Immunogenic capacities of recombinant vaccinia virus expressing CD154 : effects on CTL priming

Recombinant poxviruses expressing immuno-modulatory molecules together with specific antigens might represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in clinical trials, that co-expression of costimulatory molecules (CD80 and CD86) could increase the immunogenic capacity of a recombinant Vaccinia virus (rVV) also encoding different tumor associated antigens. In order to further investigate the capacity of these vectors to provide ligands for different co-stimulatory pathways relevant in the generation of CD8+ T cell responses, we designed a recombinant virus expressing CD40 ligand (CD154rVV). This co-receptor, expressed on activated CD4+ T cells, upon binding CD40 expressed on antigen presenting cells (APC) has been reported to increase their antigen presentation and immunomodulatory capacities. To investigate the potency of CD154rVV in CTL generation, different types of infection were performed in cultures containing APC and CD8+ T cells. Phenotypic characterization of infected iDC showed that CD154rVV enhances their activation and maturation, measured by increased expression of CD83 and CD86, as compared to wild type Vaccinia virus as control. Cytokine gene expression was evaluated by quantitative real time PCR. As expected, control virus infection triggered cytokine gene expression in APC and T-cells. However, typical APC cytokines such as GM-CSF, TNF-α and IL-15 and, on the other hand, typical T cells cytokines such as IL-2 and IFN-γ seemed to be expressed to a higher extent in CD154rVV infected cultures. Furthermore, as a landmark of the CD40/CD154 pathway, IL-12p40 gene transcription in APC was exclusively induced by CD154rVV infection. The latter factor is also known to play a major role in CTL priming. Complementary, as expected, control virus infection triggered IL-6 gene expression, which is known to render tumor specific T cells refractory to T regulatory cell activity. Nevertheless, this expression could not be further enhanced by CD154rVV. Activation of specific CD8+ T cells was investigated by using the MART-1/Melan-A27-35 epitope as model antigen and monitored by tetramer staining and cytotoxicity assays. We found that increased numbers of specific cytotoxic CD8+ T cells were induced by the specific peptide in the presence of the CD154rVV activated APCs, as compared to control virus. Finally, CD154rVV was demonstrated to enhance T cell proliferation, mainly CD4+ T cell, in culture of infected APCs with autologous T cells. Taken together, these data indicate that functional CD154 expression from recombinant Vaccinia virus infected cells induces APC activation and maturation, thereby enhancing antigen specific CD8+ T cell generation. Such recombinant vector might help bypassing the requirement for activated helper cells, thus qualifying as a potentially relevant reagent in the generation of CD8+ T cell responses in cancer immunotherapy.