key: cord-0711992-v2kv2vul authors: Petrović, Tea; Alves, Inês; Bugada, Dario; Pascual, Julio; Vučković, Frano; Skelin, Andrea; Gaifem, Joana; Villar-Garcia, Judit; Vicente, Manuel M; Fernandes, Ângela; Dias, Ana M; Kurolt, Ivan-Christian; Markotić, Alemka; Primorac, Dragan; Soares, Adriana; Malheiro, Luis; Trbojević-Akmačić, Irena; Abreu, Miguel; Sarmento e Castro, Rui; Bettinelli, Silvia; Callegaro, Annapaola; Arosio, Marco; Sangiorgio, Lorena; Lorini, Luca F; Castells, Xavier; Horcajada, Juan P; Pinho, Salomé S; Allegri, Massimo; Barrios, Clara; Lauc, Gordan title: Composition of the immunoglobulin G glycome associates with the severity of COVID-19 date: 2020-11-10 journal: Glycobiology DOI: 10.1093/glycob/cwaa102 sha: 73361f54925be294ff6245a69d33f5ce12e2c767 doc_id: 711992 cord_uid: v2kv2vul A large variation in the severity of disease symptoms is one of the key open questions in COVID-19 pandemics. The fact that only a small subset of people infected with SARS-CoV-2 develop severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the IgG glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of inter-individual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting N-acetylglucosamine (GlcNAc) in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing. was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing. The knowledge about SARS-CoV-2 virus and COVID-19 increased tremendously in the last few months, but it is still unclear why some infected patients have a very mild disease, or even no symptoms, while others develop the serious disease with considerable mortality. Over a million COVID-19 related deaths have been reported so far, but a significant number of people (even over 80% in some populations) infected with SARS-CoV-2 manage to contain infection only to their upper respiratory tract and despite being positive for the virus do not develop any visible symptoms (Oran & Topol, 2020) . Many factors have been raised as clinical predictors of worse disease progression, such as obesity, diabetes, hypertension, kidney injury and in general, the previous cardiovascular disease burden of the patient (Williamson et al., 2020) . Undoubtedly, age is the most important factor predicting severity with 100-fold difference in mortality risk in different age groups (Sehra et al., 2020) . Several independent studies suggested that environmental factors (temperature and humidity) play an important role for viral transmission, but also for severity of the disease (Kifer et al., 2020; Lauc et al., 2020) . IgG glycome composition is an essential component of the immune system that regulates inflammation at multiple levels (Nimmerjahn & Ravetch, 2008; Seeling et al., 2017) and is considered to be one of the important drivers of inflammaging (Franceschi et al., 2018) . Many observational and molecular studies of the IgG glycome identified and confirmed its role of both a biomarker and a functional effector of inflammation that contributes to the development of different inflammatory diseases (Lauc et al., 2016) . Moreover, it was shown that COVID-19 patients had substantial differences in anti-glycan antibodies, IgG and IgM, as well as unusual antibodies to self-glycans, e.g. N-glycans, LacNAc-containing glycans, blood group H, gangliosides, and sialyl Lewis X, compared to healthy controls (Butler & Gildersleeve, 2020) . IgG glycome composition has not yet been thoroughly addressed in COVID-19 infection, especially in the context of disease severity. A recent (unreviewed) small study suggested that the generation of afucosylated antigen-specific IgG may be an important element in the defense against SARS-CoV-2 and other enveloped viruses (Larsen et al., 2020) . In another unreviewed study antigen-specific IgG Fc fucosylation in PCRdiagnosed COVID-19 patients was reduced compared to SARS-CoV-2-seropositive children and relative to adults with symptomatic influenza virus infections (Chakraborty et al., 2020) . To address this question, we analysed the total IgG N-glycome composition in three independent cohorts of COVID-19 patients. IgG glycome composition was analyzed in 167 patients with mild (without need of admission to hospital intensive care unit and mechanical ventilation) and 166 patients with severe form (with need of admission to hospital intensive care unit and mechanical ventilation, or those who deceased during hospitalization) of COVID-19 from three independent cohorts. The descriptive information about included patients is presented in Table I . Total IgG N-glycome (combined Fc and Fab glycans) composition was determined by ultra-high-performance liquid chromatography (UHPLC) analysis of glycans labelled with2-aminobenzamide (2-AB) as described in the Materials and methods section. Statistical analysis was performed on main summary features of the IgG glycome composition (G0glycans without galactose, G1glycans with one galactose, G2glycans with two galactoses, Spercentage of all glycans with sialic acid, Fpercentage of fucosylated glycans, and Bpercentage of glycans with bisecting GlcNAc). The analysis of differences between severe and mild cases was performed using a logistic regression model with sex and age included as additional covariates. Significant difference in the IgG glycome composition in severe and mild COVID-19 patients was observed (Table II, Galactosylation was also consistently decreased in severe cases in all three cohorts, but the statistical significance of this difference was observed only for monogalactosylation in Barcelona cohort (effect= -0.34; p=0.016). Consistent changes in the levels of sialylated and fucosylated IgG glycan structures between mild and severe COVID-19 cases were not detected. In this study we found consistent differences in IgG glycome composition between patients with mild and severe COVID-19. The level of bisecting GlcNAc was decreased in severe patients (effect = -0.34), replicated in all three cohorts. Despite the small size of each cohort, this change was statistically significant even after adjusting for multiple testing (adjusted meta-analysis p = 0.009). Higher levels of bisecting GlcNAc on IgG are often associated with increased FcγRIII binding and enhanced antibody-dependent cell cytotoxicity (ADCC), explaining a more pro-inflammatory effector functions of IgGs (Irvine & Alter, 2020; Umana et al., 1999) . If this study didn't have a cross-sectional design, it would allow us to distinguish whether the observed associations reflect a pre-existing risk factor, or rapid changes in IgG glycosylation that occurred during the disease. This question will be addressed in a longitudinal study that we are currently setting up. A recent small study on antigen-specific antibodies found a decrease in bisecting GlcNAc specifically on anti-SARS-CoV2 antibodies (Larsen et al., 2020) , which is consistent with changes in the total IgG glycome observed in our study. Interestingly, in the same study anti-SARS-CoV2 antibodies had higher galactosylation and sialylation (relative to the total IgG glycome composition), which is the opposite of what we have observed in this study. This difference suggests that the observed differences in IgG glycosylation between mild and severe cases may not be a simple reflection of the increased proportion of newly synthetised anti-SARS-CoV2 antibodies in the total IgG pool but also of a pre-existing risk factor for more severe COVID-19. However, another study found that IgG1 against the receptor binding domain of the SARS-CoV-2 spike protein from COVID-19 patients had significantly lower core fucosylation, galactosylation and bisection when compared with total IgG1 from healthy adult controls (Chakraborty et al., 2020) , suggesting that these changes/differences may be very individual and that additional studies are needed. "Cytokine storm" and over-activation of the immune system have been suggested key features of severe COVID-19 (Catanzaro et al., 2020) , but this significantly varies from patient to patient (Calfee et al., 2014) and increased levels of pro-inflammatory cytokines are not present in all severe patients (Kox et al., 2020) . Inter-individual differences in IgG glycosylation are large and reflect both genetic and environmental contributing factors (Klarić et al., 2020; Pučić et al., 2011) . IgG glycans are known effectors of the immune system and composition of the IgG glycome associates with different diseases (Gudelj et al., 2018) . It has been shown that age and excess adiposity is a risk factor for severe disease and 7 mortality in people with SARS-CoV-2 infection (Seidu et al., 2020) . Furthermore, age (Krištić et al., 2014) and adiposity (Russell et al., 2019) IgG was isolated from plasma using a 96-well protein G monolithic plate (BIA Separations, Slovenia) (Pučić et al., 2011) , as previously described (Trbojević-Akmačić et al., 2017). After IgG isolation, IgG eluates were heated at 65 °C for 30 minutes to reduce risk from any potential residual virus in the IgG eluate, and 300 μL of each eluate was dried in a vacuum concentrator. Glycans were released, fluorescently labelled and cleaned up as previously described (Trbojević-Akmačić et al., 2017) , with a modification of using 0.2 μm Supor AcroPrep filter plate (Pall Corporation, USA) as a stationary phase. inorganic silica to improve its inertness and to mitigate problematic analyte to metal adsorption. Having a chemical composition that is highly similar to ethylene-bridged siloxane (BEH) particles (D. Wyndham et al., 2003) , this barrier layer is considerably more inert than fused silica surfaces and significantly less hydrophobic than polyether ether ketone (PEEK). That it is comprised of highly crosslinked ethylene-bridged siloxane groups also means that it is resilient to chemical and pH stress and amenable to usage between pH 1 and pH 12 mobile phase conditions. Separation method used a linear gradient of 75-62% acetonitrile (v/v) at a flow rate of 0.4 ml/min in a 29 minutes analytical run. Separation temperature was 60 °C, and samples were maintained at 10 °C before injection. Hydrolysed and 2-AB labelled glucose oligomers were used as external standard, from which the retention times for the individual glycans were converted to glucose units. The chromatograms were separated into 24 chromatographic peaks and the amount of glycans in each peak was expressed as percentage of total integrated chromatogram area (% Area). In order to remove experimental variation from measurements, normalization and batch correction were performed on UHPLC glycan data. To make measurements across samples comparable, normalization by total area was performed where the peak area of each of 24 glycan structures was divided by the total area of the corresponding chromatogram. Prior to batch correction, normalized glycan measurements were log transformed due to rightskewness of their distributions and the multiplicative nature of batch effects. Batch correction was performed on log-transformed measurements using ComBat method (R package sva), where the technical source of variation (which sample was analyzed on which plate) was modeled as a batch covariate. To get measurements corrected for experimental noise, estimated batch effects were subtracted from log-transformed measurements. In addition to 10 measured glycans. These derived traits average particular glycosylation features across different individual glycan structures and consequently they are more closely related to individual enzymatic activities and underlying genetic polymorphisms. Association analyses between disease severity status and glycan traits were performed using a regression model with age and gender included as additional covariates. Analyses were firstly performed for each cohort separately and then combined using inverse-variance weighted meta-analysis approach (R package metafor). 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Maximum Daily Temperature, Precipitation, Ultra-Violet Light and Rates of Transmission of SARS-Cov-2 in the United States The impact of obesity on severe disease and mortality in people with SARS-CoV-2: A systematic review and meta-analysis Comparative Analysis and Validation of Different Steps in Glycomics Studies Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity We would like to thank all anesthesiologists and nurses in the Emergency and Intensive Care Unit, as well as all the people who worked at ASST Papa Giovanni XXIII, during the COVID-19 pandemic in Bergamo for their invaluable and brave efforts towards patient's care. We would like to thank the ROCCO Bergamo project for its scientific support.Furthermore, we want to particularly acknowledge the patients of the Parc de Salut Mar and the MARBiobanc, integrated in the Spanish National Biobanks Network, for their collaboration. We thank Waters Corporation, USA, for supporting this work by providing ACQUITY PREMIER Glycan BEH Amide columns.