key: cord-1001465-p2lhpbnq
authors: Kreer, Christoph; Zehner, Matthias; Weber, Timm; Ercanoglu, Meryem S.; Gieselmann, Lutz; Rohde, Cornelius; Halwe, Sandro; Korenkov, Michael; Schommers, Philipp; Vanshylla, Kanika; Di Cristanziano, Veronica; Janicki, Hanna; Brinker, Reinhild; Ashurov, Artem; Krähling, Verena; Kupke, Alexandra; Cohen-Dvashi, Hadas; Koch, Manuel; Eckert, Jan Mathis; Lederer, Simone; Pfeifer, Nico; Wolf, Timo; Vehreschild, Maria J.G.T.; Wendtner, Clemens; Diskin, Ron; Gruell, Henning; Becker, Stephan; Klein, Florian
title: Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients
date: 2020-07-13
journal: Cell
DOI: 10.1016/j.cell.2020.06.044
sha: d2c81972f31dfa95bb4dba17f6e5bfb2dc39ec7b
doc_id: 1001465
cord_uid: p2lhpbnq

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and the world economy. Because approved drugs and vaccines are not available, new options for COVID-19 treatment and prevention are in high demand. To identify SARS-CoV-2-neutralizing antibodies, we analyzed the antibody response of 12 COVID-19 patients from 8 to 69 days after diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days after diagnosis. Of these, 28 potently neutralized authentic SARS-CoV-2 (IC(100) as low as 0.04 μg/mL), showing a broad spectrum of variable (V) genes and low levels of somatic mutations. Interestingly, potential precursors were identified in naive B cell repertoires from 48 healthy individuals who were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2-neutralizing antibodies are readily generated from a diverse pool of precursors, fostering hope for rapid induction of a protective immune response upon vaccination.

In a longitudinal analysis of SARS-CoV-2infected people, Kreer et al. find highly potent neutralizing antibodies that use a broad spectrum of variable (V) genes and show low levels of somatic mutations. They also isolate potential precursors of these SARS-CoV-2-neutralizing antibodies from virus-naive individuals, sampled before the COVID-19 pandemic, to provide evidence that neutralizing antibodies can be readily generated from existing germline antibody sequences found in the general population.

By June 2020, over 8.4 million severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and over 450,000 casualties of the associated coronavirus disease 2019 were reported (Dong et al., 2020; Huang et al., 2020; Zhou et al., 2020; . The exponential spread of the virus has caused countries to shut down public life with unprecedented social and economic consequences. Therefore, decoding SARS-CoV-2 immunity to promote development of vaccines as well as potent antiviral drugs is an urgent health need (Sanders et al., 2020) . Monoclonal antibodies (mAbs) targeting viral surface proteins have been demonstrated to effectively neutralize viruses such as Ebola virus (EBOV) Flyak et al., 2016; Saphire et al., 2018) , respiratory syncytial virus (RSV) (Kwakkenbos et al., 2010) , influenza virus (Corti et al., 2011; Joyce et al., 2016; Kallewaard et al., 2016) , or human immunodeficiency virus 1 (HIV-1) (Walker et al., 2009; Huang et al., 2016a Huang et al., , 2016b Scheid et al., 2011; Schommers et al., 2020; Wu et al., 2010) . The most prominent target for an antibody-mediated response on the surface of SARS-CoV-2 virions is the homotrimeric spike (S) protein. The S protein promotes cell entry through interaction of a receptor-binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2) (Hoffmann et al., 2020; Walls et al., 2020 ). Antibodies that target the S protein are therefore of particular interest to combat the current pandemic (Burton and Walker, 2020; Sempowski et al., 2020) .

SARS-CoV-2 infection induces Q8 a humoral immune response of varying magnitude Ni et al., 2020) , and antibody levels depend on several factors, including disease severity (Long et al., 2020a; Wang et al., 2020b) . The first SARS-CoV-2reactive as well as SARS-CoV-2-neutralizing antibodies have now been isolated from COVID-19 survivors (Brouwer et al., 2020; Cao et al., 2020; Hansen et al., 2020; Ju et al., 2020; Liu et al., 2020a; Robbiani et al., 2020; Seydoux et al., 2020; Shi et al., 2020; Wu et al., 2020; Zost et al., 2020) , immunized animals (Hansen et al., 2020; Wang et al., 2020a; Wrapp et al., 2020a) , and phage display libraries Liu et al., 2020b; Yuan, 2020; Zeng et al., 2020) . Such antibodies are of great value to elucidate neutralization mechanisms, inform vaccination strategies, and potentially treat and prevent SARS-CoV-2 infection, as demonstrated in animal models (Cao et al., 2020; Rogers et al., 2020; Zost et al., 2020) . The antibodies described target various sites on the S protein, including the RBD (Brouwer et al., 2020; Liu et al., 2020a; Seydoux et al., 2020) . However, the affinities and neutralization activities of the reported antibodies vary strongly, and the potential for SARS-CoV-2 escape mutations highlights the need to carefully develop antibodymediated strategies . Moreover, little is known about the likelihood of generating such neutralizing antibodies and how they evolve over time, which will be critical for development of a broadly active SARS-CoV-2 vaccine.

Here, we isolated and sequenced 4,313 S-protein-reactive memory B cells from 12 SARS-CoV-2-infected individuals as early as 8 days after diagnosis (16 days after onset of symptoms). Five patients were followed over a period of 8-69 days after diagnosis to investigate the dynamics of antibody develop-ment against SARS-CoV-2. Besides presenting 28 potent neutralizing antibodies that are currently being evaluated for clinical application, we provide evidence that antibodies develop early after SARS-CoV-2 infection with limited ongoing somatic hypermutation. Finally, we identified potential precursor sequences of potent SARS-CoV-2-neutralizing antibodies in naive B cell repertoires from healthy individuals who were sampled before the SARS-CoV-2 pandemic.

SARS-CoV-2-infected individuals develop a polyclonal memory B cell response against the S protein To investigate the antibody response against SARS-CoV-2, we collected blood samples from seven COVID-19 patients (38-59 years of age) between 8 and 36 days after diagnosis ( Figure 1A ; Table S1 ). Five patients presented with mild symptoms, including dry cough, fever, and dyspnea, whereas two patients were asymptomatic (Table S1 ). Purified plasma immunoglobulin Q9 G (IgG) of all seven individuals showed binding to the full trimeric S ectodomain , and half-maximal effective concentrations (EC 50 ) ranged from 3.1-96.1 mg/mL ( Figure 1B ; Table S2 ). Moreover, neutralizing IgG activity was determined against authentic SARS-CoV-2, showing 100% inhibitory concentrations (IC 100 ) between 78.8 and 1,500 mg/mL in five of seven patients ( Figure 1B ; Table S2 ). To decipher the SARS-CoV-2 B cell and antibody response on a molecular level, we performed single-B-cell sorting and sequence analysis of all individuals. Using flow cytometry, we detected between 0.04% (±0.06) and 1.02% (±0.11) IgG + B cells that reacted with the S ectodomain ( Figures 1C and S1 ). From these, we isolated a total of 1,751 single B cells and amplified IgG heavy and light chains using optimized PCR protocols ( Figure 1C ; Table S3 ) Schommers et al., 2020) . Sequence analysis revealed a polyclonal antibody response with 22%-45% clonally related sequences per individual and 2-29 members per identified B cell clone ( Figure 1D ; Table S3 ). We conclude that a polyclonal B cell response against the SARS-CoV-2 S protein was initiated in all studied COVID-19 patients.

Longitudinal analysis of the SARS-CoV-2 antibody response To delineate the dynamics of the SARS-CoV-2 antibody response, we obtained longitudinal blood samples from an additional five infected individuals at three time points spanning 8-69 days after diagnosis ( Figure 2A ; Table S1 ). Across the different individuals, EC 50 (S ectodomain binding) and IC 100 (SARS-CoV-2 neutralization) values of plasma IgG ranged from also Table S1 ). (B) Binding to the trimeric SARS-CoV-2 S ectodomain (ELISA, EC 50 ) and authentic SARS-CoV-2 neutralization activity (complete inhibition of VeroE6 cell infection, IC 100 ) of cross-sectional poly-IgG samples. Bar plots show arithmetic or geometric means ± SD of duplicates or quadruplicates for EC 50 and IC 100 , respectively. Abbreviation is as follows: n.n., not neutralizing. (C) Dot plots of IgG + B cell analysis. Depicted numbers (percent) indicate average frequencies of S-reactive B cells across several experiments (see also Tables S2 and S3 and Figure S1 ). (D) Clonal relationship of S ectodomain-reactive B cells. Individual clones are colored in shades of blue and green. Numbers of productive heavy-chain sequences are given. Clone sizes are proportional to the total number of productive heavy chains per clone. Please cite this article in press as: Kreer et al., Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients, Cell (2020) , https://doi.org /10.1016/j.cell.2020.06.044 1.54-129 mg/mL and 78.8-1,500 mg/mL, respectively ( Figure 2B ; Table S2 ). For each individual, however, this response remained almost unchanged over the studied period (Figures 2A and 2B) .

To investigate B cell clonality and antibody characteristics on a single-cell level, we proceeded to sort S-ectodomain-reactive IgG + B cells from all five subjects at the different time points (t1, t2, and t3). We found up to 0.65% SARS-CoV-2-reactive B cells, with higher frequencies at later time points and with the number of days after diagnosis being predictive of the fraction of SARS-CoV-2-reactive B cells (p = 0.0016) ( Figure 2C ). From a total of 2,562 B cells, we detected 254 B cell clones (Table S3 ). Fiftyone percent of these clones (129) were detected recurrently, suggesting persistence of SARS-CoV-2-reactive B cells over the investigation period of 2.5 months. When separated by individual time points, the fraction of clonally related sequences ranged from 18%-67% across patients and remained constant or showed only moderate decreases over time ( Figure 2D ).

Next, we analyzed the single-cell Ig sequences (6,587 productive heavy and light chains) from all 12 patients ( Figures 2E, 2F , and S2). Here, clonally related and non-clonal sequences similarly presented a broad spectrum of V H Q10 gene segments, normally distributed heavy-chain complementarity-determining region 3 (CDRH3) lengths, symmetrical CDRH3 hydrophobicity distributions, and a predominance of the IgG1 isotype ( Figure 2E ). However, in comparison with repertoire data from healthy individuals, IgV H 3-30 was overrepresented, and clonal sequences more often facilitated k over l light chains (3 of 4 in clonal versus 2 of 3 in nonclonal sequences, p = 0.0029) ( Figures 2F and S2 ). Finally, V H genes of S-reactive B cells were, on average, less mutated than V H genes from healthy IgG + repertoires (median identity of 98.3 versus 94.3, p < 0.0001) ( Figures 2E and S2) . We concluded that a SARS-CoV-2-reactive IgG + B cell response readily develops after infection with the same B cell clones detectable over time and a preference for facilitating V H gene segment 3-30.

Isolation of highly potent near-germline SARS-CoV-2neutralizing antibodies from COVID-19 patients To determine antibody characteristics and isolate potent neutralizing antibodies, we cloned a total of 312 matched heavy-and light-chain pairs from all 12 patients. These antibodies were primarily selected on the basis of clonality with the aim to include at least one clonal member of several (but at least three) clones per individual (Table S3 ). However, the polyclonal response (median clone size of 2) (Table S3 ) and the lack of differences between clonal and non-clonal sequences suggested the presence of weakly expanded clones in the non-clonal fraction. We therefore also included about one-third randomly selected non-clonal sequences (83 antibodies) for production. From 255 successfully produced IgG1 antibodies, 79 (31%) bound to the full trimeric S ectodomain with EC 50 values ranging between 0.02 mg/mL and 5.20 mg/mL ( Figure 3A ). Of these, 30 antibodies showed SARS-CoV-2 reactivity via a commercial diagnostic system (Euroimmun IgG detection kit) (Figures 3A and 3B; Table S4 ). Surface plasmon resonance (SPR) analyses using the RBD as an analyte for 13 SARS-CoV-2-interacting antibodies gave dissociation constant (K D ) values as low as 0.02 nM (Table  S4) . By determining the neutralization Q11 activity against authentic SARS-CoV-2, we found a total of 28 neutralizing antibodies among 9 of 12 individuals, and IC 100 values ranged between 100 mg/mL (assay limit) and 0.04 mg/mL ( Figures 3C and 3D ). Of note, neutralizing activity was mainly detected among highaffinity antibodies ( Figure 3B ; Table S4 ), and a positive correlation between neutralization and binding could be detected (r s Q12 = 0.429, p = 0.023) ( Figure 3E ).

To better characterize the interaction between SARS-CoV-2 S protein and reactive antibodies, we determined binding to a truncated N-terminal S1 subunit (including the RBD), the isolated RBD, and a monomeric S ectodomain. We found 27 of 28 neutralizing antibodies binding to the RBD but only 29% of the non-neutralizing antibodies, suggesting that the RBD is a major site of vulnerability on the S protein. Epitopes for non-neutralizing antibodies included the N-terminal S1 domain and conformational epitopes ( Figure 3F ; Table S4 ). Notably, neutralizing and non-neutralizing antibodies were characterized by a broad distribution of V H as well as V L Q13 gene segments and a preference for k light chains ( Figures 3G and S4 ). Moreover, 31 of 79 binding and 11 of 28 neutralizing antibodies demonstrated germline identities of 99%-100%, and no correlation was detected between neutralizing activity and the level of somatic mutation (Figure 3G; Table S4 ; Figure S3 ).

Finally, we performed a HEp-2 cell autoreactivity assay. Of 28 neutralizing antibodies, 4 showed low to moderate signs of autoreactivity ( Figure S5 ; Table S4 ), and 2 of them also reacted with other proteins (i.e., Ebola glycoprotein and HIV-1 gp140) (Table S4 ). In summary, these data show that SARS-CoV-2neutralizing antibodies develop from a broad set of different V genes and are characterized by a low degree of somatic mutations. Moreover, we were able to isolate highly potent neutralizing antibodies that are promising candidates for antibodymediated prevention and therapy of SARS-CoV-2 infection.

To investigate the development of somatic mutations over time, we longitudinally analyzed 129 recurring B cell clones that comprised 17 binding and 6 neutralizing antibodies. To this (B) Binding to the trimeric SARS-CoV-2 S ectodomain (ELISA, EC 50 ) and authentic SARS-CoV-2 neutralization activity (complete inhibition of VeroE6 cell infection, IC 100 ) of longitudinal poly-IgG samples. Bar plots show arithmetic or geometric means ± SD of duplicates or quadruplicates for EC 50 and IC 100 , respectively. (C) Percentage of SARS-CoV-2 S ectodomain-reactive IgG + B cells over time (mean ± SD; see also Tables S2 and S3 and Figure S1 ). (D) Clonal relationship over time. Individual clones are colored in shades of blue and green. Numbers of productive heavy-chain sequences per time point are given. (E) Frequencies of V H gene segments (top), CDRH3 length and CDRH3 hydrophobicity (bottom left), as well as V H gene germline identity and IgG isotype of clonal and non-clonal sequences (bottom right) from all 12 subjects and time points. NGS reference data from 48 healthy individuals (collected before the outbreak of SARS-CoV-2) are depicted in red (see also Tables S1 and S2). (F) Ratio of k and l light chains in non-clonal (top, gray) and clonal (bottom, blue) sequences (see also Figure S2 ). Figure S5 and Table S4 ).

(C) Authentic SARS-CoV-2 neutralization activity (complete inhibition of VeroE6 cell infection, IC 100 , in quadruplicates) of S-ectodomain-specific antibodies (red). (D) Geometric mean potencies (IC 100 ) of all neutralizing antibodies. (E) Correlation between S ectodomain binding (EC 50 ) and neutralization potency (IC 100 ). The correlation coefficient r S and approximate p value were calculated by Spearman's rank-order correlation (see also Figure S3 ). (F) Epitope mapping of SARS-CoV-2 S ectodomain-specific antibodies against the RBD, truncated N-terminal the S1 subunit (aa 14-529), and a monomeric S ectodomain construct by ELISA. S2 binding was defined by interaction with monomeric S but not RBD or S1. Antibodies interacting with none of the subdomains were specified as conformational epitopes or not defined.

(legend continued on next page) ll OPEN ACCESS end, we phylogenetically matched all members of a B cell clone at a given time point with the most closely related member at the consecutive time point (331 pairings in total). Mean mutation frequencies in either direction (i.e., toward higher or lower V gene germline identities) were 0.51% ± 0.61%, 0.08% ± 0.51%, and 0.01% ± 0.19% per week for all, binding, and neutralizing clonal members, respectively ( Figure 4A , panel). When averaging the V H gene germline identity of concurrent clonal members, we found a moderate increase in somatic mutations over time (Figure 4A, bottom) . Changes were similar for binding and neutralizing subsets, with one exception among the neutralizing antibodies that accumulated about 5% nucleotide mutations over the investigated period ( Figure 4A , bottom). In line with this finding, neutralizing antibodies isolated on days 8-17 and days 34-42 after diagnosis showed V H gene germline identities of 97.5% and 97.0%, respectively ( Figure 4B ). We concluded that SARS-CoV-2-neutralizing antibodies carry similar levels of somatic hypermutation independent of the time of isolation.

Potential precursor sequences of SARS-CoV-2neutralizing antibodies can be identified among healthy individuals The low rate of somatic mutations in the majority of binding and neutralizing antibodies emphasizes the requirement for the presence of distinct germline recombinations in the naive human B cell repertoire. To estimate the frequency of potential precursor B cells, we performed unbiased heavy-and light-chain next-generation sequencing (NGS) of the naive B cell receptor repertoires from 48 healthy donors (Table S5 ). All samples were collected before the SARS-CoV-2 outbreak and comprised a total of 1.7 million collapsed reads with 455,423 unique heavy, 170,781 k, and 91,505 l chain clonotypes (defined as identical V/J Q14 pairing and the same CDR3 amino acid sequence). Within this dataset, we searched for heavy and light chains that resemble the 79 SARS-CoV-2-binding antibodies ( Figure 5A) . For 14 of 79 tested antibodies, we found 61 heavy-chain clonotypes with identical V/ J pairs and similar (±1 amino acid [aa] in length and up to 3 aa differences) CDRH3s in 28 healthy individuals ( Figures 5B and  4C ), including one exact CDRH3 match (MnC2t1p1_C12). For light chains, we identified 1,357 k chain precursors with exact CDR3 matches that cover 41 of 62 antibodies and 109 l chain precursors that represent 7 of 17 antibodies ( Figures 5B and  4C ). All 48 naive repertoires included at least one k and one l chain precursor. When combining heavy-and light-chain data, we found both precursor sequences of 9 antibodies in 14 healthy individuals ( Figure 5C ). Importantly, among these potential precursor pairs, we found three potent neutralizing antibodies (CnC2t1p1_B4, HbnC3t1p1_G4, and HbnC3t1p2_B10). Although the NGS repertoire data did not include pairing information of heavy-and light-chain combinations, we found matched heavy-and light-chain sequences despite small sample sizes of, on average, 9,500 heavy and 2,000-3,500 light chain clonotypes per individual. We thus conclude that potential SARS-CoV-2-binding and -neutralizing antibody precursors are likely to be abundant in naive B cell repertoires. Table S4 ).

(G) Top: frequencies of V H gene segments for non-neutralizing and neutralizing antibodies. Clonal sequence groups were collapsed and treated as one sample for calculation of the frequencies. Shown on the bottom are the CDRH3 length (left) and V H gene germline identity (right) of non-neutralizing and neutralizing antibodies (see also Figure S4 ). and Marston, 2015; Mascola and Montefiori, 2010; Walker and Burton, 2018; Zolla-Pazner et al., 2019) . A detailed understanding of the human antibody response to SARS-CoV-2 is therefore critical for development of effective immune-mediated approaches against the continuing pandemic (Burton and Walker, 2020; Koff et al., 2013; Kreer et al., 2020b; Sempowski et al., 2020) . Using B cell microcultures, single-cell cloning, or highthroughput single-cell sequencing, SARS-CoV-2-neutralizing antibodies have been identified from a limited number of SARS-CoV-2-infected individuals and survivors of infection with the related sarbecovirus SARS-CoV-1. Most of these antibodies target the RBD of SARS-CoV-2 and interfere with viral binding to its host cell receptor angiotensin-converting enzyme 2 (ACE2) (Andreano et al., 2020; Brouwer et al., 2020; Cao et al., 2020; Hansen et al., 2020; Ju et al., 2020; Pinto et al., 2020; Robbiani et al., 2020; Seydoux et al., 2020; Shi et al., 2020; Wu et al., 2020; Zost et al., 2020) . Demonstrating their potential for clinical application, SARS-CoV-2-neutralizing antibodies can dampen or prevent infection in animal models (Cao et al., 2020; Rogers et al., 2020; Zost et al., 2020) , although antibody combinations might be needed to restrict the potential for viral escape and resistance . Through singlecell analysis of more than 4,000 SARS-CoV-2-reactive B cells from 12 infected individuals, we identified highly potent human monoclonal SARS-CoV-2-neutralizing antibodies that preferentially target the RBD. These antibodies fully block authentic viral infection at concentrations as low as 0.04 mg/mL and therefore provide a potential option for prevention and treatment of SARS-CoV-2 infection.

Affinity maturation of antibodies through somatic hypermutation and clonal B cell selection is a hallmark of the adaptive immune response to pathogens. Chronic infections, such as HIV-1 infection, can result in extensive somatic mutation and amino acid substitutions in more than 30% of the V-gene-encoded region compared with their germline sequences (Klein et al., 2013; Scheid et al., 2011; Schommers et al., 2020; Wu et al., 2010) . Table S5 . Rogers et al., 2020; Seydoux et al., 2020) . However, previously described SARS-CoV-2-reactive antibodies were obtained from single time points, preventing conclusions regarding the molecular dynamics of the B cell response to SARS-CoV-2. By longitudinally analyzing the memory B cell response in five individuals for up to 2.5 months after SARS-CoV-2 transmission, we reveal little additional somatic hypermutation or clonal B cell expansion over time. One explanation for this observation is the potentially limited antigenic B cell stimulation because of rapid viral clearance, to which the cellular response and additional immune factors might contribute (Grifoni et al., 2020; Ni et al., 2020) . In contrast, somatic mutation over several months despite only a brief period of systemically circulating antigen can be observed after infection with EBOV (Davis et al., 2019) or the yellow fever vaccine strain 17D (Wec et al., 2020a) . However, given the high binding affinity of near-germline IgG antibodies against SARS-CoV-2 in the range of nano-to picomolar K D values, these antibodies might limit antigen access to the germinal center, the site of affinity maturation (Zhang et al., 2013) . Although recent data suggest that neutralizing titers correlate with severity of infection (Long et al., 2020b; Wang et al., 2020b) , it remains elusive whether this effect is caused by ongoing somatic hypermutation or ongoing production of highly potent antibodies that were initially generated.

SARS-CoV-2-reactive antibodies could be isolated from three SARS-CoV-2-naive individuals, although these antibodies were only weakly binding (Wec et al., 2020b) . Whether the observation of such antibodies is a consequence of prior exposure to other human coronaviruses, as suggested for SARS-CoV-2-reactive T cells from healthy donors (Braun et al., 2020; Grifoni et al., 2020) , and whether such antibodies and cells can provide background immunity remains to be elucidated. Importantly, our deep sequencing analysis of the naive B cell receptor repertoires of 48 individuals sampled before the pandemic identified potential heavy-and/or light-chain precursors of potent SARS-CoV-2neutralizing antibodies in every single individual. In addition to the limited mutation rate and broad use of antibody gene segments across potent SARS-CoV-2-neutralizing antibodies, these results suggest that protective antibodies can be widely and readily induced by vaccination.

Detailed methods are provided in the online version of this paper and include the following: 

We thank all study participants who devoted time to our research; members of the Klein and Becker Laboratories for continuous support and helpful discussions; Jason McLellan, Nianshuang Wang, and Daniel Wrapp for sharing the SARS-CoV-2 S ectodomain plasmid; Florian Krammer for sharing the RBD plasmid; Simon Pö psel and Robert Hä nsel-Hertsch for helpful discussions and technical support; Daniela Weiland and Nadine Henn for lab management and assistance; as well as Heidrun Schö ßler and Ralf Ortmanns of the health department of Heinsberg for support with patient recruitment. This work was funded by grants from the German Center for Infection Research (DZIF) to F.K. and S.B., the German Research Foundation (DFG) (CRC 1279 and CRC 1310 to F.K., FOR2722 to M.K., and EXC 2064/1 project 390727645 to N.P.), the European Research Council (ERC-StG639961 to F.K.), the Zost, S.J., Gilchuk, P., Case, J.B., Binshtein, E., Chen, R.E., Reidy, J.X., Trivette, A., Nargi, R.S., Sutton, R.E., Suryadevara, N., et al. (2020) . Potently neutralizing human antibodies that block SARS-CoV-2 receptor binding and protect animals. bioRxiv. https://doi.org/10.1101/2020.04.19. 049643.

12 Cell 182, 1-12, August 20, 2020

Please cite this article in press as: Kreer et al., Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients, Cell (2020) Please cite this article in press as: Kreer et al., Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients, Cell (2020) Please cite this article in press as: Kreer et al., Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients, Cell (2020 ), https://doi.org/10.1016 /j.cell.2020 Article RESOURCE AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Florian Klein (florian.klein@uk-koeln.de).

There are restrictions to the availability of SARS-CoV-2-binding antibodies due to limited production capacities and ongoing consumption. Reasonable amounts of antibodies will be made available by the Lead Contact upon request under a Material Transfer Agreement (MTA) for non-commercial usage.

Nucleotide sequences of all SARS-CoV-2-neutralizing antibodies were deposited at GenBank (accession numbers MT658806 -MT658861). Further antibody sequences and NGS data of healthy individuals will be shared by the Lead Contact upon request.

Samples were obtained under a study protocol approved by the Institutional Review Board of the University of Cologne and respective local IRBs (study protocol 16-054). All 12 participants (six females, six males; Table S1 ) provided written informed consent and were recruited at hospitals or as outpatients. Sites of recruitment were Munich Clinic Schwabing for IDMnC1, IDMnC2, IDMnC4, and IDMnC5; the University Hospital of Frankfurt for patients IDFnC1 and IDFnC2; and the University Hospital Cologne for patient IDCnC2. Patients IDHbnC1-5 were recruited as outpatients in the county Heinsberg. Sample size was estimated based on previous studies (Corti et al., 2011; Ehrhardt et al., 2019; Schommers et al., 2020) to sufficiently inform on B cell receptor repertoires and yield neutralizing antibodies. Participants were enrolled and allocated to single blood draws or longitudinal follow-up based on the epidemiology of the infection and participants' availability.

Isolation of peripheral blood mononuclear cells (PBMCs), plasma and total IgG from whole blood Blood draw collection was performed using EDTA tubes and/or syringes pre-filled with heparin. PBMC isolation was performed using Leucosep centrifuge tubes (Greiner Bio-one) prefilled with density gradient separation medium (Histopaque; Sigma-Aldrich) according to the manufacturer's instructions. Plasma was collected and stored separately. For IgG isolation, 1 mL of the collected plasma was heat-inactivated (56 C for 40 min) and incubated with Protein G Sepharose (GE Life Sciences) overnight at 4 C. The suspension was transferred to chromatography columns and washed with PBS. IgGs were eluted from Protein G using 0.1 M glycine (pH = 3.0) and buffered in 0.1 M Tris (pH = 8.0). For buffer exchange to PBS, 30 kDa Amicon spin membranes (Millipore) were used. Purified IgG concentration was measured using a Nanodrop (A280) and samples were stored at 4 C.

The construct encoding the prefusion stabilized SARS-CoV-2 S ectodomain (amino acids 1À1208 of SARS-CoV-2 S; GenBank: MN908947) was kindly provided by Jason McLellan (Texas, USA) and described previously . In detail, two proline substitutions at residues 986 and 987 were introduced for prefusion state stabilization, a ''GSAS'' substitution at residues 682-685 to eliminate the furin cleavage site, and a C-terminal T4 fibritin trimerization motif. For purification, the protein is C-terminally fused to a TwinStrepTag and 8XHisTag. Protein production was done in HEK293-6E cells by transient transfection with polyethylenimine (PEI, Sigma-Aldrich) and 1 mg DNA per 1 mL cell culture medium at a cell density of 0.8 10 6 cells/mL in FreeStyle 293 medium (Thermo Fisher Scientific). After 7 days of culture at 37 C and 5% CO 2 , culture supernatant was harvested and filtered using a 0.45 mm polyethersulfone (PES) filter (Thermo Fisher Scientific). Recombinant protein was purified by Strep-Tactin affinity chromatography (IBA lifescience, Gö ttingen Germany) according to the Strep-Tactin XT manual. Briefly, filtered medium was adjusted to pH 8 by adding 100 mL 10x Buffer W (1 M Tris/HCl, pH 8.0, 1.5 M NaCl, 10 mM EDTA, IBA lifescience) and loaded with a low pressure pump at 1 mL/min on 5 mL bedvolume Strep-Tactin resin. The column was washed with 15 column volumes (CV) 1x Buffer W (IBA lifescience) and eluted with 6 3 2.5 mL 1x Buffer BXT (IBA lifescience). Elution fractions were pooled and buffer was exchanged to PBS pH 7.4 (Thermo Fisher Scientific) by filtrating four times over 100 kDa cut-off cellulose centrifugal filter (Merck).

The RBD of the SARS-CoV-2 spike protein (GenBank: MN908947; aa: 319-541), fused to a hexahistidine tag, was expressed from a plasmid kindly provided by Florian Krammer through transient calcium phosphate transfection of HEK293T cells. After 7 days of culture at 37 C and 5% CO 2 , culture supernatant was harvested and filtered using a 0.45 mm PES filter (Thermo Fisher Scientific). RBD protein was purified via the hexahistidine tag using Ni-NTA agarose (Macherey-Nagel). To this end, filtered culture supernatant was mixed with an equal volume of 2x NPI10 buffer (1x NPI10 buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8). Ni-NTA beads were equilibrated with NPI10 and added to the cell culture supernatant/NPI10 mix (1 mL bed volume per 1000 mL original cell culture supernatant), followed by incubation at 4 C over night with constant rotation. Beads were harvested by centrifugation at 500 g for 5 min at 4 C and washed twice in NPI10 (100 mL per 1000 mL original cell culture supernatant), followed by centrifugation at 500 g for 5 min at 4 C. Beads were additionally washed three times in NPI20 (50 mM NaH 2 -PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8). Beads were then transferred in NPI20 to Polyprep chromatography columns (BioRad, 2 columns per 1000 mL original cell culture supernatant) and washed with 10 mL NPI20 per column. Protein was eluted with 5 mL NPI250 (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8). Buffer was exchanged to PBS using 10 kDa Amicon spin columns (Millipore). SARS-CoV-2 S ectodomain ''monomer'' without trimerization domain (GenBank: MN908947; aa:1-1207) and S1 subunit (Gen-Bank: MN908947; aa:14-529) regions of the spike DNA were amplified from a synthetic gene plasmid (furin site mutated) (Wrapp et al., 2020 Q19 ) by PCR. PCR products were cloned into a modified sleeping beauty transposon expression vector containing a C-terminal thrombin cleavage and a double Strep II purification tag. For the S1 subunit, the tag was added at the 5 0 end and a BM40 signal peptide was included. For recombinant protein production, stable HEK293 EBNA cell lines were generated employing the sleeping beauty transposon system (Kowarz et al., 2015) . Briefly, expression constructs were transfected into the HEK293 EBNA cells using FuGENE HD transfection reagent (Promega). After selection with puromycin, cells were induced with doxycycline. Supernatants were filtered and the recombinant proteins purified via Strep-TactinâXT (IBA Lifescience) resin. Proteins were then eluted by biotin-containing TBS-buffer (IBA Lifescience), and dialyzed against TBS-buffer.

Ebola surface glycoprotein (EBOV Makona, GenBank: KJ660347; GP aa:1-651) and HIV-1 gp140 (strain YU2, GenBank: M93258; Env aa:1-683), both lacking the transmembrane domain and containing a GCN4 trimerization domain , were expressed by transient transfection of HEK293-6E cells using PEI, following the same protocol and culture conditions as for the prefusion stabilized SARS-CoV-2 S ectodomain described above. After 7 days, supernatants were filtered using a 0.45 mm polyethersulfone (PES) filter (Thermo Fisher Scientific) and proteins were purified by affinity chromatography through their hexahistidine tag with Ni-NTA agarose (Macherey-Nagel) following the same protocol as for the RBD purification described above.

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies

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China Novel Coronavirus Investigating and Research Isolation of SARS-CoV S ectodomain-specific IgG + B cells B cells were isolated from PBMCs using CD19-microbeads (Miltenyi Biotec) according to the manufacturer's instruction. Isolated B cells were stained for 20 min on ice with a fluorescence staining

anti-human CD27-PE (BD) and DyLight488-labeled SARS-CoV-2 spike protein (10mg/mL). Dapi À , CD20 + , IgG + , SARC-CoV-2 spike protein positive cells were sorted using a FACSAria Fusion (Becton Dickinson) in a single cell manner into 96-well plates. All wells contained 4 ml buffer, consisting of 0.5x PBS, 0.5 U/ml RNAsin (Promega), 0.5 U/ml RNaseOUT (Thermo Fisher Scientific), and 10 mM DTT (Thermo Fisher Scientific)

Antibody heavy/light chain amplification and sequence analysis Single cell amplification of antibody heavy and light chains was mainly performed as previously described

Unique molecular identifiers (UMIs) were extracted and paired reads were pre-annotated with IgBLAST. Based on IgBLAST pre-annotation, an additional molecular identifier (MID) was extracted by taking consecutive 18 nucleotides (nt) starting 12 nt downstream of the end of framework region (FWR) 3. For error correction, reads with the same UMIs were grouped. Reads that did not match the most abundant V gene call or had more than 1 nt difference to any other read were removed from their assigned UMI group. Assuming that ungrouped and removed reads may result from RT, PCR, or sequencing errors within the UMI, the remaining single as well as the removed reads were re-grouped by their MID. MID groups with a unique V gene call and no more than 1 nt difference between included UMIs were re-defined as a novel UMI group. Clustal omega was used to align all reads within each corrected UMI group and aligned sequences were collapsed to build consensus reads. For consensus building, base calls were weighted by their quality (1 -error probability) and bases with the highest quality-weighted frequencies were taken as the consensus. Paired consensus reads were assembled with the pRESTO AssemblePairs module and a minimal overlap set to 6 nt. Assembled sequences were annotated with IgBLAST and productive sequences were kept for analyses. To minimize the influence of sequencing and PCR errors, NGSderived sequences were only evaluated for UMI groups with at least three reads. For the identification of overlapping clonotypes in healthy individuals a maximum of one amino acid length difference and three or less differences in absolute amino acid composition of CDR3s were considered as similar. Cloning and production of monoclonal antibodies Antibody cloning from 1 st PCR products was performed as previously described

35 cycles of 98 C for 10 s, 72 C for 45 s; and 72 C for 2 min. PCR products were purified (NucleoSpin 96 PCR Clean-up, Macherey Nagel), cloned into expression vectors by SLIC using T4 DNA polymerase (NEB) and chemical competent Escherichia coli DH5a. After verification of positive colonies by colony PCR and Sanger sequencing

Following transfection, cells were maintained in FreeStyle 293 Expression Medium (Thermo Fisher) and 0.2% penicillin/streptomycin (Thermo Fisher) at 37 C and 6% CO 2 and kept under constant shaking at 90-120 rpm. Seven days after transfection, supernatants were harvested and clarified by centrifugation and subsequent filtration using PES filters. For antibody purification, Protein G-coupled Sepharose beads (GE Life Sciences) were incubated with culture supernatants

Corning 3369) were coated with 2 mg/ml of protein in PBS (SARS-CoV-2 spike ectodomain, RBD, or n-terminal truncated S1) or in 2 M Urea (SARS-CoV-2 spike ectodomain ''monomer'' lacking the trimerization domain) at 4 C overnight. For SARS-CoV-2 spike ectodomain ELISA, plates were blocked with 5% BSA in PBS for 60 min at RT, incubated with primary antibody in 1% BSA in PBS for 90 min, followed by anti-human IgG-HRP (Southern Biotech 2040-05) diluted 1:2500 in 1% BSA in PBS for 60 min at RT. SARS-CoV-2 spike subunit ELISAs were done following a published protocol

Briefly, samples were serially diluted in 96-well plates starting from a concentration of 1,500 mg/ml for poly-IgG and 100 mg/ml for monoclonal antibodies. Samples were incubated for 1 h at 37 C together with 100 50% tissue culture infectious doses (TCID 50 ) SARS-CoV-2 (BavPat1/2020 isolate, European Virus Archive Global # 026V-03883)

Surface Plasmon Resonance (SPR) measurements

For SPR measurement, the RBD was additionally purified by size exclusion chromatography (SEC) purification with a Superdex200

Purified mAbs were first immobilized at coupling densities of 800-1200 response units (RU) on a series S sensor chip protein A (GE Healthcare) in PBS and 0.02% sodium azide buffer. One of the four flow cells on the sensor chip was empty to serve as a blank. Soluble RBD was then injected at a series of concentrations (i.e., 0.8, 4, 20, 100, and 500 nM) in PBS at a flow rate of 60 mL/min. The sensor chip was regenerated using 10 mM Glycine-HCl pH 1.5 buffer. A 1:1 binding model was used to describe the experimental data and to derive kinetic parameters. For some mAbs, a 1:1 binding model did not provide an adequate description for binding. In these cases, we fitted a two-state binding model that assumes two binding constants due to conformational change

HEp-2 Cell Assay Monoclonal antibodies were tested at a concentration of 100 mg/ml in PBS using the NOVA Lite HEp-2 ANA Kit (Inova Diagnostics) according to the manufacturer's instructions, including positive and negative kit controls on each substrate slide. HIV-1-reactive antibodies with known reactivity profiles were included as additional controls. Images were acquired using a DMI3000 B microscope (Leica) and an exposure time of 3.5 s, intensity of 100%

Based on the phylogenetic tree distances, all variants of a clone at a given time point were matched to variants at the consecutive time point and the slope between the pairs was computed. Hamming distances between the pairs were determined and normalized for sequence length and time difference to calculate the mean mutation frequency per day. Given the median slope per clone, a onesided Wilcoxon Signed Rank Test was applied to test whether the slopes are equal to zero, with the alternative hypothesis that the slopes are smaller than zero. For visualizing the change of V H gene germline identity over time (Figure 4A, bottom), the germline identity for each clone was normalized by its median value at the first-time measurement and the median slope was plotted. To test for a difference in V H gene germline identity between neutralizing antibodies that were isolated at early or late time points