Humans from Wuchereria bancrofti endemic area elicit substantial immune response to proteins of the filarial parasite Brugia malayi and its endosymbiont Wolbachia

Detailed information of the individuals of bancroftian filariasis endemic area participated in the current study. (PDF 54 kb

X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication

Integrated dataset of screening hits against multiple neglected disease pathogens.

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach

Immunization with Brugia malayi Myosin as Heterologous DNA Prime Protein Boost Induces Protective Immunity against B. malayi Infection in Mastomys coucha.

The current control strategies employing chemotherapy with diethylcarbamazine, ivermectin and albendazole have reduced transmission in some filaria-endemic areas, there is growing interest for complementary approaches, such as vaccines especially in light of threat of parasite developing resistance to mainstay drugs. We earlier demonstrated recombinant heavy chain myosin of B. malayi (Bm-Myo) as a potent vaccine candidate whose efficacy was enhanced by heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) vaccination in BALB/c mice. BALB/c mouse though does not support the full developmental cycle of B. malayi, however, the degree of protection may be studied in terms of transformation of challenged infective larvae (L3) to next stage (L4) with an ease of delineating the generated immunological response of host. In the current investigation, DNA vaccination with Bm-Myo was therefore undertaken in susceptible rodent host, Mastomys coucha (M. coucha) which sustains the challenged L3 and facilitates their further development to sexually mature adult parasites with patent microfilaraemia. Immunization schedule consisted of Myo-pcD and Myo-pcD+Bm-Myo followed by B. malayi L3 challenge and the degree of protection was evaluated by observing microfilaraemia as well as adult worm establishment. Myo-pcD+Bm-Myo immunized animals not only developed 78.5% reduced blood microfilarial density but also decreased adult worm establishment by 75.3%. In addition, 75.4% of the recovered live females revealed sterilization over those of respective control animals. Myo-pcD+Bm-Myo triggered higher production of specific IgG and its isotypes which induced marked cellular adhesion and cytotoxicity (ADCC) to microfilariae (mf) and L3 in vitro. Both Th1 and Th2 cytokines were significantly up-regulated displaying a mixed immune response conferring considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a potential vaccination method against LF

<span style="font-size: 20.0pt;mso-bidi-font-size:14.0pt;font-family:"Times New Roman","serif"">Transmission efficiency of <i>Culex quinquefasciatus </i>and <i>Aedes aegypti </i>to <i>Wuchereria bancrofti </i>infection : An experimental study* </span>

98-100<span style="font-size: 14.5pt;mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif""="">Present study was undertaken to evaluate the suitability of natural (Culex quinquefascialus) and experimental (Aedes aegypti) vectors for supporting the development of W. bancrofti larvae for onward transmission. Both the species permitted development of W. bancrofti mf to infective larvae (L3) within 11 to 13 days. The mf intake by both the species of mosquitoes was directly related to mf density in donor's blood. Culex exhibited higher <span style="font-size:14.5pt;mso-bidi-font-size: 8.5pt;font-family:" arial","sans-serif""="">L3 recovery than Aedes. In Aedes maximum percent L1 development occurred after ingesting 4 mf whereas Culex exhibited best establishment at an average mf intake of 11.5. Nevertheless wide variation in mf density in donor' s blood did not significantly affect the larval establishment in Aedes mosquito while in Culex very high (>400 mf / 40µl) or low ( 40 µl) mf counts in donor's blood adversely affected <span style="font-size: 14.5pt;mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif""="">the L3, recovery. The results reveals that A. aegrpti has an edge over the natural vector, Culex in being a voracious feeder, their easy laboratory maintenance and better transmission potential.<span style="font-size: 17.0pt;mso-bidi-font-size:11.0pt"> </span

Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model.

We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo) of Brugia malayi (B. malayi) in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo) and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo) in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+) and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32) against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23) and pcD-Myo (41.6%±2.45). In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC) to B. malayi infective larvae (L3). pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of providing high degree of protection against filarial larval invasion

Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi.

The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies

Additional file 1: Figure S1. of RNA interference mediated knockdown of Brugia malayi UDP-Galactopyranose mutase severely affects parasite viability, embryogenesis and in vivo development of infective larvae

The graph bars depict percent relative intensity of bands on western blot in comparison to medium control at different time points of exposure (24, 48, 72 h) and after exposure (96, 120 h) as quantified by imageJ version 1.47 software and the blot bands have been shown below each corresponding bar. Considering the medium control intensity to be 100% at each time points the relative intensities of treated ones have been evaluated. P-value < 0.05 was considered significant (*), P < 0.01 as highly significant (**) and P < 0.001 as very highly significant (***). (TIF 4078 kb

Immunization with <i>Brugia malayi</i> Myosin as Heterologous DNA Prime Protein Boost Induces Protective Immunity against <i>B</i>. <i>malayi</i> Infection in <i>Mastomys coucha</i> - Fig 3

<p><b>Antibody-dependent cell-mediated adhesion and cytotoxicity (ADCC) to (A) L3 and (B) mf.</b> Adhesion of PECs on the surface of <i>B</i>. <i>malayi</i> L3 and mfwas observed after 48 h microscopically in the presence of sera of different groups (a) pcD (b) Adj (c) pcD+Adj and (d) Myo-pcD (e) Bm-Myo (f) Myo-pcD+Bm-Myo. Photographs were captured on phase contrast fluorescent microscope (Nikon, Japan) at 40X magnification.</p