2020 03 21 990770v2 Full PDF

bioRxiv preprint doi: https://doi.org/10.1101/2020.03.21.990770.
The copyright holder for this preprint (which was not peer-reviewed) is the
author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
1 Potent human neutralizing antibodies elicited by SARS-CoV-2 infection

2 Bin Ju1,2,*, Qi Zhang3,*, Xiangyang Ge1, Ruoke Wang3, Jiazhen Yu1, Sisi Shan3,
3 Bing Zhou1, Shuo Song1, Xian Tang1, Jinfang Yu4, Jiwan Ge4, Jun Lan4, Jing
4 Yuan5, Haiyan Wang1, Juanjuan Zhao1,2, Shuye Zhang6, Youchun Wang7,
5 Xuanling Shi3, Lei Liu1,2, Xinquan Wang4, Zheng Zhang1,2#, and Linqi Zhang3#
6
1
7 Institute for Hepatology, National Clinical Research Center for Infectious
8 Disease, Shenzhen Third People’s Hospital, Shenzhen 518112, Guangdong
9 Province, China
2
10 The Second Affiliated Hospital, School of Medicine, Southern University of
11 Science and Technology, Shenzhen 518055, Guangdong Province, China.
3
12 Center for Global Health and Infectious Diseases, Comprehensive AIDS
13 Research Center, and Beijing Advanced Innovation Center for Structural
14 Biology, School of Medicine, Tsinghua University, Beijing 100084, China
4
15 The Ministry of Education Key Laboratory of Protein Science, Beijing
16 Advanced Innovation Center for Structural Biology, Beijing Frontier Research
17 Center for Biological Structure, Collaborative Innovation Center for Biotherapy,
18 School of Life Sciences, Tsinghua University, 100084 Beijing, China
5
19 Department for Infectious Diseases, Shenzhen Third People’s Hospital,
20 Shenzhen, Guangdong Province 518112, China
6
21 Shanghai Public Health Clinical Center and Institute of Biomedical Sciences,
22 Fudan University, Shanghai 201508, China
7
23 Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes
24 for Food and Drug Control, Beijing, China.
25 *These authors contributed equally to this work.
26 #Correspondence: zhangzheng1975@aliyun.com (Z.Z) or

27 zhanglinqi@tsinghua.edu.cn (L.Z.)
28
29
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bioRxiv preprint doi: https://doi.org/10.1101/2020.03.21.990770. The copyright holder for this preprint (which was not peer-reviewed) is the
30 Abstract
31 The pandemic caused by emerging coronavirus SARS-CoV-2 presents a

32 serious global public health emergency in urgent need of prophylactic and
33 therapeutic interventions. SARS-CoV-2 cellular entry depends on binding
34 between the viral Spike protein receptor-binding domain (RBD) and the
35 angiotensin converting enzyme 2 (ACE2) target cell receptor. Here, we report
36 on the isolation and characterization of 206 RBD-specific monoclonal
37 antibodies (mAbs) derived from single B cells of eight SARS-CoV-2 infected
38 individuals. These mAbs come from diverse families of antibody heavy and light
39 chains without apparent enrichment for particular families in the repertoire. In
40 samples from one patient selected for further analyses, we found coexistence
41 of germline and germline divergent clones. Both clone types demonstrated
42 impressive binding and neutralizing activity against pseudovirus and live SARS-
43 CoV-2. However, the antibody neutralizing potency is determined by
44 competition with ACE2 receptor for RBD binding. Surprisingly, none of the
45 SARS-CoV-2 antibodies nor the infected plasma cross-reacted with RBDs from
46 either SARS-CoV or MERS-CoV although substantial plasma cross-reactivity
47 to the trimeric Spike proteins from SARS-CoV and MERS-CoV was found.
48 These results suggest that antibody response to RBDs is viral species-specific
49 while that cross-recognition target regions outside the RBD. The specificity and
50 neutralizing characteristics of this plasma cross-reactivity requires further
51 investigation. Nevertheless, the diverse and potent neutralizing antibodies
52 identified here are promising candidates for prophylactic and therapeutic
53 SARS-CoV-2 interventions.
54

55 Introduction
56 The source of the recent Coronavirus Disease 2019 (COVID-19) outbreak in

1-4
57 Wuhan, China is a novel pathogenic coronavirus, SARS-CoV-2 . Its unique
58 pathogenesis and rapid international transmission poses a serious global
59 health emergency 5-9. SARS-CoV-2 belongs to the betacoronavirus family and
60 shares substantial genetic and functional similarity with other pathogenic
61 human betacoronaviruses, including Severe Acute Respiratory Syndrome
62 Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus
63 (MERS-CoV) 2-4,10,11. The virus is believed to have originated in bats, although
64 the source and animal reservoirs of SARS-CoV-2 remain uncertain 2-4,10-12. No
65 SARS-CoV-2-specific treatments or vaccine are currently available but several
66 antiviral drugs including remdesivir are being investigated clinically.
67 SARS-CoV-2 utilizes an envelope homotrimeric Spike glycoprotein (S) to

2,13,14
68 interact with cellular receptor ACE2 . Binding with ACE2 triggers a
69 cascade of cell membrane fusion events for viral entry. Each S protomer
70 consists of two subunits: a globular S1 domain at the N-terminal region, and the
71 membrane-proximal S2 and transmembrane domains. Determinants of host
72 range and cellular tropism are found in the RBD within the S1 domain, while
73 mediators of membrane fusion have been identified within the S2 domain 15,16.
74 We and others have recently determined the high-resolution structure of SARS-
14,17
75 CoV-2 RBD bound to the N-terminal peptidase domain of ACE2 . The
76 overall ACE2-binding mechanism is virtually the same between SARS-CoV-2
77 and SARS-CoV RBDs, indicating convergent ACE2-binding evolution between
18-22
78 these two viruses . This suggests that disruption of the RBD and ACE2
79 interaction would block SARS-CoV-2 entry into the target cell. Indeed, a few
80 such disruptive agents targeted to ACE2 have been shown to inhibit SARS-
81 CoV infection 23,24. However, given the important physiological roles of ACE2 in
82 vivo 25, these agents may have undesired side effects. Anti-RBD antibodies, on
83 the other hand, are therefore more favorable. Furthermore, SARS-CoV or
84 MERS-CoV RBD-based vaccine studies in experimental animals have also
15,26
85 shown strong polyclonal antibody responses that inhibit viral entry . Such
86 critical proof-of-concept findings indicate that anti-RBD antibodies should be
3

87 able to effectively block SARS-CoV-2 entry. Here, we report on the isolation

88 and characterization of 206 RBD-specific mAbs derived from single B cells of
89 eight SARS-CoV-2 infected individuals. Using bioinformatic and biologic
90 characterization, we identified several mAbs with potent neutralizing activity
91 against pseudovirus and live SARS-CoV-2. However, no cross-activity between
92 RBDs from SARS-CoV or MERS-CoV was found, suggesting that the RBD-
93 based antibody response is viral species-specific. The potent neutralizing
94 antibodies identified here are promising candidates for prophylactic and
95 therapeutic SARS-CoV-2 interventions.
96 Results
97 Plasma and B cell responses specific to SARS-CoV-2. We collected
98 cross-sectional and longitudinal blood samples from eight
99 SARS-CoV-2-infected subjects during the early outbreak in Shenzhen (Table
100 S1). Samples were named by patient number and either A, B, or C depending
101 on collection sequence. Six patients (P#1 through P#4, P#8, and P#16) had
102 Wuhan travel history and the remaining two (P#5 and P#22) had direct contact
103 with those from Wuhan. P#1 through P#5 is a family cluster with the first
104 documented case of human-to-human transmission of SARS-CoV-2 in
105 Shenzhen 5. All subjects recovered and were discharged from the hospital
106 except for P#1 who succumbed to disease despite intensive intervention. To
107 analyze antibody binding, serial plasma dilutions were applied to enzyme-linked
108 immunosorbent assay (ELISA) plates coated with either recombinant RBD or
109 trimeric Spike derived from SARS-CoV-2, SARS-CoV, and MERS-CoV or
110 recombinant NP from SARS-CoV-2. Binding activity was visualized using
111 anti-human IgG secondary antibodies at an optical density (OD) of 450nm.
112 Varying degrees of binding were found across individuals and among samples
113 from the same individual. Samples from P#1, P#2, P#5, and P#16
114 demonstrated higher binding to both SARS-CoV-2 RBD and NP than the rest
115 (Figure 1A). Three sequential plasma samples collected from P#2 over nine
116 days during early infection showed similar binding to SARS-CoV-2 RBD and
117 NP and remained relative stable over the course of the infection. To our surprise,
118 virtually no cross-reactivity between SARS-CoV RBD and MERS-CoV RBD

119 was detected (Figure 1A), despite strong recognition by the positive control
120 antibodies (data not shown). However, strong cross-reactivity was detected
121 against trimeric Spikes from SARS-CoV and MERS-CoV in both ELISA (Figure
122 1B) and cell-surface staining (Figure S1). All samples except P#4A
123 demonstrated significant levels of cross-binding to SARS-CoV trimeric Spike
124 while only those from P#1, P#2 and P#4B cross recognized MERS-CoV trimeric
125 Spike (Figure 1B). None of the plasma samples were reactive to HIV-1
126 envelope trimer derived from strain BG505 27. The same plasma samples were
127 also evaluated for neutralization of pseudoviruses bearing the Spike proteins of
128 either SARS-CoV-2, SARS-CoV, or MERS-CoV. Consistent with the antibody
129 binding results, varying degrees of neutralizing activities against SARS-CoV-2
130 were found across individuals (Figure 1C). However, cross-neutralizing against
131 SARS-CoV and MERS-CoV is rather minimal as all plasma samples tested,
132 including healthy control plasma, had negligible levels of neutralization (Figure
133 1C). No detectable neutralization was found for any plasma sample against the
134 pseudovirus control bearing the HIV-1 envelope MG04 (Figure 1C). Taken
135 together, these results suggest that RBDs from SARS-CoV-2, SARS-CoV, and
136 MERS-CoV are likely to be immunologically distinct despite substantial
14,17
137 sequence and structural similarities . Thus, regions beyond RBDs likely
138 contribute to the observed cross-reactivity against SARS-CoV and MERS-CoV
139 Spike protein.
140 Flow cytometry with a range of gating strategies was used to study
141 SARS-CoV-2-specific B cell responses and identity B cells recognizing
142 fluorescent-labeled RBD probes (Figure 1D and Figure S2). As shown in Figure
143 1E, the RBD-specific B cells constitute about 0.005-0.065% among the total B
144 cell population and 0.023-0.329% among the memory subpopulations. The
145 number of RBD-specific B cells are relatively higher in P#2, P#5, P#16, and
146 P#22 (Figure 1E), which appeared to correlate well with binding activity of
147 corresponding plasma samples to SARS-CoV-2 RBD and trimeric Spike protein
148 (Figure 1A and 1B). However, sample P#1A demonstrated the lowest RBD-
149 specific B cell response despite high-level plasma binding. As P#1 was the only
150 patient succumb to disease, it is uncertain whether this dichotomy of high
151 plasma binding activity and low levels of RBD-specific B cells is a surrogate
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152 marker of rapid disease progression. This phenomenon needs study in a larger
153 population of samples.
154
155 Single B cell antibody cloning and heavy chain repertoire analyses. We
156 further isolated RBD-binding B cells into single cell suspension for cloning and
157 evaluation of the mAb response (Figure 1D and Figure S2). Immunoglobulin
158 heavy and light chains were amplified by RT-PCR using nested primers. The
159 amplified products were cloned into linear expression cassettes to produce full
28,29
160 IgG1 antibodies as previously described . The number of B cell clones
161 varied from 10 to 106 among the subjects and each clone has been differentially
162 represented (Figure S3). Individual IgGs were produced by transfection of
163 linear expression cassettes and tested for SARS-CoV-2 RBD reactivity by
164 ELISA. On average, fifty-eight percent of the antibody clones were reactive,
165 although great variability was found among different individuals (Figure S3).
166 Out of 358 antibodies, we obtained 206 that bound to SARS-CoV-2 RBD with
167 165 distinct sequences (Table S2). These 206 antibodies demonstrated
168 significant differences in binding activity. For example, a large number of
169 antibodies from samples P#2B, P#2C, P#4A, P4#B, P#5A, P#16A, and P#22A
170 had OD 450 values well over 4.0, while none of those from sample P#1A
171 exceeded 4.0. There were too few antibodies from P#3A and P#8A to make
172 meaningful evaluations (Figure S3). Furthermore, samples from different study
173 subjects also demonstrated substantial differences in heavy chain variable
174 gene (VH) usage (Figure 2A). For instance, P#1 samples are dominated by
175 VH3-53, 3-13, and 1-69 which constituted approximately 21.4%, 14.3%, and
176 14.3% of the entire VH repertoire, respectively. Samples from P#2 and P#5 are
177 more diverse in distribution and frequency of their VH usage. However, no
178 single or group of VH families stood out among study subjects, suggesting
179 patients have immunologically distinct responses to SARS-CoV-2 infection.
180 This hypothesis is supported by the phylogenetic analysis of all 206 VH
181 sequences superimposed with their corresponding binding activities as
182 presented in Figure 2B. The high-binding clusters (light orange circle: 80% of
183 clusters with OD 450 > 3) were widely distributed across multiple heavy chain

184 families. In fact, majority of the high-binding antibodies were derived by clonal
185 expansion of specific VH families in P#2, P#4, and P#5. Similarly, the middle-
186 (60-80% of clusters with OD 450 > 3) and low- (< 60% cluster with OD 450 > 3)
187 binding clusters were also widely distributed and each consisted of
188 disproportionally represented VH gene families.
189 Broad diversity and clonal expansion of antibodies in the repertoire. As

190 P#2 showed a large number of RBD-binding antibodies and was the only
191 patient with three sequential blood samples, we conducted more detailed
192 characterization of P#2 antibodies. Among a total of 69 antibodies from P#2,
193 the majority (59%) were scattered across various branches and the remaining
194 (41%) were clonally expanded into three major clusters (Figure 3A). Antibodies
195 from the three time points (A, B, C) do not appear to group together but rather
196 interdigitate among themselves, suggesting they are highly related during early
197 infection. Three clones were significantly enriched and each constituted
198 between 12-14% of the entire tested repertoire (Figure 3A). Their heavy-chain
199 variable regions belong to the VH1-2*06, VH3-48*02, and VH3-9*01 families.
200 The corresponding light-chain kappa (Igk) belongs to 2-40*01/2D-40*01, 3-
201 20*01, and light-chain lambda (Igl) to 2-14*02 with the respective joining
202 segment kappa 4 (Jk4), Jk5 and joining segment lambda 1 (Jl1) (Table S2).
203 More importantly, these clonally expanded antibodies were identified in all three
204 samples indicating that they are strongly selected for during infection. When
205 comparing their representation within each cluster, VH1-2*06 and VH3-9*01
206 appeared to increase from approximately 33 to 45%, whereas VH3-48*02
207 decreased from 33 to 9% over the three time points, although the number of
208 clones was too small for statistical significance. Interestingly, the somatic
209 hypermutation (SHM) or germline divergence for VH1-2*06 was 0% and this
210 cluster persisted during the study period. However, the SHM for VH3-48*02
211 reached as high as 9.6% and for VH3-9*01 reached 3.8% compared to the
212 overall average of 2.2% ± 3.3 % among the 69 VH sequences. Furthermore,
213 the CDR3 length for VH1-2*06, VH3-48*02, and VH3-9*01 was 19aa, 16aa,
214 and 23aa, respectively, compared with the overall average of 16 ± 4aa among
215 the 69 VH sequences. Close examination of the longest CDR3 from the

216 VH3-9*01 cluster revealed richness in tyrosine, indicating potential hydrogen

217 bonding and hydrophobic interactions with the surrounding residues. These
218 results shed light on the clonal expansion and broad diversity of RBD-specific
219 antibodies during early infection and their potential role in controlling SARS-
220 CoV-2 infection.
221
222 Binding and neutralizing properties of selected antibodies. We selected 13
223 of the 69 P#2 antibodies sequences based on their representation and
224 distribution on the phylogenetic tree (Figure 3A, starred). Five P#1A antibody
225 clones were used as controls. Surface plasmon resonance (SPR) with SARS-
226 CoV-2 RBD showed that P#2 antibodies had dissociation constants (Kd)
227 ranging from 10-8 to 10-9 M while those from P#1 ranged from not detectable to
228 10-9 M (Table 1 and Figure S4). SHM did not appear to correlate with Kd; some
229 germline clones with 0% divergence in both VH and VL genes (P2A-1A10, P2B-
230 2G4, P2C-1A3, and P2C-1E1) had Kd values comparable to clones with higher
231 levels of SHM. The Kd of representative clones (P2A-1A8, P2A-1A10, and P2A-
232 1B3) from the three clonally expanded clusters fell into a similar range,
233 suggesting that their expansion may not be driven by affinity maturation. Next,
234 we measured each antibody for competition with ACE2 for binding to the SARS-
235 CoV-2 RBD. Specifically, the RBD was covalently immobilized on a CM5 sensor
236 chip and first saturated by antibody and then flowed through with soluble ACE2.
237 Competing capacity of each antibody was measured as percent reduction in
238 ACE2 binding with the RBD (Table 1 and Figure S5). As shown in Table 1, the
239 evaluated antibodies demonstrated various competing capacity with ACE2. The
240 most powerful were P2C-1F11 and P2B-2F6, which reduced ACE2 binding
241 about 99.2% and 98.5%, respectively. Two of the three representative
242 antibodies from the clonal expanded clusters (P2A-1A10 and P2A-1B3) had
243 slightly over 80% and 90% reduction, respectively. The third representative
244 (P2A-1A8) only showed 57% reduction. Many antibodies had only limited
245 competing power with ACE2 despite impressive Kd values, suggesting binding
246 affinity is not predictive of ACE2 competing capacity. Control antibodies from
247 P#1 demonstrated even lower competing power with ACE2. Surprisingly, none
248 of the antibodies tested demonstrated cross-binding with SARS-CoV and
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249 MERS-CoV RBD except P1A-1C7 (Kd=4.85μM), for which only limited cross
250 reactivity with SARS-CoV RBD was detected (Figure S4).
251 We next studied antibody neutralizing activities against pseudoviruses
252 bearing the Spike protein of SARS-CoV-2. Consistent with the competing
253 capacity findings, neutralizing activity varied considerably with IC50 values
254 ranging from 0.03 to > 50 μg/ml (Figure 4B, 4A and Table 1). P2C-1F11 and
255 P2B-2F6 were the most potent, followed by P2C-1A3 and P2C-1C10. Overall,
256 ACE2 competing capacity correlated well with the neutralizing activities,
257 although this correlation was not exact in some instances. Notably, no cross-
258 neutralization was found either against pseudoviruses bearing the Spike of
259 SARS-CoV or MERS-CoV (data not shown) nor with cell-surface staining of
260 trimeric SARS-CoV and MERS-CoV Spike (Figure S6). Furthermore, we
261 selected P2C-1F11, P2B-2F6, and P2C-1A3 for neutralizing activity analyses
262 against live SARS-CoV-2. Consistent with their respective pseudovirus assay
263 findings, P2C-1F11 and P2B-2F6 demonstrated potent neutralization activity
264 while that of P2C-1A3 was somewhat lower, although it needs to be noted that
265 CPE assay is not particularly quantitative (Figure 4C). Lastly, we determined
266 whether these antibodies compete for similar epitopes on the SARS-CoV-2
267 RBD. We selected a total of six antibodies with ACE2 competitive capacities of
268 at least 70% and analyzed them in a pairwise competition fashion using SPR.
269 As shown in Table 2 and Figure S7, variable degrees of competition were found
270 among the pairs of antibodies. P2C-1A3, for instance, was competitive against
271 all antibodies tested with reduction capacity ranging from 52 to 76. P2C-1F11,
272 on the other hand, was less competitive with other antibodies and in particular,
273 only minimally competitive with P2C-1C10. P2B-2F6, another potent
274 neutralizing antibody, was broadly competitive with all antibodies tested. These
275 results indicate that the antibodies analyzed recognized both overlapping and
276 distinct epitopes. Different mAbs may therefore exert their neutralizing activity
277 through different mechanisms.
278
279 Discussion
280 We characterized antibody responses in eight COVID-19 patients and isolated
281 206 mAbs specific to the SARS-CoV-2 RBD. Bioinformatic and biologic
9

282 characterization indicates that these antibodies are derived from broad and
283 diverse families of antibody heavy and light chains. Each individual appears to
284 have unique pattern of distribution in the antibody repertoire without apparent
285 preferences for particular antibody families. Each antibody clone is also
286 differentially represented. In P#2, for whom additional analyses were conducted,
287 we found substantial variability in the distribution and frequency of each
288 antibody family. Some clones were identified only once whereas others
289 underwent high degrees of clonal expansion. Some clones were virtually
290 identical to their germline ancestors while others became more divergent during
291 the infection period. The CDR3 length also varied among the different clones.
292 These differences at the genetic levels corresponded with their binding and
293 neutralizing activities. Binding affinity (Kd) fell in the range of 10-8 to 10-9 M,
30-32
294 equivalent to many antibodies identified during acute infections but
33-35
295 significantly lower than those identified during chronic HIV-1 infections .
296 However, binding affinity alone does not predict neutralizing activity.
297 Competition with the receptor ACE2 governs antibody potency, although some
298 degree of discrepancy does exist. In particular, the most potent antibodies,
299 P2C-1F11 and P2B-2F6, out-competed ACE2 with close to 100% efficiency,
300 indicating that blocking the RBD and ACE2 interaction is a useful surrogate for
301 antibody neutralization. Among the antibodies tested, substantial variations in
302 competition for similar RBD epitopes or regions were also found. The most
303 potent antibody, P2C-1F11, did not seem target the same epitope as the
304 relatively moderate antibody P2C-1C10. Thus, these two antibodies could be
305 combined for synergistic antiviral effect. As we continue to screen more
306 antibodies from P#2 and other study subjects, more potent and diverse
307 antibodies are expected to be identified. These antibodies will serve as the best
308 candidates for the development of prophylactic and therapeutic intervention
309 against COVID-19 infection.
310 Most surprising in this study was the absence of antibody cross-reactivity
311 with RBDs from SARS-CoV and MERS-CoV. Based on the sequential and
312 structural similarities of RBDs from SARS-CoV-2 and SARS-CoV, we predicted
313 some degree of cross-binding and even cross-neutralization between the two
314 viruses. However, species-specific RBD responses in SARS-CoV-2 patients do
10

315 suggest that RBDs from SARS-CoV-2 and SARS-CoV are immunologically
316 distinct. If so, antibodies and vaccines must target each viral species differently
317 in order to achieve maximum efficacy in protecting the host from infection. Our
318 finding somewhat resolves the question of why many previously isolated
319 SARS-CoV antibodies failed to cross-neutralize SARS-CoV-2 despite
320 detectable levels of binding with Spike of SARS-CoV-236. The absence of cross-
321 recognition between RBDs was also apparent at the plasma level. Although
322 strong binding to SARS-CoV-2 RBD was identified, plasma samples from the
323 study subjects failed to demonstrate appreciable cross-reactivity with either
324 SARS-CoV or MERS-CoV RBD, highlighting the immunological distinctions
325 among the RBDs from the three viruses. However, substantial cross-reactivity
326 were found when the same plasma samples were applied to the trimeric Spike
327 proteins of SARS-CoV and MERS-CoV, although this was higher with the
328 former than the latter. This indicates that such cross-reactivity likely occurs in
329 regions outside the RBD. Determining whether this cross-reactive response
330 has any neutralizing or protection capacity against infection would require
331 further investigation. Finally, despite successfully isolating and characterizing a
332 large of number mAbs against SARS-CoV-2, we cannot draw any firm
333 correlation between antibody response and disease status at this time. In
334 particular, the three severe cases (P#1, P#2, and P#5) appear to have relatively
335 higher plasma binding and neutralizing activities against SARS-CoV-2 than
336 those with relative mild symptoms. A larger number of patients must be studied
337 to elucidate the drivers and impact of associations between antibody response
338 and disease progression, which will provide pivotal reference for our antibody-
339 based intervention as well as vaccine development.
340
341 Materials and Methods
342 Study approval. This study received approval from the Research Ethics
343 Committee of Shenzhen Third People's Hospital, China (approval number:
344 2020-084). The Research Ethics Committee waived the requirement informed
345 consent before the study started because of the urgent need to collect
346 epidemiological and clinical data. We analyzed all the data anonymously.
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347 Patients and blood samples. The study enrolled a total of eight patients aged
348 10 to 66 years old infected with SARS-CoV-2 in January 2020 (Table S1). A
349 plasma sample from a healthy control was also included. Of these eight patients,
350 six (P#1 through P#4, P#8, and P#16) had Wuhan exposure history through
351 personal visit and two had direct contact with individuals from Wuhan. Four
352 subjects (P#1 through P#4) were part of a family cluster (P#1 through P#5)
353 infected while visiting Wuhan and subsequently transmitted infection to P#5
354 after returning to Shenzhen 5. All patients were hospitalized at Shenzhen Third
355 People’s Hospital, the designated city hospital for treatment of COVID-19
356 infected patients, three to nine days after symptom onset. All patients presented
357 with fever, fatigue, and dry cough and three (P#1, P#2 and P#5) developed
358 severe pneumonia. Four patients (P#1, P#2, P#5, and P#22) were 60 years or
359 older, of which three (P#1, P#2, and P#22) had underlying disease such as
360 hypertension. SARS-CoV-2 infection status was verified by RT-PCR of
361 nasopharyngeal swab and throat swab specimens. No patient had detectable
362 influenza A, B, respiratory syncytial virus (RSV), or adenovirus co-infections.
363 Chest computed tomographic scans showed varying degrees of bilateral lung
364 patchy shadows or opacity. All patients received antiviral and corticosteroid
365 treatments, recovered and were discharged except for P#1, who succumbed to
366 disease in hospital. Single (P#1, P#3, P#5, P#8, P#16, and P#22) or sequential
367 (P#2 and P#4) blood samples were collected during hospitalization and follow-
368 up visits and separated into plasma and peripheral blood mononuclear cells
369 (PBMCs) by Ficoll-Hypaque gradient (GE Healthcare) centrifugation. All plasma
370 samples were heat-inactivated at 56 ºC for 1h before being stored at -80 ºC.
371 PBMCs were maintained in freezing media and stored in liquid nitrogen until
372 use.
373
374 Recombinant RBDs and trimeric Spike from SARS-CoV-2, SARS-CoV, and
375 MERS-CoV and receptor ACE2. Recombinant RBDs and trimeric Spike for
376 MERS-CoV, SARS-CoV, and SARS-CoV-2 and the N-terminal peptidase
377 domain of human ACE2 (residues Ser19-Asp615) were expressed using the
18,19,37-39
378 Bac-to-Bac baculovirus system (Invitrogen) as previously described .
379 SARS-CoV-2 RBD (residues Arg319-Phe541) containing the gp67 secretion
12

380 signal peptide and a C-terminal 6×His tag was inserted into pFastBac-Dual
381 vectors (Invitrogen) and transformed into DH10Bac component cells. The
382 bacmid was extracted and further transfected into Sf9 cells using Cellfectin II
383 Reagents (Invitrogen). The recombinant viruses were harvested from the
384 transfected supernatant and amplified to generate high-titer virus stock. Viruses
385 were then used to infect Hi5 cells for RBD and trimeric Spike expression.
386 Secreted RBD and trimeric Spike were harvested from the supernatant and
387 purified by gel filtration chromatography as previously reported 18,19,37-39.
388 ELISA analysis of plasma and antibody binding to RBD, trimeric Spike,
389 and NP proteins. The recombinant RBDs and trimeric Spike derived from
390 SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein
391 (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or
392 2μg/ml, then coated onto 96-well plates and incubated at 4°C overnight.
393 Samples were washed with PBS-T (PBS containing 0.05% Tween 20) and
394 blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at RT
395 for 1h. Either serially diluted plasma samples or isolated mAbs were added the
396 plates and incubated at 37°C for 1h. Wells were then incubated with secondary
397 anti-human IgG labeled with HRP (ZSGB-BIO, Beijing) and TMB substrate
398 (Kinghawk, Beijing) and optical density (OD) was measured by a
399 spectrophotometer at 450nm and 630nm. The serially diluted plasma from
400 healthy individuals or mAbs against SARS-CoV, MERS-CoV or HIV-1 were
401 used as controls.
402 Isolation of RBD-specific single B cells by FACS. RBD-specific single B

28,40
403 cells were sorted as previously described . In brief, PBMCs from infected
404 individuals were collected and incubated with an antibody and RBD cocktail for
405 identification of RBD-specific B cells. The cocktail consisted of CD19-PE-Cy7,
406 CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD27-APC-H7, IgG-
407 FITC (BD Biosciences) and the recombinant RBD-Strep or RBD-His described
408 above. Three consecutive staining steps were conducted. The first was a
409 LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) in 50µl phosphate-buffered
410 saline (PBS) applied at RT for 20 minutes to exclude dead cells. The second
411 utilized an antibody and RBD cocktail for an additional 30min at 4 °C. The third
13

412 staining at 4 °C for 30min involved either: Streptavidin-APC (eBioscience)

413 and/or Streptavidin-PE (BD Biosciences) to target the Strep tag of RBD, or anti-
414 his-APC and anti-his-PE antibodies (Abcam) to target the His tag of RBD. The
415 stained cells were washed and resuspended in PBS before being strained
416 through a 70μm cell mesh (BD Biosciences). RBD-specific single B cells were
417 gated as CD19+CD3-CD8-CD14-IgG+RBD+ and sorted into 96-well PCR
418 plates containing 20μl of lysis buffer (5 μl of 5 x first strand buffer, 0.5 μl of
419 RNase out, 1.25 μl of 0.1 M DTT (Invitrogen) per well and 0.0625 μl of Igepal
420 (Sigma). Plates were then snap-frozen on dry ice and stored at -80 °C until RT
421 reaction.
422 Single B cell PCR, cloning and expression of mAbs. The IgG heavy and
423 light chain variable genes were amplified by nested PCR and cloned into linear
424 expression cassettes or expression vectors to produce full IgG1 antibodies as
29,41
425 previously described . Specifically, all second round PCR primers
426 containing tag sequences were used to produce the linear Ig expression
427 cassettes by overlapping PCR. Separate primer pairs containing the specific
428 restriction enzyme cutting sites (heavy chain, 5’-AgeI/3’-SalI; kappa chain, 5’-
429 AgeI/3’-BsiWI; and lambda chain, 5’-AgeI/3’-XhoI) were used to amplify the
430 cloned PCR products. The PCR products were purified and cloned into the
431 backbone of antibody expression vectors containing the constant regions of
432 human IgG1. Overlapping PCR products of paired heavy and light chain
433 expression cassettes were co-transfected into 293T cells (ATCC) grown in 24-
434 well plates. Antigen-specific ELISA was used to detect the binding capacity of
435 transfected culture supernatants to SARS-CoV-2 RBD. Monoclonal antibodies
436 were produced by transient transfection of 293F cells (Life Technologies) with
437 equal amounts of paired heavy and light chain plasmids. Antibodies in the
438 culture supernatant was purified by affinity chromatography using Protein A
439 beads columns (National Engineering Research Center for Biotechnology,
440 Beijing) according to the manufacturer’s protocol. Concentrations were
441 determined by BCA Protein Assay Kits (Thermo Scientific). SARS-CoV, MERS-
442 CoV, and HIV-1 mAbs were also included as controls. SARS-CoV antibodies
42
443 (S230 and m396) previously isolated by others were synthesized and
14

444 sequences verified before expression in 293T cells and purification by protein
445 A chromatography. MERS-CoV antibodies (Mab-GD33) were derived from
43
446 previously reported . HIV-1 antibody VRC01 was a broadly neutralizing
447 antibody directly isolated from a patient targeting the CD4 binding site of
448 envelope glycoprotein 40.
449 Antibody binding kinetics, epitope mapping, and competition with

450 receptor ACE2 measured by SPR. The binding kinetics and affinity of mAbs
451 to SARS-CoV-2 RBD were analyzed by SPR (Biacore T200, GE Healthcare).
452 Specifically, purified RBDs were covalently immobilized to a CM5 sensor chip
453 via amine groups in 10mM sodium acetate buffer (pH 5.0) for a final RU around
454 250. SPR assays were run at a flow rate of 30ml/min in HEPES buffer. The
455 sensograms were fit in a 1:1 binding model with BIA Evaluation software (GE
456 Healthcare). For epitope mapping, two different antibodies were sequentially
457 injected and monitored for binding activity to determine whether the two mAbs
458 recognized separate or closely-situated epitopes. To determine competition
459 with the human ACE2 peptidase domain, SARS-CoV-2 RBD was immobilized
460 to a CM5 sensor chip via amine group for a final RU around 250. Antibodies (1
461 μM) were injected onto the chip until binding steady-state was reached. ACE2
462 (2 μM), which was produced and purified as above, was then injected for 60
463 seconds. Blocking efficacy was determined by comparison of response units
464 with and without prior antibody incubation.
465 Analysis of plasma and antibody binding to cell surface expressed

466 trimeric Spike protein. HEK 293T cells were transfected with expression
467 plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-
468 CoV and incubated at 37 °C for 36 h. The cells were digested with trypsin and
469 distributed into 96 well plates for the individual staining. Cells were washed
470 twice with 200µl staining buffer (PBS with 2% heated-inactivated FBS) between
471 each following steps. The cells were stained at room temperature for 30
472 minutes in 100 μl staining buffer with 1:100 dilutions of plasma or 20 μg/ml
473 monoclonal antibodies. The cells were then stained with PE labeled anti-human
474 IgG Fc secondary antibody (Biolegend) at a 1:20 dilution in 50 μl staining buffer
475 at room temperature for 30 minutes. Finally, the cells were re-suspended and
15

476 analyzed with FACS Calibur instrument (BD Biosciences, USA) and FlowJo 10
477 software (FlowJo, USA). HEK 293T cells without transfection were also stained
478 as background control. S230 and m396 targeting the RBD of SARS-CoV spike
42 43
479 and Mab-GD33 targeting the RBD of MERS-CoV spike were used as
40
480 positive primary antibody controls, while VRC01 targeting HIV-1 env was
481 used as an irrelevant primary antibody control.
482 Neutralization activity of mAbs against pseudovirus and live SARS-CoV-2.

483 SARS-CoV-2, SARS-CoV and MERS-CoV pseudovirus were generated by co-
484 transfection of human immunodeficiency virus backbones expressing firefly
485 luciferase (pNL43R-E-luciferase) and pcDNA3.1 (Invitrogen) expression
37,38,44,45
486 vectors encoding the respective S proteins into 293T cells (ATCC) .
487 Viral supernatants were collected 48 h later. Viral titers were measured as
488 luciferase activity in relative light units (Bright-Glo Luciferase Assay Vector
489 System, Promega Biosciences). Control envelope glycoproteins derived from
490 human immunodeficiency virus (HIV)-1 and their corresponding pseudoviruses
491 were produced in the same manner. Control mAbs included VRC01 against
40 42
492 HIV-1 ; S230 and m396 against SARS-CoV ; and Merb-GD33 against
43
493 MERS-CoV . Neutralization assays were performed by incubating
494 pseudoviruses with serial dilutions of purified mAbs at 37°C for 1h. Huh7 cells
495 (ATCC) (approximately 1.5 × 104 per well) were added in duplicate to the virus-
496 antibody mixture. Half-maximal inhibitory concentrations (IC50) of the evaluated
497 mAbs were determined by luciferase activity 48h after exposure to virus-
498 antibody mixture using GraphPad Prism 6 (GraphPad Software Inc.).
499 All experiments involving live SARS-CoV-2 followed approved Biosafety

500 Level 3 laboratory standard operating procedures. Neutralization assays
501 against live SARS-CoV-2 were conducted using a clinical isolate
502 (Beta/Shenzhen/SZTH-003/2020, EPI_ISL_406594 at GISAID) previously
503 obtained from a nasopharyngeal swab of P#3. The isolate was amplified in Vero
504 cell lines to make working stocks of the virus (1 × 105 PFU/ml). To analyze the
505 mAb neutralizing activities, Vero E6 cells were seeded at 104/well in 96-well
506 culture plates and cultured at 37 °C to form a monolayer. Serial dilutions of
507 mAbs were mixed separately with 100 PFU of SARS-CoV-2, incubated at 37 °C
16

508 for 1 h, and added to the monolayer of Vero E6 cells in duplicates. Cells either
509 unexposed to the virus or mixed with 100 PFU SARS-CoV-2 were used as
510 negative (uninfected) and positive (infected) controls, respectively. Cytopathic
511 effects (CPE) were observed daily and recorded on Day 2 post-exposure.
512 Gene family usage and phylogenetic analysis of mAbs. The program
513 IMGT/V-QUEST (http://www.imgt.org/IMGT_vquest/vquest) was used to
514 analyze germline gene, germline divergence or degree of somatic
515 hypermutation (SHM), the framework region (FR) and the loop length of the
516 complementarity determining region 3 (CDR3) for each antibody clone. The IgG
517 heavy and light chain variable genes were aligned using Clustal W in the
518 BioEdit sequence analysis package (https://bioedit.software.informer.com/7.2/).
519 Phylogenetic analyses were performed by the Maximum Likelihood method
520 using MEGA X (Molecular Evolutionary Genetics Analysis across computing
521 platforms). Several forms of the phylogenetic trees are presented for clarity.
522 Antibody production. The production of antibodies was conducted as

38,46
523 previously described . The genes encoding the heavy and light chains of
524 isolated antibodies were separately cloned into expression vectors containing
525 IgG1 constant regions and the vectors were transiently transfected into
526 HEK293T or 293F cells using polyethylenimine (PEI) (Sigma). After 72h, the
527 antibodies secreted into the supernatant were collected and captured by
528 protein A Sepharose (GE Healthcare). The bound antibodies were eluted and
529 further purified by gel-filtration chromatography using a Superdex 200 High
530 Performance column (GE Healthcare). The purified antibodies were either used
531 in binding and neutralizing assays.
532 Acknowledgments We acknowledge the work and contribution of all the health
533 providers from Shenzhen Third People's Hospital. We also thank patients for
534 their active participation. This study was supported by Bill & Melinda Gates
535 Foundation, the Science and Technology Innovation Committee of Shenzhen
536 Municipality (202002073000002), and by Tsinghua University Initiative
537 Scientific Research Program (20201080053). This work is also partially
538 supported by the National Natural Science Foundation Award (81530065),
17

539 Beijing Municipal Science and Technology Commission (171100000517-001

540 and -003), Beijing Advanced Innovation Center for Structural Biology at
541 Tsinghua University, the National Key Plan for Scientific Research and
542 Development of China (grant number 2016YFD0500307), Tencent Foundation,
543 Shuidi Foundation, and TH Capital. The funders had no role in study design,
544 data collection, data analysis, data interpretation, or writing of the report.
545 Author contributions LZ, ZZ, LL and SZ conceived and designed the study.
546 BJ and QZ performed most of the experiments together with assistance from
547 XG, RW, JY, SS, BJ, SS, and XS. XT performed live SARS-CoV-2
548 neutralization assay. JY, JG, JL, XW provided assistance in RBD and trimeric
549 Spike protein production. JY and LL played critical roles in recruitment and
550 clinical management of the study subjects. HW and JZ are in charge of sample
551 collection and processing. YW provides additional pseudovirus assay for
552 measuring neutralizing activity against SARS-CoV-2. BJ, QZ, ZZ and LZ had
553 full access to data in the study, generated figures and tables, and take
554 responsibility for the integrity and accuracy of the data presentation. LZ and ZZ
555 wrote the manuscript. All authors reviewed and approved the final version of
556 the manuscript.
557 Data availability statements We are happy to share reagents and information
558 presented in this study upon request.
559 Conflict of interests: We declare no competing interest.
560
561
18

562 Figure Legends

563
564 Figure 1. Analyses of plasma and B cell responses specific to SARS-CoV-
565 2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs
566 or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA
567 and (C) for neutralizing activity against pseudoviruses bearing envelope
568 glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-
569 CoV-2 NP protein was also evaluated (A). All results were derived from at least
570 two independent experiments. (D) Gating strategy for analysis and isolation of
571 RBD-specific memory B cells and (E) their representation among the total and
572 memory subpopulation of B cells in the eight study subjects. Samples were
573 named as either A, B, or C depending on collection sequence. FSC-W, forward
574 scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.
575
576 Figure 2. Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies
577 analyzed (A) by individual subject or (B) across the eight subjects. (A)
578 Distribution and frequency of heavy chain variable (VH) genes usage in each
579 subject shown along the horizontal bar. The same color scheme is used for
580 each VH family across all study subjects. The VHs that dominate across
581 isolated antibodies are indicated by actual frequencies in their respective color
582 boxes. The number of RBD-binding antibodies versus total antibodies isolated
583 are shown on the right. (B) Clustering of VH genes and their association with
584 ELISA binding activity across the eight subjects. Unrooted phylogenetic tree
585 depicting the genetic relationships among all VH genes of the RBD-binding
586 antibodies. Branch lengths are drawn to scale so that sequence relatedness
587 can be readily assessed. Sequences from the same study subject are shown
588 in the same color at the branch tips. Colored circles represent the proportion
589 (light orange, > 80%; light yellow, 60%-80%; light green < 60%) of VH clusters
590 that bind to SARS-CoV-2 RBD with OD 450 values larger than 3. The VH gene
591 families for the highest binding clusters are shown.
592
593 Figure 3. Clonal expansion of specific heavy and light chain families in
594 the P#2 antibody repertoire. (A) Phylogenetic analysis of VH (left) and VL
19

595 (right) genes for all RBD-binding antibodies. Clonal expanded VH and VL
596 clusters are paired and highlighted in three different colors. Branch lengths are
597 drawn to scale so that sequence relatedness can be readily assessed. (B)
598 Clonal expansion in relation to members of other VH and VL families based on
599 somatic hypermutations (SHM) and CDR3 loop lengths. For the pie charts of
600 VH (left) and VL (right) genes, the radii represent the CDR3 loop length and the
601 color scale indicates the degree of SHM. Heavy and light chain repertoires for
602 each antibody are shown along the pie circles.
603
604 Figure 4. Antibody neutralization analyzed by pseudovirus and live SARS-
605 CoV-2. (A) Quality control of antibody through ELISA analysis prior to
606 neutralization assay. A serial dilution of each antibody was evaluated against
607 SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was
608 recorded at an optical density (OD) of 450nm and 630nm. (B-C) Antibody
609 neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C). A serial
610 dilution of each antibody was tested against pseudovirus while two dilutions
611 against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and
612 recorded on Day 2 post-exposure. Selected antibodies and their concentrations
613 tested are indicated at the upper left corner.
614
615 Figure S1. Analysis of plasma binding to cell surface expressed trimeric
616 Spike protein. HEK 293T cells transfected with expression plasmid encoding
617 the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated
618 with 1:100 dilutions of plasma from the study subjects. The cells were then
619 stained with PE labeled anti-human IgG Fc secondary antibody and analyzed
620 by FACS. Positive control antibodies include S230 and m396 targeting the RBD
621 of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike.
622 VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.
623
624 Figure S2. RBD-specific memory B cells analyzed and isolated through
625 FACS. The recombinant RBD was labeled with either a Strep or His tag and
626 used alone or in combination to identify and isolate RBD-specific single B cells
627 through staining with the Streptavidin-APC and/or Streptavidin-PE, or anti-His-
20

628 APC and anti-His-PE antibodies. B cells to be isolated are highlighted in boxes
629 or ovals. Samples were named as either A, B, or C depending on collection
630 sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and
631 SSC-A side scatter area.
632
633 Figure S3. ELISA screening of SARS-CoV-2 RBD-specific antibodies in
634 the supernatant of transfected cells. The study subjects and the date of
635 sampling are indicated on the top. Samples were named as either A, B, or C
636 depending on collection sequence. Antibodies tested for each sample are
637 aligned in one vertical column whenever possible. For each evaluated antibody,
638 at least two independent measurements were performed and are presented
639 adjacently on the same row. Binding activities were assessed by OD 450 and
640 indicated by the color scheme on the right. Negatives (no binding activity) are
641 shown in gray for OD 450 values less than 0.1.
642
643 Figure S4. Binding kinetics of isolated mAbs with SARS-CoV-2 RBD
644 measured by SPR. The purified soluble SARS-CoV-2 RBD were covalently
645 immobilized onto a CM5 sensor chip followed by injection of individual antibody
646 at four or five different concentrations. The black lines indicate the
647 experimentally derived curves while the red lines represent fitted curves based
648 on the experimental data.
649
650 Figure S5. Antibody and ACE2 competition for binding to SARS-CoV-2
651 RBD measured by SPR. The sensorgrams show distinct binding patterns of
652 ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior
653 incubation with each testing antibody. The competition capacity of each
654 antibody is indicated by the level of reduction in response unit of ACE2
655 comparing with or without prior antibody incubation.
656
657 Figure S6. Analysis of antibody binding to cell surface expressed trimeric
658 Spike protein. HEK 293T cells transfected with expression plasmid encoding
659 the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated
21

660 with 20ug/ml testing antibodies. The cells were then stained with PE labeled
661 anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control
662 antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and
663 Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is the negative
664 control antibody targeting HIV-1 envelope glycoprotein.
665
666 Figure S7. Epitope mapping through competitive binding measured by
667 SPR. The sensorgrams show distinct binding patterns when pairs of testing
668 antibodies were sequentially applied to the purified SARS-CoV-2 RBD
669 covalently immobilized onto a CM5 sensor chip. The level of reduction in
670 response unit comparing with or without prior antibody incubation is the key
671 criteria for determining the two mAbs recognize the separate or closely situated
672 epitopes.
673
674
675
22

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25

A Figure 1
Plasma Binding to SARS-CoV-2 RBD Plasma Binding to SARS-CoV RBD Plasma Binding to MERS-CoV RBD Plasma Binding to SARS-CoV-2 NP
4 4 4 4 P#1A
P#2A
OD450nm-630nm
OD450nm-630nm
OD450nm-630nm
OD450nm-630nm
P#2B
3 3 3 3 P#2C
P#3A
2 2 2 2 P#4A
P#4B
P#5A
1 1 1 1 P#8A
P#16A
P#22A
0 0 0 0 Healthy donor
6
103 104 105 106 103 104 105 106 103 104 105 10 103 104 105 106
Plasma Dilution Plasma Dilution Plasma Dilution Plasma Dilution
B
Plasma Binding to SARS-CoV-2 Spike Plasma Binding to SARS-CoV Spike Plasma Binding to MERS-CoV Spike Plasma Binding to HIV-1 BG505 Trimer
4 4 4 4 P#1A
P#2A
OD450nm-630nm
OD450nm-630nm
OD450nm-630nm
OD450nm-630nm
P#2B
3 3 3 3 P#2C
P#3A
2 2 2 2 P#4A
P#4B
P#5A
1 1 1 1 P#8A
P#16A
P#22A
0 0 0 0 Healthy donor
103 104 105 106 103 104 105 106 103 104 105 106 103 104 105 106
C
Plasma Neutralizing SARS-CoV-2 Plasma Neutralizing SARS-CoV Plasma Neutralizing MERS-CoV Plasma Neutralizing HIV-MG04
P#1A
100 100 100 100 P#2A
Neutralization (%)
Neutralization (%)
Neutralization (%)
Neutralization (%)
P#2B
75 75 75 75 P#2C
P#3A
50 50 50 50 P#4A
P#4B
25 25 25 25 P#5A
P#8A
0 0 0 0 P#16A
P#22A
Healthy donor
102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105
D Lymphocytes Single cells Live CD19+CD3-CD8-CD14-

E
250K 250K RBD-specific cells in total B
105
Percent in CD19+ cells (%)
<PE-Cy7>: CD19
0.08 P#1A
200K 200K P#2A
104 P#2B
FSC-W
SSC-A
150K 150K 0.06 P#2C

P#3A
103 0.04 P#4A
100K 100K
P#4B
P#5A
50K 50K 0.02 P#8A
0
P#16A
0 0 P#22A
0.00
0 50K 150K 250K 0 50K 150K 250K -103 0 103 104 105
P# 6A
A
P# 1A
P# 3A
P# A
P# 8A
P# A
P# B
P# C
P# A
P# B
22
5
2
2
2
4
4
FSC-A
1
FSC-A <Pacific Blue>: live/dead,CD3,CD8,CD14

P#
Percent in CD19+CD27+ cells (%)
CD27+ IgG+ 2019-nCoV RBD probe+

RBD-specific cells in memory B
250K 250K 250K
0.4 P#1A
P#2A
200K 200K 200K
P#2B
0.3 P#2C
SSC-A
SSC-A
SSC-A
150K 150K 150K P#3A

0.2 P#4A
100K 100K 100K P#4B
P#5A
0.1 P#8A
50K 50K 50K
P#16A
0 0 0 0.0 P#22A
P# 6A
A
P# 1A
P# 3A
P# A
P# 8A
P# A
P# B
P# C
P# A
P# B
103 104 105 103 104 105 103 104 105

22
5
0 0 0
2
2
2
4
4
1
P#
<APC-H7>: CD27 <FITC>: IgG <APC>: RBD probe

Figure 2
A Usage of VH Gene [%]
0 25 50 75 100
P#1 21.4 14.3 14.3 11/14
P#2 13.2 7.5 11.3 69/106
P#3 10.0 10.0 2/10
P#4 18.0 6.0 21/50
P#5 5.8 6.8 6.8 78/103
P#8 4/44
2.3
2.3
2.3
2.3
P#16 9.5 9.5 14.3 13/21
P#22 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 8/10
Negative
7−4−1
6−1
5−51
4−61
4−59
4−4
4−39
4−38−2
4−34
4−31
3−9
3−74
3−7
3−66
3−64
3−53
3−48
3−43D
3−33
3−30
3−23
3−15
3−13
3−11
2−70
2−5
1−8
1−69
1−46
1−3
1−24
1−2
B
P2B-1A10HC
11HC
HC
P2C-1D7HC
P5A-1B8HC
P5A-1D2HC
C
8HC
P1A-1D1
-1D1H
P2C-1F
P2B-1F5HC
6HC
C
-1D1H
-1D5H
P5A-3A1HC
P5A-3C
-1D
P22A
P5A
P1A
1HC
P1A
1HC
-1E
P2C
-1G
P2B
-59
P8 A-3D HC
A8 2HC
HC
2
V4
C1
E1
HC
1
2H
A-2
A-2
-2A
H
P5A B1HC
P4
A-1
P5
P5
P2 A-1 P4A
P5A A7HC
IG HC
P5A -1B9H 6HC
P4 -1D 6HC
P5 2A-1E C
-3B
12
-3
P3 A8H C
P5
1E
A
-3
C
P2 E5H
C
6HC
5H
P5 D10H C
B-
8H
H
C
A-2
P2 B-1 D11 -2G
P5 A1H HC
10
2H
P2
A-2
HC
P4 B-1 5A C C
P1 -1G
C
B-1 E1
C
P5
A-2 C
C
39
8H 3D9
P4 -1C
7H
2
1
C
A-
A-
A
P2
2E
1E
-
C1
P5
A- P5A HC
B-
C
0H
P5 P2B 2B A1
4-
P5
2
2 C
3A D1H C
A- -1 -2F 2H
A-
2G F1 6H C
2 H
HV
5 -
HC 7H
P A E8
5H 0H C
P4 A-1
P
2
P2
P5
HC
C
P4 6
IG
3A
-1
A- A-
P2 C
P 2D P 5 H
P2 B-2 2A 2H A9 C
B- F1 -1B C A-1 A1H C
4
1B 1H 1 P2
9 C 0H C-1 11H HC
P2 P5A HC
4-
C P2 -2G D6
2 - 2 B
P2 A
B-1 -1D C7H P2 A-2
P5
HV
C
P5 G8 5HC C 3H
IGH
A-2 HC -1B
P1 6A C
6A
-1B P5A-2 9HC
F P1 12H C
IG
P2C B
-1F4H 12 F7 A-1 6H
C P8A P5A P5 C
H HC P5 C -1A
P5A P2
-1D -2G A-3 8HC
V3-
-2C12
HC 5HC 4HC P A -1A
P2B- P5 5A-1C 10HC
A-1
P2A 10H
C
1E4H A5 6HC -1B C
P4B-
C HC P2B C10H
1E 2B -1
P
9
11HC C
P2B-1D -1D3H
P2B
9HC 2H 4HC
P5A-3C3 P2B-
HC C
1A5H
P2C-
P5A-3C1 8HC
2HC P2C-1A
P2B-1E2HC 5 P2C-1B1
HC
2HC
P2C-1C1
P2C-1F10HC P22A-1D8HC
P2B-1F9HC
P2C-1D12HC P2B-1D6HC
IGHV1-2
P2C-1D6HC P2C-1B12HC
P2C-1B10HC P5A-1D8HC
P2C-1A10HC
P5A-2G10HC
P2B-2G12HC P5A-2H6HC
P2B-2G1HC P5A-2F1HC
P16A-1B1HC
P2B-1B2HC
P1A-1C6HC
P2B-1A4HC
0HC P5A-3B1
P2A-1A1 P5A-2G
0HC
11HC 8HC
P5A-1C P5A-2D
3HC
11HC
IGHV1-8
P3A-
P5A-2F C 1F1H
C
3B9H P5A-
P5A- 1C4H
C C
-1D2H P16
P22A A
C P1A -1C1HC
-1A2H P4B -1D
P5A -1F 3HC
P5A 10H
P2B
-2G
9HC
-1F
8HC
C
A
P
P5 5A-2G D12H
P5 -2C9H 2HC
-2
1 C
C
Percent of OD450 > 3
P2B A3H
A-2
E4
C
A-1 HC
P16 B8
HC
P5
A-3
P2
2A
-1E
10H
C
P2
B
P4 -1B
P1
A-1
C1
HC
≥80%
HC B-1 4H
C3 C P4 C
B-1 E9H A F
P4 -1H 4HC
60%-80%
P 2
A-2 C B 5
P5 8H P4 -1G HC
-1A
IG
HC A
P4 -2B 2HC
6A C8 C
P1 A-2 C7H P4 A-1 3HC
30
5
HV
P H6
A-1 P4 A-2
≤60%
P1 C HC
5H P4 A-2 D9H
3-
- 1B C B- E1 C
6A 5H 1E 0H
1-4
P1
HV
D 3H C
P4 -1C A-1
2
P8
A- HC C
B- 6H B6
P5
A
C6 C
Patient
1G C
-1 8H
5H
6
6A 1C
P5 A-2
IG
P1 A-
P2 A-1
C
P5 D7
P5
P1 C-1
2A
6
-1 5HC
P2
HC
E6
P5
P1 G9
P#1
A
HC
HC
HC
P2B A3HC
A-2 -2G1
6A HC
HC
P2C
P2
C8
11
P5
A1
-1A
P2
1F
-1D
B-1
HC
-1
A
P5A-
P5A-
-1
3
B-
12
P#2
A
B
P5
HC
F2
12H
3
P2
-2G -1B1
HC
3-
HC
10
3C1H
1D10
P5
1H
4H
P5A
G
HC
A
-2
C
P5A
HV P#3
C
11
HC
B
-1C
HC
P2
C
HC
C
1C
HC
10
C
P1A-1B2HC
H
-2H
A-
6H
10
HC
3C
10
5HC
11
P1
IG
P2B-1G
C
C
1C
C
1F
P2A-1B3
P2C-1E
7HC
-1A
A-
2H
3A
A-2 10H
P2B-1B11H
P#4
C
7H
P2B-2H7HC
P2B-1E11HC
P2B-1C4HC
P2B-1B12HC
A-
HC
B-
P5
0H
A-
A1
P1
C
6A
P4
-1A
C
G5
P5
A1
7HC
H
C
B-1
C-1
P1
12
6A
11H
B-1
9HC
B1
5HC
P#5
P2
P2
HC
-2D
HC
P1
A-1
P2
P4
-3D
-3C
P5A
C
P5
P5A
P5A
P#8
HC
HC
C
1HC
1HC
3HC
1A5H
P16A-1B8HC
2C12
P5A-3B4HC
P5A-1C9HC
-1B10
P2C-1A7HC
P5A-2E
P#16
P5A-2D1
P5A-2H
P8A-
P4A-
P5A
P#22
Figure 3
P2C-1F10HC P2C-1F10KC
A P2C-1D12HC
P2C-1D6HC
P2C-1B10HC
IGHV1-2*06
P2C-1D12KC
P2C-1D6KC
P2C-1B10KC
P2C-1A10HC P2C-1A10KC
P2B-2G12HC P2B-2G12KC
P2B-2G1HC P2B-2G1KC
P2B-1B2HC IGKV2-40*01/2D-40*01 P2B-1B2KC
P2A-1A10HC
P2B-1A4HC
P2B-2G9HC
* P2C-1D7KC *
P2B-1A4KC
P2A-1A10KC
*
P2B-1F8HC P2C-1C8KC
P2B-1C3HC P2B-2F11KC
*
P2C-1C10HC P2B-1B9KC
P2B-1F11HC P2B-1F5KC
P2B-2G10HC P2B-1F9KC
P2B-1G5HC P2B-2G10KC
P2B-1A12HC P2B-1F10KC
P2C-1A7HC P2B-1A12KC
P2C-1B12HC P2B-1B4KC
P2B-1D6HC
P2B-1F9HC
P2B-2H7HC
* P2C-1A3KC
P2B-1D12KC
P2B-1C3KC
P2B-1E11HC P2B-1G8KC
P2B-1C4HC P2B-1E2KC
P2B-1B12HC P2B-1A10KC
**
P2B-1B11HC
IGHV3-48*02
P2C-1E1KC
P2A-1B3HC
P2C-1E5HC * P2C-1C10KC
P2B-2G9KC
P2B-1G12HC P2B-1F8KC
P2C-1A3HC
* P2B-1E12KC
P2B-1D12HC
P2B-1F5HC
P2B-1A10HC
* P2C-1F11KC
P2B-1G1KC
P2B-1E11KC
IGKV3-20*01
P2C-1D7HC P2C-1E5KC
P2C-1E1HC
**
P2C-1F11HC * P2A-1B3KC
P2B-1B11KC
P2B-1G1HC P2B-1B12KC
P2C-1D5HC
* *
P2B-2G4HC
P2B-1F2HC
P2B-1C4KC
P2B-1G12KC
P2B-2H7KC
P2B-1B4HC
P2C-1C8HC
P2C-1A1HC
* * P2C-1D5LC
P2B-1G5LC
P2B-1D11LC
P2A-1A9HC
* P2C-1A7LC
P2B-2G11HC
*
P2B-1E12HC
P2C-1A6HC **
P2C-1A1LC
P2B-2G11LC
P2A-1A9LC
P2A-1A8HC
P2B-1B10HC
P2B-1C10HC
* P2A-1B10LC
P2B-1F11LC
P2B-1D9LC
P2B-1D3HC P2C-1F4LC
P2B-2H4HC P2B-1D6LC
P2C-1A5HC IGHV3-9*01 P2C-1B12LC
P2C-1A8HC P2B-1F2LC
P2C-1C12HC
* P2B-2G4LC
P2B-2F11HC
P2C-1B1HC
* P2B-2F6LC
P2B-1E4LC
P2B-1B9HC P2B-1A1LC
P2A-1B10HC P2B-1C10LC
P2B-1G8HC P2B-1D3LC
P2B-1F10HC P2B-1B10LC
IGLV2-14*02
P2B-1D11HC
P2B-1A1HC * P2A-1A8LC
P2B-2H4LC
P2B-2F6HC
* P2C-1F4HC
P2B-1D9HC
P2C-1A5LC
P2C-1A8LC
P2C-1B1LC
P2B-1E4HC P2C-1C12LC
P2B-1E2HC P2C-1A6LC
1 0.20
B 7−4−1
5−51
9
4−5
1− SHM Ka
39 2 pp SHM
4− ac
* 10.0
* ha
in: 4
2
7.5 2-4
8−
20 0/2 3
3
5.0 D-
4−
40
10 1
2
1
* 15 *
1−4
2.5 1
*
6 1−
10
* 0.0 0
* *
69
* *
: 2-14
2−5
Heavy CDR3 Length Light CDR3 Length

3−9
10
a chain
20
1 2−70
* 15
** *
Lambd
3−1
* * 10 5
* * *
15
*
3−
* 5
* *
*
23
3−
3−
66
3 30
0
* *
Kappa chain: 3-20 0
3−
5 3−
3 3−
3
3−48
Figure 4
A B
mAbs binding to SARS-CoV-2 RBD mAbs neutralizing SARS-CoV-2
Patient #2 Patient #2
3
P2C-1F11
PC2-1D5
100 P2C-1F11 IC50: < 0.1 µg/mL
P2A-1A8 P2B-2F6 IC80: < 1 µg/mL
P2C-1C10 P2C-1A3 IC50: 0.1 ~ 5 µg/mL
P2C-1C8 80 P2C-1C10 IC80: 1 ~ 10 µg/mL
P2B-2F6
Neutralization (%)
P2A-1A10 IC : 5 ~ 50 µg/mL
P2A-1A9 50
P2A-1A8
P2B-2G11 60 P2C-1D5
IC80: 10 ~ 50 µg/mL
2
OD450nm-630nm
P2C-1A3
P2B-2G4 P2A-1A9
P2A-1B3 IC : 5 ~ 50 µg/mL
P2A-1A10 40 50
P2B-2G4 IC : > 50 µg/mL
P2A-1B3 80
P2B-2G11
P2C-1E1
P2C-1C8
Patient #1 20 P2C-1E1 IC50: > 50 µg/mL
1 P1A-1C10 IC80: > 50 µg/mL
P1A-1C7 Patient #1
P1A-1D1 0 P1A-1C10
P1A-1B2 P1A-1C7
P1A-1C1 P1A-1D1
Negative Control -20 Negative Control
0 VRC01 VRC01
10-2 10-1 100 101 10-2 100 102
Antibody conc. (µg/mL) Antibody conc. (µg/mL)
C
Cell Control P2C-1F11 (50µg/mL) P2B-2F6 (50µg/mL) P2C-1A3 (50µg/mL)
Virus Control P2C-1F11 (0.016µg/mL) P2B-2F6 (0.016µg/mL) P2C-1A3 (0.016µg/mL)
Table 1. Binding capacity, neutralizing activity, and heavy chain gene family analysis of 18 monoclonal Abs isolated from Patient #1 and Patient #2.
Binding to RBD Pseudovirus neutralization Gene family analysis
Patient mAbs
Kd (nM) competing w/ ACE2 IC50 (µg/ml) IC80 (µg/ml) IGHV IGHJ IGHD CDR3 length SHM (%)
P1A-1C7 51.08 -5.88 >50 >50 1-46*01,1-46*03 4*02 2-2*01 15 0.00
P1A-1C10 8.48 24.51 >50 >50 1-69*09 4*02 3-3*01 16 10.42
P#1 P1A-1B2 n.d. n.d. n.d. n.d. 3-30*03,3-30*18,3-30-5*01 4*02 5-24*01 12 11.46
P1A-1C1 n.d. n.d. n.d. n.d. 3-33*01,3-33*05,3-33*06 4*02 3-10*01 17 6.25
P1A-1D1 260.50 6.20 >50 >50 3-53*01 4*02 6-13*01 12 4.21
P2A-1A10 4.65 80.65 8.57 39.44 1-2*06 2*01 2-2*01 19 0.00
P2C-1C10 15.23 71.17 2.62 4.64 1-69*01,1-69D*01 4*02 4-23*01 11 0.35
P2C-1A3 2.47 81.21 0.62 5.94 3-11*04 5*01,5*02 6-13*01 12 0.00
P2C-1D5 1.64 17.61 10.65 25.36 3-23*04 4*02 3-10*01 14 0.69
P2B-2G4 21.29 41.03 5.11 >50 3-33*01,3-33*06 4*02 5-18*01 11 0.00
P2C-1C8 8.76 74.45 34.38 >50 3-33*01,3-33*06 4*02 3-22*01 13 0.69
P#2 P2A-1B3 6.00 92.15 16.77 >50 3-48*02 5*02 3-10*01 16 10.07
P2C-1E1 14.99 56.61 >50 >50 3-66*01,3-66*04 4*02 5-12*01 9 0.00
P2C-1F11 2.12 99.17 0.03 0.12 3-66*01,3-66*04 6*02 2-15*01 11 1.75
P2A-1A8 8.91 57.06 7.68 26.41 3-9*01 6*02 5-12*01 23 3.82
P2A-1A9 15.18 53.60 26.27 >50 3-9*01 6*02 3-22*01 17 2.08
P2B-2G11 17.57 52.28 34.84 >50 3-9*01 6*02 1-26*01 17 2.08
P2B-2F6 5.14 98.50 0.05 0.61 4-38-2*02 3*02 2-2*01 20 0.69
The program IMGT/V-QUEST was applied to analyze gene germline, complementarity determining region (CDR) 3 length, and somatic hypermutation (SHM). The
CDR3 length was calculated from amino acids sequences. The SHM frequency was calculated from the mutated nucleotides. n.d.: not detectable.
Table 2. Epitope mapping of mAbs through competitive binding to SARS-CoV-2 RBD

mAbs P2C-1A3 P2C-1C10 P2C-1F11 P2B-2F6 P2A-1B3 P2A-1A10
P2C-1A3 68.41 57.44 76.73 65.20 n.a.
P2C-1C10 75.33 -0.32 49.69 42.98 n.a.
P2C-1F11 52.05 -2.31 42.65 5.98 n.a.
P2B-2F6 74.87 70.97 30.22 52.79 n.a.
P2A-1B3 57.94 63.83 14.35 51.88 n.a.
P2A-1A10 76.31 84.27 79.50 73.92 42.19
n.a.: not applicable
Figure S1
Healthy Donor P#1A P#2A P#2B P#2C P#3A P#4A P#4B
SARS-CoV-2
spike
SARS-CoV
spike
SSC-H
MERS-CoV
spike
Blank
Control
anti-human Fc-PE
P#5A P#8A P#16A P#22A S230 m396 Mab-GD33 VRC01
SARS-CoV-2
spike
SARS-CoV
spike
SSC-H
MERS-CoV
spike
Blank
Control
anti-human Fc-PE
Figure S2
P#1A P#2A P#2B P#2C

250K 250K 250K 250K
200K 200K 200K 200K

SSC-A
SSC-A
SSC-A
SSC-A
150K 150K 150K 150K
100K 100K 100K 100K
50K 50K 50K 50K
0 0 0 0
3 4 5 3 4 5 3 4 5 3 4 5
0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
<APC>: RBD probe <APC>: RBD probe <APC>: RBD probe <APC>: RBD probe
P#3A P#4A P#4B P#5A

250K 250K 5 5
10 10
<PE>: RBD probe
<PE>: RBD probe

200K 200K
4 4
10 10
SSC-A
SSC-A
150K 150K
3 3
100K 100K 10 10
50K 50K 0 0
3 3
0 0 -10 -10
3 4 5 3 4 5 3 4 5 3 4 5
0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
<APC>: RBD probe <APC>: RBD probe <APC>: RBD probe <APC>: RBD probe
P#8A P#16A P#22A

250K 250K 5
10
<PE>: RBD probe
200K 200K
4
10
SSC-A
SSC-A
150K 150K
3
100K 100K 10
0
50K 50K
3
0 0 -10
3 4 5 3 4 5 3 4 5
0 10 10 10 0 10 10 10 0 10 10 10
<APC>: RBD probe <APC>: RBD probe <APC>: RBD probe
Figure S3
P#1A P#2A P#2B P#2C P#3A P#4A P#4B P#5A P#8A P#16A P#22A
Patient #1 Patient #2 Patient #2 Patient #2 Patient #3 Patient #4 Patient #4 Patient #5 Patient #8 Patient #16 Patient #22
2020.01.21 2020.01.21 2020.01.27 2020.01.30 2020.01.25 2020.01.20 2020.01.30 2020.02.01 2020.01.22 2020.02.03 2020.02.03
OD 450
P1A−1B2 P2A−1A3 P2B−1A1 P2C−1A1 P3A−1F1 P4A−1H4 P4B−1E3 P5A−1A1 P5A−2C7 P8A−1A1 P16A−1A2 P22A−1D1 5
P1A−1C1 P2A−1A7 P2B−1A4 P2C−1A3 P3A−1G1 P4A−1H5 P4B−1E7 P5A−1A2 P5A−2C8 P8A−1A3 P16A−1A3 P22A−1D2
P1A−1C2 P2A−1A8 P2B−1A5 P2C−1A4 P3A−1G3 P4A−1H6 P4B−1E11 P5A−1A5 P5A−2C9 P8A−1A4 P16A−1A5 P22A−1D5 4
P1A−1C6 P2A−1A9 P2B−1A8 P2C−1A5 P3A−1G4 P4A−1H9 P4B−1E12 P5A−1A8 P5A−2C10 P8A−1A5 P16A−1A7 P22A−1D6
P1A−1C7 P2A−1A10 P2B−1A10 P2C−1A6 P3A−1G6 P4A−1H11 P4B−1F4 P5A−1A10 P5A−2C12 P8A−1A6 P16A−1A8 P22A−1D7 3
P1A−1C10 P2A−1B3 P2B−1A12 P2C−1A7 P3A−1G7 P4A−2A2 P4B−1F6 P5A−1A11 P5A−2D2 P8A−1A8 P16A−1A10 P22A−1D8
P1A−1C11 P2A−1B4 P2B−1B2 P2C−1A8 P3A−1G8 P4A−2A3 P4B−1F10 P5A−1A12 P5A−2D3 P8A−1A9 P16A−1A11 P22A−1E3 2
P1A−1D1 P2A−1B8 P2B−1B4 P2C−1A9 P3A−1G9 P4A−2A6 P4B−1F12 P5A−1B1 P5A−2D5 P8A−1A10 P16A−1A12 P22A−1E6
P1A−1D3 P2A−1B9 P2B−1B9 P2C−1A10 P3A−1G10 P4A−2A8 P4B−1G1 P5A−1B5 P5A−2D6 P8A−1A11 P16A−1B1 P22A−1E8 1
P1A−1D5 P2A−1B10 P2B−1B10 P2C−1A11 P3A−1G12 P4A−2A10 P4B−1G2 P5A−1B6 P5A−2D7 P8A−1A12 P16A−1B2 P22A−1E10
0.1
P1A−1D6 P2B−1B11 P2C−1A12 P4A−2B1 P4B−1G3 P5A−1B8 P5A−2D8 P8A−1B3 P16A−1B3
Negative
P1A−1D8 P2B−1B12 P2C−1B1 P4A−2B2 P4B−1G5 P5A−1B9 P5A−2D10 P8A−1B4 P16A−1B4
P1A−1D11 P2B−1C3 P2C−1B2 P4A−2B3 P4B−1G6 P5A−1B10 P5A−2D11 P8A−1B5 P16A−1B5
P1A−1D12 P2B−1C4 P2C−1B10 P4A−2B4 P5A−1B11 P5A−2D12 P8A−1B6 P16A−1B8
P2B−1C10 P2C−1B12 P4A−2B6 P5A−1B12 P5A−2E1 P8A−1B7 P16A−1B9
P2B−1D3 P2C−1C1 P4A−2B10 P5A−1C1 P5A−2E4 P8A−1B12 P16A−1B10
P2B−1D4 P2C−1C3 P4A−2B12 P5A−1C2 P5A−2E5 P8A−1C1 P16A−1B11
P2B−1D6 P2C−1C4 P4A−2C1 P5A−1C4 P5A−2E6 P8A−1C2 P16A−1B12
P2B−1D7 P2C−1C6 P4A−2C3 P5A−1C5 P5A−2E8 P8A−1C3 P16A−1C1
P2B−1D12 P2C−1C12 P4A−2C9 P5A−1C8 P5A−2F1 P8A−1C6
P2B−1E2 P2C−1D2 P4A−2C11 P5A−1C9 P5A−2F7 P8A−1C7
P2B−1E3 P2C−1D4 P4A−2C12 P5A−1C10 P5A−2F9 P8A−1C8
P2B−1E4 P2C−1D5 P4A−2D1 P5A−1C11 P5A−2F10 P8A−1C9
P2B−1E7 P2C−1D6 P4A−2D2 P5A−1C12 P5A−2F11 P8A−1C10
P2B−1E8 P2C−1D7 P4A−2D5 P5A−1D1 P5A−2F12 P8A−1D5
P2B−1E11 P2C−1D9 P4A−2D9 P5A−1D2 P5A−2G2 P8A−1D6
P2B−1E12 P2C−1D12 P4A−2D12 P5A−1D6 P5A−2G4 P8A−1D9
P2B−1F2 P2C−1E1 P4A−2E2 P5A−1D7 P5A−2G5 P8A−1D10
P2B−1F3 P2C−1E4 P4A−2E4 P5A−1D8 P5A−2G7 P8A−1E1
P2B−1F8 P2C−1F4 P4A−2E7 P5A−1E1 P5A−2G10 P8A−1E4
P2B−1F9 P2C−1F10 P4A−2E9 P5A−2G11 P8A−1E5
P2B−1F10 P2C−1F11 P4A−2E10 P5A−2G12 P8A−1E6
P2B−1F11 P4A−2E12 P5A−2H3 P8A−1E8
P2B−1G1 P5A−2H5 P8A−1E9
P2B−1G3 P5A−2H6 P8A−1E10
P2B−1G5 P5A−2H7 P8A−1E12
P2B−1G8 P5A−2H12 P8A−1F3
P2B−1G11 P5A−3A1 P8A−1F4
P2B−1G12 P5A−3A2 P8A−1F9
P2B−1H6 P5A−3A4 P8A−1F11
P2B−1H8 P5A−3A6
P2B−1H10 P5A−3A7
P2B−2F6 P5A−3A8
P2B−2F11 P5A−3A9
P2B−2G1 P5A−3A10
P2B−2G2 P5A−3A11
P2B−2G3 P5A−3B1
P2B−2G4 P5A−3B4
P2B−2G9 P5A−3B6
P2B−2G10 P5A−3B7
P2B−2G11 P5A−3B8
P2B−2G12 P5A−3B9
P2B−2H4 P5A−3B10
P2B−2H7 P5A−3C1
P2B−2H8 P5A−3C2
P2B−2H9 P5A−3C3
P5A−3C6
P5A−3C7
P5A−3C8
P5A−3C9
P5A−3C10
P5A−3C12
P5A−3D9
P5A−3D11
P5A−3D12
Figure S4
Patient #2
P2C-1D5 P2C-1F11 P2C-1A3 P2A-1A10 P2B-2F6 P2A-1B3
60 60 120 200 100 100

100 80 80
150
40 40 80 60 60 SARS-CoV-2
60 100
40
40 40 RBD
20 20 50
20 20 20
0 0 0 0 0 0
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
30 30 30 30 30 30
20 20 20 20 20 20
10 10 10 10 10 10
SARS-CoV
RBD
0 0 0 0 0 0
-10 -10 -10 -10 -10 -10
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
30 30 30 30 30 30
20 20 20 20 20 20
MERS-CoV
10 10 10 10 10 10
RBD
0 0 0 0 0 0
Response Unit (RU)
-10 -10 -10 -10 -10 -10

0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
P2C-1C8 P2A-1A8 P2C-1E1 P2A-1A9 P2C-1C10 P2B-2G11 P2B-2G4
100 80 30 50 60 50 80
80 40 40 60
60
20 40 30
60 30
40 40
40 20 20
10 20
20 10 20
20 10
0 0 0 0 0 0 0
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
30 30 30 30 30 30 30
20 20 20 20 20 20 20
10 10 10 10 10 10 10
0 0 0 0 0 0 0
-10 -10 -10 -10 -10 -10 -10
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
30 30 30 30
30 30 30
20 20 20 20 20
20 20
10 10 10 10 10 10 10
0 0 0 0 0 0 0
-10 -10 -10 -10 -10 -10 -10

0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
Times (S)
Patient #1
P1A-1C10 P1A-1C7 P1A-1D1 P1A-1B2 P1A-1C1
150 40 80 30 30
30 60 20 20
100
20 10
SARS-CoV-2
40 10
50 RBD
10 20 0 0
Response Unit (RU)
0 0 0 -10 -10
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
30 60 30 30 30
20 40 20 20 20
10 20 10 10 10 SARS-CoV
0
0
0 0
0 RBD
-10 -10 -10
0 50 100 150 200 -10
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
30 60 30 30 30
20 40 20 20 20
10 10 10
MERS-CoV
20 10
RBD
0 0 0 0
0
-10 -10 -10 -10
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
Times (S)
Figure S5
Patient #2
P2C-1F11 vs ACE2 P2B-2F6 vs ACE2 P2A-1B3 vs ACE2
ACE2
40 ACE2 40 ACE2 40
20
20 20
0
P2C-1F11 + ACE2 P2B-2F6 + ACE2
0 0
P2A-1B3 + ACE2
-20
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
P2C-1A3 vs ACE2 P2A-1A10 vs ACE2 P2C-1C8 vs ACE2
40 40 ACE2 40 ACE2
ACE2
20 20 20
Response Unit (RU)
P2C-1A3 + ACE2 P2A-1A10 + ACE2 0 P2C-1C8 + ACE2

0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
P2C-1C10 vs ACE2 P2A-1A8 vs ACE2 P2C-1E1 vs ACE2
40 40 ACE2 40 ACE2
ACE2
20 20 20
P2A-1A8 + ACE2 P2C-1E1 + ACE2
0 P2C-1C10 + ACE2 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
P2A-1A9 vs ACE2 P2B-2G11 vs ACE2 P2B-2G4 vs ACE2 P2C-1D5 vs ACE2
40 ACE2 40 ACE2 40 40 ACE2

ACE2
20 20 20 20 P2C-1D5 + ACE2
P2A-1A9 + ACE2 P2B-2G11 + ACE2 P2B-2G4 + ACE2
0 0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Times (S)
Patient #1
P1A-1C10 vs ACE2 P1A-1D1 vs ACE2 P1A-1C7 vs ACE2
Response Unit (RU)
40 40 ACE2 40 P1A-1C7 + ACE2

ACE2
ACE2
P1A-1D1 + ACE2
20 P1A-1C10 + ACE2 20 20
0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Times (S)
Figure S6
P2A-1A8 P2A-1A9 P2A-1A10 P2A-1B3 P2B-2F6 P2B-2G4 P2B-2G11 P2C-1A3 P2C-1C8 P2C-1C10 P2C-1D5
SARS-CoV-2
spike
SARS-CoV
spike
SSC-H
MERS-CoV
spike
Blank
Control
anti-human Fc-PE
P2C-1E1 P2C-1F11 P1A-1C7 P1A-1B2 P1A-1C1 P1A-1C10 P1A-1D1 S230 m396 Mab-GD33 VRC01
SARS-CoV-2
spike
SARS-CoV
spike
SSC-H
MERS-CoV
spike
Blank
Control
anti-human Fc-PE
Figure S7
P2C-1A3 vs P2C-1C10 P2C-1A3 vs P2C-1F11 P2C-1A3 vs P2B-2F6 P2C-1A3 vs P2A-1B3 P2C-1A3 vs P2A-1A10
200 100 200 300 100 P2A-1A10
P2C-1C10 P2C-1F11 P2B-2F6 P2A-1B3
200
100 50 100 50
P2C-1A3 + P2C-1F11 100
P2C-1A3 + P2B-2F6 P2C-1A3 + P2A-1B3
P2C-1A3 + P2C-1C10 0 0 P2C-1A3 + P2A-1A10
0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
P2C-1C10 vs P2C-1A3 P2C-1C10 vs P2C-1F11 P2C-1C10 vs P2B-2F6 P2C-1C10 vs P2A-1B3 P2C-1C10 vs P2A-1A10
200 200 200 300 200
P2B-2F6 P2A-1B3
P2C-1A3 150
200
100 100 P2C-1F11 100 100 P2A-1A10
100
50 P2C-1C10 + P2C-1F11
P2C-1C10 + P2B-2F6 P2C-1C10 + P2A-1A10
P2C-1C10 + P2C-1A3 P2C-1C10 + P2A-1B3
0 0 0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Response Unit (RU)
P2C-1F11 vs P2C-1A3 P2C-1F11 vs P2C-1C10 P2C-1F11 vs P2B-2F6 P2C-1F11 vs P2A-1B3 P2C-1F11 vs P2A-1A10
200 100 200 400 100 P2A-1A10
P2C-1C10
P2B-2F6
P2C-1A3 300
P2A-1B3
100 50 100 200 50
P2C-1F11 + P2C-1C10
P2C-1F11 + P2B-2F6 P2C-1F11 + P2A-1B3
100
P2C-1F11 + P2C-1A3
0 0 P2C-1F11 + P2A-1A10
0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
P2B-2F6 vs P2C-1A3 P2B-2F6 vs P2C-1C10 P2B-2F6 vs P2C-1F11 P2B-2F6 vs P2A-1B3 P2B-2F6 vs P2A-1A10
200 200 100 300 100 P2A-1A10
P2C-1F11
P2A-1B3
P2C-1A3
200
100 100 P2C-1C10 50 50
P2B-2F6 + P2C-1F11 100

P2B-2F6 + P2A-1B3
P2B-2F6 + P2C-1A3 P2B-2F6 + P2C-1C10 0 0 P2B-2F6 + P2A-1A10
0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
P2A-1B3 vs P2C-1A3 P2A-1B3 vs P2C-1C10 P2A-1B3 vs P2C-1F11 P2A-1B3 vs P2B-2F6 P2A-1B3 vs P2A-1A10
300 100 200 200 100 P2A-1A10
P2C-1C10
P2C-1A3 P2C-1F11 P2B-2F6
200
50 100 100 50
100 P2A-1B3 + P2C-1C10 P2A-1B3 + P2C-1F11
P2A-1B3 + P2C-1A3 P2A-1B3 + P2A-1A10
P2A-1B3 + P2B-2F6
0 0 0 0
0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Times (S)
Table S1 Clinical characterization of the study subjects

P#1 P#2 P#3 P#4 P#5 P#8 P#16 P#22
Severity severe severe mild mild severe mild mild mild
Age 66 65 36 10 63 35 42 62
Gender male female male male female male female male
Wuhan exposure history yes yes yes yes no yes yes no
Symptom onset day Jan 3, 2020 Jan 2, 2020 Jan 9, 2020 Jan 11, 2020 Jan 8, 2020 Jan 8, 2020 Jan 18, 2020 Jan 20, 2020
First symptom Fever Fever Cough Cough Fever/Cough Fever/Cough Fever/Cough Fever/Cough
Hospitalization date Jan 11, 2020 Jan 11, 2020 Jan 16, 2020 Jan 16, 2020 Jan 16, 2020 Jan 16, 2020 Jan 22, 2020 Jan 23, 2020
Chronic basic disease hypertension hypertension none none none none none hypertension
2019-nCoV + + + + + + + +
Influenza A virus - - - - - - -
Influenza B virus - - - - - - -
RSV - - - - - - -
Adenovirus - - - - - - -
Interferon atomization Jan 14, 2020 Jan 26, 2020 Jan 16, 2020 Jan 17, 2020 Jan 16, 2020 Jan 16, 2020 Jan 21, 2020 Jan 23, 2020
Ribavirin Jan 14, 2020 Jan 14, 2020 Jan 16, 2020 Jan 17, 2020 Jan 16, 2020 Jan 16, 2020 Jan 21, 2020 Jan 23, 2020
yes no no no no no no yes
Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral Bilaetral Bilateral
CT finding
pneumonia pneumonia pneumonia pneumonia pneumonia pneumonia pneumonia pneumonia
Blood Sampling date Jan 21 Jan 21, 27, 30, Jan 25 Jan 20, 30 Feb 1 Jan 22 Feb 3 Feb 3
Outcome dead cure cure cure cure cure cure cure

Table S2. Gene family analysis of monoclonal antibodies.

Heavy chain Kappa chain (KC) or Lambda chain (LC)
Patient mAbs
IGHV IGHJ IGHD CDR3 length SHM (%) IGK(L)V IGK(L)J KC or LC CDR3 length SHM (%)
P#1 P1A-1C7 1-46*01,1-46*03 4*02 2-2*01 15 0.00 1-39*01,1D-39*01 3*01 KC 10 0.00
P#1 P1A-1C10 1-69*09 4*02 3-3*01 16 10.42 1-5*03 3*01 KC 9 3.41
P#1 P1A-1C11 1-69*09 4*02 3-3*01 16 10.42 1-5*03 3*01 KC 9 3.41
P#1 P1A-1C6 3-13*01 2*01 4-23*01 19 0.35 1-39*01,1D-39*01 3*01 KC 10 0.00
P#1 P1A-1D3 3-13*01 3*02 3-10*01 18 0.00 1-39*01,1D-39*01 1*01 KC 10 0.00
P#1 P1A-1C2 3-23*03 5*02 1-26*01 10 0.00 1-36*01 3*02 LC 11 0.00
P#1 P1A-1B2 3-30*03,3-30*18,3-30-5*01 4*02 5-24*01 12 11.46 2-14*01 2*01,3*01 LC 10 9.26
P#1 P1A-1C1 3-33*01,3-33*05,3-33*06 4*02 3-10*01 17 6.25 1D-13*01 5*01 KC 9 5.68
P#1 P1A-1D1 3-53*01 4*02 6-13*01 12 4.21 2-8*01 1*01 LC 10 2.22
P#1 P1A-1D5 3-53*01 6*02 2-15*01 15 1.05 1-33*01,1D-33*01 3*01 KC 9 0.00
P#1 P1A-1D6 3-53*01 6*02 2-15*01 15 4.56 1-33*01,1D-33*01 3*01 KC 9 3.79
P#2 P2A-1A10 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2B-1A4 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2B-1B2 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2B-2G1 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2B-2G12 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2C-1A10 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2C-1B10 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2C-1D6 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2C-1D12 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2C-1F10 1-2*06 2*01 2-2*01 19 0.00 2-40*01,2D-40*01 4*01 KC 9 0.00
P#2 P2B-1F8 1-2*06 6*02 3-9*01 14 8.33 3-20*01 1*01 KC 9 4.49
P#2 P2B-2G9 1-2*06 6*02 3-9*01 14 8.33 3-20*01 1*01 KC 9 4.49
P#2 P2B-1C3 1-46*01,1-46*03 3*01 2-2*01 15 0.00 1-5*03 1*01 KC 8 0.38
P#2 P2C-1C10 1-69*01,1-69D*01 4*02 4-23*01 11 0.35 3-11*01 2*01,2*02 KC 8 0.00
P#2 P2B-2G10 1-69*04 4*02 1-26*01 11 3.47 1-39*01,1D-39*01 2*01 KC 9 2.27
P#2 P2B-1F11 1-69*09 5*02 6-13*01 17 0.00 1-40*01 3*02 LC 11 0.00
P#2 P2B-1D9 2-5*02 4*02 3-10*01 16 1.03 1-47*02 2*01,3*01 LC 11 0.00
P#2 P2B-1E2 2-5*02 4*02 6-13*01 12 0.34 1-5*03 3*01 KC 8 0.00
P#2 P2B-1E4 2-5*02 4*02 5-12*01 11 0.00 2-14*01 2*01,3*01 LC 9 0.74
P#2 P2C-1F4 2-70*15 4*02 1-26*01 14 0.00 1-44*01 2*01,3*01 LC 10 0.00
P#2 P2B-1D12 3-11*04 5*01,5*02 6-13*01 12 0.00 1-9*01 4*01 KC 9 0.00
P#2 P2C-1A3 3-11*04 5*01,5*02 6-13*01 12 0.00 1-9*01 4*01 KC 9 0.00
P#2 P2B-1D6 3-15*01 6*02 3-10*01 24 0.00 1-44*01 3*02 LC 11 0.00
P#2 P2C-1B12 3-15*01 6*02 3-10*01 13 1.02 6-57*02 1*01 LC 10 0.00
P#2 P2B-1F9 3-15*01 4*02 3-22*01 16 0.00 1-NL1*01 1*01 KC 10 0.00
P#2 P2C-1D5 3-23*04 4*02 3-10*01 14 0.69 3-21*01 1*01 LC 11 0.38
P#2 P2B-1B4 3-30*04,3-30-3*03 6*02 3-10*01 22 0.35 1-39*01,1D-39*01 3*01 KC 10 0.00
P#2 P2B-1F2 3-33*01,3-33*06 4*02 5-18*01 11 0.00 2-11*01 2*01,3*01 LC 11 0.00
P#2 P2B-2G4 3-33*01,3-33*06 4*02 5-18*01 11 0.00 2-11*01 2*01,3*01 LC 11 0.00
P#2 P2C-1C8 3-33*01,3-33*06 4*02 3-22*01 13 0.69 2D-30*01 2*01 KC 9 0.36
P#2 P2A-1B3 3-48*02 5*02 3-10*01 16 10.07 3-20*01 5*01 KC 10 3.00
P#2 P2B-1B11 3-48*02 5*02 3-10*01 16 10.07 3-20*01 5*01 KC 10 2.62
P#2 P2B-1B12 3-48*02 5*02 3-10*01 16 10.07 3-20*01 5*01 KC 10 2.62
P#2 P2B-1C4 3-48*02 5*02 3-10*01 16 10.07 3-20*01 5*01 KC 10 3.00
P#2 P2B-1E11 3-48*02 5*02 3-10*01 16 10.07 3-20*01 5*01 KC 10 3.00
P#2 P2B-2H7 3-48*02 5*02 3-10*01 16 10.07 3-20*01 5*01 KC 10 2.62
P#2 P2B-1G12 3-48*02 5*02 3-10*01 16 8.33 3-20*01 5*01 KC 10 3.00
P#2 P2C-1E5 3-48*02 5*02 3-10*01 16 8.33 3-20*01 5*01 KC 10 3.00
P#2 P2B-1A10 3-53*01 3*02 1-20*01 15 0.35 1-33*01,1D-33*01 2*01 KC 10 0.38
P#2 P2B-1F5 3-53*01 4*02 2-2*01 14 0.00 1-NL1*01 1*01 KC 9 0.00
P#2 P2C-1D7 3-53*01 4*02 1-26*01 12 0.00 2D-30*01 3*01 KC 9 0.00
P#2 P2B-1G1 3-66*01,3-66*04 5*02 4-17*01 11 0.00 3-20*01 2*02 KC 9 0.00
P#2 P2C-1E1 3-66*01,3-66*04 4*02 5-12*01 9 0.00 3-11*01 1*01 KC 10 0.00
P#2 P2C-1F11 3-66*01,3-66*04 6*02 2-15*01 11 1.75 3-20*01 2*01,2*02 KC 8 0.00
P#2 P2A-1A8 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2B-1B10 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2B-1C10 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2B-1D3 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2B-2H4 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2C-1A5 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2C-1A8 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2C-1B1 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2C-1C12 3-9*01 6*02 5-12*01 23 3.82 2-14*02 1*01 LC 10 2.59
P#2 P2C-1A6 3-9*01 6*02 5-12*01 23 3.47 2-14*02 1*01 LC 10 2.59
P#2 P2A-1A9 3-9*01 6*02 3-22*01 17 2.08 1-40*01 2*01,3*01 LC 11 1.11
P#2 P2C-1A1 3-9*01 6*02 3-22*01 17 2.08 1-40*01 2*01,3*01 LC 11 1.11
P#2 P2B-2G11 3-9*01 6*02 1-26*01 17 2.08 1-40*01 2*01,3*01 LC 11 1.11
P#2 P2B-1E12 3-9*01 3*02 6-19*01 17 0.00 3-20*01 4*01 KC 9 0.00
P#2 P2B-2F6 4-38-2*02 3*02 2-2*01 20 0.69 2-8*01 3*02 LC 10 0.00
P#2 P2A-1B10 4-39*01 3*02 2-15*01 20 0.34 1-47*01 3*02 LC 8 0.37
P#2 P2B-1B9 4-39*07 4*02 4-17*01 9 0.00 1-NL1*01 1*01 KC 10 0.00
P#2 P2B-2F11 4-39*07 4*02 4-17*01 9 0.00 1-NL1*01 1*01 KC 10 0.00
P#2 P2B-1G8 4-39*07 4*02 5-12*01 11 0.34 1-5*03 3*01 KC 9 1.14
P#2 P2B-1A1 4-59*01 3*02 1-1*01 14 0.35 2-14*01 3*02 LC 10 1.11
P#2 P2B-1D11 4-59*01 5*02 2-15*01 22 0.00 3-25*03 2*01,3*01 LC 9 0.00
P#2 P2B-1F10 4-59*01,4-59*02 4*02 3-10*01 15 1.05 1-39*01,1D-39*01 2*01 KC 9 1.14

P#2 P2C-1A7 5-51*01 4*02 3-10*01 17 0.00 3-1*01 2*01,3*01,3*02 LC 9 0.00
P#2 P2B-1A12 7-4-1*02 6*02 5-12*01 16 0.00 1-39*01,1D-39*01 4*01 KC 9 0.00
P#2 P2B-1G5 7-4-1*02 6*02 4-23*01 12 1.04 3-21*01 3*02 LC 11 0.38
P#3 P3A-1F1 3-13*04 4*02 6-19*01 17 0.00 1-39*01,1D-39*01 1*01 KC 10 0.00
P#3 P3A-1G8 3-64*05,3-64D*06 6*02 3-10*01 19 0.35 1-44*01 2*01,3*01 LC 11 0.00
P#4 P4A-2A10 1-46*01,1-46*03 6*02 2-15*01 26 7.64 1-40*01 2*01,3*01 LC 10 1.85
P#4 P4B-1F6 1-69*01,1-69D*01 1*01 1-26*01 15 3.47 2-23*02 1*01 LC 10 1.85
P#4 P4B-1E11 2-5*02 4*02 3-10*01 18 0.00 1-36*01 3*02 LC 11 0.37
P#4 P4A-2A2 3-23*04 4*02 3-10*01 14 5.90 1-51*01 3*02 LC 11 2.25
P#4 P4A-2A8 3-23*04 4*02 4-11*01 11 0.00 3-21*01 2*01,3*01 LC 11 0.00
P#4 P4A-2C1 3-23*04 6*02 6-19*01 16 2.78 2-28*01,2D-28*01 4*01 KC 11 1.08
P#4 P4A-1H5 3-30*03,3-30*18,3-30-5*01 4*02 2-2*01 21 1.74 1-39*01,1D-39*01 3*01 KC 8 3.79
P#4 P4B-1G2 3-30*03,3-30*18,3-30-5*01 4*02 2-2*01 21 1.74 1-39*01,1D-39*01 3*01 KC 8 3.79
P#4 P4A-2B3 3-30*03,3-30*18,3-30-5*01 4*02 2-2*01 21 1.39 1-39*01,1D-39*01 3*01 KC 8 3.41
P#4 P4A-1H6 3-30*03,3-30*18,3-30-5*01 4*02 2-2*01 21 1.39 1-39*01,1D-39*01 3*01 KC 8 1.52
P#4 P4B-1G5 3-30*03,3-30*18,3-30-5*01 4*02 2-15*01 22 1.39 3-21*01 1*01 LC 10 0.77
P#4 P4A-2E10 3-30*03,3-30*18,3-30-5*01 4*02 2-2*01 21 4.86 1-39*01,1D-39*01 3*01 KC 8 1.89
P#4 P4B-1E3 3-30*03,3-30*18,3-30-5*01 4*02 2-2*01 21 4.86 1-39*01,1D-39*01 3*01 KC 8 1.89
P#4 P4A-2D9 3-30*03,3-30*18,3-30-5*01 4*02 2-2*01 21 2.08 1-39*01,1D-39*01 3*01 KC 8 2.27
P#4 P4B-1F4 3-30*,3-30*18,3-30-5*01 6*02 6-13*01 22 0.35 2-30*01 2*01 KC 10 0.00
P#4 P4B-1E7 3-43D*03 6*02 4-11*01 20 0.00 3-1*01 1*01 LC 10 0.00
P#4 P4B-1F10 3-7*01 6*02 3-9*01 13 0.00 3-21*01 1*01 LC 12 0.00
P#4 P4A-2D1 3-9*01 4*02 4-23*01 13 0.00 1-12*01,1-12*02,1D-12*02 4*01 KC 9 0.00
P#4 P4A-2D2 4-39*01 6*02 3-22*01 16 0.00 3-20*01 4*01 KC 10 0.00
P#4 P4B-1E12 4-59*08 4*02 2-21*01 11 1.40 1-44*01 2*01,3*01 LC 11 0.37
P#4 P4A-2C12 5-51*01 4*02 3-22*01 15 2.43 1-44*01 1*01 LC 11 1.50
P#8 P8A-1A8 3-23*04 4*02 5-12*01 11 0.35 3-21*01 3*02 LC 11 0.77
P#8 P8A-1C6 3-30*03,3-30*18,3-30-5*01 4*02 2-15*01 20 0.00 1-33*01,1D-33*01 3*01 KC 8 0.00
P#8 P8A-1A5 5-51*01 6*03 5-18*01 18 1.74 1-47*02 1*01 LC 12 0.00
P#8 P8A-1D5 6-1*01 3*02 3-10*01 16 1.01 3-20*01 4*01 KC 9 0.37
P#5 P5A-1A1 1-24*01 5*02 3-10*01 15 0.35 2-28*01,2D-28*01 4*02 KC 9 0.00
P#5 P5A-1C8 1-46*01,1-46*03 1*01 3-22*01 22 0.00 1-33*01,1D-33*01 5*01 KC 10 0.00
P#5 P5A-2D5 1-46*01,1-46*03 3*02 3-9*01 24 0.00 1-40*01 2*01,3*01 LC 11 0.00
P#5 P5A-2C8 1-46*01,1-46*03 4*02 5-12*01 15 0.00 2-23*02 1*01 LC 10 0.00
P#5 P5A-2E9 1-46*01,1-46*03 4*02 4-17*01 22 0.00 2-14*01 1*01 LC 11 0.74
P#5 P5A-3B8 1-46*01,1-46*03 4*02 3-10*01 16 0.69 2-23*02 7*01 LC 11 0.37
P#5 P5A-3A11 1-69*01,1-69D*01 6*02 2-15*01 14 0.00 1-39*01,1D-39*01 1*01 KC 9 0.00
P#5 P5A-3C10 1-69*01,1-69D*01 5*02 2-15*01 22 0.00 6-57*02 2*01,3*01 LC 8 0.00
P#5 P5A-1A2 1-8*01 5*02 3-3*01 21 0.69 1-40*01 1*01 LC 12 0.00
P#5 P5A-1C11 1-8*01 5*02 3-10*01 17 0.00 3-21*01 2*01,3*01 LC 13 0.38
P#5 P5A-2F11 1-8*01 5*02 2-2*01 15 0.00 4-1*01 4*01 KC 9 0.00
P#5 P5A-3B9 1-8*01 5*02 2-15*01 15 0.00 1-36*01 3*02 LC 11 0.00
P#5 P5A-2C12 2-5*02 4*02 6-13*01 16 0.00 3-11*01 4*01 KC 8 0.00
P#5 P5A-3C12 2-5*02 4*02 6-13*01 19 0.00 4-1*01 2*01 KC 9 0.00
P#5 P5A-3C3 2-5*02 4*02 2-15*01 12 0.34 6-57*02 2*01,3*01 LC 9 0.00
P#5 P5A-3C1 3-11*01 5*02 6-13*01 13 1.39 3-21*01 2*01 LC 13 0.00
P#5 P5A-1C4 3-13*01 6*02 3-10*01 20 0.00 1-39*01,1D-39*01 2*01 KC 10 0.00
P#5 P5A-2G8 3-13*01 4*02 1-26*01 13 0.70 1-39*01,1D-39*01 3*01 KC 10 0.00
P#5 P5A-2D3 3-13*01 2*01 6-13*01 16 0.00 1-39*01,1D-39*01 5*01 KC 10 0.00
P#5 P5A-3B10 3-13*01 2*01 6-13*01 16 0.00 1-39*01,1D-39*01 3*01 KC 10 0.00
P#5 P5A-1D8 3-15*01 3*02 3-22*01 18 0.68 3-19*01 2*01,3*01 LC 11 0.00
P#5 P5A-2G10 3-15*01 3*02 3-22*01 18 0.00 3-19*01 2*01,3*01 LC 11 0.00
P#5 P5A-2H6 3-15*01 3*02 3-22*01 18 0.00 3-19*01 2*01,3*01 LC 11 0.00
P#5 P5A-1D6 3-23*04 4*02 1-1*01 13 0.00 3-21*01 3*02 LC 11 0.00
P#5 P5A-2E12 3-23*04 4*02 6-19*01 14 0.00 3-21*01 1*01 LC 11 0.00
P#5 P5A-3D12 3-23*04 3*02 3-22*01 24 0.35 1-47*01 1*01 LC 12 0.00
P#5 P5A-1B6 3-30*04,3-30-3*03 4*02 3-10*01 20 0.00 1-33*01,1D-33*01 2*01 KC 9 0.00
P#5 P5A-2E6 3-30*04,3-30-3*03 4*02 3-10*01 20 0.00 1-33*01,1D-33*01 2*01 KC 9 0.00
P#5 P5A-1B1 3-33*01,3-33*04,3-33*06 4*02 4-23*01 14 3.13 3-15*01 4*01 KC 9 1.89
P#5 P5A-1C5 3-33*01,3-33*04,3-33*06 4*02 4-23*01 14 3.13 3-15*01 4*01 KC 9 2.27
P#5 P5A-2H7 3-33*01,3-33*04,3-33*06 4*02 4-23*01 14 3.13 3-15*01 4*01 KC 9 1.89
P#5 P5A-2G9 3-33*01,3-33*06 4*02 3-10*01 12 0.00 5-37*01 1*01 LC 10 0.35
P#5 P5A-2G11 3-33*01,3-33*06 6*02 3-16*01 17 0.00 2-14*01 2*01,3*01 LC 11 0.74
P#5 P5A-1B8 3-53*01 4*02 2-15*01 9 1.40 1-9*01 4*01 KC 9 0.00
P#5 P5A-1D2 3-53*01 4*02 1-26*01 15 1.40 1-40*01 2*01,3*01 LC 11 1.11
P#5 P5A-1D1 3-53*01 6*02 3-16*01 11 0.35 1-9*01 5*01 KC 8 0.76
P#5 P5A-2C9 3-7*01 4*02 6-19*01 14 0.00 3-20*01 5*01 KC 10 0.00
P#5 P5A-2E4 3-7*01 4*02 6-19*01 14 0.35 3-20*01 5*01 KC 10 0.00
P#5 P5A-2G12 3-7*01 4*02 5-18*01 12 0.00 6-57*02 2*01,3*01 LC 10 0.00
P#5 P5A-2D12 3-7*01 6*02 4-11*01 18 0.00 2-28*01,2D-28*01 1*01 KC 9 0.00
P#5 P5A-2F1 3-74*02 4*02 6-19*01 12 0.00 6-57*02 2*01,3*01 LC 9 0.00
P#5 P5A-1C10 3-9*01 4*02 4-17*01 14 0.00 3-21*01 1*01 LC 12 0.00
P#5 P5A-2E8 3-9*01 4*02 4-17*01 13 0.00 3-21*01 1*01 LC 11 0.00
P#5 P5A-3A2 3-9*01 4*02 4-17*01 14 1.74 3-21*01 1*01 LC 11 0.00
P#5 P5A-2D6 3-9*01 4*02 3-10*01 14 0.35 1-40*01 2*01,3*01 LC 12 0.74
P#5 P5A-1B12 3-9*01 6*02 4-17*01 17 0.69 1-51*01 2*01,3*01 LC 11 0.37
P#5 P5A-3A6 3-9*01 6*02 3-10*01 27 0.69 2-14*01 2*01,3*01 LC 10 0.74
P#5 P5A-3D9 3-9*01 3*02 3-3*02 16 0.00 3-15*01 4*01 KC 11 0.38
P#5 P5A-1D10 3-11*01 4*02 3-16*02 21 2.43 2-14*01 2*01,3*01 LC 11 1.11

P#5 P5A-3A1 3-53*01 4*02 4-17*01 11 0.00 3-20*01 2*02 KC 9 0.00
P#5 P5A-3C8 3-53*01 6*02 4-11*01 11 1.05 1-9*01 2*01 KC 11 1.14
P#5 P5A-2D10 4-31*03 5*02 5-12*01 12 0.34 6-57*02 2*01,3*01 LC 10 0.37
P#5 P5A-2G5 4-31*03 4*02 3-16*02 14 1.37 3-21*01 2*01,3*01 LC 11 0.00
P#5 P5A-1A12 4-39*01 6*02 2-21*01 17 0.69 4-1*01 1*01 KC 9 0.00
P#5 P5A-2C7 4-39*01 4*02 4-17*01 16 0.00 2-23*02 3*02 LC 10 0.00
P#5 P5A-2F7 4-39*01 4*02 3-22*01 18 0.00 2-23*02 1*01 LC 11 0.00
P#5 P5A-2F9 4-39*01 4*02 3-9*01 14 0.00 2-23*02 2*01,3*01 LC 8 0.00
P#5 P5A-1A5 4-4*02 4*02 4-23*01 14 0.00 2-14*01 2*01,3*01 LC 10 0.74
P#5 P5A-1C6 4-4*02 5*02 2-8*02 22 0.00 1-40*01 1*01 LC 12 0.00
P#5 P5A-3A10 4-4*02 6*02 6-13*01 21 0.00 1-39*01,1D-39*01 2*01 KC 9 0.00
P#5 P5A-1B9 4-59*01 2*01 3-9*01 22 0.70 4-1*01 4*01 KC 9 0.00
P#5 P5A-3A7 4-59*01 2*01 3-9*01 22 0.00 4-1*01 4*01 KC 9 0.00
P#5 P5A-3B1 4-59*01 2*01 3-9*01 22 0.00 4-1*01 4*01 KC 9 0.00
P#5 P5A-3B6 4-59*01 2*01 3-9*01 22 0.00 4-1*01 4*01 KC 9 0.00
P#5 P5A-2C10 4-59*01 1*01 4-17*01 17 0.00 3-21*01 2*01,3*01 LC 11 0.00
P#5 P5A-2E5 4-59*01 4*02 5-12*01 12 0.00 6-57*02 2*01,3*01 LC 9 0.00
P#5 P5A-2G4 4-59*12 3*02 2-8*02 12 10.88 1D-16*01 5*01 KC 9 2.65
P#5 P5A-2G7 4-61*01 5*02 3-10*01 20 0.34 2-14*01 2*01,3*01 LC 11 0.74
P#5 P5A-1B10 5-51*01 4*02 3-16*01 12 1.04 2-28*01,2D-28*01 2*01 KC 11 0.72
P#5 P5A-1C9 5-51*01 4*02 6-19*01 11 0.00 3-19*01 1*01 LC 12 0.00
P#5 P5A-2D11 5-51*01 4*02 4-23*01 13 0.00 1-44*01 2*01,3*01 LC 11 0.00
P#5 P5A-3B4 5-51*01 4*02 4-23*01 13 0.35 1-44*01 2*01,3*01 LC 11 0.00
P#5 P5A-2H3 5-51*01 4*02 4-23*01 13 0.35 1-44*01 2*01,3*01 LC 11 0.00
P#5 P5A-2E1 5-51*01 5*02 4-11*01 12 0.00 3-21*01 2*01,3*01 LC 11 0.00
P#5 P5A-1B11 7-4-1*02 4*02 2-15*01 20 0.00 1-39*01,1D-39*01 4*01 KC 10 0.00
P#5 P5A-2D7 7-4-1*02 6*02 6-19*01 10 0.00 6-21*02 1*01 KC 8 0.00
P#5 P5A-3C9 7-4-1*02 6*02 6-19*01 10 0.00 6-21*02 1*01 KC 8 0.00
P#5 P5A-3D11 7-4-1*02 6*02 6-19*01 10 0.00 6-21*02 1*01 KC 8 0.00
P#16 P16A-1A3 1-3*01 5*02 5-18*01 11 0.00 6-57*02 2*01,3*01 LC 9 0.37
P#16 P16A-1A8 1-46*01,1-46*03 4*02 2-2*01 20 0.00 3-21*01 1*01 LC 13 0.00
P#16 P16A-1B5 1-46*01,1-46*03 4*02 3-3*01 13 0.00 3-21*02 2*01,3*01 LC 12 0.00
P#16 P16A-1C6 1-46*01,1-46*03 1*01 6-19*01 16 0.69 3-21*02 3*02 LC 12 0.38
P#16 P16A-1C1 3-13*01 6*03 6-13*01 21 0.00 1-39*01,1D-39*01 1*01 KC 10 0.00
P#16 P16A-1A5 3-33*01,3-33*06 4*02 6-25*01 15 0.00 1-33*01,1D-33*01 4*01 KC 9 0.38
P#16 P16A-1A12 3-33*01,3-33*06 4*02 2-21*02 19 0.35 1-51*01 3*02 LC 11 0.75
P#16 P16A-1B1 3-74*02 5*02 6-13*01 15 2.43 1-36*01 2*01,3*01 LC 11 3.37
P#16 P16A-1B3 3-9*01 6*02 6-13*01 24 0.35 3-1*01 1*01 LC 10 0.00
P#16 P16A-1B12 4-34*01 6*03 2-2*01 16 0.00 1-51*01 2*01,3*01 LC 11 0.37
P#16 P16A-1B8 5-51*01 4*02 3-16*02 19 0.00 3-1*01 2*01,3*01 LC 11 0.00
P#16 P16A-1A7 7-4-1*02 3*02 1-26*01 14 0.69 3-21*01 2*01,3*01 LC 12 0.00
P#16 P16A-1A10 7-4-1*02 3*02 1-20*01 15 0.00 3-21*02 3*02 LC 12 0.00
P#22 P22A-1E10 1-46*01,1-46*03 6*02 2-2*01 15 0.00 3-11*01 3*01 KC 10 0.00
P#22 P22A-1D2 1-8*01 5*02 3-3*01 21 0.00 1-40*01 1*01 LC 12 0.00
P#22 P22A-1D8 3-23*04 4*02 3-22*01 20 10.42 3-15*01 1*01 KC 10 3.03
P#22 P22A-1D7 3-33*01,3-33*06 4*02 4-17*01 13 0.35 1-39*01,1D-39*01 1*01 KC 10 0.38
P#22 P22A-1D1 3-53*01 6*02 No results 11 0.00 1-9*01 1*01 KC 8 0.38
P#22 P22A-1E8 3-9*01 4*02 6-19*01 16 0.00 3-15*01 4*01 KC 11 0.00
P#22 P22A-1D5 4-39*01 4*02 5-24*01 14 0.00 2-23*01,2-23*03 1*01 LC 8 0.00
P#22 P22A-1E6 4-59*01 4*02 3-22*01 16 0.00 3-20*01 4*01 KC 9 0.37
The program IMGT/V-QUEST was applied to analyze gene germline, complementarity determining region (CDR) 3 length, and somatic hypermutation (SHM). The CDR3 length was calculated from
amino acids sequences. The SHM frequency was calculated from the mutated nucleotides.

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bioRxiv preprint doi: https://doi.org/10.1101/2020.03.21.990770.

1 Potent human neutralizing antibodies elicited by SARS-CoV-2 infection

25 *These authors contributed equally to this work.

26 #Correspondence: zhangzheng1975@aliyun.com (Z.Z) or

31 The pandemic caused by emerging coronavirus SARS-CoV-2 presents a

56 The source of the recent Coronavirus Disease 2019 (COVID-19) outbreak in

67 SARS-CoV-2 utilizes an envelope homotrimeric Spike glycoprotein (S) to

87 able to effectively block SARS-CoV-2 entry. Here, we report on the isolation

189 Broad diversity and clonal expansion of antibodies in the repertoire. As

216 VH3-9*01 cluster revealed richness in tyrosine, indicating potential hydrogen

402 Isolation of RBD-specific single B cells by FACS. RBD-specific single B

412 staining at 4 °C for 30min involved either: Streptavidin-APC (eBioscience)

449 Antibody binding kinetics, epitope mapping, and competition with

465 Analysis of plasma and antibody binding to cell surface expressed

482 Neutralization activity of mAbs against pseudovirus and live SARS-CoV-2.

499 All experiments involving live SARS-CoV-2 followed approved Biosafety

522 Antibody production. The production of antibodies was conducted as

539 Beijing Municipal Science and Technology Commission (171100000517-001

559 Conflict of interests: We declare no competing interest.

562 Figure Legends

725 20 Kirchdoerfer, R. N. et al. Stabilized coronavirus spikes are resistant to conformational

774 39 Zhang, S. et al. Structural Definition of a Unique Neutralization Epitope on the

D Lymphocytes Single cells Live CD19+CD3-CD8-CD14-

150K 150K 0.06 P#2C

FSC-A <Pacific Blue>: live/dead,CD3,CD8,CD14

CD27+ IgG+ 2019-nCoV RBD probe+

150K 150K 150K P#3A

103 104 105 103 104 105 103 104 105

<APC-H7>: CD27 <FITC>: IgG <APC>: RBD probe

P#1 21.4 14.3 14.3 11/14

P#2 13.2 7.5 11.3 69/106

P#3 10.0 10.0 2/10

P#4 18.0 6.0 21/50

P#5 5.8 6.8 6.8 78/103

P2B-1B2HC IGKV2-40*01/2D-40*01 P2B-1B2KC

P2C-1A5HC IGHV3-9*01 P2C-1B12LC

Heavy CDR3 Length Light CDR3 Length

Table 2. Epitope mapping of mAbs through competitive binding to SARS-CoV-2 RBD

P#5A P#8A P#16A P#22A S230 m396 Mab-GD33 VRC01

P#1A P#2A P#2B P#2C

200K 200K 200K 200K

100K 100K 100K 100K

50K 50K 50K 50K

P#3A P#4A P#4B P#5A

<PE>: RBD probe

<PE>: RBD probe

P#8A P#16A P#22A

60 60 120 200 100 100

-10 -10 -10 -10 -10 -10

P2C-1C8 P2A-1A8 P2C-1E1 P2A-1A9 P2C-1C10 P2B-2G11 P2B-2G4

-10 -10 -10 -10 -10 -10 -10

P2C-1A3 vs ACE2 P2A-1A10 vs ACE2 P2C-1C8 vs ACE2

P2C-1A3 + ACE2 P2A-1A10 + ACE2 0 P2C-1C8 + ACE2

P2C-1C10 vs ACE2 P2A-1A8 vs ACE2 P2C-1E1 vs ACE2

P2A-1A9 vs ACE2 P2B-2G11 vs ACE2 P2B-2G4 vs ACE2 P2C-1D5 vs ACE2

40 ACE2 40 ACE2 40 40 ACE2

40 40 ACE2 40 P1A-1C7 + ACE2

P2B-2F6 + P2C-1F11 100

Table S2. Gene family analysis of monoclonal antibodies.

P#2 P2B-1F10 4-59*01,4-59*02 4*02 3-10*01 15 1.05 1-39*01,1D-39*01 2*01 KC 9 1.14

P#5 P5A-1D10 3-11*01 4*02 3-16*02 21 2.43 2-14*01 2*01,3*01 LC 11 1.11

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P2B-1B2HC IGKV2-4001/2D-4001 P2B-1B2KC

P#2 P2B-1F10 4-5901,4-5902 402 3-1001 15 1.05 1-3901,1D-3901 2*01 KC 9 1.14

P#5 P5A-1D10 3-1101 402 3-1602 21 2.43 2-1401 201,301 LC 11 1.11