Mucosal Delivery of Recombinant SARS-CoV-2 Spike Receptor-Binding Domain Antigen Containing Immune-Stimulating Peptides Induces Protective Immune Responses Against Viral Infection in huACE2 Mice.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through the interaction between the spike protein receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (hACE2) receptor, which is expressed on epithelial cells in various tissues, including the respiratory tract. Therefore, mucosal immunity in the respiratory tract plays a key role in protection against viral infection. Previously, we demonstrated that intranasal administration of antigens (Ags) conjugated with the M cell-targeting peptide Co4B enhances both mucosal and systemic immune responses. That conjugation with human β-defensin 2 (HBD2) increases neutralizing antibody (Ab) responses. Methods: A recombinant antigen conjugate incorporating both Co4B and HBD2 was designed to enhance immunogenicity. Its immunogenicity was evaluated in mice following intranasal immunization. Antigen-specific antibody responses were measured in serum and bronchoalveolar lavage fluid. T-cell responses were evaluated in lungs and spleens. Protective efficacy was assessed using SARS-CoV-2-susceptible hACE2 knock-in mice. Results: Ag-specific Ab levels increased in both serum and bronchoalveolar lavage fluid of mice immunized intranasally with the conjugate. Especially, T-cell responses were significantly enhanced in the lungs and spleens of immunized hACE2 knock-in mice. In challenge experiments, intranasal administration of the conjugate reduced viral load. Moreover, Siglec F was identified as a potential receptor for Co4B, a previously uncharacterized M cell-targeting ligand. Conclusions: A recombinant viral Ag containing Co4B and HBD2 induces virus-specific humoral and cellular immune responses. Although further optimization of the vaccine formulation and administration strategy is needed, this conjugate shows potential as a platform for improving mucosal and systemic immunity.