Human adenoviruses (HAdVs) cause diverse disease presentations as pathogens, and are also used as viral vectors for vaccines and gene therapy products. Preexisting adaptive immune responses to HAdV are known to influence symptom severity, viral clearance and the success of viral vectored products. Of note, approximately 50% of the UK's adult population has received at least one dose of a chimpanzee adenovirus vectored SARS-CoV-2 vaccine (ChAdOx1) since January 2021. We used FluoroSpot analysis to quantify the interferon gamma (IFNg) and interleukin-2 (IL2) responses of healthy blood donors to HAdV species A, B, C, D and F and chimpanzee adenovirus Y25, related to HAdV species E. We find that cellular immune responses to multiple species of human adenovirus are ubiquitous among healthy adult blood donors, and that stimulating PBMC with whole hexon peptide libraries induces a significantly greater IFNg and IL2 response than using selected peptide pools alone. We then compared the cellular immune responses of ChAdOx1 recipients and control donors using PBMC collected in 2021, and found that homotypic and heterotypic IFNg responses were significantly boosted in ChAdOx1 recipients but not controls. Finally, we show that in PBMC derived from blood donors, IFNg responses are made to both conserved and variable regions of the hexon protein. Future vaccination campaigns using adenoviral vectored vaccines will need to account for the pre-existing exposure of recipients to both circulating HAdVs and vaccines such as ChAdOx1, which convey polyfunctional antiviral T cell responses to even low seroprevalence HAdV types.

The authors have declared no competing interest.

This work was funded by a Royal Society research grant to CJH [RGS\R2\222009] and a Cambridge-Africa ALBORADA Trust grant to CNA and CJH. BACK was supported by a Wellcome award (225023/Z/22/Z). This work was supported by the Department of Genetics, University of Cambridge.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The UK HRA and Health and Care Research Wales (HCRW) (REC reference 22/WA/0162) gave ethical permission for the Understanding humoral and cellular immune responses to DNA viruses in healthy blood donors study. The Cambridge Human Biology Research Ethics Committee, UK, (HBREC.2014.07) gave ethical permission for the ARIA (Anti-viral Responses in Ageing, CBR53) study. All patients gave informed written consent in accordance with the Declaration of Helsinki

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