Mapping of the M72 Vaccine Identifies Conserved Immunodominant PPE18 T Cell Recognition Sequences Across Mycobacterium tuberculosis Lineages

Abstract Although M72/AS01 E induces durable, multifunctional CD4 T cell responses, the specific epitopes driving vaccine-induced immunity remain undefined. Defining these targets is essential for understanding protective mechanisms and identifying immune correlates of protection. We mapped T cell recognition across the M72 fusion protein using 178 overlapping 15-mer peptides. We stimulated PBMC collected before vaccination (Day 0) and after a two-dose regimen (Day 37) from Phase 2b trial participants with peptide pools and individual peptides, and quantified IFNg and IL-17 responses by ELISpot. Vaccine-responsive peptides were identified by comparing paired responses in vaccine and placebo recipients. Epitope conservation was assessed using whole-genome sequences from approximately 34,000 clinical Mycobacterium tuberculosis isolates, and HLA class II binding was predicted using NetMHCIIpan 4.1. Peptide deconvolution revealed that immunogenicity was concentrated within PPE18, with five PPE18-derived peptides demonstrating vaccine-associated boosting exclusively in vaccine recipients. These sequences were highly conserved across major M. tuberculosis lineages and shared substantial homology with PPE19 and PPE60, suggesting potential cross-reactivity. Multiple HLA class II alleles were predicted to present the five vaccine-responsive peptides. Our findings identify conserved PPE18 epitopes targeted by M72/AS01 E -induced T cell responses and provide a foundation for investigating their contribution to vaccine-induced immunity and developing epitope-specific correlates of protection against TB.