GP38, P28-I and P28-II: candidates for a vaccine against Schistosomiasis

Three antigens protective against Schistosoma mansoni have been extensively characterized. The schistosomulum surface antigen GP38 possesses an immunodominant carbohydrate epitope of which the structure has been defined. Protection can be achieved via the transfer of monoclonal antibodies recognizing the epitope or by immunization with anti-idiotype monoclonal antibodies. The glycan epitope is shared with the intermediate host, Biomphalaria glabrata as well as being present on other molluscs, including the Keyhole Limpet. A group of molecules at 28 kDa were initially characterized in adult worms and shown to protect rats and mice against a challenge infection. One of these molecules, P28-I, was cloned and expressed in E. coli, yeast and vaccinia virus. The recombinant antigen significantly protected rats, hamsters and baboons against a challenge infection. P28-I is a glutathione-S-transferase and the recombinant antigen produced in yeast exhibits the enzyme activity and has been purified to homogeneity by affinity chromatography. A second P28 antigen, P28-II, has also been cloned, fully sequenced and expressed. This recombinant antigen also protects against S. mansoni infection.

Role of a mouse monoclonal IgE antibody in passive transfer of immunity to Schistosoma japonicum infection

We have been able to produce a mouse monoclonal IgE antibody specific to an adult worm antigen extracted from Schistosoma japonicum (Sj). The antibody was able to elicit passive cutaneous anaphylaxis in the rat skin against Sj with the highest titer of 1:256,000 but did not cross-react with S. mansoni antigen. The antibody recognized a 97-kDa molecule expressed on the surface of mechanically transformed schistosoma of S. japonicum. Passive transfer of the antibody into mice in the early stage of challenge infection resulted in a partial but significant reduction of recovery of adult worms. Induction of eosinophilia by an oral administration of embryonated eggs of Toxocara canis prior to challenge infection enhanced the reduction.

Congenital and nursing effects on the evolution of Schistosoma mansoni infection in mice

Modification of the immune response to schistosomal infection in children or offspring born to mother R infected with Schistosoma mansoni has been demonstrated in human and in experimental schistosomiasis. One of the hypothesis to explain this fact could be the transfer of circulating antigens and antibodies from mother to foetus through the placenta or from mother to child by milk. The results of this spontaneous transference are controversial in the literature. In an attempt to investigate these questions, we studied one hundred and twenty offspring (Swiss mice), sixty born to infected-mothers (group A) and sixty born to non-infected mothers (group B). These were percutaneously infected with 50 cercariae/mouse, and divided in six sub-groups (20 mice/sub-group), according to the following schedule: after birth (sub-groups A.I and B.I), 10 days old (sub-groups A.II and B.II) and 21 days old (sub-groups A.III and B.III). After the exposure period, the young mice returned to their own mothers for nursing. Six weeks later, the mice were killed. We obtained the following results: 1) There is transference of antibody to cercariae (CAP), adult worms (SWAP) and egg antigens (SEA) from the infected mothers to the offspring, probably through placenta and milk; 2) Offspring born to infected mothers exhibit much less coagulative hepatic necrosis and show a lower number of eggs in the small intestine and a less intense and predominant exsudative stage of the hepatic granulomas when compared with the exsudative-productive stage of the control groups. The findings suggest that congenital and nursing factors can interfere on the development of the schistosomiasis infection, causing an hyporesponse to the eggs.

Autoradiographic analysis of Schistosoma mansoni migration in the NZ rabbit

The migration of larval Schistosoma mansoni was tracked by means of autoradiographic analysis in naive rabbits percutaneously exposed to L-(**75 Se) selenomethionine-labeled cercariae on serial intervals of 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40 and 50 days post-infection. Autoradiographic foci were detected from the 1st day in the skin, up to the 15th day in the liver. Adult and mature worms were recovered either paired or not 60 days after infection, by perfusion of hepatic and mesenteric veins. Morphometric analysis under optical microscopy, showed that worms were within regular dimention limits as compared to adult worms harboured by other host species. These observations extend previous informations on the S. mansoni-rabbit association and clearly demonstrate the post-liver phase of S.mansoni life-cycle in this host.

Identification and characterization of sex-linked proteins of Schistosoma mansoni

The proteins of adults worms (male and female) of two isolates (BH and RJ) of Shistosoma mansoni were extracted using Triton X-114 phase separation. The SDS-polyacrilamide gel electrophoresis profiles of the three phases (detergent, aqueous and insoluble proteins) obtained were compared after Coomassie blue and silver staining, surface radioiodination and Western blotting. No major differences were detected between the 2 isolates. Of the 25 or more proteins which partitioned into the detergent phase, only about 8 proteins could be surface radiodinated on live adult worms. A comparison was also made between the profiles of mael and females worms, isolated from bisexually infected mice. Two major female-specific and one male-specific band were detected by silver and/or Coomassie staining. The female bands, 32 KDa and 18 KDa, partitioned into the detergent and aqueous phase, respectively. The male-specific band of 42 KDa remained in the insoluble phase. Antigenic differences between male and females protins were detected by Western vlotting using a sera from infected Nectomys squamipes.

Experimental heteroxenous cycle of Lagochilascaris minor Leiper, 1909 (Nematoda: Ascarididae) in white mice and in cats

Reports of natural infections of sylvatic carnivores by adult worms of species similar to Lagochilascaris minor in the Neotropical region led to attempts to estabilish experimental cycles in laboratory mice and in cats. Also, larval development was seen in the skeletal muscle of an agouti (Dasyprocta leporina) infected per os with incubated eggs of the parasite obtained from a human case. In cats, adult worms develop and fertile eggs are expelled in the feces: in mice, larval stages of the parasite develop, and are encapsulate in the skeletal muscle, and in the adipose and subcutaneous connective tissue. From our observations, we conclude that the larva infective for the mouse is the early 3rd stage, while for the final host the infective form is the later 3rd stage. A single moult was seen in the mouse, giving rise to a small population of 4th stage larvae, long after the initial infection.

Natural infection of wild rodents by Schistosoma mansoni parasitological aspects

The evaluation of the role of rodents as natural hosts of Schistosoma mansoni was studied at the Pamparrão Valley, Sumidouro, RJ, with monthly captures and examination of the animals. Twenty-three Nectomys squamipes and 9 Akodom arviculoides with a shistosomal infection rate of 56.5% and 22.2% respectively eliminated a great majority of viable eggs. With a strain isolated from one of the naturally infected N. squamipes, we infected 75% of simpatric Biomphalaria glabrata and 100% of albino Mus musculus mice. The adult worms, isolated from N. squamipes after perfusion were located mainly in the liver (91.5%) and the mesenteric veins (8.5%). The male/female proportion was 2:1. The eggs were distributed on small intestine segments (proximal, medial and distal portions) and the large intestine without any significant differences in egg concentration of these segments. In A. arviculoides, the few eggs eliminated by the stools were viable and there was litlle egg retention on intestinal segments. Considering the ease to complete S. mansoni biological cycle in the Nectomys/Biomphalaria/Nectomys system under laboratory conditions, probably the same is likely to occur in natural conditions. In support to this hypotesis there are also the facts that human mansonic shistosomiasis has a very low prevalence in Sumidouro and endemicity among the rodents has not changed even after repetead treatments of the local patients. Based on our experiments, we conclude that N. squamipes has become a natural host of S. mansoni and possibly may participate in keeping the cycle of schistosomiasis transmission at Pamparrão Valley.

Parasite enzymes as a tool to investigate immune responses

Previous evidences reported by us and by other authors revealed the presence of IgG in sera of Schistosoma mansoni-infected patients to immunodominant antigens which are enzymes. Besides their immunological interest as possible inductors of protection, several of these enzume antigens might be also intersting markers of infection in antibody-detecting immunocapture assays which use the intrinsic catalytic property of these antigens. It was thus thought important to define some enzymatic and immunological characteristics of these molecules to better exploit their use as antigens. Four different enzymes from adult worms were partially characterized in their biochemical properties and susceptibility to react with antibodies of infected patients, namely alkaline phosphatase (AKP, Mg*+, pH 9.5), type I phosphodiesterase (PDE, pH 9.5), cysteine proteinase (CP, dithiothreitol, pH 5.5) and N-acetyl-ß-D-glucosaminidase (NAG, pH 5.5). The AKP and PDE are distinct tegumental membrane-bound enzymes whereas CP and NAG are soluble acid enzymes. Antibodies in infected human sera differed in their capacity to react with and to inhibit these enzyme antigens. Possibly, the specificity of the antibodies related to the extent of homology between the parasite and the host enzyme might be in part responsible for the above differences. The results are also discussed in view of the possible functional importance of these enzymes.

Human IgE responses to Schistosoma mansoni and resistance to reinfection

Schistosoma mansoni infected Kenyan patients were treated and the intensities of their reinfections were followed over the next two years. in addition, their pre- and six month post-treatment serum levels of IgG1-4, IgM, and IgE, specific for schistosoma, egg and adult worm, were measured in ELISA. No reinfection took place before six months post-treatment. Reinfection intensities varied with age; the younger children becoming reinfected at significantly higher intensities than older individuals. When antibody and reinfection levels were compared, only the six month post-treatment IgE response against adult worm correlated negatively with intensities of reinfection and, therefore, was predictive of resistance or immunity to reinfection. IgE and IgG specific Western Blots were carried out. The adult worm antigens recognized by IgE were restricted compared with the IgG responses of the same patients, although no individual antigen was uniquely recognized by the IgE isotype. A dominant 22 kDa antigen was recognized by most but not all high IgE responders. Patients with IgE responses against this antigen suffered significantly lower subsequent levels of reinfection, compared with non-responders. A monospecific rabbit antiserum against the 22KDa adult worm antigen showed that this antigen is specifically located in the tegument of the adult worm and of 'lung' and 'liver' stage schistosomula, but is absent from the early 'skin' schistosomula. It is possible that this antigen is a target for human IgE mediated immune effector mechanisms active against the post skin stage schistosomula and that this is boosted by the death of adult worms.

Immunological profiles of patients from endemic areas infected with Schistosoma mansoni

Crude extracts of eggs (SEA) adult worms (SWAP) or cercariae (Cerc) have been used to stimulate Peripheral Blood Mononuclear cells (PBMC) and have provided rather distinct profiles of responses in different types of patients. In genenral it is clear that patients with early infections respond strongly to SEA while response to SWAP are developed more slowly. As infection progresses into the more chronic phases, a general pattern is seen whic leads to lower anti-SEA proliferative responses in the face of higher responses to SWAP and variable anti-cerc responsiveness. Cured not re-exposed patients express very high levels of anti-SEA proliferation. It has recently been seen that those individuals who live in endemic areas and have continued water contact, but are reapeatedly stool-negative (who are presumed to have self-cured or be putatively resistant; endemic normals) are strongly responsive to antigenic extracts, particularly to SEA. Furthermore, our results show that endemic normal individuals have significantly higher IFN gamma production upon PBMC stimulation with schistosome antigens than infected individuals. With the emergence of more studies it is becoming apparent that both the intensity and the prevalence of a given area may influence or shape the general responsiveness of the population under study.

Parasite and egg burden, hepatic collagen and histologic pattern of liver granulomas in selection III high and low antibody responder mice infected with Schistosoma mansoni

Selection III mice have particular immunological characteristics: they are high (H III) or low (L III) antibody producer animals, yet both lines display similar T cell responses and macrophage activities. We submittedthese mice to infection with Schistosoma mansoni to assess in vivo parasite and egg burden, hepatic collagen and cellular composition of granulomas in both lines. Titration of anti-Schistosoma IgG by ELISA showed remarkably higher values inH III line, at both studied periods (8th and 12th weeks post-infection). Nevertheless, the number of adult worms recovered from the portal system was similar inboth lines, being not associated with anti-Schistosoma antibody levels. There isan increase in hepatic collagen from the 8th to the 12th weeks post-infection, which is paralleled by an increase in the number of eggs in the liver. This association apparently occurs at the same radio in H III and L III animals. The most important difference found between the two lines was the outstanding contrast interms of volume and eosinophil counts in the granulomas, with lesions from H IIImice clearly being larger and containing more of these cells than LIII lesions.

A molecular genetic study of the variations in metabolic function during schistosome development

During their complex life cycle schistosomes alternate between the use of stored glycogen and reliance on host glucose to provide for their energy needs. In addition, there is dramatic variation between the relative contribution of aerobic versus anaerobic glucose metabolism during development. We have cloned a set of representative cDNAs that encode proteins involved in glucose uptake, glycolysis, Kreb's cycle and oxidative phosphorylation. The different cDNAs were used as probes to examine the expression of glucose metabolism genes during the schistosome life cycle. Steady state mRNA levels from whole cercariae, isolated cercarial tails, schistosomula and adult worms were analysed on Northern blots and dot blots which were quantified using storage phosphor technology. These studies reveal: (1) Transcripts encoding glycogen metabolic enzymes are expressed to much higher levels in cercarial tails than whole cercariae or schistosomula while the opposite pattern is found for glucose transporters and hexokinase transcripts; (2) Schistosomula contain low levels of transcripts encoding respiratory enzymes but regain the capacity for aerobic glucose metabolism as they mature to adulthood; (3) Male and female adults contain similar levels of the different transcripts involved in glucose metabolism.

Schistosoma mansoni: reinfections and concomitant immunity in mice: importance of perfusion time after challenge infection for evaluation of immunoprotection

The concomitant immunity in the presence of repeated infections (with 15 cercariae) was studied in mice sacrificed on the 20th day after each infection. The comparison of the averages of immature worms, recovered from mice submitted to reinfection, with those of their respective controls (previously uninfected) showed a significantly lower worm recovery rate in the animals with previous infections (concomitant immunity). However, statiscally significant differences could not be detected among the various groups of animals, when the mice that accumulated worms in this mature stage were perfused. The theoretical projection based on the accumulation of young worms which developed to adult ones indicates a lower recovery rate of adult worms in the animals with concomitant immunity, but this projection was not corroborated by the experimental data. The visceral hemodynamic alterations that occurred in reinfections due to the pathogeny, favouring recirculation of the recent arriving worms to the other organs on the occasion of perfusion of the portal system. These results suggest that special care should be taken when one wants to investigate concomitant immunity in mice based on the distinction of the immature worms from challenge infection and the mature ones from primary infection.

Biodistribution Study of the Anaesthetic Sodium Phenobarbital Labelled with Technetium-99m in Swiss Mice Infected with Schistosoma mansoni Sambon, 1907

Technetium-99m (99mTc) is a radionuclide that has negligible enviromnental impact, is easily available, inexpensive and can be used as a radioactive tracer in biological experiences. In order to know the mode of action of sodium phenobarbital in moving adult Schistosoma mansoni worms from mesenteric veins to the liver, we labelled sodium phenobarbital (PBBT) with 99mTc and a biodistribution study in infected and non-infected Swiss mice was performed. The PBBT was incubated with stannous chloride used as reducing agent and with 99mTc, as sodium pertechnetate. The radioactivity labelling (%) was determined by paper ascending chromatography perfomed with acetone (solvent). The 99mTc-PBBT was administered by intraperitoneal route to Swiss mice infected eight weeks before. The animals were perfused after diferent periods of time (0,1,2,3,4 hr) when blood, spleen, liver, portal vein, mesenteric veins, stomach, kidneys and adult worms were isolated. The radioactivity present in these samples was counted in a well counter and the percentage was determined. The radioactivity was mainly taken up by the blood, kidney, liver and spleen. No radioactivity was found on the adult worms. We concluded that the worm shift was due to an action on the host of the sodium phenobarbital

Immunological system and Schistosoma mansoni: co-evolutionary immunobiology. What is the eosinophil role in parasite-host relationship?

Schistosomes, ancestors and recent species, have pervaded many hosts and several phylogenetic levels of immunity, causing an evolutionary pressure to eosinophil lineage expression and response. Schistosoma mansoni adult worms have capitalized on the apparent adversity of living within the mesenteric veins, using the dispersion of eggs and antigens to other tissues besides intestines to set a systemic activation of several haematopoietic lineages, specially eosinophils and monocytes/macrophages. This activation occurs in bone marrow, spleen, liver, lymph nodes, omental and mesenteric milky spots (activation of the old or primordial and recent or new lymphomyeloid tissue), increasing and making easy the migration of eosinophils, monocytes and other cells to the intestinal periovular granulomas. The exudative perigranulomatous stage of the periovular reaction, which present hystolitic characteristics, is then exploited by the parasites, to release the eggs into the intestinal lumen. The authors hypothesize here that eosinophils, which have a long phylogenic story, could participate in the parasite - host co-evolution, specially with S. mansoni, operating together with monocytes/ macrophages, upon parasite transmission.