Liver Disease and Therapy of Hepatitis B Virus Infections
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1 Liver Disease and Therapy of Hepatitis B Virus Infections University of Adelaide Catherine Scougall Arend Grosse Huey-Chi Low Allison Jilbert Fox Chase Cancer Center Chunxiao Xu Carol Aldrich Sam Litwin Jeffry Saputelli Southwest Foundation for Biomedical Research Debbie Chevaz Robert Lanford QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Chen Liu Eugene Schiff QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Matthew Yeh
2 Liver Disease and Therapy of Hepatitis B Virus Infections in HIV patients Is there a risk in delaying antiviral treatment of HBV in HIV carriers (and non-hiv infected patients)?
3 HBV causes transient and chronic infections of the liver HBV productively infects hepatocytes, the main parenchymal cell of the liver Early in the course of an infection, all hepatocytes are infected Infection is non-cytopathic Carriers may have a high titer viremia (up to per ml), but titers in long term infections are generally much lower (<10 8 )
4 The immune system can rapidly resolve transient infections even after all hepatocytes are infected Chronic infections are generally life-long
5 Hepatocytes, the target of infection Hepatocytes are a closed, largely self-renewing population
6 Liver disease in HBV carriers is the result of the antiviral immune response to infected hepatocytes Persistent killing of infected hepatocytes (0.1 to >1% per day) leads to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Cirrhosis and HCC typically appear after years of infection. What causes HCC? Hepatocytes constitute a largely closed population and may therefore evolve under these selective pressures: 1. Persistent immune killing leads to clonal hepatocyte re-population, with emergence and clonal expansion of hepatocytes that are resistant to HBV infection/expression
7 After several decades of infection, most hepatocytes no longer support HBV replication anti-hbsag, no cirrhosis
8 What causes HCC? Hepatocytes constitute a largely closed population and may therefore evolve under selective pressures: 1. Persistent immune killing leads to clonal hepatocyte re-population, with emergence and clonal expansion of hepatocytes that are resistant to HBV infection/expression 2. Cirrhosis alters blood flow and lobular structure, and also leads to clonal hepatocyte re-population
9 Cirrhosis may also develop in middle age Hepatitis Cirrhosis
10 What causes HCC? Hepatocytes constitute a closed population and may therefore evolve under selective pressures: 1. Persistent immune killing leads to clonal hepatocyte re-population, with emergence and clonal expansion of hepatocytes that are resistant to HBV infection/expression 2. Cirrhosis alters blood flow and lobular structure, and also leads to clonal hepatocyte re-population Clonal hepatocyte re-population is an HCC risk factor.
11 Chronic HBV infection worldwide: 350 million 60 million of these will likely die by middle age of cirrhosis or HCC
12 Effective antiviral therapy in the later stages of HBV infection prevents worsening of cirrhosis and reduces the risk of HCC
13 Problem: Almost everything that is known about HBV associated liver diseases and HCC prevention comes from clinical studies starting with patients with decades of chronic infection. These studies have led to two biases: 1. Chronic liver disease will be reflected by fibrosis, cirrhosis, HCC and other signs of cumulative liver damage 2. Therefore, there is no reason to treat HBV carriers lacking these signs and symptom
14 Resultant HBV treatment guidelines Delay antiviral therapy until there are evident signs of immune mediated liver damage (hepatitis, elevated liver enzymes, fibrosis, etc)--that is, until the carrier is sick Practical reasons for delaying treatment (historical) Interferon alpha therapy ineffective in absence of active hepatitis Monotherapy with early nucleosides (lamivudine, in particular) is most effective in patients with active liver disease (i.e., hepatitis) due to emergence of drug resistant virus
15 Virus replication cycle and treatment options
16 Viral gene products Core protein nucleocapsid subunit HBeAg Three viral membrane proteins Reverse transcriptase Viral genome and mrnas X protein (function unknown, non-structural protein essential for virus replication) 3.2 kbp RC DNA
17 HBV replication Infection maintained by ~30 copies of cccdna. cccdna is stable and appears to survive mitosis. Integration Thus, cccdna can only be eliminated by a combination of cell death and compensatory cell division. ~90% ~10% dsl DNA
18 Approaches to antiviral therapy Immune Stimulation: The immune system is capable of curing a short term infection even when all hepatocytes have been infected (mechanism uncertain) Inhibitors of viral DNA synthesis: cccdna will be lost as infected hepatocytes die off. Because cccdna survives mitosis, about 2.6 turnovers would be required to remove all cccdna
19 Current treatment options (HBV) Interferon alpha Only useful in the immune clearance phase of infection (~20-30% cure rate) nrti Monotherapy High probability of resistance Control of infection reduces progression of liver disease, cirrhosis, and HCC nrti Multi-drug therapy Should work better, experience still limited (FTC+Tenofovir in HIV carriers)
20 nrtis Treatment options (HIV) nnrtis (not available for HBV) Protease inhibitors (boosted or not) Uptake inhibitors Integrase inhibitors
21 Improved? treatment options for HBV Combination therapy (tenofovir and FTC) is not yet approved for HBV carriers (with no HIV co-infection). It is not yet clear if resistance to Tenofovir monotherapy will be a problem and, if so, what would be used for salvage therapy. Entecavir resistance also requires multiple RT mutations
22 Decision to treat--when is there a need: Should therapy be initiated while patients are still in the immune tolerant phase of infection? Approach: Assay for clonal expansion of hepatocytes in patients in the immuno-tolerant phase of infection
23 Hepadnavirus replication Integration Integration of linear virus DNA (DSL DNA) occurs as a by-product of virus infections ~90% ~10% dsl DNA Integration creates unique virus-cell junctions that can be detected by inverse nested PCR Virus-cell junctions can be used as genetic markers of individual hepatocytes & their progeny
24 Inverse PCR assays for integrated viral DNA detects clonal expansion of human hepatocytes
25 Inverse nested PCR assay to detect integrated HBV DNA in human liver Extract DNA from 1 mg liver fragments ~250,000 cells & assay for virus/cell junctions Invert DNA for PCR Serially dilute into 96 well trays Amplify by nested PCR Purify products on agarose gels Sequence individual products to identify virus-cell junctions
26 Hepatocyte clone size Clones of >1,000 hepatocytes detected by inverse PCR constituted ~1% of hepatocytes in 5 long-term HBV patients The percent of hepatocytes in clones of >1,000 is indicated in parentheses P1 P2 P3 P4 P Adapted from Mason et al., J. Virol. 84, 8308,
27 Clonal expansion in 5 young carriers in immunotolerant phase of infection Patient 1: Clonal expansion of hepatocytes occurs during the immune tolerance stage of infection! Clone of >1000 hepatocytes identified, as well as clones of several hundred hepatocytes.
28 Out of liver biopsy samples from 5 immunotolerant patients under 30 years of age, 3 showed similar evidence for clonal hepatocyte expansion The results imply that cumulative liver damage, as reflected by clonal hepatocyte expansion, occurs prior to more obvious clinical signs of progressive liver disease
29 Conclusions: Clonal hepatocyte re-population, an HCC risk factor, can apparently begin during the immuno-tolerant phase of infection Multi-drug therapy should be strongly considered for HBV patients in the immune tolerant phase of infection and co-infected patients no matter what the stage of infection. Emergence of foci of virus free/resistant hepatocytes would be a useful and probably easier correlate than a molecular assay for hepatocyte clones
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