Heterogeneity and immunogenicity of theTrichinellaTSL‐1 antigen gp53

Abstract

This study investigates the heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53. Western blotting analysis of several Trichinella isolates with the gp53-specific monoclonal antibodies (mAbs) US5 and US9, produced in Btkxid mice, revealed that gp53 from the species T. britovi, T. murrelli and genotype T8 had higher MW (60 kDa) than gp53 from T. spiralis, T. nelsoni and genotype T6 (53 kDa) and from T. nativa (55 kDa). mAb US5 reacted only with gp53 from T. spiralis. Experiments including immunoassays of gp53 binding by sera from T. spiralis-infected mice, in the presence of different potential inhibitors (recombinant gp53, US5, T. britovi-crude larval extract (CLE), and CLE N- and O-glycans), indicate (i) that gp53 from T. spiralis bears specific epitopes that induce antibody formation during infection; (ii) that the protein epitopes of gp53 are much more important (76 or 68% of total antibody reactivity in BALB/c and Swiss CD-1 mice, respectively) than the corresponding glycan epitopes including tyvelose (11 or 32% of total reactivity) for the induction of anti-gp53 circulating antibodies; and (iii) that the species-specific epitopes present on gp53 are differentially recognized in different mouse strains. Whereas in BALB/c mice US5- and non-US5-recognized species-specific epitopes on gp53 bind about 84% of circulating antibodies on day 80 post-infection, this percentage was only 38% in Swiss CD-1 mice. These data on the antigenicity of gp53 contrast with data for Trichinella CLE antigens, in that most circulating antibodies reactive with CLE antigens recognized tyvelose-containing epitopes (57% and 58% of circulating antibodies in BALB/c and Swiss CD-1 mice, respectively). Together these results demonstrate that gp53 is recognized during infection but is antigenically different from other Trichinella TSL-1 antigens.

Evaluation ofTrichinella spiralisLarva Group 1 Antigens for Serodiagnosis of Human Trichinellosis

“…In our view, the good specificity and sensitivity obtained with TSL-1 antigens, together with the possibility of confirming serodiagnosis by means of selective inhibition with US4 or equivalent MAbs, make this assay more useful than the recently proposed assay of Bruschi et al (6), which is based on the use of synthetic tyvelose-BSA conjugate. (6,14,38). Future studies comparing cTSL-1 antigens and tyvelose-BSA conjugates by using the same serum samples may clarify this question.…”

“…The immunoglobulin G1 (IgG1)/ MAb US4, specific for the tetrasaccha- (14,38), was obtained by fusion of spleen cells from T. spiralis-infected male NBF1 mice with P3-X63-Ag8.653 cells, as previously described (39). The MAb was partially purified by ammonium sulfate precipitation, and used to immobilize antigens in capture ELISA (see below).…”

Species-Specific Antibody Responses to the Recombinant 53-Kilodalton Excretory and Secretory Proteins in Mice Infected withTrichinellaspp

“…Muscle larvae in encapsulated species develop a thick collagen capsule, and the nonencapsulated species develop only a very thin collagen capsule (23). The current study showed that the amino acid sequence of the 53-kDa protein of T. spiralis (an encapsulated species) was highly identical to those of T. britovi and T. nativa (encapsulated species), but it showed a low sequence similarity to those of T. pseudospiralis The native 53-kDa protein in E-S products of T. spiralis shows a marked heterogeneity in glycosylation (15,16) and has multiple protein isoforms (10). The different molecular masses of the native 53-kDa proteins from the different developmental stages (Fig.…”

“…In brief, the 20-l reaction volume consisted of 3 g of the sample RNA, 1 l of 0.5 g/l oligo(dT) [12][13][14][15][16][17][18] , 1 l 10 mM deoxynucleotide triphosphate mix, 4 l First-Strand buffer (Invitrogen), 1 l 0.1 M dithiothreitol, 1 l RNase inhibitor, and 1 l SuperScript III reverse transcriptase. The reaction mixture was incubated at 50°C for 60 min and then inactivated by heating at 70°C for 15 min.…”

“…The 53-kDa glycoprotein secreted from T. spiralis is a candidate immunodiagnostic antigen for trichinellosis, because this protein is present in much greater amounts in the E-S products (25), and the homologue of the 53-kDa glycoprotein of T. spiralis is present in E-S products of other species in the genus Trichinella (15,16,22). The use of the 53-kDa recombinant protein for detection of antibodies against Trichinella antigens has already been described (9,25).…”