Identification of overlapping MHC-bound peptides presented by Mycobacterium tuberculosis and Mycobacterium bovis BCG infected cells using immunopeptidomics

Background Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis (TB) is the largest cause of death by an infectious disease worldwide. According to the latest World Health Organization report, 1.6 million people died of TB in 2021. With the emergence of drug-resistant strains, as well as co-infection with HIV, new tools to control this epidemic are urgently needed. The currently available vaccine against TB is the live-attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG), which has variable efficacy throughout the world. Even though both strains are highly genetically conserved, the DNA Region of Difference (RD1) is present in all M.tb strains but absent in BCG. Moreover, the immune response against TB relies on CD4+ T-cells, and to some extent CD8+ T-cells, therefore protective vaccines require the induction of antigen-specific T-cells through peptides presented by MHC-II and MHC-I, respectively. The identification of the peptides presented at the immunological synapse is key for understanding the immune response against TB. Methods Recent advances in immunopeptidomics, based on improvements in mass spectrometry instrumentation and data analysis, have led to the precise identification of MHC-bound peptides. Using a robust protocol of immunopeptidomics, we have identified peptides presented by MHC-I and MHC-II in BCG and M.tbinfected human macrophages, and developed nucleic acid based vaccines expressing antigens common to both strains. Results Here we report the identification of overlapping peptides presented by BCG and M.tb-infected cells. Interestingly, non-overlapping peptides including one RD1 sequence, were identified in M.tb but not BCG-infected cells, as expected. We report the efficacy and immunogenicity of vaccines expressing BCG and M.tb antigens in a murine aerosol M.tb challenge model. Conclusions This is the first report of the identification of MHC-bound peptides presented by BCG and M.tb infected human macrophages, which is of great relevance for the development of more effective vaccines against TB.