Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing.

While interference with the class I MHC pathway by pathogen-encoded gene products, especially those of viruses, has been well documented, few examples of specific interference with the MHC class II pathway have been reported. Potential targets for such interference are the proteases that remove the invariant chain chaperone and generate antigenic peptides. Indeed, recent studies indicate that immature dendritic cells express cystatin C to modulate cysteine protease activity and the expression of class II MHC molecules [1]. Here, we show that Bm-CPI-2, a recently discovered cystatin homolog produced by the filarial nematode parasite Brugia malayi (W. F. Gregory et al., submitted), inhibits multiple cysteine protease activities found in the endosomes/lysosomes of human B lymphocyte lines. CPI-2 blocked the hydrolysis of synthetic substrates favored by two different families of lysosomal cysteine proteases and blocked the in vitro processing of the tetanus toxin antigen by purified lysosome fractions. Moreover, CPI-2 substantially inhibited the presentation of selected T cell epitopes from tetanus toxin by living antigen-presenting cells. Our studies provide the first example of a product from a eukaryotic parasite that can directly interfere with antigen presentation, which, in turn, may suggest how filarial parasites might inactivate the host immune response to a helminth invader.

Characterisation of the two most abundant genes in the Haemonchus contortus expressed sequence tag dataset.

Analysis of the Haemonchus contortus Expressed sequence tag (EST) dataset revealed that almost 10% of all ESTs (1719 ESTs) belong to a family of related genes. Close analysis of the ESTs suggests that these represent two genes (called here Hc-nim-1 and Hc-nim-2) with multiple alleles of each. These genes show significant similarity to two genes from Caenorhabditis elegans, F54D5.3 (Wormbase accession WBGene00010049, corresponding protein WP:CE28033) and F54D5.4 (WBGene00010050, WP:CE03409) of unknown function. Reverse transcriptase coupled-PCR showed that both genes are transcribed from the L4 stage onwards and are transcribed in both male and female adult worms. A partial bacterial recombinant of the Hc-NIM-1 protein was made and used to raise antiserum in rabbits which recognised a 19 kDa antigen in the water soluble protein fraction of adult worms. By immunohistochemistry, the Hc-NIM-1 protein was localised in the hypodermis of the pharyngeal region of adult worms but not posterior in the hypodermis surrounding the reproductive tract. To investigate the function of this novel protein family we conducted a RNA interference experiment for the homologuous proteins in C. elegans. No visible phenotype was detected after simultaneous RNAi treatment for both Ce-F54D5.3 and Ce-F54D5.4.

Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi.

Isolation and cloning of abundant reverse transcriptase-polymerase chain reaction (RT-PCR) products from the filarial nematode Brugia malayi using the conserved nematode spliced leader sequence and poly A as amplification targets has allowed us to identify abundant, stage specific transcripts from infective and post-infective larvae. The predicted protein products of the most prominent full-length transcripts from mosquito-derived L3 parasites are: (i) Bm-ALT-1, a homologue of a Dirofilaria immitis abundant larval protein: (ii) Bm-CPI-1, a cystatin-type cysteine protease inhibitor; (iii) Bm-ALT-3, a novel predicted 6 kDa glycine/tyrosine-rich protein; and (iv) Bm-TPH-1, a homologue of a mammalian translationally-controlled tumour protein. Some transcripts were not full-length but had mis-primed at A-rich stretches of coding sequence: the most abundant of these was Bm-col-3, a which encodes a collagen homologous to Bp-COL-1 of Brugia pahangi. Similar analysis of abundant spliced leader (SL)/oligo-dT products from fourth-stage larvae 9 days post-infection yielded two dominant transcripts: (i) Bm-cdd-1, which encodes a protein with homology to cytidine deaminase, differing at only one amino acid position from its homologue described in Brugia pahangi; and (ii) the same truncated form of Bm-col-3 found in L3 preparations. Expression of the major transcripts was assessed by PCR amplification of cDNA libraries derived from each stage of the life cycle. alt1, alt-3 and cpi-1 were all found to be specific to the L3 stage, while cdd-1 was found only in the L4 cDNA library. Expression of these larval-specific transcripts was not detected in either microfilarial or adult libraries.

Expression and immune recognition of Brugia malayi VAL-1, a homologue of vespid venom allergens and Ancylostoma secreted proteins.

Several important nematode parasites have been found to express members of a gene family variously termed as venom allergen antigen homologue (vah) or Ancylostoma secreted protein (asp). In some cases these products are secreted by infective larval stages and have been suggested to be effective vaccine immunogens. We isolated the corresponding gene from the human filarial nematode, Brugia malayi, by first searching the expressed sequence tag (EST) dataset generated by the Filarial Genome Project and then using gene-specific nondegenerate primers matching the selected gene for PCR, from B. malayi cDNA libraries. We report here the full-length gene sequence, which we have designated as Bm-val-1, for vah/asp-like. The corresponding protein (Bm-VAL-1) contains 232 amino acids in a single homology unit, unlike products from some other species in which there is a tandem repeat. A putative signal sequence is present at the 5' end and there are two potential N-glycosylation sites. Murine antibodies to recombinant Bm-VAL-1 react with a 28 kDa protein in L3 extracts and recombinant Bm-VAL-1 is recognised by murine T cells primed with soluble L3 proteins. Of 82 ESTs corresponding to Bm-val-1, 72 are recorded from the infective larval (L3) stage. However, PCR on the first-strand cDNA from later mammalian stages revealed some expression at most subsequent time points. Over 95% (20/21) of microfilaraemic human filariasis patients are seropositive for antibodies to Bm-VAL-1, with particularly high levels of IgG3 and IgG4 isotypes. The IgG4 subclass may indicate stimulation by adult and/or microfilarial-derived immunogens. The association of Bm-VAL-1 with the infective stage and its recognition by humans exposed to filariasis suggests that further evaluation of this antigen as a vaccine candidate should be performed.

Cuticular localisation and turnover of the major surface glycoprotein (gp29) of adult Brugia malayi.

A polyclonal antiserum was raised to a gel purified preparation of the major water-soluble surface glycoprotein (gp29) of adult Brugia malayi, and used to define the stage specificity of expression, localisation (by immunoelectron microscopy) and the dynamics of biosynthesis and turnover via pulse-chase experiments. Gp29 was not detected in surface-labelled preparations of either pre- or post-parasitic third stage larvae (L3), but was present in fourth stage larvae (L4), where its mass was estimated to be 30 kDa by SDS-PAGE. In both L4 and adult worms, the protein resolved as 3 distinct species in 2-dimensional electrophoresis, with pIs from 6.5 to 7.5. Pulse-chase studies via metabolic labelling of adult worms with [35S]methionine in vitro indicated that gp29 was processed from a 32-kDa precursor to the mature molecule within 45 min and that it was secreted into culture medium within 5 h of synthesis. On extended culture, gp29 was converted to a 56-kDa product, presumably either by complex formation or covalent linkage with another secreted molecule. This higher molecular weight component had a more acidic pI of 4.5 and was insensitive to digestion with N-glycanase. Immunoelectron microscopy showed that gp29 was distributed throughout the cuticle and hypodermal cell layer of adult worms, suggesting that the protein was synthesised in the hypodermis, and that turnover into culture medium occurred through the cuticle. The protein appeared to concentrate at the distal cell membrane of the hypodermis, particularly at the stacked invaginations. Additional immunostaining was found on the basement membrane of the basal lamina of the intestine.

Biochemical and immunochemical characterisation of a 20-kilodalton complex of surface-associated antigens from adult Onchocerca gutturosa filarial nematodes.

Surface radioiodination of adult Onchocerca parasites reveals a restricted range of proteins associated with the cuticle. We present data to show that prominent among these is a complex of low-molecular-weight proteins which can be released in soluble form by homogenisation of surface-labelled Onchocerca gutturosa in phosphate-buffered saline (PBS). One of this groups of proteins, designated gp20, has a molecular mass of 20,000, is glycosylated with two N-linked carbohydrate side chains, and has a basic pI. Other PBS-soluble, 125I-labelled proteins of similar size appear not to be glycosylated. A distinct group of molecules are released only in the presence of reducing agents, and are likely to be cuticular collagens. The low-molecular-weight components are antigenic and cross-reactive with Onchocerca volvulus infection sera. Cross-reactions are also observed in immunoprecipitation experiments using sera from Brugia-immunised animals and infected humans. Comparative two-dimensional analyses of these immunoprecipitates reveal at least two Onchocerca specific components. As an alternative to radiolabelling and PBS homogenisation, incubation of worms in medium containing the reducing agent 2-mercaptoethanol resulted in a similar set of molecules being released into the medium. Since surface antigens of O. gutturosa from bovines and O. volvulus from humans appear similar in size and are antigenically cross-reactive, the more readily available parasite is being used to study further the properties of these molecules and to provide reagents for raising antisera reactive to the equivalent O. volvulus antigens.

Filarial surface antigens: the major 29 kilodalton glycoprotein and a novel 17-200 kilodalton complex from adult Brugia malayi parasites.

Adult Brugia malayi nematode parasites possess a range of cuticular and epicuticular molecules which may be defined by various surface-labelling techniques. We present here evidence that at least two distinct antigens are associated with the surface, a glycoprotein of 29 kDa, and a complex of twelve components forming a regular series or 'ladder' between 17 and 200 kDa (17/200 kDa) which have not previously been described. Each of these products is antigenic in infected hosts, although responses in infected humans to the 17/200 kDa are relatively weak. Digestion of the 29 kDa antigen with proteases and endoglycosidases indicates that it is closely conserved between B. malayi and B. pahangi, and that it carries at least two N-linked oligosaccharide chains each of 1.5-2 kDa. By contrast, a smaller surface-labelled antigen of 15 kDa shows no glycosylation by either lectin adherence or endoglycosidase digestion assays. Trypsin treatment of intact, labelled parasites results in cleavage of 29 kDa molecules isolated 17/200 kDa 'ladder' to trypsin abolishes all bands except the 17 kDa base unit. Both the 29 kDa and 17/200 kDa antigens can be recovered as water-soluble molecules by homogenisation of the parasite in the absence of detergent, or by disruption of the cuticle with reducing agents such as 2-mercaptoethanol. In the presence of such agents, both the 17/200 kDa series and the 29 kDa glycoprotein are shed rapidly from intact parasites. Finally, two-dimensional electrophoretic analysis shows that while the 29 kDa glycoprotein is strongly basic and the 15 kDa acidic, the 17/200 kDa antigens form a related series of neutral pI.

Immune evasion genes from filarial nematodes.

Helminth parasites have large genomes (approximately 10(8) bp) which are likely to encode a spectrum of products able to block or divert the host immune response. We have employed three parallel approaches to identify the first generation of 'immune evasion genes' from parasites such as the filarial nematode Brugia malayi. The first strategy is a conventional route to characterise prominent surface or secreted antigens. In this way we have identified a 15-kDa protein, which is located on the surface of both L3 and adult B. malayi, and secreted by these parasites in vitro, as a member of the cystatin (cysteine protease inhibitor) family. This product, Bm-CPI-2, blocks conventional cysteine proteases such as papain, but also the aspariginyl endopeptidase involved in the Class II antigen processing pathway in human B cells. In parallel, we identified the major T cell-stimulating antigen from the microfilarial stage as a serpin (serine protease inhibitor), Bm-SPN-2. Microfilariae secrete this product which blocks two key proteases of the neutrophil, a key mediator of inflammation and innate immunity. The second route involves a priori hypotheses that helminth parasites encode homologues of mammalian cytokines such as TGF-beta which are members of broad, ancient metazoan gene families. We have identified two TGF-beta homologues in B. malayi, and shown that one form (Bm-TGH-2) is both secreted by adult parasites in vitro and able to bind to host TGF-beta receptors. Likewise, B. malayi expresses homologues of mammalian MIF, which are remarkably similar in both structure and function to the host protein, even though amino acid identity is only 28%. Finally, we deployed a third method of selecting critical genes, using an expression-based criterion to select abundant mRNAs taken from key points in parasite life histories. By this means, we have shown that the major transcript present in mosquito-borne infective larvae, Bm-ALT, is a credible vaccine candidate for use against lymphatic filariasis, while a second abundantly-expressed gene, Bm-VAL-1, is similar to a likely vaccine antigen being developed against hookworm parasites.

Age-specific acquisition of immunity to infective larvae in a bancroftian filariasis endemic area of Papua New Guinea.

The development of antibodies to infective stages of the filarial parasite, Wuchereria bancrofti, with age of the host human population was studied by immunofluorescence, immunoprecipitation and immunoblotting assays. Among individuals under 20 years of age, few had detectable antibodies to the infective (L3) larval surface by IFA: only 2 out of 10 scored positive. However, all adults (over 20 years) were positive in this assay although the utilization of isotypes varied between different individuals. Whilst antibodies to the L3 surface are therefore acquired after prolonged exposure to infection (greater than 20 years), recognition patterns of L3 surface labelled antigens, measured by immunoprecipitation analysis iodinated proteins on SDS-PAGE, and of somatic L3 proteins on immunoblots, were equivalent in the two age groups. Thus, a critical surface antigen, recognised in an age-dependent manner, is present on the L6 cuticle but cannot be resolved as a conventional protein or glycoprotein constituent.

Identification of tgh-2, a filarial nematode homolog of Caenorhabditis elegans daf-7 and human transforming growth factor beta, expressed in microfilarial and adult stages of Brugia malayi.

A novel member of the transforming growth factor beta (TGF-beta) family has been identified in the filarial nematode parasite Brugia malayi by searching the recently developed Expressed Sequence Tag (EST) database produced by the Filarial Genome Project. Designated tgh-2, this new gene shows most similarity to a key product regulating dauer larva formation in Caenorhabditis elegans (DAF-7) and to the human down-modulatory cytokine TGF-beta. Homology to DAF-7 extends throughout the length of the 349-amino-acid (aa) protein, which is divided into an N-terminal 237 aa, including a putative signal sequence, a 4-aa basic cleavage site, and a 108-aa C-terminal active domain. Similarity to human TGF-beta is restricted to the C-terminal domain, over which there is a 32% identity between TGH-2 and TGF-beta1, including every cysteine residue. Expression of tgh-2 mRNA has been measured over the filarial life cycle. It is maximal in the microfilarial stage, with lower levels of activity around the time of molting within the mammal, but continues to be expressed by mature adult male and female parasites. Expression in both the microfilaria, which is in a state of arrested development, and the adult, which is terminally differentiated, indicates that tgh-2 may play a role other than purely developmental. This is consistent with our observation that TGH-2 is secreted by adult worms in vitro. Recombinant TGH-2 expressed in baculovirus shows a low level of binding to TGF-beta-receptor bearing mink lung epithelial cells (MELCs), which is partially inhibited (16 to 39%) with human TGF-beta, and activates plasminogen activator inhibitor-1 transcription in MELCs, a marker for TGF-beta-mediated transduction. Further tests will be required to establish whether the major role of B. malayi TGH-2 (Bm-TGH-2) is to modulate the host immune response via the TGF-beta pathway.

Localization, turnover and conservation of gp15/400 in different stages of Brugia malayi.

The expression of a protein complex designated gp15/400, previously identified via extrinsic iodination of adult Brugia malayi, was examined by labelling all stages found in the mammalian host and immunoprecipitation with a specific antibody raised to a recombinant protein. In this way, gp15/400 could be detected in L3, L4, adult worms and microfilariae recovered from jirds and labelled with Bolton-Hunter reagent. Metabolic labelling indicated that gp15/400 was released into culture medium when adult worms were maintained in vitro, but at a rate slower than that of gp29, the major soluble cuticular glycoprotein. Immuno-electron microscopy showed that the protein complex was broadly distributed in different tissues, although it was not detectable in the cuticle of adult worms. Dense labelling was observed in the matrix of the basal laminae bordering the hypodermis, somatic musculature and oesophagus, and lower but significant labelling was seen in the cells overlying these extracellular matrices. Hybridization of genomic DNA with a cDNA probe encoding gp15/400 indicated that homologous genes were present in Dirofilaria immitis and Acanthocheilonema viteae. The failure to detect related genes in non-filarial nematodes was presumed to be due to divergence beyond the practical limits of detection by nucleic acid probes, as antibody reagents showed that the protein cross-reacted immunologically with ABA-1, a major protein allergen from the body fluid of Ascaris.

The abundant larval transcript-1 and -2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis.

Lymphatic filariasis is a major tropical disease caused by the mosquito-borne nematodes Brugia and Wuchereria. About 120 million people are infected and at risk of lymphatic pathology such as acute lymphangitis and elephantiasis. Vaccines against filariasis must generate immunity to the infective mosquito-derived third-stage larva (L3) without accentuating immunopathogenic responses to lymphatic-dwelling adult parasites. We have identified two highly expressed genes, designated abundant larval transcript-1 and -2 (alt-1 and alt-2), from each of which mRNAs account for >1% of L3 cDNAs. ALT-1 and ALT-2 share 79% amino acid identity across 125 residues, including a putative signal sequence and a prominent acidic tract. Expression of alt-1 and alt-2 is initiated midway through development in the mosquito, peaking in the infective larva and declining sharply following entry into the host. Humans exposed to Brugia malayi show a high frequency of immunoglobulin G1 (IgG1) and IgG3 antibodies to ALT-1 and -2, distinguishing them from adult-stage antigens, which are targeted by the IgG4 isotype. Immunization of susceptible rodents (jirds) with ALT-1 elicited a 76% reduction in parasite survival, the highest reported for a single antigen from any filarial parasite. ALT-1 and the closely related ALT-2 are therefore strong candidates for a future vaccine against human filariasis.

Infection of IL-4-deficient mice with the parasitic nematode Brugia malayi demonstrates that host resistance is not dependent on a T helper 2-dominated immune response.

Resistance of intact mice to infection with the filarial nematode, Brugia malayi is dependent on the presence of T cells. To investigate the role of Th2 cells in this protection, mice with a targeted disruption of the IL-4 gene were infected with different developmental stages of B. malayi. We examined the phenotypic changes in the immune response and the survival of each stage in these mice. In wild-type mice, adult female worms induce Th2 responses, characterized by antigen-specific IgG1 production, elevated IgE, and marked IL-4 secretion by splenocytes stimulated in vitro with Brugia extract. However, first stage larvae (microfilariae), induce Th1 responses with the appearance of antigen-specific IgG2a, IgG2b, and IgG3 and IFN-gamma secretion by splenocytes. Infection of IL-4-deficient mice revealed a dramatic change in the response to adult worms, with a severe reduction in IgG1 production and a corresponding increase in the production of IgG2a, IgG2b, IgG3, and IFN-gamma release. The switch to Th1-type responses was particularly marked in IL-4-deficient recipients of female worms, which continually release live microfilariae. In the absence of IL-4, down-regulation of the microfilarial-induced Th1 response does not occur. Despite these profound alterations to the immune response in IL-4-deficient mice, survival of infective larvae, adult worms, or microfilariae in the peritoneal cavity was unaffected. In mice, therefore, the prominent Th2-type response elicited by filarial parasites may not be an essential component of the host protective immune response.

Circulating antibodies and antigens in Presbytis monkeys infected with the filarial parasite Wuchereria bancrofti.

Levels of circulating filarial antigen, and humoral antibody to other defined antigenic targets, were measured over the course of experimental infection of three Presbytis cristatus monkeys with Wuchereria bancrofti. Circulating antigen levels, measured with an anti-phosphorylcholine monoclonal antibody, varied widely although all animals were positive for some period of the infection. Circulating antigen levels tended to be inversely related to the titre of anti-phosphorylcholine antibody, and this trend was maintained even following acid dissociation and inactivation of immune complexed host antibody. Other antibody specificities were measured by Western blotting against somatic proteins and immunoprecipitation of surface antigens. Amongst somatic antigens, targets between 14,000 and 200,000 daltons were recognised by monkey antibodies, but no correlation with infection status could be discerned. Likewise when measuring the response to the 29,000 dalton major adult surface glycoprotein, one animal produced a rapid response but the others did not recognise it until late in infection. In general, the experimental findings are characterised by wide variability between individuals, as may perhaps be expected in a primate host for a human spectral disease.

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.

Onchocerca volvulus: characterization of an immunodominant hypodermal antigen present in adult and larval parasites.

Biochemical and immunological data suggest that a relatively limited number of polypeptide antigens of viable Onchocerca volvulus-infective larvae are available to be recognized by the host's immune system. A partial cDNA clone encoding one such antigen, designated lambda RAL-2, was isolated by screening an expression cDNA library with antisera raised against viable O. volvulus L3. The antigen encoded by this clone was subsequently found to be immunogenic in the majority of individuals exposed to O. volvulus. In the present study, the native antigen corresponding to lambda RAL-2 (Ov17) has been characterized. Immunolocalization and in situ hybridization techniques have been used to localize Ov17 in adult and larval stages of the parasite. In adult females, Ov17 was localized primarily in the hypodermis. Ov17 was accessible to surface labeling reagents in viable adult parasites. Full-length cDNA clones encoding Ov17 suggested that the nascent protein contains a putative leader sequence, which is almost immediately followed by a polyglutamine tract. Analysis of antibody reactivity to recombinant proteins containing and lacking the polyglutamine tract demonstrated that this structure was not a significant B cell epitope in individuals exposed to O. volvulus.