Inrernarionul Journalfor Parasirology Vol. 22. No. 7, pp. M-907. 1992 002&7519/92 $5.00 + 0.00 Primed in Grew Britain Pcrgamon Press Lrd 0 I992 Aurfrahn Society for Parasitology

INVITED REVIEWARTICLE

UNDERSTANDING CHRONIC INFECTIONS: EVOLUTIONARY CONSIDERATIONS, CURRENT HYPOTHESES AND THE WAY FORWARD

JERZY M. BEHNKE,*~ CHRISTOPHER J. BARNARD~ and DEREK WAKELIN*

*MRC Experimental Research Group and SBehaviour and Ecology Research Group, Department of Life Science, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.

(Received I3 February 1992; accepted 1 May 1992)

CONTENTS 1. INTRODUCTION 862 2. EVOLUTIONARY CONSIDERATIONS 863 2.1. The Nematoda 863 2.2. The host-parasite arms race 864 2.3. Elimination or regulation: optimal regulatory responses 865 2.4. Evolution of evasive strategies 868 3. MECHANISMS EMPLOYED BY NEMATODE PARASITES FOR EVADING 868 HOST IMMUNITY 3.1. Shedding surface components of the cuticle 869 3.1.1. Intestinal species 869 3.1.2. Tissue inhabiting species 872 3.1.2.1. L3 and L4 surface antigens 872 3.1.2.2. Adult 873 3.1.2.3. Microfilarial surface antigens 874 3.1.3. Overview 875 3.2. Antioxidant enzymes 875 3.3. Relocation in the tissues 879 3.4. Camouflage with molecules of host origin or expression of host-like molecules 879 on the surface: molecular mimicry 4. IMMUNOMODULATION 882 4.1. Characterization of immunomodulatory factors 883 4.1.1. Known factors and their modes of action 883 4. I. 1.1, Factors described to date 883 4.1.1.2. Smoke screen antigens/immune complexes 883 4.1.1.3. Prostaglandins and eicosanoids in 886 4.1.1.4. Ecdysteroids in nematodes 887 4.1.2. Possible factors and likely modes of action 887 4.1.2.1. Cytokine-like molecules 887 4.1.2.2. Other targets for parasite immunomodulatory factors 888 4.2. The lessons from host specificity 889 4.3. One system or more? 890 4.4. Stability or lability 890 4.5. Consequences of immunomodulation 891

tTo whom all correspondence should be addressed.

861 862 J. M. BEHNKE C. J. BARNARD and D. WAKELIN

5. GENETIC CONSIDERATIONS 891 5.1. Parasite genes influencing host responses 892 5.2. Host genes controlling protective responses 893 5.3. Host genes providing resistance to parasite immunomodulation 896 6. THE WAY FORWARD 896 ACKNOWLEDGEMENTS x97 REFERENCES 898

INDEX KEY WORDS: Nematoda: filariae: ; gastro-intestinal nematodes; immunity; evasion of immunity; chronic infections; genetic control of immunity; MHC genes; background genes: jmmunnmodulation; ecdysteroids, oxygen scavenging or antioxidant enzymes; host-parasite arms races: optimal regulatory responses; vaccination; surface antigens; shedding antigens; molecular mimicry; camouflage with host antigens; prostaglandins; eicosanoids; cytokines.

1. INTRODUCTION providing the vaccinee with immunologkl experience MANY of the jmportant nematode parasites of man of relevant antigens and hence enabling rapid acquired and his domestic cause long-term infections immune responses to be elicited, limiting parasite which do not seem overtly to be regulated by host survival and opportunity for the development of the resistance on primary exposure and which are slow to associated disease. However, this ideal may not prove elicit acquired resistance on repeated exposure. to be feasible in the case of nematodes. Under natural Parasitologists refer to such infections as chronic conditions acquired immunity, on which most (Behnke, 1987). Among species affecting man, Rr,eutor conventional vaccines depend, is weak and unlikely to ~~e~~cun~s is known to live for 15- I7 years following a achieve the high levels of efficacy which would be single inoculation of infective larvae and Onchocercu desirable for commercial (and medical) success. As WC volvulus takes 18 years to disappear from human will show later (see section 2.3), host immune defences populations in endemic regions, when transmission may well have evolved to restrict infections within has been abolished through the elimination of insect tolerable boundaries rather than to totally eliminate vectors (Roberts, Neumann, Gockei & Highton, 1967: offending pathogens. Plaisier. van Oortmarssen, Remme & Habbema, As in malaria, alternative strategies may need to hc 1991). In fact many filariat parasites have the capacity considered including anti-disease vaccines (aiming to to survive for long periods as adults (see Behnkc, 1987 curtail the success of the disease-causing stages of the for earlier review; lhcunculus insignis --200 days + . parasite and/or limiting host immunopathological Eberhard, Ruiz-Tiben & Wallace, 1988; Acantho- responses) and anti-transmission vaccines (anti- ~~e~~~~e~u vitrac>-2 years, Johnson. Orihel & Beaver, fecundity, aiming to limit the production of trans- 1974; ~re~n~~ahooliari-i 7 months + , Ho. Chew. Yap mission stages of the parasite within the vertebrate & Singh, 1987; Singh, Yap, Ho, Kang, Lim & Lim, host; Mitchell, 1984). One further approach which 1976; - years in patas monkeys, Orihel & may be applicable to all three of the above categories Eberhard, 1985) and as microfilariae (Dipetahnenzu has focused on so-called cryptic (also known as hidden gracile--60-100 weeks, Eberhard, 1986; Monanenla or concealed) antigens (Willadsen & Kemp. 1988: &1hu10~u-350 days -I-, Bianco, 1984). In domestic Munn, Greenwood & toadwell. 1987) in the hope that animals, ~~~~1#~2~~1u.~ ~(~~t~r~u.~ causes infections arti~ciaIly induced responses against functionally which last for 10 months and longer. ~ri~hostrttn~~,lus critical parasite molecules, not normally exposed tenuis lives for 2-3 years in infected grouse. f.~gcrprrs during infection, may provide an Achilles’ heal lagopus scoticus (see Shaw & Moss, 1989a) and birds through which immunity might be achieved. This subjected to trickle infection regimes (Shaw & Moss, latter approach aims to exploit the potential of the 1989b) or acquiring parasites naturally under field immune system to improve on naturally acquired conditions (Hudson, 1986) accumulate burdens immunity and if successful. should produce vaccines with little indication of immunity (Fig. 1). which would be independent of the limitations There is a pressing need to understand the mech- imposed on natural responses by selection (see section anisms of parasite survival, particularly because of the 2.3). However. such vaccines would be limited by the implications of evasive strategies for vaccination. nbsencc of natural reinforcement. since by definition Conventional vaccines (anti-infection) aim to exploit they target antigens which the host does not normally natural host resistance to parasitic infection by experience during infection. Understanding chronic nematode infections 863

Necator amencanus not disputed, but the means through which chronic infections are achieved remain controversial and the last 5 years have seen slow progress in this area. The purpose of this article is to provide a background to o Beaver (1988) understanding the significance of evasive strategies 8000 l Palmer (1955) employed by nematodes especially in the light of evolutionary considerations which are often neglected 6000 by ‘bench-bound’ parasite immunologists. Thus we begin by reviewing evolution of the phylum Nematoda and consider the origin of the host-parasite arms race in terms of selection pressures operating on each side. We conclude that the failure of vertebrate hosts to have evolved natural sterilizing immunity against _L nematode parasites is predictable in terms of optimal 0 60 120 180 240 resource allocation vis-ri-vis host fitness and that this in Months after infection I B turn may modulate the selection pressure for novel evasion strategies by parasites. We next review the tenus evasive strategies adopted by nematodes and show $ 60,000 that these can be understood in an evolutionary P ,m context. Finally we predict that there are many 5 50,000 strategies remaining to be discovered, none of which E g 40,000 are likely to be totally efficient in protecting parasites but which provide through an understanding of the 6 30,000 8 mechanisms involved, the possibility of altering the w” 20,000 balance of the host-parasite relationship in favour of the host. We make no apology for some of the 10,000 speculative suggestions throughout this paper, perhaps exceeding current evidence on occasion: such 600 700 0 100 200 300 400 500 was our brief. If these serve to rekindle interest in the Days after InfectIon subject, even if the hypotheses eventually prove unfounded, our objective will have been achieved. FIG. 1. Examples of chronic nematode infections. A. Longevity of americanus in two cases of self infection 2. EVOLUTIONARY CONSIDERATIONS (data redrawn from Palmer, 1955 and Beaver, 1988). B. The course of infection with Trichosbvngylus tenuis in grouse, as 2.1. The Nematoda monitored through faecal egg counts (data redrawn from The phylum Nematoda probably has its origins in Shaw & Moss, 1989a). the marine environment, although not all nema- tologists support this view. The phylum is divided into two classes, the Adenophorea and the , Two further reasons for understanding the and within each class parasitic forms arose mechanisms of chronic infections are apparent. Firstly independently (Anderson, 1988a), probably several vaccines based on defined antigens with proven lab- times (Inglis, 1965; Adamson, 1986, 1987) although oratory efficacy may never work under field conditions the exact number of occasions on which this may have since parasite evasive strategies, if not foreseen, may occurred is by no means certain. However, prove to be superior to the immunity induced through of animals [some 40% of the 16,000 recognized species vaccination. In fact susceptibility to infection rather are parasites (Anderson, 1988a)] is thought to than resistance may be increased when inappropriate have been adopted only after the terrestrial antigens are used (Harn, Cianci & Caulfield, 1989). environment had been colonized since all parasitic Secondly, and optimistically, by identifying from the forms can be traced to terrestrial ancestors (Anderson, outset the precise mechanisms of parasite survival it 1984, 1988b). At most it can be concluded, with may be possible to develop quite simple procedures to relative certainty, that present day nematode parasites improve on natural immunogenicity in the face of of vertebrates arose from several independent parasite evasion and to allow host-protective parasitic lineages. responses to develop where they would not do so The parasitic Secernentea are represented by the normally (see section 6). rhabditid-line and include taxa such as the The potential chronicity of nematode infections is (Filariae), , , and 864 J. M. BEHNKE.C. J. BARNARDand D. WAKEL~N

Rhabditida which collectively comprise some 92% of moist soil environments which were relatively stable. the nematodes parasitizing man and other vertebrates. did not have dauer larvae and there was no convenient The rhabditid-line probably evolved from free-living stage to modify into an invasive larva. Thus, in soil forms feeding on bacteria which gave rise to contrast to the Secernentea, there is no dependence on lumen-dwelling organisms feeding on the host’s infective L3 in the parasitic Adenophorea. For microflora before speciating further and adopting example in the t~chinelloids the Ll stages became the more complex life cycles and preferred tissue sites infective larvae although different larval stages are (Adamson, 1986). Parasitism may have evolved on as used by other Dorylaimids. Since none of the parasitic many as four separate occasions (Inglis, 1965) species in this taxa live entirely within the gut lumen, it although there is little certainty here. The Ascaridida is suggested that initially they invaded tissues, sub- and Spirurida may have had a common ancestor, sequently coming to be secondarily associated with the which began as a parasite of the intestine of terrestrial intestinal mucosa (Adamson, 1986). vertebrates, before radiating into two branches one of which, the Ascaridida, exploited other vertebrates 2.2. The host-parasite arms race whilst the other, the Spirurida, arthropods for inter- Evasion strategies appear to have evolved as a result mediate hosts (Anderson, 1984). The Ascaridida of selection pressure provided by the host’s immune continued primarily as parasites of the small intestine system; organisms with better evasion strategies are of vertebrates, taking advantage of food-chains to likely to have had a fitness advantage over those with provide suitable intermediate hosts and developing inferior mechanisms and, through their ability to more elaborate fife cycles, including migration survive longer, would have contributed their alleles to through host tissues, later, possibly when intermediate subsequent generations with greater frequency than hosts were dropped by genera such as . Prede- organisms contributing alleles for alternative cessors of the , Oxyurida and Strongylida strategies which succumbed earlier to the immune all adopted parasitism separately on land and system. Thus the interaction between the immune continued thereafter as parasites of terrestrial system, parasites and parasite evasive strategies invertebrates and vertebrates. The Oxyurida arose as parallels to some extent that between environmental parasites of invertebrates whilst the Strongylida may stressors, free-living animals and their adaptations for have commenced as parasites of reptiles (Inglis, 1965). life in a particular niche. It is essentially an ecological The latter taxon is still similar biologically and relationship in which the principal environmental morphologically to the primitive rhabditoids and may stressor is the host’s immune system. In each case. have been the last of the nematode lineages to take up relevant strategies evolved sequentially as animals parasitism or alternatively may have retained some of competed for survival, diversified into distinct genetic the primitive features of free-living predecessors. The lineages and refined their adaptations in the face of ancestors of all the Secernentea would have been continuing competition. The adaptability of parasites preadapted to parasitism by the possession of a to changing circumstances is all too familiar to resistant third larval stage, the dauer larva. which parasitologists and is clearly reflected in such phen- became modified into a skin-penetrating or oral omena as the onset of resistance to anthelmintics invasive stage (Anderson, 1984) and probably (Smith, 1990) and the incorporation of new hosts into secondarily invaded tissues after initially establishing parasite life cycles when infective stages are carried to as intestinal species. non-endemic regions, populated by suitable altern- The Adenophorea are subdivided into two major ative hosts (e.g. introduction of Fusciolu hepatira to groups, the marine Chromadorida and the Enoplida. Australia; Srhistosoma mansoni and Onchocercu The latter is further subdivided into two lineages, the ~~~l~ulf[sto South and Central America). Enoplina (which are predominantly freshwater and As the ancestors of modern parasites adopted a marine) and the Dorylaimina (which include many parasitic existence they would have experienced soil-dwelling species that gave rise to four parasitic primitive immune systems. Modern day cestodes, for lines, the mermithoids, the trichinelloids, diocto- example, are believed to have evolved from scavenging phymatoids and muspiceoids). Familiar taxa such as free-living Platyhelminthes which initially became ~~pi~i~ria, Trichuris and Trich~ne~~~are ail grouped in micro-predators on primitive chordates and then the Tr~chinelloidea. The parasitic taxa are believed to endoparasites (Lleweltyn, 1965). As hosts diversified have arisen from terrestrial predatory or plant so also parasites speciated in parallel, giving rise to parasitic, stylet-bearing forms such as the Dorylaimids contemporary phenomena such as the existence of (Steiner. 1917; Adamson, 1986) in which invasion of closely related cestode groups among closely related animal hosts may have been aided by a modified stylet vertebrate hosts and heirloom parasites in relict host apparatus. These taxa, originating from ancestors in species. Along with morphological and physiological Understanding chronic nematode infections 865

CHROMADORIDA (Marine and freshwater) ADENOPHOREA ENOPLINA lMar~ne and freshwater) (Enoplea or Aphasmidla) ENOPLIDA f ( k:::t?::::: ;%roood. Mollusc. \ / NEMATODA (e.g. Trichuns, Trichmella, Capillana) soil and parasitic) Dioctophymatadea

TYLENCHIDA (Soil plant and insect parasitic) (Tylenchid-line) RHABDITIDA (Insect, mollusc parasitic and so111 Rhabdltoldea (e.g. Rhabdlas, Strongykxdes)

r, OXYURIDA (AntwIld. Arthropod, Vertebrate parasitic) (e g. SECERNENTEA RHIGONEMATIDA (Insect, mollusc parasitic) (Rhabditea or Phasmidia: ASCARIDIDA (Mollusc. vertebrate parasitic) (Rhabditld-lw4 Ascarldoldea (e.g. Ascaris, Toxocaral SPIRURIDA Vertebrate parasitic) CAMALLANINA. Dracunculoidea ie g Druncunculus) SPIRURINA. Filarioldea kg , , Wucherew. Laomosofdes, Dirofilaua, Loa) STRONGYLIDA (Vertebrate parasltlcl Trichostrongylotdea (e.g. Nippostrongylus, Hel~gmosomo~des, Haemonchus. Trichostrongylusl Ancylostomatoidea (e.g. Necator, )

FIG. 2. The relationships between the higher taxa of the Nematoda. The figure is based on Anderson (1984, 1988a), the CIH keys to the nematode parasites of vetebrates (see Anderson, 1988a and Adamson, 1987). The position of genera referred to in the text is highlighted in italics. Alternative names to taxa are underlined in brackets. Note that the list of Secernentean superfamilies is not comprehensive. Only those of relevance to this article have been listed. changes taking place in both hosts and parasites, newly evolved host defence mechanism is likely to be strategies for avoiding recognition by the immune overcome sooner or later by an appropriate parasite system would have been evolved by parasites as the strategy reducing its overall contribution to protection vertebrate immune system itself became more refined of the host. On the other side of the coin, selection will in response to the threat posed by diverse micro- act on hosts refining their defensive mechanisms organisms as well as parasites. Thus the evolving further, maintaining fitness in the face of novel evasive vertebrate immune system presented an enormous strategies and exploitation by parasites. Microbiol- challenge to parasitic organisms but, like many other ogists who have succeeded in relating distinct environmental stressors, it proved susceptible to cir- mutations in plants and fungi to this arms race refer to cumvention and exploitation. In nematodes the the phenomenon as the gene-for-gene theory (Flor, distinct parasitic lineages described above would have 1971). had some intralineage similarities following evolution of evasive strategies among parasitic ancestors, but 2.3. Elimination or regulation: optimal regulatory contrasting lineage-specific evasive strategies resulting responses from different solutions among independent parasitic Parasite immunologists, particularly those working lineages for evading host defences. It is perhaps too on helminths, are often concerned with the need to early yet to test this prediction rigorously since we develop sterilizing immunity in immunoprophylactic have no clear concept of how any single nematode treatments both for treatment of the individual and for evasive strategy operates but this review will attempt control of parasites at the population level. This to piece together the existing data and an overall objective is seldom achieved (Abraham, Grieve, Mika- comparison of lineage-specific as well as lineage- Grieve & Seibert, 1988) and may be beyond the scope shared strategies will be presented. The essential of existing vertebrate immune systems as designed by comparisons will be based on the subdivisions of the nature to cope with natural exposure to parasites. The phylum Nematoda as presented in Fig. 2. In time, as drive for sterilizing immunity has arisen because further and perhaps novel strategies are unearthed, it parasitic helminths such as nematodes are consider- will be necessary to modify the scenario which we ably more obvious than viruses or bacteria and even a present. few persisting individuals are often very apparent and It is thus apparent that an arms race exists in which likely to be interpreted as indicating the failure of hosts and their parasites are involved in a series of treatment. For this reason, if anti-nematode vaccines escalating mutual counter-adaptations to exploit or are to succeed in medicine they will need to induce inhibit exploitation (Behnke & Barnard, 1990). Each sterilizing immunity, so that the efficacy of treatment J. M. BEHNKE. C. J. BARNARD and D. WAKELIN

Paraslte burden Degree of reduction

D

~ Cost 1 species - cost 1 species - Cost 2 species -Benefit - Cost 3 speues -m- lnclus~ve fitness benefit ~~~ Benefit

Degree of reduction Degree of reduction

FIG. 3. A. A hypothetical cost function curve for increasing parasite burden. The cost to the host is assumed to increase disproportionately with increasing burden (see text). B. Hypothetical costs and benefits accruing to the host from different degrees of reduction in parasite burden. R,,,,, degree of reduction conferring maximum benefit: cost ratio. C. as B but allowing for inclusive fitness advantages in reducing reservoirs of infection (see text). D. as B but taking into account effects of reducing the burden of one parasite on the ability to combat others (see text).

could not be misinterpreted by patients or health cart function of parasite burden. personnel. The failure of the vaccine against canine In malaria, especially in P. ,fdciparum, it is now disease is a case in point (Miller, 1971). recognized that much of the pathology is generated Even if a vaccine were to cause significant reduction in through overzealous production by the host of part- morbidity, nothing less than total parasite eradication icular cytokines, whose primary objective is to mediate is likely to convince sceptics that such treatment has a protective immunity (Clark, Hunt & Cowden, 1986; role to play in human and even veterinary medicine. Clark & Howell, 1990; Clark, Rockett & Cowden, This may be a vain objective, however, because the 1991). The acute intestinal response to nematode natural immune response on which vaccines depend infections also involves major alterations in organ may not be designed to produce sterilizing immunity. function (Castro, 1990; Castro & Bullick, 1983) and Indeed, the scarcity of sterilizing immunity among temporary disturbances which can be life threatening. host species in nature suggests that it is rarely favoured Rats infected with Nippostrongylus hrasiliensis or by natural selection. At first sight this may seem Trichinellu spirulis may show inappetence (Crompton, surprising since we might expect the host’s survivor- Walters & Arnold, 1981; Castro, Copeland. Dudrick ship and reproductive potential to increase in relation & Ramaswamy, 1979; Ovington, 1987) and conse- to the degree of elimination of a parasite. This is likely quent weight loss (Crompton et al., 1981; Zohar & to be a naive expectation, however, because it ignores Rau, 1984). become less active and lethargic, and in both the cost of elimination to the host and the fact extreme cases may die during the period when the that the cost of infection and thus the benefit of intestinal inflammatory response is at its peak. In the attacking the parasite may not be a simple linear wild, this would have important consequences for Understanding chronic nematode infections 867 survival: host-protective immunity may be costly. benefit of a given reduction in burden is corres- In regions where Bancroftian is endemic, pondingly high so that the slope of the benefit curve is sensitivity to parasite antigens in a minority of cases is initially steep. As the burden, and thus the cost to the associated with pathology, notably elephantiasis host, decreases, the benefit accruing from the same (Ottesen, 1984). Such patients are almost invariably degree of reduction also decreases and the slope of the amicrofilaraemic and both microfilariae and adult benefit curve begins to decline. worms are hampered if not destroyed by a combin- Simply looking at the benefit to the host of reducing ation of antibody and cell-mediated responses its parasite burden, however, ignores the cost to the (Connor, Palmieri & Gibson, 1986; Nutman, 1989). It host of the reduction effort itself. There may be several would appear, therefore, that in filarial infections, components to the cost of reducing a given parasite invoking a host-protective response to limit parasite burden. Here, we shall assume expenditure of meta- development is costly in terms of the pathology bolic resources, host-induced pathology and compro- generated by relevant host effector mechanisms. mised response to other parasite species. The cost However, many indigenous people remain clinically curve in Fig. 3b assumes that the cost in these terms of asymptomatic with minimal detectable parasite-spec- a small reduction in burden is low but increases with ific antibody and cellular activity despite the presence the degree of elimination. The ratio of the benefit and of adult worms and associated microfilariaemia cost curves at any point determines the net benefit to (Ottesen, Weller & Heck, 1977; Ottesen, Weller, the host of different degrees of reduction in the burden. Lunde & Hussein, 1982; Piessens, McGreevy, Since natural selection will favour the degree of Ratiwayanto, McGreevy, Piessens, Koiman, Saroso & reduction that maximizes net benefit, the optimal Dennis, 1980). Tolerance induced prenatally or as a response may turn out to be moderate rather than result of antigens in breast milk (Petralanda, Yarzabal total elimination (Fig. 3b). Changes in the form of the & Piessens, 1988) as well as parasite-mediated benefit and/or cost curves will shift the optimal immunodepression may play their roles in down- response towards greater or lesser reduction. regulating host responses in such patients (Nutman, Several factors, of course, may affect the form of the Kumaraswami & Ottesen, 1987) but, from the cost and benefit curves. The simplistic example evolutionary perspective, it is conceivable that the cost discussed above considered benefits and costs only to the individual of responding to infection, from the point of view of the reproductive success of particularly in terms of impaired reproductive the infected individual. While incomplete elimination potential, may be sufficient to confer a selective may be the optimal response in these terms, such a advantage on those who moderate their response with strategy leaves a residual infection which may be the resultant spread of alleles for poor responsiveness transmissible to other potential hosts. If vulnerable and/or susceptibility to tolerance induction among hosts include relatives of the infected individual, local populations. It is pertinent that few immigrants inclusive fitness (Hamilton 1964a,b) benefits may from non-endemic regions, who would not have been accrue from reduction beyond the individual host’s subject to comparable selection pressure for poor optimum, resulting in an increase in the optimal responsiveness to filarial antigens, show micro- response (Fig. 3~). Two points thus emerge here. filariaemia even after prolonged exposure but many Firstly, optimal responses for individual hosts may fall develop elephantiasis (Beaver, 1970; Trent, 1963; far short of elimination and thus maintain trans- Connor et al., 1986). missible reservoirs of infection within populations. If we consider the cost of infection by a given Secondly, this reservoir may be reduced if there are parasite to be a reduced probability of the host inclusive fitness advantages to reducing the risks to reproducing due to the loss of metabolic resources and uninfected conspecifics. parasite-induced pathology, it is reasonable to As we have already indicated, the cost of reducing suppose that, in many cases, the cost will be an the burden of one parasite may include a compromised accelerating curvilinear function of parasite burden ability to respond to others. The resources available to (Fig. 3a). Increases in burden when the overall burden a host to combat parasites are likely to be limited, is low make little difference to the host’s prospects of resulting in an adaptation budget (Dawkins & Krebs, reproducing. As the burden increases. however, 1979) which has to be allocated according to resource loss and/or parasite-induced pathology mean competing demands from different parasite species that further increases are likely to be very detrimental (Behnke & Barnard, 1990). Increasing expenditure to the host. Cost functions of this form mean that a law against one species is thus likely to detract from of diminishing returns is likely to operate on the expenditure against others, potentially increasing the benefits of attacking the parasite (Fig. 3b). Thus at cost to the host in terms of reduced reproductive high burdens, when the cost of infection is high, the success. The cost of reducing the burden of any given 868 J. M. BEHNKE.C. J. BARNARD and D. WAKELIN

parasite is therefore likely to increase with the number 2.4. Evolution ofevasive strategies of other parasites exerting demands on the response The pressures for the evolution of evasive strategies budget (Fig. 3d). Furthermore, different individuals can be compared to the development of anthelmintic are likely to have different R,,, points for any given resistance, a topic which has received considerable parasite because the costs and benefits of responding attention in recent years because of its importance to will vary with, for example, sex, age, social status, both human and veterinary medicine (Smith, 1990). other parasites, etc. Variation in parasite burdens is As with drug resistance it is assumed that evasive thus predictable in terms of phenotype-limited strategies are costly to the parasite, since they are likely optimal responses as well as other classical mech- to divert available resources away from reproduction. anisms (variation in exposure, genetic variation in The expression of alleles encoding evasive devices is innate and acquired resistance, etc.). therefore likely to be governed by the overall fitness The above argument suggests that a limited res- advantage they confer and, in the absence of a threat ponse to infection may arise because hosts trade off the from the immune system, their frequency in the costs and benefits to themselves of different degrees of re- population would decline. The rate at which the sponse. It may be, however, that a limit is imposed not frequency of alleles for resistance to anthelmintics (or by the relative costs and benefits to the host, but by the in this case evasion strategies) increases in the popula- inability of the host to reject the parasite even though it tion is known to increase as the frequency of treatment would benefit by doing so. As Behnke & Barnard and the proportion of the target population exposed to (1990) point out, the arms race between host and para- the anthelmintic increase. Thus in relation to evasion site is an asymmetric one (Dawkins & Krebs, 1979) in strategies, the stronger the immune response the more which there may frequently be a built-in advantage, likely alleles for evasion will appear sooner and spread for instance through shorter generation times, to the throughout the population. It follows from this that parasite. As a result, parasites may experience lengthy an inefficient immune system will be less encouraging periods of grace before hosts counter-adapt, causing for the evolution of evasive strategies than a more departure from the optimum host response. Attempts efficient system and therefore the larger the propor- to stimulate natural immunity by vaccination during tion of non-responders in the population (see later) the such periods of grace may well be fruitless. less the pressure for evasive strategies. The same It is therefore evident that it may be beneficial to relationship should hold true for tolerance of increas- have a selection of available defensive strategies at a ing residual worm burdens. low degree of alert which nevertheless maximize Another conclusion from studies of drug resistance reproductive output despite the penalty of carrying may also be applicable here. It appears that drug some parasites and associated loss of metabolic re- resistance develops sooner in trichostrongylid nema- sources. By aiming to limit pathology rather than to todes, which have both free-living and parasitic stages invoke mechanisms capable of sterilizing immunity, the in the life cycle, when the proportion of the worms energy budget of the host can be distributed more outside the host is low. Unfortunately, no drugs with widely leaving fewer holes in the overall repertoire of de- high efficacy have been available until recently for fence. It is this important interpretation of the role of treatment of filarial nematodes which have no external the immune system vis-ci-vis host fitness which is fre- free-living phase and there is little evidence for drug quently misunderstood by those seeking to develop vac- resistance. However, the recent introduction of iver- cines against helminths. The vaccine against Ancylo- mectin will predictably result in the rapid generation stoma caninum was totally effective in eliminating the of resistance among filariae, more so than among principal pathological consequences, haemorrhagic other species, because there is no free-living reservoir anaemia, of canine but was of infection in this group. It is likewise tempting to unsuccessful commercially because it was non-sterilizing, speculate that evasive strategies should be more vaccinated animals continuing to carry a few worms and efficient in this group for the same reasons and indeed pass eggs. This was erroneously interpreted as indicat- this generalization does appear to hold true: the ing its failure to control the disease (Miller, 1971). It is filariae are renowned for the chronicity of the infec- important to stress here that while the lack of steriliz- tions they cause and for the success of their survival ing immunity as a yardstick for failure of a vaccine can strategies in the face of host resistance. be criticized on the basis of evolutionary arguments, this has most force in disease management at the 3. MECHANISMS EMPLOYED BY NEMATODE individual level. However, if part of the goal of the PARASITES FOR EVADING HOST IMMUNITY vaccine is eradication of disease at the host population Whilst a number of potential evasive strategies have level, then non-sterilizing immunity, or at least reduction been described for nematode parasites there is still no of parasite burdens below the threshold needed to main- irrefutable evidence that any, apart from immunomod- tain transmissibility, may be a legitimate criterion. ulation, play a significant role in evading host immunity. Understanding chronic nematode infections 869

3. I. Shedding surface components of the cuticle prominent serum IgA response to surface antigens was With the application of radio-iodination to the shown to correlate with the onset of worm expulsion study of surface antigens of nematodes it has become (Almond & Parkhouse, 1986). The extent to which evident that the cuticle is not entirely inert as parasite-specific serum IgA participates in luminal previously believed (Philipp & Rumjanek, 1984). events is not clear, but T. spiralis is partially intra- Surface components are shed when labelled parasites cellular, living within tunnels created in a syncytium of are maintained in vitro and if these constitute targets cytoplasm surrounding the parasites following local for specific antibody through which cellular cytotoxic breakdown of enterocyte cell boundaries. It is possible mechanisms may surround and destroy parasites, their that serum antibody therefore has some direct access potential role in aiding survival is not difficult to to worms, without the need to recirculate through the appreciate. However, if such a mechanism is to hepatobiliary system in order to gain access from the function in vivo, it requires continuous synthesis, lumen. Secondly it has also been shown that mucosal transportation, expression on the surface and shed- mast cell proteases can digest nematode collagens ding. If this cycle were to be inhibited or merely slowed (McKean & Pritchard, 1989). On this basis it has been down, the worms should become susceptible to host proposed that parasite-surface-specific antibody may effecters. However, it is not at all clear from published encourage the loss of the outermost cuticular work on nematodes whether shed antigens are components revealing unprotected deeper underlying replaced after shedding (Table 1). A comparable elements now susceptible to proteolytic damage (Prit- mechanism is known in S. mansoni where initial in chard, McKean & Rogan, 1988). vitro estimates of the half life of surface tegumental It is easier to appreciate the significance of cuticular proteins were considerably shorter (Wilson & Barnes, shedding in the biology of tissue migratory stages of 1977) than those indicated from in vivo studies based nematodes. Hookworm larvae are often quoted as on the period necessary to allow worms to adapt to having the capacity to shed surface antigens, but to new hosts following transplantation (Smithers & date only one species, Ancylosto~a ~an~~u~ has been Terry, 1976). However, experimental studies which examined in this context. Antibodies to the surface allowed accurate quantification of shedding of determinants of A. caninum L3 bind to frozen worms isotopically labelled surface proteins in vivo, sub- but not to live parasites. However, binding can be sequently confirmed that indeed the half life of surface demonstrated on live worms which have been tegumental proteins was of the order of 5.4 days inhibited by azide (Vetter & Klaver-Wesseling, 1978). (Saunders, Wilson & Coulson, 1987), considerably Although adherence of inflammatory cells to L3 has longer than intuitively would seem to be compatible been demonstrated in v&o, there is considerable doubt with tegumental turnover as a principal defence whether the latter actually play a significant role in against offensive cellular activity. protection in vivo. Live, exsheathed larvae activate 3.1.1. Intestinal species. Surprisingly few nematode complement by the alternative pathway and bind C3 parasites have been examined with respect to shedding to their surface (Klaver-Wesseling & Vetter, 1979). of cuticular antigens. The initial studies on T. spiralis Leukocytes are attracted to C3 covered larvae and (see Philipp, Parkhouse & Ogilvie, 1980), N. ~ras~Z~en- adhere to their surface but cellular binding is sis (see Maizels, Meghji 81Ogilvie, 1983) and Toxocara considerably enhanced in the presence of antibody. canis (see Smith, Quinn, Kusel & Girdwood, 1981; This interaction between C3 and antibody is syner- Maizels, Desavigny & Ogilvie, 1984; Badley, Grieve, gistic because neither component alone is capable of Rockey & Glickman, 1987) demonstrated loss of mediating as intense cytoadherence as when both are surface label in vitro over the course of some 20 h but present (Klaver-Wesseling, Vetter & Schoeman, whether surface proteins are replaced in vivo in these 1982). It is also likely that C3 somehow stabilize the species is still not known. Moreover, the relevance of adherence of antibody to the cuticular surface since surface antigen shedding in lumen-dwelling species is antibody alone will not bind to live worms. Under questionable. Such a mechanism can conceivably optimal conditions in vitro, several layers of leukocytes serve a purpose in removing anti-cuticular antibodies, will accumulate around larvae, but the worms remain but there is no evidence that surface-specific active for 5 h or more, indicating that even when antibodies pose a threat to worms such as IV.brasilien- surrounded with adherent effector cells, they still sis or H. po~ygyrus, which as adults live entirely within remain resilient enough to resist lethal damage. These the gut lumen. in vitro studies have to be considered in the light of However, two recent studies provide support for a what is likely to take place in vivo. A. caninum larvae role of antibody in the gut lumen in host protective migrate through the tissues to the intestine and may immunity. In a comparison of immunoglobulin class- arrive there as soon as 5-6 h after penetration. On specific antibody responses to T. spiralis antigens, a route they pass through blood capillaries attracting TABLE I-SPECIES OF NEMATODE PARASITES IN WHICH SHEDD~C OF SURFACE CUTICULAR ANTIGENS AND’OR SURFACE ADHERENT SPFCIFIC OR ADSORBED NON-SPECIFIC LMMUNOGLOBUL~S HAS BEEN DESCRIBED

Species Microfilariae L2 L3 L4 Adult worms References

SECERNENTEA SPIRURIDA Mf without sheaths Onchocerca cervicalis Yes(Sp.Ig)$ NA Edwards et al., 1990 viteae Yes (Sp.Ig)f NA Gatrill et al., 199 I Yes(14.5 & 30 kDa) NA Apfel & Meyer, 1990 DirQilaria immitis NA Yes (6 & 35 kDa)t Ibrahim et al., 1989; Scott, Ibrahim & Tamashiro, 1990; Yes (Non-Sp.Ig)$ NA Hammerberg et al., 1984 No (Sp.Ig) Yes* Scott ct al.. 1988

Mt” with sheaths Yes (30, 55, 150 kDa) NA Egwang & Kazura, 1987 NA Yes (Sp.Ig)j Carlow el al., 1987 NA Yes (29 kDa) Maizels et al., 1989; Selkirk et al., 1990 NA Yes (15, 29, 51 kDa) Kwan-Lim et al., 1989 NA Yes (67 & 94 kDa) No No (30 kDa) Marshall & Howells, 1986 NA Yes (30 kDa)* Devaney, 1988 No (Sp.Ig) NA Hammerberg et al., 1984 hancrofti Yes (I 7 kDa but also NA Yes (17 kDa) Maizels et al., 1986 secreted) carinii Possibly (55 kDa Ag is also secreted) Philipp et al., 1984

ASCARIDIDA Touocara canis NA Yes$ Smith et al., 1981: Maizels. Desavigny & Ogilvie, 1984; Smith, Kusel & Girdwood. I983

STRONGYLIDA Nippostrongylus brasiliensis NA NA Yes (70-300 kDa) Maizels rl al., 1983 Heligmosomoides pol.vgyws NA NA Yes Pritchard, Crawford, Duce & Behnke. 1985 NA NA Yes: Vetter & Klaver-Wesseling, 1978 A ngiostrongylus cantomwis NA NA Yes Yes* Shih & Chen. 1990 Haemonchus con tortus NA NA NO Yes (143. 30. 5X. & 8 I Rhoads & Fetterer, 1990 kDa) RHABDITIDA ratti NA NA Yes-ensheathed infective L3 No-parasitic L3 Murreil & Graham, 1983 Strongyloides srercoralis NA NA Yes- ensheathed infective L3 (12-150 kDa) Brindley, Gam, Pearce, Poindexter & Neva, 1988

ADENOPHOREA LI Trichinefla spirulis NA Es (55, 105 kDa) Yes (20, 33,40,56 kDa) Philipp er al., 1980 Trichuris muris NA Yes Roach, 1986 (unpublished PhD thesis, University of Nottingham)

*Slow release, over several days. Non-Sp.Ig, non-parasite surface-specific immunoglobuhn; Sp.lg, parasite surface-specific antibodies. tOnce lost, not replaced. fRapid release, in hours, major loss under a day. 872 3. M. BEHNKE,C. J. BARNARD and D. WAKEI~I~

the response described above, but as parasites move from the cuticle of several species (Table 1) and through fine capillaries and force their way out of there are similarities as well as clear differences within blood vessels in the lungs, the consequent abrasion is the group of the spectrum of antigens and their likely to dislodge adherent cells although the degree to properties. which this could occur has not been evaluated. Thus 3.1.2.1. L3 and L4 surface antigens. A number of the necessity of stabilizing surface antigen via C3 in elegant studies have shown that the mammalian order to allow antibody-dependent cellular adhesion infective stages of filarial parasites undergo radical and the intense activity of the migrating worms changes within days of entry into the host and that probably combine to make the response described in these are associated with changes in their susceptibility vitro ineffective in destroying all but a few larvae, to host effecters of resistance. possibly those which have run out of energy reserves Marked differences in the surface properties of D. and become exhausted before they could recommence im~l~t~s L3 and L4 have been described by Abraham, feeding (Behnke, 1990). Once the worms pass out into Grieve & Mika-Grieve (1988) who proposed that this the trachea and migrate to the gut lumen they are likely change in the antigenicity of the parasite surface to be safe from the potentially damaging effects of serves to delay potentially destructive immune respon- adherent cells. ses (Grieve, 1990). Infective L3 shed antigens but These in vitro studies of cytoadherence to A. can- these were not resynthesized and following experimen- inum larvae have not been repeated on other hook- tal infection of rats I O-20% of the labelled molecules worm species. Cytoadherence has, however, been were shed per day so that by the time of the L3-L4 described between human peripheral blood eosin- moult, little of the original label remained. Since ophils and exsheathed larvae of the 35 kDa antigen was the dominant surface protein (Desakorn, Sunthardsamai, Pukrittayakamee, Mig- and effectively represented the only antigen recog- asena & Bunnag, 1987). Here adherence appears to be nized by immune sera, the larvae gradually lost solely C3 mediated, since heat-inactivated sera were antigenicity as they grew and prepared for the next unable to mediate significant adherence. Experiments developmenta stage. After moulting a new set of using immune sera taken from mice exposed to N. antigenically distinct molecules was presented to the americanus have extended these observations. Periton- host (Ibrahim, Tamashiro, Moraga & Scott, 1989). eal leukocytes adhered to ensheathed larvae of Not surprisingly L3 larvae of this species did not N.americanus and complement was considered to be absorb canine albumin or immunoglobulins onto the more important than antibody in mediating cytoad- surface. However, despite the labile surface, mouse herence (Wells, 1987, unpublished PhD thesis, Univer- peritoneal exudate cells were attracted to L3 in culture sity of Nottingham). Studies are currently under way and adhered to the cuticle but no killing was detected to examine other species of hookworms, since it is and most larvae survived long enough to moult to the important to know which of the above is more L4 stage which no longer attracted cells (Abraham, representative of hookworms in general. Grieve & Mika-Grieve, 1988). Once the moult had Among the most impressive changes in surface been completed, L4 reacted strongly with anti-canine structure are those described for the tissue-resident L2 Ig and anti-albumin, suggesting that host molecules stages of Toxncara canis (see Grieve, 1990). These were adsorbed onto the surface or that host-like larvae can secrete a cuticular coat of glycoconjugate epitopes were expressed by this stage. It would appear which is continually synthesized and lost from the then that D. immitis L3 have a dominant surface surface (Badley et al., 1987). Therefore antibody antigen which attracts antibody and leukocyte activity specific for this surface binds only to frozen or azide- but which is shed over the course of 48 h, presumably inhibited larvae (Smith er al., 1981). On exposure to preventing the cells from invoking their tidal mechan- host leukocytes, whole sections of surface coat may be isms long enough to impart irreversible damage and shed (Rockey, John, Donnelly, McKenzie, Stromberg providing the parasite with an opportunity to grow & Soulsby, 1983) and in the case ofeosinophils, despite into the next developmental stage. Once this crucial degranulation on the parasite surface, cells eventually stage has been successfully negotiated the evasive drop off leaving L2 apparently unharmed (Fattah, strategies switch to dependence on host-like epitopes Maizels, McLaren &Spry, 1986). The in vivo success of and camouflage. the evasion strategy of T. canis is attested by the ability Interestingly, similar observations have been made of larvae to migrate out of granulomatous murine on 0. volt&us. Although the surface of L3 is thought livers (Abo-Shehada, Al-Zubaidy & Herbert, 1991). to be poorly immunogenic in humans despite seven 3.1.2. Tissue inhabiting species. Loss of surface detectable cuticular antigens (Titanji & Mbacham, cuticular antigens has also been reported in filariasis. 1990; Taylor, Goddard & McMahon, 1986), in vitro Surface components are now known to be released eosinophils adhered to and killed L3 but not L4 and in Understanding chronic nematode infections 873

cultures containing both larvae only the former attrac- concluded that some developmental changes were ted eosinophils (Strote, Brattig & Tischendorf, 1990). required before the larvae became susceptible to To date no evasion strategy has been described for L3/ killing. Their culture system permitted L3 to develop L4 stages of 0. volvulus but the above findings suggest into advanced L3s by 7-11 days but no further. that a radical change in the antigenicity and/or However, in vivo in jirds, A. viteae moults by day 7 susceptibility to host cellular attack, such as occurs in (Johnson et al., 1974) and it is conceivable that L3 of D. immitis takes place here also. In fact major this species are resistant to cellular killing until differences in structure and chemical composition of changes associated with preparation for this moult are the cuticle of L3 and L4 stages have been described initiated. If so, the relative susceptibility of L3 and L4 (Lustigman, Huima, Brotman, Miller & Prince, 1990) stages of A. viteae appears to be exactly the opposite of but how these relate to greater resistance to host Onchocerca and Brugiu species as described above. In effecters is not known. an unrestrained environment in vivo, where the worms The infective stages of other filariae also shed migrate subcutaneously, the initial resistance of L3 to surface antigens in preparation for the L4 moult. The host effecters, providing protection for a few days L3 of B. malayi shed surface-bound immunoglobulin, after infection, may be all that is required to enable the detected through immunofluorescence, within 3 h worms \t” grow and complete the L3-L4 moult. when cultured at 37°C (Carlow, Perrone, Spielman & Worms are active in the subcutaneous site (Court, Philipp, 1987). Loss of surface label was believed to Stables, Lees, Martin-Short & Rankin, 1988) and host parallel loss of a surface epitope confined exclusively cells would have to adhere tightly enough to prevent to the late L2 and L3 stages and in vivo this was shed them being detached through abrasion as the worms within 2 days of infection of mammalian hosts migrate. In contrast in culture or in chambers implan- (Carlow et al., 1987). However attempts to immunize ted in vivo parasite mobility is greatly restricted and with this antigen have been unsuccessful (Carlow, consequently cytoadherence is more efficient. The Busto, Storey & Philipp, 1990). initial resistance of L3 is intriguing and not yet B. pahangi L3 lost 90% of the original radio- understood but surface antigen shedding, disguise iodinated surface label in vivo in the period be- with host molecules or secretion of immuno- tween infection and the L4 moult. In contrast surface- modulatory factors may all account for the reported labelled L4 and adult B. pahangi appeared to have observations. relatively stable surface components and no loss of 3.1.2.2. Adult worms. In contrast to the L3 and label was detected over a period of 7-8 days follow- microfilariae (mf) of some species, the surface of adult ing implantation into recipient jirds (Marshall & filarial parasites appears to be more stable. Scott, Howells, 1986). The infective larvae of W. bancrofti Diala, Moraga, Ibrahim, Redding & Tamashiro shed a 17 kDa antigen (Maizels, Burke, Sutanto, (1988) found that D. immitis released only 0.1% of Purnomo & Partono, 1986), but less vigorously total surface “‘1 label in 8 h of incubation. Initial than the Brugia species and consequently are easily studies on adult B. pahangi suggested that surface surrounded by human eosinophils in vitro which bind antigens were stable (Marshall & Howells, 1986), through antibody (IgG)- or complement-dependent but subsequent work revealed that the 30 kDa sur- mechanisms (Higashi & Chowdhury, 1970). Freeze- face antigen of adult worms was released albeit killed or formaldehyde-treated larvae had lower relatively slowly (Devaney, 1988). A similar mole- adhesion scores suggesting that actively secreted cule on B. malayi which is also slowly released antigens were responsible, but treatment with azide in culture (Maizels, Gregory, Kwan-Lim & Sel- had no effect and despite metabolic inhibition, suf- kirk, 1989) and shared with L4 (Selkirk, Gregory, ficient material was still present to facilitate cytoad- Yazdanbakhsh, Jenkins & Maizels, 1990) has hesion. If the 17 kDa antigen was involved in cytoad- 42% homology with mammalian glutathione perox- herence, turnover must have been sufficiently slow and idase (Cookson, E., Baxter, M. & Selkirk, M. E., the residence of the antigen on the cuticular surface unpublished data presented at the Spring Meeting must have been sufficiently firm to enable effector cells of the British Society for Parasitology, 1991, in to bind. Liverpool) and may therefore function as an oxygen In contrast to the above the L3 of A. viteae have radical scavenger. The slow release of this molecule capacity to resist host effector mechanisms. When may hence relate to its protective role against radical- implanted into immune jirds in diffusion chambers no mediated attack and not antigen shedding as an killing of L3 was observed until-day 10 post-implanta- evasive strategy. tion. In vitro in the presence of peritoneal exudate cells, The adults of D. immitis and Brugiu seem to shed no adherence was observed on fresh L3 and not until 4 surface material very slowly, do not employ masking days in culture. Abraham, Weiner & Farrell (1986) by host antigens nor do they express host-like antigens 874 J. M. BEHNKE, C. J. BARNARD and D. WAKELIN

and yet in all cases worms live for a long time. It may as removal of anti-surface antibodies by massive be that immunomodulatory strategies are involved or overproduction of mf stages. Mf with surface as yet undefined characteristics of surface lipids camouflage which has deteriorated, and others which (Kennedy, Foley, Kuo, Kusel & Garland, 1987). Scott were recognized by antibodies before managing et al. (1988) showed that labelled surface peptides were to become camouflaged, expose cuticular antigens only degraded by proteases after the surface had been which facilitate sequestration and destruction in organ treated with chloroform with consequent removal of sites. glycolipids from the surface. The mf of 0. cervicalis express a number of low Another tissue nematode canton- molecular weight surface molecules, some ofwhich are ensis, not closely related to the lilariae, lives in lung involved in anti-surface immune responses and can be capillaries of rats and this parasite also sheds its shed together with bound antibody. Using a fluo- surface at a relatively slow rate. Scanning electron rescent antibody technique Edwards, Busto, James, microscopy revealed sections of the epicuticle being Carlow & Philipp (1990) showed that loss of surface- sloughed off by preadults (Shih & Chen, 1990). bound immunoglobulin was temperature dependent. However, the rate of release was estimated at only 6% At 4°C there was little loss of surface immuno- per day, again raising doubts about the usefulness of fluorescence over the course of an hour, but at 37°C such a response in the face of a concerted host cellular most of the label was lost within 30 min. offence. When B. pahangi or D. immitis mf were cultured in 3.1.2.3. Microlilarial surface antigens. The mf of L. vitro in the presence of non-specific immunoglobulin cariniiexpress only one dominant surface antigen with (i.e. sera from uninfected ), temporary attach- a M, of 55 kDa (Philipp, Worms, McLaren, Ogilvie, ment was observed only with D. immitis. B. pahangi mf Parkhouse & Taylor, 1984) which is immunogenic in bound only trace quantities. However, both species cotton rats following immunization with live sub- reacted with sera from hyperimmunized animals cutaneously administered mf but does not induce a indicating that specific antibodies were capable of specific serum antibody response in most normally making more stable bonds with cuticular antigens infected patent animals (Lawson, Wenk & Storey, (Hammerberg, Rikihisa & King, 1984). Rzepczyk, 1989). This molecule may be shed, although this is not Bishop & Atwell (1986) observed that when D. immitis clear at present, but a molecule of identical size is mf were co-incubated with neutrophils, some mf actively excreted/secreted by mf and other stages always remained free of cells as if there were differen- (Philipp et al., 1984). The host may therefore have an ces in some respect antigenically or possibly through opportunity to recognize the antigen or cross-reacting better evasion mechanisms such as more efficient epitopes from earlier developmental stages as well as shedding of surface antigens. Interestingly, the mf of during the patent period. Its failure to respond neither species had detectable immunoglobulin on the normally with circulating mf-surface-specific anti- surface when freshly examined from dogs, although D. bodies may reflect some downregulation of the immitis mf were shown to have surface immuno- immune response or alternatively the efficient removal globulins when examined in saline containing azide, of such antibodies by the enormous quantity of new mf suggesting that in this species non-specific immuno- produced daily in the tissue and organ sites such as the globulins are temporarily adsorbed and then shed lungs (Lawson et al., 1989). During the patent period, through an active metabolically dependent process mf in the circulation did not bind significant quantities (Hammerberg et al., 1984). Perhaps surprisingly Rzep- of antibodies, i.e. considerably less than mf within czyk, Bishop & Atwell (1986) found no evidence for organs such as the spleen, lungs, liver and kidneys adsorbed albumin and Tamashiro, Ehrenberg, Levy & (Muller-Kehrmann, 1988). Scott (1986) observed that less than 10% of radioac- One explanation for this may be the adsorbed host tivity was spontaneously released in a 30 min period of albumin on their surface (see Table 3) which probably incubation following surface labelling of mf with ‘?. interferes with antibody binding (Court & Storey, It thus appears that the mf of D. immitis have a slow 1981; Philipp et al., 1984). It is conceivable that the mf surface turnover, do not bind albumin, but bind some of this species, which are short-lived, depend initially non-specific immunoglobulin transiently and still on host albumin to camouflage the dominant surface some fail to attract neutrophils in culture. Taken 55 kDa antigen, but this may be a temporary and together these observations imply that as yet unknown unstable association (but note that Court & Storey, mechanisms protect the surface of the mf of this 1981 found that mf surface-bound host components species. were not removed following seven washes). Their Recently two research groups have shown that the survival may also be facilitated by downregulatory microfilariae (mf) of Acuntho~hrilonetna viteae lose mechanisms, which are not yet clear, as well surface antigens. Apfel& Meyer (1990) found that ‘? Understanding chronic nematode infections 875

labelled surface proteins from microfilariae were shed niticance of antigen shedding by adult worms is in vitro. Four major surface-exposed proteins in the less clear. Adult filariae are extremely long-lived and range 14.5-19 kDa were observed and one of these with the exception of Loa foa generally reside in the (14.5) together with a suspected dimer (30 kDa) was close confines of their specialized sites. For IV. ban- shed and replaced continuously on the cuticle of mf. A crofti and Brugia spp. these are lymphatics. Shed larger (40 kDa) molecule was also evident on the antigen will result in immune complexes accumulating surface but was less abundant and not released in and it is possible that these cause immunodepression culture. Post-infection sera from chronically infected locally. However, the secretion, replacement and jirds did not recognize the surface of living mf, but shedding of the antigens in question would have to be bound to fixed or azide-inhibited parasites and reacted maintained for months and years in the long-lived with shed antigen. Thus A. viteae mf appear to shed species and would have to be a fairly dynamic process continuously the surface proteins against which since slow turnover, as in S. mansoni, would seem to be antibody responses are directed, and replace shed of little value as an evasive device. Whether this is the material with new protein. In another study (Gatrill, case or not remains to be determined; there are no Kee, Behnke & Wakelin, 1991) using immunofluor- data on replacement of surface antigens by adult escence to demonstrate surface-bound antibody, it worms but current studies suggest that the rate of loss was shown that sera from C57BL/lO mice, which clear of Gp29 from Brugia sp. is relatively slow (Selkirk microtilariaemia rapidly, contained surface-specific et al., 1990). IgM antibody which bound to the surface ‘of mf, From the evolutionary perspective, surface antigen whereas BALB/c mice, which tolerate a chronic shedding has been reported in both the Secernentea microtilaraemia, had surface-reactive IgM antibody and the Adenophorea, and within the former group in which could be demonstrated on the mf surface only the Rhabditida, Spirurida & Ascaridida, and Stron- when mf had been treated with azide or frozen. At gylida. By comparison the Adenophorea have been 37°C surface-bound antibody from the sera of BALB/ poorly studied with information being available for c mice was shed faster than from sera of C57BL/lO only the two related genera, Trichinella and Trichuris. mice suggesting that one explanation for the two Nevertheless antigen shedding appears to be a feature contrasting response phenotypes of these strains was a of four nematode lineages in which parasitism has difference in the avidity of their IgM responses to been adopted independently and therefore probably surface cuticular determinants. An alternative represents an adaptation of an ancestral mechanism explanation may be that C57BL/lO mice recognize the evolved before parasitism or reflects a readily adapta- more stable cuticular components whereas BALB/c ble molecular feature of the nematode cuticle shared mice respond only to the labile elements which are by all groups. However, because of the variation in continuously shed. experimental approaches adopted by research groups 3.1.3. Overview. The release of surface antigens by (antigen shedding has been inferred from studies using microtilariae is easy to appreciate as a very effective surface-radiolabelled antigens, loss of fluorescent- survival strategy. In long-lived mf such as those of A. labelled specific antibody, loss of antibodies against viteae, replacement would have to be maintained host immunoglobulins, etc.) it is still not clear whether continuously, but in addition to investing their antigen shedding is a single mechanism or a range of progeny with evasive devices, female worms replace related mechanisms. The situation will not be clarified lost larvae daily and can sustain mf production for until inter-species comparisons are completed utilizing very long periods of time. This device, however, is not each technique in turn. The most apparent common totally foolproof and specific antibodies can make feature is that antigen shedding is frequently encoun- more stable bonds with the cuticular surface despite tered in infective stages which soon moult to the L4, shedding, at least in D. immitis. In B. pahangi non- where other mechanisms take over, and in transmis- specific immunoglobuhns are not adsorbed and sion stages such as mf, although in each case there are specific antibodies cannot be easily shed (Hammer- exceptions possibly indicating loss of the mechanism berg et al., 1984). In the case of L3 stages, antigen and adoption of alternative strategies. shedding allows the parasite a window in an otherwise hostile environment, providing temporary protection 3.2. Antioxidant enzymes in the period to the L4 moult as in D. immitis. It is Oxygen radicals are produced within cells as part of tempting to speculate that resistance to L3 invasion normal oxidative metabolism and most aerobic may depend on other antibodies, inhibiting growth organisms possess a range of oxygen-scavenging and delaying the moult so that effector mechanisms enzymes which protect tissues, organs and cells by have an opportunity to recognize the underlying, removing potentially damaging radicals from sites perhaps more stable antigens. However, the sig- where they could impair organ function. Additionally, TABLE 2-ANI I-OXIDANTEN~YME~ONT~N,OFPARASITICNEMATODES

Species SOD Catalase GSH-px GSH- tase References

SECERNENTEA SPIRURIDA Spirurina, Mf without sheaths Onchocerca !JOlVUlU.P Moderate Henkle ef ul., 1991 Onchocerca cervicalis Adults High Trace Moderate Mf High Trace Moderate Callahan, James & Crouch, 1988 Onchocerca gutterosu Present Pemberton & Barrett. 1989 Dirqfiluria immiris Adults V. high Trace Moderate Callahan, James & Crouch, 1988 Moderate Jaffe & Lambert, 1986 Mf V. high Trace Moderate Callahan, James & Crouch, 1988

Spirurina, Mf with sheaths Brugia malayi Adults None None Kwan-Lim et al.. 1989, Present Cookson et al., unpublished (see text) Brqia pahrrngi Adults Moderate Jaffe & Lambert, 1986 Li/omo.wides carinii Adults Present Bhargava, Le Trang, Cerami & Eaton, 1983 ASCARIDIDA TO.WUUYI crmi.c Adults Present Maizels & Page. 1990. High None Sanchez-Moreno r/ al.. 1987 To.rocara cali Adults High None Sanchez-Moreno ct al., 1987 Tosocari.r leonina Adults High None Sanchez-Moreno et al., 1987 A.scari.r .suum Adults Present Douch & Buchanan. 1978 High None Sanchez-Moreno er al., 1987 STRONGYLIDA Nippostrongylus brasiliensis Adults Moderate Moderate High Smith & Bryant, 1986 L3 V. high Knox & Jones, 1992 Adults Moderate Knox & Jones, 1992 Nematodirus battus L3 V. high Knox & Jones, 1992 Adults Moderate Knox & Jones, 1992 Oslerlagia circumcincta L3 V. high Knox &Jones, 1992 Adults Moderate Knox & Jones, 1992 Heligmosomoides polygyrus Adults V. high Moderate High Smith & Bryant, 1986 L3 High Knox & Jones, 1992 Adults Low Knox & Jones, 1992 Adults Moderate Kawalek, Rew & Heavner, 1984 Trichostrongylus colubriformis L3 High Knox & Jones, 1992 Adults Low Knox &Jones. 1992 Trichosrrongylus vitrinus L3 Moderate Knox & Jones, 1992 L4 Moderate Knox & Jones, 1992 Adults Low Knox & Jones, 1992 ADENOPHOREA Newborn larvae Low None None Rhoads, 1983 Muscle larvae V. high None V. high Adults Moderate None Moderate

Present-amount not reported. SOD-Superoxide dismutase; GSH-tase-glutathione-S-transferase; GSH-px-glutathione peroxidase. Unless a reference is given, data taken from Callaghan, Crouch and James, 1988. 878 J. M. BEHNKE,C. J. BARNARD and D. WAKELIN free oxygen radicals are generated during the respir- content and which in vitro is more susceptible to free atory burst which follows ingestion of pathogens by oxygen attack may be unable to resist this response in phagocytic leukocytes and are released into their viva. phagosomes. There is evidence for extracellular release In a similar vein Knox & Jones (1992) have recently and indeed some of the pathology associated with demonstrated that adapted N. brasiliensis, which are parasitic diseases is now recognized as being attrib- considerably more resilient in the face of host immun- utable to overexuberant release of oxygen radicals in ity than normal adults, have significantly higher levels tissue sites (Clark et al., 1986). Free oxygen radicals of SOD (30.2 and 10.9 units mg-’ protein, respect- damage cell membranes through lipid peroxidation ively). Their study also included five species of ovine and are believed to be responsible for intraerythrocytic GI nematodes (Table 2) and perhaps surprisingly, the crisis forms in malaria and Babesia infections (Clark & highest levels of SOD were observed in infective L3 Howell, 1990). stages (range 16.3 (T. vitrinus) ~ 79.4 (0. circumcincta) Oxygen-scavenging enzymes therefore constitute a units rng-’ protein) rather than adults (range 6.1 (T. natural regulatory system enabling organisms to vitrinus) ~ 12.6 (0. circumcincta) units mg-’ protein). employ oxidative metabolism whilst being protected This finding was attributed to the closer contact with from the damaging effects of their by-products. Not the host mucosa which the larval stages exhibit and the surprisingly, pathogens have exploited the system to attendant greater risk of damage by host effecters. their own advantage. A range of parasitic organisms However, when worms (T. vitrinus and 0. circunz- including nematodes (Table 2) are now known to cincta) were assayed for release of SOD in culture, possess relatively high amounts of these enzymes adults and L4 stages released significantly higher compared to host tissues suggesting that they may be quantities than L3s, suggesting that parasites can employed additionally to protect against free oxygen release the enzyme to change the environment in which radical release during host responses (Callahan, Crouch they live. Isoenzyme polymorphisms between species & James, 1988). The most widely studied enzyme is (see also Sanchez-Moreno, Monteoliva, Fatou & super oxide dismutase (SOD) which has been detected Garcia-Ruiz, 1988 for data on Ascuris suum) and in in Secernentean (Spirurida, Ascaridida and Stron- response to immunological stress as reflected in more gylida, sometimes in extraordinarily high amounts; isoenzymes in adapted adult N. brusiliensis indicated Knox &Jones, 1992; Sanchez-Moreno, Leon, Garcia- that the SOD enzyme system may be modulated by the Ruiz & Monteoliva, 1987) and in Adenophorean parasites and may play a crucial role in survival. groups (notably T. spiralis, see Rhoads, 1983). However, values for all adult worms studied were Recently, evidence has been provided for a role for comparable or marginally lower to those reported oxygen radicals in the gut lumen (Smith & Bryant, earlier (15.3 units rng-‘) for N. hrasiliensis (Smith & 1989a,b). The demonstration that free oxygen radicals Bryant, 1986) and were all much lower than values for may be released in mucosal sites and detected in the H. polygyrus (31.6 units rng-‘), only adapted N. intestinal lumen provides yet another potential mech- brasiliensis approaching levels of the former species. anism for damaging worms in the intestinal lumen H. polygyrus is not totally resistant to host effecters prior to or as part of expulsion (Smith & Bryant, and in mice concurrently infected with T. spiralis. 1989a). Treatment of rats infected with N. hrasi/ien.sis adult H. pojJ:pI’ru.s were expelled during the acute with antioxidants reduced intestinal free oxygen inflammatory phase to T. spiralis (Behnkc. Cabaj & release and inhibited expulsion of worms (Smith & Wakelin, 1992). Expulsion was more thorough during Bryant, 1989b). In this context an interesting compar- secondary responses to T. spiralis and in fast respon- ison has been made between the oxygen-scavenging der mouse strains relative to slow responders. This enzymes of two intestinal nematodes, one of which is observation serves to emphasize that H. po1ygyru.s is expelled by rodents (N. brcrsiliensis) whilst the other probably more dependent on other mechanisms for its causes chronic infections (H. poIJ~g_vrus). In contrast to survival, notably the immunomodulatory strategy the former H. poLygl,rus had twice the SOD, four times considered below. However H. pol:gyrus were seldom the glutathione reductase and three times the catalase lost completely from concurrently Infected mice, a few levels on a per gram basis (Smith & Bryant, 1986) and worms always persisted even after T. spiralis had been was shown to be more resilient in the face of free entirely cleared from the intestine. A proportion of H. oxygen radicals generated in vitro in culture. In view of po!JyyJrus is therefore more resistant than T. spiralis. It the demonstration that free oxygen radicals are would appear that whilst H. pdygyrus clearly prevent released in the intestinal tissues, it is conceivable that the inflammatory response being generated in the first the resilience of H. po/ygJ,rus may be attributable to its place, they also have a second line of defence against ability to inactivate the radicals before tissue damage elicited host effcctors and this may be based on is sustained. N. hrasi/icnsis which has a lower enzyme oxygen-scavenging enzymes. Understanding chronic nematode infections 879

Among filarial nematodes Onchocerca cervicalis involvement of a large proportion of the intestine and and D. immitis have high SOD levels but extremely low relocation in this situation would not provide a catalase and moderate glutathione peroxidase content satisfactory escape route. in both adult and mf stages (Callahan, James & Crouch, 1988; Weller, Longworth & Jaffe, 1989). 0. 3.4. Camouflage with molecules of host origin or voZvulus releases SOD into incubation medium and the expression of host-like molecules on the surface. gene encoding the enzyme has been cloned (Henkle, molecular mimicry Liebau, Muller, Bergmann & Walter, 1991). Catalase Sprent (1962) originally proposed, on purely and SOD are both required for protection against hypothetical grounds, that parallel evolution of hosts H,O, and since 0. cervicalis has only low levels of the and parasites should lead to an antigenic similarity former it remains sensitive to H,O, as well as .OH- and between the two species, thereby minimizing antigenic O,-mediated damage. The mf of D. immitis (Rzepczyk disparity and overall immunogenicity and hence per- & Bishop, 1984) are also sensitive to H,O, but the mitting parasites to cause chronic infections. In schis- picture has been somewhat confused by observations tosomiasis two distinct mechanisms have been iden- which have established that despite the oxygen burst, tified, one involving adsorption of a variety of host neutrophil-mediated killing is effected by a tidal molecules on the tegumental surface, including blood mechanism independent of oxygen metabolites (Rzep- group antigens and MHC molecules (Smithers & czyk, Bishop, Cheung, Atwell & Ferrante, 1986). In Terry, 1976) and the other expression on the parasite’s support Hopper, Subrahmanyam, Gregory, Nelson, surface of schistosome gene products with similarity to Rao & Chandrashekar (1984) concluded that oxygen host molecules (Damian, 1987). Attempts to demon- metabohtes played no role in macrophage- and strate similar mechanisms in the Nematoda have had neutrophil-mediated cytotoxicity to B. pahangi and mixed fortunes (McGreevy, Ismail, Philips & Den- Litomosoides carinii. No details of the oxygen-scaven- ham, 1975). Whilst many studies suggest that host ging enzyme content of either of these latter species molecules are adsorbed onto the cuticular surface, have been published. However, no evidence was found none have demonstrated unequivocally that for either SOD or glutathione S transferase in B. nematodes express host-like antigens. Support for the maIayi (see Kwan-Lim, Gregory, Selkirk, Portono & latter strategy requires demonstration of active syn- Maizels, 1989) but on the other hand the major surface thesis of host-like molecules, through evidence for the antigen of adult worms is believed to be a glutathione presence of RNA message encoding relevant genes in peroxidase (see 3.1.2.2). nematode cells, as has been done in . Apart from SOD, the oxygen scavenging enzyme Among the earliest studies in this area (actually content of the tilarial worms which have been studied preceding Sprent’s hypothesis), were a series of papers is unremarkable and since most appear to be sensitive by Oliver-Gonzalez & Torregrosa (1944) and Ohver- to H,O,, it would appear that the chronicity of tilarial Gonzalez (1946) who found evidence in T. spiralis, A. infections is dependent on other strategies, perhaps suum, and N. americanus for the existence of antigens preventing contact with leukocytes or preventing their similar to the human blood group substance A. activation, possibly through an immunomodulatory Essentially these studies employed parasite extracts, mechanism. which were probably homogenates, and used these to neutralize anti-A agglutinins in human sera. Whilst 3.3. Relocation in the tissues they provide evidence for the existence of host-like Where resistance is mediated locally, as for example molecules in the three species studied, there was no at the site of attachment of a parasite, detachment and evidence that the molecules were external on the relocation to a new site may be a temporary solution to parasite surface. There remains the possibility that avoiding host resistance. The canine hookworm structural or other internal molecules provided cross- Ancylostoma caninum changes its site of attachment at reacting epitopes which had no relevance for minimiz- regular intervals, perhaps as frequently as every 4-6 h ing the antigenic disparity between the parasites and (Kalkofen, 1970). Unpublished observations from our their hosts. Of the original species studied by Oliver- own studies on hookworms in hamsters confirm that Gonzalez, only Ascaris has subsequently been con- there are considerably more lesions than worms, firmed as expressing host molecules on its surface, suggestive of regular reattachment to fresh feeding namely albumin (Kennedy & Qureshi, 1986). sites. By moving to a fresh site hookworms escape the In recent years the most commonly used assays have local cellular response and the same mechanism been immunofluorescence, employing reagents probably operates in large affected with against host serum molecules such as immuno- Haemonchus and . However, sooner or later globulins and albumin, and surface radioiodination the host response may develop into an acute followed by precipitation with specific antisera against TABLL 3--SPECIfiSOt NEMATODEPARASITtS IN W”tCH tLIDENCr.HASREEN tOUNt)~OR THrAUSORBTtONOFHOSTMOLF(‘I,LtSOR tXPRESSlON0~ HOST-LtKEMOLECULESONTHEC~TtCl.:LARSURFACt

Species Microfilariae L2 L3 L4 Adult worms References

SECERNENTEA SPIRURIDA Mf without sheaths Onchocercu volvulu.s No albumin Taylor et al., 1986 Yes, type VI collagen No. type VI collagen No, type VI collagen Titanji & Mbacham. 1990 No, albumin No, Ig Engelbrecht, Braun, Connor, Downham, Whitworth & Taylor, 1991 Onchocrrcu gihsoni Yes, albumin NA Forsyth, Copeman & Mitchell, 1984 Onchocercu lienulis Yes, albumin Conraths el al., 1991 Diwfrlaria immiti.r No. non-Sp.lg* NA Hammerberg et nl., 1984 No, albumin Rzepczyk, Bishop & Atweil, 1986 No, non-Sp.Ig Yes, non-Sp.Ig Abraham et al., 1988 Possibly albumin or host-like molecules Abraham, Grieve & Mika-Grieve, 1988 No, albumin No. Ig Scott rt al., 1988 Mfwith sheaths Brugia pahungi Yes, ensheathed, serum proteins No. exsheathed NA Sayers, Mackenzie & Denham, 1984 No, non-Sp.Ig NA Hammerberg ef al., I984 No McGreevy et al., 1975 No Maizels, Philipp, Dasgupta & Partono, 1984 Wucherrriu bancrofti Yes, albumin NA Maizels et al., 1984 Yes Ridley & Hedge, 1977 Lifomo.roide.s curinii Yes, albumin NA Philipp et al., 1984 Yes Muller-Kehrmann, 1988 Yes NA No No Yes Court & Storey, 1981 Luff ir,u Yes NA Ridiey & Hedge, 1977 ASCARIDlDA Asrari.P .murn NA Yes. albumin Yes, albumin Kennedy & Qureshi, 1986 To.roc~uru ccmis NA Yes, human A & B blood group like Ags Smith et al., 1983 .- *No immunoglobulin detected in vim unless treated with azide. /n vilro non-specific immunoglobulin bound to mf and was rapidly shed. Specific antibodies in hyperimmune sera bound more stably. Non-Sp.lg, non-parasite surface-specific ~mmunoglobulin~ Sp.Ig, parasite surface-specific antibodies. Understanding chronic nematode infections 881 host components. Since it is not easy to distinguish 1986; see also 3.1.2.3). It is possible that D. immitis between the two mechanisms (adsorption of host or employs an active metabolically dependent process to expression of host-like molecules) the following sec- ensure continuous turnover of surface molecules to tion considers these strategies collectively. The species which immunoglobulins might bind non-specifically for which relevant data are available are listed in Table (Table 1) but the rate of loss is slow. Interestingly, 3 and it is readily apparent that these mechanisms have when mf were incubated in specific anti-mf serum, only been observed in the case of the Spirurida and immunoglobulins bound in a considerably more stable Ascaridida, considered to be the most ancient of the union. Therefore, the device does not protect mf from Secernentean parasitic nematodes, sharing a common specific antibodies in hyperimmunized animals, al- parasitic ancestor. It is thus tempting to propose that though it is likely that a state equivalent to experi- molecular mimicry had a common ancestry in these mental hyperimmunization seldom arises in nature. two groups and that other Secernentean and Adeno- The value of shedding surface-bound antibody in this phorean parasitic nematode lineages have not yet case may be to give mf a temporary window of security developed comparable strategies. However, a distinc- by delaying their susceptibility to existing host tion has to be made between the two mechanisms of antibody soon after patency through minimization of molecular mimicry because if adsorption of host the antigenic surface exposed to the host. This com- components is involved, common ancestry, perhaps bined with continuous production of mf by long-lived based on the electrostatic properties of cuticular adult worms ensures that some are always available in components, is conceivable. On the other hand, if peripheral blood for transmission. molecular mimicry involves expression of host-like The adsorption of host albumin on the cuticular molecules, based on parasite gene products, common surface has been singled out for particular attention. ancestry would only be possible if all the hosts Interestingly Brugia species do not appear to express involved shared a crucial set of common epitopes, an host albumin and yet the very similar and closely unlikely situation. Having made this suggestion on the related Wuchereria does (Maizels, Philipp, Dasgupta information which is available to us, we are neverthe- & Partono, 1984). Similarly 0. gibsoni mf express host less aware that comparable studies may not yet have albumin as do 0. lienalis (see Conraths, Worms, been attempted on the other groups. Furthermore, Preece, Harnett & Parkhouse, 1991) but 0. volvulus do workers may have attempted to assess other groups, not (Table 3). These differences may be explained by a failed to find appropriate evidence and subsequently secondary loss of the mechanisms enabling adsorption not bothered to publish or had rejected what would in genera such as Brugia or alternatively changes in the have been taken for ‘negative’ findings. Thus the surface properties of the species which adsorb host absence of published data for other groups constitutes molecules but this would entail the same mechanism an impediment to our hypothesis which will only be evolving on more than one occasion within the rectified when the appropriate studies have been Spirurida. completed or when those with ‘negative’ findings A possible explanation for some species being able report their results. We hereby encourage workers to adsorb host components and others not links this with pertinent unpublished and presumably otherwise property to surface charge. B. pahangi which does not ‘unpublishable’ data to write to us to enable the adsorb host albumin has a net negative surface charge present hypothesis to be updated. whereas D. immitis which shows transient adsorption In some studies host immunoglobulin has been when metabolism has been inhibited, has a neutral demonstrated on the surface of parasites. This poses surface charge (Abraham, Grieve, Mika-Grieve & the problem as to whether surface-attached immuno- Seibert, 1988; Hammerberg et al., 1984). The infective globulin represents an evasion strategy (possibly larvae of some Rhabditid (S. ratti) and Adenophorean through Fc receptors) or a host response to surface taxa (T. spiralis) also show a net negative surface determinants (binding through Fab). In this context charge (Murrell, Graham & McGreevy, 1983). Indeed, an interesting comparison between the sheathed mf of it has been suggested that a negatively charged cuticle B. pahangi and the non-sheathed mf of D. immitis led may help nematodes resist desiccation by attracting to the conclusion that only the latter bind non-specific moisture to the surface of the cuticle, as for example immunoglobulin to their surface and only when among free-living species, infective larvae on vegeta- metabolism has been inhibited by the presence of tion and even infective larvae of tilariids released by azide. There was no evidence of host albumin on live insect intermediate hosts. Thus species which exploit mf (Rzepczyk et a/., 1986) and non-specific immuno- host albumin on their cuticle may be species in which a globulin was shed within an hour (Hammerbert et a/., common charged amino acid(s) comprising collagen 1984), although the loss of radiolabelled surface or other surface components has been replaced by antigens was considerably slower (Tamashiro et al., neutral amino acid(s). The existence of Spirurids with 882 J. M. BEHNKE. C. J. BARNARD and D. WAKELIN

neutral surfaces suggests that loss of surface charge with L. carinii, circulating IgG and IgM antibodies for may be an essential first step in adoption of adsorption mf homogenate antigens decline in intensity at the of host molecules as an evasive strategy. Species such onset of patency (Muller-Kehrmann, 1988). The most as Brugiu spp. may have retained the ancestral condi- likely explanation for this latter observation is that tion of a negatively charged cuticle and antigen antibody was mopped up by the antigens secreted and shedding, both of which we suggest originated earlier expressed by the large numbers of mf which probably among free-living species from which parasitic lin- never left the lungs. However, later on. as patency eages evolved. Certainly Bvugia mf shed more neared its end, antibody levels increased, either antigenic components than Wucherericr (in W. han- because they were no longer being removed by the crofti the 17 kDa surface antigen is also secreted and presence of mf or because an ‘immunomodulatory’ may in fact be shed only in minute quantities, Table I), influence on antibody synthesis was no longer present although no information is available for the and synthesis was now unrestrained. In B. ~u~~a~~~i- Onchocerca species in question, only 0. cerviculis infected . clearance of mf has been linked to the having been shown unequivocally to shed surface appearance of antibody to an I l-22 kDa adult worm antigens. antigen (Fletcher, Birch, Samad & Denham, 1986). Numerous studies now indicate the existence of 4. IMMUNOMOD~LATiON parasite-derived immunodepression in lilarial infec- Without doubt the single evasive strategy which has tions but there have been few attempts to show that received more attention than any other is immuno- parasite molecules (as distinct from infection by modulation in its various guises. Immunon~odulation parasites) are responsible (Table 4). In an unusual but for the purpose of this article is defined as the activity fascinating approach Prince, Albiez & van de Endc of any parasite factor which reduces the effectiveness (I 985) observed that human 0. ~o/ruh~ nodule tissue of host immunity or in some way downregulates the containing adult worms survived longer on trans- host’s attempts to express a potentially protective plantation to chimpanzees than subcu~dneous tissue response. This is a large topic which has been reviewed transplanted witbout worms. Their results imply that comprehensively in several publications in recent parasite-derived factors were involved, supporting times and it is not our intention to go over old ground. other studies which have shown immunodepression in Non-specific immunodepression is easily demon- patients with (Kilian & Nielsen. strable across both classes of Nematoda although 1989a, b). specific immunod~pression of responses to the Suppressor cell activity is now well recognized in a antigens of the infecting parasite is well documented variety of chronic infections (Piessens, Ratiwayant~~. only in the case of filarial species (Piessens, McGreevy, Tuti, Palmieri, Piessens, Koiman & Dennis, 1980: Piessens, McGreevy, Koiman, Saroso & Dennis, Piessens, Partono. Hoffman. Ratiwayanto. Piessens, 1980a; Piessens. Ratiwayanto. Tuti, Palmieri, Pies- James, Palmieri, Koiman. Dennis & Carney, 1987) sens, Koiman & Dennis, 1980) but even here the story including bacterial (Petit, Richard, Burghoffer & is not quite clear cut, since a proportion of Dauget. 1985; Jarrells, 1985; Tomai. Elmquist, War- inicro~laraemic individ~dls may respond to filarial mka & Fitzgerald, 1989) and several lilarial systems antigens (Lammie, Leiva, Ruff, Eberhard, Lowrie & (B. puhangi, see Portano, Britton & Ash. 1976; Lam- Katz, 1988). Some animal-based studies have also mic & Katz, 198&b, 1984a,b; A. wifrur. see Weiss. revealed few differences in cellular and antibody 1970). It has been established that the failure of splenic responses between microlilaraemic and amicro- T cells in jirds infected with B. prlhmgi to respond to lilaraemic hosts (e.g. ferrets infected with B. rr~&.ri. infection is attributable to a nylon/plastic adherent cell see Thompson, Crandall, Doyle, Hines & Crandall. population with histamine receptors, i.e. macrophages 1986). On the other hand, in no species other than H. (Lammie & Katz, 1984a) and evidence has been polygyrus has it been conclusively demonstrated that presented showing that the effect of these cells is to immunodepression of antiparasite responses is essen- downregulate IL-2 secretion by splenic lymphocytes tial for the chronicity of infection and adult worm (L,eiva & Lammie, 1989). IL-I secretion by splenic survival (for review see Behnke, 1987). macrophages was normal and there was no cvidencc In most filarial infections, microlilaraemia is for a defect in T cells. Rather an active suppression of associated with the absence of antibodies specilic for IL-2 secretion by both parasite-specit~c and mf surface antigens (B. mulu_ri, McGreevy, Rat- hetcrologous antigen-speciftc T cells was maintained iwayanto, Sekar, McGreevy & Dennis, 1980; D. for as long as the accompanying suppressor cells were inrvwifis. Rzepczyk & Bishop. 1984; A. vitwr,. Weiss. co-incubated with T cells. One explanation may bc that 1978; W. hanuojii, Ravindran, Satapathy. Das. Patt- the worms secrete prostaglandin-like factors through naik & Subramanyam, 1990). In cotton rats infected which IL-2 secretion is downregulated. Although to Understanding chronic nematode infections 883 our knowledge there are no data that the filariae can slow. There are several reasons for this. Firstly as has secrete prostaglandins a variety of other helminths are been argued above immunomodulatory factors are known to do so (see 4.1.1.3) and in Treponema likely to be labile, components with short half-lives in pallidum suppressor cell activity has been linked to vivo and their isolation from parasites, either extracted secretion of PGE2. However, in the closely related B. directly from hosts or maintained in vitro, is likely to mala)ri immunosuppression has been linked to T be problematic. lymphocytes (Lal, Kumaraswami, Steel & Nutman, 1990) and it is therefore necessary to consider the 4.1 .l. Known factors and their modes of action. existence of more than one active mechanism or 4.1.1.1, Factors described to date. Table 4 summarizes alternatively several routes leading to the same com- studies in which extracts of parasites, their ES mon outcome. products or specific fractions of these have been The liierature is replete with reports of non-specific assayed for their immunodepressive properties either immunodepression during particular phases or in viva or in vitro. One thing is very clear: there is still a throughout infection with a variety of different long way to go before we have even a vague idea of the nematode species, ranging from laboratory studies role of the molecules involved. The only common involving rodents (N. hrasifiensis to DNP, Haig, Lima theme from the few studies in which fractions of & Mota, 1980 and heterologous antigens, McElroy, parasite products have been investigated is that in Szewczuk & Befus, 1983; A. suum to Con Al PHA & general the molecules involved appear to be relatively PWM, Barta, Stewart, Shaffer, Huang & Simmons, small, ~50 kDa (smaller than albumin, 67 kDa). B. 1986), through large domestic animals such as cattle malayi mf appear to be the exception, since Wadee, (, Trichostrongylus axei to Con A Vickery & Piessens (1987) found suppressive activity & PHA, Snider, Williams, Karns, Romaire, Trammel in fractions of ES in the molecular range from 50 to & Kearney, 1986; reviewed by Klesius, 1988) to 190 kDa and it is likely that several molecules of observations in man (0. volv~~~ to tetanus, BCG and different size were involved or alternatively that a other recall antigens, Kilian & Nielsen, 1989a,b). relatively small molecule had the capacity to form Besides demonstrating the universality of the phen- multimeric units. However, the phosphocholine-bear- omenon these studies have contributed little to our ing antigens of adult B. malayi, which are relatively understanding of evasive strategies employed by large (>90 kDa. Maizels, Burke & Denham, 1987), nematodes. Almost all groups have failed to persist are also known to be immunosuppressive as adjudged with the investigation of their model systems long by their effect on PHA-stimulated proliferation of enough to unravel the factors involved. In short the human lymphocytes in vitro (La1 et al., 1990) and majority of such studies can be classified as superficial, molecules with PC are released by Brugia and descriptive of the phenomenon but little more. A Onchocerca spp. in vitro and in vivo (Maizels et al., crucial issue to answer here is whether the suppressor 1987). cell activity or generalized immunodepression are part 4.1.1.2. Smoke screen antigens / immune com- of a concerted evasion strategy by the parasites plexes. In culture all nematodes secrete or excrete an involved aimed at reducing the effectiveness of host array of molecules some of which are potently immun- immunity. If so how are they mediated? Do parasites ogenic (Lightowlers & Rickard, 1988). It is conceiva- release factors such as PGE2 and if so precisely what ble that others act in a smoke screen capacity diverting molecules are involved and how do they act? Alter- the immune system into unnecessary expenditure of natively are we seeing here intentional downregulation energy reserves in an effort to clear the circulation, by the host in an effort to minimize pathology? If so, through the formation of immune complexes which what conditions are required to induce such respon- cause downregulation of the host’s capacity to res- ses? What are the qualities and characteristics of pond (Barnett, 1986) or through mitogenic properties relevant parasite antigens capable of influencing the leading to polyclonal activation of host cells and host response in this way? And perhaps most challeng- consequent immunodepression through exhaustion of ing of all, can the antigens employed to downregulate reactive lymphocyte clones (Wadee & Piessens, 1986). responses generating immunopathology be dis- Although there is circumstantial evidence to support sociated from those responsible for protection and can such hypotheses, it is extraordinarily difficult to they be employed in vaccines? substantiate that relevant mechanisms may be ben- eficial for worm survival. 4.1. Characterization of immunomodulatory factors Many nematodes seem to reside in tissue sites Despite the indirect evidence that immunomod- surrounded by inflammatory cells but apparently ulatory factors must exist, progress in isolating and unaffected by their presence. 0. voivulus and W. analysing potential candidates has been extremely bancrofti are prime examples. In the fatter, inflamed 884 J. M. BEHNKE. C. J. BARNARD and D. WAKELIN

TABLE &IMMUNODEPRESSION BY EXTRACTS,ES PRODUCTSAND IMMUNOMODULATORYFACTORS OF NMATODE PARASITES

Species Immunodepression IMF Stage secreting in vivo IMF

SECERNENTEA SPIRURIDA Mf without sheaths Yes Mf or adults

Adults

Onchocerca gibsom 10 kDa Mf

Mf with sheaths Brugia malayi 50-190 kDa Mf

Serum factors Mf/host derived?

Adults

Brugia pahangi Adults

ASCARIDIDA ES & Homogenate Adult

Anisakis simplex & spp. >lOkDa L3

STRONGYLIDA Heligmosomoides polvg~nu <12kDa L4

i 25 kDa Adult

Homogenate Adult

25-35 kDa L3 & L4

ADENOPHOREA Trichinella spiralis Homogenate Muscle larvae Understanding chronic nematode infections 885

TABLE4-Continued

Effect observed References

Inhibition of mixed lymphocyte response & PHA Prince et al., 1985 induced proliferation Suppression of response to PPD but not ConA Luty, Downham. Whitworth, Morgan, McNicholas & Taylor, 1990 Suppression of ConA induced proliferation of Foo, Nowak, Copeman &McCabe, bovine lymphocytes 1983

Suppression of ConA induced proliferation of lymphocytes. Suppression of IL-2 production by mitogen Wadee et al., 1987 stimulated lymphocytes

Suppression of antigen induced lymphocyte Piessens,Ratiwayanto, Tuti, proliferation in vitro Palmieri, Piessens, Koiman & Dennis, 1980 Suppression of PHA induced proliferation of Lal et al., 1990 lymphocytes Reduced lymphocyte responsiveness to antigens Miller et ul.. 1991

Suppression of reaginic & haemagglutinating Komatsu, Nishimura, Sano & response Shinka, 1979

Suppression of mitogen induced proliferation of Raybourne. Desowitz, Kliks murine lymphocytes Deardorff. I983

Suppression of mitogen induced proliferation of Losson, Lloyd & Soulsby, 1985 murine lymphocytes Suppression of mitogen induced proliferation of Monroy, Dobson & Adams, 1989 murine lymphocytes Suppression of development and expression of Pritchard & Behnke, 1985 homologous protective response in mice Suppression of mitogen & antigen induced Gasbarre, Romanowski & proliferation of peripheral blood leukocytes at Douvres. 1985 50 ng/per culture (50%)

Suppression of response to SRBC Barriga 1975, 1978a Reduced response of cells to mitogens Barriga, 1978b 886 J. M. BEHNKE,C. J. BARNAKU and D. WAKELI~ lymph nodes may also show follicular atrophy (Con- tionai studies on peripheral blood lymphocytes have nor et al., 1986), although whether this is a con- indicated that cells from lilariasis patients with cir- sequence of the activities of the parasite or the host culating antigens are less responsive to stimulation by response to the presence of worms is not known. T. L3 antigens (Dasgupta, Bala & Dutta, 1987b) or mf ES cunis larvae not only reside within granulomatous antigens (Prasdd & Harinath, 1987) than those from murine livers but escape from these and accumulate in persons without circulating antigens. However, B. the brain, apparently shrugging off host inflammatory malayi mf also have mitogenic properties (Wadee & cells as posing no threat to them at all (Abo-Shehada et Piessens, 1986) and it could be that fewer reactive cells al., 1991). Eosinophils will attach temporarily to T. are present because they have been swamped by the canis larvae and degranulate but within l-2 h the proliferation of clones which do not recognize parasite larvae have fought free from adherent cells, and antigens. However, Wadec & Piessens ( 1986) detected having sloughed off their surf&e coat continue with- immunodepressive activity in lyophilized extracts of out loss of viability (Badley et af., 1987). mf and there remains the possibility that internal Another example is found in H. pdygyrus, the L3 molecules not normally released in the host were and L4 larvae of which develop in the muscularis involved. In a later study they showed that mf release externa of the mouse intestine for a period of 8-9 days immunodepressive factors (see 4.1.1.1). before returning to the gut lumen as adults. In 4. I. 1.3. Prostaglandins and eicosanoids in nema- immune, challenged mice, these sites of development todes. Some helminths have been found to contain become surrounded by in~ammatory ceils and despite large amounts of free fatty acids which could con- the intensity oflocal cellular activity, few if any worms ceivably be used in the manufacture of lipid-derived are actually killed within such foci of cellular activity. mediators such as prostaglandins and leukotrienes, There is a very obvious stunting of worms and their known to have potent immunomodulatory activity. capacity to survive in the gut lumen is greatly impaired Indeed, prostaglandins have been reported from a feu once they have completed development. However. helminths. e.g. Taenia t~i~n~a~~)rr~~~~ (see Leid & larvae can survive in such granulomata for weeks and McConnell, 1983a.b). In S. rn~~zs~nj pJ-ostaglandjI]s still succeed in returning to the gut lumen. Ey (1988) have been shown to be necessary during cercarial has suggested that larvae are protected, at least to a penetration but there is no evidence that they are degree, through continuous secretion of antigens actually of parasite origin as distinct from exploited which mop up specific antibody and form immune host-synthesized prostaglandins (Sdlafsky, Yu-Shang complexes. The parasites also deplete complement Wang, Fusco & Antonacci. 1984). In F. hqwticcr PGE locally. Soluble jmmune complexes confining C3 and PGF had a marked effect on glycogen phos- fragments would compete with cuticle-bound C3 and phorylase activity but again the PCs were not shown antibody for appropriate receptors on leukocytes. Ey to be of parasite origin (Simonic, Sartorclli R: has hypothesized that this may prevent attachment of Locatelli, 1983). Recent studies, however, have dctcc- effector cells to the parasite cuticle, thereby allowing ted PG E2 and F2 in S. rcltti (see Minematsu, worms sufficient time to complete development and Yamazaki. Ufi, Okabe. Korenaga & Tada. 1990) and eventually to escape from the granulonlata despite the PG A, B. C, D. E and F in T. ,~~~rf~l~.~and T. intense local accumulation of inflammatory cells. p.~~~u~~)sp~ral~.swith up to 39.28 and 41.8 ,ug ml-’ OS Immune complexes are recognized as having the parasite homogenate, respectively (Hadas & Staude- potential to cause downregulation. presumably Adamczewska, 1990). Furthermore, the L3 of N~VLIIOI through feedback circuits which turn off unnecessary mwricmus arc now known to secrete quantities of antibody secretion (Barnett, 1986). In this context K eicosanoids (including both prostaglandins and leuk- b~zn~~~~~jis associated with immune complexes (Das- otriennes) during penetration of mammali~~n skin gupta, Bala & Dutta, 1987a; Prasad, Reddy & Harin- (Salafsky, Fusco & Siddiqui, 1990). 0. ~;~.~~~~7~adults ath, 1983; Dissanayake, edpdhitiyawa & Ismail, 1982) have a non-glycosyiatcd lipid composition and ;I and a recent study has shown that the dominant substantial repertoire of glycolipids including many antigen involved was the 200 kDa phosphorylcholine with the characteristics of gangliosides (Maloney & (PC)-bearing glycoprotein often detectable in the Semprevivo. 199 I ). Adult lilariae may release some of circulation of infected persons (Lunde, Paranjape. these lipid compounds and since it is known that Hawley & Ottesen, 1988) and experimen~dll~- infected ~ly~osphingolipid and sphingolipid br~akdo~vn monkeys (Maizels, Morgan, Gregory. Selkirk. Pur- products including phospholipids can depress the nomo, Sukartono & Partono, 1988). Immune com- host’s immune response (Hannum & Bell. 1989). these plexes have also been observed in cats with B. p~hrrn~i molecules also have potential as immunomodulators. and humans with B. mcrla~i (see Au, Denham. In rabbits infected with 7rrc~~~~~~tr pd/idwn PGE Steward, Draper, Ismail, Rao 81 Mak. 1981). Func- secretion by host macrophages 3 weeks after- infection Understanding chronic nematode infections 887

has been implicated as responsible for inducing sup- been largely unsuccessful. It is therefore likely that pression of T cell activity. PGE is known to down- parasites exploit host-manufactured precursors fur- regulate IL-2 secretion and consequently blast cell ther along the biosynthetic pathway towards ecdysone activity and clonal expansion of potentially reactive T than cholesterol or alternatively that they employ cells with resultant severe overall depression of host synthetic pathways for ecdysteroids yet to be deftned. responsiveness (Salafsky & Fusco, 1987). Prostaglan- Overall, there is still too Little relevant information dins also have potent anti-inflammatory effects (Moq- for a comprehensive assessment of these molecules in be1 & MacDonald, 1990). Tomai er al. (1989) in fact nematodes and, as emphasized above, a central suggested that this sequence of events may even be problem in most studies has been to demonstrate initiated by T. pallidurn secreting PGE2 which in turn conclusively that the steroid molecules detected origin- enhances PGEZ secretion by macrophages thereby ated from the parasite’s metabolic pathways and not mediating suppression. Clearly parasites which secrete those of the host (Barker, Chitwood & Rees, 1990). PGE or similar molecules could also exploit these However, since there is evidence for the presence and immunoregulatory pathways to tone down host release of ecdysteroids in both the parasitic Sercernen- protective responses. In support of this suggestion it tea and Adenophorea [and also among free living has been shown that immunosuppression initiated by species (Dennis, 1977), where the evidence for Trypanosoma brucei infection in mice is mediated endogenous synthesis of ecdysteroids in Cnenorhab- through at Ieast two m~hanisms, one of which is ditis eieguns has also been questioned (Barker et al., prostaglandin-independent and accounts for down- 1990)], it is possible that the ability to exploit steroid regulation of IL-2 receptor expression and the other, molecules, whether de novo or xenobiotically synth- involving prostaglandins causes suppression of IL-2 esized, evolved prior to parasitism in the phylum. secretion (Sileghem, Darji & de Baetselier, 1991). Consequently it would not be surprising to discover 4.1.1.4. Ecdysteroids in nematodes. Some nema- that some parasitic nematode lineages have evolved todes have been reported to contain and release the pathways necessary to produce host-metabolism ecdysteroids. Molecules resembling ecdysone or show- regulating molecules from steroids and/or other ing ecdysone-like activity have been found in larval precursors. Lipid metabolism has been documented in (L3 of H. contorrus, Dennis, 1977) and adult Sercer- Trichurisglobulosa (see Sarwal, Sanyal& Khera, 1989) nentea (adult Ascaridida, e.g. , and 0. gibsoni (see Maloney & Semprevivo, 199 1) and Horn, Wilkie & Thomson, 1974; Fleming, 198.5; shown to involve fast turnover of several key equorum, O’Hanlon, Mercer & Rees, 199 1 molecules including cholesterol. The potential impor- and simplex, Evershed, Mercer & Rees, 1987; tance of steroid-based molecules in affecting host adult Spirurida, e.g. D. immitis and B. pahangi, immunoregulatory pathways must therefore be con- Mendis, Rose, Rees & Goodwin, 1983; Barker, Mer- sidered. In most mammals steroids and related cer, Rees & Howells, 1991; larval Strongylida, e.g. L3 molecules are potent immunosuppressants, agents of N. brasiliensis, Bottjer, Whisenton & Weinstein, such as cortisone and betamethasone having been used 1984) and also in the Adenophorea (Ll of T. spiralis, extensively in immunosuppressive therapy as well as Hitch0 & Thorson, 1971). experimental work. Furthermore, recent reports released 20-hydroxyecdysone 25-glucoside (O’Hanlon showing that ecdysone and 20-hydroxyecdysone have et al., 199 1). Endogenous ecdysone and 20-hydroxyec- dose-dependent immunosuppressive activity in mice dysone have been detected in Ascaris suum and D. (Barker, Chitwood & Rees, 1990; Barker, Mercer, immiris (Cleator, Delves, Howells & Rees, 1987). The Rees & Howells, 1990), can be detected in nodule function of steroid molecules in nematodes is not tissues immediately surrounding 0. gibsoni and 0. understood but members of the steroid famity of ~ul~~i~s (Mercer, Barker, Howells & Rees, 1989) and molecules are important growth- and reproduction- that parasite-derived ecdysteroids (from Lou loa and modulating hormones in vertebrates and invertebrates Mansoneh perstuns) may be identified in the host’s (Beckage, 1991). Barker et al. (1991) concluded that circulation emphasize that this is a realistic possibility ecdysone, an insect hormone, may play an important (Lansoud-Soukate, Gharib, Baswaid, Capron & de role in reproduction in filarial worms, by stimulating Reggi, 1990). Finally, the association of high ecdys- mf release in B. pahnngi and by inducing meiotic teroid levels in man with pathology suggests that host reinitiation in oocytes in D. immit~s. In comparison 20- immunoregulation may indeed be perturbed by these hydroxyecdysone had little effect. However, attempts molecules (Lansoud-Soukate et al., 1990). to demonstrate de novo synthesis of ecdysteroids from radiolabelled cholesterol in B. pahangi and D. immitis 4.1.2. Possible ,factors and likely modes of action. (Mercer, Barker, McCall, Howells & Rees, 1989) and 4. I .2.1. Cytokine-like molecules. In a recent paper ti. cut&ii (Koolman, Walter & Zahner. 1984) have Dopheide, Tachedjian, Phillips, Frenkel, Wagland & 888 J. M. BEHNKE. C. J. BARNARD and D. WAKELIN

Ward (1991) reported that they had cloned an 11 kDa cules have affinity for class II MHC, to which they molecule normally secreted by L4 and adult T. bind outside the conventional peptide-binding groove. colubr(formis. On sequencing, this factor showed Communication with T cells is effected through the striking homology with a human gamma interferon- MHC-T cell receptor interaction although the com- induced protein. The latter is a molecule comprising ponent of the TCR involved is also distinct from that 145 amino acids in a simple linear arrangement and is involved in conventional recognition of MHC-presen- thought to be an intermediate of gamma interferon- ted peptide fragments. Thus MHC-bound super- induced activation (Blomstrom, Fahey, Kutny, antigens interact with the V, chain of the TCR, the Korant & Knight, 1986). It is conceivable (although exact V, allele determining susceptibility to particular we concede highly speculative at this stage) that superantigens. Each superantigen therefore has the nematode parasites have evolved similar molecules capacity to activate a particular pane1 of T cells through which they might downregulate host immun- depending on the V, chain expressed. Su~rantigens ity. In rodents, where CD4’” T lymphocytes are are the most potent activators of human and murine divided into two subsets now referred to as Thl and lymphocytes yet described mediating activation at Th2 cells, the latter are believed to be instrumental in picomolar concentrations. Although the biological mediating the inflammatory response usually function of this interaction is not fully understood, it associated with worm expulsion (Mosmann & Coff- has been suggested that superantigens stimulate man, 1987). Mouse strains which respond with Thl CD4’” and CD8-” T cells which proliferate and on ceils fail to express good immunity (Else & Grencis, differentiation into cytotoxic T cells proceed to kill 1991). The Thl response is associated with increased MHC expressing antigen presenting cells (e.g. levels of gamma interferon and TNF/J in the circula- macrophages and B cells) as well as activated class I1 tion. both cytokines originating principally from the bearing autologous T cells. This results in the deple- Thl cells. Furthermore, IFN gamma is known to tion of the cells necessary for the generation and downregulate the Th2 response (Mosmann & Moore, expression of host protective mechanisms locally and 1991). Thus by secreting a molecule which functionally thereby generates an opportunity for the bacteria resembles the gamma interferon-induced protein, T. secreting superantigens to proliferate (see Dohlsten. coluhriformis may be committing the host to a non- Hedlund & Kalland, 1991 for recent review). protective Thl response and thereby avoiding immun- We concede that to the best of our knowledge no ity. such factors have as yet been described from parasitic Another molecule also cloned from T. ~~~~~r~~~r- nematodes although many helminth parasite antigen mis, 30 kDa in size, shows 28% sequence homology preparations have unde~ned nlitogen~city. In a recent with a 25 amino acid peptide called valosin from the study Behnke & Wahid (1991) showed that mice porcine upper intestine (Savin, Dopheide, Frenkel, bearing the MHC haplotype H-2’ were particularly Wagland, Grant & Ward, 1990). The latter is a slow at expelling H. polygyrus. Moreover this haplo- modulator of acid and pepsin in the stomach and type produced the slowest antibody responses to the bicarbonate and protein in the pancreas, with parasite and it was suggested that parasite factors additional ele~trophysiological effects including chan- preferentially impair H-25 bearing antigen presenting ges in gut motility. It has been proposed that the cells and/or homologous T cells through direct inter- parasite molecule may mediate some of the patho- actions. Although this interpretation is highly physiological consequences of infection with T. speculative, the precedent among bacterial super- colubr~formis (Savin et al., 1990). antigens shows that parasite factors can have affinity 4.1.2.2. Other targets for parasite immunomod- and specificity for host membrane receptors and there ulatory factors. Besides the studies discussed above, is no reason why factors capable of blocking or there is little further information at present to indicate directly impairing such molecules should not have how nematode factors might interfere with host evolved. On an even more speculative note, we predict immunity. Part of the problem is still an incomplete that future studies will reveal parasite factors which understanding of what exactly is required in order to block cytokine receptors and others which mimic kill, eliminate or expel parasitic nematodes from the cytokines, thereby causing disregulation of normal host. In bacterial, viral and plant parasitic fungi the pathways and circuits through which the immune evasive strategies of invasive organisms are much response is orchestrated. Such mechanisms arc better comprehended because a detailed molecular especially appealing because communication via description of host-protective resistance is available. cytokines is extremely local, usually expressed at the Perhaps among the most intriguing of relevant cell-cell interface. Parasites which have evolved the mechanisms are the so-called bacterial ‘superantigens’ ability to mimic or disrupt these channels of commun- such as the Staphylococcal enterotoxins. These mole- ication should impair host immune competence locally Understanding chronic nematode infections 889 and not systemically, enabling their own survival but intimately linked to the particular qualities of the leaving the host with the competence to respond to relevant immune system. Apart from domestic other infectious organisms. animals, man and laboratory rodents few other animals have been studied in sufficient detail for 4.2. The lessons from host specificity molecular differences between their receptors and Some parasites have a wide host specificity (e.g. cytokines to be understood. However, it is likely that Trichinella spiralis) but by far the majority depend for each parasite produces several molecules with greater their survival in nature on only a single host species or or lesser specificity for the particular components of at best several closely related hosts. Whilst the host species which it has evolved to parasitize, i.e. physiological/ecological incompatibilities obviously the host with which it has been involved in an arms rank as major impediments to the colonization of race. other hosts and hence serve to limit the host range in It is regrettable (but easily appreciated why) that nature, the immune system in abnormal hosts may be most laboratories concentrate their activities on one an equally formidable obstacle. Even closely related chosen host and parasite system. Comparative studies hosts, occupying similar ecological niches whilst har- of parasites in different host species as well as in host bouring similar species of parasites, will nevertheless strains of varying response phenotype can yield inter- have species of their own and will share few identical esting data which may help to explain immuno- species, e.g. , cattle and deer grazing common modulation. In Mastomys natalensis, the pattern of pasture. primary microtilaraemia was much the same for L. Whilst some evasive strategies, such as an acquired carinii. A. viteae. B. pahangi and B. malayi, all four disguise of host molecules, may be equally effective in species generating microfilaraemia which lasted for unrelated host species, in general immunomodulation well over 250 and even 350 days @anger, Lammler & is likely to be host-specific because of the degree of tine Kimmig, 1981). Thus whatever the evasive strategies tuning necessary to disrupt immunoregulatory path- of these parasites, the differences in immuno- ways. Examples of parasites which can be grown or depression or accompanying alternative mechanisms, even maintained in abnormal hosts treated with all four were successful. Perhaps these results indicate immunosuppresive drugs are too many to list. It is that M. natalensis are unable to respond with likely that immunomodulatory mechanisms function appropriate host-protective immunity against tilariae with optimum efficiency only in the hosts for which in general or they may reflect a mechanism targeting they evolved, although it is conceivable that some IMF conserved rodent molecules as explained above. may interfere with conserved host molecules. When eosinophilia was studied in three groups of Heligmosomoides polygyrus is but one species in a rodents infected with B. pahangi, an interesting con- family of nematode parasites affecting rodents trast emerged. Thus in resistant mice (BALB/c) where throughout the world. Several closely related parasites no microfilaraemia was induced, eosinophilia was affect voles in Europe, Asia and North America. confined to the first 4 weeks of infection and was Experiments with the H. polygyrus bakeri, a species probably linked to the response against L3 and L4 now maintained in laboratories around the globe in stages. In susceptible jirds, eosinophilia was observed laboratory house mice, have established that it will not only in the first 8 weeks of infection and declined with survive for long in the European field mouse the onset ofmicrotilaraemia (Nakanishi, Horii, Fujita, Apodemus sylvaticus even though when immuno- Terashima, Ueda & Kurokawa, 1987), perhaps suppressed the parasites develop normally and grow indicating that some form of immunomodulatory into fecund adults (Quinnell, Behnke & Keymer, influence was exerting a downregulation of the Th2- 1991). Essentially the only obstacle limiting the useful- IL-5 axis. Consistent with this suggestion is the ness of A. sylvaticus as a host is the immune system. recognized suppressor cell activity in jirds infected Thus the immunomodulatory strategy which is so with B. pahangi (see earlier section 4) and subsequent successful in Mus domesticus does not work to the work by the authors showing that jirds with chronic benefit of the parasite in field mice. Likewise Helig- microtilaraemia had markedly depressed eosinophil mosomoides polygyrus polygyrus, which affects A. responses to homologous but not heterologous sylvaticus in Europe and causes chronic infections in challenge (Horii, Nakanishi, Mori, Zaitsu, Ueda, the field mouse (Gregory, Keymer & Clarke, 1990), is Kurokawa, Oda & Fujita, 1989). However, in rats easily killed by M. domesticus during the tissue stage of which are also susceptible, eosinophilia remained high development. Again the immunomodulatory strategy throughout patency and there was no evidence of a fails in a closely related but different host species. decline until mf were eliminated by anthelmintic The specificity of immunomodulatory mechanisms treatment. One conclusion from this study was that for definitive hosts suggests that the factors are eosinophilia played little role in resistance, at least in 890 J. M. BEHNKE. C. J. BARNARD and D. WAKELIN

rats. In this example, does the immunomodulatory with limited scope for escalation as a specialism. In the mechanism fail in rats thereby allowing eosinophilia to context of the host’s entire parasite community. persist? If the immunomodulatory mechanism is suc- generalizing adds a tier of within-species diversity that cessful in jirds (and other studies suggest that indeed it may compound the host’s difficulties in responding to is, Lammie & Katz, 1983a,b), does this offer an parasite counter-adaptations. opportunity to identify the immunological target through comparative across-species studies? In vitro 4.4. Stability or labilitv.? experiments using leukocytes from both species may Despite considerable efforts and clear evidence that identify the point of resistance to immunomodulation parasite immunomodulatory factors must exist, there in rats and thus provide clear indication of the target of is still no single immunomodulatory molecule from parasite immunomodulatory factor (IMF). any nematode which has been identified, its structure elucidated and its biological properties understood. 4.3. One system or mure? The simplest explanation for this lack of progress is Clearly, there are several potential evasion that the factors concerned are difficult to isolate, strategies open to parasites. The question thus arises as possibly because they may be short-lived or involve to which strategy to adopt and whether to specialize or complex interactions not yet understood. Thus IMF generalize. Choice of strategy will be determined by may be intrinsically labile or, like cytokines, they may the feasible option that most reduces the host’s impact operate most efficiently when several component on the parasite’s survival and reproductive success factors can synergize. They may be rapidly meta- relative to the cost of the strategy to the parasite. bolized and probably act at short distances (i.e. Whether to specialize or generalize will depend on the membrane to membrane). If indeed parasite strategies relative advantage of each approach to the parasite in prove to be dependent on many distinct IMFs, then the host/parasite arms race. the isolation of any one such molecule would represent Specializing has the advantage that more resources only the start. As with cytokinc research in recent can be invested in the chosen strategy which may thus years the full complexity of the interactions involved afford a longer period ofgrace before the host counter- would only be understood when all the components adapts, but the disadvantage that the host can also had been identified and their interrelationships concentrate its resources. However, if the host is appreciated. threatened by a range of parasites, it may not have the From the evolutionary perspective. it would bc resources to concentrate. The effectiveness of special- expected that parasite IMFs have short half-lives. II ization is also likely to depend on the scope for the purpose of IMFs is to extend parasite survival to counter-adaptive refinement. An immunomodulatory maximize the production of transmission stages strategy. for example, could be escalated by secreting through which subsequent generations of genes might different, preferably unrelated, molecules to impair be perpetuated, it follows that the extent to which the the various cytokinc-mediated pathways which host should be suppressed becomes a dilemma of some regulate the immune system. Each step in this com- importance. Long-lived factors would accumulate in plexity is open to manipulation. A parasite molecule the host as the parasite survived and if relcascd resembling a particular cytokine may block, impair or systemically would have a progressively increasing compete with a corresponding cytokine or in turn may deleterious overall effect on the host, weakening its target the corresponding cell receptor. Parasite immune system. It would hardly be of benefit to a immunomodulatory molecules may have wide-spec- chronic species to create a space for itself initially and trum efficacy, blocking more than one vital interaction then to lose it as the host dies from other infectious between host components or may target key cytokines diseases against which it cannot respond through influencing divergent pathways in the existing hier- severe immunodepression. If such factors could be archical structure, or there may be a panel of confined locally, however, a safe haven from the molecules with individual targets. Which ever way, immune system might be created. but such a solution there would be scope for evolution of novel factors in would require the parasite to have the ability to response to refinement in the host’s defences. Such a modulate the release of IMF as and when required in strategy could be pursued for several rounds of the the parasite’s immediate vicinity. A far simpler solu- arms race before the parasite wins, succumbs or tion would be for IMFs to have short half-lives and to switches to a different strategy (e.g. camouflage). be continuously secreted, the net outcome of which Generalizing to two or more evasion strategies would also be to produce local immunodepression spreads both host and parasite resources more thinly. confined to the immediate microhabitat of the parasite However, by reducing the selection pressure against in question. much like the manner in which cytokincs each strategy, it may increase the efficacy of strategies arc now known to operate in rig). Understanding chronic nematode infections 891

4.5. Consequences of immunomodulation but often many more. B. pahangi aggregate in lym- Whilst immunomodulatory strategies are likely to phatic vessels of dogs and a recent study has shown be finely tuned to the particular characteristics of the that under infection intensities such as those normally definitive host, their mechanisms of action are likely to encountered in the field, there may be local immuno- be non-specific. A prediction of this hypothesis is that depression in popliteal lymph node cells to parasite other parasites living in proximity to the immuno- antigens whilst peripheral blood lymphocytes remain modulatory species will be able to benefit from the responsive (Miller, Schreuer & Hammerberg, 1991). local downregulation of host immunity. Gastrointestinal species are seldom uniformly dis- Earlier literature has already been reviewed (Beh- tributed along the gut. Often the majority of worms nke, 1987) but in recent years interactions of this type may be found in only a few centimetres in large hosts have been described for bovine species (Kloosterman and less than a centimetre of the small intestine in & Frankena, 1988). Thus cattle infected with both rodents. It is particularly noticeable that the degree of Ostertagia ostertagi and oncophora are more aggregation increases as the worms come under stress susceptible to infection with the Dic- from the immune response. This has been observed in tyocaulus viviparus and develop larger and more H. polygyrus, T. colubriformis and in T. vitrinus. In the fecund female (Kloosterman, Frankena & latter two species, ‘fingerprint lesions can be detected Ploeger, 1989). This effect appears to be associated in the late stages of infection. Most of the pathology is with experience of combined infections with both of confined to these lesions and most of the worms are the intestinal species but is not dependent on their located in them. Neighbouring sections of the gut may presence since increased susceptibility is still apparent have quite normal morphology and may be totally free when the gut worms are removed by anthelmintics of worms (Jackson, Angus & Coop, 1983; Angus & (Kloosterman, Ploeger & Frankena, 1990). The Coop, 1984). mechanism through which this is achieved is unknown but the observations could be explained by immuno- 5. GENETIC CONSIDERATIONS modulatory activity of the intestinal worms affecting Each host-parasite association is the product of a components of the mucosal system and consequently complex interrelationship, the outcome of which is downregulating resistance in the lungs. However, in decided by an interaction between the genetically this case a long lasting effect has been observed and it determined characteristics of the two species concer- may be that another explanation will be found but it is ned. In the examples considered here, i.e. ‘natural’ conceivable that a persistent immunodepression host-parasite relationships, the nematode is preadap- follows experience of infection with both 0. ostertagi ted to the morphological and physiological character- and C. oncophora through the synergistic interaction istics of the host and is therefore readily able to of their individual mechanisms. It is pertinent that establish in the host. No evidence is available for neither species alone was capable of enhancing the genetic variation in host molecules that might act as establishment and growth of lungworms. Other exam- ligands for parasite receptors and which, by analogy ples include enhanced establishment of A. viteae in with the situations known to exist in relation to jirds concurrently infected with B.malaJG (Court et al., infection with Escherichia co/i (see Sellwood, Gibbons, 1988) and increased establishment of Ostertagia oster- Jones & Rutter, 1975) and Plasmodium vivax (see tagi and 0. leptospicularis in mixed infections com- Miller, Mason, Clyde & McGinniss, 1976) might pared with single species infections (Al Saqur, prevent establishment in otherwise permissive hosts, Armour, Bairden, Dunn, Jennings & Murray, 1984) although these might well exist. Once established, the (see also Behnke, 1987 and Christensen, Nansen, worm will survive for a shorter or longer period, the Fagbemi & Monrad, 1987). greatest threat to long-term survival being the host’s Another consequence may be that parasite species adaptive immune response. The duration of survival accumulate in sites within hosts which they transform could be determined at one extreme, in the absence of into safe havens from the host response through their effective immunity, by the genetically programmed life collective immunomodulatory activity. Examples of span of the parasite or, at the other, by the strength parasites aggregating within infected hosts are again and speed of the host immune response. In the simplest only too familiar. There are of course other benefits terms it might therefore be considered that the prime such as the availability of the opposite sex in dioecious evolutionary considerations are for the parasite to species but often the numbers involved vastly exceed achieve the maximum possible survival by avoiding or the requirement for one representative of each sex. In downregulating the host’s immune response, whatever rats infected with Litomosoides carinii, many worms the cost, and for the host to minimize parasite survival become entangled in knots within the pleural cavity. by mounting the strongest possible response, again 0. vo/vu/us likewise accumulate two to four in a nodule whatever the cost. However, as this review makes 892 J. M. BEHNKE.C. J. BARNARD and D. WAKELIN

clear, such a simple view is misleading because it fails modulatory molecules acting as factors regulating to take account of the need for each partner to host immune and inflammatory responses. Other maximize fitness, which inevitably involves cost-ben- molecules may be involved in camouflaging the efit trade-offs in relation to subverting or increasing exposed cuticular surface. All of these molecules are immunity. Evaluation of these trade-offs is difficult, gene products and it is well known that there can be not least because comparatively little is known about developmental regulation of their expression and the theoretical maximum life-spans of parasitic release, i.e. the molecules may show stage specificity. nematodes; it is not possible in in vivo studies to Equally, as there undoubtedly is genetic polymor- exclude all influences of host origin, even in immuno- phism within all populations of worms, there will be logically anergic animals, and in vitro studies are genetically determined variation in the structure of inappropriate. The data of Robinson, Wahid, Behnke these molecules, some of which is likely to influence & Gilbert (I 989) on survival of H. polygyrus infections both their biological and their immunological activity. in mice provide one of the few detailed and well- At the present time there are data relating to molecular controlled studies in this area, showing clearly how variation within parasitic nematodes, albeit largely at life-span in this species is largely host-determined. a descriptive level, and a little is known about the Conversely, although many parameters of the host influence of such variation on the host response, but response can be used to measure the speed and almost nothing is known of the genetic basis for this strength of response to infection, it is difficult in vivo to variation. This is one area in which the research monitor the subtle degrees of difference in parasite- carried out with the model nematode derived antigenic stimulation or, more importantly, in will provide valuable pointers. immunomodulation, that ultimately determine these The belief that the molecular variation existing in parameters. Immunoparasitologists interested in the nematodes will be reflected in significant differences in biology of chronic infections must therefore interpret host response, and therefore in parasite survival, is the data provided by experimental approaches with supported by the observation that relatively few of the considerable caution if they are to reach valid con- many molecules exposed to the host appear to func- clusions about the determinants of parasite survival. tion as immunodominant antigens. Given it is likely One particular area in which this caution has to be that host recognition of these antigens is directed exercised concerns the immunogenetics of host-para- against a limited number of epitopes, it can be site relationships. There is now a substantial body of assumed that quite small changes in molecular struc- information concerning host genetic influences upon ture could result in significant differences in host immune and inflammatory responses to nematode recognition and response. If such differences resulted infection (Wakelin, 1988) and it is universally accepted in enhanced parasite survival or reproduction there that there is considerable variation within outbred would be a positive selection to maintain the highly populations in ability to respond immunologically to, variable molecules in the population. Maintenance of and thus control, infection. It must also be the case variable immunologically active molecules in the that there will be considerable genetically determined parasite population can be seen as an evolutionary variation within parasite populations in ability to elicit strategy designed to counter host-population hetero- or to modulate host responses. geneity in immune responsiveness and, while not the primary selection pressure, the effect would be to 5. I. Parasite genes influencing host responses reduce the effectiveness of long-term host immunity to Host responses to are elicited by a particular species (Allison, 1982). If it is the case that two subsets of molecules, those expressed at the nematodes release molecules whose primary function cuticular surfaces and those released (excreted/ is to interact with and modulate host responses, then it secreted) into the immediate environment of the worm is clear that molecular variations leading to more (E/S molecules). Both include protein, glycoprotein effective modulation will be strongly selected, and carbohydrate components, and it is now well- polymorphism in this case having less selective value. established that, in many species, the two sets have A similar argument applies to variation in molecules components in common. The biological functions of involved in parasite camouflage, although in both the majority of these molecules are unknown, except cases, the fitness costs of synthesizing and releasing where enzymatic properties have been defined, but it is these molecules have to be taken into account. clear that many play an important role in eliciting or Evidence that intraspecific parasite variation can modifying host responses (Almond & Parkhouse, influence the outcome of infection is limited to a few 1985; Lightowlers & Rickard, 1988). Among the nematodes and some of this evidence is observational immunologically active components will be molecules rather than experimental. Perhaps the best known acting as antigens and allergens, and immuno- observational data concern the variations in the Understanding chronic nematode infections 893

immunology and immunopathology of onchocerciasis 5.2. Host genes controlling protective responses between the forest and savanna zones (Piessens & It has been known empirically for many years, and is McKenzie, 1984). It has been proposed that these now universally accepted, that the interaction of hosts variations reflect molecular and other biological dif- with infectious organisms is profoundly influenced by ferences between populations of 0. volvulus, but, genetic factors. This is most clearly demonstrable although these are known to exist, there are few data when inbred strains of laboratory mice are exposed to available directly to support this correlation. Experi- standardized inocula under uniform experimental mental studies of intraspecific variations influencing conditions, but is equally evident when detailed immunological aspects of infection have been carried studies are made of outbred populations (both human out using isolates of Trichindu. This genus is, per- and animal) under natural conditions of exposure to haps, the least host-specific of all nematodes, and to infection. There is now an enormous literature des- some extent this lack of specificity (compounded by a cribing both the genetics of this intraspecific variation basic morphological similarity) has led to an uncertain and the components of host defensive responses . At the present time there is a view that the through which variation is expressed (Wakelin & genus contains a number of distinct gene pools, some Blackwell, 1988). In a small proportion of cases the of which are agreed to represent full species (Pozio, genes involved have been identified and sequenced, 1987). Several studies have shown that when different and the functional significance of their gene products Trichinellu isolates are used to infect particular strains determined, but in the majority of cases analysis has of laboratory hosts, there can be substantial variation not proceeded quite so far, and this is certainly true of in parameters of infection (Dick,‘l983). Among the host-parasite relationships involving nematodes. most significant variables are the length of survival of It is true to say that the existence of such extensive adult worms in the intestine and the level of worm variations in ability to respond to infection has usually reproduction. Some isolates survive for a compara- been interpreted in terms of the genetic diversity tively long period in the intestine (i.e. are relatively necessary for maintaining high levels of host popul- chronic) and generate large numbers of muscle larvae, ation resistance in the face of constantly changing others have a short survival time and produce rela- populations of pathogens. In this view, evolution is tively few larvae. Some workers have attributed seen to act by selecting the genotypes associated with variation between different isolates purely to gen- the strongest and most efficient immune responses. It etically determined differences in parasite life-span is our contention that it is equally valid to see both host and fecundity (Dick, 1983), however, both of these and parasite variation as providing the evolutionary parameters are known to be significantly influenced by material for the selection of host-parasite relation- the host’s immune response (Wakelin & Denham, ships that confer optimal fitness on both partners. 1983) and could therefore reflect not variation in Selection may well, therefore, be for less than maximal programmed capacity for worm survival and repro- host responsiveness and less than maximal parasite duction, but variation in immunogenicity. Experimen- evasive or immunomodulatory capacities, in order to tal support for this view has been obtained by produce an interaction in which both partners can comparing the outcome of infections with different survive and reproduce. isolates in inbred mice of different response pheno- Genetic variation in response to nematodes can be type, as well as intact and immune-suppressed animals measured by a number of parameters, including (Bolas-Fernandez & Wakelin, 1989). Although dif- prevalence and duration of infection. Differences in ferences in immunologically recognized molecules prevalence in populations known, or suspected, to be between Trichinellu isolates are well known, we are still exposed uniformly to infection can be assumed to a long way from being able to correlate these differ- reflect differences in duration of parasite survival ences with functional host responses, although such resulting from differences in the degree of host-protec- work is being undertaken (Wassom, Dougherty & tive responses. This assumption can only be tested in Dick, 1988; Bolas-Fernandez & Wakelin, 1990). Given studies where sequential time course assays of parasite the progress now being made in analysis of Trichinella survival are possible. Data from studies involving antigens at the immunochemical and molecular level, human hosts, and many of those on domestic or wild and our present understanding of the initiation, animals, provide only prevalence data, time course regulation and expression of anti-Trichinellu immune data come largely from experimental systems and responses, we can be optimistic that this system has the particularly those involving mice. In some systems potential to provide a real insight into the role of (e.g. with Trichinella spiralis) differences between genetically determined parasite variation in determin- individual mice or between strains of inbred mice may ing the outcome of infection and thus contribute to our be quite small, high responders retaining infection for understanding of chronicity of infection at this level. only a few days to a week or so more than low- 894 J. M. BEHNKE. C. J. BARNARD and D. WAKELI~

responders (Wakelin, 1980). In other systems differen- been studied through several volunteer exposures and ces may be very large and, on occasion absolute. For these have indicated a life-span of 15-17 years in the example, in some inbred strains of mice infections with case of N. americanus (see Beaver, 1988). However, all H. polygyrus or Trichuris murk are controlled immun- the trials with N. americanus have been carried out on ologically and the worms expelled, in others, equally Caucasian volunteers. There are data from the time ‘normal’ as far as conventional immune responses are when hookworm infection was widely distributed in concerned, the infection may persist until worm the southern states of the U.S.A. (Smilie & Augustine, senility intervenes (Robinson et al., 1989; Wahid, 1925; Leathers, Keller & Wyman, 1936) that the Robinson & Behnke, 1989; Else & Wakelin, 1988). disease and consequently heavy infections were more With the latter species such differences can also be seen common among the Caucasian inhabitants of these between individuals within strains, both outbred and regions compared with the sector of African origin in inbred. Similar data are available for filarial infections the population. It may be, therefore, that races which in mice. Infections with the jird species Acantho- have lived in endemic regions for a long time have cheilonema viteae can be established by direct transfer evolved resistance mechanisms, but to date these have of female worms. In some strains this produces a long- not been identified and there is still no clear evidence term, high-level microfilaraemia, in others that humans as a whole have any protective device to microfilaraemia is of low level and short lived (Haque, curtail hookworms (Behnke, 1991). Data from work Worms, Ogilvie & Capron, 1980; Storey, Wakelin & with domestic animals are more clear cut. Many Behnke, 1985). Strains of mice also differ in the ability authors have shown genetically determined differen- to sustain infections with Brugia spp., whether these ces within and between breeds of sheep in ability to are initiated by injection of infective larvae or micro- resist infections with gastro-intestinal trichostron- lilariae, or by transfer of adults (Howells, Devaney, gyles, and there is no doubt about the role played by Smith & Hedges, 1983; Fanning & Kazura, 1983; immune responsiveness in this resistance (Windon. Suswillo, Doenhoff & Denham, I98 1). Collectively 1991). Low responder sheep sustain heavy and chronic these experimental data provide support for the view infections with these worms, high responders can exert that genetically determined differences underlie a sig- a substantial degree of control. The genetic basis for nificant proportion of the variation in levels of infection differential resistance is still largely unknown, alth- apparent when prevalence data relating to gastro-intes- ough a single dominant gene has been proposed for tinal and filarial nematodes are collected in the field. high response to Haemonchus contortus (see Gray, In terms of the major gastro-intestinal nematodes of 1987) and detailed breeding studies have been under- man (Ascaris, hookworm, Trichuris) there is now a taken to analyse inheritance of resistance to Tricho- consensus that infections always show aggregated strongy/us colubrifi?rmis (see Gray, 1991). patterns of distribution in the host population, a One of the intriguing facets of human tilarial minority of individuals being heavily and con- infections, both those involving the lymphatic species tinuously infected, the majority being only lightly and those involving Onchocerca, is the broad spectrum infected or free of worms (Bundy, 1988). Although it is of clinical manifestations and infection levels seen in clear that many behavioural and environmental fac- endemic areas, where challenge from the bites of tors can contribute to this aggregation, it is difficult to infective vectors is likely to be common to all members believe that an absence or low level of infection in an of the community (Piessens & McKenzie, 1982; Pies- endemic area can be wholly explained by lack of sens, McGreevy, Piessens, McGreevy, Koiman, exposure. If it is accepted that variation in protective Sarosa & Dennis, 1980; Piessens, Ratiwayanto, Tuti, responses can determine whether these worms do or Palmieri, Piessens, Koiman & Dennis, 1980: Piessens, do not survive, then it can be assumed that the worm McGreevy, Ratiwayanto. McGreevy, Piessens. burdens in the chronically infected individuals arise in Koiman, Saroso & Dennis, 1980; Ottesen, 1984; one of two ways. They may result from infections in Nutman, 1989). At one extreme individuals may show which worm life span is prolonged, because of ineffec- chronic infections, with persistent microtilarial bur- tive or strategically reduced immunity, or from infec- dens, at the other individuals may be parasitologically tions where the worm population shows a regular negative, despite immunological evidence of exposure turnover, individual parasites having short life spans, to infection. Some of the parasitologically negative but weak immunity allowing frequent and successful individuals will suffer marked pathology (e.g. obstruc- reinfection to take place. Both of course may occur tive conditions such as elephantiasis and hydrocoele. simultaneously, but the net result is a chronic infec- or severe skin conditions), others will show no path- tion. For some of these gastro-intestinal species there ology. Pathological conditions may be equally varia- are data showing that intrinsic parasite longevity is ble among those who are positive for microtilaria. long. For example hookworm infections in man have Many studies have identified significant differences in Understanding chronic nematode infections 895 immunological responsiveness between these various uncertain. There are now several examples [in Ascaris groups (Piessens & MacKenzie, 1982; Ottesen, 1984; suum, B. malayi, H. polygyrus, T. spiralis and T. muris Nutman, 1989) implying a correlation between (see Kennedy, 1989)] of MHC-linked differences in the immune response capacity and the outcome of infec- patterns of serologically detectable antigen recogni- tion. Chronicity of infection here may again result tion and it is possible that differential antibody from defective responsiveness. responses may contribute to differential protective Although many associations have been made in immunity. Individual variation in antigen-recognition humans and in domestic animals between specific patterns have been described in many human immune components and high or low responsiveness nematode infections, and MHC-linked influences on to infection, detailed analysis of the immunogenetic antibody-mediated responses that might be protective basis of these differences is possible only using defined against Trichuris in humans have also been proposed experimental models. Some of these models allow (Bundy, 1988). However, it seems more likely that the identification of defects in particular protective responses most important consequences of MHC-linked genes as the cause of the low-responder phenotype, in others may be their influence on the pattern of initial T cell no direct correlation is yet possible. For example, low responses to parasite antigens and thus the cytokine responsiveness to T. spiralis appears to arise from an profile generated. In this way MHC genes would inherent inability to generate effective intestinal in- control the range of effector mechanisms ultimately flammatory responses, reflected in levels of mas- available for interaction with the parasite, although tocytosis and eosinophilia (Tuohy, Lammas, Wakelin, these would still be regulated by non-MHC genes, as Huntley, Newlands & Miller, 1990; Lammas, Mit- outlined earlier. This aspect of immunoparasitology is chell, Tuohy & Wakelin, in press). Mice capable of now a very active area of research (Finkelman, Pearce, mounting early immunity to T. murk make earlier and Urban & Sher, 1991) and one that should contribute greater specific IgGl responses (Else & Wakelin, very greatly to our understanding of the determinants 1989). Low responsiveness to filarial infections is as- of host resistance and parasite survival. In the mouse, sociated with defective IgM production and possibly with where there is convincing evidence for the existence of low antibody affinity (Gattrill, Kee, Behnke & Wak- defined T cell subsets and subset-restricted cytokine elin, 1991). In contrast, low responsiveness and chron- production (Mosmann & Coffman, 1987) it is striking ic infection in mice infected with H. polygyrus appear that, in most cases, nematode infections selectively to reflect genetically determined host susceptibility promote T helper subset 2 (Th2) responses. Th2 cells to parasite immunomodulation (Behnke & Wahid, release the cytokines 11-3, 4, 5, 9 and 10, which are 1991) although the critical protective components involved in the downregulation of Thl cytokine that are suppressed are not yet known in detail production and in the promotion of eosinophilia, All of these models, and many others, have been mastocytosis and IgE synthesis, all hallmarks of used for intense genetic analysis of response phen- nematode infections. It has been suggested that Th2 otype and particular attention has been paid to the responses may be deliberately induced by parasites as relative roles of major histocompatibility complex a strategy for downregulating host-protective Thl (MHC)-linked and non-MHC-linked genes. The responses, but the evidence that the latter are sig- reasons for this focus are three-fold: resistance to nificantly involved in resistance to nematodes is certain infectious diseases is known to show MHC equivocal. Although this interpretation is supported linkage; MHC-linked genes control the basic process by the work of Pond, Wassom & Hayes (1989) on T. of antigen recognition; MHC gene products provide spiralis, other data from work with this species show convenient markers for population studies concerned that both high and low responder mice produce Th2- with differential resistance to infection. In general, as pattern responses when infected (Grencis, Hultner & far as nematodes are concerned, MHC-linked genes Else, 1991). Additionally, work with Trichuris muris do not exert an overriding influence on resistance per shows that low responsiveness is associated with Thl se, which appears to be most strongly influenced by responses, high responsiveness with Th2 (Else & background (non-MHC) genes. Different MHC Grencis 1991). These latter data are significant in the haplotypes do have a significant effect on the outcome context of this review, because mice that are low of infection, however, when background genes are responders to T. muris not only fail to expel the worms held constant (Wassom, Brooks & Cypess, 1983; Else early in infection, but appear to become ‘tolerized’ by & Wakelin, 1988; Behnke & Wahid, 1991) suggesting exposure to later stages of the parasite and lose their that MHC-linked genes do play a role in regulating ability to expel both the existing and subsequent expression of responses necessary for effective resis- infections (Else, Wakelin & Roach, 1989). The possi- tance, the absolute levels of which are determined ble role of parasite-secreted cytokine-like molecules is elsewhere in the genome; what this role is remains discussed in section 4.1.2.1. 896 J. M. BEHNKE.C. J. BARNARD and D. WAKELIN

The major roles played by non-MHC genes in the ability to make antibodies that neutralize the determining overall levels of susceptibility and resis- activity of these factors. These aspects of the host- tance to infection are still undetermined in the parasite relationship are, of course, another facet of majority of cases. As with MHC-linked genes there is the continuing arms race between host and parasite, evidence of an effect upon the specificity, isotype and but they are of considerable practical importance. kinetics of antibody responses made to infection Identification of the immunomodulatory factors used (Almond & Parkhouse, 1986; Tomlinson, Christie, by parasites and of the mechanisms involved in host Fraser, McLaughlin McIntosh & Kennedy, 1989; counter-measures could make a substantial contribu- Else, Wakelin, Wassom & Hauda, 1990b) and these tion to the rational control of many chronic nematode may of course influence worm survival. Background infections. gene effects upon inflammatory response capacity have been clearly defined in mice infected with T. 6. THE WAY FORWARD spiralis and a good correlation established with protec- Throughout this paper we have attempted to iden- tive immunity to the intestinal stages (Wakelin, 1985). tify common principles which apply across species, There is suggestive evidence that similar correlations genera and higher taxa as well as points of distinction may exist with high and low responsiveness to tricho- between them with respect to their evasive strategies. It strongyle infections in sheep (Windon, 1991). is still early to synthesize a coherent picture, but the process has been initiated and already it is evident that 5.3. Host genes providing resistance to parasite there exist significant differences in survival strategies immunomodulation even between closely related taxa. These do not bode The emphasis in the above section has been on host well for future immunotherapy. genes that may directly control elements of the The chronic survival of adult nematodes and the protective response to nematode infection and there- associated extensive patent period must be important fore influence parasite survival. It has already been in enabling the generation of the quantities of trans- pointed out, however, that the outcome of host- mission stages required to ensure successful transmis- parasite relationships depends upon an interaction sion, otherwise they would not have evolved. In involving both host and parasite. Parasitic nematodes, filariae, the persistence of mf would maximize the like all parasites, are not simply passive vehicles for likelihood of encountering a suitable vector and would antigen presentation, many of them are known to achieve the same objective (Bayer & Wenk, 1988). interfere actively with the host response, using a However, quite different strategies may be envisaged variety of strategies (section 3). An important com- for achieving ultimately th_ese same objectives. For ponent of these strategies is the release of immuno- example L. carinii has mf which are relatively short- modulatory molecules, which downregulate, divert or lived, surviving perhaps only 2214 days at most interfere with host-protective responses. The produc- (Wegerhof & Wenk, 1979). Here the emphasis ap- tion of these molecules is likely to be variable within pears to be on overwhelming the host’s ability to clear parasite populations (see above) and the degree to mf with minimum protection for individual trans- which the host is susceptible to their effects is also mission stages mediated primarily through adsorbed likely to be variable within host populations. Hence host albumin. The daily output from one female L. some hosts may be susceptible to infection, fail to carinii in cotton rats was estimated by Haas & Wenk mount rapid host-protective immune responses, and (1981) to be 20,000-200,000 mf although later Mossin- support chronic infections, not because they lack the ger & Wenk (1986) concluded that 22,000 was a more genes necessary for the expression of an appropriate realistic figure. Mf are coated with host albumin and effector response, but because they are susceptible to there is accompanying immunodepression but large the immunomodulatory products of the worms them- numbers of mf are successfully sequestered and des- selves. This aspect of resistance and susceptibility has troyed in various organs. The daily output of Loa loa is so far received comparatively little attention, but it is estimated at IO-20,000 (Eberhard & Orihel, 1986) and clear from experimental studies with H. polygyrus and there is evidence that the mf of this species, like those with T. muris(Behnke & Wahid, 1991; Elseet al., 1989) of L. carinii, express host-like or adsorbed host not only that it exists, but that it can be an important antigens (Ridley & Hedge, 1977) but in this case mf are determinant of the outcome of infection. Evolutionary long-lived surviving 6612 months (Duke, 1960). A. considerations would predict that hosts would be viteae (7000 mf per female per day, Mossinger & under selective pressure to develop resistance to Barthold, 1988) D. perstans and Onchocerca volvulus parasite immunomodulation, either by modifying the (I-4000 per female per day, Engelbrecht & Schulz- molecules that are the targets for immunomodulatory Key, 1984; Schulz-Key & Karam, 1986) produce few, factors, or by developing counter-measures, such as relatively long-lived mf daily which can survive for the Understanding chronic nematode infections 897 best part of the year and more (see reviews by Behnke, an urgent need to Gnd in vitro ways for their mainten- 1987; Duke, 1968). In these latter species the emphasis ance or adaptation to more readily available appears to be on antigen shedding as a protective experimental animals. device ensuring the survival of individual mf. Despite the difficulties, there must be a concerted It follows from this that the precise significance of effort to isolate. characterize and to understand the each survival strategy must be evaluated in order to mode of action of human nematode 1MFs. Animal allow immunological intervention a chance to succeed. models such as H. polygyrus offer the advantage of A particular strategy may be quite crucial with others convenience but in the longer term what is learned contributing to but not solely capable of enabling through animal models will have to be used to provide survival. This is eloquently illustrated by studies with the basis for the exploration of factors from human N. polygJ)rus. Mice maintained under trickle infection nematodes. It is likely that the active molecules are to regimes and subjected to regular doses of H. ~~13?g~rus be encountered in the secretoryiexcretory products of take 8-12 weeks to develop immunity which expels nematodes and it is here that the search must continue. adult worms and ensures protection against challenge Whilst molecular techniques may be one solution, larvae (Brailsford & Behnke, 1992). The chronicity of until we have some idea of what it is we are looking for, infection is essentially determined by IMF from adult it is difficult to imagine how the relevant factors might worms. Once this was realized, short-term immunizing be identified from gene libraries, other than through infections, abbreviated with anthelmintics before the the meticulous screening of the entire genome, each larvae had developed to the adutt stage, were explored fusion product in turn. One approach worth exploring and found to induce potent acquired immunity (Beh- would be to try using DNA sequences for cytokines nke & Robinson, 1985). Even non-responder mice and vertebrate immunoregulatory molecules, many of such as C57BL/lO and CBA can be made totally which have now been cloned, to identify comple- resistant to the parasite by a 6-day primary infection. mentary sequences in the nematode genome. Under trickle-infection regimes both of these latter In recent years studies on parasite-induced strains accumulate worms and die (Brailsford & immunodepression seem to have lost their appeal and Behnke, 1992). Thus mouse strains which previously are no longer in vogue. Loss of interest in this aspect of could not be made immune were found to express nematode biology stems primarily from the picture strong resistance to challenge so long as they did not emerging in the last two decades which essentially experience adult worms during the immunization indicated that virtually all species could cause period. The lesson from these findings is that non- immunodepression and there was little relationship responsive animals and ho~fully patients as well, can between immunodepression and resistance to infec- be made resistant, once the interrelationships between tion. Much has changed in the last decade. We hope to host resistance and parasite evasive strategies are have shown here that indeed patterns are emerging understood and taken into account when designing and that these may be linked to the evolutionary immunization procedures. biology of the Nematoda. There is still a long way to If immunomodulatory mechanisms prove to be go and much of the road will involve repetitive studies host-specific, then factors produced by parasites affec- on the species which have not yet been examined. ting non-primates may well have little relevance for Ultimately we believe that the rewards will be valuable medicine, except perhaps in enabling us to compre- indeed. A thorough understanding of the scope of the hend the general principles involved. The search for mechanisms evolved by nematodes to counteract the immunomodulatory molecules of potential value in principal stressor to which they are exposed in ver- medicine should concentrate on specilic parasites tebrate hosts may provide us with a direction which causing chronic infections in man because it is these will lead to successful vaccination and ho~fully new which are most likely to target human cell receptors agents for the treatment of immunological complica- and/or immunoregulatory molecules. Here the tions and disorders in man and animals. We believe paradox: host-specific organisms affecting man are that this road is worth following and encourage others extremely difficult to study because they cannot be not to be dissuaded by the difficulties ahead. easily maintained in laboratory animals. Some of the AeknoM,ledgement.~-We xe grateful to Prof. P. N. R. most widespread species affecting man, e.g. Ascaris Usherwood for the facilities provided for our research groups ~lirnbri~oi~~s, ‘4~~.~l~~torna ~~~~enaie, T~~~h~ri~tri- in the Department of Life Science (formally Zoology) at chiura and filarial species such as Nottingham University. JMB would like to thank the and Onchocerca volvulus still cannot be maintained Trustees of the Colt Foundation, the Sir Halley Stewart Trust easily in laboratory animals, other than through the and the Medical Research Council for financial support use of powerful immunosuppressive agents or by provided in recent years. We are also grateful to Prof. R. C. exploitation of exotic host species. There is therefore Anderson, W. Piessens, H. H. Rees. Dr A. Pike and Dr E. R. 898 J. M. BEHNKE. C. J. BARNARD and D. WAKEL~

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