Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis

Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4⁺ Th1 responses, higher numbers of IFN-γ⁺ CD4⁺ T_EM and IL-2⁺ CD8⁺ T_CM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.