A. Kwame Nyame

Antibodies to Glycans Dominate the Host Response to Schistosome Larvae and Eggs: Is Their Role Protective or Subversive?

Multiple exposures of chimpanzees to the radiation-attenuated schistosome vaccine provoked a strong parasite-specific cellular and humoral immune response. Specific IgM and IgG were directed mainly against glycans on antigens released by cercariae; these were also cross-reactive with soluble antigens from larvae, adult worms, and eggs. Egg deposition was the major antigenic stimulus after challenge of vaccinated and control chimpanzees with normal parasites, eliciting strong antiglycan responses to egg secretions. Glycan epitopes recognized included LacdiNAc, fucosylated LacdiNAc, Lewis(X) (weakly), and those on keyhole limpet hemocyanin. Antibodies to peptide epitopes became prominent only during the chronic phase of infection, as glycan-specific IgM and IgG decreased. Because of their intensity and cross-reactivity, the antiglycan responses resulting from infection could be a smoke screen to subvert the immune system away from more vulnerable larval peptide epitopes. Their occurrence in humans might explain the long time required for antischistosome immunity to build up after infection.

Immunity to schistosomiasis: glycans are potential antigenic targets for immune intervention.

The major humoral immune responses in animals infected with Schistosoma mansoni are directed toward carbohydrate antigens. Among these antigens are complex-type N-glycans expressing LDN [GalNAcbeta1-4GlcNAc-R], LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R], and polymeric Lewis x (Lex) [Galbeta1-4(Fucalpha1-3)GlcNAc]n-R epitopes. We have now evaluated the potential of the three glycan antigens as targets for immune-mediated intervention of infections and serodiagnosis. A variety of approaches were employed, including ELISA, Western blot, immunohistology, and in vitro complement lysis assays, to determine the immunogenicity of the glycans in infected humans, their localization on the parasites and their efficacy as targets for parasite lysis. Our results show that S. mansoni-infected patients, with either intestinal or hepatosplenic disease, generate predominantly IgM, but also IgG and IgA, antibodies to LDN, LDNF, and Lex. However, immune responses to Lex are generally lower than responses to LDN and LDNF and less specific to schistosome infections. Western blot analysis with monoclonal antibodies (mAb) to LDN, LDNF, and Lex determinants show that the glycan antigens occur on multiple glycoproteins from cercariae, 3-h, 48-h, and lung stage schistosomula, as well as adults and eggs. Immunohistological studies demonstrate that LDN, LDNF, and Lex are expressed on the parasite surface at all stages of development in the vertebrate host. Importantly, a mAb to LDN in the presence of complement efficiently kills schistosomula in vitro, as demonstrated by flow-cytometric assays that quantify cytolysis by propidium iodide uptake into damaged parasites. These findings raise the possibility that LDN and LDNF may be targets for vaccination and/or serodiagnosis of chronic schistosomiasis in humans.

DIFFERENTIAL EXPRESSION OF LacdiNAc, FUCOSYLATED LacdiNAc, AND LEWIS X GLYCAN ANTIGENS IN INTRAMOLLUSCAN STAGES OF SCHISTOSOMA MANSONI

We report the expression of 3 well-characterized adult Schistosoma mansoni glycan antigens among molluscan stages of the parasite. These antigens are LacdiNAc (LDN; GalNAcβ1-4GlcNAc-R), fucosylated LacdiNAc (LDNF; GalNAc[Fucα1-3]β1-4GlcNAc-R), and Lewis x (Lex; Gal[Fucα1-3]β1-4GlcNAc-R). The presence of the glycans was determined by both immunoblot and immunohistological methods using monoclonal antibodies that specifically recognize each glycan epitope. Immunoblot analyses reveal that LDN and LDNF epitopes are expressed on many different glycoproteins, including eggs, mother sporocysts, daughter sporocysts, and cercariae, although LDN expression among daughter sporocysts is greatly reduced. LDN and LDNF epitopes are localized on the tegument and in the intrasporocyst cell masses of both in vitro–derived and in vivo–derived mother sporocysts and in the daughter sporocysts derived on day 16 after infection. Unexpectedly, high levels of LDN and LDNF glycans were detected in the infected, but not in the uninfected, snail hemolymph, suggesting that the infecting larvae secrete LDN and LDNF glycoconjugates into the snail hosts. In contrast, the expression of Lex antigen among the molluscan stages is highly restricted. Lex is present on a few high–molecular weight glycoproteins in eggs and cercariae but is undetectable in mother and daughter sporocysts. Taken together with our earlier studies on vertebrate stages of S. mansoni, these results show that LDN and LDNF glycans are conserved during schistosome development. The study further extends the evidence that Lex is a developmentally regulated antigen in schistosomes.

Development and characterization of a specific IgG monoclonal antibody toward the Lewis x antigen using splenocytes of Schistosoma mansoni-infected mice

The parasitic blood fluke Schistosoma mansoni synthesizes immunogenic glycans containing the human Lewis x antigen (Le(x); Galactose-β1-4(Fucα1-3)N-acetylglucosamine-β-R, also called CD15), but the biological role(s) of this antigen in the parasites and in humans is poorly understood. To develop IgG-based monoclonal antibodies (mAbs) specific for Le(x), we harvested splenocytes from S. mansoni-infected Swiss Webster mice at Week 10 postinfection, when peak IgG responses to glycan antigens occur, and generated a panel of hybridomas secreting anti-glycan IgG that recognize periodate-sensitive epitopes in soluble egg antigens of the parasites, and also recognizes a neoglycoprotein containing a pentasaccharide with the Le(x) sequence. One murine mAb, an IgG3 designated F8A1.1, bound to glycoproteins and glycolipids from schistosome adults and human promyelocytic leukemic HL-60 cells that express Le(x) antigens, as assessed by a wide variety of approaches including immunofluorescence staining, confocal microscopy, flow cytometry and western blotting, as well as overlay assays of glycolipids after thin-layer chromatography. In contrast, F8A1.1 bound weakly to cercariae, 3-h schistosomula and human Jurkat cells. We also directly compared the glycan specificity of F8A1.1 with commercially available anti-CD15 IgG1 (clone W6D3) using a defined glycan microarray. The results demonstrated that F8A1.1 recognized glycans expressing Le(x) epitopes in a terminal nonreducing position, whereas anti-CD15 bound to glycans with multiple repeats of Le(x) epitopes, but not to glycans with a single, terminal Le(x) epitope. Our results show that F8A1.1 recognizes terminal Le(x) epitopes and can be used for identification, immunolocalization, immunoprecipitation and purification of Le(x)-containing glycoconjugates from schistosomes and mammalian cells.

Differential Expression of Anti-Glycan Antibodies in Schistosome-infected Humans, Rhesus monkeys, and Mice

Schistosomiasis is a debilitating parasitic disease of humans, endemic in tropical areas, for which no vaccine is available. Evidence points to glycan antigens as being important in immune responses to infection. Here we describe our studies on the comparative humoral immune responses to defined schistosome-type glycan epitopes in Schistosoma mansoni-infected humans, rhesus monkeys and mice. Rhesus anti-glycan responses over the course of infection were screened on a defined glycan microarray comprising semi-synthetic glycopeptides terminating with schistosome-associated or control mammalian-type glycan epitopes, as well as a defined glycan microarray of mammalian-type glycans representing over 400 glycan structures. Infected rhesus monkeys generated a high immunoglobulin G (IgG) antibody response to the core xylose/core α3 fucose epitope of N-glycans, which peaked at 8-11 weeks post infection, coinciding with maximal ability to kill schistosomula in vitro. By contrast, infected humans generated low antibody levels to this epitope. At 18 months following praziquantel therapy to eliminate the parasite, antibody levels were negligible. Mice chronically infected with S. mansoni generated high levels of anti-fucosylated LacdiNAc (GalNAcβ1, 4(Fucα1, 3)GlcNAc) IgM antibodies, but lacked a robust response to the core xylose/core α3 fucose N-glycan antigens compared with other species studied, and their sera demonstrated an intermediate level of schistosomula killing in vitro. These differential responses to parasite glycan antigens may be related to the ability of rhesus monkeys to self-cure in contrast to the chronic infection seen in humans and mice. Our results validate defined glycan microarrays as a useful technology to evaluate diagnostic and vaccine antigens for schistosomiasis and perhaps other infections.

SURFACE MEMBRANE PROTEINS OF BIOMPHALARIA GLABRATA EMBRYONIC CELLS BIND FUCOSYL DETERMINANTS ON THE TEGUMENTAL SURFACE OF SCHISTOSOMA MANSONI PRIMARY SPOROCYSTS

Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of l-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host–parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval–host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (∼35–150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan-reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated glycoconjugates.

Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite

Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies.

The ruminant parasite Haemonchus contortus expresses an α1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize α1,3-fucosylated GlcNAc and terminal β-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal β-Gal, and the glycoconjugates lack the Lewis x (Lex) antigen or other related fucose-containing antigens, such as sialylated Lex, Lea, Leb Ley, or H-type 1. Direct assays of parasite extracts demonstrate the presence of an α1,3-fucosyltransferase (α1,3FT) and β1,4-N-acetylgalactosaminyltransferase (β1,4GalNAcT), but not β1,4-galactosyltransferase. The H. contortus α1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galα1→4GlcNAcβ1→3Galβ1→4Glc to form lacto-N-fucopentaose III Galβ1→ 4[Fucα1→3]GlcNAcβ1→3Galβ1→4Glc, which contains the Lex antigen, and the acceptor lacdiNAc (LDN) GalNAcβ1→4GlcNAc to form GalNAcβ1→4[Fucα1 →3]GlcNAc. The α1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 μ M and a Vmax of 10.8 nmol-mg−1 h−1. The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The α1,3FT acts best with type-2 glycan acceptors (Galβ1→4GlcNAcβ1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus α1,3FT can synthesize the Lex antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

ABSTRACT Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis.