新型冠状病毒疫苗免疫BALB/c小鼠后的T细胞表位鉴定

目的 鉴定携带新型冠状病毒S、N和M基因的疫苗在BALB/c小鼠上的T细胞表位。方法 用携带新型冠状病毒S、N和M基因的DNA疫苗和痘病毒载体疫苗免疫6~8周龄雌性BALB/c小鼠,末次免疫1个月内取小鼠脾细胞,用ELISPOT法检测其对新型冠状病毒特异性多肽的T细胞免疫反应,筛选出具有阳性反应的多肽片段,并对其表位进行预测分析。结果 S、N、M基因分别鉴定出12、2、17条阳性多肽。S基因中多肽S49、S50、S100、S101、S102、S103阳性反应率为100%,且免疫刺激指数最高;覆盖M蛋白全长的41条多肽中有7条能刺激50%以上的小鼠产生阳性反应。预测分析结果显示,S、N、M基因分别有10、1、16个MHC-1 H-2限制性表位,主要位于S基因的RBD区、N基因RBD区以及M基因的跨膜区。这些表位在SARS-CoV-2及其变异株、SARS-CoV中高度保守。结论 新型冠状病毒DNA疫苗和痘病毒载体疫苗诱导了针对多个基因、多个表位的特异性T细胞应答,未来通用型疫苗设计应包含能诱导细胞免疫的多个抗原,特别是包含保守表位的结构抗原。

Identification of T cell epitopes in BALB/c mice immunized with COVID-19 vaccine

Objective To identify the T cell epitopes of the COVID-19 vaccine carrying SARS-CoV-2 S, N and M genes in BALB/c mice. Methods Groups of 6-8 week-old female BALB/c mice were immunized with DNA vaccines and recombinant vaccinia virus carrying SARS-CoV-2 S, N and M genes. The spleen cells were harvested within 1 month after the last immunization, and the T cell immune responses to SARS-CoV-2 specific overlapping peptides were detected by enzyme-linked immunosorbent assay (ELISPOT) method. The peptides with positive reaction were screened out and MHC-1 restricted epitopes were predicted using NetMHCpan EL 4.1. Results S, N and M genes were identified 12, 2 and 17 positive peptides respectively. The positive reaction rate of peptides S49, S50, 100, S101, S102 and S103 of S gene was 100%, with the highest immune stimulation index. Among the 41 peptides covering the full length of M protein, 7 peptides could stimulate T cell responses in more than 50% of vaccinated mice. The prediction analysis showed that there were 10, 1 and 16 MHC-1 H-2 restricted epitopes in S, N and M genes respectively, which mainly located in the RBD region of S and N genes, and the transmembrane region of M gene. These epitopes were highly conserved in SARS-CoV, SARS-CoV-2 and its variants. Conclusions The COVID-19 DNA vaccine and recombinant vaccinia virus vaccine have induced SARS-CoV-2 specific T cell responses against multiple genes and multiple epitopes. The future universal vaccine design should contain multiple antigens that can induce cellular immune responses, especially the structural antigens containing conserved epitopes.