Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility.

Intracellular bacteria have been described in several species of filarial nematodes, but their relationships with, and effects on, their nematode hosts have not previously been elucidated. In this study, intracellular bacteria were observed in tissues of the rodent parasite Litomosoides sigmodontis by transmission electron microscopy and by immunohistochemistry using antiendobacterial heat shock protein-60 antisera. Molecular phylogenetic analysis of the bacterial 16S ribosomal RNA gene, isolated by PCR, showed a close relationship to the rickettsial Wolbachia endobacteria of arthropods and to other filarial intracellular bacteria. The impact of tetracycline therapy of infected rodents on L. sigmodontis development was analyzed in order to understand the role(s) these bacteria might play in filarial biology. Tetracycline therapy, when initiated with L. sigmodontis infection, eliminated the bacteria and resulted in filarial growth retardation and infertility. If initiated after microfilarial development, treatment reduced filarial fertility. Treatment with antibiotics not affecting rickettsial bacteria did not inhibit filarial development. Acanthocheilonema viteae filariae were shown to lack intracellular bacteria and to be insensitive to tetracycline. These results suggest a mutualistic interaction between the intracellular bacteria and the filarial nematode. Investigation of such a mutualism in endobacteria-containing human filariae is warranted for a potential chemotherapeutic exploitation.

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.

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.

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.

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.

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.

Onchocerca volvulus recombinant antigen: physical characterization and clinical correlates with serum reactivity.

An Onchocerca volvulus cDNA clone expressing epitopes found in adult and larval parasites, designated lambda RAL-2, was derived from a 1,000-base message present in adult O. volvulus, which encodes a protein with an apparent molecular weight of 17,000. This protein does not appear to be extensively posttranslationally modified. Serum samples from 52 individuals exposed to O. volvulus were examined for antibodies recognizing the lambda RAL-2 recombinant antigen; 77% produced such antibodies. In addition, individuals producing antibodies recognizing the recombinant antigen were significantly less likely to develop some aspects of ocular pathology associated with O. volvulus infection than were individuals who did not do so. These results suggest that recombinant antigens such as that produced by lambda RAL-2 may be useful in attempts to understand the mechanism of O. volvulus-induced ocular pathology.

Experimental interstitial keratitis induced by Onchocerca volvulus antigens.

To elucidate the pathogenic mechanisms involved in onchocercal sclerosing keratitis in humans, we developed a model of onchocercal interstitial keratitis in guinea pigs. Onchocerca volvulus antigens injected intrastromally into corneas of preimmunized Hartley guinea pigs induced an intense stromal keratitis with corneal edema, neovascularization, and infiltration with acute and chronic inflammatory cells. This reaction subsided after two weeks. Repeated intrastromal injection resulted in an exacerbation of the keratitis and ultimately in residual scarring. These findings are consistent clinically and histopathologically with the chronic interstitial keratitis observed in humans. To define which antigens induce the corneal reaction, O volvulus antigens were separated by molecular sieve chromatography and injected intrastromally. The highest activity was shown to reside in the fraction containing molecules of intermediate molecular weight. This model will be useful in defining O volvulus antigens and their role in sclerosing keratitis as well as in elucidating the immune mechanisms involved.

Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae.

The isolation of recombinant cDNA clones expressing antigens found in Onchocerca volvulus infective larvae is described. To isolate such clones, an expression cDNA library constructed from adult O. volvulus RNA was screened with antiserum raised against infective larvae. One clone, designated lambda RAL-1 was characterized further. The recombinant antigen produced by lambda RAL-1 stimulates proliferation of peripheral blood mononuclear cells from O. volvulus infected humans. Lambda RAL-1 is derived from a 1450 bases message that encodes a protein with an apparent molecular weight of 42,000 in adult O. volvulus. The inserted DNA of lambda RAL-1 contains an open reading frame of 1008 bp. The amino acid sequence predicted by this open reading frame contains three repeats of the sequence KKPEDWD. The identification of clones such as lambda RAL-1 will provide quantities of purified antigens sufficient to begin to study the immune response to and explore the development of immunity against the infectious form of the parasite.