IgG antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anti-cancer therapeutic antibodies for their elevated activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (~6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses, but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high levels of afucosylated IgG antibodies against SARS-CoV-2, amplifying pro-inflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.
Antibody function has long been considered static and mostly determined by their isotype and subclass. The presence of a conserved N-linked glycan at position 297 in the Fc domain of IgG, is essential for its effector functions (1–3). Moreover the composition of this glycan is highly variable, which has functional consequences (2–4). This is especially true for the core fucose attached to the Fc glycan. The discovery that IgG variants without core fucosylation cause elevated antibody-dependent cellular cytotoxicity (ADCC), via increased IgG-Fc receptor IIIa (FcγRIIIa) affinity (5, 6), has resulted in next-generation glyco-engineered monoclonal antibodies (mAb) lacking core fucosylation for targeting tumors (7).
Generally, changes in the Fc glycans are associated with age, sex, and autoimmune diseases, and are most pronounced for IgG-Fc galactosylation, which decreases steadily with advancing age. After a marked elevation in young women, IgG-Fc galactosylation decreases during menopause to the levels seen in men (8). IgG-Fc fucosylation is more stable, decreasing slightly from birth to approximately 94% at adulthood (9), after which it remains fairly constant, albeit with a minor reduction throughout life (8, 10).
Despite the apparent constant level of Fc fucosylation during adulthood, alloantibodies against red blood cells (RBCs) and platelets show remarkably low IgG-Fc fucosylation in most patients, even down to 10% in several cases (11–13). By contrast, overall serum IgG-Fc fucosylation is consistently high. Moreover, lowered IgG-Fc fucosylation is one of the factors determining disease severity in pregnancy-associated alloimmunizations, resulting in excessive thrombocytopenia and RBC destruction when targeted by afucosylated antibodies (12–14). In addition to the specific afucosylated IgG response against platelets and RBC antigens, this response has only been identified against HIV and dengue virus (15, 16). Interestingly, low core fucosylation of anti-HIV antibodies has been suggested to be a feature of elite controllers of infection, whereas for dengue, it has been associated with enhanced pathology due to excessive FcγRIIIa activation (15, 16). The mechanisms controlling IgG core fucosylation remain unclear however.
Similar afucosylated IgG are found in various alloimmune responses (11–13, 17), HIV (16) and dengue (15), which are all directed against surface-exposed, membrane-embedded proteins. Therefore, we analyzed IgG glycosylation in anti-human platelet responses and in natural infections by enveloped viruses, including human immunodeficiency virus (HIV), cytomegalovirus (CMV), measles virus, mumps virus, hepatitis B virus (HBV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also assessed responses to a non-enveloped virus (parvovirus B19), vaccination with a HBV-protein subunit, and live attenuated enveloped viruses, to test if the antigen context was an important determinant for IgG-Fc glycosylation.
IgG-Fc glycosylation of affinity-purified total and antigen-specific antibodies were made possible by tandem liquid chromatography-mass spectrometry (LC-MS) (Fig. 1 and fig. S1) (12, 17, 18). Fc fucosylation of antigen-specific antibodies against the alloantigen human platelet antigen (HPA)-1a were substantially reduced (14) (Fig. 2A), akin to previous findings for other alloantigens (12, 17). Analogous to platelet and RBC alloantigens (11–13, 17), the response to the enveloped viruses CMV and HIV also showed significant afucosylation of the antigen-specific IgG (Fig. 2B). By contrast, IgG against the non-enveloped virus parvovirus B19 were fucosylated (Fig. 2C). Notably, the total IgG showed high fucosylation levels throughout (Fig. 2, A to C), reaffirming previous findings that the majority of IgG responses result in fucosylated IgG (12, 18, 19). The extent of afucosylated IgG responses to the enveloped viruses was highly variable, both between individuals and between the types of antigen, similar to observations of immune responses to different RBC alloantigens (17). Afucosylation was particularly strong for CMV and less pronounced for HIV (Fig. 2B), confirming previous observation in HIV (16). Afucosylated IgG-responses were often accompanied by elevated galactosylation (fig. S2).
Fig. 1Flowchart of antibody-specific IgG1 glycosylation analysis by mass spectrometry.
(A) Antibodies were captured from sera using Protein G beads and antigen-coated 96-well plates resulting in total and antigen-specific IgG fractions, respectively. Thereafter, isolated IgG were digested with trypsin and the resulting glycopeptides were analyzed by nano liquid chromatography-mass spectrometry. (B and C) Representative mass spectra of glycopeptides encompassing the Fc glycosylation site Asn297. Neutral (B) and sialylated (C) IgG1 glycopeptides are shown from a single patient’s total (upper panel, in black) and antigen-specific (lower panel, in red) IgG1 fraction. Asterisks indicate non-Fc glycopeptides.
Fig. 2Foreign membrane protein antigens, such as envelope proteins of (attenuated) enveloped viruses or alloantigens can trigger afucosylated IgG responses.
IgG1-Fc fucosylation levels of total (filled circles) and antigen-specific (open circles) antibodies are shown for each differently color-coded group of antigens: (A) alloantigen HPA-1a; (B) viral envelope antigens from CMV and HIV; (C) non-enveloped viral antigens from parvovirus B19; (D) HBsAg – in individuals naturally infected with HBV (left) or vaccinated with recombinant soluble HBsAg (right); and (E) mumps virus antigens in individuals naturally infected with mumps virus (left) or vaccinated with live attenuated mumps virus (right). Each circle represents a biological replicate [n = 80 for anti-HPA-1a (A), n = 65 for CMV and n = 40 for HIV (B), n = 22 for B19 (C), n = 17 for naturally infected individuals for HBV and n = 17 for HBV vaccinated individuals (D) and n = 24 naturally infected individuals for mumps virus and n = 21 for mumps vaccinated individuals (E)] of a representative LC-MS run (examples of technical replicates in fig.S1C). Statistical analyses were performed as paired t tests for (A), (B), and (C) and a mixed model two-way ANOVA with Bonferroni correction of post hoc t tests for comparing Fc fucosylation between groups for (D) and (E). Only statistically significant differences are shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
To test whether some individuals had a greater intrinsic capacity to generate an afucosylated IgG response than others, we compared IgG1-Fc fucosylation levels against two different antigens within the same individual. No correlation was observed comparing the level of afucosylation between two different antigens within the same individual, neither for anti-HPA-1a and anti-CMV (fig. S3A), nor for anti-HIV and anti-CMV antibodies (fig. S3B). Thus, the level of afucosylation is not pre-determined by general host factors such as genetics but is rather stochastic or multifactorial, with the specific triggers remaining obscure.
To further investigate the immunological context by which potent afucosylated IgG is formed, we compared immune responses to identical viral antigens in different contexts. First, we compared hepatitis B surface antigen (HBsAg)-specific antibody glycosylation in humans naturally infected with HBV or vaccinated with the recombinant HBsAg protein (Fig. 2D). Total IgG1-Fc fucosylation levels were similar for the two groups, whereas anti-HBsAg IgG1-Fc fucosylation was elevated in individuals vaccinated with the HBsAg protein when compared to either total IgG- or antigen-specific IgG-Fc fucosylation of the naturally infected group (Fig. 2D). Thus, HBsAg-specific antibodies in individuals who cleared a natural infection show lowered Fc fucosylation compared to protein subunit vaccination. This strongly suggests that a specific context for the antigenic stimulus is required for afucosylated IgG responses.
We then compared antiviral IgG responses against mumps and measles viruses formed after a natural infection or vaccination with live attenuated viruses. Unlike the HBV protein subunit vaccine, both live attenuated vaccines showed a similar antigen-specific Fc fucosylation compared to their natural infection counterpart (Fig. 2E and fig. S4). The tendency to generate afucosylated IgG was weak for measles, whereas the mumps response showed clear signs of afucosylation by either route of immunization (Fig. 2E and figs. S4 and S5).
We then tested if this type of response also plays a role in patients with coronavirus disease 2019 (COVID-19). Symptoms of COVID-19 are highly diverse, ranging from asymptomatic or mild self-limiting infection to a severe airway inflammation leading to acute respiratory distress syndrome (ARDS), often with a fatal outcome (20, 21). Both extreme trajectories follow similar initial responses: patients have approximately a week of relatively mild symptoms, followed by a second wave that either resolves the disease or leads to a highly aggravated life-threatening phenotype (20, 21). Both the timing of either response type and the differential clinical outcome suggested different routes taken by the immune system to combat the disease. So far, no clear evidence has emerged that can distinguish between these two hypothetical immunological paths. In accordance with our hypothesis and responses observed against other enveloped viruses, anti-S IgG responses against SARS-CoV-2 spike protein (S), which is expressed on the cell surface and the viral envelope, were strongly skewed toward low levels of core fucosylation. By contrast, responses against the nucleocapsid protein (N), which is not expressed on cell surface/viral envelope, were characterized by high levels of fucosylation (Fig. 3A). The IgG response appeared to be highly specific for SARS-CoV-2 as there was very weak or absent reactivity to SARS-CoV-2 antigens in pre-outbreak samples, even to the more conserved N antigen (fig. S6) (22). Importantly, the anti-S IgG1 responses of patients with ARDS recently hospitalized in intensive care units (<5 days) were significantly less fucosylated than in convalescent plasma donors consisting of individuals who were asymptomatic or had relative mild symptoms (non-ARDS) (Fig. 3A).
Fig. 3Fc fucosylation levels of anti-S IgG1 are significantly decreased in critically ill COVID-19 patients.
(A) Fc fucosylation, (B) galactosylation, (C) sialylation, and (D) bisection degree of anti-S, anti-N, and total IgG1 from ARDS patients and non-ARDS donors clearing the infection asymptomatically or with mild symptoms from the initial screen. (E to L) Longitudinal IgG1-Fc fucosylation and IgG quantity for anti-S [(E) to (H)] and anti-N [(I) to (L)] in ARDS patients [(E), (F), (I), and (J)] and in non-ARDS cases [(G), (H), (K), and (L)]. (M) Correlation between anti-N and anti-S IgG1-Fc fucosylation. (N) Representative examples of IL-6 release from macrophages triggered through FcγR- by stimulation with glycoengineered IgG-complexes with or without poly(I:C). (O) Correlation between plasma IL-6 levels and degrees of anti-S IgG1-Fc fucosylation. (P) Correlation between plasma CRP levels and degrees of anti-S IgG1-Fc fucosylation. Each circle represents a biological replicate (n = 20 for ARDS, n = 23 for non-ARDS [(A) to (D)], n = 17 and n = 14 for longitudinal ARDS and non-ARDS, respectively [(E) to (L)]. Examples of technical replicates for LC-MS data are shown in fig.S1C. IgG data [(F), (H), (J), and (L)] are representative ELISA values calibrated against a standard pool from two technical experiments. All available paired data used in (M) (n = 40). IL-6 production by macrophages were measured by ELISA with each dot (n = 3) representing a technical replicate (N). All six biological replicates are shown in fig. S13. CRP and IL-6 levels were obtained from clinical parameters and IL-6 data by Mesoscale Discovery using all available paired data [n = 82 (O) and n = 53 (P)]. Statistical analyses were performed as a mixed model two-way ANOVA with Bonferroni correction of post hoc t tests for comparing glycosylation traits and cytokine secretion between groups. Spearman’s correlations were performed in (O) to (P). To test the correlation between Fc fucosylation levels for anti-S and anti-N, a Pearson’s correlation was performed. Only statistically significant differences are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
These decreased levels of Fc fucosylation of anti-S IgG were not a result of inflammation as total IgG-Fc fucosylation levels were similar between the two groups and to what has been reported in the general population (~94%) (12, 18). In addition, IgG1-Fc galactosylation and sialylation of both anti-S and anti-N responses (Fig. 3, B and C) were significantly increased compared to total IgG, consistent with reports describing increased Fc galactosylation and sialylation in active or recent immunization (18, 23). Total IgG1-Fc galactosylation and sialylation levels were significantly lowered in the ARDS patients, which was perhaps a reflection of a slight age difference between these two groups [non-ARDS donors median age (IQR) 49 (40 to 55) years versus ARDS patients 60 (55–63) years (tables S1 and S2)]. Notably, both Fc galactosylation and sialylation decrease with age (9, 19). Increased galactosylation and sialylation of antigen-specific IgG1-Fc elevates complement activity by approximately three- to fourfold. Fc galactosylation further enhances affinity of afucosylated IgG to FcγRIII by approximately twofold (24). Lastly, although Fc bisection was significantly lowered in both anti-N and anti-S responses (Fig. 3D), the biological and clinical significance of this is limited as IgG-Fc bisection affects neither Fc receptor nor complement activity (24). More importantly, accumulating evidence strongly suggests that the primary and major biologically relevant change in IgG-Fc glycosylation is the lack of core fucose. Afucosylated IgG have a 20–40-fold increase in affinity to FcγRIIIa, often accompanied by an absolute change from no cellular response to strong phagocytic and ADCC responses upon afucosylation (5, 15, 24, 25). The lowered Fc fucosylation in the anti-S responses of the ARDS patients suggests a pathological role through FcγRIIIa, similar to what has previously been proposed for dengue (15). In dengue, non-neutralizing antibodies formed to previous infections of other dengue serotypes also tend to have low levels of core-fucosylated IgG. As they are incapable of preventing infection, they lead to aggravated dengue hemorrhagic fever due to FcγRIIIa-mediated overreactions by immune cells (15).
ARDS patients were sampled within 1 week after ICU admission and non-ARDS patients were convalescent non-hospitalized individuals. In order to eliminate any possible sampling bias in the observed IgG-Fc glycosylation patterns over time, we also analyzed longitudinal samples from both groups (26). Alloantibody Fc fucosylation to platelets and RBC antigens is stable for at least a decade with or without natural booster through pregnancies (12, 14) or blood transfusion (27). This also held true for anti-CMV and anti-HIV responses (fig. S7). By contrast, changes in all glycosylation traits were already observed for SARS-CoV-2 during the first week following ICU admission (Fig. 3, E to L and figs. S8 and S9). These observed changes in Fc galactosylation (fig. S8) were in line with previous reports that recent immunizations are accompanied with a transient rise in antigen-specific IgG-Fc galactosylation and sialylation (18, 23). After seroconversion, all ARDS patients initially showed low levels of anti-S IgG fucosylation compared to non-ARDS patients. Fucosylation levels rose over time in ARDS patients, reaching levels comparable to those of the non-ARDS cohort (Fig. 3, E and G). The increases in fucose levels were associated with simultaneous rises in IgG levels (Fig. 3, E to H, and fig. S10, A and B), which were much less pronounced in the non-ARDS cohort. Similar kinetics were observed for anti-N IgG levels (Fig. 3, J and L). Reduced levels of anti-N Fc fucosylation were also present in the ARDS group, although to a lesser degree than for anti-S (Fig. 3, I and K and fig. S11). This unexpected reduction in the fucosylation of anti-N IgG seen in the ARDS cohort may have been the result of classical bystander effects (28). Namely, B cells proliferating in the same lymphoid organs receive similar environmental cues from antigen-presenting cells and T cells. The IgG1-Fc fucosylation of anti-S and anti-N correlated significantly (Fig. 3M) with higher levels of afucosylation for anti-S (P < 0.0001). Significant correlations were also observed for other glycosylation traits, with similar skewing for both anti-S and anti-N IgG (fig. S12). These elevations in antigen-specific IgG1-Fc galactosylation and sialylation agreed with earlier reports suggesting that these are general features of newly formed ongoing immune responses (18, 23). Total IgG1-Fc fucosylation remained stable throughout the observation period (figs. S8J and S9J).
We then asked how these afucosylated anti-SARS-CoV-2 antibodies might contribute to the strong inflammatory response observed in ARDS patients. Alveolar macrophages are front-line scavengers in the lung and express FcγRIIIa, the major myeloid sensory receptor for afucosylated IgG. Thus, we examined their potential to stimulate the production of the pro-inflammatory cytokine interleukin (IL)-6, the cytokine that is most critical for acute-phase responses in humans (29). Afucosylated IgG, together with Toll-like receptor (TLR)3 ligand, enhanced IL-6 production from macrophages in vitro, particularly when using afucosylated and highly galactosylated IgG, as is found prominently in the ARDS patients (Fig. 3N and figs. S8 and S13). There was a significant correlation between anti-S IgG1-Fc fucosylation and both plasma IL-6 and C-reactive protein (CRP) levels (Fig. 3, O and P). This agrees with our hypothesis that afucosylated anti-SARS-CoV-2 IgG plays an important role in COVID-19 pathogenesis. Levels of plasma IL-6 and CRP increased around the time when afucosylated anti-S IgG appeared, which suggested a direct causality (Fig. 4, A and B). Plasma D-dimer levels also shared this temporal pattern (fig. S14). Thus, the afucosylated and highly galactosylated anti-S and anti-N IgG in some patients may cause an exaggerated release of pro-inflammatory cytokines and subsequent systemic inflammation due to their enhanced binding capacity to FcγRIIIa (24) on alveolar macrophages. No increase of either IL-6 or CRP was observed in the non-ARDS cases (Fig. 4, C and D, and fig. S15).
Fig. 4Longitudinal changes of anti-SARS-CoV-2 IgG1 Fc fucosylation, CRP and IL-6.
(A and C) Anti-S IgG fucosylation and anti-N IgG fucosylation and (Band D) IL-6 and CRP levels in an ARDS cohort [(A) and (B)] and a non-ARDS cohort [(C) and (D)]. Each panel represents longitudinal biological replicates of a LC-MS run [(A) and (C), examples of technical replicates in fig.S1C)], CRP obtained from clinical parameters, and IL-6 data using a validated Mesoscale Discovery assay [(B) and (D) (n = 12 for ARDS and n = 14 for non-ARDS, with 2 to 16 longitudinal replicates per patient as indicated]. See fig. S15 for additional non-ARDS samples. Hash signs denote samples prior to these points were below the limit of detection for IgG1-glycosylation analyses. Vertical dotted lines in (A) and (B) represent the time of ICU admission and ICU discharge (black) or death (red), whereas dotted horizontal lines in (B) and (D) represent IL-6 and CRP detection limits.
In conclusion, our results show a pattern of afucosylated IgG1 immune responses against membrane-embedded antigens such as surface membrane proteins of allo-antigens on blood cells or on enveloped viruses (including attenuated enveloped virus vaccines that often complete their first round of infection). This contrasts with soluble protein antigens and non-enveloped viruses for which immune responses with high levels of IgG1-Fc fucosylation were consistently observed. Although there was afucosylated anti-N IgG in COVID-19 patients, this was no longer the case 1–2 weeks after seroconversion.
We hypothesize that antigen-presenting membranes are directly sensed by B cells by combining at least two signals provided by the B cell receptor and undescribed host receptor–ligand pair(s). This two-step mechanism would be essential for the production of long-lasting afucosylated IgG responses and would not be triggered by soluble proteins, internal proteins of enveloped viruses, or non-enveloped viruses (Fig. 5). Alternatively, differential antigen recognition may be more complex and require additional interactions from antigen-presenting cells, T cells, and/or cytokines. This notion is supported by the fact that anti-N SARS-CoV-2 responses occur concomitantly with anti-S responses, which suggests that proximal factors in the lymphoid microenvironment can influence the response.
Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity
Abstract
IgG antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anti-cancer therapeutic antibodies for their elevated activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (~6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses, but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high levels of afucosylated IgG antibodies against SARS-CoV-2, amplifying pro-inflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.
Antibody function has long been considered static and mostly determined by their isotype and subclass. The presence of a conserved N-linked glycan at position 297 in the Fc domain of IgG, is essential for its effector functions (1–3). Moreover the composition of this glycan is highly variable, which has functional consequences (2–4). This is especially true for the core fucose attached to the Fc glycan. The discovery that IgG variants without core fucosylation cause elevated antibody-dependent cellular cytotoxicity (ADCC), via increased IgG-Fc receptor IIIa (FcγRIIIa) affinity (5, 6), has resulted in next-generation glyco-engineered monoclonal antibodies (mAb) lacking core fucosylation for targeting tumors (7).
Generally, changes in the Fc glycans are associated with age, sex, and autoimmune diseases, and are most pronounced for IgG-Fc galactosylation, which decreases steadily with advancing age. After a marked elevation in young women, IgG-Fc galactosylation decreases during menopause to the levels seen in men (8). IgG-Fc fucosylation is more stable, decreasing slightly from birth to approximately 94% at adulthood (9), after which it remains fairly constant, albeit with a minor reduction throughout life (8, 10).
Despite the apparent constant level of Fc fucosylation during adulthood, alloantibodies against red blood cells (RBCs) and platelets show remarkably low IgG-Fc fucosylation in most patients, even down to 10% in several cases (11–13). By contrast, overall serum IgG-Fc fucosylation is consistently high. Moreover, lowered IgG-Fc fucosylation is one of the factors determining disease severity in pregnancy-associated alloimmunizations, resulting in excessive thrombocytopenia and RBC destruction when targeted by afucosylated antibodies (12–14). In addition to the specific afucosylated IgG response against platelets and RBC antigens, this response has only been identified against HIV and dengue virus (15, 16). Interestingly, low core fucosylation of anti-HIV antibodies has been suggested to be a feature of elite controllers of infection, whereas for dengue, it has been associated with enhanced pathology due to excessive FcγRIIIa activation (15, 16). The mechanisms controlling IgG core fucosylation remain unclear however.
Similar afucosylated IgG are found in various alloimmune responses (11–13, 17), HIV (16) and dengue (15), which are all directed against surface-exposed, membrane-embedded proteins. Therefore, we analyzed IgG glycosylation in anti-human platelet responses and in natural infections by enveloped viruses, including human immunodeficiency virus (HIV), cytomegalovirus (CMV), measles virus, mumps virus, hepatitis B virus (HBV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also assessed responses to a non-enveloped virus (parvovirus B19), vaccination with a HBV-protein subunit, and live attenuated enveloped viruses, to test if the antigen context was an important determinant for IgG-Fc glycosylation.
IgG-Fc glycosylation of affinity-purified total and antigen-specific antibodies were made possible by tandem liquid chromatography-mass spectrometry (LC-MS) (Fig. 1 and fig. S1) (12, 17, 18). Fc fucosylation of antigen-specific antibodies against the alloantigen human platelet antigen (HPA)-1a were substantially reduced (14) (Fig. 2A), akin to previous findings for other alloantigens (12, 17). Analogous to platelet and RBC alloantigens (11–13, 17), the response to the enveloped viruses CMV and HIV also showed significant afucosylation of the antigen-specific IgG (Fig. 2B). By contrast, IgG against the non-enveloped virus parvovirus B19 were fucosylated (Fig. 2C). Notably, the total IgG showed high fucosylation levels throughout (Fig. 2, A to C), reaffirming previous findings that the majority of IgG responses result in fucosylated IgG (12, 18, 19). The extent of afucosylated IgG responses to the enveloped viruses was highly variable, both between individuals and between the types of antigen, similar to observations of immune responses to different RBC alloantigens (17). Afucosylation was particularly strong for CMV and less pronounced for HIV (Fig. 2B), confirming previous observation in HIV (16). Afucosylated IgG-responses were often accompanied by elevated galactosylation (fig. S2).
(A) Antibodies were captured from sera using Protein G beads and antigen-coated 96-well plates resulting in total and antigen-specific IgG fractions, respectively. Thereafter, isolated IgG were digested with trypsin and the resulting glycopeptides were analyzed by nano liquid chromatography-mass spectrometry. (B and C) Representative mass spectra of glycopeptides encompassing the Fc glycosylation site Asn297. Neutral (B) and sialylated (C) IgG1 glycopeptides are shown from a single patient’s total (upper panel, in black) and antigen-specific (lower panel, in red) IgG1 fraction. Asterisks indicate non-Fc glycopeptides.
IgG1-Fc fucosylation levels of total (filled circles) and antigen-specific (open circles) antibodies are shown for each differently color-coded group of antigens: (A) alloantigen HPA-1a; (B) viral envelope antigens from CMV and HIV; (C) non-enveloped viral antigens from parvovirus B19; (D) HBsAg – in individuals naturally infected with HBV (left) or vaccinated with recombinant soluble HBsAg (right); and (E) mumps virus antigens in individuals naturally infected with mumps virus (left) or vaccinated with live attenuated mumps virus (right). Each circle represents a biological replicate [n = 80 for anti-HPA-1a (A), n = 65 for CMV and n = 40 for HIV (B), n = 22 for B19 (C), n = 17 for naturally infected individuals for HBV and n = 17 for HBV vaccinated individuals (D) and n = 24 naturally infected individuals for mumps virus and n = 21 for mumps vaccinated individuals (E)] of a representative LC-MS run (examples of technical replicates in fig.S1C). Statistical analyses were performed as paired t tests for (A), (B), and (C) and a mixed model two-way ANOVA with Bonferroni correction of post hoc t tests for comparing Fc fucosylation between groups for (D) and (E). Only statistically significant differences are shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
To test whether some individuals had a greater intrinsic capacity to generate an afucosylated IgG response than others, we compared IgG1-Fc fucosylation levels against two different antigens within the same individual. No correlation was observed comparing the level of afucosylation between two different antigens within the same individual, neither for anti-HPA-1a and anti-CMV (fig. S3A), nor for anti-HIV and anti-CMV antibodies (fig. S3B). Thus, the level of afucosylation is not pre-determined by general host factors such as genetics but is rather stochastic or multifactorial, with the specific triggers remaining obscure.
To further investigate the immunological context by which potent afucosylated IgG is formed, we compared immune responses to identical viral antigens in different contexts. First, we compared hepatitis B surface antigen (HBsAg)-specific antibody glycosylation in humans naturally infected with HBV or vaccinated with the recombinant HBsAg protein (Fig. 2D). Total IgG1-Fc fucosylation levels were similar for the two groups, whereas anti-HBsAg IgG1-Fc fucosylation was elevated in individuals vaccinated with the HBsAg protein when compared to either total IgG- or antigen-specific IgG-Fc fucosylation of the naturally infected group (Fig. 2D). Thus, HBsAg-specific antibodies in individuals who cleared a natural infection show lowered Fc fucosylation compared to protein subunit vaccination. This strongly suggests that a specific context for the antigenic stimulus is required for afucosylated IgG responses.
We then compared antiviral IgG responses against mumps and measles viruses formed after a natural infection or vaccination with live attenuated viruses. Unlike the HBV protein subunit vaccine, both live attenuated vaccines showed a similar antigen-specific Fc fucosylation compared to their natural infection counterpart (Fig. 2E and fig. S4). The tendency to generate afucosylated IgG was weak for measles, whereas the mumps response showed clear signs of afucosylation by either route of immunization (Fig. 2E and figs. S4 and S5).
We then tested if this type of response also plays a role in patients with coronavirus disease 2019 (COVID-19). Symptoms of COVID-19 are highly diverse, ranging from asymptomatic or mild self-limiting infection to a severe airway inflammation leading to acute respiratory distress syndrome (ARDS), often with a fatal outcome (20, 21). Both extreme trajectories follow similar initial responses: patients have approximately a week of relatively mild symptoms, followed by a second wave that either resolves the disease or leads to a highly aggravated life-threatening phenotype (20, 21). Both the timing of either response type and the differential clinical outcome suggested different routes taken by the immune system to combat the disease. So far, no clear evidence has emerged that can distinguish between these two hypothetical immunological paths. In accordance with our hypothesis and responses observed against other enveloped viruses, anti-S IgG responses against SARS-CoV-2 spike protein (S), which is expressed on the cell surface and the viral envelope, were strongly skewed toward low levels of core fucosylation. By contrast, responses against the nucleocapsid protein (N), which is not expressed on cell surface/viral envelope, were characterized by high levels of fucosylation (Fig. 3A). The IgG response appeared to be highly specific for SARS-CoV-2 as there was very weak or absent reactivity to SARS-CoV-2 antigens in pre-outbreak samples, even to the more conserved N antigen (fig. S6) (22). Importantly, the anti-S IgG1 responses of patients with ARDS recently hospitalized in intensive care units (<5 days) were significantly less fucosylated than in convalescent plasma donors consisting of individuals who were asymptomatic or had relative mild symptoms (non-ARDS) (Fig. 3A).
(A) Fc fucosylation, (B) galactosylation, (C) sialylation, and (D) bisection degree of anti-S, anti-N, and total IgG1 from ARDS patients and non-ARDS donors clearing the infection asymptomatically or with mild symptoms from the initial screen. (E to L) Longitudinal IgG1-Fc fucosylation and IgG quantity for anti-S [(E) to (H)] and anti-N [(I) to (L)] in ARDS patients [(E), (F), (I), and (J)] and in non-ARDS cases [(G), (H), (K), and (L)]. (M) Correlation between anti-N and anti-S IgG1-Fc fucosylation. (N) Representative examples of IL-6 release from macrophages triggered through FcγR- by stimulation with glycoengineered IgG-complexes with or without poly(I:C). (O) Correlation between plasma IL-6 levels and degrees of anti-S IgG1-Fc fucosylation. (P) Correlation between plasma CRP levels and degrees of anti-S IgG1-Fc fucosylation. Each circle represents a biological replicate (n = 20 for ARDS, n = 23 for non-ARDS [(A) to (D)], n = 17 and n = 14 for longitudinal ARDS and non-ARDS, respectively [(E) to (L)]. Examples of technical replicates for LC-MS data are shown in fig.S1C. IgG data [(F), (H), (J), and (L)] are representative ELISA values calibrated against a standard pool from two technical experiments. All available paired data used in (M) (n = 40). IL-6 production by macrophages were measured by ELISA with each dot (n = 3) representing a technical replicate (N). All six biological replicates are shown in fig. S13. CRP and IL-6 levels were obtained from clinical parameters and IL-6 data by Mesoscale Discovery using all available paired data [n = 82 (O) and n = 53 (P)]. Statistical analyses were performed as a mixed model two-way ANOVA with Bonferroni correction of post hoc t tests for comparing glycosylation traits and cytokine secretion between groups. Spearman’s correlations were performed in (O) to (P). To test the correlation between Fc fucosylation levels for anti-S and anti-N, a Pearson’s correlation was performed. Only statistically significant differences are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
These decreased levels of Fc fucosylation of anti-S IgG were not a result of inflammation as total IgG-Fc fucosylation levels were similar between the two groups and to what has been reported in the general population (~94%) (12, 18). In addition, IgG1-Fc galactosylation and sialylation of both anti-S and anti-N responses (Fig. 3, B and C) were significantly increased compared to total IgG, consistent with reports describing increased Fc galactosylation and sialylation in active or recent immunization (18, 23). Total IgG1-Fc galactosylation and sialylation levels were significantly lowered in the ARDS patients, which was perhaps a reflection of a slight age difference between these two groups [non-ARDS donors median age (IQR) 49 (40 to 55) years versus ARDS patients 60 (55–63) years (tables S1 and S2)]. Notably, both Fc galactosylation and sialylation decrease with age (9, 19). Increased galactosylation and sialylation of antigen-specific IgG1-Fc elevates complement activity by approximately three- to fourfold. Fc galactosylation further enhances affinity of afucosylated IgG to FcγRIII by approximately twofold (24). Lastly, although Fc bisection was significantly lowered in both anti-N and anti-S responses (Fig. 3D), the biological and clinical significance of this is limited as IgG-Fc bisection affects neither Fc receptor nor complement activity (24). More importantly, accumulating evidence strongly suggests that the primary and major biologically relevant change in IgG-Fc glycosylation is the lack of core fucose. Afucosylated IgG have a 20–40-fold increase in affinity to FcγRIIIa, often accompanied by an absolute change from no cellular response to strong phagocytic and ADCC responses upon afucosylation (5, 15, 24, 25). The lowered Fc fucosylation in the anti-S responses of the ARDS patients suggests a pathological role through FcγRIIIa, similar to what has previously been proposed for dengue (15). In dengue, non-neutralizing antibodies formed to previous infections of other dengue serotypes also tend to have low levels of core-fucosylated IgG. As they are incapable of preventing infection, they lead to aggravated dengue hemorrhagic fever due to FcγRIIIa-mediated overreactions by immune cells (15).
ARDS patients were sampled within 1 week after ICU admission and non-ARDS patients were convalescent non-hospitalized individuals. In order to eliminate any possible sampling bias in the observed IgG-Fc glycosylation patterns over time, we also analyzed longitudinal samples from both groups (26). Alloantibody Fc fucosylation to platelets and RBC antigens is stable for at least a decade with or without natural booster through pregnancies (12, 14) or blood transfusion (27). This also held true for anti-CMV and anti-HIV responses (fig. S7). By contrast, changes in all glycosylation traits were already observed for SARS-CoV-2 during the first week following ICU admission (Fig. 3, E to L and figs. S8 and S9). These observed changes in Fc galactosylation (fig. S8) were in line with previous reports that recent immunizations are accompanied with a transient rise in antigen-specific IgG-Fc galactosylation and sialylation (18, 23). After seroconversion, all ARDS patients initially showed low levels of anti-S IgG fucosylation compared to non-ARDS patients. Fucosylation levels rose over time in ARDS patients, reaching levels comparable to those of the non-ARDS cohort (Fig. 3, E and G). The increases in fucose levels were associated with simultaneous rises in IgG levels (Fig. 3, E to H, and fig. S10, A and B), which were much less pronounced in the non-ARDS cohort. Similar kinetics were observed for anti-N IgG levels (Fig. 3, J and L). Reduced levels of anti-N Fc fucosylation were also present in the ARDS group, although to a lesser degree than for anti-S (Fig. 3, I and K and fig. S11). This unexpected reduction in the fucosylation of anti-N IgG seen in the ARDS cohort may have been the result of classical bystander effects (28). Namely, B cells proliferating in the same lymphoid organs receive similar environmental cues from antigen-presenting cells and T cells. The IgG1-Fc fucosylation of anti-S and anti-N correlated significantly (Fig. 3M) with higher levels of afucosylation for anti-S (P < 0.0001). Significant correlations were also observed for other glycosylation traits, with similar skewing for both anti-S and anti-N IgG (fig. S12). These elevations in antigen-specific IgG1-Fc galactosylation and sialylation agreed with earlier reports suggesting that these are general features of newly formed ongoing immune responses (18, 23). Total IgG1-Fc fucosylation remained stable throughout the observation period (figs. S8J and S9J).
We then asked how these afucosylated anti-SARS-CoV-2 antibodies might contribute to the strong inflammatory response observed in ARDS patients. Alveolar macrophages are front-line scavengers in the lung and express FcγRIIIa, the major myeloid sensory receptor for afucosylated IgG. Thus, we examined their potential to stimulate the production of the pro-inflammatory cytokine interleukin (IL)-6, the cytokine that is most critical for acute-phase responses in humans (29). Afucosylated IgG, together with Toll-like receptor (TLR)3 ligand, enhanced IL-6 production from macrophages in vitro, particularly when using afucosylated and highly galactosylated IgG, as is found prominently in the ARDS patients (Fig. 3N and figs. S8 and S13). There was a significant correlation between anti-S IgG1-Fc fucosylation and both plasma IL-6 and C-reactive protein (CRP) levels (Fig. 3, O and P). This agrees with our hypothesis that afucosylated anti-SARS-CoV-2 IgG plays an important role in COVID-19 pathogenesis. Levels of plasma IL-6 and CRP increased around the time when afucosylated anti-S IgG appeared, which suggested a direct causality (Fig. 4, A and B). Plasma D-dimer levels also shared this temporal pattern (fig. S14). Thus, the afucosylated and highly galactosylated anti-S and anti-N IgG in some patients may cause an exaggerated release of pro-inflammatory cytokines and subsequent systemic inflammation due to their enhanced binding capacity to FcγRIIIa (24) on alveolar macrophages. No increase of either IL-6 or CRP was observed in the non-ARDS cases (Fig. 4, C and D, and fig. S15).
(A and C) Anti-S IgG fucosylation and anti-N IgG fucosylation and (Band D) IL-6 and CRP levels in an ARDS cohort [(A) and (B)] and a non-ARDS cohort [(C) and (D)]. Each panel represents longitudinal biological replicates of a LC-MS run [(A) and (C), examples of technical replicates in fig.S1C)], CRP obtained from clinical parameters, and IL-6 data using a validated Mesoscale Discovery assay [(B) and (D) (n = 12 for ARDS and n = 14 for non-ARDS, with 2 to 16 longitudinal replicates per patient as indicated]. See fig. S15 for additional non-ARDS samples. Hash signs denote samples prior to these points were below the limit of detection for IgG1-glycosylation analyses. Vertical dotted lines in (A) and (B) represent the time of ICU admission and ICU discharge (black) or death (red), whereas dotted horizontal lines in (B) and (D) represent IL-6 and CRP detection limits.
In conclusion, our results show a pattern of afucosylated IgG1 immune responses against membrane-embedded antigens such as surface membrane proteins of allo-antigens on blood cells or on enveloped viruses (including attenuated enveloped virus vaccines that often complete their first round of infection). This contrasts with soluble protein antigens and non-enveloped viruses for which immune responses with high levels of IgG1-Fc fucosylation were consistently observed. Although there was afucosylated anti-N IgG in COVID-19 patients, this was no longer the case 1–2 weeks after seroconversion.
We hypothesize that antigen-presenting membranes are directly sensed by B cells by combining at least two signals provided by the B cell receptor and undescribed host receptor–ligand pair(s). This two-step mechanism would be essential for the production of long-lasting afucosylated IgG responses and would not be triggered by soluble proteins, internal proteins of enveloped viruses, or non-enveloped viruses (Fig. 5). Alternatively, differential antigen recognition may be more complex and require additional interactions from antigen-presenting cells, T cells, and/or cytokines. This notion is supported by the fact that anti-N SARS-CoV-2 responses occur concomitantly with anti-S responses, which suggests that proximal factors in the lymphoid microenvironment can influence the response.