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.

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 < 0.05. Error bars represent mean and SD. See also Figures S1 and S4.

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 < 0.05. Error bars represent mean and SD.

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 CD14^loCD16⁺ (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 < 0.05. Error bars represent mean and SD. Data from two experiments.

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 < 0.05. Error bars represent mean and SD. Data from two experiments.

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 < 0.05. Error bars represent mean and SD.

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 < 0.02. Error bars represent mean and SD. Data from two experiments per visit.

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⁻CD138^hi) (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 < 0.04. Error bars represent mean and SD. Data from 2 experiments per visit. See also Figure S5.

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⁺), T_CM (CD45RA⁻CCR7⁺), or T_EM (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, T_CM, and T_EM cells from V1 (circles) and V2 (squares) reported as frequency of CXCR3⁺CPD^lo 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 < 0.05. Error bars represent mean and SD. Data from 2 experiments per visit. See also Figure S6.

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 (T_CM), and T effector memory (T_EM) 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 < 0.05. Error bars represent mean and SD. Data from two experiments per visit.

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 < 0.05.

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.