Mucosal Adenovirus-Vectored Rv2299c Vaccine Protects Against Tuberculosis by Inducing Trained Immunity in Dendritic Cells and Polyfunctional T Cells

<b>Background</b>: The development of effective tuberculosis (TB) vaccines beyond BCG remains an urgent global health priority, especially for prevention of pulmonary TB in adults. While most current strategies focus on enhancing T-cell immunity, the potential of trained immunity to broadly augment both innate and adaptive responses remains underexplored in TB vaccinology. Given the central role of dendritic cells (DCs) as bridges between innate and adaptive immunity, we hypothesized that inducing trained immunity in DCs could optimize subsequent T-cell responses. Previous studies have identified Rv2299c as a promising adjuvant of other antigens by promoting DC maturation; however, whether it could be used as a standalone protective antigen of TB vaccine remains unclear. <b>Methods</b>: We constructed a chimpanzee adenovirus-vectored TB vaccine candidate expressing Rv2299c (rAd-Rv2299c), and evaluated its immunogenicity and protective efficacy in murine models. <b>Results</b>: rAd-Rv2299c vaccine effectively induced a trained immunity phenotype in DCs, as evidenced by upregulated MHC-II and CD86 expression and increased pro-inflammatory cytokine (TNF-α, IL-6, IL-1β and IL-12p70) secretion. Moreover, its immunization promoted the generation of antigen-specific polyfunctional T cells, and robustly enhanced both Th1 and Th17-type immune responses. In a murine challenge model, vaccination significantly reduced bacterial loads in the lung and spleen and attenuated pulmonary inflammation, which was associated with robust recall T-cell immune responses. <b>Conclusions</b>: rAd-Rv2299c confers anti-TB protection by inducing trained immunity in DCs and promoting polyfunctional T-cell responses, thereby offering valuable experimental evidence and conceptual insights for the development of next-generation TB vaccines.