Clinical relevance of the study of hepatitis B virus covalently closed circular DNA

Hepatitis B virus (HBV) affects approximately 240 million people 1 worldwide and is one of the main causes of cirrhosis and HCC. HBV belongs to the Hepadnaviridae family that includes various small-enveloped viruses infecting primates, rodents and birds. HBV infection becomes chronic in 5% of cases when it is acquired as an adult, and in 90% of the cases when it is acquired as a child 2.

The HBV life cycle begins with the entry of the virus into the hepatocyte. Immediately following hepatocyte infection the viral genomic DNA is transferred to the nucleus, where partially double-stranded, open circular DNA is converted into a covalently closed circular DNA molecule, cccDNA 3. cccDNA remains stable in the nucleus of infected cells and serves as a transcription template for all viral RNAs. Pregenomic RNA (pgRNA) is bifunctional: on one the hand, it serves as mRNA for the viral capsid and polymerase proteins translation and on the other hand, it is the template for reverse transcription into a new relaxed circular DNA within the viral capsid assembled in the cytoplasm. The DNA-containing nucleocapsids follow two pathways: they can be either recycled into the nucleus to increase the cccDNA reservoir or be enveloped to form new virions that will be secreted via the endoplasmic reticulum (ER) 4.

The stability of nuclear cccDNA molecules is because of their chromatin-like structure wrapped in histones transforming them into a viral mini-chromosome whose transcriptional activity is regulated by epigenetic mechanisms involving viral and host transcription factors and co-factors 5, 6. Persistent cccDNA is one of the factors involved in viral escape from the effect of antiviral medications or host immunity.

At present two types of medications are available for the treatment of HBV: –nucleos(t)ide analogues (NA) – lamivudine, adefovir, entecavir, tenofovir, telbivudine – and cytokines – interferon (IFN) and pegylated-interferon (PEG-IFN). These therapies are effective in preventing, but not eliminating, liver failure, the progression of cirrhosis and in reducing the risk of HCC 7, 8.

Covalently closed circular DNA clearance or the control of its transcriptional activity are two of the main goals of antiviral therapy, because this would result in a complete or functional cure of infection respectively 9. However, in clinical practice, monitoring the quantity and/or replicative activity of cccDNA is still a challenge. Indeed, cccDNA can only be measured from liver tissue and thus requires a liver biopsy. Moreover, there is no consensus on a clinically approved method of cccDNA quantification, making it difficult to monitor cccDNA throughout the natural history and treatment of chronic HBV infection 10, 11. Indeed, the traditional method for the detection and quantification of cccDNA is based on agarose gel electrophoresis and southern blot hybridization 3, 11, making it almost impossible to apply to clinical use because of the small size of liver biopsy specimens obtained from patients. Quantitative PCR methods to analyse cccDNA have been developed, but their specificity and standardization are still a subject of debate 10, 12, 13. Furthermore, methods to quantify cccDNA in single cells are still lacking.

In this manuscript, we have reviewed the relevance of cccDNA assessment throughout the natural history of HBV infection and during antiviral therapy of chronic hepatitis B.

cccDNA persists throughout the natural history of HBV infection

Covalently closed circular DNA levels vary in the liver during the natural history of HBV depending on the phase of infection. A positive correlation has been found between serum HBV DNA levels and intrahepatic cccDNA. The highest levels of intrahepatic cccDNA are found in the HBeAg-positive phase followed by HBeAg negative chronic hepatitis, the inactive carrier phase and the least in HBsAg negative patients 10. This suggests that even after HBsAg loss, cccDNA can persist in the liver.

The risk of HCC is decreased but not eliminated with potent NA with a high barrier to resistance 14, 15. This may be related to viral integration in the host genome 16 and to persistent cccDNA as well as the subsequent activation of proto-oncogenes via a mechanism of insertional mutagenesis or by the expression of viral proteins such as HBx or truncated envelope proteins from integrated sequences or from cccDNA 16, 17.

Hepatocellular carcinoma may even develop in patients with previous exposure to HBV 18 with resolved or occult HBV infection. Occult HBV is defined as a lack of detectable HBsAg in serum with the presence intrahepatic viral DNA and low levels of cccDNA 19, 20.

The recurrence of HCC after surgery may be caused by persistent cccDNA and integrated viral sequences in the surrounding non-tumoural liver. The relationship between the amount of cccDNA and HCC has been described by Wong et al. 21, 22.

It is believed that a functional cure of HBV infection and HBsAg clearance would be associated with a decreased risk of HCC, as suggested by natural history cohort studies in Asia, where the risk of HCC was found to be reduced in patients who clear HBsAg 18. If a complete cure of infection with cccDNA eradication were obtained, this could also be beneficial to decrease the development of HCC. However, one theoretical but important consideration is whether persistent viral integrants result in a residual risk of HCC despite clearance of active viral replication and liver inflammation.

Hepatitis B reactivation occurs when immune control of infection is lost

After resolution of hepatitis B infection, HBV remains controlled by multiple immunological host responses including CD4⁺ T helper cells and CD8⁺ cytotoxic cells 23. Viral genome sequences can persist in peripheral blood mononuclear cells even with the development of antibodies to HBsAg and HBV specific cytotoxic T cells, as reported by Rehermann et al. 24. Residual amounts of cccDNA can persist in the liver, and can be the template for the re-initiation of viral replication if immune control of infection is lost.

Viral reactivation can occur in patients with HIV-induced immune suppression or during immunosuppressive therapies 24-28, even in patients who have seemingly cleared the infection. In particular, reactivation has been described with most immunosuppressive therapies and has gained more attention with the use of rituximab 29. One case of fatal HBV reactivation was described by Dervite et al. in a patient who developed hepatitis B after receiving rituximab, even though he was HBsAg negative and Anti-HBs positive before rituximab administration 30. HBV reactivation was found 2 years after rituximab administration in 41.5% of patients with previous hepatitis B exposure who were HBsAg negative before beginning treatment 31. Clinically, reactivation of HBV varies from asymptomatic re-appearance of HBV DNA and HBsAg in blood to life-threatening cases with acute liver failure 32. Therefore, screening for HBV markers in patients undergoing immunosuppressive therapy is recommended. Depending on the clinical stage of liver damage, viral status and the type of immune suppression, either specific virological monitoring or pre-emptive antiviral therapy is recommended to prevent viral reactivation 33.

Persistent cccDNA explains the virological rebound after cessation of NA therapy

Nucleos(t)ides analogues act by inhibiting viral polymerase activity. NA do not prevent de novo cccDNA formation from the incoming virus and have no significant direct effect on the established cccDNA pool 34, 35.The effect of NA on cccDNA is mainly indirect by inhibiting the intracellular recycling of nucleocapsids to the nucleus. Interferons activate the innate immunity and affect cccDNA activity by decreasing its transcriptional activity through epigenetic regulation 36. Recently, IFN-α has been shown to induce partial noncytolytic degradation of the cccDNA pool through cytidine deamination in vitro 26.

While IFN-α effectively reduces serum HBV DNA and causes HBeAg seroconversion, it leads to HBsAg (surface antigen) loss in only 10–13% of patients after long-term follow-up 27, 28. Although oral medications effectively reduce HBV replication, the effect on HBsAg loss remains low. While HBsAg loss was observed in 11.8% of HBeAg-positive patients after 7 years of tenofovir treatment, it was only observed in one of 375 HBeAg-negative patients in the same cohort 37, 38. Because of persistent cccDNA in the liver of infected patients, and the lack of effective immune control of infection, discontinuation of NA results in a virological rebound in 43–47% of patients 39.

Even with the most potent NA, intrahepatic cccDNA levels decrease slowly during NA therapy, that is, less than 1 log₁₀ after 1 year of therapy and despite the sharp decrease in serum HBV DNA, that is, usually by more than 4 log₁₀ at 1 year of therapy, 10, 13.

Loss of cccDNA may be observed via a ‘dilution effect’ as a result of hepatocyte turnover 40, 41. Studies in the duck model have shown that antiviral therapy with polymerase inhibitors induced greater cccDNA reduction in animals displaying a higher hepatocyte proliferation rate 42. Nevertheless, it has been demonstrated that very low levels of cccDNA can persist indefinitely in a few liver cells even after acute infection has resolved 43. A few studies have shown persistent intrahepatic cccDNA in chronically infected patients after the loss of serum HBsAg 10, 44.

Importance of cccDNA in liver transplant recipients

Liver transplantation (LT) remains the only alternative for end-stage liver disease. The control of HBV pre- and post-liver transplantation is essential. Despite effective prophylactic measures taken for HBV-related liver diseases during liver transplantation, HBV recurrence occurs in 5–10% of patients, based on serological markers 45. Since NA cannot eliminate cccDNA, persistent cccDNA in liver explants accounts for the high rates of viral relapse when antiviral therapy is stopped 13, 46. Although the HBV immunoglobulins and antiviral agents prevent serologically detectable HBV recurrence, they cannot eradicate occult infection in liver tissue and HBV DNA has been detected in the donor liver of graft recipients as long as 10 years after LT, even when prophylaxis is clinically successful 47.

Data on the long-term impact of intrahepatic total HBV DNA and cccDNA in the recurrence of HBV are scarce and sometimes contradictory. This may be because it is difficult to design prospective studies with large numbers of patients with a proper sample collection and many studies have used different prophylaxis protocols. The lack of standardization of the current techniques for the detection of cccDNA is also a remaining issue.

It is interesting to note than Kwon et al. detected cccDNA in 10% and 6% of reperfusion biopsies in HBeAg-positive and high viral load recipients, respectively, revealing the rapidity of HBV recurrence in the new liver following LT 48. However, persistent cccDNA during treatment with hepatitis B immune globulins and NA did not correlate with HBV recurrence.

In patients who receive a liver graft from donors with previous HBV exposure but who are negative for HBsAg or HBV DNA in serum, there is a risk of HBV reactivation in the graft. In this setting, intrahepatic viral DNA and cccDNA may be observed even though these recipients receive prophylaxis for HBV 49.

In two consecutive studies, Lenci et al. showed that non-detectable cccDNA could be a useful diagnostic tool after LT to identify patients at a low risk of recurrent HBV, further suggesting that with close monitoring, successful HBV prophylaxis could be withdrawn in these long-term survivors 50, 51. Intrahepatic cccDNA levels have been correlated with the serum HBV core-related antigen (HBcrAg) in post-LT patients with undetectable serum markers 52. In this study, the presence of both cccDNA in the liver and HbcrAg in serum was associated with more severe fibrosis 52. This highlights the importance of persistent cccDNA despite current potent prophylactic antiviral strategies.

Surrogate markers for cccDNA monitoring

For developing new antiviral approaches to achieve either a functional or complete cure of HBV, there has been increasing research to identify surrogate, non-invasive markers of the amount of cccDNA in the liver. HBeAg is not a reliable marker in this setting because precore/core mutations that are frequently selected during conversion from HBeAg to HBeAb lead to undetectable HBeAg, thus invalidating HBeAg as a marker of cccDNA in HBeAg negative chronic carriers 53. Quantification of serum HBsAg and serum HBV DNA was recently proposed to predict viral reactivation 54, 55 and guide the management of current antiviral therapies 56. Nevertheless, the role of HBsAg and viral load as surrogate markers of intrahepatic cccDNA activity is a subject of debate, especially in HBeAg negative patients 53, 57. Moreover, it is well established that when viral suppression is achieved during NA therapy, cccDNA is still present in the liver 17, 19, 20. Although the decrease in serum HBsAg during NA treatment is very slow, a trend between a parallel decrease in serum HBsAg and intrahepatic cccDNA has consistently been found. Nevertheless, this correlation is not statistically significant probably because regulation of HBsAg expression is complex and involves more than the amount of cccDNA in infected cells, for example its transcriptional regulation and the possibility that envelope proteins could be expressed from viral sequences integrated into the host genome 41. Recently, empty virions, that is, enveloped particles with no viral genome but containing viral capsids, which are observed during NA therapy, have been proposed as a better serum cccDNA surrogate marker than HBsAg 58. The hypothesis is that HBcAg expression probably does not originate from integrated viral sequences so that the level of empty virions in serum might reflect the amount and transcriptional activity of cccDNA. This observation may be consistent with that of the circulating HBV core-related antigen 52. Further studies are needed to determine the role and potential prognostic value of these observations.

Perspectives: targeting cccDNA to cure viral infection

Significant discoveries have recently been made in the biology of cccDNA that open new perspectives in drug discovery. Indeed, for the first time a key nuclear enzyme involved in DNA repair, that is, tyrosyl DNA phospdhodiesterase 2 (TDP2), was shown to be involved in one of the first steps of cccDNA formation 4, 59. Furthermore, it has also been shown that cccDNA could be partially degraded by a non-cytolytic mechanism via deamination triggered by IFN-α or Lymphotoxin beta receptor agonists 26, at least in hepatocyte cultures. Other findings suggest that IFN-α may modify the status of histone bound to cccDNA to repress its transcriptional activity 36. The progress in molecular technologies with the development of CrispR/cas9-based approaches can be applied to the field of HBV. It has been shown that specific CrispR/cas9 can induce genetic damage in cccDNA, that is, genomic deletions, making it defective for viral protein expression 60. All of these new discoveries pave the way for research programmes to degrade or silence cccDNA more effectively 9. Translation of this basic research into clinical situations is eagerly awaited.