Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.


Introduction
The COVID-19 pandemic is caused by the newly emerged SARS-Coronavirus-2 (SARS-CoV-2) and has incomparable public health, economic, and social consequences [1,2]. Generation of neutralizing antibodies (nAbs) that specifically target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein is thought to be essential in controlling SARS-CoV-2 [3][4][5]. Support of this assumption is the fact that RBD specific nAbs have been detected and isolated from convalescent patients that have recovered from COVID-19 as early as 8 days post diagnosis [6], exhibiting little clonal expansion and low numbers of somatic hyper mutations [7][8][9][10]. However, the critical survey of the humoral response to SARS-CoV-2 infection is somewhat confusing. Some of the recoverees tend to have robust and long lasting immunity while others display a waning of their neutralizing antibodies [6,11]. The correlates of effective durable antibody response are still unclear.
Infection with SARS-CoV-2 varies clinically from asymptomatic in some infected individuals to a severe disease associated with morbidity and mortality in others [12,13]. The development of an effective antiviral antibody response may limit viral dissemination, thereby preventing a severe disease. Alternatively, severe disease can be prevented by T cell-mediated immunity or innate effector mechanisms which independently of B cells restrict viral replication and disease progression. To assess the clinical profile associated with effective antibody responses following SARS-CoV-2 infection, we recruited 18 SARS-CoV-2 infected individuals 6 weeks post infection. Eight out of the 18 donors had been hospitalized due to severe COVID-19 (termed "Severe"), while the remaining 10 donors had experienced mild or no symptoms (termed "Mild"). The intention of the present study was to comprehensively analyze the cellular, molecular, and functional aspects of the antibody responses in Severe versus Mild SARS-CoV-2 convalescent donors in order to understand the potential roles of antibodies in COVID-19. Our conclusion is that severe, and not mild infection, is correlated with robust SARS-CoV-2 antibody responses. Furthermore, we isolated 6 neutralizing antibodies from two severely affected donors and characterized their binding mechanisms using peptide libraries, computational methods and mutagenesis while examining their anti-viral efficacy alone and in combination against authentic SARS-CoV-2.

Severe COVID-19 disease is accompanied by stronger IgG responses compared to mild
To characterize the antibody repertoire in COVID-19 convalescent donors, we examined B cell responses from 18 donors (S1 Table). Based on clinical information, the donors were divided into two groups; 8 donors had experienced severe disease manifestations following SARS-CoV-2 infection with >1 week of high fever (above 38.5˚C), and pneumonia that required hospitalization. These donors were defined as Severe. The remaining 10 donors were largely asymptomatic following SARS-CoV-2 infection, or had reported only mild symptoms such as loss of taste and/or smell, fatigue, or fever lasting 2 for days or less. These donors were defined as Mild. The Spike protein is responsible for viral attachment and entry into the host cells, and hence, is the target of most of the antibody responses [14][15][16]. Within the Spike protein, the receptor binding domain (RBD) is the minimal subunit that mediates viral entry [4,5] and was reported to be the main target for neutralizing antibodies elicited during infection [7,[17][18][19][20][21][22][23][24]. Therefore, we first asked whether the Mild donors had developed higher antibody plasma responses towards the SARS-CoV-2 Spike RBD (produced in house, S1 Fig). Mild donors had significantly less anti-RBD IgG than Severe donors (p = 0.0008, Figs 1A and S2A), while no differences were observed between Mild and Severe groups for anti-RBD IgM and IgA (Figs 1B, 1C and S2B and S2C). Moreover, ELISA revealed that plasma from Mild donors had significantly weaker ability to inhibit RBD binding to a soluble angiotensin converting enzyme 2 (ACE2, produced in house, S1 Fig) compared to plasma from the Severe donors' group (p < 0.0001, Figs 1D and S2D). As expected, the levels of anti-RBD IgG, but not IgM or IgA, were directly correlated with the ability of the plasma to inhibit RBD:ACE2 binding (r 2 = 0.9018, p < 0.0001, Fig 1E-1G). Antibody responses against related respiratory viruses were previously reported to improve the outcome of a SARS-CoV-2 infection [25]. We, however, did not observe differences between the Mild and Severe groups in their IgG titers against the closely related seasonal beta Coronaviruses OC43 and 229E, as well as in IgG titers against Cytomegalovirus (CMV) and Herpes Simplex Virus 1 (HSV1) (S3 Fig). We therefore conclude that, compared to Severe donors, Mild donors have lower titers of SARS-CoV-2 IgG, and a decreased ability to inhibit RBD:ACE2 binding, while no difference in IgG directed against beta Coronaviruses OC43 and 229E.

Severe Covid-19 patients exhibit distinct BCR signatures
To compare B cell responses in Mild versus Severe donors at the molecular level, we performed total B cell receptor (BCR) sequencing using a single cell 10x platform on seven Mild donors (CoV03, CoV04, CoV05, CoV07 CoV10, CoV11, and CoV14, Fig 2A), and six Severe donors (CoV01, CoV02, CoV09, CoV12 CoV13, and CoV16). Insufficient cell amounts were available for analysis from donors CoV08, CoV17, CoV15, and CoV18, and donor CoV06 was excluded due to clinical reasons. When comparing the BCR dataset from the total 13 COVID-19 donors (Mild and Severe donors) to two COVID-19 unexposed BCR datasets (7 BCR repertoires from mature B cells, and 42 BCR repertoires from naïve B cells), 6 VHJH combinations were overrepresented, and 3 VHJH combinations were under-represented in COVID-19 samples (marked in green and red, respectively, Fig 2B) Fig 2C). In addition, VH3-53 gene was significantly overexpressed in the Severe group when compared to Mild (p = 0.004, Fig 2D and 2E), whereas no significant differences in VK gene usage was found between the two groups of COVID-19 donors (S4B and S4C Fig). When comparing both heavy and light chains, 18 VHVL combinations were enriched in Severe, and 4 VHVL combinations were enriched in the Mild patients. Overall, these results indicate that severe SARS-CoV-2 infection is associated with unique B cell signatures, enrichment of VH3-53, and a greater B cell clonal expansion.

mAbs from donors CoV01 and CoV02 effectively neutralize SARS-CoV-2
While the frequencies of anti-RBD B cells were not statistically significantly different between the two groups, on average, Mild donors display lower frequencies of anti-RBD B cells compared to Severe donors (Figs 3B and S5). Taken together with the plasma and the BCR-seq profiling, this suggests that higher B cell responses are associated with severe, and not mild, SARS-CoV-2 infection. We therefore focused on the Severe group of COVID-19 convalescent donors. To investigate antibody responses in the Severe group, we used single B cell sorting method using flow cytometry as previously described [7,26] with SARS-CoV-2 RBD as a bait to derive monoclonal antibodies for analysis. We selected two donors, CoV01 and CoV02, a married couple, who were amongst the first 10 documented COVID-19 cases in Israel (in January 2021, there are aproximately 600,000 positive cases in Israel). CoV01 infected CoV02 following a visit to Italy, as well as two of their three children. Both donors were hospitalized due to severe pneumonia and prolonged high fever. While both donors had strong anti-RBD IgG responses by ELISA (Fig 1A), CoV01 had a lower frequency of anti-RBD B cells compared to CoV02 (Fig 3B). Using single B cell sorting and Sanger sequencing as previously described [26,27] we were able to sequence a total of 35 and 118 RBD-specific paired heavy and light chains from memory B cells of CoV01 and CoV02, respectively ( Fig 3C). The two donors exhibited divergent B cell responses. While 19.5% of the sequences extracted from donor CoV02 comprised 8 clonally related families, the sequences of donor CoV01 showed no clonality, possibly due to a lower number of anti-RBD B cells (Fig 3B). The degree of somatic hypermutations were higher in CoV01, while the sequences from CoV02 were similar to germline ( Fig 3D). The CDRH3 lengths of the sequences were similar to each other ( Fig 3E). Next, we focused on antibodies that were part of the expanded clones as previously described [7,26]. For sequences from donor CoV01, where we did not observe clonal expansion, we selected the most mutated sequences for production. We generated 9 and 13 monoclonal antibodies from donors CoV01 and CoV02, respectively (S2 Table). Eight of these mAbs (3 from CoV01 and 5 from CoV02) bound SARS-CoV-2 RBD (Figs 3F and S6A and S6B left panels and S7) and stabilized Spike trimer [28] protein by ELISA (Figs 3F and S6A and S6B right panels). mAb TAU-1109 exhibited 40% competition with the previously described mAb CR3022 [29], which recognizes a non-ACE2 binding site (non-ACE2bs, Fig 3G) [30]. mAbs TAU-1145, TAU-2189, TAU-2230, and TAU-2303 inhibited RBD binding to both soluble human ACE2 (hACE2) by ELISA (Fig 3H), and to hACE2 expressing cells (Figs 3I and S8).

PLOS PATHOGENS
Next, we asked whether the mAbs have neutralizing activity. We first used lentivirus-based GFP-reporter pseudo-viruses that present SARS-CoV-2 Spike on their membrane to infect hACE2-expressing HEK-293 cells (S9 Fig). All the RBD and Spike-binding mAbs had some neutralizing activity in the pseudo-viral assay with IC50 ranging between 0.04 μg/mL to 3.8 μg/mL ( Fig 4A). We then tested the neutralizing activity of the mAbs against authentic SARS-CoV-2. Vero E6 cells were infected with authentic SARS-CoV-2 in the presence of mAbs, and after 48 h virus was detected using fluorescently labeled anti-nucleocapsid antibody. Five of the nine mAbs that neutralized the pseudo-typed virus, also neutralized authentic virus when used at a concentration of 10 μg/mL (TAU-1109, -2212, -2189, -2230, and -2303, Figs 4B, 4C and S10), while four mAbs that demonstrated neutralizing activity in the pseudoviral assay (TAU-1115, -1145, -2220 and -2310) did not inhibit authentic SARS-CoV-2. To further screen for neutralizing activity of these mAbs, the viability of the cells was tracked by cell imaging for three days to examine the effects of mAb titrations on virus-induced cell death. The same mAbs (TAU-1109, -2212, -2189, -2230, and -2303) dramatically decreased the number of dead cells when used at concentrations ranging from 1-100 μg/mL (Figs 4D and S11), and two of the mAbs, TAU-2212 and TAU-2230 inhibited syncytia formation (Fig 4E and 4F). Interestingly, mAb TAU-2212 demonstrated impressive neutralizing ability in all three assays (pseudo-virus, authentic virus and syncytia formation) despite having no detectable binding to either SARS-CoV-2 RBD or the Spike trimer by ELISA ( Fig 3F). Using fluorescent microscopy and flow cytometry, we confirmed the binding of TAU-2212 to the viral Spike when the trimer was transiently expressed on the surface of HEK-293 cells (Figs 4G and S12). It is known that the SARS-CoV-2 Spike protein transitions rapidly between an open and a closed state [31], suggesting that TAU-2212 may be targeting a conformation-dependent epitope, or an epitope that involves more than one subunit.

G446 is a key residue in the binding of ACE2bs nAbs
We sought to further explore the epitopes of ACE2bs neutralizing mAbs (ACE2bs nAbs) on RBD. We focused on nAb TAU-2230 that exhibited over 80% blockade of ACE2:RBD interaction by ELISA (Fig 3H). To map the epitope of this nAb on the surface of RBD we used phagedisplayed random peptide libraries and the Mapitope algorithm [32]. In this method, one maps linear affinity selected peptides pulled from a random peptide library onto the 3-D atomic coordinates of the antigen to get an output of 2-3 epitope candidates [33]. A total of 16 peptides were isolated with TAU-2230 (Fig 5A), and these were used as input for Mapitope to generate an epitope prediction. Three epitope candidates were predicted, Clusters A-C, one of which, Cluster B, overlapped with the ACE2bs (S13 Fig). Cluster B had 4 shared contact residues with ACE2bs [4] (Fig 5B). To identify key residues for TAU-2230 recognition, we introduced a panel of point mutations in RBD in the TAU-2230 predicted epitope, as well as  outside the predicted epitope, as control. We then tested the binding of other RBD-binding mAbs, as well as ACE2, to the generated RBD mutants. Mutation of a single residue G446A RBD , that was in the core of the predicted TAU-2230 epitope, abolished completely the binding of TAU-2230, as well as the binding of other ACE2bs nAbs, TAU-2189 and 1145 ( Fig  5C and 5D). Mutating residues T500D RBD and G496A RBD reduced the binding of ACE2, but did not affect TAU-2230, suggesting that TAU-2230 binding to these residues might be through interaction with the backbone atoms rather than the sidechains ( Fig 5C). Antibody TAU-1109 that binds the RBD strongly, and binds outside the ACE2bs (does not compete with ACE2 for binding) served as a positive control indicating that the conformation of the mutated RBD proteins was intact. Based on these computational and mutagenesis analyses we conclude that residue G446 is central in the recognition of RBD by ACE2bs nAbs.

Combinations of nAbs directed against different sites effectively block virus spread
Combination therapies have now become the paradigm for antiviral therapy and to avoid viral escape mutants [2,34], and were proven effective in preventing SARS-CoV-2 escape mutations [17]. We therefore investigated whether combinations of our nAbs could synergistically block viral infection and viral spread. The nAbs we isolated are directed against at least three different sites on the virus; CR3022-proximal (TAU-1109), an unknown conformational epitope (TAU-2212) and the ACE2bs (TAU-2230, -2189, -1145, and -2303). Therefore, we combined nAbs TAU-2212 and TAU-1109 at a concentration of 1 and 10 μg/mL, with each one of the anti-ACE2bs nAbs, in a double and triple mixes (Fig 6). In general, nAb TAU-2212 exhibited a more substantial effect compared to nAb TAU-1109. Also, the combination of 1 μg/ml of three nAbs (TAU-1109 + TAU-2212 + ACE2bs nAb) was in all cases more potent than 10 μg/ml of a single ACE2bs nAb (with the exception of TAU-2230 where 10 μg/ml of the single nAb was as potent as 1 μg/ml of three nAbs, Fig 6A). When using 10 μg/ml of TAU-1109 and TAU-2212 in a triple combination with ACE2bs nAbs, complete viral inhibition was achieved in all cases, although most of the effect was contributed by TAU-2212 and not TAU-1109 ( Fig 6B). We conclude that combining TAU-2212 with either ACE2bs nAbs or with TAU-1109 is a potent formula for authentic SARS-CoV-2 inhibition.

The precursors of nAbs are frequent in the BCR repertoire
The nAbs described in this study were elicited naturally following infection and therefore represent a desired response following vaccination. We therefore examined the frequency of these nAbs in the mature and naïve BCR repertoires. We were able to identify the VHJH combinations that give rise to these nAbs in 7 COVID-19 unexposed mature BCR repertoires, as well as in 42 COVID-19 unexposed naïve BCR repertoires. Moreover, the VHJH combinations for CoV01-CoV17. No PBMCs were obtained for donor CoV18, therefore this donor was not included. Symbol code is given on the right. (C) Pie charts representing the total number of RBD-specific memory B cell sequences (heavy chains) obtained from donors CoV01 and CoV02. The numbers in the middle of the pies represent the total number of sequences, and the pink shaded slices represent B cell clonal families. The white slices represent single sequences that were not clonal. (D) Number of nucleotide substitutions in VH of the B cell sequences of CoV01 (red) and CoV02 (magenta). Statistical analysis was calculated using one-way ANOVA. (E) Amino acid length of CDRH3 from B cell sequences isolated from CoV01 (red) and CoV02 (magenta). (F) AUC in ELISA for each one of the 22 cloned mAbs against SARS-CoV-2 RBD (left) and Spike (right) proteins. Color code is given on the right of (H). (G) and (H) Degree of mAbs competition with mAb CR3022, and ACE2, as assessed by ELISA. mGO53 was used as isotype control. (I) Frequency of RBD-PE stained hACE2-expressing cells identified by flow cytometry in the presence of the 22 mAbs isolated from CoV01 and CoV02. mAbs that reduced RBD binding to hACE2 are marked with black arrows. Unlabeled RBD was used as a positive control.
https://doi.org/10.1371/journal.ppat.1009165.g003 nAbs TAU-1145, -2212, -2230, -2189, and -2310 were present within the 30 most frequent VHJH combinations. Our data indicates that SARS-CoV-2 neutralization can be achieved in many ways and that the germline gene combinations that give rise to anti-SARS-CoV-2 nAbs are relatively frequent in the healthy BCR repertoire. Therefore, given enough antigenic stimulation (which is the case during severe COVID-19), these nAbs may be produced by the majority of individuals (Fig 7).

Discussion
In the present study we analyzed the B cell responses in convalescent COVID-19 donors, while focusing on antibodies produced in two donors with severe disease manifestations. By now, several studies showed that COVID-19 severity correlates with neutralization [7,11,35], and that anti-RBD antibody response dominates SARS-CoV-2 neutralization, although neutralizing antibodies can be directed against other viral determinants besides the RBD [9,18]. In our cohort of 18 donors all patients with symptomatic SARS-CoV-2 infection exhibited high titers of anti-RBD antibodies, in contrast to donors with milder symptoms, who exhibited a wide range of responses, ranging from high to non-detectable.
In agreement with other studies, the antibodies sequenced from memory B cells of the donors we investigated demonstrated a relatively low degree of somatic hyper mutations and clonal expansion [7,8,10,18]. Within the convalescent donors, Severe donors had higher level of shared B cell clones compared to Mild donors, likely due to increased antigenic stimulation and/or prolonged viremia to drive expansion of SARS-CoV-2-specific B cells. Moreover, the Severe donors had an increased representation of VH3-53 that was previously described as dominant in responses against SARS-CoV-2 [19,31,36]. However, the nAbs we isolated from CoV01 and CoV02 donors did not originate from this VHs and therefore constitute new VH genes that can give rise to potent anti-SARS-CoV-2 responses. Similarly to what has been reported [7,18,22], both donors, CoV01 and CoV02, exhibited neutralizing activity that was multi-clonal, i.e., comprised of several neutralizing clones targeting different sites on the Spike. The contact residues of the anti-ACE2bs nAb TAU-2230 were predicted to partially overlap with ACE2bs, using similar tools to the ones we used to map the epitope of the SARS mAb 80R in 2006 [37]. This coincides with previous reports highlighting the ACE2bs as a major neutralizing determinant on the different Coronaviruses that is targeted by antibodies elicited during infection in humans [21]. Additional two groups of neutralizing determinants, outside the ACE2bs, are targeted by nAbs TAU-1109 and TAU-2212. These nAbs bind outside the ACE2bs and their epitopes, that are still to be determined, may constitute new sites of vulnerability of SARS-CoV-2, as well as targets for vaccines. The nAbs we isolated did not require substantial affinity maturation, as evidenced from the low numbers of somatic hypermutations. Moreover, bioinformatic analysis of 7 + 42 uninfected BCR repertoires revealed that the precursors that give rise to these nAbs are abundant in the naïve population. Somewhat counterintuitively, many of the mild donors in our cohort did not develop anti-RBD IgGs that were able to inhibit RBD:ACE2 interaction (CoV05, -06, -07, -11, -14, and -18). This suggests that while most individuals have SARS-CoV-2 nAb precursors in their BCR repertoires, a single dose vaccine may not be sufficient to elicit protective anti-SARS-CoV-2 immunity.
Combinations of nAbs were previously shown to effectively prevent the selection of HIV-1 escape mutants [38,39]. While the mutation rates of SARS-CoV-2 are significantly lower compared to those of HIV-1, SARS-CoV-2 has been reported to mutate in culture in the presence of nAbs [40], emphasizing the importance of combination of antibodies targeting different weak spots on the virus. Recently, a combination of two nAbs targeting non-overlapping sites on SARS-CoV-2 Spike, effectively blocked the spread of SARS-CoV-2 in culture, as well as in a humanized mice model [17,41]. The combinations examined in our study can be further tested in clinical settings as a useful means of prevention and therapy of COVID-19.

Ethics statement
All COVID-19 convalescent donors, between the ages 25 and 65 recruited for this study provided a written informed consent prior to participating in this study. All donors tested positive

Cloning, expression, and purification of SARS-CoV-2 RBD and the extracellular human ACE2 for ELISA and FACS
Codon optimized sequences encoding the SARS-CoV-2 RBD and hACE2 were downloaded from the NCBI data base, synthesized by Syntezza-Israel, and cloned into the pcDNA 3.1 mammalian expression vector. Each construct contained an N-terminal growth factor receptor signal peptide [MKAPAVLAPGILVLLFTLVQRSNG] and two C-terminal tags-a hexa-histidine tag (HHHHHH, "His-tag") for downstream protein purification and a site-specific biotinylation tag (GLNDIFEAQKIEWHE, AviTag). Each construct was individually used to transiently transfect Expi293F cells (Thermo Fisher Scientific Inc.) using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific Inc.). Seven days post transfection, the cell supernatant was collected, filtered (0.22 μm), and incubated with Ni 2+ -NTA agarose beads (GE Life Sciences) for 2 h at room temperature (RT). Proteins were eluted by 250 mM imidazole, buffer-exchanged to PBS x1, and then aliquoted and stored at -80˚C. The RBD protein was biotinylated with the BirA biotin-protein ligase kit (Avidity LLC, Colorado, USA) using the manufacturer's protocol.

Isolation of plasma and PBMCs from whole blood samples
Plasma was isolated from 10 mL of each sample and the rest was used for isolation of peripheral blood mononuclear cells (PBMCs). For plasma separation, the sample was left in the collection tube (BD Vacutainer blood collection tubes, Becton, Dickinson and Company) in an upright position for 2 h followed by centrifugation at 1500 g for 15 min. The upper layer containing the plasma was collected, aliquoted, and stored at -20˚C. PBMCs were isolated by diluting whole blood sample 3-fold with RPMI 1640 medium (Biological Industries Israel Beit-Haemek) and loading the material on Ficoll-Paque PLUS (GE Life Sciences) at a ratio of 1.8: 1 for phase separation according to the manufacturer's protocol. The lymphocyte containing buffy coat layer was transferred to a new vial, washed, resuspended in fetal bovine serum (FBS) containing 10% DMSO, and stored in liquid nitrogen.

Plasma and mAb ELISA
For plasma ELISA, high-binding 96 well ELISA plates (Corning #9018) were coated with 1 μg/ mL RBD in PBS x1 overnight at 4˚C. The following day, the coating was discarded, the wells were washed with "washing buffer" containing PBS x1 0.05% Tween 20, and the plates were blocked for 2 h at RT with 200 μL of "blocking buffer" containing PBS x1 3% BSA (MP Biomedicals) 20mM EDTA and 0.05% Tween20 (Sigma). Plasma samples were diluted 2-fold in blocking buffer starting from 1:100 with seven consecutive dilutions, and incubated for 1 h at RT. Plates were then washed 3 times with washing buffer before adding secondary anti-IgG, or -IgM (Jackson ImmmunoResearch 109-035-088 & 109-035-129) or anti-IgA (abcam ab97215) antibodies conjugated to horseradish peroxidase (HRP) diluted 1:5000 in blocking buffer, and incubated for 45 min at RT. Following four additional washes, 100 μL of TMB/E (Millipore) was added to each well and the absorbance at 650 nm was read after 20 min (BioTek 800 TS). Positive and negative controls were added to each plate. For mAb ELISA, high binding 96 well ELISA plates were coated with 1 μg/mL RBD, washed and blocked as described above. Antibodies were added at starting concentration of 10 μg/mL with nine additional 4-fold dilutions in PBS x1, and incubated for 1 hour at RT. Plates were then washed 3 times with washing buffer before adding secondary anti-IgG HRP-conjugated as described above. After four additional washes, 100 μL of TMB/E (Millipore) was added to each well and the absorbance at 650 nm was read after 20 min. For ACE2:RBD inhibition ELISA, high-binding 96 well plates were coated with 2 μg/mL human ACE2 in PBS x1 overnight at 4˚C. The next day, plates were washed and blocked with blocking buffer for 2 h at RT. Concurrently, biotinylated RBD was mixed with the 4-fold serial dilutions of plasma or mAbs. The RBD-plasma/mAb mix was then applied to the ACE2 coated plates and incubated for 30 min. Biotinylated RBD was detected via streptavidin conjugated to HRP (Jackson ImmmunoResearch 016-030-084).
For CR3022 competition, ELISA plates were coated with 0.5 μg/mL RBD as described, and then washed and blocked. Serial dilutions of each mAb were added and the plates were incubated for 30 min. Next, biotinylated CR3022 at a concentration of 0.1 μg/mL was added to each well and incubated for an additional 15 min. The plates were then washed and incubated with streptavidin-HRP for 45 min. The plates were then washed and the signal detected as described above.
ELISA to validate affinity-selected phages binding to TAU-2230 mAb was carried out as follows: High-binding 96 well ELISA plates (Corning #3590) were coated with 2 μg/mL mAb TAU-2230 overnight at 4˚C. The following day, the wells were blocked for 1 h at RT with blocking solution (5% skim milk in TBS x1). After washing once with TBS x1 0.05% Tween 20, purified phages diluted in blocking solution were applied to each well, starting from 4 x 10 8 phages per well to give a total of five consecutive 3-fold dilutions. The plates were incubated for 1 h at RT, then washed 3 times with wash buffer, and incubated with rabbit anti-m13 polyclonal sera at a dilution of 1:5000 in blocking solution for 1 h at RT. After 3 washes, the plates were incubated at RT with a secondary anti-rabbit HRP-conjugated antibody (Jackson, West Grove, PA) at a dilution of 1:2500 in blocking solution for 45 min. The wells were then washed 3 times wash buffer and once with TBS x1, followed by the addition of the TMB/E ELISA substrate (Merck Millipore, Billerica, MA). Absorbance was measured as described.

scRNA-seq library construction and BCR-seq data analysis
B cells were isolated from frozen PBMCs by using anti-CD19 immunomagnetic beads according to the manufacturer's protocol (CD19 Microbeads, human, Miltenyi Biotec). Purified B cells were eluted in PBS x1 containing 0.5% v/v fetal bovine serum (FBS) and 2 mM EDTA. Cells were centrifuged for 10 min at 300 x g and resuspended in PBS x1 containing 0.04% w/v BSA and counted. Up to 16,600 B cells were loaded on a 10x Chromium Next GEM Chip G Kit and library synthesized according to the 10x Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 according to the manufacturer's protocol (10x Genomics). BCR CDR3 sequences were enriched using the human V(D)J B cell enrichment kit (10x Genomics). Libraries were sequenced by Genewiz on a NovaSeq 6000 System using the S4 2x 150 kit (Illumina). Raw read alignment and contig assembly were performed using Cellranger V(D)J (10x Genomics, version 3.1.0), with the default compatible human reference (refdata-cellrangervdj-GRCh38-alts-ensembl-3.1.0). Contigs were aligned to human IG loci using igBLAST-1.16.0 [43], and clones were assigned based on non-exact matching of the heavy chain sequence, using the Immcantation framework. Briefly, the normalized Hamming distance from each cell to its nearest neighbor was calculated using the Shazam R package (version 1.0.0). Based on the distribution of these distances, the cutoff to define two contigs as belonging to a single clone was set to 0.11. Clones were then assigned using the DefineClones command from the Change-O toolkit (version 1.0.0 2020.05.06 of the algorithm described by Gupta et al. [44]).

Flow cytometry single B cell sorting and sequencing
PBMCs were rapidly thawed at 37˚C and washed in 50 mL RPMI 1640 medium, followed by B cell enrichment using anti-CD19 magnetic beads (Miltenyi Biotec, 130-050-301) , 130-106-792). Single CD19 + IgG + RBD + cells were sorted into a 96 well plate containing 4 μL lysis buffer (12 units of RNasin Ribonuclease Inhibitor (Promega, N2511), 10 mM DTT and PBS x0.5) per well using a FACSAria III sorter (Becton Dickinson). The plates were stored in dry ice before proceeding to RNA reverse transcription and PCR amplification. For this purpose, the lysed cells were thawed on ice and the Ig genes were amplified by RT-PCR as previously described [26,27,45]. Briefly, cDNA was synthesized using random hexamer primers (Invitrogen, 48190011), and SuperScript III Reverse Transcriptase (Invitrogen, 18080085). Antibody sequences corresponding to the gamma, kappa, and lambda chains were amplified by nested PCR as previously described [27,45]. The first round of PCR ran at 94˚C for 7 min, 50 cycles of 94˚C for 30 s, 52˚C (Gamma) 50˚C (Kappa) or 58˚C (Lambda) for 20 s, and 72˚C for 40 s. The second round of PCR ran at similar conditions but the number of cycles was reduced to 40 and the annealing temperature of the gamma and lambda chains was slightly raised to 56˚C and 60˚C respectively. Second round PCR products were purified, sequenced, and annotated with IgBLAST [43]. First round PCR products were used as a template for additional amplification with specific 5' V and 3' J primers containing restriction sites for subsequent cloning into Ig expression vectors. PCR products were purified (MACHEREY-NAGEL, 740609.250), digested with the appropriate restriction enzymes (New England Biolabs), and ligated (New England Biolabs, M0202L) into the corresponding expression vectors for IgG, IgK, or IgL.

Antibody production
Cloned mAb vectors for IgG1 heavy chain and Kappa or Lambda light chains were co-transfected at a ratio of 1:3 (H:K/L) into Expi293F cells (Thermo Fisher Scientific Inc.) using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific Inc.). Seven days post transfection, the cell supernatant was collected, filtered (0.22 μm) and incubated with protein A coated agarose beads (GE Life Sciences, 17519901) for 2 h at RT. The beads were then loaded onto chromatography columns, washed, and eluted using 50 mM sodium phosphate (pH 3.0) into 1 M Tris-HCl (pH 8.0). Antibodies were buffer exchanged to PBS x1, aliquoted, and stored at -80˚C.

Antibody inhibition of SARS-CoV-2 RBD binding to hACE2-expressing cells
PE conjugated, biotinylated RBD was incubated with each mAb for 20 min at 4˚C, and then with hACE2-expressing mouse lung cells for 20 min at 4˚C. The cells were analyzed using CytoFLEX S4 (Beckman Coulter) and the PE florescence was recorded.

Affinity selection of TAU-2230 specific peptides
Biopanning protocol was performed as previously described [33]. Briefly, approximately 1x10 11 phages from the random-peptide library were mixed with 2 μg TAU-2230 mAb in TBS x1 3% BSA solution (complete to 100 μL) in 0.5 mL vials (AXYGEN, PCR tubes, PCR-05-C), and incubated for 1 h at RT. Next, 30 μL of Protein G coated magnetic beads (Invitrogen, Dynabeads Protein G) were added and the mix was incubated for additional 30 min on a rotating mixer at RT. The vials were then placed on a magnetic stand (Promega, MagneSphere Technology Magnetic Separation Stands) for 2 min to collect the beads, and the supernatant was discarded. The beads were washed 3 times with 200 μL TBS x1 0.5% Tween 20, and the bound phages were eluted twice with 105 μL of elution buffer (0.1N HCl pH 2.2, 1 mg/mL BSA) for 10 min, followed by addition of 19 μL of 1 M Tris-HCl pH 9.1. A total of three rounds of biopanning were carried out for each screen. In order to confirm binding to affinity-selected phages, monoclonal phages were produced in E. coli DH5αF + , re-suspended in TBS x1 0.5% Tween 20, quantified and used in confirmatory dot blot analyses.

Pseudo-particle preparation and neutralization assays
SARS-CoV-2-Spike pseudo-particles were obtained by co-transfection of Expi293F cells with pCMV delta R8.2, pLenti-GFP (Genecopoeia), and pcDNA 3.1 SΔC19 (ThermoFisher Scientific) at a ratio of 1:2:1, respectively, according to the manufacturer's instructions. The supernatant was harvested 72 h post transfection, centrifuged at 1500 x g for 10 min to remove cell debris, and passed through a 0.45 μm filter (LIFEGENE, Israel). The pseudo-particle-containing supernatant was then concentrated to 5% of its original volume using an Amicon Ultra with a 100 KDa cutoff at 16˚C (Merck Millipore).
HEK-293 cells stably expressing hACE2 were seeded into 0.1% gelatin-coated 96-well plates (Greiner) at an initial density of 0.75 x 10 5 cells per well. The following day, concentrated pseudo-particles were incubated with serial dilutions of antibodies for 1 h at 37˚C and then added to the 96 well pre-seeded plates. After 48 h, the cell medium was replaced with fresh DMEM medium excluding Phenol Red, and 24 h later, the 96-well plates were imaged by the IncuCyte ZOOM system (Essen BioScience). Cells were imaged with a 10x objective using the default IncuCyte software settings, which were used to calculate number of GFP-positive cells from four 488 nm-channel images in each well (data for each antibody concentration were collected in triplicate). The number of GFP-positive cells was normalized and converted to a neutralization percentage. IC50 and IC80 were calculated by GraphPad Prism software fitting to a non-linear regression model.

SARS-CoV-2 infection
SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources) was propagated using Vero E6 (ATCC) cells and infectious units quantified by plaque assay. For antibody neutralization assays, 10 4 Vero E6 cells were seeded in wells of a 96-well plate and incubated overnight in DMEM 10% FBS with penicillin/streptomycin at 37˚C 5% CO 2 . The following day, titrations of mAbs were incubated with 10 4 PFU for 1 h in DMEM 10% FBS at room temperature, then added to Vero E6 cells for up to 48 h. Infection was measured by tracking virus-induced cell death of Vero E6 cells and by intracellular staining for SARS-CoV-2 nucleocapsid, described below.

Virus plaque assays
Virus titers were determined by plaque assays on Vero E6 monolayers. Serial dilutions of virus stocks in DMEM (Corning, #10-014-CV) were added to Vero E6 monolayers on 12-well plates and incubated 1 h at 37˚C with rocking every 10-15 min. Virus was removed, and cells were subsequently overlaid with a 1:1 mixture of 1.2% agarose (ThermoFisher Scientific, #16500-100) and supplemented 2x MEM (2x MEM (ThermoFisher Scientific #11935046) plus 8% FBS, 2x non-essential amino acids, 2x L-glutamine, and 1.5x sodium bicarbonate). Plates were incubated at 37˚C in 5% CO 2 for 48 h and fixed with a mixture of 10% formaldehyde and 2% methanol (v/v in PBS) for 24 h. Agarose overlays were removed, and the monolayer was washed once with PBS x1 and stained with 0.025% Crystal Violet prepared in 2% ethanol. After 15 min, Crystal Violet was removed, and plaques were counted to determine the virus titers. Plaque assays were performed and counted by a blinded experimenter. All work with SARS-CoV-2 was conducted in Biosafety Level-3 conditions at the University of California San Diego following the guidelines approved by the Institutional Biosafety Committee.

Immunofluorescence imaging and analysis
For viral nucleocapsid detection in Vero E6 cells by immunofluorescence, cells were washed twice with PBS x1 and fixed in 4% formaldehyde for 30 min at RT. Fixed cells were washed with PBS x1 and permeabilized for immunofluorescence using BD Cytofix/Cytoperm according to the manufacturer's protocol for fixed cells and stained for SARS-CoV-2 with a primary nucleocapsid antibody (GeneTex GTX135357) conjugated to AF594 (ThermoFisher A20185) and nuclei counterstained with Hoechst. Eight images per well were obtained using a Nikon Ti2-E microscope equipped with a Spectra III light engine (Lumencor), appropriate filter cubes (Semrock), and a Qi-2 camera. For images acquired with the Nikon Ti2-E, analysis was performed using custom-scripted code for the Fiji distribution of ImageJ (PMID 22743772) and the DeepLearning plugin StarDist as follows. Channels were separated and the Hoechststained nuclei were segmented using StarDist to generate individual masks for each nucleus. The AF594 channel (nucleocapsid) was selected by removing background with a rolling ball of 50 pixels. A median filter (sigma = 10) was applied to facilitate thresholding of AF594 signal. The resulting image was used to generate a mask representing all positive stain. Positive nuclei were selected by firstly eroding the StarDist-generated nuclei mask by 2 pixels to reduce potential overlap with non-positive stain. Binary reconstruction was then carried out between the resulting mask and the AF594 mask. Data from all fields of view was logged and graphed with GraphPad Prism [46].

Virus-induced cell death
SARS-CoV-2-triggered cell death of Vero E6 cells was monitored using an Incucyte S3. Titrations of mAbs were incubated with 10 4 PFU for 1 h in DMEM 10% FBS at RT, then added to 10 4 confluent Vero E6 cells for up to 96 h at MOI of 1. Cell viability was monitored using 1 μg/ mL propidium iodide. Five fields of view at 10x magnification representing 33.6% well coverage were monitored for changes in cell viability every 6 h. Propidium iodide-positive cells were identified using Incucyte software.

Cell-to-cell fusion assay
HEK-293T cells were plated in a 6-well plate (Greiner) at an initial density of 0.5 x 10 6 cells per well. In parallel, HEK-293 target cells stably expressing hACE2 were plated in a gelatin-covered 96-well plate (Greiner) at an initial density of 0.75 x 10 5 cell per well. After 16 h incubation, the HEK-293T cells were transfected with 3 μg of pcDNA 3.1 plasmid encoding SARS-CoV-2-Spike (MN908947.3) with 19 amino acid C-terminal truncation (pSΔC19), using polyethyleneimine (PEI, Sigma Aldrich). Six hours post-transfection, the cells were washed once with complete media and collected using a cell scraper (BIOFIL, Israel). Next, 1 x 10 4 cells were overlaid on the target cells in the presence of 10 μg/mL of the appropriate antibody. The cells were incubated for 2 h, prior to imaging by the IncuCyte ZOOM system (Essen BioScience) at hourly intervals over 12 h. The 96-well plate (Greiner) was imaged with a 10x objective utilizing the default IncuCyte software parameters, which were used to calculate the mean confluence area of single-nuclei cells from 4 bright phase images in each well. The percentage area of syncytia was calculated by subtracting the single cell area from the total field area using the following equation: Supporting information  Fig 1A) to IgG AUC against OC43, 229E, CMV or HSV-1. AUC and correlations were calculated using GraphPad Prism software. Statistical analysis was performed using one-way ANOVA test.    Table. Anti-SARS-CoV-2 monoclonal antibodies isolated and cloned in this study. The donor from whom each mAb was isolated is indicated to the left. Neutralizing mAbs are colored in red. Clonal relatives are shaded in the same color. (TIF)