SARS-CoV-2 infection elicits a rapid neutralizing antibody response that correlates with disease severity

Abstract

The protective effect of neutralizing antibodies in SARS-CoV-2 infected individuals is not yet well defined. To address this issue, we have analyzed the kinetics of neutralizing antibody responses and their association with disease severity. Between March and May 2020, the prospective KING study enrolled 72 COVID-19+ participants grouped according to disease severity. SARS-CoV-2 infection was diagnosed by serological and virological tests. Plasma neutralizing responses were assessed against replicative virus and pseudoviral particles. Multiple regression and non-parametric tests were used to analyze dependence of parameters. The magnitude of neutralizing titers significantly increased with disease severity. Hospitalized individuals developed higher titers compared to mild-symptomatic and asymptomatic individuals, which together showed titers below the detection limit in 50% of cases. Longitudinal analysis confirmed the strong differences in neutralizing titers between non-hospitalized and hospitalized participants and showed rapid kinetics of appearance of neutralizing antibodies (50% and 80% of maximal activity reached after 11 and 17 days after symptoms onset, respectively) in hospitalized patients. No significant impact of age, gender or treatment on the neutralizing titers was observed in this limited cohort. These data identify a clear association of humoral immunity with disease severity and point to immune mechanisms other than antibodies as relevant players in COVID-19 protection.

Figure 1

Neutralization activity. (A) Dose response of normalized neutralization data for all samples tested against replicative virus in Vero E6 cells (n = 130). (B) Correlation between IC₅₀ values of plasma samples in replicative virus and pseudovirus neutralization assays (n = 122). Line indicates linear regression for illustrative purposes. Correlation coefficient and p-value (Spearman correlation test) are shown. (C) Analysis of the impact of disease severity on neutralization titers (replicative virus assay) for the whole sample set. Individual values, mean values (solid lines) are shown for each group (0 = seronegative, 1 = asymptomatic, 2 = mid-symptomatic, 3 = hospitalized non severe, 4 = severe, 5 = ICU). (D) Calculated IC₅₀ (reciprocal dilution) in the replicative virus assay for all plasma samples tested grouped by SARS-CoV-2 positivity and clinical grade of symptoms. Comparison between groups was performed by Kruskal–Wallis test (p-value indicated in the Figure) with Dunn’s correction for multiple comparisons (indicated in intergroup comparisons). Top p-value indicates the comparison of the whole hospitalized and outpatient groups.

Figure 2

Longitudinal analysis and distribution of neutralization activity. (A) Neutralization titers from hospitalized patients were plotted against time from symptoms onset and fitted (solid line). Empty symbols indicate outliers. Light and dark blue arrows indicate the calculated time required to achieve the 50% and the 80% maximal neutralization titer, respectively. Non severe, severe and ICU groups are indicated by orange, maroon and red symbols, respectively. Analysis was performed with all the dataset. (B) Neutralization titers from mild-symptomatic individuals were fitted (solid line) after identification of outliers (empty symbols). The comparison of the plateau values for neutralization titers in hospitalized (light maroon line) and mild-symptomatic individuals is shown (Z test). (C) Representation of the frequency of undetectable, low, medium and high neutralizing individuals in non-hospitalized and hospitalized (All hospital) patients (p-value of Chi-square test).

Figure 3

Factors associated with neutralizing responses. (A) Correlation between maximal individual neutralizing titers and age. P-value for Spearman’s test correlation of all data is shown (solid line), red and green dotted lines indicate correlations for hospitalized and mild-symptomatic individuals, respectively. P-value for Spearman’s test correlation is shown. (B) For hospitalized patients, correlation between neutralizing activity and duration of hospital stay. P-value for Spearman’s test correlation of data is shown (solid line). (C,D) Analysis of gender differences in the maximal neutralization titer value of COVID-19 participants (n = 73, C) and in hospitalized participants (n = 40, panel D). P-values for Mann–Whitney tests is shown.

Figure 4

Effect of treatment. Maximal neutralization titers from hospitalized participants (n = 40) were analyzed according to the indicated treatments. Individual values, median and interquartile boxes (25–75) are indicated. P-values for Mann–Whitney tests are shown.