Non-equilibrium plasma prevention of Schistosoma japonicum transmission.

Schistosoma japonicum is a widespread human and animal parasite that causes intestinal and hepatosplenic schistosomiasis linked to colon, liver and bladder cancers, and anemia. Estimated 230 million people are currently infected with Schistosoma spp, with 779 million people at risk of contracting the parasite. Infection occurs when a host comes into contact with cercariae, a planktonic larval stage of the parasite, and can be prevented by inactivating the larvae, commonly by chemical treatment. We investigated the use of physical non-equilibrium plasma generated at atmospheric pressure using custom-made dielectric barrier discharge reactor to kill S. japonicum cercariae. Survival rate decreased with treatment time and applied power. Plasmas generated in O2 and air gas discharges were more effective in killing S. japonicum cercariae than that generated in He, which is directly related to the mechanism by which cercariae are inactivated. Reactive oxygen species, such as O atoms, abundant in O2 plasma and NO in air plasma play a major role in killing of S. japonicum cercariae via oxidation mechanisms. Similar level of efficacy is also shown for a gliding arc discharge plasma jet generated in ambient air, a system that may be more appropriate for scale-up and integration into existing water treatment processes.

Schistosoma japonicum: proteomics analysis of differentially expressed proteins from ultraviolet-attenuated cercariae compared to normal cercariae.

Schistosomiasis is considered the most important human helminthiasis in terms of morbidity and mortality. In this study, comparative soluble proteomic analysis of normal cercariae and ultraviolet-irradiated attenuated cercariae (UVAC) from Schistosoma japonicum were carried out in view of the high efficiency of irradiation-attenuated cercariae vaccine. The results revealed that some proteins showed significant differential expression in the parasite after treatment with ultraviolet light. Total 20 protein spots were identified by mass spectrometry, corresponded to five groups according to their functions in the main that were structural and motor proteins (actin, et al.), energy metabolism associated enzymes (glyceraldehydes-3-phosphage dehydrogenase, et al.), signaling transduction pathway-associated molecules (14-3-3 protein, et al.), heat shock protein families (HSP 70 family, et al.), and other functional proteins (20S proteasome). Furthermore, our results indicated that the differential expression of the proteins by ultraviolet irradiation may be, at least partially, acquired by regulating the mRNA levels of corresponding proteins. These results may provide new clues for further exploring the mechanism of protective immunity induced by UVAC and may shed some light on the development of vaccines against schistosomiasis.

Proteome approach for identification of schistosomiasis japonica vaccine candidate antigen.

Experimental vaccination with radiation-attenuated cercariae (RAC) confers possible practical levels of resistance to challenge infection by humoral and by cellular mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG antibody from RAC vaccinated miniature pig. Two milligrams of soluble egg antigen (SEA) or schistosomal worm antigen preparation (SWAP) was fractionated using two dimensional liquid chromatography (proteome PF 2D) consisted of high performance chromatofocusing (HPCF) and high resolution reversed phase chromatography (HPRP). Of the 42 HPCF fractions of SEA or SWAP, 26 (61.9%) or 15 (35.7%) showed positive dot blot reaction with RAC vaccinated serum respectively. The dot blot positive fractions were applied to the second HPRP column. One hundred and seven out of 26 x 96 of SEA fractions and 18 out of 15 x 96 SWAP fractions reacted with RAC vaccinated serum. From the positive fractions we chose 17 of SEA and 10 of SWAP that had no reactivity with normal cercariae infected (NCI) sera and had single peak of 214 nm; and automated N-terminal amino acid sequence based on in situ Edman Reaction was conducted. Four sequences were obtained and applied to the homology search in NCBI database. A total of eight candidate genes were listed up and their cDNA clones from schistosomula stage were obtained. Two of the recombinant proteins (AAW27472.1 and AXX25883.1) showed strong reactivity with the RAC vaccinated serum but marginal with NCI serum. This protocol using proteome PF 2D could be applicable in identifying immunoreactive proteins from crude extract for the development of vaccines or for diagnostics.

Gene transcription profile in mice vaccinated with ultraviolet-attenuated cercariae of Schistosoma japonicum reveals molecules contributing to elevated IFN-gamma levels.

Vaccination with ultraviolet-attenuated cercariae of Schistosoma japonicum induced protective immunity against challenge infection in experimental animal models. Our preliminary study on the transcription levels of IFN-gamma and IL-4 in splenic CD4+ T cells revealed that attenuated cercariae elicited predominantly a Th1 response in mice at the early stage, whereas normal cercariae stimulated primarily Th2-dependent responses. Further analysis on the gene profile of the skin-draining lymph nodes demonstrated that the levels of IFN-gamma were significantly higher in vaccinated mice than those in infected mice at day 4, 7 and 14 post-vaccination or post-infection. However, for IL-12 and IL-4, the potent inducers of Th1 and Th2 responses, respectively, as well as IL-10, there were no differences over the course of the experiment between the infected and vaccinated mice. To explore the underlying factors that may potentially contribute to elevated IFN-gamma in vaccinated mice, the mRNA profiles of the skin-draining lymph nodes at day 4 post-exposure were compared using oligonucleotide microarrays. Within the 847 probe sets with increased signal values, we focused on chemokines, cytokines and relevant receptors, which were validated by semi-quantitative RT-PCR. A comprehensive understanding of the immune mechanisms of attenuated cercariae-induced protection may contribute to developing efficient vaccination strategies against S. japonicum, especially during the early stage of infection.

[Preliminary study of cercaria antigen of Schistosoma japonicum before and after ultraviolet irradiation].

OBJECTIVE: To study the changes in the constituents of the cercaria antigen of Schistosoma japonicum before and after ultraviolet irradiation. METHODS: The cercaria of Schistosoma japonicum were exposed to ultraviolet light (UV) irradiation at a dose of 400 mgrW/cm2 for 1 min, and the UV-irradiated cercaria antigen (UVCA) and normal cercaria antigen (NCA) were simultaneously analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: At least 2 antigens with relative molecular mass (Mr) of 212 000 and 82 000 were identified in UVCA but not in NCA by SDS-PAGE analysis, and the concentrations of the antigens with Mr of 116 000, 26 000 and 16 000 in UVCA were significant higher than those in NCA. On the other hand, the antigenic molecule with Mr of 67 000 in NCA was recognized by serum from pigs vaccinated with UV-attenuated cercariae, but not by serum from pigs with Schistosoma japonicum infection. Antigens with Mr of 79 000 and 94 000 were apparently more strongly reactive with the former porcine serum than with the latter. CONCLUSION: The results suggest that all the novel antigens arising from or increased by UV exposure, or antigens specifically recognized by serum from pigs vaccinated by UV-attenuated carcariae may be the principal factors in the highly protective immunity provoked by irradiated cercariae.

Comparison of the vaccine efficacy of gamma-irradiated Schistosoma japonicum cercariae with the defined antigen Sj62(IrV-5) in pigs.

Development of a vaccine against Schistosoma japonicum which can protect both man and the domestic animal zoonotic reservoirs of infection would be an invaluable tool in attempts to control this infection in those areas in which conventional control methods have failed to break transmission. The pig is a natural host of S. japonicum and because of its anatomical and immunological similarities to humans, it is a potentially valuable host for studies on S. japonicum in particular and schistosomes in general. Radiation-attenuated cercariae are highly effective in inducing immunity in experimental schistosomosis and there are promising reports of partial protection against schistosomes with recombinant-derived individual antigens. In the present study we have set out to establish a protocol for inducing protection with gamma-irradiated cercariae in pigs and to assess the protective capacity of recombinant and naked DNA formulations of Sj62, a 62kDa region of S. japonicum myosin. The corresponding S. mansoni version or Sj62, recombinant IrV-5, has previously been implicated in irradiated vaccine immunity in S. mansoni infections and has been shown to induce high levels of immunity in a variety of hosts. Groups of pigs were immunised three times at 2-week intervals with 2000 cercariae irradiated at 20krad, with Sj62 as a recombinant (rSj62) incorporated in Freund's adjuvant, a micellar preparation, or as a naked DNA construct. Vaccination with irradiated cercariae did not induce significant anti-Sj62 antibody but following intramuscular challenge with 2000 cercariae, the vaccinated pigs showed >95% resistance as assessed by reduced faecal egg output, worm tissue egg burdens and also reduced septal fibrosis. Immunisation with each of the Sj62 formulations induced significant anti-Sj62 antibody responses, the highest titre (>12,800) being with the Freund's preparation but none of the Sj62-immunised groups showed significant resistance to challenge. The data suggest that Sj62 shows little promise as a vaccine candidate for schistosomosis.

Protective immunity to Schistosoma japonicum infection depends on the balance of T helper cytokine responses in mice vaccinated with gamma-irradiated cercariae.

Although the strain difference in protection of mice to Schistosoma mansoni infection has been described, limited information is available in the case of Schistosoma japonicum. In the present study, we compared the protective immunity to S. japonicum infection and cytokine production in various strains of mice vaccinated with gamma-irradiated cercariae. A significant reduction in worm recovery was observed in male and female mice of DBA/2 at a 6-week interval between vaccination and a challenge infection, whereas vaccinated mice of C57BL/6, C57BL/10, (C57BL/6 x DBA/2) F1 (B6D2F1) and (C57BL/10 x DBA/2) F1 (B10D2F1) showed no detectable level of protection. No sex-linked difference in development of resistance was observed in any of the strains so far examined. Vaccination with gamma-irradiated cercariae twice with a 3-week interval also induced significant protection against a challenge infection in DBA/2 but not in BALB/c or C57BL/6 strains. Further studies demonstrated that spleen cells of vaccinated C57BL/6 mice produced lower levels of IFN-gamma compared to the cells of vaccinated BALB/c and DBA/2. On the other hand, production of IL-10 by spleen cells was relatively higher in BALB/c mice than in the other two strains. Macrophages that had been stimulated with spleen cell culture supernatants derived from vaccinated DBA/2 damaged schistosomula more effectively than cells stimulated with supernatants derived from the other strains. These results suggest that different levels of protection observed among strains of mice depend on the balance of cytokine responses which consequently activate or suppress macrophage-mediated damage to schistosomula.

Vaccination of mice with gamma-irradiated Schistosoma japonicum cercariae.

gamma-irradiated cercarial vaccines induce high levels of protection in mice against Schistosoma mansoni infection, however, the same has not been well established for S. japonicum. Here we describe vaccination studies in mice with gamma-irradiated S. japonicum cercariae testing the effectiveness of different irradiation doses, number of vaccinations, and mouse strains. In CBA/Ca mice, a single percutaneous exposure to 500 S. japonicum cercariae previously attenuated by 10, 20, 30, 40 or 50 krad gamma-irradiation induced significant, but comparable levels of protection (34-46%) against challenge infection. In a repeat experiment in C57Bl/6 mice, only groups vaccinated with 10 or 20 krad gamma-irradiated cercariae showed statistically significant, but lower levels of resistance (20-24%). Multiple vaccination of CBA/Ca mice with 500 20 krad gamma-irradiated cercariae did not improve the resistance level (40%). Analysis of IgG responses showed no clear correlation between antibody levels and levels of protection. Western blot analysis suggested that recognition of a 200-kDa antigen might be correlated with protection, that antigens of 42 and 50 kDa may be involved in the protection induced by single vaccination, but that different antigens might be protective in single vs multiple vaccinations. Sera from mice vaccinated with gamma-irradiated cercariae recognized many fewer antigens than more protective sera from mice vaccinated with UV-attenuated cercariae. These results suggest that the mouse may not be a suitable host for studies involving gamma-irradiated S. japonicum vaccines.

Immunization of pigs against infection with Schistosoma japonicum using ultraviolet-attenuated cercariae.

Since pigs are important in the zoonotic transmission of schistosomiasis japonica in China, a veterinary vaccine might contribute to the control of the disease in humans. Pigs were immunized with three doses each of 10,000 cercariae of Schistosoma japonicum attenuated with ultraviolet light (400 microWatt.min/cm2). The experiment was performed with portable irradiation equipment in a rural area of the Hubei Province (P.R. China). A challenge infection of 1,000 untreated cercariae was given 2.5 or 6 months after the last immunization, and age-matched naive pigs were challenged as a control. Immunized pigs developed about 90% resistance against the challenge. The liver egg load of these animals was reduced by over 90%. Less than 0.01% of the immunizing cercariae developed to adult parasites and the vaccination had no apparent adverse influence on the pigs' health.

[Studies on the mechanism of the protective immunity against Schistosoma japonicum--cellular immune responses].

Kinetics of cellular immune responses was studied in C57BL/6 mice immunized with irradiation-attenuated vaccine, cryopreserved and irradiated vaccine or frozen-thawn vaccine. The results and analysis of correlation between cellular responses and resistance showed that cellular immune responses played an important role in the protective immunity against Schistosoma japonicum.

Acquired resistance to Schistosoma japonicum in IgE-deficient SJA/9 mice immunized with irradiated cercariae.

Participation of IgE in protective immunity against Schistosoma japonicum was examined by comparing congenital IgE-deficient SJA/9 and IgE-producing SJL/J mice. Mice immunized with 100 irradiated cercariae 7 weeks previously were infected with 50 live cercariae. In SJL/J mice at 40 days after infection, a 3-to 4-fold increase of total IgE levels and anti-S, japonicum egg IgE antibody production were observed with no significant difference between immunized and nonimmunized mice. IgE was not detected in SJA/9 mice throughout the experiments. Protective immunity evaluated by recovery of adult worms was found in SJA/9 mice and was comparable to that of SJL/J mice. These results suggest that acquired immunity in mice with irradiated cercariae of S. japonicum was not dependent on IgE in these strains of mice.

Schistosoma japonicum: some parameters affecting the development of protective immunity induced by a cryopreserved, irradiated schistosomula vaccine in guinea-pigs.

Experiments were conducted in guinea-pigs to elucidate the parameters affecting the development of protective immunity against Schistosoma japonicum induced by a cryopreserved, irradiated schistosomula vaccine such as the number of immunizations, route of injection and the use of adjuvants. Results obtained indicated that the cryopreserved, irradiated schistosomula vaccine was effective by either intradermal or intramuscular injection. One intradermal injection with BCG adjuvant resulted in an average worm reduction of 50 x 24%, only a little lower than that of a non-cryopreserved, irradiated vaccine, 53 x 55%, with no statistically significant difference between the two. By intramuscular injection the worm reduction was lower (max. 40%) whether given with or without adjuvants or in 1 or 2 injections.

Activated macrophages in highly irradiated cercariae-induced immunity to Schistosoma japonicum in rats.

The results of studies on the schistosomulicidal activity of activated peritoneal and alveolar macrophages (pM phi and aM phi) from rats immunized with highly irradiated (50 krad.) Schistosoma japonicum cercariae are reported. The authors have examined the activation of these macrophages in terms of spreading, adhesion and ingestion of sheep erythrocytes and pinocytosis of horse-radish peroxidase. Using three criteria, peritoneal macrophages and alveolar macrophages from immunized rats and from rats intraperitoneally injected with BCG were significantly more active than those from normal rats or rats stimulated with 10% proteose-peptone or 1% sodium thioglycolate. A significantly higher percentage of adhesion and ingestion was obtained with the sheep erythrocytes that were co-opsonized by heat-inactivated rat anti-sheep erythrocyte serum and fresh normal rat serum. Schistosomulicidal effects were observed with macrophages from irradiated cercariae-immunized rats in two activation systems: in vitro activation in the presence of macrophage-activating factor (MAF), and in vivo activation by the intraperitoneal challenge with sonicated cercarial antigens.

Schistosoma mansoni and S. japonicum: comparison of levels of ultraviolet irradiation for vaccination of mice with cercariae.

When cercariae of Schistosoma mansoni and of S. japonicum were irradiated with various levels of u.v. light at 254 nm, their development to perfusable worms was reduced to below 1% at about 200 microW min cm-2. Cercariae attenuated with about 300 microW min cm-2 induced partial resistance against an homologous challenge infection in mice. No differences were observed between the two schistosome species when the same treatment was given to the cercariae. Thus the same u.v. dose can confer immunizing ability to cercariae of both S. mansoni and S. japonicum.

Schistosoma japonicum: an ultraviolet-attenuated cercarial vaccine applicable in the field for water buffaloes.

Water buffaloes were vaccinated three times with 10,000 Schistosoma japonicum cercariae irradiated with ultraviolet (uv) light at a dose of 400 microW x min/cm2. The irradiation was performed with cheap, simple, and portable equipment in a rural area of Hubei Province (People's Republic of China). A challenge infection of 1000 untreated cercariae was given to six vaccinated and six naive control buffaloes, while two vaccinated animals were not challenged. The experiment was terminated 6 weeks after the challenge. Control animals had lost body weight and harbored a mean of 110 worms and 37 eggs per gram of liver. The vaccinated animals gained weight after the challenge and developed 89% resistance to infection with S. japonicum. Since schistosomiasis japonica is nowadays transmitted in China predominantly by domestic livestock, a uv-attenuated cercarial vaccine for bovines may contribute to the control of this disease.

Induction of resistance in Oncomelania hupensis nosophora against Schistosoma japonicum, but not against Paragonimus ohirai, using irradiated miracidia.

Oncomelania hupensis nosophora snails sensitized with X-irradiated Schistosoma japonicum miracidia demonstrated resistance against a following challenge infection with non-irradiated homologous miracidia. The resistance in O. h. nosophora against S. japonicum was acquired within 1 day of sensitization, and it was strongest in a group challenged at an interval of 3 days. The resistance persisted for at least 4 weeks. Histological examinations revealed amoebocyte accumulation around the challenged S. japonicum sporocysts. On the other hand, when O. h. nosophora sensitized by exposure to X-irradiated P. ohirai or S. japonicum miracidia were subsequently challenged with normal P. ohirai miracidia, no resistance was observed, although they expressed the resistance against heterologous S. japonicum infection.

Passive transfer of resistance to mice with sera from rabbits, rats or mice vaccinated with ultraviolet-attenuated cercariae of Schistosoma japonicum.

All serum transfers from donor rats or rabbits given single or multiple vaccinations of ultraviolet (u.v.)-attenuated Schistosoma japonicum cercariae conferred significant resistance against challenge to mice. Donors given 5 vaccinations, however, produced the most effective sera; rat sera giving up to 88% protection and rabbit sera up to 80%. This protective effect was species-specific and titratable. Sera from vaccinated rabbits and rats were were most effective when transferred to mice 2 h before challenge, but became progressively less effective when transferred with increasing time after challenge. These sera had no efficacy when given 6 days after challenge. Thus, sera from vaccinated rabbits and rats were effective against the early stage of migration, but did not necessarily have to act in the skin as all serum transfers were as effective against intraperitoneal as percutaneous challenge. By contrast, serum from multiply vaccinated mice had little or no protective effect when transferred to mice before challenge, but conferred 62% resistance when transferred 5 days after challenge. Further, there was an additive protective effect when vaccinated rat and mouse sera were given in combination at their optimum transfer times (days 0 and +5, respectively). Thus, there appears to be a stage-specific immune response induced by vaccination depending upon whether the vaccinated hosts are truly permissive or not. Vaccinated rats and rabbits respond to the early phase of migration and vaccinated mice make protective responses against the lung phase of migration.

The induction of species-specific immunity against Schistosoma japonicum by exposure of rats to ultra-violet attenuated cercariae.

Single percutaneous immunizations of Fischer rats with 1000 ultra-violet attenuated Schistosoma japonicum cercariae induced 52-88% resistance to challenge 4 weeks later. Increasing this to 3 immunizations induced 90% resistance to challenge, and this level of protection remained undiminished for up to 40 weeks after vaccination. Rats vaccinated with gamma-irradiated S. mansoni cercariae were resistant to challenge with S. mansoni but not S. japonicum. Similarly rats vaccinated with u.v.-attenuated S. japonicum cercariae were not resistant to heterologous challenge. Thus irradiated vaccines are species-specific in both permissive and non-permissive hosts.

Resistance against Schistosoma mansoni induced by highly irradiated infections: studies on species specificity of immunization and attempts to transfer resistance.

Significant levels of resistance against Schistosoma mansoni challenge were developed by mice exposed to highly irradiated (20 krad.) cercariae of the homologous species (53-67%), whereas vaccination with S. bovis, S. haematobium or S. japonicum failed to confer significant levels of resistance (-5-12%), thus confirming the specificity of the immunizing procedure. Attempts to transfer resistance to naive recipients by injection of serum and of spleen or lymph node cells from donor mice vaccinated with highly irradiated cercariae were largely unsuccessful. However, significant levels of resistance could be transferred to mice by injection of serum from rabbits exposed to irradiated cercariae. Comparable levels of resistance were conferred by injection of serum at the time of challenge (34-69%) or 5-6 days later (31-56%). In contrast, sera from rabbits injected with soluble egg antigen or homogenized cercariae failed to confer protection upon recipient mice. Sera from vaccinated mice, vaccinated rabbits and antigen-injected rabbits all caused cell adherence to skin-transformed schistosomula but neither the level of adherence nor the serum titre correlated with the ability to confer protection to mice.

The induction of specific immunity against Schistosoma japonicum by exposure of mice to ultraviolet attenuated cercariae.

Mice can be partially protected against Schistosoma japonicum by prior exposure to ultraviolet (UV)-attenuated infections which fail to survive to the adult stage and produce no overt pathology in the host. Optimum resistance was induced by parasites exposed to 40 seconds of UV, significantly lower levels of resistance being stimulated by both shorter and longer exposures. No consistent relationship between the degree of resistance induced and the number of irradiated cercariae given could be demonstrated and equivocal results were obtained when comparing the efficacy of single and multiple vaccinations. Vaccinations with UV-attenuated cercariae given intraperitoneally (i.p.) were as efficacious as those given percutaneously but mice were as or more resistant to challenges given by the i.p. route, the possible reasons are discussed. There was no observed delay in the migration of the challenge, vaccinated mice being as resistant when perfused 6 or 3.5 weeks after challenge. Vaccination was species specific since mice exposed to either UV-attenuated S. japonicum cercariae or gamma-attenuated S. mansoni cercariae were resistant to homologous but not heterologous challenge.