Specificity of human natural antibodies referred to as anti-Tn.

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 GalNAcα1-3Galß1-3(4)GlcNAcß. 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.

Staphylococcus aureus throat carriage is associated with ABO-/secretor status.

OBJECTIVES: In 30% of carriers, Staphylococcus aureus colonization affects exclusively the pharynx and occurs independently from its presence in the nares. This additional reservoir has implications for S. aureus transmission, infection, and decolonization. Host factors promoting colonization of the throat, however, are unknown. METHODS: We determined pharyngeal and persistent nasal carriage of S. aureus, ABO histo-blood group and ABH secretor status phenotypes in 227 individuals. RESULTS: Compared to group A/non-secretors, group O/non-secretor individuals were at increased risk of carrying S. aureus in their throat (OR 6.50, 95% confidence interval 1.28-33.03, P = 0.02) and group O/secretor individuals were protected (OR 0.24, 0.07-0.77, P = 0.02). Both associations became moderately stronger after adjusting for persistent S. aureus nasal carriage, which was found to be a risk factor for pharyngeal colonization in the univariable analysis (OR 2.41, 1.35-4.33, p = 0.003). Most simultaneous carriers (72%) had identical S. aureus genotypes in their nose and throat. CONCLUSIONS: These findings are consistent with in vitro studies that proposed a role of histo-blood group antigens as ligands for S. aureus and support their contribution to the observed population variation in nasopharyngeal S. aureus colonization. Based on their tissue specific expression histo-blood group antigens appear to modulate individual S. aureus colonization patterns.

Expression of Lea in gastric cancer cell lines depends on FUT3 expression regulated by promoter methylation.

Aberrant expression of Lewis antigens has been demonstrated in gastric lesions, namely gastritis, intestinal metaplasia (IM) and gastric carcinoma (GC), and can be partly due to overexpression of the Lewis (FUT3) enzyme. Our aim was to evaluate the role of promoter methylation in FUT3 and Le(a) expression in gastric carcinoma cell lines. MKN45 cell line showed low amounts of Le(a), in the absence of FUT3; GP220 expressed high levels of Le(a) and FUT3. After 5aza-2'deoxycytidine MKN45 showed increased levels of FUT3 and Le(a), by immunohistochemistry and Real-Time PCR, whereas GP220 showed an increase in FUT3 without increase of Le(a). Enzyme activity assays confirmed an increase in alpha-1,4 fucosyltransferase activity in both cell lines by 5aza-2'deoxycytidine. Luciferase reporter gene assays, using methylated and unmethylated deletion constructs of FUT3 promoter, showed that FUT3 expression is regulated by methylation. Summing up, we showed that FUT3 overexpression in gastric cells depends upon promoter hypomethylation and that FUT3 is responsible for overexpression of Le(a) in gastric cells, in vitro. FUT3, Lea, Methylation.

Norwalk virus infection associates with secretor status genotyped from sera.

ABO histo-blood group type and secretor status are two genetically determined factors that contribute to resistance and susceptibility to Norwalk virus (NV). Archived serum samples but not saliva samples are available from NV and many other norovirus challenge studies and outbreaks. A person's ABO phenotype is easily determined from their archived sera, but the individual's secretor phenotype cannot easily be ascertained without saliva. We now report that a person's secretor genotype can also be determined from the archived serum samples. Of the 51 volunteers who participated in a NV challenge study, all eight non-secretors were resistant to NV infection, all of the 42 NV-infected volunteers were secretor positive, and a single uninfected secretor was histo-blood group type B. In agreement with a previous report, secretor status was most predictive of risk of NV infection. The methods described in this report should rapidly improve our knowledge of the associations between carbohydrate antigen expression and susceptibility to different strains of the non-cultivatable noroviruses by enabling retrospective studies from previously collected volunteer challenge and outbreak sera.

Cellular and humoral immunity following Snow Mountain virus challenge.

Little is known about the immune response to noroviruses. To elucidate the immunobiology of norovirus infection in humans, 15 volunteers were challenged with Snow Mountain virus (SMV), a genogroup 2 norovirus. We assessed the cellular and humoral immune response and infection by analyzing stool, serum, saliva, and peripheral blood mononuclear cell (PBMC) responses pre- and postchallenge. In contrast to Norwalk virus (NV), SMV infection was not dependent upon blood group secretor status. Nine of 15 volunteers were infected and showed a >/=4-fold increase over the prechallenge anti-SMV serum immunoglobulin G (IgG) titer, mostly subclass IgG1. Although serum IgG elicited by SMV infection was cross-reactive with Hawaii virus (HV), another genogroup 2 norovirus, salivary IgA was less cross-reactive. Neither SMV-elicited serum IgG nor salivary IgA cross-reacted with NV, a genogroup 1 norovirus. Significant increases in serum gamma interferon (IFN-gamma) and IL-2, but not IL-6 or IL-10, were noted on day 2 postchallenge. For the majority of volunteers, both infected and uninfected, PBMCs stimulated with norovirus virus-like particles secreted IFN-gamma and other Th1 cytokines, suggesting previous norovirus exposure in most volunteers. Like the IgG antibodies, the SMV-activated T cells were cross-reactive with HV but not NV. IFN-gamma production was dependent upon CD4(+) cells, consistent with a predominant, but not exclusive, Th1 response. To our knowledge, this is the first report characterizing T-cell and cytokine responses following live norovirus challenge.

Noroviruses bind to human ABO, Lewis, and secretor histo-blood group antigens: identification of 4 distinct strain-specific patterns.

We characterized the binding of 8 Noroviruses (NORs) to histo-blood group antigens (HBGAs) in human saliva using recombinant NOR (rNOR) capsid proteins. Among the 8 rNORs tested, 6 formed viruslike particles (VLPs) when the capsid proteins were expressed in insect cells, all of which revealed variable binding activities with saliva; the remaining 2 rNORs did not form VLPs, and the proteins did not bind, or bound weakly, to saliva. Four distinct binding patterns were associated with different histo-blood types, defined by Lewis, secretor, and ABO types. Three patterns (VA387, NV, and MOH) recognized secretors, and 1 pattern (VA207) recognized Lewis-positive nonsecretors. The 3 secretor-recognizing patterns were defined as A/B (MOH), A/O (NV), and A/B/O (VA387) binders. Oligosaccharides containing the Lewis and ABH antigenic epitopes were involved in binding. Our findings suggest that different strains of NORs may recognize different human HBGAs on intestinal epithelial cells as receptors for infection.

Human susceptibility and resistance to Norwalk virus infection.

Infectious diseases have influenced population genetics and the evolution of the structure of the human genome in part by selecting for host susceptibility alleles that modify pathogenesis. Norovirus infection is associated with approximately 90% of epidemic non-bacterial acute gastroenteritis worldwide. Here, we show that resistance to Norwalk virus infection is multifactorial. Using a human challenge model, we showed that 29% of our study population was homozygous recessive for the alpha(1,2)fucosyltransferase gene (FUT2) in the ABH histo-blood group family and did not express the H type-1 oligosaccharide ligand required for Norwalk virus binding. The FUT2 susceptibility allele was fully penetrant against Norwalk virus infection as none of these individuals developed an infection after challenge, regardless of dose. Of the susceptible population that encoded a functional FUT2 gene, a portion was resistant to infection, suggesting that a memory immune response or some other unidentified factor also affords protection from Norwalk virus infection.