重组流感血凝素三聚体蛋白疫苗的制备及免疫原性评价

Preparation and immunogenicity evaluation of recombinant influenza hemagglutinin trimer vaccine

目的制备重组流感病毒血凝素(hemagglutinin,HA)三聚体蛋白疫苗并进行相关表征分析,研究重组HA三聚体在小鼠模型中的免疫原性。方法构建稳定表达HA三聚体蛋白的CHO细胞株,通过Western blot、单向免疫扩散试验、蛋白质粒径检测和N-糖基化位点分析对重组蛋白进行定性及定量分析。根据剂量、佐剂等处理条件的不同将BALB/c小鼠分为11个组,并进行一致的免疫程序。初次免疫后56 d检测血清中和抗体效价,以评价免疫原性。结果构建的CHO细胞株能够分泌表达HA三聚体。HA三聚体具备生物学活性能够在单向琼脂免疫扩散试验中形成沉淀环,蛋白质粒径大小约为18.79 nm,具有10个N-糖基化位点,包括高甘露糖型、复合糖型和杂合糖型;HA三聚体重组蛋白疫苗在2次免疫小鼠后,3.75μg剂量配伍RFH01佐剂与对照组单价疫苗原液15μg引起的中和抗体效价差异无统计学意义(P=0.4312,U=36),配伍佐剂组免疫后血清抗体效价均高于未加佐剂组,其中15μg HA三聚体+RFH01组效价最高,为1280。结论成功制备了流感病毒重组HA三聚体蛋白,其与RFH01佐剂配伍能够诱导BALB/c小鼠产生针对流感病毒的体液免疫应答,为流感病毒重组亚单位疫苗的研发提供了参考。

Objective To prepare a recombinant hemagglutinin trimer(HA-Tri)vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods A stable CHO cell line that could express HA-Tri was constructed.Western blot,single radial immunodiffusion,protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein.According to the different treatment conditions such as dosage and adjuvant,BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures.Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results The constructed CHO cells could secret and express HA-Tri proteins.The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion.The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected,including high mannose,complex glycoforms and heterozygous glycoforms.After prime-boost immunization,there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75μg of HA-Tri in combination with RFH01 adjuvant and 15μg of monovalent vaccine stock solution(P=0.4312,U=36).Serum antibody titers in the HA-Tri+RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant,and the highest titer was induced in the 15μg HA-Tri+RFH01 group,which was 1280.Conclusions The recombinant HA-Tri protein was successfully prepared.HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice,which would provide reference for the development of influenza virus recombinant subunit vaccines.