CpG联合铝佐剂增强RBD-Fc重组蛋白诱导小鼠免疫反应效果的研究

Enhanced Immune Response of Mice Induced by RBD-Fc Recombinant Protein Combined with CpG-Aluminum Adjuvant

由严重急性呼吸系统综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)引起的2019冠状病毒病(Coronavirus disease 2019,COVID-19)大流行对全球健康和经济构成了严重威胁,SARS-CoV-2关切变异株(Variants of concern,VOCs)的出现更增加了疫苗研发的难度,因此,优化设计对突变株具有广谱免疫反应的疫苗显得尤为重要。本研究选取具有大量显性中和表位的受体结合域(Receptor-binding domain,RBD)蛋白作为目标抗原,在SARS-CoV-2经典株RBD序列的基础上引入多个VOCs的关键突变位点,将其与人IgG1 Fc片段融合表达,并结合CpG单佐剂、氢氧化铝单佐剂或CpG与氢氧化铝复合佐剂两剂次免疫小鼠,比较CpG联合铝佐剂相比单独使用铝或CpG佐剂诱导细胞免疫和体液免疫反应的增强作用,同时观察小鼠产生抗不同VOCs活病毒的交叉中和抗体滴度。结果显示,与单佐剂相比,RBD-Fc蛋白结合CpG与氢氧化铝复合佐剂免疫小鼠可产生最高的IgG结合抗体及高水平Th1偏向的细胞免疫应答。同时,小鼠血清还可以有效地中和SARS-CoV-2贝塔株(GMT:11143)、德尔塔株(GMT:4850)、经典株(GMT:3675),甚至是奥密克戎BA.1株(GMT:1393)和BA.5株(GMT:459)的感染。这些结果表明含有突变位点的SARS-CoV-2 RBD-Fc重组蛋白具有良好的潜力,联合使用CpG和铝佐剂可明显提高和平衡小鼠的细胞免疫反应,产生对不同SARS-CoV-2高滴度的交叉中和抗体,可作为一种广谱疫苗进一步研究和开发。

The 2019 coronavirus disease(COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) poses a serious threat to global health and the economy.The emergence of variants of concern(VOCs) in SARS-CoV-2 has made vaccine development more challenging.Therefore,designing vaccines that can elicit a broad immune response against mutant strains is crucial.In this study,the receptor-binding domain(RBD) protein,which contains dominant neutralizing epitopes,was selected as the target antigen.The RBD sequence of the SARS-CoV-2 Wild-type(WT) strain was served as the basis for introducing several key mutation sites of VOCs,and fused with human IgG1 Fc fragments.The RBD-Fc protein was expressed and administered with either CpG single adjuvant,aluminum hydroxide single adjuvant,or CpG combined with aluminum hydroxide adjuvant in two doses to mice.This study compared the enhancement of cellular and humoral immune responses induced by CpG combined with aluminum hydroxide adjuvant with those induced by aluminum or CpG adjuvant alone.The cross-neutralizing antibody titers against live viruses of different VOCs were also observed.The results demonstrated that RBD-Fc protein combined with CpG and aluminum hydroxide adjuvant induced the highest IgG binding antibodies and high-level of Th1-biased cellular immune response in immunized mice compared to single adjuvant.Additionally,mouse serum could effectively neutralize infections of SARS CoV-2 beta strain(GMT:11143),Delta strain(GMT:4850),classic strain(GMT:3675),and even Omicron BA.1 strain(GMT:1393) and BA.5 strain(GMT:459).These results indicate that the SARS-CoV-2 RBD-Fc recombinant protein containing mutation sites has excellent potential,and the combined use of CpG and aluminum adjuvant can significantly improve and balance the cellular immune response of mice,produce cross-neutralizing antibodies against different SARS-CoV-2varants with high titers,which can be further studied and developed as a broad-spectrum vaccine.