Characterization of a surface glycoprotein from Echinococcus multilocularis and its mucosal vaccine potential in dogs.

Alveolar echinococcosis is a refractory disease caused by the metacestode stage of Echinococcus multilocularis. The life cycle of this parasite is maintained primarily between foxes and many species of rodents; thus, dogs are thought to be a minor definitive host except in some endemic areas. However, dogs are highly susceptible to E. multilocularis infection. Because of the close contact between dogs and humans, infection of dogs with this parasite can be an important risk to human health. Therefore, new measures and tools to control and prevent parasite transmission required. Using 2-dimensional electrophoresis followed by western blot (2D-WB) analysis, a large glycoprotein component of protoscoleces was identified based on reactivity to intestinal IgA in dogs experimentally infected with E. multilocularis. This component, designated SRf1, was purified by gel filtration using a Superose 6 column. Glycosylation analysis and immunostaining revealed that SRf1 could be distinguished from Em2, a major mucin-type antigen of E. multilocularis. Dogs (n=6) were immunized intranasally with 500 µg of SRf1 with cholera toxin subunit B by using a spray syringe, and a booster was given orally using an enteric capsule containing 15 mg of the same antigen. As a result, dogs immunized with this antigen showed an 87.6% reduction in worm numbers compared to control dogs (n=5) who received only PBS administration. A weak serum antibody response was observed in SRf1-immunized dogs, but there was no correlation between antibody response and worm number. We demonstrated for the first time that mucosal immunization using SRf1, a glycoprotein component newly isolated from E. multilocularis protoscoleces, induced a protection response to E. multilocularis infection in dogs. Thus, our data indicated that mucosal immunization using surface antigens will be an important tool to facilitate the development of practical vaccines for definitive hosts

Synthesis, Antigenicity Against Human Sera and Structure-Activity Relationships of Carbohydrate Moieties from <em>Toxocara</em> larvae and Their Analogues

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the Galβ1-3GalNAc core of the TES-glycoprotein antigen obtained from larvae of the parasite <em>Toxocara</em> and their analogues have been accomplished. Trisaccharides Fuc2Meα1-2Gal4Meβ1-3GalNAcα1-OR (<strong>A</strong>), Fucα1-2Gal4Meβ1-3GalNAcα1-OR (<strong>B</strong>), Fuc2Meα1-2Galβ1-3GalNAcα1-OR (<strong>C</strong>), Fucα1-2Galβ1-3GalNAcα1-OR (<strong>D</strong>) and a disaccharide Fuc2Meα1-2Gal4Meβ1-OR (<strong>E</strong>) (R = biotinylated probe) were synthesized by block synthesis using 5-(methoxycarbonyl)pentyl-2,3,4,6-tetra-<em>O</em>-acetyl-β-D-galactopyranosyl-(1®3)-2-azide-4-<em>O</em>-benzyl-2-deoxy-α-D-galactopyranoside as a common glycosyl acceptor. We examined the antigenicity of these five oligosaccharides by enzyme linked immunosorbent assay (ELISA). Our results demonstrate that the <em>O</em>-methyl groups in these oligosaccharides are important for their antigenicity and the biotinylated oligosaccharides <strong>A</strong>, <strong>B</strong>, <strong>C</strong> and <strong>E</strong> have high serodiagnostic potential to detect infections caused by <em>Toxocara</em> larvae

Tolerance of SRf1 against gastric protease digestion.

<p>SRf1 was digested in the presence of pepsin (1 mg/mL) at pH 2 or in the presence of trypsin (0.4 mg/mL) and chymotrypsin (1.7 mg/mL) at pH 7.4. After 1 or 4 h digestion, the reaction mixture was applied to a Superose 6 gel filtration column, and peak areas were compared to those of the control. Panel B: 1D-western blot analysis of digested SRf1s detected using sera from dogs infected 5 times. Lanes: M, molecular marker; C, controls (no proteases); 1 and 2, pepsin digestion for 1 and 4 h, respectively; 3 and 4, tryptic digestion for 1 and 4 h, respectively.</p

Localization of SRf1 in the larval and adult stages of <i>E. multilocularis</i>.

<p>Immunostaining was performed using polyclonal mouse anti-SRf1 antiserum. Panels A (40×) and B (100×) show adult worms harbored in the epithelium of the small intestine of infected dogs at 23 days postinfection. Panels C (100×) and D (200×) show protoscoleces in cysts derived from infected cotton rats. The brown color indicates specific antibody reactivity; the blue color indicates hematoxylin staining of nuclei. Anti-SRf1 antibodies were detected on the surface, including suckers, rostella, and hooks in both stages of worm development.</p

Identification and purification of vaccine antigen (SRf1) from <i>E. multilocularis</i>.

<p>Crude extracts were prepared from protoscoleces (PCE) and adult worms (ACE). A total of 120 µg protein was applied to 2D-PAGE. The proteins were blotted onto a PVDF membrane, and reactivity between proteins and intestinal IgA from dogs experimentally infected with <i>E. multilocularis</i> was examined. Panels A and B: PCE and ACE, respectively; panels 1 and 5: CBB-stained gels; panels 2 and 6: tested with intestinal IgA from dogs infected 5 times; panels 3 and 7: tested with intestinal IgA from dogs infected 3 times; and panels 4 and 8: tested with intestinal IgA from dogs infected once. Molecular size markers are indicated on the left (in kDa). Panel C: a gel filtration chromatogram of the vaccine antigen (SRf1) and 2D-western blot analysis; panels 9 and 11: CBB-stained gels of SRf1 and SRf2; panel 10: 2D-western blotting for SRf1 using intestinal swabs from dogs infected 5 times; panel 12: 2D-western blotting for SRf2 using sera from dogs infected 5 times. Panel D: SDS-PAGE analysis of SRf1; lanes 1 and 2: glycoprotein stained-gel of molecular size markers and SRf1; 3 and 4: CBB-stained gel with molecular size markers and SRf1. Glycoprotein detection was performed with a Pro-Q Emerald 300 gel stain kit and CandyCane glycoprotein molecular weight standards.</p

Estimated O-glycan compositions of SRf1 from mass spectrometric data.

*<p>δmass = [observed m/z] – [theoretical m/z]. Abbreviations: Hex, hexose (e.g., mannose, galactose); HexNAc, N-acetylhexosamine (e.g., GlcNAc, GalNAc); NeuAc, N-acetylneuraminic acid; Sulph, sulfated glycan; Pent, pentose.</p

A novel nairovirus associated with acute febrile illness in Hokkaido, Japan

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014–2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan