New therapies are urgently required for the treatment of patients with melanoma. Here we describe the generation and preclinical evaluation of 3 new recombinant ALVAC(2) poxviruses vCP2264, vCP2291, and vCP2292 for their ability to induce the desired cellular immune responses against the encoded melanoma-associated antigens. This was done either in HLA-A2/K transgenic mice or using in vitro antigen-presentation studies. These studies demonstrated that the vaccine was able to induce HLA-A*0201-restricted T-cell responses against gp100 and NY-ESO-1, detectable directly ex vivo, in HLA-A2/K-transgenic mice. The in vitro antigen presentation studies, in the absence of appropriate animal models, demonstrated that target cells infected with the vaccine construct were lysed by MAGE-1, MAGE-3 or MART-1 peptide-specific T cells. These data indicate that ALVAC(2)-encoded melanoma-associated antigens can be properly processed and presented to induce antigen-specific cytotoxic T-cell responses. To enhance the immunogenicity of the melanoma antigens, a TRIad of COstimulatory Molecules (TRICOM) were also cloned into all 3 vectors. Increased in vitro proliferation and IFN-γ production was observed with all ALVAC(2) poxviruses encoding TRICOM, confirming the immune-enhancing effect of the ALVAC-encoded TRICOM. These studies demonstrated that all components of the vaccine were functionally active and provide a rationale for moving this candidate vaccine to the clinic.