Modeling of hepatitis B virus kinetics and accumulation of cccDNA in primary human hepatocytes

Abstract

Background & Aims

Knowledge about early HBV covalently closed circular DNA (cccDNA) accumulation post infection is lacking. We characterized and mathematically modeled HBV infection kinetics during early infection and treatment in primary human hepatocytes (PHHs).

Methods

PHHs were inoculated with HBV, and infection was monitored with and without treatment with the nucleoside analog entecavir (ETV), the HBV-entry inhibitor Myr-preS1, or both ETV + Myr-preS1. Extracellular HBV DNA (exHBV), total intracellular HBV DNA (inHBV), and cccDNA were frequently measured during the 12 days post inoculation. A multicompartmental mathematical model was developed to explain HBV infection dynamics.

Results

Multiphasic exHBV and inHBV kinetics were overall similar in untreated and Myr-preS1-treated PHHs. In ETV-treated PHHs (either alone or with Myr-preS1), exHBV and inHBV initially declined and did not resurge. ETV-treated cultures had significantly (p=0.002) lower mean cccDNA levels at Day 2 post inoculation (4.3 ± 0.1 vs. 4.7 ± 0.1) and reached plateau slower (5 vs. 2 days) than untreated and Myr-preS1-treated cultures, respectively. Modeling predicts that the recycling of inHBV into cccDNA stops when cccDNA reaches a maximum and HBV secretion changes depending on the concentration of inHBV. Even when initiated at the time of inoculation, ETV did not prevent or eradicate infection but rather blocked inHBV accumulation gradually, reaching 97% efficacy by the end of the 12-day experiment and resulting in an average 44% slower cccDNA accumulation.

Conclusions

The study provides insight into the interrelationships and dynamics of cccDNA accumulation, inHBV accumulation, and secretion of exHBV containing particles. Although kinetics and modeling support the conclusion that the level of cccDNA in the cell is regulated, the mechanisms that determine HBV capsid secretion vs. recycling to the nucleus for cccDNA accumulation require further investigation.

Impact and implications:

Using primary human hepatocytes (PHHs), we characterize early HBV kinetics post infection. HBV-infected PHH were treated with an entry inhibitor to characterize the accumulation of intracellular and extracellular HBV DNA and the nuclear episomal viral genome called covalently closed circular DNA (cccDNA) in the absence of HBV spread. Kinetic and mathematical modeling in PHHs confirms that the replication inhibitor, entecavir, strongly blocks intracellular HBV DNA accumulation, which also slowed the accumulation of cccDNA. However, modeling indicates that effects on cccDNA accumulation are not directly determined by intracellular HBV DNA levels, supporting the conclusion that cccDNA levels within the cell are regulated in some yet-to-be-elucidated manner.