Detection of HBV Covalently Closed Circular DNA

Abstract

Chronic hepatitis B virus (HBV) infection affects approximately 240 million people worldwide and remains a serious public health concern because its complete cure is impossible with current treatments. Covalently closed circular DNA (cccDNA) in the nucleus of infected cells cannot be eliminated by present therapeutics and may result in persistence and relapse. Drug development targeting cccDNA formation and maintenance is hindered by the lack of efficient cccDNA models and reliable cccDNA detection methods. Southern blotting is regarded as the gold standard for quantitative cccDNA detection, but it is complicated and not suitable for high-throughput drug screening, so more sensitive and simple methods, including polymerase chain reaction (PCR)-based methods, Invader assays, in situ hybridization and surrogates, have been developed for cccDNA detection. However, most methods are not reliable enough, and there are no unified standards for these approaches. This review will summarize available methods for cccDNA detection. It is hoped that more robust methods for cccDNA monitoring will be developed and that standard operation procedures for routine cccDNA detection in scientific research and clinical monitoring will be established.

Hepatitis B Core Antibody: Role in Clinical Practice in 2020

“…In ongoing HBV infection, anti-HBc is detected together with HBsAg. While HBsAg may disappear, anti-HBc IgG commonly persists for life even in "resolved" HBV infection [22], probably due to the persistence of HBV in the form of cccDNA and low levels of core antigen production stimulating host B cells [27][28][29]. Thus, anti-HBc is useful for population-based HBV screening to determine exposure rates, and should be part of HBV testing panels for all patients in any population deemed at risk by current WHO or CDC guidelines Fig.…”

The Role of Infected Cell Proliferation in the Clearance of Acute HBV Infection in Humans

“…An inefficient and weak immune response is associated with failure to clear acute HBV infection and results in chronicity of HBV infection [ 84 , 85 , 86 ]. However, the clearance of acute HBV infection in humans is intriguing as: (i) HBV infects nearly all hepatocytes at the peak of acute infection [ 4 , 22 , 55 , 56 ], (ii) infected hepatocytes contain at least one cccDNA molecule, but possibly more due to the transport of relaxed circular DNA (rcDNA) to the nucleus [ 25 , 87 , 88 ], (iii) template (cccDNA) has a long half-life of around 7–8 weeks [ 57 ], and (iv) there is limited HBV-specific CTL precursor frequency which can clear HBV infection [ 4 ]. And, yet clearance occurs in 8 to 12 weeks post-peak of infection [ 54 , 55 ].…”

Clinical Implications of Hepatitis B Virus RNA and Covalently Closed Circular DNA in Monitoring Patients with Chronic Hepatitis B Today with a Gaze into the Future: The Field Is Unprepared for a Sterilizing Cure

“…Eliminating cccDNA could be the primary goal of antiviral therapy and one of the most important biomarkers for predicting and monitoring patients’ response to antiviral therapy, as well as assessing the risk of liver disease progression. The major obstacle to monitoring cccDNA in these settings is the lack of sensitive and unambiguous methods to detect cccDNA, which remains one of the biggest challenges in the field of hepatology [ 56 ]. All modern tools are based on detecting cccDNA in liver biopsy specimens [ 60 ], representing a small piece of the liver in which cccDNA could be extremely or even undetectably low in CHB patients, especially those that are undergoing antiviral therapy [ 97 ].…”

“…The much more sensitive PCR-based techniques (real-time PCR [ 186 ], droplet-digital PCR [ 60 ], competitive PCR, and Invader Assay) [ 187 ], coupled with novel approaches for cccDNA isolation and enrichment (modified Hirt procedure [ 188 ], T5/PSAD DNase digestion of linear and chromosome DNA [ 189 , 190 ], and magnetic separation of cccDNA [ 191 ]) are among the available tools for quantifying cccDNA from liver biopsies with a certain degree of confidence [ 56 , 69 ]. Advantages and downsides of these techniques have been described elsewhere [ 56 , 192 ], but the common concern is that they might underrepresent cccDNA levels or amplify other forms of the HBV genome, such as mature rcDNA particles or HBV DNA integrants, along with cccDNA. Recent technological developments provided an exciting opportunity to visualize cccDNA templates in specially prepared tissue samples and cell cultures, including fluorescent in situ hybridization (FISH) detection of cccDNA [ 61 , 134 ].…”

“…It is thus essential to have practical and sensitive methods for defining the point of sterilizing cure, that is, providing evidence that HBV infection is resolved and the virus is completely eliminated from the body. Until now, cccDNA could be detected using ambiguous PCR-based techniques from small liver biopsy specimens, and this detection remains challenging due to technical limitations [ 56 ]. Surrogate markers of cccDNA activity and HBV replication are useful in therapy [ 32 , 57 ], but not for determining complete viral clearance with a sterilizing cure at the horizon.…”