Yaobi Zhang

Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection.

Several defined vaccine candidate antigens of Schistosoma japonicum have shown promise in large animal vaccination experiments. However, vaccination of mice in the laboratory with either single recombinant antigens or DNA encoding forms of the individual antigens has so far failed to induce significant protection against S. japonicum cercarial challenge infection as judged by worm reduction, although specific antibodies were generated. This is in contrast to the results achieved using radiation-attenuated vaccines which are highly protective. Even in large animal vaccination experiments, the protection levels obtained with single defined antigens were far below those achieved using the attenuated vaccines. One possible interpretation is that the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. We, therefore, carried out mouse vaccination experiments using a cocktail DNA vaccine comprising four DNA plasmids encoding four different S. japonicum antigens, Sj62, Sj28, Sj23 and Sj14-3-3, respectively. We, also investigated whether co-injection of the mouse IL-12 encoding plasmid with the cocktail DNA vaccine was able to enhance the Th1 responses and hence the protective immunity. Three intramuscular injections of the cocktail DNA vaccine induced a significant Th1-type cellular response with high level of IFN-gamma production by splenocytes upon in vitro stimulation with recombinant antigens. Importantly, significant IgG antibody responses were also induced against crude worm antigens. In two out of three experiments, significant resistance (34-37 and 44-45%, respectively) was demonstrated while another experiment did not show any protection against S. japonicum cercarial challenge infection. Co-injection of the IL-12 encoding DNA did not further enhance these responses, nor the level of resistance, compared with the cocktail DNA alone.

Laboratory and field evaluation of Schistosoma japonicum DNA vaccines in sheep and water buffalo in China

Vaccines are needed to control zoonotic Schistosoma japonicum infection and several vaccine candidates have now been identified. Two of these (Sj28GST and Sj23) have shown particular promise in sheep when injected with Freunds adjuvants. The objective of the present work was to find a vaccine formulation which may have potential for widespread use in the field. DNA vaccine formulations of these antigens were produced and tested first in sheep under laboratory conditions and then in both the laboratory and the field in water buffalo. In both host species partial protection as evidenced by a reduction in parasite counts in vaccinated compared with control animals was induced by both vaccines, and in water buffalo the vaccines were shown to be partially protective in the field as well as in the laboratory. These results suggest that the two DNA vaccines tested here may have potential for large-scale field use.

Field testing of Schistosoma japonicum DNA vaccines in cattle in China

Vaccines are needed to reduce the zoonotic reservoir of Schistosoma japonicum infection in bovines in China. We have developed two experimental DNA vaccines and have already shown these to be capable of inducing partial protection in water buffalo naturally exposed to the risk of S. japonicum infection in the field. We now report a similar field trial in cattle, the other major bovine reservoir host species in China. Groups of cattle were vaccinated with the VRSj28 vaccine or the VRSj23 vaccine, or, to test whether protection could be enhanced by combination vaccination, with both these DNA vaccines together. After vaccination, the cattle were exposed to natural infection in the field for a period of 54 days. Worm and egg counts carried out at the end of the experiment showed that each of the vaccine groups showed partial resistance, and that combined vaccination was not more effective than vaccination with the individual plasmids.

Schistosoma japonicum myosin: cloning, expression and vaccination studies with the homologue of the S. mansoni myosin fragment IrV‐5

The Schistosoma japonicum homologue of the 62 kDa fragment of S. mansoni myosin (SmIrV‐5), which has proved highly protective against S. mansoni infection in mice and rats, has been cloned and expressed as the full length 62 kDa equivalent, Sj62, and a truncated 44 kDa version, Sj44. DNA sequencing showed the Sj62 sequence to be 88.4% identical at the nucleic acid level and 96% identical in deduced amino acid sequence to that of SmIrV‐5. The recombinant proteins (rSj44 and rSj62) were strongly recognized in Western blotting by sera from mice multiply vaccinated with UV‐irradiated S. japonicum cercariae and weakly recognized by S. japonicum chronic infection mouse sera. Unlike SmIrV‐5, mouse antisera against the recombinant S. japonicum proteins did not give positive recognition in immunofluorescence assay with the surface of newly transformed schistosomula of the homologous species, S. japonicum, nor did they react with S. mansoni schistosomula. However, the anti‐rSj62 sera clearly localized the native antigen to the subtegumental muscle layers in male adult worm sections by immunoelectron microscopy. Vaccination of several groups of mice and/or rats with rSj44 and rSj62 incorporated into different adjuvants induced high titres of specific IgG but in only one experimental group was there a significant reduction in worm burden (27%, P < 0.05). The possible reasons for the disparity between the vaccination results presented here and those demonstrated in experiments using rSm62 (IrV‐5) are discussed.

MOLECULAR CLONING AND CHARACTERIZATION OF A NOVEL SCHISTOSOMA JAPONICUM IRRADIATED VACCINE-SPECIFIC ANTIGEN, SJ14-3-3

A Schistosoma japonicum cDNA coding for a full length S. japonicum 14-3-3 protein was obtained by antibody screening of an adult worm cDNA library using sera taken from mice vaccinated with UV-attenuated cercariae, which are capable of transferring high levels of passive immunity to this parasite. The deduced amino acid sequence consists of 254 amino acids and is highly homologous with 14-3-3 family of proteins from a variety of species (55-69% identity). The recombinant S. japonicun 14-3-3 protein (rSj14-3-3) was expressed and purified in pGEX/E. coli, and in Western blotting was strongly recognised by sera from mice, rats and bovines vaccinated with irradiated S. japonicum cercariae. Analysis of mRNA showed that Sj14-3-3 is expressed in sporocysts and adult worms, but not in cercariae, however mouse antisera against rSj14-3-3 recognised a 29 kDa native antigen in antigen preparations made from eggs, cercariae, schistosomula and adult worms of S. japonicum indicating that this antigen is present in all life-cycle stages. The presence of the native antigen in detergent extracts of intact schistosomula suggests that it is also present in the schistosomular tegument which is the most vulnerable target for immune attack. However, antisera against rSj14-3-3 did not recognise a similar band in S. mansoni or S. haematobium antigens, indicating that, like the UV-attenuated vaccines, this protein induced species-specific immune responses. Southern blot analysis suggested that there may exist more than one gene copy and/or polymorphism for Sj14-3-3. Immunoelectron microscopy confirmed that the native antigen is present throughout the body of adult worms including the tegument, but is less abundant in the muscles. The potential of rSj14-3-3 as a vaccine is now under further investigation.

Comparison of the vaccine efficacy of γ-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 Freunds 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 Freunds 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.

Vaccination of mice with gamma-irradiated Schistosoma japonicum cercariae

γ‐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 γ‐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 γ‐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 γ‐irradiated cercariae showed statistically significant, but lower levels of resistance (20–24%). Multiple vaccination of CBA/Ca mice with 500 20 krad γ‐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 γ‐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 γ‐irradiated S. japonicum vaccines.

Immunogenicity of plasmid DNA encoding the 62 kDa fragment of Schistosoma japonicum myosin.

The recombinant Schistosoma mansoni 62 kDa myosin fragment, rIrV-5, is highly protective in experimental animals, however, vaccination of mice and rats with the recombinant Schistosoma japonicum homologue, rSj62, did not induce significant resistance against S. japonicum infection. To explore alternative ways of presenting this antigen, we further constructed a plasmid (VRSj62) which encodes Sj62 using the VR1020 vector and tested it in vaccination experiments. Four immunisations with 10 microg VRSj62 DNA alone were sufficient to induce high and progressively increasing levels of IgG antibodies against rSj62 with increasing numbers of injections in CBA/Ca mice (IgG titre > or =1:25000), and three injections with 50 microg VRSj62 DNA alone induced significant IgG responses in C57Bl/6 mice (IgG titre, 1:1600). However, vaccination with plasmid DNA entrapped in cationic liposomes or together with pUC19 DNA as a source of CpG motifs, both of which have been reported to enhance immune responses, did not enhance specific antibody production. In spite of the stimulation of specific antibodies against rSj62 with the naked DNA construct no resistance to challenge was demonstrated.

Down-regulation of specific antigen-driven cytokine production in a population with endemic Schistosoma japonicum infection.

Schistosome antigen‐driven cytokine responses and antischistosome antibody levels of residents of a Schistosoma japonicum endemic island in Poyang Lake, Jiangxi Province were studied before and 45 days after treatment with praziquantel. IL‐4, IL‐5, IL‐10 and INF‐γ were all detected in the supernatants of whole‐blood cultures after stimulation with schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP). The percentages of subjects producing detectable amounts of each cytokine assayed were higher in the group who were negative by stool examination at the start of the study than in those who were initially stool positive. After praziquantel treatment the percentages of subjects producing both type I and type II cytokines increased. This suggests that the production of both types of cytokine was down‐regulated in the presence of live, egg‐laying S. japonicum adult worms but that this was reversible by treatment. In contrast, the antibody studies showed higher levels of SWAP and SEA‐specific antibodies (IgE, total IgG, IgG4, IgM) in subjects who were originally stool‐positive than in those who were stool‐negative. After treatment specific IgE responses were elevated, but total IgG and IgG4 anti‐SEA and IgM anti‐SWAP antibody levels all fell significantly.

Cloning of Schistosoma japonicum 14-3-3 epsilon (Sj14-3-3ε), a new member of the 14-3-3 family of proteins from schistosomes.

Abstract A new member of the 14-3-3 protein family from Schistosoma japonicum has been identified. Phylogenetic analysis showed that this member belongs to the epsilon subfamily of the 14-3-3 proteins, and it is therefore named Sj14-3-3e. Consistent with the findings for the previously reported S. japonicum 14-3-3 protein (Sj14-3-3), Southern analysis suggested the presence of more than one gene, and/or introns or allelic polymorphism in this epsilon isoform. By RT–PCR, Sj14-3-3e was shown to be stage-specifically transcribed, being abundant in adults, present in sporocysts but absent in cercariae. Furthermore, mRNA of the epsilon isoform seemed to be much less abundant in the sporocyst stage, compared with Sj14-3-3. This suggests varying requirements of the different 14-3-3 isoforms at different stages of the life cycle.