Longitudinal analysis shows durable and broad immune memory after SARS-CoV-2 infection with persisting antibody responses and memory B and T cells

Abstract

Ending the COVID-19 pandemic will require long-lived immunity to SARS-CoV-2. Here, we evaluate 254 COVID-19 patients longitudinally up to eight months and find durable broad-based immune responses. SARS-CoV-2 spike binding and neutralizing antibodies exhibit a bi-phasic decay with an extended half-life of >200 days suggesting the generation of longer-lived plasma cells. SARS-CoV-2 infection also boosts antibody titers to SARS-CoV-1 and common betacoronaviruses. In addition, spike-specific IgG+ memory B cells persist, which bodes well for a rapid antibody response upon virus re-exposure or vaccination. Virus-specific CD4+ and CD8+ T cells are polyfunctional and maintained with an estimated half-life of 200 days. Interestingly, CD4+ T cell responses equally target several SARS-CoV-2 proteins, whereas the CD8+ T cell responses preferentially target the nucleoprotein, highlighting the potential importance of including the nucleoprotein in future vaccines. Taken together, these results suggest that broad and effective immunity may persist long-term in recovered COVID-19 patients.

Keywords: Antibody; B cells; CD4+ T cells; CD8+ T cells; COVID-19; Endemic coronaviruses; Immune memory; Kinetics; Neutralization; RBD; SARS-CoV-2; Spike.

Figure 1.. Longitudinal SARS-CoV-2 spike binding antibody responses.

IgG (A-C), IgA (D-F), and IgM (G-I) antibodies reactive to SARS-CoV-2 spike (A, D, G), spike receptor binding domain (RBD; B, E, H), and the spike N terminal domain (NTD; C, F, I) were measured in triplicate by an electrochemiluminescent multiplex immunoassay and reported as arbitrary units per ml (AU/ml) as normalized by a standard curve. Longitudinal antibody titers of COVID-19 patients (in blue, n=222 COVID-19+ for IgG; n=190 COVID-19+ for IgA and for IgM) are plotted over days since symptom onset, whereas longitudinal pre-pandemic donor samples (in red, n=51 for IgG, IgA and IgM) were collected in the course of a non-SARS-CoV-2 vaccine study before 2019 and plotted over days since immunization. IgG decay curves and half-lives estimated by an exponential decay model are shown in black, and the decay curves and half-lives at day 120 post symptom onset estimated by a power law model are shown in green.

Figure 2.. Longitudinal binding antibody responses to other coronavirus spike proteins.

IgG (A-E), IgA (F-J), and IgM (K-O) antibody responses in sera collected from COVID-19+ patients (in blue, n=222 for IgG; n=190 for IgA and IgM) and pre-pandemic donors (in red, n=51 for IgG, IgA and IgM) that were measured to 229E spike (A, F, K), NL63 spike (B, G, L), HKU1 spike (C, H, M), OC43 spike (D, I, N), and the SARS-CoV-1 spike protein (E, J, O) in triplicate. Longitudinal antibody titers of COVID-19 patients are plotted over days since symptom onset, whereas longitudinal pre-pandemic donor samples were collected in the course of a non-SARS-CoV-2 vaccine study before 2019 and plotted over days since immunization. Antibody responses were measured by an electrochemiluminescent multiplex immunoassay and reported as arbitrary units per ml (AU/ml) as normalized by a standard curve. IgG decay curves and half-lives estimated by an exponential decay model are shown in black. There was no significant decline in IgG reactive to endemic alpha and betacoronaviruses in longitudinal samples collected in healthy donors before the pandemic (red, A-D).

Figure 3.. Neutralizing antibody responses to SARS-CoV-2

(A) In vitro serum neutralization antibody titers to SAR-CoV-2 were measured in duplicate by focus-reduction neutralization assay COVID-19 patients (n=183). The limit of detection is indicated with a dashed line at FRNT-mNG₅₀ = 20. The half-life estimated by the exponential decay model (black) is 150 days, whereas the half-life estimated at day 120 using the power law model (green) is 254 days. IgG antibody titers reactive to SARS-CoV-2 spike (B) and RBD (C) of the matched 183 COVID-19 for whom neutralization titers were assessed. The geometric mean titer plus 3 standard deviations of pre-pandemic samples is indicated by a dashed line (B and C). SARS-CoV-2 spike (D) and RBD (E) reactive IgG levels correlated with neutralization titers at the matched time point (repeated measures correlation, p<0.0001). The limit of detection is indicated with a dashed line at FRNT-mNG₅₀ = 20.

Figure 4.. SARS-CoV-2 spike and RBD specific memory B cells.

(A) Representative memory B cell gating strategy is shown for identification of SARS-CoV-2 spike and RBD-specific IgD− IgG+, IgD− IgM+ and IgD− IgA+ memory B cells in PBMCs from a SARS-CoV-2 convalescent participant. The frequency of spike+ (B) IgG+ and (C) IgM+ memory B cells out of memory B cells (IgD− CD19+ CD20+) is displayed over time from initial symptom onset among SARS-CoV-2 infected subjects (n=105 subjects; measured in singlet replicates). The dashed line indicates the limit of detection. The bold line represents the median fitted curve from a linear mixed effects model of post-day 30 responses. (D) The median percent of spike+ memory B cells expressing IgG, IgM or IgA isotypes was assessed at monthly intervals post-symptom onset. (E) The frequency of RBD+ IgG+ of memory B cells over time (n=141). (F) The proportion of S+ IgG+ memory B cells that are specific for the receptor binding domain are depicted over time.

Figure 5.. CD4+ T cell responses to SARS-CoV-2 antigens.

(A) The sum of background-subtracted CD4+ T cells expressing ex vivo IFN-γ, IL-2 and/or CD40L to peptide pools spanning SARS-CoV-2 structural proteins: S1, S2, envelope (E), membrane (M), nucleocapsid (N), and the following ORFs: 3a, 3b, 6, 7a, 7b, and 8 (n=114; tested in singlets) for each individual/timepoint. Each sample that is ‘positive’ (by MIMOSA) for at least one SARS-CoV-2 antigen is indicated by a solid circle, whereas samples that are ‘negative’ for all of the SARS-CoV-2 antigens at that timepoint are indicated by open triangles. The bold line represents the median fitted curve from a nonlinear mixed effects model of post-day 30 responses among those with a positive response at >= 1 time point; t_1/2 is the median half-life estimated from the median slope, with 95%CI [104, 411]. (B) The proportion of SARS-CoV-2-specific CD4+ T cells expressing a specific memory phenotype over time: central memory (CCR7+ CD45RA−), effector memory (CCR7− CD45RA−) or T_EMRA (CCR7− CD45RA+); restricted to ‘positive’ responders. Polyfunctionality of SARS-CoV-2-specific CD4+ T cells are shown at (C) 21–60 days since symptom onset (median, 30 days) and (D) >180 days median post symptom onset (median, 203 days). Percentages of cytokine-expressing CD4+ T cells are background subtracted and only subsets with detectable T cells are displayed. Data shown were restricted to ‘positive’ responders and a single data point per individual per time frame. All subsets were also evaluated for expression of IL-4, IL-5, IL-13, IL-17 and perforin, and were found to be negative. (E) Bar graphs indicate the proportion of COVID-19 convalescent patients who had a positive CD4+ T cell response to the individual SARS-CoV-2. peptide pool ex vivo stimulations. Some antigens were combined for stimulation as indicated. (F) For each subject with positive SARS-CoV-2- specific CD4+ T cells, the proportion of the total SARS-CoV-2 responding CD4+ T cells that are specific for each stimulation.

Figure 6.. CD8+ T cell responses to SARS-COV-2 antigens.

(A) The sum of background-subtracted CD8+ T cells expressing IFN-γ (with or without other cytokines), in response to peptide pools covering SARS-CoV-2 structural proteins: S1, S2, envelope (E), membrane (M), nucleocapsid (N), and the following ORFs: 3a, 3b, 6, 7a, 7b, and 8 (n=114; tested in singlets) for each individual/timepoint. Each sample that is ‘positive’ (MIMOSA) for at least 1 SARS-CoV-2 antigen is indicated by a solid circle, whereas samples that are ‘negative’ for all of the SARS-CoV-2 antigens at that timepoint are indicated by open triangles. The bold black line represents the median fitted curve from a nonlinear mixed effects model of post-day 30 responses among those with a positive response to the antigen(s) under consideration at ≥ 1 time point; t_1/2 shown is the median half-life estimated from the median slope, with 95%CI [92, 417]. (B) The proportion of SARS-CoV-2-specific CD8+ T cells by memory phenotype over time: effector memory (EM; CCR7− CD45RA−), T_EMRA (CCR7− CD45RA+) and central memory (CM; CCR7+ CD45RA−). Analyses were restricted to ‘positive’ responders. Polyfunctionality of SARS-CoV-2-specific CD8 T cells at (C) 21–60 days post symptom onset (median, 30 days) and (D) >180 days median post symptom onset (median, 203 days). Percentages of cytokine expressing CD8+ T cells are background subtracted and only subsets with detectable T cells are displayed. Data shown were restricted to positive responders and a single data point per individual per time frame. All CD8+ T cell subsets were also evaluated for expression of IL-4, IL-5, IL-13, and IL-17 and were found to be negative. (E) The bar graphs indicate the proportion of COVID-19 convalescent patients who had a ‘positive’ CD8+ T cell response to the individual SARS-CoV-2 stimulations. (F) The fraction of the total SARS-CoV-2 responding CD8+ T cells per subject that are specific for each peptide pool.

Figure 7.. Correlations between SARS-CoV-2-specific immune responses and assessment of covariates.

(A) The forest plot depicts the estimated fold-change in the level of each immune response per decade of age, with 95% Wald-based CIs and p-values. (B) The forest plot shows the estimated fold-change in the level of each immune response for severe (WHO score > 4) vs. non-severe (WHO score ≤ 4) disease, with 95% Wald-based CIs and p-values. S1 CD8+ T cell responses compared moderate-severe (WHO score > 2) to mild (WHO score ≤ 2) disease as there were no participants with severe disease with at least one positive S1 CD8+ T cell response post-day 30. Estimates in (A) and (B) are from mixed effects models of post-day 30 (B and T cell responses) or post-day 42 (antibody responses) among responders that account for fixed effects of age and disease severity on the level of immune response. Univariate assessment of disease severity on the magnitude of (C) spike IgG antibodies and (D) SARS-CoV-2 neutralizing antibodies at day 120 is shown for mild (WHO score: 0–2), moderate (WHO score: 3–4), and severe disease (WHO score: 5+); p-values from one-way ANOVA. (E) The heatmap shows Spearman correlations between critical SARS-CoV-2 memory immune responses (day 30 B and T cell responses and day 180 antibody responses) with significance levels: *p<0.05, **p<0.01, ***p<0.001. The tile size and color intensity correspond to the absolute value of the Spearman rank correlation coefficient, with red or blue indicating a positive or negative correlation, respectively. Day 30, 42 and 180 immune responses were estimated from mixed effects models of the longitudinal SARS-CoV-2 binding antibodies, SARS-CoV-2 neutralizing antibodies, CD4+ and CD8+ T cell responses, and B cell responses.