The Natural History and the Staging of Chronic Hepatitis B: Time for Reevaluation of the Virus–Host Relationship Based on Molecular Virology and Immunopathogenesis Considerations?
2007; Elsevier BV; Volume: 133; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.07.038
ISSN1528-0012
AutoresA. Thompson, Stephen Locarnini, Kumar Visvanathan,
Tópico(s)Hepatitis Viruses Studies and Epidemiology
ResumoSee “Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients” by Volz T, Lutgehetmann M, Wachtler P, et al, on page 843; and “Viral quasi-species evolution during Hepatitis Be antigen seroconversion” by Lim SG, Cheng Y, Guindon S, et al on page 951.Hepatitis B is a disease with both virologic and immunologic components. The 2 key virologic events in the life cycle of the hepatitis B virus (HBV) are the generation from genomic DNA of the covalently closed circular (ccc) DNA transcriptional template and reverse transcription of the viral pregenomic (pg) RNA to form the HBV DNA genome. Because the virus utilizes reverse transcription to copy its genome, mutant viral genomes, or quasispecies, are frequently found in the blood of HBV-infected patients. Not surprisingly then, a number of immune escape mutants of HBV are selected out as dominant populations during this response within the term of the natural history of the disease. Human HBV is a highly evolved pathogen and under normal circumstances viral infection and subsequent replication within hepatocytes is not cytopathic. The liver damage associated with acute or chronic hepatitis B (CHB) occurs mainly as a result of attempts, usually unsuccessful, by the host’s immune response to clear HBV from infected hepatocytes1Chisari F.V. Ferrari C. Hepatitis B virus immunopathogenesis.Annu Rev Immunol. 1995; 13: 29-60Crossref PubMed Scopus (1460) Google Scholar as part of the “immuno-elimination” phase of the disease.2Lok A.S. Chronic hepatitis B.N Engl J Med. 2002; 346: 1682-1683Crossref PubMed Scopus (396) Google Scholar, 3Lok A.S. Lai C.L. Acute exacerbations in Chinese patients with chronic hepatitis B virus (HBV) infection Incidence, predisposing factors and etiology.J Hepatol. 1990; 10: 29-34Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 4Chu C.M. Karayiannis P. Fowler M.J. et al.Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum.Hepatology. 1985; 5: 431-434Crossref PubMed Scopus (304) Google Scholar, 5Maini M.K. Boni C. Lee C.K. et al.The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.J Exp Med. 2000; 191: 1269-1280Crossref PubMed Scopus (668) Google ScholarInnate and adaptive immunity have traditionally been considered as largely separate, although complementary, mechanisms of defense against microbial threats. The adaptive system, being evolutionally newer and having the capacity for selectivity, adaptation, amplification, and memory, has arguably been regarded as more sophisticated and potent. However, recent advances in our understanding of the nature and functions of Toll-like receptors (TLR) have sparked a new appreciation of the extensive interdependence and communication between innate and adaptive responses and have led to renewed interest and higher regard for the contribution of the innate arm of the immune network. The host response to viral (and bacterial) infections is biphasic, with innate effectors such as interferon (IFN), natural killer cells, and macrophages being critical in the early phase, with slower-developing adaptive antigen-specific T- and B-cell responses often being essential for clearance of the pathogen and establishment of immunity. It is only in recent years that it has been recognized that hepatocytes, Kupffer cells, liver sinusoidal epithelial cells, and other liver cells have functional TLRs and probably play an important role in the eradication of viral infections such as HBV. Not surprisingly, viruses have developed mechanisms to counteract the effects of the innate immune system, including the hepatitis B e antigen (HBeAg), which serves to suppress the TLR pathway, thereby allowing HBV to establish persistent infection in the host.6Visvanathan K. Skinner N.A. Thompson A.J. et al.Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.Hepatology. 2007; 45: 102-110Crossref PubMed Scopus (279) Google ScholarSpecific serology has proven to be the mainstay of diagnostic testing for hepatitis B. Several commercial immunoassays are available for the various hepatitis B markers of viral antigens and antibodies.7Bowden S. Serological and molecular diagnosis.Semin Liver Dis. 2006; 26: 97-103Crossref PubMed Scopus (21) Google Scholar The presence of large amounts of hepatitis B surface antigen (HBsAg) and HBeAg in the serum may affect the ability to detect circulating antibodies and may obscure the onset of seroconversion. The available commercial assays usually detect anti-HBs and anti-HBe antibodies only after the respective antigens have been cleared from the serum. It was recognized more than a decade ago that, by using more sensitive immunoassays, it was possible to detect antibody in the presence of excess serum antigen.8Maruyama T. McLachlan A. Iino S. et al.The serology of chronic hepatitis B infection revisited.J Clin Invest. 1993; 91: 2586-2595Crossref PubMed Scopus (134) Google Scholar Such approaches identified serologic responses in the context of active viral replication. For example, all patients with “active” CHB and the majority of patients with asymptomatic CHB (the so-called tolerant or without disease groups) demonstrated ongoing humoral immune responses including anti-HBe and anti-surface antibody (anti-HBs) production. In fact, anti-HBe seroconversion can occur many years (up to 6 years) before the actual loss of HBeAg or onset of liver injury. Similarly, anti-HBs may coexist with virions and subviral HBsAg particles for many years before viral clearance and loss of HBsAg. The concept of a relatively nonoverlapping seroconversion from HBeAg-positive to anti-HBe positive status during CHB may need amendment.The recent advances in HBV DNA testing have also provided new tools to investigate HBV infections. Molecular assays for HBV DNA can be useful for resolving ambiguous serologic patterns, but the major role of HBV DNA assays is their use in staging patients in the various phases of CHB and for monitoring patients undergoing antiviral therapy. Assays must be normalized to the World Health Organization international standard to permit expression of viral load in international units per milliliter (IU/ml), allowing results of different studies to be compared. Molecular assays are now being developed to detect intrahepatic HBV replicative forms. Assays for intrahepatic HBV cccDNA and pgRNA have now been published, although problems related to assay standardization need to be overcome before they can be classified as a routine part of the diagnostic armamentarium. However, initial studies have been promising and have shown that in patients on antiviral therapy, the intrahepatic HBV cccDNA levels at the end of treatment were actually a superior indicator of sustained response compared with serum HBV DNA levels.9Sung J.J. Wong M.L. Bowden S. et al.Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy.Gastroenterology. 2005; 128: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Furthermore, patients who underwent HBeAg seroconversion were more likely to have a lower baseline level of HBV cccDNA at the initiation of therapy.10Werle-Lapostolle B. Bowden S. Locarnini S. et al.Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.Gastroenterology. 2004; 126: 1750-1758Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar High HBV cccDNA levels have also been shown to be predictive of HBV reactivation in HBsAg-positive lymphoma patients treated with cytotoxic chemotherapy.11Hui C.K. Bowden S. Jackson K. et al.Clinical significance of intrahepatic hepatitis B virus covalently closed circular DNA in chronic hepatitis B patients who received cytotoxic chemotherapy.Blood. 2005; 105: 2616-2617Crossref PubMed Scopus (42) Google Scholar Thus, the monitoring of intrahepatic HBV intermediates may provide new insights into the natural history of hepatitis B and response to antiviral therapy, to help refine existing treatment protocols.12Lok A.S. Navigating the maze of hepatitis B treatments.Gastroenterology. 2007; 132: 1586-1594Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 13Ghany M. Liang T.J. Drug targets and molecular mechanisms of drug resistance in chronic hepatitis B.Gastroenterology. 2007; 132: 1574-1585Abstract Full Text Full Text PDF PubMed Scopus (152) Google ScholarThis issue of Gastroenterology contains 2 articles that further our understanding of the events that occur before, during, and after HBeAg seroconversion and also the natural history of CHB. Rather than being an acute immunologic event, as suggested by standard serologic assays, HBeAg seroconversion is shown to involve dynamic shifts of the serum HBV quasispecies pool, a process that begins years before a drop in the viral load is detectable.14Lim S.G. Cheng Y. Guindon S. et al.Viral quasi-species evolution during Hepatitis Be antigen seroconversion.Gastroenterology. 2007; 133: 951-958Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar This flux in peripheral viral populations reflects a shift in the intrahepatic compartment, where variants with reduced or absent HBeAg production accumulate. Furthermore, HBV replication is shown to be less efficient in the setting of HBeAg-negative disease, a process independent of the precore/basal core promoter (PC/BCP) mutations.15Volz T. Lutgehetmann M. Wachtler P. et al.Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.Gastroenterology. 2007; 133: 843-852Abstract Full Text Full Text PDF PubMed Scopus (168) Google ScholarLim et al14Lim S.G. Cheng Y. Guindon S. et al.Viral quasi-species evolution during Hepatitis Be antigen seroconversion.Gastroenterology. 2007; 133: 951-958Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar report the results of a detailed prospective study of the viral evolution occurring during perinatally acquired CHB infection, focusing on the events around and during HBeAg seroconversion. They investigated 4 groups of patients: spontaneous seroconverters (SS), nonseroconverter controls (CC), IFN-induced seroconverters (IR),16Wursthorn K. Lutgehetmann M. Dandri M. et al.Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.Hepatology. 2006; 44: 675-684Crossref PubMed Scopus (375) Google Scholar and IFN nonresponders (NR). At study entry, the SS and IFN-treated patients were in the “immunoelimination” phase of disease, marked by abnormal serum alanine aminotransferase (ALT). The CC group was selected by normal ALT and were likely still “immunotolerant.” All were infected with genotype B HBV. Serum was collected from 4–5 time points, representing on average a period of 30 months before and after HBeAg seroconversion. HBV DNA was extracted for polymerase chain reaction, cloning, and sequencing of the precore/core (PC/C) gene (20 clones per sample). More than 3386 sequences were then analyzed, comparing the clinical groups for viral sequence diversity, evolutionary rate, and positive selection (both at the nucleotide and amino acid level). The primary analysis focused on the nonoverlapping region of the PC/C gene (nt 1839–2306). The key finding in this study was that before HBeAg seroconversion, viral sequence diversity was found to be 2.4-fold higher in HBeAg seroconverters (SS, IR) compared with nonseroconverters (CC, NR). The selection pressure driving this diversity was operating up to 3 years before HBeAg seroconversion actually occurred. The viral diversity increased further after viral load reduction. The authors then determined the substitution rate in the PC/C gene, defined as the number of nucleotide substitutions per site per month. The substitution rate was 1 log higher in seroconverters compared to nonseroconverters; this was constant over the time period of the study. Phylogenetic trees of seroconverter patients were more complex, forming clusters over time with longer branch lengths. Positive selection in HBeAg and HBcAg protein sequences was detected in 14 of 20 seroconverters compared with only 1 of 20 nonseroconverters.The article extends previous work demonstrating that viral mutation rates are increased when comparing patients in the immunotolerant to the immunoelimination phase of CHB.17Bozkaya H. Akarca U.S. Ayola B. et al.High degree of conservation in the hepatitis B virus core gene during the immune tolerant phase in perinatally acquired chronic hepatitis B virus infection.J Hepatol. 1997; 26: 508-516Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 18Bozkaya H. Ayola B. Lok A.S. High rate of mutations in the hepatitis B core gene during the immune clearance phase of chronic hepatitis B virus infection.Hepatology. 1996; 24: 32-37Crossref PubMed Google Scholar, 19Hannoun C. Horal P. Lindh M. Long-term mutation rates in the hepatitis B virus genome.J Gen Virol. 2000; 81: 75-83Crossref PubMed Scopus (143) Google Scholar This is the first work that has performed such detailed virologic studies over such a prolonged time period. The second important message from this study by Lim et al is contained within the observation that nucleotide substitution rates persist at high levels after seroconversion, when viral diversity increases further. Far from the “nonreplicative state,” it is clear that there is an ongoing dynamic postseroconversion, albeit at lower levels of viral load, with viral diversity likely driven by an immune-based selection pressure.The second article in this issue compares patients in the immunoelimination phase of HBeAg-positive CHB to patients with HBeAg-negative CHB,15Volz T. Lutgehetmann M. Wachtler P. et al.Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.Gastroenterology. 2007; 133: 843-852Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar and by using molecular virologic techniques demonstrates that HBV replicates less efficiently in the HBeAg-negative phase of disease. Rather than examining viral quasispecies in peripheral blood, the authors have compared intrahepatic virologic profiles. Liver biopsies from 119 treatment-naïve patients with CHB (42 HBeAg-positive, 77 HBeAg-negative) were assayed for cccDNA (the intranuclear reservoir of HBV), and relaxed circular HBV DNA (rcDNA, used to estimate intrahepatic replicating virus). Virion productivity was defined as the ratio of rcDNA/cccDNA. pgRNA, the replicative RNA intermediate of HBV, was then measured as a marker of the transcriptional activity of cccDNA and the replication pathway. Pre-S/S mRNA was estimated as an alternative marker of cccDNA transcriptional activity. Data were compared with peripheral viral load and quantitative measures of HBsAg. Finally, the cccDNA was sequenced for analysis of genotype and PC/BCP variants.As has previously been documented,10Werle-Lapostolle B. Bowden S. Locarnini S. et al.Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.Gastroenterology. 2004; 126: 1750-1758Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, 16Wursthorn K. Lutgehetmann M. Dandri M. et al.Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.Hepatology. 2006; 44: 675-684Crossref PubMed Scopus (375) Google Scholar, 20Laras A. Koskinas J. Dimou E. et al.Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients.Hepatology. 2006; 44: 694-702Crossref PubMed Scopus (158) Google Scholar, 21Wong D.K. Yuen M.F. Yuan H. et al.Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients.Hepatology. 2004; 40: 727-737Crossref PubMed Scopus (118) Google Scholar patients with HBeAg-negative CHB had significantly lower levels of serum viral load, cccDNA (>1 log lower) and rcDNA (>2 log lower). Furthermore, virion productivity per cccDNA molecule was found to be 5-fold lower than in HBeAg-positive disease. Levels of pgRNA were correspondingly reduced, suggesting that the low virion productivity resulted from lower amounts of pgRNA. In contrast to patients with HBeAg-positive CHB, there was no correlation between serum viral load and the amount of cccDNA in the HBeAg-negative patients, consistent with the observed impairment of virion productivity. The HBsAg titres were significantly lower in the HBeAg-negative cohort, correlating with the cccDNA. However, Pre-S/S mRNA level and HBsAg titre per cccDNA molecule were similar between HBeAg-positive and negative patients, suggesting that the production of subviral particles was not impaired in the HBeAg-negative phase, but rather that the replication pathway might be selectively impaired.The authors investigated whether specific viral factors such as common PC/BCP variants or HBV genotype might explain these observations. As expected, mixed cccDNA populations containing PC/BCP variants were detectable in most HBeAg-negative patients. PC/BCP variants were also detectable in 53% of HBeAg-positive patients, consistent with the observation of Lim et al,14Lim S.G. Cheng Y. Guindon S. et al.Viral quasi-species evolution during Hepatitis Be antigen seroconversion.Gastroenterology. 2007; 133: 951-958Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar that variant viral populations are strongly selected before seroconversion. Impairment in virion productivity was not linked to the presence of these variants, however. In fact, in the HBeAg-negative patients in whom wild-type cccDNA molecules were not detectable, BCP mutations were associated with higher replication levels, consistent with previous in vitro data suggesting that it may represent a compensatory mutation.22Tacke F. Gehrke C. Luedde T. et al.Basal core promoter and precore mutations in the hepatitis B virus genome enhance replication efficacy of Lamivudine-resistant mutants.J Virol. 2004; 78: 8524-8535Crossref PubMed Scopus (102) Google Scholar Most of these Caucasian patients were found to be genotype A, D, or mixed A and D; no specific effect on productivity was identified.Volz et al15Volz T. Lutgehetmann M. Wachtler P. et al.Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.Gastroenterology. 2007; 133: 843-852Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar have described the intrahepatic molecular virologic events occurring as patients transition through seroconversion into HBeAg-negative disease, where virion productivity is reduced because of lower steady-state levels of pgRNA. The effect appears to be specific for the replication pathway; the production of subviral particles was spared. This cannot be attributed to the effect of PC/BCP mutations, because it may be observed in their absence. The mechanism reducing pgRNA levels remains unclear.Although no immunologic studies were performed in either study, it seems likely that host immune pressure is driving this viral evolution. Noncytolytic mechanisms have been demonstrated to be important in animal models of HBV, dependent on the key cytokines IFN-α/β, IFN-γ, and tumor necrosis factor (TNF)-α.23Wieland S.F. Chisari F.V. Stealth and cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar IFN-γ and TNF-α have been shown to decrease pgRNA by destabilizing the SSB/La autoantigen,24Heise T. Guidotti L.G. Cavanaugh V.J. et al.Hepatitis B virus RNA-binding proteins associated with cytokine-induced clearance of viral RNA from the liver of transgenic mice.J Virol. 1999; 73: 474-481Crossref PubMed Google Scholar, 25Heise T. Guidotti L.G. Chisari F.V. La autoantigen specifically recognizes a predicted stem-loop in hepatitis B virus RNA.J Virol. 1999; 73: 5767-5776Crossref PubMed Google Scholar, 26Heise T. Guidotti L.G. Chisari F.V. Characterization of nuclear RNases that cleave hepatitis B virus RNA near the La protein binding site.J Virol. 2001; 75: 6874-6883Crossref PubMed Scopus (51) Google Scholar and may therefore inhibit virion productivity by decreasing steady-state pgRNA. It has recently been noted that TLR2, a key stimulant of TNF-α production, is down-regulated in the setting of HBeAg-positive CHB relative to HBeAg-negative disease.6Visvanathan K. Skinner N.A. Thompson A.J. et al.Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.Hepatology. 2007; 45: 102-110Crossref PubMed Scopus (279) Google Scholar The precore protein itself appears to be critical for this effect. Increased innate immune signalling in the setting of HBeAg-negative disease, perhaps via TLR2-stimulated TNF-α, might therefore contribute to the observed phenomena. The evolving cytokine milieu within the liver may also be driving viral diversity with the positive selection of resistant variants, as observed in the first study by Lim et al; however, it may very well be that humoral responses, such as those described by Maruyama et al8Maruyama T. McLachlan A. Iino S. et al.The serology of chronic hepatitis B infection revisited.J Clin Invest. 1993; 91: 2586-2595Crossref PubMed Scopus (134) Google Scholar shape the viral quasispecies found in patients.Using this new information, it is now possible to refine the traditional view of the natural history of CHB, incorporating recent advances in the understanding of the importance of the immune system, with this molecular virologic data (Figure 1). Both studies provide new insight into the prolonged and dynamic virologic evolution that both precedes seroconversion and continues thereafter. HBeAg seroconversion is clearly not a “black and white” immunologic event, as suggested by existing commercial serologic assays. Rather, a progressive increase in the viral diversity is observed, with positive selection of viral quasispecies defective for HBeAg production, which are able to persist in the host, but at the cost of reduced replication phenotype. Identifying these selection pressures will be the critical next step for resolving the host–virus interplay in CHB, both HBeAg positive and HBeAg negative. These 2 detailed virologic studies have given new insight into the host–virus relationship and the various phases of the natural history of CHB. Similarly stringent immunologic studies, both innate and adaptive, are required to complete the picture. See “Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients” by Volz T, Lutgehetmann M, Wachtler P, et al, on page 843; and “Viral quasi-species evolution during Hepatitis Be antigen seroconversion” by Lim SG, Cheng Y, Guindon S, et al on page 951. See “Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients” by Volz T, Lutgehetmann M, Wachtler P, et al, on page 843; and “Viral quasi-species evolution during Hepatitis Be antigen seroconversion” by Lim SG, Cheng Y, Guindon S, et al on page 951. See “Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients” by Volz T, Lutgehetmann M, Wachtler P, et al, on page 843; and “Viral quasi-species evolution during Hepatitis Be antigen seroconversion” by Lim SG, Cheng Y, Guindon S, et al on page 951. Hepatitis B is a disease with both virologic and immunologic components. The 2 key virologic events in the life cycle of the hepatitis B virus (HBV) are the generation from genomic DNA of the covalently closed circular (ccc) DNA transcriptional template and reverse transcription of the viral pregenomic (pg) RNA to form the HBV DNA genome. Because the virus utilizes reverse transcription to copy its genome, mutant viral genomes, or quasispecies, are frequently found in the blood of HBV-infected patients. Not surprisingly then, a number of immune escape mutants of HBV are selected out as dominant populations during this response within the term of the natural history of the disease. Human HBV is a highly evolved pathogen and under normal circumstances viral infection and subsequent replication within hepatocytes is not cytopathic. The liver damage associated with acute or chronic hepatitis B (CHB) occurs mainly as a result of attempts, usually unsuccessful, by the host’s immune response to clear HBV from infected hepatocytes1Chisari F.V. Ferrari C. Hepatitis B virus immunopathogenesis.Annu Rev Immunol. 1995; 13: 29-60Crossref PubMed Scopus (1460) Google Scholar as part of the “immuno-elimination” phase of the disease.2Lok A.S. Chronic hepatitis B.N Engl J Med. 2002; 346: 1682-1683Crossref PubMed Scopus (396) Google Scholar, 3Lok A.S. Lai C.L. Acute exacerbations in Chinese patients with chronic hepatitis B virus (HBV) infection Incidence, predisposing factors and etiology.J Hepatol. 1990; 10: 29-34Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 4Chu C.M. Karayiannis P. Fowler M.J. et al.Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum.Hepatology. 1985; 5: 431-434Crossref PubMed Scopus (304) Google Scholar, 5Maini M.K. Boni C. Lee C.K. et al.The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.J Exp Med. 2000; 191: 1269-1280Crossref PubMed Scopus (668) Google Scholar Innate and adaptive immunity have traditionally been considered as largely separate, although complementary, mechanisms of defense against microbial threats. The adaptive system, being evolutionally newer and having the capacity for selectivity, adaptation, amplification, and memory, has arguably been regarded as more sophisticated and potent. However, recent advances in our understanding of the nature and functions of Toll-like receptors (TLR) have sparked a new appreciation of the extensive interdependence and communication between innate and adaptive responses and have led to renewed interest and higher regard for the contribution of the innate arm of the immune network. The host response to viral (and bacterial) infections is biphasic, with innate effectors such as interferon (IFN), natural killer cells, and macrophages being critical in the early phase, with slower-developing adaptive antigen-specific T- and B-cell responses often being essential for clearance of the pathogen and establishment of immunity. It is only in recent years that it has been recognized that hepatocytes, Kupffer cells, liver sinusoidal epithelial cells, and other liver cells have functional TLRs and probably play an important role in the eradication of viral infections such as HBV. Not surprisingly, viruses have developed mechanisms to counteract the effects of the innate immune system, including the hepatitis B e antigen (HBeAg), which serves to suppress the TLR pathway, thereby allowing HBV to establish persistent infection in the host.6Visvanathan K. Skinner N.A. Thompson A.J. et al.Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.Hepatology. 2007; 45: 102-110Crossref PubMed Scopus (279) Google Scholar Specific serology has proven to be the mainstay of diagnostic testing for hepatitis B. Several commercial immunoassays are available for the various hepatitis B markers of viral antigens and antibodies.7Bowden S. Serological and molecular diagnosis.Semin Liver Dis. 2006; 26: 97-103Crossref PubMed Scopus (21) Google Scholar The presence of large amounts of hepatitis B surface antigen (HBsAg) and HBeAg in the serum may affect the ability to detect circulating antibodies and may obscure the onset of seroconversion. The available commercial assays usually detect anti-HBs and anti-HBe antibodies only after the respective antigens have been cleared from the serum. It was recognized more than a decade ago that, by using more sensitive immunoassays, it was possible to detect antibody in the presence of excess serum antigen.8Maruyama T. McLachlan A. Iino S. et al.The serology of chronic hepatitis B infection revisited.J Clin Invest. 1993; 91: 2586-2595Crossref PubMed Scopus (134) Google Scholar Such approaches identified serologic responses in the context of active viral replication. For example, all patients with “active” CHB and the majority of patients with asymptomatic CHB (the so-called tolerant or without disease groups) demonstrated ongoing humoral immune responses including anti-HBe and anti-surface antibody (anti-HBs) production. In fact, anti-HBe seroconversion can occur many years (up to 6 years) before the actual loss of HBeAg or onset of liver injury. Similarly, anti-HBs may coexist with virions and subviral HBsAg particles for many years before viral clearance and loss of HBsAg. The concept of a relatively nonoverlapping seroconversion from HBeAg-positive to anti-HBe positive status during CHB may need amendment. The recent advances in HBV DNA testing have also provided new tools to investigate HBV infections. Molecular assays for HBV DNA can be useful for resolving ambiguous serologic patterns, but the major role of HBV DNA assays is their use in staging patients in the various phases of CHB and for monitoring patients undergoing antiviral therapy. Assays must be normalized to the World Health Organization international standard to permit expression of viral load in international units per milliliter (IU/ml), allowing results of different studies to be compared. Molecular assays are now being developed to detect intrahepatic HBV replicative forms. Assays for intrahepatic HBV cccDNA and pgRNA have now been published, although problems related to assay standardization need to be overcome before they can be classified as a routine part of the diagnostic armamentarium. However, initial studies have been promising and have shown that in patients on antiviral therapy, the intrahepatic HBV cccDNA levels at the end of treatment were actually a superior indicator of sustained response compared with serum HBV DNA levels.9Sung J.J. Wong M.L. Bowden S. et al.Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy.Gastroenterology. 2005; 128: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Furthermore, patients who underwent HBeAg seroconversion were more likely to have a lower baseline level of HBV cccDNA at the initiation of therapy.10Werle-Lapostolle B. Bowden S. Locarnini S. et al.Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.Gastroenterology. 2004; 126: 1750-1758Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar High HBV cccDNA levels have also been shown to be predictive of HBV reactivation in HBsAg-positive lymphoma patients treated with cytotoxic chemotherapy.11Hui C.K. Bowden S. Jackson K. et al.Clinical significance of intrahepatic hepatitis B virus covalently closed circular DNA in chronic hepatitis B patients who received cytotoxic chemotherapy.Blood. 2005; 105: 2616-2617Crossref PubMed Scopus (42) Google Scholar Thus, the monitoring of intrahepatic HBV intermediates may provide new insights into the natural history of hepatitis B and response to antiviral therapy, to help refine existing treatment protocols.12Lok A.S. Navigating the maze of hepatitis B treatments.Gastroenterology. 2007; 132: 1586-1594Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 13Ghany M. Liang T.J. Drug targets and molecular mechanisms of drug resistance in chronic hepatitis B.Gastroenterology. 2007; 132: 1574-1585Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar This issue of Gastroenterology contains 2 articles that further our understanding of the events that occur before, during, and after HBeAg seroconversion and also the natural history of CHB. Rather than being an acute immunologic event, as suggested by standard serologic assays, HBeAg seroconversion is shown to involve dynamic shifts of the serum HBV quasispecies pool, a process that begins years before a drop in the viral load is detectable.14Lim S.G. Cheng Y. Guindon S. et al.Viral quasi-species evolution during Hepatitis Be antigen seroconversion.Gastroenterology. 2007; 133: 951-958Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar This flux in peripheral viral populations reflects a shift in the intrahepatic compartment, where variants with reduced or absent HBeAg production accumulate. Furthermore, HBV replication is shown to be less efficient in the setting of HBeAg-negative disease, a process independent of the precore/basal core promoter (PC/BCP) mutations.15Volz T. Lutgehetmann M. Wachtler P. et al.Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.Gastroenterology. 2007; 133: 843-852Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar Lim et al14Lim S.G. Cheng Y. Guindon S. et al.Viral quasi-species evolution during Hepatitis Be antigen seroconversion.Gastroenterology. 2007; 133: 951-958Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar report the results of a detailed prospective study of the viral evolution occurring during perinatally acquired CHB infection, focusing on the events around and during HBeAg seroconversion. They investigated 4 groups of patients: spontaneous seroconverters (SS), nonseroconverter controls (CC), IFN-induced seroconverters (IR),16Wursthorn K. Lutgehetmann M. Dandri M. et al.Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.Hepatology. 2006; 44: 675-684Crossref PubMed Scopus (375) Google Scholar and IFN nonresponders (NR). At study entry, the SS and IFN-treated patients were in the “immunoelimination” phase of disease, marked by abnormal serum alanine aminotransferase (ALT). The CC group was selected by normal ALT and were likely still “immunotolerant.” All were infected with genotype B HBV. Serum was collected from 4–5 time points, representing on average a period of 30 months before and after HBeAg seroconversion. HBV DNA was extracted for polymerase chain reaction, cloning, and sequencing of the precore/core (PC/C) gene (20 clones per sample). More than 3386 sequences were then analyzed, comparing the clinical groups for viral sequence diversity, evolutionary rate, and positive selection (both at the nucleotide and amino acid level). The primary analysis focused on the nonoverlapping region of the PC/C gene (nt 1839–2306). The key finding in this study was that before HBeAg seroconversion, viral sequence diversity was found to be 2.4-fold higher in HBeAg seroconverters (SS, IR) compared with nonseroconverters (CC, NR). The selection pressure driving this diversity was operating up to 3 years before HBeAg seroconversion actually occurred. The viral diversity increased further after viral load reduction. The authors then determined the substitution rate in the PC/C gene, defined as the number of nucleotide substitutions per site per month. The substitution rate was 1 log higher in seroconverters compared to nonseroconverters; this was constant over the time period of the study. Phylogenetic trees of seroconverter patients were more complex, forming clusters over time with longer branch lengths. Positive selection in HBeAg and HBcAg protein sequences was detected in 14 of 20 seroconverters compared with only 1 of 20 nonseroconverters. The article extends previous work demonstrating that viral mutation rates are increased when comparing patients in the immunotolerant to the immunoelimination phase of CHB.17Bozkaya H. Akarca U.S. Ayola B. et al.High degree of conservation in the hepatitis B virus core gene during the immune tolerant phase in perinatally acquired chronic hepatitis B virus infection.J Hepatol. 1997; 26: 508-516Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 18Bozkaya H. Ayola B. Lok A.S. High rate of mutations in the hepatitis B core gene during the immune clearance phase of chronic hepatitis B virus infection.Hepatology. 1996; 24: 32-37Crossref PubMed Google Scholar, 19Hannoun C. Horal P. Lindh M. Long-term mutation rates in the hepatitis B virus genome.J Gen Virol. 2000; 81: 75-83Crossref PubMed Scopus (143) Google Scholar This is the first work that has performed such detailed virologic studies over such a prolonged time period. The second important message from this study by Lim et al is contained within the observation that nucleotide substitution rates persist at high levels after seroconversion, when viral diversity increases further. Far from the “nonreplicative state,” it is clear that there is an ongoing dynamic postseroconversion, albeit at lower levels of viral load, with viral diversity likely driven by an immune-based selection pressure. The second article in this issue compares patients in the immunoelimination phase of HBeAg-positive CHB to patients with HBeAg-negative CHB,15Volz T. Lutgehetmann M. Wachtler P. et al.Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.Gastroenterology. 2007; 133: 843-852Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar and by using molecular virologic techniques demonstrates that HBV replicates less efficiently in the HBeAg-negative phase of disease. Rather than examining viral quasispecies in peripheral blood, the authors have compared intrahepatic virologic profiles. Liver biopsies from 119 treatment-naïve patients with CHB (42 HBeAg-positive, 77 HBeAg-negative) were assayed for cccDNA (the intranuclear reservoir of HBV), and relaxed circular HBV DNA (rcDNA, used to estimate intrahepatic replicating virus). Virion productivity was defined as the ratio of rcDNA/cccDNA. pgRNA, the replicative RNA intermediate of HBV, was then measured as a marker of the transcriptional activity of cccDNA and the replication pathway. Pre-S/S mRNA was estimated as an alternative marker of cccDNA transcriptional activity. Data were compared with peripheral viral load and quantitative measures of HBsAg. Finally, the cccDNA was sequenced for analysis of genotype and PC/BCP variants. As has previously been documented,10Werle-Lapostolle B. Bowden S. Locarnini S. et al.Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.Gastroenterology. 2004; 126: 1750-1758Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, 16Wursthorn K. Lutgehetmann M. Dandri M. et al.Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.Hepatology. 2006; 44: 675-684Crossref PubMed Scopus (375) Google Scholar, 20Laras A. Koskinas J. Dimou E. et al.Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients.Hepatology. 2006; 44: 694-702Crossref PubMed Scopus (158) Google Scholar, 21Wong D.K. Yuen M.F. Yuan H. et al.Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients.Hepatology. 2004; 40: 727-737Crossref PubMed Scopus (118) Google Scholar patients with HBeAg-negative CHB had significantly lower levels of serum viral load, cccDNA (>1 log lower) and rcDNA (>2 log lower). Furthermore, virion productivity per cccDNA molecule was found to be 5-fold lower than in HBeAg-positive disease. Levels of pgRNA were correspondingly reduced, suggesting that the low virion productivity resulted from lower amounts of pgRNA. In contrast to patients with HBeAg-positive CHB, there was no correlation between serum viral load and the amount of cccDNA in the HBeAg-negative patients, consistent with the observed impairment of virion productivity. The HBsAg titres were significantly lower in the HBeAg-negative cohort, correlating with the cccDNA. However, Pre-S/S mRNA level and HBsAg titre per cccDNA molecule were similar between HBeAg-positive and negative patients, suggesting that the production of subviral particles was not impaired in the HBeAg-negative phase, but rather that the replication pathway might be selectively impaired. The authors investigated whether specific viral factors such as common PC/BCP variants or HBV genotype might explain these observations. As expected, mixed cccDNA populations containing PC/BCP variants were detectable in most HBeAg-negative patients. PC/BCP variants were also detectable in 53% of HBeAg-positive patients, consistent with the observation of Lim et al,14Lim S.G. Cheng Y. Guindon S. et al.Viral quasi-species evolution during Hepatitis Be antigen seroconversion.Gastroenterology. 2007; 133: 951-958Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar that variant viral populations are strongly selected before seroconversion. Impairment in virion productivity was not linked to the presence of these variants, however. In fact, in the HBeAg-negative patients in whom wild-type cccDNA molecules were not detectable, BCP mutations were associated with higher replication levels, consistent with previous in vitro data suggesting that it may represent a compensatory mutation.22Tacke F. Gehrke C. Luedde T. et al.Basal core promoter and precore mutations in the hepatitis B virus genome enhance replication efficacy of Lamivudine-resistant mutants.J Virol. 2004; 78: 8524-8535Crossref PubMed Scopus (102) Google Scholar Most of these Caucasian patients were found to be genotype A, D, or mixed A and D; no specific effect on productivity was identified. Volz et al15Volz T. Lutgehetmann M. Wachtler P. et al.Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.Gastroenterology. 2007; 133: 843-852Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar have described the intrahepatic molecular virologic events occurring as patients transition through seroconversion into HBeAg-negative disease, where virion productivity is reduced because of lower steady-state levels of pgRNA. The effect appears to be specific for the replication pathway; the production of subviral particles was spared. This cannot be attributed to the effect of PC/BCP mutations, because it may be observed in their absence. The mechanism reducing pgRNA levels remains unclear. Although no immunologic studies were performed in either study, it seems likely that host immune pressure is driving this viral evolution. Noncytolytic mechanisms have been demonstrated to be important in animal models of HBV, dependent on the key cytokines IFN-α/β, IFN-γ, and tumor necrosis factor (TNF)-α.23Wieland S.F. Chisari F.V. Stealth and cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar IFN-γ and TNF-α have been shown to decrease pgRNA by destabilizing the SSB/La autoantigen,24Heise T. Guidotti L.G. Cavanaugh V.J. et al.Hepatitis B virus RNA-binding proteins associated with cytokine-induced clearance of viral RNA from the liver of transgenic mice.J Virol. 1999; 73: 474-481Crossref PubMed Google Scholar, 25Heise T. Guidotti L.G. Chisari F.V. La autoantigen specifically recognizes a predicted stem-loop in hepatitis B virus RNA.J Virol. 1999; 73: 5767-5776Crossref PubMed Google Scholar, 26Heise T. Guidotti L.G. Chisari F.V. Characterization of nuclear RNases that cleave hepatitis B virus RNA near the La protein binding site.J Virol. 2001; 75: 6874-6883Crossref PubMed Scopus (51) Google Scholar and may therefore inhibit virion productivity by decreasing steady-state pgRNA. It has recently been noted that TLR2, a key stimulant of TNF-α production, is down-regulated in the setting of HBeAg-positive CHB relative to HBeAg-negative disease.6Visvanathan K. Skinner N.A. Thompson A.J. et al.Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.Hepatology. 2007; 45: 102-110Crossref PubMed Scopus (279) Google Scholar The precore protein itself appears to be critical for this effect. Increased innate immune signalling in the setting of HBeAg-negative disease, perhaps via TLR2-stimulated TNF-α, might therefore contribute to the observed phenomena. The evolving cytokine milieu within the liver may also be driving viral diversity with the positive selection of resistant variants, as observed in the first study by Lim et al; however, it may very well be that humoral responses, such as those described by Maruyama et al8Maruyama T. McLachlan A. Iino S. et al.The serology of chronic hepatitis B infection revisited.J Clin Invest. 1993; 91: 2586-2595Crossref PubMed Scopus (134) Google Scholar shape the viral quasispecies found in patients. Using this new information, it is now possible to refine the traditional view of the natural history of CHB, incorporating recent advances in the understanding of the importance of the immune system, with this molecular virologic data (Figure 1). Both studies provide new insight into the prolonged and dynamic virologic evolution that both precedes seroconversion and continues thereafter. HBeAg seroconversion is clearly not a “black and white” immunologic event, as suggested by existing commercial serologic assays. Rather, a progressive increase in the viral diversity is observed, with positive selection of viral quasispecies defective for HBeAg production, which are able to persist in the host, but at the cost of reduced replication phenotype. Identifying these selection pressures will be the critical next step for resolving the host–virus interplay in CHB, both HBeAg positive and HBeAg negative. These 2 detailed virologic studies have given new insight into the host–virus relationship and the various phases of the natural history of CHB. Similarly stringent immunologic studies, both innate and adaptive, are required to complete the picture. Viral Quasi-Species Evolution During Hepatitis Be Antigen SeroconversionGastroenterologyVol. 133Issue 3PreviewBackground & Aims: Although viral quasi-species evolution may be related to pathogenesis of disease, little is known about this in hepatitis B virus (HBV); consequently, we aimed to evaluate the evolution of HBV quasi-species in patients with well-characterized clinical phenotypes of chronic hepatitis B. Methods: Four cohorts of well-defined clinical phenotypes of chronic hepatitis B, hepatitis Be antigen (HBeAg) seroconverters (spontaneous seroconverters and interferon-induced seroconverters) and nonseroconverters (controls and interferon nonresponders) were followed during 60 months on average. Full-Text PDF
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