D.D. Heath

Vaccination against hydatidosis using a defined recombinant antigen

Echinococcus granulosus is the causative agent of hydatid disease in humans and animals. Natural transmission of the parasite occurs between dogs as definitive hosts and animal intermediate hosts. There is an urgent need for improved methods to control the parasites transmission. Here we describe the development of a vaccine based on a cloned recombinant antigen from the parasite egg (oncosphere). Sheep vaccinated with the antigen, designated EG95, are protected (mean 96–98%) against hydatidosis developing from an experimental challenge infection with E. granulosus eggs. The vaccine will provide a valuable new tool to aid in control of transmission of this important human pathogen. It also has the potential to prevent hydatid disease directly through vaccination of humans.

Vaccination trials in Australia and Argentina confirm the effectiveness of the EG95 hydatid vaccine in sheep.

Experimental vaccine trials against hydatid disease have been undertaken in sheep using the EG95 recombinant vaccine. Challenge infection was with viable Echinococcus granulosus eggs obtained from a New Zealand isolate (dog/sheep cycle), an Australian isolate (dingo/wallaby cycle) and an Argentine isolate (dog/sheep cycle). Vaccination with EG95 conferred a high degree of protection against challenge with all three parasite isolates (protection range 96-100%). Taken together, the trials demonstrated that 86% of vaccinated sheep were completely free of viable hydatid cysts when examined approximately 1 year after challenge infection. Vaccination reduced the number of viable cysts by 99.3% compared with unvaccinated controls. These results suggest that the EG95 vaccine could have wide applicability as a new tool for use in hydatid control campaigns.

Control of echinococcosis and cysticercosis: a public health challenge to international cooperation in China.

Echinococcosis, both cystic and alveolar, and Taenia solium cysticercosis are the most serious zoonotic cestodoses worldwide. Because of the emerging importance of these diseases in China, several international workshops and meetings were held in this country from 1998 to 2001. Based on round table discussions in Chengdu 2000, the proposal of a strategy to control echinococcosis and cysticercosis has been prepared in China. It includes a comprehensive approach based on a careful analysis of the local situations (particularly concerning the particularities of the cycle, ecology, and ethology of the animal hosts, and behavioral characteristics of the population at risk), the use of newly developed tools both in animals and human (immunology, molecular biology, and imaging), and the association of the traditional control measures (control of slaughtering, antiparasitic treatment and control of the definitive hosts, and health education) with more recent developments such as vaccination of the intermediate hosts. Plans on for the control of echinococcosis and cysticercosis in China are summarized.

Vaccination Against Cysticercosis and Hydatid Disease

Infections with the larval stages of taeniid cestode parasites cause substantial human morbidity as well as economic losses in domestic livestock species. Despite ongoing efforts around the world, few countries have been able substantially to reduce or eradicate these infections through the use of anthelmintics and lifestyle changes. Vaccines offer an additional potential tool to assist with the control of parasite transmission. Here, Marshall Lightowlers and colleagues review the substantial progress that has been made towards developing practical vaccines against hydatid disease in sheep and cysticercosis in sheep and cattle. Recombinant antigens have been used to induce more than 90% protection against challenge infections. Such success in animals encourages investigation of the potential use of vaccines in humans to prevent hydatid disease arising from infection with Echinococcus granulosus and cysticercosis from infection with Taenia solium.

Progress in control of hydatidosis using vaccination—a review of formulation and delivery of the vaccine and recommendations for practical use in control programmes

A vaccine to protect sheep, goats, and bovines against hydatid disease caused by the cysts of Echinococcus granulosus is prepared as a recombinant fusion protein expressed in Escherichia coli. Solubilised inclusion bodies are injected, together with Quil A, subcutaneously on two occasions 1 month or more apart, and induce protection against infection which lasts for at least 12 months. A third injection given 6-12 months after the second injection induces a high and long-lasting protection against artificial or natural challenge infections. This review describes work carried out on the formulation, safety and efficacy of the vaccine under laboratory and field conditions, using artificial or natural challenges with E. granulosus eggs, followed by necropsy. Hydatid control programmes based on regular treatment of all dogs with the correct dose of a highly-efficient anthelmintic have sometimes not been successful in Continental environments. Access to dogs is difficult in summer because of the distances to summer pastures, and is often impossible in winter because of snow. A control program using strategic twice-yearly anthelmintic treatment of dogs is likely to be successful provided grazing animals are vaccinated as well. Vaccination as a control tool only requires the veterinarians to visit two times a year, and while the veterinarian is present, the dogs can be treated with anthelmintic for little additional cost. One visit should take place after the autumn kill of animals for winter consumption, and this is a good time to vaccinate animals born in the summer, and also all other animals while they are healthy and immunologically responsive. The other visit should take place in the spring, at which time animals born during winter can be vaccinated. Although a single immunization has been shown to induce a useful degree of protection, where possible it is best to give two initial injections, 1 month apart. If it is possible for veterinarians to stay in the field for 2 months in November/December and March/April, in order to give the two injections, a more rapid onset of full protective immunity will initially be achieved than if the injections are given 6 months apart. A large-scale safety and efficacy trial involving 50,000 and 100,000 lambs in Qinghai and Xinjiang Provinces of China has taken place. Results have confirmed safety and efficacy. In most countries, prevalence of infection increases with age. The vaccine has no effect on established cysts, and therefore, in order to prevent the biomass of Echinococcus spp. from increasing, it might be an effective strategy to begin a control programme by vaccinating all animals. Because many of the older stock will already be infected, they will remain a source of infection for dogs for the average lifetime of the stock. Dogs will still be able to be infected from the older stock, and will continue to infect humans. We advocate that a vaccination programme be accompanied by education about hydatid disease, and anthelmintic treatment of dogs in late autumn and early spring.

Transmission dynamics and control options for Echinococcus granulosus

Cystic echinococcosis, caused by the larval stage of Echinococcus granulosus, is a global public health problem. Whilst in a few localities, such as New Zealand, the parasite has been effectively controlled or even eradicated, in most endemic regions it remains a persistent problem. In some areas, such as the former Soviet Union, the disease incidence in humans has increased rapidly in recent years. It is important to have an understanding of the transmission dynamics, both between dogs and domestic livestock where the parasite maintains itself and from dogs to people. It is from this knowledge that effective control measures can be devised to reduce the prevalence of the parasite in animals and hence reduce the incidence of human disease. Mathematical models to describe the transmission of the parasite and the effects of different control strategies were first proposed over twenty years ago. Since then further information has been acquired, new technology has been developed and better computing technology has become available. In this review, we summarise these developments and put together a theoretical framework on the interpretation of surveillance information, how this affects transmission and how this information can be exploited to develop new intervention strategies for the control of the parasite. In particular, the parasite remains a persistent or re-emerging problem in countries of low economic output where resources for an intensive control programme, that has been successful in rich countries, are not available. By understanding of the transmission biology, including mathematical modelling, alternative and cost-effective means of control can be developed.

Antigenic polypeptides of Echinococcus granulosus oncospheres and definition of protective molecules

Immunoblotting and in vitro oncosphere‐killing were used to identify a putative protective molecule in Echinococcus granulosus mature oncospheres. A range of sera from sheep that had been shown to be protected against E. granulosus, and from those that were not, were tested. The sera used were obtained from sheep hyperimmunized with E. granulosus oncospheres, or immunized with oncosphere non‐denatured extract, with immature oncosphere extract or with denatured extracts of oncospheres. Results indicated the involvement of native antigens of 23, 25, 30, 34 and 40 kDa in the protective response to E. granulosus infection. The rapid appearance of antibodies to the 23, 25 kDa antigens, their association with early onset of protection and in vitro oncosphere lysis by affinity‐purified antibodies obtained from these fractions, indicated that these antigens contained protective epitopes. Final confirmation was provided by immunization of sheep with fractions prepared by preparative SDS/PAGE, and challenge infection. Only the fraction containing the 23 and 25 kDa molecules was able to stimulate protection. Antisera against this pair of molecules should provide a useful probe for screening an E. granulosus oncosphere cDNA library to identify clones expressing protective molecules.

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.

Protection against hydatid disease induced with the EG95 vaccine is associated with conformational epitopes

This paper describes attempts to map the location of host-protective epitopes of a recombinant vaccine antigen by assessing the ability of truncated regions of the antigen to elicit protective immune responses in sheep. Sheep were immunised with three truncated regions (EG95-1, EG95-2 and EG95-3) of the hydatid vaccine antigen, EG95. These regions overlapped each other and corresponded to amino acids 1-70 (EG95-1), 51-106 (EG95-2) and 89-153 (EG95-3) of the full length recombinant protein. Each region elicited antibody which reacted with the parent antigen, although these reactivities were a small proportion of the level of reactivity generated by immunisation with the full length antigen. Antisera raised against each of the truncated proteins reacted with the native parasite antigen. In vaccination and parasite challenge trials in sheep, none of the truncated regions elicited significant protection against challenge infection or antibody which was lethal to the parasite in vitro. Antibodies from sheep immunised with the combination of all three overlapping truncations elicited a comparatively low but significant level of lysis of the parasite in vitro. These antigens did not inhibit anti-EG95 antibody reactivity with EG95 nor did they inhibit in vitro oncosphere killing induced by anti-EG95 antibodies. These results indicate that the major part of the immune response induced by EG95 vaccination is directed against conformational epitopes and that the host-protective epitope(s) is/are conformational.

Evaluation of three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.