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.     

Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis

Two monoclonal antibodiesWuchereria bancrofti E 33 andWuchereria bancrofli E 34 raised againstWuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility.Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. WhenWuchereria bancrofti E 34 monoclonal antibody was used alongwith immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay. 68% of microfilaraemic sera (26 out of 38). 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected byWuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.     

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.     

Wuchereria bancrofti: Diminished platelet activation in filarial patients

Blood platelets are the innate immune elements that have not been investigated in human filarial infections. Platelet activation status in the endemic normals (EN), microfilaria positive individuals (MF) and patients with chronic pathology (CP) was evaluated in whole blood, under unstimulated as well as antigen exposed (BmA, E. coli) conditions for PAC-1 expression by Flow cytometry. A diminished PAC-1 expression was observed in MF compared to CP and EN spontaneously as well as upon antigen exposure. Besides this, PAC-1 expression within the groups did not exhibit any significant difference under all the experimental conditions. However in CP patients, E. coli antigen exposure resulted in a significantly reduced PAC-1 expression compared to the spontaneous expression levels. NO release in platelet culture supernatants from EN was inversely proportional to platelet aggregation. Collagen stimulated platelets from EN, exposed to sera and immune complexes from CP and MF patients resulted in elevated Nitric Oxide (NO) release, compared to those exposed to autologous sera and fetal calf serum. In addition, under similar conditions, collagen stimulated platelets from EN, exposed to filarial antigen (BmA) exhibited increased NO compared to the E. coli antigen exposed ones and light microscopic observations of cultured platelets supported the above findings. Thus it appears from the results of the present study that filarial antigen may play a role in the loss of platelet aggregation, leading to platelet inactivation.     

Detection of filarial antigen by inhibition enzyme linked immunosorbent assay using fractionatedBrugia malayi microfilarial excretory secretory antigen

Detection of filarial antigen in blood or urine samples would provide an accurate indication of active infection. The absence of a simple, well established animal model and limitations in getting the required amount of parasite material from human sources have been the main obstacles for the diagnosis ofWuchereria bancrofti infection. An inhibition ELISA has been developed for detection of filarial antigen using a partially purifiedBrugia malayi mf ES antigen (BmE DE1) and its affinity purified antibodies. Filarial antigen was detected in the sera of 88% of microfilaraemic, 60% of chronic filarial, 17% of endemic normal and none of the non- endemic normal subjects. The sensitivity and specificity of the assay were 88% and 89% respectively. Moreover, undiluted urine samples from 82% of microfilaraemic and 17% of endemic normal, but none of the non- endemic normal samples showed the presence of filarial antigen. With the limitations on the availability of sufficient homologous parasite materials, the inhibition ELISA using BmE DE1 and anti BmE DE1 antibodies shows promise for the detection of active infection in bancroftian filariasis in man. Moreover, its detection in urine makes it more possible to test patients in field areas.     

Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.     

Identification of Wb123 as an Early and Specific Marker of Wuchereria bancrofti Infection

Background

The current antibody tests used for monitoring in lymphatic filariasis (LF) elimination programs suffer from poor specificity because of the considerable geographical overlap with other filarial infections such as Loa loa (Ll), Onchocerca volvulus (Ov), and Mansonella perstans (Mp).

Methods

Using bioinformatics to assemble into contigs 2048 expressed sequence tags (ESTs) from the L3 infective larvae of W. bancrofti (Wb), these were next assessed for homology to known proteins and nucleotides and to similar assemblies of L3 larval ESTs of B. malayi (Bm – n = 5068), Ov (n = 4166), and Ll (n = 3315). Nineteen potential L3- and Wb- and/or Bm-specific antigens were identified. Sixteen of the 19 antigens could be expressed as fusion proteins with Renilla luciferase (Ruc); these were used in a rapid Luciferase Immunopreciptation System (LIPS) assay.

Results

One of the 16 expressed antigens (Wb123) was both highly immunogenic and specific for Wb. Using Wb123-based IgG and IgG4 LIPS assays on well-defined sera from normal North Americans and those infected exclusively with intestinal helminths, we could detect all of the Wb-infected individuals (from diverse geographic regions) with 100% sensitivity and 100% specificity. Using sera from exclusively Ll-infected, Ov-infected Mp-infected or Bm-infected subjects as the negative comparator, the sensitivities were between 98–100% and the specificities ranged between 84–100% (for IgG anti-Wb123) and between 98–100% (for IgG4 anti-Wb123). Blinded assessments using panels of sera from various Wb-, Bm- or non-Wb helminth-infected subjects demonstrated equally high degrees of sensitivity and specificity.

Significance

We have identified a Wb-encoded antigen that can be used both as a rapid, high throughput tool to diagnose individual Wb infections and as a sensitive method for early detection of recrudescent infections in areas of control and for mapping new areas of Wb transmission.     

A monoclonal antibody-based immunoradiometric assay for detection of circulating antigen in Bancroftian filariasis

A monoclonal antibody designated Gib 13 has been used in an immunoradiometric assay (IRMA) to detect circulating antigen in the sera of Wuchereria bancrofti-infected subjects from an endemic area of Papua New Guinea. A clear association between the presence of patent infection and the Gib 13 target epitope in serum was established because 93% of microfilaremic individuals were antigen-positive. Moreover, there was a significant correlation between levels of serum antigen and blood microfilarial counts. Detection of circulating antigen in amicrofilaremic subjects with acute symptoms of lymphatic filariasis, and 53% of asymptomatic amicrofilaremic subjects, but not in nonendemic controls, suggests that the Gib 13 IRMA will also be of value in the diagnosis of occult filariasis. However, as in all IRMA based on detection of potentially immunogenic molecules in man, antibodies can be expected to be the major contributor to reduced sensitivity of the assay.     

Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA₂-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA₂-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.     

Detection of filarial infection usingWuchereria bancrofti microfilariae culture antigen and filter paper blood samples in enzyme linked immunosorbent assay

Blood collected on filter paper by finger-prick gave results comparable to intravenous serum samples when analysed by enzyme-linked immunosorbent assay (ELISA). All the 100 microfilaraemia, 5 out of 100 endemic normals and none of the 10 nonendemic normal filter paper blood samples showed the presence of filarial antibody when tested by this method,using culture antigen and anti-immunoglubulins, class G, M and A — penicillinase conjugate. When the same samples were screened for the presence of IgM antibody, 91 out of 100 microfilaraemia, 13 out of 100 endemic normal and none of the 10 nonendemic normal samples showed a positive reaction. Enzyme linked immunosorbent assay, using culture antigen and filter paper blood samples, appears to work in large field studies for detection of filarial infection.     

Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG

The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.     

Proinflammatory cytokines secreted by monocytes of filarial patients.

The levels of interleukin 1, granulocyte macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor (TNF)-alpha secreted by the monocytes of filarial patients, such as asymptomatic microfilaremics (MF), chronic pathology (CP), and normal individuals, residing in a Wuchereria bancrofti endemic area (EN) in response to whole Brugia malayi antigen (BmA) and Setaria digitata (Sd-cuticular) and a recombinant filarial antigen (pRJ51) were studied. Stimulation of peripheral blood adherent cells with whole parasite antigen showed marked increase in IL-1 levels in MF as compared to CP or EN. The recombinant antigen stimulation, however, resulted in similar levels of IL-1 in MF and CP. In contrast, stimulation of peripheral blood adherent cells with whole parasite antigen produced high levels of GM-CSF and TNF-alpha in CP as opposed to MF or EN. Recombinant antigen stimulation, however, produced high levels of GM-CSF in EN as compared to MF or CP, while no significant change in the release of TNF-alpha was observed in these patients. These results suggest that monocytes from filarial patients exhibit functional activity similar to that observed by the monocytes of endemic normals (control group).     

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.     

The ICT Filariasis Test: A rapid-format antigen test for diagnosis of bancroftian filariasis

Antigen testing is now recognized as the method of choice for detection of Wuchereria bancrofti infections. Unlike tests that detect microfilariae, antigen tests can be performed with blood collected during the day or night. However, existing enzyme-linked immunosorbent assay (ELISA) tests for filarial antigenemia are difficult to perform in the field, and this has limited their use in endemic countries. In this article, Gary Weil, Patrick Lammie and Niggi Weiss review their experience with a new rapid-format filarial antigen test. They found that the ICT card test was very easy to perform and that it was comparable with ELISA for the detection of filarial antigen in sera from people with microfilaremia. The introduction now of an antigen test suitable for use in the field is especially timely, in that it may facilitate implementation of new strategies proposed by the World Health Organization for control and elimination of lymphatic filariasis.     

Biological activity and diagnostic use of detergent soluble antigens fromSetaria digitata

Filarial parasite, responsible for filariasis is known to remain in the host for long periods of time. A 29 kDa protein isolated fromSetaria digitata by gel electrophoresis and electroelution of detergent soluble surface antigens showed a 70% inhibition of the cell mediated immune response. On evaluation of its diagnostic application, the same protein was found to be very sensitive in detecting antibody at an antigenic protein concentration as low as 1 ng per μl. The cross reactivity of surface antigen with bancroftian filarial patient’s sera was tested by Dot-ELISA and ELISA. Both the antigen as well as antibody detection tests showed 100% positivity with all types of filariasis cases. It did not produce any positive reaction with non-endemic control sera. However, a proportion of endemic normal subjects showed positivity and this is attributed to the fact that people in endemic areas are exposed to infective mosquito bites. The biological property of inhibition and 100% positivity of filariasis cases in both antibody and antigen detection tests point towards the bifunctional nature of the surface proteins before and after release. The same may be happening under normal conditions also, perhaps at a much lower rate     

Regulatory T cells in human lymphatic filariasis: stronger functional activity in microfilaremics

Infection with filarial parasites is associated with T cell hyporesponsiveness, which is thought to be partly mediated by their ability to induce regulatory T cells (Tregs) during human infections. This study investigates the functional capacity of Tregs from different groups of filarial patients to suppress filaria-specific immune responses during human filariasis. Microfilaremic (MF), chronic pathology (CP) and uninfected endemic normal (EN) individuals were selected in an area endemic for Brugia timori in Flores island, Indonesia. PBMC were isolated, CD4CD25(hi) cells were magnetically depleted and in vitro cytokine production and proliferation in response to B. malayi adult worm antigen (BmA) were determined in total and Treg-depleted PBMC. In MF subjects BmA-specific T and B lymphocyte proliferation as well as IFN-gamma, IL-13 and IL-17 responses were lower compared to EN and CP groups. Depletion of Tregs restored T cell as well as B cell proliferation in MF-positives, while proliferative responses in the other groups were not enhanced. BmA-induced IL-13 production was increased after Treg removal in MF-positives only. Thus, filaria-associated Tregs were demonstrated to be functional in suppressing proliferation and possibly Th2 cytokine responses to BmA. These suppressive effects were only observed in the MF group and not in EN or CP. These findings may be important when considering strategies for filarial treatment and the targeted prevention of filaria-induced lymphedema.     

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.     

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.     

IgE responses in human filariasis. II. Qualitative characterization of filaria-specific IgE

Crossed radioimmunoelectrophoresis (CRIE) was used to characterize human IgE antibody responses to filarial parasites by using antigens derived from Brugia malayi (Bm) adult worms. A reference pool of patient sera was initially used to determine the sensitivity and specificity of CRIE. Because IgG-blocking antibodies interfered with IgE binding in certain sera, all sera were preabsorbed with protein A-Sepharose. As little as 50 ng of specific IgE antibody (determined by quantitative radioallergosorbent test [RAST]) in the reference pool bound to 20 of the 35 antigen precipitates in crossed immunoelectrophoresis. Increasing IgE antibody concentration did not increase the number of IgE-binding precipitates. Six patients from each of the three major clinical groups in lymphatic filariasis (i.e., tropical pulmonary eosinophilia [TE], chronic lymphatic pathology [CP], or circulating microfilaremia [MF]) were studied by CRIE with the use of a constant amount of IgE antibody (50 ng IgE anti-BmA). Distinct patterns of allergen recognition were observed among the groups. Individuals with TE recognized both anodic and cathodic antigens as allergens, whereas the other two groups recognized predominantly anodic antigens. The greatest number of allergens was recognized by patients with TE; this number ranged from nine to 18, whereas patients with CP or circulating MF recognized from six to 11 allergens. Although potentiated IgE responses at a quantitative level in parasitic helminth infections is a well-established phenomenon, our studies showing the diversity of antigens recognized as allergens indicate for the first time potentiated IgE responses at a qualitative level as well.     

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.