A novel peptide vaccination augments cytotoxic CD8+ T-cell responses against Mycobacterium tuberculosis HspX antigen

•Alternating peptide vaccination strategy enhanced Th1-mediated CD8+ T-cell response against HspX tuberculosis antigen in C57BL/6 mice.•No changes in Tregs percentages throughout alternating peptide vaccination, showing a least hindrance of immunosuppression upon challenge.•Senescence and exhaustion markers of CD8+ T-cells were expressed at basal level after alternating peptide vaccination.•Immunisation using alternating MHC-II helper peptides stimulated a prominent cytotoxic T-lymphocyte (CTL) activity against HspX antigen. Cellular immunity is a critical factor determining the safety and efficacy of newly developed vaccines against Mycobacterium tuberculosis infection. Crosstalk between CD4+ and CD8+ T-lymphocytes plays central roles in perpetuating the cytotoxic killing to the infected cells for host clearance. Our study proposed a novel alternating MHC-class II restricted peptide vaccination strategy to enhance the antigen-specific CD8+ T-cell activity against alpha-crystalline heat-shock protein (HspX) in C57BL/6 mice. Alternating peptide vaccination significantly stimulated a prominent HspX-specific CD8+ T-cell response with elevated Th1 and Th17 responses, without interference from Tregs suppression. Heightened central and effector CD8 memory were apparent in mice receiving alternating peptide vaccine, indicating a persisting recall immunity against HspX antigen. It was unlikely for alternating peptide vaccine to cause dysregulation in CD8+ T-cells as shown by minimal expression of KLRG1, PD1, LAG3, and CTLA-4 markers. Strong cytotoxic T-lymphocyte (CTL) responses were demonstrated in mice administrated with alternating peptide vaccines, suggesting its capacity in executing killing effector function against targeted cells. In conclusion, our novel vaccination strategy delineated potential benefits of alternating MHC-II peptides to invigorate efficient cytotoxic CD8+ T-cell responses against HspX antigen. Such approach might be applicable to serve as alternative immunotherapy for latent tuberculosis infection in future.