Antiviral Properties and Mechanism of Action Studies of the Hepatitis B Virus Capsid Assembly Modulator JNJ-56136379

Abstract

Capsid assembly is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the core protein. Core is a potential target for new antiviral therapies, the capsid assembly modulators (CAMs). JNJ-56136379 (JNJ-6379) is a novel and potent CAM currently in phase II trials. We evaluated the mechanisms of action (MOAs) and antiviral properties of JNJ-6379 in vitro Size exclusion chromatography and electron microscopy studies demonstrated that JNJ-6379 induced the formation of morphologically intact viral capsids devoid of genomic material (primary MOA). JNJ-6379 accelerated the rate and extent of HBV capsid assembly in vitro JNJ-6379 specifically and potently inhibited HBV replication; its median 50% effective concentration (EC₅₀) was 54 nM (HepG2.117 cells). In HBV-infected primary human hepatocytes (PHHs), JNJ-6379, when added with the viral inoculum, dose-dependently reduced extracellular HBV DNA levels (median EC₅₀ of 93 nM) and prevented covalently closed circular DNA (cccDNA) formation, leading to a dose-dependent reduction of intracellular HBV RNA levels (median EC₅₀ of 876 nM) and reduced antigen levels (secondary MOA). Adding JNJ-6379 to PHHs 4 or 5 days postinfection reduced extracellular HBV DNA and did not prevent cccDNA formation. Time-of-addition PHH studies revealed that JNJ-6379 most likely interfered with postentry processes. Collectively, these data demonstrate that JNJ-6379 has dual MOAs in the early and late steps of the HBV life cycle, which is different from the MOA of nucleos(t)ide analogues. JNJ-6379 is in development for chronic hepatitis B treatment and may translate into higher HBV functional cure rates.

Trial registration: ClinicalTrials.gov NCT03361956.

Keywords: capsid; capsid assembly modulator; cccDNA; hepatitis; hepatitis B virus; primary human hepatocytes.

FIG 1

Antiviral properties in HBV-infected PHHs. Cryopreserved primary human hepatocytes (PHHs) were infected with purified HBV (from stably HBV-replicating HepG2.2.15 cells). Compounds were either added together with the viral inoculum (day zero) or on day 4 or 5 postinfection. Infected cells were incubated with compounds for a total of 11 days, and the compounds replenished when the culture medium was changed. The antiviral properties of compounds were tested in dose-response assays. HBV DNA (extracellular) and HBV RNA (intracellular) levels were assessed using quantitative PCR (qPCR). Extracellular HBe/cAg and HBsAg were evaluated using AlphaLISA. Cytotoxicity was evaluated by assessing extracellular human albumin levels. The graphs show the results from a representative experiment. CPD, compound; GE, genome equivalents; D, day; HBe/cAg, hepatitis B e antigen and hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen.

FIG 2

Effect of JNJ-6379 on preformed mature capsids. HBV-replicating HepG2.117 cells were cultured for 3 days without doxycycline, and on day 4, cells were treated with JNJ-6379 (1:5 serial dilution at 5 different concentrations) or ETV for 30 min in the presence of doxycycline. Cell lysates were incubated with or without DNase I. HBV DNA was assessed by Southern blotting. DMSO, dimethyl sulfoxide; ETV, entecavir; rcDNA, relaxed circular DNA; ssDNA, single-stranded DNA; DSL, double-stranded linear DNA.

FIG 3

Effect of JNJ-6379 or ETV on viral particles. HBV-infected PHHs were incubated with compound from day 5, and samples were harvested on day 12. Extracellular and intracellular HBV DNA (Southern blot), HBV core (Western blot), and HBsAg (Western blot) were assessed. DMSO, dimethyl sulfoxide; ETV, entecavir; HBsAg, hepatitis B surface antigen.

FIG 4

Time-of-addition studies in HBV-infected PHHs. Cryopreserved primary human hepatocytes (PHHs) were infected with HBV (derived from HepG2.2.15 cell culture supernatant). Compounds were added at 0, 4, 8, 24, or 48 h postinfection at the concentration ranges shown and replenished as indicated. Cells were treated until day 11 postinfection. Extracellular HBV DNA levels and intracellular HBV RNA levels were quantified by qPCR. The HBe/cAg and HBsAg levels in the cell culture supernatant were detected by AlphaLISA. The data represent mean values from at least 2 replicates. Error bars show standard deviations. CPD, compound; D, day; HBe/cAg, hepatitis B e antigen and hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; hpi, hours postinfection; TDF, tenofovir disoproxil.

FIG 5

Time-of-addition studies in HBV-infected primary human hepatocytes (PHHs). Cryopreserved PHHs were infected with HBV. Compounds were added at 0, 4, 8, 24, or 48 h postinfection and replenished when culture medium was changed. Immunofluorescence analysis of intracellular HBsAg at the single-cell level was conducted on day 12. Automated image analysis was used to determine the number of infected cells. The mean percentage of intracellular HBsAg-positive cells is listed for each compound and time point (mean of 2 wells/condition and compound). CPD, compound; D, day; hpi, hours postinfection; TDF, tenofovir disoproxil.

FIG 6

HBV cccDNA Southern blotting. HBV-infected primary human hepatocytes (PHHs) were treated with compounds either together with the viral inoculum (day zero) (top) or on day 4 or 5 postinfection (bottom). Ten-microgram amounts of DNA extracted using the Hirt method (30) were subjected to agarose gel electrophoresis and Southern blotting to detect HBV cccDNA with an HBV-specific probe. HBV cccDNA contains a single EcoRI restriction site that can be used for linearization. cccDNA, covalently closed circular DNA; D, day; DMSO, dimethyl sulfoxide; ETV, entecavir; rcDNA, relaxed circular DNA; TDF, tenofovir disoproxil.