Interaction between noroviruses and human histo-blood group antigens

Norovirus (NOV), a member of the family Caliciviridae, is a major cause of water and food-borne acute nonbacterial gastroenteritis, and forms many morphologically similar but antigenically diverse groups of viruses. The virus-like particles (VLPs) derived from the prototype strain of NoV, Norwalk virus (NV/68), bind to histo-blood group antigens (HBGAs). HBGAs are carbohydrates that contain structurally related saccharide moieties, and are found in saliva and mucosal secretions from intestinal epithelial cells of secretor individuals who have FUT2 gene encoding a fucosyltransferase. From volunteer challenge studies, there is strong evidence that the carbohydrate-binding is essential for the NV/68 infection. Non-secretors, who do not express FUT2 fucosyltransferase and consequently do not express H type 1 or Leb in the gut, were not infected after the challenge with NV/68. However, other NoV VLPs display different ABH and Lewis carbohydrate-binding profiles, and indeed epidemiological studies showed that some NoV strains could infect individuals with another ABH phenotypes. GII/4 is known to be global epidemic strain and bound more HBGAs when compared with other strains. The strength of the transmission of GII/4 strains may be linked with their wide recognition of HBGAs. It is obvious that HBGAs are important factors to determine the host specificity, although it is still unclear whether the HBGAs act as the primary receptor or enhance NoV infectivity. Further investigation is needed.     

Norwalk virus-like particle hemagglutination by binding to h histo-blood group antigens

Noroviruses are a major cause of epidemic acute nonbacterial gastroenteritis worldwide. Here we report our discovery that recombinant Norwalk virus virus-like particles (rNV VLPs) agglutinate red blood cells (RBCs). Since histo-blood group antigens are expressed on gut mucosa as well as RBCs, we used rNV VLP hemagglutination (HA) as a model system for studying NV attachment to cells in order to help identify a potential NV receptor(s). rNV VLP HA is dependent on low temperature (4 degrees C) and acidic pH. Of the 13 species of RBCs tested, rNV VLPs hemagglutinated only chimpanzee and human RBCs. The rNV VLPs hemagglutinated all human type O (11 of 11), A (9 of 9), and AB (4 of 4) RBCs; however, few human type B RBC samples (4 of 14) were hemagglutinated. HA with periodate- and neuraminidase-treated RBCs indicated that rNV VLP binding was carbohydrate dependent and did not require sialic acid. The rNV VLPs did not hemagglutinate Bombay RBCs (zero of seven) that lack H type 2 antigen, and an anti-H type 2 antibody inhibited rNV VLP HA of human type O RBCs. These data indicated that the H type 2 antigen functions as the rNV VLP HA receptor on human type O RBCs. The rNV VLP HA was also inhibited by rNV VLP-specific monoclonal antibody 8812, an antibody that inhibits VLP binding to Caco-2 cells. Convalescent-phase sera from NV-infected individuals showed increased rNV VLP HA inhibition titers compared to prechallenge sera. In carbohydrate binding assays, the rNV VLPs bound to synthetic Lewis d (Le(d)), Le(b), H type 2, and Le(y) antigens, and these antigens also inhibited rNV VLP HA of human type O RBCs. Overall, our results indicate that carbohydrate antigens in the gut are a previously unrecognized factor in NV pathogenesis.     

The distribution of type-2 chain histo-blood group antigens in normal cycling human endometrium

Summary The blood group ABO(H) determinants are major allogenic antigens in both erythrocytes and tissue of man. These antigens and related carbohydrates are markers of cellular maturation and differentiation in many epithelial tissues and have recently attracted great interest as tumor-associated antigens. Previous studies of endometrial tissues have indicated that glycosylation in this tissue may be related to hormonal stimulation. We have investigated the immunohistochemical distribution of type-2 chain histo-blood group-related carbohydrates in specimens of normal, cycling endometria obtained from hysterectomies on women with known ABO/Lewis erythrocyte type and saliva secretor status. N-acetyllactosamine and Le^x were demonstrated to be uninfluenced by the genetic background. A and Ale^y antigens were exclusively demonstrated in endometria from blood group A individuals, while Le^y was expressed in endometria from blood group 0 individuals mainly. The precursor N-acetyllactosamine as well as the terminal H, A, and ALe^y antigens were shown in only a few cells. In contrast, N-acetyllactosamine substituted by sialic acid and/or fucose residues (Le^x, sialosyl-Le^x, Le^y) were demonstrated in epithelial cells of normal, cycling endometrium, but with both quantitative and qualitative differences in staining relating to the menstrual cycle, indicating that type-2 chain antigens are expressed under both genetic and hormonal influence in human cycling endometrium.     

Norovirus capture with histo-blood group antigens reveals novel virus-ligand interactions

Noroviruses are genetically diverse, uncultivable, positive-sense RNA viruses and are the most common cause of epidemic acute gastroenteritis in humans in the United States. Recent studies of norovirus attachment in vitro by using recombinant virus-like particles (VLPs) suggest that various norovirus strains exhibit different patterns of attachment to ABH histo-blood group antigens, which are carbohydrate epitopes present in high concentrations on mucosal cell surfaces of the gut. However, attachment of live norovirus strains to histo-blood group antigens has not been investigated to date. Utilizing a newly designed magnetic bead-virus capture method, we characterized histo-blood group antigen attachment properties of various norovirus strains obtained from clinical stool specimens to compare the attachment properties of wild-type virus and VLPs and to further map norovirus attachment. Consistent with previous reports using VLPs, various strains of noroviruses exhibited different patterns of attachment to histo- blood group antigens. Norwalk virus bound specifically to H type 1, H type 3, and Le(b). Two genogroup II noroviruses, one representing the Toronto genotype and the other from a novel genotype, bound specifically to Le(b). A Desert Shield-like strain did not attach to H types 1, 2, or 3, H type 1 and 3 precursors, Le(a), or Le(b). Surprisingly, wild-type Snow Mountain virus (SMV) attached specifically to H type 3, which contradicted previous findings with SMV VLPs. On further investigation, we found that stool components promote this attachment, providing the first known observation that one or more components of human feces could promote and enhance norovirus attachment to histo-blood group antigens.     

Synthesis of ABO histo-blood group type I and II antigens

The ABO histo-blood group system is one of the most clinically important antigen families. As part of our overall goal to prepare the entire set of the A, B and H type I-VI antigens for a range of biochemical investigations, we report herein the synthesis of the type I and II antigens with a 7-octen-1-yl aglycone. This linker was chosen to facilitate not only the future conjugation of the antigens to a protein or solid support but also the synthesis of the H type I and II octyl glycosides for enzyme kinetic studies.     

Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner

Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.     

Crystallography of a Lewis-binding norovirus, elucidation of strain-specificity to the polymorphic human histo-blood group antigens

Noroviruses, an important cause of acute gastroenteritis in humans, recognize the histo-blood group antigens (HBGAs) as host susceptible factors in a strain-specific manner. The crystal structures of the HBGA-binding interfaces of two A/B/H-binding noroviruses, the prototype Norwalk virus (GI.1) and a predominant GII.4 strain (VA387), have been elucidated. In this study we determined the crystal structures of the P domain protein of the first Lewis-binding norovirus (VA207, GII.9) that has a distinct binding property from those of Norwalk virus and VA387. Co-crystallization of the VA207 P dimer with Le(y) or sialyl Le(x) tetrasaccharides showed that VA207 interacts with these antigens through a common site found on the VA387 P protein which is highly conserved among most GII noroviruses. However, the HBGA-binding site of VA207 targeted at the Lewis antigens through the α-1, 3 fucose (the Lewis epitope) as major and the β-N-acetyl glucosamine of the precursor as minor interacting sites. This completely differs from the binding mode of VA387 and Norwalk virus that target at the secretor epitopes. Binding pocket of VA207 is formed by seven amino acids, of which five residues build up the core structure that is essential for the basic binding function, while the other two are involved in strain-specificity. Our results elucidate for the first time the genetic and structural basis of strain-specificity by a direct comparison of two genetically related noroviruses in their interaction with different HBGAs. The results provide insight into the complex interaction between the diverse noroviruses and the polymorphic HBGAs and highlight the role of human HBGA as a critical factor in norovirus evolution.     

Norovirus-host interaction: implications for disease control and prevention

Noroviruses (NVs) are a major cause of acute gastroenteritis epidemics in both developing and developed countries and affect people of all ages. Three main human histo-blood group antigens (HBGAs) - the ABO, Lewis and secretor families - are involved in NV recognition and eight strain-specific receptor-binding patterns in two major binding groups have been described. The receptor-binding interface is located at the outermost surface of the P domain of the viral capsid. Each interface contains two major binding sites and each site interacts with a carbohydrate side-chain of the HBGAs via multiple hydrogen bonds. Soluble HBGAs in human milk are able to block binding of NV to HBGA receptors, suggesting a potential decoy receptor for the protection of infants from NV infection. Phylogenetic analysis has revealed limited genetic relatedness among NVs with similar receptor-binding patterns. This review summarises and discusses recent advances and highlights implications for future studies in the control and prevention of NV gastroenteritis.     

Genomic organization of human histo-blood group ABO genes

We have isolated human genomic DNA clones encompassing 30 kbpof the histo-blood group ABO locus. The locations of the exonshave been mapped and the nucleotide sequences of the exon-intronboundaries have been determined. The human ABO genes consistof at least seven exons, and the coding sequence in the sevencoding exons spans over 18 kb of the genomic DNA. The exonsrange in size from 28 to 688 bp, with most of the coding sequencelying in exon 7. ABO genomic glycosyltransferase histo-blood group transfusion     

Stomach expression of histo-blood group antigens A and B in some vertebrates

Histo‐blood group antigens (HBGA) are genetically determined glycoproteins and glycolipids expressed not only on human erythrocytes but also in vertebrate tissues. Direct evidence for the immunobiological importance of their tissue localization in the evolutionary aspect is still lacking. The present study examines the expression of A and B HBGA in the stomach of free‐living vertebrates belonging to: Chondrichthyes, Actinopterygii, Amphibia, Reptilia, Aves, and Mammalia. HBGA were detected immunohistochemically on stomach paraffin sections from 11 species. In all classes from Actinopterygii to Mammalia HBGA expression was confined to stomach mucosa only. Antigenic heterogeneity in the pattern of expression and localization was observed. Smooth muscle tissue, endothelial and red blood cells were immunonegative, except for the reptile Emys orbicularis. Our results present the first comparative evidence for the expression of HBGA in the stomach of 11 free‐living vertebrate species from six classes, some of which have never been studied so far. It could be assumed that A and B antigens are constant and conservative structures with almost similar tissue localization. Their immunobiological role in the animal gastrointestinal tract might be possibly related to cell differentiation and homeostasis maintenance which would contribute to sustain the evolutionary stable ABH antigen cellular expression.     

Differential expression of ABH histo-blood group antigens and LAMPs in infantile hemangioma

Summary Although infantile hemangioma (IH) are the most common tumors of infancy, the mechanism of their proliferation and involution remains vague. Proliferation, differentiation and death of endothelial cells are the basic processes involved in their pathobiology. Here we hypothesize that the glycoconjugates ABH histo-blood group antigens (HBGA) and lysosome-associated membrane proteins (LAMPs) might be implied in both the differentiation and death of endothelial cells during vascular remodeling in IH. Proliferating and involuting IH were examined immunohistochemically for HGBA and LAMP expression together with vWF and CD31. Proliferative and apoptotic indices were determined. LAMPs were found in immature endothelium of proliferating IH. In involution an increased number of immunopositive cells stained with higher intensity was detected. The enhanced expression might be associated with augmented autophagy required for tissue remodeling during tumor involution. HBGA presented an opposite pattern of expression – they stained intensely the endothelium of mature capillaries, while the immature ones were positive for vWF. The presence of HBGA in endothelial cells of IH may be related to the differentiation process only, as well as to endothelial adhesion and angiogenesis. Novel evidence for differential expression of HBGA and LAMPs in proliferative and involutive phases of IH is presented.     

Multiple antigenic sites are involved in blocking the interaction of GII.4 norovirus capsid with ABH histo-blood group antigens

Noroviruses are major etiological agents of acute viral gastroenteritis. In 2002, a GII.4 variant (Farmington Hills cluster) spread so rapidly in the human population that it predominated worldwide and displaced previous GII.4 strains. We developed and characterized a panel of six monoclonal antibodies (MAbs) directed against the capsid protein of a Farmington Hills-like GII.4 norovirus strain that was associated with a large hospital outbreak in Maryland in 2004. The six MAbs reacted with high titers against homologous virus-like particles (VLPs) by enzyme-linked immunoassay but did not react with denatured capsid protein in immunoblots. The expression and self-assembly of newly developed genogroup I/II chimeric VLPs showed that five MAbs bound to the GII.4 protruding (P) domain of the capsid protein, while one recognized the GII.4 shell (S) domain. Cross-competition assays and mutational analyses showed evidence for at least three distinct antigenic sites in the P domain and one in the S domain. MAbs that mapped to the P domain but not the S domain were able to block the interaction of VLPs with ABH histo-blood group antigens (HBGA), suggesting that multiple antigenic sites of the P domain are involved in HBGA blocking. Further analysis showed that two MAbs mapped to regions of the capsid that had been associated with the emergence of new GII.4 variants. Taken together, our data map antibody and HBGA carbohydrate binding to proximal regions of the norovirus capsid, showing that evolutionary pressures on the norovirus capsid protein may affect both antigenic and carbohydrate recognition phenotypes.     

Expression of histo-blood group antigens by lipopolysaccharides of Helicobacter pylori strains from asian hosts: the propensity to express type 1 blood-group antigens

Past studies have shown that the cell surface lipopolysaccharides (LPSs) of the ubiquitous human gastric pathogen Helicobacter pylori (a type 1 carcinogen) isolated from people residing in Europe and North America express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes and, infrequently, type 1 Le(a), Le(b), and Le(d) antigens. This production of Lewis blood-group structures by H. pylori LPSs, similar to those found in the surfaces of human gastric cells, allows the bacterium to mimic its human niche. In this study, LPSs of H.pylori strains extracted from patients living in China, Japan, and Singapore were chemically and serologically analyzed. When compared with Western H.pylori LPSs, these Asian strains showed a stronger tendency to produce type 1 blood groups. Of particular interest, and novel observations in H.pylori, the O-chain regions of strains F-58C and R-58A carried type 1 Le(a) without the presence of type 2 Le(x), strains R-7A and H607 were shown to have the capability of producing the type 1 blood group A antigen, and strains CA2, H507, and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Le(b) and type 2 Le(y). The apparent proclivity for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs, as compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain.     

Noroviruses distinguish between type 1 and type 2 histo-blood group antigens for binding

Norovirus (NoV) is a causative agent of acute gastroenteritis. NoV binds to histo-blood group antigens (HBGAs), namely, ABH antigens and Lewis (Le) antigens, in which type 1 and type 2 carbohydrate core structures constitute antigenically distinct variants. Norwalk virus, the prototype strain of norovirus, binds to the gastroduodenal junction, and this binding is correlated with the presence of H type 1 antigen but not with that of H type 2 antigen (S. Marionneau, N. Ruvoen, B. Le Moullac-Vaidye, M. Clement, A. Cailleau-Thomas, G. Ruiz-Palacois, P. Huang, X. Jiang, and J. Le Pendu, Gastroenterology 122:1967-1977, 2002). It has been unknown whether NoV distinguishes between the type 1 and type 2 chains of A and B antigens. In this study, we synthesized A type 1, A type 2, B type 1, and B type 2 pentasaccharides in vitro and examined the function of the core structures in the binding between NoV virus-like particles (VLPs) and HBGAs. The attachment of five genogroup I (GI) VLPs from 5 genotypes and 11 GII VLPs from 8 genotypes, GI/1, GI/2, GI/3, GI/4, GI/8, GII/1, GII/3, GII/4, GII/5, GII/6, GII/7, GII/12, and GII/14, to ABH and Le HBGAs was analyzed by enzyme-linked immunosorbent assay-based binding assays and Biacore analyses. GI/1, GI/2, GI/3, GI/4, GI/8, and GII/4 VLPs were more efficiently bound to A type 2 than A type 1, and GI/8 and GII/4 VLPs were more efficiently bound to B type 2 than B type 1, indicating that NoV VLPs distinguish between type 1 and type 2 carbohydrates. The dissociation of GII/4 VLPs from B type 1 was slower than that from B type 2 in the Biacore experiments; moreover, the binding to B type 1 was stronger than that to B type 2 in the ELISA experiments. These results indicated that the type 1 carbohydrates bind more tightly to NoV VLPs than the type 2 carbohydrates. This property may afford NoV tissue specificity. GII/4 is known to be a global epidemic genotype and binds to more HBGAs than other genotypes. This characteristic may be linked with the worldwide transmission of GII/4 strains. GI/2, GI/3, GI/4, GI/8, GII/4, and GII/7 VLPs bound to Le(a) expressed by nonsecretors, suggesting that NoV can infect individuals regardless of secretor phenotype. Overall, our results indicated that HBGAs are important factors in determining tissue specificity and the risk of transmission.     

Comparison of human saliva and synthetic histo-blood group antigens usage as ligands in norovirus-like particle binding and blocking assays

Blocking of norovirus-like particle binding to their cellular ligands, histo-blood group antigens with immune sera, is considered a surrogate norovirus neutralization assay. We compared human secretor positive saliva and synthetic biotinylated carbohydrates as a source of histo-blood group antigens in binding and blocking assays. Six norovirus capsid-derived virus-like particles belonging to genogroup I (GI-1-2001 and GI-3-2002) and genogroup II (GII-4-1999, GII-4-2010 New Orleans, GII-4-2012 Sydney and GII-12-1998) noroviruses were produced by a recombinant baculovirus expression system and binding profile to saliva type A, B and O and to synthetic antigens (A trimer, B trimer, H type 1, H type 3, Lewis^a and Lewis^b) was identified. Good correlation between virus-like particle binding to saliva type A and synthetic A trimer (r = 0.66, p < 0.05) and saliva type B and synthetic B trimer (r = 0.75, p < 0.05) was observed. Binding of each norovirus virus-like particle to the selected histo-blood group antigens was blocked by convalescent sera from NoV-infected subjects or type-specific mouse antisera. Our results support the use of either saliva or synthetic antigens in blocking assay to measure the ability of norovirus antisera to block virus-like particle binding to the carbohydrate ligands.     

Binding of Norwalk virus-like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice

Attachment of Norwalk (NV), Snow Mountain (SMV), and Hawaii (HV) virus-like particles (VLPs) to specific ABH histo-blood group antigens was investigated by using human saliva and synthetic biotinylated carbohydrates. The three distinct Norwalk-like viruses (NLVs) have various capacities for binding ABH histo-blood group antigens, suggesting that different mechanisms for NLV attachment likely exist. Importantly, antisera from NV-infected human volunteers, as well as from mice inoculated with packaged Venezuelan equine encephalitis virus replicons expressing NV VLPs, blocked the ability of NV VLPs to bind synthetic H type 1, Le(b), and H type 3, suggesting a potential mechanism for antibody-mediated neutralization of NV.     

Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-blood group family

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.     

Mutations within the P2 domain of norovirus capsid affect binding to human histo-blood group antigens: evidence for a binding pocket

Noroviruses (NORs) are an important cause of acute gastroenteritis. Recent studies of NOR receptors showed that different NORs bind to different histo-blood group antigens (HBGAs), and at least four distinct binding patterns were observed. To determine the structure-function relationship for NORs and their receptors, two strains representing two of the four binding patterns were studied. Strain VA387 binds to HBGAs of A, B, and O secretors, whereas strain MOH binds to HBGAs of A and B secretors only. Using multiple sequence alignments, homology modeling, and structural analysis of NOR capsids, we identified a plausible "pocket" in the P2 domain that may be responsible for binding to HBGA receptors. This pocket consists of a conserved RGD/K motif surrounded by three strain-specific hot spots (N(302), T(337), and Q(375) for VA387 and N(302), N(338), and E(378) for MOH). Subsequent mutagenesis experiments demonstrated that all four sites played important roles in binding. A single amino acid mutation at T(337) (to A) in VA387 or a double amino acid mutation at RN(338) (to TT) in MOH abolished binding completely. Change of the entire RGD motif to SAS abolished binding in case of VA387, whereas single amino acid mutations in that motif did not have an apparent effect on binding to A and B antigens but decreased binding to H antigen. Multiple mutations at the RGK motif of MOH (SIRGK to TFRGD) completely knocked out the binding. Mutation of N(302) or Q(375) in VA387 affected binding to type O HBGA only, while switch mutants with three amino acid changes at either site from MOH to VA387 resulted in a weak binding to type O HBGAs. A further switch mutant with three amino acid changes at E(378) from MOH to VA387 diminished the binding to type A HBGA only. Taken together, our data indicate that the binding pocket likely exists on NOR capsids. Direct evidence of this hypothesis requires crystallography studies.