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

Abstract

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.

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

“…These global profiling studies have confirmed the major glycan structures previously identified and provided new information on the diversity of glycans and the heterogeneity found among the life stages of the parasite and their secretions (27,(29)(30)(31). Several studies also have demonstrated the feasibility of profiling schistosome glycans using various microarray printing technologies and probing with glycan-binding proteins, antibodies, and antisera, demonstrating that such techniques can be powerful tools for profiling the immune response to this disease (26,(32)(33)(34) and the development or characterization of novel anti-glycan reagents. The combination of these techniques, i.e., glycomics and/or glycan structural profiling, which is guided by the use of immunologically relevant reagents, such as antisera and MAbs, has great potential to improve our understanding of the anti-glycan response and uncover novel, immunologically relevant diagnostic and vaccine candidates.…”

“…mansoni-infected Biomphalaria glabrata snails, strain NMRI NR-21962, were provided by the Schistosome Research Reagent Resource Center through BEI Resources, NIAID, NIH. Snail maintenance, collection of cercariae, transformation to schistosomula, and isolation of adult worms from mice were conducted as previously described (34,46). The schistosomula were cultured for 2 to 3 days in DMEM with 10% fetal bovine serum (FBS) and penicillin-streptomycin at a density of 500 to 1,000 organisms/ml in tissue culture dishes.…”

“…Cercariae, schistosomula, adult worms, and eggs were isolated and processed for parasite life-stage lysates or soluble egg antigen (SEA) as previously described (34,48). For SDS-PAGE and Western blotting, 1 g of SEA and 5 g parasite lysates were boiled in 1ϫ NuPAGE SDS sample buffer plus 2.5% ␤-mercaptoethanol for 10 min and then run in 10-well Mini-Protean-TGX gels at 200 V for 30 min with broad-range protein standards (Spectra multicolor).…”

“…Schistosomula were isolated as described previously (34,48). After vortex transformation, separation from tails via Percoll, and washing in DMEM, the newly transformed schistosomula were cultured for 3 h at 37°C in 96-well plates with 50 to 100 parasites in 40 l of DMEM with penicillin-streptomycin in each well.…”

“…4C). We screened with six mouse MAbs derived from schistosome-infected mice, referred to as anti-LDNF (clone L6B8), anti-LDN (clone Y1H5), anti-Le X (clone 5F1), anti-M3GN2 (clone 100-4g11-A), and two anti-LDN-dF (clones 290-2D9-A and 290-4A8) MAbs (34,(39)(40)(41)(42). Anti-LDNF bound fractions F9-4 and F11-4.…”