Altered spike IgG Fc N-linked glycans are associated with hyperinflammatory state in adult COVID and Multisystem Inflammatory Syndrome in Children

Abstract

Background: Severe COVID and multisystem inflammatory syndrome (MIS-C) are characterized by excessive inflammatory cytokines/chemokines. In adults, disease severity is associated with SARS-CoV-2-specific IgG Fc afucosylation, which induces pro-inflammatory cytokine secretion from innate immune cells. This study aimed to define spike IgG Fc glycosylation following SARS-CoV-2 infection in adults and children and following SARS-CoV-2 vaccination in adults and the relationships between glycan modifications and cytokine/chemokine levels.

Methods: We analyzed longitudinal (n=146) and cross-sectional (n=49) serum/plasma samples from adult and pediatric COVID patients, MIS-C patients, adult vaccinees, and adult and pediatric healthy controls. We developed methods for characterizing bulk and spike IgG Fc glycosylation by capillary electrophoresis (CE) and measured levels of ten inflammatory cytokines/chemokines by multiplexed ELISA.

Results: Spike IgG were more afucosylated than bulk IgG during acute adult COVID and MIS-C. We observed an opposite trend following vaccination, but it was not significant. Spike IgG were more galactosylated and sialylated and less bisected than bulk IgG during adult COVID, with similar trends observed during pediatric COVID/MIS-C and following SARS-CoV-2 vaccination. Spike IgG glycosylation changed with time following adult COVID or vaccination. Afucosylated spike IgG exhibited inverse and positive correlations with inflammatory markers in MIS-C and following vaccination, respectively; galactosylated and sialylated spike IgG inversely correlated with pro-inflammatory cytokines in adult COVID and MIS-C; and bisected spike IgG positively correlated with inflammatory cytokines/chemokines in multiple groups.

Conclusions: We identified previously undescribed relationships between spike IgG glycan modifications and inflammatory cytokines/chemokines that expand our understanding of IgG glycosylation changes that may impact COVID and MIS-C immunopathology.

Keywords: COVID; IgG; MIS-C; antibody; glycosylation; inflammation.

Figure 1:. Schematic of IgG structure with conserved N-linked complex glycan at residue 297.

IgG is composed of the antigen binding domains, F(ab)₂, and constant domain, Fc. The Fc has a highly conserved complex N-glycan at Asn297, which is composed of a core with seven GlcNAc and mannose sugars and additional, variable modifying sugars.

Figure 2:. Proinflammatory cytokines and chemokines are significantly elevated in adult COVID patients and MIS-C patients compared to healthy controls.

Differences in IL-10, IP-10, IL-8, IFNγ, IL-6, and MIP-1α levels between adult healthy controls, which includes vaccinees at baseline, patients enrolled in ACTT-1 with different COVID severities by ordinal scale (OS) at enrollment (V1), or vaccinees (7 days post-second dose, 7d psd); as well as between pediatric healthy controls or pediatric patients with SARS-CoV-2 or MIS-C. Kruskal-Wallis with Dunn’s test (* p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001)

Figure 3:. Spike-coated beads capture anti-spike IgG from purified bulk IgG of a seropositive participant.

A) After treatment three times with His Dynabeads coated with 0 ug (red) or 12 ug (blue) SARS -CoV-2 spike protein, depleted bulk IgG were analyzed for spike binding by ELISA alongside the purified bulk IgG B) Measurement of spike binding by post-enrichment IgG eluates from His Dynabeads coated with 0 ug (red) or 12 ug (blue) spike.

Figure 4:. Afucosylated anti-spike IgG are significantly increased in severe COVID and MIS-C over bulk and healthy controls, while anti-spike IgG is globally more sialylated and less bisected than bulk or healthy control IgG.

Kruskal-Wallis with Dunn’s test (* p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001).`

Figure 5:. Changes in IgG Fc-glycans between acute infection and convalescence in severe COVID patients.

Differences in afucosylated, sialylated, galacosylated, or bisected bulk (black) or anti-spike (red) IgG Fc glycan abundances between acute infection and convalescence in paired serum samples of adults with severe COVID (ordinal scale [OS] 5 and 7), who received either remdesivir or placebo. Wilcoxon matched-pairs signed rank test (* p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001)

Figure 6:. Inverse correlations observed between afucosylated, sialylated, or galactosylated glycan abundances and pro-inflammatory cytokine levels in adult COVID and MIS-C patients are not observed in vaccinees.

Spearman correlation coefficients were estimated between serum cytokine/chemokine levels and spike IgG glycan abundances in moderate (OS 4) and severe (OS5/7) COVID at enrollment (V1), mRNA vaccine recipients at 7 days after second vaccine dose (7d psd), and MIS-C patients. Gradient from blue to red indicates strength of positive or inverse correlation, respectively. Significant relationships are indicated by asterisks (* p<0.05, ** p<0.01, *** p<0.001).