Barry, Mary

Abstract Most adenovirus vaccines are replication-defective RD-Ads. In contrast, replication-competent adenovirus (RC-Ad) replicate antigen genes 10,000-fold in each cell to amplify immune responses. While RC-Ads vaccines are more potent than RD-Ad, they risk causing adenovirus infections in humans. We engineered “single-cycle” Ad (SC-Ad) vectors that still amplify transgenes, but that do not produce infectious progeny. RD-, SC-, and RC-Ads were tested by single immunization against influenza, Ebola virus, HIV-1, Zika virus, HCV, CMV, Clostridium difficile, and cancers in mice, hamsters, and macaques and antibody and T cell responses were measured. SC-Ad expressing GFP, influenza HA, or HIV gp140 generated antigens with 33 to 100-times less virus than RD-Ad. SC-Ad generated vaginal antibodies that persisted beyond 6 months after single intranasal immunization. Intramuscular injection of 1010 vp of SC-Ad6 expressing HIV-1 gp160 generated env antibodies in sera within 4 weeks of a single immunization in rhesus macaques. SC-Ad expressing a C. difficile toxin A and B immunogen generated toxin-binding antibody reciprocal titers above 105 that persisted in excess of a half of a year after a single immunization. Single immunization with SC-Ad protected vaccinated animals against toxin and against challenge with C. difficile spores. When SC-Ads were used to amplify the expression of immune stimulatory proteins in the context of cancer, single intratumoral injection of SC-Ad expressing 4-1BBL or GMCSF mediated significant reductions in tumor growth and extension of survival without the need for exogenous cancer antigen expression. These data suggest that SC-Ads may have utility against infectious diseases and cancer.

Oncolytic viruses would ideally be of use for systemic therapy to treat disseminated cancer. To do this safely, this may require multiple layers of cancer specificity. The pharmacology and specificity of oncolytic adenoviruses can be modified by (1) physical retargeting, (2) physical detargeting, (3) chemical shielding, or (4) by modifying the ability of viral early gene products to selectively activate in cancer versus normal cells. We explored the utility of these approaches with oncolytic adenovirus serotype 5 (Ad5) in immunocompetent Syrian hamsters bearing subcutaneous HaK tumors. After a single intravenous injection to reach the distant tumors, the physically hepatocyte-detargeted virus Ad5-hexon-BAP was more effective than conditionally replicating Ad5-dl1101/07 with mutations in its E1A protein. When these control or Ad5 treated animals were treated a second time by intratumoral injection, prior exposure to Ad5 did not affect tumor growth, suggesting that anti-Ad immunity neither prevented treatment nor amplified anti-tumor immune responses. Ad5-dl1101/07 was next chemically shielded with polyethylene glycol (PEG). While 5 kDa of PEG blunted pro-inflammatory IL-6 production induced by Ad5-dl1101/07, this shielding reduced Ad oncolytic activity.