Detection of SARS‐CoV‐2‐specific memory B cells to delineate long‐term COVID‐19 immunity
Authors: Constantin J. Thieme, Mohamed Abou‐el‐Enein, Enrico Fritsche, Moritz Anft, Krystallenia Paniskaki, Sarah Skrzypczyk, Adrian Doevelaar, Magdi Elsallab, Nicola Brindle, Arturo Blazquez‐Navarro, Felix S. Seibert, Toni L. Meister, Stephanie Pfaender, Eike Steinmann, Oliver Witzke, Timm H. Westhoff, Ulrik Stervbo, Guido Heine, Toralf Roch, Nina Babel
Journal: Allergy (2021)

The COVID-19 pandemic has led to devastating health outcomes with the death toll exceeding two million cases as of February 2021. However, there is still limited data on long-term immunity against SARS-CoV-2. Long-term immunity can be analysed by SARS-CoV-2-specific memory T and B cell formation. We and others have reported on SARS-CoV-2-specific memory T-cell responses in acute infection and long-term follow up.1, 2 To our knowledge, however, only studies with Australian and US-American cohorts assessed long-term BMEMORY-cells for more than 6 months.2, 3 Memory B (BMEMORY)-cells can persist lifelong, and upon reinfection can be triggered to immediately start forming plasma cells secreting neutralizing antibodies.4 Thus, quantifying BMEMORY-cell levels may be used as an indicator of long-term immunity in convalescent patients. Therefore, this study aimed to delineate SARS-CoV-2 spike (S)-protein-specific BMEMORY-cells in a well-characterized cohort of central-European COVID-19-patients up to several months after infection. Between April and October 2020, a cohort of 27 convalescent COVID-19 patients and 14 healthy donors were included in the study in three German centers (Berlin, Bochum, Essen). Baseline characteristics are provided in Table S1. All patients gave written informed consent. The ethical committee of the Ruhr-University Bochum approved the study (20–6886). A schematic presentation of the protocol is depicted in Figure S1, and materials are listed in Table S2–S3. COVID-19 patients were included at a median time of 53 days after diagnosis or onset of symptoms (range 15–214). SARS-CoV-2-specific B cell response was analysed by characterizing levels of IgD−CD27+ BMEMORY, IgD+CD27+unswitched BMEMORY, IgD+CD27− BNAIVE, and CD27++CD38++plasmablasts (Figure 1). Overall B cell composition was not affected by SARS-CoV-2-infection in convalescent patients (Figure 2A). We did not observe T-cell lymphopenia in reconvalescent COVID-19 patients (Figure 2B). For flow-cytometric analysis of specific B cells, SARS-CoV-2-S-protein was labelled with two different fluorochromes, and double-positive B cells were determined. Blocking of specific staining by excess unlabelled S protein demonstrates specificity of labeling.5, 6 Unlabelled S-protein in class-switched BMEMORY-cells could significantly block binding in COVID-19 patients, but not in healthy donor samples, indicating only minimal cross-reactivity in healthy donors (Figures 1B, 2D). Accordingly, the frequency of S-protein-specific BMEMORY-cells was significantly higher in the COVID-19-cohort compared to healthy individuals (Figure 2D). SARS-CoV-2-specific BMEMORY-cells were also detectable 200 days after COVID-19 diagnosis with a tendency of lower frequencies in samples collected at later time points (Figure 2C). Specific labelling with SARS-CoV-2 S-protein and difference between COVID-19 patients and healthy individuals was restricted to BMEMORY-cells and not observed for other B cell subsets (Figure 2D–G). S-protein-specific BMEMORY-cells have been described to be pivotal for effective antibody responses.4 SARS-CoV-2-specific BMEMORY-cells correlated moderately with anti-S-protein IgG-antibodies, but correlation with neutralizing antibodies did not achieve statistical significance (Figure 2H–I). Thus, we demonstrate specific detection of SARS-CoV-2-S-protein-binding BMEMORY-cells over 6 months post-infection. We acknowledge limitations of our study. Subsequent studies should enrol larger patient cohorts with longer follow-up periods. Using bifluorescent tetramer-based staining may increase sensitivity to detect SARS-Cov2-specific B cells.6 Control patients were significantly younger than the COVID-19-cohort. Nevertheless, we did not observe specific staining in the control cohort as evidenced by the lack of significant blocking of the staining. In conclusion, evaluating the long-term immunity in a cohort of convalescent COVID-19 patients, we demonstrated SARS-COV-2-specific BMEMORY-cells in individuals both early as well as over 6 months after infection. Thus, our study performed on a central-European cohort is in line with the data on the recently published US-American and Australian cohorts and accordingly, confirms and extends the knowledge on the B cell response against SARS-CoV-2.2, 3 Demonstrating the persistence of SARS-CoV-2-specific B cell response, our results point towards an additional hallmark of immunisation beyond specific serum antibodies. We are grateful to the patients who donated their blood samples and clinical data for this project. We would like to acknowledge the excellent technical assistance as well as the expertise of the immune diagnostic laboratory of the Center for Translational Medicine at Marien Hospital Herne. Mr. Thieme has nothing to disclose. Dr. Abou-el-Enein has nothing to disclose and received funding from the Arab-German Young Academy of Sciences (AGYA) which is supported by the German Federal Ministry of Education and Research (BMBF) grant 01DL20003. Mr. Fritsche has nothing to disclose. Dr. Anft has nothing to disclose. Ms. Paniskaki has nothing to disclose. Ms. Skrzypczyk has nothing to disclose. Mr. Doevelaar has nothing to disclose. Mr. Elsallab has nothing to disclose. Dr. Brindle has nothing to disclose. Dr. Blazquez-Navarro has nothing to disclose. Dr. Seibert has nothing to disclose. Ms. Meister has nothing to disclose. Dr. Pfänder has nothing to disclose. Dr. Steinmann has nothing to disclose. Dr. Witzke has received research grants for clinical studies, speaker's fees, honoraria and travel expenses from Amgen, Alexion, Astellas, Basilea, Biotest, Bristol-Myers Squibb, Correvio, Chiesi, Gilead, Hexal, Janssen, Dr. F. KöhlerChemie, MSD, Novartis, Roche, Pfizer, Sanofi, TEVA, and UCB. Dr. Westhoff has nothing to disclose. Dr. Stervbo has nothing to disclose. Dr. Heine reports personal fees from Allergopharma, Biotest, Eli Lilly, Sanofi, and Abbvie, outside the submitted work. Dr. Roch has nothing to disclose. Dr. Babel has nothing to disclose and received funding from the Mercator Foundation, the German Federal Ministry of Education and Research (BMBF) e:KID (01ZX1612A), and BMBF NoChro (FKZ 13GW0338B). All authors declare no competing interests. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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