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.     

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.     

Norwalk virus-like particles bind specifically to A,H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids

Noroviruses and norovirus virus-like particles (VLPs) exhibit strain specific patterns in their binding to ABH and Lewis histo-blood group antigens. In this study we demonstrate for the first time specific binding of Norwalk virus VLPs to type 1 and type 2 chain glycosphingolipids (GSLs) carrying ABH and Lewis antigens. N-succinimidyl-3-tributylstannyl benzoate (ATE) was precursor labeled with ¹²⁵I and then conjugated to VLPs. The ¹²⁵I-VLPs were used in GSL thin-layer chromatogram binding assays and displayed binding to H type 1, Lewis b, A type 1, A Lewis b GSLs but no binding to B type 1 or B Lewis b GSLs. For the type 2 chain GSLs the Norwalk VLPs bound to H type 2, Lewis y, A type 2 and A Lewis y. In addition, the VLPs bound to several complex GSLs from blood group O and A, but not from blood group B red blood cells.     

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.     

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.     

The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors

Noroviruses (NVs) are the most important pathogen of epidemic nonbacterial gastroenteritis. The recent finding that NVs recognize human histo-blood group antigens (HBGAs) as receptors provided a new approach to study the pathogenesis of NVs. Using computational and site-directed mutagenesis approaches, our investigators previously identified a plausible binding pocket in the P domain of the NV capsids. In this study, we further characterize the role of the P domain in the interaction with human HBGA receptors using three NV strains representing three binding patterns. Our results show that the isolated P domain, although it did not form virus-like particles (VLPs), formed dimers, and the dimers bound HBGAs with the same patterns as those of the intact viral capsids. In contrast, the S domain, which formed small, thin-layer VLPs, did not bind A, B, or H HBGAs. A chimera containing the S domain of VA387 and the P domain of MOH revealed a binding pattern of the P donor strain (MOH). Deletion experiments revealed that an intact P domain is necessary for receptor binding. The P domain dimers are stable over a broad range of pH (2 to 11) or under strong denaturing conditions. Taken together, our results suggest that the P domain of NV contains essential elements for strain-specific binding to receptors. Further study of the P domain will provide useful information about the virus-receptor interaction. The high yield and easy production of the recombinant P protein in the Escherichia coli expression system will provide a simple approach to this goal.     

Porcine gastric mucin binds to recombinant norovirus particles and competitively inhibits their binding to histo-blood group antigens and Caco-2 cells

AIMS: To determine if human noroviruses (NV) bind to histo-blood group antigens (HBGA) from pig gastric mucosa. METHODS AND RESULTS: An assay was developed to measure the inhibition of binding of recombinant norovirus-like particles (rNVLP) to HBGA in human saliva by porcine gastric mucin (PGM). The binding of rNVLP to HBGA could be inhibited by PGM in a dose-dependent pattern. Also, rNVLP could be captured effectively by PGM coated directly on plates and was detected by binding of polyclonal antibodies against rNVLP. Similarly, the binding of rNVLP to PGM could be inhibited effectively by HBGA in human saliva, and by Lewis b and Lewis d synthetic oligosaccharides (OS), but not inhibited effectively by an H3 OS or by purified bovine submaxillary gland mucin. Preincubation of rNVLPs with PGM completely inhibited their binding to human Caco-2 cells. CONCLUSIONS: PGM binds effectively to rNVLPs and competitively inhibits rNVLPs binding to human HBGA and Caco-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the binding of glycoproteins from animal gastric mucosa to human NVs. This study highlights the importance of further characterizing the NV incidence and infections in nonhuman animal hosts and the possibility that NV is a zoonotic disease.     

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.     

Histo-blood group antigens act as attachment factors of rabbit hemorrhagic disease virus infection in a virus strain-dependent manner

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50-90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level.     

Norovirus Binding to Intestinal Epithelial Cells Is Independent of Histo-Blood Group Antigens

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le^b-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.     

Norwalk virus RNA is infectious in mammalian cells

Human noroviruses are positive-sense RNA viruses and are the leading cause of epidemic acute viral gastroenteritis in developed countries. The absence of an in vitro cell culture model for human norovirus infection has limited the development of effective antivirals and vaccines. Human histo-blood group antigens have been regarded as receptors for norovirus infection, and expression of the α(1,2) fucosyltransferase gene (FUT2) responsible for the secretor phenotype is required for susceptibility to Norwalk virus (NV) infection. We report for the first time that transfection of NV RNA, isolated from stool samples from human volunteers, into human hepatoma Huh-7 cells leads to viral replication, with expression of viral antigens, RNA replication, and release of viral particles into the medium. Prior treatment of the RNA with proteinase K completely abolishes RNA infectivity, suggesting a key role of an RNA-protein complex. Although overexpression of the human FUT2 gene enhances virus binding to cells, it is not sufficient to allow a complete viral infection, and viral spread from NV-transfected cells to naïve cells does not occur. Finally, no differences in NV RNA replication are observed between Huh-7 and Huh-7.5.1 cells, which contain an inactivating mutation in retinoic acid-inducible gene I (RIG-I), suggesting that the RIG-I pathway does not play a role in limiting NV replication. Our results strongly suggest that the block(s) to NV replication in vitro is at the stage of receptor and/or coreceptor binding and/or uncoating, either because cells lack some specific factor or activation of cellular antiviral responses independent of RIG-I inhibits virus replication.The human pathogen Norwalk virus (NV) is the prototype strain of the Norovirus genus in the family Caliciviridae. Noroviruses are responsible for the majority of outbreaks of nonbacterial gastroenteritis in developed countries, and it is estimated that they have a significant impact in developing countries as well. Although human noroviruses were originally identified more than 30 years ago, our understanding of their replication cycle and mechanisms of pathogenicity has been limited because these viruses are noncultivatable in established cell lines and a small animal model to study viral infection is not available. Only recently, it has been reported that both genogroup I (GI) and GII strains of human noroviruses can be passaged several times with limited replication in a differentiated three-dimensional cell culture system derived from a human small intestinal cell line (40). In addition, gnotobiotic pigs can support replication of a human norovirus GII strain, with occurrence of mild diarrhea and virus shedding and immunofluorescent detection of both structural and nonstructural proteins in enterocytes (10). Although these results are promising, it remains unclear whether these systems are robust enough to be widely used to efficiently propagate human noroviruses in vitro, and the factors responsible for the block(s) of viral replication using standard cell culture systems remain unknown.The NV genome is a positive-sense, polyadenylated, single-stranded RNA molecule of 7.7 kb and contains three open reading frames (ORFs): ORF1 encodes a nonstructural polyprotein, and ORF2 and ORF3 encode the major and minor capsid proteins, VP1 and VP2, respectively (14, 24). Due to the lack of an in vitro system to propagate human noroviruses, features of their life cycle have been inferred from studies using other animal caliciviruses that can grow in mammalian cell cultures. A 3′ coterminal polyadenylated subgenomic RNA is produced within infected cells, and it is believed that both genomic and subgenomic RNAs are covalently linked to the nonstructural protein VPg at their 5′ ends. Upon infection of cells, nonstructural proteins are expressed from genomic RNA and form an RNA replication complex, which generates new genomic RNA molecules as well as subgenomic RNAs encoding VP1 and VP2. After expression of the structural proteins from subgenomic RNA molecules, the capsid is assembled and viral RNA encapsidated prior to progeny release. Some of these features have been confirmed using recombinant systems to express the native NV genome in mammalian cells by using vaccinia virus expression systems (2, 25).Studies with human volunteers have shown that some individuals are either repeatedly susceptible or resistant to NV infection (36) and led to the identification of a genetically determined factor that predicts a person''s susceptibility to infection and disease (19, 30). Binding experiments using recombinant NV virus-like particles (VLPs) demonstrated attachment of VLPs to surface epithelial cells of the gastroduodenal junction on biopsies from secretors but not to cells from nonsecretors, showing that the expression pattern of ABH histo-blood group antigens may influence susceptibility to NV (32). The gene responsible for the secretor phenotype encodes an α(1,2)fucosyltransferase (FUT2) that produces H antigens on the surface of epithelial cells and in mucosal secretions (27). Since it was observed that transfection of the FUT2 gene into nonpermissive cells enhances NV binding (31), it has been hypothesized that H antigens or related blood group antigens may function as a receptor for NV.The main goal of our study was to understand the molecular basis of the restricted growth of NV in cultured cells by transfecting wild-type NV RNA into human cells. Our studies show for the first time that transfection of wild-type NV RNA isolated from human stool samples can lead to the production of viral particles, indicating that wild-type NV RNA is infectious and replicates. However, a block to NV spread to other cells in the culture remains, indicating that the block(s) exists at the cell entry and/or uncoating steps.     

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.     

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.     

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.     

Structural basis for the recognition of lewis antigens by genogroup I norovirus

Noroviruses (NoVs) bind to histo-blood group antigens, namely, ABH antigens and Lewis antigens. We previously showed the NoVs GI/2, GI/3, GI/4, and GI/8 were able to strongly bind to Lewis a (Le^a) antigen, which is expressed by individuals who are nonsecretors. In this study, to investigate how Lewis antigens interact with GI NoV virion protein 1 (VP1), we determined the crystal structures of the P domain of the VP1 protein from the Funabashi 258 (FUV258) strain (GI/2) in complexes with Le^a, Le^b, H type 1, or A type 1 antigens. The structures were compared with those of the NV/68 strain (GI/1), which does not bind to the Le^a antigen. The four loop structures, loop P, loop S, loop A, and loop B, continuously deviated by more than 2 Å in length between the Cα atoms of the corresponding residues of the FUV258 and NV/68 P domains. The most pronounced differences between the two VP1 proteins were observed in the structures of loop P. In the FUV258 P domain, loop P protruded toward the next protomer, forming a Le^a antigen-binding site. The Gln389 residue make a significant contribution to the binding of the Le^a antigen through the stabilization of loop P as well as through direct interactions with the α4-fucosyl residue (α4Fuc) of the Le^a antigen. Mutation of the Gln389 residue dramatically affected the degree of binding of the Lewis antigens. Collectively, these results suggest that loop P and the amino acid residue at position 389 affect Lewis antigen binding.     

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: Relationship with ABH blood group determinants

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.     

Qualitative and quantitative analysis of the binding of GII.4 norovirus variants onto human blood group antigens

Noroviruses (NoVs) are one of the leading causes of gastroenteritis in children and adults. For the last 2 decades, genogroup II genotype 4 (GII.4) NoVs have been circulating worldwide. GII.4 NoVs can be divided into variants, and since 2002 they have circulated in the population before being replaced every 2 or 3 years, which raises questions about the role of their histo-blood group antigen (HBGA) ligands in their evolution. To shed light on these questions, we performed an analysis of the interaction between representative GII.4 variants and HBGAs, and we determined the role of selected amino acids in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the amino acids, directly implicated in interactions with HBGAs. However, the ablation of the threonine residue at position 395 (ΔT395), an epidemiological feature of the post-2002 variants, was not deleterious to the binding of the virus-like particle (VLP) to the H antigen, while binding to A and B antigens was severely hampered. Nevertheless, the ΔT395 VLPs gained the capacity to bind to the Lewis x and sialyl-Lewis x antigens in comparison with the wild-type VLP, demonstrating that amino acid residues outside the HBGA binding site can modify the binding properties of NoVs. We also analyzed the attachment of baculovirus-expressed VLPs from six variants (Bristol, US95/96, Hunter, Yerseke, Den Haag, and Osaka) that were isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs. We showed that the six variants could all attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, Den Haag and Osaka variants additionally bound to carbohydrates present in the saliva of Lewis-positive nonsecretors. The carbohydrate binding profile and the genetic and mutagenesis analysis suggested that GII.4 binding to Lewis x and sialyl-Lewis x antigens might be a by-product of the genetic variation of the amino acids located in the vicinity of the binding site. Analysis of the binding properties for the six variants by surface plasmon resonance showed that only post-2002 variants (i.e., Hunter, Yerseke, Den Haag, and Osaka) presented strong binding to A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post-2002 variants. The combination of increased affinity for ABH antigens and of a newly acquired ability to recognize glycans from Lewis-positive nonsecretors could have contributed to the epidemiological importance of strains such as the Den Haag GII.4 subtype.     

Immunodetection of Helicobacter sp. and the associated expression of ABO blood group antigens in the gastric mucosa of captive and free-living New World primates in the Amazon region

The histo-blood group ABH antigens were first described in humans. These antigens are only present on erythrocytes from great apes and humans, while in more primitive animals they are found in tissues and body fluids. The ABH antigens are mainly distributed in tissues exposed to the external environment and potentially serve as ligands for pathogens or inhibitors of tissue connections. The objective of this paper was two-fold: (i) to determine the presence of Helicobacter sp. in the gastric mucosa of 16 captive and 24 free-living New World monkeys and (ii) to evaluate the presence of histopathological alterations related to bacterial infection and the associated expression of ABH antigens in the tissue. Stomach tissues from 13 species of monkey were assessed using haematoxylin-eosin and modified Gram staining (Hucker) methods. An immunohistochemical analysis of the tissue revealed the presence of infectious bacteria that were characteristic of the genus Helicobacter sp. The results demonstrate that various species of monkey might be naturally infected with the Helicobacter sp. and that there is an increased susceptibility to infection. This study serves as a comparative analysis of infection between human and non-human primates and indicates the presence of a new species of Helicobacter.     

Aberrant expression of histo-blood group A type 3 antigens in vascular endothelial cells in inflammatory sites.

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1-4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.     

Trivalent Combination Vaccine Induces Broad Heterologous Immune Responses to Norovirus and Rotavirus in Mice

Rotavirus (RV) and norovirus (NoV) are the two major causes of viral gastroenteritis (GE) in children worldwide. We have developed an injectable vaccine design to prevent infection or GE induced with these enteric viruses. The trivalent combination vaccine consists of NoV capsid (VP1) derived virus-like particles (VLPs) of GI-3 and GII-4 representing the two major NoV genogroups and tubular RV recombinant VP6 (rVP6), the most conserved and abundant RV protein. Each component was produced in insect cells by a recombinant baculovirus expression system and combined in vitro. The vaccine components were administered intramuscularly to BALB/c mice either separately or in the trivalent combination. High levels of NoV and RV type specific serum IgGs with high avidity (>50%) as well as intestinal IgGs were detected in the immunized mice. Cross-reactive IgG antibodies were also elicited against heterologous NoV VLPs not used for immunization (GII-4 NO, GII-12 and GI-1 VLPs) and to different RVs from cell cultures. NoV-specific serum antibodies blocked binding of homologous and heterologous VLPs to the putative receptors, histo-blood group antigens, suggesting broad NoV neutralizing activity of the sera. Mucosal antibodies of mice immunized with the trivalent combination vaccine inhibited RV infection in vitro. In addition, cross-reactive T cell immune responses to NoV and RV-specific antigens were detected. All the responses were sustained for up to six months. No mutual inhibition of the components in the trivalent vaccine combination was observed. In conclusion, the NoV GI and GII VLPs combination induced broader cross-reactive and potentially neutralizing immune responses than either of the VLPs alone. Therefore, trivalent vaccine might induce protective immune responses to the vast majority of circulating NoV and RV genotypes.