Oxidative modifications of tuberculosis antigen Ag85B alter its T cell antigenicity

Mycobacterium tuberculosis (Mtb) remains a global health threat, necessitating innovative vaccine strategies that transcend traditional antigen delivery. While oxidative stress is a hallmark of the host-pathogen interface, the deliberate use of reactive oxygen and nitrogen species (RONS) to modulate antigen immunogenicity remains underexplored. We employed cold physical plasma, a potent source of diverse reactive species, to engineer oxidatively modified Ag85B (oxAg85B) variants. Using high-resolution mass spectrometry, we performed comprehensive oxidative protein modification mapping, identifying a landscape of 59 distinct oxPTMs. The functional impact on cellular immunity was evaluated using transgenic mice harboring Ag85B-specific CD4⁺ T-cells. Gas plasma treatment significantly enhanced the immunogenic profile of Ag85B. Compared to native controls, oxAg85B potentiated CD4⁺ T-cell activation and increased interferon-gamma secretion in an antigen-dependent manner. Systematic correlation analysis revealed that the "redox fingerprint" of the antigen was strictly dependent on the plasma operation mode. Notably, hydroxyl radical-rich plasma environments favored a pro-inflammatory Th17-linked profile. High-dimensional mapping showed that elevated IL-17α release strongly correlated with a specific cluster of modifications, including dihydroxylation, deamidation, and protein carbonylation. Crucially, vaccination with oxAg85B increased systemic inflammatory cytokines without altering anti-Ag85B antibody titers, suggesting a selective enhancement of cell-mediated immunity without compromising B-cell priming.