Differential escape of HCV from CD8+ T cell selection pressure between China and Germany depends on the presenting HLA class I molecule

Abstract

Adaptation of hepatitis C virus (HCV) to CD8⁺ T cell selection pressure is well described; however, it is unclear if HCV differentially adapts in different populations. Here, we studied HLA class I-associated viral sequence polymorphisms in HCV 1b isolates in a Chinese population and compared viral substitution patterns between Chinese and German populations. We identified three HLA class I-restricted epitopes in HCV NS3 with statistical support for selection pressure and found evidence for differential escape pathways between isolates from China and Germany depending on the HLA class I molecule. The substitution patterns particularly differed in the epitope VTLTHPITK_1635-1643 , which was presented by HLA-A*03 as well as HLA-A*11, two alleles with highly different frequencies in the two populations. In Germany, a substitution in position seven of the epitope was the most frequent substitution in the presence of HLA-A*03, functionally associated with immune escape and nearly absent in Chinese isolates. In contrast, the most frequent substitution in China was located at position two of the epitope and became the predominant consensus residue. Moreover, substitutions in position one of the epitope were significantly enriched in HLA-A*11-positive individuals in China and associated with different patterns of CD8⁺ T cell reactivity. Our study confirms the differential escape pathways selected by HCV that depended on different HLA class I alleles in Chinese and German populations, indicating that HCV differentially adapts to distinct HLA class I alleles in these populations. This result has important implications for vaccine design against highly variable and globally distributed pathogens, which may require matching antigen sequences to geographic regions for T cell-based vaccine strategies.

Figure 1

Phylogenetic tree of complete HCV NS3 sequences from China together with unrelated sequences and sequences from a single‐source outbreak from Germany. Sequences from China (red) predominantly form two separate clades suggesting a common ancestor. Sequences from the anti‐D cohort (blue) fall into three distinct clades. No clusters were formed within the German unrelated isolates (black)

Figure 2

Frequency of HLA‐A*11 and HLA‐A*03 associated sequence polymorphisms in the NS3_1635‐1643. A, The frequencies of variations from the consensus sequence in isolates from China (light grey) and Germany (dark grey) are shown. B and C, The frequency of variations from the consensus sequence in viral isolates from China from individuals carrying HLA‐A*11 (dark grey) and individuals lacking HLA‐A*11 (white) is shown for each position. In C, the frequency of variations from the amino acids isoleucine and threonine in position is shown. D and E, The frequency of variations from the consensus sequence in viral isolates from Germany from individuals carrying HLA‐A*03 (dark grey) and individuals lacking HLA‐A*03 (white) is shown for each position. In E, the frequency of variations from the amino acids isoleucine and threonine in position is shown. P‐values were calculated with a Fisher's exact test

Figure 3

Characterization of the correct length of the epitope NS3_1635‐1643 in the context of HLA‐A*11. HLA‐A*11 positive PBMCs were expanded for 10 days with the 9‐mer peptide VTLTHPITK and truncated 8‐mer variants and restimulated with different peptide variants as indicated. After intracellular cytokine staining, the frequency of IFN‐gamma positive CD8⁺ T cells was determined by flow cytometry and is shown in % of restimulated CD8⁺ T cells

Figure 4

The substitution I1641V in the epitope NS3_1635‐1643 is associated with functional immune escape in the context of HLA‐A*03. HLA‐A*03 positive PBMCs from three different donors were expanded with the peptide VTLTHPITK (German consensus) or VILTHPITK (Chinese consensus) for 10 days and then restimulated with different peptide variants as indicated. After intracellular cytokine staining, the frequency of IFN‐gamma positive CD8⁺ T cells was determined by flow cytometry and is shown in % of restimulated CD8 ⁺ T cells

Figure 5

Two different patterns of immune escape from HLA‐A*11‐associated selection pressure. HLA‐A*11 positive PBMCs from four different donors were expanded with the peptide VTLTHPITK (German consensus) or VILTHPITK (Chinese consensus) for 10 days and then restimulated with different peptide variants as indicated. After intracellular cytokine staining, the frequency of IFN‐gamma positive CD8⁺ T cells was determined by flow cytometry and is shown in % of restimulated CD8 ⁺ T cells. Two distinct patterns of CD8⁺ T cell reactivity were observed: A, In the first pattern, the response was predominantly directed against the German consensus (VTLTHPITK) and the Chinese consensus (VILTHPITK) as well as the V1635I variants were associated with lower CD8⁺ T cell responses. B and C, In the second pattern, the response was predominantly directed against the Chinese consensus (VILTHPITK), here, the V1635I variant was not associated with a lower response (B). In this pattern, rare substitutions in position two of the epitope were associated with lower CD8⁺ T cell responses (C)