Vaccination against animal parasites

veterinary parasitology ELSEVIER

Veterinary Parasitology 54 (1994) 177-204

Vaccination against animal parasites M.W. Lightowlers Faculty of VeterinaryScience, The University of Melbourne, Vic. 3030, Australia

Abstract A decade of molecular parasitology is beginning to bear fruit, with the appearance of several new, highly effective, practical vaccines against parasitic diseases. Recombinant antigen vaccines have been developed against cestode, nematode, trematode, protozoan and arthropod parasites. Greatest progress has been made with veterinary vaccines, where the ability to test numerous vaccine formulations in challenge trials has allowed more rapid identification of host-protective antigens than is possible with many medically important parasites. Several quite different approaches to vaccine development have been successful. The traditional approach using live, attenuated parasites continues to provide effective vaccines against several protozoan and nematode parasites. Recombinant DNA technology, monoclonal antibody technology, protein chemistry and immunochemistry have played critical roles in the outstanding success which has been achieved over the last 5 years in the development of defined-antigen vaccines. Two approaches have been successful in research towards defined antigen vaccines against parasites: (1) the 'natural antigen' approach where immune responses are stimulated to parasite molecules which are normally antigenic, and possibly host-protective, in infected hosts; (2) the 'naive antigen' approach where parasite molecules which are not antigenic, or of very low antigenicity, in infected hosts are used to raise immune responses capable of killing the parasite. This review examines the successful approaches taken towards the development of effective anti-parasite vaccines and the vaccines which have been produced to date. Keywords: Vaccines

1. Introduction Achievements in the field o f vaccination against parasitic infections have lagged b e h i n d the degree o f success which has been attained with vaccines against other infectious organisms. This is best exemplified by c o m p a r i s o n o f the m y r i a d o f 0304-4017/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0304-4017 ( 94 ) 03084-A

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effective vaccines which have been produced against viral and bacterial infections of man (Bittle and Muir, 1989), with the absence of a vaccine against any eukaryotic parasite of man. The situation with respect to vaccines against animal parasites is not much better. Factors which have contributed most to this relative lack of success in development of vaccines against parasites are the lack of highly effective, host-protective immune responses following an initial infection with many parasitic organisms, and the much greater degree of difficulty associated with in vitro culture of parasitic organisms in comparison with the relative simplicity with which many prokaryotes can be cultured. Chronic parasitic infections, in which the immune responses are incapable of effecting long-lasting immunity to continuing infection or re-infection, are the norm rather than the exception in parasitic diseases. Factors which contribute to the poor level of immunity to continuing infection or re-infection are many and vary widely, reflecting the wide variety of modes of infection and sites of infection of the different parasitic organisms. An important distinction between eukaryotic parasites and the prokaryotes is their genetic complexity, indicated by their substantially larger genomes (Caenorhabditis elegans 350 Mb, Plasmodium falciparum 25 Mb, Escherichia coli 5 Mb). Genetic complexity is expressed in the variety of complex methods by which parasitic organisms are able to avoid or subvert the host's immune defences (Dessaint and Capron, 1993 ), allowing some species, for example the schistosomes, piroplasms and lymphatic filariae, to parasitise sites which might be expected to favour the induction of host-protective immunity. Antigenic variation in the variant-specific surface glycoprotein (VSG) surface antigens oftrypanosomes (Borst, 1991 ) is one example of a sophisticated immune evasion mechanism, which continues to frustrate attempts to develop vaccines against this group of organisms. In some instances, immunity to re-infection is effective, but does not remove parasites established from the initial infection. The term 'concomitant immunity' has been borrowed from the field of tumour immunology to describe this form of immunity to re-infection with parasites (Mitchell, 1990). Examples are infections with schistosomes and infections with the larvae of taeniid cestodes. Host-protective immunity against parasites is rarely completely effective. A significant degree of immunologically mediated acquired immunity does develop, often reflected in partial protection, even against parasite species known to cause chronic parasitism for the life of the host, such as occurs with many intestinal nematode species, Plasmodium spp. and Schistosoma spp. Often, a degree of immunity which is somewhat less than sterilising would be sufficient to achieve a satisfactory level of parasite and parasitic disease control. This applies particularly to parasites of veterinary importance. Depending on the parasite species and circumstances, research on development of vaccines may aspire to achieve a variety of different aims, including: the induction of sterile immunity to subsequent infection; the induction of partial, but sufficient immunity, to prevent major pathological consequences of subsequent infection; the removal of a parasite burden existing prior to vaccination; blocking parasite transmission; prevention of certain disease manifestations associated with parasitic infection.

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Despite the substantial difficulties associated with research on vaccination against parasitic infections, several highly effective vaccines have been produced against parasites of veterinary importance. All have been based upon the application of vaccines containing live parasites whose virulence had been attenuated by irradiation, in vitro culture or in vivo passage (Table 1 ). Also, all of these vaccines have relied upon mimicking immunity to re-infection which occurs naturally with each of the parasite species involved. Two of these vaccines have been developed relatively recently (against Toxoplasma-induced abortion in sheep (Wilkins and O'Connell, 1992 ) and coccidiosis in chickens (Shirley, 1989 ). Prior to these, only four parasitic diseases: parasitic bronchitis, canine hookworm, theileriosis and babesiosis, had been the targets of effective vaccines. Major advances in biotechnology over the past decade are now beginning to bear fruit with the description of experimental results achieving significant protection against cestode, nematode, trematode, protozoan and arthropod parasites of veterinary importance (Table 2). Of the medically important parasites, some success has also been achieved with recombinant or peptide antigen vaccines against schistosomosis (Capron et al., 1992 ) and malaria (Good et al., 1993 ). This review concentrates on vaccines against parasitic infections in animals, with particular emphasis on recent results using vaccines based on recombinant antigens. The taeniid cestodes, and Taenia ovis in particular, are reviewed in more detail, as examples where vaccination with recombinant antigens has been especially effective. Vaccination against arthropod parasites is mentioned in the context of this general review of vaccination against animal parasites, with a more complete coverage of this particular topic provided by another paper in this seties. Reference is made to previously published reviews and the reader is referred to these for a more detailed coverage of the relevant literature, particularly that published prior to the application of recombinant DNA technology to anti-parasite vaccines. In addition, the books by Soulsby (1987), Bittle and Murphy Table 1 Anti-parasite vaccines which have been developed based on the use of living, avirulent parasites Parasite

Host

Vaccine/Antigen

References

Ancylostoma caninum Dictyocaulus viviparus Dictyocaulusfilaria Theileria annulata

Dog Cattle Sheep, goats Cattle

Irradiated L 3 Irradiated L 3 Irradiated L 3

Theileria hirci

Sheep

Cultured schizonts

Babesia bovis

Cattle

In vivo passaged

Babesia bigemina Eimeria Slap. Toxoplasrna gondii

Cattle Chicken Sheep

In vivo passaged Precocious lines In vivo passaged

Miller, 1971, 1978 Urquhart, 1985; Armour, 1987 Dhar and Sharma, 1981 Pipano and Tsur, 1966; Irvin and Morrison, 1987 Hawa et al., 1981; HooshmandRad, 1985 Callow and Mellors, 1966; Callow, 1976 Dalgliesh et al., 1981 Jeffers, 1975; Shirley, 1989 Wilkins et al., 1987; Wilkins and O'Connell, 1992

Cultured schizonts

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Table 2 Recombinant antigen vaccines which have been developed against parasitic infections of animals Parasite

Host

Antigen

% protection a

References

Taenia ovis Boophilus microplus Babesia boris

Sheep Cattle Cattle

47/52 kDa Bm86 12D3, l 1C5

94 45(91 )b 75 c

Fasciola hepatica

Sheep, cattle

26, 26.5 kDa GST

50

Theileria parva Eimeria tenella

Cattle Chicken

p67 66/200 kDa 28 kDa

67 ?~ 9d

Eimeria acervulina

Chicken

(SO7') p250

~ 9d

Johnson et al., 1989 Tellam et al., 1992 Gale et al., 1992; Wright et al., 1992 T. Spithill, personal communication, 1993 Musoke et al., 1992 Danforth et al., 1989 Miller et al., 1989; Bhogal et al., 1992 Crane et al., 1991 Kim et al., 1989; Lillehoj et al., 1990; Jenkins et al., 1991

aProportion of animals protected or mean reduction in parasite burden; highest level of protection published to date. bThe vaccine reduced the number of ticks 45% as well as the size and fecundity of the ticks, resulting in a decrease in reproductive capacity of 91%; full details of the vaccine trials are not published; the antigen used was Bm86 (Rand et al., 1989) expressed via baculovirus. CGale et al. (1992) refer to the inclusion of an additional cloned antigen (T21B4) and results of a field trial where none of 13 cattle vaccinated with a trivalent vaccine required drug treatment following field challenge with Babesia spp., whereas 13 of 19 controls required drug treatment to prevent life-threatening infection levels. dPartial protection has been described with each of these recombinant proteins and, in some cases, with live recombinant bacteria; protection is expressed in reduced lesion scores and in increased productivity (food conversion, weight gain ) compared with non-vaccinated controls; it was not possible to convert the data to percentage protection for comparison with the other vaccines.

(1989), Yong (1992a) and Warren and Agabian (1993 ) and reviews by Clegg and Smith (1978), Barbet (1989), Smith (1992) and Soulsby (1992) provide detailed coverage of immunity to, and vaccination against, parasitic infections. 2. Approaches to vaccination: live versus defined antigen vaccines To date, only vaccines based upon infection with live, attenuated parasites have been used successfully in practice. A vaccine using non-living exo-antigens of Babesia canis, Pirodog, has been marketed commercially in France. However, little published information could be found concerning this vaccine and there seems to be some doubt about whether it could be regarded as effective (discussed below). Soluble exo-antigens have also been tested under field conditions against cattle babesiosis in Venezuela. As with the Babesia canis vaccine, little information could be found about the vaccine and some doubt has been expressed concerning the effectiveness of this form of immunisation (Timms et al.,

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1983). Another method referred to as 'vaccination' has been the induction of infection with virulent parasites, sometimes with subsequent drug treatment of the infection, in order to induce immunity to re-infection. Examples of this form of immunisation have been leishmanisation in man with Leishmania major (Greenblatt, 1988 ), and the largely or entirely superseded practices of 'vaccination' against infections in animals with Theileria spp., Babesia spp. and Eimeria spp. These practices will not be considered further except where they are mentioned in relation to the development of what might be considered to be true vaccines. An indication that the time is near when new generation vaccines based on recombinant parasite antigens will be used commercially, can be gained from the granting of provisional registration of recombinant antigen vaccines against Taenia ovis in August 1990, and Boophilus microplus in July 1992. Many of the vaccines listed in Table 1 have been used extensively and very effectively. However, there are several limitations to the use of vaccines based upon live attenuated parasites. Conversely, it is likely that there may be certain advantages to the use of living parasites. These advantages and disadvantages, in comparison with those of defined antigen vaccines, are summarised in Table 3. The principal advantage of live, attenuated vaccines has been their effectiveness in comparison with non-living antigens. Each of the vaccines listed in Table 1 raises a level of immunity which is superior to that achieved experimentally by immunisation with non-viable parasites or crude parasite extracts. Defined antigen vaccines offer advantages which overcome many of the disadvantages of the live vaccines. However, a major obstacle to the development of defined-antigen vaccines has been the inability to produce sufficient quantities of antigen for any practical purpose. The introduction of recombinant DNA technology to parasitology in the early 1980s provided the tools with which this limitation in antigen supply could be overcome. Early optimism for the development Table 3 Comparison of the advantages and disadvantages of anti-parasite vaccines. (A) Vaccinesbased on the use of live, attenuated parasites. (B) Vaccinesbased on non-living defined antigens (A) Live attenuated vaccine Advantages

(B) Defined antigen vaccine

Effective Long shelflife Antigenically complex Sterile Immunity boosted by natural exposure Quality control Novel antigen targets Inexpensive Disadvantages Short shelflife Antigenically simple Storage requirements -ineffectiveagainst parasite variants Disease due to incompleteattenuation -genetically determined non-responders Reversion to virulence ? Boostingby exposure Accidental transmission of pathogens Quality control Expensive

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of anti-parasite vaccines was premature, and it was not until 1987 that significant protection was achieved with a recombinant antigen (Balloul et al., 1987a) and not until 1989 that the first highly effective vaccination trials against a parasitic infection with a recombinant antigen vaccine were published (Johnson et al., 1989 ). Together with the advances in monoclonal antibody technology, protein chemistry and immunochemistry, genetic engineering has given rise to several highly effective, experimental, defined antigen vaccines. Defined antigen vaccines may comprise complete or incomplete polypeptide copies of native protein antigens, expressed using recombinant DNA methods, or antigenic epitopes produced entirely synthetically. The first example of a synthetic peptide vaccine against a parasitic disease, which has achieved some degree of success, is the SPf66 vaccine against P. falciparum malaria in man (Patarroyo et al., 1987, 1988; reviewed by Targett, 1992 ). While the potential exists for these defined vaccines to be effective in practice, it remains for any to be used in a parasite control program. Also, the defined antigen vaccines have several potential disadvantages (Table 3) which will need to be considered in their further development and in monitoring their effectiveness in the field.

3. Vaccination against cestode parasites The publication by Johnson et al. ( 1989 ), detailing the development of a vaccine against Taenia ovis infection in sheep, was the first to describe a highly effective recombinant vaccine against a parasite. The vaccine remains the most effective of the defined antigen vaccines described to date. For these reasons, and as an example of the successful development of a defined antigen vaccine against a parasite, vaccination against cestode parasites, and against Taenia ovis in particular, is reviewed in more detail than that provided for the other parasites. Among the helminth parasites, taeniid metacestodes are unusual in that they induce an immune response which is extremely effective in preventing the establishment of parasites after those comprising the initial infection (see reviews by: Rickard and Williams, 1982; Lightowlers, 1990; Lightowlers et al., 1993). The level and duration of immunity are not the same for all taeniid species, but concomitant immunity is a feature in most, if not all, species (reviewed in Lightowlers et al., 1992 ). Numerous publications, particularly in the 1960s and 1970s by M.A. Gemmell, and through the 1970s and 1980s by D.D. Heath and M.D. Rickard, formed the scientific foundation on which more recent research towards the development of practical recombinant antigen vaccines has been based. Research in the 1930s with Taenia taeniaeformis (Miller, 1931; Kan, 1934; Campbell, 1936) and Taenia pisiformis (Kerr, 1935 ) had clearly established the potential for the use of non-living vaccines against infection with the larval stages of taeniid cestodes. As vaccine research progressed to work on livestock animals in the 1960s, it appeared that immunity could only be stimulated by the injection and subsequent development of viable parasites and not by similar material which had been killed either by freezing or ultrasonic disruption so that growth of living parasites directly from the immunisation did not occur (Gemmell, 1962; 1964a,b,

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1965, 1966, 1967, 1969, 1970). The hypothesis that immunity was only stimulated by living parasites was tested by Rickard and Bell (Rickard and Bell, 1971 a) who implanted activated oncospheres of Taenia ovis or Taenia hydatigena intraperitoneally into lambs in diffusion chambers having a pore size of 0.22 pm. The viable parasites were unable to escape from the chambers but the excretory/secretory (E/S) products released by the parasites were able to pass through the pores. Lambs in which the diffusion chambers had been implanted were solidly immune to subsequent challenge infection with eggs of the parasite per os. Inside the diffusion chambers the oncospheres had developed into cystic larvae and it was presumed that the parasites' E/S products had stimulated the host-protective immune responses. Follow-up experiments confirmed that the supernatant from in vitro cultivation of activated oncospheres contained host-protective antigens (Rickard and Bell, 197 lb; Rickard and Adolph, 1977 ). Subsequently, it was found that mice could be protected against infection with Taenia taeniaeformis by immunisation with crude, non-living extracts of oncospheres (Rajasekariah et al., 1980; Lightowlers et al., 1984). Osborn et al. (1981) and Osborn and Heath (1982) found that sheep could be vaccinated against cysticercosis and hydatidosis using antigens obtained from non-living extracts or E/S products of Taenia ovis and Echinococcus granulosus oncospheres, respectively. It is unclear why Gemmell's earlier work had failed to induce immunity with frozen or sonicated, non-living preparations from oncospheres. Rickard and his colleagues continued research on vaccination against Taenia saginata in cattle and Taenia ovis in sheep using antigens derived from in vitro culture and established the following features of vaccine-induced immunity: ( 1 ) a single immunisation can stimulate immunity against challenge infection, lasting at least 12 months (Rickard et al., 1977a); (2) antibody in colostrum provides passive immunity to infection in the neonate (Rickard et al., 1977a,b); (3) offspring from a vaccinated dam can be successfully immunised in the presence of protective colostral antibody (Rickard et al., 1977a); (4) significant levels of protection can be achieved against infection acquired in the field (Rickard and White, 1976; Rickard et al., 1982 ). Clearly, these studies had established the potential for practical application of vaccines to control infection with Taenia spp. in sheep and cattle. The obstacle which remained was that it was highly unlikely sufficient quantities of vaccine antigens could be obtained directly from tapeworms to allow practical application of the vaccines. This is particularly true of Taenia solium and Taenia saginata, where man is the obligate definitive host. Recombinant DNA technology provided the solution to the limitations in supply of host-protective antigens of taeniid cestodes. Work on the adaptation of recombinant DNA technology to expression of taeniid antigens began early, with expression of Taenia taeniaeformis antigens being the first to use recombinant DNA for production of candidate vaccine antigens for a helminth parasite (Bowtell et al., 1984). Simultaneously, evidence on the characterisation of the nature of the host-protective oncospheral antigens of Taenia taeniaeformis indicated that

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protein components were important in inducing immunity (Lightowlers et al., 1984), suggesting that recombinant DNA methods may be appropriate for their production in vitro. 3.1. Development of a recombinant vaccine against Taenia ovis

The cysticercus of Taenia ovis encysts in the musculature of sheep. At abattoirs, carcasses which are detected as infected are either trimmed to remove the lesions or condemned for human consumption. This causes losses in the sheep meat production industries. Also, detection of Taenia ouis lesions in imported sheep meat has, in the past, resulted in difficulties with the export of sheep meats from Australia and New Zealand to the USA. In New Zealand, Taenia ovis became recognised as a parasite of increasing importance owing to the initiation of a control campaign for hydatid disease (Gemmell et al., 1986 ). These various factors identified a need for a vaccine to prevent Taenia ouis infection in sheep and led to the establishment of a collaborative research program involving Coopers Animal Health New Zealand Ltd. (now Pitman-Moore New Zealand Ltd. ), the New Zealand Ministry of Agriculture and Fisheries and The University of Melbourne, towards the development of a recombinant antigen vaccine. Initial research focused on the identification of the particular antigens which induced host-protective immune responses. Prior to the initiation of the work it was known that oncospheres and their E/S products were rich sources of host protective antigens. Also, antibodies from either infected or vaccinated sheep were capable of passive protection against Taenia ovis infection. This information served as the initial basis on which research was performed to identify the host-protective antigens from oncospheres. Immunological analyses identified numerous oncosphere components as antigens in immune sheep (Harrison et al., 1993 ). Lambs vaccinated with antigens derived from immature eggs, from parts of the Taenia ouis tapeworm anterior to those containing mature eggs, were not protected against challenge infection with Taenia ovis. In comparison, lambs vaccinated with antigens from mature eggs were protected. Comparison of the components of mature oncospheres recognised by the sera of those two groups of animals revealed that antigens in the regions 31-34 kDa and a doublet running at 47/52 kDa were recognised by the protected but not the non-protected lambs. Antigens in these two regions were also detected by the sera of sheep immune through prior infection with the parasite. Oncosphere antigens which had been solubilised in sodium dodecyl sulphate (SDS) under reducing conditions, retained their ability to stimulate host-protective immunity in lambs, allowing oncospheral antigens to be fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent testing of the fractions in vaccination trials. Lambs immunised with antigens contained within a gel slice including the 47/52 kDa doublet were protected against Taenia ovis challenge, hence these antigens became the targets for in vitro production by recombinant DNA methods. In order to identify the cloned equivalents of these antigens, antibody probes with specificities restricted largely to the 47/52 kDa doublet were prepared

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(Johnson et al., 1989; Harrison et al., 1993 ). Antisera were raised in rabbits immunised with the E/S products of Taenia ovis oncospheres and the anti-47/52 kDa specificities selected by affinity purification from the doublet on Western blots (BeaU and Mitchell, 1986 ). Some evidence suggests that the preparation of antibody of high specificity for the target antigen (s) may have been critical to the successful detection of clones encoding the host protective antigen (Lightowlers and Rickard, 1991 ). In vitro activated Taenia ovis oncospheres were chosen as the source of mRNA for cDNA cloning as these were known to secrete or excrete host-protective antigens. An expression library was created in 2gt 11 (Johnson et al., 1989) and this was screened in plaque immunoassay for antigens binding the affinity purified, rabbit anti-47/52 kDa antibodies. Two classes of clones were detected, one with a relatively strong signal strength in immunoassay, designated 45S, and the other with a weak signal strength, designated 45W. DNA sequence analysis revealed these to be cDNAs from closely related genes. Neither contained what appeared to be 5' untranslated sequence nor an initiation ATG, indicating that they were incomplete copies of the mRNAs encoding the native proteins. In relation to the 45W clone, the 45S cDNA was shorter and lacked some of the protein-encoding open reading frame of 45W. The 45W encoded protein included additional polypeptide sequences at both the NH2- and COOH-termini in comparison with that encoded by 45S. The 45S and 45W cDNAs were sub-cloned into the plasmid vector pUR292 and p-galactosidase (fl-gal) fusion proteins semi-purified from SDS-PAGE gels. Vaccination trials in sheep induced specific IgG antibody against the associated native antigen 47/52 kDa doublet; however, they failed to induce immunity to challenge infection with Taenia ovis. Because the evidence implicating the 47/52 kDa doublet with host-protective immunity was strong, the cDNAs were subcloned into an alternate vector which expressed the parasite encoded proteins as fusion proteins to the COOH-terminus of glutathione S-transferase (GST). This was a forerunner of the pGEX series of plasmids (Smith and Johnson, 1988). Vaccination of sheep with the 45W-GST fusion induced 94% protection against challenge infection with Taenia ovis. Although the 45S-GST fusion protein also induced antibody which reacted against the 47/52 kDa doublet, it did not protect sheep against infection with Taenia ovis. Information concerning the differences in the protein encoded by 45W and 45S is being used in studies which aim to identify the host-protective epitopes on the 45W-encoded protein (Lightowlers and Rickard, 1993 ). It is unclear why the 45W-fl-gal fusion protein was not protective, especially since it is now known that protein capable of inducing potent host-protective immunity can be prepared from the 45W-GST fusion protein, as well as the native 47/52 kDa doublet, using the same methods as were used to isolate the 45W fl-gal fusion protein (SDS PAGE). The Taenia ovis vaccine is being developed commercially (Lightowlers et al., 1992 ). Many of the host-parasite immunological relationships are common amongst the several species of taeniid cestode which have been examined to date (reviewed in Lightowlers et al., 1993). In addition to the taeniid cestode species

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infecting laboratory animals, oncospheral antigens have been shown to be highly protective against the medically a n d / o r economically important species Echinococcus granulosus (Heath et al., 1981; Osborn and Heath, 1982 ), Taenia solium (Molinari et al., 1988) and Taenia saginata (Rickard et al., 1977b; Craig and Rickard, 1980; Rickard and Brumley, 1981; Rickard et al., 1982 ). Evidence concerning the prevalence of human Taenia solium neurocysticercosis in central America (Hisser et al., 1979), hydatidosis in China (Craig et al., 1991), and other epidemiological data, indicate that there is a need for vaccines to assist with the prevention of transmission of these important human pathogens. Information and experience gained in developing the vaccine against Taenia ovis will assist with the production of similar vaccines against the other taeniid cestode parasites.

4. Vaccines against trematode parasites 4.1. Liver fluke Nothing has been published in the scientific literature about a vaccine for fasciolosis with practical potential., However, Dr Terry Spithill has provided the following information from unpublished work. These describe substantial protection against Fasciola hepatica in sheep and cattle vaccinated with recombinant antigens (T. Spithill, personal communication, 1993). The antigens are cDNA clones expressing F. hepatica GSTs. Fluke GSTs were selected as targets for research on vaccination against F. hepatica because native GSTs (Balloul et al., 1987b; Mitchell, 1989; Wolowczuk et al., 1989) and recombinant GSTs (Smith et al., 1986; Balloul et al., 1987a; Boulanger et al., 1991 ) of schistosomes have been used successfully to induce partial, but significant immunity against infection with Schistosoma japonicum and Schistosoma mansoni in animal models. The Schistosomajaponicum 26 kDa protein was identified as a potential vaccine target because it was recognised as antigenic in mice of a strain which was naturally resistant to infection, but not by mice of a susceptible strain (Mitchell et al., 1984, 1985 ). The Schistosoma mansoni 28 kDa protein was targeted because it was recognised as an antigen exposed on the schistosomulum surface and antibodies to this antigen were cytotoxic for schistosomula in in vitro assays. Both antigens were subsequently identified as GSTs following their cloning from cDNA (Smith et al., 1986; Balloul et al., 1987a). Sheep vaccinated with native GST, affinity purified from adult F. hepatica extracts on glutathione agarose beads, had a 57% reduction in the number of fluke developing following a challenge infection with metacercariae (Sexton et al., 1990). Several of the GST isoenzymes have now been cloned (Panaccio et al., 1992; Wijffels et al., 1992 ). Vaccine trials with the recombinant antigens in collaboration with Ciba Geigy have achieved up to 48% reduction in challenge infection in sheep trials and 50% reduction in cattle trials.

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5. Vaccines against nematode parasites

5.1. Lungworm infection in cattle and sheep The first, and arguably the most effective, true vaccine against a parasitic disease was an irradiated larval vaccine against Dictyocaulus viviparus infection in cattle, Dictol. Immunity to, and vaccination against, parasitic bronchitis caused by Dictyocaulus spp. has been reviewed by Armour ( 1987 ). The vaccine was developed commercially following the discovery by Jarrett et al. (1960) that two doses of larvae attenuated by irradiation, induced up to 98% protection against challenge infection with the parasite. The vaccine, and others similar (e.g. Nobivac), continue to be used in Europe, achieving outstanding control of parasitic bronchitis in cattle. A related lungworm species, Dictyocaulus filaria, is a significant pathogen of sheep in eastern Europe, the Mediterranean area, the Middle East and India. The procedures adopted successfully in production of the D. viviparus vaccine have been applied to D.filaria, resulting in the production of a vaccine in India in 1971 (Dhar and Sharma, 1981 ). The vaccine continues to be used successfully in India for the control of disease caused by the parasite and Sharma et al. ( 1981 ) have indicated that transfer of the technology to neighbouring countries is underway.

5.2. Hookworm disease in dogs Miller ( 1971, 1978 ) has reviewed the development and commercialisation of a vaccine against disease caused by Ancylostoma caninum in dogs. The vaccine was based on the use of infective L3 larval stages which had been attenuated by irradiation. Vaccinated dogs were immune to the parasite in the sense that they did not acquire the heavy burdens ofAncylostoma caninum which would result in pathologic hookworm disease. They were, however, not immune to infection per se and acceptance of the vaccines by veterinarians suffered because vaccinated dogs were found to have Ancylostoma caninum eggs in the faeces, even though the dogs were not suffering associated pathology. This, together with the cost of production of the vaccine and short shelf-life, saw the withdrawal of the commercial vaccine after only 2 years.

6. Vaccines against protozoan parasites

6.1. Babesiosis The haemoprotozoan parasites Babesia boris and Babesia bigemina are economically important cattle pathogens transmitted by ticks. The immunology of these infections has been reviewed by De Vos et al. ( 1987 ) and Wright (1990). Cattle which have recovered from infection with the parasites show strong resistance to clinical babesiosis on subsequent infection (Wright, 1990). Early at-

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tempts to 'vaccinate' cattle used deliberate infection with virulent parasites from 'bleeder' cattle, which had recovered from babesial infection but retained some parasites in the blood (Pound, 1897 ). Commercial vaccines became available for both species of parasite after it was discovered that they could be attenuated by sequential passage through splenectomised calves (Callow and Mellors, 1966; Callow, 1976; Dalgliesh et al., 1981 ). Attenuation of Babesia boris is achieved by syringe passage between 20 and 30 times. Vaccines are prepared containing 107 parasites. The vaccines must be stored refrigerated and used within 1 week. Some progress had been made with the use of cryopreserved vaccines (Jorgensen et al., 1989; Dalgliesh et al., 1990), in an effort to overcome the constraints imposed by the short shelf life and stringent storage conditions required for the live vaccine. In Australia, where the vaccines have been used most widely, as many as 1.4 × 106 doses have been supplied in a single year (Callow, 1979 ). The live attenuated Babesia vaccines have been highly effective, but they have suffered from many of the disadvantages of live vaccines, listed in Table 3 (Wright, 1990). The potential for the development of a vaccine incorporating non-living antigens was indicated by the successful vaccination of cattle against Babesia boris by Mahoney (1967) using a preparation of killed parasites. Also, cattle were successfully immunised against Babesia boris using a culture-derived soluble antigen (Smith et al., 1981 ). This discovery led to vaccine trials with culture-derived antigens in cattle in Venezuela (Montenegro-James et al., 1989 ), and the production and commercial marketing in France of a vaccine, Pirodog, against Babesia canis infection. However, the effectiveness of these vaccines has been questioned. Timms et al. (1983) were unable to induce immunity in cattle with the culturederived Babesia boris vaccine. Lepetit ( 1988, cited by Schetters et al., 1992 ) found that under field conditions the Babesia canis vaccine was only 26.3% effective (clinical babesiosis was detected in 12.3% vaccinated dogs compared with 16.7% in the total dog population over the same period). Wright and his colleagues have pursued the goal of developing a defined-antigen vaccine against Babesia boris for more than a decade. Some 80 vaccination trials were carried out in cattle using fractionated Babesia antigens. This led to the successful isolation of several recombinant, host-protective antigens. Some information concerning the development of this vaccine has been published in various conference and workshop proceedings (Riddles et al., 1990; Gale et al., 1990, 1992) and review-type publications (Wright, 1991; Wright et al., 1992). Unfortunately, the details of vaccine trials using recombinant antigens have not yet been published in the scientific literature. Native Babesia boris antigens were fractionated, particularly via affinity chromatography with monoclonal antibodies, and vaccine trials with the fractions identified several host-protective native antigens (Wright et al., 1983, 1985; Goodger et al., 1985). Three of these antigens have been cloned and the cloned antigens shown to induce host-protective immunity in cattle against both experimental challenge infection and field-derived infections with Babesia boris (Gale et al., 1992). The cloned 1 IC5 antigen was detected by immunoassay with a monoclonal antibody. A cDNA clone expressing another antigen, designated

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12D3, was isolated by hybridisation with oligonucleotides designed using information from partial amino acid sequencing for the native antigen. Information concerning the cloning of a third host protective antigen, 21B4, has not been published in the scientific literature. Commercialisation of a vaccine based on these recombinant antigens has been delayed as Freund's complete adjuvant is needed to achieve a reliable host-protective immune response with the antigens (I. Wright, personal communication, 1993 ). Research is underway to identify an effective but commercially acceptable adjuvant for the vaccine. 6.2. Theileriosis Parasites of the genus Theileria are blood-borne protozoans transmitted by ixodid ticks. The diseases they cause are a major factor limiting the productivity of cattle industries in many parts of Africa, Asia and the Middle East. Indigenous breeds of cattle in these areas show significant natural resistance to infection and disease. Non-indigenous breeds, however, suffer severely from the parasites, with mortality at times reaching 100% (Pipano, 1981; Irvin and Morrison, 1987 ). The pathology is caused by hypertrophy of lymphoid tissues associated with intracellular parasitism with the schizont life cycle stage of the parasite (Irvin and Morrison, 1987). The most pathogenic and most important species infecting cattle are Theileria parva and Theileria annulata, the causative agents of East Coast Fever, and Tropical Theileriosis or Mediterranean Coast Fever, respectively. Vaccination against both Theileria parva and Theileria annulata has been achieved with some degree of success by deliberate controlled infection of cattle with virulent sporozoite or schizont stage parasites, sometimes followed by chemotherapy to control the vaccine-induced infection (reviewed in Irvin and Morrison, 1987 ). Methods for the continuous in vitro culture of macroschizonts of Theileria annulata were developed (Tsur, 1945; Tsur and Adler, 1963 ) and led to the production of a vaccine by Pipano and Tsur ( 1966 ) based on passage attenuated parasites. The vaccine has been used widely and successfully, is safe in all breeds of cattle and provides at least some protection against challenge with parasite isolates from different areas (Hashemi-Fesharki, 1988; Irvin and Morrison, 1987). Attempts to use non-living parasite preparations of the vaccine have, however, met with only limited success (Pipano et al., 1977; Irvin and Morrison, 1987). A sporozoite surface protein of Theileria annulata has been cloned in Escherichia coli and antibodies induced by this antigen inhibited the entry of sporozoites into erythrocytes in vitro (Williamson et al., 1989). Procedures similar to those adopted for Theileria annulata have been applied in the production of a culture-attenuated vaccine against infection with Theileria hirci, a highly pathogenic parasite of sheep and goats (Hawa et al., 1981; Hooshmand-Rad, 1985). Sheep inoculated with schizonts from parasites derived from the third passage in tissue culture developed severe febrile and parasitological reactions. However, parasites from the 30th or 63rd passage had reduced virulence but induced protection against subsequent challenge with virulent parasites.

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Much effort has been expended in research towards the culture of Theileria parva, with the aim of producing a vaccine similar to that for Theileria annulata. Success has been achieved with culture of the parasite but, for vaccine purposes, problems have arisen due to the poor transfer rate of parasites from inoculated cells in the vaccine into the host's own cells. This transfer is necessary in order to stimulate immunity in the vaccine recipient (Irvin and Morrison, 1987 ). A Theileria parva sporozoite surface antigen of 67 kDa has been identified as the target ofa monoclonal antibody that shows sporozoite neutralising activity in vitro, and this antigen has been cloned from sporoblast mRNA (Nene et al., 1992 ). The gene encoding this antigen was isolated by a combination of immunoscreening and DNA hybridisation using oligonucleotides derived from partial protein sequencing of purified p67. Sequence encoding the full recombinant protein comprising p67 was assembled on a 2.3 kb cloned DNA fragment and the antigen expressed as a GST fusion protein (Nene et al., 1992). The GST fusion protein was highly unstable so an alternative vector, in which the antigen is expressed as a fusion protein with part of the NSI protein of influenza virus A, was used (Musoke et al., 1992 ). Cattle were immunised with this antigen and saponin on five occasions and then challenged with virulent Theileria parva sporozoites. Of nine immunised cattle, six were protected against the challenge infection. Modification of the antigen and optimisation of the vaccination regime might provide protection for a larger proportion of vaccinated cattle. This could form the basis of a practical vaccine. 6.3. Coccidiosis in chickens

Seven common species of Eimeria cause substantial production losses in the poultry industry. Infections are controlled by chemotherapy, but the parasites have developed resistance to anticoccidial drugs, including the ionophorous antibiotics. Birds which have survived and recovered from infection are immune to re-infection, although this is substantially restricted to re-infection with the homologous parasite species (Rose, 1987 ). The vaccines against Eimeria spp., Coccivac and Immunocox, were developed based on the administration of a limited number of virulent oocysts. Problems with this method of vaccination arise where birds fail to ingest sufficient oocysts from the immunising dose and remain susceptible to infection with the large number of progeny from the 'vaccine' dose (Shirley, 1992 ). Similar strategies have involved infection with virulent oocysts followed by drug cure of the immunising infection (Rose, 1987 ). Problems with drug resistance and the expense involved in coccidiosis control have encouraged research into the development of both living, avirulent and nonliving parasite vaccines. Substantial success has been achieved with the production of avirulent strains of Eimeria by sequential selection of parasites which show the most rapid development in vivo. Parasite lines selected for such precocious development show greatly reduced pathogenicity (Jeffers, 1975 ), however they retain their ability to stimulate protective immunity against subsequent challenge

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with virulent parasites (Shirley, 1989; Shirley, 1992). Commercial vaccines (Paracox) based on precocious lines are becoming available (Shirley, 1992 ). Of all the parasitic infections of animals, coccidiosis in chickens has been probably the subject of the greatest monetary investment towards recombinant vaccine development. To date more papers have been published on vaccination with recombinant antigens against Eimeria in chickens than the sum total of those on all other animal parasites. Regrettably, these have not led to the degree of success which would enable them to compete effectively with either chemotherapy or vaccines such as Paracox. Recently there has been contraction of research and development effort on recombinant vaccines against coccidia. Nevertheless, there has been substantial progress made with cloning antigens capable of inducing partial protection. Jenkins and his colleagues at the US Department of Agriculture laboratories, Beltsville, characterised the immunodominant surface antigens of Eimeria acervulina sporozoites and merozoites (Jenkins et al., 1988 ). Two cDNA clones were selected from 2gt I 1, libraries expressing epitopes of a p240/p 160 sporozoite antigen (s) and p250 merozoite antigen. Each was detected in plaque immunoassay using rabbit anti-sera against sporozoite membranes and merozoite membranes, respectively. The polypeptide derived from the p250 merozoite antigen was recognised by sera from immune chickens and was shown to stimulate specific Tcell activation in vitro. This 35 kDa parasite-encoded polypeptide was able to induce partial protection in chickens against challenge infection with sporulated oocysts of Eimeria acervulina. Chickens were vaccinated per os with 109 live recombinant Escherichia coli expressing this antigen. This induced both antibody and cell-mediated immune responses to the recombinant antigen, and primed the chickens for an accelerated response to p250 following parasite challenge (Kim et al., 1989). Oocyst production in the vaccinated birds was decreased 30% compared with controls. Subsequent experiments used an expression vector and host bacterium capable of controlling in vivo expression of the antigen (Jenkins et al., 1991 ), and achieved significant protection against challenge infection in vaccination trials, reflected in reduced weight loss and lower intestinal lesion scores in vaccinated birds compared with controls. Danforth et al. (1989) used sera from chickens experimentally infected with Eimeria tenella to screen a cDNA library prepared from Eimeria tenella oocysts. One clone, designated 5401, was selected and shown to express a fl-gal fusion protein containing 31 kDa of parasite encoded protein. The corresponding native sporozoite antigen was identified in Western blots as two bands of 66 and over 200 kDa. Chickens immunised subcutaneously with the fusion protein in Freund's complete adjuvant were protected significantly against challenge infection with Eimeria tenella and had decreased lesion scores and mortality and increased individual weight gains compared with non-vaccinated controls. Researchers at the Merck Sharp and Dohme laboratories took a different approach to identify antigens of Eimeria tenella. Sporulated oocysts and isolated sporozoites were sonicated and extracted with Zwittergent 3-10. The crude solu-

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ble material was fractionated by S-200 chromatography and fractions used in immunisation trials in chickens (Karkhanis et al., 1991 ). A protective fraction containing molecules with a limited range of molecular weight was identified and antisera raised against this fraction were used to isolate cDNA clones expressing antigens in the host-protective fraction (Karkhanis et al., 1991 ). One antigen, designated S07' was able to induce partial protection against challenge infection with Eimeria tenella, and cross-protection against infection with Eimeria acervulina, Eimeria maxima and Eimeria necatrix. Birds vaccinated and protected with the recombinant antigen injected intramuscularly without adjuvant did not produce antibodies against the vaccine antigen, suggesting that cell-mediated immune mechanisms were responsible for host-protection Research teams working with A.H. Robbins Co. and Genex Corporation were also successful in achieving partial protection against challenge infection with Eimeria tenella using a recombinant sporozoite antigen (Miller et al., 1989). A monoclonal antibody raised against a sporozoite antigen of Eimeria acervulina was used to screen a cDNA library prepared from Eimeria tenella sporulated oocysts. A clone designated GX3262 was selected and found to express 12 kDa of parasite encoded protein, the native equivalent of which was a 28 kDa sporozoite antigen. Partially purified protein (a fl-gal fusion), heat killed and live recombinant Escherichia coli were tested in vaccine trials against challenge infection with Eimeria tenella. Live bacteria produced the better results. Bhogal et al. ( 1992 ) then showed that the antigen also induced cross protection against challenge infection with Eimeria acervulina. Additional data concerning the cloning of Eimeria antigens, including the vaccination trials referenced in various patents, can be found in Ellis and Johnson (1992). It is difficult to predict the future for recombinant vaccines against coccidiosis in chickens. As mentioned previously, the major animal health companies have reduced their level of research input in this area. A number of factors will need to be addressed before recombinant vaccines could rival chemotherapy or live vaccines, e.g. Paracox, for control of coccidiosis. These include improvement in the level of protection achieved with each of the antigens tested to date, difficulties associated with reliable and inexpensive delivery of the vaccine, and antigenic variation within and between species.

6.4. Toxoplasmosis in sheep A live, attenuated parasite vaccine against Toxoplasma gondii has been produced in New Zealand (O'Connell et al., 1988 ). The vaccine is used in ewes and results in a significant increase in the number of live lambs born to vaccinated animals compared with non-vaccinated control sheep (O'Connell et al., 1988; Wilkins et al., 1988) presumably through vaccine-induced prevention of Toxoplasma-associated abortion. In addition to increasing the number of live lambs, lambs from vaccinated ewes are significantly less likely to be congenitally infected with Toxoplasma gondii compared with lambs from control ewes. The vac-

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cine derives from an isolate of the parasite that originated from an aborted sheep foetus and has been maintained entirely in the tachyzoite stage since 1958 by twice weekly intraperitoneal passage through mice (Wilkins et al., 1987). The strain, designated Strain 48, has lost its capacity to form oocysts in cats. Sheep are vaccinated intramuscularly with 10 6 o r l 0 4 viable Strain 48 tachyzoites, derived from the peritoneal exudate of infected mice. The vaccine, Toxovax, has been marketed commercially in New Zealand since 1988 (Wilkins and O'Connell, 1992 ). 7. Vaccines against ectoparasites

7.1. Tropical cattle tick The tropical cattle tick, Boophilus microplus, is an economically important ectoparasite of cattle in many parts of the world. Heavy infestations result in direct production losses due to the loss of blood ingested by the parasite, and damage to the hide. Indirectly, Boophilus microplus causes life-threatening diseases through transmission of the haemoprotozoan Babesia spp. parasites and the intraerythrocytic rickettsia, Anaplasma marginale. Practical control of tick infection is achieved with chemical acaricides, with the use of cattle breeds showing natural resistance to the parasite, and by farm management practices employed to limit the accumulation of tick larvae on pasture. The development of resistance to acaricides is a major problem in the control of ticks. Peter Willadsen and his colleagues at the Commonwealth Scientific and Industrial Research Organisation and Biotechnology Australia Pty. Ltd., have investigated the control ofBoophilus microplus by vaccination. Painstaking research on fractionation of host-protective antigens resulted in the identification of a hostprotective tick glycoprotein (Willadsen et al., 1988, 1989). Subsequently, a full length cDNA clone was isolated from a 2gt 11 cDNA library prepared from adult tick mRNA (Rand et al., 1989 ). The clone was isolated using DNA hybridisation to oligonucleotide primers prepared using information from partial peptide sequencing of the native Bm86 antigen. Cattle vaccinated with a fusion protein which comprised a nearly full length recombinant copy of Bm86, were protected significantly against challenge infection with ticks, reflected in reduced tick numbers, weight and fecundity. Recently, 89% and 91% reductions have been achieved in the reproductive capacity of ticks on cattle vaccinated with Escherichia coli- or baculovirus-expressed antigen, respectively (Tellam et al., 1992). For more detailed discussion of this vaccine the reader is referred to another paper in this series. 8. Vaccination with novel antigens The Boophilus microplus vaccine is the most successful example to date of a vaccine based on immunisation with a parasite molecule which is not normally

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antigenic in animals infected with the parasites. The concept of using such antigens for the purposes of controlling haemophagus arthropods was raised some time ago, for example, for the control of mosquitoes (Alger and Cabrera, 1963 ), stable flies (Schlein and Lewis, 1976) and ticks (Allen and Humphreys, 1978). Mitchell (1984) and others (O'Donnell et al., 1989; Sexton et al., 1990 ) referred to these as 'novel antigens'. Others used the term 'concealed antigen' (Willadsen and Kemp, 1988; WiUadsen and McKenna, 1991 ). The former is preferred by the author for reasons of teleology, in that it cannot be interpreted as inferring intent on the part of the parasite. Application of this concept to vaccination against parasites expands the range of target antigens available to the immunoparasitologist (Willadsen et al., 1993 ). In addition to the outstanding success achieved with vaccination against Boophilus microplus using a novel antigen, research on other parasitic organisms, particularly haemophagus parasites, has made substantial progress towards the development of vaccines against other arthropod parasites, as well as nematode and trematode parasites. Vaccines based on novel antigens could be expected to have the disadvantage that the immunised animals would not have their immunity boosted by natural exposure to the parasite. However, the approach may have some advantages in, for example, the induction of vaccine-induced immunity in young animals at an age at which naturally acquired immunity from infection does not occur (Munn et al., 1987; Tavernor et al., 1992a; Smith, 1993).

9. Successful approaches to vaccine antigen identification and cloning The majority of the successful, defined-antigen vaccines which have been described to date have followed a series of steps in development of the vaccine: ( 1 ) demonstration of host-protective immunity with parasite extract or product; (2) identification of the host-protective native molecule ( s ); (3) cloning and expression of cDNA encoding the protein component of hostprotective antigens; (4) vaccine trials with recombinant antigens. Exceptions to this general scheme have been the p67 Theileria parva and Eimeria vaccines, where antigens were identified for cloning based on their location as surface antigens on parasite stages known to be the targets of host-protective immune responses. Two approaches have been used, with approximately equal effectiveness, in the identification of the cloned-equivalents of native antigens: immunoscreening (e.g. Taenia ovis 45W, F. hepatica GSTs) and hybridisation with redundant oligonucleotides synthesised on the basis of a knowledge of part of the protein sequence of the host-protective antigen (e.g. Boophilus microplus Bm86, Babesia boris 12D3). Introduction of polymerase chain reaction technology will provide an additional method for the isolation and cloning of DNA sequences encoding parasite vaccine antigens. As the number of recombinant antigen vaccines increases, so also will the pos-

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sibility of using knowledge of the identity of host protective antigens in one species to isolate the homologous antigens and genes from related species. The first example of the successful application of this method for identifying vaccine antigens has been provided by T. Spithill and his colleagues in the isolation of hostprotective GSTs ofF. hepatica. 10. Prospects for additional recombinant antigen vaccines Many studies have identified specific native antigens or antigen fractions which can be used to induce host-protective immunity. The reader is referred to other recent reviews (Yong, 1992b; Warren and Agabian, 1993) for detailed discussions of this topic. Some examples warrant particular mention because of the prospects they hold for extension into practical vaccines. Novel antigens of Haemonchus contortus associated with the parasite gut have been shown to be capable of inducing host protective immune responses in vaccinated sheep (Munn et al., 1987; Tavernor et al., 1992a,b; Smith, 1993 ). Possibly the most effective of these is an antigen termed H 11, an integral membrane glycoprotein present in the intestinal microvillar membrane of parasitic stages of H. contortus (Tavernor et al., 1992a,b). In SDS-PAGE the antigen runs as a doublet at Mr 110 kDa. Immunisation of sheep with a fraction called 'Concanavalin A binding proteins', 85% of which consists of H1 l, induced very high levels of protection against challenge infection with H. contortus (Tavernor et al., 1992a ). In addition, immunity was generated in young lambs at an age at which immunity does not normally occur (Tavernor et al., 1992b). Some information on the characterisation of the antigen is available from a presentation made recently at a Keystone Symposium (Munn et al., 1993 ). The full length cDNA encoding H 11 has been cloned, and the antigen found to have homology with mammalian microsomal aminopeptidase. The level of amino acid identity was about 32%, and similarity based on like residues about 51%. The native antigen fraction containing H 11 was found to possess enzymatic activity consistent with H 11 being an aminopeptidase. The conference presentation reported expression of the fulllength protein via baculovirus in Sf9 cells. Vaccine trials with baculovirusexpressed antigen are in progress at present and the results are awaited with eager expectation. Native antigens from other nematode parasites of sheep have also been identified and were capable of eliciting host-protective immune responses (Frenkel et al., 1989; O'Donnell et al., 1989; Savin et al., 1990; Dopheide et al., 1991; Frenkel et al., 1992; McGillivery et al., 1992; Tavernor et al., 1992a,b). However, no reports yet describe host-protective immunity using recombinant equivalents of these antigens. Willadsen et al. (1993) refer to vaccine trials against myosis in sheep due to Lucilia caprina. A novel antigen PM44, associated with the peritrophic membrane of the larval gut, was used to immunise sheep and the rate of growth of parasitic first instar larvae was inhibited. Trials with recombinant antigens are

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underway. Identifications of other potential host-protective antigens for vaccination against myosis are reviewed by Sandeman ( 1992 ).

11. Concluding remarks The future is bright for the control of many parasitic diseases using recombinant antigen vaccines. Within about a decade of the introduction of recombinant DNA techniques to parasitology, publications have described significant levels of protection against infections with seven species of parasites infecting animals of economic importance (Table 2) and also significant protection against human parasites causing schistosomosis (Capron et al., 1992 ) and malaria (Good et al., 1993). At least two of the vaccines for animals are near to commercial release. Additional vaccines will be developed using similar procedures to those which have led to the current recombinant vaccines and an ever increasing option to target vaccine candidates will be the use of protein homologues of those antigens that are protective against a related species of parasite. The first successful application of this method of antigen targeting is the use of F. hepatica GSTs for vaccination against liver fluke in sheep and cattle. Although many of the recombinant antigen vaccines are successful experimentally, none have yet been tested by practical application. Immunological and commercial constraints may require that many of the vaccines described to date show improved, or at least long-lasting, levels of protection in order for them to be of value as commercial vaccines. Some have particular problems with marketing. For example, the farmers who would need to use, and presumably pay for, the Taenia ovis vaccine do not generally bear the direct cost incurred as a result of the parasite being detected in carcass meat (Lightowlers et al., 1992). Novel mechanisms for marketing the vaccine (not novel antigens) will be necessary in this instance to encourage farmers to spend money on a vaccine against a disease which does not appear to cost them anything. The same problems could be expected to apply to vaccines against zoonotic parasites such as Echinococcus granulosus, Taenia solium, Taenia saginata and Trichinella spiralis. For parasites such as these, control campaigns incorporating vaccines as part of the strategy would probably require legislative backing and government subsidisation of the cost of the vaccine in order to ensure that the vaccines were used. Another example where marketing may be difficult, particularly initially, is the Boophilus microplus vaccine. Vaccinated cattle will not show an immediate loss of tick parasites. Also, even after the immune response has peaked, a significant proportion of the parasites will remain on the animals. The true benefits of the vaccine will not be realised until it has been used over several life cycle generations, after which the reduced fecundity of parasites from vaccinated cattle will result in a decreased number of infective parasites in the environment and hence, a generalised decrease in tick burden. Another major challenge to those involved in research into vaccination against parasitic infections in animals is to develop improved methods for the delivery

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of the vaccines. Delivery of vaccines without syringes would increase the likelihood of anti-parasite vaccines realising their full potential, particularly in developing countries. Vaccines against diseases such as cysticercosis in pigs and cystic hydatid disease are examples which might be adopted more widely if injection were not required. Live recombinant organisms for delivery of vaccines, e.g. attenuated Salmonella strains or Pox viruses (Esposito and Murphy, 1989; Dougan et al., 1989; Boyle and Radford, 1992) have great prospects for development of vaccines which could be administered orally. Another potential method for vaccine production could be the expression of vaccine antigens in recombinant plants (Ow et al., 1986; Mason et al., 1992). The vaccine antigen could be delivered if animals to be vaccinated were fed or grazed on the recombinant plants. Induction of immunological tolerance to antigens given via the oral route presents a formidable but not insurmountable obstacle to the use of such a strategy. The challenges for the immediate future in vaccination against animal parasites are translation of the present defined-antigen vaccines into successful commercial vaccines, creation of vaccines against other parasite species and the production of vaccine delivery systems for achieving effective, low cost, long lasting protection with a single vaccine dose. Progress in the area of vaccine delivery is likely to determine the future for practical application of many experimentally successful vaccines against animal parasites.

Acknowledgements The skilful assistance of Kerri Runnalls in word processing is gratefully acknowledged. Research in the author's laboratory is funded by the National Health and Medical Research Foundation of Australia.

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