Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is causing a global pandemic, and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine whether they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific immunoglobulin (IgG) antibodies, neutralizing plasma, and memory B and memory T cells that persisted for at least 3 months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, whereas memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.

Keywords: COVID-19; SARS-CoV2; adaptive immune response; human; memory B cell; memory T cell; monoclonal antibody; vaccine.

Conflict of interest statement

Declaration of Interests M.P., D.J.R., J.N., C.D.T., Y.C., and L.B.R. have filed a patent under the provisional serial no. 63/063,841. Other authors declare no competing interests.

Copyright © 2020 Elsevier Inc. All rights reserved.

Figures

Graphical abstract

Figure 1

Mild COVID-19 Induces Persistent, Neutralizing Anti-SARS-CoV-2 IgG Antibody (A) Study timeline. Range is indicated by box and median indicated by line for each event. (B) ELISA dilution curves and AUC for anti-RBD IgG (left), IgM (center), and IgA (right) from HC and CoV2+ plasma samples at Visit 1 (V1) and Visit 2 (V2). Dashed line indicates mean + 3 SD of the HC AUC values. (C) Comparing V1 and V2 AUC in HC and CoV2+ individuals for each antibody isotype. V2 AUC values were normalized to V1 samples run with V2 samples. (D) Percent inhibition of RBD binding to ACE2 by plasma sVNT at 1:2 plasma dilution. (E) Spearman correlation between percent RBD inhibition by sVNT at a 1:2 plasma dilution and anti-RBD IgG AUC at both visits. (F) Percent RBD inhibition at 1:2 plasma dilution at V1 and V2, paired by sample. (G) Spearman correlation between percent RBD inhibition by sVNT at a 1:2 plasma dilution and percent virus neutralization by PRNT at a 1:160 plasma dilution. (H) CoV2+ percent virus neutralization by PRNT at a 1:160 plasma dilution normalized and paired as in (C). Statistics for unpaired data determined by 2-tailed Mann-Whitney tests and, for paired data, by 2-tailed Wilcoxon signed-rank tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure S1

Healthy Controls Do Not Have SARS-CoV-2 RBD or Spike-Specific Antibodies, Related to Figure 1 ELISA dilution curves and area under the curve (AUC) for anti-RBD and anti-spike IgG (left) and IgM (right) in plasma collected from individuals prior to 2020 and the SARS-CoV-2 pandemic (historical negatives, HN, black), from healthy controls (HC, at Visit 2) and from individuals that tested PCR+ for SARS-CoV-2 (CoV2+, at Visit 1). Dashed line indicates mean + 3 SD of HN AUC values. Statistics determined by two-tailed Mann-Whitney tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure S2

PBMC Innate Populations in CoV2+ Individuals Return to Immune Quiescence by Visit 1, Related to Figure 1 (A) Flow cytometry gating for CD15-CD3-CD19-CD56-HLADR+CD14+ monocytes (purple gate), which were further divided into CD14loCD16+ (red gate), CD14+CD16+ (blue gate), and CD14+CD16- monocytes (green gate), and CD15-CD3-CD19-CD56-CD14-CD304+CD123+ plasmacytoid dendritic cells (pDCs) (pink gate). (B) Percent monocytes and pDCs of live PBMCs from healthy controls (HC) and previously SARS-CoV-2 infected (CoV2+) individuals. (C) Percent subsets of monocytes from PBMCs. Statistics determined by two-tailed Mann-Whitney tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure S3

Bulk PBMCs Return to Immune Quiescence by Visit 1, Related to Figure 1 (A and B) Representative flow cytometry plots and frequencies of αβ and γδ T cell subsets at Visit 1 (V1) in PBMCs from healthy control (HC) and SARS-CoV-2-recovered (CoV2+) individuals. (C and D) Representative flow cytometry plots and frequencies of CD4+ and CD8+ T cell effector/activation states (Ki67+, T-bet+, HLA-DR+CD38+) of total non-naive, memory CD45RA+CCR7+/− CD4+ or CD8+ T cells at V1 in HC and CoV2+ PBMCs. (E and F) Representative flow cytometry plots and frequencies of CD4+ memory and T-helper subsets at V1 in HC and CoV2+ PBMCs. (G and H) Representative flow cytometry plots and frequencies of cTfh (CXCR5+CD45RA-) and cTfh activation (ICOS+PD-1+) and helper (CXCR3+/−CCR6+/−) subsets at V1 in HC and CoV2+ PBMCs. (I) Frequency of B cells (CD19+CD3-) at V1 in HC and CoV2+ PBMCs. Statistics determined by two-tailed Mann-Whitney tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure S4

Mild COVID-19 Induces Persistent, Neutralizing Anti-SARS-CoV-2 IgG Antibody, Related to Figure 1 (A) ELISA dilution curves and area under the curve (AUC) for anti-spike IgG (left), IgM (center), and IgA (right) from healthy control (HC) and SARS-CoV-2-recovered (CoV2+) individuals plasma at Visit 1 (V1). Dashed line indicates mean + 3 SD of the HC AUC values. (B) Spearman correlation of V1 anti-RBD and anti-spike IgG (left), IgM (center), and IgA (right) AUC. Statistics determined by two-tailed Mann-Whitney tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure S5

Mild COVID-19 Induces a Sustained Enrichment of RBD-Specific IgG+ Memory B Cells, Related to Figure 2 (A) Representative flow cytometry gates for phenotyping RBD-specific B cells from PBMCs in Figure 2 set on total B cells from a healthy control (HC) (surface stain, top; intracellular stain, bottom). (B) Number of RBD-specific IgD+, IgM+ and IgA+ MBCs (CD20+RBD tetramer+decoy tetramer- CD27+CD21+/CD27+CD21-/CD27-CD21-) from healthy control (HC) and SARS-CoV-2-recovered (CoV2+) PBMCs at Visit 1 (V1) and Visit 2 (V2). Statistics for unpaired data determined by two-tailed Mann-Whitney tests and, for paired data, by two-tailed Wilcoxon signed-rank tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure 2

Mild COVID-19 Induces a Sustained Enrichment of RBD-Specific IgG+ Memory B Cells (A) Representative gating of live CD3–CD14–CD16– cells for SARS-CoV-2 RBD-specific cells (RBD tetramer+decoy tetramer–) from CoV2+ and HC PBMCs at V1 and V2. (B) Number of RBD-specific B cells (RBD tetramer+decoy tetramer–CD20+) per 1 × 106 PBMCs. (C) Representative flow cytometry plots and number of RBD-specific PBs (RBD tetramer+decoy tetramer−CD20−CD138hi) (na = could not be calculated because all values 0). (D) Representative gating of RBD-specific B cells for naive B cells (CD21+CD27−) and MBCs (CD21+CD27+/CD21–CD27+/CD21–CD27– populations outlined in green). (E) Proportion of RBD-specific B cells that are naive (CD21+CD27−), classical MBCs (CD21+CD27+), or activated MBCs (CD21−CD27+/−), statistics for the proportion that are MBCs. (F) Number of RBD-specific MBCs (classical and activated). (G) Representative gating of RBD-specific MBCs for BCR isotype (IgD, IgM, IgA, and IgG) expression. (H) Proportion of RBD-specific MBCs expressing the BCR isotypes IgD, IgM, IgA, and IgG. Statistics are for the proportion that are IgG+. (I) Number of RBD-specific IgG+ MBCs. (J) Representative gating of RBD-specific MBCs for T-bet expression and number of RBD-specific T-bet+ MBCs. Statistics for unpaired data determined by 2-tailed Mann-Whitney tests and, for paired data, by 2-tailed Wilcoxon signed-rank tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure 3

SARS-CoV-2 Infection Induces Durable, Functional Spike-Reactive CD4+ T Cells (A) Representative flow cytometry plots of ICOS and CD40L expression on antigen-experienced (non-CD45RA+CCR7+) CD4+ T cells 20 h after incubation of PBMCs from HC and CoV2+ individuals at V1 and V2 with vehicle or SARS-CoV-2 spike. (B) Number of antigen-experienced ICOS+CD40L+CD4+ T cells per 1x106 CD4+ T cells from HC and CoV2+ samples after incubation with vehicle (Veh.) or spike (S) at both time points (right) and calculated number of spike-responsive CD4+ T cells (number after incubation with spike minus number after incubation with vehicle) compared across time points (left). (C) Number of antigen-experienced CXCR5+ICOS+CD40L+CD4+ T cells (cTfh) per 1 × 106 CD4+ T cells from HC and CoV2+ samples after incubation with Veh. or S at both time points (right) and calculated number of spike-responsive cTfh cells (number after incubation with spike minus number after incubation with vehicle) compared across time points (left). (D) Representative flow cytometry plots of sorted CD4+ naive (CD45RA+CCR7+), TCM (CD45RA−CCR7+), or TEM (CD45RA−CCR7−) T cells from HC and CoV2+ PBMCs after 5–6 days of co-culture with SARS-CoV-2 spike-protein-pulsed autologous monocytes and measuring proliferation by CPD dilution. (E) SARS-CoV-2 spike-specific expansion of sorted CD4+ naive T, TCM, and TEM cells from V1 (circles) and V2 (squares) reported as frequency of CXCR3+CPDlo cells after incubation with spike minus frequency after incubation with vehicle. (F) Number of cytokine-producing, antigen-experienced CD69+CD4+ T cells per 1 × 106 CD4+ T cells after incubation with Veh. or S (right) and calculated number of spike-responsive, cytokine-producing CD4+ T cells (number after incubation with spike minus number after incubation with vehicle) (left). (G) Frequency of antigen-experienced CD69+CD4+ T cell subsets, CCR6+/− Teff (CXCR5−), and CCR6+/− cTfh (CXCR5+) producing IL-2, IFN-γ, and IL-17A effector cytokines after incubation with spike for 20 h. (H) Representative flow cytometry plots of CD69 and IFN-γ expression on antigen-experienced CD8+ T cells from HC and CoV2+ PBMCs at V2 after 20 h of incubation with vehicle or SARS-CoV-2 spike. (I) Number of antigen-experienced IFN-γ+CD69+CD8+ T cells per 1 × 106 CD8+ T cells after 20 h of incubation with Veh. or S (right) and calculated number of spike-responsive CD8+ T cells (number after incubation with spike minus number after incubation with vehicle) (left). Statistics for unpaired data determined by 2-tailed Mann-Whitney tests and, for paired data, by 2-tailed Wilcoxon signed-rank tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure S6

SARS-CoV-2 Infection Induces Durable, Functional Spike-Reactive CD4+ T Cells, Related to Figure 3 (A) Flow cytometry sorting strategy for naive, T central memory (TCM), and T effector memory (TEM) cells from HC and CoV2+ PBMCs at Visit 1 and Visit 2 before 5-6 days of culture with autologous monocytes and SARS-CoV-2 spike protein or vehicle. (B) Representative flow cytometry gating on PMA/Ionomycin-activated PBMCs for cytokine expression by antigen experienced (non-CD45RA+CCR7+) CD4+ T cells subset into CCR6+/− T effector cells (Teff, CXCR5-) and circulating T follicular helper cells (cTfh, CXCR5+). (C) Representative flow cytometry gating on antigen-experienced (non-CD45RA+CCR7+) CD4+ T cells from HC and CoV2+ V2 PBMCs following incubation with SARS-CoV-2 spike for 20 h. Gating on CD69+ CCR6+/− T effector cells (Teff, CXCR5-) and CCR6+/− circulating T follicular helper cells (cTfh, CXCR5+) for IL-2, IFN-γ and IL-17A effector cytokines expression. (D) Number of IL-4-producing, antigen-experienced CD69+CD4+ T cells per 1x106 CD4+ T cells after incubation with vehicle (Veh.) or SARS-CoV2 spike (S) (left) and calculated number of spike-responsive, cytokine-producing CD4+ T cells (number after incubation with spike minus number after incubation with vehicle)(right). Statistics determined by two-tailed Mann-Whitney tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure S7

SARS-CoV-2-Specific MBCs Can Express Neutralizing Antibodies, Related to Figure 4 and Table S1 (A) Gating strategy for sorting RBD-specific B cells. (B) IgG ELISA to confirm expression of Visit 1 antibodies in transfected cell culture supernatants. Positive control is the kit standard (std) and negative control is supernatant from untransfected cells (no trans, green). (C) RBD ELISA of purified Visit 1 monoclonal antibodies. Negative control (green) is an irrelevant Plasmodium-specific antibody. (D) IgG ELISA to confirm expression of Visit 2 antibodies in transfected cell culture supernatants. (E) RBD ELISA of purified Visit 2 monoclonal antibodies. (F) Number of mutations in variable regions of RBD-specific monoclonal antibodies. (G) Mutation frequency of variable regions of RBD-specific monoclonal antibodies. Statistics determined by two-tailed Mann-Whitney tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value 

Figure 4

SARS-CoV-2-Specific MBCs Can Express Neutralizing Antibodies (A) Representative flow plots of index-sorted RBD-tetramer specific B cells (gating scheme in Figure S7A). BCRs cloned from cells are shown in red. (B) Anti-RBD ELISA of culture supernatants from cells transfected to express one of the Visit 1 monoclonal antibodies or supernatant from untransfected cells (no trans). Antibodies that did not bind RBD are shown in orange. (C) Inhibition of RBD binding to ACE2 by Visit 1 monoclonal antibody supernatants measured by sVNT assay, compared to a known RBD-specific neutralizing antibody (Ty1). Red indicates strong inhibitors, blue moderate inhibitors and black non-inhibitors. (D) Anti-RBD ELISA of culture supernatants from cells transfected to express one of the Visit 2 monoclonal antibodies. Antibodies that did not bind RBD are shown in orange. (E) Inhibition of RBD binding to ACE2 by Visit 2 monoclonal antibodies measured by sVNT assay. Red indicates strong inhibitors and black non-inhibitors. (F) Neutralization capacity of purified monoclonal antibodies as measured by PRNT. 2B04 and 2C02 are previously identified strong and weak neutralizing murine antibodies, respectively, and MSP-003 is an irrelevant Plasmodium-specific antibody. (G) IC50 values calculated from PRNT. Dotted line represents the limit of detection. See also Figure S7.

Figure 5

Recovered Individuals Formed Multifaceted SARS-CoV-2-Specific Immune Memory Heatmap of values for independent SARS-CoV2 RBD- or spike-specific immune memory components from each HC and CoV2+ individual at Visit 2. RBD-specific IgG measured by ELISA (AUC). Percent inhibition by sVNT calculated at 1:2 plasma dilution. Number of RBD-specific IgG+ MBCs per 1 × 106 PBMCs. Number of spike-responsive (CD69+), cytokine-producing (IL-2/IFN-γ/IL-17A), antigen-experienced CD4+ T cells calculated by number after 20 h incubation with spike minus number after incubation with vehicle. Number of spike-responsive (CD69+IFN-γ+), antigen-experienced CD8+ T cells calculated by number after 20 h incubation with spike minus number after incubation with vehicle. The color scales are set for each metric (row) with the mean + 1 SD of the HC set to white.