Preparation and immunogenicity evaluation of influenza virus chimeric recombinant hemagglutinin based on Bac-to-Bac baculovirus expression system
10.13200/j.cnki.cjb.004753
- VernacularTitle:基于Bac-to-Bac杆状病毒表达系统的流感病毒嵌合重组血凝素的制备及其免疫原性评价
- Author:
Junjun ZHOU
- Publication Type:Journal Article
- Keywords:
Influenza virus;
Chimeric hemagglutinin;
Immunogenicity;
Cross-protection
- From:
Chinese Journal of Biologicals
2026;39(08):897-907+914
- CountryChina
- Language:Chinese
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Abstract:
ObjectiveTo construct chimeric antigens by grafting the head domains of H5N1(A/Vietnam/1194/2004) and H7N9(A/Anhui/1/2013) onto the conserved hemagglutinin(HA) stalk region of H1N1(A/Victoria/4897/2022), and to systematically evaluate the immunogenicity and cross-protective efficacy of the antigens formulated with a nanoemulsion(NE) adjuvant in a BALB/c mouse model, so as to address the insufficient cross-protection caused by the diversity and antigenic variability of influenza virus subtypes.MethodsThe chimeric HA fragments amplified by PCR from pOET3-cH5/1 and pOET3-cH7/1 templates were cloned into the pFastBac1 vector and transformed into Trans1-T1 competent cells to generate recombinant donor plasmids.These plasmids were then introduced into DH10 Bac competent cells, and recombinant bacmids were obtained following blue-white screening and PCR verification.The Bac-to-Bac baculovirus expression system(BEVs) was employed to express these proteins in Sf-RVN cells.After purification via affinity chromatography, the purity,specificity, and conformation of the proteins were identified using SDS-PAGE, Western blot, and transmission electron microscopy(TEM).Female BALB/c mice were immunized via intramuscular injection in the hind limb using a sequential immunization strategy.Antibody responses were detected by ELISA and hemagglutination inhibition(HI) assay.Two weeks after the final immunization, the mice were challenged with H1N1, H5N1, and H7N9 viruses.Body weight change rate and survival rate were monitored continuously for 14 days to evaluate the cross-protective ability of the chimeric proteins.ResultsRecombinant bacmids were successfully constructed, and high-titer recombinant baculoviruses(rBVs) were obtained, with titers of 1.7×10~8 ivp/mL for rBV-cH5/1 and 3.4×10~8 ivp/mL for rBV-cH7/1.The protein purity exceeded90%; Western blot verified their antigenic specificity, and electron microscopy showed that HA formed trimeric aggregates.The immunological results demonstrated that in the sequential immunization plus adjuvant group, the geometric mean titers(GMTs) of IgG antibodies against H1N1, H5N1, and H7N9 viruses reached 8 977, 13 857, and 15 477, respectively.The HI antibody titers were 1∶40, 1∶160, and 1∶160, respectively.Furthermore, the neutralizing antibody titer specifically against H1N1 reached 718(P<0.001).After viral challenge, all immunized groups showed only slight body weight loss, with a survival rate of 100%, which was significantly superior to that of the PBS control group.ConclusionThis study successfully expressed conformationally correct chimeric HA proteins cH5/1 and cH7/1 using the BEVs.The sequential immunization strategy combining these proteins with NE adjuvant can elicit a broad-spectrum and robust humoral immune response,effectively mediating complete protection against three different subtypes of influenza viruses, which provides reliable candidate antigens and immunization regimens for the development of multivalent broad-spectrum influenza vaccines.
