Welcome to the Hepatitis B Special Interest Group Program for Liver Meeting 2021. Our session today is titled Expanding Hep B Treatment Criteria, New Biomarkers, and Cure Updates. My name is Hannah Lee. I'm a hepatologist at Virginia Commonwealth University in Richmond, Virginia, and both Dr. Mindy Nguyen and I will be co-chairing today's program. During the next 90 minutes, we're going to be exploring an array of topics in the field of hepatitis B, from hep B reactivation to expanding treatment in the immune-tolerant patient. We're also going to be exploring novel biomarkers and looking at updates in hep B cure from an immunologic and virologic approach. These will be presented by well-known hep B experts in the field and we hope that you will find these talks helpful in approaching hep B management now and in the future to come. We look forward to your audience participation during our Q&A session at the end of this program. Thank you. I would like to thank this opportunity to present on this important topic. These are my disclosures. We conduct a number of clinical trials at the University of Calgary. I've also received funding from industry to the Canadian Hepatitis B Network. So, in this talk, I would like to provide an overview of the clinical presentation and definition for hepatitis B reactivation, review the pathogenesis, as well as risk stratification of those for reactivation, and highlight areas of research. So, globally, approximately 250 million people are chronic hepatitis B carriers or hepatitis B surface antigen positive. Approximately 2 billion have been exposed to hepatitis B and thus are hepatitis B surface antigen negative with or without hep B core and surface antibodies, so-called occult hepatitis B according to the teramina definitions. Reactivation of hepatitis B is defined as a sudden increase in viral load in those that were previously HPV DNA negative. It may or may not be associated with a hepatitis or ALT flare, and it can occur anywhere from two weeks up to two years after stopping immunosuppression. Since the first case of reactivation after stopping chemotherapy over four decades ago, there's been numerous reports in the literature from a variety of different immunosuppressive regimens, including the novel biological agents that have been used in the last several years. It's key to identify those at risk since there's robust data showing that reactivation and the devastating clinical consequences as occurred in this first case can be prevented with nucleoside analog prophylaxis. The reason why the hepatitis B virus can persist within the cell nucleus is to the presence of a very stable viral product called covalently closed circular DNA. CCCDNA persists within the cell nucleus. It's constantly replenished. It's considered to have had a long half-life, although there's recent data showing that it may be much lower. Of course, hepatitis B is also an immune-mediated disease, and the outcome of infection is controlled by the host immune response in that HPV-specific T cells and neutralizing antibodies can control viral replication, although they can't eradicate the CCCDNA. If a patient receives immunosuppression, then the drug-induced immune compromise or immune suppression can lead to uncontrolled viral replication. This can cause direct damage to infective hepatocytes. However, after stopping immunosuppressive drugs and the immune system recovers or is reconstituted, severe injury of liver cells can occur due to immune-mediated clearance and destruction of infected hepatocytes, which would be clinically evident by the presence of elevated ALT or AST in serum. This figure, taken from a review, provides a nice overview of the clinical course of reactivation. With the start of chemotherapy or multiple cycles of chemotherapy, there's an asymptomatic asymptomatic rise in HPV DNA. This is followed by a biochemical hepatitis or increase in ALT. In severe cases, this can progress to jaundice and liver dysfunction, and if not rescued or if doesn't spontaneously occur, rescue with a nucleoside analog therapy would then lead to reduction in viral load as well as reduction or decrease in ALT. In general, patients can be stratified according to viral host or degree of immunosuppression. Viral factors generally point to the degree of host immune control, so presence of surface antigen, E antigen, or high HPV DNA. Host factors include those of older age, if they're male, or cirrhosis, as well as the type of disease. The degree and type of immunosuppression is also a factor. Some studies have shown that the presence of hepatitis B surface antibodies could be protective in those that are surface antigen negative with core antibody. This was shown in a recent systematic review, but I believe that there's more data needed because certainly the presence of anti-surface is not protective. Patients have been generally divided into three risk groups, either low, moderate, or high. In general, those that are surface antigen positive receiving most types of immunosuppression are at moderate to high risk and thus should receive nucleoside analog prophylaxis. Those that are hep B surface antigen negative but have B core antibody positive, prophylaxis is recommended if they receive anti-CD20 or B cell depleting agents and stem cell transplantation. Regardless, all hep B surface antigen positive patients should be referred for consideration of treatment or assessed by a specialist for fibrosis stage and whether they need treatment, even if they're not immune compromised. This other review provided a nice overview of the multiple different biological agents that have been used in the last number of years and risk stratification and risk stratified both surface antigen positive or anti-core positive patients. Generally, those that receive TNF alpha inhibitors, and as I mentioned, agents targeting B cells or anti-CD20 are the highest risk. It's also useful to look at certain special populations. People co-infected with hepatitis C who have received directly acting antiviral agents and achieved a sustained virological response may also be at risk of hep B reactivation due to viral interactions, especially if they're hep B surface antigen positive. Similarly, if your hepatitis delta virus co-infected, either receiving interferon or the novel agents such as the prevention and agents such as the perennialation inhibitors may be at risk of reactivation and prophylaxis should be given, especially since many of these patients have advanced fibrosis. If you have dual hep B and HIV co-infection, the nucleoside analog should be active against both viruses since with antiretroviral therapy and immune reconstitution, there's risk of reactivation. Patients with liver cancer and hepatitis B, many have underlying cirrhosis and even with local regional or immunotherapy should receive a nucleoside analog given the risk of reactivation and decompensation. Immune checkpoint inhibitors are also a new group. In and of itself, it can be associated with an autoimmune type of hepatitis. And interestingly, there's been recent case studies and clinical trials looking at whether they could be used to restore immune control and recovery of T cell exhaustion and even lead to improved outcomes and a functional cure. And I wouldn't be able to discuss a talk now in 2021 without mentioning COVID-19. Very severe cases of COVID patients are admitted to hospital and receive high dose steroids to treat their cytokine storm and also biological therapies such as IL-6 receptor antagonists and case reports and small studies in non-COVID patients show the risk of reactivation. So, in summary, all experts from ESO, the ASLD, and Asia Pacific guidelines recommend screening all immunosuppressed patients with hep B surface antigen and hep B core antibody should also test for hep B surface antibody in those that are surface antigen negative and provide the hepatitis B vaccine if not immune. Those that are hep B surface antigen positive should be treated with most types of immunosuppression with a nucleoside analog. Those that are hep B surface antigen negative or core antibody positive treatment should be given with a high risk immunosuppression such as rituximab or stem cell transplantation. Otherwise, monitoring may be reasonable. The nucleoside analogs that should be given are the more potent ones such as tenofovir or entecovir. Tenofovir allophenamide or entecovir can be considered in patients that have metabolic bone disease or risk of renal dysfunction. Immunosuppression should the prophylaxis should be given for a minimum of six months and longer up to two years if they're receiving anti-CD20 or B cell depleting therapies. And a recent meeting from the Asia Pacific Association actually discussed whether it may be more cost effective to provide the nucleoside analogs given that we now have low cost generics available when balanced against the cost of monitoring and viral mode testing. If we look at this in a lower HPV endemic area, this was recently reviewed by my colleague Dr. Congley at the University of Calgary. And in fact, it is cost effective for screening for hepatitis B with hepatitis B surface antigen. When you look at the cost of the serological testing, it would be approximately $4,000 for one diagnosis. And this is balanced against the high cost of an ICU stay, which is which is a potential outcome in those with severe hepatitis B reactivation. In addition, the low cost nucleoside analogs are now only about $4 or $5 a day or $2,000 per year. Whereas if you compare this to the monthly viral load testing, it's also about $2,000 a day. And so perhaps we can have a lower threshold to treat instead of just monitoring just based on cost effective analysis. However, this has to be balanced against individual patient preference and adherence. So in summary, key points to highlight is that all patients receiving immunosuppression should receive, should be screened for hepatitis B, including hep B surface antigen, hep B core antibody, hep B surface antibodies, vaccinate people that are non-immune. It is cost effective to screen. Antiviral therapy with the potent nucleoside analogs prevent reactivation. Those that are hep B surface antigen positive, most of them should require prophylaxis for most types of immunosuppression. Regardless, they should be reviewed and assessed for fibrosis and need for treatment anyway. If you're surface antigen negative and hep B core antibody positive, the greatest risk is with B cell depleting therapies as well as stem cell transplantation. The lower risk group may be considered for monitoring or provide a more individualized approach based on the duration and comorbidities and the patient preference. The risk of reactivation can be protracted up to two years after completion of immunosuppression. There's evolving data with the newer immunosuppressive regimens, and it even may be more cost effective to treat versus to monitor in low to medium risk groups. Thank you, and I'm happy to take any questions. Hi, I'm Dr. Doug Dietrich. I'm here today in sunny Anaheim talking to you about expanding treatment to immune tolerant and borderline active hepatitis B. Is it really worthwhile? My background opinions here are that virtually all hepatitis B treatment trials in the past were designed to get meds approved quickly and economically. The fastest way to get those FDA approval was to show normalization of ALT, even though those patients are the vast minority of patients with hepatitis B. The gold standard of randomized clinical trial betrayed us this time. It carried evidence-based medicine to its illogical extreme. A large reveal cohort study is much more representative of the natural history of hepatitis B. Now, there are other supporting evidence that HPV DNA is the only real factor involved in the risk. This is the reveal data, which is familiar to all of you, showing a dramatically decreased mortality for people who were surface antigen positive and decreased mortality for people who are more than 2,000 IUs or more than 10,000 IUs here, both liver mortality and overall all-cause mortality. Now, this is the important part of a reveal in the so-called inactive carriers, is that compared to controls, quote, inactive carriers have a much higher likelihood of developing HCC and a much higher likelihood of developing liver-related death without having anything else. Inactive carriers, as you can see, defined as less than 10,000 IUs. This is what I believe are carriers. I don't think we should use this word for hepatitis B. The easel guidelines don't contain the word carriers anymore either. I think that's a very positive effect. They talk about chronic infection or chronic hepatitis. In chronic hepatitis, patients have elevated ALT and chronic infection doesn't have ALT. I think that's the main thing. ALT is not really pertinent, frankly, here. Here's newer data from 2019 that shows that baseline HPV DNA of more than 10,000 IUs is associated with a much higher risk of complications. You can see the Kaplan-Meier curve there. And also in this trial, ALT was not predictive of outcomes. It's a virus. Antiviral therapy delays overall disease progression. We know that. This is a proof of principle using lamivudine, an outdated drug for sure, but definitely made a difference in progression of liver disease in this study in Hong Kong. In addition, it actually also reduced the incidence of HCC by half in only three years. Actually, the study was stopped because the treatment was so effective. Of course, we have much better treatments than lamivudine. Now, our endpoints for therapy for hepatitis B, of course, we want to suppress HPV DNA. After that, liver histology improves. We can prevent cirrhosis. We can probably reduce the incidence of HCC, not prevent it. And I don't know if we can prevent death. We can prevent death from liver disease, for sure. We can also prevent mother-to-child transmission, hepatitis B reactivation, and extrahepatic manifestations of hepatitis B. Of course, during treatment, ALT normalizes and frequently we get some kind of seroconversion, e-antigen loss anyway, e-antibody production. And then sometimes we get hepatitis B-S antigen loss, which is sort of halfway to our goal. The main goal is DNA suppression. The optimal goal is seroconversion. Now, if we don't treat, what happens even if the ALT is normal? Long-term nucleoside analog therapy did reduce the extent of DNA integration in chronic hepatitis B patients. So, DNA integration is not a good thing, right? That's what's going to cause long-term complications. And here's another paper that's also just a poster that just came out. Tenofovir does the same thing, reduces the number of transcriptionally active viral integrations in chronically infected hepatitis B patients here. It's associated with reduced expressed viral integrations and dysregulated genes, all of which are risks for liver cancer. Antiviral therapy is currently recommended in the, quote, immune active phases of hepatitis B. As you can see here in the classic diagram, although at least on the bottom, there are the easel guidelines of chronic infection, chronic hepatitis with the antigen positive and negative. So, these are the criteria for ASLD, for easel, and for APOSL. The DNA levels are the same. They're taken from reveal. The ALT levels vary, actually, between the three. I'm not sure where those came from. And of course, anybody with cirrhosis should be treated or a history of liver cancer, which makes sense. And I think people are making these thresholds now a guide, not an absolute commandment because so many people were not getting treated before, but HPV DNA levels should be linked with risk. I think that's important. ALT elevations should be due to hepatitis B, but of course, you have to be able to determine that. They can't be due to fatty liver or other things. The problem with these guidelines is that most chronic hepatitis B patients fall into the gray zone, and those are the ones that are E negative, the ones that are probably most likely to run into trouble over the long term with complications. These gray zone patients are not uncommon. This is a California and Taiwan retrospective cohort of almost 4,000 patients untreated without cirrhosis at entry, and at baseline, 38% were in the indeterminate phase. By year five, 74% were still indeterminate and still not getting treated. This is using ASLD thresholds. What happens when you do that? A high proportion of chronic hepatitis B outside the treatment criteria in current international treatment guidelines develop liver cancer. The fewer patients developed HCC with the less stringent easel criteria than the ASLD and the older APOSL 2015 guidelines. The proportion of patients developing HCC is very clearly related to the number of patients getting treated that is meeting guidelines for treatment. What evidence was there behind this assumption that nothing bad happens in the early stage of infection when patients have a very high viral load? These patients have high DNA levels and are considered high risk of transmitting the virus to others. Hepatitis B, we forget, is incredibly transmissible. Based on the main modes of transmission, treatment to reduce the risk of transmission is recommended for some populations, pregnant women for sure, to prevent perinatal transmission, health care professionals performing exposure prone procedures definitely need treatment despite the Americans with Disabilities Act. What about young patients with hepatitis B? People who use drugs or people who have multiple sexual partners. I had a patient, for instance, who came to me when he was 18 years old. He was just entering a big university here in New York and he had almost a billion IUs of hepatitis B, which he had gotten close to birth from his nanny. I just didn't feel that it was ethical to unleash this guy on his college dormitory and all the other people he would be dating and having contact with in a large university with a billion IUs of hepatitis B. Treating the population viral load is another way to think about this. I was treating the population more than the patient perhaps at that point. Of course, this is high DNA levels are also associated with an increased risk of cirrhosis and HCC. He'd already had it for 18 years at this point. I think the time to treat him was obviously nigh. Untreated immune-tolerant patients have a significant risk for morbidity and mortality. In this study of untreated patients, 413 of them in the immune-tolerant phase, DNA more than 20,000, no evidence of cirrhosis with normal ALT, their 10-year risk of HCC was almost 13%, increased about two and a half times from the 6% of the treated patients who had abnormal ALTs because that's how they got treated. The P-value was highly significant there. The 10-year risk of death or transplantation was about 10%, was increased almost four times from the 3.4% of the patients who were actually treated. Now, in this matched cohort study in Hong Kong of almost 700 patients who had hepatitis B and HIV concurrently with 2,400 patients who were hepatitis B alone, they found that the co-infected patients had a much lower risk of liver cancer. Here you can see in the green line, it's a little faint, but that's why I put the arrow there. At a lower risk of HCC compared with antiviral therapy treated hepatitis B monoinfected patients. Why is that? That's because patients with HIV and hepatitis B get treated immediately for their hepatitis B. They don't wait like patients who are monoinfected. Actually having HIV is better for your hepatitis B and better for your risk of HCC because you get treated immediately regardless of your ALT level. Is this cost effective? This study from France showed that a test and treat strategy of treating all hepatitis B patients was the most cost effective in France. This study in Saudi Arabia showed that test and treat became highly cost effective in a few years. It was repeated in multiple other countries, including the Philippines, where the prevalence of hepatitis B is very high. Now, how do we go about thinking about this? This is one way. It's a little complicated. Patients who could be treated immediately, patients who could be possibly treated or patients who could be discussed later. This is a simplified algorithm that we've developed and submitted for publication recently. I think it's very reasonable that a lot of people be thinking about this and we make it simple. Basically, if you're over 2,000, you should treat. If you're a believer in 10,000, that's okay too. ALT has nothing to do with this. If you're over 30 and over 2,000, basically treat. If you're under 30 and less than 2,000, you can hold or defer treatment, but keep watching and keep screening for HCC. I think that's really important to emphasize, particularly as we expand our treater base like we have done for hepatitis C. Of course, we're using TAF as needed. Now, in conclusion, hepatitis B did not read the guidelines. Hepatitis B DNA is a continuous variable. Why do we need to create artificial categories for a continuous variable? DNA over a small number, 2,000 or 10,000, leads to much worse outcomes and a higher risk for liver cancer. That's what we're really worried about. ALT is not a significant indicator of risk. The longer you let the virus replicate, the more integration events take place, increasing the risk of HCC. Treat the virus. It's cost-effective to just test and treat, test everyone for hepatitis B, universal testing for hepatitis B, just like we do for HIV and hepatitis C, and treat over 2,000 international units. Hello, everyone. I'm very happy to present this talk on novel biomarkers, advances in disease outcome, and HPV cure prediction. I'm Anna Lok from University of Michigan, and I'm the director of hepatology there. These are my financial disclosures. And this is an outline of my talk. For this talk, I'll cover four markers, quantitative service antigen, HPV RNA, hepatitis B correlated antigen, and quantitative anti-HPC. And I'll discuss their utility in differentiating phases of chronic hepatitis B infection, predicting clinical outcomes, response to the currently available therapies, as well as some assessment of novel antiviral therapies. So as you know, the hepatitis B virus consists of the envelope and nucleocapsid. Upon entry into the hepatocyte, the relaxed circular DNA is being repaired in the hepatocyte nucleus into the CCC DNA, through which pre-genomic RNA is being transcribed. And this is then packaged into nucleocapsid, and the HPV RNA is being reverse transcribed into HPV DNA package and being secreted. But some of these nucleocapsid-containing RNA is being exported also before reverse transcription into the DNA, and that's why we can sometimes detect HPV RNA in the circulation. In addition, some of the RNA species comes from the messenger RNA as well as the splice RNA. All of us know that hepatitis B virus, in addition to the complete variants, also have sub-viral particles circulating in the form of spheres and filaments that do not contain HPV DNA and are not infectious. Among the messenger RNAs and the viral protein produced is the hepatitis B e-antigen. When we talk about the core-related antigen, it contains the consistent e-antigen as well as the core antigen and some of the pre-core protein as well. But let me get into some of the details. First, in terms of quantitative surface antigen, as I've indicated, circulating HPS antigen comes not only from infectious viral particles but also sub-viral particles. In fact, the sub-viral particles outnumber the complete variants. In the past, we think that HPS antigen comes entirely from CCC DNA, but we now know that they are also being translated and from integrated HPV DNA. In fact, the predominant source in e-antigen negative patients is from the HPV DNA, while in the e-antigen positive patients, it's predominantly from the CCC DNA. This explains why HPS antigen may persist after CCC DNA transcription is being silenced. What about quantitative HPV RNA? I've indicated that this is being detected when the nucleocapsid with the pre-genomic RNA is being exported before it is being reverse transcribed into the DNA. But some of that can also be from the messenger RNAs as well. In untreated patients, HPV DNA outnumber HPV RNA. But once you put the patients on treatment, for example, on the nukes, where you block the reverse transcription of the RNA into the DNA, then the ratio of the RNA to the DNA would be increased. What about hepatitis B-correlated antigen? This really consists of three different components. In the e-antigen, the core antigen, and a p22 protein, which is incompletely processed pre-core core protein. It is a micro-CCC DNA transcription and translation, and they're more reliable in e-antigen-negative patients because in e-antigen-positive patients, a lot of it is really coming from detection of hepatitis B e-antigen. The problem with HPV-correlated antigen is that the current assays have suboptimal sensitivity. We know less about quantitative anti-HPV. Other than that, this is a quantitative assay in contrast to the qualitative anti-HPV that we're familiar with, and it appeared to reflect immune response to core antigen. Studies show that they correlate with ALT levels and histologic inflammatory activity. I'll now discuss the utility of these markers in assessing natural history. This is a depiction of the natural history of chronic hepatitis B in four different phases. We all know that early on, patients are e-antigen-positive, and then they circumvert to anti-E. During the e-antigen-positive phase, there's an initial immune tolerance phase with high level of HPV DNA, normal liver enzyme, and also high levels of surface antigen. As the patients enter into the immune-active phase, the DNA may go down and the S antigen may go down, while the ALT level may go up. After circumversion to anti-E, many of these patients get into the inactive phase where all three levels, S antigen, DNA, and ALT are low. But later on, some of these patients progress to the e-antigen-negative chronic hepatitis phase when S antigen, DNA, and the ALT level may go up again. As I've indicated early on, the S antigen comes predominantly from the CCC DNA, whereas later on, the S antigen comes predominantly from the integrated HPV DNA. So you can see that quantitative S can help us differentiate the different phases of chronic hepatitis B infection. So what about HPV RNA? They're in some ways very similar. Early on in the e-antigen positive phase, HPV RNA level is very high, except for patients who are on the way to E-cell conversion. Once the patients become e-antigen negative, HPV RNA tends to be much lower, particularly when they get into the inactive phase. In those of e-negative chronic hepatitis, the HPV RNA level may be higher. And obviously, in the patients who become S antigen negative, the levels will be lowest. So these are data that we reported in the HPV RNA study. Similarly, using the HPV RNA cohort study, we also look at the hepatitis B correlated antigen levels. In red, it indicates high levels, and in green, it indicates low levels. So again, you see that during the e-positive phase, correlated antigen level is very high. Not surprisingly, because it also measures the presence of e-antigen, whereas in a e-negative phase, the levels become much lower, particularly in the inactive carrier phase. And by the time the patients become S antigen negative, the majority of them have very low levels. This might be easier to see, because this slide shows all the different markers together, starting from the immune-tolerant phase, to the e-antigen positive active phase, to the inactive carrier phase, e-negative chronic hepatitis, and when the patients become S antigen negative. So dark green here represents HPV DNA high initially, lower in the inactive carrier phase, higher gain in e-negative chronic hepatitis, and by the time they're S antigen negative, usually HPV DNA undetectable. And you can see that HPV RNA, as well as hepatitis B correlated antigen, tracks very similarly to HPV DNA. Now quantitative S also, to some extent, tracks similarly, except that you can see that the differentiation is really not as good. There is substantial overlap until the patients become S antigen negative. The review HPV study in Taiwan show us the importance of HPV DNA in predicting outcomes. Here they show that among the patients with low HPV DNA, where this is not a very useful marker because everyone had low DNA level, and yet some of them continue to progress to cirrhosis and HCC. Here quantitative S antigen levels become helpful. In those patients with low levels, they have a lower chance of progressing to cirrhosis and also a lower chance of progressing to HCC. Well, what about correlated antigen? Similarly, they appear to be predictive of cirrhosis as well as HCC. These are the patients of low levels of correlated antigen compared to those of higher levels. This is located on the AUROC comparing correlated antigen as well as HPV DNA, very similar AUROC, but S antigen overall has a lower level. Nonetheless, in this particular study, they found that correlated antigen would be best suited for patients of intermediate HPV DNA level between 2,000 and 20,000. Now, I'm now going to talk about these markers in relation to treatment response. Well, first looking at interferon treatment, we've all heard about the stop rule where when you put the patients on interferon therapy, even though they may drop the HPV DNA, but it's not helpful in predicting ultimate response when you stop treatment. Now, if the aim is to look for S antigen loss, then a drop in quantitative S early on during treatment is highly predictive, and that is being used as a stop rule. In this case, you look at the patients who show a good response of S antigen clearance versus those with no S antigen clearance, certainly quantitative S is predictive even early on. Similarly, quantitative correlated antigen also differentiate the patients with and those without S antigen, but it's not as sharp. What about HPV RNA? That also is helpful in predicting who might go on to S antigen loss and who might have sustained response of treatment. Those who found HPV RNA drop by at least some two log or drop to undetectable by 24 weeks of treatment are more likely to have sustained response and S antigen loss. Similarly, patients who had a substantial drop in correlated antigen by 24 weeks are more likely to have a sustained response and an S antigen loss. Well, what about new therapy? This group from China was the first to show us the impact of nucleoside nucleotide analogs on HPV RNA levels. As I've indicated very early on, in untreated patients, you have far more circulating HPV DNA than circulating HPV RNA. So this is DNA and this is RNA. But once you put a patient on mu, blocking the reverse transcription of RNA back into DNA, you find that the DNA drops substantially, but not as much of a drop in HPV RNA. And the residual detection of HPV RNA at the end of new therapy, if you're planning to stop treatment, is highly predictive of rebound. Those who have detectable HPV RNA, when treatment is stopped, all of them rebound. Whereas those with undetectable level, not all of them will rebound. So this nicely show the kinetics of these different HPV markers when you put the patients on mu. HPV DNA drop first, followed by slow declining correlated antigen and HPV RNA, but very minimal drop in S antigen, even up to five years of treatment. Now, can these markers predict clinical relapse after discontinuation of mu? And the answer is yes. Both quantitative anti-HPC, as well as some quantitative HBS antigen are predictive of whether patients would have a clinical relapse when you stop the mu. As expected, higher end of treatment S antigen level, more likely to relapse, but for quantitative core is actually a lower level that is some predictive. So what about these new markers in assessing response to the new treatment, such as some siRNA? In this particular study, the siRNA is designed to knock down S antigen production. And you can see that using quantitative S antigen, you can follow the kinetics and the dose response effect with a higher dose resulting in bigger drop in S antigen and a slow rebound after treatment. The other group of new compounds are capsid assembly modulators. They are designed to prevent proper assembly of core particles resulting in empty or aberrant particles. The red line here shows that is the capsid assembly modulator plus entacavir versus the blue line entacavir alone. The combination therapy results in a more market decline in HPV DNA, correlated antigen, as well as pre-genomic RNA in both treatment naive, as well as some virally suppressed patients. Thus in summary, what I have discussed is that these new markers can help us differentiate the different phases of chronic hepatitis B infection, HPV DNA, RNA, and correlated antigen are better than quantitative S antigen. When used to predict clinical outcomes such as cirrhosis, liver cancer, and liver related deaths, HPV DNA is the best. Quantitative surface antigen is good for antigen negative patients with low DNA and correlated antigen for patients of intermediate levels of DNA. For predicting S antigen loss, quantitative S antigen is obviously the best, but this is not truly a predictor as it is also the outcome. There are also some studies that suggest that quantitative core can predict HPV reactivation during immunosuppressive therapy. For patients on treatment with the current therapies, in terms of predicting S antigen loss of interference therapy, quantitative S is the best, followed by HPV RNA and correlated antigen, and the least is HPV DNA. For predicting S antigen loss after new withdrawal, quantitative S is the best, followed by HPV RNA and HPV DNA. And for predicting clinical relapse after new withdrawal, HPV RNA and core antibody seems to be useful. As far as new therapies are concerned, these markers can be used to confirm target engagement as well as to assess antiviral activity. Right now, we don't know how good they are in predicting S antigen loss because most of these new therapies have not achieved this outcome yet. Thank you so much for your attention. Thank you, Dr. Nguyen, Dr. Lee, ladies and gentlemen. I'm delighted to contribute to this HPV SIG. Today, I'm going to give you an update on immunological approaches to hepatitis B cure. Here are my disclosures. The ultimate goal of treatment is functional cure, a finite treatment duration, allows us to cease all treatment, have absence of surface antigen, no active disease or viral replication. We know that barriers to functional cure, not only the high viral burden, but also the very weak post-immune responses associated with lifelong chronic hepatitis B and all the deficiencies of the immunobiology of chronic hepatitis B are shown here on the left. And where we want to get to is the immune status associated with self-limited, resolved hepatitis B infection. We need effective host innate and adaptive immune responses to ensure off-treatment immune control. The three therapeutic approaches to cure include inhibition of viral replication, lowering the viral burden and boosting the host immune response. And certainly improvements in knowledge of the viral life cycle and immunology has identified many targets for new drug development. And today I'm going to concentrate on those which boost immune responses. This can really be grouped into four different strategies. Those which stimulate antiviral effector cells, those which rescue exhausted T-cells, those which redirect non-HPV specific T-cells, and finally those which can generate new T-cells. We start off with the top group and these are the TLR agonists. I'm not going to talk about the RIG-I receptor agonists which have been discontinued at this time. We know that TLR receptors are crucial really for the early host response against infections by linking innate with adaptive immune responses. The TLR7 and TLR8 pathways activate different antigen preventing cells and different effector cells. And they have very different antiviral cytokine pathways. TLR8 is IL-12 and 18 and TNF-alpha and interferon-gamma while TLR7 is largely interferon-alpha. The one TLR7 agonist which has entered clinical development, it's a Roche molecule and this is an oral prodrug which is selectively activated in the liver so it's liver targeting. And in healthy subjects, it does show evidence of target engagement. At this meeting, we're seeing the effects in patients with chronic hepatitis B not on treatment. We received TLR7 agonists orally every alternate day for six weeks. And the results are shown here. You can see modest reductions in DNA, RNA, and surface antigen, but only one patient had a greater than one log reduction in surface antigen. Six weeks treatment appeared to be safer, more tolerated with manageable flu-like symptoms and no ALG flares. And this TLR7 agonist has now moved into combination therapy with other novel agents in the phase two platform studies. The TLR8 agonist self-autonomous from Gilead is now in phase two development. I presented the data of the phase two study in new express patients with our randomized to two different doses of sulcantolamide or placebo, three for 24 weeks and then followed up. And this is weekly oral dosing. You can see evidence of target engagement with dose proportional increase in interferon gamma and reduction in CD3 positive T cells thought to represent movement from the peripheral compartment into the liver. Surface antigen declines were seen in more than 25% of patients and these continue to decline during the 24 weeks follow-up. Two patients successfully had sustained loss of surface antigen and three patients lost E antigen. If we look now on the parallel study presented by Harry Janssen earlier this year, in new naive patients, a very similar design. You can see also evidence of target engagement and larger reductions in surface antigen in this treatment naive population. Although of note, no patient lost surface antigen or lost E antigen in the new naive cohort. Let's move now to rescuing exhausted T cells through checkpoint inhibitors. We know that chronic hepatitis B is characterized by immune exhaustion. So PD-L1 blockade should really restore effective intrapathic T cell responses. I presented the proof of concept studies a couple of years ago using the monoclonal nivolumab at very low doses, 0.3 milligrams per kilogram, only for a single dose in patients who are new suppressed chronic hepatitis B. And we did show that almost all patients had a reduction in surface antigen with a mean reduction of almost 0.5 logs. And one patient successfully did achieve functional cure. This was a single dose of a very low dose, one-tenth the therapeutic dose in oncology. And we know that if we are gonna use repeated dosing long-term, there will be the risk for immune-related adverse events. And this has really driven the strategies to develop liver targeting, antisense oligos, or oral small molecule inhibitors. At this meeting, we're gonna see the results of the Roche antisense oligos. This is GalNet conjugated single strand oligo, which induces RNAase H degradation, the PD-L1 mRNA, and therefore knocking down PD-L1 expression. And this proof of concept study, chronic hepatitis B patients received five doses every two weeks of up to three milligrams per kilogram of subcutaneous injections. This appeared to be real to the patient. This appeared to be real tolerated with no immune-related adverse events. There was a dose-related decline in PD-L1 expression and in surface antigen decline with a mean decline of 0.3 logs. One patient, as shown here, did achieve a more than 0.5 log reduction associated with a parallel drop in PD-L1 expression and an increase in ALT. So this was safe, certainly up to 12 weeks. There was some evidence of targeted engagement and immune restoration at the highest dose. And the short half-life may actually be a safety advantage over the monoclonals in terms of turning off any immune-related adverse events. Let's look now at the exciting possibility of redirecting non-HPV-specific T cells. And this is the immunocore approach. And the immunotab molecules are biospecific fusion molecules. They contain a TCR-variable region that is able to recognize surface antigen-derived peptides on the HCl complex on infected hepatocytes. And this is also fused to a humanized antibody-variable region that is able to bind CD3 on T cells that are not HPV-specific. And therefore, this protein is able to activate and redirect polyclonal non-specific T cells and able to kill infected cells expressing surface peptides. And this will include both hepatocytes with replicating virus, those with integrated HPV DNA, and will also include HPV-hepatomous cells. This has been shown to activate CD8 cells, which can kill every infected hepatocyte which expresses surface antigen. And this is a dose-related kill. So we need to be able to mitigate this liver injury. So we need to look at starting off with the lowest biologically active doses, targeting patients initially with a low surface antigen T cell, avoiding those patients with severe fibrosis. Immunocore have developed this molecule to have a very short residency on CD3-positive cells, only three minutes, which should be able to have an on-off effect when you stop dosing. And you can actually turn off the kill with high-dose corticosteroids. The first patient has been successfully dosed in Hong Kong in June. One disadvantage of this platform is it is restricted to HLA-A0201 epitopes. And as we know, this is found in a minority of patients with hepatitis B, only 25% of Asians and less than 15% of Polynesians. And they have to overcome this. They have developed the HLA-A platform that should allow a universal application of this approach. Let's now come to generating new T cells. And this, of course, is therapeutic vaccines. And the point of therapeutic vaccines is to change the immunity in the patient from that with chronic hepatitis B to that of resolved hepatitis B infection. Unfortunately, therapeutic vaccine development has been a bit of the graveyard. And there have been more than 30 different programs, which unfortunately have failed to achieve the goal of functional cure. They are seeing some evidence of restored adaptive T and B cell responses and the occasional patient who achieves e-antigen seroconversion. But as shared, there's really no significant effect on HPV DNA or on surface antigen levels. So to try and improve the chances of success with therapeutic vaccines, the current programs are looking at optimizing immunogenicity, having delivery systems which target the liver, combining with checkpoint inhibitors to reverse T cell exhaustion, and trying to reduce the HPV antigen load. And this last strategy has been looked at in the mouse model, the mouse with high-level surface antigen. And you'll recall this experiment had three different groups of animals, either simply knocked down with surface antigen for three months or therapeutic vaccines for three months, or sequential knockdown followed by the therapeutic vaccine. And the results show that the monotherapies really have modest effects on surface antigen, which weren't sustained, and really little effect on HPV-specific immune responses. In contrast, sequential therapy was certainly synergistic, achieved surface antigen loss, which was sustained in all animals, and showed evidence of strong T and B cell responses. And the sequential knockdown, followed by therapeutic vaccine, was able to achieve cure in all animals. Looking at a novel immunotherapy designed to restore immunity, and this is a VR3434 engineered human antibody, and this has multiple potential mechanisms of action, not just neutralizing effect and mopping up circulating antigen, but it also presents these to antigen-presenting cells, and hopefully will promote a vaccinal effect. And this has now entered clinical development. We've seen the phase one results presented earlier this year. At this meeting, we'll see the phase one results in patients with chronic hepatitis B, just at the three lowest doses, six, 18, and 75 milligram single doses only, are going to be presented at this meeting. And certainly, this appears to be a safe approach. We're not seeing immune complex disease by giving monoclonals to infected patients. I should point out, doses up to 3,000 units have been administered to healthy subjects with no adverse events. It did promote a dose-related decline in surface antigen in patients with chronic hepatitis B. Most patients achieved a more than one log reduction, and most achieved suppression to less than 100 natural units per mil, including five of six in the highest dose of 75 milligrams. And this suppression was durable. It lasted for up to four weeks after a single dose. The question is, of course, is this just simply a mass effect of binding of the surface antigen to the antibody to form immune complexes, or are we starting to see immune stimulation or a true vaccine effect? And this will need to be explored in the next cohorts of multiple dosing. So I'd like to conclude now, but of the immunomodulatory approaches to hepatitis B cure, firstly talking about safety, and this is an issue. We are seeing adverse events related to target engagement. We're seeing flu-like symptoms of TLR7 agonists, GI side effects of TLR8 agonists, the typical side effects of checkpoint inhibitors, and the possibility of immune complexes in highly viremic patients with monoclonal antibodies. We will see ALT flares with this approach, and we need to carefully evaluate these when they arise. They could be good flares and mechanisms of action restoring the host immune responses against hepatitis B, but they could be bad flares, drug toxicity, or the immune-related hepatitises. Or with the immunocore approach, we could potentially see bad flares or with overwhelming hepatocytosis. What about efficacy of immunomodulators? We are seeing restoration of HIV-specific immune responses in some patients, but this is not predictable, and there appears to be no sustained effect with service antigen levels. We need to be able to augment the responses we're seeing by combining immunomodulators with other novel therapies, but which ones to combine is not yet clear. I think the jury is out. With the inhibition of viral replication by other nukes or CAMs, or reduction of the antigen burden by the translation inhibitors is able to restore HPV-specific T-cell responses, which would be synergistic with immuno-modulated. We just are not sure of that as yet. And I think we have really neglected the B-cell responses. Is there a specific anti-HPS profile that we need to maintain immune control post-treatment, i.e. functional cure? And what is the ideal target population? Will it be all patients who are surface antigen positive, or just those who are young, with early disease, and naive, who may have the best retained host T-cell responses? The big question, of course, will be, will we actually need an immuno-modulator as part of a combination strategy to achieve functional cure? I'd like to finish now by thanking all of those who helped me put together these slides. And I'd like to thank you for your attention. Thank you. Dear Chairs, dear ladies and gentlemen, my name is Herod Jensen. I'm Professor of Medicine at the University of Toronto, and I'm very honoured to give you the last presentation in this HPV-6 symposium, really focusing on virological approaches to get hepatitis B cured. Here are my disclosures. So if we're looking at functional cure, there's a virological approach, which I'll touch upon, an immunological approach that my colleague Ed Gaines just touched upon. And if you look at the virological approach to get cure, what you're really aiming for is blocking the viral replication at multiple steps, and try to eliminate CCCDNA, which hopefully will eventually cure hepatitis B infection with no infected hepatocytes left. That's what some people think that will happen. It is unclear that that is possible even, so we'll have to see. If you would like to do that, you need, however, assays which detect a very low level of replication, even below the current lower limit of detection, to determine its efficacy. Another option might be that intense inhibition of protein and viral production by itself might mount an immune response. And we do have some evidence in treatment. For instance, if you stop nucleoside analogs, where a few patients would have essential loss. And actually also with the newer curative agents, we have some evidence for that. Then if you go to the other side of the room, to the immunologist, they say, well, if you want to get immunological control to get HPV functionally cured, go where the money is and deliver immune modulation. The virus integrates into the host genome and will always remain to be present in hepatocytes. And we know that because if patients have an essential loss and are exposed to severe immune suppression, we get reactivation of disease. So there's both are possible and maybe we have to combine them in the future. So if you look at these antiviral and immune modulatory approaches, and if I focus on the antiviral approaches, which is the topic of my talk, there are several types of target engagement. You can really try to inhibit entry of the virus into the hepatocytes. You can try to target the CCC DNA, which is wrapped up in the core of the nucleus of the hepatocyte, really difficult to target without off-target effects. Really, I would like to say that this is pretty far away from clinical development in patients. Then there's RNA interference, where I'll touch upon in a minute, basically blocking protein production of the virus. There's capsid assembly modulators where you're producing empty capsids. There's nucleoside analogs, which we know we have that in our hand in our everyday clinical practice. And you can also try to get functional cure by inhibiting HPS antigen release. So in my talk, I have only 15 minutes. I'll talk mainly on RNA interference, capsid assembly modulators, and inhibition of HPS antigen release. So, and if you look at the different pillars of functional cure, I think three are very important. Inhibition of the replication of the virus, antigen reduction, so less protein produced, and immune stimulation. And I will touch upon the left two as the viral approaches. So if we start with the replication inhibition and we take capsid assembly modulators, which are being extensively studied, these are interesting compounds. And capsid assembly modulators disrupt the HPV lifecycle by destabilizing nucleocapsids or core particles and blocking RNA packaging. In other words, you're getting a core particles without any genetic information. And we can inhibit HPV genome replication and viral assembly. And that would, with the capsid assembly modulators, not only lead to HPV DNA decline, but also HPV RNA decline. And we don't get HPV RNA decline with nucleoside analogs. So that's an additional step really, where an HPV RNA is kind of indicative of transcriptionally active CCCDNA. So the hypothesis was, or still is, that with CAMs, you have an additional effect. The second effect of CAMs is that there could be blockage of CCCDNA replenishment right here at this particular steps, which would really lead to less CCCDNA production over time and maybe S antigen release. Or S antigen reduction. So if we look at what these compounds do, they're easy to use once daily oral regimen. And therefore, there are many of these CAMs in development. If you give monotherapy, you have both an HPV DNA and RNA decline of up to three logs in four weeks. In combination with new up to six logs of HPV DNA decline at roughly one year of therapy. There's no sustained off treatment viral response thus far. So you give these compounds, the patient becomes viral load undetectable. But once you stop, the virus pops up back. There's no or very modest HPS antigen and E antigen decline. There's second generation of more potent CAMs coming. And as I've mentioned, the HPV RNA decline suggests decreased CCCDNA activity. And the question is, if you would give these compounds, for instance, not for 24 weeks, but for two or three years, would that give an S antigen reduction and functional cure? And that's a very interesting concept. The problem, however, that in drug development, the companies want to give drugs for six months or three months. So it's very difficult to test studies where you would have to give a drug for two or three years. With CAMs, there's a definite risk of initial non-response and resistance. The resistance is really diminished if you combine these compounds with nukes. There are side effects, which are minimal thus far. So rash and ALT elevations have been reported. But I must say, two of the CAMs are recently stopped in development due to ALT flares. So here you see an example of patients being treated either with a nucleoside analog alone or a nucleoside analog and a CAM. So the nuke alone is in gray and patients being treated also with the CAM are in blue. And if you go to the left side of this slide, you see the HPV DNA reduction in patients who have not been treated before. And you see that there's really no difference between patients being treated with the nuke alone in gray or the blue lines, which represent the patient being treated with the combination therapy, both for E-positive and E-negative, same results. However, if you look at the HPV RNA reduction, you see a profound difference where if you treat with the capsid assembly modulators, you see a much more significant decline than only with the nuke, both in not treated and virally suppressed patients. So in this particular study, there was a confirmed viral breakthrough in five of the 28 patients with monotherapy of the CAM and with the T33N resistance pattern. And there was no viral breakthrough if the CAM was combined with the nuke. And there was just a tiny bit of S antigen decline in E-positive patients, but otherwise not very much at all. So if we could go to the nucleic acid polymers, these are interesting compounds where we don't know the exact mechanism, but we think that they destabilize our block release of sub-particles of HPS antigen, essentially. And in an early study in patients being treated with NAPS in combination with pack interferon tenofovir, S antigen serial conversion happened in 60% of the patients, which is unprecedented. It's really extremely high at the end of treatment. And then at the end of off-treatment follow-up, it was roughly 35%. And here you see the S levels going down in these particular patients. That S decline was accompanied by flares, some of them severe, so we have to be careful. And unfortunately, this has only been studied in pilot studies with, I think, too many compounds and too few patients in largely uncontrolled settings. So we really need better conformational studies to see how this compound would be doing. There's another compound which contains the S antigen traffic-inhibiting oligonucleotide polymers, or in other words, the stops. And these are oligonucleotides that inhibit S antigen secretion, very interesting compounds being studied, and they're safe and well-tolerated in early clinical studies. So if we go to antigen reduction, we move in to the fields of RNA interference. And that's mostly done with small interfering RNAs. And HPV is susceptible to RNA interference because it replicates through an RNA intermediate. The delivery of these compounds is easier nowadays. We have good platforms like the GalNet platform to get these drugs and to deliver, which has been really difficult over the last decades, but we're able to do it and give these drugs subcutaneously on a monthly basis. And siRNA are very powerful because they can knock down the production of all HPV genes and thereby significantly decrease the number of infectious particles of the HPV DNA, but also the antigens or the proteins of the virus, which might be needed for functional cure. Most of these siRNAs target the S and or the X protein. So they're very potent. You get more than one log of S antigen reduction in 90% of the patients, and in many of those patients, more than that, more than one log, resulting in an S level below 100 in more than half of your patients. The question, however, is whether this is a real, like lasting off treatment, sustainable S antigen decline or loss, or whether it's kind of artificial. Because if you very specifically block the S antigen production and would not mount an effective immune response, you might get S antigen to decline, even become negative. But over time, if you stop the drug, the S antigen might pop back up and you just have a relapse of disease. So we don't know that. Current studies are ongoing. And the question is whether the S decline, and we have some indication for that, as I'll show you in a minute, by itself might mount HPV specific immunity. We think that these drugs could well be used as priming therapy for immune modulators, currently mostly given in combination with CAMs and nucleoside analogs. And they're also antisense oligonucleotides targeting HPV RNA in a similar mode of action where the drugs have to be dosed weekly and actually very, very potent. So here you see what these siRNAs do. And here on the y-axis, you see the change in S antigen level. These are just different compounds that are currently in development. And over time, let's say of 16 to 24 weeks of treatment, you see that most of these compounds generate a one and a half to two log HPS antigen decline, which is quite spectacular. For many, many years, we were not able to do that. So the question is, how do we do that? We were not able to do that. This is an antisense oligonucleotide that has to be dosed weekly with a similar mode of action where you see in red here a very, very quick and profound HPS antigen decline, interestingly followed by an ALT flare, which could be suggestive of an HPV specific immune reaction, immune reactivity. You see that happening in all these three patients, and which may suggest we don't know that with an antiviral drug like antisense oligonucleotide, you could get a more lasting response. In these particular patients, there was also, if you looked at serum markers of innate and adaptive immunity in different gamma in particular, as a readout, clear, I would say evidence of immune activation. And also in another study where an siRNA was given, where they looked specifically on upregulation of HPV specific T cell activation, there were definitely signs of immune reactivation with these particular RNA interference. So if you look at two regimens of triple combination therapy that actually are presented here at this ASLD meeting, and presented by the same author, actually, on the left side, you see patients being treated with triple combination therapy of nucleoside analog, small interferon RNA, and capsid assembly modulators in the REAP1 study, which is a phase two B large study of patients not being treated and given these different compounds in different treatment arms, and with a response readout of HPV DNA undetectability, E antigen negativity, and an S level below 10, 10 international units per amount. And if you then look at these responses, you see that there's hardly any response if you just give the nuke by itself or the nuke together with the capsid assembly modulator. And then if you give the siRNA in increasing doses, you see that the amount of patients responding, so having an S level below 10, moves up from roughly five to almost 20% with 1,400 and 200 milligrams of the siRNA. So that's very good. And then the interesting part of this particular study that if you give the triple therapy of the nucleoside analog to CAM and the siRNA, your response is actually less good than if you would just give dual treatment with the same dose of the siRNA. So there is clearly evidence of an antagonistic effect here between the CAM and this particular siRNA. And in fact, with these trials, the capsid assembly modulator was taken out of the study because it was also shown in another study not to be very helpful. So these drugs do not always add up, but can also work in a detrimental way if they're combined. And on the right side, you see a study where a nuke is combined with an siRNA and Pacinterferin. Pacinterferin is an interesting drug. It has been around for a long time. And if we give it short term, 12 to 24 weeks, it might be quite helpful because it reduces the CCCDNA and also has an immune modulatory effect. And I think 12 to 24 weeks is quite well. People can tolerate that. Anyway, so if you give the combination of a nuke and the siRNA, which is VA2218 and Pacinterferin, you see an almost one log additional decline of S antigen here, the purple line, as compared to the siRNA alone, suggesting that the combination of Pacinterferin is helpful in order to get a cure potentially in the future. So what are the major remaining issues? I think one problem is that we're really lacking correct or good preclinical models. Relevant cell cultures and animal models do not always translate well into humans. And we know that particularly, for instance, from the HPV mouse model. Then there is the answer, what is the correct endpoint? Is HPS antigen loss with the new compounds similar to what we are seeing in the natural history or the licensed compounds? Or is it more artificial, as I've mentioned? Should we be more liberal than just HPS antigen loss and move to partial cure with low S levels? Because there's also S antigen produced from integrated HPV DNA, which is not really reflecting active disease. We need new biomarkers and HPV RNA and correlated antigen are being used, but they're not good as endpoint. They might help us to get a better idea on target engagement of specific drugs. And the question is, do we need to look into the serum or in the organ where all the action happens, namely in the liver? Another interesting point is the correct use of nucleoside analogs. So when should we stop a nucleoside analogs and should we use nucleoside withdrawal as a therapeutic options? And a lot of companies are struggling with how to deal with all of these issues. So in conclusion, ladies and gentlemen, direct CCC DNA inhibition may be needed, but it's very difficult to reach. Capsid assembly modulators, there is an HPV RNA decline which suggests inhibition of active CCC DNA, but very limited S antigen decline in short-term treatment. There's not always an additive effect to nukes and more potent cams are coming along. If you look at the nucleic acid polymers, they're very effective to achieve functional cure, but in small and thus far ill-designed studies, we need conformational studies and alleviate the risk of severe flares. RNA interference, very interesting and important, I think. Significant HPS antigen decline with the siRNAs and the antisense oligonucleotides, but thus far, I think insufficient really to tip the balance and give the knockout punch really to get this disease cured. So I would not be surprised if we have to combine these compounds with immune-modifying agents to reach functional cures. New biomarkers are, in my view, unlikely to be used as endpoint, but can definitely help to assess the type of target engagements of new drugs. Thank you very much for your attention.

Hepatitis B

treatment criteria

biomarkers

chronic infections

surface antigen

RNA

anti-HBC

nucleoside analog treatment

cirrhosis

siRNA

capsid assembly modulators