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Dissecting the function(s) of proteins present exclusively in Mycobacterium tuberculosis ( M.tb ) will provide important clues regarding the role of these proteins in mycobacterial pathogenesis. Using extensive computational approaches, we shortlisted ORFs/proteins unique to M.tb among 13 different species of mycobacteria and identified a hypothetical protein Rv1509 as a 'signature protein' of M.tb . This unique protein was found to be present only in M.tb and absent in all other mycobacterial species, including BCG. In silico analysis identified numerous putative T cell and B cell epitopes in Rv1509. Initial in vitro experiments using innate immune cells demonstrated Rv1509 to be immunogenic with potential to modulate innate immune responses. Macrophages treated with Rv1509 exhibited higher activation status along with substantial release of pro-inflammatory cytokines. Besides, Rv1509 protein boosts dendritic cell maturation by increasing the expression of activation markers such as CD80, HLA-DR and decreasing DC-SIGN expression and this interaction was mediated by innate immune receptor TLR2. Further, in vivo experiments in mice demonstrated that Rv1509 protein promotes the expansion of multifunctional CD4+ and CD8+T cells and induces effector memory response along with evoking a canonical Th1 type of immune response. Rv1509 also induces substantial B cell response as revealed by increased IgG reactivity in sera of immunized animals. This allowed us to demonstrate the diagnostic efficacy of this protein in sera of human TB patients compared to the healthy controls. Taken together, our results reveal that Rv1509 signature protein has immunomodulatory functions evoking immunological memory response with possible implications in serodiagnosis and TB vaccine development., Competing Interests: Author SAR was employed by company BioInceptionPvt. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 P, Ahmad, Samal, Sheikh, Arora, Khubaib, Aggarwal, Kumari, Luthra, Rahman, Hasnain and Ehtesham.)

Mycobacterium tuberculosis ( M. tb ) persists as latent infection in nearly a quarter of the global population and remains the leading cause of death among infectious diseases. While BCG is the only vaccine for TB, its inability to provide complete protection makes it imperative to engineer BCG such that it expresses immunodominant antigens that can enhance its protective potential. In-silico comparative genomic analysis of Mycobacterium species identified M. tb Rv1507A as a "signature protein" found exclusively in M. tb . In-vitro (cell lines) and in-vivo experiments carried out in mice, using purified recombinant Rv1507A revealed it to be a pro-inflammatory molecule, eliciting significantly high levels of IL-6, TNF-α, and IL-12. There was increased expression of activation markers CD69, CD80, CD86, antigen presentation molecules (MHC I/MHCII), and associated Th1 type of immune response. Rv1507A knocked-in M. smegmatis also induced significantly higher pro-inflammatory Th1 response and higher survivability under stress conditions, both in-vitro (macrophage RAW264.7 cells) and in-vivo (mice). Sera derived from human TB patients showed significantly enhanced B-cell response against M. tb Rv1507A. The ability of M. tb Rv1507A to induce immuno-modulatory effect, B cell response, and significant memory response, renders it a putative vaccine candidate that demands further exploration., (Copyright © 2020 Arora, Alam, Naqvi, Ahmad, Sheikh, Rahman, Hasnain and Ehtesham.)

Tuberculosis (TB) continues to be a major health problem due to lack of accurate, rapid, and cost-effective diagnostic tests. Serodiagnostic tests incorporating highly specific region of difference (RD) antigens (early secretory antigenic target 6 [ESAT-6], culture filtrate protein 10 [CFP-10], culture filtrate protein 21 [CFP-21], and mycobacterial protein from species tuberculosis 64 [MPT-64]) have recently been shown to be promising for specific diagnosis of TB in our lab. However, only few studies have reported the use of synthetic peptides of RD antigens, and none has used them to differentiate TB from sarcoidosis, a close mimic of smear-negative pulmonary TB (PTB) with entirely different management. The present study was conducted with an aim to study the utility of B-cell epitopes based peptides of RD1 (ESAT-6, CFP-10) and RD2 (CFP-21, MPT-64) antigens for immunodiagnosis of PTB for which sputum smear-positive PTB patients, sputum smear-negative PTB patients, sarcoidosis patients, and healthy controls (n = 24/group) were recruited. Bioinformatic software Bcepred was used to predict linear B-cell epitopes, using physico-chemical properties on a non-redundant dataset. Seven peptides as representative B-cell epitopes of ESAT-6, CFP-10, CFP-21, and MPT-64 were evaluated as targets of the antibody responses in TB patients and controls by enzyme-linked immunosorbent assay (ELISA). The current study showed sensitivity with individual peptides ranging from 37.5% to 83.3% for smear positive, 25% to 58.3% for smear negative as compared to 4.16% to 20.8% for sarcoidosis. Four out of 7 peptides that showed higher reactivity with TB patients and better discrimination from sarcoidosis patients representing ESAT-6, CFP-10, CFP-21, and MPT-64 were selected for multiepitope ELISA. The combination of peptides yielded 83.3% sensitivity for smear positive, 62.5% for smear negative, and only 4.16% for sarcoidosis. The specificity, however, for all the peptides/combination was 100%. Combination of peptides has proven to be better than individual peptides as per the latest criteria of the World Health Organization according to which a test that can replace smear microscopy with sensitivity of >90% for smear-positive patients and >65% for smear-negative TB patients with a specificity >95%, and thus, the present study suggests that a test based on combination of peptides selected from mycobacterial RD1 and RD2 antigens could be important for promoting an early diagnosis and management of otherwise difficult to diagnose smear-negative PTB patients. Moreover, it can also be used to discriminate sarcoidosis from PTB, thus preventing the misdiagnosis and mismanagement., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Tuberculin skin test (TST), an age old method is based on measuring delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). However, inspite of simplicity, ease and cost effectiveness, the usefulness of PPD test is limited due to its inability to distinguish among a protective immune response, latent infection and active tuberculosis disease. On the other hand, a skin test based on RD antigens would add advantages of a high specificity of antigens with the logistics of a skin test. However, except few reports, in vivo data of intradermal use of RD antigens for skin testing is limited. Therefore, in the present study, four M. tuberculosis (Mtb) specific antigens (ESAT6, CFP10, CFP21 and MPT64) were evaluated for their diagnostic utility based on DTH response. These antigens alone and their multiple combinations induced strong DTH response in Mtb infected guinea pigs and the response was negligible in BCG vaccinated and sham immunized animals.