key: cord-0832029-moydss0g
authors: Van Coillie, Julie; Pongracz, Tamas; Rahmöller, Johann; Chen, Hung-Jen; Geyer, Chiara; van Vlught, Lonneke A.; Buhre, Jana S.; Šuštić, Tonći; van Osch, Thijs L. J.; Steenhuis, Maurice; Hoepel, Willianne; Wang, Wenjun; Lixenfeld, Anne S.; Nouta, Jan; Keijzer, Sofie; Linty, Federica; Visser, Remco; Larsen, Mads D.; Martin, Emily L.; Künsting, Inga; Lehrian, Selina; von Kopylow, Vera; Kern, Carsten; Lunding, Hanna B.; de Winther, Menno; van Mourik, Niels; Rispens, Theo; Graf, Tobias; Slim, Marleen A.; Minnaar, René; Bomers, Marije K.; Sikkens, Jonne J.; Vlaar, Alexander P. J.; van der Schoot, C. Ellen; den Dunnen, Jeroen; Wuhrer, Manfred; Ehlers, Marc; Vidarsson, Gestur
title: The BNT162b2 mRNA SARS-CoV-2 vaccine induces transient afucosylated IgG1 in naive but not antigen-experienced vaccinees
date: 2022-02-15
journal: bioRxiv
DOI: 10.1101/2022.02.14.480353
sha: d7d24c72fa88d285940c6532a06b6d76776a94b5
doc_id: 832029
cord_uid: moydss0g

The onset of severe SARS-CoV-2 infection is characterized by the presence of afucosylated IgG1 responses against the viral spike (S) protein, which can trigger exacerbated inflammatory responses. Here, we studied IgG glycosylation after BNT162b2 SARS-CoV-2 mRNA vaccination to explore whether vaccine-induced S protein expression on host cells also generates afucosylated IgG1 responses. SARS-CoV-2 naive individuals initially showed a transient afucosylated anti-S IgG1 response after the first dose, albeit to a lower extent than severely ill COVID-19 patients. In contrast, previously infected, antigen-experienced individuals had low afucosylation levels, which slightly increased after immunization. Afucosylation levels after the first dose correlated with low fucosyltransferase 8 (FUT8) expression levels in a defined plasma cell subset. Remarkably, IgG afucosylation levels after primary vaccination correlated significantly with IgG levels after the second dose. Further studies are needed to assess efficacy, inflammatory potential, and protective capacity of afucosylated IgG responses. One sentence summary A transient afucosylated IgG response to the BNT162b2 mRNA vaccine was observed in naive but not in antigen-experienced individuals, which predicted antibody titers upon the second dose.

Immunoglobulin G (IgG) antibodies (Abs) are crucial for protective immunity in coronavirus disease 2019 through both fragment antigen binding (Fab)-mediated neutralization and fragment crystallizable (Fc)-mediated effector functions. The IgG Fcmediated effector functions mainly depend on IgG subclass and Fc N-glycosylation, of which the latter has been shown to be important for COVID-19 disease exacerbation (1) (2) (3) (4) . Human

IgG contains a single, conserved biantennary N-linked glycan at N297 of the Fc portion. This N-glycan has a common pentasaccharide core that can further be modified with a fucose, a bisecting N-acetylglucosamine (GlcNAc), as well as one or two galactose residues, of which each can further be capped by a sialic acid. Of these glycan residues, galactose and fucose have been described to modulate the activity of complement or natural killer (NK) and myeloid cell IgG-Fc gamma receptors (FcγR), respectively (5-7) (Fig. 1A) .

Fc-galactosylation levels are highly variable (40-60%) , with decreased levels being found in inflammatory diseases such as various infectious, cardiovascular, and autoimmune diseases as well as cancer (8) (9) (10) (11) (12) , whereas increased Fc-galactosylation has been shown to characterize IgG after vaccination (13, 14) and . Elevated Fcgalactosylation promotes IgG Fc-Fc interaction, leading to hexamerization, which enables docking of complement component 1q (C1q), the first component of the classical complement cascade, and ensuing complement activation (15, 16) .

Afucosylated IgG has an enhanced binding to FcγRIII, resulting in increased cytokine production and cellular responses, such as Ab-dependent cellular phagocytosis (ADCP) and cytotoxicity (ADCC) (6, 7, 17) . In healthy conditions, the majority of IgG found in plasma is fucosylated (~94%) (18, 19) , but afucosylated, antigen-specific IgG responses have been described in various pathologies, including alloimmune responses to blood cells (20) (21) (22) , as well as immune responses to Plasmodium (P) falciparum antigens expressed on erythrocytes (23) and to foreign proteins of enveloped viruses, including human immunodeficiency virus (HIV) (24) , dengue virus (25) , and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2, 3) . The common characteristic of such responses is that the corresponding pathogenspecific antigens are generally expressed on the host cell membrane, unlike most foreign antigens. Intriguingly, pathogen-specific afucosylated IgG1 responses seem to be protective in malaria (23) and HIV (24) , but can, in turn, cause massive inflammation via FcγRIII-mediated pathologies in patients with severe dengue fever (25) and have been shown to precede severe 26) .

Non-enveloped viruses, bacteria, and soluble protein-subunit vaccines, which all lack the host cell membrane context, induce almost no afucosylated IgG responses. This includes those of recombinant hepatitis B virus (HBV) and P. falciparum-proteins. On the contrary, when expressed in their natural context on host cells, afucosylated IgG responses have been observed in HBV and malaria (2, 23) . This led us to the hypothesis that antigen presentation at the surface of host cells, possibly together with host co-factors, is required for the induction of afucosylated IgG responses (2) .

The new mRNA-and adenovirus-based SARS-CoV-2 vaccines induce host cell production of the SARS-CoV-2 spike (S) protein and its subsequent presentation on the cell membrane, unlike traditional soluble protein-subunit vaccines (27) . Similar to attenuated enveloped-viral vaccines (2), mRNA-and adenoviral-based vaccines might therefore also induce an afucosylated IgG response.

Here, we investigated anti-S IgG glycosylation in both naive and antigen-experienced participants after the first and second dose of the BNT162b2 mRNA vaccine against SARS-CoV-2. Additionally, we evaluated glycosyltransferase expression in antigen-specific IgG + plasma cell (PC) subsets to obtain insights into the generation of anti-S IgG glycosylation phenotypes. We furthermore studied the potential contribution of anti-S IgG afucosylation to inflammatory responses using an in vitro macrophage activation assay.

To analyze the immune response in naive and antigen-experienced individuals upon vaccination with the mRNA vaccine BNT162b2, blood samples were collected from healthy donors at four locations: 1) the Amsterdam University Medical Center (UMC) in The Netherlands, 2) the Fatebenefratelli-Sacco University Hospital in Milan in Italy, 3) the University Medical Center of Schleswig-Holstein Lübeck in Germany, and 4) the Dutch blood bank Sanquin in The Netherlands ( Fig. 1A and Table S2 -5) .

To identify antigen-experienced individuals, anti-nucleocapsid (N) and anti-spike (S) IgG responses were investigated both prior to the first dose and during the study, together with previous positive SARS-CoV-2 PCR results ( Fig. 1B -C, S1 and Table S2 -5) . Vaccinated SARS-CoV-2 naive individuals showed a detectable anti-S IgG response around day ten after vaccination that further increased upon the second dose ( Fig. 1D and S1A, D, F). All vaccinated antigen-experienced individuals had anti-S IgG Abs before vaccination and levels increased fast upon the first dose of BNT162b2 ( Fig. 1D and Fig. S1A, D) . Both naive and antigen-experienced reached similar anti-S levels, which were dominated by IgG1 and IgG3 subclasses against both the S1 and S2 subunits of the S protein ( Fig. S1G) (28, 29) . For clarification, we have compared the vaccine-induced responses with the dynamics of mild and intensive care unit (ICU)-admitted COVID-19 patients as described by Larsen et al. (2) (Fig.   1D ). (D) Longitudinal anti-S IgG levels for naive (left, cohort 1 (n=33) and 2 (n=9)) and antigen-experienced (middle, cohort 1 (n=6) and 2 (n=0)) vaccinees and corresponding dynamics in comparison to mild (grey) and ICU hospitalized (red) COVID-19 patients (right). Similar data for cohort 3 and 4 are plotted in Fig. S1 and S5, respectively.

Next, we explored anti-S and total IgG1 Fc N-glycosylation patterns over time ( Fig. 2 and S2-3). In both naive and antigen-experienced individuals, an initial drop of anti-S IgG1 bisection levels were seen, with lowered levels as compared to total IgG1 ( Fig. 2A and S2A,   3A ). An early response of highly galactosylated and sialylated anti-S IgG1 was observed in both naive and antigen-experienced individuals, both after the first and second dose ( patients, as previously described (Fig. 2B ) (2, 30) . A high level of IgG galactosylation boosted the classical complement pathway activation capacity through enhanced C1q-binding, which was in line with previous reports (Fig. S4 ) (15) . Anti-S IgG1 sialylation follows the galactosylation trend, with an increase after the first and second dose (Fig 2C, S2C) .

We recently hypothesized that afucosylated IgG, hardly seen in responses to soluble protein or polysaccharide antigens, are specifically induced against foreign antigens on host cells (2) . In agreement with this, up to 25% of anti-S IgG1 Fc was found to be afucosylated after vaccination with the BNT162b2 SARS-CoV-2 mRNA vaccine, in comparison to ~6% of afucosylated total IgG1 found in serum or plasma ( Fig. 2D and S2D, S3D ). This pronounced afucosylation pattern was observed only early on in naive individuals after the first dose of BNT162b2, which gradually decreased to levels similar to total IgG1 at four weeks post seroconversion ( Fig. 2D and S2D, S3D ). This early, transient afucosylated response in naive vaccinees after the first dose was less prominent when compared to ICU-admitted COVID-19

patients and most individuals with mild symptoms (Fig. 2D) .

In contrast, antigen-experienced individuals had an anti-S IgG1 afucosylation level of ~2-10% and slightly increased after vaccination (Fig. 2D, S2D) . We further investigated this by expanding the vaccinated antigen-experienced through recruiting vaccinated blood donors previously infected with SARS-CoV-2 (Fig. S5 , Table S5 ). Similar anti-S IgG1 Fc bisection, galactosylation, sialylation, and fucosylation dynamics were observed ( Fig. 2E-H) . Compared to naive individuals, this antigen-experienced cohort showed a significantly lower anti-S IgG1

Fc fucosylation after vaccination (Fig. 2H) . No temporal changes were observed for total IgG glycosylation ( Fig. S2-3 ).

Longitudinal anti-S IgG1 Fc (A) bisection, (B) galactosylation, (C) sialylation, and (D) fucosylation for naive (left, blue, cohort 1 (n=33) and 2 (n=9)), antigen-experienced (middle, yellow, cohort 1 (n=6) and 2 (n=0)) in comparison to mild (grey) and ICU hospitalized (red) COVID-19 patients (right) anti-S IgG1 galactosylation from our previous study (2) . Anti-S IgG1 Fc (E) bisection, (F) galactosylation, (G) sialylation, and (H) fucosylation for the additional vaccinated antigen-experienced plasma donors (purple, cohort 4 (n=22)) before (pre) and after (post) vaccination and (H) in comparison to naive vaccinees at seroconversion (seroconv.) and after the second dose (post) (blue, cohort 1 (n=33) and 2 (n=9)). Differences were assessed using the Mann-Whitney U test (***, ****: p-value < 0.001, 0.0001, respectively). Similar data for cohort 3 are plotted in Fig. S2 . 

We assessed the effector function of the anti-S Abs induced by BNT162b2 mRNA vaccination by testing their capacity to induce macrophage-driven inflammatory responses. For this, we measured IL-6 production by human-derived, in vitro differentiated, alveolar-like macrophages. These were stimulated overnight by exposure to immune complexes (ICs) generated from S protein and vaccinees' sera in the presence and absence of virus-like costimuli (polyinosinic:polycytidylic acid (poly(I:C))) ( Fig. 3A) (31) . Notably, anti-S ICs from antigen-experienced individuals induced significantly higher IL-6 levels compared to naive individuals for all time points after the first dose, with the most pronounced difference seen at day 10 ( Fig. 3B) . IL-6 induction was similar for both groups after the second dose as IgG levels became comparable (Fig. 1D, 3B and S6 ). Despite the clear difference between both groups, IL-6 levels were relatively low for all conditions, which is in line with various previous findings showing that IgG ICs only induce pro-inflammatory cytokine production in the presence of both high afucosylation and antibody levels in the presence of viral or bacterial co-stimulus that activates receptors such as Toll-like Receptors (TLRs) (1, 32, 33) .

To further test the inflammatory capacity of anti-S IgG, we also measured IL-6 production upon TLR co-stimulation with the TLR3 ligand poly(I:C). Upon TLR costimulation, anti-S ICs strongly amplified IL-6 production by human macrophages in both groups (Fig. 3C) . Again, the difference between the vaccinated naive and antigen-experienced individuals was most pronounced around day ten post vaccination ( Fig. 3C) . In both cases, the capacity of the sera to activate these macrophages seem to be explained by antibody levels (Fig.   3D ). However, when anti-IgG levels became comparable after the second dose, the sera of antigen-experienced individuals induced only slightly higher IL-6 levels both with and without poly(I:C) ( Fig. 3B-D and S6 ), which correlated with higher afucosylation levels, but not with other IgG1 glycosylation traits (Fig. S7) . Combined, these data suggest that the transient afucosylated anti-S IgG that is produced after vaccination of naive individuals has little effect on macrophage activation, because it is accompanied by low antibody levels. Correlation between IL-6 levels and anti-S IgG levels in the absence (left) and presence (right) of poly(I:C) stimulation. All data represent a subgroup of cohort 1 (n=23, see Table S2 ).

In line with literature, a highly sialylated IgG glycosylation phenotype was observed early after vaccination, regardless of antigen experience (Fig 2C and S2C, 3C) (14, 34, 35) .

Interestingly, this early, transient, high sialylation was particularly pronounced for fucosylated anti-S IgG1, for both naive and antigen-experienced vaccinees until day fourteen after the first dose ( Fig. 4A and S8 ). Anti-S IgG1 Fc galactosylation levels of neither naive nor antigenexperienced showed a difference between fucosylated and afucosylated anti-S IgG1 ( Fig. 4B and S8). This result led to the hypothesis that early highly galactosylated and sialylated anti-S IgG1 and afucosylated anti-S IgG1 might be produced by different PC subsets.

Next, we analyzed the anti-S1 blood-derived IgG + CD38 + PC subset responses to assess whether they phenotypically diverge in their anti-S IgG1 glycosylation pattern between naive and antigen-experienced individuals (36) (37) (38) . We found CD27 low CD138 -IgG + CD38 + PCs to be dominant in naive individuals after both the first and second dose ( Fig. 4C -G, S9A-G, S10A-F). In contrast, antigen-experienced vaccinees primarily induced CD27 + CD138 -IgG + CD38 + PCs after both doses ( Fig. 4J -K, S9A-I, S10A-F), which was also the dominant subset in total IgG + PCs of the naive antigen unvaccinated controls ( Fig. S10A -F, S11).

We found that a1,6-fucosyltransferase 8 (FUT8; the glycosyltransferase responsible for core fucosylation (39)) protein expression was lowest in the CD27 low CD138 -IgG + PC subset in naive individuals after the first, but not the second dose ( Fig. 4H -I, L-M and S10H). The α2,6-sialyltransferase 1 (ST6GAL1; the glycosyltransferase responsible for α2,6-linked sialylation (35)) protein expression was highest in the CD27 + CD138 + and the lowest in CD27 low CD138 -IgG + PC subset after both doses in naive and antigen-experienced individuals, as well as in total IgG + PCs of unvaccinated healthy control individuals ( Fig. 4I , M and S10G, S11).

In naive individuals, FUT8 expression in CD27 low CD138 -IgG + PCs correlated with anti-S IgG1 fucosylation ( Fig. 4N ).

Sialylation and (B) galactosylation levels of afucosylated (grey) and fucosylated (black) anti-S IgG for naive (left) and antigen-experienced (right) vaccinated participants over time of cohort 1 (n=39) and 2 (n=9) after renormalization by setting the sum of all afucosylated glycoforms to 100% and all fucosylated glycoforms to 100%. Glycosylation levels were compared by a paired t-test. (C-N) Flow cytometry analysis of blood cells gated on single, living lymphocytes from naive and antigen-experienced vaccinees (subset of cohort 3 (n=15), see Table  S2 ) were analyzed 7-14 days upon the first (naive: n=6 and antigen-experienced: n=5) or 5-8 days upon the second (naive: n=15 and antigen-experienced: n=4) dose. (C-E) Gating strategy exemplified for a naive individual (C) pre-immunization and (D) after the first dose. S1-reactive B cells were gated and further gated for CD19 int CD38 + PCs to analyze IgG + PC subsets as defined by (E) CD27 and CD138. (F-G) Naive and (J-K) antigen-experienced vaccinees analyzed according to the gating strategy. (H, L) Relative fucosyltransferase 8 (FUT8; median (MFI)) expression per IgG + PC subset and (I, M) its correlation with relative alpha2,6-sialyltransferase (ST6GAL1) expression. (N) Relative FUT8 expression of CD27 low CD138 -IgG + PCs correlated with anti-S IgG1 Fc fucosylation found in the corresponding serum (Fig. S9C) . The median (MFI) of FUT8 or ST6GAL1 expression in CD138 + IgG+ S1-reactive PCs of each sample was set to 1 for inter-assay comparison. Dotted horizontal lines indicate corresponding values of total IgG + PC subsets from untreated healthy controls (Fig. S11) . 

The mRNA vaccine-induced presentation of the viral S protein on the membrane of host cells mimics the S protein presentation during natural SARS-CoV-2 infections. Enveloped viruses, attenuated enveloped viral vaccines, but also P. falciparum-infected erythrocytes and alloantigens on blood cells express antigens on host cells and induce persistent afucosylated IgG responses (2, (22) (23) (24) (25) 40) . Although we previously found afucosylated IgG responses to be associated with strong pro-inflammatory responses in critically ill COVID-19 patients (1, 2) , this type of response seems to be protective in HIV infections (24) and malaria (23) .

Here, we show for the first time that the BNT162b2 mRNA vaccine induces afucosylated anti-S IgG1 responses in SARS-CoV-2 naive individuals upon seroconversion, which decreases within four weeks to the level of total IgG1. Recent work from Farkash et al.

and Chakraborty et al. did not pick up this transient response due to sampling of two and four weeks after the first dose, respectively (26, 41) . This afucosylated response was similar, but less pronounced than observed in natural SARS-CoV-2 infections (2, 30) . The transient afucosylated IgG1 glycosylation pattern after vaccination suggests that a co-stimulus may be missing to induce memory B cells and long-lived IgG + PCs producing stable anti-S IgG1 afucosylation levels. Alternatively, a missing local type of inflammatory signal might provide a negative feedback steering developing B cells to produce fucosylated IgG. In contrast, SARS-CoV-2 antigen-experienced vaccinees start off with low (~2-10%), but persistent anti-S IgG1 afucosylation levels which slightly increase upon vaccination, assuming re-activation of memory B cells generating afucosylated IgG antibodies.

Our analyses revealed that the differences in the effector functions elicited by anti-S ICs on macrophages between naive and antigen-experienced vaccinees mainly depend on the titer after the first dose, with afucosylation only being a secondary factor. Our previous work has shown that exaggerated pro-inflammatory responses were only observed with serum containing high titers of considerably afucosylated IgG1 (>10%) (1, 2) . Such afucosylated IgG1 levels in this study were only observed early after seroconversion in naive individuals and not in combination with high titers. In line with this, anti-S ICs from vaccinee's sera induced very moderate pro-inflammatory cytokine levels in the absence of TLR co-stimulation, suggesting low inflammatory side effects in both groups after immunization with the BNT162b2 mRNA vaccine. Nevertheless, when comparing naive and antigen-experienced vaccinees after the second dose, when anti-S IgG levels were comparable, antigen-experienced individuals induced slightly higher IL-6 production in the macrophage activation assay, which is in agreement with their higher levels of afucosylated IgG.

Moreover, the different immune responses of naive versus antigen-experienced individuals upon vaccination were reflected in the antigen-specific PC response. Whereas naive individuals primarily induced CD27 low CD138 -IgG + CD38 + PCs, antigen-experienced individuals primarily induced CD27 + CD138 -IgG + CD38 + PCs after both doses. Furthermore, only the naive subpopulation showed reduced FUT8 expression in CD27 low CD138 -IgG + CD38 + PCs only after the first vaccination, which correlated with the amount of afucosylated IgG1 observed in these individuals (26) . The existence of an IgG + PC subset responsible for the biosynthesis of afucosylated IgG Abs in antigen-experienced individuals has yet to be identified in further studies.

In accordance with previous reports on immunization (14, 34, 35) and Fc glycosylation after BNT162b2 mRNA vaccination (26, 41) , we also observed transiently, highly sialylated anti-S IgG1 at one to two weeks after both the first and second dose, which has been suggested to facilitate antigen presentation in subsequent GC reactions for improving affinity maturation (14, 42) . Furthermore, the anti-S IgG1 for both naive and antigen-experienced vaccinees was extensively galactosylated. These high levels of IgG galactosylation have been shown to boost the capacity to activate the classical complement pathway, through enhanced C1q-binding, in line with recent findings that have shown galactosylation promotes IgG1 hexamerization ultimately leading to increased C1q-binding and ensuing classical complement activation (15, 16, 41) .

Afucosylated IgG1 may support antigen presentation on antigen-presenting cells through FcγRIIIa, as well as inducing better T-helper and memory B cell responses, and subsequent booster responses (43) (44) (45) . In support of this, we observed that early afucosylated anti-S IgG1 responses correlated significantly with anti-S IgG levels after the second dose in naive individuals (41) . At seroconversion, afucosylated anti-S IgG1 Abs in naive vaccinees might provide enhanced protection, even without high titers. Over time, when afucosylated IgG levels drop, protection in these individuals might be compensated by the increased anti-S IgG levels, which should be considered for the timing of subsequent vaccination. Furthermore, reduced levels of anti-S IgG1 afucosylation might reduce the risk of pro-inflammatory side effects, with a trade-off of dampened Fc-mediated effector functions upon pathogen contact. In antigen-experienced individuals, matters are reversed, as these individuals start off with lower afucosylated anti-S IgG levels prior to vaccination, which significantly increased after vaccination. This suggests an enhanced corresponding memory B cell response, which would be in line with stronger protection in this group (46) (47) (48) . Similarly, a gradual increase in afucosylation has been observed with repeated natural immunizations to antigens displayed on the membrane of P. falciparum-infected red blood cells (23) . This is in contrast to alloimmunization to the red blood cell RhD antigen, where the afucosylated response in hyperimmune donors is very stable over time (40) . The increased level of afucosylated anti-SARS-CoV2 IgG in vaccinated antigen-experienced individuals might have a positive impact on the therapeutic effect of convalescent plasma, as especially these donors are presently selected for clinical trials and it has been shown that increased ADCC activity of the administered antibodies is positively correlated with outcome.

Due to the limited sample size, we did not stratify study participants according to sex and age, which may influence IgG glycosylation profiles (18) . However, outside of the context of a specific pathology, total IgG fucosylation levels remain constant throughout life with the exception of an initial decrease after birth (19) . A second limitation of our study is the uneven sample size for naive and antigen-experienced vaccine recipients after the first and second dose of BNT162b2. This is largely due to lack of an accessible, high-throughput serological assay to measure antigen-specific IgG glycosylation to study both transient and stable glycosylation features in disease settings.

In summary, our data demonstrate a qualitatively and quantitatively distinct IgG 

This study was designed to investigate the effect of the BNT162b2 BioNTech/Pfizer mRNA vaccine on anti-Spike IgG1 Fc glycosylation and PC subsets. We obtained serum, plasma and/or PBMC samples from vaccinated participants from 1) healthcare works at the Amsterdam UMC,

The Netherlands ( Fig. S1 ) received the 2 nd dose between day 32 and 37 after the 1st) and 2) and 8 unvaccinated individuals without SARS-CoV-2 infection history as negative control (Table S4 ). Blood samples were collected after obtaining written informed consent under the local ethics board-approved protocols 19-019(A) and 20-123 (Ethics Committee of the University of Lübeck, Germany).

Sanquin blood donors (n=22) found seropositive for SARS-CoV-2 prior to vaccination were included in the study ( All studies complied with the latest version of the Declaration of Helsinki.

Anti-S IgG Abs levels were measured by coating MaxiSorp NUNC 96-well flat-bottom plates (Thermo Fisher Scientific, Roskilde, Denmark) overnight with 1 µg/ml recombinant, inhouse produced trimerized spike protein in PBS, as described before (49) . The following day, plates were washed five times with PBS supplemented with 0.02% polysorbate-20 (PBS-T) and

incubated for 1 hour with a dilution range of plasma from the Amsterdam UMC cohort in PBS-T supplemented with 0.3% gelatin (PTG). A serially diluted plasma pool, obtained by combining plasma from a collection of convalescent COVID-19 donors (50) (51).

measured as by an RBD and N-based bridging assay, respectively, as described previously (50, 51) .

To detect anti-S1 IgG as well as anti-NCP IgG Abs, serum samples were collected on the indicated days (Table S4) 

Anti-S IgG Abs were affinity-captured from plasma or sera using recombinant, in-house produced trimerized spike protein-coated plates (Thermo Fisher Scientific, Roskilde, Denmark) followed by a 100 mM formic acid elution step, as described elsewhere (2, 49) . Netherlands), respectively, as described elsewhere (2, 30, 52) .

Eluates from both anti-S and total IgG affinity-purification were dried by vacuum centrifugation and subjected to tryptic cleavage followed by LC-MS analysis as described previously (2, 30) .

Raw LC-MS spectra were converted to mzXML files. LaCyTools, an in-house developed software was used for the alignment and targeted extraction of raw data (53) .

Alignment was performed based on average retention time of at least three high abundant glycoforms. The analyte list for targeted extraction of the 2 + and 3 + charge states was based on manual annotation as well as on literature reports (2, 54) . Inclusion of an analyte for the final data analysis was based on quality criteria including signal-to-noise (higher than 9), isotopic pattern quality (less than 25% deviation from the theoretical isotopic pattern), and mass error (within a ±20 parts per million range) leading to a final analyte list (Table S5) . Relative intensity of each glycan species in the final analyte list was calculated by normalizing to the sum of their total areas. Normalized intensities were used to calculate fucosylation, bisection, galactosylation and sialylation (Table S6) .

Pierce™ Nickel Coated Clear 96-well plates (Thermo Fisher Scientific, #15442) were incubated with 100 µL of 1 µg/mL purified RBD-protein for 1 hour at RT. Hereafter, the plates were washed five times with 0.05% PBS-Tween20 and incubated with 100 µL glycoengineered COVA1-18 (2C1) hIgG1 mAbs for 1 hour at RT (1, 49) . A two-fold dilution series was used, with a starting concentration of 20 µg/ml. Subsequently the plates were washed and 100 µL of 1:35 pooled human serum in Veronal Buffer (5) with 0.1% poloxamer 407, 2 mM MgCl2 and 10 mM CaCl2 was added and incubated for 1 hour at RT, as described previously (15) .

Consequently, the plates were washed and 100 µL 1/1000 anti-C1q-HRP (55) (56) (57) 

Supernatants of stimulated alveolar-like monocyte-derived macrophages (MDMs) were harvested after 24 hours to determine cytokine production. IL-6 levels in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA) using IL-6 CT205-c and CT205-d antibody pair (U-CyTech, Utrecht, the Netherlands) as described previously (1).

Buffy coats from healthy donors were obtained from Sanquin Blood Supply (Amsterdam, the Netherlands). Monocytes were isolated from buffy coat by density gradient centrifugation using Lymphoprep TM (Axis-Shield, Dundee, Scotland) followed by CD14 + selection via magnetic cell separation using MACS CD14 MicroBeads and separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described (31 

96-well high affinity plates were coated with 2 µg/ml soluble perfusion stabilized Spike protein as described previously (1). After overnight incubation, plates were blocked with 10 % FCS in PBS for 1 hour at 37 °C. Diluted heat-inactivated serum ( without or supplemented with 20 µg/ml polyinosinic:polycytidylic acid (poly(I:C)) (Sigma-Aldrich, Darmstadt, Germany).

Blood samples were collected at the indicated days in EDTA-tubes and processed or frozen within the next three hours for flow cytometric analysis (Attune Nxt; Thermo Fisher Scientific) of different B cell populations (28) . Peripheral blood mononuclear cells ( 

The log10 values of the anti-spike IgG titers were used for the correlation analyses between log10 values of the measured concentrations of IL-6 (in pg/ml). The percentages of anti-S IgG1 glycosylation traits were used for the color overlay. The Pearson correlation coefficient (R) and associated P-value are stated in each graph. For the comparison of the IL-6 concentration produced by alveolar-like macrophages, an unpaired t-test was performed.

These analyses were performed in the R statistical environment (v3.6.3).

Other statistical analyses were performed using GraphPad Prism v6.0 (GraphPad, La Jolla, CA). Differences in anti-S IgG1 glycosylation for antigen-experienced vaccinees were assessed with Wilcoxon matched-pairs signed rank test. Differences between naive and antigen-experienced vaccinees two unpaired groups were assessed with Mann-Whitney U test and differences in sialylation and galactosylation for fucosylated and afucosylated anti-S IgG1 were determined by a paired t-test.The correlations between anti-S IgG1 titer and afucosylation was determined by linear regression.

P-values < 0.05 were considered as significant. Asterisks indicate the degree of significance as follows: *, **, ***, ****: p-value < 0.05, 0.01, 0.001, 0.0001, respectively.

We thank the Academic Medical Centre of the University of Amsterdam, the Sanquin

Group. We are greatly indebted to all cohort participants for their extensive participation. 

The authors declare that they have no conflicts of interest. f 22 0, 12, 22(0), 25 (3), 36(14) , 49(27) 1249 46 m 21 1, 5, 7, 9, 14, 21(0) , 26(5) , 28 (7), 30(9) , 35(14) , 50 (29) (7), 36(14) , 49(27) 1485 39 m 21 1, 5, 9, 13, 21(0) , 23 (2), 27 (6), 42 (21), 48 (27) Yes 3, 5, 10, 14, 21(9) , 24(3), 26 (5), 32 (11), 34(13) , 42 (21), 49 (28) Yes 4, 7, 11, 14, 20(0) , 22 (2), 35(15) , 49 (27) Yes 4, 11, 15, 22(0), 35(13) , 41(19) , 49(27) 1953 4, 6, 10, 14, 19, 24(3) , 27 (6), 32(11), 35(14) , 42(21) , 49 (28) Figure S1 . Anti-S and -N serum IgG Ab levels of cohort 3. (A-B) Sera of naive (blue) and antigen-experienced (yellow) individuals from a subset of cohort 3 (see Table S4 ) were analyzed by EUROIMMUN (EURO) anti-SARS-CoV-2-Spike1 (S1) and -Nucleocapsid (N) IgG ELISA. (C) Correlation between EURO the anti-S1 and -N IgG levels before vaccination. Dotted lines are reference values as determined by the company. (D) HL-1 anti-S1 IgG ELISA. (E) Correlation between the EUROIMMUN and HL-1 anti-S1 IgG ELISA data. (F) HL-1 anti-S2 IgG ELISA. (G) HL-1 anti-S1 and -S2 IgG1-4 ELISA. The data of the naive-considered individual that showed enhanced anti-N IgG levels before vaccination were marked in dark red in all graphs. All data represent a subgroup of cohort 1 (n=23, see Table S2 ). Figure S8 . Anti-S IgG1 Fc glycopeptide composition. Anti-spike (S) IgG1-Fc glycopeptides composition for naive (left, cohort 1 (n=33) and 2 (n=9)) and antigen-experienced (right, cohort 1 (n=6) and 2 (n=0)) over time. H: hexose; N: Nacetylhexosamine; F: fucose; S: N-acetylneuraminic (sialic) acid. anti-S1 IgG (Ratio to reference value) 

IgG S1 PCs (counts) + + ST6GAL1 in IgG S1 PC subsets + + FUT8 in IgG S1 PC subsets + + + + + + + IgG (%) of S1 PCs S1 B cells (%) of lymph. S1 PCs (counts) PCs (%) of S1 B cells

Cells ( Figure S10 . Counts, frequencies and glycosyltransferase expression of IgG + S1-reactive PC subsets from naive and antigen-experienced individuals described in Fig. 4C-N and S8. (A-F) Counts and frequencies of S1-reactive B cells, S1reactive CD38 + PCs and IgG + S1-reactive CD38 + PCs from naive (blue) and antigen-experienced (yellow) individuals. (G, H) IgG + S1-reactive CD38 + PCs were subdivided in CD27 + CD138 -(grey), CD27 + CD138 + (red), CD27 low CD138 -(blue) and CD27 low CD138 + (pink) subsets and analyzed for (G) ST6GAL1 or (H) FUT8 expression. Example overlay histograms and the relative glycosyltransferase expression (median (MFI) of all samples are shown. The median (MFI) of ST6GAL1 or FUT8 expression in CD138 + IgG+ S1-reactive PCs of each sample was set to 1 for inter-assay comparison. The fine dotted horizontal lines indicate corresponding values of total IgG+ PC subsets from untreated healthy controls (Fig. S10) . PCs. (B, C) Counts and frequencies of PCs, IgG + PCs and CD27 + CD138 -(grey), CD27 + CD138 + (red), CD27 low CD138 -(blue) and CD27 low CD138 + (pink) IgG + PC subsets gated as described in Fig. 4 , S8 and S9. (D) Relative ST6GAL1 or FUT8 expression median (MFI) in the four IgG + PC subsets. The median (MFI) of ST6GAL1 or FUT8 expression in CD138 + IgG+ PCs of each sample was set to 1 for inter-assay comparison. Anti-S fucosylation at seroconversion (%) Anti-S bisection at seroconversion(%) Anti-S galactosylation at seroconversion (%) Anti-S sialylation at seroconversion (%) Figure S13 . Anti-Spike IgG titer after the first and second dose correlate. (A) Correlation by linear regression between anti-Spike (S) titer from cohort 1 (n=39) and 2 (=8), when timing of sampling matched selection criteria, after the 1 st dose (on the day of the 2 nd dose up until three days prior) and after the 2 nd dose (highest titer up to 14 days after the 2 nd dose). (B-I) Correlation by linear regression between anti-Spike (S) IgG1-Fc glycoprofiling (left: bisection, middle left: galactosylation, middle right: sialylation, and right: fucosylation) and titer for naive vaccines from cohort 1 (n=33) and 2 (n=9) when the timing of sampling matched selection criteria. (B-E) Correlation of anti-S IgG1-Fc glycosylation upon seroconversion and (F-I) after the first dose (on the day of the 2 nd dose up until three days prior) with titer after the first dose (on the day of the 2 nd dose up until three days prior).

High titers and low fucosylation of early human anti-SARS-CoV-2 IgG promote inflammation by alveolar macrophages

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity

Proinflammatory IgG Fc structures in patients with severe COVID-19

Decoding the human immunoglobulin G-glycan repertoire reveals a spectrum of Fc-receptorand complement-mediated-effector activities

Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity

Unique carbohydratecarbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose

Novel concepts of altered immunoglobulin G galactosylation in autoimmune diseases

Immunoglobulin G glycosylation in aging and diseases

Glycosylation Profile of Immunoglobulin G Is Cross-Sectionally Associated With Cardiovascular Disease Risk Score and Subclinical Atherosclerosis in Two Independent Cohorts

Sialylated autoantigen-reactive IgG antibodies attenuate disease development in autoimmune mouse models of Lupus nephritis and rheumatoid arthritis

IgG Fc N-Glycosylation Translates MHCII Haplotype into Autoimmune Skin Disease

Changes in antigen-specific IgG1 Fc N-glycosylation upon influenza and tetanus vaccination

Anti-HA Glycoforms Drive B Cell Affinity Selection and Determine Influenza Vaccine Efficacy

Fc Galactosylation Promotes Hexamerization of Human IgG1, Leading to Enhanced Classical Complement Activation

Fc galactosylation follows consecutive reaction kinetics and enhances immunoglobulin G hexamerization for complement activation

Functional Attributes of Antibodies, Effector Cells, and Target Cells Affecting NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity

High-throughput IgG Fc N-glycosylation profiling by mass spectrometry of glycopeptides

Changes in healthy human IgG Fc-glycosylation after birth and during early childhood

Fc-Glycosylation in human IgG1 and IgG3 is similar for both total and Anti-Red-Blood cell anti-k antibodies

Low anti-RhD IgG-Fc-fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn

Regulated glycosylation patterns of IgG during alloimmune responses against human platelet antigens

Afucosylated Plasmodium falciparum-specific IgG is induced by infection but not by subunit vaccination

Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity

Antibody fucosylation predicts disease severity in secondary dengue infection

Early non-neutralizing , afucosylated antibody responses are associated with COVID-19 severity

Distinguishing features of current COVID-19 vaccines: knowns and unknowns of antigen presentation and modes of action, npj Vaccines

The BioNTech / Pfizer vaccine BNT162b2 induces class-switched SARS-CoV-2-specific plasma cells and potential memory B cells as well as IgG and IgA serum and IgG saliva antibodies upon the first immunization, medRxiv

Symptomatic SARS-CoV-2 infections display specific IgG Fc structures

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Meta-Analysis of in vitro-Differentiated Macrophages Identifies Transcriptomic Signatures That Classify Disease Macrophages in vivo

Physiological and Pathological Inflammation Induced by Antibodies and Pentraxins

Fc gamma receptor-TLR cross-talk elicits pro-inflammatory cytokine production by human M2 macrophages

Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface

IgG Fc sialylation is regulated during the germinal center reaction following immunization with different adjuvants

Differential transcriptome and development of human peripheral plasma cell subsets

Elucidation of seventeen human peripheral blood B-cell subsets and quantification of the tetanus response using a density-based method for the automated identification of cell populations in multidimensional flow cytometry data

Challenges and Opportunities for Consistent Classification of Human B Cell and Plasma Cell Populations

Characterizing human α-1

substrate specificity and structural similarities with related fucosyltransferases

A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy

Anti-SARS-CoV-2 antibodies elicited by COVID-19 mRNA vaccine exhibit a unique glycosylation pattern

Antigen-specific antibody Fc glycosylation enhances humoral immunity via the recruitment of complement

Viral replication in human macrophages enhances an inflammatory cascade and interferon driven chronic COVID-19 in humanized mice

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release

Immune imprinting, breadth of variant recognition and germinal center response in human SARS-CoV-2 infection and vaccination

Fc-engineered antibody therapeutics with improved anti-SARS-CoV-2 efficacy

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Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection

Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability

Development of a SARS-CoV-2 Total Antibody Assay and the Dynamics of Antibody Response over Time in Hospitalized and Nonhospitalized Patients with COVID-19

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors

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LaCyTools: A Targeted Liquid Chromatography-Mass Spectrometry Data Processing Package for Relative Quantitation of Glycopeptides

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Evidence That Complement Protein C1q Interacts with C-Reactive Protein through Its Globular Head Region

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The bacteria binding glycoprotein salivary agglutinin (SAG/gp340) activates complement via the lectin pathway

 1, 7, 14, 26(5) , 28 (7), 30 (9), 35 (14), 42 (21), 49 (28) 1561 26 f 23 1, 6, 8, 12, 14, 23(0) , 26 (3), 30 (7), 44 (21), 51 (28) Yes (28) 1675 23 f Yes 287 21 1, 4, 8, 11, 14, 21(0) , 26 (5), 28 (7), 33 (12), 42 (21), 49 (28) Yes 1897 (5), 28 (7), 32 (11), 35(14) , 43 (22), 49 (28) Yes 2, 9, 13, 21(0) , 23 (2), 28 (7), 34 (13), 49 (28) Yes 3, 7, 10, 11, 14, 21(0) , 24 (3), 29 (8), 31 (10), 36 (15), 49 (28) Yes 4, 7, 11, 15, 21 (0), 25(4), 28 (7), 33 (12), 36 (15), 43(22), 49 (28) Yes 1507 60 f 21 1, 5, 7, 9, 14, 21(0) , 28 (7), 30 (9), 35 (14), 42 (21), 49 (28) Yes -4, 2, 7, 10, 14, 21, 24(2) , 30 (8), 38 (16), 44 (22), 49 (27) Yes 5, 7, 14, 21(0) , 23 (2), 27 (6), 36 (15), 42 (21), 49 (28) 1540 43 f 22 1, 7, 9, 14, 22(0) , 26(4), 28 (6), 34 (12), 37 (15), 50 (28) (4), 29 (7), 33 (11), 36 (14), 44 (22), 50 (28) 1109 57 m 21 1, 7, 9, 14, 21(0) , 26 (5), 29 (8), 35 (14) , 42 (21), 50 (29) Yes 1073 30 f Yes 57 23 1, 8, 13, 23 (0) , 30 (7), 36 (13), 49(26) 1169 44 f 24 3, 7, 10, 14, 21, 27(3) , 31 (7), 34 (10), 38 (14), 49 (25) Yes 3, 7, 13, 21(0) , 24 (3), 28 (7), 31 (10), 35 (14), 49 (28) 1317 39 f 21 0, 3, 7, 11, 21(0) 1291 29 m 21 0, 5, 8, 13, 15, 21(0) , 27 (6), 32 (11), 48(27) Yes 4, 5, 8, 11, 15, 22(0) , 25 (3), 29 (7), 32 (10), 41(19) , 49 (27) Yes 4, 7, 14, 21(0) , 25 (4), 29 (8), 34 (13), 42 (21), 49 (28) 1219 25 f 21 1, 5, 7, 9, 21(0) , 23 (2), 28 (7), 30 (9), 35 (14), 42 (21), 49 (28) 1987 30 f 21 1, 5, 7, 14, 21(0) , 26 (5), 28 (7), 30 (9), 35 (14), 42 (21) 3, 7, 11, 14, 17, 21(0) , 24 (3), 28 (7), 32 (11), 35(14) , 39 (18) -1, 3, 7, 10, 14, 17, 20, 24(3) , 28 (7), 30 (9), 35 (14), 38 (17) 1, 2, 6, 9, 13, 16, 20, 23(2) , 27 (6), 30 (9), 34 (13) (3), 28 (7), 31 (10), 35 (14), 38 (17), 42 (21) 35 -2, 22, 29, 40 (5) , 62 (27) 40 (5) 22 22