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The abundant larval transcript-1 and -2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis.

ABSTRACT: Lymphatic filariasis is a major tropical disease caused by the mosquito-borne nematodes Brugia and Wuchereria. About 120 million people are infected and at risk of lymphatic pathology such as acute lymphangitis and elephantiasis. Vaccines against filariasis must generate immunity to the infective mosquito-derived third-stage larva (L3) without accentuating immunopathogenic responses to lymphatic-dwelling adult parasites. We have identified two highly expressed genes, designated abundant larval transcript-1 and -2 (alt-1 and alt-2), from each of which mRNAs account for >1% of L3 cDNAs. ALT-1 and ALT-2 share 79% amino acid identity across 125 residues, including a putative signal sequence and a prominent acidic tract. Expression of alt-1 and alt-2 is initiated midway through development in the mosquito, peaking in the infective larva and declining sharply following entry into the host. Humans exposed to Brugia malayi show a high frequency of immunoglobulin G1 (IgG1) and IgG3 antibodies to ALT-1 and -2, distinguishing them from adult-stage antigens, which are targeted by the IgG4 isotype. Immunization of susceptible rodents (jirds) with ALT-1 elicited a 76% reduction in parasite survival, the highest reported for a single antigen from any filarial parasite. ALT-1 and the closely related ALT-2 are therefore strong candidates for a future vaccine against human filariasis.

SUBMITTER: Gregory WF

PROVIDER: S-EPMC101719 | biostudies-literature | 2000 Jul

REPOSITORIES: biostudies-literature

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The abundant larval transcript-1 and -2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis.

Gregory W F WF Atmadja A K AK Allen J E JE Maizels R M RM

Infection and immunity 20000701 7

Lymphatic filariasis is a major tropical disease caused by the mosquito-borne nematodes Brugia and Wuchereria. About 120 million people are infected and at risk of lymphatic pathology such as acute lymphangitis and elephantiasis. Vaccines against filariasis must generate immunity to the infective mosquito-derived third-stage larva (L3) without accentuating immunopathogenic responses to lymphatic-dwelling adult parasites. We have identified two highly expressed genes, designated abundant larval t ...[more]

PMID: 10858234

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Project description:Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1 Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.

Project description:The lack of effective short-course therapies for treatment of the adult stage of filarial worms is a major limitation in the global effort to eliminate lymphatic filariasis. Studies using current small mammal models of lymphatic filariasis are limited by difficulties in quantifying adult worm numbers and in assessing lymphatic anatomy and function.Here, we re-established Brugia malayi infection of ferrets as a model for lymphatic filariasis and demonstrated parasitological, immunological, and histological parallels with human infection. Subcutaneous injection of L3 larvae into a hind-footpad resulted in a mean of 18 adult worms recovered 16 weeks post-infection, primarily from the draining inguinal and femoral lymphatics of the injected limb. Infected ferrets developed microfilaremia, with patency lasting from 12-26 weeks post-infection. Quantitative PCR assessing cytokine transcription by antigen-stimulated lymph node cells demonstrated a mixed Th1/Th2 response occurring during early infection. Immunoregulation with production of down-regulatory cytokine IL-10 occurred just prior to peak microfilaremia. Histological analysis revealed progressive inflammation of the lymphatic vessel walls, with intimal thickening and disorganization of collagen fibers. Inflammation was observed as early as 8 weeks post-infection and extended into the perivascular and subcutaneous tissues by 16 weeks post-infection. Finally, we developed a novel ferret PET/CT lymphoscintigraphy method demonstrating substantial changes in lymphatic anatomy and function as early as 3 weeks post-infection, with progression over the course of infection.B. malayi infection of ferrets is a robust model of human lymphatic filariasis that can be utilized to study efficacy of novel antifilarial agents against adult worms residing within lymphatic vessels. In conjunction with PET/CT lymphoscintigraphy, this model can also be used to investigate pathogenesis of lymphatic dysfunction in lymphatic filariasis and efficacy of medications aimed at reversing lymphatic dysfunction after clearance of adult worms.

Project description: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.

Project description:BackgroundMost 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 findingsA 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.ConclusionsImmunohistology 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.

Dataset Information

The abundant larval transcript-1 and -2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis.

Publications

The abundant larval transcript-1 and -2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis.

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