Figure 1.

Decreased recognition of the methionine variant (M_p5) of the HLA A*0201 restricted T-cell epitope NS3₁₄₀₆ is not due to loss of antigen processing and presentation or masking of low level responses. A. IFN-γ ELISpot was performed using PBMC obtained from Subject 28 one year following HCV infection when M_p5 was the dominant form of virus. Responses for PBMC incubated with the L_P5 peptide (circles), which was present upon initial viremia, and M_p5 peptide (triangles), which was dominant six months following infection demonstrate decreased recognition of the M_p5 variant. B. Constructs containing either the L_p5 or M_p5 sequence were introduced via electroporation into EBV transformed cells from Subject 28. Immunogenicity of the construct-containing EBV cells (Black bars labeled mRNA) was assessed via intracellular cytokine staining (ICS) for IFN-γ using NS3₁₄₀₆ specific-T cell lines generated from Subject 28. As a positive control, the same T cell line was tested for recognition of L_p5 and M_p5 peptides pulsed onto the surface of the autologous EBV transformed cells (Gray bars labeled peptide pulsed). There was no recognition of mock transfected EBV transformed cells (Black Control) or EBV transformed cells not pulsed with peptide (Grey Control). Recognition of the constructs as well as control constructs known not to represent processing mutations were similar to recognition when the peptides were pulsed onto the cell surface at a concentration of 0.001M, indicating that the M_p5 substitution did not prevent processing.