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HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines. Financial support: CNH was supported by the Deutsche Forschungsgemeinschaft (DFG; Emmy Noether Program, NE 1567/1-1). CNH and RT were supported by the European Union (EFRE Interreg IV Oberrhein, project A30). SV was supported by the German Ministry of Education and Research (BMBF, grant no. 01 KI 1008E). DL was supported by the Ministerio de Ciencia e Innovación, Spain. This project was funded, in part, by Federal funds from the National Institute of Allergy and Infectious Diseases (NIAID), grants R01-AI067926 (TMA) and U19 AI082630 (TMA and GML), and the Swiss HIV Cohort Study, supported by the Swiss National Science Foundation (grant #33CS30_134277). Sí

Methods: HBV-specific T cells were expanded in a 2-dimensional combinatorial expansion assay (67 different conditions from 11 factors) and the frequency of HBV-specific T cells was quantified by intra-cellular cytokine staining. Due to the exponentially increasing technical difficulties associated with analyzing multiple factors of T cell dysfunction, we coupled the expansion assay together with fluorescent cell barcoding to achieve a high throughput flow cytometric analysis. Results: By varying the concentration of an amine reactive dye, we were able to obtain 5 distinct fluorescent barcodes which were used to label different samples. This allows us to pool 5 samples into a single tube, reducing staining antibody consumption and the number of flow cytometer acquisitions to an operationally manageable quantity (from 70 to 14 tubes). The flow cytometric data of each tube was then deconvoluted into the individual samples based on the fluorescent barcodes and analyzed. Interestingly, preliminary results from 4 chronic HBV patients and 4 healthy subjects show that combinations which blocks apoptosis through the action of a pan-caspase inhibitor was able to preferentially enhance the in vitro expansion of exhausted HBV-specific T cells and not functional T cells specific for other viruses (CMV or FLU). Conclusion: The development of this assay makes it possible to simultaneously analyze combinations of multiple strategies to rescue exhausted HBV-specific T cells. Further in-depth analysis is ongoing to determine the ideal combinations to rescue exhausted HBV-specific T cells.