Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis – a multicenter trial

The development of antifilarial antibody responses is a characteristic feature of infection with filarial parasites. It should be possible to exploit this fact to develop tools to monitor the progress of the global program to eliminate lymphatic filariasis (LF); however, assays based on parasite extracts suffer from a number of limitations, including the paucity of parasite material, the difficulty of assay standardization and problems with assay specificity. In principle, assays based on recombinant filarial antigens should address these limitations and provide useful tools for diagnosis and surveillance of LF. The present multicenter study was designed to compare the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. Coded serum specimens were distributed to five participating laboratories where assays for each antigen were conducted in parallel. Assays based on Bm14, WbSXP, or BmR1 demonstrated good sensitivity (>90%) for field use and none of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections. Limitations of the assays are discussed. Well-designed field studies are now needed to assess sampling methodology and the application of antibody testing to the monitoring and surveillance of LF elimination programs.     

Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host–parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.     

Longitudinal Monitoring of the Development of Antifilarial Antibodies and Acquisition of Wuchereria bancrofti in a Highly Endemic Area of Haiti

Antifilarial antibody testing has been established as a sensitive and specific method of diagnosing lymphatic filariasis. However, the development of serological responses to specific filarial antigens and their relationship to acquisition of infection is poorly understood. In order to evaluate whether the development of antigen specific antifilarial antibodies precedes microfilaremia and antigenemia, we compared the antibody responses of serum samples collected between 1990 and 1999 from a cohort of 142 Haitian children followed longitudinally. Antigen status was determined using the Og4C3 ELISA and the presence of microfilaremia was detected using microscopy. Antibody responses to Wb123, a Wuchereria bancrofti L3 antigen, were measured using a Luciferase Immunoprecipitation System (LIPS) assay. Antibody responses to Bm14 and Bm33, Brugia malayi antigens and to a major surface protein (WSP) from Wolbachia were analyzed using a multiplex bead assay. Over follow-up, 80 (56%) of the children became antigen-positive and 30 (21%) developed microfilaremia. Detectable antibody responses to Bm14, Bm33, Wb123, and WSP developed in 95%, 100%, 92%, and 29% of children, respectively. With the exception of WSP, the development of antibody responses generally preceded detection of filarial antigen. Our results show that antifilarial antibody responses can serve as an important epidemiological indicator in a sentinel population of young children and thus, may be valuable as tool for surveillance in the context of lymphatic filariasis elimination programs.     

Comparison of IgG4 assays using whole parasite extract and BmR1 recombinant antigen in determining antibody prevalence in brugian filariasis

BACKGROUND: Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. METHODS: Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid trade mark ]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. RESULTS: There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. CONCLUSIONS: Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.     

Immunoradiometric assay for detection of filarial antigens in human serum

The immunoradiometric assay (IRMA) for detection of filarial antigens in the serum of patients infected with Brugia malayi (Bm) or the closely related filarial parasite Wuchereria bancrofti (Wb) was investigated, and its performance and clinical utility were examined. Reference sera prepared by the addition of crude Bm antigen (BmA) to negative control human sera provided a reproducible reference curve. The IRMA displayed acceptable precision and reproducibility. Agreement between dilutions (parallelism) was good in sera without specific antibody, but the presence of even modest levels of antibody resulted in nonparallelism in about one-half of the tested sera from endemic areas. Significant reduction in detectable BmA occurred when low levels of specific antibody (less than 1 microgram/ml) were added to BmA containing sera. Thus, antibody interference limited absolute quantitation of antigen in the IRMA. Results were therefore expressed in a semi-quantitative manner by using the mean + 3 SD of the binding of nonexposed human sera as the positive threshold. The frequency of reliable filarial antigen detection in individuals from the Wb endemic areas of India and the South Pacific was the following: microfilaremia, 15 out of 15; elephantiasis, 2 out of 18; tropical pulmonary eosinophilia, 2 out 8. These findings show clearly that a two-site IRMA can effectively detect circulating antigen (and thus be diagnostic of infection) in a great many patients with filariasis, but to enhance the sensitivity of the assay to the point where all patients can be diagnosed, a number of suggested modifications will be necessary.     

Immunodiagnosis of bancroftian filariasis—Problems and progress

The immunodiagnosis of bancroftian filariasis is a major challenge to the immunoparasitologist. Significant progress is yet to be made in developing convenient laboratory animal model and inin vitro cultivation of filarial parasites making it very difficult to obtain required amount of parasite material for research. Parasitological examination techniques are not useful in low microfilaraemia, occult or chronic.filarial infections. A precise and accurate immunodiagnostic technique is very much needed for successful filaria control programmes. Such a test will also avoid the need for laborious night blood examination in bancroftian filariasis. Due to comparatively easy availability, a good amount of work has been done to explore immunodiagnostic potential of heterologous filarial antigens isolated fromLitomosoide carinii, Dirofilaria immitis, Brugia malayi, Setaria digitata, Setaria cervi and number of other filarial species. However, there has been limited or no significant success due to number of false negative and false positive reactions. Extensive study has also been made with antigens isolated fromWuchereria bancrofti microfilariae. Soluble antigens of microfilariae have been used in different immunological techniques such as skin test, counter immuno electrophoresis, indirect haemagglutination test, indirect fluorescent antibody test and enzyme linked immunosorbent assay. Fractionation of Wuchereria bancrofti microfilarial soluble antigens yielded mfS3e antigen fraction which was found to be highly reactive in microfilaraemia by enzyme linked immunosorbent assay, but the yield of the purified antigen was not sufficient enough to make it a practical proposition for large scale isolation of antigen. Wuchereria bancrofti microfilarial excretory-secretory antigens were found to be specific and highly sensitive requiring as little as 0.35 ng antigen protein per well in penicillinase enzyme linked immunosorbent assay for detection of filarial antibody. One ml of culture fluid was found to be sufficient for 400,000 tests. Field evaluation of this test showed that it can replace laborious night blood examination. Assay systems have been developed for detection of filarial antigen in serum, urine, hydrocele fluid and immune complexes using immunoglobulins from chronic filarial sera and antisera to excretory filarial antigens. Further purification of excretory-secretory antigens by affinity chromatography and production of monoclonal antibodies should hopefully give suitable reagents for use in sensitive assays such as enzyme immuno assay and immuno radiometric assay, providing an ideal assay system for detection of active filarial infection in the not too distant future.     

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.     

Isolation, purification and characterization of surface antigens of the bovine filarial parasite Setaria digitata for the immunodiagnosis of bancroftian filariasis

The surface antigens of the bovine filarial parasite Setaria digitata were isolated by EDTA extraction and purified by affinity chromatography using sepharose bound human filarial (Wuchereria bancrofti) antibodies obtained from chronic human filarial sera. The purified and crude antigens were used in enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies in bancroftian filariasis. The purified antigen showed sensitive and specific reactions in ELISA for the detection of antibodies in filarial sera and showed least cross reactivity with other parasitic infections. The crude and purified antigens showed about 18 and 6 peptide bands respectively in SDS-PAGE and about 11 and 6 antigenic bands respectively in enzyme-linked immunoelectrotransfer blot (EITB). The purified antigen was observed to be glycoprotein in nature. It was possible to identify the stage-specific infection in human filariasis by using the crude and purified antigens in EITB.     

A Comprehensive Assessment of Lymphatic Filariasis in Sri Lanka Six Years after Cessation of Mass Drug Administration

BackgroundThe Sri Lankan Anti-Filariasis Campaign conducted 5 rounds of mass drug administration (MDA) with diethycarbamazine plus albendazole between 2002 and 2006. We now report results of a comprehensive surveillance program that assessed the lymphatic filariasis (LF) situation in Sri Lanka 6 years after cessation of MDA.ConclusionsComprehensive surveillance is feasible for some national filariasis elimination programs. Low-level persistence of LF was present in all study sites; several sites failed to meet provisional endpoint criteria for LF elimination, and follow-up testing will be needed in these areas. TAS was not sensitive for detecting low-level persistence of filariasis in Sri Lanka. We recommend use of antibody and MX testing as tools to complement TAS for post-MDA surveillance.     

Stage-specific and common antigens of Brugia malayi identified with monoclonal antibodies

To identify parasite antigens that trigger protective, pathogenic, and allergic immune responses during filarial infections, we generated a series of monoclonal antibodies to infective larvae, adult worms, and microfilariae of Brugia malayi, a human pathogen. Quantitative and qualitative analysis of the reaction patterns of these monoclonal antibodies indicates the existence of stage-specific antigens of B. malayi, as well as of antigens shared by different stages of this parasite and by other related and unrelated helminths. These antibodies should provide invaluable tools for the analysis of host-parasite interactions in filariasis at the molecular level.     

Diagnostic utility of fractionated urinary filarial antigen

Urinary filarial antigen isolated from urine samples of microfilaraemic patients was analysed for its antigenic activity by immunoblotting and enzyme linked immunosorbent assay techniques. SDS-PAGE fractionation of urinary filarial antigen showed 11 protein bands, of which two showed reactivity with immunoglobulin-G fraction of filarial serum immunoglobulin in immunoblotting. Antigenic analysis of SDS-PAGE fractions of urinary filarial antigen by inhibition enzyme linked immunosorbent assay using filarial serum immunoglobulin-G andWuchereria bancrofti microfilarial excretory-secretory antigen revealed 3 fractions, numbers 5, 6 and 9 with significant activity. In indirect enzyme linked immunosorbent assay using fractions 5 and 6, filarial immunoglobulin-G antibody was detected in about 90% of microfilaraemics, 80% clinical filariasis and 20% of endemic normal individuals. Further, there was no phosphorylcholine epitope in these fractions. Fractions 5 and 6 can be a candidate antigens for the immunodiagnosis of filariasis.     

Biochemical and immunologic characterization of a major IgE-inducing filarial antigen of Brugia malayi and implications for the pathogenesis of tropical pulmonary eosinophilia.

A major allergen of the human filarial parasite Brugia malayi has been identified by two-dimensional immunoblot analysis using a serum pool from patients with tropical pulmonary eosinophilia. The allergen is composed of two Ag with M(r) 23 and M(r) 25 and acidic isoelectric point (Bm23-25). Immunoblots using affinity-purified IgE antibodies to BM23-25 indicated that Bm23-25 is expressed mainly in the microfilarial stage. Digestion of the allergen with endoglycosidases indicates that it has N-linked oligosaccharide chains. Analysis of the reactivity of T cells derived from patients with lymphatic filariasis revealed that the Bm23-25 allergen was capable of stimulating T cell proliferation; Bm23-25 was also shown to induce IgE production in vitro from PBMC derived from patients with either TPE or other filarial symptoms. Bronchoalveolar lavage fluid of patients with TPE contained IgE antibodies that recognized Bm23-25 strongly, an observation suggesting that the microfilarial allergen might be involved in the pathogenesis of the TPE syndrome.     

Antibody responses to Brugia malayi antigens induced by DNA vaccination

BACKGROUND: DNA vaccination is a convenient means of immunizing animals with recombinant parasite antigens. DNA delivery methods are believed to affect the qualitative nature of immune responses to DNA vaccines in ways that may affect their protective activity. However, relatively few studies have directly compared immune responses to plasmids encoding the same antigens after injection by different routes. Therefore, the purpose of this study was to explore the influence of the route of administration on antibody responses to plasmids encoding antigens from the filarial nematode parasite Brugia malayi. METHODS: Four B. malayi genes and partial genes encoding paramyosin (BM5), heat shock protein (BMHSP-70), intermediate filament (BMIF) and a serodiagnostic antigen (BM14) were inserted in eukaryotic expression vectors (pJW4303 and pCR trade mark 3.1). BALB/c mice were immunized with individual recombinant plasmids or with a cocktail of all four plasmids by intramuscular injection (IM) or by gene gun-intradermal inoculation (GG). Antibody responses to recombinant antigens were measured by ELISA. Mean IgG1 to IgG2a antibody ratios were used as an indicator of Th1 or Th2 bias in immune responses induced with particular antigens by IM or GG immunization. The statistical significance of group differences in antibody responses was assessed by the non-parametric Kruskal-Wallis test. RESULTS: Mice produced antibody responses to all four filarial antigens after DNA vaccination by either the IM or GG route. Antibody responses to BM5 paramyosin were strongly biased toward IgG1 with lower levels of IgG2a after GG vaccination, while IM vaccination produced dominant IgG2a antibody responses. Antibody responses were biased toward IgG1 after both IM and GG immunization with BMIF, but antibodies were biased toward IgG2a after IM and GG vaccination with BMHSP-70 and BM14. Animals injected with a mixture of four recombinant plasmid DNAs produced antibodies to all four antigens. CONCLUSIONS: Our results show that monovalent and polyvalent DNA vaccination successfully induced antibody responses to a variety of filarial antigens. However, antibody responses to different antigens varied in magnitude and with respect to isotype bias. The isotype bias of antibody responses following DNA vaccination can be affected by route of administration and by intrinsic characteristics of individual antigens.     

Brugia malayi infection in Meriones unguiculatus: antibody response to recombinant BmR1

BmR1 recombinant antigen has previously been shown to demonstrate high sensitivity and specificity in the serological diagnosis of brugian filariasis in humans. In this study, the pattern of recognition of antibody to BmR1 during Brugia malayi infection was investigated by employing Meriones unguiculatus as the experimental model. Thirty two gerbils were infected subcutaneously with 120 L(3); and two control groups each comprising 25 animals were employed. ELISA using BmR1 was used to detect filaria-specific IgG antibodies elicited by the gerbils; using sera collected from the day 1 until day 150 post-inoculation (p.i.). The results showed that BmR1 detected B. malayi infection in gerbils harboring adult worms irrespective of the presence of circulating microfilaria, and was exemplified by positive ELISA results in nine a microfilaraemic animals that harbored live adult worms. The initial time of the antibody recognition was at day 8 p.i. and the antibody titre showed some correlation with adult worm burden.     

Lymphatic Filariasis Control in Tanzania: Effect of Repeated Mass Drug Administration with Ivermectin and Albendazole on Infection and Transmission

Background

In most countries of sub-Saharan Africa the control of lymphatic filariasis (LF) is based on annual mass drug administration (MDA) with a combination of ivermectin and albendazole, in order to interrupt transmission. Here we present the first detailed study on the effect of 3 repeated MDAs with this drug combination, as implemented by the Tanzanian National Lymphatic Filariasis Elimination Programme (NLFEP).

Methodology/Principal Findings

Infection and transmission was monitored during a five-year period (one pre-intervention and four post-intervention years) in a highly endemic community (Kirare village) in north-eastern Tanzania. The vectors were Anopheles gambiae, An. funestus and Cx. quinquefasciatus. After start of intervention, human microfilaraemia initially decreased rapidly and statistically significant (prevalence by 21.2% and 40.4%, and mean intensity by 48.4% and 73.7%, compared to pre-treatment values after the first and second MDA, respectively), but thereafter the effect levelled off. The initial decrease in microfilaraemia led to significant decreases in vector infection and vector infectivity rates and thus to a considerable reduction in transmission (by 74.3% and 91.3% compared to pre-treatment level after first and second MDA, respectively). However, the decrease in infection and infectivity rates subsequently also levelled off, and low-level transmission was still noted after the third MDA. The MDAs had limited effect on circulating filarial antigens and antibody response to Bm14.

Conclusion/Significance

Critical issues that may potentially explain the observed waning effect of the MDAs in the later study period include the long intervals between MDAs and a lower than optimal treatment coverage. The findings highlight the importance of ongoing surveillance for monitoring the progress of LF control programmes, and it calls for more research into the long-term effect of repeated ivermectin/albendazole MDAs (including the significance of treatment intervals and compliance), in order to optimize efforts to control LF in sub-Saharan Africa.     

Purification of recombinant antigen BmSXP used in panLF rapid kit for lymphatic filariasis

A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.     

Antibody responses of Wuchereria bancrofti patients to recombinant Brugia pahangi superoxide dismutase

Lymphatic filarial parasite Brugia malayi contains significant amount of Cu/Zn superoxide dismutase (SOD) activity in the extract of different life stages and in the excretory-secretory product of adults. In the present study recombinant SOD from B. pahangi has been used to see the antibody response in Wuchereria bancrofti infected patients. The recombinant SOD from B. pahangi reacted specifically with W. bancrofti infected sera in ELISA and immunoblotting. The reactivity of IgM subclass was more as compared to IgG subclass both in the asymptomatic microfilaraemic and symptomatic amicrofilaraemic when tested by ELISA. Serum from other helminthic infection was very low and found to be insignificant. The antibody response to rec SOD was directly proportional to the number of microfilariae in infected patients. The circulating filarial SOD was detected in filarial patients using polyclonal antibodies raised against recombinant Cu/Zn SOD in rabbits. The apparent molecular masses as determined by immunoblotting were 29 and 22 kDa. The specificity of recombinant SOD could be explored for its use in immunodiagnosis of lymphatic filariasis.     

Failure of highly immunogenic filarial proteins to provide host-protective immunity

In areas that are endemic for lymphatic filariasis, there are individuals who are parasite free and who appear not to have experienced symptoms attributable to filarial infection. These "putatively immune" individuals may recognize immunogens that could be important in host protection. We have immunoscreened expression libraries expressing epitopes encoded by filarial open reading frames and have identified three antigens that are differentially recognized by the two polar clinical groups-endemic normals and asymptomatic microfilaremics. Pre-immunization of susceptible hosts (Meriones unguiculatus) with these antigens revealed that none was able to elicit consistent host protective immunity. Our data are consistent with Waksman's conjecture that highly immunogenic antigens of parasite origin may be inappropriate candidates for prophylactic immunization.     

A major allergen of lymphatic filarial nematodes is a parasite homolog of the gamma-glutamyl transpeptidase.

BACKGROUND: Bm2325, a major IgE-inducing antigen of the filarial parasite Brugia malayi has been implicated in the pathology of tropical pulmonary eosinophilia (TPE), a pulmonary syndrome thought to result from hypersensitivity to microfilariae. MATERIALS AND METHODS: Affinity-purified IgE to Bm2325 from patients with TPE was used to identify a complementary DNA (cDNA) from a B. malayi expression library. Sequence analysis of the cDNA revealed a hitherto unknown parasite protein. Immunoblotting of the recombinant filarial protein using sera of patients with TPE determined its IgE-binding capacity. Reactivity to human lung epithelial cell proteins was analyzed using murine anti-Bm2325 antibodies and serum from patients with TPE. RESULTS: The predicted protein is a homolog of the entire precursor of the gamma-glutamyl transpeptidase (gamma-GT), a key enzyme in the synthesis and degradation of glutathione. The filarial precursor encodes both the heavy (H) and the light (L) chain subunits and shares structural similarities with the mammalian enzymes. The Bm2325 allergen was identified as the homolog of the enzyme light chain subunit. Murine antibodies against the recombinant parasite gamma-GT cross-reacted with the human enzyme present in human airway epithelial cells, and human gamma-GT is a target of antibodies present in the serum of patients with TPE. CONCLUSION: Molecular mimicry between the parasite gamma-GT homolog and the host membrane-bound gamma-GT present in lung epithelial cells likely contributes to the pathogenesis observed in tropical pulmonary eosinophilia.     

Litomosoides sigmodontis in mice: reappraisal of an old model for filarial research

Onchocerciasis and lymphatic filariasis (LF) are major causes of severe morbidity and considerable socio-economic problems throughout the tropics. Vector control and mass chemotherapy have helped to control these infections in some regions, but the temporary success of such measures argues strongly for the development of vaccines. Success in such a venture will require detailed knowledge of protective immune responses in conjunction with the identification of target antigens. By comparison with other important parasitic infections, such as schistosomiasis and leishmaniasis, work on the development of vaccines for onchocerciasis and LF has been constrained because of the difficulties of producing cyclical and patent filarial infection in laboratory mice. Wolfgang Hoffmann and colleagues here outline the opportunities presented by the rodent filaria Litomosoides sigmodontis for filarial research.