Monoclonal Antibodies against Extracellular Domains of Claudin-1 Block Hepatitis C Virus Infection in a Mouse Model

Abstract

Hepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1).Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant that is nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (MAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All MAbs produced by these hybridomas specifically bound to human CLDN1 with a very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected MAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infection in vivo. Anti-CLDN1 MAbs may hence be promising candidates as novel anti-HCV agents. IMPORTANCESafe and effective therapeutic entry inhibitors against hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs, such as direct-acting antivirals. In this study, we first showed an effective strategy for developing functional monoclonal antibodies (MAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), by using parental cells expressing the intact target membrane protein and target-defective cells. The established MAbs against CLDN1, which had a very high affinity for intact CLDN1, efficiently inhibited in vitro and in vivo HCV infections. These anti-CLDN1 MAbs are promising leads for novel entry inhibitors against HCV. W orldwide, 170 million people are infected with hepatitis C virus (HCV), which is a major cause of liver cirrhosis and hepatocellular carcinoma. Thus, overcoming HCV infection is an important global health care issue (1). HCV is an enveloped, positive-sense, single-stranded RNA virus in the Flaviviridae family (2). Recent clinical research using direct-acting antivirals that target HCV enzymes, such as sofosbuvir and simeprevir, has provided new insights into combination therapy with inhibitors of multiple targets (3-5).Preventing viral entry into hepatocytes is an attractive target for anti-HCV agents, but strategies for preventing HCV entry into host cells are clinically unavailable (6). Host factors involved in initiating infection include heparan sulfate (7), low-density lipoprotein receptor (8), CD81 (9), scavenger receptor class B type I (SRBI) (10), claudin-1 (CLDN1) (11), occludin (12, 13), epidermal growth factor receptor (EGFR) (14), and Niemann-Pick C1-like 1 (15). Among these, CLDN1 is considered a potent target because it is essential for HCV entry into cells via interaction with CD81 and for cell-to-cell HCV transmission (16,17). Anti-CLDN1 antibodies (Abs) that inhibit HCV infection in vitro were reported by Baumert et al. (18,19) and Hötzel et al. (20), but a CLDN1 binder that prevents HCV infection in vivo has not...

Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection

“…9) We have isolated various hepatic cell mutants resistant to HCV infection, among which CD81-defective 751r cells and CLDN1-defective S7-A cells have been established as clones that are non-permissive to HCV infection. 10,11) Using genetic approaches with these mutant cells, we have confirmed that CD81 and CLDN1 are essential for HCV entry into hepatic cells, which is consistent with other studies. 6,7,12) There is as yet no conclusive evidence for the necessity of OCLN for HCV infection of hepatic cells, although OCLN has been shown by RNA interference (RNAi) experiments to be importantly involved in the infection.…”

“…[21][22][23] Detection of HCV infection was performed using quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemistry as previously described. 10,11) HCV Pseudoparticle Infection HCV pseudoparticles (HCVpp) were generated as previously described. 24) Briefly, a Gag-Pol packaging construct (Gag-Pol 5349), a transfer vector construct (Luc 126), and an envelope glycoprotein (E1 and E2)-expressing vector [H77, genotype 1a (GenBank accession number JX472009.1); TH, genotype 1b (GenBank accession number AB985268.1); J6, genotype 2a (GenBank accession number AB047639); JFH1, genotype 2a (GenBank accession number AB047639.1); or VSV-G (GenBank accession number M27165)] were transfected into HEK 293T cells.…”

Novel human anti-claudin 1 mAbs inhibit hepatitis C virus infection and may synergize with anti-SRB1 mAb

“…The ability of anti-CLDN-1 human mAbs to inhibit infection by different HCV isolates was not unexpected and was in line with previous observations made with rat and mouse anti-CLDN-1 antibodies (Fofana et al, 2010;Fukasawa et al, 2015;Mailly et al, 2015;Yamashita et al, 2015). Most importantly, mAbs B9X and D10X displayed binding affinity for cell-displayed CLDN-1 and IC 50 values comparable with the rat anti-CLDN-1 mAb OM-7D3, which was recently shown to prevent HCV infection and to clear persistent infection in a human liver-chimeric mouse model without detectable toxicity .…”

Strategies to Preclude Hepatitis C Virus Entry

“…Further experiments in human liver-chimeric mouse models confirmed its potency in preventing HCV infection and eliminating persistent infection in vivo [86]. Pretreatment of another anti-CLDN1 mAb 3A2 targeting CLDN1 extracellular loop also showed protective effect in a chimeric mouse model [87]. Safety profiles of these antibodies were also assessed regarding the levels of human albumin, aspartate transaminase, alanine transaminase and total bilirubin, and potential side effects on the other organs and tight junction integrity.…”

“…Safety profiles of these antibodies were also assessed regarding the levels of human albumin, aspartate transaminase, alanine transaminase and total bilirubin, and potential side effects on the other organs and tight junction integrity. Further studies were suggested to assess potential immune-mediated adverse effects to ensure its relevance for clinical use [86,87].…”