DOI: 10.14457/tu.the.2020.939
title: Isolation and characterization of anti-EV-A71 IRES scFv antibody

description: Enterovirus A71 (EV-A71) is one of the main causative agents of hand-foot-mouth disease which is occasionally associated with neurocomplications of the central nervous system and cardiopulmonary failure, particularly in young children. So far, effective and specific treatments have not been established yet. EV-A71 RNA genome carries type I internal ribosomal entry site (IRES) at 5ʹUTR. The type I is conserved sequence among members of Enterovirus genus. Essential roles on genomic translation of enterovirus IRES have been studied, however, the detailed mechanism has not been elucidated yet. A specific antibody acting as a chaperon for chaperon-assisted crystallography to obtain information on the biological interaction and structural organization, and mechanism of the EV-A71 RNA genome translation. Although the generation of antibodies against RNA is challenging because of intrinsic instability and less immunogenicity of RNA, modern technology of antibody phage display has approved in vitro selection and isolation of anti-RNA antibodies has become more accomplishable. In this study, the domains IV, V, and VI of EV-A71 IRES were selected as target antigens since they are structurally conserved among type I IRES enteroviruses and shared high sequence identity among EV-A71. The transcripts of domains IV, V, and VI of EV-A71 IRES were produced by T7 polymerase in vitro assay. The transcripts were labelled with biotin at 3ʹ end and used as antigen to select mouse single-chain variable fragment (muscFv) from the well-established naïve phage display library by solution-phase biopanning. Phage displaying muscFv clone number 16-3 (muscFv-phage #16-3) was chosen as it showed the distinguished ELISA signals to lysate extracted from EV-A71 infected cells. The phage-displayed-muscFv #16-3 was reformatted into a soluble free-molecule. The soluble form of muscFv #16-3 antibody specifically bound biotin-labelled domains IV, V, and VI of EV-A71 IRES but not type IV IRES of HCV 5ʹUTR as determined by northern blot analysis. It bound only the relaxed EV-A71 IRES target antigen but not a native form. It was indicated that the epitope bound by muscFv #16-3 might be a short stretch of nucleotides that are hidden upon the RNA folding. The soluble muscFv #16-3 antibody could detect EV-A71 infection as determined by immunofluorescence microscopy. The immunoglobulin domains prediction of the muscFv #16-3 sequence indicated it is a complete antigen-binding domain. A preference for serine (S), tyrosine (Y), and glycine (G) amino acid residues were observed in the regions of CDR-H1, CDR-H2, CDR-H3, and CDR-L3 in the deduced amino acid sequence of the muscFv #16-3. In the CDR-H3, positively charged amino acids including arginine (R) and lysine (K) were slightly enriched. These features have been mostly found in the anti-RNA antibodies; Fab fragments. It could be concluded that the muscFv #16-3 antibody specific to structurally conserved domains of EV-A71 type I IRES was successfully produced. It warrants further investigations to develop the muscFv #16-3 useful to study the RNA biology of enteroviruses and to use as an anti-viral therapeutic tool.