Biochemical characterization and evaluation of a Brugia malayi small heat shock protein as a vaccine against lymphatic filariasis

Filarial nematodes enjoy one of the longest life spans of any human pathogen due to effective immune evasion strategies developed by the parasite. Among the various immune evasion strategies exhibited by the parasite, Interleukin 10 (IL-10) productions and IL-10 mediated immune suppression has significant negative impact on the host immune system. Recently, we identified a small heat shock protein expressed by Brugia malayi (BmHsp12.6) that can bind to soluble human IL-10 receptor alpha (IL-10R) and activate IL-10 mediated effects in cell lines. In this study we show that the IL-10R binding region of BmHsp12.6 is localized to its N-terminal region. This region has significant sequence similarity to the receptor binding region of human IL-10. In vitro studies confirm that the N-terminal region of BmHsp12.6 (N-BmHsp12.6) has IL-10 like activity and the region containing the alpha crystalline domain and C-terminus of BmHsp12.6 (BmHsp12.6αc) has no IL-10 like activity. However, BmHsp12.6αc contains B cell, T cell and CTL epitopes. Members of the sHSP families are excellent vaccine candidates. Evaluation of sera samples from putatively immune endemic normal (EN) subjects showed IgG1 and IgG3 antibodies against BmHsp12.6αc and these antibodies were involved in the ADCC mediated protection. Subsequent vaccination trials with BmHsp12.6αc in a mouse model using a heterologous prime boost approach showed that 83% protection can be achieved against B. malayi L3 challenge. Results presented in this study thus show that the N-BmHsp12.6 subunit of BmHsp12.6 has immunoregulatory function, whereas, the BmHsp12.6αc subunit of BmHsp12.6 has significant vaccine potential.     

Diagnostic value of IgG isotype responses against Brugia malayi antifilarial antibodies in the clinical spectrum of brugian filariasis

To study the diagnostic significance of antifilarial IgG subclasses in the clinical spectrum of brugian filariasis, IgG1, IgG3 and IgG4 antifilarial antibodies were determined in an exposed population comprising 74 asymptomatic amicrofilaraemics, 30 microfilaraemics, 20 lymphangitis and 16 elephantiasis patients resident in Narathiwart province, an area endemic for Brugia malayi lymphatic filariasis in southern Thailand. The dominant isotype of antifilarial antibody was IgG4. A significantly higher percentage of individuals were positive for IgG1 in the microfilaraemic and lymphangitis groups compared with the elephantiasis and endemic normal patients, while a significantly higher positive rate of IgG3 was found in those with lymphangitis. The possible role of these isotypes for diagnostic purposes and the pattern of antibody response in various clinically manifesting groups are discussed.     

Brugia malayi: intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10(6) microfilariae. Ferrets (Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 +/- 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3-4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10(6) microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (greater than or equal to 1:200) and microfilarial agglutination titers (greater than or equal to 1:40) rapidly cleared injected microfilariae (less than 24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunolocalization and serum antibody responses to Brugia malayi pepsin inhibitor homolog (Bm-33)

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. Paraffin-embedded 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.     

Rapid diagnosis of Brugia malayi and Wuchereria bancrofti filariasis by an acridine orange/microhematocrit tube technique.

A microhematocrit tube technique for diagnosis of human filariasis has been previously described. A system incorporating heparin, EDTA, and acridine orange into a microhematocrit tube (Quantitative Blood Count, QBC) has been commercially developed for the quantitation of blood counts and has been used for the diagnosis of malaria. We evaluated this test for its usefulness in the diagnosis of filariasis. Upon centrifugation, the parasites were concentrated in the area of the buffy coat and could be observed through the wall of the tube. The parasites were concentrated further by a plastic float that expands the buffy coat and confines the parasites to the periphery of the tube. Acridine orange stains the DNA of the parasite, and morphologic characteristics can be examined by fluorescence microscopy. The terminal and subterminal nuclei and long cephalic space of Brugia malayi, as well as the short cephalic space and caudal nuclei of Wuchereria bancrofti, were easily recognized and differentiated from each other. Microfilariae were detected in samples diluted to a level of approximately 50/ml.     

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.     

Effects of selected cholinergic and anticholinergic drugs on Brugia malayi (Nematoda)

1. Several drugs were tested as inhibitors of the body movements of adult Brugia malayi. 2. Atropine, carbachol, DDNS (a fluorescent acetylcholine analog), diethylcarbamazine, and physostigmine caused significant reduction in motor activity. 3. Glutamate, hexamethonium, muscarine, norepinephrine, serotonin and d-tubocurarine had no effect. Three novel phosphonium compounds were tested as inhibitors of Brugia and vertebrate acetylcholinesterase. 4. Two of these produced preferential inhibition of the enzyme from Brugia.     

Modeling analysis of GST (glutathione-S-transferases) from Wuchereria bancrofti and Brugia malayi

GST (glutathione S-transferases) are a family of detoxification enzymes that catalyze the conjugation of reduced GSH (glutathione) to xenobiotic (endogenous electrophilic) compounds. GST from Wb (Wuchereria bancrofti) and Bm (Brugia malayi) are significantly different from human GST in sequence and structure. Thus, Wb-GST and Bm-GST are potential chemotherapeutic targets for anti-filarial treatment. Comparison of modeled Wb and Bm GST with human GST show structural difference between them. Analysis of the active site residues for the binding of electrophilic co-substrates provides insight towards the design of parasite specific GST inhibitors.     

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

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 (K_b) values between 10.23?×?10³ and 6.52?×?10³ M^?1. The binding reactions were enthalpy driven with ΔH_b 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.     

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.     

Identification and cloning of a novel tetraspanin (TSP) homologue from Brugia malayi.

This is the first report of a tetraspanin (TSP)-like molecule in the lymphatic filarial parasites. Expressed sequence tag (EST) database search for TSP like molecules in the filarial genome resulted in three significant EST hits (two partial ESTs from Brugia malayi and one full length EST from Wuchereria bancrofti). The full length gene cloned from B. malayi showed significant similarity to Caenorhabditis elegans TSP and human TSP and hence the gene was named B. malayi TSP (BmTSP). Subsequent Genbank analysis with the predicted ORF of BmTSP showed additional homologous genes reported from Schistosoma mansoni and Taenia solium parasites. Structural analyses showed that BmTSP has four transmembrane domains and other conserved domains such as CCG and two other critical cysteine residues present within the large extracellular loop similar to other reported TSPs. In addition, putative post-translational modifications such as N-glycosylation, protein kinase c phosphorylation, casein kinase II phosphorylation and N-myristoylation sites have been found in BmTSP sequence. Further, PCR analyses showed that BmTSP is differentially transcribed, with highest level of expression being present in the adult stages followed by L3 and mf stages. This study thus describes a novel TSP cloned from B. malayi, its putative functions in cuticle biogenesis and role in protective immunity.     

Recombinant Wolbachia heat shock protein 60 (HSP60) mediated immune responses in patients with lymphatic filariasis

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.     

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels of microfilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in a microfilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.     

Genetic determinism of parasitic circadian periodicity and subperiodicity in human lymphatic filariasis

The larval parasites of the pantropical lymphatic filariasis exhibit two types of circadian behaviour. Typically, they only appear in the human bloodstream at nighttime, synchronised with their mosquito vectors. In Polynesia and parts of Southeast Asia, free of nocturnal vectors, they are found at all hours, and each population biorhythm differs. Through a geometrical approach, we explain this circadian diversity by a single, dominant mutation: the clocks of individual parasites are set at midnight (ubiquitous) or at 2 p.m. Compared to other circadian genes, this mutation must be very old, as it is shared by four biologically remote genera of parasites. This seniority sheds new light on several theoretical and practical aspects of vector-parasite temporal relations.     

T-cell activation and the balance of antibody isotypes in human lymphatic filariasis

Human filarial infection presents a spectrum of clinical states with two major poles: asymptomatic microfilaraemia and amicrofilaraemic chronic disease. Microfilaremia is associated with a Th1-type tolerance, and maximal IgG4 antibodies, while elephantiasis patients react across a broad range of immune parameters. In this review, Rick Maizels and his colleagues discuss recent advances in the immunology of human filariasis and present a summary of their latest studies in an endemic area of Indonesia.     

Immunoprophylactic studies with a 43 kDa human circulating filarial antigen and a cross reactive 120 kDaBrugia malayi sodium dodecyl sulphate soluble antigen in filariasis

Bancroftian filariasis is a major public health problem affecting about 120 million people all over the world. Immunoprophylaxis may serve as an additional adjunct along with chemotherapy and anti larval measures for successful filaria control. Circulating filarial antigen fraction (CFA₂-6) containing 43 kDa antigen and adultBrugia malayi sodium dodecyl sulphate (S DS) soluble antigen fraction BmA-2 with a 120 kDa molecule were earlier shown to be reactive with endemic normal sera by immunoblotting and indirect ELISA techniques. BmA-2 was found to be highly cross reactive with CFA₂-6. Sera raised against both the antigen fractions showed about 90 % cytotoxicity to the parasites in the presence of jird peritoneal cells inin vitro as well as byin situ micropore chamber implantation technique. Further inin vivo studies using animal model, jirds CFA₂-6 and BmA-2 could induce about 90% protection to infection in immunized animals. In passive transfer studies of immunity it has been observed that BmA-2 induced protection is mainly antibody mediated.     

Induction of TRAIL- and TNF-alpha-dependent apoptosis in human monocyte-derived dendritic cells by microfilariae of Brugia malayi

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.     

Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses

BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.     

A comparison of the biological activities of authentic rat GRF(1-43)OH with the analogue rat GRF(1-29)NH2

The purpose of this study was to characterize the biological activity of the synthetic rat growth hormone releasing factor analogue rGRF(1-29)NH2 and to compare its action on growth hormone (GH) release to that of authentic rGRF(1-43)OH. We first compared the concentration-response characteristics of the two peptides in static incubation, and then examined the reversibility and repeatability of the GH response in a perifusion system. Authentic rGRF(1-43)OH was significantly more potent in static incubation (EC50 = 3 x 10(-11) M) than the analogue (5 x 10(-11) M), whereas the reverse held true in perifusion. The shapes of the GH responses were similar for both peptides in the perifusion system. However, while the GH response to authentic rGRF was repeatable, the prior administration of rGRF(1-29)NH2 significantly reduced (greater than 50%) the GH response to the subsequent administration of either rGRF(1-29)NH2 or rGRF(1-43)OH. Thus authentic rGRF and the synthetic fragment may have different actions at the level of the GRF receptor or at a postreceptor (second messenger) step.     

Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29)

The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.