ÁNGELES GARCÍA PARDOsgfm.elcorteingles.es/SGFM/FRA/recursos/doc/2013/... · Web viewAnti-HBV and...

Simposio Internacional: Control o erradicación de las hepatitis virales B y CInternational Symposium: Control or eradication of viral hepatitis B and C

Barcelona, 30 y 31 de mayo de 2013 Barcelona, May 30-31, 2013

Inicio

1. Hepatitis C virus life cycle, Volker Lohmann

2. Burden of viral hepatitis in Europe and World-Wide, Heiner Wedemeyer

3. Immune Response to HBV and HCV, Kyong-Mi Chang

4. Present and future treatment of HCV using Direct Antiviral Agents (DAAs), Rafael

Esteban Mur

5. New insights into HCV replication: potential antiviral targets, Charles Rice

6. Animal models to study HBV/HDV infections, Maura Dandri

7. Variability and challenges to eradicate HBV, Fabien Zoulim

8. Anti-HCV treatment in Liver transplanted patients, Xavier Forns Bernhardt

9. Anti-HBV and HCV treatments in HIV coinfected patients, Javier García Samaniego

Rey

10.Antiviral resistance mutations to anti-HCV treatments and long-term persistence,

Christoph Sarrazin

11.Challenges of ultra-deep pyrosequencing to study clinical relevant minority mutants,

Niko Beerenwinke

12.The interplay between mutagenesis and inhibition. Novel strategies of antiviral

therapy, Esteban Domingo Solans

FUNDACIÓN RAMÓN ARECES



Hepatitis C virus life cycle, Volker Lohmann

Hepatitis C virus infections cause severe liver disease and are a major global health

burden. Accessibility of in vitro models and cell culture systems allowed a detailed

understanding of several steps of the viral replication cycle in molecular detail. HCV enters

the cell by a well defined set of receptors and entry factors, the positive strand RNA

genome is released into the cytoplasm and drives translation of a polyprotein, which is

processed by cellular and viral proteases. The viral nonstructural proteins then induce

membrane alterations harboring the sites of viral RNA replication. Viral RNA synthesis and

the formation of the viral replication sites involve a concerted action of viral nonstructural

proteins and host factors like microRNA-122, cyclophilin A, and PI4KIIIα. Almost all

antivirals approved for therapy or in late clinical development target polyprotein processing

or viral and host proteins involved in RNA synthesis. The presentation will therefore

particularly focus on our current understanding of the distinct steps involved in viral RNA

replication and summarize our current knowledge on the mode of action of some of the

inhibitors interfering with these processes.

VOLVER

Burden of viral hepatitis in Europe and World-Wide, Heiner Wedemeyer

Viral hepatitis is a major global health burden. An estimated 500 Million individuals are

carriers of a hepatitis virus and up to 1 Million deaths each year are caused by virus-

induced end-stage liver disease or hepatocellular carcinoma. In addition, acute liver failure

is not uncommon. E.g., 70.000 patients die each year from acute hepatitis E.

Overall, hepatitis B is the most prevalent chronic hepatitis virus infection with about 350

Million HBsAg carriers. Importantly, it is estimated that 15-20 Million patients are co-

infected with hepatitis delta virus (HDV). HDV is considered the most severe form of

chronic viral hepatitis. The global disease burden caused by HCV is still increasing and

may peak during this century.

Fortunately, novel infection with HBV and HCV did significantly decline during the last 20

years. HBV vaccination is recommended by most (but not all!) countries. HCV infection is




mainly present in drug user populations but medical procedures are still a main reason of

new HCV infection. Few prophylactic HCV vaccine programs are ongoing but it will be

challenging to prove efficacy. An HEV vaccine has been licensed in China in 2012. It is not

clear yet when and if this vaccine will become available in other countries.

There are several challenges to reduce the disease burden caused by viral hepatitis.

Access to screening and treatment must be ensured but hepatitis virus infections

frequently affect special populations such as drug user, migrant populations, prisoner or

individuals coinfected with other pathogens. Even though promising new therapies will

become available in the near future it is currently unclear if the new drugs will reach all

patients at need in resource-limited settings.VOLVER

Immune Response to HBV and HCV, Kyong-Mi Chang

HBV and HCV are both hepatotropic viruses that can persist and cause progressive liver

disease, including cirrhosis and hepatocellular carcinoma. The outcome of viral infection

is defined by the interplay between the virus and host immune defense. Both HBV and

HCV have multiple mechanisms to evade or subvert innate cellular immune pathways with

a delayed onset of adaptive immune response. While T cells play a key role in viral

clearance and protective immunity, both HBV and HCV persists with the induction of

multiple extrinsic and intrinsic T cell regulatory mechanisms including Tregs, regulatory

cytokines (e.g. IL-10) and inhibitory costimulatory pathways (e.g. PD-1, CTLA-4, Tim-3).

These regulatory factors may be induced by ongoing viral replication and inflammation,

thereby limiting severe liver damage, but also inhibiting effective virus control and

ultimately contributing to chronic liver damage. Viral persistence (particularly for HCV) is

further associated with the induction of viral variants that escape B and T cell recognition,

that challenges the development of effective HCV vaccine. The natural history and

therapeutic outcomes of HBV and HCV infection are influenced by the underlying host

immunogenetics (e.g. IL28 polymorphism, HLA type) although their precise mechanisms

remains to be fully defined. This presentation will provide an update of host immune




responses and their regulation in HBV and HCV infection, relevant for their natural history

and therapy.VOLVER

Present and future treatment of HCV using Direct Antiviral Agents (DAAs), Rafael Esteban

Mur

Hepatitis C virus (HCV) chronically infects 2,3 % of the world population and 2,5% of the

Spanish population. Current standard treatment (“standard of care”, SOC) with pegylated

interferon (peg-IFN) and ribavirin (RBV) eradicates infection in about 50% of HCV

infected patients, but the associated side effects limit its effectiveness in many cases.

The recent development of small molecule compounds that directly inhibit the viral life

cycle represents a major milestone for the treatment of chronic hepatitis C virus (HCV)

infection. These new drugs that are collectively termed direct-acting antivirals (DAA)

mainly include a range of inhibitors of the non-structural (NS) 3/4A protease, NS5B

polymerase and NS5A protein. Two NS3/4A protease inhibitors (boceprevir and telaprevir)

in combination with pegIFN+RBV have been approved by the FDA and EMEA, for the

treatment of chronic HCV genotype 1 infection and cure rates could be increased by 20-

30%. However, the majority of DAAs (more than 60) are still in early clinical development.

Significant improvements on SVR rates have been achieved in ongoing trials using

combinations of DAAs with or without peg-IFN and/or RBV, and in some cases using

exclusively, oral treatment regimens. This will opens the possibility to include into therapy,

difficult-to-treat patients. VOLVER

New insights into HCV replication: potential antiviral targets, Charles Rice

An estimated 200 million people have been infected with hepatitis C virus (HCV) with a

majority unable to clear the virus. Chronic HCV infection can lead to cirrhosis,




hepatocellular carcinoma, and end-stage liver disease. It is generally believed that disease

results at least in part from immune mediated inflammation. Since HCV’s discovery in

1989 significant progress has been made in establishing experimental systems and

unraveling the details of the virus lifecycle. Examples include infectious molecular clones,

RNA replicons and a robust cell culture system based on an HCV isolate from a rare case

of acute fulminant hepatitis. Definition of the human factors required for HCV entry and

blunting innate immune response pathways has led to the development of a mouse model

that supports HCV entry, replication and virus production. Together these tools have

increased our understanding of the HCV lifecycle and revealed numerous targets for

therapeutic intervention. In 2011, two direct acting antivirals targeting the HCV serine

protease were approved in combination with the previous standard of care, pegylated type

I interferon and ribavirin. Even for the most difficult to treat HCV genotype (genotype I),

more that 70% of patients can now be successfully treated. The future is even brighter with

wealth of compounds in the pipeline against both virus and host targets, raising hopes for

an all-oral interferon-free regimen with broad genotype coverage, minimal side-effects and

a high cure rate.VOLVER

Animal models to study HBV/HDV infections, Maura Dandri

The obvious limitations imposed by the use of high primates and the necessity to perform

infection studies with small well-characterized animals led to the development of human

liver chimeric mice, such as the uPA/SCID mice. In this system, transgene-induced

hepatocyte damage creates the space and the regenerative stimulus needed for

transplanted hepatocytes to reconstitute the disease mouse liver, while the absence of

adaptive immune responses enables xenogenic engraftment. Humanized mice have

shown to remain susceptible to infection with HBV, HCV and HDV. Upon infection

establishment, mice invariably become chronically infected and allow preclinical evaluation

of compounds targeting any step of the viral life-cycle. We previously demonstrated the in

vivo capacity of entry inhibitors derived from the large envelope protein of HBV to prevent




de novo HBV and HDV infection, as well as HBV spreading post infection. Moreover, by

transplanting HBV-infected hepatocytes isolated from chimeric mice into new uPA/SCID

recipients we could investigate the impact of hepatocyte proliferation on cccDNA stability.

The drastic cccDNA reduction induced by cell division in the absence of antiviral agents

underscores a weak point in HBV persistence.

Within the mouse liver, human hepatocytes maintain a functional innate immune system

and respond to stimuli induced by exogenously applied cytokines. By administering

conventional human IFN-α we found that HBV inhibited the enhancement of various

interferon stimulated genes (ISGs) and STAT nuclear accumulation, although IFN-α could

induce temporary suppression of HBV replication by mediating epigenetic repression of

the cccDNA minichromosome. Notably, sustained human hepatocyte responsiveness

could be induced upon administration of the longer-acting peg-IFNα, which led to

substantial viral antigen decline within the first 12 weeks of treatment, thus suggesting that

the involvement of host´s immune components may not be required to trigger early HBsAg

decline in IFN-treated patients. Using humanized mice we recently studied the antiviral

state of the human hepatocytes in the setting of chronic HBV/HDV co-infection compared

to HBV mono-infection. Establishment of HDV infection was accompanied by a remarkable

induction of human ISGs, while HBV mono-infection led to only moderate ISG elevations.

Interestingly, enhancement of human-specific cytokine (i.e. TGF-ß, IFN-λ) was

predominantly found in HBV/HDV infected mice. These studies suggest that elevated pre-

treatment ISG and interferon levels may directly contribute to liver damage and

inflammation, providing a rationale for the more severe course of HDV-associated liver

disease. Such antiviral state induction might also explain the lower levels of HBV activity

frequently found in co-infected hepatocytes.VOLVER

Variability and challenges to eradicate HBV, Fabien Zoulim

HBV mainly infect hepatocytes. It replicates its DNA genome via a reverse transcriptase

phase. The persistence of viral covalently closed circular (ccc) DNA, the viral




minichromosome, in the nucleus of infected cells is responsible for the chronicity of the

infection at the level of a single cell. Because of its long half-life, long-term administration

of nucleos(t)ide analogues (NUC) is necessary to prevent relapses of viral replication.

However, because of the error rate of the viral polymerase and the spontaneous viral

genome variability, antiviral therapy may be associated with the selection of drug resistant

mutants. The advent of novel NUCs with a high barrier to resistance has limited the risk of

antiviral drug resistance. It is now possible to achieve viral suppression in the majority of

infected patients. The main challenge is to develop new treatment strategies toward a cure

of the infection with a clearance of HBsAg and/or cccDNA. HBsAg clearance is obtained in

only 10% of patients treated with NUCs or Interferon alpha. Several approaches are being

evaluated in experimental models. The clearance of cccDNA might require hepatocyte turn

over induced by immune cell killing, leading to equal or unequal dilution of cccDNA

through hepatocyte mitosis. The destruction of cccDNA may also be induced by the

induction of several cell signalling pathways leading to degradation by nucleases. If

cccDNA cannot be eliminated, its silencing can be another objective by interfering with its

epigenetic control with drugs altering the acetylation/methylation status of histones or via

the induction of cytokines, including interferon alpha. In that respect, the restoration of

innate responses that are repressed by HBV represent another interesting concept.

Studies investigating the effect of TLR agonists on the intrahepatic innate responses and

the reshaping of adaptive T cell responses are ongoing in experimental models.

Immunomodulatory approaches based on therapeutic vaccine activation of specific CD4+

and CD8+ T cell responses might also represent a complement to NUC based therapy.

These new developments should also help designing early treatment intervention

strategies to prevent the development of hepatocellular carcinoma.

References




Zoulim F, Locarnini S. Hepatitis B virus resistance to nucleos(t)ide analogues.

Gastroenterology. 2009 Nov;137(5):1593-608

Zoulim F, Luangsay S, Durantel D. Targeting Innate Immunity: A New Step in the

Development of Combination Therapy for Chronic Hepatitis B. Gastroenterology 2013, in

press

Zoulim F, Testoni B, Lebossé F. Kinetics of intrahepatic cccDNA and serum HBsAg during

antiviral therapy for chronic hepatitis B - Lessons from experimental and clinical studies.

Clinical Gastroenterology and Hepatology, 2013 in press.

Belloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T, Petersen J, Raimondo G,

Dandri M, Levrero M. IFN-α inhibits HBV transcription and replication in cell culture and in

humanized mice by targeting the epigenetic regulation of the nuclear cccDNA

minichromosome. J Clin Invest. 2012 Feb 1;122(2):529-37.

Zoulim F, Mason WS. Reasons to consider earlier treatment of chronic HBV infections.

Gut 2012;61:333-336.VOLVER

Anti-HCV treatment in Liver transplanted patients, Xavier Forns Bernhardt

Liver transplantation (LTx) is the only alternative for patients with end-stage liver cirrhosis

or hepatocellular carcinoma. Hepatitis B and C (particularly C) are the most common

cause of cirrhosis and liver cancer in our environment and therefore the first indication of

LTx. Since the introduction of anti-HBV immunoglobulin and especially new oral antivirals,

the recurrence of post-transplant HBV is no longer a problem and patients have an

excellent prognosis. Conversely, post-transplant HCV is the leading cause of liver graft

loss in the long term and represents a real problem for the majority of transplant programs.

Among the strategies we have to prevent this infection in the graft include antiviral

treatment on the waiting list (only in patients with compensated cirrhosis) and treatment in

the post-transplant (when liver damage has occurred). These strategies achieve HCV cure

infection in one third of patients. However, it is likely that in the next years prevention or


http://www.ncbi.nlm.nih.gov/pubmed/22251702








cure of HCV infection will be achieved in a much larger proportion of cases using direct

antiviral agents (DAAs) that provide greater efficiency, tolerance and safety.VOLVER

Anti-HBV and HCV treatments in HIV coinfected patients, Javier García Samaniego Rey




Coinfection with hepatitis B virus (HBV) is common in patients with human

immunodeficiency virus (HIV) infection because both agents share the same routes of

transmission. Chronic hepatitis B virus (HBV) infection is recognised in nearly 10% of HIV

infected patients. Co-infected individuals show an accelerated course of HBV-associated

liver disease with faster progression to cirrhosis. The indications for HBV therapy are the

same as in HIV-negative patients, based on HBV DNA levels, serum ALT levels and

histological lesions, irrespective of the CD4 cell count. Thus, most co-infected patients

should be simultaneously treated for both HIV and HBV. Tenofovir combined with

emtricitabine or lamivudine plus a third agent active against HIV is a good strategy in this

scenario. In a small number of patients with CD4 count >500/ml, chronic hepatitis B can be

treated before the institution of anti-retroviral therapy; pegylated interferon (mostly for HBe-

positive patients), adefovir and telbivudine, which are not proven to be active against HIV,

should be the therapeutic alternatives. Lamivudine, entecavir and tenofovir have activity

against both HIV and HBV and are contraindicated as single agents for hepatitis B in co-

infected patients because of the risk of developing HIV resistance.

Chronic hepatitis C is a leading cause of morbidity and mortality in HIV population. The

prevalence of HIV/HCV coinfection is very high among intravenous drug users. Thus,

chronic liver disease due to HCV infection is a major public health problem. Therapy with

pegylated interferon alfa plus ribavirin has a limited efficacy in coinfected patients. The

recent approval of the first direct acting antivirals (DAAs), telaprevir and boceprevir, in

combination with PegIFN/RBV for the treatment of HCV genotype 1 infection has

significantly increased the rate of sustained virological response in both monoinfected and

coinfected patients. These HCV treatment regimens represent a “new paradigm” for care of

HCV disease and may lead to higher rates of HCV eradication in HIV/HCV-coinfection

setting. However, the new concerns include the adequate management of serious adverse

effects (particularly anemia), adherence and drug-drug interactions with antiretroviral agents.VOLVER




Antiviral resistance mutations to anti-HCV treatments and long-term persistence, Christoph

Sarrazin

HCV replicates with a short half-life and high viral turn-over. The non-proof-reading HCV

NS5B RNA-dependent RNA-polymerase hereby generates a large number of closely

related but different viral isolates (termed quasispecies). HCV NS3/4A protease inhibitors

Boceprevir and Telaprevir have been approved for treatment of chronic hepatitis C

genotype 1 infected patients. Both, compounds showed a medium to strong antiviral

activity in phase 1 clinical studies but cannot be used as monotherapies because of a

rapid selection of pre-existent resistant variants followed by viral break-through.

At baseline, dominant variants associated with resistance to HCV protease inhibitors are

rarely detectable in treatment naïve patients (<1%) and the presence of such variants

does not exclude virologic response to triple-therapy. This is explained by a lack of cross-

resistance of these variants to pegylated interferon alfa / ribavirin. Thus, resistance

analysis at baseline is not required.

Overall, the likeliness of virologic treatment failure is associated with the individual

sensitivity to pegylated interferon / ribavirin treatment and increases in treatment naïve

patients from approx. 10% to previous null-responders to >50%. Stopping rules are

important because continuous dosing of protease-inhibitors in the presence of persistent

viral replication should be strongly avoided, as this might induce selection of compensatory

mutations with enhanced replicative fitness and long half-life.

Mutations within the NS3 protease associated with resistance to Boceprevir and Telaprevir

are largely overlapping (positions V36, T54, V55, R155, A156 and V170 of the NS3

protease) and this excludes a switch between the two drugs in case of viral break-through.

After the end-of-treatment the frequency of resistant variants within the viral quasispecies

in patients with treatment failure declines. After approx. 2 years in the fast majority of

patients (>80%) no resistant variants are detectable anymore by population based

sequencing. Whether persistence at high levels occurs in a minority of patients and

whether residual variants may influence virologic response to future direct antiviral

therapies has to be explored in future clinical trials.




Additional NS3 protease inhibitors and DAAs targeting other HCV proteins (i.e. NS5A

inhibitors, nucleoside and non-nucleoside NS5B inhibitors) are currently in development

and will be approved in the near future. Frequencies of pre-existent resistant variants,

failure due to selection of resistance and likeliness of persistence may be different for

different drugs of the same class and different classes.

For example baseline resistance mutation Q80K is frequently observed in previously

untreated HCV subtype 1a infected patients and this variant was associated with reduced

virologic response rates to the NS3 protease inhibitor simeprevir (TMC435).

Naturally occurring variants leading to resistance to NS5A inhibitors are rarely observed

(<8%) in untreated patients. However, different to NS3 resistant variants mutations

conferring resistance to NS5A inhibitors seem to persistent more frequently as

replicational fitness is not impaired.

Various variants confer resistance to non-nucleoside NS5B inhibitors depending on the

binding site at the NS5B protein and the specific compound. Rates of pre-existence

significantly vary from variant to variant and little is known so far on rates of persistence.

Pre-existence of resistance as well as detection of resistant variants in patients with

treatment failure to nucleoside polymerase inhibitors (i.e. mericitabine, sofosbuvir) has

been observed so far in single patients only.

VOLVER

Challenges of ultra-deep pyrosequencing to study clinical relevant minority mutants, Niko

Beerenwinke

Next-generation sequencing (NGS) allows for cost-effective probing of virus populations at

an unprecedented level of detail. The massively parallel sequencing approach can detect

low-frequency alleles and it provides a snapshot of the structure of the entire virus

population. However, analyzing ultra-deep sequencing data obtained from mixed virus

populations is challenging, because (i) data sets are very large, (ii) reads contain

amplification and sequencing errors, and (iii) the read length is typically shorter than the

genomic region of interest. Thus, ultra-deep sequencing experiments provide only indirect


http://www.bsse.ethz.ch/cbg/people/nikob

http://www.bsse.ethz.ch/cbg/people/nikob



evidence of the underlying viral population structure. We will discuss computational and

statistical methods for analyzing such data, including read error correction, haplotype

reconstruction, and haplotype frequency estimation. We present several bioinformatics

approaches, analyze their performance on simulated data and on data obtained from

control experiments and from clinical viral samples. If confounding factors are identified

and treated properly, then NGS can be used to investigate, in a quantitative manner, the

genetic diversity of RNA virus populations. VOLVER

The interplay between mutagenesis and inhibition. Novel strategies of antiviral therapy,

Esteban Domingo Solans

Lethal mutagenesis aims at extinguishing viruses by increasing the mutation rate above

the maximum value compatible with maintenance of viral functions. Model studies with

several viral-host systems have indicated the feasibility of this new strategy as a means to

counteract the adaptive potential of RNA viral quasispecies. In particular, experiments with

the RNA viruses foot-and-mouth disease virus and lymphocytic choriomeningitis virus

showed that when a mutagenic agent participates in therapy, a sequential inhibitor-

mutagen administration can be more effective in reducing the viral load and avoiding

selection of escape mutants than the corresponding combined administration (Perales et

al. TIM 20-595-603, 2012). Two mechanisms have been identified as being responsible of

the advantage of the sequential treatment: (i) the dynamics of interaction of the viral

quasispecies with an inhibitor and a mutagen, and (ii) the need of the mutagen-produced

defector genomes to replicate to exert their interfering activity (lethal defection) in the

transition towards virus extinction. To explore an extension of lethal mutagenesis protocols

to HCV, we first investigated whether ribavirin (Rib) acted as a mutagenic agent for HCV in

the cell culture system based on HCVcc [virus rescued by electroporation of Lunet cells

with a transcript from plasmid Jc1FLAG2 (p7-nsGluc2A) (Marukian et al. Hepatology 48:

1843-1850, 2008)] replicating in human hepatoma Huh-7.5 cells (collaboration with C.

Rice). HCVcc replication in the presence of ribavirin resulted in significant increases of




mutation frequencies and average genetic distances in the mutant spectrum. A mutational

bias in favour of G→A and C→U transitions provided additional evidence of a mutagenic

activity of ribavirin. Concerning antiviral efficacy, there was a range of inhibitory and

mutational activities under which a sequential inhibitor (telaprevir, cyclosporine A,

daclatasvir or interferon-)-mutagen (ribavirin) administration reduced the viral load more

effectively than the corresponding combination. The parameter range under which either

combination or sequential therapies might be more effective is under investigation.

Prospects for a possible clinical application of lethal mutagenesis to control HCV and other

highly variable viruses will be briefly outlined.

VOLVER

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expresa del autor.

© FUNDACIÓN RAMÓN ARECES. Todos los derechos reservados.

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forbidden. © FUNDACIÓN RAMÓN ARECES. All rights reserved.


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