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The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order 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., Author summary Almost a billion people receive >7.7 billion doses of treatment aimed at eliminating lymphatic filariasis, which is caused by three filarial nematodes: Wuchereria bancrofti, Brugia malayi, and Brugia timori. Drug resistance and adaptation are both associated with pathogen success as well as higher levels of genetic diversity. In an examination of genetic diversity in Brugia malayi and Brugia pahangi, we observed a lack of genetic diversity over a third of the genome that is found on chromosome X. These species have neo-X chromosomes where a chromosome X fused with an autosome. Using publicly-available published data, the other filarial nematodes of greatest human significance are also found to have a similar lack of genetic diversity on their neo-X chromosomes. The two filarial nematodes with publicly-available data that lack neo-X chromosomes did not have this lack of genetic diversity. This lack of sequence diversity in B. malayi, W. bancrofti, and O. volvulus could have profound effects on all traits encoded on chromosome X.

The recombinant heavy chain myosin of Brugia malayi (Bm-Myo) has earlier been reported as a potent vaccine candidate in our lab. Subsequently, we further enhanced its efficacy employing heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) immunization approach that produced superior immune-protection than protein or DNA vaccination. In the present study, we evaluated the efficacy of heterologous prime boost vaccination in combination with CpG, synthetic oligodeoxynucleotides (ODN) adjuvant in BALB/c mice. The results showed that CpG/Myo-pcD+Bm-Myo conferred 84.5 ± 0.62% protection against B. malayi infective larval challenge which was considerably higher than Myo-pcD+Bm-Myo (75.6 ± 1.10%) following immunization. Although, both the formulations of immunization elicited robust production of specific IgG antibody and their isotypes (IgG1, IgG2a, IgG2b, and IgG3); however, CpG/Myo-pcD+Bm-Myo predominantly enhanced the level of IgG2a suggesting Th1 biased immune response in presence of CpG. Furthermore, spleen isolated from mice that immunized with CpG/Myo-pcD+Bm-Myo had greater accumulation of CD4+, CD8+, and CD19+ B cells and there was an augmented expression of co-stimulatory molecules CD40, CD86 on host dendritic cells (DCs). In contrast to Myo-pcD+Bm-Myo group, the splenocytes of CpG/Myo-pcD+Bm-Myo immunized mice developed comparatively higher pro-inflammatory cytokines IL-2 and IFN-γ leaving anti-inflammatory cytokine levels unchanged. Moreover, CpG formulation also upregulated the RNA expression of IL-12 and TNF-α in spleenocytes. The current findings suggest that the use of CpG would be more advantageous as an adjuvant predominantly in DNA/protein prime boost vaccine against Bm-Myo and presumably also for filarial infection.

Variations due to geographical location and dioecious nature have shown implications in the chemical and pharmacological properties of medicinal plants and their herbal products. Tinospora cordifolia is one of the most important dioecious plant distributed throughout India and very widely used in many herbal products and formulations. In this study a method combining direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight (TOF) mass spectrometer (MS) along with multivariate analysis was developed and applied for metabolic fingerprinting and screening of the major phytochemicals in this plant. Using this approach phytodiversity in plants due to gender and geographical distribution were studied in T. cordifolia stem cuttings without any processing. An aqueous/ethanolic stem extracts of male and female T. cordifolia were also evaluated for immunomodulatory activity in inbred strain of age and sex matched BALB/c mice. A characteristic nine and sixteen marker peaks were respectively, identified as gender and geographical markers for T. cordifolia stem. It also discriminates the herbal and polyherbal formulations of T. cordifolia stem using principal component analysis. Female plant stem extract caused a significant up regulation in the pro-inflammatory and anti-inflammatory cytokines and activated the peritoneal exudate cells leading to significant release in reactive oxygen species and enhanced the in vitro lymphocyte proliferation than male stem extract. This finding underscore the importance of gender in all dioecious medicinal plants where only vegetative parts are used as a source of drug as the pharmacological activity may vary depending on the sex of the plant used.

The resolution of racemic coumarinyl amino alcohols 5–10 was achieved by using the inexpensive and readily accessible chiral resolving agent N-carbethoxy- l -proline (S)-11. Direct esterification of rac-5–10 with (S)-11 furnished diastereomeric esters, which were easily separated by column chromatography. The obtained diastereomers yielded the desired enantiopure coumarinyl amino alcohols (S)-(+)-5–10 and (R)-(−)-5–10 in good yields with high enantiomeric excess on saponification. The absolute configurations were determined by X-ray crystal analysis and/or by comparison of the specific rotations. Furthermore, in in vitro antifilarial motility inhibition assays, enantiopure coumarins (S)-(+)-9, (R)-(−)-9 and (S)-(+)-10, (R)-(−)-10 were found to be less efficient in affecting the viability of macrofilariae of Brugia malayi than their racemic forms 9 and 10, respectively, indicating the synergistic effect of the enantiomers in evoking antifilarial action.

Background Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most important medicinal plant in the traditional Indian medical systems. Pharmacological studies have established that root extracts of W. somnifera contain several bioactive constituents called withanolides. The plant has long been used for its several beneficial properties and recently as an immunomodulator. Hypothesis/Purpose A combination therapy including a potential and safe immunostimulant with lower doses of effective drug, which can reduce the parasitic burden and simultaneously can produce an enhancement of adaptive immunity, has proven to be significantly a more effective approach than immunotherapy or drug therapy alone. Study design Evaluation of the immunostimulatory effect of W. somnifera chemotype NMITLI 101R when used in combination with ED 50 doses of antileishmanial drugs in Leishmania donovani infected hamsters. Methods Infected animals were administered with chemotype 101R(30 mg/kg × 15 days) either alone or in combination with ED 50 doses of miltefosine (10 mg/kg × 5 days), paromomycin (30 mg/kg × 5 days) or amphotericin B (0.5 mg/kg × 5 days). The treated animals were euthanized on days 30 and 60 post-treatment (p.t.) and checked for parasite clearance, delayed type hypersensitivity (DTH) response, cytokine and inducible nitric oxide synthase levels by real-time PCR, nitric oxide (NO) production, reactive oxygen species (ROS) generation, lymphoproliferative and antibody responses. Results The group of animals that received 101R and ED 50 dose of miltefosine showed optimum inhibition of parasite multiplication (∼98%) by day 60 p.t. followed by the group that received 101R plus paromomycin (∼94%) and 101R plus amphotericin B (∼93%). The efficacy was well supported by the increased inducible NO synthase mRNA transcript, strong IFN-γand IL-12 mediated Th1 immune responses and significantly suppressed levels of Th2 cytokines (IL-4, IL-10 and TGF-β). Additionally, same therapy also induced significant increase in the level of NO production, ROS generation, Leishmania specific IgG2 antibody along with profound DTH and strong T-cell responses as compared with all the other treated groups. Conclusion Our results suggest that combination of chemotype 101R with ED 50 doses of antileishmanial drugs may provide a promising alternative for the cure of visceral leishmaniasis with significant restoration of the host immune response.

The present study is aimed to evaluate the antifilarial activity of Eucalyptus globules Labill. ( Myrtaceae ) against human lymphatic filarial parasite Brugia malayi in vitro and in vivo . The ethanolic extract of the leaves was tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active sample was further evaluated in vivo in B. malayi intraperitoneally (i.p.) transplanted in the jird model ( Meriones unguiculatus) and Mastomys coucha subcutaneously infected with infective larvae (L3). The ethanolic extract of the leaves of the E. globulus was tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active sample was further evaluated in vivo in B. malayi. The ethanolic extract was active in vitro (IC 50 : adult = 62.5 μg/ml; mf = 31.2. μg/ml) where it demonstrated 66.7% adulticidal and embryostatic effect on B. malayi in Mastomys at a dose of 5 × 100 mg/kg by oral route. The antifilarial test conducted was at 5 × 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 66.7% transplanted adult B. malayi in the peritoneal cavity of jirds in addition to noticeable microfilaricidal action on the day of autopsy. The findings revealed that the extract from the leaves of E. globulus contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which may be further explored to new antifilarial agents. Bangladesh Pharmaceutical Journal 19 (1): 44-47, 2016

Systemic chemotherapeutic targeting of filarial parasites is unfocused due to their deep seated location in lymphatic vessels. This warrants a prolonged dosing regimen in high doses for an anthelmintic like doxycycline hydrochloride (DOX). In order to provide an alternative, we have constructed ultrafine PLGA nanoparticles of DOX (DPNPs), so as to exploit the peculiarity of lymphatic vasculature underneath the subcutaneous layer of skin, which preferentially allows entry of only 10-100 nm sized particles. DPNPs were constructed using a novel solvent diffusion method aided by probe sonication, which resulted in an average size 95.43 ± 0.8 nm as per DLS, PDI 0.168 ± 0.03, zeta potential -7.38 ± 0.32, entrapment efficiency 75.58 ± 1.94%, and refrigerator stability of 7 days with respect to size in the optimized batch. TEM further substantiated the spherical shape of DPNPs along with their actual nonhydrated size as being well below 100 nm. FTIR analysis of DOX, dummy nanoparticles, and freeze-dried DPNPs revealed that the formulation step did not induce prominent changes in the chemical nature of DOX. The drug release was significantly altered (p0.05) with 64.6 ± 1.67% release in 48 h from DPNPs and was dictated by Fickian diffusion. Pharmacokinetic studies in Wistar rats further revealed that DPNPs caused a 16-fold prolongation in attainment of plasma Tmax and a 2-fold extension of elimination half-life (28.569 ± 1.27 h) at a dose of 5 mg/kg when compared to native drug (DOX solution) of the same strength. Contrastingly the trend was reversed in regional lymph nodes where Cmax for DPNPs (820 ± 84 ng/mg) was 4-fold greater, and lymphatic Tmax was attained in one-fourth of what was required for DOX solution. This size based preferential lymphatic targeting resulted in significantly greater in vivo antifilarial activity of DPNPs when compared to DOX solution as gauged by several parameters in Brugia malayi infected Mastomys coucha. Interestingly, the magnification in efficacy was obtained despite equivalent in vitro antifilarial activity of DOX solution and DPNPs against B. malayi worms.

Background In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes ( Rv3111 , Rv0864 and Rv3324c ) encode for MoaC homologs. Out of these three only MoaC1 ( Rv3111 ) is secretory in nature. Methods We have characterized MoaC1 protein through biophysical, in-silico , and immunological techniques. Results We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ. Conclusion Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response. General significance This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.

Vaccination is devised/formulated to stimulate specific and prolonged immune responses for long-term protection against infection or disease. A vaccine component, namely adjuvant, enhances antigen recognition by the host immune system and thereby stimulates its cellular and adaptive responses. Especially synthetic Toll-like receptor (TLR) agonists having self-assembling properties are considered as good candidates for adjuvant development. Here, a human TLR4-derived 20-residue peptide (TR-433), present in the dimerization interface of the TLR4–myeloid differentiation protein-2 (MD2) complex, displayed self-assembly and adopted a nanostructure. Both in vitro studies and in vivo experiments in mice indicated that TR-433 is nontoxic. TR-433 induced pro-inflammatory responses in THP-1 monocytes and HEK293T cells that were transiently transfected with TLR4/CD14/MD2 and also in BALB/c mice. In light of the self-assembly and pro-inflammatory properties of TR-433, we immunized with a mixture of TR-433 and either ovalbumin or filarial antigen trehalose-6-phosphate phosphatase (TPP). A significant amount of IgG titers was produced, suggesting adjuvanting capability of TR-433 that was comparable with that of Freund's complete adjuvant (FCA) and appreciably higher than that of alum. We found that TR-433 preferentially activates type 1 helper T cell (T(h)1) response rather than type 2 helper T cell (T(h)2) response. To our knowledge, this is the first report on the identification of a short TLR4-derived peptide that possesses both self-assembling and pro-inflammatory properties and has significant efficacy as an adjuvant, capable of activating cellular responses in mice. These results indicate that TR-433 possesses significant potential for development as a new adjuvant in therapeutic application.

Lymphatic filariasis leads to profound impairment of parasite‐specific T helper type 1 (Th1) and Th2 immune responses and significantly increases the expression of regulatory networks and regulatory effectors like transforming growth factor‐β, CD25, cytotoxic T‐lymphocyte antigen 4, glucocorticoid‐induced tumour necrosis factor receptor (GITR) and regulatory T (Treg) cells, which together play an important role in immunosuppression. While Treg cells suppress the activity of effector cells, monocyte dysfunction, characterized by an alternatively activated immunoregulatory phenotype, is one hypothesis that explains the lack of an antigen‐specific T‐cell response in infected individuals. In the present study, we administered neutralizing antibodies against the Treg cell‐associated markers CD25 and GITR and observed its effects on filaria‐induced immunosuppression. Our results show that administration of anti‐CD25 and anti‐GITR in infected animals not only arrested the accumulation of Treg cells and reduced arginase activity, but also led to an increase in the percentages of Th17 cells in the secondary lymphoid organs of mice. Elevated levels of interferon‐γ and decreased levels of interleukin‐10 were also noted in the culture supernatants of mouse splenocytes that were treated with neutralizing antibodies. Furthermore, treatment with neutralizing antibodies enhanced the expression of inducible nitric oxide synthase on host macrophages and CD40 on host dendritic cells with concomitant decreased expression of alternative activation markers Arg1, Ym1 and Fizz1, which together lead to reduced parasite burden in treated animals. In summary, administration of neutralizing antibodies helps in breaking the regulatory network in mice and limits parasite‐induced immunosuppression at the earliest host–parasite interface.

A series of 4-oxycoumarin derivatives was synthesized, characterized and evaluated in vitro and in vivo for antifilarial activity against the human lymphatic filarial parasite, Brugia malayi. A majority of the compounds studied showed potent in vitro activity with low IC50 values in the micro molar (μM) range (0.014-1.73 and 0.0056-0.43) against adult worms and microfilariae, respectively. Compounds 8 and 9 were identified to be the most promising antifilarial candidate molecules exhibiting activity in the nanomolar (nM) range. The IC50 values for compound 8 were 14 nM and 5.6 nM while for compound 9 were 94 nM and 13 nM, respectively, for adult worm and microfilaria. These two compounds also displayed promising adulticidal activity (74.9 ± 4.8% and 69.4 ± 2.8%, respectively) in the primary rodent (jird) screen. This study also serves as a starting point for investigating structure-activity relationship with different amino substituents.

Lymphatic filariasis caused by tissue nematodes is a nonfatal public health disease which causes huge deformities in several tropical and subtropical countries including India leading to severe man-hour loss. Mass drug administration using single-dose treatment with diethylcarbamazine or ivermectin in combination with albendazole has brought down filarial prevalence in several geographical regions; however, reports on development of resistance to mainstay drugs especially in veterinary settings are alarming. Discovery of Wolbachia endosymbionts in filarial parasites and dependence of filariids on these microorganisms for their fertility, growth and survival suggest these bacteria to be an attractive drug target. Knowledge on Brugia malayi genome has given huge impetus on the search for new drug molecules and novel drug targets. Modern bioinformatics approaches such as in silico drug designing and screening of compounds/target enzyme inhibitors or compound libraries as well as new assay development would facilitate the new drug discovery and development programme.

In the present study chitosan (Chi) coated alginate micro particles (MPs) were prepared by a spray drying technique for the effective delivery to lymphatics via combination therapy. The optimized MPs were spherical of less than 5 μm in size with loading and entrapment efficiencies of 14.13 ± 1.1% and 41.28 ± 1.5% for diethylcarbamazine (DEC), and 14.93 ± 0.58% and 43.51 ± 1.9% for doxycycline (DOX) respectively. The physical mixture and drug entrapped MPs showed good stability and no interaction was evident from FT-IR, XRD and thermal analysis. In vitro release studies revealed that the drug release pattern was sustained beyond 16 h in simulated gastro-intestinal fluids for DEC and DOX from the MPs. In vivo ileal uptake and histopathological studies in rats divulged the uptake of the MPs by intestinal epithelium. Further in vitro antifilarial activity studies demonstrated notable killing by the combination therapy compared to individual treatments with Chi–DOX MPs and Chi–DEC MPs. A combined dose of 25 mg kg−1 and 10 mg kg−1 in a 2.5 : 1 ratio of DEC and DOX respectively, following the oral MP therapy, significantly improved the microfilaricidal and macrofilaricidal action of the in vitro estimations. In vivo experiments of infected jirds (Meriones unguiculatus) were carried out. The macrofilaricidal/adulticidal action was enhanced by 2 fold with Chi–DOX MPs in combination with Chi–DEC MPs, microfilaricidal enhancements of the Chi–DEC & Chi–DOX combination are also inferred.

Moxidectin is a macrocyclic lactone belonging to milbemycin family closely related to ivermectin and is currently progressing towards Phase III clinical trial against human infection with the filaria Onchocerca volvulus (Leuckart, 1894). There is a single report on the microfilaricidal and embryostatic activity of moxidectin in case of the human lymphatic filarial parasite Brugia malayi (Brug, 1927) in Mastomys coucha (Smith) but without any adulticidal action. In the present study, the in vitro and in vivo antifilarial efficacy of moxidectin was evaluated on, B. malayi. In vitro moxidectin showed 100% reduction in adult female worm motility at 0.6 µM concentration within 7 days with 68% inhibition in the reduction of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye) (which is used to detect viability of worms). A 50% inhibitory concentration (IC50) of moxidectin for adult female parasite was 0.242 µM, for male worm 0.186 µM and for microfilaria IC50 was 0.813 µM. In adult B. malayi-transplanted primary screening model (Meriones unguiculatus Milne-Edwards), moxidectin at a single optimal dose of 20 mg/kg by oral and subcutaneous route was found effective on both adult parasites and microfilariae. In secondary screening (M. coucha, subcutaneously inoculated with infective larvae), moxidectin at the same dose by subcutaneous route brought about death of 49% of adult worms besides causing sterilisation in 54% of the recovered live female worms. The treated animals exhibited a continuous and sustained reduction in peripheral blood microfilaraemia throughout the observation period of 90 days. The mechanism of action of moxidectin is suggested to be similar to avermectins. The in silico studies were also designed to explore the interaction of moxidectin with glutamate-gated chloride channels of B. malayi. The docking results revealed a close interaction of moxidectin with various GluCl ligand sites of B. malayi.

Although recombinant vaccines have several advantages over conventional vaccines, protection induced by single antigen vaccines is often inadequate for a multicellular helminth parasite. Therefore, immunoprophylactic efficacy of cocktail antigen vaccines comprised of several combinations of three Brugia malayi recombinant proteins BmAF-Myo, Bm-iPGM and Bm-TPP were evaluated. Myosin+TPP and iPGM+TPP provided the best protection upon B. malayi infective larval challenge with ∼70% reduction in adult worm establishment over non-vaccinated animals that was significantly higher than the protection achieved by any single antigen vaccine. Myosin+iPGM, in contrast did not provide any enhance protection over the single recombinant protein vaccines. Specific IgG, IgM level, IgG antibody subclasses levels (IgG1, IgG2a, IgG2b, IgG3), lymphocyte proliferation, reactive oxygen species level and cytokines level were also determined to elucidate the characteristics of the protective immune responses. Thus the study undertaken provided more insight into the cocktail vaccination approach to combat LF.

The endosymbiotic organism Wolbachia is an attractive antifilarial drug target. Here we report on the cloning and expression of an rsmD -like rRNA methyltransferase from the Wolbachia endosymbiont of Brugia malayi , its molecular properties, and assays for specific inhibitors. The gene was found to be expressed in all the major life stages of B. malayi . The purified enzyme expressed in Escherichia coli was found to be in monomer form in its native state. The activities of the specific inhibitors (heteroaryl compounds) against the enzyme were tested with B. malayi adult and microfilariae for 7 days in vitro at various concentrations, and NSC-659390 proved to be the most potent compound (50% inhibitory concentration [IC 50 ], 0.32 μM), followed by NSC-658343 (IC 50 , 4.13 μM) and NSC-657589 (IC 50 , 7.5 μM). On intraperitoneal administration at 5 mg/kg of body weight for 7 days to adult jirds into which B. malayi had been transplanted intraperitoneally, all the compounds killed a significant proportion of the implanted worms. A very similar result was observed in infected mastomys when inhibitors were administered. Docking studies of enzyme and inhibitors and an in vitro tryptophan quenching experiment were also performed to understand the binding mode and affinity. The specific inhibitors of the enzyme showed a higher affinity for the catalytic site of the enzyme than the nonspecific inhibitors and were found to be potent enough to kill the worm (both adults and microfilariae) in vitro as well as in vivo in a matter of days at micromolar concentrations. The findings suggest that these compounds be evaluated against other pathogens possessing a methyltransferase with a DPPY motif and warrant the design and synthesis of more such inhibitors.

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

Statistical analysis of IgG among different groups (NEN, EN, MF and CP) for each protein. Table S3. Statistical analysis of IgA among different groups (NEN, EN, MF and CP) for each protein. Table S4. Statistical analysis of IgG1 among different groups (NEN, EN, MF and CP) for each protein. Table S5. Statistical analysis of IgG2 among different groups (NEN, EN, MF and CP) for each protein. Table S6. Statistical analysis of IgG3 among different groups (NEN, EN, MF and CP) for each protein. Table S7. Statistical analysis of IgG4 among different groups (NEN, EN, MF and CP) for each protein. Table S8. Statistical analysis of IgE among different groups (NEN, EN, MF and CP) for each protein. Table S9. Statistical analysis of IgM among different groups (NEN, EN, MF and CP) for each protein. Table S10. Statistical analysis of PBMC proliferation between groups (EN, MF and CP) for each protein. Table S11. Statistical analysis of ROS generation in terms of mean fluorescence for each protein, comparison of stimulated PBMC between groups (EN, MF and CP) were done. Table S12. Statistical analysis, comparison of % killing (Mean ± SD, n = 3) between three groups at baseline (no depletion of protein specific antibodies) using Kruskal-Wallis ANOVA. Table S13. Multiple comparisons of mean ranks of % killing between three groups at baseline. Table S14. Statistical analysis; For each group, comparisons of % killing between different proteins after depletion of protein specific antibodies at baseline. (PDF 118 kb)

Currently emphasized conventional chemotherapies for the elimination of lymphatic filariasis (LF) are imperfect due to unfocused targeting of poorly water-soluble antifilarial drugs. The profound location of drug targets (filarial parasites or wolbachia) within the complex anatomy of lymphatic tissues often necessitates prolonged treatment schedules with high doses leading to undesired side effects and poor patient compliance. Therefore, we need to reformulate antifilarial drugs taking the advantages of nanotechnology through a wide range of nanomedical carriers, which improve drug efficacy, increase bioavailability, and diminish toxicity.Connotations of drug delivery systems (DDSs) to target lymphatic filaroids or wolbachia and systemic microfilaria have been discussed. The potentials of liposomes and solid lipid nanoparticles for the treatment of LF are highlighted. Various critical factors, viz optimal size range, surface properties, preferred pH, mechanism of reticuloendothelial avoidance, and control of the release of antifilarial agents for safe elimination of parasites, are enclosed to design a novel DDS for LF. The review of nanotechnological approaches to improve antifilarial chemotherapy will help to resolve existing technological gaps.Precincts in the antifilarial discovery programs can never be overcome by conventional methods. Nanomedicine encompasses wide-range solution for each single problem (i.e., from poor solubility to nonspecific targeting of antifilarial agents) for the cure of LF at low costs and may reduce the economic burden of LF diseases. Advances in nanotechnology loom will certainly come forward as silver bullets in the near future for quick diagnosis, control, and elimination of this tropically neglected disease.

SUMMARYThe role ofMycobacterium w(Mw) vaccine as an immunomodulator and immunoprophylactant in the treatment of mycobacterial diseases (leprosy and pulmonary tuberculosis) is well established. The fact that it shares common antigens with leishmanial parasites prompted its assessment as an immunostimulant and as an adjunct to known anti-leishmanials that may help in stimulating the suppressed immune status ofLeishmania donovani-infected individuals. The efficacy ofMwvaccine was assessed as an immunomodulator, prophylactically either alone or in combination with anti-leishmanial vaccine, as well as therapeutically as an adjunct to anti-leishmanial treatment inL. donovani-infected hamsters, representing a chronic human Visceral Leishmaniasis (VL) model. Similarly, its efficacy was also evaluated inL. donovani-infected BALB/c mice, representing an acute VL model. The preliminary studies revealed thatMwwas ineffective as an immunostimulant and/or immunoprophylactant in hamsters infected withL. donovani, as estimated by T-cell immunological responses. However, in the BALB/c mice-VL model it appeared as an effective immunostimulant but a futile prophylactic agent. It is therefore inferred that, contrary to its role in managing tuberculosis and leprosy infections,Mwvaccine has not been successful in controlling VL infection, emphasizing the need to find detailed explanations for the failure of this vaccine against the disease.

While the personalized medicine literature has hitherto focused on diseases in developed countries, little discussion has materialized on ways in which genomics can importantly remedy highly prevalent diseases in developing countries. This paper aims to address this important lacuna in the biomedical literature— what we refer to herein as ―developing world genomics‖ that needs recognition as an emerging subspecialty of global personalized medicine. We direct our attention on neglected tropical diseases (NTDs), a group of high prevalence chronic parasitic and related infections that affect the world's poorest people. Lymphatic filariasis (LF) is a mosquito-borne NTD caused by thread like lymphatic-dwelling nematode and causes significant disability as well as loss of productivity in developing nations. The sustainability of the ongoing efforts for vector control and chemotherapy are uncertain and new drugs are required in the absence of an adulticidal drug while some mainstay drugs are threatened with drug resistance. In this paper, we share and synthesize research findings focussed on Wolbachia, the endosymbiont bacteria of filarial parasites, which itself is a promising target for the development of personalized drugs or customized vaccines as it contributes to the normal functioning of the parasites. We analyze the recent advances in parasite and Wolbachial genomics to highlight potential applications of genomic technologies for targeted drug discovery and personalized therapeutics for this NTD with a vast burden on the global public health. Comparative genomic approaches should also be valuable for exploring genetic changes involved in resistance to antifilarial drugs and understanding the potential mechanisms of drug resistance in human parasites.

Bioassay guided fractionation of ethanolic extract of the leaves of Bauhinia racemosa led to the isolation of galactolipid and catechin class of the compounds (1-7) from the most active n-butanol fraction (F4). Among the active galactolipids, 1 emerged as the lead molecule which was active on both forms of lymphatic filarial parasite, Brugia malayi. It was found to be better than the standard drug ivermectin and diethylcarbamazine (DEC) in terms of dose and efficacy.

The present study incorporates the findings on in vitro and in vivo antifilarial activity in the marine sponge, Haliclona oculata using an experimental rodent infection of human lymphatic filarial parasite, Brugia malayi. The in vitro antifilarial action was determined on both adult female worms as well as microfilariae using two parameters viz. adverse effect on motility and inhibition in MTT reduction by the treated adult parasite over control worm. The antifilarial activity could be located in the methanol extract and one of its four fractions (chloroform). Bioactivity guided fractionation of chloroform fraction led to localization of in vitro activity in one of its eight chromatographic fractions. Methanol extract, chloroform fraction and one of the chromatographic fractions revealed IC(50) values of 5.00, 1.80, and 1.62μg/ml, respectively when adult B. malayi were exposed to these test samples for 72h at 37°C. Under similar exposure conditions, the IC(50) values for microfilariae were 1.88, 1.72 and 1.19μg/ml, respectively. The active test samples were found to be safe revealing >10 selectivity indices (SI) on the basis of cytotoxicity to Vero cells (monkey kidney cells) and therefore selected for in vivo evaluation against primary (adult B. malayi intraperitoneal transplanted jird) and secondary (subcutaneous infective larvae induced mastomys) screens. In primary jird model, the three test samples at 100mg/kg for five consecutive days by subcutaneous route demonstrated significant macrofilaricidal efficacy to the tune of 51.3%, 64% and 70.7% by methanol extract, chloroform and chromatographic fraction, respectively. The three samples demonstrated 45-50% macrofilaricidal activity with moderate embryostatic effect in secondary model at 5×500, 5×250 and 5×125mg/kg by oral route. Chromatographic fraction possessing highest antifilarial action was primarily found to be a mixture of four alkaloids Mimosamycin, Xestospongin-C, Xestospongin-D and Araguspongin-C in addition to few minor compounds.

Withania somnifera is an ayurvedic Indian medicinal plant whose immunomodulatory activities have been widely used as a home remedy for several ailments. We recently observed immunostimulatory properties in the root extracts of chemotypes NMITLI-101, NMITLI-118, NMITLI-128 and pure withanolide, withaferin A. In the present study, we evaluated the potential immunoprophylactic efficacies of these extracts against an infective pathogen. Our results show that administration of aqueous ethanol extracts (10 mg/kg) and withaferin A (0·3 mg/kg), 7 days before and after challenge with human filarial parasite Brugia malayi, offers differential protection in Mastomys coucha with chemotype 101R offering best protection (53·57%) as compared to other chemotypes. Our findings also demonstrate that establishment of B. malayi larvae was adversely affected by pretreatment with withaferin A as evidenced by 63·6% reduction in adult worm establishment. Moreover, a large percentage of the established female worms (66·2%) also showed defective embryogenesis. While the filaria-specific immunological response induced by withaferin A and NMITLI-101 showed a mixed Th1/Th2 phenotype, 118R stimulated production of IFN-γ and 128R increased levels of IL-4. Taken together, our findings reveal potential immunoprophylactic properties of W. somnifera, and further studies are needed to ascertain the benefits of this plant against other pathogens as well.

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10–100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3–30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10 mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses.

The DExD/H box families of RNA helicases are a multifunctional group of proteins involved in unwinding of inter- and intra-molecular base-paired regions. Successful knockdown of DEAD box RNA helicase gene (BmL3-Helicase) of human lymphatic filarial parasite Brugia malayi was done with specifically designed and chemically synthesized siRNA of

Annona squamosa (AS) has traditionally been used as ethnomedicine. We have earlier extracted and fractionated the twigs of AS based upon its bioactivity and observed its immune potentiating activity that was localized in its three fractions. Present communication deals with the phytochemical analysis and pharmacological investigation of the most active chloroform fraction that led to isolation and identification of a number of compounds whose structures were elucidated using 1D and 2D NMR spectroscopic analysis. Amongst the twelve pure compounds isolated, five compounds Lanuginosine (1), (+)-O-methylarmepavine (2), (+)-anomuricine (3), Isocorydine (4), and N-methyl-6, 7-dimethoxyisoquinolone (5) were evaluated in vivo for their immune modifier activities in BALB/c mice after oral administration at three log doses of 0.3, 1.0 and 3.0mg/kg for 14 consecutive days. Of these, three compounds (1, 2 and 5) showed dose dependent immune stimulating activity. However, the uppermost activity was noted in the compound N-methyl-6, 7-dimethoxyisoquinolone at the 3.0mg/kg oral dose. The activity was assessed in the form of increased splenic T and B cellular proliferation, up-regulated CD4+, CD8+ and CD19+ cell population and accentuation in the peritoneal macrophage function. The compound possibly acted modifying the expression of Th1- and Th2- cytokines via stimulation of pro-inflammatory Th1 cytokines IL-2 and IFN-γ. These results warrant the use of the above compounds as an efficient immune-stimulant or immune-adjuvant against diseases with immune suppression. The analogs of the compound may further be chemically synthesized to achieve desired immune modifying activity.

The hexane and ethanolic extracts prepared from the seeds of plant Pterospermum acerifolium were evaluated for their immunomodulatory activities by exploiting their effects on the humoral and cellular immune arms of BALB/c mice after oral administration for 14 consecutive days at different log doses. Various immune parameters viz. lymphoproliferative index, oxidative burst in peritoneal macrophages, modulation in T/B cell population and regulation of Th1/Th2 cytokines in mice were monitored to assess the immunomodulatory characteristics of the plant at 3, 10 and 30 mg/kg doses. Both the extracts exerted remarkable dose-dependent immunosuppressive effect with down-regulation of all the immune markers studied. Administration of extracts in immune-stimulated mice (by treatment with levamisole) further validated the immunosuppressive action. The findings are exceedingly encouraging and warrant bioactivity guided fractionation/purification to locate the activity in a single molecule for its future use in autoimmune disorders and organ transplant patients.

The present investigation deals with the evaluation of antifilarial efficacy of liposome entrapped antiwolbachial antibiotics doxycycline and rifampicin (5 doses at 10 mg/kg, subcutaneously for 15 days) alone and/or in combination with standard filaricide diethylcarbamazine (DEC) against human lymphatic filariid Brugia malayi in rodent host Mastomys coucha. The delivery system maintained the sustained release of antibiotics up to 48 h and significantly (P0.05) augmented the antifilarial potential of these antibiotics over their free administration. A combination of DEC with each entrapped antibiotics significantly (P0.05) improved microfilaricidal efficacy, while marginal enhancement was noticed in adulticidal activity. Combination of both antibiotics formulation with DEC demonstrated marginal increase in macrofilaricidal efficacy; however, it was highest ( approximately 75%).

We earlier reported a sizeable protection conferred by 'mitochondria rich' (MT) fraction of adult B. malayi and the present study was planned to locate the candidate protective molecule/s in the active fraction. The MT fraction was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the antigen bands showing strong immune-reactivity with the resistant mastomys sera were assayed for their lymphoproliferative response using splenocytes of protected animals. Of the eight such protein bands, one sub fraction with a molecular weight of approximately 34 kDa (BMT-5) produced utmost cellular proliferation and was therefore exploited for vaccination study. BMT-5 emulsified in Freund's adjuvant produced discernible protection causing 69 and 67% reductions in microfilaraemia and adult worm burden respectively along with sterilization of 68% of the recovered female parasites. Significant levels of filaria-specific and non-specific lymphoproliferation along with enhanced release of Th1 cytokines (TNF-alpha, IFN-gamma and IL-2) by splenocytes were observed in the vaccinated mastomys in addition to elevated levels of antigen-specific IgG, IgG2a, IgG2b and IgA. The peritoneal macrophages of immunized animals also revealed enhanced nitric oxide production in the presence of BMT-5. The findings suggest that approximately 34 kDa (BMT-5) molecules present in the MT fraction of adult B. malayi provided sizeable protection against infective larval challenge by generating a Th1 biased milieu in the host.

Japanese encephalitis is a major cause of encephalitis in Asia. Cases occur largely in rural areas of the South and East Asian region resulting in significant morbidity and mortality. Multiple vaccines exist to control Japanese encephalitis, but all suffer from problems. Envelope protein domain III of Japanese encephalitis virus is involved in binding to host receptors and it contains specific epitopes that elicit virus-neutralizing antibodies. Earlier, the protective efficacy of domain III has been evaluated in mice by some researchers, but these studies are lacking in explanation of humoral and cellular immune responses. We have earlier reported cloning, expression, purification and in vitro refolding of Japanese encephalitis virus envelope protein domain III (rJEV-DIII). Ninety percent JEV is neutralized when the serum against refolded rJEV-DIII is used at a dilution of 1:80. In the present study, we have evaluated the immunomodulatory potential of refolded rJEV-DIII protein in BALB/c mice with Freunds complete/incomplete adjuvants. Mice were tested for humoral immune response by ELISA. Cell-mediated immune response was tested by lymphocyte proliferation assay and cytokine profiling. The rJEV-DIII generated high IgG antibody and its isotypes (IgG2a and IgG3) and induced significant expression of INF-gamma and IL-2 cytokines. The rJEV-DIII induced significant lymphoproliferation of splenocytes. In conclusion rJEV-DIII induced Th1 type of immune response which plays an important role in protection for intracellular pathogens.

Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi-infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2-DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2-D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI-TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down-regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.

The present study reports on the antifilarial activity of a marine sponge Haliclona exigua (phylum Porifera). The crude methanol extract and n-butanol-soluble fraction killed adult Brugia malayi at 31.25-microg/ml concentration (both in motility and 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay) while the chloroform fraction was lethal at a lower concentration of 15.6 microg/ml. The activity could be located in a single molecule araguspongin C which brought about mortality of worm at 15.6 microg/ml. In vivo evaluation of the crude extract (5 x 500 mg/kg, orally) and the chloroform fraction (5 x 250 mg/kg, orally) in B. malayi-infected rodent host, Mastomys coucha, did not show any significant microfilaricidal actions; however, microfilarial densities in both the treated groups were significantly much lower than those of untreated group in contrast to standard filaricide diethylcarbamazine which exerted 79% microfilaricidal action on day 8 of treatment. Both these extracts also demonstrated adulticidal (macrofilaricidal) activity which was more pronounced in the chloroform fraction (50.2%). In addition, there was moderate adverse effect on the reproductive potential of female worms (crude extract 46.5%; chloroform 58.6%). The findings suggest that the marine sponge H. exigua possesses adulticidal and embryostatic action against human lymphatic filarial parasite B. malayi in experimental rodent model and this activity could be attributed to the presence of araguspongin C.

Modulation of immune functions by using herbal plants and their products has become fundamental regime of therapeutic approach. Piper betle Linn. (Piperaceae) is a widely distributed plant in the tropical and subtropical regions of the world and has been attributed as traditional herbal remedy for many diseases. We have recently reported the antifilarial and antileishmanial efficacy in the leaf extract of Bangla Mahoba landrace of P. betle which is a female plant. The present report describes the in vivo immunomodulatory efficacy of the crude methanolic extract and its n-hexane, chloroform, n-butanol fractions of the female plant at various dose levels ranging between 0.3 and 500 mg/kg in BALB/c. Attempts were also made to observe antifilarial activity of the active extracts and correlate it with the antigen specific immune responses in another rodent Mastomys coucha infected with human lymphatic filarial parasite Brugia malayi . The crude methanol extract and n-hexane fraction were found to potentiate significant ( p 0.001 ) enhancement of both humoral (plaque forming cells, hemagglutination titre) as well as cell-mediated (lymphoproliferation, macrophage activation, delayed type hypersensitivity) immune responses in mice. The flow cytometric analysis of splenocytes of treated mice indicated enhanced population of T-cells (CD4 + , CD8 + ) and B-cells (CD19 + ). The n-hexane fraction (3 mg/kg) was found to induce biased type 2 cytokine response as revealed by increased IL-4 + and decreased IFN-γ + T-cell population while the chloroform fraction (10 mg/kg) produced a predominant type 1 cytokines. Crude methanolic extract (100 mg/kg) demonstrated a mixed type 1 and type 2 cytokine responses thus suggesting a remarkable immunomodulatory property in this plant. The induction of differential T-helper cell immune response appears ideal to overcome immunosuppression as observed in case of lymphatic, filarial Brugia malayi infection which may also be extended to other infections as well.

DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. We have identified a novel immunodominant cDNA clone, BmL3-helicase, encoding DEAD box RNA helicase by immunoscreening of a larval stage cDNA library of Brugia malayi. The cDNA sequence exhibited strong sequence homology to Caenorhabditis elegans and C. briggsae RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. The clone also showed similarity with RNA helicase of Wolbachia, an endosymbiotic bacterium of filarial parasite. It was overexpressed as approximately 50 kDa His-tag fusion protein, and ATP hydrolysis assay of recombinant enzyme showed that either ATP or dATP was required for the unwinding activity, indicating BmL3-helicase as an ATP/dATP-dependent RNA helicase. The recombinant protein also demonstrated cross-seroreactivity with human bancroftian sera. The presence of BmL3-helicase in various life stages of B. malayi was confirmed by immunoblotting of parasite-life-cycle extracts with polyclonal sera against the BmL3-helicase, which showed high levels of expression in microfilaria, L(3,) and adult (both male and female) stages. In the absence of an effective macrofilaricidal agent and validated anti-filarial drug targets, RNA helicases could be utilized as a rational drug target for developing agents against the human filarial parasite.

The discovery of the endosymbiont Wolbachia, which has a mutualistic relationship with filarial nematodes, and its importance in filarial parasite biology has provided a lead for developing novel chemotherapeutic agents against human filariasis. Wolbachia also appears to be involved in immunopathological responses as well as adverse reactions after antifilarial therapy. The aim of the present study was to explore the potential of administering anti-Wolbachial therapy before antifilarial treatment to improve the filaricidal efficacy of the present-day filaricide diethylcarbamazine. An additional objective was to minimize host inflammatory reactions using a rodent model Mastomys coucha and Meriones unguiculatus infected with human lymphatic filariid Brugia malayi. We observed: (1) a 40-day treatment schedule of tetracycline alone resulted in delayed reduction in microfilaraemia and a low degree of macrofilaricidal efficacy; (2) tetracycline therapy followed by 100 mg/kg diethylcarbamazine (DEC) x5 days led to marked reduction in microfilaraemia from day 48 onward after initiation of treatment. The combination treatment also brought about approximately 70% death of adult B. malayi and sterilization of 82.3% of the surviving female worms, thus exhibiting remarkable enhancement in the antifilarial activity of DEC; (3) tissue inflammatory reactions and pathogenesis were significantly reduced as observed by histopathology, and peritoneal macrophage mediated oxidative burst shown by fluorescence-activated cell sorting (FACS) analysis using dichlorofluorescein diacetate (DCF-DA); and (4) the characteristic filarial antigen-specific and mitogen-specific cellular unresponsiveness was significantly reversed, possibly due to marked clearance of microfilaraemia. It is therefore advisable to give an anti-Wolbachial antibiotic trial before starting antifilarial therapy to achieve maximum benefits.

Currently, there is a clinical need for more effective vaccine for hepatitis B that induces robust cell-mediated immune response capable of viral clearance in chronic hepatitis B infection. In the present study, hepatitis B vaccines formulations were designed by loading the hepatitis B surface antigen into liposomes adjuvanted with rough lipopolysaccharide (Re-LPS) and lpxL1 LPS using conventional rotatory evaporation method and were characterized for various parameters, such as vesicle shape and surface morphology, size and size distribution, entrapment efficiency, turbidity, and in vitro release pattern. The immunoreactivity in mice was evaluated by measuring anti-HBs IgG titer and compared with alum-adsorbed HBsAg solution, plain HBsAg, and liposomal HBsAg formulations. The formulations were also evaluated for cell-mediated immune response by HBsAg specific proliferation of spleenocytes after secondary immunization and re-stimulation in vitro with the same antigen. Simultaneous estimation of cytokines (IL-4, IFN-gamma) was also carried out. Ex vivo cellular uptake study was performed by fluorescence microscopy. Results indicate that the serum IgG titer obtained after i.m administration of Re-LPS- and lpxL1 LPS-adjuvanted liposomal HBsAg formulation was equivalent to alum-adsorbed HBsAg formulation but was more responsive, sustained, and significantly higher than the corresponding liposomal HBsAg and plain HBsAg formulations. Incorporation of lpxL1 LPS into the liposomal HBsAg increased the stimulation index (SI) 6-10 times as compared with plain HBsAg. Re-LPS- and lpxL1 LPS-adjuvanted liposomal HBsAg formulations induced stronger cellular immune response with a predominant Interferon-gamma (IFN-gamma) level than those induced by free HBsAg alone, alum-adsorbed HBsAg, and non-adjuvanted liposomal HBsAg. Probably, the possible mechanism for the enhancement of cellular immunity in addition to humoral immunity by LPS-adjuvanted liposomal HBsAg formulation is due to marked enhancement of immunological presentation and recruitment of antigen via macrophage and antigen-presenting cells (APCs).

Aim: Wolbachia is a promising antifilarial chemotherapeutic target. Translation initiation factor-1 (Tl IF-1) is an essential factor in prokaryotes. Functional characterization of Wolbachia’s novel proteins/enzymes is necessary for the development of adulticidal drugs. Materials & methods: Mutant, Wol Tl IF-1 R45D was constructed by site directed mutagenesis. Fluorimetry and size exclusion chromatography were used to determine the biophysical characteristics. Mobility shift assay and fluorescence resonance energy transfer were used to investigate the functional aspect of Wol Tl IF-1 with its mutant. Results: Both wild and mutant were in monomeric native conformations. Wild exhibits nonspecific binding with ssRNA/ssDNA fragments under electrostatic conditions and showed annealing and displacement of RNA strands in comparison to mutant. Conclusion: Point mutation impaired RNA chaperone activity of the mutant and its interaction with nucleotides.

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.