AU741016B2 - Compounds and methods for treatment and diagnosis of mycobacterial infections - Google Patents

Description

P00011 Regulation 3.2 Revised 2/98

AUSTRALIA

Patents Act, 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT TO BE COMPLETED BY THE APPLICANT NAME OF APPLICANT: ACTUAL INVENTORS: ADDRESS FOR SERVICE: GENESIS RESEARCH DEVELOPMENT CORPORATION LIMITED PAUL TAN; JUN HIYAMA; ELIZABETH S VISSER; MARGOT A SKINNER; LINDA M SCOTT and ROSS L PRESTIDGE Peter Maxwell Associates Level 6 60 Pitt Street SYDNEY NSW 2000 COMPOUNDS AND METHODS FOR TREATMENT AND DIAGNOSIS OF MYCOBACTERIAL INFECTIONS Divisional of Australian Patent Application No. 40,365/97 filed on 28 August 1997 oooo oo INVENTION TITLE: DETAILS OF ASSOCIATED

APPLICATION(S):

The following statement is a full description of this invention including the best method of performing it known to me:- COMPOUNDS AND METHODS FOR TREATMENT AND DIAGNOSIS OF MYCOBACTERIAL

INFECTIONS

Technical Field The present invention relates generally to the detection, treatment and prevention of infectious diseases. In particular. the invention is related to compounds and methods for the treatment of mycobacterial infections including Mycobacerium tuberculosis and Mycobacterium avium. The invention is further related to compounds that function as non-specific immune response amplifiers, and the use of such non-specific immune response amplifiers as adjuvants in vaccination or immunotherapy against infectious disease, and in certain treatments for immune disorders and cancer.

Background of the Invention 15 Tuberculosis is a chronic, infectious disease, that is caused by infection with Mycobacterium tuberculosis (M tuberculosis). It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly S 20 manifested as a chronic inflammation of the lungs, resulting in fever and respiratory S symptoms. If left untreated, significant morbidity and death may result.

*i Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behaviour is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistant mycobacteria.

Inhibiting the spread of tuberculosis requires effective vaccination and accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common mycobacterium employed for this purpose is Bacillus Calmette-Guerin (3CG), an avirulent strain of Mycobacterium bovis. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public. Diagnosis of M. tuberculosis infection is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, thereby indicating exposure to mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.

A less well-known mycobacterium that has been used for immunotherapy for tuberculosis, and also leprosy, is Mycobacterium vaccae, which is non-pathogenic in humans. However, there is less information on the efficacy of M. vaccae compared with BCG, and it has not been used widely to vaccinate the general public. M. bovis BCG and M. vaccae are believed to 15 contain antigenic compounds that are recognised by the immune system of individuals exposed to infection with M. tuberculosis.

.o There thus remains a need in the art for effective compounds and methods for preventing, treating and detecting tuberculosis.

According to one aspect of the invention there is provided a composition comprising delipidated and deglycolipidated M. vaccae cells. The invention also provides a method for enhancing an immune response comprising administering such a composition.

According to another aspect of the invention there is provided a vaccine comprising delipidated and deglycolipidated M. vaccae cells. The invention also provides a method for inducing protective immunity in a patient comprising 10 administering to a patient such a vaccine.

S. These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references o o *o *oo oo *o •o disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

Brief Description of the Drawings Figs. 1A and IB illustrate the protective effects of immunizing mice with autoclaved M. vaccae or unfractionated M vaccae culture filtrates, respectively, prior to infection with live M tuberculosis H37Rv.

Figs. 2A and B show components of M vaccae and M. tuberculosis culture filtrates, respectively, as analysed by 2 -dimensional polyacrylamide gel electrophoresis.

Fig. 3 is a comparison of the Antigen 85A protein sequence obtained from MA.

vaccae with those from M. bovis, M. tuberculosis and M leprae.

Fig. 4A(i) (iv) illustrate the non-specific immune amplifying effects of pg, 1 00pg and Img autoclaved M vaccae and 7 5pg unfractionated culture filtrates of 15 M vaccae. respectively. Fig. 4B(i) and (ii) illustrate the non-specific immune amplifying effects of autoclaved M vaccae and delipidated M. vaccae, respectively.

Fig. 4C(i) illustrates the non-specific immune amplifying effects of whole autoclaved M. vaccae. Fig. 4C(ii) illustrates the non-specific immune amplifying effects of delipidated M vaccae from which glycolipids had been removed and the proteins 20 extracted with SDS. Fig. 4C(iii) illustrates that the adjuvant effect of the preparation of Fig. 4C(ii) is destroyed by treatment with the proteolytic enzyme pronase. Fig. 4D illustrates -h-e non-specific immune amplifying effects of heat-killed M. vaccae (Fig.

M tuberculosis (Fig. 4D(ii)), M. bovis BCG (Fig. 4D(iii)), M. phlei (Fig.

4D(iv)) and M. smegmatis (Fig. 4D(v)).

Fig. 5 shows the results of polyacrylamide gel electrophoresis analysis of SDS-extracted proteins derived from delipidated and deglycolipidated M. vaccae.

Fig. 6 illustrates the non-specific immune amplifying effects of different molecular weight fractions of SDS-extracted M. vaccae proteins.

Fig. 7 illustrates the non-specific immune amplifying effects of different pi fractions of SDS-extracted M. vaccae proteins.

Fig. 8 illustrates the induction of IL-12 by heat-killed M. vaccae, lyophilized M.

vaccae, delipidated and deglycolipidated M. vaccae (referred to as "delipidated M.

vaccae") and M. vaccae glycolipids.

Fig. 9 illustrates the stimulation of interferon-gamma production by different concentrations of i. vaccae recombinant proteins, heat-killed M. vaccae, delioidated and deglycolipidated M. vaccae (referred to as "delipidated 1. vaccae"), vaccae glycolipids and lipopolysaccharide in C57BL-6 peritoneal macrophages (Fig 9A); BALB/C peritoneal macrophages (Fig 9B); and C3H/HeJ peritoneal macrophages (Fig 9C).

Detailed Description of the Invention As noted above, the present invention is generally directed to compositions and methods for preventing, treating and diagnosing mycobacterial infections, including ll.

tuberculosis and M. avium infections.

Considerable research efforts have been directed towards elucidating the 15 mechanism of immune response to mycobacterial infection, in particular M.

tuberculosis infection. While macrophages have been shown to act as the principal effectors of M. tuberculosis immunity, T cells are the predominant inducers of such ~immunity. The essential role of T cells in protection against M. tuberculosis infection is illustrated by the frequent occurrence of tuberculosis in AIDS patients, due to the 20 depletion of CD4 T cells associated with human immunodeficiency virus (HIV) infection. Mycobacterium-reactive CD4 T cells have been shown to be potent S. :producers of gamma-interferon which, in turn. has been shown to trigger the anti-mycobacrerial effects of macrophages in mice. While the role of IFN-y in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-y or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis infection. Furthermore, it is known that IFN-y stimulates human macrophages to make 1,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play a role in stimulating resistance to M. tuberculosis infection. Another property of CD4' T cells and macrophages is their ability to activate CD8' cyvtoxr T cells which are capable of killing pathogen-infected cells. CD8' T cells have been shown to kill macrophages and other cells that harbour M. tuberculosis. For a review of the immunology of M. tuberculosis infection see Chan and Kaufmann in Tuberculosis: Pathogenesis. Protection and Control, Bloom ASM Press.

Washington. DC, 1994.

The compositions of the present invention include polypeptides that comprise at least one immunogenic portion of an M vaccae antigen, or a variant thereof. Such polypeptides stimulate T cell proliferation, and/or, interferon gamma secretion from T cells of individuals exposed to M tuberculosis. In certain embodiments, the inventive polypeptides comprise at least an immunogenic portion of a soluble M vaccae antigen. A "soluble M vaccae antigen" is a protein of M. vaccae origin that is present in M. vaccae culture filtrate. As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins antigens), wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M vaccae antigen or may be heterologous, 15 and such sequences may (but need not) be immunogenic.

"Immunogenic," as used herein, refers to the ability to elicit an immune response in a patient, such as a human, or in a biological sample. In particular.

immunogenic antigens are capable of stimulating cell proliferation, interleukin-12 production or interferon-y production in biological samples comprising one or more 20 cells selected from the group ofT cells, NK cells, B cells and macrophages, where the cells are derived from an M tuberculosis-immune individual. Polypeptides comprising at least an immunogenic portion of one or more M. vaccae antigens may generally be used to detect tuberculosis or to induce protective immunity against tuberculosis in a patient.

The compositions and methods of this invention also encompass variants of the above polypeptides. As used herein, the term "variant" covers any sequence which exhibits at least about 50%, more preferably at least about 70% and more preferably yet, at least about 90% identity to a sequence of the present invention.

Most preferably, a "variant" is any sequence which has at least about a 99% probability of being the same as the inventive sequence. The probability for DNA sequences is measured by the computer algorithm FASTA (version 2.0u4, February 1996; Pearson W. R. et al., Proc. Nat. Acad. Sci., 85:2444-2448, 1988), the probability for translated DNA sequences is measured by the computer algorithm TBLASTX and that for protein sequences is measured by the computer algorithm BLASTP (Altschul, S. F. et al. J. Mol. Biol.. 215:403-410, 1990). The term S -"variants" thus encompasses sequences wherein the probability of finding a match by chance (smallest sum probability), is less than about 1% as measured by any of the above tests.

A polypeptide of the present invention may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or posttranslationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

S. 15 In general, M vaccae antigens, and DNA sequences encoding such antigens, may be prepared using any of a variety of procedures. For example, soluble antigens Smay be isolated from M. vaccae culture filtrate as described below. Antigens may also be produced recombinantly by inserting a DNA sequence that encodes the antigen into an expression vector and expressing the antigen in an appropriate host.

Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring antigens, portions of; naturally occurring antigens, or other variants thereof.

DNA sequences encoding M. vaccae antigens may be obtained by screening an appropriate M. vaccae cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from partial amino acid sequences of isolated soluble antigens. Suitable degenerate oligonucleotides may be designed and synthesized, and the screen may be performed as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. As described below, polymerase chain reaction (PCR) may be employed to isolate a nucleic acid probe from a cDNA or genomic DNA library. The library screen may then be performed using the isolated probe.

DNA molecules encoding M. vaccae antigens may also be isolated by screening an appropriate M. vaccae expression library with anti-sera rabbit or monkey) raised specifically against M. vaccae antigens.

Regardless of the method of preparation, the antigens described herein have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce cell proliferation and/or cytokine production (for example, interferon-y and/or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from an M. tuberculosis-immune individual. An M. tuberculosis-immune individual is one who is considered to be resistant to the 15 development of tuberculosis by virtue of having mounted an effective T cell response to M. tuberculosis. Such individuals may be identified based on a strongly positive greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD), and an absence of any symptoms of tuberculosis infection.

s 20 The selection of cell type for use in evaluating an immunogenic response to an antigen will depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing B cells or macrophages.

T

cells, NK cells, B cells and macrophages derived from M. tuberculosis-immune individuals may be prepared using methods well known in the art. For example, a preparation of peripheral blood mononuclear cells (PBMCs) may be employed without further separation of component cells. PBMCs may be prepared, for example, using density centrifugation through FicollTM (Winthrop Laboratories,

NY).

T cells for use in the assays described herein may be purified directly from PBMCs.

Alternatively, an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins, may be employed. Such T cell clones may be generated by, for example, culturing PBMCs from M. tuberculosisimmune individuals with mycobacterial proteins for a period of 2-4 weeks. This allows expansion of only the mycobacterial protein-specific T cells, resulting in a line composed solely of such cells. These cells may then be cloned and tested with individual proteins, using methods well known in the ar, to more accurately define individual T cell specificity. Assays for cell proliferation or cytokine production in T cells, NK cells, B cells or macrophages may be performed, for example, using the procedures described below.

In general, immunogenic antigens are those antigens that stimulate proliferation or cytokine production interferon-y and/or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from at least about of M tuberculosis-immune individuals. Among these immunogenic antigens, Spolypeptides having superior therapeutic properties may be distinguished based on the *magnitude of the responses in the above assays and based on the percentage of Sindividuals for which a response is observed. In addition, antigens having superior :15 therapeutic properties will not stimulate cell proliferation or cytokine production in vitro in cells derived from more than about 25% of individuals that are not M tuberculosis-immune, thereby eliminating responses that are not specifically due to M tuberculosis-responsive cells. Thus, those antigens that induce a response in a high percentage of T cell, NK cell, B cell or macrophage preparations from 0:.9 20 M tuberculosis-immune individuals (with a low incidence of responses in cell i preparations from other individuals) have superior therapeutic properties.

:Antigens with superior therapeutic properties may also be identified based on their ability to diminish the severity of M tuberculosis infection, or other mycobacterial infection, in experimental animals, when administered as a vaccine.

Suitable vaccine preparations for use in experimental animals are described in detail below.

Antigens having superior diagnostic properties may generally be identified based on the ability to elicit a response in an intradermal skin test performed on an individual with active tuberculosis, but not in a test performed on an individual who is not infected with M tuberculosis. Skin tests may generally be performed as described below, with a response of at least about 5 mm induration considered positive.

Immunogenic portions of the antigens described herein may be prepared and identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3d ed., Raven Press, 1993, pp. 243-247. Such techniques include screening polypeptide portions of the native antigen for immunogenic properties. The representative proliferation and cytokine production assays described herein may be employed in these screens. An immunogenic portion of a polypeptide is a portion that, within such representative assays, generates an immune response cell proliferation, interferon-y production or interleukin-12 production) that is substantially similar to that generated by the full length antigen. In other words, an immunogenic portion of an antigen may generate at least about 20%, preferably about and most preferably about 100%, of the proliferation induced by the full length antigen in the model proliferation assay described herein. An immunogenic portion may also, or alternatively, stimulate the production of at least about 20%, preferably about 65% and most preferably about 100%, of the interferon-y and/or interleukin-12 15 induced by the full length antigen in the model assay described herein.

Portions and other variants of M. vaccae antigens may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such 20 polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J Am. Chem.

Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions. Variants of a native antigen may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Sections of the DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides.

In general, regardless of the method of preparation, the polypeptides disclosed herein are prepared in substantially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure. In certain preferred embodiments, described in detail below, the substantially pure polypeptides are incorporated into pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein.

SThe present invention also provides fusion proteins comprising a first and a second inventive polypeptide or, alternatively, a polypeptide of the present invention and a known M tuberculosis antigen, such as the 38 kD antigen described in Andersen and Hansen, Infect. Immun. 57:2481-2488, 1989, together with variants of such fusion proteins. The fusion proteins of the present invention may also include a linker peptide between the first and second polypeptides.

A DNA sequence encoding a fusion protein of the present invention is .constructed using known recombinant DNA techniques to assemble separate

DNA

sequences encoding the first and second polypeptides into an appropriate expression S vector. The 3' end of a DNA sequence encoding the first polypeptide is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.

A peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each polypeptide folds into I its secondary and tertiary structures. Such a peptide linker sequence is incorporated 0..i into the flsion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: their ability to adopt a flexible extended conformation; (2)their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maraea e Gene 40:346, 1985; Murphy etal., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. The linker sequence may be from 1 to about 50 amino acids in length. Peptide linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

The ligated DNA sequences encoding the fusion proteins are cloned into suitable expression systems using techniques known to those of ordinary skill in the art.

In another aspect, the present invention provides methods for using one or more of the inventive polypeptides or fusion proteins (or DNA molecules encoding such polypeptides or fusion proteins) to induce protective immunity against tuberculosis in a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may be afflicted with a disease, or may be free of detectable disease or infection. In other words, protective immunity may be induced to prevent or treat tuberculosis.

S. 15 In this aspect, the polypeptide, fusion protein or DNA molecule is generally present within a pharmaceutical composition or a vaccine. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. Vaccines may comprise one or more of the above polypeptides and a non- 20 specific immune response amplifier, such as an adjuvant or a liposome, into which the polypeptide is incorporated. Such pharmaceutical compositions and vaccines may .i also contain other mycobacterial antigens, either, as discussed above, incorporated into a fusion protein or present within a separate polypeptide.

Alternatively, a vaccine of the present invention may contain DNA encoding one or more polypeptides as described above, such that the polypeptide is generated in situ. In such vaccines, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic, or defective, replication competent virus. Techniques for incorporating DNA into such expression systems are well known in the an. The DNA may also be "naked," as described, for example, in Ulmer etal., Science 259:1745-I749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

A DNA vaccine as described above may be administered simultaneously with or sequentially to either a polypeptide of the present invention or a known mycobacterial antigen, such as the 38 kD antigen described above. For example.

administration of DNA encoding a polypeptide of the present invention, may be followed by administration of an antigen in order to enhance the protective immune 15 effect of the vaccine.

Routes and frequency of administration, as well as dosage, will vary from Sindividual to individual and may parallel those currently being used in immunization using BCG. In general, the pharmaceutical compositions ad vaccnes may be administered by injection intradermal, intramuscular, intravenous or 20 subcutaneous), intranasally by aspiration) or orally. Between 1 and 3 doses ma be administered for a 1-36 week period. Preferably, 3 doses are administered, at intervals of 3-4 months, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount ofpolypeptide or DNA that, when administered as described above, iscapable of raising an immuneresponse in a patient sufficient to protect the patient from mycobacterial infection. for at least -2 -years. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about I gg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.

Biodegradable microspheres polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.

Any of a variety of adjuvants may be employed in the vaccines of this invention to non-specifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum 15 hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis, M. tuberculosis, or, as discussed below, M. vaccae.

Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.

I'

Iniriother aspect, this invention provides methods for using one or more of the polypeptides described above to diagnose tuberculosis using a skin test. As used herein, a "skin test" is any assay performed directly on a patient in which a delayedtype hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of one -or more polypeptides as described above. Preferably, the reaction is measured at least 48 hours after injection, more preferably 48-72 hours.

The DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen the immunogenic portion of the polypeptide employed, or a variant thereof). The response may be measured visually, using a ruler. In general, a response that is greater than about cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of tuberculosis infection.

For use in a skin test, the polypeptides of the present invention are preferably S formulated, as pharmaceutical compositions containing a polypeptide and a physiologically acceptable carrier, as described above. Such compositions typically contain one or more of the above polypeptides in an amount ranging from about I .g to about 100 pg preferably from about 10 gg to about 50 .g in a volume of 0.1 mL.

Preferably, the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween In a preferred embodiment, a polypeptide employed in a skin test is of sufficient size such that it remains at the site of injection for the duration of the .reaction period. In general, a polypeptide that is at least 9 amino acids in length is sufficient. The polypeptide is also preferably broken down by macrophages or o 15 dendritic cells within hours of injection to allow presentation to T-cells. Such polypeptides may contain repeats of one or more of the above sequences or other 00 immunogenic or nonimmunogenic sequences.

:b In another aspect, methods are provided for detecting mycobacterial infection in a biological sample, using one or more of the above polypeptides, either alone or in S 20 combination In embodiments in which multiple polypeptides are employed, polypeptides other than those specifically described herein, such as the 38 kD antigen described above, may be included. As used herein, a "biological sample" is any antibody-containing sample obtained from a patient. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient or a blood supply. The polypeptide(s) are used in an assay, as described below, to determine the presence or absence of antibodies to the polypeptide(s) in the sample, relative to a predetermined cut-off value. The presence of such antibodies indicates the presence of mycobacterial infection.

In embodiments in which more than one polypeptide is employed, the polypeptides used are referably complementary one component polypeptide will tend to detect infection in samples where the infection would not be detected by another component polypeptide). Complementary polypeptides may generally be identified by using each polypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with a Mycobacterium. After determining which samples test positive (as described below) with each polypeptide, combinations of two or more polypeptides may be formulated that are capable of detecting infection in most, or all, of the samples tested. For example, approximately 25-30% of sera from tuberculosis-infected individuals are negative for antibodies to any single protein, such as the 38 kD antigen mentioned above. Complementary polypeptides may, therefore, be used in combination with the 38 kD antigen to improve sensitivity of a diagnostic test.

S. A variety of assay formats employing one or more polypeptides to detect antibodies in a sample are well known in the art. See, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a 15 preferred embodiment, the assay involves the use of polypeptide immobilized on a solid support to bind to and remove the antibody from the sample. The bound antibody may then be detected using a detection reagent that contains a reporter group. Suitable detection reagents include antibodies that bind to the antibody/polypeptide complex and free polypeptide labelled with a reporter group in a semi-competitive assay). Alternatively, a' competitive assay may be utilized, in which an antibody that binds to the polypeptide is labelled with a reporter group and allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The extent to which components of the sample inhibit the binding of the labelled antibody to the polypeptide is indicative of the reactivity of the sample with the immobilized polypeptide.

The solid support may be any solid material to which the antigen may be attached. Suitable materials are well known in the art. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.

Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S.

Patent No. 5,359,681.

The polypeptides may be bound to the solid support using a variety of techniques well known in the art. In the context of the present invention, the term S"bound" refers to both noncovalent association, such as adsorption, and covalent attachment, which may be a direct linkage between the antigen and functional groups on the support or a linkage by way of a cross-linking agent. Binding by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the polypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about I hour and I day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of polypeptide ranging from about 10 ng to about I and preferably about 100 ng, is sufficient to bind an adequate amount of antigen.

Covalent attachment of polypeptide to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the p. olypeptide. For example, the polypeptide may be bound to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde 20 group on the support with an amine and an active hydrogen on the polypeptide (see, Pierce Immunotechnology Catalog and Handbook, 1991, at A2-A13).

.i In certain embodiments, the assay is an enzyme-linked immunosorbent assa (ELISA). This assay may be performed by first contacting a polypeptide antigen that has been immobilized on a solid support, with the sample, such that antibodies to the polypeptide within the sample are allowed to bind to the immobilized polypeptide.

Unbound sample is then removed from the immobilized polypeptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific detection reagent.

IMore specifically, once the polypeptide is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20TM (Sigma Chemical Co., St. Louis, MO) may be employed. The immobilized polypeptide is then incubated with the sample, and antibody is allowed to bind to the antigen. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time, or incubation time, is that period of time that is sufficient to detect the presence of antibody within a M. tuberculosis-infected sample. Preferably, the contact time is sufficient to achieve a level of binding that is at least 95% of that achieved at equilibrium between bound and unbound antibody. The time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about minutes is generally sufficient.

Unbound sample may be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20

M

Detection reagent may 15 then be added to the solid support. An appropriate detection reagent is any compound that binds to the immobilized antibody-polypeptide complex and that can be detected by any of a variety of means known in the art. Preferably, the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of binding agent to reporter group may be achieved using standard methods known in the art. Common binding agents may also be purchased conjugated to a variety of reporter groups from many commercial sources Zymed Laboratories, San Francisco, CA, and Pierce, Rockford,

IL).

The detection reagent is incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time may generally be determined from the manufacturer's instructions or by assaying the level of binding that occurs over a period of time.

Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups.

Biotin may be detected using avidin. coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of anti-mycobacterial antibodies in the sample, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value is the average mean signal obtained when the immobilized antigen is incubated with samples from an uninfected Spatient. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett etal., Clinical 15 Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107. In general, signals higher than the predetermined cut-off value are S considered to be positive for mycobacterial infection.

The assay may also be performed in a rapid flow-through or strip test format, wherein the antigen is immobilized on a membrane, such as nitrocellulose. In the 20 flow-through test, antibodies within the sample bind to the immobilized polypeptide as the sample passes through the membrane. A detection reagent proteinA- .i colloidal gold) then binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane. The detection of bound detection reagent may then be performed as described above. In the strip test format, one end of the membrane to which polypeptide is bound is immersed in a solution containing the sample. The- sample migrates along the membrane through a region containing detection reagent and to the area of immobilized polypeptide.

Concentration of detection reagent at the polypeptide indicates the presence of antimycobacterial antibodies in the sample. Typically, the concentration of detection reagent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of polypeptide immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in an ELISA, as discussed above.

Preferably, the amount of polypeptide immobilized on the membrane ranges from about 25 ng to about 1 pg, and more preferably from about 50 ng to about 500 ng.

Such tests can typically be performed with a very small amount one drop) of patient serum or blood.

Numerous other assay protocols exist that are suitable for use with the polypeptides of the present invention. The above descriptions are intended to be exemplary only.

The present invention also provides antibodies to the inventive polypeptides.

Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an 15 immunogen comprising the antigenic polypeptide is initially injected into any of a wide variety of mammals mice, rats, rabbits, sheep and goats). The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified 20 from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells may then be immortalized by fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal, using one of a variety of techniques well known in the art.

Monoclonal antibodies may be isolated from the superatants of the resulting hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood.

Antibodies may be used in diagnostic tests to detect the presence of mycobacterial antigens using assays similar to those detailed above and other techniques well known to those of skill in the art, thereby providing a method for detecting mycobacterial infection, such as M tuberculosis infection, in a patient.

Diagnostic reagents of the present invention may also comprise

DNA

sequences encoding one or more of the above polypeptides, or one or more portions thereof. For example, primers comprising at least 10 contiguous oligonucleotides of 15 subject DNA sequences may be used for hybridizing to specific sequences Primers or probes may thus be used to detect M tuberculosis and other mycobacterial infections in biological samples, preferably sputum, blood, serum, saliva, cerebrospinal fluid or urine. DNA robes or primers comprising oligonucleotide 20 sequences described above may be used alone, in combination with each other, or :with previously identified sequences, such as the 38 kD antigen discussed above.

Asiscussed above, effective vaccines contain at least two different components. The first is a polypeptide comprising an antigen, which is processed by macrophages and other antigen-presenting cells and displayed for CD4 T cells or for CD8 T cells. This antigen forms the "specific" target of an immune response Th second component of a vaccine is a non-specific immune response amplifi such ae an adjuvant or a liposome, into which the anigen is incorporated. n aduvant amplifies immune responses to a sructurally unrelated compound or polypptide.

Several adjuvants are prepared from microbes such as Bordetella pertussis,

M.

tuberculosis and M bovis BCG. Adjuvants may also contain components designed to protect polypeptide antigens from degradation, such as aluminum hydroxide or mineral oil.

While the antigenic component of a vaccine contains polypeptides that direct the immune attack against a specific pathogen, such as M. auberculosis, the adjuvant is often capable of broad use in many different vaccine formulations.

Certain pathogens, such as M. tuberculosis, as well as certain cancers, are effectively contained by an immune attack directed by T cells, known as cellmediated immunity. Other pathogens, such as poliovirus, also require antibodies produced by B cells for containment. These different classes of immune attack (T cell or B cell) are controlled by different subpopulations of CD4" T cells, commonly referred to as Thi and Th2 cells. A.desirable property of an adjuvant is the ability to selectively amplify the function of either Thl or Th2 Dopulations of CD4' T cells. As shown below in Example 6, M. vaccae and a modified form of autoclaved M. vaccae have been found to have adjuvant properties. As used herein, the term "modified M. vaccae" includes delipidated M. vaccae cells.

deglycolipidated M. vaccae cells and M. vaccae cells that have been both delipidated and deglycolipidated (hereinafter referred to as DD-.f. vaccae).

Furthermore, it has been found that vaccae produces compounds which amplify the immune response to M. vaccae antigens, as well as to antigens from 20 other sources. The present invention thus provides methods for enhancing immune responses to an antigen comprising administering kiled M. vaccae cells, M. vaccae culture filtrate or modified M. vaccae cells. As detailed below, firther studies have demonstrated that this non-specific immune amplifying effect is.due, at least in part, to an AM. vaccae polypeptide having homoloy to heat shock protein 65 (GroEL), previously identified in M. tuberculosis.

As described below in Example 10, it has also been found that heat-killed M. vaccae and M. vaccae constituents have cytokine stimulation properties. Jn particular, it has been found that heat-killed M. vaccae, lyophilised M. vaccae and DD-M. vaccae stimulate the production of interleukin 12 (IL-12) rom macrophages. Production of IL-12 from macrophages is known to enhance stimulation of a Thl immune response.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1 EFFECT OF IMMUNIZATION OF MICE WITH

VACCAE

ON TUBERCULOSIS This example illustrates the effect of immunization with MA vaccae or M vaccae culture filtrate in mice prior to challenge with live M. tuberculosis.

M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/l; tryptone, 5 g/l; glucose, I g/l) at 37 OC. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 oC for one day. The medium was .then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 0 equivalent to 10o /m equivalent to 10 vaccae organisms per ml. The cell suspension was then 15 autoclaved for 15 min at 120 C. The culture filtrate was passaged through a 0.45 filter into sterile bottles.

As shown in Fig.lA, when mice were immunized with 1 mg, 100 gg or 10 pg SofM vaccae and infected three weeks later with 5x 05 colony forming units (CFU) of of Mcvac aond form ing units (C FU live M tuberculosis H37Rv, significant protection from infection was seen. In this 20 example, spleen, liver and lung tissue was harvested from mice three weeks after nfection, and live bacilli determined (expressed as CFU). The reduction in bacilli .numbers, vhen compared to tissue from non-immunized control mice, exceeded 2 logs in liver and lung tissue, and 1 log in spleen tissue. Immunization of mice with heat-kiled M tuberculosis H37Rv had no significant protective effects on mice subsequently infected with live M tuberculosis H37Rv.

Fig.lB shows that when mice were immunized with 100 gfM vaccae culture filtrate, and infected three weeks later with 5x l0 CFU of M tuberculosis H37Rv, significant protection was also seen. When spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli numbers

(CFU)

determined, a 1-2 log reduction in numbers, as compared to non-immunized control mice, was observed.

EXAMPLE 2 PURIFICATION AND CHARACTERIZATION OF POLYPEPTIDES FROM M VACCAE CULTURE

FILTRATE

This example illustrates the preparation of M. vaccae soluble proteins from culture filtrate. Unless otherwise noted, all percentages in the following example are weight per volume.

M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 °C.

The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 p filter into sterile bottles.

.The culture filtrate was concentrated by lyophilization, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration 15 through a 0.45p membrane. The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained a 3,000 kilodalton molecular weight cut-off (MWCO) membrane. The pressure was maintained at 50 psi using nitrogen gas. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This 20 procedure reduced the 20 1 volume to approximately 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).

*:Thedesalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech, Uppsala, Sweden) (16 X 100 mm) equilibrated with 10mM Tris HCI buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCI from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.

The pool of polypeptides eluting from the ion exchange column was concentrated in a 400 ml Amicon stirred cell which contained a 3,000 MWCO membrane. The pressure was maintained at 50 psi using nitrogen gas. The polypeptides were repeatedly concentrated by membrane filtration and diluted with 1% glycine until the conductivity of the sample was less than 0.1 mS.

The purified polypeptides were then fractionated by preparative isoelectric focusing in a Rotofor device (Bio-Rad, Hercules, CA, USA). The pH gradient was established with a mixture of Ampholytes (Pharmacia Biotech) comprising 1.6% pH 3.5-5.0 Ampholytes and 0.4% pH 5.0 7.0 Ampholytes. Acetic acid (0.5 M) was used as the anolyte, and 0.5 M ethanolamine as the catholyte. Isoelectric focusing was carried out at 12W constant power for 6 hours, following the manufacturer's instructions. Twenty fractions were obtained.

Fractions from isoelectric focusing were combined, and the polypeptides were purified on a Vydac C4 column (Separations Group, Hesperia, CA, USA) 300 Angstrom pore size, 5 micron particle size (10 x 250 mm). The polypeptides were eluted from the column with a linear gradient of acetonitrile (0-80% v/v) in 0.05% trifluoroacetic acid (TFA). The flow-rate was 2.0 ml/min and the HPLC eluent Swas monitored at 220 nm. Fractions containing polypeptides were collected to maximize the purity of the individual samples.

15 Relatively abundant polypeptide fractions were rechromatographed on a Vydac C4 column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6 x 250 mm). The polypeptides were eluted from the column with a linear gradient from 20-60% of acetonitrile in 0.05% TFA at a flow-rate of 1.0 ml/min. The column eluent was monitored at 220 nm. Fractions containing the eluted 20 polypeptides were collected to maximise the purity of the individual samples.

~Approximately 20 polypeptide samples were obtained and they were analysed for .i purity onS polyacrylamide gel according to the procedure of Laemmli (Laemmli,

U.

Nature 277:680-685, 1970).

The polypeptide fractions which were shown to contain significant contamination were further purified using a Mono Q column (Pharmacia Biotech) micron particle size (5 x 50 mm) or a Vydac Diphenyl column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6 x 250 mm). From a Mono

Q

column, polypeptides were eluted with a linear gradient from 0-0.5 M NaCI in 10 mM Tris HC1 pH 8.0. From a Vydac Diphenyl column, polypeptides were eluted with a linear gradient of acetonitrile (20-60% v/v) in 0.1% TFA. The flow-rate was ml/min and the column eluent was monitored at 220 nm for both columns. The polypeptide peak fractions were collected and analysed for purity on a polyacrylamide gel as described above.

For sequencing, the polypeptides were individually dried onto Biobrene

T

(Perkin Elmer/Applied BioSystems Division, Foster City, CA)-treated glass fiber filters. The filters with polypeptide were loaded onto a Perkin Elmer/Applied BioSystems Procise 492 protein sequencer and the polypeptides were sequenced from the amino terminal end using traditional Edman chemistry. The amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards.

Internal sequences were also determined on some antigens by digesting the antigen with the endoprotease Lys-C, or by chemically cleaving the antigen with cyanogen bromide. Peptides resulting from either of these procedures were separated by reversed-phase HPLC on a Vydac C18 column using a mobile phase of 0.05% trifluoroacetic acid with a gradient of acetonitrile containing 0.05% TFA 15 The eluent was monitored at 214 nm. Major internal peptides were identified by their UV absorbance, and their N-terminal sequences were determined as ****described above.

Using the procedures described above, six soluble M. vaccae antigens, designated GVc-1, GVc-2, GVc-7, GVc-13, GVc-20 and GVc-22, were isolated.

20 Determined N-terminal and internal sequences for GVc-1 are shown in SEQ ID NOS: 1, 2 and 3, respectively; the N-terminal sequence for GVc-2 is shown in SEQ ID NO: 4; internal sequences for GVc-7 are shown in SEQ ID NOS: 5-8; internal sequences for GVc-13 are shown in SEQ ID NOS: 9-11; internal sequence for GVc-20 is shown in SEQ ID NO: 12; and N-terminal and interal sequences for GVc-22 are shown in SEQ ID NO:56-59, respectively. Each of the internal peptide sequences provided herein begins with an amino acid residue which is assumed to exist in this position in the polypeptide, based on the known cleavage specificity of cyanogen bromide (Met) or Lys-C (Lys).

Three additional polypeptides, designated GVc-16, GVc-18 and GVc-21, were isolated employing a preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) purification step in addition to the preparative isoelectric focusing procedure described above. Specifically. fractions comprising mixtures of polypeptides from the preparative isoelectric focusing purification step previously described, were purified by preparative SDS-PAGE on a polyacrylamide gel. The samples were dissolved in reducing sample buffer and applied to the gel. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting in 10 mM 3 -(cyclohexylamino)I propanesulfonic acid (CAPS) buffer pH 11 containing 10% methanol. The transferred protein bands were identified by staining the PVDF membrane with Coomassie blue. Regions of the PVDF membrane containing the most abundant 0 polypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The N-terminal sequences for GVc- S16, GVc-18 and GVc-21 are provided in SEQ ID NOS: 13, 14 and 15, respectively Additional antigens, designated GVc-12, GVc-14, GVc-15, GVc-17 and GVc- 19, were isolated employing a preparative SDS-PAGE purification step in addition to the chromatographic procedures described above. Specifically, fractions comprising a mixture of antigens from the Vydac C4 HPLC purification step previously described were fractionated by preparative SDS-PAGE on a polyacrylamide gel. The samples were dissolved in non-reducing sample buffer and applied to the gel. The separated 20 proteins were transferred to a PVDF membrane by electroblotting in 10 mM CAPS buffer, pH 11 containing 10% methanol. The transferred protein bands were identifiedTby staining the PVDF membrane with Coomassie blue. Regions ofthe PVDF membrane containing the most abundant polypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The determined N-terminal sequences for GVc-12, GVc -14, GVc- 5, GVc- 17 and GVc-19 are provided in SEQ ID NOS: 16-20, respectively.

All of the above amino acid sequences were compared to known amino acid sequences in the SwissProt data base (version R32) using the GeneAssist system. No 30 using^ th G es syste No 30 significant homologies to the amino acid sequences GVc-2 to GVc-22 were obtained.

The amino acid sequence for GVc-1 was found to bear some similarity to sequences previously identified from M. bovis and M. tuberculosis. In particular, GVc-I was found to have some homology with ML tuberculosis MPT83. a cell surface protein, as well as MPT70. These proteins form part of a protein family (Harboe et al., Scand J.

Immunol. 42:46-51, 1995).

Subsequent studies led to the isolation of DNA sequences for GVc-14 and GVc-22 (SEQ ID NO: 107 and 108, respectively). The corresponding predicted amino acid sequences for GVc-14 and GVc-22 are provided in SEQ ID NO: 109 and 110, respectively.

Amplifications primers AD86 and ADI12 (SEQ ID NO: 60 and 61, respectively) were designed from the amino acid sequence of GVc-1 (SEQ ID NO: 1) and the M tuberculosis MPT70 gene sequence. Using these primers, a 310 bp fragment was amplified from M. vaccae genomic DNA and cloned into EcoRVdigested vector pBluescript (Stratagene) containing added dTTP residues. The sequence of the cloned insert is provided in SEQ ID NO: 62.

15 The purified polypeptides were screened for the ability to induce T-cell proliferation and IFN-y in peripheral blood cells from immune human donors. These donors were known to be PPD (purified protein derivative from M. tuberculosis) skin test positive and their T cells were shown to proliferate in response to PPD. Donor PBMCs and crude soluble proteins from M vaccae culture filtrate were cultured in medium comprising RPMI 1640 supplemented with 10% autologous serum, penicillin (60 Ag/ml), streptomycin (100 gg/ml), and glutamine (2 mM).

Afer 3 days, 50 p.l of medium was removed from each well for the determination of IFN-y levels, as described below. The plates were cultured for a further 4 days and then pulsed with 1Ci/well of tritiated thymidine for a further 18 hours, harvested and tritium uptake determined using a scintillation counter.

Fractions that stimulated proliferation in both. replicates two-fold greater than the proliferation observed in cells cultured in medium alone were considered positive.

IFN-y was measured using an enzyme-linked immunosorbent assay (ELISA).

ELISA plates were coated with a mouse monoclonal antibody directed to human IFNy (Endogen, Wobural, MA) 1 Ag/ml phosphate-buffered saline (PBS) for 4 hours at 4 Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS/0.2% Tween 20. and samples diluted 1:2 in culture medium in the ELISA plates were incubated overnight at room temperature. The plates were again washed, and a biotinylated polyclonal rabbit anti-human IFN-y serum (Endogen), diluted to 1 4g/ml in PBS, was added to each well. The plates were then incubated for 1 hour at room temperature, washed and horseradish peroxidase-coupled avidin A (Vector Laboratories, Burlingame,

CA)

was added at a 1:4,000 dilution in PBS. After a further 1 hour incubation at room temperature, the plates were washed and orthophenylenediamine (OPD) substrate added. The reaction was stopped after 10 min with 10% HC. The optical density (OD) was determined at 490 nm. Fractions that resulted in both replicates giving an OD two-fold greater than the mean OD from cells cultured in medium alone were considered positive.

Examples of polypeptides containing sequences that stimulate peripheral blood mononuclear cells (PBMC) T cells to proliferate and produce IFN-y are shown in Table 1, wherein indicates a lack of activity, indicates polypeptides having a result less than twice higher than background activity of control media, indicates Spolypeptides having activity two to four times above background, and indicates polypeptides having activity greater than four times above background.

.9e TABLE 1 Antigen Proliferation

IFN-

GVc- GVc-2 GVc-7 GVc-13 GVc-14 EXAMPLE 3 PURIFICATION AND CHARACTERISATION OF POLYPEPTIDES FROM M. VACCAE CULTURE FILTRATE

BY

2-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS M. vaccae soluble proteins were isolated from culture filtrate using 2dimensional polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.

M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 OC.

M. tuberculosis strain H37Rv (ATCC number 27294) was cultured in sterile Middlebrook 7H9 medium with Tween 80 and oleic acid/albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Michigan). The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 "C for one day. The medium was then centrifuged (leaving the bulk of the cells) 15 and filtered through a 0.45 p filter into sterile bottles. The culture filtrate was concentrated by lyophilisation, and redissolved in MilliQ water. A small amount of S insoluble material was removed by filtration through a 0.

4 5 p membrane filter.

The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained a 3,000 MWCO membrane. The pressure was maintained 20 at 60 psi using nitrogen gas. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the 20 L volume to approximately 50 mL.

Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).

The desalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech) (16 x 100 mm) equilibrated with TrisHCL buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCI from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.

The pool of polypeptides eluting from the ion exchange column were fractionated by preparative 2D gel electrophoresis. Samples containing 200-500 ug of polypeptide were made 8M in urea and applied to polyacrylamide isoelectric focusing rod gels (diameter 2mm, length 150 mm, pH After the isoelectric focusing step, the first dimension gels were equilibrated with reducing buffer and applied to second dimension gels (16% polyacrylamide). Figs. 2A and 2B are the 2-D gel patterns observed with M. vaccae culture filtrate and M. tuberculosis H37Rv culture filtrate respectively. Polypeptides from the second dimension separation were transferred to PVDF membranes by electroblotting in 10mM CAPS buffer pH 11 containing methanol. The PVDF membranes were stained for protein with Coomassie blue.

Regions of PVDF containing polypeptides of interest were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. The polypeptides were sequenced from the amino terminal end using traditional Edman chemistry. The amino acid sequence was determined for S. each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Using these procedures, eleven 15 polypeptides, designated GVs-1, GVs-3, GVs-4, GVs-5, GVs-6, GVs-8, GVs-9, GVs- GVs- 11, GV-34 and GV-35 were isolated. The determined N-terminal sequences for these polypeptides are shown in SEQ ID NOS: 21-29, 63 and 64, respectively.

Using the purification procedure described above, more protein was purified to extend the amino acid sequence previously obtained for GVs-9. The extended amino acid sequence for GVs-9 is provided in SEQ ID NO:65: Further studies resulted in the .isolation of the DNA sequence for GVs-9 (SEQ ID NO: 111). The corresponding .o T corresponding predicted amino acid sequence is provided in SEQ ID NO: 112.

All of these amino acid sequences were compared to known amino acid sequences in the SwissProt data base (version R32) using the GeneAssist system. No significant homologies were obtained, with the exceptions of GVs-3, GVs-4, and GVs-9. GVs-9 was found to bear some homology to two previously identified

M

tuberculosis proteins, namely M. tuberculosis cutinase precursor and an M.

tuberculosis hypothetical 22.6 kD protein. GVs-3, GVs-4 and GVs-5 were found to bear some similarity to the antigen 85A and 85B proteins from M. leprae (SEQ

ID

NOS: 30 and 31, respectively), M tuberculosis (SEQ ID NOS: 32 and 33, respectively) and M bovis (SEQ ID NOS: 34 and 35, respectively), and the antigen proteins from M. leprae (SEQ ID NO: 36) and M. tuberculosis (SEQ ID NO: 37).

A comparison of the inventive antigen 85A protein from M. vaccae with those from M. tuberculosis, M. bovis and M. leprae, is presented in Fig. 3.

EXAMPLE 4 DNA CLONING STRATEGY FOR THE M VACCAE ANTIGEN 85 SERIES Probes for antigens 85A. 85B, and 85C (SEQ ID NOS: 38 and 39) were prepared by the polymerase chain reaction (PCR) using degenerate oligonucleotides designed to regions of antigen 85 genomic sequence that are conserved between family members in a given mycobacterial species, and between mycobacterial species. These oligonucleotides were used under reduced stringency conditions to amplify target 15 sequences from M vaccae genomic DNA. An appropriately-sized 0.5kb band was identified, purified, and cloned into T-tailed p Bluescript II SK (Stratagene, La Jolla, CA). Twenty-four individual colonies were screened at random for the presence of the antigen 85 PCR product, then sequenced using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the MI3-based primers, T3 and T7.

20 Homology searches of the GenBank databases showed that twenty-three clones contained insert with significant homology to published antigen 85 genes from M tuberculosis and M. bovis. Approximately half were most homologous to antigen gene sequences, with the remainder being more similar to antigen sequences. In addition, these two putative M. vaccae antigen 85 genomic sequences were 80% homologous to one another. Because of this high similarity, the antigen PCR fragment was chosen to screen M vaccae genomic libraries at. low stringency for all three antigen 85 genes.

An M. vaccae genomic library was created in ZapExpress (Stratagene, La Jolla, CA) by cloning BamHI partially-digested M. vaccae genomic DNA into similarly-digested X vector, with 3.4 x 10 independent plaque-forming units resulting. For screening purposes, twenty-seven thousand plaques from this nonamplified library were plated at low density onto eight 100 cm 2 plates. For each plate, duplicate plaque lifts were taken onto Hybond-N- nylon membrane (Amersham International, United Kingdom). and hybridised under reduced-stringency conditions oC) to the radiolabelled antigen 85C PCR product. Autoradiography demonstrated that seventy-nine plaques consistently hybridised to the antigen probe under these conditions. Thirteen positively-hybridising plaques were selected at random for further analysis and removed from the library plates, with each positive clone being used to generate secondary screening plates containing about two hundred plaques. Duplicate lifts of each plate were taken using Hybond-N- nylon membrane, and hybridised under the conditions used in primary screening. Multiple positively-hybridising plaques were identified on each of the thirteen plates screened.

S* Two well-isolated positive phage from each secondary plate were picked for further analysis. Using in vitro excision, twenty-six plaques were converted into phagemid, and restriction-mapped. It was possible to group clones into four classes on the basis 15 of this mapping. Sequence data from the 5' and 3' ends of inserts from several representatives of each group was obtained using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the T3 and T7 primers. Sequence the GeneAssist software package. Two of these sets of clones were found to be homologous to M bovis andM. tuberculosis antigen 85A genes, each containing Mi eac ae a or 3 ens 8 a re he d ge t e s r ea ch c o a i n either the 5' or 3' ends of the M vaccae gene (this gene was cleaved during library Sconstruction as it contains an internal BamHIsite). The remaining clones were found to contain sequences homologous to antigens 85B and 85C from a number of mycobacterial species. To determine the remaining nucleotide sequence for each gene, appropriate subclones were constructed and sequenced. Overlapping sequences were aligned using the DNA Strider software. The determined DNA sequences for M. vaccae antigens 85A, 85B and 85C are shown in SEQ ID NOS: 40-42, respectively, with the predicted amino acid sequences being shown in SEQ ID NOS: 43-45, respectively.

The M vaccae antigens GVc-3 and GVc-5 were expressed and purified as follows. Amplification primers were designed from the insert sequences of GVc-3 34 and GVc-5 (SEQ ID NO: 40 and 42, respectively) using sequence data downstream from the putative leader sequence and the 3' end of the clone. The sequences of the primers for GVc-3 are provided in SEQ ID NO: 66 and 67. and the sequences of the primers for GVc-5 are provided in SEQ ID NO: 68 and 69. A XhoI restriction site was added to the primers for GVc-3, and EcoRI and BamHI restriction sites were added to the primers for GVc-5 for cloning convenience. Following amplification from genomic M. vaccae DNA, fragments were cloned into the appropriate site of pProEX HT prokaryotic expression vector (Gibco BRL, Life Technologies, Gaithersburg, MD) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the recombinant protein was performed according to the manufacturer's protocol.

Expression of a fragment of the M. vaccae antigen GVc-4 (antigen 85B homolog) was performed as follows. The primers AD58 and AD59, described above, were used to amplify a 485 bp fragment from M vaccae genomic DNA. This fragment was gel- 15 purified using standard techniques and cloned into EcoRV-digested pBluescript containing added dTTP residues. The base sequences of inserts from five clones were determined and found to be identical to each other. These inserts had highest homology to Ag85B from M tuberculosis. The insert from one of the clones was subcloned into the EcoRI/XhoI sites of pProEX HT prokaryotic expression vector (Gibco BRL), expressed and purified according to the manufacturer's protocol. This clone was renamed GVc-4P because only a part of the gene was expressed. The amino aci iand DNA sequences for the partial clone GVc-4P are provided in SEQ ID NO: 70 and 106, respectively.

In subsequent studies, using procedures similar to those described above, GVc-3, GVc-4P and GVc-5 were re-cloned into the alternative vector pET16 (Novagen, Madison,

WI).

The ability of purified recombinant GVc-3, GVc-4P and GVc-5 to stimulate proliferation of T cells and interferon-y production in human PBL from PPD-positive, healthy donors, was assayed as described above in Example 2. The results of this assay are shown in Table 2, wherein indicates a lack of activity, indicates polypeptides having a result less than twice higher than background activity of control media, indicates polypeptides having activity two to four times above background.

indicates polypeptides having activity greater than four times above background.

and ND indicates not determined.

Table 2 Donor Donor Donor Donor Donor Donor G97005 G97006 G97007 G9700 G97009 Donor Prolif IFN Prolif IFN Prolif IFN ProG9700 G97009 ProG970 Y- -Y -Y -Y

-Y

GVc- ND ND GVc- ND ND GVC- EXAMPLE DNA CLONING STRATEGY FOR M VACCAE ANTIGENS S 15 An 84 bp probe for the M vaccae antigen GVc-7 was amplified using Sdegenerate oligonucleotides designed to the determined amino acid sequence of GVc- S7 (SEQ ID NOS: This probe was used to screen a M vacca genomic

DNA

library as described in Example 4. The determined nucleotide sequence for GVc-7 is shown in SEQ ID NO: 46 and predicted amino acid sequence SEQ ID NO: 47.

Comparison of these adsequence in SEQ ID NO 47 Comparison of these sequences with those in the databank revealed homology to a hypothetical 15.8 kDa membrane protein of M tuberculosis.

The sequence of SEQ ID NO: 46 was used to design amplification primers (provided in SEQ ID NO: 71 and 72) for expression cloning of the GVc-7 gene using sequence data downstream from the putative leader sequence. A XhoI restriction site was added to the primers for cloning convenience. Following amplification from genomic M. vaccae DNA, fragments were cloned into the XhoI-site of pProEX

HT

prokaryotic expression vector (Gibco BRL) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the fusion protein was performed according to the manufacturer's protocol. In subsequent studies, GVc-7 was re-cloned into the vector pET16 (Novagen).

The ability of purified recombinant GVc-7 to stimulate proliferation of T-cells and stimulation of interferon-y production in human PBL. from PPD-positive, healthy donors, was assayed as described previously in Example 2. The results are shown in Table 3, wherein indicates a lack of activity, indicates polypeptides having a result less than twice higher than background activity of control media, indicates polypeptides having activity two to four times above background, and indicates polypeptides having activity greater than four times above background.

TABLE3 Donor Proliferation Interferon-y G97005 G97008 G97009 G97010

A

A redundant oligonucleotide probe was designed to the GVs-8 peptide sequence shown in SEQ ID NO: 6 and used to screen an M. vaccae genomic DNA library as described above. Positive plaques were isolated.

Four different genomic clones were identified, hereinafter referred to as GVs- 8A, GVs-8B and GVs-8C and GVs-8D. The determined DNA sequences for the clones GVs-8A, GVs-8B, GVs-8C and GVs-8D are shown in SEQ ID NOS: 48-51, respectively, with the corresponding amino acid sequences being shown in SEQ ID NOS: 52-55, respectively. The clone GVs-8A contains regions showing some similarity to known prokaryotic valyl-tRNA synthetases; GVs-8B shows some similarity to M. smegmatis aspartate semialdehyde dehydrogenase; and GVs-8C shows some similarity to the H. influenza folylpolyglutamate synthase gene. GVs-8D contains an open reading frame which shows some similarity to sequences previously identified In U' tuberculosis and MIf rzbtwoefizcjnhsntbe Identified. .lpabtwoefnto a o eI In subsequent studies, the Mvfvaccae genornic DNA library constructed in the Bamf-l -site of lambda ZAP Express vector (Stratagene) was screened with a second redundant oligonucleotjde (referred to .as MPGI 5; SEQ ID NO:73) designed from the GVs-8 sequence provided in SEQ ID NO:6. Screening of the library was performed in the presence of tetramethylammonim chloride (TiMAC), so that nucleotide base pairs would melt at a standard temperature independent of sequence A-T pairs and G-C pairs melt at the same temperature). Hybridisation stringency therefore depended only on the length and degeneracy of the oligonucleotide used 'as probe (Wood et al. Proc. Nai. Acad Scd. USA, 82:1585-1588,1985). Filters were prepared S Susing standard methods of transfer and pre-hybridised overnight at a temperature 'C b lo w th e pp r p ri te T A C ash tem perature H ybridisation w as perfo rm ed *overnight in freshly prepared hybridisation solution containing 100 pmol probe.

15 Hybridisation Solution for 01 igonucleo tides Stock: I M MNaCI 0.1lMTris PH.8 1 M Denhardt's I OX 0.05% NaPPi *SS*0 SDS 0. 1: ni/r yeast tRNA 10 gm 125 units/ml heparin The filters were washed at a temperaue calculated to allow approximately 4 mismatching in TMAC wash buffer. More specifically, the wash protocol -inclutded the following washing steps: 2 x 15 min in 6X SSC, 0.05% NaPPi at room temp; I1 min in TMAC wash (see below) at room temperature; 2 x 15 min in TMAC wash at the calculated stringent temperature; and I x 15 min in 6X SSC, 0.05% NaPPi at room temp.

TMAC wash buffer 3 M Tetramethylammonium chloride (TMAC) mM Tris pH 0.2 mM EDTA Positive plaques were picked and stored in 1 ml SM buffer with 20 ;il chloroform.

Screening was repeated until plaques were pure following the procedure described above.

The pBK-CMV phagemid containing the desired insert was excised from the lambda ZAP Express vector in the presence of ExAssist helper phage following the manufacturer's protocol. A phagemid containing an 8 kb insert (GVs-8D) was characterised by restriction mapping and sub-cloning. An open reading frame was identified at the 3' end of the insert and the antigen encoded by this open reading frame was named GV-33. Base sequence corresponding to GVs-8 was not found in 15 the insert, and it was assumed that GV-33 was obtained as a non-specific product of the TMAC screening. By further sub-cloning and base sequencing, the 3' end of the gene was determined. The determined partial DNA sequence for GV-33 is provided in SEQ ID NO:74 with the corresponding predicted amino acid sequence being oprovided in SEQ ID NO:75. Sequence data from the 3' end of the clone showed 20 homology to a previously identified 40.6 kDa outer membrane protein of M.

tuberculosis.

The partial GV-33 gene was amplified from M. vaccae genomic DNA with primers based on the determined nucleotide sequence. This DNA fragment was cloned into EcoRv-digested pBluescript (Stratagene) with additional dTTP residues, and then transferred to pProEX HT expression vector (Gibco BRL) using EcoRI and HindIII-subcloning. Recombinant protein was purified following the manufacturer's protocol. In subsequent studies, GV-33 was re-cloned into the alternative vector pET16 (Novagen).

The ability of purified recombinant antigen to stimulate proliferation of T-cells and stimulation of interferon-y production in human PBL from PPD-positive, healthy donors, was assayed as described previously in Example 2. The results are shown in Table 4, wherein indicates a lack of activity niae oyetdshvn result less than twice higher than background activity of control media, ()indicates polypeptides having activity two to four times above background. and(-)incae polypeptides having activity greater than four times above background.

TABLE 4 Donor Proliferation Interferon-Y G970o G9700 G97007+- G97008+- G97009 1 G9701 0+- EXAMPLE 6 DETECTION OF NONSPECIFIC IMMUNE AMPLIFIER FROM WHOLE V4CCAE AND THE CULTURE FILTRATE OF M. V4CCAE This example illustrates the preparation of whole M vaccae and M vaccae culture filtrate and its non-specific immune amplifying or 'adjuvant' property.

M. vaccae bacteria was cultured, pelleted and autoclaved as described in Example 1. Culture filtrates of live M vaccae refer to the supernatant from 24 hour cultures of M vaccae in 7H9 medium with glucose. A delipidated form of M vaccae was prepared by sonicating autoclaved M. vaccae for four bursts of 30 seconds on ice using the Virsonic sonicator (Virtis, Disa, USA). The material was then centrifuged (9000 rpm, 20 minutes, JA10 rotor, brake The resulting pellet was suspended in 100 ml of chloroform/methanol incubated at room temperature for 1 hour; recentrifuged, and the chloroform/methanol extraction repeated. The pellet was obtained by centrifugation, dried in vacuo, weighed and resuspended in PBS at (dry weight) per ml as delipidated M. vaccae.

:G:lycolipids were removed from the delipidated M. vaccae preparation by refluxing in 50% v/v ethanol for 2 hours. The insoluble material was collected by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake The extraction with v/v ethanol under reflux was repeated twice more. The insoluble material was collected. by centrifugation and washed in PBS. Proteins were extracted by resuspending the pellet in 2% SDS in PBS at 56 'C for 2 hours. The insoluble material was collected by centrifugation and the extraction with 2% SDS/PBS at 56 'C was repeated twice more. The pooled SDS extracts were cooled to 4- C, and precipitated SDS was removed by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake Proteins were precipitated from the supernatant by adding an equal volume of acetone and incubating at -20 'C for 2 hours. The precipitated proteins were collected by centrifugation, washed in 50% v/v acetone, dried in vacuo, and redissolved in PBS.

M. vaccae culture supernatant killed M vaccae and delipidated

M

vaccae were tested for adjuvant activity in the generation of cytotoxic T cell immune response to ovalbumin, a structurally unrelated protein, in the mouse. This antiovalbumin-specific cytotoxic response was detected as follows. C57BL/6 mice (2 per 3 group) were immn;-r 4 5 group) were i -ized by the intraperitoneai injection of 100 ,g of ovalbumin with the following test adjuvants: autoclaved MA vaccae; delipidated M vaccae delipidated M. vaccae with glycolipids also extracted and proteins extracted with SDS; the SDS protein extract treated with pronase (an enzyme which degrades protein); whole M. vaccae culture filtrate; and heat-killed M tuberculosis or heatkilled M bovis BCG, M phlei or M smegmatus or M. vaccae culture filtrate. After days, spleen cells were stimulated in vitro for a further 6 days with E.G7 cells Swhich are EL4 cells (a C5 7 BL/6-derived T cell lymphoma) transfected with the ovalbumin gene and thus express ovalbumin. The spleen cells were then assayed for teir ability to kill non-specifically EL4 Snon-specifically EL4 target cells or to kill specifically the E.G7 ovalbumin expressing cells. Killing activity was detected by the release of 51 Chromium with which the EL4 and E.G7 cells have been labelled (100 PCi per 2x106), prior to the killing assay. Killing or cytolytic activity is expressed as specific lysis using the formula: Sin tet culturesntrol cultux100% S- -total cpm cpin control cultures It is generally known that ovalbumin-specific cytotoxic cells are generated only in mice immunized with ovalbumin with an adjuvant but not in mice immunized with ovalbumin alone.

The diagrams that make up Fig. 4 show the effect of various M. vaccae derived adjuvant preparations on the generation of cytotoxic T cells to ovalbumin in C57BL/6 mice. As shown in Fig. 4A, cytotoxic cells were generated in mice immunized with 10 jig, (ii) 100 jg or (iii) 1 mg of autoclaved M. vaccae or (iv) pg ofM vaccae culture filtrate. Fig. 4B shows that cytotoxic cells were generated in 42 mice immunized with 1 mg whole autoclaved M. vaccae or (ii) 1 mg delipidated M. vaccae. As shown in Fig. 4C(i), cytotoxic cells were generated in mice immunized with 1 mg whole autoclaved M. vaccae; Fig. 4C(ii) shows the active material in 100 pg delipidated M. vaccae which then had glycolipids removed and the proteins extracted with SDS. Fig. 4C(iii) shows that active material in the adjuvant preparation of Fig. 4C(ii) was destroyed by treatment with the proteolytic enzyme pronase. By way of comparison, 100 gg of the SDS-extracted proteins had significantly stronger immune-enhancing ability (Fig. 4C(ii)) than did 1 mg whole autoclaved M. vaccae (Fig. 4C(i)).

Mice immunized with I mg heat-killed M. vaccae (Fig. 4D(i)) generated cytotoxic cells to ovalbumin, but mice immunized separately with I mg heat-killed M. tuberculosis (Fig. 4D(ii)), I mg M. bovis BCG (Fig. 4D(iii)), I mg M phlei (Fig. 4D(iv)), or 1 mg M. smegmatis (Fig. 4D(v)) failed to generate cytotoxic cells.

15 The SDS-extracted proteins derived from delipidated and deglycolipidated

M.

vaccae were analysed by polyacrylamide gel electrophoresis. As shown in Fig. Sthree major bands were observed after staining with silver.

In subsequent studies, more of the SDS-extracted proteins described above were prepared by preparative SDS-PAGE on a BioRad Prep Cell (Hercules,

CA).

20 Fractions corresponding to molecular weight ranges were precipitated by Strichloroacetic acid to remove SDS before assaying for adjuvant activity in the anti- °*ovalbumiiF-pecific cytotoxic response assay in C57BL/6 mice as described above. As seen in Fig. 6, the adjuvant activity was highest in the 60-70 kDa fraction. The most abundant protein in this size range was purified by SDS-PAGE blotted on to a polyvinylidene difluoride (PVDF) membrane and then sequenced. The sequence of the first ten amino acid residues is provided in SEQ ID NO:76. Comparison of this sequence with those in the gene bank as described above, revealed homology to the heat shock protein 65 (GroEL) gene from M tuberculosis, indicating that this protein is an M. vaccae member of the GroEL family.

An expression library of M vaccae genomic DNA in BamHI-lambda

ZAP

Express (Stratagene) was screened using sera from cynomolgous monkeys immunised with M. vaccae secreted proteins prepared as described above. Positive plaques were identified using a colorimetric system. These plaques were re-screened until plaques were pure following standard procedures. pBK-CMV phagemid 2-1 containing an insert was excised from the lambda ZAP Express (Stratagene) vector in the presence of ExAssist helper phage following the manufacturer's protocol. The base sequence of the 5' end of the insert of this clone, hereinafter referred to as GV-27, was determined using Sanger sequencing with fluorescent primers on Perkin Elmer/Applied Biosystems Dvision automatic sequencer. The determined nucleotide sequence of the partial M. vaccae GroEL-homologue clone GV-27 is provided in SEQ ID NO:77 and the predicted amino acid sequence in SEQ ID NO:78. This clone was found to have homology to M tuberculosis GroEL. A partial sequence of the 65 kDa heat shock protein of V vaccae has been published by Kapur et al. (Arch. Pathol Lab. Med. 119:131-138, 1995). The nucleotide sequence of the Kapur et al. frament Sis shown in SEQ ID NO:79 and the predicted amino acid sequence in SEQ ID In subsequent studies, a full-length (except for the predicted 51 terminal nucleotides) DNA sequence for GV-27 was obtained (SEQ ID NO: 113). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 114.

GV-

27was found to be 93.7% identical to the M tuberculosis GroEL at the amino acid level.

r20 e Two peptide fragments, comprising the N-terminal sequence (hereinafter referred to as GV-27A) and the carboxy terminal sequence of GV-27 (hereinafter referred tas GV-27B) were prepared using techniques well known in the art The nucleotide sequences for GV-27A and GV-27B are provided in SEQ ID NO: 115 and 116, 'V-27B are provided in SEQ ID NO: 115 and 116, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 117 and 118. The sequence of GV-27A is 95.8% identical to the M tuberculosis GroEL sequence and contains the shorter Mvaccae sequence of Kapr pvaccae sequence of Kapur et al. discussed above. The sequence for GV-27B shows about 92.2% identity to the corresponding region of M. tuberculosis The M. vaccae culture filtrate described above was also fractionaed by isoelectric focusing and the fractions assayed for adjuvant activity in the anti-ovalbumin.

specific cytotoxic response assay in C57BL/6 mice as described above. As shown in Fig. 7, peak adjuvant activities were demonstrated in fractions corresponding to pi of 4.2-4.32 (fraction nos. 4.49-4.57 (fraction nos. 13-17) and 4.81-5.98 (fraction nos. 23-27).

EXAMPLE 7 AUTOCLAVED M VACCAE GENERATES CYTOTOXIC CD8 T CELLS AGAINST M TUBERCULOSIS INFECTED

MACROPHAGES

This example illustrates the ability of killed M. vaccae to stimulate cytotoxic CD8 T cells which preferentially kill macrophages that have been infected with M.

tuberculosis.

Mice were immunized by the intraperitoneal injection of 500 pg of killed M.

vaccae which was prepared as described in Example 1. Two weeks after immunization, the spleen cells of immunized mice were passed through a CD8 T cell enrichment column (R&D Systems, St. Paul, MN, USA). The spleen cells recovered .from the column have been shown to be enriched up to 90% CD8 T cells. These T cells, as well as CD8 T cells from spleens of non-immunized mice, were tested for their ability to kill uninfected macrophages or macrophages which have been infected with M. tuberculosis.

Macrophages were obtained from the peritoneal cavity of mice five days after they have-been given 1 ml of 3% thioglycolate intraperitoneally. The macrophages were infected overnight with M. tuberculosis at the ratio of 2 mycobacteria per macrophage. All macrophage preparations were labelled with 5 IChromium at 2 cci per 104 macrophages. The macrophages were then cultured with CD8 T cells overnight (16 hours) at killer to target ratios of 30:1. Specific killing was detected by the release of 51Chromium and expressed as specific lysis, calculated as in Example The production of IFN-y and its release into medium after 3 days of co-culture of CD8 T cells with macrophages was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with a rat monoclonal antibody directed to mouse IFN-y (Pharmigen, San Diego, CA, USA) in PBS for 4 hours at 4 OC. Wells were blocked with PBS containing 0.2% Tween 20 for I hour at room temperature. The plates were then washed four times in PBS containing 0.2% Tween 20, and samples diluted 1:2 in culture medium in the ELISA plates were incubated overnight at room temperature The plates were again washed and a biotinylated monoclonal rat anti-mouse IFN-y antibody (Pharmigen), diluted to I .ug/ml in PBS, was added to each well. The plates were then incubated for I hour at room temperature, washed, and horseradish peroxidase-coupled avidin D (Sigma

A-

3151) was added at a 1:4,000 dilution in PBS. After a further 1 hour incubation at room temperature, the plates were washed and OPD substrate added. The reaction was stopped after 10 min with 10% HCI. The optical density was determined at 490 nm. Fractions that resulted in both replicates giving an OD two-fold greater than the mean OD from cells cultured in medium alone were considered positive.

i As shown in Table 5, CD8 T cells from spleens of mice immunized with M 15 vaccae were cytotoxic for macrophages infected with M tuberculosis and did not lyse uninfected macrophages. The CD8 T cellsfrom non-immunized mice did not lyse macrophages. CD8 T cells from naive or non-immunized mice do produce

IFN-

when cocultured with infected macrophages. The amount of IFN- produced in coculture was greater with CD8 T cells derived from M vaccae immunized mice.

20 EFFECT WITH M TUBERCULOSIS

INFECTED

AND UNINFECTED

MACROPHAGES

Specific Lysis

IF-

of Macrophages IFN-y (ng/ml) CD8 T cells uninfected infected uninfected infected Control 0 0 0.7 24.6 0.7 24.6 M. vaccae Immunized 0 952.2 43.8 46 EXAMPLE 8 DNA CLONING STRATEGY FOR THE ML V4CCAE

ANTIGENS

GV-23. GV-24. GV-25. GV-26. GV-38A AND GV-38B M. vaccae (ATCC Number 15483) was grown in sterile Medium 90 at 37 °C for 4 days and harvested by centrifugation. Cells were resuspended in 1 ml Trizol (Gibco BRL, Life Technologies, Gaithersburg, Maryland) and RNA extracted according to the standard manufacturer's protocol. M tuberculosis strain H37Rv (ATCC Number 27294) was grown in sterile Middlebrooke 7H9 medium with Tween 80 T and oleic acid/ albumin/dextrose/catalase additive (Difco Laboratories. Detroit, Michigan) at 37 OC and harvested under appropriate laboratory safety conditions.

Cells were resuspended in 1 ml Trizol (Gibco BRL) and RNA extracted according to 15 the manufacturer's standard protocol.

Total M tuberculosis and M. vaccae RNA was depleted of 16S and 23S ribosomal RNA (rRNA) by hybridisation of the total RNA fraction to oligonucleotides ADIO and AD11 (SEQ ID NO: 81 and 82) complementary to M.

tuberculosis rRNA. These oligonucleotides were designed from mycobacterial 16S 20 rRNA sequences published by Bottger (FEMS Microbiol. Lett. 65:171-176, 1989) and from sequences deposited in the databanks. Depletion was done by hybridisation of total RNAfo oligonucleotides AD10 and ADII immobilised on nylon membranes (Hybond N, Amersham International, United Kingdom). Hybridisation was repeated until rRNA bands were not visible on ethidium bromide-stained agarose gels. An oligonucleotide, AD12 (SEQ ID NO: 83), consisting of 20 dATP-residues, was ligated to the 3' ends of the enriched mRNA fraction using RNA ligase. First strand cDNA synthesis was performed following standard protocols, using oligonucleotide AD7 (SEQ ID NO:84) containing a poly(dT) sequence.

The M. tuberculosis and M. vaccae cDNA was used as template for singlesided-specific PCR (3S-PCR). For this protocol, a degenerate oligonucleotide

ADI

(SEQ ID NO:85) was designed based on conserved leader sequences and membrane protein sequences. After 30 cycles of amplification using primer ADI as and AD7 as 3'-primer, products were separated on a urea/polyacrylamide gel. DNA bands unique to M vaccae were excised and re-amplified using primers ADI and AD7. After gel purification, bands were cloned into pGEM-T (Promega) and the base sequence determined.

Searches with the determined nucleotide and predicted amino acid sequences of band 12B21 (SEQ ID NOS: 86 and 87, respectively) showed homology to the pota gene of E.coli encoding the ATP-binding protein of the spermidine/putrescine

ABC

transporter complex published by Furuchi et al. (Jn. Biol Chem. 266: 20928-20933, 1991). The spermidine/putrescine transporter complex of E.coli consists of four genes and is a member of the ABC transporter family. The ABC (ATP-binding Cassette) transporters typically consist of four genes: an ATP-binding gene, a S periplasmic, or substrate binding, gene and two transmembrane genes. The transmembrane genes encode proteins each characteristically having six membrane- 15 spanning regions. Homologues (by similarity) of this ABC transporter have been identified in the genomes of Haemophilus influenza (Fleischmann et al. Science 269 :496-512, 1995) and Mycoplasma genitalium (Fraser, et al. Science, 270:397-403, 1995).

An M. vaccae genomic DNA library constructed in BamHl-digested lambda ZAP Express (Stratagene) was probed with the radiolabelled 238 bp band 12B21 Sfollowing standard protocols. A plaque was purified to purity by repetitive screening and a phag d mid containing a 4.5 kb insert was identified by Southern blotting and hybridisation. The nucleotide sequence of the full-length M. vaccae homologue of pota (ATP-binding protein) was identified by subcloning of the 4.5 kb fragment and base sequencing. The gene consisted of 1449 bp including an untranslated 5' region of 320 bp containing putative -10 and -35 promoter elements. The nucleotide and predicted amino acid sequences of the M vaccae pota homologue are provided in SEQ ID NOS:88 and 89, respectively.

The nucleotide sequence of the M. vaccae pota gene was used to design primers EV24 and EV25 (SEQ ID NO: 90 and 91) for expression cloning. The amplified DNA fragment was cloned into pProEX HT prokaryotic expression system (Gibco BRL) and expression in an appropriate E.coli host was induced by addition of 0.6 mM isopropylthio-P-galactoside (IPTG). The recombinant protein was named GV-23 and purified from inclusion bodies according to the manufacturer's protocol.

In subsequent studies. GV-23 (SEQ ID NO: 88) was re-cloned into the alternative S vector pETI6 (Novagen).

A 322 bp Sall-BamHl subclone at the 3'-end of the 4.5 kb insert described above showed homology to the potd gene, (periplasmic protein), of the spermidine/putrescine ABC transporter complex of E. coli. The nucleotide sequence of this subclone is shown in SEQ ID NO:92. To identify the gene, the radiolabelled insert of this subclone was used to probe an M vaccae genomic DNA library constructed in the Sail-site of lambda Zap Express (Stratagene) following standard protocols. A clone was identified of which 1342 bp showed homology with the potd gene of E. coli. The potd homologue of M. vaccae was identified by sub-cloning and base sequencing. The determined nucleotide and predicted amino acid sequences are 15 shown in SEQ ID NO: 93 and 94.

For expression cloning, primers EV26 and EV27 (SEQ ID NOS:95-96) were designed from the determined M vaccae potd homologue. The amplified fragment was cloned into pProEX HT Prokaryotic expression system (Gibco BRL). Expression in an appropriate E. coli host was induced by addition of 0.6 mM IPTG and the 20 recombinant protein named GV24. The recombinant antigen was purified from inclusion bodies according to the protocol of the supplier. In subsequent studies, GV- 24 (SEQ ID'NO: 93) was re-cloned into the alternative vector pET 6 (Novagen).

The ability of purified recombinant protein GV-23 and GV-24 to stimulate proliferation of T cells and interferon- production in human PBL was determined as described in Example 2. The results of these assays are provided in Table 6, wherein indicates a lack of activity, indicates polypeptides having a result less than twice higher than background activity of control media, indicates polypeptides having activity two to four times above background, indicates polypeptides having activity greater than four times above background, and (ND) indicates not determined.

TABLE 6 Donor Donor Donor Donor Donor Donor G97005 G97006 G97007 G97008 G97009 G97010 Prolif IFN Prolif IFN Prolif IFN Prolif if Proif

FN

GY-yY I Prolif I GV-. 24GV- ND ND 24__

I

Base sequence adjacent to the l L vaccae pod gene-homologue was found to show homology to the potb gene of the spermidine/putrescine ABC transporter complex of E.coli, which is one of two transmembrane proteins in the ABC transporter complex. The M. vaccae porb homologue (referred to as GV-25) was identified through further subcloning and base sequencing. The determined nucleotide and predicted amino acid sequences for GV-25 are shown in SEQ ID 10 NOS:97 and 98, respectively.

Further subcloning and base sequence analysis of the adjacent 509 bp failed to reveal significant homology to PotC, the second transmembrane protein of E.coli, and Ssuggests that a second transmembrane protein is absent in the M vaccae homologue of the ABC transporter. An open reading frame with homology to M tuberculosis acetyl-CoA acetyl transferase, however, was identified starting 530 bp downstream of the transmembrane protein and the translated protein was named GV-26. The determined partial nucleotide sequence and predicted amino acid sequence for GV-26 are shown n SEQ ID NO:99 and 100.

Using a protocol similar to that described above for the isotion of GV-23, the 3S-PCR band 12B28 (SEQ ID NO: 119) was used to screen the M vaccae genomic library constructed in the BamHI-site of lambda ZAP Express (Stratagene). The clone isolated from the library contained a novel open reading frame and the antigen encoded by this gene was named GV-38A. The determined nucleotide sequence and predicted amino acid sequence of GV-38A are shown in SEQ ID NO: 120 and 121, respectively. Comparison of these sequences with those in the gene bank, revealed some homology to an unknown M tuberculosis protein previously identified in cosmid MTCY428.12. (SPTREMBL:P71915).

Upstream of the GV-38A gene, a second novel open reading frame was identified and the antigen encoded by this gene was named GV-38B. The determined and 3" nucleotide sequences for GV-38B are provided in SEQ ID NO: 122 and 123, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 124 and 125. respectively. This protein was found to show homology to an unknown M tuberculosis protein identified in cosmid MTCY428.11 (SPTREMBL: P71914).

Both the GV-38A and GV-38B antigens were amplified for expression cloning into pET16 (Novagen). GV-38A was amplified with primers KRI 1 and KR12 (SEQ ID NO: 126 and 127) and GV-38B with primers KRI3 and KR14 (SEQ ID NO: 128 and 129). Protein expression in the host cells BL21(DE3) was induced with 1 mM IPTG, however no protein expression was obtained from these constructs.

Hydrophobic regions were identified in the N-termini of antigens GV-38A and GV- 38B which may inhibit expression of these constructs. The hydrophobic region 15 present in GV-38A was identified as a possible transmembrane motif with six membrane spanning regions. To express the antigens without the hydrophobic regions, primers KR20 for GV-38A, (SEQ ID NO: 130) and KR21 for GV-38B

(SEQ

ID NO: 131) were designed. The truncated GV-38A gene was amplified with primers KR20 and KR12, and the truncated GV-38B gene with KR21 and KR14. The 20 determined nucleotide sequences of truncated GV38A and GV-38B are shown in SEQ ID NO: 132 and 133, respectively, with the corresponding predicted amino acid sequencesbeing shown in SEQ ID NO: 134 and 135, respectively.

EXAMPLE 9 PURIFICATION AND CHARACTERISATION OF POLYPEPTIDES FROM M VACCAE CULTURE FILTRATE BY PREPARATIVE ISOELECTRIC

FOCUSING

AND PREPARATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS M vaccae soluble proteins were isolated from culture filtrate using preparative isoelectric focusing and preparative polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.

M. vaccae (ATCC Number 15483) was cultured in 250 1 sterile Medium which had been fractionated by ultrafiltration to remove all proteins of greater than kDa molecular weight. The medium was centrifuged to remove the bacteria, and steriised by filtration through a 0.45 m filter. The sterile filtrate was concentrated by ultrafiltration over a 10 kDa molecular weight cit-off membrane.

Proteins were isolated from the concentrated culture filtrate by precipitation with 10% trichloroacetic acid. The precipitated proteins were re-dissolved in 1 00 mM Tris.HCI pH 8.0. and re-precipitated by the addition of an equal volume of acetone.

The acetone precipitate was dissolved in water, and proteins were re-precipitated by the addition of an equal volume of chloroform:methanol 2:1 The Schloroform:methanol precipitate was dissolved in water, and the solution was freezedried.

The freeze-dried protein was dissolved in iso-electric focusing buffer, containing 8 M deionised urea, 2% Triton X-100, 10 mM dithiothreitol and 2% *ampholytes (pH 2.5 The sample was fractionated by preparative iso-electric focusing on a horizontal bed of Ultrodex gel at 8 watts constant power for 16 hours.

P roteins were eluted from the gel bed fractions with water and concentrated b 20 precipitation with 10% trichloroacetic acid.

Pools of fractions containing proteins of interest were identified by analytical polyacrylaiide gel electrophoresis and fractionated by preparative polyacrylamide gel electrophoresis. Samples were fractionated on 12.5% SDS-PAGE gels, and electroblotted onto nitrocellulose membranes. Proteins were located on the membranes by staining with Ponceau Red, destained with water and eluted from the membranes with 40% acetonitrile/0. .M ammonium bicarbonate pH 8.9 and then concentrated by lyophilisation.

Eluted proteins were assayed for their ability to induce proliferation and interferon-y secretion from the peripheral blood lymphocytes of immune donors as detailed in Example 2. Proteins inducing a strong response in these assays were selected for further study.

Selected proteins were further purified by reversed-phase chromatography on a Vydac Protein C4 column, using a trifluoroacetic acid-acetonitrile system.

Purified proteins were prepared for protein sequence determination by SDSpolyacrylamide gel electrophoresis, and electroblotted onto PVDF membranes.

Protein sequences were determined as in Example 3. The proteins were named GV-41, GV-42, GV43 and GV-44. The determined N-terminal sequences for these polypeptides are shown in SEQ ID NOS:101-105, respectively.

Subsequent studies led to the isolation of a middle fragment and 3' DNA sequence for GV-42 (SEQ ID NO: 136, 137 and 138, respectively). The corresponding predicted amino acid sequences are provided in SEQ ID NO: 139, 140 and 141, respectively.

All of these amino acid sequences were compared to known amino acid *sequences in the ENMBL data base using TFASTA. No significant homologies were obtained with GV40, GV42 and GV-44. GV-41 had similarity to a putative 15 ribosome recycling factor from M. tuberculosis, a protein responsible for die release of ribosomes from mRNA at the termination of protein biosynthesis. GV- 43 showed homology (by similarity) to a previously identified unknown IM.

tuberculosis and M. leprae protein.

EXAMPLE IMMf.UNE MODULATING PROPERTIES "OF DELIPIDATED AND DEGLYCOLIPIDATED M.VACCAE AND RECOMBINANT PROTEINS FROM M.VACCAE This example illustrates the processing of different constituents of M.vaccae and their immune modulating properties.

Heat-killed M. vaccae and M. vaccae culture filtrate A M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/l; tryptone, 5 g/l; glucose 1 g/l) at 37 OC. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco 553 Laboratories, Detroit, MI, USA) with glucose at 37 OC for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10 M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 oC. Tne culture filtrate was passaged through a 0.45 pM filter into sterile bottles.

Lyophilised, Delipidated and Deglycolipidated M.vaccae To prepare delipidated iM.vaccae, the autoclaved M.vaccae was pelleted bv centrifugation, the pellet washed with water and collected again by centrifugation and then freeze-dried. An aliquor of this freeze-dried M.vaccde was set aside and referred to as lyophilised M.vaccae. When used in experiments it was resuspended in PBS to the desired concentration. Freeze-dried Mi. vaccae was extracted with chloroform/methanol for 60 mins at room temperature, and the extraction 15 was repeated once. The residue from chloroform/methanol extraction was further extracted with 50% ethanol by refluxing for two hours. The 50% ethanol extraction was repeated two times. The pooled 50% ethanol extracts were used as a source of M.vaccae giycolipids (see below). The residue from the ethanol extraction was freeze-dried and weighed. The amount of deliidated 20 M.vaccae prepared was equivalent to 11.1% of the starting wet weight ofM.vaccae used. For bioassay, the delipidated and deglycolipidated M.vaccae (DD-M.

vaccae; referred to as delipidated M.vaccae in Figures 8 and was resuspended in phosphate-buffered saline by sonication, and sterilised by autoclaving.

M.vaccae glycolipids The pooled 50% ethanol extracts described above were dried by rotary evaporation redissolved in water, and freeze-dried. The amount of lycolipid recovered was 1.2% of the starting wet weight of M.vaccae used. For bioassay, the glycolipids were dissolved in phosphate-buffered saline.

Production of Interleukin-12 from macrophages As detailed below, whole heat-killed M.vaccae and M vaccae constituents were shown to stimulate the production of interleukin-12 (IL-12) from macrophages, an important component of the Thl response. A group of C57BL/6J mice were injected intraperitoneally with DIFCO thioglycolate and after three days, peritoneal macrophages were collected and placed in cell culture with interferon-gamma for three hours. The culture medium was replaced and various concentrations of whole heat-killed M. vaccae, lyophilized M vaccae, DD-M. vaccae (referred to as delipidated M. vaccae in Figure 8) and M. vaccae glycolipids were added. After a further three days at 37 the culture supernatants were assayed for the presence of IL-12 produced by macrophages. Asshown in Figure 8, the M vaccae preparations stimulated the production of IL-12 from macrophages.

Figures 9A, B, and C show data from separate experiments in which groups of i C57BL/6 mice (Fig. 9A), BALB/C mice (Fig. 9B) or C3H/HeJ mice (Fig. 9C) were 15 given DIFCO thioglycolate intraperitoneally and, after three days, peritoneal macrophages were collected and placed in culture with interferon-gamma for three *..hours. The culture medium was replaced and various concentrations of M.vaccae recombinant proteins GVc-3 (GV GVc4P (GV 4P), GVc-7 (GV7), GV-23, GV 27, heat killed M.vaccae, DD-M.vaccae (referred to as delipidated M vaccae in Figures 20 9A, B and Mvaccae glycolipids or lipopolysaccharide were added. After three days at 37"C, the culture supernatants were assayed for the presence of IL-12 produced" macrophages. As shown in Figures 9A, B and C, the recombinant proteins and Mvaccae preparations stimulated the production of IL-12 from macrophages.

EXAMPLE 11 EFFECT OF INTRADERMAL

ROUTE

OF IMMUNISATION WITH M. VACCA4E ON TUBERCULOSIS IN CYNOMOLGOUS

MONKEYS

This example illustrates the effect of immunisation with Mvaccae or M vaccae culture filtrate intradermally in cynomolgous monkeys prior to challenge with live M tuberculosis.

M.vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast Sextact, 2 .5g/1; tryptone, 5g/l; glucose, Ig/l) at 37C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco 15 Laboratories, Detroit, MI, USA) with glucose at 37 0 C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of equivalent to 10'0 Mvaccae organisms per mi. The cell suspension was then autoclaved for 15 min at 120 0 C. The culture filtrate was passaged through a 0.45 jM 20 filter into sterile bottles.

Three groups of cynomolgous monkeys were included in this study, with each Sgroup containing 2 monkeys. One group of monkeys were immunised with whole o. heat-killed M. vaccae; one group were immunised with M vaccae culture filtrate and a control group received no immunisations. The composition employed for immunisation, amount of immunogen and route of administration for each group of monkeys are provided in Table 7.

TABLE 7 COMPARISON OF INTRADERMAL ROUTE OF IMMUNISATION Group Identification Amount of Route of Number Number of Antigen Immunisation t5 Immunisation Monkey 1 S3101-E 0 (Controls) 3144-B 0 2 4080-B 500pg intradermal (Immunised 3586-B 500gg intradermal with heat-killed Mvaccae) :(Immunised 3564-B 100.g intradermal with culture filtrate) 3815-B 100 Lg intradermal Prior to immunisation, all monkeys were weighed (Wt kgs), body temperature measured (temp), and a blood sample taken for determination of erythrocyte sedimentation rate (ESR mm/hr) and lymphocyte proliferation (LPA) to an in vitro challenge with purified protein (PPD) prepared from Mycobacterium bovis. At day 33 30 post-immunisation these measurements were repeated. At day 34, all monkeys received a second immunisation using the same amount of M.vaccae. On day 62, body weight, temperature, ESR and LPA to PPD were measured, then all monkeys were infected with 10' colony forming units of the Erdman strain of M. tuberculosis.

Twenty eight days following infection, body weight, temperature, ESR and LPA to PPD were measured in all monkeys, and the lungs were X-rayed to determine whether, infection with live M tuberculosis had resulted in the onset of pneumonia.

As shown in Tables 8A, B and C, the monkeys in the control group showed radiologic evidence of pulmonary tuberculosis by 28 days after infection with M.tuberculosis. Clinical disease was not evident 84 days after infection in monkeys immunised intradermally with two doses Of 500 Pag Of U fvaccae. The onset of clinical disease was delayed in both monkeys inmmunised itaeral vaccae culture filtrate.

TABLE 8A CONTROL

MONKEYS

1D# Days Wt.Kg-s Temp. ESR LPA LPA X-Ray Mm/hr PPD

PPD

Mpjg Ijgg 34 1.88 37.3 ND 0.5 1.4

N

InNeection 56 1.2 37.2 20 1 5.6 9.1 Positive 84 1.81 37.5 5.6

D

8.7Positive 314B1 .05 376 0 09.87 21.8 Negtive 1584 .1.82 37.3 4 45.3 23.4 pqru ND ND Not Done TABLE 8B MONKEYS IMMUNISED WITH WHOLE HEAT-KILLED M. VACCAE (500 jig) INTRADERMALLY a. a a a ID# Days Wt.Kgs Temp. -ESR LPA LP A X-Ra Mm/hr PPD PPD Slog~g 1In 4080-B 0 2.05 jf37.1 1 1.1 0.77 TNegative 1.97 138.0 ND 1.7 1.4 jND 62 12.09 136.7 ND 11.5 1.5 IND Time of Infection 28 ]2.15 137.6 0 1 2.6 Negative 12.17 137.6 0 Negativ-e 84__ ]2.25 37.3 0 2. Negative ID# Days Wt.Kgs Temp. ES LP LA XRa mm/hr PPD PPD 3586-B 0 2.29 J37.0 0 11.1 1.4 Negative 2.22 138.0 ND 11.9 1.6ND -02_ 2.39 136.0 ND 11.3 1.6. ND Time of Infection 2.31 138.2 0 3.2 26 Negative 2.32 137.2 0 7.8 4.2 Negatv 2.81 j37.4 0 3.4 1.8 JNegiv ND NtDn Not Done TABLE 8C MONKEYS IMMUNISED WITH CULTURE FILTRATE (1004g)

INTRADERMALLY

ND Not Done Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, changes and modifications can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims.

SEQUENCE

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INFECTIONS

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TELEX

INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS. single

T

OPOLOGY: linear (ii) MOLECUJLE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:1: Ala Pro Val Gly Pro Gly Xaa Ala Ala Tyr Val Gin Gln Val Pro Asp 1 5 10 1s Gly Pro Gly Ser Val Gin Gly Met Ala INFORMATION FOR SEQ ID NO:2: SEQUENCE

CHARACTERISTICS:

LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Xaa Asp Gin Leu Lys Val Asn Asp Asp 1 5 INFORMATION FOR SEQ ID NO:3: SEQUENCE

CHARACTERISTICS:

LENGTH: 11 amino acids TYPE: amino acid STRANDEDNESS: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Xaa Pro Val Pro Val Ala Thr Ala Ala Tyr S INFORMATION FOR SEQ ID NO:4: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ili.MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Thr Pro Ala Pro Ala Pro Pro Pro Tyr Val Asp His Val Glu Gln Ala 1 5 10 Lys Phe Gly Asp Leu 1 0 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 29 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Gin Ala Phe Asn Ala Asp Ala Tyr Ala Phe Ala Lys Arg Glu Lys 1 5 10 Val Ser Leu Ala Pro Gly Val Pro Xaa Val Phe Glu Thr INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ala Asp Pro Asn Xaa Ala Ile Leu Gin Val Ser Lys Thr Thr Arg 1 5 10 Gly Gly Gin Ala Ala INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Pro Ile Leu Gin Val Ser Gin Thr Gly Arg 1 5 INFORMATION FOR SEQ ID NO:8: SEQUENCE

CHARACTERISTICS:

LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Xaa Asp Pro Ile Xaa Leu Gin Leu Gin Val Ser Ser Thr 1 5 INFORMATION FOR SEQ ID NO:9: SEQUENCE

CHARACTERISTICS:

LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS. Single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Lys Ala Thr Tyr Val G in Gly Gly Leu Gly Arg Ile Glu Ala Arg Val 1 5 -O 1 INFORMATION FOR SEQ ID NO:lO: SEQUENCE

CHARACTERISTICS:

LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECUL~E TYPE: protein .06 SEQUENCE DESCRIPTION: SEQ ID N:1o: :Lys Xaa Gly Leu Ala Asp Leu Ala Pro INFORMATION FOR SEQ ID NO:li: SEQUENCE

CHARACTERISTICS:

LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS. single TOPOLOGY: linear 6*GS (ii) MOLECULE TYPE: protein SS@@ (ix) FEATURE: Ja) NAMVE/KEY: the -LOCATION: .12 OTHER INFORMATION: Residue can be either Glu or Ile (xi) SEQUENCE DESCRIPTION: SEQ ID N0:l1: Lys Xaa Tyr Ala Leu Ala Leu Met Ser Al a Val Xaa Ala'Ala 1 5 INFORMATION FOR SEQ ID NO:12: SEQUENCE

CHARACTERISTICS:

LENGTH: 11 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Lys Asn Pro Gln Val Ser Asp Glu Leu Xaa Thr 1 5 INFORMATION FOR SEQ ID NO:13: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Ala Pro Ala Pro Ala Ala Pro Ala Xaa Gly Asp Pro Ala Ala Val Val 1 5 10 Ala Ala Asn Ser Thr INFORMATION FOR SEQ ID NO:14: i) SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear- (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Glu Ala Glu Val Xaa Tyr Leu Gly Gln Pro Gly Glu Leu Val Asn 5 10 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Residue can be either Gly or Ala NAME/KEY: Other LOCATION: 15...15 OTHER INFORMATION: Residue can be either Pro or Ala (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Xaa Val Val Pro Pro Xaa Gly Pro Pro Ala Pro Gly Ala Xaa 1 5 1 0 INFORMATION FOR SEQ ID NO:16: SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Ala Pro Ala Pro Asp Leu Gln Gly Pro Leu Val Ser Thr Leu Ser INFORMATION FOR SEQ ID NO:17: SEQUENCE

CHARACTERISTICS:

A) LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Ala Thr Pro Asp Trp Ser Gly Arg Tyr Thr Val Val Thr Phe Ala Ser 1 5 10 Asp Lys Leu Gly Thr Ser Val Ala Ala *20 INFORMATION FOR SEQ ID NO:18: (i)CSEQUENCE

CHARACTERISTICS:

LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other LOCATION: 15...15 OTHER INFORMATION: Residue can be either Ala or Arg NAME/KEY: Other LOCATION: 23 23 OTHER INFORMATION: Residue can be either Val or Leu (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 66 Ala Pro Pro Tyr Asp Asp Arg Gly Tyr Val Asp Ser Thr Ala Xaa Xaa 1 5 10 Ala Ser Pro Pro Thr Leu Xaa Val Val INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Glu Pro Glu Gly Val Ala Pro Pro 1 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Pro Ala Gly Ile Pro Ala. Gly Phe Pro Asp Val Ser Ala Tyr Ala 1 5 10 Ala Val Asp Pro Xaa Xaa Tyr Val Val 20 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Ala Pro Val Gly Pro Gly Xaa Ala Ala Tyr Val Gln Gln Val Pro 1 5 10 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (x4) SEQUENCE DESCRIPTION: SEQ ID NO:22: Phe Ser Arg Pro. Gly Leu Pro Val Glu Tyr Leu Asp ValPhSe INFORMATI ON FOR SEQ ID NO:23: SEQUENCE

CHARACTERISTICS:

LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Phe Ser Arg PoGly Leu Pro Val Giu Tyr LeMtVa Pro Ser Pro Ser Met Gly INFORMATION FRSEQ ID NO:24: i)SEQUENCE

CHARACTERISTICS:

LENGTH: 15 aioacids TYPE: amino acid STRNDEDNSS single tD) TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:'24: Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Met Val Pro Ser 1 5 1 INFORMATION FOR SEQ ID Ci) SEQUJENCE

CHARACTERISTICS:

LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2S: Xaa Xaa Thr Gly Leu His Arg Leu Arg Met Met Val Pro Asn 1 5 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other LOCATION: 16...16 OTHER INFORMATION: Residue NAME/KEY: Other LOCATION: 17...17 OTHER INFORMATION: Residue can be either Ser or Val can be either Gin or Val (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Val Pro Ala Asp Pro Val Gly Ala Ala Ala 1 5 10 Xaa Arg Ile Asp Gin Ala Glu Pro Ala Xaa INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other 1B) LOCATION: 4...4 *OTHER INFORMATION: Residue NAME/KEY: Other LOCATION: 8...8 OTHER INFORMATION: Residue can be either Tyr or Pro can b-e i1 h- NAME/KEY: Other LOCATION: 9...9 OTHER INFORMATION: S e ier Val or Giy Residue can be either Ile or Tyr (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Asp Pro Xaa Xaa Asp Ile Glu Xaa Xaa Phe Ala Arg Gly Thr 1 5 INFORMATION FOR SEQ ID NO:28: SEQUENCE

CHARACTERISTICS:

IUV 1 LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Ala Pro Ser Leu Ser Val Ser Asp Tyr Ala Arg Asp Ala Gly Phe 1 5 1 0 INFORMATION FOR SEQ ID NO:29: SEQUENCE

CHARACTERISTICS:

LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other LOCATION: 2...2 Residu INcan bTIOe D) OTHER INFORMATION: Residue can be either Leu or Pro (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Xaa Xaa Leu Ala Xaa Ala Xaa Leu Gly Xaa Thr Val Asp Ala Asp Gln 1o 5 10 15 INFORMATION FOR SEQ ID (ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 330 amino acids TYPE: amino acid STRANDEDNESS: single c: D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Phe Val Asp Arg Phe Arg Gly Ala Val Ala Gly Met Leu Arg 1 5 0 Arg Leu Val Val Glu Ala Met Gly Val Ala e Ser Ala e Ile 20 25 30 Gly Val Val Gly Ser Ala Pro Ala Glu Ala Phe Ser Arg Pro Gly Leu 40 Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Se Met Gly Arg Asp 55 Se Met Gly Ag Asp Ile Lys Val Gln Phe Gln Asn Gly Gly Ala Ash Ser Pro Ala Leu Tyr Leu 70 75 Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe Ser Gly Trp Asp Ile Asn 85 90 Thr Thr Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Ile Ser Val Val Met Pro Val Gly Gly Gin Ser Ser Phe Cys Ser 145 Gly Leu Giy Ala Ser 225 Thr Lys Gly Gly Ser 305 Gin Gly 130 Giu Ser Al a Leu Met 210 Thr Leu Pro Leu Gly 290 Trp Gin Lys Leu Al a Ile Leu 195 Gly Asp Ile Thr Val 275 Gly Glu Tyr Al a Pro Ala Tyr 180 As p AspD Pro Ala Glu 260 Arg His Tyr Leu Gly Giu Val 165 His Pro Ala Ala Asn 245 Leu Thr Asn Tr-p Gly.

Cys Tyr iso Gly Pro Ser Gly T rp 230 Asn Giy Ser Alia 310 A.la Gin 135 Leu Leu Asp Asn Gly 215 Lys Thr Gly Asn Val 295 Giu Thr 120 Thr GIn Ser Gin Al a 200 Tyr Arg Arg Asn Ile 280 Phe Gin Pro 105 Tyr Tyr Ser Met~ Phe 185 Met Lys Asn Ile Asn 265 Lys Asn Leu Giy Lvs T Asn L 1.

Ala G: :70 Ile T Gvy P2 Al1a Al Aso Pi 250 Leu Pr Phe Gi Phe Pr Asn As 31 Al a 330 NO: 3 1: 125 rp Giu Thr Phe Leu Thr 140 ys Gin Ile Lys Pro Thr 110 Ser Asp Trp Tyr 55 ly :0 '5 n *0 p 0 Leu Vai Ser Asp 220 Thr Tyr Ala Asp Asp 300 Met Ser Gly Leu 205 Met Val Cys Lys Gly 285 Ser Lys Ala Ser 190 Ile T -p Asn Gly Leu 270 Tyr Gly Pro Leu 175 Met Gly Gly Val Asn 255 Leu Asn Thr Asp 160 Thr Ser Leu Pro Gly 240 Gly Glu Ala His Leu 320 INFORMVATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 327 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii-I.MOLECULE TYPE: protein Met 1 Val Al a Leu Gin Arg Glu Gin (xi) SEQUENCE Ile Asp Val Ser

S

Gly Ala Ala Ala Ala Thr Ala Ser Gin Vai Pro Ser Asn Gly Gly Asn Ala Gin Asp Asp Trp Tyr Tyr Gin 100 Ser Ser Phe Tyr DESCRIPTION: SEQ 7D Gly Lys Ile Arg Ala 10 Thr Leu Pro Ser Leu 25 Ala Phe Ser Arg Pro 40 Giu Ala Met Giy Arg 55 Gly Ser Pro Ala Val 70 Tyr Asn Gly Trp Asp 90 Ser Gly Leu Ser Val 105 Ser Asp Trp Tyr Ser NO: 3 1: Trp Gly Ile Ser Gly -Leu Thr Ile Tyr Leu 75 Ile Asn Val Met Pro Ala Arg Trp, Leu Ala Pro 'Val Lys Val Leu Asp Thr Ser Pro Vai 110 Cys Gly Leu Gly Gi u Gin Gly Al~a Gly Lys Leu Gly Tyr Phe Leu Phe Gly Ala 1.hIfL7 UUjt):, Gly Cys 130 Lys Tro 145 Val Gly His Pro Ser Ser Ala Gly 210 Ala Trp 225 Asn Asn Leu Gly

C

Gly SerA 2 Asn Ala V 290 Trp Gly A 305 :Met Ala V 11 Th Le Lei Asj Gir 195 Gly Glm rhr ;ly sn 75 al la al

S

r Thr Tyr Lys u Ser Ala Asn 150 1 Ser Met Ala 165 Gin Phe -Ile 180 Gly Ile Glu Tyr Lys Ala Arg Asn Asp 230 His Leu Trp 245 Thr Asn Val 1 260 Leu Lys Phe G Phe Asn Leu A 2 Gin Leu Asn A 310 Pro Arg Ser G.

325 Trp 135 Arg Gly Tyr Pro Ala 215 Pro lal i 'ro A 1n A 2 sn A 95 la Mi ly 12 G1 Se Se: Al Glr 20C Asp Ile 'yr ~la sp 80 la et 0 u Th r Va r Se a Gi' 18 1 Lei.

Met Leu Cys Glu 265 Ala Asp Lys .r Phe Leu 1 Lys Ser 155 r Ala Leu 170 y Ser Lieu 1 Ile Gly Trp Gly Gin Ala 235 Gly Asn C 250 Phe Leu G Tyr Asn G Gly Thr H 3 Pro Asp Li 315 Th 14 Th le Se, Leu Pro 220 31 ly ;lu ly is 00 eu.

12 r Se; 0 r Gi' Let Ala Ala 205 Pro Lys Thr Asn Ala 285 Ser Gin

S

r Glu Leu Ser Ala 2 Ala Ala 175 Leu Met 190 Met Gly Asn. Asp Leu Val Pro Ser 255 Phe Val 1 270 Gly Gly H Trp Giu T Asn Thr L 3 Pro Val 160 Tyr Asp Asp Pro Ala 240 31u ~is [is yr eu Met 1 Arg Gly Leu lie Leu Asn Met Ala INFORMATION FOR SEQ ID NO:32: SEQUENCE

CHARACTERISTICS:

LENGTH: 338 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xij SEQUENCE DESCRIPTION: SEQ ID NO:32: Gin Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly 5 10 Leu Val Val Giy Ala Val Gly Ala Ala Leu Val Ser 20 25 Ala Val Giy Gly Thr Ala Thr Ala Gly Ala Phe Ser 35 40 45 Pro Vai Glu Tyr Leu Gin Val Pro Ser Pro Ser Met s0 55 60 Lys Val Gin Phe Gin Ser Gly Gly Ala Asn Ser Pro 70 75 Leu Asp Giy Leu Arg Ala Gin Asp Asp Phe Ser Gly 90 Thr Pro Ala Phe Giu Trp Tyr Asp Gn Ser Gly Leu 100 105 Pro Val Gly Giy Gin Ser Ser Phe Tyr Ser Asp Trp 115 120 125 Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys Trp Giu Met Gly Arg Gly Ala Trp Ser 110 Tyr rhr Ser Arg Leu Val Pro Gly Arg Asp Leu. Tyr Asp Ile Val Val Gin Pro Phe Leu Thr Ser Giu Leu Pro Gly Tr-p Leu Gin Ala Asn Arg His Val Lys Pro 145 ISO 1 ;C Thr Gly Thr Leu Ser Gly Leu Ala Pro Lys 225 Gly Lys Gly Lys Glu Gly Ala Gly 290 His Ser 305 Leu Gin Gly Ala Ser Ala Ala Ile 180 Leu Leu 195 Met Gly Giu Asp Leu Ile Pro Ser 260 Phe Val 275 Gly Gly Trp Glu Arg Ala Val 165 Tyr Asp Asp Pro Al1 a 245 Asp Arg His Tyr Leu Val Gly His Pro Pro Ser Ala Gly 215 Al1a Tm 230 Asn Asn Leu Gly Thr Ser Asn Gly 295 Trp Gly 310 Gly Ala Leu Gin Gin 200 Gly Gin Thr Giy Asn 280 Val Ala Thr Ser Gin 185 Al a Tyr Arg Arg As n 265 Ile Phe Gin Pro Met 170 Phe Met LyS Asn Val 250 Asn Lys Asp Leu Asn 330 Ala Val1 Gly Al a Asp 235 TmD Leu Phe Phe Asn 315 Thr Ala Tyr Pro Ser 220 Pro Vai Pro Gin Pro 300 Al a Gly Ser Al a Thr 205 Asp Leu Tyr Aila Asp 285 Asp Met Pro Ser Gly 190 Leu Met Leu Cys Lys 270 Al a Ser Lys Al1a Al a 175 Al a le Trp Asn Gly 255 Phe Tyr Gly Pro Pro 335 Leu Met Gly Gly Val 240 Asn Leu Asn Thr Asp 320 Gin 325 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 325 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met~ Ile Gly Giu Gin Giy Al a Gly Lys (xi.1. SEQUENCE Thr Asp Val Ser.

Gly Thr Ala Ala Gly Ala Ala Thr Tyr Leu Gin Val Phe Gin Ser Giy Leu Arg Ala Gin Phe Giu Trp Tyr 100 Gly Gin Ser Ser 115 Ala Gly Cys Gin DESCRIPTION: SEQ ID Arg Lys Ile Arg Ala 10 Ala Val Val Leu Pro 25 Ala Gly Ala Phe Ser 40 Pro Ser, Pro Ser-Met 55 Gly Asn Asn Ser Pro 70 Asp Asp Tyr Asn Gly 90 Tyr Gin Ser Giy Leu 105 Phe Tyr Ser Aspo Trp 120 Thr Tyr Lys Trp Glu NO: 3 3: Trp Gly Gly Leu Arg Pro Gly Arg Ala Val 75 Trp Asp Ser Ile Tyr Ser Thr Phe Arg Val Giy Asp Tyr Ile Val Pro 125 Leu Arg Gly Leu Ile Leu Asn Met 110 Ala Thr Leu is Leu Pro Lys Leu Thr Pro Cys Ser met Al a Val Val Asp Pro Val Gly Glu Lei 14 Ala Ala Leu Gly Asp 225 Val Asn Val Gly Glu 305 Ser 130 u Pro Gln Ala Ile Tyr His Asp Pro 195 Asp Ala 210 Pro Ala Ala Asn Glu Leu Arg Ser 275 His Asn 290 Tyr Trp C Leu Gly A Tr Gly Pro 180 Ser Gly Trp Asn Gly 260 Ser Ala ;ly la p Leu r Leu 165 Gin Gin Gly Glu Thr 245 Gly Asn I Val i Ala G 3 Gly Ser 150 Ser Gin Gly Tyr Arg 230 Arg Ala Leu Phe ;In 10 13 Al Met Phe Met Lys 215 Asn Leu Asn Lys Asn 295 Leu 5 a Asn Arg Ala Val 155 t Ala Gly Ser Ser 170 Ile Tyr Ala Gly 185 Gly Pro Ser Leu 200 Ala Ala Asp Met Asp Pro Thr Gln 235 Trp Val Tyr Cys 250 Ile Pro Ala Glu 265 Phe Gln Asp Ala 280 Phe Pro Pro Asn C 3 Asn Ala Met Lys G 315 140 Lys Pro Ala Met Ser Leu Ile Gly 205 Trp Gly 220 Gin Ile Gly Asn Phe Leu Tyr Asn A 285 ;ly Thr H ;00 ily Asp L Th Ile Ser 190 Leu Pro Pro Gly Glu 270 4la is eu r Gly Ser 160 Leu Ala 175 Ala Leu Ala Met Ser Ser Lys Leu 240 Thr Pro 255 Asn Phe Ala Gly Ser Trp Gin Ser 320 9 *9 9* 9 Met 1 Arg Gly Leu Ile Leu Asn Met Ala Thr INFORMATION FOR SEQ ID NO:34: SEQUENCE

CHARACTERISTICS:

LENGTH: 338 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gin Leu Val Asp Arg Val Arg Gly Ala Val 5 10 Leu Val Val Gly Ala Val Gly Ala Ala Leu 25 Ala Val Gly Gly Thr Ala Thr Ala Gly Ala 40 Pro Val Glu Tyr Leu Gln Val Pro Ser Pro 55 Lys Val Gln Phe Gln Ser Gly Gly Ala Asn 70 75 Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe 90 Thr Pro Ala Phe Glu Trp Tyr Asp Gln Sen 100 105 Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser 115 120 Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys 130 135 Ser Glu Leu Pro Gly Trp Leu Gin Ala Asn 34: Thr Gly Met Val Ser Gly Phe Ser Arg Ser Met Gly 60 Set Pro Ala Ser Gly Trp Gly Leu Ser 110 Asp Trp Tyr 125 Trp Glu Thr 140 Arg His Val Ser Leu Pro Arg Leu- Asp Val Gin Phe Lys Arg Val Gly Asp Tyr Ile Val Pro Leu Pro 145 150 155 Thr Gly Ser Ala Val Val Gly Leu Ser Met Ala Ala Ser Ser Ala 165 Thr Ser Leu Pro 225 Gly Gly Glu Ala His 305 Leu Gly Ala 210 Lys Lys Lys Gly Gly 290 Ser Ala Leu 195 Met Glu Leu Pro Phe 275 Gly Trp Ile 180 Leu Gly.

Asp Ile Ser 260 Val Gly Glu Tyr Asp Asp Pro Ala 245 Asp Arg His Tyr His Pro Ala Ala 230 Asn Leu Thr Asn Trp Pro Ser Gly 215 Tr= Asn Gly Ser Gly 295 Gly Gin Gin 200 Gly Gin Thr Gly Asn 280 Val Ala 170 Gin Phe 185 Ala Met Tyr Lys Arg Asn Arg Vai 250 Asn Asn 265 Ile Lys Phe Asp Gin Leu Val Gly Ala AsD 235 Trp Leu Phe Phe Asn Tyr Pro Ser 220 Pro Val Pro Gin Pro 300 Ala Ala Thr 205 Asp Leu Tyr Ala Asp 285 Asp Met Gly 190 Leu Met Leu Cys Lys 270 Ala Ser Lys 175 Ala Ile Trp Asn Gly 255 Phe Tyr Gly Pro 160 Leu Met Gly Gly Val 240 Asn Leu Asn Thr Aso *r

S*

S*

S

S

S

S

*.qS 74 S. S 6S 310 315 Leu Gin Arg Ala Leu 325 Gly Ala Gly Ala Thr Pro Asn 330 Thr Gly Pro Ala Pro.

335 INFORMATION FOR SEQ ID i) SEQUENCE

CHARACTERISTICS:

LENGTH: 323 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Met Thr Asp Vai Ser 1 5 Ile Gly Thr Ala Ala Gly Gly Ala Ala Thr Glu Tyr Leu Gin Val Gin Phe Gin Ser Gly Gly Leu Arg Ala Gin Ala Phe Giu Trp Tyr 100 Gly Gly Gin Ser Ser 115 LysAla Gly Cys Gin 130 DESCRIPTION: SEQ ID Arg Lys Ile Ala Val Ala Gly Pro Ser 55 Gly Asn 70 Asp Asp Tyr Gin Phe Tyr Thr Tyr 135 Val Ala 40 Pro Asn Tyr Ser Ser 120 Lys Arg Leu 25 Phe Ser Ser Asn Gly 105 Asp Trp Ala 10 Pro Ser Met Pro.

Gly 90 Leu Trp Glu Tro Gly Arg Gly Ala 75 Trp Ser Tyr Thr Gly Leu Pro Arg Val Asp Ile Ser Leu Arg Val Gly Asp Tyr, Ile Val Pro 125 Leu Arg Gly Leu Ile Leu' Asr Met 110 Ala Thr Leu Leu Pro Lys Leu Thr Pro Cys Ser Met Ala Val Val Asp Pro Val Gly Glu Leu Leu Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser 145 Ala Al Ala Ty Leu Asr Ala Gly 210 Ala Trp 225 Asn Asn Leu Gly Ser Ser Asn Ala 290 Trp Gly 305 Gly Ala 150 a Ile Gly Leu Ser Met Ala 165 r His Pro Gin Gin Phe Ile 180 Pro Ser Gin Gly Met Gly 195 200 Gly Tyr Lys Ala Ala Asp 215 Glu Arg Asn Asp Pro Thr 230 Thr Arg Leu Trp Val Tyr 245 Gly Ala Asn Ile Pro Ala 260 Asn Leu Lys Phe Gin Asp 2 275 280 Val Phe Asn Phe Pro Pro A 295 Ala Gln Leu Asn Ala Met L 310 Gly Gly Tyr 185 Leu Met Gin C Cys G 2 Glu P 265 la T sn G; ys G Ser 170 Ala Ile G rrp G ;in I 2 ;ly A, 50 he L( yr LE ly Th Ly As 31 Met 1 Phe Gly Lys Gly Tyr Ile Ile Pro Leu 145 Pro INFORMATION FOR SEQ ID NO:36: SEQUENCE

CHARACTERISTICS:

LENGTH: 333 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Lys Phe Leu Gin Gin Met Arg Lys Leu Phe 5 10 Pro Ala Arg Leu Thr Ile Ala Val Ile Gly 25 Leu Val Gly Val Val Gly Asp Thr Ala Ile 40 Pro Gly Leu Pro Val Glu Tyr Leu Gin Val 55 His Asp Ile Lys Ile Gln Phe Gin Gly Gly 70 75 Leu Leu Asp Gly Leu Arg Ala Gin Glu- Asp 90 Asn Thr Pro Ala Phe Glu Glu Tyr Tyr His 100 105 Met Pro Val Gly Gly Gin Ser Ser Phe Tyr 115 120 Ser Gin Gly Asn Gly Gin His Tyr Thr Tyr 130 135 Thr Gin Glu Met Pro Ser Trp Leu Gin Ala 150 155 Thr Gly Asn Alala Aa Val Gly Leu Ser Met 155 Ser Ala Mel Gly Ser Let ;ly Leu Ala 205 ly Pro Ser 220 le Pro Lys 35 sn Gly Thr eu Glu Asn ys Pro Ala 285 ir His Ser 300 P Leu Gin 5 36: Gly Leu A Thr Ala L, 3' Ala Val A: Pro Ser pa Gly Gin Hi Tyr Asn Gl Ser Gly Le 11 Ser Asn Tr 125 Lys Trp G1 140 Asn Lys As: Ser Gly Se: 160 SIle Leu Ala 175 1 Ser Ala Leu 190 SMet Gly Asp Ser Asp Pro Leu Val Ala 240 Pro Asn Glu 255 Phe Val Arg 270 Gly Gly His Trp Glu Tyr Ser Ser Leu 320 I Ala Lys Leu Ala- Phe Ser Ser Met Ala Val .Trp Asp Ser Val Tyr Gin Thr Phe Val Leu 160 Ser Ala 165 170 175 Leu Ile Leu Ala Ser Tyr Tyr Pro Gin Gin Phe Pro Tyr Ala Ala Ser 180 185 190 Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Thr Met Ile 195 200 205 Gly Leu Ala Met Asn Asp Ser Gly Gly Tyr Asn Ala Asn Ser Met Trp 210 215 220 Gly Pro Ser Thr Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Gln 225 230 235 240 Iie Pro Arg Leu Val Ala Asn Asn Thr Arg Ile Trp Val Tyr Cys Gly 245 250 255 Asn Gly Ala Pro Asn Glu Leu Gly Gly Asp Asn Ile Pro Ala Lys Phe 260 265 270 Leu Glu Ser Leu Thr Leu Ser Thr Asn Glu Ile Phe Gin Asn Thr Tyr 275 280 285 Ala Ala Ser Gly Gly Arg Asn Gly Val Phe Asn Phe Pro Pro Asn Gly 290 295 300 Thr His Ser Trp Pro Tyr Trp Asn Gin Gin Leu Val Ala Met Lys Pro 305 310 315 320 Asp Ile Gin Gin Ile Leu Asn Gly Ser Asn Asn Asn Ala 325 330 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 340 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Met Thr Phe Phe Glu Gin Val Arg Arg Leu Arg Ser Ala Ala Thr Thr 1 5 10 Leu Pro Arg Arg Val Ala Ile Ala Ala Met Gly Ala Val Leu Val Tyr 20 25 S. Gly Leu V a l Gly Thr Phe Gly Gly Pro Ala Thr Ala Gly Ala Phe Ser 35 40 Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Ala Ser Met 55 Gly Arg Asp Ile Lys Val Gin Phe Gin Gly Gly Gly Pro His Ala Val 70 75 Tyr Leu Leu Asp Gly Leu Arg Ala Gin Asp Asp Tyr Asn Gly Trp Asp 90 Ile Asn Thr Pro Ala Phe- Glu Glu Tyr Tyr *Gi Ser Gly Leu Ser Val 100 105 110 Ile Met Pro Val Gly Gly Gin Ser Ser Phe Tyr Thr Asp Trp Tyr Gin 115 120 125 Pro Ser Gin Ser Asn Gly Gin Asn Tyr Thr Tyr Lys Trp Glu Thr Phe 130 135 140 Leu Thr Arg Glu Met Pro Ala Trp Leu Gin Ala Asn Lys Gly Val Ser 145 150 155 160 Pro Thr Gly Asn Ala Ala Val Gly Leu Ser Met Ser Gly Gly Ser Ala 165 170 175 Leu Ile Leu Ala Ala Tyr Tyr Pro Gin Gln Phe Pro Tyr Ala Ala Ser

-VVAUJ

180 185 Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp 195 200 Gly Leu Ala Met Asn Asp Ser Gly Gly Tyr Asn 210 215 Gly Pro Ser Ser Asp Pro Ala Trp Lys Arg Asn 225 230 235 Ile Pro Arg Leu Val Ala Asn Asn Thr Arg Ile 245 Asn Gly Thr Pro Ser Asp Leu Gly Gly Asp Asn 260 265 Leu Glu Gly Leu Thr Leu Arg Thr Asn Gin Thr 275 280 Ala la Asp Gly Gly Arg Asn Gly Val Phe Asn i 290 295 Thr His Ser Trp Pro Tyr Trp Asn Glu Gin Leu V 305 310 315 Asp Ile Gin His Val Leu Asn Gly Ala Thr Pro p 325 3 Ala Pro Ala Ala 330 340 INFORMATION FOR SEQ ID NO:38: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: AGCGGCTGGG

ACATCAACAC

INFORMATION FOR SEQ ID NO:39: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CAGACGCGGG

TGTTGTTGGC

INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 1211 base pairs TYPE: nucleic acid 190 Trp Pro Thr Leu 205 Ala Asn Ser Met 220 Asp Pro Met Val Trp Val Tyr Cys 255 Ile Pro Ala Lys 270 ?he Arg Asp Thr 285 Phe Pro Pro Asn 100 'al Ala Met Lys A 3 ro Ala Ala Pro A 335 Ile Trp Gin 240 Gly Phe ryr Sly Lla la STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA o o (Xi) SEQUENCE Dl GGTACCGGAA GCTGGAGGAT GCACGCCGTT TCGGCGTCGT 120 GCTGGAGGGT CGGCGACCGC 180 ATGGTGCCTT CGCCGTCGAT 240 AACTCGCCGG CTCTCTACCT 300 GACATCAACA CTCAGGCTTT 360 GTCGGTGGCC AGTCCAGCTT 420 ACCGTGACCT ACA-AGTGGGA 480 AACCGCGCGG TCAAGCCGAC 540 GCGCTGAACC TGGCGACCTG 600 TTCCTGAACC CCTCCGAGGG 660 GGCGGCTTCA AGGCCGACGA 720 CAGCGCAACG ATCCGATGCT 780 GTCTACTGCG GTAACGGCCA 840 CTCGAAGGTC TGACCATCCG 900 GGCCACAACG GTGTGTTCAA 960- CGCGAGCTGC AGGCGATGAA 1020 ACGAAGCCCC CGGCCGATTG 1080 TAACCGAAAT CAACGCGATG 1140 CACGAGGTGG GCGAGCAATC 1200 TGACGGTATG AGACTTCTTG ACAGGATTCG

GGCTGTCGCG

CGGAGCATTC

GGGGCGCGAC

GCTCGACGGC

CGAGTGGTTC

CTACACCGAC

GACCTTCCTG,

CGGCAGCGGC

GCACCCGGAG

CTGGTGGCCG

CATGTGGGGC

GAACATCCCG

GCCCACCGAG

CACCAACGAG

CTTCCCGGCC

GCCTGACCTG

CGGCCGAGGG

GTGGCTCATC

CTTCCTGCCC

ACAGCGATGA

TCCCGGCCAG

ATCAAGATCC

CTGCGTGCGC

CTCGACAGCG

TGGTACGCCC

ACCCAGGAGC

CCTGTCGGTC

CAGTTCATCT

TTCCTGATCA

AAGACCGAGG

ACCCTGGTCG

CTCGGCGGCG

ACCTTCCGCG

AACGGCACGC

CAGGCGCACC

TTTCGTCGTC

AGGAACGCCG

GACGGAGAGG

TGCCTGCTTT

GTCTGCCGGT

AGTTCCAGAG

AGGAGGACTI'

GCATCTCCGT

CCGCCCGTAA

TCCCGGGCTG

TGTCGATGGC

ACGCGGGCTC

ACATCTCGAT

GGATCCCAAC

CCAACAACAC

GCGACCTIGCC

ACAACTACAT

ACAACTGGGC

TTCTCTGACG

CGGGGCTACT

AGGGGGTCAT

TCAACATCCA

TGGGCCTTGG

GG-TGGGCCTG

GGAGTACCTG

CGGTGGCGAG

CAACGGCTGG

GGTGATGCCG

CAAGGGCCCG

GCTGCAGGCC

GGGTTCGGCC

GATGTCCGGC

GGGTGACGCC

AGCGGTTGGA

CCGTATCTGG

CGCCACGTTC

CGCCGCGGGT

GTACTGGGGT

GTTGCACCAA

GTGGCCGACA

TGCGCTACGA

CGTCGAGTAC

ZSCRIPTION: SEQ ID TCCAGCGTGA A 1211 INFORMATION FOR SEQ ID NO:41: C(i) SEQUENCE CHARACTERISTICS: LENGTH: 485 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

SEQUENCE DESCRIPTION: SEQ. ID NO:41: AGCGGCTGGG ACATCAACAC CGCCGCCTTC GAGTGG3TACG TCGACTCGGG

TCTCGCGGTG

ATCATGCCCG TCGGCGGGC.A.GTCCAGCTTC TACAGCGACT GGTACAGCCC

GGCCTGCGT

120 AAGGCCGGCT GCCAGACCTA CAAGTGGGAG ACGTTCCTG.A CCCAGGAGCT

GCCGGCCTAC

180 CTCGCCGCCA ACAAGGGGGT CGACCCGAC CGCAACGCGG CCGTCGGTCT

GTCCATGGCC

240 GGTTCGGCGG CGCTGACGCT GGCGATCTAC CACCCGCAGC AGTTCCAGTA

CGCCGGGTCG

300 CTGTCGGGCT ACCTGAACCC GTCCGAGGGG TGGTGGCCGA TGCTGATCA

CATCTCGATG

360 GGTGACGCGG GCGGCTACAA GGCCAACGAC ATGTGGGGTC CACCGAAGGA

CCCGAGCAGC

420 GCCTGGAAGC GCAACGACCC GATGGTCAAC ATCGGCAAGC TGGTGGCCA

CAACACCCCC

480

CTCTC

485 INFORMATION FOR SEQ ID NO:42:.

SEQUENCE

CHARACTERISTICS:

LENGTH: 1052 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GTTGATGAGA A.AGGTGGGTT GTTTGCCGTT ATGAAGTTCA CAGAGAAGTG

GCGGGGCTCC

60 GCAAAGGCQ CGATGCACCG GGTGGGCGTT GCcG.ATATG CCGCCGTTGC

GCTGCCCGGA

120 CTGATCGGCT TCGCCGGGG TTCGGCAACG GCCGGGGCAT TCTCCCGGCC

CGGTCTTCCT

180 GTCGAGTACC TCGACGTG.'D CTCGCCGTCG ATGGGCCGCG ACATCCGGGT CCAGTTCCAG. 240 GGTGGCGGTA CTCATGCGGT CTACCTGCTC GACGGTCTGC GTGCCCAGGA

CGACTACAAC

300 GGCTGGGAA TCAACACCCC TGCGTTCGAG TGGTTCTACG AGTCCGGCTT...GTCGACGATCj 360 ATGCCGGTCG GCGGACAGTC CAGCTTCTAC AGCGACTGGT ACCAGCCGTC

TCGGGGCAAC

420 GGGCAGAACT ACACCTAA GTGGGAGACG TTCCTGACCC AGGAGCTGCC

GACGTGGCTG

480 GAGGCCAACC GCGGAGTGTC GCGCACCGGC AACGCGTTCG TCGGCCTGTC

GATGGCGGGC

540 AGCGCGGCGC TGACCTACGC GATCCATCAC CCGCAGCAGT TCATCTACGC CTCGTCGCTG 600.

TCAGGCTTCC

660

GACGCAGGCG

720

CGCAACGACC

780

TACTGCGGCA

840

GCCGCGCAGT

900

ATCGCAGCCG

960

GGGTACTGGG

1020

CAGGCCACCG

1052

TGAACCCGTC

GCTTC.AACGC

CGATGGTCAA

CCGGCACCCC

TCCTCGAAGG

GCGGCACCAA

GGCAGCAGCT

CCTAGCCACC

CGAGGGCTGG

CGAGAGCATG

CATCAACCAG

GTCGGAGCTG

ATTCACGTTG

CGGTGTCTTC

GCAGCAGATG

CACCCCACAC

TGGCC!GATGC

TGGGGCCCGT

CTGGTGGCCA

GACACCGGGA

CGC-ACCAACA

A-ACTTCCCGG

AAG-CCCGACA

cc TGATCGGGCT GGCGATGAAC CCTCGGACCC GGCGTGGAAG ACAACACCCG

GATCTGGATC

CCCCGGGCCA

GAACCTGATG

TCGCCTTCCG

TGACA.ACTAC

CCTCGGGCAC CCACAGCTGG TCCAGCGGGT TCTGGGAGCT INFORM4ATION FOR SEQ ID NO:43: Ui) SEQUENCE CHARACTERISTICS: LENGT.H: 326 amino acids TYPE: amino acid STRAND)EDNESS: single TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:43: Met Val.

Gly Glu Gin 65 Giy Ala Gly Lys Leu 145 Gly Thr Leu Arg Leu Leu Asp Arg Ile Arg Gly Pro Val Ala Gly Ser 35 Tyr Leu 50 Phe Gln Leu Arg Phe Glu Gly Gln 115 Gly Pro 130 Pro Gly Pro Val Trp His Asn Pro Val Ala Met Ser Ala Trp 100 Ser Thr Trm Gly Pro 180 Ser Al a

T!=

Val Gly Gln Phe Ser Val Leu Leu 165 Glu Giu Thr Ala Pro Gly 70 Glu Leu Phe Thr Gin 150 Ser Gin Gly Al a Gly Ser 55 Glu Asp Aspo Tyr Tyr 135 Al a Met Phe Trp Lys 215 Met Ala 40 Pro Asn Phe Ser Thin 120 Lys Asn Al a Ile Trp 200 Ala Met Pro 25 Phe Ser Ser Mez Ser Pro Asn Gly 90 Gly Ile 105 Asp Trp, Trp Glu Aing Ala Gly Ser 170 Tyr Ala 185 Pro Phe Asp Asp Trp Ala Ala Leu Aing Pro Gly Arg Ala Leu 75 Trp Asp Ser Val Tyr Ala Thr Phe '140 Val Lys Ala Ala Gly Ser Leu Ile Met Trp 220 Arg Val Gly Asp Tyr Ile Val Pro 125 Leu Pro Leu Met Asn 205 Gly Arg Gly Leu Ile Leu Asn Met 110 Al a Thr Thr Asn Ser 190 Ile Lys Phe Leu Pro Lys Leu Thn Pro Aing Gin Gly Leu 175 Gly Ser Thr Gly Ala Val Ile Asp Gin Val Asn Giu Ser 160 Ala Phe Met Glu 195 Gly Asp 210 Ala Gly Gly Phe Gl225y lie Pro Thr Ala Val Gly Gn Arg Asn Asp Pro Met Leu Asn Ile 225 230 235 240 Pro Thr Leu Val Ala Asn Asn Thr Arg Ile Trp Val Tyr Cys Gly Asn 4y 5n o r G 250 255 SGn Pro Thr Gu Leu Gy Gly Gly Asp Leu Pro Ala Thr Phe Leu 260 265 270 Glu Gly Leu Thr Ile Arg Thr Asn Glu Thr Phe Arg Asp Asn Tyr Ile 275 280 285 Ala Ala Gly Gly His Asn Gy Val Phe Asn Phe Pro Ala Asn Gly Thr 290 295 300 His Asn Trp Ala Tyr Trp Gly Arg Glu Leu Gln Ala Met Lys Pro Asp 305 310 315 Met ys Po Asp Leu Gin Ala His Leu Leu 3 1 320 325 INFORMATION FOR SEQ ID NO:44: SEQUENCE

CHARACTERISTICS:

LENGTH: 161 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: .er

G

l y Trp As Ile Asn Thr Ala Ala Phe Glu Trp Tyr Val Asp Ser 1 5 0 G l y Leu Ala Val Ile Met Pro Val y Gy G Sr Ser Phe Tyr Ser 25 30Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys 40 Trp Glu Thr Phe Leu Thr Gln Glu Leu Pro Ala Tyr Le Ala Ala Asn 55a Tyr Leu Ala Ala Asn Lys Gly Val Asp Pro Asn Arg Asn Ala la Val Gly Leu Ser Met Ala 70 75 Gly Ser Ala Ala Leu Thr Leu Ala Ile Tyr His Pro Gin Gln Phe Gln 90 Asn Asp pro Met Val Asn *Il G 1 4 0 5Tyr Ala Gly SeLe Se Glyyr LeuAsn Pro Ser Glu Gy Tr Trp Leu 9 1555 16 Pro Met Le e A le Se Met Gly Asp Ala Gy Tyr Lys Ala 115 120 125 Asn Asp Met Trp Gy Pro Pro Lys Asp Pro Se Ser Ala Trp Lys Arg 130 135 140 Asn Asp Pro Met Val Asn Ie Gly Lys Leu Val Ala Asn Asn Thr Pro 145 150 .155-. 160 Leu 155 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 334 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID Met Lys Phe Thr Glu Lys Trp Arg Gly Ser Ala Lys Ala Ala Met His

I,.

a a. a a.

Arg Gly Leu Ile Asp Pro Val Gly Glu 145 Asn Ala Phe I Met z Ser 225 Leu Pro S Gin P Asn T 2 Ser G 305 Lys P Va Ph Pr Ar G1 Alz Gl Asn 130 Leu 4.la Ile 4 eu ksn ~sp ral er 'he yr 90 ly ro 1 Gly e Ala Val Val Leu Phe Gly Gly Pro Phe His Asn 295 Asp Pro Ala P Glu L 2 Leu G 275 Ile A Thr H Asp I Va

GI'

G1 G1i Arc

G'

Gir Gir Thr Tal iis LB1 ?ro la la isn eu 60 lu Ia is le 1 Ala y Gly L Tyr .i Phe Ala Trp 1 Ser Asn Trp Gly 2.65 Pro Ser Gly Trp I Asn *J 245 Asp 'I Gly P Ala G Ser T 3 Gin A 325 10 is Asp Met Ala Ala Val Ala Se: Lei Glr 70 Gir Phe Ser Tyr Leu 150 Leu Gln Glu ly -vs !30 hr 'hr 'he ;ly rp 10 rg r Al.

i Asr 55 1 Gly I Asp Tyr Phe Thr 135 Glu Ser Gin Gly Phe 215 A.rg Arg Gly Thr Gly 295 Gly Val x Th 40 Va.

Gl As 1 Gli Tyr 120 Tyr Ala Met Phe Trp 200 Asn Asn Ile Thr Leu 280 Thr Tyr Leu r Al 1 Ph Gl Ty3 I Se: 105 Ser Lys Asn Ala Ile 185 Trp Ala Asp Tro Pro 265 Arg Asn Trp Gly a Gly Ala e Ser Pro r Th.r His Asn Gly 90 Gly Leu Asp Trp Trp Glu Arg Gly 155 Gly Ser 170 Tyr Ala Pro Met I Glu Ser Pro Met 235 Ile Tyr C 250 Gly Gin P Thr Asn I Gly Val P 3 Gly Gin G 315 Ala Gin A 3'30 Leu Phe Ser Ala Pro Ser Met Val Gly Arg Gly Tyr Leu Pro Arg Leu Ile Gly Asp Leu Trp Asp Ile Asn Thr Ser Tyr Thr 140 Val Ala Ser Leu 4et lal :ys sn le 'he 00 in la Thr Ile Met 110 Gin Pro Ser 125 Phe Leu Thr Ser Arg Thr Ala Leu Thr 175 Ser Leu Ser 190 Ile Gly Leu 205 Trp Gly Pro Asn Ile Asn Gly Thr Gly 255 Leu Met Ala 270 Ala Phe Arg 285 Pro Arg Gin Gly 160 Tyr Gly Ala Ser Gin 240 rhr Ala sp

I

'I

Phe Gin Ala Pro Ala Gin Met 320 INFORMATION FOR SEQ ID NO:46: SEQUENCE

CHARACTERISTICS:

LENGTH: 795 base pairs TYPE: nucieic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE

DESCRIPTION:

CTGCCGCGGG TTTGCCATCT

CTTGGGTCCT

CCGGTACCGT CCGGCGATGT

GACCAACATG

120 CTCGGCGCAG

CAGCATTGGT'GGCCGCCACG

180 CAGGAAGGGC ACCAGGTCCG

TTACACGCTC

240 'TCTATCTGA CGACGCAGCC

GCCGAGCATG

300 GCCAAGCGGG AGAAGGTCAG

CCTCGCCCCG

360 GCCGACCCGA ACTGGGCGAT

CCTTCAGGTC

420 CCGAACGCGC ACTGCGACAT CGCCGTCGAT

C

480 CCCTACAACG TGCGGTGCCA GCTCGGTCAG 7I 540 GCGCCGGCGG CAGCGGCTCG CGGTGCAGCA

C

600 CGACGTAAAG ATCGCTGGCC CCGCGCGGCC

C

660 CCAGCACGGC GTCTAACTCC AGACCCTTGG

T

720 CGGGCGGGCC GATCACCACG CTGGTGCCCT

C

780 CGATGGCACC

GGCGA

795 SEQ ID NO: 46: GGGTCGGGAG

GC

CGAACGCGA

CA;

GGGATGGTCA

GCC

ACCTCGGCCG

GCG

CAGGCGTTCA

ACG

GGTGTGCCGT

GGG

kGCAGCACCA

CCC

;GcCAGGAGG

TGC

GGTGAGTCA

CCTC

CCCGAGGCG

CTGG

CTCGGCGAG

GATC

CTGCGTGGG

TGCC

CCGGTCCGC

CTCC

CATGTTCT

1

CGAAGCT

;CGGCGAC

~CTTACGA

CCGACGC

TCTTCGA

GCGGTGG

rCAGCCA

:GCCGAG

;GTCGCG

C

'TGCTCC

G

ACCGCG

C

GCACGC

A

GGGTAACGAT

AGGAGCGGCG

GGCGAACGCC

GTTCGAcCTG GTATGCG'1rC

AACCACGATG

GCAGGCCGCC.

3CACGACGAc

~GTCCGGCCA

:GGGTCAGCG

GGTAGACCA

CCGGGACAC

CGAAATCGT

i Met 1 Leu Glu Phe Asn Pro Ala INFORMATION FOR SEQ ID NO:47: ()SEQUENCE

CHARACTERISTICS:

LENGTH: 142 amino acids TYPE: amino acid STRANDEDNESS: sinale (DI TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Axg Thr Ala Thr Thr Lys Leu Gly Ala Ala 5 10 Val Ala Ala Thr Gly Met Val Ser Ala Ala 25 Gly His Gin Val Arg Tyr Thr Leu Thr Ser 40 Asp Leu Phe Tyr Leu Thr Thr Gin Pro Pro s0 55 Ala Asp Ala Tyr Ala Phe Ala Lys Arg Glu 70 75 Gly Val Pro Trp Val Phe Glu Thr Thr Met 90 Ile Leu Gin Val Ser Ser Thr Thr Arg Gly 47: Leu Giy Ala -Thr Ala Asnj Ala Gly Al1a Ser Met Gin 60 Lys Val Ser Al1a Asp Pro Gly Gin Ala Al a Al a Tyr Ala Leu Asn Aa Ala Gin Glu Phe Al a Trp Pro 100 105 110 Asn Ala His Cys Asp Ile Ala Val Asp Gly Gin Giu Vai Leu Ser Gln 115 120 125 His Asp Asp Pro Tyr Asn Val Arg Cys Gin Leu Giy Gin Trp 130 135 140 INFORMATION FOR SEQ ID NO:48: SEQUENCE

CHARACTERISTICS:

LENGTH: 300 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GCCAGTGCGC CAACGGTTTT CATCGATGCC GCACACAACC CCGGTGGGCC

CTGCGCTTGC

CGAAGGCTGC GCGACGAGTT CGACTTCCGG TATCTCGTCG GCGTCGTCTC

GGTGATGGGG

120 GACAAGGACG TGGACGGGAT CCGCCAGGAC CCGGGCGTGC CGGACGGGCG

CGGTCTCGCA

180 CTGTTCGTCT CGGCGACAA CCTTCGAAAG GGTGCGGCGC TCAACACGAT

CCAGATCGCC

240 GAGCTGCTGG CCGCCCAGTT GTAAGTGTTC CGCCGAAATT GCATTCCACG

CCGATAATCG

300 t o 0.0.

too.

o to "o.o to to INFORMATION FOR SEQ ID NO:49: SEQUENCE

CHARACTERISTICS.

LENGTH: 563 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GGATCCTCGG CCGGCTCAAG AGTCCGCGCC GAGGTGGATG

TGACGCTGGA

TTCAGTCGGG CCTGCGAGGC GCTGTACCAC TTCGCCTGGG

ACGAGTTCTG

120 GTCGAGCTTG CCAAAGTGCA ACTGGGTGAA GGTTTCTCGC

ACACCACGGC

180 ACCGTGCTCG ATGTGCTGCT CAAGCTTCTG CACCCGGTCA

TGCCGTTCGT

240 CTGTGGAAGG CCCTGACCGG GCGGGCCGGC GCGAGCGAAC

GTCTGGGAAA

300 CTGGTCGTCG CGGACTGGCC CACGCCCACC GGATACGCGC

TGGATCAGGC

360 CGGATCGCCG ACACCCAGAA GTTGATCACC GAGGTGCGCC

GGTTCCGCAG

420 CTGGCCGACC GCCAGCGGGT GCCTGCCCGG TTGTCCGGCA

TCGACACCGC

480

CGGCTACGAG

CGACTGGTAT

CGTGTTGGCC

CACCGAGGTG

TGTGGAGTCA

TGCCGCACAA

CGATCAGGGT

GGGTCTGGAC

GCCCATGTCC CGGCGGTGCG CGCGCTGGCC TGGCTTGACC GAGGGTGATG

AGGGCTTCAC

540 CGCGTCCGA TCGGTCGAGG

TGC

563

GGGCCGC

GCCGACG

120

ACGAACG

180

TACCTCG

240

GCCGACG,

300

ATGGAGG~

360

GAGTGCGC

420

TCGGTGGC

434 INFORMATION FOR SEQ ID Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 434 base pairs TYPE: nucleic acid STRANDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: Genornic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID GCC CGAGGATGAG CAAGTTCGAA GTCGTCACCG

GGATGGCGTT

CGC CCATCGACGT CGCCGTCGTC GAGGTCGGGC

TCGGTGGTCG

TGG TGAACGCACC GGTCGCGGTC ATCACCCCGA

TCGGGGTGGA

GTG ACACGATCGC CGAGATCGCC GGGGAGAAGG

CCGGAAATCA

PCC TGGTGCCGAC CGACACCGTC GCCGTGCTGG

CGCGGCAGGT

TGC TGCTGGCCCA GGCGGTGCGC TCGGATGCGG

CTGTAGCGCG

:GG TGCTGGGCCG TCAGGTCGCC ATCGGCGGCA

GCTGCTCCGG

GT CTAC

CGCGGCTTTC

CTGGGACGCG

CCACACCGAC

TCACCCGCCA

TCCCGAGGCC

CGAGGATTCG

TTGCAGGGGC

4* INFORMATION FOR SEQ ID NO:51: Ci SEQUENCE

CHARACTERISTICS:

CA) LENGTH: 438 base pairs TYPE: nucleic acid CC) STRANDEDNESS. single TOPOLOGY: linear Cii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCCACT CCCGCGCCGG CGGCGGCCAG CTGGTACGGC CATTCCAGCG

TGCTGATCGA

GGTCGACGGC TACCGCGTGC TGGCCGACCC GGTGTGGAGC AACAGATGTT

CGCCCTCACG

120 GGCGGTCGGA CCGCAGCGCA TGCACGACGT CCCGGTGCCG CTGGAGGCGC

TTCCCGCCGT

IS0 GGACGCGGTG GTGATCGCCA ACGACCACTA CGACCACCTC GACATCGACA

CCATCGTCGC

240 GTTGGCGCAC ACCCAGCGG CCCCGTTCGT GGTGCCGTTG GGCATCGGCG

CACACCTGCG

300 CAAGTGGGGC GTCCCCGAGG CGCGGATCGT CGAGTTGGAC TGGCACGAAG

CCCACCGCAT

360 CGACGACCTG ACGCTGGTCT GCACCCCCGC CCGGCACTTC TCCGGCCGGT

TGTTCTCCCG

420 CGACTCGACG

CTGTGGGC

438 INFORMATION FOR SEQ ID NO: 52: SEQUENCE CHARACTERISTICS: LENGTH: 87 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: Ser Ala Pro Thr Val Phe Ile Asp Ala Ala His Asn Pro Gly Gly 5 10 Cys Ala Cys Arg Arg Leu Arg Asp Giu Phe Asp Phe Arg Tyr Leu 20 25 Giv Val Val Ser Val Met Giy Asp Lys Asp Val Asp Gly Ile Arg 35 40 Asp Pro Gly Val Pro Asp Gly Arg Gly Leu Ala Leu Phe Val Ser 55 Asp Asn Leu Arg Lys Gly Ala Ala Leu Asn Thr Ile Gl1n Ile Ala 75 Leu Leu Ala Ala GIn Leu q Ala Pro Val Gin Gly Giu Gly Asp Trp Gly Val Leu Asn INFORMATION FOR SEQ ID NO:53: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 175 amino acids TYPE: amino acid STRANDEDNESS: single (Q TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: Ser Ser Ala Gly Ser Arg Val Arg Ala Giu Val 10 Gly Tyr Giu Phe Ser Arg Ala Cys Glu Aia- -Leu 25 Asp Giu Phe Cys Asp Tr-p Tyr Val Giu Leu Ala 40 Giu Gly Phe Ser His Thr Thr Ala Val Leu Ala s0 55 60 Leu Leu Lys Leu Leu His Pro Val Met Pro Phe 70 75 T Lys Ala Leu Thr Gly Arg Ala Gly Ala Ser 90 Val Giu Ser Leu Val Val Ala Asp Trp Pro Thr Asp Tyr- Lys Thr Val Glu Pro Val1 His Val Vai Thr Arg Thr Thr Phe Gin Leu Giu Leu Gly Leu Al1 a Leu Asp Val Gly Tyr 100 105 110 Ala Leu Asp Gln Ala Ala Ala Gln Arg Ile Ala Asp Thr Gln Lys Leu 115 120 125 Ile Thr Glu Val Arg Arg Phe Arg Ser Asp Gln GIy Leu Ala Asp Arg 130 135 140 Gln Arg Val Pro Ala Arg Leu Ser Gly Ile Asp Thr Ala Gly Leu Asp 145 15H v 155 1 Ala His Val Pro Ala Val Arg Ala Leu Ala Trp Leu Asp Arg Gly160 165 170 175 INFORMATION FOR SEQ ID NO:54: SEQUENCE

CHARACTERISTICS:

LENGTH: 144 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: *Gy Pro Gly Pro Arg Asn Ser Lys Phe Glu Val ValThr Gly Met Ala Phe Ala Ala Phe Ala Asp Ala Pro lie Asp Val Ala Valal Glu Val 20 25 Gl Gly Leu Gly Gly Arg Trp Asp Ala Thr Asn Val Val Asn Ala Pro Val 4O4 Ala Val Ile Thr Pro Ile Gly Val Asp His Thr Asp Tyr Leu Gly Asp 40 so 55 T h r le Ala Glu le Ala G Thr Ile Ala Glu e Gly Glu Lys Ala Gly Asn His His Pro Pro 6570 75 Ala Asp Asp Leu Val Pro Thr Asp Thr Val Ala Val Leu Ala Arg Gin 90 Val Pro Glu Ala Asn Glu Val Leu Leu Ala Gn Ala Val Ar Ser As 100 105 al A Ap Ala Ala Val Ala Arg Glu Asp Ser Glu Cys Ala L rg ln 120 115 a l A l a L l e Gly Gly Ser C 1 2 Val Ala le Gly Gly Ser Cys Ser Gly Cys Arg Gly Ser Val Ala Ser .130 135 140 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 145 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Pro Thr Pro Ala Pro Ala Ala Ala Ser Trp Tyr Gly His Ser Ser 1 5 10 Val Leu Ile Glu Val Asp Gly Tyr Arg Val Leu Ala Asp Pro Val Trp 230 Ser Asn Asp Val Ile Ser Leu Ala Ala His Asp Trp Pro Ala 130 Trp 145 Arg Pro Asn His Leu His 115 Arg Cys Val Asp Thr Arg 100 Glu His Ser Pro His Gin Lys Ala Phe Pro Leu Tyr 70 Arg Trp His Ser Ser Arg 40 Glu Ala 55 Asp His Ala Pro Gly Val Arg Ile 120 Gly Arg 135 Ala Leu Leu Phe Pro 105 Asp Leu Val Gly Pro Pro Ala Val Asp Ile Asp 75 Val Val Pro 90 Glu Ala Arg Asp Leu Thr Phe Ser Arg 140 Gin Arg Asp Ala Thr Ile Leu Gly Ile Val 110 Leu Val 125 Asp Ser Met Val Val Ile Glu Cys Thr His Val Ala Gly Leu Thr Leu .*oo 0 INFORMATION FOR SEQ ID NO:56: SEQUENCE

CHARACTERISTICS:

LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Residue can be either Gly, Ile, Leu NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Residue can be either Ile, Leu, Gly or Val or Ala (xi4-.SEQUENCE DESCRIPTION: SEQ ID NO:56: Xaa Xaa Ala Pro Xaa Gly Asp Ala Xaa Arg 1 5 INFORMATION FOR SEQ ID NO:57: SEQUENCE

CHARACTERISTICS:

LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Residue can be either Ile or Leu (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Pro Glu Ala Glu Ala Asn Xaa Arg 1 INFORMATION FOR SEQ ID NO:58: SEQUENCE

CHARACTERISTICS-

LENGTH: 11 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Residue can be either Gln or Gly NAME/KEY: Other LOCATION: S(D) OTHER INFORMATION: Residue cn be either Gly or Gln (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: Thr Ala Asn Xaa Xaa Glu Tyr Tyr Asp Asn Arg INFORMATION FOR SEQ ID NO:59: SEQUENCE

CHARACTERISTICS:

LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (iiU.MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: Asn Ser Pro Arg Ala Glu Ala Glu Ala Asn Leu Arg Gly Tyr Phe Thr 1 5 10 Ala Asn Pro Ala Glu Tyr Tyr Asp Leu Arg Gly Ile Leu Ala Pro Ile 20 25 30 Gly Asp 25 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other (xi) SEQUENCE DESCRIPTION: SEQ ID CCGGTGGGCC

CGGGCT-GCGC

INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARZACTERI

STICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECU.LE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:Gl: TGGCCGGCCA

CCACGTGGTA

INFOR.MATION FOR SEQ, ID NO:62: SEQUENCE

CHARACTERISTICS:

CA-EN 313 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE. TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: GCCGGTGGGC CCGGGCT-!GCG CGGAATACGC GGCAGCC7AT CCCACTGGGC

CGGCCTCGGT

GCAGGGAATG TCGCAGGACC CGGTCGCGGT GGCGGCCTCG AACAATCCGG

AGTTGACAAC

120 .:GCTGTACGGC .TGCACTG-LCG GGCCAGCTCA ATCCGCAAGT AAACCTGGTG

GACACCCTCA

ACAGCGGTCA GTACACGGTG TTCGCACCGA CCA.ACCGCGGC ATTTAGCAAG

CTGCCGGCAT

240 CCACGATCGA CGAGCTCAAG ACCAATTCGT CACrjGCTGAC CAGCATCCTG

ACCTACCACG

300 TGGTGGCCGG

CCA

313 INFORMATION FOR SEQ ID NO:63: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single CD) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 91 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: Glu Pro Ala Gly Pro Leu Pro Xaa Tyr Asn Glu Arg Leu His Thr Leu 1 510 Xaa Gin INFORMATION FOR SEQ ID NO:64: SEQUENCE

CHARACTERISTICS:

LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: Gly Leu Asp Asn Glu Leu Ser Leu Val Asp Gly Gln Gly Arg Thr Leu 1 5 10 Thr Val Gin Gin Xaa Asp Thr Phe Leu 0 20 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Pro Xaa Pro Asp Ile Glu Val Glu Phe Ala Arg Gly Thr Gly Ala 1 5 Glu Pro Gy Leu Xaa Xaa Val Xaa Asp Ala INFORMATION FOR SEQ ID NO:66: SEQUENCE

CHARACTERISTICS:

LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: ACCGCCCTCG AGTTCTCCCG GCCAGGTCTG

CC

32 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: AAGCACGAGC TCAGTCTCTT CCACGCGGAC

GT

32 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single 00 TOPOLOGY: linear (ii) MOLECULE TYPE: Other "0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: CATGGATCCA TTCTCCCGGC CCGGTCTTCC INFORMATION FOR SEQ ID NO:69: i) SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear *0* (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: TTTGAATTCT AGGCGGTGGC

CTGAGC

26 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 161 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Gly Trp Asp Ile Asn Thr Ala Ala Phe Glu Trp Tyr Val Asp Ser 1 510 Gly Leu Ala Val Ile Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser .25 Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys 40 Trp Glu Thr Phe Leu Thr Gln Glu Leu Pro Ala Tyr Leu Ala Ala Asn 55 60 Lys Gly Val Asp Pro Asn Arg Asn Al la a Val Gly Leu Ser Met Ala 70 75 Gly Ser Ala Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Gln Tyr Ala Gly Ser Leu Ser Gly Tyr Leu Asn Pro Ser Glu Gly Trp Tro 100 105 110 Pro Met Leu Ile Asn Ile Ser Met Gly Asp Ala Gly Gly Tyr Lys Ala 115 120 125sp Ala Gly Gly yr Lys Ala Asn Asp Met Trp Gly Arg Thr Glu Asp Pro Se Ser Ala Trp Lys Arg 130 135 P M V n 140 Asn Asp Pro Met Val Asn Ile Gly Lys Leu Val Ala Asn Asn Thr Pro 145 150 155 160 Leu 5 160 INFORMATION FOR SEQ ID NO:71: *i SEQUENCE

CHARACTERISTICS:

LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 3GAGAGACTCG AGAACGCCCA GGAAGGGCAC

CAG

INFORMATION FOR SEQ ID NO:72: SEQUENCE

CHARACTERISTICS:

LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: GAGAGACTCG AGTGACTCAC CACTGACCGA

GC

32 INFORMATION FOR SEQ ID NO:73: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: GGNGCNGCNC

ARGCNGARCC

INFORMATION FOR SEQ ID NO:74: SEQUENCE

CHARACTERISTICS:

LENGTH: 825 base pairs CB) TYPE: nucleic acid CC) STRANDEDNESS: single CD) TOPOLOGY: linear Cii) MOLECULE TYPE: Genomic

DNA

Cxi) SEQUENCE DESCRIPTION: SEQ, ID NO:74: TTGGATCCCA CTCCCGCGCC

GGCGGCGGCC

60

GAGGTCGACG

120

CGGGCGGTCG

IS0

GTGGACGCGG

240

AGCTGGTACG

CCGGTGTGGA

GCCATTCCAG

GCAACAGATG

GCTAcCGCGT

GACCGCAGCG

TGGTGATCAG

GCGTTGGCGC

ACACCCAGCG

300 CGCAAGTGGG

GCGTCCCCGA

360 ATAGACGACC

TGACGCTGGT

420 CGCGACTCGA

CGCTGTGGGC

480 GGTGGCGAGA

CCGGATACAC

540 GATCTGACCC

TGCTGCCGAT

g00 CCCGAGGAGG

CGGTGCGCGC

660 CCCATCCACT GGGCGACArr 720 CTGCTGACCG

CTGCCGACGC

780 GTGGACCCG

AGTCGACGTT

825

GCTGGCCGAC

CATGCACGAC

CCACGACCAC

GGCCCCGTTC

GGCGCGGATC

CTGCACCCCC

GTCGTGGGTG

GAAGAGCTTC

CGGGGCCTAC

CCATCTGGAC

CCGCCTCGCC

-GAGCGGGTA

:GACCCGTGG

TACGACCACC

GTGGTGCCGT

GTCGAGTTGG

GCCCGGCACT

GTCACCGGCT

GCC!GAGATCG

CATCCCGCGT

CTGACCGAGG

CCGCATCCGT

:GCCTGACCG

TGGCGGTTC!T

GTCCCGGTGC CGCTGGAGGC

TCGACATCGA

TGGGCATCGG

ACTGGCACGA

TCTCCGGACG

CGTCGCACAA

GCGACGAGTA

TCGCCGACAT

TGGACAACAG

CGTGCTGATC

TTCGCCCTCA

GCTTCCCGCC

CACCATCGTC

CGCACACCTG

AGCCCACCGC

GTTGTTCTCC

GGCGTTCTTC

CGGTCCGTTC

:CACATGAAC

:CTGATGGTG

GGTCCGAGCC CGCCGAACGC

TGCCGATTCC

GAACC

6CGGtCAGCGG- C2) INFORMATION FOR SEQ ID Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 273 amino acids CB) TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:. SEQ ID Leu Asp Pro Thr Pro Ala Pro Ala Ala Ala Ser Trp 10 Ser Val Leu Ile Giu Val Asp Gly Tyr Azg Val Leu 25 Trp Ser Asn Arg Cys Ser Pro Ser Arg Ala Val Gly 40 His Asp Vai Pro Val Pro Leu Glu Ala Leu Pro Ala 5055 60 Val Ilie Ser His Asp His Tyr Asp His Leu Aspol 70 75Il Ala Leu Ala His Thr Gn Arg Ala Pr 7heV5 a 85 90 Gly Ala His Leu Arg Lys Trp Gly Val Pro Giu Ala 100 105 Leu Asp Trp His Glu Ala His Arg Ile Asp Asp Leu 115 120 Thr Pro Ala Arg His Phe Ser Gly Arg Leu Phe Ser 130 135 140 Leu Trp Ala Ser Trp Val Vai Thr Gly Ser Ser His L 143 150 155 Gly Gly Asp Thr Gly Tyr Thr Lys Ser Phe Ala Glu

I

165 170 Tyr Gly Pro Phe Asp Leu Thr Leu Leu Pro Ile Gly A 180 185 Ala Phe Ala Asp Ile His Met Asn Pro Glu Glu Ala

V

195 2002 Leu Asp Leu Thr Giu Val Asp Asn Ser Leu Met Val P~ 210 215 220 Ala Thr Phe Arg Leu Ala Pro His Pro Trp Ser Glu P1 225 230 235 Leu Leu Thr Ala Ala Asp Ala Giu Arg Val Arg Leu Th- 245 250 Pr i GnAg Val Asp Pro Giu Ser Thr Phe Asp Pr 260 265 Phe INFORMATION FOR SEQ ID NO-76: Ci) SEQUENCE CHARACTERI

STICS:

LENGTH: 10 amino acids TYPE: amino acid STRANESS: single TOPOLOGY: linear (ii) MOLECUJLE TYPE: protein Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:76: Ala Lys Thr Ile Ala Tyr Asp Giu Glu Ala 1 5 Tyr Gly His Ser Ala Asp Pro Val Pro Gin Arg Met Val Asp Ala Val Asp Thr Ile Val Pro Leu Gly Ile Arg Ile Val Giu 110 ['hr Leu Val Cys ~rg Asp Ser Thr Ys Ala Phe Phe 160 le Gly Asp Glu 175 la Tyr His Pro 190 al Arg Ala His o Ile His Tr-D 0o Ala Glu 240 ir Val Pro Ile 255 o Trp Trp Arg- 270 9 6* INFORMATION FOR SEQ ID NO:77: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 337 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY:. linear (ii) MOLECULE. TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: GATCCCTACA TCCTGCTGGT CAGCTCCAAG GTGTCGACCG TCAAGGATCT

GCTCCCGCTG

CTGGAGAAGG TCATCCAGGC CGGCAAGCCG CTGCTGATCA TCGCCGAGGA

CGTCGAGGGC

2.20 GAGGCCCTGT CCACGCTGGT GGTCAACAAG ATCCGCGGCA CCTTCAAGTC

CGTCGCCGTC

180 AAGGCTCCGG GCTTCGGTGA CCGCCGCAAG GCGATGCTGC AGGACATGGC

CATCCTCACC

240 GGTGGTCAGG TCGTCAGCGA AAGAGTCGGG CTGTCCCTGG AGACCGCCGA

CGTCTCGCTG

300 CTGGGCCAGG CCCGCAAGGT CGTCGTCACC

AAGGACA

33.7

S.

S S *5 S S

S.

S*

S

*5 S

S

55 S S *55* 555S55

S

55 5

S

S.

Asp Leu Ile Asn Phe Gly Asp INFORMATION FOR SEQ ID NO:78: SEQUENCE

CHARACTERISTICS:

LENGTH: 112 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: Pro Tyr Ile Leu Leu Val Ser Ser Lys Vai Ser 5 10 Leu Pro Leu Leu Giu Lys Val Ile Gln Ala Gly 25 Ile Ala Giu Asp Val Giu Gly Glu Ala Leu Ser 40 Lys Ile Arg Gly Thr Phe Lys Ser Val Ala Val 55 60 Gly Asp Ar-g Arg Lys Ala 'Me t Leu Gin Asp Met 75 Gly Gin Val Val Ser Glu Arg Val Gly Leu Ser 90 Val Ser Leu Leu Gly Gin Ala Arg Lys Val Val 100 2.05 INFORMATION FOR SEQ ID NO:79: SEQUENCE

CHARACTERISTICS:

LENGTH: 360 base pairs Thr Val Lys Pro Thr Leu Lys Ala Ala lie Leu Glu Val Thr 110 Lys Leu Val Pro Leu Thr Lys Asp.

Leu Val Gly Thr Al a Asp TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECL TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: CCGTACGAGA AGATCGGCGC TGAGCTGGTC AAAGAGGTCG CCAAGAAGAC

CGACGACGTC

GCGGGCGACG GCACCACCAC CGCCACCGTG CTCGCTCAGG CTCTGGTTCG

CGAAGGCCTG

120 CGCAACGTCG CAGCCGGCGC CAACCCGCTC GGCCTCAAGC GTGGCATCGA

GAAGGCTGTC

180 GAGGCTGTCA CCCAGTCGCT GCTGAAGTCG GCCAAGGAGG TCGAGACA

GGAGCAGAT

240 TCTGCCACCG CGGCGATCTC CGCCGGCGAC ACCCAGATCG GCGAGCTCAT

CGCCGAGGCC

300 ATGGACAAGG TCGGCAACGA GGGTGTCATC ACCGTCGAGG AGTCGAACAC

CTTCGGCCTG

360

S*

00 09 ~S 0 6S S0 9 0 15 0 Si. 0 0* 00*0 0*00 0*@0 0 *0*0

S

000*

SI

0 40*0 0000 4 0000 4 0S S 0S 00 Pro Thr Gin Pro Gin.

Ser Ile Glu INFORMATION FOR SEQ ID i)SEQUENCE

CHARACTERISTICS:

LENGTH: 120 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID No:e0: Tyr Giu Lys Ile Gly Ala Giu Leu Val Lys Giu 5 10 Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala 20 25 Ala Leu Val A-rg Glu Gly Leu Arg Asn Val Ala 3,5- 40 Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val 55 60 Ser Leu Leu Lys Ser Ala Lys Glu Val Giu Thr .70 75 Ala Thr Ala Ala Ile Ser Ala Gly Asp Thr Gin 90 Ala Giu Ala Met Asp Lys Val Gly Asn GJlu Gly 100 105 Glu Ser Asn Thr Phe Gly Leu 115 120 INFORMATION FOR SEQ ID NO:8i: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS. single TOPOLOGY: linear Val Ala Lys Thr Val Leu Ala Gly Ala Giu Ala Val Lys Giu Gin Ile Gly Giu Val Ile Thr ild Lys Al a Asn Thr Ile Leu Val (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: ACTGACGCTG AGGAGCGAAA GCGTGGGGAG CGAACAGGAT

TAG

43 INFORMATION FOR SEQ ID NO:82: SEQUENCE

CHARACTERISTICS:

LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: CGACAAGGAA CTTCGCTACC TTAGGACCGT CATAGTTACG

GGC

43 INFORMATION FOR SEQ ID NO:83: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: AAAAAAAAAA

AAAAAAAAAA

12) INFORMATION FOR SEQ ID NO:84: SEQUENCE

CHARACTERISTICS:

LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: GGAAGGAAGC GGCCGCTTTT TTTTTTTTT

T

31 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNqESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQ UENCE DESCRIPTION: SEQ ID GAGAGAGAGC CCGGGCATGC TSCTSCTSCT

S

31 INFORMATION FOR SEQ ID NO:86: SEQUENCE

CHARACTERISTICS:

LENGTH: 238 base pai;rs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genornic

D)NA

(xi) SEQUENCE DESCRIPTION: SEQ ID No:eG: CTCGATGAC CCCTCGGAGC GCTCGACCTG AAGCTGCGCC ACGTCATGCA

GTTCGAGCTC

**.:AAGCGCATCC ACGAG CGGGATCACG TTCATCTACG TGACCCACGA

CICAGGAAGA

AGC0GGAGG GCGCTCACGA TGAGTGACCG CATCGCGGTG ATGAACGCCG GCAACGTCGA ACAGATCGGC 180 AGCCCGACCG AGATCTACGA CCGTCCCGCG ACGGTGTTCG TCGCCAGCTT CATCGAAT 238 INFORMATION FOR SEQ ID NO:87; SEQUENCE

CHARACTERISTICS:

LENGTH: 79amnacd TYPE: amino acid STADDES single S.B TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: Leu Asp Giu Pro Leu Gly Ala Leu Asp Leu Lys LeU Arg His Val Met 1 5 10 Gln Phe Glu Leu Lys Arg Ile Gin Arg Glu-Val Gly'lie Thr Phe"1le 25 Tyr Val Thr His Asp Gin Glu Glu Ala Leu Thr Met Ser Asp Arg Ile 40 Ala Val Met Asn Ala Gly Asn Val Glu Gin Ile Gly Ser Pro Thr Glu 55

GO

Ile Tyr Asp Arg Pro Ala Thr Val Phe Val Aia Ser Phe Ile Glu 70 INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 1518 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi)

CACTCGCCAI

ACATCCAGCI

120

CATCAGTACA

180

AATCCATTGT

240

GAAGGTTTAC

300

CACATCACCG

360

GCGCCGGTGA

420

GACGCAGACT

480

GGGAAGACGA

540

CGCCTCGAAG

600

TTCCAGCACT

660

CGCAGCAAGA

720

GTCCGGCTGA

780

CGGGTGGCGT

840

CTCGGAGCGQ

900

CGGGAGGTCG

960

AGTGACCGCA

1020

ATCTACGACC

1080

GCGGGCCGGT

1140

ACGCTGAAGG

1200

GTGCGTCCGG

1260

GCCTGCGTGC

1320

CTGGCCGCTC

1380 GGGTGT~TACA

ATACCCCAC(

GAGAAAATAT

TCACAGCGAC

AGTTCCTCGA AGTAAACGAA

*GCGCGCTTTC

CGAAATGTAA

CCCACAGCCA

CTGCAGAACC

TCGAGATCGA

TCTCCATCGC

CCACGTTGCG

GCGCCGACGT

ACGCGCTGTT

AACTCGGCAA

CCGAATTTGC

TGGCCCGGGC

-TCGACCTGAA

GGATCACGTT

TCGCGGTGAT

GTCCCGCGAC

GCACCGGCCG

CACGCCCGGG

AACGCATCCG

GTGCCACCGT

CGGACGACTC

CTGCGCGGA1

*ATTCGTTGCC

*CGACGGCTG7

TGCAGAACACG

CCATGTCACG

GCCCGGGGAG

CATGATCGCGj

GTCGAGGACC

CCCGCACATG

AGGCGAGGTC

CGAGCGCAGG

ACTGGTGAAC

GCTGCGCCAC

CATCTACGTG

GAACGCCGGC

GGTGTTCGTC

CTCCAACCGC

CGAGACCACG

GGTCACCCCG

CACCGACCTG

GACCGTGATC

GAAGCCCGGC

TCTATTGTCG

GAATCACTTG

CCCCGAGGAG

ACGGCGGATT

AAGCGCTTCG

TTCTTCTCCA

GGATTCGAGA

CCACCCAACA

ACGGTCTGGG

CGCAAGCGCG

CCCGCCCAGC

TACCCCAGCG

GTCATGCAGT

ACCCACGACC

AACGTCGAAC

GCCAGCTTCA

GATTACGTCG

ATCGAGCCCG

GGCTCCCAGG

ACCTTCCAAG

GCCCACGTCG

CGATGCCTGA

AGTCCGGGGT

CATAGG7TCCG GACC7GCCCT

CCGCGGCACC

GCGACTACCT

TGCTCGGCCC

CCCCGACTGA

AGCGCAACGT

ACAACGTCGC

TCGACGAGCT

TGTCCGGCGG

CGCTGCTGCT

TCGAGCTCAA

AGGAAGAGGC

AGATCGGCAG

TCGGACAGGC

AGATCGACGT

GCGGGCACGC

ACGCGCCGAC

GTCCGGTGGT

C

GCCCCGAGCA

C

CAGAACCGTG

TGGGGTCCGG

GTGACGAAGG

TCAGATCCGC

GACCGGCACA

GCCCAAGGGC

GGCCGTCGCG

GTCCGGGTGT

AGGGGCGATC

CA6ACACGGTG

GTACGGCCCG

GCTGGAGATC

GCAGCAGCAG

CGATGAACCG

GCGCATCCAG

GCTCACGATG

:CCGACCGAG

CAACCTCTGG

rCTCGGCTCG

:ACCCTGATG

:GGTGACGTC

;CGGCTCTCG

;GATCTGCCG

SEQUENCE DESCRIPTION: SEQ ID N0:88: CTGCTGCGCC CCGGCGACGA CGTGTACGTC AGCTGGGCAC CGGAAGCCTC

CCTGGTGCTT

1440 CCCGGCGACG ACATCCCcAc CACCGAGGAC CTCGAAGAGA TGCTCGACGA

CTCCTGAGTC

1500 ACGCTTCCCG

ATTGCCGA

1518 Gi

LE

Gl Va 65 Hi G1 As Pr( Alz 145 Ala Ile Glu Asn Thr 225 Arg Gly Gly Gly INFORMATION FOR SEQ ID NO:89: SEQUENCE

CHARACTERISTICS:

LENGTH: 376 amino acids TYPE: amino acid STRANDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N:89: al Ile Glu Ile Asp His Val Thr Lys Arg Phe Gly 1 5 10 il Ala Asp Ala Asp Phe Ser Ile Ala Pro Gly Giu 20 25 .u Gly Pro Ser Gly Cys Gly Lys Thr Thr Thr Leu 40 y Phe Giu Thr Pro Thr Glu Gly Ala Ile Arg Leu 50 55 60 .1 Ser Arg Thr Pro Pro Asn Lys Arg Asn Val Asn 70 75 s Tyr Ala Leu Phe Pro His Met Thr Val Trp Asp 85 90 y Pro Arg Ser Lys Lys Leu Giy Lys Gly Giu Val 100 105 p Giu Leu Leu Giu Ile Val Arg Leu Thr Giu Phe 115 120 3 Ala Gin Leu Ser Gly Gly Gin Gin Gin Arg Val 130 135 140 Leu Val Asn Tyr Pro Ser Ala Leu Leu Leu Asp C 150 155 Leu Asp Leu Lys Leu Arg His Val Met Gin Phe

G

165 170 Gin Arg Glu Val Gly Ile Thr Phe Ile Tyr Val

T

180 185 Glu Ala Leu Thr Met Ser Asp Arg Ile Ala Vai

M

195 200. 2 Vai Giu Gin Ile Gly Ser Pro Thr Glu Ile Tyr A 210 215 220 Val Phe Val Ala Ser Phe Ile Gly Gin Ala Asn

L

230 235 Cys Thr Giy Arg Ser Asn Arg Asp Tyr Val Giu 1 245 250 Ser Thr Leu Lys Ala Arg Pro Gly Giu Thr Thr

II

260 265 His Ala Thr Leu Met Val Arg Pro Giu Arg Ile Az 275 280 Ser Gin Asp Ala Pro Thr Gly Asp Val Ala Cys Va 290 295 300 As Ph Ar Gi Th As Are klz Ua ;lu lu 'hr et sp Le Le L1 jp Tyr Leu is Le Phe Ser 9 Met Ile u Gly Ala r Val Phe n Val Ala 3 Lys Arg 110 Glu Arg Leu Ala Pro Leu C 1 Leu Lys pn 175 His Asp G 190 Asn Ala G Arg Pro A Trp Ala G 2 Asp Val L 255 Glu Pro G 270 Val Thr P2 Arg Ala Tk Ala Met Ala Asp Gin Tyr Val Arg krg ;ly 160 Lrg ln I y la ly eu Ly o ir a. Val Thr Asp Leu Thr Phe Gin Gly Pro Val Val Arg Leu Ser Leu Ala 305 310 315 320 Ala Pro Asp Asp Ser Thr Val Ile Ala His Val Gly Pro Glu Gin Asp 325 330 335 Leu Pro Leu Leu Arg Pro Gly Asp Asp Val Tyr Val Ser Trp Ala Pro 340 345 350 Glu Ala Ser Leu Val Leu Pro Gly Asp Asp Ile Pro Thr Thr Glu Asp 355 360 365 Leu Glu Glu Met Leu Asp Asp Ser 370 375 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID GAGAGACTCG AGGTGATCGA GATCGACCAT

GTC

33 INFORMATION FOR SEQ ID NO:91: SEQUENCE

CHARACTERISTICS:

LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: AGAGACTCGA-GCAATCGGGA AGCGTGACTC

A

31 INFORMATION FOR SEQ ID NO:92: SEQUENCE

CHARACTERISTICS:

LENGTH: 323 base pairs TYPE: nucleic acid STRANDEDNESS:. single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: GTCGACTACA AAGAAGACTT CAACGACAAC GAGCAGTGGT TCGCCAAGGT

CAAGGAGCCG

TTGTCGCGCA AGCAGGACAT AGGCGCCGAC CTGGTGATCC CCACCGAGTT

CATGGCCGCG

103 103 CGCGTCAAGG GCCTGGGATG GCTCAATGAG ATCAGCGAAG CCGGCGTGCC

CAATCGCAAG

180 AATCTGCGTC AGGACCTGTT GGACTCGAGC ATCGACGAGG GCCGCAAGTT

CACCGCGCCG

240 TACATGACCG GCATGGTCGG TCTCGCCTAC AACAAGGCAG CCACCGGACG

CGATATCCGC

300 ACCATCGACG ACCTCTGGGA

TCC

323 INFORMATION FOR SEQ ID NO:93: SEQUENCE

CHA.RACTERISTICS:

LENGTH: 1341 base pairs TYPE: nucleic acid STRANDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: Genornic

DNA

(xi) SEQUENCE DESCRIPTION:' SEQ ID No:'93: CCCCACCCCC TTCCCTGGAG CCGACGAAAG GCACCCGCAC ATGTCCCGTG

ACATCGATCC

60 CCACCTGCTG GCCCGAATGA CCGCACGCCG CACCTTGCGT CGCCGCTTCA TCGGCGGTGG 120 CGCCGCGGCC GCCGCGGGCC TGACCCTCGG TTCGTCGT-rC CTGGCGGCGT GCGGGTCCGA 180 CAGTGGGACC TCGAGCACCA CGTCACAGGA CAGCGGCCCC GCCAGCGGCG CCCTGCGCGT 240 CTCCAACTGG CCGCTCTATA TGGCCGACGG TTTCATCGA GCGTTCCAGA CCGCCTCGGG 300 CATCACGGTC GACTACAAG AAGACTTCA CGACAACGAG CAGTGGTICG CCAAGGTCA 360 GGAGCCGTTG TCGCGCAAGC AGGACATAGG CGCCGACCTG GTGATCCCCA CCGAGTTAT 420 GGCCGCGCGC GTCAAGGGCC TGGGATGGCT CAATGAGATC AGCGAAGCCG GCGTGCCCA 480 TCGCAAGAAT CTGCGTCAGG ACCTGTTGA CTCGAGCATC GACGAGGGCC GCAAGTTCAC 540 CGCGCCGTAC ATGACCGGCA TGGTCGGTCT CGCCTACAC AAGGCAGCCA CCGGACGCGA 600 TATCCGCACC ATCGACGACC TCTGGGATCC CGCGTTCAAG GGCCGCGTCA GTCTGTTCTC 660 CGACGTCCAG GACGGCCTCG GCATGATAT GCTCTCGCAG

GGCAACTCGC'CGGAGAATCC

720 GACCACCGAG TCCATTCAGC AGGCGGTCGA TCTGGTCCGC GAACAGAACG ACAGGGGGTC 780 AGATCCGTCG CTTCACCGGC AACGACTACG CCGACGACCT GGCCGCAGA ACATCGCCAT 840 CGCGCAGGCG TACTCCGGTG ACGTCGTGCA GCTGCAGGCG GACAACCCCG ATCTGCAGTT 900 CATCGTTCCC GAATCCGGCG GCGACTGGT CGTCGACACG ATGGTGATCC CGTACACcAC 960 .GCAGAACCAG A.AGGCCGCCG AGGCGTGGAT CGACTACATC TACGACCGAG CCAACTACGC 1020 CAAGCTGGTC GCGTTCACCC AGTTCGTGCC CGCACTCTCG GACATGACCG ACGAACTCGC 1080 *t CAAGGTCGAT CCTGCATCGG CGGAGAACCC GCTGATCAAC CCGTCGGCCG

AGGTGCAGGC

1140 GAACCTGAAG TCGTGGGCGG CACTGACCGA CGAGCAGACG CAGGAGTTCA

ACACTGCGTA

1200 CGCCGCCGTC ACCGGCGGCT GACGCGGTGG TAGTGCCGAT GCGAGGGGCA

TAAATGGCCC

1260 TGCGGACGCG AGGAGCATAA ATGGCCGGTG TCGCCACCAG CAGCCGTCAG

CGGACAAGGT

1320 CGCTCCGTAT CTGATGGTCC

T

1341 INFORMATION FOR SEQ ID NO:94: SEQUENCE

CHARACTERISTICS:

LENGTH: 393 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: Met Ara Gi y Gly Leu Ala Asn, Lys Al a Val 145 As~o Leu Asp Val Glu 225 Glu Ser Th r Leu Thr s0 Arg Phe Asp Gin Arg 130 Pro Glu Al- a Leu Gin 210 Asn Gin *Arg Asp Ile Leu A-rg Arg Thr Leu Gly 35 Ser Ser Thr Val Ser Asn Gin Thr Ala 85 Asn Glu Gln 100 Asp Ile Gly "s& Val Lys Gly.

Asn Arg Lys Gly Arg Lys 165 Tyr Asn Lys 180 Trp Asp Pro 195 Asp Gly Leu Pro Thr Thr Asn Asp Arg 245 Asp Pro His Leu Leu Ala Arg Ser Thr Trp 70 Ser Trp Ala Leu Asn 150 Phe Ala Al a Gly Giu 230 Gly Phe Ser Ser 55 Pro Gly Phe Asp Gly 135 Leu Thr Ala Phe Met 215 Ser Ser Ile Phe 40 Gin Leu Ile Al a Leu 120 Trp Arg Ala Thr Lys 200 Ile Ile Asp 10 Gly Gly Gly Arg Met Thr Ala Arg Ala Ala Ala Ala Ala Leu Ala Ala Cys Gly Asp Tyr Thr Lys 105 Val Leu Gin Pro Giy 185 Gly Gin Pro Ile 165 Ser *Met *Val 90 Val Ile Asn Asp Tyr 170 Arg Arg Leu Gin Ser Gly Ala 75 Asp Lys Pro Glu Leu 155 Met Asp Val Ser Ala 235 Leu *Pro *Asp Tyr Glu Thr Ile 140 Leu Thr Ile Ser Gin 220 Val1 His Al a Giy Lys Pro Giu 125 .Ser Asp Gly Arg Leu 205 Gly Asp Ser Ser Phe Glu Leu 110 Phe Giu Ser Met Thr 190 Phe Asn Leu *Asp *Gly Asp Ser Met Ala Ser Val 175 Ile Ser Ser Vai Ser Al a Al a Phe Arg Al a Gly Ile 160 Gly Asp As p Pro Arg 240 Arg Arg Arg Pro Gly Arg Arg Asn 260 Ala Ile Ala Gin 255 Ala Tyr Ser 270 Gly Asp Val Val Gin Leu Gin Ala Asp Asn Pro Asp Leu Gin Phe Ile 275 280 285 Val Pro Glu Ser Gly Gly Asp Trp Phe Val Asp Thr Met Val Ile Pro 290 295 300 Tyr Thr Thr Gin Asn Gln Lys Ala Ala Glu Ala Trp Ile Asp Tyr Ile 305 310 315 320 Tyr Asp Arg Ala Asn Tyr Ala Lys Leu Val Ala Phe Thr Gln Phe Val 325 330 335 Pro Ala Leu Ser Asp Met Thr Asp Glu Leu Ala Lys Val Asp Pro Ala 340 345 350 Ser Ala Glu Asn Pro Leu Ile Asn Pro Ser Ala Glu Val Gln Ala Asn 355 360 365 Leu Lys Ser Trp Ala Ala Leu Thr Asp Glu Gln Thr Gin Glu Phe Asn 370 375 380 Thr Ala Tyr Ala Ala Val Thr Gly Gly 385 390 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single o TOPOLOGY: linear S(ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID ATGTCCCGTG ACATCGATCC

CC

22 INFORMATION FOR SEQ ID NO:96: i) SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single L TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: ATCGGCACTA CCACCGCGTC

A

21 INFORMATION FOR SEQ ID NO:97: SEQUENCE

CHARACTERISTICS:

LENGTH: 861 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: GCCGGCGCTC GCATATCTCG CGATCTTCTT CCGTGGTGCC

GTTCTTCTCG

CCTCGTTGTC

120

TCGGCAACT.A

180

ACGCGTTCGT

240

CC-TTCAAGGC

300

TCACGTTCCT

360

TCACCGCGCT

420

GGGCGGTCAT

480

TCAGCCTGGA

540

CGCCGCGCAG

600

GCATGCTGGT

660

CCCAGACCAC

720

CGGCGGCGGC

780

ACACACGGGC

840

CACCGCCIGCC

861

GGAGACCGGC

CGTCGACGCG

CGCCACGGTG

CGGCCGGTTC

GATCCGCACC

GGGCGCCATC

CGGCGGTCTG

GAAGATCGAC

CTTCGGCAAG

GTTCATCCCG

CATGATCGGC

GGCTCGGTGT

TTCACGATGT

TCATGCCGAC

ACCACGAGCA

GCTGACGTTC

CTGTGCCTGT TGCTGGCGTT

CCCGCTGGCC

A.AGAACCTGA

ATTGCGTGG.A

GGGCTGCTGC

ACCTACAAT

CCGCGTCTGC

GTGATCCTGC

GCCGTCGGCG

AACGTGATCC

TCCTGGGGCT

AGACGATCCT

CTGACGAGGG

GGATCATCT

TGGAGGCCTC

CGATGGCGAT

ACTTCATCA

AGAAGCAGTT

GGTGATCCTG

GGCCGACGAA

CCGGCTGCTG

CATGATCCTG

CCAGGACCTC

GCCCGGGGTG

CGCCGACTAT

CCTGGTCGTC

CTGGCACGCA

GCCTGGGACT

TCGTTCGGCT

TACGTCATCG

CCGTTCTTCG

GGCTGGGTGG

TCCACCAGCT

CCGCTGTACG

rACTCGTCGG

:TGGCCGGGA

TCGGCAGTA

a*

S

CGCGCTGAGT CTGGGGCTGA TGTTGCTGAT CCTGATCGGC

GTGCTCCTCT

GCTGGGTTCG GAGGATCTGG TATGACCACC CAGGCAGGCG

CCGCACTGGC

CAGCAGGATC C INFORMATION FOR SEQ ID NO:98: SEQUENCE

CHARACTERISTICS:

LENGTH: 259 amino acids TYPE: amino acid (G.STRANflEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCR.IPTION:. SEQ ID NO: 98:.

Val Pro Phe Phe Ser Leu Ala Arg Thr Ser Leu 10 Ser Val Phe Met Pro Thr Leu Thr Phe Ala Trp 25 Val Asp Ala Phe Thr Met Tyr His Glu. Gin Ile 40 Tyr Ala Phe Val Ala Thr Val Leu Cys Leu Leu s0 55 60 Ala Tyr Val Ile Ala Phe Lys Ala Gly Arg Phe 70 75 Gly Leu Val Ile Leu Pro Phe Phe Val Thr Phe a Val Gly Tyr Gly Leu Leu Ser Asp Phe Leu Lys Leu Glu Phe Arg Al a Asn Ile Thr Gly Gly Asn Ser Phe Phe Pro Leu Ile Arg Thr 107 Ile Ala Trp Thr Ile LeI Ala Asp Glu G1ly Trp Val Val Thr Ala Leu 100 105 110 Gly Ala Ile Gly Leu Leu Pro Asp Glu Gly Arg Leu Leu Ser Thr Ser 115 120 125 Trp Ala Val Ile Gly Giy Leu Thr Tyr Asn Trp Ile Ile Phe Met Ile 130 135 140 Leu Pro Leu Tyr Val Ser Leu Giu Lys Ile Asp Pro Arg Leu Leu Glu 145 150 155 160 Ala Ser Gin Asp Leu Tyr Ser Ser Ala Pro Arg Ser Phe Gly Lys Val 165 170 175 Ile Leu Pro Met Ala Met Pro Gly Val Leu Aia Giy Ser Met Leu Val 180 iss 190 Phe le Pro Ala .Vai Gly Asp.Phe Ile Asn Ala Asp Tyr Leu Gly Ser 195 200 205 Thr Gin Thr Thr Met Ile Gly Asn Val Ile Gin Lys Gin Phe Leu Val 210 215 220 Val Lys Asp Tyr Pro Ala Ala Ala Ala Leu Ser Leu Giy Leu Met Leu 225 230 235 240 Leu Ile Leu Ile Gly Val Leu Leu.Tyr Thr Arg Ala Leu Gly Ser Giu 245 250 255 0eAsp Leu Val INFORMATION FOR SEQ ID NO:99:- SEQUENCE~

CHA.RACTERISTICS:

LENGTH: 277 base pairs TYPE: nucleic acid STPANDEDNESS: single TOPOLOGY: linear (i)MOLECULE TP:Genomic

DN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: GTAATCTTTG CTGGAGCCCG TACGCCGGTA GGCAAACTCA TGGGTTCGCT

CAAGGACTTC

AAGGGCAGCG ATCTCGGTGC CGTGGCGATC AAGGGCGCCC TGGAGAAAGC CTTCCCCGGC *.120 GTCGACGACC CTGCTCGTCT CGTCGAGTAC GTGATCATGG GCCAAGTGCT

CTCCGCCGGC

180 GCCGGCCAGA TGCCCGCCCG CCAGGCCGCC GTCGCCGCCG GCATCCCGTG

GGACGTCGCC

240 TCGCTGACGA TCAACAAGAT GTGCCTGTCG

GGCATCG

277 INFORMATION FOR SEQ ID NO:iOO.:: Ci)-SEQUENCE

CHARACTERISTICS:

LENGTH: 92 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:iOO: Val1 Leu Al a Glu Pro Ser Ile Lys Leu Tyr so Al a Leu Phe Asp Glu Val Arg Thr Ala Phe Lys Ile Gin le Gly Ala Lys Gly Ala Phe Met Gly Ala Ala 70 Asn Lys Arg Ser Pro Gin 55 Val Met Thr Asp Gly 40 Val Al a Cys Pro Leu 25 Val Leu Ala Leu Val Gly 10 Gly Ala Asp Asp Ser Ala Gly Ile Ser Gly Lys Val Pro Gly Pro Ile Leu Met Gly Ser i-s Ala Ile Lys Gly Ala Arg Leu Val Ala Gly Gin Met Trp Asp Val Ala S S

SS

0 S

S.

6*

S

S

*5 S 5e 0S *59* INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids (B).TY'PE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Other LOCATION: 1 .1 OTHER INFORMATION: Residue NAME/KEY: Other LOCATION: .2 OTHER INFORMATION: Residue (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Xaa Ala ASP Arg Gly Xaa Ser Lys Tyr 1 r 101: can be either Glu or Pro can be either Pro or Glu NO :101: Arg Xaa eve* C S 6664 42) INFORMATION FOR SEQ ID NO:102: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDMESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: Xaa Ile Asp Glu Ser Leu Phe Asp Ala Glu Glu Lys Met Giu Lys Ala 1 5 10 is Val Ser Val Ala Arg Asp Ser Ala INFORMATION FOR SEQ ID NO:103: SEQUENCE

CHARACTERISTICS:

LENGTH: 23 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: Xaa Xaa Ile Ala Pro Ala Thr Ser Gly Thr Leu Ser Glu Phe Xaa Ala 1 S 10 15 Xaa Lys Gly Val Thr Met Glu INFORMATION FOR SEQ ID NO:10 4 SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10 4 Pro Asn Val Pro Asp Ala Phe Ala Val Leu Ala Asp Arg Val Gly 1 5 10 INFORMATION FOR SEQ ID N0:105: SEQUENCE

CHARACTERISTICS:

LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii 4 -MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: Xaa Ile Arg Val Gly Val Asn Gly Phe 1 5 INFORMATION FOR SEQ ID NO:106: SEQUENCE

CHARACTERISTICS:

LENGTH: 485 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: AGCGGCTGGG ACATCAACAC CGCCGCCTTC GAGTGGTACG TCGACTCGGG

TCTCGCGGTG

ATCATGCCCG TCGGCGGGCA. GTCCAGC'rTC TACAGCGACT GGTACAGCCC

GGCCTGCGGT

120 AAGGCCGGCT GCCAGACCTA CAAGTGGGAG ACGTTCCTGA CCCAGGAGCT

GCCGGCCTAC

180 CTCGCCGCCA ACAAGGGGGT CGACCCGAAC CGCAACGCGG CCGTCGGTCT

GTCCATGGCC

240 GGTTCGGCGG CGCTGACGCT GGCGATCTAC CACCCGCAGC AGTTCCAGTA

CGCCGGGTCG

300 CTGTCGGGCT ACCTGAACCC GTCCGAGGGG TGGTGGCCGA TGCTGATCAA

CATCTCGATG

360 GGTGACGCGG GCGGCAC.A GGCCAACGAC ATGTGGGGTC GCACCGAGGA

CCCGAGCAGC

420 GCCTGGAAGC GCAACGACCC GATGGTC.C ATCGGCAAGC TGGTCGCCAA

CAACACCCCC

480

CTCTC

485

S

*5 S S

S.

S

S

*SS.

*SSS

S

S INFORMATION FOR SEQ ID NO: 107: SEQUENCE

CHARACTERISTICS:

LENGTH: 501 base pairs TYPE: nucleic acid STRUANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE

DESCRIPTION:

ATGCCGGTGC GACGTGCGCuG

CAGTGCGCTT

60 GTGGGCGCTG AGGGCACCGC

ACTGGCGGCG

120 GTGACGTTCG CCTCCGACAA

ACTCGGCACG

180 TTCAGCGGTC AGTACACCTT

CAGCACGTCC

240 GACGGCCCGG CGCCGTCGAA~

CCCGACGATT

300 AGGCAGTGco"'TGTTCAACTA

CAACTGGCAG

360 CGCGAGTACG CCGCCGCGCG

TTCGCTGGTG

420 TTCGGCACCT GGCGCACCGA

NATCCTGGAN

480 GTCGCGGCCT ATCCGGCGTA

G

501 SEQ ID NO:107: GCGTCCGOTGA

CCTTCGTCGC

ACGCCGGACT

GGAGCGGGCG

AGTGTGGCCG

CCCGCCAGCC

TGTGTGGGCA

CCTGCGTGGC

CCGCAGCCCG

CGCGCTACAC

TGGGAGTGCT

TCCGCGGCGC

TTCTACGCCC

CGACCGCCGA

GGCCTCTGCA AGGGCACCGT

GGCCGCGTGC

CTACACGGTG

AGAACCCGAC

CACCGCGTCC

CTGGGACGGC

CGACGTCCCG

CGGGTCGATG

GATCATGCCG

INFORMATION FOR SEQ ID*NO:108': (i)SEQUENCE

CHARACTERISTICS:

LENGTH: 180 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: ATGAACCAGC CGCGGCCCGA GGCCGAGGCG AACCTCGGG GCTACTTCAC

CGCCAAC

CGCCAACCCG

GCGGAGTACT ACGACCTGCG GGGCATCCTC GCCCCGATCG GTGACGCGCA

GCGCAACTGC

120 TAGG

GGICC

AACATACCG TGCTGCCGT AGAGCTGG

ACZCTC

180ACATCACC TGCTGCCGT AGAGCTGCAG ACGGCCTACG ACACGTTCAT

GGCCGGCTGA

180 INFORMATION FOR SEQ ID NO:109: SEQUENCE

CHARACTERISTICS:

LENGTH: 166 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: Met Prb Val Arg Arg Ala Arg Ser Ala Leu Ala Ser Val Thr Phe Val 1 5 10 15 Ala Ala Ala Cys Val Gly Ala Glu Gly Thr Ala Leu Ala Ala Thr Pro 20 25 Asp Trp Ser Gly Arg Tyr Thr Val Val Thr Phe Ala Se Ly Leu 35 40 45 Gly Thr Ser Val Ala Ala 40AerspLse y Thr Ser Val Al a Arg Gin Pro Glu Pro Asp Phe Ser Gly Gin 50 55 60 Tyr Thr Phe Ser Thr Ser Cys Val Gly Thr Cys Val Ala Thr Ala Ser 75 Asp Gly Pro Ala Pro Ser Asn Pro Thr Ile Pro5 Gin Pro Ala Arg Tyr80 90 95 yr Cys Phe Asprg Gy Arg laGn Trp Val Phe Asn Tyr Asn Trp Gin Tro Glu 100 .105 110 Cys Phe Ar Ala Val Pro Arg Glu Tyr Ala Ala Ala Arg Ser 115 120 125 Leu Val Phe Tyr Ala Pro Thr Ala Asp Gly Ser Met Phe Gly Thr Trp 130 135 140 Arg Thr Xaa Ile Leu Xaa Gly Leu Cys Lys Gly Thr Val Ile Met Pro S145 150 155 160 Val Ala Ala Tyr Pro Ala 155 165 .I INFORMATION FOR SEQ ID NO:110: SEQUENCE

CHARACTERISTICS:

LENGTH: 74 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110.: Pro Arg Asp Thr His Pro Gly Ala Asn Gin Ala Val Thr Ala Ala Met 1 5 10 15 Asn Gin Pro Arg Pro Glu Ala Glu Ala Asn Leu Arg Gly Tyr Phe Thr 20 25 30 Ala Asp Pro Ala Glu Tyr Tyr Asp Leu Arg Gly Ile Leu Ala Pro Ile 35 40 Gly Asp Ala Gin Arg Asn Cys Asn Ile Thr Val Leu Pro Val Glu Leu 55 '60 Gin Thr Ala Tyr Asp Thr Phe Met Ala Gly 70 INFORMATION FOR SEQ ID NO:111: SEQUENCE

CHARACTERISTICS:

LEN'GTH: 503 base pairs TYPE: nucleic acid STRANDEDNESS: single TO POL OGY: linear (iMOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIfPTION: ATGCAGG TGC GGCGTGTTCT

TGGCAGACGG

120

ATCTTCGCGC

180

AACGCGCTGC

240

GCAGGATTCG

~300

GCAGTGGATG

360

CGCCGGCGTC

420

CCCGATGCCG

480

GCGCGACATC

503

GGGCTTTCGAT

GCGGGACCGG

GGCCCAAGGT

GACTTCGACA

GCCGACAACT

ATCGACCTGA

CCCCGCGTCG

CC-TGGTGGCG

GGGCAG TGTC

ACCGACCGCC

TGCGGAACCC

CGGTGAGCAG

AATCGGCGCC

GCCCGGACAC

TCACCGTCGA

CCGACCACGT

GTC

SEQ ID NO:ilI: GGTGCAGCAG

TCGCGGTTTC

TCAGCGGAT C CGTGTCCGGA GGCCTCGGGT

GGG-TCGGTGA

T-GGTGGGCA

CCTACGCGGT

CATGGGCc3CG

GCCGACGCAT

CAAGCTTGTC

CTGGGCGGCA

TCCGCGACCG

CTGGGCCGGT

GGCCGCCGTT

GTGGTCTTCG

GGCCGCGTTA

CATCGAGGTG

TGCGTTCGTC

GAACTACCCG

CGGGGCGGGT

TGTCGCANGG

TCACCCCCAC

GAAATCCGTT

INFORMATION FOR SEQ ID NO: 112: i)SEQUENCE

CHARACTERISTICS:

LENGTH: 167 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:112: Met Gin Val Arg 1 Ser Ala Asp Pro Glu Pro Pro Lys Ala Gly Ser Gly Val Leu Val Asp 130 Arg Val 145 Al a Cys Gly Val1 Phe Arg Gly 115 Pro Ala Leu Pro Leu Gly Asp Val 100 Gly Arg Asp Arg 5 T rp Asp Gly Glu Phe 85 Gin Met Pro His Val Gin Ile T rp Gln 70 Asp Trp Ser Leu ValI Leu Gly Ser Thr Glu ValI 55 Ser Lys Met Xa a Gly 135 Ala Gly Val1 40 Gly ValI Ser Al a Gly 120 Arg Ala ValI 25 Ile Asp Gly Al a Asp 105 Al a Phe Val1 Val 10 Ser Phe Al a Thr Pro 90 As n Gly Thr Gly Ile Al a Phe Tyr 75 met Cys ValI Pro Al a Pro Arg Val 60 Ala Gly Pro Ile Thr Al a Thr Gl y As n Val Ala" Asp Asp 125 Pro Val1 Al a Thr Al a As n Al a Thr 110 Leu Met Al a Ser Gly Leu Tyr Asp Lys Ile Pro ValI Al a Al a Arg Pro Ala Leu Thr Val Val Phe Gly Asn Pro Leu Arg Asp Ile Arg Gly Gly Gly 165 INFORMATION FOR SEQ 1D NO:JJ3: SEQUENCE

CHARACTERISTICS:

LENGTH: 1569 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLE:CUT TYPE: Genornic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: ATGGCCAAGA CAATTGCGTA TGACGAAGAG GCCCGCCGTG

GCCTCGAGCGJ

GCCCTCGCAG ACGCCGTA.A GGTGACGTTG GGCCC-GAACG

GTCGCAACG-.

120 AAGAAGTGGG GCGC-CCCCAC GATCACCAAC GATGGTGTGT

CCATCGCCA

180 CTGGAGGACC CGTACGAGAA. GATCGGCGCT GAGCTGGTCA

AAGAGGTCGC

240

GACGACGT(

300

GAAGGCCTC

360

AAGGCTGTC

420

GAGCAGATT

480

GCCGAGGCC

540

TTCGGCCTG

600

TACT-TCGTG,

660

GTCAGCTCCA

720

GCCGGCAAG(

780

GTGGTCAAC;

840

GACCGCCGC;

900

GAAAGAGTCG.

960

GTCGTCGTCA

1020

GCCGGCCGGG

108b

GAGAAGCTGC

1140

GCTGCCACCG

1200

GCGAAGGCTG

1260

GCTCC-TGCGC

1320

CGCGTGGCGC

1380

GTCGTTGCCG

1440

GAGTACGAGG

1500

CTGCAGAACG

1560 'G CGGGCGAC( ;C GCAACGTCC :G AGGCTGTC; 'T CTGCCACCC A TGGACAAGG C AGCTCGAGC k CCGACGCCG k AGGTGTCGA 2CGCTGCTGA'

~AGATCCGCG(

SAGGCGATGC

J

;GGCTGTCCCI

*CCAAGGACGP

TGGCTCAGAT

AGGAGCGCCT

AGGTGGAGCT

CCGTCGAAGA

TGGACGACCT

TGTCGGCTCC

AGAAGGTGTC

ACCTGCTCMA

CGGCGTCCAT

;G CACCACCACC ;C AGCCGGCGCC C CCAGTCGCTG C GGCGATTTCC T CGGCAACGAG r CACCGAGGGT

GCGCCAGGA

2CGTCAAGGAT

CATCGCCGAG

CACCTTCAAG

GCAGGACATG

GGAGACCGCCC

GACCACCATC

C

CCGCGCCGAG

P~

GGCCAAGCTG

G

CAAGGAGCGC

GGGCATCGTC

G

CGGCCTGACG

G

.GCTCAAGCAG

A

GCCACCG-TGC

AACC-CGCTCG

CTGAAGTCGG

GCCGGCGACA

GGTGTCATCA

ATGCGCTTCG

GCCGTCCTGG

CTGCTCCCGC

GACGTCGAGG

TCCGTCGCCG

IJ

GCCATCCTCA

C

3ACGTCTCGC

T

;TCGAGGGCT

C

~TCGAGAACA

G

CCGGCGGTG

T

AGCACCGCA

T(

CCGGTGGCG

CC

GCGACGAGG

CC

TCGCTCAGGC

GCCTCAAGCG

CCAAGGAGGT

CCCAGATCGG

CCGTCGAGGA

ACAAGGGCTA

AGGATCCCTA

rGC-TGGAGA 3CGAGGCCCT rCAAGGCTCC

C

CGGTGGTCA

C

GCTGGGCCA

G

GGGCGATTC

C

CGACTCCGA

C

TGCGGTGAT

C.

CGAGGACGC

C

:GTGGCTCT

G(

ACCGGTGC

C

GGGCCTCAAC

CGTGCTGGAG

GGAGATCGAG

CAAGAAGACC

TCTGGTTCGC

TGGCATCGAG

CGAGACCAAG

CGAGCTCATC

GTCGAACACC

CATCTCGGGT

:ATCCTGCTG

;GTCATCCAG

;TCCACGCTG

GGCTTCGGT

GTCGTCAGC

GCCCGCAAG

GATGCCATC

rACGACCGC kIAGGCCGGA

;TCCGCAAC

TGCAGTCG

ACATCGTC

AGCCCGGC

CGACCGGT

GCTCGGCG

TCGCCGAC

CAACCTGCCC GCGGGTCACG

GCCTCAACGC

GGCCGGCGTC

CGCGGCTCTG

GCCGACCCGG

TTCCTCACCA

TGAAGGTCAC

CCGAGGCCGT

AAGCCGGAG

1569 INFORMATION FOR SEQ ID NO:114: SEQUENCE

CHARACTERISTICS:

LENGTH: 523 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu 1 5 10 Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro 25 Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile 40 Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro 55 Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr 70 75 Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gin 85 90 Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro l 100 105 110 .Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Ala Val Thr Gin 115 120 125 Ser Leu Leu Lys Ser Ala Lys Glu Val Glu Thr Lys Glu Gin Ile Ser 130 135 140 Ala Thr Ala Ala Ile Ser Ala Gly Asp Thr Gin Ile Gly Glu Leu Ile 0145 150 155 160 Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu 165 170 175 Glu Ser Asn Thr Phe Gly Leu Gin Leu Glu Leu Thr Glu Gly Met Arg 180 185 190 Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Ala Glu Arg 195 200 205 CGln Glu Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys 210 215 220 Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gln 225 230 235 240 Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala 245 250 255 Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val 260 265 270 Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gin 275 280 285 Asp Met Ala Ile Leu Thr Gly Gly Gin Val Val Ser Glu Arg Val Gly 290 295 .300 Leu Ser Leu Glu Thr Ala Asp Val Ser Leu Leu Gly Gin Ala Arg Lys 305 310 315 320 Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ser Gly Asp 325 330 335 Ser Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Ala Glu Ile Glu 340 345 350 Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gin Glu Arg Leu Ala 355 360 365 Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Glu 370 375 380 Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn 385 390 395 400 Al a Le u Glu Lys L ys 465 G1u Th r Th r Lys Le u Al a G In 450 ValI T yr Arg Thr Ala Gin Thr 435 -le Ser Ser 515 Ala Ser 420 Al a As-n Al a 300 Aa ValI 405 Ala Ala Phe Leu Le u 485 Leu Val1 Glu Pro As n.

As n Pro 470 Leu G-in Val1 Glu Gly Ile Val 410 Ala Leu Asp Asp 425 Ile Val Arg Val 440 Gly Gly Leu Glu 455 Ala Gly His Gly Lys Ala Gly Val 490 Asn Ala Ala Ser 505 Ala Asp Lys Pro Al a Le u Al a Pro Le u 475 Ala 'le Sly Gly Leu Gly 460 Asn Asp Ala GI y Le u Ser 445 Va 1 Ala Pro Al a Gly Th r 430 Ala ValI Ala Val Leu *Va I Ala 415 Gly Asp Pro Leu Al a Glu Thr Gly 4 83n Lys Vai 495 Phe Leu INFORMATION FOR SEQ'ID SEQUENCE

CHARACTERISTICS:

LENGTH: 647 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

RNA

(xi) SEQUENCE DESCRIPTION: SEQ 1D NO:115: ATGGCCAAGA CAATTGCGTA TGACGAAGAG GCCCGCCGT .G GCCTCGAGCG

GGGCCTCAAC

GCCCTCGCAG ACGCCGTAA GGTGACGtTG GGCCCGAAGG GTCGCAACGT CGTGCTGGAG 120 AAGAAGTGGG GCGCCCCCAC GATCACCAC GATGGTGTGT CCATCGCCAA GGAGATCGAG 180 CTGGAGGACC CGTACGAGA GATCGGCGCT GAGCTGGTCA AAGAGGTCGC CAAGAAGACC 240 GACGACGTCG CGGGCGACGG CACCACCACC GCCACCGTGC TCGCTCAGGC TCTGGTTCGC 300 GAAGGCCTGC GCAACGTCGC AGCCGGCGCC AACCCGCTCG GCCTCAAGCG TGGCATCGAG 360 AAGGCTGTCG AGGCTGTCAC CCAGTCGCTG CTGAAGTCGG CCAAGGAGGT CGAGACCAAG 420 GAGCAGATT-TCTGCCACCGC GGCGATTTCC GCCGGCGACA CCCAGATCGG CGAGCTCATC 480 GCCGAGGCCA TGGACAAGGT CGGCAACGAG GGTGTCATCA CCGTCGAGGA GTCGAACACC 540 TTCGGCCTGC AGCTCGAGCT CACCGAGGGT ATGCGCTTCG ACAAGGGCA CATCTCGGGT 600 TACTTtGTGA CCGACGCCGA GCGCCAGGAjA GCCGTCCTGG

AGGATCC

647 INFORMATION FOR SEQ ID NO:116: SEQUENCE

CHARACTERISTICS.

LENGTH: 927 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genornic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:

GATCCCTACA

TC-CTGCTGGT- CAGCTCCAAG GTGTCrG ACCG TC;AGG.ATCT GC-TCCCGCTC CTGGAGAAGG

TCATCCAGGC

GAGGC!-CTGJT

.180

AAGGCTCC-GG

240

GGTGGTCAGG

300

CTGGGCCAGG

360 GGCC-A TTCCG 420

GACTCCGACT

480

GCGGTGATCA

540

GAGGACGCCG

600

GTGGCTCTGC

660

AC'%CGGTGCCA

GGCGGCCTGG

7180

CTCAACGCCG

840

AAGGTCACCC

900

GAGGCCGTCG

927 CCACGCTGG7

GCTTCGGTGA

TCGTCAGCGA

CCOGCAAGGT

ATGCCA'rTCGC

ACGACCGCGA

AGGCCGGAGC

TCCGCA:ACGC

TGCAGTCGGC

ACATCGTCCG

AGCCCGGCcGT

CGACCGGTGA

GCTCGGCGCT

'rCGCCGACAA

*CGGCAAGCCG

*GGTCAACAAG

CCGC-CGCAAG

A.AGAGTCGGG

CGTCGTCACC

CGGCCGGGTG

GAAGCTGCAG

TGCCACCGAG

GAAGGCTGCC

TCCTGCGCTG

CGTGGCGCTG

CGTTGCCGAG

GTACGAGGAC

GCAGAACGCG

GCCGGAG

CTGCTGATC;

ATCCGCGGC;

GCGATGC

TGC

CT r -ICt CT GG

AAGGA'CGAGA

GCTCAGATCC

GAGCGCC-TGG

GTGGAGCTCA

GTCGAAGAGG

GACC-ACCTCG

TCGGCTCCGC

AAGGTGTCCA

CTGCTCAAGG

GCGTCCATCG

TCGCCO-AGGA

C~C7-A A GT C

AGGACATGGC

AGACCGCCGA

CCACCATCu-T

GCGCCGAGAT

CCAAGCTGGC

AGGAGCGCA

GCATCGTCGC

GCC-TGACGGG

TCAAGCAGAT

ACCTGCCCGC

CCGGCGTCGC

CGGCTCTGTT(.

CGTCGAGGGC

CG-TCGCCGTC

CATCCTCACC--

CGTCTCGCTG

CGAGGGCTCG

CGAGAACAC

CGGCGGTGTT

GCACCGCATC

CGGTGGCGGC

CGACGAGGCC-

CGCCTTCAAC

GGGTCACGGC

CGACCCGGTG

CCTCACCACC

0* INFORMYATION FOR SEQ ID NO:l117: SEQUENCE

CHARACTERISTICS:

LENGTH: 215 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE (x14 -SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:117: Met Ala Lys Thr 1 Arg Lys Thr Tyr Asp Al a Leu S er Ala 145 Gly Gly Asn Glu Asp Leu Gly Leu 130 Thr Leu Arg Asp Lys V al Val1 Leu 115 Leu Ala As n As n Gly I le Ala Arg 100 Lys Lys Al a Ile Al a ValI ValI Gly Gly 85 Glu Ar g Ser Ile Ala Leu Val Ser Al a 70 Asp GI y Gly Ala Ser 150 Tyr Al a Leu Ile 55 Gi u Gi y Leu le Lys 135 Al a Asp Asp Glu 40 Al a Leu Thr Arq Glu 120 Glu Gly Glu Ala 25 Lys Lys Val Thr As n 105 Lys Val1 Asp Glu Ala 10 Val Lys Lys Trp Giu Ile Lys Glu 75 Thr Ala 90 Val Ala Ala Val Giu Thr Thr Gin 155 Arg Val1 Gly Giu 60 Val1 Tb r Al a Giu Lys 140 Ile Arg Thr Ala Leu Al a Val Gly Ala 125 Giu Gly Gly Le u Pro Glu Lys Le u Al a 110 Val1 Gin Glu Leu Glu Gly Pro Thr Ile Asp Pro Lys Thr Ala Gin Asn Pro Thr Gin Ile Ser Leu Ile 160 Ala Glu Ala Me: Aso Lys Val Gly Asn Gu Gly Val le Thr Val Glu 16u Ile 170 175 Glu Ser As Thr Phe Glv Leu Gin Leu Glu Leu Thr Glu Gly Me Ar 180 185 Phe Asp Lys Gly Tyr Ile Ser GlyO Tyr Phe Val Thr Asp Ala Glu Arg 195200 Gin Glu Ala Val Leu Glu Aso 205 210 215 INFORMATION FOR SEQ ID NO:118: SEQUENCE

CHARACTERISTICS

LENGTH: 309 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGy: linear (ii) MOLECULE TypE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: Asp Pro Tyr Ile Leu Leu Val SerSer Lys Val Ser Thr Val Lys Asp 1 5 10 Leu Leu Pro Leu Leu Glu Lys Val Ile Gin Ala Gly Lys Pro Leu Leu 2 5 Ile Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr 3Leu Val Val 35999 40 Asn Lys Ile Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly 50 55 60 Phe Gly Asp Arg Arg Lys Ala Met Leu Gin Asp Met Ala Ile Leu Thr 65970655780 Gly Gly Gln Val Val Ser Glu Arg Val Gly.Leu Ser Leu Glu Thr Ala 75.85- y.e euGoTh 85 90 95 AsoVal Ser Leu Leu Gly Gin Ala Arg Lys Val Val Val Thr Lys Asp 100 105 110 Gu Thr Thr Ile Val Gu Gly Ser Gly Asp Ser Asp Ala Ile Ala Gly 115 120 125 Arg Val Ala Gin lie Arg Ala Glu Ile Glu Asn Ser Asp Ser Aso Tyr 130 135 140 Asp Arg Glu Lys Leu Gn Glu Arg Leu Ala Lys Leu Ala Gly Gly Val .9.145 15015 1 0 9 9 1 6 0 Ala Val Ile Lys Ala Gly Ala Ala Thr Glu Va Glu Leu Lys Glu rg 165 170 175 Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val5 Glu 9180 185 190 Glu Gly Ile Val Ala Gly Gly Gly Val Ala Leu Leu Gin Ser Ala Pro 195 200 205 Ala Leu Asp Asp Leu Gly Leu Thr Gly Asp Glu Ala Thr Gly Ala Asn 210 215 220 Ile Val Arg Val Ala Leu Ser Ala Pro Leu Lys Gin Ile Ala Phe Asn 225 230 235 240 Gly Gly Leu Glu Pro Gly Val Val Ala Glu Lys Val Ser Asn Leu Pro 245 250 255 Ala Gly His Gly Leu Asn Ala Ala Thr Gly 2u Tyr Giu Asp Leu Leu 260 265 270 Lys Ala Gly Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu Gin 275 280 285 Asn Ala Ala Ser lie 2la Ala Leu Phe Leu Thr Thr Glu Ala Val Val 290 295 300 Ala Asp Lys Pro Glu 300 305 INFORMATION FOR SEQ ID NO:119: SEQUENCE

CHARACTERISTICS:

LENGTH: 162 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear xii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119: CTCGTACAGG CGACGGAGAT CTCCC-ACGAC CCCACGTCGG TACGGTTCGT

CGCCACCCTAG

TTCGGCGTCG TGTTGTTGAC GTTGG'rGCTG TCCGGGCTCA ACGCCACCCT1

CATCCAGGGC

.120 GCACCAGAAG ACAGCTGGCG CAGGCGGATT CCGTCGATCT

TC

162 INFORMATION FOR SEQ ID. NO:*120: SEQUENCE

CHARACTERISTICS:

LENGTH: 1366 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (i4i) MOLECULE TYPE: Genomic DNA ease 9* 90 (xi)

GATGAGCAGC

60

CCCGGTGCTG

120

GCTGGCCCGC

180

CCTGCTGGTA

240

CCTGTTCGGC

300

GGGCGCACCA

360

CTTCGCGCTG

420

GGGGGGCCTG

480

TTCGGTCGGT

540

CGACTGGATC

600

AGTCAACTGG

660

CGAACTCGCC

720

CGTCGTCACC

780

GGTCGCGGCG

840

TGCGGCCGAA

900

GAGCACGTAC

960

CGTCGCCGAC

1020

CACACTGCGC

1080 SEQUENCE DESCRIPTION: SEQ ID NO:120: GTGCTGAACT CGACCTGGTT

GGCCTGGGCC

CTGGTCGTGC

CCGGTGCAAC

CAGGCGATGG

GTCGTGTTGT

GAAGACAGCT

ATCGCGGTCG

TTCACCGCAC

CAGATCATCT

ACCGTCCCCA

CGTGCAACAC

GGCGCGTCGT

ACCTTCAACG

TCGCTGCCCG

TACGAGAAGT

CTGCGATGGG

GANTTCGACA

TTGGCAGACG

TGACCGAGGT

TCCTGCGTAC

AGATCTCCGA

TGACGTTGGT

GGdGCAGGCG

GTATCACCGT

TGGGCGTCAC

CGGGTCTGCT

CCGCGGCGGG

ATATCGACAC

TCACCAATTA

CCGCGGACAC

AACTGCGCAC

CGATCCCGTT

TCTGGTACGC

CGCCGGAACG

ACGAACAGCA

GCACAACGCG

CTACATCCTG

CGACGCCACG

GCTGTCCGGG

GATTCCGTCG

GATCATGGCC

TTCCATCGTT

GCTGCTGTTC

CCGGCCGTCC

CGGCGGCAAC

CAG CC GGCC C

CCCCGATGAT.

CGACGGACAG

GCACACACCC

CGCGCGCCGG

GATCGCCTCG

GGAGATCGCC

GTCGCGGTCG

TTGCGTCGGC

CCGCTGGGCG

TCGGTACGGT

CTCAACGCCA

ATCTTCCTCG

TATGTCTGGG

CTTGGCCTGG

GAGCAACCGT

GCCCACGGCC

CTGCTGGTA.A

GTGGGAGAGC

GTCTGCGAGA

ATCGCCACGC

GCGGTGGACG

CAGGAACTTC

GCCATGCGGG

GACGTGGTGC

CGGTCGGGTT

GCGGCAGCGC

CGTTGCTGCT

TGGTCGCCAC

CCCT'CATCCA

ACGTCGCGCG

GCGCGAACGT

CTCTGCAGAA.

TCCGGCTCGG

GCGTGGTGGA

TGCCCAACGC

ACCGGCTGAC

*TGCTGTCGTC

TCTATCTCGG

ACTCGGTCAG

GCCTNAACGG

CTGTGGCGTC

GTCTGGTCCG

TTACGGCA.AC GGGGAACGCc 7TCCAGCAGCC GGGTCAGGTA CCGAC-GGGA

TGAGGTTCA-'

CGTAGACGGC AGGGTGAGTC TGTCCGTGAT CGATCAGGAC GGCC-ACGTGA

TCCCGGCGCG-

GGTGCTC'QAG CGTOGGCGACT T-CTGGG.GCA GACCACGCTG ACGCGGGAAC

CGGTACTGGC

1260 GACCGCGCAC GCGCTGGAGG AAGTCACCGT GCTGGAGA-G GCCCGTGACr: AGATC-GAGCG 1320 CC-TGGTGCAC CGAAAGCC%-A TCCTGCTGCA CGTGA'TC-GGG

GCCGTG

1366 INFORMAT TON FOR SEQ ID NO:121: SEQUENCE

CHARLACTERISTICS:

LENGTH: 455 amino acids TYPE: amino acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein SEQUENCE DESCRIPTION:' SEQ ID NO:121: Met Ser Ser Val Leu Asn Ser Thr Trp Leu Ala Trp Ala Val Ala Val .1 5 10 Ala Val Gly Phe.Pro Val Leu Leu Val Val Leu Thr Giu Val His Asn .20 23 30 Ala Leu Arg Arg Arg Gly Ser Ala Leu Ala Arg Pro Val Gin Leu Leu 35 40 4 :Arg Thr Tyr Ile Leu Pro Leu Gly Ala Leu Leu Leu Leu Leu Val Gin 55 6 Ala Met Giu Ile Ser Asp Asp Ala Thr Ser Va 1 Arg Leu Val Ala Thr 70 75 Leu Phe Gly Val Val Leu Leu Thr Leu Val Leu Ser Gly Leu Asn Ala 90 Thr Leu Ile Gin Gly Ala Pro GiluAs eTrAgAgAgIlPo 10105 110 Ser Ile Phe Leu Asp Val Ala 'Arg Phe Ala Leu Ile Ala Val Gly Ile .*115 120 125 Thr Val Ile Met Ala Tyr Val Trp Gly Ala Asn Vai Giy Gly Leu Phe 130 135 140 Thr Ala Leu Gly Vai Thr Ser le Val Leu Gly Leu Ala Leu Gin Asn 145 150 15510 Ser Val GU y Gi le I e 1yLeu Leu Leu Leu Phe Giu Gin Pro *165 170 175 16 Phe Arg Leu Gly Asp Trp Ile Thr Val Pro Thr Ala Ala Giy Arg Pro 180 185 190 Ser Ala His Gly Arg Val Val Giu Val Asn Trp Arg Ala Thr His Ile 195 200 205 Asp Thr Gly Gly Asn Leu Leu Val Met Pro Asn Ala Glu Leu Ala Gly 210 215 220 Ala Ser Phe Thr Asn Tyr Ser Arg Pro Val Gly Glu His Arg Leu Thr 225 230 235 240 Val Val Thr Thr Phe Asn Ala Ala Asp* Thr Pro Asp Asp Val Cys Giu 245 250 255 Met Leu Ser Ser Val Ala Ala Ser Leu Pro Glu Leu Arg Thr Asp Gly 260 265 270 Gin lie Ala Thr Leu Tyr Leu Giy Ala Ala Giu Tyr Giu Lys Ser Ile .275 280 285 Pro Leu His Thr Pro Ala Val Asp Asp Ser Val Arg Ser Thr Tyr Leu 290 295 300 Arg Trp Val Trp Tyr Ala Ala Arg Arg Gin Giu Leu Arg Xaa Asn Gly 305 310 315 320 Val Ala Asp Xaa Phe Asp Thr Pro Giu Arg Ile Ala Ser Ala Met Arg 325 330 335 Ala Ala Gi',n Val 385 ValI Met Le u Val2 As p Pro 370 Ser Leu Al a H 4 450 Al a Val1 353 GlIy Le u Glu Le Arg 433 Val Ser Thr Leu 340 Val Arg Leu Gln Val Pro Ser Val Ile 390 Ar; Gly Asp 405 Al ±n7,,r A_-a 420 Asp C-lu Ile lie Gly Ala Ar; Val Thr 375 Asp Phe his Glu ValI 455 Leu

A--

360 Gly Gin Leu n.

Arg 440 Al a 345 Tyr MetZ Asp Gly 425 Leu Asp Asp Gly Asn Arg Phe Gly Asp 395 Gln Thr 410 u.U G 1Ql Val H is Clu Gly Ile 380 ValI T hr Val Arg Gln GI n 350 Glu Arg 30-5 Val Aso Ile Pro Leu Thr Thr Vai 430 Lys Pro 445 G I u Le u Gly Al a Ar; 415 Leu I Ie Ile Arc Arg 400 Glu G 1.U Leu INFORMATION FOR SEQ ID NO:l22: SEQUENCE

CHARACTERISTICS:

LENGTH: 898 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE

T'

(xi) SEQUENCE

DI

ATGACAATTC

TGCCCTGGAA

CGCTACCACC

TCCTGTCGCG

120 ACCAGCATTC

TCTCGGCTGC

180 CTGCGCGCAT

CGGTGTTCGA

240 GAGAATCAGT

TCGCGGACCT

300 ACGGAGGCGA

TCGGCGCGTT

360 ACCGGGCAGG

CGGCGTCATT

420 GACGACAGCZ

.GAAACCGCGT

480 TATCTGCAGG

CGCTCTATAC

540 GACGCGCGCG

ACGGCAGCGC

600 GAGATCGTGC

ACCGCTTCA

660 GTGTACTCCG

CCTACAAGGG

720 AACCGGGAAC

'TGTCGGAAGC

780 GGTGTCACCG

ACTTCGGGTG

840 rPE: Genomic DNA :.SCRI PT ION:

TGCGCGAACG

GATGAGCATC

GGTGGTCGGT

CCGCCTCACC

GAAGAACTCG

CAGCGACGGT

GCGCCGTTAC

CGACGTCCGC

CCCGCCGTTT

CTGGTCGGCC

CTTCGAGGAT

GCCGGATCTC

CTACGAGAAG

GTACCTGCCT

C

G

G

SEQ 1D NO:122: TCTGAACACC

CGACGCGAAA

CAGTCCAAGT

TGCTGCTGAT

TTCATCGGCT

ATCAGTCCGG

GACATCCGCG

AGTCGCAGTC

ATGGTGATTT ACTCdCGCGG TTCCGTCAGC

TCGGCGATGC

1'ACGACCGGA

CGTTCGCCAA

3CGCTCATCC

CGAAATCCAA

:AGAACTGGG

AGAAGGCGAT

~CCAATGCCA

GATTCAACGA

:TGATGCTGC

TCGACCTCGA

;GGACAAACA

TCGTCAACGG

;CGGTCGCGT

CGAACTCGAT

;CCGAGGAAC

CGACCGCCTG

AAGACGCGGG

GCTGCTTCTG

ACGGTCCTCG

GCGCGGGTTG

CAGCACTGC

GACGATCAAT

CACCACCC'rC

CCCCCAGCGC

CGCGTTCGAC

GTTCTTCCGC

GGGCAACGTG

CCCCTATCGC

CGACTATGTC

CCGGTCGGGT TGAAGGACCG

AGTCGACGGT

898 GTGATGGCGG TCCAGTTCCC

CGGAATTC

INFORMATION FOR SEQ ID NO:123: SEQUENCE

CHARACTERISTICS:

LENGTH: 1259 base pairs TYPE: nucleic acid

CGCAAT

120

GGGAG-A)

180 CG GT TG;z 240

CCCAACG

300

ACGCGTA

360

CCGACGA

420 STRANDEDNTESS: single TOPOLOGY: linear MOLECULE TYPE: G enomic

DNA

(xil SEQUENCE DESCRIPTION: SEQ ID NO:123: TGAT GACGGCGCGG GGACAGTGGC CTCACACCGG

GAT

TGG'T CC-GACCGGAC AATCTGATGC GCTCGG?,CTC

CCG

~GTT CCTGGCCGAC GTCGTCGAGG GGGGAACCCC

GCC-

~CCG CCGCGGCACC ACGCTGGTGC AGCCGGTGAC

CACC

;CGG CAACACCGGG ACGACGATCG AGGACGACTA

TCTC

.CTC ACCGGTGGAC CTGCCGGGAC TGCACTGG-GT

GATC

CCC GTTCGCCCCG GTGGCGCAGT TCACCAGGAC

CCTG

GGGAG;z

~CTGTI

GGAGG7

-CGCTC

:GGCCA

GTGGCi

TCATCATCT:

480

TCCGGCGGTT

543C

GGTCTTGTC

600

ATCTGTCGAT

660

TGTCCCTGAT

720

AGGACCACA

780

GCATGTTGAC

840

CCGCCGCCGA

CGGCGTGTCG

GCAGGCMCGG

TCGTGACGAA

CAAGGACGAG

GCCCGAACCG

GAACGTCACC

CTCCGAGGAA

GAGTCTCGGG

CTGGCCGCC;

GCCCAGCAG;

TTCGGCGATC-

CTGCTCGGCG

GTGATGCAGC

GTGATCTTCG

CTGATGGTGG

GTCGACCACG

CGGCTGGACA

CGGCACGCCG

GCGGCCAGCG

SATGTCGCCT

;TGCACGAGG

GCGGCGTCG

kTGCTGCTGG,

TCAGCGGCG,

TGACAACAGC

AGGACCGCG%,

GCTACCTCG?

CCGACATGAT

TGGTCAACGA

TGCGGACGCT

ACGTCCGCG

CCGAGTCCGG

GGCTGGTGGG

ACCAGCTGCA

TCATGCAGGA

C GCGCTTCTTC

TGACTACCGC

TT'TCAACGAC

CGAGAACCAA

CGGGGAGGAG

GGGCCTCGAC

CCTGACCCGC

GCACGACGGG

CACGCTCAAT

CCACGACCTG

GCGGTCCACG

GCGCGGCTCC

AACTCTCGAC

k.C ACCCGTGAGA 'C CGCGAGA.ACC 'C GCCGACGAAT C GTCGAGGAGG C GAGGCGTTAC

CAAGATCGACA

3TCGACGGTGA

-GTCCGTCCGA

-CTCGCTCTGC

*ATGAGTCGCA

CGGCTGATGC

ACGATCGCCC

GAGTTGTCGC

CAGTTCGACG

TACCTGGCCA

TTCGCGAtCG

CGGCTCCGCG

TTGGCC--TACG

CCCCAGCCCG

TTCGTCGCCG

GCTGCGGGTT

AGGCGTGCCG

960 AAATGGACCG

CATCATCGAC

1020 CGGGCATCGA

CACCGGGTCG

1080

ACATGTGGC.TTCGGCGGTC

1140 GCTCTACCT

CACCTCGCGG

1200 CCGGGGAGGT

CGTCGGCGAG

1259 INFORMATION FOR SEQ ID NO:124: SEQUENCE

CHARACTERISTICS:

LENGTH: 299 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N'O:124: Met Thr Ile Leu Pro TrP Asn Ala Arg Thr Ser Glu His Pro Thr Arg 1 5 10 Lys Arg Arg Gly Arg Tyr His Leu Leu Ser Arg Met Ser Ile Gin Ser 122 122 Lys Leu Leu Met Leu Leu Leu Va Val GIl Glv Arg Asn 145 Tyr lie Ala Gu Tyr 225 Asn lie Glu Gly Phe Asn Ser Leu Tvr 30 Arg Leu Ala Arg Asp 210 Lys Arg Asp Pro Phe Aso Gin Thr Gly 115 Tyr Val Gin Phe Phe 195 Leu Gly Glu Tyr Thr 275 Ile Arg Phe Al a Asp Aso Asp Ala Asp 180 Asn M'et Pro Leu lal.

kla Gly Leu Ala Thr Al a Arg VaI Leu 165 Asp Glu Leu Asp Ser 245 Gly Trp Tyx Thr 70 Asp Glu Thr Thr Arg 150 Tyr Ala Phe Leu Leu 230 Glu VaI Phe Gin 55 Aso Leu Ala 7le Phe 135 Ala Thr Arg Phe Aso 215 Gly Ala Thr Leu Ser Ile Lys Tie Asn 120 Ala Leu Pro Asp Arg 200 Leu Thr Tyr Asp Ser 280 ?he Thr Gly Arg Asn Gly 105 Thr Asn Ile Pro Gly 185 Glu Glu Asn Glu i Phe 265 Pro Pro C A I Ser Ile Leu Ser Ala Val Arg Glu Ser 90 Ala Gly Thr Pro Phe 170 Ser Ile ly Ile Lys 250 31y lal ly Ser Ser 75 Met Phe Gin Thr Lys 155 Gin Ala Val Asn Val 235 Ala Trv Gly Ile Sei Gir Val Ser Ala Leu 140 Ser Asn Trp His Val 220 Asn Vial Tyr Leu Leu Ser Ile Asp Al a 125 Aso Asn Trp Ser Arg 205 Val Gly Ala Leu Lys 4 285 Arg Arg Tvr Gly 110 Ser Asp Pro Giu Ala 190 Phe Tyr Pro Ser ?ro 270 ksp SAla

G]

Ser Phe Leu Ser Gln Lys 175 Al a Asn Ser Tyr Asn 255 Ala Arg Ser Leu Arg A rc Arg Gly Arg 160 Ala Asn ?he Al a Ara 240 Ser Glu Val Asp Gly 290 Val Met Ala Val Gin INFORMATION FOR SEQ ID NO:125: SEQUENCE

CHARACTERISTICS:

LENGTH: 419 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Gin 1 Thr Ser Glu Gly Gin Glu Val Gin (ii) MOLECULE (xi) SEQUENCE Leu Met Thr Ala 5 Gly Glu Thr Ile Arg Leu Phe Arg Gly Gly Thr Pro Thr Thr Leu Val Arg Gly Asn Thr Ala Leu Gin Ala 100 Ile Val Ala Lys 115 Phe Thr Arg Thr TYPE: protein DESCRIPTION: SEQ ID Arg Gly Gin Trp Arg 10 Leu Val Gly Pro Asp 25 Glu Asn Arg Glu Lys 40 Pro Glu Val Ala Asp 55 Gin Pro Val Thr Thr 70 Gly Thr Thr Ile Glu 90 Tyr Se: Pro Val Asp 105 lie Asp Thr Asp Glu 120 Leu Val Leu Se: Thr NO: 125: Asp Thr Asn Leu Phe Leu Glu Ser 60 Arg Ser 75 Asp Asp Leu Pro Ala Phe Val Ile Gly Met Ala Val Va1 Tyr Gly Ala 125 Ile Met Arg Asp Asp Glu Leu Leu 110 Pro Ile Gly Ser V~al Arg Glu Gly His Val Phe Asp Asp Val Arg Ala His Trp Ala Gly Val 145 Arg Leu Ala GLy Gu 225 Asp Glu Asp His Val I 305 Met A S. Arg L Giy A Ala T 3 Ser A: 385 Gly G Gly Hj 13 Se Ar Al Ph Gl Pr His Leu Leu Val 290 Pro Isp .eu rg yr 70 rg lu is !0 r Leu g Leu a Leu e Asn 195 u Glu o Val Lys Ser Thr 275 Arg Arg I Arg I Arg A 3 Ser T 355 Gin V Val H: Val V< Pro

A;

GJ

Pr 18 As Ar Me Asi Ar 26( Arc rhr Leu le .la 40 hr al is al La Ala in Ala 165 *o Val ;0 p Met g Ala t Gin n Val 245 g Met I SGin E Leu H Asp A 3 le A 325 Gly I Leu A.

Gin AJ Glu VE 3S Gly G1 405 Me 15 Gl Le Se Gli Ar 23C Thi Leu Phe lis ,sn 10 sp le La rg 9 )0 .u 13 -t Le 0 y Al u Se r Ar u Asi 21; g Tyr SVal Thr Asp Asp 295 Val Arg Asp Tyr Gly 375 Met Arg 35 -u Leu A.

a Gin G! r Arg As 18 g Asn Le 200 n Gin Ar 5 r Leu As SIle Ph SSer Gb! 26; Ala Ala 280 Gly Tyr Arg Arg His Ala Thr Gly 345 Asp Met 360 Ser Pro Gin Glu Gly Val 14C La Arg Leu Phe 155 Ln lie Ser Gly 170 ~p Giu ?he Gly 15 u Ser lie Lvs g Leu Me: Leu 220 p Giy Glu Glu 235 e Ala Asp Met 250 Giu Leu Met 5 Ala Glu Ser L 2 Leu Ala Ser C 300 Thr Val As P 315 Ala Glu Ser G 330 Ser Ala Ala S Trp Gly Ser AJ 3( Gin Pro Gly

II

380 Thr Leu Asp Ph 395 Glu Thr Val Tr 410

V;

G1 As As 20 CSe Th Mle Val *eu ys he ly er La le e p al Arg Pro y Asp Tyr 175 p Leu Thr 190 p Glu Leu 5 Seu Met r Ile Ala t Gly Leu 255 SVal Val 270 Gly Val Gly Leu C Ala Ile G 3 His Asp L 335 Gly Leu V 350 Val Asp V Tyr Val T] Val Ala A 4( Arg Leu GI 415 lie 160 Arg Thr Leu 0-- Gin 240 Asp Asn Aso ly ;lu eu al al hr La )0 in INFORMATION FOR SEQ ID NO:126: SEQUENCE

CHARACTERISTICS:

LENGTH: 27 base pairs TYPE: nucleic acid LC1 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: CCGGATCCGA TGAGCAGCGT

GCTGAAC

27 2 INFORMATION FOR SEQ ID NO:127: SEQUENCE

CHARACTERISTICS:

LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127: GCGGATCCCA CGGCCCCGAT

CACGTG

26 INFORMATION FOR SEQ ID NO:128: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: CCGGATCCAA TGACATTTCT GCCCTGGAAT

GCG

33 INFORMATION FOR SEQ ID NO:129: SEQUENCE

CHARACTERISTICS:

LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other SEQUENCE DESCRIPTION: SEQ ID NO:129: CCGGATCCAT TCGGTGGCCC TGCAACCGCC

AG

32 INFORMATION FOR SEQ ID NO:130: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xil.SEQUENCE DESCRIPTION: SEQ ID NO:130: CCGGATCCGG AGCAACCGTT

CCGGCTC

27 INFORMATION FOR SEQ ID NO:131: SEQUENCE

CHARACTERISTICS:

LENGTH: 27 base pairs TYPE: nucleic acid.

STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: CCGGATCCCG GCTATCAGTC

CGGACGG

27 INFORMATION FOR SEQ ID NO:132:

GAGCAAC

GCCC-ACG

120

CTGCTGG

180

GTGGGAG,

240

GTCTGCG

300

ATCGCCAC

J 60 GCG GTGG; 420

CAGGAACI

480

CATGCGGG

540

CGTGGTGC

600

GACCGGGA

660

CGACGTGA

720

GCGGGAAC(

780 CCGTGACG2 840

CGTG

844 SEQUENCE

CHARACTERISTICS:

LENGTH: 844 base pairs TYPE: nucleic acid STRANDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: Genoric

DNA

'xi) SEQUENCE DESCRIPTION: SEQ ID NO:132: .CGT TCCGGCTCGG CGACTGGATC ACCGTCCCA

CCGCGGCGGG

GCC GC-G'T'GGTGGA AGTCAACTGG CGTGCAACAC

ATATCGACAC

TAA TGCCCAACGC CGAACTCGCC GGCGCGTCGT

TCACCAATTA

A\GC ACCGGCTGAC CGTCGJTCACC ACCTTCAACG

CCGCGGACAC

%.GA TGCTGTCGTC GGTCGCGGCG TCGCT-QCCCG

AACTGCGCAC

GC TCTATCTCG-G TGCGGCCG. TACGAGAAGT

CGATCCCGTT

~CG ACTCGGTCAG GAGCACGTAC CTGCGATGGG

.TCTGGTACGC

'TC GCCTAACGGC GTCGCCGACG A7TTCGACACG

CCGGAACGGA

CT GTGGCGTCCA CACTGCGCTT GGCAGACGAC

GAACAGCAGG

GT CTGGTCCGTT ACGGCAACGG GGAACGCCTC

CAGCAGCCGG

TG AGGTTCATCG TAGACGGCAG GGTGAGTCTG TCCGTGATCG TC CCGGCGCGGG TGCTCGAGCG TGGCGJACTTC CTGGGGCAGA

I

CG GTACTGGCGA CCGCGCACGC GCTGGAGGAA GTCACCGTGC

TI

kG ATCGAGCGCC TGGTGCACCG AAAGCCGATC CTGCTGCACG

T

INFORMATION FOR SEQ ID NO:133: EQUENCE

CHARACTERISTICS:

LENGTH: 742 base pairs TYPE: nucleic ac-id STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION:. SEQ ID NO:133: GGCTATCAGT CCGGACGGTC CTCGCTGCGC GCATCGGTGT

TCGACCGCCT

CGCGAGTCGC *AGTCGCGCGG GTTGGAGAAT CAGTTCGCGG

ACCTGAAGA

120 ATTTACTCGC GCGGCAGCAC TGCCACGGAG GCGATCGGCG

CGTTCAGCGA

180 CAGCTCGGCG ATGCGACGAT CAATACCGGG CAGGCGGCGT

CATTGCGCCG

240 CGGACGTTCG -CCAACACCAC CCTCGACGAC AGCGGWACC

GCGTCGACGT

300 ATCCCGWAT CCAACCCCCA GCGCTATCTG CAGGCGCTCT

ATACCCCGCC

360

CA

CT

CG

TT.

CC'

GT'

CCGGCCGTCC

CGGCGGCAAC

CAGCCGGCCC

CC CC G AT G r-

CGACGGACAG

GCACACACCC

CGCGCGCCGG

TCCCCTCGGC

A.GATCGCC-A

3TCAGGTACC

TCAGGACGG

.CACGCTGAC

GGAGATGGC

'GATCGGGGC

CCGACATC

CGATGGTG

GTTTCCGT

ACTACGAC

GCGCGCTC

rTCAGAAC

TGGGAGAAGG

420

GCCAGATTCA

480'

CTGCTCGACC

540

AACATCGTCA

600

GCGTCCAACT

660

GAACCGACCG

720

GCGGTCCAGT

742

CGATCGCGTT

ACGAGTTCTT

TCGAGGGCAA

ACGGCCCCTA

CGATCGACTA

CCTGGTTCCT

TCCCCGGAAT

CGACGACGCG

CCGCGAGATC

CGTGGTGT1AC

TCGCAACCGG

TGTCGGTGTC

GTCCCCGGTC

TC

CGCGACGGCA

GTGCACC-GCT

TCCGC!CTACA

GAACTGTCGG

ACCGACTTCG

GGGTT GAAGG

GCGCCTGGTC

TCAACTTCGA.

AGGGGCCGGA

AAGCCTACGA

CCTGGTACCT-

ACCGAGTCGA

GGCCGCCAAT

GGATCTGATG

TCTCGGGACA

GAAGGCGGTC

GCCTGCCGAG

CGGTGTGATG

INFORMATION FOR SEQ ID NO:134: SEQUENCE

CHARACTERISTICS:

LENGTH: 282 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear

S

*5 S *5 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:134: Giu Gin Pro Phe 1 Gi y Thr Le u Ar g ValI Thr Lys Thr Xaa 145 Ala Gin Arg Asp Pro 225 Thr ValI Pro Arg His Al a Leu Cys Asp Se r T yr 130 Asn Met Giu Leu Gi y 210 Al a Arg Leu Ile Pro Ile Giy Thr Giu Gly Ile 115 Gly Arg I le Gin 195 Arg Arg Glu Glu Leu 275 Ser Asp Ala Val1 Met Gin 100 Pro Arg Val Ala Ala 180 Gin Val1 Val1 Pro Met 260 Leu Arg 5 Al a Thr Ser Val1 Leu 85 Ile Leu Trp Al a Val1 165 Asp Pro Ser Leu Val1 245 Al a His Leu His Gly Phe Thr 70 Ser Al a His Val Asp 150 Al a Val1 Gly Leu Giu 230 Leu Arg Val1 IGiy Gly Gly Thr 55 Thr Ser Thr Thr T rp 135 Xaa Set Val1 Gin Ser 215 Arg Ala Asp Ile Asp Arg As n 40 As n Phe ValI Leu Pro 120 Tyr Phe Thr Arg Val1 200 ValI Gly I'hr Glu Gly 280 Trp Val1 25 Leu Tyr Asn Al a Tyr 105 Al a Al a Asp Leu Leu 185 Pro Ile Asp Al a Ile 265 Ala li e 10 ValI Leu Se r Al a Al, a 90 Leu Va I Al a Thr Arg 170 Val1 Thr Asp P-he His 250 Giu Val1 Thr Giu Val1 Arg Ala 75 Ser Giy Asp Arg Pro 155 Le u Arg Giy Gin Leu 235 Al a Arg *Val Val Met Pro Asp Leu Al a Asp Arg 140 Giu Al a Tyr Met Asp 220 Gly Leu Le u P rc As n Pro Val1 Thr Pro Ala Ser 125 Gin Arg Asp Gly Arg 205 Gly Gin Giu Val1 Thr T rp As n Gly Pro Giu Giu 110 Val1 Giu Ile Asp As n 190 Phe Asp Thr Glu His 270 Al a Arq Al a Giu Asp Leu T yr Arg Leu Al a Glu 175 Gi y Ile ValI Thr Vali 255 Arg Al a Ala Giu His Asp Arg Giu.

Ser Ar g Ser 160 Gin Glu Val Ile Leu 240 Thr Lys INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 247 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCR:?TION: SEQ ID NO:135: Gly Tyr Gin Ser Gly Arg Ser Ser Leu Arg Ala Ser Val Phe Asp Arg 1 5 10 Leu Thr Asp lie Arg Glu Ser Gin Ser Arg Gly Leu Glu Asn Gin Phe 20 25 30 Ala Asp Leu Lys Asn Ser Met Val Ile Tyr Ser Arg Gly Ser Thr Ala 40 45 Thr Gu Ala Ile Gly Ala Phe Ser Asp Gly Phe Arg Gin Leu Gly Asp 55 60 Ala Thr Ile Asn Thr Gly Gin Ala Ala Ser Leu Arg Arg Tyr Tyr Asp 65 70 75 Arg Thr Phe Ala Asn Thr Thr Leu Asp Asp SeGly Asn Arg Val Asp 90 Val Arg Ala Leu Ile Pro Lys Ser Asn Pro Gin Arg Tyr Leu Gin Ala 100 105 Leu Tyr Thr Pro Pro Phe GIn Asn Trp Glu Lys Ala Ile Ala Phe Asp 115 120 125 Asp Ala Arg Asp Gly Ser Ala Trp Ser Ala Ala Asn Ala Arg Phe Asn ArgP A s Gl y Phe V HA 130 135 140 Glu Phe Phe Arg Glu Ile Val His Arg Phe Asn Phe Glu Asp Leu Met 145 150 155 160 Leu Leu Asp Leu Glu Gly Asn Val Val Tyr Ser Ala Tyr Lys Gly Pro 165 170 175 Asp Leu Gly Thr Asn Ile Val Asn Gly Pro Tr Arg An Arg Gu Lu 180 185 o r g Asn Arg Glu Le Ser Glu Ala Tyr Glu Lys Ala Val Ala Ser Asn Ser Ile Asp Tyr Val 195 200 205 Gly Val Thr Asp Phe Gly Trp Tyr Leu Pro Ala Glu Glu Pro Thr Ala 210 215 220 Pey Leu Pro Gly Lys Asp Arg Val Asp Gly Val Met 225 230 235 240 Ala Val Gin Phe Pro Gly Ile 2 3 5 245 INFORMATION FOR SEQ ID NO:136:

SEQUENCE.CHARACTERISTICS:

LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:136: ATGAGCGAAA TCGCCCGNCC CTGGCGGGTT CTGGCATGTG

GCATC

INFORMATION FOR SEQ ID NO:137: SEQUENCE

CHARACTERISTICS:

LENGTH: 340 base pairs TYPE: nucleic acid STRANDEDNESS: single

GCCACC

CCCGCG,

120 AG TiT 180

CTCAAC

240

GACGCG

300

CACGTG(

340 TOPOLOGY: linear (iMOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137: GGCG GCGCCGCCGC GGTGCCCGCC GGGGTGACCS

CCC

ATGC CCGCCCGCCC GGTGTCCACG ATCGCGCCGG

CGA

-CGCG AAGGG CGTCACGATG GAGCCCGCAG7

CCA

k.TCG TGCTGCCGAA GCCGCGGGGC TGGGAGCACA

TCC(

rTCG CGGTGC-TGGC CGACCGGGTC AGNGGTAAAG

GTCJ

;TGG TCGACAAACA CGTAGGCGAG

TTLCGACGGCA

GGCGGT CGCGCCGGCC- CCTCGGG

CACGCTCAGC

GCCGCGA

CTTCCGCGCC

CGGACCC GAACGTGCCG kGNAGT C GACAAACGCC- 0* INFORMATION FOR SEQ ID NO: 138: SEQUENCE CHARACTERISTICS: LENGTH: 235 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic

DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:138: GGTGACCACC AGCGTNGAAC AGGTCGTTGC CGAAGCCGCG GAGGCCACCG

ACGCGATTGT

CAACGGCTTC AAGGTCAGCG TTCCGGGTCC GGGTCCGGCC GCACCGCCAC

CTGCACCCGG

120 TGCCCCCGGT GTCCCGCCCG CCCCCGGCGC CCCGGCGCTG CCGCTGGCCG

TCGCACCACC

180 CCCGGCTCCC GCTGTTCCCG CCGTGG;CGCC CGCGCCACAG CTGCTGGGAC

TGCAG

235 INFORMATION FOR SEQ ID NO:139: SEQUENCE

CHARACTERISTICS:

(LI LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139: Met Ser Glu Ile Ala Arg Pro Trp Arg-Val Leu Ala* Cys Gly Ile- 1 5 10 INFORMATION FOR SEQ ID NO:140: SEQUENCE

CHARACTERISTICS:

LENGTH: 113 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear.

(ii) MOLECULE TYPE: protein (xi) SEQUENCE Ala Thr Gly Glv Ala 1 5 Val Ala Pro Ala Pro Pro Ala Thr Ser Gly Thr Met Glu Pro Gin Leu Pro Lys Pro Arg Asp Ala Phe Ala Val Ser Thr Asn Ala His 100 Gly DESCRIPTION: SEQ ID NO:140: Ala Ala Val Pro Ala Gly Val 10 Ala Met Pro Ala Arg Pro Val 25 Thr Leu Ser Glu Phe Phe Ala 40 Ser Ser Arg Asp Phe Arg Ala 55 60 Gly Trp Glu His Ile Pro Asp 70 75 Leu Ala Asp Arg Val Gly Gly 90 Val Val Val Asp Lys His Val 105 Ser Ala Ser Thr Ala Lvs 45 Leu Asn Pro Asn Lys Gly Gly Glu 110 Pro Ala lie Ala Gly Val Ile Val Val Pro Gin Xaa Phe Asp

S.

*5 S S

**SS

S

S

S

INFORMATION FOR SEQ ID NO:141: SEQUENCE

CHARACTERISTICS:

LENGTH: 73 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141: Val Thr Thr Ser Val Glu Gin Val Val Ala Ala Ala Asp Ala Thr Glu 1 5 10 Ala Ile Val Asn Gly Phe Lys Val Ser Val Pro Gly Pro Gly Pro Ala Ala 20 25 Ala Pro Pro Pro Ala Pro Gly Ala Pro Gly Val Pro Pro Ala Pro Gly 35 40 45 Ala Pro Ala Leu Pro Leu Ala Val Ala Pro Pro Pro Ala Pro Ala Val 55 60 Pro Ala Val Ala Pro Ala Pro Gln Leu 130

Claims (4)

1. cells. A composition comprising delipidated and deglycolipidated M. vaccae 0 0 0

2. A method for enhancing an immune response, comprising administering a composition according to claim 1.

3. A vaccine comprising delipidated and deglycolipidated M. vaccae cells.

4. A method for inducing protective immunity in a patient, comprising administering to a patient a vaccine according to claim 3. Dated this 20 day of Septeniber 2001 GENESIS RESEARCH DEVELOPMENT CORPORATION LIMITED Patent Attorneys for the Applicant PETER MAXWELL ASSOCIATES