| Abstract|| |
Hepatitis B is the leading cause of viral hepatitis in the developing world; about 400 million people are infected with chronic hepatitis B worldwide. Continuing research on hepatitis B over the last few decades has added considerably to our understanding of its replication, pathology, natural history, diagnosis and treatment. This section highlights a few selected landmark research articles published during the past year in the field of Hepatitis B. These articles enhance our understanding of the basic nature of this infection and help us to treat patients with chronic infection better. Comments have been added after each abstract to highlight the reasons for selecting these articles.
Keywords: Hepatitis B, ccc DNA, HBV DNA, toll like receptor, TLR, natural history, treatment, lamivudine resistance
|How to cite this article:|
Piramanayagam P, Choudhuri G. Hepatitis B: News from the research world. Hep B Annual 2007;4:133-45
| Introduction|| |
Hepatitis B virus infection continues to be a major health problem across the globe. Around 40 million people in India alone are estimated to be harboring the infection. Research on this subject over the last few decades has added considerably to our understanding of its replication, pathology, natural history, diagnosis and treatment.
Ongoing research on this topic continues to throw up new knowledge that helps to understand and control this infection. In this article, we have selected 7 from over 10,000 articles published in 2007 on this topic for review; in our view they enhance our understanding of the basic nature of this infection, throw new light on the natural history of this infection or help us to treat patients with chronic infection better. We have added our comments after each abstract to highlight the reasons why we want to share the research information with you. Happy reading!!
| Basic Sciences|| |
Volz T, Lutgehetmann M, Wachtler P, Jacob A, Quaas A, Murray JM, et al . Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg negative patients. Gastroenterology 2007;133:843-52.
Background and Aims: Knowledge of factors regulating transcriptional activity of hepatitis B virus covalently closed circular DNA (ccc DNA) may help in understanding mechanisms of viral decay and how these processes are thwarted in chronically HBV infected patients. Methods: Liver biopsies from 119 treatment naοve chronically infected patients (42 HBeAg positive and 77 HBeAg negative) were assessed for HBV transcriptional and replicative activity. Results: Significantly lower median serum HBV DNA (-4 log), intrahepatic HBV DNA (-2 log) and ccc DNA (-1 log) amounts were measured in HBeAg negative compared with HBeAg positive patients. Despite a good correlation found between intrahepatic amounts of progeny virions and serum HBV DNA in all patients, ccc DNA levels did not correlate with serum titers in HBeAg negative individuals. Analysis of HBV RNA transcripts showed that impaired virion productivity in HBeAg negative individuals was due to lower steady state levels of pregenomic RNA produced per ccc DNA. Interestingly, pre S/S RNA levels and serum HBsAg concentrations did not differ between HBeAg negative patients when normalized for ccc DNA contents showing that subviral particle production was not impaired in HBeAg negative patients and correlated with ccc DNA levels. Although majority of HBeAg negative individuals harbored ccc DNA with common precore and/or basal core promoter mutations, occurrence of these variants was not responsible for reduced viral replication. Instead, replacement of wild type ccc DNA with core promoter mutants reestablished high virion productivity. Conclusions: Lower viremia in HBeAg negative individuals is not only due to lower ccc DNA content but also impaired virion productivity, which can arise without emergence of HBeAg variants and without affecting HBsAg production.
| Comments|| |
It has been common clinical observation that HBeAg negative patients have lower viral loads than HBeAg positive patients. This is also reflected in the lower viral cutoffs used for initiation of treatment in HBeAg negative patients [>4 log copies/ml] as compared with HBeAg positive patients [>5 log copies/ml].The reason for the lower HBV DNA observed in HBeAg negative patients was not clear. This study has tried to provide an answer.
HBV replication is a complex multi-step pathway which includes formation of closed circular covalent [ccc] DNA followed by its transcription to pregenomic RNA which is reverse transcribed into DNA [Figure 1]
Liver biopsies from HBeAg +ve and HBeAg -ve chronic HBV infected patients were studied for intrahepatic HBV DNA levels, ccc DNA levels, pregenomic RNA levels. It was found that significantly lower ccc DNA levels were found in HBeAg -ve patients as compared with HBeAg +ve patients. Moreover, each ccc DNA produced fewer pregenomic RNA in HBeAg -ve patients as compared with HBeAg +ve patients. The serum levels of HBV DNA were consequently lower in HBeAg -ve patients.
Visvanathan K, Skinner NA, Thompson AJ, Riordan SM, Sozzi V, Edwards R, et al . Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology 2007;45:102-10.
Toll-like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells and peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison with HBeAg-negative CHB and controls, whereas it was significantly increased in HBeAg-negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild-type HBV (HBeAg-positive), precore stop codon (G1896A) mutant HBV (HBeAg-negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF-a) and phospho-p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up-regulation of the TLR2 pathway leading to increased TNF-a production. Conclusion: This study demonstrates a potentially important interaction between HBeAg, HBV and the innate immune response.
| Comments|| |
Innate immunity has an important role in controlling viral infection. Toll like receptors (TLR) are important for identification of viral and bacterial pathogens. They are trans-membrane receptors which on activation induce transcription of antiviral cytokines that result in control of infection. Decreased expression of TLR may lead to decreased immune responsiveness.
HBV infection becomes chronic by escaping from such antiviral cytokines. Presence of the "e" antigen is associated with HBV replication. "e"+ patients have higher HBV DNA loads than "e" -ve patients. The mechanism may include escape from immunological pressure. Could HBeAg mask HBV from TLR mediated immune pressure?
This study has investigated whether the "e" antigen might be playing a role in protecting HBV from recognition by the Toll like receptors. The authors found decreased TLR 2 expression in monocytes, liver cells and Kupffer cells in HBe Ag positive patients as compared with HBeAg negative patients. They postulated that HBeAg may decrease TLR expression. Moreover, antiviral cytokine [TNF -α] production is decreased in HBeAg positive patients as compared with HBeAg negative patients. These findings suggest that HBeAg decreased TLR expression and thereby protected HBV from immune response, leading to higher HBV DNA viral load in HBeAg positive patients.
| Natural History|| |
Fattovich G, Olivari N, Pasino M, D'Onofrio M, Martone E, Donato F. Long-term outcome of chronic hepatitis B in Caucasian patients: Mortality after 25 years. Gut 2008;57:84-90.
Objective: To assess risk factors for liver-related death, we re-evaluated, after a median follow-up of 25 years, a cohort of 70 Caucasian patients with hepatitis B e antigen (HBeAg) positive chronic hepatitis (CH) at presentation. Methods: Follow-up studies included clinical and ultrasound examinations, biochemical and virologic tests and cause of death. Results: Sixty one (87%) patients underwent spontaneous HBeAg seroconversion. During a median period of 22.8 years after HBeAg seroclearance, 40 (66%) patients became inactive carriers, whereas the remaining 21 (34%) showed alanine aminotransferase elevation: 1 (1%) had HBeAg reversion, 9 (15%) detectable serum HBV-DNA but were negative for HBeAg, 8 (13%) concurrent virus(es) infection and 3 (5%) concurrent non alcoholic fatty liver disease. Liver-related death occurred in 11 (15.7%) patients, caused by hepatocellular carcinoma in 5 and liver failure in 6. The 25-year survival probability was 40% in patients persistently HBeAg positive, 50% in patients with HBeAg negative CH or HBeAg reversion and 95% in inactive carriers. Older age, male sex, cirrhosis at entry and absence of sustained remission predicted liver-related death independently. The adjusted hazard ratios (95% CI) for liver related death were 33 (3.01-363) for persistently HBeAg positive patients and 38.73 (4.65-322) for those with HBeAg negative CH or HBeAg reversion relative to inactive carriers. Conclusion: Most patients with HBeAg seroconversion became inactive carriers with very good prognosis. The risk of liver-related mortality in Caucasian adults with CH is strongly related with sustained disease activity and ongoing high level of HBV replication independently of HBeAg status.
| Comments|| |
Studies on long term clinical outcome of patients chronically infected with HBV help in understanding the natural history of this infection. Chronic HBV infection passes through 3 phases: the immunotolerant phase, the immunoactive phase and the seroconverted phase. During the immunotolerant phase, HBV replicates quickly and is characterized by expression of the "e" antigen. Over time, the body's immune system recognizes and targets the virus with mounting immunological pressure and tries to clear HBV infected hepatocytes marking the onset of immunoactive phase.
Traditionally, the immune-active phase has been associated with liver cell injury and determines clinical outcome of HBV infection. Outcome is dependent on the equilibrium between immune pressure and HBV replication. Heightened immune response leads to suppression of HBV replication, but seldom complete HBV clearance. Repeated cycles of HBV replication and immune mediated liver cell injury lead to liver cirrhosis and its attendant complications. HBV is oncogenic and may lead to HCC formation even without progression to liver cirrhosis.
In this retrospective, long-term [mean follow-up: 22 years] study, the outcome of chronically infected HBV patients gets clearer. Of the 70 HBeAg positive Caucasian patients studied, >80% achieved spontaneous seroconversion. Of these, 2/3 rd remained inactive while 15% had persisitent low level HBV viral replication. 15% had liver related mortality over 20 year follow-up with liver decompensation and HCC responsible in equal proportions. Old age, male and cirrhosis were found to be independent predictors of liver related mortality. Details regarding clinically relevant outcomes like progression of cirrhosis, incidence of varices/portal hypertension have not been addressed in this study.
The finding suggest that the majority of Caucasian patients with initial "e"+ disease undergo "e" seroconversion over time and in most of those who do so, the infection remains inactive and inconsequential; in only a small proportion is there active viral replication and a bad liver-related outcome. Reassuring as it may sound, the observations may apply to only Caucasians, who usually have genotypes A or D and in whom the infection is often acquired later in life. Whether these observations would apply to South Asians with different genotypes (B/C) perinatally acquired, remains to be validated.
Chu CM, Liaw YF. Predictive factors for reactivation of hepatitis B following hepatitis B e antigen seroconversion in chronic hepatitis B. Gastroenterology 2007;133:1458-65.
Background and Aims: Predictors of reactivation of hepatitis B following hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B have rarely been reported before and deserve further study. Methods: A total of 133 HBeAg-positive asymptomatic carriers who have undergone HBeAg seroconversion were studied. Reactivation of hepatitis B was defined as elevation of alanine aminotransferase >2 x upper normal limit accompanied by serum hepatitis B virus DNA detectable by hybridization assays. Results: The samples consisted of 75 men and 58 women and the mean age at entry was 28.2 ± 6.9 years. One hundred eight subjects had genotype B and 25 had genotype C. The maximal alanine aminotransferase levels during the HBeAg-positive phase were <2, 2-5 and >5 x upper normal limit in 49, 40 and 44 subjects, respectively. HBeAg seroconversion occurred after a mean follow-up of 4.6 ± 3.7 years. During a mean follow-up of 5.8 ± 4.6 years following HBeAg seroconversion, reactivation of hepatitis B occurred in 26 patients at 3.3% per year. Multivariate analyses demonstrated that reactivation of hepatitis B correlated significantly with genotype C (P =0.003), male sex (P =0.03), alanine aminotransferase levels >5 x upper normal limit during the HBeAg-positive phase (P =0.02) and age at HBeAg seroconversion >/=40 years (P =0.002). Conclusions: At baseline, genotype C and male sex are independent factors predictive of reactivation of hepatitis B. Additionally, the likelihood of reactivation of hepatitis B is increased if more rigorous immune-mediated hepatocytolysis or more prolonged immune clearance phase is necessary to eliminate the virus.
In the immune active phase, HBV replication is determined by the dynamic equilibrium between HBV replicative potency and the host's immune pressure. After a phase of low HBV replication post HBeAg seroconversion, a variable proportion of patients switch gear and demonstrate reactivation with high HBV viral load. Such reactivation and subsequent attempts at immune clearance leads to progressive liver injury.
Who are the patients who are likely to exhibit such reactivation? This prospective study has tried to identify factors predictive of HBV reactivation in HBeAg seroconverted patients. Reactivation after seroconversion occurred in 20% [26/133] patients during a mean follow-up of 5 years. Genotype C was associated with higher incidence of reactivation. Elderly males and high ALT during immune active phase were predictive of reactivation.
Why is identification of this subset important and what is the clinical importance of this observation? Patients who are likely to have reactivation may benefit from prolonged therapy or close monitoring post seroconversion. This study had included predominantly Genotype B and C patients. In India, Genotype A and D are the commonest genotypes. Whether the same predictive factors apply in Indian setting needs to be studied further.
| Treatment|| |
Hellstrom U, Lindh M, Krogsgaard K, Sylvan S. Demonstration of an association between detection of IgG antibody reactivity towards the C-terminal region of the preS1 protein of hepatitis B virus and the capacity to respond to interferon therapy in chronic hepatitis B. J Gastroenterol Hepatol 2008 May;23(5):804-10. Epub 2007 Oct 10.
Background and Aim: The treatment of hepatitis B virus (HBV) remains complex, with somewhat unpredictable responses. The aim of this study was to determine the predictive value of the pretreatment presence of circulatory antibodies towards a synthetic peptide mimicking the amino acids 94-117 of the preS1 protein of HBV and the capacity to respond to alpha-inteferon (IFN-alpha) treatment. Methods: The anti preS1(94-117) antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA) and the response to IFN-alpha therapy was judged by the effect on the viral kinetics as measured by an assay based on quantitative polymerase chain reaction during the treatment and follow up. Results: We found a significant (p< 0.001) correlation between the pretreatment presence of anti preS1(94-117) antibodies and a decrease in viral levels on follow up after the end of IFN-alpha therapy. The combined response of HBV DNA suppression (P < 0.001), hepatitis B e antigen (HBeAg) loss (P < 0.0001), anti-HBe seroconversion (P < 0.005) and AST aminotransferase normalization (P < 0.01) was also highly associated with the pretreatment presence of anti preS1(94-117) antibodies. Conclusion: The positive predictive value (PPV) of anti preS1(94-117) in determining a virological response was 83% and the negative predictive value (NPV) was 100%, indicating that in the absence of pretreatment anti preS1 reactivity virtually no patient has the capacity to respond to IFN-alpha therapy. Our findings may help to improve the efficacy of IFN-alpha therapy for chronic hepatitis B (CHB) by guiding the selection of patients for treatment and optimizing the clinical management of the individual patient.
| Comment|| |
Although IFN therapy for HBV infection has the advantage of finite duration and perhaps a more stable suppression of the infection, it achieves HBeAg clearance in only 30% of patients. It is also associated with significant side-effects, which include liver decompensation, fever, myalgia, bone marrow suppression, hypothyroidism, that limit its utility. Hence, being able to identify those who are likely to have a favorable response despite these hardships has been a long felt need. Few established predictors include low serum HBV DNA level and high serum ALT levels at initiation of therapy. Infection with HBV at birth or early in the patient's life (as is often the case in countries where HBV infection is hyperendemic, such as South East Asia) is predictive of poor response to IFN. These predictors have poor reliability in a given patient.
Serological markers for determining response to interferon may be applicable in individual patients and may be helpful in a clinical setting. In this study, the correlation between the presence of circulatory antibodies to preS1 protein of HBV and response to interferon was examined. This study demonstrated 83% positive predictive value of the pretreatment presence of circulatory antibodies [towards a synthetic peptide mimicking the amino acids 94-117 of the preS1 protein of HBV] and the capacity to respond to alpha-inteferon (IFN-alpha) treatment. More important to note is that 100% of those without these antibodies did not respond to interferon. Such serological markers need to be validated in large prospective studies and if proven may be a significant advancement in guiding therapy in HBV infection.
Lampertico P, Vigaṇ M, Manenti E, Iavarone M, Sablon E, Colombo M. Low resistance to adefovir combined with lamivudine: A 3-year study of 145 lamivudine-resistant hepatitis B patients. Gastroenterology 2007;133:1445-51.
Background and Aims: Adefovir monotherapy is an established treatment modality for lamivudine-experienced patients with chronic hepatitis B, but it carries a significant risk of resistance in the long-term. We assessed whether this risk could be overcome by adefovir-lamivudine combination therapy. Methods: A total of 145 lamivudine-resistant patients with chronic hepatitis B (73% cirrhotics, 86% hepatitis B e antigen negative, 92% genotype D) were treated with adefovir 10 mg in addition to lamivudine 100 mg. Liver function tests and hepatitis B virus (HBV) DNA (Versant 3.0) were assessed bimonthly, whereas adefovir-related mutations were searched by INNOLiPA assay at baseline and at yearly intervals. Results: During 42 months (range, 12-74), 116 patients (80%) cleared serum HBV DNA, 67 (84%) had normalized alanine aminotransferase levels and 145 (100%) remained free of virologic and clinical breakthroughs, independently of the degree of HBV suppression. The rtA181V/T was the only adefovir-related mutation detected, which occurred in 6 patients at baseline (4%; 1 rtA181V and 5 rtA181T) and in an additional 3 patients (2%; all rtA181T) during treatment. In all these 9 patients, HBV DNA levels progressively declined during therapy to become undetectable in 7 (78%). The 1-, 2-, 3- and 4-year cumulative rates of de novo rtA181T were 1%, 2%, 4% and 4%, respectively. None of the cirrhotic patients clinically decompensated, but 11 (12%) developed hepatocellular carcinoma. Conclusions: Under prolonged adefovir-lamivudine therapy, patients with lamivudine-resistant hepatitis B were unlikely to develop genotypic resistance to adefovir and had durable prevention of virologic and clinical breakthrough.
| Comment|| |
In resource limited countries with high HBV prevalence, such as India, lamivudine has been in common use due to its availability and affordability. Long-term lamivudine therapy is however associated with high rates of resistance, with 70% of patients develop resistance over 4 years. Lamivudine resistant HBV hepatitis is a major health concern in the present decade. Treatment options in such patients include "add on" adefovir or a change to other nucleoside analogues [adefovir, entecavir]. Based on small randomized trials, "add on" therapy was shown to develop low levels of adefovir resistance and was preferred to "switch over" therapy with adefovir in lamivudine resistant patients.
The present study describes the results of 145 lamivudine resistant patients treated with adefovir and lamivudine combination therapy followed up over a period of 42 months with HBV DNA and LFT done bi monthly. Presence of adefovir related mutation was detected by Inno-LiPA; 86% of patients were HBeAg positive. None of these patients on adefovir and lamivudine combination developed virological or clinical breakthrough during follow up; 80% of patients cleared HBV DNA while only 4% showed de novo adefovir resistance.
What is the clinical implication of this study? This is the largest reported follow-up cohort study of lamivudine resistant chronic HBV hepatitis patients treated with combination of adefovir and lamivudine. Apart from Entecavir, this combination may be considered as an alternative. Randomized controlled trial comparing entecavir with combination of adefovir and lamivudine will prove if this combination therapy is as effective, while being immensely affordable as well.
Hatakeyama T, Noguchi C, Hiraga N, Mori N, Tsuge M, Imamura M, et al . Serum HBV RNA is a predictor of early emergence of the YMDD mutant in patients treated with lamivudine. Hepatology 2007;45:1179-86.
Lamivudine (LAM) is a nucleoside analogue widely used for the treatment of chronic hepatitis B virus (HBV) infection. Emergence of resistant strains with amino acid substitutions in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of reverse transcriptase is a serious problem in patients on LAM therapy. The amount of covalently closed circular DNA in the serum is reported to be higher in patients who develop YMDD mutants than in those without mutants. However, there is no useful serum marker that can predict early emergence of mutants during LAM therapy. Analysis of patients who were treated with entecavir (n = 7) and LAM (n = 36) showed some patients had high serum levels of HBV RNA. Median serum levels of HBV RNA were significantly higher in patients in whom the YMDD mutant had emerged within 1 year (n = 6, 1.688 log copies/ml) than in those in whom the YMDD mutant emerged more than 1 year after treatment (n = 12, 0.456 log copies/ml, P = 0.0125) or in whom the YMDD mutant never emerged (n = 18, 0.688 log copies/ml, P = 0.039). Our results suggest that HBV RNA is a valuable predictor of early occurrence of viral mutation during LAM therapy.
| Comment|| |
Lamivudine resistance is a common clinical problem encountered in hepatology practice. HBV DNA polymerase gene mutations associated with lamivudine resistance include L180M and M204I. Detection of these mutants is the earliest indicator for future clinical deterioration. This, in effect, predates clinical deterioration but is an indicator of established resistance mutation. But, prevention of development of resistance is the main aim of long term treatment with nucleoside analogues. This requires that we identify factors that can predict the development of resistant mutation at a later period.
This study examined the relation of HBV RNA levels in serum and development of YMDD mutation. Median HBV RNA levels were significantly higher in whom YMDD resistant mutants emerged within 1 year of lamivudine therapy.
Why is early identification of YMDD mutants important? The clinical outcome of emergence of resistant stains depends on the interaction between wild and mutant stains. Early predictors of YMDD mutants will alert the clinician to have close monitoring of HBV viral load and liver functions. Whether such monitoring changes therapeutic decisions awaits future clinical research.
Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow-226 014
Source of Support: None, Conflict of Interest: None