Immune Response Profiling of Cocktails of Brugia malayi Vaccine Candidates DIM-1, Calponin and Troponin 1 in BALB/c Mice.

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-1ß, IL-2, IFN-γ and TNF-α) and Th2 (IL-4, IL-6, IL-10 and TGF-ß) 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.

Immunoreactivity of Brugia malayi Calreticulin and Its Domains with Sera of Different Categories of Bancroftian Filarial Patients.

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.

Strategies to Control Human Lymphatic Filarial Infection: Tweaking Host's Immune System.

Human lymphatic filariasis (LF), a parasitic infection caused by the nematodes Wuchereria bancrofti, Brugia malayi and B. timori, and transmitted by mosquito, results in a debilitating disease commonly identified as 'elephantiasis'. LF affects millions of people in India and several other tropical and sub-tropical countries imposing a huge economic burden on governments due to disability associated loss of man-hours and for disease management. Efforts to control the infection by WHO's mass drug administration (MDA) strategy using three antifilarials diethylcarbamazine, albendazole and ivermectin are only partly successful and therefore, there is an immediate need for alternative strategies. Some of the alternative strategies being explored in laboratories are: enhancing the immune competence of host by immunomodulation, combining immunomodulation with antifilarials, identifying immunoprophylactic parasite molecules (vaccine candidates) and identifying parasite molecules that can be potential drug targets. This review focuses on the advances made in this direction.

Leishmania donovani molecules recognized by sera of filaria infected host facilitate filarial infection.

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-ß. 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.

Antifilarial diarylheptanoids from Alnus nepalensis leaves growing in high altitude areas of Uttarakhand, India.

Lymphatic filariasis continues to be a major health problem in tropical and subtropical countries. A macrofilaricidal agent capable of eliminating adult filarial parasites is urgently needed. Platyphyllenone (A), alusenone (B), hirustenone (C) and hirsutanonol (D) are important biologically active diarylheptanoids present in Alnus nepalensis. In the present study, we report the antifilarial activity in diarylheptanoids isolated from the leaves of A. nepalensis. Out of four compounds (A-D) tested in vitro one has shown promising anti-filarial activity both in vitro and in vivo studies. This is the first ever report on antifilarial efficacy of a compound of the plant and warrants further studies around this scaffold. In addition, a sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for the above four biomarker compounds. The separation was performed on silica gel 60F(254) high-performance thin layer chromatography plates using chloroform:methanol (9:1, v/v) as mobile phase. The quantitation of marker compounds was carried out using densitometric reflection/absorption mode at 600 nm after post-chromatographic derivatization using vanillin-sulfuric acid reagent. The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines.

Inflammatory mediator release by Brugia malayi from macrophages of susceptible host Mastomys coucha and THP-1 and RAW 264.7 cell lines.

OBJECTIVE: To investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages. METHODS: The human macrophage/monocyte cell line THP-1, the mouse macrophage cell line RAW 264.7 and naive peritoneal macrophages (PM) from the rodent host Mastomys coucha (M. coucha) were incubated at 37 °C in 5% CO(2) atmosphere with extracts of microfilariae (Mf), third stage infective larvae (L(3)) and adult worms (Ad) of Brugia malayi. After 48 hr post exposure, IL-1ß, IL-6, TNF-α, IL-10 and nitric oxide (NO) in cell-free supernatants were estimated. RESULTS: Extracts of all the life stages of the parasite were capable of stimulating pro- (IL-1ß, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines in both the cell lines and peritoneal macrophages of M. coucha. Mf was the strongest stimulator of pro-inflammatory cytokines followed by L(3) and Ad; however, Ad was a strong stimulator of IL-10 release. Mf was found to have potential to modulate LPS-induced NO release in RAW cells. Ad-induced NO release was concentration dependent with maximum at 20 µg/mL in both RAW and PMs. CONCLUSIONS: The results show that parasites at all life stages were capable of stimulating pro- (IL-1ß, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines and NO release from macrophages of susceptible host M. coucha, human and mouse macrophage cell lines. Mf can suppress the LPS-induced NO release in RAW cells. The findings also show that the two cell lines may provide a convenient in vitro system for assaying parasite-induced inflammatory mediator release.

Plant products in the treatment and control of filariasis and other helminth infections and assay systems for antifilarial/anthelmintic activity.

Lymphatic filariasis, onchocerciasis, loaisis, and other helminth infections cause serious health problems especially in resource-limited tropical and subtropical developing countries of the world, and more than 2 billion people are infected with at least one helminth species. From times immemorial, man looked up to the plant kingdom in search of anthelmintics, antifilarials, and remedies for parasite-induced health problems. Although more than 50 % of drugs in modern medicine are derived from plants or leads from plants, a success story of plant-based anthelminthics or antifilarials is yet to be told. In the last 5 decades, more than 100 plant products were reported to be beneficial in the treatment or control of these parasitic infections but they could not be developed into viable drugs for a variety of reasons. This review focuses on the plant products reported to be useful in the control and treatment of human helminth infections with the main emphasis on filariasis and the in vitro and in vivo systems available for assaying anthelmintic activity.