Albert E. Bianco

Susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to nitric oxide and hydrogen peroxide in cell-free culture and from IFNγ-activated macrophages

The susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to H2O2 and NO either in cell-free culture or from IFN gamma-activated macrophages was examined. In cell-free culture, O. lienalis microfilariae were highly susceptible to H2O2 induced toxicity, exhibiting rapid reductions in motility and viability. The addition of exogenous catalase abrogated H2O2-induced killing. In contrast, B. malayi microfilariae were relatively resistant to H2O2, with concentrations as high as 50 microM having no effect on motility or viability. On exposure to NO, both species showed reductions in motility within 5-30 min, but longer was required to see effects on the viability of microfilariae. Parasites incubated with IFN gamma-activated macrophages also exhibited marked reductions in motility and viability. In cultures with B. malayi and activated macrophages, inhibition of these effects was achieved by the addition of either L-NMMA, to abolish NO production, or neutralizing anti-TNF alpha antibodies. Attempts to inhibit parasite killing by the addition of catalase to macrophage cultures were ineffective. The results of this study show that B. malayi and O. lienalis microfilariae have different susceptibility to H2O2, but are equally affected by exposure to NO. Moreover both species are killed by IFN gamma-activated macrophages and in the case of B. malayi, killing is dependent on the generation of NO via TNF alpha.

Thioredoxin peroxidase from Onchocerca volvulus: a major hydrogen peroxide detoxifying enzyme in filarial parasites.

Random screening of an Onchocerca volvulus third-stage (L3) cDNA library identified a highly abundant cDNA encoding a newly discovered antioxidant enzyme, thioredoxin peroxidase (TPx), a member of the peroxidoxin superfamily. This TPx cDNA (Ov-tpx-2) encodes a polypeptide of 199 amino acid residues with a calculated molecular weight of 21,890 Da. The Ov-tpx-2 cDNA represents roughly 2.5% of the total cDNAs from the L3 cDNA library. The gene was expressed in Escherichia coli and the protein product was shown to have antioxidant activity. Antiserum raised against Ov-TPX-2 recognized a native protein from extracts of both the L3 and adult-stages with a molecular weight of 22 kD. The localization and stage-specificity of Ov-TPX-2 protein was analyzed by immunocytochemistry and immunoelectron microscopy using monospecific antibodies. Expression was detected in late first-stage larvae during development in the vector and increased in intensity during differentiation to the infective L3-stage. The antigen was also detected in post-infective larvae and adult worms. In larvae, Ov-TPX-2 protein was predominantly localized to the hypodermis and cuticle, with additional sites in the hypodermal chords and multivesicular bodies. In adult worms, the primary sites of expression were the uterine epithelium and intestine, with additional labeling of the body wall and cuticle. Developing embryos and microfilariae in utero were bathed in Ov-TPX-2 protein discharged from epithelial cells. These results suggest that Ov-TPX-2 may protect the parasites from being damaged by host-generated oxidative stress and that Ov-TPX-2 protein provides the H2O2-detoxifying activity predicted but not previously identified in filarial parasites. Its highly upregulated expression in infective larvae may aid in parasite establishment following transmission to the definitive host.

Onchocerca ochengi infections in cattle as a model for human onchocerciasis: recent developments.

The bovine parasite Onchocerca ochengi is a nodule-dwelling filarial nematode, closely related to O. volvulus, the causal agent of human River Blindness, and, sharing with it, the same vector. This brief review, based on a presentation at the BSP Autumn Symposium 1999, describes recent work supported by the WHO Drug Development Research Macrofil programme and the Edna McConnell Clark Foundation vaccine development programme, to research the chemotherapy and immunology of onchocerciasis utilising this model system, with experimental infections in Liverpool and field infections in northern Cameroon. In a series of chemotherapeutic trials involving 10 compounds in 20 treatment regimes, the comparability of drug efficacy against O. ochengi with that described against O. volvulus has been demonstrated. Repeated, long-term treatment with oxytetracycline has been shown to be macrofilaricidal and the effect is hypothesized to be related to action on Wolbachia endobacteria, abundant in O. ochengi. Avermectins/milbemycins are not macrofilaricidal (even in high and repeated long-term treatments) but induce sustained abrogation of embryogenesis. In prospective, field exposure experiments with naive calves, prophylactic treatments with ivermectin and moxidectin prevented the development of adult worm infection, raising the possibility that drug-attenuated larval challenge infections may induce immunity. Putatively immune adult cattle exist in endemically exposed populations, and these have been shown to be significantly less susceptible to challenge than age-matched naive controls, whereas radically drug-cured, previously patently-infected cattle were not. Experimental infections with O. ochengi have revealed the kinetics of the immune response in relation to parasite development and demonstrate analogous responses to those reported in O. volvulus infection in humans and chimpanzees. In an immunization experiment with irradiated L3 larvae, cattle were significantly protected against experimental challenge--the first such demonstration of the experimental induction of immunity in a natural Onchocerca host-parasite system. Taken collectively, these studies not only demonstrate the similarity between the host-parasite relationships of O. ochengi in cattle and O. volvulus in humans, but promise to advance options for the control of human onchocerciasis.

Eosinophils are the major effector cells of immunity to microfilariae in a mouse model of onchocerciasis

Mice inoculated with microfilariae of the filarial nematode Onchocerca lienalis clear their parasites over a period of 3-4 months and are highly resistant to re-infection. We have investigated the comparative roles of the eosinophil, macrophage and neutrophil in effecting this parasite clearance, employing agents specifically to perturb cell function in vivo. Using the anti-IL-5 monoclonal antibody TRFK-5, we show that eosinophils are of primary importance in effecting resistance to re-infection. Ablation of macrophages (with carbon) and neutrophils (with the monoclonal antibody NIMP-R14) had no effect on parasite clearance following re-infection. Neutralization of these 3 cell types during a primary infection showed that while the removal of both eosinophils and macrophages caused a small but significant delay in parasite clearance, the depletion of neutrophils had no effect. This report describes the first direct evidence for eosinophil-mediated killing of microfilariae in vivo, and is consistent with Th-2 cell responses previously described in this model.

Distinct Response Kinetics of Gamma Interferon and Interleukin-4 in Bovine Tuberculosis

ABSTRACT This study shows that gamma interferon (IFN-γ) and interleukin-4 (IL-4) cytokine responses are produced by peripheral blood cells in cattle infected with Mycobacterium bovis. The different kinetics of the IFN-γ and IL-4 responses to bovine tuberculin and to ESAT-6 following experimental intratracheal infection with M. bovis are described. An early increase in IFN-γ was observed that was maintained throughout the period studied. In contrast, the IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a lower dose of M. bovis which did not result in the development of lesions, positive DTH skin test, or substantial IFN-γ responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infected M. bovis field reactors showed increased IFN-γ and IL-4 responses compared to uninfected cattle and that both of these cytokines were equally able to differentiate infected from uninfected animals. The magnitude of theM. bovis-induced IL-4 responses were found to be similar to the antigen-specific IL-4 responses of cattle infected with the parasitic nematode Onchocerca ochengi, further supporting the presence of this type 2 cytokine in bovine tuberculosis.

DNA immunisation with Onchocerca volvulus chitinase induces partial protection against challenge infection with L3 larvae in mice.

The parasitic nematode, Onchocerca volvulus is a major cause of blindness and dermal pathology in tropical regions. A vaccine directed to infective larvae would provide a valuable control tool alongside the current methods of chemotherapy and vector control. Previously we have described the identification of a chitinase cDNA that is expressed in a stage specific manner by O. volvulus infective third stage (L3) larvae. To evaluate its host protective potential, the complete open reading frame was cloned into the eukaryotic expression plasmid pJW4303 and used to vaccinate mice by DNA immunisation with the Accell GeneGun. The survival of challenge infective larvae was monitored using implanted micropore chambers. In the first trial, mice immunised 3 times over 4 months with 1 microg O. volvulus chitinase DNA responded with modest antibody responses dominated by IgG2a and exhibited a 36% (p=0.189, NS) reduction in parasite survival compared with challenge controls. In the second trial, an increased dose of DNA (5 microg) and more frequent immunisations (5 times over 6 months) stimulated an IgG1 dominant response and a 53% reduction in parasite survival (p=0.042). Antibodies from the vaccinated mice reacted with the cuticle of post-infective L3 larvae, implying that this may be the site of immune attack following secretion of chitinase.

Protective immunity induced by vaccination with Onchocerca volvulus tropomyosin in rodents

A cDNA clone of Onchocerca volvulus, designated MOv14, and encoding 136 amino‐acid residues from the C‐terminus of O. volvulus tropomyosin, was evaluated as a protective immunogen in two complimentary rodent models of onchocerciasis. Vaccination of BALB/c mice with the recombinant fusion of MOv14 coupled to Maltose‐Binding Protein (MBP) induced significant reductions (48–62%) in the recovery of Onchocerca lienalis microfilariae from the skin, compared to control groups immunized with MBP alone. The predominant antibody response generated to MOv14 by vaccination was of IgG1. Following a similar vaccination protocol in Mongolian jirds, two independent experiments demonstrated that 16 weeks after infection with Acanthocheilonemaviteae there was a 46% reduction in the recovery of adult worms in vaccinated animals compared to control groups. Antibodies generated by vaccination recognized a product released during culture of A. viteae infective larvae which migrated at a distinct molecular mass from native tropomyosin from somatic tissues.

Characterization of a secreted antigen of Onchocerca volvulus with host-protective potential

A cDNA designated MOv2 was isolated from an Onchocerca volvulus library on the basis of its products recognition by an antiserum raised against the infective stage. Immunogold electron microscopy revealed a high density of antigenic sites associated with the annulae of the L3 cuticle and with the uterine wall of the adult female: a general, low density of labell ing occurred in all developmental forms. Western blotting confirmed the presence of the antigen throughout the life cycle and the existence of an immunologically cross‐reactive homologue in the related filaria, Acanthocheilonema viteae. The antigen was shown to be secreted by infective larvae and adult females of A. viteae. Sequence comparisons revealed two homologues of MOv2 (Ov‐20, Ov‐9) which had been selected independently by other laboratories on the basis of their specific recognition by human onchocerciasis infection sera. The IgG antibody response against MOv2 in cattle experimentally infected with O. lienalis revealed the induction of a response during the prepatent period that was strongly boosted at the onset of patency. However, only a proportion of infected cattle responded with a detectable level of anti‐MOv2 antibodies. The appearance of MOv2 in larval cuticle and secretions prompted us to evaluate it as a candidate molecule for prophylactic immunization. Trials performed in the A. viteae/Mongolian jird model of filariasis revealed that recombinant MOv2 induced a host‐protective response, reducing worm recoveries by 36–55% following a challenge infection.

Chitinase genes expressed by infective larvae of the filarial nematodes, Acanthocheilonema viteae and Onchocerca volvulus.

State-specific products of 220 and 75 kDa were identified by metabolic labelling of infective larvae of the filarial nematode Acanthocheilonema viteae in ticks. Synthesis was temperature sensitive, occurring at 27 degrees C but not at 37 degrees C. These products were secreted 3-6 days after leaving the vector during post-infective development, but subsequent expression was not detected. The smaller protein with a pI of 6.2, was purified by two-dimensional electrophoresis and the N-terminal amino acid sequence was derived. This provisionally identified the protein as a chitinase, which was confirmed biochemically by glycol-chitin substrate gel electrophoresis. The polymerase chain reaction was used to amplify a product from a cDNA library of A. viteae infective larvae. The nucleotide sequence codes for a putative signal peptide of 20 amino acids and a mature protein of 504 residues (Mr 56 kDa), exhibiting 69% identity (81% similarity allowing for conservative substitutions) with the MF1 chitinase described from microfilariae of Brugia malayi. N-linked glycosylation may account for some, or all, of the discrepancy in Mr between the predicted polypeptide and the native parasite product (75 kDa). Primers based on the A. viteae sequence were used to amplify a related sequence from a cDNA library of Onchocerca volvulus infective larvae. The O. volvulus cDNA codes for a 20-amino acid signal peptide followed by 477 residues with an Mr of 54 kDa, and shares 67% identity with the A. viteae chitinase (80% similarity allowing for conservative substitutions) and 69% identity with the B. malayi MF1 molecule. It is proposed that chitinases expressed by infective stages of these filarial nematodes may play a role in ecdysis during post-infective development.

Down-Regulated Lymphoproliferation Coincides with Parasite Maturation and with the Collapse of Both Gamma Interferon and Interleukin-4 Responses in a Bovine Model of Onchocerciasis

ABSTRACT Onchocerciasis is a debilitating parasitic infection caused by the filarial nematode Onchocerca volvulus. Infections are chronic, and persistence of the parasites for several years argues for highly adapted mechanisms of immune evasion. Due to the restricted host repertoire of O. volvulus, we have used the cattle parasite Onchocerca ochengi to investigate the nature of immunomodulation underpinning these long-term infections. Cattle were infected with a single inoculation of 350 infective-stage larvae under laboratory conditions (n = 6). Intradermal nodules containing immature adult worms were detected from 110 days postinfection, and microfilariae in skin were detected from day 280 postinfection. Parasite-specific responses during early infection were nonpolarized with respect to the major Th cytokines (interleukin-4 [IL-4], IL-2, and gamma interferon [IFN-γ]) produced by antigen-stimulated peripheral blood mononuclear cells (PBMC) or serum antibody isotypes. Antigen-induced proliferation of PBMC peaked shortly after exposure and remained high during the prepatent infection. As the parasites matured and animals developed patent infections, there was a profound down-regulation of lymphoproliferation, accompanied by sharp falls in the expression of both IL-4 and IFN-γ and a gradual decline in IL-2. Levels of immunoglobulin G2 (IgG2) fell, while those of IgG1 remained high. We conclude that neither a classical Th2 response nor a simple Th1-to-Th2 switch is sufficient to explain the immunomodulation associated with patent Onchocerca infections. Instead, there is an initial Th0 response, which matures into a response with some, but not all of the features of a Th2 response. The natural host-parasite relationship of O. ochengi in cattle may be useful as both a descriptive and predictive tool to test more refined models of immunomodulation in onchocerciasis.