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Progress in recombinant vaccine development against coccidiosis A review and prospects into the next millennium
International Journal for Parasitology 17 "0887# 0010Ð0029
Progress in recombinant vaccine development against coccidiosis A review and prospects into the next millennium A[N[ Vermeulen Department of Parasitology\ Intervet International BV\ PO Box 20 4729 AA Boxmeer\ The Netherlands Received 2 December 0886^ accepted 09 March 0887
Abstract The increasing problems encountered by the poultry industry\ despite the extensive use of drugs\ have emphasised the need for an immunological solution for the economic damage caused by the Eimeria parasite[ Although immunity develops relatively fast following a natural infection\ to induce protection by using parasite extracts or single antigens appears more di.cult[ Nevertheless\ the development of a vaccine based on de_ned antigens seems the best solution in the long run[ At the VIth International Coccidiosis Conference in 0882 the _rst promising results were reported from small!scale experiments using recombinant antigens[ This review summarises the advances in this _eld of research from 0882 onwards[ Although since then not many reports have been published about the e}ects of using recombinant antigens as a vaccine against coccidiosis\ a number of interesting new proteins which could be considered good targets for such a vaccine have been described and are referred to herein[ Proteins involved in the process of invasion of the host cell by the extracellular parasite are regarded as key components in the developmental cycle of the parasite[ These components possibly bind to receptors on the host cell[ Interference with this process could be a target of the protective immune response[ Progress has also been made in characterising the immune mechanisms activated by infection with the parasite[ From experimental mouse models and from studies in chickens\ a better insight has been obtained towards the involvement of CD3! and CD7!type T cells in\ respectively\ the inductor and the e}ector branch of the immune response\ although not all questions have been answered[ Several antigens have been selected using T!cell stimulation and cytokine assays and these are reviewed[ In a third section\ mostly unpublished results of our own experiments dealing with the use of live vectors to present de_ned antigens such as Ea0A and EaSC1\ a parasite refractile body transhydrogenase and a lactate dehydrogenase\ respectively\ are summarised[ Partial protection could be induced using Salmonella typhimurium as a carrier for these antigens\ in that the oocyst output was reduced by up to 49) after challenge and weight gain could be improved by 4Ð09) over non!vaccinated challenged chickens\ when tested in a ~oor!pen trial[ Similar results were obtained when these antigens were presented by viral vectors such as Fowlpox virus or Herpes virus of turkey[ These data seem to o}er good prospects for the accomplishment of a safe and e.caceous vaccine based on recombinant DNA technology[ These expectations are corroborated by recent breakthroughs in transfection of related parasites such as Plasmodium and Toxoplasma gondii\ and by the increasing amount of genomic information becoming available every day\ the impact of which cannot even be estimated yet[ These new technologies will allow us to solve the complex problems that we once created ourselves[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Key words] Eimeria^ Coccidiosis^ Recombinant DNA^ Salmonella typhimurium^ Herpes virus of turkey^ Fowlpox^ Vaccine^ Review
Tel[] ¦20[374[476269^ fax] ¦20[374[476228^ e!mail] arno[vermeulenÝintervet[akzonobel[nl[ S9919!6408:87 ,08[99¦9[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Printed in Great Britain PII] S9919!6408"87#99979!9
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0[ Introduction Ending a comprehensive review at the VIth Inter! national Coccidiosis Conference in Guelph\ Canada in 0882\ Dr Harry Danforth felt positive about the future development of a recombinant vaccine ð0Ł[ This was based on the partial "cross! species# protection that could be induced using gen! etically engineered subunit material in the presence of adjuvants and the assumption that\ by using live! vector systems\ the antigenic presentation would become more optimal and protection could be improved[ This optimism\ however\ would not exclude that more antigens should be screened in order to obtain better protective candidates[ Now\ 3 years later\ we have to conclude that the e}orts to obtain such a vaccine have become less and only a few publications have dealt with the induction of protective immunity by the admin! istration of recombinant vaccines[ This reduced level of progress is caused by the complexity of the matter and the high costs involved in performing such studies[ On the other hand\ there is certainly a future for a recombinant vaccine[ The increasing number of reports about lowered e.cacy of the available drugs "reviewed by Chapman ð1Ł# and the apparent success of live "attenuated# vaccines "reviewed by Shirley and Bedrnik ð2Ł# warrant further inves! tigations towards a safe\ easy to produce\ cost! e}ective vaccine[ Since live vaccines still bear the risk of inducing pathology and have the dis! advantage of high production costs\ this new vac! cine should be based on recDNA technology that is available to date and will be developed further in the next millennium[ In this review\ a summary will be given of the _ndings that have been made public\ and some of the work that has been performed in our laboratory concerning these aspects will be highlighted[ Progress has been made in the _eld of cell biology\ where new proteins have been dis! covered*some of which showed homology to known proteins and others appeared unique but were targeted to speci_c organelles\ which could suggest aspects of their function[ Although most of these proteins were discovered without the intention of developing a vaccine\ their
speci_c function or localisation could make them good vaccine targets\ for example\ proteins involved in the essential steps of invading the host cell[ These proteins are localised either on the sur! face of the extracellular parasite\ the sporozoite or merozoite\ or secreted by the rhoptries and micro! nemes during the process of invasion[ Dense gran! ule "DG# proteins are also thought to be good targets\ since the contents of DG are deposited into the vacuolar space or inserted in the parasito! phorous vacuole membrane "the actual interface between parasite and host#[ These proteins might be presented on the surface of the parasitised cell in the context of major histocompatibility complex molecules[ In the past 3Ð4 years\ emphasis has also been given to studies of the immune mechanisms under! lying the protective immune response to infection[ T!Cell mediated immune mechanisms are essential for solid protection ð3Ł[ Both CD3¦ and CD7¦ T! cell subsets are involved\ whereby the former is mainly responsible for the proliferative response that leads to an expanding memory cell pool and for the production of helper cytokines to stimulate CD7¦ cells to act as e}ector cells[ Interferon!g is one of the cytokines produced by both subsets and may act directly on the developing parasite ð4Ł[ By applying this knowledge\ methods have been developed to study antigens involved in stimulating the potentially relevant pathways of the immune system and improvement in aa and DNA sequ! encing have facilitated their characterisation[ Some of the antigens that have been identi_ed from the application of these techniques will be mentioned below[ In the last part of this review attention will be given to the vaccine carrier systems examined in our laboratory and their possible applicability for presenting Eimeria genes to the immune system[ Di}erent antigen preparations and vector sys! tems will be discussed in the context of the level of protection being induced[ The parameters to mea! sure protection have always been a matter of debate[ An obvious parameter would be the reduction of the proliferation of the parasite to be measured by the reduction of oocyst numbers in the faeces[ This\ however\ is a very laborious e}ort due to large variation between individual chickens[
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Furthermore\ it is not clear what the impact of a partial reduction in oocyst output would mean for the practical situation in the _eld[ Thus many inves! tigators use the extent of pathology "lesion scores#\ or weight gain of meat!type chickens over a speci_c time period as alternative readouts[ These par! ameters are more indirect indicators and could be biased by secondary factors ð5Ł[ Due to this incon! sistency in the methods to validate the usefulness of individual antigens or vector!based vaccines\ it is hard to compare results published by di}erent investigators[
1[ Identi_cation of Eimeria antigens 1[0[ Proteins involved in host!cell invasion Following advances in the description of Toxo! plasma gondii invasion!related proteins\ Eimeria organelles\ such as rhoptries\ micronemes and dense granules have been studied in more detail and di}erent proteins have been described which were targeted speci_cally to any of these sites ð6Ł[ A mic! roneme!associated\ thrombospondin!related pro! tein of Eimeria tenella\ designated as Etmic!0 "Et099#\ was described by Tomley et al[ ð7Ł[ This protein had homologues in other species and even genera[ Pasamontes et al[ ð8Ł described a similar protein in Eimeria maxima with a high aa sequence homology to Et099\ and similar proteins are present in Plasmodium\ Cryptosporidium and tachyzoites of T[ gondii ð6\ 09Ł[ The Eimeria proteins are expressed in sporozoites and late in schizogony and\ apart from their role in invasion\ are possibly also involved in movement and escape of the merozoite from the schizont!infected cell "Vermeulen\ unpub! lished observations#[ A second microneme!associated protein\ desig! nated as Etmic!1\ was described recently as being a 49!kDa protein from E[ tenella sporozoites ð00Ł[ This protein could also be detected on the surface of the sporozoite prior to invasion and\ intriguingly\ also on the surface of recently invaded host cells "Tomley\ see this volume#[ This may therefore be an interesting candidate to follow up[ Tomley ð01Ł characterised rhoptry proteins of E[
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tenella and found that the pattern of proteins pre! sent in these organelles was much more complex than in micronemes[ The rhoptry protein pattern of di}erent developmental stages of this species was highly variable[ These aspects have complicated the characterisation of rhoptry!associated proteins in Eimeria[ Although the function of dense granules in Eimeria has not been studied as much as in Toxoplasma\ they are considered to be involved in exocytosis of parasite material after invasion[ Entzeroth et al[ ð02Ł described a 054!kDa protein of Eimeria papillata[ Monoclonal antibodies localised the protein in dense granules of the sporozoites\ but it was also apparent in the host!cell cytoplasm[ No nucleotide or aa sequence data were published[ A _bronectin!related protein was described by Lopez!Bernad et al[ ð03Ł[ The 009!kDa protein was localised on the surface of E[ tenella sporozoites and could be involved in attachment or invasion of the host cell[ A similar protein was found in Leishmania parasites ð04Ł[ None of these proteins has been really tested for immunogenicity and e.cacy as a vaccine[ These antigens\ however\ are potentially good candidates for a future recombinant vaccine based on their function and associated localisation and should therefore be evaluated more closely[
1[1[ Refractile body proteins Several proteins have been described that are associated with the refractile bodies of the sporo! zoite[ The function of these bodies is not known and it has not even been shown yet that the refractile body is an organelle with a distinct membrane[ The twin bodies in the sporozoite fuse shortly after invasion of the host cell and the formed body gradu! ally disintegrates during the process of schizogony[ Every _rst!stage merozoite carries some of the material\ but after this stage it cannot be detected[ The _rst refractile body!associated protein described was the 15!kDa RB0\ also known as GX2151 ð05Ł or EtSO6 ð06Ł\ having homologues in di}erent avian Eimeria species but also in Eimeria bovis\ designated as Eb14:49 ð07Ł[ The function of this family of proteins is not known\ but vaccination
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with the native or recombinant protein signi_cantly reduced the pathology by about 49)\ as measured by lesion scores\ after challenge with four di}erent species ð0\ 08Ł[ O|Lorcain et al[ ð19Ł have described a few possible B!cell epitopes of GX2151[ These could be used within a vaccine composed of a com! bination of di}erent oligopeptides derived from several antigens[ Vermeulen et al[ ð10Ł described an Eimeria acer! vulina 099!kDa "designated as Ea0A# refractile body protein which demonstrated sequence hom! ology with a nicotinamide!nucleotide!dependent transhydrogenase[ The partial gene could be expre! ssed in di}erent hosts and appeared to signi_cantly reduce oocyst output of vaccinated chickens ð11Ł and reduce weight gain loss of vaccinated chickens after challenge "see below#[ The protein is probably also involved in carbohydrate transport as it con! tained a sequence signature for hexose! trans! porters[ This assumption is corroborated by the observed strong association of amylopectin gran! ules with the refractile bodies[ This\ therefore\ sug! gests a role of refractile bodies in energy storage and metabolism[ Kramer et al[ ð12Ł described the full!length sequence for the homologue of this pro! tein in E[ tenella[ An aspartyl proteinase of 32 kDa "known as 5S1# has been shown to be associated with the refractile bodies of E[ acervulina sporozoites ð13Ł[ The protein has homologues in other species\ such as E[ bovis[ Until now\ these proteins have not been used in vaccination trials[ mRNA of 5S1\ Eb14:49 and Ea0A was found to be transcribed during the pro! cess of sporulation\ but the proteins could be detected up to the _rst merozoite stage "Clark and Vermeulen\ unpublished observations#[ From these results it is clear that refractile body! related proteins\ like microneme!associated proteins\ also show a highly conserved antigenic repertoire among Eimeria spp[ from mammals to avians[ In those cases where protection data have been published\ immunity appeared to cross the species barrier ð0\ 08Ł[ Apparently the immunity induced by these antigens is di}erent from the immunity induced by a live infection\ which is spec! ies!speci_c[ Therefore these proteins should be con! sidered as very promising candidates for a recombinant vaccine[
2[ Developmental stage antigens 2[0[ Asexual stage antigens From earlier observations it was shown that developmental!stage antigens could be crucial for the induction of protective immunity\ especially fol! lowing a live infection ð3\ 14Ł[ Di}erent inves! tigators have pursued proteins from merozoites or trophozoiteÐschizont stages[ Binger et al[ ð15Ł described a 22!kDa merozoite protein "Mzp4Ð6# in E[ tenella which was expressed by recombinant Vaccinia virus and the construct was shown to induce antibodies after vaccination of chickens[ No protection data were published[ From our own work using E[ acervulina\ a set of four merozoite!associated antigens "Eam199\ Eam099\ Eam34 and Eam19# was found[ The latter two antigens were detected on the surface of the 1ndÐ2rd generation merozoites "harvested 61 h p[i[#\ whereas the _rst two antigens were soluble cytoplasmic proteins as determined by TX003 extraction and phase separation as described by Bordier ð16Ł[ Vaccination of chickens using the native proteins Eam34 or Eam19 puri_ed by mAb! immuno!a.nity chromatography and adjuvanted with Quil A "Superfos\ Vedbaek# resulted in sig! ni_cant reduction of oocyst output "Table 0#[ In this experiment groups of 19 SPF White Leghorn chickens were vaccinated three times s[c[ at weekly intervals and challenged 1 weeks after the _nal booster with 199Ð499 E[ acervulina sporulated oocysts[ Oocyst output was measured from each chicken individually and compared with the controls[ The mean oocyst outputs of vaccinated
Table 0 Immunisation of chickens using E[ acervulina developmental stage antigens
Vaccine
) oocyst output of controls2S[D[
Eam34 19 mg¦049 mg Quil A Eam19 19 mg¦049 mg Quil A EaSC1 1 mg¦049 mg Quil A EaSC1 9[1 mg¦049 mg Quil A
54211a 55207a 53211a 89216 "NS#
a
Marks statistical signi_cance "P ³ 9[94#^ NS not signi_cant[
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chickens was about 24) lower than from the con! trols\ which was signi_cantly di}erent at P ³ 9[94[ In Table 0\ results of an additional experiment are given using the antigen EaSC1*shown to be a lactate dehydrogenase "LDH#[ This 26!kDa protein is obtained from developmental stages of E[ acer! vulina harvested from the chicken 31 h p[i[ It is dominantly present in these stages[ Puri_cation of the native protein and subsequent immunisation of chickens resulted in signi_cant reduction of oocyst output which appeared to be dose related "Table 0#[ The EaSC1 gene sequence demonstrated high homology with known LDH sequences and especially with Toxoplasma and Plasmodium\ including the conservation of a _ve!aa insertion "Fig[ 0# which might be a typical apicomplexan feature[ The functional implication of this insertion is not known[ Antibodies from rabbits reactive against the E[ acervulina protein appeared to recog! nise a homologue in E[ tenella sporozoites and tro! phozoites\ which demonstrates that cross!reactive epitopes are present[ Following the in!vitro culture of E[ tenella sporozoites in a _broblast!like cell line\ Brake and co!workers ð17Ł described a number of antigens which were secreted into the medium at 37Ð61 h of intracellular development[ In subsequent experi! ments 0Ð3!day!old broiler chicks were given crude supernatant antigens in the presence of oil!based adjuvant "Amphigen# and 2 days later chicks were boosted and 5 days later orally challenged with 24 999 E[ tenella oocysts[ Vaccinated birds were protected from weight!gain loss measured 5 days
Fig[ 0[ Alignment of partial stretch of lactate dehydrogenase from Eimeria acervulina "EaSC1#\ Plasmodium falciparum "PFLDH#\ Toxoplasma gondii tachyzoites "TOXLDH0# and bradyzoites "TOXLDH1# and compared with some other lactate dehydrogenase of mammalian "LDHH*Human#\ avian LDHH*chick or rickettsian LDHH*ANAPL[
0014
following challenge\ and in some experiments lesion scores were slightly "09)# lower after challenge[ Some genes encoding the secreted proteins of inter! est have been cloned and expressed in Escherichia coli[ Recombinant proteins were also used as a vac! cine and induced partial protection as measured by reduced weight loss after challenge[ 2[1[ Sexual!stage antigens Apart from asexual!stage antigens\ studies were also done on sexual!stage antigens\ such as those detectable in gametocytes and zygotes or unsporu! lated oocysts[ The well!documented work on maternal immun! ity against E[ maxima is related to a set of three glycoproteins of 129 kDa\ 71 kDa and 45 kDa iso! lated from the total gametocyte extract by either a lectin!a.nity column or using a mAb as ligand ð18Ł[ Using this combination of proteins with Freund|s adjuvant\ breeder animals were vaccinated[ Anti! bodies to the gametocyte antigens were detectable in the yolk of eggs and appeared to reduce the infection of challenged o}spring chicks[ After chal! lenge with E[ maxima a 59Ð79) reduction of oocyst output was reported and after challenge with E[ tenella or E[ acervulina a 39) reduction of oocyst output was found[ A gene expressed predominantly in macrogametocytes:zygotes encoded a 129!kDa antigen\ but the relationship to any of the proteins described above is not clear ð29Ł[ The gene was selected from a gametocyte cDNA library using antibodies against the 71!kDa antigen[ Antibodies against the E[ coli fusion protein recognised a ladder of bands on gametocyte blots[ These antibodies recognised the forming oocyst wall in zygotes[ Oocyst!wall glycoproteins have been described by Karim et al[ ð20Ł and Eschenbacher et al[ ð21Ł[ Karim et al[ reported an IgM mAb raised against a 01!kDa protein that was able to reduce oocyst out! put 39Ð59) when given to birds that were chal! lenged subsequently with E[ tenella or E[ maxima[ The mAb cross!reacted with seven other Eimeria spp[ Eschenbacher et al[ ð21Ł probably identi_ed the same protein[ These authors used polyclonal IgG raised in rats against a 03!kDa oocyst wall protein from E[ tenella and E[ acervulina[ The IgG anti! bodies showed only species!speci_c reactivity[ Par!
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tial N!terminal aa determination demonstrated the presence of a high percentage of hydroxylated aa[ Binding studies to peanut agglutinin indicated the addition of O!linked oligosaccharides[ The developmental stage!speci_c antigens com! prise a set of interesting candidates to be evaluated further[ It should be realised\ however\ that approaches to use these antigens as vaccines can reduce the numbers of oocysts in the poultry houses but will not necessarily protect against the asexual replication of the parasite\ which is largely respon! sible for the damage of the gut epithelium and the associated negative e}ects on the feed uptake[
3[ T!Cell stimulatory antigens T!Cell stimulatory antigens have been selected as being more relevant to protection in comparison with antibody!selected antigens[ Dunn et al[ ð22Ł described protein kinases con! taining a calmodulin!binding domain from E[ tenella and E[ maxima[ The proteins are thought to play a role in the invasion process\ being detectable at the apical complex of the sporozoite around time of invasion[ A related patent mentioned the ability of this antigen speci_cally to stimulate primed T cells from infected animals to proliferate[ Vaccin! ation of 2!week!old Leghorn chickens using 14 mg of the puri_ed His!tagged recombinant antigen pro! duced by E[ coli resulted in a moderate 19) reduction of oocyst output after an E[ maxima chal! lenge[ A similar result was obtained when the pro! tein was given as a live Salmonella gallinarum recombinant vaccine ð23Ł[ T!Cell proliferation and interferon!g induction were used as criteria to select candidate E[ tenella antigens from sporozoites ð4Ł[ Antigen preparations were allowed to stimulate primed T cells from chickens recovering from a primary infection[ Those fractions that induced the highest lympho! proliferation and interferon!g production were selected for an immunisation experiment[ Figure 1 shows the results of such a vaccination study using some of these fractionated antigen prep! arations "Breed et al[\ in preparation#[ Groups of 09 chickens were vaccinated twice with 4Ð04 mg pro! tein of the selected fractions in the presence of
Fig[ 1[ Vaccine potential of antigen fractions selected for their capacity to stimulate T cells to proliferate and produce g!inter! feron in vitro[ Stimulation of T cells and g!interferon production "measured as concentration of NO in interferon!stimulated mac! rophages indicated as nitrite index# were performed in vitro with cells taken prior to challenge[ "A# Mean proliferative PBL responses[ "B# Mean macrophage activating activity[ "C# Mean caecal lesion scores after challenge[ Groups 0\ 1\ 2 and 7 indicate di}erent fractions from puri_cation regime[ Statistical sig! ni_cance] P ³ 9[94\ P ³ 9[994\ P ³ 9[9994[
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049 mg Quil A "Superfos Biosector\ Vedbaek# and challenged 1 weeks after the booster with 3499 E[ tenella oocysts[ Lesions were scored 6 days later[ Figure 1 demonstrates clearly that fraction 2\ being the most immunogenic and inducing the highest interferon response\ reduces the lesion score signi! _cantly "P ³ 9[94# after a high!dose E[ tenella infec! tion[ Fraction 2 contained several proteins\ which are now being analysed further[ Concluding this part of the review\ several inter! esting candidate antigens have been described during the past 3Ð4 years[ Di}erent strategies have all been successful to some extent\ but so far no single antigen has demonstrated the capacity to induce complete protection[ However\ this may not be a realistic target at all since\ in common with so many other infections\ the best immunity will be induced by a "repeated# live infection[ The com! plexity of the life!cycle\ the di}erent stages develop! ing and the array of antigens being exposed all provide the immune system with enough tools to attack the organism from either side[ None the less\ the parasite only induces a species!speci_c pro! tection after one or more passages through the same host[ Subunit vaccines comprising single proteins have so far been shown to induce about a 49) reduction of oocyst output or gross lesion scores[ This should enable the host to withstand the _rst negative e}ects of infection and subsequently to develop immunity[ Vaccine carriers in terms of bac! teria\ viruses or even protozoa might play a decisive role in the appropriate delivery of the antigens and presentation to the immune system[
4[ Progress in development of recombinant vaccine carriers From the time of using Vaccinia virus as carrier for heterologous genes\ di}erent prokaryotic and viral vectors have been described which were able to elicit some immune response against the hetero! logous gene!encoded protein[ For protozoal genes\ vectors like Salmonella typhimurium and Poxviruses have been used with some success in promoting T! cell mediated immunity "reviewed by Tomley et al[ ð24Ł#[ In the _eld of Eimeria recombinant vaccines\ a
0016
limited set of reports is available on this subject[ Binger et al[ ð15Ł used Vaccinia virus to express the E[ tenella Mzp4Ð6 gene[ Although the Vaccinia virus is not a pathogen for chickens\ it was able to induce some degree of infection in chickens such that antibodies against the heterologous protein were raised[ Protection after subsequent challenge was not reported[ From our own work\ S[ galli! narum\ S[ typhimurium\ Fowlpox virus "FPV# and Herpes virus of turkeys "HVT# have been used with the Ea0A refractile body associated antigen to induce protection against challenge ð25Ł[ The Sal! monella vectors were used because they present the relevant antigens at the intestine\ which is the nor! mal site of Eimeria infection[ A major problem related to the use of bacterial vector systems is their instability in combination with the high mutation frequency[ Escherichia coli or Salmonella con! situtively expressing heterologous genes become less viable so that these populations will be over! grown rapidly by phenotypes that have lost the heterologous genes[ To account for this\ new Salmonella strains have been developed which harbour the characteristic of switchable expression by inverting the promoter that drives the expression of the heterologous protein[ This results in a sustained growth of the total population with continuous stimulation of the immune response ð26Ł[ Viral vectors are important because they su}er less from stability problems as mentioned above for Salmonella[ Herpes virus of turkeys is especially interesting since it induces a viraemia in chickens and the virus is used already to vaccinate broilers at day 0 or even in ovo against Marek|s disease[ In order to evaluate the e.cacy of these vectors\ the Ea0A partial gene was subcloned in all vectors[ It had already been found that the Ea0A gene enco! des a good candidate for a recombinant vaccine\ able to reduce oocyst output by 49Ð69) when applied as a subunit vaccine to white Leghorn chickens ð11Ł[ It was thus important to assess what this would mean for broiler chickens kept in a ~oor pen*a situation more closely resembling con! ditions in the _eld[ Weight gain was chosen as the performance parameter[ Broiler chickens were vac! cinated with 0999 p[f[u[ of HVT!Ea0A given s[c[ at
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day 0 or with 4×097 Salmonella!Ea0A combined with an equal dose of Salmonella!EaSC1 given orally on day 2[ Control animals were inoculated with the respective vectors without the insert[ Chickens were raised in ~oor pens and challenged at day 12 with 049 999 oocysts of E[ acervulina\ 24 999 oocysts of E[ maxima and 0499 E[ tenella oocysts[ Weight gain was assessed weekly until day 32[ Table 1 shows the results of these two experi! ments[ These data indicate that these vector systems can be used to present antigens e.ciently and that these combinations are able to induce protection in terms of reduced weight!gain loss after a heavy challenge with three di}erent species[ Previous results also showed that S[ gallinarum and FPV induced a par! tial protection with similar e}ects using the same "Ea0A# antigen ð25Ł[ In summary\ these reports clearly demonstrate the feasibility of using either subunit antigens or more complex heterologous gene:vector systems to induce protective responses in chickens that are able to reduce either the pathological e}ects or the replication of the parasite following a challenge infection[ The extent of the e}ects measured should be evaluated on a larger scale to evaluate the econ! omical impact of applying such a strategy in the control of the disease[
5[ Recombinant vaccines into the next millennium When looking over the edge of the new millen! nium\ one should be optimistic about the practical application of a recombinant vaccine in the _rst
years of the new era[ Some of the genes described above may prove to be good candidates for inclusion in a future cocktail recombinant vaccine[ Since no other criteria are available but e.cacy\ the major task will be to select the right candidates\ which will require predominantly in!vivo testing of individual genes and combinations in practical vector systems[ The vector systems described have been validated using only a limited set of antigens and need further investigation to ensure safe and e.cacious appli! cation in the _eld[ Certainly some of the vectors can be used relatively safely\ for example HVT\ but for others\ safety could become a major issue in terms of persistence[ The latter is the main reason why Salmonella is so far only used as a research tool in this _eld[ Developments such as transfection of parasites as described recently for Toxoplasma ð27Ł have changed the type of research dramatically and have created new possibilities for selection of vital com! ponents of the organism[ These kinds of studies have been initiated for Eimeria and could lead to new live vectors as well ð28Ł[ The impact of DNA! based vaccines cannot be foreseen as yet\ but the technology will allow more rapid and easier selec! tion of vaccine candidates[ Di}erential display PCR\ expressed sequence tags and phage display libraries are amongst the new tools that molecular biologists can apply to tackle the problems of tomorrow[ Altogether\ this should lead to a de_nitive solu! tion for the increasing problems in the poultry industry caused by the beautiful creatures named Eimeria[
Table 1 Weight gain "g# from day of challenge to day 32 of Ea0A!vaccinated broilers using di}erent vectors to present the antigen
Vaccine
Weight gain vaccinates2S[D[
Weight gain controls2S[D[
S[ typhimurium Ea0A¦EaSC1 HVT Ea0A
03372046 "n 028# 03312126 "n 78#
03932037 "n 025# 02102278 "n 76#
Student|s t!test\ two!tailed[
P valuea 9[90 9[90
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Acknowledgements I am greatly indebted to my colleagues Drs D[ Lutticken\ N[ Visser\ T[ Schetters\ D[ Clercx!Breed\ D[ Schaap\ P[ van den Boogaart\ R[ Dijkema\ P[ Sondermeyer\ M[ Cronenberg and E[ Rijke for their scienti_c input over the past years[ I wish to thank my colleagues from the Department of Parasitology at Intervet International for their excellent technical assistance and scientists from the Institute of Animal Health\ Compton\ U[K[ and the Max! Planck Institute\ Tubingen\ Germany for their collaborative support[
References ð0Ł Danforth HD\ Augustine PC\ Jenkins MC[ A review of progress in coccidial vaccine development[ In] Proceedings of the VIth International Coccidiosis Conference[ Guelph\ Ont[\ Canada\ 10Ð14 June 0882^38Ð59[ ð1Ł Chapman HD[ Resistance to anticoccidial drugs in fowl[ Parasitol Today 0882^8]048Ð051[ ð2Ł Shirley MW\ Bedrnik P[ Live attenuated vaccines against avian coccidiosis] success with precocious and egg!adapted lines of Eimeria[ Parasitol Today 0886^02"01#]370Ð373[ ð3Ł Rose ME[ Immunity to coccidia[ In] Davison TF\ Morris TR\ Payne LN\ editors[ Poultry immunology[ Oxford] Car! fax Publishing 0885^154Ð88[ ð4Ł Breed DGJ\ Schetters TPM\ Verhoeven NAP\ Vermeulen AN[ Characterisation of phenotype related responsiveness of peripheral blood lymphocytes from Eimeria tenella infected chickens[ Parasite Immunol 0886^08]452Ð458[ ð5Ł Conway DP\ McKenzie ME\ Dayton AD[ Relationship of coccidial lesion scores and weight gain in infections of Eimeria acervulina\ E[ maxima and E[ tenella in broilers[ Avian Pathol 0889^08"2#]378Ð385[ ð6Ł Dubremetz JF[ Apical organelles and host cell invasion by Apicomplexa zoites[ In] Proceedings of the COST 719 Annual Workshop[ Copenhagen\ Denmark\ 0885^06Ð08[ ð7Ł Tomley FM\ Clarke LE\ Kawazoe U\ Dijkema R\ Kok JJ[ Sequence of the gene encoding an immunodominant microneme protein of Eimeria tenella[ Mol Biochem Par! asitol 0880^38"1#]166Ð177[ ð8Ł Pasamontes L\ Hug D\ Humbelin M\ Weber G[ Sequence of a major Eimeria maxima antigen homologous to the Eimeria tenella microneme protein Etp099[ Mol Biochem Parasitol 0882^46"0#]060Ð063[ ð09Ł Spano F\ Putignani L\ Ricci I\ Di Cristina M\ Puri C\ Crisanti A[ The {{thrombospondin|| and {{sag|| protein fam! ilies of Cryptosporidium parvum and Toxoplasma gondii] cloning of three new molecules potentially involved in para! siteÐhost cell interaction[ In] Proceedings of the COST 719 Annual Workshop[ Copenhagen\ Denmark\ 0885^13Ð4[
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ð00Ł Tomley FM\ Bumstead JM\ Billington KJ\ Dunn PPJ[ Molecular cloning and characterisation of a novel acidic microneme protein "Etmic!1# from the apicomplexan proto! zoan parasite\ Eimeria tenella[ Mol Biochem Parasitol 0885^68"1#]084Ð195[ ð01Ł Tomley FM[ Characterisation of rhoptry proteins of Eimeria tenella sporozoites] antigenic diversity of rhoptry epitopes within species of the genus Eimeria and among three asexual generations of a single species\ E[ tenella[ Infect Immun 0883^51"09#]3545Ð3547[ ð02Ł Entzeroth R\ Stefen C\ Chobotar B\ Dubremetz JF[ Evi! dence of secretion of an internal antigen of Eimeria papillata into cultured cells[ Arch Protistenk 0882^032"3#]236Ð240[ ð03Ł Lopez!Bernad F\ Delcacho E\ Gallego M\ Quilez J\ San! chez!Acedo C[ Identi_cation of a _bronectin!like molecule on Eimeria tenella[ Parasitology 0885^002"5#]494Ð409[ ð04Ł Delcacho E\ Quilez J\ Lopezbernad F\ Sanchezacedo C\ Gallego M[ Identi_cation of a _bronectin!like molecule on the surface of Leishmania amastigotes[ Vet Parasitol 0885^55"0!1#]02Ð07[ ð05Ł Miller GA\ Bhogal BS\ McCandliss R[\ et al[ Charac! terisation and vaccine potential of a novel recombinant coccidial antigen[ Infect Immun 0878^46"6#]1903Ð1919[ ð06Ł Karkhanis YD\ Nollstadt KA\ Bhogal B[S[\ et al[ Puri! _cation and characterisation of a protective antigen from Eimeria tenella[ Infect Immun 0880^48"2#]872Ð878[ ð07Ł Abrahamsen MS\ Johnson RR\ Jutila MA\ Speer CA\ White MW[ Eimeria bovis] expression of a related group of refractile body!associated proteins during schizogony[ Exp Parasitol 0883^67"2#]220Ð224[ ð08Ł Crane MSJ\ Goggin B\ Pellegrino R[M[\ et al[ Cross!pro! tection against 3 species of chicken Coccidia with a single recombinant antigen[ Infect Immun 0880^48"3#]0160Ð0166[ ð19Ł O|Lorcain P\ Talebi A\ Mulcahy G[ Mapping for B!cell epitopes in the GX2151 antigenic sequence derived from Eimeria tenella sporulated oocysts[ Vet Parasitol 0885^55"2! 3#]048Ð058[ ð10Ł Vermeulen AN\ Kok JJ\ van den Boogaart P\ Dijkema R\ Claessens JAJ[ Eimeria refractile body proteins contain 1 potentially functional characteristics*transhydrogenase and carbohydrate transport[ FEMS Microbiol Lett 0882^009"1#]112Ð129[ ð11Ł Vermeulen A\ Dijkema R\ Kok JJ[ Coccidiosis vaccine[ European Patent speci_cation 9238960B0\ granted 12 March 0883[ ð12Ł Kramer RA\ Tomchak LA\ McAndrew S[J[\ et al[ An Eimeria tenella gene encoding a protein with homology to the nucleotide transhydrogenases of Escherichia coli and bovine mitochondria[ Mol Biochem Parasitol 0882^59"1#]216Ð221[ ð13Ł Laurent F\ Bourdieu C\ Kaga M[\ et al[ Cloning and charac! terisation of an Eimeria acervulina sporozoite gene hom! ologous to aspartyl proteinases[ Mol Biochem Parasitol 0882^51"1#]292Ð201[ ð14Ł McDonald V\ Rose ME\ Je}ers TK[ Eimeria tenella] immu! nogenicity of the _rst generation of schizogony[ Para! sitology 0875^82]0Ð6[ ð15Ł Binger MH\ Hug D\ Weber G\ Schildknecht E\ Humbelin
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A[N[ Vermeulen : International Journal for Parasitolo`y 17 "0887# 0010Ð0029 M\ Pasamontes L[ Cloning and characterisation of a surface antigen of Eimeria tenella merozoites and expression using a recombinant Vaccinia virus[ Mol Biochem Parasitol 0882^50"1#]068Ð076[ Bordier C[ Phase separation of integral membrane proteins in Triton X003 solution[ J Biol Chem 0870^145]0593Ð0596[ Brake DA\ Strang G\ Lineberger J[E[\ et al[ Immunogenic characterisation of a tissue culture!derived vaccine that a}ords partial protection against avian coccidiosis[ Poultry Sci 0886^65"6#]863Ð872[ Wallach M\ Smith NC\ Petracca M\ Miller CMD\ Eckert J\ Braun R[ Eimeria maxima gametocyte antigens] potential use in a subunit maternal vaccine against coccidiosis in chickens[ Vaccine 0884^02"3#]236Ð243[ Fried M\ Mencher D\ Sarshalom O\ Wallach M[ Devel! opmental gene expression of a 129!kilodalton mac! rogamete!speci_c protein of the avian coccidial parasite\ Eimeria maxima[ Mol Biochem Parasitol 0881^40]140Ð151[ Karim MJ\ Basak SC\ Trees AJ[ Characterisation and immunoprotective properties of a monoclonal antibody against the major oocyst wall protein of Eimeria tenella[ Infect Immun 0885^53"3#]0116Ð0121[ Eschenbacher KH\ Eggli P\ Wallach M\ Braun R[ Charac! terisation of a 03 kda oocyst wall protein of Eimeria tenella and E[ acervulina[ Parasitology 0885^001"1#]058Ð065[ Dunn PPJ\ Bumstead JM\ Tomley FM[ Sequence\ expression and localization of calmodulin!domain protein
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kinases in Eimeria tenella and Eimeria maxima[ Para! sitology 0885^002"4#]328Ð337[ Bumstead JM\ Dunn PPJ\ Tomley FM\ Vermeulen AN[ Coccidiosis poultry vaccine[ US Patent 4503084[ 14 March 0886[ Tomley F\ Vermeulen AN\ van den Boogaart P[ The use of live vectors for presentation of recombinant antigens to chickens[ In] Proceedings of the VIth International Coc! cidiosis Conference[ Guelph\ Ont[\ Canada\ 10Ð14 June 0882^013Ð5[ Van den Boogaart P\ Kok JJ\ Ross R et al[ Eimeria acer! vulina refractile body antigen "Ea0a#] functional charac! teristics and immunogenicity of live recombinants in chickens[ In Proceedings of the VIth International Coc! cidiosis Conference[ Guelph\ Ont[\ Canada[ 10Ð14 June 0882^046[ Tijhaar EJ\ Karlas JA\ Van Els A[C[M[\ et al[ Plasmids that allow the continuous expression of antigens in Salmonella typhimurium at toxic levels[ Vaccines 0881^81]268Ð273[ Donald RGK\ Carter D\ Ullman B\ Roos DS[ Insertional tagging\ cloning\ and expression of the Toxoplasma gondii hypoxanthine!xanthine!guanine phosphoribosyltransfer! ase gene*use as a selectable marker for stable trans! formation[ J Biol Chem 0885^160"13#]03909Ð03908[ Kelleher M\ Dunn PP\ Tomley FM[ Transient transfection of sporozoites of Eimeria tenella[ In] Proceedings of the COST 719 Annual Workshop[ Copenhagen\ Denmark[ 0885^17[
Progress in recombinant vaccine development against coccidiosis A review and prospects into the next millennium A[N[ Vermeulen Department of Parasitology\ Intervet International BV\ PO Box 20 4729 AA Boxmeer\ The Netherlands Received 2 December 0886^ accepted 09 March 0887
Abstract The increasing problems encountered by the poultry industry\ despite the extensive use of drugs\ have emphasised the need for an immunological solution for the economic damage caused by the Eimeria parasite[ Although immunity develops relatively fast following a natural infection\ to induce protection by using parasite extracts or single antigens appears more di.cult[ Nevertheless\ the development of a vaccine based on de_ned antigens seems the best solution in the long run[ At the VIth International Coccidiosis Conference in 0882 the _rst promising results were reported from small!scale experiments using recombinant antigens[ This review summarises the advances in this _eld of research from 0882 onwards[ Although since then not many reports have been published about the e}ects of using recombinant antigens as a vaccine against coccidiosis\ a number of interesting new proteins which could be considered good targets for such a vaccine have been described and are referred to herein[ Proteins involved in the process of invasion of the host cell by the extracellular parasite are regarded as key components in the developmental cycle of the parasite[ These components possibly bind to receptors on the host cell[ Interference with this process could be a target of the protective immune response[ Progress has also been made in characterising the immune mechanisms activated by infection with the parasite[ From experimental mouse models and from studies in chickens\ a better insight has been obtained towards the involvement of CD3! and CD7!type T cells in\ respectively\ the inductor and the e}ector branch of the immune response\ although not all questions have been answered[ Several antigens have been selected using T!cell stimulation and cytokine assays and these are reviewed[ In a third section\ mostly unpublished results of our own experiments dealing with the use of live vectors to present de_ned antigens such as Ea0A and EaSC1\ a parasite refractile body transhydrogenase and a lactate dehydrogenase\ respectively\ are summarised[ Partial protection could be induced using Salmonella typhimurium as a carrier for these antigens\ in that the oocyst output was reduced by up to 49) after challenge and weight gain could be improved by 4Ð09) over non!vaccinated challenged chickens\ when tested in a ~oor!pen trial[ Similar results were obtained when these antigens were presented by viral vectors such as Fowlpox virus or Herpes virus of turkey[ These data seem to o}er good prospects for the accomplishment of a safe and e.caceous vaccine based on recombinant DNA technology[ These expectations are corroborated by recent breakthroughs in transfection of related parasites such as Plasmodium and Toxoplasma gondii\ and by the increasing amount of genomic information becoming available every day\ the impact of which cannot even be estimated yet[ These new technologies will allow us to solve the complex problems that we once created ourselves[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Key words] Eimeria^ Coccidiosis^ Recombinant DNA^ Salmonella typhimurium^ Herpes virus of turkey^ Fowlpox^ Vaccine^ Review
Tel[] ¦20[374[476269^ fax] ¦20[374[476228^ e!mail] arno[vermeulenÝintervet[akzonobel[nl[ S9919!6408:87 ,08[99¦9[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Printed in Great Britain PII] S9919!6408"87#99979!9
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0[ Introduction Ending a comprehensive review at the VIth Inter! national Coccidiosis Conference in Guelph\ Canada in 0882\ Dr Harry Danforth felt positive about the future development of a recombinant vaccine ð0Ł[ This was based on the partial "cross! species# protection that could be induced using gen! etically engineered subunit material in the presence of adjuvants and the assumption that\ by using live! vector systems\ the antigenic presentation would become more optimal and protection could be improved[ This optimism\ however\ would not exclude that more antigens should be screened in order to obtain better protective candidates[ Now\ 3 years later\ we have to conclude that the e}orts to obtain such a vaccine have become less and only a few publications have dealt with the induction of protective immunity by the admin! istration of recombinant vaccines[ This reduced level of progress is caused by the complexity of the matter and the high costs involved in performing such studies[ On the other hand\ there is certainly a future for a recombinant vaccine[ The increasing number of reports about lowered e.cacy of the available drugs "reviewed by Chapman ð1Ł# and the apparent success of live "attenuated# vaccines "reviewed by Shirley and Bedrnik ð2Ł# warrant further inves! tigations towards a safe\ easy to produce\ cost! e}ective vaccine[ Since live vaccines still bear the risk of inducing pathology and have the dis! advantage of high production costs\ this new vac! cine should be based on recDNA technology that is available to date and will be developed further in the next millennium[ In this review\ a summary will be given of the _ndings that have been made public\ and some of the work that has been performed in our laboratory concerning these aspects will be highlighted[ Progress has been made in the _eld of cell biology\ where new proteins have been dis! covered*some of which showed homology to known proteins and others appeared unique but were targeted to speci_c organelles\ which could suggest aspects of their function[ Although most of these proteins were discovered without the intention of developing a vaccine\ their
speci_c function or localisation could make them good vaccine targets\ for example\ proteins involved in the essential steps of invading the host cell[ These proteins are localised either on the sur! face of the extracellular parasite\ the sporozoite or merozoite\ or secreted by the rhoptries and micro! nemes during the process of invasion[ Dense gran! ule "DG# proteins are also thought to be good targets\ since the contents of DG are deposited into the vacuolar space or inserted in the parasito! phorous vacuole membrane "the actual interface between parasite and host#[ These proteins might be presented on the surface of the parasitised cell in the context of major histocompatibility complex molecules[ In the past 3Ð4 years\ emphasis has also been given to studies of the immune mechanisms under! lying the protective immune response to infection[ T!Cell mediated immune mechanisms are essential for solid protection ð3Ł[ Both CD3¦ and CD7¦ T! cell subsets are involved\ whereby the former is mainly responsible for the proliferative response that leads to an expanding memory cell pool and for the production of helper cytokines to stimulate CD7¦ cells to act as e}ector cells[ Interferon!g is one of the cytokines produced by both subsets and may act directly on the developing parasite ð4Ł[ By applying this knowledge\ methods have been developed to study antigens involved in stimulating the potentially relevant pathways of the immune system and improvement in aa and DNA sequ! encing have facilitated their characterisation[ Some of the antigens that have been identi_ed from the application of these techniques will be mentioned below[ In the last part of this review attention will be given to the vaccine carrier systems examined in our laboratory and their possible applicability for presenting Eimeria genes to the immune system[ Di}erent antigen preparations and vector sys! tems will be discussed in the context of the level of protection being induced[ The parameters to mea! sure protection have always been a matter of debate[ An obvious parameter would be the reduction of the proliferation of the parasite to be measured by the reduction of oocyst numbers in the faeces[ This\ however\ is a very laborious e}ort due to large variation between individual chickens[
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Furthermore\ it is not clear what the impact of a partial reduction in oocyst output would mean for the practical situation in the _eld[ Thus many inves! tigators use the extent of pathology "lesion scores#\ or weight gain of meat!type chickens over a speci_c time period as alternative readouts[ These par! ameters are more indirect indicators and could be biased by secondary factors ð5Ł[ Due to this incon! sistency in the methods to validate the usefulness of individual antigens or vector!based vaccines\ it is hard to compare results published by di}erent investigators[
1[ Identi_cation of Eimeria antigens 1[0[ Proteins involved in host!cell invasion Following advances in the description of Toxo! plasma gondii invasion!related proteins\ Eimeria organelles\ such as rhoptries\ micronemes and dense granules have been studied in more detail and di}erent proteins have been described which were targeted speci_cally to any of these sites ð6Ł[ A mic! roneme!associated\ thrombospondin!related pro! tein of Eimeria tenella\ designated as Etmic!0 "Et099#\ was described by Tomley et al[ ð7Ł[ This protein had homologues in other species and even genera[ Pasamontes et al[ ð8Ł described a similar protein in Eimeria maxima with a high aa sequence homology to Et099\ and similar proteins are present in Plasmodium\ Cryptosporidium and tachyzoites of T[ gondii ð6\ 09Ł[ The Eimeria proteins are expressed in sporozoites and late in schizogony and\ apart from their role in invasion\ are possibly also involved in movement and escape of the merozoite from the schizont!infected cell "Vermeulen\ unpub! lished observations#[ A second microneme!associated protein\ desig! nated as Etmic!1\ was described recently as being a 49!kDa protein from E[ tenella sporozoites ð00Ł[ This protein could also be detected on the surface of the sporozoite prior to invasion and\ intriguingly\ also on the surface of recently invaded host cells "Tomley\ see this volume#[ This may therefore be an interesting candidate to follow up[ Tomley ð01Ł characterised rhoptry proteins of E[
0012
tenella and found that the pattern of proteins pre! sent in these organelles was much more complex than in micronemes[ The rhoptry protein pattern of di}erent developmental stages of this species was highly variable[ These aspects have complicated the characterisation of rhoptry!associated proteins in Eimeria[ Although the function of dense granules in Eimeria has not been studied as much as in Toxoplasma\ they are considered to be involved in exocytosis of parasite material after invasion[ Entzeroth et al[ ð02Ł described a 054!kDa protein of Eimeria papillata[ Monoclonal antibodies localised the protein in dense granules of the sporozoites\ but it was also apparent in the host!cell cytoplasm[ No nucleotide or aa sequence data were published[ A _bronectin!related protein was described by Lopez!Bernad et al[ ð03Ł[ The 009!kDa protein was localised on the surface of E[ tenella sporozoites and could be involved in attachment or invasion of the host cell[ A similar protein was found in Leishmania parasites ð04Ł[ None of these proteins has been really tested for immunogenicity and e.cacy as a vaccine[ These antigens\ however\ are potentially good candidates for a future recombinant vaccine based on their function and associated localisation and should therefore be evaluated more closely[
1[1[ Refractile body proteins Several proteins have been described that are associated with the refractile bodies of the sporo! zoite[ The function of these bodies is not known and it has not even been shown yet that the refractile body is an organelle with a distinct membrane[ The twin bodies in the sporozoite fuse shortly after invasion of the host cell and the formed body gradu! ally disintegrates during the process of schizogony[ Every _rst!stage merozoite carries some of the material\ but after this stage it cannot be detected[ The _rst refractile body!associated protein described was the 15!kDa RB0\ also known as GX2151 ð05Ł or EtSO6 ð06Ł\ having homologues in di}erent avian Eimeria species but also in Eimeria bovis\ designated as Eb14:49 ð07Ł[ The function of this family of proteins is not known\ but vaccination
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with the native or recombinant protein signi_cantly reduced the pathology by about 49)\ as measured by lesion scores\ after challenge with four di}erent species ð0\ 08Ł[ O|Lorcain et al[ ð19Ł have described a few possible B!cell epitopes of GX2151[ These could be used within a vaccine composed of a com! bination of di}erent oligopeptides derived from several antigens[ Vermeulen et al[ ð10Ł described an Eimeria acer! vulina 099!kDa "designated as Ea0A# refractile body protein which demonstrated sequence hom! ology with a nicotinamide!nucleotide!dependent transhydrogenase[ The partial gene could be expre! ssed in di}erent hosts and appeared to signi_cantly reduce oocyst output of vaccinated chickens ð11Ł and reduce weight gain loss of vaccinated chickens after challenge "see below#[ The protein is probably also involved in carbohydrate transport as it con! tained a sequence signature for hexose! trans! porters[ This assumption is corroborated by the observed strong association of amylopectin gran! ules with the refractile bodies[ This\ therefore\ sug! gests a role of refractile bodies in energy storage and metabolism[ Kramer et al[ ð12Ł described the full!length sequence for the homologue of this pro! tein in E[ tenella[ An aspartyl proteinase of 32 kDa "known as 5S1# has been shown to be associated with the refractile bodies of E[ acervulina sporozoites ð13Ł[ The protein has homologues in other species\ such as E[ bovis[ Until now\ these proteins have not been used in vaccination trials[ mRNA of 5S1\ Eb14:49 and Ea0A was found to be transcribed during the pro! cess of sporulation\ but the proteins could be detected up to the _rst merozoite stage "Clark and Vermeulen\ unpublished observations#[ From these results it is clear that refractile body! related proteins\ like microneme!associated proteins\ also show a highly conserved antigenic repertoire among Eimeria spp[ from mammals to avians[ In those cases where protection data have been published\ immunity appeared to cross the species barrier ð0\ 08Ł[ Apparently the immunity induced by these antigens is di}erent from the immunity induced by a live infection\ which is spec! ies!speci_c[ Therefore these proteins should be con! sidered as very promising candidates for a recombinant vaccine[
2[ Developmental stage antigens 2[0[ Asexual stage antigens From earlier observations it was shown that developmental!stage antigens could be crucial for the induction of protective immunity\ especially fol! lowing a live infection ð3\ 14Ł[ Di}erent inves! tigators have pursued proteins from merozoites or trophozoiteÐschizont stages[ Binger et al[ ð15Ł described a 22!kDa merozoite protein "Mzp4Ð6# in E[ tenella which was expressed by recombinant Vaccinia virus and the construct was shown to induce antibodies after vaccination of chickens[ No protection data were published[ From our own work using E[ acervulina\ a set of four merozoite!associated antigens "Eam199\ Eam099\ Eam34 and Eam19# was found[ The latter two antigens were detected on the surface of the 1ndÐ2rd generation merozoites "harvested 61 h p[i[#\ whereas the _rst two antigens were soluble cytoplasmic proteins as determined by TX003 extraction and phase separation as described by Bordier ð16Ł[ Vaccination of chickens using the native proteins Eam34 or Eam19 puri_ed by mAb! immuno!a.nity chromatography and adjuvanted with Quil A "Superfos\ Vedbaek# resulted in sig! ni_cant reduction of oocyst output "Table 0#[ In this experiment groups of 19 SPF White Leghorn chickens were vaccinated three times s[c[ at weekly intervals and challenged 1 weeks after the _nal booster with 199Ð499 E[ acervulina sporulated oocysts[ Oocyst output was measured from each chicken individually and compared with the controls[ The mean oocyst outputs of vaccinated
Table 0 Immunisation of chickens using E[ acervulina developmental stage antigens
Vaccine
) oocyst output of controls2S[D[
Eam34 19 mg¦049 mg Quil A Eam19 19 mg¦049 mg Quil A EaSC1 1 mg¦049 mg Quil A EaSC1 9[1 mg¦049 mg Quil A
54211a 55207a 53211a 89216 "NS#
a
Marks statistical signi_cance "P ³ 9[94#^ NS not signi_cant[
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chickens was about 24) lower than from the con! trols\ which was signi_cantly di}erent at P ³ 9[94[ In Table 0\ results of an additional experiment are given using the antigen EaSC1*shown to be a lactate dehydrogenase "LDH#[ This 26!kDa protein is obtained from developmental stages of E[ acer! vulina harvested from the chicken 31 h p[i[ It is dominantly present in these stages[ Puri_cation of the native protein and subsequent immunisation of chickens resulted in signi_cant reduction of oocyst output which appeared to be dose related "Table 0#[ The EaSC1 gene sequence demonstrated high homology with known LDH sequences and especially with Toxoplasma and Plasmodium\ including the conservation of a _ve!aa insertion "Fig[ 0# which might be a typical apicomplexan feature[ The functional implication of this insertion is not known[ Antibodies from rabbits reactive against the E[ acervulina protein appeared to recog! nise a homologue in E[ tenella sporozoites and tro! phozoites\ which demonstrates that cross!reactive epitopes are present[ Following the in!vitro culture of E[ tenella sporozoites in a _broblast!like cell line\ Brake and co!workers ð17Ł described a number of antigens which were secreted into the medium at 37Ð61 h of intracellular development[ In subsequent experi! ments 0Ð3!day!old broiler chicks were given crude supernatant antigens in the presence of oil!based adjuvant "Amphigen# and 2 days later chicks were boosted and 5 days later orally challenged with 24 999 E[ tenella oocysts[ Vaccinated birds were protected from weight!gain loss measured 5 days
Fig[ 0[ Alignment of partial stretch of lactate dehydrogenase from Eimeria acervulina "EaSC1#\ Plasmodium falciparum "PFLDH#\ Toxoplasma gondii tachyzoites "TOXLDH0# and bradyzoites "TOXLDH1# and compared with some other lactate dehydrogenase of mammalian "LDHH*Human#\ avian LDHH*chick or rickettsian LDHH*ANAPL[
0014
following challenge\ and in some experiments lesion scores were slightly "09)# lower after challenge[ Some genes encoding the secreted proteins of inter! est have been cloned and expressed in Escherichia coli[ Recombinant proteins were also used as a vac! cine and induced partial protection as measured by reduced weight loss after challenge[ 2[1[ Sexual!stage antigens Apart from asexual!stage antigens\ studies were also done on sexual!stage antigens\ such as those detectable in gametocytes and zygotes or unsporu! lated oocysts[ The well!documented work on maternal immun! ity against E[ maxima is related to a set of three glycoproteins of 129 kDa\ 71 kDa and 45 kDa iso! lated from the total gametocyte extract by either a lectin!a.nity column or using a mAb as ligand ð18Ł[ Using this combination of proteins with Freund|s adjuvant\ breeder animals were vaccinated[ Anti! bodies to the gametocyte antigens were detectable in the yolk of eggs and appeared to reduce the infection of challenged o}spring chicks[ After chal! lenge with E[ maxima a 59Ð79) reduction of oocyst output was reported and after challenge with E[ tenella or E[ acervulina a 39) reduction of oocyst output was found[ A gene expressed predominantly in macrogametocytes:zygotes encoded a 129!kDa antigen\ but the relationship to any of the proteins described above is not clear ð29Ł[ The gene was selected from a gametocyte cDNA library using antibodies against the 71!kDa antigen[ Antibodies against the E[ coli fusion protein recognised a ladder of bands on gametocyte blots[ These antibodies recognised the forming oocyst wall in zygotes[ Oocyst!wall glycoproteins have been described by Karim et al[ ð20Ł and Eschenbacher et al[ ð21Ł[ Karim et al[ reported an IgM mAb raised against a 01!kDa protein that was able to reduce oocyst out! put 39Ð59) when given to birds that were chal! lenged subsequently with E[ tenella or E[ maxima[ The mAb cross!reacted with seven other Eimeria spp[ Eschenbacher et al[ ð21Ł probably identi_ed the same protein[ These authors used polyclonal IgG raised in rats against a 03!kDa oocyst wall protein from E[ tenella and E[ acervulina[ The IgG anti! bodies showed only species!speci_c reactivity[ Par!
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tial N!terminal aa determination demonstrated the presence of a high percentage of hydroxylated aa[ Binding studies to peanut agglutinin indicated the addition of O!linked oligosaccharides[ The developmental stage!speci_c antigens com! prise a set of interesting candidates to be evaluated further[ It should be realised\ however\ that approaches to use these antigens as vaccines can reduce the numbers of oocysts in the poultry houses but will not necessarily protect against the asexual replication of the parasite\ which is largely respon! sible for the damage of the gut epithelium and the associated negative e}ects on the feed uptake[
3[ T!Cell stimulatory antigens T!Cell stimulatory antigens have been selected as being more relevant to protection in comparison with antibody!selected antigens[ Dunn et al[ ð22Ł described protein kinases con! taining a calmodulin!binding domain from E[ tenella and E[ maxima[ The proteins are thought to play a role in the invasion process\ being detectable at the apical complex of the sporozoite around time of invasion[ A related patent mentioned the ability of this antigen speci_cally to stimulate primed T cells from infected animals to proliferate[ Vaccin! ation of 2!week!old Leghorn chickens using 14 mg of the puri_ed His!tagged recombinant antigen pro! duced by E[ coli resulted in a moderate 19) reduction of oocyst output after an E[ maxima chal! lenge[ A similar result was obtained when the pro! tein was given as a live Salmonella gallinarum recombinant vaccine ð23Ł[ T!Cell proliferation and interferon!g induction were used as criteria to select candidate E[ tenella antigens from sporozoites ð4Ł[ Antigen preparations were allowed to stimulate primed T cells from chickens recovering from a primary infection[ Those fractions that induced the highest lympho! proliferation and interferon!g production were selected for an immunisation experiment[ Figure 1 shows the results of such a vaccination study using some of these fractionated antigen prep! arations "Breed et al[\ in preparation#[ Groups of 09 chickens were vaccinated twice with 4Ð04 mg pro! tein of the selected fractions in the presence of
Fig[ 1[ Vaccine potential of antigen fractions selected for their capacity to stimulate T cells to proliferate and produce g!inter! feron in vitro[ Stimulation of T cells and g!interferon production "measured as concentration of NO in interferon!stimulated mac! rophages indicated as nitrite index# were performed in vitro with cells taken prior to challenge[ "A# Mean proliferative PBL responses[ "B# Mean macrophage activating activity[ "C# Mean caecal lesion scores after challenge[ Groups 0\ 1\ 2 and 7 indicate di}erent fractions from puri_cation regime[ Statistical sig! ni_cance] P ³ 9[94\ P ³ 9[994\ P ³ 9[9994[
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049 mg Quil A "Superfos Biosector\ Vedbaek# and challenged 1 weeks after the booster with 3499 E[ tenella oocysts[ Lesions were scored 6 days later[ Figure 1 demonstrates clearly that fraction 2\ being the most immunogenic and inducing the highest interferon response\ reduces the lesion score signi! _cantly "P ³ 9[94# after a high!dose E[ tenella infec! tion[ Fraction 2 contained several proteins\ which are now being analysed further[ Concluding this part of the review\ several inter! esting candidate antigens have been described during the past 3Ð4 years[ Di}erent strategies have all been successful to some extent\ but so far no single antigen has demonstrated the capacity to induce complete protection[ However\ this may not be a realistic target at all since\ in common with so many other infections\ the best immunity will be induced by a "repeated# live infection[ The com! plexity of the life!cycle\ the di}erent stages develop! ing and the array of antigens being exposed all provide the immune system with enough tools to attack the organism from either side[ None the less\ the parasite only induces a species!speci_c pro! tection after one or more passages through the same host[ Subunit vaccines comprising single proteins have so far been shown to induce about a 49) reduction of oocyst output or gross lesion scores[ This should enable the host to withstand the _rst negative e}ects of infection and subsequently to develop immunity[ Vaccine carriers in terms of bac! teria\ viruses or even protozoa might play a decisive role in the appropriate delivery of the antigens and presentation to the immune system[
4[ Progress in development of recombinant vaccine carriers From the time of using Vaccinia virus as carrier for heterologous genes\ di}erent prokaryotic and viral vectors have been described which were able to elicit some immune response against the hetero! logous gene!encoded protein[ For protozoal genes\ vectors like Salmonella typhimurium and Poxviruses have been used with some success in promoting T! cell mediated immunity "reviewed by Tomley et al[ ð24Ł#[ In the _eld of Eimeria recombinant vaccines\ a
0016
limited set of reports is available on this subject[ Binger et al[ ð15Ł used Vaccinia virus to express the E[ tenella Mzp4Ð6 gene[ Although the Vaccinia virus is not a pathogen for chickens\ it was able to induce some degree of infection in chickens such that antibodies against the heterologous protein were raised[ Protection after subsequent challenge was not reported[ From our own work\ S[ galli! narum\ S[ typhimurium\ Fowlpox virus "FPV# and Herpes virus of turkeys "HVT# have been used with the Ea0A refractile body associated antigen to induce protection against challenge ð25Ł[ The Sal! monella vectors were used because they present the relevant antigens at the intestine\ which is the nor! mal site of Eimeria infection[ A major problem related to the use of bacterial vector systems is their instability in combination with the high mutation frequency[ Escherichia coli or Salmonella con! situtively expressing heterologous genes become less viable so that these populations will be over! grown rapidly by phenotypes that have lost the heterologous genes[ To account for this\ new Salmonella strains have been developed which harbour the characteristic of switchable expression by inverting the promoter that drives the expression of the heterologous protein[ This results in a sustained growth of the total population with continuous stimulation of the immune response ð26Ł[ Viral vectors are important because they su}er less from stability problems as mentioned above for Salmonella[ Herpes virus of turkeys is especially interesting since it induces a viraemia in chickens and the virus is used already to vaccinate broilers at day 0 or even in ovo against Marek|s disease[ In order to evaluate the e.cacy of these vectors\ the Ea0A partial gene was subcloned in all vectors[ It had already been found that the Ea0A gene enco! des a good candidate for a recombinant vaccine\ able to reduce oocyst output by 49Ð69) when applied as a subunit vaccine to white Leghorn chickens ð11Ł[ It was thus important to assess what this would mean for broiler chickens kept in a ~oor pen*a situation more closely resembling con! ditions in the _eld[ Weight gain was chosen as the performance parameter[ Broiler chickens were vac! cinated with 0999 p[f[u[ of HVT!Ea0A given s[c[ at
0017
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day 0 or with 4×097 Salmonella!Ea0A combined with an equal dose of Salmonella!EaSC1 given orally on day 2[ Control animals were inoculated with the respective vectors without the insert[ Chickens were raised in ~oor pens and challenged at day 12 with 049 999 oocysts of E[ acervulina\ 24 999 oocysts of E[ maxima and 0499 E[ tenella oocysts[ Weight gain was assessed weekly until day 32[ Table 1 shows the results of these two experi! ments[ These data indicate that these vector systems can be used to present antigens e.ciently and that these combinations are able to induce protection in terms of reduced weight!gain loss after a heavy challenge with three di}erent species[ Previous results also showed that S[ gallinarum and FPV induced a par! tial protection with similar e}ects using the same "Ea0A# antigen ð25Ł[ In summary\ these reports clearly demonstrate the feasibility of using either subunit antigens or more complex heterologous gene:vector systems to induce protective responses in chickens that are able to reduce either the pathological e}ects or the replication of the parasite following a challenge infection[ The extent of the e}ects measured should be evaluated on a larger scale to evaluate the econ! omical impact of applying such a strategy in the control of the disease[
5[ Recombinant vaccines into the next millennium When looking over the edge of the new millen! nium\ one should be optimistic about the practical application of a recombinant vaccine in the _rst
years of the new era[ Some of the genes described above may prove to be good candidates for inclusion in a future cocktail recombinant vaccine[ Since no other criteria are available but e.cacy\ the major task will be to select the right candidates\ which will require predominantly in!vivo testing of individual genes and combinations in practical vector systems[ The vector systems described have been validated using only a limited set of antigens and need further investigation to ensure safe and e.cacious appli! cation in the _eld[ Certainly some of the vectors can be used relatively safely\ for example HVT\ but for others\ safety could become a major issue in terms of persistence[ The latter is the main reason why Salmonella is so far only used as a research tool in this _eld[ Developments such as transfection of parasites as described recently for Toxoplasma ð27Ł have changed the type of research dramatically and have created new possibilities for selection of vital com! ponents of the organism[ These kinds of studies have been initiated for Eimeria and could lead to new live vectors as well ð28Ł[ The impact of DNA! based vaccines cannot be foreseen as yet\ but the technology will allow more rapid and easier selec! tion of vaccine candidates[ Di}erential display PCR\ expressed sequence tags and phage display libraries are amongst the new tools that molecular biologists can apply to tackle the problems of tomorrow[ Altogether\ this should lead to a de_nitive solu! tion for the increasing problems in the poultry industry caused by the beautiful creatures named Eimeria[
Table 1 Weight gain "g# from day of challenge to day 32 of Ea0A!vaccinated broilers using di}erent vectors to present the antigen
Vaccine
Weight gain vaccinates2S[D[
Weight gain controls2S[D[
S[ typhimurium Ea0A¦EaSC1 HVT Ea0A
03372046 "n 028# 03312126 "n 78#
03932037 "n 025# 02102278 "n 76#
Student|s t!test\ two!tailed[
P valuea 9[90 9[90
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Acknowledgements I am greatly indebted to my colleagues Drs D[ Lutticken\ N[ Visser\ T[ Schetters\ D[ Clercx!Breed\ D[ Schaap\ P[ van den Boogaart\ R[ Dijkema\ P[ Sondermeyer\ M[ Cronenberg and E[ Rijke for their scienti_c input over the past years[ I wish to thank my colleagues from the Department of Parasitology at Intervet International for their excellent technical assistance and scientists from the Institute of Animal Health\ Compton\ U[K[ and the Max! Planck Institute\ Tubingen\ Germany for their collaborative support[
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kinases in Eimeria tenella and Eimeria maxima[ Para! sitology 0885^002"4#]328Ð337[ Bumstead JM\ Dunn PPJ\ Tomley FM\ Vermeulen AN[ Coccidiosis poultry vaccine[ US Patent 4503084[ 14 March 0886[ Tomley F\ Vermeulen AN\ van den Boogaart P[ The use of live vectors for presentation of recombinant antigens to chickens[ In] Proceedings of the VIth International Coc! cidiosis Conference[ Guelph\ Ont[\ Canada\ 10Ð14 June 0882^013Ð5[ Van den Boogaart P\ Kok JJ\ Ross R et al[ Eimeria acer! vulina refractile body antigen "Ea0a#] functional charac! teristics and immunogenicity of live recombinants in chickens[ In Proceedings of the VIth International Coc! cidiosis Conference[ Guelph\ Ont[\ Canada[ 10Ð14 June 0882^046[ Tijhaar EJ\ Karlas JA\ Van Els A[C[M[\ et al[ Plasmids that allow the continuous expression of antigens in Salmonella typhimurium at toxic levels[ Vaccines 0881^81]268Ð273[ Donald RGK\ Carter D\ Ullman B\ Roos DS[ Insertional tagging\ cloning\ and expression of the Toxoplasma gondii hypoxanthine!xanthine!guanine phosphoribosyltransfer! ase gene*use as a selectable marker for stable trans! formation[ J Biol Chem 0885^160"13#]03909Ð03908[ Kelleher M\ Dunn PP\ Tomley FM[ Transient transfection of sporozoites of Eimeria tenella[ In] Proceedings of the COST 719 Annual Workshop[ Copenhagen\ Denmark[ 0885^17[