SARS-CoV-2细菌样颗粒对小鼠的免疫原性及攻毒保护效果评价

Evaluation of immunogenicity and protection of SARS-CoV-2 bacterial-like particles vaccine in mice

目的本研究以C57BL/6N小鼠为模型,评价SARS-CoV-2细菌样颗粒(Bacterium-Like Particles,BLPs)的免疫原性和攻毒保护效果,为新型冠状病毒感染(Corona Virus Disease 2019,COVID-19)疫苗研发提供新思路。方法使用SARS-CoV-2细菌样颗粒Trim-RBD-GEM分别在第0、21 d滴鼻免疫C57BL/6N小鼠,在第7、14、21、28、35 d采小鼠血,分离血清,采用ELISA检测特异性IgG、IgG1和IgG2a抗体水平。在免疫后第35 d使用50 LD50的C57MA14毒株对C57BL/6N小鼠进行攻毒,记录攻毒后14 d内小鼠体重变化及存活情况;在攻毒后第3 d取小鼠的鼻甲骨和肺脏组织,测定鼻甲骨和肺脏组织中的病毒滴度和病毒载量。另取肺脏组织,使用4%多聚甲醛固定后制作病理切片并染色,观察肺脏组织病理变化,免疫组化法检测病毒蛋白的表达。结果Trim-RBD-GEM滴鼻免疫小鼠后诱导产生特异性抗体,IgG、IgG1和IgG2a抗体水平均显著升高。从攻毒后第1 d开始Trim-RBD-GEM组小鼠体重出现下降,第4 d体重逐步回升,观察期小鼠均存活,存活率为100%;Mock组小鼠在攻毒后第7 d体重均下降至75%以下,且所有小鼠均死亡,存活率为0。攻毒后Trim-RBD-GEM组小鼠肺脏组织中病毒滴度显著低于mock组(P<0.05)。攻毒后第3 d取各组小鼠肺脏做组织病理学检查,Trim-RBD-GEM组小鼠局部支气管管腔内可见少量巨噬细胞浸润,mock组小鼠肺脏切片中多处支气管管腔内可见坏死细胞碎片;免疫组化试验显示mock组小鼠肺脏切片检测到SARS-CoV-2的N蛋白。结论Trim-RBD-GEM滴鼻免疫小鼠可诱导产生特异性IgG抗体,可为COVID-19疫苗的研发提供参考。

Objective To evaluate the immunogenicity and protective efficacy of SARS-CoV-2 bacterium-like particles(BLPs),our study used C57BL/6N mice as theanimal model and provide new ideas for the development of COVID-19 vaccine.Methods C57BL/6N mice were immunized intranasally with SARS-CoV-2 BLPs Trim-RBD-GEM on day O and 21,respectively.Blood was collected from mice on days 7,14,21,28 and 35,and the serum was isolated for the detection of specific IgG,IgG1 and IgG2a antibodies by ELISA.On day 35 after immunization,C57BL/6N mice were challenged with the 50 LDso of C57MA14 strain,and the body weight changes and survival of the mice were recorded for 14 days after the challenge.The lungs were fixed with 4%paraformaldehyde and stained to observe the pathological changes of lung tissues,and the expression of viral proteins was detected by immunohistochemistry.ResultsTrim-RBD-GEM intranasally immunized mice induced specific antibody levels,and the levels of IgG,IgGl and IgG2a antibodies were significantly increased.The body weight of the mice in the Trim-RBD-GEM group decreased from day 1 after the challenge and gradually increased on day 4,and all mice survived during the observation period,with a survival rate of 100%.The virus titer in the lungs of Trim-RBD-GEM group was significantly lower than that of the mock group(P<0.05).A small number of macrophages were found in the local bronchial lumen of the mice in the Trim-RBD-GEM group,while necrotic cell fragments were seen in the lung sections of the mock grou.pC.onclusion Trim-RBD-GEM intranasal immunization of mice induces the production of specific IgG antibodies,which may provide a reference for COVID-19 vaccine development.