猴痘病毒mRNA疫苗的构建及免疫评价

Construction,expression,identification,and immune evaluation of a monkeypox virus mRNA vaccine

目的设计猴痘病毒(MPXV)抗原,选择多种方案设计抗猴痘病毒mRNA候选疫苗,并完成其表达鉴定及小鼠体内的免疫效果评价。方法选取猴痘病毒M1R、A35R、A29L、H3L基因设计三组候选抗原基因(命名为:M1RA35R、A29L-H3L-A35R、A35R-Fc),并设置一组痘苗病毒(VACV)基因(L1R-A33R)作为对照,分别连接至mRNA制备专用载体pGEM-3Zf-n3,通过线性化、体外转录、纯化和加帽获得功能性mRNA,利用Western blot、IFA对目的抗原进行鉴定。通过微流控芯片制备LNP-mRNAs疫苗,制定免疫程序并完成小鼠的免疫及特异性抗体水平监测。结果成功构建3组抗猴痘病毒的mRNA疫苗和1组抗痘苗病毒的mRNA疫苗,四组疫苗均能表达抗原蛋白,并且制备的4组LNP-mRNAs疫苗在免疫后84 d仍维持较高的特异性抗体水平。结论成功完成猴痘病毒mRNA疫苗的构建、表达及鉴定,并通过对不同猴痘病毒抗原设计的验证,为以后猴痘疫苗的研发提供数据支撑。

Objective Selects several options to design the mRNA vaccine against monkeypox virus,and completes its expression identification and evaluation of its immune effect in mice.Methods Monkeypox(Mpox)is a zoonotic viral disease caused by the monkeypox virus(MPXV),divided into type I and type II,the current outbreak of monkeypox outbreaks are mainly caused by type IIb,although the branch of the type II has a lower virulence,but do not rule out the possibility of mutation,and now there is no monkeypox vaccine for immunisation against monkeypox virus for the general population,leading to a general susceptibility of humans to the virus,resulting in monkeypox outbreaks of the This has led to a global outbreak of monkeypox.Therefore,from the perspective of antigen design,this paper selects several options to design the mRNA vaccine against monkeypox virus,and completes its expression identification and evaluation of its immune effect in mice.Results Three groups of mRNA vaccines against monkeypox virus and one group of mRNA vaccines against poxvirus were successfully constructed.All four groups of vaccines were able to express antigenic proteins,and the four groups of LNP-mRNAs vaccines were able to maintain a high level of specific antibodies at 84 d after immunisation.The LNP/mA29L-H3L-A35R vaccine group had the fastest specific antibody production time(day 28)and its A450 value was still greater than 2 on day 84,followed by the LNP/mA35R-Fc vaccine group,and lastly,among the three groups of monkeypox vaccine candidates,the one with the longest time of onset of action and the lowest A450 value was the LNP/mM1R-A35R vaccine group,but its A450 value was still greater than 1.Conclusion The construction,expression and identification of monkeypox virus mRNA vaccine were successfully completed,and the validation of different monkeypox virus antigen designs in this paper provides data support for the development of monkeypox vaccine in the future.