M. G. Taylor

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.

Inter-species variation of schistosome 28-kDa glutathione S-transferases

The 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) is a candidate vaccine antigen. To evaluate the antigenic and phylogenetic variations between the 28-kDa GSTs from 4 species of schistosome, we have cloned and sequenced the 28-kDa GSTs from Schistosoma haematobium (Sh28GST) and Schistosoma bovis (Sb28GST). Sb28GST and Sh28GST are more similar to each other (97%) than to Sm28GST (90%) and particularly to the 28-kDa GST from Schistosoma japonicum (Sj28GST, 77%). Antisera directed against the major Sm28GST epitopes revealed differences in the recognition of the 28-kDa GSTs from the other schistosome species suggesting that these regions have been subjected to evolutionary pressure. The consequences of such species-specific epitopes on the development of a multi-species anti-schistosome vaccine are discussed.

Immunization of mice with gamma-irradiated intramuscularly injected schistosomula of Schistosoma mansoni.

The parameters involved in the induction of resistance against Schistosoma mansoni by injection of irradiated, artificially transformed schistosomula were studied in mice. Single intramuscular injections of 500 schistosomula exposed to radiation doses in the range 2.3 to 160 krad. resulted in significant protection (in the range 20 to 50% as assessed by reduced worm burdens) against a challenge infection administered at intervals from 3 to 24 weeks post-vaccination. However, schistosomula irradiated with 20 krad. consistently resulted in better protection than those exposed to either higher or lower radiation doses despite the persistence of stunted adults from the infections irradiated with 2.3 krad. Vaccination with 40 krad. schistosomula resulted in significant protection in terms of reduced worm and tissue egg burdens and increased survival following lethal challenge. Varying the number of irradiated schistosomula, the frequency and route of their administration, the site of challenge and the strain of host all failed to enhance the level of resistance. However, percutaneously applied, irradiated cercariae were found to be more effective in stimulating resistance (60%) than intramuscularly injected, irradiated schistosomula (40%).

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.

Suppression of Schistosoma bovis egg production in cattle by vaccination with either glutathione S-transferase or keyhole limpet haemocyanin

Two of the antigens which have shown vaccine potential in animal experiments against Schistosoma mansoni are glutathione‐S‐transferase (GST) and GP38, protective epitopes of which are shared with keyhole limpet haemocyanin (KLH). We therefore tested S. bovis GST and KLH for vaccine efficacy against S. bovis in the natural Zebu cattle host. In a preliminary experiment three vaccinations with a total of 1.39 mg of native GSTs of S. bovis induced specific antibody at the time of challenge as detected by Western blotting and ELISA and mean faecal egg counts between weeks 6‐10 post‐challenge were reduced by 56.4 to 82.5% compared to non‐vaccinated controls. Mean adult worm recoveries and tissue egg densities in large intestine and liver samples were also reduced in the vaccinated group, but these differences were not statistically significant. In a subsequent experiment one group of calves was vaccinated with a similar schedule to that used above; a second group of calves was given only two injections of GST (total 0.48 mg protein); a third group of calves was vaccinated twice with a total of 2.0 mg KLH in PBS. All three vaccination schedules induced specific antibody. Both GST vaccination schedules induced significant reductions in faecal egg counts compared to non‐vaccinated controls and in this experiment tissue egg densities were also significantly reduced. A striking finding, however, was that adult worm counts were not reduced by vaccination. An essentially similar outcome resulted from KLH vaccination, since there were significant reductions in faecal and tissue egg counts in the absence of a reduction in adult worm numbers. This type of immunity mimics that induced by natural or experimental infections in the calf and clearly has implications for vaccine design.

Schistosoma mansoni: characterization of two protective monoclonal antibodies

Summary Two monoclonal antibodies against the surface of S. mansoni schistosomula were found to confer significant passive protection to mice (M7B3A, range 28–70%; M22H12C, range 14–58%). No additive effect was observed when both were transferred together. Neither McAb bound to the cercarial surface but both bound to the surface of in vitro derived schistosomula and schistosomula recovered from mouse skin up to 3 days after infection. The McAbs were species specific, but not S. mansoni strain specific. M22H12C immunoprecipitated an 125I‐labelled surface antigen of relative molecular weight (mol. wt) 32 000. In Western blotting of an NP40 schistosomular extract, M7B3A recognized an antigen smear of 13000–18000 with a dominant band at 16000. This 16000 antigen was recognized by serum from demonstrably immune mice and rats vaccinated with highly irradiated carcariae but not by sera from mice with chronic single sex or bisexual infections.

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.

Immunization of rats against Schistosoma mansoni using irradiated cercariae, lung schistosomula and liver-stage worms

In PVG rats a single immunizing infection with Schistosoma mansoni cercariae exposed to 0, 5, 10 or 20 krad. gamma radiation failed to induce more than minimal resistance (17-29%) to challenge 4 weeks later, whereas 4 immunizations with 20 krad.-irradiated cercariae, over several months, induced substantial resistance (75%). In contrast, significant protection was induced in Fischer rats by a single immunization with unirradiated cercariae or cercariae irradiated with up to 80 krad. Comparable resistance was induced by unirradiated and by 2, 5 and 20 krad.-irradiated cercariae (67-74%) and lower levels by 10, 40 and 80 krad.-irradiated infections (57, 48 and 33%, respectively). Although the resistance induced by a single dose of 1000 20 krad.-irradiated cercariae could be boosted by a second (88%), further immunizations failed to enhance this resistance. Also, increasing the number of immunizing cercariae in single or multiple vaccinations from 1000 to 3000 failed to increase the resistance. While the resistance induced by 20 krad.-irradiated cercariae persisted undiminished for at least 25 weeks (67%), the resistance induced by normal cercariae declined to insignificant levels by 25 weeks (21%). Comparison of the migration and survival of unirradiated and of 20 and 40 krad.-irradiated cercariae revealed dramatic differences in their fate: parasites exposed to 40 krad. remained in the skin, while the majority of 20 krad.-irradiated parasites died in the lungs after a sojourn of at least 14 days. Thus, although skin schistosomula alone could induce significant protection, optimal resistance was induced only by parasites which migrated to the lungs or beyond. The immunizing potential of these older parasites was investigated by exposing rats to lung- and liver-stage larvae injected into the tail and mesenteric veins, respectively. Irradiated 4-day lung schistosomula induced comparable resistance (79%) to that induced by a complete unattenuated cercarial infection (78%), whereas less resistance was induced by irradiated and unirradiated 11-day-old liver worms (30 and 27%) and by 25-day-old pre-adult worms (48%).

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.