RESUMEN
Filarial parasites release macromolecules into their environment both in vitro and in vivo. These excretory-secretory products (E-S) have been studied with respect to function, vaccination potential, pathogenicity, and ability to serve as antigen targets for diagnostic tests. We have recently described monoclonal antibody OV-1 which binds to an intermediate filament in E-S and circulating antigens of Onchocerca volvulus. OV-1 also binds to cross-reactive antigens of Brugia malayi. Therefore, OV-1 was used to immunoscreen a B. malayi adult worm cDNA library in an attempt to clone a homologue (BMIF). BMIF is a 1664-bp full-length transcript which codes for 505 amino acids. BMIF has 95% sequence homology at the amino-acid level to OV1CF, an O. volvulus intermediate filament that was also selected with OV-1, and 75% homology to Ascaris intermediate filament A. Southern blot analysis suggests that BMIF is confined to a single location in the genomic DNA of B. malayi. Antibodies raised to BMIF identified native antigens in immunoblots of B. malayi adult worms, infective larvae and adult E-S. In addition, the antibody also bound to a 60-kDa antigen in immunoblots of poly(ethylene glycol)-precipitated immune complexes in sera from B. malayi infected patients. Localization studies showed that the antigen encoded by BMIF is present in the hypodermis, developing embryos and muscle of adult B. malayi. These studies show that BMIF is an E-S product of B. malayi adult worms which is detectable in sera from patients with brugian filariasis.
Asunto(s)
Brugia Malayi/genética , Brugia Malayi/fisiología , Proteínas del Helminto/genética , Proteínas del Helminto/fisiología , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/genética , Antígenos Helmínticos/fisiología , Secuencia de Bases , Brugia Malayi/inmunología , Clonación Molecular , ADN Complementario/genética , ADN de Helmintos/genética , Femenino , Filariasis/inmunología , Proteínas del Helminto/inmunología , Humanos , Proteínas de Filamentos Intermediarios/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Immunological crossreactivity among nematodes has hampered development of specific serodiagnostic assays for lymphatic filariasis. In the present study, we report the molecular cloning and characterization of two filaria-specific recombinant clones (BmM5 and BmM14) with immunodiagnostic potential. BmM5 is a 505-bp cDNA which codes for a protein of 130 residues that ends with an endoplasmic reticulum targeting sequence. BmM14 is closely related to a recently reported clone (SXP-1), and it has 62% homology (deduced amino acid sequence) with a previously described Onchocerca volvulus clone, lambda RAL-2. Glutathione S-transferase fusion proteins of BmM5 and BmM14 were tested in various ELISA formats. The best results were obtained by measuring IgG4 antibodies to the fusion proteins. ELISA studies showed that approximately 90% of 111 sera from Indian and Egyptian patients with brugian and bancroftian filariasis were reactive with both antigens. Nonendemic sera as well as sera from patients with schistosomiasis or intestinal helminths were uniformly nonreactive. Assays based on BmM5 and BmM14 may be useful for large scale screening as an alternative to microfilaria or filarial antigen detection as a means of obtaining a rough index of filariasis endemicity in previously unstudied areas.
Asunto(s)
Antígenos Helmínticos/genética , Brugia Malayi/genética , Brugia Malayi/inmunología , Filariasis Linfática/diagnóstico , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/sangre , Clonación Molecular , ADN de Helmintos/genética , Filariasis Linfática/inmunología , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Vectores Genéticos , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Pruebas Inmunológicas , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Homología de Secuencia de AminoácidoAsunto(s)
Antioxidantes , Proteínas de Neoplasias , Onchocerca volvulus/genética , Peroxidasas/genética , Peroxidasas/metabolismo , Secuencia de Aminoácidos , Animales , Antioxidantes/química , Clonación Molecular , ADN Superhelicoidal/metabolismo , Genes de Helminto , Datos de Secuencia Molecular , Onchocerca volvulus/enzimología , Peroxidasas/química , Peroxirredoxinas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Onchocerca volvulus is a nematode that causes severe dermatitis and blindness in humans. Prior studies have shown that immune complexes in sera from onchocerciasis patients contain parasite antigens with M(r) of 23,000 and 65,000-70,000. Monoclonal antibody OV-1 binds to these antigens and to corresponding antigens in adult worm extracts and in vitro culture supernatants. OV-1 was used to immunoscreen an O. volvulus adult worm cDNA library. Clone OV1CF contains a 1632-bp open-reading frame that codes for a protein with a predicted M(r) of 63,000. The deduced protein sequence of OV1CF has 78% identity with Ascaris lumbricoides intermediate filament A and 40%-50% identity with several mammalian intermediate filaments. Antibodies raised to OV1CF bind to human keratin, and some sera from onchocerciasis patients contain antibodies to OV1CF and to keratin. Thus, immune complexes from onchocerciasis patients contain a parasite antigen that is immunologically cross-reactive with human intermediate filament proteins.
Asunto(s)
Queratinas/inmunología , Oncocercosis/inmunología , Adolescente , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/inmunología , Niño , Clonación Molecular , Reacciones Cruzadas , ADN Complementario/genética , Humanos , Proteínas de Filamentos Intermediarios/genética , Datos de Secuencia Molecular , Onchocerca volvulus/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Piel/inmunologíaRESUMEN
Immunization with recombinant Brugia malayi paramyosin protein (BM5) induces partial immunity to this filarial nematode in jirds. The present study examined the effects of intramuscular immunization with plasmid DNA that encodes BM5. DNA-immunized mice produced strong antibody and cell-mediated responses to paramyosin. The protective activity of DNA vaccination with BM5 was tested in jirds. Vaccinated jirds produced strong antibody responses to paramyosin, but adult worm recoveries after challenge were not decreased in vaccinated animals relative to controls. These studies show that DNA vaccination can induce immune responses to filarial antigens in rodents. Further efforts will be needed to achieve the goal of inducing protective immunity to filariasis with this promising new technology.
Asunto(s)
Brugia Malayi/inmunología , Proteínas del Helminto/inmunología , Tropomiosina/inmunología , Vacunas de ADN/inmunología , Vacunas Sintéticas/inmunología , Animales , Gerbillinae , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Plásmidos , Proteínas Recombinantes/inmunología , VacunaciónRESUMEN
Immunological cross-reactivity among fungi has hampered the development of specific serodiagnostic assays for histoplasmosis. We report the molecular cloning and characterization of a Histoplasma capsulatum cDNA (GH17) that encodes an antigen with immunodiagnostic potential. GH17 is an 810-bp cDNA which encodes a protein of 211 amino acid residues. The GH17 sequence has almost no significant homology with other sequences in GenBank. Southern blot analysis suggests that GH17 is confined to a single location in the genomic DNA of H. capsulatum. Immunoblots indicated that the protein product of GH17 (expressed as a 140-kDa beta-galactosidase fusion protein) was recognized by antibodies in 18 of 18 sera from histoplasmosis patients, but not by antibodies in sera from patients or animals infected with other fungi. GH17 was expressed in a prokaryotic expression vector, pPROEX-1, and recombinant protein was purified by preparative electrophoresis. Antibodies raised to this protein bound to a 60-kDa native antigen in immunoblots of H. capsulatum yeast antigen extract. These results suggest that GH17 encodes an H. capsulatum antigen that may be useful for the diagnosis of histoplasmosis in humans.