Barbara L. Doughty

Cloning of a cDNA encoding an egg antigen homologue from Schistosoma mansoni

The egg-induced granulomas are closely associated with pathology in schistosomiasis patients. The soluble components of the eggs, called soluble egg antigens (SEA), were found to be mainly responsible for granuloma formation [l]. Several antigens have been identified from eggs including a prominent 40-kDa protein, p40 [2,3]. The p40 elicited a strong immune response in over 90% of patients and shared a sequence homology with 0t-crystallins and small heat shock proteins (SHSP). The possibility of the existence of a multi-gene family encoding p40 was also proposed [3]. In our effort to characterize SEA, we cloned two cDNAs. One was a 98% match to the p40 reported previously. Another one encoded a peptide which had a 57% identity to p40. Here we present the data for this new clone. A rabbit polyclonal anti-SEA antiserum was used to screen a cDNA expression library constructed from Schistosoma mansoni eggs in the 2Zap II vector [4,5]. Positive clones were plaque purified and transformed into phage-

Production and characterization of human monoclonal antibodies against Schistosoma mansoni

Summary We have produced a panel of human monoclonal antibodies (MoAb) from patients infected with Schistosoma mansoni in order to analyse more carefully the human immune response to this helminth infection. This study describes the production, characterization and analysis of these MoAbs. Briefly, peripheral blood mononuclear cells from chronically infected patients were (1) isolated and stimulated with parasite antigens in vitro, (2) positively selected for B–cells on anti–Ig columns, and (3) then transformed with Epstein–Barr virus (EBV). Once EBV cell lines were established, they were selected for anti–5. mansoni antibodies using an ELISA, cloned, retested and then fused with the mouse–human heteromyeloma SHM–D33. In this study, we describe five MoAbs which have different antigenic specificities for life–cycle stages based on ELISA to soluble crude antigen preparations, membrane immunofluorescence on whole intact organisms, and immunofluorescent staining of cryostat frozen sections. The importance of these reagents with regard to the human immune response to S. mansoni is currently being evaluated.

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human Schistosomiasis: I. Granuloma formation and modulation around polyacrylamide antigen-conjugated beads

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of delayed type hypersensitivity granuloma formation in patients infected with Schistosoma mansoni. Our data show that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding the antigen conjugated beads; 2) this reactivity is a CD4 cell dependent, macrophage dependent, B cell independent response and 3) the in vitro granuloma response is antigenically specific for parasite egg antigens. Studies designed to investigate the immune regulation of granulomatous hypersensitivity using purified populations of either CD4 or CD8 T cells have demonstrated the complexity of cellular interactions in the suppression of granulomatous hypersensitivity. The anti-S. mansoni egg immune responses of individual patients with chronic intestinal schistosomiasis can be classified either as soluble egg antigen (SEA) hypersensitive with maximal granulomatous hypersensitivity or SEA suppressive with activation of the T cell suppressor pathway with effective SEA granuloma modulation. Our data suggest that T cell network interactions are active in the generation of effective granuloma modulation in chronic intestinal schistosomiasis patients.

Granulomatous hypersensitivity to Schistosoma mansoni EGG antigens in human schistosomiasis. IV. A role for prostaglandin‐induced inhibition of in vitro granuloma formation

The prostaglandins (PG) are known to regulate immune cell function(s) and participate in the progression of both acute and chronic inflammatory reactions. Using an in vitro model of Schistosoma mansoni egg‐induced hypersensitivity granulomas, we have delineated the role of immune complexes (IC) in the induction and release of PG and their inhibitory effects on granuloma development. The hypersensitivity‐type granuloma reaction to soluble egg antigen (SEA) was examined using a model of in vitro granuloma formation. Our results show that granuloma formation was dramatically suppressed by the addition to the granuloma cultures of IC, PGE1, PGE2, while PGF2 alpha had no significant effect. The inhibition of the PG function was achieved by the introduction of anti‐PG antibodies that blocked suppression of granuloma formation. It appears in this model system that IC may inhibit the activity of granuloma formation by stimulating the monocyte‐macrophage lineage to release inhibitory mediators. Our results suggest that the prostaglandins E series may be important in the generation and maintenance of suppression of the granulomatous inflammatory response to S. mansoni egg antigens.

Characterization of Schistosoma mansoni 44.756.8 kDa egg antigens recognized by human monoclonal antibodies which induce protection against experimental infection and proliferation of peripheral blood mononuclear cells from schistosomiasis patients

We described here the characterization of Schistosoma mansoni egg antigens recognized by human monoclonal antibodies B10 (HmAb-B10) and D5 (HmAb-D5). SDS-PAGE and Western blot analysis revealed that these monoclonals recognized two antigens of M W 44.7/56.8 kDa, with pI of 7.0 and 7.8, respectively. The passive transfer of B10 and D5 induced a significant protection of 48% and 54% in Balbic mice. Results of in vitro cytotoxicity assay showed that both monoclonals were able to kill schistosomula in the presence of rabbit complement. These monoclonals mediated 48% and 74% of schistosomula cytotoxicity, respectively. Egg antigens were purified by affinity chromatography using monoclonal antibodies B10 and D5. Treatment of purified antigens with periodate, galactose oxidase and trifluoromethane sulphonic acid did not prevent binding by B10 and D5 in ELISA assay. However, the treatment with protease K and 2-mercaptoethanol affects the antibodies binding, showing that the HmAbs B10 and D5 recognize polypeptide epitopes. Vaccination of mice with these antigens in Freunds adjuvant induced 43% reduction in worms burden after challenge with S. mansoni cercariae. In vitro blastogenesis assays with peripheral blood mononuclear cells from patients infected with S. mansoni revealed that purified antigens were able to induce significant cell proliferation.

HPLC fractionated soluble egg antigen from Schistosoma mansoni elicits a heterogeneous human immune response

Peripheral blood mononuclear cells (PBMC) from five chronic schistosomiasis patients, three former patients, a SEA sensitized individual, and normal controls were tested in lymphoblastogenesis assays for their ability to proliferate in response to soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP) from Schistosoma mansoni. Cells from all patients and the SEA sensitized individual gave significantly higher responses than the normal controls when stimulated with SEA and SWAP. However, the chronic patients’ SEA responses were much lower than those of the former patients and the SEA sensitized individual. When cells from the same donors were tested in the in vitro granuloma assay, all produced significant granulomatous responses except the normal controls. Once it was established that all individuals in the study gave significant lymphoproliferative responses and granulomatous reactions, SEA was subjected to HPLC fractionation to identify immunogenic protein components of SEA. HPLC separation yielded 25 major fractions. SEA responses from the sensitized individual and former patients exhibited broad, unregulated responsiveness including fractions with neutral, less charged proteins while the chronic patients demonstrated a more restricted range of responsiveness. SEA‐HPLC fractions 14, 21, and 22 contain the most immunodominant proteins based on cellular proliferation data from reactive individuals tested.