Compounds for treatment of infectious and immune system disorders and methods for their use Number:7,041,295 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides polypeptides comprising an immunogenic epitope of a M. vaccae protein, polynucleotides encoding such polypeptides, and fusion proteins comprising at least one such polypeptide, together with genetic constructs comprising at least one inventive polynucleotide. Compositions comprising such polypeptides, polynucleotides, fusion proteins and/or genetic constructs may be employed in the treatment of infectious diseases and immune disorders.<H infectious, disorders, Compounds, for, t, 7041295.html, Patent, pto, 7041295

Title: Compounds for treatment of infectious and immune system disorders and methods for their use

Abstract: The present invention provides polypeptides comprising an immunogenic epitope of a M. vaccae protein, polynucleotides encoding such polypeptides, and fusion proteins comprising at least one such polypeptide, together with genetic constructs comprising at least one inventive polynucleotide. Compositions comprising such polypeptides, polynucleotides, fusion proteins and/or genetic constructs may be employed in the treatment of infectious diseases and immune disorders.

Patent Number: 7,041,295 Issued on 05/09/2006 to Delcayre

Inventors: Delcayre; Alain (San Jose, CA)
Assignee: Genesis Research & Development Corporation LTD (Auckland, NZ)

Appl. No.: 100679

Filed: March 14, 2002

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09450072	Nov., 1999	6358734
09351348	Jul., 1999	6436898

Current U.S. Class: 424/184.1 ; 424/190.1; 424/9.34; 514/2; 530/300; 530/388.4

Current International Class: A01N 37/18 (20060101); A61B 5/055 (20060101); A61K 38/00 (20060101); A61K 39/00 (20060101); C07K 16/00 (20060101)

Field of Search: 514/2 530/300,388.4,350 424/9.34,184.1,190.1

References Cited [Referenced By]

U.S. Patent Documents


6410720	June 2002	Tan et al.
6436898	August 2002	Delcayre
2004/0072224	April 2004	Delcayre

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Primary Examiner: Nguyen; Dave Trong
Assistant Examiner: Marvich; Maria
Attorney, Agent or Firm: Sleath; Janet Speckman Law Group PLLC

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 09/450,072 filed Nov. 29, 1999, now U.S. Pat. No. 6,358,734, which is a continuation-in-part of U.S. patent application Ser. No. 09/351,348 filed Jul. 12, 1999, and claims priority to PCT International Patent Application PCT/NZ00/00121 filed Jul. 10, 2000.

Claims

I claim:

1. An isolated polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 71, wherein the polypeptide possesses at least one property selected from the group consisting of: (i) an ability to stimulate proliferation of T cells from individuals exposed to Mycobacterium tuberculosis, and (ii) an ability to stimulate interferon-gamma secretion from T cells from individuals exposed to Mycobacterium tuberculosis.

2. A composition comprising at least one polypeptide according to claim 1, and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants.

3. A fusion protein comprising at least one polypeptide according to claim 1.

4. A composition comprising at least one fusion protein according to claim 3 and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants.

5. The composition of claim 4, wherein the composition further comprises a flt3 ligand.

Description

TECHNICAL FIELD

The present invention relates generally to the detection, treatment and prevention of infectious diseases. In particular, the invention is related to compounds comprising immunogenic epitopes isolated from Mycobacterium vaccae, and the use of such compounds in vaccination or immunotherapy against infectious disease, including mycobacterial infections such as infection with Mycobacterium tuberculosis or Mycobacterium avium, and in certain treatments for immune disorders and cancer.

BACKGROUND OF THE INVENTION

The present invention relates generally to the treatment and prevention of infectious diseases, and to the treatment of certain immune disorders and cancers. In particular, the invention is related to compounds and methods for the treatment and prevention of mycobacterial infections, including infection with Mycobacterium tuberculosis or Mycobacterium avium.

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

Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behaviour is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistant 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 (BCG), an avirulent strain of Mycobacterium bovis. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States of America, do not vaccinate the general public. Diagnosis of M. tuberculosis infection is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48 72 hours after injection, thereby indicating exposure to mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.

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

Several patents and other publications disclose treatment of various conditions by administering mycobacteria, including M. vaccae, or certain mycobacterial fractions. International Patent Publication WO 91/02542 discloses treatment of chronic inflammatory disorders in which a patient demonstrates an abnormally high release of IL-6 and/or TNF or in which the patient's IgG shows an abnormally high proportion of agalactosyl IgG. Among the disorders mentioned in this publication are psoriasis, rheumatoid arthritis, mycobacterial disease, Crohn's disease, primary biliary cirrhosis, sarcoidosis, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, Guillain-Barre syndrome, primary diabetes mellitus, and some aspects of graft rejection. The therapeutic agent preferably comprises autoclaved M. vaccae administered by injection in a single dose.

U.S. Pat. No. 4,716,038 discloses diagnosis of, vaccination against and treatment of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae. U.S. Pat. No. 4,724,144 discloses an immunotherapeutic agent comprising antigenic material derived from M. vaccae for treatment of mycobacterial diseases, especially tuberculosis and leprosy, and as an adjuvant to chemotherapy. International Patent Publication WO 91/01751 discloses the use of antigenic and/or immunoregulatory material from M. vaccae as an immunoprophylactic to delay and/or prevent the onset of AIDS. International Patent Publication WO 94/06466 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for therapy of HIV infection, with or without AIDS and with or without associated tuberculosis.

Traditional vaccines contain the disease-causing organism (or a component thereof) in either attenuated or killed form. As an alternative approach to traditional vaccines, DNA vaccines have been developed for diseases as diverse as AIDS, influenza, cancer and malaria. Clinical trials of DNA vaccines are in progress for a number of these diseases. A typical DNA vaccine consists of DNA encoding an antigen cloned in a non-active plasmid carrier. Expression of the antigen encoded by the vaccine DNA is usually under control of a strong promoter, such as human .beta.-actin, Rous sarcoma virus (RSV) or CMV promoter (Ramsay A J, et al. Immunology and Cell Biology 75:360 363, 1997). The first experimental evidence that DNA vaccines were able to induce the desired immune response was produced by Tang et al. (Tang D-C, et al. Nature 356:152 154, 1992). In these experiments, mice inoculated with plasmids containing the gene encoding for human growth hormone developed specific primary antibody responses.

Immune responses to two DNA vaccines containing genes from M. tuberculosis have been evaluated in animal models. The first vaccine contained the gene coding for the GroEL stress protein (65 kDa protein; Tascon R E, et al. Nature Med. 2:888 892, 1996). Mice injected with this DNA vaccine were protected at a level equivalent to mice receiving the traditional BCG vaccine. The second DNA vaccine against M. tuberculosis contained DNA encoding an antigen from the antigen 85 complex and similar results to the study by Tang et al. were obtained (Huygen K, et al. Nature Med. 2:893 898, 1996). U.S. Pat. No. 5,736,524 discloses vaccination of domestic mammals or livestock against infection by M. tuberculosis or M. bovis by administering a polynucleotide tuberculosis vaccine comprising the M. tuberculosis antigen 85 gene operably linked to transcription regulatory elements.

The first human DNA vaccine trial was reported recently (Wang R, et al. Science 282:476 80, 1998). In this trial, an antigen from Plasmodium falciparum, the causative agent of malaria, was injected into healthy volunteers. The desired immune response was elicited, as demonstrated by the presence of cytotoxic T (CD8.sup.+) lymphocytes (CTL), suggesting that the immune system would be able to clear parasites from infected patients. Safety and immunogenicity of a human DNA vaccine against HIV-1 infection was determined in a trial performed by McGregor et al. (J. Infect. Dis. 178:92 100, 1998). Experimental data from other DNA vaccine experiments has also suggested that antibodies, MHC class 1-restricted CD8.sup.+ CTL and class II-restricted CD4.sup.+ helper T cells are produced following injection with DNA vaccines (Ramsay, A J et al. Immunology and Cell Biology 75:360 363, 1997).

DNA vaccines have distinct advantages over more traditional vaccines containing killed or attenuated organisms. DNA vaccination induces immune responses that are long-lived and therefore only a single inoculation may be required. DNA encoding a number of antigens may be incorporated into a single plasmid thereby providing protection against a number of diseases. The technology for DNA vaccine production is relatively simple and the same technology can be used to produce all vaccines, with a resulting cheaper production cost. Delivery of efficacious traditional vaccines to the patient are dependent on maintaining an unbroken "cold chain" from manufacturer to clinic. DNA vaccines produced in solution or in dried form are not sensitive to storage conditions.

Recently, alternative ways of constructing and applying DNA vaccines have been developed. In one of the techniques, called Somatic Transgene Immunization (STI), the plasmid DNA carrying an immunoglobulin heavy chain gene under the control of tissue-specific regulatory elements was inoculated directly into the spleen of mice, with subsequent expression of the antigen on the surface of B-cells (Xiong S, et al. Proc. Natl. Acad. Sci. USA 94:6352 6357, 1997). These B cells produced antibodies against the expressed antigen, leading to an immune response. Subsequent studies showed that STI induced persistent immunologic memory for up to 2 years (Gerloni M, et al. Vaccine (2 3):293 297, 1998).

Expression Library Immunization (ELI) is another technique employing DNA vaccines (Barry M A, et al. Nature 377:632 635, 1995). In this technique, fragments of the complete genome of a pathogen are cloned into a vector and used as vaccine. Selection of protective antigen(s), particularly those inducing CTL, is done by screening and re-screening pools of clones until single clones can be identified. The polynucleotide or polypeptide identified may then be incorporated into a proven delivery system.

Progress on the development of an epitope-based vaccine for the treatment and prevention of HIV infection by scientists at Epimmune Inc. (San Diego, Calif.), has recently been published (Ishioka G Y, et al., Journal of Immunology 162:3915 3925, 1999).

There remains a need in the art for effective compounds and methods for preventing and treating infectious disorders, such as tuberculosis and other mycobacterial infections in humans and in domestic mammals or livestock, and for the treatment of certain immune system-related disorders.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compounds and methods for the prevention and treatment of infectious diseases, such as mycobacterial infections, and for the treatment of immune disorders and cancers.

In a first aspect, isolated polynucleotides are provided that are derived from the M. vaccae genome. These polynucleotides encode polypeptide epitopes selected on the basis of their immunogenic properties as illustrated by results from a number of immunological assays. In specific embodiments, the inventive polynucleotides comprise a sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 8 21; (b) sequences having at least 75%, 90% or 95% identical residues to a sequence of SEQ ID NO: 8 21 as determined using the computer algorithm BLASTN; and (c) complements of the sequences of (a) and (b).

In a second aspect, the invention provides isolated polypeptides comprising an immunogenic epitope of a M. vaccae antigen. In specific embodiments, the inventive polypeptides comprise a sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 64 77; and (b) sequences having at least 75%, 90% or 95% identical residues to a sequence of SEQ ID NO: 64 77 as determined using the computer algorithm FASTX.

Genetic constructs comprising at least one of the inventive polynucleotides, and host cells transformed or transfected with such genetic constructs are also provided.

In another aspect, the present invention provides fusion proteins comprising at least one polypeptide of the present invention. In specific embodiments, such fusion proteins comprise a sequence of SEQ ID NO: 79 81 or 90 97. Polynucleotides encoding such fusion proteins are also provided. In certain embodiments, such polynucleotides comprise a sequence of SEQ ID NO: 56 58 or 82 89.

Within other aspects, the present invention provides compositions that comprise at least one of the inventive polypeptides, polynucleotides, fusion proteins or genetic constructs, and a physiologically acceptable carrier. The invention also provides compositions comprising at least one of the above polypeptides, polynucleotides, fusion proteins or genetic constructs and an immunostimulant.

In yet another aspect, methods are provided for enhancing an immune response in a patient, comprising administering to a patient an effective amount of one or more of the above compositions. In one embodiment, the immune response is a Th1 response.

In further aspects of this invention, methods are provided for the treatment of a disorder in a patient, comprising administering to the patient a composition of the present invention. In certain embodiments, the disorder is selected from the group consisting of immune disorders, infectious diseases and cancer.

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

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A F illustrate the induction of protective immunity, measured as a decrease in M. tuberculosis CFU in lung and spleen homogenates of BALB/cByJ mice, by vaccination with M. bovis BCG (FIGS. 1A and D, respectively), with ME/D DNA (FIGS. 1B and E, respectively), or with rME/D (FIGS. 1C and F, respectively).

FIGS. 2A D show the proliferative responses of lymph node cells from BALB/cByJ mice immunized subcutaneously with rME/A (FIG. 2A), rME/B (FIG. 2B) or rME/D (FIG. 2C). Control mice were immunized with PBS (FIG. 2D).

FIGS. 3A D illustrate IFN-.gamma. secretion by lymph node cells from BALB/cByJ mice immunized subcutaneously with recombinant multi-epitope constructs rME/A (FIG. 3A), rME/B (FIG. 3B) or rME/D (FIG. 3C). Control mice were immunized with PBS (FIG. 3D).

FIG. 4A demonstrates the proliferative responses of lymph node cells from BALB/cByJ mice immunized with rME/A, rME/D or ME/D DNA by three different routes of immunization. The proliferative response of lymph node cells from control mice immunized with PBS is shown in FIG. 4B.

FIG. 5A demonstrates the level of IFN-.gamma. secretion by lymph node cells from BALB/cByJ mice immunized with rME/A, rME/D or ME/D DNA by three different routes of immunization. The level of IFN-.gamma. secretion by control mice immunized with PBS is shown in FIG. 5B.

FIG. 6A show the contribution of single epitopes to the proliferative responses of lymph node cells from BALB/cByJ mice immunized with rME/A, rME/D or ME/D DNA by three different routes of immunization. The proliferative response of lymph node cells from control mice immunized with PBS is shown in FIG. 6B.

FIG. 7A demonstrates contribution of single epitopes to the level of IFN-.gamma. secretion by lymph node cells from BALB/cByJ mice immunized with rME/A, rME/D or ME/D DNA by three different routes of immunization. The level of IFN-.gamma. secretion by control mice immunized with PBS is shown in FIG. 7B.

FIGS. 8A and B illustrate the titre and subclass of anti-ME antibodies in the serum of mice immunized with ME/D DNA that reacted with rME/A and rME/D in vitro. The titres of IgG1 antibodies are shown in FIG. 8A, with the titre of IgG2a antibodies being shown in FIG. 8B.

FIGS. 9A C show the IFN-.gamma. secretion by memory splenocytes from BALB/cByJ mice immunized with recombinant single epitopes (FIG. 9B) or rME/D (FIG. 9C). IFN-.gamma. secretion by splenocytes after stimulation with controls is shown in FIG. 9A.

FIGS. 10A and B demonstrate the IFN-.gamma. secretion (FIG. 10A) and proliferative response (FIG. 10B) by human PBMC after stimulation in vitro with rME/A, rME/B or rME/D.

FIGS. 11A and B demonstrate the IFN-.gamma. secretion (FIG. 11A) and proliferative response (FIG. 11B) by human PBMC after stimulation in vitro with eight recombinant single epitopes.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to compositions and methods for preventing and treating disorders including infectious diseases and certain immune disorders and cancers. Examples of such disorders include, but are not limited to, mycobacterial infections, including M. tuberculosis and M. avium infections, and disorders in which the stimulation of a Th1 immune response is beneficial, including (but not limited to) psoriasis and allergic rhinitis.

Certain pathogens, such as M. tuberculosis, as well as certain cancers, are effectively contained by an immune attack directed by CD4.sup.+ T cells, known as cell-mediated immunity. Other pathogens, such as poliovirus, also require antibodies, produced by B cells, for containment. These different classes of immune attack (T cell or B cell) are controlled by different subpopulations of CD4.sup.+ T cells, commonly referred to as Th1 and Th2 cells.

The two types of Th cell subsets have been well characterized in a murine model and are defined by the cytokines they release upon activation. The Th1 subset secretes IL-2, IFN-.gamma. and tumor necrosis factor, and mediates macrophage activation and delayed-type hypersensitivity response. The Th2 subset releases IL-4, IL-5, IL-6 and IL-10, which stimulate B cell activation. The Th1 and Th2 subsets are mutually inhibiting, so that IL-4 inhibits Th1-type responses, and IFN-.gamma. inhibits Th2-type responses. Similar Th1 and Th2 subsets have been found in humans, with release of the identical cytokines observed in the murine model. Amplification of Th1-type immune responses is central to a reversal of disease state in many disorders, including disorders of the respiratory system such as tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancers.

In one aspect, the compositions of the present invention include polypeptides that comprise at least one immunogenic epitope of a M. vaccae antigen, or a variant thereof. In specific embodiments, the inventive polypeptides comprise a sequence provided in SEQ ID NO: 61 77. Such polypeptides stimulate T cell proliferation, and/or interferon gamma secretion from T cells of individuals exposed to M. tuberculosis.

As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising an immunogenic epitope of one of the above antigens may consist entirely of the immunogenic epitope, or may contain additional sequences. The additional sequences may be derived from the native M. vaccae antigen or may be heterologous, and such sequences may (but need not) be immunogenic.

"Immunogenic," as used herein, refers to the ability to elicit an immune response in a patient, such as a human, or in a biological sample. In particular, an immunogenic epitope is that portion of a polypeptide that is capable of stimulating cell proliferation, interleukin-12 production or interferon-.gamma. production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from a mycobacteria-immune individual. In general, an immunogenic epitope will stimulate proliferation of PBMC from mycobacteria-immune individuals at levels at least two-fold greater than that observed in control PBMC, determined using assay techniques detailed below in Example 1. Alternatively, or additionally, an immunogenic epitope will stimulate the production of interferon-.gamma. in PBMC from mycobacteria-immune individuals at levels that are at least two-fold greater than those observed in control cells as determined by at least a two-fold increase in OD in an ELISA assay as detailed in Example 1. A mycobacteria-immune individual is one who is considered to be resistant to the development of mycobacterial infection by virtue of having mounted an effective T cell response to M. tuberculosis, to environmental saprophytes, or to BCG. Such individuals may be identified based on a strongly positive (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD), and an absence of any symptoms of tuberculosis infection. Polypeptides comprising at least an immunogenic epitope of one or more M. vaccae antigens may generally be used to induce protective immunity against tuberculosis in a patient and/or to stimulate an immune response in a patient.

In another aspect, the compositions of the present invention comprise isolated polynucleotides that encode polypeptides and/or fusion proteins comprising an immunogenic epitope of a M. vaccae antigen. In specific embodiments, the inventive polynucleotides comprise a sequence of SEQ ID NO: 8 21, 56 58 and 82 89. Complements of the inventive isolated polynucleotides, reverse complements of such isolated polynucleotides and reverse sequences of such isolated polynucleotides are also provided, together with variants of such sequences. The present invention also encompasses polynucleotide sequences that differ from the disclosed sequences but which, due to the degeneracy of the genetic code, encode a polypeptide which is the same as that encoded by a polynucleotide sequence disclosed herein.

The term "polynucleotide(s)," as used herein, means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides. An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner. An mRNA molecule corresponds to an HnRNA and/or DNA molecule from which the introns have been excised. A polynucleotide may consist of an entire gene, or any portion thereof. Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of "polynucleotide" therefore includes all such operable anti-sense fragments. Anti-sense polynucleotides and techniques involving anti-sense polynucleotides are well known in the art and are described, for example, in Robinson-Benion et al. "Antisense techniques," Methods in Enzymol. 254:363 375, 1995; and Kawasaki et al. Artific. Organs 20:836 848, 1996.

The definition of the terms "complement", "reverse complement" and "reverse sequence", as used herein, is best illustrated by the following example. For the sequence 5' AGGACC 3', the complement, reverse complement and reverse sequence are as follows:

TABLE-US-00001 complement 3' TCCTGG 5' reverse complement 3' GGTCCT 5' reverse sequence 5' CCAGGA 3'.

Preferably, sequences that are complements of a specifically recited polynucleotide sequence are complementary over the entire length of the specific polynucleotide sequence.

All the polynucleotides and polypeptides provided by the present invention are isolated and purified, as those terms are commonly used in the art. Preferably, the inventive polypeptides and polynucleotides are at least about 80% pure, more preferably at least about 90% pure, and most preferably at least about 99% pure.

The compositions and methods of this invention also encompass variants of the above polypeptides and polynucleotides. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. As used herein, the term "variant" comprehends nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 50%, more preferably at least 75%, more preferably yet at least 90%, and most preferably at least 95% identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100. By way of example only, assume a queried polynucleotide having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the default parameters as described below. The 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the queried polynucleotide to the hit in the EMBL database is thus 21/220 times 100, or 9.5%. The percentage identity of polypeptide sequences may be determined in a similar fashion.

Polynucleotide and polypeptide sequences may be aligned, and percentages of identical residues in a specified region may be determined against another polynucleotide or polypeptide sequence, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. Polynucleotides may also be analyzed using the BLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The percentage identity of polypeptide sequences may be examined using the BLASTP algorithm. The BLASTN, BLASTP and BLASTX algorithms are available on the NCBI anonymous FTP server under /blast/executables/ and are available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894, USA. The BLASTN algorithm Version 2.0.11 [Jan. 20, 2000], set to the parameters described below, is preferred for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm, set to the parameters described below, is preferred for use in the determination of polypeptide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP and BLASTX, is described in the publication of Altschul, et al., Nucleic Acids Res. 25: 3389 3402, 1997.

The FASTA and FASTX algorithms are available on the Internet, and from the University of Virginia by contacting the Vice Provost for Research, University of Virginia, P.O. Box 9025, Charlottesville, Va. 22906 9025, USA. The FASTA algorithm, set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of polynucleotide variants. The readme files for FASTA and FASTX Version 1.0x that are distributed with the algorithms describe the use of the algorithms and describe the default parameters. The use of the FASTA and FASTX algorithms is described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 2448, 1988; and Pearson, Methods in Enzymol. 183:63 98, 1990.

The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotides: Unix running command with the following default parameters: blastall -p blastn -d embldb -e 10 -G O-E O -r 1 -v 30 -b 30 -i queryseq results; and parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File In]; -o BLAST report Output File [File Out] Optional.

The following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprotdb -e 10 -G O-E O -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional.

The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence. The BLASTN, FASTA and BLASTP algorithms also produce "Expect" values for polynucleotide and polypeptide alignments. The Expect value (E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and matched portions of the sequences then have a probability of 90% of being related. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN algorithm. E values for polypeptide sequences may be determined in a similar fashion using various polypeptide databases, such as the SwissProt database.

According to one embodiment, "variant" polynucleotides and polypeptides, with reference to each of the polynucleotides and polypeptides of the present invention, preferably comprise sequences having the same number or fewer nucleic or amino acids than each of the polynucleotides or polypeptides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA or BLASTP algorithms set at the default parameters. According to a preferred embodiment, a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN algorithm set at the default parameters. Similarly, according to a preferred embodiment, a variant polypeptide is a sequence having the same number or fewer amino acids than a polypeptide of the present invention that has at least a 99% probability of being the same as the polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the default parameters.

In addition to having a specified percentage identity to an inventive polynucleotide or polypeptide sequence, variant polynucleotides and polypeptides preferably have additional structure and/or functional features in common with the inventive polynucleotide or polypeptide. Polypeptides having a specified degree of identity to a polypeptide of the present invention share a high degree of similarity in their primary structure and have substantially similar functional properties. In addition to sharing a high degree of similarity in their primary structure to polynucleotides of the present invention, polynucleotides having a specified degree of identity to, or capable of hybridizing to, an inventive polynucleotide preferably have at least one of the following features: (i) they contain an open reading frame or partial open reading frame encoding a polypeptide having substantially the same functional properties as the polypeptide encoded by the inventive polynucleotide; or (ii) they contain identifiable domains in common.

In certain embodiments, variant polynucleotides hybridize to a polynucleotide of the present invention under stringent conditions. As used herein, "stringent conditions" refers to prewashing in a solution of 6.times.SSC, 0.2% SDS; hybridizing at 65.degree. C., 6.times.SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1.times.SSC, 0.1% SDS at 65.degree. C. and two washes of 30 minutes each in 0.2.times.SSC, 0.1% SDS at 65.degree. C.

The present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the discrepancy of the genetic code, encode a polypeptide having similar enzymatic activity as a polypeptide encoded by a polynucleotide of the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences disclosed herein (or complements, reverse sequences, or reverse complements of those sequences) as a result of conservative substitutions are encompassed within the present invention. Additionally, polynucleotides comprising sequences that differ from the inventive polynucleotide sequences or complements, reverse complements, or reverse sequences as a result of deletions and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention. Similarly, polypeptides comprising sequences that differ from the inventive polypeptide sequences as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by and encompassed within the present invention, provided the variant polypeptide has similar activity to the inventive polypeptide.

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 post-translationally 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 (e.g., 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.

As used herein, the term "x-mer," with reference to a specific value of "x," refers to a polynucleotide comprising at least a specified number ("x") of contiguous residues of any of the polynucleotides identified as SEQ ID NO: 8 21, 56 58 and 82 89. The value of x may be from about 20 to about 600, depending upon the specific sequence.

Polynucleotides of the present invention comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NO: 8 21, 56 58 and 82 89 or their variants. According to preferred embodiments, the value of x is preferably at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80. Thus, polynucleotides of the present invention include polynucleotides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer a 250-mer, or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide identified as SEQ ID NO: 8 21, 56 58 and 82 89 or a variant of one of the polynucleotides identified as SEQ ID NO: 8 21, 56 58 and 82 89.

In general, the inventive polypeptides and polynucleotides, may be prepared using any of a variety of procedures. For example, polypeptides may be produced recombinantly by inserting a polynucleotide that encodes the polypeptide into an expression vector and expressing the polypeptide 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 polynucleotide 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 polynucleotides expressed in this manner may encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.

Polynucleotides of the present invention may be isolated by screening a M. vaccae genomic DNA library as described below in Example 1. Alternatively, polynucleotides encoding M. vaccae epitopes may be obtained by screening an appropriate M. vaccae cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from amino acid sequences of isolated epitopes. Suitable degenerate oligonucleotides may be designed and synthesized, and the screen may be performed as described, for example in Sambrook et al. Molecular cloning: a laboratory manual CSHL Press: Cold Spring Harbor, N.Y., 1989. Polymerase chain reaction (PCR) may be employed to isolate a nucleic acid probe from genomic DNA, or a cDNA or genomic DNA library, using techniques well known in the art. The library screen may then be performed using the isolated probe.

Regardless of the method of preparation, the epitopes described herein have the ability to induce an immunogenic response. More specifically, as discussed above, the epitopes have the ability to induce cell proliferation and/or cytokine production (for example, interferon-.gamma. and/or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from a mycobacteria-immune individual.

The selection of cell type for use in evaluating an immunogenic response to an epitope will depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing T cells, NK cells, B cells and/or macrophages derived from mycobacteria-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 Ficol.TM. (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 mycobacteria-immune individuals with mycobacterial proteins for a period of 2 4 weeks. This allows expansion of only the mycobacterial protein-specific T cells, resulting in a line composed solely of such cells. These cells may then be cloned and tested with individual proteins, using methods well known in the art, to more accurately define individual T cell specificity. Assays for cell proliferation or cytokine production in T cells, NK cells, B cells or macrophages may be performed, for example, using the procedures described below.

Among the immunogenic epitopes, polypeptides and/or polynucleotides of the present invention, those having superior therapeutic properties may be distinguished based on the magnitude of the responses in the above assays and based on the percentage of individuals for which a response is observed. In addition, epitopes having superior therapeutic properties will not stimulate cell proliferation or cytokine production in vitro in cells derived from more than about 25% of individuals that are not mycobacteria-immune, thereby eliminating responses that are not specifically due to mycobacteria-responsive cells. Thus, those antigens that induce a response in a high percentage of T cell, NK cell, B cell or macrophage preparations from mycobacteria-immune individuals (with a low incidence of responses in cell preparations from other individuals) have superior therapeutic properties.

Epitopes 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.

Portions and other variants of the inventive polypeptides may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149 2154, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions. Variants of a native epitope 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.

The 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 kDa antigen described in Andersen and Hansen, Infect. Immun. 57:2481 2488, 1989, together with variants of such fusion proteins. In a related aspect, genetic constructs comprising a first and a second inventive polynucleotide are also provided. Preparation of a construct comprising multiple epitopes of the present invention and expression of the corresponding recombinant protein is detailed below in Example 4.

In general, a polynucleotide encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides into an appropriate expression vector. The 3' end of a DNA sequence encoding the first 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 fold into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the 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 Maratea et al., Gene 40:39 46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262, 1986; and U.S. Pat. Nos. 4,935,233 and 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 polynucleotides encoding such polypeptides or fusion proteins) to induce protective immunity against disorders such as 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 disorders.

In related aspects, the M. vaccae polynucleotides and polypeptides of the present invention may be employed to activate T cells and NK cells; to stimulate the production of cytokines (in particular Th1 class of cytokines) in human PBMC; to produce anti-epitope antibodies; to induce long-term memory cells and/or to enhance an immune response against an antigen.

For use in such methods, the polypeptide, fusion protein or polynucleotide is generally present within a composition, such as a pharmaceutical composition or an immunogenic composition. 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. Immunogenic compositions may comprise one or more of the above polypeptides and an immunostimulant, such as an adjuvant or a liposome, into which the polypeptide is incorporated. Such compositions may also contain other antigens and/or polypeptides, either incorporated into a fusion protein or present within a separate polypeptide. Examples of polypeptides which may be usefully employed in combination with the polypeptides of the present invention include other mycobacterial antigens and flt3 ligands, as disclosed in U.S. Pat. No. 5,554,512, the disclosure of which is hereby incorporated in its entirety.

Alternatively, a composition of the present invention may contain a polynucleotide encoding one or more polypeptides as described above, such that the polypeptide is generated in situ. In such compositions, the polynucleotide 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/RNA 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-Guerin) that expresses an immunogenic epitope of the polypeptide on its cell surface. In a preferred embodiment, the DNA and/or RNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), that may involve the use of a non-pathogenic, or defective, replication competent virus. Techniques for incorporating DNA and/or RNA into such expression systems are well known in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745 1749, 1993 and reviewed by Cohen, Science 259:1691 1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. Methods for the administration of polynucleotide sequences comprising DNA and/or RNA include those disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466.

A polynucleotide composition 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 kDa antigen from M. tuberculosis. 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 effect of the composition.

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

While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

Any of a variety of immunostimulants, such as an adjuvant, may be employed in the compositions of this invention to non-specifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis, M. tuberculosis, or, as discussed below, M. vaccae. Suitable adjuvants are commercially available as, for example, Incomplete Freund's Adjuvant (IFA) and Complete Freund's Adjuvant (Difco Laboratories, Detroit, Mich.), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.

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

EXAMPLE 1

Cloning and Selection of Immunogenic M. Vaccae Epitopes

M. vaccae (ATCC Number 15483, Manassas, Va.) was cultured in medium 90 (yeast extract, 2.5 g/l; tryptone, 5 g/l; glucose, 1 g/l) at 37.degree. C. for four days. Genomic DNA was isolated from these cells following standard protocols and then digested with restriction endonuclease Sau3A under conditions that produced DNA fragments of approximately 0.25 kb. The fragments were purified using the QIAquick PCR clean-up system (Qiagen, Venlo, The Netherlands).

To express the cloned M. vaccae DNA in three different reading frames, the pcDNA3 expression vector (Invitrogen, Carlsbad, Calif.) was modified by insertion of a human growth hormone signal peptide (to facilitate recombinant protein secretion) amplified with three different 3' primers. These primers allowed the insertion of one or two extra base pairs into the PCR product to shift the reading frame of the expressed polypeptide. The primers were AD105 (human growth hormone 5' primer; SEQ ID NO: 1) and the three human growth hormone (hGH) 3' primers AD106, AD107 and AD108 (SEQ ID NO: 2 4, respectively). From these PCR fragments, most of the hGH sequence downstream of the leader sequence cleavage site was removed by digestion with the restriction endonuclease BsgII. The hGH PCR fragments were then cloned into the pcDNA3 expression vector following digestion with the restriction endonucleases HindIII and BamHI. The nucleotide sequence of the inserted fragments are given in SEQ ID NO: 5 7, with the corresponding amino acid sequences being provided in SEQ ID NO: 61 63, respectively. Three expression libraries (one for each of the three reading frames) were constructed by cloning the 0.25 kb M. vaccae PCR fragments, prepared as described above, into the BamHI cloning site of the chimeric pcDNA3/human growth hormone vectors (pcDNA3-hGH1', pcDNA3-hGH2' and pcDNA3-hGH3'). Replica lift master plates were made of bacterial colonies transformed with the library constructs and stored. Plasmid DNA, prepared from these colonies, was divided into 500 pools, each containing DNA from 40 to 50 plasmids. The DNA was transfected into COS7 cells using lipofectamine (BRL Life Technologies, Gaithersburg Md.) and the immunogenic properties of the products of each group were determined by a spleen cell assay, wherein the production of IFN-.gamma. in cultures of spleen cells obtained from mice primed with heat-killed M. vaccae was determined by ELISA as described below.

Plasmid pools that encoded recombinant polypeptides eliciting an immune response (as determined by the ability to increase IFN-.gamma. production in the spleen cell assay), were subdivided into smaller pools containing 10 plasmids each and these pools were again transfected into COS7 cells. The culture supernatants of these cells were subjected to the spleen cell assay as described above.

After three rounds of screening, 120 plasmids were identified that encoded recombinant polypeptides stimulating spleen cells of heat-killed M. vaccae-immunised mice to produce IFN-.gamma.. The 120 supernatants of COS7 cells transfected with these plasmids were screened in two addit