Puvvada Kalpana Murthy

Purpose In search of a vaccine for the control of human lymphatic filariasis (LF) caused by Wuchereria bancrofti, Brugia malayi and B. timori, we identified three parasite-specific potential candidates: the disorganized muscle protein-1 (D), calponin (C) and troponin 1 (T) in B. malayi adult worm. In the present study, we investigated the immune response profile of the cocktails of the recombinant D, T and C proteins. Methods Groups of BALB/c mice were immunized with individual rproteins or their cocktails DT, TC, DC and DTC, and the immunogen-specific IgG and its subclasses and IgE were determined. Cells from the immunized animals were challenged in vitro with the respective rproteins and cocktails and the release of nitric oxide (NO) from macrophages and Th1 and Th2 cytokines from splenocytes were determined. Results Among the immunized groups, DTC elicited comparatively a stronger response which included augmented release of NO, Th1 (IL-1β, IL-2, IFN-γ and TNF-α) and Th2 (IL-4, IL-6, IL-10 and TGF-β) cytokines, and increased levels of immunogen-specific IgG, IgG1 and IgG2b and low levels of immunogen-specific IgG2a and IgE and the Th2 cytokine IL-13. Conclusion Immune responses that play important role in host protection were elicited strongly by DTC cocktail compared to the individual rproteins or DT, TC and DC cocktails. The findings provide a sound rationale for further studies on DTC cocktail as a vaccine candidate for the control of LF.

Purpose We identified calreticulin in human filaria Brugia malayi (BmCRT) that shares 97% homology with Wuchereria bancrofti calreticulin (WbCRT), but only 56% with human calreticulin. We found that BmCRT binds C1q and prevents complement-mediated parasite death; immunization with BmCRT leads to parasite death in a rodent model of the infection. BmCRT could, therefore, be a potential vaccine candidate. In the present study, we determined the levels of BmCRT-reactive IgG and its isotype in bancroftian filarial subjects. Methods Recombinant BmCRT (rBmCRT) was prepared, and the sera of endemic normal subjects (EN), microfilaraemics (Mf+) and chronic amicrofilaraemics (ChMf-) from a bancroftian filaria-endemic area and normal subjects from filaria-non-endemic area (NEN) were probed for IgG and its isotypes reacting with rBmCRT and its domains rN, rP and rC. Results rBmCRT and its rN domain-reactive IgG levels were high in EN and Mf+ groups; rC domain and rP domain showed moderate and very little reactivity, respectively. NEN sera were non-reactive. Moderate levels of rBmCRT-reactive IgG1, IgG3 and IgG4 in EN and Mf+ groups and low levels of IgG2 in Mf+ were found; IgG1 and IgG3 reactivity was found for rBmCRT and its rN domain only, while IgG4 reactivity was moderate for rN domain and low for rP and rC domains. While IgG reactivity was seen in all the endemic subjects, IgG isotype reactivity was found mostly in EN and Mf+ subjects. Conclusions Moderate levels of rBmCRT (and its rN domain)-reactive IgG and its isotypes are present in bancroftian subjects. Preponderance of IgG1 and IgG3 isotypes which bind and activate complement has relevance to vaccine potential of BmCRT.

We recently identified disorganized muscle protein-1 of Brugia malayi (DIM-1bm) as a vaccine candidate for human lymphatic filariasis. The present study wa

In the search for immunoprophylactics for the control of human lymphatic filariasis, we recently identified troponin 1 (Tn1) in Brugia malayi adult worms. The present study reports the cloning and expression of the B. malayi Tn1 (Tn1bm), its immunoprophylactic efficacy against B. malayi infection, and the immunological responses of the host. The Tn1bm gene was cloned (Acc no. JF912447) and expressed, and the purified recombinant Tn1bm (rTn1bm) presented a single ~ 27 kDa band. Parasite load in rTn1bm-immunized BALB/c mice challenged with B. malayi infective larvae (L₃) was assessed. In rTn1bm-immunized animals, IgE, IgG, and IgG subclasses in the serum, cell proliferative response, Th1 and Th2 cytokine secretion (from splenocytes), and NO release (from peritoneal macrophages) were determined. Antibody-dependent cell-mediated cytotoxicity (ADCC) to L₃ was assayed using rTn1bm-immune serum. The innate immune response markers MHC class-I, MHC class-II, TLR2, TLR4, and TLR6 expression in peritoneal macrophages and CD3+, CD4+, CD8+, and CD19+ in the splenocyte population were determined in Tn1bm-exposed cells from naïve mice. rTn1bm-immunized L₃-challenged animals showed a 60% reduction in parasite burden. Immunization upregulated cellular proliferation, cytokine (IFN-γ, TNF-α, IL-1β, IL-4, IL-6, and IL-10) secretion, NO release, and antigen-specific IgG, IgG1, and IgG2b antibody levels. rTn1bm-immune serum killed > 65% of L₃ in the ADCC assay. Increased MHC class-II, TLR2, and TLR6 expression and the relative CD4+ and CD19+ cell populations of naïve animal cells indicated the ability of rTn1bm to mobilize innate immune responses. This is the first report of the immunoprophylactic potential of rTn1bm against B. malayi.

We earlier found that F6 fraction of human filaria Brugia malayi cross-reacted with sera of Leishmania donovani infected hamsters and immunization with F6 inhibited both filarial and leishmanial infections. In the present study, we identified a 52.9–93.6 kDa fraction (Ld1) of L. donovani that cross-reacted with sera of B. malayi infected animals and investigated effect of Ld1 on filarial infection. Immunization of BALB/c mice with Ld1 facilitated B. malayi infection with remarkable increase in parasite burden. Facilitation of filarial infection was associated with downregulated cell proliferation, IL-5, IL-13, IFN-γ, TNF-α, and IL-2 levels and upregulated IL-4 and TGF-β. Ld1 exposure also suppressed MHC class-I, MHC class-II, and FcεR1 expression, and phagocytosis in naive mouse macrophages, and CD4+, CD8+, and CD19+ cell population in mouse spleen. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization–time of flight-mass spectrometry revealed eight proteins in Ld1: putative heat shock protein (HSP) 70-related protein 1, HSP70 mitochondrial precursor, alanine aminotransferase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, protein disulfide isomerase, putative ATPase beta subunit, trypanothione reductase, and a hypothetical protein. HSP70 protein mitochondrial precursor and trypanothione reductase showed homology with Trypanosoma cruzi and L. donovani, respectively, and the rest 6 proteins including hypothetical protein bear homology with L. infantum. In conclusion, the present study for the first time shows that immunization with filarial cross-reactive Ld1 fraction of L. donovani facilitates filarial infection by modulating Th1 and Th2 responses. Ld1 molecules may therefore facilitate filarial infection in filaria-leishmania co-infection.

Lymphatic filariasis caused by Wuchereria bancrofti, Brugia malayi and B. timori, is a debilitating disease with an adverse social and economic impact. The infection remains unabated in spite of treatment with existing antifilarial drugs diethylcarbamazine (DEC) and ivermectin which are chiefly microfilaricides. There is therefore, need for macrofilaricides, embryostatic agents and better microfilaricides. In the present study we explored the antifilarial potential of crude extract and its molecular fractions of the plant Taxodium distichum using in vitro assay systems and rodent models of B. malayi infection. Ethanolic extract (A001) of aerial parts of T. distichum was solvent fractionated and sub-fractionated. Four molecules, 3-Acetoxylabda-8(20), 13-diene-15-oic acid (K001), Beta-sitosterol (K002), labda-8(20),13-diene-15-oic acid (K003) and Metasequoic acid A (K004) were isolated from the fractions and their structure determined by spectroscopic analysis. The extract, subfractions and molecules were evaluated for antifilarial activity against B. malayi by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) reduction and motility assays in vitro and in two animal models, Meriones unguiculatus and Mastomys coucha, harbouring B. malayi infection. A001 was effective in killing microfilariae (mf) and adult worms in vitro. The diterpenoid K003 produced 100 % reduction in motility of both mf and adult worms and > 80 % inhibition in MTT reduction potential of adult female worms. In B. malayi-M. unguiculatus model, A001 killed all the adult worms in > 80 % of infected animals. K003 was embryostatic (> 95 %) in this model. In the B. malayi-M. coucha model, K003 killed ~54 % of adult worms (macrofilaricidal activity) and rendered > 36 % female worms sterile; it also stopped any further rise in microfilaraemia after day 42 post-initiation of treatment. Ethanolic extract of aerial parts of the plant T. distichum possesses potent antifilarial activity and the active principle was localised to K003 which showed significant macrofilaricidal activity and late suppression of peripheral microfilaraemia and some embryostatic activity. These findings indicate that labdane diterpenoid molecule(s) may provide valuable leads for design and development of new macrofilaricidal agent(s). To the best of our knowledge, this is the first report on antifilarial efficacy of products from the plant T. distichum.