This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Biological Sciences - Article
Michael Diamond
Washington University School of Medicine
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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 pseudoviruses2-4. 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.
Keywords
Antibody-based Countermeasures, Vaccine Efficacy, Pseudoviruses, Chimeric Washington Strain, South African Spike Gene, Assay Standardization
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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.
This is a list of supplementary files associated with this preprint. Click to download.
- 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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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Biological Sciences - Article
Michael Diamond
Washington University School of Medicine
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 10 Feb, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Virology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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 pseudoviruses2-4. 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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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.
This is a list of supplementary files associated with this preprint. Click to download.
- 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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