针对SARS-CoV2不同突变株广谱特异性中和抗体的筛选与鉴定

Screening and identification of broadly neutralizing antibodies against different mutant strains of SARS-CoV2

目的筛选对严重急性呼吸综合征冠状病毒2(SARS-CoV2)不同突变株具有广谱特异性的、高亲和性中和抗体,并进行功能验证。方法用脂质体包被的SARS-CoV2刺突蛋白(S蛋白)mRNA疫苗对Balb/c小鼠进行2次免疫,测定小鼠血清中特异性抗体含量,选取抗体含量较高的小鼠进行冲击免疫;取冲击免疫后小鼠脾细胞与骨髓瘤细胞融合,用有限稀释法和ELISA筛选出上清能特异性结合S蛋白及S蛋白受体结合域(RBD)的杂交瘤细胞克隆;纯化单克隆抗体,进行假病毒中和实验,验证抗体中和活性;并通过ELISA筛选对4种SARS-CoV2突变株的重组RBD蛋白具有广谱结合特性的单克隆抗体。结果经2次免疫,检测到高剂量组4号小鼠血清中针对S蛋白的抗体含量最高;经过冲击免疫、细胞融合后,共筛选到12株与S蛋白具有结合活性的杂交瘤细胞,其中9株特异性结合RBD蛋白;假病毒中和实验结果显示,41、46和47这3株抗体在体外有显著的中和病毒活性(P<0.05);与另2株抗体相比,46号抗体显示出与SARS-CoV24种包括Omicron在内的突变株的RBD蛋白的广谱结合活性。结论46号抗体是对SARS-CoV2四种不同突变株具有广谱特异性的中和抗体,有望进一步进行人源化改造,用于新型冠状病毒肺炎(COVID-19)的治疗。

Objective To screen high-affinity neutralizing antibodies with broad spectrum specificity against different SARS-CoV2 mutants and verify their functions.Methods Balb/c mice were immunized twice with SARS-CoV2 S protein mRNA vaccine coated with liposome,and the specific antibody content in serum was determined.The mice with higher antibody titer were selected for shock immunization.The spleen cells of mice after shock immunization were fused with myeloma cells to prepare fusion cells,and the clones that produces monoclonal antibodies against different parts of S protein were screened by limited dilution and ELISA.Monoclonal antibody was purified and neutral-ization experiments were performed to verify the neutralization activity of the antibody.Monoclonal antibodies with broad spectrum binding properties against recombinant receptor binding domain(RBD)proteins of four SARS-CoV2 mutants were screened by ELISA.Results After twice immunizations,the serum antibody titer against S protein in high dose group 4 was the highest.After shock immunity and cell fusion,12 hybridoma cell clones with binding activity to S protein were screened,among which 9 strains specifically bound to RBD protein.The neutralization test results showed that the antibodies 41,46 and 47 had significant neutralization activity in vitro.Compared with the other two antibodies,antibody 46 showed broad-spectrum binding activity to the RBD proteins of four SARS-CoV2 mutants including Omicron.Conclusions Antibody 46 is a broad-spectrum specific neutralizing antibody against four different SARS-CoV2 mutants,which can be further humanized and potentially used for the treatment of COVID-19.