cDNA coding for Brugia malayi pepsin inhibitor homolog (Bm-33) from the human filarial parasite was cloned in pRSET for large-scale expression and functional characterization. The pRSET-B cloned gene did not yield recombinant protein expression and the reason was attributed to the presence of an N-terminal signal peptide. The gene was subcloned in pRSET-A without a signal peptide and the 33 kDa histidine-tagged recombinant protein was purified by IMAC. All individuals from an endemic area generated IgG responses against Bm-33 in the order MF>CP>EN. Isotype analysis indicated an elevated IgG4 reactivity in the order MF>EN>CP. Bm-33-specific IgE levels were elevated in MF, CP and EN compared to non-endemic normals with no significant differences among the groups. Paraffinembedded sections of Setaria digitata (cattle filarial parasite) stained with mouse anti-Bm-33 antibodies exhibited the hypodermal nature of Bm-33. These findings suggest that Bm-33 is an immunodominant antigen and contributes to filarial pathogenesis. Key words antibody, Brugia malayi, ELISA, immunolocalization, isotype.Human lymphatic filariasis is caused by lymph-dwelling, parasitic nematodes such as Wuchereria bancrofti, Brugia malayi and Brugia timori which are prevalent in tropical and subtropical countries of the world. The initiatives of the Filarial Genome Project to combat this nematode scourge have enabled the identification and characterization of genes with diagnostic and prophylactic capabilities such as WbSXP-1, Bm-ALT etc. (1, 2). Despite these and a few more, there is an urgent need to identify and characterize more antigens involved in parasite-specific immune modulation, a major factor that contributes to filarial pathogenesis.The cDNA coding for B. malayi, pepsin inhibitor homolog (Bm-33), a 33 kDa protein (3, 4), was cloned Correspondence Rangarajan B. Narayanan, Centre For Biotechnology, Anna University, Chennai-600 025, Tamil Nadu, India. Tel: +91 044 22350772; fax: +91 044 22350299; email: rbn@annauniv.edu List of Abbreviations ALP, alkaline phosphatase; anti-BmA, antibodies against Brugia malayi crude protein; BCIP, bromochloroindoloyl phosphate; CP, chronic pathology; DAB, diaminobenzidine; EN, endemic normal; HLA, human leukocyte antigen; HRP, horseradish peroxidase; IgG, immunoglobulin G; IMAC, immobilized metal affinity chromatography; IPTG, isopropyl beta thiogalactosidase; MF, microfilaremic; NBT, nitroblue tetrazolium; NEN, non-endemic normal; pNPP, para nitrophenol phosphate. and serologically characterized. Earlier, a similar homolog from Onchocerca volvulus (Ov33) was cloned in pGEX-2T and the GST tagged-recombinant protein was used for immunodiagnosis based on its elevated IgG4 reactivity in microfilaremic patients (5, 6). Homologs were also identified from related parasites such as Dirofilaria immitis and Acanthocheilonema viteae and the respective recombinant proteins (Di33 and Av33) were used for the development of immunodiagnostic assays (7,8). In the case of the Brugian homolog, the 750 bp cDNA cod...

Wolbachia, an endosymbiont present in filarial nematodes, have been implicated in a variety of roles, including the worm development and survival. Elucidation of the role of Wolbachia in filarial nematode biology and pathogenesis has become the focus of many studies and its contribution to parasite survival or immune response is still unclear. Recombinant Wolbachia HSP60 decreases T cell activation and lymphoproliferation in filarial infected people compared to endemic controls as observed by the assessment of T cell activation markers and cytokine responses in the peripheral blood mononuclear cells. Reduced T cell activation may be linked to T regulatory cell activity since it is associated with increased expression of CTLA4 and CD25 on CD4+ T cells in filarial infected group upon stimulation with recombinant Wolbachia HSP60. In addition, elevated interleukin-10 and TGF-β cytokines corroborate the reduced CD4+ T cell activation and interferon-γ observed upon recombinant Wolbachia HSP60 stimulation in filarial patients. Hence, these findings indicate that Wolbachia HSP60 may also contribute to the immune modulation seen in filarial patients.

Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B-cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T-cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR-amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T-7 expression vector pRSET-A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP-specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP-specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of Band T-cell epitopes. Hence, a combinatorial approach of in silico predictions and wet-lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.

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Immune responses to recombinant Brugia malayi pepsin inhibitor homolog (rBm-33) were investigated in patients with human lymphatic filariasis (microfilaremics (MF) and chronic pathology (CP)) along with endemic normals (EN). Flow cytometric analysis (24 h) revealed CD4+ T cell activation in patients (MF and CP) compared to normals (EN), with increased expression of CD69 and diminished levels of CD62L and CD127. This was associated with an elevated expression of CD154 but not CD28 and CTLA4 in CP patients. However, Bm-33-induced cytokine expression profile (IL-1β, IL-12, IL-8, IFN-γ, IL-10 and TGF-β) did not exhibit any significant difference between normals and patients at the same time point. Although CD4+ T cell activation was observed initially in filarial patients (24 h), lymphoproliferation studies (96 h) suggested diminished proliferation compared to normals, indicating functional inactivation in the former upon prolonged antigen exposure. This indicates that rBm-33 induces an early T cell activation in MF and CP patients followed by a decreased lymphoproliferation that might contribute to immune suppression in these individuals.

Human lymphatic filariasis is a debilitating parasitic disease characterized by downregulation of the host’s immune response in asymptomatic carriers along with profound hyperreactivity in chronic patients apart from putatively immune endemic normals. The endosymbiont Wolbachia, a bacterium of filarial nematodes has received much attention as possible chemotherapeutic target and its involvement in disease pathogenesis. The role of recombinant Wolbachia surface protein (rWSP), one of the most abundantly expressed proteins of the endosymbiont, in modulating cell-mediated immune responses in patients harboring Wuchereria bancrofti infections was evaluated in the current study. rWSP-induced lymphoproliferation with peripheral blood mononuclear cells suggested an impaired proliferative response in asymptomatic microfilaremic (MF) and symptomatic chronic pathology (CP) patients compared to endemic normals (EN). This was further supported by a significantly diminished expression of CD69 along with elevated levels of CD127 and CD62L in filarial patients (MF and CP) compared to EN. Further, rWSP induced the expression of regulatory T cell markers CTLA-4 and CD25 along with suppressor cytokines IL-10 and TGF-β in MF and CP patients compared to EN. However, the rWSP-stimulated expression of IFN-γ was diminished significantly in filarial patients compared to endemic normals. Thus, these findings suggest that WSP may also contribute to the suppression of immune responses seen in filarial patients.

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable 'enzyme-product' complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 10(3) and 6.52 × 10(3) M(-1). The binding reactions were enthalpy driven with ΔHb values between -50.99 and -46.07 kJ mol(-1). From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.

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