Identification of Brugia malayi immunogens by an immunoproteomics approach

Filariasis remains a health problem in tropical countries. Identification of immunogens from its causative organism would lead to development of a better diagnostic test, as well as vaccine discovery to effectively prevent this disease. We applied immunoproteomics to define potential immunogens of adult Brugia malayi that were recognized by IgM, IgG1 and IgG4 in sera of patients with four distinct clinical spectra of filariasis, including endemic asymptomatic, lymphangitis, elephantiasis and microfilaremia (n=5/group). Sera of healthy individuals (n=5) from non-endemic area served as the negative control. Brugian proteins were resolved by 2-DE and subjected to 2-D Western blot analysis probed with these sera. A total of 30 immunoreactive proteins recognized by IgM, IgG1 and IgG4 in sera from all four filarial groups were identified by Q-TOF MS and MS/MS analyses. Interestingly, only three immunogens were recognized by IgM in lymphangitis, elephantiasis and microfilaremia, but not in endemic asymptomatic group. IgG1 recognized 20 immunogens in endemic asymptomatic, lymphangitis and microfilaremia (mostly in endemic asymptomatic group), but not in elephantiasis, whereas IgG4 recognized 28 immunogens in all four filarial groups (mostly in microfilaremia). This large data set is an important resource for further development of a new diagnostic test and/or vaccine for filariasis.     

Brugia malayi: microfilarial polyunsaturated fatty acid composition and synthesis

The polyunsaturated fatty acid composition of Brugia malayi microfilariae was analyzed by gas chromatography and compared to that of sera from B. malayi-infected jirds. The essential fatty acid, linoleic acid (18:2 omega 6), was the most abundant fatty acid present in both microfilarial total lipids and phospholipids as well as in jird sera. In contrast, arachidonic acid (20:4 omega 6), as well as the 18:3 omega 6, 20:2 omega 6, and 20:3 omega 6 intermediates that are formed in the enzymatic conversion of linoleic acid to arachidonic acid, were proportionally more abundant in microfilariae than in jird sera. To assess the capacity of microfilariae to transform linoleic acid into arachidonic acid, B. malayi microfilariae were incubated with [14C]linoleic acid. Microfilarial lipids were extracted and resolved by high-pressure liquid chromatography and thin-layer chromatography. A portion of [14C]linoleic acid incorporated by microfilariae was converted to [14C]arachidonic acid. Thus, microfilariae can not only incorporate exogenous arachidonic acid, as previously demonstrated, but can also synthesize arachidonic acid from exogenous linoleic acid. The capacity of microfilariae to utilize specific host polyunsaturated fatty acids raises the possibility that intravascular filarial parasites may synthesize eicosanoid metabolites of arachidonic acid that could mediate filarial-host cell interactions.     

Brugia malayi: clearance of microfilaremia induced by diethylcarbamazine independently of antibody

The microfilaricidal effect of diethylcarbamazine was studied in the murine model of Brugia malayi microfilaremia. CFI mice with circulating microfilariae were treated with either diethylcarbamazine (6 mg kg-1 day-1) for 10 days or with normal saline. Although antibodies against crude microfilarial antigen were detected in both groups at the time of completion of therapy, the chemically induced microfilarial clearance occurred prior to the development of these antibodies. In addition, the passive transfer of antibody just prior to chemical treatment did not enhance diethylcarbamizine-induced microfilarial clearance. In vitro studies confirmed previous observations that diethylcarbamazine enhanced antibody-dependent neutrophil adherence. Microfilarial clearance was found to be due to trapping and lysis in the liver on pathologic examination. These studies demonstrate that diethylcarbamazine leads to microfilarial clearance in the liver by mechanisms that are not dependent on host antibody.     

Molecular cloning and characterization of Brugia malayi hexokinase

5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.     

Antibody responses to Brugia malayi antigens induced by DNA vaccination

BACKGROUND: DNA vaccination is a convenient means of immunizing animals with recombinant parasite antigens. DNA delivery methods are believed to affect the qualitative nature of immune responses to DNA vaccines in ways that may affect their protective activity. However, relatively few studies have directly compared immune responses to plasmids encoding the same antigens after injection by different routes. Therefore, the purpose of this study was to explore the influence of the route of administration on antibody responses to plasmids encoding antigens from the filarial nematode parasite Brugia malayi. METHODS: Four B. malayi genes and partial genes encoding paramyosin (BM5), heat shock protein (BMHSP-70), intermediate filament (BMIF) and a serodiagnostic antigen (BM14) were inserted in eukaryotic expression vectors (pJW4303 and pCR trade mark 3.1). BALB/c mice were immunized with individual recombinant plasmids or with a cocktail of all four plasmids by intramuscular injection (IM) or by gene gun-intradermal inoculation (GG). Antibody responses to recombinant antigens were measured by ELISA. Mean IgG1 to IgG2a antibody ratios were used as an indicator of Th1 or Th2 bias in immune responses induced with particular antigens by IM or GG immunization. The statistical significance of group differences in antibody responses was assessed by the non-parametric Kruskal-Wallis test. RESULTS: Mice produced antibody responses to all four filarial antigens after DNA vaccination by either the IM or GG route. Antibody responses to BM5 paramyosin were strongly biased toward IgG1 with lower levels of IgG2a after GG vaccination, while IM vaccination produced dominant IgG2a antibody responses. Antibody responses were biased toward IgG1 after both IM and GG immunization with BMIF, but antibodies were biased toward IgG2a after IM and GG vaccination with BMHSP-70 and BM14. Animals injected with a mixture of four recombinant plasmid DNAs produced antibodies to all four antigens. CONCLUSIONS: Our results show that monovalent and polyvalent DNA vaccination successfully induced antibody responses to a variety of filarial antigens. However, antibody responses to different antigens varied in magnitude and with respect to isotype bias. The isotype bias of antibody responses following DNA vaccination can be affected by route of administration and by intrinsic characteristics of individual antigens.     

Brugia malayi: patterns of expression of surface-associated antigens.

Patterns of expression of surface-associated antigens were analyzed in the filarial nematode Brugia malayi immediately prior, and during development in the vertebrate host. Two surface-associated protein molecules, i.e., accessible to surface radioiodination and soluble in aqueous buffers, were investigated: Mrs 29-30,000 and 16,000, both of which are antigenic in infected animals. The Mr 29-30,000 glycoprotein is expressed in a surface-associated manner by adult worms and by fourth-stage larvae, but is not detectable in preparasitic third-stage larvae. The 16,000 component, which appears not to be glycosylated, is surface-associated in adult worms and fourth-stage larvae. In contrast to the 29-30,000 glycoprotein, the 16,000 protein is also expressed both by pre- and postparastic third-stage larvae. However, it becomes surface-associated only after infection. Thus, immediately prior, and during development within the vertebrate host, B. malayi displays at least two different patterns of expression of surface-associated antigens: (i) de novo, intiated either immediately after infection (phase specific) or during genesis of the fourth-stage larva (stage specific); (ii) continuous, but with phase-dependent surface exposure of previously cryptic antigens, during the transition from intermediate to definitive host.     

Fluorescent diethylcarbamazine analogues: sites of accumulation in Brugia malayi

New fluorescein and rhodamine B-labeled antifilarial drug DEC analogues for use in drug localization studies with confocal microscopy have been prepared by a high-yield three-step synthesis. The resulting beta-amine-substituted DEC analogue has a single ethyl substituent which is beta-aminated to accommodate the fluorophore of either fluorescein isothiocyananate or rhodamine B. Confocal microscopy is used to show that the drug accumulates in the adult filarial worms in the pharynx, esophagus, and near the nerve ring of all adults, as well as in the uteri and vulva and the testes of the females and males.     

Tissue and stage-specific distribution of Wolbachia in Brugia malayi

Background

Most filarial parasite species contain Wolbachia, obligatory bacterial endosymbionts that are crucial for filarial development and reproduction. They are targets for alternative chemotherapy, but their role in the biology of filarial nematodes is not well understood. Light microscopy provides important information on morphology, localization and potential function of these bacteria. Surprisingly, immunohistology and in situ hybridization techniques have not been widely used to monitor Wolbachia distribution during the filarial life cycle.

Methods/Principal Findings

A monoclonal antibody directed against Wolbachia surface protein and in situ hybridization targeting Wolbachia 16S rRNA were used to monitor Wolbachia during the life cycle of B. malayi. In microfilariae and vector stage larvae only a few cells contain Wolbachia. In contrast, large numbers of Wolbachia were detected in the lateral chords of L4 larvae, but no endobacteria were detected in the genital primordium. In young adult worms (5 weeks p.i.), a massive expansion of Wolbachia was observed in the lateral chords adjacent to ovaries or testis, but no endobacteria were detected in the growth zone of the ovaries, uterus, the growth zone of the testis or the vas deferens. Confocal laser scanning and transmission electron microscopy showed that numerous Wolbachia are aligned towards the developing ovaries and single endobacteria were detected in the germline. In inseminated females (8 weeks p.i.) Wolbachia were observed in the ovaries, embryos and in decreasing numbers in the lateral chords. In young males Wolbachia were found in distinct zones of the testis and in large numbers in the lateral chords in the vicinity of testicular tissue but never in mature spermatids or spermatozoa.

Conclusions

Immunohistology and in situ hybridization show distinct tissue and stage specific distribution patterns for Wolbachia in B. malayi. Extensive multiplication of Wolbachia occurs in the lateral chords of L4 and young adults adjacent to germline cells.     

Guanidine hydrochloride and urea-induced unfolding of Brugia malayi hexokinase

Guanidine hydrochloride and urea-induced unfolding of B. malayi hexokinase (BmHk), a tetrameric protein, was examined in detail by using various optical spectroscopic techniques, enzymatic activity measurements, and size-exclusion chromatography. The equilibrium unfolding of BmHk by guanidine hydrochloride (GdmCl) and urea proceeded through stabilization of several unique oligomeric intermediates. In the presence of low concentrations of GdmCl, stabilization of an enzymatically active folded dimer of BmHk was observed. However an enzymatically inactive dimer of BmHk was observed for urea-treated BmHk. This is the first report of an enzymatically active dimer of hexokinase from any human filarial parasite. Furthermore, although complete recovery of the native enzyme was observed on refolding of BmHk samples denatured by use of low concentrations of GdmCl or urea, no recovery of the native enzyme was observed for BmHk samples denatured by use of high concentrations of GdmCl or urea.     

Brugia malayi and Brugia pahangi: transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti

Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L2, and L3 larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L3 larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L3's recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microfilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microfilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microfilariae to develop to the L3 stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microfilariae also failed to develop to L3 in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 ng/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microfilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Brugia malayi and Brugia pahangi: inherent difference in immune activation in the mosquitoes Armigeres subalbatus and Aedes aegypti

The inherent ability of Brugia malayi and Brugia pahangi (Nematoda) to establish successful relationships with the mosquitoes Armigeres subalbatus and Aedes aegypti Liverpool strain was evaluated. Brugia pahangi microfilariae (mff) avoided the immune response and developed normally in A. subalbatus exposed to the parasite by an infective bloodmeal, whereas nearly 85% of B. malayi were destroyed by the immune response. Because A. aegypti supports the development of both filarial worm species but destroys intrathoracically inoculated B. pahangi isolated from jird blood, blood-isolated B. malayi were inoculated into A. aegypti, and the immune response was compared with that observed against B. pahangi. The response against B. malayi was significantly more rapid and effective than the response against B. pahangi. Similar results were obtained when blood-isolated B. pahangi or B. malayi were inoculated into A. subalbatus. Microfilariae of both species were able to avoid immune destruction in A. aegypti if they were allowed to penetrate the Liverpool midgut in vitro prior to inoculation. Most B. pahangi that had first penetrated an Armigeres midgut prior to inoculation into A. subalbatus were able to avoid the immune response, but by day 3 postinoculation, less than 40% of the B. malayi, treated in the same manner, were able to escape the immune response. Genetic susceptibility of mosquitoes to infection by filarial worms and potential mechanisms of immune evasion/suppression are discussed regarding B. malayi and B. pahangi.     

Stage- and gender-specific proteomic analysis of Brugia malayi excretory-secretory products

Introduction

While we lack a complete understanding of the molecular mechanisms by which parasites establish and achieve protection from host immune responses, it is accepted that many of these processes are mediated by products, primarily proteins, released from the parasite. Parasitic nematodes occur in different life stages and anatomical compartments within the host. Little is known about the composition and variability of products released at different developmental stages and their contribution to parasite survival and progression of the infection.

Methodology/Principal Findings

To gain a deeper understanding on these aspects, we collected and analyzed through 1D-SDS PAGE and LC-MS/MS the Excretory-Secretory Products (ESP) of adult female, adult male and microfilariae of the filarial nematode Brugia malayi, one of the etiological agents of human lymphatic filariasis. This proteomic analysis led to the identification of 228 proteins. The list includes 76 proteins with unknown function as well as also proteins with potential immunoregulatory properties, such as protease inhibitors, cytokine homologues and carbohydrate-binding proteins. Larval and adult ESP differed in composition. Only 32 proteins were shared between all three stages/genders. Consistent with this observation, different gene ontology profiles were associated with the different ESP.

Conclusions/Significance

A comparative analysis of the proteins released in vitro by different forms of a parasitic nematode dwelling in the same host is presented. The catalog of secreted proteins reflects different stage- and gender-specific related processes and different strategies of immune evasion, providing valuable insights on the contribution of each form of the parasite for establishing the host–parasite interaction.     

Brugia malayi: arachidonic acid uptake into lipid bodies of adult parasites

Lipid bodies are non-membrane bound intracellular organelles, which have been recognized morphologically in a diversity of mammalian and nonmammalian cells, but are of uncertain function. In mammalian cells, in addition to serving as a storage site of cholesterol and triglyceride, lipid bodies can be a repository of esterified arachidonic acid. Adult worms of the human filarial parasite Brugia malayi have been found to esterify exogenous [3H]arachidonic acid into parasite phospholipids and neutral lipids. Electron microscopic autoradiography demonstrated that [3H]arachidonate was preferentially incorporated into filarial lipid bodies. The dominant incorporation of arachidonate into lipid bodies of a nematode establishes that lipid bodies are a site of arachidonic acid accumulation in nonmammalian, as well as mammalian, cells.     

Live Brugia malayi Microfilariae Inhibit Transendothelial Migration of Neutrophils and Monocytes

Lymphatic filariasis is a major tropical disease caused by the parasite Brugia malayi. Microfilariae (Mf) circulate in the peripheral blood for 2–3 hours in synchronisation with maximal feeding of the mosquito vector. When absent from the peripheral blood, Mf sequester in the capillaries of the lungs. Mf are therefore in close contact with vascular endothelial cells (EC) and may induce EC immune function and/or wound repair mechanisms such as angiogenesis. In this study, Mf were co-cultured with human umbilical vein EC (HUVEC) or human lung microvascular EC (HLMVEC) and the transendothelial migration of leukocyte subsets was analysed. In addition, the protein and/or mRNA expression of chemokine, cytokine and angiogenic mediators in endothelial cells in the presence of live microfilariae were measured by a combination of cDNA arrays, protein arrays, ELISA and fluorescence antibody tests.Surprisingly, our findings indicate that Mf presence partially blocked transendothelial migration of monocytes and neutrophils, but not lymphocytes. However, Mf exposure did not result in altered vascular EC expression of key mediators of the tethering stage of extravasation, such as ICAM-1, VCAM-1 and various chemokines. To further analyse the immunological function of vascular EC in the presence of Mf, we measured the mRNA and/or protein expression of a number of pro-inflammatory mediators. We found that expression levels of the mediators tested were predominantly unaltered upon B. malayi Mf exposure. In addition, a comparison of angiogenic mediators induced by intact Mf and Wolbachia-depleted Mf revealed that even intact Mf induce the expression of remarkably few angiogenic mediators in vascular EC. Our study suggests that live microfilariae are remarkably inert in their induction and/or activation of vascular cells in their immediate local environment. Overall, this work presents important insights into the immunological function of the vascular endothelium during an infection with B. malayi.     

Brugia malayi: localization of nitric oxide synthase in a lymphatic filariid

Nitric oxide synthase converts L-arginine to citrulline and nitric oxide, a gaseous signaling molecule critical to multiple physiological responses. Nitric oxide synthase was detected by Western blot analysis of Brugia malayi extracts using an antibody raised against a peptide from murine brain nitric oxide synthase. Using NADPH diaphorase staining and immunohistochemistry, nitric oxide synthase was localized in the parasitic nematode B. malayi. As in Ascaris suum, nitric oxide synthase was detected in the body wall muscles of adult B. malayi. This localization pattern is in agreement with the role of nitric oxide in the control of muscle tone in other invertebrates and in vertebrates. A novel finding was the localization of nitric oxide synthase in the oocytes, in developing embryos, and in spermatozoa. B. malayi nitric oxide synthase may play a role in developmental signaling, as has been suggested for Drosophila and Ilyanassa, a marine mud snail.     

Brugia malayi: whole genome amplification for genomic characterization of filarial parasites

Genetic characterization of field isolates and clinical specimens of filarial nematodes is often limited by a shortage of DNA; therefore, we evaluated a multiple displacement amplification (MDA) based whole genome amplification method. The quality of amplified DNA was examined by conventional PCR, real-time PCR, and DNA hybridization. MDA of 5.0 ng of adult Brugia malayi DNA and one-fifteenth of the DNA isolated from a single microfilaria resulted in 6.3 and 4.2 μg of amplified DNA, respectively. Amplified DNA was equivalent to native genomic DNA for hybridization to B. malayi BAC library clones or to an oligonucleotide microarray with approximately 18,000 filarial DNA sequences. MDA is useful for whole genome amplification of filarial DNA from very small amounts of starting material. This technology will permit detailed studies of genetic diversity that were not previously feasible.