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PreprintCited by 285SARS-CoV-2 and COVID-19 ResearchAnimal Virus Infections Studiesvaccines and immunoinformatics approaches

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Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection

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Song, Ge, He, Wan-ting, Callaghan, Sean, Anzanello, Fabio, Huang, Deli, Ricketts, James, Torres, Jonathan L., Beutler, Nathan, Peng, Linghang, Vargas, Sirena, Cassell, Jon, Parren, Mara, Yang, Linlin, Ignacio, Caroline, Smith, Davey M., Voss, James E., Nemazee, David, Ward, Andrew B, Rogers, Thomas, Burton, Dennis R., Andrabi, Raiees·2020·Nature Communications
DOI 10.1101/2020.09.22.308965PMC PMC7523096

Preprint — published as 10.1038/s41467-021-23074-3

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Abstract

Pre-existing immune responses to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, either induced in natural infection or through vaccination. Such consequences are well established in the influenza and flavivirus fields. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. We compared serum antibody and memory B cell responses to coronavirus spike (S) proteins from pre-pandemic and SARS-CoV-2 convalescent donors using a series of binding and functional assays. We found weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we found stronger evidence of pre-existing cross-reactive memory B cells that were activated on SARS-CoV-2 infection. Monoclonal antibodies (mAbs) isolated from the donors showed varying degrees of cross-reactivity with betacoronaviruses, including SARS and endemic coronaviruses. None of the cross-reactive mAbs were neutralizing except for one that targeted the S2 subunit of the S protein. The results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.

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Figures

Fig. 1. Reactivity of COVID and pre-pandemic human sera with cell surface-expressed human coronaviruses spikes and their soluble S-protein versions. A. Heatmap showing cell-based flow cytometry binding (CELISA) of COVID and pre-pandemic donor sera with 293T cell surface-expressed full-length spike proteins from β-(SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43) and α-(HCoV-NL63 and HCoV-229E) human coronaviruses (HCoVs). Sera were titrated (6 dilutions-starting at 1:30 dilution) and the extent of binding to cell surface-expressed HCoVs was recorded by % positive cells, as detected by PE-conjugated anti-human-Fc secondary Ab using flow cytometry. Area-under-the-curve (AUC) was calculated for each binding titration curve and the antibody titer levels are color-coded as indicated in the key. Binding of sera to vector-only plasmid (non-spike) transfected 293T cells served as a control for non-specific binding. B. ELISA binding of COVID and pre-pandemic donor sera to soluble S-proteins from β-(SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43) and α-(HCoV-NL63 and HCoV-229E) HCoVs. Serum dilutions (8 dilutions- starting at 1:30 dilution) were titrated against the S-proteins and the binding was detected as OD405 absorbance. AUC representing the extent of binding was calculated from binding curves of COVID (left) and pre-pandemic (right) sera with S-proteins and comparisons of antibody binding titers are shown. Binding to BSA served as a control for non-specific binding by the sera. Statistical comparisons between two groups were performed using a Mann-Whitney test, (**p <0.01; ***p < 0.001, ****p < 0.0001; ns- p >0.05).
Fig. 1. Reactivity of COVID and pre-pandemic human sera with cell surface-expressed human coronaviruses spikes and their soluble S-protein versions. A. Heatmap showing cell-based flow cytometry binding (CELISA) of COVID and pre-pandemic donor sera with 293T cell surface-expressed full-length spike proteins from β-(SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43) and α-(HCoV-NL63 and HCoV-229E) human coronaviruses (HCoVs). Sera were titrated (6 dilutions-starting at 1:30 dilution) and the extent of binding to cell surface-expressed HCoVs was recorded by % positive cells, as detected by PE-conjugated anti-human-Fc secondary Ab using flow cytometry. Area-under-the-curve (AUC) was calculated for each binding titration curve and the antibody titer levels are color-coded as indicated in the key. Binding of sera to vector-only plasmid (non-spike) transfected 293T cells served as a control for non-specific binding. B. ELISA binding of COVID and pre-pandemic donor sera to soluble S-proteins from β-(SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43) and α-(HCoV-NL63 and HCoV-229E) HCoVs. Serum dilutions (8 dilutions- starting at 1:30 dilution) were titrated against the S-proteins and the binding was detected as OD405 absorbance. AUC representing the extent of binding was calculated from binding curves of COVID (left) and pre-pandemic (right) sera with S-proteins and comparisons of antibody binding titers are shown. Binding to BSA served as a control for non-specific binding by the sera. Statistical comparisons between two groups were performed using a Mann-Whitney test, (**p <0.01; ***p < 0.001, ****p < 0.0001; ns- p >0.05).CC BY-NC-ND · PubMed Central
Fig. 2. BioLayer Interferometry binding of COVID and pre-pandemic serum antibodies to SARS-CoV-2 and endemic HCoV S-proteins. A. Heatmap summarizing the apparent BLI binding off-rates (koff (1/s)) of the COVID and pre-pandemic human serum antibodies to SARS-CoV-2 S and endemic β-HCoV, HCoV-HKU1 and α-HCoV, HCoV-NL63 S-proteins. Biotinylated HCoV S-proteins (100nM) were captured on streptavidin biosensors to achieve binding of at least 1 response unit. The S-protein-immobilized biosensors were immersed in 1:40 serum dilution solution with serum antibodies as the analyte and the association (120 s; 180–300) and dissociation (240 s; 300–540) steps were conducted to detect the kinetics of antibody-protein interaction. koff (1/s) dissociation rates for each antibody-antigen interaction are shown. B. Off-rates for binding of serum antibodies from COVID donors and from pre-pandemic donors to SARS-CoV-2 S and endemic HCoV, HCoV-HKU1 and HCoV-Nl63, S proteins. Significantly lower dissociation off-rates are observed for COVID compared to pre-pandemic sera. Statistical comparisons between the two groups were performed using a Mann-Whitney test.
Fig. 2. BioLayer Interferometry binding of COVID and pre-pandemic serum antibodies to SARS-CoV-2 and endemic HCoV S-proteins. A. Heatmap summarizing the apparent BLI binding off-rates (koff (1/s)) of the COVID and pre-pandemic human serum antibodies to SARS-CoV-2 S and endemic β-HCoV, HCoV-HKU1 and α-HCoV, HCoV-NL63 S-proteins. Biotinylated HCoV S-proteins (100nM) were captured on streptavidin biosensors to achieve binding of at least 1 response unit. The S-protein-immobilized biosensors were immersed in 1:40 serum dilution solution with serum antibodies as the analyte and the association (120 s; 180–300) and dissociation (240 s; 300–540) steps were conducted to detect the kinetics of antibody-protein interaction. koff (1/s) dissociation rates for each antibody-antigen interaction are shown. B. Off-rates for binding of serum antibodies from COVID donors and from pre-pandemic donors to SARS-CoV-2 S and endemic HCoV, HCoV-HKU1 and HCoV-Nl63, S proteins. Significantly lower dissociation off-rates are observed for COVID compared to pre-pandemic sera. Statistical comparisons between the two groups were performed using a Mann-Whitney test.CC BY-NC-ND · PubMed Central
Fig. 3. SARS-CoV-2 S and endemic HCoV S-protein specific cross-reactive IgG+ memory B cells from COVID donors and isolation of and characterization of mAbs. A-B. Flow cytometry analysis showing the single B cell sorting strategy for COVID representative donor CC9 and frequencies of SARS-CoV-2 S and endemic β-HCoV, HCoV-HKU1 and α-HCoV, HCoV-NL63 S-protein specific memory B cells in 8 select COVID donors. The B cells were gated as sSl, CD4−, CD8−, CD11C−, IgD−, IgM−, CD19+, IgG+. The frequencies of HCoV S-protein-specific IgG memory B cells were as follows; SARS-CoV-2 S (up to ~8% - range = ~1.6–8%), HCoV-HKU1 S (up to ~4.3% - range = ~0.2–4.3%), HCoV-NL63 S (up to ~0.6% - range = ~0.4–0.6%) protein single positive and SARS-CoV-2/HCoV-HKU1 S (up to ~2.4% - range = ~0.02–2.4%) and SARS-CoV-2/HCoV-NL63 S-protein (up to ~0.09% - range = ~0–0.09%) double positives. SARS-CoV-2 infected donors showed the presence of SARS-CoV-2/HCoV-HKU1 S-protein cross-reactive IgG memory B cells. A Mann-Whitney test was used to compare the levels of HCoV S-protein specific IgG memory B cells and the p-values for each comparison are indicated. **p <0.01; ***p < 0.001. C. Pie plots showing immunoglobulin heavy chain distribution of mAbs isolated from 4 COVID donors, CC9, CC10, CC36 and CC40. The majority of the mAbs were encoded by the IgVH3 immunoglobulin gene family. D. Plots showing % nucleotide mutations in heavy (VH) and light (VL) chains of isolated mAbs across different individuals. The VH and VL mutations ranged from 0–17% and 0–4.5%, respectively. E. CELIsA binding curves of isolated mAbs from 4 COVID donors with SARS-CoV-2 and HCoV-HKU1 spikes expressed on 293T cells. Binding to HCoV spikes is recorded as % positive cells using a flow cytometry method. 5 mAbs, 3 from the CC9 donor and 2 from the CC40 donor show cross-reactive binding to SARS-CoV-2 and HCoV-HKU1 spikes. F. Neutralization of SARS-CoV-2 by mAbs isolated from COVID donors. 4 mAbs, 2 each from donors, CC36 and CC40, show neutralization of SARS-CoV-2. G. Heatmap showing CELISA binding of COVID mAbs to 7 HCoV spikes. Binding represented as area-under-the-curve (AUC) is derived from CELISA binding titrations of mAbs with cell surface-expressed HCoV spikes and the extent of binding is color-coded. 5 mAbs show cross-reactive binding across β-HCoV spikes.
Fig. 3. SARS-CoV-2 S and endemic HCoV S-protein specific cross-reactive IgG+ memory B cells from COVID donors and isolation of and characterization of mAbs. A-B. Flow cytometry analysis showing the single B cell sorting strategy for COVID representative donor CC9 and frequencies of SARS-CoV-2 S and endemic β-HCoV, HCoV-HKU1 and α-HCoV, HCoV-NL63 S-protein specific memory B cells in 8 select COVID donors. The B cells were gated as sSl, CD4−, CD8−, CD11C−, IgD−, IgM−, CD19+, IgG+. The frequencies of HCoV S-protein-specific IgG memory B cells were as follows; SARS-CoV-2 S (up to ~8% - range = ~1.6–8%), HCoV-HKU1 S (up to ~4.3% - range = ~0.2–4.3%), HCoV-NL63 S (up to ~0.6% - range = ~0.4–0.6%) protein single positive and SARS-CoV-2/HCoV-HKU1 S (up to ~2.4% - range = ~0.02–2.4%) and SARS-CoV-2/HCoV-NL63 S-protein (up to ~0.09% - range = ~0–0.09%) double positives. SARS-CoV-2 infected donors showed the presence of SARS-CoV-2/HCoV-HKU1 S-protein cross-reactive IgG memory B cells. A Mann-Whitney test was used to compare the levels of HCoV S-protein specific IgG memory B cells and the p-values for each comparison are indicated. **p <0.01; ***p < 0.001. C. Pie plots showing immunoglobulin heavy chain distribution of mAbs isolated from 4 COVID donors, CC9, CC10, CC36 and CC40. The majority of the mAbs were encoded by the IgVH3 immunoglobulin gene family. D. Plots showing % nucleotide mutations in heavy (VH) and light (VL) chains of isolated mAbs across different individuals. The VH and VL mutations ranged from 0–17% and 0–4.5%, respectively. E. CELIsA binding curves of isolated mAbs from 4 COVID donors with SARS-CoV-2 and HCoV-HKU1 spikes expressed on 293T cells. Binding to HCoV spikes is recorded as % positive cells using a flow cytometry method. 5 mAbs, 3 from the CC9 donor and 2 from the CC40 donor show cross-reactive binding to SARS-CoV-2 and HCoV-HKU1 spikes. F. Neutralization of SARS-CoV-2 by mAbs isolated from COVID donors. 4 mAbs, 2 each from donors, CC36 and CC40, show neutralization of SARS-CoV-2. G. Heatmap showing CELISA binding of COVID mAbs to 7 HCoV spikes. Binding represented as area-under-the-curve (AUC) is derived from CELISA binding titrations of mAbs with cell surface-expressed HCoV spikes and the extent of binding is color-coded. 5 mAbs show cross-reactive binding across β-HCoV spikes.CC BY-NC-ND · PubMed Central
Fig. 4. Binding, ADE and epitope specificities of SARS-CoV-2/HCoV-HKU1 S-protein specific cross-reactive mAbs. A. BLI of SARS-CoV-2 and HCoV-HKU1 S-protein-specific cross-reactive mAbs. BLI binding of both IgG and Fab versions of 3 cross-reactive mAbs (CC9.2, CC9.3 and CC40.8) to SARS-CoV-2 and HCoV-HKU1 S-proteins was tested and the binding curves show association (120 s; 180–300) and dissociation rates (240 s; 300–540). BLI binding of antibody-S-protein combinations shows more stable binding (higher binding constants (KDs)) of cross-reactive mAbs HCoV-HKU1 compared to the SARS-CoV-2 S protein. B. Antibody Dependent Enhancement (ADE) activities of cross-reactive mAbs, CC9.2, CC9.3 and CC40.8 bonding to SARS-CoV-2 live virus using FcγRIIa (K562) and FcγRIIb (Daudi)-expressing target cells. A dengue antibody, DEN3, was used as a control. C-D. Epitope mapping of the mAbs binding to domains and subdomains of SARS-CoV-2 S-protein, NTD, RBD, RBD-SD1 and RBD-SD1–2 and heatmap showing BLI responses for each protein. The extent of binding responses is color coded. 5 mAbs were specific for RBD, 2 for NTD and the remaining mAbs displayed binding only to the whole S protein. E-F. Negative stain electron microscopy of HCoV-HKU1 S-protein + Fab CC40.8 complex and comparison to MERS-CoV S + Fab G4 complex. (E) Raw micrograph of HCoV-HKU1 S in complex with Fab CC40.8. (F) Select reference-free 2D class averages with Fabs colored in orange for Fab CC40.8 and blue for Fab G4, which in 2D appear to bind a proximal epitope at the base of the trimer. 2D projections for MERS-CoV S-protein in complex with Fab G4 were generated in EMAN2 from PDB 5W9J.
Fig. 4. Binding, ADE and epitope specificities of SARS-CoV-2/HCoV-HKU1 S-protein specific cross-reactive mAbs. A. BLI of SARS-CoV-2 and HCoV-HKU1 S-protein-specific cross-reactive mAbs. BLI binding of both IgG and Fab versions of 3 cross-reactive mAbs (CC9.2, CC9.3 and CC40.8) to SARS-CoV-2 and HCoV-HKU1 S-proteins was tested and the binding curves show association (120 s; 180–300) and dissociation rates (240 s; 300–540). BLI binding of antibody-S-protein combinations shows more stable binding (higher binding constants (KDs)) of cross-reactive mAbs HCoV-HKU1 compared to the SARS-CoV-2 S protein. B. Antibody Dependent Enhancement (ADE) activities of cross-reactive mAbs, CC9.2, CC9.3 and CC40.8 bonding to SARS-CoV-2 live virus using FcγRIIa (K562) and FcγRIIb (Daudi)-expressing target cells. A dengue antibody, DEN3, was used as a control. C-D. Epitope mapping of the mAbs binding to domains and subdomains of SARS-CoV-2 S-protein, NTD, RBD, RBD-SD1 and RBD-SD1–2 and heatmap showing BLI responses for each protein. The extent of binding responses is color coded. 5 mAbs were specific for RBD, 2 for NTD and the remaining mAbs displayed binding only to the whole S protein. E-F. Negative stain electron microscopy of HCoV-HKU1 S-protein + Fab CC40.8 complex and comparison to MERS-CoV S + Fab G4 complex. (E) Raw micrograph of HCoV-HKU1 S in complex with Fab CC40.8. (F) Select reference-free 2D class averages with Fabs colored in orange for Fab CC40.8 and blue for Fab G4, which in 2D appear to bind a proximal epitope at the base of the trimer. 2D projections for MERS-CoV S-protein in complex with Fab G4 were generated in EMAN2 from PDB 5W9J.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Song, G., He, W.-T., Callaghan, S., Anzanello, F., Huang, D., Ricketts, J., Torres, J. L., Beutler, N., Peng, L., Vargas, S., Cassell, J., Parren, M., Yang, L., Ignacio, C., Smith, D. M., Voss, J. E., Nemazee, D., Ward, A. B., Rogers, T., Burton, D. R., & Andrabi, R.
Affiliations
  • Scripps Research Institute (US)
  • University of California San Diego (US)
Corresponding author
  • Burton, Dennis R.
  • Andrabi, Raiees
ORCID
Venue
Nature Communications
Publisher
openRxiv
Published
2020-09-23
Volume
12
Issue
1
Pages
2938-2938
ISSN
2041-1723 (ISSN-L)
DOI
10.1101/2020.09.22.308965
PMC
PMC7523096
OpenAlex
W3161511556
Cited by
285
References
40
Citations by year
2026: 15 · 2025: 17 · 2024: 30 · 2023: 53 · 2022: 78 · 2021: 85 · 2020: 7
FWCI
19.48
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • Bill and Melinda Gates Foundation (OPP1170236, UM1 AI44462)
  • International AIDS Vaccine Initiative
  • National Institutes of Health (UM1 AI44462, COVID-19)
  • National Institute of Allergy and Infectious Diseases (R01AI132317, R01AI073148, R56AI073148)
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
Keywords
SARS-CoV-2, cross-reactive antibodies, memory B cells, spike protein, endemic coronaviruses, pre-existing immunity, SARS-CoV-1, betacoronaviruses, neutralizing antibodies, S2 subunit, monoclonal antibodies, antibody cross-reactivity, convalescent donors

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