Epitope mapping of Brugia malayi ALT-2 and the development of a multi-epitope vaccine for lymphatic filariasis.

Human lymphatic filariasis is a neglected tropical disease, causing permanent and long-term disability with severe immunopathology. Abundant larval transcript (ALT) plays a crucial role in parasite establishment in the host, due to its multi-faceted ability in host immune regulation. Although ALT protein is a key filarial target, its exact function is yet to be explored. Here, we report epitope mapping and a structural model of Brugia malayi ALT-2, leading to development of a multi-epitope vaccine. Structural analysis revealed that ALT represents unique parasitic defence proteins belonging to a toxin family that carries a 'knottin' fold. ALT-2 has been a favourite vaccine antigen and was protective in filarial models. Due to the immunological significance of ALT-2, we mapped B-cell epitopes systematically and identified two epitope clusters, 1-30 and 89-128. To explore the prophylactic potential of epitope clusters, a recombinant multi-epitopic gene comprising the epitopic domains was engineered and the protective efficacy of recombinant ALT epitope protein (AEP) was tested in the permissive model, Mastomys coucha. AEP elicited potent antibody responses with predominant IgG1 isotype and conferred significantly high protection (74.59%) compared to ALT-2 (61.95%). This proved that these epitopic domains are responsible for the protective efficacy of ALT-2 and engineering protective epitopes as a multi-epitope protein may be a novel vaccine strategy for complex parasitic infections.

Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.

Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis.

Lymphatic filariasis, a mosquito-borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria-specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi-epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi-epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript-2 (ALT-2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi-epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential vaccine candidate.

Immunodiagnostic Properties of Wucheraria bancrofti SXP-1, a Potential Filarial Diagnostic Candidate Expressed in Tobacco Plant, Nicotiana tabacum.

Transgenic tobacco plants were developed expressing WbSXP-1, a diagnostic antigen isolated from the cDNA library of L3 stage larvae of Wucheraria bancrofti. This antigen produced by recombinant Escherichia coli has been demonstrated by to be successful as potential diagnostic candidate against lymphatic filariasis. A rapid format simple and qualitative flow through immune-filtration diagnostic kit has been developed for the identification of IgG antibodies to the recombinant WbSXP-1 and is being marketed by M/S Span Diagnostics Ltd in India and Africa. Here, we present the results of experiments on the transformation and expression of the same filarial antigen, WbSXP-1, in tobacco plant, Nicotiana tabacum, to produce plant-based diagnostic antigen. It was possible to successfully transform the tobacco plant with WbSXP-1, the integration of the parasite-specific gene in plants was confirmed by PCR amplification and the expression of the filarial protein by Western blotting. The immunoreactivity of the plant-produced WbSXP-1 was assessed based on its reaction with the monoclonal antibodies developed against the E. coli-produced protein. Immunological screening using clinical sera from patients indicates that the plant-produced protein is comparable to E. coli-produced diagnostic antigen. The result demonstrated that plants can be used as suitable expression systems for the production of diagnostic proteins against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. This is the first report of the integration, expression and efficacy of a diagnostic candidate of lymphatic filariasis in plants.Key MessageTransgenic tobacco plants with WbSXP-1, a filarial diagnostic candidate, were developed. The plant-produced protein showed immunoreactivity on par with the E. coli product.

An evaluation of antigen capture assays for detecting active filarial antigens.

Lymphatic filariasis is a parasitic disease of tropical countries. This is a disfiguring and painful disease contracted in childhood, but the symptoms become apparent only in later years. Diagnosis of filarial infection is very crucial for the management of the disease. The main objective of this study was to develop a filarial antigen-based immunological assay for the diagnosis and surveillance of the disease. Monoclonal and polyclonal antibodies were raised to the recombinant protein Brugia malayi vespid allergen homologue (VAH). Capture enzyme-linked immunosorbent assay (ELISA) was standardized utilizing various combinations of antibodies and evaluated with serum samples of endemic normal (EN, n= 110), microfilaraemic (MF, n= 65), chronic pathology (CP, n= 45) and non-endemic normal (NEN, n= 10) individuals. Of the 230 samples tested, VAH capture assay detected circulating antigen in 97.91% of bancroftian and 100% of brugian microfilaraemic individuals, and 5% of endemic normal individuals, comparable to the earlier reported SXP-1 antigen detection assay. However, the combination of VAH and SXP-1 (VS) capture ELISA was found to be more robust, detecting 100% of microfilaraemic individuals and with higher binding values. Thus an antigen capture immunoassay has been developed, which can differentiate active infection from chronic infection by detecting circulating filarial antigens in clinical groups of endemic areas.

Evaluation of immune response elicited by inulin as an adjuvant with filarial antigens in mice model.

Filariasis caused by infectious parasitic nematodes has been identified as the second leading source of permanent and long-term disability in Sub-Saharan Africa, Asia and Latin America. Several vaccine candidates were identified from infective third-stage larvae (L3) which involves in the critical transition from arthropod to human. Hitherto studies of these antigens in combination with alum adjuvant have shown to elicit its characteristic Th2 responses. Inulin is a safe, non-toxic adjuvant that principally stimulates the innate immune response through the alternative complement pathway. In the present study, the immune response elicited by inulin and alum as adjuvants were compared with filarial antigens from different aetiological agents: secreted larval acidic protein 1 (SLAP1) from Onchocerca volvulus and venom allergen homologue (VAH) from Brugia malayi as single or as cocktail vaccines in mice model. The study revealed that inulin can induce better humoral response against these antigens than alum adjuvant. Antibody isotyping disclosed inulin's ability to elevate the levels of IgG2a and IgG3 antibodies which mediates in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC), respectively, in mice. Splenocyte analysis showed that T cells prestimulated with inulin have higher stimulation index (P < 0.05) than alum except for BmVAH antigen. In vitro ADCC assay showed that inulin formulation had induced higher cytotoxicity with filarial antigens (as single P < 0.01 and as cocktail P < 0.05, respectively) than alum. The results had confirmed the capability of inulin to deplete the levels of Treg and brought a balance in Th1/Th2 arms against filarial antigens in mice.

Wuchereria bancrofti 20/22 a homologue of abundant larval transcript L3 stage filarial antigen: molecular and immunological characterization.

The chromadorea abundant larval transcript (ALT) family of proteins contains ALT one of the most studied putative vaccine candidate in experimental filariasis. This study reports the characterization of Wuchereria bancrofti 20/22 (Wb20/22) as a member of chromadorea, the ALT family of proteins from the L3 stage of W. bancrofti. The high reactivity with serum from the endemic normal (EN) population suggests that Wb20/22 could be a target of elicit protective immunity. The glutamic acid-rich region of Wb20/22 was predicted to harbour the longest linear B-cell epitope by insilico prediction tools. The significance of this region was revealed by studying the mutant form of Wb20/22, without acidic domain (WOAD) which was cloned, and the immune response was compared with Wb20/22. The signal sequence of Wb20/22 was also an immunodominant region, and mutant construct without signal sequence (WOSS) was cloned and characterized. The peak antibody titre elicited by WOAD was higher than Wb20/22 or WOSS, which pointed to the immunomodulatory role of glutamic acid-rich region. Wb20/22 elicited very high levels of IL-10 and diminished levels of IL-4 and IL-5 which could be the reason for low antibody titre. The prophylactic efficacy of WOAD conferred protection (62·26%) which was higher than Wb20/22 (49·82%) and WOSS (54·78%).

Tandem antioxidant enzymes confer synergistic protective responses in experimental filariasis.

Helminth parasites use antioxidant defence strategies for survival during oxidative stress due to free radicals in the host. Accordingly, tissue-dwelling filarial parasites counteract host responses by releasing a number of antioxidants. Targeting these redox regulation proteins together, would facilitate effective parasite clearance. Here, we report the combined effect of protective immune responses trigged by recombinant Wuchereria bancrofti thioredoxin (WbTRX) and thioredoxin peroxidase (WbTPX) in an experimental filarial model. The expression of WbTRX and WbTPX in different stages of the parasite and their cross-reactivity were analysed by enzyme-linked immunosorbent assay (ELISA). The immunogenicity of recombinant proteins and their protective efficacy were studied in animal models when immunized in single or cocktail mode. The antigens showed cross-reactive epitopes and induced high humoral and cellular immune responses in mice. Further, parasite challenge against Brugia malayi L3 larvae in Mastomys coucha conferred significant protection of 57% and 62% against WbTRX and WbTPX respectively. The efficacy of L3 clearance was significantly higher (71%) (P <  0.001) when the antigens were immunized together, showing a synergistic effect in multiple-mode vaccination. Hence, the study suggests WbTRX and WbTPX to be attractive vaccine candidates when immunized together and provides a tandem block for parasite elimination in the control of lymphatic filariasis.

Bacterial lipid modification enhances immunoprophylaxis of filarial abundant larval transcript-2 protein in Mastomys model.

As in many other parasitic diseases, efficacious vaccine for lymphatic filariasis has been elusive for want of new approaches leaving billions of people either debilitated or at risk. With multiple B- and T-cell epitopes, the abundant larval transcript-2 (ALT-2) of the filarial worm, Brugia malayi, has been shown to be a promising immunoprophylactic target. To enhance its efficacy, it was lipid modified using our recently developed protein engineering tool, which then offered 30% more immunoprotection (49 vs. 79%) in Mastomys coucha model. Sustained high levels of IFN-γ (about 100 times) and high antibody titres (10-fold) elicited by lipid-modified ALT-2, as compared to the native form, indicated the maintenance of Th1/Th2 balance that is impaired in filariasis. Thus, this study provides the basis for developing efficacious vaccines for filariasis and other parasitic diseases by exploiting bacterial lipid modification.

Molecular and functional characterization of macrophage migration inhibitory factor (MIF) homolog of human from lymphatic filarial parasite Wuchereria bancrofti.

The ability of nematode parasites to survive in a highly complex immune system involves diverse strategies including production of a variety of host immune modulators. Various parasite-associated surface antigens or excretory and secretory products may possibly play a role in the host-parasite interactions and successful survival of parasite in their respective host. One among these molecules is a human cytokine homolog, macrophage migration inhibitory factor-1 (MIF-1) in various parasites. We identified a homolog of this cytokine from human lymphatic filarial parasite, Wuchereria bancrofti, expression cloned and investigated its molecular characteristics and catalytic properties. We also assessed the humoral reactivity of the recombinant MIF-1 of W. bancrofti (rWb-MIF-1) against sera belonging to different categories of individuals viz. microfilaremic, chronic patients, endemic normal, and non-endemic normal. Our results showed that the complete coding sequence of W. bancrofti is 1,078 bp, comprising two introns and three exons: first and second introns being 577 and 153 bp long, while the three exons I, II, and III being 108, 173, and 67 bp long, respectively. The rWb-MIF-1 was overexpressed in a salt-inducible host, Escherichia coli GJ 1158, and its functional activity was determined by dopachrome tautomerase and insulin reduction assays. The results of both the assays showed that the purified protein is functionally active and hence folded appropriately. The rWb-MIF-1 protein did not show elevation of specific IgG4 antibodies in microfilaremic cases, a hallmark in case of lymphatic filariasis, while it showed IgE reactivity in some of these cases (five out of ten).

A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and BmVAH) enhances immune responses and protection in jirds.

In this study filarial recombinant protein or DNA vaccine constructs encoding BmALT-2 and BmVAH as single or as cocktail antigens were evaluated. Male jirds were immunized intramuscularly with DNA vaccine constructs or were immunized intraperitoneally with protein vaccine. The single and bicistronic DNA constructs induced substantial interferon-γ responses in spleen cells; antigen-specific responses were higher following immunization with the bicistronic cocktail construct and evoked a significant protective response of 57% in jirds challenged with Brugia malayi that was similar in the antibody-dependent cellular cytotoxicity (ADCC) assay and micropore chamber experiment. The cocktail protein vaccines induced a mixture of IgG2a (Th1) and IgG1 (Th2) responses with 80% protective response when challenged with B. malayi infective larvae. However, the single protein vaccine rALT-2 induced Th2 (IgG1/IgG3) with a 70% protective response and rVAH induced Th1 (IgG2a) with a lower proliferative response with 60% protection following challenge with B. malayi infective larvae. These results suggest that filarial cocktail protein vaccines are able to elicit substantial immune and protective responses when compared with single antigen vaccination in suitably vaccinated jirds.

Development and characterization of monoclonal antibodies against WbSXP-1 for the detection of circulating filarial antigens.

The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the World Health Organization. Presently, few immunodiagnostics are available for filarial monitoring programmes. The Wuchereria bancrofti (Wb) SXP-1 parasite protein, with 84% homology to Brugia malayi (Bm) SXP-1, was found to be highly immunogenic. WbSXP-1 is one among the diagnostic candidate molecules that were used for developing a rapid-antibody-flow-through diagnostic kit for filariasis. Studies were initiated with the aim of developing monoclonal antibodies against recombinant WbSXP-1 and prospective applications for the detection of both circulating Wb and Bm antigens in serum samples from infected individuals. The monoclones 1A6C2 of subclass IgG1k, and 2A12F8 of class IgM, specifically detected Wb and Bm microfilaria isolated from patients and did not show cross-reactivity with other filarial recombinant antigens. We anticipate that this work will address the problems faced in the rapid diagnosis of human lymphatic filariasis in endemic areas in developing countries.

Dominant T-cell epitopes of filarial BmALT-2 and their cytokine profile in BALB/c mice.

Filarial nematodes down-regulate the host immune response to establish infection by inducing IL-10-mediated T-cell suppression.Abundant larval transcript (ALT) proteins are of major interest as they are expressed abundantly in the L3 stage, implicated in protective immunity and may play a role in immune evasion. The T-cell epitopes of BmALT-2 and the cytokine responses induced by these peptides were investigated in a mouse model using synthetic peptides, which could be exploited in the design of a potent epitope-based vaccine. Five regions were found to carry T-cell epitopes inducing high levels of cellular proliferation. The regions 55­68 and 73­91 of ALT-2 induced very high levels of IL-10 secretion and hence could be involved in immunomodulation in the host.

Crucial epitopes of Wuchereria bancrofti abundant larval transcript recognized in natural infection.

Lymphatic filariasis is a tropical disease, with over 40 million people seriously incapacitated due to lymphangitis and elephantiasis. Over 99% of infections are caused by the nematode Wuchereria bancrofti. Expressed sequence tag (EST) analysis of filarial genome identified novel infective larval (L3) stage-specific antigen, abundant larval transcript (ALT-2), which was shown to be highly essential for parasite establishment and survival in the host. The unique structural features and immunological characteristics of ALT-2 indicate the presence of critical motifs that needs to be explored in natural human infection for understanding and management of the disease. In order to dissect the epitopes of ALT protein recognized in humans, eight peptides spanning the entire protein sequence were selected based on in-silico epitope prediction and synthesized. Analysis of the reactivity of W. bancrofti ALT-2 synthetic peptides with clinical sera (n = 40) from endemic areas identified epitopes recognized by putatively immune sera, of which two comprise the highly variable acidic domain (21-60). Interestingly, our study also revealed crucial epitopes recognized by microfilaremic (MF) sera with significantly high IgG4 isotype (p < 0.001), implicated in immunomodulation. The epitope peptides identified were highly specific for MF sera and, thus, have the potential to be exploited as diagnostic markers.

Production, purification and diagnostic application of filarial recombinant protein WbSXP-1 expressed in salt inducible Escherichia coli.

Wuchereria bancrofti protein WbSXP-1 was identified and established as a potential candidate for the diagnosis of lymphatic filariasis. For the economic production of rWbSXP-1, osmotically (salt) inducible Escherichia coli GJ1158 was preferred. Cultivation and expression was optimized in 3 L airlift bioreactor (ALB) and was successfully extended to 30 L ALB. Purification of rWbSXP-1 his-tag protein was optimized in technical scale using FPLC and the maximal recovery of rWbSXP-1 with significant level of purity was achieved using the combination of IMAC and gel filtration. Quality criteria for immuno-reactivity of purified rWbSXP-1 were established for diagnostic applications. Enhancement of sensitivity in rapid diagnostic format was optimized to effectively detect weak to strong antibody reactivity in individuals exposed to lymphatic filariasis. Performance of the rapid format during field evaluation was successful. The accelerated stability assessment of the rapid format satisfied the requirements of WHO-cGMP norms. This investigation presents a successful technical scale production and purification of rWbSXP-1 considering the future industrial application and an enhanced rapid flow through antibody assay for the diagnosis of human lymphatic filariasis.

Diminished monocyte function in microfilaremic patients with lymphatic filariasis and its relationship to altered lymphoproliferative responses.

Antigen-specific hyporesponsiveness to filarial antigens is a phenomenon observed in patent infection with lymph-dwelling filarial parasites of humans. This phenomenon has been attributed to a multitude of factors, one of which is altered monocyte function. To examine the role played by monocytes in filarial infection, we assessed the responses of monocytes obtained from normal and filarial parasite-infected individuals to both crude filarial antigen and purified recombinant filarial antigen WbSXP-1 and attempted to relate these to the altered lymphoproliferative responses seen in filarial infection. Monocytes from microfilaremic (MF) patients demonstrated an inability to respond to lipopolysaccharide compared to monocytes from endemic normal persons or from lymphedema patients. Indeed, interleukin 1beta (IL-1beta) production was severely limited, a finding that did not extend to monocyte responses to filarial antigens. Serum from MF patients reduced adherence and spreading of normal monocytes, a finding not seen with serum from the other clinical groups. Interestingly, there was a significant correlation between the production of IL-1beta and adherence. Moreover, the levels of spontaneous production of IL-1beta correlated with high levels of spontaneous secretion of IL-10. The effects observed were not a result of diminished viability or alteration in the expression of the cell surface markers CD14 and HLA-DR. These data suggest that monocyte function is dampened in MF patients, a finding which could alter lymphoproliferative responses during patent infection.

Wuchereria bancrofti: cloning and characterization of heat shock protein 70 from the human lymphatic filarial parasite.

Heat shock protein 70 (HSP70) was identified as an immunodominant antigen by screening a Wuchereria bancrofti (Wb) microfilarial cDNA library with pooled Wb-infected sera, with 28% of the immunopositive clones coding for Wb-HSP70. The deduced amino acid sequence showed greater than 97 and 85% identity with HSP70 from filarial nematodes and humans, respectively. Recombinant HSP70 (74 kDa) and a recombinant protein from the C-terminal portion (43 kDa) also reacted with pooled Wb-infected sera, suggesting that the C-terminal region of HSP70 contains at least one antibody epitope. Brugia malayi L3 larvae showed increasing levels of HSP70 with increasing temperatures. Further, a polyclonal mouse anti-Wb-HSP70 antibody had reactivity to the HSP70 of cattle filarial parasite Settaria digitata and to human HSP70 derived from a Hep-2 cell line. Immune reactivity to Wb-HSP70 was strong, with uninfected non-endemic normal sera showing significantly greater reactions than sera from filaria-infected individuals. Both immunodominant self-HSP70 and HSP70 from other microbial infections may be primary targets for developing autoantibodies naturally.

Thioredoxin from Brugia malayi: defining a 16-kilodalton class of thioredoxins from nematodes.

Thioredoxins are a family of small redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase. This results in a supply of reducing equivalents that cells use in a wide variety of biological reactions, which include maintaining reduced forms of the enzymes important for protection against damage from high-energy oxygen radicals, the regulation of transcription factor activity, and the inhibition of apoptosis. Here we report on a new member of the thioredoxin family of proteins from the filarial nematode Brugia malayi, Bm-TRX-1, which defines a new subclass of 16-kDa thioredoxins that occur widely in nematodes, including Caenorhabditis elegans. In addition to being larger than the thioredoxins found in mammalian and bacterial species, the putative active site sequence of Bm-TRX-1, WCPPC, does not conform to the highly conserved WCGPC reported for thioredoxins from mammals to bacteria. Interestingly, an allelic form of Bm-TRX-1 was identified with an active site sequence WCPQC, which appears to be unique to the thioredoxins from filarial species. Bm-TRX-1 was between 98% and 35% identical to thioredoxins from other nematodes and approximately 20% identical to the thioredoxins from mammals and Escherichia coli. Bm-TRX-1 was constitutively transcribed throughout the B. malayi life cycle, and Bm-TRX protein was detectable in somatic extracts and excretory-secretory products from adults and microfilariae. Recombinant Bm-TRX-1 had thiodisulfide reductase activity, as measured by the reduction of insulin, and protected DNA from the nicking activity of oxygen radicals. Overexpression of Bm-TRX-1 in a human monocyte cell line negatively regulated tumor necrosis factor alpha-induced p38 mitogen-activated protein kinase activity, suggesting a possible role of the 16-kDa Bm-TRX-1 in immunomodulation.

Analysis of Wuchereria bancrofti infections in a village community in northern Nigeria: increased prevalence in individuals infected with Onchocerca volvulus.

Infections with Wuchereria bancrofti causing lymphatic filariasis still represent one of the major health problems in the tropics, with 120 million people infected and over 750 million exposed to this filarial parasite. We have studied lymphatic filariasis infections as part of a multi-parasite survey in a village community in the savannah of northern Nigeria. We analysed serum samples from 341 individuals aged 5-70 years, detecting a W. bancrofti circulating antigen using the commercially available ICT Filariasis card test. The prevalence of infections was 10% and clearly age-dependent, increasing from below 2% in children to over 20% in subjects older than 40 years. Measuring IgG4 antibodies against the recombinant W. bancrofti antigen SXP1 showed that 36% of all tested individuals had been at least exposed to the parasite. Antibody levels also increased very significantly with age. A further analysis measuring Onchocerca volvulus-specific IgG4 antibodies showed a very significant association between infections with O. volvulus and those with W. bancrofti. Our data show that infections with W. bancrofti in Nigeria are still a frequently occurring health problem, since they are more prevalent than previously reported, and that individuals with an O. volvulus infection are more often infected with W. bancrofti than expected statistically.

Molecular characterization of a calcium binding translationally controlled tumor protein homologue from the filarial parasites Brugia malayi and Wuchereria bancrofti.

We have cloned homologues of the mammalian translationally controlled tumor protein (TCTP) from the human filarial parasites Wuchereria bancrofti and Brugia malayi. TCTP genes from B. malayi and W. bancrofti were expressed in a T7 promoter vector as histidine tagged fusion proteins. Both the recombinant B. malayi TCTP (rBm-TCTP) and recombinant W. bancrofti TCTP (rWb-TCTP) have a molecular mass of approximately 28 kDa with the histidine tag. Sequence analyses showed that there is a 98% similarity between the two filarial TCTPs at amino acid levels and are immunologically cross-reactive. Analysis of soluble proteins from various lifecycle stages of B. malayi suggested that the expression of Bm-TCTP might be differentially regulated and occurs in multimeric form. Recombinant TCTP were found to form multimers in solution under non-reducing conditions. The tendency for filarial TCTPs to become multimers was predicted by the presence of the Lupas coiled coil structure in their sequence. Despite the absence of a signal sequence, Bm-TCTP is present abundantly in the excretory/secretions (ES) of microfilariae. Characterization studies showed that both Bm- and Wb-TCTPs are calcium-binding proteins and have histamine-releasing function in vitro. When injected intraperitoneally both the filarial TCTPs induced inflammatory infiltration of eosinophils into the peritoneal cavity of mice suggesting that the filarial TCTPs may have a role in the allergic inflammatory responses associated with filarial infections.