Impact of sequence variation in a dominant HLA-A*02-restricted epitope in hepatitis C virus on priming and cross-reactivity of CD8+ T cells

Abstract

CD8+ T cells are an essential component of successful adaptive immune responses against hepatitis C virus (HCV). A major obstacle to vaccine design against HCV is its inherent viral sequence diversity. Here, we test the hypothesis that different sequence variants of an immunodominant CD8+ T cell epitope, all binding with high affinity to HLA class I, target different T cell receptor repertoires and thereby influence the quality of the CD8+ T cell response. The impacts of sequence differences in the HLA-A*02-restricted HCV NS31406-1415 epitope on in vitro priming of naive CD8+ T cells from seronegative donors and cross-reactivity of primed T cells with other epitope variants were characterized. Although the six epitope variants tested were all high-affinity binders to HLA-A*02:01, substantial differences in priming and cross-reactivity of CD8+ T cells were observed. The variant associated with the most reproducible priming and induction of T cells with broad cross-reactivity was a genotype 1b variant (KLSALGLNAV) that is more common in HCV isolates collected in Asia but is rare in sequences from Europe and North America. The superior immunogenicity and cross-reactivity of this relatively rare epitope variant were confirmed by using HCV-specific memory CD8+ T cells from people who inject drugs, who are frequently exposed to HCV. Collectively, the data suggest that sequence differences at the epitope level between HCV isolates substantially impact CD8+ T cell priming and the degree of cross-reactivity with other epitope variants.

Importance: The results have important implications for vaccine design against highly variable pathogens and suggest that evidence-based selection of the vaccine antigen sequence may improve immunogenicity and T cell cross-reactivity. Cross-reactive CD8+ T cells are likely beneficial for immune control of transmitted viruses carrying epitope variants and for prevention of immune escape during acute infection. To this end, rare epitope variants and potentially even altered epitope sequences associated with priming of broadly cross-reactive T cell receptors should be considered for vaccine design and need further testing.

FIG 1

Peptide binding affinity for HLA-A*02:01. Variants of the NS3_1406–1415 epitope were analyzed for their specific binding to HLA-A*02:01 by a peptide-exchange assay. After induction of degradation of a conditional reference peptide in complex with HLA-A*02:01 by UV light, the peptide-exchange rate with selected ligands was determined by an ELISA that detects intact pHLA complexes. A high-affinity binder to HLA-A*02:01 (NLVPMVATV) and an HLA-A*01:01 ligand with no substantial affinity for HLA-A*02:01 (CTELKLSDY) served as positive (Pos) and negative (Neg) controls, respectively. “UV” indicates the ELISA results for pHLA complexes upon exposure to UV light in the absence of peptide. The absorbance of all peptides was evaluated relative to that of the high-affinity binder, which was set at 100%.

FIG 2

Different epitope variants show distinct priming capacities. Naive CD8⁺ T cells were primed with different epitope variants, as indicated, for 28 days. All experiments were started in duplicate with cells from 11 different donors. Results from the same donor are presented with the same symbol, as indicated in the key. (A) Cultures from donors D5 to D11 were analyzed by peptide-specific MHC class I multimers. (B) Cultures from donors D1 to D11 were analyzed by intracellular cytokine staining for IFN-γ. (C) Success rate of CD8⁺ T cell priming, defined as a minimum of 0.5% IFN-γ-secreting CD8⁺ T cells upon peptide restimulation. (D) Polyfunctionality (secretion of IFN-γ, TNF-α, IL-2, and MIP1β and degranulation) of CD8⁺ T cells from one donor primed with the gt1a_1 and gt1b_3 variants as determined by Boolean gating (FlowJo software). Pie charts were generated by using Pestle and SPICE software.

FIG 3

Primed peptide-specific CD8⁺ T cells show distinct cross-reactivity patterns. (A) Examples of cross-reactivity of CD8⁺ T cells primed with either gt1b_3 (left) or gt1b_AD (right) with all other epitope variants upon restimulation after 28 days. CD8⁺ T cells primed with gt1b_3 were cross-reactive with other genotype 1b epitope variants, whereas CD8⁺ T cells primed with gt1b_AD were not cross-reactive. (B) Summary of the cross-reactivity profiles from all priming experiments. The results of 9 to 11 independent experiments utilizing cells of different donors are shown.

FIG 4

Distinct T cell receptor Vβ repertoire of gt1b_3-primed CD8⁺ T cells. Naive CD8⁺ T cells (donor D8) were primed in parallel with gt1b_3 (A and B) or with gt1b_AD (C and D) for 28 days and restimulated with the peptides gt1b_3, gt1b_1, and gt1b_AD in two separate experiments (I and II). (A and C) The frequency of IFN-γ⁺ CD8⁺ T cells upon peptide restimulation was determined. (B and D) TCR Vβ⁺ usage of IFN-γ⁺ CD8⁺ T cells upon peptide restimulation was analyzed with a panel of 24 antibodies covering approximately 70% of the TCR Vβ repertoire.

FIG 5

NS3_1406–1415 variant-specific CD8⁺ T cell responses in 12 HLA-A*02-positive patients from a cohort of PWID. PBMCs from PWID were expanded in the presence of the epitope variant gt1a_1, gt1b_1, or gt1b_3, as indicated at the bottom of the graph. After 10 days, cultures were restimulated with all three epitope variants and analyzed by intracellular cytokine staining for IFN-γ. The percent frequencies of IFN-γ⁺ CD8⁺ T cells upon peptide restimulation are shown.

FIG 6

The epitope variant gt1b_3 is enriched in HCV genotype 1b isolates from Asia. All available HCV genotype 1b NS3 sequences were retrieved from the public HCV sequence database. Identical sequences and sequences of unknown origin were removed, and additional sequences obtained in Germany and China were added and aligned. A phylogenetic tree was calculated by utilizing the neighbor-joining method and the Jukes-Cantor distance model. Branches were transformed into a cladogram, and sequences from Japan and China are shown in red. Sequences carrying the epitope variant gt1b_3 are indicated by black dots.