Vaccine · 2026
Optimized flagellin enhances systemic and mucosal immune responses induced by SARS-CoV-2 virus-like particle vaccines.
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Song Li, He Guoyu, Cui Yaodan, Tan Ruimeng, Wang Yue, Zhang Hui, Qian Wen, Geng Shizhong, Pan Zhiming, Jiao Xinan
Аннотация
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.
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.