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.     

IgG antibody subclasses in human filariasis. Differential subclass recognition of parasite antigens correlates with different clinical manifestations of infection

The four subclasses of IgG are distinct in structure, function, and degree of participation in the antibody response to complex antigens. Looking for differential responsiveness of potential pathogenetic significance, we have analyzed both quantitatively and qualitatively the filaria-specific IgG subclass responses of 20 patients with lymphatic filariasis presenting either with chronic lymphatic obstructive pathology and elephantiasis (CP) or with asymptomatic microfilaremia (MF). Subclass-specific monoclonal antibodies were used in an enzyme-linked immunosorbent assay to study IgG filarial antibodies quantitatively and in immunoblot analyses to determine qualitatively the subclass antibody specificities. Quantitatively, the most significant differences among patient groups were in levels of IgG4, which were more than 17 times higher in MF patients (geometric mean, 64.7 micrograms/ml) than in those with CP (mean, 3.7 micrograms/ml). When qualitative analyses were done on the same sera, major differences were noted, particularly in the recognition profiles of the IgG1, IgG3, and IgG4 responses. IgG1 and IgG3 responses to antigens were seen especially to antigens with m.w. greater than 68,000 in all patients with elephantiasis, whereas MF patients showed most of their reactivity to antigens less than 68,000. For IgG4, the MF patients showed prominent recognition of antigens throughout the entire range of m.w., whereas those with CP had very little IgG4 recognition of antigens of any m.w. Interestingly, this relationship was essentially reversed in the IgG3 antibody responses (especially to antigens greater than 68,000) and, to a lesser extent, the IgG1 responses. These findings demonstrate correlations of potential cause/effect significance between IgG4 antibody responsiveness and the immunomodulated asymptomatic MF form of clinical filariasis and between IgG3/IgG1 antibody responsiveness and the clinical presentation of CP.     

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.     

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.     

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.     

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.     

Distribution of filarial elephantiasis and hydrocele in Matara district, Sri Lanka, as reported by local leaders, and an immunological survey in areas with relatively high clinical rates

To eliminate lymphatic filariasis by means of mass drug administration, it is essential to have reliable data on the disease distribution and prevalence in targeted areas. In Matara district, Sri Lanka, self-administered questionnaires were mailed to 2105 local leaders questioning the presence and the numbers of elephantiasis and hydrocele cases. The information provided by them revealed that elephantiasis was clearly aggregated in the southern part of the district along the coast, while hydrocele was distributed rather evenly in the whole district, including Deniyaya region where no endemic filariasis had been known. To confirm active transmission of filariasis in Deniyaya, Wuchereria bancrofti antigen and filaria-specific urinary IgG4 antibody were measured with 2436 subjects. The positive rates for antigen and antibody were 0.6% and 4.3%, respectively. The titer analysis of IgG4 according to age revealed that the youngest IgG4 positive was 3 years old, and that in 10 years old or less, there were 16 positives out of 607 children examined (2.6%). It was concluded that filarial transmission at a low level was going on in the region.     

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.     

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.     

Onchocerca volvulus and Wuchereria bancrofti: fluorescent antibody staining of frozen homologous sections for diagnosis

Antibody responses to two filarial diseases of man, onchocerciasis and bancroftian filariasis, were evaluated with the indirect fluorescent antibody technique (IFAT), using antigens derived from the appropriate etiologic agent. Antigenic preparations consisted of frozen cut sections of the adult Onchocerca volvulus and stage III larvae of Wuchereria bancrofti fixed to glass slides. Little difference between the preparations was demonstrated in tests for the diagnosis of onchocerciasis. Of 105 sera from individuals with biopsy-proven infections, 102 (97%) reacted with homologous O. volvulus antigen, and 19 of 22 (86%) with W. bancrofti antigen. In bancroftian filariasis, however, the homologously derived antigen was superior for diagnosis, and the highest seropositive rates occurred in acute, symptomatic infections. All such sera (8) reacted with homologous antigen. In contrast, only 75% (6) reacted with onchocercal antigen. Of those with chronic disease, characterized by long-standing elephantiasis or lymphedema without microfilaremia, 79% (22) were reactors to homologous antigen and 32% (9) to heterologous. The lowest seropositive rates occurred where microfilaremia was unaccompanied by local or systemic symptoms: 38% (3) were positive to homologous antigen and none to onchocercal antigen.Of sera from seven individuals apparently free of bancroftian filariasis, but living in a hyperendemic area, five reacted with bancroftian antigen and four with onchocercal antigen. These reactions could be attributed to occult infections, but more likely resulted from repeated exposure to nondeveloping infective larvae.Cross-reactions in nonfilarial infections were rare with either antigen, and no positive reactions occurred in sera from healthy controls.     

A survey of Brugia malayi infection on the Heugsan Islands,Korea

Lymphatic filariasis due to Brugia malayi infection was endemic in several areas of South Korea. The infection was controlled, or disappeared, in most areas, with the exception of the remote southwestern islands of Jeonranam-do, including the Heugsan Islands. To discover its current situation, a small-scale survey was performed on the Heugsan Islands in September 2000. A total of 378 people, 151 male and 227 female, living in 8 villages (6 on Daeheugsan-do, 1 on Daejang-do, and 1 on Yeongsan-do) were subjected to a night blood survey for microfilaremia, and physical examination for elephantiasis on the extremities. There were 6 (1.6%) microfilaria positive cases, all in females aged 57-72 years, and from only two villages of the Daeheugsan-do area. There were 4 patients with lower leg elephantiasis, but they showed no microfilaremia. The results show that a low-grade endemicity of filariasis remains on the Daeheugsan-do.     

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.     

Immune response to an allergenic fraction ofSetaria digitata in human filariasis

A low molecular weight antigenic fraction has been isolated from saline-soluble extracts of cattle filarial parasiteSetaria digitata. This glycoprotein fraction (Fr III) which appears to have low phosphorylcholine content cross-reacted with infective larval (L₃) antigens ofWuchereria bancrofti. Binding of human chronic filarial serum with L₃ antigens could be inhibited partially by Fr III. The fraction elicited immediate cutaneous hypersensitivity reaction in people living inWuchereria bancrofti endemic regions. The proportion of skin test positive cases was found to be highest in endemic normals in contrast to infected cases. IgE levels were however not different in chronic filariasis and in endemic normals (or in asymptomatic microfilaraemic carriers). On the other hand, specific IgG level was considerably enhanced only in chronic filariasis     

An analysis of in vitro B cell immune responsiveness in human lymphatic filariasis

The immunoregulatory mechanisms involved in B cell function in patients with varying clinical manifestations of bancroftian filariasis were examined by studying the ability of peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from patients with elephantiasis, asymptomatic microfilaremia (MF), and acute tropical pulmonary eosinophilia (TPE) to produce polyclonal and parasite-specific antibody in vitro, both spontaneously and in response to a mitogen (PWM) and to parasite antigen. When the spontaneous or mitogen-driven polyclonal responses were examined, all groups produced significant amounts of IgM and IgG; those with TPE produced extremely high levels. However, when in vitro parasite antigen-specific responses were examined, those with MF were unable to produce filaria-specific antibody either spontaneously or in response to PWM or parasite antigen; in contrast, patients with chronic lymphatic obstruction or TPE produced large quantities. Removal of neither adherent cells nor T8+ T cells affected the parasite-specific B cell anergy seen in those with MF. This absent or severely diminished capacity to produce antibody on parasite antigenic stimulation in patients with MF is likely responsible for the low levels of parasite-specific antibody seen in this most common clinical manifestation of bancroftian filariasis. Its inability to be reversed by the removal of "suppressor elements" suggests a state of B cell unresponsiveness to the parasite.     

Neonatal tolerance and patent filarial infection

Lymphatic filariasis occurs in endemic pockets. Patent infections with long-term, high-grade microfilaremia do not develop in nonendemic individuals. It is tempting to speculate that individuals with intact immune responses to filarial antigens are capable of dealing with filarial exposure without developing persistent infection. There are published data that support the idea that only those individuals who are impaired in their immune defense against these parasites owing to neonatal tolerization become productively infected with the filarial parasites. If the model is correct, there are profound implications for global eradication.     

Cytodiagnosis of filarial infections from an endemic area

OBJECTIVE: To throw light on cytologic findings as a possible mode of diagnosis of lymphatic filariasis. STUDY DESIGN: Filariasis has worldwide distribution, but lymphatic filariasis predominantly affects tropical and subtropical regions. Demonstration of microfilaremia, the specific test for diagnosis of lymphatic filariasis, often shows false negative results in endemic areas. The present study, done in an endemic area, showed the presence of microfilariae or adult worms of Wuchereria bancrofti in fine needle aspirates collected from amicrofilariaemic cases. In a few cases the discovery was incidental. A total 4,534 cases undergoing cytologic evaluation were carefully screened for the presence of adult worms or larvae, irrespective of clinical diagnosis. Microfilariae were demonstrated in both clinically suspected cases of filariasis and asymptomatic cases. RESULTS: A total of 1 positive cases were found; in 4 cases the clinical diagnosis was lymphatic filariasis, and 7 cases were asymptomatic. All 11 cases were amicrofilariaemic. CONCLUSION: Various sophisticated investigations are used for diagnosis of lymphatic filariasis without microfilaremia. Fine needle aspiration cytology, being a cheap, simple and easy procedure, may have some role in this field, but further detailed studies are needed before any final claim.     

Circulating Microbial Products and Acute Phase Proteins as Markers of Pathogenesis in Lymphatic Filarial Disease

Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag−) active infection; with clinically asymptomatic infections (INF); and in those without infection (endemic normal [EN]). Comparisons between the two actively infected groups (CP Ag+ compared to INF) and those without active infection (CP Ag− compared to EN) were used preliminarily to identify markers of pathogenesis. Thereafter, we tested for group effects among all the four groups using linear models on the log transformed responses of the markers. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein), acute phase proteins (haptoglobin and serum amyloid protein-A), and inflammatory cytokines (IL-1β, IL-12, and TNF-α) are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins.     

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.     

Large Extracellular Loop of Tetraspanin as a Potential Vaccine Candidate for Filariasis

Lymphatic filariasis affects nearly 120 million people worldwide and mass preventive chemotherapy is currently used as a strategy to control this infection. This has substantially reduced the incidence of the infection in several parts of the world. However, a prophylactic vaccine would be more effective in preventing future infections and will supplement the mass chemotherapy efforts. Unfortunately, there is no licensed vaccine available currently to prevent this infection. Molecules expressed on the surface of the parasite are potential candidates for vaccine development as they are exposed to the host immune system. In this study we show that the large extracellular loop of tetraspanin (TSP LEL), a protein expressed on the cuticle of Brugia malayi and Wuchereria bancrofti is a potential vaccine candidate. Our results showed that BmTSP LEL is expressed on the surface of B. malayi infective third stage larvae (L3) and sera from human subjects who are putatively immune to lymphatic filariasis carry high titer of IgG1 and IgG3 antibodies against BmTSP LEL and WbTSP LEL. We also showed that these antibodies in the sera of human subjects can participate in the killing of B. malayi L3 in an antibody dependent cell-mediated cytotoxicity mechanism. Vaccination trials in mice showed that close to 64% protection were achieved against challenge infections with B. malayi L3. Immunized animals showed high titer of anti-WbTSP LEL IgG1, IgG2a and IgG2b antibodies in the sera and IFN-γ secreting cells in the spleen. Onchocerca volvulus another filarial parasite also expresses TSP LEL. Cross-reactivity studies showed that IgG1 antibody in the sera of endemic normal subjects, recognize OvTSP LEL. Similarly, anti-OvTSP LEL antibodies in the sera of subjects who are immune to O. volvulus were also shown to cross-react with rWbTSP LEL and rBmTSP LEL. These findings thus suggested that rTSP LEL can be developed as a potential vaccine candidate against multiple filarial infections.     

Immune dynamics in the pathogenesis of human lymphatic filariasis

Despite the longstanding recognition of the spectral nature of human disease due to lymphatic filariasis, immunologists interested in pathogenesis have mostly examined patients classified as being at either one extreme pole or the other. While the clinically asymptomatic individuals with microfilaremia who sit at one pole always have active infection, it has been difficult to define who else on the clinical spectrum is actively infected with living adult worms. In this review, David Freedman discusses how the ability to measure circulating filarial antigen in patient serum has advanced our ability to understand the immunopathogenesis of lymphatic filariasis by improving the precision of patient classification. Recent work suggests that the presence (or absence) of antigenemia, rather than overt clinical manifestations of disease, is closely associated with specific cytokine responses. A framework for patient classification based on these findings is proposed.