Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans

Abstract

Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10-15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID₅₀ > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID₅₀ had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection.

Figure 1. Kinetics of antibody development against SARS-CoV-2 antigens over time.

A) A cumulative frequency analysis describing the point of seroconversion for each person in the cohort. Graph shows the percentage of individuals in the cohort that become IgM, IgA or IgG positive to S, RBD and N each day post onset of symptoms. A serum sample is considered positive when the OD is 4-fold above background. B) OD values at 1:50 serum dilution for IgM, IgA and IgG against S, RBD and N overtime. Each line represents one individual. Severity 0-3 are shown in black and severity 4/5 are shown in red. Development of the ELISA assay is described in Pickering et al .

Figure 2. Kinetics of neutralizing antibody responses in SARS-CoV-2 infection.

a) nAb ID₅₀ change related to days POS. ID₅₀ measured using HIV-1 based virus particles (PV), pseudotyped with SARS-CoV-2 S. Each line represents one individual. Severity 0-3 are shown in black and severity 4/5 are shown in red. b) Example kinetics of Ab responses (IgM, IgA, IgG binding to S, RBD and N, and ID₅₀ against PV and wild type virus) for four individuals during acute infection and the convalescent phase. Graphs show comparison between severity 0 (left) and severity 4 (right) rated disease. The cut-off for the pseudovirus and wild-type virus neutralization assays are 1:50 and 1:20 respectively. Error bars for OD values represent the range of the value for experiments performed in duplicate (not shown when smaller than symbol size).

Figure 3. Impact of disease severity on Ab responses in SARS-CoV-2 infection.

Comparison for individuals with 0-3 or 4/5 disease severity for a) peak ID₅₀ of neutralization (p<0.0001), b) the time POS to reach peak ID₅₀ (p=0.674), and c) the time POS to detect neutralizing activity (p=0.9156). ID₅₀ measured using HIV-1 based virus particles, pseudotyped with SARS-CoV-2 S. Comparison in OD values for individuals with 0-3 or 4/5 disease severity for d) IgG (p=0.0635), e) IgM (p=0.0003) and f) IgA (p=0.0018) against S measured at peak ID₅₀. G) Comparison of the peak ID₅₀ value for individuals who were treated for hyperinflammation or not, and had 4/5 disease severity (p>0.999). Statistical significance was measured using a Mann-Whitney test. ns = not significant.

Figure 4. Longevity of the nAb response.

a) ID₅₀ at peak neutralization (measured using HIV-1 based virus particles, pseudotyped with SARS-CoV-2 S) is plotted against the donor matched ID₅₀ at the last time point sera was collected. Only individuals where the peak ID₅₀ occurs before the last time point, and where the last time point is >30 days POS are included in this analysis. The dotted line represents the cut-off for the pseudovirus neutralization assay. b-c) EC₅₀ values for IgG binding to S, RBD and N were calculated at time points with peak ID₅₀ and the final time point sera was collected. EC₅₀ at peak neutralization is plotted with the donor matched EC₅₀ at the last time point sera was collected. Individuals with a disease severity 0-3 are shown in black and those with 4/5 are shown in red. The dotted line represents the cut-off for EC₅₀ measurement. e) Correlation of ID₅₀ with IgG EC₅₀ against S (r ²=0.8293), RBD (r ²=0.7128) and N (r ²=0.4856) (Spearman correlation, r. A linear regression was used to calculate the goodness of fit, r ²). f) Change in IgG EC₅₀ measured against S, RBD and N and ID₅₀ using pseudovirus and wild type virus over time for 4 example patients (all severity 4). The lowest dilution used for the pseudovirus and wild-type virus neutralization assays are 1:50 and 1:20 respectively.

Figure 5. Ab responses in a healthcare worker cohort.

a) ID₅₀ values plotted against the time post onset of symptoms (POS) at which sera was collected. Each line represents one individual. ID₅₀ measured using HIV-1 based virus particles, pseudotyped with SARS-CoV-2 S. Asymptomatic individuals shown in green, symptomatic individuals shown in black and PCR+ HCW shown in red (for comparsion). The dotted line represents day 0 post onset of symptoms. b) Comparison of the peak ID₅₀ between asymptomatic individuals (n=10, includes 7 HCW and 3 hospital patients), healthcare workers (n=24 symptomatic HCW with no PCR test), and PCR+ individuals with either severity 0-3 (n=28) or 4/5 (n=32). The 2 PCR+ individuals sampled at early time points (<8 days POS) and did not seroconvert were not included in this analysis. c) ID₅₀ at peak neutralization is plotted with the donor matched ID₅₀ at the last time point sera was collected. The dotted line represents the cut-off for the SARS-CoV-2 pseudotyped virus particles neutralization assay. Asymptomatic donors are shown in green and symptomatic donors shown in black.