David S. Newburg

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 milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis

Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens.

Human Milk Oligosaccharide Blood Group Epitopes and Innate Immune Protection against Campylobacter and Calicivirus Diarrhea in Breastfed Infants

Human milk contains a variety of bioactive agents, including oligosaccharides, which are part of the innate defense system (Hanson et al. 1991; Zopf & Roth 1996; Newburg et al. 1998; Erney et al. 2000; Hamosh 2001; Chaturvedi et al. 2001). Oligosaccharides are the third largest solid constituent of human milk after lactose and lipids (Stahl et al. 1994; Zopf & Roth 1996; Erney et al. 2000; Chaturvedi et al. 2001; Hamosh 2001). α 1,2-Linked fucosylated oligosaccharides in human milk have been shown to inhibit host ligand binding to Campylobacter, caliciviruses, and other enteric pathogens (Newburg et al. 1990; Crane et al. 1994; Marionneau et al. 2002; Ruiz-Palacios et al. 2003; Huang et al. 2003). Significant variation exists between mothers in the quantity of α 1,2-linked fucosylated oligosaccharides in their milk. We conducted this study in breastfed infants to determine the clinical relevance of the observed variation in human milk fucosylated oligosaccharides in relation to protection offered infants against diarrhea due to Campylobacter jejuni and caliciviruses.

Human Milk α1,2-Linked Fucosylated Oligosaccharides Decrease Risk of Diarrhea Due to Stable Toxin of E. Coli in Breastfed Infants

Breastfed infants have a lower risk of diarrhea than infants fed artificial formula. Human milk components, including oligosaccharides, inhibit pathogens in vitro and are postulated to protect infants from disease. Most human milk oligosaccharides are fucosylated. The fucose terminus may be connected by an a 1,2 linkage catalyzed by a fucosyltransferase produced by the secretor gene (FUT2), or by an α1,3 or α1,4 linkage catalyzed by fucosyltransferases produced by the Lewis gene (FUT3) or other α1,3 transferase genes (FUT4,5,6,7,9) of this family. The secretor and Lewis genes also control expression of the Lewis blood group type (Erney et al. 2000, Henry et al. 1995). Some women are nonsecretors, i.e., they do not secrete 2-linked fucosyloligosaccharides into their milk and other bodily fluids. Even among secretors, however, the expression of milk fucosyloligosaccharides varies significantly (Chaturvedi et al. 2001, Erney et al. 2000, Thurl et al. 1997, Viverge et al. 1990). Enterotoxigenic Escherichia coli that produce stable toxin (ST-E. coli) is a common cause of diarrhea; the ability of stable toxin to cause diarrhea is inhibited by human milk α1,2-linked fucosyloligosaccharides in vitro and in vivo.

Fucosylated Oligosaccharides in Human Milk in Relation to Gestational Age and Stage of Lactation

Breastfeeding is known to offer significant protection against infectious diarrhea. Oligosaccharides, the third largest solid component of human milk, constitute a major mechanism of innate defense of breastfed infants against infectious diarrhea (Morrow et al. 2004; Jiang et al. 2004; Newburg et al. 2004; Ruiz-Palacios et al. 2003). The fucosyl-ated oligosaccharides of human milk comprise the majority of milk oligosaccharides and have been shown to inhibit host cell binding to specific enteric pathogens (Crane et al. 1994; Newburg et al. 1990; Ruiz-Palacios et al. 2003). Mothers vary in the quantity and type of oligosaccharides found in their milk, which may influence the degree to which their milk offers protection of their infants against specific pathogens. Human milk fucosylated oligosaccharide synthesis is controlled by the same fucosyltransferase genes (FUT2 and FUT3) that control secretor and Lewis blood group types (Newburg et al. 2004; Viverge et al. 1990). Oligosaccharide expression in human milk also may be affected by gestational age and stage of lactation.

α1–2 Fucosylated Chains (H-2, H-1, and Lewisb) are the Main Human Milk Receptor Analogs for Campylobacter

The pathogenesis of Campylobacter diarrhoea involves a series of complex events, in which toxin production and adherence and invasion to gut mucosa are of primary importance. A possible strategy for the prevention of gastrointestinal infections is the inhibition of enteropathogen attachment to mucosal surfaces. It has been reported that human milk contributes to the protection against gastrointestinal infections in infants, presumably by acting as receptor analogues for pathogens. To determine the role of factors present in human milk that may act as receptor analogues for C. jejuni, we used isolates from children participating in a cohort study, where the clinical outcome of infection was well defined; we developed an in vitro inhibition assay for bacterial association using HEp-2 cells and 4 oligosaccharide (OS) fractions of human milk, and an experimental model for colonization in mice. Strong inhibition of cell association was observed with the crude OS fractions. When these were further purified, only the neutral fraction retained inhibitory activity, and, when further purified, only the fucosylated OS were inhibitory. To identify the fucose-containing determinants that inhibited the attachment of Campylobacter in vitro and in vivo, we developed two bacterial-binding assays, a Western blot and an ELISA, with a group of commercially available fucosylated carbohydrate chains linked to proteins. With these methods we were able to characterize three main human milk receptor analogs that inhibited Campylobacter attachment to epithelial cells.

Inhibition of HIV-1 infection in vitro by human milk sulfated glycolipids and glycosaminoglycans.

Sulfated glycolipids (SG) and glycosaminoglycans (GAG) present on the surface of colonic, vaginal epithelial, and neuroglial cells bind to HIV gpl20, suggesting that these glycoconjugates may have a role in HIV infection. The major goal of our study was to test the ability of SG and GAG from human milk to inhibit HIV-1 infection in vitro. SG and GAG were purified from pooled human milk and characterized by high pressure liquid chromatography (HPLC) and mass spectrometry. Eight different preparations of SG and one of GAG were tested for inhibition of infection. Two laboratory isolates, HIV-1Ada (macrophage-tropic virus) and HIV-1SF2 (lymphotropic virus), were used for inhibition assays using peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). Inhibition assays were performed by preincubation of serial dilutions of glycoconjugates with each virus before infecting the monolayer of cultured MDM and PBMC. After 4 days, HIV p24 antigen was quantified by enzyme immunoassay (EIA) in culture supernatants. Significant inhibition of viral infectivity was defined as >80% reduction in p24 concentration. GAG showed a low inhibitory effect (8–44%) in HIV-1 infection of PBMC. Table 1 shows the minimal amounts of the most active SG fractions, expressed as volumes of human milk from which they were extracted, that inhibit >80% HIV-1 infection.