Immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector ex pressing two forms of the melanoma-associated MART-1/Melan-A antigen. The f irst recombinant virus expressed a minigene encoding a fusion product betwe en an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan -A protein. The capacity of HLA-A201 cells infected with either viral const ruct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T -lymphocytes (CTL), was comparatively characterized. The results obtained h erewith a tumor antigen confirmed the capacity of vaccinia virus-encoded ER -minigene to generate a very strong antigenic signal. In cytotoxicity assay s, recognition of target cells infected with high amounts of both recombina nt viruses with activated specific CTL clones, resulted in similar lytic ac tivity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10 - to 1000-fold stronger responses. Remarkably, the immunogenic difference b etween the two formulations, in their respective capacity to generate CTL f rom naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressi ng complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MAR T-1/Melan-A immunodominant epitope, non replicative vaccinia virus expressi ng ER-targeted minigenes appear to represent a significantly more immunogen ic epitope vaccine formulation.