Designing of a novel multi-epitope peptide based vaccine against Brugia malayi: An in silico approach.

In spite of the tremendous efforts of the World Health Organization, scientific and medical community to eradicate lymphatic filariasis (LF) within 2020, the disease is still taking a huge toll on mankind throughout the globe. The current therapeutic strategies and solution measures against this alarming condition are suffering from a number of limitations such as inadequate effectiveness of the drugs against the adult-stage parasites, low bioavailability, and emergence of resistance. Considering this situation, development of the new therapeutics are urgently needed to combat human LF, especially targeting the adult filarial nematodes. Brugia malayi, the causative parasite for the human brugian filariasis majorly found in the countries of the South-Asia. In this study, we have designed a vaccine candidate using B-cell and T-cell epitopes derived from the aspartic protease of B. malayi (BmASP-1) and found to display significant humoral and cell mediated immune responses using in-silico approaches. Protein-protein docking between the human Toll-like receptor 4 (TLR4) and the vaccine candidate helped us to predict the way of inductive signaling that leads to immune-response. Molecular dynamics (MD) simulation studies further confirmed the proper docking between the TLR4 and vaccine candidate. Moreover, in-silico cloning of the vaccine element within the expression vector was found useful to optimize the restriction sites as well as to determine the primer location. Taken together, the in-silico vaccine candidate depicted in this study promises could be a useful therapeutic option for treating LF and experimental validation of this study is expected to strengthen the candidature of the said vaccine in the future.

Effects of cross-mating on susceptibility of synonymous mosquitoes, Anopheles paraliae and Anopheles lesteri to infection with nocturnally subperiodic Brugia malayi.

In Southeast Asia, Anopheles lesteri (recently synonymized with An. paraliae) is a competent vector for Plasmodium parasites, but its ability to transmit parasites that cause lymphatic filariasis has yet to be determined. In this study, the susceptibility of An. lesteri and An. paraliae to Brugia malayi parasites was determined by comparing with the control mosquito, Aedes togoi. We found that the infection prevalence per infected mosquito in An. paraliae was significantly lower than that in Ae. togoi in all experiments (p < 0.05). Reciprocal crosses (female An. paraliae x male An. lesteri) produced highly susceptible F1-hybrid progeny, with increased infection prevalence when compared to parental stocks (p < 0.05). Subsequently, the possibilities of introgression between high and low/moderate parasite susceptibility genes were investigated by cross-mating experiments (parental, reciprocal crosses, back crosses and repeated backcrosses). The results showed the possibility of introgression of B. malayi-susceptible genes between An. paraliae (low/moderate susceptibility) and An. lesteri (high susceptibility) based on increasing or decreasing susceptibility and normal larval development in the thoracic muscles of F3-hybrids. Additionally, melanization, an innate immune response with proven involvement in the susceptibility or refractoriness of mosquitoes to B. malayi parasites, was examined. Parasite degeneration and cell aggregation, and melanization were observed for first-stage larvae in the thoracic muscle fibers of hybrid mosquitoes.

Microfilariae of Brugia malayi Inhibit the mTOR Pathway and Induce Autophagy in Human Dendritic Cells.

Immune modulation is a hallmark of patent filarial infection, including suppression of antigen-presenting cell function and downmodulation of filarial antigen-specific T cell responses. The mammalian target of rapamycin (mTOR) signaling pathway has been implicated in immune regulation, not only by suppressing T cell responses but also by regulating autophagy (through mTOR sensing amino acid availability). Global proteomic analysis (liquid chromatography-tandem mass spectrometry) of microfilaria (mf)-exposed monocyte-derived dendritic cells (DC) indicated that multiple components of the mTOR signaling pathway, including mTOR, eIF4A, and eIF4E, are downregulated by mf, suggesting that mf target this pathway for immune modulation in DC. Utilizing Western blot analysis, we demonstrate that similar to rapamycin (a known mTOR inhibitor), mf downregulate the phosphorylation of mTOR and its regulatory proteins, p70S6K1 and 4E-BP1, a process essential for DC protein synthesis. As active mTOR signaling regulates autophagy, we examined whether mf exposure alters autophagy-associated processes. mf-induced autophagy was reflected in marked upregulation of phosphorylated Beclin 1, known to play an important role in both autophagosome formation and autolysosome fusion, in induction of LC3II, a marker of autophagosome formation, and in induced degradation of p62, a ubiquitin-binding protein that aggregates protein in autophagosomes and is degraded upon autophagy that was reduced significantly by mf exposure and by rapamycin. Together, these results suggest that Brugia malayi mf employ mechanisms of metabolic modulation in DC to influence the regulation of the host immune response by downregulating mTOR signaling, resulting in increased autophagy. Whether this is a result of the parasite-secreted rapamycin homolog is currently under study.

Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

Lymphatic filariasis and onchocerciasis are parasitic helminth diseases, which cause severe morbidities such as elephantiasis, skin disease and blindness, presenting a major public health burden in endemic communities. The anti-Wolbachia consortium (A·WOL: http://www.a-wol.com/) has identified a number of registered antibiotics that target the endosymbiotic bacterium, Wolbachia, delivering macrofilaricidal activity. Here we use pharmacokinetics/pharmacodynamics (PK/PD) analysis to rationally develop an anti-Wolbachia chemotherapy by linking drug exposure to pharmacological effect. We compare the pharmacokinetics and anti-Wolbachia efficacy in a murine Brugia malayi model of minocycline versus doxycycline. Doxycycline exhibits superior PK in comparison to minocycline resulting in a 3-fold greater exposure in SCID mice. Monte-Carlo simulations confirmed that a bi-daily 25-40 mg/Kg regimen is bioequivalent to a clinically effective 100-200 mg/day dose for these tetracyclines. Pharmacodynamic studies showed that minocycline depletes Wolbachia more effectively than doxycycline (99.51% vs. 90.35%) after 28 day 25 mg/Kg bid regimens with a more potent block in microfilarial production. PK/PD analysis predicts that minocycline would be expected to be 1.7 fold more effective than doxycycline in man despite lower exposure in our infection models. Our findings warrant onward clinical investigations to examine the clinical efficacy of minocycline treatment regimens against lymphatic filariasis and onchocerciasis.

Seasonal prevalence of mosquitoes, including vectors of Brugian filariasis, in southern islands of the Republic of Korea.

A survey of mosquitoes, including the vector status of Brugia malayi filariasis and their relative larval density, was conducted from 2002 to 2005 at several southern remote islands of Jeollanam-do (province), Gyeongsangnam-do, and Jeju-do, Korea, where filariasis was previously endemic. Overall, a total of 9 species belonging to 7 genera were collected. Ochlerotatus togoi (formerly known as Aedes togoi), Anopheles (Hyrcanus) group, and Culex pipiens were the predominant species captured at all areas. Oc. togoi larvae were most frequently collected at salinity levels <0.5% during June and July, with densities decreasing sharply during the rainy season in August. The most likely explanation for the eradication of filariasis in these areas is suggested to be an aggressive treatment program executed during the 1970s and the 1990s. However, high prevalence of the vector mosquitoes may constitute a potential risk for reemerging of brugian filariasis in these areas.

Seasonal Prevalence of Mosquitoes, Including Vectors of Brugian Filariasis, in Southern Islands of the Republic of Korea

A survey of mosquitoes, including the vector status of Brugia malayi filariasis and their relative larval density, was conducted from 2002 to 2005 at several southern remote islands of Jeollanam-do (province), Gyeongsangnam-do, and Jeju-do, Korea, where filariasis was previously endemic. Overall, a total of 9 species belonging to 7 genera were collected. Ochlerotatus togoi (formerly known as Aedes togoi), Anopheles (Hyrcanus) group, and Culex pipiens were the predominant species captured at all areas. Oc. togoi larvae were most frequently collected at salinity levels <0.5% during June and July, with densities decreasing sharply during the rainy season in August. The most likely explanation for the eradication of filariasis in these areas is suggested to be an aggressive treatment program executed during the 1970s and the 1990s. However, high prevalence of the vector mosquitoes may constitute a potential risk for reemerging of brugian filariasis in these areas.

Isolation of an antigen fraction from Setaria cervi adults having potential for immunodiagnosis of human filariasis.

Crude antigenic preparations from heterologous filarial parasites gave false positive results because of complex nature of these antigens and their cross-reactivity with other helminth parasites. In the present study, efforts have been made to isolate and characterize the antigens from Setaria cervi important for diagnostic purposes. The fractionation of S. cervi somatic antigenic preparation on Sephacryl S-200 resulted in separation of three major antigenic peak fractions. Crossed immunoelectrophoretic analysis, using immune rabbit serum, revealed 13-14 antigens in SFP-I pool fraction, which showed high reactivity with filarial patients sera as compared to other two pool fractions. This SFP-I fraction was further purified by DEAE-Cellulose column chromatography. Out of the 4 antigen pool fractions, DFP-IV fraction showed high ELISA reactivity with filarial patient serum pool (Wuchereria bancrofti and Brugia malayi) as compared to other fractions. The SDS-PAGE analysis of DFP-IV fraction revealed 2 major and 1 minor protein bands (mol. wt. range 65-70 kDa). Crossed immunoelectrophoresis also showed the presence of 3 antigenic peaks in DFP-IV fraction. The purified DFP-IV fraction showed high reactivity with filarial patients sera but did not cross-react with sera from ascaris and hookworm infections thereby suggesting the filaria-specificity and potential for immunodiagnosis of human filariasis.

Cystatins from filarial parasites: evolution, adaptation and function in the host-parasite relationship.

Cystatins, together with stefins and kininogens, are members of the cystatin superfamily of cysteine protease inhibitors (CPI) present across the animal and plant kingdoms. Their role in parasitic organisms may encompass both essential developmental processes and specific interactions with the parasite's vector and/or final host. We summarise information gathered on three cystatins from the human filarial nematode Brugia malayi (Bm-CPI-1, -2 and -3), and contrast them those expressed by other parasites and by the free-living nematode Caenorhabditis elegans. Bm-CPI-2 differs from C. elegans cystatin, having acquired the additional function of inhibiting asparaginyl endopeptidase (AEP), in a manner similar to some human cystatins. Thus, we propose that Bm-CPI-2 and orthologues from related filarial parasites represent a new subset of nematode cystatins. Bm-CPI-1 and CPI-3 share only 25% amino acid identity with Bm-CPI-2, and lack an evolutionarily conserved glycine residue in the N-terminal region. These sequences group distantly from the other nematode cystatins, and represent a second novel subset of filarial cystatin-like genes. Expression analyses also show important differences between the CPI-2 and CPI-1/-3 groups. Bm-cpi-2 is expressed at all time points of the parasite life cycle, while Bm-cpi-1 and -3 expression is confined to the late stages of development in the mosquito vector, terminating within 48h of infection of the mammalian host. Hence, we hypothesise that CPI-2 has evolved to block mammalian proteases (including the antigen-processing enzyme AEP) while CPI-1 and -3 function in the milieu of the mosquito vector necessary for transmission of the parasite.

Susceptibility of Mansonia uniformis to Brugia malayi microfilariae from infected domestic cat.

Microfilariae of Brugia malayi is transmitted to man and other susceptible hosts via mosquito. The transmission of B. malayi from cat to man by Ma. uniformis bite has never been reported. The Ma. uniformis mosquito is the normal vector for Wuchereria bancrofti but has never been reported as a vector for B. malayi, or a susceptible host for the growth and development of the microfilariae of B. malayi. The purpose of this study was to examine the development of B. malayi in Mansonia uniformis after feeding on the blood of an infected cat in the laboratory. The B. malayi infected cat was identified using PCR with the primers Bm-1/Bm-2 on DNA (at 10 ng/50 microl) extracted from the WBC of the cat. W. bancrofti was employed as a negative control. The sensitivity of the B. malayi DNA detection by PCR was 0.0001 ng. Adult Ma. uniformis mosquitos at the ages of 5, 10, and 15 days, 100 mosquitos in each group, were fed on the infected cat blood. Recovery of third stage microfilariae was found to be the highest in the 5-day old mosquito group (48%), followed by the 10- and 15-day old mosquito groups (32% and 18%, respectively). The mean number of B. malayi microfilariae found in thorax, head, and abdomen of the mosquitos were composed. The 5-day old (40.3%) and 10-day old (41.9%) mosquitos were significantly more susceptible to microfilariae than the 15-day old mosquitos (17.8%) (p-values using the Scheffe method: 0.027 and 0.039, respectively). There was no significant difference in the mean number of microfilariae in the thorax (p = 0.482) by age, but the mean numbers of microfilariae in the heads, and abdomens were significantly different by age between the 5- and10-, and the 15-day old mosquitos (p < 0.001 and p = 0.004, respectively).

Use of RNA interference to investigate gene function in the human filarial nematode parasite Brugia malayi.

We describe the successful use of the reverse genetic technique RNA interference (RNAi) to investigate gene function in the human filarial nematode parasite Brugia malayi. We used fluorescently labelled double stranded RNA (dsRNA) to demonstrate that 300 bp molecules are able to enter adult females in culture while they remain excluded from microfilariae (mf). We have developed an optimised microvolume culture system to allow the exposure of parasites to high concentrations of dsRNA for extended periods. Culturing of adult female parasites in this system for 24h does not significantly reduce parasite lifespan or mf release in culture. Three B. malayi genes, beta-tubulin (Bm-tub-1), RNA polymerase II large subunit (Bm-ama-1) and B. malayi mf sheath protein 1/mf22 (Bm-shp-1) were targeted by soaking adult female B. malayi in dsRNA complementary to these transcripts in the optimised culture system. Targeting of the two housekeeping genes Bm-tub-1 and Bm-ama-1 led to a reduction in the levels of their transcripts, as assessed by reverse transcriptase coupled PCR (RT-PCR), and resulted in parasite death in culture. In contrast, targeting of the Bm-shp-1 gene was not lethal to adult females in culture. A marked reduction in mf release was observed for shp-1 RNAi parasites compared to controls and in addition 50% of mf released did not have fully elongated sheaths. This "short" phenotype correlated with the loss of the stockpiled shp-1 transcript from developing mf in treated adult female gonads. From these data we conclude that RNAi may be a useful method for assessment of drug target potential of genes identified in filarial gene discovery projects.

The Brugia malayi genome project: expressed sequence tags and gene discovery.

To advance and facilitate molecular studies of Brugia malayi, one of the causative agents of human lymphatic filariasis, an expressed sequence tag (EST)-based gene discovery programme has been carried out. Over 22,000 ESTs have been produced and deposited in the public databases by a consortium of laboratories from endemic and non-endemic countries. The ESTs have been analysed using custom informatic tools to reveal patterns of individual gene expression that may point to potential targets for future research on anti-filarial drugs and vaccines. Many genes first discovered as ESTs are now being analysed by researchers for immunodiagnostic, vaccine and drug target potential. Building on the success of the B. malayi EST programme, significant EST datasets are being generated for a number of other major parasites of humans and domesticated animals, and model parasitic species.

Cytochemical analysis of the sheath of microfilariae of Wuchereria bancrofti and Brugia malayi.

Thin sections of Epon and Lowicryl embedded microfilariae of Wuchereria bancrofti and Brugia malayi were analyzed by transmission electron microscopy aiming at topochemical characterization of the sheath. Three layers could be distinguished. Some of the layers were labeled when incubated in the presence of antibodies, lectins and enzymes which recognize extracellular matrix components usually associated with the basal laminae lining epithelial cells.