Rapid Generation of Neutralizing Antibody Responses in COVID-19 Patients

Abstract

SARS-CoV-2, the virus responsible for COVID-19, is causing a devastating worldwide pandemic, and there is a pressing need to understand the development, specificity, and neutralizing potency of humoral immune responses during acute infection. We report a cross-sectional study of antibody responses to the receptor-binding domain (RBD) of the spike protein and virus neutralization activity in a cohort of 44 hospitalized COVID-19 patients. RBD-specific IgG responses are detectable in all patients 6 days after PCR confirmation. Isotype switching to IgG occurs rapidly, primarily to IgG1 and IgG3. Using a clinical SARS-CoV-2 isolate, neutralizing antibody titers are detectable in all patients by 6 days after PCR confirmation and correlate with RBD-specific binding IgG titers. The RBD-specific binding data were further validated in a clinical setting with 231 PCR-confirmed COVID-19 patient samples. These findings have implications for understanding protective immunity against SARS-CoV-2, therapeutic use of immune plasma, and development of much-needed vaccines.

Keywords: COVID-19; SARS-CoV-2; coronavirus; humoral immune response; neutralizing antibody; protective immunity; receptor-binding protein; serology test; spike protein.

Graphical abstract

Figure 1

Antibody Responses against SARS-CoV-2 RBD in PCR-Confirmed Acutely Infected COVID-19 Patients (A) Structure of a SARS-CoV-2 spike protein (single monomer is shown) with the RBD highlighted in red. (B) Sequence homology analysis of SARS-CoV-2 spike protein RBD compared to SARS, MERS, and seasonal alpha- and beta-CoVs. (C) ELISA endpoint titers for SARS-CoV-2 RBD-specific IgG, IgA, and IgM in PCR confirmed acute COVID-19 patients (n = 44) and healthy controls collected in early 2019. Endpoint cutoff values were calculated using the average plus 3 standard deviations of the 32 healthy controls at 1/100 dilution (shown as a dotted line). (D) Representative ELISA assays for 10 patients and 12 healthy controls. (E) Direct comparison of IgM and IgG for individual donors. A number of the IgG negative or low early samples were IgM positive (shown in green). (F) Endpoint titer analysis of IgG subclass distribution. Each experiment was performed at least twice, and representative donors were selected to display the dynamic range observed in the dataset.

Figure 2

COVID-19 Patient Plasma Neutralizes SARS-CoV-2 (A) Neutralization activity of serum samples against SARS-CoV-2. The FRNT₅₀ titers of COVID-19 patients (n = 44) and healthy controls (n = 21) sera were determined by a FRNT assay using an immunostain to detect infected foci. Each circle represents one serum sample. The dotted line represents the maximum concentrations of the serum tested (1/50). (B) Representative sample showing a reduction in foci from a neutralization assay with sera from an infected COVID-19 patient. (C) Representative FRNT₅₀ curves were selected to display the dynamic range observed in the dataset (n = 22). The dotted line represents 50% neutralization. (D) Comparison of PRNT₅₀ against FRNT₅₀ titers (n = 9). Each experiment was performed at least twice, and a representative dataset is shown.

Figure 3

SARS-CoV-2 Antibody Responses Correlate with the Progression of Acute SARS-CoV-2 Infection Comparison of RBD-specific IgG titers and neutralization titers with (A and B) days after symptom onset or (C and D) days after PCR positive confirmation for each patient. Correlation analysis was performed by log transformation of the endpoint ELISA titers followed by linear regression analysis.

Figure 4

RBD-Specific Antibody Titers as a Surrogate of Neutralization Potency in Acutely Infected COVID-19 Patients (A) Comparison of RBD-specific IgG endpoint titers with SARS-CoV-2-specific FRNT₅₀ titers. Correlation analysis was performed by log transformation of the endpoint ELISA or FRNT₅₀ titers followed by linear regression analysis. (B) The RBD-specific ELISA was validated for high-throughput clinical testing in Emory Medical Laboratories. Sera (n = 231) were collected from COVID-19 patients within the first 22 days after PCR-confirmation (Table S1). Sera (n = 490) collected in 2019 were used as negative controls. ROC curves are shown comparing the true-positive and false-negative rates of the ELISA using different OD cutoffs and sera collected at different times post-infection. Whereas the RBD ELISA produced an area under the curve (AUC) of 0.89 when samples were collected close to the time of infection (within 3 days of positive PCR; n = 76), longer sampling times resulted in better performance. Assay performance was nearly perfectly discriminatory (AUC = 1.00) when samples were collected at least 7 days after the positive PCR (n = 83).