The attenuatedMycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine is currently the only validated vaccine against tuberculosis (TB). In a previous study, we conducted anin-silicoselection of four peptides (G1, G2, H1, and H2) derived from the mycobacterial protein antigens TB10.9·TB10.4 (EsxG·EsxH). Bioinformatic analysis and molecular dynamic simulations predicted these epitopes could be loaded into a MHC-II complex, inducing T and B cell activation. The present study aimed to experimentally validate these peptides as subunit vaccines by determining their cytotoxicity, immunogenicity, and protective efficacy againstMycobacterium tuberculosis(Mtb) in mice when administered as a booster to BCG vaccination. Mice were vaccinated with BCG and, two months later, were subcutaneously immunized with either peptide G1, G2, H1, or H2. One-month post-immunization, mice were challenged with the reference strain H37Rv of moderate virulence or the hypervirulent clinical isolate 09005186. After vaccination and before the challenge, the spleen and lung cells were harvested and stimulatedin vitrowith the corresponding peptide to measure cytokine expression in CD4+, and CD8+T cells, as well as the phenotypes of activated effector T cells, proliferative senescence, central and periphery memory CD4+and CD8+cells. Additionally, specific IgG antibody titers elicited by each peptide were measured using ELISA. Compared with animals vaccinated only with BCG, boosting BCG vaccination with these peptides provided enhanced protection by significantly prolonging the mice survival, reducing the bacillary load, and decreasing tissue damage (pneumonia). These findings contribute to the broader understanding of peptide-based subunit vaccines and highlight the potential for tailored approaches to enhance protective immunity.
