CA2223610A1 - Nucleic acid respiratory syncytial virus vaccines - Google Patents
Nucleic acid respiratory syncytial virus vaccines Download PDFInfo
- Publication number
- CA2223610A1 CA2223610A1 CA002223610A CA2223610A CA2223610A1 CA 2223610 A1 CA2223610 A1 CA 2223610A1 CA 002223610 A CA002223610 A CA 002223610A CA 2223610 A CA2223610 A CA 2223610A CA 2223610 A1 CA2223610 A1 CA 2223610A1
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- rsv
- protein
- nucleotide sequence
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- 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
- A61P31/12—Antivirals
-
- 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
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
- C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Abstract
Vectors containing a nucleotide sequence coding for an F protein of respiratory syncytial virus (RSV) and a promoter for such sequence, preferably a cytomegalovirus promoter, are described. Such vectors also may contain a further nucleotide sequence located adjacent to the RSV F protein encoding sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo. Such vectors may be used to immunize a host, including a human host, by administration thereto. Such vectors also may be used to produce antibodies for detection of RSV infection in a sample.
Description
w o 9{'10345 PCT/CA96/00398 NUCLEIC ACID RESPIRATORY SYNCYTIAL YIRUS VACCINES
FIE~D OF 1NV~N11ON
The present invention is related to the field of Respiratory Syncytial Virus (RSV) vaccines and is particularly concerned with vaccines comprising nucleic acid sequences encoding the fusion (F) protein of RSV.
REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of copending United States Patent Application No.
08/476,397, filed June 7, 1995.
BACKGROUND OF lNv~NllON
Respiratory syncytial virus (RSV), a negative-strand RNA virus belonging to the Paramyxoviridae family of viruses, is the major viral pathogen responsible for bronchiolitis and pneumonia in infants and young children (ref. 1 - Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Acute respiratory tract - infections caused by RSV result in approximately 90,000 hospitalizations and 4,500 deaths per year in the United States (ref. 2). Medical care costs due to RSV infection are greater than $340 M annually in the United States alone (ref. 3). There is currently no licensed vaccine against RSV. The main approaches for developing an RSV
vaccine have included inactivated virus, live-attenuated viruses and subunit vaccines.
The F protein of RSV is considered to be one of the most important protective antigens of the virus. There is a significant similarity (89~ identity) in the amino acid sequences of the F proteins from RSV subgroups A and WO3~'1091' PCT/CA96/~398 B (ref. 3) and anti-F antibodies can cross-neutralize viruses of both subgroups as well as protect ;~mlln;zed ~n;m~l S against infection with viruses from both subgroups (ref. 4). Furthermore, the ~ protein has been identified as a major target for RSV-specific cytotoxic T-lymphocytes in mice and hl~m~n5 (ref. 3 and ref. 5).
The use of RSV proteins as vaccines may have obstacles. Parenterally ~m;n;stered vaccine candidates have so far proven to be poorly ;mm-~nogenic with regard to the induction of neutralizing antibodies in seronegative hllm~nc or chimpanzees. The serum antibody response induced by these antigens may be further diminished in the presence of passively acquired antibodies, such as the transplacentally acquired maternal antibodies which most young infants possess. A
subunit vaccine candidate for RSV consisting of purified fusion glycoprotein from RSV infected cell cultures and purified by ;mmllno~ffinity or ion-exchange chromatography has been described (ref. 6). Parenteral immunization of seronegative or seropositive chimpanzees with this preparation was performed and three doses of 50 ~g were required in seronegative animals to induce an RSV serum neutralizing titre of approximately 1:50. Upon subsequent challenge of these ~nim~ls with wild-type RSV, no effect of immunization on virus shedding or clinical disease could be detected in the upper respiratory tract.
The effect of ;mmlln;zation with this vaccine on virus shedding in the lower respiratory tract was not investigated, although this is the site where the serum antibody induced by parenteral immunization may be expected to have its greatest effect. Safety and immunogenicity studies have been performed in a small number of seropositive individuals. The vaccine was found to be safe in seropositive children and in three seronegative children (all ~ 2.4 years of age). The effects of immunization on lower respiratory tract WO~6~10~1, PCT/CA96/00398 -disease could not be determined because of the small number of children ;~ nlzed. One immunizing dose in seropositive children induced a 4-fold increase in virus neutralizing antibody titres in 40 to 60~ of the vaccinees. Thus, insufficient information is available from these small studies to evaluate the efficacy of this vaccine against RSV-induced disease. A further problem facing subunit RSV vaccines is the possibility that inoculation of seronegative subjects with immunogenic preparations might result in disease enhancement (sometimes referred to as immunopotentiation), similar to that seen in formalin inactivated RSV vaccines. In some studies, the immune response to immunization with RSV F
protein or a synthetic RSV FG fusion protein resulted in a disease enhancement in rodents resembling that induced by a formalin-inactivated RSV vaccine. The association of immunization with disease enhancement using non-replicating antigens suggests caution in their use as vaccines in seronegative h11m~nc.
Live attenuated vaccines against disease caused by RSV may be promising for two main reasons. Firstly, infection by a live vaccine virus induces a balanced immune response comprising mucosal and serum antibodies and cytotoxic T-lymphocytes. Secondly, infection of infants with live attenuated vaccine candidates or naturally acquired wild-type virus is not associated with enhanced disease upon subsequent natural reinfection. It will be challenging to produce live attenuated vaccines that are ~mm-1nogenic for younger infants who possess maternal virus-neutralizing antibodies and yet are attenuated for seronegative infants greater than or equal to 6 months of age. Attenuated live virus vaccines also have the risks of residual virulence and genetic instability.
Injection of plasmid DNA containing sequences encoding a foreign protein has been shown to result in w0~6,~c9t~ PCT/CA96/00~98 -expression of the foreign protein and the induction of antibody and cytotoxic T-lymphocyte responses to the antigen in a number of studies (see, for example, refs 7, 8, 9). The use of plasmid DNA inoculation to express ~iral proteins for the purpose of ;m~lln;zation may offer several advantages over the strategies summarized above Firstly, DNA encoding a viral antigen can be introduced in the presence of antibody to the virus itself, without loss of potency due to neutralization of virus by the antibodies. Secondly, the antigen expressed in vivo should exhibit a native conformation and, therefore, should induce an antibody response similar to that induced by the antigen present in the wild-type virus infection. In contrast, some processes used in purification of proteins can induce conformational changes which may result in the loss of ;m~l~nogenicity of protective epitopes and possibly immunopotentiation.
Thirdly, the expression of proteins from injected plasmid DNAs can be detected in vivo for a considerably longer period of time than that in virus-infected cells, and this has the theoretical advantage of prolonged cytotoxic T-cell induction and enhanced antibody responses.
Fourthly, in vivo expression of antigen may provide protection without the need for an extrinsic adjuvant.
The ability to immunize against disease caused by RSV by administration of a DNA molecule encoding an RSV
F protein was unknown before the present invention. In particular, the efficacy of immunization against RSV
induced disease using a gene encoding a secreted form of the RSV F protein was unknown. Infection with RSV leads to serious disease. It would be useful and desirable to provide isolated genes encoding RSV F protein and vectors for in vivo administration for use in ;m~llnogenic preparations, including vaccines, for protection against disease caused by RSV and for the generation of diagnostic reagents and kits. In particular, it would be WO96/4091, PCT/CA96/00398 s desirable to provide vaccines that are ~ nogenic and protective in h-lm~nq, including seronegative infants, that do not cause disease enhancement (;mmllnopotentiation).
SU~D$ARY OF INV~N11ON
The present invention relates to a method of immunizing a host against disease caused by respiratory syncytial virus, to nucleic acid molecules used therein, and to diagnostic procedures utilizing the nucleic acid molecules. In particular, the present invention is directed towards the provision of nucleic acid respiratory syncytial virus vaccines.
In accordance with one aspect of the invention, there is provided a vector, comprising:
a first nucleotide sequence encoding an RSV F
protein or a protein capable of inducing antibodies that specifically react with RSV F protein;
a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV F protein, and a second nucleotide sequence located adjacent the first nucleotide sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo from the vector in a host.
The first nucleotide sequence may be that which encodes a full-length RSV F protein, as seen in Figure 2 (SEQ ID No: 2). Alternatively, the first nucleotide sequence may be that which encodes an RSV F protein from which the tr~nq~emhrane region is absent. The latter embodiment may be provided by a nucleotide sequence which encodes a full-length RSV F protein but contains a translational stop codon immediately upstream of the start of the transmembrane coding region, thereby preventing expression of a transmembrane region of the RSV F protein, as seen in Figure 3 (SEQ. ID No. 4). The lack of expression of the transmembrane region results in W096/40945 PCT/CA96/0~98 a secreted form of the RSV F protein.
The second nucleotide sequence may ~;G~ ise a pair of splice sites to prevent aberrant mRNA splicing, whereby substantially all transcribed mRNA encodes the RSV protein. Such second nucleotide sequence may be located between the first nucleotide sequence and the promoter sequence. Such second nucleotide sequence may be that of rabbit ~-globin intron II, as shown in Figure 8 (SEQ ID No: 5).
A vector enCo~;n~ the F protein and provided by this aspect of the invention may specifically be pXL2 or pXL4, as seen in Figures 5 or 7.
The promoter sequence may be an imme~-ate early cytomegalovirus (CMV) promoter. Such cytomegalovirus promoter has not previously been employed in vectors containing nucleotide sequences encoding an RSV F
protein.
Accordingly, in another aspect of the invention, there is provided a vector, comprising:
a first nucleotide sequence encoding an RSV F
protein or a protein capable of generating antibodies that specifically react with RSV F protein, and a cytomegalovirus promoter operatively coupled to the first nucleotide sequence for expression of the RSV
F protein.
The first nucleotide sequence may be any of the alternatives described above. The second nucleotide sequence, included to enhance the immunoprotective ability of the RSV F protein when expressed in vivo from the vector in a host, described above also may be present located adjacent a first nucleotide sequence in a vector provided in accordance with this second aspect of the invention.
Certain of the vectors provided herein may be used to immunize a host against RSV infection or disease by in vivo expression of RSV F protein lacking a trAncm~mhrane WO~ C31~ PCT/CA96/00398 region following ~m; n;stration of the vectors. In accordance with a further aspect of the present invention, therefore, there is provided a method of ;~ml~nizing a host against disease caused by infection with respiratory syncytial virus, which comprises administering to the host an effective amount of a vector comprising a first nucleotide sequence encoding an RSV F
protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F protein lacking a tr~nc~emhrane region and a promoter seguence operatively coupled to the first nucleotide sequence for expression of the RSV F protein in the host, which may be a human. The promoter may be an immediate early cytomegalovirus promoter.
The nucleotide sequence encoding the truncated RSV
F protein lacking the transmembrane region may be that as described above.
A vector cont~; n; ng a second nucleotide sequence located adjacent a first nucleotide sequence encoding an RSV F protein, a protein capable of inducing antibodies that specifically react with RSV F protein or an RSV F
protein lacking a transmembrane region and effective to enhance the immunoprotective ability of the RSV F protein expressed by the first nucleotide sequence may be used to immunize a host. Accordingly, in an additional aspect of the present invention, there is provided a method of immunizing a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises administering to the host an effective amount of a vector comprising a first nucleotide sequence encoding an RSV F
protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F protein lacking a transmembrane region, a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV F protein, and a second nucleotide sequence located adjacent the first sequence to Pnh~nce W0~ 3t5 PCT/CA96t~398 the immunoprotective ability of the RSV-F protein when expressed in vivo from said vector in said host.
Specific vectors which may be used in this aspect of the invention are those identified as pXL2 and pXL4 in Figures 5 and 7.
The present invention also includes a novel method of using a gene enco~i ng an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F protein lacking a transmembrane region to protect a host against disease caused by infection with respiratory syncytial virus, which comprises:
isolating the gene;
operatively linking the gene to at least one control sequence to produce a vector, said control sequence directing expression of the RSV F protein when said vector is introduced into a host to produce an immune response to the RSV F protein, and introducing the vector into the host.
The procedure provided in accordance with this aspect of the invention may further include the step of:
operatively linking the gene to an immunoprotection enhancing sequence to produce an enhanced immunoprotection by the RSV F protein in the host, preferably by introducing the immunoprotection enhancing sequence between the control sequence and the gene.
In addition, the present invention includes a method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus, which comprises:
isolating a first nucleotide sequence encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F
protein lacking a transmembrane region;
operatively linking the first nucleotide sequence to at least one control sequence to produce a vector, the W O 96/40945 PC~r/CA96/00398 control sequence directing expression of the RS V F
protein when introduced into a host to produce an lmmltne response to the RSV F protein when expressed in vivo from the vector in a host, and formulating the vector as a vaccine for in vivo administration.
The first nucleotide sequence further may be operatively linked to a second nucleotide sequence to enhance the lmm~lnoprotective ability of the R SV F protein when expressed in vivo from the vector in a host. The vector may be selected from pXL1, pXL2 and pXL4. The invention further includes a vaccine for A~m; n; stration to a host, including a human host, produced by this method as well as immunogenic compositions comprising an immunoeffective amount of the vectors described herein.
As noted previously, the vectors provided herein are useful in diagnostic applications. In a further aspect of the invention, therefore, there is provided a method of determining the presence of an RSV F protein in a sample, comprising the steps of:
(a) immunizing a host with a vector comprising a first nucleotide sequence encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RS V F protein or an RS V F
protein lacking a transmembrane region and a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RS V F
protein in the host to produce antibodies specific for the RSV F protein;
(b) isolating the RS V F protein specific antibodies;
(c) contacting the sample with the isolated antibodies to produce complexes comprising any RSV
F protein present in the sample and the RS V F
protein- specific antibodies; and (d) determining production of the complexes.
.
WOgG,J~9-~ PCT/CA96/00398 ~ The vector employed to elicit the antibodies may be pXLl, pXL2, pXL3 or pXI4.
The inYention also includes a diagnostic kit for detecting the presence of an RSV F protein in a sample, comprising:
(a) a vector comprising a first nucleotide sequence encoding an RSV F protein, a protein capahle of generating antibodies that specifically react with RSV F protein, or a RSV F protein lacking a transmembrane region and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein in a host ;~mtlnlzed therewith to produce antibodies specific for the RSV F protein;
(b) isolation means to isolate said RSV F protein specific antibodies;
(c) contacting means to contact the isolated RSV F
specific antibodies with the sample to produce a complex comprising any RSV F protein present in the sample and RSV F protein specific antibodies; and (d) identifying means to determine production of the complex.
The present invention is further directed to immunization wherein the polynucleotide is an RNA
molecule which codes for an RSV F protein, a protein capable of inducing antibodies that specifically react with RSV F protein or an RSV F protein lacking a tr~nsm~mhrane region.
The present invention is further directed to a method for producing RSV F protein specific polyclonal antibodies comprising the use of the ;mmllnization method described herein, and further comprising the step of isolating the RSV F protein specific polyclonal antibodies from the ;mmllnt zed ~n;m~1 , The present invention is also directed to a method for producing monoclonal antibodies specific for an F
WO~G/4C915 PCT/CA96/~398 protein of RSV, comprising the steps of:
(a) constructing a vector comprising a first nucleotide seguence encoding a RSV F protein and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F
protein; and, optionally, a second nucleotide sequence located adjacent said first nucleotide sequence to ~nh~nce the imTl-noprotective ability of said RSV F protein when expressed in vi~o from said vector in a host.
(b) administering the vector to at least one mouse to produce at least one ;mmlln;zed mouse;
(c) ~e,.,~ving B-lymphocytes from the at least one ;m~lln; zed mouse;
(d) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;
(e) cloning the hybridomas;
(f) selecting clones which produce anti-F protein antibody;
(g) culturing the anti-F protein antibody-producing clones; and (h) isolating anti-F protein monoclonal antibodies.
In this application, the term "RSV F protein~ is used to define a full-length RSV F protein, such proteins having variations in their amino acid sequences including those naturally occurring in various strains of RSV, a secreted form of RSV F protein lacking a transmembrane region, as well as functional analogs of the RSV F
protein. In this application, a first protein is a "functional analog" of a second protein if the first protein is immunologically related to and/or has the same function as the second protein. The functional analog may be, for example, a fragment of the protein or a substitution, addition or deletion mutant thereof.
WO~,/4~91r PCT/CA96/00398 BRIEF DESCRIPTION OF THE FIGURES
The present invention will be further understood from the following General Description and Examples with reference to the Figures in which:
Figure 1 illustrates a restriction map of the gene encoding the F protein of Respiratory Syncytial Virus;
Figure 2 illustrates the nucleotide sequence of the gene encoding the membrane attached form of the F protein of Respiratory Syncytial Virus (SEQ ID No: 1) as well as the amino acid sequence of the RSV F protein encoded thereby (SEQ ID No: 2);
Figure 3 illustrates the nucleotide sequence of the gene encoding the secreted form of the RSV F protein lacking the trAn~m~mhrane region (SEQ ID No: 3) as well as the amino acid sequence of the truncated RSV F protein lacking the tr~ncmemhrane region encoded thereby (SEQ ID
No: 4)i Figure 4 shows the construction of plasmid pXLl containing the gene encoding a secreted form of the RSV
F protein lacking the transmembrane region;
Figure 5 shows the construction of plasmid pXL2 containing a gene encoding a secreted form of the RSV F
protein lacking the transmembrane region and containing the rabbit ~-globin Intron II sequence;
Figure 6 shows the construction of plasmid pXL3 containing the gene encoding a full length membrane attached form of the RSV F protein;
Figure 7 shows the construction of plasmid pXL4 containing a gene encoding a membrane attached form of the RSV F protein and containing the rabbit ~-globin Intron II sequencei and Figure 8 shows the nucleotide sequence for the rabbit ~-globin Intron II sequence (SEQ ID No. 5).
W09"~031~ PCT/CA96100398 GENERAL DESCRIPTION OF INV~N 110N
AS described above, the present invention relates generally to polynucleotide, including DNA, immunization to obtain protection against infection by respiratory syncytial virus ~RSV) and to diagnostic procedures using particular vectors. In the present invention, several recombinant vectors were constructed to contain a nucleotide sequence encoding an RSV F protein.
The nucleotide sequence of the full length RSV F
gene is shown in Figure 2 (SEQ ID No: 1). Certain constructs provided herein include the nucleotide sequence encoding the full-length RSV F (SEQ ID NO 2) protein while others include an RSV F gene modified by insertion of termination codons immediately upstream of the tr~nc~emhrane coding region (see Figure 3, SEQ ID No:
3), to prevent expression of the tr~ncm~mhrane portion of the protein and to produce a secreted or truncated RSV F
protein lacking a transmembrane region (SEQ ID No. 4).
The nucleotide sequence encoding the RSV F protein is operatively coupled to a promoter sequence for expression of the encoded RSV F protein. The promoter sequence may be the immediately early cytomegalovirus (CMV) promoter. This promoter is described in ref. 13.
Any other convenient promoter may be used, including constitutive promoters, such as, Rous Sarcoma Virus LTRs, and inducible promoters, such as metallothionine promoter, and tissue specific promoters.
The vectors provided herein, when ~;n;stered to an animal, effect in vivo RSV F protein expression, as demonstrated by an antibody response in the ~n;~l to which it is administered. Such antibodies may be used herein in the detection of RSV protein in a sample, as described in more detail below. When the encoded RSV F
protein is in the form of an RSV F protein from which the 3~ transmembrane region is absent, such as plasmid pXL1 ~Figure 4), the administration of the vector conferred CA 022236l0 l997-l2-04 WO 9G,'l~Y1, PCT/CA96/00398 protection in mice and cotton rats to challenge by live RSV virus neutralizing antibody and cell mediated 1~mtln responses and an absence of immunopotentiation in ;mml-n;zed ~n;~l S, as seen from the Examples below.
The recombinant vector also may include a second nucleotide sequence located adjacent the RSV F protein encoding nucleotide sequence to ~nh~nce the ;~mllnoprotective ability of the RSV F protein when expressed in ~ivo in a host. Such enhancement may be provided by increased in ~ivo expression, for example, by increased mRNA stability, enhanced transcription and/or translation. This additional sequence preferably is located between the promoter sequence and the RSV F
protein-encoding sequence.
This enhancement sequence may comprise a pair of splice sites to prevent aberrant mRNA splicing during transcription and translation so that substantially all transcribed mRNA encodes an RSV F protein. Specifically, rabbit ~-globin Intron II sequence shown in Figure 7 ~SEQ
ID No: 5) may provide such splice sites, as also described in ref. 15.
The constructs cont~;n1ng the Intron II sequence, CMV promoter and nucleotide sequence coding for the truncated RSV F protein lacking a transmembrane region, i.e. plasmid pXL2 ~Figure 5), induced complete protection in mice against challenge with live RSV, as seen in the Examples below. In addition, the constructs cont~; n; ng the Intron II sequence, CMV promoter and nucleotide sequence coding for the full-length RSV F protein, i.e.
plasmid pXI.4 (Figure 7), also conferred protection in mice to challenge with live RSV, as seen from the Examples below.
The vector provided herein may also comprise a third nucleotide sequence encoding a further antigen from RSV, an antigen from at least one other pathogen or at least one immunomodulating agent, such as cytokine. Such vector may contain said third nucleotide sequence in a ch;~eric or a bicistronic structure. Alternatively, vectors containing the third nucleotide sequence may be separately constructed and co~m;nlstered to a host, with the nucleic acid molecule provided herein.
The vector may further comprise a nucleotide sequence encoding a heterologous signal peptide, such as human tissue plasminogen activator ~TPA), in place of the endogenous signal peptide.
It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis and treatment of RSV infections. A further non-limiting discussion of such uses is further presented below.
1. Vaccine Preparation and ~se Immunogenic compositions, suitable to be used as vaccines, may be prepared from the RSV F genes and vectors as disclosed herein. The vaccine elicits an immune response in a subject which includes the production of anti-F antibodies. Immunogenic compositions, including vaccines, containing the nucleic acid may be prepared as injectables, in physiologically-acceptable liquid solutions or emulsions for polynucleotide administration. The nucleic acid may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a nucleic acid liposome (for example, as described in WO 9324640, ref.
17) or the nucleic acid may be associated with an adjuvant, as described in more detail below. Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture up to 100~
of the polynucleotide. In addition, the polycationic complexes fuse with cell membranes, resulting in an intracellular delivery of polynucleotide that bypasses wo~6~as~ PCT/CA96/00398 .
the degradative enzymes of the lysosomal compartment.
Published PCT application WO 94/27435 describes compositions for genetic ;~ nization comprising cationic lipids and polynucleotides. Agents which assist in the cellular uptake of nucleic acid, such as calcium ions, viral proteins and other transfection facilitating agents, may advantageously be used.
Polynucleotide ;~llnogenic preparations may also be formulated as microcapsules, including biodegradable time-release particles. Thus, U.S. Patent 5,151,264 describes a particulate carrier of a phospholipid/glycolipid/polysaccharide nature that has been termed Bio Vecteurs Supra Moléculaires (BVSM). The particulate carriers are intended to transport a variety of molecules having biological activity in one of the layers thereof.
U.S. Patent 5,075,109 describes encapsulation of the antigens trinitrophenylated keyhole limpet hemocyanin and staphylococcal enterotoxin B in 50:50 poly ~DL-lactideco-glycolide). Other polymers for encapsulation aresuggested, such as poly(glycolide), poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone, poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters and poly(8-hydroxybutyric acid), and polyanhydrides.
Published PCT application WO 91/06282 describes a delivery vehicle comprising a plurality of bioadhesive microspheres and antigens. The microspheres being of starch, gelatin, dextran, collagen or albumin. This delivery vehicle is particularly intended for the uptake of vaccine across the nasal mucosa. The delivery vehicle may additionally contain an absorption enhancer.
The RSV F genes and vectors may be mixed with pharmaceutically acceptable excipients which are compatible therewith. Such excipients may include, water, saline, dextrose, glycerol, ethanol, and -wos6!lo91~ PCT/CA96/00398 combinations thereof. The ;mm~1nogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enh~n~e the effectiveness thereof.
Immunogenic compositions and vaccines may be ~m;nl stered parenterally, by injection subcutaneously, intravenously, intradermally or intramuscularly, possibly following pretreatment of the injection site with a local anesthetic. Alternatively, the ;mmllnogenic compositions formed according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces. Thus, the immunogenic composition may be ~mi n; stered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes.
Alternatively, other modes of A~m; n; stration including suppositories and oral formulations may be desirable.
For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. Oral formulations may include normally employed incipients, such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.
The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and ;mm1lnogenic.
The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize the RSV F
protein and antibodies thereto, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Howe~er, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of about 1 ~g to about 1 mg of the RSV F genes and vectors. Suitable regimes for initial administration and booster doses are also -variable, but may include an initial A~min;stratio followed by subseguent A~mi n; strations. The dosage may also depend on the route of a~m;n;stration and will vary according to the size of the host. A vaccine which protects against only one pathogen is a monovalent vaccine. Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.
Immunogenicity can be significantly improved if the vectors are co-A~mt n; stered with adjuvants, commonly used as 0.05 to 0.1 percent solution in phosphate-buffered saline. Adjuvants ~nhAnce the imm~nQgenicity of an antigen but are not necessarily immunogenic themselves.
Adjuvants may act by retA;n;ng the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Thus, adjuvants have been identified that enhance the immune response to antigens.
Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many An;m~ls. Indeed, only all-m;nllm hydroxide and al~m;nl~m phosphate (collectively commonl y referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.
A wide range of extrinsic adjuvants and other immunomodulating material can provoke potent ;mml~ne responses to antigens. These include saponins complexed to membrane protein antigens to produce immune WOg~'a915 PCT/CA96/00398 stimulating complexes tISCOMS), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as monophoryl lipid A, QS 21 and polyphosphazene.
In particular embodiments of the present invention, the vector comprising a first nucleotide sequence encoding an F protein of RSV may be delivered in conjunction with a targeting molecule to target the vector to selected cells including cells of the immllne system.
The polynucleotide may be delivered to the host by a variety of procedures, for example, Tang et al. (ref.
10) disclosed that introduction of gold microprojectiles coated-with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH
antibodies in the mice, while Furth et al. (ref. 11) showed that a jet injector could be used to transfect skin, muscle, fat and m~mm~ry tissues of living animals.
20 2. Immunoa8~ay8 The RSV F genes and vectors of the present invention are useful as immunogens for the generation of anti-F
antibodies for use in immunoassays, including enzyme-linked immtlnosorbent assays (ELISA), RIAs and other non-25 enzyme linked antibody binding assays or procedures knownin the art. In ELISA assays, the vector first is administered to a host to generate antibodies specific to the RSV F protein. These RSV F-specific antibodies are immobilized onto a selected surface, for example, a surface capable of binding the antibodies, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed antibodies, a nonspecific protein such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample may be bound to the selected surface.
This allows for blocking of nonspecific adsorption sites -w0961~c9~5 pcTlcAs6l~398 on the immobilizing surface and thus reduces the background caused by nonspecific bindings of antisera onto the surface.
The immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a m~n~er conducive to j~mlln~ complex (antigen/antibody) formation. This procedure may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from about 2 to 4 hours, at temperatures such as of the order of about 20~ to 37~C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound RS~ F specific antibodies, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined.
BIOLOGICAL MATERIALS
Certain plasmids that contain the gene encoding RSV
F protein and referred to herein have been deposited with the America Type Culture Collection ~ATCC) located at 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., pursuant to the Budapest Treaty and prior to the filing of this application.
Samples of the deposited plasmids will become available to the public upon grant of a patent based upon this United States patent application and all restrictions on access to the deposits will be ,el..o~ed at that time. The invention described and claimed herein is not to be limited in scope by plasmids deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar plasmids that encode similar or equivalent antigens as WOg6,4C915 PCT/CA96/00398 described in this application are within the scope of the invention.
Plasmid ATCC Desiqnation Date DeDosited pXLl 97167 May 30, 199 S pXL2 97168 May 30, 1995 pXL3 97169 May 30, 1995 pXL4 97170 May 30, 1995.
EXANPLES
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.
Methods of molecular genetics, protein biochemistry, and immunology used but not explicitly described in this disclosure and these Examples are amply reported in the scientific literature and are well within the a~ility of those skilled in the art.
Exam~le 1 This Example describes the construction of vectors containing the RSV F gene.
Figure 1 shows a restriction map of the gene encoding the F protein of Respiratory Syncytial Virus and Figure 2 shows the nucleotide sequence of the gene encoding the full-length RSV F protein (SEQ ID No: 1) and the deduced amino acid sequence (SEQ ID No: 2). Figure 3 shows the gene encoding the secreted RSV F protein ~SEQ
ID No: 3) and the deduced amino acid sequence (SEQ ID No:
4).
A set of four plasmid DNA constructs were made (as WO 96' 'C9 ~r PCT/CA96/00398 shown schematically in Figures 4 to 7) in which cDNA
encoding the RSV-F was subcloned downstream of the immediate-early promoter, enhancer and intron A sequences of human cytomegalovirus (CMV) and upstream of the bovine growth hormone (BGH) poly-A site. The 1.6 Kb Sspl-PstI
fragment cont~; n; ng the promoter, Pnh~ncer and intron sequences of CMV Towne strain were initially derived from plasmid pRL43a obtained from Dr. G.S. Hayward of Johns Hopkins University (ref. 20) and subcloned between EcoRV
and PstI sites of pBluescript 11 SK +/- (Stratagene).
For the construction of plasmids expressing the secretory form of the F protein (pXL1 and pXL2 in Figs. 4 and 5), the 1.6 Kb ~coRI-BamHI fragment cont~;n;ng the truncated form of the F cDNA originally cloned from a clinical isolate belonging to subgroup A was excised from pRSVF
(ref. 18 and WO 93/14207) and subcloned between EcoRI and BamHI sites of pSG5 (Strategene, ref. 14). Either the 1.6 kb EcoRI-BamHI fragment or the 2.2-kb ClaI-BamHI
fragment was then excised from the pSG5 construct, filled-in with Klenow and subcloned at the SmaI site of the pBluescript II SK +/- construct containing the promoter and intron A sequences. The 0.6 kb ClaI-EcoRI
fragment derived from pSG5 contained the intron II
sequences from rabbit ~-globin. Subsequently, the plasmids were diges~ed with HindIII, filled-in with Klenow, and digested with XbaI to yield either a 3.2 or a 3.8 Kb fragment. These fragments were used to replace the 0.8 kb NruI-XbaI fragment cont~;n;ng the CMV promoter in pRc/CMV (Invitrogen), resulting in the final pXL1 and pXL2 constructs, respectively.
For the construction of plasmids expressing the full-length F protein (pXL3 and pXL4 - Figs. 6 and 7), the full length RSV F cDNA was excised as a 1.9 kb EcoRI
fragment from a recombinant pBluescript M13-SK
(Stratagene) containing the insert (ref. 18 and WO
93/14207) and subcloned at the EcoRI site of pSG5 W096/4094S PCTtCA96/00398 - ~Stratagene). Either the 1.9 Kb EcoRI fragment or the 2.5 Kb ClaI-BamHI fragment was then excised from the pSG5 construct, filled-in with Klenow and subcloned at the SmaI site of the pBluescript II SK +/- construct S contA;n;ng the promoter and intron A sequences. The rest of the construction for pXL3 and pXL4 was identical to that for pXL1 and pXL2, as described above. Therefore, except for the CMV promoter and intron A sequences, the rest of the vector components in pXLl-4 were derived from plasmid pRc/CMV. Plasmids pXLl and pXL2 were made to express a truncated/secretory form of the F protein which carried stop codons resulting in a C-terminal deletion of 48 amino acids including the transmembrane (TM) and the C-terminal cytosolic tail as compared to the intact molecule. In contrast, pXL3 and pXL4 were made to express the intact membrane-attached form of the RSV F
molecule containing the TM and the cytosolic C-terminal tail. The rationale for the presence of the intron II
sequences in pXL2 and pXL4 was that this intron was reported to mediate the correct splicing of RNAs. Since mRNA for the RSV-F has been suspected to have a tendency towards aberrant splicing, the presence of the intron II
sequences might help to overcome this. All four plasmid constructs were confirmed by DNA sequencing analysis.
Plasmid DNA was purified using plasmid mega kits from Qiagen (Chatsworth, CA, USA) according to the manufacturer's instructions.
- Example 2 This Example describes the immunization of mice.
Mice are susceptible to infection by RSV as described in ref. 16.
For intramuscular (i.m) ;~mllnization, the anterior tibialis anterior muscles of groups of 9 BALB/c mice (male, 6-8 week old) (Jackson Lab., Bar Harbor, ME, USA) were bilaterally injected with 2 x 50 ~g (1 ~g/~L in PBS) of pXLl-4, respectively. Five days prior to DNA
CA 022236l0 l997-l2-04 W096~9~ PCTICA96/00398 - injection, the muscles were treated with 2 x 50 ~L (10 ~M
in PBS) of cardiotoxin (Latoxan, France). Pretreatment of the muscles with cardiotoxin has been reported to increase DNA uptake and to PnhAnce the subsequent immune responses by the intramuscular route (ref. 24). These ~ni~-l S were similarly boosted a month later. Mice in the control group were ; mml-n; zed with a placebo plasmid cont~; n; ng identical vector backbone sequences without the RSV F gene according to the same sche~-lle. For intradermal (i.d.) immunization, 100 ~g of pXL2 (2 ~g/~L
in PBS) were injected into the skin 1-2 cm distal from the tall base. The ~n;m~l S were similarly boosted a month later.
Seventy-five days after the second immunization, mice were challenged intr~n~s~lly with 105-4 plaque forming units (pfu) of mouse-adapted RSV, A2 subtype (obtained from Dr. P. Wyde, Baylor College of Medicine, Houston, TE, USA). Lungs were aseptically ~e,uuved 4 days later, weighed and homogenized in 2 mL of complete culture medium. The number of pfu in lung homogenates was determined in duplicates as previously described (ref.
19) using vaccine quality Vero cells. These data were subjected to statistic analysis using SigmaStat (Jandel Scientific Software, Guelph, Ont. Canada).
Sera obtained from immlln;zed mice were analyzed for anti-RSV F antibody titres (IgG, IgG1 and IgG2a, respectively) by enzyme-linked immunosorbent assay (ELISA) and for RSV-specific pla~ue-reduction titres.
ELISA were performed using 96-well plates coated with i~l~no~ffinity purified RSV F protein ~50 ng/mL) and 2-fold serial dilutions of immune sera. A goat anti-mouse IgG antibody conjugated to alkaline phosphatase (Jackson ImmunoRes., Mississauga, Ont., G~n~ ) was used as secondary antibody. For the measurement of IgG1 and IgG2a antibody titres, the secondary antibodies used were monospecific sheep anti-mouse IgG1 (Serotec, Toronto, WO96J4C~1, PCT/CA96/00398 ~ Ont., Canada) and rat anti-mouse IgG2a (Zymed, San Francisco, CA, USA) antibodies conjugated to alkaline phosphatase, respectively. Plaque reduction titres were determined according to Prince et al ~ref. 19) using vaccine quality Vero cells. Four-fold serial dilutions of immune sera were incubated with 50 pfu of RSV, Long strain (ATCC) in culture medium at 37~C for 1 hr in the presence of 5~ CO2. Vero cells were then infected with the mixture. Plaques were fixed with 80~ methanol and developed 5 days later using a mouse anti-RSV-F
monoclonal IgG1 antibody and donkey antimouse IgG
antibody conjugated to peroxidase (Jackson ImmunoRes., Mississauga, Ont. Canada). The RSV-specific plaque reduction titre was defined as the dilution of serum sample yielding 60~ reduction in the number of plaques.
Both ELISA and plaque reduction assays were performed in duplicates and data are expressed as the means of two determinations. These data were subjected to statistic analysis using SigmaStat (Jandel Scientific Software, Guelph, Ont. Canada).
To ~x~mine the induction of RSV-specific CTL
following DNA immunization, spleens from 2 immunized mice were removed to prepare single cell suspensions which were pooled. Splenocytes were incubated at 2.5 x 106 cells/mL in complete RPMI medium containing 10 U/mL
murine interleukin 2 (IL-2) with ~-irradiated (3,000 rads) syngeneic splenocytes (2.5 x 106 cells/mL) infected with 1 TCIDs0/cell RSV (Long strain) for 2 hr. The source of murine IL-2 was supernatant of a mouse cell line constitutively secreting a high level of IL-2 obtained from Dr. H. Karasuyama of Basel Institute for Immunology (ref. 20). CTL activity was tested 5 days following the in vitro re-stimulation in a standard 4 hr chromium release assay. Target cells were 5 slCr-labelled uninfected BALB/c fibroblasts (BC cells) and persistently RSV-infected BCH14 fibroblasts, respectively. Washed w096,~(c915 PCTICA96/00398 responder cells were incubated with 2 x 103 target cells at varying effector to target ratios in 200 ~L in 96-well V-bottomed tissue-culture plates for 4 hr at 37~C
Spontaneous and total chromium releases were determined by incubating target cells with either medium or 2.5 Triton-X 100 in the absence of responder lymphocytes Percentage specific chromium release was calculated as (counts-spontaneous counts)/(total counts-spontaneous counts) X 100. Tests were performed in triplicates and data are expressed as the means of three determinations.
For antibody blocking studies in CTL assays, the effector cells were incubated for 1 hr with 10 ~g/ml final of purified mAb to CD4 (GK1.5) (ref. 21) or mAb against murine CD8 (53-6.7) (ref. 22) before adding chromium labelled BC or BCH4 cells. To determine the effect of anti-class I MHC antibodies on CTL killing, the chromium labelled target cells BC or BCH4 were incubated with 20 ~L of culture supernate of hybridoma that secretes a mAb that recognizes Kd and Dd of class I MHC (34-1-2S) (ref.
23) prior to the addition of effector cells.
Exam~le 3 This Example describes the immunogenicity and protection by polynucleotide immunization by the intramuscular route.
To characterize the antibody responses following i.m. DNA administration, immune sera were analyzed for anti-RSV F IgG antibody titre by ELISA and for RSV-specific plaque reduction titre, respectively. All four plasmid constructs were found to be immunogenic. Sera obtained from mice ;mml-n;zed with pXL1-4 demonstrated significant anti-RSV F IgG titres and RSV-specific plague reduction titres as compared to the placebo group (Table 1 below) (P<0.0061 and cO.0001, respecti~ely, Mann-Whitney Test). Howe~er, there is no significant difference in either anti-RSV F IgG titre or RSV-specific plaque reduction titre among mice ;mml-n;zed with either WO9~ PCT/CA96/00398 - pXLl, pXL2, pXL3 or pXL4.
To evaluate the protective ability of pXLl-4 against primary RSV infection of the lower respiratory tract, ; mmlm; zed mice were challenged intr~n~s~lly with mouse-adapted RSV and viral lung titres post challenge wereassessed. All four plasmid constructs were found to protect An;m~ls against RSV infection. A significant reduction in the viral lung titre was observed in mice immunized with pXLl-4 as compared to the placebo group ~Pc0.0001, Mann-Whitney Test). However, varying degrees of protection were observed depending on the plasmid. In particular, PXLl was more protective than pXL3 (P=0.00109, Mann-Whitney Test), and pXL4 more than pXL3 (P=0.00125), whereas only pXL2 induced complete protection. This conclusion was confirmed by another analysis with number of fully protected mice as end point (Fisher Exact Test). Constructs pXLl, pXL2 or pXL4 conferred a higher degree of protection than pXL3 (P~0.004, Fisher Exact Test) which was not more effective than placebo. Only pXL2 conferred full protection in all immunized mice.
The above statistical analysis revealed that PXLl conferred more significant protection than pXL3. The former expresses the truncated and secretory form and the latter the intact membrane anchored form of the RSV F
protein. Furthermore, pXL4 was shown to be more protective than pXL3. The difference between these two constructs is the presence of the intron II sequence in pXL4. Construct pXL2 which expresses the secretory form of the RSV-F in the context of the intron II sequence was the only plasmid that confers complete protection in all immnnized mice.
Exam~le 4 This Example describes the influence of the route of administration of pXL2 on its immll~ogenicity and protective ability.
WO9C,14091~ PCT/CA96/00398 ~ The i.m. and i.d. routes of DNA ~min;stration were compared for imm~lno~enicity in terms of anti-RSV F
antibody titres and RSV-specific plaque reduction titres.
Analyses of the imml~ne sera (Table 2 below) revealed that the i.d. route of DNA ~m; n; stration was as ;~ nogenic as the i.m. route as judged by anti-RSV F IgG and IgG1 antibody responses as well as RSV-specific plaque reduction titres. However, only the i.m. route induced significant anti-RSV F IgG2a antibody responses, whereas the IgG2a isotype titre was negligible when the i.d.
route was used. The i.m. and i.d. routes were also compared with respect to the induction of RSV-specific CTL. Significant RSV-specific CTL activity was detected in mice ;mmllnized intramuscularly. In contrast, the cellular response was significantly lower in mice inoculated intradermally ~Table 3 below). In spite of these differences, protection against primary RSV
infection of the lower respiratory tract was observed in both groups of mice immunized via either route (Table 4 below). The CTL induced by RSV-F DNA are classical CD8+
class I restricted CTL. The target cells, BCH4 fibroblasts express class I MHC only and do not express class II MHC. Further, prior incubation of BCH4 target cells with anti class-I MHC antibodies significantly blocked the lytic activity of RSV-F DNA induced CTL line.
While anti-CD8 antibody could partially block lysis of BCH4 cells, antibody to CD4 molecule had no effect at all ~Table 5 below). Lack of total blocking by mAb to CD8 could either be due to CTL being CD8 independent (me~n;ng that even though they are CD8+ CTL, their TCR has enough affinity for class I MHC+peptide and it does not require CD8 interaction with the alpha 3 of class I MHC) or the amount of antibody used in these experiments was limiting. There was no detectable lysis of YAC-1 (NK
sensitive target) cells (data not shown).
WO~f~C91~ PCT/CA96/00398 ~ ExamPle 5 This Example describes i~mnnization studies in cotton rats using pXL2.
The immune response of cotton rats to DNA
immtlnization was analyzed by the protocol shown in Table 6 below. On day -5, 40 cotton rats were randomly selected and divided into 8 groups of 5. Cotton rats in groups 1 and 7 were inoculated intramuscularly (i.m.) into the tiberlia anteria (TA) muscles bilaterally with cardiotoxin (1.0 ~M). On day -1, the cotton rats in group 8 were inoculated in the TA muscles with bupivacaine (0.25%). On day 0, several animals in each group were bled to determine levels of RSV-specific antibodies in the serum of the test animals prior to administration of vaccines. All of the ~nim~ls were then inoculated i.m. or intradermally ~i.d.) with 200 ~g of plasmid DNA, placebo (non-RSV-specific ~NA), 100 median cotton rat infectious doses (CRID50; positive control) of RSV, or of formalin inactivated RSV prepared in Hep-2 tissue culture cells and adjuvanted in alum. Forty-four days later the cotton rats in groups 1 & 7 were reinoculated with cardiotoxin in the TA muscles. Four days later (48 days after priming with vaccine), the animals in group 8 were reinoculated with bupivacains in the TA muscle of the right leg. The next day, (seven weeks after priming with vaccine) all of the ~nimAl5 were bled and all, except those in the group given live RSV, were boosted with the same material and doses used on day 0. 29 days later, each cotton rat was bled and then challenged intranasally ~i.n.) with 100 CRID50 RSV A2 grown in Hep-2 tissue culture cells. Four days after this virus challenge (day +88) all of the cotton rats were killed and their lungs removed. One lobe from each set of lungs was fixed in formalin and then processed for histologic evaluation of pulmonary histopathology. The rem~ining lobes of lung will be assessed for the presence WO96J1091~ PCT/CA96/00398 ~ and levels of RSV. Each of the sera collected on days 0, 49 and 78 were tested for RSV-neutralizing activity, anti-RSV fusion activity and RSV-specific ELISA antibody.
The RSV neutralizing titres on day +49 and +78 are shown in Tables 7(a) below and 7(b) below respectively.
As can be seen from the results shown in Table 7(a), on day +49 the ~n;m-l S ;mmlln; zed with live RSV and DNA
immunization had substantial RSV serum neutralizing titres. The ~n;m~ls ;~mt1n;zed with formalin-inactivated RSV had a neutralizing titre equivalent to the placebo group on day +49 but following boosting titres by day +78 had reached 5.8 (log10/0.05). Boosting had no significant effect upon ~n;m~l S immunized with live RSV or by i.m.
plasmid ;mmllnization~
RSV titres in nasal washes ~upper respiratory tract) on day +82 are shown in Table 8 below. RSV titres in the lungs (lower respiratory tract) on day +82 are shown in Table 9 below. All of the vaccines pro~ided protection against lung infection but under these conditions, only live virus provided total protection against upper respiratory tract infection.
The lungs from the cotton rats were ~m; n~d histologically for pulmonary histopathology and the results are shown in Table lO below. With the exception of lung sections obtained from Group 9 which were essentially free of inflammatory cells or evidence of inflammation, and those from Group 3, which exhibited the maximal plll~on~ry pathology seen in this study, all of the sections of lung obtained from the other groups looked familiar, i.e. scattered inflammatory cells were present in most fields, and there was some thickening of septae. These are evidence of mild inflammatory diseases. Large numbers of inflammatory cells and other evidence of inflammation were present in sections of lung from Group 3 (in which formalin-inactivated [FI] RSV
vaccine was given prior to virus challenge). This result -indicated that immunization with plasmid DNA expressing the RSV F protein does not result in pulmonary histopathology different from the placebo, whereas FI-RSV
caused more severe pathology.
SI~ARY OF THE DISCLOSURE
In summary of this disclosure, the present invention provides certain novel vectors containing genes encoding an RSV F proteins, methods of ;mmtln;zation using such vectors and methods of diagnosis using such vectors.
Modifications are possible within the scope of this invention.
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c o ~ - ~ ~ ~ 'o z WO 9~'1û3 15 PCT/CA9~ /~0338 Table 10. .c~ of ~Iistopatholo~ Results Seen in Sections of Cotton Rat Lung.
Group TlCA~ Major Ol,~.valions & C~.. - .. 1~
1. Placebo + RSV S~,a~,d individual and groups of L~ ,~h ges and pOl,~LuOll~h~ llrl~A~ l;)pileS (PMN) in all fields. Overt Ih.~ ;n~ of septae. OCCA~:;QTI~1 .oLic cells seen. Overall: ild to moderate ;--n~------~l;~n 2. Live RSV TCC~lAt~d LUal,lUph ges seen in most fields.
Sca~ d PMN. Overall: minimAI infl,.."".AI;~"
3. FI-RSV + RSV Vir~ally every field COIIIA;IIC ~ C,uus ...n~ f~r cells & PMN. Pyknotic cells and debris CCJL~ On. Th;rL~ nlcd septae. Evidence of e~ac~ àL~d disease.
. Plasmid + RSV T!;o1AtJ~ LuaClùl)hag~.S seen in most fields.
OCC~;OnA1 PMN seen. Very similar to live virus gTOUp.
6. Plasmid i.d. + Isolated Lllae.uphages seen in most fields.
RSV OCC'A';OI.A1 PMN seen.
7. Plas_id + CT Isolated n~t~n~.,rleAr cells and PMN seen in most + RSV fields.
8. Plasmid + Biv Scal~lcd mononuclear cells and PMN seen in + RSV most fields.
9. Normal CR Few l~uko~;yL~s evidence. Airy, open appeal~Luce.
Lung T_in septae.
CT = caldilu~
Biv = ~upivacaine REFEREN OES
1. McIntosh K., C~nock, R.M. In: Fields BN, Knipe, DM, editors. Virology. New York: Raven Press: 1990:
2. Katz SL., In: New Vaccine De~elopment establishing priorities. Vol. 1. W;~.Ch; n~ton: National Academic Press: 1985: 397-409.
3. Wertz GW, Sullender WM., Biotechnology 1992; 20:
FIE~D OF 1NV~N11ON
The present invention is related to the field of Respiratory Syncytial Virus (RSV) vaccines and is particularly concerned with vaccines comprising nucleic acid sequences encoding the fusion (F) protein of RSV.
REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of copending United States Patent Application No.
08/476,397, filed June 7, 1995.
BACKGROUND OF lNv~NllON
Respiratory syncytial virus (RSV), a negative-strand RNA virus belonging to the Paramyxoviridae family of viruses, is the major viral pathogen responsible for bronchiolitis and pneumonia in infants and young children (ref. 1 - Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Acute respiratory tract - infections caused by RSV result in approximately 90,000 hospitalizations and 4,500 deaths per year in the United States (ref. 2). Medical care costs due to RSV infection are greater than $340 M annually in the United States alone (ref. 3). There is currently no licensed vaccine against RSV. The main approaches for developing an RSV
vaccine have included inactivated virus, live-attenuated viruses and subunit vaccines.
The F protein of RSV is considered to be one of the most important protective antigens of the virus. There is a significant similarity (89~ identity) in the amino acid sequences of the F proteins from RSV subgroups A and WO3~'1091' PCT/CA96/~398 B (ref. 3) and anti-F antibodies can cross-neutralize viruses of both subgroups as well as protect ;~mlln;zed ~n;m~l S against infection with viruses from both subgroups (ref. 4). Furthermore, the ~ protein has been identified as a major target for RSV-specific cytotoxic T-lymphocytes in mice and hl~m~n5 (ref. 3 and ref. 5).
The use of RSV proteins as vaccines may have obstacles. Parenterally ~m;n;stered vaccine candidates have so far proven to be poorly ;mm-~nogenic with regard to the induction of neutralizing antibodies in seronegative hllm~nc or chimpanzees. The serum antibody response induced by these antigens may be further diminished in the presence of passively acquired antibodies, such as the transplacentally acquired maternal antibodies which most young infants possess. A
subunit vaccine candidate for RSV consisting of purified fusion glycoprotein from RSV infected cell cultures and purified by ;mmllno~ffinity or ion-exchange chromatography has been described (ref. 6). Parenteral immunization of seronegative or seropositive chimpanzees with this preparation was performed and three doses of 50 ~g were required in seronegative animals to induce an RSV serum neutralizing titre of approximately 1:50. Upon subsequent challenge of these ~nim~ls with wild-type RSV, no effect of immunization on virus shedding or clinical disease could be detected in the upper respiratory tract.
The effect of ;mmlln;zation with this vaccine on virus shedding in the lower respiratory tract was not investigated, although this is the site where the serum antibody induced by parenteral immunization may be expected to have its greatest effect. Safety and immunogenicity studies have been performed in a small number of seropositive individuals. The vaccine was found to be safe in seropositive children and in three seronegative children (all ~ 2.4 years of age). The effects of immunization on lower respiratory tract WO~6~10~1, PCT/CA96/00398 -disease could not be determined because of the small number of children ;~ nlzed. One immunizing dose in seropositive children induced a 4-fold increase in virus neutralizing antibody titres in 40 to 60~ of the vaccinees. Thus, insufficient information is available from these small studies to evaluate the efficacy of this vaccine against RSV-induced disease. A further problem facing subunit RSV vaccines is the possibility that inoculation of seronegative subjects with immunogenic preparations might result in disease enhancement (sometimes referred to as immunopotentiation), similar to that seen in formalin inactivated RSV vaccines. In some studies, the immune response to immunization with RSV F
protein or a synthetic RSV FG fusion protein resulted in a disease enhancement in rodents resembling that induced by a formalin-inactivated RSV vaccine. The association of immunization with disease enhancement using non-replicating antigens suggests caution in their use as vaccines in seronegative h11m~nc.
Live attenuated vaccines against disease caused by RSV may be promising for two main reasons. Firstly, infection by a live vaccine virus induces a balanced immune response comprising mucosal and serum antibodies and cytotoxic T-lymphocytes. Secondly, infection of infants with live attenuated vaccine candidates or naturally acquired wild-type virus is not associated with enhanced disease upon subsequent natural reinfection. It will be challenging to produce live attenuated vaccines that are ~mm-1nogenic for younger infants who possess maternal virus-neutralizing antibodies and yet are attenuated for seronegative infants greater than or equal to 6 months of age. Attenuated live virus vaccines also have the risks of residual virulence and genetic instability.
Injection of plasmid DNA containing sequences encoding a foreign protein has been shown to result in w0~6,~c9t~ PCT/CA96/00~98 -expression of the foreign protein and the induction of antibody and cytotoxic T-lymphocyte responses to the antigen in a number of studies (see, for example, refs 7, 8, 9). The use of plasmid DNA inoculation to express ~iral proteins for the purpose of ;m~lln;zation may offer several advantages over the strategies summarized above Firstly, DNA encoding a viral antigen can be introduced in the presence of antibody to the virus itself, without loss of potency due to neutralization of virus by the antibodies. Secondly, the antigen expressed in vivo should exhibit a native conformation and, therefore, should induce an antibody response similar to that induced by the antigen present in the wild-type virus infection. In contrast, some processes used in purification of proteins can induce conformational changes which may result in the loss of ;m~l~nogenicity of protective epitopes and possibly immunopotentiation.
Thirdly, the expression of proteins from injected plasmid DNAs can be detected in vivo for a considerably longer period of time than that in virus-infected cells, and this has the theoretical advantage of prolonged cytotoxic T-cell induction and enhanced antibody responses.
Fourthly, in vivo expression of antigen may provide protection without the need for an extrinsic adjuvant.
The ability to immunize against disease caused by RSV by administration of a DNA molecule encoding an RSV
F protein was unknown before the present invention. In particular, the efficacy of immunization against RSV
induced disease using a gene encoding a secreted form of the RSV F protein was unknown. Infection with RSV leads to serious disease. It would be useful and desirable to provide isolated genes encoding RSV F protein and vectors for in vivo administration for use in ;m~llnogenic preparations, including vaccines, for protection against disease caused by RSV and for the generation of diagnostic reagents and kits. In particular, it would be WO96/4091, PCT/CA96/00398 s desirable to provide vaccines that are ~ nogenic and protective in h-lm~nq, including seronegative infants, that do not cause disease enhancement (;mmllnopotentiation).
SU~D$ARY OF INV~N11ON
The present invention relates to a method of immunizing a host against disease caused by respiratory syncytial virus, to nucleic acid molecules used therein, and to diagnostic procedures utilizing the nucleic acid molecules. In particular, the present invention is directed towards the provision of nucleic acid respiratory syncytial virus vaccines.
In accordance with one aspect of the invention, there is provided a vector, comprising:
a first nucleotide sequence encoding an RSV F
protein or a protein capable of inducing antibodies that specifically react with RSV F protein;
a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV F protein, and a second nucleotide sequence located adjacent the first nucleotide sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo from the vector in a host.
The first nucleotide sequence may be that which encodes a full-length RSV F protein, as seen in Figure 2 (SEQ ID No: 2). Alternatively, the first nucleotide sequence may be that which encodes an RSV F protein from which the tr~nq~emhrane region is absent. The latter embodiment may be provided by a nucleotide sequence which encodes a full-length RSV F protein but contains a translational stop codon immediately upstream of the start of the transmembrane coding region, thereby preventing expression of a transmembrane region of the RSV F protein, as seen in Figure 3 (SEQ. ID No. 4). The lack of expression of the transmembrane region results in W096/40945 PCT/CA96/0~98 a secreted form of the RSV F protein.
The second nucleotide sequence may ~;G~ ise a pair of splice sites to prevent aberrant mRNA splicing, whereby substantially all transcribed mRNA encodes the RSV protein. Such second nucleotide sequence may be located between the first nucleotide sequence and the promoter sequence. Such second nucleotide sequence may be that of rabbit ~-globin intron II, as shown in Figure 8 (SEQ ID No: 5).
A vector enCo~;n~ the F protein and provided by this aspect of the invention may specifically be pXL2 or pXL4, as seen in Figures 5 or 7.
The promoter sequence may be an imme~-ate early cytomegalovirus (CMV) promoter. Such cytomegalovirus promoter has not previously been employed in vectors containing nucleotide sequences encoding an RSV F
protein.
Accordingly, in another aspect of the invention, there is provided a vector, comprising:
a first nucleotide sequence encoding an RSV F
protein or a protein capable of generating antibodies that specifically react with RSV F protein, and a cytomegalovirus promoter operatively coupled to the first nucleotide sequence for expression of the RSV
F protein.
The first nucleotide sequence may be any of the alternatives described above. The second nucleotide sequence, included to enhance the immunoprotective ability of the RSV F protein when expressed in vivo from the vector in a host, described above also may be present located adjacent a first nucleotide sequence in a vector provided in accordance with this second aspect of the invention.
Certain of the vectors provided herein may be used to immunize a host against RSV infection or disease by in vivo expression of RSV F protein lacking a trAncm~mhrane WO~ C31~ PCT/CA96/00398 region following ~m; n;stration of the vectors. In accordance with a further aspect of the present invention, therefore, there is provided a method of ;~ml~nizing a host against disease caused by infection with respiratory syncytial virus, which comprises administering to the host an effective amount of a vector comprising a first nucleotide sequence encoding an RSV F
protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F protein lacking a tr~nc~emhrane region and a promoter seguence operatively coupled to the first nucleotide sequence for expression of the RSV F protein in the host, which may be a human. The promoter may be an immediate early cytomegalovirus promoter.
The nucleotide sequence encoding the truncated RSV
F protein lacking the transmembrane region may be that as described above.
A vector cont~; n; ng a second nucleotide sequence located adjacent a first nucleotide sequence encoding an RSV F protein, a protein capable of inducing antibodies that specifically react with RSV F protein or an RSV F
protein lacking a transmembrane region and effective to enhance the immunoprotective ability of the RSV F protein expressed by the first nucleotide sequence may be used to immunize a host. Accordingly, in an additional aspect of the present invention, there is provided a method of immunizing a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises administering to the host an effective amount of a vector comprising a first nucleotide sequence encoding an RSV F
protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F protein lacking a transmembrane region, a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV F protein, and a second nucleotide sequence located adjacent the first sequence to Pnh~nce W0~ 3t5 PCT/CA96t~398 the immunoprotective ability of the RSV-F protein when expressed in vivo from said vector in said host.
Specific vectors which may be used in this aspect of the invention are those identified as pXL2 and pXL4 in Figures 5 and 7.
The present invention also includes a novel method of using a gene enco~i ng an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F protein lacking a transmembrane region to protect a host against disease caused by infection with respiratory syncytial virus, which comprises:
isolating the gene;
operatively linking the gene to at least one control sequence to produce a vector, said control sequence directing expression of the RSV F protein when said vector is introduced into a host to produce an immune response to the RSV F protein, and introducing the vector into the host.
The procedure provided in accordance with this aspect of the invention may further include the step of:
operatively linking the gene to an immunoprotection enhancing sequence to produce an enhanced immunoprotection by the RSV F protein in the host, preferably by introducing the immunoprotection enhancing sequence between the control sequence and the gene.
In addition, the present invention includes a method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus, which comprises:
isolating a first nucleotide sequence encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F
protein lacking a transmembrane region;
operatively linking the first nucleotide sequence to at least one control sequence to produce a vector, the W O 96/40945 PC~r/CA96/00398 control sequence directing expression of the RS V F
protein when introduced into a host to produce an lmmltne response to the RSV F protein when expressed in vivo from the vector in a host, and formulating the vector as a vaccine for in vivo administration.
The first nucleotide sequence further may be operatively linked to a second nucleotide sequence to enhance the lmm~lnoprotective ability of the R SV F protein when expressed in vivo from the vector in a host. The vector may be selected from pXL1, pXL2 and pXL4. The invention further includes a vaccine for A~m; n; stration to a host, including a human host, produced by this method as well as immunogenic compositions comprising an immunoeffective amount of the vectors described herein.
As noted previously, the vectors provided herein are useful in diagnostic applications. In a further aspect of the invention, therefore, there is provided a method of determining the presence of an RSV F protein in a sample, comprising the steps of:
(a) immunizing a host with a vector comprising a first nucleotide sequence encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RS V F protein or an RS V F
protein lacking a transmembrane region and a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RS V F
protein in the host to produce antibodies specific for the RSV F protein;
(b) isolating the RS V F protein specific antibodies;
(c) contacting the sample with the isolated antibodies to produce complexes comprising any RSV
F protein present in the sample and the RS V F
protein- specific antibodies; and (d) determining production of the complexes.
.
WOgG,J~9-~ PCT/CA96/00398 ~ The vector employed to elicit the antibodies may be pXLl, pXL2, pXL3 or pXI4.
The inYention also includes a diagnostic kit for detecting the presence of an RSV F protein in a sample, comprising:
(a) a vector comprising a first nucleotide sequence encoding an RSV F protein, a protein capahle of generating antibodies that specifically react with RSV F protein, or a RSV F protein lacking a transmembrane region and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein in a host ;~mtlnlzed therewith to produce antibodies specific for the RSV F protein;
(b) isolation means to isolate said RSV F protein specific antibodies;
(c) contacting means to contact the isolated RSV F
specific antibodies with the sample to produce a complex comprising any RSV F protein present in the sample and RSV F protein specific antibodies; and (d) identifying means to determine production of the complex.
The present invention is further directed to immunization wherein the polynucleotide is an RNA
molecule which codes for an RSV F protein, a protein capable of inducing antibodies that specifically react with RSV F protein or an RSV F protein lacking a tr~nsm~mhrane region.
The present invention is further directed to a method for producing RSV F protein specific polyclonal antibodies comprising the use of the ;mmllnization method described herein, and further comprising the step of isolating the RSV F protein specific polyclonal antibodies from the ;mmllnt zed ~n;m~1 , The present invention is also directed to a method for producing monoclonal antibodies specific for an F
WO~G/4C915 PCT/CA96/~398 protein of RSV, comprising the steps of:
(a) constructing a vector comprising a first nucleotide seguence encoding a RSV F protein and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F
protein; and, optionally, a second nucleotide sequence located adjacent said first nucleotide sequence to ~nh~nce the imTl-noprotective ability of said RSV F protein when expressed in vi~o from said vector in a host.
(b) administering the vector to at least one mouse to produce at least one ;mmlln;zed mouse;
(c) ~e,.,~ving B-lymphocytes from the at least one ;m~lln; zed mouse;
(d) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;
(e) cloning the hybridomas;
(f) selecting clones which produce anti-F protein antibody;
(g) culturing the anti-F protein antibody-producing clones; and (h) isolating anti-F protein monoclonal antibodies.
In this application, the term "RSV F protein~ is used to define a full-length RSV F protein, such proteins having variations in their amino acid sequences including those naturally occurring in various strains of RSV, a secreted form of RSV F protein lacking a transmembrane region, as well as functional analogs of the RSV F
protein. In this application, a first protein is a "functional analog" of a second protein if the first protein is immunologically related to and/or has the same function as the second protein. The functional analog may be, for example, a fragment of the protein or a substitution, addition or deletion mutant thereof.
WO~,/4~91r PCT/CA96/00398 BRIEF DESCRIPTION OF THE FIGURES
The present invention will be further understood from the following General Description and Examples with reference to the Figures in which:
Figure 1 illustrates a restriction map of the gene encoding the F protein of Respiratory Syncytial Virus;
Figure 2 illustrates the nucleotide sequence of the gene encoding the membrane attached form of the F protein of Respiratory Syncytial Virus (SEQ ID No: 1) as well as the amino acid sequence of the RSV F protein encoded thereby (SEQ ID No: 2);
Figure 3 illustrates the nucleotide sequence of the gene encoding the secreted form of the RSV F protein lacking the trAn~m~mhrane region (SEQ ID No: 3) as well as the amino acid sequence of the truncated RSV F protein lacking the tr~ncmemhrane region encoded thereby (SEQ ID
No: 4)i Figure 4 shows the construction of plasmid pXLl containing the gene encoding a secreted form of the RSV
F protein lacking the transmembrane region;
Figure 5 shows the construction of plasmid pXL2 containing a gene encoding a secreted form of the RSV F
protein lacking the transmembrane region and containing the rabbit ~-globin Intron II sequence;
Figure 6 shows the construction of plasmid pXL3 containing the gene encoding a full length membrane attached form of the RSV F protein;
Figure 7 shows the construction of plasmid pXL4 containing a gene encoding a membrane attached form of the RSV F protein and containing the rabbit ~-globin Intron II sequencei and Figure 8 shows the nucleotide sequence for the rabbit ~-globin Intron II sequence (SEQ ID No. 5).
W09"~031~ PCT/CA96100398 GENERAL DESCRIPTION OF INV~N 110N
AS described above, the present invention relates generally to polynucleotide, including DNA, immunization to obtain protection against infection by respiratory syncytial virus ~RSV) and to diagnostic procedures using particular vectors. In the present invention, several recombinant vectors were constructed to contain a nucleotide sequence encoding an RSV F protein.
The nucleotide sequence of the full length RSV F
gene is shown in Figure 2 (SEQ ID No: 1). Certain constructs provided herein include the nucleotide sequence encoding the full-length RSV F (SEQ ID NO 2) protein while others include an RSV F gene modified by insertion of termination codons immediately upstream of the tr~nc~emhrane coding region (see Figure 3, SEQ ID No:
3), to prevent expression of the tr~ncm~mhrane portion of the protein and to produce a secreted or truncated RSV F
protein lacking a transmembrane region (SEQ ID No. 4).
The nucleotide sequence encoding the RSV F protein is operatively coupled to a promoter sequence for expression of the encoded RSV F protein. The promoter sequence may be the immediately early cytomegalovirus (CMV) promoter. This promoter is described in ref. 13.
Any other convenient promoter may be used, including constitutive promoters, such as, Rous Sarcoma Virus LTRs, and inducible promoters, such as metallothionine promoter, and tissue specific promoters.
The vectors provided herein, when ~;n;stered to an animal, effect in vivo RSV F protein expression, as demonstrated by an antibody response in the ~n;~l to which it is administered. Such antibodies may be used herein in the detection of RSV protein in a sample, as described in more detail below. When the encoded RSV F
protein is in the form of an RSV F protein from which the 3~ transmembrane region is absent, such as plasmid pXL1 ~Figure 4), the administration of the vector conferred CA 022236l0 l997-l2-04 WO 9G,'l~Y1, PCT/CA96/00398 protection in mice and cotton rats to challenge by live RSV virus neutralizing antibody and cell mediated 1~mtln responses and an absence of immunopotentiation in ;mml-n;zed ~n;~l S, as seen from the Examples below.
The recombinant vector also may include a second nucleotide sequence located adjacent the RSV F protein encoding nucleotide sequence to ~nh~nce the ;~mllnoprotective ability of the RSV F protein when expressed in ~ivo in a host. Such enhancement may be provided by increased in ~ivo expression, for example, by increased mRNA stability, enhanced transcription and/or translation. This additional sequence preferably is located between the promoter sequence and the RSV F
protein-encoding sequence.
This enhancement sequence may comprise a pair of splice sites to prevent aberrant mRNA splicing during transcription and translation so that substantially all transcribed mRNA encodes an RSV F protein. Specifically, rabbit ~-globin Intron II sequence shown in Figure 7 ~SEQ
ID No: 5) may provide such splice sites, as also described in ref. 15.
The constructs cont~;n1ng the Intron II sequence, CMV promoter and nucleotide sequence coding for the truncated RSV F protein lacking a transmembrane region, i.e. plasmid pXL2 ~Figure 5), induced complete protection in mice against challenge with live RSV, as seen in the Examples below. In addition, the constructs cont~; n; ng the Intron II sequence, CMV promoter and nucleotide sequence coding for the full-length RSV F protein, i.e.
plasmid pXI.4 (Figure 7), also conferred protection in mice to challenge with live RSV, as seen from the Examples below.
The vector provided herein may also comprise a third nucleotide sequence encoding a further antigen from RSV, an antigen from at least one other pathogen or at least one immunomodulating agent, such as cytokine. Such vector may contain said third nucleotide sequence in a ch;~eric or a bicistronic structure. Alternatively, vectors containing the third nucleotide sequence may be separately constructed and co~m;nlstered to a host, with the nucleic acid molecule provided herein.
The vector may further comprise a nucleotide sequence encoding a heterologous signal peptide, such as human tissue plasminogen activator ~TPA), in place of the endogenous signal peptide.
It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis and treatment of RSV infections. A further non-limiting discussion of such uses is further presented below.
1. Vaccine Preparation and ~se Immunogenic compositions, suitable to be used as vaccines, may be prepared from the RSV F genes and vectors as disclosed herein. The vaccine elicits an immune response in a subject which includes the production of anti-F antibodies. Immunogenic compositions, including vaccines, containing the nucleic acid may be prepared as injectables, in physiologically-acceptable liquid solutions or emulsions for polynucleotide administration. The nucleic acid may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a nucleic acid liposome (for example, as described in WO 9324640, ref.
17) or the nucleic acid may be associated with an adjuvant, as described in more detail below. Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture up to 100~
of the polynucleotide. In addition, the polycationic complexes fuse with cell membranes, resulting in an intracellular delivery of polynucleotide that bypasses wo~6~as~ PCT/CA96/00398 .
the degradative enzymes of the lysosomal compartment.
Published PCT application WO 94/27435 describes compositions for genetic ;~ nization comprising cationic lipids and polynucleotides. Agents which assist in the cellular uptake of nucleic acid, such as calcium ions, viral proteins and other transfection facilitating agents, may advantageously be used.
Polynucleotide ;~llnogenic preparations may also be formulated as microcapsules, including biodegradable time-release particles. Thus, U.S. Patent 5,151,264 describes a particulate carrier of a phospholipid/glycolipid/polysaccharide nature that has been termed Bio Vecteurs Supra Moléculaires (BVSM). The particulate carriers are intended to transport a variety of molecules having biological activity in one of the layers thereof.
U.S. Patent 5,075,109 describes encapsulation of the antigens trinitrophenylated keyhole limpet hemocyanin and staphylococcal enterotoxin B in 50:50 poly ~DL-lactideco-glycolide). Other polymers for encapsulation aresuggested, such as poly(glycolide), poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone, poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters and poly(8-hydroxybutyric acid), and polyanhydrides.
Published PCT application WO 91/06282 describes a delivery vehicle comprising a plurality of bioadhesive microspheres and antigens. The microspheres being of starch, gelatin, dextran, collagen or albumin. This delivery vehicle is particularly intended for the uptake of vaccine across the nasal mucosa. The delivery vehicle may additionally contain an absorption enhancer.
The RSV F genes and vectors may be mixed with pharmaceutically acceptable excipients which are compatible therewith. Such excipients may include, water, saline, dextrose, glycerol, ethanol, and -wos6!lo91~ PCT/CA96/00398 combinations thereof. The ;mm~1nogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enh~n~e the effectiveness thereof.
Immunogenic compositions and vaccines may be ~m;nl stered parenterally, by injection subcutaneously, intravenously, intradermally or intramuscularly, possibly following pretreatment of the injection site with a local anesthetic. Alternatively, the ;mmllnogenic compositions formed according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces. Thus, the immunogenic composition may be ~mi n; stered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes.
Alternatively, other modes of A~m; n; stration including suppositories and oral formulations may be desirable.
For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. Oral formulations may include normally employed incipients, such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.
The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and ;mm1lnogenic.
The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize the RSV F
protein and antibodies thereto, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Howe~er, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of about 1 ~g to about 1 mg of the RSV F genes and vectors. Suitable regimes for initial administration and booster doses are also -variable, but may include an initial A~min;stratio followed by subseguent A~mi n; strations. The dosage may also depend on the route of a~m;n;stration and will vary according to the size of the host. A vaccine which protects against only one pathogen is a monovalent vaccine. Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.
Immunogenicity can be significantly improved if the vectors are co-A~mt n; stered with adjuvants, commonly used as 0.05 to 0.1 percent solution in phosphate-buffered saline. Adjuvants ~nhAnce the imm~nQgenicity of an antigen but are not necessarily immunogenic themselves.
Adjuvants may act by retA;n;ng the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Thus, adjuvants have been identified that enhance the immune response to antigens.
Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many An;m~ls. Indeed, only all-m;nllm hydroxide and al~m;nl~m phosphate (collectively commonl y referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.
A wide range of extrinsic adjuvants and other immunomodulating material can provoke potent ;mml~ne responses to antigens. These include saponins complexed to membrane protein antigens to produce immune WOg~'a915 PCT/CA96/00398 stimulating complexes tISCOMS), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as monophoryl lipid A, QS 21 and polyphosphazene.
In particular embodiments of the present invention, the vector comprising a first nucleotide sequence encoding an F protein of RSV may be delivered in conjunction with a targeting molecule to target the vector to selected cells including cells of the immllne system.
The polynucleotide may be delivered to the host by a variety of procedures, for example, Tang et al. (ref.
10) disclosed that introduction of gold microprojectiles coated-with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH
antibodies in the mice, while Furth et al. (ref. 11) showed that a jet injector could be used to transfect skin, muscle, fat and m~mm~ry tissues of living animals.
20 2. Immunoa8~ay8 The RSV F genes and vectors of the present invention are useful as immunogens for the generation of anti-F
antibodies for use in immunoassays, including enzyme-linked immtlnosorbent assays (ELISA), RIAs and other non-25 enzyme linked antibody binding assays or procedures knownin the art. In ELISA assays, the vector first is administered to a host to generate antibodies specific to the RSV F protein. These RSV F-specific antibodies are immobilized onto a selected surface, for example, a surface capable of binding the antibodies, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed antibodies, a nonspecific protein such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample may be bound to the selected surface.
This allows for blocking of nonspecific adsorption sites -w0961~c9~5 pcTlcAs6l~398 on the immobilizing surface and thus reduces the background caused by nonspecific bindings of antisera onto the surface.
The immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a m~n~er conducive to j~mlln~ complex (antigen/antibody) formation. This procedure may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from about 2 to 4 hours, at temperatures such as of the order of about 20~ to 37~C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound RS~ F specific antibodies, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined.
BIOLOGICAL MATERIALS
Certain plasmids that contain the gene encoding RSV
F protein and referred to herein have been deposited with the America Type Culture Collection ~ATCC) located at 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., pursuant to the Budapest Treaty and prior to the filing of this application.
Samples of the deposited plasmids will become available to the public upon grant of a patent based upon this United States patent application and all restrictions on access to the deposits will be ,el..o~ed at that time. The invention described and claimed herein is not to be limited in scope by plasmids deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar plasmids that encode similar or equivalent antigens as WOg6,4C915 PCT/CA96/00398 described in this application are within the scope of the invention.
Plasmid ATCC Desiqnation Date DeDosited pXLl 97167 May 30, 199 S pXL2 97168 May 30, 1995 pXL3 97169 May 30, 1995 pXL4 97170 May 30, 1995.
EXANPLES
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.
Methods of molecular genetics, protein biochemistry, and immunology used but not explicitly described in this disclosure and these Examples are amply reported in the scientific literature and are well within the a~ility of those skilled in the art.
Exam~le 1 This Example describes the construction of vectors containing the RSV F gene.
Figure 1 shows a restriction map of the gene encoding the F protein of Respiratory Syncytial Virus and Figure 2 shows the nucleotide sequence of the gene encoding the full-length RSV F protein (SEQ ID No: 1) and the deduced amino acid sequence (SEQ ID No: 2). Figure 3 shows the gene encoding the secreted RSV F protein ~SEQ
ID No: 3) and the deduced amino acid sequence (SEQ ID No:
4).
A set of four plasmid DNA constructs were made (as WO 96' 'C9 ~r PCT/CA96/00398 shown schematically in Figures 4 to 7) in which cDNA
encoding the RSV-F was subcloned downstream of the immediate-early promoter, enhancer and intron A sequences of human cytomegalovirus (CMV) and upstream of the bovine growth hormone (BGH) poly-A site. The 1.6 Kb Sspl-PstI
fragment cont~; n; ng the promoter, Pnh~ncer and intron sequences of CMV Towne strain were initially derived from plasmid pRL43a obtained from Dr. G.S. Hayward of Johns Hopkins University (ref. 20) and subcloned between EcoRV
and PstI sites of pBluescript 11 SK +/- (Stratagene).
For the construction of plasmids expressing the secretory form of the F protein (pXL1 and pXL2 in Figs. 4 and 5), the 1.6 Kb ~coRI-BamHI fragment cont~;n;ng the truncated form of the F cDNA originally cloned from a clinical isolate belonging to subgroup A was excised from pRSVF
(ref. 18 and WO 93/14207) and subcloned between EcoRI and BamHI sites of pSG5 (Strategene, ref. 14). Either the 1.6 kb EcoRI-BamHI fragment or the 2.2-kb ClaI-BamHI
fragment was then excised from the pSG5 construct, filled-in with Klenow and subcloned at the SmaI site of the pBluescript II SK +/- construct containing the promoter and intron A sequences. The 0.6 kb ClaI-EcoRI
fragment derived from pSG5 contained the intron II
sequences from rabbit ~-globin. Subsequently, the plasmids were diges~ed with HindIII, filled-in with Klenow, and digested with XbaI to yield either a 3.2 or a 3.8 Kb fragment. These fragments were used to replace the 0.8 kb NruI-XbaI fragment cont~;n;ng the CMV promoter in pRc/CMV (Invitrogen), resulting in the final pXL1 and pXL2 constructs, respectively.
For the construction of plasmids expressing the full-length F protein (pXL3 and pXL4 - Figs. 6 and 7), the full length RSV F cDNA was excised as a 1.9 kb EcoRI
fragment from a recombinant pBluescript M13-SK
(Stratagene) containing the insert (ref. 18 and WO
93/14207) and subcloned at the EcoRI site of pSG5 W096/4094S PCTtCA96/00398 - ~Stratagene). Either the 1.9 Kb EcoRI fragment or the 2.5 Kb ClaI-BamHI fragment was then excised from the pSG5 construct, filled-in with Klenow and subcloned at the SmaI site of the pBluescript II SK +/- construct S contA;n;ng the promoter and intron A sequences. The rest of the construction for pXL3 and pXL4 was identical to that for pXL1 and pXL2, as described above. Therefore, except for the CMV promoter and intron A sequences, the rest of the vector components in pXLl-4 were derived from plasmid pRc/CMV. Plasmids pXLl and pXL2 were made to express a truncated/secretory form of the F protein which carried stop codons resulting in a C-terminal deletion of 48 amino acids including the transmembrane (TM) and the C-terminal cytosolic tail as compared to the intact molecule. In contrast, pXL3 and pXL4 were made to express the intact membrane-attached form of the RSV F
molecule containing the TM and the cytosolic C-terminal tail. The rationale for the presence of the intron II
sequences in pXL2 and pXL4 was that this intron was reported to mediate the correct splicing of RNAs. Since mRNA for the RSV-F has been suspected to have a tendency towards aberrant splicing, the presence of the intron II
sequences might help to overcome this. All four plasmid constructs were confirmed by DNA sequencing analysis.
Plasmid DNA was purified using plasmid mega kits from Qiagen (Chatsworth, CA, USA) according to the manufacturer's instructions.
- Example 2 This Example describes the immunization of mice.
Mice are susceptible to infection by RSV as described in ref. 16.
For intramuscular (i.m) ;~mllnization, the anterior tibialis anterior muscles of groups of 9 BALB/c mice (male, 6-8 week old) (Jackson Lab., Bar Harbor, ME, USA) were bilaterally injected with 2 x 50 ~g (1 ~g/~L in PBS) of pXLl-4, respectively. Five days prior to DNA
CA 022236l0 l997-l2-04 W096~9~ PCTICA96/00398 - injection, the muscles were treated with 2 x 50 ~L (10 ~M
in PBS) of cardiotoxin (Latoxan, France). Pretreatment of the muscles with cardiotoxin has been reported to increase DNA uptake and to PnhAnce the subsequent immune responses by the intramuscular route (ref. 24). These ~ni~-l S were similarly boosted a month later. Mice in the control group were ; mml-n; zed with a placebo plasmid cont~; n; ng identical vector backbone sequences without the RSV F gene according to the same sche~-lle. For intradermal (i.d.) immunization, 100 ~g of pXL2 (2 ~g/~L
in PBS) were injected into the skin 1-2 cm distal from the tall base. The ~n;m~l S were similarly boosted a month later.
Seventy-five days after the second immunization, mice were challenged intr~n~s~lly with 105-4 plaque forming units (pfu) of mouse-adapted RSV, A2 subtype (obtained from Dr. P. Wyde, Baylor College of Medicine, Houston, TE, USA). Lungs were aseptically ~e,uuved 4 days later, weighed and homogenized in 2 mL of complete culture medium. The number of pfu in lung homogenates was determined in duplicates as previously described (ref.
19) using vaccine quality Vero cells. These data were subjected to statistic analysis using SigmaStat (Jandel Scientific Software, Guelph, Ont. Canada).
Sera obtained from immlln;zed mice were analyzed for anti-RSV F antibody titres (IgG, IgG1 and IgG2a, respectively) by enzyme-linked immunosorbent assay (ELISA) and for RSV-specific pla~ue-reduction titres.
ELISA were performed using 96-well plates coated with i~l~no~ffinity purified RSV F protein ~50 ng/mL) and 2-fold serial dilutions of immune sera. A goat anti-mouse IgG antibody conjugated to alkaline phosphatase (Jackson ImmunoRes., Mississauga, Ont., G~n~ ) was used as secondary antibody. For the measurement of IgG1 and IgG2a antibody titres, the secondary antibodies used were monospecific sheep anti-mouse IgG1 (Serotec, Toronto, WO96J4C~1, PCT/CA96/00398 ~ Ont., Canada) and rat anti-mouse IgG2a (Zymed, San Francisco, CA, USA) antibodies conjugated to alkaline phosphatase, respectively. Plaque reduction titres were determined according to Prince et al ~ref. 19) using vaccine quality Vero cells. Four-fold serial dilutions of immune sera were incubated with 50 pfu of RSV, Long strain (ATCC) in culture medium at 37~C for 1 hr in the presence of 5~ CO2. Vero cells were then infected with the mixture. Plaques were fixed with 80~ methanol and developed 5 days later using a mouse anti-RSV-F
monoclonal IgG1 antibody and donkey antimouse IgG
antibody conjugated to peroxidase (Jackson ImmunoRes., Mississauga, Ont. Canada). The RSV-specific plaque reduction titre was defined as the dilution of serum sample yielding 60~ reduction in the number of plaques.
Both ELISA and plaque reduction assays were performed in duplicates and data are expressed as the means of two determinations. These data were subjected to statistic analysis using SigmaStat (Jandel Scientific Software, Guelph, Ont. Canada).
To ~x~mine the induction of RSV-specific CTL
following DNA immunization, spleens from 2 immunized mice were removed to prepare single cell suspensions which were pooled. Splenocytes were incubated at 2.5 x 106 cells/mL in complete RPMI medium containing 10 U/mL
murine interleukin 2 (IL-2) with ~-irradiated (3,000 rads) syngeneic splenocytes (2.5 x 106 cells/mL) infected with 1 TCIDs0/cell RSV (Long strain) for 2 hr. The source of murine IL-2 was supernatant of a mouse cell line constitutively secreting a high level of IL-2 obtained from Dr. H. Karasuyama of Basel Institute for Immunology (ref. 20). CTL activity was tested 5 days following the in vitro re-stimulation in a standard 4 hr chromium release assay. Target cells were 5 slCr-labelled uninfected BALB/c fibroblasts (BC cells) and persistently RSV-infected BCH14 fibroblasts, respectively. Washed w096,~(c915 PCTICA96/00398 responder cells were incubated with 2 x 103 target cells at varying effector to target ratios in 200 ~L in 96-well V-bottomed tissue-culture plates for 4 hr at 37~C
Spontaneous and total chromium releases were determined by incubating target cells with either medium or 2.5 Triton-X 100 in the absence of responder lymphocytes Percentage specific chromium release was calculated as (counts-spontaneous counts)/(total counts-spontaneous counts) X 100. Tests were performed in triplicates and data are expressed as the means of three determinations.
For antibody blocking studies in CTL assays, the effector cells were incubated for 1 hr with 10 ~g/ml final of purified mAb to CD4 (GK1.5) (ref. 21) or mAb against murine CD8 (53-6.7) (ref. 22) before adding chromium labelled BC or BCH4 cells. To determine the effect of anti-class I MHC antibodies on CTL killing, the chromium labelled target cells BC or BCH4 were incubated with 20 ~L of culture supernate of hybridoma that secretes a mAb that recognizes Kd and Dd of class I MHC (34-1-2S) (ref.
23) prior to the addition of effector cells.
Exam~le 3 This Example describes the immunogenicity and protection by polynucleotide immunization by the intramuscular route.
To characterize the antibody responses following i.m. DNA administration, immune sera were analyzed for anti-RSV F IgG antibody titre by ELISA and for RSV-specific plaque reduction titre, respectively. All four plasmid constructs were found to be immunogenic. Sera obtained from mice ;mml-n;zed with pXL1-4 demonstrated significant anti-RSV F IgG titres and RSV-specific plague reduction titres as compared to the placebo group (Table 1 below) (P<0.0061 and cO.0001, respecti~ely, Mann-Whitney Test). Howe~er, there is no significant difference in either anti-RSV F IgG titre or RSV-specific plaque reduction titre among mice ;mml-n;zed with either WO9~ PCT/CA96/00398 - pXLl, pXL2, pXL3 or pXL4.
To evaluate the protective ability of pXLl-4 against primary RSV infection of the lower respiratory tract, ; mmlm; zed mice were challenged intr~n~s~lly with mouse-adapted RSV and viral lung titres post challenge wereassessed. All four plasmid constructs were found to protect An;m~ls against RSV infection. A significant reduction in the viral lung titre was observed in mice immunized with pXLl-4 as compared to the placebo group ~Pc0.0001, Mann-Whitney Test). However, varying degrees of protection were observed depending on the plasmid. In particular, PXLl was more protective than pXL3 (P=0.00109, Mann-Whitney Test), and pXL4 more than pXL3 (P=0.00125), whereas only pXL2 induced complete protection. This conclusion was confirmed by another analysis with number of fully protected mice as end point (Fisher Exact Test). Constructs pXLl, pXL2 or pXL4 conferred a higher degree of protection than pXL3 (P~0.004, Fisher Exact Test) which was not more effective than placebo. Only pXL2 conferred full protection in all immunized mice.
The above statistical analysis revealed that PXLl conferred more significant protection than pXL3. The former expresses the truncated and secretory form and the latter the intact membrane anchored form of the RSV F
protein. Furthermore, pXL4 was shown to be more protective than pXL3. The difference between these two constructs is the presence of the intron II sequence in pXL4. Construct pXL2 which expresses the secretory form of the RSV-F in the context of the intron II sequence was the only plasmid that confers complete protection in all immnnized mice.
Exam~le 4 This Example describes the influence of the route of administration of pXL2 on its immll~ogenicity and protective ability.
WO9C,14091~ PCT/CA96/00398 ~ The i.m. and i.d. routes of DNA ~min;stration were compared for imm~lno~enicity in terms of anti-RSV F
antibody titres and RSV-specific plaque reduction titres.
Analyses of the imml~ne sera (Table 2 below) revealed that the i.d. route of DNA ~m; n; stration was as ;~ nogenic as the i.m. route as judged by anti-RSV F IgG and IgG1 antibody responses as well as RSV-specific plaque reduction titres. However, only the i.m. route induced significant anti-RSV F IgG2a antibody responses, whereas the IgG2a isotype titre was negligible when the i.d.
route was used. The i.m. and i.d. routes were also compared with respect to the induction of RSV-specific CTL. Significant RSV-specific CTL activity was detected in mice ;mmllnized intramuscularly. In contrast, the cellular response was significantly lower in mice inoculated intradermally ~Table 3 below). In spite of these differences, protection against primary RSV
infection of the lower respiratory tract was observed in both groups of mice immunized via either route (Table 4 below). The CTL induced by RSV-F DNA are classical CD8+
class I restricted CTL. The target cells, BCH4 fibroblasts express class I MHC only and do not express class II MHC. Further, prior incubation of BCH4 target cells with anti class-I MHC antibodies significantly blocked the lytic activity of RSV-F DNA induced CTL line.
While anti-CD8 antibody could partially block lysis of BCH4 cells, antibody to CD4 molecule had no effect at all ~Table 5 below). Lack of total blocking by mAb to CD8 could either be due to CTL being CD8 independent (me~n;ng that even though they are CD8+ CTL, their TCR has enough affinity for class I MHC+peptide and it does not require CD8 interaction with the alpha 3 of class I MHC) or the amount of antibody used in these experiments was limiting. There was no detectable lysis of YAC-1 (NK
sensitive target) cells (data not shown).
WO~f~C91~ PCT/CA96/00398 ~ ExamPle 5 This Example describes i~mnnization studies in cotton rats using pXL2.
The immune response of cotton rats to DNA
immtlnization was analyzed by the protocol shown in Table 6 below. On day -5, 40 cotton rats were randomly selected and divided into 8 groups of 5. Cotton rats in groups 1 and 7 were inoculated intramuscularly (i.m.) into the tiberlia anteria (TA) muscles bilaterally with cardiotoxin (1.0 ~M). On day -1, the cotton rats in group 8 were inoculated in the TA muscles with bupivacaine (0.25%). On day 0, several animals in each group were bled to determine levels of RSV-specific antibodies in the serum of the test animals prior to administration of vaccines. All of the ~nim~ls were then inoculated i.m. or intradermally ~i.d.) with 200 ~g of plasmid DNA, placebo (non-RSV-specific ~NA), 100 median cotton rat infectious doses (CRID50; positive control) of RSV, or of formalin inactivated RSV prepared in Hep-2 tissue culture cells and adjuvanted in alum. Forty-four days later the cotton rats in groups 1 & 7 were reinoculated with cardiotoxin in the TA muscles. Four days later (48 days after priming with vaccine), the animals in group 8 were reinoculated with bupivacains in the TA muscle of the right leg. The next day, (seven weeks after priming with vaccine) all of the ~nimAl5 were bled and all, except those in the group given live RSV, were boosted with the same material and doses used on day 0. 29 days later, each cotton rat was bled and then challenged intranasally ~i.n.) with 100 CRID50 RSV A2 grown in Hep-2 tissue culture cells. Four days after this virus challenge (day +88) all of the cotton rats were killed and their lungs removed. One lobe from each set of lungs was fixed in formalin and then processed for histologic evaluation of pulmonary histopathology. The rem~ining lobes of lung will be assessed for the presence WO96J1091~ PCT/CA96/00398 ~ and levels of RSV. Each of the sera collected on days 0, 49 and 78 were tested for RSV-neutralizing activity, anti-RSV fusion activity and RSV-specific ELISA antibody.
The RSV neutralizing titres on day +49 and +78 are shown in Tables 7(a) below and 7(b) below respectively.
As can be seen from the results shown in Table 7(a), on day +49 the ~n;m-l S ;mmlln; zed with live RSV and DNA
immunization had substantial RSV serum neutralizing titres. The ~n;m~ls ;~mt1n;zed with formalin-inactivated RSV had a neutralizing titre equivalent to the placebo group on day +49 but following boosting titres by day +78 had reached 5.8 (log10/0.05). Boosting had no significant effect upon ~n;m~l S immunized with live RSV or by i.m.
plasmid ;mmllnization~
RSV titres in nasal washes ~upper respiratory tract) on day +82 are shown in Table 8 below. RSV titres in the lungs (lower respiratory tract) on day +82 are shown in Table 9 below. All of the vaccines pro~ided protection against lung infection but under these conditions, only live virus provided total protection against upper respiratory tract infection.
The lungs from the cotton rats were ~m; n~d histologically for pulmonary histopathology and the results are shown in Table lO below. With the exception of lung sections obtained from Group 9 which were essentially free of inflammatory cells or evidence of inflammation, and those from Group 3, which exhibited the maximal plll~on~ry pathology seen in this study, all of the sections of lung obtained from the other groups looked familiar, i.e. scattered inflammatory cells were present in most fields, and there was some thickening of septae. These are evidence of mild inflammatory diseases. Large numbers of inflammatory cells and other evidence of inflammation were present in sections of lung from Group 3 (in which formalin-inactivated [FI] RSV
vaccine was given prior to virus challenge). This result -indicated that immunization with plasmid DNA expressing the RSV F protein does not result in pulmonary histopathology different from the placebo, whereas FI-RSV
caused more severe pathology.
SI~ARY OF THE DISCLOSURE
In summary of this disclosure, the present invention provides certain novel vectors containing genes encoding an RSV F proteins, methods of ;mmtln;zation using such vectors and methods of diagnosis using such vectors.
Modifications are possible within the scope of this invention.
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Group TlCA~ Major Ol,~.valions & C~.. - .. 1~
1. Placebo + RSV S~,a~,d individual and groups of L~ ,~h ges and pOl,~LuOll~h~ llrl~A~ l;)pileS (PMN) in all fields. Overt Ih.~ ;n~ of septae. OCCA~:;QTI~1 .oLic cells seen. Overall: ild to moderate ;--n~------~l;~n 2. Live RSV TCC~lAt~d LUal,lUph ges seen in most fields.
Sca~ d PMN. Overall: minimAI infl,.."".AI;~"
3. FI-RSV + RSV Vir~ally every field COIIIA;IIC ~ C,uus ...n~ f~r cells & PMN. Pyknotic cells and debris CCJL~ On. Th;rL~ nlcd septae. Evidence of e~ac~ àL~d disease.
. Plasmid + RSV T!;o1AtJ~ LuaClùl)hag~.S seen in most fields.
OCC~;OnA1 PMN seen. Very similar to live virus gTOUp.
6. Plasmid i.d. + Isolated Lllae.uphages seen in most fields.
RSV OCC'A';OI.A1 PMN seen.
7. Plas_id + CT Isolated n~t~n~.,rleAr cells and PMN seen in most + RSV fields.
8. Plasmid + Biv Scal~lcd mononuclear cells and PMN seen in + RSV most fields.
9. Normal CR Few l~uko~;yL~s evidence. Airy, open appeal~Luce.
Lung T_in septae.
CT = caldilu~
Biv = ~upivacaine REFEREN OES
1. McIntosh K., C~nock, R.M. In: Fields BN, Knipe, DM, editors. Virology. New York: Raven Press: 1990:
2. Katz SL., In: New Vaccine De~elopment establishing priorities. Vol. 1. W;~.Ch; n~ton: National Academic Press: 1985: 397-409.
3. Wertz GW, Sullender WM., Biotechnology 1992; 20:
4. Johnson et al., J. Virol 1987, 61: 3163-3166 5. Pemberton et al., J. Gen Virol. 1987, 68: 2177-2182 6. Crowe, J.E., Vaccine l9g5, 13: 415-421 7. WO 90/11092 8. WO 94/21797 9. Ulmer, Current Opinion, Invest Drugs, 1993, 2: 983-10. Tang et al., Nature 1992, 356: 152-154 11. Furth et al. Analytical Biochemistry, 1992, 205:
12. Pizzorno et al., J. Virol. 1988, 62: 1167-1179 13. Chapman, B.S.; Thayer, R.M.; Vincent, K.A. and Haigwood, N.L., Nucl. Acids. Res. 1991, 19: 3979-3986.
14. Green, S. Isseman, I., and Sheer, E., Nucl. Acids.
Res. 1988, 16: 369 15. Breathnack, R. and Harris, B.A., Nucl. Acids Res.
1983, 11: 7119-7136 16. Graham, B.S.; Perkins M.D.; Wright, P.F. and Karzon, D.T. J. Mod. Virol. 1988 26: 153-162.
Res. 1988, 16: 369 15. Breathnack, R. and Harris, B.A., Nucl. Acids Res.
1983, 11: 7119-7136 16. Graham, B.S.; Perkins M.D.; Wright, P.F. and Karzon, D.T. J. Mod. Virol. 1988 26: 153-162.
17. Nabel, G.J. 1993, Proc. Natl. Acad. Sci. USA 90:
11307-11311.
11307-11311.
18. Du, R.P et al. 1994., Biotechnology 12: 813-818.
19. Prince, G.A. et al, 1978. Ame. J. Pathol. 93: 771-790.
20. Karasuyama & Melchers, Eur. J. Immunol. 18, 97-104, 21. Wild~ David B., et al. 1983 J. Immunol-. 131: 2178- -2183.
22. Ledbetter, J.A., Rouse R., Micklem, ~.- 1980, J.
Exp. Med. 152: 280-295.
Exp. Med. 152: 280-295.
23._ Ozato Keiko, et al, 1982, Transplantation 34: 113-118.
24. Davis et al., Vaccine 1994, 12: 1503-1509.
AMEI~D'D SHEET
AMEI~D'D SHEET
Claims (42)
1. A non-replicating vector, comprising:
a first nucleotide sequence encoding an RSV F
protein or a protein capable of inducing antibodies that specifically react with RSV F protein;
a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F
protein, and a second nucleotide sequence located adjacent said first nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from said vector in a host.
a first nucleotide sequence encoding an RSV F
protein or a protein capable of inducing antibodies that specifically react with RSV F protein;
a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F
protein, and a second nucleotide sequence located adjacent said first nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from said vector in a host.
2. The vector of claim 1 wherein said first nucleotide sequence encodes a full-length RSV F protein.
3. The vector of claim 1 wherein said first nucleotide sequence encodes a RSV F protein from which the transmembrane region is absent.
4. The vector of claim 1 wherein said first nucleotide sequence encodes a full-length RSV F protein and contains a translatianal stop codon immediately upstream of the start of the transmembrane coding region to prevent translation of the transmembrane coding region.
5. The vector of claim 1 wherein said promoter sequence is an immediate eariy cytomegalovirus promoter.
6. The vector of claim 1 wherein said second nucleotide sequence comprises a pair of splice sites to prevent aberrant mRNA splicing, whereby substantially all RNA transcribed encodes an RSV F protein.
7. The vector of claim 6 wherein said second nucleotide sequence is located between said first nucleotide sequence and said promoter sequence.
8. The vector of claim 7 wherein said second nucleotide sequence is that of rabbit .beta.-globin intron II.
9. The vector of claim 1 which is pXL2 as shown in Figure 5.
10. The vector of claim 1 which is pXL4- as shown in Figure 7.
11. A method of immunizing a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises administering to said host an effective amount of a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein or a protein capable of inducing antibodies that specifically react with RSV F protein or an RSV F
protein lacking a transmembrane region, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein in said host, and a second nucleotide sequence located adjacent said first nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from said vector in said host.
protein lacking a transmembrane region, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein in said host, and a second nucleotide sequence located adjacent said first nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from said vector in said host.
12. The method of claim 11, wherein the first nucleotide sequence encodes a full-length RSV F protein and contains a translational stop codon immediately upstream of the start of the transmembrane coding region to prevent translation of the transmembrane coding region.
13. The method of claim 11 wherein said host is a human.
14. The method of claim 13 wherein said promoter sequence is an immediate early cytomegalovirus promoter.
15. The method of claim 11 wherein said vector is pXL2 as shown in Figure 5.
16. The method of claim 11 wherein said vector is pXL3 as shown in Figure 6.
17. The method of claim 11 wherein said first nucleotide sequence encodes a full-length RSV F protein.
18. The method of claim 11 wherein said first nucleotide sequence encodes an RSV F protein from which the transmembrane region is absent.
19. The method of claim 11 wherein said first nucleotide sequence encodes a full-length RSV F protein and contains a translational stop codon immediately upstream of the-start of the transmembrane coding region to prevent translation of the transmembrane coding region.
20.- The method of claim 11 wherein said promoter sequence is an immediate early cytomegalovirus promoter.
21. The method of claim 20 wherein said second-nucleotide sequence comprises a pair of splice sites to prevent aberrant mRNA splicing, whereby substantially all transcribed mRNA encodes an RSV F protein.
22. The method of claim 21 wherein said second nucleotide sequence is located between said first nucleotide sequence and said promoter sequence.
23. The method of claim 22 wherein said second nucleotide sequence is that of rabbit .beta.-globulin intron II.
24. A method of using a gene encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F
protein lacking a transmembrane region to produce an immune response in a host, which comprises:
isolating said gene;
operatively linking said gene to at least one control sequence to produce a non-replicating vector, said control sequence directing expression of said RSV F
protein when said vector is introduced into a host to produce an immune response to said RSV F protein;
operatively linking said gene in said non-replicating vector to an immunoprotective enhancing sequence to produce an enhanced immunoprotection to said RSV F protein in a host, and introducing said non-replicating vector into the host.
protein lacking a transmembrane region to produce an immune response in a host, which comprises:
isolating said gene;
operatively linking said gene to at least one control sequence to produce a non-replicating vector, said control sequence directing expression of said RSV F
protein when said vector is introduced into a host to produce an immune response to said RSV F protein;
operatively linking said gene in said non-replicating vector to an immunoprotective enhancing sequence to produce an enhanced immunoprotection to said RSV F protein in a host, and introducing said non-replicating vector into the host.
25. The method of claim 24 wherein said gene encoding an RSV F protein encodes an RSV F protein lacking the transmembrane region.
26. The method of claim 25 wherein said at least one control sequence comprises the immediate early cytomegalovirus promoter.
27. The method of claim 24 wherein said immunoprotective enhancing sequence is-introduced into said vector between said control sequence and said gene.
28. The method of claim 27 wherein said immunoprotection enhancing sequence comprises a pair of splice sites to prevent aberrant mRNA splicing whereby substantially cell transcribed mRNA encodes an RSV F
protein.
protein.
29. The method of claim 28 wherein said immunoprotection enhancing sequence is that of rabbit .beta.-globin intron II.
30. The method of claim 24 wherein said gene is contained within a plasmid selected from the group consisting of pXL2 and pXL4.
31. A method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises:
isolating a first nucleotide sequence encoding an RSV F protein or a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F protein lacking a transmembrane region;
operatively linking said first nucleotide sequence to at least one control sequence to produce a non-replicating vector, the control sequence directing expression of said RSV F protein when introduced into a host to produce an immune response to said RSV F
protein;
operatively linking said first nucleotide sequence to a second nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from the non-replicating vector in a host, and formulating said non-replicating vector as a vaccine for in vivo administration.
isolating a first nucleotide sequence encoding an RSV F protein or a protein capable of generating antibodies that specifically react with RSV F protein or an RSV F protein lacking a transmembrane region;
operatively linking said first nucleotide sequence to at least one control sequence to produce a non-replicating vector, the control sequence directing expression of said RSV F protein when introduced into a host to produce an immune response to said RSV F
protein;
operatively linking said first nucleotide sequence to a second nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from the non-replicating vector in a host, and formulating said non-replicating vector as a vaccine for in vivo administration.
32. The method of claim 31 wherein said vector is selected from the group consisting of pXL2 and pXL4.
33. A vaccine produced by the method of claim 31.
34. A method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises:
isolating a first nucleotide sequence encoding an RSV F protein from which the transmembrane region is absent;
operatively linking said first nucleotide sequence to at least one control sequence to produce a non-replicating vector, the control sequence directing expression of said RSV F protein when introduced into a host to produce an immune response to said RSV F
protein; and formulating said vector as a vaccine for in vivo administration.
isolating a first nucleotide sequence encoding an RSV F protein from which the transmembrane region is absent;
operatively linking said first nucleotide sequence to at least one control sequence to produce a non-replicating vector, the control sequence directing expression of said RSV F protein when introduced into a host to produce an immune response to said RSV F
protein; and formulating said vector as a vaccine for in vivo administration.
35. The method of claim 31 wherein said vector is selected from group consisting of pXL1 and pXL2.
36. A vaccine produced by the method of claim 34.
37. A method of determining the presence of a respiratory syncytial virus (RSV) F protein in a sample, comprising the steps of:
(a) immunizing a host with a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein, or a RSV F protein lacking a transmembrane region, and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein in said host to produce antibodies specific for the RSV F protein;
(b) isolating the RSV F protein specific antibodies;
(c) contacting the sample with the isolated antibodies to produce complexes comprising any RSV
F protein present in the sample and said isolated RSV F protein-specific antibodies; and (d) determining production of the complexes.
(a) immunizing a host with a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein, or a RSV F protein lacking a transmembrane region, and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein in said host to produce antibodies specific for the RSV F protein;
(b) isolating the RSV F protein specific antibodies;
(c) contacting the sample with the isolated antibodies to produce complexes comprising any RSV
F protein present in the sample and said isolated RSV F protein-specific antibodies; and (d) determining production of the complexes.
38. The method of claim 37 wherein said vector is selected from the group consisting of pXL1, pXL2, pXL3 and pXL4.
39. A diagnostic kit for detecting the presence of an RSV F protein in a sample, comprising:
(a) a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein, or a RSV F
protein lacking a transmembrane region, and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F
protein in a host immunized therewith to produce antibodies specific for the RSV F protein;
(b) isolation means to isolate said RSV F
protein-specific antibodies;
(c) contacting means to contact the isolated RSV
F specific antibodies with the sample to produce a complex comprising any RSV F protein present in the sample and RSV F protein specific antibodies, and (d) identifying to determine production of the complex.
(a) a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein, a protein capable of generating antibodies that specifically react with RSV F protein, or a RSV F
protein lacking a transmembrane region, and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F
protein in a host immunized therewith to produce antibodies specific for the RSV F protein;
(b) isolation means to isolate said RSV F
protein-specific antibodies;
(c) contacting means to contact the isolated RSV
F specific antibodies with the sample to produce a complex comprising any RSV F protein present in the sample and RSV F protein specific antibodies, and (d) identifying to determine production of the complex.
40. The diagnostic kit of claim 39 wherein said non-replicating vector is selected from the group consisting of pXL1, pXL2, pXL3 and pXL4.
41. A method for producing antibodies specific for an F
protein of RSV comprising:
(a) immunizing a host with an effective amount of a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein lacking a transmembrane region and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F
protein in said host to produce antibodies specific for the F protein; and (b) isolating the antibodies from the host.
protein of RSV comprising:
(a) immunizing a host with an effective amount of a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein lacking a transmembrane region and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F
protein in said host to produce antibodies specific for the F protein; and (b) isolating the antibodies from the host.
42. A method of producing monoclonal antibodies specific for an F protein of RSV comprising the steps of:
(a) constructing a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein; and, optionally, a second nucleotide sequence located adjacent said first nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from said vector in a host.
(b) administering the vector to at least one mouse to produce at least one immunized mouse;
(c) removing B-lymphocytes from the at least one immunized mouse;
(d) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;
(e) cloning the hybridomas;
(f) selecting clones which produce anti-F protein antibody;
(g) culturing the anti-F protein antibody-producing clones; and (h) isolating anti-F protein monoclonal antibodies from the cultures.
(a) constructing a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein; and, optionally, a second nucleotide sequence located adjacent said first nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from said vector in a host.
(b) administering the vector to at least one mouse to produce at least one immunized mouse;
(c) removing B-lymphocytes from the at least one immunized mouse;
(d) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;
(e) cloning the hybridomas;
(f) selecting clones which produce anti-F protein antibody;
(g) culturing the anti-F protein antibody-producing clones; and (h) isolating anti-F protein monoclonal antibodies from the cultures.
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US08/476,397 US6019980A (en) | 1995-06-07 | 1995-06-07 | Nucleic acid respiratory syncytial virus vaccines |
US08/476,397 | 1995-06-07 |
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CA (1) | CA2223610A1 (en) |
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- 1996-06-07 AU AU61176/96A patent/AU695527B2/en not_active Ceased
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- 1996-06-07 AT AT96918542T patent/ATE267258T1/en not_active IP Right Cessation
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AU695527B2 (en) | 1998-08-13 |
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ATE267258T1 (en) | 2004-06-15 |
EP0832253B9 (en) | 2004-12-22 |
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US5843913A (en) | 1998-12-01 |
WO1996040945A2 (en) | 1996-12-19 |
EP0832253B1 (en) | 2004-05-19 |
WO1996040945A3 (en) | 1997-01-23 |
EP0832253A2 (en) | 1998-04-01 |
DE69632536D1 (en) | 2004-06-24 |
AU6117696A (en) | 1996-12-30 |
US6019980A (en) | 2000-02-01 |
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