RESUMEN
Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Proteínas del Envoltorio Viral/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/genética , Especificidad de Anticuerpos/genética , Especificidad de Anticuerpos/inmunología , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Clonación Molecular , Humanos , Ratones , Estructura Terciaria de ProteínaRESUMEN
An elevated plasma fibrinogen level is an independent risk factor for cardiovascular disease. Therefore, an understanding of the regulation of fibrinogen expression is important. Inflammation and genetic variation of the fibrinogen beta gene regulate plasma fibrinogen levels, and there are indications that inflammation and genetic variation interact. The aim of our study was to gain more understanding of the regulation of the inflammatory response of the fibrinogen beta gene and to determine the effects of genetic variation. Luciferase reporter gene assays in hepatoma cells, mutation analysis, and electrophoretic mobility shift assays were used to investigate the transcriptional regulation of the fibrinogen beta promoter. We identified a hepatocyte nuclear factor-3 (HNF-3) site located just upstream of previously identified interleukin-6 (IL6)-responsive sequences. This HNF-3 site is essential for a full response of the promoter to IL6, which is a new function for HNF-3. The activity of the CCAAT box/enhancer-binding protein site (located 18 nucleotides downstream of the HNF-3 site and important to the IL6 response) depends on the integrity of the HNF-3 site and vice versa, explaining the necessity of HNF-3 in the IL6 response of the fibrinogen beta promoter. Furthermore, small interfering RNA to HNF-3 reduces the fibrinogen beta mRNA levels. The rare T allele of the -148C/T polymorphism, which is present between the binding sites of HNF-3 and CCAAT box/enhancer-binding protein, interferes with this mechanism, and this polymorphism is in our assay system the only genetic determinant of IL6-induced promoter activity among six polymorphisms in the fibrinogen beta promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fibrinógeno/química , Fibrinógeno/genética , Interleucina-6/biosíntesis , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Alelos , Secuencias de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Núcleo Celular/metabolismo , Análisis Mutacional de ADN , Cartilla de ADN/química , Proteínas de Unión al ADN/química , Relación Dosis-Respuesta a Droga , Electroforesis , Fibrinógeno/metabolismo , Genes Reporteros , Variación Genética , Vectores Genéticos , Factor Nuclear 3-alfa del Hepatocito , Factor Nuclear 3-beta del Hepatocito , Factor Nuclear 3-gamma del Hepatocito , Humanos , Inflamación , Interleucina-6/metabolismo , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/química , Unión Proteica , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Riesgo , Factores de Transcripción/química , Activación TranscripcionalRESUMEN
Antibody phage display technology was used to identify human monoclonal antibodies that neutralize rabies virus (RV). A phage repertoire was constructed using antibody genes harvested from the blood of vaccinated donors. Selections using this repertoire and three different antigen formats of the RV glycoprotein (gp) resulted in the identification of 147 unique antibody fragments specific for the RV gp. Analysis of the DNA sequences of these antibodies demonstrated a large variation in the heavy- and light-chain germ-line gene usage, suggesting that a broad antibody repertoire was selected. The single-chain variable fragment (scFv) antibodies were tested in vitro for RV neutralization, resulting in 39 specificities that neutralize the virus. Of the scFv clones, 21 were converted into full-length human IgG(1) format. Analysis of viral escape variants and binding competition experiments indicated that the majority of the neutralizing antibodies are directed against antigenic site III of the RV gp. The obtained specificities expand the set of human anti-RV antibodies eligible for inclusion in an antibody cocktail aimed for use in rabies post-exposure prophylaxis.