Comparative analysis of Ad5, mRNA, and protein vaccines reveals context-dependent immune responses

Despite the widespread use of adenovirus, mRNA, and protein-based vaccines during the COVID-19 pandemic, their relative immunological profiles and protective efficacies remain incompletely defined. Here, we compared antigen kinetics, innate and adaptive immune responses, and protective efficacy following Ad5, mRNA, and protein vaccination in mice. Ad5 induced the most sustained antigen expression, but mRNA induced the most potent interferon responses, associated with robust antigen presentation and costimulation. Unlike Ad5 vaccines, which were hindered by pre-existing vector immunity, mRNA vaccines retained efficacy after repeated use. As a single-dose regimen, Ad5 vaccines elicited superior immune responses. However, as a prime-boost regimen, and particularly in Ad5 seropositive mice, mRNA vaccines outperformed the other vaccine platforms. These findings highlight strengths of each vaccine platform and underscore the importance of vaccination context in determining optimal vaccine performance. C57BL/6 mice were immunized intramuscularly with each respective vaccine (Ad5, mRNA, and protein vaccines). At day 1 post-vaccination, lymph nodes from five mice were pooled and MACS-sorted with a CD45 MACS positive selection kit (STEMCELL) and used for single-cell sequencing. A naive group with no vaccination was used as control.