Natural killer cell-mediated ADCC in SARS-CoV-2-infected individuals and vaccine recipients

Abstract

COVID-19, caused by SARS-CoV-2, has emerged as a global pandemic. While immune responses of the adaptive immune system have been in the focus of research, the role of NK cells in COVID-19 remains less well understood. Here, we characterized NK cell-mediated SARS-CoV-2 antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 spike-1 (S1) and nucleocapsid (NC) protein. Serum samples from SARS-CoV-2 resolvers induced significant CD107a-expression by NK cells in response to S1 and NC, while serum samples from SARS-CoV-2-negative individuals did not. Furthermore, serum samples from individuals that received the BNT162b2 vaccine induced strong CD107a expression by NK cells that increased with the second vaccination and was significantly higher than observed in infected individuals. As expected, vaccine-induced responses were only directed against S1 and not against NC protein. S1-specific CD107a responses by NK cells were significantly correlated to NK cell-mediated killing of S1-expressing cells. Interestingly, screening of serum samples collected prior to the COVID-19 pandemic identified two individuals with cross-reactive antibodies against SARS-CoV-2 S1, which also induced degranulation of NK cells. Taken together, these data demonstrate that antibodies induced by SARS-CoV-2 infection and anti-SARS-CoV-2 vaccines can trigger significant NK cell-mediated ADCC activity, and identify some cross-reactive ADCC-activity against SARS-CoV-2 by endemic coronavirus-specific antibodies.

Keywords: ADCC; COVID-19; Innate immunity; NK cells; Vaccine.

Figure 1

Antibodies against the SARS‐CoV‐2 spike 1 (S1) and nucleocapsid (NC) protein induce NK cell degranulation. (A) Spike and NC antibody titers were determined by Ig sandwich assays. Results in confirmed COVID‐19 resolvers were compared to serum samples of healthy individuals. Squared data points represent results outside the assay detection limit (3.8 and 400 AU/mL). Cut off for positive results was set to 15 AU/mL and 1 COI. (B) Statistical comparison of CD107a expression by NK cells in response to SARS‐CoV‐2 S1 and NC protein‐directed serum antibodies between confirmed SARS‐CoV‐2 resolvers (n = 15) and SARS‐CoV‐2‐negative (n = 15) individuals. The experimental setup contained duplicates and triplicates and median values including the IRQ are shown. For resolver and healthy individuals, replicates were combined to single data points. Statistical analysis was performed using nonparametric Mann–Whitney test (****p < 0.0001).

Figure 2

Anti‐SARS‐CoV‐2 cross‐reactive antibodies found in pre‐COVID‐19 serum are capable of inducing NK cell degranulation. (A) Anti‐SARS‐CoV‐2 Spike 1 ELISA confirmed cross‐reactive IgG antibodies in two pre‐COVID‐19 collected serum samples, with decreasing antibody titers over time. Dotted lines mark the distributer's range for negative (0.8) and positive (1.1) results (n = 85). (B) Pre‐COVID‐19 cross‐reactive antibodies stimulated CD107a expression by NK cells. Dotted lines mark the mean value and the gray area represents the range of results observed in SARS‐CoV‐2‐negative serum samples. Experiments were performed using triplicates and replicates were combined into single data points.

Figure 3

BNT162b2 vaccine‐induced SARS‐CoV‐2 spike 1‐directed serum antibodies trigger NK cell degranulation. (A) Longitudinal antibody titers against spike, RBD and NC were determined by Ig sandwich assays in BNT162b2‐vaccinated health‐care workers (n = 10). (B) CD107a expression by NK cells in response to SARS‐CoV‐2 S1 and NC protein‐directed serum antibodies in vaccinated individuals compared to SARS‐CoV‐2 resolvers (n = 3) and healthy control individuals (n = 3). Samples of vaccinated individuals were each collected on the day of the first vaccination (week 0), the day of the second vaccination (week 3), and 2 weeks after the second vaccination (week 5). As CD107a levels remain constant between weeks 3 and 5, a selection of donors (n = 3, marked in red) was titrated in a follow‐up CD107a degranulation assay. The experimental setup contained triplicates and median values and IRQ are shown. For resolver, healthy, and vaccinated individuals, replicates were combined into single data points. Statistical analysis was performed using nonparametric Mann–Whitney test (****p < 0.0001). (C) CD107a expression by NK cells after serial dilution (1:20, 1:50, 1:100, 1:500, 1:1000) of vaccinated (n = 3), SARS‐CoV‐2 resolver (n = 2), and SARS‐CoV‐2‐negative (n = 1) serum samples.

Figure 4

SARS‐CoV‐2‐RBD‐directed antibodies induce ADCC‐mediated target cell killing by NK cells. (A) RBD expression was determined in mock‐ and RBD‐nucleofected Raji cells. In addition, binding capability of SARS‐CoV‐2‐directed antibodies toward the RBD expressed on nucleofected Raji cells was analyzed in serum samples of SARS‐CoV‐2 resolver and healthy individuals. (B) RBD‐mediated antibodies induce CD107a expression in SARS‐CoV‐2 resolvers (n = 3) and vaccinated individuals (n = 3) at 3 and 5 weeks after their initial vaccination. Raji cells alone induce low CD107a‐expression levels comparable to those seen in healthy individuals (n = 3) and on the day of the first vaccination (week 0, n = 3). Replicates of the same serum donor are indicated by shape and color. (C) Killing of target cells increased after incubation with serum of SARS‐CoV‐2 resolver and vaccinated (weeks 3 and 5) individuals, while it remains low in healthy individuals. Replicates of the same serum donor are indicated by shape and color. (D) CD107a expression by NK cells shows a positive correlation to target cell killing. Mean value of replicates is shown. A linear regression was calculated and statistical analysis was performed using nonparametric Mann–Whitney test.