Radiation-attenuated schistosome vaccination--a brief historical perspective.

The high level of protection which can be induced by vaccination of a range of hosts, from rodents to primates, with live radiation-attenuated schistosome larvae offers great promise for development of a human schistosome vaccine. Studies of the irradiated vaccine models benefitted from significant funding during the 1970-90s and much was learned concerning the inducers, targets and mechanisms of immunity. Less progress was made in definition of the protective antigens involved. The application of new techniques for identifying membrane and secreted antigens has recently provided new vaccine candidates and a new impetus for schistosome vaccine development. This article is intended as an overview of some of the main lessons learned from the studies of the irradiated vaccines as a backdrop to renewed interest in schistosome vaccine development.

Altered patterns of gene expression underlying the enhanced immunogenicity of radiation-attenuated schistosomes.

BACKGROUND: Schistosome cercariae only elicit high levels of protective immunity against a challenge infection if they are optimally attenuated by exposure to ionising radiation that truncates their migration in the lungs. However, the underlying molecular mechanisms responsible for the altered phenotype of the irradiated parasite that primes for protection have yet to be identified. METHODOLOGY/PRINCIPAL FINDINGS: We have used a custom microarray comprising probes derived from lung-stage parasites to compare patterns of gene expression in schistosomula derived from normal and irradiated cercariae. These were transformed in vitro and cultured for four, seven, and ten days to correspond in development to the priming parasites, before RNA extraction. At these late times after the radiation insult, transcript suppression was the principal feature of the irradiated larvae. Individual gene analysis revealed that only seven were significantly down-regulated in the irradiated versus normal larvae at the three time-points; notably, four of the protein products are present in the tegument or associated with its membranes, perhaps indicating a perturbed function. Grouping of transcripts using Gene Ontology (GO) and subsequent Gene Set Enrichment Analysis (GSEA) proved more informative in teasing out subtle differences. Deficiencies in signalling pathways involving G-protein-coupled receptors suggest the parasite is less able to sense its environment. Reduction of cytoskeleton transcripts could indicate compromised structure which, coupled with a paucity of neuroreceptor transcripts, may mean the parasite is also unable to respond correctly to external stimuli. CONCLUSIONS/SIGNIFICANCE: The transcriptional differences observed are concordant with the known extended transit of attenuated parasites through skin-draining lymph nodes and the lungs: prolonged priming of the immune system by the parasite, rather than over-expression of novel antigens, could thus explain the efficacy of the irradiated vaccine.

Effect of UV-irradiation, gamma irradiation and praziquantel on infected Biomphalaria alexandrina snails.

Schistosomiasis control represent the main target of many research programme allover the world. Effect of physical (UV- and gamma-irradiation) treatment as well as chemical (PZQ) treatment on infected laboratory bred Biomphalria alexandrina snails have been studied. Gamma irradiation and PZQ-treatment induced high rate of mortality than UV-irradiated and non-treated control snails. The cercarial production was severely reduced as a result of UV-, gamma-irradiation and PZQ-treatment. Pre-patent period was significantly increased in gamma-irradiated and PZQ-treated groups than UV-irradiated and control groups. Gamma irradiation and PZQ-treatment showed severe effects on hermaphrodite gland and cercariae. Trials of experimental infection in swiss albino mice revealed that low rate of infection (9.7% and 1.3%) was induced by cercarial shedding from UV-irradiated and PZQ-treated snails respectively. Whereas, cercarial production of gamma-irradiated snails failed to induce infection in swiss mice.

Schistosomiasis vaccines: Farewell to the God of Plague?

Control and even eradication of schistosomiasis have been achieved in some countries using integrated measures but this disease remains endemic in 74 countries with 600 million at risk of whom 200 million are currently infected. The application of control measures, particularly population-based chemotherapy, has in many countries greatly reduced the incidence of serious disease manifestations, but vaccines are urgently needed to supplement existing control measures. Great advances are being made in vaccine development and the first clinical trials are expected to begin shortly.

PIP: By 1915 the very complex life cycles of all the major schistosomes of man had been completely worked out. The disease could be attacked by removing the adult worms with drugs, by eliminating the snail hosts by habitat modifications, and by the provision of safe water supplies. Effective drugs, e.g., trivalent antimonials, were introduced in 1918. In the 1920s copper sulphate was shown to be lethal to the aquatic vectors of S. mansoni and S. haematobium and lime was first used to attack the amphibious vectors of S. japonicum. In the search for antischistosome vaccines several protective antigens have been defined at the molecular level and are being evaluated in animal models. The characteristic decline in age-specific prevalence and intensity of infection seen in S. haematobium and S. mansoni endemic areas is largely due to the gradual development of acquired immunity. In the 1970s, work involving immunization with irradiation attenuated or heterologous, normal cercariae, using several different animal models demonstrated that short-lived larval infections could reliably induce significant levels of resistance to challenge infection with normal cercariae. The concomitant immunity premise led to the purification of the first such antigen, the 38 kDa S. mansoni surface glycoprotein GP38. Vaccination with KLH also protected rats against S. mansoni infection and elicited anti-GP38 antibodies which could passively protect rats; deglycosylation of KLH abolished these properties. S. mansoni GST was identified for protective antigens suitable for gene cloning. The protective efficacy of combined GST/KLH S. japonicum vaccines and the efficacy of recombinant-derived GSTs, and of S. japonicum paramyosin are being explored in sheep and bovines in China. Two irradiated vaccine dominant antigens have been cloned and sequenced, IVGS3 and IVRB4, which are now being tested in protection experiments in rodents prior to trials on large animals in China.

The susceptibility of adult schistosomes to immune attrition.

Mouse infection models are described that demonstrate reduction of egg production in Schistosoma haematobium infections and both worm loss and reduced fecundity in S. bovis infections. Neither phenomenon could be shown in S. mansoni infected mice. The immunological basis for these anti-adult responses was inferred by comparison with infections in T-cell deprived mice and by serum transfer of the ability to reduce a S. bovis worm burden into immunocompromised hosts. Vaccination with irradiation attenuated parasites was also shown to have consequences for the adults of a challenge infection of S. haematobium and S. bovis specifically. Prior vaccination resulted in an abrogation of the anti-fecundity and adult worm elimination that occurred in non-vaccinated similarly infected mice. These models are being used to define the targets and mechanisms involved in anti-adult attrition. A serological assay, quantitation of a circulating antigen (CAA) has been assessed for its ability to measure worm burdens of different species of schistosome in mice. This assay will be used to question whether anti-adult immunity contributes to the pattern of infection with S. mansoni and S. haematobium in man.

Variable species and stage specificity of schistosomulum surface epitopes recognized by mice vaccinated with highly irradiated cercariae.

Antibodies from mice vaccinated with highly irradiated Schistosoma mansoni or S. haematobium cercariae were used to characterize schistosomulum surface epitopes which were found to be diverse in their species and stage specificities. The epitopes recognized on the Mr greater than 200,000 and 15,000 schistosomulum surface antigens of S. mansoni and the Mr greater than 200,000 schistosomulum surface antigen of S. haematobium were found to be cross-specific whereas those on the Mr 38,000, 32,000 and 20,000 schistosomulum surface antigens of S. mansoni and the Mr 35,000, 30,000 and 24,000 schistosomulum surface antigens of S. haematobium were only immunoprecipitated by homologous antibody and are thus possible targets of the protective species-specific immunity stimulated by highly irradiated cercariae. The epitopes recognized on the Mr greater than 200,000 and 38,000 antigens of S. mansoni were shown to cross-react with both the egg and the adult worm whereas those on the Mr 32,000 and 20,000 antigens only cross-reacted with the adult worm, and those on the Mr 15,000 antigen cross-reacted with neither the adult worm nor the egg. In addition the epitopes on the Mr 38,000 and 32,000 antigens were demonstrated to be polypeptide in nature. Those on the Mr greater than 200,000, 20,000 and 15,000 antigens, on the other hand, could not be conclusively defined.

Protective effect of irradiated metacercariae of Fasciola gigantica and irradiated cercariae of Schistosoma bovis against fascioliasis in goats.

Sensitisation of goats for eight weeks with metacercariae of Fasciola gigantica gamma-irradiated at 3 kr resulted in significant resistance to an homologous challenge with normal metacercariae. However, serum sorbitol dehydrogenase assay suggested that, whereas little damage was produced by immunising infections, considerable hepatic damage was caused by flukes remaining from challenge infections. On the other hand sensitisation with cercariae of Schistosoma bovis gamma-irradiated at 3 kr did not stimulate any resistance against heterologous challenge with F. gigantica.

Protection of sheep against Schistosoma bovis using cryopreserved radiation-attenuated schistosomula.

Three sheep were vaccinated with two doses of 3 krad-irradiated cryopreserved Schistosoma bovis schistosomula containing 20,000 and 17,000 organisms respectively, injected intramuscularly 23 days apart after storage in liquid nitrogen for between 9 and 46 days. A challenge of 5360 S. bovis cercariae was administered percutaneously approximately four weeks after the last vaccine dose to these animals and to three controls. Post-challenge the vaccinated animals gained significantly more weight (27% v. 9%), produced fewer eggs in their faeces, showed a smaller reduction in PCV values (-18% v. -27%) and were over-all in better condition than control animals. At perfusion 49.1% fewer adult worms were found in the vaccinated sheep than in controls. The tissue egg burdens were similar in both groups. Histopathologically both groups were similar except that fewer and smaller egg lesions were observed in the livers of vaccinated animals.

Observations on cattle schistosomiasis in the Sudan, a study in comparative medicine. III. Field testing of an irradiated Schistosoma bovis vaccine.

Previous work has shown that cattle can acquire a strong resistance to Schistosoma bovis infection following repeated natural exposure. Partial resistance to a laboratory challenge with S. bovis has also been demonstrated in calves after immunization with an irradiated schistosomular or cercarial vaccine. The aim of the present study was to see whether this type of vaccine could protect calves under the very different conditions of natural exposure to S. bovis in the field. Thirty 6- to 9-month-old calves were each immunized with 10,000 irradiated S. bovis schistosomula by intramuscular injection and 8 weeks later were released into an enzootic area along with 30 unvaccinated animals. The calves were followed up for 10 months, during which period protection was evidenced by a lower mortality rate, a slower rate of acquisition of infection, and lower fecal egg counts in the vaccinated calves. Necropsy of the survivors showed 60--70% reductions in worm and tissue egg counts of the vaccinated calves as compared to those not vaccinated.

Histopathology of experimental Schistosoma incognitum infection in mice following exposure to normal and irradiated cercariae.

The tissue reactions in mice, experimentally infected with normal and irradiated cercariae of S. incognitum were studied. The lesions observed in the skin, liver, lungs and the intestine of mice infected with normal cercariae are briefly described, and compared with those observed with cercariae irradiated at 3000 r of gamma rays. In general, the reactions in mice exposed to normal cercariae were more intense than in those infected with irradiated cercariae. The severity of the reactions appeared largely due to the deposition of eggs in the tissues of the mice infected with normal cercariae. The experimental evidence suggested that most of the flukes from the irradiated cercariae are destroyed in the liver by tissue reaction.

Further observations on immunization of sheep against Schistosoma mansoni and S. bovis using irradiation-attenuated schistosomula of homologous and heterologous species.

This paper describes further characteristics of the immunization of sheep against schistosomes using live, irradiation schistosomula. Sheep immunized with a non-virulent strain of Schistosoma mattheei were protected against a more virulent strain of the same species for over a year. As there was no evidence that the irradiated parasites were able to persist this long, it was concluded that the vaccine had induced a sterile resistance. Heterologous vaccination, using irradiated S. mattheei schistosomula to immunize against S. bovis or irradiated S. mansoni schistosomula to immunize against S. mattheei, failed to induce any protection.

In vitro effect of the lethal antibody on schistosomula in sera of rhesus monkeys immunized with highly x-irradiated cercariae.

The in vitro effect of the lethal antibody on schistosomula in sera of rhesus monkeys immunized with highly X-irradiated cercariae of Schistosoma mansoni or S. japonicum was studied. In all 6 experimental monkeys, 4 unchallenged and 2 challenged, the effects of lethal antibody on schistosomula were demonstrated. The sera of the challenged monkeys had no stronger lethal effect than those of the unchallenged monkeys. This shows that the lethal antibody can be produced by the antigenic stimulation of schistosomula alone. The mortality rates of schistosomula in immune sera were already high at day 1, increased to a certain extent from day 1 to day 4, but showed no significant further increase in days 5 and 6. Two kinds of immunological reactions were observed: perischistosomular precipitate (PSP) and perischistosomular envelope (PSE). Schistosomula surrounded with PSP were usually dying or dead and those enclosed in PSE were usually alive and motile. Thus PSP may be related with the lethal antibody and PSE with a kind of enhancing antibody. Schistosomula with PSP showed a positive fluorescent reaction when stained with fluorescein-labeled rabbit anti-rhesus IgG. Scanning electron micrographs of schistosomula with PSP showed a highly degenerated tegument. These facts indicate that the antibody in PSP contains a fraction of IgG which acts on the tegument of schistosomula.