Comparative evaluation of Schistosoma mansoni, Schistosoma intercalatum, and Schistosoma haematobium alkaline phosphatase antigenicity by the alkaline phosphatase immunoassay (APIA).

To know if alkaline phosphatase (AP) from schistosomes other than Schistosoma mansoni can be used as diagnostic marker for schistosomiasis in alkaline phosphatase immunocapture assay (APIA), we comparatively tested n-butanol extracts of adult worm membranes from a Venezuelan (JL) strain of S. mansoni (Ven/AWBE/Sm); a Cameroonian (EDEN) strain of Schistosoma intercalatum (Cam/AWBE/Si) and a Yemeni strain of Schistosoma haematobium (Yem/AWBE/Sh). APIA was evaluated with sera of patients from Venezuela, Senegal, and Gabon infected with S. mansoni, from Gabon infected with S. intercalatum or S. haematobium, from Chine infected with Schistosoma japonicum and from Cambodian patients infected with Schistosoma mekongi. Results indicate that 92.5% (37/40) of Venezuela sera, 75% (15/20) of Senegal sera, 39.5% (17/43) of S. haematobium sera, and 19.2% (5/26) S. intercalatum sera were APIA-positive with the Ven/AWBE/Sm preparation. APIA with the Cam/AWBE/Si preparation showed that 53.8% of S. intercalatum-positive sera had anti-AP antibodies, and 51.2% S. haematobium-positive sera cross-immunocapturing the S. intercalatum AP. APIA performed with Yem/AWBE/Sh showed that 55.8% S. haematobium sera were positive. Only two out of nine S. japonicum sera were APIA-positive with the Ven/AWBE/Sm and Cam/AWBE/Si, and no reaction was observed with Cambodian S. mekongi-positive sera. AP activity was shown to be present in all the schistosome species/strains studied. The use of APIA as a tool to explore the APs antigenicity and the presence of Schistosoma sp. infections through the detection of anti-Schistosoma sp. AP antibodies in a host, allowed us to demonstrate the antigenicity of APs of S. mansoni, S. intercalatum, and S. haematobium.

Detection of the Sm31 antigen in sera of Schistosoma mansoni- infected patients from a low endemic area.

Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14-18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113-122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83.3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis.

Detection of schistosomiasis cases in low-transmission areas based on coprologic and serologic criteria The Venezuelan experience.

Low and very-low intensities of infection hinder the diagnosis of schistosomiasis. Therefore, new parameters should be established in order to more accurately identify active cases and true infection prevalence, for the adequate implementation of a control program. After the survey and analysis of the epidemiological characteristics of five Venezuelan communities, we propose three criteria for the definition of a "schistosomiasis case", based on different diagnostic methods: stool examination, ELISA-soluble egg antigen with sodium metaperiodate (SMP-ELISA), alkaline phosphatase immunoassay (APIA) and the circumoval precipitin test (COPT). Briefly, criterion I: persons with Schistosoma mansoni eggs in stools; criterion II: persons without eggs in stools, with positive COPT, without previous antischistosome chemotherapy in the last year; and criterion III: persons without eggs in stools, with negative COPT, with two positive immunoenzymatic tests (SMP-ELISA and APIA), and with no previous chemotherapy. The incorporation of serological tests to epidemiologic surveillance in areas of low-transmission tries to compensate the underestimation of prevalence based only on parasitological diagnosis.

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni.

This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complement-dependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule.

Immunogenicity of polymerizable synthetic peptides derived from a vaccine candidate against schistosomiasis: the asparaginyl endopeptidase (Sm32).

The asparaginyl endopeptidase (Sm32) is expressed in the gastrodermal cells of the schistosome gut and in the head glands of the cercariae. Possibly, Sm32 hydrolyzes pro-proteins involved in the degradation of host hemoglobin [Parasitol. Today 12 (1996) 125]. Preliminary evidences using an Sj32/Sm32 murine vaccine have shown a profound effect on oviposition and worm burden [Chin. J. Schist. Control. 7 (1995) 72; Bull. Human Med. Univ. 24 (1999) 225; Vaccine 20 (2002) 439]. The importance of Sm32 as a novel vaccine candidate is based on the possibility of preventing the maturation of other cathepsins and/or preventing schistosome skin invasion. We studied the immunogenicity of polymerizable peptides derived from Sm32 to select potential protective epitopes. Sm32 prediction of T and B epitopes and homology studies with human legumain were performed. Among the variety of factors that influence the antibody response, we specifically examined the effect of: (i) genetic background of mouse strain, inbred (C57BL/6) versus outbred (Swiss) mice; and (ii) vaccination with a single peptide versus pool of peptides. Swiss mice raised antibodies to three different regions of the Sm32, as tested by the Multiple Antigen Blot Assay (MABA): 182-215 (peptides IMT-70 and 72), 244-273 (IMT-64) and 336-355 (IMT-66). None of these regions were immunogenic for C57BL/6. On the contrary, other peptides, IMT-4 (21-40), IMT-12 (101-120) and IMT-26 (292-313) were highly immunogenic for this inbred strain. Only Swiss mice immunized with a single peptide (IMT-64 and 72) or with three different pools of IMT-peptides (Pool A-II: 14, 16, 18, 70, 72, 89; pool A-III: 22, 64, 24, 26, 28 and pool A-V: 64, 66, 28, 70, 72) recognized the original protein in a crude extract of the worm antigen by Western blot. Peptides IMT-64, 14 and 26 were responsible for this recognition. In general, the vaccination with pool of peptides was more immunogenic for both mouse strains. Predicted B cell epitopes, with hydrophilicity scores over +10 (IMT-12, 64, 26) were always immunogenic after either single or combined peptide vaccination. Sm32 sequences 41-80 (IMT-6 and 8), 141-160 (IMT-16) and 182-215 (IMT-70 and 72) were nearly identical to the corresponding human legumain regions and should be excluded from the human vaccine. We can conclude that the regions of Sm32 that were recognized by antibodies of mice immunized with polymerizable peptides depended on the mice strain and on the hydrophilicity score of the peptides.

Specificity of the solid phase alkaline phosphatase immunocapture assay for the diagnosis of human Schistosoma mansoni infection.

The species specificity of the solid phase alkaline phosphatase immunocapture assay (APIA) for the immunological detection of human immunoglobulin G antibodies to the alkaline phosphatase of adult Schistosoma mansoni was evaluated. Sera from schistosomiasis patients from South America, West Africa, south-east Asia and uninfected control subjects were compared. Only the sera of patients infected with S. mansoni gave positive results. There was no apparent difference between 2 populations infected with S. mansoni, one from South America and the other from West Africa. The results with sera from various regions of West Africa were also indistinguishable. Although the APIA was not able to discriminate the geographical origin of the S. mansoni-infected subjects, the method appeared to be specific for S. mansoni and suitable for use in the immunodiagnosis of schistosomiasis mansoni, particularly in endemic areas where mixed infections of Schistosoma spp. occur.

Natural Schistosoma mansoni infection in wild rats from Guadeloupe: parasitological and immunological aspects.

Rattus rattus is the predominant rodent in the mangrove area of Guadeloupe. Between 1990 and 1991 we found 73 R. rattus and five R. norvegicus. Among the infected rats with Schistosoma mansoni, 59% for R. rattus and 80% for R. norvegicus, the comparison of the median of the worm load was not statistically different. Both species of infected rats showed adult worms and eggs in the lungs and 20% of them showed, at the same time, two and even three generations of worms. Neither adults nor eggs were seen in the intestinal wall or stools of R. norvegicus, instead R. rattus had eggs in the liver, in the intestinal wall and the stools. Therefore, R. norvegicus gets infection as well as R. rattus, but does not participate in the transmission of the schistosomiasis. In order to elucidate this difference, we looked at the humoral recognition of these two rats, to the molecular antigens of the three stages of the parasite: cercaria, adult worm (AWA) and egg (SEA). In general, R. norvegicus recognized cercarial antigens more frequently than R. rattus, 73, 81 and 172 kDa being statistically different. Regarding AWA, molecules 82, 86, 117 and 150 kDa were recognized more often by R. rattus as compared to R. norvegicus. The reverse was true for the 18, 33 and 61 kDa. Only the differences between 61 and 150 kDa molecules were statistically significant. With respect to SEA, R. norvegicus recognized more 28, 45, 47, 49, 64 and 92 kDa molecules than R. rattus, but the latter recognized the 140 kDa molecules of SEA to a higher degree (95 and 140 kDa were significantly different). It is plausible that the immune response to cercarial invasion is more effective in R. norvegicus in allowing the parasites to reach adulthood, but it does not let them live in the mesenteric veins and therefore to lay their eggs in the intestinal wall and feces.

Evaluation of alkaline phosphatase immunoassay and comparison with other diagnostic methods in areas of low transmission of schistosomiasis.

The alkaline phosphatase immunoassay (APIA) is an antibody detection technique which permits the diagnosis of schistosomiasis using a butanolic extract preparation from adult worms. APIA has demonstrated high sensitivity and specificity in previous reports with well characterized human sera. Its potential as a diagnostic tool for epidemiological surveillance was assessed in comparison with three other diagnostic tests: stool examination, ELISA with soluble egg antigen (SEA) and the circumoval precipitin test (COPT). APIA was 100% specific in an area without Schistosoma mansoni transmission and had 89% sensitivity in an endemic area where 69% of the infected subjects excreted less than 100 eggs g of faeces. It was found to be less sensitive in children under 5 years of age who were positive by the COPT test. APIA can be applied as an initial screening test, based on its high sensitivity, specificity, absence of cross-reactivity with intestinal parasites and the fact that it is a technique suitable for use in epidemiological surveillance.

A simplified methodology for purification of peanut (Arachis hypogaea) agglutinin.

The agglutinin from peanut (Arachis hypogaea) was readily isolated by affinity chromatography on acid-treated Sepharose 6B. The recovered lectin (50 mg/100 g seeds) appeared as a single band of Mr 32,000 on gel electrophoresis and its specific haemagglutination titre on desialylated human A red blood cells was very high (2(15)).

Aminopeptidase activity in adult Schistosoma mansoni.

An aminopeptidase activity capable of hydrolysing leucine 4-nitroanilide and alanine 4-nitroanilide at pH 7.0 was detected in saponin-CaCl2 extracts and homogenates of adult Schistosoma mansoni. The extracts were also capable of acting on synthetic dipeptides at the same pH, preferentially hydrolysing peptide bonds following leucine, alanine, or proline N-terminal residues. Imide bonds were not hydrolysed. The hydrolysis of leucine 4-nitroanilide was apparently stimulated by thiols, strongly inhibited by 1 mM 4-chloromercuric benzene sulfonic acid, and partially inhibited by 1 mM 1,10-phenanthroline.

Enzyme activities in Schistosoma mansoni soluble egg antigen.

Schistosoma mansoni egg antigens are mostly responsible for the granulomatous pathology in human intestinal schistosomiasis. Several previous studies have indicated that the induction of an immune response against some parasite enzymes may protect against pathology. The present work was designed to identify enzyme activities present in a standard soluble egg antigen (SEA) preparation. Simple colorimetric analyses were performed incubating SEA with 2-naphthyl, 2-naphthylamide (2NA), or p-nitrophenyl substrates at different pHs in the absence of added effectors. Results showed prominent acid phosphatase (pH 5.4), alkaline phosphatase (pH 8.5), and N-acetyl-beta-glucosaminidase (pH 5.4) activities. Relevant peptidase activities were also detected at pH 6.5-7.5 against 2NA derivatives of (1) aliphatic (alpha-Ala > beta-Ala > Leu > Met > S-benzyl-Cys), polar (Ser > Gln), basic (Arg > Lys > ornithine), and acidic (Glu) amino acids; (2) dipeptides: X-Ala (X = Gly > Leu > Lys > Asp), X-Arg (X = Ala > Arg > Phe > Gly > Pro > Asp), Ser-Met, and Phe-Pro; and (3) tripeptides (Ala-Phe-Pro > Phe-Pro-Ala). The data demonstrated that S. mansoni SEA contains a rich set of hydrolases with different specificities that might play a role in the egg physiology and possibly also in the host-parasite relationships.

Schistosoma mansoni: surface membrane isolation with lectin-coated beads.

Lectins from Lens culinaris and Arachis hypogaea immobilized on polyacrylamide beads were used for selective isolation of glycosylated surface membrane domains of adult Schistosoma mansoni worms, and the method was compared with the membrane isolation procedure developed with polycationic (Affi-Gel) beads. The lentil lectin proved to be suitable for interaction with surface membrane components: an increment in the specific activities of tegumental phosphohydrolases was observed in the bound fraction with respect to that observed in a total worm homogenate. A characteristic polypeptide pattern on gel electrophoresis was also seen, more restricted than that obtained with the bound Affi-Gel fraction. Immobilized peanut lectin was not successful as a method for isolating membrane material from the tegument of adult worms. Solubilization and dissociation of the lentil lectin-bound enzyme markers was achieved after addition of detergent and competing sugars. Glycosylation of the solubilized enzymes was further confirmed by affinity chromatography with fresh lentil lectin-coated beads. These results, together with histochemical evidences, suggest that the active sites of some of these enzymes are located within or close to the cytoplasmic leaflet of the surface tegumental membranes, and allow us to propose a model for the double surface membrane complex where some proteins may be crossing the two bilayers.

Mouse lambda-chain sequences.

Amino-acid sequences of the variable regions of three lambda chains produced by plasmacytomas of BALB/c mice are compared. Two are almost certainly identical and one differs from these by three amino acids. These findings extend our earlier conclusion on the relative uniformity of sequences in this type of immunoglobulin light chain. With amino-acid sequence data on two additional lambda chains, eight mouse lambda chains studied to date are indistinguishable and four probably differ from these by one, two, or three amino acids.

Antigenicity of adult Schistosoma mansoni alkaline phosphatase.

Antibodies to the alkaline phosphatase (AP) of Schistosoma mansoni in infected human and mice sera were evaluated by a direct solid-phase AP immunoadsorption assay (APIA) and by Western blot and immunostaining. APIA consisted of (a) solid-phase capture of immunoglobulins from infected human or mice, (b) immunoadsorption of the enzyme antigen by the antibodies, and (c) detection of the enzymatic activity. By this procedure the appearance of the anti-AP response in mice was detected around 50 days post-infection; this response was not specific for an AP of a given schistosome strain and it was not induced by an autoimmunity phenomenon. Fourteen out of 15 sera from infected people tested by APIA showed a clear antibody response against this enzyme. Immunoblots in non-reducing conditions supported APIA results indicating that the parasite AP was specifically recognized by the antibodies present in infected human and mice sera. These results suggest the possible usefulness of the schistosome AP as a marker for S. mansoni infection.

Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms.

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized within the tegument of fixed worms on the cytoplasmic leaflet of both the double surface membrane and the basement membrane; this reaction was inhibited by 1 microM chlorpromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosomes are discussed.

Isoenzyme studies in one Brazilian and two Venezuelan strains of Schistosoma mansoni.

1. Enzyme polymorphism, analyzed by starch gel electrophoresis, was found to be zero for acid phosphatase, phosphoglucomutase, phosphoglucose isomerase, glucose 6-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase and malic enzyme, in one Brazilian and two Venezuelan strains of Schistosoma mansoni. 2. All loci studied were monomorphic within strains, but the isoenzymic patterns were, however, different among the strains. 3. Results suggest a drastic loss of the genetic variability usually found in natural populations.