Enhancing tuberculosis vaccine efficacy with a heterologous mRNA-ChAdOx1 prime-pull strategy targeting lung-resident memory T cells

Introduction Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide, and Bacillus Calmette–Guérin (BCG) offers inconsistent protection against adult pulmonary TB. We previously showed that homologous mRNA vaccination encoding the mycobacterial antigen PPE15 (mRNA.PPE15) enhanced immunogenicity but did not improve protection over BCG alone. We hypothesised that a heterologous “prime-pull” strategy, systemic mRNA priming followed by mucosal adenoviral boosting, would enrich lung-resident memory T cells (TRM) and improve efficacy. Methods Female C57BL/6 mice received BCG prime followed by subunit regimens combining intramuscular mRNA.PPE15 and intranasal ChAdOx1.PPE15 in different administration orders, alongside homologous controls. Cellular responses in spleen and lung were quantified by intracellular cytokine staining after PPE15 peptides stimulation. Intravascular staining was used to distinguish parenchymal (IV-) from vascular (IV+) cells and combined with tetramer staining to identify PPE15-specific CD4+ and CD8+ TRM-phenotype in the lung parenchymal following vaccination. PPE15-specific serum antibodies were measured by ELISA. Protective efficacy was assessed four weeks after aerosol Mycobacterium tuberculosis ( M.tb ) challenge by lung and spleen CFU enumeration. Results Heterologous vaccination induced robust spleen CD4+ and CD8+ responses and PPE15-specific IgG. In the lung, mRNA.PPE15-ChAdOx1.PPE15 induced IFN-γ+ CD4+ and CD8+ T cells in the parenchyma and PPE15-specific TRM-like cells. Following M.tb challenge, both heterologous regimens reduced lung CFU compared to naïve controls, but only mRNA.PPE15-ChAdOx1.PPE15 significantly decreased CFU in both lungs and spleen. When used to boost BCG, BCG-mRNA.PPE15-ChAdOx1.PPE15 achieved 0.8 log10 CFU reductions in lungs and spleen compared to BCG control group and induced the greatest numbers of lung TRM-phenotype cells. Discussion A heterologous prime-pull strategy that combines intramuscular mRNA.PPE15 priming with intranasal ChAdOx1.PPE15 boosting effectively directs PPE15-specific T cells to the lung parenchyma, enriches TRM-like populations, and improves protection over homologous regimens. There was a trend for the mRNA.PPE15-ChAdOx1.PPE15 regimen to outperform the reverse order, particularly as a BCG booster. These data support heterologous platform vaccination and prime-pull strategy as a novel strategy for TB vaccines and indicate potential to progress to the next stages of vaccine development.