Chapter 3: Results
3.3. HDV immune evasion
3.3.2. Amino acid substitutions and their impact on viral-specific CD8+ T cell response
Next, we asked whether the identified substitutions within HLA-B*27-restricted epitope (L-HD Ag99-108) would have an impact on the magnitude of HDV-specific CD8+ T cell response. To address this question, we performed a T cell assay using synthetic peptides from the identified HLA-B*27 epitope with all observed substitutions. For this assay HDV-specific T cell lines (driven from PBMCs of a patient with resolved HDV infection) were stimulated and the induction of HDV-specific CD8+ T cells to produce IFN-γ was evaluated. Amino acid analysis at this region indicated that the substitutions are divided into two groups: 1) the polymorphisms at position 100 (L-HD Ag100) which are apparently not associated with HLA-B*27 (RRDHRRRKAL, RQDHRRRKAL, REDHRRRKAL and RKDHRRRKAL) and 2) substitutions at residues 105 (R105K) and 106 (K106M) which are significantly associated with the presence of HLA-B*27 and seem to be selected under immune selection pressure restricted by HLA-B*27. Figure 3.15 demonstrates the impact of sequence polymorphism at second position of the epitope (L-HD Ag100) on the HDV-specific T cell response. As shown in Figure 3.15, the 4 observed amino acid residues (R, Q, K and E) at the second position of the novel HDV epitope do not have a significant impact on the induction of CD8+ T cells to produce IFN-γ; that is, regardless of which of these 4 amino acids (R, Q, K and E) are located at this position, the magnitude of the intra cellular IFN-γ response show no differences and would be between 0.50-0.59%. To investigate whether these substitutions at the 2nd residue of the epitope RRDHRRRKAL are present in previously studied genotype 1
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isolates or other genotypes (2 to 8), we performed a comprehensive analysis of all available sequences from HDV genotypes 1 to 8 with respect to this region.
Figure 3.15 Sequence variations at 2nd position of the identified HLA-B*27-restricted epitope (L-HD Ag99-108) and their impact on CD8+ T cell response.
The role of variations at the second position of HLA-B*27-restricted epitope was determined by re-stimulation of HDV-specific T cell lines with all 4 possible variants and compared with the negative control. The frequency of IFN-γ producing CD8+ T cells, determined by ICS, is indicated as percentage in each panel.
Table 3.7 demonstrates the most prevalent amino acid residues at L-HD Ag100 and indicates that, similar to our cohort, this position can accept any of the four amino acid residues (Q, R, K and E) regardless of the genotype. Nevertheless, the prevalence of these residues is not equal between different HDV genotypes. An analysis of 543 isolates at this region, from a local data base including all available HDV genotypes submitted in the GenBank as well as isolates from this study showed a combination of Glutamine (Q), Arginine (R), Glutamic acid (E), Lysine (K) and Glycine (G) in 57, 34, 6, 2 and 0.2% of isolates, respectively.
Table 3.7 Polymorphism of L-HD Ag in the region restricted to HLA-B*27 among all HDV genotypes (1 to 8) with respect to amino acid residue at L-HD Ag100.
98 99 100 101 102 103 104 105 106 107 108 109 110 111 112
67 Next, we addressed whether R105K and K106M substitutions, observed in HLA-B*27 positive patients, have an impact on the function of CD8+ T cells. To address this issue, we used synthetic peptides having these substitutions to stimulate PBMCs of a patient with resolved HDV infection (patient B) and compared with result of the same cells when they are stimulated with the “wild type” epitope (this patient had already reacted to the “wild type”
epitope, L-HD Ag99-108). As for the “wild type” epitope, we used both Arginine (R) and Glutamine (Q) at L-HD Ag100, RRDHRRRKAL and RQDHRRRKAL, respectively, since these two were the most prevalent residues compared to other two substitutions, Glutamic acid (E) and Lysine (K). Therefore, the corresponding mutant variants for R105K and K106M were synthetized for both “wild type” epitopes, RRDHRRRKAL and RQDHRRRKAL (Table 3.8).
Table 3.8 List of synthetic peptides corresponding to the identified HLA-B*27-restricted epitope with all observed substitutions.
# Position Sequence Length HLA Comment 1 HDV 99-108 RQDHRRKKAL 10 B*27 R100Q, R105K 2 HDV 99-108 RQDHRRRMAL 10 B*27 R100Q, K106M 3 HDV 99-108 RRDHRRKKAL 10 B*27 Q100R, R105K 4 HDV 99-108 RRDHRRRMAL 10 B*27 Q100R, K106M 5 HDV 99-108 RKDHRRRKAL 10 B*27 Q100K Wild Type 6 HDV 99-108 REDHRRRKAL 10 B*27 Q100E Wild Type 7 HDV 99-108 RRDHRRRKAL 10 B*27 Q100R Wild Type 8 HDV 99-108 RQDHRRRKAL 10 B*27 R100Q Wild Type
To test the functional impact of amino acid substitutions within this region on T cell response, the epitope-specific cell lines were stimulated using all possible combination of mutations in a same T cell assay. The results of this assay are demonstrated in Figure 3.16. Analysis confirmed that the “wild type” epitope (aa 99-108 RRDHRRRKAL or RQDHRRRKAL) is able to reproducibly induce CD8+ T cells to produce IFN-γ; however, the mutated variants (R105K and K106M) are not able to induce IFN-γ production when PBMCs are stimulated with these mutated epitopes (Figure 3.16). Taken together, strong statistical evidence as well as the experimental findings indicate a clear viral selection in HLA-B*27 positive patients for variants which can evade an effective immune response and probably supports a persistent HDV infection in those patients.
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Figure 3.16 Functional impact of HLA-B*27 selected variants on the HDV-specific CD8+ T cell response.
A 10-day antigen-specific expansion of the PBMCs in the presence of the prototype epitope was performed. Then the cultures were re-stimulated with the prototype or mutated epitopes and the number of IFNγ+/CD8+ T cells was
measured (A) RRDHRRRKAL (frequently seen in genotype 1 and other genotypes) was applied as the prototype along with the corresponding variants (R105K and K106M): RRDHRRKKAL and RRDHRRRMAL. (B) RQDHRRRKAL (prevalent sequence in this cohort) was used as the wild type epitope and the corresponding mutated epitopes at residues 105 and 106: RQDHRRKKAL and RQDHRRRMAL.
Next, we asked whether isolates from HLA-B*27 patients show more variations at the regions restricted by HLA-B*27 when we apply UDPS (deep sequencing) technology. Therefore, 5 isolates from HLA-B*27 positive patients were compared with 6 isolates from HLA-B*27 negative patients as control samples. The data from this study indicated that indeed, in addition to the mutations observed in conventional sequencing, there are minor variations within this region in the HLA-B*27 positive isolates which are absent in the control (HLA-B*27 negative) group at the same region. This may indicate additional HLA-B*27-restricted T cell pressure on other residues at lower level.
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