Human Filarial Infections Research Articles

Comparative development of human filariae Loa loa, Onchocerca volvulus and Mansonella perstans in immunocompromised mouse strains.

Mouse models of human filarial infections are not only urgently needed to investigate the biology of the nematodes and their modulation of the host's immunity, but will also provide a platform to screen and test novel anti-filarial drugs. Recently, murine Loa loa infection models have been stablished using immunocompromised mouse strains, whereas murine Mansonella perstans infections have not been implemented until now. Therefore, we aim to establish experimental M. perstans infections using the immunocompromised mouse strains RAG2IL-2Rγ-/- (lack B, T and natural killer cells), IL-4Rα/IL-5-/- (impaired IL-4/5 signalling and eosinophil activation) and NOD.Cg-PrkdcscidIl2rgtm1Wj l/SzJ (NOD scid gamma, NSG) BALB/c mice (lack mature lymphocytes) through subcutaneous (s.c.) or intraperitoneal (i.p.) inoculation of infective stage 3 larvae (L3) isolated from engorged vectors. In total, 145 immunocompromised mice have been inoculated with 3,250 M. perstans, 3,337 O. volvulus, and 2,720 Loa loa L3 to comparatively analyse which immunocompromised mouse strain is susceptible to human filarial infections. Whereas, no M. perstans and O. volvulus L3 could be recovered upon 2-63 days post-inoculation, a 62-66% Loa loa L3 recovery rate could be achieved in the different mouse strains. Gender of mice, type of inoculation (s.c. or i.p.) or time point of analysis (2-63 days post inoculation) did not interfere with the success of L3 recovery. In addition, administration of the immune suppressants hydrocortisone, prednisolone and cyclophosphamide did not restore M. perstans L3 recovery rates. These findings show that RAG2IL-2Rg-/-BALB/c and C57BL/6, IL-4Rα/IL-5-/- BALB/c and NSG mice were not susceptible to M. perstans and O. volvulus L3 inoculation using the applied methods, whereas Loa loa infection could be maintained. Further studies should investigate if humanized immunocompromised mice might be susceptible to M. perstans. and O. volvulus.

Single-cell molecular signature of pathogenic T helper subsets in type 2-associated disorders in humans.

To unravel the heterogeneity and molecular signature of effector memory Th2 cells (Tem2), we analyzed 23 individuals' PBMCs of filaria-infected (Filaria+) and 24 healthy volunteers (Filaria-), with or without coincident house dust mite (HDM) allergic sensitization. Flow cytometry revealed 3 CD4+ Tem subsets - CCR4+CCR6+CRTH2- Tem17, CCR4+CCR6-CRTH2+ Tem2, and CCR6+CCR4+CRTH2+ Tem17.2 - markedly enriched in Filaria+ individuals. These subsets were sorted and analyzed by multiomic single-cell RNA immunoprofiling. SingleR-annotated Th2 cells from Tem2 and Tem17.2 cell subsets had features of pathogenic Th2 effector cells based on their transcriptional signatures, with downregulated CD27 and elevated expression levels of ITGA4, IL17RB, HPGDS, KLRB1, PTGDR2, IL9R, IL4, IL5, and IL13 genes. When the Filaria+ individuals were subdivided based on their allergic status, Tem2 cells in HDM+Filaria+ individuals showed an overall reduction in TCR diversity, suggesting the occurrence of antigen-driven clonal expansion. Moreover, HDM+Filaria+ individuals showed not only an expansion in the frequency of both Tem2 and Tem17.2 cell subsets, but also a change in their molecular program by overexpressing GATA3, IL17RB, CLRF2, and KLRB1, as well as increased antigen-induced IL-4, IL-5, and IL-13 production, suggesting that aeroallergens reshape the transcriptional and functional programming of Th2 cell subsets in human filarial infection toward a pathogenic immunophenotype.

Eosinophil: An innate immune cell with anti-filarial vaccine and biomarker potential.

Filarial infections continue to pose a great challenge in endemic countries. One of the central goals in the fight against human filarial infections is the development of strategies that will lead to the inhibition of microfilariae (mf) transmission. Keeping mf under a certain threshold within endemic populations will stop transmission and eliminate the infection. A narrative review was carried out to identify the possibilities and limitations of exploring the use of eosinophil responses as an anti-filarial vaccine, and biomarker for the detection of filarial infections. An extensive literature search was performed in online scientific databases including PubMed Central, PubMed, BioMed Central, with the use of predefined search terms. A better understanding of the parasite-host interactions will lead to the development of improved and better treatment or vaccine strategies that could eliminate filariasis as soon as possible. Highlighted in this review is the explorative use of eosinophil-producing CLC/Galectin-10 as a potential biomarker for filarial infections. Also discussed are some genes, and pathways involved in eosinophil recruitments that could be explored for anti-filarial vaccine development. In this short communication, we discuss how eosinophil-regulated genes, pathways, and networks could be critical in providing more information on how reliably a front-line immune player could be exploited for anti-filarial vaccine development and early infection biomarker.

Diethylcarbamazine mediated potentiation of emodepside induced paralysis requires TRP-2 in adult Brugia malayi

Human and veterinary filarial nematode infections are a major health concern in tropical countries. They are transmitted by biting insects and mosquitoes. Lymphatic filariasis, a group of filarial infections caused by Brugia spp. and Wucheria bancrofti affect more than 120 million people worldwide. Infected individuals develop swollen limbs and disfigurement, leading to an inability to work and ostracization from society. Control and prophylaxis for these infections involve mass drug administration combinations of anthelmintics including diethylcarbamazine (DEC). DEC has actions on microfilariae, but its effects on adult worms are less pronounced. The SLO-1 (BK) channel activator, emodepside, kills adults of many filarial species. However, the in vivo efficacy of emodepside is suboptimal against B. malayi, possibly due to reduced bioavailability in the lymphatic system. Expressing different slo-1 splice variants in B. malayi also affects sensitivity to emodepside. This study explores the potentiation of emodepside mediated paralysis by DEC in adult female B. malayi. Worminator motility measurements show that co-application of DEC and emodepside increases the potency of emodepside 4-fold. The potentiation of the emodepside effect persists even after the worms recover (desensitize) from the initial effects of DEC. RNAi knock-down demonstrates that the DEC-mediated potentiation of emodepside requires the presence of TRP-2 channels. Our study demonstrates that the addition of DEC could enhance the effect of emodepside where bioavailability or activity against a specific species may be low.

Discovery of Substituted Di(pyridin-2-yl)-1,2,4-thiadiazol-5-amines as Novel Macrofilaricidal Compounds for the Treatment of Human Filarial Infections.

Filarial diseases, including lymphatic filariasis and onchocerciasis, are considered among the most devastating of all tropical diseases, affecting about 145 million people worldwide. Efforts to control and eliminate onchocerciasis are impeded by a lack of effective treatments that target the adult filarial stage. Herein, we describe the discovery of a series of substituted di(pyridin-2-yl)-1,2,4-thiadiazol-5-amines as novel macrofilaricides for the treatment of human filarial infections.

Pre-existing helminth infection impairs the efficacy of adjuvanted influenza vaccination in mice.

The world health organization estimates that more than a quarter of the human population is infected with parasitic worms that are called helminths. Many helminths suppress the immune system of their hosts to prolong their survival. This helminth-induced immunosuppression “spills over” to unrelated antigens and can suppress the immune response to vaccination against other pathogens. Indeed, several human studies have reported a negative correlation between helminth infections and responses to vaccinations. Using mice that are infected with the parasitic nematode Litomosoides sigmodontis as a model for chronic human filarial infections, we reported previously that concurrent helminth infection impaired the vaccination-induced protection against the human pathogenic 2009 pandemic H1N1 influenza A virus (2009 pH1N1). Vaccinated, helminth-infected mice produced less neutralizing, influenza-specific antibodies than vaccinated naïve control mice. Consequently helminth-infected and vaccinated mice were not protected against a challenge infection with influenza virus but displayed high virus burden in the lung and a transient weight loss. In the current study we tried to improve the vaccination efficacy using vaccines that are licensed for humans. We either introduced a prime-boost vaccination regimen using the non-adjuvanted anti-influenza vaccine Begripal or employed the adjuvanted influenza vaccine Fluad. Although both strategies elevated the production of influenza-specific antibodies and protected mice from the transient weight loss that is caused by an influenza challenge infection, sterile immunity was not achieved. Helminth-infected vaccinated mice still had high virus burden in the lung while non-helminth-infected vaccinated mice rapidly cleared the virus. In summary we demonstrate that basic improvements of influenza vaccination regimen are not sufficient to confer sterile immunity on the background of helminth-induced immunosuppression, despite amelioration of pathology i.e. weight loss. Our findings highlight the risk of failed vaccinations in helminth-endemic areas, especially in light of the ongoing vaccination campaign to control the COVID-19 pandemic.

Adoptive Transfer of Immune Cells Into RAG2IL-2Rγ-Deficient Mice During Litomosoides sigmodontis Infection: A Novel Approach to Investigate Filarial-Specific Immune Responses.

Despite long-term mass drug administration programmes, approximately 220 million people are still infected with filariae in endemic regions. Several research studies have characterized host immune responses but a major obstacle for research on human filariae has been the inability to obtain adult worms which in turn has hindered analysis on infection kinetics and immune signalling. Although the Litomosoides sigmodontis filarial mouse model is well-established, the complex immunological mechanisms associated with filarial control and disease progression remain unclear and translation to human infections is difficult, especially since human filarial infections in rodents are limited. To overcome these obstacles, we performed adoptive immune cell transfer experiments into RAG2IL-2Rγ-deficient C57BL/6 mice. These mice lack T, B and natural killer cells and are susceptible to infection with the human filaria Loa loa. In this study, we revealed a long-term release of L. sigmodontis offspring (microfilariae) in RAG2IL-2Rγ-deficient C57BL/6 mice, which contrasts to C57BL/6 mice which normally eliminate the parasites before patency. We further showed that CD4+ T cells isolated from acute L. sigmodontis-infected C57BL/6 donor mice or mice that already cleared the infection were able to eliminate the parasite and prevent inflammation at the site of infection. In addition, the clearance of the parasites was associated with Th17 polarization of the CD4+ T cells. Consequently, adoptive transfer of immune cell subsets into RAG2IL-2Rγ-deficient C57BL/6 mice will provide an optimal platform to decipher characteristics of distinct immune cells that are crucial for the immunity against rodent and human filarial infections and moreover, might be useful for preclinical research, especially about the efficacy of macrofilaricidal drugs.

IL-10 and Its Related Superfamily Members IL-19 and IL-24 Provide Parallel/Redundant Immune-Modulation in Loa loa Infection.

Interleukin-10 (IL-10) has been implicated as the major cytokine responsible for the modulation of parasite-specific responses in filarial infections; however, the role of other IL-10 superfamily members in filarial infection is less well studied. Peripheral blood mononuclear cells from loiasis patients were stimulated with or without filarial antigen. Cytokine production was quantified using a Luminex platform and T-cell expression patterns were assessed by flow cytometry. All patients produced significant levels of IL-10, IL-13, IL-5, IL-4, and IL-9 in response to filarial antigen, indicating a common infection-driven response. When comparing microfilaria (mf)-positive and mf-negative patients, there were no significant differences in spontaneous cytokine nor in parasite-driven IL-10, IL-22, or IL-28a production. In marked contrast, mf-positive individuals had significantly increased filarial antigen-driven IL-24 and IL-19 compared to mf-negative subjects. mf-positive patients also demonstrated significantly higher frequencies of T cells producing IL-19 in comparison to mf-negative patients. T-cell expression of IL-19 and IL-24 was positively regulated by IL-10 and IL-1β. IL-24 production was also regulated by IL-37. These data provide an important link between IL-10 and its related family members IL-19 and IL-24 in the modulation of the immune response in human filarial infections. NCT00001230.

Macrofilaricides: An Unmet Medical Need for Filarial Diseases.

Neglected parasitic helminth diseases such as onchocerciasis and lymphatic filariasis affect an estimated 145 million people worldwide, creating a serious health burden in endemic areas such as sub-Saharan Africa and India. Although these diseases are not usually lethal, these filarial nematodes, transmitted by blood-feeding insect vectors, cause severe debilitation and cause chronic disability to infected individuals. The adult worms can reproduce from 5 to up to 14 years, releasing millions of microfilariae, juvenile worms, over an infected individual's lifetime. The current treatments for controlling human filarial infections is focused on killing microfilariae, the earliest larval stage. Currently, there is an unmet medical need for treatments consisting of a macrofilaricidal regimen, one that targets the adult stage of the parasite, to increase the rate of elimination, allow for safe use in coendemic regions of Onchocerca volvulus and Loa loa, and to provide a rapid method to resolve reinfections. Herein, recent approaches for targeting human filarial diseases are discussed, including direct acting agents to target parasitic nematodes and antibacterial approaches to target the endosymbiotic bacteria, Wolbachia.

IL-4 receptor dependent expansion of lung CD169+ macrophages in microfilaria-driven inflammation.

Lung disease is regularly reported in human filarial infections but the molecular pathogenesis of pulmonary filariasis is poorly understood. We used Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity responsible for pleural inflammation, to model responses to human filarial infections and probe the mechanisms. Wild-type and Th2-deficient mice (ΔdblGata1 and Il-4receptor(r)a-/-/IL-5-/-) were infected with L. sigmodontis. Survival and growth of adult filariae and prevalence and density of microfilariae were evaluated. Cells and cytokines in the pleural cavity and bronchoalveolar space were characterized by imaging, flow cytometry and ELISA. Inflammatory pathways were evaluated by transcriptomic microarrays and lungs were isolated and analyzed for histopathological signatures. 40% of WT mice were amicrofilaremic whereas almost all mutant mice display blood microfilaremia. Microfilariae induced pleural, bronchoalveolar and lung-tissue inflammation associated with an increase in bronchoalveolar eosinophils and perivascular macrophages, production of mucus, visceral pleura alterations and fibrosis. Inflammation and pathology were decreased in Th2-deficient mice. An IL-4R-dependent increase of CD169 was observed on pleural and bronchoalveolar macrophages in microfilaremic mice. CD169+ tissue-resident macrophages were identified in the lungs with specific localizations. Strikingly, CD169+ macrophages increased significantly in the perivascular area in microfilaremic mice. These data describe lung inflammation and pathology in chronic filariasis and emphasize the role of Th2 responses according to the presence of microfilariae. It is also the first report implicating CD169+ lung macrophages in response to a Nematode infection.

Susceptibility to L. sigmodontis infection is highest in animals lacking IL-4R/IL-5 compared to single knockouts of IL-4R, IL-5 or eosinophils

BackgroundMice are susceptible to infections with the rodent filarial nematode Litomosoides sigmodontis and develop immune responses that resemble those of human filarial infections. Thus, the L. sigmodontis model is used to study filarial immunomodulation, protective immune responses against filariae and to screen drug candidates for human filarial diseases. While previous studies showed that type 2 immune responses are protective against L. sigmodontis, the present study directly compared the impact of eosinophils, IL-5, and the IL-4R on the outcome of L. sigmodontis infection.MethodsSusceptible wildtype (WT) BALB/c mice, BALB/c mice lacking eosinophils (dblGATA mice), IL-5−/− mice, IL-4R−/− mice and IL-4R−/−/IL-5−/− mice were infected with L. sigmodontis. Analyses were performed during the peak of microfilaremia in WT animals (71 dpi) as well as after IL-4R−/−/IL-5−/− mice showed a decline in microfilaremia (119 dpi) and included adult worm counts, peripheral blood microfilariae levels, cytokine production from thoracic cavity lavage, the site of adult worm residence, and quantification of major immune cell types within the thoracic cavity and spleen.ResultsOur study reveals that thoracic cavity eosinophil numbers correlated negatively with the adult worm burden, whereas correlations of alternatively activated macrophage (AAM) numbers with the adult worm burden (positive correlation) were likely attributed to the accompanied changes in eosinophil numbers. IL-4R−/−/IL-5−/− mice exhibited an enhanced embryogenesis achieving the highest microfilaremia with all animals becoming microfilariae positive and had an increased adult worm burden combined with a prolonged adult worm survival.ConclusionsThese data indicate that mice deficient for IL-4R−/−/IL-5−/− have the highest susceptibility for L. sigmodontis infection, which resulted in an earlier onset of microfilaremia, development of microfilaremia in all animals with highest microfilariae loads, and an extended adult worm survival.

IL-6 is required for protective immune responses against early filarial infection

IL-6 has a wide range of biological activities that includes anti- and pro-inflammatory aspects. In this study, we investigated the role of IL-6 in immune responses to the rodent filarial nematode Litomosoides sigmodontis, a model for human filarial infections. IL-6−/− mice had a significantly increased worm burden after natural infection compared with wild type controls at early time points p.i. Given that the worm burden in IL-6−/− mice was already increased at the time point the infective larvae reached the pleural cavity, immune responses that may facilitate the migration from the site of infection (skin) via the lymphatics to the pleural cavity were analysed. Increased vascular permeability may facilitate larval migration, but blocking of histamine receptors had no effect on worm burden and vascular permeability was similar between IL-6−/− mice and wild type controls. In contrast, blocking mast cell degranulation reduced the worm burden in IL-6−/− mice partially, suggesting that release of mast cell-derived mediators improves larval migration to some degree. Protective immune responses within the skin were involved, as bypassing the skin barrier by inoculating infective L3s subcutaneously resulted in a comparable worm recovery in both mouse strains. Analysis of the cellular composition by flow cytometry and PCR array in the skin after exposure to filarial extract or L3s, respectively, indicate that the absence of IL-6 results in a delayed recruitment of neutrophils and macrophages to the site of initial infection. These results demonstrate that IL-6 is essentially involved in protective immune responses within the skin that impair migration of infective L3s.

Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™.

Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA. Here, we report use of Cappable-seq™ to specifically enrich, in a single step, Wolbachia endobacterial mRNA transcripts from total RNA prepared from the parasitic filarial nematode, Brugia malayi. The obligate Wolbachia endosymbiont is a proven drug target for many human filarial infections, yet the precise nature of its symbiosis with the nematode host is poorly understood. Insightful analysis of the expression levels of Wolbachia genes predicted to underpin the mutualistic association and of known drug target genes at different life cycle stages or in response to drug treatments is typically challenged by low transcriptomic coverage. Cappable-seq resulted in up to ~ 5-fold increase in the number of reads mapping to Wolbachia. On average, coverage of Wolbachia transcripts from B. malayi microfilariae was enriched ~40-fold by Cappable-seq. Additionally, this method has an additional benefit of selectively removing abundant prokaryotic ribosomal RNAs.The deeper microbial transcriptome sequencing afforded by Cappable-seq facilitates more detailed characterization of gene expression levels of pathogens and symbionts present in animal tissues.

Lessons from the genomes and transcriptomes of filarial nematodes

Human filarial infections are a leading cause of morbidity in the developing world. While a small arsenal of drugs exists to treat these infections, there remains a tremendous need for the development of additional interventions. Recent genome sequences and transcriptome analyses of filarial nematodes have provided novel biological insight and allowed for the prediction of novel drug targets as well as potential vaccine candidates. In this review, we discuss the currently available data, insights gained into the metabolism of these organisms, and how the filaria field can move forward by leveraging these data.

The Potentials and Pitfalls of Microarrays in Neglected Tropical Diseases: A Focus on Human Filarial Infections

Data obtained from expression microarrays enables deeper understanding of the molecular signatures of infectious diseases. It provides rapid and accurate information on how infections affect the clustering of gene expression profiles, pathways and networks that are transcriptionally active during various infection states compared to conventional diagnostic methods, which primarily focus on single genes or proteins. Thus, microarray technologies offer advantages in understanding host-parasite interactions associated with filarial infections. More importantly, the use of these technologies can aid diagnostics and helps translate current genomic research into effective treatment and interventions for filarial infections. Studying immune responses via microarray following infection can yield insight into genetic pathways and networks that can have a profound influence on the development of anti-parasitic vaccines.

How do the macrocyclic lactones kill filarial nematode larvae?

The macrocyclic lactones (MLs) are one of the few classes of drug used in the control of the human filarial infections, onchocerciasis and lymphatic filariasis, and the only one used to prevent heartworm disease in dogs and cats. Despite their importance in preventing filarial diseases, the way in which the MLs work against these parasites is unclear. In vitro measurements of nematode motility have revealed a large discrepancy between the maximum plasma concentrations achieved after drug administration and the amounts required to paralyze worms. Recent evidence has shed new light on the likely functions of the ML target, glutamate-gated chloride channels, in filarial nematodes and supports the hypothesis that the rapid clearance of microfilariae that follows treatment involves the host immune system.

Concurrent transcriptional profiling of Dirofilaria immitis and its Wolbachia endosymbiont throughout the nematode life cycle reveals coordinated gene expression.

BackgroundDirofilaria immitis, or canine heartworm, is a filarial nematode parasite that infects dogs and other mammals worldwide. Current disease control relies on regular administration of anthelmintic preventives, however, relatively poor compliance and evidence of developing drug resistance could warrant alternative measures against D. immitis and related human filarial infections be taken. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont thought to be involved in providing certain critical metabolites to the nematode. Correlations between nematode and Wolbachia transcriptomes during development have not been examined. Therefore, we detailed the developmental transcriptome of both D. immitis and its Wolbachia (wDi) in order to gain a better understanding of parasite-endosymbiont interactions throughout the nematode life cycle.ResultsOver 215 million single-end 50 bp reads were generated from total RNA from D. immitis adult males and females, microfilariae (mf) and third and fourth-stage larvae (L3 and L4). We critically evaluated the transcriptomes of the various life cycle stages to reveal sex-biased transcriptional patterns, as well as transcriptional differences between larval stages that may be involved in larval maturation. Hierarchical clustering revealed both D. immitis and wDi transcriptional activity in the L3 stage is clearly distinct from other life cycle stages. Interestingly, a large proportion of both D. immitis and wDi genes display microfilarial-biased transcriptional patterns. Concurrent transcriptome sequencing identified potential molecular interactions between parasite and endosymbiont that are more prominent during certain life cycle stages. In support of metabolite provisioning between filarial nematodes and Wolbachia, the synthesis of the critical metabolite, heme, by wDi appears to be synchronized in a stage-specific manner (mf-specific) with the production of heme-binding proteins in D. immitis.ConclusionsOur integrated transcriptomic study has highlighted interesting correlations between Wolbachia and D. immitis transcription throughout the life cycle and provided a resource that may be used for the development of novel intervention strategies, not only for the treatment and prevention of D. immitis infections, but of other closely related human parasites as well.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1041) contains supplementary material, which is available to authorized users.

Potential involvement of Brugia malayi cysteine proteases in the maintenance of the endosymbiotic relationship with Wolbachia

Brugia malayi, a parasitic nematode that causes lymphatic filariasis, harbors endosymbiotic intracellular bacteria, Wolbachia, that are required for the development and reproduction of the worm. The essential nature of this endosymbiosis led to the development of anti-Wolbachia chemotherapeutic approaches for the treatment of human filarial infections. Our study is aimed at identifying specific proteins that play a critical role in this endosymbiotic relationship leading to the identification of potential targets in the adult worms. Filarial cysteine proteases are known to be involved in molting and embryogenesis, processes shown to also be Wolbachia dependent. Based on the observation that cysteine protease transcripts are differentially regulated in response to tetracycline treatment, we focused on defining their role in symbiosis. We observe a bimodal regulation pattern of transcripts encoding cysteine proteases when in vitro tetracycline treated worms were examined. Using tetracycline-treated infertile female worms and purified embryos we established that the first peak of the bimodal pattern corresponds to embryonic transcripts while the second takes place within the hypodermis of the adult worms. Localization studies of the native proteins corresponding to Bm-cpl-3 and Bm-cpl-6 indicate that they are present in the area surrounding Wolbachia, and, in some cases, the proteins appear localized within the bacteria. Both proteins were also found in the inner bodies of microfilariae. The possible role of these cysteine proteases during development and endosymbiosis was further characterized using RNAi. Reduction in Bm-cpl-3 and Bm-cpl-6 transcript levels was accompanied by hindered microfilarial development and release, and reduced Wolbachia DNA levels, making these enzymes strong drug target candidates.

Disorganized muscle protein-1 (DIM-1) of filarial parasite Brugia malayi: cDNA cloning, expression, purification, structural modeling and its potential as vaccine candidate for human filarial infection

We have recently identified disorganized muscle protein-1 (DIM-1) in one of the proinflammatory fractions of the human filaria Brugia malayi adult worm. The present study was undertaken to characterize B. malayi DIM-1 (DIM-1bm) and explore its vaccine potential. In this study we cloned and expressed the DIM-1bm gene, investigated its sequence homology with other nematodes, constructed in silico structural model, purified the recombinant DIM-1bm (rDIM-1bm) protein, and studied the effect of immunization with rDIM-1bm on the establishment of B. malayi infection in Mastomys coucha. DIM-1bm showed similarity with DIM-1 of Caenorhabditis elegans, Ascaris suum and Loa loa. Structural modeling revealed three immunoglobulin domains in DIM-1bm indicating that it is a member of immunoglobulin superfamily (IgSF) and ‘blastn’ results showed that DIM-1bm coding sequence (CDS) have almost no homology with human and mouse nucleotide sequences. Immunization with rDIM-1bm partially protected M. coucha against establishment of infection as inferred by a low recovery of microfilariae (37–64%) and parasite burden (∼50%). The enhanced activity of macrophages, and IFN-γ and NO responses, and elevated levels of specific IgG, IgG1, IgG2a and IgG2b correlated with parasitological findings. This is the first report on cloning, expression, structural modeling and purification of rDIM-1bm and its ability to partially prevent establishment of B. malayi infection. DIM-1bm's almost complete lack of homology with the human counterpart makes it an attractive protein for exploring its vaccine potential.

Recombinant translation initiation factor-1 of Wolbachia is an immunogenic excretory secretory protein that elicits Th2 mediated immune protection against Brugia malayi

Wolbachia, the intracellular alpha-proteobacteria are required for the development, fertility and survival of filarial parasites. Wolbachia Translation initiation factor-1 (Wol Tl IF-1) is one of the factors required for Wolbachia growth and viability. In the present study, we cloned, over expressed and purified Wol Tl IF-1 that exhibited strong immuno-reactivity with various categories of bancroftian sera. Immunization with the recombinant protein resulted into significant reduction in microfilarial density (70-72%) and adult worm establishment (61-63%) in susceptible Mastomys coucha. Protection offered by Wol Tl IF-1 was found associated with humoral immune arm as observed by an increased antibody level with preponderance of IgE, IgM, IgG1 and IgG2a isotypes. The anti-Wol Tl IF-1 antibodies promoted profound adherence of peritoneal exudates cells to the surface of microfilariae and infective larvae causing cytotoxicity and their death. The present study indicates potential of recombinant Wol Tl IF-1 as a promising vaccine candidate against human lymphatic filarial infection.