EASL 2015 Hepatitis Research: Dr A. Ploss

Posted on May 18, 2015

For more information on the selected EASL 2015 Hepatitis abstracts below and others to be presented at The International Liver Congress™, please click here to review the Congress abstract e-book.

Abstract G09
Targeting a Host-Cell Entry Factor Barricades Antiviral Resistant HCV Variants From On-Therapy Breakthrough In Human-Liver Mice
Vercauteren K et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

The study discussed in abstract G09 focused on the possibility that conserved host factors used by the hepatitis C virus for its propagation could be alternative targets for antiviral intervention, and may be complimentary to the action of DAAs. Could you please explain for our audience why it’s necessary to keep looking for alternative targets for antiviral intervention, despite the reported success rates of DAAs?

Well, this is a tough question. Clearly there are a number of very important combinations of drugs that have already been approved, and many more candidates in the pipeline. So realistically, introducing a new treatment modality like an entry inhibitor is questionable at this point. There is a remote possibility that some very small sub-fraction of individuals may develop resistance against certain directed antivirals, and thus exploring the use of an additional antiviral such as an entry inhibitor may be of potential benefit.

The authors of abstract G09 don’t really make any strong statements in the abstract about whether the entry inhibitor mAb1671 should be added to clinical therapy, and I think this group is realistic enough to know that already very potent combinations of two, three, or four distinct DAAs are on the market. They are just saying that when you treat with a combination of DAAs and an entry inhibitor, you have lower chances of developing resistance to the DAAs.

Abstract O012

Hepatitis Delta Virus Can Survive Liver Regeneration and is Amplified through Human Cell Division Both In Vitro and In Vivo
Giersch K, et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

To help understand the objectives of the study discussed in abstract O012, could you please explain how the lifecycles of HDV and HBV are intertwined? 

Hepatitis B virus is a hepadnavirus that causes chronic infection in humans. It enters the cell and forms a covalently closed circular DNA (cccDNA), which is the transcription template for all viral RNAs. This cccDNA produces the structural proteins that eventually leads to formation of new virions, which are then released by the virus. Hepatitis Delta virus is a very small satellite virus, which requires hepatitis B virus envelope proteins for packaging new virions. So while you can have a hepatitis B virus monoinfection, usually, you have a hepatitis B and Delta co-infection because of the dependency of hepatitis Delta on the hepatitis B virus. Hepatitis Delta is a very different virus, and has a rather complicated replication cycle. But the key to understanding the results in abstract O012 is that hepatitis Delta requires hepatitis B virus for genome propagation.

The study discussed in abstract O012 focused on how HDV, but not HBV, is able to persist in quiescent hepatocytes after liver transplantation and during replication in HBV/HDV-infected humanized mice. Could you please discuss the clinical implications of these findings?

The data presented here are somewhat surprising, as the authors claim that HDV appears to be able to persist even in the absence of an underlying HBV co-infection. It is commonly assumed that if there is no co-infection with hepatitis B virus, hepatitis Delta virus is basically at a dead-end and cannot propagate. Although HDV does not stably integrate its genome into the host cell, the nuclear replication complexes appear to be rather stable and may be maintained for longer periods of time. If the data presented here can be further substantiated, it may be warranted to not only treat the HBV infection directly but also use specific treatments to eliminate all HDV reservoirs.

Abstract O014
A Targeted RNAi Screen Using a High-Throughput Infectious Model System Uncovers Glypican GPC5 as a Host Factor For Hepatitis B And D Virus Entry
Verrier ER, et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

In the study discussed in abstract O014, a targeted RNAi screen was used to investigate the role of heparan sulfate proteoglycan core proteins in HBV/HDV entry. Could you please explain what an RNAi screen is and how this screen was used to identify the glypican GPC5 as being important to HBV/HDV infection?

siRNA or shRNA screens are loss of function screens. Basically, you have a platform into which you introduce small interfering RNAs (short RNA sequences that can bind to complementary target sequences of host genes). When this happens, the host RNA is then subjected to degradation, and you can selectively eliminate certain host genes. But if you do it genome-wide, you obviously have tens of thousands of these siRNAs, each targeting a specific host gene. And then you can basically analyze the impact of this loss of gene expression on a particular biological phenomenon, in this case, hepatitis B virus infection.

Here, the investigators used a cell line that is permissive to hepatitis B virus infection, specifically a human hepatoma cell line that overexpresses sodium taurocholate cotransporting polypeptide (NTCP), and then they subjected it to a selected pool of siRNAs that would target members of the heparan sulfate proteoglycan family. They then measured how the efficiency of hepatitis B and Delta virus uptake is affected by this selective knockdown.

How do results of this study enhance understanding of the pathogenesis of HBV- and HBV/HDC virus-induced liver disease?

I don’t think they are necessarily providing new insights into hepatitis Delta, or hepatitis B/Delta co-infection, or hepatitis B pathogenesis, but I think it’s an important study because it sheds light on a new host molecule that is implicated in hepatitis B and Delta virus uptake. These two viruses share the same envelope proteins, so they probably follow, at least in the initial steps, a similar entry pathway. Until very recently, we knew very little about hepatitis Delta virus or hepatitis B virus uptake, and consequently, Delta virus uptake. Then a molecule was identified called NTCP, a bile acid transporter clearly required for uptake of both of these viruses into cell lines in vitro, but also in vivo. But besides this molecule, we still have a very poor understanding of the host factors that are required for the entry of these viruses into their host cell, the hepatocyte. So this study basically puts a new player on the map and adds to our understanding of the uptake of these two viruses.

Abstract O024
Cyclophilin and NS5A Inhibitors, But Not Other Anti-HCV Agents, Preclude HCV-Mediated Formation Of Double Membrane Vesicle Viral Factories
Chatterji U et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015. 

The results of an experimental study discussed in abstract O024 suggest that a possible host targeted anti-HCV therapy (a cyclophilin A inhibitor) and a specific direct-acting antiviral therapy (NS5Ai) act in concert to prevent viral replication from occurring. Could you please explain the importance of these findings to understanding the lifecycle of HCV? Could these therapies be used together to eliminate HCV?

Both of these classes of inhibitors, NS5A inhibitors as well as inhibitors that target host factors, have been considered or are actively being pursued or are already part of recently-approved therapies. As the authors stated, it is important to understand the mechanism of action of these drugs, specifically, inhibitors of the NS5A hepatitis C unstructured proteins and cyclophilin A inhibitors. Both of these inhibitors have been tested in clinical trials, and I think this study adds to our understanding of how these inhibitors work. Besides the utility for clinical treatment, these kinds of inhibitors are also great tools to investigate the lifecycle of hepatitis C virus because they can interfere with the lifecycle at specific points. We can see how the cell biology changes in the presence of a particular inhibitor, which provides clues about the lifecycle and teaches you something new about hepatitis C virus biology.

Abstract O059
Long-Term Persistence of HCV NS5A Variants After Treatment With NS5A Inhibitor Ledipasvir
Dvory-Sobol H et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

The study discussed in abstract O059 examines the persistence of resistance-associated variants in subjects who did not achieve SVR following ledipasvir. Could you please explain the clinical relevance of this study and its conclusion that NS5A variants can persist for >96 weeks posttreatment in subjects who relapse to regimens containing NS5A inhibitor?

I think that all the new therapeutic regimens now being approved and used in clinical practice usually contain combinations of direct-acting antivirals. Certainly, the exclusive use of an NS5A inhibitor is not really considered. So in some sense, this study is not accurately modeling clinical practice. However, it is important to understand the stability of viral variants that contain resistant mutations.

Let’s say you give a combination of an NS5A and an NS5B inhibitor, and the virus within the patient already harbors resistance mutations against the NS5A inhibitor, you basically only have the NS5B inhibitor that is acting on repressing viral replication, which could potentially lead to problems if resistant mutations were to arise that render a virus insensitive to NS5B treatment. In this particular study, they only looked exclusively at an NS5A inhibitor, ledipasvir, which is very potent but has probably a lower barrier of resistance.

The point is that these resistance mutations do not only emerge, become unstable and disappear, but instead, emerge and are maintained even after cessation of treatment, as stable mutations within the viral population. So a patient who has this set of mutations will not respond to this particular inhibitor, and other combinations of direct-acting antivirals would have to be tested. Again, this study depicts an unusual clinical scenario because NS5A inhibitors are, to my understanding, not being considered as a single treatment model.

Abstract O102
Galectin-1 Expression is Essential For an Effective Liver Regeneration
Potikha T et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

The results of the preclinical study discussed in abstract O102 demonstrate that the Galectin-1 protein is essential for efficient liver regeneration following partial hepatectomy in mouse models. Could you please explain the hypothesis behind this study?

The investigators performed partial hepatectomies in wild type mice and noticed that in the early phase during liver regeneration, there is an up-regulation of transcript encoding for galactin-1. Galectin-1 has been proposed to be an inflammatory regulator, and therefore, the investigators wanted to test whether the absence of galectin-1 would affect liver regeneration in a simple hepatectomy mouse model. So they performed partial hepatectomies in both wild type mice and mice with a targeted disruption of the gene, Lgals1, which encodes for galectin-1. When you remove up to 70% of the liver in the wild type mouse, the liver grows back very quickly, but in the galectin-1 deficient mice, liver regeneration was, according to the authors, substantially slowed down.

Abstract P0483
Changes in Liver Immunity Upon HBV Infection: a Comparison of Humanised Mice and Humans
Wai LE et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

The lack of robust animal models to study chronic HBV infection and immunopathogenesis has been a roadblock to the development of effective therapies. Could you comment on the findings of the preclinical study discussed in abstract P0483 showing that while inflammatory events triggered by HBV infection in humanized mice are similar to those detected in HBV chronically infected humans, intrahepatic lymphocyte populations and virus-specific immunity could not be fully recapitulated in the mouse model?

As you stated, there are currently no good preclinical models that allow us to model the complicated condition occurring during chronic hepatitis B virus infection. Clearly, in individuals who have acute chronic hepatitis B and an ongoing inflammatory response in the liver, the immune system tries to exert pressure on the virus and eliminate infected hepatocytes, which requires a combination of different immune cell subsets during the acute as well as chronic infection.

What several groups have pursued as a potential solution to the challenge of a lack of an experimental system, is the development of different types of humanized mice. Historically, the best established models are mice that are engrafted with human hepatocytes, “human liver chimeric mice”, and they are great for challenge studies. We can inject them with hepatitis B virus clinical isolates, or cell culture-derived virus, and they will develop chronic infection. However, this needs to be done in animals that do not have any adaptive immune system to prevent graft rejection.

In this study (abstract P0483), using an approach has been attempted by and published by other groups, the investigators co-transplanted human blood-forming stem cells and fetal liver cells into an immune-deficient mouse and infected it with hepatitis B virus. The investigators wanted to see how similar the immune response in this mouse model is to the one observed in infected humans (ie, do similar human immune cell populations infiltrate the mouse liver). Overall, the data show that the immune response in this mouse model and humans is quite different. The investigators do make inferences that certain monocyte populations and dendritic cells may be similar, but overall, many of the lymphocyte populations that you would expect to see are not present in the mouse model.

I think one issue here is that these investigators are not necessarily using the most cutting-edge models for this kind of study, but these results are still important because it shows that all preclinical models need to be validated and compared with clinical samples to make sure that they actually reflect what is seen in patients. 

Do you see similar issues in HCV infected humanized mice?

These systems are not easy to work with, and there are very few published studies. I am only aware of one or two studies that have investigated or characterized hepatitis C virus specific immune responses in animals that are co-engrafted with human immune cell populations and human hepatocytes. Overall, you do see activation of the engrafted human immune system, and it has been reported that these engrafted mice develop signs of liver disease (ie, fibrosis), but overall, the immune response still differs considerably between what is seen in humans versus what can currently be modeled in these humanized mice. I think there is a lot more work that needs to be put into this to improve immune function and immune reconstitution to make this a better model.

How does the lifecycle of HBV and HCV differ?

Again, these are two completely different viruses. Hepatitis B virus is a mostly double-stranded DNA virus, whereas hepatitis C virus is a single-stranded RNA virus. What they have in common is that they both infect probably exclusively human hepatocytes in the liver. Again, for hepatitis B virus, the major mechanism of establishing persistence is through the formation of covalently closed circular DNA (cccDNA), which is a stable DNA template that is present as a “mini chromosome” in the nucleus; so hepatitis B has a nuclear phase. In this case, viral mRNAs are generated that give rise to viral proteins, which are important for production of new virions.

For hepatitis C virus, you have an RNA genome that is released from the nuclear capsid of this virus. It is translated using the cellular machinery. The resulting polyprotein is processed, which releases the individual mature proteins, many of which are, for example, being targeted for antiviral therapy. The replication complex that produces new RNAs is formed within the cytoplasm near host-derived membranes.

So the major difference between viruses is that hepatitis C exclusively replicates in the cytoplasm, whereas hepatitis B virus has a nuclear stage. They use completely different sets of host molecules for going through their lifecycles starting at the level of cell entry, and certainly also for replication and likely also for assembly of the infectious virions. Basically, they have completely different mechanisms of infecting a human hepatocyte, despite the fact that they cause clinically-similar disease and infect the same cells.

Abstract P0530
Myrcludex-B Inhibits Establishment of HDV Super-Infection In HBV Infected Mice And Reduces HDV Viremia In Stably HBV/HDV Co-Infected Mice
Volz T et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

Regarding the study discussed in abstract P0530, is there a possibility that mono- or co-infected humans could one day be treated with entry inhibitors like Myrcludex-B?

It’s my understanding that Myrcludex-B, which is a short peptide sequence derived from the hepatitis virus pre-S1 protein, is being pursued in preclinical studies as a novel treatment modality for hepatitis B and Delta virus infections. I am aware of a number of preclinical reports, and I think some of this may have also led to the initiation of clinical studies in individuals who are infected with hepatitis B or potentially co-infected with hepatitis B and Delta virus. So yes, Myrcludex-B, to my understanding, is being actively pursued as a potentially new treatment therapy. Just to be clear, it’s not anywhere close to being approved, but at least it’s being considered as a new drug to interfere with the hepatitis B and Delta virus lifecycles.

The results of the study discussed in abstract P0530 indicate high infection efficiency and great survival capacities of HDV in humanized mice treated with Myrcludex-B. Does this reflect the difficulties encountered when treating HBV/HDV co-infected patients?

I think the main take-home message from this study is that Myrcludex-B may be very effective in preventing hepatitis B/Delta co-infection. There are many patients who are chronically infected with hepatitis B, and if they not treated, they are at risk of being superinfected with hepatitis Delta virus. The problem is that when they are co-infected with both viruses, the ensuing liver disease is usually much more aggressive than what is observed in patients with a hepatitis B virus monoinfection. So treatment with Myrcludex-B may actually help to prevent hepatitis Delta virus superinfection. How Myrcludex-B could be administered over long periods of time is not addressed in this abstract, and I’m also not sure how this would be accomplished practically. But this study does show that you can effectively prevent superinfection in a mice model.

The other part of this study is geared towards showing the effects of Myrcludex-B treatment on mice with an established co-infection with hepatitis B and Delta virus. The investigators took humanized mice (mice that are engrafted with a human liver), and infected them with both hepatitis B and Delta virus. The investigators waited until the mice were stably infected with both viruses, and then treated them with Myrcludex-B. What the investigators observe is reduced hepatitis Delta viremia in the mice subjected to the Myrcludex-B treatment. This is certainly of significance since this regimen could potentially be added to currently available drug combinations that mostly targeting hepatitis B virus, and could help to potentially eliminate Delta virus infection from a co-infected individual.

Abstract P0532
HBx–DLEU2 lncRNA Complex Affects Transcription of New Target Promoters
Guerrieri F et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015. 

The results of the cell culture study discussed in abstract P0534 highlight a newly discovered part of the lifecycle of HBV, in which the HBx–DLEU2 lncRNA complex affects transcription of new target promoters. Could you please explain the potential clinical relevance of these findings to chronic HBV infection in humans?

It is difficult to say how these results may affect future therapy, as they stem from a very basic study. Here, the authors identify a long noncoding RNA or combinations of long noncoding RNAs (link RNAs), which seem to be regulated by the hepatitis B virus X protein. These RNAs do not encode specific proteins that could be directly targeted by therapeutic interventions. What these specific link RNAs may do, conceivably, is regulate important processes required for maintaining hepatitis B virus chronicity in an infected hepatocyte. Future hepatitis B therapies could potentially focus on interfering with this particular pathway, but I think a lot more work needs to be done to determine what influence HBx protein has on link RNA expression, and what this actually means to the lifecycle of hepatitis B.

Abstract P0534
Hepatitis E Virus RNA Replication is Ensured by Unprocessed ORF1 Protein
Loan Dao Thi V et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

HEV is a non-enveloped positive-strand RNA virus whose genome encodes 3 open reading frames, namely ORF1, ORF2 and ORF3. The cell culture study discussed in abstract P0534 shows that HEV RNA replication is ensured by unprocessed ORF1 protein. What do these findings mean to developers of HEV therapies?

Hepatitis E virus is a virus that causes an acute infection in most individuals who may not even become clinically symptomatic. Few patient populations can develop chronic infections, which include HIV/HEV co-infected individuals. There are a few case studies that have shown that individuals who are HIV-infected develop this unusual clinical disease course, where you have a chronic hepatitis E infection. Most patients who become acutely infected with hepatitis E do not require any kind of specific treatment because the infection is self-limiting, and hepatitis E infection can also be fairly effectively prevented with a vaccine, which is, I believe, approved in China. I don’t think this vaccine has been approved in the US or in Europe.

I do not know how much active effort is being put into developing specific drugs that would interfere with chronic hepatitis E virus infection, but clearly, we need to learn a lot more about the hepatitis E virus lifecycle. It’s very different from all of the other hepatitis viruses that we have discussed before, B, C, Delta, also A. Until a few years ago, there was no cell culture system available to study the individual gene products encoded by the hepatitis E virus, but this has changed now. Infectious clones are available that are currently being refined, and now, we can start more systematic studies to figure out what these individual proteins are doing. ORF1 protein encodes for a number of different enzymatic activities, and therefore is particularly interesting as a potential drug target. Further investigations of the role of ORF1 could lead to a better understanding of what ORF1 is doing during the viral lifecycle, and this could potentially be exploited down the road to devise assays and later screens to identify compounds that would interfere directly with HEV infection. Again, I am not sure if this is a main priority right now given the fairly infrequent rate of chronic infections, and the fact that HEV is usually a self-limited acute infection.

Abstract P0545
Targeting Viral DNA With CRISPR/Cas9 Robustly Suppresses Hepatitis B Virus
Shlomai A et al. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

Current anti-HBV drugs efficiently suppress viral replication but do not clear viral episomal DNA. What is the importance of viral episomal DNA?

Covalently closed circular DNA (cccDNA) produced by hepatitis B virus is the transcription template for all the viral RNAs, and is responsible for chronicity in an infected cell. You can find cccDNA in patients even when they are taking direct-acting antivirals (DAAs) to suppress HBV active viremia. This is illustrated by the fact that when you discontinue DAAs in HBV carriers, the HBV viremia usually flares and returns if the immune system is not able to contain the infection on its own. The episomal DNA addressed in this study (abstract P0545) is the cccDNA, and a number of academic groups, and also industry, are currently trying to find ways to either prevent the formation of cccDNA, destabilize it, or to actually actively destroy it. In this study, this group has utilized a system called CRISPR/Cas9 in which you can guide a nuclease, the Cas9 nuclease, to specific sites in the genome, in this case, the hepatitis B virus genome, to induce cuts in the viral DNA that will inactivate it, and thereby prevent or eliminate persistent infection.

The results of the preclinical study discussed in abstract P0545 indicate the possible therapeutic importance of specific guide RNAs, which may one day suppress HBV replication in humans and possibly eliminate viral episomal DNA. Could you please explain the following to our audience: what are guide RNAs, and how did they suppress HBV replication and eliminate viral episomal DNA in the preclinical study?

The guide RNA itself does not suppress HBV infection. The guide RNA is basically bringing the nuclease, the Cas9 enzyme, to a specific site in the genome. This is to ensure that the nuclease cuts at defined sites within the hepatitis B virus genome, and the guide RNA is a sequence which has a complementary sequence within the hepatitis B virus genome. So the guide RNA targets the nuclease to the site, and at this site, the nuclease will basically introduce a cut in the cccDNA.

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