Specificity of predominant IgG4 antibodies to adult and microfilarial stages of Brugia malayi.

Human infections with filarial nematodes such as Brugia malayi are accompanied by unusually high titres of parasite-specific IgG4 antibodies. We have compared the profile of antigens recognised by filarial-specific IgG1 and IgG4 isotypes by Western blotting. Serum samples were collected from 120 subjects exposed to Brugia malayi, divided into three groups of asymptomatic amicrofilaraemic (endemic normal), microfilaraemic, and elephantiasis patients. Antigen preparations were tested from both adult B. malayi parasites, and from microfilariae; 24 distinct bands were analysed from the former, and 19 from the latter. Both qualitative scoring for band reactivity, and densitometric scanning of major bands, were employed. The consistent result was one of high and preferential IgG4 reactivity to a set of low molecular weight bands, of 15, 17, 20, 31 and 33 kDa; most of the 19 other bands showed higher reactivity with IgG4. Analysis of Western blot patterns showed an overall tendency for stronger IgG4 responses in microfilaraemic cases, and higher IgG1 responses in elephantiasis patients, consistent with published studies using ELISA on unfractionated parasite extracts. This study has defined an array of filarial antigens from each stage, and relative levels of IgG4 recognition, which will be important in unravelling distinct immune responses to this complex parasite.

Antibody responses to filarial infective larvae are not dominated by the IgG4 isotype.

The humoral immune response in humans to filarial parasites is generally dominated by the IgG4 isotype, when measured by ELISA against somatic adult worm extract. In contrast, as we report here, antibodies reactive to somatic extracts of infective larvae are more equally represented by IgG1 and IgG4. Moreover, binding to surface exposed epitopes in immunofluorescence on larval stages is mediated foremost by IgG1 and IgM, secondarily by IgG2 and IgG3, and very little by IgG4. Both anti-L3 surface and somatic antibodies are strongest in elephantiasis patients, and tend to increase with age. Antibody to the L3 surface is also present in most microfilaraemic individuals who bear no detectable antibodies to the surface of the microfilarial stage. These results demonstrate that a stage- and isotype-specific response is mounted to the L3 surface which should be considered as a possible mediator of concomitant immunity in filariasis.

Differential decline in filaria-specific IgG1, IgG4, and IgE antibodies in Brugia malayi-infected patients after diethylcarbamazine chemotherapy.

In human filariasis, the predominant serum antibody is IgG4, accompanied by significant IgE production. The ratio of IgG4 to IgE is highest in asymptomatic microfilaremic carriers, while chronic disease is associated with elevated IgG1-3. The changes in isotypes following chemotherapy with diethylcarbamazine (DEC) were studied in 2 groups of Brugia malayi-infected patients from Sumatra and South Kalimantan, Indonesia. Similar results were obtained from each group. IgG4 levels decreased sharply (65%-78%) within 12 months. IgG1 levels declined in a less consistent and extreme manner, and levels of IgG2 and IgG3 declined only in patients with elephantiasis, who also had the highest initial levels of these antibodies. IgE responses were relatively stable to therapy in microfilaremic patients (7%-28% reduction) and showed only moderate decline (56% over 2 years) in elephantiasis patients. Active filarial infection is thus associated with specific IgG4 antibodies, but there is independent expression of the IgE and IgG4 isotypes in filariasis.

Specific T cell unresponsiveness in human filariasis: diversity in underlying mechanisms.

In an attempt to overcome T cell unresponsiveness to filarial antigens, 65 individuals belonging to the three clinical groups of elephantiasis patients, microfilaraemics, and asymptomatic amicrofilaraemics who exhibited unresponsiveness to Brugia malayi adult worm antigen (BmA) were studied. Peripheral blood mononuclear cells were cocultured with antigen and one of the following reagents that have been reported to be effective in reconstituting T cell proliferation: interleukin-2 (IL-2), interleukin-7 (IL-7), anti-interleukin-4, anti-interleukin-10, anti-CD2, anti-CD27, anti-CD28, indomethacin, phorbol myristate acetate (PMA), or calcium ionophore (A23187). We were able to overcome antigen-specific unresponsiveness in only a minority of the individuals studied. Co-culture with IL-2, IL-7, indomethacin and PMA were the only conditions which resulted in enhanced proliferation to BmA in these individuals. In general, unresponsiveness in elephantiasis patients was easier to reverse than in other clinical groups: in 50% of elephantiasis patients, in 12.5% of microfilaraemics and in 20% of asymptomatic amicrofilaraemics. The results indicate that more than one distinct immunological mechanism may account for the antigen-specific unresponsiveness in individuals exposed to and infected with brugian filariasis.

Differential antibody isotype reactivity to specific antigens in human lymphatic filariasis: gp15/400 preferentially induces immunoglobulin E (IgE), IgG4, and IgG2.

Lymphatic filarial infection in humans is associated with a strong skewing of the immune response towards the TH2 arm, with prominent interleukin 4-producing cells and elevated levels of immunoglobulin G4 (IgG4) and IgE antibodies in peripheral blood. To determine how such a generalized TH2 imbalance governs responses to individual parasite antigens, the profiles of isotypes of antibodies to two recombinant proteins of Brugia spp. were studied. One molecule was the C-terminal portion of the filarial heat shock protein 70 (Bpa-26), representative of a cytoplasmic protein, and the second antigen was a single unit of the tandem repeats of a Brugia polypeptide (BpL-4), a secreted product which is prominently exposed to the immune system. Serum samples from 146 individuals resident in areas in which brugian filariasis is endemic were used, and it was found that whereas the levels of IgG1 and IgG3 responses to both Bpa-26 and BpL-4 were high, IgG4 and IgE antibodies to only BpL-4, not to Bpa-26, were prominent. Thus, an antigen which is chronically exposed to the immune system elicited a TH2-dependent isotype switch, as manifested by increased IgG4 and IgE responses. Moreover, IgG4 and IgE responses to BpL-4 showed a strong negative association, suggesting that mediators other than interleukin 4 must be responsible for such differential regulation of these two isotypes. When the data were analyzed as a function of clinical status, a striking association between elevated levels of IgG3 antibodies to Bpa-26 and manifestation of chronic obstructive disease was found; elephantiasis patients showed significantly higher levels of IgG3 antibodies to Bpa-26 than microfilaremics and asymptomatic amicrofilaremics. This indicates that an imbalance of isotypes of antibodies to particular filarial antigens might play a role in the pathogenesis of chronic disease.

HLA and elephantiasis in lymphatic filariasis.

Lymphatic filariasis presents a spectrum of manifestations with infection-free asymptomatics at one end and elephantiasis at the other. In order to determine if any HLA antigens are associated with the development of elephantiasis, we compared the HLA frequencies in 55 elephantiasis patients with those in 40 controls consisting of individuals older than 45 years of age without any signs of elephantiasis. The only significant difference in class I antigen frequencies was observed for B27, which was present in 11% of the patients and absent in the controls. More differences were observed in HLA class II antigen frequencies. Both DR3 and the 2B3 epitope (on DQ6, DQ8, and DQ9 molecules) were significantly decreased in patients with elephantiasis whereas the DQ5 frequency was significantly higher in patients than in controls. Analysis of specific antibody isotype profiles revealed that DQ5-positive individuals had increased levels of antifilarial IgG3, an isotype known to be involved in tissue damage. These data suggest that HLA class II genes may control the course of Brugian filariasis by influencing the T-cell-dependent antibody repertoire.

Elevated cellular immune responses and interferon-gamma release after long-term diethylcarbamazine treatment of patients with human lymphatic filariasis.

Cellular immune responses to filarial antigens were examined in persons before and 1 year after beginning treatment with diethylcarbamazine (DEC). The subjects (17 microfilaremics, 13 asymptomatic amicrofilaremics, and 13 with elephantiasis) had not responded to Brgia malayi adult worm antigen (BmA) before chemotherapy. T cell proliferative responses to BmA improved significantly after therapy in the 3 clinical groups (P < .05) but was highest in the elephantiasis patients and asymptomatic amicrofilareimics. Cytokine release profiles after stimulation with parasite antigen were analyzed. Production of interferon (IFN)-gamma by BmA-stimulated mononuclear cells increased significantly after DEC treatment (geometric mean, 39.6-55.7 U/mL; P < .05), largely due to improved responses in elephantiasis patients and asymptomatic amicrofilaremics. In contrast, BmA-induced interleukin (IL)-4 release did not change significantly in these same patients after treatment. Thus, both microfilaremic and amicrofilaremic infections with B. malayi are associated with similar down-regulation of proliferative T cell function and IFN-gamma release.

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.

A polymerase chain reaction assay for the detection of Brugia malayi in blood.

There is need for sensitive, rapid, species-specific diagnosis of Brugia filarial parasites because traditional methods are tedious and time-consuming, with little guarantee of species specificity. A polymerase chain reaction (PCR)-based assay was developed using the Hha I family of highly repeated DNA sequences from Brugia. The assay was tested on 124 human blood samples collected in a field study in Indonesia. These included 66 microfilaria-positive samples from patients in an area endemic for Brugia, 30 from healthy individuals from the same endemic area, and 28 from healthy individuals from a nonendemic area. Twenty-eight blood samples collected in a village in French Polynesia endemic for Wuchereria bancrofti, but not B. malayi, were also tested. The blood samples were screened using the traditional blood smear and membrane filtration methods, which served as the gold standards to which the PCR assay was compared. The samples were digested with proteinase K, extracted with phenol and chloroform, and dialyzed. A fraction of the dialyzed product was used in PCRs using Hha I-specific primers. The PCR assay correctly identified all of the microfilaria-positive samples as PCR positive and all of the nonendemic samples as PCR negative. Additionally, 26 of 30 samples from healthy individuals in the endemic area were also identified as PCR negative, while four were PCR positive. It is likely that these four individuals had very low-level or cryptic infections, and that the PCR assay detected circulating DNA released from dead filariae. The results indicate that the Hha I PCR detection system is rapid, species-specific, and sensitive.

Elevated levels of T cell activation antigen CD27 and increased interleukin-4 production in human lymphatic filariasis.

To assess the immunological changes occurring during filarial infection with or without elephantiasis, 145 patients in different clinical groups from an endemic area in Indonesia were compared with respect to plasma levels of both soluble CD25 (sCD25) and sCD27; interleukin-4 (IL-4) and interferon-gamma release by peripheral blood mononuclear cells was also measured in a smaller subset of individuals. Levels of sCD27 were significantly elevated in elephantiasis and microfilaremic patients compared with endemic normals (p < 0.002), whereas sCD25 levels remained low in microfilaremics and was only slightly elevated in elephantiasis patients compared with endemic normals (p < 0.02). As activated T cell populations release both sCD27 and sCD25, these findings imply that there is filarial-driven activation of a T cell subset that releases sCD27 rather than sCD25. The expansion of a particular T cell population by filarial parasites is further suggested by the enhancement in both IL-4-producing and CD4+CD27-T cells in PBMC from elephantiasis and microfilaremic patients compared with endemic normals. More detailed characterization and comparison of CD27-lymphocytes from these individuals may identify mechanisms involved in the pathogenesis of lymphatic filariasis.

Primary structure of and immunoglobulin E response to the repeat subunit of gp15/400 from human lymphatic filarial parasites.

We have isolated and sequenced clones encoding the repeated subunit of the surface-associated glycoprotein gp15/400 from the two nematode species predominantly responsible for lymphatic filariasis in humans: Brugia malayi and Wuchereria bancrofti. The amino acid sequence of the 15-kDa subunit, derived from the nucleotide sequence of the gene fragment from B. malayi, is identical to that previously reported for B. pahangi, whereas the derived W. bancrofti protein sequence differs in only 7 of 132 residues. The identity of the protein in the two Brugia species allowed us to use a recombinant from B. pahangi to examine the serological response of adult Indonesian subjects infected with B. malayi. The polymerase chain reaction-amplified subunit was expressed in Escherichia coli via the pDS56/RBS11 plasmid and purified by nickel-chelating chromatography. A significant proportion of individuals produced antigen-specific immunoglobulin E (IgE). This was most pronounced in the individuals with elephantiasis, with 14 of 15 showing elevated titers and a mean of 3.2 ng of specific IgE ml-1. Only 2 of 15 microfilaremic individuals possessed elevated titers of specific IgE, with a mean of 0.045 ng ml-1 for the group as a whole. Asymptomatic amicrofilaremic residents showed approximately equal numbers of responders (defined as having a value in the radioimmunoassay greater than two standard deviations above controls) and nonresponders, with a group mean of 1.2 ng of antigen-specific IgE ml-1.

Differential expression of IgE and IgG4 specific antibody responses in asymptomatic and chronic human filariasis.

A population of 164 adult individuals resident in an area endemic for Brugia malayi lymphatic filariasis has been studied for humoral immune responses to filarial parasites. Antibody levels to Ag extracted from adult worms were determined for each of the IgG subclasses, for IgM and for IgE. The dominant isotype of antifilarial antibody was IgG4, which represented 88% of total IgG in asymptomatic microfilaremics, most of whom possessed 100 to 1000 micrograms/ml of specific antibody of this subclass (geometric mean 762 micrograms/ml). Patients with chronic disease (elephantiasis), who were generally amicrofilaremic, had substantially higher levels of IgG1, IgG2, and IgG3, but a 3.4-fold lower geometric mean level of specific IgG4 (222 micrograms/ml) than asymptomatics with or without microfilaremia. In contrast, specific IgE antibody levels in cases of elephantiasis were on average 4.5 times higher than those found in the asymptomatic carrier state. The majority of microfilaremics were therefore typified by extremely high specific IgG4 concentrations and relatively low IgE reactivities, whereas clinical cases tended to show the reverse relationship. The possible roles of these isotypes and the implications of changing IgG4/IgE balances in disease are discussed.

T cell responsiveness correlates differentially with antibody isotype levels in clinical and asymptomatic filariasis.

To establish the relationships among T and B cell responses, active infection, and clinical manifestations in lymphatic filariasis, filarial-specific lymphocyte proliferation, IgG antibody isotypes, and IgE levels were determined in an exposed population: 31 asymptomatic amicrofilaremics, 43 microfilaremics, 12 symptomatic amicrofilaremics, and 52 elephantiasis patients. Lymphocyte proliferation was higher in elephantiasis patients and asymptomatic amicrofilaremics than in microfilaremics (P < .004). A proportion of asymptomatic amicrofilaremics (32%), elephantiasis patients (37%), and symptomatic amicrofilaremics (58%) showed antigen-specific lymphocyte unresponsiveness, and lymphocyte proliferation to filarial antigens correlated negatively with specific IgG4 levels (rho = -0.315, P < .001). As elevated specific IgG4 is an indicator of active infection, it is argued that active infection may result in lymphocyte hyporesponsiveness irrespective of clinical category. Of those with elevated specific IgE levels and high T cell proliferative responses, 70% had elephantiasis, suggesting these factors have a role in pathology. However, the existence of a proportion of elephantiasis patients with low anti-filarial IgE and T cell unresponsiveness to filarial antigens suggests that elephantiasis can be caused by distinct processes.

Recent advances in the application of molecular biology in filariasis.

Monitoring of filarial parasites in the host and vector has traditionally depended on morphological identification. Recently, species-specific DNA probes have been developed for Brugia malayi, Brugia pahangi and Wuchereria bancrofti. Repeated DNA sequences are useful in developing DNA probes because they evolve more rapidly then coding sequences and their high copy number increases the sensitivity of detection. The Hhal repeated DNA family represents 12% of the total B. malayi DNA. This DNA family is present in species of Brugia (B. malayi, B. timori and B. pahangi) but not W. bancrofti. Sequence analysis of the repeated DNA in B. malayi and B. pahangi has allowed construction of two species-specific DNA probes. These probes were used in a double blind field study in Indonesia. Microfilariae (mf) from infected cats and humans were identified by classical morphological methods and DNA probes. Agreement was found in 98.6% of the 642 samples tested by the two different techniques. Besides mf identification DNA probes can be used to determine the species of infective larvae (L3s) in infected mosquitos. This is useful because the L3s have similar morphology. DNA probes for the identification of W. bancrofti have recently been developed and are in the initial stages of testing in China (Piessens, personal communication) and Egypt (Williams, personal communication). An alternative approach for identification of infected individuals is to detect specific parasite antigens in circulation. A WHO initiative to use either an antigen or antibody assay to replace night blood is presently underway. This approach, if successful would not require the presence of microfilariae, but could detect occult infections.

Ivermectin and diethylcarbamazine trials in leaf monkeys (Presbytis cristatus) infected with Wuchereria kalimantani.

Clinical trials of Ivermectin in single oral doses of 200, 400, and 1,000 mg/kg body weight or in multiple doses of 200 mg/kg body weight for 5 consecutive days were performed in leaf monkeys (Presbytis cristatus) infected with Wuchereria kalimantani. Optimal microfilaricidal effect occurred at 200 mg/kg body weight. The drug was less effective than diethylcarbamazine in this animal model for human filariasis but had no adverse effects.

Towards a filariasis-free community: evaluation of filariasis control over an eleven year period in Flores, Indonesia.

A population of 202 residents in an area endemic for Brugia timori lymphatic filariasis was treated in a diethylcarbamazine control programme commencing in 1977. All individuals were treated twice with diethylcarbamazine on a mass basis with additional selected treatment for cases with manifestations of infection. Clinical features of lymphatic filariasis were recorded annually until 1982, and the population re-assessed in 1988, six years after the completion of chemotherapy. Microfilarial counts were made on each occasion, and circulating filarial antigen levels measured for 1982 and 1988. The results showed a dramatic and sustained reduction in the rate of elephantiasis and adenolymphangitic disease, and of circulating antigenaemia, and the prevalence of microfilaraemia was reduced to zero by the end of the study.

Secreted antigens of filarial nematodes: a survey and characterization of in vitro excreted/secreted products of adult Brugia malayi.

We report here a broad analysis of the excretory/secretory (E/S) products of adult Brugia malayi, collected by in-vitro cultivation of the parasite. Culture media and conditions were optimized, and non-essential amino acids were found to be crucial for efficient protein synthesis under cell- and serum-free culture conditions. A close correlation was found between total protein secretion, phosphorylcholine-bearing antigen release and lactate production on each day of culture, indicating that E/S molecules are actively secreted. Parasites cultured in vitro take 2-3 days to adjust to the new environment, and show peak levels of secretion at days 3 and 4. The active secretion of phosphorylcholine by the parasite therefore justifies the measurement of this molecule as an indication of active infection, possibly reflecting total worm burdens. By comparing metabolically labelled E/S from male and female worms, several molecules of low mol. wt, namely 10,000, 13,000, 14,000 and 22,000, together with high mol. wt components of above 12,000 were found to be female specific. Tracing the origin of the E/S products, several molecules were also found to be associated with the surface. Among these, there are at least two glycoproteins, 29,000 and 51,000 of which the 29,000 molecule is a major surface protein. The immunogenicity of the E/S was examined and antigenic cross-reactivity was found with sera from most filarial infections but not with non-filarial nematodiases such as hookworm or Trichinella. However, two molecules of low mol. wt, 15,000 and 19,000, were not recognized by anti-Onchocerca sera and appeared to be potential Brugia-specific diagnostic molecules. Possible functional roles of the adult E/S products were examined but we could find no evidence of protease activity in the E/S or glutathione S-transferase activity in either the E/S or in whole somatic extract.

Heat shock cognate 70 is a prominent immunogen in Brugian filariasis.

A cDNA expression library constructed from RNA derived from adult stage Brugia pahangi (mixed sexes) was screened with pooled sera from chronic, amicrofilaremic cases of human lymphatic filariasis from the Indonesian island of Tanjungpinang, where Brugia malayi is endemic. Polyclonal antisera raised to purified beta-galactosidase fusion proteins from two of the most highly reactive clones identified a protein of Mr 70,000 in all stages examined (microfilariae, L3 and adults) of both B. malayi and Brugia pahangi. Derivation of the amino acid sequence from these two overlapping cDNAs identified the encoded protein as a member of the heat shock protein 70 family, and showed the closest similarity to the constitutively expressed "heat shock cognate 70" (hsc70) protein. Hybridization of hsc70 cDNAs to RNA and DNA from B. pahangi under stringent conditions identified a major transcript of 2.4 kb and revealed the existence of a family of related genes. In vitro culture of larval stages of B. pahangi at elevated temperatures (43 degrees C) resulted in increased expression of hsc70, and a classic heat shock response in which five proteins (mr 18,500, 22,000, 62,000, 70,000, and 85,000) were exclusively synthesized in microfilariae. Analysis of cross-reactivities by Western blotting implied that antibody generated by infection with B. malayi was directed at filarial-specific determinants of Brugia hsc70. However, ELISA with recombinant fusion proteins for both Plasmodium falciparum and Schistosoma mansoni hsc70 indicated that some individuals with Brugian or Bancroftian filariasis did produce antibodies which cross-reacted with plasmodial and schistosomal homologs. Thus filarial-specific antibody responses were not generated in all individuals, indicating that this molecule would not be suitable for diagnostic purposes. ELISA with a purified beta-galactosidase fusion protein from B. pahangi showed antibody responses to hsc70 across the clinical spectrum of filariasis. Alignment of the derived amino acid sequences from B. pahangi, P. falciparum, S. mansoni and rat hsc70 homologs, and comparison of the immunologic reactivity of the products of the two cDNA clones by Western blotting and ELISA suggested that these determinants were located primarily at the C terminus of the protein.

Identification, synthesis and immunogenicity of cuticular collagens from the filarial nematodes Brugia malayi and Brugia pahangi.

The major structural proteins of the cuticle of the filarial nematode parasites Brugia malayi and Brugia pahangi were identified by extrinsic iodination and sensitivity to clostridial collagenase. At least 16 acidic components were identified in adult worms by 2-dimensional electrophoresis, with molecular weights ranging from 35,000 to 160,000. These proteins appear to be cross-linked by disulphide bonds, and localised in the basal and inner cortical layers of the cuticle. The outer cortex, containing the epicuticle, is insoluble in 1% sodium dodecyl sulphate and 5% 2-mercaptoethanol, and can be isolated free of cellular material. Despite their inaccessibility to the immune system in intact worms, antibodies to the cuticular collagens are provoked in humans infected with a variety of filarial parasites. Immunological cross-reactivity was demonstrated between a 35 kDa component and human type IV (basement membrane) collagen. Autoantibodies to type IV collagen were detected in a number of individuals with lymphatic filariasis, although no correlation could be drawn with observed pathology. Synthesis of cuticular collagens is discontinuous, occurs at negligible levels in mature adult male worms, and does not appear to involve the production of small molecular weight precursors, in contrast to Caenorhabditis elegans. Hybridisation with a heterologous cDNA probe coding for the alpha 2 chain of chicken type 1 collagen suggests that they are encoded by a multigene family.