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37 Lorenzo, S. et al. (2000) O-glycans as a source of cross-reactivity in determinations of human serum antibodies to Anisakis simplex antigens. Clin. Exp. Allergy 30, 551–559 38 Lorenzo, S. et al. (1999) Human immunoglobulin isotype profiles produced in response to antigens recognized by monoclonal antibodies specific to Anisakis simplex. Clin. Exp. Allergy 29, 1095–1101 39 Metcalfe, D.D. and Sampson, H.A. (1990) Workshop on experimental methodology for clinical studies of adverse reactions to foods and food additives. J. Allergy Clin. Immunol. 86, 421–442 40 Iglesias, R. et al. (1996) Antigenic cross-reactivity in mice between third stage larvae of Anisakis simplex and other nematodes. Parasitol. Res. 82, 378–381 41 Ardusso, D.D. et al. (1996) Hipersensibilidad inmediata al parásito del pescado Anisakis simplex. Estudio de reactividad cruzada. Rev. Esp. Alergologia Inmunol Clin. 11, 280–286 42 Pascual, C. et al. (1997) Cross-reactivity between IgE-binding proteins from Anisakis, German cockroach, and chironomids. Allergy 52, 514–520 43 Leung, P.S.C. et al. (1996) IgE reactivity against a cross-reactive allergen in crustacea and mollusca: Evidence for tropomyosin as the common allergen. J. Allergy Clin. Immunol. 98, 954–961 44 Moneo, I. et al. (1997) Periodate treatment of Anisakis simplex allergens. Allergy 52, 565–569 45 Romarís, F. et al. (1996) Free and bound biotin molecules in helminths: a source of artifacts for avidin biotin-based immunoassays. Parasitol. Res. 82, 617–622

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46 Petithory, J.C. et al. (1991) Données séroepidémiologiques sur l’anisakiase: conséquences prophylactiques pour les produits de la pêche. Bull. Acad. Nat. Med. 175, 273–279 47 Asaishi, K. et al. (1980) Studies on the etiologic mechanism of anisakiasis. II. Epidemiological study of inhabitants and questionnaire survey in Japan. Gastroenterol. Jpn. 15, 128–134 48 Akao, N. et al. (1990) Immunoblot analysis of serum IgG, IgA and IgE responses against larval excretorysecretory antigens of Anisakis simplex in patients with gastric anisakiasis. J. Helminthol. 64, 310–318 49 Tsuji, M. (1989) Serological and immunological studies. In Gastric Anisakiasis in Japan. Epidemiology, Diagnosis, Treatment (Ishikura, H. and Namiki, M., ed.), pp. 89–95, Springer-Verlag 50 Sakanari, J.A. et al. (1988) Intestinal anisakiasis. A case diagnosed by morphologic and immunologic methods. Am. J. Clin. Pathol. 90, 107–113 51 Takahashi, S. et al. (1990) Serodiagnosis of intestinal anisakiasis using micro-ELISA – diagnostic significance of patients’ IgE. In Intestinal Anisakiasis in Japan. Infected Fish, Sero-immunological Diagnosis, and Prevention (Ishikura, H. and Kikuchi, K., eds), pp. 221–223, Springer-Verlag 52 Poggensee, U. et al. (1989) Immunodiagnosis of human anisakiasis by use of larval excretorysecretory antigen. Zentralbl. Bakt. Microbiol. Hyg. A270, 503–510 53 Yamamoto, Y. et al. (1990) Detection of anti-Anisakis antibody of IgE type in sera of patients with intestinal anisakiasis. In Intestinal Anisakiasis in Japan. Infected Fish, Sero-immunological Diagnosis, and Prevention (Ishikura, H. and Kikuchi, K., eds), pp. 205–216, Springer-Verlag

54 Dell, A. (2001) Novel carbohydrate structures. In Parasitic Nematodes: Molecular Biology, Biochemistry and Immunology (Kennedy, M.W. and Harnett, W. eds), pp. 285–307, CABI Publishing 55 Minamoto, T. et al. (1991) Anisakiasis of the colon: report of two cases with emphasis on the diagnosis and therapeutic value of colonoscopy. Endoscopy 23, 50–52 56 Kikuchi, Y. et al. (1989) Pathology of gastric anisakiasis. In Gastric Anisakiasis in Japan. Epidemiology, Diagnosis, Treatment (Ishikura, H. and Namiki, M. eds), pp. 117–127, Springer-Verlag 57 Van Thiel, P.H. (1976) The present state of anisakiasis and its causative worms. Trop. Geo. Med. 28, 75–85 58 Ishikura, H. et al. (1993) Anisakidae and anisakidosis. In Progress in Clinical Parasitology (Vol. 3) (Sun, T., ed.), pp. 43–102, Springer-Verlag 59 Nagasawa, K. (1993) Review of human pathogenic parasites in the Japanese common squid (Todarodes pacificus). In Recent Advances in Fisheries Biology (Okutani, T. et al., eds), pp. 293–312, Tokai University Press 60 Verhamme, M.A.M. and Ramboer, C.H.R. (1988) Anisakiasis caused by herring in vinegar: a little known medical problem. Gut 29, 843–847 61 Kasuya, S. and Koga, K. (1992) Significance of detection of specific IgE in Anisakis-related diseases. Jap. J. Allergol. 41, 106–110 62 Deardorff, T.L. et al. (1991) Human anisakiasis transmitted by marine food products. Hawaii Med. J. 50, 9–16 63 Anderson, R.C., ed. (2000) Nematode Parasites of Vertebrates, their Development and Transmission, CABI Publishing

Resistance and susceptibility in human onchocerciasis – beyond Th1 vs Th2 C

ol

Oncho o n tr

Achim Hoerauf and Norbert Brattig As research progress has led to programs for the elimination of onchocerciasis as a public health problem, research must now be intensified to protect elimination efforts. A profound understanding of the immunology in the human–parasite relationship is required for predicting the impacts of an altered immune response in a population post-microfilaricide treatment, and for the development of a vaccine against onchocerciasis, a highly desirable tool to guarantee sustained elimination success. This article summarizes the recent advancements in understanding the human immune mechanisms against onchocerciasis, and focuses on the new concept of T-cell suppressor responses as a major counterbalance mechanism for effector responses driven by T helper 1 and T helper 2 cells against the filarial worms.

Onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is endemic in 37 countries and affects >17.7 million people. The infection is transmitted by blackfly (Simulium) species that acquire microfilariae (larval stage 1, L1) from infected http://parasites.trends.com

humans during blood feeding. The L1 larvae mature into infective larvae stage 3 (L3) within 10–12 days in the vector. Within several months, L3 develop into female or male adult worms, which reside in the subcutaneous (s.c.) nodules (onchocercomas) for 10–15 years and produce millions of microfilaria (Mf). It is this Mf stage that is the cause of substantial morbidity. Blindness – the most devastating disease manifestation – is caused by inflammation in the eye as a result of Mf dying in the cornea. With visual impairment in 500 000 and blindness in 270 000 humans, onchocerciasis has remained the second most frequent cause of preventable blindness in Africa. Second in its impact on life, but a much more frequent disease manifestation is dermatitis, ranging from acute episodes of papular inflammation to chronic atrophy, lichenification and leopard skin.

1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)02173-0

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The world community aims to eliminate onchocerciasis as public health problem [1]. The first approach, the Onchocerciasis Control Programme in West Africa (OCP), has targeted transmission by controlling blackflies and distributing the microfilaricidal drug ivermectin. This successful program will end in 2002. The second strategy, the African Programme for Onchocerciasis Control (APOC), relies on community-based mass distribution of ivermectin once a year. It is accepted that APOC, in its current form, might not stop transmission completely [2,3]. Hence, a complementary approach has been adopted for developing a vaccine against onchocerciasis, supported by the Edna McConnell Clark Foundation [4]. Identification of target antigens has been a major activity of this program and this will be reviewed in other articles of this series. This work has demonstrated that protective immunity can be induced in animals. However, a prerequisite for vaccine development is an improved understanding of the mechanisms that govern protective immunity to onchocerciasis in humans. Clinical presentations

Achim Hoerauf* Norbert Brattig Bernhard Nocht Institute of Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany. *e-mail: hoerauf@ bni.uni-hamburg.de

There are different types of immune reactions that must be considered when studying immunity to onchocerciasis because O. volvulus develops in several stages within its host: (1) vaccine-induced immunity against L3, (2) protective immunity without vaccination, which impedes the development of patent infection in individuals in endemic areas (designated putative immunity), (3) hyperreactive immune response leading to destruction of Mf (sowda) and (4) concomitant immunity in patently infected individuals (in a hyperendemic area, most L3 are prevented from development, thus a stable, adult worm load comprising a few worms is maintained in such individuals [5]). A key to understanding immunity against O. volvulus involves analyzing the different clinical presentations following exposure to the parasite. In endemic areas, generalized infection with onchocerciasis (GEO) is most frequent and characterized by adult worms in the s.c. nodules and millions of dermal Mf. Despite this high antigen load, individuals with GEO often present weak skin inflammation and exhibit relatively mild alterations, such as atrophy and pigmentary changes. By contrast to GEO, there are a few individuals in hyperendemic areas who remain free from infection despite equivalent heavy exposure to parasites and assumed carriage of L3. These people are termed putatively immune individuals (PI). The term putative describes the prospective nature of analysis because PI should be re-examined for a correct diagnosis to exclude prepatency at the time point of immune characterization. The alternative term, endemic normal, has also been used to describe PI, and thus implies exposure to infection, by contrast to studies of http://parasites.trends.com

other infections where the term endemic normal is used to describe individuals who are not infected with the infective agent under investigation. Whereas the existence of PI was originally established in Latin America [6], there has been concern about their existence in Africa. In one study, nine out of ten cases of skin snips that were microscopically negative for Mf were positive when analyzed by polymerase chain reaction (PCR) [7]. However, even if PI should have some degree of productive infection (PCR cannot prove the existence of live Mf), they clearly have a higher degree of protection compared with individuals with GEO, which is immunologically distinct. Therefore, the analysis of immune protection can be done validly by using the PI group. Putative immunity to bovine onchocerciasis (Onchocerca ochengi) has also been reported [8], yet the hypothesis that putative immunity might result from low vector-attractiveness of respective individuals has not been supported [9]. Sowda includes the minority of individuals with chronic hyperreactive onchodermatitis presented unilaterally. Sowda patients suffer from papular dermatitis, hyperpigmented lichenified lesions, pruritus and lymphadenitis. They have low Mf loads and low numbers of adult worms resident in large nodules [10]. This condition is assumed to be the result of a hyperreactive inflammatory response, leading to the killing of Mf at the expense of skin integrity. Immune effector pathways associated with the different clinical presentations

The immune effector mechanisms against Mf and L3 are complex (Table 1). In untreated GEO, eosinophilic granulocytes and macrophages are found in the tissue around degenerating or dead Mf [10]. Increased eosinophil accumulation in the nodules (onchocercomas) is dependent on the presence of Mf [11]. An attack against vital, non-degenerated Mf in GEO is observed in histological skin biopsies taken from patients after microfilaricidal treatment, but is usually not seen in untreated GEO [12]. Apart from eosinophils and macrophages, the participation of neutrophils in the cellular reaction to skin Mf has been documented [13]. There is evidence for a constant participation of the immune system in the daily turnover of Mf (up to 50 000 Mf) [14], demonstrated by elevated blood eosinophilia and increased levels of circulating, cationic eosinophil proteins [15]. This immune effector response, however, mainly involves the elimination of dead Mf. In sowda, by contrast to GEO, the killing of Mf without previous antifilarial treatment is regularly observed in infected patients by histology [10]. Sowda patients have a restricted number of parasites and a few, large onchocercomas, characterized by massive inflammatory infiltrates of lymphocytes (including several plasma cells), mast cells, eosinophils,

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Table 1. Parasitological, clinical and immununological differences between the generalized form of onchocerciasis, sowda form and putative immunesa

Suspected prevalence (%) Mf load (per mg of skin) Skin lesions Onchocercoma Weight ratio Host tissue/female worm nodule weight Lymph nodes

Generalized form of onchocerciasis

Hyperreactive form of onchocerciasis

Putatively immune individuals

Refs

<98%b 1−>500 Subclinical or intermittent dermatitis Moderate inflammatory reaction 20

<1% <10 Severe dermatitis

<1–5%b 0 None

[1] [6,10] [6,10]

Strong inflammatory reaction 200

None

[6,10,11,16]

None No specific reaction

600 mg Often fibrotic

None

T-cell response

Th2, Th3/Tr1

6000 mg Usually severe lymphadenitis Strong Th2 response

Proliferative response to OvAg B-cell response Levels of OvAg-reactive IgG1 (U ml–1) IgG3 (U ml–1) IgG4 (U ml–1) Ov20/S1-reactive IgE (OD) Total IgE (IU ml–1) Effector response Eosinophils (cells µl–1) Serum EDN (µg l–1)

Weak

Pronounced

Weak Th1 and [6,21–25,31, strong Th2 response 36,40] Pronounced [6,21,22,31]

40 (20–70)c 2 (1–3)c 170 (25–320)c <0.15 5800 (3000–10 000)c

210 (70–400)c 30 (5–100)c 170 (30–380)c 0.25–1.25 20 000 (15 000–40 000)c

20 (15–25)c 1 (1–2)c 1 NT 2000 (1500–5000)c

1200 (700–1800)c 120 (80–200)c

1400 (1500–2500)c 330 (300–360)c

550 (300–1000)c NT

[10]

[23,26,27,32,34]

[33]

[32] [15]

aAbbreviations: EDN, eosinophil-derived neurotoxin; Ig, immunoglobulin; Mf, microfilaria; NT, not tested; OvAg, Onchocerca volvulus antigen; Th2, T helper 2 cells; Th3/Tr1; T helper 3 cells/ T regulatory 1 cells. bThe suspected prevalence is dependent on the intensity of transmission. cThe numbers enclosed in parentheses indicate the range.

neutrophils and macrophages [16]. The eosinophils, neutrophils and macrophages all possess the capacity to kill Mf [10,16]. A similar pattern is also seen in the enlarged lymph nodes. The difference in immune attack against Mf between sowda and GEO suggests that Mf are killed in vivo by an interplay between inflammatory cells, which are strongly activated in sowda but are partially suppressed in GEO. T cells and antigen-presenting cells apparently play important regulatory roles in directing these responses. Several in vitro studies confirmed the capacity of eosinophils and neutrophils to kill Mf and to immobilize or kill L3 in infected patients [17,18]. Whereas L3 were immobilized by eosinophils after complement activation by normal serum, an opsonization by specific antibodies is needed for immobilizing Mf [19]. By contrast to Mf killing, the mechanisms for immune destruction of L3 are elusive to analysis when using human material ex vivo (e.g. by histology). In animal models, antibody-dependent eosinophils mediate the destruction of L3 shortly after host entry and during the molt stages to L4, but not of the later developmental stages of the parasite (D. Abraham, unpublished), in accordance with in vitro observations [19]. The presence of antibodies and effector cells seems essential, and the dependence on interleukin (IL)-4 and IL-5 qualify this reaction as T helper (Th) 2 cells. http://parasites.trends.com

Th cell and antibody responses associated with PI, sowda and GEO

In the 1980s, it was demonstrated that, first in mice, Th cells divide into two major mutually crossregulating subgroups: Th1 and Th2. This paradigm was later applied to humans, and results from individuals infected with helminths, including O. volvulus, served as early examples for studying human Th2 responses [20]. Given the potential importance of T cells in regulating the immune effector pathways against Mf, and L3 in PI, sowda and GEO patients, the possibility of a Th1 and Th2 dichotomy associated with different clinical presentations was explored. In one report, a Th1-type response was observed in a subgroup of PI [21]. However, in most other reports, peripheral blood mononuclear cells (PBMC) from PI or endemic normals displayed a mixed Th1 and Th2 response [22–25], which was elevated when compared with those from GEO, and interferon γ (IFN- γ) and IL-5 were the major cytokines detected in PI. The involvement of a Th2 response in immunity is feasible given the results from animal models, but it is still unclear how a Th1 response can account for immunity against invading L3, the most probable target stage in PI. The levels of Fc receptor- and complement-binding IgG3-type antibody levels are higher in PI [26] compared with those in GEO individuals, who characteristically produce elevated IgG4 antibodies against a water-soluble extract from

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O. volvulus worms (OvAg [27]) that are of a blocking isotype [28]. By contrast to the reported impact of IL-4 [29] and IL-12 [30] on IgG4 regulation, the relationship between IgG3 and Th1 has not been documented. An explanation for increased IFN-γ in PI was brought forward – potentially different levels of co-infections. Infections with gastrointestinal nematodes, however, have often been found in PI and GEO individuals in equivalent frequencies [23,24,31]. The suggestions that other co-infections potentially reduce a Th1 response against onchocerciasis (e.g. Th2 induction by schistosomiasis) were largely excluded [24,31]. By contrast to PI characterized by an overall mixed Th1 and Th2 response, a pronounced Th2-type response against OvAg is observed in sowda individuals [25]. In addition, sowda patients produced elevated levels of IgG1, IgG3 [32] and IgE [33] against O. volvulus. The levels of IgG3 antibodies against OvAg correlate negatively with Mf loads [34]. It can thus be assumed that a Th2-mediated, antibody-dependent and granulocyte-dependent response leads to Mf destruction but also to skin inflammation in sowda. Th1 and Th2 effector function counterbalance

Although immune effector mechanisms are involved in clearing the daily Mf turnover in GEO, there is clearly a downregulation of the immune system in GEO compared with the other two clinical presentations, particularly in individuals with high worm loads [35]. Lower proliferative responses to OvAg from PBMC in GEO patients have been found consistently when compared with either sowda (A. Doetze, PhD thesis, Universität Hamburg, 1999) or PI and endemic normals [21,22,31,36]. In accordance with the low proliferative responses to OvAg in GEO patients, a diminished delayed-type hypersensitivity (DTH) response following injection of OvAg was observed in GEO patients but not in PI and sowda patients [37]. Reduction of DTH response was antigen specific because the DTH response to tuberculin was equivalent to that in PI and sowda (G. Burchard and A. Hoerauf, unpublished). Whereas OvAg-induced DTH reactions have been interpreted as Th1-mediated [37], recent reports show that in Th2-dependent reactions, a DTH type II reaction can occur which produces a clinical presentation similar to that from a classical DTH. However, the DTH type II reaction is Th2-induced and leads to migration of different cell types (see Ref. [38]). Thus, it remains to be determined if the OvAg-induced DTH is Th1-mediated or Th2-mediated. Early attempts to ascribe the hyporesponsiveness in GEO to a Th2 deviation are now considered as an oversimplification, not only because of the mixed type of effector response seen in PI and sowda, but also because the recovery in the cellular proliferation after anti-Mf treatment by ivermectin increases both Th1 and Th2 responses, rather than inducing an overall shift from Th2 to Th1 [22,39]. Instead, there is a third arm of a Th cell reaction, which is associated with http://parasites.trends.com

OvAg-specific hypoproliferation and is able to suppress both Th1 and Th2 responses. Production of IL-10 was associated with cellular hyporesponsiveness [21–23,31,35,39], and this production was reduced after restoration of cellular proliferation following microfilaricidal treatment of GEO individuals by ivermectin [39]. However, for in vitro enhancement of lymphocyte proliferation of PBMC in GEO to levels seen in PI, neutralization of IL-10 by antibodies in the culture alone was insufficient [31,40]. Additional neutralization of another downregulatory cytokine, transforming growth factor β (TGF-β), was required [31]. Cellular hyporesponsiveness in GEO is at least partially antigen-specific because proliferation and IL-5 or IFN-γ production in response to antigen from Ascaris and tuberculin were observed equivalent to PI [31]. Therefore, it was imperative to look for the role of T cells in maintaining this O. volvulus-specific suppression. Onchocerca volvulus-specific T-cell clones could be generated from PBMC in GEO individuals with a cytokine profile that is characteristic of a new type of suppressor cell [31], which has been found in tolerization against autoimmunity [41,42] but not in infections so far. T-cell clones of this type were also obtained from onchocercomas – the likely location of suppression induction in onchocerciasis. Importantly, these T-cell clones block proliferation in co-culture, different from that by Th1 and Th2 clones (J. Satoguina and A. Hoerauf, unpublished). Antigen-specific suppression by these T-cell clones would not strictly argue against nonspecific suppression of immune responses (mainly observed in heavily infected individuals [35,43]) because suppressor T cells can spread tolerance by deviating from antigen-presenting cells, a phenomenon called infectious tolerance [44]. T cells of this type have been termed either Th3 or T regulatory 1 (Tr1). It has been suggested to define the downregulatory response as Th3/Tr1 in onchocerciasis [31] in order to underscore that downregulation in chronic helminth infection requires a novel, non-Th1 and non-Th2 type of response (Fig. 1). In lymphatic filariasis and schistosomiasis, T-cell effector pathways also comprise mixed Th1 and Th2 cells [45,46], whereas cellular hypoproliferation to specific antigen is mediated by IL-10 and TGF-β [47,48]. It is highly likely that Th3/Tr1 cells are also found in lymphatic filariasis and schistosomiasis. Given that IL-10 seems to have a role in downregulating Th2-driven allergic responses [49,50], it is worthwhile to consider this type of response as a negative regulator of Th2-driven immune pathways (including future analysis of other members of the IL-10 family, such as IL-19, IL-20 and IL-22). Importantly, a downregulatory molecule, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) was a key marker for this new type of T cell (J. Satoguina and A. Hoerauf, unpublished), is upregulated spontaneously in Mf-infected individuals, and is induced by antigen from Mf but not from other parasite stages (C. Steel, pers. commun.). Recently, dendritic cells and macrophages have been subdivided into distinct functional subpopulations [51].

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Fig. 1. Model of the balance between effector and suppressor mechanisms in onchocerciasis. (a) In generalized onchocerciasis, the Th1and Th2-dependent effector reactions are suppressed by IL-10 and TGF-β, and antigenspecific T-regulatory cells (Th3/Tr1) can be found [31]. (b) In putatively immune individuals and patients with the hyperreactive form of disease (sowda), the T helper (Th) 1 and Th2 effector mechanisms prevail, leading to immune attack against L3 (putative immunity) or Mf (sowda, histologically documented in Refs [10,16]). Abbreviations: IFN-γ, interferon γ; IL , interleukin; Th3, T helper 3 cells; TGF-β, transforming growth factor β ; Tr1, T regulatory cell 1.

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(a) Generalized onchocerciasis Suppressor mechanisms

Effector mechanisms IL-2 IL-4, Th1/2 IL-5, IL-13

IL-10, TGF-β Th3/Tr1

(b) Putatively immune individuals and hyperreactive onchocerciasis (sowda) Suppressor mechanisms

Effector mechanisms

IL-10, TGF-β Th3/Tr1 Th1/2

IL-2 (IFN-γ), IL-4, IL-5, IL-13

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Type 1 dendritic cells and macrophages produce IL-12 and promote Th1 responses, and respective cells of type 2 generate less IL-12 but IL-10, TGF-β or other negative regulators. The occurrence of suppressive macrophages that produce IL-10 has been reported for lymphatic filariasis [48]. The demonstration of IL-10 in elutriated (>96% pure) macrophage cultures [52] after stimulation with OvAg confirmed this for onchocerciasis and indicates that macrophages can express a type 2 phenotype in response to O. volvulus and thus influence the Th cell dichotomy by IL-10. Thus, in addition to regulatory suppressor Th3 and Tr1 cells, antigen-presenting cells might contribute to cellular hyporeactivity. What are the final effector pathways that are actually suppressed along with cellular hyporeactivity in peripheral blood? T-cell suppression of Th1 and Th2 responses probably diminishes the presence of both effector cells and antibodies, and alters the antibody subclass, thus preventing the attack against Mf in GEO. How are the different forms of the immune response induced?

Complex, multicellular organisms such as O. volvulus present a plethora of antigens to their hosts. Immunomodulatory proteins [53,54] or proteins homologous to mammalian downregulatory cytokines [55] have been cloned from filarial species. However, non-protein molecules, such as bacterial DNA [56], lipopolysaccharide (LPS) [57] or oligosaccharides [58], also drive T-cell responses into the different effector pathways. Interestingly, all these molecules are found in filarial species. Molecules derived from Wolbachia intracellular mutualistic [59] bacteria are found in most human and animal filarial species including O. volvulus [60]. Bacterial products (both LPS related [52,61] and LPS unrelated [52]) stimulated the http://parasites.trends.com

29

release of pro-inflammatory and anti-inflammatory cytokines. These bacterial products were also the main inducers of the neutrophil response around adult worms [62], which is Th1 dependent in mice [63]. Sugar moieties and phosphorylcholine, present on filarial L3 and Mf surfaces, promote Th2 responses [58,64,65]. Xid mice, which do not recognize phosphorylcholine, produce significantly lower levels of IL-4 and IL-5 in response to worm antigen in vitro [64]. Thus, the worm can provide the host with both Th1- and Th2-inducing molecules, and supposedly Th3/Tr1-inducing molecules [54,55]. In addition, the individual immune response is likely to be influenced, to a major part, by host genetics plus environmental factors (see Table 2). Genetic studies suggest a link between HLA-DQ molecules and a low or high immune reactivity in onchocerciasis [66,67]. Cytokine or cytokine-receptor allelism also influences immune reactivity to O. volvulus: a variant of human IL-13 that correlates with asthma and atopy was strongly associated with immunological hyperreactivity (sowda) in populations from two west African countries [68]. A major environmental factor for the development of tolerance to filarial antigens appears to be exposure in utero when mothers are already infected [69]. Accordingly, the chronicity and intensity of infection might account for the different clinical presentations and immunological status observed when infected visitors to endemic areas were compared with long-term endemic subjects [70]. Finally, crossreactivity to other filariae and nematodes could still influence the extent of tolerization or suppression seen in GEO. Conclusive remarks

Research over the past decades has led to a thorough understanding of effector and suppressor immune reactions in onchocerciasis. Further analysis of the immunosuppression generated by this chronic helminth infection could also yield a better understanding of the pathways in other fields of immunology, and might continue to trespass a strict dogma of Th1 and Th2 dichotomies (e.g. by further establishing Th3/Tr1 or other types of suppressor cells as a counterregulator of Th2-driven effector responses). The local immune response in onchocercomas and skin will be another necessary focus of research, which probably provides a clearer picture than PBMC analysis. Further avenues of research to be dealt with, such as the impact of onchocerciasis on co-infection (HIV, malaria and tuberculosis) and the success of vaccination against other diseases [35], are summarized in a final paper in this onchocerciasis series (T. Nutman, unpublished). With our improved understanding of the immune system and having defined many potential vaccine candidates, probably the most important remaining question is: do we really believe that the development of a vaccine against onchocerciasis holds promise for helping in disease elimination? This is argued strongly

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Table 2. Factors which influence the development of different cellular responses after exposition to Onchocerca volvulus a Group of factors

Factors

Worm-derived molecules biasing immune responses

Immunomodulatory proteins

Host genetics

Timepoint of exposure

Molecules

Cellular response

Cystatin MIF TGF-β-like molecule Products derived from DNA Wolbachia endobacteria LPS-like molecule Oligosaccharides, Fucosyl-, phosphorylcholinephosphorylcholines residues HLA-DQ molecules associated DQA1*0501 with immune activity DQB1*0301 Allelism in cytokines R110Q variant of IL-13 associated with hyperreactive sowda In utero exposure to parasite ND antigens associated with tolerization Infected expatriates show less Lack of in utero exposure tolerance to infection than the infected endemic population

Refs

Downregulatory (Th3/Tr-1?) [53–55]

Proinflammatory (Th1?)

[52,61]

Th2 ND

[58,64, 65] [66,67]

Th2

[68]

Downregulatory (Th3/Tr-1?, [69] type 2 antigen-presenting cells?) Lack of downregulation [70]

aThe

factors can influence the development of different cellular responses including Th1 cells, Th2 cells, Th3/Tr1, and type 1 and 2 antigenpresenting cells. Abbreviations: HLA-DQ, human leukocyte antigen-DQ; IL-13, interleukin 13; LPS, lipopolysaccharide; MIF, migration inhibitory factor; ND, not determined; TGF-β, transforming growth factor β; Th1, T helper 1 cells; Th2, T helper 2 cells; Th3/Tr1, T helper 3 cells/T regulatory 1 cells.

Acknowledgements We thank Dietrich W. Büttner for suggestions and comments. Support from the German Research Foundation (grants Ho 2009/1-3 and Br 1020/1-3), and to A.H. by the European Community (grant ICA4Ct-1999-10002), the Wellcome Trust Foundation and previously from the Edna McConell Clark foundation is gratefully acknowledged.

in favor for the following reasons: (1) in veterinary medicine, anthelmintic vaccines have been successfully developed for field use and are being considered for human trials [71] therefore the proof-of-principle has been already performed; (2) in bovine onchocerciasis, putative immunity has been reported under conditions of natural exposure [8]; (3) concomitant immunity in humans [5] could cease gradually when ivermectin is widely used by the APOC. Therefore, additional vaccine-induced immunity might be required for final disease eradication eventually. The research community is left with the problem of rendering particular O. volvulus molecules, which induce

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