DC populations incubated with rAd5, rAd28, rAd35, TLR7/8-ligand, or unexposed

Recombinant adenovirus vectors (rAds) are being investigated as vaccine delivery vehicles in pre-clinical and clinical studies. rAds constructed from different serotypes differ in receptor usage, tropism, and ability to activate cells, aspects of which likely contribute to their different immunogenicity profiles. Here, we compared the infectivity and cell stimulatory capacity of rAds of serotype 5 (rAd5), 28 (rAd28) and 35 (rAd35) in association with their immunogenicity. We found that rAd28 and rAd35 infected, and led to the in vitro maturation and activation of both human and mouse dendritic cells (DCs) more efficiently than did rAd5. In stark contrast to rAd5, rAd28 and rAd35 induced production of interferon-alpha (IFNα) and stimulated interferon-related intracellular pathways. However, the in vivo immunogenicity of rAd28 and rAd35 was significantly lower than that of rAd5. Deletion of IFNα signaling during vaccination with rAd28 and rAd35 vectors increased the magnitude of the insert-specific T-cell response to levels induced by vaccination with rAd5 vector. The negative impact of IFNα signaling on the magnitude of the T cell response could be overcome by increasing the vaccine dose, which was also associated with greater polyfunctionality and a more favorable long-term memory phenotype of the CD8 T cell response in the presence of IFNα signaling. Taken together, our results demonstrate that rAd-induced IFNα production has multiple effects on T cell immunogenicity, the understanding of which should be considered in the design of rAd vaccine vectors. FACSAria-purified DC populations were incubated with rAd5, rAd28, rAd35, TLR7/8-ligand, or unexposed, for 24 hours. RNA samples from incubated myeloid DCs (mDCs; n=5 for Ad5, Ad35 and mock; n=4 for Ad28 and TLR7/8-ligand) and plasmacytoid DCs (pDCs; n=5 for Ad28; n=4 for mock; n=3 for Ad35 and TLR7/8-ligand; n=2 for Ad5) were prepared using the Illumina BeadStation assay and hybridized to Illumina RefSeq-8 V3 BeadChips.