Optimized flagellin enhances systemic and mucosal immune responses induced by SARS-CoV-2 virus-like particle vaccines.

BACKGROUND

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutates continuously, and current injectable vaccines induce limited mucosal immunity, while clinical subunit mucosal vaccines and safe adjuvants are lacking. Virus-like particles (VLPs) are promising vaccine candidates, and flagellin is a potential TLR5 agonist adjuvant, yet its high intrinsic antigenicity remains a challenge.

METHODS

SARS-CoV-2 S, E, M proteins were expressed in Sf9 cells via the baculovirus expression system to prepare self-assembled VLPs. Full-length Salmonella flagellin (FliC) and its truncated variant NCFliC (ΔD2/D3) were expressed and purified. TLR5 ligand activity of FliC/NCFliC was verified in HEK293-mTLR5 cells. BALB/c mice were intranasally immunised with VLP alone or combined with FliC/NCFliC. The humoral, mucosal and cellular immune responses were evaluated by ELISA, splenocyte proliferation assay and cytokine detection assays.

RESULTS

The prepared VLPs (≈80 nm) exhibited native structure and specific ACE2-binding activity. FliC and NCFliC both retained robust TLR5 ligand activity, activating NF-κB and promoting IL-8 secretion. Intranasal immunization showed NCFliC and FliC both significantly enhanced VLP-specific humoral immunity (IgG), Th1-type responses (IgG2a/IFN-γ), mucosal IgA (nasal/bronchoalveolar lavage fluid) and splenocyte proliferation, with no significant difference in adjuvant efficacy between them. Notably, compared with FliC, NCFliC induced significantly lower levels of FliC-specific serum IgG, IgG subclasses (IgG1, IgG2a), and IgA in nasal and bronchoalveolar lavage fluid.

CONCLUSIONS

NCFliC retains potent adjuvant activity while having reduced intrinsic immunogenicity, and intranasal immunization with VLP + NCFliC elicits robust systemic and mucosal immunity in mice. NCFliC is a promising mucosal adjuvant for SARS-CoV-2 VLP vaccines, providing a potential strategy for developing next-generation anti-coronavirus mucosal vaccines.