RESUMEN
UNLABELLED: Dengue virus (DENV) infection is an arthropod-borne disease with increasing prevalence worldwide. Attempts have been made to develop therapeutic molecules for treatment for DENV infection. However, most of potentially therapeutic DENV monoclonal antibody was originated from mouse, which could cause undesirable effects in human recipients. Thus, fully human antibody is preferable for therapeutic development. Human single-chain variable fragments (HuScFv) with inhibitory effect to DENV infection were generated in this study. HuScFv molecules were screened and selected from the human antibody phage display library by using purified recombinant DENV full-length envelope (FL-E) and its domain III (EDIII) proteins as target antigens for biopanning. HuScFv molecules were then tested for their bindings to DENV particles by indirect ELISA and immunofluorescent microscopy. EDIII-specific HuScFv exhibited neutralizing effect to DENV infection in Vero cells in a dose-dependent manner as determined by plaque formation and cell ELISA. Epitope mapping and molecular docking results concordantly revealed interaction of HuScFv to functional loop structure in EDIII of the DENV E protein. The neutralizing HuScFv molecule warrants further development as a therapeutic biomolecule for DENV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: No approved vaccine and specific drug for dengue virus (DENV) infection are available; thus, their developments are urgently required. The human single-chain variable antibody fragments (HuScFv) specific to DENV envelope (E) protein are potential to be developed as therapeutic biomolecules. HuScFv that bound specifically to recombinant full-length DENV E (FL-E) and its domain III (EDIII) were generated and testified for its inhibitory effect in DENV infection. EDIII-specific HuScFv inhibited DENV infection in a dose-dependent manner and has potential to be further developed as a therapeutic biomolecule for DENV infection.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Anticuerpos de Cadena Única/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/administración & dosificación , Chlorocebus aethiops , Dengue/inmunología , Dengue/prevención & control , Dengue/virología , Virus del Dengue/genética , Mapeo Epitopo , Humanos , Inmunización Pasiva , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/genética , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Influenza A virus (IAV) subtype H5N1 is associated with the re-emergence of severe human influenza. The virus is highly virulent and viral non-structural protein 1 (NS1) is believed to play a crucial role in the viral pathogenesis. A screening for human proteins interacting with NS1 was performed by a yeast two-hybrid system (Y2H). Two bait plasmids that expressed DNA binding domain (BD) fused to either RNA binding domain or to effector domain of NS1 were constructed and transformed into yeast. The bait yeast was mated with a prey yeast containing human macrophage cDNA library fused to DNA activation domain. Obtained clones were interacting with interleukin-6 receptor (IL-6R), MHC class I HLA-B, cathepsin B, ubiquitin, and adenosine deaminase acting on RNA (ADAR1). These proteins play important role in the immune response, targeting for proteosomal degradation, and RNA editing. Thus, IAV H5N1 may use NS1 to manipulate these host proteins for its own benefit and in that way confer an unusual severity to the infection.