Human CD4+ T cell epitopes from vaccinia virus induced by vaccination or infection

Abstract

Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4(+) T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4(+) T cell responses have been poorly characterized, and CD4(+) T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens.

Figure 1. Predicted Binding and Antigen Presentation Scores for Known HLA-DR1-Restricted T Cell Epitopes

(A–D) T cell epitopes for which the MHC-binding register has been definitively established. Filled circles (red) show 9-mer binding frames established by crystallographic analysis or truncation and alanine scanning mutagenesis, black dots indicate all possible 9-mer epitopes present in the entire proteins. (A–C) Binding frame identified by crystallographic analysis. (A) HA(306–318) from influenza virus heamagglutinin (A/New AYork/383/2004(H3N2)) [47]; (B) mutated TPI(23–37) from human triose phophate isomerase containing Ile instead of Thr at position 28 [77,78], with filled blue square indicating mutated peptide recognized as a tumor antigen; (C) PP16 p24(161–181) from HIV-1 strain NY-5 gag p24 protein; (D) TT(830–843) from tetanus toxin [46]; (E–H) T cell epitopes identified by overlapping peptides for which a small minimal peptide epitope has been characterized by truncation analysis. For each minimal peptide epitope, all of the possible 9-mer binding frames are shown as open circles, with the likely MHC binding frame, identified as a 9-residue sequence starting with a hydrophobic residue near the N-terminus of the peptide [23,38,70], indicated by a filled symbol. (E) N. meningitidis outer membrane porin A protein (91–108) [79]; (F) EBV nuclear antigen EBNA-1([78–88) [80]; (G) hepatitis C virus strain LIV23 polyprotein. Two epitopes have been characterized: NS4(1809–1817) and NS4(1879–1888) [81], shown in red circles and in blue squares, respectively. For the NS4 epitope, the minimal 10-mer peptide contains two likely binding frames, indicated by half-filled squares. (H) Dengue virus type 4 virus D4V capsid(84–92) [82]. (I–L) T cell epitopes identified by overlapping peptide analysis with larger minimal peptides. As in (E–H), presumptive binding frames are indicated by solid symbols with other potential epitopes from the same minimal peptide shown with open symbols. (I) HIV-1 (strain HXB2) p24 gag epitopes: AEWDRVHPVHAG(210–221) [83] in red circles; NKIVRMYSPTSI(271–282) [83] in blue squares, as in (G) two possible binding frames are indicated with half-filled squares; HIV-1 (strain NY5) p24 gag epitope PEVIPMFSALSEGATP(167–182) in green diamonds [84], for this epitope the binding frame is known exactly as shown in (C); PIVQNIQGQMVHQ(133–145) in magenta up-facing triangles; QEQIGMTNNPPIP(244–256) [83] in cyan down-facing triangles; KRWIILGLNKIVRMVSP(264–280) [85,86] in brown left-facing triangles; FRDYVDRFYTLRAEQAS(294–311) in aquamarine right-facing triangles [86,87]. (J) Mycobacterium leprae heat shock protein hsp65(61–75) [88]. (K) Human cytomegalovirus (strain AD169) pp65(115–127) [89], as above with two potential binding frames shown in half-filled symbols, (L), EBV BZLF-1 protein(198–210) [90,91].

Figure 2. Prediction of Vaccinia-Derived HLA-DR1-Binding Peptides and Experimental Validation of Binding Affinity

(A) HLA-DR1 predicted binding score and predicted antigen presentation score for each 9-mer potential peptide epitope are indicated by dots. Circles show peptides selected for further analysis (Table 1), filled circles indicate peptides for which T cell responses were observed. Inset shows high-scoring region. High-scoring peptides that were not included in the study because of synthetic difficulty are indicated by x. (B) Competition binding assay to evaluate MHC–peptide affinity. The ability of various concentrations of unlabelled MVA peptide to compete with biotinylated test HA peptide for binding to recombinant soluble HLA-DR1 was determined. Values shown (error bars) represent average (standard deviation) of three independent binding experiments. IC₅₀ values were determined by fitting to a competition binding equation. Peptides were grouped as indicated into seven pools based on observed IC₅₀ values.

Figure 3. TCLs Raised from Vaccinia Immune Donors Recognize Pools of Predicted HLA-DR1-Binding Peptides

(A) IFN-γ ELISPOT response of vaccinia-specific TCLs from DR1⁺ donors to pools of predicted peptides. TCL were generated from PBMCs obtained from a non-immunized (gray bars) or immunized (dark bars) DR1⁺ donor by stimulation with a crude preparation of heat-inactivated vaccinia virus. After 15 to 20 d of in vitro expansion, cells were tested in an IFN-γ ELISPOT assay using autologous PBMCs as APCs and the peptide pools as antigen. T cell assays were performed in cRPMI+10% human serum (see Materials and Methods). (B) Kinetics of the IFN-γ ELISPOT response in the immunized DR1 donor after a boosting immunization: shown are the T cell responses to the peptide pools for TCLs generated from blood samples obtained at the indicated time points. (C) Recognition of peptide pools by TCLs generated from PBMCs obtained from five non-DR1 donors: three immunized (SL101, SL107, and SL135), one accidentally infected with the vaccinia virus WR strain (SL136), and one non-immunized (LS 137).

Figure 4. Fine Specificity of TCLs Generated in Vaccinated and Infected Donors

Fine specificities were determined by IFN-γ ELISPOT using as antigen individual peptides included in a given pool. (A) Peptide pool 1; (B) peptide pool 2; (C) peptide pool 3; (D) peptide pool 4; (E) peptide pool 5; and (F) peptide pool 6. Background responses (wells in which peptide was not added) were on average less than 22 spots per 10⁶ cells (range 2 to 34).

Figure 5. Validation of the Prediction Algorithm and Recognition of Vaccinia Peptides That Fell outside the Boundaries of the High Score Region for Binding and Presentation

(A) HLA-DR1 predicted binding and predicted antigen presentation scores are shown for each 9-mer potential peptide epitope from the vaccinia genome, Open circles indicate peptides tested and closed red circles indicate peptides for which T cell responses were observed. This graph includes the 36 peptides presented in Figure 2 and the set of 53 peptides used to validate the prediction algorithm. Peptides are clustered according to their binding and presentation scores into groups H, P, B, and N. Only three peptides outside the H region are recognized by T cells. (B) IFN-γ ELISPOT responses of SL131, SL135, and SL136 TCLs to pools of peptides included in P, B, or N regions. (C) Deconvolution of the IFN-γ response of SL131 TCL day 0 (gray bars) and day 13 (solid bars) to positive pools.

Figure 6. Ex Vivo IFN-γ Response of T Cells in Peripheral Blood to Vaccinia Peptides

Response in PBMCs isolated from vaccinia-exposed donors and from non-exposed donors to pools of vaccinia peptides or individual peptides, measured by IFN-γ ELISPOT. (A) IFN-γ responses of PBMCs in an immunized donor (SL135) and in an infected donor (SL136). No spots were observed in control wells (cRPMI+10% HS, no peptide). (B) Kinetics of the IFN-γ response after a boosting immunization of donor SL131, a DR1 donor. Spots for control wells (cRPMI+10% HS, no peptide) day 7 4+/−1, day 13 1+/−1, and day 39 0+/−0 (C–F) IFN-γ responses in PBMCs after depletion of CD8⁺ T and overnight incubation in cRPMI+10% HS (gray bars) or incubation in cRPMI+10% HS supplemented with a crude preparation of heat-inactivated vaccinia virus (solid bars). (C, D) non-immunized donors SL139 and SL140. (E, F) vaccinia-immunized donors SL135 and SL136. Spots in control wells (Medium, no peptide) 25+/−20. Statistically significant responses (p <0.05) indicated by an asterisk.