Preparation and immunogenicity evaluation of monkeypox virus A29L protein, A29L neutralization epitope tandem protein and A29L mRNA vaccine
10.13200/j.cnki.cjb.004516
- VernacularTitle:猴痘病毒A29L蛋白、A29L中和表位串联蛋白、A29L mRNA疫苗的制备及免疫原性评价
- Author:
WU Peixuan
- Publication Type:Journal Article
- Keywords:
Monkeypox virus(MPXV);
A29L protein;
Neutralization epitope tandem;
mRNA vaccine;
Immunogenicity
- From:
Chinese Journal of Biologicals
2025;38(07):769-775+780
- CountryChina
- Language:Chinese
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Abstract:
Objective To prepare A29L protein, A29L neutralization epitope tandem protein(Neu6) and A29L-mRNA-lipid nanoparticles(LNPs) vaccine based on A29L antigen, a key neutralizing target of monkeypox virus(MPXV), and evaluate their immunogenicity, so as to provide experimental basis for the development of MPXV vaccine and antibody drugs.Methods The A29L and Neu6 proteins were expressed by E.coli prokaryotic system and purified. Female BALB/c mice were immunized at five sites by subcutaneous injection in the extremities and intraperitoneal injection with five mice in each group. After three times of immunization, blood samples were collected from posterior orbital venous plexus and serum was separated. The template sequence of A29L-mRNA was cloned into plasmid pBluescript Ⅱ SK(+), and the template plasmid pBlueScriptⅡSK(+)-A29L-mRNA was constructed. A29L-mRNA was synthesized by transcription in vitro and purified by capping. A29L-mRNA were encapsulated by INano~(TM)L nano-drug preparation system to prepare A29L-mRNA-LNPs, which wereinjectedintramuscularlyintothebackoffivefemale BALB/c mice. After three times of immunization, blood samples were collected from orbital venous plexus and serum was separated. The serum IgG titers were detected by indirect ELISA and the serum neutralizing antibody titers were detected by plaque reduction neutralization test(PRNT).Results A29L and Neu6proteins were obtained successfully, with the purity of more than 90%, which were expressed mainly in inclusion bodies and showed specific binding to mouse anti-A29L monoclonal antibodies. The particle size of A29L-mRNA-LNPs was uniform(71. 35 nm), the polymer dispersity index(PDI) was 0. 097, and the encapsulation efficiency was 93%. HEK-293T cells transfected with A29L-mRNA-LNPs in vitro effectively expressed A29L protein. The serum IgG antibody titers of mice in A29L and Neu6 groups were significantly higher than that of mRNA group(t = 5. 692 and 5. 774, respectively, each P < 0. 001),while the serum neutralizing antibody titer of Neu6 group was significantly higher than those of A29L and mRNA groups(t =5. 202 and 4. 475, respectively, each P < 0. 01).Conclusion The immunogenicity of A29L-mRNA-LNPs is weak, suggesting that the effect of single antigen mRNA vaccine is limited. A29L and Neu6 proteins expressed in prokaryotic cells have good immunogenicity, and Neu6 can significantly improve the neutralizing antibody level, indicating that multi-antigen combination strategy should be explored in the future, and Neu6 protein is expected to become a highly effective candidate immunogen for MPXV vaccine and neutralizing antibody development.
