Francisella tularensis T-Cell Antigen Identification Using Humanized HLA-DR4 Transgenic Mice

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-γ) 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-γ recall assay with purified CD4⁺ T cells from F. tularensis subsp. novicida ΔiglC-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.Francisella tularensis is a Gram-negative bacterium and the etiological agent of the zoonotic disease tularemia. F. tularensis is classified into four subspecies, namely, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida (F. novicida) on the basis of their biochemical and genetic profiles, virulence properties, and geographical origins (51). To this end, F. tularensis subsp. tularensis (type A) is the most virulent subspecies, with the inhalation of as few as 10 organisms causing disease and mortality rates of between 30 and 60% in untreated cases of pneumonic tularemia (53). The live vaccine strain (LVS) derived from F. tularensis subsp. holarctica has been used as a prophylactic vaccine against tularemia (48). Millions of individuals in the Soviet Union were immunized with live vaccine strains between 1946 and 1960 (52). However, LVS has not been licensed for use in the United States due to a lack of understanding of the genetic mutations that are responsible for attenuation of this strain, although it is used as an investigational new drug (IND) to immunize at-risk workers, primarily tularemia researchers. F. novicida, which causes disease only in immunocompromised humans but which is highly virulent for mice, has been used as a comparative model organism due to the high degree of genetic similarity with type A strains (98.1% homology between sequences common to strains U112 and SCHU S4 [45]). We recently reported that a defined vaccine strain (ΔiglB) generated in strain U112 was effective in inducing heterologous protection against various Francisella strains in a mouse model of pulmonary tularemia, suggesting the conservation of protective antigens (12).Cell-mediated immunity has been documented to play an important role in protection against tularemia (2, 18, 19, 49, 56). The role of antibodies, via neutralization and Fc receptor-mediated clearance (43, 44) in response to infection, has also gained significant attention. Therefore, the availability of a combination of multiple Francisella antigens containing T-cell and/or B-cell epitopes would be desirable for formulating an effective multivalent vaccine against this organism. However, the use of conventional mouse strains to identify protective antigens may not be feasible, given the differences in the major histocompatibility complex (MHC) binding properties between murine and human MHCs. These constraints can be overcome with the use of engineered humanized mice, such as the HLA-DR4 transgenic (tg) mouse. This mouse was generated to express the extracellular human α1 and β1 domains of the HLA-DRA and HLA-DRB1*0401 haplotypes, which form the peptide binding sites for antigen presentation, in conjunction with the murine α2 and β2 domains (29). These chimeric molecules have been shown to exhibit the same antigen-binding specificity as HLA-DRB1*0401 and to be functional in presenting antigens to T cells (29). The frequency of the HLA-DR allele in humans is 29% in Caucasian individuals, 10% in African American individuals, and 34% in other individuals (38), underscoring the translational value of the epitopes identified in these mice for humans. We recently demonstrated the feasibility of using the HLA-DR4 tg mouse for the identification of vaccine antigens against genital Chlamydia infection (39), demonstrating the value of the use of these animals in the rational selection of vaccine candidates.In this study, we utilized a robust biochemical membrane protein fractionation method, cytokine recall assays, and humanized HLA-DR4 tg mice to identify putative CD4⁺ T-cell-reactive antigens from U112. Moreover, using bioinformatics tools, we further validated one of the identified antigens (FTN_0119), designated Francisella outer membrane protein B (FopB), as a potential subunit vaccine candidate against pneumonic tularemia in HLA-DR4 tg mice.