Afucosylated immunoglobulin G responses are a hallmark of enveloped virus infections and show an exacerbated phenotype in COVID-19

Abstract

27IgG antibodies are crucial for protection against invading pathogens. A highly conserved N-28 linked glycan within the IgG-Fc-tail, essential for IgG function, shows variable composition in 29 humans. Afucosylated IgG variants are already used in anti-cancer therapeutic antibodies for 30 their elevated binding and killing activity through Fc receptors (FcγRIIIa). Here, we report that 31 afucosylated IgG which are of minor abundance in humans (~6% of total IgG) are specifically 32 formed against surface epitopes of enveloped viruses after natural infections or 33 immunization with attenuated viruses, while protein subunit immunization does not elicit 34 this low fucose response. This can give beneficial strong responses, but can also go awry, 35 resulting in a cytokine-storm and immune-mediated pathologies. In the case of COVID-19, 36 the critically ill show aggravated afucosylated-IgG responses against the viral spike protein. In 37 contrast, those clearing the infection unaided show higher fucosylation levels of the anti-38 spike protein IgG. Our findings indicate antibody glycosylation as a potential factor in 39 inflammation and protection in enveloped virus infections including COVID-19.40Main Text: 41 Antibodies have long been considered functionally static, mostly determined by their isotype 42 and subclass. The presence of a conserved N-linked glycan at position 297, in the so called 43 constant Fc-domain of IgG, is essential for effector functions (1-3). Moreover, it is now 44 generally accepted that the composition of this glycan is highly variable and has functional 45 consequences (2-4). This is especially true for the core fucose attached to the Fc glycan. The 46 discovery that IgG variants without core fucosylation cause elevated antibody dependent 47 cellular cytotoxicity (ADCC), via increased IgG-Fc-receptor IIIa (FcγRIIIa) affinity (5, 6), 48 resulted in next-generation glyco-engineered monoclonal antibodies (mAb) without core 49 fucosylation for targeting tumors (7). 50Generally, changes in the Fc glycans are associated with age, sex and autoimmune diseases 51 (8). Serum IgG are highly fucosylated at birth and slightly decrease to ~94% fucosylation at 52 adulthood (9). Until now, no strong clues on how IgG core fucosylation is controlled have 53 come forward. 54 We have previously observed that alloantibodies against red blood cells (RBC) and platelets 55 show remarkably low IgG-Fc-fucosylation in most patients, even down to 10% in several 56 cases (10-12), whereas the overall serum IgG Fc-fucosylation show consistently normal high 57 levels. Moreover, we have reported the lowered IgG-Fc fucosylation to be one of the factors 58 determining disease severity in pregnancy associated alloimmunizations, resulting in 59 excessive thrombocytopenia's and blood cell destruction when targeted by afucosylated 60 antibodies (11)(12)(13). In addition to the specific afucosylated-IgG response against platelets 61 and RBC antigens, this response has also been identified against HIV and Dengue virus (...

Anti-SARS-CoV-2 IgG from severely ill COVID-19 patients promotes macrophage hyper-inflammatory responses

“…Previously, we and others have shown that anti-Spike IgG of severe COVID-19 patients has aberrant fucose and galactose expression, both compared to the total IgG within these individual patients, as well as compared to anti-Spike IgG from mild or asymptomatic patients (26,27). For a subset of COVID-19 serum samples in the present study, the glycosylation pattern of anti-Spike IgG1 had been determined previously for the study of Larsen et al (26), which showed significantly decreased fucosylation and increased galactosylation of anti-Spike IgG compared to total IgG within the tested patients (IgG glycosylation of the sera used in this study is depicted in Figure S2A). When plotting the percentage of anti-Spike IgG fucosylation against pro-inflammatory cytokine production, we observed an inverse correlation for IgG fucosylation and production of IL-1β, IL-6, and TNF ( Figure 3B), while this was not seen for IL-8 and IL-10 ( Figure S2B).…”

“…One of the key characteristics that determines IgG pathogenicity is glycosylation of the IgG Fc tail at position 297 (24,25). Previously, we and others have shown that anti-Spike IgG of severe COVID-19 patients has aberrant fucose and galactose expression, both compared to the total IgG within these individual patients, as well as compared to anti-Spike IgG from mild or asymptomatic patients (26,27). For a subset of COVID-19 serum samples in the present study, the glycosylation pattern of anti-Spike IgG1 had been determined previously for the study of Larsen et al (26), which showed significantly decreased fucosylation and increased galactosylation of anti-Spike IgG compared to total IgG within the tested patients (IgG glycosylation of the sera used in this study is depicted in Figure S2A).…”

“…In severely ill COVID-19 patients the glycosylation of anti-Spike IgG is changed, which we here show amplifies the IgG effector function to promote pro-inflammatory cytokine production, of which COVID-19-associated cytokines such as IL-1β, IL-6, and TNF (5,32) are most pronounced. Decreased IgG fucosylation, as observed in severe cases of COVID-19, has so far only been observed in patients infected with HIV and Dengue virus (33,34), but may actually be a general phenomenon in a response to enveloped viruses (26). While decreased fucosylation increases the infection of cells by a process known as antibody-dependent enhancement (ADE)(35), there is little evidence for antibody-enhanced infection in COVID-19 (36).…”

“…To determine the glycosylation of COVA1-18, aliquots of the mAb samples (5µg) were subjected to acid denaturation (100 mM formic acid, 5 minutes), followed by vacuum centrifugation. Subsequently, samples were trypsinized, and Fc glycopeptides were measured as described previously (26). Relative abundances of Fc glycopeptides were determined, and levels of bisection, fucosylation, galactosylation and sialylation were determined as described before (26).…”

“…Subsequently, samples were trypsinized, and Fc glycopeptides were measured as described previously (26). Relative abundances of Fc glycopeptides were determined, and levels of bisection, fucosylation, galactosylation and sialylation were determined as described before (26).…”