A comprehensive, longitudinal analysis of humoral responses specific to four recombinant antigens of SARS-CoV-2 in severe and non-severe COVID-19 patients

Abstract

There is an urgent need for effective treatment and preventive vaccine to contain this devastating global pandemic, which requires a comprehensive understanding of humoral responses specific to SARS-CoV-2 during the disease progression and convalescent phase of COVID-19 patients. We continuously monitored the serum IgM and IgG responses specific to four SARS-CoV-2 related antigens, including the nucleoprotein (NP), receptor binding domain (RBD), S1 protein, and ectodomain (ECD) of the spike protein among non-severe and severe COVID-19 patients for seven weeks since disease onset. Most patients generated humoral responses against NP and spike protein-related antigens but with their distinct kinetics profiles. Combined detection of NP and ECD antigens as detecting antigen synergistically improved the sensitivity of the serological assay, compared to that of using NP or RBD as detection antigen. 80.7% of convalescent sera from COVID-19 patients revealed that the varying extents of neutralization activities against SARS-CoV-2. S1-specific and ECD-specific IgA responses were strongly correlated with the neutralization activities in non-severe patients, but not in severe patients. Moreover, the neutralizing activities of the convalescent sera were shown to significantly decline during the period between 21 days to 28 days after hospital discharge, accompanied by a substantial drop in RBD-specific IgA response. Our data provide evidence that are crucial for serological testing, antibody-based intervention, and vaccine design of COVID-19.

Fig 1. Establishment of the serological assay for SARS-CoV-2.

(A) Schematic representation of RBD, S1, and ectodomain (ECD) of the SARS-CoV-2 spike protein. (B) The expression and purification of RBD, S1, ECD, and NP protein. (C) ELISA assay for detecting IgM and IgG humoral responses against RBD, S1, ECD and NP protein was performed using sera samples diluted at 1:200. The dashed blue line indicates the cutoff value for each assay, which was determined based on the archived serum samples collected before the COVID-19 outbreak. Sera from 46 healthy controls (HC) collected in 2019 served as the negative control, whereas 72 samples collected at different time points from COVID-19 patients were used as the positive control.

Fig 2. Representative dynamic IgM or IgG responses specific to NP, RBD, S1, and ECD for three severe COVID-19 patients and three non-severe COVID-19 patients over time since symptom onset, determined by ELISA assay.

Fig 3. Correlation analysis of IgM or IgG responses specific to NP, RBD, S1, and ECD.

The Bonferroni correction was used and the adjusted significance level of p value was 0.05/3 = 0.016.

Fig 4. The IgM, IgG, IgA, IgG1, and IgG3 binding tiers specific with RBD, S1, ECD, and NP, respectively, in the convalescent sera of non-severe and severe COVID-19 patients.

Fig 5. Neutralization activities of convalescent sera from COVID-19 recovered patients.

(A) The neutralization titers (ID₅₀) were determined between severe and non-severe patients. (B) The percentage of convalescent serum samples with undetectable neutralization titers (ID₅₀ titer less than 20), low level of neutralization titers (ID₅₀ titer between 20 to 270), moderate level of neutralization titers (ID₅₀ titer between 270 to 1000), high level of neutralization titers (ID₅₀ titer over 1000). (C) Correlation analysis of neutralization titers with the binding titers of S1-specific IgA, RBD-specific IgA, RBD-specific IgG, RBD-specific IgG3, N-specific IgM, ECD-specific IgA, ECD-specific IgG1 and ECD-specific IgG3.

Fig 6. The dynamic humoral responses at the time point of hospital discharge and follow-up visit between 21 days and 28 days after discharge.

(A) The comparison between the IgM, IgG, and IgA titer specific to RBD, ECD, S1, and NP protein and the neutralization activities at the time point of hospital discharge and follow up visit between 21 days and 28 days after discharge. (B) The reduced folds of IgM, IgG and IgA titer specific to RBD, ECD, S1, and NP protein and neutralization activities at the time point of hospital discharge and follow-up visit between 21 days and 28 days after discharge were analyzed between severe patients and non-severe patients.