Enhanced Immunogenicity and Cross-Neutralizing Activity of a Trivalent Adenoviral Vector-Based SARS-CoV-2 Vaccine in Mice.

The emergence of diverse SARS-CoV-2 Omicron subvariants has reduced the efficacy of existing vaccines, highlighting the need for multivalent vaccines with broader neutralizing capacity. We developed a trivalent adenoviral vector-based vaccine (Ad5-CoV19-prototype/Delta/BF.7) encoding the spike proteins of the ancestral strain, Delta variant, and Omicron BF.7 strain, and evaluated its immunogenicity in BALB/c mice. Four- to 6-week-old female BALB/c mice were intramuscularly immunized with 10 µg of monovalent or trivalent vaccines using single-dose or two-dose (2-week interval) regimens. Neutralizing antibody (nAb) titers against the ancestral strain, Omicron BA.5, and XBB.1.5, as well as IFN-γ+ T-cell responses, were measured. The trivalent vaccine elicited significantly higher nAb titers against the ancestral strain (GMT 2183.6 at week 8) and Omicron subvariants (BA.5: GMT 680.0; XBB.1.5: GMT 161.3) after two doses compared to monovalent vaccines. Two doses also enhanced IFN-γ+ CD8+ T-cell responses (p < 0.0001). These results demonstrate that the trivalent vaccine induces enhanced immunogenicity and cross-neutralizing activity, including improved cross-neutralizing antibody titers and IFN-γ+ T-cell responses against multiple SARS-CoV-2 variants compared to monovalent formulations. These findings suggest potential for broader immune coverage against antigenically drifted variants, although protective efficacy remains to be confirmed in viral challenge models and further clinical evaluation.