DataSheet_1_Identification of mouse CD4+ T cell epitopes in SARS-CoV-2 BA.1 spike and nucleocapsid for use in peptide:MHCII tetramers.docx

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice.

阐明针对严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）的适应性免疫应答，是开发新型冠状病毒肺炎（COVID-19）有效疫苗与治疗方案的关键前提。CD4+ T淋巴细胞（CD4+ T cells）在该过程中发挥不可或缺的核心作用，其主要机制为分泌抗病毒细胞因子，并辅助抗体产生性B细胞的功能活化。为支持小鼠模型中针对SARS-CoV-2特异性CD4+ T细胞应答的精细化研究，本研究通过γ干扰素酶联免疫斑点试验（IFNγ ELISpot），全面绘制了BA.1关切变异株的刺突蛋白（spike）与核衣壳蛋白（nucleocapsid）所对应的I-Ab限制性表位图谱。在此基础上，我们制备了对应的肽段-主要组织相容性复合体II类分子四聚体（peptide:MHCII tetramer）试剂，以直接标记表位特异性T淋巴细胞。通过这套严谨的验证策略，本研究在刺突蛋白中鉴定出6个免疫原性表位，在核衣壳蛋白中鉴定出3个，所有表位在原始武汉毒株中均高度保守。此外，我们验证了一个此前在武汉毒株中发现、但在BA.1变异株中缺失的表位。上述表位与四聚体试剂将成为小鼠模型中SARS-CoV-2抗原特异性CD4+ T细胞研究的宝贵工具。