RESUMEN
Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-borne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independent regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDa tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-borne encephalitis virus, determination of domain C was more problematic; however, at least four epitopes had biochemical characteristics consistent with C-domain epitopes.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Virus del Dengue/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Virales/química , Sitios de Unión , Unión Competitiva , Línea Celular , Epítopos de Linfocito B/química , Pruebas de Inhibición de Hemaglutinación , Humanos , Jamaica , Masculino , Fusión de Membrana , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Pruebas de Neutralización , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Mapeo Peptídico , Conformación Proteica , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/químicaRESUMEN
Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (Mabs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-bourne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independently regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus haemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDA tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-bourne encephalitis virus, determination of domain C was more problematic: however, at least four epitopes and biochemical characteristics consistent with C-domain epitopes(AU)
Asunto(s)
21003 , Humanos , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Virus del Dengue/inmunología , Mapeo Epitopo , Proteínas del Envoltorio Viral/inmunología , Pruebas de Inhibición de Hemaglutinación , Jamaica , Fusión de Membrana , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Pruebas de Neutralización , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Mapeo Peptídico , Conformación Proteica , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/química , Anticuerpos Monoclonales/inmunología , Antígenos Virales/química , Sitios de Unión , Unión Competitiva , Línea CelularRESUMEN
We used previously characterized murine monoclonal antibodies to develop a panel useful in subtyping Venezuelan equine encephalitis (VEE) viruses by an indirect fluorescent antibody assay. This panel worked well with either prototype VEE viruses or a series of more recent VEE virus isolates. The panel is particularly useful for rapidly differentiating VEE viruses with epidemic-epizootic potential from other endemic varieties of this virus. Using this panel, we identified an antigenic variant of prototype VEE subtype 1E virus currently present in Mexico. This antigenic change in the E2 glycoprotein was confirmed by enzyme-linked immunosorbent assay. Because VEE virus virulence has been associated in part with the E2 glycoprotein, this observed antigenic change in the 1E virus E2 glycoprotein may explain the apparent equine virulence of this unusual VEE 1E virus.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Encefalitis Equina Venezolana/aislamiento & purificación , Encefalomielitis Equina Venezolana/virología , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Animales , Virus de la Encefalitis Equina Venezolana/inmunología , Caballos , HumanosRESUMEN
A peptide composed of the amino-terminal 25 amino acids of the E2 glycoprotein of the virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalomyelitis virus was found to protect peptide-immunized mice from lethal TRD virus challenge (Hunt et al., 1990). Viral growth in peptide-immunized animals was found to be limited in comparison to that in nonimmunized controls. Although both treated and control groups of mice responded to virus challenge by producing neutralizing antibody, only immunized mice with preexisting antipeptide antibody survived. Polyclonal antipeptide sera as well as a monoclonal antipeptide antibody were able to passively protect naive mice from TRD virus challenge, despite the fact that these antibodies were nonneutralizing. Passive transfer of antipeptide antibody to immunosuppressed recipients was not protective, thus indicating that survival of TRD virus challenge required an in situ immune response as well as preexisting antipeptide antibody. Binding studies of both polyclonal and monoclonal antipeptide antibodies indicated that they recognize only epitopes present on virus-infected cells or denatured virus.
Asunto(s)
Anticuerpos Antivirales , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/inmunología , Péptidos/síntesis química , Replicación Viral , Animales , Formación de Anticuerpos , Complejo Antígeno-Anticuerpo/análisis , Línea Celular , Virus de la Encefalitis Equina Venezolana/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Pruebas de Inhibición de Hemaglutinación , Caballos , Inmunización Pasiva , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Ratones , Ratones Endogámicos , Péptidos/inmunologíaRESUMEN
We have prepared a murine monoclonal antibody (MAb) capable of distinguishing between wild-type Venezuelan equine encephalomyelitis (VEE) virus and the TC-83 vaccine derivative. This MAb, 1A2B-10, was derived from immunization with a synthetic peptide corresponding to the first 19 amino acids of the E2 glycoprotein of Trinidad donkey VEE virus. The MAb reacts with prototype viruses from all naturally occurring VEE subtypes except subtype 6 in an enzyme-linked immunosorbent assay. It does not react with TC-83 virus or members of the western and eastern equine encephalitis virus complex or with Semliki Forest virus. This antibody will also differentiate between TC-83 and Trinidad donkey VEE virus in indirect immunofluorescence assays with virus-infected Vero cells.