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The potency of CD8⁺ cytotoxic T lymphocyte (CTL) responses toward core antigen has been shown to affect the outcomes of hepatitis B virus (HBV) infection. Since single‐chain trimers (SCT) composed of peptide epitope β₂‐microglobulin (β₂m) and major histocompatiblity complex (MHC) class I heavy chain covalently linked together in a single molecule have been shown to stimulate efficient CTL responses, we investigated the properties of human leucocyte antigen (HLA)‐A2 SCTs encoding the HBV core antigen (HBcAg) epitopes C₁₈₋₂₇ and C_107−115. Transfection of NIH‐3T3 cells with pcDNA3.0‐SCT‐C₁₈₋₂₇ and SCT‐C_107−115 leads to stable presentation of HBcAg epitopes at the cell surface. HLA‐A2.1/Kb transgenic mice vaccinated with the SCT constructs, either as a DNA vaccine alone or followed by a boost with recombinant vaccinia virus, were shown to generate HBcAg‐specific CTL responses …