Search

Observations were made on the development of acquired resistance to Schistosoma haematobium in the baboon following immunization with cercariae by the percutaneous route and by the transplantation of adult worms into the mesenteric veins. In the first experiment six baboons were immunized with 1000 S. haematobium cercariae given percutaneously. They were challenged with 10 000 cercariae given 73 weeks later and the results were compared with a similar infection in non-immunized animals. The results showed that the baboon can develop a strong resistance to reinfection with S. haematobium. The manifestations of the immunity were (i) the absence of any increase in egg output after challenge (ii) the substantially lower level of adult worms and eggs in the tissues of the immunized baboons compared with the challenge control animals (iii) a reduction in the egg laying capacity of the residual worms and (iv) the virtual absence of gross pathology and the mild lesions seen in the tissue sections of all the immunized animals. The depression in egg laying of the worms was confirmed by transplanting them into non-immune baboons. This experiment indicated that the non-egg-laying worms in the immune baboons were not irreversibly damaged since they survived, some even migrating to the vesical and ureteric vessels, and egg-laying was rapidly resumed after transplantation. A further experiment was designed to see if a similar degree of immunity could be produced by an adult worm infection without previous exposure to cercariae or schistosomula. The immunization dose consisted of 50-100 S. haematobium worm pairs which were transplanted into the mesenteric veins of each of six baboons and the animals were challenged percutaneously with 7000 cercariae 35-55 weeks later. There was little difference in the worm burdens of the immunized and control animals but the worms in the immunized baboons produced fewer eggs and the pathology seen in these animals was much milder than in the challenge control animals suggesting that some degree of resistance to reinfection was produced by the transplanted worms.

The potential sites of attrition of a challenge population of schistosomes have been investigated in mice, rats and guinea pigs vaccinated with irradiated cercariae of Schistosoma mansoni, by the use of challenge regimens that permit sequential site elimination. Vaccinated mice showed significant immunity to a percutaneous cercarial challenge, but were only marginally resistant to an i.v. challenge with healthy lung stage worms. Vaccinated rats and guinea pigs differed from mice, in that they were able to mediate significant challenge attrition at post-skin sites. Healthy lung worms were subject to immune elimination by rats in the lungs, or perhaps en route to the liver, but not in the liver itself. In contrast, guinea pigs had the capacity to kill challenge lung worms injected into either the lungs or the liver. Interestingly, lung worms harvested by extended incubation were shown to be sub-optimal in terms of viability, since they were eliminated in significant numbers when injected i.v. into vaccinated mice. These data show that different hosts vaccinated in essentially the same manner differ in terms of their site potential for challenge attrition. It is emphasised however, that sites implicated by these experiments as having the capacity to mediate immune elimination are not necessarily the sites at which challenge attrition occurs under normal circumstances.

Serum taken from mice 12-15 weeks after a primary infection of Schistosoma mansoni transfers to normal recipients a partial resistance to subsequent schistosome challenge. The transfer of immunity is evident not only in the reduced recovery of mature parasites from the liver, but also in the diminished numbers of invading schistosomula recovered of resistance equivalent on average to 47% of that found in actively immunized animals, the results suggest that humoral factors play a major role in the effector mechanism of schistosome immunity.

Protective immunity has been demonstrated in experimental schistosomiasis and is also believed to occur in man. It can be mediated by antibodies from infected animals or animals immunized with attenuated organisms. Recombinant Escherichia coli synthesizing antigenic polypeptides from the three principal species of schistosome that infect man, Schistosoma mansoni, S. japonicum and S. haematobium, have been constructed. Libraries of adult worm cDNA were prepared from each species in the expression vector lambda gt 11 and directly screened with antibodies from animals experimentally immunized with S. mansoni and S. japonicum and from humans infected with S. haematobium. The S. mansoni clones have been analysed in greatest detail. At least four different types of clones were identified. All the detected recombinant polypeptide antigens were recognised by antibodies from chronically infected mice and most were also recognised by antibodies from mice immunized with attenuated cercariae and anti-surface membrane antibodies. Clones synthesizing species-specific antigens for both S. mansoni and S. japonicum were identified by simultaneous screening of both libraries. At least three types of S. haematobium clones were identified by screening with human infection serum, most of which were species-specific. All the antigens were in the form of fusion peptides with E. coli beta-galactosidase and their expression was induced by isopropylthiogalactopyranoside. Since known protective monoclonal antibodies recognise highly glycosylated membrane proteins which cannot be identified in the form of nascent polypeptides, the direct identification of polypeptide antigens defined by their reactivity, as reported here, is an essential step in producing reagents by recombinant DNA technology, suitable for vaccination and diagnosis.

Techniques for recovering Schistosoma mansoni from the skin, lungs, and liver have enabled us to trace the attrition of a challenge infection in immune mice. Two separate phases of attrition were demonstrated. In mice which had developed immunity following exposure to 20 cercariae a major phase of attrition occurred between days 6 and 13, after the parasites had left the lungs, and only in mice which had been infected for 12 weeks or more did a minor phase of attrition occur in the skin. In contrast, mice which had been immunized by exposure to highly irradiated (20 kr) cercariae destroyed the majority of their reinfecting population in the skin. These results suggest that there are two distinct mechanisms of immunity to S. mansoni in mice. This view was strengthened by experiments in which both types of immune mice were challenged intravenously with 5-day-old lung schistosomula (a challenge which bypassed the skin). While the immunized mice were susceptible to the lung worm challenge, the infected mice were substantially resistant to it.

Serum harvested from Sprague-Dawley rats twice vaccinated with gamma-irradiated cercariae of Schistosoma mansoni is able to protect naive recipients against a challenge infection, but the challenge parasites are susceptible to immune elimination over a very short period of time. Thus, vaccinated rat serum protects recipients against a percutaneous cercarial challenge when transferred on Day +5 but not Day 0 and protects recipients against a tail vein challenge with 5-day-old lung worms when transferred on Days 0 or +1, but not Days +4 or +5. Rats challenged with lung worms via the tail vein and given serum on Day +3 exhibit approximately half the protection expressed by counterparts that received serum on Day 0. However, vaccinated rat serum does not protect naive recipients against a lung worm challenge introduced directly into the liver. These data indicate that immune elimination of challenge parasites in the vaccinated rat model is site-dependent rather than stage-dependent, and most probably occurs during the lung phase of parasite migration.

Eosinophils from the peritoneal cavity of normal rats, in the presence of fresh normal rat serum (NRS), adhered to schistosomula of Schistosoma mansoni in vitro and killed the majority of parasites within 18 h. The reaction differed from the previously described antibody-mediated eosinophil adherence to schistosomula which occurs in heat-inactivated immune rat serum (IRS) and where adherence is mediated through Fc receptors. Adherence of eosinophils in fresh NRS was shown to be due to the activation of complement at the schistosomular surface by the alternative pathway, and it was effected through C3 receptors. The ability of eosinophils to kill in Fc-mediated adherence. This enhancement of killer activity may be due to the generation by complement activation of eosinophil chemotactic factors which increase the concentration of cells at the target surface. It is suggested that eosinophil adherence mediated through complement activation could be the principla mechanism of destroying schistosomula in the host.

The level of immunity to a percutaneous cercarial challenge with Schistosoma mansoni was assayed 4-6 weeks after immunization of mice with highly irradiated (20 krad.) cercariae or schistosomula. When immunization and challenge occurred through the same skin site, resistance, particularly that which occurred in the skin, was greater than that observed when immunization and challenge occurred in different sites. The enhanced resistance is believed to be due to localized changes in the skin; 4 weeks after exposure to irradiated cercariae, abdominal skin is characterized by a thickened epidermis, changes in the ground substance and a cellular infiltration of the dermis. A convenient mouse model is described in which one or both ear pinnae are exposed to irradiated cercariae and a percutaneous challenge is given via the abdomen, thus eliminating the effects of local skin changes. In this model, the majority of the challenge infection which succumbs to the immune response appears to be killed in the skin.

The carrier effect, using TNP-labelled schistosomula was used to measure the helper T-cell activity against the schistosomula surface in CBA mice exposed to 30 cercariae of Schistosoma mansoni. After infection the helper T-cell activity reached a peak in 8--10 days, but by 6 weeks it had declined to background levels. Five x 10(7) spleen cells from chronically (12-week) infected mice when injected into 9-day infected mice caused a specific suppression of the helper T-cell response to schistosomula. Subsequent fractionation of the spleen cell population using a nylon wool column and specific depletion of T cells from the spleen cell population with anti-Thy 1.2 antisera and complement, showed that the suppressive activity was due to T cells. We conclude that during infection of mice with S. mansoni a population of suppressor T cells is generated which partially regulates antibody production against schistosome surface antigens.

Antigenic proteins on the surfaces of different developmental stages of Schistosoma mansoni were radio-iodinated by the Iodogen-catalysed method and identified by immunoprecipitation with a panel of antisera. The sera comprised specific immune serum from mice harbouring a chronic schistosome infection or vaccinated with gamma-irradiated cercariae; serum from rabbits immunized with adult schistosome tegumental outer membranes or a partially purified Mr 32 K glycoprotein from adult worm membranes; and a monoclonal antibody recognizing an Mr 20 K antigen on the surface of schistosomula. The Mr 38-32 K glycoproteins were the major antigens identified in surface-labelled cercariae and their probable association with the glycocalyx is discussed. Schistosomula transformed from cercariae either mechanically or by penetration of host skin in vitro, expressed a similar pattern of surface antigens to that identified for cercariae, but low molecular weight antigens of Mr 20, 17 and 15 K were also detected. The Mr 38-32 K glycoproteins, although present on newly transformed schistosomula, were progressively replaced with time, by a single dominant glycoprotein (Mr 32 K) expressing identical epitopes to those on the Mr 38-32 K complex. Moreover, the data confirm that the Mr 32 K glycoprotein persists on the tegument after in vivo maturation and is conserved, together with Mr 20 and 15 K antigens, through to the adult stage. New antigens (Mr 97 and 25 K) were also detected during in vivo maturation and were present in late-stage schistosomes recovered from infected hosts. In addition, the enzyme alkaline phosphatase is expressed on the surfaces of 3-week-old liver worms as a dominant antigen (Mr 65 K); this feature may be related to nutritional and/or physiological processes in the tegument of this metabolically active stage of development.

Protein A-sepharose affinity chromatography was used to isolate IgG subclasses from the serum of CBA mice chronically infected with Schistosoma mansoni. The subclasses were tested for the presence of two antibodies which are responsible for the death of young schistosomula in vitro; 'lethal antibody' (LA), which kills schistosomula in co-operation with complement and 'eosinophil adherence antibody' (EAA) which causes the death of schistosomula by promoting the adherence of eosinophils to the parasite. LA and EAA were detected only in the IgG fraction of the serum. LA was concentrated in the IgG2a fraction and EAA in the IgG1 fraction. The development of IgG subclasses specific for schistosomula was followed in mice exposed to twenty cercariae by the fluorescent antibody technique. IgG1, IgG2a and IgG2b antibodies were detected 2 weeks after infection and their titres rose steadily to reach high levels by weeks 12 or 14. IgM antibody was not detected until week 6 and IgA until week 10; both were present at lower concentrations than the IgG1 antibodies.

The production of antibodies against the schistosomulum surface antigens of Schistosoma mansoni in response to immunization with highly irradiated cercariae was followed. Four antigens were reproducibly identified by 125I surface labelling using Iodogen and immunoprecipitation; they had mol. wts of 38, 32, 20 and 15 kD. In addition a 92 kD antigen was also evident in most experiments. It was demonstrated that the 20 kD antigen was the same as that recognized by the monoclonal antibody NIMP/M.47 and that this antigen like the 38 and 32 kD antigens was thus identified during both chronic infection and following vaccination with irradiated cercariae. Two weeks following immunization with irradiated cercariae antibody was produced only against the 15 kD antigen but at 4 weeks the major response was against the 32 kD antigen. A second immunization with irradiated cercariae boosts the antibody response so that all four antigens were strongly precipitated. Further vaccinations did not lead to the identification of further antigens. Immunization of rats with highly irradiated cercariae also resulted in antibody production against the 38, 32, 20 and 15 kD antigens. Surface labelling of schistosomula transformed from irradiated cercariae resulted in the same four antigens being precipitated as from normal cercariae indicating that irradiation did not affect transformation nor antigen expression on 3h schistosomula. Furthermore, antibodies against the same surface antigens were detectable 4 weeks after immunization with equal numbers of cercariae irradiated with 0, 5, 25 or 50 krad. Vaccination of mice with irradiated, cloned cercariae resulted in identical antibody production and similar levels of immunity directed at both an homologous or heterogeneous challenge. Thus all parasites within our laboratory population appear to express the same antigens and there was no evidence for a genetically defined variation that could account for the partial resistance to reinfection exhibited by mice vaccinated with irradiated cercariae.

New or improved techniques for recovering Schistosoma mansoni from the skin, lungs and liver have enabled us to trace the attrition of a challenge infection in naive (i.e. previously uninfected) and chronically infected mice. Within each experiment, numbers of schistosomes recovered from the skin of naive mice on day 2 after challenge or from the skin and lungs on days 3, 4 or 5, did not differ significantly from the numbers recovered from the liver on days 14, 21, 28 or 35. Approximately 65% of cercariae which penetrated the skin failed to be recovered from naive mice by any of the assays and it appeared that these schistosomes had already died in the skin in the first 24 h. No further significant loss of the infection was detected in naive mice. In chronically infected mice a further attrition of the challenge infection was demonstrated in two distinct phases. An 'early phase' occurred within the first 3 days of exposure and accounted for the death of 30% of the remaining parasites. A 'late phase' occurred between days 6 and 14 and accounted for an additional 43% of deaths. Thus, the two phases of attrition accounted for a loss of approximately 73% of the infection that would have survived in a naive mice. The late phase of attrition could be demonstrated before the primary infection had matured, in contrast to the early phase of attrition which was seen only after egg laying had commenced. We believe that the early phase of attrition takes place in the skin and the late phase occurs after the schistosomes have left the lungs, either en route for the liver or as soon as they arrive in that organ. The results suggest that there are two distinct mechanisms of immunity against re-infection with S. mansoni in mice.