key: cord-0290556-m89wmmxd
authors: Ju, Bin; Zhang, Qi; Ge, Xiangyang; Wang, Ruoke; Yu, Jiazhen; Shan, Sisi; Zhou, Bing; Song, Shuo; Tang, Xian; Yu, Jinfang; Ge, Jiwan; Lan, Jun; Yuan, Jing; Wang, Haiyan; Zhao, Juanjuan; Zhang, Shuye; Wang, Youchun; Shi, Xuanling; Liu, Lei; Wang, Xinquan; Zhang, Zheng; Zhang, Linqi
title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
date: 2020-03-26
journal: bioRxiv
DOI: 10.1101/2020.03.21.990770
sha: 3af33a7ac2bffc8e944d65efbfbf954ec82c861d
doc_id: 290556
cord_uid: m89wmmxd

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

Single B cell antibody cloning and heavy chain repertoire analyses. We 155 further isolated RBD-binding B cells into single cell suspension for cloning and 156 evaluation of the mAb response ( Figure 1D and Figure S2 ). Immunoglobulin 157 heavy and light chains were amplified by RT-PCR using nested primers. The 158 amplified products were cloned into linear expression cassettes to produce full 159 IgG1 antibodies as previously described 28, 29 . The number of B cell clones 160 varied from 10 to 106 among the subjects and each clone has been differentially 161 represented ( Figure S3 ). Individual IgGs were produced by transfection of 162 linear expression cassettes and tested for SARS-CoV-2 RBD reactivity by 163 ELISA. On average, fifty-eight percent of the antibody clones were reactive, 164 although great variability was found among different individuals ( Figure S3 ). 165 Out of 358 antibodies, we obtained 206 that bound to SARS-CoV-2 RBD with 166 165 distinct sequences (Table S2) (60-80% of clusters with OD 450 > 3) and low-(< 60% cluster with OD 450 > 3) 186 binding clusters were also widely distributed and each consisted of 187 disproportionally represented VH gene families. (Table S2) . 202 More importantly, these clonally expanded antibodies were identified in all three 203 samples indicating that they are strongly selected for during infection. When 204 comparing their representation within each cluster, VH1-2*06 and VH3-9*01 205 appeared to increase from approximately 33 to 45%, whereas VH3-48*02 206 decreased from 33 to 9% over the three time points, although the number of 207 clones was too small for statistical significance. Interestingly, the somatic 208 hypermutation (SHM) or germline divergence for VH1-2*06 was 0% and this 209 cluster persisted during the study period. However, the SHM for VH3-48*02 210 reached as high as 9.6% and for VH3-9*01 reached 3.8% compared to the 211 overall average of 2.2% ± 3.3 % among the 69 VH sequences. Furthermore, 212 the CDR3 length for VH1-2*06, VH3-48*02, and VH3-9*01 was 19aa, 16aa, CoV-2 RBD showed that P#2 antibodies had dissociation constants (Kd) 226 ranging from 10 -8 to 10 -9 M while those from P#1 ranged from not detectable to 227 10 -9 M (Table 1 and Figure S4 ). SHM did not appear to correlate with Kd; some 228 germline clones with 0% divergence in both VH and VL genes (P2A-1A10, P2B- suggesting that their expansion may not be driven by affinity maturation. Next, 233 we measured each antibody for competition with ACE2 for binding to the SARS- 234 CoV-2 RBD. Specifically, the RBD was covalently immobilized on a CM5 sensor 235 chip and first saturated by antibody and then flowed through with soluble ACE2. 236 Competing capacity of each antibody was measured as percent reduction in 237 ACE2 binding with the RBD (Table 1 and Figure S5 ). As shown in Table 1 MERS-CoV RBD except P1A-1C7 (Kd=4.85μM), for which only limited cross 249 reactivity with SARS-CoV RBD was detected ( Figure S4 ). 250 We next studied antibody neutralizing activities against pseudoviruses 251 bearing the Spike protein of SARS-CoV-2. Consistent with the competing 252 capacity findings, neutralizing activity varied considerably with IC 50 values 253 ranging from 0.03 to > 50 μg/ml ( Figure 4B , 4A and Table 1 ). P2C-1F11 and 254 P2B-2F6 were the most potent, followed by P2C-1A3 and P2C-1C10. Overall, 255 ACE2 competing capacity correlated well with the neutralizing activities, 256 although this correlation was not exact in some instances. Notably, no cross- while that of P2C-1A3 was somewhat lower, although it needs to be noted that 264 CPE assay is not particularly quantitative ( Figure 4C ). Lastly, we determined 265 whether these antibodies compete for similar epitopes on the SARS-CoV-2 266 RBD. We selected a total of six antibodies with ACE2 competitive capacities of 267 at least 70% and analyzed them in a pairwise competition fashion using SPR.

As shown in Table 2 have unique pattern of distribution in the antibody repertoire without apparent 284 preferences for particular antibody families. Each antibody clone is also 285 differentially represented. In P#2, for whom additional analyses were conducted, 286 we found substantial variability in the distribution and frequency of each 287 antibody family. Some clones were identified only once whereas others 288 underwent high degrees of clonal expansion. Some clones were virtually 289 identical to their germline ancestors while others became more divergent during 290 the infection period. The CDR3 length also varied among the different clones.

These differences at the genetic levels corresponded with their binding and 292 neutralizing activities. Binding affinity (Kd) fell in the range of 10 -8 to 10 -9 M, 293 equivalent to many antibodies identified during acute infections 30-32 but 294 significantly lower than those identified during chronic HIV-1 infections 33-35 . 295 However, binding affinity alone does not predict neutralizing activity.

Competition with the receptor ACE2 governs antibody potency, although some 297 degree of discrepancy does exist. In particular, the most potent antibodies, 298 P2C-1F11 and P2B-2F6, out-competed ACE2 with close to 100% efficiency, 299 indicating that blocking the RBD and ACE2 interaction is a useful surrogate for 300 antibody neutralization. Among the antibodies tested, substantial variations in 301 competition for similar RBD epitopes or regions were also found. The most 302 potent antibody, P2C-1F11, did not seem target the same epitope as the 303 relatively moderate antibody P2C-1C10. Thus, these two antibodies could be 304 combined for synergistic antiviral effect. As we continue to screen more 305 antibodies from P#2 and other study subjects, more potent and diverse 306 antibodies are expected to be identified. These antibodies will serve as the best 307 candidates for the development of prophylactic and therapeutic intervention 308 against COVID-19 infection.

Most surprising in this study was the absence of antibody cross-reactivity 310 with RBDs from SARS-CoV and MERS-CoV. Based on the sequential and 311 structural similarities of RBDs from SARS-CoV-2 and SARS-CoV, we predicted 312 some degree of cross-binding and even cross-neutralization between the two 313 viruses. However, species-specific RBD responses in SARS-CoV-2 patients do 314 11 suggest that RBDs from SARS-CoV-2 and SARS-CoV are immunologically 315 distinct. If so, antibodies and vaccines must target each viral species differently 316 in order to achieve maximum efficacy in protecting the host from infection. Our 317 finding somewhat resolves the question of why many previously isolated 318 SARS-CoV antibodies failed to cross-neutralize SARS-CoV-2 despite 319 detectable levels of binding with Spike of SARS-CoV-2 36 . The absence of cross-320 recognition between RBDs was also apparent at the plasma level. Although 321 strong binding to SARS-CoV-2 RBD was identified, plasma samples from the 322 study subjects failed to demonstrate appreciable cross-reactivity with either 323 SARS-CoV or MERS-CoV RBD, highlighting the immunological distinctions 324 among the RBDs from the three viruses. However, substantial cross-reactivity 325 were found when the same plasma samples were applied to the trimeric Spike 326 proteins of SARS-CoV and MERS-CoV, although this was higher with the 327 former than the latter. This indicates that such cross-reactivity likely occurs in 328 regions outside the RBD. Determining whether this cross-reactive response 329 has any neutralizing or protection capacity against infection would require 330 further investigation. Finally, despite successfully isolating and characterizing a 331 large of number mAbs against SARS-CoV-2, we cannot draw any firm 332 correlation between antibody response and disease status at this time. In 333 particular, the three severe cases (P#1, P#2, and P#5) appear to have relatively 334 higher plasma binding and neutralizing activities against SARS-CoV-2 than 335 those with relative mild symptoms. A larger number of patients must be studied 336 to elucidate the drivers and impact of associations between antibody response 337 and disease progression, which will provide pivotal reference for our antibody-338 based intervention as well as vaccine development. Patients and blood samples. The study enrolled a total of eight patients aged 347 10 to 66 years old infected with SARS-CoV-2 in January 2020 (Table S1) . A 348 plasma sample from a healthy control was also included. Of these eight patients, Bac-to-Bac baculovirus system (Invitrogen) as previously described 18, 19, [37] [38] [39] .

SARS-CoV-2 RBD (residues Arg319-Phe541) containing the gp67 secretion Table 1 . Binding capacity, neutralizing activity, and heavy chain gene family analysis of 18 monoclonal Abs isolated from Patient #1 and Patient #2. 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. 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.

P#1A P#2A P#2B P#2C P#3A P#4A P#4B P#5A P#8A P#16A P#22A P#1A P#2A P#2B P#2C P#3A P#4A P#4B P#5A P#8A P#16ANeutralization (%) P#1A P#2A P#2B P#2C P#3A P#4A P#4B P#5A P#8A P#16A P#22A Healthy donor P2B-1A10HC C H 8 B 1 - A 5 P P2C-1D7HC P5A-3A1H C P2B-1F 5HC P5A-1 D2HC P1A-1D1H C P2C -1F1 1HC P5A -3C8 HC P22 A-1 D1H C P5A -1D 1HC P1 A-1 D5 HC P1 A-1 D6 HC P2 B-1 G1 HC P2 C-1E 1H C P5 A-2E 12 HC P5 A-3D 12 HC P8 A-1A 8H C P4 A-2C 1H C P4 A- 2A 2H C P5 A- 1D 6H C P 2C -1 D 5H C P 4A -2 A 8H C P 3A -1 G 8H C P 1A -1 C 2H C P 4B -1 E 7H C P 2 B -1 E 1 2 H C P 5 A -1 C 1 0 H C P 5 A -2 E 8 H C P 5A -3 D 9H C P 5A -3 A 2H C P 4A -2 D 1H C P 22 A -1 E 8H C P 5A -3 A 6H C P2 A-1A 9H C P2 C -1 A1 H C P2 B-2G 11 HC P5 A-2D 6H C P1 6A -1 B3 HC P5 A-1B 12 HC P2 C-1A 6H C P2 A-1 A8 HC P2 B-1 B1 0H C P2 B-1 C1 0H C P2B -1D 3HC P2B -2H 4HC P2C -1A5 HC P2C -1A8 HC P2C-1B1H C P2C-1 C12H C P22A-1 D8HC P2B-1F9H C P2B-1D6HC P2C-1B12HC P5A-1D8HC P5A-2G10HC P5A-2H6HC P5A-2F1HC P16A-1B 1HC P1A-1 C6HC P5A-3B10 HC P5A-2G8H C P5A -2D3 HC P3A -1F1 HC P5A -1C 4HC P1 6A -1C 1H C P1 A-1 D3 HC P4 B-1 F1 0H C P5 A-2D 12 HC P5 A-2G 12 HC P5 A-2C 9H C P5 A-2E 4H C P1 A-1C 1H C P2 B-1B 4H C P4 B-1F 4H C P4 A-1H 5H C P 4B -1 G 2H C P 4A -2 B 3H C P 4A -1 H 6H C P 4A -2 D 9H C P 4 A -2 E 1 0 H C P 4 B -1 E 3 H C P 4 B -1 G 5 H C P 8A -1 C 6H C P 5A -1 B 6H C P 5A -2 E 6H C P 22 A -1 D 7H C P 16 A -1 A 5H C P2 C -1 C 8H C P1 6A -1 A1 2H C P5 A- 2G 9H C P5 A- 2G 11 HC P2 B- 1F 2H C P2 B- 2G 4H C P5 A- 1B 1H C P5 A-1 C5 HC P5 A-2 H7 HC P2 B-1 D1 2H C P2 C-1 A3 HC P5A -3C 1HC P5A -1D 10H C P2B -1G1 2HC P2C- 1E5H C P2A-1 B3HC P2B-1 B11HC P2B-1B1 2HC P2B-1C4HC C H 1 1 E 1 - B 2 P C H 7 H 2 - B 2 P C H 2 B 1 - A 1 P C H 7 A 1 - C 2 P P16A-1B8HC P5A-1C9 HC P5A-3B 4HC P5A-2 D11H C P5A-2H3H C P5A -2E1 HC P8A -1A5 HC P4A -2C 12H C P5A -1B 10H C P5 A-3 C9 HC P5 A-3 D1 1H C P5 A-2 D7 HC P5 A-1B 11 HC P1 6A -1 A7 HC P1 6A -1 A1 0H C P2 B-1A 12 HC P2 B- 1G 5H C P4 A- 2A 10 H C P2 C -1 C 10 H C P 5A -3 C 10 H C P 5A -3 A 11 H C P 4B -1 F 6H C P 2B -2 G 10 H C P 2B -1 F 11 H C P 1 A -1 C 1 0 H C P 1 A -1 C 1 1 H C P 5 A -1 A 1 H C P 5A -1 C 8H C P 16 A -1 C 6H C P 5A -2 D 5H C P 16 A -1 B 5H C P1 A- 1C 7H C P5 A-2C 8H C P1 6A -1 A8 H C P5 A-2E 9H C P2 B-1C 3H C P2 2A -1 E1 0H C P5 A-3B 8H C P1 6A -1A 3H C P2 B-1 F8 HC P2 B-2 G9 HC P5 A-1 A2 HC P22 A-1 D2H C P5A -3B 9HC P5A -2F1 1HC P5A-1C11 HC P2A-1A10 HC P2B-1 A4HC P2B-1B2 HC P2B-2G1H C P2B-2G12HC P2C-1A10HC P2C-1B10HC P2C-1D6HC P2C-1D12H C P2C-1F10 HC P2B-1E 2HC P5A-3 C12H C P5A-3C3H C P2B -1D9P 5A -2 G 5H C P 5A -2 G 7H C P 2B -1 D 11 H C P 4B -1 E 12 H C P2 B- 1A 1H C P5 A- 2C 10 H C P5 A- 2D 10 HC P5 A- 2E 5H C P2 2A -1 E6 HC P5 A- 1B 9H C P5 A- 3A 7H C P5 A-3 B1 HC P5 A-3 B6 HC 5 P#1 P#2 P#3 P#4 P#5 P#8 P#16 P#22P2C-1D12HC P2C-1D6HC P2C-1B10HC P2C-1A10HC P2B-2G12HC P2B-2G1HC P2B-1B2HC P2A-1A10HC P2B-1A4HC P2B-2G9HC P2B-1F8HC P2B-1C3HC P2C-1C10HC P2B-1F11HC P2B-2G10HC P2B-1G5HC P2B-1A12HC P2C-1A7HC P2C-1B12HC P2B-1D6HC P2B-1F9HC P2B-2H7HC P2B-1E11HC P2B-1C4HC P2B-1B12HC P2B-1B11HC P2A-1B3HC P2C-1E5HC P2B-1G12HC P2C-1A3HC P2B-1D12HC P2B-1F5HC P2B-1A10HC P2C-1D7HC P2C-1E1HC P2C-1F11HC P2B-1G1HC P2C-1D5HC P2B-2G4HC P2B-1F2HC P2C-1C8HC P2B-1B4HC P2C-1A1HC P2A-1A9HC P2B-2G11HC P2B-1E12HC P2C-1A6HC P2A-1A8HC P2B-1B10HC P2B-1C10HC P2B-1D3HC P2B-2H4HC P2C-1A5HC P2C-1A8HC P2C-1C12HC P2C-1B1HC P2B-2F11HC P2B-1B9HC P2A-1B10HC P2B-1G8HC P2B-1F10HC P2B-1D11HC P2B-1A1HC P2B-2F6HC P2C-1F4HC P2B-1D9HC P2B-1E4HC P2B-1E2HC 1 P2C-1F10KC P2C-1D12KC P2C-1D6KC P2C-1B10KC P2C-1A10KC P2B-2G12KC P2B-2G1KC P2B-1B2KC P2B-1A4KC P2A-1A10KC P2C-1D7KC P2C-1C8KC P2B-2F11KC P2B-1B9KC P2B-1F5KC P2B-1F9KC P2B-2G10KC P2B-1F10KC P2B-1A12KC P2B-1B4KC P2C-1A3KC P2B-1D12KC P2B-1C3KC P2B-1G8KC P2B-1E2KC P2B-1A10KC P2C-1E1KC P2C-1C10KC P2B-2G9KC P2B-1F8KC P2B-1E12KC P2C-1F11KC P2B-1G1KC P2B-1E11KC P2C-1E5KC P2A-1B3KC P2B-1B11KC P2B-1B12KC P2B-1C4KC P2B-1G12KC P2B-2H7KC P2C-1D5LC P2B-1G5LC P2B-1D11LC P2C-1A7LC P2C-1A1LC P2B-2G11LC P2A-1A9LC P2B-1F11LC P2A-1B10LC P2B-1D9LC P2C-1F4LC P2B-1D6LC P2C-1B12LC P2B-1F2LCP1A-1B2 P1A-1C1 P1A-1C7 P1A-1C10 P1A-1D1 P2A-1A8 P2A-1A9 P2A-1A10 P2A-1B3 P2B-2F6 P2B-2G4 P2B-2G11 P2C-1A3 P2C-1C8 P2C-1C10 PC2-1D5 P2C-1E1 P2C-1F11 VRC01 Patient #2 Patient #1

P2A-1A8 P2A-1A9 P2A-1A10 P2A-1B3 P2B-2F6 P2B-2G4 P2B-2G11 P2C-1A3 P2C-1C8 P2C-1C10 P2C-1D5 P2C-1E1 P2C-1F11 P1A-1C7 P1A-1C10 P1A-1D1 VRC01−1A2 P16A−1A3 P16A−1A5 P16A−1A7 P16A−1A8 P16A−1A10 P16A−1A11 P16A−1A12 P16A−1B1 P16A−1B2 P16A−1B3 P16A−1B4 P16A−1B5 P16A−1B8 P16A−1B9 P16A−1B10 P16A−1B11 P16A−1B12 P16A−1C1 P16A−1C2 P16A−1C6 1 2 3 4 5 0.1 P22A−1D1 P22A−1D2 P22A−1D5 P22A−1D6 P22A−1D7 P22A−1D8 P22A−1E3 P22A−1E6 P22A−1E8 P22A−1E10 OD

P2B−2G9 P2B−2G10 P2B−2G11 P2B−2G12

P2C−1C8 P2C−1C10 P2C−1C12