基于登革病毒prME序列mRNA候选疫苗的构建与评价

Construction and evaluation of a dengue mRNA vaccine candidate based on the prME sequence of dengue virus type 1

目的利用mRNA结合脂质纳米颗粒(lipid nanoparticle,LNP)包裹递送技术,设计构建mRNA疫苗,以期为登革热疫苗研发提供新思路。方法以DENV1 Hawaii株prME序列为靶基因,体外合成D1ME-mRNA,利用流式细胞术及免疫荧光染色鉴定其在细胞内表达后,用LNP包裹mRNA,制备D1ME mRNA-LNP候选疫苗;利用C57BL/6小鼠模型,肌肉注射10μg进行二剂次免疫接种,评价疫苗的保护作用及免疫原性。结果流式细胞术及免疫荧光检测证实D1ME-mRNA在细胞内高效表达;经颅内注射DENV1攻毒后,疫苗组小鼠体重下降幅度明显小于对照组,且无明显临床症状;疫苗组小鼠的中和抗体平均效价为1∶20,高于对照组;疫苗组小鼠的脾淋巴细胞经特异性抗原刺激可分泌高水平的Th1细胞相关的细胞因子IFN-γ、TNF-α。结论本研究成功构建了针对DENV1的mRNA-LNP疫苗,二剂次免疫接种可诱导小鼠产生DENV1特异性的、以Th1型细胞免疫为主的免疫应答,并可为小鼠提供保护作用以抵抗DENV1的感染。

Dengue virus(DENV),the pathogen causing dengue fever,is responsible for one of the fastest-growing disease burdens according to the WHO.At present,no specific therapeutics are available for dengue fever and the development of safe and effective dengue vaccines is urgently needed.The structural proteins of DENV precursor membrane protein(prM)and envelope protein(E)include B-cell and T-cell epitopes,and are the main target antigens of dengue vaccines.In this study,we designed and constructed an mRNA vaccine with lipid nanoparticle(LNP)encapsulation and delivery technology,aiming to provide new ideas for the development of dengue vaccines.The prME sequence of the DENV1 Hawaii strain was selected as the target gene,and D1ME-mRNA was synthesized in vitro.After expression analysis,the mRNA was encapsulated with LNP to manufacture the D1ME mRNA-LNP vaccine candidate.A prime-boost vaccination with two doses of 10μg was delivered intramuscularly into C57BL/6 model mice.Immunogenicity and protective effects were subsequently evaluated.Flow cytometry and immunofluorescence staining confirmed the high expression of D1ME-mRNA in cells.After intracranial challenge with DENV1,the mean weight loss of mice in the vaccinated group was significantly less than that in the control group.The mean titer of neutralizing antibody in the vaccine group was 1∶20,and the splenocytes in the vaccine group showed abundant secretion of the Th1 cell-associated cytokines IFN-γand TNF-α.In conclusion,the mRNA-LNP vaccine candidate against DENV1 was successfully constructed,and prime-boost vaccination with 10μg had a protective effect eliciting robust Th1 cellular immune responses.Thus,we developed a laboratory manufacturing platform for a dengue nonreplicating mRNA vaccine and confirmed its efficacy and immunogenicity.Our findings may provide a reference for future dengue vaccine development.