RESUMO
The threat of West Nile virus (WNV) epidemics necessitates the development of a technology platform that can produce reagents to support detection and diagnosis rapidly and inexpensively. A plant expression system is attractive for protein production due to its low-cost and high-scalability nature and its ability to make appropriate posttranslational modifications. Here, we investigated the feasibility of using plants to produce two WNV detection and diagnostic reagents to address the current cost and scalability issues. We demonstrated that WNV DIII antigen and E16 monoclonal antibody are rapidly produced at high levels in two plant species and are easily purified. Furthermore, they are effective in identifying WNV and in detecting human IgM response to WNV infection. E16 mAb does not cross-react with other flaviviruses, therefore, is valuable for improving diagnostic accuracy. This study provides a proof of principle for using plants as a robust and economical system to produce diagnostic reagents for arboviruses.
Assuntos
Anticorpos Monoclonais/biossíntese , Antígenos Virais/biossíntese , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/biossíntese , Febre do Nilo Ocidental/diagnóstico , Vírus do Nilo Ocidental/isolamento & purificação , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Antígenos Virais/genética , Antígenos Virais/isolamento & purificação , Estudos de Viabilidade , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Lactuca/química , Lactuca/genética , Lactuca/metabolismo , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Nicotiana/química , Nicotiana/genética , Nicotiana/metabolismo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope protein has been established.