Anti-schistosomal immunity to core xylose/fucose in N-glycans

Abstract

Schistosomiasis is a globally prevalent, debilitating disease that is poorly controlled by chemotherapy and for which no vaccine exists. While partial resistance in people may develop over time with repeated infections and treatments, some animals, including the brown rat (Rattus norvegicus), are only semi-permissive and have natural protection. To understand the basis of this protection, we explored the nature of the immune response in the brown rat to infection by Schistosoma mansoni. Infection leads to production of IgG to parasite glycoproteins with complex-type N-glycans that contain a non-mammalian-type modification by core α2-Xylose and core α3-Fucose (core Xyl/Fuc). These epitopes are expressed on the surfaces of schistosomula and adult worms. Importantly, IgG to these epitopes can kill schistosomula by a complement-dependent process in vitro. Additionally, sera from both infected rhesus monkey and infected brown rat were capable of killing schistosomula in a manner inhibited by glycopeptides containing core Xyl/Fuc. These results demonstrate that protective antibodies to schistosome infections in brown rats and rhesus monkeys include IgG responses to the core Xyl/Fuc epitopes in surface-expressed N-glycans, and raise the potential of novel glyco-based vaccines that might be developed to combat this disease.

Keywords: N-glycans; antigens; core fucose; core xylose; immunity; schistosomiasis.

FIGURE 1

Rats generate IgG to core xylose/core α3 fucose epitope. Schistosoma mansoni twice-infected rat serum (A, B) and mouse sera from various infection time points (C, D), diluted 1:50 and screened for IgG (A, C) and IgM (B, D) binding to the Defined Schistosome-type Array (DSA), and detected with goat anti-mouse IgG-Alexa 488, anti-mouse IgM-Alexa 633, goat anti-rat IgG-Alexa 546, and anti-rat IgM-Alexa 488. Each bar represents an average of binding intensity (relative fluorescence units, RFU) to tetra-replicate spots for each glycan ID number (listed on X-axis), +/− 1 standard deviation. Relevant glycan structures depicted, and full list of structures and glycan IDs in Supplementary Figure S1. Glycans are either N-linked (asparagine-linked) or AEAB-linked (bifunctional fluorescent linker, 2-amino-N-(2-aminoethyl)-benzamide); N-glycopeptides contain 1-4 amino acids.

FIGURE 2

Rat antisera and rabbit anti-HRP share similar specificity for CX/CF on the DSA. (A) Rabbit anti-HRP screened on the DSA at 20 μg/mL and detected with goat anti-rabbit IgG-Alexa 488. (B) Rat serum at 1:50 screened for IgG on the DSA with or without pre-incubation with 100 µg HRP in the binding buffer.

FIGURE 3

Anti-HRP kills schistosomula in vitro. (A) Rabbit anti-HRP and normal rabbit IgG at indicated doses incubated with 3h transformed schistosomula and active guinea pig complement, and percentage of non-viable out of total schistosomula quantified at 48 h. 2-way repeated measures ANOVA showed a significant effect of antibody type and dose. *p < 0.05; **p < 0.01. (B) Rabbit anti-HRP at 2 mg/mL incubated with 3h transformed schistosomula and active or heat-inactivated (HIA) guinea pig complement, and percentage of non-viable out of total schistosomula quantified at 24–72 h. 2-way repeated measures ANOVA showed a significant effect of time and complement activity. ****p < 0.0001. (C) Rabbit anti-HRP at 2 mg/mL, pre-incubated with or without the indicated blockers (50 μg per well), incubated with 3h transformed schistosomula and active guinea pig complement, and percentage of non-viable out of total schistosomula quantified at 24–48 h. 2-way repeated measures ANOVA with Sidak’s multiple comparisons test showed a significant effect of time and blocker, with the level of significance for each blocker compared to control shown in the table. ns, not significant; **p < 0.01, ***p < 0.001 ****p < 0.0001. BSA, Bovine serum albumin, HRP, Horseradish peroxidase, HRP gps, HRP glycopeptides, PHA, phytohemagglutinin-E, BRO, Pineapple bromelain.

FIGURE 4

Core xylose/core fucose expressed on surface and secretions of S. mansoni larval life stages. (A) Fixed S. mansoni cercaria stained with 100 μg/mL anti-HRP or control (normal rabbit IgG), anti-rabbit-Alexa488, mounted on slides and visualized at 100x (exposure time 207.8 ms, FITC 539.4 ms). (B) S. mansoni cercaria, in vitro isolated schistosomula, and murine-isolated adult worms stained with rabbit anti-HRP or control (normal rabbit IgG) and visualized in 96-well plates; larvae imaged at 10x (exp time 1.051 s, FITC 490.3 ms), adults imaged at 4x (exp time 6.115 s, FITC 717.9 ms). (C) Cercaria stained with anti-HRP and imaged at 20x using deconvolution microscopy. (D) 3 h schistosomula stained with anti-HRP, (left), or anti-HRP pre-incubated with HRP glycopeptides (right) and imaged at 20x (exp time 1.051 s, FITC 490.3 ms). (E) Western blot of rabbit anti-HRP staining: S. mansoni soluble egg antigen (Egg), RIPA extracts of adult worms (Adult), 3 h schistosomula (Som) and whole cercaria (Cerc) each with 8 μg loaded per well (left panel); Concentrated supernatant of cercarial secretions (Cerc. Sec., 1.4 μg), secretion-depleted cercarial pellet (Cerc. Pel., 6.7 μg) (middle panel); aquarium water concentrate as negative control (control), cercarial secretions (1.4 μg) and secretion-depleted cercarial pellet (6.7 μg) (right panel) at indicated exposure times.

FIGURE 5

Killing of schistosomula by infected rat and rhesus serum with glycoprotein blockers. 3 h schistosomula were incubated with active guinea pig complement and pooled rat serum (A–C) or rhesus serum (D, E) from secondary infection, which had been pre-incubated with the indicated blocking glycoproteins or glycopeptides. Percentage of non-viable out of total schistosomula were quantified at 48h. Paired t-tests were performed for sera with or without HRP blocking with the indicated p-values (A,B, and D). In (C, E), concentrations of blocker are 50 μg (asialofetuin, BSA, HRP, PHA, BRO) or 25 μg (HRP glycopeptides). One-way ANOVA with Dunnett’s multiple comparisons was performed to compare each blocker to control (no blocker) *p < 0.05; **p < 0.01, ***p < 0.001 ****p < 0.0001. In (F), 10 samples from individual infected rhesus monkeys were stratified by high (>1,000 RFU) or low (≤1,000 RFU) reactivity to CX/CF epitope on the DSA, and tested for schistosomula killing with or without HRP blocking (100 μg/well). 2-way ANOVA with Sidak’s multiple comparisons showed significant effect of blocking for rhesus samples with high CX/CF reactivity, but not for blocking of samples with low CX/CF reactivity.