Immunity to Onchocerca spp. in animal hosts

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Immunity to Onchocerca spp. in animal hosts

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David Abraham, Richard Lucius and Alexander J. Trees This review summarizes research using Onchocerca spp. in chimpanzees, cattle and mice to gain insight into the protective immune response to the filarial worm Onchocerca volvulus in humans. In addition, Acanthocheilonema viteae has been evaluated as a surrogate filarial worm for studying immunity to the infection. Immune mechanisms controlling these infections are described and initial success using recombinant antigen vaccines in these models is reviewed.

David Abraham* Dept of Microbiology and Immunology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA. *e-mail: david.abraham@ mail.tju.edu Richard Lucius Lehrstuhl für Molekulare Parasitologie, Institut für Biologie, HumboldtUniversität zu Berlin, Philippstr. 13, 10115 Berlin, Germany. Alexander J. Trees Dept of Veterinary Parasitology, Liverpool School of Tropical Medicine and Faculty of Veterinary Science, University of Liverpool, Pembroke Place, Liverpool, UK L3 5QA.

Onchocerciasis remains a significant health problem in countries within Africa and South America. Whereas effective vector control measures are in place in many areas and chemotherapy is in widespread use, a vaccine that reduced parasite burden or the development of pathology would be a valuable tool to complement existing disease control programs. Vaccine development against Onchocerca volvulus has focused on two larval stages, the infective third-stage larvae (L3) and the microfilariae (Mf). Interest in immunity to L3 is based on the premise that preventing this stage from surviving would block the infection; hence, prevent the development of disease. Immunity to Mf is of interest because elimination of this stage would remove the major source of pathology in humans and would prevent transmission of infection. Human exposure to infection with Onchocerca volvulus can result in a range of disease states from generalized to hyperreactive disease to putatively immune individuals (PI). PI are of particular interest in that they represent individuals living in an endemic area who have developed resistance to infection. The immune response appears to be responsible for these different disease states and roles have been described for T helper (Th) 1, Th2 cells, antibodies and granulocytes in the different states. In addition, Wolbachia, the endosymbiont bacteria found in O. volvulus, clearly has an effect on the immune response [1]. Therefore, vaccine development against this infection must take into account the complex nature of the immune response of humans to the parasite and find a means to dissect discrete components of the host–parasite interaction for analysis. There are several significant experimental limitations that have hindered vaccine development against O. volvulus. A major obstacle stems from the fact that, in Nature, the parasite infects only humans and has rarely been observed in other primates [2]. Experimental infections have been attempted in several animals, with only chimpanzees and mangabey monkeys shown to be susceptible to http://parasites.trends.com

infection [3–7]. Another obstacle has been the difficulty in procuring parasites for laboratory studies. The limited host range for the parasite and the necessity for blackflies to serve as the source of L3 have made this parasite very difficult to study in the laboratory. This obstacle was partially overcome by the demonstration that both L3 [8] and Mf [9–11] can be cryopreserved in a manner that allowed their continued development after defrosting. The development of a clinically useful vaccine requires antigens that can be efficiently and reproducibly produced. Complementary DNA (cDNA) libraries have been prepared for the adult and larval forms of this parasite and these libraries have served as the source of recombinant antigens for use in vaccine trials [12]. Because of the host restriction of O. volvulus, research has followed three approaches: (1) experimental infection of chimpanzees with O. volvulus; (2) use of bovine Onchocerca spp. in their natural host and (3) investigation of Onchocerca spp. in surrogate mouse hosts paralleled by studies with the rodent filarial parasite Acanthocheilonema viteae. Given the ethical constraints on chimpanzee use, much work has used options (2) and (3). Each has advantages and disadvantages. Using surrogate murine hosts allows several, well replicated experiments and detailed dissection of the immune responses, which have been very informative. Importantly, it also permits work using the target parasite, O. volvulus. However, the immune responses might not completely reflect those of significance in a naturally evolved host–parasite relationship. With the exception of O. volvulus, Onchocerca spp. are parasites of ungulates, thus the onchocercids of cattle provide appropriate, natural infections analogous to O. volvulus. However, experimental infections in cattle are logistically challenging, and the paucity of bovine immunological reagents has been somewhat limiting (Table 1). The goal of this review is to summarize what is known about immunity to Onchocerca spp. in natural and surrogate hosts, and how this information might be applicable to vaccine development for use in humans. O. volvulus in chimpanzees

Studies have been performed in chimpanzees to determine the immunological responses that develop following primary and challenge infections. Antibody responses were measured after primary infections

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Table 1. Animal models used for studying protective immunity to Onchocerca volvulusa Host

Parasite

Natural Stages in infectionb host

Stages monitored Time for results Availability of Refs for infection immunoreagentsc

Chimpanzee Cow Cow Jird

Onchocerca volvulus Onchocerca lienalis Onchocerca ochengi Acanthocheilonema viteae

± + + +

All All All All

Mf Mf and adults Mf and adults All stages

Mouse Mouse

O. lienalis O. volvulus

– –

L3, L4, Mf L3, L4, Mf

L3, L4, Mf L3, L4, Mf

9–31 mths 4–20 mths 4–20 mths 7 days for L3; 84 days for adults 7–21 days 7–21 days

+ ± ± ±

[13–19] [21,22] [23–30,32] [35–37]

+ +

[41–45,47–54] [46,54–62, 66–68]

aAbbreviations:

L3, third-stage larvae; L4, fourth-stage larvae; Mf, microfilariae. of natural infections is indicated: +, common; ±, rare; –, never. cAvailability of immunoreagents for each host is indicated: +, many immunoreagents; ±, few immunoreagents. bFrequency

with L3 against antigens solubilized from adult worms and against recombinant O. volvulus antigens [13–17]. When the infections developed, the number of individual antigens recognized increased as determined by western blot analyses. In addition, antibody responses in animals with Mf in the skin could be distinguished from responses in non-patent animals [17]. Lymphocyte proliferation assays were performed with cells from infected chimpanzees and it was observed that, after a strong early response to parasite antigens, a reversible cellular hyporesponsiveness developed [16,18]. A single study was performed to determine if chimpanzees could be immunized against infection with O. volvulus. Four chimpanzees were immunized with irradiated L3 (XL3) followed by infection with L3. After 11–26 months, three out of the four immunized chimpanzees developed Mf in the skin, whereas all four of the control animals became patent. There were no significant differences in the number of Mf found in the skin of the immunized chimpanzees when compared with the infected controls [19]. This experiment is difficult to interpret because adult worm burdens cannot be determined in live chimpanzees; hence, protective effects could be obscured by Mf production from very few adult worms. The reason why one vaccinated animal did

Fig. 1. (a) Microinjection of Simulium with microfilaria. In this way, the third-stage larvae of Onchocerca ochengi were generated for experimental infection. (b) Adult, female O. ochengi worms were dissected from their nodules. One female worm is found per nodule. Each worm mass measures ~0.5cm in diameter.

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not develop Mf in the skin could have been a result of a failure to infect (not all chimpanzees develop patent infections after primary challenge [14,18]), or could have indicated some immune protection because this animal developed an antibody response to 11–12 kDa L3 antigens of A. viteae [16]. Bovine Onchocerca spp. infections Onchocerca lienalis in cattle

Onchocerca lienalis is common in cattle in temperate latitudes, and thousands of Mf can be extracted from bovine skin collected from abattoirs. Moreover, L3 have been produced by intrathoracic injection of Simulium flies [20], which has permitted studies of the dynamics of the antibody response after experimental infection. The response was characterized by three steps coincident with L3 to fourth-stage larvae (L4), L4 to fifth-stage larvae (L5) molts and with patency [21]. The antibodies cross-reacted with antigens derived from O. volvulus adult worms, with the same sequential pattern against homologous antigens [22]. Protective immunity to O. ochengi

Recently, O. ochengi infection of cattle has emerged as an excellent analog of human onchocercal infection [23,24] (Fig. 1). Not only is O. ochengi closely related to O. volvulus, but the superficial location of the adult worm inhabiting the nodules has enabled studies of adult parasite intensity in the context of immunity and exposure to infection. There is extensive antigen cross-reactivity between O. ochengi and O. volvulus native and recombinant antigens [25,26], and there is epidemiological evidence that this cross-reactivity extends to cross-protection of humans against O. volvulus, by exposure to infected larvae of O. ochengi [27]. Exploiting the cross-reactivity of antigens, the kinetics of recognition of recombinant O. volvulus antigen during experimental cattle infection with O. ochengi has been studied [28]. This did not always reflect the developmentally regulated expression of the relevant gene, possibly reflecting cross-reactive epitopes on different parasite proteins. The kinetics of

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response have also been compared between O. volvulus-infected chimpanzees and O. ochengiinfected cattle [26]. Whereas there were similarities in the kinetics of the responses, there were differences in the relative immunodominance of certain recombinant antigens. Cross-sectional field studies have provided evidence consistent with immunity both to Mf and to pre-adult stages. The parasite burden in age-cohorts was compared and when adult burdens increased with age, Mf loads decreased in older cattle despite the continuing fecundity of excised, adult, female worms [23]. Whereas no immunological responses were measured in those field studies, they are consistent with the experimental induction of immunity against O. lienalis Mf implanted into cattle [29]. Moreover, proportions of cattle that have been maintained in an endemic area are not infected, based on nodule palpation and skin biopsy. These endemic normals have been observed in several studies [23,25,30]. Their lack of parasitosis is remarkable given the huge annual transmission potentials of O. ochengi that could exist (e.g. see Ref. [27]) and the fact that cattle, unlike humans, are permanently exposed, outdoors and without clothes! Such animals are analogous to human PI, although, as yet, their immune responses have not been well characterized. However, unlike humans, it has been possible to challenge bovine PI. Naïve age-matched cattle and PI were exposed to natural challenge and the PI were significantly less susceptible to infection than the naïve controls (V.L. Tchakouté, unpublished). The differences in acquiring infection were not related to differences in attracting vectors. Interestingly, in the same experiment, previously infected cattle, in which infections had been eradicated by melarsomine treatment, were susceptible to challenge. These results are consistent with field studies in humans conducted by Duke, who found that humans re-examined several years after adulticidal therapy had acquired infections which were of equal or higher intensity than those observed in co-endemic children of the age equivalent to the post-treatment period [31]. In both cases, the results suggested that, far from pre-infected individuals being immunized, they were of heightened susceptibility because their burdens were higher than expected (V.L. Tchakouté, unpublished). Although infection that develops to patency clearly does not confer protection to challenge, it has been possible to induce protective immunity experimentally using XL3. In a controlled experiment, vaccinated cattle were significantly protected against challenge and their total worm burden was greatly reduced (S.P. Graham, unpublished). This experiment demonstrates, for the first time, the feasibility of vaccinating a natural host against Onchocerca infection. Using experimental infection, the kinetics of the host immune response has been studied in this http://parasites.trends.com

host–parasite system [32]. Following single-dose infection, antigen-specific in vitro proliferation of peripheral blood mononuclear cells (PBMC) and cytokine production of interferon (IFN) γ, interleukin (IL) 2 and IL-4 increased rapidly and remained high throughout the pre-patent period, but declined at patency (Fig. 2). Antigen-specific immunoglobulin (Ig) G responses showed a stepped rise associated with infection and then patency, but IgG2 isotype levels slowly fell after patency whereas the predominant isotype, IgG1, remained elevated (Fig. 2). These results demonstrated a downregulation of T-cell responses in association with patency, which parallels cross-sectional observations on humans and longitudinal infection studies in chimpanzees with O. volvulus [16,18,33,34]. These studies demonstrate the potential of this host–parasite system to investigate mechanisms of parasite modulation of the host response, the nature of protective mechanisms and the induction of protective immunity. Taken collectively, they allow the construction of a descriptive model of immunity against bovine onchocerciasis. A subset of the heterogeneous host population is able to effectively control pre-adult infections and remain aparasitotic, even in the face of huge challenge (the PI). However, protection against infection can be induced in a much larger proportion of the population by exposure to attenuated L3. Once infection is established, a hyporesponsive state ensues. This permits the survival and reproduction of established adults, but a slow accumulation of new infection (adult burdens increase with age). This rate of increase of cumulative worm burden is, however, much below the rate of exposure to infective larvae, suggesting a degree of concomitant protection. This is ablated by adulticidal therapy, but the downregulation is hypothesized to continue for a period, rendering drug-cured, previously infected animals highly susceptible to re-infection. Such a hypothesis is based on the parasitological observations to date and is largely conjectural in terms of immunology. The challenge now is to investigate the immunology of this infection in relation to these parasitological states. Immunization of jirds against A. viteae

Studies on the protective effect of immunization with XL3 and immunization studies with recombinant O. volvulus proteins were performed in Meriones unguiculatus (jird) infected with the filarial nematode A. viteae [35]. This natural host–parasite association allows the study of resistance to challenge infections, but the lack of specific immune reagents limits the analysis of immune mechanisms. Therefore, the analysis of parasitological parameters was focused in this model. Immunization of jirds with A. viteae XL3 induced immunity that killed 90% of the challenge infection [36]. The target of the immune response was the L3. Histological and electron micrograph studies revealed

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(d) Mean OD (405 nm)

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2000 1500 1000 500 0 100

200 300 400 Days post-infection

(e) Mean OD (405 nm)

Stimulation index

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0.5 0.25

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1.25 1 0.75 0.5 0.25 0

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(f)

0.3 Mean OD (405 nm)

10 IL-2 (U ml–1)

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0.1

0 0

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Fig. 2. The kinetics of parasite-specific cytokine production and antibody responses after experimental infection of cattle (n = 6) with Onchocerca ochengi. Mean levels of (a) interferon (IFN) γ, (b) interleukin (IL) 4 and (c) IL-2 produced from peripheral blood mononuclear cells (PBMC) stimulated by parasite antigens. The yellow shaded area in (a–c) indicates the mean levels of cytokines +2 standard error (SE) (n = 18) for an uninfected animal. Mean levels of antigen-specific (d) immunoglobulin (Ig) G, (e) IgG1 and (f) IgG2 measured by ELISA. The yellow shaded area in (d–f) represents mean optical density (OD) reading +2 SE (n = 24) for an uninfected animal. Error bars represent SE. Microfilariae were detected in the skin from 280 days post-infection (Reproduced, with permission, from Ref. [32].)

that in the subcutaneous connective tissues of jirds, four days after challenge, dead L3 were observed with eosinophils flattened against the surface of the larvae, and degranulated electron-dense material could be detected on the worm surface. These data suggest that the newly invading L3 are the main target of protective immune response in vaccinated jirds and that eosinophils are integral to this process [37]. In addition, it was shown that L3 excretory or secretory products contained proteins or proteinassociated carbohydrates that were capable of inducing protection (R. Lucius, unpublished). http://parasites.trends.com

Immunization of rodents with one species of filariae has been shown to induce cross-protection against other filarial worms [38,39]. Therefore, immunization of jirds with recombinant O. volvulus proteins and subsequent challenge infection with A. viteae L3 was considered as a possible approach for screening recombinant O. volvulus proteins for protection in a natural host–parasite relationship. Immunization experiments were performed with 31 recombinant O. volvulus proteins. Although immunization together with a synthetic adjuvant [40] or with alum generally induced specific antibodies, the animals were not protected against challenge infection with A. viteae L3 (R. Lucius, unpublished). This lack of cross protection could be a result of: (1) lack of protective epitopes common to both filarial species; (2) absence of inducing the appropriate immune effector mechanisms in jirds or (3) lack of protective properties by the chosen antigens. However, studies of the A. viteae infection of jirds do reveal that immunization with XL3 induces resistance against homologous challenge infection. The effector mechanisms are directed against early L3 stages and

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the animal, and the humoral and cellular immune components in the parasite microenvironment can be identified. In calves, although the parasites could survive for extended periods of time inside diffusion chambers, their development was impaired when compared with larvae found free in the tissues [54]. It was demonstrated that mice immunized with O. lienalis XL3 developed protective immunity to challenge infections contained within diffusion chambers [52]. Protective immunity to O. volvulus L3

Fig. 3. Granulocytes attaching and degranulating on the surface of larval Onchocerca volvulus recovered from diffusion chamber, implanted in mouse immunized with irradiated O. volvulus third-stage larvae. Scale bar = 20 µm.

could be induced by proteins or protein-associated carbohydrates. Onchocerca spp. in mice Immunity against Mf

Mice have been used as surrogate hosts to investigate immunity against Mf of Onchocerca spp. Mice will support the survival of inoculated Mf for 3–4 months. Mf injected subcutaneously at the nape of the neck, concentrate in the ears, therefore recovery of Mf from the ears post-mortem can be used as a measure of parasite survival [41,42]. Using this model with O. lienalis, significant protective immunity was conferred by live, homologous primary infection, immunization with Mf extracts and with live infections that were abbreviated by drug treatment [42,43]. Some protection was also conferred by heterologous stages and species but at a lower level [44,45]. Significantly, protection against O. volvulus Mf was also demonstrated in surrogate mice immunized with live homologous or heterologous (O. lienalis) Mf [46]. The mechanisms of protection have been extensively studied using O. lienalis. Eosinophils are crucial and IL-5 is dominant in the expression of microfilarial immunity [47,48]. Using gene knockout mice, clearance of Mf following primary and secondary infections was independent of IL-4 [49,50]. Exogenous IL-12 was used to ablate parasite-induced IL-4 responses without eliminating the protective IL-5 dependent responses [48]. This could have significance for any vaccination strategy directed against Mf in view of the role of IL-4 in pathogenesis. Protective immunity to O. lienalis L3

Attempts to infect laboratory animals with O. lienalis were unsuccessful [51]. However, mice, rats, jirds and multimammate rats could serve as surrogate hosts for L3 contained in diffusion chambers, implanted subcutaneously [20,52,53]. The advantages of using diffusion chambers include that they allow complete recovery of larvae introduced into http://parasites.trends.com

Onchocerca volvulus L3 were implanted in diffusion chambers in chimpanzees, mangabey monkeys, rhesus monkeys, squirrel monkeys, six strains of inbred mice, inbred jirds, four strains of inbred rats and multimammate rats for 3–63 days. Survival and growth rates did not differ among the primate and rodent hosts tested, with the exception of squirrel monkeys and rats that were resistant to the infection. Molting from L3 to L4 began on Day three and continued through Day 14 in primate and rodent species. It was concluded that the L3 would develop equally well, for a short period of time, in the susceptible chimpanzee host as they did in resistant hosts (such as mice) when implanted within diffusion chambers. It was clear from the parasite survival rates in all hosts that there was decrease in parasite survival over time, suggesting that either there is natural death of some of the larvae or that innate immune responses were capable of killing a proportion of the population [55–57]. Mice were immunized by subcutaneous injection of normal, irradiated or freeze-thaw-killed Onchocerca spp. larvae, and then received challenge infections of L3 contained in diffusion chambers to study protective immunity in this host. There was a significant reduction of ~40–80% in the survival of challenge parasites in mice immunized with normal, irradiated or freeze-thaw-killed O. volvulus L3 or O. lienalis XL3 [58]. Studies were performed to describe the mechanism of immune-mediated killing of O. volvulus L3 in diffusion chambers in mice. L3 were implanted in mice in diffusion chambers that either permitted or restricted cellular influx into the diffusion chamber. In these experiments, it was determined that direct contact between host cells and the parasites was required for killing of larvae in immunized hosts. The only cell type that was found to increase in diffusion chambers in immunized mice was eosinophils; maximal levels of eosinophils were coincident with the time of parasite killing [59] (Fig. 3). The observation that eosinophils increased in immunized animals suggested that immunity would be dependent on a Th2 response because eosinophils require IL-5 for many of their functions and IL-5 is one of the cytokines associated with Th2-dependent immunity. This hypothesis was confirmed in spleen cell cultures from immunized mice in which, after stimulation with O. volvulus antigens, IL-4, IL-5 and

Review

Acknowledgements We are grateful to the Edna McConnell Clark Foundation (EMCF) for support. We thank Milan Trpis and Sara Lustigman for their efforts to produce the L3 used in these studies. We also acknowledge the support of Peter Enyong (Tropical Medicine Research Station, Kumba, Cameroon). A.J.T. thanks Simon Graham, Ginny Tchakouté, Ted Bianco, Sara Lustigman, Vincent Tanya, Mark Taylor, Goetz Wahl, Alfons Renz and Sabine Klager for their collaboration in the O. ochengi work and permission to quote unpublished results; many other collaborators, notably Steve Williams, Sandra Laney, Tom Unnasch, Kim HenckleDuehrsen and Gabby Braun for the generation of recombinant antigens; and a special thanks to the late Bob Collins. Finally, our thanks to the members of the Onchocerciasis Task Force from the EMCF and, in particular, to Joe Cook for his guidance and support throughout these studies.

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IFN-γ were produced [60]. IL-5 was also found in diffusion chambers of immunized mice coincident with the time of parasite killing and not in diffusion chambers recovered from control mice. Significant levels of IFN-γ, however, were absent in the diffusion chambers of both groups. Elimination of either IL-5 or IL-4 by monoclonal antibody (mAb) treatment significantly reduced the protective effects of vaccination against larval O. volvulus [59]. The dependency on Th2 cytokines was further corroborated in cytokine-deficient mice. Immunized IL-4-deficient mice did not develop protective immunity, whereas animals deficient in IFN-γ developed immunity comparable to immunized wild-type mice [61]. Apart from the presence of cellular contact, it was determined that immunity to larval O. volvulus in mice was dependent on antibody. This conclusion was based on the inability of mice deficient in B cells to mount protective immune responses to the infection (D. Abraham, unpublished). The finding that immunity was dependent on IL-4 and IL-5 suggested a dependency on IgG1 or IgE. Measurements of total serum antibody levels, and identification of specific antibody responses to surface antigens, internal antigens and soluble antigens in western blots revealed responses by IgM, IgE and all IgG subclasses. Although it was clear from the mice deficient in B cells that antibody was essential, the complex pattern of recognition of parasite antigens by antibodies found in immunized mice made it difficult to discern the protective antibody isotypes and their antigenic targets [62]. Although irradiated larvae served as successful immunogens for inducing protective immunity in mice, it was clear that these larvae could never be used in a vaccine for use in humans because of safety and practicality issues. A more reasonable approach for immunization would be to use recombinant antigens as the basis for the vaccine. To test the potential of this approach, 44 recombinant antigens produced by 12 independent laboratories were tested. Fourteen of these antigens were able to induce partial but significant protection in the presence of Block Copolymer, alum or Freund’s complete adjuvant (FCA) [12]. Three recombinant antigens, Ov7, Ov64 and OvB8, were selected for further study based on their expression in the early larval stages of the parasite, and following their identification by screening adult and larval cDNA libraries with antibodies from immune humans or chimpanzees. When mice were immunized with the three individual recombinant antigens, statistically significant

References 1 Hoerauf, A. et al. (2002) Resistance and susceptibility in humans onchocerciasis beyond Th1vs Th2. Trends Parasitol. 18, 25–31 2 van den Berghe, L. et al. (1964) The filarial parasites of the eastern gorilla in the Congo. J. Helminthol. 38, 349–368 http://parasites.trends.com

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reductions in parasite survival were induced in mice when administered in alum, but not when injected in FCA. The finding that immunity was only induced after vaccination with alum suggested that the immune response induced with the recombinant antigens had to be a Th2 response induced by alum and not a Th1 response induced by FCA [63–65]. Other protective antigens, including fructose-1,6biphosphate aldolase, have been identified, which function in the presence of FCA and not alum, suggesting that these antigens require a Th1 response to function [66]. The level of protective immunity induced with the recombinant antigens was comparable to that seen in mice immunized with XL3 [67]. An alternative approach for immunization with recombinant antigens is DNA immunization. Mice, immunized with O. volvulus chitinase DNA, developed protective immunity again at levels comparable to those achieved with irradiated larvae immunization [68]. These two studies therefore demonstrate that recombinant antigens are effective at inducing protective immunity in mice and thereby suggest the potential of this mode of vaccination against infection in other hosts. Conclusions

Field studies of natural bovine infections showing the existence of PI and Mf burdens that were lower than predicted in older cattle suggested that naturally occurring immunity occurs against pre-adult and Mf stages, respectively. In parallel, it has been possible to immunize surrogate animal hosts against L3 and Mf experimentally. Crucially, protection against L3 challenge has been induced by XL3 in a fully permissive host and in surrogate hosts. Whereas these studies used immunization protocols unsuitable for human use, they have created a favorable scientific background to pursue subunit vaccination strategies. Concentrating on anti-L3 immunity, the mouse model has proved invaluable for identifying and selecting recombinant vaccine candidates generated by several collaborating laboratories. Antigens have been identified that apparently require a Th1 response whereas others require a Th2 response. This duality of protective responses is different from that observed in mice immunized with XL3, but reflects the situation seen in human PI. Finally, a selected subset of vaccine candidates, which were identified in mice, is currently being field tested in cattle against natural challenge. Together, the animal studies reviewed here and the studies of natural human infections [1] provide a knowledge base for the development of a vaccine for onchocerciasis.

3 Duke, B.O.L. (1962) Experimental transmission of Onchocerca volvulus from man to chimpanzee. Trans. R. Soc. Trop. Med. Hyg. 56, 271 4 Suswillo, R.R. et al. (1977) Attempts to infect jirds (Meriones unguiculatus) with Wuchereria bancrofti, Onchocerca volvulus, Loa loa and Mansonella ozzardi. J. Helminthol. 51, 132–134

5 Kozek, W.J. et al. (1982) Attempts to establish Onchocerca volvulus infection in primates and small laboratory animals. Acta Trop. 39, 317–324 6 Taylor, H.R. et al. (1988) Ivermectin prophylaxis against experimental Onchocerca volvulus infection in chimpanzees. Am. J. Trop. Med. Hyg. 39, 86–90

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7 Duke, B.O. (1980) Observations on Onchocerca volvulus in experimentally infected chimpanzees. Tropenmed. Parasitol. 31, 41–54 8 Trpis, M. et al. (1993) Cryopreservation of infective larvae of Onchocerca volvulus (Filarioidea: Onchocercidae). J. Parasitol. 79, 695–700 9 Tagboto, S.K. et al. (1991) Onchocerca lienalis: comparison of techniques for the cryopreservation of microfilariae within skin-snips or free of host tissues. J. Helminthol. 65, 301–309 10 Ham, P.J. et al. (1982) Separation of viable and non-viable Onchocerca microfilariae using an ion exchanger. Trans. R. Soc. Trop. Med. Hyg. 76, 758–767 11 Ham, P.J. et al. (1981) An improved technique for the cryopreservation of Onchocerca microfilariae. Parasitology 83, 139–146 12 Lustigman, S. et al. (2002) Towards a recombinant antigen vaccine against Onchocerca volvulus. Trends Parasitol. 18, 95–143 13 Weiss, N. et al. (1986) Detection of serum antibodies and circulating antigens in a chimpanzee experimentally infected with Onchocerca volvulus. Trans. R. Soc. Trop. Med. Hyg. 80, 587–591 14 Eberhard, M.L. et al. (1991) Experimental Onchocerca volvulus infections in mangabey monkeys (Cercocebus atys) compared to infections in humans and chimpanzees (Pan troglodytes). Am. J. Trop. Med. Hyg. 44, 151–160 15 Eberhard, M.L. et al. (1995) Onchocerca volvulus: parasitologic and serologic responses in experimentally infected chimpanzees and mangabey monkeys. Exp. Parasitol. 80, 454–462 16 Luder, C.G. et al. (1993) Experimental onchocerciasis in chimpanzees: cellular responses and antigen recognition after immunization and challenge with Onchocerca volvulus infective third-stage larvae. Parasitology 107, 87–97 17 Soboslay, P.T. et al. (1992) Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus. Exp. Parasitol. 74, 367–380 18 Soboslay, P.T. et al. (1991) Experimental onchocerciasis in chimpanzees. Cell-mediated immune responses, and production and effects of IL-1 and IL-2 with Onchocerca volvulus infection. J. Immunol. 147, 346–353 19 Prince, A.M. et al. (1992) Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae. Exp. Parasitol. 74, 239–250 20 Bianco, A.E. et al. (1989) A semi-automated system of intrathoracic injection for the largescale production of Onchocerca lienalis infective larvae. Trop. Med. Parasitol. 40, 57–64 21 Kuo, Y.M. et al. (1995) Stage-specific and species cross-reactive antibody responses in experimental Onchocerca infections of cattle. Am. J. Trop. Med. Hyg. 53, 624–632 22 Kuo, Y.M. et al. (1995) Temporal changes in the humoral immune response of cattle during experimental infections with Onchocerca lienalis. Parasite Immunol. 17, 393–404 23 Trees, A.J. et al. (1992) Age-related differences in parasitosis may indicate acquired immunity against microfilariae in cattle naturally infected with Onchocerca ochengi. Parasitology 104, 247–252

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24 Trees, A.J. et al. (2000) Onchocerca ochengi infections in cattle as a model for human onchocerciasis: recent developments. Parasitology 120, S133–S142 25 Hoch, B. et al. (1992) Serological recognition of specific and crossreactive of O. ochengi and O. volvulus by infected cattle and humans. Trop. Med. Parasitol. 43, 206–207 26 Graham, S.P. et al. (2000) Onchocerca volvulus: comparative analysis of antibody responses to recombinant antigens in two animal models of onchocerciasis. Exp. Parasitol. 94, 158–162 27 Wahl, G. et al. (1998) Onchocerca ochengi: epidemiological evidence of cross-protection against Onchocerca volvulus in man. Parasitology 116, 349–362 28 Graham, S.P. et al. (1999) Patterns of Onchocerca volvulus recombinant antigen recognition in a bovine model of onchocerciasis. Parasitology 119, 603–612 29 Townson, S. et al. (1982) Immunization of calves against the microfilariae of Onchocerca lienalis. J. Helminthol. 56, 297–303 30 Wahl, G. et al. (1994) Bovine onchocercosis in north Cameroon. Vet. Parasitol. 52, 297–311 31 Duke, B.O.L. (1968) Reinfections with Onchocerca volvulus in cured patients exposed to continuing transmission. Bull. WHO 39, 307–309 32 Graham, S.P. et al. (2001) Down-regulated lymphoproliferation coincides with parasite maturation and with the collapse of both gamma interferon and interleukin-4 responses in a bovine model of onchocerciasis. Infect. Immun. 69, 4313–4319 33 Greene, B.M. et al. (1985) Humoral and cellular immune responses to Onchocerca volvulus infection in humans. Rev. Infect. Dis. 7, 789–795 34 Ward, D.J. et al. (1988) Onchocerciasis and immunity in humans: enhanced T cell responsiveness to parasite antigen in putatively immune individuals. J. Infect. Dis. 157, 536–543 35 Lucius, R. et al. (1995) Acanthocheilonema viteae: rational design of the life cycle to increase production of parasite material using less experimental animals. Appl. Parasitol. 36, 22–33 36 Lucius, R. et al. (1991) Acanthocheilonema viteae: vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens. Exp. Parasitol. 73, 184–196 37 Bleiss, W. et al. Protective immunity induced by irradiated third-stage larvae of the filaria Acanthocheilonema viteae is directed against challenge third-stage larvae before molting. J. Parasitol. (in press) 38 Storey, D.M. et al. (1982) Vaccination of jirds, Meriones unguiculatus, against Litomosoides carinii and Brugia pahangi using irradiate larvae of L. carinii. Tropenmed. Parasitol. 33, 23–24 39 Geiger, S.M. et al. (1996) Filariidae: crossprotection in filarial infections. Exp. Parasitol. 83, 352–356 40 Byars, N.E. et al. (1987) Adjuvant formulation for use in vaccines to elicit both cell-mediated and humoral immunity. Vaccine 5, 223–228 41 Townson, S. et al. (1982) Experimental infection of mice with the microfilariae of Onchocerca lienalis. Parasitology 85, 283–293 42 Carlow, C.K. et al. (1986) Further studies on the resistance to Onchocerca microfilariae in CBA mice. Trop. Med. Parasitol. 37, 276–281

43 Townson, S. et al. (1984) Immunity to Onchocerca lienalis microfilariae in mice. I. Resistance induced by the homologous parasite. Tropenmed. Parasitol. 35, 202–208 44 Townson, S. et al. (1985) Immunity to Onchocerca lienalis microfilariae in mice. II. Effects of sensitization with a range of heterologous species. J. Helminthol. 59, 337–346 45 Carlow, C.K. et al. (1987) Resistance to Onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous Onchocerca species. Parasitology 94, 485–496 46 Bianco, A.E. et al. (1991) Immunity to Onchocerca volvulus microfilariae in mice and the induction of cross-protection with O. lienalis. Trop. Med. Parasitol. 42, 188–190 47 Folkard, S.G. et al. (1996) Eosinophils are the major effector cells of immunity to microfilariae in a mouse model of onchocerciasis. Parasitology 112, 323–329 48 Hogarth, P.J. et al. (1999) Interleukin-12 modulates T-cell responses to microfilariae but fails to abrogate interleukin-5-dependent immunity in a mouse model of onchocerciasis. Immunology 98, 406–412 49 Hogarth, P.J. et al. (1995) Accelerated clearance of Onchocerca microfilariae and resistance to reinfection in interleukin-4 gene knockout mice. Parasite Immunol. 17, 653–657 50 Hogarth, P.J. et al. (1998) IL-5-dependent immunity to microfilariae is independent of IL-4 in a mouse model of onchocerciasis. J. Immunol. 160, 5436–5440 51 Townson, S. et al. (1981) Attempts to infect small laboratory animals with the infective larvae of Onchocerca lienalis. J. Helminthol. 55, 247–249 52 Abraham, D. et al. (1992) Identification of surrogate rodent hosts for larval Onchocerca lienalis and induction of protective immunity in a model system. J. Parasitol. 78, 447–453 53 Taylor, M.J. et al. (1992) Host strain, H-2 genotype and immunocompetence do not affect the survival or development of Onchocerca lienalis infective larvae implanted within micropore chambers into mice or rats. Parasitology 105, 445–451 54 Bianco, A.E. et al. (1989) Fate of developing larvae of Onchocerca lienalis and O. volvulus in micropore chambers implanted into laboratory hosts. J. Helminthol. 63, 218–226 55 Strote, G. (1989) Studies on the activity of the Ciba Geigy compounds CGP 6140, 20376, 20309 and 21833 against third and fourth stage larvae of Onchocerca volvulus. Trop. Med. Parasitol. 40, 51–56 56 Strote, G. (1985) Development of infective larvae of Onchocerca volvulus in diffusion chambers implanted into Mastomys natalensis. Trop. Med. Parasitol. 36, 120–122 57 Abraham, D. et al. (1993) Survival and development of larval Onchocerca volvulus in diffusion chambers implanted in primate and rodent hosts. J. Parasitol. 79, 571–582 58 Lange, A.M. et al. (1993) Induction of protective immunity against larval Onchocerca volvulus in a mouse model. Am. J. Trop. Med. Hyg. 49, 783–788 59 Lange, A.M. et al. (1994) IL-4- and IL-5dependent protective immunity to Onchocerca volvulus infective larvae in BALB/cBYJ mice. J. Immunol. 153, 205–211

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Emerging Chagas disease in Amazonian Brazil José Rodrigues Coura, Angela C.V. Junqueira, Octavio Fernandes, Sebastiao A.S. Valente and Michael A. Miles In the Amazon Basin, Trypanosoma cruzi infection is enzootic, involving a variety of wild mammals and at least 10 of the 16 reported silvatic triatomine bug species. Human cases of Chagas disease are increasing, indicating that the disease may be emerging as a wider public health problem in the region: 38 cases from 1969 to 1992, and 167 in the past eight years. This article reviews the status of Chagas disease in Amazonian Brazil, including known reservoirs and vectors, and the genetic diversity of T. cruzi. At least three subspecific groups of T. cruzi – T. cruzi I Z1, T. cruzi Z3 and T. cruzi Z3/Z1 ASAT – are present. It appears that T. cruzi I has an extant capacity for genetic exchange. Attention is also drawn to the risk of domestic endemicity, in addition to the tasks facing the disease control authorities.

José Rodrigues Coura* Angela C.V. Junqueira Octavio Fernandes Dept of Tropical Medicine, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil. *e-mail: coura@ ioc.fiocruz.br Sebastiao A.S. Valente Instituto Evandro Chagas, Fundação Nacional de Saude, Belém, Pará State, Brazil. Michael A. Miles Dept of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK WC1E 7HT.

The greatest risks underlying the establishment of Chagas disease in the Amazonian region of Brazil are human migration and uncontrolled deforestation. Human migration, with carriage of triatomine vectors from endemic areas, could transport the domestic cycle from established areas of domestic transmission to other areas. Deforestation can encourage adaptation of silvatic triatomine vectors to human dwellings. Since the beginning of the 1900s, it has been known that there are abundant reservoir hosts for Trypanosoma cruzi among wild animals [1,2] and several triatomine insect vector species [3,4] in Amazonian Brazil. However, the first autochthonous human cases of Chagas disease in the region were reported only in 1969, from the city of Belém, Pará State [5]. From a recent survey of case reports assembled at the Instituto Evandro Chagas, a total of 205 cases of Chagas disease have now been registered, between 1968 and 2000 [6] (Fig. 1). Of these, 178 were acute cases, nine of which were fatal, and 27 were chronic. Geographical distribution by State was as follows: 121 in Pará , 53 in Amapa, 14 in Amazonas, nine in Maranhao and eight in Acre. This is considered to be a small proportion of the real http://parasites.trends.com

number of cases, as the distribution reflects the research interest in Chagas disease and the presence of local diagnostic facilities in Belém. The national serological survey carried out by the Brazilian Ministry of Health from 1975 to 1980 showed a 1.88% seroprevalence in the State of Amazonas and 2.4% in the State of Acre [7]. Three additional surveys performed in 1991, 1993 and 1997 involving 2254 individuals from Barcelos, State of Amazonas, showed that 2.8–5% of this number had positive

Brazilian Amazon

Key: Endemic area Enzootic area Isolated human cases TRENDS in Parasitology

Fig. 1. Distribution of Chagas disease in Brazil: enzootic areas with isolated human cases or small outbreaks and endemic regions [6,8,15].

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