G.B.L. Harrison

Identification and cDNA cloning of two novel low molecular weight host-protective antigens from Taenia ovis oncospheres

Oncosphere antigens of Taenia ovis were solubilised in sodium dodecyl sulphate and separated by electrophoresis in polyacrylamide gels (SDS-PAGE). Antigen-containing gel fractions cut from the region covering 18-12 kDa were shown to be highly immunogenic in sheep challenge experiments. Specific antisera against 2 candidate antigens at 18 and 16 kDa were used to screen a cDNA library prepared from T. ovis oncosphere mRNA. Recombinant proteins selected with antibody to the 16 and 18 kDa native antigens were expressed as GST fusion proteins. Vaccination trials using either of the 2 fusion proteins To16.17-GST and To18-GST, revealed that each was capable of inducing high levels of immunity in sheep against challenge infection with T. ovis eggs. Antibodies induced by vaccination with the recombinant antigens reacted specifically with their respective 18 or 16 kDa native oncosphere antigens. There was no apparent homology between the T. ovis cDNA coding for To18 and To16.17, or with another host-protective antigen, To45W, described previously. These additional host-protective antigens should prove a valuable adjunct to To45W and permit the development of effective vaccination strategies.

Taenia ovis recombinant vaccine--'quo vadit'.

Several years have elapsed since the publication by Johnson et al. (1989) of the cloning of a recombinant antigen from the cestode parasite Taenia ovis which stimulated high levels of protective immunity in sheep. A great deal of subsequent research and development was necessary to bring the fledgling vaccine to the point of being a registered commercial product. The results of these subsequent studies are dealt with briefly in this paper, including the results of field trials. The T. ovis vaccine was registered by the New Zealand Animal Remedies Board in February 1994. Where then is the commercial product? This paper gives a background to market problems which have emerged through the politics (and realities) of the NZ T. ovis control campaign. It serves as notice that the best science dedicated to producing vaccines or products for parasitic, or other, diseases often faces significant hurdles in the real world of commerce and politics.

Echinococcus granulosus: Development of an intermediate host mouse model for use in vaccination studies

A mouse model has been developed to evaluate potential protective antigens which could render intermediate hosts resistant to a challenge infection with Echinococcus granulosus eggs. DBA/2J, CBA/J, Balb/cJ, C57/B16J and CF-1 mice were initially infected orally and parenterally with eggs, hatched eggs or activated oncospheres. Generally less than 1% of the oral dose established as cysts. Mean cysts counts were increased when Balb/cJ mice were injected intraperitoneally or intravenously with activated oncospheres. A challenge regime using 600 activated oncospheres injected intraperitoneally into adult Balb/cJ mice was subsequently adopted yielding means of 15-51 cysts per mouse. When activated oncospheres were injected intraperitoneally into Balb/cJ, DBA/2J and CF-1 mice, cysts were restricted to the peritoneal cavity. Activated oncospheres injected intravenously, however, lodged almost exclusively in the lung and thoracic cavity, except in DBA/2J mice where 55% lodged in the liver. This anatomical localization enabled the outcome of prior infection and challenge to be monitored separately. Prior infection rendered Balb/cJ mice fully resistant to subsequent challenge.

IMMUNE RESPONSES ASSOCIATED WITH PROTECTION IN SHEEP VACCINATED WITH A RECOMBINANT ANTIGEN FROM TAENIA OVIS

This paper describes the evaluation of the protective antibody response of sheep to vaccination against Taenia ovis infection with a defined recombinant antigen (45W). Sera from 181 vaccinated sheep, collected prior to experimental challenge with T.ovis, were assessed for 45W specific IgA, IgG, IgG1, IgG2 and IgM levels and these results correlated with protection data. There were significant relationships (P < 0.001) between IgG, IgG1 and IgG2 titres and protection. Serum IgA levels did not correlate with protection and there were no significant levels of 45W specific IgM detected. Killing of several other taeniid cestodes has been shown to be complement mediated and the findings in this study are consistent with the involvement of this immune mechanism in 45W vaccinated sheep. A comparison of the adjuvants used in this study (saponin and oil in water) demonstrated that whereas both adjuvants stimulated the production of similar levels of 45W specific IgG1, the IgG2 response was significantly higher in sheep vaccinated with oil adjuvant.

IDentification of host-protective antigens of taenia ovis oncospheres

Sheep were fully protected against challenge infection following immunization with a homogenate of T. ovis oncospheres. Ultracentrifugation of sonicated oncospheres either alone or in the presence of a range of detergents did not reduce the immunogenicity of the extracts. Solubilization of oncosphere extracts in non-ionic detergents or sodium dodecyl sulphate (SDS) enabled analysis of host-protective antigens by isoelectric focusing (IEF) and electrophoresis in polyacrylamide gels (SDS-PAGE), respectively. Immunoblotting analysis of oncosphere antigens with immune sheep sera identified predominantly two groups of antigens with relative mobilities of 31-34 kDa and 47-52 kDa with a common isoelectric point of 5.8. The immunogenicity of these antigens was confirmed in vaccination trials using appropriate fractions cut from SDS-PAGE gels and agarose IEF gels. Affinity-purified antibodies prepared against the candidate antigens were used to select the corresponding recombinant DNA-derived polypeptides, one of which was subsequently found to be host-protective.

Echinococcus granulosus: Use of an intermediate host mouse model to evaluate sources of protective antigens and a role for antibody in the immune response

A Balb/cJ mouse model was used to determine which stage of the E. granulosus life cycle possessed the most potent protective antigens. Mice were immunized with crude extracts of protoscoleces, brood capsules, cyst fluid, adult worm tissue, eggs or oncospheres and then challenged intraperitoneally with 600 activated oncospheres. Sonically disrupted oncospheres induced the highest levels of protection (greater than 90%) at doses greater than or equal to 10(3) oncosphere equivalents per mouse. High levels of protection were maintained when these preparations were solubilized in SDS. Immunization with Taenia ovis or T. hydatigena oncosphere preparations induced a maximum of 62 and 40% cross-protection, respectively. In passive transfer experiments, serum from triple-infected immune donors that were completely resistant to subsequent challenge induced 69% protection in naive recipients (P less than 0.01). Serum from mice that had been immunized with oncosphere sonicates that were shown to be highly immune, failed to induce statistically significant protection in recipients. A sheep trial confirmed the protective ability of prior infections. Immunization of sheep with a SDS solubilized oncosphere preparation produced 91% protection (P less than 0.01).

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.

Pilot field trial of a recombinant Taenia ovis vaccine in lambs exposed to natural infection

Previous trials of an experimental Taenia ovis vaccine using the recombinant antigen GST--45W(B/X) established that it was possible to achieve >90% protection against a single artificial challenge of T. ovis eggs. This trial was undertaken to assess vaccine efficacy against artificial challenge and natural infection acquired by lambs grazing contaminated pasture. Two hundred Romney lambs were vaccinated at 6 and 12 weeks of age. One hundred control lambs were not vaccinated but were allowed to run with the vaccinated mob. At 15 weeks of age, 10 controls and 18 vaccinated lambs were artificially challenged with 2000 T. ovis eggs. The remaining control and vaccinated lambs were allowed to graze contaminated pasture for 3 weeks and were then moved to clean pasture for 5 months. The artificially challenged lambs plus 24 of the field-infected lambs were slaughtered and the carcasses dissected to obtain cyst counts. The remaining field-infected lambs were slaughtered at a commercial processing plant and the carcasses examined by conventional meat inspection. The results showed that the vaccine provided a high level of protection against artificial challenge (92%) and natural infection (98%) when assessed by carcass dissection. The data from commercial meat inspection showed that vaccination provided 89% efficacy against downgrading or condemnation compared to non-vaccinated control lambs. The average difference in carcass values between vaccinated and non-vaccinated groups was 4.36 dollars, representing a 35% loss in value due to T.ovis infection in non-vaccinated lambs.