Abstract / Summary
Background: Continuous antigenic evolution of SARS-CoV-2 has reduced the effectiveness of vaccines based on a single ancestral strain, highlighting the need for multivalent vaccine strategies. We developed a trivalent whole-virion inactivated vaccine containing ancestral (ERAGEM), Delta, and Omicron BA.5 antigens and evaluated its immunogenicity and protective efficacy in K18-hACE2 transgenic mice. Methods: β-propiolactone-inactivated ERAGEM, Delta, and Omicron BA.5 antigens were combined at 1 µg each (3 µg total) with aluminium hydroxide and administered intraperitoneally on days 0 and 21. Variant-specific binding and neutralizing antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA) and microneutralization assays. On day 42, mice were challenged with ERAGEM, Delta, or Omicron BA.5. Survival, body weight, body temperature, infectious viral titers, and viral RNA loads were evaluated. Results: The trivalent vaccine induced binding and neutralizing antibodies against all three vaccine components, and titers rose after the booster dose. Neutralizing responses were highest against the BA.5 component. Vaccinated mice showed 100% survival following challenge with each vaccine-matched variant and maintained stable body weight and temperature throughout the observation period. Unvaccinated controls challenged with Omicron BA.5 showed no mortality, so protection against this component could be assessed only virologically. No infectious virus was detected in the lungs of vaccinated animals, while viral titers and RNA loads in the nasal turbinates were significantly reduced compared with unvaccinated controls. Conclusions: A trivalent whole-virion inactivated SARS-CoV-2 vaccine containing ancestral, Delta, and Omicron BA.5 antigens elicited variant-specific humoral immunity and protected mice against challenge with each vaccine-matched component: against lethal challenge for the ancestral and Delta components, and against viral burden for Omicron BA.5, which was not lethal in unvaccinated controls. These outcomes were obtained at a lower per-antigen dose than in our previously reported monovalent and bivalent formulations; because those were independent studies rather than contemporaneous comparators, this suggests a dose-sparing potential that warrants formal evaluation rather than demonstrating one. Cross-protection against variants not included in the formulation was not assessed.