WO1998008542A2 - Compounds and methods for treatment and diagnosis of mycobacterial infections - Google Patents

Compounds and methods for treatment and diagnosis of mycobacterial infections Download PDF

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Publication number
WO1998008542A2
WO1998008542A2 PCT/NZ1997/000105 NZ9700105W WO9808542A2 WO 1998008542 A2 WO1998008542 A2 WO 1998008542A2 NZ 9700105 W NZ9700105 W NZ 9700105W WO 9808542 A2 WO9808542 A2 WO 9808542A2
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ala
gly
leu
val
pro
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PCT/NZ1997/000105
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French (fr)
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WO1998008542A3 (en
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Paul Tan
Jun Hiyama
Elizabeth S. Visser
Margot A. Skinner
Linda M. Scott
Ross L. Prestidge
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Genesis Research & Development Corporation Limited
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Priority to EP97937915A priority Critical patent/EP0939646A2/en
Priority to BR9711457-0A priority patent/BR9711457A/en
Priority to JP51151698A priority patent/JP2001503969A/en
Priority to NZ334358A priority patent/NZ334358A/en
Priority to CA002263885A priority patent/CA2263885A1/en
Priority to AU40365/97A priority patent/AU723606B2/en
Publication of WO1998008542A2 publication Critical patent/WO1998008542A2/en
Publication of WO1998008542A3 publication Critical patent/WO1998008542A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/863—Mycobacterium

Definitions

  • the present invention relates generally to the detection, treatment and prevention of infectious diseases.
  • the invention is related to compounds and methods for the treatment of mycobacte ⁇ al infections including Mycobacterium tuberculosis and Mycobacterium avium
  • the invention is further related to compounds that function as non-specific immune response amplifiers, and the use of such non-specific immune response amplifiers as adjuvants in vaccination or lmmunotherapy against infectious disease, and in certain treatments for immune disorders and cancer.
  • Tuberculosis is a chronic, infectious disease, that is caused by infection with
  • Mycobacterium tuberculosis (M tuberculosis) It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as a chronic inflammation of the lungs, resulting in fever and respiratory symptoms. If left untreated, significant morbidity and death may result.
  • tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behaviour is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistant mycobacte ⁇ a
  • BCG Bacillus Calmette-Gue ⁇ n
  • PPD protein-purified derivative
  • Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, thereby indicating exposure to mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.
  • M. bovis BCG and M. vaccae are believed to contain antigenic compounds that are recognised by the immune system of individuals exposed to infection with M. tuberculosis.
  • the present invention provides compounds and methods for the prevention, treatment and diagnosis of mycobacterial infection, together with adjuvants Tor use in vaccines or immunotherapy of infectious diseases and cancers.
  • polypeptides derived from Mycobacterium vaccae comprising an immunogenic portion of an antigen, or a variant of such an antigen.
  • the antigen includes an amino acid sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 1-4, 9-16, 18- 21, 23, 25, 26, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100-105, 109, 110, 112, 1 14, 1 17, 1 18, 121, 124, 125, 134, 135, 140 and 141 ; and (b) sequences having at least about a 99% probability of being the same as a sequence recited in SEQ ID NOS: 1-4, 9-16, 18-21, 23, 25, 26, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100-105, 109, 1 10, 1 12, 1 14. 1 17, 1 18, 121. 124.
  • the invention provides polypeptides comprising an immunogenic portion of an M. vaccae antigen wherein the antigen comprises an amino acid sequence encoded by a DNA molecule selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 40-42, 46, 48-51 , 74, 88, 93, 97, 99, 106-
  • DNA sequences encoding the inventive polypeptides, expression vectors comprising these DNA sequences, and host cells transformed or transfected with such expression vectors are also provided.
  • the present invention provides fusion proteins comprising a first and a second inventive poiypeptide or, alternatively, an inventive poiypeptide and a known M. tuberculosis antigen.
  • the present invention provides pharmaceutical compositions that comprise at least one of the inventive polypeptides, or a DNA molecule encoding such a poiypeptide, and a physiologically acceptable carrier.
  • the invention also provides vaccines comprising at least one of the above polypeptides and a nonspecific immune response amplifier, together with vaccines comprising at least one DNA sequence encoding such polypeptides and a non-specific immune response amplifier.
  • methods are provided for inducing protective immunity in a patient, comprising administering to a patient an effective amount of one or more of the above polypeptides together with an immune response amplifier.
  • the method comprises contacting dermal cells of a patient with one or more of the above polypeptides and detecting an immune response on the patient's skin.
  • the method comprises contacting a biological sample with at least one of the above polypeptides; and detecting in the sample the presence of antibodies that bind to the poiypeptide or polypeptides, thereby detecting M. tuberculosis infection in the biological sample.
  • suitable biological samples include whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine.
  • Diagnostic kits comprising one or more of the above polypeptides in combination with an apparatus sufficient to contact the poiypeptide with the dermal cells of a patient are provided.
  • the present invention also provides diagnostic kits comprising one or more of the inventive polypeptides in combination with a detection reagent.
  • the present invention provides antibodies, both polyclonal and monoclonal, that bind to the polypeptides described above, as well as methods for their use in the detection of M. tuberculosis infection.
  • the present invention also provides methods for enhancing a non-specific immune response to an antigen.
  • such methods comprise administering a composition comprising a component selected from the group consisting of : (a) delipidated M. vaccae cells, (b) deglycosylated M. vaccae cells; (c) delipidated and deglycosylated M. vaccae cells and (d) M. vaccae culture filtrate.
  • such methods comprise administering a poiypeptide, the poiypeptide comprising an immunogenic portion of an antigen, wherein said antigen includes a sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 114, 1 17 and 118; and (b) sequences having at least about 97% identity to a sequence recited in SEQ ID NOS: 114, 1 17 and 118.
  • compositions comprising a component selected from the group consisting of delipidated M. vaccae cells, deglycosylated M. vaccae cells, and delipidated and deglycosylated M. vaccae cells are provided, together with vaccines comprising such components and methods of using such compositions and vaccines to induce protective immunity in a patient.
  • Figs. 1A and IB illustrate the protective effects of immunizing mice with autoclaved M. vaccae or unfractionated M. vaccae culture filtrates, respectively, prior to infection with live M. tuberculosis H37Rv.
  • Figs. 2A and B show components of M. vaccae and M. tuberculosis culture filtrates, respectively, as analysed by 2-dimensional polyacrylamide gel electrophoresis.
  • Fig. 3 is a comparison of the Antigen 85A protein sequence obtained from M. vaccae with those from M. bovis, M. tuberculosis and M. leprae.
  • Fig. 4A(i) - (iv) illustrate the non-specific immune amplifying effects of 10 ⁇ g, XOO ⁇ g and lmg autoclaved M. vaccae and 15 ⁇ g unfractionated culture filtrates of M. vaccae, respectively.
  • Fig. 4B(i) and (ii) illustrate the non-specific immune amplifying effects of autoclaved M. vaccae and delipidated M. vaccae, respectively.
  • Fig. 4C(i) illustrates the non-specific immune amplifying effects of whole autoclaved M. vaccae.
  • Fig. 4C(ii) illustrates the non-specific immune amplifying effects of delipidated M. vaccae from which glycolipids had been removed and the proteins extracted with SDS.
  • Fig. 4A(i) - (iv) illustrate the non-specific immune amplifying effects of 10 ⁇ g, XOO ⁇ g and lmg autoclaved M. vaccae and 15 ⁇ g unfractionated culture
  • FIG. 4C(iii) illustrates that the adjuvant effect of the preparation of Fig. 4C(ii) is destroyed by treatment with the proteolytic enzyme pronase.
  • Fig. 4D illustrates The non-specific immune amplifying effects of heat-killed M. vaccae (Fig. 4D(i)), M. tuberculosis (Fig. 4D(ii)), M. bovis BCG (Fig. 4D(iii)), M. phlei (Fig. 4D(iv)) and M. smegmatis (Fig. 4D(v)).
  • Fig. 5 shows the results of polyacrylamide gel electrophoresis analysis of
  • Fig. 6 illustrates the non-specific immune amplifying effects of different molecular weight fractions of SDS-extracted M. vaccae proteins.
  • Fig. 7 illustrates the non-specific immune amplifying effects of different pi fractions of SDS-extracted M. vaccae proteins.
  • Fig. 8 illustrates the induction of IL-12 by heat-killed M. vaccae, lyophilized M. vaccae, delipidated and deglycosylated M. vaccae (referred to as "delipidated M. vaccae”) and M. vaccae glycolipids.
  • Fig. 9 illustrates the stimulation of interferon-gamma production by different concentrations of M. vaccae recombinant proteins, heat-killed M. vaccae, delipidated and deglycosylated M. vaccae (referred to as "delipidated M. vaccae"), M. vaccae glycolipids and lipopolysaccharide in C57BL-6 peritoneal macrophages (Fig 9A); BALB/C peritoneal macrophages (Fig 9B); and C3H/HeJ peritoneal macrophages (Fig 9C).
  • delipidated M. vaccae M. vaccae glycolipids and lipopolysaccharide in C57BL-6 peritoneal macrophages
  • Fig 9B BALB/C peritoneal macrophages
  • Fig 9C C3H/HeJ peritoneal macrophages
  • the present invention is generally directed to compositions and methods for preventing, " treating and diagnosing mycobacterial infections, including M. tuberculosis and M. avium infections.
  • T cells are the predominant inducers of such immunity.
  • the essential role of T cells in protection against M. tuberculosis infection is illustrated by the frequent occurrence of M. tuberculosis in AIDS patients, due to the depletion of CD4 T cells associated with human immunodeficiency virus (HIV) infection.
  • HIV human immunodeficiency virus
  • Mycobacterium-reactive CD4 T cells have been shown to be potent producers of gamma-interferon (IFN- ⁇ ), which, in turn, has been shown to trigger the anti-myco acterial effects of macrophages in mice. While the role of IFN- ⁇ in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN- ⁇ or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis infection. Furthermore, it is known that IFN- ⁇ stimulates human macrophages to make 1 ,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play a role in stimulating resistance to M. tuberculosis infection.
  • IFN- ⁇ gamma-interferon
  • CD8 * T cells Another property of CD4" T cells and macrophages is their ability to activate CD8 * cytotoxic T cells which are capable of killing pathogen-infected cells.
  • CD8 * T cells have been shown to kill macrophages and other cells that harbour M. tuberculosis.
  • compositions of the present invention include polypeptides that comprise at least one immunogenic portion of an M. vaccae antigen, or a variant thereof. Such polypeptides stimulate T cell proliferation, and/or, interferon gamma secretion from T cells of individuals exposed to M. tuberculosis.
  • inventive polypeptides comprise at least an immunogenic portion of a soluble M. vaccae antigen.
  • a "soluble M. vaccae antigen” is a protein of M. vaccae origin that is present in M. vaccae culture filtrate.
  • poiypeptide encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
  • a poiypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences.
  • the additional sequences may be derived from the native M. vaccae antigen or may be heterologous, and such sequences may (but need not) be immunogenic.
  • immunogenic refers to the ability to elicit an immune response in a patient, such as a human, or in a biological sample.
  • immunogenic antigens are capable of stimulating cell proliferation, interleukin-12 production or interferon- ⁇ production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from an M. tuberculosis-immune individual.
  • Polypeptides comprising at least an immunogenic portion of one or more M. vaccae antigens may generally be used to detect tuberculosis or to induce protective immunity against tuberculosis in a patient.
  • compositions and methods of this invention also encompass variants of the above polypeptides.
  • variant covers any sequence which exhibits at least about 50%, more preferably at least about 70% and more preferably yet, at least about 90% identity to a sequence of the present invention. Most preferably, a "variant” is any sequence which has at least about a 99% probability of being the same as the inventive sequence. The probability for DNA sequences is measured by the computer algorithm FASTA (version 2.0u4, February 1996; Pearson W. R. et al., Proc. Natl. Acad.
  • a poiypeptide of the present invention may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post- translationally directs transfer of the protein.
  • the poiypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the poiypeptide (e.g., poly-His), or to enhance binding of the poiypeptide to a solid support.
  • a poiypeptide may be conjugated to an immunoglobulin Fc region.
  • M. vaccae antigens, and DNA sequences encoding such antigens may be prepared using any of a variety of procedures. For example, soluble antigens may be isolated from M.
  • Antigens may also be produced recombinantly by inserting a DNA sequence that encodes the antigen into an expression vector and expressing the antigen in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant poiypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.
  • DNA sequences encoding M. vaccae antigens may be obtained by screening an appropriate M. vaccae cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from partial amino acid sequences of isolated soluble antigens. Suitable degenerate oligonucleotides may be designed and synthesized, and the screen may be performed as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. As described below, polymerase chain reaction (PCR) may be employed to isolate a nucleic acid probe from a cDNA or genomic DNA library. The library screen may then be performed using the isolated probe.
  • PCR polymerase chain reaction
  • DNA molecules encoding M. vaccae antigens may also be isolated by screening an appropriate M. vaccae expression library with anti-sera (e.g., rabbit or monkey) raised specifically against M. vaccae antigens.
  • anti-sera e.g., rabbit or monkey
  • the antigens described herein have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce cell proliferation and/or cytokine production (for example, interferon- ⁇ and/or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from an M. tuberculosis-immune individual.
  • cytokine production for example, interferon- ⁇ and/or interleukin-12 production
  • M. tuberculosis-immune individual is one who is considered to be resistant to the development of tuberculosis by virtue of having mounted an effective T cell response to M. tuberculosis.
  • Such individuals may be identified based on a strongly positive (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD), and an absence of any symptoms of tuberculosis infection.
  • PPD tuberculosis proteins
  • the selection of cell type for use in evaluating an immunogenic response to an antigen will depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing B cells or macrophages. T cells, NK cells, B cells and macrophages derived from M.
  • tuberculosis-immune individuals may be prepared using methods well known in the art. For example, a preparation of peripheral blood mononuclear cells (PBMCs) may be employed without further separation of component cells. PBMCs may be prepared, for example, using density centrifugation through FicollTM (Winthrop Laboratories, NY). T cells for use in the assays described herein may be purified directly from PBMCs. Alternatively, an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins, may be employed. Such T cell clones may be generated by, for example, culturing PBMCs from M. tuberculosis- immune individuals with mycobacterial proteins for a period of 2-4 weeks.
  • PBMCs peripheral blood mononuclear cells
  • T cells This allows expansion of only the mycobacterial protein-specific T cells, resulting in a line composed solely of such cells. These cells may then be cloned and tested with individual proteins, using methods well known in the art, to more accurately define individual T cell specificity. Assays for cell proliferation or cytokine production in T cells, NK cells, B cells or macrophages may be performed, for example, using the procedures described below.
  • immunogenic antigens are those antigens that stimulate proliferation or cytokine production (i.e. , interferon- ⁇ and or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from at least about
  • polypeptides having superior therapeutic properties may be distinguished based on the magnitude of the responses in the above assays and based on the percentage of individuals for which a response is observed.
  • antigens having superior therapeutic properties will not stimulate cell proliferation or cytokine production in vitro in cells derived from more than about 25% of individuals that are not
  • M. tuberculosis-responsive cells M. tuberculosis-responsive cells.
  • those antigens that induce a response in a high percentage of T cell, NK cell, B cell or macrophage preparations from M. tuberculosis-immune individuals have superior therapeutic properties.
  • Antigens with superior therapeutic properties may also be identified based on their ability to diminish the severity of M. tuberculosis infection, or other mycobacterial infection, in experimental animals, when administered as a vaccine. Suitable vaccine preparations for use in experimental animals are described in detail below.
  • Antigens having superior diagnostic properties may generally be identified based on the ability to elicit a response in an intradermal skin test performed on an individual with active tuberculosis, but not in a test performed on an individual who is not infected with M. tuberculosis. Skin tests may generally be performed as described below, with a response of at least about 5 mm induration considered positive. Immunogenic portions of the antigens described herein may be prepared and identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3d ed., Raven Press, 1993, pp. 243-247. Such techniques include screening poiypeptide portions of the native antigen for immunogenic properties. The representative proliferation and cytokine production assays described herein may be employed in these screens.
  • An immunogenic portion of a poiypeptide is a portion that, within such representative assays, generates an immune response (e.g. , cell proliferation, interferon- ⁇ production or interleukin-12 production) that is substantially similar to that generated by the full length antigen.
  • an immunogenic portion of an antigen may generate at least about 20%, preferably about 65%, and most preferably about 100%, of the proliferation induced by the full length antigen in the model proliferation assay described herein.
  • An immunogenic portion may also, or alternatively, stimulate the production of at least about 20%, preferably about 65% and most preferably about 100%, of the interferon- ⁇ and/or interleukin-I2 induced by the full length antigen in the model assay described herein.
  • Portions and other variants of M. vaccae antigens may be generated by synthetic or recombinant means.
  • Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids may be generated using techniques well known to those of ordinary skill in the art.
  • such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J Am. Chem. Soc. 55:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems, Inc.
  • the polypeptides disclosed herein are prepared in substantially pure form.
  • the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure.
  • the substantially pure polypeptides are incorporated into pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein.
  • the present invention also provides fusion proteins comprising a first and a second inventive poiypeptide or, alternatively, a poiypeptide of the present invention and a known M tuberculosis antigen, such as the 38 kD antigen described in Andersen and Hansen, Infect. Immun. 57:2481-2488. 1989, together with variants of such fusion proteins.
  • the fusion proteins of the present invention may also include a linker peptide between the first and second polypeptides.
  • a DNA sequence encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides into an appropriate expression vector.
  • the 3' end of a DNA sequence encoding the first poiypeptide is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second poiypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.
  • a peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each poiypeptide folds into its secondary and tertiary structures.
  • Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the poiypeptide functional epitopes.
  • Preferred peptide linker sequences contain
  • linker sequence Gly, Asn and Ser residues.
  • Other near neutral amino acids such as Thr and Ala may also be used in the linker sequence.
  • Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-4 , 1985;
  • the linker sequence may be from 1 to about 50 amino acids in length. Peptide linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • the ligated DNA sequences encoding the fusion proteins are cloned into suitable expression systems using techniques known to those of ordinary skill in the art.
  • the present invention provides methods for using one or more of the inventive polypeptides or fusion proteins (or DNA molecules encoding such polypeptides or fusion proteins) to induce protective immunity against tuberculosis in a patient.
  • a "patient” refers to any warm-blooded animal, preferably a human.
  • a patient may be afflicted with a disease, or may be free of detectable disease or infection.
  • protective immunity may be induced to prevent or treat tuberculosis.
  • the poiypeptide, fusion protein or DNA molecule is generally present within a pharmaceutical composition or a vaccine.
  • compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier.
  • Vaccines may comprise one or more of the above polypeptides and a non-specific immune response amplifier, such as an adjuvant or a liposome, into which the poiypeptide is incorporated.
  • Such pharmaceutical compositions and vaccines may also contain other mycobacterial antigens, either, as discussed above, incorporated into a fusion protein or present within a separate poiypeptide.
  • a vaccine of the present invention may contain DNA encoding one or more polypeptides as described above, such that the poiypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the poiypeptide on its cell surface.
  • the DNA may be introduced using a viral expression system (e.g.
  • vaccinia or other pox virus, retrovirus, or adenovirus which may involve the use of a non-pathogenic, or defective, replication competent virus.
  • Techniques for incorporating DNA into such expression systems are well known in the art.
  • the DNA may also be "naked,” as described, for example, in Ulmer et al., Science 259:1745-1749. 1993 and reviewed by Cohen, Science 259: 1691-1692, 1993.
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
  • a DNA vaccine as described above may be administered simultaneously with or sequentially to either a poiypeptide of the present invention or a known mycobacterial antigen, such as the 38 kD antigen described above.
  • administration of DNA encoding a poiypeptide of the present invention may be followed by administration of an antigen in order to enhance the protective immune effect of the vaccine.
  • compositions and vaccines may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 3 doses may be administered for a 1-36 week period. Preferably, 3 doses are administered, at intervals " of 3-4 months, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients.
  • a suitable dose is an amount of poiypeptide or DNA that, when administered as described above, is capable of raising an immune response in a patient sufficient to protect the patient from mycobacterial infection for at least 1-2 years.
  • the amount of poiypeptide present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 ⁇ g.
  • Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • the type of carrier will vary depending on the mode of administration.
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactic galactide
  • suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
  • adjuvants may be employed in the vaccines of this invention to non-specifically enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis, M. tuberculosis, or, as discussed below, M. vaccae.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ).
  • Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.
  • this invention provides methods for using one or more of the polypeptides described above to diagnose tuberculosis using a skin test.
  • a skin test is any assay performed directly on a patient in which a delayed- type hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of one or more polypeptides as described above.
  • DTH delayed- type hypersensitivity
  • the reaction is measured at least 48 hours after injection, more preferably 48-72 hours.
  • the DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen (i.e., the immunogenic portion of the poiypeptide employed, or a variant thereof).
  • the response may be measured visually, using a ruler.
  • a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of tuberculosis infection.
  • the polypeptides of the present invention are preferably formulated, as pharmaceutical compositions containing a poiypeptide and a physiologically acceptable carrier, as described above.
  • Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 ⁇ g to about 100 ⁇ g, preferably from about 10 ⁇ g to about 50 ⁇ g in a volume of 0.1 mL.
  • the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80TM.
  • a poiypeptide employed in a skin test is of sufficient size such that it remains at the site of injection for the duration of the reaction period.
  • a poiypeptide that is at least 9 amino acids in length is sufficient.
  • the poiypeptide is also preferably broken down by macrophages or dendritic cells within hours of injection to allow presentation to T-cells.
  • Such polypeptides may contain repeats of one or more of the above sequences or other immunogenic or nonimmunogenic sequences.
  • a biological sample is any antibody-containing sample obtained from a patient.
  • the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient or a blood supply.
  • polypeptide(s) are used in an assay, as described below, to determine the presence or absence of antibodies to the polypeptide(s) in the sample, relative to a predetermined cut-off value. The presence of such antibodies indicates the presence of mycobacterial infection.
  • the polypeptides used are preferably complementary (i.e., one component poiypeptide will tend to detect infection in samples where the infection would not be detected by another component poiypeptide).
  • Complementary polypeptides may generally be identified by using each poiypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with a Mycobacterium.
  • combinations of two or more polypeptides may be formulated that are capable of detecting infection in most, or all, of the samples tested. For example, approximately 25-30% of sera from tuberculosis-infected individuals are negative for antibodies to any single protein, such as the 38 kD antigen mentioned above. Complementary polypeptides may, therefore, be used in combination with the 38 kD antigen to improve sensitivity of a diagnostic test.
  • the assay involves the use of poiypeptide immobilized on a solid support to bind to and remove the antibody from the sample. The bound antibody may then be detected using a detection reagent that contains a reporter group. Suitable detection reagents include antibodies that bind to the antibody/polypeptide complex and free poiypeptide labelled with a reporter group (e.g., in a semi-competitive assay).
  • a competitive assay may be utilized, in which an antibody t at binds to the poiypeptide is labelled with a reporter group and allowed to bind to the immobilized antigen after incubation of the antigen with the sample.
  • the extent to which components of the sample inhibit the binding of the labelled antibody to the poiypeptide is indicative of the reactivity of the sample with the immobilized poiypeptide.
  • the solid support may be any solid material to which the antigen may be attached. Suitable materials are well known in the art.
  • the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
  • the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
  • the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
  • polypeptides may be bound to the solid support using a variety of techniques well known in the art.
  • bound refers to both noncovalent association, such as adsorption, and covalent attachment, which may be a direct linkage between the antigen and functional groups on the support or a linkage by way of a cross-linking agent. Binding by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the poiypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day.
  • contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of poiypeptide ranging from about 10 ng to about 1 ⁇ g, and preferably about 100 ng, is sufficient to bind an adequate amount of antigen.
  • Covalent attachment of poiypeptide to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the poiypeptide.
  • the poiypeptide may be bound to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the poiypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991 , at A12-A13).
  • the assay is an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • This assay may be performed by first contacting a poiypeptide antigen that has been immobilized on a solid support, with the sample, such that antibodies to the poiypeptide within the sample are allowed to bind to the immobilized poiypeptide. Unbound sample is then removed from the immobilized poiypeptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific detection reagent. More specifically, once the poiypeptide is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked.
  • any suitable blocking agent known to those of ordinary skill in the art such as bovine serum albumin or Tween 20TM (Sigma Chemical Co., St. Louis, MO) may be employed.
  • the immobilized poiypeptide is then incubated with the sample, and antibody is allowed to bind to the antigen.
  • the sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation.
  • PBS phosphate-buffered saline
  • an appropriate contact time, or incubation time is that period of time that is sufficient to detect the presence of antibody within a M. tuberculosis-infected sample.
  • the contact time is sufficient to achieve a level of binding that is at least 95% of that achieved at equilibrium between bound and unbound antibody.
  • the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
  • Unbound sample may be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM.
  • Detection reagent may then be added to the solid support.
  • An appropriate detection reagent is any compound that binds to the immobilized antibody-polypeptide complex and that can be detected by any of a variety of means known in the art.
  • the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group.
  • Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • binding agent to reporter group may be achieved using standard methods known in the art. Common binding agents may also be purchased conjugated to a variety of reporter groups from many commercial sources (e.g., Zymed Laboratories, San Francisco, CA, and Pierce, Rockford, IL).
  • the detection reagent is incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound antibody.
  • An appropriate amount of time may generally be determined from the manufacturer's instructions or by assaying the level of binding that occurs over a period of time.
  • Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group.
  • the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin. coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
  • the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value.
  • the cut-off value is the average mean signal obtained when the immobilized antigen is incubated with samples from an uninfected patient.
  • the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107. In general, signals higher than the predetermined cut-off value are considered to be positive for mycobacterial infection.
  • the assay may also be performed in a rapid flow-through or strip test format, wherein the antigen is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • a detection reagent e.g., protein A- colloidal " gold
  • a detection reagent then binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane.
  • the detection of bound detection reagent may then be performed as described above.
  • the strip test format one end of the membrane to which poiypeptide is bound is immersed in a solution containing the sample.
  • the sample migrates along the membrane through a region containing detection reagent and to the area of immobilized poiypeptide.
  • Concentration of detection reagent at the poiypeptide indicates the presence of anti- mycobacterial antibodies in the sample.
  • concentration of detection reagent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result.
  • the amount of poiypeptide immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in an ELISA, as discussed above.
  • the amount of poiypeptide immobilized on the membrane ranges from about 25 ng to about 1 ⁇ g, and more preferably from about 50 ng to about 500 ng.
  • Such tests can typically be performed with a very small amount (e g , one drop) of patient serum or blood.
  • the present invention also provides antibodies to the inventive polypeptides.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g.. Harlow and Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory. 1988.
  • an immunogen comprising the antigenic poiypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep and goats).
  • the immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
  • Polyclonal antibodies specific for the poiypeptide may then be purified from such antisera by, for example, affinity chromatography using the poiypeptide coupled to a suitable solid support.
  • Monoclonal antibodies specific for the antigenic poiypeptide of interest may be prepared, for example, using the technique of Kohler and Mil stein, Eur. J Immunol. 6:5 ⁇ 1-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the poiypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells may then be immortalized by fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal, using one of a variety of techniques well known in the art.
  • Monoclonal antibodies may be isolated from the supernatants of the resulting hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
  • Antibodies may be used in diagnostic tests to detect the presence of mycobacterial antigens using assays similar to those detailed above and other techniques well known to those of skill in the art, thereby providing a method for detecting mycobacterial infection, such as M. tuberculosis infection, in a patient.
  • Diagnostic reagents of the present invention may also comprise DNA sequences encoding one or more of the above polypeptides, or one or more portions thereof. For example, primers comprising at least 10 contiguous oligonucleotides of the subject DNA sequences may be used in polymerase chain reaction (PCR) based tests. Similarly, probes comprising at least 18 contiguous oligonucleotides of the subject DNA sequences may be used for hybridizing to specific sequences.
  • PCR polymerase chain reaction
  • Primers or probes may thus be used to detect M. tuberculosis and other mycobacterial infections in biological samples, preferably sputum, blood, serum, saliva, cerebrospinal fluid or urine.
  • DNA probes or primers comprising oligonucleotide sequences described above may be used alone, in combination with each other, or with previously identified sequences, such as the 38 kD antigen discussed above.
  • effective vaccines contain at least two different components.
  • the first is a poiypeptide comprising an antigen, which is processed by macrophages and other antigen-presenting cells and displayed for CD4 + T cells or for CD8 + T cells.
  • This antigen forms the "specific" target of an immune response.
  • the second component of a vaccine is a non-specific immune response amplifier, such as an adjuvant or a liposome, into which the antigen is incorporated.
  • An adjuvant amplifies immune responses to a structurally unrelated compound or poiypeptide.
  • adjuvants are prepared from microbes such as Bordetella pertussis, M. tuberculosis and M. bovis BCG.
  • Adjuvants may also contain components designed to protect poiypeptide antigens from degradation, such as aluminum hydroxide or mineral oil.
  • the antigenic component of a vaccine contains polypeptides that direct the immune attack against a specific pathogen, such as M. tuberculosis
  • the adjuvant is often capable of broad use in many different vaccine formulations.
  • Certain pathogens, such as M. tuberculosis, as well as certain cancers, are effectively contained by an immune attack directed by T cells, known as cell-mediated immunity.
  • Other pathogens, such as poliovirus, also require antibodies produced by B cells for containment.
  • T cell or B cell are controlled by different subpopulations of CD4 + T cells, commonly referred to as Thl and Th2 cells.
  • a desirable property of an adjuvant is the ability to selectively amplify the function of either Thl or Th2 populations of CD4 * T cells.
  • M. vaccae and a modified form of autoclaved M. vaccae have been found to have adjuvant properties.
  • the term "modified M. vaccae” includes delipidated M. vaccae cells, deglycosylated M. vaccae cells and M. vaccae cells that have been both delipidated and deglycosylated (hereinafter referred to as DD- ⁇ . vaccae).
  • M. vaccae produces compounds which amplify the immune response to M. vaccae antigens, as well as to antigens from other sources.
  • the present invention thus provides methods for enhancing immune responses to an antigen comprising administering killed M. vaccae cells, M. vaccae culture filtrate or modified M. vaccae cells.
  • this non-specific immune amplifying effect is due, at least in part, to an M. vaccae poiypeptide having homology to heat shock protein 65 (GroEL), previously identified in M. tuberculosis.
  • GroEL heat shock protein 65
  • heat-killed M. vaccae and M. vaccae constituents have cytokine stimulation properties.
  • vaccae and DD- vaccae stimulate the production of interleukin 12 (IL-12) from macrophages.
  • IL-12 interleukin 12
  • Production of IL-12 from macrophages is known to enhance stimulation of a Thl immune response.
  • the following examples are offered by way of illustration and not by way of limitation.
  • This example illustrates the effect of immunization with M vaccae or M. vaccae culture filtrate in mice prior to challenge with live M. tuberculosis.
  • M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/1; tryptone, 5 g/1; glucose, 1 g/1) at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10'° M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 °C. The culture filtrate was passaged through a 0.45 ⁇ M filter into sterile bottles.
  • sterile Medium 90 yeast extract, 2.5 g/1; tryptone, 5 g/1; glucose, 1 g/1
  • the cells were harvested by centrifugation
  • mice when mice were immunized with 1 mg, 100 ⁇ g or 10 ⁇ g of M. vaccae and infected three weeks later with 5x10 s colony forming units (CFU) of live M. tuberculosis H37Rv, significant protection from infection was seen.
  • CFU colony forming units
  • spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli determined (expressed as CFU).
  • the reduction in bacilli numbers when compared to tissue from non-immunized control mice, exceeded 2 logs in liver and lung tissue, and 1 log in spleen tissue.
  • Immunization of mice with heat-killed M. tuberculosis H37Rv had no significant protective effects on mice subsequently infected with live M. tuberculosis H37Rv.
  • Fig.1 B shows that when mice were immunized with 100 ⁇ g of M. vaccae culture filtrate, and infected three weeks later with 5x10 5 CFU of M. tuberculosis H37Rv, significant protection was also seen.
  • CFU live bacilli numbers
  • M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 ⁇ filter into sterile bottles.
  • the culture filtrate was concentrated by lyophilization, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45 ⁇ membrane.
  • the culture filtrate was desalted by membrane filtration in a 400 ml A icon stirred cell which contained a 3,000 kilodalton molecular weight cut-off (MWCO) membrane. The pressure was maintained at 50 psi using nitrogen gas.
  • the culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the 20 I volume to approximately 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).
  • the " esalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech, Uppsala, Sweden) (16 X 100 mm) equilibrated with lOmM Tris HCl buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCl from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.
  • the pool of polypeptides eluting from the ion exchange column was concentrated in a 400 ml Amicon stirred cell which contained a 3,000 MWCO membrane. The pressure was maintained at 50 psi using nitrogen gas. The polypeptides were repeatedly concentrated by membrane filtration and diluted with
  • the purified polypeptides were then fractionated by preparative isoelectric focusing in a Rotofor device (Bio-Rad, Hercules, CA, USA). The pH gradient was established with a mixture of Ampholytes (Pharmacia Biotech) comprising 1.6% pH 3.5-5.0 Ampholytes and 0.4% pH 5.0 - 7.0 Ampholytes. Acetic acid (0.5 M) was used as the anolyte, and 0.5 M ethanolamine as the catholyte. Isoelectric focusing was carried out at 12W constant power for 6 hours, following the manufacturer's instructions. Twenty fractions were obtained.
  • the poiypeptide fractions which were shown to contain significant contamination were further purified using a Mono Q column (Pharmacia Biotech) 10 micron particle size (5 x 50 mm) or a Vydac Diphenyl column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6 x 250 mm).
  • Mono Q column polypeptides were eluted with a linear gradient from 0-0.5 M NaCl in 10 mM Tris HCl pH 8.0.
  • polypeptides were eluted with a linear gradient of acetonitrile (20-60% v/v) in 0.1% TFA.
  • the flow-rate was 1.0 ml/min and the column eluent was monitored at 220 nm for both columns.
  • the poiypeptide peak fractions were collected and analysed for purity on a 15% polyacrylamide gel as described above.
  • polypeptides were individually dried onto BiobreneTM (Perkin Elmer/Applied BioSystems Division, Foster City, CA)-treated glass fiber filters.
  • the filters with poiypeptide were loaded onto a Perkin Elmer/Applied BioSystems Procise 492 protein sequencer and the polypeptides were sequenced from the amino terminal end using traditional Edman chemistry.
  • the amino acid sequence was determined for each poiypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Internal sequences were also determined on some antigens by digesting the antigen with the endoprotease Lys-C, or by chemically cleaving the antigen with cyanogen bromide.
  • GVc-1 six soluble M. vaccae antigens, designated GVc-1, GVc-2, GVc-7, GVc-13, GVc-20 and GVc-22, were isolated.
  • N-terminal and internal sequences for GVc-1 are shown in SEQ ID NOS. 1, 2 and 3, respectively; the N-terminal sequence for GVc-2 is shown in SEQ ID NO: 4; internal sequences for GVc-7 are shown in SEQ ID NOS: 5-8; internal sequences for GVc-13 are shown in SEQ ID NOS: 9-11; internal sequence for GVc-20 is shown in SEQ ID NO: 12; and N-terminal and internal sequences for GVc-22 are shown in SEQ ID NO:56-59, respectively.
  • Each of the internal peptide sequences provided herein begins with an amino acid residue which is assumed to exist in this position in the poiypeptide, based on the known cleavage specificity of cyanogen bromide (Met) or Lys-C (Lys).
  • GVc-16, GVc-18 and GVc-21 Three additional polypeptides, designated GVc-16, GVc-18 and GVc-21, were isolated employing a preparative sodium dodecyl sulfate-poiyacrylamide gel electrophoresis (SDS-PAGE) purification step in addition to the preparative isoelectric focusing procedure described above. Specifically, fractions comprising mixtures of polypeptides from the preparative isoelectric focusing purification step previously described, were purified by preparative SDS-PAGE on a 15% polyacrylamide gel. The samples were dissolved in reducing sample buffer and applied to the gel.
  • SDS-PAGE preparative sodium dodecyl sulfate-poiyacrylamide gel electrophoresis
  • the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting in 10 mM 3-(cyclohexylamino)-l- propanesulfonic acid (CAPS) buffer pH 1 1 containing 10% (v/v) methanol.
  • PVDF polyvinylidene difluoride
  • CAPS 3-(cyclohexylamino)-l- propanesulfonic acid
  • the transferred protein bands were identified by staining the PVDF membrane with Coomassie blue. Regions of the PVDF membrane containing the most abundant poiypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above.
  • the N-terminal sequences for GVc- 16, GVc-18 and GVc-21 are provided in SEQ ID NOS: 13, 14 and 15, respectively.
  • Additional antigens designated GVc-12, GVc-14, GVc-15, GVc-17 and GVc- 19, were isolated employing a preparative SDS-PAGE purification step in addition to the chromatographic procedures described above. Specifically, fractions comprising a mixture of antigens from the Vydac C4 HPLC purification step previously described were fractionated by preparative SDS-PAGE on a polyacrylamide gel. The samples were dissolved in non-reducing sample buffer and applied to the gel. The separated proteins were transferred to a PVDF membrane by electroblotting in 10 mM CAPS buffer, pH 1 1 containing 10% (v/v) methanol. The transferred protein bands were identified "" by staining the PVDF membrane with Coomassie blue.
  • Regions of the PVDF membrane containing the most abundant poiypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/ Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The determined N-terminal sequences for GVc-12, GVc-14, GVc-15, GVc- 17 and GVc-19 are provided in SEQ ID NOS: 16-20, respectively.
  • GVc-1 The amino acid sequence for GVc-1 was found to bear some similarity to sequences previously identified from M. bovis and M. tuberculosis. In particular, GVc-1 was found to have some homology with M. tuberculosis MPT83, a cell surface protein, as well as MPT70. These proteins form part of a protein family (Harboe et al., Scand. J. Immunol. 42:46-51 , 1995). Subsequent studies led to the isolation of DNA sequences for GVc-14 and
  • GVc-22 (SEQ ID NO: 107 and 108, respectively).
  • the corresponding predicted amino acid sequences for GVc-14 and GVc-22 are provided in SEQ ID NO: 109 and 1 10, respectively.
  • Amplifications primers AD86 and AD1 12 (SEQ ID NO: 60 and 61 , respectively) were designed from the amino acid sequence of GVc-1 (SEQ ID NO: 1) and the M. tuberculosis MPT70 gene sequence. Using these primers, a 310 bp fragment was amplified from M. vaccae genomic DNA and cloned into £coRV- digested vector pBluescript (Stratagene) containing added dTTP residues. The sequence of the cloned insert is provided in SEQ ID NO: 62. The purified polypeptides were screened for the ability to induce T-cell proliferation and IFN- ⁇ in peripheral blood cells from immune human donors. These donors were known to be PPD (purified protein derivative from M.
  • PPD purified protein derivative from M.
  • Donor PBMCs and crude soluble proteins from M. vaccae culture filtrate were cultured in medium comprising RPMI 1640 supplemented with 10% (v/v) autologous serum, penicillin (60 ⁇ g/ml), streptomycin (100 ⁇ g/ml), and glutamine (2 mM).
  • IFN- ⁇ was measured using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA plates were coated with a mouse monoclonal antibody directed to human IFN- ⁇ (Endogen, Wobural, MA) 1 ⁇ g/ml phosphate-buffered saline (PBS) for 4 hours at 4 °C.
  • Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS/0.2% Tween 20. and samples diluted 1 :2 in culture medium in the ELISA plates were incubated overnight at room temperature.
  • polypeptides containing sequences that stimulate peripheral blood mononuclear cells (PBMC) T cells to proliferate and produce IFN- ⁇ are shown in Table 1, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, and (++) indicates polypeptides having activity greater than four times above background.
  • M. vaccae soluble proteins were isolated from culture filtrate using 2- dimensional polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
  • M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 °C.
  • M. tuberculosis strain H37Rv (ATCC number 27294) was cultured in sterile Middlebrook 7H9 medium with Tween 80 and oleic acid/albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Michigan). The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 ⁇ filter into sterile bottles. The culture filtrate was concentrated by lyophilisation, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45 ⁇ membrane filter.
  • the culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained a 3,000 MWCO membrane. The pressure was maintained at 60 psi using nitrogen gas. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the 20 L volume to approximately 50 mL. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The desalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech) (16 x 100 mm) equilibrated with lOmM TrisHCL buffer pH 8.0.
  • Polypeptides were eluted with a linear gradient of NaCl from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm. The pool of polypeptides eluting from the ion exchange column were fractionated by preparative 2D gel electrophoresis. Samples containing 200-500 ug of poiypeptide were made 8M in urea and applied to polyacrylamide isoelectric focusing rod gels (diameter 2mm, length 150 mm, pH 5-7). After the isoelectric focusing step, the first dimension gels were equilibrated with reducing buffer and applied to second dimension gels (16% polyacrylamide). Figs.
  • 2A and 2B are the 2-D gel patterns observed with M. vaccae culture filtrate and M. tuberculosis H37Rv culture filtrate, respectively.
  • Polypeptides from the second dimension separation were transferred to PVDF membranes by electroblotting in lOmM CAPS buffer pH 11 containing 10% (v/v) methanol.
  • the PVDF membranes were stained for protein with Coomassie blue. Regions of PVDF containing polypeptides of interest were cut out and directly introduced into the sample cartridge of the Perkin Elmer/ Applied BioSystems Procise 492 protein sequencer. The polypeptides were sequenced from the amino terminal end using traditional Edman chemistry.
  • the amino acid sequence was determined for each poiypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Using these procedures, eleven polypeptides, designated GVs-1 , GVs-3, GVs-4, GVs-5, GVs-6, GVs-8, GVs-9, GVs- 10, GVs-1 1, GV-34 and GV-35 were isolated. The determined N-terminal sequences for these polypeptides are shown in SEQ ID NOS: 21-29, 63 and 64, respectively. Using the purification procedure described above, more protein was purified to extend the amino acid sequence previously obtained for GVs-9. The extended amino acid sequence for GVs-9 is provided in SEQ ID NO:65. Further studies resulted in the isolation of the DNA sequence for GVs-9 (SEQ ID NO: 111). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 1 12.
  • Probes for antigens 85A, 85B, and 85C were prepared by the polymerase chain reaction (PCR) using degenerate oligonucleotides designed to regions of antigen 85 genomic sequence that are conserved between family members in a given mycobacterial species, and between mycobacterial species. These oligonucleotides were used under reduced stringency conditions to amplify target sequences from M. vaccae genomic DNA. An appropriately-sized 0.5kb band was identified, purified, and cloned into T-tailed p Bluescript II SK (Stratagene, La Jolla, CA).
  • An M. vaccae genomic library was created in ⁇ ZapExpress (Stratagene, La Jolla, CA) by cloning BamHl partially-digested M. vaccae genomic DNA into similarly-digested ⁇ vector, with 3.4 x 10 5 independent plaque-forming units resulting.
  • Twenty-seven thousand plaques from this non- amplified library were plated at low density onto eight 100 cm : plates.
  • duplicate plaque lifts were taken onto Hybond-N " nylon membrane (Amersham International. United Kingdom), and hybridised under reduced-stringency conditions (55 °C) to the radiolabelled antigen 85C PCR product. Autoradiography demonstrated that seventy-nine plaques consistently hybridised to the antigen 85C probe under these conditions.
  • Sequence data from the 5' and 3' ends of inserts from several representatives of each group was obtained using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the T3 and T7 primers. Sequence homologies were determined using FASTA analysis of the GenBank databases with the GeneAssist software package. Two of these sets of clones were found to be homologous to M. bovis and M. tuberculosis antigen 85A genes, each containing either the 5' or 3' ends of the M. vaccae gene (this gene was cleaved during library construction as it contains an internal Bam l site). The remaining clones were found to contain sequences homologous to antigens 85B and 85C from a number of mycobacterial species.
  • M. vaccae antigens 85 A, 85B and 85C are shown in SEQ ID NOS: 40-42, respectively, with the predicted amino acid sequences being shown in SEQ ID NOS: 43-45, respectively.
  • the M. vaccae antigens GVc-3 and GVc-5 were expressed and purified as follows. Amplification primers were designed from the insert sequences of GVc-3 and GVc-5 (SEQ ID NO: 40 and 42, respectively) using sequence data downstream from the putative leader sequence and the 3' end of the clone.
  • the sequences of the primers for GVc-3 are provided in SEQ ID NO: 66 and 67. and the sequences of the primers for GVc-5 are provided in SEQ ID NO: 68 and 69.
  • a Xhol restriction site was added to the primers for GVc-3, and EcoRl and Bamlil restriction sites were added to the primers for GVc-5 for cloning convenience.
  • fragments were cloned into the appropriate site of pProEX HT prokaryotic expression vector (Gibco BRL, Life Technologies, Gaithersburg, MD) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the recombinant protein was performed according to the manufacturer's protocol.
  • M. vaccae antigen GVc-4 (antigen 85B homolog) was performed as follows.
  • the primers AD58 and AD59, described above, were used to amplify a 485 bp fragment from M. vaccae genomic DNA. This fragment was gel- purified using standard techniques and cloned into £c ⁇ RV-digested pBluescript containing added dTTP residues. The base sequences of inserts from five clones were determined and found to be identical to each other. These inserts had highest homology to Ag85B from M. tuberculosis.
  • the insert from one of the clones was subcloned into the EcoRI/Xhol sites of pProEX HT prokaryotic expression vector (Gibco BRL), expressed and purified according to the manufacturer's protocol.
  • This clone was renamed GVc-4P because only a part of the gene was expressed.
  • the amino aci " and DNA sequences for the partial clone GVc-4P are provided in SEQ ID NO: 70 and 106, respectively.
  • GVc-3, GVc-4P and GVc-5 were re-cloned into the alternative vector pET16 (Novagen, Madison, WI).
  • An 84 bp probe for the M vaccae antigen GVc-7 was amplified using degenerate oligonucleotides designed to the determined amino acid sequence of GVc- 7 (SEQ ID NOS: 5-8) This probe was used to screen a M vaccae genomic DNA library as described in Example 4.
  • the determined nucleotide sequence fo: GVc-7 is shown in SEQ ID NO 1 46 and predicted amino acid sequence in SEQ ID NO 47 Comparison of these sequences with those in the databank revealed homology to a hypothetical 15.8 kDa membrane protein of M tuberculosis
  • SEQ ID NO 46 The sequence of SEQ ID NO 46 was used to design amplification primers (provided in SEQ ID NO 71 and 72) for expression cloning of the GVc-7 gene using sequence data downstream from the putative leader sequence A Xhol restriction site was added to the primers for cloning convenience Following amplification from genomic M vaccae DNA, fragments were cloned into the of pProEX HT prokaryotic expression vector (Gibco BRL) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the fusion protein was performed according to the manufacturer ' s protocol. In subsequent studies, GVc-7 was re-cloned into the vector pET16 (Novagen).
  • a redundant oligonucleotide probe was designed to the GVs-8 peptide sequence shown in SEQ ID NO: 6 and used to screen an M. vaccae genomic DNA library as described above. Positive plaques were isolated.
  • GVs- 8A Four different genomic clones were identified, hereinafter referred to as GVs- 8A, GVs-8B and GVs-8C and GVs-8D.
  • the determined DNA sequences for the clones GVs-8A, GVs-8B, GVs-8C and GVs-8D are shown in SEQ ID NOS: 48-51, respectively, with the corresponding amino acid sequences being shown in SEQ ID NOS: 52-55, respectively.
  • the clone GVs-8A contains regions showing some similarity to known prokaryotic valyi-tRNA synthetases; GVs-8B shows some similarity to M.
  • GVs-8C shows some similarity to the H. influenza folylpolyglutamate synthase gene.
  • GVs-8D contains an open reading frame which shows some similarity to sequences previously identified in M. tuberculosis and M. leprae, but whose function has not been identified.
  • MPG15 BamHl -site of lambda ZAP Express vector (Stratagene) was screened with a second redundant oligonucleotide (referred to as MPG15; SEQ ID NO:73) designed from the
  • GVs-8 sequence provided in SEQ ID NO:6.
  • Screening of the library was performed in the presence of tetramethylammonium chloride (TMAC), so that nucleotide base pairs would melt at a standard temperature independent of sequence (i.e. A-T pairs and G-C pairs melt at the same temperature).
  • TMAC tetramethylammonium chloride
  • Hybridisation was performed overnight in freshly prepared hybridisation solution containing 100 pmol probe.
  • yeast tRNA 10 mg/ml
  • the filters were washed at a temperature calculated to allow approximately 4 % mismatching in TMAC wash buffer. More specifically, the wash protocol included the following washing steps: 2 x 15 min in 6X SSC, 0.05% NaPPi at room temp; 1 x 15 min in TMAC wash (see below) at room temperature; 2 x 15 min in TMAC wash at the calculated stringent temperature; and 1 x 15 min in 6X SSC, 0.05% NaPPi at room temp.
  • TMAC wash buffer included the following washing steps: 2 x 15 min in 6X SSC, 0.05% NaPPi at room temp; 1 x 15 min in TMAC wash (see below) at room temperature; 2 x 15 min in TMAC wash at the calculated stringent temperature; and 1 x 15 min in 6X SSC, 0.05% NaPPi at room temp.
  • TMAC Tetramethylammonium chloride
  • the pBK-CMV phagemid containing the desired insert was excised from the lambda ZAP Express vector in the presence of ExAssist helper phage following the manufacturer's protocol.
  • a phagemid containing an 8 kb insert (GVs-8D) was characterised by restriction mapping and sub-cloning. An open reading frame was identified at the 3' end of the insert and the antigen encoded by this open reading frame was named GV-33. Base sequence corresponding to GVs-8 was not found in the insert, and it was assumed that GV-33 was obtained as a non-specific product of the TMAC screening. By further sub-cloning and base sequencing, the 3' end of the gene was determined.
  • the determined partial DNA sequence for GV-33 is provided in SEQ ID NO:74 with the corresponding predicted amino acid sequence being provided in SEQ ID NO:75. Sequence data from the 3' end of the clone showed homology to a previously identified 40.6 kDa outer membrane protein of M. tuberculosis.
  • TKe " partial GV-33 gene was amplified from M. vaccae genomic DNA with primers based on the determined nucleotide sequence. This DNA fragment was cloned into EcoRv-digested pBluescript (Stratagene) with additional dTTP residues, and then transferred to pProEX HT expression vector (Gibco BRL) using EcoRl and Hindlll-subcloning. Recombinant protein was purified following the manufacturer's protocol. In subsequent studies, GV-33 was re-cloned into the alternative vector pET16 (Novagen).
  • This example illustrates the preparation of whole M. vaccae and M. vaccae culture filtrate and its non-specific immune amplifying or 'adjuvant' property.
  • M. vaccae bacteria was cultured, pelleted and autoclaved as described in Example 1.
  • Culture filtrates of live M. vaccae refer to the supernatant from 24 hour cultures of M. vaccae in 7H9 medium with glucose.
  • the resulting pellet was suspended in 100 ml of chloro form/methanol (2:1), incubated at room temperature for 1 hour; recentrifuged, and the chloroform methanol extraction repeated.
  • the pellet was obtained by centrifugation, dried in vacuo, weighed and resuspended in PBS at 50mg (dry weight) per ml as delipidated M. vaccae.
  • Glycolipids were removed from the delipidated M. vaccae preparation by refluxing in 50% v/v ethanol for 2 hours.
  • the insoluble material was collected by centrifugation and washed in PBS. Proteins were extracted by resuspending the pellet in 2% SDS in PBS at 56 °C for 2 hours. The insoluble material was collected by centrifugation and the extraction with 2% SDS/PBS at 56 °C was repeated twice more.
  • M. vaccae culture supernatant (S N) killed M. vaccae and delipidated M. vaccae were tested for adjuvant activity in the generation of cytotoxic T cell immune response to ovalbumin, a structurally unrelated protein, in the mouse.
  • This anti- ovalbumin-specific cytotoxic response was detected as follows. C57BL/6 mice (2 per group) were immunized by the intraperitoneal injection of 100 ⁇ g of ovalbumin with the following test adjuvants: autoclaved M. vaccae; delipidated M. vaccae; delipidated M. vaccae with glycolipids also extracted and proteins extracted with SDS; the SDS protein extract treated with pronase (an enzyme which degrades protein); whole M. vaccae culture filtrate; and heat-killed M. tuberculosis or heat- killed M. bovis BCG, M. phlei or M. smegmatus or M. vaccae culture filtrate.
  • E.G7 cells which are EL4 cells (a C57BL/6-derived T cell lymphoma) transfected with the ovalbumin gene and thus express ovalbumin.
  • the spleen cells were then assayed for their ability to kill non-specifically EL4 target cells or to kill specifically the E.G7 ovalbumin expressing cells. Killing activity was detected by the release of 5 I Chromium with which the EL4 and E.G7 cells have been labelled (100 ⁇ Ci per 2xl0 6 ), prior to the killing assay. Killing or cytolytic activity is expressed as % specific lysis using the formula:
  • ovalbumin-specific cytotoxic cells are generated only in mice immunized with ovalbumin with an adjuvant but not in mice immunized with ovalbumin alone.
  • FIG. 4 show the effect of various M. vaccae derived adjuvant preparations on the generation of cytotoxic T cells to ovalbumin in C57BL/6 mice.
  • cytotoxic cells were generated in mice immunized with (i) 10 ⁇ g, (ii) 100 ⁇ g or (iii) 1 mg of autoclaved M. vaccae or (iv) 75 ⁇ g of M. vaccae culture filtrate.
  • Fig. 4B shows that cytotoxic cells were generated in mice immunized with (i) 1 mg whole autoclaved M. vaccae or (ii) 1 mg delipidated M. vaccae.
  • Fig. 4A shows that cytotoxic cells were generated in mice immunized with (i) 1 mg whole autoclaved M. vaccae or (ii) 1 mg delipidated M. vaccae.
  • cytotoxic cells were generated in mice immunized with 1 mg whole autoclaved M. vaccae; Fig. 4C(ii) shows the active material in 100 ⁇ g delipidated M. vaccae which then had glycolipids removed and the proteins extracted with SDS. Fig. 4C(iii) shows that active material in the adjuvant preparation of Fig. 4C(ii) was destroyed by treatment with the proteolytic enzyme pronase.
  • 100 ⁇ g of the SDS-extracted proteins had significantly stronger immune-enhancing ability (Fig. 4C(ii)) than did 1 mg whole autoclaved M. vaccae (Fig. 4C(i)). Mice immunized with 1 mg heat-killed M.
  • Fig. 4D(i) generated cytotoxic cells to ovalbumin, but mice immunized separately with 1 mg heat-killed M. tuberculosis (Fig. 4D(ii)), 1 mg M. bovis BCG (Fig. 4D(iii)), 1 mg M. phlei (Fig. 4D(iv)), or 1 mg M smegmatis (Fig. 4D(v)) failed to generate cytotoxic cells.
  • the SDS-extracted proteins derived from delipidated and deglycolipidated M. vaccae were analysed by polyacrylamide gel electrophoresis. As shown in Fig. 5, three major bands were observed after staining with silver.
  • GV-27 The base sequence of the 5' end of the insert of this clone, hereinafter referred to as GV-27, was determined using Sanger sequencing with fluorescent primers on Perkin Elmer/Applied Biosystems Dvision automatic sequencer.
  • the determined nucleotide sequence of the partial M. vaccae GroEL-homologue clone GV-27 is provided in SEQ ID NO:77 and the predicted amino acid sequence in SEQ ID NO:78. This clone was found to have homology to M. tuberculosis GroEL.
  • a partial sequence of the 65 kDa heat shock protein of M. vaccae has been published by Kapur et al. (Arch. Pathol. Lab. Med. 7/9 :131-138, 1995).
  • the nucleotide sequence of the Kapur et al. fragment is shown in SEQ ID NO:79 and the predicted amino acid sequence in SEQ ID NO:80.
  • GV-27 was found to be 93.7% identical to the M. tuberculosis GroEL at the amino acid level.
  • Two peptide fragments, comprising the N-terminal sequence hereinafter referred to as GV-27A
  • t ⁇ as GV-27B were prepared using techniques well known in the art.
  • the nucleotide sequences for GV-27A and GV-27B are provided in SEQ ID NO: 1 15 and 116, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 1 17 and 1 18.
  • the sequence of GV-27A is 95.8% identical to the M. tuberculosis GroEL sequence and contains the shorter M. vaccae sequence of Kapur et al. discussed above.
  • the sequence for GV-27B shows about 92.2% identity to the corresponding region of M. tuberculosis HSP65.
  • the M. vaccae culture filtrate described above was also fractionated by iso- electric focusing and the fractions assayed for adjuvant activity in the anti-ovalbumin- specific cytotoxic response assay in C57BL/6 mice as described above. As shown in Fig. 7, peak adjuvant activities were demonstrated in fractions corresponding to pi of 4.2-4.32 (fraction nos. 7-9), 4.49-4.57 (fraction nos. 13-17) and 4.81-5.98 (fraction nos. 23-27).
  • CD8 T cells which preferentially kill macrophages that have been infected with M tuberculosis.
  • mice were immunized by the intraperitoneal injection of 500 ⁇ g of killed M. vaccae which was prepared as described in Example 1.
  • the spleen cells of immunized mice were passed through a CD8 T cell enrichment column (R&D Systems, St. Paul, MN, USA).
  • the spleen cells recovered from the column have been shown to be enriched up to 90% CD8 T cells.
  • These T cells, as well as CD8 T cells from spleens of non-immunized mice were tested for their ability to kill uninfected macrophages or macrophages which have been infected with M. tuberculosis.
  • Macrophages were obtained from the peritoneal cavity of mice five days after they have ⁇ been given 1 ml of 3% thioglycolate intraperitoneally.
  • the macrophages were infected overnight with M. tuberculosis at the ratio of 2 mycobacteria per macrophage. All macrophage preparations were labelled with 51 Chromium at 2 ⁇ ci per 10 4 macrophages.
  • the macrophages were then cultured with CD8 T cells overnight (16 hours) at killer to target ratios of 30:1. Specific killing was detected by the release of 51 Chromium and expressed as % specific lysis, calculated as in Example 5.
  • ELISA enzyme-linked immunosorbent assay
  • CD8 T cells from spleens of mice immunized with M. vaccae were cytotoxic for macrophages infected with M. tuberculosis and did not lyse uninfected macrophages.
  • the CD8 T cells from non-immunized mice did not lyse macrophages.
  • CD8 T cells from naive or non-immunized mice do produce IFN- ⁇ when cocultured with infected macrophages. The amount of IFN- ⁇ produced in coculture was greater with CD8 T cells derived from M. vaccae immunized mice.
  • M. vaccae (ATCC Number 15483) was grown in sterile Medium 90 at 37 °C for 4 days and harvested by centrifugation. Cells were resuspended in 1 ml Trizol (Gibco BRL, Life Technologies, Gaithersburg, Maryland) and RNA extracted according to the standard manufacturer ' s protocol. M. tuberculosis strain H37Rv (ATCC Number 27294) was grown in sterile Middlebrooke 7H9 medium with Tween 80TM and oleic acid/ albumin dextrose/catalase additive (Difco Laboratories, Detroit, Michigan) at 37 °C and harvested under appropriate laboratory safety conditions. Cells were resuspended in 1 ml Trizol (Gibco BRL) and RNA extracted according to the manufacturer's standard protocol.
  • Total M. tuberculosis and M. vaccae RNA was depleted of 16S and 23 S ribosomal RNA (rRNA) by hybridisation of the total RNA fraction to oligonucleotides AD 10 and ADH (SEQ ID NO: 81 and 82) complementary to M. tuberculosis rRNA.
  • oligonucleotides were designed from mycobacterial 16S rRNA sequences published by Bottger (FEMS Microbiol. Lett. 65: 171 - 176, 1989) and from sequences deposited in the databanks.
  • RNA ⁇ fo oligonucleotides AD10 and ADH immobilised on nylon membranes Hybond N, Amersham International, United Kingdom. Hybridisation was repeated until rRNA bands were not visible on ethidium bromide-stained agarose gels.
  • First strand cDNA synthesis was performed following standard protocols, using oligonucleotide AD7 (SEQ ID NO:84) containing a poly(dT) sequence.
  • the M. tuberculosis and M. vaccae cDNA was used as template for single- sided-specific PCR (3S-PCR).
  • a degenerate oligonucleotide AD1 (SEQ ID NO:85) was designed based on conserved leader sequences and membrane protein sequences. After 30 cycles of amplification using primer AD 1 as 5'-primer and AD7 as 3'-primer, products were separated on a urea/polyacrylamide gel. DNA bands unique to M vaccae were excised and re-amplified using primers AD1 and AD7. After gel purification, bands were cloned into pGEM-T (Promega) and the base sequence determined.
  • Cassette transporters typically consist of four genes: an ATP-binding gene, a periplasmic, or substrate binding, gene and two transmembrane genes.
  • the transmembrane genes encode proteins each characteristically having six membrane- spanning regions. Homologues (by similarity) of this ABC transporter have been identified in the genomes of Haemophilus influenza (Fleischmann et al. Science 269
  • the nucleotide sequence of the full-length M. vaccae homologue of pota (ATP-binding protein) was identified by subcloning of the 4.5 kb fragment and base sequencing.
  • the nucleotide and predicted amino acid sequences of the M. vaccae pota homologue are provided in SEQ ID NOS:88 and 89, respectively.
  • the nucleotide sequence of the M. vaccae pota gene was used to design primers EV24 and EV25 (SEQ ID NO: 90 and 91) for expression cloning.
  • the amplified DNA fragment was cloned into pProEX HT prokaryotic expression system (Gibco BRL) and expression in an appropriate E.coli host was induced by addition of 0.6 mM isopropylthio- ⁇ -galactoside (IPTG).
  • IPTG isopropylthio- ⁇ -galactoside
  • the recombinant protein was named GV-23 and purified from inclusion bodies according to the manufacturer ' s protocol. In subsequent studies, GV-23 (SEQ ID NO: 88) was re-cloned into the alternative vector pETl 6 (Novagen).
  • a 322 bp Sall-BamHl subclone at the 3'-end of the 4.5 kb insert described above showed homology to the potd gene, (periplasmic protein), of the spermidine/putrescine ABC transporter complex of E. coll
  • the nucleotide sequence of this subclone is shown in SEQ ID NO:92.
  • the radiolabelled insert of this subclone was used to probe an M. vaccae genomic DNA library constructed in the Sail -site of lambda Zap Express (Stratagene) following standard protocols.
  • a clone was identified of which 1342 bp showed homology with the potd gene of E. coli.
  • the potd homologue of M. vaccae was identified by sub-cloning and base sequencing. The determined nucleotide and predicted amino acid sequences are shown in SEQ ID NO: 93 and 94.
  • primers EV26 and EV27 were designed from the determined M. vaccae potd homologue.
  • the amplified fragment was cloned into pProEX HT Prokaryotic expression system (Gibco BRL). Expression in an appropriate E. coli host was induced by addition of 0.6 mM IPTG and the recombinant protein named GV24.
  • the recombinant antigen was purified from inclusion bodies according to the protocol of the supplier.
  • GV- 24 SEQ ID ' NO: 93 was re-cloned into the alternative vector pET16 (Novagen).
  • M. vaccae potb homologue Base sequence adjacent to the M vaccae potd gene-homologue was found to show homology to the potb gene of the spermidine/putrescine ABC transporter complex of E.coli, which is one of two transmembrane proteins in the ABC transporter complex.
  • the M. vaccae potb homologue (referred to as GV-25) was identified through further subcloning and base sequencing. The determined nucleotide and predicted amino acid sequences for GV-25 are shown in SEQ ID NOS :97 and 98, respectively.
  • the 3S-PCR band 12B28 (SEQ ID NO: 119) was used to screen the M. vaccae genomic library constructed in the BamHI-site of lambda ZAP Express (Stratagene).
  • the clone isolated from the library contained a novel open reading frame and the antigen encoded by this gene was named GV-38A.
  • the determined nucleotide sequence and predicted amino acid sequence of GV-38A are shown in SEQ ID NO: 120 and 121 , respectively. Comparison of these sequences with those in the gene bank, revealed some homology to an unknown M. tuberculosis protein previously identified in cosmid MTCY428.12. (SPTREMBL:P71915).
  • GV-38B Upstream of the GV-38A gene, a second novel open reading frame was identified and the antigen encoded by this gene was named GV-38B
  • the determined 5' and 3' nucleotide sequences for GV-38B are provided in SEQ ID NO 122 and 123, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 124 and 125, respectively
  • This protein was found to show homology to an unknown M tuberculosis protein identified in cosmid MTCY428 11 (SPTREMBL: P71914).
  • GV-38A and GV-38B antigens were amplified for expression cloning into pET16 (Novagen).
  • GV-38A was amplified with primers KR1 1 and KR12 (SEQ ID NO 126 and 127) and GV-38B with primers KR13 and KR14 (SEQ ID NO- 128 and 129).
  • Protein expression in the host cells BL21(DE3) was induced with 1 M IPTG. however no protein expression was obtained from these constructs
  • Hydrophobic regions were identified in the N-termini of antigens GV-38A and GV- 38B which may inhibit expression of these constructs
  • the hydrophobic region present in GV-38A was identified as a possible transmembrane motif with six membrane spanning regions.
  • primers KR20 for GV-38A, (SEQ ID NO: 130) and KR21 for GV-38B (SEQ ID NO. 131 ) were designed.
  • the truncated GV-38A gene was amplified with primers KR20 and KR12, and the truncated GV-38B gene with KR21 and KR14
  • the determined nucleotide sequences of truncated GV38A and GV-38B are shown in SEQ ID NO. 132 and 133, respectively, with the corresponding predicted amino acid sequenceS"be ⁇ ng shown in SEQ ID NO: 134 and 135, respectively.
  • M. vaccae soluble proteins were isolated from culture filtrate using preparative isoelectric focusing and preparative polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
  • M. vaccae (ATCC Number 15483) was cultured in 250 1 sterile Medium 90 which had been fractionated by ultrafiltration to remove all proteins of greater than 10 kDa molecular weight. The medium was centrifuged to remove the bacteria, and sterilised by filtration through a 0.45 m filter. The sterile filtrate was concentrated by ultrafiltration over a 10 kDa molecular weight cut-off membrane.
  • Proteins were isolated from the concentrated culture filtrate by precipitation with 10% trichloroacetic acid. The precipitated proteins were re-dissolved in 100 mM Tris. HCl pH 8.0. and re-precipitated by the addition of an equal volume of acetone. The acetone precipitate was dissolved in water, and proteins were re-precipitated by the addition of an equal volume of chloroform:methanol 2: 1 (v/v). The chloroform:methanol precipitate was dissolved in water, and the solution was freeze - dried.
  • the freeze-dried protein was dissolved in iso-electric focusing buffer, containing 8 M deionised urea, 2% Triton X-100, 10 mM dithiothreitol and 2% ampholytes (pH 2.5 - 5.0).
  • the sample was fractionated by preparative iso-electric focusing on a horizontal bed of Ultrodex gel at 8 watts constant power for 16 hours. Proteins were eluted from the gel bed fractions with water and concentrated by precipitation with 10% trichloroacetic acid.
  • Eluted proteins were assayed for their ability to induce proliferation and interferon- ⁇ secretion from the peripheral blood lymphocytes of immune donors as detailed in Example 2. Proteins inducing a strong response in these assays were selected for further study. Selected proteins were further purified by reversed-phase chromatography on a Vydac Protein C4 column, using a trifluoroacetic acid-acetonitrile system. Purified proteins were prepared for protein sequence determination by SDS-polyacrylamide gel electrophoresis, and electroblotted onto PVDF membranes. Protein sequences were determined as in Example 3. The proteins were named GV-40, GV-41 , GV-42, GV-43 and GV-44.
  • GV-40 had similarity to a putative ribosome recycling factor from M. tuberculosis, a protein responsible for the release of ribosomes from mRNA at the termination of protein biosynthesis.
  • GV-43 showed homology (by similarity) to a previously identified unknown M. tuberculosis and M. leprae protein.
  • This example illustrates the processing of different constituents of M.vaccae and their immune modulating properties.
  • M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/1; tryptone, 5 g/1; glucose 1 g/1) at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10 10 M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 °C. The culture filtrate was passaged through a 0.45 ⁇ M filter into sterile bottles.
  • sterile Medium 90 yeast extract, 2.5 g/1; tryptone, 5 g/1; glucose 1 g/1
  • the cells were harvested by centrifugation, and transferred
  • M.vaccae To prepare delipidated M.vaccae, the autoclaved M. vaccae was pelleted by centrifugation, the pellet washed with water and collected again by centrifugation and then freeze-dried. An aliquot of this freeze-dried M. vaccae was set aside and referred to as lyophilised M.vaccae. When used in experiments it was resuspended in PBS to the desired concentration. Freeze-dried M vaccae was extracted with chloroform/methanol (2:1) for 60 mins at room temperature, and the extraction was repeated once. The residue from chloroform/methanol extraction was further extracted with 50% ethanol by refluxing for two hours. The 50% ethanol extraction was repeated two times. The pooled 50% ethanol extracts were used as a source of
  • delipidated M.vaccae glycolipids see below.
  • the residue from the 50% ethanol extraction was freeze-dried and weighed.
  • the amount of delipidated M.vaccae prepared was equivalent to 11.1% of the starting wet weight of M.vaccae used.
  • the delipidated and deglycosylated M.vaccae DO-M. vaccae referred to as delipidated
  • the culture supematants were assayed for the presence of IL-12 produced by macrophages.
  • the M. vaccae preparations stimulated the production of IL-12 from macrophages.
  • FIGS 9A, B, and C show data from separate experiments in which groups of C57BL/6 mice (Fig. 9A), BALB/C mice (Fig. 9B) or C3H/HeJ mice (Fig. 9C) were given DIFCO thioglycolate intraperitoneally and, after three days, peritoneal macrophages were collected and placed in culture with interferon-gamma for three hours.
  • the culture medium was replaced and various concentrations of M.vaccae recombinant proteins GVc-3 (GV 3), GVc4P (GV 4P), GVc-7 (GV7), GV-23, GV 27, heat killed M.vaccae, OD-M.vaccae (referred to as delipidated M.
  • This example illustrates the effect of immunisation with M.vaccae or M.vaccae culture filtrate intradermally in cynomolgous monkeys prior to challenge with live M. tuberculosis.
  • M.vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5g/l; tryptone, 5g/l; glucose, lg/I) at 37°C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37°C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of lOmg/ml, equivalent to 10'° M.vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120°C. The culture filtrate was passaged through a 0.45 ⁇ M filter into sterile bottles.
  • sterile Medium 90 yeast extract, 2.5g/l; tryptone, 5g/l; glucose, lg/I
  • the cells were harvested by centrifug
  • the monkeys in the control group showed radiologic evidence of pulmonary tuberculosis by 28 days after infection with M. tuberculosis.
  • Clinical disease was not evident 84 days after infection in monkeys immunised intradermally with two doses of 500 ⁇ g of M. vaccae. The onset of clinical disease was delayed in both monkeys immunised intradermally with 100 ⁇ g of M vaccae culture filtrate.
  • MOLECULE TYPE protein
  • xi SEQUENCE DESCRIPTION SEQ ID NO : 5 ⁇
  • Residue can be either Glu or He
  • MOLECULE TYPE protein ( xi ) SEQUENCE DESCRIPTION - SEQ ID NO - 12
  • Residue can be either Gly or Ala
  • Residue can be either Pro or Ala
  • Residue can be either Ala or Arg
  • Residue can be either Val or Leu
  • Residue can be either Ser or Val
  • Residue can be either Gin or Val
  • Residue can be either Tyr or Pro
  • Residue can be either Val or Gly
  • Residue can be either He or Tyr
  • Residue can be either Leu or Pro
  • Trp Gly Ala Gin Leu Asn Ala Met Lys Gly Asp Leu Gin Ser Ser Leu 305 310 315 320
  • MOLECULE TYPE Genomic DNA
  • GGTACCGGAA GCTGGAGGAT TGACGGTATG AGACTTCTTG ACAGGATTCG TGGGCCTTGG 60
  • CTCGCCGCCA ACAAGGGGGT CGACCCGAAC CGCAACGCGG CCGTCGGTCT GTCCATGGCC 240

Abstract

The present invention provides polypeptides comprising an immunogenic portion of a M. vaccae protein and DNA molecules encoding such polypeptides, together with methods for their use in the diagnosis and treatment of mycobacterial infection. Methods for enhancing the immune response to an antigen including administration of M. vaccae culture filtrate or delipidated M. vaccae cells are also provided.

Description

COMPOUNDS AND METHODS FOR TREATMENT AND DIAGNOSIS OF MYCOBACTERIAL INFECTIONS
Technical Field The present invention relates generally to the detection, treatment and prevention of infectious diseases. In particular, the invention is related to compounds and methods for the treatment of mycobacteπal infections including Mycobacterium tuberculosis and Mycobacterium avium The invention is further related to compounds that function as non-specific immune response amplifiers, and the use of such non-specific immune response amplifiers as adjuvants in vaccination or lmmunotherapy against infectious disease, and in certain treatments for immune disorders and cancer.
Background of the Invention Tuberculosis is a chronic, infectious disease, that is caused by infection with
Mycobacterium tuberculosis (M tuberculosis) It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as a chronic inflammation of the lungs, resulting in fever and respiratory symptoms. If left untreated, significant morbidity and death may result.
Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time In addition, although compliance with the treatment regimen is critical, patient behaviour is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistant mycobacteπa
Inhibiting the spread of tuberculosis requires effective vaccination and accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common mycobacterium employed for this purpose is Bacillus Calmette-Gueπn (BCG), an avirulent strain of Mycobacterium bovis. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public. Diagnosis of M. tuberculosis infection is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, thereby indicating exposure to mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals. A less well-known mycobacterium that has been used for immunotherapy for tuberculosis, and also leprosy, is Mycobacterium vaccae, which is non-pathogenic in humans. However, there is less information on the efficacy of M. vaccae compared with BCG, and it has not been used widely to vaccinate the general public. M. bovis BCG and M. vaccae are believed to contain antigenic compounds that are recognised by the immune system of individuals exposed to infection with M. tuberculosis.
There thus remains a need in the art for effective compounds and methods for preventing, treating and detecting tuberculosis.
Summary of the Invention Briefly stated, the present invention provides compounds and methods for the prevention, treatment and diagnosis of mycobacterial infection, together with adjuvants Tor use in vaccines or immunotherapy of infectious diseases and cancers.
In a first aspect, polypeptides derived from Mycobacterium vaccae are provided comprising an immunogenic portion of an antigen, or a variant of such an antigen. In one embodiment, the antigen includes an amino acid sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 1-4, 9-16, 18- 21, 23, 25, 26, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100-105, 109, 110, 112, 1 14, 1 17, 1 18, 121, 124, 125, 134, 135, 140 and 141 ; and (b) sequences having at least about a 99% probability of being the same as a sequence recited in SEQ ID NOS: 1-4, 9-16, 18-21, 23, 25, 26, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100-105, 109, 1 10, 1 12, 1 14. 1 17, 1 18, 121. 124. 125, 134. 135, 140 and 141 as measured by the computer algorithm BLASTP.
In a second aspect, the invention provides polypeptides comprising an immunogenic portion of an M. vaccae antigen wherein the antigen comprises an amino acid sequence encoded by a DNA molecule selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 40-42, 46, 48-51 , 74, 88, 93, 97, 99, 106-
108, 1 1 1 , 1 13, 1 15, 1 16, 120, 122, 123, 132, 133 and 136-138; (b) complements of the sequences recited in SEQ ID NOS: 40-42, 46, 48-51, 74, 88, 93, 97, 99, 106-108,
1 1 1, 1 13, 115, 1 16, 120, 122, 123, 132, 133 and 136-138; and (c) sequences having at least about a 99% probability of being the same as a sequence of (a) or (b) as measured by the computer algorithm FASTA.
DNA sequences encoding the inventive polypeptides, expression vectors comprising these DNA sequences, and host cells transformed or transfected with such expression vectors are also provided. In another aspect, the present invention provides fusion proteins comprising a first and a second inventive poiypeptide or, alternatively, an inventive poiypeptide and a known M. tuberculosis antigen.
Within other aspects, the present invention provides pharmaceutical compositions that comprise at least one of the inventive polypeptides, or a DNA molecule encoding such a poiypeptide, and a physiologically acceptable carrier. The invention also provides vaccines comprising at least one of the above polypeptides and a nonspecific immune response amplifier, together with vaccines comprising at least one DNA sequence encoding such polypeptides and a non-specific immune response amplifier. In yet another aspect, methods are provided for inducing protective immunity in a patient, comprising administering to a patient an effective amount of one or more of the above polypeptides together with an immune response amplifier.
In further aspects of this invention, methods and diagnostic kits are provided for detecting tuberculosis in a patient. In a first embodiment, the method comprises contacting dermal cells of a patient with one or more of the above polypeptides and detecting an immune response on the patient's skin. In a second embodiment, the method comprises contacting a biological sample with at least one of the above polypeptides; and detecting in the sample the presence of antibodies that bind to the poiypeptide or polypeptides, thereby detecting M. tuberculosis infection in the biological sample. Suitable biological samples include whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine.
Diagnostic kits comprising one or more of the above polypeptides in combination with an apparatus sufficient to contact the poiypeptide with the dermal cells of a patient are provided. The present invention also provides diagnostic kits comprising one or more of the inventive polypeptides in combination with a detection reagent.
In yet another aspect, the present invention provides antibodies, both polyclonal and monoclonal, that bind to the polypeptides described above, as well as methods for their use in the detection of M. tuberculosis infection.
The present invention also provides methods for enhancing a non-specific immune response to an antigen. In one embodiment, such methods comprise administering a composition comprising a component selected from the group consisting of : (a) delipidated M. vaccae cells, (b) deglycosylated M. vaccae cells; (c) delipidated and deglycosylated M. vaccae cells and (d) M. vaccae culture filtrate. In a second embodiment, such methods comprise administering a poiypeptide, the poiypeptide comprising an immunogenic portion of an antigen, wherein said antigen includes a sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 114, 1 17 and 118; and (b) sequences having at least about 97% identity to a sequence recited in SEQ ID NOS: 114, 1 17 and 118.
In yet a further aspect, compositions comprising a component selected from the group consisting of delipidated M. vaccae cells, deglycosylated M. vaccae cells, and delipidated and deglycosylated M. vaccae cells are provided, together with vaccines comprising such components and methods of using such compositions and vaccines to induce protective immunity in a patient.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
Brief Description of the Drawings Figs. 1A and IB illustrate the protective effects of immunizing mice with autoclaved M. vaccae or unfractionated M. vaccae culture filtrates, respectively, prior to infection with live M. tuberculosis H37Rv.
Figs. 2A and B show components of M. vaccae and M. tuberculosis culture filtrates, respectively, as analysed by 2-dimensional polyacrylamide gel electrophoresis.
Fig. 3 is a comparison of the Antigen 85A protein sequence obtained from M. vaccae with those from M. bovis, M. tuberculosis and M. leprae.
Fig. 4A(i) - (iv) illustrate the non-specific immune amplifying effects of 10 μg, XOOμg and lmg autoclaved M. vaccae and 15μg unfractionated culture filtrates of M. vaccae, respectively. Fig. 4B(i) and (ii) illustrate the non-specific immune amplifying effects of autoclaved M. vaccae and delipidated M. vaccae, respectively. Fig. 4C(i) illustrates the non-specific immune amplifying effects of whole autoclaved M. vaccae. Fig. 4C(ii) illustrates the non-specific immune amplifying effects of delipidated M. vaccae from which glycolipids had been removed and the proteins extracted with SDS. Fig. 4C(iii) illustrates that the adjuvant effect of the preparation of Fig. 4C(ii) is destroyed by treatment with the proteolytic enzyme pronase. Fig. 4D illustrates The non-specific immune amplifying effects of heat-killed M. vaccae (Fig. 4D(i)), M. tuberculosis (Fig. 4D(ii)), M. bovis BCG (Fig. 4D(iii)), M. phlei (Fig. 4D(iv)) and M. smegmatis (Fig. 4D(v)). Fig. 5 shows the results of polyacrylamide gel electrophoresis analysis of
SDS-extracted proteins derived from delipidated and deglycolipidated M. vaccae.
Fig. 6 illustrates the non-specific immune amplifying effects of different molecular weight fractions of SDS-extracted M. vaccae proteins.
Fig. 7 illustrates the non-specific immune amplifying effects of different pi fractions of SDS-extracted M. vaccae proteins. Fig. 8 illustrates the induction of IL-12 by heat-killed M. vaccae, lyophilized M. vaccae, delipidated and deglycosylated M. vaccae (referred to as "delipidated M. vaccae") and M. vaccae glycolipids.
Fig. 9 illustrates the stimulation of interferon-gamma production by different concentrations of M. vaccae recombinant proteins, heat-killed M. vaccae, delipidated and deglycosylated M. vaccae (referred to as "delipidated M. vaccae"), M. vaccae glycolipids and lipopolysaccharide in C57BL-6 peritoneal macrophages (Fig 9A); BALB/C peritoneal macrophages (Fig 9B); and C3H/HeJ peritoneal macrophages (Fig 9C).
Detailed Description of the Invention As noted above, the present invention is generally directed to compositions and methods for preventing," treating and diagnosing mycobacterial infections, including M. tuberculosis and M. avium infections.
Considerable research efforts have been directed towards elucidating the mechanism of immune response to mycobacterial infection, in particular M. tuberculosis infection. While macrophages have been shown to act as the principal effectors of M. tuberculosis immunity, T cells are the predominant inducers of such immunity. The essential role of T cells in protection against M. tuberculosis infection is illustrated by the frequent occurrence of M. tuberculosis in AIDS patients, due to the depletion of CD4 T cells associated with human immunodeficiency virus (HIV) infection. Mycobacterium-reactive CD4 T cells have been shown to be potent producers of gamma-interferon (IFN-γ), which, in turn, has been shown to trigger the anti-myco acterial effects of macrophages in mice. While the role of IFN-γ in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-γ or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis infection. Furthermore, it is known that IFN-γ stimulates human macrophages to make 1 ,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play a role in stimulating resistance to M. tuberculosis infection. Another property of CD4" T cells and macrophages is their ability to activate CD8* cytotoxic T cells which are capable of killing pathogen-infected cells. CD8* T cells have been shown to kill macrophages and other cells that harbour M. tuberculosis. For a review of the immunology of M. tuberculosis infection see Chan and Kaufmann in Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASM Press. Washington, DC, 1994.
The compositions of the present invention include polypeptides that comprise at least one immunogenic portion of an M. vaccae antigen, or a variant thereof. Such polypeptides stimulate T cell proliferation, and/or, interferon gamma secretion from T cells of individuals exposed to M. tuberculosis. In certain embodiments, the inventive polypeptides comprise at least an immunogenic portion of a soluble M. vaccae antigen. A "soluble M. vaccae antigen" is a protein of M. vaccae origin that is present in M. vaccae culture filtrate. As used herein, the term "poiypeptide" encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. Thus, a poiypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M. vaccae antigen or may be heterologous, and such sequences may (but need not) be immunogenic.
"Immunogenic," as used herein, refers to the ability to elicit an immune response in a patient, such as a human, or in a biological sample. In particular, immunogenic antigens are capable of stimulating cell proliferation, interleukin-12 production or interferon-γ production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from an M. tuberculosis-immune individual. Polypeptides comprising at least an immunogenic portion of one or more M. vaccae antigens may generally be used to detect tuberculosis or to induce protective immunity against tuberculosis in a patient. The compositions and methods of this invention also encompass variants of the above polypeptides. As used herein, the term "variant" covers any sequence which exhibits at least about 50%, more preferably at least about 70% and more preferably yet, at least about 90% identity to a sequence of the present invention. Most preferably, a "variant" is any sequence which has at least about a 99% probability of being the same as the inventive sequence. The probability for DNA sequences is measured by the computer algorithm FASTA (version 2.0u4, February 1996; Pearson W. R. et al., Proc. Natl. Acad. Sci., 85:2444-2448, 1988), the probability for translated DNA sequences is measured by the computer algorithm TBLASTX and that for protein sequences is measured by the computer algorithm BLASTP (Altschul, S. F. et al. J. Mol. Biol.. 215:403-410, 1990). The term "variants" thus encompasses sequences wherein the probability of finding a match by chance (smallest sum probability), is less than about 1% as measured by any of the above tests.
A poiypeptide of the present invention may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post- translationally directs transfer of the protein. The poiypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the poiypeptide (e.g., poly-His), or to enhance binding of the poiypeptide to a solid support. For example, a poiypeptide may be conjugated to an immunoglobulin Fc region. In general, M. vaccae antigens, and DNA sequences encoding such antigens, may be prepared using any of a variety of procedures. For example, soluble antigens may be isolated from M. vaccae culture filtrate as described below. Antigens may also be produced recombinantly by inserting a DNA sequence that encodes the antigen into an expression vector and expressing the antigen in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant poiypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.
DNA sequences encoding M. vaccae antigens may be obtained by screening an appropriate M. vaccae cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from partial amino acid sequences of isolated soluble antigens. Suitable degenerate oligonucleotides may be designed and synthesized, and the screen may be performed as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. As described below, polymerase chain reaction (PCR) may be employed to isolate a nucleic acid probe from a cDNA or genomic DNA library. The library screen may then be performed using the isolated probe.
DNA molecules encoding M. vaccae antigens may also be isolated by screening an appropriate M. vaccae expression library with anti-sera (e.g., rabbit or monkey) raised specifically against M. vaccae antigens.
Regardless of the method of preparation, the antigens described herein have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce cell proliferation and/or cytokine production (for example, interferon-γ and/or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from an M. tuberculosis-immune individual. An
M. tuberculosis-immune individual is one who is considered to be resistant to the development of tuberculosis by virtue of having mounted an effective T cell response to M. tuberculosis. Such individuals may be identified based on a strongly positive (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD), and an absence of any symptoms of tuberculosis infection. The selection of cell type for use in evaluating an immunogenic response to an antigen will depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing B cells or macrophages. T cells, NK cells, B cells and macrophages derived from M. tuberculosis-immune individuals may be prepared using methods well known in the art. For example, a preparation of peripheral blood mononuclear cells (PBMCs) may be employed without further separation of component cells. PBMCs may be prepared, for example, using density centrifugation through Ficoll™ (Winthrop Laboratories, NY). T cells for use in the assays described herein may be purified directly from PBMCs. Alternatively, an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins, may be employed. Such T cell clones may be generated by, for example, culturing PBMCs from M. tuberculosis- immune individuals with mycobacterial proteins for a period of 2-4 weeks. This allows expansion of only the mycobacterial protein-specific T cells, resulting in a line composed solely of such cells. These cells may then be cloned and tested with individual proteins, using methods well known in the art, to more accurately define individual T cell specificity. Assays for cell proliferation or cytokine production in T cells, NK cells, B cells or macrophages may be performed, for example, using the procedures described below.
In general, immunogenic antigens are those antigens that stimulate proliferation or cytokine production (i.e. , interferon-γ and or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from at least about
25% of M. tuberculosis-immune individuals. Among these immunogenic antigens, polypeptides having superior therapeutic properties may be distinguished based on the magnitude of the responses in the above assays and based on the percentage of individuals for which a response is observed. In addition, antigens having superior therapeutic properties will not stimulate cell proliferation or cytokine production in vitro in cells derived from more than about 25% of individuals that are not
M. tuberculosis-immune, thereby eliminating responses that are not specifically due to
M. tuberculosis-responsive cells. Thus, those antigens that induce a response in a high percentage of T cell, NK cell, B cell or macrophage preparations from M. tuberculosis-immune individuals (with a low incidence of responses in cell preparations from other individuals) have superior therapeutic properties.
Antigens with superior therapeutic properties may also be identified based on their ability to diminish the severity of M. tuberculosis infection, or other mycobacterial infection, in experimental animals, when administered as a vaccine. Suitable vaccine preparations for use in experimental animals are described in detail below.
Antigens having superior diagnostic properties may generally be identified based on the ability to elicit a response in an intradermal skin test performed on an individual with active tuberculosis, but not in a test performed on an individual who is not infected with M. tuberculosis. Skin tests may generally be performed as described below, with a response of at least about 5 mm induration considered positive. Immunogenic portions of the antigens described herein may be prepared and identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3d ed., Raven Press, 1993, pp. 243-247. Such techniques include screening poiypeptide portions of the native antigen for immunogenic properties. The representative proliferation and cytokine production assays described herein may be employed in these screens. An immunogenic portion of a poiypeptide is a portion that, within such representative assays, generates an immune response (e.g. , cell proliferation, interferon-γ production or interleukin-12 production) that is substantially similar to that generated by the full length antigen. In other words, an immunogenic portion of an antigen may generate at least about 20%, preferably about 65%, and most preferably about 100%, of the proliferation induced by the full length antigen in the model proliferation assay described herein. An immunogenic portion may also, or alternatively, stimulate the production of at least about 20%, preferably about 65% and most preferably about 100%, of the interferon-γ and/or interleukin-I2 induced by the full length antigen in the model assay described herein.
Portions and other variants of M. vaccae antigens may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J Am. Chem. Soc. 55:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions. Variants of a native antigen may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Sections of the DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides. In general, regardless of the method of preparation, the polypeptides disclosed herein are prepared in substantially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure. In certain preferred embodiments, described in detail below, the substantially pure polypeptides are incorporated into pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein. The present invention also provides fusion proteins comprising a first and a second inventive poiypeptide or, alternatively, a poiypeptide of the present invention and a known M tuberculosis antigen, such as the 38 kD antigen described in Andersen and Hansen, Infect. Immun. 57:2481-2488. 1989, together with variants of such fusion proteins. The fusion proteins of the present invention may also include a linker peptide between the first and second polypeptides.
A DNA sequence encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides into an appropriate expression vector. The 3' end of a DNA sequence encoding the first poiypeptide is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second poiypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.
A peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each poiypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the poiypeptide functional epitopes. Preferred peptide linker sequences contain
Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-4 , 1985;
Murphy et al., Proc. Natl. Acad. Sci. USA 55:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4.751 ,180. The linker sequence may be from 1 to about 50 amino acids in length. Peptide linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. The ligated DNA sequences encoding the fusion proteins are cloned into suitable expression systems using techniques known to those of ordinary skill in the art.
In another aspect, the present invention provides methods for using one or more of the inventive polypeptides or fusion proteins (or DNA molecules encoding such polypeptides or fusion proteins) to induce protective immunity against tuberculosis in a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may be afflicted with a disease, or may be free of detectable disease or infection. In other words, protective immunity may be induced to prevent or treat tuberculosis. In this aspect, the poiypeptide, fusion protein or DNA molecule is generally present within a pharmaceutical composition or a vaccine. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. Vaccines may comprise one or more of the above polypeptides and a non- specific immune response amplifier, such as an adjuvant or a liposome, into which the poiypeptide is incorporated. Such pharmaceutical compositions and vaccines may also contain other mycobacterial antigens, either, as discussed above, incorporated into a fusion protein or present within a separate poiypeptide.
Alternatively, a vaccine of the present invention may contain DNA encoding one or more polypeptides as described above, such that the poiypeptide is generated in situ. In such vaccines, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the poiypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g. , vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic, or defective, replication competent virus. Techniques for incorporating DNA into such expression systems are well known in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749. 1993 and reviewed by Cohen, Science 259: 1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. A DNA vaccine as described above may be administered simultaneously with or sequentially to either a poiypeptide of the present invention or a known mycobacterial antigen, such as the 38 kD antigen described above. For example, administration of DNA encoding a poiypeptide of the present invention, may be followed by administration of an antigen in order to enhance the protective immune effect of the vaccine.
Routes and frequency of administration, as well as dosage, will vary from individual to individual and may parallel those currently being used in immunization using BCG. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 3 doses may be administered for a 1-36 week period. Preferably, 3 doses are administered, at intervals "of 3-4 months, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of poiypeptide or DNA that, when administered as described above, is capable of raising an immune response in a patient sufficient to protect the patient from mycobacterial infection for at least 1-2 years. In general, the amount of poiypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 μg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
Any of a variety of adjuvants may be employed in the vaccines of this invention to non-specifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis, M. tuberculosis, or, as discussed below, M. vaccae. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.
InΕriother aspect, this invention provides methods for using one or more of the polypeptides described above to diagnose tuberculosis using a skin test. As used herein, a "skin test" is any assay performed directly on a patient in which a delayed- type hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of one or more polypeptides as described above. Preferably, the reaction is measured at least 48 hours after injection, more preferably 48-72 hours.
The DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen (i.e., the immunogenic portion of the poiypeptide employed, or a variant thereof). The response may be measured visually, using a ruler. In general, a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of tuberculosis infection.
For use in a skin test, the polypeptides of the present invention are preferably formulated, as pharmaceutical compositions containing a poiypeptide and a physiologically acceptable carrier, as described above. Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 μg to about 100 μg, preferably from about 10 μg to about 50 μg in a volume of 0.1 mL. Preferably, the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80™.
In a preferred embodiment, a poiypeptide employed in a skin test is of sufficient size such that it remains at the site of injection for the duration of the reaction period. In general, a poiypeptide that is at least 9 amino acids in length is sufficient. The poiypeptide is also preferably broken down by macrophages or dendritic cells within hours of injection to allow presentation to T-cells. Such polypeptides may contain repeats of one or more of the above sequences or other immunogenic or nonimmunogenic sequences.
In another aspect, methods are provided for detecting mycobacterial infection in a biological sample, using one or more of the above polypeptides, either alone or in combination. In embodiments in which multiple polypeptides are employed, polypeptides other than those specifically described herein, such as the 38 kD antigen described above, may be included. As used herein, a "biological sample" is any antibody-containing sample obtained from a patient. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient or a blood supply. The polypeptide(s) are used in an assay, as described below, to determine the presence or absence of antibodies to the polypeptide(s) in the sample, relative to a predetermined cut-off value. The presence of such antibodies indicates the presence of mycobacterial infection. In embodiments in which more than one poiypeptide is employed, the polypeptides used are preferably complementary (i.e., one component poiypeptide will tend to detect infection in samples where the infection would not be detected by another component poiypeptide). Complementary polypeptides may generally be identified by using each poiypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with a Mycobacterium. After determining which samples test positive (as described below) with each poiypeptide, combinations of two or more polypeptides may be formulated that are capable of detecting infection in most, or all, of the samples tested. For example, approximately 25-30% of sera from tuberculosis-infected individuals are negative for antibodies to any single protein, such as the 38 kD antigen mentioned above. Complementary polypeptides may, therefore, be used in combination with the 38 kD antigen to improve sensitivity of a diagnostic test.
A variety of assay formats employing one or more polypeptides to detect antibodies in a sample are well known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment, the assay involves the use of poiypeptide immobilized on a solid support to bind to and remove the antibody from the sample. The bound antibody may then be detected using a detection reagent that contains a reporter group. Suitable detection reagents include antibodies that bind to the antibody/polypeptide complex and free poiypeptide labelled with a reporter group (e.g., in a semi-competitive assay). Alternatively, a competitive assay may be utilized, in which an antibody t at binds to the poiypeptide is labelled with a reporter group and allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The extent to which components of the sample inhibit the binding of the labelled antibody to the poiypeptide is indicative of the reactivity of the sample with the immobilized poiypeptide.
The solid support may be any solid material to which the antigen may be attached. Suitable materials are well known in the art. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
The polypeptides may be bound to the solid support using a variety of techniques well known in the art. In the context of the present invention, the term "bound" refers to both noncovalent association, such as adsorption, and covalent attachment, which may be a direct linkage between the antigen and functional groups on the support or a linkage by way of a cross-linking agent. Binding by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the poiypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of poiypeptide ranging from about 10 ng to about 1 μg, and preferably about 100 ng, is sufficient to bind an adequate amount of antigen. Covalent attachment of poiypeptide to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the poiypeptide. For example, the poiypeptide may be bound to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the poiypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991 , at A12-A13).
In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). This assay may be performed by first contacting a poiypeptide antigen that has been immobilized on a solid support, with the sample, such that antibodies to the poiypeptide within the sample are allowed to bind to the immobilized poiypeptide. Unbound sample is then removed from the immobilized poiypeptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific detection reagent. More specifically, once the poiypeptide is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20™ (Sigma Chemical Co., St. Louis, MO) may be employed. The immobilized poiypeptide is then incubated with the sample, and antibody is allowed to bind to the antigen. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time, or incubation time, is that period of time that is sufficient to detect the presence of antibody within a M. tuberculosis-infected sample. Preferably, the contact time is sufficient to achieve a level of binding that is at least 95% of that achieved at equilibrium between bound and unbound antibody. The time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
Unbound sample may be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20™. Detection reagent may then be added to the solid support. An appropriate detection reagent is any compound that binds to the immobilized antibody-polypeptide complex and that can be detected by any of a variety of means known in the art. Preferably, the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of binding agent to reporter group may be achieved using standard methods known in the art. Common binding agents may also be purchased conjugated to a variety of reporter groups from many commercial sources (e.g., Zymed Laboratories, San Francisco, CA, and Pierce, Rockford, IL).
The detection reagent is incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time may generally be determined from the manufacturer's instructions or by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin. coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
To determine the presence or absence of anti-mycobacterial antibodies in the sample, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value is the average mean signal obtained when the immobilized antigen is incubated with samples from an uninfected patient. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107. In general, signals higher than the predetermined cut-off value are considered to be positive for mycobacterial infection.
The assay may also be performed in a rapid flow-through or strip test format, wherein the antigen is immobilized on a membrane, such as nitrocellulose. In the flow-through test, antibodies within the sample bind to the immobilized poiypeptide as the sample passes through the membrane. A detection reagent (e.g., protein A- colloidal "gold) then binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane. The detection of bound detection reagent may then be performed as described above. In the strip test format, one end of the membrane to which poiypeptide is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing detection reagent and to the area of immobilized poiypeptide. Concentration of detection reagent at the poiypeptide indicates the presence of anti- mycobacterial antibodies in the sample. Typically, the concentration of detection reagent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of poiypeptide immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in an ELISA, as discussed above. Preferably, the amount of poiypeptide immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount (e g , one drop) of patient serum or blood.
Numerous other assay protocols exist that are suitable for use with the polypeptides of the present invention. The above descriptions are intended to be exemplary only.
The present invention also provides antibodies to the inventive polypeptides. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g.. Harlow and Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory. 1988. In one such technique, an immunogen comprising the antigenic poiypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep and goats). The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the poiypeptide may then be purified from such antisera by, for example, affinity chromatography using the poiypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for the antigenic poiypeptide of interest may be prepared, for example, using the technique of Kohler and Mil stein, Eur. J Immunol. 6:5 \ 1-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the poiypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells may then be immortalized by fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal, using one of a variety of techniques well known in the art. Monoclonal antibodies may be isolated from the supernatants of the resulting hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
Antibodies may be used in diagnostic tests to detect the presence of mycobacterial antigens using assays similar to those detailed above and other techniques well known to those of skill in the art, thereby providing a method for detecting mycobacterial infection, such as M. tuberculosis infection, in a patient. Diagnostic reagents of the present invention may also comprise DNA sequences encoding one or more of the above polypeptides, or one or more portions thereof. For example, primers comprising at least 10 contiguous oligonucleotides of the subject DNA sequences may be used in polymerase chain reaction (PCR) based tests. Similarly, probes comprising at least 18 contiguous oligonucleotides of the subject DNA sequences may be used for hybridizing to specific sequences. Techniques for both PCR based tests and hybridization tests are well known in the art. Primers or probes may thus be used to detect M. tuberculosis and other mycobacterial infections in biological samples, preferably sputum, blood, serum, saliva, cerebrospinal fluid or urine. DNA probes or primers comprising oligonucleotide sequences described above may be used alone, in combination with each other, or with previously identified sequences, such as the 38 kD antigen discussed above.
As discussed above, effective vaccines contain at least two different components. The first is a poiypeptide comprising an antigen, which is processed by macrophages and other antigen-presenting cells and displayed for CD4+ T cells or for CD8+ T cells. This antigen forms the "specific" target of an immune response. The second component of a vaccine is a non-specific immune response amplifier, such as an adjuvant or a liposome, into which the antigen is incorporated. An adjuvant amplifies immune responses to a structurally unrelated compound or poiypeptide. Several adjuvants are prepared from microbes such as Bordetella pertussis, M. tuberculosis and M. bovis BCG. Adjuvants may also contain components designed to protect poiypeptide antigens from degradation, such as aluminum hydroxide or mineral oil.
While the antigenic component of a vaccine contains polypeptides that direct the immune attack against a specific pathogen, such as M. tuberculosis, the adjuvant is often capable of broad use in many different vaccine formulations. Certain pathogens, such as M. tuberculosis, as well as certain cancers, are effectively contained by an immune attack directed by T cells, known as cell-mediated immunity. Other pathogens, such as poliovirus, also require antibodies produced by B cells for containment. These different classes of immune attack (T cell or B cell) are controlled by different subpopulations of CD4+ T cells, commonly referred to as Thl and Th2 cells. A desirable property of an adjuvant is the ability to selectively amplify the function of either Thl or Th2 populations of CD4* T cells. As shown below in Example 6, M. vaccae and a modified form of autoclaved M. vaccae have been found to have adjuvant properties. As used herein, the term "modified M. vaccae" includes delipidated M. vaccae cells, deglycosylated M. vaccae cells and M. vaccae cells that have been both delipidated and deglycosylated (hereinafter referred to as DD-Λ . vaccae). Furthermore, it has been found that M. vaccae produces compounds which amplify the immune response to M. vaccae antigens, as well as to antigens from other sources. The present invention thus provides methods for enhancing immune responses to an antigen comprising administering killed M. vaccae cells, M. vaccae culture filtrate or modified M. vaccae cells. As detailed below, further studies have demonstrated that this non-specific immune amplifying effect is due, at least in part, to an M. vaccae poiypeptide having homology to heat shock protein 65 (GroEL), previously identified in M. tuberculosis. As described below in Example 10, it has also been found that heat-killed M. vaccae and M. vaccae constituents have cytokine stimulation properties. In particular, it has been found that heat-killed M. vaccae, lyophilised M. vaccae and DD- vaccae stimulate the production of interleukin 12 (IL-12) from macrophages. Production of IL-12 from macrophages is known to enhance stimulation of a Thl immune response. The following examples are offered by way of illustration and not by way of limitation. EXAMPLE 1
EFFECT OF IMMUNIZATION OF MICE WITH M VACCAE ON TUBERCULOSIS
This example illustrates the effect of immunization with M vaccae or M. vaccae culture filtrate in mice prior to challenge with live M. tuberculosis.
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/1; tryptone, 5 g/1; glucose, 1 g/1) at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10'° M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 °C. The culture filtrate was passaged through a 0.45 μM filter into sterile bottles.
As shown in Fig.lA, when mice were immunized with 1 mg, 100 μg or 10 μg of M. vaccae and infected three weeks later with 5x10s colony forming units (CFU) of live M. tuberculosis H37Rv, significant protection from infection was seen. In this example, spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli determined (expressed as CFU). The reduction in bacilli numbers, when compared to tissue from non-immunized control mice, exceeded 2 logs in liver and lung tissue, and 1 log in spleen tissue. Immunization of mice with heat-killed M. tuberculosis H37Rv had no significant protective effects on mice subsequently infected with live M. tuberculosis H37Rv.
Fig.1 B shows that when mice were immunized with 100 μg of M. vaccae culture filtrate, and infected three weeks later with 5x105 CFU of M. tuberculosis H37Rv, significant protection was also seen. When spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli numbers (CFU) determined, a 1-2 log reduction in numbers, as compared to non-immunized control mice, was observed. EXAMPLE 2
PURIFICATION AND CHARACTERIZATION OF POLYPEPTIDES FROM M. VACCAE CULTURE FILTRATE
This example illustrates the preparation of M. vaccae soluble proteins from culture filtrate. Unless otherwise noted, all percentages in the following example are weight per volume.
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 μ filter into sterile bottles.
The culture filtrate was concentrated by lyophilization, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45μ membrane. The culture filtrate was desalted by membrane filtration in a 400 ml A icon stirred cell which contained a 3,000 kilodalton molecular weight cut-off (MWCO) membrane. The pressure was maintained at 50 psi using nitrogen gas. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the 20 I volume to approximately 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).
The" esalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech, Uppsala, Sweden) (16 X 100 mm) equilibrated with lOmM Tris HCl buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCl from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.
The pool of polypeptides eluting from the ion exchange column was concentrated in a 400 ml Amicon stirred cell which contained a 3,000 MWCO membrane. The pressure was maintained at 50 psi using nitrogen gas. The polypeptides were repeatedly concentrated by membrane filtration and diluted with
1% glycine until the conductivity of the sample was less than 0.1 mS. The purified polypeptides were then fractionated by preparative isoelectric focusing in a Rotofor device (Bio-Rad, Hercules, CA, USA). The pH gradient was established with a mixture of Ampholytes (Pharmacia Biotech) comprising 1.6% pH 3.5-5.0 Ampholytes and 0.4% pH 5.0 - 7.0 Ampholytes. Acetic acid (0.5 M) was used as the anolyte, and 0.5 M ethanolamine as the catholyte. Isoelectric focusing was carried out at 12W constant power for 6 hours, following the manufacturer's instructions. Twenty fractions were obtained.
Fractions from isoelectric focusing were combined, and the polypeptides were purified on a Vydac C4 column (Separations Group, Hesperia, CA, USA) 300 Angstrom pore size, 5 micron particle size (10 x 250 mm). The polypeptides were eluted from the column with a linear gradient of acetonitrile (0-80% v/v) in 0.05% (v/v) trifluoroacetic acid (TFA). The flow-rate was 2.0 ml/min and the HPLC eluent was monitored at 220 nm. Fractions containing polypeptides were collected to maximize the purity of the individual samples. Relatively abundant poiypeptide fractions were rechromatographed on a Vydac
C4 column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6 x 250 mm). The polypeptides were eluted from the column with a linear gradient from 20-60% (v/v) of acetonitrile in 0.05% (v/v) TFA at a flow-rate of 1.0 ml/min. The column eluent was monitored at 220 nm. Fractions containing the eluted polypeptides were collected to maximise the purity of the individual samples. Approximately 20 poiypeptide samples were obtained and they were analysed for purity on "a polyacrylamide gel according to the procedure of Laemmli (Laemmli, U. K.. Nature 277:680-685. 1970).
The poiypeptide fractions which were shown to contain significant contamination were further purified using a Mono Q column (Pharmacia Biotech) 10 micron particle size (5 x 50 mm) or a Vydac Diphenyl column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6 x 250 mm). From a Mono Q column, polypeptides were eluted with a linear gradient from 0-0.5 M NaCl in 10 mM Tris HCl pH 8.0. From a Vydac Diphenyl column, polypeptides were eluted with a linear gradient of acetonitrile (20-60% v/v) in 0.1% TFA. The flow-rate was 1.0 ml/min and the column eluent was monitored at 220 nm for both columns. The poiypeptide peak fractions were collected and analysed for purity on a 15% polyacrylamide gel as described above.
For sequencing, the polypeptides were individually dried onto Biobrene™ (Perkin Elmer/Applied BioSystems Division, Foster City, CA)-treated glass fiber filters. The filters with poiypeptide were loaded onto a Perkin Elmer/Applied BioSystems Procise 492 protein sequencer and the polypeptides were sequenced from the amino terminal end using traditional Edman chemistry. The amino acid sequence was determined for each poiypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Internal sequences were also determined on some antigens by digesting the antigen with the endoprotease Lys-C, or by chemically cleaving the antigen with cyanogen bromide. Peptides resulting from either of these procedures were separated by reversed-phase HPLC on a Vydac C18 column using a mobile phase of 0.05% (v/v) trifluoroacetic acid with a gradient of acetonitrile containing 0.05% (v/v) TFA (1%/min). The eluent was monitored at 214 nm. Major internal peptides were identified by their UV absorbance, and their N-terminal sequences were determined as described above.
Using the procedures described above, six soluble M. vaccae antigens, designated GVc-1, GVc-2, GVc-7, GVc-13, GVc-20 and GVc-22, were isolated. Determined N-terminal and internal sequences for GVc-1 are shown in SEQ ID NOS. 1, 2 and 3, respectively; the N-terminal sequence for GVc-2 is shown in SEQ ID NO: 4; internal sequences for GVc-7 are shown in SEQ ID NOS: 5-8; internal sequences for GVc-13 are shown in SEQ ID NOS: 9-11; internal sequence for GVc-20 is shown in SEQ ID NO: 12; and N-terminal and internal sequences for GVc-22 are shown in SEQ ID NO:56-59, respectively. Each of the internal peptide sequences provided herein begins with an amino acid residue which is assumed to exist in this position in the poiypeptide, based on the known cleavage specificity of cyanogen bromide (Met) or Lys-C (Lys).
Three additional polypeptides, designated GVc-16, GVc-18 and GVc-21, were isolated employing a preparative sodium dodecyl sulfate-poiyacrylamide gel electrophoresis (SDS-PAGE) purification step in addition to the preparative isoelectric focusing procedure described above. Specifically, fractions comprising mixtures of polypeptides from the preparative isoelectric focusing purification step previously described, were purified by preparative SDS-PAGE on a 15% polyacrylamide gel. The samples were dissolved in reducing sample buffer and applied to the gel. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting in 10 mM 3-(cyclohexylamino)-l- propanesulfonic acid (CAPS) buffer pH 1 1 containing 10% (v/v) methanol. The transferred protein bands were identified by staining the PVDF membrane with Coomassie blue. Regions of the PVDF membrane containing the most abundant poiypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The N-terminal sequences for GVc- 16, GVc-18 and GVc-21 are provided in SEQ ID NOS: 13, 14 and 15, respectively. Additional antigens, designated GVc-12, GVc-14, GVc-15, GVc-17 and GVc- 19, were isolated employing a preparative SDS-PAGE purification step in addition to the chromatographic procedures described above. Specifically, fractions comprising a mixture of antigens from the Vydac C4 HPLC purification step previously described were fractionated by preparative SDS-PAGE on a polyacrylamide gel. The samples were dissolved in non-reducing sample buffer and applied to the gel. The separated proteins were transferred to a PVDF membrane by electroblotting in 10 mM CAPS buffer, pH 1 1 containing 10% (v/v) methanol. The transferred protein bands were identified""by staining the PVDF membrane with Coomassie blue. Regions of the PVDF membrane containing the most abundant poiypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/ Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The determined N-terminal sequences for GVc-12, GVc-14, GVc-15, GVc- 17 and GVc-19 are provided in SEQ ID NOS: 16-20, respectively.
All of the above amino acid sequences were compared to known amino acid sequences in the SwissProt data base (version R32) using the GeneAssist system. No significant homologies to the amino acid sequences GVc-2 to GVc-22 were obtained.
The amino acid sequence for GVc-1 was found to bear some similarity to sequences previously identified from M. bovis and M. tuberculosis. In particular, GVc-1 was found to have some homology with M. tuberculosis MPT83, a cell surface protein, as well as MPT70. These proteins form part of a protein family (Harboe et al., Scand. J. Immunol. 42:46-51 , 1995). Subsequent studies led to the isolation of DNA sequences for GVc-14 and
GVc-22 (SEQ ID NO: 107 and 108, respectively). The corresponding predicted amino acid sequences for GVc-14 and GVc-22 are provided in SEQ ID NO: 109 and 1 10, respectively.
Amplifications primers AD86 and AD1 12 (SEQ ID NO: 60 and 61 , respectively) were designed from the amino acid sequence of GVc-1 (SEQ ID NO: 1) and the M. tuberculosis MPT70 gene sequence. Using these primers, a 310 bp fragment was amplified from M. vaccae genomic DNA and cloned into £coRV- digested vector pBluescript (Stratagene) containing added dTTP residues. The sequence of the cloned insert is provided in SEQ ID NO: 62. The purified polypeptides were screened for the ability to induce T-cell proliferation and IFN-γ in peripheral blood cells from immune human donors. These donors were known to be PPD (purified protein derivative from M. tuberculosis) skin test positive and their T cells were shown to proliferate in response to PPD. Donor PBMCs and crude soluble proteins from M. vaccae culture filtrate were cultured in medium comprising RPMI 1640 supplemented with 10% (v/v) autologous serum, penicillin (60 μg/ml), streptomycin (100 μg/ml), and glutamine (2 mM).
After" 3 days, 50 μl of medium was removed from each well for the determination of IFN-γ levels, as described below. The plates were cultured for a further 4 days and then pulsed with lμCi/well of tritiated thymidine for a further 18 hours, harvested and tritium uptake determined using a scintillation counter. Fractions that stimulated proliferation in both replicates two-fold greater than the proliferation observed in cells cultured in medium alone were considered positive.
IFN-γ was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with a mouse monoclonal antibody directed to human IFN- γ (Endogen, Wobural, MA) 1 μg/ml phosphate-buffered saline (PBS) for 4 hours at 4 °C. Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS/0.2% Tween 20. and samples diluted 1 :2 in culture medium in the ELISA plates were incubated overnight at room temperature. The plates were again washed, and a biotinylated polyclonal rabbit anti-human IFN-γ serum (Endogen), diluted to 1 μg/ml in PBS, was added to each well. The plates were then incubated for 1 hour at room temperature, washed, and horseradish peroxidase-coupled avidin A (Vector Laboratories, Burlingame, CA) was added at a 1 :4,000 dilution in PBS. After a further 1 hour incubation at room temperature, the plates were washed and orthophenylenediamine (OPD) substrate added. The reaction was stopped after 10 min with 10% (v/v) HCl. The optical density (OD) was determined at 490 nm. Fractions that resulted in both replicates giving an OD two-fold greater than the mean OD from cells cultured in medium alone were considered positive.
Examples of polypeptides containing sequences that stimulate peripheral blood mononuclear cells (PBMC) T cells to proliferate and produce IFN-γ are shown in Table 1, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, and (++) indicates polypeptides having activity greater than four times above background.
Figure imgf000032_0001
EXAMPLE 3 PURIFICATION AND CHARACTERISATION OF POLYPEPTIDES
FROM M. VACCAE CULTURE FILTRATE BY 2-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS
M. vaccae soluble proteins were isolated from culture filtrate using 2- dimensional polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 °C. M. tuberculosis strain H37Rv (ATCC number 27294) was cultured in sterile Middlebrook 7H9 medium with Tween 80 and oleic acid/albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Michigan). The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 μ filter into sterile bottles. The culture filtrate was concentrated by lyophilisation, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45μ membrane filter.
The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained a 3,000 MWCO membrane. The pressure was maintained at 60 psi using nitrogen gas. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the 20 L volume to approximately 50 mL. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The desalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech) (16 x 100 mm) equilibrated with lOmM TrisHCL buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCl from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm. The pool of polypeptides eluting from the ion exchange column were fractionated by preparative 2D gel electrophoresis. Samples containing 200-500 ug of poiypeptide were made 8M in urea and applied to polyacrylamide isoelectric focusing rod gels (diameter 2mm, length 150 mm, pH 5-7). After the isoelectric focusing step, the first dimension gels were equilibrated with reducing buffer and applied to second dimension gels (16% polyacrylamide). Figs. 2A and 2B are the 2-D gel patterns observed with M. vaccae culture filtrate and M. tuberculosis H37Rv culture filtrate, respectively. Polypeptides from the second dimension separation were transferred to PVDF membranes by electroblotting in lOmM CAPS buffer pH 11 containing 10% (v/v) methanol. The PVDF membranes were stained for protein with Coomassie blue. Regions of PVDF containing polypeptides of interest were cut out and directly introduced into the sample cartridge of the Perkin Elmer/ Applied BioSystems Procise 492 protein sequencer. The polypeptides were sequenced from the amino terminal end using traditional Edman chemistry. The amino acid sequence was determined for each poiypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Using these procedures, eleven polypeptides, designated GVs-1 , GVs-3, GVs-4, GVs-5, GVs-6, GVs-8, GVs-9, GVs- 10, GVs-1 1, GV-34 and GV-35 were isolated. The determined N-terminal sequences for these polypeptides are shown in SEQ ID NOS: 21-29, 63 and 64, respectively. Using the purification procedure described above, more protein was purified to extend the amino acid sequence previously obtained for GVs-9. The extended amino acid sequence for GVs-9 is provided in SEQ ID NO:65. Further studies resulted in the isolation of the DNA sequence for GVs-9 (SEQ ID NO: 111). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 1 12.
All of these amino acid sequences were compared to known amino acid sequences in the SwissProt data base (version R32) using the GeneAssist system. No significant homologies were obtained, with the exceptions of GVs-3, GVs-4, GVs-5 and GVs-9. GVs-9 was found to bear some homology to two previously identified M. tuberculosis proteins, namely M. tuberculosis cutinase precursor and an M. tuberculosis hypothetical 22.6 kD protein. GVs-3, GVs-4 and GVs-5 were found to bear some similarity to the antigen 85A and 85B proteins from M. leprae (SEQ ID NOS: 30 and 31, respectively), M. tuberculosis (SEQ ID NOS: 32 and 33, respectively) and M. bovis (SEQ ID NOS: 34 and 35, respectively), and the antigen 85C proteins from M. leprae (SEQ ID NO: 36) and M. tuberculosis (SEQ ID NO: 37). A comparison of the inventive antigen 85A protein from M. vaccae with those from M. tuberculosis. M. bovis and M. leprae, is presented in Fig. 3.
EXAMPLE 4
DNA CLONING STRATEGY FOR THE M. VACCAE ANTIGEN 85 SERIES
Probes for antigens 85A, 85B, and 85C (SEQ ID NOS: 38 and 39) were prepared by the polymerase chain reaction (PCR) using degenerate oligonucleotides designed to regions of antigen 85 genomic sequence that are conserved between family members in a given mycobacterial species, and between mycobacterial species. These oligonucleotides were used under reduced stringency conditions to amplify target sequences from M. vaccae genomic DNA. An appropriately-sized 0.5kb band was identified, purified, and cloned into T-tailed p Bluescript II SK (Stratagene, La Jolla, CA). Twenty-four individual colonies were screened at random for the presence of the antigen 85 PCR product, then sequenced using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the M13-based primers, T3 and T7. Homology searches of the GenBank databases showed that twenty-three clones contained insert with significant homology to published antigen 85 genes from M. tuberculosis' and M. bovis. Approximately half were most homologous to antigen 85C gene sequences, with the remainder being more similar to antigen 85B sequences. In addition, these two putative M. vaccae antigen 85 genomic sequences were 80% homologous to one another. Because of this high similarity, the antigen 85C PCR fragment was chosen to screen M. vaccae genomic libraries at low stringency for all three antigen 85 genes.
An M. vaccae genomic library was created in λ ZapExpress (Stratagene, La Jolla, CA) by cloning BamHl partially-digested M. vaccae genomic DNA into similarly-digested λ vector, with 3.4 x 105 independent plaque-forming units resulting. For screening purposes, twenty-seven thousand plaques from this non- amplified library were plated at low density onto eight 100 cm: plates. For each plate, duplicate plaque lifts were taken onto Hybond-N" nylon membrane (Amersham International. United Kingdom), and hybridised under reduced-stringency conditions (55 °C) to the radiolabelled antigen 85C PCR product. Autoradiography demonstrated that seventy-nine plaques consistently hybridised to the antigen 85C probe under these conditions. Thirteen positively-hybridising plaques were selected at random for further analysis and removed from the library plates, with each positive clone being used to generate secondary screening plates containing about two hundred plaques. Duplicate lifts of each plate were taken using Hybond-N+ nylon membrane, and hybridised under the conditions used in primary screening. Multiple positively-hybridising plaques were identified on each of the thirteen plates screened. Two well-isolated positive phage from each secondary plate were picked for further analysis. Using in vitro excision, twenty-six plaques were converted into phagemid, and restriction-mapped. It was possible to group clones into four classes on the basis of this mapping. Sequence data from the 5' and 3' ends of inserts from several representatives of each group was obtained using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the T3 and T7 primers. Sequence homologies were determined using FASTA analysis of the GenBank databases with the GeneAssist software package. Two of these sets of clones were found to be homologous to M. bovis and M. tuberculosis antigen 85A genes, each containing either the 5' or 3' ends of the M. vaccae gene (this gene was cleaved during library construction as it contains an internal Bam l site). The remaining clones were found to contain sequences homologous to antigens 85B and 85C from a number of mycobacterial species. To determine the remaining nucleotide sequence for each gene, appropriate subclones were constructed and sequenced. Overlapping sequences were aligned using the DNA Strider software. The determined DNA sequences for M. vaccae antigens 85 A, 85B and 85C are shown in SEQ ID NOS: 40-42, respectively, with the predicted amino acid sequences being shown in SEQ ID NOS: 43-45, respectively. The M. vaccae antigens GVc-3 and GVc-5 were expressed and purified as follows. Amplification primers were designed from the insert sequences of GVc-3 and GVc-5 (SEQ ID NO: 40 and 42, respectively) using sequence data downstream from the putative leader sequence and the 3' end of the clone. The sequences of the primers for GVc-3 are provided in SEQ ID NO: 66 and 67. and the sequences of the primers for GVc-5 are provided in SEQ ID NO: 68 and 69. A Xhol restriction site was added to the primers for GVc-3, and EcoRl and Bamlil restriction sites were added to the primers for GVc-5 for cloning convenience. Following amplification from genomic M. vaccae DNA, fragments were cloned into the appropriate site of pProEX HT prokaryotic expression vector (Gibco BRL, Life Technologies, Gaithersburg, MD) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the recombinant protein was performed according to the manufacturer's protocol.
Expression of a fragment of the M. vaccae antigen GVc-4 (antigen 85B homolog) was performed as follows. The primers AD58 and AD59, described above, were used to amplify a 485 bp fragment from M. vaccae genomic DNA. This fragment was gel- purified using standard techniques and cloned into £cσRV-digested pBluescript containing added dTTP residues. The base sequences of inserts from five clones were determined and found to be identical to each other. These inserts had highest homology to Ag85B from M. tuberculosis. The insert from one of the clones was subcloned into the EcoRI/Xhol sites of pProEX HT prokaryotic expression vector (Gibco BRL), expressed and purified according to the manufacturer's protocol. This clone was renamed GVc-4P because only a part of the gene was expressed. The amino aci " and DNA sequences for the partial clone GVc-4P are provided in SEQ ID NO: 70 and 106, respectively.
In subsequent studies, using procedures similar to those described above, GVc-3, GVc-4P and GVc-5 were re-cloned into the alternative vector pET16 (Novagen, Madison, WI).
The ability of purified recombinant GVc-3, GVc-4P and GVc-5 to stimulate proliferation of T cells and interferon-γ production in human PBL from PPD-positive, healthy donors, was assayed as described above in Example 2. The results of this assay are shown in Table 2, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, (++) indicates polypeptides having activity greater than lour times above background, and ND indicates not determined
Table 2
Figure imgf000038_0002
EXAMPLE 5
DNA CLONING STRATEGY FOR M VACCAE ANTIGENS
An 84 bp probe for the M vaccae antigen GVc-7 was amplified using degenerate oligonucleotides designed to the determined amino acid sequence of GVc- 7 (SEQ ID NOS: 5-8) This probe was used to screen a M vaccae genomic DNA library as described in Example 4. The determined nucleotide sequence fo: GVc-7 is shown in SEQ ID NO1 46 and predicted amino acid sequence in SEQ ID NO 47 Comparison of these sequences with those in the databank revealed homology to a hypothetical 15.8 kDa membrane protein of M tuberculosis
The sequence of SEQ ID NO 46 was used to design amplification primers (provided in SEQ ID NO 71 and 72) for expression cloning of the GVc-7 gene using sequence data downstream from the putative leader sequence A Xhol restriction site was added to the primers for cloning convenience Following amplification from genomic M vaccae DNA, fragments were cloned into the
Figure imgf000038_0001
of pProEX HT prokaryotic expression vector (Gibco BRL) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the fusion protein was performed according to the manufacturer's protocol. In subsequent studies, GVc-7 was re-cloned into the vector pET16 (Novagen).
The ability of purified recombinant GVc-7 to stimulate proliferation of T-cells and stimulation of interferon-γ production in human PBL, from PPD-positive, healthy donors, was assayed as described previously in Example 2. The results are shown in Table 3, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, and (++) indicates polypeptides having activity greater than four times above background.
TABLE 3
Figure imgf000039_0001
A redundant oligonucleotide probe was designed to the GVs-8 peptide sequence shown in SEQ ID NO: 6 and used to screen an M. vaccae genomic DNA library as described above. Positive plaques were isolated.
Four different genomic clones were identified, hereinafter referred to as GVs- 8A, GVs-8B and GVs-8C and GVs-8D. The determined DNA sequences for the clones GVs-8A, GVs-8B, GVs-8C and GVs-8D are shown in SEQ ID NOS: 48-51, respectively, with the corresponding amino acid sequences being shown in SEQ ID NOS: 52-55, respectively. The clone GVs-8A contains regions showing some similarity to known prokaryotic valyi-tRNA synthetases; GVs-8B shows some similarity to M. smegmatis aspartate semialdehyde dehydrogenase; and GVs-8C shows some similarity to the H. influenza folylpolyglutamate synthase gene. GVs-8D contains an open reading frame which shows some similarity to sequences previously identified in M. tuberculosis and M. leprae, but whose function has not been identified.
In subsequent studies, the M. vaccae genomic DNA library constructed in the
BamHl -site of lambda ZAP Express vector (Stratagene) was screened with a second redundant oligonucleotide (referred to as MPG15; SEQ ID NO:73) designed from the
GVs-8 sequence provided in SEQ ID NO:6. Screening of the library was performed in the presence of tetramethylammonium chloride (TMAC), so that nucleotide base pairs would melt at a standard temperature independent of sequence (i.e. A-T pairs and G-C pairs melt at the same temperature). Hybridisation stringency therefore depended only on the length and degeneracy of the oligonucleotide used as probe
(Wood et al. Proc. Nad. Acad. Sci. USA, 52: 1585- 1588,1985). Filters were prepared using standard methods of transfer and pre-hybridised overnight at a temperature -15
°C below the appropriate TMAC wash temperature. Hybridisation was performed overnight in freshly prepared hybridisation solution containing 100 pmol probe.
Hybridisation Solution for Oligonucleotides Stock:
1 M NaCl 5 M
0.1 M Tris pH 8 1 M
5X Denhardt's l00X 0.05% NaPPi 5%
0.1% SDS 10%
0. fmg/ml yeast tRNA 10 mg/ml
125 units/ml heparin
The filters were washed at a temperature calculated to allow approximately 4 % mismatching in TMAC wash buffer. More specifically, the wash protocol included the following washing steps: 2 x 15 min in 6X SSC, 0.05% NaPPi at room temp; 1 x 15 min in TMAC wash (see below) at room temperature; 2 x 15 min in TMAC wash at the calculated stringent temperature; and 1 x 15 min in 6X SSC, 0.05% NaPPi at room temp. TMAC wash buffer
3 M Tetramethylammonium chloride (TMAC)
50 mM Tris pH 8.0
0.2 mM EDTA
Positive plaques were picked and stored in 1 ml SM buffer with 20 μl chloroform. Screening was repeated until plaques were pure following the procedure described above.
The pBK-CMV phagemid containing the desired insert was excised from the lambda ZAP Express vector in the presence of ExAssist helper phage following the manufacturer's protocol. A phagemid containing an 8 kb insert (GVs-8D) was characterised by restriction mapping and sub-cloning. An open reading frame was identified at the 3' end of the insert and the antigen encoded by this open reading frame was named GV-33. Base sequence corresponding to GVs-8 was not found in the insert, and it was assumed that GV-33 was obtained as a non-specific product of the TMAC screening. By further sub-cloning and base sequencing, the 3' end of the gene was determined. The determined partial DNA sequence for GV-33 is provided in SEQ ID NO:74 with the corresponding predicted amino acid sequence being provided in SEQ ID NO:75. Sequence data from the 3' end of the clone showed homology to a previously identified 40.6 kDa outer membrane protein of M. tuberculosis.
TKe "partial GV-33 gene was amplified from M. vaccae genomic DNA with primers based on the determined nucleotide sequence. This DNA fragment was cloned into EcoRv-digested pBluescript (Stratagene) with additional dTTP residues, and then transferred to pProEX HT expression vector (Gibco BRL) using EcoRl and Hindlll-subcloning. Recombinant protein was purified following the manufacturer's protocol. In subsequent studies, GV-33 was re-cloned into the alternative vector pET16 (Novagen).
The ability of purified recombinant antigen to stimulate proliferation of T-cells and stimulation of interferon-γ production in human PBL from PPD-positive, healthy donors, was assayed as described previously in Example 2. The results are shown in Table 4, wherein (-) indicates a lack of activity. (+/-) indicates polypeptides having a result less than twice higher than background activity oϊ control media, (+) indicates polypeptides having activity two to four times above background, and (++) indicates polypeptides having activity greater than four times above background.
TABLE 4
Figure imgf000042_0001
EXAMPLE 6
DETECTION OF NONSPECIFIC IMMUNE AMPLIFIER FROM WHOLE M. VACCAE AND THE CULTURE FILTRATE OF M. VACCAE
This example illustrates the preparation of whole M. vaccae and M. vaccae culture filtrate and its non-specific immune amplifying or 'adjuvant' property.
M. vaccae bacteria was cultured, pelleted and autoclaved as described in Example 1. Culture filtrates of live M. vaccae refer to the supernatant from 24 hour cultures of M. vaccae in 7H9 medium with glucose. A delipidated form of M. vaccae was prepared by sonicating autoclaved M. vaccae for four bursts of 30 seconds on ice using the Virsonic sonicator (Virtis, Disa, USA). The material was then centrifuged (9000 rpm, 20 minutes, JA10 rotor, brake = 5). The resulting pellet was suspended in 100 ml of chloro form/methanol (2:1), incubated at room temperature for 1 hour; recentrifuged, and the chloroform methanol extraction repeated. The pellet was obtained by centrifugation, dried in vacuo, weighed and resuspended in PBS at 50mg (dry weight) per ml as delipidated M. vaccae.
Glycolipids were removed from the delipidated M. vaccae preparation by refluxing in 50% v/v ethanol for 2 hours. The insoluble material was collected by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake = 5). The extraction with 50% v/v ethanol' under reflux was repeated twice more. The insoluble material was collected by centrifugation and washed in PBS. Proteins were extracted by resuspending the pellet in 2% SDS in PBS at 56 °C for 2 hours. The insoluble material was collected by centrifugation and the extraction with 2% SDS/PBS at 56 °C was repeated twice more. The pooled SDS extracts were cooled to 4 °C, and precipitated SDS was removed by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake = 5). Proteins were precipitated from the supernatant by adding an equal volume of acetone and incubating at -20 °C for 2 hours. The precipitated proteins were collected by centrifugation, washed in 50% v/v acetone, dried in vacuo, and redissolved in PBS. M. vaccae culture supernatant (S N), killed M. vaccae and delipidated M. vaccae were tested for adjuvant activity in the generation of cytotoxic T cell immune response to ovalbumin, a structurally unrelated protein, in the mouse. This anti- ovalbumin-specific cytotoxic response was detected as follows. C57BL/6 mice (2 per group) were immunized by the intraperitoneal injection of 100 μg of ovalbumin with the following test adjuvants: autoclaved M. vaccae; delipidated M. vaccae; delipidated M. vaccae with glycolipids also extracted and proteins extracted with SDS; the SDS protein extract treated with pronase (an enzyme which degrades protein); whole M. vaccae culture filtrate; and heat-killed M. tuberculosis or heat- killed M. bovis BCG, M. phlei or M. smegmatus or M. vaccae culture filtrate. After 10 days, spleen cells were stimulated in vitro for a further 6 days with E.G7 cells which are EL4 cells (a C57BL/6-derived T cell lymphoma) transfected with the ovalbumin gene and thus express ovalbumin. The spleen cells were then assayed for their ability to kill non-specifically EL4 target cells or to kill specifically the E.G7 ovalbumin expressing cells. Killing activity was detected by the release of 5 I Chromium with which the EL4 and E.G7 cells have been labelled (100 μCi per 2xl06), prior to the killing assay. Killing or cytolytic activity is expressed as % specific lysis using the formula:
cpm in test cultures - cpm in control cultures xl00%
_ . total cpm - cpm in control cultures
It is generally known that ovalbumin-specific cytotoxic cells are generated only in mice immunized with ovalbumin with an adjuvant but not in mice immunized with ovalbumin alone.
The diagrams that make up Fig. 4 show the effect of various M. vaccae derived adjuvant preparations on the generation of cytotoxic T cells to ovalbumin in C57BL/6 mice. As shown in Fig. 4A, cytotoxic cells were generated in mice immunized with (i) 10 μg, (ii) 100 μg or (iii) 1 mg of autoclaved M. vaccae or (iv) 75 μg of M. vaccae culture filtrate. Fig. 4B shows that cytotoxic cells were generated in mice immunized with (i) 1 mg whole autoclaved M. vaccae or (ii) 1 mg delipidated M. vaccae. As shown in Fig. 4C(i), cytotoxic cells were generated in mice immunized with 1 mg whole autoclaved M. vaccae; Fig. 4C(ii) shows the active material in 100 μg delipidated M. vaccae which then had glycolipids removed and the proteins extracted with SDS. Fig. 4C(iii) shows that active material in the adjuvant preparation of Fig. 4C(ii) was destroyed by treatment with the proteolytic enzyme pronase. By way of comparison, 100 μg of the SDS-extracted proteins had significantly stronger immune-enhancing ability (Fig. 4C(ii)) than did 1 mg whole autoclaved M. vaccae (Fig. 4C(i)). Mice immunized with 1 mg heat-killed M. vaccae (Fig. 4D(i)) generated cytotoxic cells to ovalbumin, but mice immunized separately with 1 mg heat-killed M. tuberculosis (Fig. 4D(ii)), 1 mg M. bovis BCG (Fig. 4D(iii)), 1 mg M. phlei (Fig. 4D(iv)), or 1 mg M smegmatis (Fig. 4D(v)) failed to generate cytotoxic cells. The SDS-extracted proteins derived from delipidated and deglycolipidated M. vaccae were analysed by polyacrylamide gel electrophoresis. As shown in Fig. 5, three major bands were observed after staining with silver.
In subsequent studies, more of the SDS-extracted proteins described above were prepared by preparative SDS-PAGE on a BioRad Prep Cell (Hercules, CA). Fractions corresponding to molecular weight ranges were precipitated by trichloroacetic acid to remove SDS before assaying for adjuvant activity in the anti- ovalbumin^pecific cytotoxic response assay in C57BL/6 mice as described above. As seen in Fig. 6, the adjuvant activity was highest in the 60-70 kDa fraction. The most abundant protein in this size range was purified by SDS-PAGE blotted on to a polyvinylidene difluoride (PVDF) membrane and then sequenced. The sequence of the first ten amino acid residues is provided in SEQ ID NO:76. Comparison of this sequence with those in the gene bank as described above, revealed homology to the heat shock protein 65 (GroEL) gene from M. tuberculosis, indicating that this protein is an M. vaccae member of the GroEL family. An expression library of M. vaccae genomic DNA in BamHl -lambda ZAP
Express (Stratagene) was screened using sera from cynomolgous monkeys immunised with M. vaccae secreted proteins prepared as described above. Positive plaques were identified using a colorimetric system. These plaques were re-screened until plaques were pure following standard procedures. pBK-CMV phagemid 2-1 containing an insert was excised from the lambda ZAP Express (Stratagene) vector in the presence of ExAssist helper phage following the manufacturer's protocol. The base sequence of the 5' end of the insert of this clone, hereinafter referred to as GV-27, was determined using Sanger sequencing with fluorescent primers on Perkin Elmer/Applied Biosystems Dvision automatic sequencer. The determined nucleotide sequence of the partial M. vaccae GroEL-homologue clone GV-27 is provided in SEQ ID NO:77 and the predicted amino acid sequence in SEQ ID NO:78. This clone was found to have homology to M. tuberculosis GroEL. A partial sequence of the 65 kDa heat shock protein of M. vaccae has been published by Kapur et al. (Arch. Pathol. Lab. Med. 7/9 :131-138, 1995). The nucleotide sequence of the Kapur et al. fragment is shown in SEQ ID NO:79 and the predicted amino acid sequence in SEQ ID NO:80. In subsequent studies, a full-length (except for the predicted 51 terminal nucleotides) DNA sequence for GV-27 was obtained (SEQ ID NO: 1 13). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 1 14. GV- 27 was found to be 93.7% identical to the M. tuberculosis GroEL at the amino acid level. Two peptide fragments, comprising the N-terminal sequence (hereinafter referred to as GV-27A) and the carboxy terminal sequence of GV-27 (hereinafter referred tό~as GV-27B) were prepared using techniques well known in the art. The nucleotide sequences for GV-27A and GV-27B are provided in SEQ ID NO: 1 15 and 116, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 1 17 and 1 18. The sequence of GV-27A is 95.8% identical to the M. tuberculosis GroEL sequence and contains the shorter M. vaccae sequence of Kapur et al. discussed above. The sequence for GV-27B shows about 92.2% identity to the corresponding region of M. tuberculosis HSP65.
The M. vaccae culture filtrate described above was also fractionated by iso- electric focusing and the fractions assayed for adjuvant activity in the anti-ovalbumin- specific cytotoxic response assay in C57BL/6 mice as described above. As shown in Fig. 7, peak adjuvant activities were demonstrated in fractions corresponding to pi of 4.2-4.32 (fraction nos. 7-9), 4.49-4.57 (fraction nos. 13-17) and 4.81-5.98 (fraction nos. 23-27).
EXAMPLE 7
AUTOCLAVED M. VACCAE GENERATES CYTOTOXIC CD8 T CELLS AGAINST M. TUBERCULOSIS INFECTED MACROPHAGES
This example illustrates the ability of killed M. vaccae to stimulate cytotoxic
CD8 T cells which preferentially kill macrophages that have been infected with M tuberculosis.
Mice were immunized by the intraperitoneal injection of 500 μg of killed M. vaccae which was prepared as described in Example 1. Two weeks after immunization, the spleen cells of immunized mice were passed through a CD8 T cell enrichment column (R&D Systems, St. Paul, MN, USA). The spleen cells recovered from the column have been shown to be enriched up to 90% CD8 T cells. These T cells, as well as CD8 T cells from spleens of non-immunized mice, were tested for their ability to kill uninfected macrophages or macrophages which have been infected with M. tuberculosis.
Macrophages were obtained from the peritoneal cavity of mice five days after they have~been given 1 ml of 3% thioglycolate intraperitoneally. The macrophages were infected overnight with M. tuberculosis at the ratio of 2 mycobacteria per macrophage. All macrophage preparations were labelled with 51 Chromium at 2 μci per 104 macrophages. The macrophages were then cultured with CD8 T cells overnight (16 hours) at killer to target ratios of 30:1. Specific killing was detected by the release of 51 Chromium and expressed as % specific lysis, calculated as in Example 5.
The production of IFN-γ and its release into medium after 3 days of co-culture of CD8 T cells with macrophages was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with a rat monoclonal antibody directed to mouse IFN-γ (Pharmigen, San Diego, CA, USA) in PBS for 4 hours at 4 °C. Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS containing 0.2% Tween 20, and samples diluted 1 :2 in culture medium in the ELISA plates were incubated overnight at room temperature. The plates were again washed, and a biotinylated monoclonal rat anti-mouse IFN-γ antibody (Pharmigen), diluted to 1 μg/ml in PBS, was added to each well. The plates were then incubated for 1 hour at room temperature, washed, and horseradish peroxidase-coupled avidin D (Sigma A- 3151) was added at a 1 :4,000 dilution in PBS. After a further 1 hour incubation at room temperature, the plates were washed and OPD substrate added. The reaction was stopped after 10 min with 10% (v/v) HCl. The optical density was determined at 490 nm. Fractions that resulted in both replicates giving an OD two-fold greater than the mean OD from cells cultured in medium alone were considered positive.
As shown in Table 5, CD8 T cells from spleens of mice immunized with M. vaccae were cytotoxic for macrophages infected with M. tuberculosis and did not lyse uninfected macrophages. The CD8 T cells from non-immunized mice did not lyse macrophages. CD8 T cells from naive or non-immunized mice do produce IFN-γ when cocultured with infected macrophages. The amount of IFN-γ produced in coculture was greater with CD8 T cells derived from M. vaccae immunized mice.
TABLE 5
EFFECT WITH M. TUBERCULOSIS INFECTED
AND UNINFECTED MACROPHAGES
% Specific Lysis IFN-γ (ng ml) of Macrophages
CD8 T cells uninfected infected uninfected infected
Control 0 0 0.7 24.6
M. vaccae Immunized 0 95 2.2 43.8 EXAMPLE 8
DNA CLONING STRATEGY FOR THE M. VACCAE ANTIGENS GV-23. GV-24. GV-25. GV-26. GV-38A AND GV-38B
M. vaccae (ATCC Number 15483) was grown in sterile Medium 90 at 37 °C for 4 days and harvested by centrifugation. Cells were resuspended in 1 ml Trizol (Gibco BRL, Life Technologies, Gaithersburg, Maryland) and RNA extracted according to the standard manufacturer's protocol. M. tuberculosis strain H37Rv (ATCC Number 27294) was grown in sterile Middlebrooke 7H9 medium with Tween 80™ and oleic acid/ albumin dextrose/catalase additive (Difco Laboratories, Detroit, Michigan) at 37 °C and harvested under appropriate laboratory safety conditions. Cells were resuspended in 1 ml Trizol (Gibco BRL) and RNA extracted according to the manufacturer's standard protocol.
Total M. tuberculosis and M. vaccae RNA was depleted of 16S and 23 S ribosomal RNA (rRNA) by hybridisation of the total RNA fraction to oligonucleotides AD 10 and ADH (SEQ ID NO: 81 and 82) complementary to M. tuberculosis rRNA. These oligonucleotides were designed from mycobacterial 16S rRNA sequences published by Bottger (FEMS Microbiol. Lett. 65: 171 - 176, 1989) and from sequences deposited in the databanks. Depletion was done by hybridisation of total RNA~fo oligonucleotides AD10 and ADH immobilised on nylon membranes (Hybond N, Amersham International, United Kingdom). Hybridisation was repeated until rRNA bands were not visible on ethidium bromide-stained agarose gels. An oligonucleotide, AD 12 (SEQ ID NO: 83), consisting of 20 dATP-residues, was ligated to the 3' ends of the enriched mRNA fraction using RNA ligase. First strand cDNA synthesis was performed following standard protocols, using oligonucleotide AD7 (SEQ ID NO:84) containing a poly(dT) sequence.
The M. tuberculosis and M. vaccae cDNA was used as template for single- sided-specific PCR (3S-PCR). For this protocol, a degenerate oligonucleotide AD1 (SEQ ID NO:85) was designed based on conserved leader sequences and membrane protein sequences. After 30 cycles of amplification using primer AD 1 as 5'-primer and AD7 as 3'-primer, products were separated on a urea/polyacrylamide gel. DNA bands unique to M vaccae were excised and re-amplified using primers AD1 and AD7. After gel purification, bands were cloned into pGEM-T (Promega) and the base sequence determined.
Searches with the determined nucleotide and predicted amino acid sequences of band 12B21 (SEQ ID NOS: 86 and 87, respectively) showed homology to the pota gene of E.coli encoding the ATP-binding protein of the spermidine/putrescine ABC transporter complex published by Furuchi et al. (Jnl Biol Chem. 266: 20928-20933, 1991 ). The spermidine/putrescine transporter complex of E.coli consists of four genes and is a member of the ABC transporter family. The ABC (ATP-binding
Cassette) transporters typically consist of four genes: an ATP-binding gene, a periplasmic, or substrate binding, gene and two transmembrane genes. The transmembrane genes encode proteins each characteristically having six membrane- spanning regions. Homologues (by similarity) of this ABC transporter have been identified in the genomes of Haemophilus influenza (Fleischmann et al. Science 269
:496-512, 1995) and Mycoplasma genitalium (Fraser, et al. Science, 270:391-403,
1995).
An M. vaccae genomic DNA library constructed in BamHl -digested lambda ZAP Express (Stratagene) was probed with the radiolabelled 238 bp band 12B21 following standard protocols. A plaque was purified to purity by repetitive screening and a phagemid containing a 4.5 kb insert was identified by Southern blotting and hybridisation. The nucleotide sequence of the full-length M. vaccae homologue of pota (ATP-binding protein) was identified by subcloning of the 4.5 kb fragment and base sequencing. The gene consisted of 1449 bp including an untranslated 5' region of 320 bp containing putative -10 and -35 promoter elements. The nucleotide and predicted amino acid sequences of the M. vaccae pota homologue are provided in SEQ ID NOS:88 and 89, respectively.
The nucleotide sequence of the M. vaccae pota gene was used to design primers EV24 and EV25 (SEQ ID NO: 90 and 91) for expression cloning. The amplified DNA fragment was cloned into pProEX HT prokaryotic expression system (Gibco BRL) and expression in an appropriate E.coli host was induced by addition of 0.6 mM isopropylthio-β-galactoside (IPTG). The recombinant protein was named GV-23 and purified from inclusion bodies according to the manufacturer's protocol. In subsequent studies, GV-23 (SEQ ID NO: 88) was re-cloned into the alternative vector pETl 6 (Novagen).
A 322 bp Sall-BamHl subclone at the 3'-end of the 4.5 kb insert described above showed homology to the potd gene, (periplasmic protein), of the spermidine/putrescine ABC transporter complex of E. coll The nucleotide sequence of this subclone is shown in SEQ ID NO:92. To identify the gene, the radiolabelled insert of this subclone was used to probe an M. vaccae genomic DNA library constructed in the Sail -site of lambda Zap Express (Stratagene) following standard protocols. A clone was identified of which 1342 bp showed homology with the potd gene of E. coli. The potd homologue of M. vaccae was identified by sub-cloning and base sequencing. The determined nucleotide and predicted amino acid sequences are shown in SEQ ID NO: 93 and 94.
For expression cloning, primers EV26 and EV27 (SEQ ID NOS.95-96) were designed from the determined M. vaccae potd homologue. The amplified fragment was cloned into pProEX HT Prokaryotic expression system (Gibco BRL). Expression in an appropriate E. coli host was induced by addition of 0.6 mM IPTG and the recombinant protein named GV24. The recombinant antigen was purified from inclusion bodies according to the protocol of the supplier. In subsequent studies, GV- 24 (SEQ ID'NO: 93) was re-cloned into the alternative vector pET16 (Novagen).
The ability of purified recombinant protein GV-23 and GV-24 to stimulate proliferation of T cells and interferon- production in human PBL was determined as described in Example 2. The results of these assays are provided in Table 6, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, (++) indicates polypeptides having activity greater than four times above background, and (ND) indicates not determined. TABLE 6
Figure imgf000052_0001
Base sequence adjacent to the M vaccae potd gene-homologue was found to show homology to the potb gene of the spermidine/putrescine ABC transporter complex of E.coli, which is one of two transmembrane proteins in the ABC transporter complex. The M. vaccae potb homologue (referred to as GV-25) was identified through further subcloning and base sequencing. The determined nucleotide and predicted amino acid sequences for GV-25 are shown in SEQ ID NOS :97 and 98, respectively.
Further subcloning and base sequence analysis of the adjacent 509 bp failed to reveal significant homology to PotC, the second transmembrane protein of E.coli, and suggests that a second transmembrane protein is absent in the M. vaccae homologue of the ABC transporter. An open reading frame with homology to M. tuberculosis acetyl-CoA acetyl transferase, however, was identified starting 530 bp downstream of the transmembrane protein and the translated protein was named GV-26. The determined partial nucleotide sequence and predicted amino acid sequence for GV-26 are shown SEQ ID NO:99 and 100.
Using a protocol similar to that described above for the isolation of GV-23, the 3S-PCR band 12B28 (SEQ ID NO: 119) was used to screen the M. vaccae genomic library constructed in the BamHI-site of lambda ZAP Express (Stratagene). The clone isolated from the library contained a novel open reading frame and the antigen encoded by this gene was named GV-38A. The determined nucleotide sequence and predicted amino acid sequence of GV-38A are shown in SEQ ID NO: 120 and 121 , respectively. Comparison of these sequences with those in the gene bank, revealed some homology to an unknown M. tuberculosis protein previously identified in cosmid MTCY428.12. (SPTREMBL:P71915). Upstream of the GV-38A gene, a second novel open reading frame was identified and the antigen encoded by this gene was named GV-38B The determined 5' and 3' nucleotide sequences for GV-38B are provided in SEQ ID NO 122 and 123, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 124 and 125, respectively This protein was found to show homology to an unknown M tuberculosis protein identified in cosmid MTCY428 11 (SPTREMBL: P71914).
Both the GV-38A and GV-38B antigens were amplified for expression cloning into pET16 (Novagen). GV-38A was amplified with primers KR1 1 and KR12 (SEQ ID NO 126 and 127) and GV-38B with primers KR13 and KR14 (SEQ ID NO- 128 and 129). Protein expression in the host cells BL21(DE3) was induced with 1 M IPTG. however no protein expression was obtained from these constructs Hydrophobic regions were identified in the N-termini of antigens GV-38A and GV- 38B which may inhibit expression of these constructs The hydrophobic region present in GV-38A was identified as a possible transmembrane motif with six membrane spanning regions. To express the antigens without the hydrophobic regions, primers KR20 for GV-38A, (SEQ ID NO: 130) and KR21 for GV-38B (SEQ ID NO. 131 ) were designed. The truncated GV-38A gene was amplified with primers KR20 and KR12, and the truncated GV-38B gene with KR21 and KR14 The determined nucleotide sequences of truncated GV38A and GV-38B are shown in SEQ ID NO. 132 and 133, respectively, with the corresponding predicted amino acid sequenceS"beιng shown in SEQ ID NO: 134 and 135, respectively.
EXAMPLE 9
PURIFICATION AND CHARACTERISATION OF POLYPEPTIDES FROM M
VACCAE CULTURE FILTRATE BY PREPARATIVE ISOELECTRIC FOCUSING
AND PREPARATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS
M. vaccae soluble proteins were isolated from culture filtrate using preparative isoelectric focusing and preparative polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
M. vaccae (ATCC Number 15483) was cultured in 250 1 sterile Medium 90 which had been fractionated by ultrafiltration to remove all proteins of greater than 10 kDa molecular weight. The medium was centrifuged to remove the bacteria, and sterilised by filtration through a 0.45 m filter. The sterile filtrate was concentrated by ultrafiltration over a 10 kDa molecular weight cut-off membrane.
Proteins were isolated from the concentrated culture filtrate by precipitation with 10% trichloroacetic acid. The precipitated proteins were re-dissolved in 100 mM Tris. HCl pH 8.0. and re-precipitated by the addition of an equal volume of acetone. The acetone precipitate was dissolved in water, and proteins were re-precipitated by the addition of an equal volume of chloroform:methanol 2: 1 (v/v). The chloroform:methanol precipitate was dissolved in water, and the solution was freeze - dried. The freeze-dried protein was dissolved in iso-electric focusing buffer, containing 8 M deionised urea, 2% Triton X-100, 10 mM dithiothreitol and 2% ampholytes (pH 2.5 - 5.0). The sample was fractionated by preparative iso-electric focusing on a horizontal bed of Ultrodex gel at 8 watts constant power for 16 hours. Proteins were eluted from the gel bed fractions with water and concentrated by precipitation with 10% trichloroacetic acid.
Pools of fractions containing proteins of interest were identified by analytical polyacrylaTnide gel electrophoresis and fractionated by preparative polyacrylamide gel electrophoresis. Samples were fractionated on 12.5% SDS-PAGE gels, and electroblotted onto nitrocellulose membranes. Proteins were located on the membranes by staining with Ponceau Red, destained with water and eluted from the membranes with 40% acetonitrile/O.lM ammonium bicarbonate pH 8.9 and then concentrated by lyophilisation.
Eluted proteins were assayed for their ability to induce proliferation and interferon-γ secretion from the peripheral blood lymphocytes of immune donors as detailed in Example 2. Proteins inducing a strong response in these assays were selected for further study. Selected proteins were further purified by reversed-phase chromatography on a Vydac Protein C4 column, using a trifluoroacetic acid-acetonitrile system. Purified proteins were prepared for protein sequence determination by SDS-polyacrylamide gel electrophoresis, and electroblotted onto PVDF membranes. Protein sequences were determined as in Example 3. The proteins were named GV-40, GV-41 , GV-42, GV-43 and GV-44. The determined N-terminal sequences for these polypeptides are shown in SEQ ID NOS: 101 -105. respectively. Subsequent studies led to the isolation of a 5', middle fragment and 3' DNA sequence for GV-42 (SEQ ID NO: 136, 137 and 138, respectively). The corresponding predicted amino acid sequences are provided in SEQ ID NO: 139, 140 and 141, respectively.
All of these amino acid sequences were compared to known amino acid sequences in the EMBL data base using TFASTA. No significant homologies were obtained with GV-40, GV42 and GV-44. GV-41 had similarity to a putative ribosome recycling factor from M. tuberculosis, a protein responsible for the release of ribosomes from mRNA at the termination of protein biosynthesis. GV-43 showed homology (by similarity) to a previously identified unknown M. tuberculosis and M. leprae protein.
EXAMPLE 10
IMMUNE MODULATING PROPERTIES
OF DELIPIDATED AND DEGLYCOSYLATED M. VACCAE AND
RECOMBINANT PROTEINS FROM M. VACCAE
This example illustrates the processing of different constituents of M.vaccae and their immune modulating properties.
Heat-killed M. vaccae and M. vaccae culture filtrate
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/1; tryptone, 5 g/1; glucose 1 g/1) at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 1010 M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 °C. The culture filtrate was passaged through a 0.45 μM filter into sterile bottles.
Lyophilised, Delipidated and Deglycosylated M.vaccae
To prepare delipidated M.vaccae, the autoclaved M. vaccae was pelleted by centrifugation, the pellet washed with water and collected again by centrifugation and then freeze-dried. An aliquot of this freeze-dried M. vaccae was set aside and referred to as lyophilised M.vaccae. When used in experiments it was resuspended in PBS to the desired concentration. Freeze-dried M vaccae was extracted with chloroform/methanol (2:1) for 60 mins at room temperature, and the extraction was repeated once. The residue from chloroform/methanol extraction was further extracted with 50% ethanol by refluxing for two hours. The 50% ethanol extraction was repeated two times. The pooled 50% ethanol extracts were used as a source of
M.vaccae glycolipids (see below). The residue from the 50% ethanol extraction was freeze-dried and weighed. The amount of delipidated M.vaccae prepared was equivalent to 11.1% of the starting wet weight of M.vaccae used. For bioassay, the delipidated and deglycosylated M.vaccae DO-M. vaccae; referred to as delipidated
M.vaccae~ Figures 8 and 9), was resuspended in phosphate-buffered saline by sonication, and sterilised by autoclaving.
M.vaccae glycolipids
The pooled 50% ethanol extracts described above were dried by rotary evaporation , redissolved in water, and freeze-dried. The amount of glycolipid recovered was 1.2% of the starting wet weight of M.vaccae used. For bioassay, the glycolipids were dissolved in phosphate-buffered saline. Production of Intcrleukin-12 from macrophages
As detailed below, whole heat-killed M.vaccae and M. vaccae constituents were shown to stimulate the production of interleukin-12 (IL-12) from macrophages, an important component of the Thl response. A group of C57BL/6J mice were injected intraperitoneally with DIFCO thioglycolate and after three days, peritoneal macrophages were collected and placed in cell culture with interferon-gamma for three hours. The culture medium was replaced and various concentrations of whole heat-killed M. vaccae, lyophilized M. vaccae, DD- vaccae (referred to as delipidated M. vaccae in Figure 8) and M. vaccae glycolipids were added. After a further three days at 37 °C, the culture supematants were assayed for the presence of IL-12 produced by macrophages. As shown in Figure 8, the M. vaccae preparations stimulated the production of IL-12 from macrophages.
Figures 9A, B, and C show data from separate experiments in which groups of C57BL/6 mice (Fig. 9A), BALB/C mice (Fig. 9B) or C3H/HeJ mice (Fig. 9C) were given DIFCO thioglycolate intraperitoneally and, after three days, peritoneal macrophages were collected and placed in culture with interferon-gamma for three hours. The culture medium was replaced and various concentrations of M.vaccae recombinant proteins GVc-3 (GV 3), GVc4P (GV 4P), GVc-7 (GV7), GV-23, GV 27, heat killed M.vaccae, OD-M.vaccae (referred to as delipidated M. vaccae in Figures 9A, B and C), M.vaccae glycolipids or lipopolysaccharide were added. After three days at 37°C, the culture supematants were assayed for the presence of IL-12 produced "By macrophages. As shown in Figures 9A, B and C, the recombinant proteins and M.vaccae preparations stimulated the production of IL-12 from macrophages.
EXAMPLE 1 1
EFFECT OF INTRADERMAL ROUTE
OF IMMUNISATION WITH M. VACCAE ON TUBERCULOSIS IN CYNOMOLGOUS MONKEYS
This example illustrates the effect of immunisation with M.vaccae or M.vaccae culture filtrate intradermally in cynomolgous monkeys prior to challenge with live M. tuberculosis.
M.vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5g/l; tryptone, 5g/l; glucose, lg/I) at 37°C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37°C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of lOmg/ml, equivalent to 10'° M.vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120°C. The culture filtrate was passaged through a 0.45 μM filter into sterile bottles.
Three groups of cynomolgous monkeys were included in this study, with each group containing 2 monkeys. One group of monkeys were immunised with whole heat-killed M. vaccae; one group were immunised with M. vaccae culture filtrate and a control group received no immunisations. The composition employed for immunisation, amount of immunogen and route of administration for each group of monkeys are provided in Table 7. TABLE 7
COMPARISON OF INTRADERMAL ROUTE OF IMMUNISATION
Figure imgf000059_0001
with culture filtrate) 38I5-B lOOμg intradermal
Prior to immunisation, all monkeys were weighed (Wt kgs), body temperature measured (temp), and a blood sample taken for determination of erythrocyte sedimentation rate (ESR mm/hr) and lymphocyte proliferation (LPA) to an in vitro challenge with purified protein (PPD) prepared from Mycobacterium bovis. At day 33 post-immunisation these measurements were repeated. At day 34, all monkeys received a second immunisation using the same amount of M.vaccae. On day 62, body weight, temperature, ESR and LPA to PPD were measured, then all monkeys were infected with 103 colony forming units of the Erdman strain of M. tuberculosis. Twenty eight days following infection, body weight, temperature, ESR and LPA to PPD were measured in all monkeys, and the lungs were X-rayed to determine whether infection with live M. tuberculosis had resulted in the onset of pneumonia.
As shown in Tables 8A, B and C, the monkeys in the control group showed radiologic evidence of pulmonary tuberculosis by 28 days after infection with M. tuberculosis. Clinical disease was not evident 84 days after infection in monkeys immunised intradermally with two doses of 500 μg of M. vaccae. The onset of clinical disease was delayed in both monkeys immunised intradermally with 100 μg of M vaccae culture filtrate.
TABLE 8A
CONTROL MONKEYS
Figure imgf000060_0001
ND = Not Done TABLE 8B
MONKEYS IMMUNISED WITH WHOLE HEAT- KILLED M. VACCAE (500 μg) INTRADERMALLY
Figure imgf000061_0001
Figure imgf000061_0002
ND = Not Done TABLE 8C
MONKEYS IMMUNISED WITH CULTURE FILTRATE (lOOμg)
INTRADERMALLY
Figure imgf000062_0001
ND Not Done
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, changes and modifications can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims. SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT
(n) TITLE OF THE INVENTION COMPOUNDS AND METHODS FOR TREATMENT AND DIAGNOSIS OF MYCOBACTERIAL INFECTIONS
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(ix) TELECOMMUNICATION INFORMATION. (A) TELEPHONE +64 4 499 9058 (BJ .TELEFAX: +64 4 499 9306 (C) TELEX.
( 2 ) INFORMATION FOR SEQ ID NO : l :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 25 amino acids
(B) TYPE : amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY : linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ala Pro Val Gly Pro Gly Xaa Ala Ala Tyr Val Gin Gin Val Pro Asp
1 5 ιo 15
Gly Pro Gly Ser Val Gin Gly Met Ala 20 25 (2) INFORMATION FOR SEQ ID NO .2
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH- 10 amino acids
(B) TYPE, ammo acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(u) MOLECULE TYPE: protein
(x ) SEQUENCE DESCRIPTION: SEQ ID NO 2 •
Met Xaa Asp Gin Leu Lys Val Asn Asp Asp 1 5 10
(2) INFORMATION FOR SEQ ID NO .3
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(u) MOLECULE TYPE protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
Met Xaa Pro Val Pro Val Ala Thr Ala Ala Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO : •
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii .MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Thr Pro Ala Pro Ala Pro Pro Pro Tyr Val Asp His Val Glu Gin Ala
1 5 10 15
Lys Phe Gly Asp Leu 20
(2) INFORMATION FOR SEQ ID NO : 5 -
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single <D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION SEQ ID NO : 5 ■
Met Gin Ala Phe Asn Ala Asp Ala Tyr Ala Phe Ala Lys Arg Glu Lys
1 5 10 15
Val Ser Leu Ala Pro Gly Val Pro Xaa Val Phe Glu Thr 20 25
(2) INFORMATION FOR SEQ ID NO : 6 •
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 ammo acids
(B) TYPE, amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Ala Asp Pro Asn Xaa Ala lie Leu Gin Val Ser Lys Thr Thr Arg
1 5 10 IS
Gly Gly Gin Ala Ala 20
(2) INFORMATION FOR SEQ ID NO : 7 :
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 11 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i i ) MOLECULE TYPE : protein
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 7 :
Met Pro He Leu Gin Val Ser Gin Thr Gly Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO : 8 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Xaa Asp Pro He Xaa Leu Gin Leu Gin Val Ser Ser Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO : 9 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH 16 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY, linear
(11) MOLECULE TYPE, protein
(XI ) SEQUENCE DESCRIPTION SEQ ID NO .9
Lys Ala Thr Tyr Val Gin Gly Gly Leu Gly Arg He Glu Ala Arg Val 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 10.
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH . 9 amino acids
(B) TYPE, amino acid
(C) STRANDEDNESS. single
(D) TOPOLOGY- linear
(n) MOLECULE TYPE, protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.10
Lys Xaa Gly Leu Ala Asp Leu Ala Pro 1 5
(2) INFORMATION FOR SEQ ID NO: 11-
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
Figure imgf000066_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE: protein (ix) FEATURE:
JA) NAME/KEY: Other (B) LOCATION: 12...12 (D) OTHER INFORMATION: Residue can be either Glu or He
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Lys Xaa Tyr Ala Leu Ala Leu Met Ser Ala Val Xaa Ala Ala 1 5 10
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein ( xi ) SEQUENCE DESCRIPTION - SEQ ID NO - 12
Lys Asn Pro Gin Val Ser Asp Glu Leu Xaa Thr 1 5 10
( 2 ) INFORMATION FOR SEQ ID NO . 13 .
( l ) SEQUENCE CHARACTERISTICS
(A) LENGTH . 21 amino acids
Figure imgf000067_0001
(C) STRANDEDNESS. single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO: 13.
Ala Pro Ala Pro Ala Ala Pro Ala Xaa Gly Asp Pro Ala Ala Val Val
1 5 10 15
Ala Ala Asn Ser Thr 20
(2) INFORMATION FOR SEQ ID NO: 14
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 15 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 14:
Glu Ala Glu Val Xaa Tyr Leu Gly Gin Pro Gly Glu Leu Val Asn 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 2...2
(D) OTHER INFORMATION: Residue can be either Gly or Ala
(A) NAME/KEY: Other
(B) LOCATION. 15...15
(D) OTHER INFORMATION: Residue can be either Pro or Ala
(XI ) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Ala Xaa Val Val Pro Pro Xaa Gly Pro Pro Ala Pro Gly Ala Xaa
1 5 10 15
(2) INFORMATION FOR SEQ ID NO.16
(I) SEQUENCE CHARACTERISTICS
(A) LENGTH 15 ammo acids
Figure imgf000068_0001
(C) STRANDEDNESS. single
(D) TOPOLOGY- linear
(II) MOLECULE TYPE: protein
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 16-
Ala Pro Ala Pro Asp Leu Gin Gly Pro Leu Val Ser Thr Leu Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 17
(l) SEQUENCE CHARACTERISTICS-
(A) LENGTH: 25 amino acids
(B) TYPE: am o ac d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO.17:
Ala Thr Pro Asp Trp Ser Gly Arg Tyr Thr Val Val Thr Phe Ala Ser
1 5 10 15
Asp Lys Leu Gly Thr Ser Val Ala Ala 20 25
(2) INFORMATION FOR SEQ ID NO: 18:
<i)_S.EQUENCE CHARACTERISTICS.
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(u) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 15...15
(D) OTHER INFORMATION: Residue can be either Ala or Arg
(A) NAME/KEY: Other
(B) LOCATION: 23...23
(D) OTHER INFORMATION: Residue can be either Val or Leu
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Ala Pro Pro Tyr Asp Asp Arg Gly Tyr Val Asp Ser Thr Ala Xaa Xaa
1 5 10 15
Ala Ser Pro Pro Thr Leu Xaa Val Val 20 25
( 2 ) INFORMATION FOR SEQ ID NO . 19
( l ) SEQUENCE CHARACTERISTICS
(A) LENGTH - 8 amino acids
( B ) TYPE , ammo acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY linear
(n) MOLECULE TYPE, protein
(xi) SEQUENCE DESCRIPTION- SEQ ID NO 19
Glu Pro Glu Gly Val Ala Pro Pro 1 5
(2) INFORMATION FOR SEQ ID NO 20
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH. 25 am o acids
Figure imgf000069_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
( l ) SEQUENCE DESCRIPTION SEQ ID NO.20
Glu Pro Ala Gly He Pro Ala Gly Phe Pro Asp Val Ser Ala Tyr Ala
1 5 10 15
Ala Val Asp Pro Xaa Xaa Tyr Val Val 20 25
(2) INFORMATION FOR SEQ ID NO: 21:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21.
Ala Pro Val Gly Pro Gly Xaa Ala Ala Tyr Val Gin Gin Val Pro 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 22:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
Figure imgf000069_0002
(C) STRANDEDNESS Single
(D) TOPOLOGY linear
(a) MOLECULE TYPE, protein
(Xl) SEQUENCE DESCRIPTION SEQ ID NO.22-
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Asp Val Phe Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO 23
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 19 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE- protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO 23
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Met Val Pro Ser Pro
1 5 10 15
Ser Met Gly
(2) INFORMATION FOR SEQ ID NO: 24-
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 ammo acids
Figure imgf000070_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 24
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Met Val Pro Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 25:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 14 amino acids
Figure imgf000070_0002
(C) STRANDEDNESS: single
(D) TOPOLOGY- linear
( i) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Xaa Xaa Thr Gly Leu His Arg Leu Arg Met Met Val Pro Asn
1 5 10 ( 2 ) INFORMATION FOR SEQ ID NO : 26 :
( l ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 20 amino acids
( B ) TYPE : ammo acid
( C ) STRANDEDNESS : singl e
(D ) TOPOLOGY : l inear
(ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 16...16
(D) OTHER INFORMATION: Residue can be either Ser or Val
(A) NAME/KEY: Other
(B) LOCATION: 17...17
(D) OTHER INFORMATION: Residue can be either Gin or Val
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Val Pro Ala Asp Pro Val Gly Ala Ala Ala Gin Ala Glu Pro Ala Xaa
1 5 10 15
Xaa Arg He Asp 20
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Other .IB) LOCATION: 4...4 (D) OTHER INFORMATION: Residue can be either Tyr or Pro
(A) NAME/KEY: Other
(B) LOCATION: 8...8
(D) OTHER INFORMATION: Residue can be either Val or Gly
(A) NAME/KEY: Other
(B) LOCATION: 9...9
(D) OTHER INFORMATION: Residue can be either He or Tyr
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Asp Pro Xaa Xaa Asp He Glu Xaa Xaa Phe Ala Arg Gly Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS- single
(D) TOPOLOGY, linear
(11) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28
Ala Pro Ser Leu Ser Val Ser Asp Tyr Ala Arg Asp Ala Gly Phe 1 5 10 15
(2) INFORMATION FOR SEQ ID NO : 29 ■
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH 16 amino acids
Figure imgf000072_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY, linear
(ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 2...2
(D) OTHER INFORMATION: Residue can be either Leu or Pro
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29
Xaa Xaa Leu Ala Xaa Ala Xaa Leu Gly Xaa Thr Val Asp Ala Asp Gin 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 30:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 330 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single CDΛ TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Met Lys Phe Val Asp Arg Phe Arg Gly Ala Val Ala Gly Met Leu Arg
1 5 10 15
Arg Leu Val Val Glu Ala Met Gly Val Ala Leu Leu Ser Ala Leu He
20 25 30
Gly Val Val Gly Ser Ala Pro Ala Glu Ala Phe Ser Arg Pro Gly Leu
35 40 45
Pro Val Glu Tyr Leu Gin Val Pro Ser Pro Ser Met Gly Arg Asp He
50 55 60
Lys Val Gin Phe Gin Asn Gly Gly Ala Asn Ser Pro Ala Leu Tyr Leu 65 70 75 80
Leu Asp Gly Leu Arg Ala Gin Asp Asp Phe Ser Gly Trp Asp He Asn
85 90 95
Thr Thr Ala Phe Glu Trp Tyr Tyr Gin Ser Gly He Ser Val Val Met 100 105 HO
Pro Val Gly Gly Gin Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala
115 120 125
Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys Trp Glu Thr Phe Leu Thr
130 135 140
Ser Glu Leu Pro Glu Tyr Leu Gin Ser Asn Lys Gin He Lys Pro Thr 145 150 155 160
Gly Ser Ala Ala Val Gly Leu Ser Met Ala Gly Leu Ser Ala Leu Thr
165 170 175
Leu Ala He Tyr His Pro Asp Gin Phe He Tyr Val Gly Ser Met Ser
180 185 190
Gly Leu Leu Asp Pro Ser Asn Ala Met Gly Pro Ser Leu He Gly Leu
195 200 205
Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro
210 215 220
Ser Thr Asp Pro Ala Trp Lys Arg Asn Asp Pro Thr Val Asn Val Gly 225 230 235 240
Thr Leu He Ala Asn Asn Thr Arg He Trp Met Tyr Cys Gly Asn Gly
245 250 255
Lys Pro Thr Glu Leu Gly Gly Asn Asn Leu Pro Ala Lys Leu Leu Glu
260 265 270
Gly Leu Val Arg Thr Ser Asn He Lys Phe Gin Asp Gly Tyr Asn Ala
275 280 285
Gly Gly Gly His Asn Ala Val Phe Asn Phe Pro Asp Ser Gly Thr His
290 295 300
Ser Trp Glu Tyr Trp Gly Glu Gin Leu Asn Asp Met Lys Pro Asp Leu 305 310 315 320
Gin Gin Tyr Leu Gly Ala Thr Pro Gly Ala 325 330
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 327 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii .MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Met He Asp Val Ser Gly Lys He Arg Ala Trp Gly Arg Trp Leu Leu
1 5 10 15
Val Gly Ala Ala Ala Thr Leu Pro Ser Leu He Ser Leu Ala Gly Gly
20 25 30
Ala Ala Thr Ala Ser Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr
35 40 45
Leu Gin Val Pro Ser Glu Ala Met Gly Arg Thr He Lys Val Gin Phe
50 55 60
Gin Asn Gly Gly Asn Gly Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu 65 70 75 80
Arg Ala Gin Asp Asp Tyr Asn Gly Trp Asp He Asn Thr Ser Ala Phe
85 90 95
Glu Trp Tyr Tyr Gin Ser Gly Leu Ser Val Val Met Pro Val Gly Gly
100 105 110
Gin Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala 115 120 125
Gly Cys Thr Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro
130 135 140
Lys Trp Leu Ser Ala Asn Arg Ser Val Lys Ser Thr Gly Ser Ala Val 145 150 155 160
Val Gly Leu Ser Met Ala Gly Ser Ser Ala Leu He Leu Ala Ala Tyr
165 170 175
His Pro Asp Gin Phe He Tyr Ala Gly Ser Leu Ser Ala Leu Met Asp
180 185 190
Ser Ser Gin Gly He Glu Pro Gin Leu He Gly Leu Ala Met Gly Asp
195 200 205
Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Pro Asn Asp Pro
210 215 220
Ala Trp Gin Arg Asn Asp Pro He Leu Gin Ala Gly Lys Leu Val Ala 225 230 235 240
Asn Asn Thr His Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Ser Glu
245 250 255
Leu Gly Gly Thr Asn Val Pro Ala Glu Phe Leu Glu Asn Phe Val His
260 265 270
Gly Ser Asn Leu Lys Phe Gin Asp Ala Tyr Asn Gly Ala Gly Gly His
275 280 285
Asn Ala Val Phe Asn Leu Asn Ala Asp Gly Thr His Ser Trp Glu Tyr
290 295 300
Trp Gly Ala Gin Leu Asn Ala Met Lys Pro Asp Leu Gin Asn Thr Leu 305 310 315 320
Met Ala Val Pro Arg Ser Gly 325
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTE ISTICS:
(A) LENGTH: 338 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xU..SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Met Gin Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly Met Ser Arg
1 5 10 15
Arg Leu Val Val Gly Ala Val Gly Ala Ala Leu Val Ser Gly Leu Val
20 25 30
Gly Ala Val Gly Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly
35 40 45
Leu Pro Val Glu Tyr Leu Gin Val Pro Ser Pro Ser Met Gly Arg Asp
50 55 60
He Lys Val Gin Phe Gin Ser Gly Gly Ala Asn Ser Pro Ala Leu Tyr 65 70 75 80
Leu Leu Asp Gly Leu Arg Ala Gin Asp Asp Phe Ser Gly Trp Asp He
85 90 95
Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gin Ser Gly Leu Ser Val Val
100 105 110
Met Pro Val Gly Gly Gin Ser Ser Phe Tyr Ser Asp Trp Tyr Gin Pro
115 120 125
Ala Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys Trp Glu Thr Phe Leu 130 135 140
Thr Ser Glu Leu Pro Gly Trp Leu Gin Ala Asn Arg His Val Lys Pro 145 150 155 160
Thr Gly Ser Ala Val Val Gly Leu Ser Met Ala Ala Ser Ser Ala Leu
165 170 175
Thr Leu Ala He Tyr H s Pro Gin Gin Phe Val Tyr Ala Gly Ala Met
180 185 190
Ser Gly Leu Leu Asp Pro Ser Gin Ala Met Gly Pro Thr Leu He Gly
195 200 205
Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ser Asp Met Trp Gly
210 215 220
Pro Lys Glu Asp Pro Ala Trp Gin Arg Asn Asp Pro Leu Leu Asn Val 225 230 235 240
Gly Lys Leu He Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly Asn
245 250 255
Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu Pro Ala Lys Phe Leu
260 265 270
Glu Gly Phe Val Arg Thr Ser Asn He Lys Phe Gin Asp Ala Tyr Asn
275 280 285
Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp Ser Gly Thr
290 295 300
His Ser Trp Glu Tyr Trp Gly Ala Gin Leu Asn Ala Met Lys Pro Asp 305 310 315 320
Leu Gin Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro Gin
325 330 335
Gly Ala
(2) INFORMATION FOR SEQ ID NO: 33:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 325 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xii.SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Met Thr Asp Val Ser Arg Lys He Arg Ala Trp Gly Arg Arg Leu Met
1 5 10 15
He Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala
20 25 30
Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val
35 40 45
Glu Tyr Leu Gin Val Pro Ser Pro Ser Met Gly Arg Asp He Lys Val
50 55 60
Gin Phe Gin Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp 65 70 75 80
Gly Leu Arg Ala Gin Asp Asp Tyr Asn Gly Trp Asp He Asn Thr Pro
85 90 95
Ala Phe Glu Trp Tyr Tyr Gin Ser Gly Leu Ser He Val Met Pro Val
100 105 110
Gly Gly Gin Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly
115 120 125
Lys Ala Gly Cys Gin Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu 130 135 140
Leu Pro Gin Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser 145 150 155 160
Ala Ala He Gly Leu Ser Met Ala Gly Ser Ser Ala Met He Leu Ala
165 170 175
Ala Tyr His Pro Gin Gin Phe He Tyr Ala Gly Ser Leu Ser Ala Leu
180 185 190
Leu Asp Pro Ser Gin Gly Met Gly Pro Ser Leu He Gly Leu Ala Met
195 200 205
Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Ser
210 215 220
Asp Pro Ala Trp Glu Arg Asn Asp Pro Thr Gin Gin He Pro Lys Leu 225 230 235 240
Val Ala Asn Asn Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro
245 250 255
Asn Glu Leu Gly Gly Ala Asn He Pro Ala Glu Phe Leu Glu Asn Phe
260 265 270
Val Arg Ser Ser Asn Leu Lys Phe Gin Asp Ala Tyr Asn Ala Ala Gly
275 280 285
Gly His Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp
290 295 300
Glu Tyr Trp Gly Ala Gin Leu Asn Ala Met Lys Gly Asp Leu Gin Ser 305 310 315 320
Ser Leu Gly Ala Gly 325
(2) INFORMATION FOR SEQ ID NO: 34.
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 338 amino acids
Figure imgf000076_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Met Gin Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly Met Ser Arg
1 5 10 15
Arg Leu Val Val Gly Ala Val Gly Ala Ala Leu Val Ser Gly Leu Val
20 25 30
Gly Ala Val Gly Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly
35 40 45
Leu Pro Val Glu Tyr Leu Gin Val Pro Ser Pro Ser Met Gly Arg Asp
50 55 60
He Lys Val Gin Phe Gin Ser Gly Gly Ala Asn Ser Pro Ala Leu Tyr 65 70 75 80
Leu Leu Asp Gly Leu Arg Ala Gin Asp Asp Phe Ser Gly Trp Asp He
85 90 95
Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gin Ser Gly Leu Ser Val Val
100 105 110
Met Pro Val Gly Gly Gin Ser Ser Phe Tyr Ser Asp Trp Tyr Gin Pro
115 120 125
Ala Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys Trp Glu Thr Phe Leu
130 135 140
Thr Ser Glu Leu Pro Gly Trp Leu Gin Ala Asn Arg His Val Lys Pro
Figure imgf000077_0002
(2) INFORMATION FOR SEQ ID NO: 35:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 323 amino acids
Figure imgf000077_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Met Thr Asp Val Ser Arg Lys He Arg Ala Trp Gly Arg Arg Leu Met
1 5 10 15
He Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala
20 25 30
Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val
35 40 45
Glu Tyr Leu Gin Val Pro Ser Pro Ser Met Gly Arg Asp He Lys Val
50 55 60
Gin Phe Gin Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp 65 70 75 80
Gly Leu Arg Ala Gin Asp Asp Tyr Asn Gly Trp Asp He Asn Thr Pro
85 90 95
Ala Phe Glu Trp Tyr Tyr Gin Ser Gly Leu Ser He Val Met Pro Val
100 105 no
Gly Gly Gin Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly
115 120 125
Lys Ala Gly Cys Gin Thr Tyr Lys Trp Glu Thr Leu Leu Thr Ser Glu
130 135 140
Leu Pro Gin Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser 145 150 155 160
Ala Ala He Gly Leu Ser Met Ala Gly Ser Ser Ala Met He Leu Ala
165 170 175
Ala Tyr His Pro Gin Gin Phe He Tyr Ala Gly Ser Leu Ser Ala Leu
180 185 190
Leu Asp Pro Ser Gin Gly Met Gly Leu He Gly Leu Ala Met Gly Asp
195 200 205
Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Ser Asp Pro
210 215 220
Ala Trp Glu Arg Asn Asp Pro Thr Gin Gin He Pro Lys Leu Val Ala 225 230 235 240
Asn Asn Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu
245 250 255
Leu Gly Gly Ala Asn He Pro Ala Glu Phe Leu Glu Asn Phe Val Arg
260 265 270
Ser Ser Asn Leu Lys Phe Gin Asp Ala Tyr Lys Pro Ala Gly Gly His
275 280 285
Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr
290 295 300
Trp Gly Ala Gin Leu Asn Ala Met Lys Gly Asp Leu Gin Ser Ser Leu 305 310 315 320
Gly Ala Gly
(2) INFORMATION FOR SEQ ID NO.36:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 333 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Met Lys Phe Leu Gin Gin Met Arg Lys Leu Phe Gly Leu Ala Ala Lys
1 5 10 15
Phe Pro Ala Arg Leu Thr He Ala Val He Gly Thr Ala Leu Leu Ala
20 25 30
Gly Leu Val Gly Val Val Gly Asp Thr Ala He Ala Val Ala Phe Ser
35 40 45
Lys Pro Gly Leu Pro Val Glu Tyr Leu Gin Val Pro Ser Pro Ser Met
50 55 60
Gly His Asp He Lys He Gin Phe Gin Gly Gly Gly Gin His Ala Val 65 70 75 80
Tyr Leu Leu Asp Gly Leu Arg Ala Gin Glu Asp Tyr Asn Gly Trp Asp
85 90 95
He Asn Thr Pro Ala Phe Glu Glu Tyr Tyr His Ser Gly Leu Ser Val
100 105 110
He Met Pro Val Gly Gly Gin Ser Ser Phe Tyr Ser Asn Trp Tyr Gin
115 120 125
Pro Ser Gin Gly Asn Gly Gin His Tyr Thr Tyr Lys Trp Glu Thr Phe
130 135 140
Leu Thr Gin Glu Met Pro Ser Trp Leu Gin Ala Asn Lys Asn Val Leu 145 150 155 160
Pro Thr Gly Asn Ala Ala Val Gly Leu Ser Met Ser Gly Ser Ser Ala 165 170 175
Leu He Leu Ala Ser Tyr Tyr Pro Gin Gin Phe Pro Tyr Ala Ala Ser
180 185 190
Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Thr Met He
195 200 205
Gly Leu Ala Met Asn Asp Ser Gly Gly Tyr Asn Ala Asn Ser Met Trp
210 215 220
Gly Pro Ser Thr Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Gin 225 230 235 240
He Pro Arg Leu Val Ala Asn Asn Thr Arg He Trp Val Tyr Cys Gly
245 250 255
Asn Gly Ala Pro Asn Glu Leu Gly Gly Asp Asn He Pro Ala Lys Phe
260 265 270
Leu Glu Ser Leu Thr Leu Ser Thr Asn Glu He Phe Gin Asn Thr Tyr
275 280 285
Ala Ala Ser Gly Gly Arg Asn Gly Val Phe Asn Phe Pro Pro Asn Gly
290 295 300
Thr His Ser Trp Pro Tyr Trp Asn Gin Gin Leu Val Ala Met Lys Pro 305 310 315 320
Asp He Gin Gin He Leu Asn Gly Ser Asn Asn Asn Ala 325 330
(2) INFORMATION FOR SEQ ID NO: 37:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 340 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(i ) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION. SEQ ID NO : 37 :
Met Thr Phe Phe Glu Gin Val Arg Arg Leu Arg Ser Ala Ala Thr Thr
1 5 10 15
Leu Pro Arg Arg Val Ala He Ala Ala Met Gly Ala Val Leu Val Tyr
20 25 30
Gly Leu Val Gly Thr Phe Gly Gly Pro Ala Thr Ala Gly Ala Phe Ser
35 40 45
Arg Pro Gly Leu Pro Val Glu Tyr Leu Gin Val Pro Ser Ala Ser Met
50 55 60
Gly Arg Asp He Lys Val Gin Phe Gin Gly Gly Gly Pro His Ala Val 65 70 75 80
Tyr Leu Leu Asp Gly Leu Arg Ala Gin Asp Asp Tyr Asn Gly Trp Asp
85 90 95
He Asn Thr Pro Ala Phe Glu Glu Tyr Tyr Gin Ser Gly Leu Ser Val
100 105 110
He Met Pro Val Gly Gly Gin Ser Ser Phe Tyr Thr Asp Trp Tyr Gin
115 120 125
Pro Ser Gin Ser Asn Gly Gin Asn Tyr Thr Tyr Lys Trp Glu Thr Phe
130 135 140
Leu Thr Arg Glu Met Pro Ala Trp Leu Gin Ala Asn Lys Gly Val Ser 145 150 155 160
Pro Thr Gly Asn Ala Ala Val Gly Leu Ser Met Ser Gly Gly Ser Ala
165 170 175
Leu He Leu Ala Ala Tyr Tyr Pro Gin Gin Phe Pro Tyr Ala Ala Ser 180 185 190
Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Thr Leu He
195 200 205
Gly Leu Ala Met Asn Asp Ser Gly Gly Tyr Asn Ala Asn Ser Met Trp
210 215 220
Gly Pro Ser Ser Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Gin 225 230 235 240
He Pro Arg Leu Val Ala Asn Asn Thr Arg He Trp Val Tyr Cys Gly
245 250 255
Asn Gly Thr Pro Ser Asp Leu Gly Gly Asp Asn He Pro Ala Lys Phe
260 265 270
Leu Glu Gly Leu Thr Leu Arg Thr Asn Gin Thr Phe Arg Asp Thr Tyr
275 280 285
Ala Ala Asp Gly Gly Arg Asn Gly Val Phe Asn Phe Pro Pro Asn Gly
290 295 300
Thr His Ser Trp Pro Tyr Trp Asn Glu Gin Leu Val Ala Met Lys Ala 305 310 315 320
Asp He Gin His Val Leu Asn Gly Ala Thr Pro Pro Ala Ala Pro Ala
325 330 335
Ala Pro Ala Ala 340
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
AGCGGCTGGG ACATCAACAC 0
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
CAGACGCGGG TGTTGTTGGC 20
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1211 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS- single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: Genomic DNA
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 40-
GGTACCGGAA GCTGGAGGAT TGACGGTATG AGACTTCTTG ACAGGATTCG TGGGCCTTGG 60
GCACGCCGTT TCGGCGTCGT GGCTGTCGCG ACAGCGATGA TGCCTGCTTT GGTGGGCCTG 120
GCTGGAGGGT CGGCGACCGC CGGAGCATTC TCCCGGCCAG GTCTGCCGGT GGAGTACCTG 180
ATGGTGCCTT CGCCGTCGAT GGGGCGCGAC ATCAAGATCC AGTTCCAGAG CGGTGGCGAG 240
AACTCGCCGG CTCTCTACCT GCTCGACGGC CTGCGTGCGC AGGAGGACTT CAACGGCTGG 300
GACATCAACA CTCAGGCTTT CGAGTGGTTC CTCGACAGCG GCATCTCCGT GGTGATGCCG 360
GTCGGTGGCC AGTCCAGCTT CTACACCGAC TGGTACGCCC CCGCCCGTAA CAAGGGCCCG 420
ACCGTGACCT ACAAGTGGGA GACCTTCCTG ACCCAGGAGC TCCCGGGCTG GCTGCAGGCC 480
AACCGCGCGG TCAAGCCGAC CGGCAGCGGC CCTGTCGGTC TGTCGATGGC GGGTTCGGCC 540
GCGCTGAACC TGGCGACCTG GCACCCGGAG CAGTTCATCT ACGCGGGCTC GATGTCCGGC 600
TTCCTGAACC CCTCCGAGGG CTGGTGGCCG TTCCTGATCA ACATCTCGAT GGGTGACGCC 660
GGCGGCTTCA AGGCCGACGA CATGTGGGGC AAGACCGAGG GGATCCCAAC AGCGGTTGGA 720
CAGCGCAACG ATCCGATGCT GAACATCCCG ACCCTGGTCG CCAACAACAC CCGTATCTGG 780
GTCTACTGCG GTAACGGCCA GCCCACCGAG CTCGGCGGCG GCGACCTGCC CGCCACGTTC 840
CTCGAAGGTC TGACCATCCG CACCAACGAG ACCTTCCGCG ACAACTACAT CGCCGCGGGT 900
GGCCACAACG GTGTGTTCAA CTTCCCGGCC AACGGCACGC ACAACTGGGC GTACTGGGGT 960 —.
CGCGAGCTGC AGGCGATGAA GCCTGACCTG CAGGCGCACC TTCTCTGACG GTTGCACGAA 1020
ACGAAGCCCC CGGCCGATTG CGGCCGAGGG TTTCGTCGTC CGGGGCTACT GTGGCCGACA 1080
TAACCGAAAT CAACGCGATG GTGGCTCATC AGGAACGCCG AGGGGGTCAT TGCGCTACGA 1140
CACGAGGTGG GCGAGCAATC CTTCCTGCCC GACGGAGAGG TCAACATCCA CGTCGAGTAC 1200
TCCAGCGTGA A 1211
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 485 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41.
AGCGGCTGGG ACATCAACAC CGCCGCCTTC GAGTGGTACG TCGACTCGGG TCTCGCGGTG 60
ATCATGCCCG TCGGCGGGCA GTCCAGCTTC TACAGCGACT GGTACAGCCC GGCCTGCGGT 120
AAGGCCGGCT GCCAGACCTA CAAGTGGGAG ACGTTCCTGA CCCAGGAGCT GCCGGCCTAC 180
CTCGCCGCCA ACAAGGGGGT CGACCCGAAC CGCAACGCGG CCGTCGGTCT GTCCATGGCC 240
GGTTCGGCGG CGCTGACGCT GGCGATCTAC CACCCGCAGC AGTTCCAGTA CGCCGGGTCG 300
CTGTCGGGCT ACCTGAACCC GTCCGAGGGG TGGTGGCCGA TGCTGATCAA CATCTCGATG 360
GGTGACGCGG GCGGCTACAA GGCCAACGAC ATGTGGGGTC CACCGAAGGA CCCGAGCAGC 420
GCCTGGAAGC GCAACGACCC GATGGTCAAC ATCGGCAAGC TGGTGGCCAA CAACACCCCC 480
CTCTC 485
(2) INFORMATION FOR SEQ ID NO:42:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1052 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(i ) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
GTTGATGAGA AAGGTGGGTT GTTTGCCGTT ATGAAGTTCA CAGAGAAGTG GCGGGGCTCC 60 αCAAAGGCGC-CGATGCACCG GGTGGGCGTT GCCGATATGG CCGCCGTTGC GCTGCCCGGA 120
CTGATCGGCT TCGCCGGGGG TTCGGCAACG GCCGGGGCAT TCTCCCGGCC CGGTCTTCCT 180
GTCGAGTACC TCGACGTGTT CTCGCCGTCG ATGGGCCGCG ACATCCGGGT CCAGTTCCAG 240
GGTGGCGGTA CTCATGCGGT CTACCTGCTC GACGGTCTGC GTGCCCAGGA CGACTACAAC 300
GGCTGGGACA TCAACACCCC TGCGTTCGAG TGGTTCTACG AGTCCGGCTT GTCGACGATC 360
ATGCCGGTCG GCGGACAGTC CAGCTTCTAC AGCGACTGGT ACCAGCCGTC TCGGGGCAAC 420
GGGCAGAACT ACACCTACAA GTGGGAGACG TTCCTGACCC AGGAGCTGCC GACGTGGCTG 480
GAGGCCAACC GCGGAGTGTC GCGCACCGGC AACGCGTTCG TCGGCCTGTC GATGGCGGGC 540
AGCGCGGCGC TGACCTACGC GATCCATCAC CCGCAGCAGT TCATCTACGC CTCGTCGCTG 600 TCAGGCTTCC TGAACCCGTC CGAGGGCTGG TGGCCGATGC TGATCGGGCT GGCGATGAAC 660
GACGCAGGCG GCTTCAACGC CGAGAGCATG TGGGGCCCGT CCTCGGACCC GGCGTGGAAG 720
CGCAACGACC CGATGGTCAA CATCAACCAG CTGGTGGCCA ACAACACCCG GATCTGGATC 780
TACTGCGGCA CCGGCACCCC GTCGGAGCTG GACACCGGGA CCCCGGGCCA GAACCTGATG 840
GCCGCGCAGT TCCTCGAAGG ATTCACGTTG CGGACCAACA TCGCCTTCCG TGACAACTAC 900
ATCGCAGCCG GCGGCACCAA CGGTGTCTTC AACTTCCCGG CCTCGGGCAC CCACAGCTGG 960
GGGTACTGGG GGCAGCAGCT GCAGCAGATG AAGCCCGACA TCCAGCGGGT TCTGGGAGCT 1020
CAGGCCACCG CCTAGCCACC CACCCCACAC CC 1052
(2) INFORMATION FOR SEQ ID NO: 43:
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 326 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Met Arg Leu Leu Asp Arg He Arg Gly Pro Trp Ala Arg Arg Phe Gly
1 5 10 15
Val Val Ala Val Ala Thr Ala Met Met Pro Ala Leu Val Gly Leu Ala
20 25 30
Gly Gly Ser Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val
35 40 45
Glu Tyr Leu Met Val Pro Ser Pro Ser Met Gly Arg Asp He Lys He
50 55 60
Gin Phe Gin Ser Gly Gly Glu Asn Ser Pro Ala Leu Tyr Leu Leu Asp 65 70 75 80
Gly Leu Arg Ala Gin Glu Asp Phe Asn Gly Trp Asp He Asn Thr Gin
85 90 95
Ala Phe Glu Trp Phe Leu Asp Ser Gly He Ser Val Val Met Pro Val
100 105 110
Gly Gly Gin Ser Ser Phe Tyr Thr Asp Trp Tyr Ala Pro Ala Arg Asn
115 120 125
Lys Gly Pro Thr Val Thr Tyr Lys Trp Glu Thr Phe Leu Thr Gin Glu
130 135 140
Leu Pro Gly Trp Leu Gin Ala Asn Arg Ala Val Lys Pro Thr Gly Ser 145 150 155 160
Gly Pro Val Gly Leu Ser Met Ala Gly Ser Ala Ala Leu Asn Leu Ala
165 170 175
Thr Trp His Pro Glu Gin Phe He Tyr Ala Gly Ser Met Ser Gly Phe
180 185 190
Leu Asn Pro Ser Glu Gly Trp Trp Pro Phe Leu He Asn He Ser Met
195 200 205
Gly Asp Ala Gly Gly Phe Lys Ala Asp Asp Met Trp Gly Lys Thr Glu 210 215 220 Gly He Pro Thr Ala Val Gly Gin Arg Asn Asp Pro Met Leu Asn He 225 230 235 240
Pro Thr Leu Val Ala Asn Asn Thr Arg He Trp Val Tyr Cys Gly Asn
245 250 255
Gly Gin Pro Thr Glu Leu Gly Gly Gly Asp Leu Pro Ala Thr Phe Leu
260 265 270
Glu Gly Leu Thr He Arg Thr Asn Glu Thr Phe Arg Asp Asn Tyr He
275 280 285
Ala Ala Gly Gly His Asn Gly Val Phe Asn Phe Pro Ala Asn Gly Thr
290 295 300
His Asn Trp Ala Tyr Trp Gly Arg Glu Leu Gin Ala Met Lys Pro Asp 305 310 315 320
Leu Gin Ala His Leu Leu 325
(2) INFORMATION FOR SEQ ID NO: 44.
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 161 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION. SEQ ID NO:44:
Ser Gly Trp Asp He Asn Thr Ala Ala Phe Glu Trp Tyr Val Asp Ser
1 5 10 15
Gly Leu Ala Val He Met Pro Val Gly Gly Gin Ser Ser Phe Tyr Ser
20 25 30
Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys
35 40 45
Trp Glu Thr Phe Leu Thr Gin Glu Leu Pro Ala Tyr Leu Ala Ala Asn
50 55 60
Lys Gly Val Asp Pro Asn Arg Asn Ala Ala Val Gly Leu Ser Met Ala 65 70 75 80
Gly Ser Ala Ala Leu Thr Leu Ala He Tyr His Pro Gin Gin Phe Gin
85 90 95
Tyr Ala Gly Ser Leu Ser Gly Tyr Leu Asn Pro Ser Glu Gly Trp Trp
100 105 no
Pro Met Leu He Asn He Ser Met Gly Asp Ala Gly Gly Tyr Lys Ala
115 120 125
Asn Asp Met Trp Gly Pro Pro Lys Asp Pro Ser Ser Ala Trp Lys Arg
130 135 140
Asn Asp Pro Met Val Asn He Gly Lys Leu Val Ala Asn Asn Thr Pro 145 150 155 160
Leu
(2) INFORMATION FOR SEQ ID NO: 5:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 334 amino acids
(B) TYPE : ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE- protein
(xi ) SEQUENCE DESCRIPTION. SEQ ID NO 45
Met Lys Phe Thr Glu Lys Trp Arg Gly Ser Ala Lys Ala Ala Met His
1 5 10 15
Arg Val Gly Val Ala Asp Met Ala Ala Val Ala Leu Pro Gly Leu He
20 25 30
Gly Phe Ala Gly Gly Ser Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly
35 40 45
Leu Pro Val Glu Tyr Leu Asp Val Phe Ser Pro Ser Met Gly Arg Asp
50 55 60
He Arg Val Gin Phe Gin Gly Gly Gly Thr His Ala Val Tyr Leu Leu 65 70 75 80
Asp Gly Leu Arg Ala Gin Asp Asp Tyr Asn Gly Trp Asp He Asn Thr
85 90 95
Pro Ala Phe Glu Trp Phe Tyr Glu Ser Gly Leu Ser Thr He Met Pro
100 105 110
Val Gly Gly Gin Ser Ser Phe Tyr Ser Asp Trp Tyr Gin Pro Ser Arg
115 120 125
Gly Asn Gly Gin Asn Tyr Thr Tyr Lys Trp Glu Thr Phe Leu Thr Gin
130 135 140
Glu Leu Pro Thr Trp Leu Glu Ala Asn Arg Gly Val Ser Arg Thr Gly 145 150 155 160
Asn Ala Phe Val Gly Leu Ser Met Ala Gly Ser Ala Ala Leu Thr Tyr
165 170 175
Ala He His His Pro Gin Gin Phe He Tyr Ala Ser Ser Leu Ser Gly
180 185 190
Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Met Leu He Gly Leu Ala
195 200 205
Met Asn Asp Ala Gly Gly Phe Asn Ala Glu Ser Met Trp Gly Pro Ser
210 215 220
Ser Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Asn He Asn Gin 225 230 235 240
Leu Val Ala Asn Asn Thr Arg He Trp He Tyr Cys Gly Thr Gly Thr
245 250 255
Pro Ser Glu Leu Asp Thr Gly Thr Pro Gly Gin Asn Leu Met Ala Ala
260 265 270
Gin Phe Leu Glu Gly Phe Thr Leu Arg Thr Asn He Ala Phe Arg Asp
275 280 285
Asn Tyr He Ala Ala Gly Gly Thr Asn Gly Val Phe Asn Phe Pro Ala
290 295 300
Ser Gly Thr His Ser Trp Gly Tyr Trp Gly Gin Gin Leu Gin Gin Met 305 310 315 320
Lys Pro Asp He Gin Arg Val Leu Gly Ala Gin Ala Thr Ala 325 330
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 795 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: Genomic DNA ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CTGCCGCGGG TTTGCCATCT CTTGGGTCCT GGGTCGGGAG GCCATGTTCT GGGTAACGAT 60
CCGGTACCGT CCGGCGATGT GACCAACATG CGAACAGCGA CAACGAAGCT AGGAGCGGCG 120
CTCGGCGCAG CAGCATTGGT GGCCGCCACG GGGATGGTCA GCGCGGCGAC GGCGAACGCC 180
CAGGAAGGGC ACCAGGTCCG TTACACGCTC ACCTCGGCCG GCGCTTACGA GTTCGACCTG 240
TTCTATCTGA CGACGCAGCC GCCGAGCATG CAGGCGTTCA ACGCCGACGC GTATGCGTTC 300
GCCAAGCGGG AGAAGGTCAG CCTCGCCCCG GGTGTGCCGT GGGTCTTCGA AACCACGATG 360
GCCGACCCGA ACTGGGCGAT CCTTCAGGTC AGCAGCACCA CCCGCGGTGG GCAGGCCGCC 420
CCGAACGCGC ACTGCGACAT CGCCGTCGAT GGCCAGGAGG TGCTCAGCCA GCACGACGAC 480
CCCTACAACG TGCGGTGCCA GCTCGGTCAG TGGTGAGTCA CCTCGCCGAG AGTCCGGCCA 540
GCGCCGGCGG CAGCGGCTCG CGGTGCAGCA CCCCGAGGCG CTGGGTCGCG CGGGTCAGCG 600
CGACGTAAAG ATCGCTGGCC CCGCGCGGCC CCTCGGCGAG GATCTGCTCC GGGTAGACCA 660
CCAGCACGGC GTCTAACTCC AGACCCTTGG TCTGCGTGGG TGCCACCGCG CCCGGGACAC 720
CGGGCGGGCC GATCACCACG CTGGTGCCCT CCCGGTCCGC CTCCGCACGC ACGAAATCGT 780
CGATGGCACC GGCGA 795
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 142 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single D)L TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Met Arg Thr Ala Thr Thr Lys Leu Gly Ala Ala Leu Gly Ala Ala Ala
1 5 10 15
Leu Val Ala Ala Thr Gly Met Val Ser Ala Ala Thr Ala Asn Ala Gin
20 25 30
Glu Gly His Gin Val Arg Tyr Thr Leu Thr Ser Ala Gly Ala Tyr Glu
35 40 45
Phe Asp Leu Phe Tyr Leu Thr Thr Gin Pro Pro Ser Met Gin Ala Phe
50 55 60
Asn Ala Asp Ala Tyr Ala Phe Ala Lys Arg Glu Lys Val Ser Leu Ala
65 70 75 80
Pro Gly Val Pro Trp Val Phe Glu Thr Thr Met Ala Asp Pro Asn Trp
85 90 95
Ala He Leu Gin Val Ser Ser Thr Thr Arg Gly Gly Gin Ala Ala Pro 100 105 HO
Asn Ala His Cys Asp He Ala Val Asp Gly Gin Glu Val Leu Ser Gin
115 120 125
His Asp Asp Pro Tyr Asn Val Arg Cys Gin Leu Gly Gin Trp 130 135 140
(2) INFORMATION FOR SEQ ID NO 48
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 300 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE. Genomic DNA
(xi) SEQUENCE DESCRIPTION SEQ ID NO 48:
GCCAGTGCGC CAACGGTTTT CATCGATGCC GCACACAACC CCGGTGGGCC CTGCGCTTGC 60
CGAAGGCTGC GCGACGAGTT CGACTTCCGG TATCTCGTCG GCGTCGTCTC GGTGATGGGG 120
GACAAGGACG TGGACGGGAT CCGCCAGGAC CCGGGCGTGC CGGACGGGCG CGGTCTCGCA 180
CTGTTCGTCT CGGGCGACAA CCTTCGAAAG GGTGCGGCGC TCAACACGAT CCAGATCGCC 240
GAGCTGCTGG CCGCCCAGTT GTAAGTGTTC CGCCGAAATT GCATTCCACG CCGATAATCG 300
(2) INFORMATION FOR SEQ ID NO -49.
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 563 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(II) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
GGATCCTCGG CCGGCTCAAG AGTCCGCGCC GAGGTGGATG TGACGCTGGA CGGCTACGAG 60
TTCAGTCGGG CCTGCGAGGC GCTGTACCAC TTCGCCTGGG ACGAGTTCTG CGACTGGTAT 120
GTCGAGCTTG CCAAAGTGCA ACTGGGTGAA GGTTTCTCGC ACACCACGGC CGTGTTGGCC 180
ACCGTGCTCG ATGTGCTGCT CAAGCTTCTG CACCCGGTCA TGCCGTTCGT CACCGAGGTG 240
CTGTGGAAGG CCCTGACCGG GCGGGCCGGC GCGAGCGAAC GTCTGGGAAA TGTGGAGTCA 300
CTGGTCGTCG CGGACTGGCC CACGCCCACC GGATACGCGC TGGATCAGGC TGCCGCACAA 360
CGGATCGCCG ACACCCAGAA GTTGATCACC GAGGTGCGCC GGTTCCGCAG CGATCAGGGT 420
CTGGCCGACC GCCAGCGGGT GCCTGCCCGG TTGTCCGGCA TCGACACCGC GGGTCTGGAC 480 GCCCATGTCC CGGCGGTGCG CGCGCTGGCC TGGCTTGACC GAGGGTGATG AGGGCTTCAC 540
CGCGTCCGAA TCGGTCGAGG TGC 563
(2) INFORMATION FOR SEQ ID NO: 50-
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 434 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
GGGCCGGGCC CGAGGATGAG CAAGTTCGAA GTCGTCACCG GGATGGCGTT CGCGGCTTTC 60
GCCGACGCGC CCATCGACGT CGCCGTCGTC GAGGTCGGGC TCGGTGGTCG CTGGGACGCG 120
ACGAACGTGG TGAACGCACC GGTCGCGGTC ATCACCCCGA TCGGGGTGGA CCACACCGAC 180
TACCTCGGTG ACACGATCGC CGAGATCGCC GGGGAGAAGG CCGGAAATCA TCACCCGCCA 240
GCCGACGACC TGGTGCCGAC CGACACCGTC GCCGTGCTGG CGCGGCAGGT TCCCGAGGCC 300
ATGGAGGTGC TGCTGGCCCA GGCGGTGCGC TCGGATGCGG CTGTAGCGCG CGAGGATTCG 360
GAGTGCGCGG TGCTGGGCCG TCAGGTCGCC ATCGGCGGCA GCTGCTCCGG TTGCAGGGGC 420
TCGGTOGCGT CTAC 434
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 438 base pairs (JB). TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
GGATCCCACT CCCGCGCCGG CGGCGGCCAG CTGGTACGGC CATTCCAGCG TGCTGATCGA 60
GGTCGACGGC TACCGCGTGC TGGCCGACCC GGTGTGGAGC AACAGATGTT CGCCCTCACG 120
GGCGGTCGGA CCGCAGCGCA TGCACGACGT CCCGGTGCCG CTGGAGGCGC TTCCCGCCGT 180
GGACGCGGTG GTGATCGCCA ACGACCACTA CGACCACCTC GACATCGACA CCATCGTCGC 240
GTTGGCGCAC ACCCAGCGGG CCCCGTTCGT GGTGCCGTTG GGCATCGGCG CACACCTGCG 300 CAAGTGGGGC GTCCCCGAGG CGCGGATCGT CGAGTTGGAC TGGCACGAAG CCCACCGCAT 360
CGACGACCTG ACGCTGGTCT GCACCCCCGC CCGGCACTTC TCCGGCCGGT TGTTCTCCCG 420
CGACTCGACG CTGTGGGC 438
(2) INFORMATION FOR SEQ ID NO: 52
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 87 amino acids
Figure imgf000089_0001
(C) STRANDEDNESS: Single
(D) TOPOLOGY, linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO: 52-
Ala Ser Ala Pro Thr Val Phe He Asp Ala Ala His Asn Pro Gly Gly
1 5 10 15
Pro Cys Ala Cys Arg Arg Leu Arg Asp Glu Phe Asp Phe Arg Tyr Leu
20 25 30
Val Gly Val Val Ser Val Met Gly Asp Lys Asp Val Asp Gly He Arg
35 40 45
Gin Asp Pro Gly Val Pro Asp Gly Arg Gly Leu Ala Leu Phe Val Ser
50 55 60
Gly Asp Asn Leu Arg Lys Gly Ala Ala Leu Asn Thr He Gin He Ala 65 70 75 80
Glu Leu Leu Ala Ala Gin Leu 85
(2) INFORMATION FOR SEQ ID NO: 53:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 175 ammo acids
(B) TYPE, ammo acid
(C) STRANDEDNESS: single (β). TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Gly Ser Ser Ala Gly Ser Arg Val Arg Ala Glu Val Asp Val Thr Leu
1 5 10 15
Asp Gly Tyr Glu Phe Ser Arg Ala Cys Glu Ala Leu Tyr His Phe Ala
20 25 30
Trp Asp Glu Phe Cys Asp Trp Tyr Val Glu Leu Ala Lys Val Gin Leu
35 40 45
Gly Glu Gly Phe Ser His Thr Thr Ala Val Leu Ala Thr Val Leu Asp
50 55 60
Val Leu Leu Lys Leu Leu His Pro Val Met Pro Phe Val Thr Glu Val 65 70 75 80
Leu Trp Lys Ala Leu Thr Gly Arg Ala Gly Ala Ser Glu Arg Leu Gly
85 90 95
Asn Val Glu Ser Leu Val Val Ala Asp Trp Pro Thr Pro Thr Gly Tyr 100 105 HO
Ala Leu Asp Gin Ala Ala Ala Gin Arg He Ala Asp Thr Gin Lys Leu
115 120 125
He Thr Glu Val Arg Arg Phe Arg Ser Asp Gin Gly Leu Ala Asp Arg
130 135 140
Gin Arg Val Pro Ala Arg Leu Ser Gly He Asp Thr Ala Gly Leu Asp 145 150 155 160
Ala His Val Pro Ala Val Arg Ala Leu Ala Trp Leu Asp Arg Gly 165 170 175
(2) INFORMATION FOR SEQ ID NO : 54
(l) SEQUENCE CHARACTERISTICS-
(A) LENGTH- 144 ammo acids
(B) TYPE, ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY, linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION SEQ ID NO 54
Gly Pro Gly Pro Arg Asn Ser Lys Phe Glu Val Val Thr Gly Met Ala
1 5 10 15
Phe Ala Ala Phe Ala Asp Ala Pro He Asp Val Ala Val Val Glu Val
20 25 30
Gly Leu Gly Gly Arg Trp Asp Ala Thr Asn Val Val Asn Ala Pro Val
35 40 45
Ala Val He Thr Pro He Gly Val Asp His Thr Asp Tyr Leu Gly Asp
50 55 60
Thr He Ala Glu He Ala Gly Glu Lys Ala Gly Asn His His Pro Pro 65 70 75 80
Ala Asp Asp Leu Val Pro Thr Asp Thr Val Ala Val Leu Ala Arg Gin
85 90 95
Val Pro Glu Ala Asn Glu Val Leu Leu Ala Gin Ala Val Arg Ser Asp
100 105 110
Ala Ala Val Ala Arg Glu Asp Ser Glu Cys Ala Val Leu Gly Arg Gin
115 120 125
Val Ala Lie Gly Gly Ser Cys Ser Gly Cys Arg Gly Ser Val Ala Ser 130 135 140
(2) INFORMATION FOR SEQ ID NO .-55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 145 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55.
Asp Pro Thr Pro Ala Pro Ala Ala Ala Ser Trp Tyr Gly His Ser Ser
1 5 10 15
Val Leu He Glu Val Asp Gly Tyr Arg Val Leu Ala Asp Pro Val Trp 20 25 30 Ser Asn Arg Cys Ser Pro Ser Arg Ala Val Gly Pro Gin Arg Met His
35 40 45
Asp Val Pro Val Pro Leu Glu Ala Leu Pro Ala Val Asp Ala Val Val
50 55 60
He Ser Asn Asp His Tyr Asp His Leu Asp He Asp Thr He Val Ala 65 70 75 80
Leu Ala His Thr Gin Arg Ala Pro Phe Val Val Pro Leu Gly He Gly
85 90 95
Ala His Leu Arg Lys Trp Gly Val Pro Glu Ala Arg He Val Glu Leu
100 105 110
Asp Trp His Glu Ala His Arg He Asp Asp Leu Thr Leu Val Cys Thr
115 120 125
Pro Ala Arg His Phe Ser Gly Arg Leu Phe Ser Arg Asp Ser Thr Leu
130 135 140
Trp
145
(2) INFORMATION FOR SEQ ID NO: 56:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 10 am o acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii ) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 1...1
(D) OTHER INFORMATION: Residue can be either Gly, He, Leu or Val
(A) NAME/KEY: Other
(B) LOCATION: 2...2
(D) OTHER INFORMATION: Residue can be either He, Leu, Gly or Ala
(xi-V -SEQUENCE DESCRIPTION: SEQ ID NO: 56
Xaa Xaa Ala Pro Xaa Gly Asp Ala Xaa Arg 1 5 10
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 7...7
(D) OTHER INFORMATION: Residue can be either He or Leu (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Pro Glu Ala Glu Ala Asn Xaa Arg 1 5
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 4...4
(D) OTHER INFORMATION: Residue can be either Gin or Gly
(A) NAME/KEY: Other
(B) LOCATION: 5...5
(D) OTHER INFORMATION: Residue en be either Gly or Gin
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Thr Ala Asn Xaa Xaa Glu Tyr Tyr Asp Asn Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ijj. -MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Asn Ser Pro Arg Ala Glu Ala Glu Ala Asn Leu Arg Gly Tyr Phe Thr
1 5 10 15
Ala Asn Pro Ala Glu Tyr Tyr Asp Leu Arg Gly He Leu Ala Pro He
20 25 30
Gly Asp
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
CCGGTGGGCC CGGGCTGCGC 0
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
TGGCCGGCCA CCACGTGGTA 0
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
GCCGGTGGGC CCGGGCTGCG CGGAATACGC GGCAGCCAAT CCCACTGGGC CGGCCTCGGT 60
GCAGGGAATG TCGCAGGACC CGGTCGCGGT GGCGGCCTCG AACAATCCGG AGTTGACAAC 120
GCTGTACGGC -TGCACTGTCG GGCCAGCTCA ATCCGCAAGT AAACCTGGTG GACACCCTCA 180
ACAGCGGTCA GTACACGGTG TTCGCACCGA CCAACGCGGC ATTTAGCAAG CTGCCGGCAT 240
CCACGATCGA CGAGCTCAAG ACCAATTCGT CACTGCTGAC CAGCATCCTG ACCTACCACG 300
TGGTGGCCGG CCA 313
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Glu Pro Ala Gly Pro Leu Pro Xaa Tyr Asn Glu Arg Leu His Thr Leu
1 5 10 15
Xaa Gin
(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
Gly Leu Asp Asn Glu Leu Ser Leu Val Asp Gly Gin Gly Arg Thr Leu
1 5 10 15
Thr Val Gin Gin Xaa Asp Thr Phe Leu 20 25
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Asp Pro Xaa Pro Asp He Glu Val Glu Phe Ala Arg Gly Thr Gly Ala
1 5 10 15
Glu Pro G-ϊy Leu Xaa Xaa Val Xaa Asp Ala 20 25
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
ACCGCCCTCG AGTTCTCCCG GCCAGGTCTG CC 32
(2) INFORMATION FOR SEQ ID NO: 67: ( l ) SEQUENCE CHARACTERISTICS :
(A) LENGTH 32 base pairs
(B) TYPE : nucleic acid
( C ) STRANDEDNESS . s ingle
( D ) TOPOLOGY , l inear
(a) MOLECULE TYPE: Other
( i) SEQUENCE DESCRIPTION. SEQ ID NO:67
AAGCACGAGC TCAGTCTCTT CCACGCGGAC GT 2
(2) INFORMATION FOR SEQ ID NO -68.
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS- single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION- SEQ ID NO:68:
CATGGATCCA TTCTCCCGGC CCGGTCTTCC 0
(2) INFORMATION FOR SEQ ID NO: 69-
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
TTTGAATTCT AGGCGGTGGC CTGAGC 6
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 161 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: Ser Gly Trp Asp He Asn Thr Ala Ala Phe Glu Trp Tyr Val Asp Ser 1 5 10 15
Gly Leu Ala Val He Met Pro Val Gly Gly Gin Ser Ser Phe Tyr Ser
20 25 30
Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys
35 40 45
Trp Glu Thr Phe Leu Thr Gin Glu Leu Pro Ala Tyr Leu Ala Ala Asn
50 55 60
Lys Gly Val Asp Pro Asn Arg Asn Ala Ala Val Gly Leu Ser Met Ala 65 70 75 80
Gly Ser Ala Ala Leu Thr Leu Ala He Tyr His Pro Gin Gin Phe Gin
85 90 95
Tyr Ala Gly Ser Leu Ser Gly Tyr Leu Asn Pro Ser Glu Gly Trp Trp
100 105 110
Pro Met Leu He Asn He Ser Met Gly Asp Ala Gly Gly Tyr Lys Ala
115 120 125
Asn Asp Met Trp Gly Arg Thr Glu Asp Pro Ser Ser Ala Trp Lys Arg
130 135 140
Asn Asp Pro Met Val Asn He Gly Lys Leu Val Ala Asn Asn Thr Pro 145 150 155 160
Leu
(2) INFORMATION FOR SEQ ID NO.71:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
GAGAGACTCG AGAACGCCCA GGAAGGGCAC CAG 3 ) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
GAGAGACTCG AGTGACTCAC CACTGACCGA GC 32
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS. single
(D) TOPOLOGY, linear
(n) MOLECULE TYPE- Other
(xi ) SEQUENCE DESCRIPTION- SEQ ID NO: 73.
GGNGCNGCNC ARGCNGARCC 0
(2) INFORMATION FOR SEQ ID NO.74
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH 825 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY linear
(a) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 74
TTGGATCCCA CTCCCGCGCC GGCGGCGGCC AGCTGGTACG GCCATTCCAG CGTGCTGATC 60
GAGGTCGACG GCTACCGCGT GCTGGCCGAC CCGGTGTGGA GCAACAGATG TTCGCCCTCA 120
CGGGCGGTCG GACCGCAGCG CATGCACGAC GTCCCGGTGC CGCTGGAGGC GCTTCCCGCC 180
GTGGACGCGG TGGTGATCAG CCACGACCAC TACGACCACC TCGACATCGA CACCATCGTC 240
GCGTTGGCGC ACACCCAGCG GGCCCCGTTC GTGGTGCCGT TGGGCATCGG CGCACACCTG 300
CGCAAGTGGG GCGTCCCCGA GGCGCGGATC GTCGAGTTGG ACTGGCACGA AGCCCACCGC 360
ATAGACGACC TGACGCTGGT CTGCACCCCC GCCCGGCACT TCTCCGGACG GTTGTTCTCC 420
CGCGACTCGA CGCTGTGGGC GTCGTGGGTG GTCACCGGCT CGTCGCACAA GGCGTTCTTC 480
GGTGGCGAGA-CCGGATACAC GAAGAGCTTC GCCGAGATCG GCGACGAGTA CGGTCCGTTC 540
GATCTGACCC TGCTGCCGAT CGGGGCCTAC CATCCCGCGT TCGCCGACAT CCACATGAAC 600
CCCGAGGAGG CGGTGCGCGC CCATCTGGAC CTGACCGAGG TGGACAACAG CCTGATGGTG 660
CCCATCCACT GGGCGACATT CCGCCTCGCC CCGCATCCGT GGTCCGAGCC CGCCGAACGC 720
CTGCTGACCG CTGCCGACGC CGAGCGGGTA CGCCTGACCG TGCCGATTCC CGGTCAGCGG 780
GTGGACCCGG AGTCGACGTT CGACCCGTGG TGGCGGTTCT GAACC 825
(2) INFORMATION FOR SEQ ID NO: 75:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 273 ammo acids
Figure imgf000097_0001
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(11) MOLECULE TYPE: protein
(Xl ) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Leu Asp Pro Thr Pro Ala Pro Ala Ala Ala Ser Trp Tyr Gly His Ser
1 5 10 15
Ser Val Leu He Glu Val Asp Gly Tyr Arg Val Leu Ala Asp Pro Val
20 25 30
Trp Ser Asn Arg Cys Ser Pro Ser Arg Ala Val Gly Pro Gin Arg Met
35 40 45
His Asp Val Pro Val Pro Leu Glu Ala Leu Pro Ala Val Asp Ala Val
50 55 60
Val He Ser His Asp His Tyr Asp His Leu Asp He Asp Thr He Val 65 70 75 80
Ala Leu Ala His Thr Gin Arg Ala Pro Phe Val Val Pro Leu Gly He
85 90 95
Gly Ala His Leu Arg Lys Trp Gly Val Pro Glu Ala Arg He Val Glu
100 105 110
Leu Asp Trp His Glu Ala His Arg He Asp Asp Leu Thr Leu Val Cys
115 120 125
Thr Pro Ala Arg His Phe Ser Gly Arg Leu Phe Ser Arg Asp Ser Thr
130 135 140
Leu Trp Ala Ser Trp Val Val Thr Gly Ser Ser His Lys Ala Phe Phe 145 150 155 160
Gly Gly Asp Thr Gly Tyr Thr Lys Ser Phe Ala Glu He Gly Asp Glu
165 170 175
Tyr Gly Pro Phe Asp Leu Thr Leu Leu Pro He Gly Ala Tyr His Pro
180 185 190
Ala Phe Ala Asp He His Met Asn Pro Glu Glu Ala Val Arg Ala His
195 200 205
Leu Asp Leu Thr Glu Val Asp Asn Ser Leu Met Val Pro He His Trp
210 215 220
Ala Thr Phe Arg Leu Ala Pro His Pro Trp Ser Glu Pro Ala Glu Arg 225 230 235 240
Leu Leu Thr Ala Ala Asp Ala Glu Arg Val Arg Leu Thr Val Pro He
245 250 255
Pro Gly Gin Arg Val Asp Pro Glu Ser Thr Phe Asp Pro Trp Trp Arg
260 265 270
Phe
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Ala Lys Thr He Ala Tyr Asp Glu Glu Ala 1 5 10 (2) INFORMATION FOR SEQ ID NO: 77:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 337 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE: Genomic DNA
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
GATCCCTACA TCCTGCTGGT CAGCTCCAAG GTGTCGACCG TCAAGGATCT GCTCCCGCTG 60
CTGGAGAAGG TCATCCAGGC CGGCAAGCCG CTGCTGATCA TCGCCGAGGA CGTCGAGGGC 120
GAGGCCCTGT CCACGCTGGT GGTCAACAAG ATCCGCGGCA CCTTCAAGTC CGTCGCCGTC 180
AAGGCTCCGG GCTTCGGTGA CCGCCGCAAG GCGATGCTGC AGGACATGGC CATCCTCACC 240
GGTGGTCAGG TCGTCAGCGA AAGAGTCGGG CTGTCCCTGG AGACCGCCGA CGTCTCGCTG 300
CTGGGCCAGG CCCGCAAGGT CGTCGTCACC AAGGACA 337
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
Asp Pro T-ys He Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp
1 5 10 15
Leu Leu Pro Leu Leu Glu Lys Val He Gin Ala Gly Lys Pro Leu Leu
20 25 30
He He Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val Val
35 40 45
Asn Lys He Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly
50 55 60
Phe Gly Asp Arg Arg Lys Ala Met Leu Gin Asp Met Ala He Leu Thr 65 70 75 80
Gly Gly Gin Val Val Ser Glu Arg Val Gly Leu Ser Leu Glu Thr Ala
85 90 95
Asp Val Ser Leu Leu Gly Gin Ala Arg Lys Val Val Val Thr Lys Asp 100 105 110
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 360 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
CCGTACGAGA AGATCGGCGC TGAGCTGGTC AAAGAGGTCG CCAAGAAGAC CGACGACGTC 60
GCGGGCGACG GCACCACCAC CGCCACCGTG CTCGCTCAGG CTCTGGTTCG CGAAGGCCTG 120
CGCAACGTCG CAGCCGGCGC CAACCCGCTC GGCCTCAAGC GTGGCATCGA GAAGGCTGTC 180
GAGGCTGTCA CCCAGTCGCT GCTGAAGTCG GCCAAGGAGG TCGAGACCAA GGAGCAGATT 240
TCTGCCACCG CGGCGATCTC CGCCGGCGAC ACCCAGATCG GCGAGCTCAT CGCCGAGGCC 300
ATGGACAAGG TCGGCAACGA GGGTGTCATC ACCGTCGAGG AGTCGAACAC CTTCGGCCTG 360
(2) INFORMATION FOR SEQ ID NO: 80:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 120 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Pro Tyr Glu Lys He Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys
1 5 10 15
Thr Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala
20 25 30
Gin Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn
3-5" 40 45
Pro Leu Gly Leu Lys Arg Gly He Glu Lys Ala Val Glu Ala Val Thr
50 55 60
Gin Ser Leu Leu Lys Ser Ala Lys Glu Val Glu Thr Lys Glu Gin He 65 70 75 80
Ser Ala Thr Ala Ala He Ser Ala Gly Asp Thr Gin He Gly Glu Leu
85 90 95
He Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val He Thr Val
100 105 110
Glu Glu Ser Asn Thr Phe Gly Leu 115 120
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: Other
(xi ) SEQUENCE DESCRIPTION- SEQ ID NO: 81:
ACTGACGCTG AGGAGCGAAA GCGTGGGGAG CGAACAGGAT TAG 3
(2) INFORMATION FOR SEQ ID NO: 82:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
CGACAAGGAA CTTCGCTACC TTAGGACCGT CATAGTTACG GGC 3
(2) INFORMATION FOR SEQ ID NO: 83:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.-83:
AAAAAAAAAA AAAAAAAAAA 0
__2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
GGAAGGAAGC GGCCGCTTTT TTTTTTTTTT T 1
(2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE. Other
(xi) SEQUENCE DESCRIPTION. SEQ ID NO 85
GAGAGAGAGC CCGGGCATGC TSCTSCTSCT S 1
(2) INFORMATION FOR SEQ ID NO -86
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH- 238 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
In) MOLECULE TYPE. Genomic DNA
(Xl ) SEQUENCE DESCRIPTION: SEQ ID NO -86
CTCGATGAAC CGCTCGGAGC GCTCGACCTG AAGCTGCGCC ACGTCATGCA GTTCGAGCTC 60
AAGCGCATCC AGCGGGAGGT CGGGATCACG TTCATCTACG TGACCCACGA CCAGGAAGAG 120
GCGCTCACGA TGAGTGACCG CATCGCGGTG ATGAACGCCG GCAACGTCGA ACAGATCGGC 180
AGCCCGACCG AGATCTACGA CCGTCCCGCG ACGGTGTTCG TCGCCAGCTT CATCGAAT 238
(2) INFORMATION FOR SEQ ID NO: 87.
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 79 ammo acids
Figure imgf000102_0001
(C) STRANDEDNESS: single (■© TOPOLOGY: linear
(a) MOLECULE TYPE: protein
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
Leu Asp Glu Pro Leu Gly Ala Leu Asp Leu Lys Leu Arg His Val Met
1 5 10 15
Gin Phe Glu Leu Lys Arg He Gin Arg Glu Val Gly He Thr Phe He
20 25 30
Tyr Val Thr His Asp Gin Glu Glu Ala Leu Thr Met Ser Asp Arg He
35 40 45
Ala Val Met Asn Ala Gly Asn Val Glu Gin He Gly Ser Pro Thr Glu
50 55 60
He Tyr Asp Arg Pro Ala Thr Val Phe Val Ala Ser Phe He Glu 65 70 75
(2) INFORMATION FOR SEQ ID NO: 88: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1518 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
CACTCGCCAT GGGTGTTACA ATACCCCACC AGTTCCTCGA AGTAAACGAA CAGAACCGTG 60
ACATCCAGCT GAGAAAATAT TCACAGCGAC GAAGCCCGGC CGATGCCTGA TGGGGTCCGG 120
CATCAGTACA GCGCGCTTTC CTGCGCGGAT TCTATTGTCG AGTCCGGGGT GTGACGAAGG 180
AATCCATTGT CGAAATGTAA ATTCGTTGCG GAATCACTTG CATAGGTCCG TCAGATCCGC 240
GAAGGTTTAC CCCACAGCCA CGACGGCTGT CCCCGAGGAG GACCTGCCCT GACCGGCACA 300
CACATCACCG CTGCAGAACC TGCAGAACAG ACGGCGGATT CCGCGGCACC GCCCAAGGGC 360
GCGCCGGTGA TCGAGATCGA CCATGTCACG AAGCGCTTCG GCGACTACCT GGCCGTCGCG 420
GACGCAGACT TCTCCATCGC GCCCGGGGAG TTCTTCTCCA TGCTCGGCCC GTCCGGGTGT 480
GGGAAGACGA CCACGTTGCG CATGATCGCG GGATTCGAGA CCCCGACTGA AGGGGCGATC 540
CGCCTCGAAG GCGCCGACGT GTCGAGGACC CCACCCAACA AGCGCAACGT CAACACGGTG 600
TTCCAGCACT ACGCGCTGTT CCCGCACATG ACGGTCTGGG ACAACGTCGC GTACGGCCCG 660
CGCAGCAAGA AACTCGGCAA AGGCGAGGTC CGCAAGCGCG TCGACGAGCT GCTGGAGATC 720
GTCCGGCTGA CCGAATTTGC CGAGCGCAGG CCCGCCCAGC TGTCCGGCGG GCAGCAGCAG 780
CGGGTGGCGT TGGCCCGGGC ACTGGTGAAC TACCCCAGCG CGCTGCTGCT CGATGAACCG 840
CTCGGAGCG€ -TCGACCTGAA GCTGCGCCAC GTCATGCAGT TCGAGCTCAA GCGCATCCAG 900
CGGGAGGTCG GGATCACGTT CATCTACGTG ACCCACGACC AGGAAGAGGC GCTCACGATG 960
AGTGACCGCA TCGCGGTGAT GAACGCCGGC AACGTCGAAC AGATCGGCAG CCCGACCGAG 1020
ATCTACGACC GTCCCGCGAC GGTGTTCGTC GCCAGCTTCA TCGGACAGGC CAACCTCTGG 1080
GCGGGCCGGT GCACCGGCCG CTCCAACCGC GATTACGTCG AGATCGACGT TCTCGGCTCG 1140
ACGCTGAAGG CACGCCCGGG CGAGACCACG ATCGAGCCCG GCGGGCACGC CACCCTGATG 1200
GTGCGTCCGG AACGCATCCG GGTCACCCCG GGCTCCCAGG ACGCGCCGAC CGGTGACGTC 1260
GCCTGCGTGC GTGCCACCGT CACCGACCTG ACCTTCCAAG GTCCGGTGGT GCGGCTCTCG 1320
CTGGCCGCTC CGGACGACTC GACCGTGATC GCCCACGTCG GCCCCGAGCA GGATCTGCCG 1380 CTGCTGCGCC CCGGCGACGA CGTGTACGTC AGCTGGGCAC CGGAAGCCTC CCTGGTGCTT 1440
CCCGGCGACG ACATCCCCAC CACCGAGGAC CTCGAAGAGA TGCTCGACGA CTCCTGAGTC 1500
ACGCTTCCCG ATTGCCGA 1518
(2) INFORMATION FOR SEQ ID NO: 89.
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 376 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Val He Glu He Asp His Val Thr Lys Arg Phe Gly Asp Tyr Leu Ala
1 5 10 15
Val Ala Asp Ala Asp Phe Ser He Ala Pro Gly Glu Phe Phe Ser Met
20 25 30
Leu Gly Pro Ser Gly Cys Gly Lys Thr Thr Thr Leu Arg Met He Ala
35 40 45
Gly Phe Glu Thr Pro Thr Glu Gly Ala He Arg Leu Glu Gly Ala Asp
50 55 60
Val Ser Arg Thr Pro Pro Asn Lys Arg Asn Val Asn Thr Val Phe Gin 65 70 75 80
His Tyr Ala Leu Phe Pro His Met Thr Val Trp Asp Asn Val Ala Tyr
85 90 95
Gly Pro Arg Ser Lys Lys Leu Gly Lys Gly Glu Val Arg Lys Arg Val
100 105 110
Asp Glu Leu Leu Glu He Val Arg Leu Thr Glu Phe Ala Glu Arg Arg
115 120 125
Pro Ala Gin Leu Ser Gly Gly Gin Gin Gin Arg Val Ala Leu Ala Arg
130 135 140
Ala Leu Val Asn Tyr Pro Ser Ala Leu Leu Leu Asp Glu Pro Leu Gly 145 150 155 160
Ala Leu Asp Leu Lys Leu Arg His Val Met Gin Phe Glu Leu Lys Arg
165 170 175
He Gin Arg Glu Val Gly He Thr Phe He Tyr Val Thr His Asp Gin
180 185 190
Glu Glu Ala Leu Thr Met Ser Asp Arg He Ala Val Met Asn Ala Gly
195 200 205
Asn Val Glu Gin He Gly Ser Pro Thr Glu He Tyr Asp Arg Pro Ala
210 215 220
Thr Val Phe Val Ala Ser Phe He Gly Gin Ala Asn Leu Trp Ala Gly 225 230 235 240
Arg Cys Thr Gly Arg Ser Asn Arg Asp Tyr Val Glu He Asp Val Leu
245 250 255
Gly Ser Thr Leu Lys Ala Arg Pro Gly Glu Thr Thr He Glu Pro Gly
260 265 270
Gly His Ala Thr Leu Met Val Arg Pro Glu Arg He Arg Val Thr Pro
275 280 285
Gly Ser Gin Asp Ala Pro Thr Gly Asp Val Ala Cys Val Arg Ala Thr 290 295 300 Val Thr Asp Leu Thr Phe Gin Gly Pro Val Val Arg Leu Ser Leu Ala 305 310 315 320
Ala Pro Asp Asp Ser Thr Val He Ala His Val Gly Pro Glu Gin Asp
325 330 335
Leu Pro Leu Leu Arg Pro Gly Asp Asp Val Tyr Val Ser Trp Ala Pro
340 345 350
Glu Ala Ser Leu Val Leu Pro Gly Asp Asp He Pro Thr Thr Glu Asp
355 360 365
Leu Glu Glu Met Leu Asp Asp Ser 370 375
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
GAGAGACTCG AGGTGATCGA GATCGACCAT GTC 3
(2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
AGAGACTCGA -GCAATCGGGA AGCGTGACTC A
31
(2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 323 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO : 92 :
GTCGACTACA AAGAAGACTT CAACGACAAC GAGCAGTGGT TCGCCAAGGT CAAGGAGCCG 60
TTGTCGCGCA AGCAGGACAT AGGCGCCGAC CTGGTGATCC CCACCGAGTT CATGGCCGCG 120 CGCGTCAAGG GCCTGGGATG GCTCAATGAG ATCAGCGAAG CCGGCGTGCC CAATCGCAAG
180 AATCTGCGTC AGGACCTGTT GGACTCGAGC ATCGACGAGG GCCGCAAGTT CACCGCGCCG
240
TACATGACCG GCATGGTCGG TCTCGCCTAC AACAAGGCAG CCACCGGACG CGATATCCGC 300
ACCATCGACG ACCTCTGGGA TCC 323
(2) INFORMATION FOR SEQ ID NO -93:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 1341 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3
CCCCACCCCC TTCCCTGGAG CCGACGAAAG GCACCCGCAC ATGTCCCGTG ACATCGATCC 60
CCACCTGCTG GCCCGAATGA CCGCACGCCG CACCTTGCGT CGCCGCTTCA TCGGCGGTGG 120
CGCCGCGGCC GCCGCGGGCC TGACCCTCGG TTCGTCGTTC CTGGCGGCGT GCGGGTCCGA 180
CAGTGGGACC TCGAGCACCA CGTCACAGGA CAGCGGCCCC GCCAGCGGCG CCCTGCGCGT 240
CTCCAACTGG CCGCTCTATA TGGCCGACGG TTTCATCGCA GCGTTCCAGA CCGCCTCGGG 300
CATCACGGTC GACTACAAAG AAGACTTCAA CGACAACGAG CAGTGGTTCG CCAAGGTCAA 360
GGAGCCGTTG TCGCGCAAGC AGGACATAGG CGCCGACCTG GTGATCCCCA CCGAGTTCAT 420
GGCCGCGCGC GTCAAGGGCC TGGGATGGCT CAATGAGATC AGCGAAGCCG GCGTGCCCAA 480
TCGCAAGAAT CTGCGTCAGG ACCTGTTGGA CTCGAGCATC GACGAGGGCC GCAAGTTCAC 540
CGCGCCGTAC ATGACCGGCA TGGTCGGTCT CGCCTACAAC AAGGCAGCCA CCGGACGCGA 600
TATCCGCACC ATCGACGACC TCTGGGATCC CGCGTTCAAG GGCCGCGTCA GTCTGTTCTC 660
CGACGTCCAG GACGGCCTCG GCATGATCAT GCTCTCGCAG GGCAACTCGC CGGAGAATCC 720
GACCACCGAG TCCATTCAGC AGGCGGTCGA TCTGGTCCGC GAACAGAACG ACAGGGGGTC 780
AGATCCGTCG CTTCACCGGC AACGACTACG CCGACGACCT GGCCGCAGAA ACATCGCCAT 840
CGCGCAGGCG TACTCCGGTG ACGTCGTGCA GCTGCAGGCG GACAACCCCG ATCTGCAGTT 900
CATCGTTCCC GAATCCGGCG GCGACTGGTT CGTCGACACG ATGGTGATCC CGTACACCAC 960
GCAGAACCAG AAGGCCGCCG AGGCGTGGAT CGACTACATC TACGACCGAG CCAACTACGC 1020
CAAGCTGGTC GCGTTCACCC AGTTCGTGCC CGCACTCTCG GACATGACCG ACGAACTCGC 1080 CAAGGTCGAT CCTGCATCGG CGGAGAACCC GCTGATCAAC CCGTCGGCCG AGGTGCAGGC 1140
GAACCTGAAG TCGTGGGCGG CACTGACCGA CGAGCAGACG CAGGAGTTCA ACACTGCGTA 1200
CGCCGCCGTC ACCGGCGGCT GACGCGGTGG TAGTGCCGAT GCGAGGGGCA TAAATGGCCC 1260
TGCGGACGCG AGGAGCATAA ATGGCCGGTG TCGCCACCAG CAGCCGTCAG CGGACAAGGT 1320
CGCTCCGTAT CTGATGGTCC T 1341
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
Figure imgf000107_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
Met Ser Arg Asp He Asp Pro His Leu Leu Ala Arg Met Thr Ala Arg
1 5 10 15
Arg Thr Leu Arg Arg Arg Phe He Gly Gly Gly Ala Ala Ala Ala Ala
20 25 30
Gly Leu Thr Leu Gly Ser Ser Phe Leu Ala Ala Cys Gly Ser Asp Ser
35 40 45
Gly Thr Ser Ser Thr Thr Ser Gin Asp Ser Gly Pro Ala Ser Gly Ala
50 55 60
Leu Arg Val Ser Asn Trp Pro Leu Tyr Met Ala Asp Gly Phe He Ala 65 70 75 80
Ala Phe Gin Thr Ala Ser Gly He Thr Val Asp Tyr Lys Glu Asp Phe
85 90 95
Asn Asp Asn Glu Gin Trp Phe Ala Lys Val Lys Glu Pro Leu Ser Arg
100 105 110
Lys Gin Asp He Gly Ala Asp Leu Val He Pro Thr Glu Phe Met Ala
U5 120 125
Ala Arg Val Lys Gly Leu Gly Trp Leu Asn Glu He Ser Glu Ala Gly
130 135 140
Val Pro Asn Arg Lys Asn Leu Arg Gin Asp Leu Leu Asp Ser Ser He 145 ISO 155 160
Asp Glu Gly Arg Lys Phe Thr Ala Pro Tyr Met Thr Gly Met Val Gly
165 170 175
Leu Ala Tyr Asn Lys Ala Ala Thr Gly Arg Asp He Arg Thr He Asp
180 185 190
Asp Leu Trp Asp Pro Ala Phe Lys Gly Arg Val Ser Leu Phe Ser Asp
195 200 205
Val Gin Asp Gly Leu Gly Met He Met Leu Ser Gin Gly Asn Ser Pro
210 215 220
Glu Asn Pro Thr Thr Glu Ser He Gin Gin Ala Val Asp Leu Val Arg 225 230 235 240
Glu Gin Asn Asp Arg Gly Ser Asp Pro Ser Leu His Arg Gin Arg Leu
245 250 255
Arg Arg Arg Pro Gly Arg Arg Asn He Ala He Ala Gin Ala Tyr Ser 260 265 270 Gly Asp Val Val Gin Leu Gin Ala Asp Asn Pro Asp Leu Gin Phe He
275 280 285
Val Pro Glu Ser Gly Gly Asp Trp Phe Val Asp Thr Met Val He Pro
290 295 300
Tyr Thr Thr Gin Asn Gin Lys Ala Ala Glu Ala Trp He Asp Tyr He 305 310 315 320
Tyr Asp Arg Ala Asn Tyr Ala Lys Leu Val Ala Phe Thr Gin Phe Val
325 330 335
Pro Ala Leu Ser Asp Met Thr Asp Glu Leu Ala Lys Val Asp Pro Ala
340 345 350
Ser Ala Glu Asn Pro Leu He Asn Pro Ser Ala Glu Val Gin Ala Asn
355 360 365
Leu Lys Ser Trp Ala Ala Leu Thr Asp Glu Gin Thr Gin Glu Phe Asn
370 375 380
Thr Ala Tyr Ala Ala Val Thr Gly Gly 385 390
(2) INFORMATION FOR SEQ ID NO 95
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 22 base pairs
(B) TYPE, nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(u) MOLECULE TYPE Other
(xi ) SEQUENCE DESCRIPTION SEQ ID NO: 95
ATGTCCCGTG ACATCGATCC CC 2
(2) INFORMATION FOR SEQ ID NO 96
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single CD). TOPOLOGY, linear
(n) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
ATCGGCACTA CCACCGCGTC A
21
(2) INFORMATION FOR SEQ ID NO : 97
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH- 861 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS- single
(D) TOPOLOGY linear
(a) MOLECULE TYPE. Genomic DNA (xi) SEQUENCE DESCRIPTION SEQ ID NO 97
GCCGGCGCTC GCATATCTCG CGATCTTCTT CCGTGGTGCC GTTCTTCTCG CTGGCACGCA 60
CCTCGTTGTC GGAGACCGGC GGCTCGGTGT TCATGCCGAC GCTGACGTTC GCCTGGGACT 120
TCGGCAACTA CGTCGACGCG TTCACGATGT ACCACGAGCA GATCTTCCGC TCGTTCGGCT 180
ACGCGTTCGT CGCCACGGTG CTGTGCCTGT TGCTGGCGTT CCCGCTGGCC TACGTCATCG 240
CGTTCAAGGC CGGCCGGTTC AAGAACCTGA TCCTGGGGCT GGTGATCCTG CCGTTCTTCG 300
TCACGTTCCT GATCCGCACC ATTGCGTGGA AGACGATCCT GGCCGACGAA GGCTGGGTGG 360
TCACCGCGCT GGGCGCCATC GGGCTGCTGC CTGACGAGGG CCGGCTGCTG TCCACCAGCT 420
GGGCGGTCAT CGGCGGTCTG ACCTACAACT GGATCATCTT CATGATCCTG CCGCTGTACG 480
TCAGCCTGGA GAAGATCGAC CCGCGTCTGC TGGAGGCCTC CCAGGACCTC TACTCGTCGG 540
CGCCGCGCAG CTTCGGCAAG GTGATCCTGC CGATGGCGAT GCCCGGGGTG CTGGCCGGGA 600
GCATGCTGGT GTTCATCCCG GCCGTCGGCG ACTTCATCAA CGCCGACTAT CTCGGCAGTA 660
CCCAGACCAC CATGATCGGC AACGTGATCC AGAAGCAGTT CCTGGTCGTC AAGGACTATC 720
CGGCGGCGGC CGCGCTGAGT CTGGGGCTGA TGTTGCTGAT CCTGATCGGC GTGCTCCTCT 780
ACACACGGGC GCTGGGTTCG GAGGATCTGG TATGACCACC CAGGCAGGCG CCGCACTGGC 840
CACCGCCGCC CAGCAGGATC C 861
(2) INFORMATION FOR SEQ ID NO.98
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH. 259 ammo acids
(B) TYPE: amino acid Sr STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 98
Val Val Pro Phe Phe Ser Leu Ala Arg Thr Ser Leu Ser Glu Thr Gly
1 5 ιo 15
Gly Ser Val Phe Met Pro Thr Leu Thr Phe Ala Trp Asp Phe Gly Asn
20 25 30
Tyr Val Asp Ala Phe Thr Met Tyr His Glu Gin He Phe Arg Ser Phe
35 40 45
Gly Tyr Ala Phe Val Ala Thr Val Leu Cys Leu Leu Leu Ala Phe Pro
50 55 60
Leu Ala Tyr Val He Ala Phe Lys Ala Gly Arg Phe Lys Asn Leu He 65 70 75 80
Leu Gly Leu Val He Leu Pro Phe Phe Val Thr Phe Leu He Arg Thr 85 90 95 He Ala Trp Thr He Leu Ala Asp Glu Gly Trp Val Val Thr Ala Leu
100 105 °
Gly Ala He Gly Leu Leu Pro Asp Glu Gly Arg Leu Leu Ser Thr Ser
115 120 125
Trp Ala Val He Gly Gly Leu Thr Tyr Asn Trp He He Phe Met He
130 135 140
Leu Pro Leu Tyr Val Ser Leu Glu Lys He Asp Pro Arg Leu Leu Glu 145 150 155 160
Ala Ser Gin Asp Leu Tyr Ser Ser Ala Pro Arg Ser Phe Gly Lys Val
165 170 175
He Leu Pro Met Ala Met Pro Gly Val Leu Ala Gly Ser Met Leu Val
180 185 190
Phe He Pro Ala Val Gly Asp Phe He Asn Ala Asp Tyr Leu Gly Ser
195 200 205
Thr Gin Thr Thr Met He Gly Asn Val He Gin Lys Gin Phe Leu Val
210 215 220
Val Lys Asp Tyr Pro Ala Ala Ala Ala Leu Ser Leu Gly Leu Met Leu 225 230 235 240
Leu He Leu He Gly Val Leu Leu Tyr Thr Arg Ala Leu Gly Ser Glu
245 250 255
Asp Leu Val
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 277 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
GTAATCTTTG CTGGAGCCCG TACGCCGGTA GGCAAACTCA TGGGTTCGCT CAAGGACTTC 60
AAGGGCAGCG ATCTCGGTGC CGTGGCGATC AAGGGCGCCC TGGAGAAAGC CTTCCCCGGC 120
GTCGACGACC CTGCTCGTCT CGTCGAGTAC GTGATCATGG GCCAAGTGCT CTCCGCCGGC 180
GCCGGCCAGA TGCCCGCCCG CCAGGCCGCC GTCGCCGCCG GCATCCCGTG GGACGTCGCC 240
TCGCTGACGA TCAACAAGAT GTGCCTGTCG GGCATCG 277
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: Val He Phe Ala Gly Ala Arg Thr Pro Val Gly Lys Leu Met Gly Ser
1 5 10 15
Leu Lys Asp Phe Lys Gly Ser Asp Leu Gly Ala Val Ala He Lys Gly
20 25 30
Ala Leu Glu Lys Ala Phe Pro Gly Val Asp Asp Pro Ala Arg Leu Val
35 40 45
Glu Tyr Val He Met Gly Gin Val Leu Ser Ala Gly Ala Gly Gin Met
50 55 60
Pro Ala Arg Gin Ala Ala Val Ala Ala Gly He Pro Trp Asp Val Ala 65 70 75 80
Ser Leu Thr He Asn Lys Met Cys Leu Ser Gly He 85 90
(2) INFORMATION FOR SEQ ID NO.101
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 12 ammo acids
(B) TYPE amino acid
(C) STRANDEDNESS- single
(D) TOPOLOGY linear
In) MOLECULE TYPE: protein (ix) FEATURE.
(A) NAME/KEY- Other
(B) LOCATION: 1...1
(D) OTHER INFORMATION: Residue can be either Glu or Pro
(A) NAME/KEY. Other
(B) LOCATION: 2...2
(D) OTHER INFORMATION: Residue can be either Pro or Glu
(xi) SEQUENCE DESCRIPTION. SEQ ID NO.101:
Xaa Xaa Ala Asp Arg Gly Xaa Ser Lys Tyr Arg Xaa 1 5 10
<2) INFORMATION FOR SEQ ID NO: 102:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: am o acid
(C) STRANDEDNESS- single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE: protein
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:102.
Xaa He Asp Glu Ser Leu Phe Asp Ala Glu Glu Lys Met Glu Lys Ala
1 5 10 15
Val Ser Val Ala Arg Asp Ser Ala 20
( 2 ) INFORMATION FOR SEQ ID NO . 103 . (I) SEQUENCE CHARACTERISTICS
(A) LENGTH 23 ammo acids
(B) TYPE amino ac d
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(II) MOLECULE TYPE protein
(Xl ) SEQUENCE DESCRIPTION SEQ ID NO 103
Xaa Xaa He Ala Pro Ala Thr Ser Gly Thr Leu Ser Glu Phe Xaa Ala
1 5 10 15
Xaa Lys Gly Val Thr Met Glu 20
(2) INFORMATION FOR SEQ ID NO 104
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 15 ammo acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE protein
( i) SEQUENCE DESCRIPTION SEQ ID NO 104
Pro Asn Val Pro Asp Ala Phe Ala Val Leu Ala Asp Arg Val Gly 1 5 10 15
(2) INFORMATION FOR SEQ ID NO 105
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 9 ammo acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ιι+ -MOLECULE TYPE protein
(XI ) SEQUENCE DESCRIPTION SEQ ID NO 105
Xaa He Arg Val Gly Val Asn Gly Phe 1 5
(2) INFORMATION FOR SEQ ID NO: 106
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 485 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ll) MOLECULE TYPE cDNA
(xi) SEQUENCE DESCRIPTION SEQ ID NO 106
10 AGCGGCTGGG ACATCAACAC CGCCGCCTTC GAGTGGTACG TCGACTCGGG TCTCGCGGTG
60 ATCATGCCCG TCGGCGGGCA GTCCAGCTTC TACAGCGACT GGTACAGCCC GGCCTGCGGT
120 AAGGCCGGCT GCCAGACCTA CAAGTGGGAG ACGTTCCTGA CCCAGGAGCT GCCGGCCTAC
180
CTCGCCGCCA ACAAGGGGGT CGACCCGAAC CGCAACGCGG CCGTCGGTCT GTCCATGGCC 240
GGTTCGGCGG CGCTGACGCT GGCGATCTAC CACCCGCAGC AGTTCCAGTA CGCCGGGTCG 300
CTGTCGGGCT ACCTGAACCC GTCCGAGGGG TGGTGGCCGA TGCTGATCAA CATCTCGATG 360
GGTGACGCGG GCGGCTACAA GGCCAACGAC ATGTGGGGTC GCACCGAGGA CCCGAGCAGC 420
GCCTGGAAGC GCAACGACCC GATGGTCAAC ATCGGCAAGC TGGTCGCCAA CAACACCCCC 480
CTCTC 485
(2) INFORMATION FOR SEQ ID NO: 107:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
ATGCCGGTGC GACGTGCGCG CAGTGCGCTT GCGTCCGTGA CCTTCGTCGC GGCCGCGTGC 60
GTGGGCGCTG AGGGCACCGC ACTGGCGGCG ACGCCGGACT GGAGCGGGCG CTACACGGTG 120
GTGACGTTCG CCTCCGACAA ACTCGGCACG AGTGTGGCCG CCCGCCAGCC AGAACCCGAC 180
TTCAGCGGTC AGTACACCTT CAGCACGTCC TGTGTGGGCA CCTGCGTGGC CACCGCGTCC 240
GACGGCCCGG CGCCGTCGAA CCCGACGATT CCGCAGCCCG CGCGCTACAC CTGGGACGGC 300
AGGCAGTGGT; "TGTTCAACTA CAACTGGCAG TGGGAGTGCT TCCGCGGCGC CGACGTCCCG 360
CGCGAGTACG CCGCCGCGCG TTCGCTGGTG TTCTACGCCC CGACCGCCGA CGGGTCGATG 420
TTCGGCACCT GGCGCACCGA NATCCTGGAN GGCCTCTGCA AGGGCACCGT GATCATGCCG 480
GTCGCGGCCT ATCCGGCGTA G 501
(2) INFORMATION FOR SEQ ID NO: 108:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:
11 ATGAACCAGC CGCGGCCCGA GGCCGAGGCG AACCTGCGGG GCTACTTCAC CGCCAACCCG 60
GCGGAGTACT ACGACCTGCG GGGCATCCTC GCCCCGATCG GTGACGCGCA GCGCAACTGC 120
AACATCACCG TGCTGCCGGT AGAGCTGCAG ACGGCCTACG ACACGTTCAT GGCCGGCTGA 180
(2) INFORMATION FOR SEQ ID NO: 109:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:
Met Pro Val Arg Arg Ala Arg Ser Ala Leu Ala Ser Val Thr Phe Val
1 5 10 15
Ala Ala Ala Cys Val Gly Ala Glu Gly Thr Ala Leu Ala Ala Thr Pro
20 25 30
Asp Trp Ser Gly Arg Tyr Thr Val Val Thr Phe Ala Ser Asp Lys Leu
35 40 45
Gly Thr Ser Val Ala Ala Arg Gin Pro Glu Pro Asp Phe Ser Gly Gin
50 55 60
Tyr Thr Phe Ser Thr Ser Cys Val Gly Thr Cys Val Ala Thr Ala Ser 65 70 75 80
Asp Gly Pro Ala Pro Ser Asn Pro Thr He Pro Gin Pro Ala Arg Tyr
85 90 95
Thr Trp Asp Gly Arg Gin Trp Val Phe Asn Tyr Asn Trp Gin Trp Glu
100 105 110
Cys Phe Arg Gly Ala Asp Val Pro Arg Glu Tyr Ala Ala Ala Arg Ser
115 120 125
Leu Val Phe Tyr Ala Pro Thr Ala Asp Gly Ser Met Phe Gly Thr Trp
130 135 140
Arg Thr Xaa He Leu Xaa Gly Leu Cys Lys Gly Thr Val He Met Pro 145 150 155 160
Val Ala Ala Tyr Pro Ala 165
(2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 74 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:
Pro Arg Asp Thr His Pro Gly Ala Asn Gin Ala Val Thr Ala Ala Met
1 5 10 15
Asn Gin Pro Arg Pro Glu Ala Glu Ala Asn Leu Arg Gly Tyr Phe Thr
20 25 30
Ala Asn Pro Ala Glu Tyr Tyr Asp Leu Arg Gly He Leu Ala Pro He
35 40 45
Gly Asp Ala Gin Arg Asn Cys Asn He Thr Val Leu Pro Val Glu Leu
50 55 60
Gin Thr Ala Tyr Asp Thr Phe Met Ala Gly 65 70 (2) INFORMATION FOR SEQ ID NO : 111 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 503 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID MO : 111 :
ATGCAGGTGC GGCGTGTTCT GGGCAGTGTC GGTGCAGCAG TCGCGGTTTC GGCCGCGTTA 60
TGGCAGACGG GGGTTTCGAT ACCGACCGCC TCAGCGGATC CGTGTCCGGA CATCGAGGTG 120
ATCTTCGCGC GCGGGACCGG TGCGGAACCC GGCCTCGGGT GGGTCGGTGA TGCGTTCGTC 180
AACGCGCTGC GGCCCAAGGT CGGTGAGCAG TCGGTGGGCA CCTACGCGGT GAACTACCCG 240
GCAGGATTCG GACTTCGACA AATCGGCGCC CATGGGCGCG GCCGACGCAT CGGGGCGGGT 300
GCAGTGGATG GCCGACAACT GCCCGGACAC CAAGCTTGTC CTGGGCGGCA TGTCGCANGG 360
CGCCGGCGTC ATCGACCTGA TCACCGTCGA TCCGCGACCG CTGGGCCGGT TCACCCCCAC 420
CCCGATGCCG CCCCGCGTCG CCGACCACGT GGCCGCCGTT GTGGTCTTCG GAAATCCGTT 480
GCGCGACATC CGTGGTGGCG GTC 503
(2) INFORMATION FOR SEQ ID NO: 112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 167 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
Met Gin V.al Arg Arg Val Leu Gly Ser Val Gly Ala Ala Val Ala Val
1 5 10 15
Ser Ala Ala Leu Trp Gin Thr Gly Val Ser He Pro Thr Ala Ser Ala
20 25 30
Asp Pro Cys Pro Asp He Glu Val He Phe Ala Arg Gly Thr Gly Ala
35 40 45
Glu Pro Gly Leu Gly Trp Val Gly Asp Ala Phe Val Asn Ala Leu Arg
50 55 60
Pro Lys Val Gly Glu Gin Ser Val Gly Thr Tyr Ala Val Asn Tyr Pro 65 70 75 80
Ala Gly Phe Asp Phe Asp Lys Ser Ala Pro Met Gly Ala Ala Asp Ala
85 90 95
Ser Gly Arg Val Gin Trp Met Ala Asp Asn Cys Pro Asp Thr Lys Leu
100 105 110
Val Leu Gly Gly Met Ser Xaa Gly Ala Gly Val He Asp Leu He Thr
115 120 125
Val Asp Pro Arg Pro Leu Gly Arg Phe Thr Pro Thr Pro Met Pro Pro
130 135 140
Arg Val Ala Asp His Val Ala Ala Val Val Val Phe Gly Asn Pro Leu 145 150 155 160
Arg Asp He Arg Gly Gly Gly 165 ( 2 ) I N FORMAT ION FOR SEQ I D NO : 113 :
( l ) S EQUENCE CHARACTERI ST ICS :
( A ) LENGTH : 1569 base pai rs
( B ) TYPE : nucle i c acid
( C ) STRANDEDNESS : s ingl e
( D ) TOPOLOGY : l inear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:
ATGGCCAAGA CAATTGCGTA TGACGAAGAG GCCCGCCGTG GCCTCGAGCG GGGCCTCAAC 60
GCCCTCGCAG ACGCCGTAAA GGTGACGTTG GGCCCGAAGG GTCGCAACGT CGTGCTGGAG 120
AAGAAGTGGG GCGCCCCCAC GATCACCAAC GATGGTGTGT CCATCGCCAA GGAGATCGAG 180
CTGGAGGACC CGTACGAGAA GATCGGCGCT GAGCTGGTCA AAGAGGTCGC CAAGAAGACC 240
GACGACGTCG CGGGCGACGG CACCACCACC GCCACCGTGC TCGCTCAGGC TCTGGTTCGC 300
GAAGGCCTGC GCAACGTCGC AGCCGGCGCC AACCCGCTCG GCCTCAAGCG TGGCATCGAG 360
AAGGCTGTCG AGGCTGTCAC CCAGTCGCTG CTGAAGTCGG CCAAGGAGGT CGAGACCAAG 420
GAGCAGATTT CTGCCACCGC GGCGATTTCC GCCGGCGACA CCCAGATCGG CGAGCTCATC 480
GCCGAGGCCA TGGACAAGGT CGGCAACGAG GGTGTCATCA CCGTCGAGGA GTCGAACACC 540
TTCGGCCTGC AGCTCGAGCT CACCGAGGGT ATGCGCTTCG ACAAGGGCTA CATCTCGGGT 600
TACTTCGTGA CCGACGCCGA GCGCCAGGAA GCCGTCCTGG AGGATCCCTA CATCCTGCTG 660
GTCAGCTCCA AGGTGTCGAC CGTCAAGGAT CTGCTCCCGC TGCTGGAGAA GGTCATCCAG 720
GCCGGCAAGC CGCTGCTGAT CATCGCCGAG GACGTCGAGG GCGAGGCCCT GTCCACGCTG 780
GTGGTCAACA AGATCCGCGG CACCTTCAAG TCCGTCGCCG TCAAGGCTCC GGGCTTCGGT 840
GACCGCCGCA AGGCGATGCT GCAGGACATG GCCATCCTCA CCGGTGGTCA GGTCGTCAGC 900
GAAAGAGTCG GGCTGTCCCT GGAGACCGCC GACGTCTCGC TGCTGGGCCA GGCCCGCAAG 960 "~"
GTCGTCGTCA CCAAGGACGA GACCACCATC GTCGAGGGCT CGGGCGATTC CGATGCCATC 1020
GCCGGCCGGG TGGCTCAGAT CCGCGCCGAG ATCGAGAACA GCGACTCCGA CTACGACCGC 1080
GAGAAGCTGC AGGAGCGCCT GGCCAAGCTG GCCGGCGGTG TTGCGGTGAT CAAGGCCGGA 1140
GCTGCCACCG AGGTGGAGCT CAAGGAGCGC AAGCACCGCA TCGAGGACGC CGTCCGCAAC 1200
GCGAAGGCTG CCGTCGAAGA GGGCATCGTC GCCGGTGGCG GCGTGGCTCT GCTGCAGTCG 1260
GCTCCTGCGC TGGACGACCT CGGCCTGACG GGCGACGAGG CCACCGGTGC CAACATCGTC 1320
CGCGTGGCGC TGTCGGCTCC GCTCAAGCAG ATCGCCTTCA ACGGCGGCCT GGAGCCCGGC 1380
GTCGTTGCCG AGAAGGTGTC CAACCTGCCC GCGGGTCACG GCCTCAACGC CGCGACCGGT 1440
GAGTACGAGG ACCTGCTCAA GGCCGGCGTC GCCGACCCGG TGAAGGTCAC CCGCTCGGCG 1500
CTGCAGAACG CGGCGTCCAT CGCGGCTCTG TTCCTCACCA CCGAGGCCGT CGTCGCCGAC 1560 AAGCCGGAG 1569
(2; INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 523 amino acids
(B) TYPE: am o acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 114 :
Met Ala Lys Thr He Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu
1 5 10 15
Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro
20 25 30
Lvs Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr He
35 40 45
Thr Asn Asp Gly Val Ser He Ala Lys Glu He Glu Leu Glu Asp Pro
50 55 60
Tyr Glu Lys He Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr 65 70 75 * 80
Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gin
85 90 95
Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro
100 105 110
Leu Gly Leu Lys Arg Gly He Glu Lys Ala Val Glu Ala Val Thr Gin
115 120 125
Ser Leu Leu Lys Ser Ala Lys Glu Val Glu Thr Lys Glu Gin He Ser
130 135 140
Ala Thr Ala Ala He Ser Ala Gly Asp Thr Gin He Gly Glu Leu He 145 150 155 160
Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val He Thr Val Glu
165 170 175
Glu Ser Asn Thr Phe Gly Leu Gin Leu Glu Leu Thr Glu Gly Met Arg
180 185 190
Phe Asp Lys Gly Tyr He Ser Gly Tyr Phe Val Thr Asp Ala Glu Arg
195 200 205
Gin Glu Ala Val Leu Glu Asp Pro Tyr He Leu Leu Val Ser Ser Lys
210 215 220
Val Ser liar Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val He Gin 225 230 235 240
Ala Gly Lys Pro Leu Leu He He Ala Glu Asp Val Glu Gly Glu Ala
245 250 255
Leu Ser Thr Leu Val Val Asn Lys He Arg Gly Thr Phe Lys Ser Val
260 265 270
Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gin
275 280 285
Asp Met Ala He Leu Thr Gly Gly Gin Val Val Ser Glu Arg Val Gly
290 295 300
Leu Ser Leu Glu Thr Ala Asp Val Ser Leu Leu Gly Gin Ala Arg Lys 305 310 315 320
Val Val Val Thr Lys Asp Glu Thr Thr He Val Glu Gly Ser Gly Asp
325 330 335
Ser Asp Ala He Ala Gly Arg Val Ala Gin He Arg Ala Glu He Glu
340 345 350
Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gin Glu Arg Leu Ala
355 360 365
Lys Leu Ala Gly Gly Val Ala Val He Lys Ala Gly Ala Ala Thr Glu
370 375 380
Val Glu Leu Lys Glu Arg Lys His Arg He Glu Asp Ala Val Arg Asn 385 390 395 400 Ala Lys Ala Ala Val Glu Glu Gly He Val Ala Gly Gly Gly Val Ala
405 410 415
Leu Leu Gin Ser Ala Pro Ala Leu Asp Asp Leu Gly Leu Thr Gly Asp
420 425 430
Glu Ala Thr Gly Ala Asn He Val Arg Val Ala Leu Ser Ala Pro Leu
435 440 445
Lys Gin He Ala Phe Asn Gly Gly Leu Glu Pro Gly Val Val Ala G1J
450 455 460
Lys Val Ser Asn Leu Pro Ala Gly His Gly Leu Asn Ala Ala Thr Gly 465 470 475 480
Glu Tyr Glu Asp Leu Leu Lys Ala Gly Val Ala ^sp Pro Val Lys Val
485 490 495
Thr Arg Ser Ala Leu Gin Asn Ala Ala Ser lie Ala Ala Leu Phe Leu
500 505 510
Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu 515 520
(2) INFORMATION FOR SEQ ID NO: 115:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 647 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE: Genomic RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:
ATGGCCAAGA CAATTGCGTA TGACGAAGAG GCCCGCCGTG GCCTCGAGCG GGGCCTCAAC 60
GCCCTCGCAG ACGCCGTAAA GGTGACGTTG GGCCCGAAGG GTCGCAACGT CGTGCTGGAG 120
AAGAAGTGGG GCGCCCCCAC GATCACCAAC GATGGTGTGT CCATCGCCAA GGAGATCGAG 180
CTGGAGGACC CGTACGAGAA GATCGGCGCT GAGCTGGTCA AAGAGGTCGC CAAGAAGACC 240
GACGACGTCG CGGGCGACGG CACCACCACC GCCACCGTGC TCGCTCAGGC TCTGGTTCGC 300
GAAGGCCTGC GCAACGTCGC AGCCGGCGCC AACCCGCTCG GCCTCAAGCG TGGCATCGAG 360
AAGGCTGTCG AGGCTGTCAC CCAGTCGCTG CTGAAGTCGG CCAAGGAGGT CGAGACCAAG 420
GAGCAGATXI .CTGCCACCGC GGCGATTTCC GCCGGCGACA CCCAGATCGG CGAGCTCATC 480
GCCGAGGCCA TGGACAAGGT CGGCAACGAG GGTGTCATCA CCGTCGAGGA GTCGAACACC 540
TTCGGCCTGC AGCTCGAGCT CACCGAGGGT ATGCGCTTCG ACAAGGGCTA CATCTCGGGT 600
TACTTCGTGA CCGACGCCGA GCGCCAGGAA GCCGTCCTGG AGGATCC 647
(2) INFORMATION FOR SEQ ID NO : 116 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 927 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: GATCCCTACA TCCTGCTGGT CAGCTCCAAG GTGTCGACCG TCAAGGATCT GCTCCCGCTG 60
CTGGAGAAGG TCATCCAGGC CGGCAAGCCG CTGCTGATCA TCGCCGAGGA CGTCGAGGGC 120
GAGGCCCTGT CCACGCTGGT GGTCAACAAG ATCCGCGGCA CCTTCAAGTC CGTCGCCGTC 180
AAGGCTCCGG GCTTCGGTGA CCGCCGCAAG GCGATGCTGC AGGACATGGC CATCCTCACC 240
GGTGGTCAGG TCGTCAGCGA AAGAGTCGGG CTGTCCCTGG AGACCGCCGA CGTCTCGCTG 300
CTGGGCCAGG CCCGCAAGGT CGTCGTCACC AAGGACGAGA CCACCATCGT CGAGGGCTCG 360
GGCGATTCCG ATGCCATCGC CGGCCGGGTG GCTCAGATCC GCGCCGAGAT CGAGAACAGC 420
GACTCCGACT ACGACCGCGA GAAGCTGCAG GAGCGCCTGG CCAAGCTGGC CGGCGGTGTT 480
GCGGTGATCA AGGCCGGAGC TGCCACCGAG GTGGAGCTCA AGGAGCGCAA GCACCGCATC 540
GAGGACGCCG TCCGCAACGC GAAGGCTGCC GTCGAAGAGG GCATCGTCGC CGGTGGCGGC 600
GTGGCTCTGC TGCAGTCGGC TCCTGCGCTG GACGACCTCG GCCTGACGGG CGACGAGGCC 660
ACCGGTGCCA ACATCGTCCG CGTGGCGCTG TCGGCTCCGC TCAAGCAGAT CGCCTTCAAC 720
GGCGGCCTGG AGCCCGGCGT CGTTGCCGAG AAGGTGTCCA ACCTGCCCGC GGGTCACGGC 780
CTCAACGCCG CGACCGGTGA GTACGAGGAC CTGCTCAAGG CCGGCGTCGC CGACCCGGTG 840
AAGGTCACCC GCTCGGCGCT GCAGAACGCG GCGTCCATCG CGGCTCTGTT CCTCACCACC 900
GAGGCCGTCG TCGCCGACAA GCCGGAG 927
(2) INFORMATION FOR SEQ ID NO: 117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
( -SEQUENCE DESCRIPTION: SEQ ID NO: 117:
Met Ala Lys Thr He Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu
1 5 10 15
Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro
20 25 30
Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr He
35 40 45
Thr Asn Asp Gly Val Ser He Ala Lys Glu He Glu Leu Glu Asp Pro
50 55 60
Tyr Glu Lys He Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr 65 70 75 80
Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gin
85 90 95
Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro
100 105 110
Leu Gly Leu Lys Arg Gly He Glu Lys Ala Val Glu Ala Val Thr Gin
115 120 125
Ser Leu Leu Lys Ser Ala Lys Glu Val Glu Thr Lys Glu Gin He Ser
130 135 140
Ala Thr Ala Ala He Ser Ala Gly Asp Thr Gin He Gly Glu Leu He 145 150 155 160
17 Ala Glu Ala Me:: ASP Lys Val Gly Asn Glu Gly Val Ho Thr Val Glu
165 170 175
Glu Ser Asn Thr Phe Gly Leu Gin Leu Glu Leu Thr Glu Gly Mer Arg
180 185 190
Phe Asp Lvs Gly Tyr He Ser Gly Tyr Phe Val Thr Asp Ala Glu Arg
195 200 205
Gin Glu Ala Val Leu Glu Asp 210 215
(2) INFORMATION FOR SEQ ID NO : 11 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:
Asp Pro Tyr He Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp
1 5 10 15
Leu Leu Pro Leu Leu Glu Lys Val He Gin Ala Gly Lys Pro Leu Leu
20 25 30
He He Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val Val
35 40 45
Asn Lys He Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly
50 55 60
Phe Gly Asp Arg Arg Lys Ala Met Leu Gin Asp Met Ala He Leu Thr 65 70 75 80
Gly Gly Gin Val Val Ser Glu Arg Val Gly Leu Ser Leu Glu Thr Ala
85 90 95
Asp Val Ser Leu Leu Gly Gin Ala Arg Lys Val Val Val Thr Lys Asp
100 105 110
Glu Thr Thr He Val Glu Gly Ser Gly Asp Ser Asp Ala He Ala Gly
115 120 125
Arg Val Ala Gin He Arg Ala Glu He Glu Asn Ser Asp Ser Asp Tyr
130 135 140
Asp Arg Glu Lys Leu Gin Glu Arg Leu Ala Lys Leu Ala Gly Gly Val 145 150 155 160
Ala Val He Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu Arg
165 170 175
Lys His Arg He Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val Glu
180 185 190
Glu Gly He Val Ala Gly Gly Gly Val Ala Leu Leu Gin Ser Ala Pro
195 200 205
Ala Leu Asp Asp Leu Gly Leu Thr Gly Asp Glu Ala Thr Gly Ala Asn
210 215 220
He Val Arg Val Ala Leu Ser Ala Pro Leu Lys Gin He Ala Pne Asn 225 230 235 240
Gly Gly Leu Glu Pro Gly Val Val Ala Glu Lys Val Ser Asn Leu Pro
245 250 255
Ala Gly His Gly Leu Asn Ala Ala Thr Gly Glu Tyr Glu Asp Leu Leu
260 265 270
Lys Ala Gly Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu Gin
275 280 285
Asn Ala Ala Ser He Ala Ala Leu Phe Leu Thr Thr Glu Ala Val Val
290 295 300
Ala Asp Lys Pro Glu 305
(2) INFORMATION FOR SEQ ID NO : 119 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 162 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:
CTCGTACAGG CGACGGAGAT CTCCGACGAC GCCACGTCGG TACGGTTGGT CGCCACCCTG 60
TTCGGCGTCG TGTTGTTGAC GTTGGTGCTG TCCGGGCTCA ACGCCACCCT CATCCAGGGC 120
GCACCAGAAG ACAGCTGGCG CAGGCGGATT CCGTCGATCT TC 162
(2) INFORMATION FOR SEQ ID NO: 120:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1366 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:
GATGAGCAGC GTGCTGAACT CGACCTGGTT GGCCTGGGCC GTCGCGGTCG CGGTCGGGTT 60
CCCGGTGCTG CTGGTCGTGC TGACCGAGGT GCACAACGCG TTGCGTCGGC GCGGCAGCGC 120
GCTGGCCCGC CCGGTGCAAC TCCTGCGTAC CTACATCCTG CCGCTGGGCG CGTTGCTGCT 130
CCTGCTGGTA CAGGCGATGG AGATCTCCGA CGACGCCACG TCGGTACGGT TGGTCGCCAC 240
CCTGTTCGGC GTCGTGTTGT TGACGTTGGT GCTGTCCGGG CTCAACGCCA CCCTCATCCA 300
GGGCGCACCA GAAGACAGCT GGCGCAGGCG GATTCCGTCG ATCTTCCTCG ACGTCGCGCG 360
CTTCGCGCTG ATCGCGGTCG GTATCACCGT GATCATGGCC TATGTCTGGG GCGCGAACGT 420
GGGGGGCCTG TTCACCGCAC TGGGCGTCAC TTCCATCGTT CTTGGCCTGG CTCTGCAGAA 480 _.
TTCGGTCGGT CAGATCATCT CGGGTCTGCT GCTGCTGTTC GAGCAACCGT TCCGGCTCGG 540
CGACTGGATC ACCGTCCCCA CCGCGGCGGG CCGGCCGTCC GCCCACGGCC GCGTGGTGGA 600
AGTCAACTGG CGTGCAACAC ATATCGACAC CGGCGGCAAC CTGCTGGTAA TGCCCAACGC 660
CGAACTCGCC GGCGCGTCGT TCACCAATTA CAGCCGGCCC GTGGGAGAGC ACCGGCTGAC 720
CGTCGTCACC ACCTTCAACG CCGCGGACAC CCCCGATGAT GTCTGCGAGA TGCTGTCGTC 780
GGTCGCGGCG TCGCTGCCCG AACTGCGCAC CGACGGACAG ATCGCCACGC TCTATCTCGG 840
TGCGGCCGAA TACGAGAAGT CGATCCCGTT GCACACACCC GCGGTGGACG ACTCGGTCAG 900
GAGCACGTAC CTGCGATGGG TCTGGTACGC CGCGCGCCGG CAGGAACTTC GCCTNAACGG 960
CGTCGCCGAC GANTTCGACA CGCCGGAACG GATCGCCTCG GCCATGCGGG CTGTGGCGTC 1020
CACACTGCGC TTGGCAGACG ACGAACAGCA GGAGATCGCC GACGTGGTGC GTCTGGTCCG 1080 TTACGGCAAC GGGGAACGCC TCCAGCAGCC GGGTCAGGTA CCGACCGGGA TGAGGTTCAT 1140
CGTAGACGGC AGGGTGAGTC TGTCCGTGAT CGATCAGGAC GGCGACGTGA TCCCGGCGCG 1200
GGTGCTCGAG CGTGGCGACT TCCTGGGGCA GACCACGCTG ACGCGGGAAC CGGTACTGGC 1260
GACCGCGCAC GCGCTGGAGG AAGTCACCGT GCTGGAGATG GCCCGTGACG AGATCGAGCG 1320
CCTGGTGCAC CGAAAGCCGA TCCTGCTGCA CGTGATCGGG GCCGTG 1366
(2) INFORMATION FOR SEQ ID NO: 121:
( ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 455 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:
Met Ser Ser Val Leu Asn Ser Thr Trp Leu Ala Trp Ala Val Ala Val
1 5 10 15
Ala Val Gly Phe Pro Val Leu Leu Val Val Leu Thr Glu Val His Asn
20 25 30
Ala Leu Arg Arg Arg Gly Ser Ala Leu Ala Arg Pro Val Gin Leu Leu
35 40 45
Arg Thr Tyr He Leu Pro Leu Gly Ala Leu Leu Leu Leu Leu Val Gin
50 55 60
Ala Met Glu He Ser Asp Asp Ala Thr Ser Val Arg Leu Val Ala Thr 65 70 75 80
Leu Phe Gly Val Val Leu Leu Thr Leu Val Leu Ser Gly Leu Asn Ala
85 90 95
Thr Leu He Gin Gly Ala Pro Glu Asp Ser Trp Arg Arg Arg He Pro
100 105 110
Ser He Phe Leu Asp Val Ala Arg Phe Ala Leu He Ala Val Gly He
115 120 125
Thr Val He Met Ala Tyr Val Trp Gly Ala Asn Val Gly Gly Leu Phe
130 135 140
Thr Ala Leu Gly Val Thr Ser He Val Leu Gly Leu Ala Leu Gin Asn 145 150 155 160
Ser Val GJy Gin He He Ser Gly Leu Leu Leu Leu Phe Glu Gin Pro
165 170 175
Phe Arg Leu Gly Asp Trp He Thr Val Pro Thr Ala Ala Gly Arg Pro
180 185 190
Ser Ala His Gly Arg Val Val Glu Val Asn Trp Arg Ala Thr His He
195 200 205
Asp Thr Gly Gly Asn Leu Leu Val Met Pro Asn Ala Glu Leu Ala Gly
210 215 220
Ala Ser Phe Thr Asn Tyr Ser Arg Pro Val Gly Glu His Arg Leu Thr 225 230 235 240
Val Val Thr Thr Phe Asn Ala Ala Asp Thr Pro Asp Asp Val Cys Glu
245 250 255
Met Leu Ser Ser Val Ala Ala Ser Leu Pro Glu Leu Arg Thr Asp Gly
260 265 270
Gin He Ala Thr Leu Tyr Leu Gly Ala Ala Glu Tyr Glu Lys Ser He
275 280 285
Pro Leu His Thr Pro Ala Val Asp Asp Ser Val Arg Ser Thr Tyr Leu
290 295 300
Arg Trp Val Trp Tyr Ala Ala Arg Arg Gin Glu Leu Arg Xaa Asn Gly 305 ' 310 315 320
Val Ala Asp Xaa Phe Asp Thr Pro Glu Arg He Ala Ser Ala Met Arg 325 330 335 Ala Val Ala Ser Thr Leu Arg Leu Ala Asp Asp Glu Gin Gin Glu lie
340 345 50
Ala Asp Val Val Arg Leu Val Arg Tyr Gly Asn Gly Glu Arg LeL. Gm
355 360 365
Gin Pro Gly GIr. Val Pro Thr Gly Mer Arg Phe He Val Asp Gly Arg
370 375 380
Val Ser Leu Ser Val He Asp Gin Asp Gly Asp Val He Pro Ala Arg 385 390 395 400
Val Leu Glu Arg Gly Asp Phe Leu Gly Gin Thr Thr Leu Thr Arg Glu
405 410 415
Pro Val Lea Ala Thr Ala His Ala Leu Glu Glu Val Thr Va Leu Glu
420 425 430
Met Ala Arg Asp Glu lie Glu Arg Leu Val his Arg Lys Pro lie Leu
435 440 445
Figure imgf000123_0001
(2) INFORMATION FOR SEQ ID NO: 122:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 898 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:
ATGACAATTC TGCCCTGGAA TGCGCGAACG TCTGAACACC CGACGCGAAA AAGACGCGGG 60
CGCTACCACC TCCTGTCGCG GATGAGCATC CAGTCCAAGT TGCTGCTGAT GCTGCTTCTG 120
ACCAGCATTC TCTCGGCTGC GGTGGTCGGT TTCATCGGCT ATCAGTCCGG ACGGTCCTCG 180
CTGCGCGCAT CGGTGTTCGA CCGCCTCACC GACATCCGCG AGTCGCAGTC GCGCGGGTTG 240
GAGAATCAGT TCGCGGACCT GAAGAACTCG ATGGTGATTT ACTCGCGCGG CAGCACTGCC 300
ACGGAGGCGA TCGGCGCGTT CAGCGACGGT TTCCGTCAGC TCGGCGATGC GACGATCAAT 360
ACCGGGCAGG CGGCGTCATT GCGCCGTTAC TACGACCGGA CGTTCGCCAA CACCACCCTC 420
GACGACAGCC.GAAACCGCGT CGACGTCCGC GCGCTCATCC CGAAATCCAA CCCCCAGCGC 480
TATCTGCAGG CGCTCTATAC CCCGCCGTTT CAGAACTGGG AGAAGGCGAT CGCGTTCGAC 540
GACGCGCGCG ACGGCAGCGC CTGGTCGGCC GCCAATGCCA GATTCAACGA GTTCTTCCGC 600
GAGATCGTGC ACCGCTTCAA CTTCGAGGAT CTGATGCTGC TCGACCTCGA GGGCAACGTG 660
GTGTACTCCG CCTACAAGGG GCCGGATCTC GGGACAAACA TCGTCAACGG CCCCTATCGC 720
AACCGGGAAC TGTCGGAAGC CTACGAGAAG GCGGTCGCGT CGAACTCGAT CGACTATGTC 780
GGTGTCACCG ACTTCGGGTG GTACCTGCCT GCCGAGGAAC CGACCGCCTG GTTCCTGTCC 840
CCGGTCGGGT TGAAGGACCG AGTCGACGGT GTGATGGCGG TCCAGTTCCC CGGAATTC 898
(2) INFORMATION FOR SEQ ID NO: 123:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1259 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ 13 NO.1/3
CGCAATTGAT GACGGCGCGG GGACAGTGGC GTGACACCGC CATGGGAGAC ACCGGTGAGA 60
CCATCCTGGT CGGACCGGAC AATCTGATGC GCTCGGACT^ CCGGCTGTTC CGCGAGAACC 120
GGC-AGAAGTT CCTGGCCGAC GTCGTCGAGG GGGGAACCCC CCGGAGGTC GCCGACGAAT 180
CGGTTGACCG CCGCGGCACC ACGCTGGTGC AGCCGGTGAC CACCCGCTCC GTCGAGGAGG 240
CCCAACGCGG CAACACCGGG ACGACGATCG AGGACGACTA TCTCGGCCAC GAGGCGTTAC 300
AGGCGTACTC ACCGGTGGAC CTGCCGGGAC TGCACTGGGT GATCGTGGCC AAGATCGACA 360
CCGACGAGGC GTTCGCCCCG GTGGCGCAGT TCACCAGGAC CCTGGTGCTG TCGACGGTGA 420
TCATCATCTT CGGCGTGTCG CTGGCGGCCA TGCTGCTGGC GCGGTTGTTC GTCCGTCCGA 480
TCCGGCGGTT GCAGGCCGGC GCCCAGCAGA TCAGCGGCGG TGACT^CCGC CTCGCTCTGC 540
CGGTGTTGTC TCGTGACGAA TTCGGCGATC TGACAACAGC TTTCAACGAC ATG^GTCGCA 600
ATCTGTCGAT CAAGGACGAG CTGCTCGGCG AGGAGCGCGC CGAGAACCAA CGGCTGATGC 660
TGTCCCTGAT GCCCGAACCG GTGATGCAGC GCTACCTCGA CGGGGAGGAG ACGATCGCCC 720
AGGACCACAA GAACGTCACG GTGATCTTCG CCGACATGAT GGGCCTCGAC GAGTTGTCGC 780
GCATGTTGAC CTCCGAGGAA CTGATGGTGG TGGTCAACGA CCTGACCCGC CAGTTCGACG 840
CCGCCGCCGA GAGTCTCGGG GTCGACCACG TGCGGACGCT GCACGACGGG TACCTGGCCA 900
GCTGCGGGTT AGGCGTGCCG CGGCTGGACA ACGTCCGGCG CACGGTCAAT TTCGCGATCG 960
AAATGGACCG CATCATCGAC CGGCACGCCG CCGAGTCCGG GCACGACCTG CGGCTCCGCG 1020
CGGGCATCGA CACCGGGTCG GCGGCCAGCG GGCTGGTGGG GCGGTCCACG TTGGCGTACG 1080
ACATGTGGGC -TTCGGCGGTC GATGTCGCCT ACCAGGTGCA GCGCGGCTCC CCCCAGCCCG 1140
GCATCTACGT CACCTCGCGG GTGCACGAGG TCATGCAGGA AACTCTCGAC TTCGTCGCCG 1200
CCGGGGAGGT CGTCGGCGAG CGCGGCGTCG AGACGGTCTG GCGGTTGCAG GGCCACCCG 1259
(2) INFORMATION FOR SEQ ID NO: 124:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 299 ammo acids
Figure imgf000124_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:
Met Thr He Leu Pro Trp Asn Ala Arg Thr Ser Glu His Pro Thr Arg
1 5 10 15
Lys Arg Arg Gly Arg Tyr His Leu Leu Ser Arg Met Ser He Gin Ser 20 25 30
Lys Leu Leu Leu Met Leu Leu Leu Thr Ser He Leu Ser A^a Ala Val
35 40 45
Val Gly Phe He Gly Tyr Gin Ser Gly Arg Ser Ser Leu Arg Ala Ser
50 55 60
Val Phe Asp Arg Leu Thr Asp He Arg Glu Ser Gin Ser Arg Gly Leu 65 70 /5 80
Glu Asn Gin Phe Ala Asp Leu Lys Asn Ser Met Val He Tyr Ser Arg
85 90 95
Gly Ser Thr Ala Thr Glu Ala He Gly Ala Phe Ser Asp Gly Phe Arg
100 105 110
Gin Leu Gly Asp Ala Thr He Asn Thr Gly Gin Ala Ala Ser Leu Arg
115 120 125
Arg Tyr Tyr Asp Arg Thr Phe Ala Asn Thr Thr Leu Asp Asp Ser Gly
13C 135 140
Asn Arg Val Asp Val Arg Ala Leu He Pro Lys Ser Asn Pro Gin Arg 145 150 155 160
Tyr Leu Gin Ala Leu Tyr Thr Pro Pro Phe Gin Asn Trp Glu Lys Ala
165 170 175
He Ala Phe Asp Asp Ala Arg Asp Gly Ser Ala Trp Ser Ala Ala Asn
180 185 190
Ala Arg Phe Asn Glu Phe Phe Arg Glu He Val His Arg Phe Asn Phe
195 200 205
Glu Asp Leu Met Leu Leu Asp Leu Glu Gly Asn Val Val Tyr Ser Ala
210 215 220
Tyr Lys Gly Pro Asp Leu Gly Thr Asn He Val Asn Gly Pro Tyr Arg 225 230 235 240
Asn Arg Glu Leu Ser Glu Ala Tyr Glu Lys Ala Val Ala Ser Asn Ser
245 250 255
He Asp Tyr Val Gly Val Thr Asp Phe Gly Trp Tyr Leu Pro Ala Glu
260 265 270
Glu Pro Thr Ala Trp Phe Leu Ser Pro Val Gly Leu Lys Asp Arg Val
275 280 285
Asp Gly Val Met Ala Val Gin Phe Pro Gly He 290 295
(2) INFORMATION FOR SEQ ID NO: 125:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125:
Gin Leu Met Thr Ala Arg Gly Gin Trp Arg Asp Thr Gly Met Gly Asp
1 5 10 15
Thr Gly Glu Thr He Leu Val Gly Pro Asp Asn Leu Met Arg Ser Asp
20 25 30
Ser Arg Leu Phe Arg Glu Asn Arg Glu Lys Phe Leu Ala Asp Val Val
35 40 45
Glu Gly Gly Thr Pro Pro Glu Val Ala Asp Glu Ser Val Asp Arg Arg
50 55 60
Gly Thr Thr Leu Val Gin Pro Val Thr Thr Arg Ser Val Glu Glu Ala 65 70 75 80
Gin Arg Gly Asn Thr Gly Thr Thr He Glu Asp Asp Tyr Leu Gly His
85 90 95
Glu Ala Leu Gin Ala Tyr Ser Pro Val Asp Leu Pro Gly Leu His Trp
100 105 110
Val He Val Ala Lys He Asp Thr Asp Glu Ala Phe Ala Pro Val Ala
115 120 125
Gin Pne Thr Arg Thr Leu Val Leu Ser Thr Val He He He Phe Gly
Figure imgf000126_0001
Gly His Pro
(2) INFORMATION FOR SEQ ID NO: 126:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid {£ 1 STRANDEDNESS: single (D) TOPOLOGY: linear
(n) MOLECULE TYPE: Other
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 126:
CCGGATCCGA TGAGCAGCGT GCTGAAC 27
(2) INFORMATION FOR SEQ ID NO: 127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127: GCGGATCCCA CGGCCCCGAT CACGTG 6
(2) INFORMATION FOR SEQ ID NO: 128-
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION SEQ ID NO: 128:
CCGGATCCAA TGACATTTCT GCCCTGGAAT GCG 3
(2) INFORMATION FOR SEQ ID NO: 129:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129:
CCGGA1CCAT TCGGTGGCCC TGCAACCGCC AG 2
(2) INFORMATION FOR SEQ ID NO: 130:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(a) MOLECULE TYPE: Other
(x ..SEQUENCE DESCRIPTION: SEQ ID NO: 130:
CCGGATCCGG AGCAACCGTT CCGGCTC 7
(2) INFORMATION FOR SEQ ID NO: 131:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131:
CCGGATCCCG GCTATCAGTC CGGACGG 7
(2) INFORMATION FOR SEQ ID NO: 132: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 844 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ 10 NO: 132:
GAGCAACCGT TCCGGCTCGG CGACTGGATC ACCGTCCCCA CCGCGGCGGG CCGGCCGTCC 60 GCCCACGGCC GCGTGGTGGA AGTCAACTGG CGTGCΛACAC ATATCGACAC CGGCGGCAAC
120
CTGCTGGTAA TGCCCAACGC CGAACTCGCC GGCGCGTCGT TCACCAATTA CAGCCGGCCC 180
GTGGGAGAGC ACCGGCTGAC CGTCGTCACC ACCTTCAACG CCGCGGACAC CCCCGATGAT 240
GTCTGCGAGA TGCTGTCGTC GGTCGCGGCG TCGCTGCCCG AACTGCGCAC CGACGGACAG 300
ATCGCCACGC TCTATCTCGG TGCGGCCGAA TACGAGAAGT CGATCCCGTT GCACACACCC 360
GCGGTGGACG ACTCGGTCAG GAGCACGTAC CTGCGATGGG TCTGGTACGC CGCGCGCCGG 420
CAGGAACTTC GCCTAACGGC GTCGCCGACG ATTCGACACG CCGGAACGGA TCGCCTCGGC 480
CATGCGGGCT GTGGCGTCCA CACTGCGCTT GGCAGACGAC GAACAGCAGG AGATCGCCGA 540
CGTGGTGCGT CTGGTCCGTT ACGGCAACGG GGAACGCCTC CAGCAGCCGG GTCAGGTACC 600
GACCGGGATG AGGTTCATCG TAGACGGCAG GGTGAGTCTG TCCGTGATCG ATCAGGACGG 660
CGACGTGATC CCGGCGCGGG TGCTCGAGCG TGGCGACTTC CTGGGGCAGA CCACGCTGAC 720
GCGGGAACCG GTACTGGCGA CCGCGCACGC GCTGGAGGAA GTCACCGTGC TGGAGATGGC 780
CCGTGACGAG ATCGAGCGCC TGGTGCACCG AAAGCCGATC CTGCTGCACG TGATCGGGGC 840
CGTG 844
(2) INFORMATION FOR SEQ ID NO: 133:
(i)...SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133:
GGCTATCAGT CCGGACGGTC CTCGCTGCGC GCATCGGTGT TCGACCGCCT CACCGACATC 60
CGCGAGTCGC AGTCGCGCGG GTTGGAGAAT CAGTTCGCGG ACCTGAAGAA CTCGATGGTG 120
ATTTACTCGC GCGGCAGCAC TGCCACGGAG GCGATCGGCG CGTTCAGCGA CGGTTTCCGT 180
CAGCTCGGCG ATGCGACGAT CAATACCGGG CAGGCGGCGT CATTGCGCCG TTACTACGAC 240
CGGACGTTCG CCAACACCAC CCTCGACGAC AGCGGAAACC GCGTCGACGT CCGCGCGCTC 300
ATCCCGAAAT CCAACCCCCA GCGCTATCTG CAGGCGCTCT ATACCCCGCC GTTTCAGAAC 360 TGGGAGAAGG CGATCGCGTT CGACGACGCG CGCGACGGCA GCGCCTGGTC GGCCGCCAAT 420
GCCAGATTCA ACGAGTTCTT CCGCGAGATC GTGCACCGCT TCAACTTCGA GGATCTGATG 480
CTGCTCGACC TCGAGGGCAA CGTGGTGTAC TCCGCCTACA AGGGGCCGGA TCTCGGGACA 540
AACATCGTCA ACGGCCCCTΛ TCGCAACCGG GAACTGTCGG AAGCCTACGA GAAGGCGGTC 600
GCGTCGAACT CGATCGACTA TGTCGGTGTC ACCGACTTCG CGTGGTACCT GCCTGCCGAG 660
GAACCGACCG CCTGGTTCCT GTCCCCGGTC GGGTTGAAGG ΛCCGAGTCGA CGGTGTGATG 720
GCGGTCCAGT TCCCCGGAAT TC 742
(2) INFORMATION FOR SEQ ID NO: 134:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:
Glu Gin Pro Phe Arg Leu Gly Asp Trp He Thr Val Pro Thr Ala Ala
1 5 10 15
Gly Arg Pro Ser Ala His Gly Arg Val Val Glu Val Asn Trp Arg Ala
20 25 30
Thr His He Asp Thr Gly Gly Asn Leu Leu Val Met Pro Asn Ala Glu
35 40 45
Leu Ala Gly Ala Ser Phe Thr Asn Tyr Ser Arg Pro Val Gly Glu His
50 55 60
Arg Leu Thr Val Val Thr Thr Phe Asn Ala Ala Asp Thr Pro Asp Asp 65 70 75 80
Val Cys Glu Met Leu Ser Ser Val Ala Ala Ser Leu Pro Glu Leu Arg
85 90 95
Thr Asp Gly Gin He Ala Thr Leu Tyr Leu Gly Ala Ala Glu Tyr Glu
100 105 110
Lys Ser He Pro Leu His Thr Pro Ala Val Asp Asp Ser Val Arg Ser
115 120 125
Thr Tyr Leu Arg Trp Val Trp Tyr Ala Ala Arg Arg Gin Glu Leu Arg
130 135 140
Xaa Asn Gly Val Ala Asp Xaa Phe Asp Thr Pro Glu Arg He Ala Ser 145 150 155 160
Ala Met Arg Ala Val Ala Ser Thr Leu Arg Leu Ala Asp Asp Glu Gin
165 170 175
Gin Glu He Ala Asp Val Val Arg Leu Val Arg Tyr Gly Asn Gly Glu
180 185 190
Arg Leu Gin Gin Pro Gly Gin Val Pro Thr Gly Met Arg Phe He Val
195 200 205
Asp Gly Arg Val Ser Leu Ser Val He Asp Gin Asp Gly Asp Val He
210 215 220
Pro Ala Arg Val Leu Glu Arg Gly Asp Phe Leu Gly Gin Thr Thr Leu 225 230 235 240
Thr Arg Glu Pro Val Leu Ala Thr Ala His Ala Leu Glu Glu Val Thr
245 250 255
Val Leu Glu Met Ala Arg Asp Glu He Glu Arg Leu Val His Arg Lys
260 265 270
Pro He Leu Leu His Val He Gly Ala Val 275 280
(2) INFORMATION FOR SEQ ID NO: 135: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 247 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135:
Gly Tyr Gin Ser Gly Arg Ser Ser Leu Arg Ala Ser Val Phe Asp Arg
1 5 10 15
Leu Thr Asp He Arg Glu Ser Gin Ser Arg Gly Leu Glu Asn Gin Phe
20 25 30
Ala Asp Leu Lys Asn Ser Met Val He Tyr Ser Arg Gly Ser Thr Ala
35 40 45
Tnr Glu Ala He Gly Ala Phe Ser Asp Gly Phe Arg Gin Leu Gly Asp
50 55 60
Ala Thr He Asn Thr Gly Gin Ala Ala Ser Leu Arg Arg Tyr Tyr Asp 65 70 75 80
Arg Thr Phe Ala Asn Thr Thr Leu Asp Asp Ser Gly Asn Arg Val Asp
85 90 95
Val Arg Ala Leu He Pro Lys Ser Asn Pro Gin Arg Tyr Leu Gin Ala
100 105 110
Leu Tyr Thr Pro Pro Phe Gin Asn Trp Glu Lys Ala He Ala Phe Asp
115 120 125
Asp Ala Arg Asp Gly Ser Ala Trp Ser Ala Ala Asn Ala Arg Phe Asn
130 135 140
Glu Phe Phe Arg Glu He Val His Arg Phe Asn Phe Glu Asp Leu Met 145 150 155 160
Leu Leu Asp Leu Glu Gly Asn Val Val Tyr Ser Ala Tyr Lys Gly Pro
165 170 175
Asp Leu Gly Thr Asn He Val Asn Gly Pro Tyr Arg Asn Arg Glu Leu
180 185 190
Ser Glu Ala Tyr Glu Lys Ala Val Ala Ser Asn Ser He Asp Tyr Val
195 200 205
Gly Val Thr Asp Phe Gly Trp Tyr Leu Pro Ala Glu Glu Pro Thr Ala
210 215 220
Trp Phe Leu Ser Pro Val Gly Leu Lys Asp Arg Val Asp Gly Val Met 225 230 235 240
Ala Val Gin Phe Pro Gly He 245
(2) INFORMATION FOR SEQ ID NO: 136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136:
ATGAGCGAAA TCGCCCGNCC CTGGCGGGTT CTGGCATGTG GCATC 45
(2) INFORMATION FOR SEQ ID NO: 137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 340 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (i ) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137:
GCCACCGGCG GCGCCGCCGC GGTGCCCGCC GGGGTGAGCG CCCCGGCGGT CGCGCCGGCC
60 CCCGCGATGC CCGCCCGCCC GGTGTCCACG ATCGCGCCGG CGACCTCGGG CACGCTCAGC
120 GAGTTTTTCG CCGCCAAGGG CGTCACGATG GAGCCGCAGT CCAGCCGCGA CTTCCGCGCC
180 CTCAACATCG TGCTGCCGAA GCCGCGGGGC TGGGAGCACA TCCCGGACCC GAACGTGCCG
240
GACGCGTTCG CGGTGCTGGC CGACCGGGTC AGNGGTAAAG GTCAGNAGTC GACAAACGCC 300
CACGTGGTGG TCGACAAACA CGTAGGCGAG TTCGACGGCA 340
(2) INFORMATION FOR SEQ ID NO: 138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 235 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138:
GGTGACCACC AGCGTNGAAC AGGTCGTTGC CGAAGCCGCG GAGGCCACCG ACGCGATTGT 60
CAACGGCTTC AAGGTCAGCG TTCCGGGTCC GGGTCCGGCC GCACCGCCAC CTGCACCCGG 120
TGCCCCCGGT GTCCCGCCCG CCCCCGGCGC CCCGGCGCTG CCGCTGGCCG TCGCACCACC 180
CCCGGCTCCC GCTGTTCCCG CCGTGGCGCC CGCGCCACAG CTGCTGGGAC TGCAG 235
(2) INFORMATION FOR SEQ ID NO: 139:
(i) SEQUENCE CHARACTERISTICS: (A). LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139:
Met Ser Glu He Ala Arg Pro Trp Arg Val Leu Ala Cys Gly He 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 140 :
Ala Thr Glv Glv Ala Ala Ala Val Pro Ala Gly Val Ser Ala Pro Ala
1 5 10 15
Val Ala Pro Ala Pro Ala Met Pro Ala Arq Pro Val Ser Thr He Ala
20 25 30
Pro Ala Thr Ser Gly Thr Leu Ser Glu Phe Phe Ala Ala Lys Gly Val
35 40 45
Thr Met Glu Pro Gin Ser Ser Arg Asp Phe Arg Ala Leu Asn He Val
50 55 60
Leu Pro Lys Pro Arg Gly Trp Glu His He Pro Asp Pro Asn Val Pro 65 70 75 80
Asp Ala Phe Ala Val Leu Ala Asp Arg Val Gly Gly Lys Gly Gin Xaa
85 90 95
Ser Thr Asn Ala His Val Val Val Asp Lys His Val Gly Glu Phe Asp
100 105 110
Gly
(2) INFORMATION FOR SEQ ID NO : 1 1 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 amino acids
(B) TYPE: am o acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:
Val Thr Thr Ser Val Glu Gin Val Val Ala Ala Ala Asp Ala Thr Glu
1 5 10 15
Ala He Val Asn Gly Phe Lys Val Ser Val Pro Gly Pro Gly Pro Ala
20 25 30
Ala Pro Pro Pro Ala Pro Gly Ala Pro Gly Val Pro Pro Ala Pro Gly
35 40 45
Ala Pro Ala Leu Pro Leu Ala Val Ala Pro Pro Pro Ala Pro Ala Val
50 55 60
Pro Ala Val Ala Pro Ala Pro Gin Leu 65 70

Claims

Claims
1. A poiypeptide comprising an immunogenic portion of a soluble M. vaccae antigen, or a variant thereof, wherein the antigen induces an immune response in patients previously exposed to a mycobacterium.
2. A poiypeptide comprising an immunogenic portion of an M. vaccae antigen, wherein the antigen includes a sequence selected from the group consisting of:
(a) sequences recited in SEQ ID NOS: 1-4, 9-16, 18-21, 23, 25, 26, 28, 29. 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100-105, 109, 1 10. 1 12, 121 , 124, 125, 134, 135, 140 and 141 ; and
(b) sequences having at least about a 99% probability of being the same as a sequence recited in SEQ ID NOS: 1 -4, 9-16, 18-21 , 23, 25. 26, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100-105, 109, 1 10, 1 12, 121 , 124, 125, 134, 135, 140 and 141 as measured by computer algorithm BLASTP.
3. A poiypeptide comprising an immunogenic portion of an M. vaccae antigen, wherein the antigen comprises an amino acid sequence encoded by a DNA molecule selected from the group consisting of:
(a) -sequences recited in SEQ ID NOS: 40-42, 46, 48-51 , 74, 88, 93, 97,
99, 106-108, 1 1 1, 120, 122, 123, 132, 133 and 136-138;
(b) complements of the sequences recited in SEQ ID NOS:40-42, 46, 48- 51 , 74, 88, 93, 97, 99, 106-108, 1 1 1 , 120, 122, 123, 132, 133 and 136- 138; and
(c) sequences having at least about a 99% probability of being the same as a sequence of (a) or (b) as measured by computer algorithm FASTA.
4. A DNA molecule comprising a nucleotide sequence encoding a poiypeptide according to any one of claims 1-3.
5. An expression vector comprising a DNA molecule according to claim 4.
6. A host cell transformed with an expression vector according to claim 5.
7. The host cell of claim 6, wherein the host cell is selected from the group consisting of E. coli, mycobacteria, insect, yeast and mammalian cells.
8. A fusion protein comprising two or more polypeptides according to any one of claims 1-3.
9. A pharmaceutical composition comprising one or more polypeptides according to any one of claims 1-3, and a physiologically acceptable carrier.
10. A pharmaceutical composition comprising a DNA molecule according to claim 4 and a physiologically acceptable carrier.
1 1. A pharmaceutical composition comprising one or more components secreted by M. vaccae cells in culture and a physiological acceptable carrier.
12. A pharmaceutical composition comprising a fusion protein according to claim 8 a»d-a physiologically acceptable carrier.
13. A vaccine comprising one or more polypeptides according to any one of claims 1-3, and a non-specific immune response amplifier.
14. A vaccine comprising a DNA molecule according to claim 4 and a non-specific immune response amplifier.
15. A vaccine comprising one or more components secreted by M. vaccae cells in culture and a non-specific immune response amplifier.
16. A vaccine comprising a fusion protein according to claim 8 and a non¬ specific immune response amplifier.
17. A vaccine according to any one of claims 13-16 wherein the nonspecific immune response amplifier is an adjuvant.
18. A vaccine according to any one of claims 13-16 wherein the nonspecific immune response amplifier comprises delipidated M. vaccae cells.
19. A vaccine according to any one of claims 13-16 wherein the nonspecific immune response amplifier comprises culture filtrate from M. vaccae.
20. A method for inducing protective immunity in a patient, comprising administering to a patient a pharmaceutical composition according to any one of claims 9-12.
21. A method for inducing protective immunity in a patient, comprising administering to a patient a vaccine according to any one of claims 13-19.
22. A method for detecting mycobacterial infection in a patient, comprising:
(a) — -contacting dermal cells of a patient with one or more polypeptides according to any one of claims 1 -3; and
(b) detecting an immune response on the patient's skin.
23. The method of claim 22 wherein the immune response is induration.
24. A diagnostic kit comprising:
(a) a poiypeptide according to any one of claims 1-3; and
(b) apparatus sufficient to contact the poiypeptide with the dermal cells of a patient.
25. A method for detecting mycobacterial infection in a biological sample, comprising:
(a) contacting the biological sample with one or more polypeptides according to any one of claims 1 -3; and
(b) detecting in the sample the presence of antibodies that bind to at least one of the polypeptides.
26. The method of claim 25 wherein step (a) additionally comprises contacting the biological sample with a 38 kD M tuberculosis antigen and step (b) additionally comprises detecting in the sample the presence of antibodies that bind to the 38 kD M. tuberculosis antigen.
27. The method of claim 25 wherein the polypeptide(s) are bound to a solid support.
28. The method of claim 25 wherein the biological sample is selected from the group consisting of whole blood, serum, plasma, saliva, cerebrospinal fluid and urine.
29. A method for detecting mycobacterial infection in a biological sample, comprising:
(a) contacting the biological sample with a binding agent which is capable of binding to a poiypeptide according to any one of claims 1 -3 ; and
(b) detecting in the sample a protein or poiypeptide that binds to the binding agent.
30. The method of claim 29 wherein the binding agent is a monoclonal antibody.
31. The method of claim 29 wherein the binding agent is a polyclonal antibody.
32. A diagnostic kit comprising:
(a) at least one poiypeptide according to any one of claims 1 -3; and
(b) a detection reagent.
33. The kit of claim 32 wherein the poiypeptide is immobilized on a solid support.
34. The kit of claim 32 wherein the detection reagent comprises a reporter group conjugated to a binding agent.
35. The kit of claim 34 wherein the binding agent is selected from the group consisting of anti-immunoglobulins, Protein G, Protein A and lectins.
36. The kit of claim 34 wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.
37. A monoclonal antibody that binds to a poiypeptide according to any one of claims 1-3.
38. A polyclonal antibody that binds to a poiypeptide according to any one of claims 1-3-,
39. A method for enhancing a non-specific immune response to an antigen comprising administering a component selected from the group consisting of:
(a) delipidated M. vaccae cells;
(b) deglycosylated M. vaccae cells; and
(c) delipidated and deglycosylated M. vaccae cells
40. A method for enhancing a non-specific immune response to an antigen comprising administering culture filtrate from M. vaccae cells.
41. A composition comprising a component selected from the group consisting of:
(a) delipidated M. vaccae cells;
(b) deglycosylated M. vaccae cells; and
(c) delipidated and deglycosylated M. vaccae cells.
42. A method for inducing protective immunity in a patient comprising administering a composition according to claim 41.
43. A vaccine comprising a non-specific immune response amplifier and a component selected from the group consisting of:
(a) delipidated M. vaccae cells;
(b) deglycosylated M. vaccae cells; and
(c) delipidated and deglycosylated M. vaccae cells.
44. A method for inducing protective immunity in a patient, comprising administering to a patient a vaccine according to claim 43.
45. A method for enhancing a non-specific immune response to an antigen comprising administering a poiypeptide, the poiypeptide comprising an immunogenic portion of an antigen, wherein the antigen includes a sequence selected from the group consisting of:
(a) sequences recited in SEQ ID NO: 1 14, 1 17 and 1 18; and
(b) sequences having at least about 97% identity to a sequence recited in SEQ ID NO: 1 14, 1 17 and 118.
46. A vaccine comprising a poiypeptide, the poiypeptide comprising an immunogenic portion of an antigen, wherein the antigen includes a sequence selected from the group consisting of:
(a) sequences recited in SEQ ID NOS: 1 14, 1 17 and 1 18; and
(b) sequences having at least about 97% identity to a sequence of SEQ ID NOS: 1 14, 117 and 1 18.
47. An immunologically reactive fragment of an M. vaccae antigen consisting essentially of an amino acid sequence selected from the group consisting of:
(a) sequences recited in SEQ ID NOS: 1 14 and 1 18; and
(b) sequences having at least about 97% identity to a sequence recited in SEQ ID NOS: 1 14 and 1 18.
48. A method for enhancing protective immunity against a mycobacterial infection comprising administering a poiypeptide, the poiypeptide comprising an immunogenic portion of an antigen, wherein the antigen includes a sequence selected from the group consisting of:
(a) sequences recited in SEQ ID NO: 1 14, 1 17 and 1 18; and
(b) sequences having at least about 97% identity to a sequence recited in SEQ ID NO: 1 14, 1 17 and 1 18.
49. A poiypeptide comprising an immunogenic portion of an M. vaccae antigen, wherein the antigen includes a sequence selected from the group consisting of:
(a) sequences recited in SEQ ID NO: 1 14, 1 17 and 1 18; and
(b) sequences having at least about 97% identity to a sequence recited in SEQ ID NO: 1 14, 1 17 and 1 18.
PCT/NZ1997/000105 1996-08-29 1997-08-28 Compounds and methods for treatment and diagnosis of mycobacterial infections WO1998008542A2 (en)

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EP97937915A EP0939646A2 (en) 1996-08-29 1997-08-28 Compounds and methods for treatment and diagnosis of mycobacterial infections
BR9711457-0A BR9711457A (en) 1996-08-29 1997-08-28 Compounds and process for the treatment and diagnosis of mycobacterial infections.
JP51151698A JP2001503969A (en) 1996-08-29 1997-08-28 Compounds and methods for the treatment and diagnosis of mycobacterium infection
NZ334358A NZ334358A (en) 1996-08-29 1997-08-28 isolated polypeptide comprising an immunogenic portion of a M vaccine antigen, use in the treatment and diagnosis of mycobacterial infections
CA002263885A CA2263885A1 (en) 1996-08-29 1997-08-28 Compounds and methods for treatment and diagnosis of mycobacterial infections
AU40365/97A AU723606B2 (en) 1996-08-29 1997-08-28 Compounds and methods for treatment and diagnosis of mycobacterial infections

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US08/705,347 1996-08-29
US08/873,970 US6001361A (en) 1996-08-29 1997-06-12 Mycobacterium vaccae antigens
US08/873,970 1997-06-12

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US6284255B1 (en) 2001-09-04
US6410720B1 (en) 2002-06-25
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