Recombinant vaccinia viruses (rVACV) expressing various tumor-associated antigens have been shown to elicit anti-tumor effect in numerous experimental models and clinical trials. We tested the hypotheses that rVACV expressing biologically active fms-like tyrosine kinase 3 ligand (Flt3L) would show higher immunogenicity than control viruses expressing only model antigen and that coexpression of Flt3L would influence anti-tumor activity of rVACV in the preventive and therapeutic arrangements of the in vivo experiment. To answer these questions, we took advantage of the well-described model of transplanted tumor cells expressing HPV16 E6 and E7 oncoproteins. To determine the effects of hFlt3L on the induction of anti-tumor immunity, we generated live vaccinia viruses that express human Flt3L regulated by the early H5 or strong synthetic E/L promoter together with fusion protein SigE7LAMP, which is a highly immunogenic form of HPV E7 oncoprotein. We tested Flt3L production in vitro and in vivo. Despite higher expression of Flt3L from the synthetic E/L promoter in vitro, the P13-E/L-FL-SigE7LAMP induced lower levels of Flt3L in the serum of mice than P13-H5-FL-SigE7LAMP. The Flt3L expression under the strong early VACV H5 promoter is able to inhibit expansion of CD11b+Gr-1+ myeloid suppressor cells (MSC) and increase the amount of CD11b+ CD11c+ dendritic cells in the spleen of mice immunized with vaccinia virus. Determination of viral DNA isolated from the ovaries of infected animals did not reveal differences in replication between rVACVs in this organ. Coexpression of Flt3L by replication-competent virus P13-FL-SigE7LAMP induced enhancement of the cellular immune response against HPV16 E7 and VACV E3 proteins as well as increased anti-tumor efficacy in both the protective and therapeutic immunization schemes. On the other hand, the short-time Flt3L coexpression by MVA-H5-FL-SigE7LAMP was not sufficient to enhance anti-tumor effect of immunization.

Institute of Hematology and Blood Transfusion Department of experimental virology CZ 128 20 Prague 2 Czech Republic

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