Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle.

The development of novel vaccine strategies supplementing Mycobacterium bovis BCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identified Mycobacterium tuberculosis antigens in M. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized in M. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.

Tracking antigen-specific CD8 T lymphocytes in the lungs of mice vaccinated with the Mtb72F polyprotein.

This study used a major histocompatibility complex class I tetramer reagent to track antigen-specific CD8 T cells in the lungs of mice immunized with the tuberculosis vaccine candidate Mtb72F. The results show that CD8 T cells recognizing an immunodominant Mtb32-specific epitope could be detected in significant numbers over the course of infection in mice exposed to low-dose aerosol challenge with Mycobacterium tuberculosis and that prior vaccination substantially increased the numbers of these cells early in the lungs. The effector phenotype of the cells was shown by the demonstration that many secreted gamma interferon, but very few contained granzyme B. As the course of the infection progressed, many activated CD8 T cells down-regulated expression of CD45RB and upregulated expression of the interleukin-7 receptor alpha chain, indicating a transition of these cells to a state of memory. These data support the hypothesis that M. tuberculosis-specific CD8 T cells can be targeted by vaccination with the Mtb72F polyprotein.

In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis.

The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell-mediated immune responses. In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette-Guérin (BCG)-vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen-induced proliferation and interferon (IFN)-gamma secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8.4, Mtb9.8, Mtb9.9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M. tuberculosis. The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen-induced proliferation and IFN-gamma secretion assays. However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9.8 and Mtb39A in proliferation assays (median SI = 6.2 and 6.4, respectively) and of Mtb9.8, Mtb39A, Mtb40 and Ag85B in IFN-gamma assays (median delta IFN-gamma= 15.5, 10.8, 7.8 and 8.1 U/ml, respectively). BCG-vaccinated healthy donors showed weak (<30% responders) to moderate (31-50% responders) responses to all of the recombinant antigens in both assays. When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines [IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12] and the immunosuppressive cytokine IL-10, the complex CF and CW antigens as well as the recombinant Mtb9.8, Mtb9.9, Mtb40 and Ag85B induced the secretion of both types of cytokines. On the other hand, Mtb41 induced only IL-10, while Mtb8.4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL-10. In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.

Prospects for a better vaccine against tuberculosis.

There have been many new promising approaches to developing human vaccines against tuberculosis (TB). Advances in gene and antigen identification, availability of genome sequences, a greater understanding of immune mechanisms in resistance to TB, the development of adjuvants and delivery systems to stimulate T-cell immunity, and increased funding from public and private agencies are some of the reasons for progress in this area. Dozens of vaccine candidates have been tested in animal models in recent years, and several of these are poised to move into clinical trials in the next several years. Thus, there is renewed optimism for the potential of developing new and improved TB vaccines.

Chlamydial antigens colocalize within IncA-laden fibers extending from the inclusion membrane into the host cytosol.

Chlamydial IncA localizes to the inclusion membrane and to vesicular fibers extending away from the inclusion. Chlamydial outer membrane components, in the absence of developmental forms, are found within these fibers. This colocalization may explain how chlamydial developmental form antigens are localized outside of the inclusion within infected cells.

Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice.

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge with L. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.

Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein.

The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.

Serological expression cloning and immunological evaluation of MTB48, a novel Mycobacterium tuberculosis antigen.

Improved diagnostics are needed for the detection of Mycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity with M. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing the esat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.

The potency and durability of DNA- and protein-based vaccines against Leishmania major evaluated using low-dose, intradermal challenge.

DNA- and protein- based vaccines against cutaneous leishmaniasis due to Leishmania major were evaluated using a challenge model that more closely reproduces the pathology and immunity associated with sand fly-transmitted infection. C57BL/6 mice were vaccinated s.c. with a mixture of plasmid DNAs encoding the Leishmania Ags LACK, LmSTI1, and TSA (AgDNA), or with autoclaved L. major promastigotes (ALM) plus rIL-12, and the mice were challenged by inoculation of 100 metacyclic promastigotes in the ear dermis. When challenged at 2 wk postvaccination, mice receiving AgDNA or ALM/rIL-12 were completely protected against the development of dermal lesions, and both groups had a 100-fold reduction in peak dermal parasite loads compared with controls. When challenged at 12 wk, mice vaccinated with ALM/rIL-12 maintained partial protection against dermal lesions and their parasite loads were no longer significantly reduced, whereas the mice vaccinated with AgDNA remained completely protected and had a 1000-fold reduction in dermal parasite loads. Mice vaccinated with AgDNA also harbored few, if any, parasites in the skin during the chronic phase, and their ability to transmit L. major to vector sand flies was completely abrogated. The durable protection in mice vaccinated with AgDNA was associated with the recruitment of both CD8(+) and CD4(+) T cells to the site of intradermal challenge and with IFN-gamma production by CD8(+) T cells in lymph nodes draining the challenge site. These data suggest that under conditions of natural challenge, DNA vaccination has the capacity to confer complete protection against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs.

Assessment of the serodiagnostic potential of nine novel proteins from Mycobacterium tuberculosis.

To identify antigens that would improve the accuracy of serological diagnosis of active tuberculosis, we cloned the genes encoding nine potentially immunogenic secreted or surface-associated proteins of Mycobacterium tuberculosis. Recombinant proteins were reacted with sera from HIV-negative individuals with extrapulmonary tuberculosis (EP-TB) or HIV-positive individuals with pulmonary tuberculosis (TBH). Specific and high level antibody responses were obtained for four recombinant proteins, of which antigen GST-822 was recognized by 60% of EP-TB and 42% of TBH and antigen MBP-506 was recognized by 45% of EP-TB and 61% of TBH. These results suggest that these proteins are strong candidates as subunits in a polyvalent serodiagnostic test.

WT1-specific serum antibodies in patients with leukemia.

WT1 is an oncogenic protein expressed by the Wilms' tumor gene and overexpressed in the majority of acute myelogenous leukemias (AMLs) and chronic myelogenous leukemias (CMLs). The current study analyzed the sera of patients with AML and CML for the presence of antibodies to full-length and truncated WT1 proteins. Sixteen of 63 patients (25%) with AML had serum antibodies reactive with WT1/full-length protein. Serum antibodies from all 16 were also reactive with WT1/NH2-terminal protein. By marked contrast, only 2 had reactivity to WT1/COOH-terminal protein. Thus, the level of immunological tolerance to the COOH terminus may be higher than to the NH2 terminus. The WT1/COOH-terminal protein contains four zinc finger domains with homology to other self-proteins. By implication, these homologies may be related to the increased immunological tolerance. Results in patients with CML were similar with antibodies reactive to WT1/full-length protein detectable in serum of 15 of 81 patients (19%). Antibodies reactive with WT1/NH2-terminal protein were present in the serum of all 15, whereas antibodies reactive with WT1/COOH-terminal protein were present in only 3. By contrast to results in leukemia patients, antibodies reactive with WT1/full-length protein were detected in only 2 of 96 normal individuals. The greater incidence of antibody in leukemia patients provides strong evidence that immunization to the WT1 protein occurred as a result of patients bearing malignancy that expresses WT1. These data provide further stimulus to test therapeutic vaccines directed against WT1 with increased expectation that the vaccines will be able to elicit and/or boost an immune response to WT1.

Evaluation of DPPD, a single recombinant Mycobacterium tuberculosis protein as an alternative antigen for the Mantoux test.

Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult particularly because sensitization with non-tuberculous mycobacteria leads to false positive tests. Using the guinea pig model of tuberculosis, we have recently described a recombinant antigen (DPPD) that could circumvent this problem. The DPPD gene is unique to the M. tuberculosis complex organisms and is absent in the organisms representative of all other members of the Mycobacterium genus. Moreover, DPPD induced strong DTH in 100% of the guinea pigs infected with M. tuberculosis and in none of the guinea pigs immunized with nine different species of Mycobacterium. Here we present results of a clinical investigation using DPPD. Mantoux test using both PPD and DPPD was initially performed in 26 patients with confirmed pulmonary tuberculosis and in 25 healthy PPD negative individuals. The results indicated that both PPD and DPPD elicited DTH in 24 out of the 26 patients. No DTH was observed in any of the PPD negative individuals. In addition, a small clinical trial was performed in a population of 270 clinically healthy and randomly selected individuals. DPPD produced a bimodal histogram of skin reaction size and PPD produced a skewed histogram. Because the DPPD gene is not present in non-tuberculous bacilli, these results suggest that this molecule can be an additional tool for a more specific diagnosis of tuberculosis.

T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection.

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.

Molecular and immunological characterization of Mycobacterium tuberculosis CFP-10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG.

In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis 38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis.

Cloning of a Mycobacterium tuberculosis gene encoding a purifed protein derivative protein that elicits strong tuberculosis-specific delayed-type hypersensitivity.

The purified protein derivative (PPD) skin test has been used for the diagnosis of tuberculosis for more than 75 years. However, the test lacks specificity because all mycobacteria share antigens present in PPD. Therefore, sensitization with nontuberculous pathogenic or with environmental nonpathogenic mycobacteria can lead to positive skin tests. This communication describes a novel PPD protein present only in tuberculous complex mycobacteria. A recombinant protein was obtained and named DPPD on the basis of the first 4 amino acids of its N-terminus sequence. DPPD elicited delayed-type hypersensitivity (DTH) in 100% of Mycobacterium tuberculosis-infected guinea pigs but in no animals sensitized with several organisms representative of all members of the Mycobacterium genus. Preliminary results indicate that DPPD induces strong and specific DTH in humans. This work points to the definition of a single recombinant M. tuberculosis protein that may be an alternative to the PPD test.

Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon gamma production from healthy purified protein derivative (PPD)(+) donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4(+) T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-gamma production by peripheral blood mononuclear cells from PPD(+) but not PPD(-) individuals. Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes. A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

Multiepitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in patients with treated or untreated Chagas' disease.

A tetrapeptide and a recombinant protein, each representing 4 immunodominant epitopes of Trypanosoma cruzi, were tested by use of ELISA for the detection of serum antibodies. Sera from individuals with Chagas' disease, including persons untreated and successfully or unsuccessfully treated, were tested. These assays detected antibody in 100% of the parasitemias. The antibody reactivity decreased based on the success of treatment. Higher sensitivity was observed for tetrapeptide/recombinant protein assays than for lysate-based ELISA, and specificity was improved, particularly with Leishmania sera. The results indicate that multiepitope antigens provide a more sensitive and specific alternative to lysate for detection of anti-T. cruzi antibodies, as required for developing blood screening assays.

Cloning, characterization and serological evaluation of K9 and K26: two related hydrophilic antigens of Leishmania chagasi.

We report here the molecular cloning and characterization of two related hydrophilic antigens of Leishmania chagasi. These two antigens have predicted molecular weights of approximately 9 and 26 kDa and detect antibodies in sera of patients with kala-azar (k). Thus, to maintain consistency with nomenclature of the previously described 39 kDa diagnostic antigen of L. chagasi (k39 [1]), these antigens are being referred to as k9 and k26. A significant difference between k9 and k26 is the presence of 11 copies of a 14 amino acid repeat in the open reading frame of k26. The region flanking the repeats of k26 shares a 69% identity with the open reading frame of k9. The recombinant proteins encoded by both antigens are very hydrophilic and show aberrant migration on SDS PAGE. Results of Southern blot analysis reveal that k9 and k26 are conserved to varying degrees among various Leishmania species. Interestingly, the repeat region of k26 is specific to L. chagasi and L. donovani while the flanking region is conserved among several other species. Transcript levels of k26 are significantly upregulated in the amastigote stage of the parasite. Our results show that recombinant K26 is specific in detecting antibodies in infection sera from visceral leishmaniasis (VL) patients. Thus rK26 may complement rK39 in a more accurate diagnosis of VL in the old and the new world.

Cloning, expression, and immunological evaluation of two putative secreted serine protease antigens of Mycobacterium tuberculosis.

Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity against Mycobacterium infection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M. tuberculosis genomic expression library in Escherichia coli. In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are approximately 32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins. Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% identity. MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development. Finally, the possible role of MTB32 serine proteases as a virulence factor(s) during Mycobacterium spp. infection is discussed.

Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family.

We have used expression screening of a genomic Mycobacterium tuberculosis library with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termed mtb39a, that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a family of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A in M. tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculosis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine for M.tuberculosis.