呼吸道合胞病毒融合蛋白(RSV-F)mRNA疫苗设计及其在小鼠中细胞和体液免疫应答评估

Design of respiratory syncytial virus fusion protein(RSV-F)mRNA vaccines and their cellular and humoral immune responses in mice

目的开发候选的呼吸道合胞病毒融合蛋白(Respiratory syncytial virus fusion protein,RSV-F)信使核糖核酸(Messenger ribonucleic acid,mRNA)疫苗,初步评价在小鼠中的细胞和体液免疫应答。方法选择4个编码RSV A2毒株全长F蛋白的基因序列,在体外设计RSV_(A2)-F突变体(WT_(A2)-F、DS-CAV1-Tric、SC-DM和DS2)mRNA疫苗,利用免疫印迹法鉴定在人胚肾(HEK-293)细胞中的表达;在采用该4种mRNA疫苗和RSV-F疫苗免疫小鼠2剂次(间隔2周)后12d检测血清γ干扰素(IFN-γ)、白细胞介素4(IL-4)、特异性免疫球蛋白(IgG)抗体和中和抗体水平。结果4种RSV_(A2)F突变体mRNA疫苗均在HEK-293细胞中检测到表达。在小鼠免疫2剂次后,WT_(A2)-F mRNA疫苗组IFN-γ和IL-4水平显著高于RSV-F疫苗组;WT_(A2)-F mRNA疫苗组和RSV-F疫苗组均诱导特异性IgG且抗体水平无显著性差异,但前者诱导的中和抗体水平显著高于后者;post-F构象的WT_(A2)-F mRNA疫苗组与pre-F构象的mRNA疫苗组(DS-CAV1-Tric、SC-DM和DS2)均诱导特异性IgG抗体,但各mRNA疫苗组IFN-γ、IL-4和中和抗体水平均无显著性差异。结论RSV_(A2)F突变体mRNA疫苗在小鼠体内可诱导强烈的细胞和体液免疫应答,可作为潜在的RSV候选疫苗进一步研究。

Objective To develop candidate respiratory syncytial virus fusion protein(RSV-F)messenger ribonucleic acid(mRNA)vaccines and evaluate their cellular and humoral immune response in mice.Methods Based on four sequences encoding the full-length F protein of RSV A2 strain,we designed mRNA vaccines of RSV_(A2)-F mutants(WTaz-F,DS-CAV1-Tric,SC-DM,and DS2)in vitro and detected expression in human embryonic kidney(HEK-293)cells using Western blotting.We tested sera for interferon-gamma(IFN-),interleukin-4(IL-4),specific immunoglobulin G(IgG)antibody,and neutralizing antibody in mice 12 days after two doses(2 weeks apart)of the four mRNA vaccines and an RSF-V vaccine.Results Expression of each RSV_(A2)-F mRNA vaccine in HEK-293 cells was detected.Levels of IFN-and IL-4 in the WT_(A2)-F mRNA vaccine group were higher than those in the RSV-F vaccine group;the two vaccines could induce specific IgG with no significant difference in IgG levels,while the neutralizing antibody titer induced by WTAz-F mRNA vaccine was significantly higher than that induced by RSV-F vaccine.Both the post-F mRNA vaccine(WT_(A2)-F)and the pre-F mRNA vaccines(DS-CAV1-Tric,SC-DM,and DS2)induced IgG,but no significant difference was found in levels of IFN-,IL-4,and neutralizing antibodies.Conclusions RSV_(A2) F mutant mRNA vaccines induced strong cellular and humoral immune responses in mice,providing potential RSV vaccine candidates.