基于柯萨奇病毒-A5复制子的SARS-CoV-2自扩增RNA疫苗的构建及其免疫原性评价  

Construction and immunogenicity evaluation of SARS-Co V-2 self-amplifying RNA vaccine based on Coxsackievirus-A5 replicon

作　　者：付烈 靳卫平 杨洁 吴杰 卢佳 郭靖 孟胜利 王泽鋆 于代冠 申硕 FU Lie;JIN Weiping;YANG Jie;WU Jie;LU Jia;GUO Jing;MENG Shengli;WANG Zejun;YU Daiguan;SHEN Shuo(Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430207

出　　处：《中国生物制品学杂志》2023年第3期257-262,268,共7页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2020YFC0842100)。

摘　　要：目的 构建基于柯萨奇病毒-A5(Coxsackievirus-A5,CV-A5)复制子的严重急性呼吸综合征冠状病毒2(severe acute respiratory symptom coronavirus 2,SARS-CoV-2)Delta突变株(B.1.617.2)自扩增RNA(self-amplifying RNA,saRNA)疫苗,并评价其免疫原性。方法 以含有CV-A5全长基因组序列的质粒为基础,通过In-fusion克隆将SARS-CoV-2Delta突变株S蛋白基因替换不同长度的CV-A5 P1结构蛋白基因,分别构建重组质粒pDelta-S10、pDelta-S5和pDeltaS1(融合多聚蛋白的S-VP1/2A酶切位点上游的氨基酸分别为10、5和1个)。通过线性化重组质粒体外转录获得3种RNA分子Delta-S10、Delta-S5和Delta-S1。将3种RNA分子分别转染HEK-293T细胞,Western blot分析S蛋白的表达,筛选出S蛋白表达量最高的RNA分子,qPCR检测其在HEK-293T细胞中的自扩增。将筛选出的Delta-S用脂质纳米颗粒包装后,通过肌肉注射方式,采用两种剂量(0.5和2.5μg)免疫BALB/c小鼠(雌性,6~8周龄,每组5只),于第1和14天各免疫1次,第14和28天采血,ELISA法检测血清结合抗体滴度,微量中和法检测中和抗体滴度;第42天摘取脾脏,酶联免疫斑点技术(enzyme linked immunospot assay,ELISPOT)检测小鼠脾细胞分泌IFNγ水平。结果构建的重组RNA分子S蛋白表达量最高的为Delta-S10,其可在HEK-293T细胞中进行自扩增。Delta-S10高低剂量组均可诱导小鼠产生抗SARS-CoV-2 Delta S1蛋白特异性结合抗体,且剂量效应和加强免疫效应明显;Delta-S10高剂量组可诱导小鼠产生中和抗体,且可在小鼠体内诱导细胞免疫应答。结论 成功构建了基于CV-A5复制子的SARSCoV-2 Delta突变株(B.1.617.2)saRNA疫苗Delta-S10,其可诱导小鼠产生体液免疫和细胞免疫应答,为肠道病毒复制元件构建新冠saRNA疫苗的深入研究奠定了基础。ObjectiveTo construct self-amplifying RNA(saRNA)vaccine of severe acute respiratory syndrome coronavirus2(SARS-CoV-2)Delta mutant strain(B.1.617.2)based on Coxsackievirus-A5(CV-A5)replicon and evaluate its immunogenicity.MethodsThe recombinant plasmids pDelta-S10,pDelta-S5 and pDelta-S1(10,5 and 1 amino acid residues at the upstream of S-VP1/2A cleavage site of the fusion polyprotein respectively)were constructed by In-fusion cloning of the plasmids containing the full-length genome sequence of CV-A5 and substituting the S protein gene of SARS-CoV-2 Delta mutant for the P1 structural protein gene of CV-A5 with different lengths.Three RNA molecules,Delta-S10,Delta-S5 and Delta-S1,were obtained by in vitro transcription of linearized recombinant plasmids and transfected into HEK-293T cells respectively,which were analyzed for the expression of S protein by Western blot.The RNA molecule with the highest expression of S protein was screened out and detected for the self-amplification in HEK-293T cells by qPCR.BALB/c mice(female,6 ~ 8 weeks old and five for each group)were immunized i.m.with two doses(0.5 and 2.5 μg)of the screened Delta-S packaged with lipid nanoparticles for once on day 1 and day 14 seperately.Blood samples were collected on days 14and 28,detected for serum binding antibody titers by ELISA,and detected for neutralizing antibody titers by micro neutralization method.The spleens were harvested on day 42 and detected for the level of IFNγ secreted by mouse spleen cells by enzyme linked enzyme linked immunospot assay(ELISPOT).ResultsThe recombinant RNA molecule Delta-S10showed the highest expression of S protein and self-amplified in HEK-293T cells,which of both high and low doses induced specific binding antibody against SARS-CoV-2 Delta S1 protein in mice with obvious dose effect and enhanced immune effect;The high dose of Delta-S10 induced neutralizing antibodies and cellular immune responses in mice.ConclusionThe SARS-CoV-2 Delta mutant(B.1.617.2)saRNA vaccine Delta-S10 based on CV-A5 replicon was

关 键 词：严重急性呼吸综合征冠状病毒2 Delta株 自扩增RNA疫苗 柯萨奇病毒-A5 免疫原性 

分 类 号：R373.1[医药卫生—病原生物学]