Mycobacterial EsxG·EsxH (TB9.8·TB10.4) peptides as a subunit vaccine to booster BCG vaccination in an experimental model of pulmonary Tuberculosis

<b>Background/Objectives</b>: The attenuated <i>Mycobacterium bovis</i> bacillus Calmette-Guérin (BCG) vaccine remains the only validated vaccine against tuberculosis (TB), but its efficiency is limited because it induces wide variability in protection and has limited immunologic memory. A prior<i>in silico</i>study with bioinformatic analysis and molecular dynamic simulations identified four peptides (G1, G2, H1, and H2) derived from the mycobacterial antigens TB10.9·TB10.4 (EsxG·EsxH) with ability to bind MHC-II molecules that potentially activate T and B cells.The present study aimed to experimentally validate these peptides as candidates for subunit vaccines. <b>Methods</b>: We assessed the cytotoxicity and protective efficacy ofthese peptides against <i>Mycobacterium tuberculosis</i> (Mtb) in a murine model, administered as a booster to BCG vaccination. Two months after BCG vaccination, mice received a subcutaneous boost with G1, G2, H1, or H2 peptides. One-month post-immunization, mice were challenged with thereference strain H37Rv. Specific IgG antibody titers were quantified via ELISA. <b>Results</b>: Boosting BCG with these peptides significantly enhanced protection compared to BCG alone, as evidencedby reduced pulmonary bacterial loads and specific IgG antibody production. <b>Conclusions:</b> These findings underscore the potential of epitope-based subunit vaccines to boost immunity against TB whenused as booster of BCG. This approach lays the groundwork for the development of tailored vaccine strategies targeting TB.