Analysis of the DosR regulon genes to select cytotoxic T lymphocyte epitope specific vaccine candidates using a reverse vaccinology approach

Objective/background: There is an urgent need for a more effective vaccine against Mycobacterium tuberculosis [Mtb]. Although CD4+ T cells play a central role in host immunity to Mtb, recent evidence suggests a critical role of CD8+ T cells in combating Mtb. In the present study, we have predicted HLA antigen class I binding peptides of DosR operon using an in-silico approach. This method is useful as an initial computational filtration of probable epitopes based on their binding ability and antigenicity

Methods: CD8+ epitopes were predicted by software NetMHC 3.4 and BIMAS. Self-peptides were found and excluded by indigenously developed Perl script. Antigenicity of promiscuous peptides was predicted using a Vaxijen server. The top Vaxijen scoring antigenic peptides were docked to globally relevant HLA allele using CABS dock and Hex program

Results: A total of 1436 overlapping nonamer peptides were generated which gave 46 promiscuous epitopes, 25 were predicted to be antigenic. Rv2627 epitope "SAFRPPLV" which gave the highest Vaxijen score of 1.9157 and showed binding to all the three HLA loci. The top Vaxijen scoring antigenic peptides were docked and had significant interactions with residues of the HLA class I molecule indicating them to be good cytotoxic T lymphocyte epitopes

Conclusion: Our study has generated several promiscuous antigenic peptides capable of binding to major histocompatibility complex class I with high affinity. These epitopes can become part of a postexposure multivalent subunit vaccine upon experimental validation

Differential signaling of inducible nitric oxide synthase induction in Mycobacterium tuberculosis infected alveolar epithelial cell line A549 in response to cytokines IFN-gamma, TNF-alpha and IL-1beta

In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected type II alveolar epithelial cells generate de novo nitric oxide [NO], but the mycobactericidal quantity of NO was released only by stimulation of these cells with proinflammatory cytokines, i.e. IFN-gamma, TNF-alpha and IL-1beta. In the present communication, it was demonstrated that M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and NF-kappaB pathways for the induction of inducible Nitric Oxide Synthase [iNOS] mRNA and NO production. Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells. Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-gamma or a combination of three cytokines [IFN-gamma, TNF-alpha and IL-1beta] formed DNA protein complexes with probes from both -5.2 kb region [specific for binding of STAT-1 protein] and -5.8 kb region [specific for binding of both STAT-1 and NF-kappaB] of the iNOS promoter. However, TNF-alpha or IL-1beta stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from -5.2 kb region. This differential response indicated that TNF-alpha/IL-1beta does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-gamma. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly.