There is no licensed vaccine against the intracellular pathogen Francisella tularensis. The use of conventional mouse strains to screen protective vaccine antigens may be problematic, given the differences in the major histocompatibility complex (MHC) binding properties between murine and human antigen-presenting cells. We used engineered humanized mice that lack endogenous MHC class II alleles but that express a human HLA allele (HLA-DR4 transgenic [tg] mice) to identify potential subunit vaccine candidates. Specifically, we applied a biochemical and immunological screening approach with bioinformatics to select putative F. tularensis subsp. novicida T-cell-reactive antigens using humanized HLA-DR4 tg mice. Cell wall- and membrane-associated proteins were extracted with Triton X-114 detergent and were separated by fractionation with a Rotofor apparatus and whole-gel elution. A series of proteins were identified from fractions that stimulated antigen-specific gamma interferon (IFN-gamma) production, and these were further downselected by the use of bioinformatics and HLA-DR4 binding algorithms. We further examined the validity of this combinatorial approach with one of the identified proteins, a 19-kDa Francisella tularensis outer membrane protein (designated Francisella outer membrane protein B [FopB]; FTN_0119). FopB was shown to be a T-cell antigen by a specific IFN-gamma recall assay with purified CD4(+) T cells from F. tularensis subsp. novicida DeltaiglC-primed HLA-DR4 tg mice and cells of a human B-cell line expressing HLA-DR4 (DRB1*0401) functioning as antigen-presenting cells. Intranasal immunization of HLA-DR4 tg mice with the single antigen FopB conferred significant protection against lethal pulmonary challenge with an F. tularensis subsp. holarctica live vaccine strain. These results demonstrate the value of combining functional biochemical and immunological screening with humanized HLA-DR4 tg mice to map HLA-DR4-restricted Francisella CD4(+) T-cell epitopes.