Abstract 472: Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus vaccine vectors.

Cancer vaccines continue to be of global importance. In the HIV/SIV non-human primate models replicating adenovirus has shown promise as a vaccine vector. Priming with replicating recombinant host-range mutant adenovirus (Ad5hr) followed by envelope (Env) protein boosting, elicits strong cellular, humoral and mucosal immunity, and protection in non-human primates. Based on the results using Ad5hr in the HIV/SIV non-human primate models replicating Ad4 vaccines are being developed for HIV/AIDS, and Influenza. These vectors may also be of value for cancer. A limitation of replicating Ad vectors is the transgene carrying capacity which is about 3000 base pairs (bp). Using an Ad5hr encoding full-length single-chain HIVBal gp120 linked to rhesus CD4 D1D2 domains (rhFLSC; DeVico et al., PNAS 2007) we systematically deleted the genes encoding E4-orf1 to E4-orf4. The latter virus with a 1100 bp deletion expands the total carrying capacity to ∼4000 bp. Using plaque assays we were unable to determine any differences in viral progeny production that could be attributed to the deletions. We assessed the virus-host interactions by evaluating the cell cycle profile of infected cells over time. Here too no differences could be attributed to the deletions. The Ad replication cycle is divided into early and late phases with regard to viral DNA synthesis. Western blots confirmed that the parental and deleted viruses expressed similar transgene levels in a temporally regulated manner similar to Ad late proteins. Splenocytes and sera from mice vaccinated with the parental, the E4-deleted variants, or with an Ad5hr empty vector at 0 and 4 weeks were collected at week 6 and used to evaluate whether the deletions altered transgene or vector immunogenicity. Levels of Env-specific cytokines responses by T-cells were negligible for mice immunized with the empty vector yet positive for those immunized with the parental and E4-deleted viruses. Ad responses were similar in all 6 groups. Interestingly, while the 5 immunization groups yielded similar binding antibody titers against rhFLSC and gp120 the sera showed differential binding patterns for Ad antigens. Thus while little to no host-cell or cellular immunogenicity differences distinguish the E4 deleted viruses from the parent, the differential binding patterns for Ad antigens opens the possibility that the E4 gene products may influence Ad antigen recognition. These differences may prove useful in the design of replicating Ad vaccine vectors. Citation Format: Michael A. Thomas, Rui Song, Marjorie Robert-Guroff. Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus vaccine vectors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2013-472