Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan 19;2(1):100189.
doi: 10.1016/j.xcrm.2020.100189.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses

Jason T Ladner  1 , Sierra N Henson  2 , Annalee S Boyle  2 , Anna L Engelbrektson  2 , Zane W Fink  1 , Fatima Rahee  2 , Jonathan D'ambrozio  3 , Kurt E Schaecher  3 , Mars Stone  4 , Wenjuan Dong  5 , Sanjeet Dadwal  6 , Jianhua Yu  5 , Michael A Caligiuri  5 , Piotr Cieplak  7 , Magnar Bjørås  8 , Mona H Fenstad  8   9 , Svein A Nordbø  8   10 , Denis E Kainov  8 , Norihito Muranaka  11 , Mark S Chee  11 , Sergey A Shiryaev  7 , John A Altin  2
Affiliations

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses

Jason T Ladner et al. Cell Rep Med. .

Abstract

The SARS-CoV-2 proteome shares regions of conservation with endemic human coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly multiplexed peptide assay (PepSeq) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have the potential to raise broadly neutralizing responses.

Keywords: SARS-CoV-2; antibody response; cross-reactivity; endemic CoVs; highly multiplexed serology.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Epitope-resolved CoV serology using a highly multiplexed peptide-based assay (PepSeq) (A) Platform for customizable highly multiplexed peptide-based serology, comprising the following steps: (1) in silico design, (2–3) generation of a library of DNA-barcoded peptides from oligonucleotide templates using bulk in vitro reactions (transcription, ligation of a puromycin [P]-containing adaptor, translation, reverse transcription), (4) serum binding assay and protein G capture, and (5) sequencing and analysis of the distribution of binders using their DNA barcodes. (B) Peptide coverage depth across the SARS-CoV-2 Spike (S) and Nucleocapsid (N) proteins within the SCV2 peptide library. Peptide coverage depth (blue) correlates well with amino acid sequence diversity within the target SARS-CoV-2 sequences (green), calculated as the number of unique 30-mers. (C) Number of peptides within the HV library that were designed from each of the 6 human CoVs (HCoVs) known before 2019. (D) Example scatterplot illustrating SCV2 PepSeq assay results for a single serum sample. This plot shows normalized sequence read counts (log10 scale) for each peptide in the SCV2 library. Assay results using antibody-free negative controls are shown on the x axis (average of 8 replicates shown), while the results from a COVID-19 convalescent serum sample are shown on the y axis (average of 2 replicates shown). Gray circles represent unenriched peptides, with a strong correlation between the 2 assays, based on the starting abundance of the different peptides. Colored circles represent SARS-CoV-2 (orange) and non-SARS-CoV-2 control (blue) peptides that have been enriched through interaction with serum antibodies.
Figure 2
Figure 2
PepSeq identifies recurrent reactivities to SARS-CoV-2 peptides and classifies exposure status (A) Boxplots showing the number of enriched SCV2 library peptides from assays with negative control (blue, n = 68) and COVID-19 convalescent (orange, n = 55) samples, divided into 3 different categories: non-SARS-CoV-2 control peptides (Control), and SARS-CoV-2 Spike (S) and Nucleocapsid (N) peptides. ***t test with p < 1e−5, NS, not significant). Individual data points are shown as circles, the limits of the boxes correspond to the 1st and 3rd quartiles, the black line inside each box corresponds to the median, and the whiskers extend to points that lie within 1.5 interquartile ranges of the 1st and 3rd quartiles. (B and C) Heatmaps showing the locations of enriched SARS-CoV-2 peptides within the S and N proteins, respectively. Each row represents a single serum/plasma sample and each plot includes only samples with at least 1 enriched peptide from the focal protein. Each position is colored according to the number of enriched peptides that overlap that position. The horizontal dashed line separates COVID-19 convalescent samples (top) from negative control samples (bottom). The vertical dashed lines in (B) represent the S1–S2 and S2′ cleavage sites, respectively. The gray boxes indicate selected functional regions: receptor binding domain (RBD), fusion peptide (FP), and heptad repeat 2 (HR2). (D) Boxplots showing the distribution of Z scores across all assayed samples for the 6 most common epitope reactivities observed in (B) and (C). For each sample/epitope combination, the Z score of the most enriched, overlapping peptide is presented. Boxplots were drawn as described in (A), with convalescent samples in orange and negative controls in blue. t test: *p < 0.05, **p < 0.01, NS, not significant. (E) Receiver-operating curves showing sensitivity/specificity across a range of thresholds with which logistic regression models trained on randomly selected subsets of 70% of the donors were able to classify the remaining 30% of donors as either negative control or convalescent, using log-transformed Z scores for the 6 epitopes described in (D) as features. The red curve shows the average of 100 individual runs. Each patient sample was assayed in duplicate. Enriched peptides were determined based on consistent signal across replicates and Z scores shown as averages across replicates.
Figure 3
Figure 3
Epitope-level SARS-CoV-2 reactivity strongly correlates with reactivity to endemic CoVs at 2 highly conserved regions of Spike S2 (A) Correlations between reactivity to the 6 recurrent SARS-CoV-2 epitopes identified in Figure 2 and each of the 244,000 human virome-wide peptides in the HV library. Each dot represents the −log10(p value) of a Pearson correlation of log-transformed Z scores across all convalescent and control donors for the focal pair. Peptides corresponding to the CoV species in HV are colored as indicated, and filled circles represent peptides that are homologous to the focal SARS-CoV-2 epitope. (B and C) Multispecies sequence alignments for each of the 6 most commonly observed SARS-CoV-2 epitopes. In each case, 15 amino acids are shown, and the minimally reactive region for each (inferred from the data presented in Figures 2B and 2C) is highlighted with a black box. Residues are colored according to amino acid properties: small non-polar (orange), hydrophobic (green), polar (pink), negatively charged (red), and positively charged (blue). Positions that are identical to the SARS-CoV-2 sequence are indicated with “.”. Accessions for sequences in (B) Uniprot: P0DTC2, P59594, P36334, P15423 and (C) Uniprot: P0DTC9, P59595, P33469, P15130. (D and E) Heatmaps illustrating the relative locations of enriched HV library peptides within the S (D) and N (E) proteins and across all HCoVs. Results have been aggregated across all of the tested samples, and the color at each location indicates the proportion of tested samples with enriched peptides covering that position. Results from convalescent (orange, top) and negative control (blue, bottom) are presented individually and are separated by the dashed line. Vertical gray bars indicate the locations of the 6 epitopes highlighted in (A), (B), and (C). SARS2, SARS-CoV-2; SARS, SARS-CoV; MERS, MERS-CoV; Beta1, Betacoronavirus 1; OC43, HCoV-OC43; HKU1, HCoV-HKU1; 229E, HCoV-229E; NL63, HCoV-NL63; Alpha1, Alphacoronavirus 1. Each patient sample was assayed in duplicate. Enriched peptides were determined based on consistent signal across replicates.
Figure 4
Figure 4
SARS-CoV-2 elicits cross-reactive Spike S2 antibodies that preferentially recognize homologs from the endemic CoVs (A) Line plots comparing peptide-specific patterns of enrichment before and after targeted depletion of antibodies binding the SARS-CoV-2 FP or HR2 antigens. Each plot compares peptide enrichment (Z score) from the same samples before (left) and after (right) antibody depletions. Each line represents a single peptide found to be enriched before antibody depletion, and the color of each line indicates the species from which the peptide was designed. Each plot includes results from 3–6 convalescent donors (peptides from each donor are depicted with different shapes). For each depletion experiment (FP and HR2 targeted), on- and off-target peptides are plotted separately. Dashed horizontal lines represent a Z score of 8. (B) Scatterplots comparing enrichment (Z score) between SARS-CoV peptides (x axis) and endemic HCoV peptides (y axis) across 3 epitopes (S:FP, S:HR2, and N:166, respectively) and all samples assayed in duplicate using the HV library (average Z score is shown). Convalescent and negative control samples are represented by orange and blue shapes, respectively. The type of shape indicates the endemic CoV species from which the most highly enriched peptide was designed (circle, HCoV-OC43; square, HCoV-229E; upright triangle, HCoV-HKU1; and upside-down triangle, HCoV-NL63). Shapes with black outlines indicate the samples included in the antibody-depletion experiments shown in (A). (C) Ternary plot showing the relative signal across HV library peptides from 3 HCoVs (SARS-CoV, HCoV-OC43, and HCoV-229E) at 3 commonly reactive epitopes in COVID-19 convalescent patients. Each point represents a single convalescent sample that exhibited at least 1 enriched SCV2 library peptide at the relevant epitope. Position within the triangle was determined by normalizing the maximum peptide Z score (averaged across replicates) observed for each of the 3 focal species.
See this image and copyright information in PMC

Update of

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Zhu N., Zhang D., Wang W., Li X., Yang B., Song J., Zhao X., Huang B., Shi W., Lu R., China Novel Coronavirus Investigating and Research Team A Novel Coronavirus from Patients with Pneumonia in China, 2019. N. Engl. J. Med. 2020;382:727–733. - PMC - PubMed
    1. Piñana J.L., Xhaard A., Tridello G., Passweg J., Kozijn A., Polverelli N., Heras I., Perez A., Sanz J., Berghuis D., Infectious Diseases Working Party of the European Society for Blood and Marrow Transplantation and Infectious Complications Subcommittee of the Spanish Hematopoietic Stem Cell Transplantation and Cell Therapy Group (GETH) Seasonal human coronaviruses respiratory tract infection in recipients of allogeneic hematopoietic stem cell transplantation. J. Infect. Dis. 2020 doi: 10.1093/infdis/jiaa553. - DOI - PMC - PubMed
    1. Dijkman R., Jebbink M.F., El Idrissi N.B., Pyrc K., Müller M.A., Kuijpers T.W., Zaaijer H.L., van der Hoek L. Human coronavirus NL63 and 229E seroconversion in children. J. Clin. Microbiol. 2008;46:2368–2373. - PMC - PubMed
    1. Callow K.A., Parry H.F., Sergeant M., Tyrrell D.A. The time course of the immune response to experimental coronavirus infection of man. Epidemiol. Infect. 1990;105:435–446. - PMC - PubMed
    1. Ni L., Ye F., Cheng M.-L., Feng Y., Deng Y.-Q., Zhao H., Wei P., Ge J., Gou M., Li X. Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals. Immunity. 2020;52:971–977.e3. - PMC - PubMed

Publication types