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Clin Exp Immunol. 2007 March; 147(3): 504–512. doi: 10.1111/j.1365-2249.2006.03312.x. | PMCID: PMC1810490 |
Copyright © 2007 British Society for Immunology Onchocerca volvulus-specific antibody and cytokine responses in onchocerciasis patients after 16 years of repeated ivermectin therapy C S Mai,* D M Hamm,* M Banla,† A Agossou,‡ H Schulz-Key,* C Heuschkel,* and P T Soboslay* *Institute for Tropical Medicine, University of Tübingen, Tübingen, Germany †Université de Lomé, Faculté Mixte de Médecine et Pharmacie, Lomé, Togo ‡Centre Hospitalier Regional, Service Pédiatrie, Sokodé, Togo Accepted December 11, 2006. The recommended control option against onchocerciasis is repeated ivermectin treatment, which will need to be implemented for decades, and it remains unknown how repeated ivermectin therapy might affect immunity against Onchocerca volvulus in the long term. O. volvulus-specific antibody reactivity and cellular cytokine production were investigated in onchocerciasis patients receiving ivermectin (150 µg/kg) annually for 16 years. In treated patients, the T helper type 2 (Th2) cytokine interleukin (IL)-5 and T regulatory IL-10 in response to O. volvulus antigen (OvAg) and bacteria-derived Streptolysin O (SL-O) diminished to levels found in infection-free endemic controls; also, cellular release of Th1-type interferon (IFN)-γ at 16 years post initial ivermectin treatment (p.i.t.) approached control levels. In ivermectin-treated onchocerciasis patients, IL-5 production in responses to the mitogen phytohaemagglutinin (PHA) decreased, but IL-10 in response PHA increased, and neither attained the cytokine production levels of endemic controls. At 16 years p.i.t., O. volvulus-specific IgG1 and IgG4 subclass reactivity still persisted at higher levels in onchocerciasis patients than in O. volvulus exposed but microfilariae-free endemic controls. In addition, cytokine responses remained depressed in onchocerciasis patients infected concurrently with Mansonella perstans and Necator americanus or Entamoeba histolytica/dispar. Thus, long-term ivermectin therapy of onchocerciasis may not suffice to re-establish fully a balanced Th1 and Th2 immune responsiveness in O. volvulus microfilariae-negative individuals. Such deficient reconstitution of immune competence may be due to an as yet continuing and uncontrolled reinfection with O. volvulus, but parasite co-infections can also bias and may prevent the development of such immunity. Keywords: antibody, cytokines, immune responses, ivermectin, Onchocerca volvulus Infection with the filarial nematode Onchocerca volvulus causes skin pathology and ocular lesions induced by microfilaria (mf), the first larval stage of the parasite, which invades and migrates through the skin and ocular tissues [ 1]. Despite onchocerciasis having been successfully controlled in parts of West Africa by the Onchocerciasis Control Program (OCP) [ 2, 3, 4], infection with O. volvulus remains an obstacle to public health in many other endemic countries [ 5, 6, 7]. The likelihood of the eradication of O. volvulus is limited [ 8, 9, 10] as the only currently available and recommended control option is repeated ivermectin treatment. Such treatment will efficiently reduce O. volvulus microfilaria in the skin and ocular tissues of patients and thus notably lessen onchocercal skin diseases [ 11] and prevent the evolution of eye pathology [ 12]. Ivermectin is now distributed in onchocerciasis endemic countries on a large scale, and it has undoubtedly lessened intensities of O. volvulus infection in the human population [ 2, 13], decreased parasite transmission by black fly vectors ( Simulium spp.) [ 13], and ultimately may eliminate onchocerciasis as an obstacle to public health [ 14], however, global eradication of the parasite by means of ivermectin alone would not appear feasible [ 15]. While ivermectin will not exert direct embryotoxic or embryostatic effects in gravid female O. volvulus [ 16], the drug suppresses for months the release of microfilariae from gravid female O. volvulus. Furthermore, ivermectin influences the delicate parasite-host equilibrium and changes immunological processes in the host, notably, cellular unresponsiveness to onchocercal antigen will increase in patients after ivermectin treatment, cytokine responses of both Th1- as well as Th2-type will become re-activated shortly after treatment while parasite-specific antibody isotype responses will gradually decrease [ 17, 18, 19]. An ivermectin-facilitated amplification of anti- Onchocerca immunity in exposed or infected individuals may help to generate an immune competence which confers protection against infection and disease – as described in individuals putatively immune to O. volvulus infection [ 1, 20, 21, 22]. Regarding the future prospects of ivermectin treatment-based onchocerciasis control, which will have to be implemented for decades [ 23], it remains unknown whether repeated ivermectin therapy might promote anti- Onchocerca protective immunity, or in the worst case, may even increase susceptibility to new or re-infection. These aspects have to be considered in detail, since experimental studies in Onchocerca ochengi-infected cattle disclosed that drug treatment or ivermectin withdrawal will result in heightened susceptibility to infection [ 24, 25]. In the present study, antibody and cytokine profiles in onchocerciasis patients treated annually for 16 years with ivermectin were investigated. As observed, patients with 16 years of ivermectin therapy did not present any acute or adverse clinical features of onchocerciasis; their antibody and cytokine profiles approached those of onchocerciasis-free endemic controls, although an important restricting factor to full recovery of O. volvulus-specific cellular immune responsiveness was lymphatic filaria and intestinal parasite co-infections. Location of study This study was conducted in central Togo in West Africa, within the previously vector-controlled area of the former Onchocerciasis Control Programme (OCP), where transmission of infective third-stage larvae (L3) of O. volvulus has been reduced through control of the Simulium spp. black fly vectors. In central Togo the risk of reinfection still remains [ 3]. Concurrent Mansonella perstans infection was observed frequently in onchocerciasis patients [ 26, 27], and treatment of onchocerciasis patients with ivermectin did not reduce significantly microfilariae of M. perstans [ 26, 27]. The onchocerciasis patients and endemic controls were from the Prefecture Tchaoudjo in the Central Region of Togo, from the Villages Bougabou, Bounakou, Bouzalo (Bas and Montagne), Sagbadai, Tabalo I and II, Salimde and Kouwaou Woro; the distance between the villages does not exceed 10 km. All villages are borderline to the rivers Mo and Kpondjo. Up to the present, the river Mo is treated continuously with insecticides by the National Onchocerciasis Control Programme (NOCP; helicopter spraying) against black fly larvae even after the major vector control activities of the West African OCP have stopped. The Mo river basin is a ‘special intervention zone’, where vector control and intensified ivermectin distribution need to be continued for the coming years – this is because of a continuous and uncontrollable transmission of infective third-stage larvae (L3) of O. volvulus, despite 25 years of vector control and annual ivermectin distribution. The persistent and uninterrupted transmission of L3 of O. volvulus in the study area has been described by Borsboom et al. [ 13]. Study population and examinations The study participants originated from villages in central Togo where onchocerciasis was meso- to hyperendemic [ 3]. In the study area O. volvulus vector control by the OCP began in 1977 and is being continued until the present; this is due to the persistent transmission of O. volvulus L3 in the central and northern regions of Benin and Togo [ 3, 13]. Authorization and approval for this study was granted by the Ministry of Health in Togo and reapproved every 4–5 years (no. 1999: 292//MS/CAB 2003431989; no. 261//MSP/DGSP/DRSP-RC). At each time-point of examination all participants gave their informed consent, and for correct and complete understanding explanations were always given in the local language. Patients were apparently healthy males and non-pregnant women with a body weight over 30 kg, without history of multiple allergies or drug intolerance, with skin biopsies positive for microfilaria of O. volvulus and with palpable nodules (onchocercomata). All onchocerciasis patients participated in regular surveys conducted by the NOCP. All patients have received annual ivermectin treatment (150 µg/kg) as a single dose distributed by the NOCP in Togo. At regular intervals thorough physical, parasitological and ophthalmological examinations were conducted and the density of O. volvulus microfilariae (mf) was determined in skin biopsies (mf/mg skin) taken from the right and left hip [ 19, 28]. Concurrent intestinal helminth and protozoa infections were diagnosed in fresh stool samples collected from all participants by standard methodology. Fresh stool samples (0·5 g) were mixed with saline and dispersed on two microscope slides, then covered with a 24 × 48 mm slides and the samples examined by two laboratory technicians. Intestinal protozoa species and helminth eggs were identified and recorded semi-quantitatively. In addition, all stool samples were investigated for helminth eggs by the Kato–Katz methodology [ 29] (helm-TEST; Labmaster, Belo Horizonte, MG, Brazil). From each participant 10 ml of urine were filtered (polycarbonate 0·8 µm; Sartorius AG, Göttingen, Germany) and the filtrate examined under a microscope for eggs of Schistosoma haematobium. M. perstans infection was detected after Ficoll-Paque (Pharmacia, Freiburg, Germany) gradient centrifugation of 20 ml of whole blood in the peripheral blood mononuclear cells (PBMC) fraction and pellets, as described previously [ 30]. PBMC were dispensed in equal volumes into 96-well culture plates, and after overnight incubation plate wells were examined under a microscope for the presence of blood-dwelling microfilariae of M. perstans. O. volvulus antigen-specific enzyme-linked immunosorbent assay (ELISA) O. volvulus antigen-specific (OvAg-specific) IgG isotypes were determined by ELISA as described by Soboslay et al. [ 28]. Isolation of O. volvulus and adult worm-derived antigen (OvAg) preparation was effected as described previously by Schulz-Key et al. [ 31] and Greene et al. [ 32]. Briefly, adult worms were isolated as described and then washed in phosphate-buffered saline (PBS), transferred into a Ten-Broek tissue grinder and then homogenized extensively on ice. The homogenate was then sonicated twice (30% intensity) for 3 min on ice and centrifuged at 16 000 g for 30 min at 4°C. The supernatants were collected then sterile-filtered (0·22 µm), and the protein concentration determined with a BCA protein assay (Pierce, Rockford, USA). Isolation of PBMC, cell culture experiments and determination of cytokine production Heparinized venous blood was collected from onchocerciasis patients and endemic controls, and PBMC were isolated by Ficoll-Paque density gradient centrifugation. Cell culture experiments were conducted as described previously by Soboslay et al. [ 27, 28]. Briefly, PBMC were adjusted to 1 × 10 7/ml in RPMI (G ibco, Eching, Germany) supplemented with 25 mM HEPES buffer, 100 U/ml penicillin and 100 µg/ml streptomycin, 0·25 µg/ml amphotericin B (Sigma, St Louis, MO, USA); they were then used immediately to stimulate cytokine secretion. Freshly isolated PBMC were cultured at a concentration of 2·5 × 10 6 PBMC/ml in RPMI (as above) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Biochrom, Berlin, Germany) in the presence of either O. volvulus-derived antigen (OvAg, 35 µg/ml), phytohaemagglutinin (PHA) (1 : 100, Sigma) or Streptolysin-O (SL-O, 1 : 50; Difco, Augsburg, Germany) in 5% CO 2 at 37°C and saturated humidity. Cell culture supernatants were collected after 48 h and stored below –20°C until further use. Cytokine secretion by stimulated PBMC was quantified by sandwich ELISA using cytokine-specific monoclonal and polyclonal antibodies for interferon (IFN)-γ, interleukin (IL)-2, IL-5, IL-10 and IL-12 (BioSource, Rating, Germany; R&D Systems, Minneapolis, MN, USA), as recommended by the manufacturers and as described previously [ 28, 30]. The detection limit of the BioSource CytoSets was 10 pg/ml for IFN-γ, IL-10 and IL-12; for the R&D Systems DuoSets the detection limit was 20 pg/ml for IL-2 and IL-5. Statistical data analyses For data analyses the statistical package jmp 5.0.1.2 was used. Significant differences between groups were determined after logarithmic transformation to stabilize the variance of data [log (pg/ml + 1)]. The level of significance was adjusted according to Bonferroni–Holm. Data presented in box plots show the median with the 25% and 75% quartiles and 1·5 × of the interquartile range. Paired data from patients were evaluated by sign test and significant differences are given as #P = 0·05 or ##P = 0·01. Unpaired data of patient groups were compared using the Wilcoxon rank sum test and significant differences are given as *P = 0·05 or **P = 0·01. For analysis of cytokine (IFN-γ, IL-2, IL-10 and IL-12) production in ivermectin-treated onchocerciasis patients co-infected with M. perstans, Necator americanus or Entamoeba histolytica/dispar multiple regression analysis was applied. The cytokine production [least squares means (LS means) and their 95% confidence intervals] was analysed by Tukey's test with the predictors: infection groups, cytokine, antigen, patients number × infection groups (i.e. the random factor is patients' number) and their corresponding interaction of degree 2. Study groups and patients' characteristics The demographic data for the study groups are shown in Table 1. All onchocerciasis patients were treated annually with 150 µg/kg ivermectin from 1989 and were negative for microfilariae of O. volvulus at follow-up examinations for the last 7 years. Individuals were considered as endemic controls when microfilariae of O. volvulus had never been detected in skin biopsies. In onchocerciasis patients concurrent infection with M. perstans was detected despite repeated ivermectin treatment. At 16 years post-initial ivermectin treatment (p.i.t.), onchocerciasis patients presented with lower lymphocyte but higher eosinophil granulocyte ( P = 0·005) numbers than endemic controls. | Table 1Demographic data on study groups. |
Cellular cytokine production IL-5 and IL-10 were studied in onchocerciasis patients at 1 month, 12 months and 16 years p.i.t. with ivermectin (150 µg/kg) and compared to O. volvulus exposed but mf-free endemic controls (). From 1 month p.i.t. to 16 years p.i.t. IL-5 decreased significantly in treated patients (P = 0·0005). At 16 years p.i.t., in response to OvAg and bacterial SL-O, PBMC from treated patients and endemiccontrols did not differ in their capacity to release IL-5, but following PHA activation IL-5 production by PBMC from patients remained higher (although not significantly) than in controls. The IL-10 production in response to OvAg increased at 12 months p.i.t. (P = 0·013) and was found diminished at 16 years p.i.t., although onchocerciasis patients still produced more IL-10 (P = 0·004) in response to OvAg than measured in endemic controls. When PBMC from treated onchocerciasis patients were activated with bacterial SL-O, IL-10 decreased until 16 years p.i.t. (P = 0·023) and was produced similarly to that of the controls. In contrast, PBMC from treated patients increased continuously in their capacity to release IL-10 in response to mitogen activation with PHA at 1 month (P < 0·0001 versus Con), 12 months (P = 0·0001 versus Con) and 16 years p.i.t., but their IL-10 did not entirely attain the levels detected in controls. In onchocerciasis patients treated repeatedly for 16 years with ivermectin, IFN-γ responses to OvAg and SL-O were the same as in the controls, but following mitogen activation with PHA, IFN-γ was low in patients (P < 0·0001)(). The T helper 1 (Th1) : Th2 ratios of the net cytokine production values at each time-point for each patient, including endemic controls, were calculated on net production values according to the formula Exp[(IFN-γ in log pg/ml)-(IL-5 in log pg/ml)] and data are shown in . The ratios for IFN-γ/IL-5 were depressed in onchocerciasis patients at 1 month p.i.t. when compared to endemic controls; thereafter they tended to increase over time, but the Th1 : Th2 cytokine ratios in ivermectin-treated onchocerciasis patients did not attain the values found in endemic controls (P = 0·007). Similarly, at 16 years p.i.t., the Th1 : Th2 ratios of IL-12/IL-5 were found depressed (P = 0·002) in onchocerciasis patients when compared to endemic controls (data not shown). Cytokine responses with concurrent necatoriasis and mansonelliasis After 16 years of repeated ivermectin therapy, onchocerciasis patients were found to be co-infected with the hookworm N. americanus (94%) and the lymphatic filaria M. perstans (73%) ( Table 1). Cytokine responses in controls and ivermectin-treated onchocerciasis patients were analysed according to the presence or absence of M. perstans infection and intestinal parasite co-infections. Differences in IFN-γ, IL-2, IL-10 and IL-12 net production in response to OvAg, PHA and SL-O between controls (Con +) and patient groups (G0 + 1, G2, G3) are shown in . In patients positive for M. perstans and co-infected with intestinal parasites, cellular release of IFN-γ, IL-12, IL-2 and IL-10 were depressed after helminth-, bacteria- and mitogen-induced stimulation. Multiple regression analyses of the cytokine production disclosed that PBMC from onchocerciasis patients concurrently positive for M. perstans and intestinal parasite infections (patient groups G2 and G3) had a significantly diminished ( P < 0·001) capacity to produce cytokines than PBMC from O. volvulus exposed but mf-free endemic controls (). O. volvulus-specific antibody reactivity in onchocerciasis patients and controls Antibody subclass responses to OvAg were investigated in onchocerciasis patients before treatment with ivermectin, at 1 week p.i.t. and at 16 years p.i.t. and compared to O. volvulus exposed but mf-free endemic controls (). In ivermectin-treated onchocerciasis patients, the mean antibody reactivity to OvAg increased significantly at 1 week after the initial ivermectin treatment for IgG1 (P = 0·0189) and IgG3 (P = 0·007, data not shown), and a similar increase, although not significant, was also observed for IgG2 (data not shown) and IgG4 subclasses. From 1 week p.i.t. until 16 years p.i.t. O. volvulus-specific antibody responses in onchocerciasis patients diminished for IgG1 (P = 0·023), IgG3 (P = 0·0013) and IgG4 (P = 0·003). At 16 years p.i.t., IgG responses approached the levels as measured in endemic controls, but still the OvAg-specific IgG1–4 reactivity remained significantly higher (IgG1, P = 0·008; IgG2, P = 0·01; IgG3, P = 0·01, IgG4, P = 0·008) in onchocerciasis patients than in controls. Despite that OvAg-specific reactivity tended to diminish in onchocerciasis patients, no significant difference were observed between before and at 16 years p.i.t. with ivermectin. In onchocerciasis patients, cellular anergy and defective cytokine production reverses following initial ivermectin treatment of patients [ 17, 19, 33, 34], several proinflammatory and regulatory chemokines increase [ 35– 37] and eventually parasite-specific Th1-type cytokines become reactivated in patients negative for O. volvulus microfilariae [ 28]. As observed in the present study, in annually retreated patients Th1 (IFN-γ), Th2 (IL-5) as well as regulatory T cell (IL-10) cytokine responses approached but did not entirely attain the levels as observed in O. volvulus exposed but mf-free endemic controls. Also, the Th1 : Th2 (IFN-γ/IL-5) ratios of the cytokine production remained depressed in patients at 16 years p.i.t. when compared to the ratios in controls, which indicated that a Th1/Th2 imbalance still persisted after 16 years of repeated ivermectin treatment. The Th2 cytokine IL-5 and T regulatory cytokine IL-10 production in response to O. volvulus antigen and bacteria-derived SL-O diminished in ivermectin-treated patients to levels found in controls, but the release of IL-10 following mitogen stimulation (PHA) increased and approached the higher levels of responsiveness of endemic controls. Furthermore, production of IL-5 to O. volvulus antigen decreased significantly between 1 month and 12 months p.i.t., but production in response to mitogen PHA and bacteria-derived SL-O took longer to decrease. Such slower changes of IL-5 in response to PHA suggest that in patients a ‘residual predisposition’ for a Th2-type responsiveness may have persisted even though microfilaria of O. volvulus have largely been eliminated. Such a persistent and parasite non-specific IL-5 responsiveness may predispose onchocerciasis patients upon exposure to microbial pathogens or other parasites for a biased Th2-type immune response. Also, residual and potentially non-reversible hyporesponsiveness and unbalanced Th1/Th2 cytokine profiles may be due to an early-life or even in utero-exposure to O. volvulus infection, which would cause a tolerization to the parasite. The observed gradual increase of IL-10 to PHA suggests that regulatory processes in onchocerciasis patients may have gained in capacity and strength, and such patients may respond to ubiquitous challenges with an appropriately balanced and better-regulated T cell response. None the less, it should be kept in mind that a reduced capacity to release IL-5 may render patients more susceptible to O. volvulus infection, as IL-5 and eosinophil granulocytes mediate significant cytotoxic effects against infective third-stage larvae of O. volvulus [ 1]. The elevated number of eosinophil granulocytes and the distinctly higher O. volvulus-specific IgG subclass responses in onchocerciasis patients than in endemic controls may indicate a not yet fully resolved O. volvulus infection. While eosinophilia could have been caused by intestinal hookworm infections, which were indeed more prevalent in patients than in controls, the higher IgG isotype responses to O. volvulus antigens could signify that reinfection with infective third-stage larvae of O. volvulus had occurred in patients. Notably, the persistent IgG4 reactivity to O. volvulus antigens could specify a cryptic O. volvulus infection where microfilariae will not be detectable in subcutaneous tissues due to repeated ivermectin treatment. One further cause for a persisting O. volvulus-specific IgG1 and IgG4 reactivity may be a continuous and uncontrollable low level transmission of infective third-stage larvae of O. volvulus, as indeed present in the study area [ 13]. Since the beginning of the black fly control measures, as applied by the OCP, the transmission of infective third-stage larvae (L3) of O. volvulus has never been interrupted completely in central and northern Togo, and even after ivermectin mass distribution a continuous transmission of L3 of O. volvulus has been observed in the area of our study during epidemiological surveys [ 13]. Therefore, patients could have been re-exposed to infective third-stage larvae, may harbour developing or even mature adult O. volvulus, and due to repeated ivermectin therapy they will remain microfilaria-negative. While ivermectin has no sterilizing or toxic effects on adult O. volvulus [ 16], it eliminates and suppresses microfilariae in the skin of patients for months, and also blocks the release of microfilariae from gravid female O. volvulus.The unbalanced cytokine responses in onchocerciasis patients, despite O. volvulus microfilaria clearance, may have been caused by either ‘cryptic’ O. volvulus infection or by an additional persistent infectious challenge. The 16-year ivermectin therapy is expected to exceed the reproductive life span and natural life expectancy of adult O. volvulus [ 1, 2, 38, 39], and unless re-exposed and reinfected individuals should no longer present viable adult worms of O. volvulus. However, more than 70% of the formerly O. volvulus microfilariae-positive patients presented with concurrent necatoriasis and mansonelliasis. In onchocerciasis patients with concurrent mansonelliasis cellular proliferation to O. volvulus was suppressed [ 27], and as observed in the present study, depressed Th1-type cytokine responses in patients concurrently positive for the lymphatic filaria M. perstans, disclosed that parasite co-infections may bias and prevent the development of a fully re-established and appropriately balanced Th1 and Th2 immune responsiveness in O. volvulus microfilariae-negative individuals. Even though onchocerciasis patients and endemic controls were from rural villages borderline to rivers where the risk of infection with O. volvulus still remains [ 3], a differing exposure to infective third-stage larvae of O. volvulus between controls and patients may have had consequences on the type of immune response observed to parasite antigen. Also, past infection versus no past infection may act on the expression and the type of immune response observed in onchocerciasis patients and endemic controls. Furthermore, our comparison between infection-free endemic controls and onchocerciasis patients may still contain confounding variables, such as geographical location, nutrition, socio-economic level, etc. and as there are no data on these confounding parameters available, it could be that the results on different levels of cytokine responses may not be due causally to hookworm and M. perstans infection. With the currently available tools complete control or elimination of onchocerciasis in the near future may be achievable only with high rates of treatment coverage (> 85%) of the population with ivermectin being administered twice annually. The possible development of ivermectin drug resistance in O. volvulus, as observed in other parasitic nematode species [ 40, 41], or unresponsiveness to treatment, as recently found in onchocerciasis [ 42, 43], is a further concern as to whether global elimination of onchocerciasis is achievable. Therefore, new filaricidal chemotherapy with long-lasting efficacy is needed and the recently discovered depletion of Wolbachia endosymbionts in adult O. volvulus by tetracycline antibiotics [ 44, 45], which will temporarily sterilize female O. volvulus, may open up new avenues of control. There appears no obstacle to the continuation of mass treatment with ivermectin [ 14, 23, 46], as there is no macrofiliricidal drug available for mass treatment distribution ivermectin will need to be distributed for decades in order to prevent microfilaria repopulation of dermal, ocular tissues, disease progression in O. volvulus exposed and infected individuals, and also to alleviate the clinical manifestations of onchocerciasis. From our data and the present expression of the cytokine profile it may be concluded that long-term ivermectin therapy of onchocerciasis patients may not suffice to fully re-establish an appropriately balanced Th1 and Th2 immune responsiveness in O. volvulus microfilariae-negative individuals. Today, it remains unknown whether annual ivermectin treatment of endemic populations may change their susceptibility to infection upon primary exposure or drug removal, as suggested by experimental studies in ivermectin-treated cattle re-exposed to O. ochengi infection after drug withdrawal [ 24]. There is experimental and epidemiological evidence that protective immunity exists naturally, both in humans [ 1, 20] as well as in experimental filarial infection studies [ 25], and that ivermectin will attenuate patent infections in those challenged with L3 of O. volvulus and may boost the development of appropriate immunity. Therefore, onchocerciasis patients receiving singly ivermectin, or in co-administration with other anti-helminthics, should be examined in order to identify those factors which are responsible for the persisting immune response bias in patients despite repeated ivermectin therapy. For this, concurrent parasite infections should be considered and their impact on expression and strength of specific immunity taken into account. This study was supported and authorized by the Togolese Ministry of Health. This work was supported by the Commission of the European Community (CEC-TS*-CT92-0057 and FP6-INCO-SCOOTT contract no. 032321), the Edna McConnell Clark Foundation (grant 13694), the UNDP/World Bank/WHO-TDR (ID 930543), Professor Dr P. Stingl Afrika Fonds and the DFG (So 367/1–2). D. M. Hamm was supported by the Reinhold-und-Maria-Teufel-Stiftung. 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