Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

Abstract

The hepatitis C virus (HCV) NS3 protein possesses both protease and helicase activities and is essential for virus replication and maturation. Specific inhibition of NS3 enzymatic activity can be achieved by antibody binding. Transduction of hepatocytes with encoding cDNA leading to intracellular expression of antibody fragments is expected to terminate HCV replication in infected cells. The objective of the present study was the generation of human antibody fragments that neutralize the viral NS3 helicase activity for gene therapeutic applications and drug design. A human immunoglobulin phage-display library cloned from bone marrow aspirate of patients infected with HCV was used for affinity selection against HCV NS3 helicase. Antibody fragments with high affinity to HCV helicase were isolated. To evaluate the inhibitory potential of isolated single-chain antibody fragments, a helicase-mediated, DNA-unwinding enzymatic assay was developed in ELISA format. Recombinant protein comprising the full-length HCV NS3 helicase domain was expressed in the baculovirus expression system. Recombinant antibodies that inhibit the HCV helicase at nanomolar concentrations, with efficacies ranging from 20 % to complete abrogation of enzymatic unwinding activity, were identified. These antibody fragments may be useful for novel gene therapeutic strategies that employ intracellular immunization and may provide new insights into the design of small molecule inhibitors of essential HCV proteins.

Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus

“…Since the helicase domain of HCV NS3 is probably crucial for the proliferation of HCV, identification of inhibitors for the helicase has been employed mainly through random screening of small molecules (Borowski et al 2003) or phage-display antibody screening (Artsaenko et al 2003). However, potential limitations of those molecules would stem from nonspecificity or low affinity to the target protein.…”

RNA helicases in infection and disease

“…Screening of chemical libraries has identified limited numbers of weak helicase inhibitors. 212 HCV helicase activity is inhibited in vitro by recombinant antibodies, 213,214 by RNA aptamers 215,216 and by peptides. 217,218 Targeting the nucleotide binding site of HCV-NS3 has proven exceedingly difficult, 219 although nucleotide-mimicking competitive inhibitors have been identified.…”

Understanding Helicases as a Means of Virus Control

“…Once antibodies are isolated with a high avidity for a desired antigen, a gene encoding that antibody can be cloned and introduced into infected cells. This process, called "cellular immunization, " has been used successfully with HIV [200], and such an approach has been used to target the HCV helicase [201][202][203].…”

“…A ScF is composed of the immunoglobulin variable domains, of both the heavy and light chains, connected by a polypeptide linker, and can be constructed using PCR. Several high affinity ScFs that specifically interact with HCV NS3 helicase have been cloned [201,204] and shown to inhibit HCV helicase catalyzed DNA unwinding [202,204]. One ScF has been expressed in HCV infected hepatocytes and shown to reduce HCV RNA synthesis [204].…”