RESUMO
The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP-VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.
Assuntos
Antivirais/farmacologia , Filoviridae/fisiologia , Nucleoproteínas/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Sequência Conservada , Avaliação Pré-Clínica de Medicamentos , Filoviridae/efeitos dos fármacos , Filoviridae/genética , Polarização de Fluorescência , Ensaios de Triagem em Larga Escala , Técnicas In Vitro , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.
Assuntos
Eletroporação , Filoviridae/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença do Vírus de Marburg/prevenção & controle , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Códon , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Filoviridae/genética , Glicoproteínas/genética , Glicoproteínas/imunologia , Imunoglobulina G/sangue , Injeções Intramusculares , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Macaca fascicularis , Masculino , Testes de Neutralização , Plasmídeos , Análise de Sobrevida , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Filoviruses cause severe hemorrhagic fevers with case fatality rates of up to 90%, for which no antivirals are currently available. Their categorization as biosafety level 4 agents restricts work with infectious viruses to a few maximum containment laboratories worldwide, which constitutes a significant obstacle for the development of countermeasures. Reverse genetics facilitates the generation of recombinant filoviruses, including reporter-expressing viruses, which have been increasingly used for drug screening and development in recent years. Further, reverse-genetics based lifecycle modeling systems allow modeling of the filovirus lifecycle without the need for a maximum containment laboratory and have recently been optimized for use in high-throughput assays. The availability of these reverse genetics-based tools will significantly improve our ability to find novel antivirals against filoviruses.