Immunopeptidomics reveals conserved and abundant Francisella tularensis proteins as immunogenic CD4 T cell antigens in mice.

Human tularemia is caused by infection with the intracellular bacterium Francisella tularensis. The ability of the bacterium to cause a severe pneumonic form of the disease has led to concerns in the field of biodefense and efforts to develop a vaccine. F. tularensis LVS provides partial protection against fully virulent strains, with CD4+ T cells playing a significant role in this protection, however the recognized bacterial antigens remain largely unknown. To identify these antigens, we screened MHC class II peptides presented by infected dendritic cells using mass spectrometry-based immunopeptidomics. We identified 57 MHC-II peptides from 23 different F. tularensis LVS proteins. Most of these proteins were highly expressed in the bacterial proteome. Subsequent screening for IFN-γ responses identified ten peptides as immunogenic epitopes of CD4+ T cells following immunization of mice with F. tularensis LVS, thereby revealing previously unknown specificity and breadth of the induced immune responses. Five epitopes, derived from the antigens FTL_1678, DacD, DnaK, FTL_1498, and MinD, represented the dominant epitopes that elicited a response in at least 80% of immunizations. Finally, a peptide vaccine containing these epitopes proved to be immunogenic in mice and contributed to the early control of bacterial replication following challenge with the virulent F. tularensis FSC200 strain, although this alone was insufficient to ensure complete protection. Using immunopeptidomics, we have thus identified several immunogenic CD4+ T cell epitopes and antigens of F. tularensis LVS. Beyond their potential role in the protective immune response, the identified epitopes may also enable the development of tools for characterizing antigen-specific CD4+ T cells in vaccination studies or T cell biology research.