实验目的：适应性免疫系统识别病原体和启动保护反应的能力取决于大量多样化表达的抗原特异性受体，尤其是T细胞受体(TCR)和免疫球蛋白(Ig)分子。其中TCR是T细胞呈递抗原并启动适应性免疫反应的关键免疫分子，在清除病毒及病变细胞的过程中发挥着关键作用。在新冠疫苗开发中，不同抗原可引起的不同特异性T细胞反应，TCR谱系特征的分析对于疫苗的设计和评价具有重要意义。本实验探究新冠蛋白疫苗与灭活疫苗免疫恒河猴后，不同抗原的细胞免疫应答的差异，恒河猴抗原特异性TCR α/β谱系特征，TCR分布偏向性，以及疫苗诱导产生的恒河猴TCR谱系与人的异同，可为后续疫苗的研发提供参考信息。

方法：本研究在恒河猴接种两针新冠灭活疫苗或蛋白疫苗后第10天采集外周血，分离PBMC作为实验样本。随后，通过ELISpot验证PBMC对不同新冠蛋白抗原(S/N肽段)的细胞免疫反应强度。再通过流式细胞术分选新冠S/N蛋白特异性的单个T细胞。进一步， 通过多重PCR扩增得到TCR α链及β链的可变区基因。随后，通过IMGT库基因打靶分析不同疫苗免疫后特异性TCR库进化谱系特征，并与V(D)J公共数据库对比分析。最后，使用ERGO计算机预测配对的TCR与不同新冠抗原表位的结合。

结果：两种疫苗免疫组的PBMC具有不同的抗原偏向性。在新冠蛋白疫苗组中，S1肽段刺激可以诱导PBMC产生较强的细胞免疫反应。而新冠灭活疫苗免疫组，S1、S2、N肽段均可以刺激PBMC产生较强的细胞免疫反应，且免疫反应更加多样。之后通过对不同疫苗免疫组中特异性TCR谱系的分析发现，TRAV6-TRAJ42谱系的组合在蛋白疫苗及灭活疫苗组均高频表达，频率分别达到43 %、24 %。而两疫苗组的特异性TCR β链V、J区没有明显的V-J配对优势组合，且TCR CDR3区多样性和长度未表现出明显的差异。同时，通过进化树分析发现两种疫苗免疫组S/N特异性TCR之间有高同源性的公共TCR克隆序列。此外，我们结合V((D)J公共数据库分析发现，在人群中SARS-CoV-2 S/N蛋白特异性TCR α链有明显表达占优势的V、J谱系组合，而β链表达占优势的谱系间出现频率相差不大，该现象与恒河猴一致。本研究中还发现，灭活疫苗免疫后特异性TCR库分布情况与自然感染状况下更接近。最后计算机预测结果发现，不同TCR CDR3区功能偏向不同。

结论：新冠灭活疫苗与蛋白疫苗免疫后，诱导的抗原特异性TCR分布特征不同。其中，灭活疫苗免疫后与自然感染相似，TCR谱系更加丰富，免疫反应更多样。而蛋白疫苗免疫后，TCR谱系偏向性明显，主要对S1抗原刺激有较强的应答反应。对TCR库特征的进一步研究，可为疫苗的研发和评价提供信息。

Objective: The ability of the adaptive immune system to recognize pathogens and initiate protective responses depends on a large and diversely expressed antigen-specific receptor, especially T-cell receptor (TCR) and immunoglobulin (Ig) molecules. Among them, TCR is a key immune molecule for T cells to present antigens and initiate adaptive immune responses, and plays a key role in the process of clearing viruses and diseased cells. In cases of SARS-CoV-2 infection, T cell levels correlated with pneumonia severity, and the strength of specific T cell responses to antigens correlated with antibody titers. In the development of SARS-CoV-2 vaccines, the analysis of TCR gene expression profiles with different specificity of T cell responses elicited by different antigens is of great significance for vaccine design and evaluation. In this experiment, after immunizing rhesus monkeys with SARS-CoV-2 protein vaccine and SARS-CoV-2 inactivated vaccine, the intensity of immune response to different antigens, TCR α/β characteristics and specific TCR bias differences were analyzed，and providing reference information for vaccine research and development.

Methods: In this study, rhesus monkeys were immunized with SARS-CoV-2 inactivated vaccine and SARS-CoV-2 protein vaccine. Peripheral blood was collected on the 10th day after two doses of vaccine, and PBMCs were isolated as experimental samples. Subsequently, the specific cellular immune response strength of PBMC to different SARS-CoV-2 antigens (S/ N peptides) was verified by ELISpot. Single T cells specific for SARS-CoV-2 S/ N protein were then sorted by flow cytometry. The variable region genes of TCR α chain and β chain were obtained by multiplex PCR amplification. The characteristics and evolution expression profiles of specific TCR repertoires after immunization with different vaccines were analyzed by the IMGT, and compared with the VDJ public database. Finally, using ERGO, the computer predicted the binding of paired TCRs to different SARS-CoV-2 epitopes.

Result: The PBMCs of the two vaccine-immunized groups had different antigenic biases. In the SARS-CoV-2 protein vaccine group, S1 peptide stimulation could induce a strong cellular immune response in PBMCs. In the SARS-CoV-2 inactivated vaccine immunization group, S1, S2, and N peptides can stimulate PBMC to produce strong cellular immune responses, and the immune responses are more diverse. Later, through the analysis of specific TCR expression profiles in different vaccine immunization groups, it was found that the combination of TRAV6-TRAJ42 genes was highly expressed in both protein vaccine and inactivated vaccine groups, with frequencies reaching 43 % and 24 %, respectively. However, the V and J regions of the specific TCR β chains of the two vaccine groups did not have obvious V-J pairing advantage combinations, and the diversity and length of the TCR CDR3 regions did not show significant differences. At the same time, the common TCR clone sequences with high homology between the S/N-specific TCRs of the two vaccine immunization groups were found by phylogenetic tree analysis. In addition, combined with the V-(D)-J public database analysis, we found that in the population, the SARS-CoV-2 S/N protein-specific TCR α chain has a significantly dominant combination of V and J spectrums, while the β chain does not. The phenomenon is consistent with that of rhesus monkeys. In this study, it was also found that the distribution of specific TCR repertoires after inactivated vaccine immunization was closer to that of natural infection. Finally, the computer prediction results found that different TCR CDR3 regions have different functional biases.

Conclusion: The distribution characteristics of antigen-specific TCRs induced by SARS-CoV-2 inactivated vaccine and protein vaccine were different. Among them, inactivated vaccine immunization was similar to natural infection, with more abundant TCR expression profiles and more diverse immune responses. After protein vaccine immunization, the expression profile of TCR was obviously biased, and it mainly responded strongly to S1 antigen stimulation. Further studies on the characteristics of the TCR repertoire could inform vaccine development and evaluation.