Combined use of two separate but protective vaccine antigens provides protection against Taenia ovis infection in lambs in the presence of protective maternal antibody.

Three recombinant Taenia ovis antigens (To45, To16, To18) each induce protective immunity in lambs or ewes against infection with T. ovis metacestodes. The degree and duration of immunity were assessed in lambs born from vaccinated ewes. Treatment group sizes varied, typically not fewer than 5 animals per group. Ewes were immunised with one T. ovis recombinant protein prior to lambing and the degree and duration of passive immunity in their lambs was assessed by challenge infection up to 18 weeks. Lambs were fully protected up to 6 weeks of age but immunity waned from 6 to 12 weeks and there was no protection when lambs were challenged at 15 weeks. Immunisation of lambs with the homologous recombinant antigen was not effective when vaccinations were given when maternal antibody was high. Lambs were effectively immunised in the presence of passively protective antibody when vaccinated with an antigen that was different to that given to ewes. Vaccination of lambs with a combination of two proteins, To16 and To18, was more effective than giving these single antigens and gave a significant reduction of cyst numbers when lambs were challenged 12 months after immunisation. These results indicate that the use of combinations of T. ovis recombinant antigens could enable complete protection of lambs against infection, if a delivery system becomes available that will maintain antibody at protective levels for 12 months. Alternatively, a third injection given at 6 months may promote the anamnestic response to give long lasting protection.

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.

Approaches to managing aquatic animal health in Australia.

Despite a rapid and continuous expansion in aquaculture industries, Australia has not experienced significant disease emergencies in farmed aquatic animal populations. However, recent events in relation to wild, farmed, native and introduced aquatic animals have provided warning signals. The development of a national response mechanism for fisheries and aquaculture emergencies became a high priority following the pilchard mortality outbreak in 1995. In terms of more general policy, a special Task Force has provided a framework for managing exotic pests, weeds and diseases and identifying key principles and issues. This Task Force also recommended closer consultation between relevant industry organisations and government agencies. The authors describe the framework of the comprehensive five-year national strategic plan for aquatic animal health ('AQUAPLAN') developed by Australia, and the aquatic animal disease veterinary emergency plan developed within this framework ('AQUAVETPLAN').

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.

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.

Vaccination of mice against Taenia taeniaeformis using antigen fractions partitioned with Triton X-114.

Taenia taeniaeformis oncosphere and metacestode antigens were fractioned using Triton X-114 into insoluble, aqueous and detergent rich fractions. These fractions were analysed in SDS-PAGE and immunoblots and used in vaccination trials against infection with T. taeniaeformis in mice. Qualitative differences were apparent in the spectrum of antigens partitioning into the different detergent phases but host-prospective antigens were present in all three fractions. The presence of individual antigenic components in the phases did not correlate with the degree of protection afforded by these fractions in the vaccination trials. Host protective immunogenicity of T. taeniaeformis oncosphere and metacestode extracts may be due to multiple protective antigens which partition into the different Triton X-114 fractions.

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.

Cellular responses during liver fluke infection in sheep and its evasion by the parasite.

The cellular immune response in sheep to an acute and chronic primary and an acute secondary liver fluke infection were examined by immunohistology of liver tissue and flowcytometry of lymphocytes from the draining hepatic lymph nodes. Ten days after primary infection, portal tract areas surrounding migratory tunnels were infiltrated with CD4+ and CD8+ lymphocytes with fewer B cells and T19+ T cells. Micro abscesses were distributed sporadically in the liver parenchyma and young flukes could be easily observed in the liver tissue free from inflammatory cells. More intensive infiltration of the portal tract areas was observed during a secondary liver fluke infection characterized by a pronounced increase in eosinophils, B cells and CD4+ T cells. In addition, there was an increase in MHC class II+ fibroblastic-like cells surrounding the migratory tracts. In contrast to the primary infection, no young flukes were observed in the same tissue areas during the secondary infection. Chronic primary infections were characterized by perilobular fibrosis and a predominance of CD8+ and gamma delta-TCR+T19- T cells distributed within fibrotic strands. Distinct B cell follicles were observed in the fibrotic strands and near major bile ducts and necrotic patches. Pronounced lymphocyte infiltration could occasionally be observed surrounding liver fluke eggs lodged in liver tissue. A progressive increase in lymph node weight, cell number and CD4/CD8 ratio was observed in the acute and chronic primary infections. The role of the infiltrating cell populations and possible mechanism of immune evasion by the parasite are discussed.

Vaccination against cestode parasites.

Cestodes are tapeworm parasites. Infection in the intermediate host with larval (metacestode) parasites causes medically and economically important diseases known as hydatidosis and cysticercosis. Immunization against experimental infection with metacestode parasites has been highly successful, in marked contrast with the relative ineffectiveness of vaccines against infection with most parasitic organisms. High levels of immunity against a challenge infection with taeniid cestode eggs can be stimulated by immunization with extracts of the parasites, particularly with extracts of the oncosphere life-cycle stage. This led to the production of a recombinant antigen vaccine against infection in sheep with the parasite Taenia ovis, the first highly effective, non-living vaccine against a parasitic infection in animals or humans. This paper reviews immunity to the adult and metacestode life-cycle stages of cestode parasites, development and application of the T. ovis vaccine, and prospects for vaccines against other cestode infections.

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.

Analysis of taeniid antigens using monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B.

Antigens derived from Echinococcus granulosus, Taenia hydatigena and T. pisiformis cyst fluids, T. solium cysticerci, E. multilocularis protoscoleces and E. vogeli cyst membranes were examined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis (IEP) using four monoclonal antibodies (mAb) to E. granulosus antigen 5 (Ag5) and antigen B (AgB). Anti-Ag5 mAbs 24.14 and 61A12 reacted strongly with T. hydatigena and T. pisiformis cyst fluids and, to a lesser degree, anti-AgB MAbs 31.15 and 39B3 also displayed some reaction with these antigens in ELISA. The formation of a modified arc 5 band between Anti-Ag5 mAbs and T. hydatigena cyst fluid (THCF) in IEP further confirms the existence of Ag5 in T. hydatigena cyst fluid. However, the inability of THCF and T. pisiformis cyst fluid (TPCF) to form an AgB band as well as that of TPCF to form an arc 5 band with mAbs in IEP does not exclusively prove the lack of AgB in THCF and TPCF or the lack of Ag5 in TPCF. The absence of a reaction of mAbs with T. solium, E. multilocularis and E. vogeli antigen preparations in ELISA or IEP would suggest that these mAbs may recognise epitopes different from those of T. solium, E. multilocularis and E. vogeli parasites; this might be exploited for specific differentiation of E. granulosus.

Assessment of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B for detection of human hydatid circulating antigens.

Four monoclonal antibodies (MAb) to Echinococcus granulosus Antigen 5 (Ag5) and Antigen B (AgB) were assessed in an enzyme-linked immunosorbent assay (ELISA) for detection of circulating antigens (CAg) in sera of human patients with E. granulosus infection. Around 5.5-8% of 200 sera from 42 surgically proven hydatid patients contained detectable CAg by individual MAb. The combined detection rate for CAg, using four MAb, was 19% (38/200). Although hydatid CAg was detected by MAb in at least one serum sample from 21 of 42 patients, some patients remained negative in the assay regardless of the time when serum samples were taken (pre- or post-operatively), or of the continuing presence of hydatid cysts, their location or fertility. In addition, it was observed that the binding capacity of MAb for sheep hydatid cyst fluid antigen (SHCF) was somewhat reduced in the presence of normal human serum. The CAg detection assay would only be useful for assessment of hydatid infection status in patients with detectable CAg in serum samples.

A strategy for production of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B.

Serum antibody responses to sheep hydatid cyst fluid (SHCF) and a purified Antigen 5 (Ag5) were examined in ELISA, immunoelectrophoresis (IEP) and immunoprecipitation (IP) to facilitate production of monoclonal antibodies (MAb) to E. granulosus Ag5 and Antigen B (AgB). Although sera from mice immunized with SHCF contained antibodies of various classes, the fusions using these donor mice resulted in mainly anti-AgB MAb, possibly due to the preferential selection of MAb to AgB by the SHCF-based ELISA screening system. Donor mice immunized with Ag5 also produced several classes of antibodies, and the resultant fusions enabled selection of IgG MAb to Ag5.

Comparative immunoelectrophoretic analysis of Echinococcus granulosus, Taenia hydatigena and Taenia pisiformis cyst fluid antigens by hyperimmune rabbit sera.

Cyst fluid antigens of Echinococcus granulosus, Taenia hydatigena and T pisiformis were examined by electrophoresis using homologous and heterologous hyperimmune rabbit sera to these antigens. While arc 5 forming antibodies were identified in sera from rabbits immunised with E granulosus and T hydatigena cyst fluids, antibodies responsible for forming precipitating antigen B band were detected in rabbit antisera to E granulosus, T hydatigena and T pisiformis antigens. T hydatigena cyst fluid appears to contain antigen similar to E granulosus antigen 5 and probably antigen B while T pisiformis cyst fluid has mainly an antigen close to hydatid antigen B.

Examination of murine antibody response to secondary hydatidosis using ELISA and immunoelectrophoresis.

Antibody responses in mice with up to 64 weeks of secondary Echinococcus granulosus hydatidosis were examined by ELISA using hydatid protoscolex antigen (Px), Antigen 5 (Ag5) and Antigen B (AgB), and by immunoelectrophoresis (IEP) using sheep hydatid cyst fluid (SHCF). Anti-Px IgG antibodies, evident from 3-5 days post infection (p.i.), increased steadily until 16 weeks and maintained a high level afterwards. Anti-Ag5 IgG antibodies were negligible up to two weeks, but they showed a small increase around 2-3 weeks which was followed by a big increase around 16 weeks p.i. The high level of anti-Ag5 IgG antibodies persisted to the end of experiment. The level of anti-AgB IgG antibodies remained relatively low throughout infection. Anti-Px IgM antibodies appeared in the early period of infection, but became insignificant as the infection proceeded. Specific IgM antibodies to Ag5 and AgB showed two waves of increase, one between 3 days to 4 weeks p.i. and the other between 16 weeks to 46 weeks p.i. The level of IgA antibodies to Ag5 and AgB was low and only a moderate amount of anti-Px IgA antibodies was detected. Generally, a higher level of serum antibodies are associated with a larger number of mature cysts. Serum samples from 5 of 8 mice harbouring hydatid cysts formed 1-3 bands with SHCF in IEP, including Arc 5, but a precipitation are with AgB was not observed. Analysis of hydatid cyst fluid from the infected mice (MHCF) in IEP also failed to demonstrate AgB. Despite the high levels of antihydatid antibodies generated in the infected mice, protoscoleces appeared to be unhindered in their growth to mature cysts.

Further characterization of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B.

Two monoclonal antibodies (24.14, 61A12) to Echinococcus granulosus Antigen 5 and two (31.15 and 39B3) to Antigen B were further characterized using modified sheep hydatid cyst fluid antigens (SHCF) in ELISA. None of these four monoclonals were directed against carbohydrate or lipid epitopes of SHCF antigens since they all reacted strongly with periodate or lipase-treated SHCF. On the other hand, they appeared to recognize SHCF determinants of protein nature as protease treatment of SHCF destroyed binding with the monoclonals. Anti-Antigen B monoclonals 31.15 and 39B3 showed strong reaction with boiled SHCF and anti-Antigen 5 monoclonal 24.14 did not. However, the second anti-Antigen 5 monoclonal 61A12 also reacted with boiled SHCF suggesting that some epitopes of Antigen 5 are heat stable. 24.14 and 61A12 may recognize a similar epitope of Antigen 5 whereas 39B3 may be against an epitope of Antigen B different from that recognized by 31.15.

Evaluation of a monoclonal antibody-based competition ELISA for the diagnosis of human hydatidosis.

An antibody competition enzyme-linked immunosorbent assay (ELISA) using 4 different monoclonal antibodies (MAb) raised against major antigens (Antigen 5 and Antigen B) of Echinococcus granulosus was evaluated for the diagnosis of human hydatidosis. The competition assay, using anti-Ag5 MAb 24.14, detected specific antibodies in 70% (131/188) of sera from patients with surgically confirmed E. granulosus infection and 38.5% (10/26) of sera from patients with E. multilocularis infection. None of the sera from patients with Taenia solium cysticercosis (10), T. saginata (2), filariasis (22), strongyloidiasis (19), fascioliasis (4), bilharziasis (4) and amoebiasis (2) tested were positive using a cut-off point established through reaction between MAb 24.14 and normal human sera. The combined use of the MAb 24.14-based competition ELISA with the conventional antibody-binding assay provides a highly sensitive (92.8%) and specific screening system for human hydatid disease diagnosis.

Cestode vaccines.

Studies over the past 20 years have clearly shown the potential for developing vaccines against larval cestode infections of man and animals. The important larval cestode infections of man (Echinococcus granulosus--hydatidosis: Taenia solium--cysticercosis) involve domesticated animals as intermediate hosts in their natural life-cycles. These animals develop strong immunity against reinfection, and immunity can be artificially induced by vaccination with oncosphere antigens. A major stumbling block in developing commercial vaccines against cestodes has been the difficulty in obtaining adequate supplies of these antigens. Recent studies with Taenia ovis, a larval cestode causing cysticercosis in sheep, have demonstrated the feasibility of developing commercial vaccines against cestodes using recombinant DNA technology. A cDNA library prepared using mRNA obtained from T. ovis oncospheres was used to isolate a clone which expressed T. ovis polypeptide antigen 45W as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-45W). GST-45W gave up to 94% protection against challenge infection when used to vaccinate sheep with saponin as adjuvant. The vaccine antigen was shown by SDS PAGE to be unstable, a major disadvantage in subsequent attempts to obtain high yields of antigen for commercial production. The fusion protein has now been stabilized by reducing the size of GST-45W cDNA through deleting 19 carboxyl terminal hydropathic acids, and the resultant fusion protein GST-45W (B/X) was highly host-protective. Another experiment showed that the 45W T. ovis polypeptide cleaved enzymatically from GST-45W was still host-protective, suggesting that GST had no influence on the immunogenicity of GST-45W fusion protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Echinococcus granulosus: antigenic proteins in oncospheres and on the surface of protoscoleces identified by serum antibodies from infected dogs.

Proteins present in oncospheres and on the surface of living protoscoleces of Echinococcus granulosus were radioiodinated by the lodogen technique and immunoprecipitated with sera from dogs with E granulosus infection and several categories of control sera. Analysis of immunoprecipitates was performed using sodium dodecyl-sulphate polyacrylamide gel electrophoresis to identify antigenic protein components specific for E granulosus. Sera from dogs with E granulosus infection identified antigenic proteins of around Mr 37,000, 30,000 or 22,000 in oncospheres, and proteins of around Mr 70,000, 43,000, 36,000, 27,000 (triplet), 20,000 or 14,000 on the surface of protoscoleces. These antigens appear to be both species- and stage-specific and may be useful for serological discrimination between 'current' and 'recent past' prepatent and patent E granulosus infections in dogs.

Molecular cloning of Taenia taeniaeformis oncosphere antigen genes.

Infection of mice with the cestode Taenia taeniaeformis exhibits several important features common to other cestode infections, including the ability to vaccinate with crude antigen mixtures. Partial purification of the protective oncosphere antigens has been reported with a cutout from deoxycholate (DOC) acrylamide gels; this cutout was called fraction II (FII), and comprises approximately 10% of total DOC-soluble oncosphere antigen. Western blots of DOC gels probed with anti-FII antisera revealed a series of 3-5 discrete bands within the FII region. Further fractionation of the FII antigens on DOC gels was impractical due to limitations in supply of oncospheres, so a cDNA library was constructed from 150 ng of oncosphere mRNA and screened with alpha-FII antisera. Two distinct clone families were identified, oncA and oncB. Antibodies affinity-purified on either of two representative members, oncA1 and oncB1, recognised all the FII bands. Individual FII bands excised from a DOC gel resolved into an overlapping series of molecules when re-run on SDS-PAGE, indicating that each FII band consisted of several polypeptides of differing molecular weight. Immunoprecipitates resolved on SDS-PAGE revealed that alpha-FII recognised 3 major oncosphere antigens, of 62, 34 and 25 kDa; antisera against oncB precipitated both the 34- and 25-kDa antigens, whereas alpha-oncA antisera precipitated the 62-kDa antigen. We conclude that oncA and oncB encode the major antigens in the FII complex. The 62-kDa antigen encoded by oncA1 was the only common antigen precipitated by anti-FII and two other antisera raised against different protective extracts, suggesting that it may be a protective component in all three. Southern blot results indicate that oncA and oncB are distinct genes present at low copy number in the genome. Evidence is also presented suggesting that some cestode mRNAs, including oncA, may use variant polyadenylation signals.