The Papain-like Protease Domain of Severe Acute Respiratory Syndrome Coronavirus 2 Conjugated with Human Beta-Defensin 2 and Co1 Induces Mucosal and Systemic Immune Responses against the Virus

Most of the licensed vaccines against SARS-CoV-2 target spike proteins to induce viral neutralizing antibodies. However, currently prevalent SARS-CoV-2 variants contain many mutations, especially in their spike proteins. The development of vaccine antigens with conserved sequences that cross-react with variants of SARS-CoV-2 is needed to effectively defend against SARS-CoV-2 infection. Given that viral infection is initiated in the respiratory mucosa, strengthening the mucosal immune response would provide effective protection. We constructed a mucosal vaccine antigen using the papain-like protease (PLpro) domain of non-structural protein 3 of SARS-CoV-2. To potentiate the mucosal immune response, PLpro was combined with human beta-defensin 2, an antimicrobial peptide with mucosal immune adjuvant activity, and Co1, an M-cell-targeting ligand. Intranasal administration of the recombinant PLpro antigen conjugate into C57BL/6 and hACE2 knock-in (KI) mice induced antigen-specific T-cell and antibody responses with complement-dependent cytotoxic activity. Viral challenge experiments using the Wuhan and Delta strains of SARS-CoV-2 provided further evidence that immunized hACE2 KI mice were protected against viral challenge infections. Our study shows that PLpro is a useful candidate vaccine antigen against SARS-CoV-2 infection and that the inclusion of human beta-defensin 2 and Co1 in the recombinant construct may enhance the efficacy of the vaccine.