key: cord-0814449-1m0fjdx3
authors: Wu, F.; Yan, R.; Liu, M.; Liu, Z.; Wang, Y.; Luan, D.; Wu, K.; Song, Z.; Sun, T.; Ma, Y.; Zhang, Y.; Wang, Q.; Li, X.; Ji, P.; Li, Y.; Li, C.; Wu, Y.; Ying, T.; Wen, Y.; Jiang, S.; Zhu, T.; Lu, L.; Zhou, Q.; Huang, J.
title: Antibody-dependent enhancement (ADE) of SARS-CoV-2 infection in recovered COVID-19 patients: studies based on cellular and structural biology analysis
date: 2020-10-13
journal: nan
DOI: 10.1101/2020.10.08.20209114
sha: e687a9855e3e0837f694b0cd2ae018003a1a2059
doc_id: 814449
cord_uid: 1m0fjdx3

Antibody-dependent enhancement (ADE) has been reported in several virus infections including dengue fever virus, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronavirus infection. To study whether ADE is involved in COVID-19 infections, in vitro pseudotyped SARS-CoV-2 entry into Raji cells, K562 cells, and primary B cells mediated by plasma from recovered COVID-19 patients were employed as models. The enhancement of SARS-CoV-2 entry into cells was more commonly detected in plasma from severely-affected elderly patients with high titers of SARS-CoV-2 spike protein-specific antibodies. Cellular entry was mediated via the engagement of Fc{gamma}RII receptor through virus-cell membrane fusion, but not by endocytosis. Peptide array scanning analyses showed that antibodies which promote SARS-CoV-2 infection targeted the variable regions of the RBD domain. To further characterize the association between the spike-specific antibody and ADE, an RBD-specific monoclonal antibody (7F3) was isolated from a recovered patient, which potently inhibited SARS-Cov-2 infection of ACE-2 expressing cells and also mediated ADE in Raji cells. Site-directed mutagenesis the spike RBD domain reduced the neutralization activity of 7F3, but did not abolish its binding to the RBD domain. Structural analysis using cryo-electron microscopy (Cryo-EM) revealed that 7F3 binds to spike proteins at a shift-angled pattern with one up and two down RBDs, resulting in partial overlapping with the receptor binding motif (RBM), while a neutralizing monoclonal antibody that lacked ADE activity binds to spike proteins with three up RBDs, resulting in complete overlapping with RBM. Our results revealed that ADE mediated by SARS-CoV-2 spike-specific antibodies could result from binding to the receptor in slightly different pattern from antibodies mediating neutralizations. Studies on ADE using antibodies from recovered patients via cell biology and structural biology technology could be of use for developing novel therapeutic and preventive measures for control of COVID-19 infection.

previously 24 . Plasma from 16 (8%) of the recovered patients with mild COVID-19 and 117 13 (76%) of the recovered patients with severe COVID-19 (N=17, median age 66) 118 showed a concentration-dependent enhancement of SARS-CoV-2 infection of Raji cells, 119 indicated by the increase of luciferase expression in Raji cells ( Figure S1,1A) . The 120 enhancement of virus infection was significantly higher in plasma from COVID-19 121 patients compared with plasma from uninfected controls (P < 0.0001, Figure 1B , S1). 122 Moreover, plasma from these 29 patients also showed detectable enhanced infection of 123 Raji cells of pseudotyped bat-origin SARS-like coronavirus, either RS3367 or WIV1 124 (P = 0.0108 or P = 0.0046, Figure 1B ), while none of the plasma showed enhancement 125 of SARS-CoV infection ( Figure 1B) . 126 The enhancement of SARS-CoV-2 infection by patient plasma was also observed when 127 K562 cells derived from human monocytes were used as targets (P = 0.0006, Figure S2 , 128 1C). Furthermore, the enhancement of SARS-CoV-2 infection was also confirmed 129 when cultured primary B cells were used as targets. As shown in Figure S3 Figure 1D ). 135 In the following studies, Raji cells were used as targets on the mechanism of 136 enhancement of SARS-CoV2 infection because they were easily maintained and 137 generated higher luciferase reading than K562 cells and primary B cells. 158 We investigated the clinical characteristics of 29 recovered patients whose plasma 159 showed ADE effect. The median age of these patients (65 [58-72] years) was 160 significantly higher than the patients without ADE effect (50 [37-64] years, P < 0.0001, 161 Figure 1H ). The median disease duration time and the length of hospital stays of 162 patients whose plasma showed ADE was significantly longer than patients without 163 ADE effect (35 Figure 1I and 1J). These results indicated that ADE is more 165 likely to develop in elderly patients with severe and critical condition, longer hospital 166 stays and disease duration, suggesting a possible association of ADE with disease 167 severity in COVID-19 patients. 168 To evaluate whether the ADE effect resulted from pre-exposure to other pathogens in 169 elderly patients, we collected plasma from 18 uninfected elderly donors aged 60 to 80 170 years and tested them for ADE. None of the plasma from uninfected control donors 171 showed an ADE effect (P = 0.3085, Figure S4 ), confirming that ADE appeared to be the result of SARS-CoV-2 infection. 173 174 ADE is more likely to develop in patients with high titers of SARS-CoV-2 RBD-175 and S1-specific antibodies 176 Next, we evaluated the relationship between ADE effect and SARS-CoV-2-specific 177 antibodies. Significantly higher titers of SARS-CoV-2 NAbs (P < 0.0001, Figure 2A ), 178 as well as RBD-specific (P < 0.0001) and S1-specific binding antibodies (P < 0.0001) 179 ( Figure 2B ), were found in plasma with ADE effect compared to plasma without ADE 2C and Figure S5A ). The kinetics of titers of antibodies binding to RBD and S1 ( Figure   186 2D and Figure S5B ) was similar to the kinetics of ADE ( Figure 2C and Figure S5B 195 To further determine the role of spike-specific antibodies in mediating ADE of SARS-( Figure 3A) , and pre-incubation with S1 protein at the concentration of 1 μg/ml could 201 also block the ADE effect ( Figure 3B ). However, pre-incubation with SARS-CoV RBD 202 or S1 protein did not change the ADE activity in plasma ( Figure 3C and 3D). The 203 inhibition of ADE effect by SARS-CoV-2 RBD protein was also observed in the plasma 204 from other patients. As shown in Figure 3E , pre-incubation of 10 μg/ml SARS-CoV-2 205 RBD significantly reduced ADE effect mediated by plasma from six tested patients (P 206 = 0.009). These results indicated that the ADE of SARS-CoV-2 infection was mediated 207 by antibodies targeting SARS-CoV-2 spike RBD subunits. 4B). The inhibition of ADE effect by EK1 peptide, but not chloroquine, was observed 219 for six tested plasma samples with ADE effect (P = 0.0064, Figure 4C ). These results 220 indicated that ADE of SARS-CoV-2 infection was mediated through virus-to-cell 221 membrane fusion, not phagocytosis.

The ability of plasma to promote virus-to-cell membrane fusion was confirmed by an 223 in vitro syncytium formation assay, using HEK-293T cells expressing the SARS-CoV- Figure 4E ). The syncytium formation induced by plasma 228 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint from COVID-19 patients was specifically inhibited by the addition of EK1 peptide, but 229 not chloroquine ( Figure 4D , right; Figure 4E ). These results again confirmed that ADE 230 of SARS-CoV-2 infection by plasma was mainly through cell-to-cell membrane fusion, 231 a pathway involved in the formation of the six-helix bundle (6HB) of SARS-CoV-2 S2 232 protein, which could be inhibited by EK1 peptide. SARS-CoV-2 RBD and S1 proteins, but not S2, in the ELISA assay ( Figure 5B ). Figure 5E ) or blocking FcɤRII receptor with anti-CD32 antibody ( Figure 5F ). 252 We also compared the ADE effect of different isotypes of 7F3 antibodies which were 253 generated by linking different heavy-chain constant regions to the same variable region 254 of 7F3 antibody. The IgG1 isotype showed the strongest ADE effect, IgG4 isotype 255 showed detectable ADE effect, while IgG2 and IgG3 did not show any detectable ADE 256 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint ( Figure 5G ), possibly resulting from the different binding affinity to FcɤRII receptor on 257 Raji cells. Consistent with the observations for plasma samples from recovered patients, 258 antibody 7F3-mediated enhancement of SARS-CoV-2 infection could be specifically 259 reduced by pre-incubation with RBD protein of SARS-CoV-2, but not from SARS-CoV 260 virus ( Figure 5H ), and the ADE could also be blocked by fusion-inhibitor EK1 peptide, 261 but not chloroquine ( Figure 5I ), suggesting that antibody 7F3-mediated ADE of SARS-

CoV-2 infection of Raji cells also occurred through virus-to-cell membrane fusion.

Peptide scanning for hot spots in RBD associated with ADE effect 265 Next, we explored the epitopes in RBD to which patient plasma and NAb 7F3 bound 266 and induced ADE. We synthesized a series of 20-mer peptides with 10 amino acid 267 overlap spanning the RBD region (304-593) to block the ADE of patient plasma and 268 antibody 7F3. Peptides from the S1 region, i.e., 304-323, 364-383, 544-563, 564-583, 269 and 574-593, dramatically blocked the ADE of both patient plasma ( Figure 6A ) and 270 7F3 ( Figure 6B ) and decreased >70 % of AUC ( Figure 6C ). Peptides from S1 regions perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint complete resistance to the neutralization of both 7F3 and 4L12 (IC50 >50 μg/ml), 286 suggesting overlapping between the epitopes of 7F3 and 4L12. 287 We expressed the RBD protein mutants and measured the binding ability of 7F3 to these 288 mutants relative to wild type RBD. None of these mutations except P491A affected 7F3 289 and 4L12 binding to spike protein ( Figure 7B and 7C). A mutant with single mutation 290 D427A and four mutants with three amino acid alanine substitutions in RBD showed 291 decreased binding to 4L12 but had no effect on 7F3 binding ( Figure 7B and 7C). Even 292 though mutations F342L and E516A in the RBD region affected 7F3 neutralization, 293 they did not impact 7F3 binding, which may play an important role in ADE. Table S1 ). Details of cryo-EM sample 300 preparation, data collection and processing, as well as model building, can be found in 301 the Materials and Methods section in Supplementary Information (SI).

The overall resolution for S-ECD was good enough for model building, whereas the 303 resolution at the interface between 7F3 and S-ECD was worse owing to the flexibility. 304 We only docked the light chain and heavy chain of 7F3 into the cryo EM map. The S 305 protein bound with 7F3 exhibits a conformation with one "up" and two "down" RBDs, 306 among which the "up" RBD and one of two "down" RBDs were bound by 7F3, whereas 307 the other "down" RBD was not bound by 7F3 ( Figure 8A ). In contrast to the S/7F3 308 complex, all three RBDs of S protein were in "up" conformation and bound with 4L12 309 in the S/4L12 complex ( Figure 8B ). Additionally, the interfaces between antibodies and 310 RBD in both antibodies are overlapped with binding to ACE2 ( Figure S9 ).

The resolution at the interface between 4L12 and RBD was improved to 3.5 Å by 312 focused refinement, allowing detailed analysis (Fig. S7 ). When compared with 4L12 313 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint bound structure, 7F3 bound to RBD with a shift of about 28.5 angstroms ( Figure 8C perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint suggesting that ADE may be associated with worse clinical outcomes during SARS- It is interesting that antibody-mediated viral entry into Fc receptor-bearing cells was 364 not through phagocytosis, but rather, through virus-to-cell membrane fusion. However, 365 the molecular mechanism that regulates the interaction among spike protein, antibody 366 and Fc receptors in order to initiate virus-cell membrane fusion remains unknown. It 367 should be noted that not all RBD-specific antibodies will induce ADE effect. Antibodies 368 that can induce ADE in this study bind to the spike with one "up" and two "down" RBD 369 domains, while the antibodies that cannot induce ADE bind to the spike with three "up" 370 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint RBD domains. Therefore, the different binding pattern to spike proteins may result in 371 different abilities to promote ADE, but the detailed mechanism requires further studies. 372 Our results revealed that antibodies mediating ADE of SARS-CoV-2 infection were not However, these results suggest that vaccine candidates should be evaluated for 397 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint induction of ADE in addition to induction of neutralizing antibodies. A vaccine that can 398 induce high titers of neutralizing antibodies should be safer than one inducing low titers 399 since 1) most the newly invaded virions are neutralized before the ADE occurs and 2) 400 neutralizing antibodies mediate ADE only at the suboptimal neutralizing concentration.

Furthermore, these results also suggested that plasma and antibodies from patients who 402 recovered from COVID-19 should be tested for potential ADE effect before clinical 403 usage. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint constructed as previously described 18 Briefly, 50 μl of Raji cells or K562 cells were seeded into a 96-well plate pre-coated 452 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint then mixed with the effector cells and serially diluted ADE patient plasma at 37°C for 509 30 min. Then, the mixture was applied to the Raji cell as described above. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint

The kinetics of monoclonal antibody binding to SARS-CoV-2 RBD protein was 537 measured by biolayer interferometry binding assay on a FortéBio OctetRED96 538 instrument using anti-human IgG (AHC) biosensors as previously described 38 The 539 assay followed sequential steps at 30℃ as follows. First, the biosensor was immersed 540 in sterile water for 60s, and 10 μg/ml of antibody was loaded on the biosensors. The Particles for all samples were automatically picked using Relion 3.0.6 43-46 from 565 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint manually selected micrographs. After 2D classification with Relion, good particles 566 were selected and subjected to two cycles of heterogeneous refinement without 567 symmetry using cryoSPARC 47 .The good particles were selected and subjected to Non-568 uniform Refinement (beta) with C1 symmetry, resulting in 3D reconstruction for the 569 whole structures, which were further subjected to 3D auto-refinement and post-570 processing with Relion. For interface between SARS-CoV-2 S protein and mAb, the 571 dataset was subjected to focused refinement with adapted mask on each RBD-mAb sub- Table S1 for details of data collection and processing. Structural refinement was performed in Phenix 52 with secondary structure and 592 geometry restraints to prevent overfitting. To monitor the potential overfitting, the 593 model was refined against one of the two independent half maps from the gold standard 594 3D refinement approach. Then, the refined model was tested against the other maps.

Statistics associated with data collection, 3D reconstruction, and model building were 596 summarized in Table S1 . comparisons test was used to correct for multiple comparisons. All tests were two-tailed, 604 and P < .05 was considered statistically significant. 606 The funders of the study had no role in study design, data collection, data analysis, data 607 interpretation, or writing of the report. The corresponding authors had full access to all 608 data in the study and had final responsibility for the decision to submit for publication. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

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

The copyright holder for this this version posted October 13, 2020. 

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint Figure S5 . Kinetics of SARS-CoV-2 ADE, spike-binding antibodies and NAbs 38 (A) Kinetics of SARS-CoV-2 ADE effect of six ADE patients are shown. Kinetics of 39 spike-binding antibodies (right Y axis), targeting RBD (brown), S1 (green), and S2 40 (blue) (B) and kinetics of NAbs (right Y axis, blue) (C) in six COVID-19 patient plasma 41 are shown and compared with the kinetics of ADE effect (left Y axis, red) in the same 42 patient. 1:400 diluted plasma was incubate with RBD, S1, or S2 protein. 43 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint is refined (black); of the model refined against the first half-map versus the same map 54 (red); and of the model refined against the first half-map versus the second half-map 55 (green). The small difference between the red and green curves indicates that the 56 refinement of the atomic coordinates lacks sufficient overfitting. (H) FSC curve of the 57 refined model of RBD-Ab7F3 subcomplex, which is the same as (G). (I)-(N) is the 58 same as (C)-(H), except for SARS-CoV-2 bound with Ab4L12 complex. 59 60 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint Processing' section in Methods for details. 66 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint Ab7F3 is shown at threshold of 6 σ. 73 74 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.08.20209114 doi: medRxiv preprint

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