Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-19 Subjects

Abstract

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. Anti-SARS-CoV-2 immunoglobulin (Ig)G/A/M antibodies against spike (S) protein and its receptor-binding domain (RBD) were characterized in recovered subjects who were RT-PCR-positive (n = 153) and RT-PCR-negative (n = 55) using an enzyme-linked immunosorbent assay (ELISA). These antibodies were also further assessed for their ability to neutralize live SARS-CoV-2 virus. Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r = 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values > 27. These high Ct values raise the possibility that these indeterminate results are from individuals who were not infected or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR.

Keywords: COVID-19; SARS-CoV-2; antibody; serology.

Figure 1

Ct values were variable in resolved subjects who test SARS-CoV-2 antibody negative or positive. Ct values of a subset of resolved subjects (n = 54) were compared to their corresponding SARS-CoV-2 (A) anti-S protein IgG and (B) anti- receptor-binding domain (RBD) IgG. (C) Ct values were then compared to the subjects’ respective time since initial RT-PCR test. (D) Ct values for RT-PCR-positive in antibody-positive and antibody-negative were compared. Values are shown as a ratio of observed optical density to the determined assay cut-off optical density or time since RT-PCR test until blood donation compared to absolute Ct values. Red circles indicate resolved samples who were RT-PCR-positive/antibody-negative.

Figure 2

Persistence of SARS-CoV-2 antibodies in resolved subjects based on time of blood collection post-symptom onset. Resolved subjects (n = 153) were grouped based on days post-symptom onset showing levels of (A) anti-S protein and anti-RBD IgG, (B) IgA, and (C) IgM displayed as dot plots. Days post-symptom onset were binned in 60-day increments and are compared to asymptomatic resolved subjects. Asymptomatic subjects were collected between 27 and 160 days after their RT-PCR test. Circles represent anti-S protein antibodies and squares represent anti-RBD antibodies. Anti-S protein IgG was found in all resolved participants collected between 60 and 120 days (23 of 153 total resolved subjects). In the resolved patients collected between 120 and 180 days from symptom onset, there was a decrease in the percentage of antibody-positive samples when compared to the previous time bin (55 of 60 samples, 91.7%).

Figure 3

Neutralizing SARS-CoV-2 IgG, IgA, and IgM antibodies against S protein and RBD were found in variable levels in resolved and RT-PCR-negative study participants. Neutralizing SARS-CoV-2 antibody titers from resolved subjects (n = 153) were measured in the microneutralization assay and compared to (A) anti-S protein and (B) anti-RBD IgG antibody levels as measured in the SARS-CoV-2 ELISA. Neutralizing antibody titers are expressed as geometric MNT50 values (y-axis). ELISA values are shown as a ratio of observed optical density to the determined assay cut-off optical density (x-axis). (C) Neutralizing SARS-CoV-2 antibody titers from resolved subjects (n = 153) were measured in the microneutralization assay and were compared to their days since symptom onset. X-axis is expressed in log₁₀. Values above 1 ratio are considered positive in the SARS-CoV-2 ELISA.

Figure 4

Comparing anti-SARS-CoV-2 IgG, IgA, and IgM responses to S protein and RBD antigens in asymptomatic, non-hospitalized, and hospitalized resolved subjects. (A) Anti-S protein IgG, IgA, IgM, and (B) anti-RBD IgG, IgA, and IgM of the asymptomatic (n = 11), non-hospitalized resolved subjects (n = 134) and hospitalized resolved subjects (n = 8) were profiled using the SARS-CoV-2 ELISA. Values are shown as a ratio of observed optical density to the determined assay cut-off optical density. All hospitalized subjects had detectable anti-S protein IgG, anti-RBD IgG, and anti-S protein IgA antibodies in their serum. The levels of anti-S protein and anti-RBD IgG (mean OD_405nm ratio) in resolved subjects who were hospitalized were significantly higher than the non-hospitalized resolved population. Values above 1 ratio are considered positive in the SARS-CoV-2 ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0005.