Although mRNA vaccine efficacy against severe coronavirus disease 2019 remains high, variant emergence has prompted booster immunizations. However, the effects of repeated exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens on memory T cells are poorly understood. Here, we utilize major histocompatibility complex multimers with single-cell RNA sequencing to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two or three antigen exposures, including vaccination, primary infection and breakthrough infection. Exposure order determined the distribution between spike-specific and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7⁻CD45RA⁺ effectors. In contrast, individuals after breakthrough infection mount vigorous non-spike-specific responses. Analysis of over 4,000 epitope-specific T cell antigen receptor (TCR) sequences demonstrates that all exposures elicit diverse repertoires characterized by shared TCR motifs, confirmed by monoclonal TCR characterization, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and current vaccination protocols continue to expand and differentiate spike-specific memory.

尽管 mRNA 疫苗针对 2019 年严重冠状病毒病的功效仍然很高，但变种的出现促使加强免疫。然而，反复暴露于严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 抗原对记忆 T 细胞的影响尚不清楚。在这里，我们利用主要组织相容性复合物多聚体和单细胞 RNA 测序来分析来自暴露于一种、两种或三种抗原（包括疫苗接种、初次感染和突破性感染）的人类的 SARS-CoV-2 反应性 T 细胞。暴露顺序决定了尖峰特异性和非尖峰特异性反应之间的分布，感染后接种疫苗会导致尖峰特异性 T 细胞扩增并分化为 CCR7 ^- CD45RA ⁺效应细胞。相比之下，突破性感染后的个体会产生强烈的非尖峰特异性反应。对超过 4,000 个表位特异性 T 细胞抗原受体 (TCR) 序列的分析表明，所有暴露都会引发以共享 TCR 基序为特征的多样化库，并通过单克隆 TCR 表征得到证实，没有证据表明重复暴露会导致库缩小。我们的研究结果表明，突破性感染使 T 细胞记忆库多样化，并且当前的疫苗接种方案继续扩大和分化尖峰特异性记忆。