Replication deficient (RD) adenoviruses (Ad) are the most widely administered viral vectors, with licensed SARS-CoV-2 vaccines using vectors derived from human Ad type 5 (Ad5) and 26 (Ad26), and chimpanzee Ad “ChAdOx1”. Ad vectored vaccines generate robust cellular and humoral immunity, against both the transgene-encoded protein and the Ad vector itself. It’s unclear how many times a single Ad vector can be re-administered before this anti-vector immunity impairs generation of the desired transgene-specific adaptive responses. Anti-vector immunity also arises from naturally acquired Ad infections. In the absence of anti-Ad5 immunity, Ad5 is a gold-standard vector with robust vaccine immunogenicity, however widespread Ad5 seroprevalence hampers its use for the global population.

We developed novel pseudotyped Ads as RD vectored vaccines encoding SARS-CoV-2 spike protein. These vectors exhibit fiber knob swaps from low seroprevalence Ads grafted onto an Ad5 backbone. We characterised innate immune responses following intramuscular administration in mice, in addition to spike-specific adaptive responses three weeks later. We also quantified the effects of anti-vector humoral immunity against these vectors in an in vitro transduction assay with serum co-culture. The pseudotyped vectors exhibit many desirable vaccine characteristics as the equivalent Ad5 vector, including CD4⁺ and CD8⁺ T cell responses against multiple spike epitopes. Importantly, Ad fiber knob pseudotyping can substantially circumvent the direct anti-vector, humoral immunity induced through natural Ad exposure and Ad vaccination. These data indicate the adenovirus fiber knob plays a substantial role in anti-vector immunity, and can be manipulated for evasion of such responses without hampering vaccine immunogenicity.