US20130149324A1 - Therapeutic tb vaccine - Google Patents

Therapeutic tb vaccine Download PDF

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US20130149324A1
US20130149324A1 US13/369,411 US201213369411A US2013149324A1 US 20130149324 A1 US20130149324 A1 US 20130149324A1 US 201213369411 A US201213369411 A US 201213369411A US 2013149324 A1 US2013149324 A1 US 2013149324A1
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polypeptide
polypeptides
mycobacteria
tuberculosis
vaccine
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Peter Andersen
Ida Rosenkrands
Anette Stryhn
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Statens Serum Institut SSI
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Statens Serum Institut SSI
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A

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  • the present invention discloses a therapeutic vaccine against latent or active tuberculosis infection caused by the tuberculosis complex microorganisms ( Mycobacterium tuberculosis, M. bovis, M. africanum ).
  • the invention furthermore discloses a multi-phase vaccine that can be administered either prophylactically or therapeutically as well as a diagnostic reagent for the detection of latent stages of tuberculosis.
  • M. tuberculosis Human tuberculosis caused by Mycobacterium tuberculosis ( M. tuberculosis ) is a severe global health problem, responsible for approx. 3 million deaths annually, according to the WHO.
  • the worldwide incidence of new tuberculosis (TB) cases had been falling during the 1960s and 1970s but during recent decades this trend has markedly changed in part due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis.
  • Organisms of the tuberculosis complex can cause a variety of diseases, but the commonest route of invasion is by inhalation of bacteria. This initiates an infection in the lung, which can ultimately spread to other parts of the body. Normally, this infection is restricted in growth by the immune system, so that the majority of infected individuals show few signs apart from cough and fever, which eventually abates. Approximately 30% of individuals are unable to contain the infection and they will develop primary disease, which in many cases will eventually prove fatal. However, it is believed that even those individuals who apparently control the infection remain infected, probably for the rest of their life. Certainly, individuals who have been healthy for years or even decades can suddenly develop tuberculosis, which has proven to be caused by the same organism they were infected with many years previously. M.
  • tuberculosis and other organisms of the TB complex are unique in that the mycobacteria can evade the immune response and survive for long periods in a refractory non-replicating or slowly-replicating stage. This is referred to as latent TB and is at present a very significant global health problem that is estimated to affect approximately 1 ⁇ 3 of the world's population (Anon., 2001).
  • M. tuberculosis The course of a M. tuberculosis infection runs essentially through 3 phases, as illustrated in FIG. 1 .
  • the bacteria proliferate in the organs, until the immune response increases to the point at which it can control the infection, whereupon the bacterial load peaks and starts declining.
  • a latent phase is established where the bacterial load is kept stable at a low level.
  • M. tuberculosis goes from active multiplication to dormancy, essentially becoming non-replicating and remaining inside the granuloma.
  • the infection goes to the reactivation phase, where the dormant bacteria start replicating again.
  • BCG The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. Although BCG consistently performs well in animal models of primary infection, it has clearly failed to control the TB epidemic. Consistent with that, BCG vaccination appears to provide protection against pediatric TB (which is due to primary infection), while offering little or no protection against adult disease (which is often reactivation of latent infection acquired in childhood). It has also been shown that vaccination of individuals who are currently sensitized to mycobacteria or latently infected is ineffective. Thus, current vaccination strategies, while effective against primary disease, fail to activate immune responses that efficiently control surviving dormant bacteria.
  • the present invention provides therapeutic vaccines based on molecules that are induced or upregulated under the conditions of low oxygen transmission and restricted nutrients found in the granuloma (i.e., the location of latent TB infection). These vaccines are therapeutic and contrast with prior art vaccines which are designed to elicit protective immune responses prior to infection (prophylactic vaccination) that are only effective against primary infection.
  • the immune responses elicited are powerless against the latent stage of the disease, because the bacteria have changed the antigens that they produce so that in essence they have altered their appearance and the immune system can no longer recognize them.
  • latency is a dynamic process, maintained by the immune response, as indicated by the dramatic increase in the risk of reactivation of TB after HIV infection or other events that compromise immunity. Therefore, an effective vaccination strategy to protect infected individuals (therapeutic vaccination) is possible, but only if it is directed against those antigens expressed in the latent stage.
  • the present invention provides a multiphase vaccine that combines components with prophylactic and therapeutic activity.
  • existing TB vaccines do not result in sterilizing immunity but rather control the infection at a subclinical level (thereby resulting in the subsequent establishment of latent infection.
  • the evasion of the primary immune response and the subsequent development of latent disease are probably at least in part due to the change in the antigenic profile of the invading bacteria.
  • vaccinating with antigens associated with latent TB prevents or reduces the establishment of latent infection and therefore, a vaccine incorporating antigens expressed by the bacteria both in the first logarithmic growth phase and during latent disease improve long-term immunity when used as a prophylactic vaccine.
  • a multiphase vaccine of the invention will also be efficient as a therapeutic vaccine thereby addressing the problem that the majority of the population in the third world who would receive a future TB vaccine could be already latently infected.
  • the immunodominant antigens identified in this invention may be used as diagnostic reagents.
  • We has abundantly demonstrated that antigens expressed by mycobacteria during the early stages of the infection, such as ESAT-6 (Early Secretory Antigen Target-6) are recognized in individuals who are in the process of developing primary TB, even though they are healthy at the time of diagnosis (Doherty 2002).
  • ESAT-6 Early Secretory Antigen Target-6
  • Doherty 2002 the large numbers of contacts who are exposed, and almost certainly infected, remain negative to this antigen. Since those individuals latently infected remain healthy by making an immune response against the latent bacteria, they must be making an immune response to those antigens expressed by the latent bacteria.
  • the antigens of the invention may also be used to diagnose latent infection and differentiate it from primary acute TB.
  • FIGS. 1A and 1B illustrate the results of testing in TB vaccination models.
  • Each square on the time axis represents one week.
  • Three prophylactic vaccinations two weeks apart are given 6 weeks prior to an aerosol infection.
  • the protective effect of the vaccines is measured 6 weeks after infection, in the acute phase of the infection.
  • a reactivation model is established, where aerosol infected mice are treated with anti- M tuberculosis drugs for 8 weeks from the peak of infection (6 weeks after infection). This induces a latent infection phase with a low bacterial load.
  • Four to five weeks into the latency phase three therapeutic vaccinations are given two weeks apart and the protective effect of the vaccines is measured as bacterial load in the reactivation phase, seven weeks after the last immunization.
  • FIGS. 2A and 2B illustrate prophylactic and therapeutic vaccine induced protection.
  • C57Bl/6j mice were immunized 3 times with a 2-week interval with recombinant ESAT6, BCG or recombinant Rv2031c.
  • the immunization was given as a prophylactic vaccine 6 weeks before the mice were given a M. tuberculosis infection (approx. 250 bacilli) through the aerosol route with.
  • Bacterial numbers in the lung was enumerated 6 weeks post infection.
  • FIG. 2B the immunization was given as a therapeutic vaccine after a latent infection had been established.
  • Bacterial numbers in the lung was enumerated 8 weeks after the last immunization.
  • the data represents the mean of 5 individual mice.
  • FIG. 3 illustrates Rv2031c specific IFN- ⁇ responses.
  • Latent infected C57Bl/6j mice were either not immunized or immunized with 3 ⁇ g recombinant Rv2031 3 times with a two-week interval.
  • One and two weeks post immunization mice were bleed and PBMCs isolated.
  • the frequency of IFN- ⁇ producing cells specific for either ESAT6 or Rv2031c was determined for both the rRv2031c immunized and the unimmunized group.
  • ELIspot plate precoated with anti-IFN- ⁇ antibodies graded numbers of PBMCs were incubated with either 2 ⁇ g/ml rRv2031c or 2 ⁇ g/ml rESAT6.
  • FIGS. 4A and 4B illustrate the results of epitope screening of Rv2031c.
  • PBMCs from rRv2031c immunized latently infected C57Bl/6j mice were analyzed for recognition of 20′ mer overlapping peptides scanning through Rv2031c.
  • the peptides were analyzed in pools of 3-4 peptides.
  • PBMCs (2 ⁇ 10 5 ) were incubated for 72 h with the peptide pools at 5 ⁇ g/ml per peptide. Supernatant was harvested and secreted IFN- ⁇ was quantitated by ELISA.
  • FIG. 4B individual peptides of positive pools were reanalyzed.
  • PBMCs (2 ⁇ 10 5 ) were incubated for 72 h with 1 ⁇ g/ml of each peptide. Secreted IFN- ⁇ in the supernatant was quantitated.
  • FIGS. 5A and 5B illustrate protection against reactivation conferred by therapeutic vaccine given during latent infection.
  • Latent infected C57Bl/6j mice were immunized 3 times with or without rRv2031c.
  • Bacterial numbers in lung ( FIG. 5A ) and spleen ( FIG. 5B ) was enumerated 8 weeks after the last immunization. The data represents the mean of 8 individual mice.
  • FIGS. 6A and 6B illustrate Rv0569 specific IFN- ⁇ responses.
  • Latent infected C57Bl/6j mice were vaccinated with 3 ⁇ g of either recombinant Rv0569 or recombinant ESAT6 in a DDA/MPL adjuvant.
  • the vaccines were given as 3 s.c. injections with a two-week interval and the induced immune response were evaluated 7 weeks after the last vaccination.
  • Isolated splenocytes (2 ⁇ 10 5 ) were incubated for 72 h with antigen at 1 ⁇ g/ml.
  • FIG. 6A Rv0569 specific response is measured in Rv0569-vaccinated and un-vaccinated latently infected mice; in FIG. 6B , the ESAT6 specific response is measured in ESAT6-vaccinated and un-vaccinated latently infected mice
  • FIGS. 7A and 7B illustrate therapeutic vaccine induced protection against reactivation.
  • Latently infected C57Bl/6j mice were vaccinated once with BCG or 3 times with a 2-week interval with either recombinant Rv0569 or recombinant ESAT6.
  • the bacterial numbers was enumerated in FIG. 7A , the lung and in FIG. 7B , the spleen of vaccinated and un-vaccinated mice.
  • the data represents the mean of Log CFU per organ of 6-8 individual mice.
  • the invention is related to preventing, treating and detecting infections caused by species of the tuberculosis complex ( Mycobacterium tuberculosis, M. bovis, M. africanum ) by the use of a polypeptide comprising a M. tuberculosis antigen or an immunogenic portion or other variant thereof, or by the use of a DNA sequence encoding a M. tuberculosis antigen or an immunogenic portion or other variant thereof.
  • the invention discloses a new therapeutic vaccine against tuberculosis comprising antigens induced during the latent stage of TB-infection. It also discloses a multiphase vaccine incorporating a combination of prophylactic and therapeutic antigens as well as diagnostic reagents for the detection of the latent stage of M. tuberculosis infection.
  • the present invention discloses the use of one or more polypeptides, nucleic acids encoding these polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, for a therapeutic vaccine against tuberculosis.
  • the polypeptides comprises one or more amino acid sequences selected from
  • immunogenic portions are selected from the group consisting of the sequences presented in Table 1 and the nucleic acid sequences are selected from the sequences presented in Table 2.
  • the vaccine is a multiphase vaccine, where the polypeptides or fragments hereof are fused to other antigens with efficacy as prophylactic vaccines, where the fusion partner is selected from e.g. the group consisting of ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32.
  • the fusion partner is selected from e.g. the group consisting of ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32.
  • the invention further discloses a therapeutic vaccine against tuberculosis comprising one or more polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, or nucleic acids encoding these polypeptides.
  • the therapeutic and multiphase vaccine comprises an additional delivery system selected from among, live recombinant vaccines, that is gene-modified organisms such as bacteria or viruses expressing mycobacteria genes, or immunogenic delivery systems such as, DNA vaccines, that is plasmids expressing genes or gene fragments for the proteins described above, or protein vaccines, that is the proteins themselves or synthetic peptides derived from the proteins themselves delivered in a delivery system such as an adjuvant.
  • live recombinant vaccines that is gene-modified organisms such as bacteria or viruses expressing mycobacteria genes
  • immunogenic delivery systems such as, DNA vaccines, that is plasmids expressing genes or gene fragments for the proteins described above, or protein vaccines, that is the proteins themselves or synthetic peptides derived from the proteins themselves delivered in a delivery system such as an adjuvant.
  • the invention further discloses a therapeutic vaccine in which the amino acid sequence is lipidated so as to allow a self-adjuvanting effect of the polypeptide.
  • the invention also discloses a method for treating an animal, including a human being, with tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , comprising administering to the animal the above-mentioned vaccine.
  • virulent mycobacteria e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis
  • the invention also discloses a method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , comprising administering to the animal the above mentioned vaccine.
  • the invention discloses an immunogenic composition
  • an immunogenic composition comprising a polypeptide as defined above, preferably in the form of a vaccine or in the form of a diagnostic reagent.
  • the diagnostic reagent can be in the form of a skin test reagent (administered by the transcutaneous, subcutaneous or intradermal routes), a serological reagent or a reagent for stimulating a cell-mediated reaction.
  • the invention discloses a nucleic acid fragment in isolated form which
  • the nucleic acid fragment is preferably a DNA fragment.
  • the fragment can be used as a pharmaceutical.
  • the invention discloses a vaccine comprising a nucleic acid fragment according to the invention, optionally inserted in a vector, the vaccine effecting in vivo expression of antigen by a human being or other mammal or animal, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , in an animal, including a human being.
  • the invention discloses the use of a nucleic acid fragment according to the invention for the preparation of a composition for the diagnosis of tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , and the use of a nucleic acid fragment according to the invention for the preparation of a pharmaceutical composition for the vaccination against tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis.
  • the invention discloses a vaccine for immunizing an human being or other mammal or animal, against tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , comprising as the effective component a non-pathogenic microorganism, wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a polypeptide as defined above has been incorporated into the microorganism (e.g., placed on a plasmid or in the genome) in a manner allowing the microorganism to express and optionally secrete the polypeptide.
  • virulent mycobacteria e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis
  • a non-pathogenic microorganism wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a polypeptide as defined above has been incorporated into
  • the invention discloses a replicable expression vector, which comprises a nucleic acid fragment according to the invention, and a transformed cell harboring at least one such vector.
  • the invention discloses a method for producing a polypeptide as defined above, comprising
  • the invention also discloses a method of diagnosing tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide as defined above or an immunogenic composition as defined above, a positive skin response at the location of injection being indicative of the animal having tuberculosis, and a negative skin response at the location of injection being indicative of the animal not having tuberculosis.
  • the invention discloses a method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , comprising administering to the animal the polypeptide as defined above, the immunogenic composition according to the invention, or the vaccine according to the invention.
  • virulent mycobacteria e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis
  • Another embodiment of the invention discloses a monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide as defined above in an immuno assay, or a specific binding fragment of said antibody.
  • said antibody is for use as a diagnostic reagent, e.g. for detection of mycobacteria antigens in sputum, urine or other body fluids of an infected animal, including a human being.
  • the vaccine, immunogenic composition and pharmaceutical composition according to the invention can be used therapeutically in a subject infected with a virulent mycobacterium combined with a prophylactic composition in a subject to prevent further infection with a virulent mycobacterium.
  • the invention also discloses a method for diagnosing previous or ongoing infection with a virulent mycobacterium, said method comprising
  • the invention discloses a method of diagnosing Mycobacterium tuberculosis infection in a subject comprising:
  • polypeptide in the present invention should have its usual meaning. That is an amino acid chain of any length, including a full-length protein, oligopeptides, short peptides and fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.
  • the polypeptide may be chemically modified by being glycosylated, by being lipidated (e.g. by chemical lipidation with palmitoyloxy succinimide as described by Mowat et al. 1991 or with dodecanoyl chloride as described by Lustig et al. 1976), by comprising prosthetic groups, or by containing additional amino acids such as e.g. a his-tag or a signal peptide.
  • Each polypeptide may thus be characterized by specific amino acids and be encoded by specific nucleic acid sequences. It will be understood that such sequences include analogues and variants produced by recombinant or synthetic methods wherein such polypeptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide and still be immunogenic in any of the biological assays described herein. Substitutions are preferably “conservative”. These are defined according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. The amino acids in the third column are indicated in one-letter code.
  • a preferred polypeptide within the present invention is an immunogenic antigen from M. tuberculosis produced when the organism is subjected to the stresses associated with latent infection.
  • antigen can for example also be derived from the M. tuberculosis cell and/or M. tuberculosis culture filtrate.
  • a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences.
  • the additional sequences may be derived from the native M. tuberculosis antigen or be heterologous and such sequences may, but need not, be immunogenic.
  • Each polypeptide is encoded by a specific nucleic acid sequence. It will be understood that such sequences include analogues and variants hereof wherein such nucleic acid sequences have been modified by substitution, insertion, addition or deletion of one or more nucleic acids. Substitutions are preferably silent substitutions in the codon usage that will not lead to any change in the amino acid sequence, but may be introduced to enhance the expression of the protein.
  • substantially pure polypeptide fragment means a polypeptide preparation which contains at most 5% by weight of other polypeptide material with which it is natively associated (lower percentages of other polypeptide material are preferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most 1 ⁇ 2%). It is preferred that the substantially pure polypeptide is at least 96% pure, i.e. that the polypeptide constitutes at least 96% by weight of total polypeptide material present in the preparation, and higher percentages are preferred, such as at least 97%, at least 98%, at least 99%, at least 99.25%, at least 99.5%, and at least 99.75%.
  • the polypeptide fragment is in “essentially pure form”, i.e. that the polypeptide fragment is essentially free of any other antigen with which it is natively associated, i.e. free of any other antigen from bacteria belonging to the tuberculosis complex or a virulent mycobacterium.
  • This can be accomplished by preparing the polypeptide fragment by means of recombinant methods in a non-mycobacterial host cell as will be described in detail below, or by synthesizing the polypeptide fragment by the well-known methods of solid or liquid phase peptide synthesis, e.g. by the method described by Merrifield, 1963, or variations thereof.
  • virulent mycobacterium is understood a bacterium capable of causing the tuberculosis disease in an animal or in a human being.
  • examples of virulent mycobacteria include but are not limited to M. tuberculosis, M. africanum , and M. bovis .
  • relevant animals are cattle, possums, badgers and kangaroos.
  • a TB patient an individual with culture or microscopically proven infection with virulent mycobacteria, and/or an individual clinically diagnosed with TB and who is responsive to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB are well known by any person skilled in the art.
  • PPD-positive individual an individual with a positive Mantoux test or an individual where PPD induces a positive in vitro recall response determined by release of IFN- ⁇ .
  • a latently infected individual an individual, who has been infected by a virulent mycobacterium, e.g. M. tuberculosis , but shows no sign of active tuberculosis. It is likely that individuals who have been vaccinated, e.g. by BCG, or treated for TB may still retain the mycobacteria within their bodies, although this is currently impossible to prove since such individuals would be expected to be positive if tested for PPD reactivity. Nonetheless, in its most accurate sense, “latently-infected” may be used to describe any individual who has M. tuberculosis residing in their tissues but who is not clinically ill.
  • DTH delayed type hypersensitivity reaction
  • IFN- ⁇ interferon-gamma.
  • the measurement of IFN- ⁇ is used as an indication of an immunological response.
  • nucleic acid fragment and “nucleic acid sequence” are understood any nucleic acid molecule including DNA, RNA, LNA (locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included are nucleic acid molecules comprising non-naturally occurring nucleosides. The term includes nucleic acid molecules of any length e.g. from 10 to 10000 nucleotides, depending on the use. When the nucleic acid molecule is for use as a pharmaceutical, e.g.
  • a molecule encoding at least one epitope is preferably used, having a length from about 18 to about 1000 nucleotides, the molecule being optionally inserted into a vector.
  • a molecule having a length of 10-100 is preferably used.
  • molecule lengths can be used, for instance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotide derivatives), or a molecule having at most 10000, 5000, 4000, 3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nucleotides (or nucleotide derivatives).
  • stringent when used in conjunction with hybridization conditions is as defined in the art, i.e. the hybridization is performed at a temperature not more than 15-20° C. under the melting point T m , cf. Sambrook et al, 1989, pages 11.45-11.49.
  • the conditions are “highly stringent”, i.e. 5-10° C. under the melting point T m .
  • sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. The two sequences to be compared must be aligned to best possible fit allowing the insertion of gaps or alternatively, truncation at the ends of the protein sequences.
  • sequence identity can be calculated as
  • N dif is the total number of non-identical residues in the two sequences when aligned and wherein N ref is the number of residues in one of the sequences.
  • Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson, 1988, or online through the NIH website).
  • alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al. 1994 and as available through online sources.
  • a preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.
  • the polypeptide comprises an immunogenic portion of the polypeptide, such as an epitope for a B-cell or T-cell.
  • the immunogenic portion of a polypeptide is a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays described herein.
  • the immunogenic portion of a polypeptide may be a T-cell epitope or a B-cell epitope.
  • Immunogenic portions can be related to one or a few relatively small parts of the polypeptide, they can be scattered throughout the polypeptide sequence or be situated in specific parts of the polypeptide.
  • epitopes have even been demonstrated to be scattered throughout the polypeptide covering the full sequence (Ravn et al 1999).
  • overlapping oligopeptides for the detection of MHC class II epitopes, preferably synthetic, having a length of e.g. 20 amino acid residues derived from the polypeptide.
  • These peptides can be tested in biological assays (e.g. the IFN- ⁇ assay as described herein) and some of these will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide.
  • B-cell epitopes can be determined by analyzing the B cell recognition to overlapping peptides covering the polypeptide of interest as, e.g., described in Harboe et al 1998.
  • the polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues.
  • the polypeptide fragment has a length of at most 50 amino acid residues, such as at most 40, 35, 30, 25, and 20 amino acid residues.
  • the peptides having a length of from 10 to 20 amino acid residues will prove to be most efficient as MHC class II epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 18, such as 15, 14, 13, 12 and even 11 amino acid residues. It is expected that the peptides having a length of from 7 to 12 amino acid residues will prove to be most efficient as MHC class I epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 11, such as 10, 9, 8 and even 7 amino acid residues.
  • Immunogenic portions of polypeptides may be recognized by a broad part (high frequency) or by a minor part (low frequency) of the genetically heterogeneous human population. In addition some immunogenic portions induce high immunological responses (dominant), whereas others induce lower, but still significant, responses (subdominant). High frequency or low frequency can be related to the immunogenic portion binding to widely distributed MHC molecules (HLA type) or even by multiple MHC molecules (Sinigaglia, 1988, Kilgus, 1991).
  • the subdominant epitopes are however as relevant as are the dominant epitopes since it has been shown (Olsen, 2000) that such epitopes can induce protection regardless of the fact that they are not as strongly or broadly recognized.
  • polypeptides of the invention are their capability to induce an immunological response as illustrated in the examples. It is understood that a variant of a polypeptide of the invention produced by substitution, insertion, addition or deletion may also be immunogenic as determined by any of the assays described herein.
  • An immune individual is defined as a person or an animal, which has cleared or controlled an infection with virulent mycobacteria or has received a vaccination with M. bovis BCG.
  • the immune response may be monitored by one of the following methods:
  • M. tuberculosis antigens and DNA sequences encoding such antigens, may be prepared using any one of a variety of procedures.
  • Immunogenic antigens may also be produced recombinantly using a DNA sequence encoding the antigen, which has been inserted into an expression vector and expressed in an appropriate host. Examples of host cells are E. coli .
  • the polypeptides or immunogenic portion hereof can also be produced synthetically having fewer than about 100 amino acids, and generally fewer than 50 amino acids and may be generated using techniques well known to those ordinarily skilled in the art, such as commercially available solid-phase techniques where amino acids are sequentially added to a growing amino acid chain.
  • Plasmid DNA can then be prepared from cultures of the host strain carrying the plasmid of interest, and purified using e.g. the Qiagen Giga-Plasmid column kit (Qiagen, Santa Clarita, Calif., USA) including an endotoxin removal step. It is preferred that plasmid DNA used for DNA vaccination is endotoxin free.
  • the immunogenic polypeptides may also be produced as fusion proteins, by which methods superior characteristics of the polypeptide of the invention can be achieved. For instance, fusion partners that facilitate export of the polypeptide when produced recombinantly, fusion partners that facilitate purification of the polypeptide, and fusion partners which enhance the immunogenicity of the polypeptide fragment of the invention are all interesting possibilities. Therefore, the invention also pertains to a fusion polypeptide comprising at least one polypeptide or immunogenic portion defined above and at least one fusion partner.
  • the fusion partner can, in order to enhance immunogenicity, be another polypeptide derived from M.
  • tuberculosis such as of a polypeptide fragment derived from a bacterium belonging to the tuberculosis complex, such as ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32 and alpha-crystalline, or at least one T-cell epitope of any of the above mentioned antigens (Skj ⁇ t et al 2000; Danish Patent application PA 2000 00666; Danish Patent application PA 1999 01020; U.S. patent application Ser. No.
  • the invention also pertains to a fusion polypeptide comprising mutual fusions of two or more of the polypeptides (or immunogenic portions thereof) of the invention.
  • fusion partners which could enhance the immunogenicity of the product, are lymphokines such as IFN- ⁇ , IL-2 and IL-12.
  • the fusion partner can e.g. be a bacterial fimbrial protein, e.g. the pilus components pilin and papA; protein A; the ZZ-peptide (ZZ-fusions are marketed by Pharmacia in Sweden); the maltose binding protein; glutathione S-transferase; ⁇ -galactosidase; or poly-histidine. Fusion proteins can be produced recombinantly in a host cell, which could be E. coli , and it is a possibility to induce a linker region between the different fusion partners.
  • polypeptides which are lipidated so that the immunogenic polypeptide is presented in a suitable manner to the immune system.
  • This effect is e.g. known from vaccines based on the Borrelia burgdorferi OspA polypeptide as described in e.g. WO 96/40718 A or vaccines based on the Pseudomonas aeruginosa OprI lipoprotein (Cote-Sierra J 1998).
  • Another possibility is N-terminal fusion of a known signal sequence and an N-terminal cystein to the immunogenic polypeptide. Such a fusion results in lipidation of the immunogenic polypeptide at the N-terminal cystein, when produced in a suitable production host.
  • Another part of the invention pertains to a vaccine composition
  • a vaccine composition comprising a polypeptide (or at least one immunogenic portion thereof) or fusion polypeptide according to the invention.
  • a vaccine composition comprises an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant.
  • An effective vaccine wherein a polypeptide of the invention is recognized by the animal, will in an animal model be able to decrease bacterial load in target organs, prolong survival times and/or diminish weight loss after challenge with a virulent Mycobacterium , compared to non-vaccinated animals
  • Suitable carriers are selected from the group consisting of a polymer to which the polypeptide(s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the polypeptide(s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin.
  • Suitable vehicles are selected from the group consisting of a diluent and a suspending agent.
  • the adjuvant is preferably selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), Quil A, poly I:C, aluminum hydroxide, Freund's incomplete adjuvant, IFN- ⁇ , IL-2, IL-12, monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), Trehalose Dibehenate and muramyl dipeptide (MDP).
  • DDA dimethyldioctadecylammonium bromide
  • Quil A Quil A
  • poly I:C aluminum hydroxide
  • Freund's incomplete adjuvant IFN- ⁇
  • IL-2 poly I:C
  • MPL monophosphoryl lipid A
  • TDM Trehalose Dimycolate
  • Trehalose Dibehenate and muramyl dipeptide
  • agents such as aluminum hydroxide or phosphate (alum), synthetic polymers of sugars (Carbopol), aggregation of the protein in the vaccine by heat treatment, aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
  • Other possibilities involve the use of immune modulating substances such as cytokines or synthetic IFN- ⁇ inducers such as poly I:C in combination with the above-mentioned adjuvants.
  • a relevant antigen such as an antigen of the present invention can be conjugated to an antibody (or antigen binding antibody fragment) against the Fc ⁇ receptors on monocytes/macrophages.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g to 1000 ⁇ g, such as in the range from about 1 ⁇ g to 300 ⁇ g, and especially in the range from about 10 ⁇ g to 50 ⁇ g.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and advantageously contain 10-95% of active ingredient, preferably 25-70%.
  • vaccines can be administered to prevent an infection with virulent mycobacteria, a prophylactic vaccine, and/or to treat established mycobacterial infection, a therapeutic vaccine.
  • the vaccine When administered to prevent an infection, the vaccine is given prophylactically, before definitive clinical signs, diagnosis or identification of an infection TB are present. Since the current vaccine BCG appears to induce an effective, but short-lived immune response, prophylactic vaccines may also be designed to be used as booster vaccines. Such booster vaccines are given to individuals who have previously received a vaccination, with the intention of prolonging the period of protection.
  • the previous vaccination may have provided sufficient immunity to prevent primary disease, but as discussed previously, boosting this immune response will not help against the latent infection.
  • the vaccine will necessarily have to be a therapeutic vaccine designed for efficacy against the latent stage of infection.
  • the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response.
  • the vaccine may comprise two or more polypeptides or immunogenic portions, where all of the polypeptides are as defined above, or some but not all of the peptides may be derived from virulent mycobacteria.
  • the polypeptides not necessarily fulfilling the criteria set forth above for polypeptides may either act due to their own immunogenicity or merely act as adjuvants.
  • the vaccine may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides.
  • the invention also pertains to a method for immunizing an animal, including a human being, against TB caused by virulent mycobacteria, comprising administering to the animal the polypeptide of the invention, or a vaccine composition of the invention as described above, or a living vaccine described above.
  • the invention also pertains to a method for producing an immunologic composition according to the invention, the method comprising preparing, synthesizing or isolating a polypeptide according to the invention, and solubilizing or dispersing the polypeptide in a medium for a vaccine, and optionally adding other M. tuberculosis antigens and/or a carrier, vehicle and/or adjuvant substance.
  • nucleic acid fragments of the invention may be used for effecting in vivo expression of antigens, i.e. the nucleic acid fragments may be used in so-called DNA vaccines as reviewed in Ulmer et al 1993, which is included by reference.
  • the invention also relates to a vaccine comprising a nucleic acid fragment according to the invention, the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to infections caused by virulent mycobacteria in an animal, including a human being.
  • the efficacy of such a DNA vaccine can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a polypeptide that has the capability of modulating an immune response.
  • One possibility for effectively activating a cellular immune response for a vaccine can be achieved by expressing the relevant antigen in a vaccine in a non-pathogenic microorganism or virus.
  • a non-pathogenic microorganism or virus are Mycobacterium bovis BCG, Salmonella and Pseudomona and examples of viruses are Vaccinia Virus and Adenovirus.
  • Another important aspect of the present invention is an improvement of the living BCG vaccine presently available, wherein one or more copies of a DNA sequence encoding one or more polypeptide as defined above has been incorporated into the genome of the micro-organism in a manner allowing the micro-organism to express and secrete the polypeptide.
  • the incorporation of more than one copy of a nucleotide sequence of the invention is contemplated to enhance the immune response.
  • Another possibility is to integrate the DNA encoding the polypeptide according to the invention in an attenuated virus such as the vaccinia virus or Adenovirus (Rolph et al 1997).
  • the recombinant vaccinia virus is able to replicate within the cytoplasma of the infected host cell and the polypeptide of interest can therefore induce an immune response, which is envisioned to induce protection against TB.
  • the invention also relates to the use of a polypeptide or nucleic acid of the invention for use as therapeutic vaccines as have been described by D. Lowrie (Lowrie, 1999) using DNA vaccine encoding HSP65 from M. leprae .
  • Antigens with therapeutic properties may be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals or prevent reactivation of previous infection, when administered as a vaccine.
  • the composition used for therapeutic vaccines can be prepared as described above for vaccines.
  • the invention also relates to a method of diagnosing latent TB caused by a virulent mycobacterium in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide according to the invention, a positive skin response at the location of injection being indicative of the animal having TB, and a negative skin response at the location of injection being indicative of the animal not having TB.
  • a blood sample comprising mononuclear cells (i.e. T-lymphocytes) from a patient is contacted with a sample of one or more polypeptides of the invention.
  • This contacting can be performed in vitro and a positive reaction could e.g. be proliferation of the T-cells or release of cytokines such as IFN- ⁇ into the extracellular phase.
  • a positive reaction could e.g. be proliferation of the T-cells or release of cytokines such as IFN- ⁇ into the extracellular phase.
  • cytokines such as IFN- ⁇ into the extracellular phase.
  • the invention therefore also relates to an in vitro method for diagnosing latent infection in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and contacting the sample from the animal with the polypeptide of the invention, a significant release into the extracellular phase of at least one cytokine by mononuclear cells in the blood sample being indicative of the animal being sensitized.
  • a positive response being a response more than release from a blood sample derived from a patient without the TB diagnosis plus two standard deviations.
  • the invention also relates to the in vitro method for diagnosing ongoing or previous sensitization in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and by contacting the sample from the animal with the polypeptide of the invention demonstrating the presence of antibodies recognizing the polypeptide of the invention in the serum sample.
  • the immunogenic composition used for diagnosing may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides.
  • nucleic acid probes encoding the polypeptide of the invention can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample.
  • Such a method of diagnosing TB might involve the use of a composition comprising at least a part of a nucleotide sequence as defined above and detecting the presence of nucleotide sequences in a sample from the animal or human being to be tested which hybridize with the nucleic acid fragment (or a complementary fragment) by the use of PCR technique.
  • a monoclonal or polyclonal antibody which is specifically reacting with a polypeptide of the invention in an immunoassay, or a specific binding fragment of said antibody, is also a part of the invention.
  • the antibodies can be produced by methods known to a person skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of a polypeptide according to the present invention and, if desired, an adjuvant.
  • the monoclonal antibodies according to the present invention may, for example, be produced by the hybridoma method first described by Kohler and Milstein (Kohler and Milstein, 1975), or may be produced by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies may also be isolated from phage libraries generated using the techniques described by McCafferty et al (McCafferty, 1990), for example. Methods for producing antibodies are described in the literature, e.g. in U.S. Pat. No. 6,136,958.
  • a sample of a potentially infected organ or body fluid from an infected individual may be contacted with such an antibody recognizing a polypeptide of the invention.
  • the demonstration of the reaction by means of methods well known in the art between the sample and the antibody will be indicative of an ongoing infection. It is of course also a possibility to demonstrate the presence of anti-mycobacterial antibodies in serum or other body fluids by contacting a serum sample from a subject with at least one of the polypeptide fragments of the invention and using well-known methods for visualizing the reaction between the antibody and antigen.
  • an antibody, a nucleic acid fragment and/or a polypeptide of the invention can be used either alone, or as a constituent in a composition.
  • Such compositions are known in the art, and comprise compositions in which the antibody, the nucleic acid fragment or the polypeptide of the invention is coupled, preferably covalently, to at least one other molecule, e.g. a label (e.g. radioactive or fluorescent) or a carrier molecule.
  • M. tuberculosis genes are induced under low oxygen conditions.
  • the up-regulation of the genes listed in table 2 has been determined at either the mRNA (Sherman, 2001) or protein (Boon, 2001, Rosenkrands, 2002) level.
  • the coding region of these selected antigens is amplified by PCR using the primer sets listed in Table 3.
  • PCR reactions were carried out using Platinum Tag DNA Polymerase (GIBCO BRL) in a 50 ⁇ l reaction volume containing 60 mM Tris-SO 4 (pH 8.9), 18 mM Ammonium Sulfate, 0.2 mM of each of the four nucleotides, 0.2 ⁇ M of each primer and 10 ng of M. tuberculosis H37Rv chromosomal DNA.
  • the reaction mixtures were initially heated to 95° C. for 5 min., followed by 35 cycles of: 95° C. for 45 sec, 60° C. for 45 sec and 72° C. for 2 min.
  • the amplification products were precipitated by PEG/MgCl 2 , and dissolved in 50 ⁇ L TE buffer.
  • DNA fragments were cloned and expressed in Gateway Cloning system (Life Technology).
  • 5 ⁇ L of DNA fragment was mixed with 1 ⁇ L of pDONR201, 2 ⁇ L of BP CLONASE enzyme mix and 2 ⁇ L of BP reaction buffer. The recombination reactions were carried out at 25° C. for 60 min. After Proteinase K treatment at 37° C. for 10 min., 5 ⁇ L of each sample was used to transform E. coli DH5 ⁇ competent cells. Transformants were selected on LB plates containing 50 ⁇ g/mL kanamycin. One bacterial clone from each transformation was grown in 3 mL LB medium containing 50 ⁇ g/mL kanamycin and plasmid DNA was isolated (Qiagen).
  • each entry clone DNA was mixed with ⁇ L of His-tagged expression vector (pDest17), 2 ⁇ L LR reaction buffer, 2 ⁇ L LR CLONASE enzyme mix and 3 ⁇ L TE. After recombination at 25° C. for 60 min. and Proteinase K treatment at 37° C. for 10 min., 5 ⁇ L of each sample was used to transform E. coli BL21-SI competent cells. Transformants were selected on LBON (LB without NaCl) plates containing 100 ⁇ g/mL ampicillin. The resulting E. coli clones express recombinant proteins carrying a 6-histine tag at the N-terminal. All clones were confirmed by DNA sequencing.
  • lysozyme 0.1 mg/mL
  • DNase I 2.5 ⁇ g/mL
  • the recombinant protein forms inclusion bodies and can be pelleted by centrifugation at 27.000 ⁇ g for 15 min.
  • Protein pellets were solubilized by adding 20 ml of sonication buffer (8 M urea, 50 mM Na 2 HPO 4 , 100 mM Tris-HCl, pH 8.0) and sonicating 5 ⁇ 30 sec pulses interrupted by a 30 sec pause. After another centrifugation at 27.000 ⁇ g for 15 min., supernatants were applied to 10 mL TALON columns (Clontech). The columns were washed with 50 mL sonication buffer.
  • Bound proteins were eluted by lowering pH (8 M urea, 50 mM Na 2 HPO 4 , 100 mM Tris-HCl, pH 4.5). 5 mL fractions were collected and analyzed by SDS-PAGE. Fractions containing recombinant protein were pooled. Further purification was achieved by anion- or cation-exchange chromatography on Hitrap columns (Pharmacia). Bound protein was eluted using a NaCl gradient from 0-500 mM in 3 M urea, 10 mM Tris-HCl, pH 8.0. All fractions were collected and analyzed on SDS-PAGE using Coomassie staining. Fractions containing recombinant protein were pooled. Final protein concentrations were determined by micro BCA (Pierce).
  • a prophylactic vaccine given prior to infection should induce an immune response sufficiently strong to prevent or dampen the initial proliferation of the bacteria in the acute phase and thereby reduce the ensuing disease.
  • nai ⁇ umlaut over (v) ⁇ e mice are immunized 3 times, 2 weeks apart with recombinant antigens. Six weeks after the last immunization, the mice are given an aerosol infection with approximately 250 M. tuberculosis bacilli. The protective capacity of the vaccine is evaluated by enumeration of the bacteria in spleen and lung 6 weeks post-infection.
  • a murine reactivation model of latent TB has been established (van Pinxteren, 2000) ( FIG. 1B ).
  • An aerosol infection with approximately 250 M. tuberculosis bacilli is given and at the peak of infection 6 weeks later, the mice receive an 8-week course of anti-mycobacterial drug treatment of isoniazid and rifabutin given in the drinking water. This reduces the bacterial load in spleen and lung to a low level (about 500 bacteria per organ).
  • This latent phase of low chronic infection is stable for 9-10 weeks after which a slow spontaneous reactivation occurs.
  • the therapeutic vaccine is given as 3 subcutaneous (s.c.) immunizations about 5 weeks after cessation of drug treatment. The effect of the therapeutic vaccine is evaluated as protection against reactivation determined by enumeration of bacteria in spleen and lung 7 weeks after the last immunization.
  • BCG, ESAT6, and Rv2031c one of the most prominent proteins induced under low oxygen conditions (Rosenkrands, 2002), were analyzed for their prophylactic and therapeutic vaccine potential.
  • Na ⁇ ve or latently infected C57Bl mice were immunized with one s.c. injection of 2.5 ⁇ 10 5 BCG, or 3 s.c. immunizations of 10 ⁇ g of either recombinant ESAT6 or recombinant Rv2031c in a DDA/MPL adjuvant.
  • the vaccinations were done in groups of 5 mice and protective capacity of the vaccines was evaluated as described above.
  • ESAT6 offers protection against acute phase infection at the level of BCG ( FIG. 2A ). However, neither of the two shows any protective effect against reactivation of the infection when given during latent infection ( FIG. 2B ).
  • Rv2031c the low oxygen induced antigen, offers no protection against the acute phase of the infection when given as a prophylactic vaccine, but gives some protection against reactivation when given as a therapeutic vaccine. That is, some antigens, here exemplified by ESAT6, though potent as prophylactic vaccines have no effect as therapeutic vaccines. In contrast, other antigens, here exemplified by Rv2031c, can be efficient therapeutic vaccines although they have no effect or only negligible effect as prophylactic vaccines.
  • Rv2031c As a therapeutic vaccine was therefore repeated in groups of eight mice. As in the previous experiments the mice were given 3 s.c. immunizations of 10 ⁇ g rRv2031c in DDA/MPL. The induced immune responses were analyzed one week post immunization. The mice were partially bled and the PBMC from the blood purified and analyzed for Rv2031c- and ESAT6 specific recall responses.
  • the frequency of Rv2031c-specific and ESAT6-specific IFN- ⁇ -producing cells were determined in both the rRv2031c immunized and the unimmunized group ( FIG. 3 ).
  • the rRv2031c immunization has increased the frequency of Rv2031c-specific IFN- ⁇ producing cells by a factor of 43 as compared to the unimmunized group.
  • the frequency of ESAT6-specific IFN- ⁇ producing cells is significantly higher in the unimmunized group.
  • ESAT6 is an antigen produced in high amounts by the actively-growing M. tuberculosis bacteria.
  • the level of the ESAT6 specific immune response in infected mice could therefore be indicative the degree of actively-growing infection in the animals.
  • Recent reports have in fact demonstrated such a correlation between the level of ESAT6 response and degree of disease in both M. tuberculosis -infected humans and M. bovis -infected cattle (Doherty, 2002, Vordermeier, 2002). Therefore, the higher ESAT6 response in the unimmunized group of latently-infected mice could be indicative of a transition into the reactivation phase, where the bacteria are again beginning to multiply.
  • Rv2031c fourteen overlapping peptides (each 20 amino acids long) covering the whole Rv2031c protein were synthesized. Initially the peptides were analyzed in 4 pools of 3-4 peptides. PBMCs from rRv2031c immunized latently-infected mice were incubated with the peptide pools (5 ⁇ g/ml per peptide) for 72 h. The specific peptide-induced IFN- ⁇ production was quantitated in the supernatant in a standard sandwich ELISA using paired anti-murine IFN- ⁇ antibodies (PharMingen) and recombinant IFN- ⁇ (PharMingen) as standard.
  • PharMingen paired anti-murine IFN- ⁇ antibodies
  • PharMingen recombinant IFN- ⁇
  • FIG. 5 shows the bacterial load in the lung (A) and the spleen (B) of both rRv2031c-immunized and unimmunized mice.
  • Rv0569 is also a low oxygen induced antigen described in WO0179274 and illustrates very well the potential as a therapeutic vaccine.
  • Rv0569 which is highly up regulated under low oxygen growth conditions [Rosenkrands et al, 2002, 184(13): 3485-91], was analyzed for its ability to protect against reactivation given as a therapeutic vaccine in the latent phase of TB infection.
  • Latent infected C57Bl mice were vaccinated with 3 s.c. injections of 3 ⁇ g recombinant Rv0569 and for comparison with 3 s.c. injections of 3 ⁇ g recombinant ESAT6 or one s.c. injection of BCG.
  • the effect of the vaccine is evaluated 7 weeks after the last immunization.
  • the induced immune responses were analyzed for Rv0569 or ESAT6 specific responses in an in vitro recall assay.
  • FIG. 6 shows a nice Rv0569 specific IFN ⁇ response induced in the Rv0569 vaccinated group with practically no response in the un-vaccinated group.
  • the ESAT6 vaccination enhanced the ESAT6 specific response though a significant ESAT6 response persisted in the un-vaccinated latent infected group.
  • FIG. 7 shows the bacterial load in the lung and the spleen of both Rv0569-vaccinated, ESAT6-vaccinated, BCG vaccinated and un-vaccinated latently infected mice.

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Abstract

Therapeutic vaccines comprising polypeptides expressed during the latent stage of mycobacteria infection are provided, as are multiphase vaccines, and methods for treating and preventing tuberculosis.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a division of U.S. patent application Ser. No. 12/785,053, filed May 21, 2010, which is a continuation of U.S. patent application Ser. No. 10/617,038, filed Jul. 11, 2003, now abandoned, which claims the benefit of the priority of U.S. Provisional Patent Application No. 60/401,725, filed Aug. 7, 2002, now expired, and the priority of Danish Patent Patent Application No. PA 2002 01098, filed Jul. 13, 2002, which applications are incorporated herein by reference.
    • BACKGROUND OF THE INVENTION
  • The present invention discloses a therapeutic vaccine against latent or active tuberculosis infection caused by the tuberculosis complex microorganisms (Mycobacterium tuberculosis, M. bovis, M. africanum). The invention furthermore discloses a multi-phase vaccine that can be administered either prophylactically or therapeutically as well as a diagnostic reagent for the detection of latent stages of tuberculosis.
  • Human tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is a severe global health problem, responsible for approx. 3 million deaths annually, according to the WHO. The worldwide incidence of new tuberculosis (TB) cases had been falling during the 1960s and 1970s but during recent decades this trend has markedly changed in part due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis.
  • Organisms of the tuberculosis complex can cause a variety of diseases, but the commonest route of invasion is by inhalation of bacteria. This initiates an infection in the lung, which can ultimately spread to other parts of the body. Normally, this infection is restricted in growth by the immune system, so that the majority of infected individuals show few signs apart from cough and fever, which eventually abates. Approximately 30% of individuals are unable to contain the infection and they will develop primary disease, which in many cases will eventually prove fatal. However, it is believed that even those individuals who apparently control the infection remain infected, probably for the rest of their life. Certainly, individuals who have been healthy for years or even decades can suddenly develop tuberculosis, which has proven to be caused by the same organism they were infected with many years previously. M. tuberculosis and other organisms of the TB complex are unique in that the mycobacteria can evade the immune response and survive for long periods in a refractory non-replicating or slowly-replicating stage. This is referred to as latent TB and is at present a very significant global health problem that is estimated to affect approximately ⅓ of the world's population (Anon., 2001).
  • The course of a M. tuberculosis infection runs essentially through 3 phases, as illustrated in FIG. 1. During the acute phase, the bacteria proliferate in the organs, until the immune response increases to the point at which it can control the infection, whereupon the bacterial load peaks and starts declining. After this, a latent phase is established where the bacterial load is kept stable at a low level. In this phase M. tuberculosis goes from active multiplication to dormancy, essentially becoming non-replicating and remaining inside the granuloma. In some cases, the infection goes to the reactivation phase, where the dormant bacteria start replicating again. The full nature of the immune response that controls latent infection and the factors that lead to reactivation are largely unknown. However, there is some evidence for a shift in the dominant cell types responsible. While CD4 T cells are essential and sufficient for control of infection during the acute phase, studies suggest that CD8 T cell responses are more important in the latent phase. It is also likely that changes in the antigen-specificity of the response occur, as the bacterium modulates gene expression during its transition from active replication to dormancy.
  • The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. Although BCG consistently performs well in animal models of primary infection, it has clearly failed to control the TB epidemic. Consistent with that, BCG vaccination appears to provide protection against pediatric TB (which is due to primary infection), while offering little or no protection against adult disease (which is often reactivation of latent infection acquired in childhood). It has also been shown that vaccination of individuals who are currently sensitized to mycobacteria or latently infected is ineffective. Thus, current vaccination strategies, while effective against primary disease, fail to activate immune responses that efficiently control surviving dormant bacteria.
  • At this point no vaccine has been developed that confers protection against reactivation whether given as a prophylactic vaccine prior to infection or as a therapeutic vaccine given to already latently infected individuals.
  • This makes the development of a new and improved vaccine against TB an urgent matter, which has been given a very high priority by the WHO. Many attempts to define protective mycobacterial substances have been made, and different investigators have reported increased resistance after experimental vaccination. However, these efforts have almost exclusively focused on the development of prophylactic vaccines for the prevention of disease (Doherty, 2002), and such vaccines have not been demonstrated to work if given in an immunotherapeutic fashion (J. Turner et al., Infect and Immunity, 2000, pp. 1706-1709).
  • It has been suggested that the transition of M. tuberculosis from primary infection to latency is accompanied by changes in gene expression (see, for example, Honer zu Bentrup, 2001, which is incorporated herein by reference). In vitro hypoxic culture conditions, which mimic the conditions of low oxygen tension and restricted nutrients found in the granuloma (the location of the latent infection), have been used to analyze changes in gene expression and a number of antigens have been found that are induced or markedly upregulated under these conditions e.g. the 16 kDa antigen α-crystalline (Boon, 2001, Monahan, 2001, Florczyk 2001, Sherman 2001, Manganelli, 2001, all of which are incorporated herein by reference) and Rv0569 as described in Rosenkrands, 2002, and which is described in WO0179274.
  • As noted in the references cited above, it is already known that some genes are upregulated under conditions that mimic latency. However, these are a limited subset of the total gene expression during latent infection. Moreover, as one skilled in the art will readily appreciate, expression of a gene is not sufficient to make it a good vaccine candidate. The only way to determine if a protein is recognized by the immune system during latent infection with M. tuberculosis is to produce the given protein and test it in an appropriate assay as described herein. Of the more than 200 hundred antigens known to be expressed during primary infection, and tested as vaccines, less than a half dozen have demonstrated significant potential. So far only one antigen has been shown to have any potential as a therapeutic vaccine (Lowrie, 1999). However this vaccine only worked if given as a DNA vaccine, an experimental technique so far not approved for use in humans. Moreover, the technique has proved controversial, with other groups claiming that vaccination using this protocol induces either non-specific protection or even worsens disease (J. Turner et al., Infect and Immunity, 2000, pp. 1706-1709).
  • What are needed are therapeutic vaccines that treat latent TB infection.
    • SUMMARY OF THE INVENTION
  • The present invention provides therapeutic vaccines based on molecules that are induced or upregulated under the conditions of low oxygen transmission and restricted nutrients found in the granuloma (i.e., the location of latent TB infection). These vaccines are therapeutic and contrast with prior art vaccines which are designed to elicit protective immune responses prior to infection (prophylactic vaccination) that are only effective against primary infection. The immune responses elicited are powerless against the latent stage of the disease, because the bacteria have changed the antigens that they produce so that in essence they have altered their appearance and the immune system can no longer recognize them. However, latency is a dynamic process, maintained by the immune response, as indicated by the dramatic increase in the risk of reactivation of TB after HIV infection or other events that compromise immunity. Therefore, an effective vaccination strategy to protect infected individuals (therapeutic vaccination) is possible, but only if it is directed against those antigens expressed in the latent stage.
  • Further, the present invention provides a multiphase vaccine that combines components with prophylactic and therapeutic activity. In contrast, existing TB vaccines do not result in sterilizing immunity but rather control the infection at a subclinical level (thereby resulting in the subsequent establishment of latent infection. After conventional prophylactic vaccination, the evasion of the primary immune response and the subsequent development of latent disease are probably at least in part due to the change in the antigenic profile of the invading bacteria. Thus, vaccinating with antigens associated with latent TB prevents or reduces the establishment of latent infection and therefore, a vaccine incorporating antigens expressed by the bacteria both in the first logarithmic growth phase and during latent disease improve long-term immunity when used as a prophylactic vaccine. A multiphase vaccine of the invention will also be efficient as a therapeutic vaccine thereby addressing the problem that the majority of the population in the third world who would receive a future TB vaccine could be already latently infected.
  • For a number of years, a major effort has been put into the identification of protective antigens for the development of novel prophylactic vaccines against TB and today a few antigens with demonstrated protective activity in prophylactic vaccines have been identified (e.g. ESAT-6, the 38 kDa antigen, Ag85A and Ag85B). Such molecules are useful components, which in combination with latency associated antigens such as α-crystalline, form a multiphase vaccine of the invention. Advantageously and in contrast to antigens in the art, the antigens described in the invention are incorporated in vaccines through the use of well-recognized vaccination technology, as demonstrated in provided examples.
  • Finally, the immunodominant antigens identified in this invention may be used as diagnostic reagents. Our group has abundantly demonstrated that antigens expressed by mycobacteria during the early stages of the infection, such as ESAT-6 (Early Secretory Antigen Target-6) are recognized in individuals who are in the process of developing primary TB, even though they are healthy at the time of diagnosis (Doherty 2002). However, the large numbers of contacts who are exposed, and almost certainly infected, remain negative to this antigen (Doherty 2002). Since those individuals latently infected remain healthy by making an immune response against the latent bacteria, they must be making an immune response to those antigens expressed by the latent bacteria. Thus, the antigens of the invention may also be used to diagnose latent infection and differentiate it from primary acute TB.
  • Other aspects and advantages of the invention will be readily apparent to one of skill in the art.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1A and 1B illustrate the results of testing in TB vaccination models. A schematic time schedule of the models for FIG. 1A, prophylactic vaccination and FIG. 1B, therapeutic vaccination. Each square on the time axis represents one week. Three prophylactic vaccinations two weeks apart are given 6 weeks prior to an aerosol infection. The protective effect of the vaccines is measured 6 weeks after infection, in the acute phase of the infection. For analysis of therapeutic vaccinations a reactivation model is established, where aerosol infected mice are treated with anti-M tuberculosis drugs for 8 weeks from the peak of infection (6 weeks after infection). This induces a latent infection phase with a low bacterial load. Four to five weeks into the latency phase three therapeutic vaccinations are given two weeks apart and the protective effect of the vaccines is measured as bacterial load in the reactivation phase, seven weeks after the last immunization.
  • FIGS. 2A and 2B illustrate prophylactic and therapeutic vaccine induced protection. C57Bl/6j mice were immunized 3 times with a 2-week interval with recombinant ESAT6, BCG or recombinant Rv2031c. In FIG. 2A, the immunization was given as a prophylactic vaccine 6 weeks before the mice were given a M. tuberculosis infection (approx. 250 bacilli) through the aerosol route with. Bacterial numbers in the lung was enumerated 6 weeks post infection. In FIG. 2B, the immunization was given as a therapeutic vaccine after a latent infection had been established. Bacterial numbers in the lung was enumerated 8 weeks after the last immunization. The data represents the mean of 5 individual mice.
  • FIG. 3 illustrates Rv2031c specific IFN-γ responses. Latent infected C57Bl/6j mice were either not immunized or immunized with 3 μg recombinant Rv2031 3 times with a two-week interval. One and two weeks post immunization mice were bleed and PBMCs isolated. The frequency of IFN-γ producing cells specific for either ESAT6 or Rv2031c was determined for both the rRv2031c immunized and the unimmunized group. In an ELIspot plate precoated with anti-IFN-γ antibodies graded numbers of PBMCs were incubated with either 2 μg/ml rRv2031c or 2 μg/ml rESAT6. After 32 h the plate was washed and incubated with biotinylated anti-INF-γ antibodies followed by a streptavidin-alkalinephosphatase incubation. The INFγ spots, representing individual IFN-γ producing cells were visualized using BCIP/NBT substrate. The results are shown as number Rv2031c specific IFN-γ producing cell (black bars) and number of ESAT6 specific IFN-γ producing cell (hatch bars) per 106 PBMCs.
  • FIGS. 4A and 4B illustrate the results of epitope screening of Rv2031c. PBMCs from rRv2031c immunized latently infected C57Bl/6j mice were analyzed for recognition of 20′ mer overlapping peptides scanning through Rv2031c. In FIG. 4A, the peptides were analyzed in pools of 3-4 peptides. PBMCs (2×105) were incubated for 72 h with the peptide pools at 5 μg/ml per peptide. Supernatant was harvested and secreted IFN-γ was quantitated by ELISA. In FIG. 4B, individual peptides of positive pools were reanalyzed. PBMCs (2×105) were incubated for 72 h with 1 μg/ml of each peptide. Secreted IFN-γ in the supernatant was quantitated.
  • FIGS. 5A and 5B illustrate protection against reactivation conferred by therapeutic vaccine given during latent infection. Latent infected C57Bl/6j mice were immunized 3 times with or without rRv2031c. Bacterial numbers in lung (FIG. 5A) and spleen (FIG. 5B) was enumerated 8 weeks after the last immunization. The data represents the mean of 8 individual mice.
  • FIGS. 6A and 6B illustrate Rv0569 specific IFN-γ responses. Latent infected C57Bl/6j mice were vaccinated with 3 μg of either recombinant Rv0569 or recombinant ESAT6 in a DDA/MPL adjuvant. The vaccines were given as 3 s.c. injections with a two-week interval and the induced immune response were evaluated 7 weeks after the last vaccination. Isolated splenocytes (2×105) were incubated for 72 h with antigen at 1 μg/ml. Supernatant was harvested and secreted IFN-γ was quantitated by ELISA using paired anti-murine IFN-γ antibodies (PharMingen) and recombinant IFN-γ (PharMingen) as standard. In FIG. 6A, Rv0569 specific response is measured in Rv0569-vaccinated and un-vaccinated latently infected mice; in FIG. 6B, the ESAT6 specific response is measured in ESAT6-vaccinated and un-vaccinated latently infected mice
  • FIGS. 7A and 7B illustrate therapeutic vaccine induced protection against reactivation. Latently infected C57Bl/6j mice were vaccinated once with BCG or 3 times with a 2-week interval with either recombinant Rv0569 or recombinant ESAT6. Seven weeks after the last vaccination the bacterial numbers was enumerated in FIG. 7A, the lung and in FIG. 7B, the spleen of vaccinated and un-vaccinated mice. The data represents the mean of Log CFU per organ of 6-8 individual mice.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The invention is related to preventing, treating and detecting infections caused by species of the tuberculosis complex (Mycobacterium tuberculosis, M. bovis, M. africanum) by the use of a polypeptide comprising a M. tuberculosis antigen or an immunogenic portion or other variant thereof, or by the use of a DNA sequence encoding a M. tuberculosis antigen or an immunogenic portion or other variant thereof. The invention discloses a new therapeutic vaccine against tuberculosis comprising antigens induced during the latent stage of TB-infection. It also discloses a multiphase vaccine incorporating a combination of prophylactic and therapeutic antigens as well as diagnostic reagents for the detection of the latent stage of M. tuberculosis infection.
  • The present invention discloses the use of one or more polypeptides, nucleic acids encoding these polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, for a therapeutic vaccine against tuberculosis.
  • The polypeptides comprises one or more amino acid sequences selected from
      • (a) The sequences presented in Table 1.
      • (b) an immunogenic portion, e.g. a T-cell epitope, of any one of the sequences in (a); and/or
      • (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic.
  • TABLE 1
    Amino acid sequences of selected low oxygen induced antigens
    Rv no. SEQ ID NO: Sequence
    Rv0569 91 MKAKVGDWLVIKGATIDQPDHRGLIIEVRSSDGSPPYVVRWLETDHVATV
    IPGPDAVVVTAEEQNAADERAQHRFGAVQSAILHARGT
    Rv0079 1 VEPKRSRLVVCAPEPSHAREFPDVAVFSGGRANASQAERLARAVGRVLAD
    RGVTGGARVRLTMANCADGPTLVQINLQVGDTPLRAQAATAGIDDLRPAL
    IRLDRQIVRASAQWCPRPWPDRPRRRLTTPAEALVTRRKPVVLRRATPLQ
    AIAAMDAMDYDVHLFTDAETGEDAVVYRAGPSGLRLARQHHVFPPGWSRC
    RAPAGPPVPLIVNSRPTPVLTEAAAVDRAREHGLPFLETTDQATGRGQLL
    YSRYDGNLGLITPTGDGVADGLA
    Rv0080 2 MSPGSRRASPQSAREVVELDRDEAMRLLASVDHGRVVFTRAALPAIRPVN
    HLVVDGRVIGRTRLTAKVSVAVRSSADAGVVVAYEADDLDPRRRTGWSVV
    VTGLATEVSDPEQVARYQRLLHPWVNMAMDTVVAIEPEIVTGIRIVADSR
    TP
    Rv0081 3 VESEPLYKLKAEFEKTLAHPARIRILELLVERDRSVGELLSSDVGLESSN
    LSQQLGVLRRAGVVAARRDGNAMIYSIAAPDIAELLAVARKVLARVLSDR
    VAVLEDLRAGGSAT
    Rv0363c 4 MPIATPEVYAEMLGQAKQNSYAFPAINCTSSETVNAAIKGFADAGSDGII
    QFSTGGAEFGSGLGVKDMVTGAVALAEFTHVIAAKYPVNVALHTDHCPKD
    KLDSYVRPLLAISAQRVSKGGNPLFQSHMWDGSAVPIDENLAIAQELLKA
    AAAAKIILEIEIGVVGGEEDGVANEINEKLYTSPEDFEKTIEALGAGEHG
    KYLLAATFGNVHGVYKPGNVKLRPDILAQGQQVAAAKLGLPADAKPFDFV
    FHGGSGSLKSEIEEALRYGVVKMNVDTDTQYAFTRPIAGHMFTNYDGVLK
    VDGEVGVKKVYDPRSYLKKAEASMSQRVVQACNDLHCAGKSLTH
    Rv0572c 5 MGEHAIKRHMRQRKPTKHPLAQKRGARILVFTDDPRRSVLIVPGCHLDSM
    RREKNAYYFQDGNALVGMVVSGGTVEYDADDRTYVVQLTDGRHTTESSFE
    HSSPSRSPQSDDL
    Rv0574c 6 VAGNPDVVTVLLGGDVMLGRGVDQILPHPGKPQLRERYMRDATGYVRLAE
    RVNGRIPLPVDWRWPWGEALAVLENTATDVCLINLETTITADGEFADRKP
    VCYRMHPDNVPALTALRPHVCALANNHILDFGYQGLTDTVAALAGAGIQS
    VGAGADLLAARRSALVTVGHERRVIVGSVAAESSGVPESWAARRDRPGVW
    LIRDPAQRDVADDVAAQVLADKRPGDIAIVSMHWGSNWGYATAPGDVAFA
    HRLIDAGIDMVHGHSSHHPRPIEIYRGKPILYGCGDVVDDYEGIGGHESF
    RSELRLLYLTVTDPASGNLISLQMLPLRVSRMRLQRASQTDTEWLRNTIE
    RISRRFGIRVVTRPDNLLEVVPAANLTSKE
    Rv1264 7 VTDHVREADDANIDDLLGDLGGTARAERAKLVEWLLEQGITPDEIRATNP
    PLLLATRHLVGDDGTYVSAREISENYGVDLELLQRVQRAVGLARVDDPDA
    VVHMRADGEAAARAQRFVELGLNPDQVVLVVRVLAEGLSHAAEAMRYTAL
    EAIMRPGATELDIAKGSQALVSQIVPLLGPMIQDMLFMQLRHMMETEAVN
    AGERAAGKPLPGARQVTVAFADLVGFTQLGEVVSAEELGHLAGRLAGLAR
    DLTAPPVWFIKTIGDAVMLVCPDPAPLLDTVLKLVEVVDTDNNFPRLRAG
    VASGMAVSRAGDWFGSPVNVASRVTGVARPGAVLVADSVREALGDAPEAD
    GFQWSFAGPRRLRGIRGDVRLFRVRRGATRTGSGGAAQDDDLAGSSP
    Rv1592c 8 MVEPGNLAGATGAEWIGRPPHEELQRKVRPLLPSDDPFYFPPAGYQHAVP
    GTVLRSRDVELAFMGLIPQPVTATQLLYRTTNMYGNPEATVTTVIVPAEL
    APGQTCPLLSYQCAIDAMSSRCFPSYALRRRAKALGSLTQMELLMISAAL
    AEGWAVSVPDHEGPKGLWGSPYEPGYRVLDGIRAALNSERVGLSPATPIG
    LWGYSGGGLASAWAAEACGEYAPDLDIVGAVLGSPVGDLGHTFRRLNGTL
    LAGLPALVVAALQHSYPGLARVIKEHANDEGRQLLEQLTEMTTVDAVIRM
    AGRDMGDFLDEPLEDILSTPEISHVFGDTKLGSAVPTPPVLIVQAVHDYL
    IDVSDIDALADSYTAGGANVTYHRDLFSEHVSLHPLSAPMTLRWLTDRFA
    GKPLTDHRVRTTWPTIFNPMTYAGMARLAVIAAKVITGRKLSRRPL
    Rv1733c 9 MIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAV
    TVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNT
    TATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQL
    VDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQH
    DIDSLFCTQR
    Rv1734c 10 MTNVGDQGVDAVFGVIYPPQVALVSFGKPAQRVCAVDGAIHVMTTVLATL
    PADHGCSDDHRGALFFLSINELTRCAAVTG
    Rv1736c 11 VTVTPRTGSRIEELLARSGRFFIPGEISADLRTVTRRGGRDGDVFYRDRW
    SHDKVVRSTHGVNCTGSCSWKIYVKDDIITWETQETDYPSVGPDRPEYEP
    RGCPRGAAFSWYTYSPTRVRHPYARGVLVEMYREAKARLGDPVAAWADIQ
    ADPRRRRRYQRARGKGGLVRVSWAEATEMIAAAHVHTISTYGPDRVAGFS
    PIPAMSMVSHAAGSREVELIGGVMTSFYDWYADLPVASPQVFGDQTDVPE
    SGDWWDVVWQCASVLLTYPNSRQLGTAEELLAHIDGPAADLLGRTVSELR
    RADPLTAATRYVDTFDLRGRATLYLTYWTAGDTRNRGREMLAFAQTYRST
    DVAPPRGETPDFLPVVLEFAATVDPEAGRRLLSGYRVPTAALCNALTEAA
    LPYAHTVAAVCRTGDMMGELFWTVVPYVTMTIVAVGSWWRYRYDKFGWTT
    RSSQLYESRLLRIASPMFHFGILVVIVGHGIGLVIPQSWTQAAGLSEGAY
    HVQAVVLGSIAGITTLAGVTLLIYRRRTRGPVFMATTVNDKVMYLVLVAA
    IVAGLGATALGSGVVGEAYNYRETVSVWFRSVWVLQPRGDLMAEAPLYYQ
    IHVLIGLALFALWPFTRLVHAFSAPIGYLFRPYIIYRSREELVLTRPRRR
    GW
    Rv1737c 12 MRGQAANLVLATWISVVNFWAWNLIGPLSTSYARDMSLSSAEASLLVATP
    ILVGALGRIVTGPLTDRFGGRANLIAVTLASILPVLAVGVAATMGSYALL
    VFFGLFLGVAGTIFAVGIPFANNWYQPARRGESTGVEGMGMVGTALSAFE
    TPREVRWEGLFTTHAIVAAALASTAVVAMVVLRDAPYFRPNADPVLPRLK
    AAARLPVTWEMSFLYAIVEGGEVAFSNYLPTYITTIYGESTVDAGARTAG
    FALAAVLARPVGGWLSDRIAPRHVVLASLAGTALLAFAAALQPPPEVWSA
    ATFITLAVCLGVGTGGVFAWVARRAPAASVGSVTGIVAAAGGLGGYFPPL
    VMGATYDPVDNDYTVGLLLLVATALVACTYTALHAREPVSEEASR
    Rv1738c 13 MCGDQSDHVLQHWTVDISIDEHEGLTRAKARLRWREKELVGVGLARLNPA
    DRNVPEIGDELSVARALSDLGKRMLKVSTHDIEAVTHQPARLLY
    Rv1739c 14 MIPTMTSAGWAPGVVQFREYQRRWLRGDVLAGLTVAAYLIPQAMAYATVA
    GLPPAAGLWASIAPLAIYALLGSSRQLSIGPESATALMTAAVLAPMAAGD
    LRRYAVLAATLGLLVGLICLLAGTARLGFLASLRSRPVLVGYMAGIALVM
    ISSQLGTITGTSVEGNEFFSEVHSFATSVTRVHWPTFVLAMSVLALLTML
    TRWAPRAPGPIIAVLAATMLVAVMSLDAKGIAIVGRIPSGLPTPGVPPVS
    VEDLRALIIPAAGIAIVTFTDGVLTARAFAARRGQEVNANAELRAVGACN
    IAAGLTHGFPVSSSSSRTALADVVGGRTQLYSLIALGLVVIVMVFASGLL
    AMFPTAALGALVVYAALRLIDLSEFRRLARFRRSELMLALATTAAVLGLG
    VFYGVLAAVALSILELLRRVAHPHDSVLGFVPGIAGMHDIDDYPQAKRVP
    GLVVYRYDAPLCFANAEDFRRRALTVVDQDPGQVEWFVLNAESNVEVDLT
    ALDALDQLRTELLRRGIVFAMARVKQDLRESLRAASLLDKIGEDHIFMTL
    PTAVQAFRRR
    Rv1813c 15 MITNLRRRTAMAAAGLGAALGLGILLVPTVDAHLANGSMSEVMMSE
    IAGLPIPPIIHYGAIAYAPSGASGKAWHQRTPARAEQVALEKCGDK
    TCKVVSRFTRCGAVAYNGSKYQGGTGLTRRAAEDDAVNRLEGGRIV
    NWACN
    Rv1997c 16 LSASVSATTAHHGLPAHEVVLLLESDPYHGLSDGEAAQRLERFGPNTLAV
    VTRASLLARILRQFHHPLIYVLLVAGTITAGLKEFVDAAVIFGVVVINAI
    VGFIQESKAEAALQGLRSMVHTHAKVVREGHEHTMPSEELVPGDLVLLAA
    GDKVPADLRLVRQTGLSVNESALTGESTPVHKDEVALPEGTPVADRRNIA
    YSGTLVTAGHGAGIVVATGAETELGEIHRLVGAAEVVATPLTAKLAWFSK
    FLTTATLGLAALTFGVGLLRRQDAVETFTAAIALAVGAIPEGLPTAVTIT
    LAIGMARMAKRRAVIRRLPAVETLGSTTVICADKTGTLTENQMTVQSIWT
    PHGEIRATGTGYAPDVLLCDTDDAPVPVNANAALRWSLLAGACSNDAALV
    RDGTRWQIVGDPTEGAMLVVAAKAGFNPERLATTLPQVAAIPFSSERQYM
    ATLHRDGTDHVVLAKGAVERMLDLCGTEMGADGALRPLDRATVLRATEML
    TSRGLRVLATGMGAGAGTPDDFDENVIPGSLALTGLQAMSDPPRAAAASA
    VAACHSAGIAVKMITGDHAGTATAIATEVGLLDNTEPAAGSVLTGAELAA
    LSADQYPEAVDTASVFARVSPEQKLRLVQALQARGHVVAMTGDGVNDAPA
    LRQANIGVAMGRGGTEVAKDAADMVLTDDDFATIEAAVEEGRGVFDNLTK
    FITWTLPTNLGEGLVILAAIAVGVALPILPTQILWINMTTAIALGLMLAF
    EPKEAGIMTRPPRDPDQPLLTGWLVRRTLLVSTLLVASAWWLFAWELDNG
    AGLHEARTAALNLFVVVEAFYLFSCRSLTRSAWRLGMFANRWIILGVSAQ
    AIAQFATTYLPAMNMVFDTAPIDIGVWVRIFAVATAITIVVATDTLLPRI
    RAQPP
    Rv1998c 17 MSFHDLHHQGVPFVLPNAWDVPSALAYLAEGFTAIGTTSFGVSSSGGHPD
    GHRATRGANIALAAALAPLQCYVSVDIEDGYSDEPDAIADYVAQLSTAGI
    NIEDSSAEKLIDPALAAAKIVAIKQRNPEVFVNARVDTYWLRQHADTTST
    IQRALRYVDAGADGVFVPLANDPDELAELTRNIPCPVNTLPVPGLTIADL
    GELGVARVSTGSVPYSAGLYAAAHAARAVSDGEQLPRSVPYAELQARLVD
    YENRTSTT
    Rv2003c 18 VVKRSRATRLSPSIWSGWESPQCRSIRARLLLPRGRSRPPNADCCWNQLA
    VTPDTRMPASSAAGRDAAAYDAWYDSPTGRPILATEVAALRPLIEVFAQP
    RLEIGVGTGRFADLLGVREGLDPSRDALMFARRRGVLVANAVGEAVPFVS
    RHFGAVLMAFTLCFVTDPAAIFRETRRLLADGGGLVIGELPRGTPWADLY
    ALRAARGQPGYRDARFYTAAELEQLLADSGFRVIARRCTLHQPPGLARYD
    IEAAHDGIQAGAGFVAISAVDQAHEPKDDHPLESE
    Rv2005c 19 MSKPRKQHGVVVGVDGSLESDAAACWGATDAAMRNIPLTVVHVVNADVAT
    WPPMPYPETWGVWQEDEGRQIVANAVKLAKEAVGADRKLSVKSELVFSTP
    VPTMVEISNEAEMVVLGSSGRGALARGLLGSVSSSLVRRAGCPVAVIHSD
    DAVIPDPQHAPVLVGIDGSPVSELATAVAFDEASRRGVELIAVHAWSDVE
    VVELPGLDFSAVQQEAELSLAERLAGWQERYPDVPVSRVVVCDRPARKLV
    QKSASAQLVVVGSHGRGGLTGMLLGSVSNAVLHAARVPVIVARQS
    Rv2007c 20 VTYVIGSECVDVMDKSCVQECPVDCIYEGARMLYINPDECVDCGACKPAC
    RVEAIYWEGDLPDDQHQHLGDNAAEFHQVLPGRVAPLGSPGGAAAVGPIG
    VDTPLVAAIPVECP
    Rv2028c 21 MNQSHKPPSIVVGIDGSKPAVQAALWAVDEAASRDIPLRLLYAIEPDDPG
    YAAHGAAARKLAAAENAVRYAFTAVEAADRPVKVEVEITQERPVTSLIRA
    SAAAALVCVGAIGVHHFRPERVGSTAAALALSAQCPVAIVRPHRVPIGRD
    AAWIVVEADGSSDIGVLLGAVMAEARLRDSPVRVVTCRQSGVGDTGDDVR
    ASLDRWLARWQPRYPDVRVQSAAVHGELLDYLAGLGRSVHMVVLSASDQE
    HVEQLVGAPGNAVLQEAGCTLLVVGQQYL
    Rv2029c 22 MTEPAAWDEGKPRIITLTMNPALDITTSVDVVRPTEKMRCGAPRYDPGGG
    GINVARIVHVLGGCSTALFPAGGSTGSLLMALLGDAGVPFRVIPIAASTR
    ESFTVNESRTAKQYRFVLPGPSLTVAEQEQCLDELRGAAASAAFVVASGS
    LPPGVAADYYQRVADICRRSSTPLILDTSGGGLQHISSGVFLLKASVREL
    RECVGSELLTEPEQLAAAHELIDRGRAEVVVVSLGSQGALLATRHASHRF
    SSIPMTAVSGVGAGDAMVAAITVGLSRGWSLIKSVRLGNAAGAAMLLTPG
    TAACNRDDVERFFELAAEPTEVGQDQYVWHPIVNPEASP
    Rv2030c 23 VLMTAAADVTRRSPRRVFRDRREAGRVLAELLAAYRDQPDVIVLGLARGG
    LPVAWEVAAALHAPLDAFVVRKLGAPGHDEFAVGALASGGRVVVNDDVVR
    GLRITPQQLRDIAEREGRELLRRESAYRGERPPTDITGKTVIVVDDGLAT
    GASMFAAVQALRDAQPAQIVIAVPAAPESTCREFAGLVDDVVCATMPTPF
    LAVGESFWDFRQVTDEEVRRLLATPTAGPSLRRPAASTAADVLRRVAIDA
    PGGVPTHEVLAELVGDARIVLIGESSHGTHEFYQARAAMTQWLIEEKGFG
    AVAAEADWPDAYRVNRYVRGLGEDTNADEALSGFEREPAWMWRNTVVRDF
    VEWLRTRNQRYESGALRQAGFYGLDLYSLHRSIQEVISYLDKVDPRAAAR
    ARARYACFDHACADDGQAYGFAAAFGAGPSCEREAVEQLVDVQRNALAYA
    RQDGLLAEDELFYAQQNAQTVRDAEVYYRAMFSGRVTSWNLRDQHMAQTL
    GSLLTHLDRHLDAPPARIVVWAHNSHVGDARATEVWADGQLTLGQIVRER
    YGDESRSIGESTYTGTVTAASEWGGIAQRKAVRPALHGSVEELFHQTADS
    FLVSARLSRDAEAPLDVVRLGRAIGVVYLPATERQSHYLHVRPADQFDAM
    IHIDQTRALEPLEVTSRWIAGENPETYPTGL
    Rv2031c 24 MATTLPVQRHPRSLFPEESELFAAFPSFAGLRPTFDTRLMRLEDEMKEGR
    YEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVR
    TVSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN
    Rv2032 25 MPDTMVTTDVIKSAVQLACRAPSLHNSQPWRWIAEDHTVALFLDKDRVLY
    ATDHSGREALLGCGAVLDHFRVAMAAAGTTANVERFPNPNDPLHLASIDF
    SPADFVTEGHRLRADAILLRRTDRLPFAEPPDWDLVESQLRTTVTADTVR
    IDVIADDMRPELAAASKLTESLRLYDSSYHAELFWWTGAFETSEGIPHSS
    LVSAAESDRVTFGRDEPVVANTDRRPEFGHDRSKVLVLSTYDNERASLLR
    CGEMLSAVLLDATMAGLATCTLTHITELHASRDLVAALIGQPATPQALVR
    VGLAPEMEEPPPATPRRPIDEVFHVRAKDHR
    Rv2428 26 MPLLTIGDQFPAYQLTALIGGDLSKVDAKQPGDYFTTITSDEHPGKWRVV
    FEWPKDFTEVCPTEIAAFSKLNDEFEDRDAQILGVSIDSEFAHFQWRAQH
    NDLKTLPFPMLSDIKRELSQAAGVLNADGVADRVTFIVDPNNEIQFVSAT
    AGSVGRNVDEVLRVLDALQSDELCACNWRKGDPTLDAGELLKASA
    Rv2624c 27 MSGRGEPTMKTIIVGIDGSHAAITAALWGVDEAISRAVPLRLVSVIKPTH
    PSPDDYDRDLAHAERSLREAQSAVEAAGKLVKIETDIPRGPAGPVLVEAS
    RDAEMICVGSVGIGRYASSILGSTATELAEKAHCPVAVMRSKVDQPASDI
    NWIVVRMTDAPDNEAVLEYAAREAKLRQAPILALGGRPEELREIPDGEFE
    RRVQDWHHRHPDVRVYPITTHTGIARFLADHDERVQLAVIGGGEAGQLAR
    LVGPSGHPVFRHAECSVLVVRR
    Rv2625c 28 MRDAIPLGRIAGFVVNVHWSVLVILWLFTWSLATMLPGTVGGYPAVVYWL
    LGAGGAVMLLASLLAHELAHAVVARRAGVSVESVTLWLFGGVTALGGEAK
    TPKAAFRIAFAGPATSLALSATFGALAITLAGVRTPAIVISVAWWLATVN
    LLLGLFNLLPGAPLDGGRLVRAYLWRRHGDSVRAGIGAARAGRVVALVLI
    ALGLAEFVAGGLVGGVWLAFIGWFIFAAAREEETRISTQQLFAGVRVADA
    MTAQPHTAPGWINVEDFIQRYVLGERHSAYPVADRDGSITGLVALRQLRD
    VAPSRRSTTSVGDIALPLHSVPTARPQEPLTALLERMAPLGPRSRALVTE
    GSAVVGIVTPSDVARLIDVYRLAQPEPTFTTSPQDADRFSDAG
    Rv2727c 29 MASSASGDTHERSAFRLSPPVLSGAMGPFMHTGLYVAQSWRDYLGQQPDK
    LPTARPTIALAAQAFRDEIVLLGLKARRPVSNHRVFERISQEVAAGLEFY
    GNRRWLEKPSGFFAQPPPLTEVAVRKVKDRRRSFYRIFEDSGFTPHPGEP
    GSQRWLSYTANNREYALLLRHPEPRPWLVCVHGTEMGRAPLDLAVFRAWK
    LHDELGLNIVMPVLPMHGPRGQGLPKGAVFPGEDVLDDVHGTAQAVWDIR
    RLLSWIRSQEEESLIGLNGLSLGGYIASLVASLEEGLACAILGVPVADLI
    ELLGRHCGLRHKDPRRHTVKMAEPIGRMISPLSLTPLVPMPGRFIYAGIA
    DRLVHPREQVTRLWEHWGKPEIVWYPGGHTGFFQSRPVRRFVQAALEQSG
    LLDAPRTQRDRSA
    Rv2628 30 MSTQRPRHSGIRAVGPYAWAGRCGRIGRWGVHQEAMMNLAIWHPRKVQSA
    TIYQVTDRSHDGRTARVPGDEITSTVSGWLSELGTQSPLADELARAVRIG
    DWPAAYAIGEHLSVEIAVAV
    Rv2629 31 MRSERLRWLVAAEGPFASVYFDDSHDTLDAVERREATWRDVRKHLESRDA
    KQELIDSLEEAVRDSRPAVGQRGRALIATGEQVLVNEHLIGPPPATVIRL
    SDYPYVVPLIDLEMRRPTYVFAAVDHTGADVKLYQGATISSTKIDGVGYP
    VHKPVTAGWNGYGDFQHTTEEAIRMNCRAVADHLTRLVDAADPEVVFVSG
    EVRSRTDLLSTLPQRVAVRVSQLHAGPRKSALDEEEIWDLTSAEFTRRRY
    AEITNVAQQFEAEIGRGSGLAAQGLAEVCAALRDGDVDTLIVGELGEATV
    VTGKARTTVARDADMLSELGEPVDRVARADEALPFAAIAVGAALVRDDNR
    IAPLDGVGALLRYAATNRLGSHRS
    Rv2630 32 MLHRDDHINPPRPRGLDVPCARLRATNPLRALARCVQAGKPGTSSGHRSV
    PHTADLRIEAWAPTRDGCIRQAVLGTVESFLDLESAHAVHTRLRRLTADR
    DDDLLVAVLEEVIYLLDTVGETPVDLRLRDVDGGVDVTFATTDASTLVQV
    GAVPKAVSLNELRFSQGRHGWRCAVTLDV
    Rv2659c 33 VTQTGKRQRRKFGRIRQFNSGRWQASYTGPDGRVYIAPKTFNAKIDAEAW
    LTDRRREIDRQLWSPASGQEDRPGAPFGEYAEGWLKQRGIKDRTRAHYRK
    LLDNHILATFADTDLRDITPAAVRRWYATTAVGTPTMRAHSYSLLRAIMQ
    TALADDLIDSNPCRISGASTARRVHKIRPATLDELETITKAMPDPYQAFV
    LMAAWLAMRYGELTELRRKDIDLHGEVARVRRAVVRVGEGFKVTTPKSDA
    GVRDISIPPHLIPAIEDHLHKHVNPGRESLLFPSVNDPNRHLAPSALYRM
    FYKARKAAGRPDLRVHDLRHSGAVLAASTGATLAELMQRLGHSTAGAALR
    YQHAAKGRDREIAALLSKLAENQEM
    Rv2780 34 MRVGIPTETKNNEFRVAITPAGVAELTRRGHEVLIQAGAGEGSAITDADF
    KAAGAQLVGTADQVWADADLLLKVKEPIAAEYGRLRHGQILFTFLHLAAS
    RACTDALLDSGTTSIAYETVQTADGALPLLAPMSEVAGRLAAQVGAYHLM
    RTQGGRGVLMGGVPGVEPADVVVIGAGTAGYNAARIANGMGATVTVLDIN
    IDKLRQLDAEFCGRIHTRYSSAYELEGAVKRADLVIGAVLVPGAKAPKLV
    SNSLVAHMKPGAVLVDIAIDQGGCFEGSRPTTYDHPTFAVHDTLFYCVAN
    MPASVPKTSTYALTNATMPYVLELADHGWRAACRSNPALAKGLSTHEGAL
    LSERVATDLGVPFTEPASVLA
    Rv3126c 35 MVIRFDQIGSLVLSMKSLASLSFQRCLRENSSLVAALDRLDAAVDELSAL
    SFDALTTPERDRARRDRDHHPWSRSRSQLSPRMAHGAVHQCQWPKAVWAV
    IDNP
    Rv3127 36 VLKNAVLLACRAPSVHNSQPWRWVAESGSEHTTVHLFVNRHRTVPATDHS
    GRQAIISCGAVLDHLRIAMTAAHWQANITRFPQPNQPDQLATVEFSPIDH
    VTAGQRNRAQAILQRRTDRLPFDSPMYWHLFEPALRDAVDKDVAMLDVVS
    DDQRTRLVVASQLSEVLRRDDPYYHAELEWWTSPFVLAHGVPPDTLASDA
    ERLRVDLGRDFPVRSYQNRRAELADDRSKVLVLSTPSDTRADALRCGEVL
    STILLECTMAGMATCTLTHLIESSDSRDIVRGLTRQRGEPQALIRVGIAP
    PLAAVPAPTPRRPLDSVLQIRQTPEKGRNASDRNARETGWFSPP
    Rv3128c 37 VWSASGGQCGKYLAASMVLQLDGLERHGVLEFGRDRYGPEVREELLAMSA
    ASIDRYLKTAKAKDQISGVSTTKPSPLLRNSIKVRRAGDEVEAEPGFFEG
    DTVAHCGPTLKGEFAHTLNLTDVHIGWVFTRTVRNNARTHILAGLKASVT
    EIPHGITGLDFDNGTVELNKPVISWAGDNGIYFTRFRPYKKNH*ATIESK
    NNHLVRKYAFYYRYDTAEERAVLNRMWKLVNDRLNYLTPTIKPIGYASSA
    DGRRRRLYDAPQTPLDRPLAARVLSAAQQADLITYRDSLNPAQIGRKIAD
    LQNRLLILAKEKTEQLYLANIPTALPDIHKGILIKAG
    Rv3129 38 VVQGRTVLFRTAEGAKLFSAVAKCAVAFEADDHNVAEGWSVIVKVRAQVL
    TTDAGVREAERAQLLPWTATLKRHCVRVIPWEITGRHFREGPEPDRSQTF
    ACEASSHNQR
    Rv3130c 39 MNHLTTLDAGFLKAEDVDRHVSLAIGALAVIEGPAPDQEAFLSSLAQRLR
    PCTRFGQRLRLRPFDLGAPKWVDDPDFDLGRHVWRIALPRPGNEDQLFEL
    IADLMARRLDRGRPLWEVWVIEGLADSKWAILTKLHHCMADGIAATHLLA
    GLSDESMSDSFASNIHTTMQSQSASVRRGGFRVNPSEALTASTAVMAGIV
    RAAKGASEIAAGVLSPAASSLNGPISDLRRYSAAKVPLADVEQVCRKFDV
    TINDVALAAITESYRNVLIQRGERPREDSLRTLVPVSTRSNSALSKTDNR
    VSLMLPNLPVDQENPLQRLRIVHSRLTRAKAGGQRQFGNTLMAIANRLPF
    PMTAWAVGLLMRLPQRGVVTVATNVPGPRRPLQIMGRRVLDLYPVSPIAM
    QLRTSVAMLSYADDLYFGILADYDVVADAGQLARGIEDAVARLVAISKRR
    KVTRRRGALSLVV
    Rv3131 40 MNTHFPDAETVRTVLTLAVRAPSIHNTQPWRWRVCPTSLELFSRRDMQLR
    STDPDGRELILSCGVALHHCVVALASLGWQAKVNRFPDPKDRCHLATIGV
    QPLVPDQADVALAAAIPRRRTDRRAYSCWPVPGGDIALMAARAARGGVML
    RQVSALDRMKAIVAQAVLDHVTDEEYLRELTIWSGRYGSVAGVPARNEPP
    SDPSAPIPGRLFAGPGLSQPSDVLPADDGAAILALGTETDDRLARLRAGE
    AASIVLLTATAMGLACCPITEPLETAKTRDAVRAEVEGAGGYPQMLLRVG
    WAPINADPLPPTPRRELSQVVEWPEELLRQRC
    Rv3132 41 MTTGGLVDENDGAAMRPLRHTLSQLRLHELLVEVQDRVEQIVEGRDRLDG
    LVEAMLVVTAGLDLEATLRAIVHSATSLVDARYGAMEVHDRQHRVLHEVY
    EGIDEETVRRIGHLPKGLGVIGLLIEDPKPLRLDDVSAHPASIGFPPYHP
    PMRTFLGVPVRVRDESEGTLYLTDKTNGQPFSDDDEVLVQALAAAAGIAV
    ANARLYQQAKARQSWIEATRDTATELLSGTEPATVFRLVAAEALKLTAAD
    AALVAVPVDEDMPAADVGELLVIETVGSAVASIVGRTIPVAGAVLREVFV
    NGIPRRVDRVDLEGLDELADAGPALLLPLRARGTVAGVVVVLSQGGPGAF
    TDEQLEMMAAFADQAALAWQLATSQRRMRELDVLTDRDRIARDLHDHVIQ
    RLFATGLALQGAVPHERNPEVQQRLSDVVDDLQDVIQEIRTTIYDLHGAS
    QGITRLRQRIDAAVAQFADSGLRTSVQFVGPLSVVDSALADQAEAVVREA
    VSNAVRHAKASTLTVRVKVDDDLCIEVTDNGRGLPDEFTGSGLTNLRQRA
    EQAGGEFTLASVPGASGTVLRWSAPLSQ
    Rv3134c 42 MSDPRPARAVVVGIDGSRAATHAALWAVDEAVNRDIPLRLVYVIDPSQLS
    AAGEGGGQSAARAALHDASRKVEATGQPVKIETEVLCGRPLTKLMQESRS
    AAMLCVGSVGLDHVRGRRGSVAATLAGSALCPVAVIHPSPAEPATTSQVS
    AVVAEVDNGVVLRHAFEEARLRGVPLRAVAVHAAETPDDVEQGSRLAHVH
    LSRRLAHWTRLYPEVRVDRAIAGGSACRHLAANAKPGQLFVADSHSAHEL
    CGAYQPGCAVLTVRSANL
    Rv3841 43 MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYS
    QAVEERNHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPREALALALDQER
    TVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVALMATLVRVADRAG
    ANLFELENFVAREVDVAPAASGAPHAAGGRL
    Rv3842c 44 MTWADEVLAGHPFVVAHRGASAARPEHTLAAYDLALKEGADGVECDVRLT
    RDGHLVCVHDRRLDRTSTGAGLVSTMTLAQLRELEYGAWHDSWRPDGSHG
    DTSLLTLDALVSLVLDWHRPVKIFVETKHPVRYGSLVENKLLALLHRFGI
    AAPASADRSRAVVMSFSAAAVWRIRRAAPLLPTVLLGKTPRYLTSSAATA
    VGATAVGPSLPALKEYPQLVDRSAAQGRAVYCWNVDEYEDIDFCREVGVA
    WIGTHHPGRTKAWLEDGRANGTTR
    Rv3908 45 VSDGEQAKSRRRRGRRRGRRAAATAENHMDAQPAGDATPTPATAKRSRSR
    SPRRGSTRMRTVHETSAGGLVIDGIDGPRDAQVAALIGRVDRRGRLLWSL
    PKGHIELGETAEQTAIREVAEETGIRGSVLAALGRIDYWFVTDGRRVHKT
    VHHYLMRFLGGELSDEDLEVAEVAWVPIRELPSRLAYADERRLAEVADEL
    IDKLQSDGPAALPPLPPSSPRRRPQTHSRARHADDSAPGQHNGPGPGP
  • TABLE 2
    DNA sequences of selected low oxygen induced antigens
    Rv no. SEQ ID NO: Sequence
    Rv0079c 46 gtggaaccgaaacgcagtcgcctcgtcgtatgtgcacccgagccatcgca
    cgcgcgggaattcccggatgtcgccgtattctccggcggccgggctaacg
    catcccaggccgaacggttggctcgtgccgtgggtcgcgtgttggccgat
    cggggcgtcaccgggggtgctcgggtgcggctgaccatggcgaactgcgc
    cgatgggccgacgctggtgcagataaacctgcaggtaggtgacaccccat
    taagggcgcaggccgccaccgcgggcatcgatgatctgcgacccgcactg
    atcagactggatcgacagatcgtgcgggcgtcggcacagtggtgcccccg
    gccttggccggatcggccccgccggcgattgaccacgccggccgaggcgc
    tagtcacccgccgcaaaccggtcgtgctaaggcgcgcaaccccgttgcag
    gcgattgccgctatggacgccatggactacgacgtgcatttgttcaccga
    cgccgagacgggggaggacgctgtggtctatcgggctggaccgtcggggc
    tgcggctggcccgccagcaccacgtatttcccccaggatggtcacgttgt
    cgcgccccagccgggccgccggtgccgctgattgtgaattcgcgtccgac
    accggttctcacggaggccgccgcggtggaccgggcgcgcgaacatggac
    tgccattcctgtttttcaccgaccaggccaccggccgcggccagctgctc
    tactcccgctacgacggcaacctcgggttgatcaccccgaccggtgacgg
    cgttgccgacggtctggca
    Rv0080 47 atgagcccgggctcgcggcgcgccagcccgcaaagcgcccgggaggtggt
    cgagctcgaccgtgacgaggcgatgcggttgctggccagcgttgaccatg
    ggcgtgtggtgttcacccgcgcggcgctgccggcgatccgtccagtcaat
    cacctcgtggtcgacggtcgggtgatcgggcgcacccgcctgacggccaa
    ggtgtccgttgcggtgcgatcgagcgccgatgccggtgtcgtggtcgcct
    acgaagccgacgaccttgatccgcggcgtcggacggggtggagtgtggtg
    gtgacgggactggcgaccgaggtcagcgatcccgagcaggttgcccgcta
    ccagcggctgctacacccgtgggtgaacatggcgatggacaccgtggtcg
    cgatcgaacccgagatcgtcaccggcatccgcatcgttgctgactcgcgt
    acgccg
    Rv0081 48 gtggagtccgaaccgctgtacaagctcaaggcggagttcttcaaaaccct
    tgcgcatccggcgcggatcaggattttggagctgctggtcgagcgggacc
    gttcggtcggtgagttgctgtcctcggacgtcggcctggagtcgtcgaac
    ctgtcccagcagctgggtgtgctacgccgggcgggtgttgtcgcggcacg
    tcgtgacggcaacgcgatgatctattcgattgccgcacccgatatcgccg
    agctgctggcggtggcacgcaaggtgctggccagggtgctcagcgaccgg
    gtggcggtgctagaggacctccgcgccggcggctcggccacg
    Rv0363c 49 atgcctatcgcaacgcccgaggtctacgcggagatgctcggtcaggccaa
    acaaaactcgtacgctttcccggctatcaactgcacctcctcggaaaccg
    tcaacgccgcgatcaaaggtttcgccgacgccggcagtgacggaatcatc
    cagttctcgaccggtggcgcagaattcggctccggcctcggggtcaaaga
    catggtgaccggtgcggtcgccttggcggagttcacccacgttatcgcgg
    ccaagtacccggtcaacgtggcgctgcacaccgaccactgccccaaggac
    aagttggacagctatgtccggcccttgctggcgatctcggcgcaacgcgt
    gagcaaaggtggcaatcctttgttccagtcgcacatgtgggacggctcgg
    cagtgccaatcgatgagaacctggccatcgcccaggagctgctcaaggcg
    gcggcggccgccaagatcattctggagatcgagatcggcgtcgtcggcgg
    cgaagaggacggcgtggcgaacgagatcaacgagaagctgtacaccagcc
    cggaggacttcgagaaaaccatcgaggcgctgggcgccggtgagcacggc
    aaatacctgctggccgcgacgttcggcaacgtgcatggcgtctacaagcc
    cggcaacgtcaagcttcgccccgacatccttgcgcaagggcaacaggtgg
    cggcggccaagctcggactgccggccgacgccaagccgttcgacttcgtg
    ttccacggcggctcgggttcgcttaagtcggagatcgaggaggcgctgcg
    ctacggcgtggtgaagatgaacgtcgacaccgacacccagtacgcgttca
    cccgcccgatcgccggtcacatgttcaccaactacgacggagtgctcaag
    gtcgatggcgaggtgggtgtcaagaaggtctacgacccgcgcagctacct
    caagaaggccgaagcttcgatgagccagcgggtcgttcaggcgtgcaatg
    acctgcactgcgccggaaagtccctaacccac
    Rv0572c 50 atgggtgagcacgccatcaagcggcacatgcggcaacggaagcctacgaa
    gcatcccctagcccagaaacggggcgcgcggattctggtcttcaccgacg
    atccccgcaggagcgtcctcatagtgcccggttgccacctggattccatg
    cgccgagaaaagaacgcgtactacttccaggacggcaatgcgttggttgg
    gatggttgtctcgggcggcacggttgagtacgacgccgacgaccgcacat
    atgtcgtgcagctcaccgacggaaggcacaccactgagtcatctttcgaa
    cactcatcgccgagtcgatcacctcaatccgatgaccta
    Rv0574c 51 gtggctggcaatcctgatgtggtgacggtgctgctgggcggtgacgtcat
    gctcggccgtggcgtcgatcagatcctgcctcatcccggcaaaccgcaat
    tgcgcgaacggtatatgcgggatgcgaccggctatgttcgcctggccgag
    cgggtgaacgggcgcattccgctccccgtggattggcgctggccctgggg
    cgaggcgttggcggtccttgagaacaccgcgaccgacgtctgtttgatca
    atctggagacgacgatcaccgccgacggtgaattcgccgaccgcaaaccg
    gtctgctaccggatgcacccggataacgtgccggcgctgacggcattgcg
    gccgcacgtgtgcgcgctggccaacaaccacattctcgatttcggctacc
    aggggctgaccgatacggtcgcggctctcgccggtgcggggatccagagt
    gtcggggcgggagccgatttgctcgccgctcgccgctcggcgctagtcac
    ggttggccatgaacgccgggtgatcgtcggctcggtagcggcggaatcca
    gcggcgtccccgaatcctgggccgcccgccgcgaccggcccggagtgtgg
    ttgatccgggatccggcgcaacgcgacgtcgccgacgatgtggcggcaca
    ggtgctggcggacaaacgccccggcgatatcgccatagtctcgatgcatt
    ggggatccaattggggctatgcgaccgcacccggcgacgtcgcgttcgcg
    caccgactgatcgacgccggcatcgacatggtccacggacattcctcgca
    ccatccgcggccaatcgagatatatcgcggtaaaccgatcctgtacggat
    gcggtgacgtcgttgacgactacgaaggcatcggcgggcacgagtcgttc
    cgcagtgaactgcgactgctgtatctgaccgtcaccgatcccgccagcgg
    gaacctgatctcgctgcagatgcttccactgcgagtgtcgcggatgcgcc
    tacagcgtgcctcccagaccgacaccgaatggctccgcaacaccattgag
    cgcatcagccgccggttcgggattcgagtcgtgactcgacccgacaacct
    gctggaggtcgttcccgctgccaacctaacgagcaaggag
    Rv1264 52 gtgacagaccacgtgcgcgaggcggacgacgcgaacatcgacgatctgtt
    gggcgacctgggcggtaccgcgcgcgccgagcgtgcgaagcttgtcgagt
    ggttgctcgagcagggcatcacccccgacgagattcgggcgaccaacccg
    ccgttgctgctggccacccgccacctcgtcggcgacgacggcacctacgt
    atccgcaagggagattagcgagaactatggcgttgacctcgagctgctgc
    agcgggtgcagcgcgctgtcggtctggccagagtggatgatcctgacgcg
    gtggtgcacatgcgtgccgacggtgaggcggccgcacgcgcacagcggtt
    cgttgagctggggctgaatcccgaccaagtcgtgctggtcgtgcgtgtgc
    tcgccgagggcttgtcacacgccgccgaggccatgcgctacaccgcgctg
    gaggccattatgcggccgggggctaccgagttggacatcgcgaaggggtc
    gcaggcgctggtgagccagatcgtgccgctgctggggccgatgatccagg
    acatgctgttcatgcagctgcggcacatgatggagacggaggccgtcaac
    gccggagagcgtgcggccggcaagccgctaccgggagcgcgacaggtcac
    cgttgccttcgccgacctggtcggtttcacccagctaggcgaagtggtgt
    cggccgaagagctagggcacctcgccgggcggctggccggcctcgcgcgt
    gacctgaccgctccgccggtgtggttcattaagacgatcggcgacgcggt
    catgttggtctgtcctgatccggcgccattgctggacaccgtgctgaagc
    tggtcgaggtcgtcgacaccgacaacaactttccccggctgcgagccggc
    gtcgcctccgggatggcggttagccgggccggcgactggttcggcagccc
    ggtcaacgtggcaagccgggtgaccggggtggcgcgcccgggtgccgtgc
    tggtcgcggattcggtgcgggaggcccttggtgatgcccccgaagccgac
    ggatttcagtggtccttcgccggcccccgtcgcctcaggggaatccgggg
    tgacgtcaggctttttcgagtccggcgaggggccactcgcaccggctccg
    gcggcgcggcccaagacgacgatttggccggctcgtcaccg
    Rv1592c 53 atggtagagcccggcaatttggcaggcgcgaccggcgccgaatggatcgg
    ccggccaccgcacgaggaattgcagcgcaaagtgcgcccgctgctgccat
    ccgacgatccgttctacttcccacctgccggctaccagcatgccgtgccc
    ggaacggtgttgcgctcgcgcgatgtcgaactggcgtttatgggcttgat
    tccgcagcccgtcaccgctacccagctgctgtaccggaccacgaacatgt
    acggcaaccccgaggcgacggtgaccacggtgatcgtcccagcggagctt
    gccccgggtcagacctgccccttgctgtcgtaccagtgtgcgatcgatgc
    catgtcgtcgcgctgttttccgtcatatgccctgcgacgacgggccaagg
    ccctggggtcactgacccaaatggagctgttgatgatcagcgccgcactt
    gccgaaggatgggcggtatcagtacccgaccatgaagggccgaaagggct
    gtgggggtcgccgtatgaacccggttaccgagtcctcgacggaatccggg
    ctgccttgaattccgagcgtgtcgggttgtccccggcaacgccgatcggg
    ctgtggggctactccggcggcgggctggccagcgcgtgggccgccgaagc
    atgcggcgagtacgcaccggacctagacatcgtcggcgccgtgctgggat
    cacccgtcggtgaccttggtcacacgttccgccggctcaatggcactctt
    cttgccggtctgcccgcgttggtggtggccgcgctgcaacacagctaccc
    cggcctggcccgggtgatcaaggagcacgccaacgacgaaggacgtcagc
    tgctggagcaactgacggagatgacaacggtagacgcagtgatccggatg
    gccggcagggacatgggtgacttcctcgacgaaccccttgaggacattct
    gtcgacgccggaaatttcccatgtcttcggcgacaccaagctgggtagcg
    cggtgcccaccccgccggtattgatcgtgcaggccgtgcatgactacctc
    atcgacgtctctgacatcgacgcgctcgctgacagctatacagccggcgg
    cgccaacgtcacctaccaccgcgacctgttcagcgaacatgtgtccctgc
    acccgctgtcggccccaatgacgcttcgctggctcaccgaccggttcgcc
    ggcaagccactgaccgaccaccgcgtccggaccacgtggccgaccatctt
    caacccgatgacctacgccggcatggcgagactggccgtgatcgcggcca
    aggtgatcaccggcaggaagttgagccgccgtccgctc
    Rv1733c 54 atgatcgccacaacccgcgatcgtgaaggagccaccatgatcacgtttag
    gctgcgcttgccgtgccggacgatactgcgggtgttcagccgcaatccgc
    tggtgcgtgggacggatcgactcgaggcggtcgtcatgctgctggccgtc
    acggtctcgctgctgactatcccgttcgccgccgcggccggcaccgcagt
    ccaggattcccgcagccacgtctatgcccaccaggcccagacccgccatc
    ccgcaaccgcgaccgtgatcgatcacgagggggtgatcgacagcaacacg
    accgccacgtcagcgccgccgcgcacgaagatcaccgtgcctgcccgatg
    ggtcgtgaacggaatagaacgcagcggtgaggtcaacgcgaagccgggaa
    ccaaatccggtgaccgcgtcggcatttgggtcgacagtgccggtcagctg
    gtcgatgaaccagctccgccggcccgtgccattgcggatgcggccctggc
    cgccttgggactctggttgagcgtcgccgcggttgcgggcgccctgctgg
    cgctcactcgggcgattctgatccgcgttcgcaacgccagttggcaacac
    gacatcgacagcctgttctgcacgcagcgg
    Rv1734c 55 atgaccaacgtcggtgaccagggggttgacgcggtcttcggggtgatcta
    cccacctcaggtcgcgctggtcagtttcggcaagccggcacaacgagttt
    gcgccgtcgacggcgcgatccacgtcatgacgaccgtgctggctacgctg
    cccgctgaccacggctgcagcgatgaccatcgcggcgcgctgttcttcct
    gtcgatcaacgagctgacgcggtgcgccgcagtaacagga
    Rv1736c 56 gtgacggtgacaccacggaccggcagccgcatcgaggagctgcttgcacg
    cagcggccggttcttcatcccgggtgagatctcggcggatctgcgtaccg
    tgacccgccgcggcggccgcgacggcgacgtgttctatcgagaccggtgg
    agccacgacaaggtggtccgctccacacacggggtgaattgcaccgggtc
    gtgttcttggaagatctacgtcaaagacgacatcatcacctgggagacgc
    aggagaccgactatccgtcggtgggcccggaccggcccgagtatgagccc
    cgcggctgcccgcgcggcgcggcgttttcctggtacacgtattcgccgac
    gcgggtgcgccatccgtacgcccgcggcgtgcttgtcgagatgtatcggg
    aggcgaaggcacgtttgggtgatccggtggcggcctgggccgacatccag
    gccgacccgcggcggcgccgccgctaccagcgcgcccgcggcaagggcgg
    gctggtccgggtcagctgggccgaggccaccgagatgatcgccgccgccc
    acgtgcacaccatctccacatacggcccggaccgggttgccggcttctcc
    cccatcccggcgatgtccatggtgagccacgccgcggggtcgcggttcgt
    ggagctaatcggcggggtgatgacgtcgttctacgactggtacgccgacc
    tgccggtggcctccccgcaggtgttcggcgaccagaccgacgtgccggag
    tccggagattggtgggacgtggtgtggcaatgcgcctcggtgctgctgac
    ctacccgaactcacggcaactcggcaccgcagaggaattgctggcccaca
    tcgacggtccggccgcggatctgttggggcgcacggtctctgagctgcgc
    cgtgccgatccgctgaccgcggcgacccgctacgtcgacaccttcgacct
    gcgaggccgcgccaccctgtacctgacctactggaccgccggcgacaccc
    gcaaccgcggccgggagatgctggccttcgcccagacctaccgcagcacc
    gacgtcgcaccaccgcgcggcgagaccccggacttcctgccggtggtgct
    cgaattcgccgcgaccgtcgaccccgaggcggggcgacggttgctgagcg
    ggtaccgggtgcccatcgccgcgctgtgcaatgccctgaccgaggccgca
    ttgccatacgcacacacggtggccgcggtatgccggacgggtgacatgat
    gggcgaactcttctggaccgtcgtgccgtatgtgacgatgacgatcgtcg
    cggtcggctcctggtggcgctaccgctatgacaaattcggctggaccacc
    cgctcgtcccagctgtacgagtcgcggctgctgcggatcgccagcccgat
    gtttcatttcggcatcctggtggtcatcgtcggccacggtatcgggctcg
    tgatcccgcagtcgtggactcaggccgccggtttgagcgagggcgcatat
    cacgtgcaggccgtcgtgctggggtcgatcgccggcatcaccaccttggc
    cggcgttaccctgctgatctaccggcggcgcacccgcgggccggtgttca
    tggctaccaccgtcaacgacaaggtgatgtacctcgtgctggtggcggcg
    atcgtcgcgggactgggtgcgacggcgttgggctccggcgttgtcggcga
    ggcgtacaactaccgcgagacggtgtcggtgtggttccgctcggtgtggg
    tactgcaaccgcgcggggacctgatggccgaggctccgctgtattaccag
    atccatgtgctgatcgggttggcgttgttcgcgttgtggccgttcacccg
    gctggtacacgcgttcagcgccccgatcggctatctgttccgcccgtaca
    tcatctaccgcagccgcgaggagctggtgctaacgcggccgcggcggcgc
    gggtgg
    Rv1737c 57 atgagagggcaagcggccaatctcgtgctggccacctggatctcggtggt
    caacttctgggcgtggaacctgatcggcccgctgtcgaccagctacgcgc
    gtgacatgtcactgtccagcgccgaggcgtcgctgctcgtcgccaccccg
    atcctggtgggtgcccttggccgcatcgtcaccgggccgctcaccgaccg
    cttcggcgggcgcgccatgctcatcgcggtgacgctggcgtcgatcctcc
    cggtgctcgcggtcggggtcgcggcaaccatgggctcctacgcgttgctg
    gtgtttttcgggctcttcctgggcgttgccggcacgatcttcgccgtcgg
    catcccgttcgccaacaactggtaccagccggcgcggcgcggtttctcca
    ccggcgtgttcggtatgggcatggtcggcaccgcgctctcggcgttcttc
    accccgcggtttgtacggtggttcggcctgttcaccacccacgccatcgt
    cgcggccgcgctcgcgtcgaccgccgtggtggccatggtcgtgcttcgtg
    atgcaccctactttcggcccaacgccgacccggtgctgcccaggctcaag
    gccgcggcacggttgccggtgacctgggagatgtcgtttctgtacgcgat
    cgtgttcggcgggttcgtggcgttcagcaactacctgcccacctacatca
    ccacgatctacgggttctccacggtcgacgcgggcgctcgcaccgccggg
    ttcgccctggcggcggtgctggcccggccggtgggcgggtggctctccga
    ccggatcgcaccgaggcacgtggtgctggcctcgctcgccgggaccgcgc
    tgctggcgttcgccgcggcgttgcagccgccgccggaggtgtggtcggcg
    gccaccttcatcaccctggcggtctgcctcggcgtgggcaccggcggcgt
    gttcgcgtgggtggcccgccgcgccccggccgcatcggtcggctcggtca
    ccggaatcgtcgccgcggcaggcggattgggcggttacttcccgccgctg
    gtgatgggcgcgacctacgacccggtcgacaacgactacacggtcgggtt
    gctgctgctggtggcgaccgcgctggtcgcgtgtacctacaccgcgctgc
    acgcgcgggagccggtgagtgaggaggcgtccagg
    Rv1738 58 atgtgcggcgaccagtcggatcacgtgctgcagcactggaccgtcgacat
    atcgatcgacgaacacgaaggattgactcgggcgaaggcacggctgcgtt
    ggcgggaaaaggaattggtgggtgttggcctggcaaggctcaatccggcc
    gaccgcaacgtccccgagatcggcgatgaactctcggtcgcccgagcctt
    gtccgacttggggaagcgaatgttgaaggtgtcgacccacgacatcgaag
    ctgttacccatcagccggcgcgattgttgtat
    Rv1739c 59 atgattcccacgatgacatcggccggctgggcaccaggggtggtgcagtt
    ccgcgaataccaacggcgttggctgcgcggcgatgtcctcgccggcctga
    ccgtggccgcctatctgatcccgcaagcgatggcgtatgcgaccgtggcg
    ggcctaccgccggcagccgggctgtgggcgtcgatcgcgccgcttgccat
    ttacgcactgctcggatcgtcccggcagctttcaatcggcccggaatccg
    ccaccgccttgatgacggcggccgtgctcgctccgatggccgccggggat
    cttcgacgctatgccgttctggcggcaaccctcggattgctagtcggcct
    tatctgcctactcgctggcacggcgcgactaggtttcctcgccagcctgc
    gatcgcggccggtgctcgtcggatacatggccggcatcgcgcttgtcatg
    atctccagccaactcggcactatcaccggcacctcggtcgaaggcaacga
    attcttcagcgaagtacactctttcgcgactagcgtcacgcgagttcact
    ggccgacttttgtgttagccatgtctgtcctagcgctgctaactatgctc
    acgcggtgggcgccgcgcgcccccggaccgatcatcgcggttcttgcggc
    cacgatgctagtggccgttatgtccttggatgccaaaggtattgcgattg
    tgggtcggataccttccggtctgccgacgccgggtgtgccgcccgtttcg
    gtggaagacttgcgggcactgatcattccggctgccgggatcgcgattgt
    taccttcaccgacggtgtgttgaccgcacgcgccttcgccgctcgtcgag
    gtcaggaagtcaatgccaacgccgagctgcgcgcggtcggggcctgcaac
    atcgccgccgggctgacacacggttttccggtgagttccagcagcagccg
    taccgccctcgccgacgtcgtcggtggccgcacccagctgtactcgctga
    tcgcgttggggcttgttgtcatcgtgatggttttcgcgagtgggctgctg
    gccatgtttccgatcgccgctctgggcgctttggtggtatatgccgcgct
    acgcttgatcgacttgtcagaattccggcgactggcgcggtttcggcgca
    gcgaactcatgctggcactagccaccacagcagccgtgttaggcctagga
    gtgttctatggagtcctcgccgcggttgccctgtccatcctcgaactgct
    tcgtcgggtcgcacatccgcatgacagcgttctcgggttcgtgccgggca
    ttgccggcatgcacgacatcgatgactatccgcaggccaagcgcgtgccc
    gggctggtggtgtatcgctatgacgcgccgttgtgcttcgccaatgccga
    agacttccgcaggcgagcactgaccgtggtcgatcaggatccggggcaag
    tcgagtggttcgtactcaacgccgaatccaatgtggaggtcgacctgact
    gcgctggatgcgctcgaccaactccgcaccgagctgctgcgtcggggaat
    agtgttcgccatggcccgggtcaaacaagacttgcgtgaatcactcaggg
    ccgccagtcttctcgataagattggcgaagaccatatctttatgacattg
    cctaccgcagtgcaggcgttccgtcggcgc
    Rv1813c 60 atgatcacaaacctccgacgccgaaccgcgatggcagccgccggcctagg
    ggctgctctcgggctgggcatcctgctggttccgacggtggacgcccatc
    tcgccaacggttcgatgtcggaagtcatgatgtcggaaattgccgggttg
    cctatccctccgattatccattacggggcgattgcctatgcccccagcgg
    cgcgtcgggcaaagcgtggcaccagcgcacaccggcgcgagcagagcaag
    tcgcactagaaaagtgcggtgacaagacttgcaaagtggttagtcgcttc
    accaggtgcggcgcggtcgcctacaacggctcgaaataccaaggcggaac
    cggactcacgcgccgcgcggcagaagacgacgccgtgaaccgactcgaag
    gcgggcggatcgtcaactgggcgtgcaac
    Rv1997 61 ttgtcggcgtcagtgtctgccacgacggctcatcatggcttgccagcaca
    tgaagtggtgctgctgctggagagcgatccatatcacgggctgtccgacg
    gcgaggccgcccaacgactagaacgcttcgggcccaacaccttggcggtg
    gtaacgcgcgctagcttgctggcccgcatcctgcggcagtttcatcaccc
    gctgatctacgttctgctcgttgccgggacgatcaccgccggtcttaagg
    aattcgttgacgccgcagtgatcttcggtgtggtggtgatcaatgcgatc
    gtgggtttcattcaagaatccaaggcagaggccgcactgcagggcctgcg
    ctccatggtgcacacccacgccaaggtggtgcgcgagggtcacgagcaca
    caatgccatccgaagagctggttcccggtgaccttgtgctgttagcggcc
    ggtgacaaggttcccgccgatttgcggctggtgcgacagaccggattgag
    cgtgaacgagtcagcacttaccggcgagtcgacgccggttcacaaggacg
    aggtggcgttgccggagggcacaccggtcgctgatcgtcgcaatatcgcg
    tattccggcacattggtaaccgcgggccatggcgccgggatcgtcgtcgc
    gaccggcgccgaaaccgaactcggtgagattcatcggctcgttggggccg
    ccgaggttgtcgccacaccgctgaccgcgaagctggcgtggttcagcaag
    tttctgaccatcgccatcctgggtctggcagcgctcacgttcggcgtggg
    tttgctgcgccggcaagatgccgtcgaaacgttcaccgctgcgatcgcgc
    tggcggtcggggcaattcccgaaggtctgcccaccgccgtgaccatcacc
    ttggccatcggcatggcccggatggccaagcgccgcgcggtcattcgacg
    tctacccgcggtggaaacgctgggcagcaccacggtcatctgcgccgaca
    agaccggaacgctgaccgagaatcagatgacggtccagtcgatctggaca
    ccccacggtgagatccgggcgaccggaacgggctatgcacccgacgtcct
    cctgtgcgacaccgacgacgcgccggttccggtgaatgccaatgcggccc
    ttcgctggtcgctgctggccggtgcctgcagcaacgacgccgcactggtt
    cgcgacggcacacgctggcagatcgtcggcgatcccaccgagggcgcgat
    gctcgtcgtggccgccaaggccggcttcaacccggagcggctggcgacaa
    ctctgccgcaagtggcagccataccgttcagttccgagcggcaatacatg
    gccaccctgcatcgcgacgggacggatcatgtggtgctggccaagggtgc
    tgtggagcgcatgctcgacctgtgcggcaccgagatgggcgccgacggcg
    cattgcggccgctggaccgcgccaccgtgttgcgtgccaccgaaatgttg
    acttcccgggggttgcgggtgctggcaaccgggatgggtgccggcgccgg
    cactcccgacgacttcgacgaaaacgtgataccaggttcgctggcgctga
    ccggcctgcaagcgatgagcgatccaccacgagcggccgcggcatcggcg
    gtggcggcctgccacagtgccggcattgcggtaaaaatgattaccggtga
    ccacgcgggcaccgccacggcgatcgcaaccgaggtggggttgctcgaca
    acactgaaccggcggcaggctcggtcctgacgggtgccgagctggccgcg
    ctgagcgcagaccagtacccggaggccgtggatacagccagcgtgtttgc
    cagggtctctcccgagcagaagctgcggttggtgcaagcattgcaggcca
    gggggcacgtcgtcgcgatgaccggcgacggcgtcaacgacgccccggcc
    ttgcgtcaggccaacattggcgtcgcgatgggccgcggtggcaccgaggt
    cgccaaggatgccgccgacatggtgttgaccgacgacgacttcgccacca
    tcgaagccgcggtcgaggaaggccgcggcgtattcgacaatctgaccaag
    ttcatcacctggacgctgcccaccaacctcggtgagggcctagtgatctt
    ggccgccatcgctgttggcgtcgccttgccgattctgcccacccaaattc
    tgtggatcaacatgaccacagcgatcgcgctcggactcatgctcgcgttc
    gagcccaaggaggccggaatcatgacccggccaccgcgcgaccccgacca
    accgctgctgaccggctggcttgtcaggcggactcttctggtttccacct
    tgctcgtcgccagcgcgtggtggctgtttgcatgggagctcgacaatggc
    gcgggcctgcatgaggcgcgcacggcggcgctgaacctgttcgtcgtcgt
    cgaggcgttctatctgttcagctgccggtcgctgacccgatcggcctggc
    ggctcggcatgttcgccaaccgctggatcatcctcggcgtcagtgcgcag
    gccatcgcgcaattcgcgatcacatatctacccgcgatgaatatggtgtt
    cgacaccgcgccaatcgatatcggggtgtgggtgcgcatattcgctgtcg
    cgaccgcaatcacgattgtggtggccaccgacacgctgctgccgagaata
    cgggcgcaaccgcca
    Rv1998c 62 atgagtttccacgatcttcatcaccaaggtgttccgttcgtgttgcccaa
    cgcctgggatgtgccgtcggccctggcctacctcgcggagggcttcacgg
    ctatcggcacaaccagtttcggggtctcgtccagcggcgggcacccggac
    gggcaccgcgccactcgcggcgccaacatcgcactggcggccgccctggc
    accgctgcaatgctacgtcagcgtcgacatcgaggacggatacagcgacg
    aacccgacgccattgctgactacgtcgcacaactgtcgacagccggaatc
    aatatcgaggacagtagcgccgaaaagctcatcgaccccgccctggcagc
    cgctaaaatcgttgcgatcaaacaacgtaaccccgaggtgttcgtcaacg
    cccgcgtcgacacctattggttgcgccagcacgccgataccaccagcacg
    atccagcgcgcacttcgctacgtcgatgccggcgccgacggcgtctttgt
    cccactggccaacgatcccgacgaacttgctgagctcactcgcaacattc
    cgtgcccggttaacacgttgcccgtgcccggcttgacgatcgccgacctt
    ggtgagctcggggtggcccgggtgtcaaccggttcagtgccctacagcgc
    ggggttgtatgcagcggcccacgcggctcgggccgtgagcgacggagagc
    agctgccacggtccgtaccgtacgccgaactgcaggcacgcttggttgac
    tacgagaaccgcacgagtacaacg
    Rv2003c 63 gtggtcaagcgctctcgggcaacccgactttcgccgagcatctggtccgg
    atgggaatcacctcagtgtcggtccattcgggcgcgattgctgctacccc
    ggggtcggtcgcggccgccgaacgccgattgttgctggaatcagctcgcg
    gtgacgcctgacacccggatgccggcatcgtcggccgccgggcgcgacgc
    ggcggcctacgacgcctggtatgactcacccaccgggcggccgatcctgg
    cgaccgaggtcgccgcgttgcggccgctcatcgaggtctttgcccagcca
    cgcttggaaatcggtgtcggtacaggacgtttcgccgacctgctcggcgt
    gcggttcggactcgatccatcccgtgatgcgctgatgttcgcacgccggc
    gcggcgtcctggtcgccaatgccgtcggcgaggcggtccctttcgtcagc
    cggcacttcggggcggtcctcatggcattcacgctctgtttcgtcaccga
    cccggccgccatattccgggaaacgcggcgtctgctcgccgacggcggcg
    gccttgttatcgggttcttgcctcgcgggacaccgtgggccgacctgtac
    gctctgcgcgcggcccgcggacagccaggctaccgcgacgcccgcttcta
    caccgcggccgaactcgaacaactgctcgcagactcgggattccgggtca
    tcgcccgccgctgcacgctgcaccaaccgccgggactcgcccggtacgac
    atcgaagccgcccatgacggtatccaagccggcgccggcttcgttgctat
    ctcggcggtcgaccaagcgcacgagcctaaggatgatcacccactcgagt
    cggaa
    Rv2005c 64 atgtctaaaccccgcaagcagcacggagttgtcgtcggggtagatggttc
    gctcgaatcggatgccgccgcctgttggggtgccaccgatgcggcgatga
    ggaacattccgctgaccgtggtccacgtggtgaacgccgatgtagcgacg
    tggccgccgatgccgtatccggagacctggggggtttggcaggaggacga
    gggtcgccagatcgtcgccaacgccgtcaagctcgccaaagaggcggttg
    gagcggatcgaaagctcagcgtaaagagcgagctcgtattttccacgccg
    gtacctaccatggttgaaatctccaacgaggcagagatggtggtgttggg
    cagctcgggccggggagcgctggcccgaggcttgctcggttcggtcagct
    cgagcctggtgcgacgcgccgggtgcccggtcgcggtcatccacagcgat
    gatgcggtgatccctgatccgcagcacgctcccgtgctggtgggaatcga
    cggttcgccggtttcggagcttgcgacggcggtggcatttgacgaggcgt
    cgcgccgcggcgtcgaactgatcgccgtgcacgcgtggagtgacgtcgaa
    gtggtggaacttccgggtttggacttctcggctgtacagcaggaagcgga
    gcttagtctcgccgaacgcttggcaggttggcaagaacgctatcccgatg
    tgccggtgagccgggttgtcgtttgcgatcgcccggcgcggaagctggtg
    caaaagtcggcgtccgcccagcttgtcgtcgttggcagtcatggccgagg
    tggcttgaccggcatgcttctggggtcggtcagtaacgcggtcttacacg
    ccgcgcgggtgccagtgatcgtggcacggcagtcg
    Rv2007c 65 gtgacctatgtgatcggtagtgagtgcgtggatgtgatggacaagtcctg
    tgtgcaggagtgtccggtcgactgtatctatgagggcgcccgaatgctct
    acatcaaccccgacgagtgcgtggattgtggtgcgtgcaaaccggcctgc
    cgcgtcgaggcgatctactgggaaggcgatctacccgacgatcaacacca
    gcatctgggggacaacgccgcctttttccaccaagtcctgccgggccgag
    tggctccgctgggttcgccgggtggtgccgcagcggtgggcccgatcgga
    gtcgacacgcctctggtcgcggctatcccggtggagtgccct
    Rv2028c 66 atgaaccaatcacacaaacccccatcgatcgtcgtcggtattgatggctc
    gaagccggccgtgcaagccgcactgtgggcggtcgacgaggcagccagcc
    gtgacatcccgctgcgtctgctgtacgcgatcgaacccgacgatcccggg
    tacgccgcacacggcgcggcggctcgcaaactcgccgccgccgagaacgc
    ggtgcgctacgcgttcacagcggtcgaggcggcggaccggccggtcaagg
    tcgaggtggagatcacccaggagcggccggtcacctcgttgatccgcgct
    tcggcggctgctgccctggtgtgcgttggcgctatcggcgtgcaccactt
    ccgaccggagcgggtgggatctaccgcagcggccctggcgttatcggcgc
    agtgcccagtggcgatcgtgcgaccccaccgggtccccatcggacgcgac
    gccgcatggatcgtcgtcgaggcggacgggtcgtccgatatcggtgtttt
    gctgggggcggtgatggccgaagcacggctgcgcgactcgccggttcggg
    tggtcacctgccggcaatccggagtgggcgataccggggacgacgtccgt
    gccagcctggaccgctggcttgcccgttggcaaccacggtatcccgatgt
    gcgggtgcaatcggcggcagtgcacggcgagctgctggattatctggctg
    ggctgggtcgatcggtacacatggtggtgctcagcgcgagcgaccaggag
    catgtggagcaacttgtgggagcgccgggcaacgccgtgttgcaggaggc
    cggctgcaccctgctggtcgtcggtcagcagtatctg
    Rv2029c 67 atgacggagccagcggcgtgggacgaaggcaagccgcgaatcatcacttt
    gaccatgaaccccgccttggacatcacgacgagcgtcgacgtggtgcgcc
    cgaccgagaaaatgcgttgtggcgcacctcgctacgatcccggcggcggc
    ggtatcaatgtcgcccgcattgtgcatgtcctcggcggttgctcgacagc
    actgttcccggccggcgggtcgaccgggagcctgctgatggcgctgctcg
    gtgatgcgggagtgccatttcgcgtcattccgatcgcggcctcgacgcgg
    gagagcttcacggtcaacgagtccaggaccgccaagcagtatcgtttcgt
    gcttccggggccgtcgctgaccgtcgcggagcaggagcaatgcctcgacg
    aactgcgcggtgcggcggcttcggccgcctttgtggtggccagtggcagc
    ctgccgccaggtgtggctgccgactactatcagcgggttgccgacatctg
    ccgccgatcgagcactccgctgatcctggatacatctggtggcgggttgc
    agcacatttcgtccggggtgtttcttctcaaggcgagcgtgcgggaactg
    cgcgagtgcgtcggatccgaactgctgaccgagcccgaacaactggccgc
    cgcacacgaactcattgaccgtgggcgcgccgaggtcgtggtggtctcgc
    ttggatctcagggcgcgctattggccacacgacatgcgagccatcgattt
    tcgtcgattccgatgaccgcggttagcggtgtcggcgccggcgacgcgat
    ggtggccgcgattaccgtgggcctcagccgtggctggtcgctcatcaagt
    ccgttcgcttgggaaacgcggcaggtgcagccatgctgctgacgccaggc
    accgcggcctgcaatcgcgacgatgtggagaggttcttcgagctggcggc
    cgaacccaccgaagtcgggcaggatcaatacgtttggcacccgatcgtta
    acccggaagcctcggca
    Rv2030c 68 gtgctgatgaccgcagcggctgatgtcacccggcgctcgccgcggcgcgt
    gttccgtgaccgccgcgaggccggccgggtgctggcggaattactcgccg
    cctatcgggaccagccggacgtgattgtgctcggcttggcccggggtggc
    ctcccggtcgcatgggaggttgccgcggcactgcatgccccgctagacgc
    cttcgtcgtgcgcaaacttggtgccccggggcatgacgagttcgccgttg
    gtgcactggccagcggcggccgcgtcgtggtcaatgacgacgtcgtgcgg
    ggcctgcggatcacaccgcagcaactgcgcgacatcgccgaacgtgaggg
    tcgggaactgcttcggcgcgagtccgcctaccgcggcgagcgcccgccca
    ccgatatcaccggcaagacggtcattgtcgtcgatgacggtttggccacc
    ggcgcaagcatgttcgcggcggtacaggcattgcgcgatgcgcaaccagc
    gcagatcgtgattgccgtgccggcggcgccggagtccacgtgccgggagt
    tcgccggcctcgtcgacgacgttgtgtgcgcgaccatgccgaccccgttc
    ctggccgtcggtgagtcgttttgggacttccggcaggtcaccgacgagga
    ggtccgccggctcctggccaccccgaccgctgggccgtcgctgcgccggc
    ccgcggcgtcaacggcggccgatgttctgcgcagagtcgcgatcgacgcc
    cccgggggtgttccgacgcacgaggtgttggcggagctggtcggcgatgc
    acgaatcgtgttgatcggcgaaagctcgcacggcacacacgagttctacc
    aggcccgggccgccatgacacagtggctgatcgaggagaagggctttggt
    gcggtagccgccgaggcggactggcccgacgcctaccgggtcaatcggta
    cgttcgcggcctcggcgaggacaccaacgctgacgaggcgcttagcggat
    tcgagcggtttcccgcctggatgtggcgcaacaccgtggtccgagatttt
    gtggaatggctgcgcacacgcaaccagcgctacgagtcgggcgcgctgcg
    gcaagccggcttctacggtctggatctttacagcctgcatcggtcgatcc
    aagaggtgatcagctatctcgacaaggtcgacccgcgtgcggcggcacgg
    gcgcgggcccggtatgcgtgcttcgaccatgcctgcgccgatgacggtca
    ggcgtacggattcgcggccgcattcggcgccggtccgtcgtgcgaacgtg
    aagccgtcgagcaactggtcgacgttcagcgcaatgccctggcgtatgcg
    cgccaagacgggctgcttgccgaggacgaactgttctacgcccagcaaaa
    cgcgcagacggtgcgcgacgcagaggtgtattaccgggccatgttcagtg
    gacgcgttacctcgtggaacctgcgcgaccagcacatggcgcagaccctt
    ggcagtttgctgacgcatttggaccgacacctcgatgcgccgccggcgcg
    aatagtggtgtgggctcataactcccacgtgggtgacgcacgcgctaccg
    aggtgtgggccgacgggcagctcaccctcggccagatagtccgtgagcga
    tacggtgacgagtcgcgcagcatcggattcagcacgtacacgggcaccgt
    caccgcggccagcgaatggggtggtatcgcccaacgcaaagcggttcggc
    cggcactgcacggcagtgtcgaggagctcttccaccagactgcagacagt
    ttcctggtgtcagcgcggctaagccgcgacgccgaagccccgctggacgt
    tgtccggttgggacgtgccatcggcgtcgtttatctaccggcaacggaac
    ggcaaagtcactacttgcacgtgcggcccgccgaccagttcgacgccatg
    atccacatcgatcagacccgtgccctggaacctctcgaggtgacgagccg
    gtggatcgccggcgagaacccggaaacctacccgaccggtctg
    Rv2031c 69 Atggccaccacccttcccgttcagcgccacccgcggtccctcttccccga
    gttttctgagctgttcgcggccttcccgtcattcgccggactccggccca
    ccttcgacacccggttgatgcggctggaagacgagatgaaagaggggcgc
    tacgaggtacgcgcggagcttcccggggtcgaccccgacaaggacgtcga
    cattatggtccgcgatggtcagctgaccatcaaggccgagcgcaccgagc
    agaaggacttcgacggtcgctcggaattcgcgtacggttccttcgttcgc
    acggtgtcgctgccggtaggtgctgacgaggacgacattaaggccaccta
    cgacaagggcattcttactgtgtcggtggcggtttcggaagggaagccaa
    ccgaaaagcacattcagatccggtccaccaac
    Rv2032 70 atgccggacaccatggtgaccaccgatgtcatcaagagcgcggtgcagtt
    ggcctgccgcgcaccgtcgctccacaacagccagccctggcgctggatag
    ccgaggaccacacggttgcgctgttcctcgacaaggatcgggtgctttac
    gcgaccgaccactccggccgggaagcgctgctggggtgcggcgccgtact
    cgaccactttcgggtggcgatggcggccgcgggtaccaccgccaatgtgg
    aacggtttcccaaccccaacgatcctttgcatctggcgtcaattgacttc
    agcccggccgatttcgtcaccgagggccaccgtctaagggcggatgcgat
    cctactgcgccgtaccgaccggctgcctttcgccgagccgccggattggg
    acttggtggagtcgcagttgcgcacgaccgtcaccgccgacacggtgcgc
    atcgacgtcatcgccgacgatatgcgtcccgaactggcggcggcgtccaa
    actcaccgaatcgctgcggctctacgattcgtcgtatcatgccgaactct
    tttggtggacaggggcttttgagacttctgagggcataccgcacagttca
    ttggtatcggcggccgaaagtgaccgggtcaccttcggacgcgacttccc
    ggtcgtcgccaacaccgataggcgcccggagtttggccacgaccgctcta
    aggtcctggtgctctccacctacgacaacgaacgcgccagcctactgcgc
    tgcggcgagatgctttccgccgtattgcttgacgccaccatggctgggct
    tgccacctgcacgctgacccacatcaccgaactgcacgccagccgagacc
    tggtcgcagcgctgattgggcagcccgcaactccgcaagccttggttcgc
    gtcggtctggccccggagatggaagagccgccaccggcaacgcctcggcg
    accaatcgatgaagtgtttcacgttcgggctaaggatcaccgg
    Rv2428 71 atgccactgctaaccattggcgatcaattccccgcctaccagctcaccgc
    tctcatcggcggtgacctgtccaaggtcgacgccaagcagcccggcgact
    acttcaccactatcaccagtgacgaacacccaggcaagtggcgggtggtg
    ttcttttggccgaaagacttcacgttcgtgtgccctaccgagatcgcggc
    gttcagcaagctcaatgacgagttcgaggaccgcgacgcccagatcctgg
    gggtttcgattgacagcgaattcgcgcatttccagtggcgtgcacagcac
    aacgacctcaaaacgttacccttcccgatgctctccgacatcaagcgcga
    actcagccaagccgcaggtgtcctcaacgccgacggtgtggccgaccgcg
    tgacctttatcgtcgaccccaacaacgagatccagttcgtctcggccacc
    gccggttcggtgggacgcaacgtcgatgaggtactgcgagtgctcgacgc
    cctccagtccgacgagctgtgcgcatgcaactggcgcaagggcgacccga
    cgctagacgctggcgaactcctcaaggcttcggcc
    Rv2624c 72 atgtctgggagaggagagccgacgatgaaaacaatcattgttggtatcga
    tggttcgcacgcggcgattacggccgcattgtggggggttgacgaggcca
    tcagccgagcggtgccgctgcgactggtctcagtgatcaagccgacacat
    ccgtccccggacgactacgaccgcgaccttgcgcatgctgaaagatcgct
    tcgggaagcgcagtccgctgttgaggccgcgggcaagctcgtcaagatcg
    aaaccgacatcccccgcgggccagccggcccggtgcttgtggaggcatcg
    cgcgacgccgagatgatctgcgtcggctccgtgggaatcgggcgctacgc
    cagctcgatcttgggttcgacggcaaccgagctggccgaaaaggcgcatt
    gcccggtcgccgtcatgcgctcaaaagtggaccagccagcgtctgacatc
    aactggatcgtggtgcgcatgaccgacgcaccggataacgaggccgtgct
    ggaatacgctgcccgggaagcgaagttgcggcaagcgcccatactggcac
    tcggcgggcgaccggaggagctccgggagattccggacggcgaattcgaa
    cgtcgcgtgcaggattggcaccaccgtcatcccgatgtgcgcgtctaccc
    gatcaccactcacacgggtattgcccggttcctggccgaccacgacgagc
    gcgtacagctggcagtgatcggcggtggtgaggccggtcagctagcgcgg
    ctggtcgggccatccggacatccggtgttccgtcacgccgagtgttcggt
    gcttgtcgttcgccgc
    Rv2625c 73 atgcgtgatgcgatcccgcttgggcggatcgccgggtttgtggtgaacgt
    ccactggagcgtgttggtgatcctgtggttgttcacctggagtctggcga
    ccatgttgccgggtaccgtcggaggctacccggccgtggtctattggctt
    ctcggcgcaggtggcgcggtcatgttgctggcgtcgctgttggctcatga
    gctcgcgcacgccgtcgtcgctcgtcgcgccggggtatccgttgagagcg
    tgacgttgtggctgttcggcggggtgaccgcgcttggcggcgaggcaaag
    acgcccaaagccgctttccggatcgcgttcgcgggtccggctaccagcct
    ggcgctgtcggcgacattcggtgcgttggccatcacgctcgccggcgtgc
    ggaccccggccatcgtgatcagcgttgcttggtggttggctactgtcaac
    ctgctgctggggctgttcaatctgctgcctggcgcgccgttggacggtgg
    gcggttggtccgggcctatctgtggcgccgccacggcgatagtgtgcgcg
    ccgggatcggtgcggcgcgggccggacgggtggttgcgctggtcttgatc
    gcgttgggattggccgagtttgtggctggtggcctcgtcggtggggtctg
    gttagccttcattggctggtttatcttcgctgccgctcgcgaggaggaga
    cccggatttcgacccagcagctgtttgccggggtgcgtgtggccgatgcg
    atgaccgcccaaccgcatacggctcccggatggatcaatgtcgaggattt
    catccagcgttacgtgcttggtgaacggcactcggcatatccggttgccg
    atcgggacggatcgatcacgggcctggtggcattgcggcagctgcgcgat
    gttgcgcctagccggcgcagcactaccagcgtaggtgacattgcgctgcc
    gctgcacagcgtgccgaccgcccgaccacaagagccgctgaccgcgctcc
    tagagcggatggcaccgctcggcccgcgcagccgtgcgctggtcaccgaa
    gggagcgcggtggtcggcatcgtcactcccagcgatgtcgcgcggctgat
    tgacgtctaccggttggcccagccggaaccgacctttaccacgagtcccc
    aagatgcggacaggttttccgatgcgggg
    Rv2627c 74 atggcaagttctgcgagcgacggcacccacgaacgctcggcttttcgcct
    gagtccaccggtcttgagcggcgccatgggaccgttcatgcacaccggtc
    tgtacgtcgctcaatcgtggcgcgactatctgggtcaacagcccgataaa
    ctgccgatcgcacggcccactattgccttagcggcgcaagcctttcgaga
    cgaaatcgtcctgctgggcctcaaggcacgacgtccggtcagcaatcatc
    gagtgttcgagcgcatcagccaagaagtggccgctggactggagttctat
    gggaatcgcagatggctggagaagcctagcggattttttgcccagccccc
    accgctcaccgaggtcgcggtccgaaaggtcaaggaccgcagacgctcct
    tttatcgcatcttcttcgacagtgggtttacgccgcatccgggtgaaccg
    ggcagccaacggtggctctcatacactgcgaacaatcgcgagtacgccct
    gttactgcggcacccagagccgcgtccctggctggtttgtgtacacggca
    ccgagatgggcagggccccgttggatctcgcggtgttccgcgcctggaag
    ctgcatgacgaactcggcctgaacattgtcatgccggttcttccgatgca
    tggtccccgcgggcaaggtctgccgaagggcgccgtttttcccggagaag
    atgttctcgacgatgtgcatgggacggctcaagcggtgtgggatatccgg
    cggctgttgtcctggatacgatcgcaggaggaggagtcgctgatcgggtt
    gaacggtctctcgctgggcggctacatcgcgtcattggtcgccagcctcg
    aagaaggtctcgcctgcgcgattctcggtgtcccagtggctgatctgatc
    gagttgttgggccgccactgcggtcttcggcacaaagacccccgccgcca
    caccgtcaagatggccgaaccgatcggccgaatgatctcgccgctctcac
    ttacgccactggtgcccatgccgggccgctttatctacgcgggcattgcc
    gaccgactcgtgcatccacgcgaacaggtgactcgcctctgggagcactg
    gggcaaacccgaaatcgtgtggtatccaggcggtcacactggcttcttcc
    agtcgcggccggtacgacggtttgtccaggctgcgctggagcagtcgggc
    ctgttggacgcgccacggacacagcgcgaccgttccgcc
    Rv2628 75 Atgtccacgcaacgaccgaggcactccggtattcgggctgttggccccta
    cgcatgggccggccgatgtggtcggataggcaggtggggggtgcaccagg
    aggcgatgatgaatctagcgatatggcacccgcgcaaggtgcaatccgcc
    accatctatcaggtgaccgatcgctcgcacgacgggcgcacagcacgggt
    gcctggtgacgagatcactagcaccgtgtccggttggttgtcggagttgg
    gcacccaaagcccgttggccgatgagcttgcgcgtgcggtgcggatcggc
    gactggcccgctgcgtacgcaatcggtgagcacctgtccgttgagattgc
    cgttgcggtc
    Rv2629 76 atgcgatcagaacgtctccggtggctggtagccgcagaaggtccgttcgc
    ctcggtgtatttcgacgactcgcacgacactcttgatgccgtcgagcgcc
    gggaagcgacgtggcgcgatgtccggaagcatctcgaaagccgcgacgcg
    aagcaggagctcatcgacagcctcgaagaggcggtgcgggattctcgacc
    ggccgtcggccagcgtggccgcgcgctgatcgcgaccggcgagcaagtac
    tggtcaacgagcatctgatcggcccaccaccggctacggtgattcggctg
    tcggattatccgtacgtcgtgccattgatagaccttgagatgcggcgacc
    gacgtatgtatttgccgcggttgatcacaccggcgccgacgtcaagctgt
    atcagggggccaccatcagttccacgaaaatcgatggggtcggctacccg
    gtgcacaagccggtcaccgccggctggaacggctacggcgacttccagca
    caccaccgaagaagccatccgaatgaactgccgcgcggtcgccgaccatc
    tcacccgactggtagacgctgccgaccccgaggtggtgttcgtgtccggc
    gaggtgcggtcacgcacagacctgctttccacattgccgcagcgggtggc
    ggtccgggtgtcgcagctgcatgccggaccgcgcaaaagcgccttagacg
    aggaagagatctgggacctgacatccgcggagttcacccggcggcggtac
    gccgaaatcaccaatgtcgcacaacaatttgaggcggagatcggacgcgg
    atcggggctggcggcccaagggttggcggaggtgtgtgcggctctgcgtg
    acggcgacgtcgacacgctgatcgtcggagagctaggcgaggccaccgtg
    gtcaccggtaaagcgcgtactacggtcgcgcgggatgccgacatgttgtc
    cgaactcggcgaaccggtagatcgcgtggcaagggccgatgaggcgttgc
    cattcgccgcgatcgcggtaggtgccgcattggtccgtgacgacaaccgg
    atcgcgccactagatggggtgggcgcattgctgcgttatgccgccaccaa
    ccgactcggcagccatagatcc
    Rv2630 77 Atgctgcaccgcgacgatcacatcaatccgccgcggccccgcgggttgga
    tgttccttgcgcccgcctacgagcgacaaatcccctgcgcgccttggcgc
    gttgcgttcaggcgggcaagccgggcaccagttcagggcatcggtccgtg
    ccgcatacggcggacttgcgaatcgaagcctgggcaccgacccgtgacgg
    ctgtatccggcaggcggtgctgggtaccgtcgagagcttcctcgacctgg
    aatccgcgcacgcggtccatacccggctgcgccggctgaccgcggatcgc
    gacgacgatctactggtcgcggtgctcgaggaggtcatttatttgctgga
    caccgtcggtgaaacgcctgtcgatctcaggctgcgcgacgttgacgggg
    gtgtcgacgtcacattcgcaacgaccgatgcgagtacgctagttcaggtg
    ggtgccgtgccgaaggcggtgtcactcaacgaacttcggttctcgcaggg
    tcgccacggctggcgatgtgcggtaacgctcgatgtg
    Rv2659c 78 Gtgacgcaaaccggcaagcgtcagagacgcaaattcggtcgcatccgaca
    gttcaactccggccgctggcaagccagctacaccggccccgacggccgcg
    tgtacatcgcccccaaaaccttcaacgccaagatcgacgccgaagcatgg
    ctcaccgaccgccgccgcgaaatcgaccgacaactatggtccccggcatc
    gggtcaggaagaccgccccggagccccattcggtgagtacgccgaaggat
    ggctgaagcagcgtggaatcaaggaccgcacccgcgcccactatcgcaaa
    ctgctggacaaccacatcctggccaccttcgctgacaccgacctacgcga
    catcaccccggccgccgtgcgccgctggtacgccaccaccgccgtgggca
    caccgaccatgcgggcacactcctacagcttgctgcgcgcaatcatgcag
    accgccttggccgacgacctgatcgactccaacccctgccgcatctcagg
    cgcgtccaccgcccgccgcgtccacaagatcaggcccgccaccctcgacg
    agctggaaaccatcaccaaagccatgcccgacccctaccaggcgttcgtg
    ctgatggcggcatggctggccatgcgctacggcgagctgaccgaattacg
    ccgcaaagacatcgacctgcacggcgaggttgcgcgggtgcggcgggctg
    tcgttcgggtgggcgaaggcttcaaggtgacgacaccgaaaagcgatgcg
    ggagtgcgcgacataagtatcccgccacatctgatacccgccatcgaaga
    ccaccttcacaaacacgtcaaccccggccgggagtccctgctgttcccat
    cggtcaacgaccccaaccgtcacctagcaccctcggcgctgtaccgcatg
    ttctacaaggcccgaaaagccgccggccgaccagacttacgggtgcacga
    ccttcgacactccggcgccgtgttggctgcatccaccggcgccacactgg
    ccgaactgatgcagcggctaggacacagcacagccggcgccgcactccgc
    taccagcacgccgccaagggccgggaccgcgaaatcgccgcactgttaag
    caaactggccgagaaccaggagatg
    Rv2780 79 Atgcgcgtcggtattccgaccgagaccaaaaacaacgaattccgggtggc
    catcaccccggccggcgtcgcggaactaacccgtcgtggccatgaggtgc
    tcatccaggcaggtgccggagagggctcggctatcaccgacgcggatttc
    aaggcggcaggcgcgcaactggtcggcaccgccgaccaggtgtgggccga
    cgctgatttattgctcaaggtcaaagaaccgatagcggcggaatacggcc
    gcctgcgacacgggcagatcttgttcacgttcttgcatttggccgcgtca
    cgtgcttgcaccgatgcgttgttggattccggcaccacgtcaattgccta
    cgagaccgtccagaccgccgacggcgcactacccctgcttgccccgatga
    gcgaagtcgccggtcgactcgccgcccaggttggcgcttaccacctgatg
    cgaacccaagggggccgcggtgtgctgatgggcggggtgcccggcgtcga
    accggccgacgtcgtggtgatcggcgccggcaccgccggctacaacgcag
    cccgcatcgccaacggcatgggcgcgaccgttacggttctagacatcaac
    atcgacaaacttcggcaactcgacgccgagttctgcggccggatccacac
    tcgctactcatcggcctacgagctcgagggtgccgtcaaacgtgccgacc
    tggtgattggggccgtcctggtgccaggcgccaaggcacccaaattagtc
    tcgaattcacttgtcgcgcatatgaaaccaggtgcggtactggtggatat
    agccatcgaccagggcggctgtttcgaaggctcacgaccgaccacctacg
    accacccgacgttcgccgtgcacgacacgctgttttactgcgtggcgaac
    atgcccgcctcggtgccgaagacgtcgacctacgcgctgaccaacgcgac
    gatgccgtatgtgctcgagcttgccgaccatggctggcgggcggcgtgcc
    ggtcgaatccggcactagccaaaggtctttcgacgcacgaaggggcgtta
    ctgtccgaacgggtggccaccgacctgggggtgccgttcaccgagcccgc
    cagcgtgctggcc
    Rv3126c 80 Atggtcatccggtttgatcaaatagggtcattggtcctctcaatgaaatc
    ccttgcgtcactgtcgtttcagcggtgtctgcgcgagaattctagtttgg
    tcgcggcgctggaccggctcgatgctgcggtcgatgagctgagcgctttg
    tcgtttgatgcgttgaccactccggagcgggatcgcgcccgtcgcgaccg
    ggaccatcatccttggtcccgctcccgctcgcagttgtcgccacgaatgg
    cgcacggtgcagtgcaccaatgccagtggccgaaggcggtttgggctgtc
    attgacaatcca
    Rv3127 81 Gtgctcaagaacgcagtcttgctggcatgccgggcgccgtcggtgcacaa
    cagccagccctggcgttgggtggccgaaagcggctccgagcacactactg
    tgcacctgttcgtcaaccgccaccgaacggtgccggccaccgaccattcc
    ggccggcaagcgatcatcagttgcggtgccgtactcgatcaccttcgcat
    cgccatgacggccgcgcactggcaggcgaatatcactcgctttccccagc
    cgaaccaacctgaccagttggccaccgtcgaattcagtcccatcgatcac
    gtcacggcgggacagcgaaaccgcgcccaggcgattctgcagcgccgaac
    cgatcggcttccgtttgacagcccgatgtactggcacctgtttgagcccg
    cgctgcgcgacgccgtcgacaaagacgttgcgatgcttgatgtggtatcc
    gacgaccagcgaacacgactggtggtagcgtcacaactcagcgaagtcct
    gcggcgggacgatccgtactatcacgccgaactcgaatggtggacttcac
    cgttcgtgctggcccatggtgtgccgccggatacgctggcatcagacgcc
    gaacgcttgcgggttgacctgggccgtgacttcccggtccggagctacca
    gaatcgccgtgccgagctagctgatgaccgatcgaaagtccttgtgctgt
    cgacccctagcgacacgcgagccgacgcactgaggtgtggcgaagtgctg
    tcgaccatcctactcgagtgcaccatggccggcatggctacctgcacgtt
    gacccatctgatcgaatccagtgacagtcgtgacatcgtgcggggcctga
    cgaggcagcgaggcgagccgcaagccttgatccgggtagggatagccccg
    ccgttggcagcagttcccgcccccacaccacggcggccgctggacagcgt
    cttgcagattcgccagacgcccgagaaagggcgtaatgcctcagatagaa
    atgcccgtgaaacgggttggttcagcccgcct
    Rv3128c 82 gtgtggtccgcctcgggtgggcagtgcgggaagtatcttgccgcctcgat
    ggtgctgcagcttgatgggttggaacgtcacggtgtgttggagtttgggc
    gtgaccgctatggccccgaggtgcgtgaggagctgttggcgatgagtgcg
    gccagcatcgatcgttatctgaagaccgcgaaggccaaagaccagatatc
    gggtgtgtcgacgacgaaaccctcaccactgctgcgtaattcgatcaagg
    ttcgcagggccggcgatgaggtcgaggcggagccggggttcttcgagggc
    gacaccgtcgcccattgcggtccgacgctcaaaggcgagttcgcccacac
    cctgaacttgaccgacgtgcacatcggatgggtgttcacccgcaccgtcc
    gcaacaacgcccgtacccacatcctcgccgggctcaaagcttctgtcacc
    gagatcccgcatgggataacgggtttagatttcgacaacggcaccgtgtt
    tctcaacaagccggtcatcagctgggccggcgacaacggtatctacttca
    cccgctttcgcccgtacaagaaaaaccactaggccaccatcgagtccaag
    aacaaccacctggtccgcaagtacgcgttctactaccgctatgacaccgc
    cgaggaacgcgccgtgctcaaccggatgtggaagctggtcaacgaccgcc
    tcaactacctcaccccgaccatcaaaccgatcgggtatgccagcagcgcc
    gacggccgccgccgacgcctctacgatgccccacagacgccgctggaccg
    gccactggccgcaagggtgctctccgcggcccagcaggccgacctgatca
    cctaccgagacagcctcaaccccgcccagatcggccgcaaaatcgccgac
    ctgcagaaccgactcctcatcttggccaaggagaaaaccgagcagctcta
    cctcgctaacatccccaccgccctacccgacatccacaaaggcatcctga
    tcaaggcgggc
    Rv3129 83 Gtggtgcaaggccgcaccgtgctgtttcgtaccgcggagggcgccaaatt
    attttcagccgtcgcgaagtgcgcggtggctttcgaggcggacgaccaca
    acgttgccgagggctggagcgtgatcgtcaaggttcgcgcccaggtgctg
    acgaccgacgcgggggtccgcgaagccgaacgcgcccagttactaccgtg
    gaccgcgacgctgaaacgtcactgtgtgcgggtgatcccgtgggagatca
    ccggccgccacttcaggttcggtccggaaccggaccgcagccagaccttt
    gcctgcgaggcctcgtcacacaaccagcga
    Rv3130c 84 atgaatcacctaacgacacttgacgccgggtttctcaaggcagaagacgt
    ggatcggcacgtgagtctggcaatcggcgctctggcggtcatcgaggggc
    cggctcccgatcaggaagccttcttatcgtcgctcgctcaacgcctacgt
    ccctgtacccggttcgggcagcggttacgcctgcgcccgttcgacctcgg
    tgcacccaaatgggtggacgatcccgacttcgatcttggccgtcatgtgt
    ggcgcatcgccttgccgcggcctggcaacgaagaccagttattcgagctg
    atcgccgatctgatggcgcgtcgtttggaccggggtcgaccgctgtggga
    ggtctgggtcatcgaaggcctggcggacagcaagtgggcgatcctgacca
    aactgcaccactgcatggccgacggaatcgcggcgactcacctgctagct
    gggctctccgatgaaagtatgagcgacagcttcgcgagcaacatccacac
    gaccatgcagtcgcaatccgcatctgtgcggcggggtggattccgtgtca
    atccaagcgaggcgttgaccgcgtcgaccgccgtgatggcaggcatcgtt
    cgcgcggccaagggtgccagtgagatcgcggccggcgtgctaagtcccgc
    cgcgtcgtcgttgaacgggccgatcagtgatttgcgtcgctacagcgcag
    caaaggtccctctcgccgacgtcgaacaggtgtgccggaaattcgacgtc
    accatcaatgatgttgcgcttgccgcgattacggaaagctaccgcaacgt
    cctcatccagcggggtgagcggcctaggtttgattcgctgcgtacgctag
    tgccggtctcgacgcgttccaacagcgctttgagcaagaccgataaccgt
    gtttcgttaatgctgcccaacctgccggtggatcaagagaacccgctgca
    gcggctgcggatcgtgcactcgcggctgactcgggccaaggcggggggac
    agagacaattcggaaatactttgatggcgattgccaaccgccttccgttc
    cccatgaccgcatgggcggtcgggctgttgatgcggctgccgcagcgtgg
    tgttgtcaccgtggcgacaaatgtgccgggtccacgacggccgctgcaga
    ttatgggcagacgggtgcttgacctatacccggtttcgccgatcgcgatg
    caactgcgcaccagtgtcgcgatgctcagctacgccgacgacctgtactt
    cgggatcctggccgactacgacgtggtagcagatgccggccagctggcgc
    gaggaattgaagacgccgtcgcacggctggtggcgatcagtaagcggcgc
    aaggtgactcgcaggcgcggagcgctatcgctggttgtg
    Rv3131 85 atgaacacccatttcccggacgccgaaaccgtgcgaacggttctcaccct
    ggccgtccgggccccctccatccacaacacgcagccgtggcggtggcggg
    tatgcccgacgagtctggagctgttctctagacccgatatgcagctgcgt
    agcaccgatccggacgggcgtgagttgatcctcagctgtggtgtggcatt
    gcaccactgcgtcgtcgctttggcgtcgctgggctggcaggccaaggtaa
    accgtttccccgatcccaaggaccgctgccatctggccaccatcggggta
    caaccgcttgttcccgatcaggccgatgtcgccttggcggcggccatacc
    gcggcgacgcaccgatcggcgcgcctacagttgctggccggtgccaggag
    gtgacatcgcgttgatggccgcaagagcagcccgtggcggggtcatgctg
    cggcaggtcagtgccctagaccgaatgaaagccattgtggcgcaggctgt
    cttggaccacgtgaccgacgaggaatatctgcgcgagctcaccatttgga
    gtgggcgctacggttcagtggccggggttcccgcccgcaacgagccgcca
    tcagaccccagtgccccgatccccggtcgcctgttcgccgggcccggtct
    gtctcagccgtccgacgtcttacccgctgacgacggcgccgcgatcctgg
    cactaggcaccgagacagacgaccggttggcccggctgcgcgccggcgag
    gccgccagcatcgtcttgttgaccgcgacggcaatggggctggcgtgctg
    cccgatcaccgaaccgctggagatcgccaagacccgcgacgcggtccgtg
    ccgaggtgttcggcgccggcggctacccccagatgctgctgcgagtgggt
    tgggcaccgatcaatgccgacccgttgccaccgacgccacggcgcgaact
    gtcccaggtcgttgagtggccggaagagctactgcgacaacggtgc
    Rv3132c 86 atgacaacagggggcctcgtcgacgaaaacgacggcgccgcaatgcgtcc
    actgcgtcacacgctctcccaactacgcctgcacgagctgctggtcgagg
    tgcaggaccgggtcgagcagatcgtcgagggccgggaccgcctcgatggt
    ctggtggaggccatgctcgtggtcacagcgggcctggacctggaggcaac
    cctacgcgctatcgtgcattcagcgaccagccttgtcgatgcgcgctatg
    gcgctatggaggtgcacgaccggcagcatcgggtattgcactttgtctat
    gaaggcatcgacgaggagaccgttcggcggatcggccacctaccgaaagg
    cctaggcgtcatcgggctgctcatcgaagatcccaaaccgttacggctgg
    acgatgtttctgcgcacccggcctcgattggttttccgccgtatcatccg
    ccgatgcgtaccttcctcggggtaccggttcgggtgcgcgatgaatcgtt
    cggcactctgtacctgactgacaagaccaacgggcaaccgttcagcgacg
    acgacgaggttctggtccaggcgctggcggccgccgcgggtatcgcagtc
    gcgaatgcccggctctaccagcaggctaaggcgcgtcagtcgtggatcga
    ggccacccgtgacatcgccaccgagttgttgtccggcaccgaacccgcga
    cggtgttccggcttgtcgccgcggaggcgctcaagctgacggcggctgac
    gctgccctggtagccgttcccgtcgacgaggacatgcctgccgctgacgt
    gggggagctgctggtgattgaaacagtcggcagcgctgtggcttccattg
    ttgggcgaacgattccggtggcgggcgcggtgctgcgggaggtcttcgtc
    aacggcattccgcgacgggtcgaccgggtcgatttggaaggcctggacga
    actggccgacgcaggtccggcgctgctgttgccgctgcgggccagaggta
    ccgtagcgggtgtcgttgttgtgctgagtcaaggcggtccaggggctttc
    accgacgaacaactcgagatgatggccgcgttcgccgaccaggccgcgct
    ggcttggcaattggccacttcgcaacgtcggatgcgcgaactcgacgtac
    tgaccgaccgggatcgtatcgcccgtgacctccatgaccatgtcatccat
    cggctcttcgcgattggcctggctttgcagggtgctgtcccgcacgaacg
    taatcctgaagtgcagcaacgactctcggacgtggtagacgatctgcaag
    acgttatacaggaaatccggaccaccatttatgacctgcacggagcatcg
    cagggtatcactcggctccggcagcgaatcgatgcggccgtagcccaatt
    tgccgactcggggttgcgcaccagcgttcaattcgtgggtccattgtcgg
    tggtcgacagcgcgctcgccgatcaggccgaggcggtggttcgggaagcg
    gtcagcaacgcggttcgccatgcgaaggccagcacgttgaccgtccgggt
    caaagtcgacgacgacttgtgcatcgaggtgaccgacaacggccgcgggc
    tgcccgacgagttcaccggaagcggcttaacgaacctgcggcagcgggca
    gagcaggccggcggcgaattcaccctcgcgagcgtaccgggcgcgagcgg
    aacagtgctgcgatggtcagcaccgttgtcgcag
    Rv3134c 87 atgagcgatcctcggccagctcgggcagtggtcgttggtatcgacgggtc
    aagggcggcaacgcatgcggcgttgtgggcggtcgatgaggcggtgaacc
    gagacattccgctgcgactggtgtacgtcatcgatccgtcccaactgtcc
    gccgccggcgagggcggtgggcaatcagcggcccgagcggcgctgcacga
    cgcctctcggaaggtcgaggccaccgggcaaccggtcaagatcgaaacgg
    aggttctgtgcggcaggccgctcaccaagctgatgcaggagtccaggtcc
    gcggcgatgctgtgcgtcggttcggtggggcttgatcatgtccgcggtcg
    ccggggttcggtcgcggcgaccctggctgggtcggccttatgccccgtgg
    cggtgattcacccgtcgccggccgagccagcgacaacctcccaggtcagc
    gcggttgtcgcggaggtggacaatggtgtggtgctgcggcacgcattcga
    ggaggccaggctgcgcggagttccgctgcgggccgtggctgtccacgctg
    ctgaaacacccgatgacgtcgaacagggcagccggttggcgcatgtacac
    ctgagccgtcggctcgcccactggacccggctctaccccgaggtgcgggt
    ggatcgggccatcgccggcggcagtgcgtgccgtcatctggccgccaacg
    caaagccgggtcagctgttcgtcgcggactcacactccgcgcacgaattg
    tgcggtgcataccagcccggatgcgccgtacttacggtacgcagtgccaa
    cttg
    Rv3841 88 atgacagaatacgaagggcctaagacaaaattccacgcgttaatgcagga
    acagattcataacgaattcacagcggcacaacaatatgtcgcgatcgcgg
    tttatttcgacagcgaagacctgccgcagttggcgaagcatttttacagc
    caagcggtcgaggaacgaaaccatgcaatgatgctcgtgcaacacctgct
    cgaccgcgaccttcgtgtcgaaattcccggcgtagacacggtgcgaaacc
    agttcgacagaccccgcgaggcactggcgctggcgctcgatcaggaacgc
    acagtcaccgaccaggtcggtcggctgacagcggtggcccgcgacgaggg
    cgatttcctcggcgagcagttcatgcagtggttcttgcaggaacagatcg
    aagaggtggccttgatggcaaccctggtgcgggttgccgatcgggccggg
    gccaacctgttcgagctagagaacttcgtcgcacgtgaagtggatgtggc
    gccggccgcatcaggcgccccgcacgctgccgggggccgcctc
    Rv3842c 89 atgacatgggccgacgaggtgctcgccggacatccctttgtggttgctca
    ccgtggtgcgtcggcggctcggccggagcatacccttgccgcctacgacc
    tggcgctcaaagagggcgccgacggcgtggaatgtgatgtgcggttgacc
    cgggacgggcatctggtctgtgtgcatgaccgccgcctggaccgaacctc
    gacgggagccggcttggtcagcacgatgacgctggcccagctacgcgagc
    tggagtacggcgcgtggcacgacagctggcgccccgacggttcgcacggc
    gacaccagtctgctgaccctggacgcgcttgtttcgctggttttggactg
    gcaccggccggtgaagatcttcgtcgagaccaagcatcccgtccgatacg
    gctcgctggtggaaaacaagctgctggcgctgctacaccggttcggtatt
    gccgcacccgcctccgcagatcgatcccgtgcggtggtgatgtcgttttc
    ggccgccgcggtctggcggatccggcgggctgcaccgctgctgccgacgg
    tgttgctcggcaagaccccccgatacctgaccagcagtgcggccacggcg
    gtcggggcaaccgccgtgggaccctcactgcctgcgttaaaggaatatcc
    gcaactcgttgaccgctcggcagctcagggccgggcggtgtactgctgga
    acgtcgatgagtacgaggacatcgacttttgccgggaggtcggggtggcc
    tggattggtactcaccaccccggccgcaccaaggcctggctggaagacgg
    gcgggcgaacgggaccactcgc
    Rv3908 90 gtgtccgacggcgaacaagccaaatcacgtcgacgccgggggcggcgccg
    cgggcggcgcgctgcggctacagccgagaatcacatggacgcccaaccgg
    ccggcgacgccaccccgaccccggcaacggcgaagcggtcccggtcccgc
    tcacctcgtcgcgggtcgactcggatgcgcaccgtgcacgaaacatcggc
    tggagggttggtcattgacggtatcgacggtccacgagacgcgcaggtcg
    cggctctgatcggccgcgtcgaccggcgcggccggctgctgtggtcgcta
    cccaaggggcacatcgagttgggcgagaccgccgagcagaccgccatccg
    cgaggtcgccgaggagaccggcatccgcggcagtgtgctcgccgcgctgg
    ggcgcatcgactactggttcgtcaccgacggccggcgggtgcacaagacc
    gtccaccattatttgatgcggtttttaggcggagagctgtccgacgaaga
    cctcgaggtagccgaggtagcctgggtgccgatccgggaactgccgtctc
    gactggcctacgccgacgaacgtcgactagccgaggtggccgacgaactg
    atcgacaagctgcagagcgacggccccgccgcgcttccgccgctaccacc
    cagctcgcctcgtcgacggccgcaaacgcattcacgcgctcgtcatgccg
    atgactcagcaccgggtcagcacaacggtcccgggccggggccg
  • Preferably the immunogenic portions are selected from the group consisting of the sequences presented in Table 1 and the nucleic acid sequences are selected from the sequences presented in Table 2.
  • In another embodiment, the vaccine is a multiphase vaccine, where the polypeptides or fragments hereof are fused to other antigens with efficacy as prophylactic vaccines, where the fusion partner is selected from e.g. the group consisting of ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32.
  • The invention further discloses a therapeutic vaccine against tuberculosis comprising one or more polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, or nucleic acids encoding these polypeptides.
  • Preferably, the therapeutic and multiphase vaccine comprises an additional delivery system selected from among, live recombinant vaccines, that is gene-modified organisms such as bacteria or viruses expressing mycobacteria genes, or immunogenic delivery systems such as, DNA vaccines, that is plasmids expressing genes or gene fragments for the proteins described above, or protein vaccines, that is the proteins themselves or synthetic peptides derived from the proteins themselves delivered in a delivery system such as an adjuvant.
  • The invention further discloses a therapeutic vaccine in which the amino acid sequence is lipidated so as to allow a self-adjuvanting effect of the polypeptide.
  • The invention also discloses a method for treating an animal, including a human being, with tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the above-mentioned vaccine.
  • The invention also discloses a method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the above mentioned vaccine.
  • In a still further embodiment, the invention discloses an immunogenic composition comprising a polypeptide as defined above, preferably in the form of a vaccine or in the form of a diagnostic reagent. The diagnostic reagent can be in the form of a skin test reagent (administered by the transcutaneous, subcutaneous or intradermal routes), a serological reagent or a reagent for stimulating a cell-mediated reaction.
  • In another embodiment, the invention discloses a nucleic acid fragment in isolated form which
    • (a) comprises a nucleic acid sequence which encodes a polypeptide as defined above, or comprises a nucleic acid sequence complementary thereto; or
    • (b) has a length of at least 10 nucleotides and hybridizes readily under stringent hybridization conditions with a nucleotide sequence selected from the nucleotide sequences presented in Table 2 or a sequence complementary thereto, or with a nucleotide sequence selected from a sequence in (a)
  • The nucleic acid fragment is preferably a DNA fragment. The fragment can be used as a pharmaceutical.
  • In another embodiment, the invention discloses a vaccine comprising a nucleic acid fragment according to the invention, optionally inserted in a vector, the vaccine effecting in vivo expression of antigen by a human being or other mammal or animal, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in an animal, including a human being.
  • In a further embodiment, the invention discloses the use of a nucleic acid fragment according to the invention for the preparation of a composition for the diagnosis of tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, and the use of a nucleic acid fragment according to the invention for the preparation of a pharmaceutical composition for the vaccination against tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis.
  • In a still further embodiment, the invention discloses a vaccine for immunizing an human being or other mammal or animal, against tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising as the effective component a non-pathogenic microorganism, wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a polypeptide as defined above has been incorporated into the microorganism (e.g., placed on a plasmid or in the genome) in a manner allowing the microorganism to express and optionally secrete the polypeptide.
  • In another embodiment, the invention discloses a replicable expression vector, which comprises a nucleic acid fragment according to the invention, and a transformed cell harboring at least one such vector.
  • In another embodiment, the invention discloses a method for producing a polypeptide as defined above, comprising
    • (a) inserting a nucleic acid fragment according to the invention into a vector that is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell, culturing the host cell in a culture medium under conditions sufficient to effect expression of the polypeptide, and recovering the polypeptide from the host cell or culture medium;
    • (b) isolating the polypeptide from a whole mycobacterium, e.g. Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, from culture filtrate or from lysates or fractions thereof; or
    • (c) synthesizing the polypeptide e.g. by solid or liquid phase peptide synthesis.
  • The invention also discloses a method of diagnosing tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide as defined above or an immunogenic composition as defined above, a positive skin response at the location of injection being indicative of the animal having tuberculosis, and a negative skin response at the location of injection being indicative of the animal not having tuberculosis.
  • In another embodiment, the invention discloses a method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the polypeptide as defined above, the immunogenic composition according to the invention, or the vaccine according to the invention.
  • Another embodiment of the invention discloses a monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide as defined above in an immuno assay, or a specific binding fragment of said antibody. Preferably, said antibody is for use as a diagnostic reagent, e.g. for detection of mycobacteria antigens in sputum, urine or other body fluids of an infected animal, including a human being.
  • In a further embodiment the invention discloses a pharmaceutical composition that comprises an immunologically responsive amount of at least one member selected from the group consisting of:
    • (a) a polypeptide selected from the sequences presented in Table 1, or an immunogenic portion thereof;
    • (b) an amino acid sequence which has a sequence identity of at least 70% to any one of said polypeptides in (a) and is immunogenic;
    • (c) a fusion polypeptide comprising at least one polypeptide or amino acid sequence according to (a) or (b) and at least one fusion partner;
    • (d) a nucleic acid sequence which encodes a polypeptide or amino acid sequence according to (a), (b) or (c);
    • (e) a nucleic acid sequence which is complementary to a sequence according to (d);
    • (f) a nucleic acid sequence which has a length of at least 10 nucleotides and which hybridizes under stringent conditions with a nucleic acid sequence according to (d) or (e); and
    • (g) a non-pathogenic micro-organism which has incorporated (e.g. placed on a plasmid or in the genome) therein a nucleic acid sequence according to (d), (e) or (f) in a manner to permit expression of a polypeptide encoded thereby.
  • In a still further embodiment the invention discloses a method for stimulating an immunogenic response in an animal which comprises administering to said animal an immunologically stimulating amount of at least one member selected from the group consisting of:
    • (a) a polypeptide selected from the sequences presented in Table 1, or an immunogenic portion thereof;
    • (b) an amino acid sequence which has a sequence identity of at least 70% to any one of said polypeptides in (a) and is immunogenic;
    • (c) a fusion polypeptide comprising at least one polypeptide or amino acid sequence according to (a) or (b) and at least one fusion partner;
    • (d) a nucleic acid sequence which encodes a polypeptide or amino acid sequence according to (a), (b) or (c);
    • (e) a nucleic acid sequence that is complementary to a sequence according to (d);
    • (f) a nucleic acid sequence which has a length of at least 10 nucleotides and which hybridizes under stringent conditions with a nucleic acid sequence according to (d) or (e); and
    • (g) a non-pathogenic micro-organism which has incorporated therein (e.g. placed on a plasmid or in the genome) a nucleic acid sequence according to (d), (e) or (f) in a manner to permit expression of a polypeptide encoded thereby.
  • The vaccine, immunogenic composition and pharmaceutical composition according to the invention can be used therapeutically in a subject infected with a virulent mycobacterium combined with a prophylactic composition in a subject to prevent further infection with a virulent mycobacterium.
  • The invention also discloses a method for diagnosing previous or ongoing infection with a virulent mycobacterium, said method comprising
    • (a) contacting a sample, e.g. a blood sample, with a composition comprising an antibody according to the invention, a nucleic acid fragment according to the invention and/or a polypeptide as defined above, or
    • (b) contacting a sample, e.g. a blood sample comprising mononuclear cells (e.g. T-lymphocytes), with a composition comprising one or more polypeptides as defined above in order to detect a positive reaction, e.g. proliferation of the cells or release of cytokines such as IFN-γ.
  • Finally, the invention discloses a method of diagnosing Mycobacterium tuberculosis infection in a subject comprising:
    • (a) contacting a polypeptide as defined above with a bodily fluid of the subject;
    • (b) detecting binding of a antibody to said polypeptide, said binding being an indication that said subject is infected by Mycobacterium tuberculosis or is susceptible to Mycobacterium tuberculosis infection.
    DEFINITIONS Polypeptides
  • The word “polypeptide” in the present invention should have its usual meaning. That is an amino acid chain of any length, including a full-length protein, oligopeptides, short peptides and fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.
  • The polypeptide may be chemically modified by being glycosylated, by being lipidated (e.g. by chemical lipidation with palmitoyloxy succinimide as described by Mowat et al. 1991 or with dodecanoyl chloride as described by Lustig et al. 1976), by comprising prosthetic groups, or by containing additional amino acids such as e.g. a his-tag or a signal peptide.
  • Each polypeptide may thus be characterized by specific amino acids and be encoded by specific nucleic acid sequences. It will be understood that such sequences include analogues and variants produced by recombinant or synthetic methods wherein such polypeptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide and still be immunogenic in any of the biological assays described herein. Substitutions are preferably “conservative”. These are defined according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. The amino acids in the third column are indicated in one-letter code.
  •
    ALIPHATIC Non-polar GAP
    ILV
    Polar-uncharged CSTM
    NQ
    Polar-charged DE
    KR
    AROMATIC HFWY
  • A preferred polypeptide within the present invention is an immunogenic antigen from M. tuberculosis produced when the organism is subjected to the stresses associated with latent infection. Such antigen can for example also be derived from the M. tuberculosis cell and/or M. tuberculosis culture filtrate. Thus, a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M. tuberculosis antigen or be heterologous and such sequences may, but need not, be immunogenic.
  • Each polypeptide is encoded by a specific nucleic acid sequence. It will be understood that such sequences include analogues and variants hereof wherein such nucleic acid sequences have been modified by substitution, insertion, addition or deletion of one or more nucleic acids. Substitutions are preferably silent substitutions in the codon usage that will not lead to any change in the amino acid sequence, but may be introduced to enhance the expression of the protein.
  • In the present context the term “substantially pure polypeptide fragment” means a polypeptide preparation which contains at most 5% by weight of other polypeptide material with which it is natively associated (lower percentages of other polypeptide material are preferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most ½%). It is preferred that the substantially pure polypeptide is at least 96% pure, i.e. that the polypeptide constitutes at least 96% by weight of total polypeptide material present in the preparation, and higher percentages are preferred, such as at least 97%, at least 98%, at least 99%, at least 99.25%, at least 99.5%, and at least 99.75%. It is especially preferred that the polypeptide fragment is in “essentially pure form”, i.e. that the polypeptide fragment is essentially free of any other antigen with which it is natively associated, i.e. free of any other antigen from bacteria belonging to the tuberculosis complex or a virulent mycobacterium. This can be accomplished by preparing the polypeptide fragment by means of recombinant methods in a non-mycobacterial host cell as will be described in detail below, or by synthesizing the polypeptide fragment by the well-known methods of solid or liquid phase peptide synthesis, e.g. by the method described by Merrifield, 1963, or variations thereof.
  • By the term “virulent mycobacterium” is understood a bacterium capable of causing the tuberculosis disease in an animal or in a human being. Examples of virulent mycobacteria include but are not limited to M. tuberculosis, M. africanum, and M. bovis. Examples of relevant animals are cattle, possums, badgers and kangaroos.
  • By “a TB patient” is understood an individual with culture or microscopically proven infection with virulent mycobacteria, and/or an individual clinically diagnosed with TB and who is responsive to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB are well known by any person skilled in the art.
  • By the term “PPD-positive individual” is understood an individual with a positive Mantoux test or an individual where PPD induces a positive in vitro recall response determined by release of IFN-γ.
  • By “a latently infected individual” is understood an individual, who has been infected by a virulent mycobacterium, e.g. M. tuberculosis, but shows no sign of active tuberculosis. It is likely that individuals who have been vaccinated, e.g. by BCG, or treated for TB may still retain the mycobacteria within their bodies, although this is currently impossible to prove since such individuals would be expected to be positive if tested for PPD reactivity. Nonetheless, in its most accurate sense, “latently-infected” may be used to describe any individual who has M. tuberculosis residing in their tissues but who is not clinically ill.
  • By the term “delayed type hypersensitivity reaction” (DTH) is understood a T-cell mediated inflammatory response elicited after the injection of a polypeptide into, or application to, the skin, said inflammatory response appearing 72-96 hours after the polypeptide injection or application.
  • By the term “IFN-γ” is understood interferon-gamma. The measurement of IFN-γ is used as an indication of an immunological response.
  • By the terms “nucleic acid fragment” and “nucleic acid sequence” are understood any nucleic acid molecule including DNA, RNA, LNA (locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included are nucleic acid molecules comprising non-naturally occurring nucleosides. The term includes nucleic acid molecules of any length e.g. from 10 to 10000 nucleotides, depending on the use. When the nucleic acid molecule is for use as a pharmaceutical, e.g. in DNA therapy, or for use in a method for producing a polypeptide according to the invention, a molecule encoding at least one epitope is preferably used, having a length from about 18 to about 1000 nucleotides, the molecule being optionally inserted into a vector. When the nucleic acid molecule is used as a probe, as a primer or in antisense therapy, a molecule having a length of 10-100 is preferably used. According to the invention, other molecule lengths can be used, for instance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotide derivatives), or a molecule having at most 10000, 5000, 4000, 3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nucleotides (or nucleotide derivatives).
  • The term “stringent” when used in conjunction with hybridization conditions is as defined in the art, i.e. the hybridization is performed at a temperature not more than 15-20° C. under the melting point Tm, cf. Sambrook et al, 1989, pages 11.45-11.49. Preferably, the conditions are “highly stringent”, i.e. 5-10° C. under the melting point Tm.
  • Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations thereof such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
    • Sequence Identity
  • The term “sequence identity” indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. The two sequences to be compared must be aligned to best possible fit allowing the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as
  • ( N ref - N dif ) 100 N ref ,
  • wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC [SEQ ID NO: 184] will have a sequence identity of 75% with the sequence AATCAATC, SEQ ID NO: 185 (Ndif=2 and Nref=8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC [SEQ ID NO: 186] will have a sequence identity of 75% with the DNA sequence AGTCAGTC, SEQ ID NO: 187, (Ndif=2 and Nref=8). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson, 1988, or online through the NIH website). In one aspect of the invention, alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al. 1994 and as available through online sources.
  • A preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.
    • Immunogenic Portion
  • In a preferred embodiment of the invention, the polypeptide comprises an immunogenic portion of the polypeptide, such as an epitope for a B-cell or T-cell. The immunogenic portion of a polypeptide is a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays described herein. The immunogenic portion of a polypeptide may be a T-cell epitope or a B-cell epitope. Immunogenic portions can be related to one or a few relatively small parts of the polypeptide, they can be scattered throughout the polypeptide sequence or be situated in specific parts of the polypeptide. For a few polypeptides, epitopes have even been demonstrated to be scattered throughout the polypeptide covering the full sequence (Ravn et al 1999). In order to identify relevant T-cell epitopes which are recognized during an immune response, it is possible to use overlapping oligopeptides for the detection of MHC class II epitopes, preferably synthetic, having a length of e.g. 20 amino acid residues derived from the polypeptide. These peptides can be tested in biological assays (e.g. the IFN-γ assay as described herein) and some of these will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide. For the detection of MHC class I epitopes it is possible to predict peptides that will bind (Stryhn et al. 1996) and hereafter produce these peptides synthetic and test them in relevant biological assays, e.g. the IFN-γ assay as described herein. The peptides preferably having a length of, e.g., 8 to 11 amino acid residues derived from the polypeptide. B-cell epitopes can be determined by analyzing the B cell recognition to overlapping peptides covering the polypeptide of interest as, e.g., described in Harboe et al 1998.
  • Although the minimum length of a T-cell epitope has been shown to be at least 6 amino acids, it is normal that such epitopes are constituted of longer stretches of amino acids. Hence, it is preferred that the polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues. Hence, in important embodiments of the inventive method, it is preferred that the polypeptide fragment has a length of at most 50 amino acid residues, such as at most 40, 35, 30, 25, and 20 amino acid residues. It is expected that the peptides having a length of from 10 to 20 amino acid residues will prove to be most efficient as MHC class II epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 18, such as 15, 14, 13, 12 and even 11 amino acid residues. It is expected that the peptides having a length of from 7 to 12 amino acid residues will prove to be most efficient as MHC class I epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 11, such as 10, 9, 8 and even 7 amino acid residues.
  • Immunogenic portions of polypeptides may be recognized by a broad part (high frequency) or by a minor part (low frequency) of the genetically heterogeneous human population. In addition some immunogenic portions induce high immunological responses (dominant), whereas others induce lower, but still significant, responses (subdominant). High frequency or low frequency can be related to the immunogenic portion binding to widely distributed MHC molecules (HLA type) or even by multiple MHC molecules (Sinigaglia, 1988, Kilgus, 1991).
  • In the context of providing candidate molecules for a new vaccine against tuberculosis, the subdominant epitopes are however as relevant as are the dominant epitopes since it has been shown (Olsen, 2000) that such epitopes can induce protection regardless of the fact that they are not as strongly or broadly recognized.
    • Variants
  • A common feature of the polypeptides of the invention is their capability to induce an immunological response as illustrated in the examples. It is understood that a variant of a polypeptide of the invention produced by substitution, insertion, addition or deletion may also be immunogenic as determined by any of the assays described herein.
    • Immune Individual
  • An immune individual is defined as a person or an animal, which has cleared or controlled an infection with virulent mycobacteria or has received a vaccination with M. bovis BCG.
    • Immune Response
  • The immune response may be monitored by one of the following methods:
      • An in vitro cellular response is determined by induction of the release of a relevant cytokine such as IFN-γ from, or the induction of proliferation in lymphocytes withdrawn from an animal or human being currently or previously infected with virulent mycobacteria or immunized with the relevant polypeptide. The induction being performed by the addition of the polypeptide or the immunogenic portion of the polypeptide to a suspension comprising from 2×105 cells to 4×105 cells per well. The cells being isolated from either the blood, the spleen, the liver or the lung and the addition of the polypeptide or the immunogenic portion resulting in a concentration of not more than 20 μg per ml suspension and the stimulation being performed from two to five days. For monitoring cell proliferation the cells are pulsed with radioactive labeled Thymidine and after 16-22 hours of incubation detecting the proliferation by liquid scintillation counting. A positive response is defined as being a response more than background plus two standard deviations. The release of IFN-γ can be determined by the ELISA method, which is well known to a person skilled in the art. A positive response being a response more than background plus two standard deviations. Other cytokines than IFN-γ could be relevant when monitoring the immunological response to the polypeptide, such as IL-12, TNF-α, IL-4, IL-5, IL-10, IL-6, TGF-β. Another and more sensitive method for detecting the immune response is the ELISpot method, in which the frequency of IFN-γ producing cells is determined. In an ELIspot plate (MAHA, Millipore) precoated with anti-murine IFN-γ antibodies (PharMingen) graded numbers of cells isolated from either blood, spleen, or lung (typically between 1 to 4×105 cells/well) are incubated for 24-32 hrs in the presence of the polypeptide or the immunogenic portion resulting in a concentration of not more than 20 μg per ml. The plates are subsequently incubated with biotinylated anti-IFN-γ antibodies followed by a streptavidin-alkaline phosphatase incubation. The IFN-γ producing cells are identified by adding BCIP/NBT (Sigma), the relevant substrate giving rise to spots. These spots can be enumerated using a dissection microscope. It is also a possibility to determine the presence of mRNA coding for the relevant cytokine by the use of the PCR technique. Usually one or more cytokines will be measured utilizing for example PCR, ELISPOT or ELISA. It will be appreciated by a person skilled in the art that a significant increase or decrease in the amount of any of these cytokines induced by a specific polypeptide can be used in evaluation of the immunological activity of the polypeptide.
      • An in vitro cellular response may also be determined by the use of T cell lines derived from an immune individual or an M. tuberculosis-infected person where the T cell lines have been driven with either live mycobacteria, extracts from the bacterial cell or culture filtrate for 10 to 20 days with the addition of IL-2. The induction being performed by addition of not more than 20 μg polypeptide per ml suspension to the T cell lines containing from 1×105 cells to 3×105 cells per well and incubation being performed from two to six days. The induction of IFN-γ or release of another relevant cytokine is detected by ELISA. The stimulation of T cells can also be monitored by detecting cell proliferation using radioactively labeled Thymidine as described above. For both assays a positive response being a response more than background plus two standard deviations.
      • An in vivo cellular response may be determined as a positive DTH response after intradermal injection or local application patch of at most 100 μg of the polypeptide or the immunogenic portion to an individual who is clinically or subclinically infected with a virulent mycobacterium, a positive response having a diameter of at least 5 mm 72-96 hours after the injection or application.
      • An in vitro humoral response is determined by a specific antibody response in an immune or infected individual. The presence of antibodies may be determined by an ELISA technique or a Western blot where the polypeptide or the immunogenic portion is absorbed to either a nitrocellulose membrane or a polystyrene surface. The serum is preferably diluted in PBS from 1:10 to 1:100 and added to the absorbed polypeptide and the incubation being performed from 1 to 12 hours. By the use of labeled secondary antibodies the presence of specific antibodies can be determined by measuring the OD e.g. by ELISA where a positive response is a response of more than background plus two standard deviations or alternatively a visual response in a Western blot.
      • Another relevant parameter is measurement of the protection in animal models induced after vaccination with the polypeptide in an adjuvant or after DNA vaccination. Suitable animal models include primates, guinea pigs or mice, which are challenged with an infection of a virulent Mycobacterium. Readout for induced protection could be decrease of the bacterial load in target organs compared to non-vaccinated animals, prolonged survival times compared to non-vaccinated animals and diminished weight loss compared to non-vaccinated animals.
    Preparation Methods
  • In general, M. tuberculosis antigens, and DNA sequences encoding such antigens, may be prepared using any one of a variety of procedures.
  • They may be purified as native proteins from the M. tuberculosis cell or culture filtrate by procedures such as those described above. Immunogenic antigens may also be produced recombinantly using a DNA sequence encoding the antigen, which has been inserted into an expression vector and expressed in an appropriate host. Examples of host cells are E. coli. The polypeptides or immunogenic portion hereof can also be produced synthetically having fewer than about 100 amino acids, and generally fewer than 50 amino acids and may be generated using techniques well known to those ordinarily skilled in the art, such as commercially available solid-phase techniques where amino acids are sequentially added to a growing amino acid chain.
  • In the construction and preparation of plasmid DNA encoding the polypeptide as defined for DNA vaccination a host strain such as E. coli can be used. Plasmid DNA can then be prepared from cultures of the host strain carrying the plasmid of interest, and purified using e.g. the Qiagen Giga-Plasmid column kit (Qiagen, Santa Clarita, Calif., USA) including an endotoxin removal step. It is preferred that plasmid DNA used for DNA vaccination is endotoxin free.
    • Fusion Proteins
  • The immunogenic polypeptides may also be produced as fusion proteins, by which methods superior characteristics of the polypeptide of the invention can be achieved. For instance, fusion partners that facilitate export of the polypeptide when produced recombinantly, fusion partners that facilitate purification of the polypeptide, and fusion partners which enhance the immunogenicity of the polypeptide fragment of the invention are all interesting possibilities. Therefore, the invention also pertains to a fusion polypeptide comprising at least one polypeptide or immunogenic portion defined above and at least one fusion partner. The fusion partner can, in order to enhance immunogenicity, be another polypeptide derived from M. tuberculosis, such as of a polypeptide fragment derived from a bacterium belonging to the tuberculosis complex, such as ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32 and alpha-crystalline, or at least one T-cell epitope of any of the above mentioned antigens (Skjøt et al 2000; Danish Patent application PA 2000 00666; Danish Patent application PA 1999 01020; U.S. patent application Ser. No. 09/505,739; Rosenkrands et al 1998; Nagai et al 1991). The invention also pertains to a fusion polypeptide comprising mutual fusions of two or more of the polypeptides (or immunogenic portions thereof) of the invention.
  • Other fusion partners, which could enhance the immunogenicity of the product, are lymphokines such as IFN-γ, IL-2 and IL-12. In order to facilitate expression and/or purification, the fusion partner can e.g. be a bacterial fimbrial protein, e.g. the pilus components pilin and papA; protein A; the ZZ-peptide (ZZ-fusions are marketed by Pharmacia in Sweden); the maltose binding protein; glutathione S-transferase; β-galactosidase; or poly-histidine. Fusion proteins can be produced recombinantly in a host cell, which could be E. coli, and it is a possibility to induce a linker region between the different fusion partners.
  • Other interesting fusion partners are polypeptides, which are lipidated so that the immunogenic polypeptide is presented in a suitable manner to the immune system. This effect is e.g. known from vaccines based on the Borrelia burgdorferi OspA polypeptide as described in e.g. WO 96/40718 A or vaccines based on the Pseudomonas aeruginosa OprI lipoprotein (Cote-Sierra J 1998). Another possibility is N-terminal fusion of a known signal sequence and an N-terminal cystein to the immunogenic polypeptide. Such a fusion results in lipidation of the immunogenic polypeptide at the N-terminal cystein, when produced in a suitable production host.
    • Uses Protein Vaccine
  • Another part of the invention pertains to a vaccine composition comprising a polypeptide (or at least one immunogenic portion thereof) or fusion polypeptide according to the invention. In order to ensure optimum performance of such a vaccine composition it is preferred that it comprises an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant.
  • An effective vaccine, wherein a polypeptide of the invention is recognized by the animal, will in an animal model be able to decrease bacterial load in target organs, prolong survival times and/or diminish weight loss after challenge with a virulent Mycobacterium, compared to non-vaccinated animals
  • Suitable carriers are selected from the group consisting of a polymer to which the polypeptide(s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the polypeptide(s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin. Suitable vehicles are selected from the group consisting of a diluent and a suspending agent. The adjuvant is preferably selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), Quil A, poly I:C, aluminum hydroxide, Freund's incomplete adjuvant, IFN-γ, IL-2, IL-12, monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), Trehalose Dibehenate and muramyl dipeptide (MDP).
  • Preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231 and 4,599,230, all incorporated herein by reference.
  • Other methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), synthetic polymers of sugars (Carbopol), aggregation of the protein in the vaccine by heat treatment, aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Other possibilities involve the use of immune modulating substances such as cytokines or synthetic IFN-γ inducers such as poly I:C in combination with the above-mentioned adjuvants.
  • Another interesting possibility for achieving adjuvant effect is to employ the technique described in Gosselin et al., 1992 (which is hereby incorporated by reference herein). In brief, a relevant antigen such as an antigen of the present invention can be conjugated to an antibody (or antigen binding antibody fragment) against the Fcγ receptors on monocytes/macrophages.
  • The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 μg to 1000 μg, such as in the range from about 1 μg to 300 μg, and especially in the range from about 10 μg to 50 μg. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated.
  • The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and advantageously contain 10-95% of active ingredient, preferably 25-70%.
  • In many instances, it will be necessary to have multiple administrations of the vaccine. Especially, vaccines can be administered to prevent an infection with virulent mycobacteria, a prophylactic vaccine, and/or to treat established mycobacterial infection, a therapeutic vaccine. When administered to prevent an infection, the vaccine is given prophylactically, before definitive clinical signs, diagnosis or identification of an infection TB are present. Since the current vaccine BCG appears to induce an effective, but short-lived immune response, prophylactic vaccines may also be designed to be used as booster vaccines. Such booster vaccines are given to individuals who have previously received a vaccination, with the intention of prolonging the period of protection. In instances where the individual has already become infected or is suspected to have become infected, the previous vaccination may have provided sufficient immunity to prevent primary disease, but as discussed previously, boosting this immune response will not help against the latent infection. In such a situation, the vaccine will necessarily have to be a therapeutic vaccine designed for efficacy against the latent stage of infection. A combination of a prophylactic vaccine and a therapeutic vaccine, which is active against both primary and latent infection, constitutes a multiphase vaccine.
  • Due to genetic variation, different individuals may react with immune responses of varying strength to the same polypeptide. Therefore, the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response. The vaccine may comprise two or more polypeptides or immunogenic portions, where all of the polypeptides are as defined above, or some but not all of the peptides may be derived from virulent mycobacteria. In the latter example, the polypeptides not necessarily fulfilling the criteria set forth above for polypeptides may either act due to their own immunogenicity or merely act as adjuvants.
  • The vaccine may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides.
  • The invention also pertains to a method for immunizing an animal, including a human being, against TB caused by virulent mycobacteria, comprising administering to the animal the polypeptide of the invention, or a vaccine composition of the invention as described above, or a living vaccine described above.
  • The invention also pertains to a method for producing an immunologic composition according to the invention, the method comprising preparing, synthesizing or isolating a polypeptide according to the invention, and solubilizing or dispersing the polypeptide in a medium for a vaccine, and optionally adding other M. tuberculosis antigens and/or a carrier, vehicle and/or adjuvant substance.
    • DNA Vaccine.
  • The nucleic acid fragments of the invention may be used for effecting in vivo expression of antigens, i.e. the nucleic acid fragments may be used in so-called DNA vaccines as reviewed in Ulmer et al 1993, which is included by reference.
  • Hence, the invention also relates to a vaccine comprising a nucleic acid fragment according to the invention, the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to infections caused by virulent mycobacteria in an animal, including a human being.
  • The above mentioned definitions and distinctions of prophylactic-, booster-, therapeutic- and multiphase vaccines also applies for DNA vaccines
  • The efficacy of such a DNA vaccine can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a polypeptide that has the capability of modulating an immune response.
    • Live Recombinant Vaccines
  • One possibility for effectively activating a cellular immune response for a vaccine can be achieved by expressing the relevant antigen in a vaccine in a non-pathogenic microorganism or virus. Well-known examples of such microorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomona and examples of viruses are Vaccinia Virus and Adenovirus.
  • Therefore, another important aspect of the present invention is an improvement of the living BCG vaccine presently available, wherein one or more copies of a DNA sequence encoding one or more polypeptide as defined above has been incorporated into the genome of the micro-organism in a manner allowing the micro-organism to express and secrete the polypeptide. The incorporation of more than one copy of a nucleotide sequence of the invention is contemplated to enhance the immune response.
  • Another possibility is to integrate the DNA encoding the polypeptide according to the invention in an attenuated virus such as the vaccinia virus or Adenovirus (Rolph et al 1997). The recombinant vaccinia virus is able to replicate within the cytoplasma of the infected host cell and the polypeptide of interest can therefore induce an immune response, which is envisioned to induce protection against TB.
    • Therapeutic Vaccine.
  • The invention also relates to the use of a polypeptide or nucleic acid of the invention for use as therapeutic vaccines as have been described by D. Lowrie (Lowrie, 1999) using DNA vaccine encoding HSP65 from M. leprae. Antigens with therapeutic properties may be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals or prevent reactivation of previous infection, when administered as a vaccine. The composition used for therapeutic vaccines can be prepared as described above for vaccines.
    • Diagnostic Protein
  • The invention also relates to a method of diagnosing latent TB caused by a virulent mycobacterium in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide according to the invention, a positive skin response at the location of injection being indicative of the animal having TB, and a negative skin response at the location of injection being indicative of the animal not having TB.
  • When diagnosis of latent infection with virulent mycobacteria is the aim, a blood sample comprising mononuclear cells (i.e. T-lymphocytes) from a patient is contacted with a sample of one or more polypeptides of the invention. This contacting can be performed in vitro and a positive reaction could e.g. be proliferation of the T-cells or release of cytokines such as IFN-γ into the extracellular phase. It is also conceivable to contact a serum sample from a subject with a polypeptide of the invention, the demonstration of a binding between antibodies in the serum sample and the polypeptide being indicative of previous or ongoing infection.
  • The invention therefore also relates to an in vitro method for diagnosing latent infection in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and contacting the sample from the animal with the polypeptide of the invention, a significant release into the extracellular phase of at least one cytokine by mononuclear cells in the blood sample being indicative of the animal being sensitized. A positive response being a response more than release from a blood sample derived from a patient without the TB diagnosis plus two standard deviations. The invention also relates to the in vitro method for diagnosing ongoing or previous sensitization in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and by contacting the sample from the animal with the polypeptide of the invention demonstrating the presence of antibodies recognizing the polypeptide of the invention in the serum sample.
  • The immunogenic composition used for diagnosing may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides.
    • Diagnostic DNA
  • The nucleic acid probes encoding the polypeptide of the invention can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample.
  • A method of determining the presence of mycobacterial nucleic acids in an animal, including a human being, or in a sample, comprising administering a nucleic acid fragment of the invention to the animal or incubating the sample with the nucleic acid fragment of the invention or a nucleic acid fragment complementary thereto, and detecting the presence of hybridized nucleic acids resulting from the incubation (by using the hybridization assays which are well-known in the art), is also included in the invention. Such a method of diagnosing TB might involve the use of a composition comprising at least a part of a nucleotide sequence as defined above and detecting the presence of nucleotide sequences in a sample from the animal or human being to be tested which hybridize with the nucleic acid fragment (or a complementary fragment) by the use of PCR technique.
    • Antibodies
  • A monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide of the invention in an immunoassay, or a specific binding fragment of said antibody, is also a part of the invention. The antibodies can be produced by methods known to a person skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of a polypeptide according to the present invention and, if desired, an adjuvant. The monoclonal antibodies according to the present invention may, for example, be produced by the hybridoma method first described by Kohler and Milstein (Kohler and Milstein, 1975), or may be produced by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described by McCafferty et al (McCafferty, 1990), for example. Methods for producing antibodies are described in the literature, e.g. in U.S. Pat. No. 6,136,958.
  • A sample of a potentially infected organ or body fluid from an infected individual may be contacted with such an antibody recognizing a polypeptide of the invention. The demonstration of the reaction by means of methods well known in the art between the sample and the antibody will be indicative of an ongoing infection. It is of course also a possibility to demonstrate the presence of anti-mycobacterial antibodies in serum or other body fluids by contacting a serum sample from a subject with at least one of the polypeptide fragments of the invention and using well-known methods for visualizing the reaction between the antibody and antigen.
  • In diagnostics, an antibody, a nucleic acid fragment and/or a polypeptide of the invention can be used either alone, or as a constituent in a composition. Such compositions are known in the art, and comprise compositions in which the antibody, the nucleic acid fragment or the polypeptide of the invention is coupled, preferably covalently, to at least one other molecule, e.g. a label (e.g. radioactive or fluorescent) or a carrier molecule.
  • It will be understood that the following examples are illustrative of the present invention and are not a limitation thereof. A number of variations on the techniques, reagents, and conditions described in the following examples will be readily apparent to one of skill in the art.
    • EXAMPLES Example 1 Cloning and Expression of Low Oxygen Induced M. Tuberculosis Antigens in E. coli
  • A number of M. tuberculosis genes are induced under low oxygen conditions. The up-regulation of the genes listed in table 2 has been determined at either the mRNA (Sherman, 2001) or protein (Boon, 2001, Rosenkrands, 2002) level. The coding region of these selected antigens is amplified by PCR using the primer sets listed in Table 3.
  • TABLE 3
    Primer sequences for PCR amplification of selected
    low oxygen induced antigens
    SEQ
    Rv no. ID NO: Primer sequence
    Rv0079 Fwd 92 CACCGTGGAACCGAAACGCAGTCG
    Rvs 93 TTATGCCAGACCGTCGGCA
    Rv0080 Fwd 94 CACCATGAGCCCGGGCTCG
    Rvs 95 TTACGGCGTACGCGAGTCAG
    Rv0081 Fwd 96 CACCGTGGAGTCCGAACCGCTGTA
    Rvs 97 TTACGTGGCCGAGCCGC
    Rv0363c Fwd 98 CACCATGCCTATCGCAACGCCC
    (fba) Rvs 99 TTAGTGGGTTAGGGACTTTCCGG
    Rv0569 Fwd 100 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAAGGCAAAGGTCGGGGAC
    Rvs 101 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACGTTCCCCTGGCATGGA
    Rv0572c Fwd 102 CACCATGGGTGAGCACGCCATC
    Rvs 103 TTATAGGTCATCGGATTGAGGTGATC
    Rv0574c Fwd 104 CACCGTGGCTGGCAATCCTGATGT
    Rvs 105 TTACTCCTTGCTCGTTAGGTTGGC
    Rv1264 Fwd 106 CACCGTGACAGACCACGTGCGC
    Rvs 107 TTACGGTGACGAGCCGGC
    Rv1592c Fwd 108 CACCATGGTAGAGCCCGGCAATTTG
    Rvs 109 TTAGAGCGGACGGCGGCT
    Rv1733c Fwd 110 CACCATGATCGCCACAACCCGC
    Rvs 111 TTACCGCTGCGTGCAGAACA
    Rv1734c Fwd 112 CACCATGACCAACGTCGGTGACCA
    Rvs 113 TTATCCTGTTACTGCGGCGCA
    Rv1736c Fwd 114 CACCGTGACGGTGACACCACGGAC
    (narX) Rvs 115 TTACCACCCGCGCCGC
    Rv1737c Fwd 116 CACCATGAGAGGGCAAGCGGC
    (narK2) Rvs 117 TTACCTGGACGCCTCCTCACTC
    Rv1738 Fwd 118 CACCATGTGCGGCGACCAGTC
    Rvs 119 TTAATACAACAATCGCGCCGG
    Rv1739c Fwd 120 CACCATGATTCCCACGATGACATCG
    Rvs 121 TTAGCGCCGACGGAACG
    Rv1813c Fwd 122 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATCACAAACCTCCGACGC
    Rvs 123 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGTTGCACGCCCAGTTGA
    C
    Rv1997 Fwd 124 CACCTTGTCGGCGTCAGTGTCTGC
    (ctpF) Rvs 125 TTATGGCGGTTGCGCCC
    Rv1998c Fwd 126 CACCATGAGTTTCCACGATCTTCATCACC
    Rvs 127 TTACGTTGTACTCGTGCGGTTCTC
    Rv2003c Fwd 128 CACCGTGGTCAAGCGCTCTCGG
    Rvs 129 TTATTCCGACTCGAGTGGGTGA
    Rv2005c Fwd 130 CACCATGTCTAAACCCCGCAAGCA
    Rvs 131 TTACGACTGCCGTGCCACG
    Rv2007c Fwd 132 CACCGTGACCTATGTGATCGGTAGTGAGTG
    (fdxA) Rvs 133 TTAAGGGCACTCCACCGGGA
    Rv2028c Fwd 134 CACCATGAACCAATCACACAAACCCC
    Rvs 135 TTACAGATACTGCTGACCGACGACC
    Rv2029c Fwd 136 CACCATGACGGAGCCAGCGG
    (PfkB) Rvs 137 TTATGGCGAGGCTTCCGG
    Rv2030c Fwd 138 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACTGATGACCGCAGCGGCT
    Rvs 139 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACAGACCGGTCGGGTAGG
    TTT
    Rv2031c Fwd 140 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGCCACCACCCTTCCCGT
    (hspX) Rvs 141 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGTTGGTGGACCGGATCT
    GAAT
    Rv2032 Fwd 142 CACCATGCCGGACACCATGGTG
    Rvs 143 TTAGTGATCCTTAGCCCGAACGTG
    Rv2428 Fwd 144 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCCACTGCTAACCATTG
    GC
    (ahpC) Rvs 145 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGGCCGAAGCCTTGAGGA
    GT
    Rv2624c Fwd 146 CACCATGTCTGGGAGAGGAGAGCCG
    Rvs 147 TTAGCGAACGACAAGCACCGA
    Rv2625c Fwd 148 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACGTGATGCGATCCCGCT
    Rvs 149 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACCCCGCATCGGAAAACC
    Rv2627c Fwd 150 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCAAGTTCTGCGAGCG
    A
    Rvs 151 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGGAACGGTCGCGCTGTG
    T
    Rv2628 Fwd 152 CACCATGTCCACGCAACGACCG
    Rvs 153 TTAACCGCAACGGCAATCTCA
    Rv2629 Fwd 154 CACCATGCGATCAGAACGTCTCCG
    Rvs 155 TTAGGATCTATGGCTGCCGAGTC
    Rv2630 Fwd 156 CACCATGCTGCACCGCGACGA
    Rvs 157 TTACACATCGAGCGTTACCGCAC
    Rv2659c Fwd 158 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGTGACGCAAACCGGCAA
    Rvs 159 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACATCTCCTGGTTCTCGG
    CC
    Rv2780 Fwd 160 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACGCGTCGGTATTCCGACC
    Rvs 161 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACACGCTGGCGGGCTC
    Rv3126c Fwd 162 CACCATGGTCATCCGGTTTGATCAAATA
    Rvs 163 TTATGGATTGTCAATGACAGCCCA
    Rv3127 Fwd 164 CACCGTGCTCAAGAACGCAGTCTTGC
    Rvs 165 TTAAGGCGGGCTGAACCAACC
    Rv3128c Fwd 166 CACCGTGTGGTCCGCCTCGG
    Rvs 167 TTAGCCCGCCTTGATCAGGA
    Rv3129 Fwd 168 CACCGTGGTGCAAGGCCGCA
    Rvs 169 TTATCGCTGGTTGTGTGACGAG
    Rv3130c Fwd 170 CACCATGAATCACCTAACGACACTTGACG
    Rvs 171 TTACACAACCAGCGATAGCGCTC
    Rv3131 Fwd 172 CACCATGAACACCCATTTCCCGG
    Rvs 173 TTAGCACCGTTGTCGCAGTAGCT
    Rv3132c Fwd 174 CACCATGACAACAGGGGGCCTCG
    Rvs 175 TTACTGCGACAACGGTGCTGAC
    Rv3134c Fwd 176 CACCATGAGCGATCCTCGGCCA
    Rvs 177 TTACAAGTTGGCACTGCGTACCG
    Rv3841 Fwd 178 CCGGCTGAGATCTATGACAGAATACGAAGGGC
    (bfrB) Rvs 179 CCCCGCCAGGGAACTAGAGGCGGC
    Rv3842c Fwd 180 CACCATGACATGGGCCGACGAG
    (glpQ1) Rvs 181 TTAGCGAGTGGTCCCGTTCG
    Rv3908 Fwd 182 CACCGTGTCCGACGGCGAACAA
    Rvs 183 TTACGGCCCCGGCCC
  • PCR reactions were carried out using Platinum Tag DNA Polymerase (GIBCO BRL) in a 50 μl reaction volume containing 60 mM Tris-SO4 (pH 8.9), 18 mM Ammonium Sulfate, 0.2 mM of each of the four nucleotides, 0.2 μM of each primer and 10 ng of M. tuberculosis H37Rv chromosomal DNA. The reaction mixtures were initially heated to 95° C. for 5 min., followed by 35 cycles of: 95° C. for 45 sec, 60° C. for 45 sec and 72° C. for 2 min. The amplification products were precipitated by PEG/MgCl2, and dissolved in 50 μL TE buffer.
  • DNA fragments were cloned and expressed in Gateway Cloning system (Life Technology). First, to create Entry Clones, 5 μL of DNA fragment was mixed with 1 μL of pDONR201, 2 μL of BP CLONASE enzyme mix and 2 μL of BP reaction buffer. The recombination reactions were carried out at 25° C. for 60 min. After Proteinase K treatment at 37° C. for 10 min., 5 μL of each sample was used to transform E. coli DH5α competent cells. Transformants were selected on LB plates containing 50 μg/mL kanamycin. One bacterial clone from each transformation was grown in 3 mL LB medium containing 50 μg/mL kanamycin and plasmid DNA was isolated (Qiagen).
  • Second, to create expression clones, 2 μL of each entry clone DNA was mixed with μL of His-tagged expression vector (pDest17), 2 μL LR reaction buffer, 2 μL LR CLONASE enzyme mix and 3 μL TE. After recombination at 25° C. for 60 min. and Proteinase K treatment at 37° C. for 10 min., 5 μL of each sample was used to transform E. coli BL21-SI competent cells. Transformants were selected on LBON (LB without NaCl) plates containing 100 μg/mL ampicillin. The resulting E. coli clones express recombinant proteins carrying a 6-histine tag at the N-terminal. All clones were confirmed by DNA sequencing.
  • Recombinant proteins were purified from transformed E. coli BL21-SI cells cultured in 900 mL LBON medium containing 100 μg/mL at 30° C. until OD600=0.4-0.6. At this point 100 mL 3 M NaCl was added and 3 hours later bacteria were harvested by centrifugation. Bacteria pellets were resuspended in 20 mL bacterial protein extraction reagent (Pierce) incubated for 10 min. at room temperature and pelleted by centrifugation. Bacteria were lysed and their DNA digested by treating with lysozyme (0.1 mg/mL) and DNase I (2.5 μg/mL) at room temperature for 30 minutes, with gentle agitation. The recombinant protein forms inclusion bodies and can be pelleted by centrifugation at 27.000×g for 15 min. Protein pellets were solubilized by adding 20 ml of sonication buffer (8 M urea, 50 mM Na2HPO4, 100 mM Tris-HCl, pH 8.0) and sonicating 5×30 sec pulses interrupted by a 30 sec pause. After another centrifugation at 27.000×g for 15 min., supernatants were applied to 10 mL TALON columns (Clontech). The columns were washed with 50 mL sonication buffer. Bound proteins were eluted by lowering pH (8 M urea, 50 mM Na2HPO4, 100 mM Tris-HCl, pH 4.5). 5 mL fractions were collected and analyzed by SDS-PAGE. Fractions containing recombinant protein were pooled. Further purification was achieved by anion- or cation-exchange chromatography on Hitrap columns (Pharmacia). Bound protein was eluted using a NaCl gradient from 0-500 mM in 3 M urea, 10 mM Tris-HCl, pH 8.0. All fractions were collected and analyzed on SDS-PAGE using Coomassie staining. Fractions containing recombinant protein were pooled. Final protein concentrations were determined by micro BCA (Pierce).
    • Example 2 Prophylactic Versus Therapeutic Vaccine Murine Vaccination Models.
  • A prophylactic vaccine given prior to infection should induce an immune response sufficiently strong to prevent or dampen the initial proliferation of the bacteria in the acute phase and thereby reduce the ensuing disease. In the murine prophylactic vaccine model outlined in FIG. 1A, nai{umlaut over (v)}e mice are immunized 3 times, 2 weeks apart with recombinant antigens. Six weeks after the last immunization, the mice are given an aerosol infection with approximately 250 M. tuberculosis bacilli. The protective capacity of the vaccine is evaluated by enumeration of the bacteria in spleen and lung 6 weeks post-infection.
  • To define the optimal components for a therapeutic vaccine, a murine reactivation model of latent TB has been established (van Pinxteren, 2000) (FIG. 1B). An aerosol infection with approximately 250 M. tuberculosis bacilli is given and at the peak of infection 6 weeks later, the mice receive an 8-week course of anti-mycobacterial drug treatment of isoniazid and rifabutin given in the drinking water. This reduces the bacterial load in spleen and lung to a low level (about 500 bacteria per organ). This latent phase of low chronic infection is stable for 9-10 weeks after which a slow spontaneous reactivation occurs. The therapeutic vaccine is given as 3 subcutaneous (s.c.) immunizations about 5 weeks after cessation of drug treatment. The effect of the therapeutic vaccine is evaluated as protection against reactivation determined by enumeration of bacteria in spleen and lung 7 weeks after the last immunization.
  • The effect of the antigens in a prophylactic or a therapeutic vaccine.
  • BCG, ESAT6, and Rv2031c, one of the most prominent proteins induced under low oxygen conditions (Rosenkrands, 2002), were analyzed for their prophylactic and therapeutic vaccine potential. Naïve or latently infected C57Bl mice were immunized with one s.c. injection of 2.5×105 BCG, or 3 s.c. immunizations of 10 μg of either recombinant ESAT6 or recombinant Rv2031c in a DDA/MPL adjuvant. The vaccinations were done in groups of 5 mice and protective capacity of the vaccines was evaluated as described above. FIG. 2 shows the bacterial load in the lung in the acute phase (A) and in the reactivation phase (B), after prophylactic and therapeutic vaccination respectively. ESAT6 (as previously described by Brandt, 2000) offers protection against acute phase infection at the level of BCG (FIG. 2A). However, neither of the two shows any protective effect against reactivation of the infection when given during latent infection (FIG. 2B). In contrast, Rv2031c, the low oxygen induced antigen, offers no protection against the acute phase of the infection when given as a prophylactic vaccine, but gives some protection against reactivation when given as a therapeutic vaccine. That is, some antigens, here exemplified by ESAT6, though potent as prophylactic vaccines have no effect as therapeutic vaccines. In contrast, other antigens, here exemplified by Rv2031c, can be efficient therapeutic vaccines although they have no effect or only negligible effect as prophylactic vaccines.
    • Example 3 Low Oxygen Induced Antigens, Rv2031c, as Therapeutic Vaccines
  • There is a high variability in bacterial load intrinsic to the reactivation model in the latent and reactivation phase. The analysis of Rv2031c as a therapeutic vaccine was therefore repeated in groups of eight mice. As in the previous experiments the mice were given 3 s.c. immunizations of 10 μg rRv2031c in DDA/MPL. The induced immune responses were analyzed one week post immunization. The mice were partially bled and the PBMC from the blood purified and analyzed for Rv2031c- and ESAT6 specific recall responses. Using ELIspot technique, the frequency of Rv2031c-specific and ESAT6-specific IFN-γ-producing cells were determined in both the rRv2031c immunized and the unimmunized group (FIG. 3). The rRv2031c immunization has increased the frequency of Rv2031c-specific IFN-γ producing cells by a factor of 43 as compared to the unimmunized group. In contrast, the frequency of ESAT6-specific IFN-γ producing cells is significantly higher in the unimmunized group. ESAT6 is an antigen produced in high amounts by the actively-growing M. tuberculosis bacteria. The level of the ESAT6 specific immune response in infected mice could therefore be indicative the degree of actively-growing infection in the animals. Recent reports have in fact demonstrated such a correlation between the level of ESAT6 response and degree of disease in both M. tuberculosis-infected humans and M. bovis-infected cattle (Doherty, 2002, Vordermeier, 2002). Therefore, the higher ESAT6 response in the unimmunized group of latently-infected mice could be indicative of a transition into the reactivation phase, where the bacteria are again beginning to multiply.
  • To analyze the epitope recognition pattern of Rv2031c, fourteen overlapping peptides (each 20 amino acids long) covering the whole Rv2031c protein were synthesized. Initially the peptides were analyzed in 4 pools of 3-4 peptides. PBMCs from rRv2031c immunized latently-infected mice were incubated with the peptide pools (5 μg/ml per peptide) for 72 h. The specific peptide-induced IFN-γ production was quantitated in the supernatant in a standard sandwich ELISA using paired anti-murine IFN-γ antibodies (PharMingen) and recombinant IFN-γ (PharMingen) as standard. Both peptide pool 1-4 and 8-10 stimulated a significant IFN-γ response (FIG. 4A). The individual peptides of these two pools were re-analyzed (FIG. 4B). This clearly shows that the response to Rv2031c contains a dominant epitope, peptide 2 (PRSLFPEFSELFAAFPSFAG, aa 11-30 of SEQ ID NO:24), and a subdominant epitope, peptide 9 (RTEQKDFDGRSEFAYGSFVR, aa 81-100 of SEQ ID NO:24).
  • The therapeutic effect of the rRv2031c immunizations was analyzed 7 weeks after the last immunization. FIG. 5 shows the bacterial load in the lung (A) and the spleen (B) of both rRv2031c-immunized and unimmunized mice. There is a clear reduction in the level of bacteria in both organs in the rRv2031c-immunized group. That is, the induction of Rv2031c T cell responses can participate in keeping the latent infection in check.
    • Example 4 Low Oxygen Induced Antigens, Rv0569, as Therapeutic Vaccines
  • Rv0569 is also a low oxygen induced antigen described in WO0179274 and illustrates very well the potential as a therapeutic vaccine.
  • We have established a murine reactivation model of latent TB [van Pinxteren et al, 2000, 30:3689-98], which is a variant of the so-called Cornell model. An aerosol infection is allowed to be established and at the peak of infection 6 weeks after, the mice receive an 8-week course of anti-mycobacterial drug treatment of isoniazid and rifabutin given in the drinking water. This reduces the bacterial load in spleen and lung to a low level. This latent phase of low chronic infection is stable for 9-10 weeks after which a slow spontaneous reactivation can be detected. This model is used to analyze the protective effect of a post exposure vaccine on reactivation.
  • Rv0569, which is highly up regulated under low oxygen growth conditions [Rosenkrands et al, 2002, 184(13): 3485-91], was analyzed for its ability to protect against reactivation given as a therapeutic vaccine in the latent phase of TB infection. Latent infected C57Bl mice were vaccinated with 3 s.c. injections of 3 μg recombinant Rv0569 and for comparison with 3 s.c. injections of 3 μg recombinant ESAT6 or one s.c. injection of BCG. The effect of the vaccine is evaluated 7 weeks after the last immunization. The induced immune responses were analyzed for Rv0569 or ESAT6 specific responses in an in vitro recall assay. Isolated splenocytes were incubated with 1 μg/ml of Rv0569 or 1 μg/ml of ESAT6 for 72 h. The produced IFNγ in the culture supernatant was quantitated in a standard sandwich ELISA. FIG. 6 shows a nice Rv0569 specific IFNγ response induced in the Rv0569 vaccinated group with practically no response in the un-vaccinated group. The ESAT6 vaccination enhanced the ESAT6 specific response though a significant ESAT6 response persisted in the un-vaccinated latent infected group.
  • The Rv0569 induced protection against reactivation was determined by enumeration of bacteria in spleen and lung 7 weeks after the last immunization. FIG. 7 shows the bacterial load in the lung and the spleen of both Rv0569-vaccinated, ESAT6-vaccinated, BCG vaccinated and un-vaccinated latently infected mice. There is a clear reduction in the level of viable bacteria in both spleen and lungs of the Rv0569 vaccinated mice, whereas neither ESAT6 nor BCG are able to inhibit the growth of the M. tuberculosis bacteria when given as a vaccine during latent infection. That is, the induction of Rv0569 T cell responses can participate in keeping the latent infection in check.
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  • All documents listed above, and the sequence listing, are incorporated by reference herein. A variety of modifications and variations on the processes, conditions, reagents and compositions described herein will be readily apparent to one of skill in the art given the teachings of the present invention. Such modifications and variations are within the scope of the invention as set forth in the following claims.

Claims (20)

1. A method for inducing an immune response in an animal to tuberculosis, comprising delivering a composition comprising a mycobacteria polypeptide having amino acid sequence SEQ ID NO: 33 and a pharmaceutically acceptable polymeric carrier bound to the polypeptide or a pharmaceutically acceptable adjuvant.
2. The method according to claim 1, wherein said polypeptide is fused to at least one fusion partner which is an antigen expressed by bacteria within the mycobacteria family, wherein said at least one fusion partner is heterologous to said polypeptide.
3. The method according to claim 1, wherein said polypeptide is lipidated thereby providing a self-adjuvanting effect of the polypeptide.
4. The method according to claim 1, which comprises a polymeric carrier bound by covalent or non-covalent interactions to said polypeptide.
5. The method according to claim 4, wherein the polymeric carrier is a polystyrene.
6. The method according to claim 2, wherein the fusion partner is selected from the polypeptides of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45.
7. The method according to claim 6, wherein the polypeptides are recombinant or synthetic and are delivered in a delivery system comprising an adjuvant.
8. The method according to claim 2, wherein said at least one fusion partner is an antigen expressed by bacteria within the mycobacteria family other than a polypeptide selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45.
9. The method according to claim 8, wherein said at least one fusion partner is selected from the group consisting of ESAT-6, ESAT-6-Ag85B, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, and MPT32.
10. The method according to claim 8, wherein the polypeptides are recombinant or synthetic and are delivered in a delivery system comprising an adjuvant.
11. The method according to claim 1, wherein said animal is a mammal.
12. The method according to claim 11, wherein said mammal is a human.
13. A method for inducing an immune response in an animal to tuberculosis, comprising delivering one or more mycobacteria polypeptides which are upregulated or expressed during the latent stage of the mycobacteria infection which is characterized by low-oxygen tension in the microenvironment of the mycobacteria and (i) a pharmaceutically acceptable polymeric carrier bound to the one or more polypeptides or (ii) a pharmaceutically acceptable adjuvant, wherein said one or more polypeptides has the amino acid sequence of SEQ ID NO: 33,
wherein said one or more polypeptides is fused to at least one mycobacteria fusion partner.
14. The method according to claim 13, where the polypeptides are recombinant or synthetic and are delivered in a delivery system comprising an adjuvant.
15. The method according to claim 13, wherein the fusion partner is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45.
16. The method according to claim 15, wherein the fusion partner is an antigen expressed by bacteria within the mycobacteria family other than a polypeptide selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45.
17. The method according to claim 16, wherein the fusion partner is selected from the group consisting of ESAT-6, ESAT-6-Ag85B, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, and MPT32.
18. A method for inducing an immune response in an animal to latent tuberculosis, comprising delivering a composition comprising one or more mycobacteria polypeptides which are upregulated or expressed during the latent stage of the mycobacteria infection, wherein one polypeptide has the amino acid sequence of SEQ ID NO: 33.
19. The method according to claim 18, wherein the polypeptides are recombinant or synthetic and are delivered in a delivery system comprising an adjuvant.
20. The method according to claim 18, wherein the fusion partner is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45.
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