Parasite-specific anergy in human filariasis. Insights after analysis of parasite antigen-driven lymphokine production.

The antigen-specific immune unresponsiveness seen in bancroftian filariasis was studied by examining lymphokine production in peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from 10 patients with asymptomatic microfilaremia, 13 patients with elephantiasis and 6 normal North Americans. In each group of patients, the kinetics of the lymphokine response and the response to mitogens and nonparasite antigens did not differ significantly. In marked contrast, when antigen-induced lymphokine production was examined, most patients with microfilaremia were unable to produce either interleukin 2 (IL-2) or gamma-interferon (i.e., were nonresponders), and the few who could (hyporesponders, generally with quite low microfilaremia levels) did so at levels significantly less than those of patients with elephantiasis, all of whom showed strong responses to parasite antigen. Removal of neither adherent cells or T8+ cells affected the parasite-specific anergy seen in those with microfilaremia, suggesting a state of T cell tolerance to the parasite in patients with this most common clinical manifestation of bancroftian filariasis.

Analysis of salivary gland proteins of the mosquito Armigeres subalbatus.

Quantitative studies of total salivary gland protein of Armigeres subalbatus mosquito revealed that the total salivary gland protein increased dramatically during the five days after emergence as adults. The amount of salivary gland protein of female and male mosquitos at day five after adult emergence were on the average 11.55 and 1.32 microg/pair gland respectively. SDS-PAGE studies showed that salivary gland protein profiles of Armigeres subalbatus demonstrated 9 major polypeptide bands of 68, 65, 60, 55, 40, 30, 28, 21, and 15 kDa. The 21 and 65 kDa bands were found only in the distal lateral region of the mosquito salivary gland and were depleted after the female mosquito took a blood meal.

Secreted and surface antigens from larval stages of Wuchereria bancrofti, the major human lymphatic filarial parasite.

Antigenic proteins of microfilariae and infective larvae of Wuchereria bancrofti have been identified by intrinsic and extrinsic radiolabelling, and specific immunoprecipitation with sera from filarial patients. From 125I surface-labelling experiments, the most prominent antigen on both stages is of relative molecular mass (Mr) 17 000, while a molecule of similar size is both synthesized and released in vitro following labelling with [35S]methionine. A second similarity between the two stages is the production and secretion of a Mr 21 000 component, which is, however, not detected on the worm surfaces. A series of additional proteins from larval W. bancrofti are described from each parasite compartment (secreted, surface and somatic) and the antigenicity and specificity of these components explored with serum from patients with filariasis due to W. bancrofti or Brugia species, and with onchocerciasis. Among additional molecules released in vitro we have found a Mr 51 000 antigen from both stages, and also several proteins which are not recognised by antibody from human filarial patients.

Secreted and circulating antigens of the filarial parasite Brugia pahangi: analysis of in vitro released components and detection of parasite products in vivo.

A range of excretory-secretory (ES) antigens have been characterised following in vitro culture of adult Brugia pahangi filarial nematodes in serum-free medium. Analysis by radioiodination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and immunoprecipitation of purified macromolecules with antibodies from human and experimental animal infections reveals both host and parasite components. Two host molecules appear by molecular weight and immunoprecipitation analysis to be immunoglobulin and serum albumin, presumed to be taken up from the jird host from which adult worms were recovered. A further prominent component, of 19 kDa, reacts with neither anti-host nor anti-filarial antibodies, and may represent a non-immunogenic parasite product. Three additional bands, although less intensely radiolabelled, did prove to be consistently antigenic, with apparent molecular weights of 15, 29 and 40 kDa. A further ES antigen, which does not take up radio-iodine or lend itself to electrophoretic analysis, has also been detected. This molecule reacts in a immunoradiometric assay in which monoclonal antibody directed against a repetitive epitope acts both to capture and indicate antigen presence. The same antibody, Bp-1, may also be employed to detect circulating antigen in the serum of animals experimentally infected with Brugia pahangi, and in the serum of patients with each of the three species of human lymphatic filariasis, Brugia malayi, Brugia timori and Wuchereria bancrofti.

Differential recognition of two cloned Brugia malayi antigens by antibody class.

The humoral and cellular immune response to filarial parasites is complex. Numerous studies have shown that antibodies to a large number of protein and non-protein antigens may be produced over the course of infection and that immune recognition of any given antigen may vary by disease manifestation and by immunoglobulin class. We have used the techniques of molecular cloning to attempt to dissect this complex interaction, and describe here two clones, isolated from an expression library constructed from Brugia malayi genomic DNA, whose products are recognized by distinct immunoglobulin classes. A lambda gt11 fusion protein containing part of the B. malayi myosin tail region is recognized by antibodies of the IgG class from a high percentage of bancroftian filariasis patients. A fusion protein containing a collagen-like sequence is less frequently and weakly recognized under the same experimental conditions, but is almost universally recognized when the developing reagent is specific for IgE. We thus identify specific filarial proteins against which the infected human host responds preferentially with antibodies of a specific immunoglobulin class.

Antibodies to diethylcarbamazine cross-react with microfilariae of Wuchereria bancrofti.

Antibodies directed against the microfilarial sheath have been instrumental in the immune elimination of circulating microfilariae in human lymphatic filariasis. We report here that antibodies to diethylcarbamazine (DEC, the most commonly used anti-filarial drug) cross-react with the sheath of Wuchereria bancrofti microfilariae. Antibodies with reactivity to DEC were raised in rabbits by immunization with a conjugate of methylpiperazine carboxylic acid (MPCA, an acid hydrolysis product of DEC) coupled to bovine serum albumin. The reactivity of these antibodies with microfilarial sheath of W. bancrofti was demonstrated by indirect immunofluorescent assay and indirect immunoperoxidase assay. This reactivity could be effectively inhibited by pre-incubation of the antisera with different haptens such as DEC, MPCA or piperazine citrate.

Parasite antigens in sera and urine of patients with bancroftian and brugian filariasis detected by sandwich ELISA with monoclonal antibodies.

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigens in human lymphatic filariasis. The assay utilizes a polyclonal rabbit antifilarial antiserum to capture, and a monoclonal antibody to identify, circulating parasite antigen. Using this assay, we found that greater than 95% of sera from microfilaremic donors with bancroftian or brugian filariasis, approximately 60% of sera from microfilaremic patients with hydroceles, chyluria, or elephantiasis, and 15%-20% of sera from asymptomatic residents of filariasis-endemic areas evidently contain filarial antigens. Antigen was also detected in the urine of some microfilaremic patients. Serum levels of antigen detected with one monoclonal antibody, ES34, correlated well with microfilarial density in night blood. In contrast, less than 5% of sera from residents of areas where lymphatic filariasis is not endemic reacted in the assay, even though approximately one-third of the donors whose sera were tested were known to be infected with intestinal nematodes. The assay was designed to be flexible enough to allow the parallel use of multiple monoclonal antibodies with different specificities and simple enough to be applicable in most areas where lymphatic filariasis is endemic.

Comparison of Dot-ELISA with Sandwich-ELISA for the detection of circulating antigens in patients with bancroftian filariasis.

We compared the performance of a newly developed Dot-ELISA with that of a previously described Sandwich-ELISA to detect parasite antigens in sera from patients with bancroftian filariasis. The same monoclonal antibody and the same sera were used in both tests. In the Dot-ELISA, 67 of 70 sera from microfilaremic donors were deemed to contain filarial antigens when screened at a dilution of 1:50. End titers were 1:80-1:1280. With the Sandwich-ELISA, 64 of the same sera were positive at a dilution of 1:10 and 42 were positive at a dilution of 1:50. End titers were 1:10-1:320. The specificity of both assays was greater than 95%, but their sensitivity was remarkably different. The Dot-ELISA could detect as little as 0.055 ng/ml microfilarial antigen added to normal human sera, whereas the lower limit with the Sandwich-ELISA was 10 ng/ml parasite antigen. Additionally, the Dot-ELISA does not require radioactivity or sophisticated equipment and, therefore, can be performed in virtually all filariasis-endemic areas.

Serological diagnosis of bancroftian and malayan filariasis.

A study was undertaken to define the sensitivity and specificity of serological tests in bancroftian and malayan filariasis and correlate the findings with clinical disease. Sera were collected from subjects on three different islands in the Philippines: one endemic for bancroftian filariasis, another endemic for malayan filariasis and the third without endemic filariasis. Antibodies were measured, using rugia malayi as the source of antigen. Antibodies against adult worms measured by indirect immunofluorescence were found at a titer of 1:8 or greater in all patients with bancroftian or malayan filariasis but not in the control subjects. There was no relationship of antibody titer to clinical status. Antibodies against microfilariae were measured by indirect immunofluorescence and microfilarial agglutination. A high correlation was observed between the two methods. These antibodies were found in only one quarter, approximately, of patients with filariasis. Microfilarial antibodies were found more commonly in those patients with chronic lymphatic obstruction. It is concluded that measurement of antibodies to adult worms is a useful indicator of infection while microfilarial antibodies are correlated with disease.

Filarial antigen in circulating immune complexes from patients with Wuchereria bancrofti filariasis.

Detection of filarial antigen in circulating immune complexes from patient sera was performed by an enzyme immunoassay in which the immune complexes were precipitated in the cold with polyethylene glycol and then dissociated in an acid pH buffer before being added to an ELISA plate. The dissociated antigen bound to the plate where it could be detected by peroxidase-labeled polyclonal rabbit antifilarial antiserum. Control sera used for defining the specificity of the assay included sera with immune complexes not related to parasite infection with and without free parasite antigen added prior to polyethylene glycol precipitation as well as sera from normal individuals. Filarial antigen was detected in the circulating immune complexes from 10 of 28 patients with bancroftian filariasis residing in either the Cook Islands (subperiodic Wuchereria bancrofti) or India (periodic W. bancrofti). By immunoblotting, the most frequently identified filarial antigen in these complexes was an approximately equal to 200 kDa circulating antigen.

Detection of circulating parasite antigen in bancroftian filariasis by counterimmunoelectrophoresis.

We used counterimmunoelectrophoresis (CIEP) with rabbit antibodies to Dirofilaria immitis and Brugia malayi to detect soluble filarial antigen in sera collected in a Wuchereria bancrofti-endemic area in South India. Filarial antigen was detected in 38 of 38 sera from microfilaremic patients, 3 of 48 sera from amicrofilaremic patients with lymphatic pathology, and 3 of 5 sera from former microfilaria carriers with negative blood examinations 6 months or more after diethylcarbamazine therapy. One of 32 endemic control sera, 0 of 35 nonendemic sera, and 0 of 20 B. malayi sera were positive. Antigenemia was equally detectable in sera collected at night or during the day (when microfilariae are absent from the blood). Parasite antigen was also detected in the urine of patients with positive serum tests. Antibodies to circulating filarial antigen (also detected by CIEP) were absent in all but 2 antigen-positive sera but present in 22 of 45 antigen-negative sera from clinical filariasis patients and in 9 of 31 antigen-negative sera from endemic controls. Parasite antigen detection by CIEP appears to be a sensitive, specific, and practical diagnostic test for active W. bancrofti infection.

Persistence of parasite antigenemia following diethylcarbamazine therapy of bancroftian filariasis.

This study was designed to reexamine the efficacy of diethylcarbamazine for bancroftian filariasis with special reference to changes in serum parasite antigen levels and antifilarial antibody titers after treatment. Patients with asymptomatic microfilaremia were treated with 6 mg/kg diethylcarbamazine daily for 12 days. Microfilaria counts fell dramatically after treatment, as expected. IgG antibody titers to adult and microfilarial antigens of B. malayi were increased 1 month after treatment in most patients. Titers fell slowly to or below pretreatment levels, but remained positive during subsequent months. Parasite antigen levels, measured by monoclonal antibody-based enzyme immunoassay, decreased to 72%, 58%, 53%, and 48% of pretreatment values 1, 3, 6, and 12 months after diethylcarbamazine treatment, respectively. Parasite antigen levels decreased similarly in subjects with and without residual microfilaremia after treatment. These results suggest that diethylcarbamazine has only partial macrofilaricidal activity against W. bancrofti with this dosage schedule. The sustained, impressive reductions in microfilaria counts after treatment despite significant persistence of parasite antigenemia may be explained by sublethal effects of the drug on adult worms. We believe that parasite antigen detection represents a valuable new approach for monitoring the efficacy of antifilarial drug therapy which we hope will lead to improved use of existing drugs and aid in the evaluation of new drugs for filariasis.

Efficiency of papain-treated microfilariae of Wuchereria bancrofti (var. pacifica) as antigen for serodiagnosis of bancroftian filariasis in French Polynesia.

Papain-treated microfilariae of Wuchereria bancrofti have been used as antigen for indirect fluorescent assay: 0% of non-endemic sera, 8% of healthy exposed Polynesians, 48% of clinical patients and 96% of microfilaraemic subjects were positive by this test. The geometric mean titres were 22, 41, 147 and 605 respectively. Untreated microfilariae were unsuitable for diagnostic purposes. Dirofilaria immitis adult sections showed low reactivity, giving poor discrimination between non-endemic and microfilaraemic sera. The geometric mean titres were 6 and 61 respectively.

Fractionation, characterization and diagnostic potential of filarial antigens isolated from hydrocoele fluid in bancroftian filariasis.

Filarial antigen was isolated from hydrocoele fluid and fractionated on Ultrogel AcA 44. Six protein fractions (HFA C1 to HFA C6) were chromatographically separated from the column. Of the 4 antigenic fractions (HFA C1, HFA C2, HFA C3 and HFA C5), HFA C3 was a major reactive fraction with filarial serum immunoglobulin G. Analysis of HFA C3 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded 18 bands of Mr 18,000-160,000. Two fractions, HFA C3-7 and HFA C3-9 of Mr 48,000-55,000 and 32,000-39,000 obtained by gel elution, were antigenic in an enzyme-linked immunosorbent assay (ELISA). SDS-PAGE immunoblotting confirmed the ELISA by identifying 3 immunoreactive bands of Mr 51,000, 38,000 and 35,000. The diagnostic potential of HFA C3-7 and HFA C3-9 was compared in serodiagnosis of active infections and diseased states. HFA C3-9 showed greater sensitivity in detection of filarial specific IgM antibody in cases with microfilaraemia. Physicochemical characterization indicated the protein nature of HFA C3-9.

Microfilaraemia, serum antibody and development of clinical disease in microfilaraemic subjects infected with Wuchereria bancrofti and treated with diethylcarbamazine citrate.

A seroepidemiological survey of bancroftian filariasis was carried out in 2 townships in Sri Lanka with the objectives of determining the microfilaraemia rates, dependence on age and sex, susceptibility to re-infection, effect of diethylcarbamazine therapy on serum antibodies to microfilarial surface antigens, and the predictive value of the indirect fluorescent antibody test. The mean microfilaraemia rate was 5.4%. Microfilaraemia was not sex-dependent but a marginally elevated incidence was seen in the 6-35 year age groups. In up to 58% of the microfilaraemic patients who had been treated for microfilaraemia previously, a second phase of microfilaraemia was seen 2-7 years after treatment. This was unlikely to have been due to incomplete parasite elimination. Antibodies to microfilarial surface were found in 24-35% of microfilaraemic patients and in 14-63% of amicrofilaraemic symptomatic subjects. Serum anti-microfilarial surface antibody levels did not alter with chemotherapy with diethylcarbamazine citrate. The findings of follow-up investigations of microfilaraemic subjects were compatible with the notion that microfilaraemia does not necessarily lead to clinical disease.

Wuchereria bancrofti in Tanzania: immune reactions to the microfilarial surface, and the effect of diethylcarbamazine upon these reactions.

Immune reactions to the surface of microfilariae (mff) of Wuchereria bancrofti were investigated among persons living in an endemic filariasis area of Tanzania. The sera from a proportion of mff-negative persons contained antibodies to the microfilarial sheath, and a clear correlation was seen between presence of anti-sheath antibodies and the ability of the sera to mediate leucocyte attack on the mff in vitro. The anti-sheath antibodies were mainly of the IgG class and to a lesser degree of the IgM class. Only in some sera did the binding of antibody to the microfilarial sheath lead to deposition of C3 on the sheath. The cuticle of some of the sheathless mff activated complement without presence of anti-cuticle antibody. Further, the effect of diethylcarbamazine (DEC) administration in vivo (i.e., as full course treatment or as Day Provocative Test) and in vitro (i.e., applied directly to the immune tests) on the reactions to the microfilarial surface was examined. No interference of this anti-filarial drug was, however, observed, neither on the ability of sera to mediate leucocyte attack on mff in vitro, nor on complement activation and C3 deposition on the microfilarial sheath.

Microfilaraemia, filarial antibody, antigen and immune complex levels in human filariasis before, during and after DEC therapy. A two-year follow-up.

A study on the effect of DEC therapy on microfilaraemia and the immune status in 27 patients with W. bancrofti infection was carried out for two years. Persistence of microfilaraemia was observed in 4 out of 27 cases after one course of DEC therapy and were treated again for one week. On further follow-up, none was microfilaraemic upto Day 60. The mean filarial antibody titres of IgM and IgG showed a gradual decrease as assessed by enzyme linked immunosorbent assay (ELISA). The mean titres of circulating microfilarial excretory-secretory (ES) antigens and immune complexes (ICs) showed an initial increase during therapy, followed by a gradual fall upto Day 60. Filarial antigen was detected in urine of all the carriers during therapy. Excretion pattern of antigen in urine showed correlation with DEC dose. Reappearance of microfilariae (mf) in circulation in 12 patients after a year showed that DEC had temporary attenuating effect on adult worms or no effect on developing larvae, suggesting further treatment and follow-up of patients. Parasitological and immunoscreening at the end of 2 years showed that the presence of mf ES antigen in blood correlated with the appearance of microfilariae in blood.

Differential recognition of Brugia malayi antigens by bancroftian filariasis sera.

Individuals residing in an area endemic to Wuchereria bancrofti infection were broadly categorised as endemic normals (EN), microfilaraemics (mf + ve) and elephantoids i.e., chronic lymphatic filariasis (EL). The immune status of these three groups was examined in terms of (i) specific antibody levels; (ii) ability to induce antibody dependent cellular cytotoxicity (ADCC) to microfilariae; and (iii) ability to recognise different microfilarial antigens by immunoblotting. All three groups of endemic residents were indistinguishable in their antibody levels as measured by ELISA with B. malayi microfilarial antigen. Many endemic normal sera and most elephantoid sera exerted strong cytotoxicity against W. bancrofti microfilariae whereas none of the mf + ve sera had any such activity. Immunoblotting studies revealed that a protein with mol. wt of 79 KDa was the only one among the proteins of B. malayi microfilarial extracts that was consistently recognised by sera from all endemic residents. Endemic normal sera and elephantoid sera, which exerted maximum cytotoxicity, together specifically recognised three proteins with molecular weights 25, 58 and 68 KDa and these three proteins could be among the candidate antigens that induce resistance to filarial infection.

Immunocytochemical localization of surface antigens in microfilariae of Wuchereria bancrofti.

Antigenic sites were localized on the surface structure (sheath and cuticle) of microfilariae of Wuchereria bancrofti using sera from microfilaraemic and amicrofilaraemic patients using antibodies associated to colloidal gold particles and transmission electron microscopy. No labeling was observed on the surface of intact parasites. Microfilariae without the sheath and isolated sheath were obtained by ultrasound treatment. Using these preparations labeling of the inner portion of the sheath was observed when sera from amicrofilaraemic patients were used. Labeling of the cuticle surface was observed mainly in the regions of the cuticular annulations.

Evaluation of specific humoral immune responses in human filariasis.

Using the antigens prepared from adult worms of B. malayi and D. immitis, a total of 37 malayan and 150 bancroftian serum samples were analysed for the presence of filarial specific antibodies by counterimmunoelectrophoresis and indirect enzyme linked immunosorbent assay. It was observed that microfilaraemic cases do show good specific humoral immune responses against homologous as well as heterologous antigen. Of the two antigens used, B. malayi adult antigen was found to be superior to D. immitis antigen in the detection of filarial antibodies.