Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily targets respiratory mucosa, causing coronavirus disease 2019 (COVID-19). Susceptibility and severity of COVID-19 may be influenced by predisposing factors including blood groups. In this study, we investigated whether natural anti-carbohydrate antibodies provide innate protection against SARS-CoV-2 and influence disease severity. We used samples (plasma and saliva) from a longitudinal cohort study in Bangladesh that enrolled 100 COVID-19 symptomatic and asymptomatic patients. We also enrolled 21 and 38 healthy controls during the pandemic period and pre-pandemic period, respectively. We phenotype ABO blood grouping from blood and determined Lewis and secretor status (H antigen) from the saliva samples. We quantified natural anti-carbohydrate antibodies (anti-A, anti-B, anti-Tn-Mono and anti-αGal IgG, IgA, and IgM) from plasma collected at enrollment. We also explored the trend of natural anti-carbohydrate antibodies until 3 months of convalescence period among the COVID-19 patients (day 14 and day 90 from enrollment). Antibody quantification and ABH/Lewis phenotyping were performed using enzyme-linked immunosorbent assay (ELISA). We included 99 COVID-19 patients and 59 healthy controls assessing the differences of natural antibody titer during enrollment, while 95 patients were analyzed exploring Lewis and secretor status with natural antibody titer and disease status. We did not find significant difference in the distribution for neither ABO blood groups nor non-secretors and Lewis-negative individuals among asymptomatic or symptomatic patients and healthy controls. Nonetheless, we observed lower anti-A antibody titers among symptomatic patients compared to healthy controls. We also identified slight differences in antibody titers linked to age and gender. Anti-A and anti-B antibodies among asymptomatic patients had a higher trend up to 3 months from infection compared to symptomatic patients. Higher natural anti-A and anti-B antibody titers may offer protection against symptomatic COVID-19 infections. Gender and blood group differences indicate potential innate immune factors influencing disease severity, but larger studies are needed to confirm these findings.

Recently, Breiman et al. (2021) reported that the level of anti-Tn in the blood of healthy donors and COVID-19 patients is significantly lower in individuals of blood group A than B. This prompted us to look for qualitative differences in the repertoire of anti-Tn like specificity in individuals of different blood groups (BG). To this end, we isolated antibodies from the pooled sera of BG A and BG B healthy donors using GalNAcα-sepharose, followed by Printed Glycan Array analysis. As expected, antibodies affinity isolated from BG A donors completely lack species directed to canonical (GalNAcα-transferase dependent) A-glycans, such as A (types 1, 2 and 4), GalNAcα1–3(Fucα1–2)Gal, GalNAcα1-3Galβ1-4GlcNAc (linear A), and ALeY. Unexpectedly, GalNAcα1-4Galβ1-4GlcNAc, glycan with an unnatural 1–4 bond fell into the same group, i.e., antibodies to it were found only in BG B donors. Other unexpected results include the following: (1) for GalNAcα1-OCH2CH(COOH)NH2 (GalNAcα-OSer, immobilized by NH2 group) the opposite result was observed, i.e. affinity isolated anti-Tn antibodies of BG A donors demonstrated significantly higher titer than of BG B; (2) in BG A donors, the level of antibodies to GalNGcα1-3GalNAcα (i.e. disaccharide in N-glycolyl form) is close to background, while there is a significant level of these antibodies in the BG B donors. Since antiglycan antibodies are known to play both a protective role in antimicrobial immunity and to promote infectivity, the knowledge gained about the difference in the specificity profiles of anti-Tn antibodies signals the need to take blood group into account in developing therapeutic strategies.

Human rotaviruses attach to histo-blood group antigens glycans and null alleles of the ABO, FUT2 and FUT3 genes seem to confer diminished risk of gastroenteritis. Yet, the true extent of this protection remains poorly quantified. Here, we conducted a prospective study to evaluate the risk of consulting at the hospital in non-vaccinated pediatric patients according to the ABO, FUT2 (secretor) and FUT3 (Lewis) polymorphisms, in Metropolitan France and French Guiana. At both locations, P genotypes were largely dominated by P [8]-3, with P [6] cases exclusively found in French Guiana. The FUT2 null (nonsecretor) and FUT3 null (Lewis negative) phenotypes conferred near full protection against severe gastroenteritis due to P [8]-3 strains (OR 0.03, 95% CI [0.00–0.21] and 0.1, 95% CI [0.01–0.43], respectively in Metropolitan France; OR 0.08, 95% CI [0.01–0.52] and 0.14, 95%CI [0.01–0.99], respectively in French Guiana). Blood group O also appeared protective in Metropolitan France (OR 0.38, 95% CI [0.23–0.62]), but not in French Guiana. The discrepancy between the two locations was explained by a recruitment at the hospital of less severe cases in French Guiana than in Metropolitan France. Considering the frequencies of the null ABO, Secretor and Lewis phenotypes, the data indicate that in a Western European population, 34% (95% CI [29%; 39%]) of infants are genetically protected against rotavirus gastroenteritis of sufficient severity to lead to hospital visit.

The FUT2 α1,2fucosyltransferase contributes to the synthesis of fucosylated glycans used as attachment factors by several pathogens, including noroviruses and rotaviruses, that can induce life-threatening gastroenteritis in young children. FUT2 genetic polymorphisms impairing fucosylation are strongly associated with resistance to dominant strains of both noroviruses and rotaviruses. Interestingly, the wild-type allele associated with viral gastroenteritis susceptibility inversely appears to be protective against several inflammatory or autoimmune diseases for yet unclear reasons, although a FUT2 influence on microbiota composition has been observed. Here, we studied a cohort of young healthy adults and showed that the wild-type FUT2 allele was associated with the presence of anti-RVA antibodies, either neutralizing antibodies or serum IgA, confirming its association with the risk of RVA gastroenteritis. Strikingly, it was also associated with the frequency of gut microbiota-induced regulatory T cells (Tregs), so-called DP8α Tregs, albeit only in individuals who had anti-RVA neutralizing antibodies or high titers of anti-RVA IgAs. DP8α Tregs specifically recognize the human symbiont Faecalibacterium prausnitzii, which strongly supports their induction by this anti-inflammatory bacterium. The proportion of F. prausnitzii in feces was also associated with the FUT2 wild-type allele. These observations link the FUT2 genotype with the risk of RVA gastroenteritis, the microbiota and microbiota-induced DP8α Treg cells, suggesting that the anti-RVA immune response might involve an induction/expansion of these T lymphocytes later providing a balanced immunological state that confers protection against inflammatory diseases.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

ABO blood groups appear to be associated with the risk of SARS-CoV-2 infection, but the underlying mechanisms and their real importance remain unclear. Two hypotheses have been proposed: ABO compatibility-dependence (neutralization by anti-ABO antibodies) and ABO-dependent intrinsic susceptibility (spike protein attachment to histo-blood group glycans). We tested the first hypothesis through an anonymous questionnaire addressed to hospital staff members. We estimated symptomatic secondary attack rates (SAR) for 333 index cases according to spouse ABO blood group compatibility. Incompatibility was associated with a lower SAR (28% vs. 47%; OR 0.43, 95% CI 0.27-0.69), but no ABO dependence was detected in compatible situations. For the second hypothesis, we detected no binding of recombinant SARS-CoV-2 RBD to blood group-containing glycans. Thus, although no intrinsic differences in susceptibility according to ABO blood type were detected, ABO incompatibility strongly decreased the risk of COVID-19 transmission, suggesting that anti-ABO antibodies contribute to virus neutralization.

For the last 30 years, molecular surveys have shown that human norovirus (HuNoV), predominantly the GII.4 genotype, is one of the main causative agents of gastroenteritis. However, epidemiological surveys have revealed the worldwide emergence of GII.17 HuNoVs. Genetic analysis confirmed that GII.17 strains are distributed into three variants (i.e., Kawasaki 308, Kawasaki 323, and CS-E1). Here, virus-like particles (VLPs) were baculovirus-expressed from these variants to study putative interactions with HBGA. Qualitative analysis of the HBGA binding profile of each variant showed that the most recent and predominant GII.17 variant, Kawasaki 308, possesses a larger binding spectrum. The retrospective study of GII.17 strains documented before the emergence of the dominant Kawasaki 308 variant showed that the emergence of a new GII.17 variant could be related to an increased binding capacity toward HBGA. The use of duodenal histological sections confirmed that recognition of enterocytes involved HBGA for the three GII.17 variants. Finally, we observed that the relative affinity of recent GII.17 VLPs for HBGA remains lower than that of the GII.4-2012 variant. These observations suggest a model whereby a combination of virological factors, such as polymerase fidelity and increased affinity for HBGA, and immunological factors was responsible for the incomplete and non-persistent replacement of GII.4 by new GII.17 variants.

Human serum contains large amounts of anti-carbohydrate antibodies, some of which may recognize epitopes on viral glycans. Here, we tested the hypothesis that such antibodies may confer protection against COVID-19 so that patients would be preferentially found among people with low amounts of specific anti-carbohydrate antibodies since individual repertoires vary considerably. After selecting glycan epitopes commonly represented in the human anti-carbohydrate antibody repertoire that may also be expressed on viral glycans, plasma levels of the corresponding antibodies were determined by ELISA in 88 SARS-CoV-2 infected individuals, including 13 asymptomatic, and in 82 non-infected controls. We observed that anti-Tn antibodies levels were significantly lower in patients as compared to non-infected individuals. This was not observed for any of the other tested carbohydrate epitopes, including anti-αGal antibodies used as a negative control since the epitope cannot be synthesized by humans. Owing to structural homologies with blood groups A and B antigens, we also observed that anti-Tn and anti-αGal antibodies levels were lower in blood group A and B, respectively. Analyses of correlations between anti-Tn and the other anti-carbohydrates tested revealed divergent patterns of correlations between patients and controls, suggesting qualitative differences in addition to the quantitative difference. Furthermore, anti-Tn levels correlated with anti-S protein levels in the patients’ group, suggesting that anti-Tn might contribute to the development of the specific antiviral response. Overall, this first analysis allows to hypothesize that natural anti-Tn antibodies might be protective against COVID-19.

Since the emergence of COVID-19, many publications have reported associations with ABO blood types. Despite between-study discrepancies, an overall consensus has emerged whereby blood group O appears associated with a lower risk of COVID-19, while non-O blood types appear detrimental. Two major hypotheses may explain these findings: First, natural anti-A and anti-B antibodies could be partially protective against SARS-CoV-2 virions carrying blood group antigens originating from non-O individuals. Second, O individuals are less prone to thrombosis and vascular dysfunction than non-O individuals and therefore could be at a lesser risk in case of severe lung dysfunction. Here, we review the literature on the topic in light of these hypotheses. We find that between-study variation may be explained by differences in study settings and that both mechanisms are likely at play. Moreover, as frequencies of ABO phenotypes are highly variable between populations or geographical areas, the ABO coefficient of variation, rather than the frequency of each individual phenotype is expected to determine impact of the ABO system on virus transmission. Accordingly, the ABO coefficient of variation correlates with COVID-19 prevalence. Overall, despite modest apparent risk differences between ABO subtypes, the ABO blood group system might play a major role in the COVID-19 pandemic when considered at the population level.

BACKGROUND:Susceptibility to Covid-19 has been found to be associated with the ABO blood group, with O type individuals being at a lower risk. However, the underlying mechanism has not been elucidated. Here, we aimed to test the hypothesis that Covid-19 patients might have lower levels of ABO antibodies than non-infected individuals as they could offer some degree of protection.METHODS:After showing that the viral spike protein harbors the ABO glycan epitopes when produced by cells expressing the relevant glycosyltransferases, like upper respiratory tract epithelial cells, we enrolled 290 patients with Covid-19 and 276 asymptomatic controls to compare their levels of natural ABO blood group antibodies.RESULTS:We found significantly lower IgM anti-A + anti-B agglutination scores in blood group O patients (76.93 vs 88.29, P-value = 0.034) and lower levels of anti-B (24.93 vs 30.40, P-value = 0.028) and anti-A antibodies (28.56 vs 36.50, P-value = 0.048) in blood group A and blood group B patients, respectively, compared to controls.CONCLUSION:In this study, we showed that ABO antibody levels are significantly lower in Covid-19 patients compared to controls. These findings could indicate that patients with low levels of ABO antibodies are at higher risk of being infected.

In Tunisia, we observed that rotavirus P[8]-3 and P[4] strains in young children with gastroenteritis associate with secretor histo-blood group phenotype. In contrast, the emerging P[8]-4 strain, representing 10% of cases, was exclusively found in nonsecretor patients. Unlike VP8* from P[8]-3 and P[4] strains, the P[8]-4 VP8* protein attached to glycans from saliva samples regardless of the donor's secretor status. Interestingly, a high frequency of FUT2 enzyme deficiency (nonsecretor phenotype) was observed in the population. This may allow cocirculation of P[8]-3 and P[8]-4 strains in secretor and nonsecretor children, respectively.

First recognized as highly pathogenic viruses, hare lagoviruses belonging to genotype GII.1 (EBHSV) infect various Lepus species. Genetically distinct benign lagoviruses (Hare Calicivirus, HaCV) have recently been identified but few data have been available so far on these strains. The analysis of 199 samples from hunted hares collected throughout France allowed the detection of 20 HaCV and showed that they were widely distributed in this country. Ten HaCV capsid protein gene sequences were characterized. A first HaCV capsid protein structural model was proposed, revealing a global structure similar to that of a pathogenic GII.1 strain. The HaCV sequences showed an even higher genetic diversity than previously appreciated, with the characterization of two genotypes (GII.2, GII.3) and several additional putative genotypes. The most recent common ancestor for HaCV VP60 gene was estimated to be much older than that for GII.1 pathogenic strains. These results give new insights into the phylogenetic relationships of HaCV within the Lagovirus genus.

Enveloped viruses are glycosylated, meaning that their envelope proteins are post-translationally modified by the addition of glycans. Some glycosyltransferases that contribute to terminate glycan chains synthesis are differentially expressed between cell types within or between species, which bears important immunological consequences [1]. A prime example concerns the GGTA1 gene encoding a galactosyltransferase that catalyzes the transfer of a galactose in α1,3 linkage onto subterminal N-acetyllactosamines. This glycosyltransferase is expressed by all mammals except Old World monkeys, apes and humans, due to inactivating mutations that occurred in a common primate ancestor some 20 to 40 million years ago. Consequently, all humans lack the αGal glycan motif and possess natural anti-αGal antibodies generated in response to bacteria of the microbiota that express similar glycans [2]. Likewise, humans lack expression of the N-glycolyl form of sialic acid (NeuGc) due to a pseudogenization event of the CMAH gene that occurred about 2 million years ago. In most other animal species, the orthologous gene encodes the cytidine monophosphate (CMP)-NeuAc hydroxylase that converts NeuAc into NeuGc from the nucleotide form CMP-NeuAc. As a result of our inability to synthesize NeuGc, natural anti-NeuGc are also present in humans (reviewed in [3,4]). Another example concerns the enzymes that are involved in the synthesis of the ABO histo-blood group antigens. The A and B enzymes catalyze the transfer of an N-acetylgalactosamine and a galactose, respectively, in α1,3 linkage on a precursor structure called the H antigen, generating the corresponding A or B antigens. They are encoded by distinct alleles at the ABO locus. The O alleles are null alleles responsible for a lack of transferase, in which case the H antigen remains unchanged. O alleles in the homozygote state confer blood group O, which is characterized by a complete absence of A or B antigens [5]. Under stimulation by bacteria of the microbiota that present glycan motifs similar to either A or B antigens, blood group O people develop so-called “natural” anti-A and anti-B antibodies, whilst blood group A and B individuals develop either anti-B or anti-A antibodies, respectively [6]. Only people of the AB subgroup lack such antibodies. In humans, besides their expression on red blood cells, ABH antigens are widely expressed on many other cell types, including vascular endothelial cells and epithelial cells of many organs [7]. Importantly, the titers of anti-αGal, anti-NeuGc, and anti-A/B antibodies are highly variable between individuals, ranging from 100to 1000-fold [8,9]. When enveloped viruses are produced by cells expressing these glycan epitopes, they can be effectively neutralized by anti-αGal or anti-A and anti-B antibodies as shown for several animal and human enveloped viruses (reviewed in[2]). Coronavirus S protein trimers are covered by an extensive glycan shield made of N-linked glycans that surrounds the receptor-binding PLOS PATHOGENS

The level of human natural antibodies of immunoglobulin M isotype against LeC in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors’ blood using LeC-Sepharose (LeC is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with LeC, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10−9 M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galβ1-3GlcNAcβ disaccharide, indicates that the target epitope of anti-LeC antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan.

TheMacrobrachium rosenbergiinodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-bindingAleuria Aurantialectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extendedN-linked andO-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference forN-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylatedN-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections.

Host susceptibility according to human histo-blood group antigens (HBGAs) is widely known for norovirus infection, but is less described for rotavirus. Due to the variable HBGA polymorphism among populations, we aimed to evaluate the association between HBGA phenotypes (ABH, Lewis and secretor status) and susceptibility to rotavirus and norovirus symptomatic infection, and the polymorphisms of FUT2 and FUT3, of children from southeastern Brazil. Paired fecal-buccal specimens from 272 children with acute diarrhea were used to determine rotavirus/norovirus genotypes and HBGAs phenotypes/genotypes, respectively. Altogether, 100 (36.8%) children were infected with rotavirus and norovirus. The rotavirus P[8] genotype predominates (85.7%). Most of the noroviruses (93.8%) belonged to genogroup II (GII). GII.4 Sydney represented 76% (35/46) amongst five other genotypes. Rotavirus and noroviruses infected predominantly children with secretor status (97% and 98.5%, respectively). However, fewer rotavirus-infected children were Lewis-negative (8.6%) than the norovirus-infected ones (18.5%). FUT3 single nucleotide polymorphisms (SNP) occurred mostly at the T59G > G508A > T202C > C314T positions. Our results reinforce the current knowledge that secretors are more susceptible to infection by both rotavirus and norovirus than non-secretors. The high rate for Lewis negative (17.1%) and the combination of SNPs, beyond the secretor status, may reflect the highly mixed population in Brazil.

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1-3Galβ1-3(4)GlcNAcβ. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα-OSer and do not bind a wide variety of GalNAcα-OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.