key: cord-0943984-7ati571p
authors: Seydoux, Emilie; Homad, Leah J.; MacCamy, Anna J.; Parks, K. Rachael; Hurlburt, Nicholas K.; Jennewein, Madeleine F.; Akins, Nicholas R.; Stuart, Andrew B.; Wan, Yu-Hsin; Feng, Junli; Whaley, Rachael E.; Singh, Suruchi; Boeckh, Michael; Cohen, Kristen W.; McElrath, M. Juliana; Englund, Janet A.; Chu, Helen Y.; Pancera, Marie; McGuire, Andrew T.; Stamatatos, Leonidas
title: Analysis of a SARS-CoV-2 infected individual reveals development of potent neutralizing antibodies to distinct epitopes with limited somatic mutation
date: 2020-06-08
journal: Immunity
DOI: 10.1016/j.immuni.2020.06.001
sha: ad680ced7553a475ea001d4f7d30d9004c72cca0
doc_id: 943984
cord_uid: 7ati571p

Abstract Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject twenty-one days after the onset of clinical disease. Forty-five S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation, with limited clonal expansion and three bound the receptor binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine-design.

The WHO declared the 2020 COVID-19 to be a global pandemic on March 11 th , 2020

To identify B cells specific to the SARS-CoV-2 S protein that were circulating at this 118 timepoint, fluorescently labeled S2P and RBD probes were used as baits. S2P was 119 labeled with either phycoerythrin (PE) or brilliant violet 711 (BV711) and used to stain B 120 cells concurrently. This double labeling strategy helped to discriminate between bona 121 fide S2P-specific B cells and non-specific background staining to the fluorophores. were also positive for the RBD. 131 We hypothesized that the class-switched SARS-CoV-2-specific B cells were more likely had normal distributions of CDRH3 and CDRL3 lengths (Fig. 2 F-G) . Consistent with the relatively short time of infection the majority of BCR sequences showed low levels of 141 somatic mutation (Fig. 2H ). We conclude that the early B cell response to the SARS- 142 CoV-2 S protein is polyclonal and directed at epitopes mostly outside of the RBD.

Determining the binding specificities of anti-S monoclonal antibodies. 144 Among all successfully sequenced VH and VL transcripts, we obtained paired 145 sequences from forty-five. These were produced as recombinant monoclonal antibodies 146 (mAbs) of the IgG1 isotype and tested for binding to recombinant S-derived proteins 147 (Fig. 3) . 148 Most of the mAbs represented unique rearrangements, however we identified at least 4 149 expanded clones. CV4 and CV7 were identical and unmutated. CV1 and CV35 differed 150 by one synonymous LC mutation, CV40 and CV47 differed by 5 amino acids (Table S1 ).

CV12, CV39 and CV46 were much more mutated, up to 15 heavy chain and 6 light 152 chain mutations for CV12 (Table S1 ). Due to the relatively short window of time 153 between SARS-CoV-2 infection and PBMC collection, these mAbs may represent an 154 expansion of a memory B cell from a previous infection with an endemic coronavirus.

All the mAbs bound to the stabilized SARS-CoV-2 ectodomain, S2P ( Fig. 3A and E).

Consistent with the B cell staining that revealed very few RBD-specific B cells (Fig. 2B) , cell surface S. The fact that a subset of the mAbs bound to a stabilized ectodomain 163 variant of the closely related SARS-CoV S protein ( Fig. 3D and E), demonstrated that 164 there are conserved epitopes among the two viruses. Consistent with the lower degree 165 of conservation of the S1 subunit between SARS-CoV and SARS-CoV-2, the anti-RBD 166 mAbs CV30 and CV43 did not cross react with SARS-CoV S2P, while CV5 showed 167 weak binding. These results confirmed that the antibody responses to the spike protein 168 are largely outside of the RBD, and that the conformation of the stabilized ectodomain 169 variant may differ from the membrane anchored SARS-CoV-2 spike.

The early antibody response to SARS-CoV-2 infection is largely non-neutralizing.

The S2P-binding mAbs were evaluated for their ability to neutralize SARS CoV-2 172 pseudovirus infection of 293T cells stably expressing ACE2. All but three of the mAbs 173 were non-neutralizing ( Fig. 4A and Table S1 ). Although they did not achieve 100% infection by blocking the S-ACE2 interaction through an interaction that is slightly higher The S2P variants were then further purified using a Superose 6 10/300 GL column pre- mAbs that showed >50% neutralization at 50µg/ml, or sera were further analyzed to 562 determine neutralizing potency by preparing serial dilutions and conducting the 563 neutralization assay as described above. The antibody concentration or plasma dilution 564 that neutralized 50% of infectivity (IC 50 or ID 50 , respectively) was interpolated from the 565 neutralization curves determined using the log(inhibitor) vs. response --Variable slope 566 (four parameters) fit using automatic outlier detection in Graphpad Prism Software. As a 567 control for specificity SARS CoV2-mAbs were tested for neutralizing activity against 568 HIV-1 derived virions pseudotyped with murine leukemia virus envelope (MLV).

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