US20140335536A1

US20140335536A1 - Vaccine for the treatment of mycobaterium related disorders

Info

Publication number: US20140335536A1
Application number: US14/288,861
Authority: US
Inventors: Leif Kirsebom; Santanu Dasgupta; Pontus Larsson; Jaydip Ghosh
Original assignee: Marfl AB
Current assignee: Marfl AB
Priority date: 2007-12-17
Filing date: 2014-05-28
Publication date: 2014-11-13
Also published as: EP2231181A1; EP2231181B1; US8779088B2; WO2009078799A1; RU2010129856A; US20110104180A1; RU2559782C2; EP2231181A4

Abstract

The invention relates to genes and proteins involved in the sporulation of Mycobacteria and the use thereof in the technical fields of immunology and medicine.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Stage of International application no. PCT/SE2008/051486, filed Dec. 17, 2008, which claims priority from Swedish application no. 0702802-0, filed Dec. 17, 2007.

TECHNICAL FIELD

The invention relates to genes involved in the sporulation of Mycobacteria in the technical fields of immunology and medicine.

BACKGROUND

The genus Mycobacterium includes highly successful pathogens e.g. Mycobacterium tuberculosis and Mycobacterium leprae, the etiological agents of tuberculosis and leprosy, respectively. The M. tuberculosis (Mtb) is one of the most successful human pathogens and estimations suggest that one third of the human population carry this bacteria. Other Mycobacterium species worth mentioning are Mycobacterium marinum (Mm), Mycobacterium avium and Mycobacterium ulcerans.
Today approximately eight million people per year develop active disease and two million die. Pathogenic mycobacteria have the ability to persist for a long time in the host without causing any symptoms. This latency poses an additional hidden threat to human health but is not clearly understood. Mycobacteria can colonize their hosts without the hosts showing any adverse signs. For example, billions of people around the world are infected with M. tuberculosis but will never know it because they will not develop symptoms.
Mycobacterial infections are notoriously difficult to treat. The organisms are hardy due to their cell wall, which is neither truly Gram negative nor positive, and unique to the family, they are naturally resistant to a number of antibiotics that work by destroying cell walls, such as penicillin. Most mycobacteria are susceptible to antibiotics e.g. clarithromycin, isoniazid and rifamycin, but antibiotic-resistant strains are known to exist, and the emergence of multi resistant strains is an increasing problem.
An attenuated strain of Mycobacterium bovis is currently the most widely used and safest attenuated human vaccine, but controversy concerning its efficacy has emerged.
Consequently, there is a need for improved methods and medicaments to prevent and treat diseases which are caused by Mycobacteria.

SUMMARY OF THE INVENTION

It has now, for the first time, been shown that Mycobacteria have the ability to form spores. This is contrary to text book descriptions of mycobacteria as non-sporulating, gram-positive species. This introduces a new perspective into the life style of pathogenic mycobacteria and its potential for spreading, as well as persistence, of mycobacterial infections. Consequently, the present invention is directed to methods and compounds which are useful to treat and/or prevent diseases which are caused by Mycobacteria, such as tuberculosis. In particular, the invention relates to the treatment and/or prevention of such diseases wherein the Mycobacteria are, or will be, sporulating. Such methods and compounds may be based on active or passive immunization, wherein active immunization is performed by administering a vaccine, and passive immunization is performed by administering an antibody.
In one aspect, the invention provides methods of preventing or treating a disease which is caused by Mycobacteria. Such diseases are e.g. tuberculosis, leprosy and diseases caused by non-tuberculous Mycobacteria (NTM). Also included is Crohns disease and the livestock disease Johne's disease. It is well known that individuals with an impaired immune system, such as AIDS patients, and patients who receive immunosuppressing treatments are sensitive to Mycobacterial infections. Such patients may benefit from treatment according to this invention.
The invention also provides compounds which may be useful for treating the above mentioned diseases, either in the form of vaccines, or in the form of antibodies. Also contemplated are binding molecules other than antibodies.
Methods for treating such diseases entail inducing an immune response against a peptide component from one of the gene products involved in sporulation, in the patient. Such induction can be active by administration of an immunogen (such as a vaccine), or passive by administration of an antibody or an active fragment or derivative of the antibody. In some methods, the patient is asymptomatic. In some methods, treatment is prophylactic.
For treatment of patients suffering from a disease caused by Mycobacteria, one treatment regime entails administering a dose of a peptide derived from a protein component in the spore coating to the patient to induce the immune response. In some methods, the spore coating peptide is administered with an adjuvant that enhances the immune response to the spore coating peptide. Examples of adjuvants are alum and MPL. The dose of spore coating peptide administered to the patient is typically at least 1 or 10 μg, if administered with adjuvant, and at least 50 μg if administered without adjuvant. In some methods, the dose is at least 100 μg.
In some methods, the spore coating peptide is a fragment of the gene products shown in tables 1-4.
In some methods, the therapeutic agent is an effective dose of a nucleic acid encoding a spore coating peptide or an active fragment or derivative thereof. The nucleic acid encoding spore coating or a fragment thereof is expressed in the patient to produce a spore coating peptide or the active fragment thereof, which induces the immune response. In some such methods, the nucleic acid is administered through the skin, optionally via a patch. In some methods, a therapeutic agent is identified by screening a library of compounds to identify a compound reactive with antibodies to the spore coating peptide, and administering the compound to the patient to induce the immune response.
In some methods, the immune response comprises T-cells that bind to spore coating peptide complexed with MHC1 or MHCII on CD8 or CD4 cells. In other methods, the immune response is induced by administering an antibody to the spore coating peptide to the patient. In some methods, the immune response is induced by removing T-cells from the patient, contacting the T-cells with spore coating peptide under conditions in which the T-cells are primed, and replacing the T-cells in the patient.
The invention also encompasses molecules other than antibodies which are capable of binding to the spore coating peptides. Collectively, the molecules, e.g. antibodies and other molecules capable of binding to the spore coating peptides are termed “binding molecules”. Binding molecules of the invention are preferably antibodies, as defined herein. The term “antibody” refers to an intact antibody, or a binding fragment thereof. An antibody may comprise a complete antibody molecule (including polyclonal, monoclonal, chimeric, humanized, or human versions having full length heavy and/or light chains), or comprise an antigen binding fragment thereof. Antibody fragments include F(ab′)₂, Fab, Fab′, Fv, Fc, and Fd fragments, and can be incorporated into single domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (See e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). Antibody polypeptides are also disclosed in U.S. Pat. No. 6,703,199, including fibronectin polypeptide monobodies. Other antibody polypeptides are disclosed in U.S. Patent Publication 2005/0238646, which are single-chain polypeptides.
Antigen binding fragments derived from an antibody can be obtained, for example, by proteolytic hydrolysis of the antibody, for example, pepsin or papain digestion of whole antibodies according to conventional methods. By way of example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment termed F(ab′)₂. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab′ monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al, in Methods in Enzymology 1:422 (Academic Press 1967); and by Andrews, S. M. and Titus, J. A. in Current Protocols in Immunology (Coligan J. E., et al., eds), John Wiley & Sons, New York (2003). pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating heavy chains to form monovalent light-heavy chain fragments (Fd), further cleaving of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
An antibody fragment may also be any synthetic or genetically engineered protein. For example, antibody fragments include isolated fragments consisting of the light chain variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (scFv proteins).
Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also termed “minimal recognition units”, or “hypervariable region”) can be obtained by constructing polynucleotides that encode the CDR of interest. Such polynucleotides may be prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et al, Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al, “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al, (eds.), page 137 (Wiley-Liss, Inc. 1995)).
Thus, in one embodiment, the binding molecule comprises at least one CDR as described herein. The binding molecule may comprise at least two, three, four, five or six CDR's as described herein. The binding molecule further may comprise at least one variable region domain of an antibody described herein. The variable region domain may be of any size or amino acid composition and will generally comprise at least one CDR sequence responsible for binding to a spore forming peptide, for example CDR-H1, CDR-H2, CDR-H3 and/or the light chain CDRs specifically described herein and which is adjacent to or in frame with one or more framework sequences. In general terms, the variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (V_H) and/or light (V_L) chain variable domains Thus, for example, the V region domain may be monomeric and be a V_Hor V_Ldomain, which is capable of independently binding spore forming peptide with an affinity at least equal to 1×10⁻⁷M or less as describedbelow. Alternatively, the V region domain may be dimeric and contain V_H-V_H, V_H-V_L, or V_L-V_L, dimers. The V region dimer comprises at least one V_Hand at least one V_Lchain that may be non-covalently associated (hereinafter referred to as F_V). If desired, the chains may be covalently coupled either directly, for example via a disulfide bond between the two variable domains, or through a linker, for example a peptide linker, to form a single chain Fv (scF_V).
The variable region domain may be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain that has been created using recombinant DNA engineering techniques. Such engineered versions include those created, for example, from a specific antibody variable region by insertions, deletions, or changes in or to the amino acid sequences of the specific antibody. Particular examples include engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.
Other binding molecules which may be used include e.g. those described in WO03/080653, camelids, affibodies, and diabodies.
The variable region domain may be covalently attached at a C-terminal amino acid to at least one other antibody domain or a fragment thereof. Thus, for example, a VH domain that is present in the variable region domain may be linked to an immunoglobulin CH1 domain, or a fragment thereof. Similarly a V_Ldomain may be linked to a C_Kdomain or a fragment thereof. In this way, for example, the antibody may be a Fab fragment wherein the antigen binding domain contains associated V_uand V_Ldomains covalently linked at their C-termini to a CH1 and C_Kdomain, respectively. The CH1 domain may be extended with further amino acids, for example to provide a hinge region or a portion of a hinge region domain as found in a Fab′ fragment, or to provide further domains, such as antibody CH2 and CH3 domains.
As described herein, binding molecules comprise at least one of these CDRs. For example, one or more CDR may be incorporated into known antibody framework regions (IgG1, IgG2, etc.), or conjugated to a suitable vehicle to enhance the half-life thereof. Suitable vehicles include, but are not limited to Fc, polyethylene glycol (PEG), albumin, transferrin, and the like. These and other suitable vehicles are known in the art. Such conjugated CDR peptides may be in monomeric, dimeric, tetrameric, or other form. In one embodiment, one or more water-soluble polymer may be linked to one or more specific positions, for example at the amino terminus, of a binding molecule.
In certain preferred embodiments, a binding molecule comprises one or more water soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337. In certain embodiments, a derivative binding molecule comprises one or more of monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of such polymers. In certain embodiments, one or more water-soluble polymer is randomly attached to one or more side chains. In certain embodiments, PEG can act to improve the therapeutic capacity for a binding molecule, such as an antibody. Certain such methods are discussed, for example, in U.S. Pat. No. 6,133,426, which is hereby incorporated by reference for any purpose.
It will be appreciated that a binding molecule of the present invention may have at least one amino acid substitution, providing that the binding molecule retains binding specificity. Therefore, modifications to the binding molecule structures are encompassed within the scope of the invention. These may include amino acid substitutions, which may be conservative or non-conservative, that do not destroy the spore coating peptides capability of binding to a binding molecule. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. A conservative amino acid substitution may also involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
The binding molecules according to this invention may also be used as a diagnostic tool to detect the presence, or absence, of Mycobacteria in the form of spores in an animal, such as humans or livestock. Detection is preferably performed on a sample from said animal or human subject that is suspected of containing spores derived from Mycobacteria, such as body fluids, e.g. blood, serum, bronchoalveolar lavage fluid, saliva, urine, feaces, tears and the like. Also contemplated is the non-diagnostic detection of spores derived from Mycobacteria in food products, such as milk, cheese, butter, vegetables, and meat, or in products intended as animal feed. The binding agents according to the invention may also be used to detect Mycobacteria in environmental samples from environments suspected of being contaminated with spores derived from Mycobacteria, e.g. fish and bird farms. Methods for such detection are well known and include e.g. ELISA assays and other immunological assays.
The therapeutic agent, or binding molecule, can typically be administered orally, intranasally, intradermally, subcutaneously, intramuscularly, topically or intravenously. In some methods, the patient is monitored followed administration to assess the immune response. If the monitoring indicates a reduction of the immune response over time, the patient can be given one or more further doses of the agent.
In another aspect, the invention provides pharmaceutical compositions comprising a spore coating peptide and an excipient suitable for oral and other routes of administration. The invention also provides pharmaceutical compositions comprising an agent effective to induce an immunogenic response against spore coating peptide in an animal, and a pharmaceutically acceptable adjuvant. In some such compositions, the agent is spore coating peptide or an active fragment thereof. In some compositions, the adjuvant comprises alum.
In some compositions, the adjuvant comprises an oil-in-water emulsion. In some compositions, the spore coating peptide or active fragment thereof is a component of a polylactide polyglycolide copolymer (PLPG) or other particle. The invention further provides compositions comprising spore coating peptide or an active fragment linked to a conjugate molecule that promotes delivery of spore coating peptide to the bloodstream of a patient and/or promotes an immune response against spore coating peptide. For example, the conjugate can serve to promote an immune response against spore coating peptide. In some compositions, the conjugate is cholera toxin. In some compositions, the conjugate is an immunoglobulin. In some compositions, the conjugate is attenuated diphtheria toxin CRM 197 (Gupta, Vaccine 15, 1341-3 (1997).
The invention also provides pharmaceutical compositions comprising an agent effect to induce an immunogenic response against spore coating peptide in a patient with the proviso that the composition is free of Complete Freund's adjuvant. The invention also provides compositions comprising a viral vector encoding spore coating peptide or a an active fragment thereof effective to induce an immune response against spore coating peptide. Suitable viral vectors include herpes, adenovirus, adenoassociated virus, a retrovirus, sindbis, semiliki forest virus, vaccinia or avian pox.
The invention further provides methods of preventing or treating a disease which is caused by Mycobacteria. In such methods, an effective dose of spore coating peptide can be administered to a patient. The invention further provides for the use of spore coating peptide, or an antibody thereto, in the manufacture of a medicament for prevention or treatment of a disease caused by Mycobacteria.
In another aspect, the invention provides methods of assessing efficacy of a treatment method, described above, in a patient. In these methods, a baseline amount of antibody specific for a spore coating peptide is determined in a tissue sample from the patient before treatment with an agent. An amount of antibody specific for spore coating peptide in the tissue sample from the patient after treatment with the agent is compared to the baseline amount of spore coating peptide-specific antibody. An amount of spore coating peptide-specific antibody measured after the treatment that is significantly greater than the baseline amount of spore coating peptide-specific antibody indicates a positive treatment outcome.
In other methods of assessing efficacy of a treatment method in a patient, a baseline amount of antibody specific for a spore coating peptide in a tissue sample from a patient before treatment with an agent is determined. An amount of antibody specific for spore coating peptide in the tissue sample from the subject after treatment with the agent is compared to the baseline amount of spore coating peptide-specific antibody. A reduction or lack of significant difference between the amount of spore coating peptide-specific antibody measured after the treatment compared to the baseline amount of spore coating peptide-specific antibody indicates a negative treatment outcome.
In other methods of assessing efficacy of a treatment method in a patient a control amount of antibody specific for spore coating peptide is determined in tissue samples from a control population. An amount of antibody specific for spore coating peptide in a tissue sample from the patient after administering an agent is compared to the control amount of spore coating peptide-specific antibody. An amount of spore coating peptide-specific antibody measured after the treatment that is significantly greater than the control amount of spore coating peptide-specific antibody indicates a positive treatment outcome.
In other methods of assessing efficacy of a treatment method in a patient, a control amount of antibody specific for spore coating peptide in tissues samples from a control population is determined. An amount of antibody specific for spore coating peptide in a tissue sample from the patient after administering an agent is compared to the control amount of spore coating peptide-specific antibody. A lack of significant difference between the amount of spore coating peptide-specific antibody measured after beginning said treatment compared to the control amount of spore coating peptide-specific antibody indicates a negative treatment outcome.
Other methods of monitoring disease or susceptibility thereto in a patient, comprise detecting an immune response against spore coating peptide in a sample from the patient. In some such methods, the patient is being administered an agent effective to treat or prevent diseases caused by Mycobacteria, and the level of the response determines the future treatment regime of the patient.
In other methods of assessing efficacy of a treatment method in a patient a value for an amount of antibody specific for spore coating peptide in tissue sample from a patient who has been treated with an agent is determined. The value is compared with a control value determined from a population of patient experiencing amelioriation of, or freedom from, symptoms of Mycobacterial disease due to treatment with the agent. A value in the patient at least equal to the control value indicates a positive response to treatment.
The invention further provides diagnostic kits for performing the above methods. Such kits typically include a reagent that specifically binds to antibodies to spore coating peptide or which stimulates proliferation of T-cells reactive with spore coating peptide.

DEFINITIONS

Proteins which are important for spore formation include those which are encoded by: a) genes for formation of spore coat, cortex and outer layer; b) genes involved in chromosome partitioning and translocation of DNA from mother cell to spore; and c) transcription factors regulating putative sporulation genes. Such proteins are termed “spore related proteins” or “spore related peptides” in the present application.
The term “spore related peptides” includes the peptides which are coded for by one of the genes listed in Tables 1-4, or a fragment thereof. Preferred such peptides are e.g. CotA (MM1618), CotD (MM4853), CotT (MM4208), SpoVK, CotSA (MM4226), YrbC (MM2098), SpoVE, Soj, and SpoIIIE A spore forming peptide or fragment thereof should preferably be an immunogenic agent. Most preferred are CotA (MM1618), CotD (MM4853), CotT (MM4208), CotSA (MM4226), and YrbC (MM2098).
The term “spore forming peptide” indicates a spore related peptide that forms part of the spore. Similarly, “spore coating peptide” indicates a spore related peptide that forms part of the spore coat. The group “spore coating peptides” is thus a subgroup of the group “spore forming peptides” which in turn is a subgroup of “spore related peptides”. When use is made of a spore related peptide in the present invention, the use of spore forming peptides and especially spore coating peptides are preferred embodiments, if not specifically indicated otherwise.
The term “patient” is meant to include animals such as humans and livestock such as cows, sheep, pigs, dogs, chicken, goat, kangaroo, ostrich, and buffalo and domestic animals such as cats, dogs, fish and birds. Also included are wild animals such as deer, reindeer, moose, wild boar.
The term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 65 percent sequence identity, preferably at least 80 or 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity or higher). Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
The terms “binding molecule” and “therapeutic agent” can be used interchangeably.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.
When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., supra). One example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www. ncbi. nlm. nih. gov/). Typically, default program parameters can be used to perform the sequence comparison, although customized parameters can also be used. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1989)) For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group W (basic side chains): asn, gln, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe.
Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another.
Therapeutic agents of the invention are typically substantially pure. This means that an agent is typically at least about 50% w/w (weight/weight) purity, as well as being substantially free from interfering proteins and contaminants.
The agents may be at least about 80% w/w and, more preferably at least 90% w/w or about 95% w/w purity. However, using conventional protein purification techniques, homogeneous peptides of at least 99% w/w can be obtained.
Affinity between two entities means an affinity of at least 10⁶, 10⁷, 10⁸10⁹M⁻¹, or 10¹⁰M. Affinities greater than 10⁸M⁻¹are preferred.
The term“antibody” is used to include intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen. Optionally, antibodies or binding fragments thereof, can be chemically conjugated to, or expressed as, fusion proteins with other proteins.
The term“epitope” or “antigenic determinant” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. T-cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by 3H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156, 3901-3910) or by cytokine secretion.
The term “immunological” or “immune” response is the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a spore coating peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4+T helper cells and/or CD8+ cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity.
The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+T cells) or CTL (cytotoxic T lymphocyte) assays (see Burke, supra; Tigges, supra). The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.
The term “specific for” indicates that the variable regions of the antibodies, or binding molecules, recognize and bind spore coat peptides according to the invention exclusively (i.e., able to distinguish spore coating peptides from other similar polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays in which one can determine binding specificity of an anti-spore coat peptide antibody are well known and routinely practiced in the art. (Chapter 6, Antibodies A Laboratory Manual, Eds. Harlow, et I al., Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), herein incorporated by i reference in its entirety).
An “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a patient, optionally in conjunction with an adjuvant.
The term “naked polynucleotide” refers to a polynucleotide not complexed with colloidal materials. Naked polynucleotides are sometimes cloned in a plasmid vector.
The term “adjuvant” refers to a compound that when administered in conjunction with an antigen augments the immune response to the antigen, but when administered alone does not generate an immune response to the antigen.
Adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages.
The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
Compositions or methods “comprising” one or more recited elements may include other elements not specifically recited.
For example, a composition that comprises a spore coating peptide encompasses both an isolated spore coating peptide and spore coating peptide as a component of a larger polypeptide sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

Flow cytometric profiles covering the complete life cycle of M. marinum. An old M. marinum stock was used to inoculate fresh medium and the progress of the culture to stationary phase through exponential growth was followed by flow cytometry. A 2-month old stock was spread on 7H10 agar plates with necessary supplements; cells were harvested at different times over a period of 2 months. The harvested cells were fixed in 70% ethanol, washed and resuspended in 0.1 volume TM (10 mM Tris-HCl pH 7.8; 10 mM MgCl₂), stained with mithramycin A and ethidium bromide and run in the flow cytometer (see Methods). The profiles are histograms of cell numbers plotted against their DNA content (column A: fluorescence calibrated in chromosome number equivalents) or size (column B: light scattering measured in arbitrary scale units kept constant for all histograms, not shown). The ages of the cultures, in h(ours) after inoculation into fresh medium, are shown on the left side of each row. The profiles on the top row, showing a stationary phase culture of the laboratory strain of the gram-negative bacterium Escherichia coli K12 (1-3 μm×1 μm cylinders containing 4.6 Mbp DNA per chromosome), were used as a calibration standard for estimating DNA-content and cell size in the M. marinum profiles.

FIG. 2

Fluorescence microscopy of M. marinum at different stages of growth from fresh inoculum to stationary phase. 2-month old stock was spread onto plates and aliquots fixed in ethanol as described in FIG. 1 legend. The fixed cells were washed in PBS, layered onto thin films of agarose (1% in 0.9% NaCl) containing 0.5 μg/mlDAPI on a microscope slide and examined under a Zeiss (Axioplan 2) microscope with a CCD camera (see Methods). Panels a-g show cells harvested at Oh, 6 h, 12 h, 72 h (3d), 120 h (5d), 168 h (1 w) and 336 h (2 w) after inoculation into fresh medium. Sizebars: 5 μm.

FIG. 3

Surface morphologies and internal structures of M. marinum cells at different stages of sporulation by scanning electron microscopy (SEM) and thin section transmission electron microscopy (TEM), respectively. Cells at different times after inoculation into fresh medium were prepared for SEM and TEM as described in Methods. (A) SEM images: (a) isolated spores; (b) cells at germination; (c) putative endospores (5 days after inoculation) and (d) spore with a vegetative cell (7-day old culture). Sizebar: 1 μm. (B) TEM images: (a) isolated spores; (b, c) mature spores; (d-f) different stages of germinating spores (6 h after inoculation); (g) a forespore at left and a mature endospore at right (5 day old culture). Magnifications: (a) 6000×; (b,c) 60000×; (d-f) 40000×; (g) 30000×.

FIG. 4

Surface biochemistry and heat tolerance of putative spores from M. marinum

(a) Spore-specific differential staining of M. marinum cells from exponential and stationary phase cultures. Cells grown and harvested at different stages of growth were heat-fixed, stained and counterstained with malachite green and safranin, respectively (see Methods) and examined under the 100× objective of an Olympus CH30RF200 microscope.

(b) Colony forming ability after heat treatment. Plates containing exponential (12 h after inoculation) and stationary (after 14 days of growth) phase cells with and without exposure to wet-heat treatment (15 min, 65° C.).

(c) Rate of killing by wet-heat for M. marinum cells from exponential and stationary phases. Cells in exponential phase (12 h, diamonds) and stationary phase (14d, squares) were exposed to wet-heat for different times, surviving colonies were expressed as percent against identical, unexposed cell population and plotted as a function of exposure times. Each point on the survival plot represents an average of at least three measurements (Table 5) with experimental variations indicated by error bars.

FIG. 5

Relative expression levels of putative sporulation genes from M. marinum genome at different stages of the life cycle. The mRNA levels of 9 genes from M. marinum genome, homologues of known sporulation genes from Bacillus subtilis and Streptomyces coelicolor, were compared using dot-blot hybridization as the culture progressed from exponential to stationary phase. Specific oligonucleotide probes (Table 6 for sequences) were used for each mRNA candidate. The relative intensities of the dots were normalized using corresponding 5S rRNA signals as internal standards and plotted in arbitrary units (Y-axis) as M. marinum-mRNA signals (identified as their homologues, X-axis) from cultures of different ages (bars of different patterns). All samples were analyzed at least three times and estimated experimental variations are indicated by error bars. The numerical values (Table 7) were obtained from Phosphor-Imager (Image Quant, Molecular Dynamics 400) analysis of the dot signals.

FIG. 6

Presence of spore particles in a culture of Mycobacterium bovis (BCG).

(a) Phase fluorescence image of M. bovis cells from a six-month old culture. Sizebar: 5 μm.

(b) Differentially stained purified spore particles from BCG culture.

(c) SEM image of purified BCG spores. Sizebar: 1 μm.

(d) TEM image of thin sectioned purified BCG spores. Magnification: 60000×.

FIG. 7.

Morphologic comparison of M. marinum colonies from cultures germinated from single purified (see Methods) independent spore particles (a-d) with that from an exponentially growing culture inoculated from an old stock (e). Size bar: 1 cm.

FIG. 8.

RNase P RNA gene sequences from independent clones originating from isolated single spore particles. Alignment of residues 89-315 of the RNase P RNA gene from the chromosomes of clonally isolated colonies. (1-4; SEQ ID NO: 27): Sequences from genomic DNA isolated from single colonies grown out of isolated single spore particle. (5; SEQ ID NO:27) Sequence from RNaseP RNA gene of M. marinum cloned in plasmid pIGn. (6; SEQ ID NO:28) M. marinum RNaseP RNA gene sequence obtained by blast search in the Singer database 24 using the M. tuberculosis H37Rv RNase P RNA gene sequence (www.sanger.ac.uk./Projects/M. tuberculosis) reference. Sequences 1-5 are identical to those reported in the database except for two positions, 93 and 158. This could be due to the difference between the lab strain (ATCC 927) and the strain used to build the database (ATCC BAA-535), or an error in the database. Oligos used to PCR-amplify region 89-315 were:

(SEQ ID NO: 29; comprising region 65-88)

Forward: 5′-ATCGAGGAAAGTCCGGACTTCACA-3′

(SEQ ID NO: 30; complementary to region 294-315)

Reverse: 5′-ACCCGCAGACTCGGGCGAGCAG-3′

The forward oligo was used to obtain the sequences 1-5.

FIG. 9.

Life cycle of M. marinum from purified spores by microscopy.

a, pure spore population, 0 h. b, germinating spores, 6 h. c, germinated cells, 8 h. d, germinated cells, 24 h (1 d). e, same as d, 2 d. f, same as e, 3 d. g and h, appearance of endospores, 5 d. i, appearance of mature spore, 7 d. j, mature spores with cell debris, 14 d. Size bars: 5 μm.

FIG. 10.

Life cycle of M. marinum from purified spores by flow cytometry. A M. marinum culture was started from a purified spore suspension and followed through germination, exponential growth to sporulation in late stationary phase by flow cytometry. Columns (A) and (B) show DNA content (fluorescence calibrated in chromosome numbers) and cell size (light scattering in arbitrary units) distributions, respectively. The top panels refer to stationary phase LB cultures of E. coli used as calibration standards for DNA content and cell size. The nine lower panels correspond to M. marinum samples taken at: 0, 6, 8, 24, 48, 72, 120, 168 and 336 hours after suspension of the purified spores into the growth medium. Fixation and staining procedures were as described in Methods and in FIG. 1 legend.

The narrow size distribution and the shift of the peak as a whole with little widening implied synchronous transition of spores into vegetative cells. At late stationary stage (after 120 hours of growth), the cell size distribution implies emergence of endospores from the narrowing of the peak and its shift towards smaller size (Column B: 168 h and 336 h). The fluorescence profiles show the onset of DNA synthesis by 6 h after addition of growth medium; replication continues for 72 h after which non-replicative cell division is followed by sporulation.

DETAILED DESCRIPTION

The invention provides pharmaceutical compositions and methods for prophylactic and therapeutic treatment of diseases caused by a Mycobacterium infection. Furthermore, the invention provides such compositions and methods for treating such diseases wherein the Mycobacterium have formed spores.
The genus Mycobacterium includes non-pathogenic environmental bacteria as well as highly successful pathogens e.g. Mycobacterium tuberculosis (Mtb) and Mycobacterium leprae the etiological agents of tuberculosis and leprosy, respectively. While Mtb is human-specific, the closely related Mycobacterium bovis can infect both humans and animals and lead to tuberculosis regardless of host. Moreover, the mycobacteria Mycobacterium avium supsp. paratuberculosis (MAP), the causative agent of chronic diarrhea among ruminants (Johne's disease), can be present in food derived from cattle and it has been discussed that MAP is a causative agent of Crohn's disease in humans.
Another closely related Mycobacterium, Mycobacterium marinum causes a systemic tuberculosis-like infection and disease in ectothermic hosts e.g. fish and frogs. In comparison to Mtb, M. marinum grows with a relatively short doubling time and growth is limited at higher temperatures (see below), which makes M. marinum easier to handle than e.g. Mtb. M. marinum has recently emerged as model system to identify and study factors important for mycobacterial infections, disease development and its persistence. Pathogenic mycobacteria are invasive slow growing bacteria that can grow and multiply within macrophages. Moreover, most of them can establish long-term infections that can cause an acute or chronic stage of the disease or a stage that is clinically asymptomatic. In the latter situation the bacteria has the potential to resume growth after several decades of dormancy resulting in active disease.
The innate immune system is essential in defense against bacterial infections. Macrophages are important actors in this process and they play a significant role in the front line defense. Pathogenic mycobacteria e.g. Mtb are ingested by phagocytosis by alveolar macrophages at the site of entry and inside the macrophage the bacteria reside within a membrane-bound cytoplasmic vacuole referred to as the Mycobacterium phagosome. Pathogenic mycobacteria avoid phagolysosomal degradation by: 1) blocking acidification of the phagosome, 2) altering the protein content of the vacuole and 3) preventing interactions with other endosomal compartments. This ensures survival of the bacteria within the macrophage and subsequent spread of the pathogen to other cells e.g. dendrite cells and neutrophiles.
Complete Life Cycle of M. marinum: Cell Size and DNA Content Distribution
Changes in cell size and DNA content distributions of M. marinum cultures on plates through the full life cycle was followed by flow cytometry, Maisnier-Patin, S., Dasgupta, S., Krabbe, M., Nordström, K. Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli. Mol. Microbiol. 30, 1067-1079 (1998). FIG. 1 shows the flow cytometric profiles for DNA content and cell size distributions of a culture from 2 h after inoculation to 2 months. Measurements were taken every 2 h for 12 h, at 24 h, every 24 h till 96 h, 196 h and at 2 months. The overnight stationary culture of Escherichia coli K12, used as a calibration standard, showed a uniform population with a narrow size distribution (single sharp symmetrical peak with normal distribution; top row, column B) containing either one or two full-sized chromosomes (two sharp peaks indicated by numbers 1 and 2 in the fluorescence histogram pre-calibrated in terms of genome size using E. coli chromosome as standard; see top row, column A). The flow cytometric profiles of M. marinum cells from the 2 months old stock remain unchanged from 0 h (not shown) up to 4 h after inoculation into fresh medium. They showed two distinct classes of cell population: the majority consisted of very small size cells with fluorescence corresponding to one M. marinum genome (6.6 Mbp, www.sanger.ac.uk/Projects/M_marinum/) or less, and a subpopulation with fluorescence corresponding to 2-genome equivalent DNA (indicated by the size bars labeled 1 and 2 in the 2 month-DNA content profile). At 6 h, the light scattering-peak representing cell size started to broaden suggesting increased in cell size accompanied with higher than 2-genome equivalent DNA content in some cells (start of DNA synthesis). With progression of time, the cell size and DNA content distributions underwent drastic changes—separate peaks representing very small and big cells were replaced by a single, broader peak corresponding to a continuous size distribution of exponentially growing population of cells (10 h after inoculation). The fluorescence peak corresponding to 2 chromosome equivalent of DNA broadened into a continuity of 2 to 4 or more chromosomes indicating ongoing DNA synthesis. In keeping with this, the 1-chromosome peak disappeared gradually as more cells entered the replicative phase from the “non-replicating, small-size” spore-like state after 10 h of growth in fresh medium. The very small cells with one genome equivalent of DNA persisted until about 6 h after inoculation into fresh media and then both disappeared to be replaced by cells of bigger size containing 2-4 chromosome equivalents of DNA (see 10 h panels). The very small size cells are believed to represent the non-replicating spore-like particles. With further incubation, the cell size distribution reached its highest average value with broadest distribution (24 h) and then divided into smaller, more uniform size distributions; the DNA content indicated that ongoing DNA synthesis was gradually ending, and cells with 2 and 4 chromosome equivalents of DNA started to appear (24 h). Replication in a subpopulation continued while non-replicative division was evident from the increasing proportion of 2-chromosome cells after 96 h of growth (see 48 h, 72 h and 96 h).
After about a week, almost all cells were in a single, smaller size distribution with either 1 or 2 full size chromosome equivalents of DNA: at two months, the majority of the cells had been converted to the very small size 1-chromosome form with a small subpopulation of bigger cells containing 2 chromosome equivalents of DNA, similar to the initial 2 month old stock culture at 2 h (compare the flow cytometric profiles for 2 h and 2 month in FIG. 1).
Single Spore Develops into a Single M. marinum Colony
To eliminate the possibility that these spore-like particles were a contamination from another spore former, pure spores free of vegetative cells were isolated (Methods, FIG. 9 a). A batch of pure spores was diluted serially such that there would be one or less viable spore in each diluted fraction (see Methods). Samples from 10 replicates of dilutions were plated. After a week of incubation, four of the ten plates showed a single colony that had germinated and grown. Each of these colonies, originating from four independent single spores, was morphologically identical to a colony of M. marinum (FIG. 7). To verify that these colonies were indeed M. marinum, part of the RNase P RNA gene was PCR-amplified using total DNA extracted from each one of them. DNA sequencing (FIG. 8) confirmed that the colonies were M. marinum. The isolated M. marinum spores were also freshly plated, and their life cycles were followed with phase-fluorescence microscopy and flow cytometry (FIGS. 9 and 10). They were observed to germinate to vegetative cells, which again sporulated in the late stationary phase. Thus, the purified spore particles germinated into M. marinum cells and were therefore not contaminants.

Electron Microscopic Visualization of the Spore Particles, Sporulation and Germination

The very distinct sizes and morphologies of cells in sporulating and germinating states were examined by scanning electron micrograph (SEM), Lackner P. et al. Scanning electron microscopy of the neuropathology of murine cerebral malaria. Malar J. 5, 116 (2006). FIG. 3 (panel A; a-d) shows SEM images of M. marinum at various stages of sporulation. Different types of cells seen are: a) isolated spores; b) a vegetative cell germinating out from a spore (6 h); c) cells with probable endospores showing bulges near one pole (day 5); and d) a spore with a vegetative cell (day 7). The internal structures of the spores were examined by transmission electron microscopy (TEM) of thin section of fixed, isolated spores (FIG. 3 panel B; a-c) and M. marinum cells from cultures at different stages (FIG. 3 panel B; d-g). By comparing with the TEM images of well-characterized Bacillus spores, Wang, L., Perpich, J., Driks, A., and Kroos, L Maintaining the transcription factor SpoIIID level late during sporulation causes spore defects in Bacillus subtilis. J. Bacteriol. 189, 7302-7309 (2007) with that of isolated M. marinum spores, the outer coat, inner coat, cortex and core of the mature spore were clearly identified (FIG. 3 panel B; b and c). The TEM image cells 6 hours after inoculation of spores into fresh media, showed vegetative cells gradually germinating out of empty spores with disrupted surface (FIG. 3 panel B; d-f). In the TEM images of the day 5 cells that showed endospore-like species in the phase-fluorescence microscopy (FIG. 2 e), structures that looked like endospores in their different stages of maturation were seen (FIG. 3 panel B; g, forespore and mature endospore, side by side). These are morphologically very similar to published images of Bacillus spp. endospores, Erlendsson, L. S., Möller, M., and Hederstedt, L. Bacillus subtilis StoA is a thiol-disulfide oxidoreductase important for spore cortex synthesis. J. Bacteriol. 186, 6230-6308 (2004). No such structure was observed in the TEM images of a batch of vegetatively growing cells in exponential phase (not shown). These data suggest that M. marinum indeed forms spores and that the sporulation pathway proceeds via endospore formation.
Physical and Biochemical Properties of M. marinum Spore Particles and Presence of Genomic Informations Necessary for Sporulation in Mycobacteria
If the ‘spore-like’ particles visualized microscopically in the stationary M. marinum culture are true spores they should exhibit: (i) special surface-biochemistry that allows staining by spore-specific stains; (ii) high resistance towards physical and chemical stresses; and (iii) presence of the genetic apparatus necessary for entry into and exit from the spore state. Differential spore staining and a heat tolerance test, respectively, were performed to check the first two requirements, and bioinformatics for the third.
The classical differential spore staining was done using malachite green as the primary stain, water as the decolorizing agent and safranin as the counterstain Cappuccino, J. G., Sherman, N. in Microbiology, a Laboratory Manual (Benjamin Cummings, San Fransisco, sixth edition, 2001). As shown in FIG. 4 a, the vegetative cells appeared to have lost the primary stain and taken the counterstain and appeared red. In contrast, the spores retained the primary stain and appeared green, indicating that the surface of the spore-like particles in M. marinum cultures have similar biochemical properties as the well-known B. subtilis spores.
Next, the effect of wet heat treatment (65° C., 15 minutes) on exponentially growing (12 h) and stationary phase (14 days) cells was compared, Chakrabarty, K. et al., Infection and Immunity. 74, 4430-4438 (2006). While the exponentially growing cells were almost completely killed after 10 min of heat treatment, nearly 40% of the cells in stationary phase survived even after 30 min of treatment (FIGS. 4, b and c). This is qualitatively proportional to the spore population seen in stationary cultures. The spores were also demonstrated to be more resistant to glutaraldehyde treatment compared to cells grown in exponential phase (not shown).
Finally, the mycobacterial genome was scanned to look for genes relevant to spore formation. Genes essential for sporulation have been identified in the gram-positive bacteria e.g. B. subtilis, genolist.pasteur.fr/SubtiList/, and Streptomyces coelicolor, streptomyces.org.uk/sco/index.html. A reciprocal best hit (RBH) approach was used, Rivera, M. C., Jain, R., Moore, J. E. & Lake, J. A. Genomic evidence for two functionally distinct gene classes. Proc. Natl. Acad. Sci. USA 95, 62396244 (1998), to identify putative orthologues of well-known sporulation genes in the M. marinum, ftp.sanger.ac.uk/pub/pathogens/mm/, and M. tuberculosis, cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi, genomes (Tables 1-4). Based on the functional annotation of the B. subtilis genes, these were divided into three classes and include: a) genes for formation of spore coat, cortex, and outer layer; b) genes involved in chromosome partitioning and translocation of DNA from mother cell to spore; and c) transcription factors regulating putative sporulation genes. Some of these genes, from all three classes, were selected to check their relative levels of mRNA expression by dot blot analysis,' Sambrook, J., Fritsch, E. F., Maniatis, T. Molecular Cloning, a Laboratory Manual. (Cold Spring Harbor Laboratory Press, New York, third edition, 2001). As shown in FIG. 5 the selected genes were transcribed in M. marinum. Significantly, the expression of the sporulation gene-homologues depended on the age of the culture. The mRNA levels of the homologues, genolist.pasteur.fr/SubtiList/, of SpoVK (disruption of this gene leads to immature spore formation, stage V), CotSA (Spore coat protein), YrbC (similar to spore coat protein), and SpoVE (required for spore cortex peptidoglycan synthesis, stage V) showed a sharp increase from day 5, continuing to day 7. Homologues of Soj (centromere-like function, involved in forespore chromosome partitioning/negative regulation of sporulation initiation) and SpoIIIE (DNA translocase required for chromosome partitioning through the septum into the forespore) showed a similar pattern but more modest increase compared to the former four genes. Some weaker hits were obtained in the bioinformatic search for some major transcription factors, and some of them were probed for, e.g. Spo0A (master regulator for commitment to sporulation) sharp increase from day 5, which continues to day 7. The top scoring Spo0A hit in Mtb strain CDC1551 is the trcR gene that encodes a DNA binding response regulator, Haydel, S. E., Clark-Curtiss, J. E. The Mycobacterium tuberculosis TrcR response regulator represses transcription of the intracellularly expressed Rv1057 gene. J. Bacteriol. 188, 150-159 (2006), (TrcR in FIG. 5). The other factors, SigF (late sporulation specific RNA polymerase sigma factor) showed a modest increase at day 7 while the homologue to the anti-SigF, SpoIIAB, showed a modest increase at days 5 and 7. The “housekeeping” sigma factor, SigA (s⁷⁰) mRNA, Gruber, T. M., Gross, C. A. Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol. 57, 441-466 (2003), of M. marinum does not play any direct role in sporulation, Piggot, P. J., Hilbert, D. W. Sporulation of Bacillus subtilis. Current Opinion in Microbiology. 7, 579 (2004), and its mRNA level decreased as the culture entered and progressed through stationary phase. These data support the presence of a molecular pathway for sporulation in M. marinum and conceivably also in Mtb. The expressions of these genes at protein level, and their further characterizations, such as phosphorylation/dephosphorylation patterns, are yet to be examined. The fact that no strong hits were found for the major transcription factor specific for sporulation might indicate that either: a) the corresponding genes have diverged in their sequences while retaining their functions, or b) these factors are unique to mycobacteria and have to be identified using other methods. Note that the RNA samples prepared from the day 7 cells represented genes expressed in a mixed population of vegetative cells and spores (FIGS. 2 f and 3 panel A; d).
Spore Particles in Old Cultures of the M. bovis BCG Strain
We also observed spore-like species in late stationary cultures of the Myocbacterium bovis BCG strain. In phase fluorescence microscopy, BCG cells from a six-month-old plate showed bright, refractive particles (FIG. 6 a) similar to the M. marinum spores. These particles were purified (see Methods) and subjected to spore-specific staining (FIG. 6 b) and electron microscopy (both SEM and TEM, FIGS. 6 c and d, respectively). On the basis of these data, it appears that these particles are similar to M. marinum spores. Hence, these data provide experimental evidences in favor of the possibility that sporulation is a more general feature of mycobacteria, and not limited to M. marinum. Consequently, sporulation might be considered as a general strategy for survival under stress.
Therapeutic agents for use in the present invention induce an immune response against a spore coating peptide, or other proteins or peptides which are involved in spore formation. These agents include the spore coating peptide itself and variants thereof, analogs and mimetics of the spore coating peptide that induce and/or crossreact with antibodies to spore coating peptides, and antibodies or T-cells reactive with spore coating peptides. Induction of an immune response can be active as when an immunogen is administered to induce antibodies or T-cells reactive with a spore coating peptide in a patient, or passive, as when an antibody is administered that itself binds to a spore coating peptide in patient.
The therapeutic agent used in the claimed methods can be any of the naturally occurring forms of a protein involved in spore formation, such as those proteins derived from the genes in Tables 1-4. Such a gene include in particular: a) genes for formation of spore coat, cortex and outer layer; b) genes involved in chromosome partitioning and translocation of DNA from mother cell to spore; and c) transcription factors regulating putative sporulation genes. A number of these genes were chosen from all three classes (a, b, or c) to check their relative levels of mRNA expression by dot blot analysis. The selected genes may be transcribed in M. marinum. The expression of the sporulation gene-homologues depends on the age of the culture. The mRNA levels of the homologues of SpoVK (disruption of this gene leads to immature spore formation, stage V), CotSA (Spore coat protein), YrbC (similar to spore coat protein), and SpoVE (required for spore cortex peptidoglycan synthesis, stage V) showed a sharp increase from day 5, which continued to day 7. Homologues of Soj (centromere-like function, involved in forespore chromosome partitioning/negative regulation of sporulation initiation) and SpoIIIE (DNA translocase required for chromosome partitioning through the septum into the forespore)
The therapeutic agent may be an immunogenic peptide, fragment or analog of a protein encoded by a gene shown in Tables 1-4.

TABLE 1

Table 1: Putative sporulation gene orthologues of B. subtilis in Mycobacterium spp. and S. Coelicolor

Putative ortholog^b

B. subtilis 168

M. Tuberculosis

M. tuberculosis

S. coelicolor

Gene

M. marinum

CDC1551

H37Rv

A3(2)

id^a	name	Annotation	RBH	RSD	RBH	RSD	RBH	RSD	RBH	RSD

BG10054	SpoOJ	chromosome positioning	MM5481	MT_4036	Rv3917c	SCO3887
		near the pole and
		transport through the
		polar septum/antagonist
		of Soj-dependent
		inhibition of
		sporulation initiation
BG10055	Soj	centromere-like	MM5482	MT_4037	Rv391Sc	SCO3886
		function involved in
		forespore chromosome
		partitioning/negative
		regulation of
		sporulation initiation
BG10061	Jag	SpoIIIJ-associated	MM5484	MT_4039	Rv3920c	SCO3884
		protein
BG10062	SpoIIIJ	essential for sigma-G	MM5485	MT_4040	Rv3921c	SCO3883
		activity at stage III

BG10087	BofA	inhibition of the	MM0678
		pro-sigma-K
		processing machinery

BG10116	SpoVC	probable peptidyl-tRNA	MM4473	MT_1042	Rv1014c	SCO3125
		hydrolase

BG10119	SpoVT	transcriptional	Rv2595
		positive and negative
		regulator of
		sigma-G-dependent
		genes

BG10127	SpoIIE	serine phosphatase	SCO0717
		(sigma-F activation)/
		asymmetric septum
		formation

BG10203	SplB	spore photoproduct							SCO7402
		lyase

BG10226	SpoVE	required for spore	MM3194	MT_2213	Rv2154c	SCO2607
		cortex
		peptidoglycan
		synthesis

BG10296

SpoIIAA

anti-anti-sigma factor

MM5181

MM0737

MT_3789

Rv3687c

SCO3067

BG10297*	SpoIIAB	anti-sigma factor/	MM5186			SCO7322
		serine kinase
BG10298*	SigF	RNA polymerase	MM1248	MT_3385	Rv3286c	SCO5243	SCO0600
		sporulation
		forespore-
		specific (early)
		sigma factor

BG10332	SpoIVFB	membrane	MM2075	SCO1652
		metalloprotease
		required for the
		processing of
		pro-sigma-K to
		active sigma-K

BG10337	Obg	GTP-binding protein	MM3765	MT_2516	Rv2440c	SCO2595
		involved in
		initiation of
		sporulation (SpoOA
		activation)

BG10346	SpoVID	required for assembly		MM4057
		of the spore coat

BG10458	SpoIVCA	site-specific DNA	MM2129	pMM23-15	MT_3573	Rv1586c	SCO6405
		recombinase required
		for creating the sigK
		gene (excision of
		the skin element)

BG10490	CotA	spore coat protein	MM1618					SCO3440
		(outer)

BG10493	CotD	spore coat protein	MM4853
		(inner)

BG10495	CotT	spore coat protein	MM4208			MT_1271		Rv1233c		SCO4822
		(inner)

BG10611	SpsC	spore coat	MM2320	Rv1504c	SCO5746
		polysaccharide
		synthesis

BG10613	SpsE	spore coat	SCO4881
		polysaccharide
		synthesis

BG10617	SpsI	spore coat	MM0606	MT_0348	Rv0334	SCO1388
		polysaccharide
		synthesis

BG10618	SpsJ	spore coat	MM1082	MT_3570	Rv3464	SCO0749
		polysaccharide
		synthesis
BG10619	SpsK	spore coat	MM1275	MT_3366	Rv3266c	SCO7194
		polysaccharide
		synthesis

BG10763	SpoIIIE	DNA translocase	MM1967	MT_2819	Rv2748c	SCO5750
		required for
		chromosome
		partitioning through
		the septum into the
		forespore

BG10765*	SpoOA	two-component response	MM4455	MT_1062	Rv1033c	SCO2013
		regulator central for
		the initiation of
		sporulation

BG11039	SpoVK	disruption leads to	MM0541	MT_0295	Rv0282	SCO1024
		the production of
		immature spores

BG11197	CgeE	maturation of the	MT_2818	Rv2747
		outermost layer of
		the spore

BG11231

KipI

inhibitor of KinA

MM0524

MT_0277

Rv0264c

SCO0442

BG11381

CotSA

spore coat protein

MM4226

MM0812

MT_0504

Rv0486

SCO2132

SCO4204

BG11687	YqgT	unknown; similar to	SCO1948
		gamma-D-glutamyl-L-
		diamino acid
		endopeptidase I

BG11806	Tlp	small acid-soluble	MM3184
		spore protein
		(thioredoxin-like
		protein)

BG12181	YdhD	unknown; similar to	SCO1429
		unknown proteins from
		B. subtilis

BG12229	Spo0M	sporulation-control	SCO0247
		gene
BG12358	KapD	inhibitor of the	SCO7397
		KinA pathway to
		sporulation

BG13288	YkuD	unknown; similar to			MT_0125	Rv0116c
		unknown proteins

BG13781	SafA	morphogenetic protein	MM0022	MT_1271	Rv1233c
		associated with
		SpoVID

BG13783	YrbC	unknown; similar to	MM2098	MT_2678		Rv2603c	SCO1521
		spore coat protein

BG13907	YtpT	unknown; similar to	MM1967	MT_2819	Rv2748c
		DNA translocase
		stage III
		sporulation protein
		(SpoIIIE)

BG14193	KipA	antagonist of KipI	MM0523	MT_0276	Rv0263c	SCO0443
BG14195	SpsL	spore coat	MM1081	MT_3571	Rv3465	SCO0400
		polysaccharide
		synthesis

Distance measure

M. tuberculosis

S. coelicolor

B. subtilis 168

M. marinum

CDC1551

H37Rv

A3(2)

Gene	Gene			Dis-		Dis-		Dis-		Dis-
id^a	name	Annotation	Expect^c	tance^d	Expect^c	tance^d	Expect^c	tance^d	Expect^c	tance^d

BG10054	SpoOJ	chromosome positioning	3.7E−51	1.4	1.3E−48	1.4	1.2E−48	1.4	4.5E−52	1.5
		near the pole and
		transport through the
		polar septum/antagonist
		of Soj-dependent
		inhibition of
		sporulation initiation
BG10055	Soj	centromere-like	4.1E−68	1.0	1.6E−66	1.0	1.5E−66	1.0	2.3E−71	0.9
		function involved in
		forespore chromosome
		partitioning/negative
		regulation of
		sporulation initiation
BG10061	Jag	SpoIIIJ-associaled	1.5E−13	2.9	3.2E−13	3.1	2.9E−13	3.1	3.4E−15	2.6
		protein
BG10062	SpoIIIJ	essential for sigma-G	1.5E−24	2.4	1.4E−23	2.3	1.3E−23	2.3	1.0E−29	2.2
		activity at stage III
BG10087	BofA	inhibition of the		2.8
		pro-sigma-K
		processing machinery
BG10116	SpoVC	probable peptidyl-tRNA	3.3E−36	1.6	3.2E−36	1.6	3.0E−36	1.6	4.8E−32	1.7
		hydrolase
BG10119	SpoVT	transcriptional					4.2E−05	2.3
		positive and negative
		regulator of
		sigma-G-dependent
		genes
BG10127	SpoIIE	serine phosphatase								3.6
		(sigma-F activation)/
		asymmetric septum
		formation
BG10203	SplB	spore photoproduct							1.8E−25	3.0
		lyase
BG10226	SpoVE	required for spore	7.9E−58	2.2	6.1E−58	2.1	5.6E−58	2.1	5.0E−60	1.9
		cortex
		peptidoglycan
		synthesis
BG10296	SpoIIAA	anti-anti-sigma factor	3.2E−06	3.3	8.6E−04	3.7	8.0E−04	3.7	6.5E−12	2.5
BG10297*	SpoIIAB	anti-sigma factor/	4.3E−07						3.3E−10
		serine kinase
BG10298*	SigF	RNA polymerase	2.4E−33		2.4E−31		2.2E−31		1.2E−37	1.9
		sporulation
		forespore-
		specific (early)
		sigma factor
BG10332	SpoIVFB	membrane		3.8					7.0E−09	4.0
		metalloprotease
		required for the
		processing of
		pro-sigma-K to
		active sigma-K
BG10337	Obg	GTP-binding protein	1.8E−92	1.3	2.4E−88	1.4	2.2E−88	1.4	1.7E−91	1.3
		involved in
		initiation of
		sporulation (SpoOA
		activation)
BG10346	SpoVID	required for assembly		4.8
		of the spore coat
BG10458	SpoIVCA	site-specific DNA	8.6E−14	3.7	2.0E−12	4.1	2.0E−12	4.1	5.5E−14	3.9
		recombinase required
		for creating the sigK
		gene (excision of
		the skin element)
BG10490	CotA	spore coat protein	1.0E−23	3.6					6.2E−15	2.7
		(outer)
BG10493	CotD	spore coat protein	2.9E−05	3.9
		(inner)
BG10495	CotT	spore coat protein	1.2E−06			3.6		4.1		5.4
		(inner)
BG10611	SpsC	spore coat	3.3E−50	2.3			4.1E−39	1.6	1.5E−20	2.8
		polysaccharide
		synthesis
BG10613	SpsE	spore coat							2.2E−34	2.4
		polysaccharide
		synthesis
BG10617	SpsI	spore coat	1.2E−38	1.8	4.3E−41	1.7	4.0E−41	1.7	3.4E−25
		polysaccharide
		synthesis
BG10618	SpsJ	spore coat	3.4E−73	1.2	1.0E−71	1.2	9.4E−72	1.2	1.3E−75	1.1
		polysaccharide
		synthesis
BG10619	SpsK	spore coat	1.8E−42	2.0	2.2E−46	1.8	2.0E−46	1.8	1.1E−18	3.2
		polysaccharide
		synthesis
BG10763	SpoIIIE	DNA translocase	5.4E−120		3.1E−118		2.9E−118		4.5E−122	1.9
		required for
		chromosome
		partitioning through
		the septum into the
		forespore
BG10765*	SpoOA	two-component response	2.4E−17		1.6E−15		3.6E−15		1.6E−17
		regulator central for
		the initiation of
		sporulation
BG11039	SpoVK	disruption leads to	7.2E−48	1.9	2.3E−49	1.9	2.2E−49	1.9	2.1E−53	1.6
		the production of
		immature spores
BG11197	CgeE	maturation of the			7.4E−04	4.7	8.3E−04	4.5
		outermost layer of
		the spore
BG11231	KipI	inhibitor of KinA	7.3E−23	3.3	4.4E−16	3.4	4.1E−16	2.8	1.2E−26	2.3
BG11381	CotSA	spore coat protein	3.1E−22	3.0		3.2		3.2	3.4E−24	3.0
BG11687	YqgT	unknown; similar to							1.2E−04	4.0
		gamma-D-glutamyl-L-
		diamino acid
		endopeptidase I
BG11806	Tlp	small acid-soluble	3.5E−04	14.2
		spore protein
		(thioredoxin-like
		protein)
BG12181	YdhD	unknown; similar to							2.8E−05
		unknown proteins from
		B. subtilis
BG12229	SpoOM	sporulation-control							2.9E−32	2.1
		gene
BG12358	KapD	inhibitor of the							1.2E−10	3.7
		KinA pathway to
		sporulation
BG13288	YkuD	unknown; similar to			3.6E−04	3.4	4.9E−04	3.5
		unknown proteins
BG13781	SafA	morphogenetic protein	1.2E−05	4.6	5.5E−07		4.7E−07
		associated with
		SpoVID
BG13783	YrbC	unknown; similar to	2.5E−52	1.3	4.0E−54	1.3	3.7E−54	1.3	7.8E−55	1.2
		spore coat protein
BG13907	YtpT	unknown; similar to		1.9		2.0		2.0
		DNA translocase
		stage III
		sporulation protein
		(SpoIIIE)
BG14193	KipA	antagonist of KipI	8.9E−22	2.7	1.7E−19	2.9	8.4E−20	2.9	2.0E−25	2.5
BG14195	SpsL	spore coat	1.8E−09	2.9	3.0E−10	3.0	2.7E−10	3.0	1.1E−09	2.8
		polysaccharide
		synthesis

^aIds marked with asterisk indicates a weak putative ortholog (see Methods)
^bPutative ortholog gene identifier. Gene identifiers justified to the left or right are identified using the RBH or RSD approaches, respectively. A centered gene identifier was identified by both approaches.
^cExpect value of putative RBH ortholog from forward BLAST search using B. subtilis sporulation gene as query.
^dDistance estimated by RSD approach

TABLE 2

Table 2: Putative sporulation gene orthologues of S. coelicolor in Mycobacterium spp. and B. Subtilis

Putative ortholog^a

S. coelicolor A3(2)

M. tuberculosis

B. subtilis

Gene

M. marinum

CDC1551

H37Rv

168

id	name	Annotation	RBH	RSD	RBH	RSD	RBH	RSD	RBH	RSD

SCO0506	nadE2	NH(3)-dependent		BG10694
		NAD(+)synthetase
		(nadE1)
SCO1024	SCG20A.04	conserved	MM0541	BG11039
		hypothetical
		protein

SCO1177	SCG11A.08	putative gntR-famly	MT_0514	Rv0494
		transcriptional
		regulator

SCO1434	SC6D7.05c	putative CbxX/CfqX	MM5443	MT_3981	Rv3868
		family protein
SCO1772	SCI51.12c	putative partitioning	MM2523	MT_1749	Rv1708
		or sporulation protein

SCO1950

SCC54.10c

hypothetical protein

MM2230

MT_1466

Rv1423

BG12402

SCO2083	ftsQ	sporulation protein	MM3191	MT_2210	Rv2151c
SCO2085	ftsW	putative cell division	MM3194	MT_2213	Rv2154c
		protein

SCO2607

sfr

Sfr protein

BG10226

SCO2805	2SCC13.13	conserved hypothetical			MT_1099		Rv1069c
		protein

SCO3034	whiB	sporulation regulatory	MM1282	MT_3358	Rv3260c
		protein
SCO3579	SCH17.13c	putative regulatory	MM5170	MT_3783	Rv3681c
		protein

SCO3846	SCH69.16	putative	MM0019	MT_0020	Rv0017c
		FtsW/RodA/SpoVE family
		cell cycle protein

SCO3883	StH24.05	putative membrane	MM5485	MT_4040	Rv3921c	BG10062
		protein
SCO3886	StH24.08	putative partitioning	MM5482	MT_4037	Rv3918c	BG10055
		or sporulation protein
SCO3887	StH24.09	putative partitioning	MM5481	MT_4036	Rv3917c	BG10054
		or sporulation protein

SCO3934	SCQ11.17	ftsK/spoIIIE family	MM3126
		protein 4328870:
		4330171 forward
		MW: 45953

SCO4767	SC6G4.45c	putative regulatory	MM1132	MT_3525	Rv3416
		protein

SCO5302	SC5G9.31	putative integral	MM0019	MT_0020	Rv0017c
		membrane cell-cycle
		protein

SCO5320

SC6G9.13

whiE protein I

BG12784

SCO5321

SC6G9.12c

polyketide hydroxylase

MM3384

SCO5621	whiG	RNA polymerase sigma							BG10751
		factor WhiG

SCO5633	traSA	integrase fusion				MT_2962		Rv2894c		BG12099
		protein

SCO5750

SC7C7.05

ftsK homolog

MM1967

MT_2819

Rv2748c

BG10763

SCO6435	SC9B5.02	hypothetical protein	MM4895

Distance measure

M. tuberculosis

B. subtilis

S. coelicolor A3(2)

M. marinum

CDC1551

H37Rv

168

Gene	Gene			Dis-		Dis-		Dis-		Dis-
id	name	Annotation	Expect^b	tance^c	Expect^b	tance^c	Expect^b	tance^c	Expect^b	tance^c

SCO0506	nadE2	NH(3)-dependent							2.7E−69	1.0
		NAD(+)synthetase
		(nadE1)
SCO1024	SCG20A.04	conserved		2.9					1.1E−53	1.6
		hypothetical
		protein
SCO1177	SCG11A.08	putative gntR-famly				2.6		2.6
		transcriptional
		regulator
SCO1434	SC6D7.05c	putative CbxX/CfqX	1.7E−64	2.6	3.8E−65	2.5	3.5E−65	2.5
		family protein
SCO1772	SCI51.12c	putative partitioning	2.6E−98	0.7	2.3E−90	0.5	4.8E−100	0.6
		or sporulation protein
SCO1950	SCC54.10c	hypothetical protein	3.5E−119	0.4	2.7E−119	0.4	2.5E−119	0.4	1.1E−26	2.9
SCO2083	ftsQ	sporulation protein	2.3E−19	3.1	4.1E−14	3.1	3.8E−14	3.1
SCO2085	ftsW	putative cell division	1.8E−76	1.6	5.1E−77	1.6	4.7E−77	1.6
		protein
SCO2607	sfr	Sfr protein							2.8E−60	1.9
SCO2805	2SCC13.13	conserved hypothetical			3.8E−04		4.3E−04	5.2
		protein
SCO3034	whiB	sporulation regulatory	4.4E−32	0.4	5.6E−32	0.3	5.2E−32	0.5
		protein
SCO3579	SCH17.13c	putative regulatory	2.6E−27	0.7	2.2E−28	0.7	2.1E−28	0.7
		protein
SCO3846	SCH69.16	putative	2.3E−106		2.5E−107		2.3E−107
		FtsW/RodA/SpoVE family
		cell cycle protein
SCO3883	StH24.05	putative membrane	2.4E−34	2.2	3.5E−35	2.0	3.2E−35	2.0	5.5E−30	2.2
		protein
SCO3886	StH24.08	putative partitioning	1.0E−87	0.6	1.9E−86	0.6	1.8E−86	0.6	1.3E−71	0.9
		or sporulation protein
SCO3887	StH24.09	putative partitioning	5.3E−75	0.9	5.9E−76	0.9	5.4E−76	0.9	2.4E−52	1.5
		or sporulation protein
SCO3934	SCQ11.17	ftsK/spoIIIE family	2.7E−21
		protein 4328870:
		4330171 forward
		MW: 45953
SCO4767	SC6G4.45c	putative regulatory	2.1E−32	0.8	1.9E−31	0.8	1.7E−31	0.8
		protein
SCO5302	SC5G9.31	putative integral		1.1		1.2		1.2
		membrane cell-cycle
		protein
SCO5320	SC6G9.13	whiE protein I							1.3E−06	2.6
SCO5321	SC6G9.12c	polyketide hydroxylase	8.5E−40	3.1
SCO5621	whiG	RNA polymerase sigma							6.9E−48	1.6
		factor WhiG
SCO5633	traSA	integrase fusion				3.0		2.8		3.6
		protein
SCO5750	SC7C7.05	ftsK homolog	1.1E−219	1.0	1.35−218	1.4	1.2E−218	0.9	2.5E−122	1.9
SCO6435	SC9B5.02	hypothetical protein	1.5E−04

^aPutative ortholog gene identifier. Gene identifiers justified to the left or right are identified using the RBH or RSD approaches, respectively. A centered gene identifier was identified by both approaches.
^bExpect value of putative RBH ortholog from forwand BLAST search using S. coelicolor sporulation gene as query
^cDistance estimated by RSD approach

TABLE 3

Table 3: Functional annotations for putative sporulation gene orthologues of B. subtilis in Mycobacterium spp. and S. Coelicolor

B. subtilis 168

M. marinum

M. tuberculosis CDC1551

	Gene		Putative		Putative
Gene id^a	name	Annotation	ortholog	Annotation	ortholog	Annotation

BG10054	SpoOJ	chromosome positioning near	MM5481	undefined product	MT_4036	ParB family protein
		the pole and transport
		through the polar septum/
		antagonist of Soj-dependent
		inhibition of sporulation
		initiation
BG10055	Soj	centromere-like function	MM5482	undefined product	MT_4037	Soj family protein
		involved in forespore
		chromosome partitioning/
		negative regulation of
		sporulation initiation
BG10061	Jag	SpoIIIJ-associated protein	MM5484	undefined product	MT_4039	R3H domain-containing protein
BG10062	SpoIIIJ	essential for sigma-G	MM5485	undefined product	MT_4040	conserved hypothetical protein
		activity at stage III
BG10087	BofA	inhibition of the	MM0678	undefined product
		pro-sigma-K processing
		machinery
BG10116	SpoVC	probable peptidyl-tRNA	MM4473	undefined product	MT_1042	peptidyl-tRNA hydrolase (pth)
		hydrolase				[3.1.1.29]
BG10119	SpoVT	transcriptional positive
		and negative regulator of
		sigma-G-dependent genes
BG10203	SplB	spore photoproduct lyase
BG10226	SpoVE	required for spore cortex	MM3194	undefined product	MT_2213	cell division protein FtsW (ftsW-2)
		peptidoglycan synthesis
BG10296	SpoIIAA	anti-anti-sigma factor	MM5181	undefined product	MT_3789	anti-anti-sigma factor
			MM0737	undefined product
BG10297*	SpoIIAB	anti-sigma factor/serine	MM5186	undefined product
		kinase
BG10298*	SigF	RNA polymerase sporulation	MM1248	undefined product	MT_3385	stress response/stationary phase
		forespore-specific (early)				sigma factor SigF (sigF)
		sigma factor
BG10332	SpoIVFB	membrane metalloprotease	MM2075	undefined product
		required for the processing
		of pro-sigma-K to active
		sigma-K
BG10337	Obg	GTP-binding protein	MM3765	undefined product	MT_2516	GTP-binding protein (obg)
		involved in initiation of
		sporulation (SpoOA
		activation)
BG10346	SpoVID	required for assembly of	MM4057	undefined product	MT_3573	integrase, putative
		the spore coat
BG10458	SpoIVCA	site-specific DNA	MM2129	undefined product
		recombinase required for
		creating the sigK gene	pMM23-15	undefined product
		(excision of the skin
		element)
BG10490	CotA	spore coat protein (outer)	MM1618	undefined product
BG10493	CotD	spore coat protein (inner)	MM4853	undefined product
BG10495	CotT	spore coat protein (inner)	MM4208	undefined product	MT_1271	hypothetical protein
BG10611	SpsC	spore coat polysaccharide	MM2320	undefined product
		synthesis
BG10613	SpsE	spore coat polysaccharide
		synthesis
BG10617	SpsI	spore cost polysaccharide	MM0606	undefined product	MT_0348	glucose-1-phosphate
		synthesis				thymidylyltransferase
						(rfbA) [2.7.7.24]
BG10618	SpsJ	spore coat polysaccharide	MM1082	undefined product	MT_3570	dTDP-glucose-4,6-dehydratase
		synthesis				(rfbB) [4.2.1.46]
BG10619	SpsK	spore coat polysaccharide	MM1275	undefined product	MT_3366	dTDP-4-dehydrorhamnose reductase
		synthesis				(strL)
BG10763	SpoIIIE	DNA translocase required	MM1967	undefined product	MT_2819	cell division protein FtsK (ftsK)
		for chromosome partitioning
		through the septum into the
		forespore
BG10765*	SpoOA	two-component response	MM4455	undefined product	MT_1062	DNA-binding response regulator
		regulator central for the				TrcR (trcR)
		initiation of sporulation
BG11039	SpoVK	disruption leads to the	MM0541	undefined product	MT_0295	ATPase, AAA family
		production of immature
		spores
BG11197	CgeE	maturation of the outermost			MT_2818	acetyltransferase, GNAT family
		layer of the spore
BG11231	KipI	inhibitor of KinA	MM0524	undefined product	MT_0277	conserved hypothetical protein
BG11381	CotSA	spore coat protein	MM4226	undefined product	MT_0504	glycosyl transferase
			MM0812	undefined product
BG11687	YqgT	unknown; similar to
		gamma-D-glutamyl-L-diamino
		acid endopeptidase I
BG11806	Tlp	small acid-soluble spore	MM3184	undefined product
		protein (thioredoxin-like
		protein)
BG12181	YdhD	unknown; similar to unknown
		proteins from B. subtilis
BG12229	SpoQM	sporulation-control gene
BG12358	KapD	inhibitor of the KinA
		pathway to sporulation
BG13288	YkuD	unknown; similar to unknown			MT_0125	conserved hypothetical protein
		proteins
BG13781	SafA	morphogenetic protein	MM0022	undefined product	MT_1271	hypothetical protein
		associated with SpoVID
BG13783	YrbC	unknown; similar to spore	MM2098	undefined product	MT_2678	conserved hypothetical protein
		coat protein
BG13907	YtpT	unknown; simitar to DNA	MM1967	undefined product	MT_2819	cell division protein FtsK (ftsK)
		translocase stage III
		sporulation protein
		(SpoIIIE)
BG14193	KipA	antagonist of KipI	MM0523	undefined product	MT_0276	urea amidolyase-related protein
8G14195	SpsL	spore coat polysaccharide	MM1081	undefined product	MT_3571	dTDP-4-dehydrorhamnose
		synthesis				3,5-epimerase (strM)
						[5.1.3.13]

B. subtilis 168

M. tuberculosis H37Rv

S. coelicolor A3(2)

	Gene		Putative		Putative
Gene id^a	name	Annotation	ortholog	Annotation	ortholog	Annotation

BG10054	SpoOJ	chromosome positioning near	Rv3917c	(MTV028.08c), len: 344.	SCO3887	putative partitioning or
		the pole and transport		Unknown, similar to TR: O0519D		sporulation protein
		through the polar septum/		CHROMOSOME PARTITIONING
		antagonist of Soj-dependent		PROTEIN PARB from
		inhibition of sporulation		CAULOBACTER CRESCENTUS
		initiation		(293 aa). fasta scores; opt:
				564 z-score: 784.2 E( ): 0,
				38.6% identity in 308 aa
				overlap; and to SPOJ_BACSU
				P26497 stage 0 sporulation pr
				(parA)
BG10055	Soj	centromere-like function	Rv3918c	(MTV028.09c), len: 347.	SCO3886	putative partitioning or
		involved in forespore		Unknown, similar to		sporulation protein
		chromosome partitioning/		TR:O05189(EMBL: U87804)
		negative regulation of		CHROMOSOME PARTITIONING
		sporulation initiation		PROTEIN PARA from
				CAULOBACTER CRESCENTUS
				(266 aa), fasta scores; opt:
				787 z-score: 990.9 E( ): 0,
				50.6% identity in 261 aa
				overlap and to SOJ_BACSU
				P37522 soj protei (parB)
BG10061	Jag	SpoIIIJ-associated protein	Rv3920c	hypothetical protein	SCO3884	conserved hypothetical
						protein
BG10062	SpoIIIJ	essential for sigma-G	Rv3921c	hypothetical protein	SCO3883	putative membrane
		activity at stage III				protein
BG10087	BofA	inhibition of the
		pro-sigma-K processing
		machinery
BG10116	SpoVC	probable peptidyl-tRNA	Rv1014c	(MTCY10G2.35), pth, len: 191.	SCO3125	peptidyl-tRNA
		hydrolase		Probable peptidyl-tma		hydrolase
				hydrolase, similar to eg
				PTH_ECOLI P23932 peptidyl-tma
				hydrolase (ec 3.1.1.29)
				(194 aa), fasta scores, opt:
				472, E( ): 2.3e−25, (39.6%
				identity in 187 aa overlap)
				(pth)
BG10119	SpoVT	transcriptional positive	Rv2595	hypothetical protein
		and negative regulator of
		sigma-G-dependent genes
BG10203	SplB	spore photoproduct lyase			SCO7402	putative lyase
BG10226	SpoVE	required for spore cortex	Rv2154c	(MTCY270.14), len: 524. ftsW,	SCO2607	Sfr protein
		peptidoglycan synthesis		probable cell division
				protein FtsW, related to
				MTCY10H4.17C, 3.2e−17. FASTA
				best: SP5E_BACSU P07373 stage
				v sporulation protein
				e(366 aa) opt: 755 z-score:
				727.2 E( ): 1.6e−33; (38.4%
				identity in 357 aa overlap)
				(ftsW)
BG10296	SpoIIAA	anti-anti-sigma factor	Rv3687c	hypothetical protein	SCO3067	putative anti anti sigma
						factor
BG10297*	SpoIIAB	anti-sigma factor/serine			SCO7322	putative anti-sigma
		kinase				factor (fragment)
BG10298*	SigF	RNA polymerase sporulation	Rv3286c	(MTCY71.26), len: 261, sigF,	SCO5243	RNA polymerase sigma
		forespore-specific (early)		almost identical to		factor
		sigma factor		M. tuberculosis MTU41061_1
				Q50547, stress response/
				stationary phase sigma factor
				f (261 aa). Also similar to
				U00012_11
				Mycobacterium leprae Q49668
				RPSB (118 aa, 71.2% identity
				in 111 aa overlap) Contains p
				(sigF)
BG10332	SpoIVFB	membrane metalloprotease			SCO1652	conserved hypothetical
		required for the processing				protein
		of pro-sigma-K to active
		sigma-K
BG10337	Obg	GTP-binding protein	Rv2440c	(MTCY428.06), len: 479.	SCO2595	GTP-binding protein
		involved in initiation of		Probable obg, nucleotide-
		sporulation (SpoOA		binding protein, equivalent
		activation)		to Streptomyces griseus
				D87916\|D87916_3 (478 aa)
				fasta scores, opt 1328
				z-score: 1443.0 E( ): 0;
				58.9% identity in 479 aa
				overlap; also similar to eg
				OBG_BACSU P20964 spoOb-ass
				(obg)
BG10346	SpoVID	required for assembly of	Rv1586c	hypothetical protein	SCO6405	putative DNA
		the spore coat				recombinase
BG10458	SpoIVCA	site-specific DNA
		recombinase required for
		creating the sigK gene
		(excision of the skin
		element)
BG10490	CotA	spore coat protein (outer)			SCO3440	hypothetical protein
BG10493	CotD	spore coat protein (inner)
BG10495	CotT	spore coat protein (inner)	Rv1233c	hypothetical protein
BG10611	SpsC	spore coat polysaccharide	Rv1504c	hypothetical protein	SCO5746	hypothetical protein
		synthesis
BG10613	SpsE	spore coat polysaccharide			SCO4881	putative polysaccharide
		synthesis				biosynthesis related
						protein
BG10617	SpsI	spore cost polysaccharide	Rv0334	(MTCY279.01), len: 288. rfbA,	SCO1388	putative mannose-1-
		synthesis		glucose-1-phosphate		phosphate
				thymidylyl-transferase highly		guanyltransferase
				similar to many eg.
				RBA1_ECOLI P37744 glucose-1-
				phosphate thymidylyltransferase
				(293 aa), fasta scores; opt:
				1199 z-score: 1884.1 E( ): 0,
				62.0% identity in 284 aa
				overlap (rmlA)
BG10618	SpsJ	spore coat polysaccharide	Rv3464	(MTCY13E12.17), len: 331.	SCO0749	putative dehydratase
		synthesis		rmlB. Function: probable
				DTDP-GLUCOSE 4,6-DEHYDRATASE
				nearly identical to
				Mycobacterium tuberculosis
				rhamnose biosynthesis protein
				(previously rfbB, now known
				as rmlB) gene. FASTA results:
				Q50556 RHAMNOSE BIOSYNTHESIS
				PROTEIN ((rmlB)
BG10619	SpsK	spore coat polysaccharide	Rv3266c	(MTCY71.08c), len: 304, rfbD,	SCO7194	putative polysaccharide
		synthesis		similar to eg STRL_STRGR		biosynthesis protein
				P29781 didp-4-dehydrorhamnose
				reductase (304 aa), fasta
				scores, opt: 788, E( ): 0,
				(47.4% identity in 304 aa
				overlap) (rmlD)
BG10763	SpoIIIE	DNA translocase required	Rv2748c	(MTV002.13c), len: 883 aa.	SCO5750	ftsK homolog
		for chromosome partitioning		Possible ftsK, member of the
		through the septum into the		spoIIIE/ftsK family; similar
		forespore		in C-terminal half to
				SP3E_BACSU P21458 stage iii
				sporuation protein e
				(787 aa), fasta scores;
				opt: 1294 z-score: 1488.5
				E( ): 0, 50.9% identity in
				485 aa overlap, and (ftsK)
BG10765*	SpoOA	two-component response	Rv1033c	hypothetical protein	SCO2013	putative two-component
		regulator central for the				system response
		initiation of sporulation				regulator
BG11039	SpoVK	disruption leads to the	Rv0282	hypothetical proiein	SCO1024	conserved hypothetical
		production of immature				protein
		spores
BG11197	CgeE	maturation of the outermost	RV2747	hypothetical profein
		layer of the spore
BG11231	KipI	inhibitor of KinA	Rv0254c	hypothetical protein	SCO0442	conserved hypothetical
						protein
BG11381	CotSA	spore coat protein	Rv0486	hypothetical protein	SCO2132	putative glycosyl
						transferaseputative
					SCO4204	glycosyltransferase
BG11687	YqgT	unknown; similar to			SCO1948	putative zinc-binding
		gamma-D-glutamyl-L-diamino				carboxypeptidase
		acid endopeptidase I
BG11806	Tlp	small acid-soluble spore
		protein (thioredoxin-like
		protein)
BG12181	YdhD	unknown; similar to unknown			SCO1429	chitinase (putative
		proteins from B. subtilis				secreted protein) (chiD)
BG12229	SpoQM	sporulation-control gene			SCO0247	hypothetical protein
BG12358	KapD	inhibitor of the KinA			SCO7397	conserved hypothetical
		pathway to sporulation				protein
BG13288	YkuD	unknown; similar to unknown	Rv0116c	hypothetical protein
		proteins
BG13781	SafA	morphogenetic protein	Rv1233c	hypothetical protein
		associated with SpoVID
BG13783	YrbC	unknown; similar to spore	Rv2603c	hypothetical protein	SCO1521	conserved hypothetical
		coat protein				protein
BG13907	YtpT	unknown; simitar to DNA
		translocase stage III
		sporulation protein
		(SpoIIIE)
BG14193	KipA	antagonist of KipI	Rv0263c	hypothetical protein	SCO0443	conserved hypothetical
						protein
8G14195	SpsL	spore coat polysaccharide	Rv3465	(MTCY13E12.16), len: 202.	SCO0400	putative epimerase
		synthesis		rmlC. Probable
				dtdp-4-dehydrorhamnose
				3,5-epimerase. Nearly
				identical to M. tuberculosis
				sp\|O33170\|O33170
				RMLC PROTEIN
				(203 aa) opt: 1171 z-score;
				1465.1 E( ): 0; 89.5%
				identity in 200 aa overlap.
				Previously known as rfbC,
				FASTA (rmlC)

^aIds marked with asterisk indicates a weak putative ortholog (see Methods)

TABLE 4

Table 4: Functional annotations for putative sporulation gene orthologues
of S. coelicolor in Mycobacterium spp. and B. subtilis

M. marinum

M. tuberculosis CDC1551

S. coelicolor A3(2)

Putative

Gene id	Gene name	Annotation	ortholog	Annotation	ortholog	Annotation

SCO0506	nadE2	NH(3)-dependent
		NAD(+)synthetase
		(nadE1)
SCO1024	SCG20A.04	conserved hypothetical	MM0541	undefined product
		protein
SCO1177	SCG11A.08	putative gntR-famly			MT_0514	transcriptional regulator, GntR family
		transcriptional
		regulator
SCO1434	SC6D7.05c	putative CbxX/CfqX	MM5443	undefined product	MT_3981	ATPase, AAA family
		family protein
SCO1772	SCIS1.12c	putative partitioning	MM2523	undefined product	MT_1749	Soj family protein
		or sporulation
		protein
SCO1950	SCC54.10c	hypothetical protein	MM2230	undefined product	MT_1466	conserved hypothetical protein
SCO2083	ftsQ	sporulation protein	MM3191	undefined product	MT_2210	cell division protein FtsQ (ftsQ)
SCO2085	ftsW	putative cell	MM3194	undefined product	MT_2213	cell division protein FtsW (ftsW-2)
		division protein
SCO2607	sfr	Sfr protein
SCO2805	2SCC13.13	conserved hypothetical			MT_1099	hypothetical protein
		protein
SCO3034	whiB	sporulation regulatory	MM1282	undefined product	MT_3358	WhiB-related protein
		protein
SCO3579	SCH17.13c	putative regulatory	MM5170	undefined product	MT_3783	WhiB-related protein
		protein
SCO3846	SCH69.16	putative	MM0019	undefined product	MT_0020	cell division protein FtsW (ftsW-1)
		FtsW/RodA/SpoVE
		family cell
		cycle protein
SCO3883	StH24.05	putative membrane	MM5485	undefined product	MT_4040	conserved hypothetical protein
		protein
SCO3886	S1H24.08	putative	MM5482	undefined product	MT_4037	Soj family protein
		partitioning or
		sporulation protein
SCO3887	StH24.09	putative	MM5481	undefined product	MT_4036	ParB family protein
		partitioning or
		speculation protein
SCO3934	SCQ11.17	ftsK/spoIIIE family	MM3126	undefined product
		protein 4328870:
		4330171 forward
		MW: 45953
SCO4767	SC6G4.45c	putative regulatory	MM1132	undefined product	MT_3525	WhiB-related protein
		protein
SCO5302	SC6G9.31	putative integral	MM0019	undefined product	MT_0020	cell division protein FtsW (ftsW-1)
		membrane
		cell-cycle
		protein
SCO5320	SC6G9.13	whiE protein I
SCO5321	SC6G9.12c	polyketide	MM3384	undefined product
		hydroxylase
SCO5621	whiG	RNA polymerase
		sigma factor
		WhiG
SCO5633	traSA	integrase fusion			MT_2962	tyrosine recombinase XerC (xerC)
		protein
SCO5750	SC7C7.05	ftsK homolog	MM1967	undefined product	MT_2819	cell division protein FtsK (ftsK)
SCO6435	SC9B5.02	hypothetical protein	MM4895	undefined product

M. tuberculosis H37Rv

B. subtilis 168

S. coelicolor A3(2)

Putative

Gene id	Gene name	Annotation	ortholog	Annotation	ortholog	Annotation

SCO0506	nadE2	NH(3)-dependent			BG10694	NadE: NH3-dependent
		NAD(+)syntnetase				NAD* synthetase
		(nadE1)
SCO1024	SCG20A.04	conserved hypothetical			BG11039	SpoVK: disruption
		protein				leads to the
						production of
						immature spores
SCO1177	SCG11A.08	putative gntR-famly	Rv0494	hypothetical protein
		transcriptional
		regulator
SCO1434	SC6D7.05c	putative CbxX/CfqX	RV3868	hypothetical protein
		family protein
SCO1772	SCI51.12c	putative partitioning	Rv1708	hypothetical protein
		or sporulation
		protein
SCO1950	SCC54.10c	hypothetical protein	Rv1423	hypothetical protein	BG12402	YvcL: unknown;
						similar to
						unknown proteins
SCO2083	ftsQ	sporulation protein	Rv2151c	(MTCY270.17), len: 314.
				Function ftsQ: possible
				cell division protein,
				some homology to
				FTSQ_STRGR P45503
				cell division protein
				ftsq homolog (208 aa),
				fasta scores; opt: 204
				z-score: 217.5
				E( ): 4e−05;
				(30.6% identity
				in 193 aa overlap)
				(ftsQ)
SCO2085	ftsW	putative cell	Rv2154c	(MTCY270.14), len: 524.
		division protein		ftsW, probable cell
				division protein FtsW,
				related to
				MTCY10H4.17c,
				3.2e−17. FASTA
				best SP5E_BACSU
				P07373 stage v
				sporulation protein
				e(366 aa) opt:
				755 z-score: 727.2
				E( ): 1.6e−33;
				(38.4% identity
				in 357 aa overlap)
				(ftsW)
SCO2607	sfr	Sfr protein			BG10226	SpoVE: required
						for spore
						cortex
						peptidoglycan
						synthesis
SCO2805	2SCC13.13	conserved hypothetical	Rv1069c	hypothetical protein
		protein
SCO3034	whiB	sporulation regulatory	Rv3260c	(MTV015.05c), len: 89.
		protein		whiB, Probable
				regulatory protein
				very similar to
				S. griseocarneum
				WhiB SGWHIB_1
				(87 aa) and
				other Streptomyces
				and mycobacterial
				WhiB homologues.
				Start chosen by
				homology but orf
				continues to ATG
				upstream at 3754.
				FASTA scores:
				gp (whiB2)
SCO3579	SCH17.13c	putative regulatory	Rv3681c	(MTV025.029c), len: 100.
		protein		Probable regulatory
				protein, similar to
				many e.g.
				gp\|X62287\|SCWHIB_1
				S. coelicolor whiBgene
				(87 aa), FASTA scores:
				opt: 237 z-score:
				332.1 E():
				5.5e−11; 45.2%
				identity in 73 aa
				overlap. Also
				similar to several
				mycobacterial
				putativ (whiB4)
SCO3846	SCH69.16	putative	Rv0017c	(MTCY10H4.17c), len: 469,
		FtsW/RodA/SpoVE		highly similar to
		family cell		M. leprae MLCB1770_12,
		cycle protein		(465 aa), opt: 2475
				z-score: 3029.6E( ):
				0; E235744 cell
				division protein,
				(81.9% identity in
				469 aa overlap).
				Also similar to
				MTCY270_14, (524,
				32.2% identity in
				369 aa overlap)
				(rodA)
SCO3883	StH24.05	putative membrane	Rv3921c	hypothetical protein	BG10062	SpoIIIJ: essential
		protein				for sigma-G
						activity at
						stage III
SCO3886	S1H24.08	putative	Rv3918c	(MTV028.09c), len: 347.	BG10055	Soj: centromere-
		partitioning or		Unknown, similar to		like function
		sporulation		TR: 005189(EMBL:		involved in
		protein		U87804) CHROMOSOME		forespore
				PARTITIONING		chromosome
				PROTEIN PARA from		partitioning/
				CAULOBACTER		negative
				CRESCENTUS		regulation of
				(266 aa), fasta		sporulation
				scores; opt: 787		initiation
				z-score: 990.9 E( ):
				0, 50.6% identity in
				261 aa overlap and to
				SOJ_BACSU P37522
				soj protei (parB)
SCO3887	StH24.09	putative	Rv3917c	(MTV028.08c), len: 344,	BG10054	SpoOJ:
		partitioning or		Unknown, similar to		chromosome
		speculation		TR: 005190 CHROMOSOME		positioning
		protein		PARTITIONING PROTEIN		near the
				PARB from		pole and
				CAULOBACTER		transport
				CRESCENTUS		through the
				(293 aa), fasta scores;		polar septum/
				opt: 564 z-score:		antagonist of
				784.2 E( ): 0,		Soj-dependent
				38.6% identity in		inhibition of
				308 aa overlap; and		sporulation
				to SPOJ_BACSU		initiation
				P26497 stage 0
				sporulation pr (parA)
SCO3934	SCQ11.17	ftsK/spoIIIE family
		protein 4328870:
		4330171 forward
		MW: 45953
SCO4767	SC6G4.45c	putative regulatory	Rv3416	(MTCY7B.13c), len: 102.
		protein		whiB, regulatory
				protein, highly
				similar to U00020_2
				Mycobacterium leprae
				cosmid B229 and
				M. leprae
				U00015_26 cosmid
				B1620(102 aa) opt:
				657, z-score: 807.1,
				E( ): 0, (86.3%
				identity in 102 aa
				overlap) (whiB3)
SCO5302	SC6G9.31	putative integral	Rv0017c	(MTCY10H4.17c), len: 469,
		membrane		highly similar to
		cell-cycle		M. leprae
		protein		MLCB1770_12, (465 aa),
				opt: 2475 z-score:
				3029.6E( ): 0;
				E235744 cell division
				protein, (81.9%
				identity in 469 aa
				overlap). Also similar
				to MTCY270_14, (524,
				32.2% identity in
				369 aa overlap) (rodA)
SCO5320	SC6G9.13	whiE protein I			BG12784	YczJ: unknown;
						similar to
						unknown proteins
SCO5321	SC6G9.12c	polyketide
		hydroxylase
SCO5621	whiG	RNA polymerase			BG10751	SigD: RNA
		sigma factor				polymerase
		WhiG				flagella,
						motility,
						chemotaxis and
						autolysis
						sigma factor
SCO5633	traSA	integrase fusion	Rv2894c	(MTCY274,25c), len: 298.	BG12099	YdcL: unknown;
		protein		xerC, integrase/		similar to
				recombinase, most		integrase
				similar to
				XERC_HAEIN P44818
				integrase/recombinase
				xerc, (295 aa), opt:
				627, z-score: 726.6,
				E( ): 6.3e−34,
				(39.9% identity in
				296 aa overlap).
				M. leprae
				equivalent is
				sp\|O33037\|O33037
				XERC PROTE (xerC)
SCO5750	SC7C7.05	ftsK homolog	Rv2748c	(MTV002.13c), len: 883 aa.	BG10763	SpoIIIE; DNA
				Possible ftsK, member of		translocase
				the spoIIIE/ftsK family;		required for
				similar in C-terminal		chromosome
				half to SP3E_BACSU		partitioning
				P21458 stage iii		through the
				sporulation protein		septum into
				e (787 aa), fasta		the forespore
				scores; opt: 1294
				z-score: 1488.5
				E( ): 0, 50.9%
				identity in
				485 aa overlap, and
				(ftsK)
SCO6435	SC9B5.02	hypothetical protein

It is also contemplated that the immunogenic peptides can be derived from homologues of the above mentioned proteins from other Mycobacteria, e.g. M. tuberculosis, M. leprae, M. bovis, M. avium, M. microti, M. intracellulare, M. paratuberculosis, M. ulcerans, or M. africanum.
Immunogenic fragments typically have a sequence of at least 3, 5, 6, 10 or 20 contiguous amino acids from a natural peptide.
Analogs include allelic, species and induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80% or 90% sequence identity with natural peptides. Some analogs also include unnatural amino acids or modifications of N or C terminal amino acids. Examples of unnatural amino acids are a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,- carboxyglutamate, e-N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, —N-methylarginine
Fragments and analogs can be screened for prophylactic or therapeutic efficacy in disease models as described below.
A protein chosen from one of those encoded by one of the genes in Tables 1-4, their fragments, analogs and spore forming peptides can be synthesized by solid phase peptide synthesis or recombinant expression, or can be obtained from natural sources. Automatic peptide synthesizers are commercially available from numerous suppliers, such as Applied Biosystems, Foster City, Calif. Recombinant expression can be in bacteria, such as E. coli, yeast, insect cells or mammalian cells. Procedures for recombinant expression are described by Sambrook et al., Molecular Cloning: A Laboratory Manual (C. S. H. P. Press, NY 2d ed., 1989).
Therapeutic agents also include longer polypeptides that include, for example, an spore forming peptide, active fragment or analog together with other amino acids. Therapeutic agents also include multimers of monomeric immunogenic agents.
In a further variation, an immunogenic peptide can be presented as a viral or bacterial vaccine. A nucleic acid encoding the immunogenic peptide is incorporated into a genome or episome of the virus or bacteria. Optionally, the nucleic acid is incorporated in such a manner that the immunogenic peptide is expressed as a secreted protein or as a fusion protein with an outer surface protein of a virus or a transmembrane protein of a bacteria so that the peptide is displayed. Viruses or bacteria used in such methods should be nonpathogenic or attenuated. Suitable viruses include adenovirus, HSV, vaccinia and fowl pox. Fusion of an immunogenic peptide to HBsAg of HBV is particularly suitable.
Random libraries of peptides or other compounds can also be screened for suitability. Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates. Large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which is incorporated by reference for all purposes). Peptide libraries can also be generated by phage display methods. See, e.g., Devlin, WO 91/18980.
Combinatorial libraries and other compounds are initially screened for suitability by determining their capacity to bind to antibodies or lymphocytes (B or T) known to be specific for spore forming peptides. For example, initial screens can be performed with any polyclonal sera or monoclonal antibody to a spore forming peptide.
Compounds identified by such screens are then further analyzed for capacity to induce antibodies or reactive lymphocytes to a spore forming peptide. For example, multiple dilutions of sera can be tested on microtiter plates that have been precoated with a spore forming peptide and a standard ELISA can be performed to test for reactive antibodies to a spore forming peptide. Compounds can then be tested for prophylactic and therapeutic efficacy in animal models
Therapeutic agents of the invention also include antibodies that specifically bind to a spore forming peptide. Such antibodies can be monoclonal or polyclonal. The production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a spore forming peptide. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression.
Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861 (incorporated by reference for all purposes).
Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outersurfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to a spore forming peptide, or fragments thereof. Human antibodies against a spore forming peptide can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991) (each of which is incorporated by reference in its entirety for all purposes). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies.
Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent.
Optionally, such polyclonal antibodies can be concentrated by affinity purification using a spore forming peptide as an affinity reagent.
Human or humanized antibodies can be designed to have IgG, IgD, IgA and IgE constant region, and any isotype, including IgG1, IgG2, IgG3 and IgG4. Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab′F (ab′)2, and Fv, or as single chain antibodies in which heavy and light chain variable domains are linked through a spacer.
Therapeutic agents for use in the present methods also include T-cells that bind to a spore forming peptide. For example, T-cells can be activated against a spore forming peptide by expressing a human MHC class I gene and a human β-2-microglobulin gene from an insect cell line, whereby an empty complex is formed on the surface of the cells and can bind to a spore forming peptide. T-cells contacted with the cell line become specifically activated against the peptide. See Peterson et al., U.S. Pat. No. 5,314,813. Insect cell lines expressing an MHC class II antigen can similarly be used to activate CD4 T cells.
Some agents for inducing an immune response contain the appropriate epitope for inducing an immune response against a spore coating peptide but are too small to be immunogenic. In this situation, a peptide immunogen can be linked to a suitable carrier to help elicit an immune response. Suitable carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria, E. coli, cholera, or H. pylori, or an attenuated toxin derivative. Other carriers for stimulating or enhancing an immune response include cytokines such as IL-1, IL-1a and peptides, IL-2, zINF, IL-10, GM-CSF, and chemokines, such as MlPla and 0 and RANTES Immunogenic agents can also be linked to peptides that enhance transport across tissues, as described in O'Mahony, WO 97/17613 and WO 97/17614.
Immunogenic agents can be linked to carriers by chemical crosslinking. Techniques for linking an immunogen to a carrier include the formation of disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue). These reagents create a disulfide linkage between themselves and peptide cysteine resides on one protein and an amide linkage through the ε-amino on a lysine, or other free amino group in other amino acids. A variety of such disulfide/amide-forming agents are described by Immun Rev. 62, 185 (1982). Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid.
The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
Immunogenic peptides can also be expressed as fusion proteins with carriers. The immunogenic peptide can be linked at the amino terminus, the carboxyl terminus, or internally to the carrier. Optionally, multiple repeats of the immunogenic peptide can be present in the fusion protein.
Immune responses against spore forming peptides can also be induced by administration of nucleic acids encoding a spore forming peptide. Such nucleic acids can be DNA or RNA. A nucleic acid segment encoding the immunogen is typically linked to regulatory elements, such as a promoter and enhancer, that allow expression of the DNA segment in the intended target cells of a patient. For expression in blood cells, as is desirable for induction of an immune response, promoter and enhancer elements from light or heavy chain immunoglobulin genes or the CMV major intermediate early promoter and enhancer are suitable to direct expression. The linked regulatory elements and coding sequences are often cloned into a vector.
A number of viral vector systems are available including retroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol. 67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox family including vaccinia virus and the avian pox viruses, viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)).
DNA encoding an immunogen, or a vector containing the same, can be packaged into liposomes. Suitable lipids and related analogs are described by U.S. Pat. No. 5,208,036, U.S. Pat. No. 5,264,618, U.S. Pat. No. 5,279,833 and U.S. Pat. No. 5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly (lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap. (1996).
Gene therapy vectors or naked DNA can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, nasal, gastric, intradermal, intramuscular, subdermal, or intracranial infusion) or topical application (see e.g., U.S. Pat. No. 5,399,346). DNA can also be administered using a gene gun. See Xiao & Brandsma, supra. The DNA encoding an immunogen is precipitated onto the surface of microscopic metal beads. The microprojectiles are accelerated with a shock wave or expanding helium gas, and penetrate tissues to a depth of several cell layers. For example, The Accel™ Gene Delivery Device manufactured by Agacetus, Inc. Middleton Wis. is suitable. Alternatively, naked DNA can pass through skin into the blood stream simply by spotting the DNA onto skin with chemical or mechanical irritation (see WO 95/05853).
Patients amenable to treatment include individuals at risk of disease but not showing symptoms, as well as patients presently showing symptoms.
Therefore, the present methods can be administered prophylactically to the general population without any assessment of the risk of the subject patient. The present methods are especially useful for individuals who do have a known risk of being inflicted by a Mycobacterium infection. Such individuals include those having a weakened immune system.
In prophylactic applications, pharmaceutical compositions or medicants are administered to a patient susceptible to, or otherwise at risk of, a particular disease in an amount sufficient to eliminate or reduce the risk or delay the outset of the disease. In therapeutic applications, compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a therapeutically- or pharmaceutically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient immune response has been achieved. Typically, the immune response is monitored and repeated dosages are given if the immune response starts to fade.
Effective doses of the compositions of the present invention, for the treatment of the above described conditions can vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but the patient can be a nonhuman mammal, such as bovine. Treatment dosages need to be titrated to optimize safety and efficacy. The amount of immunogen depends on whether adjuvant is also administered, with higher dosages being required in the absence of adjuvant. The amount of an immunogen for administration sometimes can vary from 1 μg-500 μg per patient and more usually from 5-500 μg per injection for human administration. Occasionally, a higher dose of 0.5-5 mg per injection can be used. Typically about 10, 20, 50 or 100 μg is used for each human injection. The timing of injections can vary significantly from once a day, to once a year, to once a decade. On any given day that a dosage of immunogen is given, the dosage is greater than 1 μg/patient and usually greater than 10 μg/patient if adjuvant is also administered, and greater than 10 μg/patient and usually greater than 100 μg/patient in the absence of adjuvant. A typical regimen consists of an immunization followed by booster injections at 6 weekly intervals. Another regimen consists of an immunization followed by booster injections 1, 2 and 12 months later. Another regimen entails an injection every two months for life. Alternatively, booster injections can be on a regular or irregular basis as indicated by monitoring the immune response.
For passive immunization with an antibody, the dosage can range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg of the host body weight. Doses for nucleic acids encoding immunogens can range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viral vectors can vary from 10-100 virions or more per dose.
Agents for inducing an immune response can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment. The most typical route of administration is subcutaneous although others can be equally effective. The next most common is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles. Intravenous injections as well as intraperitoneal injections, intraarterial, intracranial, or intradermal injections are also effective in generating an immune response. In some methods, agents are injected directly into a particular tissue where deposits have accumulated.
Immunogenic agents of the invention, such as peptides, are sometimes administered in combination with an adjuvant. A variety of adjuvants can be used in combination with a spore forming peptide, to elicit an immune response. Preferred adjuvants augment the intrinsic response to an immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Preferred adjuvants include alum, 3 de-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211). QS21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al., in Vaccine Design: The Subunit and Ajuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540). The spore forming peptides according to the invention are themselves hydrophobic and may also be used as adjuvants.
Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today, Nov. 15, 1998). Alternatively, spore forming peptide can be coupled to an adjuvant. For example, a lipopeptide version of spore forming peptide can be prepared by coupling palmitic acid or other lipids directly to the N-terminus of spore forming peptide as described for hepatitis B antigen vaccination (Livingston, J. Immunol. 159, 1383-1392 (1997)). However, such coupling should not substantially change the conformation of spore forming peptide so as to affect the nature of the immune response thereto. Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent.
A preferred class of adjuvants is aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulating agents such as MPL or 3-DMP, QS21, polymeric or monomeric amino acids such as polyglutamic acid or polylysine. Another class of adjuvants is oil-in-water emulsion formulations. Such adjuvants can be used with or without other specific immunostimulating agents such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′ dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP) Theramide™), or other bacterial cell wall components. Oil-in-water emulsions include (a) MF59 (WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80.5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox).
Another class of preferred adjuvants is saponin adjuvants, such as Stimulons (QS21, Aquila, Worcester, Mass.) or particles generated therefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvants include Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA). Other adjuvants include cytokines, such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF).
An adjuvant can be administered with an immunogen as a single composition, or can be administered before, concurrent with or after administration of the immunogen Immunogen and adjuvant can be packaged and supplied in the same vial or can be packaged in separate vials and mixed before use Immunogen and adjuvant are typically packaged with a label indicating the intended therapeutic application. If immunogen and adjuvant are packaged separately, the packaging typically includes instructions for mixing before use. The choice of an adjuvant and/or carrier depends on the stability of the vaccine containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, Complete Freund's adjuvant is not suitable for human administration. Alum, MPL and QS21 are preferred. Optionally, two or more different adjuvants can be used simultaneously. Preferred combinations include alum with MPL, alum with QS21, MPL with QS21, and alum, QS21 and MPL together. Also, Incomplete Freund's adjuvant can be used (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998)), optionally in combination with any of alum, QS21, and MPL and all combinations thereof.
Agents of the invention can be administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. However, some reagents suitable for administration to animals, such as Complete Freund's adjuvant are not typically included in compositions for human use.
Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).
For parenteral administration, agents of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water, oils, saline, glycerol, or ethanol.
Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
Typically, compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation can also be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see Langer, Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997). The agents of the invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.
For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5 k to 10 k, preferably 1%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.
Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins (See Glenn et al., Nature 391, 851 (1998)). Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
Alternatively, transdermal delivery can be achieved using a skin path or using transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).
The invention provides methods of detecting an immune response against a spore forming peptide in a patient suffering from or susceptible a disease caused by a Mycobacterium. The methods are particularly useful for monitoring a course of treatment being administered to a patient. The methods can be used to monitor both therapeutic treatment on symptomatic patients and prophylactic treatment on asymptomatic patients.
Some methods entail determining a baseline value of an immune response in a patient before administering a dosage of agent, and comparing this with a value for the immune response after treatment. A significant increase (i.e., greater than the typical margin of experimental error in repeat measurements of the same sample, expressed as one standard deviation from the mean of such measurements) in value of the immune response signals a positive treatment outcome (i.e., that administration of the agent has achieved or augmented an immune response). If the value for immune response does not change significantly, or decreases, a negative treatment outcome is indicated. In general, patients undergoing an initial course of treatment with an agent are expected to show an increase in immune response with successive dosages, which eventually reaches a plateau. Administration of agent is generally continued while the immune response is increasing.
Attainment of the plateau is an indicator that the administered of treatment can be discontinued or reduced in dosage or frequency.
In other methods, a control value (i.e., a mean and standard deviation) of immune response is determined for a control population. Typically the individuals in the control population have not received prior treatment. Measured values of immune response in a patient after administering a therapeutic agent can then be compared with the control value. A significant increase relative to the control value (e.g., greater than one standard deviation from the mean) signals a positive treatment outcome. A lack of significant increase or a decrease signals a negative treatment outcome.
Administration of agent is generally continued while the immune response is increasing relative to the control value.
As before, attainment of a plateau relative to control values is an indicator that the administration of treatment can be discontinued or reduced in dosage or frequency.
In other methods, a control value of immune response (e.g., a mean and standard deviation) can be determined from a control population of individuals who have undergone treatment with a therapeutic agent and whose immune responses have plateaued in response to treatment. Measured values of immune response in a patient are compared with the control value. If the measured level in a patient is not significantly different (e.g., more than one standard deviation) from the control value, treatment can be discontinued. If the level in a patient is significantly below the control value, continued administration of agent is warranted. If the level in the patient persists below the control value, then a change in treatment regime, for example, use of a different adjuvant may be indicated.
In other methods, a patient who is not presently receiving treatment but has undergone a previous course of treatment is monitored for immune response to determine whether a resumption of treatment is required. The measured value of immune response in the patient can be compared with a value of immune response previously achieved in the patient after a previous course of treatment. A significant decrease relative to the previous measurement (i.e., greater than a typical margin of error in repeat measurements of the same sample) is an indication that treatment can be resumed. Alternatively, the value measured in patient can be compared with a control value (mean plus standard deviation) determined in population of patients after undergoing a course of treatment.
Alternatively, the measured value in a patient can be compared with a control value in populations of prophylactically treated patients who remain free of symptoms of disease, or populations of therapeutically treated patients who show amelioration of disease characteristics. In all of these cases, a significant decrease relative to the control level (i.e., more than a standard deviation) is an indicator that treatment should be resumed in a patient.
The tissue sample for analysis is typically blood, plasma, serum, mucus or cerebral spinal fluid from the patient. The sample is analyzed for indicia of an immune response to any form of a spore forming peptide, typically SpoVK, CotSA, YrbC, SpoVE, Soj, and SpoIIIE The immune response can be determined from the presence of, e.g., antibodies or T-cells that specifically bind to such a peptide. ELISA methods of detecting antibodies specific to a spore forming peptide are described in the Examples section. Methods of detecting reactive T-cells have been described above (see Definitions).
The invention further provides diagnostic kits for performing the diagnostic methods described above. Such diagnostic methods include detecting the presence of Mycobacteria in patients, food products and in the environment, such as in fish or poultry farms. Typically, kits for such diagnosis or detection contain an agent that specifically binds to antibodies to a spore forming peptide or reacts with T-cells specific for a spore forming peptide. The kit can also include a label. For detection of antibodies to a spore forming peptide, the label is typically in the form of labelled anti-idiotypic antibodies. For detection of antibodies, the agent can be supplied prebound to a solid phase, such as to the wells of a microtiter dish. For detection of reactive T-cells, the label can be supplied as ³H-thymidine to measure a proliferative response. Kits also typically contain labelling providing directions for use of the kit. The labelling may also include a chart or other correspondence regime correlating levels of measured label with levels of antibodies to a spore forming peptide or T-cells reactive with a spore forming peptide. The term labelling refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, the term labelling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.
The binding molecules described herein may be used as diagnostic tools within the field of human and veterinary medicine.
To summarize, the invention provides:
A binding molecule which is capable of binding to a peptide chosen from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, for use as a medicament.
In one embodiment of the invention, the binding molecule is for use in treating, diagnosing or preventing a disease caused by a Mycobacterium.
In one embodiment of the invention, the binding molecule is for treating, diagnosing or preventing a disease caused by a Mycobacterium, wherein the disease is tuberculosis, leprosy, Johnes disease, Crohn's disease, or a disease caused by a non-tuberculous Mycobacterium.
Furthermore, the invention provides an agent effective to induce an immunogenic response against a spore forming peptide, or an antibody thereto, for use as a medicament.
In one embodiment of the invention, the agent effective to induce an immunogenic response against a spore forming peptide, or an antibody thereto, is a peptide chosen from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or a binding molecule capable of binding thereto.
In one embodiment of the invention, an agent effective to induce an immunogenic response against a spore forming peptide, or an antibody thereto, is for treating or preventing a disease caused by a Mycobacterium.
In one embodiment of the invention, an agent effective to induce an immunogenic response against a spore forming peptide, or an antibody thereto, is for treating or preventing a disease selected from tuberculosis, leprosy, Johnes disease, Crohn's disease, or a disease caused by a non-tuberculous Mycobacterium.
In another aspect the invention provides pharmaceutical composition comprising an agent effective to induce an immunogenic response against a spore forming peptide, or an antibody thereto, in a patient, and a pharmaceutically acceptable adjuvant.
Furthermore, the invention provides a pharmaceutical composition according to the above, wherein the agent comprises a spore forming peptide or an active fragment thereof.
Furthermore, the invention provides a pharmaceutical composition as defined herein above, wherein the agent is chosen from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or an antibody thereto.
Furthermore, the invention provides a pharmaceutical composition as defined herein above, wherein the spore forming peptide is chosen from the group comprising CotA, CotD, CotT, CotSA, and YrbC.
Furthermore, the invention provides a pharmaceutical composition according to the above, for use as a medicament.
In another aspect, the invention provides a pharmaceutical composition according to the above, for treating or preventing a disease caused by a Mycobacterium.
In yet another aspect, the invention provides a method of preventing or treating a Mycobacterial infection in a patient, comprising: administering an agent effective to induce an immune response against a peptide component of an spore coating of a Mycobacterium in a mammal.
Furthermore, the invention provides a method according to the above wherein the patient is a human.
Furthermore, the invention provides a method according to the above, wherein the disease is tuberculosis, leprosy, Johnes disease, Crohns disease, or a disease caused by non-tuberculosous Mycobacteria.
Furthermore, the invention provides a method according to the above, wherein the agent comprises a spore forming peptide or an active fragment thereof.
Furthermore, the invention provides a method according to the above, wherein the spore forming peptide is chosen from the group comprising CotA, CotD, CotT, CotSA, YrbC.
Furthermore, the invention provides a method according to the above, wherein the immune response comprises antibodies that bind to the spore forming peptide.
In another aspect, the invention provides a kit comprising a binding molecule which is capable of binding to a peptide chosen from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, and instructions for use.
The kit may be used to detect the presence of Mycobacteria in various samples, such as samples taken from animals, or from the environment around animals, such as from fish or poultry farms.

EXAMPLES

Strains, Media and Growth Conditions

Mycobacterium marinum T CCUG 20998 (ATCC 927) was grown at 30° C. on 7H10 agar plates supplemented with 0.5% glycerol, 10% OADC (oleic acid-albumin-dextrose complex), and 0.01% cycloheximide Mycobacterium bovis BCG was grown at 37° C. on 7H10 agar plates supplemented with 0.5 % Tween 80, 10% OADC, and 0.01% cycloheximide. The Escherichia coli K12 strain MG1655 (Maisnier-Patin, S., Dasgupta, S., Krabbe, M., Nordström, K. Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli. Mol. Microbiol. 30, 1067-1079 (1998)) was grown in M9 media supplemented with 0.2% glucose and 0.2% casamino acids at 37° C.

Flow Cytometry

Cells were harvested at different growth times, fixed, stained, and run in the flow cytometer. The data analyzed as described previously (Maisnier-Patin, S., Dasgupta, S., Krabbe, M., Nordström, K. Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli. Mol. Microbiol. 30, 1067-1079 (1998)). Briefly, the cells were fixed in 70% (final concentration) ice-cold ethanol and stored at 4° C. For flow cytometry, the fixed cells were washed with particle-free (passed through 0.2 micron nitrocellulose filters) 10 mM Tris-HCl (pH 7.9) buffer supplemented with 10 mM MgCl₂. The cells were subsequently resuspended into 0.1× vol of the same buffer. Washed cells were stained with ethidium bromide (0.4 μg/ml, final concentration) and mithramycin A (16 μg/ml, final concentration) and kept on ice for 30 min before flow cytometry (Compact A440 analyser Unit, Apogee Flow Systems, in FIG. 10 BioRad Bryte HS was used). At least 20,000 cells were counted for each flow histogram.

Fluorescence and Phase Contrast Microscopy

The cells were collected and fixed as described above, washed with phosphate buffered saline (PBS) and spotted onto a thin layer of 1% agarose in 0.9% saline containing 0.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) on a microscope slide. A Zeiss (Axioplan 2) microscope with a CCD camera (AxioCam, Zeiss) linked to a computerized image analysis system (Program: Axiovision 4.3) was used for all microscopy. The filter used for DAPI fluorescence was D360/40 (360±20 nm). The phase and fluorescence images were superimposed using the program Axiovision 4.3.

Isolation of Pure Spores

Pure M. marinum spores were isolated as described in previous methods, with slight variations (Lewis P. J., & Errington, J. Mol. Microbia, 25, 945-954 (1997), Long S. K., & Williams, O. B. J. Bacteria 76, 332 (1958), Powers, E. M., Appl. Microbiol. 16, 180-181 (1968)). Cells were collected and harvested as described above from two week (M. marinum) and six month old plates (M. bovis BCG), washed in 0.9% NaCl, and then resuspended in TE-buffer (10 mM Tris-HCl pH 7.9 and 1 mM EDTA). Silica beads (0.1 mm, Q-Biogene, Lysing matrix B) were added to the cells and shaken vigorously. The two suspensions were kept at 4° C. for 5 minutes to allow the glass beads to sediment. The supernatants were collected and then filtered through a sterile 2 micron filter (Millipore). The filtrate was centrifuged at 10,000 r.p.m., for 10 minutes at 4° C. The pellet was resuspended in TE-buffer and lysozyme was added to 3 mg/ml final and incubated at 37° C. for 1 hour. Then SDS was added to 6.25% (v/v final concentration) and incubated at 37° C. for at least 30 min. The lysate was centrifuged at 10,000 r.p.m., for 10 min at 4° C. The pellet was washed 5 times in sterile water at 4° C., and finally resuspended in sterile water. The presence of spores was verified microscopically (FIGS. 6 and 9).

Serial Dilution of Pure Spores to Isolate Single Spore Particle

The idea of serial dilution to isolate a single spore was based on a previous report (Lister, J., Transactions of the Pathological Society of London 29, 425-467 (1878)). A batch of isolated spores was split into two fractions; one was plated freshly and after a week of growth the colonies were counted to estimate the number of viable spore particles in the spore preparation. This number was of the order of 10³/ml. The other fraction was serially diluted 10⁴-fold such that there would be no more than one viable spore in each tube. The so obtained spore solutions were plated on 7H10 plates with necessary supplements (see above) and the plates were incubated at 30° C.

Scanning Electron Microscopy (SEM)

Samples for SEM were prepared as described previously (Lackner P. et al., Malar J. 5, 116 (2006). Briefly, cells were grown and collected as described above, fixed in 2.5% glutaraldehyde, washed 3× in 0.1 M PBS followed by fixation in 1% osmium tetraoxide. The fixed cells were washed 3× in 0.1 M PBS, dehydrated in ethanol (successively in 50%, 70%, 90%, 95%, and absolute ethanol), injected through Millipore filters (2 micron) and dried. The filters with samples were mounted on 12 mm Cambridge stubs using Carbon tape and sputtered with gold. Examinations of the samples were conducted using a Zeiss Supra 35-VP Field Emission SEM at 4 kV.

Transmission Electron Microscopy (TEM)

Samples for TEM were prepared as described previously (Reynolds, E. S., 1963. J. Cell Biol. 17, 208-212). Briefly, cells were grown and collected as described above, fixed in 2.5% glutaraldehyde, washed in 0.1 M sodium caccodylate buffer pH 7.2, and fixed with 1% osmium tetraoxide in sodium caccodylate buffer. The fixed cells were dehydrated in ethanol (successively in 70%, 95%, and absolute ethanol), treated with propylene oxide (transitional solvent), infiltrated in a mixture of propylene oxide and resin (Epon) and then embedded in pure resin mixture. This was followed by curing at 60° C. Thin sections of 50 nm were generated using an LKB 2088 ultrotom V (V=5), applied on copper slot grids and stained separately with 5% uranyl acetate and 3% lead citrate. Images were examined and photographed in a TEM Hitachi H-7100 at 75 KV.
Differential Staining of M. marinum Spores and Vegetative Cells
This was done as described previously (Cappuccino, J. G., Sherman, N. Microbiology, a Laboratory Manual (Benjamin Cummings, San Francisco, sixth edition, 2001)). Cells were grown as described above and harvested at different stages of growth. The cells were resuspended in water, heat-fixed on microscopic slides, heat-stained with malachite green (5% w/v in water), washed with water, and then counterstained with safranin (2.5% w/v in ethanol). After counterstaining the samples were washed with water and cells were visualized with an Olympus CH30RF200 microscope. Photographs were taken with a FujiFilm FinePix 2400Z camera.

Wet-Heat Treatment of Cells

This was performed as described previously (Chakrabarty, K. et al., Infection and Immunity. 74, 4430-4438 (2006). Cells grown as described above were harvested at different times during growth, resuspended in water and divided in two equal aliquots. One aliquot was exposed to heat, 65° C., for different times and then plated on 7H10 agar plates with necessary supplements (see above) and grown at 30° C. for 5 days; the other was without exposure to heat and plated as a control.

TABLE 5

Percent survival of M. marinum cells after wet heat treatment

time of heat	% survival	% survival
treatment(min)	1 day cells	7 day cells

Set1

0	100	100
2	49	95
5	17	63
10	2	44
15	0	39
30	0	34

Set2

0	100	100
2	47	97
5	19	59
10	2	49
15	0	36
30	0	37

Set3

0	100	100
2	46	90
5	22	69
10	3	40
15	0	41
30	0	28

	% survival		% survival
	1 day cells		7 day cells
time of heat	calculated	calculated	calculated	calculated
treatment(min)	average	error	average	error

0	100	0	100	0
2	47.33	1.53	94	3.61
5	19.33	2.52	63.67	5.03
10	2.33	0.58	44.33	4.51
15	0	0	38.67	2.52
30	0	0	33	4.58

Extraction of Total RNA from M. marinum

Total RNA were extracted as described previously (Raynaud, C. et al, Mol. Microbiol. 45, 203 (2002)). Cells were broken in a 1 ml solution of Trizol (Invitrogen) and 0.3 ml chloroform with 0.1 mm silica beads (Q-Biogene, Lysing matrix B) using a BIO101/Savant FastPrep FP120 machine (6.5 speed, 30 sec pulse, four cycles). RNA was extracted with 300 μl of chloroform. After 10 min of centrifugation at 13000 g, the aqueous phase was re-extracted with 300 μl of chloroform and then transferred to a tube containing 250 μl of isopropanol. Total RNA was precipitated overnight at 4° C. and washed with 1 ml of a 75% ethanol before resuspension in RNase free water. Contaminating DNA was removed by digestion with RQ1 DNase (Ambion) according to the manufacturer's instructions.

RNA Dot Blot

Dot blot and probing of RNA were done as described previously (Sambrook, J., Fritsch, E. F., Maniatis, T. Molecular Cloning, a Laboratory Manual. (Cold Spring Harbor Laboratory Press, New York, third edition, 2001)). DNase-treated RNA samples prepared from day 1, 3, 5 and 7 M. marinum cells were denatured at 90° C. for 5 min and then spotted (10 μg in each spot) on Hybond-N+ nylon membranes (Amersham Biosciences) and air-dried. The RNA was immobilized by ultraviolet cross-linking using a Bio-Rad cross-linker, followed by hybridization with specific ³²P-5′-end-labeled oligonucleotides at 42° C. The membranes were washed as described previously (Sambrook, J., Fritsch, E. F., Maniatis, T. Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, third edition, 2001)). After probing for the mRNAs, the membranes were stripped with hot 0.01% SDS, and then re-hybridized with 5S rRNA probes and washed as above. Hybridization signals were analyzed using a Phosphor Imager (ImageQuant, Molecular Dynamics). E. coli K12 stationary phase RNA samples were also spotted on the same membranes and probed with the same oligonucleotides to calculate the background signals of each dot arising from unspecific binding, which were very low in the hybridization conditions. The background values were subtracted from the corresponding values of the mRNA and 5S rRNA signals. Oligonucleotide probes were labeled at their 5′-ends with γ³²P-ATP using T4 polynucleotide kinase (PNK) according to standard procedures. The nucleotide sequences of the oligonucleotide probes are listed in Table 6. To probe for M. marinum 5S rRNA the oligonucleotide 5′-GCTGACAGGCTTAGCTTCCG (SEQ ID NO:31) was used.

TABLE 6

Homologs Oligo-probes used in dot blot

SpoVK	AGTCGTTCTTGGCGCCGGTTG (SEQ ID NO: 32)

SigF	GGAACCTTGACCGACCAAC (SEQ ID NO: 33)

SpoIIAB	AGTTGGTGCACGCCTCGCTG (SEQ ID NO: 34)

Spo0A	GTGTGTATGCGTCAGAGTC (SEQ ID NO: 35)

CotSA	ACGAGTGCTGGCCAGTTGAG (SEQ ID NO: 36)

YrbC	CAGTACGGCCACACCGTTG (SEQ ID NO: 37)

SpoVE	GCAAATGAGCTCATGAAAAC (SEQ ID NO: 38)

Soj	AGCGTTGCGCAAGCGATTCTC (SEQ ID NO: 39)

SpoIIIE	GTTGACGAAGCTGGACTTTC (SEQ ID NO: 40)

SigA	AGGCTTGCCTTCCTGGATG (SEQ ID NO: 41)

TABLE 7

Numerical values of dot blot signals

	SpoVK	CotSA	YrbC	SpoVE	Soj	SpoIIIE	SigF	SpoIIAB	SpoOA(TrcR)	SigA

Set 1

1 d	1234	714	1405	1103	908	1315	732	1044	1302	5698
3 d	1677	813	861	878	1234	1008	411	983	974	1983
5 d	9432	9126	10023	9553	2251	3984	654	1706	8905	1309
7 d	19821	14271	20017	20127	8093	7139	2037	1938	18337	890

Set 2

1 d	1098	615	1233	993	789	1578	689	977	1173	6605
3 d	1541	798	792	803	1402	1239	321	912	862	2474
5 d	8790	8773	9510	8902	2009	4037	614	1611	8001	1721
7 d	16889	13043	18121	17977	9124	7802	1799	1745	17002	756

Set 3

1 d	1363	810	1601	1187	974	1147	823	1100	1527	5004
3 d	1805	867	917	1014	1307	980	465	1004	1108	1879
5 d	10017	1033	11053	10765	2511	3315	711	1892	9403	2043
7 d	21680	15302	21976	22331	8672	6984	2199	2076	19319	1007

Calculated average values:

1 d	1231.67	713	1413	1094.33	890.33	1346.67	748	1040.33	1334	5769
3 d	1674.33	826	856.67	898.33	1314.33	1075.67	399	966.33	981.3333333	2112
5 d	9413	6310.67	10195.33333	9740	2257	3778.67	659.67	1736.33	8769.666667	1691
7 d	19463.33333	14205.33333	20038	20145	8629.67	7308.33	2011.666667	1919.67	18219.33333	884.33

Calculated errors:

1 d	132.52	97.50	184.13	97.29	93.76	217.24	68.42	61.58	179.16	802.86
3 d	132.02	36.29	62.61	106.96	84.24	142.14	72.75	48.21	123.16	317.78
5 d	613.72	4574.000036	785.80	945.47	251.05	402.42	48.75	142.93	710.73	367.92
7 d	2415.44	1130.93	1927.59	2177.06	516.80	434.50	201.20	166.26	1162.97	125.60

Bioinformatics

Sequence Data

Protein sequences from Bacillus subtilis were downloaded from the SubtiList web server, genolist.pasteur.fr/SubtiList/(Moszer, I., Glaser P., & Danchin, A., Microbiol. 141, 261-268 (1995), Moszer, I., FEBS Letters 430, 28-36 (1998)).
Sporulation-specific proteins were obtained according to the SubtiList functional category 1.8. In addition, protein sequences for sporulation related factors SigE, SigF, SigG, Spo0A (TrcR in FIG. 5) and SpoVT were also downloaded Amino acid sequences of identified open-reading frames from M. tuberculosis CDC1551, M. tuberculosis H37Rv, and S. coelicolor A3 (2) were downloaded from the TIGR Comprehensive Microbial Resource (TIGR-CMR, cmr.tigr.org/tigr-scripts/CMR/cmrHomePage.cgi) Peterson, J. D., Umayam, L. A., Dickinson, T. M., Hickey E. K., & White, 0., Nucleic Acids Res. 29, 123-125 (2001), Cole S. T. et al., Nature 393, 537-544 (1998), Fleischmann, R. D. Whole-Genome Comparison of Mycobacterium tuberculosis Clinical and Laboratory Strains. J. Bacteriol. 184, 5479-5490 (2002), cmr.tigr.org/tigr-scripts/CMR/cmrHomePage.cig Amino acid sequences of the initial gene predictions for M. marinum were downloaded from the Sanger Institute ftp server, ftp.sanger.ac.uk/pub/pathogens/mm/Proteins involved in S. coelicolor sporulation were obtained by querying the ScoDB database (streptomyces.org.uk/sco/index.html) for genes with annotations containing any of the words “sporulation”, “whiA”, “whiB”, “whiD”, “whiE”, “whiG”, “whiH”, “whiI” or “whiJ” as well as genes with gene names matching “whi*” (‘*’ corresponding to any letter). In addition, the S. coelicolor gene for sigF was included.

Ortholog Search

The Washington University BLAST (WU-BLAST) package (version 2.0, release date 10 May 2005, Gish, W. [1996-2004] blast.wustl.edu) was used in a reciprocal best hit approach (RBH) (Rivera, M. C., Jain, R., Moore, J. E. & Lake, J. A. Genomic evidence for two functionally distinct gene classes. Proc. Natl. Acad. Sci. USA 95, 62396244 (1998)) to identify putative orthologues for sporulation associated B. subtilis and S. coelicolor proteins in the mycobacteria species listed above. A pair of genes was considered, a and b from genomes A and B, respectively, as putative orthologues if a search with a against B identifies b as the most significant match and a search with b against A results in a as the most significant match. The blastp program was used with default parameters and an expect cutoff at 0.001. Putative orthologues are listed in Tables 1-4. In addition, top scoring hits for the B. subtilis transcription factors Spo0A, SpoIIAB and SigF were used. These hits did not return the initial B. subtilis query protein as the top scoring hit in the RBH search and are therefore considered as weak hits. As a complement to the RBH approach, putative orthologs were identified with the reciprocal smallest distance algorithm (RSD) implemented in the software package available for download at cbi.med.harvard.edu/RSD.tar.gz (Wall, D. P., Fraser, H. B. & Hirsh, A. E. Detecting putative orthologs. Bioinformatics 19 (13), 1710-1711 (2003)). The program was run with default settings except for an E-value threshold of 0.001 and a divergence threshold of 0.5. The results are listed in Tables 1-4.

Measurement of Antibody Titers

Measurement of antibody titers mice can be performed as follows. Mice are bled four to seven days following each immunization starting after the second immunization, for a total of five bleeds. Antibody titers may be measured as spore forming peptide-binding antibody using a sandwich ELISA with plastic multi-well plates coated with a spore forming peptide.
Spore forming peptide-dependent lymphoproliferation can be measured using spleen cells harvested approximately one week following the final, sixth, immunization. Freshly harvested cells, 105 per well, may e.g. be cultured for 5 days in the presence of spore forming peptide at a concentration of 5 μM for stimulation.
Preparation of polyclonal antibodies for passive protection can be performed as follows. Twenty non-transgenic mice may be immunized with a spore forming peptide or other immunogen, optionally plus adjuvant, and then euthanized at 4-5 months. Blood may then be collected from immunized mice. Optionally, IgG is separated from other blood components. Antibody specific for the immunogen may be partially purified by affinity chromatography. An average of about 0.5-1 mg of immunogen-specific antibody may be obtained per mouse, giving a total of 5-10 mg.
Passive immunization with antibodies to a spore forming peptide can be performed as follows. Groups of 7-9 month old mice each can be injected with 0.5 mg in PBS of polyclonal anti-spore forming peptide or specific anti-spore forming peptide monoclonals as shown below. All antibody preparations should be purified to have low endotoxin levels. Monoclonals can be prepared against a fragment by injecting the fragment or longer form of a spore forming peptide into a mouse, preparing hybridomas and screening the hybridomas for an antibody that specifically binds to a desired fragment of a spore forming peptide without binding to other nonoverlapping fragments of a spore forming peptide.
Mice can be injected intraperitoneally (i.p.) as needed over a 4 month period to maintain a circulating antibody concentration measured by ELISA titer of greater than 1/1000 defined by ELISA to a CotSA or other immunogen according to this invention. Titers can be monitored as above and mice can be euthanized at the end of 4 months of injections.

Claims

1. A method for detecting spores derived from Mycobacteria in a sample comprising determining the presence in said sample of a peptide chosen from the group consisting of mycobacterial CotA, CotD, CotT, CotSA, SpoVK, SpoVE, Soj, SpoIIIE, YrbC and peptides having an amino acid sequence according to SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, and 26, and immunogenic fragments thereof.

2. The method according to claim 1, wherein the Mycobacteria is selected from Mycobacterium marinum, Mycobacterium bovis, Mycobacterium tuberculosis, and Mycobacterium avium subspecies paratuberculosis.

3. The method according to claim 1, wherein the sample is a body fluid sample from a human or animal, a food product sample, a feed product sample, or an environmental sample.

4. The method according to claim 3, wherein the sample is a body fluid sample selected from the group consisting of blood, serum, bronchoalveolar lavage fluid, saliva, urine, feces, and tears.

5. The method according to claim 3, wherein the sample is a food product sample selected from the group consisting of milk, cheese, butter, vegetables, and meat.

6. The method according to claim 1, wherein the detection is performed through an immunological assay.

7. The method according to claim 4, wherein the immunological assay is an ELISA assay.

8. The method according to claim 2, wherein the peptide is CotA.

9. The method according to claim 2, wherein the peptide is CotD.

10. The method according to claim 2, wherein the peptide is CotT.

11. The method according to claim 2, wherein the peptide is CotSA.

12. The method according to claim 2, wherein the peptide is SpoVK.

13. The method according to claim 2, wherein the peptide is SpoVE.

14. The method according to claim 2, wherein the peptide is Soj.

15. The method according to claim 2, wherein the peptide is SpoIIIE.

16. The method according to claim 2, wherein the peptide is YrbC.

17. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 2.

18. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 4.

19. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 6.

20. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 8.

21. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 10.

22. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 12.

23. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 16.

24. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 18.

25. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 20.

26. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 22.

27. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 24.

28. The method according to claim 2, wherein the peptide has an amino acid sequence according to SEQ ID NO: 26.