The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneumonia and other severe disease manifestations known as COVID-19. This health crisis has resulted in a significant effort to stop the spread of this new coronavirus. However, while propagating itself in the human population, the virus accumulates mutations and generates new variants with increased fitness and the ability to escape the human immune response. Here we describe a color-based barcoded spike flow cytometric assay (BSFA) that is particularly useful to evaluate and directly compare the humoral immune response directed against either wild type (WT) or mutant spike (S) proteins or the receptor-binding domains (RBD) of SARS-CoV-2. This assay employs the human B lymphoma cell line Ramos, transfected for stable expression of WT or mutant S proteins or a chimeric RBD-CD8 fusion protein. We find that the alpha and beta mutants are more stably expressed than the WT S protein on the Ramos B cell surface and/or bind with higher affinity to the viral entry receptor ACE2. However, we find a reduce expression of the chimeric RBD-CD8 carrying the point mutation N501Y and E484K characteristic for the alpha and beta variant, respectively. The comparison of the humoral immune response of 12 vaccinated probands with 12 COVID-19 patients shows that after the boost, the S-specific IgG class immune response in the vaccinated group is similar to that of the patient group. However, in comparison to WT the specific IgG serum antibodies bind less well to the alpha variant and only poorly to the beta variant S protein. This is in line with the notion that the beta variant is an immune escape variant of SARS-CoV-2. The IgA class immune response was more variable than the IgG response and higher in the COVID-19 patients than in the vaccinated group. In summary, we think that our BSFA represents a useful tool to evaluate the humoral immunity against emerging variants of SARS-CoV-2 and to analyze new vaccination protocols against these variants.

SARS-CoV-2 大流行已蔓延到世界各地，并可能导致危及生命的肺炎和其他称为 COVID-19 的严重疾病表现。这场健康危机导致为阻止这种新型冠状病毒的传播做出了重大努力。然而，在人群中自我传播的同时，病毒会积累突变并产生新的变种，这些变种具有更高的适应性和逃避人类免疫反应的能力。在这里，我们描述了一种基于颜色的条形码尖峰流式细胞术 (BSFA)，它对于评估和直接比较针对野生型 (WT) 或突变体尖峰 (S) 蛋白或受体结合域的体液免疫反应特别有用。 RBD) 的 SARS-CoV-2。该测定采用人 B 淋巴瘤细胞系 Ramos，转染稳定表达 WT 或突变 S 蛋白或嵌合 RBD-CD8 融合蛋白。我们发现 alpha 和 beta 突变体在 Ramos B 细胞表面比 WT S 蛋白更稳定地表达和/或以更高的亲和力与病毒进入受体 ACE2 结合。然而，我们发现分别带有 alpha 和 beta 变体特征的点突变 N501Y 和 E484K 的嵌合 RBD-CD8 的表达降低。12 名接种疫苗的先证者与 12 名 COVID-19 患者体液免疫反应的比较表明，加强后，接种组的 S 特异性 IgG 类免疫反应与患者组相似。然而，与 WT 相比，特异性 IgG 血清抗体与 α 变体的结合较差，与 β 变体 S 蛋白的结合也很差。这与 beta 变体是 SARS-CoV-2 的免疫逃逸变体的观点一致。COVID-19 患者的 IgA 类免疫反应比 IgG 反应更易变，并且高于接种疫苗组。总之，我们认为我们的 BSFA 是一种有用的工具，可用于评估针对 SARS-CoV-2 新变种的体液免疫并分析针对这些变种的新疫苗接种方案。