key: cord-0774157-park7t3g
authors: Winklmeier, S.; Eisenhut, K.; Taskin, D.; Ruebsamen, H.; Schneider, C.; Eichhorn, P.; Keppler, O. T.; Klein, M.; Mader, S.; Kuempfel, T.; Meinl, E.
title: Persistence of functional memory B cells recognizing SARS-CoV-2 variants despite loss of specific IgG
date: 2021-05-18
journal: nan
DOI: 10.1101/2021.05.15.21257210
sha: 30c6bf4de996cb6a658bad1d50edc5a24a1e6990
doc_id: 774157
cord_uid: park7t3g

While some COVID-19 patients maintain SARS-CoV-2-specific serum IgGs for more than 6 months post-infection, others, especially mild cases, eventually lose IgG levels. We aimed to assess the persistence of SARS-CoV-2-specific B cells in patients who have lost specific IgGs and analyzed the reactivity of the immunoglobulins produced by these B cells. Circulating IgG memory B cells specific for SARS-CoV-2 were detected in all 16 patients 1-8 months post-infection, and 11 participants had specific IgA B cells. Four patients lost specific serum IgG after 5-8 months but had SARS-CoV-2-specific-B-cell levels comparable to those of seropositive donors. Immunoglobulins produced after in vitro differentiation blocked receptor-binding domain (RBD) binding to the cellular receptor ACE-2, indicating neutralizing activity. Memory-B-cell-derived IgGs recognized the RBD of B.1.1.7 similarly to the wild-type, while reactivity to B.1.351 and P.1. decreased by 30% and 50%, respectively. Memory-B-cell differentiation into antibody-producing cells is a more sensitive method for detecting previous infection than measuring serum antibodies. Circulating SARS-CoV-2 IgG memory B cells persist, even in the absence of specific serum IgG; produce neutralizing antibodies; and show differential cross-reactivity to emerging variants of concern. These features of SARS-CoV-2-specific memory B cells will help to understand and promote long-term protection.

While some COVID-19 patients maintain SARS-CoV-2-specific serum IgGs for more than 6 months post-infection, others, especially mild cases, eventually lose IgG levels. We aimed to assess the persistence of SARS-CoV-2-specific B cells in patients who have lost specific IgGs and analyzed the reactivity of the immunoglobulins produced by these B cells. Circulating IgG memory B cells specific for SARS-CoV-2 were detected in all 16 patients 1-8 months postinfection, and 11 participants had specific IgA B cells. Four patients lost specific serum IgG after 5-8 months but had SARS-CoV-2-specific-B-cell levels comparable to those of seropositive donors. Immunoglobulins produced after in vitro differentiation blocked receptor-binding domain (RBD) binding to the cellular receptor ACE-2, indicating neutralizing activity. Memory-B-cellderived IgGs recognized the RBD of B.1.1.7 similarly to the wild-type, while reactivity to B.1.351 and P.1. decreased by 30% and 50%, respectively. Memory-B-cell differentiation into antibody-producing cells is a more sensitive method for detecting previous infection than measuring serum antibodies. Circulating SARS-CoV-2 IgG memory B cells persist, even in the absence of specific serum IgG; produce neutralizing antibodies; and show differential crossreactivity to emerging variants of concern. These features of SARS-CoV-2-specific memory B cells will help to understand and promote long-term protection.

The development of adaptive immunity to SARS-CoV-2 may provide protection against re-infection and allows the identification of patients that have had a previous infection. Adaptive immunity to SARS-CoV-2 involves antibody (Ab)-producing cells, memory B cells, and several T cell subsets. Analysis of immune responses to different viruses, including other coronaviruses, has shown that the lifespans of the adaptive immune system components vary (1, 2) .

Details of the kinetics of immune responses to SARS-CoV-2 are beginning to be uncovered (3) (4) (5) (6) (7) (8) (9) . Antibody (Ab) responses peak at about 2-3 weeks after infection, at which point the Ab-levels decline (4, 10, 11) . In most individuals, anti-SARS-CoV-2 serum Abs persist for more than 6 months after primary infection, but some patients rapidly lose their specific Abs, especially those that experienced a mild disease course (2, 4, 6, (10) (11) (12) (13) . It has been proposed that, in addition to serum antibody titers, the memory B cell pool should be evaluated to estimate humoral immunity as an indicator of immune protection (4, (14) (15) (16) .

Initial Ab responses are made by short-lived plasmablasts that develop in extrafollicular sites (14, 17, 18) , and the subsequent development of high-affinity and persistent Abs involves affinity maturation and the expansion of B cells in germinal centers (15, (19) (20) (21) (22) . Two types of B cells exit the germinal center: memory B cells and plasmablasts (14, 19) . Many of these plasmablasts are short-lived and die within a few weeks, but some find survival niches in the bone-marrow and persist as long-lived plasma cells (23) . The extent to which these long-lived plasma cells develop differs between different viruses and vaccines (24, 25) . Different mechanisms regulate the survival of long-lived plasma cells (23, 26) and memory B cells (19) .

of the RBD (27, 28) . These rapidly spreading VoCs are currently causing serious concerns regarding the increased frequency of re-infection, utility of convalescent plasma, and limited vaccine responses (29, 30) .

In this study, we analyzed the persistence of IgA and IgG memory B cells specific for SARS-CoV-2 in COVID-19 patients. We specifically investigated donors who had lost circulating IgG to SARS-CoV-2 and analyzed whether they still harbored specific memory B cells in their blood. To study the patient B cells, we adopted a functional approach, converting blood-derived B cells into Ab-secreting cells in vitro (31) (32) (33) . Having identified the SARS-CoV-2-specific memory B cells in the blood, we analyzed whether the secreted Abs have neutralizing activity and show cross-reactivity to the recently emerged SARS-CoV-2 VoCs B.1.1.7, B.1.351, and P.1. The findings of the study revealed functional properties of persisting memory B specific to SARS-CoV-2 and this could help to understand and promote protection.

Persistence of IgG memory B cells specific for SARS-CoV-2 in the presence and absence of specific IgG

We analyzed, in parallel, the presence of memory B cells specific for SARS-CoV-2 in blood and specific IgG in serum ( Figure 1 shows our approach). Our study included 16 COVID-19 patients that had undergone a mild or asymptomatic disease course (Table 1) , and pre-pandemic blood samples from six HC donors served as the control group. We detected B cells that could be developed into SARS-CoV-2-specific-IgG-secreting plasmablasts in the blood of all COVID-19 patients analyzed. The reactivity to SARS-CoV-2 of these in vitro differentiated plasmablasts and the patient sera from the same blood withdrawal was investigated ( Figure 2A) . Remarkably, the sera from three COVID-19 patients were negative in the ELISA, and a fourth was borderline.

These four donors (HC=1, MS=2, SLE=1; #5, #11, #12, #15) had been seropositive 1-2 months after acute infection ( Table 1 ) but had lost their specific IgG 5-8 months post-infection (HC=1, MS=2, SLE=1). Two of these four donors were under immunotherapeutic regimens at the time their blood was sampled for this study ( Table 1) .

Based on these findings, we grouped the patients into those with (COVID19-IgG + ) or without (COVID19-IgG − ) serum IgG to SARS-CoV-2. We calculated the mean of the total IgG secreted into the cell culture supernatant and the SARS-CoV-2-specific IgG levels of the samples from each donor and compared the three groups ( Figure 2 , B and C). The COVID19-IgG + and COVID19-IgG − patients had significantly more SARS-CoV-2-specific B cells in their blood than HC donors (p < 0.0001), even though the amount of total secreted IgG was not significantly different between the groups. Remarkably, the levels of SARS-CoV-2-specific IgG produced in vitro were similar between the COVID19-IgG − and COVID19-IgG + subgroups ( Figure 2C ).

The method of seeding PBMCs into individual wells and testing each well for the development of specific IgG yields a high sensitivity. We noted that all wells containing samples from 15 of the 16 COVID-19 patients were positive for SARS-CoV-2-specific IgG, and for one donor, 6 of the 8 wells were positive, which demonstrated the high frequency of SARS-CoV-2specific B cells in these patients. We subsequently performed limiting dilution assays on PBMC from five COVID-19 patients (Figure 2 , D and E). We calculated the frequency of specific B cells according to the Poisson distribution and the individual percentage of B cells within the PBMC (between 5% and 15%) and obtained thereby the following rates of B cells that gave rise to Abs to SARS-CoV-2: Patient #6 (seropositive): 1:13,000; patient #8 (seropositive): 1:11,000; patient #14 (seropositive): 1:20,000; patient #5 (seronegative): 1:22,000; patient #12 (seronegative) 1:7,000. Thus, in accordance with our calculation of SARS-CoV-2-reactivity in the bulk cultures ( Figure 2C ), our limiting dilution analysis indicated that the seronegative and seropositive COVID-19 patients had similar frequencies of circulating RBD-specific B cells. The frequencies observed lay within the reactivity ranges previously reported for measles virus and tetanus toxoid (32, 33) . IgG memory B cells constitute about 15% of peripheral B cells, and only 30% to 40% of IgG memory B cells are capable of antibody production under these culture conditions (31) . We found in one well of one HC sample B cells giving rise to SARS-CoV-2recognizing Abs ( Figure 2A) ; however, these Abs did not show neutralizing activity (see below).

We also analyzed the presence of anti-SARS-CoV-2 IgA in serum and of specific B cells in blood secreting IgA (Supplemental Figure 1 , A-C). The COVID-19 patients had significantly more SARS-CoV-2-specific IgA B cells in their blood than the healthy controls (p = 0.022, Mann-Whitney U test). We noted that two COVID-19 patients had serum IgA, but no detectable specific IgA B cells. In healthy controls, SARS-CoV-2-specific IgA B cells were seen in 2 out of 6 donors and one was borderline, and no specific serum IgA was detected in 5 of the 6 healthy controls and 1 had borderline levels (Supplemental Figure 1 , A-C). While all COVID-19 patients tested had specific IgG B cells in their blood, 11 patients had at least one well with specific IgA B cells. The difference between the occurrence of IgG-and IgA-positive B cells became clearer when we considered the number of positive wells. Specific IgG was detected in 115 out of 117 wells for COVID-19-patients, while specific IgA was detected in only 33 wells (p < 0.0001; p-values were determined using Fisher's exact test). Therefore, IgA B cells specific for SARS-CoV-2 were detected in the blood of the COVID-19 patients but were less abundant than specific IgG B cells.

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To analyze whether circulating peripheral B cells specific for SARS-CoV-2 can give rise to SARS-CoV-2-neutralizing Abs, we used a surrogate assay to analyze secreted-Ab inhibition of RBD binding to the viral entry receptor ACE-2 (34) (Figure 1 ). The significantly higher neutralizing potency of the COVID-19-patient-derived Abs produced in vitro was evident when compared to the neutralizing activity of the HC-derived Abs ( Figure 3 ; p < 0.0001, Mann-Whitney U test). Thus, the SARS-CoV-2-specific B cells from COVID-19-patients released substantial amounts of neutralizing Abs after differentiation into Ab-secreting cells.

We analyzed the cross-reactivity of SARS-CoV-2-specific memory B cells ( Figure 4A ) against the RBD of three major VoCs in current circulation: B.1.1.7, B.1.351, and P.1 (28) . When we examined the memory B cells from each of the 16 COVID patients, we found that Ab-recognition of the RBD of B.1.1.7 variant was quantitatively unaltered, while recognition of the B.1.351 RBD was reduced by approximately 30% (p < 0.0001), and recognition of the P.1 RBD was 50% lower than that for the WT (p < 0.0001) ( Figure 4B ). The reactivity pattern SARS-CoV-2 (WT, D614G) = B1.1.7 > B1.351 > P.1 was seen in each of the COVID-19 patients analyzed.

In this study, we robustly detected the persistence of memory B cells specific for SARS-CoV-2 in all COVID-19 patients in our cohort that had undergone mild or asymptomatic acute infections, even if their specific serum IgG had declined to undetectable levels ( Figure 5 ). This has two All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 18, 2021. ; https://doi.org/10.1101/2021.05.15.21257210 doi: medRxiv preprint implications: Firstly, the persistence of specific memory B cells, which gave rise to neutralizing Abs and showed differential cross-reactivity to VoCs, helps us to understand and start to develop models for predicting long-term protection against SARS-CoV-2. Secondly, the assay employed to differentiate B cells into Ab-producing cells in vitro is a more sensitive method for detecting previous infection than measuring serum levels of SARS-CoV-2 IgG.

We demonstrated the persistence of specific IgG memory B cells by differentiating them into Ab-secreting cells in vitro, which also facilitated our functional analysis of the Abs secreted by the cells. Previous studies have also detected COVID-19 patients' memory B cells by staining them with labelled antigens (4, 15, 21, (35) (36) (37) (38) (39) and using the enzyme-linked immune absorbent spot (ELISPOT) assay (8, (40) (41) (42) . Sorting the SARS-CoV-2-specific B cells and cloning their antigen-receptors provided important insights into clonal turnover and the ongoing somatic hypermutation of SARS-CoV-2-specific B cells associated with antigen persistence (15, 21) .

We also detected SARS-CoV-2 IgA memory B cells in the blood of the recovered COVID-19 patients, but in contrast to the specific circulating IgG memory B cells, these were only seen in a subset of the cohort and less abundant. While IgA is best known for its role in the immune response at mucosal sites (43, 44) , a systemic IgA immune response also occurs that includes the expansion of circulating IgA plasmablasts specific for SARS-CoV-2 (43). Mucosal IgA secretion continues for longer than the serum IgA response (43) , but was completely lost after 189 days (43) . Circulating IgA declined more rapidly than IgG and decayed by ~90 days in most COVID-19 cases to levels indistinguishable from controls (4). We robustly detected circulating IgG memory B cells more than 6 months after infection. Taken together, the picture is emerging from this study and previous work that when circulating IgG and IgA, mucosal IgA, and circulating IgA memory B cells are gone, circulating IgG memory B cells persist ( Figure 5 ). All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We found that the Abs produced by these memory B cells had high neutralizing activity, indicating their functional importance upon exposure to the same or mutated SARS-CoV-2.

In general, persisting IgG memory B cells can rapidly differentiate into antibody-secreting cells upon re-exposure (14, 19) . The importance of memory B cells for protection against reinfection is evident from immune responses to other human viruses: protective immunity against hepatitis B was observed despite the loss of Abs, (45) and circulating memory B cells to viruses can be sustained for many decades after exposure, well into the tenth decade of life (46) . In a mouse model of cytomegalovirus, the activation of virus-specific memory B cells to secrete IgG was independent on cognate or bystander T cell help (47) . Thus, the persisting memory B cells are expected to be capable of providing relevant functional protection upon subsequent reexposure with SARS-CoV-2, even if the relevant Abs have vanished.

The global spread of VoCs such as B.1.1.7, B.1.351, and P.1 may increase re-infection rates and compromise the success of current vaccines (48) . We found that the polyclonal memory B cells recognized the RBD of B.1.1.7 (UK variant) to a similar degree as they did the WT, while reactivity to B.1.351 (SA variant) was reduced by 30% and to P.1 (BR variant) by as much as 50%. These findings are in accordance with the molecular signatures of these VoCs, as the UK variant contains one mutation, and the SA and BR variants have three mutations in the RBD (28) .

A recent study reported a more pronounced drop in cross-reactivity following infection with B.1.1.7 than after infection with the WT, indicating asymmetric heterotypic immunity is induced by SARS-CoV-2 variants (49) . Another study showed that some monoclonal (m)Abs showed greatly reduced binding to the UK variant; however, patient serum showed little change in reactivity (50) and unaltered neutralizing activity to the UK variant, but reduced reactivity to the SA variant was reported (51) . This is exactly in line with our observations of memory B cells from our cohort. We extended our analysis to show that cross-reactivity for the BR variant was All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (28) . To what extent the reduced humoral activity is associated with an increased susceptibility to these strains is not yet clear. Re-infection with SARS-CoV-2 is apparently rare on a global scale (52) but was a possible reason for the resurgence of COVID-19 in Manaus (53) . It remains to be analyzed whether patients become reinfected because they have lost a certain type of anti-SARS-CoV-2 immunity or are confronted with a new variant, against which they do not yet have a protective immunity.

Because the consequences of infection with SARS-CoV-2 range from asymptomatic to lethal, accurate confirmation of previous infections is of great epidemiological and prognostic significance. Serological testing has made great advances (54) (55) (56) (57) , but the specific IgG response wanes over time, and some donors with previous infections score negative in current serology tests (4, 16) . We showed that the donors who lost IgG to SARS-CoV-2 still had specific IgG memory B cells. It has been previously noted that about 5% of infected donors are "nonresponders" who remain seronegative (58, 59) . Using our method, we can analyze whether such donors have memory B cells with potential protective activity.

Our assay, involving the differentiation of B cells into Ab-secreting cells in vitro, identifies circulating memory B cells. We distributed the blood cells into different cell-cultureplate wells and analyzed the wells individually to perform an assay with high sensitivity that allows the identification of even rare autoreactive B cells (33) . By considering all wells containing samples from each donor, a clear distinction can be made between rare and abundant responses. We noted that, for one HC, one well showed ELISA reactivity to SARS-CoV-2 without neutralizing activity; as the blood sample was obtained before the pandemic, we can probably exclude a previous infection with SARS-CoV-2. The cross-reactivity of memory IgG B All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (60) . However, in our assay, these cross-reactive antibodies did not show neutralizing activity. Nevertheless, such cross-reactivity could be clinically relevant because a recent infection with endemic coronavirus is associated with less severe COVID-19 (61) . Thus, the assay we present here extends our methodological armamentarium to evaluate if seronegative individuals have already been infected with SARS-CoV-2. This is of relevance in epidemiology and for optimizing urgently vaccination has yet to be analyzed in detail. We analyzed the persistence of B cells reactive to the S1 protein, but the maintenance of B cells against other SARS-CoV-2 proteins remains to be analyzed; although the response to the RBD is of paramount importance, as the RBD is targeted by neutralizing Abs (3). We analyzed circulating memory B cells but are aware that both systemic and mucosal immunity are relevant for protection. We did not analyze the persistence of B cells in patients very severely affected by COVID-19, but it has been reported that the serum Ig response is even greater and longer-lasting in these patients (6, 13) .

In this study, we showed that circulating IgG memory B cells specific for SARS-CoV-2 persist in the blood after infection despite the loss of systemic IgG. These persisting B cells can be harnessed to identify a previous infection. Furthermore, these B cells gave rise to neutralizing All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Abs and showed high cross-reactivity against the emerging UK variant, suggesting patients have protection against re-exposure with this variant even after the loss of specific Abs. In contrast to reactivity against the UK variant, the reactivity of memory B cells against the SA variant and, particularly, the BR variant was greatly reduced. This warrants further attention and indicates a possible need for follow-up vaccinations covering these mutants (62) . Thus, our study has added to the efforts to fully uncover the features of SARS-CoV-2-specific memory B cells, which will help us to understand and promote long-term protection. 

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We analyzed the B-cellular responses to SARS-COV-2 of 16 COVID-19-patients (HC=11, MS=4 and SLE=1) that had a mild or asymptomatic disease course ( Table 1) . Five of them were identified in a survey of health care workers (63) . Infection was confirmed by SARS-CoV-2specific PCR (64) and/or SARS-CoV-2 serology, as indicated in Table 1 . Three patients did not receive a PCR test at the beginning of the pandemic but displayed typical symptoms (fever, respiratory symptoms, severe anosmia) and subsequently tested positive for SARS-CoV-2 Abs.

All study participants tested positive for SARS-CoV-2 antibodies at least once after they were infected. As a reference, we analyzed pre-pandemic blood samples from six healthy adults (HC), two males and four females, with a mean age of 28 years.

Our experimental scheme is outlined in Figure 1 . PBMCs were obtained by a standard density gradient method using SepMate-50 tubes (STEMCELL Technologies, Vancouver, Canada) and frozen. After thawing, the PBMCs were seeded at 1 × 10 6 cells into 1 mL of culture medium (RPMI + 10 % FCS) in 24-well plates and differentiated into Ab-secreting cells using the TLR7/8 ligand resiquimod (2.5 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) and IL-2 (1,000 IU/mL; R&D Systems, Minneapolis, MN, USA) during 11 days of culturing, essentially as previously described (31) (32) (33) . This culture system differentiated the memory B cells into Ab-secreting cells (31) . The total IgG and IgA levels of the culture supernatants were measured using human IgG and IgA ELISA development kits (Mabtech, Nacka Strand, Sweden). We considered an IgG All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 18, 2021. ; https://doi.org/10.1101/2021.05.15.21257210 doi: medRxiv preprint supernatants (50 µL) were incubated at room temperature for 2 h. Abs were then detected with 50 µL of anti-human IgG horseradish peroxidase (1:5000, 109-036-003, Jackson ImmunoResearch, West Grove, PA, USA) and 50 µL of tetramethylbenzidin (TMB, Sigma-Aldrich) as the substrate. The reaction was stopped by adding 25 µL of 1 M sulfuric acid. The optical density (OD) of the chromogenic reaction was measured at 450 nm, and the plate background was measured at 540 nm. The OD cutoff value for the recognition of RBD was 0.52, which was calculated using the mean + 3 SD of the control cell culture supernatants from stimulated wells with 10 6 PBMCs per 1 ml.

To assess the SARS-CoV-2 inhibitory and neutralizing activity of the Abs, we analyzed whether they blocked the binding of the RBD to ACE-2, the cellular receptor for SARS-CoV-2 according to (34) (Figure 1) . ELISA plates were coated with RBD and blocked as described above. Then 50 µL of the undiluted cell culture supernatants were added and maintained for 2 h at room temperature, followed by the addition of 50 µL of biotin-tagged ACE-2 (1 µg/mL, SAE0171, Sigma-Aldrich). The binding of ACE-2 was detected by 50 µL of streptavidin conjugated with horseradish peroxidase (1:200, 890803, R&D Systems). ELISAs were developed with TMB as described above, and OD values were normalized to those ACE-2 without cell culture supernatant.

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The cross-reactivities of Abs recognizing SARS-CoV-2 wild type (WT) to RBDs of VoCs overnight at 4°C. The subsequent procedure was as described for the RBD ELISA.

Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA).

The study was approved by the ethical committee of the medical faculty of the Ludwig-Maximilians-Universität Munich. Written informed consent was obtained from each donor prior to their inclusion in the study.

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. PBMCs from each donor were separated into individual wells and stimulated with the TLR7/8 agonist R848 and IL-2 to differentiate to them into Ab-secreting plasmablasts. This was used to compare the serum response to SARS-CoV-2 with that of specific Abs produced in vitro. The frequency of SARS-CoV-2-specific B cells that differentiated into Ab secreting cells was determined. The cross-reactivity to RBDs of emerging variants was tested. The ability of in vitroproduced Abs to block the binding of RBD to its receptor ACE-2 was determined as outlined.

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Ab-secreting cells. The cell culture supernatants (each dot represents an individual well) were added to ELISA plates coated with the RBD. Biotinylated ACE-2 was then added, and its binding was detected with streptavidin-horseradish peroxidase. For calibration, the binding of biotinylated ACE-2 to RBD in the presence of buffer was set as 1. Then, the mean OD of the wells of each donor was calculated to compare the Abs binding to ACE-2 from COVID-19 patients with those from HCs. The Abs from COVID-19 patients reduced ACE-2 binding (p < 0.0001; Mann-Whitney U; HC=6, COVID-19=16).

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. however, all three patients displayed symptoms pathognomonic for COVID-19, including fever, respiratory symptoms, and severe anosmia, and tested positive for SARS-CoV-2 antibodies All rights reserved. No reuse allowed without permission.

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A Cell-Based ELISA to Improve the Serological Analysis of Anti-SARS-CoV-2 IgG

Multicenter nationwide comparison of seven serology assays reveals a SARS-CoV-2 nonresponding seronegative subpopulation

Serum-IgG responses to SARS-CoV-2 after mild and severe COVID-19 infection and analysis of IgG non-responders

Preexisting and de novo humoral immunity to SARS-CoV-2 in humans

Recent endemic coronavirus infection is associated with less-severe COVID-19

mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection

Prospective Longitudinal Serosurvey of Health Care Workers in the First Wave of the SARS-CoV-2 Pandemic in a Quaternary Care Hospital in

Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin

A minimal common outcome measure set for COVID-19 clinical research

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