Humoral responses to a secretory Onchocerca volvulus protein: differences in the pattern of antibody isotypes to recombinant Ov20/OvS1 in generalized and hyperreactive onchocerciasis.

The Onchocerca volvulus secretory protein Ov20/OvS1 represents a dominant antigen expressed in the infective larvae, microfilariae and adult stages of the parasite. The humoral responses to this protein have not yet been analysed in the polar clinical and immunological forms of onchocerciasis. Analysis by ELISA of class and subclass antibodies to Ov20/OvS1 in persons with the generalized or the hyperreactive form of onchocerciasis revealed similar strong responses of IgG1, IgG4 and IgM antibody levels in both forms of onchocerciasis and significant differences were observed in the IgE and IgA antibody classes. Computation of the ratios of antibodies showed that persons with the generalized form exhibited significantly higher ratios of IgG4 to IgG1, IgG4 to IgE, and IgM to IgE than patients with the hyperreactive form. To investigate the isotype recognition of antigenic sites on Ov20/OvS1 protein, three recombinantly expressed fragments (F1-3) of Ov20/OvS1 were probed using sera which strongly reacted with intact recombinant Ov20/OvS1. Epitope(s) on F1 comprising amino acid residues 1-63 were significantly recognized by IgG1 and IgE, while IgM recognized epitopes on all three fragments. The strongest reaction of IgM occurred with epitope(s) formed by residues 108-171 (F3). In contrast, IgG4 type antibodies were not reactive with either of the three OvS1 fragments, but they reacted with intact Ov20/OvS1 protein. Generalized onchocerciasis, unable to eliminate microfilariae, and hyperreactive onchocerciasis, with a high potency to eliminate or to reduce parasite loads, can be distinguished by a distinct pattern of isotype responses to Ov20/OvS1.

The secretory Onchocerca volvulus protein OvS1/Ov20 exhibits the capacity to compete with serum albumin for the host's long-chain fatty acids.

The mechanism by which filarial parasites derive fatty acids bound to the host's carrier protein is poorly understood. The capacity of a secretory protein of Onchocerca volvulus (OvS1/Ov20) to compete with serum albumin for arachidonic and other fatty acids was investigated in this study. Binding affinities of the two proteins for the long-chain fatty acids were determined using displacement assays. The fluorescent probes used included 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino) undecanoic acid (DAUDA) and cis-parinaric acid. OvS1 protein bound arachidonic acid with an affinity five-fold greater than the affinity exhibited by serum albumin. Oleic acid was bound by the parasite protein with an affinity two-fold greater than the affinity shown by serum albumin. Furthermore, the affinities exhibited by OvS1 protein in binding arachidonic and linoleic acid were about two times higher than the affinity for oleic acid. The results suggest that the OvS1 protein has the capacity to compete with the main host's fatty acid carrier protein for the long-chain fatty acids, in particular arachidonic acid, the precursor for eicosanoids.

Use of the recombinant Onchocerca volvulus protein Ov20/OvS1 for the immunodiagnostic differentiation between onchocerciasis and mansonelliasis and for the characterization of hyperreactive onchocerciasis (sowda).

The protein Ov20/OvS1 was used as antigen in ELISA and Western blot in order to differentiate onchocerciasis from African mansonelliasis and to characterize the hyperreactive form of Onchocerca volvulus infection (sowda). The specificity of the IgG4 Western blot was 98% for the differentiation between persons with onchocerciasis and Mansonella microfilariae (mf) carriers (125 persons with M. perstans and 92 with M. streptocerca), whereas the IgG4 ELISA showed a specificity of 81% in 137 M. perstans mf carriers and 85% in 94 M. streptocerca mf carriers. The sensitivity of Ov20/OvS1 in identifying onchocerciasis using the IgG4 ELISA was 75% for 103 O. volvulus mf carriers with the generalized and 89% for 44 patients with the sowda form of onchocerciasis. IgE antibodies against OvS1 were found in 95% of 39 patients with hyperreactive onchocerciasis but only in 15% of 47 persons with the generalized form. Thus, Ov20/-OvS1 appears a promising candidate antigen for the diagnosis of onchocerciasis and in particular for the detection of the sowda type of disease.

HLA DRB1-DQA1-DQB1 haplotype diversity in two African populations.

HLA class II DRB1-DQA1-DQB1 haplotypic polymorphism was determined in 120 Liberian and 230 Gabonese individuals. In our study groups, the number of allelic variants observed for each locus was similar to that found in non-African populations. However, 39 novel haplotypes and several yet unrecognized DRB1-DQA1 and DQA1-DQB1 combinations were identified. The extent of HLA-haplotypic variability in Africans appears to result from the high degree of allele combinations rather than from allelic polymorphism.

Production of both IFN-gamma and IL-5 by Onchocerca volvulus S1 antigen-specific CD4+ T cells from putatively immune individuals.

Protective immunity to the parasitic nematode Onchocerca volvulus (Ov) appears to be directed against molecules of invading L3 larvae. In this study, the cellular immune reaction to such an Ov L3 protein (S1) which is protective in an animal model was analyzed using peripheral blood mononuclear cells (PBMC) of individuals from a hyperendemic area in West Africa who were exposed to Ov but remained free from disease ('putatively immune individuals'). Despite seronegativity of these individuals against S1, proliferation of PBMC was inducible, allowing generation of an S1-specific T cell line which produced IFN-gamma upon stimulation with both Ov lysate and S1. However, S1 induced significantly more IL-5 than Ov lysate. S1-specific, DQ6 (DQA1*0103/DQB1*0603)-restricted T cell clones were generated which reacted against synthetic peptides comprising amino acids 99-111 of S1. These clones, which are the first generated against a recombinant fllarial antigen, produced both IFN-gamma and IL-5 as well as little IL-4, suggestive of a Th0-like phenotype. In conclusion, in putative immunity, reactivity against a particular parasite protein can be detectable on the level of T but not B cells. Induction of both IFN-gamma and IL-5 by S1 suggests that it may trigger macrophage plus eosinophil dependent killing of L3 in vivo. The identification of a likely DQ6 (DQA1*0103/DQB1*0603)-restricted T cell epitope may be of more general relevance, given that allele combinations of DQ6, including DQA1*0103/DQB1*0603, are negatively associated with diabetes mellitus.

Immunohistological studies on an Onchocerca volvulus ankyrin (EI).

The distribution of an Onchocerca volvulus ankyrin, designated E1, was studied in different O. volvulus stages and other helminths by immunohistochemistry using rabbit antibodies raised against the recombinant E1 protein. In adult O. volvulus the protein designated E1 was localized to the extracellular clefts as well as to the cytoplasm adjacent to the cell membrane in the area of the basal labyrinth in hypodermis, intestine and uterus and to a lesser extent in oviduct and vas deferens. Neuronal cell bodies were also labelled. No labelling of the basal laminae, muscles or epithelia of ovary or testis was observed. Detection of the E1 protein was associated with embryonic development. Germ cells and early morulae showed no reaction; labelling was first seen in late morulae, corresponding to the stage of gastrulation, and increased in the following embryonic stages. In microfilariae the nerve ring and the cephalic space, which represents the anterior nerve-enriched portion of the body, were labelled. In third-stage larvae of O. volvulus labelling was associated with the hypodermis, and in those of Anisakis sp. the cytoplasm adjacent to the membrane of the excretory gland cell and the basal labyrinth of the hypodermis were labelled. Following anthelminthic treatment a disruption of the labelling pattern of the E1 protein was observed in adult O. volvulus with leakage of the protein into neighbouring areas. Damage to the worm was associated with reduction and finally loss of E1 protein labelling. No E1 protein was detected in dead adult worms, embryos or microfilariae. Labelling of the same organs was observed in 8 other Onchocerca species and in several other nematodes, but no reaction was seen in trematodes. The results indicate that the EI protein is associated with neuronal structures of O. volvulus, that its presence is developmentally regulated and that it has cross-reactive homologues in other nematodes. The results suggest that E1 is a functional protein. It may be useful for the assessment of parasite damage and death as well as in the characterization of the filarial nervous system.

Onchocerca volvulus: identification of cDNAs encoding a putative phosphatidyl-ethanolamine-binding protein and a putative partially processed mRNA precursor.

We have identified and sequenced cDNAs of the parasitic nematode Onchocerca volvulus which encode a homologue of phosphatidylethanolamine-binding proteins from mammals. These clones are also closely related to the O. volvulus Ov16 cDNA (Lobos et al., 1990). One identified cDNA clone appears to represent a partially processed precursor. These results suggest that these cDNAs are derived from a complex genomic locus, raising the possibility of polycistronic transcription in this nematode.

The putatively protective Onchocerca volvulus neuronal protein E1 is a member of the death domain protein family.

Here we show that E1, an ankyrin-related, potentially protective, neuronal protein of the human filarial nematode Onchocerca volvulus contains a death domain (DD), most similar to that of human Mort1/FADD (39% identity). In addition, sequence comparison of E1 to its homologue from Litomosoides sigmodontis and to Caenorhabditis elegans ankyrin defines two further putative functional domains. One represents the end of the spectrin-binding domain of ankyrins, the other an unique domain, most highly conserved between these nematodes, containing a calpain sequence motif. Thus, E1 may be involved in apoptosis, raising the possibility that protection against this parasitic helminth may be induced by apoptotic processes.

Host-parasite interaction in human onchocerciasis: identification and sequence analysis of a novel human calgranulin.

A novel S100 ca2+-binding protein, termed calgranulin-related protein (CGRP), was purified to homogeneity from extracts of adult Onchocerca volvulus, a human tissue-dwelling parasite. Its complete amino acid sequence was determined using microanalytical techniques. The primary structure of CGRP consists of 91 residues and displays identity with the recently reported partial sequence of an S100 protein present in human neutrophils. The human origin of CGRP is supported by the occurrence in O. volvulus extracts of additional human neutrophil proteins, including migration inhibitory factor-related protein 8 and defensins. The results suggest that these proteins interact with the worm surface following their release by activated neutrophils in the course of inflammatory reactions caused by O. volvulus infection.

Molecular cloning, expression, and localization of E1, an Onchocerca volvulus antigen with similarity to brain ankyrin.

Protective immunity against human onchocerciasis may best be reflected by the existence of individuals who in spite of exposure to the filarial nematode Onchocerca volvulus do not develop disease (putatively immune). We observed preferential recognition of an O. volvulus antigen of approximately 90 kDa by sera from putatively immune individuals compared with sera from diseased individuals. Screening of an adult worm cDNA library with one serum recognizing this antigen almost exclusively led to the identification of a full length clone of 2043 base pairs designated E1. The open reading frame of 462 amino acid residues shows similarity to human brain ankyrin. E1 appears to represent a small transcript of the O. volvulus ankyrin gene. The nonfusion protein obtained by expression of the complete E1 cDNA exhibits an apparent molecular mass of 90 kDa on SDS-polyacrylamide gel electrophoresis. An antiserum against the recombinant protein reacts with the 90-kDa antigen in O. volvulus extract. In O. volvulus, E1 was localized in the neuronal cell bodies, the nerve ring, and the extracellular clefts of the basal labyrinth. These results identify an ankyrin-related O. volvulus protein as an immunogen to putatively immune individuals, suggesting that neuronal proteins may be important targets for immunity against O. volvulus in vivo.

Impaired antibody responses and loss of reactivity to Onchocerca volvulus antigens by HIV-seropositive onchocerciasis patients.

The impact of concomitant human immunodeficiency virus (HIV) infection on the antibody response of onchocerciasis patients to Onchocerca volvulus antigens (OvAg) was studied by Western blotting and enzyme linked immunosorbent assay (ELISA). Immunoglobulin G (IgG) antibodies in sera from 45 HIV-sero-positive O. volvulus microfilariae (mf) carriers (HIV+/Ov+) recognized significantly fewer distinct O. volvulus antigenic bands, and responded less frequently to all detected bands compared to sera from 61 matched HIV-seronegative mf carriers (HIV-/Ov+). 29% of 31 follow-up sera from the HIV+/Ov+ patients failed to react to many of the antigenic bands recognized by initial sera from the same patients. Among 4 HIV+/Ov+ persons examined for total CD4+ cells, loss of reactivity corresponded with low CD4+ total cell counts. In an OvAg ELISA, sera from the HIV+/Ov+ individuals had significantly lower IgG+IgM antibody levels than sera from the HIV-/Ov+ persons, and the sensitivity of the assay was 87% for the HIV+/Ov+ subjects compared to 100% for those who were HIV-/Ov+. It is concluded that HIV-infected onchocerciasis patients exhibit significantly impaired antibody responses to O. volvulus antigens, and tend to lose their reactivity to these antigens over time due to immune response abnormalities caused by the concomitant HIV infection.

Human autoantibody to defensin: disease association with hyperreactive onchocerciasis (sowda).

Chronic hyperreactive onchodermatitis (sowda) is a severe form of onchocerciasis observed in a subset of individuals infected with the filarial nematode Onchocerca volvulus. SDS-PAGE and immunoblot analyses of O. volvulus adult worm extracts were used to characterize the antigens of the marked antibody response of sowda patients. One 2.5-kD antigen was recognized by sera from all 35(100%) sowda patients that were studied. In comparison, only 7 of 44 (16%) patients with generalized onchocerciasis and 11 of 21 (52%) of exposed individuals with no microfilariae in skin snips and no signs of disease showed reactivity to this antigen. Microfilaricidal treatment of sowda patients with improvement of the clinical status was associated with a decrease or disappearance of antibodies to the 2.5-kD antigen. Amino acid sequencing of the antigen indicated identity to human defensins 1-3 of neutrophils. Defensin was demonstrated by immunohistochemical staining in onchocercal nodules on the surface of adult filariae and in the surrounding tissue. A similar staining pattern was observed for other proteins present in neutrophils such as myeloperoxidase, elastase, and the L-1 protein complex (MRP 8/MRP 14), indicating that neutrophils, macrophages, and their proteins predominate in the environment adjacent to the worms. These results demonstrate an association between the presence of autoantibodies to defensins and an infectious disease of known etiology. The association with a particular form of onchocerciasis, sowda, suggests a link between formation of autoantibodies to defensin and enhanced immune reactivity towards the parasite.

A putative protein related to human chemokines encoded antisense to the cDNA of an Onchocerca volvulus antigen.

Chronic hyperactive dermatitis (sowda) in humans infected with the filarial parasite Onchocerca volvulus appears to reflect a hyperresponsiveness to parasite antigens. To identify antigens which play a role in this hyperresponsiveness an expression cDNA library of adult O. volvulus was screened with sera from patients with sowda. One further characterized cDNA clone, S1, consisting of 723 bp, surprisingly shows open reading frames (ORF) in both orientations. While a single ORF of 171 amino acids is present in sense orientation, a putative ORF of 95 AA is found in antisense orientation (aS1). Whereas no homologies to known proteins are found in S1, the sequence of aS1 shows a striking structural homology to human CC chemokines. The genomic organization of the coding region of aS1 shows the conserved three exon/two intron structure of the CC chemokine family. In adult worms transcription of mRNA corresponding to S1 but not to aS1 was detected. Expression of S1 as a non fusion protein and Western blot analysis revealed antibody recognition by all sera from patients with sowda, by 60% of sera from patients with the generalized form of onchocerciasis, but not by sera of exposed individuals with no evidence of onchocerciasis. IgG subclass analysis showed that IgG3 reactivity was restricted to sowda sera. In adult worms the S1 protein was localized to the hypodermis. Here we present the cloning and characterization of an O. volvulus antigen, which may be useful in the diagnosis of onchocerciasis. Furthermore, the results suggest the presence of a gene structurally related to human inflammatory cytokines in antisense orientation, raising the question of bidirectional transcription in O. volvulus.

Immunodiagnostic studies on Onchocerca volvulus and Mansonella perstans infections using a recombinant 33 kDa O. volvulus protein (Ov33).

A total detergent-soluble extract of adult female Onchocerca volvulus (OvAg) and a recombinant O. volvulus protein (Ov33) linked to glutathione-S-transferase (GST) were compared with regard to their serodiagnostic suitability for differentiating between O. volvulus and Mansonella perstans infections in a region endemic for both filarial worms in western Uganda. Using OvAg in an enzyme-linked immunosorbent assay (ELISA), 98.8% sensitivity was obtained examining 84 O. volvulus microfilariae (mf) carriers living in the hyperendemic area. However, 5 of 18 (28%) sera from M. perstans mf carriers without O. volvulus mf, from another area hypoendemic for O. volvulus, cross-reacted with OvAg. Using the recombinant antigen Ov33-GST in an ELISA and Western blot assay, sensitivity for O. volvulus remained high (97.2% and 98.8% respectively) while none of 90 sera from M. perstans mf carriers reacted positively. Both antigens were used to examine a batch of sera from 260 persons living in the onchocerciasis hyperendemic area who did not have mf in their skin snips (9.5% of 2728 persons examined); 116 of these sera (44.6%) were positive in the OvAg ELISA, compared to 85 (32.7%) and 69 (26.5%) which were positive in Ov33-GST ELISA and Ov33-GST Western blot, respectively. Reaction with GST alone was minimal. The recombinant antigen Ov33 efficiently differentiates between O. volvulus and M. perstans infections, and is sensitive when used to detect patent and prepatent or low-level O. volvulus infections.

Strong IgG isotypic antibody response in sowdah type onchocerciasis.

Persons exposed to Onchocerca volvulus express differences in manifestations of onchocerciasis ranging from hyporeactive (generalized) to hyperreactive (sowdah) forms; absence of disease is seen in endemic normal persons. Analysis of the IgG isotypic antibody response to O. volvulus extracts and nonparasite ubiquitous antigens in 92 West Africans revealed highest anti-O. volvulus IgG1 and IgG2 responses in sowdah, high levels in the generalized form, and low antibody levels in endemic normal persons. Nonexposed persons had no antibodies. A significant IgG3 antibody response was detected only in sowdah, while high IgG4 levels occurred in both polar groups but were absent in both control groups. Isotypic responses to antigens unrelated to O. volvulus were similar in all groups but showed higher IgG1 and IgG2 levels in sowdah. Sowdah patients had high levels of Ro/SS-A antibodies, circulating immune complexes, and eosinophil cationic protein. These results document a strong B cell response in African sowdah and indicate variations in immune responsiveness.

HLA-D alleles associated with generalized disease, localized disease, and putative immunity in Onchocerca volvulus infection.

Human infections with the tissue nematode Onchocerca volvulus result in a variety of clinical conditions that possibly include protective immunity. In a West African area hyperendemic for human onchocerciasis, 120 residents were classified according to clinical and laboratory findings as presenting with generalized onchocerciasis, localized onchocerciasis, or as being putatively immune. The three groups differed in the distribution of HLA-D variants as determined by DNA typing. The most pronounced differences were found among alleles of the DQ loci. The haplotype DQA1*0501-DQB1*0301 was significantly more frequent among putatively immune individuals than among patients with generalized or localized disease. Conversely, DQA1*0101-DQB1*0501 and, independently, the allele DQB1*0201 were more frequent in generalized disease than in localized disease or putative immunity. In these correlations, the frequencies of allelic variants were in localized disease intermediate to those of the two other groups. The only distinct association found with localized disease was that of the DP allele DPB1*0402. The findings indicate that HLA-D variants influence the course of O. volvulus infection and help to define a state that may reflect protective immunity.

Epidemiological studies of onchocerciasis in southern Benin.

We studied the prevalence of human onchocerciasis in four geographically different regions of the southern part of Benin in West Africa. In a total of thirteen villages 1596 individuals were examined for clinical and parasitological signs of onchocerciasis. Prevalence of microfilariae of Onchocerca volvulus in skin snips was 29% in region I (lower Oueme river), 64% in region II (Mono river), 56% in region III (upper Oueme river) and 70% in region IV (Okpara river). Based on endemicity criteria of the WHO regions II and IV were found to be hyperendemic, region III mesoendemic and region I hypoendemic for onchocerciasis. The community microfilarial load ranged from 4 mf/mg skin in the hypoendemic region to 10.5 mf/mg skin in the hyperendemic regions. The prevalence of nodules was 21% in region I, 30% in region II, 17% in region III and 41% in region IV. The overall prevalence of chronic onchocercal dermatitis was 12%. Of 689 individuals infected with O. volvulus 388 were treated with a single dose of ivermectin.