Live versus killed Listeria monocytogenes immunization differentially programs Hup-specific CD4+ T cell responses with distinct protective outcomes.

The histone-like protein Hup is a highly conserved DNA-stabilizing and compacting protein found across bacterial families. We previously demonstrated that the Hup protein of Staphylococcus and Streptococcus species harbors a shared CD4+ T cell epitope, and that Hup77-88-specific CD4+ T cells confer cross-species protection against pulmonary infection with these bacteria. Here, we investigated whether exposure to the Gram-positive intracellular pathogen Listeria monocytogenes, that expresses an orthologous Hup protein, induces a Hup-specific CD4+ T cell response. Live L. monocytogenes infection in mice generated detectable but modest Hup78-89-specific CD4+ T cell responses. In contrast, immunization with heat-killed L. monocytogenes or infection with a listeriolysin O-deficient strain, induced robust Hup78-89-specific Th17 responses. Despite their expansion, heat-killed L. monocytogenes-induced Hup-specific CD4+ T cells did not protect against secondary L. monocytogenes challenge, consistent with the requirement for T helper 1 and CD8+ T cell immunity for anti-Listeria protection. However, these CD4+ T cells conferred protection against pulmonary infection with Staphylococcus aureus expressing the L. monocytogenes Hup epitope. These findings establish Hup as conserved CD4+ T cell antigen target across bacterial species and demonstrates that T cell polarization and pathogen lifecycle are critical considerations when designing pan-antibacterial vaccines targeting shared bacterial antigens.