Early Human Infection with Onchocerca volvulus Is Associated with an ...

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Philip J. Cooper,1,a Tamara Mancero,2 Mauricio Espinel,2 ..... 92:153–6. 13. Rodriguez-Perez MA, Danis-Lozano R, Rodriguez MH, Bradley JE. Com- parison of ...
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Early Human Infection with Onchocerca volvulus Is Associated with an Enhanced Parasite-Specific Cellular Immune Response Philip J. Cooper,1,a Tamara Mancero,2 Mauricio Espinel,2 Carlos Sandoval,2 Raquel Lovato,2 Ronald H. Guderian,2 and Thomas B. Nutman1

1

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; 2Department of Clinical Investigations, Hospital Vozandes, Quito, Ecuador

The immune response after early exposure to or infection with Onchocerca volvulus was investigated in an autochthonous focus caused by the migration of infected persons to a previously unaffected area in Ecuador. Peripheral blood mononuclear cell (PBMC) proliferative and cytokine responses (interferon [IFN]–g and interleukin [IL]–5) to filarial antigens were measured in 14 subjects with serologic evidence of exposure and in 7 subjects with evidence of dermal microfilarial DNA and were compared with responses in 43 subjects with chronic O. volvulus infections. PBMC proliferative and cytokine responses (IFN-g and IL-5) to parasite antigens were elevated in the early exposure/infection group, compared with those in the chronic infection group. Addition of an IL-10–neutralizing antibody to filaria antigen–stimulated cultures resulted in significantly elevated proliferative responses in the chronic infection group. The findings suggest that early exposure and early parasite patency are associated with a vigorous cellular response, but, as infections become chronic, the cellular response becomes down-regulated, partly through an IL-10–dependent mechanism.

Immunoepidemiologic studies of Onchocerca volvulus infection have identified differences in cellular immune responsiveness in persons living in endemic regions that depend on parasitologic status. Subjects with patent infection (e.g., microfilariae in the skin) tend to have a parasite-specific cellular immune response characterized by poor lymphocyte proliferation, impaired production of Th1 cytokines (interleukin [IL]–2 and interferon [IFN]–g), and prominent production of Th2 cytokines (IL-4, IL-5, and IL-10), whereas those without clinical or parasitologic evidence of infection, despite evidence of exposure to the parasite (e.g., O. volvulus–specific antibody response), have enhanced lymphocyte proliferation and production of both Th1 cytokines (IL-2 and IFN-g) [1–8] and Th2 cytokines (IL-5) [5, 6, 8]. The latter group has been termed putative immune (PI) or endemic normal (EN), and subjects in this group are assumed to have protective immunity to O. volvulus infection. These relatively distinct immune and parasitologic phenotypes are characteristic of adults in endemic regions and are Received 8 December 2000; revised 27 February 2001; electronically published 27 April 2001. Informed verbal consent was obtained from all subjects. Procedures were explained in the local language. The study followed protocols approved by the National Institutes of Health and Hospital Vozandes, Quito, Ecuador. Financial support: Edna McConnell Clark Foundation. a Present affiliation: Division of Infectious Diseases, St. George’s Hospital Medical School, London, United Kingdom. Reprints or correspondence: Dr. Philip J. Cooper, Division of Infectious Diseases, St. George’s Hospital Medical School, Cranmer Terrace, Tooting, London SW17 ORE, United Kingdom ([email protected]). The Journal of Infectious Diseases 2001; 183:1662–8 䉷 2001 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2001/18311-0015$02.00

thought to develop after long-term exposure to infection. Very little is known of the early immune response to O. volvulus. Most data derive from a few studies of infected subjects with a relatively short exposure history (e.g., expatriates) [9] or children living in endemic areas [1, 10]. The development of highly sensitive and specific polymerase chain reaction (PCR)–based assays has enabled the identification of low infection intensities [11] not detected by classical parasitologic methods and, at the same time, has provided a diagnostic tool to permit investigation of the parasite-specific cellular response near the time of microfilarial patency. We identified a group of subjects with evidence of recent exposure to infection with O. volvulus and early microfilarial patency after the migration of infected persons from an area of Ecuador in which the infection is highly endemic to a previously nonendemic area where vector species of black fly exist. We describe the phenotype of the parasite-specific cellular immune responses in this group and compare these with those in a comparable O. volvulus–infected group from the original endemic focus.

Subjects and Methods Study population and recruitment procedures. Infected subjects were recruited in communities of Chachi Amerindians along the Rio Cayapas in the Santiago River basin of Esmeraldas Province, Ecuador, where onchocerciasis is hyperendemic. Communities were screened for infected persons by using annually updated census data compiled by the Ecuadorian Onchocerciasis Control Programme. Healthy inhabitants ⭓5 years old were invited to enter the study. Subjects with suspected recent exposure to or infection with O. volvulus were recruited from 2 large communities of Tsachila Amerindians in Santo Domingo de Los Colorados in Pichincha Prov-

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ince. The establishment of a new focus of onchocerciasis was suspected in these communities because of in migration of Chachi Amerindians over the previous 10 years (1986–1996) and the presence of the vector species complex Simulium exiguum [12]. A survey to detect new infections among Tsachila Amerindians was done by using censuses compiled by the Ecuadorian Onchocerciasis Control Programme and Tsachila community leaders. O. volvulus infections were identified by using a combination of 3 methods: microscopic examination of skin snips for the presence of O. volvulus microfilariae; detection of O. volvulus DNA in skin snips by a highly sensitive and specific PCR-based assay [11]; and detection of O. volvulus–specific IgG antibodies from blood spots by use of a highly sensitive and specific ELISA [13]. The communities were revisited, and Tsachilas ⭓5 years old who had the following characteristics were recruited into the study: positive PCR assay and/or serologic responses for O. volvulus and no travel history to the Esmeraldas focus of onchocerciasis (i.e., infection was acquired locally). The studies were done in 1996 before the start of community-based ivermectin treatment of the study communities in Santo Domingo de Los Colorados and Rio Cayapas. Parasitologic examination. Skin snips were taken from both iliac crests and were examined for the presence of microfilariae after incubation in saline for 24 h. Skin snips negative for the presence of microfilariae were tested for the presence of O. volvulus DNA by a PCR-based assay, as described elsewhere [11]. Serologic examination. Capillary fingerprick blood was collected, and a blood spot was placed on Whatman no. 2 filter paper and was allowed to air dry before storage at ⫺20⬚C in sealed plastic bags containing silica gel. A 6-mm-diameter disk was punched from the blood spots and was analyzed for the presence of O. volvulus–specific IgG antibodies by a highly sensitive and specific ELISA, in which microtiter plates were coated with a cocktail of O. volvulus–recombinant antigens, as described elsewhere [13]. The blood spots were processed within 3 months of collection. Peripheral blood mononuclear cell (PBMC) proliferation assays. Thirty milliliters of blood was drawn form the antecubital vein into a syringe containing heparin, and PBMC were isolated and cultured, as described elsewhere [14]. PBMC were stimulated with filarial antigens at concentrations of 1–5 mg/mL, purified protein derivative of tuberculin at 10 mg/mL (PPD; Statens Serum Institute), and pokeweed mitogen (PWM; Life Technologies GIBCO BRL) at a dilution of 1:200. Filarial parasite antigens from adult O. volvulus worms (Ovag), O. volvulus L3 (OvL3) parasites, and Brugia malayi microfilariae (Bmmf) were prepared, as described elsewhere [2, 15, 16]. All stimulations were done in triplicate. For purposes of exogenous neutralization of cytokine and costimulatory molecule function, specific antibodies and appropriate isotype control antibodies were added to PBMC cultures stimulated with Ovag: rat anti–human IL-10 (10 mg/mL; PharMingen), polyclonal rabbit anti–human transforming growth factor (TGF)–b (5 mg/mL; R&D Systems), and human CTLA4Ig fusion protein (15 mg/mL; provided by Peter Linsley, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle). Cytokine ELISA. Supernatant fluids were harvested from the PBMC cultures at 5 days. Cytokine ELISAs for IFN-g and IL-5 were done, as described elsewhere [17]. Cytokine levels are shown in picograms per milliliter as the absolute difference between protein levels in antigen-stimulated cultures and control medium cultures.

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Statistical analysis. Skin infection intensities are expressed as the geometric mean number of microfilariae per milligram of skin. Data variables for each group are expressed either as geometric means or as medians, in which data include negative values. We analyzed the effect of the addition of cytokine-neutralizing antibodies or CTLA4Ig on proliferative or cytokine responses, compared with control antibodies, by using the 1-sample sign test. Data variables between the study groups were compared by the MannWhitney U test.

Results Demographic and parasitologic details of study population. Of a total census population of 961 persons, 609 (63.4%) presented for sampling. The census population included 32 migrant Chachis, of whom 21 (65.6%) provided samples during the survey. The survey detected microscopically positive skin snips in 4 (geometric mean infection intensity, 2.1 microfilariae [mf]/mg; range, 0.5–7.5 mf/mg) of the 21 Chachis sampled (19.0% of Chachis and 0.7% of sample population). None of the Tsachila Amerindians screened had skin snips containing microfilariae by microscopic examination. The presence of O. volvulus DNA was detected in 50 subjects (8.2% of sample population; 43 Tsachilas and 7 Chachis) and positive serologic results in 144 subjects (23.6%; 130 Tsachilas and 14 Chachis). All subjects with positive skin snips had both a positive PCR reaction and positive serologic results, and all subjects with a positive PCR reaction had positive serologic results. Demographic and parasitologic details of study sample. From the surveyed study population, we recruited 21 Tsachila Indians (13 males and 8 females; mean age, 26 years; range, 6–65 years) with positive skin snips for O. volvulus by PCR (33% with positive reactions) or positive serologic responses (100% with positive tests). From the O. volvulus–hyperendemic communities, 43 subjects were recruited (25 males and 18 females; mean age, 38 years; range, 11–69 years), all of whom had positive skin snips for O. volvulus microfilariae both by microscopy (geometric mean, 16.9 mf/mg) and by PCR assay. Since there were no significant differences in any of the cellular immunologic parameters measured between subjects with serologic evidence of exposure alone and those with DNA evidence of dermal microfilariae, all subjects were combined into a single group (early exposure/infection) for the purpose of analysis. PBMC proliferation. There were no significant differences in PBMC proliferative responses between the 2 study groups to nonparasite antigen (PPD) or mitogen (PWM; table 1). Lymphocyte proliferative responses to OvL3 were modest and did not differ between the 2 groups (table 1). In contrast, significantly greater responses to Ovag (P ! .0001) and Bmmf (P ! .0001) were observed in the early exposure/infection group than in the chronic infection group. Cytokine-neutralizing antibodies and a CD28/B7 costimulation antagonist (or appropriate antibody controls) were added to Ovag-stimulated cultures, to determine the relative impor-

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Table 1. Peripheral blood mononuclear cell proliferation in response to mitogen, nonparasite antigens, and parasite antigens in the early exposure/infection and chronic Onchocerca volvulus infection groups. Group Early exposure/infection Chronic infection

PPD 10.1 (1.7–87.0) 5.9 (1.1–54.0)

PWM

Ovag

70.7 (16.0–167.9) 55.6 (16.0–198.8)

a

15.1 (1.6–44.9) 1.9 (0.8–8.5)

OvL3 2.0 (1.0–4.8) 1.7 (0.9–6.7)

Bmmf a

26.8 (6.8–63.9) 3.1 (1.3–9.6)

NOTE. Data are geometric mean stimulation indices (range). Bmmf, Brugia malayi microfiliariae; Ovag, O. volvulus adult worm antigen; OvL3, O. volvulus L3 antigen; PPD, purified protein derivative; PWM, pokeweed mitogen. a P ! .0001.

tance of each in modulating PBMC proliferative responses to parasite antigen. The results are shown in figure 1. Addition of neutralizing antibodies to IL-10 resulted in increased lymphocyte proliferative responses in the chronic infection group (geometric mean stimulation ratio, 1.4; range, 0.8–2.4; P ! .0001) but not in the early exposure/infection group (geometric mean stimulation ratio, 1.2; range, 0.5–2.2). Addition of TGFb–neutralizing antibodies did not significantly enhance lymphoproliferative responses in either the early exposure/infection (geometric mean stimulation ratio, 1.1; range, 0.6–1.6) or the chronic infection (geometric mean stimulation ratio, 1.3; range, 0.8–2.1) group; however, addition of CTLA4Ig had a significant inhibitory effect on proliferative responses in the early exposure/ infection group (geometric mean stimulation ratio, 0.2; range, 0.1–0.8; P ! .0001) but had no effect on responses in the chronic infection group (geometric mean stimulation ratio, 1.1; range, 0.7–2.3; figure 1). The differences in PBMC proliferative responses in CTLA4Ig-supplemented cultures between early exposure/infection and chronic infection groups were highly significant (P ! .0001). Cytokine responses to filarial antigen preparations. There were no significant differences in the production of IL-5 and IFN-g by PBMC in the infection groups stimulated with mitogen (PWM) or nonparasite antigen (PPD) (data not shown). There was negligible production of both IL-5 and IFN-g in response to OvL3 antigen in both groups (table 2). PBMC from the early exposure/infection group produced significantly more IL-5 (P p .0001) and IFN-g (P p .008) in response to Ovag than did the chronic infection group. Similarly, the early exposure/infection group produced more IL-5 (P p .001) in response to Bmmf antigen than did the chronic infection group. IFN-g production by Bmmf-stimulated PBMC also was greater in the early exposure/infection group, although this did not achieve statistical significance. Effect of IL-10 and TGF-b or costimulatory blockade on cytokine production. Addition of IL-10– or TGF-b–neutralizing antibodies to PBMC cultures stimulated with Ovag did not have a significant impact on the production of either IL-5 or IFN-g in either the early exposure/infection or the chronic infection group (figure 2). In the early exposure/infection group, addition of the CD28/B7 costimulatory antagonist CTLA4Ig to Ovag-stimulated cultures inhibited the production of both IL-5 (P ! .0001) and IFN-g (P p .01), compared with that in control cultures (figure 2). No effect of CTLA4Ig was observed in PBMC cultures from the chronic infection group.

Discussion We identified a group of subjects with relatively short-term exposure to O. volvulus transmission (e.g., !10 years) and with serologic evidence of exposure and evidence of microfiladermia detectable only by specific PCR, indicating early infections with very low parasite burdens. The establishment of autochthonous transmission of O. volvulus in a previously unaffected region of Pichincha Province, Ecuador, resulted from the migration and settlement of relatively few infected persons from the hyperendemic focus. Rapid transmission of O. volvulus probably was facilitated by the presence of the Cayapa cytotype of the vector, S. exiguum, in Santo Domingo de Los Colorados [12]. This is a highly efficient vector at low dermal microfilarial densities [18]. The migration was of infected indigenous Indians of the Chachi tribe to communities in Santo Domingo de Los Colorados, which are populated by the related Tsachila tribe. This recent 1-way migration has resulted from the ecologic and social disruption caused by extensive deforestation and immigration into the hyperendemic focus in Esmeraldas Province over the last 20 years and has led to the emigration of Chachis from the focus to other less affected regions of Esmeraldas Province. Although the number of Chachis included in the census populations of the 2 Tsachila communities was low, there also has been, over the same 10-year period, much larger temporary migration of Chachi families from the hyperendemic focus to the same communities to work on Tsachila fincas (plantations/ farms). The Chachi dispersion has established new potential foci in Esmeraldas Province, where human infection reservoirs and vector species of black fly coexist [12, 19], but this is the first satellite focus where autochthonous transmission has been demonstrated. We identified the autochthonous transmission in a new area by using 2 highly specific and sensitive diagnostic tools. The serologic assay, which uses a cocktail of 3 recombinant peptides, is very sensitive and specific for O. volvulus infection [20]. Although this assay is considerably more sensitive than standard diagnostic techniques (e.g., skin snips), it cannot differentiate between early exposure and active infections. The use of PCRbased assays to identify O. volvulus DNA in skin snips is much more sensitive than standard microscopy [11, 21], is species specific, and indicates the recent presence of O. volvulus microfilariae in the skin, since DNA will remain in the skin only for a few weeks after microfilaria death [22]. Because of the exten-

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Figure 1. Peripheral blood mononuclear cell proliferation in early exposure/infection (shaded boxes) and chronic Onchocerca volvulus infection (open boxes) groups in O. volvulus antigen–stimulated cultures supplemented with neutralizing antibodies (a) to interleukin (IL)–10, transforming growth factor (TGF)–b, and CTLA4Ig or appropriate controls. Y-axis variable is expressed as a stimulation ratio of cultures stimulated with adult O. volvulus antigen (Ovag) in the presence of cytokineneutralizing antibodies or CTLA4Ig, compared with cultures stimulated with Ovag supplemented with control antibodies. Box plots represent median values (central line), interquartile range (box margins), 95% confidence intervals (bars), and outlying values (circles). P value represents significant intergroup differences.

sive specificity testing performed on both assays [20], it is extremely unlikely that the results represent false positives. The Tsachila Amerindians with evidence of early exposure/ infection had markedly enhanced lymphoproliferative responses to both Ovag and Bmmf antigen, compared with those in a comparable group of infected control subjects. Likewise, production of the cytokines IFN-g and IL-5 by PBMC cultures stimulated with adult and microfilarial antigens was greater in the early exposure/infection group. Furthermore, there were no

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differences in the cellular responses of persons with serologic evidence of infection alone and of those with DNA evidence of dermal microfilariae. These findings indicate that early exposure to infection is associated with a pronounced cellular response characterized by the production of both Th1 and Th2 cytokines and that the magnitude of these responses is not altered by the presence of small numbers of O. volvulus microfilariae in the skin, but that, as infections become more chronic and parasite burdens greater, these responses may be actively down-regulated. The cellular responses described for the early exposure/infection group do not differ qualitatively from those described for EN/PI persons from the Ecuador onchocerciasis focus [4] and from other endemic regions [8, 23]. There appears to be significant heterogeneity in cytokine responses within PI/EN study groups; some studies suggest enhanced production of IFN-g [3–5], whereas others indicate that IL-5 [5, 6, 8, 23, 24] production by PBMC stimulated with adult-derived O. volvulus antigen is enhanced in EN/PI persons, compared with that in infected control subjects. Such differences may be interpreted to suggest that both of these cytokines contribute to protective immunity against O. volvulus infection. Evidence from experimental murine infections with Onchocerca microfilariae or L3 larvae would support this [25–27]. The similarities in cellular immune responsiveness between early exposure/infection and EN/PI study groups indicate that there probably is considerable overlap between these groups. Of interest, experimental infections of chimpanzees with O. volvulus showed that PBMC proliferated well to parasite antigen at the start of infection but declined with the onset of patency [28]. The chimpanzee model seems to parallel the findings in human populations and would support the assumption that early exposure and very light infections (i.e., not detectable by standard parasitologic methods) induce a similar cellular immune response in the host that becomes down-modulated as infections become patent and parasite burdens increase. Chronic infections with O. volvulus are associated with parasite-specific cellular hyporesponsiveness. A number of mechanisms have been proposed to explain this phenomenon, including the induction of prenatal and neonatal tolerance [29, 30] and the enhanced secretion of immunosuppressive cytokines. The observation that cellular hyporesponsiveness is reversible would favor the latter hypothesis—cellular reactivity is restored after clearance of microfilariae with chemotherapy [3]. IL-10 and TGF-b may be important as the modulators of the immune response to O. volvulus, since elevated levels of IL10 have been reported in patent human infections [4, 5, 8], and neutralization of IL-10 [7, 8] and/or TGF-b [8] in vitro can partially reverse impaired cellular reactivity in a proportion of affected persons. Likewise, in this study, we demonstrated that IL-10 neutralization resulted in significantly enhanced lymphoproliferation in the chronically infected group but not in persons with relatively early exposure/infection; however, although IL-

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Table 2. Production of interferon (IFN)–g and interleukin (IL)–5 by peripheral blood mononuclear cells (PBMC) stimulated with parasite antigens in the early exposure/infection and chronic Onchocerca volvulus infection groups. Ovag Group Early exposure/infection Chronic infection

OvL3

IFN-g a

97 (0–1173) 6 (0–1040)

IL-5 a

61 (0–15.2) 5 (0–610)

IFN-g 2 (0–473) 8 (0–8437)

Bmmf IL-5

0 (0–26) 2 (0–145)

IFN-g 54 (0–2937) 10 (0–3115)

IL-5 a

146 (29–617) 2 (0–328)

NOTE. Data are geometric mean cytokine levels in pg/mL (range). PBMC were stimulated with adult O. volvulus antigen (Ovag), O. volvulus L3 antigen (OvL3), and Brugia malayi microfilarial antigen (Bmmf). a P p .001.

10 neutralization lacked a significant effect on lymphocyte proliferation in the early exposure/infection group as a whole, there was some evidence of an enhancement of proliferative responses in this group, which indicates that IL-10–mediated suppression may occur even at an early stage of infection. Neutralization of TGF-b had less effect but did enhance lymphocyte reactivity in some subjects in both study groups (figure 1). Induction of cellular hyporeactivity to parasite antigens probably occurs gradually after microfilarial patency and an increase in the parasite burden and may serve to prevent potentially damaging inflammatory responses in the host. The development of cellular hyporesponsiveness is probably determined by both the duration of infection and the parasite burden, both of which are a function of the intensity of transmission. In areas of intense transmission, infections are acquired at a young age, and higher parasite burdens develop; under such circumstances, cellular hyporesponsiveness would be expected to develop earlier in the course of infection. Microfilaricidal chemotherapy with ivermectin is capable of reversing cellular hyporesponsiveness [31] through the immediate reduction of the larval parasite load. This effect is temporary [31], and cellular hyporesponsiveness returns as microfilariae repopulate the skin after treatment; however, immune responsiveness may be restored in the long term with repeated treatments [3]. Likewise, in areas where transmission has been interrupted (e.g., by larvicidal spraying), cellular responsiveness would be expected to be restored over a period of years as adult parasites die through natural attrition, resulting in declining microfilarial burdens. Costimulatory signals are required, in addition to signals through the T cell receptor, for the development of cytokineproducing T helper effector cells. CD28 is a potent costimulatory molecule on T cells that binds B7-1 and B7-2 on professional antigen-presenting cells. CTLA4Ig is a chimeric fusion protein, comprising the extracellular domain of CTLA4 linked to immunoglobulin, that can inhibit the interaction of both CD28 and CTLA4 with B7 molecules. In this study, the antagonism of B7CD28/CTLA4 interactions with CTLA4Ig markedly impaired lymphocyte proliferation and cytokine production in the early exposure/infection group but not in the chronic infection control group. These results indicate the importance of CD28/CTLA4 costimulation in the parasite-specific adaptive immune response. Although there is evidence for suppression of B7 expression on antigen-presenting cells in subjects with chronic helminth infec-

tions [7, 32], the differential effects of B7-CD28/CTLA4 blockade between the 2 study groups probably are a reflection of the differences in magnitude of intergroup PBMC proliferative and cytokine responses rather than of a difference in the relative

Figure 2. Production of interferon (IFN)–g and interleukin (IL)–5 by peripheral blood mononuclear cells in early exposure/infection (shaded boxes) and chronic Onchocerca volvulus infection (open boxes) groups in O. volvulus antigen–stimulated cultures supplemented with neutralizing antibodies (a) to IL-10, transforming growth factor (TGF)–b, and CTLA4Ig or appropriate controls. Shown are median values of absolute differences in cytokine production between cultures stimulated with adult O. volvulus antigen (Ovag) in the presence of cytokine-neutralizing antibodies or CTLA4Ig and cultures stimulated with Ovag supplemented with control antibodies. Box plots represent median values (central line), interquartile range (box margins), 95% confidence intervals (bars), and outlying values (circles). P values represent significant intergroup differences.

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importance of B7-CD28/CTLA4 costimulation in the parasitespecific cellular response between the 2 groups. In conclusion, we identified communities in Pichincha Province, Ecuador, where O. volvulus transmission has been initiated within the past 10 years after the migration of a small group of infected persons from a known endemic focus. We identified a group of subjects within these communities with serologic evidence of exposure to O. volvulus transmission and some with evidence of O. volvulus DNA in skin snips, which indicates early parasite patency. There were no differences in the magnitude of the proliferative and cytokine response to parasite antigens between these 2 groups. This mixed group demonstrated highly reactive cellular responses to both adult and microfilarial antigen preparations that were characterized by enhanced lymphocyte proliferation and the production of both Th1 (IFN-g) and Th2 (IL-5) cytokines, compared with that in an infected control group from the original endemic focus. Furthermore, in contrast to the infected control group, the early exposure/infection group did not demonstrate significant inhibition of lymphocyte reactivity induced by IL-10. These findings indicate that early exposure to and infection with O. volvulus are associated with activation of a mixed cytokine response and that the characteristic parasite-specific cellular hyporesponsiveness associated with patent infections probably is likely to develop gradually as infections become more chronic and parasite burdens increase.

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Acknowledgments 17.

We thank the study communities for their cooperation; Kenneth Farr (US Agency for International Development, Quito) for assistance; and Brenda Rae Marshall for help in preparing the manuscript.

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