168 Parasitology Today, vol. 8, no. 5, 1992

Animal Models for the Study of Immunity in Human Filariasis

J,B. Lok and D, Abraham

A major challenge to the development of vaccines against human lymphatic filari- asis and onchocerciasis is to direct the immune response toward elimination of the early, prepathogenic larval stages and away from responses that mediate path- ology. In this review, James Lok and David Abraham discuss the various animal models that have been used to investigate the pathways leading to immunity, im- munological tolerance and chronic path- ology in these diseases. Owing to the strict host specificities of the human-dwelling filariae, no single model serves to duplicate exactly all these aspects. Nevertheless, it has been possible to demonstrate a protective immune response invoked by and directed against incoming third-stage larvae of both lymphatic and skin-dwelling filariae. The fact that subsets of the sequelae of human filarial infection can be duplicated in animal systems should also aid in unravelling the mechanisms de- termining the course of infection and in ensuring that vaccine candidates do not produce an inappropriate immunopatho- logical response. A proposed scheme for using animal models in screening candi- dates for a vaccine against Onchocerca volvulus is presented.

Human lymphatic filariasis and oncho- cerciasis cause chronic, debilitating dis- ease in over 100 million people world- wide. These diseases are characterized by a spectrum of clinical manifestations ranging from an asymptomatic carrier state to severe dermal and ocular complications in the case of onchocer- ciasis or elephantiasis and tropical pul- monary eosinophilia in the case of lymphatic filariasis 1'2. The nematode agents are the lymphatic-dwelling Wuchereria bancrofti and Brugia malayi in the case of filariasis, and Onchocerca volvulus in the case of onchocerciasis. These worms are viviparous, releasing motile embryonic stages called micro- filariae (mr) which migrate through the host skin in the case of O. volvulus, or the blood in the case of the lymphatic filariae. Completion of the filarial life cycle involves an obligatory period of development comprising the first through third (L3) larval stages in a hematophagous arthropod vector.

Efforts to combat other widespread parasitic diseases have benefitted from the availability of animal models, but the complex life cycles and exquisite host specificities of the pathogenic filariae have severely hampered the develop- ment of such models for human oncho- cerciasis and lymphatic filariasis. In a recent review, the ideal animal model of a human parasitosis was aptly described as one faithfully recapitulating the dis- ease's parasitic, immunologic and patho- logic attributes, making available large quantities of parasite material and lend- ing itself to detailed immunological analysis 3. To these criteria we would add that a model for filariasis must also provide for a means of accurately evaluating burdens of all life stages either by postmortem examination, immunodiagnosis or some noninvasive imaging technique.

None of the existing animal models for the human filariases fulfills all of these criteria. However, animal hosts that support parasite development and/ or mimic some aspect of the relevant human infections and diseases have been identified and used to investigate the relationships between infection status, immunity and disease. Our dis- cussion of these models will proceed from animals supporting development of the worms through to reproductive maturity with the release of mf, ie. patency, to those that allow only limited development or survival but which nonetheless provide insight into specific pathogenic or immunologic mechan- isms. Relevant findings on filariae that are natural parasites of rodents or larger domestic animals will also be presented. We will conclude with a discussion of possible vaccine objec- tives and the models relevant to those goals.

Models Involving Patent Infection

The only nonhuman host in which W. bancrofti is known to develop to patency is the silvered leaf monkey, Presbytis cristatus. This animal exhibits histopathological changes resembling those in humans with filarial lym-

phadenopathy although macroscopic lesions have not been observed 4's,

Both the subperiodic form of the human filaria, B. malayi, and the related animal parasite, B. pahangi, develop to patency in mongolian jirds, Meriones unguiculatus. The sequence of immune and pathologic responses to Brugia spp infection in jirds is essentially the reverse of that postulated for humans with lymphatic filariasis 6. In the acute phase of infection, jirds exhibit lymphatic changes, including thrombi and lym- phatic dilatations, more frequently associated with chronically infected humans exhibiting lymphadenopathy and amicrofilaremia 7. In contrast, chronically infected jirds exhibit certain features of interim-stage asymptomatic, microfilaremic infection in humans in- cluding a stabile microfilaremia and a state of immunological tolerance marked by suppression of antigen-specific re- sponsiveness 3'8. Immunization of jirds with irradiated L3 (Ref. 9) or with aqueous extracts of microfilariae ~° con- fers partial protection against L3 chal- lenge. In addition to suppression of adult worm burdens, ]irds immunized with microfilarial extracts exhibit diminished microfilaremia accompanied by an elevated titer of antimicrofilarial anti- body.

Subperiodic B. malayi also develops to patency in ferrets (Mustela putorius furo), and the course of infection in these animals more closely mimics that believed to occur in humans 6'~ i. The majority of ferrets clear circulating mf around the eighth month of infection and subsequently exhibit signs remi- niscent of chronic human infections, namely, heightened immune responsive- ness, interstitial changes in the liver and lungs in response to trapped mf and marked lymphadenopathy in animals receiving multiple inoculations I i.

Domestic cats and dogs are natural hosts for B. pahangi and have been proposed as models of human lym- phatic filariasis. Unlike jirds, cats have the advantage that, during B. pahangi infection, they spontaneously progress from microfilaremia and susceptibility to incoming L3 to a postmicrofilaremic state characterized by mild lymph-

(~) 1992, Elsevier Science Publishers Ltd, {UK)

Parasitology Today, vol. 8, no. 5, 1992 169

adenopathy, killing of adult worms, in situ, and persisting immunity to re- infection ~2, Protective immunity has also been induced in cats under exper- imental conditions ~3, Similarly, dogs exhibit a range of manifestations from putative immunity to asymptomatic microfilaremic infection and, finally, to varying states of lymphad,~nopathy 14. In general, the hypothetical course of in- fection and symptomatology in human lymphatic filariasis 6 is consistent with the progress of B. pahangi infection in cats and dogs.

Filariae in the genus Onchocerca are less amenable to establishment in labora- tory animals than their lymphatic-dwell- ing counterparts. Despite numerous attempts, no member of the genus has been found to develop to patency in laboratory rodents ~5. However, O. volvulus will develop to patency in chimpanzees ~6 and the mangabey monkey, Cercocebus atys tT. However, neither chimpanzees nor mangabeys exhibit ocular or dermal changes re- sembling those occurring in human onchocerciasis patients.

Natural onchocercal infections are common in cattle and horses in tem- perate regions. O. lienalis evokes no gross pathology in cattle but, in addition to its wide availability, it has the advan- tage that susceptible blackfly vectors can be reared in the laboratory, thus providing a source of L3 for laboratory study ~8. This has made possible exper- imental infections of cattle 19. Adult O. gibsoni in cattle form subdermal nodules reminiscent of those formed by O. volvulus in humans but mf of this species cause none of the ocular and dermal lesions attributed to mf of O. volvulus in humans 2°, A controversy exists in the literature over a causal relationship between O. cervicalis mf and observed dermal 21 and ocular 22 pathology in horses. To our knowledge, there is as yet no successful system for large-scale pro- duction of O. gibsoni or C'. cervicalis L3.

Other filarial parasitisms that occur naturally in small and medium-sized animals provide useful models for basic research, which may ultimately find application in human filariasis. One such model involves the canine heartworm, Dirofilaria immitis. A high level of pro- tective immunity to this parasite may be induced by administration of radiation- attenuated L3 (Ref. 23) or by means of a primary infection interrupted by treat- ment with filaricidal drugs 24. The rodent filariid Acanthocheilonema viteae is a natural parasite of certain jirds and has been studied in laboratory colonies of the jird M. unguiculatus. Radiation- attenuated L3 or L3 exoantigens confer

protection against this parasite 25. Lito- mosoides carinii infection in multi- mammate rats (Mastomys ndtalensts) 26 and A. viteae infection in Syrian hamsters 27 mimic some aspects of human lymphatic filariasis in that they progress to a 'postpatent' stage, charac- terized by suppression of microfilaremia. Drug-abbreviated infections with L. carinii in multimammates also result in partial protection against L3 challenge 26,

Models Involving Dead-end Infections

None of the rodents that are avail- able as inbred, immunologically charac- terized strains supports development of the human-dwelling filariae to patency. However, in many cases they will allow extended survival of mf or limited development of early larval stages. Such abortive and 'dead-end' infections have provided clues to mechanisms of pro- tective immunity and pathology in ex- perimental filarial infections. Intravenous injection of Brugia malayi mf into BALB/c mice results in a persistent micro- filaremia, the kinetics of which provide the basis for evaluating potential im- munogens. Repeated immunization by intraperitoneal injection of microfilariae or of a recombinant antigen results in accelerated clearance of challenge mf inoculated intravenously 2a, Mice immunized in this manner also exhibit peripheral blood and pulmonary eosinophilia as well as elevated levels of microfilaria-specific immunoglobulin G (IgG) and IgE. This system is therefore proposed as a useful model for human tropical pulmonary eosinophilia 29,

Intraperitoneal inoculation of Brugia spp L3 into intact mice or implantation of these stages within semipermeable micropore chambers allows survival of the parasites for at least two weeks and development to the fourth larval stage. Using such systems, BALB/c mice have been shown to mount an early pro- tective immune response after sensitiz- ation with live or radiation-attenuated larvae or with soluble fractions of adults and microfilariae 3°-32,

Although laboratory mice are largely resistant to Brugia infection, some genetically defined immunodeficient strains allow development to patency. Innate resistance in mice is known to be immunologically based insofar as con- genitally athymic (nu/nu) mice or surgi- cally thymectomized mice support development of a patent infection while syngeneic, normal controls do not 33'34. Reconstitution of athymic mice by in- oculation of thymocytes or by grafting

thymus tissue confers heightened resist- ance 3s. Similarly, scid mice, which lack both functional T and B cells, allow development of B. malayi to repro- ductive maturity and develop significant tymphadenopathy. Results obtained with this strain argue against an immune-mediated mechanism for the development of gross pathologic changes in lymphatics harboring adult worms. Scid mice have the added potential to allow reconstitution with components of the human as well as murine immune system. Early results indicate that reconstitution with murine spleen cells does not promote rejection of an established B. malayi infection 36.

There has also been a trend in onchocerciasis research towards studies of short-term infections with isolated life cycle stages in genetically defined rodents. Analogous to the murine model of Brugia sp. microfilaremia, inbred mice will also establish a stabile microfiladermia when injected sub- cutaneously with O. lienalis or O. volvulus mf 37'38. Immunity to dermal mf infection has been demonstrated in this system 39. Recently, it has been determined that sensitization with O. lienalis mf confers crossprotection against subcutaneous challenge with O. volvulus mf 38.

Third and fourth larval stages of O. lienalis and O. volvulus undergo ex- tended survival and limited develop- ment within subcutaneous micropore chamber implants in inbred rodent hosts 4e~42, Protective immunity in terms of reduced survival of O. lienalis can be induced by subcutaneous injection of radiation-attenuated L3 (Ref. 42).

Similar studies on canine heartworm showed that the L3 of D. immitis, confined within micropore chambers, will undergo limited development in selected strains of inbred mice. Mice can be immunized against such an infection either actively, by inoculation of L3, or passively, by transfer of serum from immune dogs 43.

The absence of ocular changes in response to onchocercat infection in nonhuman primates or large domestic animals has prompted attempts to re- produce ocular onchocerciasis in labora- tory animals by direct inoculation of microfilariae into the eye and surround- ing tissues. In a guinea pig model, subconjunctivally injected O. lienalis mf elicit punctate keratitis along with local and systemic antibody and local cellular responses 44. Cynomolgus monkeys ex- hibit symptoms of onchocercal retin- apathy in response to intravitreal injec- tion of O. volvulus mf. These changes precede a local humoral and cellular im- mune response and are more pronounced

170 Parasitology Today, vol. 8, no. 5. 1992

Box I. Screening for Candidate Vaccines

The figure illustrates a scheme for the use of animal models in screening candidates for a vaccine against Onchocerca volvulus infec- tion, as proposed by the Edna McConnell Clark Foundation's Program for Tropical Disease Research. In stage I, antigen screening will be performed simultaneously in two rodent models, A. viteae in the jird and O. volvulus L3 inoculated within micropore chambers implanted into mice. This approach will allow relatively rapid evaluation of a large number of candidates for their ability to block patent A. viteae infection while ensuring activity against the putative target stage of the human parasite. In stage 2, testing of candidate vaccines in patency models will proceed to the bovine oncho- cerciases. Further evaluation of candidate vaccines' stage and species specificity and of host responses will be carried out in rhesus and mangabey monkeys using O. volvulus L3 in micropore chambers. Stage 3 will involve screening against O. volvulus in chimpanzees. Efficacy will be de- termined by presence of dermal microfilariae, by a circulating anti- gen assay yet to be developed or by some noninvasive imaging technique. Identification of circu- lating antigens that reliably predict patent infection would provide the basis for evaluating a vaccine candidate far in advance of patency some one to two years after infection. (Adapted, with permission, from Ref. 47.)

( . viteae JlaO)

/ ~0. lienalis

(CATTLE)

STAGE 1

O. volvulus 1 (MOUSE / CHAMBER)

\ STAGE 2

O. volvulus

(MONKEY / CHAMBER)

O. gibsoni

(CATTLE)

STAGE 3

(CHIMPANZEE)

STAGE 4

I ( . volvulus

HUMANS) 1

implantation of Onchocerca sp. L3 in micropore chambers. Use of sub- cutaneous micropore chambers has the added advantage of allowing presen- tation of challenge larvae in the natural location while retaining the benefit of ease of recovery. This method also allows the investigator to evaluate the relative contributions of cellular and humoral effectors in the parasite's microenvironment.

(2) Immunization against disease in onchocerciasis would target mf of O. volvulus. Models involving inoculation of O. volvulus or O. lienalis mf into inbred mice used in conjunction with the small animal models for ocular pathology would be useful in ascertaining pro- tection and guarding against an inappro- priate immunopathological response. The objective of a disease-preventing vaccine in lymphatic filariasis would be prevention of development to the adult stage. Here, L3-restricted models could be used in conjunction with models allowing development of Brugia sp. to patency such as the ferret, jird, cat or dog.

(3) Finally, vaccines that prevent the elaboration of mf, either by direct action on that stage or by interrupting development earlier in the life cycle, would have the added benefit of sup- pressing transmission. Murine models. involving intravenous infusion of Brugia sp. mf or dermal inoculation of Oncho- cerca sp. mf would be useful in ident- ifying antigens that evoke an antimicro- fllarial response. The critical problem again will be direction of the immune response toward elimination of the parasite without exacerbating pathology associated with mf.

in response to O. volvulus than to O. lienalis 45. A murine model has also been used to study routes of migration of Onchocerca mf into the eye from the surrounding subcutis and vasculature 46. Subcutaneous mf of O. cervicalis invaded the cornea via the eyebrow skin and subsequently the scleral surface. Retinal migration of O. cervicalis mf in mice only occurred after intravenous infusion of the parasites.

Strategies for Vaccine Development

The primary objective of vaccination against filarial infection is prevention of infection and thus disease. The goal is, therefore, to interrupt the life cycle at a point prior to the emergence of patho- genic stages. As is apparent from this

and other discussions 3,6, the L3 is both the stimulus for and target of the protective responses demonstrated in many of the animal systems investi- gated. One problem inherent in target- ing the L3 is that it is a short-lived stage. It is therefore not surprising that there is a consistent lack of sterile immunity in the aforementioned studies of im- munoprophylaxis. It is for this reason that vaccination against life cycle stages responsible for disease and/or trans- mission is also worthy of attention. The models described above can be applied to these goals in the following manner:

(I) Screening for antigens that protect against incoming L3 could be accomplished in models that only per- mit development and survival of the early larval stages, such as the murine models involving intraperitoneal infec- tions with Brugia sp. or subcutaneous

Conclusions

The lack of all-encompassing small animal models for the major human filariases calls for screening programs for protective antigens involving a multi- plicity of models used in tandem and in sequence. A tentative protocol for screening candidate vaccines against O. volvulus has recently been formulated along these lines (Box I and Ref. 47). The primate models alluded to above will be useful for terminal evaluation of vaccine candidates prior to testing in humans. However, the use of such scarce animals for which there are ethical constraints against euthanasia will not be practical for the initial phases of vaccine candidate screening. These con- cerns, along with the exceedingly long developmental period of O. volvulus, will dictate that alternatives to necropsy as a

Parasitology Today, voL 8, no. 5, 1992 171

method of evaluating parasite burdens be developed. Similar concerns will necessitate that appropr ia te infections in small animals that are more suitable for rapid, h igh-volume screening be adopted in the earl ier phases of vaccine deve lopment programs,

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37, 359-360 47 The Edna McConnell Clark Foundation ( 1991 )

Strategic Plan for Onchocerciasis p. I I

James Lok ~s at the Department of Patho- biology, School of Veterinary Medicine, Univer- sity of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA and David Abraham is at the Department of Microbiology and Immunology, School of Medicine, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.

Genetics of Biomphalaria glabrata and its Effect on the Outcome of Schistosoma

mansoni Infection C.S. Richards, M. Knight and F.A. Lewis

The genetics o f the snail Biomphalaria glabrata is better characterized than that o f any other intermediate host o f schisto- somes o f humans. Using techniques of selective breeding, several snail stocks have been developed that consistently display resistant or susceptible pheno- types. Investigators using these stocks have learned that several snail and para- site genes influence the course o f parasite development. Here, Charles Richards, Mat ty Knight and Fred Lewis discuss the importance of the snail's genetics in categorizing resistance in this complex invertebrate, some recent molecular evi- dence that may help us understand several o f the problems that still remain, and some challenges lying ahead for investigators in this field.

For many years, biologists have been intr igued by wha t happens to a snail's

internal defense system when tr iggered by an encounter wi th invading t rema- tode larvae, Recently, several reviews on t rematode-snai l compatibi l i ty have appeared that describe, at some length, possible mechanisms that affect the outcome of a t rematode invasion of the snail host ~ . Rather than discussing mechanisms, this rev iew will focus on the genetics o f a much-studied snail, B. glabrata, in relation to infection by the medically impor tant schistosome, Schistosoma rnansoni,

Being a hermaphrod i te species wi th the ability to self- and cross-ferti l ize, B. glabrata is ideally suited as a subject for investigating the genetic determinants for compatibi l i ty between parasite and snail. Major advances in understanding the resistance or susceptibil i ty of B. glabrata to S. mansoni infections have been possible because snail lines wi th

adequately def ined phenotypes can be generated.

Genetics of Susceptibility

That the genetic make-up of the snail influences the outcome of infection was suspected f rom the studies of Files and Cram, w h o showed variat ion in sus- ceptibi l i ty of B. glabrata s. Since then, investigators have shown unequivocal ly that the genetics o f the snail plays a major role in determining the outcome of parasite invasion and results in a variety o f consequences. Among these are: ( I ) parasite deve lopment w i thou t host reaction in susceptible snails; (2) failure of the parasite to develop de- spite a lack of host reaction in unsuitable snails; (3) recognit ion, encapsulation and destruct ion of the parasite in resistant

~) 1992. Elsevier Science Publishers Ltd. (UK)