BACKGROUND: With SARS-CoV-2 continuing to evolve, there is a need to adapt COVID-19 vaccines to enhance mucosal immunity and better address immune-evasive variants. This pilot study was performed in mice and rhesus macaques to compare Advax-adjuvanted monovalent and bivalent recombinant spike protein vaccines, including when delivered via a combination of intramuscular (IM) and intrapulmonary (IPM) or oral routes. METHODS: Mice were first used to compare the immunogenicity of monovalent and bivalent vaccines containing a variety of spike protein variants. Then, rhesus macaques (n = 23) were divided into 5 groups to receive COVID-19 vaccines via different routes. Clinical signs, local vaccination site reactions, body weight, food consumption, serum, alveolar lavage, nasal and oral antibody levels, and nasal and alveolar lavage virus loads were assessed in response to a heterologous Omicron BA.5 virus challenge. RESULTS: The Wuhan + Mu bivalent vaccine gave the most broadly cross-neutralizing antibody responses. Robust serum neutralizing antibodies against Wuhan, Delta and Lambda variants were obtained, but BA.5 neutralizing antibodies were not detectable pre-challenge. Overall, the IM x3 and the IM x2 plus oral x2 vaccines delivered the best protection, with reduced lung virus load versus unimmunized controls across Days 2, 4 and 7. CONCLUSIONS: Advax-adjuvanted monovalent or bivalent recombinant spike protein vaccines given via parenteral and/or mucosal routes protected against a heterologous BA.5 challenge, despite absent serum BA.5 neutralizing antibody, pre-challenge. The possibility of using an oral Advax-adjuvanted protein booster to provide broad protection against newer SARS-CoV-2 variants warrants further investigation.