Fc-effector functional antibody assays for SARS-CoV-2 variants of concern

Abstract

Background: The Fc regions of antibodies mediate important effector cell functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and complement-dependent cytotoxicity (CDC). These functions enhance immune defense and infected cell clearance. This study evaluated the effect COVID-19 XBB.1.5 booster vaccination on Fc-effector antibody responses to SARS-CoV-2.

Methods: We developed four assays to evaluate the Fc-effector functions of SARS-CoV-2 antibodies. ADCC and CDC assays utilized stably transfected luciferase-based target cell lines expressing SARS-CoV-2 spike variants (Ancestral, Wu-1 Omicron XBB.1.5, and EG.5) to measure antibody-mediated lysis by effector cells. ADCP and ADNP were assessed by flow cytometry to measure phagocytosis of fluorescently labeled virus-like particles that display SARS-CoV-2 variant spike proteins. Serum samples from 20 healthy adult volunteers pre- and post-monovalent XBB.1.5 COVID-19 vaccine were analyzed for pseudovirus neutralizing and Fc-effector antibodies.

Results: Prior to administration of the COVID-19 XBB.1.5 booster vaccination, cross-neutralizing antibodies against XBB.1.5 and EG.5 variants were minimally detectable, while cross-functional Fc-effector antibodies were present at higher baseline levels. The COVID-19 XBB.1.5 booster vaccination significantly boosted both neutralizing and Fc-effector antibodies in magnitude and breadth. The greatest increase in neutralizing antibodies was against the XBB.1.5 strain, while Fc-effector functional antibodies had similar fold-increases in antibody titers against the breadth of SARS-CoV-2 variants tested. Neutralizing and Fc-effector antibodies were most highly correlated at baseline (prior to booster vaccination) but were less correlated post-vaccination, consistent with differential boosting of neutralizing vs Fc-effector antibodies by the monovalent vaccine.

Conclusion: The COVID-19 XBB.1.5 booster vaccination significantly improved the magnitude, breadth, and quality of antibody responses to SARS-CoV-2. Combining Fc-mediated functional and neutralizing antibody assays provides a more comprehensive model for understanding vaccine-induced immunity and optimizing vaccination strategies.

Keywords: ADCC; ADCP; ADNP; complement deposition; cytotoxicity; non-neutralizing antibodies; phagocytosis.

Figure 1

Characterization and recognition of HIV gag virus-like particles functionated with SARS-Cov-2 spike protein by SARS-CoV-2 polyclonal serum. (A) Schematic representation of the amino acid change in the spike protein of XBB.1.5 and EG.5 compared to ancestral variant Wu-1. (B) Flow cytometry analysis of G protein expression on the VLP production cell surface. (C) Western blot analysis of purified VLPs using COVID-19 vaccinated serum as primary antibody for detection of spike protein. Full lengths protein was used as control. (D) An example of flowcytometry analysis of Antibody-Dependent Cellular Phagocytosis (ADCP) activity (left). Microscopy images of THP1 cells demonstrate phagocytotic activity induced by COVID 19 vaccinated serum compares to healthy serum (right). (E) An example of flow cytometry analysis of Antibody-Dependent Neutrophile Phagocytosis (ADNP) activity (left). Microscopy images of HL60 cells demonstrate phagocytotic activity induced by COVID 19 vaccinated serum compares to healthy serum (right).

Figure 2

Representative data of Fc effector and pseudovirus neutralizing antibody responses in serially diluted sera from a participant (#8) with pre- and post-XBB1.5 booster serum samples, from two donors collected prior to the COVID-19 pandemic with an unknown history of coronavirus infection (pre-pandemic) noted as healthy serum #1 and #2, and from two reference sera samples obtained from BEI Resources (NRH-28557 and NRH-28563) for (A) ADCP and ADNP, (B) ADCC and CDC, and (C) pseudovirus neutralizing antibody responses against SARS-CoV-2 variant spike proteins (Ancestral Wu-1, Omicron XBB.1.5, and EG.5). The red dashed line indicates 50% reduction in viral infection in neutralization assay, and cut-off was calculated from 12 serum samples collected prior to the COVID-19 pandemic in ADCC and CDC.

Figure 3

SARS-CoV-2-specific antibody responses against Wu-1, XBB.1.5, and EG.5 in adults pre- and post-COVID-19 XBB.1.5 booster vaccination. (A) SARS-CoV-2 S protein–specific neutralization IC50 titers were determined using a pseudotyped virus neutralization assay. (B) Functional antibody-dependent cell-mediated cytotoxicity (ADCC) responses were evaluated, measuring the capacity of antibodies to mediate the destruction of target cells by natural killer cells. (C) Antibody-dependent cellular phagocytosis (ADCP) activity and (D) antibody-dependent neutrophil phagocytosis (ADNP) activity were assessed, indicating the ability of antibodies to promote phagocytosis of fluorescent virus-like particles by phagocytes and neutrophils. (E) Complement-dependent cytotoxicity (CDC) responses were measured, reflecting the ability of antibodies to induce complement-mediated lysis of target cells. Statistical comparisons were made using paired T-tests (**P < 0.01, ***P < 0.001, ****P < 0.0001). The fold changes between pre- and post-COVID-19 XBB.1.5 booster vaccination are shown in brackets. F to J show the individual spike-specific neutralization IC50 titers (F), ADCC end-point titers (G), ADCP phagocytosis percentage (H), ADNP phagocytosis percentage (I) and CDC end-point titers (J) against Wu-1, XBB.1.5 and EG.5 pre- and post-COVID-19 XBB.1.5 booster vaccination.

Figure 4

Pearson’s correlation matrix analysis was performed to analyze the correlation between different functional antibody responses against SARS-CoV-2 variants Wu-1, XBB.1.5, and EG.5 pre- and post-COVID-19 XBB.1.5 booster vaccination. The colored squares show the correlation coefficient between two variables, with darker blue color representing a stronger correlation. The red asterisks indicate the level of statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001).

Figure 5

(A) Heatmap analysis of pseudovirus neutralizing antibody and Fc effector functional antibody responses against SARS-CoV-2 variants Wu-1, XBB.1.5, and EG.5 pre- and post-COVID-19 XBB.1.5 booster vaccination. Data was normalized in each assay by defining the smallest value as equal to 0% and the largest value equal to 100%. (B, C) Principal component analysis (PCA) was conducted on pseudovirus neutralization antibody and Fc effector functional antibody responses pre- and post-COVID-19 XBB.1.5 booster vaccination. (B) highlights the variables contributing to the spread of the point. (C) shows individual plot with the pre-booster serum in blue and the post-booster serum in red.