Prospects for vaccines of helminth parasites of grazing ruminants

ELSEVIER

International Journal for Parasitology 29 (1999) I T--24

Prospects for vaccines of helminth parasites of grazing ruminants W.D. Smith* Moredun

Research

Institute,

Penrland

Science Park,

Bush Loan, Penicuik

EHXi

OPZ.

C’K

Received 26 March 1998; accepted 7 October 1998

Abstract Definedmolecularvaccinesfor severalruminanthelminthparasitesarebeingpursuedat severaldifferent Iaboratories. The most fruitful sourcesof antigen have beenoncospheresurfaceproteins, excretory/secretoryproducts and integral gut membraneproteins. Nematodegut membraneproteins are unconventional in that they do not come into contact with the host immuneresponseduring infection, a feature which bringsadvantagesaswell asdisadvantages.The genes encodingseveralof the protective antigenshave beencloned,but only in the caseof the oncospheresutfaeeproteinshas substantialprotection been reported with recombinantversions. In addition to the problem of identifying suitable expressionsystems,issues suchaschoiceof adjuvant and/or the possibleuseof a vaccinevector haveto be solvedbefore molecularvaccinesfor the economicallyimportant helminthscan be launched.Of the latter, it seemsthat vatines for Huemonchus andFasciola are the brightestprospects.0 1998Australian Society for Parasitology.Publishedby Elsevier ScienceLtd. All rights reserved. Keywords: Helm&h; vaccines; Ruminants; Oncosphere: Excretory/secretory and gut membrane proteins; Natural and hidden antigens: Recombinant protective antigens; Adjuvants

--.-_.__

1. Introduction

With the notable exception of that for the bovine lungworm, Dictyocaulus viviparus, there are no commercially available vaccines for the control of helminth infections in ruminants. Liver flukes and gastrointestinal nematodes are nearly always controlled by a combination of anthelmintic drugs and pasture management. Although this practice can be extremely effective, it is not sustainable, as the situation is threatened by the advent of strains of worms resistant to anthelmintics. *Corresponding author. Tel: 44-131-445-6131; Fax: 44-131445-611 1; e-mail: smitdiii mri.sari.ac.uk.

Partly

because of the expense involved

in

developing new anthelmintic compounds, several laboratories have been investigating the potential of vaccines against ruminant helminths. In the Iast decade, considerable progress has been made in identifying candidate vaccine antigens for several important helminth species and several reviews have recently been published [l-6]. This review concentrates on some of the strategies that are being used to achieve this goal and provides an up to date list of the various protective antigens that have been discovered for the different species of ruminant helminth. Several antigens have been claimed to be protective previously, but the supporting evidence has apparently not been published, presumably for commercial reasons (e.g. [3]). For further antigens.

0020-75 19!99!$ - see front matter lcz 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights rerervrd. PII:s0020-7519(98)00195-7

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the published protection data is either unconvincing (e.g. [7, S]), not statistically significant (e.g. [9]). not reproducible [lo], or from experiments with laboratory animals (e.g. [I I]). None of this work is included in this review.

2. Vaccine strategies

In the 1960s it was discovered that infection with larvae which had been attenuated by irradiation could stimulate a high degree of protection against challenge with normal infective larvae. This discovery led to the development of “Dictol”, a vaccine which is still sold today. Attempts were immediately made to extend the principle to the gastrointestinal helminths, but unfortunately it was found that while the method worked well under experimental conditions for Haemonchus contortus and Trichostrongylus colubriformis in sexually mature sheep, in field infections the protective effect was either too weak or too variable to be a practical proposition, particularly in young lambs. Of course, no helminth vaccine can be commercially successful if it is not efficacious in young, highly susceptible ruminants on pasture. For reasons that are still unclear, immunity to Dictyocaulus spp. can develop rapidly and effectively in the calf or lamb, whereas immunity to the gastrointestinal nematodes is acquired much more slowly. Much research has been and is still being conducted into the mechanisms of naturally acquired immunity to gastrointestinal helminth infections of sheep and cattle, but the final effector mechanisms are not known. Unfortunately the situation seems to be complex, involving a combination of local hypersensitivity, cell mediated, antibody and inflammatory responses and is complicated further by the natural unresponsiveness which exists in the young lamb or calf and in the dam around parturition. Only if and when these mechanisms and the respective antigens which trigger them are finally unravelled, can rational attempts be made to artificially stimulate them. The vaccine strategy currently enjoying most success largely ignores the mechanisms of natural Dictyocaulus

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immunity, but attempts to direct responses towards potentially susceptible targets on or secreted by the parasite. For example, with the cestodes Taenia ozk, Taenia saginata and Echinococcus granulosus, molecules on the surface of the delicate oncosphere stage have proved to be an Achilles heel, whereas for Fasciola hepatica proteases excreted by the parasite are also candidate protective antigens. These are termed “natural” or “conventional” antigens because they are recognised by the host during the course of infection. However, in the case of bloodfeeding H. contortus, the luminal surface of the intestine has been a particularly rich source of suitable target molecules. Because molecules on the luminal surface of the parasite intestinal cells are not normally recognised by the host during infection, these antigens are classified as “hidden”, a feature which raises some unconventional concepts relating to vaccination strategy. Examples of “hidden” and “natural” protective antigens for ruminant helminths are described in more detail below. The general approach for either type of antigen has been first to screen candidate protective fractions enriched for the parasite target in preliminary protection trials, second to purify the protective components as much as possible, and finally to isolate and express the genes which encode these so that a functional recombinant protein can be produced. As the adult stages of the economically important species of ruminant helminth cannot be cultured satisfactorily in vitro, it can be relatively difficult and/or expensive to obtain sufficient quantity of worms for the first two of these steps, and large numbers of parasite donor animals are required. This partially explains why so much more work has been done with ovine rather than bovine parasites, the exception being adult Fasciola which can be obtained relatively cheaply from the abattoir. It is generally assumed that if protective antigens can be identified in a nematode species which infects sheep, it should be relatively straightforward to identify the homologous proteins in the equivalent bovine parasite. Of course, the final step of producing a functional recombinant antigen is crucial to the whole approach, because a helminth vaccine which consists of native worm antigen can never be a commercially viable proposition.

W.D. Smithjinternarional

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3. Natural antigens Technically, the most advanced defined antigen vaccine for ruminant helminths, indeed for any helminth parasite, is that for T. ovis, where three distinct, highly protective recombinant antigens have been synthesised. All three are oncosphere antigens which are recognised serologically by naturally infected sheep. The first one to be discovered, designated To45W, [ 121 has been tested extensively with great success in field trials in New Zealand (Table 1). The protective effect is antibody mediated and is passively transferred from ewe to lamb by maternal antibody. Two further antigens known as To 18 and To 16.17 appear to be just as promising [ 131. However, T. ovis is not a human or sheep pathogen. Although harmless, the cystic stage in meat has occasionally been a trade barrier for the export of lamb from New Zealand, as it presents an aesthetic problem to the diner. Therefore, for political and marketing reasons, commercialisation of the vaccine has not yet occurred [14]. The same approach is showing similar promise for T. saginata and E. granulosus where recombinant oncosphere antigens have also been highly effective in immunising cattle and sheep, respectively (Table 1). As the importance of both parasites is largely zoonotic and mainly a problem of developing countries, it remains to seen whether these scientific successes will be translated into commercially practical control measures, especially as the diseases can be prevented by a combination of meat inspection, improved sanitation and other conventional control measures. Cathepsin L from F. hepatica is an example of an

Table 1 Candidate Parasite

recombinant

vaccine

antigens

from

cestodes which

Antigen

Mol.

To To To Tsa EC

45 16 18

45W 16.17 18 9fTsa 95

18 23

wt (kDa)

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enzyme which is recognised by the host immune response following infection, and which can induce high levels of protection when used as a vaccine antigen. Vaccination of sheep with native enzyme reduced F. hepatica egg production by 70% and egg viability by more than 80% [ 151 (Table 2). Whereas one report in cattle claimed 50% reduction in fluke numbers, this effect was improved to 70% in cattle immunised with cathepsin L combined with fluke haemoglobin, and it was found that almost 100% of the eggs shed by the vaccinated animals failed to embryonate, suggesting that parasite transmission would be enormously reduced 1161. Curiously, cathepsin L did not seem to work as a protective antigen for Fasciola gigantic-a [ 171. Interest in this protease was aroused originally when secretions from F. hepatica were found to degrade immunoglobulin and it was suggested that if the enzyme responsible could be neutwlised by antibody, the flukes would become more susceptible to the host response [18]. Flukes recovered from cattle vaccinated with cathepsin L were retarded, as might be expected if this hypothesis were true, but the mechanism which inhibited egg embryonation was not clear [ 16]. The most recent results suggest that antibodies of the IgC2 isotype correlated best with protection [6]. Striking protective effects have recently been reported against I-I. contortus, using two proteins of 15 and 24 kDa from adult worm ES products which are recognised serologically by infected sheep 119, 201 (Table 3). The nature and biological function of these molecules has yet to be determ,ined, but it will be interesting to see whether these antigens are as effective in the young lamb and per-

have given protection ___Host (cysts)

Sheep Cattle Sheep

in cattle or sheep

-.-

% Protection

Reference

94 13 99 99

II4 [I31 L131 [W [531

97

20

W. D. Smithilniernational

Table 2 Candidate vaccine antigens from Fmciola

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spp. which, in native form, have given protection in cattle or sheep -~ % Protection Mol. wt Genes Type” isolated Host Egg viability Worms &Da)

Parasite

Antigen

F. hepatica

Glutathione S-Transferase Haemoglobin Catbepsins Cathepsin L 1 Cathepsin Ll + haemoglobin Cathepsin L2 -I- haemoglobin Fatty acid binding protein Fatty acid binding protein

F. gigantica

Journal

H

23-27

N N N N

Several

> 200 27

‘I ‘I

12 12-14

1 1 1

Sheep Cattle Cattle Sheep Cattle Cattle Cattle Cattle Cattle

51

19-69 60 80

41 39 98

44 0 43 52 72 55 31

Reference [38. 501 [401

[I61 1151 I161 [16,511

[I63521 [44, 491 [I71

“H, hidden; N. natural; ?. not known.

iparturient ewe where natural immunity is poor. Interestingly, the protective effect did not correlate

with antibody titre.

4. Hiddea~antigens

The gut membrane approach was first applied successfully to ticks in research which culminated in the launch of the recombinant vaccine against Buophilus microplus, the Australian cattle tick [21]. The principle is straightforward. The host is immunised with appropriate gut membrane proteins from a haematophagous parasite and a high titre circulating antibody response is raised. When the parasite subseqnently feeds on the host, it ingests antibodies which bind to functional proteins on the brush border of its intestinal cells, so that its digestive processes are compromised, leading to starvation, loss of fecundity and weakness. Eventually the parasite becomes detached and, in the case of the gastrointestinal species, is swept out of the gut by peristalsis. This technique is showing great promise as a method for controlling the blood feeder H. contortus, from which several different gut membrane proteins or protein complexes have been isolated, giving more than 80% reduction in eggs together with greater than 50% protection against

worm

numbers

when

tested under

experi-

mental conditions [22-251 (Table 3). So far most of these antigens have been identified as various

proteases which, it is presumed, are involved in the digestion of the blood meal [26-281. The thrust of current research is to produce these antigens in recombinant form. A major advantage of the hidden antigen approach is that, because the mechanism of immunity is quite different, it works in situations where natural immunity to H. contortus is weak or ineffective. Thus it has been shown that young lambs [29, 301, goat kids [31] and periparturient ewes [32] can be successfully immunised and that some protective immunity is even transferred by maternal antibody [32]. On the other hand, the fact that the antigens are hidden means that, unlike conventional vaccines, immunity is not boosted by infection. At first sight this seems a serious disadvantage, because it might seem that frequent immunisations would be required for an effective level of protection to be maintained. However, experimental evidence suggests otherwise. The mechanism of protection in lambs immunised by this method mainly affects late fourth stage and older worms, whereas incoming larval stages which are not yet blood feeders are largely unaffected [33]. Thus, when immunised lambs are subjected to repeated daily infections of larvae, to mimic the situation in the field, faecal egg counts and adult worm numbers are controlled by the vaccine and the continued presence and activity of the early larval stages stimulates a natural immunity which is capable of replacing the effects of vaccine immunity

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when this wanes [33]. Therefore, the hope, is that transmission of haemonchosis can be controlled both by immunising lambs before weaning and ewes during pregnancy to prevent the periparturient rise in egg count. Another potential advantage of hidden over natural antigens is that, because the host does not respond to them during natural infection, the parasite has not needed to adapt to counteract the host response. Thus hidden antigens are likely to be conserved, both within and probably between genera. Certainly, the protective gut membrane aminopeptidase from H. contortus, often referred to as Hll, has been shown to be effective against geographically distant isolates of worms and, on an equally practical note, against anthelmintic-resistant strains [33, 341. It is not entirely clear whether the gut antigen principle can be employed successfully against species of nematode which are not direct blood feeders. The precise diet of economically important genera like Ostertagia and Dictyocauius is not known, but they contain host immunoglobulin, presumably ingested with mucus, tissue fluids and/or serous exudates [35]. It remains to be determined whether the amounts consumed are adequate for this vaccination strategy to be effective, but there are at least two encouraging results with Ostertagia circumcincta [36] Table 2. Incidentally, even though “Dictol” is effective, because it is live and attenuated, it is cumbersome to manufacture and distribute, so that a vaccine for D. viviparus based on a defined antigen would be an attractive alternative. The glutathione S-transferases (GSTs) of F. hepatica were chosen as candidate vaccine antigens because homologous proteins from Schistosoma mansoni and Schistosoma japonicum had been shown to be protective in laboratory animal model infections [37]. Glutathione S-transferases are hidden antigens because they are not recognised serologically by sheep or cattle infected by this parasite [38]. Although they are present on the surface of the gut cells of F. hepatica, GSTs are also distributed in the parenchema and tegument of the parasite [39], and so it is not clear whether the mode of protection is analogous to that described for the nematode gut membrane enzymes. Glutathione S-transferases are thought to be involved in the flukes’ metabolism of

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xenobiotics, transport of anionic compounds and the detoxification of lipid peroxides. Sheep and cattle immunised with native GSTs isolated from F. hepatica have been protected on average by 49 and 29%, respectively, although the results from individual trials have been quite variable [38,40] Table 2. The effect depended very much on the adjuvant employed, but the formulation most effective against F. hepatica did not work against F. gigantica [17], suggesting that it was not possible to extrapolate results from one Fasciola species to another. It is not clear by what mechanism protection is induced, but the simplest possibility that anti-GST antibody neutralises these enzymes seems to have been discounted [40].

5. Vaccine formulation and mode of delivery

The choice of adjuvant or delivery system can clearly be vital for the success of any vaccine. In the case of the cestode oncosphere antigens, an adjuvant which stimulates a high titre antibody response is clearly what is needed. In the case of the nematode hidden antigens the requirement is the same, except that the response should also be prolonged as much as possible. Because the mechanisms of protection are not yet understood, it is not yet clear what the requirements of adjuvants for the fluke antigens are, but experiments to date have shown that the choice is likely to be critical [40]. For those species of nematode which reside in the small intestine, it may be necessary for a recombinant vaccine to stimulate facets of the mucosal response for effective protection to be induced. The possibility of using Salmonella vectors for achieving this is actively being pursued [41]. The concept of naked DNA vaccines is attractive, particularly because it could short circuit the need for producing functional recombinant proteins. Unfortunately, immunising sheep with DNA alone was not encouraging in the case of the 45W T. ouisantigen, since only low titre antibody responses were invoked and no protection against challenge was observed. However, sheep primed by this naked DNA and boosted by the same gene in an adenovirus vector had much higher antibody titres and

showed correspondingly high levels of protection following challenge [42].

6. How good do worm vaccines have to be?

Conventional wisdom suggests that a worm vaccine would have to approach the efficacy of either anthelmintics or bacterial and viral vaccines to be effective. In the case of the gastrointestinal nematodes of ruminants, it is more appropriate to consider a vaccine as an epidemiological tool to maintain low-level pasture contamination, rather than a weapon to abolish infection completely. Barnes, Dobson, and Barger developed a mathematical model for simulating Trichostrongylus populations in grazing sheep and compared theoretical vaccines of nominal efficacy with conventional control methods based on anthelmintic treatment [43]. They concluded that if the vaccine consisted of a natural antigen, only 60% efficacy in 80% of the flock would bring substantial benefits; and if the vaccine was based on a hidden antigen, then protection of 80% of the flock with 80% efficacy would give better control than a conventional anthelmintic programme.

7. Conclusions

Tremendous advances towards worm vaccines for grazing ruminants have been made in the last decade. Several highly protective antigens have been discovered and cDNAs encoding several of these have been cloned. Scientifically, the concept of a recombinant antigen worm vaccine has been proven for T. ovis. However, there are still considerable hurdles to be overcome before monovalent vaccines for Haemonchusand Fasciola reach the marketplace and the prospects for a multivalent nematode vaccine are more distant. Specific obstacles include the production of cost-effective recombinant versions of the protective antigens and appropriate adjuvants. On the other hand, if the rate of progress which has been achieved over the last 10 years can be sustained, then the next decade should see the launch of the first defined antigen helminth vaccine.

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intestinal nematodes. International Patent WO 9801550. 1998. [20] Schalhg HDFH, Van Leeuwen MAW, Comelissen WCA. Protective immunity induced by vaccination with two Haemonchus contortus excretory secretory proteins cn sheep. Parasite immunol 1997;19:447-453. [21] Willadsen P, Bird P, Cobon GS, Hungerford .I. Commercialisation of a recombinant vaccine against Roophilus microplus. Parasitology 1995;l lOS43S50. [22] Jasmer DP, Perryman LE. Conder GA, Crow S. Mctiuire TC. Protective immunity to Haemonchus conforms induced by immunoaffinity isolated antigens that share a phylogenetically conserved carbohydrate gut surface epirope. J Immunol 1993:151:5450-5460. [23] Smith TS. Munn EA. Graham M, Tavernor AS. Greenwood CA. Purification and evaluation of the integral membrane protein Hll as a protective antigen against Haemonchus contortus. Int J Parasitol 1993;23:271-280. [24] Smith WD, Smith SK, Murray JM. Protection studies with integral membrane fractions of Haemonckuicontortu.s. Parasite Immunol 1994;16:231-241. [25] Knox DP. Smith SK, Smith WD, Murray JM. Redmond DL Vaccines against helmintic parasites. International Patent Application No WO 95;/26402. 199.5. [26] Smith TS, Graham M, Munn EA, et al. Clontngltnd characterization of a microsomal aminopeptidase from the intestine of the parasitic nematode, Haemonckus. Biochim Biophys Acta 1997; 1338:295-306. [27] Redmond DL, Knox DP, Newlands 0, Smith WD. Moiecular cloning of a developmentally regulated putative metallopeptidase present in a host protective extract of Haemonchus contortus. Mol Biochem Parasitoi 1997;85:77 87. [28] Longbottom D. Redmond DL, Russell M, Liddell S, Smith WD. Knox DP. Molecular cloning and characterisation of an aspartate proteinase associated with a highly protective gut membrane protein complex from adult Ffaemnnchus contortus. Mol Biochem Parasitol 1997;88:63---7?. [29] Tavernor AS. Smith TS. Langford F. Munn EA. Graham M. Vaccination of young Dorset Iambs against haemonchosis. Parasite Immunol 1992; 14645656. [30] Smith WD. Protection in lambs immunised with Hurnron&us wntortus gut membrane proteins. RCs Vet Sci 1993:54:94-101. [31] Jasmer DP. McGuire TC. Protective immunity XI a bloodfeeding nematode (Haemonchus contortus) induced by parasite gut antigens. Infect Immun 1991;59:4412--417 [32] Andrews SJ. Hole NJK. Munn EA, Rolph TP. Vaccination of sheep against haemonchosis with Hll, a gut membrane derived protective antigen from the adult parasite: prevention of the peripartuient rise and colostral transfer of protective immunity. Int J Parasitol 1995;25:83Y X46. [33] Smith WD, Smith SK. Evaluation of aspects qrf the protection afforded to sheep immunised with a gut membrane protein of Haemonchus comortus. Res Vet Sci 1993:55: 1 9. [34] Newton SE. Morrish LE. Martin PJ, Montague PE. Ralph TP. Protection against multiply drug-resistani and geographically distant strains of Huemonchrrs contc~ ru\ by \wc-

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[35] [36] [37] [38] [39]

[40] [41]

[42]

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[45]

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