基于甲型流感病毒H1N1亚型血凝素的mRNA疫苗制备和加强免疫策略研究

Construction of an mRNA vaccine encoding hemagglutinin of influenza A H1N1 virus and investigation on booster immunization strategy

目的制备甲型流感病毒H1N1亚型血凝素(hemagglutinin,HA)mRNA疫苗,并探讨不同加强免疫策略的免疫保护作用。方法以荧光素酶(firefly luciferase,Fluc)为报告基因,构建Fluc mRNA-脂质纳米颗粒(lipid nanoparticle,LNP)疫苗,通过小鼠活体成像实验鉴定Fluc mRNA-LNP疫苗肌内注射后在体内的表达情况。进一步构建H1N1亚型(A/Michigan/45/2015)HA(M15-HA)的mRNA-LNP疫苗,将20、10、5和1μg M15-HA mRNA-LNP疫苗通过肌内注射分别免疫不同剂量组小鼠2次(间隔3周),酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定小鼠第2次免疫2周和4周后血清抗体滴度,血凝抑制试验检测功能性抗体水平。第2次免疫后40 d,采用1μg mRNA疫苗或10μg HA蛋白亚单位疫苗对1μg剂量免疫组小鼠进行加强免疫,接种2周和4周后用ELISA和血凝抑制试验分别检测特异性抗体及功能性抗体水平。结果活体成像实验结果显示,小鼠接种Fluc mRNA-LNP疫苗1 d后即能在小鼠体内检测到荧光素酶活性。制备获得的M15-HA mRNA-LNP疫苗2次免疫小鼠2周和4周后,所有剂量组小鼠的特异性抗体水平均较免疫前显著上升(均P=0.000);血凝抑制试验结果显示,20μg和10μg剂量组的功能性抗体水平均较PBS对照组显著升高(均P<0.05)。对1μg低剂量组小鼠进行HA蛋白或M15-HA mRNA-LNP的加强免疫后,均诱导产生了更高水平的特异性抗体和功能性抗体,并能维持较长时间;2种不同加强免疫策略之间差异无统计学意义。结论成功制备M15-HA mRNA-LNP疫苗,显示出良好的免疫原性和抗体中和活性;低剂量mRNA疫苗免疫2次后,同源mRNA疫苗和异源蛋白疫苗加强免疫均可以诱导更强的免疫反应。

Objective To construct an mRNA vaccine encoding hemagglutinin(HA)of influenza A H1N1 virus,and explore the protective effects of different booster vaccination strategies.Methods Firefly luciferase(Fluc)was used as the reporter gene to construct Fluc mRNA vaccine enveloped in lipid nanoparticles(LNP).The in vivo expression of Fluc mRNA-LNP after intramuscular injection was determined by live imaging assay in mice.Furthermore,M15-HA mRNA-LNP derived from H1N1 subtype(A/Michigan/45/2015)was constructed.Mice were immunized with 20,10,5,or 1μg doses of M15-HA mRNA-LNP twice(with an interval of 3 weeks)through intramuscular injection.Serum antibody titers were measured by enzyme-linked immunosorbent assay(ELISA)at 2 weeks and 4 weeks after the second immunization,and functional antibody levels were detected by hemagglutination inhibition test.The third booster vaccination was performed 40 d after the second immunization in 1μg dose group with 1μg M15-HA mRNA-LNP or 10μg HA subunit vaccine.The levels of specific antibody and functional antibody were detected by ELISA and hemagglutination inhibition test,respectively 2 weeks and 4 weeks later.Results Live imaging assay showed that luciferase activity could be detected in mice 1 d after injection of Fluc mRNA-LNP.At 2 weeks and 4 weeks after the second immunization of M15-HA mRNA-LNP,HA-specific antibodies were significantly higher than those before the immunization in all vaccination groups at different doses(P=0.000).The hemagglutination inhibition test showed that the levels of functional antibodies in the 20μg dose and 10μg dose groups were significantly higher than those in the PBS control group(P<0.05).After 1μg dose group mice were immunized with HA protein or M15-HA mRNA-LNP,higher levels of HA-specific antibody and functional antibody were induced and maintained for a long time.There was no significant difference between the two different booster immunization strategies.Conclusion M15-HA mRNA-LNP vaccine is constructed with immunogenicity and antibody neutralization activity.Low-dose mRNA priming vaccination followed by both homologous mRNA vaccine and heterologous protein subunit vaccine booster vaccination can induce stronger immune recall responses.