key: cord-323041-wf0hlhim
authors: Larsen, Mads Delbo; de Graaf, Erik L.; Sonneveld, Myrthe E.; Plomp, H. Rosina; Linty, Federica; Visser, Remco; Brinkhaus, Maximilian; Šuštić, Tonći; de Taeye, Steven W.; Bentlage, Arthur E.H.; Nouta, Jan; Natunen, Suvi; Koeleman, Carolien A. M.; Sainio, Susanna; Kootstra, Neeltje A.; Brouwer, Philip J.M.; Sanders, Rogier W.; van Gils, Marit J.; de Bruin, Sanne; Vlaar, Alexander P.J.; Zaaijer, Hans L.; Wuhrer, Manfred; van der Schoot, C. Ellen; Vidarsson, Gestur
title: Afucosylated immunoglobulin G responses are a hallmark of enveloped virus infections and show an exacerbated phenotype in COVID-19
date: 2020-05-18
journal: bioRxiv
DOI: 10.1101/2020.05.18.099507
sha: 
doc_id: 323041
cord_uid: wf0hlhim

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 binding and killing activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG which are of minor abundance in humans (∼6% of total IgG) are specifically formed against surface epitopes of enveloped viruses after natural infections or immunization with attenuated viruses, while protein subunit immunization does not elicit this low fucose response. This can give beneficial strong responses, but can also go awry, resulting in a cytokine-storm and immune-mediated pathologies. In the case of COVID-19, the critically ill show aggravated afucosylated-IgG responses against the viral spike protein. In contrast, those clearing the infection unaided show higher fucosylation levels of the anti-spike protein IgG. Our findings indicate antibody glycosylation as a potential factor in inflammation and protection in enveloped virus infections including COVID-19.

Generally, 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 (11-13). 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 (14, 62 Inspired by the similarities between the unique afucosylated IgG responses in various 68 alloimmune responses (10-12, 18), HIV (15) and Dengue(14) -all being directed against 69 surface exposed and membrane embedded proteins -we analyzed IgG glycosylation in anti- To investigate the Fc-glycosylation of total-and antigen-specific antibodies, first IgG from 76 >400 human serum samples was affinity-purified using protein G affinity beads and 77 immobilized antigens, respectively. Thereafter, isolated IgG was digested with trypsin and 78 resulting IgG1-Fc-glycopeptides were analyzed with liquid chromatography-mass 79 spectrometry (LC-MS) ( Fig. 2A) (11, 16, 18 ). Subsequently, intensities were extracted and IgG-80 glycosylation profiles were calculated ( Fig. 1B-C) . t-test for A,B, and C and as a one-way ANOVA Sidak's multiple comparisons test comparing 106 total IgG to antigen-specific IgG within groups, and same specificity IgG between groups, for 107 D and E. Only statistically significant differences are shown. *= p<0.1, **= p<0.01, ***= 108 p<0.001, ****= p<0.0001. 109 Analogous to the platelet and Red Blood cell alloantigens (10-12, 18), the response to these 110 enveloped viruses also showed significant afucosylation of the antigen-specific IgG (Fig.2B) , 111 while the afucosylation was absent against the non-enveloped virus Parvo B19 (Fig.2C ). Of note, total IgG showed high fucosylation levels throughout ( Fig.2A-C) , underlining that the 113 majority of human IgG responses consists of fucosylated IgG responses (11, 16, 19) . The 114 extent of the response to the enveloped viruses was highly variable, both between 115 individuals and between the types of antigen, which is in agreement with the variable 116 tendency of different RBC-alloantigens to induce an afucosylated response (18).

Afucosylation was particularly strong for CMV and to a lesser degree for HIV (Fig. 2B) . The to their natural infection counterpart (Fig. 2E, Fig. S3 ). Both showed reduction, with a more 144 prominent difference for the mumps response (Fig. 2E, Fig S3) . Other glycan traits for anti-145 measles and anti-mumps are shown in Fig. S1 . 146 We then tested if this type of response also plays a role in patients with SARS-CoV-2 (COVID- or non-enveloped viruses (Fig. 4) . The Netherlands were used as controls. Washing and eluting specific antibodies was done as 290 described above for CMV-specific antibodies. (Table S1 ) was at least 351 higher than background plus 10 times its standard division, otherwise the data was excluded (11). The total level of glycan traits was calculated as described in Table S2 . No correlation was found between the level of IgG1 Fc-fucosylation made during alloimmunization against HPA1a in pregnancy (y-axis) and CMV (x-axis) in the same individual. b. Also no correlation was found between the level of IgG1 Fc-fucosylation made against HIV (y-axis) and CMV (x-axis) in the same individual. Statistical analysis was performed using Pearson correlation.

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 Fig. S3 : Fucosylation of anti-Measles. Compared to total IgG fucosylation, the antigen-specific IgG fucosylation, of anti-measle antibodies was only significantly lowered in the younger vaccinated cohort (mean age 19.5). This is likely to be masked by the natural tendency of lowered total IgG-fucosylation with increasing age (9), as the naturally infected cohort (before introduction of the MeV/MuV vaccination program in 1980s in the Netherlands) is older than the vaccine cohort (average 63.5 vs 19.5 years, respectively). In line with this, the total IgG fucosylation of the older cohort showed significantly lowered total-IgG fucosylation compared to the younger vaccinated cohort / Statistical analysis was performed as paired t-test for A,B, and C and as a one-way ANOVA Sidak's multiple comparisons test comparing total IgG to antigenspecific IgG within groups, and same specificity IgG between groups, for D and E. Only statistically significant differences are shown. (*= p<0.1 )