International research efforts developed recommendations to optimize, control, and validate quantitative viral DNA measurements of chronic hepatitis B in the liver.
Worldwide, more than 300 million people are chronically infected with hepatitis B virus (HBV). Chronic HBV, an infection that lasts 6 months or longer, is not susceptible to hepatitis B vaccination. People living with chronic HBV are at high risk of liver cirrhosis or hepatocellular carcinoma, and in urgent need of new therapies.
Supported by the International Coalition to Eliminate HBV (ICE-HBV), investigators from the “German Center for Infection Research” (DZIF) have been developing recommendations to optimize, control, and validate quantitative viral DNA measurements.
DZIF scientists from the University Medical Center Hamburg-Eppendorf, Heidelberg University, and the Technical University of Munich collaborated with the French L'Agence nationale de recherches sur le sida et les hépatites virales (ANRS) and Gilead Sciences to establish protocols and best practices to quantify covalently closed circular DNA (cccDNA) through PCR-based testing.
A primary HBV treatment objective is to reduce or inactivate intrahepatic viral cccDNA, making cccDNA quantification vital in clinical studies. After HBV infection, the virus’s DNA genome is converted into cccDNA by cellular enzymes. In this stable form, the viral DNA is mimicked by new HBV particles
“A major limitation of preclinical and clinical HBV research is the lack of standardized PCR-based methods for the specific quantification of viral DNA present as cccDNA in HBV-infected samples,” the DZIF scientists said in a statement.
The variety of samples, preservation conditions, DNA extraction, nuclease digestion methods, and qPCR strategies has complicated viral DNA standardization. Thus, the ICE-HCV consortium utilized 6 laboratories to compare protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures. The resulting evidence-based guidelines were recently published in the BMJ journal Gut.
The investigators exchanged reference materials, including HBV-infected humanized mouse livers and HepG2-NTCP cells, for cross-validation. The scientists compared different DNA extraction methods and nuclease digestion protocols to observe reduction of viral replicative intermediates (RI).
For DNA extraction, each group of investigators compared Hirt extraction versus total DNA extraction with or without proteinase K treatment (+PK/-PK). For nuclease digestion protocols, they compared plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, and exonucleases I/III.
The results showed that Hirt and -PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by quantitative PCR (qPCR) testing.
T5 and Exo I/III nucleases eliminated all RI forms. Comparatively, PSD did not digest pf-rcDNA, though it was less susceptible to cccDNA overdigestion. In stabilized tissues, nucleases negatively affected cccDNA.
After their research, the DZIF investigators were confident their informed guidance would optimize, control, and validate cccDNA measurements calculated with available qPCR assays. “The study will assist the HBV cure research programs aiming at assessing the impact of therapies on the cellular HBV reservoir in preclinical studies and clinical trials,” said lead investigator Maura Dandri, PhD, a DZIF scientist at the University Medical Center Hamburg-Eppendorf.