Immunological approaches for the control of fasciolosis

Pergamon PII:

Immunological TERRY Department

W. SPITHILL,* of Biochemistry

Inrernariona/ Journalfor Parasirology, Vol. 27, No. 10, pp. 1221-1235, 1997 0 1997 Australian Society for Parasitology. Published by Elsevier Science Lid Pnnted in Great Britain 002&7519/97 Sl7.00+0.00 SOO20-7519(97)00120-3

Approaches for the Control of Fasciolosis DAVID

PIEDRAFITAT

and Molecular

and PETER

Biology, Monash University,

M. SMOOKER Clayton, Australia

Abstract-SpithiU T. W., Piedrafita D. & Smooker P. M. Immunological approaches for the control of fasciolosis. ZnternationaZ Journal for Parasitology 27: 1221-1235. The immunological relationship between liver flukes and their mammalian hosts is being unravelied by in viuo and in vitro studies. Vaccine studies in cattle and sheep with puritied antigens (fatty acid binding protein, FARP; glutathione S-transferase, GST; cathepsin L, CatL; hemoglobin) have shown that high reductions in worm burdens (31-72%) and egg production (69-98%) can be achieved, raising the realistic possibility that immunological control of Fuscioh infection is a commercially achievable goal. Combination vaccines may also be feasible since a cocktail of CatL and hemoglobin elicits a significant 72% protection in cattle. Analysis of immune responses to Fasciolu during infection in ruminants suggests that chronic infection correlates with a type 2 helper T cell response, implying that type 1 helper T ceil responses are down-regulated in fasciolosis. Recent results studying the resistance of Indonesian Thin Tail (ITT) sheep to F. gigantica have shown that this breed exhibits high innate (or rapidly acquired) resistance to infection and acquires a higher level of resistance after a primary challenge. Initial studies suggest that the resistance of ITT sheep to F. gigantica may be determined by a major gene. Merino sheep also acquire resistance to F. gigantica. In contrast, ITT and Merino sheep do not exhibit resistance to F. Zzepuficu. These results suggest that there are fundamental differences between these two species of Fasciola in the biology of their interaction with the sheep immune system. In uitro studies on immune mechanisms of kiiiing of juvenile guke have shown that juvenile larvae of F. hepafica are susceptible to antibody-dependent kiliing by activated rat macrophages in vitro which is mediated by nitric oxide. Future studies on the immune effector mechanisms expressed by resistant sheep which control infection by F. gigantica will lead to new knowledge which may allow the design of more effective vaccines for fasciolosis. 0 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.

Ke.v words; Fmciola; vaccine; GST; cathepsin; fatty acid binding protein; hemoglobin; sheep; immune response; genetic resistance; macrophage; nitric oxide.

INTRODUCTION

Recent developments in studies of the immunobiology of Fasciola infection in ruminants have provided new insights into the nature of the relationship between liver flukes and their mammalian hosts. Molecular studies aimed at developing defined vaccines for controlling fluke infection have raised the realistic possibility that immunological control of *Correspondence to: Dr Terry Spithill, Dept of Biochemistry and Molecular Biology. Monash University, Clayton, 3168. Australia. E-mail: [email protected]; Tel: 03-99053778; Fax: 03-99054699. t Present address: Department of Immunology, Infirmary, Glasgow Gl 1 6NT. Scotland, U.K.

Western

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Fasciola infection is a commercially achievable goal. Before 1987, attempts to vaccinate agricultural animals against F. hepatica had generally used crude somatic parasite extracts, mixtures of secreted parasite proteins or radiation attenuated parasites (Haroun & Hillyer, 1986). Since then, there have been several reports of the identification and characterisation of proteins from F. hepatica some of which have been tested in vaccine trials with positive results. This review will discuss the strategies being used to develop new vaccines for F. hepatica, the results to date with candidate antigens, the molecular biology of vaccine molecules and the prospects for the near future. The earlier literature has been reviewed by Rickard & Howell (1982) and Haroun & Hillyer (1986). Recent

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T. W. Spithill et al.

results studying the resistance of Indonesian Thin Tail sheep to F. gigantica and in vitro studies on immune mechanisms of killing of juvenile fluke are also discussed as these studies shed new light on the nature of the FascioZu/host relationship. VACCINE

STRATEGIES AND ANTIGENS

CANDIDATE

Three basic strategies have been used to identify candidate vaccine molecules of F. hepatica and F. gigantica: Cross protective antigens F hepatica antigens, recognised by cross reactive antibodies raised against the trematode Schistosoma mansoni, which are capable of cross protecting against S. mansoni. The prototype antigen is Fatty acid binding protein (FABP). Homologous antigens F. hepatica molecules homologous to an antigen previously shown to protect animals against S. mansoni and S. japonicum infection. The prototype antigen is Glutathione S-transferase (GST). Essential antigens F. hepatica molecules predicted to perform functions essential for parasite infection or survival. The prototype antigen is cathepsin L (CatL). Four candidate antigens from F. hepatica and/or F. gigantica have shown efficacy in vaccine trials conducted in cattle and/or sheep. CROSS

PROTECTIVE

ANTIGENS

Fatty acid binding proteins The first family listed in Table 1. FABP, is a classic example of the pathway of vaccine discovery. The history of this protein and its’ association with research into Fasciola dates to a series of cross-protection experiments performed in Hillyer’s laboratory. A soluble fraction of proteins was isolated from F. hepatica by affinity to rabbit antisera raised to S. mansoni whole-worm extract (Hillyer et al., 1977). Thus, only proteins which share epitopes between the two trematode species were isolated. This preparation, termed FhSmIlltM,, was shown to protect mice and calves against infection by F. hepatica (Hillyer, 1985; Hillyer et al., 1987). Moreover, preservation of epitopes was confirmed when the mixture of Fasciola proteins was shown to significantly protect mice and hamsters against infection by schistosomula of S. mansoni (Hillyer, 1979; Hillyer et al., 1988a). Hence, this protein preparation protects against both homologous and heterologous trematode chal-

lenge. Subsequently, Fhs,,,,,~, was further fractionated and a purified protein of size 12 kDa (termed Fh12) was demonstrated to significantly reduce the ability of S. mansoni to infect mice (Hillyer et al., 1988b). In addition, mice given a primary infection of S. mansoni developed low titres of antisera against the 12 kDa Fusciolu protein, demonstrating cross-reactivity between Fh12 and a protein antigenic in the schistosome infection (Hillyer et al., 1988a). An F. hepatica cDNA library was screened with antisera to Fh12 and several clones were isolated and characterised. The clones were shown to encode a member of the cytoplasmic FABP family (Rodriguez-Perez et al., 1992). The S. mansoni homologue to this cDNA clone had been isolated the previous year by Moser et a/. (199 l), and hence the relationship between the two predicted proteins and the basis for the cross-protection could be examined. The predicted proteins are 44% identical and differ by only one amino acid in length. The obvious question to be answered was that, since Fh12 can protect against both S. mansoni and F. hepatica challenge in mice, can the Schistosome homologue (Sm14) do the same? This was answered in the affirmative in a recent paper by Tendler et al. (1996) where a recombinant form of Sm14 protected mice up to 67% from S. mansoni (homologous) challenge, and 100% against F. hepatica (heterologous) challenge. This unusual efficacy in mice should be noted with caution since mice are highly susceptible to infection with F. hepatica and are killed by a burden of a few parasites; thus, the challenge in mouse vaccine trials involves infection with 3 metacercariae which may bias vaccine efficacy in favour of the host. Nevertheless, Fh12 and Sm14 appear to represent homologous, cross protective antigens from the liver and blood flukes, respectively. To date, there has been only one report of the efficacy of recombinant Fusciola FABP in vaccine trials (Estuningsih et al., 1997: see below). There have been two further reports of FABP proteins in F. hepatica. The first, a protein sequence deposited in the data base in 1994 (Chicz, 1994) describes the sequence of an FABP protein with 72% identity to that reported by Rodriguez-Perez et al. (1992). Further, Bozas & Spithill (1996) described the partial sequence of a third FABP homologue. It is thus apparent that F. hepatica expresses a family of FABPs and, indeed, we have shown that the same is probably true of F. gigantica (Smooker et al., 1997). We have isolated and expressed a cDNA clone (FgFABPlc) encoding the F. gigantica homologue of that reported by Rodriguez-Perez et al. (1992) and also isolated a native protein fraction containing two proteins which cross-react with anti-FABP sera. This

immunological Table l-Known Protein Family

Database entries Nucleotide Protein

approaches for the control of fasciolosis

1223

candidate vaccine molecules from Fasciula Proposed Function

Host

Species

Vaccine Protection

References Hillyer et al., 1987 Hiliyer, 1985 Estuningsih et al., 1997 Sexton et al., 1990 Morrison ef al., 1996 Estuningsih et al., 1997 Wijffels et al.. 1994b

FABP

2

Transport of Fatty-acids

cattle mice cattle

F. hepatica F. hepatica F. gigantica

55% 69-78% 31%

GST

6

Detoxification

sheep cattle

F. hepatica F. hepatica

57% 19-69%

cattle

F. gigantica

18% (ns)

sheep

F. hepatica

69% (FEC)

cattle cattle

F. hepa f ica F. gigantica

cattle

F. hepatira

44%

Dalton et al., 1996 Estuningsih et nl., 1997 Dalton ei al., 1996

cattle

F. hepatica

52s72%

Dalton et al.. 1996

F. hepatiea

ND

Heussler & Dobbelaere. 1994 Tkalcevic et al., 1995 Wilson et al.. 1997

Cathepsin L

Hemoglobin Cathepsin L + Hemoglobin Cathepsin B

11

0

Extracellular protease, egg production?

Unknown: see above

See above 2

Intra/extracellularprotease

42-69% 0%

The entries of both cDNA and protein sequences in the ENTREZ databases are exclusively of F. hepatira (or unspecified and assumed to be F. hepatica) origin, except for a single F. gigantica sequence (Smooker et al., 1997, see below). There are 55 protein sequences from Fascida currently listed in the protein database. Of these, 22 are derived from the corresponding DNA sequences listed in the nucleotide database, in which there are 76 entries. Most of the remainder of the entries in the protein database are partial sequences, derived from the N-terminal or peptide sequencing of purified proteins. ns: not significant; ND, not determined: FEC, effect on FEC only. native protein fraction elicits low but significant protection (3 1%) against F. giguntica infection in cattle (Estuningsih et al., 1997): however, the recombinant FgFABPlc protein failed to protect cattle in the same trial which may be a result of the expression of the recombinant construct as a fusion protein. Despite this, it is fair to say that FABPs represent one of the most promising vaccine candidates against trematodes and future results with other recombinant antigens are awaited with interest. HOMOLOGOUS

ANTIGENS

GST The glutathione S-transferases (GST) of F, hepatica (FhGST) were chosen as candidate vaccine antigens on the basis that homologous GST proteins from S. nzansorzi (Sm28) and S. japonicum (Sj26) were shown previously to induce significant reductions in worm burdens in vaccinated laboratory animals (Smith et al., 1986; Balloul ef al., 1987; Mitchell et al., 1988; reviewed in Brophy & Pritchard, 1994). GSTs com-

prise a large family of isoenzymes, the most studied of which are the cytosolic classes which are comprised of dimers of 24-29 kDa monomeric units and are found widely in the plant and animal kingdoms. In mammalian tissues there are at least four main classes of GST (Mannervik ef al., 1985; Meyer et al., 1991). The GST functions include the initial steps of detoxification of xenobiotics and endogenous compounds toxic to the organism. GSTs are proposed to play a major role in these reactions in helminths (Precious & Barrett, 1989; Brophy et al., 1990a). The GSTs of adult F. hepatica were purified and shown to consist of a mixture of at least 5 isoenzymes of size 23-26.5 kD which showed N-terminal sequence heterogeneity (Howell et a/., 1988; Brophy et al., 1990b; Wijffels et al.. 1992). GSTs of F. gigantica have also been isolated (Estunings~h ef al., 1997). This heterogeneity in Fasciola GSTs is reflected in the isolation and characterisation of several cDNA clones encoding GSTs. In the GenBank database there are six cDNA sequences encoding full-length (or near fulllength) GSTs (see Table 1) and in the GenPep database are the 6 corresponding peptide sequences, along

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T. W. Spithill et al.

with several partial sequences. Two of these cDNA sequences, the GSTS 1 sequence reported in Panaccio et al. (1992) and the sequence of Muro et al. (1993), are 98% identical. GSTSl is one of the four cDNA clones (GSTl, GST7, GST47, GST51) encoding isoenzymes of F. hepatica identified in our laboratory (Panaccio et al., 1992). The protein sequences predicted by these clones have between 71 and 89% identity with each other and are members of the p class of GSTs. The biochemical properties of the four F. hepatz’ca GST isoenzymes (expressed as recombinant proteins in E. coli) have been examined (Salvatore et al., 1995). Each recombinant protein formed a homodimer, and all were recognised by antisera raised to native GST. Enzymatic analysis of the proteins showed that they all conjugate glutathione to the universal substrate, I-chloro-2,4-dinitrobenzene. Further analysis using specific substrates demonstrated that the recombinant GSTs had overlapping but differing substrate specificities and exhibited differing sensitivity to inhibitors. Differences in substrate specificity and inhibitor sensitivity are also exhibited by native GST isoenzymes (Brophy et al., 1990b). The X-ray crystal structure of GST47 has recently been solved (Rossjohn et ul., 1997). Immunolocalisation studies. using antisera generated to native GST. initially showed that GSTs were distributed in the parenchyma, tegument and gut tissues of adult F. hepatica (Howell et al., 1988; Wijffels et al., 1992). Further isoenzyme-specific immunolocalisation studies were performed using antisera generated to synthetic peptides of regions unique to each of the four GST proteins predicted by the cDNA sequences. This showed that GSTl was localised to the parenchyma of adult fluke but not to the lamellae of the intestinal caeca (Creaney et al., 1995a). The antiserum to a GST 51 peptide, which cross-reacted with GST 7 and GST 47 but not GST 1, localised the other GSTs not only to the parenchyma but also to the intestinal lamellae of adult fluke. This appears to be the first evidence of tissue specific expression of GST isoenzymes in trematodes. In contrast to adult fluke, immunolocalisation of the GSTs in juvenile F. heputica revealed the binding of both the GST 1 and GST 51 antisera to the parenchymal cytoplasm, to cytoplasmic extensions of the parenchyma cells in the subtegumental area, as well as the excretory ducts. No labelling was observed in the intestinal epithelium of the juvenile fluke (Creaney et al., 1995a). These results demonstrate that adult F. hepaticu, in contrast to juvenile flukes, contain a GST, that is not GSTl, associated with the lamellae of the gut and suggest that GSTs in adult fluke may play a role in the absorptive function of the adult gut (Creaney et al., 1995a). GSTs have been proposed as ideal targets for

immunotherapy in a variety of hostparasite systems (reviewed in Brophy & Pritchard, 1994), including F. heputica (Howell et al., 1988; Sexton et al., 1990; Hillyer et al., 1992). In the first vaccine trial reported with FhGST, Howell et al. (1988) showed that vaccination of rats with 2 doses of 25Opg of FhGST in Freund’s adjuvant did not induce protection. In our laboratory, we have tested the ability of native FhGST isoenzymes to confer resistance to F. heputicu in both sheep (Sexton et ul., 1990) and cattle (Morrison et al., 1996) and significant levels of protection were achieved in both species (Table 1). Linear epitopes recognised by IgG 1 and IgG 2 antibodies from sheep vaccinated with GST have been mapped on a homology model of GST5 1 as well as the crystal structure of GST47 (Sexton et al., 1994; Rossjohn et al., 1997). In cattle. protection was dependent on the choice of adjuvant and did not correlate with total antibody titre to GST or the induction of neutralising antibodies to GST. Protection was shown to persist for at least 6 months post challenge. Interestingly, the degree of responsiveness to the GST vaccine varied between animals with some cattle showing levels of protection of >90% (Morrison et al., 1996). Recently. the efficacy of GST from F. gigantica was assessed in cattle vaccine trials (Estuningsih et al., 1997); surprisingly, no significant reduction in worm burdens or faecal egg counts was observed despite the use of adjuvants which were previously shown to induce protection against F. heputicu with FhGST (Morrison et ul., 1996). The basis for this difference in efficacy of homologous GST formulations from two related species of Fusciolu is unclear. One curious observation from Hillyer’s laboratory should be noted. Hillyer et al. (1992) have studied the antibody response to GST and the Fhs,,,,+,, fraction in infected and immunised animals and described the presence of antibodies to GST in rabbits and calves vaccinated with Fh,,,,,(,,. This suggests that the Fhsm,,,,,, fraction, which is clearly predominantly FABP, may contain low levels of GST. Whether the protective efficacy of the Fh,,,,io, fraction is influenced by the presence of this low GST component is open to question but worth consideration in view of the positive protection elicited by both purified Fh12 FABP and GST in animals (Hillyer et al., 1987; Sexton et al., 1990; Morrison et ul., 1996; Estuningsih et al., 1997).

ESSENTIAL

ANTlGENS

Cuthepsin proteases The notion that proteases produced by parasites are important for virulence is well established. Several

Immunological approaches for the control of fasciolosis reports have demonstrated that proteases from parasites may be useful as protective vaccines (reviewed in Knox, 1994). It was recognised very early in Fusciolu studies that the organism, like many parasites. secretes a variety of proteins some of which exhibit proteolytic activity against gelatin (Thorsell & Bjorkman, 1965; Howell, 1966). It was found that the secreted proteases were able to cleave immunoglobulin, a finding which generated great excitement as it implied a role for the proteases in immune evasion by the parasite and strengthened the case for targeting these proteases as potential vaccine antigens for controlling infection with F. hepatica (Chapman & Mitchell, 1982). Characterisation of the requirements for enzymatic activity and the use of specific protease inhibitors demonstrated that in adult fluke excretory/secretory (E/S) material all proteases detectable in substrate gels were of the cysteine family (see for example Rege et al., 1989: Dalton & Heffernan, 1989; Yamasaki et al., 1989; Smith et al., 1993; McGinty et al., 1993; Wijffels et al., 1994a. 1994b). When the E/S material is electrophoresed in substrate (generally gelatin) gels, proteins of a variety of molecular mass appear to have proteolytic activity. In our laboratory this activity is predominant at apparent molecular weight of 2628 kDa, corresponding to the most abundant proteins visible by either Coomassie or silver staining (Wijffels et ul., 1994b). In some reports cysteine protease activity has been observed at lower (Simpkin et al., 1980) and higher (Dalton & Heffernan, 1989; Smith et al., 1993; Carmona et al.. 1993; McGinty et al., 1993) apparent molecular weight. Several laboratories have screened cDNA libraries and isolated full-length clones encoding adult Fusciolu cysteine proteases (Yamasaki & Aoki, 1993; Heussler & Dobbelaere, 1994; Wijffels et al., 1994a; Panaccio & Spithill, unpublished data). This has revealed that the secreted adult proteases are of the cathepsin L (CatL) class, and to date a total of 6 complete CatL sequences of F. hepatica (FhCatL) are deposited in the databases (see Table 1). They are all similar in that they predict a protein of 326 amino acids (325 in one case) with a 17 amino acid signal peptide and a 90 amino acid pro-region. The mature protease is 219 amino acids in length. In our laboratory, we have isolated two cDNA sequence classes, termed FhCatLl (Wijffels et al., 1994a) and FhCatL2 (Panaccio & Spithill, unpublished data), which share 87% amino acid sequence identity. We have also isolated two CatL cDNAs from a F. gigantica cDNA library which encode two homologues of F. hepatica FhCatLl (Smooker & Spithill, unpublished data). These cDNA clones predict proteins with 94.2% identity to each other, and 92.9% and 94.2% identity to F. hepatica CatLI. In addition, proteolytic enzymes have been

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isolated from F. gigantica whole worms (Fagbemi & Hillyer, 1991) and the 2628 kDa component shown to be a cysteine protease (Fagbemi & Hillyer, 1992; Estuningsih et a/., 1997). The cathepsin L proteases have been used in attempts to develop diagnostic tests for Fusciolu infection. It was shown by Coles & Rubano (1988) that the 27 kDa proteases are immunogenic during an infection of rats; however in this experiment the sera of mice infected with S. mansoni also reacted and hence the cathepsins were not considered to be a promising diagnostic tool. The proteases were also immunogenic during a F. hepatica infection of sheep (Wijffels et al., 1994b) and have been used to diagnose infection in ruminants (Fagbemi & Guobadia, 1995). In an extensive survey, Yamasaki et al. (1989) tested the reactivity of human sera from patients infected with a variety of parasites to the 27 kDa protease. All sera from patients infected with F. hepatica reacted to the antigen, and serum from only one patient with another infection (the fellow trematode S. juponicum) yielded an absorbance value greater than any of the F. hepatica patients. Hence. the 27 kDa protease was a sensitive and relatively specific antigen. A later study by Silva et al. (1996) yielded similar results. Cathepsin L proteases have been tested as vaccines, with some surprising results. In the first report of the vaccine efficacy of FhCatL, Wijffels et al. (1994b) vaccinated sheep with purified FhCatL proteases in Freund’s complete adjuvant and found that, rather than a reduction in worm numbers, faecal egg counts were significantly reduced. The proteases therefore induce an anti-fecundity effect on F. hepatica. Recently, Dalton’s laboratory has demonstrated that vaccination with two different FhCatL proteins (also termed CatLl and CatL2), together with Freund’s adjuvant. can reduce both worm burdens (up to 69%) and egg production and viability (up to 65%) after challenge in cattle, particularly in concert with a large heme-containing protein complex (termed hemoglobin) (Dalton et al., 1996. see below). It may be that such a multivalent vaccine, targeting both fluke survival and fecundity, will be commercially viable as discussed previously (Spithill & Morrison 1996). CatL proteins from F. gigantica have also been tested as vaccines in cattle, using DEAE in Squalene Montanide’” 80 as adjuvant (Estuningsih et al.. 1997). Despite the induction of high antibody titres to CatL, no reduction in worm burdens or faecal egg counts was observed. This may be due to the use of a different adjuvant and it may be that Freund’s adjuvant induces critical immune effector responses required to inhibit fluke infection and fecundity. The challenge now is to show that the FhCatL/hemoglobin combination has efficacy using commercially acceptable adjuvants.

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T. W. Spithill et al

Cathepsin B cDNA sequences have also been identified in adult (Heussler & Dobbelaere, 1994) and juvenile fluke cDNA (Wilson et al., 1997), and a juvenile Cathepsin B protein has been identified in both somatic (Tkalcevic et al., 1995) and ES products (Wilson et al., 1997). Cathepsin B has been immunolocalised to the parenchyma in adult fluke tissues (Creaney, J.C., Irradiated Fasciola hepatica in protection and immunolocalisation studies, PhD thesis, Latrobe University, 1995) and to the gut lumen and to secretory granules within the gut epithelia in juvenile flukes (Creaney et al.. 1996). An effect of irradiation on the expression of cathepsin B in newly excysted juveniles (NEJ) of F. hepatica has been reported (Creaney et al., 1996). Irradiation of fluke with 3, 10 and 40 krad of y-rays significantly reduced the tissue expression of cathepsin B at 8-24 h post-irradiation in an apparently dose-dependent manner. Protease activity of ES samples collected over a 24 h period from irradiated and non-irradiated NEJ cultured in vitro was reduced when compared to ES from nonirradiated controls. This study shows that yirradiation of NEJ alters the expression of cathepsin B protease which may be detrimental to parasite invasion since the recovery of mature flukes from lt& 40 krad irradiated metacercariae is significantly reduced in sheep (Creaney et al., 1995b). Hemoglobin Recently a heme containing protein was isolated and characterised from the E/S material of adult F. hepatica (McGonigle & Dalton, 1995). This protein, with an apparent molecular weight greater than 200 kDa, was shown to have an absorption spectra similar to hemoglobins, although N-terminal sequence analysis did not reveal any similarity to known hemoglobin sequences. The protein was highly immunogenic in cattle after liver fluke infection; antihemoprotein antibodies were generated within a week of infection. The precise function of the Fasciola hemoprotein is unknown although a role in oxygen storage and/or transport is reasonably suspected. As proposed by the authors, the protein may provide oxygen for the oxidative metabolic processes in the low oxygen bile duct environment (Fasciola is primarily an anaerobic organism in the bile duct, see Tielens et al., 1984). Dalton et al. (1996) have performed a set of vaccination trials using FhCatLl, FhCatL2 and the hemoprotein. As mentioned above, FhCatLl alone yielded up to 69% protection, hemoprotein alone gave 44% protection and combination of the two antigens gave 52% protection. However, immunising with the hemoprotein and FhCatL2 increased efficacy to give 72% reduction in worm burdens (Table 1). Impor-

tantly, this combination also resulted in a 98% decrease in fecundity, perhaps due to a reduction in oxygen delivery by the hemoprotein. Egg shell production requires oxidative metabolism (Bjorkman Kc Thorsell, 1963) and interference with this process may affect the fecundity of the liver fluke. Thus, using such a combination vaccine will not only reduce damage to cattle livers, but contamination of pasture and subsequent infections will be dramatically lowered, potentially leading to elimination of the parasite from vaccinated areas (Spithill & Morrison, 1996). IMMUNE

RESPONSES IN INFECTED DURING FASCIOLA INFECTION

ANIMALS

In mice, and probably in humans, there is a functional dichotomy in the helper T (CD4+) response. Thl (Type 1) cells secrete y-interferon, IL2 and IL12 which stimulate inflammatory immune responses, such as DTH, and the production of complement fixing antibodies: this suggests that the primary function of Thl cells is to elicit phagocyte-mediated defence against infections since the Thl cytokines promote the ability of macrophages to phagocytose and destroy microbes. Th2 (Type 2) cells secrete IL4, IL5 and IL6 which regulate the humoral immune response (amount and isotype of antibody); Th2 cells promote B cell switching to IgE production, eosinophil activation and the production of non-complement fixing IgG isotypes (reviewed in Sher & Coffman, 1992; Abbas et al., 1996). Th cells progress through a ThO phenotype, a subset of Th cells which secrete both Thl and Th2 cytokines, before differentiating to Thl or Th2 cells. Thl and Th2 cells produce cytokines which act as autocrine growth factors as well as crossregulating each other’s development. For example, y-interferon amplifies Thl development, stimulates macrophage microbicidal activity and inhibits Th2 cell proliferation: IL4 antagonises the effects of y-interferon on macrophages, induces Th2 cell development and blocks activation of Thl cells. During the course of an immune response, both Thl and Th2 responses are induced and the final degree of polarization of any response is determined by several factors including the nature of the antigenic stimulus and the microenvironment in which the responding Th cells find themselves. For example, in mice infected with Leishmania major, a Thl response predominates in BlO.D2 mice whereas a Th2 response predominates in BALB/c mice; in contrast, chronic helminth infections tend to stimulate Th2 responses which may or may not be protective (Abbas et a/., 1996). The immunological mechanisms of resistance to helminth infections, which have been identified in

~mmunoiogi~alapproachesfor the controf of fasciolasis studies in animal models or in humans, have been reviewed (Wakelin, 1992; Sher & Coffman, 1992; Maizels et al., 1993; King & Nutman, 1993; Capron LIZCapron, 1994; Abbas et ab., 1996). The ability of animals to mount Thl or Th2 responses appears to be a critical determinant of the outcome of parasitic infection with Thl responses being important in certain systems and Th2 responses in other models (Wakelin, 1992; Sher & Coffman, 1992; Abbas eb al., 1996). Although the ThljTh2 cytokine network described for mice may not necessarily be identical in ruminants, this paradigm provides a biolagical framework with which to begin to understand the immune responses expressed during Fasciola infections,asdiscussedbelow(Brown et nl., 1994a,Brown et al., 1994b). The nature of the immune responseto Faxida infection in sheepand cattle is not yet clearIy defined (reviewedin Clery et al., 1996).Evidenceto date suggeststhat in animalschronically infectedwith Fuscioiu a non-Thl responseisdominant.IgGl isthe dominant antibody isotype in chronically infected cattle (Clery et al,, 1996 and referencestherein), sheep(Movsesijan et al., 1975;Sexton et al., 1994)and rats (Poitou ef ai., 1993).1gE(a Th2~ependent isotype) to Fir.&& antigenshasbeenidentified in infected cattle (Pfister, 1984),rats (Pfister el al., 1983; Poitou et al., 1993; Meeusen& Brandon, 1994)and sheep(Spithill et al., unpublisheddata). In acute F. hepatice infection of sheep infiltration of CD4+ T cells, B cells and inflammatory eeils(neutrophils, eosinophils,macrophages)is observedin the liver; in chronic primary infections, CD8+ and ySTCR+ cellsincreasein number in the liver. During secondaryinfection, CD4+ cellsincreasedrelative to CDS+ cellsand eosinophil and B cell infiltration was more pronouncedin the liver (Meeusenef at., 1995;Chauvin & Boulard, 1996). Systemiceosinophiliais observedfrom 3-13 weeks after primary infection of sheepwith I;: hepatica (Chauvin et al., 1995)and F. gigantica (Roberts et al., 1997a).Theseobservationssuggesta Th2-type of responseduring the hepatic stageof infection with

1227

type and Thl-type cellswere not detected.SinceThl cellswereisolatedfrom other genetically-identicalcattle infected with Buksia hovis, thesedata are consistentwith a down regulation of Thl responsesin ~~~~cjo~~-~~~~d cattle (Brown ef af., 1994a).CIery ef al. (1996) have shown that, in cattle chronically infected with F. hepatica,lymphocytes proliferating in responseto F. hepatica antigensfail to produce y interferon and suggestedthat a putative Th2 lymphocyte subset is induced as infections become chronic. Theseresultssuggestan inversecorrelation betweenchronic F. hepatica infection and induction of parasite-specificThl cellsand are consistentwith the hypothesisthat Th2 responsesare promoted in chronic fasciolosis.The corollary of this hypothesisis that Tht responses may beimportant determinantsof resistanceto Fasciola infection. A study of the parasite molecules which are pres~p~vely involved in inducing the apparently biasedTh responseduring chronic Fusciola infection will be informative. ACQUIRED ~~G~~~~C~

RESISTANCE INFECTION

TO FASCIOLA IN SHEEP

The yields of mature parasitesfrom primary and secondaryinfections of sheepwith F. heppatica show that sheepdo not acquireresistanceagainstthis parasite (Table 2; reviewed in Rickard & Howell, 1982; Haroun & Hillyer, 1986;Boyce et al., 1987).In the studieswith European fleecesheep,yields of F. hepaticu rangedfrom 16to 38% after primary infection, and From 13 to 31% after secondaryinfection and suchhigh yields of parasitesindicate that resistance to F. hepatica doesnot developin thesesheepbreeds (Boyce et al., 1987).In contrast, the situation with F. giganldcais curiousin that acquiredresistanceto this parasitehas been reported in sheep.A’Gadir et ad. (1987) reported a significant reduction in parasite numbersin Sudanesedesert sheepvaccinated with irradiated metacercariaeof F. gigantica; following a secondarychallenge with F. gigantica, recovery of adult parasiteswasreducedfrom 17% in control aniFUXi&. mafs to 3.4%. The study of Wiedosari & Copeman Uldham & Williams (1985)showedthat T cell pro- (1990)showedthat IndonesianThin Tail (ITT) sheep Iiferation and IL2 (a Thl cytokinef production in were highly resistantto F. gigantica basedon a comcattle was suppressed during the courseof infection parison of the yield of parasitesfrom primary infecwith t;l hepatica, suggestingthat F. hepatica immuno- tions, compared with the yields publishedin other modulatesits host. Similar suppressiveeffects on T studieswith this parasitein other breedsof sheep.In cellproliferation have beenobservedin infected sheep view of the failure to showacquired resistanceto F. (~imme~an ef af., 1983) and rats (Poitou et at., ~e~~~~~~ in sheep,theseresultsencouragedusto study 1992).ES products from ad&t F. hepatica suppress the resistanceof ITT sheepto F. gigantica. DTI-I responses in rats (Cervi et al., 1996)and modulate sheeplymphocyte responses in vitro (Jeffrieset al., Indanesian Thin Tail sheep acquire high resistance to 1996).Brown et al. (1994a)have shownthat antigen- F. gigantica specificT cell clonesisolatedfrom cattle chronically Recentstudieson the susceptibilityof ITT sheepto infectedwith i;. hepatica expressa ThOor Th2 pheno- challengewith F. gigantica showedthat the recovery

T. W. Spithill et al.

1228 Table 2-Summary

of recoveries of flukes in sheep infected with F. gigantica or F. hepatica

Breed ITTb W. African Dwarf Sudanese Desertd Merino St. Croix Finn/Rambouilleth Florida Nativeh Barbados Blackbellyh

F. gigantica” Primary Secondary l.l-5% l&17% 17% 25%’ 6.3, 6.7%9

0.32-1.1% 3.4% 12%’

F. hepaticaa Primary Secondary 31%

39%

23-53%’ 16%h 24% 27% 38%

23-53%’ 13.7%h 31% 16% 30%

a: mean % recoveries of flukes b: Wiedosari & Copeman (1990); Roberts et al. (1997a, 1997b); Spithill et al. (unpublished data) c: Ogunrinade (1984) d: A’Gadir et al. (1987) e: Roberts et al. (1996) f. Boray (1969); Sexton et al. (1990); Wijffels et al. (1994b); Creaney et al. (1996) g: Roberts et al. (1997a.b) h: Boyce et al. (1987) of adult parasites in naive ITT sheep at 21 weeks after challenge was only 1.1% and that this was significantly reduced further (to 0.32%) in sheep previously exposed to F. gigantica (Roberts et al., 1997a). Similar recoveries of parasites in naive ITT sheep were observed in a second experiment (Roberts et al., 1997b). Thus, naive ITT sheep are highly resistant to infection with F. gigantica and acquire a further degree of resistance following a primary infection. Further studies recovering immature parasites at different times post challenge showed that, at 68 weeks post infection, only 0.5 and 3% of parasites were present in livers of exposed or naive ITT sheep, respectively (Table 2) (Wiedosari & Copeman, 1990; Spithill et al., unpublished data). These results show that naive ITT sheep have an innate (or rapidly acquired) capacity to resist a primary immature F. gigantica infection within 6 weeks of challenge and that exposed ITT sheep acquire an additional degree of resistance. Table 2 summarises the recovery of parasites in various breeds of sheep. A challenge trial to assess the susceptibility of Merino sheep to a secondary infection with F. gigantica showed that Merino sheep also acquire a significant level of resistance to F. gigantica after exposure to a primary infection; recovery of flukes was reduced by 54% in the exposed sheep (Table 2) (Roberts et al., 1996). These results are intriguing in view of the extensive previous studies which failed to demonstrate acquired resistance in Merino and other sheep breeds to F. heputica (Rickard & Howell, 1982; Haroun & Hillyer, 1986; Boyce et al., 1987). In order to test whether the resistance of ITT sheep to F. gigantica extended to F. hepatica, naive ITT sheep and ITT

sheep exposed to a primary F. heputicu infection were challenged with F. heputica. No resistance to F. heputica was observed (Roberts et al., 1997a; Table 2). These results show that ITT and Merino sheep are competent to mount a protective acquired immune response against F. gigantica. The fact that ITT and Merino sheep cannot acquire resistance against F. hepUtica implies that either: 1. this immune response is not induced by F. hepaticu due to differences in antigen expression in the two fluke species 2. this response is ineffective against F. heputicu due to some defence mechanism operating in this species 3. this response is suppressed by F. heputica as a result of some factor(s) released by the parasite during infection. Clearly, the biology of the two Fusciolu species in sheep is different, suggesting that these two species differ in some fundamental way. Support for this conclusion comes from comparative infection studies in rats which show that the recovery of adult F. heputica (20-30%) and Japanese Fusciolu spp. (3647%) is high whereas the recovery of F. gigantica is low (O-5%) (Itagaki et al., 1995; Mango et al., 1972). Taken together, these results suggest that a comparative study of antigen expression and defence systems in juvenile/ immature F. gigunticu/F. heputica is warranted. In view of the known ability of F. hepaticu to modulate immune responses (Rickard & Howell, 1982; Zimmerman et al., 1983; Oldham & Williams, 1985; Poitou et al., 1992; Cervi et al., 1996) a study of the relative ability of F. giganticu/F. heputica infection to immunosuppress sheep may be informative.

Immunological

approaches

for the control

Mechanisms of immunity to F. gigantica in ITT sheep In light of these results, a key question now is: what are the innate and acquired mechanisms of resistance operating in ITT sheep? There are at least two possible mechanisms: 1.

(i)

(ii)

Innate mechanism The major component of the resistance in ITT sheep is innate ie. is expressed in naive animals on first contact with the parasite. Innate resistance could result from several mechanisms as discussed by Wakelin (1992) but two mechanisms warrant attention: Constitutive expression of a biochemical response which either directly debilitates the parasite or which initiates a cascade that leads to a “downstream“ rapidly induced effector response which allows ITT sheep to control infection. The Nramp gene is an example of a genetically programmed innate biochemical response which regulates the level of macrophage activation (phagocytosis, respiratory burst, expression of activation markers) following microbial invasion (Schurr et al., 1991; Skamene, 1994). A role for inflammatory reactions in the resistance of rats to infection with F. hepatica has been demonstrated (Baeza et al., 1994a, 1994b). It is now clear that the (early) innate cytokine response occurring at the outset of infection is a critical component of the acquired immune response (Kaufmann, 1995). For example, divergence of ThO cells into Thl or Th2 cells is regulated by the early innate response to infection which controls the production of IL4 or IL12 in the microenvironment of the responding lymph node (LN) (Reiner, 1994; Abbas et al., 1996). Genetically-determined differences in innate IL4 production or IL12 responsiveness by T cells would dramatically alter the cascade which leads to the acquired Thl/ThZ response; precedents exist for such genetically determined responses in mice (Pond et aI., 1992; Abbas et al., 1996). Whatever the mechanism, the innate response in naive ITT sheep must be expressed and manifested against the juvenile/immature fluke since most parasites are killed within 8 weeks of infection (Roberts et al., 1997a) and the recovery of parasites is reduced within 4-6 weeks of infection (Wiedosari & Copeman, 1990; Spithill et al., unpublished data). Some unknown physiological difference between ITT and other sheep (eg. a difference in vascularity or gut physiology) which inhibits migration of the juvenile flukes. For example,

2.

of fasciolosis

1229

the resistance of 129/J mice to infection with Schistosoma has been shown to correlate with the “leakiness“ of the hepato-portal system in these mice relative to susceptible mice, resulting in the shunting of the parasite to the lungs and parasite elimination (Mitchell et at., 1990). Ford et al. (1987) described shunting of microspheres from the portal system to the systemic circulation in F. hepatica -infected rats, indicating that physiological changes occur in animals infected with Fasciola. Acquired immune mechanism Acquired resistance could result from some immune response (antibody, cell mediated or antibody-dependent cellular cytotoxicity) which is induced and expressed rapidly (within a few weeks) following a primary or secondary infection. Wakelin (1992) and Gray & Gill (1993) have reviewed the literature on the genetic variation in animals of resistance to parasite infection. It is clear that genetically determined differences between animals can effect the rate of induction of protective immune responses to parasite infection. Precedents exist to support the hypothesis that a rapidly induced immune response could be responsible for the resistance expressed early by ITT sheep. Resistance of different mouse strains to infection with Taenia taeniaeformis correlates with the rate of appearance of protective antibody in resistant mice relative to susceptible mice (Mitchell et al., 1980). In mice, resistance to Trichinella spiralis correlates with the early activation of yIFN secreting T cells and little activation of IL4/IL5 secreting T cells (Pond er al., 1992).

Further studies on the basis for the high resistance of ITT sheep to F. gigantica are in progress to determine whether the resistance is innate or results from a rapidly acquired immune response and to shed light on the nature of the immune effector mechanisms which are able to eliminate this parasite.

IN VITRO STUDIES ON MECHANISMS ANTIBODY-DEPENDENT KILLING JUVENILE F. HEPA TICA

OF OF

Many studies on the host immune effector mechanisms directed against trematode parasites have focussed on the immature stages of S. mansoni. These studies identified several effector mechanisms in vitro and their potential roles in vivo are currently under investigation (James et al., 1990; Gazzinelli et al., 1992; Oswald et al., 1994; Wynn et al., 1994; reviewed in Sher & Coffman, 1992; Maizels et al., 1993; Capron

1230

T. W. Spithill et al.

& Capron, 1994). In contrast, similar in vitro studies using NEJ of F. hepatica have been unable to demonstrate irreversible damage to this parasite when incubated with sera and eosinophils or neutrophils (Doy et al., 1980; Duffus & Franks, 1980; Doy & Hughes, 1982). However, NEJ of F. hepaticu are killed when injected intraperitoneally into resistant rats, suggesting effector mechanism(s) present in the peritoneal cavity alone are able to kill the migrating parasite (Rajasekariah & Howell, 1977; Kelly et al., 1980; Davies & Goose, 1981; Doy & Hughes, 1982). Excysted juveniles of F. heputica which penetrate the gut wall of resistant rats are, prior to their destruction within the peritoneal cavity, coated with antibody and host cells, including eosinophils, neutrophils, macrophages and mast cells (Hughes, 1987). Monocytes/macrophages have not been specifically studied in the killing of NEJ liver fluke but are one of the major effector cells involved in the killing of parasites by the production of free radicals (James & Glaven, 1989; Smith, 1989; Golenser &Chevion, 1993; Liew & O’Donnell, 1993; Wynn et al., 1994). Such nonspecific defense mechanisms may be important in the resistance of various hosts to infection by F. hepUtica (Doy et al., 1981; Oldham & Hughes, 1982; Oldham, 1983; Hughes, 1987; Smith et al., 1992; Baeza et al., 1994a, 1994b). Resident peritoneal lavage cells (PLCs) of naive rats are a source rich in monocytes/macrophages and passive transfer of sera from F. heputicu-infected sheep, cattle or rats protect naive rats from F. heputica infection when injected intraperitoneally (Armour & Dargie, 1974; Hughes, 1987; Boyce et al., 1986). The juvenile parasite is killed before reaching the liver, suggesting resident PLCs such as monocytes/macrophages could be involved in the killing of NEJ liver fluke by a mechanism dependent on parasite-specific antibody. We have begun to study antibody-dependent cell cytotoxicity based on the production of free radicals by resident rat cell populations containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed in the peritoneal cavity of rats. Resident rat peritoneal, lavage cells were able to mediate significant ( > 60%) killing of newly excysted juvenile liver fluke in vitro (Piedrafita, D.M. Immune mechanisms of killing of juvenile Fusciola heputicu, PhD Thesis, La Trobe University, 1995; Piedrafita, Spithill &Parsons, unpublished data). The mechanism of cytotoxicity was dependent on the production of nitric oxide and attachment of effector cells to the NEJ tegument, which occurred following the addition of sera from F. hepaticu infected animals. The level of killing of NEJ liver fluke by LPS-stimulated rat PLCs was significantly correlated with the levels of nitrite

in culture supematants suggesting that greater than 40 PM nitric oxide is necessary to attain a biologically significant level of killing of NEJ liver fluke. These findings, therefore, suggest that killing of NEJ liver fluke by LPS-stimulated rat PLCs requires both attachment of effector cells to the NEJ liver fluke tegument and the production of high levels of nitric oxide at the parasite surface. This suggestion is consistent with the view that, due to the short half-life of nitric oxide, susceptible parasites are required to be in close contact with the effector cells producing the nitric oxide (McLaren & James, 1985; Liew & Cox, 1991). This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke in vitro and suggests that the ability of PLCs from F. hepaticu-naive rats to produce high levels of nitric oxide could be a mechanism by which migrating juvenile flukes are killed within the peritoneal cavity of rats (Piedrafita, D.M. Immune mechanisms of killing of juvenile Fusciola hepaticu, PhD Thesis, La Trobe University, 1995; Piedrafita, Spithill & Parsons, unpublished data). The importance of non-specific effector mechanisms, such as free radicals, in the resistance of both ruminants and rodents to Fusciolu infection are currently under investigation. FUTURE

PROSPECTS

The future prospects for the control of fasciolosis by immunological intervention appear to be bright. The high levels of protection seen in vaccine trials with several antigens of F. heputica suggest that successful development of a vaccine for Fusciola spp. is feasible but two key barriers need to be overcome. Firstly, the use of Freund’s adjuvant in the FABP and CatL/ hemoglobin formulations is not commercially acceptable and the discovery of other adjuvants which will elicit equivalent levels of efficacy is required. An understanding of the immune responses induced by Freund’s adjuvant which correlate with induction of immunity in cattle will clearly help in the choice of a new adjuvant. Interestingly, Freund’s adjuvant was not useful with the GST vaccine in cattle (Morrison et al., 1996), suggesting that the protective effector response induced by GST may be distinct from the response induced by the FABP and CatL/hemoglobin vaccines. Secondly, the production of recombinant molecules which induce levels of protection comparable to those induced by the native antigens is needed. This may not be a straightforward task as shown by the inability of a recombinant F. gigantica FABP construct to mimic the protection observed in cattle with the native FABP mixture (Estuningsih et al., 1997). The successful development of the recom-

Immunological

approaches

binant Tickguard@ vaccine for cattle tick and the Tueniu ouis vaccine for sheep demonstrates that the path to a commercial recombinant vaccine for ruminants is feasible (Lightowlers, 1994; Willadsen er OZ., 1995). A study of the innate and acquired effector responses in ITT sheep which are responsible for the high resistance to F. gigantica exhibited by this breed is clearly a priority and will provide new insights into the immune mechanisms which are capable of killing this parasite. A preliminary study of the genetics of resistance in ITT sheep suggests that resistance may result from the activity of a major gene with incomplete dominance (Roberts et al., 1997b). Abel et al. (1991) have described evidence for a major gene determining resistance to S. mansoni in humans. Recently, the genetic locus for this gene (SM 1) was mapped by a genomewide genotype screen (Marquet et al., 1996): significant linkage was obtained on chromosome 5q3 1-q33 with the colony stimulating factor 1 receptor (CSFlR) gene. The observation that SMl is closely linked to the CSFl R gene may suggest that resistance to S. rnan~o~~ infection in humans involves genetic polymorphisms in the CSFI receptor which, through modifying macrophage production and/or activity, contributes to enhanced killing of schistosome larvae. Taken together, these observations imply that resistance to trematodes may result from the effects of major genes, a somewhat surprising suggestion in view of the known complexity of genetic resistance to nematode parasites (Gray & Gill, 1993). Further work unravelling the immunology of resistance to F. gigantica in ITT sheep, together with molecular genetic mapping of the segregation of resistance in crossbred sheep, may lead to the identification of a candidate gene (s) involved in dete~ining resistance. A similar strategy was applied to successfully identify the Nranzp gene in mice (Schurr et al., 199 1; Skamene, 1994). Further in vitro studies on the effector responses which kill juvenile Fasciola in rats and sheep are clearly important as these will provide insights into the mechanisms which may be effective in viva. Questions to be answered include the mechanisms by which activated macrophages kill juvenile flukes, the nature of the antibody participating in the ADCC reactions and whether other inflammatory cells such as mast cells can also play a role in vivo. Knowledge of these effector responses will assist vaccine design and allow a rational choice of adjuvants which can promote the appropriate effector arm responsible for parasite killing. The observations that T cell responses are suppressed during the course of an F. hepatica infection in sheep (Zimmerman et al., 1983), cattle (Oldham & Williams, 1985) and rats (Poitou ef af., 1992). and that

for the control

of fasciolosis

1231

ES products from adult F. hepatica suppress DTH responses in rats (Cervi et al., 1996) and modify sheep lymphocyte responses in ritro (Jeffries ea cal., 1996), suggest that the developing parasite actively releases factors which interact with the host immune network. This parasite-mediated suppression may be responsible for the non-Thl phenotype observed in the overall immune response to F. hepatica infection of cattle (Brown et al., 1994a; Clery et al., 1996). Studies in mice have shown that helminth infections selectively induce Th2 responses; in mice infected with S. mansoni, it has been shown that the parasite releases antigens which induce Th2 responses as the infection matures (Sher & Coffman, 1992). A detailed study of the suppressor factors released by Fasciola (Cervi et al., 1996) will allow a better understanding of the Fascioia-host relationship and may identify molecuIes which could be targeted for immunoneutralisation which will benefit the host to the detriment of the parasite.

acknowledgements-This work was supported by the Australian Centre for International Agricultural Research, Canberra, and Monash University. David Piedrafita’s PhD studies were supported by a PhD scholarship from the Victorian Education Foundation.

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