptide that comprises 6 consecutive amino acid residues may comprise residues 1 to 6, 2 to 7, 3 to 8 and so on of the UspAl or UspA2 protein. Such peptides may be represented by the formula
x to (x + n) = 5' to 3' the positions of the first and last consecutive residues
where x is equal to any number from 1 to the full length of a UspAl or UspA2 protein and n is equal to the length of the peptide minus 1. So, for UspAl , x = 1 to 831, for UspA2, x = 1 to 576. Where the peptide is 10 residues long (n = 10-1), the formula represents every 10-mer possible for each antigen. For example, where x is equal to 1 the peptide would comprise residues 1 to (1 + [10-1]), or 1 to 10. Where x is equal to 2, the peptide would comprise residues 2 to (2 + [10-2]), or 2 to 1 1, and so on.
Syntheses of peptides are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of a commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptides synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of 7.0 to 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or
SUBSTTTUTE SHEET (RULE 25) Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4°C, or more preferably, frozen. Of course, where the peptide(s) are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled, deionized) or buffer prior to use.
Of particular interest are peptides that represent epitopes that lie within the UspA antigen and are encompassed by the UspAl and UspA2 proteins of the present invention. An "epitope" is a region of a molecule that stimulates a response from a T-cell or B-cell, and hence, elicits an immune response from these cells. An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is structurally "complementary" to, and therefore will bind to, binding sites on antibodies or T-cell receptors. It will be understood that, in the context of the present disclosure, the term "complementary" refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitopic core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the corresponding UspA antigen to the corresponding UspA- directed antisera.
The identification of epitopic core sequences is known to those of skill in the art. For example U.S. Patent 4,554,101 teaches identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity, and by Chou-Fasman analyses. Numerous computer programs are available for use in predicting antigenic portions of proteins, examples of which include those programs based upon Jameson- Wolf analyses (Jameson and Wolf, 1988; Wolf et al, 1988), the program PepPlot® (Brutlag et al, 1990; Weinberger et al, 1985), and other new programs for protein tertiary structure prediction (Fetrow & Bryant, 1993) that can be used in conjunction with computerized peptide sequence analysis programs.
In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence anticipated by the present disclosure would be on the order of about 6 amino acids in length. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
SUBSTITUTE SHEET (RULE 25) 2.0 UspAl and UspA2 Nucleic Acids
In addition to polypeptides, the present invention also encompasses nucleic acids encoding the UspAl (SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO: 10 and SEQ ID NO: 14) and UspA2 (SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 12 and SEQ ID NO: 16) proteins from the exemplary M. catarrhalis strains 035E, 046E, TTA24 and TTA37, respectively. Because of the degeneracy of the genetic code, many other nucleic acids also may encode a given UspAl or UspA2 protein. For example, four different three-base codons encode the amino acids alanine, glycine, proline, threonine and valine, while six different codons encode arginine, leucine and serine. Only methionine and tryptophan are encoded by a single codon. Table I provides a list of amino acids and their corresponding codons for use in such embodiments. In order to generate any nucleic acid encoding UspAl or UspA2, one need only refer to the codon table provided herein. Substitution of the natural codon with any codon encoding the same amino acid will result in a distinct nucleic acid that encodes UspAl or UspA2. As a practical matter, this can be accomplished by site-directed mutagenesis of an existing uspAl or uspA2 gene or de novo chemical synthesis of one or more nucleic acids.
These observations regarding codon selection, site-directed mutagenesis and chemical synthesis apply with equal force to the discussion of substitutional mutant UspAl or UspA2 peptides and polypeptides, as set forth above. More specifically, substitutional mutants generated by site-directed changes in the nucleic acid sequence that are designed to alter one or more codons of a given polypeptide or epitope may provide a more convenient way of generating large numbers of mutants in a rapid fashion. The nucleic acids of the present invention provide for a simple way to generate fragments (e.g., truncations) of UspAl or UspA2, UspAl-UspA2 fusion molecules (discussed above) and UspAl or UspA2 fusions with other molecules. For example, utilization of restriction enzymes and nuclease in the uspAl or uspA2 gene permits one to manipulate the structure of these genes, and the resulting gene products.
The nucleic acid sequence information provided by the present disclosure also allows for the preparation of relatively short DNA (or RNA) sequences that have the ability to specifically hybridize to gene sequences of the selected uspAl or uspA2 gene. In these aspects nucleic acid probes of an appropriate length are prepared based on a consideration of the coding sequence of the uspAl or uspA2 gene, or flanking regions near the uspAl or uspA2 gene, such as regions downstream and upstream in the M. catarrhalis chromosome. The ability of such
SUBSTITUTE SHEET RULE 25) nucleic acid probes to specifically hybridize to either uspAl or uspA2 gene sequences lends them particular utility in a variety of embodiments. For example, the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample. In addition, these oligonucleotides can be inserted, in frame, into expression constructs for the purpose of screening the corresponding peptides for reactivity with existing antibodies or for the ability to generate diagnostic or therapeutic reagents.
To provide certain of the advantages in accordance with the invention, the preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 10 to 20, or so, nucleotide stretch of the sequence, although sequences of 30 to 60 or so nucleotides are also envisioned to be useful. A size of at least 9 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Though molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of the specific hybrid molecules obtained. Thus, one will generally prefer to design nucleic acid molecules having either uspAl or uspA2 gene-complementary stretches of 15 to 20 nucleotides, or even longer, such as 30 to 60, where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR™ technology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant production.
The probes that would be useful may be derived from any portion of the sequences of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16. Therefore, probes are specifically contemplated that comprise nucleotides 1 to 9, or 2 to 10, or 3 to 11 and so forth up to a probe comprising the last 9 nucleotides of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or
SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO:16. Thus, each probe would comprise at least about 9 linear nucleotides of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16., designated by the formula "n to n + 8," where n is an integer from 1 to the number of nucleotides in the sequence. Longer probes that hybridize to the uspAl or uspA2 gene under low, medium, medium-high and high stringency conditions are
SUBSTITUTE 5HEET RULE 25 also contemplated, including those that comprise the entire nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO: 12 or SEQ ID NO.T4 or SEQ ID NO: 16. This hypothetical may be repeated for probes having lengths of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 and greater bases.
In that the UspA antigenic epitopes of the present invention are believed to be indicative of pathogenic Moraxella species as exemplified by strains O35E, O46E, TTA24 and TTA37, the probes of the present invention will find particular utility as the basis for diagnostic hybridization assays for detecting UspAl or UspA2 DNA in clinical samples. Exemplary clinical samples that can be used in the diagnosis of infections are thus any samples which could possibly include Moraxella nucleic acid, including middle ear fluid, sputum, mucus, bronchoalveolar fluid, amniotic fluid or the like. A variety of hybridization techniques and systems are known which can be used in connection with the hybridization aspects of the invention, including diagnostic assays such as those described in Falkow et al, U.S. Patent 4,358,535. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe toward the target sequence. For applications requiring a high degree of selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, for example, one will select relatively low salt and/or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C to 70°C. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent hybridization conditions are called for in order to allow formation of the heteroduplex. In these circumstances, one would desire to employ conditions such as 0.15M-0.9M salt, at temperatures ranging from 20°C to 55°C. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and the method of choice will generally depend on the desired results.
SUBSTHUTE SHEET RULE 25) In certain embodiments, one may desire to employ nucleic acid probes to isolate variants from clone banks containing mutated clones. In particular embodiments, mutant clone colonies growing on solid media which contain variants of the UspAl and/or UspA2 sequence could be identified on duplicate filters using hybridization conditions and methods, such as those used in colony blot assays, to obtain hybridization only between probes containing sequence variants and nucleic acid sequence variants contained in specific colonies. In this manner, small hybridization probes containing short variant sequences of either the uspAl or uspA2 gene may be utilized to identify those clones growing on solid media which contain sequence variants of the entire uspAl or uspA2 gene. These clones can then be grown to obtain desired quantities of the variant UspAl or UspA2 nucleic acid sequences or the corresponding UspA antigen.
In clinical diagnostic embodiments, nucleic acid sequences of the present invention are used in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred diagnostic embodiments, one will likely desire to employ an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with pathogen nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridizations as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) from suspected clinical samples, such as exudates, body fluids (e.g., amniotic fluid, middle ear effusion, bronchoalveolar lavage fluid) or even tissues, is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove
SUBSTTTUTE SHEET (RULE 25) nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
The nucleic acid sequences which encode for the UspAl and/or UspA2 epitopes, or their variants, may be useful in conjunction with PCR™ methodology to detect M. catarrhalis. In general, by applying the PCR™ technology as set out, e.g. , in U.S. Patent 4,603,102, one may utilize various portions of either the uspAl or uspA2 sequence as oligonucleotide probes for the PCR™ amplification of a defined portion of a uspAl or uspA2 nucleic acid in a sample. The amplified portion of the uspAl or uspA2 sequence may then be detected by hybridization with a hybridization probe containing a complementary sequence. In this manner, extremely small concentrations of M. catarrhalis nucleic acid may detected in a sample utilizing uspAl or uspA2 sequences.
3.0 Vectors, Host Cells and Cultures for Producing UspAl and/or UspA2 Antigens
In order to express a UspAl and/or UspA2 polypeptide, it is necessary to provide an uspAl and/or uspA2 gene in an expression cassette. The expression cassette contains a UspAl and/or UspA2-encoding nucleic acid under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. Those promoters most commonly used in prokaryotic recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al, 1977; Goeddel et al, 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1980; EPO Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors
(EPO Appl. Publ. No. 0036776). Additional examples of useful promoters are provided in Table IV below.
SUBSTHUTE SHEET RULE 25 TABLE IV
Figure imgf000028_0001
SUBSTITUTE SHEET RULE 25) TABLE IV (Continued)
Figure imgf000029_0001
SUBSTITUTE SHEET RULE 25) TABLE IV (Continued)
Figure imgf000030_0001
SUBSTHUTE SHEET RULE 25) The appropriate expression cassette can be inserted into a commercially available expression vector by standard subcloning techniques. For example, the E. coli vectors pUC or pBluescript™ may be used according to the present invention to produce recombinant UspAl and/or UspA2 polypeptide in vitro. The manipulation of these vectors is well known in the art. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar et al, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins.
In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as a transforming vector in connection with these hosts. For example, the phage lambda GEM -1 1 may be utilized in making recombinant phage vector which can be used to transform host cells, such as E. coli LE392.
In one embodiment, the UspA antigen is expressed as a fusion protein by using the pGEX4T-2 protein fusion system (Pharmacia LKB, Piscataway, NJ), allowing characterization of the UspA antigen as comprising both the UspAl and UspA2 proteins. Additional examples of fusion protein expression systems are the glutathione S-transferase system (Pharmacia,
Piscataway, NJ), the maltose binding protein system (NEB, Beverley, MA), the FLAG system
(IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA). Some of these fusion systems produce recombinant protein bearing only a small number of additional amino acids, which are unlikely to affect the functional capacity of the recombinant protein. For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the protein to its native conformation. Other fusion systems produce proteins where it is desirable to excise the fusion partner from the desired protein. In another embodiment, the fusion partner is linked to the
SUBSTITUTE SHEET (RULE 25) recombinant protein by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA).
E. coli is a preferred prokaryotic host. For example, E. coli strain RR1 is particularly useful. Other microbial strains which may be used include E. coli strains such as E. coli LE392, E. coli B, and E. coli X 1776 (ATCC No. 31537). The aforementioned strains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may be used. These examples are, of course, intended to be illustrative rather than limiting. Recombinant bacterial cells, for example E. coli, are grown in any of a number of suitable media, for example LB, and the expression of the recombinant polypeptide induced by adding IPTG to the media or switching incubation to a higher temperature. After culturing the bacteria for a further period of between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual media. The bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a selective speed.
If the recombinant protein is expressed in the inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g. 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents such as β- mercaptoethanol or DTT (dithiothreitol).
Under some circumstances, it may be advantageous to incubate the polypeptide for several hours under conditions suitable for the protein to undergo a refolding process into a conformation which more closely resembles that of the native protein. Such conditions generally include low protein concentrations less than 500 μg/ml, low levels of reducing agent, concentrations of urea
SUBSTITUTE SHEET RULE 25) less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulfide bonds within the protein molecule.
The refolding process can be monitored, for example, by SDS-PAGE or with antibodies which are specific for the native molecule (which can be obtained from animals vaccinated with the native molecule isolated from bacteria). Following refolding, the protein can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns.
There are a variety of other eukaryotic vectors that provide a suitable vehicle in which recombinant UspA proteins can be produced. In various embodiments of the invention, the expression construct may comprise a virus or engineered construct derived from a viral genome.
The ability of certain viruses to enter cells via receptor-mediated endocytosis and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The first viruses used as vectors were DNA viruses including the papovaviruses (simian virus 40 (SV40), bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986) and adeno-associated viruses. Retroviruses also are attractive gene transfer vehicles (Nicolas and Rubenstein, 1988; Temin, 1986) as are vaccina virus (Ridgeway, 1988) adeno-associated virus (Ridgeway, 1988) and herpes simplex virus (HSV) (Glorioso et al, 1995). Such vectors may be used to (i) transform cell lines in vitro for the purpose of expressing proteins of interest or (ii) to transform cells in vitro or in vivo to provide therapeutic polypeptides in a gene therapy scenario.
With respect to eukaryotic vectors, the term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
SUBSTITUTE SHEET (RULE 25) At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a
TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell.
Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter. Preferred promoters include those derived from HSV, including the α4 promoter. Another preferred embodiment is the tetracycline controlled promoter.
In various other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
Table IV lists several promoters which may be employed, in the context of the present invention, to regulate the expression of a transgene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of transgene expression but, merely, to be exemplary thereof.
SUBSTTTUTE SHEET RULE 25) Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Table V lists several enhancers, of course, this list is not meant to be limiting but exemplary.
TABLE V
Figure imgf000035_0001
SUBSTITUTE SHEET RULE 25) TABLE V (Continued)
Figure imgf000036_0001
Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data
Base EPDB) could also be used to drive expression of a transgene. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
Host cells include eukaryotic microbes, such as yeast cultures may also be used.
Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid already contains the
SUBSTITUTE SHEET (RULE 25) trp\ gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trp\ lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (Hitzeman et al, 1980) or other glycolytic enzymes (Hess et al, 1968; Holland et al, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.
In addition to eukaryotic microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973). Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
SUBSTITUTE SHEET (RULE 25) 4.0 Preparation of Antibodies to UspA Proteins
Antibodies to UspAl or UspA2 peptides or polypeptides may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., purified or partially purified protein, synthetic protein or fragments thereof, as discussed in the section on vaccines. Animals to be immunized are mammals such as cats, dogs and horses, although there is no limitation other than that the subject be capable of mounting an immune response of some kind. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is possible. The use of rats may provide certain advantages, but mice are preferred, with the BALB/c mouse being most preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions.
For generation of monoclonal antibodies (MAbs), following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer removed. Spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5
7 R x 10 to 2 x 10 lymphocytes.
The antibody-producing B cells from the immunized animal are then fused with cells of an immortal myeloma cell line, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells, called "hybridomas."
SUBSTITUTE SHEET (RULE 25) Any one of a number of myeloma cells may be used and these are known to those of skill in the art. For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NSl/l .Ag 4 1 , Sp210-Agl4, FO, NSO/U, MPC-1 1, MPC1 1-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell line is the NS-1 myeloma cell line (also termed
P3-NS-l-Ag4-l), which is readily available from the NIGMS Human Genetic Mutant Cell
Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler & Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate.
Fusion procedures usually produce viable hybrids at low frequencies, about 1 10" to 1 10 . This does not pose a problem, however, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culture in a selective medium. The selective medium generally is one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
SUBSTTTUTE SHEET (RULE 25) The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
Monoclonal antibodies of the present invention also include anti-idiotypic antibodies produced by methods well-known in the art. Monoclonal antibodies according to the present invention also may be monoclonal heteroconjugates, i.e., hybrids of two or more antibody molecules. In another embodiment, monoclonal antibodies according to the invention are chimeric monoclonal antibodies. In one approach, the chimeric monoclonal antibody is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region
SUBSTITUTE SHEET (RULE 25) sequences from a mouse antibody producing cell and the constant-region exons from a human antibody gene. The antibody encoded by such a recombinant gene is a mouse-human chimera. Its antibody specificity is determined by the variable region derived from mouse sequences. Its isotype, which is determined by the constant region, is derived from human DNA.
In another embodiment, the monoclonal antibody according to the present invention is a
"humanized" monoclonal antibody, produced by techniques well-known in the art. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse Ig into a human V-domain, followed by the replacement of some human residues in the framework regions of their murine counterparts. "Humanized" monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and therapeutic methods for treating Moraxella infections.
As stated above, the monoclonal antibodies and fragments thereof according to this invention can be multiplied according to in vitro and in vivo methods well-known in the art. Multiplication in vitro is carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like. In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for large scale hybridoma cultivation under tissue culture conditions are known in the art and include homogenous suspension culture, e.g., in an airlift reactor or in a continuous stirrer reactor or immobilized or entrapped cell culture.
Large amounts of the monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo. Cell clones are injected into mammals which are histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to injection.
In accordance with the present invention, fragments of the monoclonal antibody of the invention can be obtained from monoclonal antibodies produced as described above, by methods which include digestion with enzymes such as pepsin or papain and/or cleavage of
SUBSTITUTE SHEET (RULE 25) disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or they may be produced manually using techniques well known in the art.
The monoclonal conjugates of the present invention are prepared by methods known in the art. e.g., by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as 3H, l25I, I311 32P, 35S, 14C, Cr, Cl, 3 Co, Co, Fe, Se, Eu, and mTc, are other useful labels which can be conjugated to antibodies. Radio-labeled monoclonal antibodies of the present invention are produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium- m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNC12, a buffer solution such as sodium-potassium phthalate solution, and the antibody.
5.0 Use of Peptides and Monoclonal Antibodies in Immunoassays
It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and western blot methods, as well as other procedures which may utilize antibodies specific to CopB epitopes. While ELISAs are preferred, it will be readily appreciated that such assays include RIAs and other non-enzyme linked antibody binding assays or procedures. Additionally, it is proposed that monoclonal antibodies specific to the particular UspA epitope may be utilized in other useful applications. For example, their use in immunoabsorbent protocols may be useful in purifying native or recombinant UspA proteins or variants thereof.
It also is proposed that the disclosed UspAl and UspA2 peptides of the invention will find use as antigens for raising antibodies and in immunoassays for the detection of anti-UspA
SUBSTITUTE SHEET (RULE 25) antigen-reactive antibodies. In a variation on this embodiment, UspAl and UspA2 mutant peptides may be screened, in immunoassay format, for reactivity against UspAl- or UspA2-specific antibodies, such as MAb 17C7. In this way, a mutational analysis of various epitopes may be performed. Results from such analyses may then be used to determine which additional UspAl or UspA2 epitopes may be recognized by antibodies and useful in the preparation of potential vaccines for Moraxella.
Diagnostic immunoassays include direct culturing of bodily fluids, either in liquid culture or on a solid support such as nutrient agar. A typical assay involves collecting a sample of bodily fluid from a patient and placing the sample in conditions optimum for growth of the pathogen. The determination can then be made as to whether the microbe exists in the sample.
Further analysis can be carried out to determine the hemolyzing properties of the microbe.
Immunoassays encompassed by the present invention include, but are not limited to those described in U.S. Patent No. 4,367,1 10 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.
Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELI S As) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.
In one exemplary ELISA, the anti-UspA antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the desired antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen, that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a
SUBSTITUTE SHEET (RULE 25) third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
In another exemplary ELISA, the samples suspected of containing the UspA antigen are immobilized onto the well surface and then contacted with the anti-UspA antibodies. After binding and appropriate washing, the bound immune complexes are detected. Where the initial antigen specific antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antigen specific antibody, with the second antibody being linked to a detectable label.
Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the primary antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if desired.
Competition ELISAs are also possible in which test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells. (Antigen or antibodies may also be linked to a solid support, such as in the form of beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody.) The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.
Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
SUBSTITUTE SHEET (RULE 25) In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin
(BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C. Following incubation, the antisera- contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity in general for human IgG. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
SUBSTITUTE SHEET (RULE 25) After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid [ABTS] and H202, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. Alternatively, the label may be a chemilluminescent one. The use of such labels is described in U.S. Patent Nos. 5,310,687, 5,238,808 and 5,221,605.
6.0 Prophylactic Use of UspA Peptides and UspA-Specific Antibodies
In a further embodiment of the present invention, there are provided methods for active and passive immunoprophylaxis. Active immunoprophylaxis will be discussed first, followed by a discussion on passive immunoprophylaxis. It should be noted that the discussion of formulating vaccine compositions in the context of active immunotherapy is relevant to the raising antibodies in experimental animals for passive immunotherapy and for the generation of diagnostic methods.
6.1 Active Immunotherapy
According to the present invention, UspAl or UspA2 polypeptides or UspAl- or UspA2-derived peptides, as discussed above, may be used as vaccine formulations to generate protective anti- catarrhalis antibody responses in vivo. By protective, it is only meant that the immune system of a treated individual is capable of generating a response that reduces, to any extent, the clinical impact of the bacterial infection. This may range from a minimal decrease in bacterial burden to outright prevention of infection. Ideally, the treated subject will not exhibit the more serious clinical manifestations of M. catarrhalis infection.
Generally, immunoprophylaxis involves the administration, to a subject at risk, of a vaccine composition. In the instant case, the vaccine composition will contain a UspAl and/or UspA2 polypeptide or immunogenic derivative thereof in a pharmaceutically acceptable carrier, diluent or excipient. As stated above, those of skill in the art are able, through a variety of mechanisms, to identify appropriate antigenic characteristics of UspAl and UspA2 and , in so doing, develop vaccines that will achieve generation of immune responses against M. catarrhalis.
SUBSTITUTE 5HEET (RULE 25) The stability and immunogenicity of UspAl and UspA2 antigens may vary and, therefore, it may be desirable to couple the antigen to a carrier molecule. Exemplary carriers are KLH, BSA, human serum albumin, myoglobin, β-galactosidase, penicillinase, CRM197 and bacterial toxoids, such as diphtheria toxoid and tetanus toxoid. Those of skill in the art are aware of proper methods by which peptides can be linked to carriers without destroying their immunogenic value. Synthetic carriers such as multi-poly-DL-alanyl-poly-L-lysine and poly-L- lysine also are contemplated. Coupling generally is accomplished through amino or carboxyl- terminal residues of the antigen, thereby affording the peptide or polypeptide the greatest chance of assuming a relatively "native" conformation following coupling.
It is recognized that other protective agents could be coupled with either a UspAl or
UspA2 antigen such that the UspAl or UspA2 antigen acts as the carrier molecule. For example, agents which protect against other pathogenic organisms, such as bacteria, viruses or parasites, could be coupled to either a UspAl or UspA2 antigen to produce a multivalent vaccine or pharmaceutical composition which would be useful for the treatment or inhibition of both M. catarrhalis infection and other pathogenic infections. In particular, it is envisioned that either UspAl or UspA2 proteins or peptides could serve as immunogenic carriers for other vaccine components, for example, saccharides of pneumococcus, menigococcus or hemophylus influenza and could even be covalently coupled to these other components.
It also may be desirable to include in the composition any of a number of different substances referred to as adjuvants, which are known to stimulate the appropriate portion of the immune system of the vaccinated animal. Suitable adjuvants for the vaccination of subjects
(including experimental animals) include, but are not limited to oil emulsions such as Freund's complete or incomplete adjuvant (not suitable for livestock use), Marcol 52:Montanide 888
(Marcol is a Trademark of Esso, Montanide is a Trademark of SEPPIC, Paris), squalane or squalene, Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), MPL™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research
Inc., Hamilton, Utah), Stimulon™ (QS-21 ; Aquila Biopharmaceuticals Inc., Wooster, MA), mineral gels such as aluminum hydroxide, aluminum phosphate, calcium phosphate and alum, surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyl- dioctadecylammonium bromide, N,N-dioctadecyl-N,N'-bis(2-hydroxyethyl)-propanediamine, methoxyhexadecylglycerol and pluronic polyols, polyanions such as pyran, dextran sulfate,
SUBSTITUTE SHEET (RULE 25) polyacrylic acid and carbopol, peptides and amino acids such as muramyl dipeptide, dimethylglycine, tuftsin and trehalose dimycolate. Agents include synthetic polymers of sugars (Carbopol), emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution, of a perfluorocarbon (Fluosol-DA) also may be employed.
The preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601 ,903; 4,599,231; 4,599,230; 4,596,792; and 4.578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables. Either as liquid solutions or suspensions: Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
The vaccine preparations of the present invention also can be administered following incorporation into non-toxic carriers such as liposomes or other microcarrier substances, or after conjugation to polysaccharides, proteins or polymers or in combination with Quil-A to form "iscoms" (immunostimulating complexes). These complexes can serve to reduce the toxicity of the antigen, delay its clearance from the host and improve the immune response by acting as an adjuvant. Other suitable adjuvants for use this embodiment of the present invention include INF, IL-2, IL-4, IL-8, IL-12 and other immunostimulatory compounds. Further, conjugates comprising the immunogen together with an integral membrane protein of prokaryotic origin, such as TraT (see PCT/AU87/00107) may prove advantageous.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient
SUBSTTTUTE SHEET (RULE 25) in the range of 0.5% to 10%, preferably 1 -2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate. sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
The peptides may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as. for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and
SUBSTITUTE SHEET (RULE 25) preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.
6.2 Passive Immunotherapy Passive immunity is defined, for the purposes of this application, as the transfer to an organism of an immune response effector that was generated in another organism. The classic example of establishing passive immunity is to transfer antibodies produced in one organism into a second, immunologically compatible animal. By "immunologically compatible," it is meant that the antibody can perform at least some of its immune functions in the new host animal. More recently, as a better understanding of cellular immune functions has evolved, it has become possible to accomplish passive immunity by transferring other effectors, such as certain kinds of lymphocytes, including cytotoxic and helper T cells, NK cells and other immune effector cells. The present invention contemplates both of these approaches.
Antibodies, antisera and immune effector cells are raised using standard vaccination regimes in appropriate animals, as discussed above. The primary animal is vaccinated with at least a microbe preparation or one bacterial product or by-product according to the present invention, with or without an adjuvant, to generate an immune response. The immune response may be monitored, for example, by measurement of the levels of antibodies produced, using standard ELISA methods.
Once an adequate immune response has been generated, immune effector cells can be collected on a regular basis, usually from blood draws. The antibody fraction can be purified from the blood by standard means, e.g., by protein A or protein G chromatography. In an alternative preferred embodiment, monoclonal antibody-producing hybridomas are prepared by standard means (Coligan et al, 1991). Monoclonal antibodies are then prepared from the hybridoma cells by standard means. If the primary host's monoclonal antibodies are not
SUBSTTTUTE SHEET (RULE 25) compatible with the animal to be treated, it is possible that genetic engineering of the cells can be employed to modify the antibody to be tolerated by the animal to be treated. In the human context, murine antibodies, for example, may be "humanized" in this fashion.
Antibodies, antisera or immune effector cells, prepared as set forth above, are injected into hosts to provide passive immunity against microbial infestation. For example, an antibody composition is prepared by mixing, preferably homogeneously mixing, at least one antibody with at least one pharmaceutically or veterinarally acceptable carrier, diluent, or excipient using standard methods of pharmaceutical or veterinary preparation. The amount of antibody required to produce a single dosage form will vary depending upon the microbial species being vaccinated against, the individual to be treated and the particular mode of administration. The specific dose level for any particular individual will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the individual, time of administration, route of administration, rate of excretion, drug combination and the severity of the microbial infestation.
The antibody composition may be administered intravenously, subcutaneously, intranasally, orally, intramuscularly, vaginally, rectally, topically or via any other desired route. Repeated dosings may be necessary and will vary, for example, depending on the clinical setting, the particular microbe, the condition of the patient and the use of other therapies.
6.3 DNA Immunization HC The invention also relates to a vaccine comprising a nucleic acid molecule encoding a
UspAl , UspA2 protein or a peptide comprsing SEQ ID NO: 17 wherein said UspAl , UspA2 protein or peptide retains immunogenicity and, when incorporated into an immunogenic composition or vaccine and administered to a vertebrate, provides protection without inducing enhanced disease upon subsequent infection of the vertebrate with M. catarrhalis, and a physiologically acceptable vehicle. Such a vaccine is referred to herein as a nucleic acid vaccine or DNA vaccine and is useful for the genetic immunization of vertebrates.
The term, "genetic immunization", as used herein, refers to inoculation of a vertebrate, particularly a mammal such as a mouse or human, with a nucleic acid vaccine directed against a pathogenic agent, particularly M. catarrhalis, resulting in protection of the vertebrate against M.
SUBSTTTUTE SHEET (RULE 25) catarrhalis. A "nucleic acid vaccine" or "DNA vaccine" as used herein, is a nucleic acid construct comprising a nucleic acid molecule encoding UspAl , UspA2 or an immunogenic epitope comprising SEQ ID NO: 17. The nucleic acid construct can also include transcriptional promoter elements, enhancer elements, splicing signals, termination and polyadenylation signals, and other nucleic acid sequences.
The nucleic acid vaccine can be produced by standard methods. For example, using known methods, a nucleic acid (e.g., DNA) encoding UspAl or UspA2 can be inserted into an expression vector to construct a nucleic acid vaccine (see Maniatis et al, 1989). The individual vertebrate is inoculated with the nucleic acid vaccine (i.e., the nucleic acid vaccine is administered), using standard methods. The vertebrate can be inoculated subcutaneously, intravenously, intraperitoneally, intradermally, intramuscularly, topically, orally, rectally, nasally, buccally, vaginally, by inhalation spray, or via an implanted reservoir in dosage formulations containing conventional non-toxic, physiologically acceptable carriers or vehicles. Alternatively, the vertebrate is inoculated with the nucleic acid vaccine through the use of a particle acceleration instrument (a "gene gun"). The form in which it is administered (e.g., capsule, tablet, solution, emulsion) will depend in part on the route by which it is administered. For example, for mucosal administration, nose drops, inhalants or suppositories can be used.
The nucleic acid vaccine can be administered in conjunction with any suitable adjuvant. The adjuvant is administered in a sufficient amount, which is that amount that is sufficient to generate an enhanced immune response to the nucleic acid vaccine. The adjuvant can be administered prior to (e.g., 1 or more days before) inoculation with the nucleic acid vaccine; concurrently with (e.g., within 24 hours of) inoculation with the nucleic acid vaccine; contemporaneously (simultaneously) with the nucleic acid vaccine (e.g., the adjuvant is mixed with the nucleic acid vaccine, and the mixture is administered to the vertebrate); or after (e.g., 1 or more days after) inoculation with the nucleic acid vaccine. The adjuvant can also be administered at more than one time (e.g., prior to inoculation with the nucleic acid vaccine and also after inoculation with the nucleic acid vaccine). As used herein, the term "in conjunction with" encompasses any time period, including those specifically described herein and combinations of the time periods specifically described herein, during which the adjuvant can be administered so as to generate an enhanced immune response to the nucleic acid vaccine
(e.g., an increased antibody titer to the antigen encoded by the nucleic acid vaccine, or an
SUBSTTTUTE SHEET (RULE 25) increased antibody titer to M. catarrhalis). The adjuvant and the nucleic acid vaccine can be administered at approximately the same location on the vertebrate; for example, both the adjuvant and the nucleic acid vaccine are administered at a marked site on a limb of the vertebrate.
In a particular embodiment, the nucleic acid construct is co-administered with a transfection-facilitating agent. In a preferred embodiment, the transfection-facilitating agent is dioctylglycylspermine (DOGS) (as exemplified in published PCT application publication no. WO 96/21356 and incorporated herein by reference). In another embodiment, the transfection- facilitating agent is bupivicaine (as exemplified in U.S. Patent 5,593,972 and incorporated herein by reference).
6.4 Animal Model for Testing Efficacy of Therapies
The evaluation of the functional significance of antibodies to surface antigens of M. catarrhalis has been hampered by the lack of a suitable animal model. The relative lack of virulence of this organism for animals rendered identification of an appropriate model system difficult (Doern, 1986). Attempts to use rodents, including chinchillas, to study middle ear infections caused by M. catarrhalis were unsuccessful, likely because this organism cannot grow or survive in the middle ear of these hosts (Doyle, 1989).
Murine short-term pulmonary clearance models have now been developed (Unhanand et al, 1992; Verghese et al, 1990) which permit an evaluation of the interaction of M. catarrhalis with the lower respiratory tract as well as assessment of pathologic changes in the lungs. This model reproducibly delivers an inoculum of bacteria to a localized peripheral segment of the murine lung. Bacteria multiply within the lung, but are eventually cleared as a result of (i) resident defense mechanisms, (ii) the development of an inflammatory response, and/or (iii) the development of a specific immune response. Using this model, it has been demonstrated that serum IgG antibody can enter the alveolar spaces in the absence of an inflammatory response and enhance pulmonary clearance of nontypable H. influenzae (McGehee et al, 1989), a pathogen with a host range and disease spectrum nearly identical to those of M. catarrhalis.
SUBSTITUTE SHEET (RULE 25) 7.0 Screening Assays
In still further embodiments, the present invention provides methods for identifying new M. catarrhalis inhibitory compounds, which may be termed as "candidate substances," by screening for immunogenic activity with peptides that include one or more mutations to the identified immunogenic epitopic region. It is contemplated that such screening techniques will prove useful in the general identification of any compound that will serve the purpose of inhibiting, or even killing, M. catarrhalis, and in preferred embodiments, will provide candidate vaccine compounds.
It is further contemplated that useful compounds in this regard will in no way be limited to proteinaceous or peptidyl compounds. In fact, it may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assays will be non-peptidyl in nature and, e.g. , which will serve to inhibit bacterial protein transcription through a tight binding or other chemical interaction. Candidate substances may be obtained from libraries of synthetic chemicals, or from natural samples, such as rain forest and marine samples.
To identify a M. catarrhalis inhibitor, one would simply conduct parallel or otherwise comparatively controlled immunoassays and identify a compound that inhibits the phenotype of M. catarrhalis. Those of skill in the art are familiar with the use of immunoassays for competitive screenings (for example refer to Sambrook et al. 1989).
Once a candidate substance is identified, one would measure the ability of the candidate substance to inhibit M. catarrhalis in the presence of the candidate substance. In general, one will desire to measure or otherwise determine the activity of M. catarrhalis in the absence of the added candidate substance relative to the activity in the presence of the candidate substance in order to assess the relative inhibitory capability of the candidate substance.
7.1 Mutagenesis Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence
SUBSTTTUTE SHEET RULE 25) variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art. as will be appreciated, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single- stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For
SUBSTTTUTE SHEET (RULE 25) example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydrox ylamine, to obtain sequence variants.
7.2 Second Generation Inhibitors In addition to the inhibitory compounds initially identified, the inventor also contemplates that other sterically similar compounds may be formulated to mimic the key portions of the structure of the inhibitors. Such compounds, which may include peptidomimetics of peptide inhibitors, may be used in the same manner as the initial inhibitors.
Certain mimetics that mimic elements of protein secondary structure are designed using the rationale that the peptide backbone of proteins exists chiefly to orientate amino acid side chains in such a way as to facilitate molecular interactions. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
Some successful applications of the peptide mimetic concept have focused on mimetics of β-turns within proteins, which are known to be highly antigenic. Likely β-turn structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
The generation of further structural equivalents or mimetics may be achieved by the techniques of modeling and chemical design known to those of skill in the art. The art of computer-based chemical modeling is now well known. Using such methods, a chemical that specifically inhibits viral transcription elongation can be designed, and then synthesized, following the initial identification of a compound that inhibits RNA elongation, but that is not specific or sufficiently specific to inhibit viral RNA elongation in preference to human RNA elongation. It will be understood that all such sterically similar constructs and second generation molecules fall within the scope of the present invention.
SUBSTTTUTE SHEET RULE 25 8.0 DiagnosingM catarrhalis Infections
8.1 Amplification and PCR™
Nucleic acid sequence used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where
RNA is used, it may be desired to convert the RNA to a cDNA.
Pairs of primers that selectively hybridize to nucleic acids corresponding to UspAl or UspA2 protein or a mutant thereof are contacted with the isolated nucleic acid under conditions that permit selective hybridization. The term "primer", as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template- dependent process. Typically, primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.
Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
Next, the amplification product is detected. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).
A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, and each incorporated herein by reference in entirety.
SUBSTTTUTE SHEET RULE 25 Briefly, in PCR™, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
A reverse transcriptase PCR™ (RT-PCR™) amplification procedure may be performed in order to quantify the amount of mRNA amplified or to prepare cDNA from the desired mRNA. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed December 21 , 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.
Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPA No. 320 308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
Qbeta Replicase, described in PCT Application No. PCT US87/00880, incorporated herein by reference, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.
SUBST E 25 An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]- triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.
Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.
Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR™-like, template- and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically.
After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR
Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.
Davey et al, EPA No. 329 822 (incorporated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
Miller et al, PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a
SUBSTTT T RULE 25 promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, 1990, incorporated by reference).
Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention.
Following any amplification, it may be desirable to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al, 1989.
Alternatively, chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.
Amplification products must be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled, nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
S BST E 25 In one embodiment, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non- covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
One example of the foregoing is described in U.S. Patent No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
All the essential materials and reagents required for detecting P-TEFb or kinase protein markers in a biological sample may be assembled together in a kit. This generally will comprise preselected primers for specific markers. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
Such kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker primer pair. Preferred pairs of primers for amplifying nucleic acids are selected to amplify the sequences specified in SEQ ID
NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16 such that, for example, nucleic acid fragments are prepared that include a contiguous stretch of nucleotides identical to for example about 15, 20, 25, 30, 35, etc. ; 48, 49, 50, 51, etc. ; 75, 76, 77, 78, 79, 80 etc. ; 100, 101, 102, 103 etc. ; 1 18, 1 19, 120, 121 etc.; 127, 128, 129, 130, 131, etc.; 316, 317, 318, 319, etc. ; 322, 323, 324, 325, 326, etc.; 361, 362, 363, 364, etc.; 372, 373, 374, 375, etc. of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16, so long as the selected contiguous stretches are from spatially distinct regions. Similar fragments may be prepared which are identical or complimentary to, for example, SEQ ID NO: 1 such that the fragments do not hybridize to, for example, SEQ ID NO:3.
In another embodiment, such kits will comprise hybridization probes specific for UspAl or UspA2 proteins chosen from a group including nucleic acids corresponding to the sequences specified in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16 or to intermediate lengths of the sequences specified. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker hybridization probe.
8.2 Other Assays
Other methods for genetic screening to accurately detect M. catarrhalis infections that alter normal cellular production and processing, in genomic DNA, cDNA or RNA samples may be employed, depending on the specific situation.
For example, one method of screening for genetic variation is based on RNase cleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes. As used herein, the term "mismatch" is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single and multiple base point mutations.
U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as +.
SUBSTTT ULE 25 Currently available RNase mismatch cleavage assays, including those performed according to U.S. Patent No. 4,946,773, require the use of radiolabeled RNA probes. Myers and Maniatis in U.S. Patent No. 4,946,773 describe the detection of base pair mismatches using RNase A. Other investigators have described the use of E. coli enzyme, RNase I, in mismatch assays. Because it has broader cleavage specificity than RNase A, RNase I would be a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches. The use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufficiently high.
The RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution. The assay relies on being able to easily generate high specific activity radiolabeled RNA probes complementary to the mRNA of interest by in vitro transcription.
Originally, the templates for in vitro transcription were recombinant plasmids containing bacteriophage promoters. The probes are mixed with total cellular RNA samples to permit hybridization to their complementary targets, then the mixture is treated with RNase to degrade excess unhybridized probe. Also, as originally intended, the RNase used is specific for single- stranded RNA, so that hybridized double-stranded probe is protected from degradation. After inactivation and removal of the RNase, the protected probe (which is proportional in amount to the amount of target mRNA that was present) is recovered and analyzed on a polyacrylamide gel.
The RNase Protection assay was adapted for detection of single base mutations. In this type of RNase A mismatch cleavage assay, radiolabeled RNA probes transcribed in vitro from wild type sequences, are hybridized to complementary target regions derived from test samples. The test target generally comprises DNA (either genomic DNA or DNA amplified by cloning in plasmids or by PCR™), although RNA targets (endogenous mRNA) have occasionally been used. If single nucleotide (or greater) sequence differences occur between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position ("mismatch") can be recognized and cleaved in some cases by single-strand specific
SUBSTITUTE SHEET RULE 25 ribonuclease. To date, RNase A has been used almost exclusively for cleavage of single-base mismatches, although RNase I has recently been shown as useful also for mismatch cleavage. There are recent descriptions of using the MutS protein and other DNA-repair enzymes for detection of single-base mismatches.
9.0 Examples
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE I: Sequence Analysis and Characterization i uspAl
Bacterial strains and culture conditions. M. catarrhalis strains 035E, 046E, TTA24,
012E, FR2682, and B21 have been previously described (Helminen et al, 1993a; Helminen et al, 1994; Unhanand et al, 1992). M. catarrhalis strains FR3227 and FR2336 were obtained from Richard Wallace, University of Texas Health Center, Tyler, TX. M. catarrhalis strain B6 was obtained from Elliot Juni, University of Michigan, Ann Arbor, MI. M. catarrhalis strain TTA1 was obtained from Steven Berk, East Tennessee State University, Johnson City, TN.
M. catarrhalis strain 25240 was obtained from the American Type Culture Collection, Rockville, MD. M. catarrhalis strains were routinely cultured in Brain Heart Infusion (BHI) broth (Difco Laboratories, Detroit, MI) at 37°C or on BHI agar plates in an atmosphere of 95% air-5% C02. Escherichia coli strains LE392 and XLl-Blue MRF' (Stratagene, La Jolla, CA) were grown on Lubria-Bertani medium (Maniatis et al, 1982) supplemented with maltose
(0.2% w/v) and 10 mM MgSO4 at 37°C, with antimicrobial supplementation as necessary.
Monoclonal antibodies (MAbs). MAb 17C7 is a murine IgG antibody reactive with the
UspA proteinaceous material of all M. catarrhalis strains tested to date (Helminen et al, 1994).
Additional MAbs specific for UspA material (i.e., 16A7, 17B1 , and 5C12) were produced for this study by fusing spleen cells from mice immunized with outer membrane vesicles from
SUBSTITU E SHEET RULE 25 M. catarrhalis 035E with the SP2/0-Agl4 plasmacytoma cell line, as described (Helminen et al, 1993a). These MAbs were used in the form of hybridoma culture supernatant fluid in western blot and dot blot analyses.
Cloning vectors. Plasmid and bacteriophage cloning vectors utilized in this work and the recombinant derivatives of these vectors are listed in Table VI.
TABLE VI Bacteriophages And Plasmids
Bacteriophage or plasmid Description Source
Bacteriophage
LambdaGEM-11 Cloning vector Promega Corp. (Madison, WI)
MEH200 LambdaGEM- 11 containing an (Helminen et al, 1 1 kb insert of M. catarrhalis 1994) strain 035 E DNA encoding the UspA proteinaceous material
ZAP Express Cloning vector Stratagene
USP100 ZAP Express with a 2.7 kb This study fragment of DNA (containing the uspAl) amplified from the chromosome of M. catarrhalis strain 035E
Plasmids pBluescript II SK+ Cloning vector, Ampκ Stratagene (pBS) pJL501.6 pBS containing the 1.6 kb This study 5g II-EcoRJ fragment from MΕH200 pJL500.5 pBS containing the 600-bp Bglll This study fragment from MEH200
SUBSTTTUTE SHEET RULE 25) MEH200, the original recombinant bacteriophage clone that produced plaques reactive with the UspA-specific MAb 17C7, has been described previously (Helminen et al, 1994).
Genetic techniques. Standard recombinant DNA techniques including plasmid isolation, restriction enzyme digestions, DNA modifications, ligation reactions and transformation of E. coli are familiar to those of skill in the art and were performed as previously described
(Maniatis et al, 1982; Sambrook et al, 1989).
Polymerase Chain Reaction (PCR™). PCR™ was performed using the GeneAmp kit (Perkin-Elmer, Branchberg, NJ). All reaction were carried out according to the manufacturer's instructions. To amplify products from total genomic DNA, 1 μg of M. catarrhalis chromosomal DNA and 100 ng of each primer were used in each 100 μl reaction.
Nucleotide sequence analysis. Nucleotide sequence analysis of DNA fragments in recombinant plasmids, in bacteriophage, or derived by PCR™ was performed using an Applied Biosystems Model 373 A automated DNA sequencer (Applied Biosystems, Foster City, CA). DNA sequence information was analyzed using the Intelligenetics suite package and programs from the University of Wisconsin Genetics Computer Group software analysis package
(Devereux et al, 1984). Analysis of protein hydrophilicity using the method of Kyte and Doolittle (1982) and analysis of repeated amino acid sequences within the UspA protein was performed using the MacVector™ software protein matrix analysis package (Eastman Kodak Company, Rochester, NY).
Identification of recombinant bacteriophage. Lysates were generated from E. coli cells infected with recombinant bacteriophage by using the plate lysis method as described (Helminen et al, 1994). MAb-based screening of plaques formed by recombinant ZAP Express bacteriophage on E. coli XL 1 -Blue MRF' cells was performed according to the manufacturer's instructions (Stratagene, La Jolla, CA). Briefly, nitrocellulose filters soaked in 10 mM IPTG were applied to the surface of agar plates five hours after bacteriophage infection of the bacterial lawn. After overnight incubation at 37°C, the nitrocellulose pads were removed, washed with PBS containing 0.5% (v/v) Tween 20 and 5% (w/v) skim milk (PBS-T) and incubated with hybridoma culture supernatant containing the MAb for 4 hours at room
125 temperature. After four washes with PBS-T, PBS-T containing I-labeled goat anti-mouse
SUBSTTTUTE SHEET RULE 25) IgG was applied to each pad. After overnight incubation at 4°C, the pads were washed four times with PBS-T, blotted dry, and exposed to film.
Characterization of M. catarrhalis protein antigens. Outer membrane vesicles were prepared from BHI broth-grown M. catarrhalis cells by the EDTA-buffer method (Murphy and Loeb, 1989). Proteins present in these vesicles were resolved by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) using 7.5% (w/v) polyacrylamide separating gels. These SDS-PAGE-resolved proteins were electrophoretically transferred to nitrocellulose and western blot analysis was performed as described using MAb 17C7 as the primary antibody (Kimura et al, 1985). For western blot analysis of proteins encoded by DNA inserts in recombinant bacteriophage, one part of a lysate from bacteriophage-infected E. coli cells was mixed with one part SDS-digestion buffer (Kimura et al, 1985) and this mixture was incubated at 37°C for 15 minutes prior to SDS-PAGE.
Features of the uspA 1 gene and its encoded protein product. The nucleotide sequence of the M. catarrhalis 035E uspAl gene and the deduced amino acid sequence of the UspAl protein are provided in SEQ ID NO:2 and SEQ ID NOT, respectively. The open reading frame (ORF), containing 2,493 nucleotides , encoded a protein product of 831 amino acids , with a calculated molecular mass of 88,271 daltons.
The predicted protein product of the uspAl ORF had a pi or 4.7, was highly hydrophilic, and was characterized by extensively repeated motifs. The first motif consists of the consensus sequence NXAXXYSXIGGGXN (SEQ ID NO:24), which is extensively repeated between amino acid residues 80 and 170. The second region, from amino acid residues 320 to 460, contains a long sequence which is repeated three times in its entirety, but which also contains smaller units which are repeated several times themselves. This "repeat within a repeat" arrangement is also true of the third region, which extends from amino acid residues 460 to 600 . This last motif consists of many repeats of the small motif QADI (SEQ ID NO:25) and two large repeats which contain the QADI (SEQ ID NO:25) motif within themselves.
Similarity of UspAl to other proteins. A BLAST-X search (Altschul et al, 1990; Gish and States, 1993) of the available databases for proteins with significant homology to UspAl indicated that the prokaryotic proteins that were most similar to this M. catarrhalis antigen were
SUBSTITUTE SHEET (RULE 25) a putative adhesin of H. influenzae Rd (GenBank accession number U32792) (Fleischmann et al, 1995), the Ηia adhesin from nontypable H. influenzae (GenBank accession number U38617) (Barenkamp and St. Geme III, 1996), and the YadA invasin of Yersinia enter ocolitica (Skurnik and Wolf-Watz, 1989) (SwissProt:P31489). When the GAP alignment program (Devereux et al, 1984) was used to compare the UspAl sequence to that of these and closely related bacterial adhesins, UspAl proved to be 25% identical and 47% similar to the E. coli AIDA-I adhesin from enteropathogenic E. coli (Benz and Schmidt, 1989; Benz and Schmidt, 1992b), 23% identical and 46% similar to Ηia (Barenkamp and St. Geme III, 1996), and 24% identical and 43% similar to YadA (Skurnik and Wolf-Watz, 1989). Other proteins retrieved from database searches as having homology with UspAl included myosin heavy chains from a number of species.
EXAMPLE II: Two Genes Encode the Proteins UspAl and UspA2
MAb 17C7 binds to a very high molecular weight proteinaceous material of M. catarrhalis, designated UspA, that migrates with an apparent molecular weight (in SDS-PAGE) of at least 250 kDa. This same MAb also reacts with another antigen band of approximately 100 kDa, as described in U.S. Patent No. 5,552,146 and incorporated herein by reference, and it is bound by a phage lysate from E. coli infected by a recombinant bacteriophage that contained a fragment of M. catarrhalis chromosomal DNA. The M. catarrhalis proteinaceous material in the phage lysate that binds this MAb migrates at a rate similar or indistinguishable from that of the native UspA material (Helminen et al, 1994).
Analysis of uspAl. Nucleotide sequence analysis of the M. catarrhalis strain O35E gene expressed by the recombinant bacteriophage, designated uspAl, revealed the presence of an ORF encoding a predicted protein product with a molecular mass of 88,271 (SEQ ID NOT). The use of the uspAl ORF in an in vitro DNA-directed protein expression system revealed that the protein encoded by the uspAl gene migrated in SDS-PAGE with an apparent molecular weight of about
120 kDa. (Those of skill in the art will be aware that denaturing processes, such as SDS-PAGE, can alter the migration rate of proteins such that the apparent molecular weight of the denatured protein is somewhat different than the predicted molecular weight of the non-denatured protein.) In addition, when the uspAl ORF was introduced into a bacteriophage vector, the recombinant E. coli strain containing this recombinant phage expressed a protein that migrated in SDS-PAGE apparently at the same rate as the native UspA protein from M. catarrhalis.
SUBSTTTUTE SHEET (RULE 25) Southern blot analysis of chromosomal DNA from several M. catarrhalis strains, using a
0.6 kb BgHl-Pvull fragment derived from the cloned uspAl gene as the probe, revealed that, with several strains, there were two distinct restriction fragments that bound this uspA 1 -derived probe (FIG. 1 ), indicating that M. catarrhalis possessed a second gene had some similarity to the uspAl gene.
Native very high molecular weight UspA proteinaceous material from M. catarrhalis strain 035E was resolved by SDS PAGE, electroeluted, and digested with a protease. N-terminal acid sequence analysis of some of the resultant peptides revealed that the amino acid sequences of several peptides did not match that of the deduced amino acid sequence of UspAl . Other peptides obtained from this experiment were similar to those present in the deduced amino acid sequence but not identical.
Protease and cyanogen bromide (CNBr) Cleavage of High Molecular Weight UspA
Proteinaceous Material: Three tenths (0.3) mg of purified very high molecular weight UspA proteinaceous material (at the time of the purification this material was thought to be a single protein) was precipitated with 90% ethanol and the pellet was resuspended in 100 ml of 88% formic acid containing 12M urea. Following resuspension, 100 ml of 88% formic acid containing 2M CNBr was added and the mixture was incubated in the dark overnight at room temperature. One ml (2.0 mg) of purified UspA material was added directly to a vial containing 25 mg of either trypsin or chymotrypsin. The reaction mixtures were incubated for -48 hours, at 37°C. One ml (2.0 mg) of purified UspA material was added directly to a vial containing 15 mg of endoproteinase Lys-C. The reaction mixtures were incubated for about 48 hours at 37°C.
The cleavage reaction mixtures were clarified by centrifugation in an Eppendorf M centrifuge at 12,000 rpm for 5 minutes. The clarified supernatant was loaded directly onto a Vydac C4 HPLC column using a mobile phase of 0.1% (v/v) aqueous trifiuoroacetic acid (Solvent A) and acetonitrile:H20:trifluoroacetic acid, 80:20:0.1 (v/v/v) (Solvent B) at a flow rate of 1.0 ml/min. The reaction mixtures were washed onto the column with 100% Solvent A followed by elution of cleavage fragments using a 30 minutes linear gradient (0-100%) of Solvent B. Fractions were collected manually, dried overnight in a Speed- Vac and resuspended
SUBSTTTUTE SHEET (RULE 25) in House Pure Water. The resuspended HPLC-separated fractions were subjected to SDS- PAGE analysis using 10-18% gradient gels in a Tris-Tricine buffer system. The fractions which exhibited a single peptide band were submitted for direct N-terminal sequence analysis. Fractions displaying multiple peptide bands were transferred from SDS-PAGE onto a PVDF membrane and individual bands excised and submitted for N-terminal sequence analysis.
The N-terminal amino acid sequences of these fragments then were determined using an Applied Biosystems Model 477A PTH Analyzer (Applied Biosystems, Foster City, CA, U.S.A.). A summary of these sequences is given in Table VII. About half of the sequences were found to match the sequence deduced from the uspAl gene, while the other half did not.
Attempts at shifting the reading frame of the uspAl gene sequence failed to account for the non- matching peptide sequences, indicating that the high molecular weight UspA protein may comprise either a multimer of more than one distinct protein or distinct multimers of two different proteins.
SUBSTTTUTE SHEET RULE 25) TABLE VII Summary of the N-terminal Sequences of Internal Peptide Fragments
Figure imgf000072_0001
SUBSTTTUTE SHEET (RULE 25) TABLE VII (Continued)
Figure imgf000073_0001
a Certain residues of several peptides could not be verified and these ambiguities are shown by an "X" in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:49 and SEQ ID NO:50. In SEQ ID NO:29 the ambiguous residue is likely to be a serine; in SEQ ID NO:33, position 13 is likely to be aspartic acid, position 14 is likely to be glycine and position 15 is likely to be arginine; in SEQ ID NO:35 both positions 13 and 19 are likely to be serines; in SEQ ID NO:49 the ambiguous residue is likely to be an asparagine; and in SEQ ID NO:50 position 4 is likely to be serine and position 8 is likely to be threonine.
Additional attempts to resolve the very high molecular weight UspA protein band from M. catarrhalis strain O35E by SDS-PAGE, followed by electroelution and digestion with proteases or with cyanogen bromide, again yielded a number of peptides which were sequenced. Several peptides (peptides 1-6, Table VIII) were obtained. The amino acid sequence of which was identical or very similar to that deduced from the nucleotide sequence of the uspAl gene. However, several additional peptides, peptides 7-10, Table VIII, were not present in the deduced amino acid sequence. This finding substantiated the suggestion that a second protein was present in the UspA antigen preparation.
SUBSTTTUTE SHEET (RULE 25) TABLE VIII
Matching or closely matching peptides:
Peptide # Amino acid sequence
Peptide 1 KALESNVEEGLLDLSGR (SEQ ID NO:55) Peptide 2 ALESNVEEGLLELSGRTIDQR (SEQ ID NO:56) Peptide 3 NQAHIANNINXIYELAQQQDQK (SEQ ID NO:57) Peptide 4 NQADIAQNQTDIQDLAAYNELQ (SEQ ID NO:58) Peptide 5 ATHDYNERQTEA (SEQ ID NO:59) Peptideό KASSENTQNIAK (SEQ ID NO:60)
Noήmatching peptides:
Peptide # Amino acid sequence
Peptide 7 MILGDTAIVSNSQDNKTQLKFYK (SEQ ID NO:61)
Peptide 8 AGDTIIPLDDDXXP (SEQ ID NO:62)
Peptide 9 LLHEQQLXGK (SEQ ID NO:63)
Peptide 10 IFFNXG (SEQ ID NO:64)
Certain residues of several peptides could not be verified and these ambiguities are shown by an "X" in SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64.
Further evidence corroborating the assertion that the high molecular weight UspA proteinaceous material was either a multimer of more than one distinct protein or distinct multimers of two different proteins was derived from earlier electrospray mass spectroscopic analysis which predicted that a monomer of the UspA material had a molecular weight of 59,500. This approximately 60 kDa protein reacted immunogenically with the MAbs 17C7, 45- 2, 13-1, and 29-31, in contrast to the UspAl protein which only cross-reacted with MAb 17C7. The fact that MAb 17C7 reacted with both isolated proteins suggested that this Mab recognized an epitope common to both proteins.
Preparation of mutant uspAl construct. The nucleotide sequence of the cloned uspAl gene was used to construct an isogenic uspAl mutant. Oligonucleotide primers (ifømHI-ended P 1 and PI 6 in Table IX) were used to amplify a truncated version of the uspAl ORF from M. catarrhalis strain O35E chromosomal DNA; this PCR™ product was cloned into the BamUl site of the
SUBSTTTUTE SHEET RULE 25) plasmid vector pBluescript II SK+. A 0.6 kb BgHl fragment from the middle of this cloned fragment was excised and was replaced by a BamUl-endeά cassette encoding kanamycin resistance. This new plasmid was grown in E. coli DH5α, purified by column chromatography, linearized by digestion with EcoRI, precipitated, and then dissolved in water. This linear DNA molecule was used to electroporate the wild-type M. catarrhalis strain 035Ε, using a technique described previously (Helminen et al, 1993b). Approximately 5,000 kanamycin-resistant transformants were obtained; several picked at random were found to be still reactive with MAb 17C7. One of these kanamycin-resistant clones was randomly chosen for further examination and Southern blot analysis confirmed that this mutant was isogenic.
Analysis of products expressed by the uspAl mutant When whole cell lysates of both the wild-type M. catarrhalis strain and this mutant were subjected to SDS-PAGE, both the wild-type strain and the mutant strain still expressed the very high-molecular-weight band originally designated as UspA. However, a protein of approximately 120 kDa was found to be missing in the mutant strain (FIG. 2A). The fact that both this mutant and the wild-type parent strain still expressed a very high molecular weight antigen reactive with MAb 17C7 (FIG. 2B) indicated that there had to be a second gene in M. catarrhalis strain O35E that encoded a MAb 17C7-reactive antigen. Furthermore, it should be noted that EDTA-extracted outer membrane vesicles of both the wild-type strain (FIG. 2C, lanes 5 and 7) and mutant strain (FIG. 2C, lanes 6 and 8) possessed a protein of approximately 70-80 kDa that was reactive with MAb 17C7. This approximately 70- 80 kDa band likely represents one form, perhaps the monomeric form, of the product of a second gene encoding the MAb 17C7-reactive epitope.
It is important to note that, when chromosomal DNA from both the wild-type parent strain and the mutant were digested with vuII and probed in Southern blot analysis with a 0.6 kb BgHl- Pvull fragment derived from the uspAl gene, the wild-type strain exhibited a 2.6 kb band and a 2.8 kb band which bound this probe (FIG. 3). In contrast, the mutant strain had a 2.6 kb band and a 3.4 kb band that bound this probe. The presence of the 3.4 kb band was the result of the insertion of the kan cartridge into the deletion site in the uspAl gene.
SUBSTTTUTE SHEET (RULE 25) EXAMPLE III: Characterization of UspA2 and uspA2
Construction of fusion proteins. The epitope which binds MAb 17C7 was localized by using the nucleotide sequence of the uspAl gene described above to construct fusion proteins. First, fusion proteins containing five peptides spanning the UspAl protein were constructed by using the pGEX4T-2 protein fusion system (Pharmacia LKB). The oligonucleotide primers used in PCR™ to amplify the desired nucleotide sequences from M. catarrhalis strain 035E chromosomal DNA are listed in Table IX. Each of these had either a BamUl site or a Xhol site at the 5' end, thereby allowing directional in-frame cloning of the amplified product into the BamUl- and TioI-digested vector. When recombinant E. coli strains expressing each of these five fusion proteins were used in a colony blot radioimmunoassay, only fusion protein MF-4 readily bound MAb 17C7. Further analysis of the uspA 1 -derived nucleotide sequence in the MF-4 fusion construct involved the production of fusion proteins containing 79 amino acid residues (MF-4-1) and 123 amino acid residues (MF-4-2) derived from the MF-4 fusion protein (Table IX). These two fusion proteins both bound MAb 17C7 (Table IX). FIG. 4 depicts the western blot reactivity of MAb 17C7 with the MF-4-1 fusion protein. These two fusion proteins had in common only a 23-residue region NNINNIYELAQQQDQHSSDIKTL (SEQ ID NO:65), suggesting that this 23-residue region, designated as the "3Q" peptide, contains the epitope that binds MAb 17C7.
SUBSTITUTE SHEET RULE 25) TABLE IX
PCR™ primers used for the production of usp A 1 gene fragments for use in the construction of fusion proteins and mutagenesis and the reactivity of the resulting fusion protein with MAb 17C7
Fragment Generated: Primer Pair3 Reactivity with MAb 17C7
MF-3 P5-P8
MF-4 P6-P13 +
MF-4.1 P7-P12 +
MF-4.2 P11-P13 + a primer sequences are as follows:
P5 GGTGCAGGTCAGATCAGTGAC SEQ ID NO:66
P6 GCCACCAACCAAGCTGAC SEQ ID NO:67
P7 AGCGGTCGCCTGCTTGATCAG SEQ ID NO:68
P8 CTGATCAAGCAGGCGACCGCT SEQ ID NO:69 Pll CAAGATCTGGCCGCTTACAA SEQIDNO:70
P12 TTGTAAGCGGCCAGATCTTG SEQIDNO:71
P13 TGCATGAGCCGCAAACCC SEQIDNO:72
Elucidation of the MAb 17C7 Epitope. It is important to note that the nucleotide sequence encoding this 23-residue polypeptide (i.e., the 3Q peptide) was present in the 0.6 kb Bglll-Pvull fragment used in the Southern blot analysis described in Example II. This finding suggested that the epitope that bound MAb 17C7 might be encoded by DNA present in both the 2.6 and 2.8 kb Pvull fragments, with the 2.8 kb Pvull fragment being derived from the cloned uspAl gene and the 2.6 kb Pvull fragment representing all or part of another gene encoding this same epitope.
A ligation-basedPCR™ system was used to verify this finding. Chromosomal DNA from the mutant strain was digested to completion with Pvull and was resolved by agarose gel electrophoresis. Fragments ranging in size from 2-3 kb were excised from the agarose, blunt- ended, and ligated into the Ec RV site in pBluescript II SK+ This ligation reaction mixture was precipitated and used in a PCR™ amplification reaction. Each PCR™ reaction contained either the T3 or T7 primer derived from the DNA encoding the 3Q peptide. This approach yielded a 1.7
SUBSTIT T EET RULE 25 kb product with the T3 and P10 primers and a 0.9 kb product from the T7 and P9 primers (FIG. 5). The sum of these two bands is the same as the 2.6 kb size of the desired DNA fragment.
Nucleotide sequence analysis of these two PCR™ products revealed two incomplete ORFs which, when joined at the region encoding the 3Q peptide, formed a 1,728-bp ORF encoding a protein with a calculated molecular weight of 62,483 daltons (SEQ ID NO:3). The amino acid sequence of this protein had 43% identity with that of UspAl . Closer examination revealed that a region extending from amino acids 278-41 1 in this second protein, designated UspA2, was nearly identical to the region in UspAl between amino acids 505-638 (SEQ ID NOT). Furthermore, these two regions both contain the 23-mer (the 3Q peptide) that likely contains the epitope that binds MAb 17C7. It should also be noted that the four peptides from Table IX (Peptides 7-10) that were not found in UspA 1 were found to be identical or very similar to peptides in the deduced amino acid sequence of UspA2. In addition, the first six peptides listed in Table IX, which matched or were very similar to peptides in the deduced amino acid sequence of UspAl , also matched peptides found in the deduced amino acid sequence of UspA2.
Oligonucleotide primers PI and P2 (Table IX) were used to amplify a 2.5-2.6 kb fragment from M. catarrhalis strain O35E chromosomal DNA. Nucleotide sequence analysis of this PCR™ product was used to confirm the nucleotide sequence of the uspA2 ORF determined from the ligation-based PCR™ study. These results proved that M. catarrhalis strain 035E contains two different ORFs (i.e., uspAl and uspAT) which encode the same peptide (i.e., the 3Q peptide) which likely binds MAb 17C7. This 3Q peptide appears twice in UspAl and once in UspA2
(SEQ ID NO: 1 and SEQ ID NO:3).
The nucleotide sequences of the two DNA segments encoding these 3Q peptides in uspAl are nearly identical, with three nucleotides being different. These nucleotide differences did not cause a change in the amino acid sequence. The nucleotide sequence of the DNA segment encoding the 3Q peptide in uspA2 is identical to the DNA encoding the first 3Q peptide in UspAl .
As seen in FIG. 2C, lane 7, the three dominant MAb 17C7-reactive bands present in M. catarrhalis strain O35E outer membrane vesicles have apparent molecular weights of greater than
200 kDa, approximately 120 kDa, and approximately 70-80 kDa. It should be noted that the existence of several MAb 17C7-reactive bands, with apparent molecular weights of greater than 200 kDa, approximately 120 kDa, and approximately 70-80 kDa was also apparent in U.S. Patent
SUBSTTTUTE HEET RULE 26 5,552,146 (FIG. 1, lane H). Therefore, the existence of at least more than one M. catarrhalis antigens reactive with MAb 17C7 was apparent as early as 1991. It is now apparent that the approximately 120 kDa band likely represents the monomeric form of the UspAl antigen and the approximately 70-80 kDa band likely represents the monomeric form of the UspA2 antigen from M. catarrhalis strain 035E. One or more than one of these species may aggregate to form the very high molecular weight proteinaceous material (i.e. greater than 200 kDa) of the UspA antigen.
A new M. catarrhalis strain 035E genomic library was constructed in the bacteriophage vector ZAP Express (Stratagene, La Jolla, CA). Chromosomal DNA from this strain was partially digested with Sau2>A\ and 4-9 kb DNA fragments were ligated into the vector arms according to the instructions obtained from the manufacturer. This library was amplified in E. coli MRF'. An aliquot of this library was diluted and plated and the resultant plaques were screened for reactivity with MAb 17C7. Approximately 24 plaques which bound this MAb were detected; the responsible recombinant bacteriophage were purified by the single plaque isolation method, and the DNA insert from one of these bacteriophage was subjected to nucleotide sequence analysis.
Nucleotide sequence of the 2.6 kb DNA fragment present in this recombinant bacteriophage revealed that, on one end, it contained an incomplete ORF that encoded the 3Q peptide. Until its truncation by the vector cloning site, the sequence of this incomplete ORF was identical or nearly identical to that of the uspA2 ORF derived from the ligation-based PCR™ study described immediately above, providing further evidence that two genes which share a common epitope encode the UspA antigen.
EXAMPLE IV: Purification of and Immunological Properties of the Proteins UspAl and UspA2
Materials and Methods Bacteria. TTA24 and O35E isolates were as previously described in Example I.
Additional isolates were obtained from the University of Rochester and the American Type Culture Collection (ATCC). The bacteria were routinely passaged on Mueller-Hinton agar (Difco, Detroit, Ml) incubated at 35°C with 5% carbon dioxide. The bacteria used for the purification of the protein were grown in sterile broth containing 10 g casamino acids (Difco, Detroit, Ml) and 15 g yeast extract (BBL, Cockeysville, MD) per liter. The isolates were stored at -70°C in Mueller-Hinton broth containing 40% glycerol.
SUBSTTTU EET RULE 25 Purification of UspA2. Bacterial cells (-400 g wet wt. of M. catarrhalis 035E) were washed twice with 2 liters of pH 6.0, 0.03 M sodium phosphate (NaPO4) containing 1.0% Triton® X-100 (TX-100) (J.T. Baker Inc., Philipsburg, NJ) (pH 6.0) by stirring at room temperature for 60 min. Cells containing the UspA2 protein were pelleted by centrifugation at 13,700 x g for 30 min at 4°C. Following centrifugation, the pellet was resuspended in 2 liters of pH 8.0, 0.03 M Tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) containing 1.0% TX-100 and stirred overnight at 4°C to extract the UspA2 protein. Cells were pelleted by centrifugation at 13,700 x g for 30 min at 4°C. The supernatant, containing the UspA2 protein, was collected and further clarified by sequential microfiltration through a 0.8 μm membrane (CN.8, Nalge, Rochester, NY) then a 0.45 μm membrane (cellulose acetate, low protein binding, Corning,
Corning, NY).
The entire filtered crude extract preparation was loaded onto a 50 x 217 mm (-200 ml) TMAE column [650(S), 0.025-0.4 mm, EM Separations, Gibbstown, NJ] equilibrated with pH 8.0, 0.03 M Tris-HCl buffer containing 0.1% TX-100 (THT). The column was washed with 400 ml of equilibration buffer followed by 600 ml of 0.25 M NaCl in 0.03 M THT. UspA2 was subsequently eluted with 800 ml of 1.0 M NaCl in 0.03 M THT. Fractions were screened for UspA2 by SDS-PAGE and pooled. Pooled fractions (-750 ml), containing UspA2, were concentrated approximately two-fold by ultrafiltration using an Amicon stirred cell (Amicon Corp., Beverly, MA) with a YM-100 membrane under nitrogen pressure. The TMAE concentrate was split into two 175 ml aliquots and each aliquot buffer exchanged by passage over a 50 x 280 mm (-550 ml) Sephadex G-25 (Coarse) column (Pharmacia Biotech, Piscataway, NJ) equilibrated with pH 7.0, 10 mM NaP04 containing 0.1% TX-100 (10 mM PT). The buffer exchanged material was subsequently loaded onto a 50 x 217 mm (-425 ml) ceramic hydroxyapatite column (Type I, 40 μm, Bio-Rad) equilibrated with 10 mM PT. The column was washed with 450 ml of the equilibration buffer followed by 900 ml of pH 7.0,
0.1M NaP04 containing 0.1% TX-100. UspA2 was then eluted with a linear pH 7.0 NaPO4 concentration gradient between 0.1 and 0.2 M NaP04 containing 0.1% TX-100. An additional volume of pH 7.0, 0.2 M NaPO4 containing 0.1% TX-100 was applied to the column and collected to maximize the recovery of UspA2. Fractions were screened for UspA2 by SDS- PAGE and pooled. The column was then washed with 900 ml of pH 7.0, 0.5 M NaPO4
SUBSTTTUTE SHEET RULE 26) containing 0.1% TX-100. The fractions from this wash were screened for UspAl by SDS- PAGE, pooled, and stored at 4°C. This pool was used for the purification of UspAl .
Purification of UspAl. The UspAl enriched fractions collected during four separate purifications of UspA2 were pooled. The combined UspAl pools were concentrated approximately threefold by ultrafiltration using an Amicon stirred cell with a YM-100 membrane under nitrogen pressure. The UspAl concentrate was split into two 175 ml aliquots and the buffer exchanged by passage over a 50 x 280 mm (-550 ml) Sephadex G-25 column equilibrated with 10 mM PT. The buffer exchanged material was subsequently loaded onto a 50 x 217 mm (-425 ml) ceramic hydroxyapatite column (Bio-Rad) equilibrated with 10 mM PT. The column was washed with 450 ml of the equilibration buffer followed by 900 ml of pH
7.0, 0.25 M NaPO4 containing 0.1% TX-100. UspAl was subsequently eluted with a linear NaPO4 gradient of pH 7.0, 0.25-0.5 M NaPO4 containing 0.1% TX-100. The fractions containing UspAl were identified by SDS-PAGE and pooled.
SDS-PAGE and Western blot Analysis. SDS-PAGE was carried out as described by Laemmli (1970) using 4 to 20% (w/v) gradient acrylamide gels (Integrated Separation Systems
(ISS), Natick, MA). Proteins were visualized by staining the gels with Coomassie Brilliant
Blue R250. Gels were scanned using a Personal Densitometer SI (Molecular Dynamics Inc.,
Sunnyvale, CA) and molecular weights were estimated with the Fragment Analysis software
(version 1.1) using the prestained molecular weight markers from ISS as standards. Transfer of proteins to polyvinylidene difluoride (PVDF) membranes was accomplished with a semi-dry electroblotter and electroblot buffers (ISS). The membranes were probed with protein specific antisera or MAb's followed by goat anti-mouse alkaline phosphatase conjugate as the secondary antibody (BioSource International, Camarillo, CA). Western blots were developed with the
BCIP/NBT Phosphatase Substrate System (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Protein Estimation. Protein concentrations were estimated by the BCA assay (Pierce, Rockford, IL), using bovine serum albumin as the standard.
SUBSTTTUT ET RULE 26 Enzymatic and Chemical Cleavages of UspA2 and UspAl .
(i) CNBr Cleavage. Approximately 0.3 mg of the purified protein was precipitated with
90% (v/v) ethanol and the pellet resuspended in 100 μl of 88% (v/v) formic acid containing 12
M urea. Following resuspension, 100 μl of 88% (v/v) formic acid containing 2 M CNBr (Sigma, St. Louis, MO) was added and the mixture incubated overnight at room temperature in the dark.
(ii) Trypsin and Chymotrvpsin Cleavage. Approximately 2 mg of the purified protein was precipitated with 90% (v/v) ethanol and the pellet resuspended in a total volume of 1 ml of phosphate-buffered saline (PBS) containing 0.1%) TX-100. This preparation was added directly to a vial containing 25 μg of either trypsin or chymotrypsin (Boehringer Mannheim,
Indianapolis, IN). The reaction mixture was incubated for 48 h at 37°C.
(iii) Endoproteinase Lys-C Cleavage. Approximately 2 mg of the purified protein was precipitated with 90% (v/v) ethanol and the pellet resuspended in a total volume of 1.0 ml of
PBS containing 0.1% TX-100. This preparation was added directly to a vial containing 15 μg of endoproteinase Lys-C (Boehringer Mannheim). The reaction mixture was incubated for 48 h at 37°C.
(iv) Separation of Peptides. The above cleavage reaction mixtures were centrifuged in an Eppendorf centrifuge at 12,000 rpm for 5 min and the supernatant loaded directly onto a Vydac Protein C4 HPLC column (The Separations Group, Hesperia, CA). The solvents used were 0.1% (v/v) aqueous trifluoroacetic acid (TFA) [Solvent A] and acetonitrile:H20:TFA,
80:20:0.1 (v/v/v) [Solvent B] at a flow rate of 1.0 ml/min. Following the initial wash with Solvent A, the peptides were eluted with a linear gradient between 0 and 100% of Solvent B and detected by absorbance at 220 nm. Suitable fractions were collected, dried in a Speed- Vac concentrator (Jouan Inc., Winchester, VA) and resuspended in distilled water. The fractions were separated by SDS-PAGE in 10 to 18% (w/v, acrylamide) gradient gels (ISS) in a Tris-
Tricine buffer system (Schagger and von Jagow, 1987). The fractions containing a single peptide band were submitted directly for N-terminal sequence analysis. Fractions displaying multiple peptide bands in SDS-PAGE were electrophoretically transferred onto a PVDF membrane as described above. The membrane was stained with Coomassie Brilliant Blue R-
SUBSTTTU EET RULE 26 250 and the individual bands excised before submitting them for N-terminal sequence analysis (Matsudaira, 1987).
Determination of subunit size. Determination of molecular weight by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry (Hillenkamp and Karas, 1990) was done on a Lasermat 2000 Mass Analyzer (Finnigan Mat, Hemel Hempstead,
UK) with 3,5-dimethoxy-4-hydroxy-cinnamic acid as the matrix. Cold ethanol precipitation was done on samples containing >0.1% (v/v) TX-100 to remove the detergent. The final ethanol concentration was 90% (v/v). The precipitated protein was resuspended in water.
Determination of aggregate sizes by gel filtration chromatography. Approximately 1 mg of the purified protein was precipitated with 90% (v/v) ethanol and the pellet resuspended in a total volume of 1.0 ml of PBS containing 0.1% TX-100. Two hundred microliters of the preparation were applied to a Superose-6 HR 10/30 gel filtration column (10 x 30 mm, Pharmacia) equilibrated in PBS /0.1%> TX-100 at a flow rate of 0.5 ml/min. The column was calibrated using the HMW Calibration Kit (Pharmacia) which contains aldolase with a size of 158,000, catalase with a size of 232,000; ferritin with a size of 440,000; thyroglobulin with a size of 669,000; and blue dextran with sizes between 2000 and 2,000,000.
Amino Acid Sequence Analysis. N-terminal sequence analysis was carried out using an
Applied Biosystems Model 477A Protein/Peptide Sequencer equipped with an on-line Model
120A PTH Analyzer (Applied Biosystems, Foster City, CA). The phenylthiohydantoin (PTH) derivatives were identified by reversed-phase HPLC using a Brownlee PTH C-18 column
(particle size 5 μm, 2.1 mm i.d. x 22 cm 1.; Applied Biosystems).
Immunizations. Female BALB/c mice (Taconic Farms, Germantown, NY), age 6-8 weeks, were immunized subcutaneously with two doses of UspAl or UspA2 four weeks apart. To prepare the vaccine, purified UspAl or UspA2 was added to aluminum phosphate, and the mixture rotated overnight at 4°C. 3-O-deacylated monophosphoryl lipid A (MPL) (Ribi
ImmunoChem Research, Inc.) was added just prior to administration. Each dose of vaccine contained 5 μg of purified protein, 100 μg of aluminum phosphate and 50 μg of MPL resuspended in a 200 μl volume. Control mice were injected with 5 μg of CRM197 with the same adjuvants. Serum samples were collected before the first vaccination and two weeks after
SUBSTTTUTE SHEET RULE 26 the second immunization. Mice were housed in a specific-pathogen free facility and provided water and food ad libitum.
Monoclonal antibodies. The 17C7 MAb was secreted by a hybridoma (ATCC HB 11093). MAbs 13-1, 29-31, 45-2, and 6-3 were prepared as previously described (Chen et al, 1995).
Murine model of M. catarrhalis pulmonary clearance. This model was performed as described previously (Chen et al, 1995).
Enzyme linked immunosorbent assay (ELISA) procedures. Two different ELISA procedures were used. One was used to examine the reactivity of sera to whole bacterial cells and the other the reactivity to the purified proteins.
For the whole cell ELISA, the bacteria were grown overnight on Mueller-Hinton agar and swabbed off the plate into PBS. The turbidity of the cells was adjusted to 0.10 at 600 nm and 100 μl added to the wells of a 96 well Nunc F Immunoplate (Nunc, Roskilde, Denmark). The cells were dried overnight at 37°C, sealed with a mylar plate sealer and stored at 4°C until needed. On the day of the assay, the residual protein binding sites were blocked by adding 5% non-fat dry milk in PBS with 0.1% Tween 20 (Bovine Lacto Transfer Technique Optimizer [BLOTTO]) and incubating 37°C for one hour. The blocking solution was then removed and 100 μl of sera serially diluted in the wells with blotto. The sera were allowed to incubate for 1 h at 37°C. The plate wells were soaked with 300 ml PBS containing 0.1% Tween 20 for 30 seconds and washed 3 times for 5 seconds with a Skatron plate washer and then incubated 1 hr at 37°C with goat anti-mouse IgG conjugated to alkaline phosphatase (BioSource) diluted 1 : 1000 in blotto. After washing, the plates were developed at room temperature with 100 μl per well of 1 mg/ml p-nitrophenyl phosphate dissolved in diethanolamine buffer. Development was stopped by adding 50 μl of 3N NaOH to each well. The absorbance of each well was read at 405 nm and titers calculated by linear regression. The titer was reported as the inverse of the dilution extrapolated to an absorption value of 0.10 units.
For the ELISA against the purified proteins, the proteins were diluted to a concentration of 5 μg/ml in a 50 mM sodium carbonate buffer (pH 9.8) containing 0.02% sodium azide (Sigma Chemical Co.). One hundred microliters were added to each well of a 96 well
SUBSTTTUTE SHEET RULE 26) E.I.A./R.I.A medium binding ELISA plate (Costar Corp., Cambridge, MA) and incubated for 16 hours at 4°C. The plates were washed and subsequently treated the same as described for whole cell ELISA procedure.
Complement-dependent bactericidal assay. For this assay, 20 μl of the bacterial suspension containing approximately 1200 cfu bacteria in PBS supplemented with 0.1 mM
CaCl2:, MgCl2 and 0.1% gelatin (PCMG) were mixed with 20 μl of serum diluted in PCMG and incubated for 30 min at 4°C. Complement, prepared as previously described (Chen et al, 1996), was added to a concentration of 20%, mixed, and incubated 30 min at 35°C. The assay was stopped by diluting with 200 μl of cold, 4°C, PCMG. 50 μl of this suspension was spread onto Mueller-Hinton plates. Relative killing was calculated as the percent reduction in cfu in the sample relative to that in a sample in which heat inactivated complement replaced active complement.
Inhibition of bacterial adherence to HEp-2 cells. The effect of specific antibodies on bacterial adherence to HEp-2 cells was examined. A total of 5 x 10 HEp-2 cells in 300 μl of RPMI-10 were added to a sterile 8-well Lab-Tek chamber slide (Nunc, Inc., Naperville, 111) and incubated overnight in a 5% CO2 incubator to obtain a monolayer of cells on the slide. The slide was washed with PBS and incubated with 300 μl of bacterial suspension (A550=0.5) or with a bacterial suspension that had been incubated with antisera (1 :100) at 37°C for 1 h. The slides were then washed with PBS and stained with the Difco quick stain following the manufacturer's instructions. The slide was viewed and photographed using a light microscope equipped with a camera (Nikon Microphot-SA, Nikon, Tokyo, Japan).
Protein interaction with fibronectin and vitronectin. The interactions of purified UspAl and UspA2 with fibronectin were examined by dot blot. Human plasma fibronectin (Sigma Chemical Co., St. Louis, MO) was applied to a nitrocellulose membrane, and the membrane blocked with blotto for 1 h at room temperature. The blot was then washed with PBS and incubated with purified UspAl or UspA2 (2 μg/ml in blotto) overnight at 4°C. After three washes with PBS, the membrane was incubated with the MAb 17C7 diluted in blotto for 2 h at room temperature and then with goat anti-mouse immunoglobulin conjugated to alkaline phosphatase (BIO-RAD Lab. Hercules, Calif.) (1 :2,000 in PBS with 5% dry milk, 2 h, room temperature). The membrane was finally developed with a substrate solution containing
SUBSTTTUTE SHEET RULE 25) nitroblue tetrazolium and 5-bromo-chloro-3-indolyl phosphate in 0.1 M tris-HCl buffer (pH 9.8).
Interaction with vitronectin was examined by a similar procedure. The purified UspAl and UspA2 were spotted onto the nitrocellulose membrane and the membrane blocked with blotto. The membrane was then incubated sequentially with human plasma vitronectin (GIBCO
BRL, Grand Island, N.Y., 1 μg/ml in blotto), rabbit anti-human vitronectin serum (GIBCO
BRL), goat anti-rabbit IgG-alkaline phosphatase conjugate and substrate.
Interaction with HEp-2 cells by the purified protein. Each well of a 96 well cell culture plate (Costar Corp., Cambridge, Mass.) was seeded with 5 x 10 HEp-2 cells in 0.2 ml RPMI containing 10% fetal calf serum and the plate incubated overnight in a 37°C incubator containing 5% C02. Purified UspAl or UspA2 (1 to 1,000 ng) in blotto was added and incubated at 37°C for 2 h. The plate was washed with PBS, and incubated with the 1 : 1 mixed mouse antisera to either UspAl or UspA2 (1 : 1000 dilution in PBS containing 5% dry milk), the plate was washed and incubated with rabbit anti-mouse IgG conjugated to horseradish peroxidase (1 :5,000 in PBS containing 5% dry milk) (Brookwood Biomedical, Birmingham,
AL) at room temperature for 1 h. Finally, the plate was washed and developed with a substrate solution containing 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) at 0.3 mg/ml in pH 4.0 citrate buffer containing 0.03% hydrogen peroxide (KPL, Gaithersburg, MD). Whole bacteria of strain 035E were included as a positive control. The highest concentration of the bacteria tested had an optical density of A550=1.0. The abscissa for the bacterial data shown in
FIG. 7 plots the values for three fold dilutions of the bacterial suspension.
Results
Purification of UspAl and UspA2. The inventors developed a large-scale, high yield process for extracting and purifying UspA2 from a pellet of M. catarrhalis cells. The method consisted of three critical steps. First the UspA2 protein was extracted from the bacteria with pH 8.0, 0.03 M THT. Second, the cell extract was applied to a TMAE column and the UspA2 protein eluted with NaCl. Finally, the enriched fractions from the TMAE chromatography were applied to a ceramic hydroxyapatite column and the UspA2 eluted with a linear NaPO4 gradient. A yield of 250 mg of purified UspA2 was typically obtained from -400 g wet weight of M. catarrhalis O35E strain cells. A single band was seen for the UspA2 in SDS-PAGE gels
SUBSTTTUTE SHEET RULE 26) by Coomassie blue staining. It corresponded to a molecular size of -240,000 and contained greater than 95% of the protein based on scanning densitometry (FIG. 6A). A second band reacting with the 17C7 MAb at approximately 125,000 could be detected in the UspA2 preparation by western but not by Coomassie blue staining (FIG. 6C). The cells need not be lysed to achieve this high yield, which suggested this protein is present in large amounts on the surface of the bacterium.
A method for the purification of the UspAl protein was also developed. This protein co-purified with UspA2 through the initial extraction and TMAE chromatography steps. Following hydroxyapatite chromatography, however, UspAl remained bound to the column and had to be eluted at the higher salt concentration of 500 mM NaPO4. The crude UspAl preparation obtained in this step was reapplied and eluted from the hydroxyapatite column using a linear sodium phosphate gradient. A total of 80 mg of purified UspAl was isolated from -1.6 kg wet wt. of M. catarrhalis O35E strain cells. UspAl purified using this method migrated at three different apparent sizes on SDS-PAGE depending on the method of sample preparation. Unheated samples exhibited a single band at -280,000, whereas samples heated at
100°C for 3 min resulted in an apparent molecular weight shift to -350,000. Prolonged heating at 100°C resulted in a shift of the 350,000 band to one at 100,000 (FIG. 6B). Following heating of the sample for 7 min at 100°C, the band at 100,000 contained greater than 95% of the protein based on scanning densitometry of the Coomassie stained gel. In contrast, UspA2 migrated at 240,000 regardless of the duration of the heating when examined by SDS-PAGE. The different migration behaviors indicated the preparations contained two distinctly different proteins
Molecular Weight Determinations. MALDI-TOF mass spectrometric analysis for determination of molecular weight of UspA2 using 3,5-dimethoxy-4-hydroxy-cinnamic acid matrix in presence of 70% (v/v) aqueous acetonitrile and 0.1% TFA resulted in the identification of a predominant species with average molecular mass of 59,518 Da. In addition to the expected [M+H]+ and [M+2H]2+ molecular ions, the [2M+H] + and [3M+H] + ions were also observed. The latter two ions were consistent with the dimer and the trimer species. Using similar conditions, the inventors were unable to determine the mass of UspAl.
To determine the molecular sizes of the purified proteins in solution, UspAl and UspA2 were independently run on a Superose-6 HR 10/30 gel filtration column (optimal separation
SUBSTTTUTE SHEET RULE 26 range: 5,000-5,000,000) calibrated with molecular weight standards. Purified UspAl exhibited a native molecular size of 1,150,000 and UspA2 a molecular size of 830,000. These sizes, however, may be affected by the presence of TX-100.
N-terminal Sequence Analysis of Internal UspAl and UspA2 Peptides. All attempts to determine the N-terminal sequences of both UspA and UspAl proved unsuccessful. No sequence could be determined. This suggested two things. First, the N-terminus of both proteins were blocked, and, second, neither protein preparation contained contaminating proteins that were not N-terminally blocked.
Thus, to confirm that the primary sequence of purified UspAl and UspA2 corresponded to that deduced from their respective gene sequences, internal peptide fragments were generated and subjected to N-terminal sequence analysis. Tables X and XI show the N-terminal sequences obtained for fragments generated from the digestion of the UspA2 and UspAl proteins, respectively. The sequences matching the primary amino acid sequence deduced from the respective gene sequences are indicated for each fragment. The UspA2 fragments #3 and #4 exhibited sequence similarity with residues 505-515 and 605-614 respectively of the amino acid sequence deduced from the UspAl gene. In Table XII, UspAl fragment #3 exhibited sequence similarity with residues #278-294 of the UspA2 primary sequence. These sequences corresponded with the domains within UspAl and UspA2 that share 93% sequence identity.
The remainder of the sequences, however, were unique to the respective proteins.
TABLE X
N-terminal sequences of internal UspA2 peptide cleavage fragments
UspA2 Fragment Sequence3 Match Cleavage
1 ) LLΔEQQLNG SEQ ID NO:73 92-100 Trypsin 2) ALESNVEEGL SEQ ID NO:74 216-225 Lys-C
245-254
274-283
3) ALESNVEEGLLDLS SEQ ID NO:75 274-288 Trypsin
*505-515
SUBSTTTUTE SHEET RULE 26) TABLE X cont'd
UspA2 Fragment Sequence Match Cleavage
4) AKASAANTDR SEQ ID NO:76 378-387 Chymotrypsin * 605-614
5) AATAADAITKNGN SEQ ID NO:77 439-450 Chymotrypsin
6) SITDLGTKVDGFDGR SEQ ID NO:78 458-472 Lys-C
7) VDALXTKVNALDXKVN SEQ ID NO:79 473-488 Trypsin
8) AAQAALSGLFVPYSVGKFNATAALGGYGSK 506-535 CNBr SEQ ID NO:80 aUnderlined residues denote mismatch with the nucleotide derived amino acid sequence.
Ambiguous residues whose identity could not be verified are denoted by the letter X.
Asterisk (*) indicates match with UspAl . Without asterisk indicates matches with nucleotide derived amino acid sequence of UspA2.
TABLE XI N-terminal sequences of internal UspAl peptide cleavage fragments
UspAl Fragment Sequence Match' Cleavage
1) LENNVEE£XLNLS 456-468 Lys-C
2) DQKADI 473-478 Trypsin
3) NNVEEGLLDLSGRLIDQK 504-521 Lys-C
* 278-294
4) VAEGFEIF 690-697 Trypsin
5) AGIATNKQELILQNDRLNRI 701-720 Lys-C
aAs per Table X. X denotes an unidentified amino acid residue,
As stteerriisskk ((**)) iinnddiiccaatteess mmaattcchh wwiitthh UUssppAA22.. Without asterisk indicates matches with nucleotide derived amino acid sequence of UspAl .
Reactivity of MAbs with UspAl and UspA2. The western blot analysis of purified UspAl and UspA2 revealed that both proteins reacted strongly with the MAb 17C7 described by Helminen et al. (1994) (FIG. 7). The reactivity of the proteins with other MAbs was also investigated. The data in Table XII show that, whether assayed by ELISA or western, the
SUBSTTT T RULE 25 MAbs 13-1, 29-31 and 45-2 only reacted with UspA2, the MAbs 7D7, 29C6, 11A6 and 12D5 only reacted with UspAl , while 17C7 and 6-3 reacted with both UspAl and UspA2. All the MAbs shown in Table XIII bind to whole bacteria when examined by ELISA. These results indicated that UspA2 was exposed on the surface of the bacterium.
TABLE XII
Summary of reactivity of monoclonal antibodies with purified UspAl,
UspA2 and whole bacteria of strain O35E
Reactivity mAb Isotype Whole Purified Purified UspA2" bacterium" UspAlb
13-1 IgGlκ + - +
29-31 IgGlλ + - +
45-2 IgG2a + - +
17C7 IgG2a + + +
6-3 IgM + + +
7D7 IgG2b + + -
29C6 IgGl + + -
1 1A6 IgA + + -
12D5 IgGl + + -
aDetermined by whole cell ELISA. Determined by ELISA and western blot.
SUBSTTTUTE SHEET RULE 25) TABLE XIII
Cross-reactivity of antibodies to UspAl and UspA2 proteins
Antiserum to Geometric mean ELISA titer" to
UspAl UspA2
UspAla 740,642c 10,748c UspA2a 19,120d 37,615d
aThe preparation of the sera are described in the text.
ELISA titers are for total IgG and IgM antibodies for sera pooled from ten mice. cThe difference in titer of the anti-UspA 1 with the two purified proteins was statistically different by the Wilcoxon signed rank test (/?=0.0002). The difference in titer of the anti-UspA2 with the two purified proteins was statistically different by the Wilcoxon signed rank test (p=0.0\ ).
Immunogenicity and antibody cross-reactivity. Antisera to the purified UspAl and
UspA2 proteins were generated in mice. The titers of antigen specific antibodies (IgG and IgM) as well as the cross-reactive antibodies in these sera were determined by an ELISA assay using each of the purified proteins (Table XIII). Both proteins elicited antibody titers that were greater against themselves than against the heterologous protein. Thus, the reactivities of both the MAbs (Table XII) as well as the polyclonal antibodies indicate that the proteins possessed both shared and non-shared B-cell epitopes.
Antibody reactivity to whole bacterial cells and bactericidal activity. Antisera to the
UspAl and UspA2 were assayed by whole cell ELISA against the homologous 035E strain and several heterologous isolates (Table XIV). The antibodies to UspAl and to UspA2 reacted strongest with the 035E strain. The reactivity of the sera toward the heterologous isolates indicated they bound antibodies elicited by both UspAl and UspA2. TABLE XIV
ELISA and complement mediated bactericidal titers toward whole bacterial cells of multiple isolates of M. catarrhalis elicited by purified UspAl and purified UspA2
Whole cell ELISA- Bactericidal titer12
Isolate anti-UspAla anti-UspA2a anti-UspAl anti -UspA2
035E 195,261 133,492 400 800
430-345 12,693 18,217 400 400
1230-359 7,873 13,772 400 400
TTA24 14,341 7,770 800 800 aTiter determined for pool of sera from ten mice. The titer of the sera drawn before the first immunization was less than 50 for all isolates. bBactericidal titers were determined as the inverse of the highest serum dilution killing greater than 50% of the bacteria. The titers for the sera from mice immunized contemporaneously with CRMI97 were less than 100.
The bactericidal activities of the antisera to UspAl and UspA2 were determined against
O35E and other isolates as well (Table XIV). Both sera had bactericidal titers ranging from 400-800 against 035E and the disease isolates. Anti-CRM197 serum, the negative control, as well as sera drawn before immunization, had a titers of < 100 against all the strains. These results were consistent with the previous observation that the epitopes shared by the two proteins are highly conserved among isolates and the antibodies toward those isolates are bactericidal.
Pulmonary challenge. Immunized mice were given a pulmonary challenge with the homologous O35E strain or the heterologous TTA24 strain. Relative to the control mice immunized with CRM197, enhanced clearance of both strains was observed regardless of whether the mice were immunized with UspAl or UspA2 (Table XV). No statistical difference
(p> 0.05) was seen between the groups of mice immunized with UspAl and with UspA2.
E 25 TABLE XV
Pulmonary clearance of M. catarrhalis by mice immunized with purified UspAl and UspA2
Study Immunogen Challenge strain % clearance8 a
P
1 UspAl 035E 49.0 0.013
UspA2 31.8 0.05
CRM197 0 -
2 UspAl TTA24 54.6 0.02
UspA2 66.6 0.0003
CRM197 0 - aChallenge method described in text. Numbers are the percentage of bacteria cleared from the immunized mice compared to control mice which were immunized with CRM,97.
Interaction of purified proteins with HEp-2 cells. The purified UspA 1 and UspA2 were tested for their ability to interact with HEp-2 cell monolayer in a 96-well plate using an ELISA. Protein binding to the HEp-2 cells was detected with a 1 :1 mix of the mouse antisera to UspAl and UspA2. Purified UspAl bound to HEp-2 cells at concentrations above 10 ng. A weak binding by the UspA2 was detected at concentrations above 100 ng (FIG. 7). The attachment of O35E bacteria to HEp-2 cells was used as a positive control. This result, plus the data showing that the anti-UspAl antibodies inhibited attachment of the bacteria to HEp2 cells, suggests UspAl plays an important role in bacterial attachment which also suggested that UspAl was exposed on the bacterial surface.
Interaction of purified proteins with fibronectin and vitronectin. The purified proteins were assayed for their ability to interact with fibronectin and vitronectin by dot blot assays. Human plasma fibronectin immobilized on a nitrocellulose membrane bound purified UspAl but not UspA2 (FIG. 8), while UspA2 immobilized on the nitrocellulose membrane was capable of binding vitronectin (FIG. 8). Vitronectin binding by the UspAl was also detected, but the reactivity was weaker. Collagen (type IV), porcine mucin (type III), fetuin and heparin were also tested for interaction with purified UspAl and purified UspA2, but these did not exhibit detectable binding.
SUBSTTTUTE HEET RULE 26 Discussion
Previous UspA purification attempts yielded preparations containing multiple high molecular weight protein bands by SDS-PAGE and western blot. Because each of the bands reacted with the "UspA specific" MAb 17C7, it was thought they represented multiple forms of the UspA protein (Chen et al, 1996). However, the inventors have discovered that there are two distinct proteins, UspAl and UspA2, that share an epitope recognized by the 17C7 MAb. These two proteins are encoded by different genes. This study shows that UspAl and UspA2 can be separated from one another. The isolated proteins had different SDS-PAGE mobility characteristics, different reactivity with a set of monoclonal antibodies, and different internal peptide sequences. The results, however, were consistent with the proteins sharing a portion of their peptide sequences, including the MAb 17C7 epitope. The separation of the proteins from one another has allowed the inventors to further demonstrate how the proteins were different as well as examine their biochemical, functional, and immunological characteristics.
In solution, the purified proteins appear to be homopolymers of their respective subunits held together by strong non-covalent forces. This is indicated by the fact that UspA2 lacks any cysteines and treatment of both proteins with reducing agents did not alter their mobilities in SDS-PAGE. Both gene sequences possess leucine zipper motifs that might mediate coil-coil interactions (O'Shea et al, 1991). Even so, it was surprising that the non-covalent bonds of both proteins were not only strong enough to resist dissociation by the conditions normally used to prepare samples for SDS-PAGE, but also high concentrations of chaotropic agents such as urea (Klingman and Murphy, 1994) and guanidine HC1. Of the two proteins, UspA2 appeared to be less tightly aggregated, this was indicated by the fact that its subunit size of 59,500 Da could be determined by mass spectrometry. UspAl, however, was recalcitrant to dissociation by all the methods tried, and this may be the reason its size could not be determined by mass spectrometry. In SDS-PAGE, the dominant UspA2 migrated with an apparent size of 240,000 while a far smaller portion migrated at about 125,000 and could only be detected by western analysis. The mobility of UspAl, however, varied depending on how long the sample was heated. The smallest form was about 100,000. This was consistent with the size of the gene product missing from the uspAl mutant but not with the size predicted from the gene sequence of 88,000 Da. In solution, both proteins formed larger aggregates than those seen by SDS-
PAGE. Their sizes, as measured by gel filtration chromatography, were 1,150,000 and 830,000
SUBSTTT T EET RULE 25 for UspAl and UspA2 respectively. If the proteins behave this way in vivo, UspAl and UspA2 likely occur as large molecular complexes on the bacterial surface of the bacterium.
The results of the N-terminal amino acid sequence analyses of the UspA2 and UspAl derived peptides (Tables X and XI) were in agreement with the protein sequences derived from the respective gene sequences. This confirmed that the purified UspAl and UspA2 proteins were the products of the respective uspAl and uspA2 genes. Further, the experimental and theoretical amino acid compositions of UspAl and UspA2 were consistent, given the size of the proteins and the accuracy of the amino acid determination. There was, however, a discrepancy between the size determined by mass spectrometry of 59,518 and the size indicated from the gene sequence for UspA2 of 62,483. This discrepancy suggested that this protein either undergoes post-translational processing or proteolytic degradation.
The data also suggest that both proteins are exposed on the bacterial surface. That at least one of the proteins is exposed is evident from the finding that the MAb 17C7 and polyclonal sera react with whole cells. The reactivities of the UspA2 specific monoclonal antibodies 13-1, 29-31 and 45-2 with the bacterial cells in the whole cell ELISA provided evidence that the UspA2 is a surface protein (Table XII). The reactivities of the UspAl specific MAbs 7D7, 29C6, 11A6 and 12D5 with the bacterial cells in the whole cell ELISA provided evidence that the UspAl is a surface protein (Table XII). Further evidence for surface exposure of UspAl was indicated by the inhibitory effect of the antiserum on bacterial attachment to HEp-2 cells. The sera to the UspA2 lacked this activity. Thus, both UspAl and UspA2 appeared to be surface exposed on the bacterium.
Surface exposure of the proteins is probably important for the two proteins' functions. One function for UspAl appears to be meditation of adherence to host tissues. The evidence for this was that UspAl antibodies inhibited bacterial binding to HEp-2 cells and the purified protein itself bound to the cells. The relevance of binding to HEp-2 cells is that they are epithelial cells derived from the larynx, a common site of M. catarrhalis colonization (Schalen et al, 1992). This confirms the inventors findings that mutants that do not express UspAl fail to bind epithelial cells. The inventors' also showed that UspAl binds fibronectin. Fibronectin has been reported to be a host receptor for other pathogens (Ljungh and Wadstrόm, 1995; Westerlund and Korhonen, 1993). Examination of the gene sequence, however, failed to reveal
SUBSTTTUTE SHEET RULE 26) any similarity with the fibronectin binding motifs reported for Gram positive organisms (Westerlund and Korhonen, 1993). Thus, it is fairly clear that UspAl plays a role in host adherence, possibly via cell associated fibronectin.
The function of UspA2 is less certain. Antibodies toward it did not block adherence to the HEp-2 or Chang cell lines, nor did the purified protein bind to those cells. Yet, UspA2 bound vitronectin strongly. Pathogen binding of vitronectin has been linked to host cell adherence (Gomez-Duarte et al, 1997; Limper et al, 1993); however, van Dijk and his co- workers have reported that vitronectin binding by M. catarrhalis may be used by the bacteria to subvert host defenses (Verdiun et al, 1994). The soluble form of vitronectin, known as complement factor S, regulates formation of the membrane attack complex (Su, 1996). They suggest that the binding of vitronectin to the M. catarrhalis surface inhibits the formation of the membrane attack complex, rendering the bacteria resistant to the complement dependent killing activity of the sera. They have also described two types of human isolates: one that binds vitronectin and is resistant to the lytic activity of the serum and the other that does not bind vitronectin and is serum sensitive (Hoi et al, 1993). It must be noted, however, that vitronectin, like all the extracellular matrix proteins, has many forms and serves multiple functions in the host (Preissner, 1991 ; Seiffert, 1997). Thus, the interaction of both UspAl and UspA2 with the extracellular matrix proteins fibronectin and vitronectin may serve the bacterium in ways beyond subverting host defenses or as receptors for bacterial adhesion.
Even though the two proteins share epitopes and sequences, they have different biochemical activities and likely serve different biological functions. If an immune response to the respective protein interferes with its function, it ought to be considered as a vaccine candidate. The results of the immunological studies in mice indicated that both proteins would be good vaccine candidates. Mice immunized with either UspAl or UspA2 developed high antibody titers toward the homologous and heterologous bacterial isolates. Further, the sera from these mice had complement dependent bactericidal activity toward all the isolates tested. In addition, immunized mice exhibited enhanced pulmonary clearance of the homologous isolate and heterologous isolates. It is important to note that antibodies elicited by the proteins were partially cross-reactive. This was expected since both react with the 17C7 MAb and share amino acid sequence.
SUBSTTTUTE HEET RULE 26 EXAMPLE V: The Level and Bactericidal Capacity of Child and Adult Human Antibodies Directed against the Proteins UspAl and UspA2
To determine if humans have naturally acquired antibodies to the UspAl and UspA2 of the M. catarrhalis and the biological activity of these antibodies if present, sera from healthy humans of various ages was examined using both ELISA and a bactericidal assay. It was found that healthy people have naturally acquired antibodies to both UspAl and UspA2 in their sera, and the level of these antibodies and their bactericidal capacity were age-dependent. These results also indicate that naturally acquired antibodies to UspAl and UspA2 are biologically functional, and thus support their use as vaccine candidates to prevent M. catarrhalis disease.
Material and methods
Bacteria. The M. catarrhalis strains 035E and TTA24 were as described in Example I. An ATCC strain (ATCC 25238) and three other clinical isolates from the inventors' collection were also used.
Human sera. Fifty-eight serum samples were collected from a group of ten children at 2, 4, 6, 7, 15 and 18 months of age who had received routine childhood immunizations. Individual sera from twenty-six adults and fifteen additional children 18-36 months of age were also assayed. All sera were obtained from clinically healthy individuals. Information on M. catarrhalis colonization and infection of these subjects was not collected. The sera were stored at -70°C.
Purification of UspAl and UspA2. Purified UspAl and UspA2 were made from the
035E strain of M. catarrhalis as described in Example IV herein. Each protein preparation contained greater than 95% of the specific protein based on densitometric scanning of Coomassie brilliant blue stained SDS-PAGE. Based on western blot analysis using monoclonal antibodies, each purified protein contained no detectable contamination of the other.
Purification of UspAl and UspA2 specific antibodies from human plasma. Human plasmas from two healthy adults were obtained from the American Red Cross (Rochester, N.Y.) and pooled. The antibodies were precipitated by adding ammonium sulfate to 50% saturation. The precipitate was collected by centrifugation and dialyzed against PBS. A nitrocellulose
SUBSTTTUTE SHEET RULE 26) membrane (2 x 3 inches) was incubated with UspAl or UspA2 at 0.5 mg/ml in PBS containing 0.1% (vol/vol) Triton X-100 for 1 h at room temperature, washed twice with PBS and residual binding sites on the membrane blocked with 5% (wt/vol) dry milk in PBS for 2 h at room temperature. The membrane was then sequentially washed twice with PBS, 100 mM glycine (pH 2.5) and finally with PBS before incubation with the dialyzed antibody preparation. After incubating for 4 h at 4°C, the membrane was washed again with PBS, and then 10 mM Tris buffer (pH 8.0) containing 1 M sodium chloride to remove non-specific proteins. The bound antibodies were eluted by incubation in 5 ml of 100 mM glycine (pH 2.5) for 2 min with shaking. One ml of Tris-HCl (1M, pH 8.0) was immediately added to the eluate to neutralize the pH. The eluted antibodies were dialyzed against PBS and stored at -20°C.
Enzyme-linked immunosorbent assay (ELISA). Antibody titers to the 035E and other M. catarrhalis strains were determined by a whole-cell ELISA as previously described using biotin-labeled rabbit anti-human IgG or IgA antibodies (Brookwood Biomedical. Birmingham. Alabama) (Chen et al, 1996). Antibody titers to UspAl and UspA2 were determined by a similar method except that the plates were coated with 0.1 μg of purified protein in 100 μg of
PBS per well overnight at room temperature. The IgG subclass antibodies to UspAl or UspA2 were determined using sheep anti-human IgG subclass antibodies conjugated to alkaline phosphatase (The Binding Site Ltd., San Diego, Calif). The antibody end point titer was defined as the highest serum dilution giving an A415 greater than three times that of the control. The control wells received all treatments except human sera and usually had absorbance values ranging from 0.03 to 0.06.
The specificity of biotin-labeled rabbit anti-human IgG and IgA antibodies was determined against purified human IgG, IgM and IgA (Pierce, Rockford, IL) by ELISA. No cross-reactivity was found. The assay sensitivity determined by testing against purified human antibodies of appropriate isotype in an ELISA was 15 and 60 ng/ml in the IgG and IgA assays, respectively. Likewise, the specificity of the human IgG subclass antibody assays was confirmed in ELISA against purified human myeloma IgG subclass proteins (ICN Biomedicals, Inc., Irvine, CA), and the assay sensitivity was 15 ng/ml in the IgGl , IgG3 and IgG4 assays, and 120 ng/ml in the IgG2 assay. Two control sera were included to control for assay to assay variation.
SUBSTTTUTE SHEET RULE 25 Complement dependent bactericidal assay. The bactericidal activity of the human sera was determined as described previously (Chen et al, 1996). In some studies, the sera were absorbed with purified UspAl or UspA2 prior to the assay. The absorption of specific antibodies from these sera was accomplished by adding the purified proteins to 20 or 50 μg/ml final concentration. The final serum dilution was 1 : 10. The mixtures were incubated for 2 h at
4°C and the precipitate removed by micro-centrifugation. The purified human antibodies specific for UspAl and UspA2 were assayed against five M. catarrhalis strains in a similar manner.
Statistics. Statistical analysis was performed on logarithmic transformed titers using JMP software (SAS institute, Cary, N.C.). To allow transformation, a value of one half the lowest serum dilution was assigned to sera which contained no detectable titers. Comparison of IgG levels among the age groups was done by analysis of variance, and the relationship of antibody titer and the bactericidal titer was determined by logistic regression. A p value less than 0.05 was considered significant.
Results
Comparison of serum IgG and IgA titers to UspAl and UspA2 in children and adults. The IgG and IgA antibody titers in the sera from ten children collected longitudinally between 2-18 months of age, as well as the random samples from fifteen 18-36 month old children and twenty-six adults were determined against the whole bacterial cells of the 035E strain, the purified UspAl and the purified UspA2 by ELISA. IgG titers to all three antigens were detected in almost all the sera (FIG. 9). The IgG titers to UspAl and UspA2 exhibited strong age-dependent variation when compared to IgG titers to the 035E bacterium (FIG. 9). The adult sera had significantly higher IgG titers to the purified proteins than sera from children of various age groups(/?<0.01). Sera from children at 6-7 months of age had the lowest IgG titers to UspA proteins and the mean titer at this age was significantly lower than that at 2 months .of age (p<0.05).
The level of IgA antibodies to UspAl , UspA2 and 035E bacterial cells were age dependent (FIG. 9). A serum IgA titer against the UspAl and UspA2 was detected in all twenty-six adults and children of 18-36 months of age. For children less than 18 months of age, the proportion exhibiting antigen specific IgA titers increased with age. The mean IgA titers to
SUBSTTTUTE SHEET RULE 25) UspAl , UspA2 or 035E bacterium in these sera were low for the first 7 months of age but gradually increased thereafter (FIG. 9).
Age-dependent subclass distribution of IgG antibodies to UspAl and UspA2. The IgG subclass titers to the UspAl and UspA2 antigens were determined on sera from ten adult sera and thirty-five children's sera. The subclass distribution was found to be age-dependent. The most prominent antibodies to the UspAl and UspA2 antigens were of the IgGl and IgG3 subclasses, which were detected in almost all sera. The IgG2 and IgG4 titers were either undetectable or extremely low. Therefore, only data on IgGl and IgG3 subclasses are reported (FIG. 10). The IgG3 titers against UspAl or UspA2 in the adult sera were significantly higher than the IgGl titers (p<0.05). The same subclass profile was seen in the sera from the 2 month old children, although the difference between IgGl and IgG3 titers did not reach statistical significance, probably because of the smaller sample size. Sera from children between 4 and 36 months of age all had a similar subclass profile which was different from that of the adults and 2 month old children. The IgGl titers in children's sera were either higher than or equivalent to the IgG3 titers. The mean IgGl titer to either UspAl or UspA2 was significantly higher than
IgG3 titer to the same antigens in these children's sera (p<0.05).
Bactericidal activity. The bactericidal titers of seventeen sera representing different age groups were determined (Table XVI). All the adult sera and three out of five sera from the two month old children which had high IgG titers to the UspA proteins had strong bactericidal activity. Sera from 6 month old children had the least bactericidal activity. All five sera from this age group had a marginal bactericidal titer of 50, the lowest dilution assayed. The bactericidal activity of the sera from 18 to 36 month old children was highly variable with titers ranging from less than 50 to 500. There was a significant linear relationship between the bactericidal titers and the IgG antibody titers against both UspAl and UspA2 by logistic regression analysis (p<0.0\) (FIG. 11).
SUBSTTTUTE SHEET RULE 26) TABLE XVI
The level of IgG antibodies to UspAl and UspA2 from normal human serum and the serum bactericidal activity
Subject' Age ELISA IgG titer BC titer UspAl UspA2
1 2 month 17,127 6,268 500
6 month 4,273 1,363 50
15 month 798 250 <50
2 2 month 12,078 12,244 500
6 month 1,357 878 50
18 month 14,041 14,488 200
3 2 month 30,283 20,362 500
6 month 1 ,077 1,947 50
18 month 2,478 1,475 <50
4 2 month 2,086 869 <50
6 month 530 802 50
18 month 9,767 8,591 200
5 2 month 3,233 2,655 <50
6 month 2,246 360 50
18 month 26,693 43,703 500
6 1.5-3 year 4,036 2,686 50
7 1.5-3 year 2,037 1,251 50
8 1.5-3 year 341 251 <50
9 1.5-3 year 2,538 1,200 500
10 1.5-3 year 1078 1,370 500
1 1 1.5-3 year 1,265 953 50
SUBSTTTUT SHEET RULE 26 TABLE XVI (Continued)
Subject'1 Age ELISA Ij gG titer" BC titerc UspAl UspA2
12 adult 161,750 87,180 450
13 adult 873,680 248,290 >1350
14 adult 154,650 146,900 450
15 adult 10,330 7,860 50
16 adult 35,780 31,230 150
17 adult 19,130 132,200 450 aThree consecutive samples from subjects 1 through 5 were collected at the stated ages. ELISA end point titers to purified UspAl or UspA2 from the 035E strain were determined as the highest serum dilution giving an A415 greater than three times the background. CBC titers: bactericidal titer assayed against the 035E strain. Sera were assayed at 1 :50,
100, 200, and 500. Bactericidal titer was determined as the highest serum dilution resulting in killing of 50% or more of the bacteria relative to the control. Control bacteria were incubated with test serum and heat inactivated complement serum.
Bactericidal activity of sera absorbed with purified UspAl or UspA2. Because normal human sera contain antibodies to numerous antigens of M. catarrhalis as indicated by western blot, an absorption method was used to determine the contribution of UspAl and UspA2 specific antibodies towards the bactericidal activity. Six adult sera were absorbed with purified UspAl or UspA2, and the change in ELISA reactivity to UspA proteins determined. A reduction in ELISA reactivity was seen for all the sera after absorption (Table XVII). Further, absorption with one protein resulted in a reduction of IgG titers to the other protein. Reduction of UspA2 reactivity was of the same degree regardless of whether the absorbent was UspAl or UspA2. In contrast, there was less reduction in UspAl reactivity after absorption with UspA2 than with UspAl (Table XVII). This indicated that antibodies to UspAl and UspA2 were partially cross-reactive.
SUBSTTTUTE SHEET RULE 25) TABLE XVII ELISA titer of adult sera before and after absorption"
Absorbent I? *G titers to UspAl in sampli e b
#1 #2 #3 #4 #5 #6 saline 161,750 873,680 154,650 10,330 35,780 19,130
UspAl 2,450 2,210 3,160 1 ,650 <500 3,010
UspA2 42,620 90,150 33,570 6,420 3,490 4, 130 IgG titers to UspA2b saline 87,180 248,290 146,900 7,860 31,230 13,200
UspAl 2,800 2,120 2,700 2,220 <500 <500
UspA2 <500 1 ,820 3,010 2,960 <500 <500 aAbsorption: An aliquot of adult serum was diluted and added with purified UspAl or UspA2 from 035E strain to a final 50 μg/ml protein concentration and final 1 : 10 serum dilution. The mixtures were incubated at 4°C for 2 h, and precipitates removed by microcentrifugation.
IgG titers against the UspAl and UspA2 proteins were end point titers determined with a starting serum dilution of 1 :500.
The bactericidal titers of the absorbed sera were determined and compared with those seen before absorption (Table XVIII). Absorption with either UspAl or UspA2 resulted in complete loss of bactericidal activity (<50) for all six sera when assayed against the 035E strain, the strain from which the purified proteins were made (Table XVIII). The bactericidal activity of the absorbed sera was also reduced by at least three fold when assayed against the a heterologous strain 1230-359. Absorption using UspAl resulted in greater reduction of the bactericidal titer against the heterologous strain in 3 out of 6 samples compared to absoφtions using UspA2 (Table XVIII). This result was consistent with the difference in the reductions of
ELISA titers to the UspAl after absorption with the two proteins. Absoφtion using the combined proteins UspAl and UspA2 did not result in further reduction of the bactericidal activity compared to UspAl alone. All six human sera contained antibodies to a 74 kDa OMP from M. catarrhalis as determined by western blot analysis, and absorption using the purified
74 kDa protein did not affect the bactericidal activity of either the 035E strain or the 1230-357
S E 25 strain. This indicated that antibodies to the UspA proteins were the major source of the bactericidal activity against M. catarrhalis in adult sera.
TABLE XVIII Bactericidal titer of the adult human sera before and after absorption"
Adsorbent Bactericidal titer to O35E strain in sample"
#1 #2 #3 #4 #5 #6 saline 450 >1350 450 50 150 450
UspAl <50 <50 <50 <50 <50 <50
UspA2 <50 150 <50 <50 <50 <50
Bactericidal titer to 1230-359 strain saline 450 4050 >1350 150 150 450
UspAl 50 150 <50 <50 50 150
UspA2 150 1350 450 <50 50 50
aSera were the same as those described in Table XVII. bBactericidal titer: The bactericidal activity was measured against the 035E or 1230-359 strains with 3-fold diluted sera starting at 1:50. The highest serum dilution resulting in 50% or greater killing was determined as the bactericidal titer. The purified UspAl and UspA2 proteins used for absoφtion were made from the 035 E strain.
Because only small volumes of the children sera were available, absoφtion of these sera was done using a mixture of UspAl and UspA2 proteins. Absoφtion resulted in the complete loss or a significant reduction of bactericidal activity in four out of seven sera (Table XIX). The four sera including three from two month old children all had an initial bactericidal titer of 200 or greater prior to absoφtion. The other three sera, which did not show a change in bactericidal titer upon absoφtion, all had a marginal titer of 50 before absoφtion. The reduction in ELISA reactivity to the UspA proteins after absoφtion confirmed that the antibody concentration had been reduced. This suggested that antibodies specific for the UspAl and UspA2 proteins in children's sera were also a major source of the bactericidal activity towards M. catarrhalis.
SUBSTTT ULE 25 TABLE XIX
Bactericidal activity of children's sera before and after absorption with pooled purified UspAl and UspA2a
Sample Age Unabsorbed serum Absorbed serum (months) A415" BC titerc Λ A415 b BC titerc
1 2 0.84 200 0.29 <50
2 2 0.93 200 0.19 <50
3 2 0.98 500 0.38 50
4 18 0.88 200 0.43 50
5 15 0.66 50 0.25 50
6 18 0.62 50 0.32 50
7 15 0.68 50 0.35 50 aAbsoφtion: Each serum was absorbed with a mixture of UspAl and UspA2 proteins from 035E strain at final protein concentrations of 200, 50 or 20 μg/ml. The same result was seen for all three absorptions of each sample. Only the data from the assay using 20 μg/ml of protein are shown. bA415: The absorbance at 415 nm in ELISA using the mixture of UspAl and UspA2 as detection antigen. Sera were tested at a 1 :300 dilution. CBC titer: Highest serum dilution resulting in 50% or greater killing of the 035E strain in the assay. Sera were assayed at dilutions 1 :50, 200, and 500.
Affinity purified antibodies to UspAl and UspA2: To confirm their cross-reactivity and bactericidal activity, antibodies to UspAl or UspA2 from adult plasma were isolated by an affinity purification procedure. The purified antibodies reacted specifically with the UspAl and the UspA2 proteins but not with non-UspA proteins in the 035E lysates in a western blot assay. The purified antibodies to one protein also reacted to the other with almost equivalent titer in ELISA (Table XX). Both antibody preparations exhibited reactivity with five M. catarrhalis strains in the whole-cell ELISA and bactericidal assay (Table XXI). The bactericidal titers against all five M. catarrhalis strains ranged between 400 and 800, which was equivalent to
0.25-0.50 μg/ml of the protein in the purified antibody preparations (Table XXI).
SUBSTTTUTE SHEET RULE 25) TABLE XX Cross-reactivity of affinity purified human antibodies to UspAl and UspA2 in
ELISA
Antibodies purified to" IgG titers against"
UspAl UspA2
UspAl 50,468 20,088 UspA2 53,106 52,834
"The antibodies were purified from plasma pooled from two healthy adults by immune elution using purified UspAl or UspA2 from the 035E strain immobilized on nitrocellulose membrane. bELISA end point titers are the highest antibody dilutions giving an A4] 5 greater than three times the background.
TABLE XXI Whole cell ELISA titer and bactericidal titer of affinity purified human antibodies to UspAl and UspA2"
Assay Whole cell ELISA titer" BC titerc strain Ab to UspAl Ab to UspA2 Ab to UspAl Ab to UspA2
035E 12,553 9,939 400 800
ATCC25238 30,843 29,512 400 400
TTA24 51,51 1 57,045 800 800
216:96 31,140 23,109 400 400
1230-359 8,495 16,458 800 800 a T. he purified antibody preparations were the same as described in Table XX. The specific reactivities of the purified antibodies to UspA proteins, but not other outer membrane proteins, were confirmed by western blots. ELISA end point titers are the highest antibody dilutions giving an A415 greater than three times the background when assayed against whole bacterial cells. °BC titer: Highest antibody dilution resulting in 50% or greater killing of the bacterial inoculum in the assay. Antibodies (120 μg/ml) were assayed at dilutions 1 : 100, 200, 400, and 800.
SUBSTTT ET RULE 25 Discussion
Previous studies examining human antibodies to M. catarrhalis whole cells or outer membrane proteins usually focused on a single age group. Further, the biological function of the antibodies was left largely undetermined (Chapman et al, 1985), and the antigens eliciting the functional antibodies were not identified. Thus, these previous studies did not provide information as to the role of naturally acquired antibodies in protection against M. catarrhalis diseases, nor did they provide clear information as to what antigens are suitable for vaccine development. The data from this study indicate that the IgG antibodies to UspAl and UspA2 are present in normal human sera and their levels are age-dependent. These antibodies are an important source of serum bactericidal activity in both children and adults.
These data indicated that most children had serum IgG antibodies to both UspAl and UspA2 at two months of age although the level varied from individual to individual, and the IgG subclass profile in these infant sera was similar to that in adult sera. The infant sera had bactericidal activity. The absoφtion studies suggested that the bulk of the bactericidal antibodies in these sera were directed against the UspAl and the UspA2 proteins. These results suggest that the IgG antibodies detected in the two month old children are of maternal origin. This is consistent with the report that umbilical cord serum contains high titers of antibodies to an extract of M. catarrhalis whole cells (Ejlertsen et al, 1994b).
Due to the lack of clinical information on the study subjects and small number of subjects examined in this study, it could not be determined whether maternal antibodies against UspA, although bactericidal in vitro, were protective in young children. However, at two months of age the children had significantly higher serum IgG titers against the UspA proteins and only a few of these children had a low level of IgA antibodies to M. catarrhalis as compared to children at 15-18 months of age. If serum IgA reflects prior mucosal exposure to the bacterium, then most of the children are not infected by M. catarrhalis in the first few months of age. One of the reasons may be that the maternal antibodies present in the young children protect them from infection at this age. This is consistent with the finding that young children seldom carry this bacterium and do not develop M. catarrhalis disease during the first months of life (Ejlertsen et al, 1994a).
SUBSTTTUTE SHEET RULE 25 Children may become susceptible to M. catarrhalis infection as maternal antibodies wane. In this study, the sera from 6 to 7 month old children had the lowest level of IgG antibodies to the UspA proteins and barely detectable bactericidal titers against whole cells of M. catarrhalis. By 15 months of age, nearly all children had serum IgA antibodies to the UspA proteins, and the level of IgA antibodies had significantly increased along with the level of IgG antibodies and bactericidal activity when compared with children of 6 to 7 months of age. This suggested that these children had been exposed to the bacterium and mounted an antibody response. The fifteen sera from the group of 18-36 month old children all had IgG and IgA titers to the UspA proteins and the bactericidal titers varied greatly. The UspA specific IgG antibodies in the older children's sera had different characteristics than the antibodies from the two month old children. First, the IgGl antibody titer was significantly higher than the IgG3 titer in children's sera, while the opposite was true for the 2 month old children (FIG. 10). Second, most sera from 2 month old children had bactericidal activity, while bactericidal activity was barely detectable in the sera from children of 6 months or older. The low antibody level and the low serum bactericidal activity seen in children between 6-36 months of age is consistent with the epidemiological findings that children of this age group have the highest colonization rate and highest incidence of M. catarrhalis disease (Bluestone, 1986; Ejlertsen et al, 1994b; Leinonen et al , 1981 ; Roitt et al, 1985; Ruuskanen and Heikkinen, 1994; Sethi et al, 1995; Teele et al, 1989).
Adults, a population usually resistant to M. catarrhalis infections (Catlin, 1990;
Ejlertsen et al, 1994a), were found to have consistently higher levels of IgG antibodies to the UspA proteins as well as higher serum bactericidal activity than children. The bactericidal activity of the adult sera was clearly antibody-mediated since immunoglobulin depleted sera had no activity (Chen et al, 1996), and the antibodies purified from adult plasma exhibited complement dependent bactericidal activity. The antibodies purified from human sera using
UspAl or UspA2 from a single isolate exhibited killing against multiple strains. This result indicates that humans developed bactericidal antibodies toward the conserved epitopes of UspA proteins in response to natural infections.
In all adult samples, the IgG antibodies were primarily of the IgGl and IgG3 subclasses with IgG3 being higher. This is consistent with previous reports that the IgG3 subclass is a major constituent of the immune response to M. catarrhalis in adults and children greater than 4
SUBSTTTUTE EET RULE 25 years of age, but not in younger children (Carson et al, 1994; Goldblatt et al, 1990). Of the four IgG subclasses in humans, IgG3 constitutes only a minor component of the total immunoglobulin in serum. However, IgG3 antibody has the highest affinity to interact with Clq, the initial step in the classic complement pathway leading to elimination of the bacterium by both complement-dependent killing and opsono-phagocytosis (Roitt et al, 1985). Since
IgG3 antibody is efficiently transferred across the placenta, it may also confer protective immunity to infants. The data from this study indicate that IgG3 antibody to the UspA proteins is an important component of the immune response to natural infection and has in vitro biological activity.
As clinical information related to M. catarrhalis infection was not collected for the study subjects, it is unknown how the antibodies to UspAl or UspA2 were induced. When antibodies made against the UspA proteins in guinea pigs were tested for reactivity with other bacterial species, including Pseudomonas aeruginosa, Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Escherichia coli, and nontypable Haemophilus influenzae by western blot, no reactivity was detected. This suggests that the antibodies were elicited as a specific response to the UspA antigens of M. catarrhalis. This is consistent with the high colonization rate and the endemic nature of this organism in human populations. Since the affinity purified antibodies to the two UspA proteins were cross-reactive, it could not be determined whether the human antibodies were elicited by one or both proteins. It seemed clear that the shared sequence between these two proteins was the main target of the bactericidal antibodies.
In summary, this study demonstrated that antibodies to the two UspA proteins are present in nearly all humans regardless of age. The overall level and subclass distribution of these antibodies, however, were age-dependent. IgG antibodies against UspAl and UspA2 were cross-reactive, and are a major source of serum bactericidal activity in adults. The level of these antibodies and serum bactericidal activity appears to correlate with age-dependent resistance to M. catarrhalis infection. Since humans make an antibody response to many other M. catarrhalis antigens in addition to UspAl and UspA2 after natural infection, it remains to be determined if immunization with one or both UspA proteins will confer adequate protection in susceptible populations.
SUBSTTTUTE SHEET RULE 26) EXAMPLE VI: UspA2 as a Carrier for Oligosaccharides
UspA2 as a pneumococcal saccharide carrier.
This study demonstrates that UspA2 can serve as a carrier for a pneumococcal saccharide. A seven valent pneumococcal polysaccharide was conjugated to UspA2 by reductive amination. Swiss Webster mice were immunized on wk 0 and wk 4 and a final bleed taken on wk 6. Each mouse was immunized subcutaneously (s.c.) in the abdomen with 1 μg carbohydrate per dose with aluminum phosphate as the adjuvant. A group of mice was immunized with the PP7F- CRM conjugate as a control. The data for the sera from the 6 wk bleed are shown in Table XXII, Table XXIII, and Table XXIV. The conjugate elicited antibodies against both the polysaccharide as well as bactericidal antibodies to M. catarrhalis. These results demonstrate that UspA2 can serve a carrier for eliciting antibodies to this pneumococcal saccharide and retain its immunogenicity to UspA2.
TABLE XXII Titers elicited by 7F conjugates to the pneumococcal polysaccharide 7F
Antigen IgG ELISA titer to Pn Ps 7F*
PP7F-UspA2 mix <100 PP7F-UspA2 conjugate 9,514 PP7F-CRM conjugate 61 ,333
*Pool of sera from five mice.
TABLE XXIII ELISA titers of sera against whole cells of three M. catarrhalis isolates
Immunogen Strain Tested
Group 035E 430-345 1230-359
PP7F-UspA2z mix 51 ,409 4,407 9,124
PP7F CRM conjugate 56 49 47
PP7F UspA2 conjugate 31,1 1 1 3,529 8,310
Vaccine group consists of 5 Swiss- Webster mice. Each group immunized at wk 0 and wk 3 and serum collected at wk 6.
SUBSTTTUTE SHEET (RULE 25) Vaccine composed of 1 μg Pneumo Type 7F and 1 μg UspA2 adjuvanted with aluminum phosphate.
TABLE XXIV
Complement dependent bactericidal antibodies against three M. catarrhalis isolates
Immunogen Strain Tested
Group 035E 430- 345 1230- 359
PP7F- UspA2 mix 400 400 400
PP7F CRM conjugate <100 <100 <100
PP7F UspA2 conjugate 400 400 200
BC50 titer is highest serum dilution at which >50% of bacteria were killed as compared to serum from wk 0 mice. The most concentrated serum tested was a 1 TOO dilution.
UspA2 as an Haemophilus b Oligosaccharide Carrier. This study demonstrates that UspA2 can serve as a carrier for an Haemophilus influenzae type b oligosaccharide (HbO). An HbO sample (average DP=24) was conjugated to UspA2 by aqueous reductive amination in the presence of 0.1% Triton X-100. The ratio of the HbO to UspA2 was 2: 1 by weight. Conjugation was allowed to proceed for 3 days at 35°C and the conjugate diafiltered using an Amicon 100K cutoff membrane. The conjugate ratio (mg carbohydrate/mg UspA2) was 0.43: 1. The carbohydrate was determined by orcinal assay and the protein by Lowry. The number of hydroxy-ethyl lysines was determined by amino acid analysis and found to be 12.6.
The immunogenicity of the conjugate was examined by immunizing Swiss- Webster mice. The mice were immunized twice on wk 0 and wk 4 with 1 μg of carbohydrate. No adjuvant was used with the conjugate, but was used with UspA2. The sera were pooled and titered. The reactivity toward HbPS by the radioantigen binding assay (RABA) was similar to that seen when HbO is conjugated to CRM197 (Table XXV). The whole cell titer toward the homologous M. catarrhalis isolate (035E) was similar to that seen for non-conjugated USpA2 (Table XXVI), as were the bactericidal titers (Table XXVII). Thus, when a carbohydrate
SUBSTTTUTE SHEET RULE 25) antigen that typically elicits a RABA titer less than 0.10 is conjugated to UspA2, it becomes immunogenic.
TABLE XXV Comparison of immunogenicity of HbO conjugated to UspA2 to HbO conjugated to
CRM, 7 to Haemophilus b polysaccharide by Radioantigen Binding Assay (RABA)
Week HbO-CRM197 Hbo-UspA2 0 <0.10 <0.10
3 2.51 2.87
4 4.46 3.56 6 58.66 18.92
TABLE XXVI
Comparison of immunogenicity of HbO-UspA2 conjugate with non-conjugated UspA2 by ELISA against whole cell of the O35E isolate to M. catarrhalis
Week UspA2a Hbo-UspA2
0 <50 <50
4 54,284 17,424
6 345,057 561,513
a5 μg UspA2 adjuvanted with 500 μg aluminum phosphate.
TABLE XXVII Bactericidal of sera toward two M. catarrhalis isolates.
Isolate UspA2a Hbo-UspA2
035E 4,500 >4,500
345 n.d. 450
a5 μg UspA2 adjuvanted with 500 μg aluminum phosphate. n.d. = not determined
SUBSTTTUTE EET RULE 25 EXAMPLE VII: Association of mouse serum sensitivity with expression of mutant forms of UspA2
When bacteria are killed in the presence of serum that lack specific antibodies toward them, it is called "serum sensitivity." In the case of M. catarrhalis, the mutants lacking an intact UspA2 protein have been found to be serum sensitive. These mutants were constructed so that one (035E.1 ; refer to Example IX for a description of isolates 035E.1 , 035E.2 and 035E.12) did not express UspAl , one (035E.2) did not express UspA2, and one (035E.12) did not express either protein based on a lack of reactivity with the 17C7 monoclonal antibody. The 035E.2 and 035E.12, however, expressed a smaller truncated form UspA2 (tUspA2) that reacts with antibodies prepared by immunizing mice with purified UspA2. The tUspA2 could be detected in a western blot of bacterial lysates using either polyclonal anti-UspA2 sera or the MAb 13-1. The size of the smaller form was consistent with the gene truncation used for the construction of the two mutants.
This bactericidal capacity was tested by mixing the non-immune mouse sera, a 1 :5 dilution of human complement and a suspension of bacteria (Approx. 1000 cfu) in the wells of a microtiter plate. The mouse sera were tested at both a 1 :50 and 1 TOO dilution. The number of surviving bacteria was then determined by spreading a dilution of this bacterial suspension on agar growth medium. The killing was considered significant when fewer than 50% viable bacteria as cfu's were recovered relative to the samples without mouse sera. Killing by the non-immune sera was seen only for the mutants lacking a "complete" UspA2 (Table XXVIII).
TABLE XXVIII Bactericidal activity of the pre- immune sera from Balb/c mice
Mutant Proteins Expressed Bactericidal Activity of Normal Mouse Sera
035E UspAl & UspA2 -
035E.1 UspA2 -
035E.2 UspAl & tUspA2 +
035E.12 tUspA2 +
SUBSTTTUTE SHEET RULE 25) EXAMPLE VIII: Identification of a Decapeptide Epitope in UspAl that Binds MAb 17C7
It was clear from the work with different strains of M. catarrhalis and analyses of their protein sequences of UspAl that certain epitopic regions must exist which are similar.if not identical, in all of the strains and provide the basis of the immunogenic response in humans. In order to identify such immunogenic epitope(s), peptides spanning the UspAl region known to contain the binding site for MAb 17C7 were prepared and examined for their ability to bind to MAb 17C7.
Specifically, overlapping synthetic decapeptides, as shown in Table XXIX and FIG. 12, that were N-terminally bound to a membrane composed of derivatized cellulose were obtained from Research Genetics Inc. (Huntsville. AL). After five washes with PBS-Tween containing 5% (w/v) non-fat dry milk, the membrane was subsequently incubated with MAb 17C7 (in the form of hybridoma culture supernatant) overnight at 4°C. Following three washes with PBS- Tween, the membrane was incubated overnight at 4°C with gentle rocking with 10 cpm of radioiodinated (specific activity 2 x 10 cpm/μg protein), affinity-purified goat anti-mouse immunoglobulin. The membrane was then washed as before and exposed to X-ray film (Fuji RX safety film, Fuji Industries, Tokyo, Japan).
SUBSTTTUTE SHEET (RULE 25) TABLE XXIX
Decapeptides Used to Identify Binding Site for MAb 17C7
PEPTIDE # P PEEP1 TIDE SEQUENCE
9 SGRLLDQKAD SEQ ID NO:81
10 QKADIDNNIN SEQ ID NO:82
1 1 NNINNIYELA SEQ ID NO:83
12 NNIYELAQQQ SEQ ID NO:84
13 YELAQQQDQH SEQ ID NO: 18
14 AQQQDQHSSD SEQ ID NO:85
15 QDQHSSDIKT SEQ ID NO:86
16 HSSDIKTLKN SEQ ID NO:87
17 DIKTLKNNVE SEQ ID NO:88
18 TLKNNVEEGL SEQ ID NO:89
19 EEGLLDLSGR SEQ ID NO:90
20 LSGRLIDQKA SEQ ID NO:91
21 DQKADIAKNQ SEQ ID NO:92
22 AKNQADIAQN SEQ ID NO:93
23 IAQNQTDIQD SEQ ID NO:94
24 DIQDLAAYNE SEQ ID NO:95
It is clear from the dot blot results shown in the autoradiograph (FIG. 13) that peptide 13, YELAQQQDQH (SEQ ID NO : 18) exhibited optimal binding of MAb 17C7 with peptide 14
(SEQ ID NO:85) exhibiting less than optimal binding. This same peptide (SEQ ID NOT 8) is present in UspA2 which explains why both proteins bind to MAb 17C7.
Interestingly, peptide 12 shows no binding and binding by peptides 15, 16, 19, 22, 23 is probably non-specific. Thus, a comparison of peptides 12, 13, and 14 yields the conclusion that the 7-mer AQQQDQH (SEQ ID NO: 17) is an essential epitope for MAb 17C7 to bind to
UspAl and UspA2. This conclusion is in agreement with the current understanding that an immunogenic epitope may comprise as few as five, six or seven amino acid residues.
SUBSTTTUTE SHEET (RULE 25) Example IX: Phenotypic Effect of Isogenic uspAl and uspA2 Mutations on
M. catarrhalis Strain O35E
Materials and Methods
Bacterial strains, plasmids and growth conditions. The bacterial strains and plasmids used in this study are listed in Table XXX. M. catarrhalis strains were routinely grown at 37°C on Brain-Heart Infusion (BHI) agar plates (Difco Laboratories, Detroit, MI) in an atmosphere of 95% air-5% C02 supplemented, when necessary, with kanamycin (20 μg/ml) (Sigma Chemicals Co., St. Louis, MO) or chloramphenicol (0.5 μg/ml) (Sigma), or in BHI broth. The BHI broth used to grow M. catarrhalis cells for attachment assays was sterilized by filtration. Escherichia coli strains were cultured on Luria-Bertani (LB) agar plates (Maniatis et al, 1982) supplemented, when necessary, with ampicillin (100 μg/ml), kanamycin (30 μg/ml), or chloramphenicol (30 μg/ml).
TABLE XXX Bacterial Strains and Plasmids Used in this Study
Strain or plasmid Description Source or reference
M catarrhalis 035E Wild-type isolate from Helminen et al. , 1994 middle ear fluid
035E.1 Isogenic mutant of 035 E Aebi e/ α/., 1997 with a kan cartridge in the uspAl structural gene
035E.2 Isogenic mutant of 035E Aebi e/ α/., 1997 with a kan cartridge in the uspA2 structural gene
SUBSTTTUTE SHEET RULE 26) TABLE XXX (Continued)
Strain or plasmid Description Source or reference
035E.12 Isogenic mutant of 035E This study with a kan cartridge in the uspA2 structural gene and a cat cartridge in the uspAl structural gene
P-44 Wild-type isolate that Soto-Hernandez et al. , 1989 exhibits rapid hemagglutination
P-48 Wild-type isolate that Soto-Hernandez et al , 1989 exhibits slow hemagglutination
Escherichia coli
DH5α Host for cloning studies Stratagene
Plasmids pBluescript II Cloning vector; Ampr Stratagene pUSPAl pBluescript II SK+ with a Aebi e/ α/., 1997
2.7 kb insert containing most of the uspAl gene of M. catarrhalis strain 035 E pUSPA I CAT pUSPAl with a cat cartridge This study replacing the 0.6 kb Bglϊl fragment of the uspAl gene
Characterization of outer membrane proteins. Whole cell lysates and outer membrane vesicles of M. catarrhalis strains were prepared as described (Murphy and Loeb, 1989; Patrick et al, 1987). Proteins present in these preparations were resolved by SDS-PAGE and detected
SUBSTTTUTE EET RULE 26 by staining with Coomassie blue or by western blot analysis as described (Helminen et al, 1993a).
Monoclonal antibodies (MAbs). MAb 17C7, a murine IgG antibody that reacts with a conserved epitope of both UspAl and UspA2 from M. catarrhalis strain 035E, as described in earlier examples herein, was used for immunologic detection of these proteins. MAb 17C7 was used in the form of hybridoma culture supernatant fluid in western blot analysis and in the indirect antibody-accessibility assay. MAb 3F12, an IgG MAb specific for the major outer membrane protein of Haemophilus ducreyi (Klesney-Tait et al. , 1997), was used as a negative control in the indirect antibody-accessibility assay.
Molecular cloning methods. Chromosomal DNA of M. catarrhalis strain 035E was used as the template in a polymerase chain reaction (PCR™) system together with oligonucleotide primers derived from either just after the start of the strain 035E uspAl open reading frame (i.e., PI in FIG. 14) or just after the end of this open reading frame (i.e., P2 in
FIG. 14). These primers were designed to contain a BamUl restriction site at their 5'-end. The sequence of these primers was:
PI - 5'-CGGGATCCGTGAAGAAAAATGCCGCAGGT-3' (SEQ ID NO:96); P2 - 5'-CGGGATCCCGTCGCAAGCCGATTG-3' (SEQ ID NO:97). DNA fragments were amplified using a PTC 100 Programmable Thermal Controller (MJ
Research, Inc., Cambridge, MA) and the GeneAmp PCR™ kit (Roche Molecular Systems, Inc., Branchburg, NJ). PCR™ products were extracted from 0.7% agarose gel slices using the Qiaex Gel Extraction Kit (Qiagen, Inc., Chadsworth, CA) and digested with BamUl (New England Biolabs, Inc., Beverly, MA) for subsequent ligation into the BamUl site of pBluescript II SK+ (Stratagene, La Jolla, CA). Ligation reactions were performed with overnight incubation at
16°C using T4 DNA ligase (Gibco BRL, Inc., Gaithersburg, MD). Competent E. coli DH5α cells were transformed with the ligation reaction mixture according to a standard heat-shock procedure (Sambrook et l, 1989) and the desired recombinants were selected by culturing in the presence of an appropriate antimicrobial compound. The 1.3 kb chloramphenicol (cat) resistance cartridge was prepared by excision (using BamUl) from pUCΔECAT (Wyeth-
Lederle, Rochester, NY). The cat cartridge was subsequently ligated into Bglll restriction sites located in the mid-portion of cloned segment from the uspAl gene and, after transformation of
SUBSTTTUTE SHEET RULE 25 competent E. coli DH5 cells, recombinant clones were identified by selection on solidified media containing chloramphenicol.
Transformation of M. catarrhalis. The electroporation method used for transformation of M. catarrhalis strain 035Ε has been described in detail (Helminen et al , 1993b). Briefly, a
30-ml portion of a logarithmic-phase broth culture (10 colony forming units [cfu]/ml) was harvested by centrifugation, washed three times with 10% (v/v) glycerol in distilled water, and resuspended in 100 μl of the same solution. A 20-μl portion of these cells was electroporated with 5 μg of linear DNA (i.e., the truncated uspAl gene containing the cat cartridge) in 5 μl of water in a microelectroporation chamber (Cel-Porator Electroporation system; Bethesda
Research Laboratories, Gaithersburg, MD) by applying a field strength of 16.2 kV over a distance of 0.15 cm. Following electroporation, the cell suspension was transferred to 1 ml of BHI broth and incubated with shaking at 37°C for 90 min. Ten 100-μl portions were then spread on BHI agar plates containing the appropriate antimicrobial compound.
Southern blot analysis. Chromosomal DNA purified from wild-type and mutant M. catarrhalis strains strains was digested with either Pvull or Hindlll (New England Biolabs) and Southern blot analysis was performed as described (Sambrook et al, 1989). Double- stranded DNA probes were labeled with P by using the Random Primed DNA Labeling Kit (Boehringer-Mannheim, Indianapolis, IN).
Indirect antibody-accessibility assay. Overnight BHI broth cultures of M. catarrhalis strain 035E and its isogenic mutants were diluted in PBS buffer containing 10% (v/v) fetal bovine serum and 0.025% (w/v) sodium azide (PBS-FBS-A) to density of 110 Klett units (ca. 10 cfu/ml) as measured with a Klett-Summerson colorimeter (Klett Manufacturing Co., New
York, NY). Portions (100 μl) of this suspension were added to 1 ml of MAb 17C7 or MAb 3F12 culture supernatant. After incubation at 4°C for one hour with gentle agitation, the bacterial cells were washed once and suspended in 1 ml of PBS-FBS-A. Affinity-purified goat anti-mouse immunoglobulin, radiolabeled with I to a specific activity of 10 cpm per μg, was added and the mixture was incubated for one hour at 4°C with gentle agitation. The cells were then washed four times with 1 ml of PBS-FBS-A, suspended in 500 μl of triple detergent
SUBSTTTUTE SHEET RULE 26) (Helminen et al , 1993a) and transferred to glass tubes. The radioactivity present in each sample was measured by using a gamma counter.
Autoagglutination and hemagglutination assays. The ability of M. catarrhalis strains to autoagglutinate was assessed using bacterial cells grown overnight on a BHI agar plate. These cells were resuspended in PBS to a turbidity of 400 Klett units in a glass tube and subsequently allowed to stand at room temperature for ten minutes at which time the turbidity of this suspension was again determined. Rapid and slow autoagglutination were defined as turbidities of less that and greater than 200 Klett units, respectively, after 10 minutes. The hemagglutination slide assay using heparinized human group O Rh+ erythrocytes was performed as previously described (Soto-Hernandez et al, 1989).
Serum bactericidal assay. Complement-sufficient normal adult human serum was prepared by standard methods. Complement inactivation was achieved by heating the serum for 30 min at 56°C. A M. catarrhalis broth culture in early logarithmic phase was diluted in
Veronal-buffered saline containing 0.10% (w/v) gelatin (GVBS) to a concentration of 1-2 x 103 cfu/ml, and 20 μl portions were added to 20 μl of native or heat-inactivated normal human serum together with 160 μl of Veronal-buffered saline containing 5 mM MgCl2 and 1.5 mM CaCl2. This mixture was incubated at 37°C in a stationary water bath. At time 0 and at 15 and 30 min, 10 μl aliquots were removed, suspended in 75 μl of BHI broth and spread onto prewarmed BHI agar plates.
Adherence assay. A method used to measure adherence of Haemophilus influenzae to Chang conjunctival cells in vitro (St. Geme III and Falkow, 1990) was adapted for use with M. catarrhalis. Briefly, 2-3 x 10 HEp-2 cells (ATCC CCL 23) or Chang conjunctival cells
(ATCC CCL 20.2) were seeded into each well in a 24-well tissue culture plate (Corning-Costar) and incubated for 24 h before use. A 0.3 ml volume from an antibiotic-free overnight culture of M. catarrhalis was inoculated into 10 ml of fresh BHI medium lacking antibiotics and this culture was subsequently allowed to grow to a concentration of approximately 5 x 10 cfu/ml (120 Klett units) with shaking in a gyratory water bath. The culture was harvested by centrifugation at 6,000 x g at 4-8°C for 10 min. The supernatant was discarded and a Pasteur pipet was used to gently resuspend the bacterial cells in 5 ml of pH 7.4 phosphate-buffered
SUBSTTTUTE SHEET (RULE 26) saline (PBS) or PBS containing 0.15% (w/v) gelatin (PBS-G). The bacterial cells were centrifuged again and this final pellet was gently resuspended in 6-8 ml of PBS or PBS-G.
Portions (25 μl) of this suspension (10 CFU) were inoculated into the wells of a 24- well tissue culture plate containing monolayers of HEp-2 or Chang cells. These tissue culture plates were centrifuged for 5 min at 165 x g and then incubated for 30 min at 37°C. Non- adherent bacteria were removed by rinsing the wells gently five times with PBS or PBS-G, and the epithelial cells were then released from the plastic support by adding 200 μl of PBS containing 0.05% trypsin and 0.02% EDTA. This cell suspension was serially diluted in PBS or PBS-G and spread onto BHI plates to determine the number of viable M. catarrhalis present.
Adherence was expressed as the percentage of bacteria attached to the human cells relative to the original inoculum added to the well.
Results Construction of an isogenic M. catarrhalis mutant lacking expression of both UspAl and UspA2. Construction of M. catarrhalis mutants lacking the ability to express either UspAl (mutant strain 035E.1) or UspA2 (mutant strain 035E.2) has been described in previous examples (Aebi et al, 1997). For constructing a double mutant that lacked expression of both UspAl and UspA2, the 0.6 kb Bglll fragment of pUSPAl (FIG. 14A) was replaced by a cat cassette, yielding the recombinant plasmid pUSPAl CAT. Using the primers PI and P2, the 3.2 kb insert of pUSPAJCAT was amplified by PCR™. This PCR™ product was used to electroporate the kanamycin-resistant uspA2 strain 035E.2 and yielded the chloramphenicol - and kanamycin-resistant transformant 035E.12, a putative uspAl uspA2 double mutant.
Southern blot analysis was used to confirm that strains 035E.1, O35E.2, and 035E.12 were isogenic mutants and that allelic exchange had occurred properly, resulting in replacement of the wild-type uspAl or uspA2 gene, or both, with the mutated allele. Chromosomal DNA preparations from the wild-type parent strain 035E, the uspAl mutant 035E.1, the uspA2 mutant 035E.2, and the putative uspAl uspA2 mutant strain 035E.12 were digested to completion with Pvull and probed in Southern blot analysis with DNA fragments derived from these two M. catarrhalis genes or with the kan cartridge. For probing with the cat cartridge, chromosomal DNA from strain 035E.12 was digested with Hindlll.
SUBSTTT EET RULE 25 The uspA 1 -specific DNA probe was obtained by PCR™-based amplification of M. catarrhalis strain 035E chromosomal DNA using the primers P3 and P4 (FIG. 14A). A 500-bp
Figure imgf000122_0001
DNA fragment was amplified from 035E chromosomal DNA by PCR™ with the primers P5 and P6 (FIG. 14B). Use of these two gene-specific probes together with the kan and cat cartridges in Southern blot analysis confirmed that strain 035E.12 was a uspAl uspA2 double mutant.
Characterization of selected proteins expressed by the wild-type and mutant M. catarrhalis strains. Proteins present in outer membrane vesicles extracted from the the wild- type and these three mutant strains were resolved by SDS-PAGE and either stained with Coomassie blue (FIG. 15 A) or probed with MAb 17C7 in western blot analysis (FIG. 15B). The wild-type parent strain 035E possessed a very high molecular weight band detectable by Coomassie blue staining (FIG. 15 A, lane 1, closed arrow) that was also similarly abundant in the uspAl mutant 035E.1 (FIG. 15A, lane 2). The uspA2 mutant 035E.2 (FIG. 15A, lane 3) had a much reduced level of expression of a band in this same region of the gel; this band was not visible at all in the uspAl uspA2 double mutant 035E.12 (FIG. 2, panel A, lane 4).
Western blot analysis revealed that the wild-type strain (FIG. 15B, lane 1) expressed abundant amounts of MAb 17C7-reactive antigen, most of which had a very high molecular weight, in excess of 220,000. The wild-type strain also exhibited discrete antigens with apparent molecular weights of approximately 120.000 and 85,000 which bound this MAb (FIG.
15B, lane 1 , open and closed arrows, respectively). The uspAl mutant 035E.1 (FIG. 15B, lane
2) lacked expression of the 120 kDa antigen, which was proposed to be the monomeric form of UspAl , but still expressed the 85 kDa antigen. The amount of very high molecular weight
MAb 17C7-reactive antigen expressed by this uspAl mutant appeared to be equivalent to that expressed by the wild-type strain. The uspA2 mutant 035E.2 (FIG. 15B, lane 3) expressed the
120 kDa antigen but lacked expression of the 85 kDa antigen which was proposed to be the monomeric form of the UspA2 protein. In contrast to the uspAl mutant, the uspA2 mutant had relatively little very high molecular weight antigen reactive with MAb 17C7. Finally, the uspAl uspA2 double mutant 035E.12 (FIG. 15B, lane 4) expressed no detectable MAb 17C7- reactive antigens.
SUBSTTTUTE SHEET (RULE 26) Binding of MAb 17C7 to whole cells of the wild-type and mutant strains. The indirect antibody-accessibility assay was used to determine whether both UspAl and UspA2 are exposed on the surface of M. catarrhalis and accessible to antibody. Whole cells of both the wild-type strain 035E and the uspAl mutant 035E.1 bound similar amounts of MAb 17C7
(Table XXXI). This result suggested that UspA2 is expressed on the surface of M. catarrhalis, or at least on the surface of the uspAl mutant. The uspA2 mutant 035E.2 bound substantially less MAb 17C7 than did the wild-type strain, but the level of binding was still at least an order of magnitude greater than that obtained with an irrelevant IgG Mab directed against a H. ducreyi outer membrane protein (Table XXXI). As expected from the western blot analysis, the uspAl uspA2 double mutant 035E.12 did not bind MAb 17C7 at a level greater than obtained with the negative controls involving the H. wcrev -specifιc MAb (Table XXXI).
TABLE XXXI
Binding of MAb 17C7 to the Surface of Wild-Type and Mutant Strains of M. catarrhalis
Binding" of
Strain MAb 17C7 MAb 3F12b
035E (wild-type) 145,583° 4,924
035E.1 (uspAl mutant) 154,1 19 4,208
035E.2 (uspA2 mutant) 96,721 4,455
035E.12 (uspAl uspA2 double mutant) 6,081 3,997
Counts per min of I-labeled goat anti-mouse immunoglobulin bound to MAbs attached to the bacterial cell surface, as determined in the indirect antibody-accessibility assay. MAb 3F12, a murine IgG antibody specific for a Η. ducreyi outer membrane protein (Klesney-Tait et al, 1997), was included as a negative control.
The values represent the mean of two independent studies.
Characterization of the growth, autoagglutination, and hemagglutination properties of the wild-type and mutant strains. The colony morphology of these three mutant strains grown on BΗI agar plates did not differ from that of the wild-type strain parent strain. Similarly, the
SUBSTTTUTE SHEET (RULE 26) rate and extent of growth of all four of these strains in BHI broth were very similar if not identical (FIG. 16). In an autoagglutination assay performed as described in above in the Materials and Methods section of this example, all four strains exhibited the same rate of autoagglutination. Finally, there was no detectable difference between the wild-type parent and the three mutants in a hemagglutination assay using human group O erythrocytes (Soto-
Hernandez et al, 1989). Control hemagglutination studies were performed using a pair of M. catarrhalis isolates (i.e. , strains P-44 and P48) previously characterized as having rapid or slow rates, respectively, of hemagglutination (Soto-Hernandez et al, 1989).
Effect of the uspAl and uspA2 mutations on the ability of M. catarrhalis to adhere to human cells. Preliminary studies revealed that the wild-type M. catarrhalis strain 035E adhered readily to HeLa cells, HEp-2 cells, and Chang conjunctival cells in vitro. To determine whether lack of expression of UspAl or UspA2 affected this adherence ability, the wild-type and the three mutant strains were first used in an attachment assay with Hep-2 cells. In this set of studies, PBS was used as the diluent for washing the HEp-2 cell monolayers and for serial dilution of the trysinized HEp-2 cell monolayer at the completion of the assay. Both the wild- type strain and the uspA2 mutant 035E.2 exhibited similar levels of attachment to HEp-2 monolayers (Table XXXI). The uspAl mutant 035E.1 , however, was less able to adhere to these HEp-2 cells; lack of expression of UspAl reduced the level of attachment by approximately six-fold (Table XXXII). The uspAl uspA2 double mutant 035E.12 exhibited a similarly reduced level of attachment (Table XXXII).
SUBSTTTU ET RULE 26 TABLE XXXII
Adherence of Wild-Type and Mutant Strains of M. catarrhalis to HEp-2 and Chang Conjunctival Cells in vitro
Adherence" to Strain HEp-2 cells Chang cells0
035E (wild-type) 14.7 ± 4.9 51.4 ± 30.8
035E.1 (uspAl mutant) 2.4 ± 0.9 (0.006d) 0.8 ± 0.5 (0.002d)
O35E.2 (uspA2 mutant) 19.1 ± 7.0 (0.213d) 55.9 ± 16.7 (0.728d) 25 Λ2 (uspAl uspA2 double mutant) 2.3 ± 1.8 (0.01 ld) 0.6 ± 0.2 (0.002d) a Adherence is expressed as the percentage of the original inoculum that was adherent to the human epithelial cells at the end of the 30 min incubation period. Each number represents the mean (± S.D.) of two independent studies.
PBS was used for washing of the monolayers and for serial dilutions of adherent M. catarrhalis. c PBS-G was used for washing of the monolayers and for serial dilutions of adherent M. catarrhalis. d P value when compared to the wild-type strain O35E using the two-tailed Student t- test.
Control studies revealed, however, that M. catarrhalis cells did not survive well in the PBS used for washing of the HEp-2 monolayer and serial dilution of the attached M. catarrhalis organisms. When 10 CFU of the wild-type and mutant M. catarrhalis strains were suspended in PBS, serially diluted, and allowed to stand for 30 min on ice, the viable number of bacteria decreased to 107 CFU. In contrast, when PBS containing 0.15% (w/v) gelatin (PBS-G) was used for this same type of experiment, there was no reduction in the viability of these M. catarrhalis strains over the duration of the experiment. When the HEp-2 cell-based attachment studies were repeated using PBS-G for washing the HEp-2 cell monolayer and as the diluent, there was only a three-fold reduction in adherence of the uspAl mutant relative to that obtained with the wild-type parent strain. This finding suggested that the original six-fold difference in attachment ability observed between the wild-type and uspAl mutant strain may
SUBSTTTUTE SHEET RULE 26) have been attributable in part to viability problems caused by the use of the PBS wash and diluent.
Subsequent studies using Chang conjunctival cells as the target for bacterial attachment together with a PBS-G wash and diluent revealed a substantial difference in the attachment abilities of the wild-type strain and the uspAl mutant (Table XXXII). Whereas the wild-type and uspA2 mutant exhibited similar levels of attachment to the Chang cells, the extent of attachment of the uspAl mutant was nearly two orders of magnitude less than that of the wild- type parent strain. The uspAl uspA2 double mutant also exhibited a much reduced level of attachment similar to obtained with the uspAl mutant (Table XXXII).
Effect of the uspAl and uspA2 mutations on serum resistance of M. catarrhalis. Similar to the majority of disease isolates of M. catarrhalis (Hoi et al, 1993; 1995; Verduin et al, 1994), the wild-type strain 035E was resistant to killing by normal human serum in vitro (Helminen et al, 1993b). To examine the effect of the lack of expression of UspAl or UspA2 on serum resistance, the wild-type strain and the three mutant strains were tested in a serum bactericidal assay. Both the wild-type strain (FIG. 17, closed diamonds) and the uspAl mutant O35E.1 (FIG. 17, closed triangles) were able to grow in the presence of normal human serum, indicating that lack of expression of UspAl did not adversely affect the ability of strain 035E.1 to resist killing by normal human serum. However, both the uspA2 mutant O35E.2 (FIG. 17, closed circles) and the uspAl uspA2 double mutant O35E.12 (FIG. 17, closed squares), having in common the lack of expression of UspA2, were readily killed by normal human serum. Heat-based inactivation of the complement system present in this normal human serum eliminated the ability of this serum to kill these latter two mutants (FIG. 17, open circles and squares).
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically,
SUBSTTTUTE SHEET RULE 25 it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incoφorated herein by reference.
EPO Appl. Publ. No. 0036776 U.S. PatentNo. 5,552,146 U.S. Patent No. 5,310,687 U.S. Patent No. 5,238,808 U.S. Patent No. 5,221,605
U.S. Patent No. 4,608,251 U.S. Patent No. 4,603,102 U.S. Patent No. 4,601,903 U.S. Patent No. 4,599,231 U.S. Patent No. 4,599,230
U.S. Patent No. 4,596,792 U.S. Patent No. 4,578,770 U.S. Patent No. 4,554,101 U.S. Patent No. 4,452,901 U.S. Patent No. 4,367,1 10
U.S. Patent No. 4,358,535 U.S. Patent No. 4,196,265 U.S. Patent No. 4,174,384 U.S. Patent No. 3,949,064 U.S. Patent No. 3,791,932
SUBSTTTUTE SHEET (RULE 25) Aebi, Stone, Beucher, Cope, Maciver, Thomas, McCracken Jr., Sparling, Hansen, "Expression of the CopB outer membrane protein by Moraxella catarrhalis is regulated by iron and affects iron acquisition from transferrin and lactoferrin," Infect. Immun., 64:2024-2030,
1996. Altschul, Gish, Miller, Myers and Lipman. "Basic local alignment search tool," J. Mol. Biol,
215:403-410, 1990. Baichwal and Sugden, In: GENE TRANSFER Kucherlapati, R., ed. New York: Plenum Press, pp.
1 17-148, 1986. Barenkamp and St. Geme III, "Identification of a second family of high-molecular-weight adhesion proteins expressed by nontypable Haemophilus influenzae, " Mol. Microbial,
19: 1215-1223, 1996. Bartos and Muφhy, "Comparison of the outer membrane proteins of 50 strains of Branhamella catarrhalis, "! Infect. Dis., 158:761-765, 1988. Benz and Schmidt, "Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathic Escherichia coli, " Infect. Immun. , 57:1506- 1511, 1989.
Benz and Schmidt, "Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787
(0126:H27)," Infect. Immun., 60:13-18, 1992b. Berk, Arch. Intern. Med, 150:2254-2257, 1990. Bliska, Copass, Falkow, "The Yersinia pseudotuberculosis adhesin YadA mediates intimate bacterial attachment to and entry into HEp-2 cells," Infect. Immun., 61 :3914-3921, 1993. Bluestone, "Otitis media and sinusitis in children: role of Branhamella catarrhalis," Drugs,
31(Suppl. 3): 132-141 , 1986. Bluestone, Stephenson, Martin, "Ten-year review of otitis media pathogens," Pediatr. Infect. Dis. J, 1 1 :S7-S1 1, 1992.
Bolivar et al, Gene, 2:95, 1977. Brutlag et α/., CABIOS, 6:237-245, 1990. Campagnari, Shanks, Dyer, "Growth of Moraxella catarrhalis with human transferrin and lactoferrin: Expression of iron-repressible proteins without siderophore production," Infect. Immun., 62:4909-4914, 1994.
Catlin, "Branhamella catarrhalis: an organism gaining respect as a pathogen," Clin. Microbiol
Rev, 3:293-320, 1990.
SUBSTTTUTE SHEET RULE 26 Chang et al, Nature, 375:615, 1978.
Chapman et al, J. Infect. Dis., 151 :878-882, 1985.
Chen, McMichael, Vandermeid, Hahn, Smith, Eldridge, Cowell, "Antibodies to the UspA outer membrane protein of Moraxella catarrhalis block bacterial attachment in vitro and are protective in a murine pulmonary challenge model," Abstracts General Meeting Amer.
Soc. Microbiol, E-53:290, 1995. China, Sory, N'Guyen, de Bruyere, Cornells, "Role of YadA protein in prevention of osponization of Yersinia enterocolitica by C3b molecules," Infect. Immun., 61 :3129-
3136, 1993. Christensen, Renneberg, Bruun, Forsgren, "Serum antibody response to proteins of Moraxella
(Branhamella) catarrhalis in patients with lower respiratory tract infection." Clin.
Diagn. Lab. Immunol, 2: 14-17, 1995. Coligan et al. (eds), In: CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley, New York, ch. 2.5, 1991. Consensus, Pediater. Infect. Dis. J. , 8 : S94-S97, 1989.
Davies and Maesen, "The epidemiology of respiratory tract pathogens in Southern
Netherlands," Eur. Respir. J., 1 :415-420, 1988. Devereux, Haeberli and Smithies, "A comprehensive set of sequence analysis programs for the
VAX," Nucleic Acids Res., 12:387-395, 1984. Doern, Diag. Microbiol. Infect. Dis. , 4: 191-201, 1986.
Doyle, Peditr. Infect. Dis. 1, 8(Suppl):S45-7, 1989. Faden et al, Ann. Otol Rhinol Laryngol, 100:612-615, 1991. Faden et al, Pediatr. Infect. Dis. J, 9:623-626, 1990.
Faden, "Comparison of the local immune response to nontypeable Haemophilus influenzae (nHI) and Moraxella catarrhalis (MC) during otitis media," In: Advances in Mucosal
Immunology, J. Mestecky and et al. (ed.), Plemun Press, New York, p. 733-736, 1995. Faden, Duffy, Wasielewski, Wolf, Krystofik, Tung, Tonawanda/Williamsburg Pediatrics,
"Relationship between nasopharyngeal colonization and the development of otitis media in children," J. Infect. Dis., 175:1440-1445, 1997. Faden, Harabuchi, Hong, Tonawanda/Williamsburg Pediatrics, "Epidemiology of Moraxella catarrhalis in children during the first 2 years of life: Relationship to otitis media," J.
Infect. Dis., 169:1312-1317, 1994.
SUBSTTTUT S EET RULE 26 Faden, Hong and Muφhy, "Immune response to outer membrane antigens of
Moraxella catarrhalis in children with otitis media," Infect. Immun., 60:3824-3829,
1992. Fetrow & Bryant, Biotechnology, 11 :479-483, 1993. Fitzgerald, Mulcahy, Muφhy, Keane, Coakley, Scott, "A 200 kDa protein is associated with haemagglutinating isolates of Moraxella (Branhaemella) catarrhalis," FEMS Immunol.
Med. Microbiol, 18:209-216, 1997. Fleischmann, Adams, White, Clayton, Kirkness, Kerlavage, Bult, Tomb, Dougherty, Merrick,
McKenney, Sutton, FitzHugh, Fields, Gocayne, Scott, Shirley, Liu, Glodek, Kelley, Weidman, Phillips, Spriggs, Hedblom, Cotton, Utterback, Hanna, Nguyen, Saudek,
Brandon, Fine, Frichman, Fuhrmann, Geoghagen, Gnehm, McDonald, Small, Fraser,
Smith and Venter, "Whole-genome random sequencing and assembly of
Haemophilus influenzae Rd., Science, 269:496-512, 1995. Gefter et al, Somatic Cell Genet., 3:231-236, 1977. Gish and States, "Identification of protein coding regions by database similarity search," Nat. genet., 3:266-272, 1993. Glorioso et α/., w?. Rev. Microbiol. 49:675-710, 1995. Goeddel et al. , Nature 281 :544, 1979. Goeddel et al, Nucleic Acids Res., 8:4057, 1980. Goldblatt, Scadding, Lund, Wade, Turner, Pandey, "Association of Gm allotypes with the antibody response to the outer membrane proteins of a common upper respiratory tract organism, Moraxella catarrhalis," J. Immunol, 153:5316-5320, 1994. Goldblatt, Turner, and Levinsky, "Branhamella catarrhalis: antigenic determinants and the development of the IgG subclass response in childhood," J. Infect. Dis., 162: 1128-1 135, 1990.
Hager, Verghese, Alvarez, Berk, "Branhamella catarrhalis respiratory infections," Rev. Infect.
Dis., 9:1 140-1 149, 1987. Helminen, Beach, Maciver, Jarosik, Hansen, Leinonen, "Human immune response against outer membrane proteins of Moraxella (Branhamella) catarrhalis determined by immunoblotting and enzyme immunoassay," Clin. Diagn. Lab. Immunol, 2:35-39,
1995.
SUBSTTTUTE SHEET RULE 25 Helminen, Maciver, Latimer, Cope. McCracken Jr., and Hansen, "A major outer membrane protein of Moraxella catarrhalis is a target for antibodies that enhance pulmonary clearance of the pathogen in an animal model," Infect. Immun., 61 :2003-2010, 1993a. Helminen, Maciver, Latimer, Klesney-Tait, Cope, Paris, McCracken Jr., and Hansen, "A large, antigenically conserved protein on the surface of Moraxella catarrhalis is a target for protective antibodies," J. Infect. Dis., 170:867-872, 1994. Helminen, Maciver, Latimer, Lumbley, Cope, McCracken, Jr., and Hansen,. "A mutation affecting expression of a major outer membrane protein of Moraxella catarrhalis alters serum resistance and survival of this organism in vivo," J. Infect. Dis., 168:1194-1201, 1993b. Hess et al. , J. Adv. Enzyme Reg. , 7: 149, 1968.
Hitzeman et α/., J Biol Chem., 255:2073, 1980.
Hoi, Verduin, Van Dijke, Verhoef, Fleer, van Dijk, "Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis," FEMS Immunol. Med. Microbiol,
1 1 :207-212, 1995. Hoi, Verduin, van Dijke, Verhoef, van Dijk, "Complement resistance in Branhamella
(Moraxella) catarrhalis," Lancet, 341 :1281, 1993. Holland et al, Biochemistry, 17:4900, 1978. Horstmann, Sievertsen, Knobloch, Fischetti, "Antiphagocytic activity of streptococcal M protein: selective binding of complement control protein factor H," Proc. Natl. Acad. Sci. USA, 85:1657-1661, 1988.
Hsiao, Sethi, Muφhy, "Outer membrane protein CD of Branhamella catarrhalis -Sequence conservation in stains recovered from the human respiratory tract," Microb. Pathog,
19:215-225, 1995. Itakura et al, Science, 198:1056, 1977. Jamesonand Wolf, Comput. Appl. Biosci, 4(1): 181-186, 1988.
Jones, Genetics, 85:12, 1977. Jordan, Berk, Berk, "A comparison of serum bactericidal activity and phenotypic characteristics of bacteremic, pneumonia-causing strains, and colonizing strains of Branhamella catarrhalis," Am. J. Med, 88(5A):28S-32S, 1990. Kimura, Gulig, McCracken, Loftus and Hansen, "A minor high-molecular- weight outer membrane protein of Haemophilus influenzae type b is a protective antigen," Infect.
Immun., 47:253-259, 1985.Kingsman et al, Gene, 7: 141, 1979.
SUBSTTTUTE SHEET RULE 25) Klesney-Tait, Hiltke, Spinola, Radolf, Hansen, "The major outer membrane protein of
Haemophilus ducreyi consists of two OmpA homologs," J. Bacteriol, 179:1764-1773,
1997. Klingman and Muφhy, "Purification and characterization of a high-molecular-weight outer membrane protein of Moraxella (Branhamella) catarrhalis, " Infect. Immun., 62:1 150-
1 155, 1994. Klingman, Pye, Muφhy, Hill, "Dynamics of respiratory tract colonization by Branhamella catarrhalis in bronchiectasis," Am. J. Respir. Crit. Care Med., 152:1072-1078, 1995. Kohler & Milstein, Eur. J. Immunol, 6:511-519, 1976. Kohler & Milstein, Nature, 256:495-497, 1975.
Kovatch, Wald, Michaels, "β-Lactamase-producing Branhamella catarrhalis causing otitis media in children," J Pediatr., 102:261-264, 1983. Kyte & Doolittle, J. Mol. Biol, 157: 105-132, 1982.
Leininger, Bowen, Renauld-Mongenie, Rouse, Menozzi, Locht, Heron, Brennan, "Immunodominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin," J. Infect. Dis., 175:1423-1431, 1997. Leinonen et al., J. Infect. Dis., 144:570-574, 1981. Maniatis, Fritsch and Sambrook, Molecular Cloning: A Laboratory Manual Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York, 1982. Marchant, Am. J. Med, 88(Suppl. 5A):15S-19S, 1990.
McGehee, m. J. Respir. Cell Mol. Biol, 1 :201-210, 1989.
McLeod, Ahmad, Capewell, Croughan, Calder, Seaton, "Increase in bronchopulmonary infection due to Branhamella catarrhalis," Br. Med. J. [Clin. Res]., 292: 1 103-1 105,
1986. Melendez & Johnson, Rev. Infect. Dis. , 13 :428-429, 1990.
Muφhy and Bartos, "Surface exposed and antigenically conserved determinants of outer membrane proteins of Branhamella catarrhalis, " Infect. Immun., 57:2938-2941 , 1989. Muφhy and Loeb, "Isolation of the outer membrane of Branhamella catarrhalis, " Microb.
Pathog, 6:159-174, 1989. Murphy et al, Am. Jrn Med , 88:5A-41S-5A-45S, 1990.
Muφhy, Kirkham and Lesse, "The major heat-modifiable outer membrane protein CD is highly conserved among strains of Branhamella catarrhalis, "Mol Microbiol, 10:87-97, 1993.
SUBSTTTUTE SHEET RULE 26) Muφhy, Pediat. Infect. Dis. J, 8: S75-S77, 1989.
Nicolas and Rubenstein, In: Vectors: A survey of molecular cloning vectors and their uses,
Rodriguezand Denhardt(eds.), Stoneham: Butterworth,pp. 494-513, 1988. Nicotra, Rivera, Liman, Wallace, "Branhamella catarrhalis as a lower respiratory tract pathogen in patients with chronic lung disease," Arch. Intern. Med., 146:890-893, 1986.
Patrick, Kimura, Jackson, Hermanstorfer, Hood, McCracken Jr., Hansen, "Antigenic characterization of the oligosaccharide portion of the lipooligosaccharide of nontypable
Haemophilus influenzae," Infect. Immun., 55:2902-291 1, 1987. Pilz, Vocke, Heesemann, Brade, "Mechanism of YadA-mediated serum resistance of Yersinia enterocolitica serotype 03." Infect. Immun., 60:189-195, 1992.
Reddy, Muφhy, Faden, Bernstein, "Middle ear mucin glycoprotein; Purification and interaction with nontypeable Haemophilus influenzae and Moraxella catarrhalis," Otolaryngol
Head Neck Surg, 1 16: 175-180, 1997. Ridgeway, In: Vectors: A survey of molecular cloning vectors and their uses. Stoneham: Butterworth, Rodriguez RL, Denhardt DT, ed., pp. 467-492, 1988.
Sambrook, Fritsch and Maniatis, Molecular Cloning - A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Sarubbi et al, Am. J. Med, 88(Suppl. 5A):9S-14S, 1990. Schonheyder & Ejlertsen, Eur. J. Clin. Microbiol Infect. Dis., 8:299-300, 1989. Shine and Dalgarno, "Determinant of cistron specificity in bacterial ribosomes," Nature 254:34-
38, 1975. Skurnik and Wolf-Watz, "Analysis of the yopA gene encoding the Yopl virulence determinants of Yersinia spp.," Mol. Microbiol, 3:517-529, 1989. Soto-Hernandez, Holtsclaw-Berk, Harvill, Berk, "Phenotypic characteristics of Branhamella catarrhalis strains," J. Clin. Microbiol, 27:903-908, 1989.
St.Geme III and Falkow, "Haemophilus influenzae adheres to and enters cultured human epithelial cells," Infect. Immun. , 58:4036-4044, 1990. St.Geme III, Cutter, Barenkamp, "Characterization of the genetic locus encoding Haemophilus influenzae type b surface fibrils," J. Bacteriol, 178:6281-6287, 1996. Stinchcomb et al. , Nature, 282:39, 1979.
Temin, /??: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986. Tissue Culture, Academic Press, Kruse and Patterson, editors, 1973.
SUBSTTTUTE SHEET RULE 25) Tschemper et al. , Gene, 10: 157, 1980.
Unhanand et a/., J Infect. Dis. , 165:644-650, 1992.
Verduin, Bootsma, Hoi, Fleer, Jansze, Klingman, Muφhy, van Dijk, "Complement resistance in
Moraxella (Branhamella) catarrhalis is mediated by a high-molecular-weight outer membrane protein (HMW-OMP)," Abstracts General Meeting Amer. Soc. Microbiol,
B137:189(Abstract), 1995. Verduin, Jansze, Hoi, Mollnes, Verhoef, van Dijk, "Differences in complement activation between complement-resistant and complement-sensitive Moraxella (Branhamella) catarrhalis strains occur at the level of membrane attack complex formation," Infect. Immun. , 62:589-595, 1994.
Verghese et α/., J Infect. Dis., 162:1189-92, 1990. Weinberger et al, Science, 228:740-742, 1985. Wolf et al, Comput. Appl. Biosci., 4(1): 187-191, 1988. Wright & Wallace, Semin. Respir. Infect., 4:40-46, 1989.
SUBSTTTUTE SHEET RULE 26) SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT:
(A) NAME: Board of Regents, The University of Texas
System
(B) STREET: 201 W. 7th Street
(C) CITY: Austin (D) STATE: Texas
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP) : 78701
(ii) TITLE OF INVENTION: UspAl AND UspA2 ANTIGENS OF MORAXELLA CATARRHALIS
(iii) NUMBER OF SEQUENCES: 98
(iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/033,598
(B) FILING DATE: 20-DEC-1996
(2) INFORMATION FOR SEQ ID NO : 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 831 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS :
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1: Met Asn Lys lie Tyr Lys Val Lys Lys Asn Ala Ala Gly His Leu Val 1 5 10 15
Ala Cys Ser Glu Phe Ala Lys Gly His Thr Lys Lys Ala Val Leu Gly 20 25 30
Ser Leu Leu lie Val Gly Ala Leu Gly Met Ala Thr Thr Ala Ser Ala 35 40 45
Gin Ala Thr Asn Ser Lys Gly Thr Gly Ala His lie Gly Val Asn Asn 50 55 60
Asn Asn Glu Ala Pro Gly Ser Tyr Ser Phe lie Gly Ser Gly Gly Tyr 65 70 75 80 Asn Lys Ala Asp Arg Tyr Ser Ala lie Gly Gly Gly Leu Phe Asn Lys
85 90 95
SUBSTTTUTE SHEET RULE 25 Ala Thr Asn Glu Tyr Ser Thr lie Val Gly Gly Gly Tyr Asn Lys Ala 100 105 110 Glu Gly Arg Tyr Ser Thr lie Gly Gly Gly Ser Asn Asn Glu Ala Thr
115 120 125
Asn Glu Tyr Ser Thr lie Val Gly Gly Asp Asp Asn Lys Ala Thr Gly 130 135 140
Arg Tyr Ser Thr lie Gly Gly Gly Asp Asn Asn Thr Arg Glu Gly Glu 145 150 155 160
Tyr Ser Thr Val Ala Gly Gly Lys Asn Asn Gin Ala Thr Gly Thr Gly 165 170 175
Ser Phe Ala Ala Gly Val Glu Asn Gin Ala Asn Ala Glu Asn Ala Val 180 185 190 Ala Val Gly Lys Lys Asn He He Glu Gly Glu Asn Ser Val Ala He
195 200 205
Gly Ser Glu Asn Thr Val Lys Thr Glu His Lys Asn Val Phe He Leu 210 215 220
Gly Ser Gly Thr Thr Gly Val Thr Ser Asn Ser Val Leu Leu Gly Asn 225 230 235 240
Glu Thr Ala Gly Lys Gin Ala Thr Thr Val Lys Asn Ala Glu Val Gly 245 250 255
Gly Leu Ser Leu Thr Gly Phe Ala Gly Glu Ser Lys Ala Glu Asn Gly 260 265 270 Val Val Ser Val Gly Ser Glu Gly Gly Glu Arg Gin He Val Asn Val
275 280 285
Gly Ala Gly Gin He Ser Asp Thr Ser Thr Asp Ala Val Asn Gly Ser 290 295 300
Gin Leu His Ala Leu Ala Thr Val Val Asp Asp Asn Gin Tyr Asp He 305 310 315 320
Val Asn Asn Arg Ala Asp He Leu Asn Asn Gin Asp Asp He Lys Asp 325 330 335
Leu Gin Lys Glu Val Lys Gly Leu Asp Asn Glu Val Gly Glu Leu Ser 340 345 350 Arg Asp He Asn Ser Leu His Asp Val Thr Asp Asn Gin Gin Asp Asp
355 360 365
He Lys Glu Leu Lys Arg Gly Val Lys Glu Leu Asp Asn Glu Val Gly
370 375 380
Val Leu Ser Arg Asp He Asn Ser Leu His Asp Asp Val Ala Asp Asn
385 390 395 400
SUBSTTTUTE SHEET RULE 25) Gin Asp Asp He Ala Lys Asn Lys Ala Asp He Lys Gly Leu Asn Lys 405 410 415
Glu Val Lys Glu Leu Asp Lys Glu Val Gly Val Leu Ser Arg Asp He 420 425 430
Gly Ser Leu His Asp Asp Val Ala Thr Asn Gin Ala Asp He Ala Lys 435 440 445
Asn Gin Ala Asp He Lys Thr Leu Glu Asn Asn Val Glu Glu Glu Leu 450 455 460
Leu Asn Leu Ser Gly Arg Leu Leu Asp Gin Lys Ala Asp He Asp Asn 465 470 475 480
Asn He Asn Asn He Tyr Glu Leu Ala Gin Gin Gin Asp Gin His Ser 485 490 495 Ser Asp He Lys Thr Leu Lys Asn Asn Val Glu Glu Gly Leu Leu Asp
500 505 510
Leu Ser Gly Arg Leu He Asp Gin Lys Ala Asp He Ala Lys Asn Gin 515 520 525
Ala Asp He Ala Gin Asn Gin Thr Asp He Gin Asp Leu Ala Ala Tyr 530 535 540
Asn Glu Leu Gin Asp Gin Tyr Ala Gin Lys Gin Thr Glu Ala He Asp 545 550 555 560
Ala Leu Asn Lys Ala Ser Ser Glu Asn Thr Gin Asn He Ala Lys Asn 565 570 575 Gin Ala Asp He Ala Asn Asn He Asn Asn He Tyr Glu Leu Ala Gin
580 585 590
Gin Gin Asp Gin His Ser Ser Asp He Lys Thr Leu Ala Lys Val Ser 595 600 605
Ala Ala Asn Thr Asp Arg He Ala Lys Asn Lys Ala Glu Ala Asp Ala 610 615 620
Ser Phe Glu Thr Leu Thr Lys Asn Gin Asn Thr Leu He Glu Gin Gly 625 630 635 640
Glu Ala Leu Val Glu Gin Asn Lys Ala He Asn Gin Glu Leu Glu Gly
645 650 655 Phe Ala Ala His Ala Asp He Gin Asp Lys Gin He Leu Gin Asn Gin
660 665 670
Ala Asp He Thr Thr Asn Lys Thr Ala He Glu Gin Asn He Asn Arg 675 680 685
Thr Val Ala Asn Gly Phe Glu He Glu Lys Asn Lys Ala Gly He Ala
690 695 700
SUBSTTTUTE SHEET RULE 25) Thr Asn Lys Gin Glu Leu He Leu Gin Asn Asp Arg Leu Asn Arg He 705 710 715 720
Asn Glu Thr Asn Asn Arg Gin Asp Gin Lys He Asp Gin Leu Gly Tyr 725 730 735
Ala Leu Lys Glu Gin Gly Gin His Phe Asn Asn Arg He Ser Ala Val 740 745 750
Glu Arg Gin Thr Ala Gly Gly He Ala Asn Ala He Ala He Ala Thr 755 760 765
Leu Pro Ser Pro Ser Arg Ala Gly Glu His His Val Leu Phe Gly Ser 770 775 780
Gly Tyr His Asn Gly Gin Ala Ala Val Ser Leu Gly Ala Ala Gly Leu 785 790 795 800 Ser Asp Thr Gly Lys Ser Thr Tyr Lys He Gly Leu Ser Trp Ser Asp
805 810 815
Ala Gly Gly Leu Ser Gly Gly Val Gly Gly Ser Tyr Arg Trp Lys 820 825 830
(2) INFORMATION FOR SEQ ID NO : 2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3349 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2:
ATCAGCATGT GAGCAAATGA CTGGCGTAAA TGACTGATGA GTGTCTATTT AATGAAAGAT 60
ATCAATATAT AAAAGTTGAC TATAGCGATG CAATACAGTA AAATTTGTTA CGGCTAAACA 120
TAACGACGGT CCAAGATGGC GGATATCGCC ATTTACCAAC CTGATAATCA GTTTGATAGC 180
CATTAGCGAT GGCATCAAGT TGTGTTGTTG TATTGTCATA TAAACGGTAA ATTTGGTTTG 240 GTGGATGCCC CATCTGATTT ACCGTCCCCC TAATAAGTGA GGGGGGGGGG GAGACCCCAG 300
TCATTTATTA GGAGACTAAG ATGAATAAAA TTTATAAAGT GAAGAAAAAT GCCGCAGGTC 360
ACTTGGTGGC ATGTTCTGAA TTTGCCAAAG GTCATACCAA AAAGGCAGTT TTGGGCAGTT 420
TATTGATTGT TGGGGCGTTG GGCATGGCAA CGACGGCGTC TGCACAAGCA ACCAACAGCA 480
AAGGCACAGG CGCGCACATC GGTGTTAACA ATAACAACGA AGCCCCAGGC AGTTACTCTT 540 TCATCGGTAG TGGCGGTTAT AACAAAGCCG ACAGATACTC TGCCATCGGT GGTGGCCTTT 600
TTAACAAAGC CACAAACGAG TACTCTACCA TCGTTGGTGG CGGTTATAAC AAAGCCGAAG 660
SUBSTTTUTE SHEET RULE 25) GCAGATACTC TACCATCGGT GGTGGCAGTA ACAACGAAGC CACAAACGAG TACTCTACCA 720
TCGTTGGTGG CGATGACAAC AAAGCCACAG GCAGATACTC TACCATCGGT GGTGGCGATA 780
ACAACACACG CGAAGGCGAA TACTCAACCG TCGCAGGGGG CAAGAATAAC CAAGCCACAG 840
GTACAGGTTC ATTTGCCGCA GGTGTAGAGA ACCAAGCCAA TGCCGAAAAC GCCGTCGCCG 900 TGGGTAAAAA GAACATTATC GAAGGTGAAA ACTCAGTAGC CATCGGCTCT GAGAATACCG 960
TTAAAACAGA ACACAAAAAT GTCTTTATTC TTGGCTCTGG CACAACAGGT GTAACGAGTA 1020
ACTCAGTGCT ACTGGGTAAT GAGACCGCTG GCAAACAGGC GACCACTGTT AAGAATGCCG 1080
AAGTGGGTGG TCTAAGCCTA ACAGGATTTG CAGGGGAGTC AAAAGCTGAA AACGGCGTAG 1140
TTTCTGTGGG TAGTGAAGGC GGTGAGCGTC AAATCGTTAA TGTTGGTGCA GGTCAGATCA 1200 GTGACACCTC AACAGATGCT GTTAATGGCT CACAGCTACA TGCTTTGGCC ACAGTTGTTG 1260
ATGACAACCA ATATGACATT GTTAACAACC GAGCTGACAT TCTTAACAAC CAAGATGATA 1320
TCAAAGATCT TCAGAAGGAG GTGAAAGGTC TTGATAATGA GGTGGGTGAA TTAAGCCGAG 1380
ACATTAATTC ACTTCATGAT GTTACTGACA ACCAACAAGA TGACATCAAA GAGCTTAAGA 1440
GGGGGGTAAA AGAGCTTGAT AATGAGGTGG GTGTATTAAG CCGAGACATT AATTCACTTC 1500 ATGATGATGT TGCTGACAAC CAAGATGACA TTGCTAAAAA CAAAGCTGAC ATCAAAGGTC 1560
TTAATAAGGA GGTGAAAGAG CTTGATAAGG AGGTGGGTGT ATTAAGCCGA GACATTGGTT 1620
CACTTCATGA TGATGTTGCC ACCAACCAAG CTGACATTGC TAAAAACCAA GCGGATATCA 1680
AAACACTTGA AAACAATGTC GAAGAAGAAT TATTAAATCT AAGCGGTCGC CTGCTTGATC 1740
AGAAAGCGGA TATTGATAAT AACATCAACA ATATCTATGA GCTGGCACAA CAGCAAGATC 1800 AGCATAGCTC TGATATCAAA ACACTTAAAA ACAATGTCGA AGAAGGTTTA TTGGATCTAA 1860
GCGGTCGCCT CATTGATCAA AAAGCAGATA TTGCTAAAAA CCAAGCTGAC ATTGCTCAAA 1920
ACCAAACAGA CATCCAAGAT CTGGCCGCTT ACAATGAGCT ACAAGACCAG TATGCTCAAA 1980
AGCAAACCGA AGCGATTGAC GCTCTAAATA AAGCAAGCTC TGAGAATACA CAAAACATTG 2040
CTAAAAACCA AGCGGATATT GCTAATAACA TCAACAATAT CTATGAGCTG GCACAACAGC 2100 AAGATCAGCA TAGCTCTGAT ATCAAAACCT TGGCAAAAGT AAGTGCTGCC AATACTGATC 2160
GTATTGCTAA AAACAAAGCT GAAGCTGATG CAAGTTTTGA AACGCTCACC AAAAATCAAA 2220
ATACTTTGAT TGAGCAAGGT GAAGCATTGG TTGAGCAAAA TAAAGCCATC AATCAAGAGC 2280
TTGAAGGGTT TGCGGCTCAT GCAGATATTC AAGATAAGCA AATTTTACAA AACCAAGCTG 2340
SUBSTTTUTE SHEET RULE 26) ATATCACTAC CAATAAGACC GCTATTGAAC AAAATATCAA TAGAACTGTT GCCAATGGGT 2400
TTGAGATTGA GAAAAATAAA GCTGGTATTG CTACCAATAA GCAAGAGCTT ATTCTTCAAA 2460
ATGATCGATT AAATCGAATT AATGAGACAA ATAATCGTCA GGATCAGAAG ATTGATCAAT 2520
TAGGTTATGC ACTAAAAGAG CAGGGTCAGC ATTTTAATAA TCGTATTAGT GCTGTTGAGC 2580
GTCAAACAGC TGGAGGTATT GCAAATGCTA TCGCAATTGC AACTTTACCA TCGCCCAGTA 2640
GAGCAGGTGA GCATCATGTC TTATTTGGTT CAGGTTATCA CAATGGTCAA GCTGCGGTAT 2700
CATTGGGCGC GGCTGGGTTA AGTGATACAG GAAAATCAAC TTATAAGATT GGTCTAAGCT 2760 GGTCAGATGC AGGTGGATTA TCTGGTGGTG TTGGTGGCAG TTACCGCTGG AAATAAAGCC 2820
TAAATTTAAC TGCTGTGTCA AAAAATATGG TCTGTATAAA CAGACCATAT TTTTATCCAA 2880
AAAAATTATC TTAACTTTTA TAAAGTATTA TAAGCCAAAG CTGTAATAAT AAGAGATGTT 2940
GAAATAAGAG ATGTTAAAGC TGCTAGACAA TCGGCTTGCG ACGATAAAAT AAGATACCTG 3000
GAATGGACAG CCCCAAAACC AATGCTGAGA TGATAAAAAT CGCCTCAAAA AAATGACGCA 3060 TCATAACGAT AAATAAATCC ATATCAAATC CAAAATAGCC AATTTGTACC ATGCTAACCA 3120
TGGCTTTATA GGCAGCGATT CCCGGCATCA TACAAATCAA GCTAGGTACA ATCAAGGCTT 3180
TAGGTGGCAG GCCATGACGC TGAGCAAAAT GTACACCCAA AAAGCTACCC GCCATCGCCC 3240
CAAAGAATGT TGCCACAACC AAATGCACAC CAAAAATTAC CATCACTTGT TTTAAACCAA 3300
AACCAAGTGG TGTTACCATC ATGCAATGCA TGATGTATTG CTTTGTCAA 3349
(2) INFORMATION FOR SEQ ID NO : 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 573 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Lys Leu Leu Pro Leu Lys He Ala Val Thr Ser Ala Met He Val 1 5 10 15
Gly Leu Gly Ala Thr Ser Thr Val Asn Ala Gin Val Val Glu Gin Phe 20 25 30
Phe Pro Asn He Phe Phe Asn Glu Asn His Asp Glu Leu Asp Asp Ala 35 40 45 Tyr His Asn Met He Leu Gly Asp Thr Ala He Val Ser Asn Ser Gin
50 55 60
SUBSTTTUTE SHEET RULE 25) Asp Asn Ser Thr Gin Leu Lys Phe Tyr Ser Asn Asp Glu Asp Ser Val 65 70 75 80
Pro Asp Ser Leu Leu Phe Ser Lys Leu Leu His Glu Gin Gin Leu Asn 85 90 95
Gly Phe Lys Ala Gly Asp Thr He He Pro Leu Asp Lys Asp Gly Lys 100 105 110 Pro Val Tyr Thr Lys Asp Thr Arg Thr Lys Asp Gly Lys Val Glu Thr
115 120 125
Val Tyr Ser Val Thr Thr Lys He Ala Thr Gin Asp Asp Val Glu Gin 130 135 140
Ser Ala Tyr Ser Arg Gly He Gin Gly Asp He Asp Asp Leu Tyr Asp 145 150 155 160
He Asn Arg Glu Val Asn Glu Tyr Leu Lys Ala Thr His Asp Tyr Asn 165 170 175
Glu Arg Gin Thr Glu Ala He Asp Ala Leu Asn Lys Ala Ser Ser Ala 180 185 190 Asn Thr Asp Arg He Asp Thr Ala Glu Glu Arg He Asp Lys Asn Glu
195 200 205
Tyr Asp He Lys Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu Glu 210 215 220
Leu Ser Gly His Leu He Asp Gin Lys Ala Asp Leu Thr Lys Asp He 225 230 235 240
Lys Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu Glu Leu Ser Gly 245 250 255
His Leu He Asp Gin Lys Ala Asp Leu Thr Lys Asp He Lys Ala Leu 260 265 270 Glu Ser Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu Leu
275 280 285
Asp Gin Lys Ala Asp He Ala Lys Asn Gin Ala Asp He Ala Gin Asn 290 295 300
Gin Thr Asp He Gin Asp Leu Ala Ala Tyr Asn Glu Leu Gin Asp Ala 305 310 315 320
Tyr Ala Lys Gin Gin Thr Glu Ala He Asp Ala Leu Asn Lys Ala Ser 325 330 335
Ser Glu Asn Thr Gin Asn He Ala Lys Asn Gin Ala Asp He Ala Asn 340 345 350 Asn He Asn Asn He Tyr Glu Leu Ala Gin Gin Gin Asp Gin His Ser
355 360 365
SUBSTTTUTE SHEET RULE 26) Ser Asp He Lys Thr Leu Ala Lys Ala Ser Ala Ala Asn Thr Asp Arg 370 375 380
He Ala Lys Asn Lys Ala Asp Ala Asp Ala Ser Phe Glu Thr Leu Thr 385 390 395 400
Lys Asn Gin Asn Thr Leu He Glu Lys Asp Lys Glu His Asp Lys Leu 405 410 415 He Thr Ala Asn Lys Thr Ala He Asp Ala Asn Lys Ala Ser Ala Asp
420 425 430
Thr Lys Phe Ala Ala Thr Ala Asp Ala He Thr Lys Asn Gly Asn Ala 435 440 445
He Thr Lys Asn Ala Lys Ser He Thr Asp Leu Gly Thr Lys Val Asp 450 455 460
Gly Phe Asp Gly Arg Val Thr Ala Leu Asp Thr Lys Val Asn Ala Leu 465 470 475 480
Asp Thr Lys Val Asn Ala Phe Asp Gly Arg He Thr Ala Leu Asp Ser 485 490 495 Lys Val Glu Asn Gly Met Ala Ala Gin Ala Ala Leu Ser Gly Leu Phe
500 505 510
Gin Pro Tyr Ser Val Gly Lys Phe Asn Ala Thr Ala Ala Leu Gly Gly 515 520 525
Tyr Gly Ser Lys Ser Ala Val Ala He Gly Ala Gly Tyr Arg Val Asn 530 535 540
Pro Asn Leu Ala Phe Lys Ala Gly Ala Ala He Asn Thr Ser Gly Asn 545 550 555 560
Lys Lys Gly Ser Tyr Asn He Gly Val Asn Tyr Glu Phe 565 570
(2) INFORMATION FOR SEQ ID NO : 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2596 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CTGGTGGTCG CAGGGGGCGT CTCTGCCAAT CAGTACACTA CGCCGCACCC TGACCGAAAC 60
GCTCCGCCAA ATCGATGCGT CGGTGTACCA TGCCCCGACC GAGCTATGCA CGGATAATGG 120 TGCGATGATC GCCTATGCTG GCTTTTGTCG GCTAATCCGT GGACAGTCGG ATGACTTGGT 180
GGTTCGCTGC ATTCCCCGAT GGGATATGAC GACGCTTGGC GTATCTGCTC ATAAATAGCC 240
SUBSTTTUTE SHEET RULE 25) ACATCAATCA TACCAACCAA ATCATACCAA CCAAATCGTA CAAACGGTTG ATACATGCCA 300
AAAATACCAT ATTGAAAGTA GGGTTTGGGT ATTATTTATG TAACTTATAT CTAATTTGGT 360
GTTGATACTT TGATAAAGCC TTGCTATACT GTAACCTAAA TGGATATGAT AGAGATTTTT 420
CCATTTATGC CAGCAAAAGA GATAGATAGA TAGATAGATA GATAGATAGA TAGATAGATA 480 GATAGATAGA TAGATAGATA AAACTCTGTC TTTTATCTGT CCGCTGATGC TTTCTGCCTG 540
CCACCGATGA TATCATTTAT CTGCTTTTTA GGCATCAGTT ATTTCACCGT GATGACTGAT 600
GTGATGACTT AACTACCAAA AGAGAGTGCT AAATGAAAAC CATGAAACTT CTCCCCCTAA 660
AAATCGCTGT AACCAGTGCC ATGATTGTTG GCTTGGGTGC GACATCTACT GTGAATGCAC 720
AAGTAGTGGA ACAGTTTTTT CCGAATATCT TTTTTAATGA AAACCATGAT GAATTAGATG 780 ATGCATACCA TAATATGATC TTAGGGGATA CTGCGATTGT ATCTAATTCA CAAGATAATA 840
GTACTCAATT GAAATTTTAT TCTAATGATG AAGATTCAGT TCCTGACAGC CTACTCTTTA 900
GTAAACTACT TCATGAGCAG CAACTTAATG GTTTTAAAGC AGGTGACACA ATCATTCCTT 960
TGGATAAGGA TGGCAAACCT GTTTATACAA AGGACACGAG AACAAAGGAT GGTAAAGTAG 1020
AAACAGTTTA TTCGGTCACC ACCAAAATCG CTACCCAAGA TGATGTTGAA CAAAGTGCAT 1080 ATTCACGAGG CATTCAAGGT GATATCGATG ATCTGTATGA CATTAACCGT GAAGTCAATG 1140
AATACTTAAA AGCAACACAT GATTATAATG AAAGACAAAC TGAAGCAATT GACGCTCTAA 1200
ACAAAGCAAG CTCTGCGAAT ACTGATCGTA TTGATACTGC TGAAGAGCGT ATCGATAAAA 1260
ACGAATATGA CATTAAAGCA CTTGAAAGCA ATGTCGAAGA AGGTTTGTTG GAGCTAAGCG 1320
GTCACCTCAT TGATCAAAAA GCAGATCTTA CAAAAGACAT CAAAGCACTT GAAAGCAATG 1380 TCGAAGAAGG TTTGTTGGAG CTAAGCGGTC ACCTCATTGA TCAAAAAGCA GATCTTACAA 1440
AAGACATCAA AGCACTTGAA AGCAATGTCG AAGAAGGTTT GTTGGATCTA AGCGGTCGTC 1500
TGCTTGATCA AAAAGCAGAT ATCGCTAAAA ACCAAGCTGA CATTGCTCAA AACCAAACAG 1560
ACATCCAAGA TCTAGCCGCT TACAACGAGC TACAAGATGC CTATGCCAAA CAGCAAACCG 1620
AAGCGATTGA CGCTCTAAAC AAAGCAAGCT CTGAGAATAC ACAAAACATT GCTAAAAACC 1680 AAGCGGATAT TGCTAATAAC ATCAACAATA TCTATGAGCT GGCACAACAG CAAGATCAGC 1740
ATAGCTCTGA TATCAAAACC TTGGCAAAAG CAAGTGCTGC CAATACTGAT CGTATTGCTA 1800
AAAACAAAGC CGATGCTGAT GCAAGTTTTG AAACGCTCAC CAAAAATCAA AATACTTTGA 1860
TTGAAAAAGA TAAAGAGCAT GACAAATTAA TTACTGCAAA CAAAACTGCG ATTGATGCCA 1920
SUBSTTTUTE 5HEET RULE 26) ATAAAGCATC TGCGGATACC AAGTTTGCAG CGACAGCAGA CGCCATTACC AAAAATGGAA 1980
ATGCTATCAC TAAAAACGCA AAATCTATCA CTGATTTGGG TACTAAAGTG GATGGTTTTG 2040
ACGGTCGTGT AACTGCATTA GACACCAAAG TCAATGCCTT AGACACCAAA GTCAATGCCT 2100
TTGATGGTCG TATCACAGCT TTAGACAGTA AAGTTGAAAA CGGTATGGCT GCCCAAGCTG 2160
CCCTAAGTGG TCTATTCCAG CCTTATAGCG TTGGTAAGTT TAATGCGACC GCTGCACTTG 2220
GTGGCTATGG CTCAAAATCT GCGGTTGCTA TCGGTGCTGG CTATCGTGTG AATCCAAATC 2280
TGGCGTTTAA AGCTGGTGCG GCGATTAATA CCAGTGGTAA TAAAAAAGGC TCTTATAACA 2340 TCGGTGTGAA TTACGAGTTT TAATTGTCTA TCATCACCAA AAAAAAGCAG TCAGTTTACT 2400
GGCTGCTTTT TTATGGGTTT TTGTGGCTTT TGGTTGTGAG TGATGGATAA AAGCTTATCA 2460
AGCGATTGAT GAATATCAAT AAATGATTGG TAAATATCAA TAAAGCGGTT TAGGGTTTTT 2520
GGATATCTTT TAATAAGTTT AAAAACCCCT GCATAAAATA AAGCTGGGCA TCAGAGCTGC 2580
GAGTAGCGGC ATACAG 2596
(2) INFORMATION FOR SEQ ID NO : 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 892 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Asn Lys He Tyr Lys Val Lys Lys Asn Ala Ala Gly His Leu Val 1 5 10 15
Ala Cys Ser Glu Phe Ala Lys Gly His Thr Lys Lys Ala Val Leu Gly 20 25 30
Ser Leu Leu He Val Gly Ala Leu Gly Met Ala Thr Thr Ala Ser Ala
35 40 45 Gin Ala Thr Lys Gly Thr Gly Lys His Val Val Asp Asn Lys Asp Asn
50 55 60
Lys Ala Lys Gly Asp Tyr Ser Thr Ala Ser Gly Gly Lys Asp Asn Glu 65 70 75 80
Ala Lys Gly Asn Tyr Ser Thr Val Gly Gly Gly Asp Tyr Asn Glu Ala 85 90 95
Lys Gly Asn Tyr Ser Thr Val Gly Gly Gly Ser Ser Asn Thr Ala Lys 100 105 110
SUBSTTTUTE SHEET RULE 26) Gly Glu Lys Ser Thr He Gly Gly Gly Asp Thr Asn Asp Ala Asn Gly 115 120 125
Thr Tyr Ser Thr He Gly Gly Gly Tyr Tyr Ser Arg Ala He Gly Asp 130 135 140
Ser Ser Thr He Gly Gly Gly Tyr Tyr Asn Gin Ala Thr Gly Glu Lys 145 150 155 160 Ser Thr Val Ala Gly Gly Arg Asn Asn Gin Ala Thr Gly Asn Asn Ser
165 170 175
Thr Val Ala Gly Gly Ser Tyr Asn Gin Ala Thr Gly Asn Asn Ser Thr 180 185 190
Val Ala Gly Gly Ser His Asn Gin Ala Thr Gly Glu Gly Ser Phe Ala 195 200 205
Ala Gly Val Glu Asn Lys Ala Asn Ala Asn Asn Ala Val Ala Leu Gly 210 215 220
Lys Asn Asn Thr He Asp Gly Asp Asn Ser Val Ala He Gly Ser Asn 225 230 235 240 Asn Thr He Asp Ser Gly Lys Gin Asn Val Phe He Leu Gly Ser Ser
245 250 255
Thr Asn Thr Thr Asn Ala Gin Ser Gly Ser Val Leu Leu Gly His Asn 260 265 270
Thr Ala Gly Lys Lys Ala Thr Ala Val Ser Ser Ala Lys Val Asn Gly 275 280 285
Leu Thr Leu Gly Asn Phe Ala Gly Ala Ser Lys Thr Gly Asn Gly Thr 290 295 300
Val Ser Val Gly Ser Glu Asn Asn Glu Arg Gin He Val Asn Val Gly 305 310 315 320 Ala Gly Asn He Ser Ala Asp Ser Thr Asp Ala Val Asn Gly Ser Gin
325 330 335
Leu Tyr Ala Leu Ala Thr Ala Val Lys Ala Asp Ala Asp Glu Asn Phe 340 345 350
Lys Ala Leu Thr Lys Thr Gin Asn Thr Leu He Glu Gin Gly Glu Ala 355 360 365
Gin Asp Ala Leu He Ala Gin Asn Gin Thr Asp He Thr Ala Asn Lys 370 375 380
Thr Ala He Glu Arg Asn Phe Asn Arg Thr Val Val Asn Gly Phe Glu 385 390 395 400 He Glu Lys Asn Lys Ala Gly He Ala Lys Asn Gin Ala Asp He Gin
405 410 415
SUBSTTTUTE SHEET RULE 26) Thr Leu Glu Asn Asn Val Gly Glu Glu Leu Leu Asn Leu Ser Gly Arg 420 425 430
Leu Leu Asp Gin Lys Ala Asp He Asp Asn Asn He Asn Asn He Tyr 435 440 445
Asp Leu Ala Gin Gin Gin Asp Gin His Ser Ser Asp He Lys Thr Leu 450 455 460 Lys Lys Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu He
465 470 475 480
Asp Gin Lys Ala Asp Leu Thr Lys Asp He Lys Thr Leu Glu Asn Asn
485 490 495
Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu He Asp Gin Lys 500 505 510
Ala Asp He Ala Lys Asn Gin Ala Asp He Ala Gin Asn Gin Thr Asp 515 520 525
He Gin Asp Leu Ala Ala Tyr Asn Glu Leu Gin Asp Gin Tyr Ala Gin 530 535 540 Lys Gin Thr Glu Ala He Asp Ala Leu Asn Lys Ala Ser Ser Ala Asn
545 550 555 560
Thr Asp Arg He Ala Thr Ala Glu Leu Gly He Ala Glu Asn Lys Lys 565 570 575
Asp Ala Gin He Ala Lys Ala Gin Ala Asn Glu Asn Lys Asp Gly He 580 585 590
Ala Lys Asn Gin Ala Asp He Gin Leu His Asp Lys Lys He Thr Asn 595 600 605
Leu Gly He Leu His Ser Met Val Ala Arg Ala Val Gly Asn Asn Thr
610 615 620 Gin Gly Val Ala Thr Asn Lys Ala Asp He Ala Lys Asn Gin Ala Asp
625 630 635 640
He Ala Asn Asn He Lys Asn He Tyr Glu Leu Ala Gin Gin Gin Asp 645 650 655
Gin His Ser Ser Asp He Lys Thr Leu Ala Lys Val Ser Ala Ala Asn 660 665 670
Thr Asp Arg He Ala Lys Asn Lys Ala Glu Ala Asp Ala Ser Phe Glu 675 680 685
Thr Leu Thr Lys Asn Gin Asn Thr Leu He Glu Gin Gly Glu Ala Leu
690 695 700 Val Glu Gin Asn Lys Ala He Asn Gin Glu Leu Glu Gly Phe Ala Ala
705 710 715 720
SUBSTT ET RULE 25 His Ala Asp Val Gin Asp Lys Gin He Leu Gin Asn Gin Ala Asp He 725 730 735
Thr Thr Asn Lys Ala Ala He Glu Gin Asn He Asn Arg Thr Val Ala 740 745 750
Asn Gly Phe Glu He Glu Lys Asn Lys Ala Gly He Ala Thr Asn Lys 755 760 765 Gin Glu Leu He Leu Gin Asn Asp Arg Leu Asn Gin He Asn Glu Thr
770 775 780
Asn Asn Arg Gin Asp Gin Lys He Asp Gin Leu Gly Tyr Ala Leu Lys
785 790 795 800
Glu Gin Gly Gin His Phe Asn Asn Arg He Ser Ala Val Glu Arg Gin
805 810 815
Thr Ala Gly Gly He Ala Asn Ala He Ala He Ala Thr Leu Pro Ser 820 825 830
Pro Ser Arg Ala Gly Glu His His Val Leu Phe Gly Ser Gly Tyr His 835 840 845 Asn Gly Gin Ala Ala Val Ser Leu Gly Ala Ala Gly Leu Ser Asp Thr
850 855 860
Gly Lys Ser Thr Tyr Lys He Gly Leu Ser Trp Ser Asp Ala Gly Gly 865 870 875 880
Leu Ser Gly Gly Val Gly Gly Ser Tyr Arg Trp Lys 885 890
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3381 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6: TGTGAGCAAA TGACTGGCGT AAATGACTGA TGAATGTCTA TTTAATGAAA GATATCAATA 60
TATAAAAGTT GACTATAGCG ATGCAATACA GTAAAATTTG TTACGGCTAA ACATAACGAC 120
GGTCCAAGAT GGCGGATATC GCCATTTACC AACCTGATAA TCAGTTTGAT AGCCATTAGC 180
GATGGCATCA AGTTGTGTTG TTGTATTGTC ATATAAACGG TAAATTTGGT TTGGTGGATG 240
CCCCATCTGA TTTACCGTCC CCCTAATAAG TGAGGGGGGG GGAGACCCCA GTCATTTATT 300 AGGAGACTAA GATGAACAAA ATTTATAAAG TGAAAAAAAA TGCCGCAGGT CACTTGGTGG 360
CATGTTCTGA ATTTGCCAAA GGCCATACCA AAAAGGCAGT TTTGGGCAGT TTATTGATTG 420
SUBSTT RULE 26 TTGGGGCATT GGGCATGGCA ACGACGGCGT CTGCACAAGC AACCAAAGGC ACAGGCAAGC 480
ACGTTGTTGA CAATAAGGAC AACAAAGCCA AAGGCGATTA CTCTACCGCC AGTGGTGGCA 540
AGGACAACGA AGCCAAAGGC AATTACTCTA CCGTCGGTGG TGGCGATTAT AACGAAGCCA 600
AAGGCAATTA CTCTACCGTC GGTGGTGGCT CTAGTAATAC CGCCAAAGGC GAGAAATCAA 660 CCATCGGTGG TGGCGATACT AACGACGCCA ACGGCACATA CTCTACCATC GGTGGTGGCT 720
ATTATAGCCG AGCCATAGGC GATAGCTCTA CCATCGGTGG TGGTTATTAT AACCAAGCCA 780
CAGGCGAGAA ATCAACGGTT GCAGGGGGCA GGAATAACCA AGCCACAGGC AACAACTCAA 840
CGGTTGCAGG CGGCTCTTAT AACCAAGCCA CAGGCAACAA CTCAACGGTT GCAGGTGGCT 900
CTCATAACCA AGCCACAGGT GAAGGTTCAT TTGCAGCAGG TGTAGAGAAC AAAGCCAATG 960 CCAACAACGC CGTCGCTCTA GGTAAAAATA ACACCATCGA TGGCGATAAC TCAGTAGCCA 1020
TCGGCTCTAA TAATACCATT GACAGTGGCA AACAAAATGT CTTTATTCTT GGCTCTAGCA 1080
CAAACACAAC AAATGCACAA AGCGGCTCCG TGCTGCTGGG TCATAATACC GCTGGCAAAA 1140
AAGCAACCGC TGTTAGCAGT GCCAAAGTGA ACGGCTTAAC CCTAGGAAAT TTTGCAGGTG 1200
CATCAAAAAC TGGTAATGGT ACTGTATCTG TCGGTAGTGA GAATAATGAG CGTCAAATCG 1260 TCAATGTTGG TGCAGGTAAT ATCAGTGCTG ATTCAACAGA TGCTGTTAAT GGCTCACAGC 1320
TATATGCTTT GGCCACAGCT GTCAAAGCCG ATGCCGATGA AAACTTTAAA GCACTCACCA 1380
AAACTCAAAA TACTTTGATT GAGCAAGGTG AAGCACAAGA CGCATTAATC GCTCAAAATC 1440
AAACTGACAT CACTGCCAAT AAAACTGCCA TTGAGCGAAA TTTTAATAGA ACTGTTGTCA 1500
ATGGGTTTGA GATTGAGAAA AATAAAGCTG GTATTGCTAA AAACCAAGCG GATATCCAAA 1560 CGCTTGAAAA CAATGTCGGA GAAGAACTAT TAAATCTAAG CGGTCGCCTG CTTGATCAAA 1620
AAGCGGATAT TGATAATAAC ATCAACAATA TCTATGATCT GGCACAACAG CAAGATCAGC 1680
ATAGCTCTGA TATCAAAACA CTTAAAAAAA ATGTCGAAGA AGGTTTGTTG GATCTAAGTG 1740
GTCGCCTCAT TGATCAAAAA GCAGATCTTA CGAAAGACAT CAAAACACTT GAAAACAATG 1800
TCGAAGAAGG TTTGTTGGAT CTAAGCGGTC GCCTCATTGA TCAAAAAGCA GATATTGCTA 1860 AAAACCAAGC TGACATTGCT CAAAACCAAA CAGACATCCA AGATCTGGCC GCTTACAACG 1920
AGCTACAAGA CCAGTATGCT CAAAAGCAAA CCGAAGCGAT TGACGCTCTA AATAAAGCAA 1980
GCTCTGCCAA TACTGATCGT ATTGCTACTG CTGAATTGGG TATCGCTGAG AACAAAAAAG 2040
ACGCTCAGAT CGCCAAAGCA CAAGCCAATG AAAATAAAGA CGGCATTGCT AAAAACCAAG 2100
SUBSTTT E 26 CTGATATCCA GTTGCACGAT AAAAAAATCA CCAATCTAGG TATCCTTCAC AGCATGGTTG 2160
CAAGAGCGGT AGGAAATAAC ACACAAGGTG TTGCTACCAA TAAAGCTGAC ATTGCTAAAA 2220
ACCAAGCAGA TATTGCTAAT AACATCAAAA ATATCTATGA GCTGGCACAA CAGCAAGATC 2280
AGCATAGCTC TGATATCAAA ACCTTGGCAA AAGTAAGTGC TGCCAATACT GATCGTATTG 2340
CTAAAAACAA AGCTGAAGCT GATGCAAGTT TTGAAACGCT CACCAAAAAT CAAAATACTT 2400
TGATTGAGCA AGGTGAAGCA TTGGTTGAGC AAAATAAAGC CATCAATCAA GAGCTTGAAG 2460
GGTTTGCGGC TCATGCAGAT GTTCAAGATA AGCAAATTTT ACAAAACCAA GCTGATATCA 2520 CTACCAATAA GGCCGCTATT GAACAAAATA TCAATAGAAC TGTTGCCAAT GGGTTTGAGA 2580
TTGAGAAAAA TAAAGCTGGT ATTGCTACCA ATAAGCAAGA GCTTATTCTT CAAAATGATC 2640
GATTAAATCA AATTAATGAG ACAAATAATC GTCAGGATCA GAAGATTGAT CAATTAGGTT 2700
ATGCACTAAA AGAGCAGGGT CAGCATTTTA ATAATCGTAT TAGTGCTGTT GAGCGTCAAA 2760
CAGCTGGAGG TATTGCAAAT GCTATCGCAA TTGCAACTTT ACCATCGCCC AGTAGAGCAG 2820 GTGAGCATCA TGTCTTATTT GGTTCAGGTT ATCACAATGG TCAAGCTGCG GTATCATTGG 2880
GTGCGGCTGG GTTAAGTGAT ACAGGAAAAT CAACTTATAA GATTGGTCTA AGCTGGTCAG 2940
ATGCAGGTGG ATTATCTGGT GGTGTTGGTG GCAGTTACCG CTGGAAATAG AGCCTAAATT 3000
TAACTGCTGT ATCAAAAAAT ATGGTCTGTA TAAACAGACC ATATTTTTAT CTAAAAACTT 3060
ATCTTAACTT TTATGAAGCA TCATAAGCCA AAGCTGAGTA ATAATAAGAG ATGTTAAAAT 3120 AAGAGATGTT AAAACTGCTA AACAATCGGC TTACGACGAT AAAATAAAAT ACCTGGAATG 3180
GACAGCCCCA AAACCAATGC TGAGATGATA AAAATCGCCT CAAAAAAATG ACGCATCATA 3240
ACGATAAATA AATCCATATC AAATCCAAAA TAGCCAATTT GTACCATGCT AACCATGGCT 3300
TTATAGGCAG CGATTCCCGG CATCATACAA ATCAAGCTAG GTACAATCAA GGCTTTAGGC 3360
GGCAGGCCAT GACGCTGAGC A 3381
(2) INFORMATION FOR SEQ ID NO : 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 624 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7:
Val Asn Lys He Tyr Lys Val Lys Lys Asn Ala Ala Gly His Ser Val 1 5 10 15
SUBSTTTUTE SHEET RULE 25) Ala Cys Ser Glu Phe Ala Lys Gly His Thr Lys Lys Ala Val Leu Gly 20 25 30
Ser Leu Leu He Val Gly Ala Leu Gly Met Ala Thr Thr Ala Ser Ala 35 40 45
Gin Thr Gly Ser Thr Asn Ala Ala Asn Gly Asn He He Ser Gly Val 50 55 60
Gly Ala Tyr Val Gly Gly Gly Val He Asn Gin Ala Lys Gly Asn Tyr 65 70 75 80
Pro Thr Val Gly Gly Gly Phe Asp Asn Arg Ala Thr Gly Asn Tyr Ser 85 90 95
Val He Ser Gly Gly Phe Asp Asn Gin Ala Lys Gly Glu His Ser Thr 100 105 110
He Ala Gly Gly Glu Ser Asn Gin Ala Thr Gly Arg Asn Ser Thr Val 115 120 125
Ala Gly Gly Ser Asn Asn Gin Ala Val Gly Thr Asn Ser Thr Val Ala 130 135 140
Gly Gly Ser Asn Asn Gin Ala Lys Gly Ala Asn Ser Phe Ala Ala Gly 145 150 155 160
Val Gly Asn Gin Ala Asn Thr Asp Asn Ala Val Ala Leu Gly Lys Asn 165 170 175
Asn Thr He Asn Gly Asn Asn Ser Ala Ala He Gly Ser Glu Asn Thr 180 185 190 Val Asn Glu Asn Gin Lys Asn Val Phe He Leu Gly Ser Asn Thr Thr
195 200 205
Asn Ala Gin Ser Gly Ser Val Leu Leu Gly His Glu Thr Ser Gly Lys 210 215 220
Glu Ala Thr Ala Val Ser Arg Ala Arg Val Asn Gly Leu Thr Leu Lys 225 230 235 240
Asn Phe Ser Gly Val Ser Lys Ala Asp Asn Gly Thr Val Ser Val Gly 245 250 255
Ser Gin Gly Lys Glu Arg Gin He Val His Val Gly Ala Gly Gin He 260 265 270 Ser Asp Asp Ser Thr Asp Ala Val Asn Gly Ser Gin Leu Tyr Ala Leu
275 280 285
Ala Thr Ala Val Asp Asp Asn Gin Tyr Asp He Glu He Asn Gin Asp
290 295 300
Asn He Lys Asp Leu Gin Lys Glu Val Lys Gly Leu Asp Lys Glu Val
305 310 315 320
SUBSTTTUTE SHEET (RULE 26) Gly Val Leu Ser Arg Asp He Gly Ser Leu His Asp Asp Val Ala Asp
325 330 335
Asn Gin Ala Asp He Ala Lys Asn Lys Ala Asp He Lys Glu Leu Asp
340 345 350
Lys Glu Met Asn Val Leu Ser Arg Asp He Val Ser Leu Asn Asp Asp
355 360 365
Val Ala Asp Asn Gin Ala Asp He Ala Lys Asn Gin Ala Asp He Lys 370 375 380
Thr Leu Glu Asn Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg 385 390 395 400
Leu He Asp Gin Lys Ala Asp He Asp Asn Asn He Asn His He Tyr 405 410 415 Glu Leu Ala Gin Gin Gin Asp Gin His Ser Ser Asp He Lys Thr Leu
420 425 430
Ala Lys Ala Ser Ala Ala Asn Thr Asp Arg He Ala Lys Asn Lys Ala 435 440 445
Asp Ala Asp Ala Ser Phe Glu Thr Leu Thr Lys Asn Gin Asn Thr Leu 450 455 460
He Glu Lys Asp Lys Glu His Asp Lys Leu He Thr Ala Asn Lys Thr 465 470 475 480
Ala He Asp Ala Asn Lys Ala Ser Ala Asp Thr Lys Phe Ala Ala Thr 485 490 495 Ala Asp Ala He Thr Lys Asn Gly Asn Ala He Thr Lys Asn Ala Lys
500 505 510
Ser He Thr Asp Leu Gly Thr Lys Val Asp Gly Phe Asp Gly Arg Val 515 520 525
Thr Ala Leu Asp Thr Lys Val Asn Ala Phe Asp Gly Arg He Thr Ala 530 535 540
Leu Asp Ser Lys Val Glu Asn Gly Met Ala Ala Gin Ala Ala Leu Ser 545 550 555 560
Gly Leu Phe Gin Pro Tyr Ser Val Gly Lys Phe Asn Ala Thr Ala Ala 565 570 575 Leu Gly Gly Tyr Gly Ser Lys Ser Ala Val Ala He Gly Ala Gly Tyr
580 585 590
Arg Val Asn Pro Asn Leu Ala Phe Lys Ala Gly Ala Ala He Asn Thr 595 600 605
Ser Gly Asn Lys Lys Gly Ser Tyr Asn He Gly Val Asn Tyr Glu Phe 610 615 620
SUBSTTTUTE SHEET (RULE 25) (2) INFORMATION FOR SEQ ID NO : 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3295 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8:
GCCGCACCTG ACCGAGACGC TCCGCCAAAT CAATGCGTCG GTGTACTATG CCCCGACCGA 60 GCTATGCACG GATAATGGTG CGATGATCGC CTATGCTGGC TTTTGTCGGC TAAGCCGTGG 120
ACAGTCGGAT GACTTGGCGG TTCGCTGCAT TCCCCGATGG GATATGACAA CGCTTGGTAT 180
CGAATATGAT AATTAGGCTG TGGTATTTGA GTTTTGAGTA ATGTACCTAC TACCACTAAT 240
TTATCATACA ATACATAAAC ATAAAAAACA TCGGTATTGT TAAAAAACAA TACCCAAGTT 300
AAAATAGCTC AATACTTTAC CATAGCACAA AGAAACTTGT GAACGAAACA TTTAATAATT 360 GCCCAAAATG TCACTGCACA CACTTTGTAA AAGCAGGTTT GGGCAATGGC AAACAACGAT 420
ACAAATGCAA AGGTTACCAT CACTATTTTT CTGTGAAGCA ACGAAGCAAC CAAAAAAGTA 480
ATGACATTAA AAAAACAAGC CATTGATACA AACAGTAAAC AAATCTTAGG CTTTGTCTGT 540
GGTAAAACAG ACACTAACAC CTTTAAACGA CTTTATCAGC AGTTAAATAC CCATAACATT 600
CAACTGTTTT TTAGTGACTA CTGGAAATCT TATCGTCAAG TCATTTTAAA GCCAAAACAT 660 ATAACAAGCA AAGCTCAAAC TTTTACCATA GAGGACTATA ATAGTCTCAT TGGGCATTTC 720
ATAGCAAGAT TTACAAGAAA GTCAAAGTAT TATTCTAAAT CCGAAAAAAT GATAGAAAAC 780
ACGTTGAATT TATTATTTGC TAAGTGGAAT GGTAGCTTAA GATATGTATT TTAATTTAAC 840
AATGCCAAAA ACATCAATTA CAGTAAGATT TTAGGCGTTT TGCAGTTGCT ACTTTAGTAA 900
AGCTTTGTTA TACTAGCTGT TAATATACTC AAGCTTGTTT GTGTTTGAGC TATGTTTATT 960 TTATAGCAGT AGTTGGTTAT AAAATATAAA TAAAGCTAAG CTCGAGGGTT TGGTAATGGT 1020
TTTTTATGTT TATAATACCA ACAGAGTATC TATACAGCTA AAATAGCTAA TACCTTAGGT 1080
GTATTACAAG TAAAAATCCT TTGTTAATCA GGGAGTGTAT TATATGTATA TTTCCTTTGT 1140
ATTTGGTTAT AGCAATCCCT TGGTAAGAAA TCATATCTAT TTTTTATTGT TCAATTATTC 1200
AGGAGACTAA GGTGAACAAA ATTTATAAAG TGAAAAAAAA TGCCGCAGGT CATTCGGTGG 1260 CATGTTCTGA ATTTGCCAAA GGCCATACCA AAAAGGCAGT TTTGGGCAGT TTATTGATTG 1320
TTGGGGCATT GGGCATGGCA ACGACAGCGT CTGCACAAAC AGGCAGTACA AATGCAGCCA 1380
SUBSTTTUTE SHEET (RULE 26) ACGGCAATAT AATCAGCGGC GTAGGCGCGT ACGTCGGTGG TGGCGTTATA AACCAAGCCA 1440
AAGGCAATTA CCCTACCGTC GGTGGTGGCT TTGATAACCG AGCCACAGGC AATTACTCTG 1500
TCATCAGTGG TGGCTTTGAT AACCAAGCCA AAGGCGAGCA CTCTACCATC GCAGGGGGTG 1560
AGAGTAACCA AGCTACAGGT CGTAACTCAA CGGTTGCAGG GGGTTCTAAT AACCAAGCCG 1620 TGGGTACAAA CTCAACGGTT GCAGGGGGTT CTAATAACCA AGCCAAAGGT GCAAATTCAT 1680
TTGCAGCAGG TGTAGGTAAC CAAGCCAATA CCGACAACGC CGTCGCTCTA GGTAAAAATA 1740
ACACCATCAA TGGCAATAAC TCAGCAGCCA TCGGCTCTGA GAATACCGTT AACGAAAATC 1800
AAAAAAATGT CTTTATTCTT GGCTCTAACA CAACAAATGC ACAAAGCGGC TCAGTACTGC 1860
TAGGTCATGA AACCTCTGGT AAAGAAGCGA CCGCTGTTAG CAGAGCCAGA GTGAACGGCT 1920 TAACCCTAAA AAATTTTTCA GGCGTATCAA AAGCTGATAA TGGTACTGTA TCTGTCGGTA 1980
GTCAGGGTAA AGAGCGTCAA ATCGTTCATG TTGGTGCAGG TCAGATCAGT GATGATTCAA 2040
CAGATGCTGT TAATGGCTCA CAGCTATATG CTTTGGCTAC AGCTGTTGAT GACAACCAAT 2100
ATGACATTGA AATAAACCAA GATAATATCA AAGATCTTCA GAAGGAGGTG AAAGGTCTTG 2160
ATAAGGAAGT GGGTGTATTA AGCCGAGACA TTGGTTCACT TCATGATGAT GTTGCTGACA 2220 ACCAAGCTGA TATTGCTAAA AACAAAGCTG ACATCAAAGA GCTTGATAAG GAGATGAATG 2280
TATTAAGCCG AGACATTGTC TCACTTAATG ATGATGTTGC TGATAACCAA GCTGACATTG 2340
CTAAAAACCA AGCGGATATC AAAACACTTG AAAACAATGT CGAAGAAGGT TTATTGGATC 2400
TAAGCGGTCG CCTCATTGAT CAAAAAGCAG ATATTGATAA TAACATCAAC CATATCTATG 2460
AGCTGGCACA ACAGCAAGAT CAGCATAGCT CTGATATCAA AACCTTGGCA AAAGCAAGTG 2520 CTGCCAATAC TGATCGTATT GCTAAAAACA AAGCCGATGC TGATGCAAGT TTTGAAACAC 2580
TCACCAAAAA TCAAAATACT TTGATTGAAA AAGATAAAGA GCATGACAAA TTAATTACTG 2640
CAAACAAAAC TGCGATTGAT GCCAATAAAG CATCTGCGGA TACCAAGTTT GCAGCGACAG 2700
CAGACGCCAT TACCAAAAAT GGAAATGCTA TCACTAAAAA CGCAAAATCT ATCACTGATT 2760
TGGGTACTAA AGTGGATGGT TTTGACGGTC GTGTAACTGC ATTAGACACC AAAGTCAATG 2820 CCTTTGATGG TCGCATCACA GCTTTAGACA GTAAAGTTGA AAACGGTATG GCTGCCCAAG 2880
CTGCCCTAAG TGGTCTATTC CAGCCTTATA GCGTTGGTAA GTTTAATGCG ACCGCTGCAC 2940
TTGGTGGCTA TGGCTCAAAA TCTGCGGTTG CTATCGGTGC TGGCTATCGT GTGAATCCAA 3000
ATCTGGCGTT TAAAGCTGGT GCGGCGATTA ATACCAGTGG CAATAAAAAA GGCTCTTATA 3060
SUBSTTTUTE SHEET (RULE 26) ACATCGGTGT GAATTACGAG TTCTAATTGT CTATCATCAC CAAAAAAAGC AGTCAGTTTA 3120
CTGGCTGCTT TTTTATGGGT TTTTATGGCT TTTGGTTGTG AGTGATGGAT AAAAGCTTAT 3180 CAAGCGATTG ATGAATATCA ATAAATGATT GGTAAATATC AATAAAGCGG TTTAGGGTTT 3240
TTGGATATCT TTTAATAAGT TTAAAAACCC CTGCATAAAA TAAAGCTGGC ATCAG 3295
(2) INFORMATION FOR SEQ ID NO : 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 941 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9: Met Asn Lys He Tyr Lys Val Lys Lys Asn Ala Ala Gly His Leu Val
1 5 10 15
Ala Cys Ser Glu Phe Ala Lys Gly His Thr Lys Lys Ala Val Leu Gly 20 25 30
Ser Leu Leu He Val Gly He Leu Gly Met Ala Thr Thr Ala Ser Ala 35 40 45
Gin Met Ala Thr Thr Pro Ser Ala Gin Val Val Lys Thr Asn Asn Lys 50 55 60
Lys Asn Gly Thr His Pro Phe He Gly Gly Gly Asp Tyr Asn Thr Thr 65 70 75 80 Lys Gly Asn Tyr Pro Thr He Gly Gly Gly His Phe Asn Thr Ala Glu
85 90 95
Gly Asn Tyr Ser Thr Val Gly Gly Gly Phe Thr Asn Glu Ala He Gly 100 105 110
Lys Asn Ser Thr Val Gly Gly Gly Phe Thr Asn Glu Ala Met Gly Glu 115 120 125
Tyr Ser Thr Val Ala Gly Gly Ala Asn Asn Gin Ala Lys Gly Asn Tyr 130 135 140
Ser Thr Val Gly Gly Gly Asn Gly Asn Lys Ala He Gly Asn Asn Ser 145 150 155 160 Thr Val Val Gly Gly Ser Asn Asn Gin Ala Lys Gly Glu His Ser Thr
165 170 175
He Ala Gly Gly Lys Asn Asn Gin Ala Thr Gly Asn Gly Ser Phe Ala 180 185 190
Ala Gly Val Glu Asn Lys Ala Asp Ala Asn Asn Ala Val Ala Leu Gly 195 200 205
SUBSTTTUTE SHEET (RULE 26) Asn Lys Asn Thr He Glu Gly Thr Asn Ser Val Ala He Gly Ser Asn 210 215 220 Asn Thr Val Lys Thr Gly Lys Glu Asn Val Phe He Leu Gly Ser Asn
225 230 235 240
Thr Asn Thr Glu Asn Ala Gin Ser Gly Ser Val Leu Leu Gly Asn Asn 245 250 255
Thr Ala Gly Lys Ala Ala Thr Thr Val Asn Asn Ala Glu Val Asn Gly 260 265 270
Leu Thr Leu Glu Asn Phe Ala Gly Ala Ser Lys Ala Asn Ala Asn Asn 275 280 285
He Gly Thr Val Ser Val Gly Ser Glu Asn Asn Glu Arg Gin He Val 290 295 300 Asn Val Gly Ala Gly Gin He Ser Ala Thr Ser Thr Asp Ala Val Asn
305 310 315 320
Gly Ser Gin Leu His Ala Leu Ala Lys Ala Val Ala Lys Asn Lys Ser 325 330 335
Asp He Lys Gly Leu Asn Lys Gly Val Lys Glu Leu Asp Lys Glu Val 340 345 350
Gly Val Leu Ser Arg Asp He Asn Ser Leu His Asp Asp Val Ala Asp 355 360 365
Asn Gin Asp Ser He Ala Lys Asn Lys Ala Asp He Lys Gly Leu Asn 370 375 380 Lys Glu Val Lys Glu Leu Asp Lys Glu Val Gly Val Leu Ser Arg Asp
385 390 395 400
He Gly Ser Leu His Asp Asp Val Ala Asp Asn Gin Asp Ser He Ala 405 410 415
Lys Asn Lys Ala Asp He Lys Gly Leu Asn Lys Glu Val Lys Glu Leu 420 425 430
Asp Lys Glu Val Gly Val Leu Ser Arg Asp He Gly Ser Leu His Asp 435 440 445
Asp Val Ala Thr Asn Gin Ala Asp He Ala Lys Asn Gin Ala Asp He 450 455 460 Lys Thr Leu Glu Asn Asn Val Glu Glu Glu Leu Leu Asn Leu Ser Gly
465 470 475 480
Arg Leu He Asp Gin Lys Ala Asp He Asp Asn Asn He Asn Asn He 485 490 495
Tyr Glu Leu Ala Gin Gin Gin Asp Gin His Ser Ser Asp He Lys Thr 500 505 510
SUBSTTTUTE SHEET (RULE 25) Leu Lys Asn Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu 515 520 525
He Asp Gin Lys Ala Asp Leu Thr Lys Asp He Lys Thr Leu Lys Asn 530 535 540
Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu He Asp Gin 545 550 555 560
Lys Ala Asp He Ala Lys Asn Gin Ala Asp He Ala Gin Asn Gin Thr 565 570 575
Asp He Gin Asp Leu Ala Ala Tyr Asn Glu Leu Gin Asp Gin Tyr Ala 580 585 590
Gin Lys Gin Thr Glu Ala He Asp Ala Leu Asn Lys Ala Ser Ser Ala 595 600 605
Asn Thr Asp Arg He Ala Thr Ala Glu Leu Gly He Ala Glu Asn Lys 610 615 620
Lys Asp Ala Gin He Ala Lys Ala Gin Ala Asn Glu Asn Lys Asp Gly 625 630 635 640
He Ala Lys Asn Gin Ala Asp He Gin Leu His Asp Lys Lys He Thr 645 650 655
Asn Leu Gly He Leu His Ser Met Val Ala Arg Ala Val Gly Asn Asn 660 665 670
Thr Gin Gly Val Ala Thr Asn Lys Ala Asp He Ala Lys Asn Gin Ala 675 680 685 Asp He Ala Asn Asn He Lys Asn He Tyr Glu Leu Ala Gin Gin Gin
690 695 700
Asp Gin His Ser Ser Asp He Lys Thr Leu Ala Lys Val Ser Ala Ala 705 710 715 720
Asn Thr Asp Arg He Ala Lys Asn Lys Ala Glu Ala Asp Ala Ser Phe 725 730 735
Glu Thr Leu Thr Lys Asn Gin Asn Thr Leu He Glu Gin Gly Glu Ala 740 745 750
Leu Val Glu Gin Asn Lys Ala He Asn Gin Glu Leu Glu Gly Phe Ala 755 760 765 Ala His Ala Asp Val Gin Asp Lys Gin He Leu Gin Asn Gin Ala Asp 770 775 780
He Thr Thr Asn Lys Thr Ala He Glu Gin Asn He Asn Arg Thr Val 785 790 795 800
Ala Asn Gly Phe Glu He Glu Lys Asn Lys Ala Gly He Ala Thr Asn 805 810 815
SUBSTTTUTE SHEET (RULE 26) Lys Gin Glu Leu He Leu Gin Asn Asp Arg Leu Asn Gin He Asn Glu 820 825 830
Thr Asn Asn His Gin Asp Gin Lys He Asp Gin Leu Gly Tyr Ala Leu 835 840 845
Lys Glu Gin Gly Gin His Phe Asn Asn Arg He Ser Ala Val Glu Arg 850 855 860
Gin Thr Ala Gly Gly He Ala Asn Ala He Ala He Ala Thr Leu Pro 865 870 875 880
Ser Pro Ser Arg Ala Gly Glu His His Val Leu Phe Gly Ser Gly Tyr 885 890 895
His Asn Gly Gin Ala Ala Val Ser Leu Gly Ala Ala Gly Leu Ser Asp 900 905 910
Thr Gly Lys Ser Thr Tyr Lys He Gly Leu Ser Trp Ser Asp Ala Gly 915 920 925
Gly Leu Ser Gly Gly Val Gly Gly Ser Tyr Arg Trp Lys 930 935 940
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3538 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
TTCTGTGAGC AAATGACTGG CGTAAATGAC TGATGAGTGT CTATTTAATG AAAGATATCA 60
ATATATAAAA GTTGACTATA GCGATGCAAT ACAGTAAAAT TTGTTACGGC TAAACATAAC 120
GACGGTCCAA GATGGCGGAT ATCGCCATTT ACCAACCTGA TAATCAGTTT GATAGCCATT 180
AGCGATGGCA TCAAGTTGTG TTGTTGTATT GTCATATAAA CGGTAAATTT GGTTTGGTGG 240 ATGCCCCATC TGATTTACCG TCCCCCTAAT AAGTGAGGGG GGGGGGGAGA CCCCAGTCAT 300
TTATTAGGAG ACTAAGATGA ACAAAATTTA TAAAGTGAAA AAAAATGCCG CAGGTCACTT 360
GGTGGCGTGT TCTGAATTTG CCAAAGGTCA TACCAAAAAG GCAGTTTTGG GCAGTTTATT 420
GATTGTTGGA ATATTGGGTA TGGCAACGAC AGCATCTGCA CAAATGGCAA CGACGCCGTC 480
TGCACAAGTA GTCAAGACAA ACAATAAAAA AAACGGCACG CACCCTTTCA TCGGTGGTGG 540 CGATTATAAT ACCACCAAAG GCAATTACCC TACCATCGGT GGTGGCCATT TTAATACCGC 600
CGAAGGCAAT TACTCTACCG TCGGTGGTGG CTTTACTAAC GAAGCCATAG GCAAGAACTC 660
SUBSTTTUTE SHEET (RULE 26) TACCGTCGGT GGTGGCTTTA CTAACGAAGC CATGGGCGAA TACTCAACCG TCGCAGGCGG 720
TGCTAACAAC CAAGCCAAAG GCAATTACTC TACCGTCGGT GGTGGCAATG GCAACAAAGC 780
CATAGGCAAC AACTCAACGG TTGTAGGTGG TTCTAACAAC CAAGCCAAAG GCGAGCACTC 840
TACCATCGCA GGGGGCAAGA ATAACCAAGC TACAGGTAAT GGTTCATTTG CAGCAGGTGT 900 AGAGAACAAA GCCGATGCTA ACAACGCCGT CGCTCTAGGT AACAAGAACA CCATCGAAGG 960
TACAAACTCA GTAGCCATCG GCTCTAATAA TACCGTTAAA ACTGGCAAAG AAAATGTCTT 1020
TATTCTTGGC TCTAACACAA ACACAGAAAA TGCACAAAGT GGCTCCGTGC TGCTGGGTAA 1080
TAATACCGCT GGCAAAGCAG CGACCACTGT TAACAATGCC GAAGTGAACG GCTTAACCCT 1140
AGAAAATTTT GCAGGTGCAT CAAAAGCTAA TGCTAATAAT ATTGGTACTG TATCTGTCGG 1200 TAGTGAGAAT AATGAGCGTC AAATCGTTAA TGTTGGTGCA GGTCAGATCA GTGCCACCTC 1260
AACAGATGCT GTTAATGGCT CACAGCTACA TGCTTTAGCC AAAGCTGTTG CTAAAAACAA 1320
ATCTGACATC AAAGGTCTTA ATAAGGGGGT GAAAGAGCTT GATAAGGAGG TGGGTGTATT 1380
AAGCCGAGAC ATTAATTCAC TTCATGATGA TGTTGCTGAC AACCAAGATA GCATTGCTAA 1440
AAACAAAGCT GACATCAAAG GTCTTAATAA GGAGGTGAAA GAGCTTGATA AGGAGGTGGG 1500 TGTATTAAGC CGAGACATTG GTTCACTTCA TGATGATGTT GCTGACAACC AAGATAGCAT 1560
TGCTAAAAAC AAAGCTGACA TCAAAGGTCT TAATAAGGAG GTGAAAGAGC TTGATAAGGA 1620
GGTGGGTGTA TTAAGCCGAG ACATTGGTTC ACTTCATGAT GATGTTGCCA CCAACCAAGC 1680
TGACATTGCT AAAAACCAAG CGGATATCAA AACACTTGAA AACAATGTCG AAGAAGAATT 1740
ATTAAATCTA AGCGGTCGCC TCATTGATCA AAAAGCGGAT ATTGATAATA ACATCAACAA 1800 TATCTATGAG CTGGCACAAC AGCAAGATCA GCATAGCTCT GATATCAAAA CACTTAAAAA 1860
CAATGTCGAA GAAGGTTTGT TGGATCTAAG CGGTCGCCTC ATTGATCAAA AAGCAGATCT 1920
TACGAAAGAC ATCAAAACAC TTAAAAACAA TGTCGAAGAA GGTTTATTGG ATCTAAGCGG 1980
TCGCCTCATT GATCAAAAAG CAGATATTGC TAAAAACCAA GCTGACATTG CTCAAAACCA 2040
AACAGACATC CAAGATCTGG CCGCTTACAA CGAGCTACAA GACCAGTATG CTCAAAAGCA 2100 AACCGAAGCG ATTGACGCTC TAAATAAAGC AAGCTCTGCC AATACTGATC GTATTGCTAC 2160
TGCTGAATTG GGTATCGCTG AGAACAAAAA AGACGCTCAG ATCGCCAAAG CACAAGCCAA 2220
TGAAAATAAA GACGGCATTG CTAAAAACCA AGCTGATATC CAGTTGCACG ATAAAAAAAT 2280
CACCAATCTA GGTATCCTTC ACAGCATGGT TGCAAGAGCG GTAGGAAATA ATACACAAGG 2340
SUBSTTTUTE SHEET (RULE 26) TGTTGCTACC AACAAAGCTG ATATTGCTAA AAACCAAGCA GATATTGCTA ATAACATCAA 2400
AAATATCTAT GAGCTGGCAC AACAGCAAGA TCAGCATAGC TCTGATATCA AAACCTTGGC 2460 AAAAGTAAGT GCTGCCAATA CTGATCGTAT TGCTAAAAAC AAAGCTGAAG CTGATGCAAG 2520
TTTTGAAACG CTCACCAAAA ATCAAAATAC TTTGATTGAG CAAGGTGAAG CATTGGTTGA 2580
GCAAAATAAA GCCATCAATC AAGAGCTTGA AGGGTTTGCG GCTCATGCAG ATGTTCAAGA 2640
TAAGCAAATT TTACAAAACC AAGCTGATAT CACTACCAAT AAGACCGCTA TTGAACAAAA 2700
TATCAATAGA ACTGTTGCCA ATGGGTTTGA GATTGAGAAA AATAAAGCTG GTATTGCTAC 2760 CAATAAGCAA GAGCTTATTC TTCAAAATGA TCGATTAAAT CAAATTAATG AGACAAATAA 2820
TCATCAGGAT CAGAAGATTG ATCAATTAGG TTATGCACTA AAAGAGCAGG GTCAGCATTT 2880
TAATAATCGT ATTAGTGCTG TTGAGCGTCA AACAGCTGGA GGTATTGCAA ATGCTATCGC 2940
AATTGCAACT TTACCATCGC CCAGTAGAGC AGGTGAGCAT CATGTCTTAT TTGGTTCAGG 3000
TTATCACAAT GGTCAAGCTG CGGTATCATT GGGCGCGGCT GGATTAAGTG ATACAGGAAA 3060 ATCAACTTAT AAGATTGGTC TAAGCTGGTC AGATGCAGGT GGATTATCTG GTGGTGTTGG 3120
TGGCAGTTAC CGCTGGAAAT AGAGCCTAAA TTTAACTGCT GTATCAAAAA ATATGGTCTG 3180
TATAAACAGA CCATATTTTT ATCTAAAAAA CTTATCTTAA CTTTTATGAA GCATCATAAG 3240
CCAAAGCTGA GTAATAATAA GAGATGTTAA AATAAGAGAT GTTAAAACTG CTAAACAATC 3300
GGCTTGCGAC GATAAAATAA AATACCTGGA ATGGACAGCC CCAAAACCAA TGCTGAGATG 3360 ATAAAAATCG CCTCAAAAAA ATGACGCATC ATAACGATAA ATAAATCCAT ATCAAATCCA 3420
AAATAGCCAA TTTGTACCAT GCTAACCATG GCTTTATAGG CAGCGATTCC CGGCATCATA 3480
CAAATCAAGC TAGGTACAAT CAAGGCTTTA GGCGGCAGGC CATGACGCTG AGCAAAAA 3538
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 610 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Met Lys Leu Leu Pro Leu Lys He Ala Val Thr Ser Ala Met He He 1 5 10 15 Gly Leu Gly Ala Ala Ser Thr Ala Asn Ala Gin Ser Arg Asp Arg Ser
20 25 30
SUBSTTTUTE SHEET (RULE 25) Leu Glu Asp He Gin Asp Ser He Ser Lys Leu Val Gin Asp Asp He 35 40 45
Asp Thr Leu Lys Gin Asp Gin Gin Lys Met Asn Lys Tyr Leu Leu Leu 50 55 60
Asn Gin Leu Ala Asn Thr Leu He Thr Asp Glu Leu Asn Asn Asn Val 65 70 75 80 He Lys Asn Thr Asn Ser He Glu Ala Leu Gly Asp Glu He Gly Trp
85 90 95
Leu Glu Asn Asp He Ala Asp Leu Glu Glu Gly Val Glu Glu Leu Thr 100 105 110
Lys Asn Gin Asn Thr Leu He Glu Lys Asp Glu Glu His Asp Arg Leu 115 120 125
He Ala Gin Asn Gin Ala Asp He Gin Thr Leu Glu Asn Asn Val Val 130 135 140
Glu Glu Leu Phe Asn Leu Ser Gly Arg Leu He Asp Gin Glu Ala Asp 145 150 155 160 He Ala Lys Asn Asn Ala Ser He Glu Glu Leu Tyr Asp Phe Asp Asn
165 170 175
Glu Val Ala Glu Arg He Gly Glu He His Ala Tyr Thr Glu Glu Val 180 185 190
Asn Lys Thr Leu Glu Asn Leu He Thr Asn Ser Val Lys Asn Thr Asp 195 200 205
Asn He Asp Lys Asn Lys Ala Asp He Asp Asn Asn He Asn His He 210 215 220
Tyr Glu Leu Ala Gin Gin Gin Asp Gin His Ser Ser Asp He Lys Thr
225 230 235 240 Leu Lys Asn Asn Val Glu Glu Gly Leu Leu Glu Leu Ser Gly His Leu
245 250 255
He Asp Gin Lys Ala Asp Leu Thr Lys Asp He Lys Ala Leu Glu Ser
260 265 270
Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu Leu Asp Gin
275 280 285
Lys Ala Asp Leu Thr Lys Asp He Lys Ala Leu Glu Ser Asn Val Glu 290 295 300
Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu Leu Asp Gin Lys Ala Asp
305 310 315 320 He Ala Gin Asn Gin Thr Asp He Gin Asp Leu Ala Ala Tyr Asn Glu
325 330 335
SUBSTTTUTE SHEET (RULE 25) Leu Gin Asp Gin Tyr Ala Gin Lys Gin Thr Glu Ala He Asp Ala Leu 340 345 350
Asn Lys Ala Ser Ser Glu Asn Thr Gin Asn He Glu Asp Leu Ala Ala 355 360 365
Tyr Asn Glu Leu Gin Asp Ala Tyr Ala Lys Gin Gin Thr Glu Ala He 370 375 380 Asp Ala Leu Asn Lys Ala Ser Ser Glu Asn Thr Gin Asn He Ala Lys
385 390 395 400
Asn Gin Ala Asp He Ala Asn Asn He Asn Asn He Tyr Glu Leu Ala
405 410 415
Gin Gin Gin Asp Gin His Ser Ser Asp He Lys Thr Leu Ala Lys Ala
420 425 430
Ser Ala Ala Asn Thr Asn Arg He Ala Thr Ala Glu Leu Gly He Ala 435 440 445
Glu Asn Lys Lys Asp Ala Gin He Ala Lys Ala Gin Ala Asn Ala Asn 450 455 460 Lys Thr Ala He Asp Glu Asn Lys Ala Ser Ala Asp Thr Lys Phe Ala
465 470 475 480
Ala Thr Ala Asp Ala He Thr Lys Asn Gly Asn Ala He Thr Lys Asn 485 490 495
Ala Lys Ser He Thr Asp Leu Gly Thr Lys Val Asp Gly Phe Asp Gly 500 505 510
Arg Val Thr Ala Leu Asp Thr Lys Val Asn Ala Phe Asp Gly Arg He 515 520 525
Thr Ala Leu Asp Ser Lys Val Glu Asn Gly Met Ala Ala Gin Ala Ala 530 535 540 Leu Ser Gly Leu Phe Gin Pro Tyr Ser Val Gly Lys Phe Asn Ala Thr
545 550 555 560
Ala Ala Leu Gly Gly Tyr Gly Ser Lys Ser Ala Val Ala He Gly Ala 565 570 575
Gly Tyr Arg Val Asn Pro Asn Leu Ala Phe Lys Ala Gly Ala Ala He 580 585 590
Asn Thr Ser Gly Asn Lys Lys Gly Ser Tyr Asn He Gly Val Asn Tyr 595 600 605
Glu Phe 610
(2 ) INFORMATION FOR SEQ ID NO : 12
SUBSTTTUTE SHEET (RULE 25) (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2673 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CCATCAGTAC ATACGCCGCA CCTGACCGAG ACGCTCCGCC AAATCAATGC GTCGGTGTAC 60
TACGCCCCGA CCGAGCTATG CACGGATAAT GGTGCGATGA TCGCTTACGC TGGCTTTTGT 120
CGGCTAAGCC GTGGACAGTC GGATGACTTG GCGGTTCGCT GCATTCCCCG ATGGGATATG 180 ACAACGCTTG GCGTATCTGC TCATAGATAG CCACATCAAT CATACCAACG ATATTGGTAT 240
ATACCAAATT GATACCTGCC AAAAATACCA TATTGAAAGT AGGGTTTGGG TATTATTTAT 300
GTAACTTATA TCTAATTTGG TGTTGATACT TTGATAAAGC CTTGCTATAC TGTAACCTAA 360
ATGGATATGA TAGAGATTTT TCCATTTATG CCAGCAAAAG AGATAGATAG ATAGATAGAT 420
AGATAGATAG ATAGATAGAT AGATAGATAG ATAGATAAAA CTCTGTCTTT TATCTGTCCA 480 CTGATGCTTT CTGCCTGCCA CCGATGATAT CGTTTATCTG CTTTTTTAGG CATCAGTTAT 540
TTCACCGTGA TGACTGATGT GATGACTTAA CCACCAAAAG AGAGTGCTAA ATGAAAACCA 600
TGAAACTTCT CCCTCTAAAA ATCGCTGTAA CCAGTGCCAT GATTATTGGT TTGGGTGCGG 660
CATCTACTGC GAATGCACAG TCTCGGGATA GATCTTTAGA AGATATACAA GATTCAATTA 720
GTAAACTTGT TCAAGATGAT ATAGATACAC TAAAACAAGA TCAGCAGAAG ATGAACAAGT 780 ATCTGTTGCT CAACCAGTTA GCTAATACTT TAATTACAGA CGAGCTCAAC AATAATGTTA 840
TAAAAAACAC CAATTCTATT GAAGCTCTTG GTGATGAGAT TGGATGGCTT GAAAATGATA 900
TTGCAGACTT GGAAGAAGGT GTTGAAGAAC TCACCAAAAA CCAAAATACT TTGATTGAAA 960
AAGATGAAGA GCATGACAGA TTAATCGCTC AAAATCAAGC TGATATCCAA ACACTTGAAA 1020
ACAATGTCGT AGAAGAACTA TTCAATCTAA GCGGTCGCCT AATTGATCAA GAAGCGGATA 1080 TTGCTAAAAA TAATGCTTCT ATTGAAGAGC TTTATGATTT TGATAATGAG GTTGCAGAAA 1140
GGATAGGTGA GATACATGCT TATACTGAAG AGGTAAATAA AACTCTTGAA AACTTGATAA 1200
CAAACAGTGT TAAGAATACT GATAATATTG ACAAAAACAA AGCTGATATT GATAATAACA 1260
TCAACCATAT CTATGAGCTG GCACAACAGC AAGATCAGCA TAGCTCTGAT ATCAAAACAC 1320
TTAAAAACAA TGTCGAAGAA GGTTTGTTGG AGCTAAGCGG TCACCTCATT GATCAAAAAG 1380 CGGATCTTAC AAAAGACATC AAAGCACTTG AAAGCAATGT CGAAGAAGGT TTGTTGGATC 1440
TAAGCGGTCG TCTGCTTGAT CAAAAAGCGG ATCTTACAAA AGACATCAAA GCACTTGAAA 1500
SUBSTTTUTE SHEET (RULE 25) GCAATGTCGA AGAAGGTTTG TTGGATCTAA GCGGTCGTCT GCTTGATCAA AAAGCGGATA 1560
TTGCTCAAAA CCAAACAGAC ATCCAAGATC TGGCCGCTTA CAACGAGCTA CAAGACCAGT 1620
ATGCTCAAAA GCAAACCGAA GCGATTGACG CTCTAAATAA AGCAAGCTCT GAGAATACAC 1680
AAAACATCGA AGATCTGGCC GCTTACAATG AGCTACAAGA TGCCTATGCC AAACAGCAAA 1740 CCGAAGCGAT TGACGCTCTA AATAAAGCAA GCTCTGAGAA TACACAAAAC ATTGCTAAAA 1800
ACCAAGCGGA TATTGCTAAT AACATCAACA ATATCTATGA GCTGGCACAA CAGCAAGATC 1860
AGCATAGCTC TGATATCAAA ACCTTGGCAA AAGCAAGTGC TGCCAATACT AATCGTATTG 1920
CTACTGCTGA ATTGGGCATC GCTGAGAACA AAAAAGACGC TCAGATCGCC AAAGCACAAG 1980
CGAATGCCAA CAAAACTGCG ATTGATGAAA ACAAAGCATC TGCGGATACC AAGTTTGCAG 2040 CAACAGCAGA CGCCATTACC AAAAATGGAA ATGCTATCAC TAAAAACGCA AAATCTATCA 2100
CTGATTTGGG CACTAAAGTG GATGGTTTTG ACGGTCGTGT AACTGCATTA GACACCAAAG 2160
TCAATGCCTT TGATGGTCGT ATCACAGCTT TAGACAGTAA AGTTGAAAAC GGTATGGCTG 2220
CCCAAGCTGC CCTAAGTGGT CTATTCCAGC CTTATAGCGT TGGTAAGTTT AATGCGACCG 2280
CTGCACTTGG TGGCTATGGC TCAAAATCTG CGGTTGCTAT CGGTGCTGGC TATCGTGTGA 2340 ATCCAAATCT GGCGTTTAAA GCTGGTGCGG CGATTAATAC CAGTGGCAAT AAAAAAGGCT 2400
CTTATAACAT CGGTGTGAAT TACGAGTTCT AATTGTCTAT CATCACCAAA AAAAGCAGTC 2460
AGTTTACTGG CTGCTTTTTT ATGGGTTTTT GTGGCTTTTG GTTGTGAGTG ATGGATAAAA 2520
GCTTATCAAG CGATTGATGA ATATCAATAA ATGATTGGTA AATATCAATA AAGCGGTTTA 2580
GGGTTTTTGG ATATCTTTTA ATAAGTTTAA AAACCCCTGC ATAAAATAAA GCTGGGCATC 2640 AGAGCTGCGA GTAGCGGCAT ACAGCGGGAG ATC 2673
(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 873 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Met Asn Lys He Tyr Lys Val Lys Lys Asn Ala Ala Gly His Leu Val 1 5 10 15
Ala Cys Ser Glu Phe Ala Lys Gly His Thr Lys Lys Ala Val Leu Gly 20 25 30
SUBSTTTUTE SHEET RULE 25) Ser Leu Leu He Val Gly He Leu Gly Met Ala Thr Thr Ala Ser Ala 35 40 45
Gin Gin Thr He Ala Arg Gin Gly Lys Gly Met His Ser He He Gly 50 55 60
Gly Gly Asn Asp Asn Glu Ala Asn Gly Asp Tyr Ser Thr Val Ser Gly 65 70 75 80
Gly Asp Tyr Asn Glu Ala Lys Gly Asp Ser Ser Thr He Gly Gly Gly 85 90 95
Tyr Tyr Asn Glu Ala Asn Gly Asp Ser Ser Thr He Gly Gly Gly Phe 100 105 110
Tyr Asn Glu Ala Lys Gly Glu Ser Ser Thr He Gly Gly Gly Asp Asn 115 120 125 Asn Ser Ala Thr Gly Met Tyr Ser Thr He Gly Gly Gly Asp Asn Asn
130 135 140
Ser Ala Thr Gly Arg Tyr Ser Thr He Ala Gly Gly Trp Leu Asn Gin 145 150 155 160
Ala Thr Gly His Ser Ser Thr Val Ala Gly Gly Trp Leu Asn Gin Ala 165 170 175
Thr Asn Glu Asn Ser Thr Val Gly Gly Gly Arg Phe Asn Gin Ala Thr 180 185 190
Gly Arg Asn Ser Thr Val Ala Gly Gly Tyr Lys Asn Lys Ala Thr Gly 195 200 205 Val Asp Ser Thr He Ala Gly Gly Arg Asn Asn Gin Ala Asn Gly He
210 215 220
Gly Ser Phe Ala Ala Gly He Asp Asn Gin Ala Asn Ala Asn Asn Thr 225 230 235 240
Val Ala Leu Gly Asn Lys Asn He He Lys Gly Lys Asp Ser Val Ala 245 250 255
He Gly Ser Asn Asn Thr Val Glu Thr Gly Lys Glu Asn Val Phe He 260 265 270
Leu Gly Ser Asn Thr Lys Asp Ala His Ser Asn Ser Val Leu Leu Gly 275 280 285 Asn Glu Thr Thr Gly Lys Ala Ala Thr Thr Val Glu Asn Ala Lys Val
290 295 300
Gly Gly Leu Ser Leu Thr Gly Phe Val Gly Ala Ser Lys Ala Asn Thr 305 310 315 320
Asn Asn Gly Thr Val Ser Val Gly Lys Gin Gly Lys Glu Arg Gin He 325 330 335
SUBSTTTUTE SHEET RULE 25 Val Asn Val Gly Ala Gly Gin He Arg Ala Asp Ser Thr Asp Ala Val 340 345 350
Asn Gly Ser Gin Leu His Ala Leu Ala Thr Ala Val Asp Ala Glu Phe 355 360 365
Arg Thr Leu Thr Gin Thr Gin Asn Ala Leu He Glu Gin Gly Glu Ala 370 375 380
He Asn Gin Glu Leu Glu Gly Leu Ala Asp Tyr Thr Asn Ala Gin Asp 385 390 395 400
Glu Lys He Leu Lys Asn Gin Thr Asp He Thr Ala Asn Lys Thr Ala 405 410 415
He Glu Gin Asn Phe Asn Arg Thr Val Thr Asn Gly Phe Glu He Glu 420 425 430 Lys Asn Lys Ala Gly He Ala Lys Asn Gin Ala Asp He Gin Thr Leu
435 440 445
Glu Asn Asp Val Gly Lys Glu Leu Leu Asn Leu Ser Gly Arg Leu Leu 450 455 460
Asp Gin Lys Ala Asp He Asp Asn Asn He Asn Asn He Tyr Glu Leu 465 470 475 480
Ala Gin Gin Gin Asp Gin His Ser Ser Asp He Lys Thr Leu Lys Asn 485 490 495
Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu He Asp Gin 500 505 510 Lys Ala Asp Leu Thr Lys Asp He Lys Ala Leu Glu Asn Asn Val Glu
515 520 525
Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu He Asp Gin Lys Ala Asp
530 535 540
He Ala Lys Asn Gin Ala Asp He Gin Asp Leu Ala Ala Tyr Asn Glu
545 550 555 560
Leu Gin Asp Gin Tyr Ala Gin Lys Gin Thr Glu Ala He Asp Ala Leu 565 570 575
Asn Lys Ala Ser Ser Ala Asn Thr Asp Arg He Ala Thr Ala Glu Leu 580 585 590
Gly He Ala Glu Asn Lys Lys Asp Ala Gin He Ala Lys Ala Gin Ala 595 600 605
Asn Glu Asn Lys Asp Gly He Ala Lys Asn Gin Ala Asp He Ala Asn
610 615 620
Asn He Lys Asn He Tyr Glu Leu Ala Gin Gin Gin Asp Gin His Ser
625 630 635 640
SUBSTTTUTE SHEET (RULE 25) Ser Asp He Lys Thr Leu Ala Lys Val Ser Ala Ala Asn Thr Asp Arg 645 650 655
He Ala Lys Asn Lys Ala Glu Ala Asp Ala Ser Phe Glu Thr Leu Thr 660 665 670
Lys Asn Gin Asn Thr Leu He Glu Gin Gly Glu Ala Leu Val Glu Gin 675 680 685
Asn Lys Ala He Asn Gin Glu Leu Glu Gly Phe Ala Ala His Ala Asp 690 695 700
Val Gin Asp Lys Gin He Leu Gin Asn Gin Ala Asp He Thr Ala Asn 705 710 715 720
Lys Thr Ala He Glu Gin Asn He Asn Arg Thr Val Ala Asn Gly Phe 725 730 735 Glu He Glu Lys Asn Lys Ala Gly He Ala Thr Asn Lys Gin Glu Leu
740 745 750
He Leu Gin His Asp Arg Leu Asn Arg He Asn Glu Thr Asn Asn Arg 755 760 765
Gin Asp Gin Lys He Asp Gin Leu Gly Tyr Ala Leu Lys Glu Gin Gly 770 775 780
Gin His Phe Asn Asn Arg He Ser Ala Val Glu Arg Gin Thr Ala Gly 785 790 795 800
Gly He Ala Asn Ala He Ala He Ala Thr Leu Pro Ser Pro Ser Arg 805 810 815 Ala Gly Glu His His Val Leu Phe Gly Ser Gly Tyr His Asn Gly Gin
820 825 830
Ala Ala Val Ser Leu Gly Ala Ala Gly Leu Ser Asp Thr Gly Lys Ser 835 840 845
Thr Tyr Lys He Gly Leu Ser Trp Ser Asp Ala Gly Gly Leu Ser Gly 850 855 860
Gly Val Gly Gly Ser Tyr Arg Trp Lys 865 870
(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3292 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
SUBSTTTUTE SHEET (RULE 26) GTAAATGACT GATGAGTGTC TATTTAATGA AAGATACAAT ATATAAAAGT TGACTATAGC 60
GATGCAATAC AGTAAAATTT GTTACGGCTA AACATAACGA CGGTCCAAGA TGGCGGATAT 120
CGCCATTTAC CAACCTGATA ATCAGTTTGA TAGCCATTAG CGATGGCATC AAGTTGTGTT 180
GTTGTATTGT CATATAAACG GTAAATTTGG TTTGGTGGAT GCCCCATCTG ATTTACCGTC 240
CCCCTAATAA GTGAGAGGGG GGGGGAGACC CCAGTCATTT ATTAGGAGAC TAAGATGAAC 300
AAAATTTATA AAGTGAAAAA AAATGCCGCA GGTCACTTGG TGGCATGTTC TGAATTTGCC 360
AAAGGCCATA CCAAGAAGGC AGTTTTGGGC AGTTTATTGA TTGTTGGAAT ATTGGGTATG 420 GCAACGACAG CATCTGCACA ACAAACAATC GCACGCCAAG GCAAAGGCAT GCACTCTATC 480
ATCGGTGGTG GCAATGACAA CGAAGCCAAC GGCGATTACT CTACCGTCAG TGGTGGCGAT 540
TATAACGAAG CCAAAGGCGA TAGCTCTACC ATCGGTGGTG GCTATTATAA CGAAGCCAAC 600
GGCGATAGCT CTACCATCGG TGGTGGCTTT TATAACGAAG CCAAAGGCGA GAGCTCTACC 660
ATCGGTGGTG GCGATAACAA CTCAGCCACA GGCATGTACT CTACCATCGG TGGTGGCGAT 720 AACAACTCAG CCACAGGCAG GTACTCTACC ATCGCAGGGG GTTGGCTTAA CCAAGCTACA 780
GGTCATAGCT CAACGGTTGC AGGGGGTTGG CTTAACCAAG CTACAAACGA GAATTCTACC 840
GTTGGTGGCG GCAGGTTTAA CCAAGCTACA GGTCGTAACT CAACGGTTGC AGGGGGCTAT 900
AAAAACAAAG CCACAGGCGT AGACTCTACC ATCGCAGGGG GCAGGAATAA CCAAGCCAAC 960
GGTATAGGTT CATTTGCAGC AGGTATAGAC AACCAAGCCA ATGCCAACAA CACCGTCGCT 1020 CTAGGTAACA AGAACATCAT CAAAGGTAAA GACTCAGTAG CCATCGGCTC TAATAATACC 1080
GTTGAAACTG GCAAAGAAAA TGTCTTTATT CTTGGCTCTA ACACAAAAGA TGCACATAGT 1140
AACTCAGTGC TACTGGGTAA TGAGACCACT GGCAAAGCAG CGACCACTGT TGAGAATGCC 1200
AAAGTGGGTG GTCTAAGCCT AACAGGATTT GTAGGTGCAT CAAAAGCTAA TACTAATAAT 1260
GGTACTGTAT CTGTCGGTAA GCAGGGTAAA GAGCGTCAAA TCGTTAATGT TGGTGCAGGT 1320 CAGATCCGTG CTGATTCAAC AGATGCTGTT AATGGCTCAC AGCTACATGC TTTGGCCACA 1380
GCTGTCGATG CAGAATTTAG AACACTCACC CAAACTCAAA ATGCTTTGAT TGAGCAAGGT 1440
GAAGCCATCA ATCAAGAGCT TGAAGGTTTG GCAGATTATA CAAATGCTCA AGATGAGAAA 1500
ATTCTAAAAA ACCAAACTGA CATCACTGCC AATAAAACTG CTATTGAGCA AAATTTTAAT 1560
AGAACTGTTA CCAATGGGTT TGAGATTGAG AAAAATAAAG CTGGTATTGC TAAAAACCAA 1620 GCGGATATCC AAACACTTGA AAACGATGTC GGAAAAGAAC TATTAAATCT AAGCGGTCGC 1680
CTGCTTGATC AAAAAGCAGA TATTGATAAT AACATCAACA ATATCTATGA GCTGGCACAA 1740
SUBSTTTUTE SHEET RULE 25 CAGCAAGATC AGCATAGCTC TGATATCAAA ACACTTAAAA ACAATGTCGA AGAAGGTTTG 1800
TTGGATCTAA GCGGTCGCCT CATTGATCAA AAAGCAGATC TTACGAAAGA CATCAAAGCA 1860
CTTGAAAACA ATGTCGAAGA AGGTTTATTG GATCTAAGCG GTCGCCTCAT TGATCAAAAA 1920
GCAGATATTG CTAAAAACCA AGCAGACATC CAAGATTTGG CCGCTTACAA CGAGCTACAA 1980 GACCAGTATG CTCAAAAGCA AACCGAAGCG ATTGACGCTC TAAATAAAGC AAGCTCTGCC 2040
AATACTGATC GTATTGCTAC TGCTGAATTG GGTATCGCTG AGAACAAAAA AGACGCTCAG 2100
ATCGCCAAAG CACAAGCCAA TGAAAATAAA GACGGCATTG CTAAAAACCA AGCAGATATT 2160
GCTAATAACA TCAAAAATAT CTATGAGCTG GCACAACAGC AAGATCAGCA TAGCTCTGAT 2220
ATCAAAACCT TGGCAAAAGT AAGTGCTGCC AATACTGATC GTATTGCTAA AAACAAAGCT 2280 GAAGCTGATG CAAGTTTTGA AACGCTCACC AAAAATCAAA ATACTTTGAT TGAGCAAGGT 2340
GAAGCATTGG TTGAGCAAAA TAAAGCCATC AATCAAGAGC TTGAAGGGTT TGCGGCTCAT 2400
GCAGATGTTC AAGATAAGCA AATTTTACAA AACCAAGCTG ATATCACTGC CAATAAGACC 2460
GCTATTGAAC AAAATATCAA TAGAACTGTT GCCAATGGGT TTGAGATTGA GAAAAATAAA 2520
GCTGGTATTG CTACCAATAA GCAAGAGCTT ATTCTTCAAC ATGATCGATT AAATCGAATT 2580 AATGAGACAA ATAATCGTCA GGATCAGAAG ATTGATCAAT TAGGTTATGC ACTAAAAGAG 2640
CAGGGTCAGC ATTTTAATAA TCGTATTAGT GCTGTTGAGC GTCAAACAGC TGGAGGTATT 2700
GCAAATGCTA TCGCAATTGC AACTTTACCA TCGCCCAGTA GAGCAGGTGA GCATCATGTC 2760
TTATTTGGTT CAGGTTATCA CAATGGTCAA GCTGCGGTAT CATTGGGTGC GGCTGGGTTA 2820
AGTGATACAG GAAAATCAAC TTATAAGATT GGTCTAAGCT GGTCAGATGC AGGTGGATTA 2880 TCTGGTGGTG TTGGTGGTAG TTACCGCTGG AAATAGAGCC TAAATTTAAC TGCTGTATCA 2940
AAAAATATGG TCTGTATAAA CAGACCATAT TTTTATCTAA AAACTTATCT TAACTTTTAT 3000
GAAGCATCAT AAGCCAAAGC TGAGTAATAA TAAGAGATGT TAAAATAAGA GATGTTAAAA 3060
CTGCTAAACA ATCGGCTTAC GACGATAAAA TAAAATACCT GGAATGGACA GCCCCAAAAC 3120
CAATGCTGAG ATGATAAAAA TCGCCTCAAA AAAATGACGC ATCATAACGA TAAATAAATC 3180 CATATCAAAT CCAAAATAGC CAATTTGTAC CATGCTAACC ATGGCTTTAT AGGCAGCGAT 3240
TCCCGGCATC ATACAAATCA AGCTAGGTAC AATCAAGGCT TTAGGCGGCA GG 3292
(2) INFORMATION FOR SEQ ID NO : 15: (i) SEQUENCE CHARACTERISTICS:
SUBSTTTUTE 5HEET (RULE 25) (A) LENGTH: 889 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Val Asn Lys He Tyr Lys Val Lys Lys Asn Ala Ala Gly His Leu Val 1 5 10 15
Ala Cys Ser Glu Phe Ala Lys Gly His Thr Lys Lys Ala Val Leu Gly 20 25 30
Ser Leu Leu He Val Gly Ala Leu Gly Met Ala Thr Thr Ala Ser Ala 35 40 45
Gin Pro Leu Val Ser Thr Asn Lys Pro Asn Gin Gin Val Lys Gly Tyr 50 55 60 Trp Ser He He Gly Ala Gly Arg His Asn Asn Val Gly Gly Ser Ala
65 70 75 80
His His Ser Gly He Leu Gly Gly Trp Lys Asn Thr Val Asn Gly Tyr 85 90 95
Thr Ser Ala He Val Gly Gly Tyr Gly Asn Glu Thr Gin Gly Asp Tyr 100 105 110
Thr Phe Val Gly Gly Gly Tyr Lys Asn Leu Ala Lys Gly Asn Tyr Thr 115 120 125
Phe Val Gly Gly Gly Tyr Lys Asn Leu Ala Glu Gly Asp Asn Ala Thr 130 135 140 He Ala Gly Gly Phe Ala Asn Leu Ala Glu Gly Asp Asn Ala Thr He
145 150 155 160
Ala Gly Gly Phe Glu Asn Arg Ala Glu Gly He Asp Ser Val Val Ser 165 170 175
Gly Gly Tyr Ala Asn Gin Ala Thr Gly Glu Ser Ser Thr Val Ala Gly 180 185 190
Gly Ser Asn Asn Leu Ala Glu Gly Lys Ser Ser Ala He Gly Gly Gly 195 200 205
Arg Gin Asn Glu Ala Ser Gly Asp Arg Ser Thr Val Ser Gly Gly Tyr 210 215 220 Asn Asn Leu Ala Glu Gly Lys Ser Ser Ala He Gly Gly Gly Glu Phe
225 230 235 240
Asn Leu Ala Leu Gly Asn Asn Ala Thr He Ser Gly Gly Arg Gin Asn 245 250 255
Glu Ala Ser Gly Asp Arg Ser Thr Val Ala Gly Gly Glu Gin Asn Gin 260 265 270
SUBSTTTUTE SHEET RULE 25) Ala He Gly Lys Tyr Ser Thr He Ser Gly Gly Arg Gin Asn Glu Ala 275 280 285
Ser Gly Asp Arg Ser Thr Val Ala Gly Gly Glu Gin Asn Gin Ala He 290 295 300
Gly Lys Tyr Ser Thr Val Ser Gly Gly Tyr Arg Asn Gin Ala Thr Gly 305 310 315 320
Lys Gly Ser Phe Ala Ala Gly He Asp Asn Lys Ala Asn Ala Asp Asn 325 330 335
Ala Val Ala Leu Gly Asn Lys Asn Thr He Glu Gly Glu Asn Ser Val 340 345 350
Ala He Gly Ser Asn Asn Thr Val Lys Lys Asn Gin Lys Asn Val Phe 355 360 365 He Leu Gly Ser Asn Thr Asp Thr Lys Asp Ala Gin Ser Gly Ser Val
370 375 380
Leu Leu Gly Asp Asn Thr Ser Gly Lys Ala Ala Thr Ala Val Glu Asp 385 390 395 400
Ala Thr Val Gly Asp Leu Ser Leu Thr Gly Phe Ala Gly Val Ser Lys 405 410 415
Ala Asn Ser Gly Thr Val Ser Val Gly Ser Glu Gly Lys Glu Arg Gin 420 425 430
He Val His Val Gly Ala Gly Arg He Ser Asn Asp Ser Thr Asp Ala 435 440 445 Val Asn Gly Ser Gin Leu Tyr Ala Leu Ala Ala Ala Val Asp Asp Asn
450 455 460
Gin Tyr Asp He Glu Lys Asn Gin Asp Asp He Ala Lys Asn Gin Ala 465 470 475 480
Asp He Ala Lys Asn Gin Ala Asp He Gin Thr Leu Glu Asn Asp Val 485 490 495
Gly Lys Glu Leu Leu Asn Leu Ser Gly Arg Leu He Asp Gin Lys Ala 500 505 510
Asp He Asp Asn Asn He Asn His He Tyr Glu Leu Ala Gin Gin Gin 515 520 525 Asp Gin His Ser Ser Asp He Lys Thr Leu Lys Lys Asn Val Glu Glu
530 535 540
Gly Leu Leu Glu Leu Ser Gly His Leu He Asp Gin Lys Ala Asp Leu 545 550 555 560
Thr Lys Asp He Lys Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu 565 570 575
SUBSTTTUTE SHEET (RULE 25) Asp Leu Ser Gly Arg Leu He Asp Gin Lys Ala Asp He Ala Gin Asn 580 585 590
Gin Ala Asn He Gin Asp Leu Ala Ala Tyr Asn Glu Leu Gin Asp Gin 595 600 605
Tyr Ala Gin Lys Gin Thr Glu Ala He Asp Ala Leu Asn Lys Ala Ser 610 615 620
Ser Glu Asn Thr Gin Asn He Glu Asp Leu Ala Ala Tyr Asn Glu Leu 625 630 635 640
Gin Asp Ala Tyr Ala Lys Gin Gin Thr Glu Ala He Asp Ala Leu Asn 645 650 655
Lys Ala Ser Ser Glu Asn Thr Gin Asn He Ala Lys Asn Gin Ala Asp 660 665 670 He Ala Asn Asn He Asn Asn He Tyr Glu Leu Ala Gin Gin Gin Asp
675 680 685
Gin His Ser Ser Asp He Lys Thr Leu Ala Lys Ala Ser Ala Ala Asn 690 695 700
Thr Asp Arg He Ala Lys Asn Lys Ala Asp Ala Asp Ala Ser Phe Glu 705 710 715 720
Thr Leu Thr Lys Asn Gin Asn Thr Leu He Glu Lys Asp Lys Glu His 725 730 735
Asp Lys Leu He Thr Ala Asn Lys Thr Ala He Asp Ala Asn Lys Ala 740 745 750 Ser Ala Asp Thr Lys Phe Ala Ala Thr Ala Asp Ala He Thr Lys Asn
755 760 765
Gly Asn Ala He Thr Lys Asn Ala Lys Ser He Thr Asp Leu Gly Thr 770 775 780
Lys Val Asp Gly Phe Asp Gly Arg Val Thr Ala Leu Asp Thr Lys Val 785 790 795 800
Asn Ala Phe Asp Gly Arg He Thr Ala Leu Asp Ser Lys Val Glu Asn 805 810 815
Gly Met Ala Ala Gin Ala Ala Leu Ser Gly Leu Phe Gin Pro Tyr Ser 820 825 830 Val Gly Lys Phe Asn Ala Thr Ala Ala Leu Gly Gly Tyr Gly Ser Lys
835 840 845
Ser Ala Val Ala He Gly Ala Gly Tyr Arg Val Asn Pro Asn Leu Ala 850 855 860
Phe Lys Ala Gly Ala Ala He Asn Thr Ser Gly Asn Lys Lys Gly Ser 865 870 875 880
SUBSTTTUTE SHEET RULE 26) Tyr Asn He Gly Val Asn Tyr Glu Phe 885
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4228 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GCCGCACCCT GACCGAGACG CTCCGCCAAA TCGATGCGTC GGTGTACTAT GCCCCGACCG 60
AGCTATGCAC GGATAATGGT GCGATGATCG CCTATGCTGG CTTTTGTCGG CTAAGCCGTG 120 GACAGTCGGA TGACTTGGTG GTTCGCTGTA TTCCCCGATG GGATATGACG ACGCTTGGTA 180
TCGAATATGA TAATTAGGCT GTGGTATTTG AGTTTTGAGT AATGTACCTA CTACCACTAA 240
TTTATCATAC AATACATAAA CATAAAAAAC ATCGGTATTG TTAAAAAACA ATACCCAAGT 300
TAAAATAGCT CAATACTTTA CCATAGCACA AAGAAACTTG TGAACGAAAC ATTTAATAAT 360
TGCCCAAAAT GTTACTGCAC ACACTTTGTA AAAGCAGGCT TGGGCAATGG CAAACAACGA 420 TACAAATGCA AAGGTTGCCA TCACTATTTT TCTGTGAAGC AACGAAGCAA CCAAAAAAGT 480
AATGACATTA AAAAAACAAG CCATTGATAC AAACAGTAAA CAAATCTTAG GCTTTGTCTG 540
TGGTAAAACA GACACTAACA CCTTTAAACG ACTTTATCAG CAGTTAAATA CCCATAGCAT 600
TCAACTGTTT TTTAGTGACT ACTGGAAATC TTATCGTCAA GTCATTTTAA AGCCAAAACA 660
TATAACAAGC AAAGCTCAAA CTTTTACCAT AGAGGGCTAT AATAGTCTCA TTAGGCATTT 720 CATAGCAAGA TTTACAAGAA AGTCAAAGTG TTATTCTAAA TCCGAAAAAA TGATAGAAAA 780
CACGTTGAAT TTATTATTTG CTAAGTGGAA TGGTAGCTTA AGATATGTAT TTTAATTTAA 840
CAATGCCAAA AACATCAATT ACAGTAAGAT TTTAGGCGTT TTGCAGTTGC TACTTTAGTA 900
AAGCTTTGTT ATACTAGCTG TTAGTATACT CAAGCTTGTT TGTGTTTGAG CTATATTTAT 960
TTTATAGCAG TAGTTGGTTA TAAAATATAA ATAAAGCTAA GCTCGAGGGT TTGGTAATGG 1020 TTTTTTATGT TTATAATACC AACAGAGTCT ATACAGCTAA AATAGCTAAT ACCTTAGGTG 1080
TATTACAAGT AAAAATCCTT TGGTTAATCA GGGGGTGTAT TATATGTATA TTTCCTTTGT 1140
ATTTGGTTAT AGCAATCCCT TGGTAAGAAA TCATATCTAT TTTTTATTGT TCAATTATTT 1200
AGGAGACTAA GGTGAACAAA ATTTATAAAG TGAAAAAAAA TGCCGCAGGT CACTTGGTGG 1260
SUBSTTTUTE SHEET (RULE 26) CATGTTCTGA ATTTGCCAAA GGCCATACCA AAAAGGCAGT TTTGGGCAGT TTATTGATTG 1320
TTGGGGCGTT GGGCATGGCA ACGACGGCGT CTGCACAGCC ATTAGTAAGT ACAAATAAGC 1380
CTAATCAGCA GGTAAAGGGT TATTGGTCTA TTATTGGTGC AGGTCGTCAT AATAACGTAG 1440
GTGGATCCGC TCATCACTCA GGGATTCTTG GTGGTTGGAA AAATACAGTC AATGGCTATA 1500
CCTCAGCCAT TGTAGGTGGT TATGGTAACG AAACTCAGGG TGATTATACA TTCGTCGGTG 1560
GTGGTTATAA AAACTTGGCA AAGGGTAATT ATACATTCGT CGGTGGTGGT TATAAAAACT 1620
TGGCAGAGGG TGATAATGCA ACCATCGCTG GTGGTTTTGC AAACTTGGCA GAGGGTGATA 1680 ATGCAACCAT CGCTGGTGGT TTTGAAAACC GTGCAGAGGG TATCGACTCA GTAGTTTCTG 1740
GTGGTTATGC CAACCAAGCT ACAGGAGAAA GCTCAACCGT CGCAGGTGGT TCTAATAACC 1800
TAGCAGAGGG CAAAAGCTCA GCCATTGGTG GTGGCCGTCA AAATGAGGCG TCTGGTGACC 1860
GATCTACTGT CTCAGGTGGT TATAATAACC TAGCAGAGGG CAAAAGCTCA GCCATTGGTG 1920
GCGGTGAGTT TAACTTAGCA TTAGGGAATA ACGCTACCAT TAGTGGTGGC CGTCAAAATG 1980 AGGCGTCTGG TGACCGATCT ACTGTCGCAG GTGGTGAACA AAACCAAGCC ATAGGCAAGT 2040
ATTCTACCAT TAGTGGTGGC CGTCAAAATG AGGCGTCTGG TGACCGATCT ACTGTCGCAG 2100
GTGGTGAACA AAACCAAGCC ATAGGCAAGT ATTCTACCGT TAGTGGTGGC TATCGAAACC 2160
AAGCCACAGG TAAAGGTTCA TTTGCAGCAG GTATAGATAA CAAAGCCAAT GCCGACAACG 2220
CCGTCGCTCT AGGTAACAAG AACACCATCG AAGGTGAAAA CTCAGTAGCC ATCGGCTCTA 2280 ATAATACCGT TAAAAAAAAT CAAAAAAATG TCTTTATTCT TGGCTCTAAC ACAGACACAA 2340
AAGATGCACA AAGCGGCTCA GTACTGCTAG GTGATAATAC CTCTGGTAAA GCAGCGACCG 2400
CTGTTGAGGA TGCCACAGTG GGTGATCTAA GCCTAACAGG ATTTGCAGGC GTATCAAAAG 2460
CTAATAGTGG TACTGTATCT GTCGGTAGTG AGGGTAAAGA GCGTCAAATC GTTCATGTTG 2520
GTGCAGGTCG GATCAGTAAT GATTCAACAG ATGCTGTTAA TGGCTCACAG CTATATGCTT 2580 TGGCCGCAGC TGTTGATGAC AACCAATATG ACATTGAAAA AAACCAAGAT GACATTGCTA 2640
AAAACCAAGC TGACATTGCT AAAAACCAAG CTGACATCCA AACACTTGAA AACGATGTCG 2700
GAAAAGAACT ATTAAATCTA AGCGGTCGCC TCATTGATCA AAAAGCAGAT ATTGATAATA 2760
ACATCAACCA TATCTATGAG CTGGCACAAC AGCAAGATCA GCATAGCTCT GATATCAAAA 2820
CACTTAAAAA AAATGTCGAA GAAGGTTTGT TGGAGCTAAG CGGTCACCTC ATTGATCAAA 2880 AAGCAGATCT TACAAAAGAC ATCAAAGCAC TTGAAAGCAA TGTCGAAGAA GGTTTGTTGG 2940
ATCTAAGCGG TCGCCTCATT GATCAAAAAG CAGATATTGC TCAAAACCAA GCTAACATCC 3000
SUBSTTTUTE SHEET (RULE 25) AAGATTTGGC TGCTTACAAC GAGCTACAAG ACCAGTATGC TCAAAAGCAA ACCGAAGCGA 3060
TTGACGCTCT AAATAAAGCA AGCTCTGAGA ATACACAAAA CATCGAAGAT CTGGCCGCTT 3120
ACAACGAGCT ACAAGATGCC TATGCCAAAC AGCAAACCGA AGCCATTGAC GCTCTAAATA 3180
AAGCAAGCTC TGAGAATACA CAAAACATTG CTAAAAACCA AGCGGATATT GCTAATAACA 3240 TCAACAATAT CTATGAGCTA GCACAACAGC AAGATCAGCA TAGCTCTGAT ATCAAAACCT 3300
TGGCAAAAGC AAGTGCTGCC AATACTGATC GTATTGCTAA AAACAAAGCC GATGCTGATG 3360
CAAGTTTTGA AACGCTCACC AAAAATCAAA ATACTTTGAT TGAAAAAGAT AAAGAGCATG 3420
ACAAATTAAT TACTGCAAAC AAAACTGCGA TTGATGCCAA TAAAGCATCT GCGGATACCA 3480
AGTTTGCAGC GACAGCAGAC GCCATTACCA AAAATGGAAA TGCTATCACT AAAAACGCAA 3540 AATCTATCAC TGATTTGGGT ACTAAAGTGG ATGGTTTTGA CGGTCGTGTA ACTGCATTAG 3600
ACACCAAAGT CAATGCCTTT GATGGTCGTA TCACAGCTTT AGACAGTAAA GTTGAAAACG 3660
GTATGGCTGC CCAAGCTGCC CTAAGTGGTC TATTCCAGCC TTATAGCGTT GGTAAGTTTA 3720
ATGCGACCGC TGCACTTGGT GGCTATGGCT CAAAATCTGC GGTTGCTATC GGTGCTGGCT 3780
ATCGTGTGAA TCCAAATCTG GCGTTTAAAG CTGGTGCGGC GATTAATACC AGTGGCAATA 3840 AAAAAGGCTC TTATAACATC GGTGTGAATT ACGAGTTCTA ATTGTCTATC ATCACCAAAA 3900
AAAGCAGTCA GTTTACTGGC TGCTTTTTTA TGGGTTTTTG TGGCTTTTGG TTGTGAGTGA 3960
TGGATAAAAG CTTGTCAAGC GATTGATGAA TATCAATAAA TGATTGGTAA ATATCAATAA 4020
AGCGGTTTAG GGTTTTTGGA TATCTTTTAA TAAGTTTAAA AACCCCTGCA TAAAATAAAG 4080
CTGGCATCAG AGCTGCGAAG TAGCGGCATA CAGCTGGCAA TGCACGCCTG TGCCTAGGGG 4140 GCGTGAGACC ACCCAGCCTT TGCGTTCGTA TTCTAAAATT ACCCAATCAG GCAGAGCGGC 4200
AACTCCATGT TCGGAGGCGA CCAGCTGA 4228
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Ala Gin Gin Gin Asp Gin His
1 5
SUBSTTTUTE SHEET (RULE 25) (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 18
Tyr Glu Leu Ala Gin Gin Gin Asp Gin His 1 5 10
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Tyr Asp Leu Ala Gin Gin Gin Asp Gin His 1 5 10
(2) INFORMATION FOR SEQ ID NO : 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 20: GACGCTCAAC AGCACTAATA CG 22
(2) INFORMATION FOR SEQ ID NO : 21: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 21: CCAAGCTGAT ATCACTACC 19
SUBSTTTUTE SHEET (RULE 25) (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: TCAATGCCTT TGATGGTC 18
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: TGTATGCCGC TACTCGCAGC T 21
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATIONS..13
(D) OTHER INFORMATION :/note= "X = any" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Asn Xaa Ala Xaa Xaa Tyr Ser Xaa He Gly Gly Gly Xaa Asn 1 5 10
(2) INFORMATION FOR SEQ ID NO : 25:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 25:
SUBSTTTUTE SHEET (RULE 25) Gin Ala Asp He
1
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Ala Ala Gin Ala Ala Leu Ser Gly Leu Phe Val Pro Tyr Ser Val Gly
1 5 10 15
Lys Phe Asn Ala Thr Ala Ala Leu Gly Gly Tyr Gly Ser Lys 20 25 30
(2) INFORMATION FOR SEQ ID NO : 27:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY : l inear (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 27 :
Gly Lys He Thr Lys Asn Ala Ala Arg Gin Glu Asn Gly 1 5 10
(2) INFORMATION FOR SEQ ID NO : 28:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 28:
Val He Gly Asp Leu Gly Arg Lys Val 1 5
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
SUBSTTTUTE SHEET (RULE 25) ( ix) FEATURE :
(A) NAME/KEY: Modified- site
(B) LOCATIONS
(D) OTHER INFORMATION :/note= "X = any"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 29:
Ala Leu Glu Xaa Asn Val Glu Glu Gly Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO : 30:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified- site
(B) LOCATION: 11..12
(D) OTHER INFORMATION :/note= "X = any" (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 30:
Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Xaa Xaa Leu Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO : 31;
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 31:
Ala Leu Glu Phe Asn Gly Glu 1 5
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
SUBSTTTUTE SHEET (RULE 26) ( ix) FEATURE :
(A) NAME/KEY: Modified- site
(B) LOCATION: 7
(D) OTHER INFORMATION :/note= "X = any"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Ser He Thr Asp Leu Gly Xaa Lys Val 1 5
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified- site
(B) LOCATION: 13..15
(D) OTHER INFORMATION: /note= "X = any" (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 33:
Ser He Thr Asp Leu Gly Thr He Val Asp Gly Phe Xaa Xaa Xaa 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Ser He Thr Asp Leu Gly Thr He Val Asp 1 5 10
(2) INFORMATION FOR SEQ ID NO : 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 5..19
(D) OTHER INFORMATION: /note= "X = any"
SUBSTTTUTE SHEET RULE 26) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Val Asp Ala Leu Xaa Thr Lys Val Asn Ala Leu Asp Xaa Lys Val Asn 1 5 10 15
Ser Asp Xaa Thr 20
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Leu Leu Ala Glu Gin Gin Leu Asn Gly Lys Thr Leu Thr Pro Val
1 5 10 15
(2) INFORMATION FOR SEQ ID NO : 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 37:
Ala Lys His Asp Ala Ala Ser Thr Glu Lys Gly Lys Met Asp 1 5 10
(2) INFORMATION FOR SEQ ID NO : 38: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 38:
Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly 1 5 10 15
SUBSTTTUTE SHEET (RULE 25) (2) INFORMATION FOR SEQ ID NO : 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 39:
Asn Gin Asn Thr Leu He Glu Lys Thr Ala Asn Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO : 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: He Asp Lys Asn Glu Tyr Ser He Lys
1 5
(2) INFORMATION FOR SEQ ID NO : 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 41: Ser He Thr Asp Leu Gly Thr Lys l 5
(2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42
Asn Gin Asn Thr Leu He Glu Lys 1 5
(2) INFORMATION FOR SEQ ID NO: 43:
SUBSTTTUTE SHEET (RULE 25) (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: Ala Leu His Glu Gin Gin Leu Glu Thr Leu Thr Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44 Asn Ser Ser Asp i
(2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Asn Lys Ala Asp Ala Asp Ala Ser Phe Glu Thr Leu Thr Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D ) TOPOLOGY : linear (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 46
Phe Ala Ala Thr Ala He Ala Lys Asp Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO: 47:
SUBSTTTUTE SHEET (RULE 25) (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 47:
Lys Ala Ser Ser Glu Asn Thr Gin Asn He Ala Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Arg Leu Leu Asp Gin Lys 1 5
(2) INFORMATION FOR SEQ ID NO : 49:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS :
(D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(D) OTHER INFORMATION : /note= "X = any" (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 49 :
Ala Ala Thr Ala Asp Ala He Thr Lys Asn Gly Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO : 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
SUBSTTTUTE SHEET (RULE 25) ( ix) FEATURE :
(A) NAME/KEY: Modified-site
(B) LOCATIONS..8
(D) OTHER INFORMATION: /note= "X = any"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 50:
Ala Lys Ala Xaa Ala Ala Asn Xaa Asp Arg 1 5 10
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 51:
Asn Gin Ala Asp He Ala Gin Asn Gin Thr Asp He Gin Asp Leu Ala 1 5 10 15 Ala Tyr Asn Glu Leu Gin
20
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Asn Gin Ala Asp He Ala Asn Asn He Asn Asn He Tyr Glu Leu Ala 1 5 10 15
Gin Gin Gin Asp Gin 20
(2) INFORMATION FOR SEQ ID NO : 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53
SUBSTTTUTE SHEET (RULE 26) Tyr Asn Glu Arg Gin Thr Glu Ala He Asp Ala Leu Asn 1 5 10
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: He Leu Gly Asp Thr Ala He Val Ser Asn Ser Gin Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO : 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Lys Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly 1 5 10 15
Arg
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu Glu Leu Ser Gly Arg 1 5 10 15
Thr He Asp Gin Arg 20
SUBSTTTUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID NO : 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ix) FEATURE: (A) NAME/KEY: Modified-site
(B) LOCATION: 11
(D) OTHER INFORMATION :/note= "X = any"
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 57 :
Asn Gin Ala His He Ala Asn Asn He Asn Xaa He Tyr Glu Leu Ala 1 5 10 15
Gin Gin Gin Asp Gin Lys 20
(2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS :
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Asn Gin Ala Asp He Ala Gin Asn Gin Thr Asp He Gin Asp Leu Ala 1 5 10 15
Ala Tyr Asn Glu Leu Gin 20
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: Ala Thr His Asp Tyr Asn Glu Arg Gin Thr Glu Ala
1 5 10
SUBSTTTUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID NO : 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Lys Ala Ser Ser Glu Asn Thr Gin Asn He Ala Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO : 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: Met He Leu Gly Asp Thr Ala He Val Ser Asn Ser Gin Asp Asn Lys
1 5 10 15
Thr Gin Leu Lys Phe Tyr Lys 20
(2) INFORMATION FOR SEQ ID NO: 62
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12..13
(D ) OTHER INFORMATION : /note= "X = any" (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 62 :
Ala Gly Asp Thr He He Pro Leu Asp Asp Asp Xaa Xaa Pro 1 5 10
SUBSTTTUTE SHEET (RULE 25) (2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ix) FEATURE: (A) NAME/KEY: Modified-site
(B) LOCATION: 8
(D) OTHER INFORMATION :/note= "X = any"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Leu Leu His Glu Gin Gin Leu Xaa Gly Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATIONS
(D) OTHER INFORMATION :/note= "X = any"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: He Phe Phe Asn Xaa Gly
1 5
(2) INFORMATION FOR SEQ ID NO : 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Asn Asn He Asn Asn He Tyr Glu Leu Ala Gin Gin Gin Asp Gin His 1 5 10 15
Ser Ser Asp He Lys Thr Leu 20
SUBSTTTUTE SHEET (RULE 25) (2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: GGTGCAGGTC AGATCAGTGA C 21
(2) INFORMATION FOR SEQ ID NO : 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 67:
GCCACCAACC AAGCTGAC 18
(2) INFORMATION FOR SEQ ID NO : 68:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
AGCGGTCGCC TGCTTGATCA G 21
(2) INFORMATION FOR SEQ ID NO : 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 69: CTGATCAAGC AGGCGACCGC T 21
SUBSTTTUTE SHEET (RULE 25) (2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: CAAGATCTGG CCGCTTACAA 20
(2) INFORMATION FOR SEQ ID NO : 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
TTGTAAGCGG CCAGATCTTG 20
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
TGCATGAGCC GCAAACCC 18
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: Leu Leu Ala Glu Gin Gin Leu Asn Gly
1 5
SUBSTTTUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Ala Leu Glu Ser Asn Val Glu Glu Gly Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO : 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu Asp Leu Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 76
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3788 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Thr His Glu Phe He Arg Ser Thr Ser Glu Gin Glu Asn Cys Glu Ser 1 5 10 15
Ala Arg Glu Asn Thr His Glu Asp He Ser Lys Glu Thr Thr Glu Ser 20 25 30 Ala Asn Asp Ala Thr Leu Glu Ala Ser Thr Ser Met Glu Phe Thr His
35 40 45
Glu Ser Glu Ala Leu Arg Glu Ala Asp Tyr Asn Thr His Glu Thr Asp 50 55 60
Arg He Val Glu Cys His Glu Cys Lys Trp He Thr His Gly Glu Arg 65 70 75 80
Arg He Thr His Glu Ser Glu Ser Glu Gin Glu Asn Cys Glu Ser Ala 85 90 95
Arg Glu Asn Ala Met Glu Asp Asn Thr His Glu Asp He Ser Lys Glu
SUBSTTTUTE SHEET (RULE 26) 100 105 110
Thr Thr Glu Ser Ala Asn Asp His Ala Arg Asp Cys Pro He Glu Ser 115 120 125
Ala Thr Thr Ala Cys His Glu Asp Ala Ser Phe Leu Leu Trp Ser Ala 130 135 140
Asn Asp Asp Asn Thr His Ala Val Glu Ala Asn Tyr Ser Pro Glu Cys 145 150 155 160
He Ala Leu Cys His Ala Arg Ala Cys Thr Glu Arg Ser Asp Asn Thr 165 170 175 Thr Arg Ala Asn Ser Leu Ala Thr Glu Ala Asn Tyr Ser Glu Gin Glu
180 185 190
Asn Cys Glu Ser Thr His Ala Thr He Ser Thr His Glu Arg Glu He 195 200 205
Ser Asn Ser Thr Ala Arg Thr Cys Asp Asn Ser Glu Gin He Asp Asn 210 215 220
Phe He Leu Glu Asn Ala Met Glu Thr Tyr Pro Glu Ser Thr Arg Ala 225 230 235 240
Asn Asp Thr Pro Leu Gly Tyr Ser Glu Gin He Asp Asn Glu Ser Pro 245 250 255 Ala Ala Ala Pro Arg Thr Glu He Asn Asn Ala Leu He Asn Glu Ala
260 265 270
Arg Ser Glu Gin He Asp Asn Glu Ser Pro Ala Asn Ala Asp Asn Ala 275 280 285
Asp Asx Leu Glu Leu He Asn Glu Ala Arg Ser Glu Gin He Asp Asn 290 295 300
Glu Ser Pro Ala Ala Ala Pro Arg Thr Glu He Asn Asn Ala Leu He 305 310 315 320
Asn Glu Ala Arg Ser Glu Gin He Asp Asn Glu Ser Pro Ala Asn Ala 325 330 335 Asp Asn Ala Asp Asx Leu Glu Leu He Asn Glu Ala Arg Ser Glu Gin
340 345 350
He Asp Asn Glu Ser Pro Ala Ala Ala Pro Ala Thr Pro Arg Thr Glu 355 360 365
He Asn Asn Ala Leu He Asn Glu Ala Arg Ser Glu Gin He Asp Asn 370 375 380
Glu Ser Pro Ala Asn Ala Pro Ala Thr Asp Asn Ala Asp Asx Leu Glu 385 390 395 400
SUBSTTTUTE SHEET (RULE 25) Leu He Asn Glu Ala Arg Ser Glu Gin He Asp Asn Glu Ser Pro Ala 405 410 415
Ala Ala Pro Ala Thr Pro Arg Thr Glu He Asn Asn Ala Leu He Asn 420 425 430
Glu Ala Arg Ser Glu Gin He Asp Asn Glu Ser Pro Ala Asn Ala Pro 435 440 445 Ala Thr Asp Asn Ala Asp Asx Leu Glu Leu He Asn Glu Ala Arg Ser
450 455 460
Glu Gin He Asp Asn Thr Thr Ala Ser Pro Ala Ala Ala Pro Ala Thr 465 470 475 480
Pro Arg Thr Glu He Asn Asn Ala Leu He Asn Glu Ala Arg Ser Glu 485 490 495
Gin He Asp Asn Thr Thr Ala Ser Pro Ala Asn Ala Pro Ala Thr Asp 500 505 510
Asn Ala Asp Asx Leu Glu Leu He Asn Glu Ala Arg Ser Glu Gin He 515 520 525 Asp Asn Thr Thr Ala Ser Pro Ala Ala Ala Pro Ala Thr Pro Arg Thr
530 535 540
Glu He Asn Asn Ala Leu He Asn Glu Ala Arg Ser Glu Gin He Asp 545 550 555 560
Asn Thr Thr Ala Ser Pro Ala Asn Ala Pro Ala Thr Asp Asn Ala Asp 565 570 575
Asx Leu Glu Leu He Asn Glu Ala Arg Ser Glu Gin He Asp Asn Thr 580 585 590
Thr Ala Ser Pro Ala Ala Ala Pro Ala Thr Pro Arg Thr Glu He Asn 595 600 605 Asn Ala Leu He Asn Glu Ala Arg Ser Glu Gin He Asp Asn Thr Thr
610 615 620
Ala Ser Pro Ala Asn Ala Pro Ala Thr Asp Asn Ala Asp Asx Leu Glu
625 630 635 640
Leu He Asn Glu Ala Arg Ser Glu Gin He Asp Asn Thr Thr Ala Ser 645 650 655
Pro Ala Ala Ala Pro Ala Thr Pro Arg Thr Glu He Asn Asn Ala Leu 660 665 670
He Asn Glu Ala Arg Ser Glu Gin He Asp Asn Thr Thr Ala Ser Pro 675 680 685 Ala Asn Ala Pro Ala Thr Asp Asn Ala Asp Asx Leu Glu Leu He Asn
690 695 700
SUBSTTTUTE SHEET RULE 25) Glu Ala Arg Ser Glu Gin He Asp Asn Thr His Arg Gly His Ser Glu 705 710 715 720
Gin He Asp Asn He Ser Gly He Val Glu Asn Asx Glu Leu Trp Ala 725 730 735
Asn Asp Ala Arg Glu Asn Thr Asn Thr His Glu Asp He Ser Lys Glu 740 745 750 Thr Thr Glu Ser Ser Glu Gin Glu Asn Cys Glu Ser Glu Gin He Asp
755 760 765
Asn Thr Tyr Pro Glu Thr Pro Leu Gly Tyr Ser Thr Arg Ala Asn Asp 770 775 780
Ser Pro Glu Cys He Ala Leu Ala Gin Gin Gin Asp Gin His Ser Glu 785 790 795 800
Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg 805 810 815
Asn Ala Tyr Glu Leu Ala Gin Gin Gin Asp Gin His Ser Glu Gin He 820 825 830 Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala
835 840 845
Tyr Asp Leu Ala Gin Gin Gin Asp Gin His Ser Glu Gin He Asp Asn 850 855 860
Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Gly Ala 865 870 875 880
Cys Gly Cys Thr Cys Ala Ala Cys Ala Gly Cys Ala Cys Thr Ala Ala 885 890 895
Thr Ala Cys Gly Ser Glu Gin He Asp Asn Asp Asn Ala Leu He Asn 900 905 910 Glu Ala Arg Asp Asx Leu Glu Cys Cys Ala Ala Gly Cys Thr Gly Ala
915 920 925
Thr Ala Thr Cys Ala Cys Thr Ala Cys Cys Ser Glu Gin He Asp Asn 930 935 940
Asp Asn Ala Leu He Asn Glu Ala Arg Asp Asx Leu Glu Thr Cys Ala 945 950 955 960
Ala Thr Gly Cys Cys Thr Thr Thr Gly Ala Thr Gly Gly Thr Cys Ser 965 970 975
Glu Gin He Asp Asn Asp Asn Ala Leu He Asn Glu Ala Arg Asp Asx 980 985 990 Leu Glu Thr Gly Thr Ala Thr Gly Cys Cys Gly Cys Thr Ala Cys Thr
995 1000 1005
SUBSTTTUTE SHEET (RULE 26) Cys Gly Cys Ala Gly Cys Thr Ser Glu Gin He Asp Asn Asp Asn Ala 1010 1015 1020
Leu He Asn Glu Ala Arg Asp Asx Leu Glu Asn Xaa Ala Xaa Xaa Tyr 1025 1030 1035 1040
Ser Xaa He Gly Gly Gly Xaa Asn Ser Glu Gin He Asp Asn Pro Arg 1045 1050 1055 Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr
1060 1065 1070
Ala Thr Pro Ser He Thr He Asn Ser Gin Ala Asp He Ser Glu Gin 1075 1080 1085
He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn
1090 1095 1100
Ala Ala Ala Gin Ala Ala Leu Ser Gly Leu Phe Val Pro Tyr Ser Val 1105 1110 1115 1120
Gly Lys Phe Asn Ala Thr Ala Ala Leu Gly Gly Tyr Gly Ser Lys Ser 1125 1130 1135 Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala
1140 1145 1150
Arg Asn Ala Gly Lys He Thr Lys Asn Ala Ala Arg Gin Glu Asn Gly 1155 1160 1165
Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu 1170 1175 1180
Ala Arg Asn Ala Val He Gly Asp Leu Gly Arg Lys Val Ser Glu Gin 1185 1190 1195 1200
He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn 1205 1210 1215 Ala Ala Leu Glu Xaa Asn Val Glu Glu Gly Leu Ser Glu Gin He Asp
1220 1225 1230
Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Xaa 1235 1240 1245
Ala Asn Tyr Ala Thr Pro Ser He Thr He Asn Ala Leu Glu Ser Asn 1250 1255 1260
Val Glu Glu Gly Leu Xaa Xaa Leu Ser Ser Glu Gin He Asp Asn Pro 1265 1270 1275 1280
Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Xaa Ala Asn 1285 1290 1295 Tyr Ala Thr Pro Ser He Thr He Asn Ser Ala Leu Glu Phe Asn Gly
1300 1305 1310
SUBSTTTUTE SHEET (RULE 25) Glu Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn 1315 1320 1325
Glu Ala Arg Asn Ala Ser He Thr Asp Leu Gly Xaa Lys Val Ser Glu 1330 1335 1340
Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg 1345 1350 1355 1360 Asn Ala Xaa Ala Asn Tyr Ala Thr Pro Ser He Thr He Asn Ser He
1365 1370 1375
Thr Asp Leu Gly Thr He Val Asp Gly Phe Xaa Xaa Xaa Ser Glu Gin 1380 1385 1390
He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn 1395 1400 1405
Ala Xaa Ala Asn Tyr Ala Thr Pro Ser He Thr He Asn Ser Ser He 1410 1415 1420
Thr Asp Leu Gly Thr He Val Asp Ser Glu Gin He Asp Asn Pro Arg 1425 1430 1435 1440 Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Val Asp Ala Leu
1445 1450 1455
Xaa Thr Lys Val Asn Ala Leu Asp Xaa Lys Val Asn Ser Asp Xaa Thr 1460 1465 1470
Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu 1475 1480 1485
Ala Arg Asn Ala Xaa Ala Asn Tyr Ala Thr Pro Ser He Thr He Asn 1490 1495 1500
Ser Leu Leu Ala Glu Gin Gin Leu Asn Gly Lys Thr Leu Thr Pro Val
1505 1510 1515 1520 Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu
1525 1530 1535
Ala Arg Asn Ala Ala Lys His Asp Ala Ala Ser Thr Glu Lys Gly Lys 1540 1545 1550
Met Asp Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He 1555 1560 1565
Asn Glu Ala Arg Asn Ala Ala Leu Glu Ser Asn Val Glu Glu Gly Leu 1570 1575 1580
Leu Asp Leu Ser Gly Ser Glu Gin He Asp Asn Pro Arg Thr Glu He 1585 1590 1595 1600 Asn Leu He Asn Glu Ala Arg Asn Ala Asn Gin Asn Thr Leu He Glu
1605 1610 1615
SUBSTTTUTE SHEET (RULE 25) Lys Thr Ala Asn Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He 1620 1625 1630
Asn Leu He Asn Glu Ala Arg Asn Ala He Asp Lys Asn Glu Tyr Ser 1635 1640 1645
He Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He 1650 1655 1660 Asn Glu Ala Arg Asn Ala Ser He Thr Asp Leu Gly Thr Lys Ser Glu
1665 1670 1675 1680
Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg 1685 1690 1695
Asn Ala Asn Gin Asn Thr Leu He Glu Lys Ser Glu Gin He Asp Asn 1700 1705 1710
Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Ala Leu 1715 1720 1725
His Glu Gin Gin Leu Glu Thr Leu Thr Lys Ser Glu Gin He Asp Asn 1730 1735 1740 Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Asn Ser
1745 1750 1755 1760
Ser Asp Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He 1765 1770 1775
Asn Glu Ala Arg Asn Ala Asn Lys Ala Asp Ala Asp Ala Ser Phe Glu 1780 1785 1790
Thr Leu Thr Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn 1795 1800 1805
Leu He Asn Glu Ala Arg Asn Ala Phe Ala Ala Thr Ala He Ala Lys 1810 1815 1820 Asp Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He
1825 1830 1835 1840
Asn Glu Ala Arg Asn Ala Lys Ala Ser Ser Glu Asn Thr Gin Asn He 1845 1850 1855
Ala Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He 1860 1865 1870
Asn Glu Ala Arg Asn Ala Arg Leu Leu Asp Gin Lys Ser Glu Gin He 1875 1880 1885
Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala 1890 1895 1900 Ala Ala Thr Ala Asp Ala He Thr Lys Asn Gly Xaa Ser Glu Gin He
1905 1910 1915 1920
SUBSTTTUTE SHEET (RULE 25) Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala 1925 1930 1935
Ala Lys Ala Xaa Ala Ala Asn Xaa Asp Arg Ser Glu Gin He Asp Asn 1940 1945 1950
Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Xaa Ala 1955 1960 1965 Asn Tyr Ala Thr Pro Ser He Thr He Asn Ser Asn Gin Ala Asp He
1970 1975 1980
Ala Gin Asn Gin Thr Asp He Gin Asp Leu Ala Ala Tyr Asn Glu Leu 1985 1990 1995 2000
Gin Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn 2005 2010 2015
Glu Ala Arg Asn Ala Asn Gin Ala Asp He Ala Asn Asn He Asn Asn 2020 2025 2030
He Tyr Glu Leu Ala Gin Gin Gin Asp Gin Ser Glu Gin He Asp Asn 2035 2040 2045 Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Tyr Asn
2050 2055 2060
Glu Arg Gin Thr Glu Ala He Asp Ala Leu Asn Ser Glu Gin He Asp 2065 2070 2075 2080
Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala He 2085 2090 2095
Leu Gly Asp Thr Ala He Val Ser Asn Ser Gin Asp Ser Glu Gin He 2100 2105 2110
Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala 2115 2120 2125 Lys Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly
2130 2135 2140
Arg Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn 2145 2150 2155 2160
Glu Ala Arg Asn Ala Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu
2165 2170 2175
Glu Leu Ser Gly Arg Thr He Asp Gin Arg Ser Glu Gin He Asp Asn 2180 2185 2190
Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Asn Gin 2195 2200 2205 Ala His He Ala Asn Asn He Asn Xaa He Tyr Glu Leu Ala Gin Gin
2210 2215 2220
SUBSTTTUTE SHEET (RULE 26) Gin Asp Gin Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn 2225 2230 2235 2240
Leu He Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr Ala Thr Pro Ser 2245 2250 2255
He Thr He Asn Asn Gin Ala Asp He Ala Gin Asn Gin Thr Asp He 2260 2265 2270 Gin Asp Leu Ala Ala Tyr Asn Glu Leu Gin Ser Glu Gin He Asp Asn
2275 2280 2285
Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Ala Thr 2290 2295 2300
His Asp Tyr Asn Glu Arg Gin Thr Glu Ala Ser Glu Gin He Asp Asn 2305 2310 2315 2320
Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Lys Ala 2325 2330 2335
Ser Ser Glu Asn Thr Gin Asn He Ala Lys Ser Glu Gin He Asp Asn 2340 2345 2350 Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Met He
2355 2360 2365
Leu Gly Asp Thr Ala He Val Ser Asn Ser Gin Asp Asn Lys Thr Gin 2370 2375 2380
Leu Lys Phe Tyr Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He 2385 2390 2395 2400
Asn Leu He Asn Glu Ala Arg Asn Ala Ala Gly Asp Thr He He Pro 2405 2410 2415
Leu Asp Asp Asp Xaa Xaa Pro Ser Glu Gin He Asp Asn Pro Arg Thr 2420 2425 2430 Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr Ala
2435 2440 2445
Thr Pro Ser He Thr He Asn Ser Leu Leu His Glu Gin Gin Leu Xaa 2450 2455 2460
Gly Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He 2465 2470 2475 2480
Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr Ala Thr Pro Ser He Thr 2485 2490 2495
He Asn He Phe Phe Asn Xaa Gly Ser Glu Gin He Asp Asn Pro Arg 2500 2505 2510 Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr
2515 2520 2525
SUBSTTTUTE SHEET (RULE 26) Ala Thr Pro Ser He Thr He Asn Asn Asn He Asn Asn He Tyr Glu 2530 2535 2540
Leu Ala Gin Gin Gin Asp Gin His Ser Ser Asp He Lys Thr Leu Ser 2545 2550 2555 2560
Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala 2565 2570 2575 Arg Asn Ala Gly Gly Thr Gly Cys Ala Gly Gly Thr Cys Ala Gly Ala
2580 2585 2590
Thr Cys Ala Gly Thr Gly Ala Cys Ser Glu Gin He Asp Asn Asp Asn 2595 2600 2605
Ala Leu He Asn Glu Ala Arg Asp Asx Leu Glu Gly Cys Cys Ala Cys 2610 2615 2620
Cys Ala Ala Cys Cys Ala Ala Gly Cys Thr Gly Ala Cys Ser Glu Gin 2625 2630 2635 2640
He Asp Asn Asp Asn Ala Leu He Asn Glu Ala Arg Asp Asx Leu Glu 2645 2650 2655 Ala Gly Cys Gly Gly Thr Cys Gly Cys Cys Thr Gly Cys Thr Thr Gly
2660 2665 2670
Ala Thr Cys Ala Gly Ser Glu Gin He Asp Asn Asp Asn Ala Leu He 2675 2680 2685
Asn Glu Ala Arg Asp Asx Leu Glu Cys Thr Gly Ala Thr Cys Ala Ala 2690 2695 2700
Gly Cys Ala Gly Gly Cys Gly Ala Cys Cys Gly Cys Thr Ser Glu Gin 2705 2710 2715 2720
He Asp Asn Asp Asn Ala Leu He Asn Glu Ala Arg Asp Asx Leu Glu 2725 2730 2735 Cys Ala Ala Gly Ala Thr Cys Thr Gly Gly Cys Cys Gly Cys Thr Thr
2740 2745 2750
Ala Cys Ala Ala Ser Glu Gin He Asp Asn Asp Asn Ala Leu He Asn 2755 2760 2765
Glu Ala Arg Asp Asx Leu Glu Thr Thr Gly Thr Ala Ala Gly Cys Gly 2770 2775 2780
Gly Cys Cys Ala Gly Ala Thr Cys Thr Thr Gly Ser Glu Gin He Asp 2785 2790 2795 2800
Asn Asp Asn Ala Leu He Asn Glu Ala Arg Asp Asx Leu Glu Thr Gly 2805 2810 2815 Cys Ala Thr Gly Ala Gly Cys Cys Gly Cys Ala Ala Ala Cys Cys Cys
2820 2825 2830
SUBSTTTUTE SHEET (RULE b) Ser Glu Gin He Asp Asn Asp Asn Ala Leu He Asn Glu Ala Arg Asp 2835 2840 2845
Asx Leu Glu Leu Leu Ala Glu Gin Gin Leu Asn Gly Ser Glu Gin He 2850 2855 2860
Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala 2865 2870 2875 2880 Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Ser Glu Gin He Asp Asn
2885 2890 2895
Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Ala Leu 2900 2905 2910
Glu Ser Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Ser Glu Gin He
2915 2920 2925
Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala 2930 2935 2940
Asn Ala Lys Ala Ser Ala Ala Asn Thr Asp Arg Ser Glu Gin He Asp 2945 2950 2955 2960 Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Ala
2965 2970 2975
Ala Thr Ala Ala Asp Ala He Thr Lys Asn Gly Asn Ser Glu Gin He 2980 2985 2990
Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala 2995 3000 3005
Ser He Thr Asp Leu Gly Thr Lys Val Asp Gly Phe Asp Gly Arg Ser 3010 3015 3020
Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala 3025 3030 3035 3040 Arg Asn Ala Val Asp Ala Leu Xaa Thr Lys Val Asn Ala Leu Asp Xaa
3045 3050 3055
Lys Val Asn Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu 3060 3065 3070
He Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr Ala Thr Pro Ser He 3075 3080 3085
Thr He Asn Ser Ala Ala Gin Ala Ala Leu Ser Gly Leu Phe Val Pro 3090 3095 3100
Tyr Ser Val Gly Lys Phe Asn Ala Thr Ala Ala Leu Gly Gly Tyr Gly 3105 3110 3115 3120 Ser Lys Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He
3125 3130 3135
SUBSTTTUTE SHEET (RULE 25) Asn Glu Ala Arg Asn Ala Ser Gly Arg Leu Leu Asp Gin Lys Ala Asp 3140 3145 3150
Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu 3155 3160 3165
Ala Arg Asn Ala Gin Lys Ala Asp He Asp Asn Asn He Asn Ser Glu 3170 3175 3180 Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg
3185 3190 3195 3200
Asn Ala Asn Asn He Asn Asn He Tyr Glu Leu Ala Ser Glu Gin He 3205 3210 3215
Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala 3220 3225 3230
Asn Asn He Tyr Glu Leu Ala Gin Gin Gin Ser Glu Gin He Asp Asn 3235 3240 3245
Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Ala Gin 3250 3255 3260 Gin Gin Asp Gin His Ser Ser Asp Ser Glu Gin He Asp Asn Pro Arg
3265 3270 3275 3280
Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Gin Asp Gin His 3285 3290 3295
Ser Ser Asp He Lys Thr Ser Glu Gin He Asp Asn Pro Arg Thr Glu 3300 3305 3310
He Asn Leu He Asn Glu Ala Arg Asn Ala His Ser Ser Asp He Lys 3315 3320 3325
Thr Leu Lys Asn Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn 3330 3335 3340 Leu He Asn Glu Ala Arg Asn Ala Asp He Lys Thr Leu Lys Asn Asn 3345 3350 3355 3360
Val Glu Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He 3365 3370 3375
Asn Glu Ala Arg Asn Ala Thr Leu Lys Asn Asn Val Glu Glu Gly Leu 3380 3385 3390
Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu 3395 3400 3405
Ala Arg Asn Ala Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Ser Glu 3410 3415 3420 Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg
3425 3430 3435 3440
SUBSTTTUTE SHEET (RULE 25) Asn Ala Leu Ser Gly Arg Leu He Asp Gin Lys Ala Ser Glu Gin He 3445 3450 3455
Asp Asn Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala 3460 3465 3470
Asp Gin Lys Ala Asp He Ala Lys Asn Gin Ser Glu Gin He Asp Asn 3475 3480 3485 Pro Arg Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala Ala Lys
3490 3495 3500
Asn Gin Ala Asp He Ala Gin Asn Ser Glu Gin He Asp Asn Pro Arg 3505 3510 3515 3520
Thr Glu He Asn Leu He Asn Glu Ala Arg Asn Ala He Ala Gin Asn 3525 3530 3535
Gin Thr Asp He Gin Asp Ser Glu Gin He Asp Asn Pro Arg Thr Glu 3540 3545 3550
He Asn Leu He Asn Glu Ala Arg Asn Ala Asp He Gin Asp Leu Ala 3555 3560 3565 Ala Tyr Asn Glu Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn
3570 3575 3580
Leu He Asn Glu Ala Arg Asn Ala Cys Gly Gly Gly Ala Thr Cys Cys 3585 3590 3595 3600
Gly Thr Gly Ala Ala Gly Ala Ala Ala Ala Ala Thr Gly Cys Cys Gly 3605 3610 3615
Cys Ala Gly Gly Thr Ser Glu Gin He Asp Asn Asp Asn Ala Leu He 3620 3625 3630
Asn Glu Ala Arg Asp Asx Leu Glu Cys Gly Gly Gly Ala Thr Cys Cys 3635 3640 3645 Cys Gly Thr Cys Gly Cys Ala Ala Gly Cys Cys Gly Ala Thr Thr Gly
3650 3655 3660
Ser Glu Gin He Asp Asn Asp Asn Ala Leu He Asn Glu Ala Arg Asp 3665 3670 3675 3680
Asx Leu Glu Ser Gly Arg Leu Leu Asp Gin Lys Ala Asp He Asp Asn 3685 3690 3695
Asn He Asn Asn He Tyr Glu Leu Ala Gin Gin Gin Asp Gin His Ser 3700 3705 3710
Ser Asp He Lys Thr Leu Lys Asn Asn Val Glu Glu Gly Leu Leu Asp 3715 3720 3725 Leu Ser Gly Arg Leu He Asp Gin Lys Ala Asp He Ala Lys Asn Gin
3730 3735 3740
SUBSTTTUTE SHEET (RULE 25) Ala Asp He Ala Gin Asn Gin Thr Asp He Gin Asp Leu Ala Ala Tyr 3745 3750 3755 3760
Asn Glu Ser Glu Gin He Asp Asn Pro Arg Thr Glu He Asn Leu He 3765 3770 3775
Asn Glu Ala Arg Asn Ala Ala Trp Glx Xaa Asp Cys 3780 3785
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Ala Ala Thr Ala Ala Asp Ala He Thr Lys Asn Gly Asn 1 5 10
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: Ser He Thr Asp Leu Gly Thr Lys Val Asp Gly Phe Asp Gly Arg
1 5 10 15
(2) INFORMATION FOR SEQ ID NO : 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATIONS..13 (D) OTHER INFORMATION :/note= "X = any"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 79:
Val Asp Ala Leu Xaa Thr Lys Val Asn Ala Leu Asp Xaa Lys Val Asn 1 5 10 15
SUBSTTTUTE SHEET (RULE 25) (2) INFORMATION FOR SEQ ID NO : 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Ala Ala Gin Ala Ala Leu Ser Gly Leu Phe Val Pro Tyr Ser Val Gly 1 5 10 15
Lys Phe Asn Ala Thr Ala Ala Leu Gly Gly Tyr Gly Ser Lys 20 25 30
(2) INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
Ser Gly Arg Leu Leu Asp Gin Lys Ala Asp 1 5 10
(2) INFORMATION FOR SEQ ID NO : 82:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
Gin Lys Ala Asp He Asp Asn Asn He Asn 1 5 10
(2) INFORMATION FOR SEQ ID NO: 83
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY : linear
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 83 :
Asn Asn He Asn Asn He Tyr Glu Leu Ala 1 5 10
SUBSTTTUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY : l inear
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 84 :
Asn Asn He Tyr Glu Leu Ala Gin Gin Gin 1 5 10
(2) INFORMATION FOR SEQ ID NO : 85:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 85:
Ala Gin Gin Gin Asp Gin His Ser Ser Asp 1 5 10
(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
Gin Asp Gin His Ser Ser Asp He Lys Thr 1 5 10
(2) INFORMATION FOR SEQ ID NO : 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: His Ser Ser Asp He Lys Thr Leu Lys Asn
1 5 10
SUBSTTTUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID NO : 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 88:
Asp He Lys Thr Leu Lys Asn Asn Val Glu 1 5 10
(2) INFORMATION FOR SEQ ID NO : 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 89:
Thr Leu Lys Asn Asn Val Glu Glu Gly Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg 1 5 10
(2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
Leu Ser Gly Arg Leu He Asp Gin Lys Ala 1 5 10
SUBSTTTUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
Asp Gin Lys Ala Asp He Ala Lys Asn Gin 1 5 10
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: Ala Lys Asn Gin Ala Asp He Ala Gin Asn
1 5 10
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
He Ala Gin Asn Gin Thr Asp He Gin Asp 1 5 10
(2) INFORMATION FOR SEQ ID NO: 95: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
Asp He Gin Asp Leu Ala Ala Tyr Asn Glu 1 5 10
(2) INFORMATION FOR SEQ ID NO: 96:
SUBSTTTUTE SHEET (RULE 25) (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96: CGGGATCCGT GAAGAAAAAT GCCGCAGGT 29
(2) INFORMATION FOR SEQ ID NO: 97: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: CGGGATCCCG TCGCAAGCCG ATTG 24
(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
Ser Gly Arg Leu Leu Asp Gin Lys Ala Asp He Asp Asn Asn He Asn 1 5 10 15
Asn He Tyr Glu Leu Ala Gin Gin Gin Asp Gin His Ser Ser Asp He 20 25 30
Lys Thr Leu Lys Asn Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly 35 40 45 Arg Leu He Asp Gin Lys Ala Asp He Ala Lys Asn Gin Ala Asp He 50 55 60
Ala Gin Asn Gin Thr Asp He Gin Asp Leu Ala Ala Tyr Asn Glu 65 70 75
SUBSTTTUTE SHEET (RULE 25)

Claims

1. An isolated peptide of about 7 to about 60 amino acids comprising the amino acid sequence AQQQDQH (SEQ ID NO: 17).
2. The isolated peptide of claim 1, wherein said peptide is about 10 amino acids in length.
3. The isolated peptide of claim 1, wherein said peptide is about 20 amino acids in length.
4. The isolated peptide of claim 1, wherein said peptide is about 30 amino acids in length.
5. The isolated peptide of claim 1, wherein said peptide is about 40 amino acids in length.
6. The isolated peptide of claim 1, wherein said peptide is about 50 amino acids in length.
7. The isolated peptide of claim 1, wherein said peptide is about 60 amino acids in length.
The isolated peptide of claim 1 , wherein said peptide is at least about 16 consecutive residues of the amino acid sequence YELAQQQDQH (SEQ ID NO: 18).
SUBSTTTUTE SHEET (RULE 26) An antigenic composition comprising (a) an isolated peptide of about 7 to about 60 amino acids comprising the amino acid sequence AQQQDQH (SEQ ID NO: 17) and (b) a pharmaceutically acceptable buffer or diluent.
10. The antigenic composition of claim 9, wherein said antigenic composition further comprises a carrier conjugated to said peptide.
11. The antigenic composition of claim 10, wherein said carrier is KLH, diphtheria toxoid, tetanus toxoid or CRM|97.
12. The antigenic composition of claim 9, further comprising an adjuvant.
13. The antigenic composition of claim 12, wherein said adjuvant comprises a lipid.
14. The antigenic composition of claim 9 wherein said peptide is covalently linked to a second antigen.
15. The antigenic composition of claim 14, wherein said second antigen is a peptide antigen.
16. The antigenic composition of claim 14, wherein said second antigen is a non-peptide antigen.
SUBSTTTUTE SHEET (RULE 25)
17. The antigenic composition of claim 9, wherein said isolated peptide comprises at least about 16 consecutive residues of the amino acid sequence YELAQQQDQH (SEQ ID NO: 18).
18. A vaccine composition comprising an isolated peptide of about 7 to about 60 amino acids comprising the amino acid sequence AQQQDQH (SEQ ID NO: 17) and a pharmaceutically acceptable buffer or diluent.
19. The vaccine composition of claim 18, wherein said isolated peptide is further defined as comprising at least about 16 consecutive residues of the amino acid sequence YELAQQQDQH (SEQ ID NO: 18).
20. A method for inducing an immune response in a mammal comprising the step of providing to said mammal an antigenic composition comprising (a) an isolated peptide of about 7 to about 60 amino acids comprising the amino acid sequence AQQQDQH
(SEQ ID NO: 17) and (b) a pharmaceutically acceptable buffer or diluent.
21. The method of claim 20, wherein said isolated peptide is further defined as comprising at least about 16 consecutive residues of the amino acid sequence YELAQQQDQH (SEQ ID NO: 18).
22. A nucleic acid encoding the UspAl antigen (SEQ ID NOT) of the M. catarrhalis isolate O35E.
SUBSTTTUTE SHEET (RULE 26)
23. A nucleic acid having the uspAl DNA sequence (SEQ ID NO:2) of the M. catarrhalis isolate 035E.
24. A nucleic acid encoding the UspA2 antigen (SEQ ID NO:3) of the M. catarrhalis isolate O35E.
25. A nucleic acid having the uspA2 DNA sequence (SEQ ID NO:4) of the M. catarrhalis isolate 035E.
26. A nucleic acid encoding the UspAl antigen (SEQ ID NO:5) of the M. catarrhalis isolate
O46E.
27. A nucleic acid having the uspAl DNA sequence (SEQ ID NO:6) of the M. catarrhalis isolate O46E.
28. A nucleic acid encoding the UspA2 antigen (SEQ ID NO:7) of the M. catarrhalis isolate O46E.
29. A nucleic acid having the uspA2 DNA sequence (SEQ ID NO:8) of the M. catarrhalis isolate O46E.
30. A nucleic acid encoding the UspAl antigen (SEQ ID NO:9) of the M. catarrhalis isolate TTA24.
SUBSTTTUTE SHEET (RULE 25)
31. A nucleic acid having the uspAl DNA sequence (SEQ ID NO TO) of the M. catarrhalis isolate TTA24.
32. A nucleic acid encoding the UspA2 antigen (SEQ ID NOT 1) of the M. catarrhalis isolate TTA24.
33. A nucleic acid having the uspA2 DNA sequence (SEQ ID NO: 12) of the M. catarrhalis isolate TTA24.
34. A nucleic acid encoding the UspAl antigen (SEQ ID NO:13) of the M. catarrhalis isolate TTA37.
35. A nucleic acid having the uspAl DNA sequence (SEQ ID NO: 14) of the M. catarrhalis isolate TTA37.
36. A nucleic acid encoding the UspA2 antigen (SEQ ID NO: 15) of the M. catarrhalis isolate TTA37.
37. A nucleic acid having the uspA2 DNA sequence (SEQ ID NO: 16) of the M. catarrhalis isolate TTA37.
SUBSTTTUTE SHEET RULE 26)
38. A method for diagnosing M. catarrhalis infection comprising the step of determining the presence, in a sample, of an M. catarrhalis amino acid sequence corresponding to residues of epitopic core sequences of said UspAl or UspA2 antigen.
39. The method of claim 38, wherein said determining comprises PCR.
40. The method of claim 38, wherein said determining comprises immunologic reactivity of an antibody to an M. catarrhalis antigen.
41. A method for treating an individual having an M. catarrhalis infection comprising providing to said individual an isolated peptide of about 7 to about 60 amino acids comprising the amino acid sequence AQQQDQH (SEQ ID NO: 17).
42. The isolated peptide of claim 41, wherein the said peptide comprises at least about 16 consecutive residues of the amino acid sequence YELAQQQDQH (SEQ ID NOT 8).
43. A method for preventing or limiting an M. catarrhalis infection comprising providing to a subject an antibody that reacts immunologically with an epitope formed by the amino acid sequence AQQQDQH (SEQ ID NO: 17).
44. The method of claim 42, wherein said epitope is further defined as comprising at least about 16 consecutive residues of the amino acid sequence YELAQQQDQH (SEQ ID NOT 8).
SUBSTTTUTE SHEET RULE 26)
45. A method for screening a peptide for reactivity with an antibody that bind immunologically to UspAl or UspA2 comprising the steps of:
a) providing said peptide;
b) contacting said peptide with said antibody; and
c) determining the binding of said antibody to said peptide.
46. The method of claim 45, wherein said antibody is 17C7, 45-2, 13-1, 29-31, 16A7, 17B1 or 5C12.
47. The method of claim 46, wherein said antibody is 17C7.
48. The method of claim 46, wherein said antibody is 45-2.
49. The method of claim 46, wherein said antibody is 13-1.
50. The method of claim 46, wherein said antibody is 29-31.
51. The method of claim 46, wherein said antibody is 16A7.
52. The method of claim 46, wherein said antibody is 5C 12.
53. The method of claim 46, wherein said antibody is 17B1.
54. The method of claim 45, wherein said determining comprises an immunoassay selected from the group consisting of a western blot, an ELISA, and RIA and immunoaffinity separation.
55. A method for screening a UspAl or UspA2 peptide for the ability to induce a protective immune response against M. catarrhalis comprising the steps of:
a) providing said peptide;
b) administering a peptide in a suitable form to an experimental animal;
c) challenging said animal with M. catarrhalis; and
d) assaying the infection of said animal with M. catarrhalis.
56. The method of claim 56, wherein said animal is a mouse, said challenging is a pulmonary challenge, and said assaying comprises assessing the degree of pulmonary clearance by said mouse.
57. The method of claim 56, wherein said UspAl peptide encompasses about residues 582-604 (SEQ ID NOT) of M. catarrhalis or the analogous position thereof when compared to M. catarrhalis strain 035E.
58. The method of claim 56, wherein said UspA2 peptide encompasses about residues 355-377 (SEQ ID NO:3) of M. catarrhalis or the analogous position thereof when compared to M. catarrhalis strain O35E.
SUBSTTTUTE SHEET (RULE 26)
59. The method of claim 57, wherein said UspAl peptide includes about residues 452-642 (SEQ ID NOT) of M. catarrhalis or the analogous positions thereof when compared to M. catarrhalis strain 035E.
60. The method of claim 58, wherein said UspA2 peptide includes about residues 242-415 (SEQ ID NO:3) of M. catarrhalis, or the analogous position thereof when compared to M. catarrhalis strain 035E.
61. An isolated peptide having at least about 7 consecutive amino acids from the UspAl or
UspA2 protein of M. catarrhalis, wherein said peptide includes residues located within the region defined by about residues 582-604 of said UspAl protein (SEQ ID NOT), or by about residues 355-377 of said UspA2 protein (SEQ ID NO:3), or the analogous positions thereof when compared to strain O35E.
62. The isolated peptide of claim 61, wherein said peptide is between 7 and 60 amino acids in length.
63. The isolated peptide of claim 61, wherein said peptide comprises non-UspAl or non-UspA2 sequences.
64. The isolated peptide of claim 61, wherein said peptide comprises non- catarrhalis sequences.
SUBSTTTUTE SHEET (RULE 25)
65. An antigenic composition comprising
a) an isolated peptide having at least about 7 consecutive amino acids from the UspAl or UspA2 protein of M. catarrhalis, wherein said amino acids include residues located within the region defined by about residues 582-604 of said UspAl protein (SEQ ID NOT), or by about residues 355-377 of said UspA2 protein (SEQ ID NO:3), or the analogous positions thereof when compared to strain 035E.
b) a pharmaceutically acceptable buffer or diluent.
66. An antigenic composition comprising
a) an isolated peptide of about 7 to about 60 amino acids comprising at least 7 consecutive residues of the amino acid sequence of UspAl or UspA2 wherein said isolated peptide acts as a carrier covalently linked to a second antigen; and
b) a pharmaceutically acceptable buffer or diluent.
67. The antigenic composition of claim 66, wherein said second antigen is a peptide antigen.
68. The antigenic composition of claim 66, wherein said second antigen is a non-peptide antigen.
SUBSTTTUTE SHEET (RULE 26)
PCT/US1997/023930 1996-12-20 1997-12-19 USPA1 AND USPA2 ANTIGENS OF $i(MORAXELLA CATARRHALIS) WO1998028333A2 (en)

Priority Applications (13)

Application Number Priority Date Filing Date Title
AT97953461T ATE497004T1 (en) 1996-12-20 1997-12-19 MORAXELLA CATARRHALIS ANTIGENS USPA1 AND USPA2
CA002274495A CA2274495C (en) 1996-12-20 1997-12-19 Uspa1 and uspa2 antigens of moraxella catarrhalis
JP52907598A JP2001515467A (en) 1996-12-20 1997-12-19 USPA1 and USPA2 antigens of Moraxella catarrhalis
DE69740108T DE69740108D1 (en) 1996-12-20 1997-12-19 MORAXELLA CATARRHALIS ANTIGENE USPA1 AND USPA2
BR9714160-7A BR9714160A (en) 1996-12-20 1997-12-19 Morapaella catarrhalis uspa1 and uspa2 antigens
EP97953461A EP0948625B1 (en) 1996-12-20 1997-12-19 Uspa1 and uspa2 antigens of moraxella catarrhalis
AU57201/98A AU746442B2 (en) 1996-12-20 1997-12-19 UspA1 and UspA2 antigens of Moraxella catarrhalis
US09/336,447 US6310190B1 (en) 1996-12-20 1999-06-21 USPA1 and USPA2 antigens of Moraxella catarrhalis
US09/952,267 US6753417B2 (en) 1996-12-20 2001-09-12 UspA1 and UspA2 antigens of Moraxella catarrhalis
US10/872,768 US7288646B2 (en) 1996-12-20 2004-06-21 USPA2 nucleic acid of Moraxella catarrhalis
US10/872,769 US7344724B2 (en) 1996-12-20 2004-06-21 UspA1 and UspA2 antigens of Moraxella catarrhalis
US12/044,805 US7576194B2 (en) 1996-12-20 2008-03-07 UspA2 nucleic acid of moraxella catarrhalis
US12/044,818 US7569683B2 (en) 1996-12-20 2008-03-07 UspA2 nucleic acid of Moraxella catarrhalis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3359896P 1996-12-20 1996-12-20
US60/033,598 1996-12-20

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US09/336,447 Continuation US6310190B1 (en) 1996-12-20 1999-06-21 USPA1 and USPA2 antigens of Moraxella catarrhalis

Publications (3)

Publication Number Publication Date
WO1998028333A2 true WO1998028333A2 (en) 1998-07-02
WO1998028333A9 WO1998028333A9 (en) 1998-10-22
WO1998028333A3 WO1998028333A3 (en) 1999-01-07

Family

ID=21871324

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/023930 WO1998028333A2 (en) 1996-12-20 1997-12-19 USPA1 AND USPA2 ANTIGENS OF $i(MORAXELLA CATARRHALIS)

Country Status (11)

Country Link
US (6) US6310190B1 (en)
EP (1) EP0948625B1 (en)
JP (2) JP2001515467A (en)
KR (1) KR100615109B1 (en)
CN (1) CN1159441C (en)
AT (1) ATE497004T1 (en)
AU (2) AU746442B2 (en)
BR (1) BR9714160A (en)
CA (1) CA2274495C (en)
DE (1) DE69740108D1 (en)
WO (1) WO1998028333A2 (en)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009694A1 (en) * 1998-08-14 2000-02-24 Smithkline Beecham Biologicals S.A. Cloning of basb023 antigen from moraxella catarrhalis
WO2000015802A1 (en) * 1998-09-14 2000-03-23 Smithkline Beecham Biologicals S.A. Moraxella catarrhalis basb034 polypeptides and uses thereof
WO2000052042A1 (en) * 1999-02-26 2000-09-08 Smithkline Beecham Biologicals S.A. Immunogenic compounds
WO2000061165A1 (en) * 1999-04-13 2000-10-19 Smithkline Beecham Corporation Conserved adhesin motif and methods of use thereof
WO2000071724A2 (en) * 1999-05-24 2000-11-30 Smithkline Beecham Biologicals S.A. Novel compounds from moraxella catarrhalis
WO2004031236A2 (en) * 2002-10-02 2004-04-15 University Of Bristol Therapeutic uspa1-derived peptides
WO2005099337A2 (en) * 2004-04-15 2005-10-27 The University Of Bristol Therapeutic peptides
WO2007018463A2 (en) * 2005-08-10 2007-02-15 Arne Forsgren Ab Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
US7449302B2 (en) 2002-10-02 2008-11-11 The University Of Bristol, University Walk Therapeutic peptides
EP2261340A3 (en) * 1998-05-01 2012-01-04 Novartis Vaccines and Diagnostics, Inc. Neisseria meningitidis antigens and compositions
WO2015125118A1 (en) * 2014-02-24 2015-08-27 Glaxosmithkline Biologicals Sa Uspa2 protein constructs and uses thereof
US9267163B2 (en) 2000-02-28 2016-02-23 Glaxosmithkline Biologicals Sa Hybrid expression of neisserial proteins
WO2019034575A1 (en) * 2017-08-14 2019-02-21 Glaxosmithkline Biologicals Sa Methods of boosting immune responses
WO2019170702A1 (en) * 2018-03-08 2019-09-12 Glaxosmithkline Biologicals Sa Immunogenic composition comprising uspa2 epitope
CN110540964A (en) * 2018-12-20 2019-12-06 湖北诺美华抗体药物技术有限公司 monoclonal antibody of surface protein of Moraxella catarrhalis and application thereof
US10730917B2 (en) 2011-04-13 2020-08-04 Glaxosmithkline Biologicals Sa Fusion proteins and combination vaccines comprising haemophilus influenzae Protein E and Pilin A
WO2021048159A1 (en) * 2019-09-11 2021-03-18 Glaxosmithkline Biologicals Sa Assay

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6335018B1 (en) * 1995-05-01 2002-01-01 Aventis Pasteur Limited High molecular weight major outer membrane protein of moraxella
US6440425B1 (en) * 1995-05-01 2002-08-27 Aventis Pasteur Limited High molecular weight major outer membrane protein of moraxella
MY129765A (en) 2000-12-19 2007-04-30 Wyeth Corp Improved mycoplasma hyopneumoniae bacterin vaccine
WO2002083710A2 (en) * 2001-04-13 2002-10-24 Wyeth Holdings Corporation Removal of bacterial endotoxin in a protein solution by immobilized metal affinity chromatography
US7407672B2 (en) * 2002-11-04 2008-08-05 National Heart Center Composition derived from biological materials and method of use and preparation
US7709001B2 (en) 2005-04-08 2010-05-04 Wyeth Llc Multivalent pneumococcal polysaccharide-protein conjugate composition
CN113198013B (en) 2005-04-08 2024-02-20 惠氏有限责任公司 Multivalent pneumococcal polysaccharide-protein conjugate composition
US8298544B2 (en) * 2006-02-27 2012-10-30 Gal Markel CEACAM based antibacterial agents
WO2008000918A1 (en) * 2006-06-29 2008-01-03 Suomen Punainen Risti, Veripalvelu Novel cellular glycan compositions
WO2009057763A1 (en) * 2007-11-02 2009-05-07 Nippon Zenyaku Kogyo Co., Ltd. Novel adjuvant
US9120859B2 (en) 2012-04-04 2015-09-01 Zoetis Services Llc Mycoplasma hyopneumoniae vaccine
UA114503C2 (en) 2012-04-04 2017-06-26 Зоетіс Сервісіз Ллс PCV AND MYCOPLASMA HYOPNEUMONIA COMBINED VACCINE
UA114504C2 (en) 2012-04-04 2017-06-26 Зоетіс Сервісіз Ллс PCV, MYCOPLASMA HYOPNEUMONIA AND PRRS COMBINED VACCINE
CN105203754B (en) * 2014-08-18 2017-02-01 董俊 Method and kit for fast detection of moraxella catarrhalis based on magnetic resolution and quantum dot labelling
CN110540594B (en) * 2018-12-20 2021-05-11 湖北工业大学 Preparation method of latex microsphere immunochromatographic test paper based on Moraxella catarrhalis surface protein

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5279721A (en) 1993-04-22 1994-01-18 Peter Schmid Apparatus and method for an automated electrophoresis system

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4554101A (en) * 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
US5552146A (en) * 1991-08-15 1996-09-03 Board Of Regents, The University Of Texas System Methods and compositions relating to useful antigens of Moraxella catarrhalis
US6335018B1 (en) * 1995-05-01 2002-01-01 Aventis Pasteur Limited High molecular weight major outer membrane protein of moraxella
US6440425B1 (en) 1995-05-01 2002-08-27 Aventis Pasteur Limited High molecular weight major outer membrane protein of moraxella

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5279721A (en) 1993-04-22 1994-01-18 Peter Schmid Apparatus and method for an automated electrophoresis system

Cited By (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2261340A3 (en) * 1998-05-01 2012-01-04 Novartis Vaccines and Diagnostics, Inc. Neisseria meningitidis antigens and compositions
WO2000009694A1 (en) * 1998-08-14 2000-02-24 Smithkline Beecham Biologicals S.A. Cloning of basb023 antigen from moraxella catarrhalis
WO2000015802A1 (en) * 1998-09-14 2000-03-23 Smithkline Beecham Biologicals S.A. Moraxella catarrhalis basb034 polypeptides and uses thereof
US7432366B2 (en) 1998-09-14 2008-10-07 Smithkline Beecham Biologicals S.A. Moraxella catarrhalis BASB034 polypeptides and used thereof
AU752667B2 (en) * 1998-09-14 2002-09-26 Smithkline Beecham Biologicals (Sa) Moraxella catarrhalis BASB034 polypeptides and uses thereof
US6770459B1 (en) 1999-02-26 2004-08-03 Glaxosmithkline Biologicals S.A. Immunogenic compounds
WO2000052042A1 (en) * 1999-02-26 2000-09-08 Smithkline Beecham Biologicals S.A. Immunogenic compounds
WO2000061165A1 (en) * 1999-04-13 2000-10-19 Smithkline Beecham Corporation Conserved adhesin motif and methods of use thereof
WO2000071724A3 (en) * 1999-05-24 2001-02-15 Smithkline Beecham Biolog Novel compounds from moraxella catarrhalis
WO2000071724A2 (en) * 1999-05-24 2000-11-30 Smithkline Beecham Biologicals S.A. Novel compounds from moraxella catarrhalis
US9267163B2 (en) 2000-02-28 2016-02-23 Glaxosmithkline Biologicals Sa Hybrid expression of neisserial proteins
WO2004031236A3 (en) * 2002-10-02 2004-06-24 Univ Bristol Therapeutic uspa1-derived peptides
WO2004031236A2 (en) * 2002-10-02 2004-04-15 University Of Bristol Therapeutic uspa1-derived peptides
JP2006516952A (en) * 2002-10-02 2006-07-13 ザ ユニバーシティ オブ ブリストル Therapeutic peptide
JP4763291B2 (en) * 2002-10-02 2011-08-31 ザ ユニバーシティ オブ ブリストル Therapeutic peptide
CN1305899C (en) * 2002-10-02 2007-03-21 布里斯托尔大学 Therapeutic peptides
KR101057587B1 (en) 2002-10-02 2011-08-19 더 유니버시티 오브 브리스톨 Therapeutic Peptides
EP2305700A1 (en) * 2002-10-02 2011-04-06 University Of Bristol Therapeutic USPA1-derived peptides
US7449302B2 (en) 2002-10-02 2008-11-11 The University Of Bristol, University Walk Therapeutic peptides
AU2003299131B2 (en) * 2002-10-02 2010-04-22 University Of Bristol Therapeutic USPA1-derived peptides
WO2005099337A2 (en) * 2004-04-15 2005-10-27 The University Of Bristol Therapeutic peptides
EP2468863A1 (en) * 2004-04-15 2012-06-27 The University of Bristol Therapeutic peptides
WO2005099337A3 (en) * 2004-04-15 2006-08-10 Univ Bristol Therapeutic peptides
AU2006277076B2 (en) * 2005-08-10 2012-03-29 Arne Forsgren Ab Interaction of Moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
US8895030B2 (en) 2005-08-10 2014-11-25 Arne Forsgren Ab Interaction of Moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
EA014352B1 (en) * 2005-08-10 2010-10-29 Арне Форсгрен Аб Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
WO2007018463A3 (en) * 2005-08-10 2007-05-10 Arne Forsgren Ab Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
US8323667B2 (en) 2005-08-10 2012-12-04 Arne Forsgren Ab Interaction of Moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
EP2548885A3 (en) * 2005-08-10 2013-05-01 Arne Forsgren AB Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
EP2548884A3 (en) * 2005-08-10 2013-05-01 Arne Forsgren AB Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
US10183071B2 (en) 2005-08-10 2019-01-22 Arne Forsgren Ab Interaction of moraxella catarrhalis with epithellal cells, extracellular matrix proteins and the complement system
US8092811B2 (en) 2005-08-10 2012-01-10 Arne Forsgren Ab Interaction of Moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
US9255127B2 (en) 2005-08-10 2016-02-09 Arne Forsgren Ab Interaction of Moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
WO2007018463A2 (en) * 2005-08-10 2007-02-15 Arne Forsgren Ab Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
EP3305807A1 (en) * 2005-08-10 2018-04-11 Arne Forsgren AB Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
US10786562B2 (en) 2005-08-10 2020-09-29 Arne Forsgren Ab Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system
US11198707B2 (en) 2011-04-13 2021-12-14 Glaxosmithkline Biologicals Sa Fusion proteins and combination vaccines comprising Haemophilus influenzae Protein E and Pilin A
US10730917B2 (en) 2011-04-13 2020-08-04 Glaxosmithkline Biologicals Sa Fusion proteins and combination vaccines comprising haemophilus influenzae Protein E and Pilin A
WO2015125118A1 (en) * 2014-02-24 2015-08-27 Glaxosmithkline Biologicals Sa Uspa2 protein constructs and uses thereof
US10947280B2 (en) 2014-02-24 2021-03-16 Glaxosmithkline Biologicals Sa UspA2 protein constructs and uses thereof
EP3498292A1 (en) * 2014-02-24 2019-06-19 GlaxoSmithKline Biologicals SA Uspa2 protein constructs and uses thereof
BE1022345B1 (en) * 2014-02-24 2016-03-25 Glaxosmithkline Biologicals S.A. USPA2 PROTEIN CONSTRUCTS AND USES THEREOF
AU2015220369B2 (en) * 2014-02-24 2018-02-01 Glaxosmithkline Biologicals Sa UspA2 protein constructs and uses thereof
EA034352B1 (en) * 2014-02-24 2020-01-30 Глэксосмитклайн Байолоджикалз Са UspA2 PROTEIN CONSTRUCTS AND USES THEREOF
US10040832B2 (en) 2014-02-24 2018-08-07 Glaxosmithkline Biologicals Sa UspA2 protein constructs and uses thereof
CN111499701A (en) * 2014-02-24 2020-08-07 葛兰素史密斯克莱生物公司 USPA2 protein constructs and uses thereof
US10745449B2 (en) 2014-02-24 2020-08-18 Glaxosmithkline UspA2 protein constructs and uses thereof
WO2019034575A1 (en) * 2017-08-14 2019-02-21 Glaxosmithkline Biologicals Sa Methods of boosting immune responses
US11723966B2 (en) 2017-08-14 2023-08-15 Glaxosmithkline Biologicals Sa Methods of boosting immune responses
WO2019170702A1 (en) * 2018-03-08 2019-09-12 Glaxosmithkline Biologicals Sa Immunogenic composition comprising uspa2 epitope
CN110540964A (en) * 2018-12-20 2019-12-06 湖北诺美华抗体药物技术有限公司 monoclonal antibody of surface protein of Moraxella catarrhalis and application thereof
WO2021048159A1 (en) * 2019-09-11 2021-03-18 Glaxosmithkline Biologicals Sa Assay

Also Published As

Publication number Publication date
US7288646B2 (en) 2007-10-30
JP2001515467A (en) 2001-09-18
AU746442B2 (en) 2002-05-02
US6753417B2 (en) 2004-06-22
US6310190B1 (en) 2001-10-30
US7576194B2 (en) 2009-08-18
AU5720198A (en) 1998-07-17
EP0948625A2 (en) 1999-10-13
ATE497004T1 (en) 2011-02-15
US20050137131A1 (en) 2005-06-23
CA2274495C (en) 2009-06-02
AU2011201228A1 (en) 2011-04-14
BR9714160A (en) 2000-05-02
US20090137788A1 (en) 2009-05-28
CN1251611A (en) 2000-04-26
WO1998028333A3 (en) 1999-01-07
CN1159441C (en) 2004-07-28
KR20000057575A (en) 2000-09-25
US20030032772A1 (en) 2003-02-13
EP0948625B1 (en) 2011-01-26
US20050131221A1 (en) 2005-06-16
US7344724B2 (en) 2008-03-18
KR100615109B1 (en) 2006-08-22
US7569683B2 (en) 2009-08-04
JP2009142276A (en) 2009-07-02
CA2274495A1 (en) 1998-07-02
DE69740108D1 (en) 2011-03-10
US20090118486A1 (en) 2009-05-07

Similar Documents

Publication Publication Date Title
US6753417B2 (en) UspA1 and UspA2 antigens of Moraxella catarrhalis
WO1998028333A9 (en) USPA1 AND USPA2 ANTIGENS OF $i(MORAXELLA CATARRHALIS)
Helminen et al. A major outer membrane protein of Moraxella catarrhalis is a target for antibodies that enhance pulmonary clearance of the pathogen in an animal model
US6420134B1 (en) Vaccines for nontypable haemophilus influenzae
WO1993003761A1 (en) METHODS AND COMPOSITIONS RELATING TO USEFUL ANTIGENS OF $i(MORAXELLA CATARRHALIS)
US20090053292A1 (en) Moraxella catarrhalis protein, nucleic acid sequence and uses thereof
EP0832238A1 (en) Streptococcal heat shock proteins members of the hsp70 family
AU749382B2 (en) Novel surface exposed proteins from chlamydia pneumoniae
US5993826A (en) Methods and compositions relating to useful antigens of moraxella catarrhalis
AU740481B2 (en) Defining epitopes of the outer membrane protein CopB of moraxella catarrhalis
WO2001009334A1 (en) Basb118 polypeptide and polynucleotide from moraxella catarrhalis
Hiltke et al. Antigenic Structure of Outer Membrane

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 97180843.0

Country of ref document: CN

AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
CFP Corrected version of a pamphlet front page

Free format text: ADD INID NUMBER (63) "RELATED BY CONTINUATION (CON) OR CONTINUATION-IN-PART (CIP) TO EARLIER APPLICATION" WHICH WAS INADVERTENTLY OMITTED FROM THE FRONT PAGE

COP Corrected version of pamphlet

Free format text: PAGES 1/18-18/18, DRAWINGS, REPLACED BY NEW PAGES 1/20-20/20

121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2274495

Country of ref document: CA

Ref document number: 2274495

Country of ref document: CA

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1019997005332

Country of ref document: KR

ENP Entry into the national phase

Ref document number: 1998 529075

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 09336447

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 57201/98

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1997953461

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1997953461

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1019997005332

Country of ref document: KR

WWG Wipo information: grant in national office

Ref document number: 57201/98

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1019997005332

Country of ref document: KR

WWG Wipo information: grant in national office

Ref document number: 1019997005332

Country of ref document: KR