Functional diversification of innate and inflammatory immune responses mediated by antibody fragment crystallizable activities against SARS-CoV-2

Abstract

Monoclonal antibodies (mAb) targeting the SARS-CoV-2 Spike (S) glycoprotein have been exploited for the treatment of severe COVID-19. In this study, we evaluated the immune-regulatory features of two neutralizing anti-S mAbs (nAbs), named J08 and F05, with wild-type (WT) conformation or silenced Fc functions. In the presence of D614G SARS-CoV-2, WT nAbs enhance intracellular viral uptake in immune cells and amplify antiviral type I Interferon and inflammatory cytokine and chemokine production without viral replication, promoting the differentiation of CD16⁺ inflammatory monocytes and innate/adaptive PD-L1⁺ and PD-L1⁺CD80⁺ plasmacytoid Dendritic Cells. In spite of a reduced neutralizing property, WT J08 nAb still promotes the IL-6 production and differentiation of CD16⁺ monocytes once binding Omicron BA.1 variant. Fc-mediated regulation of antiviral and inflammatory responses, in the absence of viral replication, highlighted in this study, might positively tune immune response during SARS-CoV-2 infection and be exploited also in mAb-based therapeutic and prophylactic strategies against viral infections.

Keywords: Immune response; Immunology; Virology.

Graphical abstract

Figure 1

Inflammatory cytokine and chemokine production in PBMC treated with D614G virus in the presence of J08 nAbs (A) Schematic representation of the in vitro experimental setting, culturing conditions and read-outs used in this study. Peripheral blood mononuclear cells (PBMC) and isolated CD14⁺ monocytes or BDCA4⁺ plasmacytoid dendritic cells (pDC) were collected from buffy coats of healthy volunteers. Cells were adsorbed for 1 hour (h) with D614G SARS-CoV-2 at 0.04 multiplicity of infection alone or after opsonization for 1 h with two classes of anti-SARS-CoV-2 Spike neutralizing IgG1 antibodies (nAbs) (i.e., J08 and F05), then cells were washed, and new complete medium added for cell culture. Cells were then analyzed at 24 or 72 h post-infection (hpi). J08 and F05 nAbs were used as wild-type version (WT), as a version engineered in the Fc receptor binding domain (MUT), as well as a version truncated in the complete constant Fc region (Fab). In particular, D614G SARS-CoV-2 was opsonized with three increasing doses of J08 and F05 nAbs in the WT, MUT, and Fab versions: one-tenth of the virus neutralizing dose assessed for both the nAbs at a concentration of 7.81 ng nAb/mL for 100 TCID₅₀ (Dose N/10), the neutralizing dose itself (Dose N) and twice the neutralizing dose (Dose 2N). (B‒E) PBMC isolated from healthy volunteers (n = 8) were left untreated (not stimulated, ns) or stimulated with D614G SARS-CoV-2 at a multiplicity of the infection of 0.04 alone or in the presence of J08 nAbs in the WT, MUT, and Fab versions. Production of interferon (IFN)-αs (B), as well as of the inflammatory cytokines interleukin (IL)-6 (C), tumor necrosis factor (TNF)-α (D) and the chemokine IL-8 (E) were measured in culture supernatants harvested at 24 (B) or 72 hpi (C‒E). p-values were calculated by two-way ANOVA and assigned as follows: ∗ ≤0.05; ∗∗ ≤0.01.

Figure 2

FcγR crosslinking with J08 nAb immune complexes and Fc-mediated viral entry in PBMC (A) Schematic representation of the in vitro experimental setting used to mimc FcγR crosslinking. Biotynilated recombinant trimeric Spike (B-Spike) protein was coupled to streptavidin (SA)-labeled magnetic beads. Immune complexes (IC) were formed between anti-SARS-CoV-2 Spike neutralizing IgG1 antibody J08 in the wild-type (WT) version at the neutralizing dose and trimeric Spike coupled to SA-magnetic beads. IC were added to peripheral blood mononuclear cells (PBMC) to crosslink Fcγ Receptors (FcγR) on different FcγR-expressing PBMC subsets. (B‒E) PBMC isolated from healthy volunteers (n = 3) were left untreated (not stimulated, ns) or stimulated with J08 WT neutralizing antibody (nAb) and biotinylated recombinant (rec) trimeric Spike protein alone or coupled to SA-magnetic beads. As positive control biotynilated goat anti-human CD16, CD32 and CD64 antibodies (anti-FcγR Abs) were also coupled to SA-magnetic beads and then mixed together in equal quantity to crosslink all the FcγR expressed by PBMC subsets. Biotynilated goat anti-human immunoglobulin G (Isotype Ab) coupled to SA-magnetic beads was used as a negative control. Cells were also stimulated with the toll-like receptor (TLR)-7/8 agonist R848 and the TLR-9 agonist class C CpG. Production of interferon-alphas (IFN-αs) (B), interleukin (IL)-6 (C), tumor necrosis factor (TNF)-α (D) and IL-8 (E) was measured in culture supernatants. (F and G) PBMC were adsorbed for 1 h with virus-like particles (VLP) expressing green-fluorescent protein (GFP) pseudotyped with membrane-tethered SARS-CoV-2 (D614G) Spike (S) protein alone or immunocomplexed with J08 nAbs in the WT or MUT versions. (F) Representative dot plots of the different experimental conditions derived from 1 experiment, out of 3 independently performed, are shown. (G) Results shown are the mean values of the percentage (%) of GFP⁺ cells in total single live PBMC derived from the 3 experiments independently performed. p-values were calculated by two-way ANOVA and assigned as follows: ∗ ≤0.05; ∗∗∗∗ ≤0.0001.

Figure 3

Regulation of cytokine release and immunophenotype in plasmacytoid dendritic cells treated with D614G in the presence of J08 nAbs BDCA4⁺ plasmacytoid dendritic cells (pDC), purified from peripheral blood mononuclear cells (PBMC) of healthy volunteers (n = 4), were left untreated (not stimulated, ns) or stimulated with D614G virus at a multiplicity of infection of 0.04 alone or in the presence of the neutralizing dose of the anti-SARS-CoV-2 Spike neutralizing IgG1 antibody J08 in the wild-type (WT), Fc mutated (MUT) and Fc truncated (Fab) versions. Cells were harvested at 24 h post-infection (hpi). (A‒D) Production of interferon-alphas (IFN-αs) (A) and the inflammatory cytokines interleukin (IL)-6 (B) and tumor necrosis factor (TNF)-α (C) as well as of the chemokine IL-8 (D) were measured in culture supernatants. (E‒G) PDC were studied by flow cytometry for cell subset differentiation. In particular, innate P1-pDC (PDL1⁺CD80⁻, in blue), innate/adaptive P2-pDC (PDL1⁺CD80⁺, in red), adaptive P3-pDC (PDL1⁻CD80⁺, in green) as well as resting P4-pDC (PDL1⁻CD80⁻, in gray) were analyzed. (E) Representative dot plots at the different experimental conditions displaying different pDC subsets derived from 1 experiment, out of 4 independently performed, are shown. (F) Results shown in the pie charts are mean values of the percentage (%) of the different pDC subsets analyzed in the parental single-live BDCA4⁺-gated cells in the 4 experiments independently performed. (G) Surface expression of the costimulatory marker CD86 as well as of the Fc receptors CD32 and CD64 was determined as mean fluorescence intensity (MFI) by cytofluorimetric analysis in single-live BDCA4⁺ pDC. Results were shown as median values ± Interquartile range of 4 independent experiments. p-values were calculated by two-way ANOVA and assigned as follows: ∗ ≤0.05.

Figure 4

Regulation of cytokine release and immunophenotype in monocytes treated with D614G in the presence of J08 nAbs CD14⁺ monocytes, purified from peripheral blood mononuclear cells (PBMC) of healthy volunteers (n = 4), were left untreated (not stimulated, ns) or stimulated with D614G virus at a multiplicity of infection of 0.04 alone or upon opsonization with the neutralizing dose of the anti-SARS-CoV-2 Spike neutralizing IgG1 antibody J08 in the wild-type (WT), Fc mutated (MUT) and Fc truncated (Fab) versions. Cells were harvested at 24 and 72 h post-infection (hpi). (A‒C) Production of the inflammatory cytokines interleukin (IL)-6 (A), tumor necrosis factor (TNF)-α (B) as well as of the chemokine IL-8 (C) were measured in culture supernatants. (D‒F) Monocytes were studied by flow cytometry for cell subset differentiation. In particular, classical [CD14^hiCD16^neg] monocytes are indicated in blue, inflammatory [CD14⁺CD16^int] monocytes in aqua blue, and atypical [CD14^low-negCD16^hi] monocytes in red. (D) Representative dot plots at the different experimental conditions displaying different monocyte subsets derived from 1 experiment, out of 4 independently performed, are shown. (E) Results shown in the pie charts are mean values of the percentage (%) of the different monocyte subsets analyzed in the parental single-live CD14⁺-gated cells in the 4 experiments independently performed. (F) Surface expression of the costimulatory marker CD86 as well as of the Fc receptors CD16, CD32, and CD64 was determined as mean fluorescence intensity (MFI) by cytofluorimetric analysis in single-live CD14⁺ monocytes. Results were shown as median values ± Interquartile range of 4 independent experiments. p-values were calculated by two-way ANOVA and assigned as follows: ∗ ≤0.05.

Figure 5

Impact of Omicron BA.1 infection on monocyte-driven inflammatory response in the presence of J08 nAbs Peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers (n = 4) were left untreated (not stimulated, ns) or stimulated for 24 or 72 h with Omicron BA.1 virus at a multiplicity of infection of 0.04 alone or upon opsonization with the neutralizing dose (N) of the anti-SARS-CoV-2 Spike neutralizing IgG1 antibody J08 (nAb) in the wild-type (WT), Fc mutated (MUT) and Fc truncated (Fab) versions. (A) A schematic representation of experimental settings is depicted. (B) Production of the inflammatory cytokine interleukin (IL)-6 was measured in culture supernatants harvested at 72 h post-infection. Results were shown as median values ± Interquartile range of 4 independent experiments. p-values were calculated by two-way ANOVA and assigned as follows: ∗ ≤0.05; ∗∗ ≤0.01. (C) Cells were harvested at 72 h post-infection and studied by flow cytometry for monocyte subset differentiation. In particular, classical [CD14^hiCD16^neg] monocytes are indicated in blue, inflammatory [CD14⁺CD16^int] monocytes in aqua blue, and atypical [CD14^low-negCD16^hi] monocytes in red. Results shown in the pie charts are mean values of the percentage (%) of the different monocyte subsets analyzed in the parental single-live CD14⁺-gated cells in the 4 experiments independently performed.