SARS-CoV-2 variants show resistance to neutralization by many monoclonal and serum-derived polyclonal antibodies

doi:10.21203/rs.3.rs-228079/v1

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Biological Sciences - Article

SARS-CoV-2 variants show resistance to neutralization by many monoclonal and serum-derived polyclonal antibodies

https://doi.org/10.21203/rs.3.rs-228079/v1

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published 04 Mar, 2021

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic infecting more than 106 million people and causing 2.3 million deaths. The rapid deployment of antibody-based countermeasures has provided hope for curtailing disease and ending the pandemic¹. However, the emergence of rapidly-spreading SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B.1.351), and elsewhere with mutations in the spike protein has raised concern for escape from neutralizing antibody responses and loss of vaccine efficacy based on preliminary data with pseudoviruses^2-4. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera, and human sera from recipients of the Pfizer-BioNTech (BNT162b2) mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, a chimeric Washington strain with a South African spike gene (Wash SA-B.1.351), and isogenic recombinant variants with designed mutations or deletions at positions 69-70, 417, 484, 501, and/or 614 of the spike protein. Several highly neutralizing mAbs engaging the receptor binding domain (RBD) or N-terminal domain (NTD) lost inhibitory activity against Wash SA-B.1.351 or recombinant variants with an E484K spike mutation. Most convalescent sera and virtually all mRNA vaccine-induced immune sera tested showed markedly diminished neutralizing activity against the Wash SA-B.1.351 strain or recombinant viruses containing mutations at position 484 and 501. We also noted that cell line selection used for growth of virus stocks or neutralization assays can impact the potency of antibodies against different SARS-CoV-2 variants, which has implications for assay standardization and congruence of results across laboratories. As several antibodies binding specific regions of the RBD and NTD show loss-of-neutralization potency in vitro against emerging variants, updated mAb cocktails, targeting of highly conserved regions, enhancement of mAb potency, or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

Virology

Antibody-based Countermeasures

Vaccine Efficacy

Pseudoviruses

Chimeric Washington Strain

South African Spike Gene

Assay Standardization

ACKNOWLEDGEMENTS

This study was supported by contracts and grants from NIH (75N93019C00062, 75N93019C00051, 75N93019C00074, HHSN272201400006C, HHSN272201400008C, R01 AI157155, U01 AI151810, R01 AI142759, R01 AI134907, R01 AI145617, UL1 TR001439, P30 AR073752, U01AI141990), and the Defense Advanced Research Project Agency (HR001117S0019), the Dolly Parton COVID-19 Research Fund at Vanderbilt University, Fast Grants (Mercatus Center, George Mason University), and the Future Insight Prize (Merck KGaA; to J.E.C). J.B.C. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship, E.S.W. is supported by F30 AI152327, and J.S.T. is supported by 5T32CA009547. P-Y.S. is supported by awards from the Sealy & Smith Foundation, the Kleberg Foundation, the John S. Dunn Foundation, the Amon G. Carter Foundation, the Gilson Longenbaugh Foundation, and the Summerfield Robert Foundation. P.D. is supported by a Junior Faculty Development Award from the American College of Gastroenterology. We thank Rachel Nargi and Robert Carnahan for assistance with mAb purification, Lisa Purcell for critical comments on experiments and the manuscript, Adrian Creanga and Barney Graham for the Vero-hACE2-TMPRSS2 cells, and the Laboratory of Virology, Division of Intramural Research, NIAID, NIH for the NHP immune sera.

AUTHOR CONTRIBUTIONS

R.E.C. and J.B.C. performed and analyzed neutralization assays. D.P. carried out pseudovirus neutralization and flow cytometry assays. X.X., Y.L., J.L., and X.Z. designed and generated the isogenic SARS-CoV-2 variant viruses. S.T. L.D., and D.W. performed the deep sequencing analysis. A.H.E., R.M.P., J.A.O., A.K.J.K, and P.D. designed and supervised the clinical studies and J.S.T., W.K., M.T. and A.J.S. obtained and characterized clinical samples. A.C.M.B. provided hamster immune sera. D.C., N.S., P.G., J.E.C., A.E. and H.W.V. provided mAbs. P.Y.S., A.H.E., D.C., and M.S.D. obtained funding and supervised the research. R.E.C, J.B.C., E.S.W., H.W.V., and M.S.D. wrote the initial draft, with the other authors providing editorial comments.

COMPETING FINANCIAL INTERESTS

M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. J.E.C. has served as a consultant for Eli Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from AstraZeneca and IDBiologics. Vanderbilt University (J.E.C.) and Washington University (A.H.E., A.C.M.B., M.S.D., and D.H.F.) have applied for patents related to antibodies described in this paper. The Ellebedy laboratory has received funding support in sponsored research agreements from AbbVie and Emergent BioSolutions. The Boon laboratory has received funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences Inc., AbbVie Inc., and Nano targeting & Therapy Biopharma Inc. The Shi laboratory has received sponsored research agreements from Pfizer, Gilead, Merck, and IGM Sciences Inc. D.P., D.C., and H.W.V. are employees of Vir Biotechnology and may hold equity in Vir Biotechnology. H.W.V. is a founder of Casma Therapeutics and PierianDx. P.D. has served as an advisory board member for Janssen, Pfizer, Prometheus Biosciences, and Arena Pharmaceuticals and has received funding support in sponsored research agreements from Takeda Pharmaceuticals.

Cells. Vero E6 (CRL-1586, American Type Culture Collection (ATCC), Vero-TMPRSS2, and Vero-hACE2-TMPRSS2 cells were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of penicillin–streptomycin. Vero-TMPRSS2 were generated after lentivirus transduction. Briefly, human TMPRSS2 was cloned into a pLX304 lentiviral vector (gift of S. Ding, Washington University) with a C-terminal V5 tag and a blasticidin selection marker. TMPRSS2-V5-encoding vectors were packaged as lentiviruses and Vero E6 cells were transduced. Vero E6 cells stably expressing TMPRSS2 were selected under blasticidin (5 mg/mL), and surface TMPRSS2 expression was confirmed using an anti-V5 antibody (Thermo Fisher 2F11F7) or anti-TMPRSS2 mAb (Abnova, Clone 2F4) and APC-conjugated goat anti-mouse IgG (BioLegend, 405308). Vero-hACE2-TMPRSS2 were obtained as a generous gift (A. Creanga and B. Graham, NIH).

Viruses. The 2019n-CoV/USA_WA1/2020 isolate of SARS-CoV-2 was obtained from the US Centers for Disease Control (CDC). The B.1.1.7 isolate was obtained from an infected individual. Individual point mutations in the spike gene (D614G, K417N/D614G, E484K/D614G, N501Y/D614G, P681H/D614G, del69-70/N501Y/D614G, and E484K/N501Y/D614G) were introduced into an infectious cDNA clone of the 2019n-CoV/USA_WA1/2020 (WA1/2020) strain as described previously⁴¹. Nucleotide substitutions were introduced into a subclone puc57-CoV-2-F5-7 containing the spike gene of the SARS-CoV-2 wild-type infectious clone⁴². The South African variant spike gene (B.1.351) was produced synthetically by Gibson assembly. The full-length infectious cDNA clones of the variant SARS-CoV-2 viruses were assembled by in vitro ligation of seven contiguous cDNA fragments following the previously described protocol⁴². In vitro transcription was then performed to synthesize full-length genomic RNA. To recover the mutant viruses, the RNA transcripts were electroporated into Vero E6 cells. The viruses from the supernatant of cells were collected 40-h later and served as p0 stocks⁴³. All viruses were passaged once in Vero-hACE2-TMPRSS2 or Vero-TMPRSS2 cells and subjected to deep sequencing after RNA extraction to confirm the introduction and stability of substitutions (Supplementary Table 1). Viral RNA from cell culture supernatants was used to generate next generation sequencing libraries using either the Illumina TruSeq Stranded Total RNA Library Prep with Ribo-Zero kit or the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit per the manufacturer's protocol. The final indexed libraries were quantified using Agilent's Bioanalyzer and pooled at an equal molar concentration. Illumina's NextSeq sequencer was used to generate paired end 150 base pair reads. Raw sequencing data was processed using fastp⁴⁴ v.0.20.1 (https://github.com/OpenGene/fastp) to trim adapters and filter out sequence with < Q30. Alignment to the SARS-CoV-2 reference genome (MN908947.3) was performed using BWA⁴⁵ v0.7.17-r1188 (http://bio-bwa.sourceforge.net). DeepVariant⁴⁶ v1.1.0 (https://github.com/google/deepvariant) was used to call variants with an allele frequency >= 50%. Variants were annotated using SNPEff⁴⁷ 5.0c (https://sourceforge.net/projects/snpeff/). All virus preparation and experiments were performed in an approved Biosafety level 3 (BSL-3) facility.

Monoclonal antibodies. The human mAbs studied in this paper (COV2-2196, COV2-2072, COV2-2050, COV2-2381, COV2-2130, COVOX-384, COVOX-40, S309, S2E12, S2H58, S2X333, VIR-7381, and S2X259) were isolated from blood samples from individuals in North America or Europe with previous laboratory-confirmed symptomatic SARS-CoV or SARS-CoV-2 infection. The original clinical studies to obtain specimens after written informed consent were previously described^10,13,14,25 and approved by the Institutional Review Board of Vanderbilt University Medical Center, the Institutional Review Board of the University of Washington, the Research Ethics Board of the University of Toronto, and the Canton Ticino Ethics Committee (Switzerland). Chimeric mAb 1B07 with a murine Fv and human Fc (human IgG1) were isolated from C57BL/6 mice immunized with recombinant spike and RBD proteins and described previously¹². Murine mAbs were generated in BALB/c or C57BL/6 mice immunized with recombinant spike and RBD proteins and described previously^28,30.

Human immune sera. Multiple sources of human serum samples were used in this study: Convalescent serum samples were obtained from a cohort recruited from the St. Louis metropolitan area who experienced mild SARS-CoV-2 infection. None of those patients required intubation, and the study was approved by Washington University School of Medicine Institutional Review Board (202003186 (WU353)). The serum samples from individuals immunized with the Pfizer-BioNTech (BNT162b2) mRNA vaccine were obtained prior to primary immunization or one week after boosting from young adults, and the studies were approved by Washington University School of Medicine Institutional Review Board (202012081 (WU368) and 202012084 (COVaRiPAD))

Mouse, hamster, and NHP immune sera. The mouse, hamster, and NHP immune sera were obtained one month after intranasal immunization with ChAd-SARS-CoV-2, a chimpanzee adenoviral vectored vaccine encoding for a prefusion stabilized form of the spike protein. Details of the immunization protocol and functional analyses have been described elsewhere^21-23.

Focus reduction neutralization test. Serial dilutions of mAbs or serum were incubated with 10² focus-forming units (FFU) of different strains or variants of SARS-CoV-2 for 1 h at 37°C. Antibody-virus complexes were added to Vero-hACE2-TMPRSS2 or Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 24 h later by removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with an oligoclonal pool of SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-71 anti-S antibodies and HRP-conjugated goat anti-mouse IgG in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).

ELISA. Assays were performed in 96-well plates (MaxiSorp; Thermo) coated with 100 μL of recombinant spike or RBD protein¹² in PBS, and plates were incubated at 4 °C overnight. Plates were then blocked with 10% FBS and 0.05% Tween20 in PBS. Serum were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween-20 in PBS. Goat anti-human IgG-HRP (Jackson ImmunoResearch, 1:2,500) was diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween-20 in PBS, and then washed 3 times with PBS before the addition of peroxidase substrate (SigmaFAST o-Phenylenediamine dihydrochloride, Sigma-Aldrich). Reactions were stopped by the addition of 1 M HCl. Optical density measurements were taken at 490 nm. The half-maximal binding dilution for each serum or plasma sample was calculated using nonlinear regression. The limit of detection was defined as 1:30.

Transient expression of recombinant SARS-CoV-2 spike proteins and flow cytometry. The full-length S gene of SARS-CoV-2 strain (SARS-CoV-2-S) isolate BetaCoV/Wuhan-Hu-1/2019 (accession number MN908947) carrying D614G was codon-optimized for expression in hamster cells and cloned into the pcDNA3 expression vector. Amino acid substitutions for B.1.1.7, P.1 (Brazilian lineage: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1167F), and B.1.351 variants were introduced by overlap extension PCR. Briefly, DNA fragments with overlap sequences were amplified by PCR (step 1).  Mutations were introduced by amplification with primers with similar melting points (Tm).  Deletion of the C-terminal 21 amino acids was introduced to increase surface expression of the recombinant spike. Next, three contiguous overlapping fragments were fused by a first overlap PCR (step 2) using the utmost external primers of each  set, resulting in three larger fragments with overlapping sequences. A final overlap PCR (step 3) was performed on the three large fragments using the utmost external primers to amplify the S gene and the flanking sequences including the restriction sites KpnI and NotI. This fragment was digested and cloned into the expression plasmid phCMV1.  For all PCR reactions, the Q5 Hot Start High fidelity DNA polymerase was used (New England Biolabs), according to the manufacturer’s instructions and adapting the elongation time to the size of the amplicon. After each PCR step, the amplified regions were separated on agarose gel and purified using Illustra GFX™ PCR DNA and Gel Band Purification Kit (Merck KGaA).

Expi-CHO cells were transiently transfected with SARS-CoV-2-S expression vectors using Expifectamine CHO Enhancer. Two days later, cells were collected for immunostaining with mAbs. An Alexa647-labelled secondary antibody anti-human IgG Fc was used for detection. Binding of mAbs to transfected cells was analyzed by flow-cytometry using a ZE5 Cell Analyzer (Biorard) and FlowJo software (TreeStar). Positive binding was defined by differential staining of CoV-S-transfectants versus mock-transfectants.

SARS-CoV-2 pseudotyped virus production. 293T/17 cells were seeded in 10-cm dishes for 80% next day confluency. The next day, cells were transfected with the plasmid pcDNA3.1(+)-spike-D19 (encoding the SARS-CoV-2 spike protein) or pcDNA3.1(+)-spike-D19 variants using the transfection reagent TransIT-Lenti according to the manufacturer’s instructions. One day post-transfection, cells were infected with VSV-luc(VSV-G) at an MOI of 3. The cell supernatant containing SARS-CoV-2 pseudotyped virus was collected at day 2 post-transfection, centrifuged at 1,000 x g for 5 min to remove cellular debris, aliquoted and frozen at -80°C. The SARS-CoV-2 pseudotyped virus preparation was quantified using Vero E6 cells seeded at 20,000 cells/well in clear bottom black 96 well plates the previous day. Cells were inoculated with 1:10 dilution series of pseudotyped virus in 50 μL DMEM for 1 h at 37°C. An additional 50 μL of DMEM was added, cells were incubated overnight at 37°C. Luciferase activity was quantified with Bio-Glo reagent by adding 100 μL of Bio-Glo (diluted 1:1 in PBS), incubated at room temperature for 5 min, and relative light units (RLU) were read on an EnSight or EnVision plate reader.

Neutralization of SARS-CoV-2 pseudotyped virus. Vero E6 cells were seeded into clear bottom black-walled 96-well plates at 20,000 cells/well in 100 μL medium and cultured overnight at 37°C. Twenty-four hours later, 1:3 8-point serial dilutions of mAb were prepared in medium, with each dilution tested in duplicate on each plate (range: 10 μg/mL to 4 ng/mL final concentration). Pseudovirus was diluted 1:25 in medium and added 1:1 to 110 μL of each antibody dilution. Pseudovirus:antibody mixtures were incubated for 1 h at 37°C. Media was removed from the Vero E6 cells and 50 μL of pseudovirus:antibody mixtures were added to the cells. One hour post-infection, 100 μL of medium was added to wells containing pseudovirus:antibody mixtures and incubated for 17 h at 37°C. Media then was removed and 100 μL of Bio-Glo reagent (diluted 1:1 in DPBS) was added to each well. The plate was shaken on a plate shaker at 300 RPM at room temperature for 20 min, and RLUs were read on an EnSight or EnVision plate reader.

Data availability. All data supporting the findings of this study are available within the paper and are available from the corresponding author upon request. Deep sequencing datasets of viral stocks are available at NCBI BioProject PRJNA698378 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA698378?reviewer=g0mic4v5t4e1tpssk63p990suu).

Statistical analysis. All statistical tests were performed as described in the indicated figure legends. Non-linear regression curve fitting was performed to calculate EC₅₀ values. Statistical significance was calculated using a non-parametric Wilcoxon matched-pairs signed rank test and Prism 8.0. The number of independent experiments used are indicated in the relevant Figure legends.

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Table 1. Monoclonal antibodies used in this study

	MAb	Region	Species	EC50 value (ng/ml)^a	block ACE2 binding	Binds RBM	Structural contacts	Functionally important residues ^b	Other mapping data	Reference
CLASS 1	COV2-2196	RBD - RBM	human	15	yes	yes		F486A, N487A (LOB)		(Dong et al., 2021; Zost et al., 2020a; Zost et al., 2020b)
	COV2-2130	RBD - RBM	human	107	yes	yes		K444A, G447R (LOB); and R346I, K444R, K444E (NE)		(Dong et al., 2021; Zost et al., 2020a; Zost et al., 2020b)
	COV2-2072	RBD - RBM	human	26	yes	yes			COV2-2196 competitive	(Zost et al., 2020a; Zost et al., 2020b)
	COV2-2050	RBD - RBM	human	80	yes	yes		E484K (LOB and NE)	COV2-2196 and COV2-2130 competitive	(Greaney et al., 2021; Zost et al., 2020a; Zost et al., 2020b)
	COV2-3025	RBD - RBM	human	37	yes	yes			COV2-2196 competitive	(Zost et al., 2020a; Zost et al., 2020b)
	COV2-2381	RBD - RBM	human	42	yes	yes			COV2-2196 competitive	(Zost et al., 2020a; Zost et al., 2020b)
	1B07	RBD - RBM	mouse-human chimera	279	yes	yes		E484A/D/G/K, F486Y (NE)		(Alsoussi et al., 2020; Liu et al., 2020)
	S2E12	RBD - RBM	human	4.2	yes	yes	455 to 458, 473 to 493	G476S, F486A (LOB)		(Tortorici et al., 2020), Starr, Corti et al. unpublished
	COVOX-384	RBD - RBM	human	2	yes	yes	L455, F456, G482-F486			Dejnirattisai and Screaton, unpublished
	COVOX-40	RBD - RBM	human	24	yes	yes	K417, Q409, Y505			Dejnirattisai and Screaton, unpublished
	S2H58	RBD-RBM	human	5	yes	yes		E484K, F490L, S494P (LOB)		Starr, Corti et al. unpublished
	S2X259	RBD	human	55	yes	no		G504D (LOB)		McCallum, Corti et al, unpublished
CLASS 2	S309	RBD- BASE	human	79	no	no	T333-L335,P337,G339-V341,N343R346,N354,K356-C361,N440L441,K444,R509			(Pinto et al., 2020)
	SARS2-31	RBD- BASE	mouse	246	yes	no		K378E/Q, R408K, G504D (NE)	CR3022 competitive	VanBlargan and Diamond, unpublished
	SARS2-10	RBD- BASE	mouse	694	yes	no			CR3022 competitive	VanBlargan and Diamond, unpublished
	SARS2-54	RBD- BASE	mouse	32	yes	no			CR3022 competitive	VanBlargan and Diamond, unpublished
	SARS2-44	RBD- BASE	mouse	258	yes	no			CR3022 competitive	VanBlargan and Diamond, unpublished
	SARS2-3	RBD- BASE	mouse	670	no	no			CR3022 competitive	VanBlargan and Diamond, unpublished
	SARS2-65	RBD- BASE	mouse	145	no	no			CR3022 competitive	VanBlargan and Diamond, unpublished
CLASS 3	COV2-2676	NTD	human	501	no	no		Y144A, N164A (LOB) and F140S (NE)		(Suryadevara et al., 2021; Zost et al., 2020a; Zost et al., 2020b)
CLASS 3	COV2-2489	NTD	human	199	no	no		G142A, Y144A, F157A, N164A (LOB); and G142D, R158S (NE)		(Suryadevara et al., 2021; Zost et al., 2020a; Zost et al., 2020b)

^aNeutralization potency determined by FRNT assay with WA1/2020 isolate.

^bLOB, loss-of-binding to spike protein, and NE, neutralization escape mutants.

Yes there is potential Competing Interest. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. J.E.C. has served as a consultant for Eli Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from AstraZeneca and IDBiologics. Vanderbilt University (J.E.C.) and Washington University (A.H.E., A.C.M.B., M.S.D., and D.H.F.) have applied for patents related to antibodies described in this paper. The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences Inc., AbbVie Inc., and Nano targeting & Therapy Biopharma Inc. The Shi laboratory has received sponsored research agreements from Pfizer, Gilead, Merck, and IGM Sciences Inc.

SupplementaryTable1.xlsx
Table S1. List of mutations in the variant SARS-CoV-2 viruses as defined by next generation sequencing. Comparisons are made to the Wuhan-Hu-1 reference virus (MN908947.3).
EDF1.tif
Extended Data Figure 1. MAb-spike structures. Structures of the SARS-CoV-2 RBD in complex with a representative neutralizing antibody from (a) class 1 (S2E12, PDB: 7K45), or (b) class 2 (S309, PDB: 6WPS). c, Structure of the SARS-CoV-2 spike N-terminal domain (NTD) in complex with a representative class 3 neutralizing antibody (4A8, PDB: 7C2L). All structural analysis and figures were generated with UCSF ChimeraX40.
EDF2mAbFRNTFINAL.tif
Extended Data Figure 2. Neutralization curves with mAbs and variant SARS-CoV-2 strains. Anti-SARS-CoV-2 human mAbs were tested for neutralization of infection of the indicated viral variants and isolates using a FRNT on Vero-hACE2-TMPRSS2 or Vero-TMPRSS2 cells. One representative experiment of two performed in duplicate is shown.
EDF3BindingDataFINAL.tif
Extended Data Figure 3. Binding and neutralizing activity of mAbs to SARS-CoV-2 variants. a, Binding of mAbs S2E12 (class 1, RBM), S309 (class 2, RBD base), VIR-7381 (class 2, RBD-base), and S2X333 (class 3, NTD) to SARS-CoV-2 spike proteins from indicated strains when expressed on the surface of expiCHO cells (symbols, mean of duplicates from one experiment). b, Neutralization of VSV-SARS-CoV-2 pseudotyped viruses (with indicated spike proteins) on Vero E6 cells. Mean ± standard deviation of sextuplicates is shown for all pseudoviruses, except for SARS-CoV-2 WT (mean of triplicates). WT, Wuhan-1 + D614G.
EDF4ConvalescentHumanSeraFRNTs.tif
Extended Data Figure 4. Neutralization curves with convalescent human sera from longitudinal cohort and variant SARS-CoV-2 strains. Serum from individuals (n = 19) who had been infected with SARS-CoV-2 (samples obtained at ~1-month post-infection) were tested for neutralization of the indicated viral variants and isolates in Vero-hACE2-TMPRSS2 cells using a FRNT. One experiment performed in duplicate is shown.
EDF5AnimalVaccineSeraFRNTs.tif
Extended Data Figure 5. Neutralization curves with animal sera from ChAd-CoV-2 vaccinated animals and variant SARS-CoV-2 strains. Serum samples were collected from mice (n = 10), hamsters (n = 8), or rhesus macaques (NHP, n = 6) ~30 days after a single intranasal immunization with ChAd-SARS-CoV-2-S. Sera were tested for neutralization of infection of the indicated viral variants and isolates in Vero-hACE2-TMPRSS2 cells using a FRNT. One experiment performed in duplicate is shown.
EDF6VaccineELISAspikeandRBD.tif
Extended Data Figure 6. S and RBD binding activity of human sera from individuals vaccinated with BNT162b2 mRNA vaccine. Individuals were vaccinated and boosted with the Pfizer-BioNTech mRNA vaccine. At seven days after boosting, sera were collected and tested for binding to S or RBD proteins (WA1/2020 strain) by ELISA. One experiment performed in duplicate is shown.
EDF7HumanVaccineSeraFRNTS.tif
Extended Data Figure 7. Neutralization curves in Vero-hACE2-TMPTSS2 cells with human sera from subjects vaccinated with the BNT162b2 mRNA vaccine and variant SARS-CoV-2 strains. Individuals were vaccinated and boosted with the Pfizer-BioNTech mRNA vaccine. Sera were collected and tested for neutralization of infection of the indicated viral variants and isolates using a FRNT and Vero-hACE2-TMPRSS2 cells. One experiment performed in duplicate is shown.
EDF8HumanConvalescentandVaccineTMPRSS2FRNTs.tif
Extended Data Figure 8. Neutralization curves in Vero-TMPRSS2 cells with human sera from convalescent subjects or those vaccinated with the BNT162b2 mRNA vaccine and variant SARS-CoV-2 strains. Serum from individuals (n = 20) who had been infected with SARS-CoV-2 (~ 1-month time point) or vaccinated with the Pfizer-BioNTech mRNA vaccine (n = 10) were tested for neutralization of the indicated SARS-CoV-2 strains (WA1/2020, B.1.1.7, or Wash SA-B.1.351) using a FRNT and Vero-TMPRSS2 cells. One experiment performed in duplicate is shown.
EDF9P0vsP1SA3x.tif
Extended Data Figure 9. Differential serum neutralization of SARS-CoV-2 produced in Vero E6 and Vero-hACE2-TMPRSS2 cells. (Top panels) Immune or vaccine-derived sera from mice, hamsters, NHP, or humans (see Fig 2 and 3) were incubated with deep-sequenced confirmed p0 (Vero cell-produced) or p1 (Vero-hACE2-TMPRSS2 cell-produced) versions of K417N/E484K/N501Y/D614G virus and then subjected to a FRNT in Vero-hACE2-TMPRSS2 recipient cells. EC50 values were calculated from one experiment performed in duplicate (Wilcoxon matched-pairs signed rank test, *, P < 0.05; **, P < 0.01). GMT values are shown above each graph. Dotted line represents the limit of detection of the assay. (Middle panels) Serum neutralization curves with K417N/E484K/N501Y/D614G virus (p0, generated in Vero E6 cells; p1, generated in Vero-hACE2-TMPRSS2 cells) using a FRNT and Vero-hACE2-TMPRSS2 cells. One experiment performed in duplicate is shown. (Bottom panel) Neutralization curves and EC50 values with COV2-2050 and COV2-2196 mAbs using the p0 (Vero cell-produced) or p1 (Vero-hACE2-TMPRSS2 cell-produced) viruses and recipient Vero-hACE2-TMPRSS2 cells.

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SARS-CoV-2 variants show resistance to neutralization by many monoclonal and serum-derived polyclonal antibodies

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