Development of LepReact, a defined skin test for paucibacillary leprosy and low-level M. leprae infection.

The persistence of new leprosy cases in endemic areas such as India, Brazil, Bangladesh, and the Philippines has encouraged studies of chemoprophylaxis among contacts of patients. Epidemiological screening tools to enable early detection of infected individuals in endemic populations would be critical to target individuals most in need of intervention. Despite decades of attempts, however, there still are no tests available for the early detection of low-level infection with Mycobacterium leprae. In this report, we describe the development of a leprosy skin test using M. leprae-specific antigens. We selected the chimeric LID-1 fusion protein, formulated to achieve maximum performance at a minimal dose, as a skin test candidate based on its ability to elicit delayed-type hypersensitivity (DTH) reactions in M. leprae immune guinea pigs in a sensitive and specific manner, i.e., with no cross-reactivity observed with other mycobacterial species. Importantly, evaluations in armadillos indicated that intradermal inoculation of formulated LID-1 could distinguish uninfected from M. leprae-infected animals manifesting with symptoms distinctly similar to the PB presentation of patients. Together, our data provide strong proof-of-concept for developing an antigen-specific skin test to detect low-level M. leprae infection. Such a test could, when applied with appropriate use of chemo- and/or immunoprophylaxis, be instrumental in altering the evolution of clinical disease and M. leprae transmission, thus furthering the objective of zero leprosy.

Molecular characterization and immunogenic function of ML1899 (LipG) of Mycobacterium leprae.

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.

Invasion of human microvascular endothelial cells by Mycobacterium leprae through Mce1A protein.

In patients with lepromatous leprosy, Mycobacterium leprae is often observed inside the human microvascular endothelial cells (HMVEC) surrounding Schwann cells (SC) at the site of lesions in the peripheral nerves. Based on this observation, it is considered that the nasal mucous may be the invasion pathway for M. leprae and HMVEC serve as an important reservoir for the bacteria before they invade SC. In light of previous research which revealed that Mce1A protein mediates bacterial invasion into nasal epithelial cells and HMVEC, we conducted a study to determine whether the invasion of M. leprae into HMVEC can be suppressed by blocking the Mce1A protein. In this study, we analyzed bacterial invasive activity by adding recombinant Escherichia coli, which express the active region (InvX:72 a.a.) of Mce1A protein on their external membrane, into cultured HMVEC, using the adhesin involved in the diffuse adherence mechanism. The number of bacteria that invaded into the cells was then measured by a colony counting method. The active region of Mce1A was divided into four sections, and hyperimmune antisera was prepared for each section for analyzing the inhibitory effect against invasion. The invasive activity was suppressed by antibodies against InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. This suggests that the InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. of Mce1A protein play an important role in the invasion of M. leprae into HMVEC and that it may be possible to suppress entry of M. leprae in HMVEC with antibodies against these regions.

Infect and Inject: How Mycobacterium tuberculosis Exploits Its Major Virulence-Associated Type VII Secretion System, ESX-1.

Mycobacterium tuberculosis is an ancient master of the art of causing human disease. One important weapon within its fully loaded arsenal is the type VII secretion system. M. tuberculosis has five of them: ESAT-6 secretion systems (ESX) 1 to 5. ESX-1 has long been recognized as a major cause of attenuation of the FDA-licensed vaccine Mycobacterium bovis BCG, but its importance in disease progression and transmission has recently been elucidated in more detail. This review summarizes the recent advances in (i) the understanding of the ESX-1 structure and components, (ii) our knowledge of ESX-1's role in hijacking macrophage function to set a path for infection and dissemination, and (iii) the development of interventions that utilize ESX-1 for diagnosis, drug interventions, host-directed therapies, and vaccines.

Mycobacterium indicus pranii protein MIP_05962 induces Th1 cell mediated immune response in mice.

Utility of Mycobacterium indicus pranii (MIP) as a multistage vaccine against mycobacterial infections demands identification of its protective antigens. We explored antigenicity and immunogenicity of a candidate protein MIP_05962 that depicts homology to HSP18 of M. leprae and antigen1 of Mycobacterium tuberculosis. This protein elicited substantial antibody response in immunized mice along with modulation of cellular immune response towards protective Th1 type. Both CD4+ and CD8+ subsets from immunized mice produced hallmark protective cytokines, IFN-γ, TNF-α and IL-2. This protein also enhanced the CD4+ effector memory that could act as first line of defence during infections. These results point to MIP_05962 as a protective antigen that contributes, in conjunction with others, to the protective immunity of this live vaccine candidate.

Vaccines for Leprosy and Tuberculosis: Opportunities for Shared Research, Development, and Application.

Tuberculosis (TB) and leprosy still represent significant public health challenges, especially in low- and lower middle-income countries. Both poverty-related mycobacterial diseases require better tools to improve disease control. For leprosy, there has been an increased emphasis on developing tools for improved detection of infection and early diagnosis of disease. For TB, there has been a similar emphasis on such diagnostic tests, while increased research efforts have also focused on the development of new vaccines. Bacille Calmette-Guérin (BCG), the only available TB vaccine, provides insufficient and inconsistent protection to pulmonary TB in adults. The impact of BCG on leprosy, however, is significant, and the introduction of new TB vaccines that might replace BCG could, therefore, have serious impact also on leprosy. Given the similarities in antigenic makeup between the pathogens Mycobacterium tuberculosis (Mtb) and M. leprae, it is well possible, however, that new TB vaccines could cross-protect against leprosy. New TB subunit vaccines currently evaluated in human phase I and II studies indeed often contain antigens with homologs in M. leprae. In this review, we discuss pre-clinical studies and clinical trials of subunit or whole mycobacterial vaccines for TB and leprosy and reflect on the development of vaccines that could provide protection against both diseases. Furthermore, we provide the first preclinical evidence of such cross-protection by Mtb antigen 85B (Ag85B)-early secretory antigenic target (ESAT6) fusion recombinant proteins in in vivo mouse models of Mtb and M. leprae infection. We propose that preclinical integration and harmonization of TB and leprosy research should be considered and included in global strategies with respect to cross-protective vaccine research and development.

Determination of Lipoprotein Z-Specific IgA in Tuberculosis and Latent Tuberculosis Infection.

Tuberculosis (TB) remains one of the most severe infectious diseases. It is still of paramount importance to establish more accurate, rapid, and efficient diagnostic methods. Since infection with Mycobacterium tuberculosis (M. tb) is largely mediated through the respiratory tract, IgA responses against mycobacterial proteins are worthy of investigation for their potential clinical utility. In this study, the IgA response targeting lipoprotein Z (LppZ) was determined by using a homemade ELISA with plasma of TB patients (N = 125), LTBI individuals (N = 92), healthy controls (HCs) (N = 165), as well as TB patients undergoing anti-TB treatment (N = 9). In parallel the antigen-specific IFN-γ release from PBMCs triggered by LppZ and M. tb-specific ESAT-6 or CFP-10 was detected by using an ELISPOT assay. It was found that the LppZ-specific IgA level was dramatically higher in TB patients than in HCs (p < 0.0001). Compared to that before anti-TB treatment, the LppZ-specific IgA level decreased substantially after 2 months of anti-TB treatment (p = 0.0297) and remained at low levels until the end of the treatment. What is more, pulmonary TB patients exhibited significantly higher LppZ-specific IgA-values than extra-pulmonary TB patients (p = 0.0296). Interestingly, the LppZ-specific IgA-values were negatively correlated to the amounts of IFN-γ released in response to LppZ with statistical significance (r = -0.5806, p = 0.0002). LppZ-specific IgA level was also higher in LTBI individuals than in HCs (p < 0.0001). Additionally there were some PPD+ HC individuals with high LppZ-specific IgA levels but the potential of this assay for identifying leaky LTBI in PPD+ HCs needs to be further investigated through follow-up studies. The sensitivity of detecting TB solely with ESAT-6 or CFP-10-specific IFN-γ release was increased by including the LppZ-specific IgA results, respectively, from 86.11 to 100% and 88.89 to 100%; the sensitivity of screening for LTBI was increased from 80.36 to 83.93% and 57.14 to 69.64%, respectively. The higher LppZ-specific IgA responses in TB and LTBI populations than in controls indicated high immunoreactivity to LppZ upon M. tb infection. Although the assay was not efficient enough for independent application in sero-diagnosis, LppZ-specific IgA might become a complementary biomarker for the improvement of TB and LTBI screening.

ELISA-based assay of immunoglobulin G antibodies against mammalian cell entry 1A (Mce1A) protein: a novel diagnostic approach for leprosy.

BACKGROUND: Leprosy is a chronic infectious disease caused by the obligate intracellular bacillus Mycobacterium leprae. Because leprosy diagnosis is complex and requires professional expertise, new tools and methodologies are needed to detect cases in early stages and prevent transmission. The M. leprae genome contains mce1A, which encodes a putative mammalian cell entry protein (Mce1A). We hypothesised that the presence of Mce1A on the cell surface could be detected by the host's immune system. OBJECTIVE: The aim of this study was to evaluate antibody responses against the Mce1A protein in leprosy patients, household contacts of patients, and the general population to present an addition tool for leprosy diagnosis. METHODS: A cross-sectional study involving 89 volunteers [55 leprosy cases, 12 household contacts (HHC) and 22 endemic controls (EC)] was conducted at Couto Maia Hospital, in Salvador, Bahia (BA), Brazil. RESULTS: The median anti-Mce1A IgA was significantly higher in multibacillary (MB) and paucibacillary (PB) cases than in EC (p < 0.0001). A similar trend was observed in IgM levels, which were significantly higher in both MB (p < 0.0001) and PB (p = 0.0006) groups compared to in EC individuals. The greatest differences were observed for IgG class-specific antibodies against Mce1A. The median levels of MB and PB were significantly higher compared to both controls HHC and EC (MB or PB vs EC, MB vs HHC p < 0.0001; PB vs HHC, p = 0.0013). Among leprosy cases, IgG enzyme-linked immunosorbent assay sensitivity and specificity were 92.7% and 97.1%, respectively. IgG positivity was confirmed in 92.1% and 94.1% of MB and PB patients, respectively. CONCLUSION: This novel diagnostic approach presents an easy, non-invasive, and inexpensive method for leprosy screening, which may be applicable in endemic areas.

ELISA-based assay of immunoglobulin G antibodies against mammalian cell entry 1A (Mce1A) protein: a novel diagnostic approach for leprosy

BACKGROUND Leprosy is a chronic infectious disease caused by the obligate intracellular bacillus Mycobacterium leprae. Because leprosy diagnosis is complex and requires professional expertise, new tools and methodologies are needed to detect cases in early stages and prevent transmission. The M. leprae genome contains mce1A, which encodes a putative mammalian cell entry protein (Mce1A). We hypothesised that the presence of Mce1A on the cell surface could be detected by the host's immune system. OBJECTIVE The aim of this study was to evaluate antibody responses against the Mce1A protein in leprosy patients, household contacts of patients, and the general population to present an addition tool for leprosy diagnosis. METHODS A cross-sectional study involving 89 volunteers [55 leprosy cases, 12 household contacts (HHC) and 22 endemic controls (EC)] was conducted at Couto Maia Hospital, in Salvador, Bahia (BA), Brazil. RESULTS The median anti-Mce1A IgA was significantly higher in multibacillary (MB) and paucibacillary (PB) cases than in EC (p < 0.0001). A similar trend was observed in IgM levels, which were significantly higher in both MB (p < 0.0001) and PB (p = 0.0006) groups compared to in EC individuals. The greatest differences were observed for IgG class-specific antibodies against Mce1A. The median levels of MB and PB were significantly higher compared to both controls HHC and EC (MB or PB vs EC, MB vs HHC p < 0.0001; PB vs HHC, p = 0.0013). Among leprosy cases, IgG enzyme-linked immunosorbent assay sensitivity and specificity were 92.7% and 97.1%, respectively. IgG positivity was confirmed in 92.1% and 94.1% of MB and PB patients, respectively. CONCLUSION This novel diagnostic approach presents an easy, non-invasive, and inexpensive method for leprosy screening, which may be applicable in endemic areas.

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed ß-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

DNAhsp65 Vaccine as Therapy against Paracoccidioidomycosis.

The conventional treatment for fungal diseases usually shows long periods of therapy and the high frequency of relapses and sequels. New strategies of the treatment are necessary. We have shown that the Mycobacterium leprae HSP65 gene can be successfully used as therapy against murine Paracoccidioidomycosis (PCM). Here, we described the methodology of DNAhsp65 immunotherapy in mice infected with the dimorphic fungus Paracoccidioides brasiliensis, one of PCM agent, evaluating cytokines levels, fungal burden, and lung injury. Our results provide a new prospective on the immunotherapy of mycosis.

Synthesis and immunogenicity of PG-tb1 monovalent glycoconjugate.

A PG-tb1 hapten from the West Beijing strains of Mycobacterium tuberculosis cell wall has been efficiently synthesized and conjugated to CRM197 in a simple way as linker-equipped carbohydrate by applying squaric acid chemistry for an original neoglycoprotein, creating a potent T-dependent conjugate vaccine. The intermediate monoester can be easily purified and the degree of incorporation can be monitored by MALDI-TOF mass spectrometry. After administered systemically in mice without any adjuvant, the conjugate induced high antigen-specific IgG levels in serum. Furthermore, following the third immunization, significant antibody titers frequently exceeding 0.8 million were observed in the sera of mice vaccinated with PG-CRM197 conjugate which showed the potential for preparation of TB vaccine.

Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients.

Previously we showed that 65-kDa Mycobacterium leprae heat shock protein (Hsp65) is a target for the development of a tuberculosis vaccine. Here we evaluated peripheral blood mononuclear cells (PBMC) from healthy individuals or tuberculosis patients stimulated with two forms of Hsp65 antigen, recombinant DNA that encodes Hsp65 (DNA-HSP65) or recombinant Hsp65 protein (rHsp65) in attempting to mimic a prophylactic or therapeutic study in vitro, respectively. Proliferation and cytokine-producing CD4+ or CD8+ cell were assessed by flow cytometry. The CD4+ cell proliferation from healthy individuals was stimulated by DNA-HSP65 and rHsp65, while CD8+ cell proliferation from healthy individuals or tuberculosis patients was stimulated by rHSP65. DNA-HSP65 did not improve the frequency of IFN-gamma+ cells from healthy individuals or tuberculosis patients. Furthermore, we found an increase in the frequency of IL-10-producing cells in both groups. These findings show that Hsp65 antigen activates human lymphocytes and plays an immune regulatory role that should be addressed as an additional antigen for the development of antigen-combined therapies.

Excavating the surface-associated and secretory proteome of Mycobacterium leprae for identifying vaccines and diagnostic markers relevant immunodominant epitopes.

For centuries, Mycobacterium leprae, etiological agent of leprosy, has been afflicting mankind regardless of extensive use of live-attenuated vaccines and antibiotics. Surface-associated and secretory proteins (SASPs) are attractive targets against bacteria. We have integrated biological knowledge with computational approaches and present a proteome-wide identification of SASPs. We also performed computational assignment of immunodominant epitopes as coordinates of prospective antigenic candidates in most important class of SASPs, the outer membrane proteins (OMPs). Exploiting the known protein sequence and structural characteristics shared by the SASPs from bacteria, 17 lipoproteins, 11 secretory and 19 novel OMPs (including 4 essential proteins) were identified in M. leprae As OMPs represent the most exposed antigens on the cell surface, their immunoinformatics analysis showed that the identified 19 OMPs harbor T-cell MHC class I epitopes and class II epitopes against HLA-DR alleles (54), while 15 OMPs present potential T-cell class II epitopes against HLA-DQ alleles (6) and 7 OMPs possess T-cell class II epitopes against HLA-DP alleles (5) of humans. Additionally, 11 M. leprae OMPs were found to have B-cell epitopes and these may be considered as prime candidates for the development of new immunotherapeutics against M. leprae.

Modulation of human T cell cytokines by the Mycobacterium tuberculosis-secreted protein Wag31.

Mycobacterium tuberculosis secretes a number of proteins into the extracellular milieu during growth. Several of these proteins have been associated with modulation of the host immune response. Antigen 84, or Wag31, is one such protein that is conserved among all mycobacterial species and is recognized by the sera from tuberculosis and leprosy patients. Here, we examined the effect of Wag31 on the ability of activated human T cells to produce cytokines such as IL-10, IL-17 and IFN-γ in response to combined anti-CD3 and anti-CD28 stimulation. Purified recombinant Wag31 inhibited the secretion of IL-10 and IL-17, but not IFN-γ, by human T cells stimulated with plate-bound anti-CD3 and anti-CD28 monoclonal antibodies. Furthermore, the C-terminal domain, but not the N-terminal domain, inhibited the production of IL-10 and IL-17 without a significant effect on the production of IFN-γ. These data suggest that Wag31 may modulate human T cell immune responses during tuberculosis infection through its C-terminal domain.

Immunoglobulin G response to mammalian cell entry 1A (Mce1A) protein as biomarker of active tuberculosis.

Cell wall components are major determinants of virulence of Mycobacterium tuberculosis and they contribute to the induction of both humoral and cell-mediated immune response. The mammalian cell entry protein 1A (Mce1A), in the cell wall of M. tuberculosis, mediates entry of the pathogen into mammalian cells. Here, we examined serum immunoglobulin levels (IgA, IgM and total IgG) against Mce1A as a potential biomarker for diagnosis and monitoring tuberculosis (TB) treatment response. Serum samples of 39 pulmonary TB patients and 65 controls (15 healthy household contacts, 19 latently infected household contacts, 13 non-TB and 18 leprosy patients) were screened by ELISA. The median levels of all immunoglobulin classes were significantly higher in TB patients when compared with control groups. The positive test results for IgA, IgM and total IgG were 62, 54 and 82%, respectively. For comparison, routine sputum smear examination diagnosed only 26 (67%) of 39 TB cases. Sensitivities of IgA, IgM and IgG test were 59, 51.3 and 79.5%, respectively, while the specificities observed were 77.3, 83.3 and 84.4%, respectively. A significant decrease compared with baseline was also shown after TB treatment. These results suggest that circulating total IgG antibody to Mce1A could be a complementary tool to diagnosis pulmonary TB.

Mycobacterial r32-kDa antigen-specific T-cell responses correlate with successful treatment and a heightened anti-microbial response in human leprosy patients.

BACKGROUND: Immunological characterization of mycobacterial peptides may help not only in the preparation of a vaccine for leprosy but also in developing in vitro T-cell assays that could perhaps be used as an in vitro correlate for treatment outcome. The main goal of this study was to evaluate the use of Mycobacterium bovis recombinant 32-kDa protein (r32-kDa) antigen-stimulated T-cell assay as a surrogate marker for treatment outcome and monitor vitamin D receptor (VDR)-mediated anti-microbial responses during multidrug therapy (MDT) in leprosy. METHODS: Newly diagnosed tuberculoid and lepromatous leprosy patients were enrolled and followed up during their course of MDT at 6 and 12 months. IFN-γ, IL-10, IL-17 and IL-23 levels in culture supernatants and expression of VDR, TLR2, LL37 and DEFB in r32-kDa-stimulated PBMCs were measured. Controls comprised household contacts (HHCs) and healthy endemic subjects (HCs). RESULTS: Significant differences were observed in the levels of IFN-γ, IL-17, IL-23, VDR and anti-microbial peptides LL37 and DEFB after treatment and when compared with that of HHCs and HCs, respectively. CONCLUSIONS: These findings suggest that responses to r32-kDa antigen reflect an improved immunological and anti-microbial response in leprosy patients during therapy, thereby indicating its potential use as an immune correlate in the treatment of leprosy patients.

Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis.

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0(+)CD69(+)CD4(+) memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.

Characterization of a cross-reactive, immunodominant and HLA-promiscuous epitope of Mycobacterium tuberculosis-specific major antigenic protein PPE68.

PPE68 (Rv3873), a major antignic protein encoded by Mycobacteriun tuberculosis-specific genomic region of difference (RD)1, is a strong stimulator of peripheral blood mononuclear cells (PBMCs) obtained from tuberculosis patients and Mycobacterium bovis bacillus Calmette Guerin (BCG)-vaccianted healthy subjects in T helper (Th)1 cell assays, i.e. antigen-induced proliferation and interferon-gamma (IFN-γ) secretion. To confirm the antigen-specific recognition of PPE68 by T cells in IFN-γ assays, antigen-induced human T-cell lines were established from PBMCs of M. Bovis BCG-vaccinated and HLA-heterogeneous healthy subjects and tested with peptide pools of RD1 proteins. The results showed that PPE68 was recognized by antigen-specific T-cell lines from HLA-heteregeneous subjects. To further identify the immunodominant and HLA-promiscuous Th1-1 cell epitopes present in PPE68, 24 synthetic peptides covering the sequence of PPE68 were indivdually analyzed for HLA-DR binding prediction analysis and tested with PBMCs from M. bovis BCG-vaccinated and HLA-heterogeuous healthy subjects in IFN-γ assays. The results identified the peptide P9, i.e. aa 121-VLTATNFFGINTIPIALTEMDYFIR-145, as an immunodominant and HLA-DR promiscuous peptide of PPE68. Furthermore, by using deletion peptides, the immunodominant and HLA-DR promiscuous core sequence was mapped to aa 127-FFGINTIPIA-136. Interestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced strains/species of M. tuberculosis and M. tuberculosis complex, and several other pathogenic mycobacterial species, including M. leprae and M. avium-intracellulalae complex. These results suggest that the peptide aa 121-145 may be exploited as a peptide-based vaccine candidate against tuberculosis and other mycobacterial diseases.