Francisella tularensis T-cell antigen identification using humanized HLA-DR4 transgenic mice

Abstract

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.

FIG. 1.

Fractionation of TX-114-extracted F. novicida proteins. Cell wall- and membrane-associated proteins partitioned in the TX-114 aqueous phase were fractionated (pI 3.7 to 9.3) with a Rotofor apparatus. The protein profiles of the Rotofor fractions separated by SDS-PAGE were visualized by Coomassie blue staining. Std., molecular mass standard.

FIG. 2.

Analysis of cell-mediated and humoral responses induced by Rotofor fractions. (A) Rotofor fractions 5 to 10 were used to stimulate spleen cells from ΔiglC (KKF24)-primed or mock (PBS)-treated HLA-DR4 tg mice using a cytokine recall assay; (B) sera were collected at day 28 to determine the antibody profiles for the respective pooled proteins.

FIG. 3.

Screening of respective whole-gel elution fractions that contain potential T-cell antigens. (A) Rotofor fraction 5 was fractionated into 14 subfractions (subfractions 1 to 14), and the protein bands were visualized by Coomassie blue staining. (B) Rotofor fractions 6 and 7 were subfractionated by whole-gel elution in a fashion identical to that described for Rotofor fraction 5. Subfractions of Rotofor fraction 5 (C) and subfraction 6.5 and 7.3 (D) were used to stimulate spleen cells from ΔiglC (KKF24)-primed or mock (PBS)-treated HLA-DR4 tg mice in an IFN-γ recall assay. Total TX-114-extracted proteins (TP) were used as a positive control for the cytokine recall assay. *, fractions selected for protein identification.

FIG. 4.

Diagrammatic representation of the sequence of in silico steps used to select putative CD4⁺ T-cell-reactive antigens.

FIG. 5.

Expression and isolation of recombinant FopB protein. Total protein was prepared from pET28a-FopB-transformed E. coli BL21(DE3) cells or from the pET28a vector with or without IPTG induction and was visualized by Coomassie blue staining. Also shown are the culture medium (Med) prepared from the IPTG-induced pET28a-FopB transformant and the rFopB purified by nickel-affinity chromatography.

FIG. 6.

FopB is a T-cell reactive antigen. CD4⁺ T cells were enriched from KKF24- or mock (PBS)-vaccinated HLA-DR4 tg mice and stimulated with increasing concentrations of rFopB in the presence of human APCs expressing HLA-DR4. The level of induction of IFN-γ was measured after stimulation for 72 h. Differences in IFN-γ stimulation between KKF24- and mock-vaccinated CD4⁺ T cells was significant at P values of <0.001 for rFopB (*) and <0.0001 for UV-inactivated KKF24 (**).

FIG. 7.

Intranasal immunization with rFopB-protected mice against pulmonary LVS challenge. C57BL/6 and HLA-DR4 tg mice (6/group) were i.n. or s.c. primed and boosted with rFopB. The mice were rested for 16 days after boosting, challenged i.n. with 35,000 CFU of LVS (∼5 LD₅₀s), and monitored for survival. LVS-challenged mock-vaccinated mice were used as controls. The difference in the rate of survival between i.n. rFopB-immunized and mock-vaccinated mice was significant at a P value of <0.05 in both strains of mice.

FIG. 8.

Immune response induced in HLA-DR4 tg mice by intranasal rFopB immunization. (A) Spleens were collected at 14 days postvaccination, and single cells were prepared and assayed for FopB- and LVS-induced IFN-γ production. Differences in IFN-γ stimulation between rFopB- and mock-vaccinated mice were significant at P values of <0.0001 for rFopB (**) and <0.01 for UV-LVS (*). (B) Sera were analyzed for their antibody profiles 14 days postboosting.