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Vaccine
. 2026 May 28:87:128767.
doi: 10.1016/j.vaccine.2026.128767. Online ahead of print.
Optimized flagellin enhances systemic and mucosal immune responses induced by SARS-CoV-2 virus-like particle vaccines
Li Song 1 , Guoyu He 1 , Yaodan Cui 1 , Ruimeng Tan 1 , Yue Wang 1 , Hui Zhang 1 , Wen Qian 1 , Shizhong Geng 1 , Zhiming Pan 2 , Xinan Jiao 3
Affiliations
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.
Keywords: Adjuvant; Mucosal vaccine; Optimized flagellin; SARS-CoV-2; Toll-like receptor 5; Virus-like particles.
. 2026 May 28:87:128767.
doi: 10.1016/j.vaccine.2026.128767. Online ahead of print.
Optimized flagellin enhances systemic and mucosal immune responses induced by SARS-CoV-2 virus-like particle vaccines
Li Song 1 , Guoyu He 1 , Yaodan Cui 1 , Ruimeng Tan 1 , Yue Wang 1 , Hui Zhang 1 , Wen Qian 1 , Shizhong Geng 1 , Zhiming Pan 2 , Xinan Jiao 3
Affiliations
- PMID: 42208338
- DOI: 10.1016/j.vaccine.2026.128767
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.
Keywords: Adjuvant; Mucosal vaccine; Optimized flagellin; SARS-CoV-2; Toll-like receptor 5; Virus-like particles.