Kinetics of antibody responses dictate COVID-19 outcome

Abstract

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se, but rather with the delayed kinetics of NAb production.

Fig. 1 |. COVID-19 severity correlates with anti-S antibodies.

a-b, Plasma reactivity to S protein and RBD by ELISA. a, Anti-S IgM and IgG comparison in non-hospitalized, moderate, severe and deceased COVID-19 hospitalized patients. IgM (HCW, n = 21; non-hospitalized, n=21; moderate, n = 94; severe, n = 25; deceased, n=14). IgG (HCW, n = 87; non-hospitalized, n=21; moderate, n = 96; severe, n = 23; deceased, n=14). b, Anti-RBD IgM and IgG comparison in non-hospitalized, moderate, severe and deceased COVID-19 patients. IgM (HCW, n = 21; non-hospitalized, n=7; moderate, n = 75; severe, n = 13; deceased, n=11). IgG (HCW, n = 21; non-hospitalized, n=6; moderate, n = 74; severe, n = 13; deceased, n=11). Negative controls (HCWs) are shown in black. OD, optical density at 450 nm (OD_{450 nm}). N-hospitalized, non-hospitalized. For a and b, each dot represents a single individual at its maximum antibody titer over the disease course. Horizontal bars indicate mean values. c, Correlation and linear regression of virus-specific IgG (OD_{450 nm}) and length of hospitalization measured over time. Left, Total patients regardless of disease severity. Regression lines are shown as dark blue, continuous line (Anti -S) or dashed line (Anti-RBD). Right, Patients grouped by disease severity. Regression lines are shown as dark purple (moderate) or pink (severe), continuous line (Anti -S) or dashed line (Anti-RBD). d–e, Correlation of virus-specific IgG (OD_{450 nm}) and (d) length of intubation and patients’ maximum levels of (e) ferritin, D-dimer, and CRP. f, Heat map correlation of the levels of virus-specific IgG (OD_{450 nm}) and the major immune cell populations within PBMCs in COVID-19 patients. Subjects are arranged across rows based on their levels of IgG anti-S (upper) or anti- RBD (lower) with each coloured unit indicating the relative distribution of an immune cell population normalized against the same population across all subjects. K-means clustering was used to arrange patients and measurements. Eosi, eosinophils. Neut, neutrophils. Mono, monocytes. CD8act, CD4act, CD4Tfh, follicular helper T cells. g, Spectral clustering of COVID-19 patients with S1 IgG+ samples at their first collection time point into IgG low, IgG high, and Convalescent clusters (main, below). Dashed vertical line represents threshold of S1 IgG positivity, and was generated by calculating the 95th percentile of S1 IgG O.D. 450 levels from SARS-CoV-2 negative individuals + 1 standard deviation. Solid horizontal line represents the lower limit of detection for our RT-qPCR assay previously described. (insets, above) i, Graphical schematic of transitions between disease states for COVID-19 patients, colored by disease state (acute, sub-acute, convalescent) ii, Comparison of mean viral loads between Sub-acute clusters using two sample t-test. iii, Violin plots of clinical scores for each cluster. Solid black lines are means of each cluster, whereas dashed lines represent the median value. No significant difference was noted, significance was assessed by Kruskal-Wallis testing corrected for multiple comparisons using Dunn’s method. iv, Days from symptom onset compared for each cluster. Significance was assessed by Kruskal-Wallis testing corrected for multiple comparisons using Dunn’s method. *** p < .001; ** p < .01; *p < .05.

Fig. 2 |. Serum antibody kinetics reveals distinct COVID-19 outcomes.

a, Patients plasma reactivity to S protein and RBD by ELISA. Anti-S and Anti-RBD IgM and IgG comparison in discharged or deceased patients. Longitudinal data plotted over time continuously. Regression lines are shown as light blue (discharged), purple (deceased) and red (High neutralizers). Shading represents 95% CI and are coloured accordingly. Anti-S IgM, (Discharged, n=127; Deceased, n=14). Anti-S IgG, (Discharged, n=128; Deceased, n=14). Anti-RBD IgM, (Discharged, n=88; Deceased, n=11). Anti-S RBD, (Discharged, n=87; Deceased, n=11). b–c, Viral loads measured by nasopharyngeal swabs are plotted as log₁₀ of genome equivalents (GE). b, Viral loads against time after symptom onset accordingly with patient’s outcome. b, Regression lines are shown as light blue (discharged) or purple (deceased). c, Viral load measured in discharged, deceased and high neutralizer patients. (HN, n=6; discharged, n = 53; deceased, n = 12). Each dot represents the viral load of a single individual at its maximum antibody titer over the disease course. d, Heat map correlation of the levels of Anti-S IgG (OD_{450 nm}) and plasma cytokines/chemokines measurements within discharged (n=146) or deceased patients (n=26). Subjects are arranged across rows based on their levels of anti-S IgG with each coloured unit indicating the relative cytokine concentration (log₁₀) normalized against the same population across all subjects. K-means clustering was used to arrange patients and measurements.

Fig. 3 |. Neutralizing antibodies temporal dynamics distinguishes discharged and deceased COVID-19 patients.

a–e, Longitudinal neutralization assay using wild-type SARS-CoV-2. a, Frequency of neutralizers among patients with high anti-S IgG levels (O.D.>1,2), n=65. b, Neutralization capacity between discharged (light blue), deceased (purple) and high neutralizers (red) at the experimental sixfold serially dilutions (from 1:3 to 1:2430). Health care workers, below the threshold for anti-S/RBD ELISA, were used as negative controls. HN, high neutralizers. (HCW, n = 22; discharged, n=35; deceased, n=17; high neutralizers, n=13). c–d, Logistic Regression and neutralization titer (PRNT50) according to clinical severity scale as described in Methods. CS, clinical score. e, Longitudinal data plotted over time of neutralization capacity between discharged (light blue), deceased (purple) and high neutralizers (red) at the experimental sixfold serially dilutions (from 1:3 to 1:2430). f, Average of days from symptom onset to reach 50% of neutralization at each experimental serum dilution between groups.

Fig. 4 |. Early neutralizing antibodies correlates with better COVID-19 clinical trajectory.

Patients stratification by early NAb capacity, based on levels of Anti-S IgG, PRNT50 titers and days from symptom onset. a, Cohort overview by IgG Anti-S titers (3 external circles) and NAb production (internal circle). Frequency of patients in each level are indicated in light grey. Frequency of early and late neutralizers stratified based on days from symptom onset at 1:90 dilution is indicated in black. NAb, neutralizing antibodies. DfSO, days from symptom onset. * Frequency of patients with NAb capacity over the disease. course within patients with high levels of anti-S IgG. b, Frequency of neutralizers with >50% neutralization activity until 14 days after symptom onset. c, Distribution of age, BMI and frequency of males and females among early (>50% neutralization activity in 1:90 titer before day 14 after symptom onset) or late (<50% neutralization activity in 1:90 titer before day 14 after symptom onset) neutralizers length, as determined in (a). d, Disease progression measured by clinical severity score for patients in each group. Data (mean ± s.e.m) are ordered by the collection time points for each patient, with regular collection intervals of 3–4 days. e, Percentage of mortality in each group. (Early neutralizers, n=24; Late neutralizers, n=38). f, Viral load measured by nasopharyngeal swabs plotted as log10 of genome equivalents in early and late neutralizers. (Early neutralizers, n=21; Late neutralizers, n=28). Each dot represents a single individual at its maximum antibody titer over the disease course. Box analysis with minimum and maximum represented for each group.