Heterologous immunization combining vector- and mRNA-based vaccine elicits both robust innate and adaptive immune responses

Heterologous vaccination regimens are increasingly recommended for their potential benefits, including reduced side effects and enhanced immunogenicity. In this study, we investigated the innate and adaptive immune responses elicited by heterologous prime-boost vaccination using adenoviral vector and mRNA vaccines against SARS-CoV-2 in a mouse model. Our findings show that heterologous vaccination induced superior serum neutralizing and binding antibody titers, as well as enhanced cellular immune responses against the Delta and Omicron (BA.5) variants, compared to homologous vaccination. Single-cell transcriptomic analysis at the injection site, conducted 16 hours post-vaccination, revealed that the lipid nanoparticle (LNP)-based mRNA vaccine—and even empty LNP injection—triggered significant immune cell infiltration, while adenoviral vector vaccination caused minimal changes in injection-site cell composition following the first dose. Notably, spike mRNA was predominantly expressed in fibroblasts and macrophages across both vaccine platforms, suggesting these cells’ role in local immune responses. Further analysis demonstrated that the adenoviral vector booster induced amplified immune responses, marked by increased transcriptional changes, particularly in stromal pro-inflammatory pathways. Heterologous vaccination (adenoviral prime, mRNA boost) further intensified these responses compared to homologous mRNA vaccination, indicating that adenoviral priming enhances inflammatory responses when followed by an mRNA boost. In summary, our results demonstrate that heterologous vaccination strategies elicit stronger innate and adaptive immune responses compared to homologous regimens, supporting their use as an effective approach for enhanced protection against variants. In this study, we evaluated the immunogenicity of SARS-CoV-2 spike adenoviral vector-based and LNP-mRNA vaccines, administered in both homologous and heterologous prime-boost regimens. Saline (phosphate-buffered saline; PBS) and empty lipid nanoparticle (LNP, formulated without the mRNA component) injections served as controls. To create a single-cell transcriptome atlas of injection-site responses, BALB/c mice were immunized intramuscularly with either mRNA or adenoviral vector vaccines, with a three-week interval between the prime and boost shots.