CccDNA Persistence during Natural Evolution of Chronic VHB Infection

Transcription

1 cccdna persistence in chronic viral B hepatitis REVIEWS CccDNA Persistence during Natural Evolution of Chronic VHB Infection Florin Alexandru Cãruntu 1, Violeta Molagic 2 1) Infectious Diseases Clinic Prof.dr.Matei Balº, University of Medicine and Pharmacy Carol Davila. 2) Infectious Diseases Clinic Prof.dr.Matei Balº, Bucharest Abstract HBV infection remains a public health issue. CccDNA HBV is a unique, intermediate replicative episome, responsible for persistence of infection in hepatocytes. The clearance of cccdna may be explained by cellular death. CccDNA detection in chronic infected human livers may represent an important marker in monitoring antiviral therapy. Short term therapy is followed by viral rebound in the majority of chronic HBV hepatitis patients. Based on mathematical models, it is considered that the period needed to achieve a complete intrahepatic cccdna clearance is 14.5 years. Key words cccdna hepatitis B virus natural history chronic viral B hepatitis Rezumat Infecþia cu HBV rãmâne o problemã de sãnãtate publicã. CccDNA este un intermediar replicativ episomal, unic, responsabil pentru persistenþa infecþiei HBV în hepatocite. Clearance-ul cccdna poate fi explicat prin moarte celularã. Detecþia cccdna în ficatul infectat cronic poate reprezenta un important marker în monitorizarea terapiei antivirale. Terapia de scurtã duratã este urmatã de rebound viral la majoritatea pacienþilor cu hepatitã cronicã HBV. Bazat pe modele matematice, se considerã cã sunt necesari 14,5 ani de tratament pentru a obþine un clearance complet cccdna intrahepatic. Introduction Worldwide, HBV infection represents a public health Romanian Journal of Gastroenterology December 2005 Vol.14 No.4, Address for correspondence: Florin Alexandru Cãruntu Clinica de Boli Infecþioase Str.Grozovici no.1, Sector 2 Bucureºti, Romania drcaruntu@mateibals.ro issue, being a frequent cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (1). Hepatitis B virus belongs to the Hepadnaviridae family. It is a DNA virus with hepatic tropism and its replication is performed by a reverse-transcriptase. HBV is spherical, with a 42 nm diameter, a lipid coating and a 32 nm nuclear capside which contains HBcAg, the viral genome and the viral polymerases. The viral genome is composed of a circular, partial double stranded DNA molecule. Viral replication cycle Direct cytopathic role of HBV is not very well defined at this moment, the important liver lesions which appear during chronic infection with HBV being attributed mostly to the host immunologic response. It is known that chronic HBV infection has several distinct serologic and virologic phases (2). Thus, one of the objectives of antiviral therapy is to accelerate the evolution of chronic hepatitis patients to stages when viral replication decreases and the incidence of severe liver lesions is significantly reduced. After binding to the hepatocytes and penetration into the cells through endocytosis, the virus is decapsidated and the genome enters the nucleus. Inside the nucleus, the short strand is completed by the endogenous DNA Pol and the long strand is covalently closed so the rcdna (relaxed circular DNA) is converted to cccdna (covalently closed circular DNA) which represents the replicative form of HBV DNA. CccDNA together with cellular histones and other nuclear proteins form minicromosomes and serve as a matrix for mrna transcription (3,4). It is considered that this is methylated in order to be active for transcription. Persistence of cccdna in hepatocyte plays a key role in viral persistence, reactivation of viral replication after cessation of antiviral therapy and resistance to therapy. Viral transcript is translated in capside together with polymerase proteins. Then, they are subsequentially

2 374 encapsidated and retranscripted into new partial double stranded viral genomes. The new nuclear capside with partial double stranded DNA may re-entry into hepatocyte nucleus to produce new cccdna molecules or may be secreted as a complete virion after the attachment of coating proteins. CccDNA may persist in hepatocyte nucleus as long as the infected cells survive. Chronic infection of hepatocytes is maintained by the viral cccdna pool (5). Short term antiviral therapy cannot completely exhaust the viral pool, which is stable and constitutes the source of viral production renewal after cessation of therapy (5,6). Chronic infection is maintained in the nucleus by copies/nucleus (genomic copies) (4,7). Complete inhibition of HBV polymerase should prevent the restoration of cccdna pool by blocking synthesis of rcdna precursors. If the production of new virions can be blocked, cccdna is eliminated rather as a result of hepatocyte death than through cccdna clearance. Two immune mechanisms have been proposed for cccdna clearance from hepatocyte nucleus (8-11): - non-cytolytic cytokine dependent mechanism; - cytolytic mechanism cytotoxic T lymphocyte dependent, by which the infected cells are eliminated and replaced with non-infected cells. The nature of the mechanism that eliminates cccdna remains controversial. It is considered that its elimination is a result of hepatocyte turnover. The latest studies raise several questions about cccdna half life and development of antiviral treatment strategies to prevent its formation or to reduce cccdna pool in an already infected liver. Experimental tissue cultures proved the importance of cccdna in persistence of HBV infection in a single cell. Use of nucleoside analogs made the analysis of cccdna half life in undivided cells possible. Several studies have proved cccdna stability in non-dividable hepatocyte cultures with a half life > 30 days, while others have shown a shorter half life (12). It seems that in non-dividable hepatocytes with a 3-5 days half life cccdna is unstable. It is estimated that in ducks and in duck hepatocyte cultures, cccdna half life is 3-5 days, and this value is greater in vivo (35-50 days) (6). De novo synthesis of cccdna has been proven in human infected hepatocytes. Several studies of HBV clearance kinetics during antiviral therapy indicate that cccdna half life in chronic HBV infection is determined by the turn-over of infected cells. Thus, the question rises if de novo formation of cccdna can be prevented. Use of nucleoside analogs which inhibit viral DNA dependent DNA polymerase in therapeutic regimes raises the issue of preventing de novo formation of cccdna by the initial virions. It has been shown in duck hepatocyte cultures inoculated with DHBV and in Tupaia hepatocytes that the presence of Lamivudine or Adefovir cannot prevent the formation of initial cccdna. These results suggest that Cãruntu and Molagic during antiviral therapy, remaining circulating virions may infect new hepatocytes despite the presence of antiviral drugs. Studies on animal models (ducks and woodchucks) have shown the absence of cccdna clearance from HBV infected hepatocytes during antiviral therapy. Although literature cites different opinions on the mechanisms involved in cccdna clearance, it becomes clear that during acute HBV infection a concentrated action on both non-cytolytic Th1 immune response which exerts a direct antiviral effect and cytolytic cellular Th 1 response is required. In order to achieve this, a few factors are required (13): 1. production of neutralizing antibodies for prevention of hepatocyte infection by the circulating virions; 2. hepatocyte cellular regeneration by the precursor cells; 3. hepatocyte proliferative response directed towards killer cells, leading to cccdna clearance after one or more cell divisions; 4. cccdna clearance by non-cytolytic Th1 response; 5. blocking new cccdna synthesis in already infected hepatocytes. Studies on animal models have shown that the use of a potent HBV polymerase inhibitor which may produce a partial cccdna clearance does not explain either the viral replication rebound after therapy cessation, or the emergence of therapy resistant virus during prolonged therapy. One hypothesis sustains that the polymerase inhibitor, by inhibiting viral DNA synthesis, would block both the intracellular recirculation of new nucleocapsides and the maintaining of cccdna intranuclear pool. Thus, cccdna clearance can be explained by cellular death (14). CccDNA study in humans Although the crucial role of cccdna in the persistence of HBV infection is well known and despite the importance of comprehending its clearance mechanism, most of the information comes from studies done on animal models (infected woodchucks and ducks). Difficulties in studying cccdna in humans included: 1. necessity for liver biopsy, difficult to perform and to accept, especially by the patients in the inactive phase of natural evolution of HBV infection; 2. lack of specific and quantitative methods for cccdna determination in liver specimens. Nevertheless, there are several studies that have analyzed HBV-DNA in human infected livers. Using the Southern Blot method, Miller et al (15) identified the presence of cccdna in the nucleus of infected hepatocytes and its persistence despite antiviral therapy. Werle et al (16) reported in a study the development of a new real time PCR method, which allows cccdna quantification in liver biopsies from chronic HBV hepatitis patients. Method s specificity is based on the use of two major stages: 1. DNAse plasmidic therapy that digests noncovalently closed circular DNA (replicative intermediate forms);

3 cccdna persistence in chronic viral B hepatitis use of a primer localized on both sides of rcdna gap, which preferentially amplifies cccdna. In Hong Kong, Wong Danny Ka Ho et al (17) tried to quantify cccdna in liver specimens and in serum, using the Invader method (non PCR method). Specificity of this method has not been completely proven. Although levels of cccdna copies /ml serum have been reported, it is considered that this method detects rcdna rather than cccdna. CccDNA persistence during natural evolution of HBV infection and its decline during Adefovir therapy In a study published in 2004, Werle et al (16) analyzed cccdna levels in liver specimens from patients in different stages of the natural evolution of HBV infection and from patients included in a placebo-controlled, clinical trial using real time PCR method. Liver samples from 98 patients with chronic HBV infection were obtained by hepatic needle biopsy. There were: - 63 patients with positive HBeAg - 18 patients with negative HBeAg - 10 inactive chronic carriers of HBsAg - 7 patients who achieved HBs seroconversion (negative HBsAg, positive antihbe, positive anti HBc IgG) - 32 patients with chronic hepatitis, positive HBeAg, treated as follows: 10 received placebo, 22 received Adefovir (10 mg or 30 mg daily). CccDNA and total intrahepatic DNA levels were analyzed. The study showed that cccdna levels were significantly higher in positive HBeAg patients (1.4 copies/cell) than in negative HBeAg patients (0.01 copies/cell) or chronic HBsAg carriers (0.02 copies/cell). In patients that become HBsAg negative, cccdna may persist at a very low level (0.002 copies/cell). These results correlate with the existence of higher levels of intrahepatic replicative intermediate HBV forms and serum DNA in positive HBeAg patients. It is surprising that the identified cccdna level in negative HBeAg patients is not significantly different (p=0.68) from that of inactive HBV carriers. This finding may be attributed to the natural variation of viral replication in patients infected with precore mutant HBV and to the relatively small number of samples taken from inactive carrier patients. It is considered that the main difference between a negative HBeAg chronic hepatitis patient and a chronic HBV carrier is related to immunologic factors rather than virologic ones (2,18). The fact that cccdna remains detectable in all negative HBeAg patients explains the observation of viral reactivation in these patients. Fluctuations of viral replication and acute episodes of hepatitis are common in patients infected with precore mutant HBV (18). During natural evolution of inactive chronic HBsAg carriers viral reactivation and acute episodes of hepatitis were noted. Even the patients that achieved HBV clearance may develop active illness during severe immunosuppresion as after a transplant, chemotherapy or cortisone use. CccDNA persistence at low levels may explain the possibility of de novo HBV infections in patients with liver transplant from a donor who had a HBV infection and got cured (19). These observations regarding natural evolution of HBV infection correlate with the theory according to which resolution of hepadnaviridae infections is achieved through the efficient control of viral replication by the host s immune system (20). The mean level of total intrahepatic HBV-DNA was significantly higher in positive HBeAg patients (155 copies/ cell) than in negative HBeAg patients (0.6 copies/cell), inactive chronic carriers (2 copies/cell) and in negative HBsAg patients (0.19 copies/cell). CccDNA evolution inside the liver during antiviral therapy A recent study showed that Adefovir dipivoxil may reduce cccdna levels(16). After 48 weeks of therapy, an 84% reduction of cccdna and a 98% reduction of total intrahepatic DNA levels were noted. With Adefovir, cccdna levels decreased 11.5 times compared to placebo, not statistically significant in patients that achieved HBe seroconversion. The decline of cccdna levels was smaller than the decline of intrahepatic DNA and that of the viral load. The 48 weeks Adefovir therapy resulted in (13,16): - reduction of mean serum viral load with 4,7 log 10 copies/ ml; - reduction of intrahepatic HBV DNA with 1,63 log 10 copies/ml; - reduction of cccdna with 0,80 log 10 copies/ml; - modification of HBsAg serum levels = - 0,6 log 10 ng/ml (73% reduction). Clinical studies with nucleoside analogs indicate that short term therapy is followed by viral rebound in the majority of cases (21). Studies on animal models suggest that long viral cccdna half life is responsible for viral resistance and reactivation after cessation of therapy. In this study, the authors showed that Adefovir significantly reduces the cccdna levels by potently inhibiting the viral polymerase. This reduction is correlated and similar in magnitude with the reduction of serum levels of HBsAg. As a significant reduction of viral cccdna has been associated with the reduction of HBsAg levels beyond the detection limit of usual methods, quantification of serum HBsAg could represent a surrogate marker of intrahepatic cccdna pool. Using immunohistochemistry, the number of HBcAg and HBsAg positive cells in liver tissue samples from study patients treated with Adefovir was determined. A correlation has been found between the number of cccdna copies, total intrahepatic DNA level and the number of HBcAg positive cells.

4 376 This study showed a slow cccdna clearance during Adefovir therapy. The factors contributing to the slow cccdna clearance during therapy are the following (4,13,16,22,23): 1. cccdna existing as a stable chromatin episome; 2. the nature of asymmetric replication of Hepadnaviridae that protects cccdna from being directly eliminated by the polymerase inhibitors; 3. the preferential use of mature viral nucleocapsides to rebuild cccdna pool during the phase of reduced replication; 4. the potential of hepatocyte re-infection by residual circulating viruses despite therapy/ It is well known that the viral load decreases in biphasic manner (24): - one rapid phase corresponds to the inhibition of de novo production of viral particles in infected cells and their elimination into the plasma; - a second, slow phase, secondary to infected cells clearance. The slow cccdna clearance observed by the authors seems to contribute to the second, slow phase of viral kinetics. CccDNA longevity brings into discussion the need for long term antiviral therapy in order to control HBV replication, if a strong immune response of the host is not present. The studies conducted until present concluded that Lamivudine and Adefovir therapy may produce a partial restoration of the immune response necessary for a lasting control of HBV infection (25). Conclusions Based on the results obtained on hepadnaviridae infected chimpanzees and woodchucks, it is still unclear if the primary clearance mechanism of infection is mediated by non-cytolytic immune response or by killer T cells, followed by cell division. It is considered that the clearance of intrahepatic cccdna is mainly due to potent suppression of viral DNA synthesis, which effectively depletes mature cytoplasmic nucleocapside pool available for conversion into cccdna. During antiviral therapy, cccdna clearance was not significantly correlated with the main indicators of immune-mediated hepatocyte lysis (elevated ALAT, increased necroinflammatory activity). This may suggest that during therapy, cccdna clearance may be achieved through another mechanism rather than cellular death. Based on mathematical models which used data regarding cccdna levels during antiviral therapy, it is considered that the period needed to achieve a complete intrahepatic cccdna clearance is 14.5 years (26). As intrahepatic cccdna remains detectable even in patients that achieved HBs seroconversion, it may not be necessary to completely eradicate it in order to control HBV infection on the long term. CccDNA detection in chronic infected human livers may represent an important marker for monitoring antiviral therapy. Even there is no routine quantification method, the Cãruntu and Molagic quantification of cccdna decline during antiviral therapy may represent an important therapy endpoint in clinical trials using new antiviral drugs or combinations. Because significant reduction of cccdna during antiviral therapy is correlated with the reduction of HBsAg and HBeAg levels, it is necessary to routinely monitor these non-invasive markers (quantification of serum HBsAg and HBeAg). Discovery of new cellular and virologic factors involved in cccdna production and regulation may identify new molecular targets for antiviral therapy. In the future, antiviral therapy should be adapted to each infected patient s clinical situation. A long term therapy with potent antiviral drugs to induce a low incidence of viral resistance and development of new therapeutic regimens to minimize resistance to therapy, thus increasing cccdna clearance and intensifying antihbv immune response are required. References 1. 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