Dynamic landscape mapping of humoral immunity to SARS-CoV-2 identifies non-structural protein antibodies associated with the survival of critical COVID-19 patients

Abstract

A comprehensive analysis of the humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential in understanding COVID-19 pathogenesis and developing antibody-based diagnostics and therapy. In this work, we performed a longitudinal analysis of antibody responses to SARS-CoV-2 proteins in 104 serum samples from 49 critical COVID-19 patients using a peptide-based SARS-CoV-2 proteome microarray. Our data show that the binding epitopes of IgM and IgG antibodies differ across SARS-CoV-2 proteins and even within the same protein. Moreover, most IgM and IgG epitopes are located within nonstructural proteins (nsps), which are critical in inactivating the host's innate immune response and enabling SARS-CoV-2 replication, transcription, and polyprotein processing. IgM antibodies are associated with a good prognosis and target nsp3 and nsp5 proteases, whereas IgG antibodies are associated with high mortality and target structural proteins (Nucleocapsid, Spike, ORF3a). The epitopes targeted by antibodies in patients with a high mortality rate were further validated using an independent serum cohort (n = 56) and using global correlation mapping analysis with the clinical variables that are associated with COVID-19 severity. Our data provide fundamental insight into humoral immunity during SARS-CoV-2 infection. SARS-CoV-2 immunogenic epitopes identified in this work could also help direct antibody-based COVID-19 treatment and triage patients.

Fig. 1

Detection of SARS-CoV-2 serum antibodies in critical COVID-19 patients. a Workflow of the longitudinal analyses of SARS-CoV-2 proteome antibodies in the serum of critical COVID-19 patients. b Characteristics of the COVID-19 patients in this study. c The reproducibility of serological antibody detection using the SARS-CoV-2 proteome microarray within an experiment and between different experiments. The r correlation was calculated within an array and between different arrays. d Representative fluorescent image of antibody detection using serum from a COVID-19 patient who did not survive at different days post symptom onset

Fig. 2

Longitudinal changes of SARS-CoV-2 proteome antibodies in critical COVID-19 patients. a Longitudinal detection of SARS-CoV-2 IgM and IgG antibodies in critical COVID-19 patients. The raw data for each peptide and protein on the array were normalized to the z-score. The left y-axis represents the different SARS-CoV-2 proteins. The right y-axis represents the amino acids of each protein sequence, from the N-terminus (left) to C-terminus (right). The top x-axis shows the number of days post symptom onset in 10-day intervals. b Schematic illustration of epitope identification via epitope mapping. c Validation of reported peptide epitopes with data from this study. d Number of IgM and IgG peptide epitopes identified within each SARS-CoV-2 protein

Fig. 3

The landscape of antibody epitopes identified in critical COVID-19 patients. a, b Landscapes of IgM and IgG antibody epitopes identified within the SARS-CoV-2 proteome, respectively. The left y-axis is the protein name and the right y-axis is the percent coverage of peptide epitopes on each protein sequence. c Longitudinal changes of IgM and IgG antibody epitopes identified within the SARS-CoV-2 nsp1 protein. d Structural analyses of IgG antibody epitopes within the SARS-CoV-2 nsp1 protein. The epitopes within the functional domain that may interfere with protein activity are labeled in red

Fig. 4

Dynamic changes of antibody epitopes within SARS-CoV-2 proteases. a Longitudinal changes of IgM and IgG antibody epitopes identified within the SARS-CoV-2 nsp3 protein. b, c Structural analyses of IgM and IgG epitopes within the ADP ribose phosphatase and papain-like protease, respectively. d Longitudinal changes of IgM and IgG antibody epitopes identified within the SARS-CoV-2 nsp5 protein. e Structural analyses of IgM and IgG epitopes within the nsp5 protein. The epitopes within the functional domain that may interfere the protein activity are labeled in red. ADRP ADP-ribose phosphatase domain, PLpro papain-like protease domain

Fig. 5

Dynamic changes of antibody epitopes within the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). a–c Longitudinal changes of IgM and IgG antibody epitopes identified within the SARS-CoV-2 nsp7, 8, and 12 proteins, respectively. d–h Structural analyses of IgM epitopes on the RdRp complex, nsp8-RNA interaction interface, Thumb and Palm domain, Fingers domain, and N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain, respectively. i–k Structural analyses of IgG epitopes on the RdRp complex, nsp8-RNA interaction interface, Fingers domain and NiRAN domain, respectively. The epitopes within the functional domain that may interfere the protein activity are labeled in red

Fig. 6

Dynamic changes of antibody epitopes within the SARS-CoV-2 nsp10/nsp16 2′-O-methylase complex. a, b Longitudinal changes of IgM and IgG antibody epitopes identified within the nsp10/nsp16 2′-O-methylase complex, respectively. c, d Structural analyses of IgM and IgG epitopes within the nsp10/nsp16 2′-O-methylase complex. The epitopes within the functional domain that may interfere the protein activity are labeled in red

Fig. 7

Association between SARS-CoV-2 antibodies and the survival of critical COVID-19 patients. a, b Identification of SARS-CoV-2 IgM and IgG antibodies associated with the survival and non-survival of critical COVID-19 patients, respectively. The antibody candidates were identified using a t-test with a p-value < 0.05 and an immunogenicity with a z-score > 1.96 in ≥3 serum samples. b IgM and IgG peptide epitopes within SARS-CoV-2 proteins associated with the survival and non-survival of COVID-19 patients, respectively. c Validation of epitopes associated with survival and non-survival in an independent serum cohort. d Kaplan–Meier curve analyses of SARS-CoV-2 antibodies as potential risk factors of COVID-19 mortality. The Kaplan–Meier curves show the survival probability of patients with high versus low antibody levels since symptom onset. Grouping criteria (cutpoints) are provided in the graphs. Hazard ratios (HR) for high versus low antibody levels are provided with p-values from log-rank tests

Fig. 8

Correlations between antibodies to structural proteins and clinical variables associated with COVID-19 survival. a Global correlation map of the SARS-CoV-2 proteome IgG antibodies and clinical variables in the survival and non-survival COVID-19 patient groups. b Differential correlations of the clinical variables and SARS-CoV-2 IgG antibodies between survival and non-survival COVID-19 groups in the cluster #2 group. The rainbow color from blue to red corresponds to the correlation of two variables from −1 (low correlation; blue) to +1 (high correlation; red)