Multiple Routes of Antibody-Dependent Enhancement of SARS-CoV-2 Infection

Abstract

Antibody-dependent enhancement (ADE) of infection is generally known for many viruses. A potential risk of ADE in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has also been discussed since the beginning of the coronavirus disease 2019 (COVID-19) pandemic; however, clinical evidence of the presence of antibodies with ADE potential is limited. Here, we show that ADE antibodies are produced by SARS-CoV-2 infection and the ADE process can be mediated by at least two different host factors, Fcγ receptor (FcγR) and complement component C1q. Of 89 serum samples collected from acute or convalescent COVID-19 patients, 62.9% were found to be positive for SARS-CoV-2-specific IgG. FcγR- and/or C1q-mediated ADE were detected in 50% of the IgG-positive sera, whereas most of them showed neutralizing activity in the absence of FcγR and C1q. Importantly, ADE antibodies were found in 41.4% of the acute COVID-19 patients. Neutralizing activity was also detected in most of the IgG-positive sera, but it was counteracted by ADE in subneutralizing conditions in the presence of FcγR or C1q. Although the clinical importance of ADE needs to be further investigated with larger numbers of COVID-19 patient samples, our data suggest that SARS-CoV-2 utilizes multiple mechanisms of ADE. C1q-mediated ADE may particularly have a clinical impact since C1q is present at high concentrations in plasma and its receptors are ubiquitously expressed on the surfaces of many types of cells, including respiratory epithelial cells, which SARS-CoV-2 primarily infects. IMPORTANCE Potential risks of antibody-dependent enhancement (ADE) in the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been discussed and the proposed mechanism mostly depends on the Fc gamma receptor (FcγR). However, since FcγRs are exclusively expressed on immune cells, which are not primary targets of SARS-CoV-2, the clinical importance of ADE of SARS-CoV-2 infection remains controversial. Our study demonstrates that SARS-CoV-2 infection induces antibodies that increase SARS-CoV-2 infection through another ADE mechanism in which complement component C1q mediates the enhancement. Although neutralizing activity was also detected in the serum samples, it was counteracted by ADE in the presence of FcγR or C1q. Considering the ubiquity of C1q and its cellular receptors, C1q-mediated ADE may more likely occur in respiratory epithelial cells, which SARS-CoV-2 primarily infects. Our data highlight the importance of careful monitoring of the antibody properties in COVID-19 convalescent and vaccinated individuals.

Keywords: ADE; C1q; COVID-19; Fc receptor; FcR; SARS-CoV-2; antibody-dependent enhancement; complement.

FIG 1

SARS-CoV-2-specific IgG, IgM, and neutralizing antibodies detected in serum samples collected from COVID-19 acute and convalescent patients. (A) IgG and (B) IgM antibodies reactive to the SARS-CoV-2 whole virus antigens were measured in ELISA. (A, B) The cutoff values (dashed lines) were determined as averages ± 3×SD of the OD values of healthy volunteers. (C) Neutralizing titers and ELISA OD values of the samples are plotted with a regression line (dotted line). The detection limit of neutralizing titers was 10 (reciprocal dilution) as shown by the dashed line. Samples below the limit of detection are not shown (C, right panel). Each box with a horizontal black line represents the IQR and median. Symbols represent outlying plots located over 1.5 × IQR from the upper quartile. Whiskers are shown from the highest and lowest values within a fence to the 3rd and 1st quartiles, respectively. Asterisks indicate significant differences (P < 0.05) determined using the Kruskal-Wallis test followed by Dunn’s multiple-comparison test.

FIG 2

ADE mechanisms and assay validation for FcγR- and C1q-mediated ADE using VSV-EBOV and an EBOV glycoprotein-specific monoclonal antibody. Infectious titers of VSV-EBOV mixed with indicated concentrations of monoclonal antibody ZGP12/1.1 (27) were measured in (A) Vero E6/FcγRIIa cells, (B) Vero E6 cells in the presence of C1q, and (C) Vero E6 cells in the absence of C1q. The relative numbers of infected cells were calculated by setting the number of GFP-positive cells in the absence of the antibody to 100%. Dots and error bars indicate the means and standard deviations of triplicate wells, respectively. Right panels show schematics of the respective conditions.

FIG 3

Neutralization and ADE activities found in serum samples against pseudotyped virus. Fifty representative serum samples from COVID-19 convalescents (numbers 26–46), acute patients with mild (numbers 48, 51, 56, 57, 60, 62, 67, and 70), moderate (numbers 74, 76, 80–82, 84–87, 91, 92, 94, and 95), and severe (numbers 96–98, 101, 105, 108, 111, and 114) symptoms were investigated. The relative numbers of infected cells were calculated by setting the number of GFP-positive cells in the absence of the serum to 100%. Each experiment was done twice and results are shown as means and standard deviations. ADE-positive sample numbers are shown in red.

FIG 3

Neutralization and ADE activities found in serum samples against pseudotyped virus. Fifty representative serum samples from COVID-19 convalescents (numbers 26–46), acute patients with mild (numbers 48, 51, 56, 57, 60, 62, 67, and 70), moderate (numbers 74, 76, 80–82, 84–87, 91, 92, 94, and 95), and severe (numbers 96–98, 101, 105, 108, 111, and 114) symptoms were investigated. The relative numbers of infected cells were calculated by setting the number of GFP-positive cells in the absence of the serum to 100%. Each experiment was done twice and results are shown as means and standard deviations. ADE-positive sample numbers are shown in red.

FIG 4

Comparison of optimal ADE conditions between serum samples with high and low neutralizing activities. Data shown in Fig. 3 are reconstituted for the comparison of FcγR-mediated ADE (A), C1q-mediated ADE (B), and neutralization (C) curves of each sample. The ADE-positive serum samples are divided into 2 groups based on their neutralizing titers; high (1:320–1:2560) and low (<1:160). Serum samples from convalescents, patients with mild, moderate, and severe symptoms are shown by circles, squares, triangles, and diamonds, respectively.

FIG 5

Correlation between IgG/IgM antibody levels and ADE activities. Serum samples that showed FcR- and/or C1q-mediated ADE activities (numbers 29, 32, 33, 34, 35, 36, 37, 41, 42, 43, 44, 45, 46, 60, 62, 67, 70, 76, 81, 84, 85, 86, 97, 105, and 111) were analyzed. ELISA OD values and peak relative infectivity (%) were obtained from the data shown in Fig. 1 and 3, respectively.

FIG 6

ADE and reduced neutralization of SARS-CoV-2 by serum samples of COVID-19 patients. SARS-CoV-2 strain JPN/TY/WK-521 was inoculated into Vero E6 and Vero E6/FcγRIIa cells and grown in the presence of serum samples of numbers 41, 45, 46, 62, 111, and a negative control (NC) diluted at 1280, 320, 1280, 320, 640, and 320, respectively. For C1q-mediated ADE, the virus was grown in Vero E6 cells with the sera and the medium supplemented with C1q during the incubation. The data are shown as means and standard deviations of three independent experiments. Significant differences (P < 0.05) compared to the serum (-) in the respective conditions (i.e., Vero E6/FcγRIIa, Vero E6 + C1q, or Vero E6) were determined using one-way ANOVA followed by Dunnett's multiple-comparison test and are shown with asterisks. Daggers indicate significant differences (P < 0.05) between the 2 indicated groups determined using Student's t test.