Characterization of native pathogenic antigens of Onchocerca volvulus: identification of high molecular mass protein antigens eliciting interstitial keratitis in a guinea pig model.

Sclerosing keratitis is the predominant cause of blindness due to onchocerciasis which is a major human parasitic disease caused by the filarial parasite Onchocerca volvulus. In the present investigation, native pathogenic antigens of O. volvulus which are particularly potent in causing interstitial keratitis were characterized utilizing a guinea pig model. Following demonstration of the protein nature of these antigens using pronase digestion, the crude O. volvulus antigen extract was subjected to stepwise procedures of protein purification. At each stage of purification, pooled antigen fractions were injected into one cornea of presensitized guinea pigs followed by clinical evaluation of stromal inflammation and vascularization at different intervals of time after intrastromal challenge. Initial purification of the pathogenic antigens was carried out in the following order: molecular sieve chromatography on Bio-gel A-5m. anion exchange chromatography on Mono Q followed by DEAE-Sepharose CL-6B and cation exchange chromatography on Mono S. Two out of six different pools from the Mono S column (pool a eluted unbound at 10 mM-NaCl and pool e eluted between 130 mM and 475 mM-NaCl) were found to be most pathogenic. Further purification of Mono S pool a and pool e separately by gel filtration chromatography using Superose 12 demonstrated that the fractions which were most potent in inducing interstitial keratitis contained proteins with approximate molecular masses between 100 and 200 kDa. These results show that minor subfractions of total crude antigens of O. volvulus are largely responsible for induction of experimental interstitial keratitis. We have demonstrated the presence of these antigens in O. volvulus microfilariae by their cross-reactivities with anti-microfilarial antibodies, and hence the relevance of the purified antigens to ocular onchocerciasis in man since sclerosing keratitis is associated with invasion of the cornea by O. volvulus microfilariae. Isolation of these two pathogenic antigen pools represents the practical limits of purification and subsequent animal experiments possible with the available amounts of native parasite material obtained from infected human individuals in the absence of a suitable non-human host or of an in vitro culture system for O. volvulus.

Infiltration of CD4+ T cells into cornea during development of Onchocerca volvulus-induced experimental sclerosing keratitis in mice.

Sclerosing keratitis is the major cause of blindness due to onchocerciasis caused by the parasite Onchocerca volvulus. Although the importance of T cells in the pathogenesis of onchocerciasis has been suggested, their precise role in onchocercal sclerosing keratitis has not yet been defined. Using immunohistological techniques and a murine model of onchocercal sclerosing keratitis, we have performed a temporal analysis of the inflammatory T cells infiltrating into the cornea at Days 4, 7, and 21 following intrastromal challenge with soluble O. volvulus antigens into presensitized mice. The maximum number of CD3+ T cells were observed in the corneal stroma at Day 21 when sclerosing keratitis was most severe. The majority (> 85%) of the CD3+ T cells were CD4+ at all time points. A few infiltrating cells bore IL-2 receptors indicating possible activation of a small fraction of the T cells. These results suggest that CD4+ T cells play an important role in onchocercal sclerosing keratitis.

Prevalence of hypertension in rural west Africa.

The prevalence of hypertension in rural African communities has been reported previously to be relatively low, with prevalence rates ranging between 2.5% and 10.9%. The authors obtained blood pressure recordings in 3,588 adult residents (age > 20 years) of the Liberian Agricultural Company rubber plantation, located in rural Liberia, West Africa. Blood pressures were obtained using automated digital blood pressure machines in a house-to-house survey. Individuals with a systolic blood pressure greater than 139 mm Hg or a diastolic blood pressure greater than 89 mm Hg were referred to a central site, where three subsequent blood pressure determinations were made, the mean of which was used to determine whether people were hypertensive. The overall prevalence of hypertension (blood pressure > 139/89 mm Hg) was 12.5%. Hypertension was most common in the older women (prevalence 5.7% in women age 20-24 years old, 50% in women older than 55). Using more stringent criteria for hypertension, systolic blood pressure greater than 159 mm Hg or diastolic blood pressure greater than 94 mm Hg, 20% of women between 50-54 had hypertension and 26% of the women older than 55 had hypertension. In some ethnic groups, the prevalence of hypertension was as high as 25%. Based on these results, the prevalence of hypertension in some rural African communities may be higher than reported previously, particularly in older women and among certain ethnic groups, possibly suggesting a strong hereditary component.

The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase.

Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a cDNA encoding the PDI homologue of Onchocerca volvulus is described. This cDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.

Immune-mediated Onchocerca volvulus sclerosing keratitis in the mouse.

Sclerosing keratitis is the major cause of blindness due to onchocerciasis; its pathogenesis is poorly understood. We have previously reported an immune-mediated model of experimental interstitial keratitis in guinea pigs following intrastromal challenge with soluble antigens from Onchocerca volvulus. This model system is ideal for evaluation of pathogenicity of multiple purified antigen preparations; however, reagents necessary for detailed immunologic analysis of the inflammatory cellular infiltrate are not yet available for guinea pigs. Because of the ready availability of these reagents for mice, a mouse model has been developed. The inflammatory response observed in this model was analogous to that seen in human onchocercal sclerosing keratitis as well as in the guinea pig model of onchocercal sclerosing keratitis. Granulocytes were present in the acute inflammatory response, whereas the chronic response showed lymphocytes, plasma cells, and histiocytes. Neovascularization and scarring of the corneal stroma was also observed. This model will be helpful in examining the mechanisms of immunopathogenesis and the contribution of the host genetic background to the disease.

Experimental onchocerciasis in chimpanzees: cellular responses and antigen recognition after immunization and challenge with Onchocerca volvulus infective third-stage larvae.

Immunization of chimpanzees with radiation-attenuated infective 3rd-stage larvae (L3) of Onchocerca volvulus did not induce strong protective immunity against a subsequent challenge infection; only 1 out of 4 immunized animals remained non-patent (i.e. microfilariae-negative) after challenge, and may have been protected. However, during immunization and before challenge, a broad range of adult O. volvulus-derived antigens (OvAg) and also uterus-derived OvAg were recognized by circulating antibodies; moreover, the repertory of antigens recognized increased further in subsequently patent animals after challenge, particularly in the range of M(r) 12-42 kDa. In the immunized and non-patent chimpanzee, by contrast, serological recognition of uterus-derived OvAg with M(r) 14 kDa and 105 kDa disappeared by 19 months post-challenge (p.c.). During immunization, Acanthocheilonema viteae L3 antigens of M(r) 11-12 kDa were strongly recognized only by the non-patent animal, suggesting that recognition of these antigens may have supported resistance to the subsequent challenge infection. In immunized chimpanzees, a substantial increase in the cellular reactivity to OvAg was induced; this, however, declined by 19 months p.c. to levels similar to those seen prior to immunization. At that time, 3 out of 4 immunized animals were patently infected. The effect of exogenous cytokines on in vitro-reactivity of PBMC to OvAg was examined. Addition of exogenous IL-2 alone, IFN-gamma alone, and IFN-gamma in combination with IL-2, did not augment net cellular responses to OvAg by PBMC from infected and control chimpanzees. In the presence of IL-4 alone, IL-6 alone, IL-2 with IL-4, IL-2 with IL-4 and IFN-gamma, or IL-2 with IL-4 and IL-6, the net cellular reactivity to OvAg increased significantly in patent chimpanzees and reached levels similar to non-patent animals. Thus, non-patent chimpanzees maintain high cellular reactivity to OvAg and in vitro cellular unresponsiveness to OvAg on the part of patent chimpanzees is reversible after addition of several cytokines which act individually or synergistically.

Human infection by genetically diverse SIVSM-related HIV-2 in west Africa.

Our understanding of the biology and origins of human immunodeficiency virus type 2 (HIV-2) derives from studies of cultured isolates from urban populations experiencing epidemic infection and disease. To test the hypothesis that such isolates might represent only a subset of a larger, genetically more diverse group of viruses, we used nested polymerase chain reactions to characterize HIV-2 sequences in uncultured mononuclear blood cells of two healthy Liberian agricultural workers, from whom virus isolation was repeatedly unsuccessful, and from a culture-positive symptomatic urban dweller. Analysis of pol, env and long terminal repeat regions revealed the presence of three highly divergent HIV-2 strains, one of which (from one of the healthy subjects) was significantly more closely related to simian immunodeficiency viruses infecting sooty mangabeys and rhesus macaques (SIVSM/SIVMAC) than to any virus of human derivation. This subject also harboured multiply defective viral genotypes that resulted from hypermutation of G to A bases. Our results indicate that HIV-2, SIVSM and SIVMAC comprise a single, highly diverse group of lentiviruses which cannot be separated into distinct phylogenetic lineages according to species of origin.

Modern medicine versus an ancient scourge: progress toward control of onchocerciasis.

Control of human onchocerciasis has been problematic. Vector control is practical and effective in only a circumscribed region of West Africa. Even in this area, control depends on meticulous monitoring of the vector and associated parasite species and precise application of requisite larvicide on a regular basis. As an alternative to vector control, chemotherapy-based control has been revolutionized by the finding that ivermectin, a novel semisynthetic macrocyclic lactone, is acceptable for mass distribution at the community level and that it represents an effective means of disease control when given once yearly. Efforts are underway to develop a vaccine to prevent infection.

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.

Characterization of the autoantigen calreticulin.

Anti-Ro/SS-A antibodies are commonly found in the sera of patients with Sjögren's syndrome and SLE. These antibodies also occur in the mothers of children with neonatal lupus and congenital heart block. Ro/SS-A is a ribonucleoprotein complex whose cellular function remains unknown. To study its cellular function and to characterize its immunoreactivity, we have used an oligonucleotide designed after the published amino terminal sequence of a putative 60-kDa Ro/SS-A autoantigen to isolate its cDNA. This cDNA encodes a polypeptide that is the human homologue of calreticulin, a calcium binding protein of the endoplasmatic reticulum. The encoded polypeptide also shows a 64.4% identity with RAL-1, an Ag of the river blindness pathogen Onchocerca volvulus. Contrary to the data published by other authors, our results indicate that calreticulin is not a Ro/SS-A autoantigen. Moreover, we show that anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis.

Experimental onchocerciasis in chimpanzees. Cell-mediated immune responses, and production and effects of IL-1 and IL-2 with Onchocerca volvulus infection.

Nine of eighteen chimpanzees inoculated with infective third-stage larvae of Onchocerca volvulus developed patent infection with microfilariae in skin biopsies. In all infected chimpanzees the in vitro cellular reactivity to O. volvulus adult worm-derived Ag (OvAg) increased significantly after exposure to third-stage larvae. However, during prepatency the in vitro cellular responses to OvAg decreased gradually in subsequently mf positive (patent) animals, and returned with patency to values not different to those before infection. In non-patent chimpanzees cellular responses remained significantly higher than before infection. Stimulation of PBMC in vitro with bacterial Ag and mitogen did not show any differences between the experimental groups through 20 months p.i. The addition of exogenous IL-2 did not restore the impaired responses of PBMC to OvAg in patent animals. Exogenous IL-2 elicited an additive increase of the cellular response to OvAg in nonpatent, and a mitogenic effect to OvAg in patent animals. Selective depletion of adherent, suppressor/cytotoxic (CD8+), NK cells (CD16+) and the use of autologous serum had no effect on antigenic and mitogenic cellular responsiveness. OvAg-induced IL-2 production decreased after patency, whereas, IL-1 production was significantly greater in both patent and nonpatent than in control chimpanzees. In summary, these data demonstrate that experimental O. volvulus infection in chimpanzees stimulated a substantial cell-mediated immune response. In patent chimpanzees an OvAg-specific cellular hyporesponsiveness occurred before onset of patency, possibly due to decreased IL-2 production and responsiveness.

Improvement in severe onchocercal skin disease after a single dose of ivermectin.

PURPOSE: Skin disease is the most common clinically important manifestation of onchocerciasis. Ivermectin, a newly available drug, is well tolerated and effective in Onchocerca volvulus infection. However, little information is available regarding its effect on onchocercal skin disease. The purpose of this study was to examine, in patients with well-characterized onchodermatitis, the effect of a single dose of ivermectin. SUBJECTS AND METHODS: Twenty-one persons with severe onchodermatitis were followed over a 6-month period. In order to evaluate the effect of ivermectin on their skin lesions, photographic transparencies were made before treatment and at 3 and 6 months after treatment. These were then evaluated in a blinded fashion. RESULTS: Following a single dose of 150 micrograms/kg, there was a significant improvement in dermatitis in the first 3 months after treatment. All 14 persons with the worst skin disease showed improvement. The drug had no demonstrable effect on depigmented lesions over the period of observation. Treatment was well tolerated. CONCLUSION: Single-dose ivermectin shows promise as the first acceptable treatment for severe onchocercal dermatitis.

Ocular manifestations of onchocerciasis in a rain forest area of west Africa.

The epidemiology and natural history of onchocerciasis and its ocular complications in rain forest areas are poorly understood. The present study was conducted on a rubber plantation in a hyperendemic area in the rain forest of Liberia, West Africa, where 800 persons were examined. The prevalence of infection was 84% overall 29% had intraocular microfilariae, and 2.4% were blind in one or both eyes. Onchocerciasis was the cause of all binocular blindness and one-third of all visual impairment. Over half of the visual impairment caused by onchocerciasis was due to posterior segment diseases. Chorioretinal changes were present in 75% of people, and included intraretinal pigment clumping in 52% and retinal pigment epithelium atrophy in 32%. Atrophy of the retinal pigment epithelium was associated with increasing age and severity of infection. Intraretinal pigment was strongly associated with anterior uveitis. There was a strong correlation between uveitis and the inflammatory chorioretinal sequelae: retinitis, intraretinal pigment, subretinal fibrosis, and optic neuropathy. These findings indicate that considerable visual impairment associated with rain forest onchocerciasis is common and is due largely to chorioretinal disease.

A comparison of 6-, 12-, and 24-monthly dosing with ivermectin for treatment of onchocerciasis.

This study was designed to examine the optimal dose and interval of administration of ivermectin, the now-accepted drug of choice for onchocerciasis. Two hundred Liberians with Onchocerca volvulus infection received 100, 150, or 200 micrograms/kg ivermectin or placebo and were followed for 36 months. The reaction after the second dose of ivermectin was significantly less than after the initial dose, although it was still significant in the 200-micrograms/kg group. The skin microfilaria counts in the group treated 6-monthly with 150 micrograms/kg was significantly less than in the group treated yearly (12 and 24 months after initial therapy). Prevalence of microfilariae in the anterior chamber and punctate corneal opacities decreased progressively in all groups over 3 years. There appears to be a slight advantage, in terms of antiparasitic effect over the first 2 years, of therapy given 6-monthly compared with yearly.

Community-based treatment of onchocerciasis with ivermectin: safety, efficacy, and acceptability of yearly treatment.

The safety, acceptability, and efficacy of ivermectin during community-based mass treatment of onchocerciasis was assessed. Ivermectin was distributed three times, 1 year apart, to the population of a rubber plantation (14,000 people) in Liberia where greater than 80% of adults have Onchocerca volvulus infection. In a sample of adults, the microfilarial density was reduced by 84% after 2 years of treatment. Acceptance was as high as 98% during the third year. Adverse reactions occurred in 6-13 in 1000 people after first treatment and in only 3-4 in 1000 receiving their second or third annual dose of ivermectin; severe adverse reactions were not seen. These data show that community-based treatment with ivermectin is safe, well accepted, and effective in reducing microfilarial loads and suggest that ivermectin is the first practical drug suitable for mass treatment campaigns to control human onchocerciasis.

Viability of adult Onchocerca volvulus after six 2-weekly doses of ivermectin.

Ivermectin is a safe, effective microfilaricide and microfilarial suppressant for Onchocerca volvulus; but in single doses of 100-200 micrograms/kg body weight it has no macrofilaricidal action. The present trial aimed to determine whether 6 doses of 100 micrograms/kg ivermectin, given at 2-week intervals, would kill the adult worms. Eighty-two nodules from 28 otherwise healthy adult male Liberian patients treated with this ivermectin schedule, and 102 nodules from a similar group of 25 control patients, were removed four months after the last dose of ivermectin. They were coded and assessed in a masked fashion either by routine histology or by examination of whole worms extracted from the nodules after collagenase digestion. The drug had no visible effect on adult male worms. More adult female worms were assessed as moribund or dead in the ivermectin-treated group than in the control group (for the collagenase digests P = 0.09; for the histological assessment P = 0.47). The data suggest that repeated dosage with ivermectin may lead to a slow attrition of some female worms and this possibility should be investigated in patients receiving regular doses every 3, 6 or 12 months as part of onchocerciasis control programmes.