Tuberculosis (TB) remains the leading cause of death from a single infectious agent worldwide, highlighting the need for improved vaccines beyond the limited protection currently provided by the Bacillus Calmette-Guérin (BCG) vaccine. Here we investigated the immunological impact of incorporating alpha-Galactosylceramide (αGC) into mRNA lipid nanoparticles (mRNA-LNPs) as a vaccination strategy against Mycobacterium tuberculosis (Mtb), the pathogen causing TB. Using Mtb antigens ESAT-6 and Ag85B, we demonstrate that αGC-adjuvanted mRNA-LNPs induce controlled activation of invariant natural killer T (iNKT) cells, thereby reshaping vaccine-induced immunity. Specifically, αGC incorporation promoted the generation of CD44+CD69+CD62L-CD4+ T cells in the spleen and altered the CD4+ T -helper (TH) profile through the addition of modest TH2 and TH17 responses alongside robust TH1 immunity. Furthermore, antigen-specific CD8+ T-cell responses against ESAT-6 and Ag85B were observed exclusively in the genetically diverse CB6F1/J mouse model, highlighting the importance of host genetic background in evaluating mRNA vaccine immunogenicity after homologous vaccination strategies. Finally, we demonstrate that using a heterologous prime-pull strategy combining intramuscular mRNA vaccination with local antigen instillation increased the frequency of antigen-specific lung-associated CD4+ T cells displaying a tissue residency-associated phenotype, with additional contribution of αGC adjuvantation to this response. In summary, these findings provide novel insights into αGC adjuvant-mediated immune modulation of mRNA vaccines and support further testing of protective efficacy of this approach against TB.
