Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

Abstract

dodecyl sulfate; SOC, standard-of-care; STAT-C, specifically targeted anti-viral therapy for hepatitis C; SVR, sustained virologic response; VH, variable heavy chain domain of conventional four-chain IgG; V H H, variable heavy chain domain of heavy chain antibody; VL, variable light chain domainA new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.

A Nanobody Targeting Viral Nonstructural Protein 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication

“…CPPs provide an ideal means for intracellular delivery of the growing number of pharmaceutically relevant proteins. Previous studies reported that nanobodies fused with CPPs could pass through the cell membrane and interfere with hepatitis C virus replication (38)(39)(40).…”

Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

“…Cell penetrating ability of the PEN-nanobodies was determined by indirect ELISA and confocal microscopy as described previously [ 26 , 27 ]. The Huh7 cells were cultured in DMEM supplemented with 10% heat inactivated fetal calf serum (Hyclone, South Logan, TX, USA), penicillin (50 units/mL) and streptomycin (50 μg/mL).…”

“…CytoTox96® non-radioactive cytotoxicity (lactate dehydrogenase, LDH) assay kit (Promega, Madison, WI, USA) was used for determining toxicity of the HCV protease specific-transbodies [ 25 , 26 , 27 ]. The Huh7 cells were cultured as above; 2 × 10 4 cells were added to individual wells of a 96 well tissue culture plate and incubated at 5% CO 2 incubator at 37 °C for 24 h. The monolayer were washed twice with PBS before incubating with 25 µg PEN-V H Hs for 24 h. Cells incubated with medium alone and 10% SDS were included as negative and positive cytotoxic controls, respectively.…”

“…Genomic HCV RNA was produced from the pJFH-1 containing full-length cDNA of JFH-1 HCV genotype 2a (GenBank AB047639) as described previously [ 25 , 27 ]. The pJFH-1 was linearized with Xba I and one μg was subjected to transcription in vitro by using MEGAscript® T7 transcription kit (Ambion, Calsbad, CA, USA).…”

“…Total RNA was extracted from the culture supernatants and the transfected cells by using Trizol TM reagent (Invitrogen). Copy number of HCV 5' UTR (230 bp) in each sample was determined by quantitative real-time RT-PCR [ 25 , 27 ].…”