Great, so it's 11 o'clock Eastern Standard Time. Thank you all for joining us at the ASLB HPV SIG webinar on Immune Tolerance and Chronic Hepatitis B Mechanisms, Management and Controversies. I'm Kyungmi Chang from Philadelphia VA and University of Pennsylvania in Philadelphia, USA. And my co-moderator and co-organizer is Dr. Carla Coffin from University of Calgary in Canada. Today, we hope to provide you with some updates on the evolving understanding of immune tolerance in chronic hepatitis B and clinical implications with our team of experts shown here on the video. And our speakers will present in sequence without interruption, but with Q&A opportunity at the end of the webinar. We encourage the audience to post the questions in the chat box, preferably directed to the specific speaker or speakers, and we will do our best to provide a response during the given time or thereafter. So once again, thank you all for joining. And without any further ado, I'd like to introduce our very first speaker, Dr. Anna Lok, who requires really no introduction, and she's going to give us the overview, the historical perspective on immune tolerance. Thank you, Anna. Take it away. All right. Thank you very much, Kyungmi and Carla for including me. So I was asked to share with you the background about immune tolerance. When I started my career in hepatology back in the early 1980s, we actually didn't have this term because back then our understanding of hepatitis B was very limited and the ability to test for HPV DNA was some beginning. We thought of hepatitis B as consisting only of two phases, the antigen positive, the antigen negative, DNA being detected in the antigen positive phase, and DNA not detected in the antigen negative phase. But things evolved very quickly with the availability of initially hybridization assay for HPV DNA and subsequently PCR assays. So I was just trying to figure out who was the first one to call the term immune tolerance. And it turned out it was Professor Leo and Professor Howard Thomas back in 1985 in this hepatology article, where they tested some samples from 79 patients in Taiwan who had liver biopsy as well as blood samples on the same day. And they found that HPV DNA was higher in the antigen positive compared to the antigen negative patients. And the highest DNA was actually in the youngest patient. But more interestingly, these young patients who had the highest DNA level actually had the least affected histology. They tend to have what we call then chronic persistent hepatitis in contrast to chronic active hepatitis, or simply nonspecific, just a little bit of inflammatory cells, not much. And therefore they said the first phase of chronic hepatitis B infection with high level virus, but very minimal liver inflammation, reflects immune tolerance to hepatitis B. So anyone who doesn't like that term, you can now find a culprit to call that term. Now, there's a lot of debate whether this really truly exists, immune tolerance. But regardless, as a clinician, and I'm not an immunologist, I would say that these patients do behave very differently when they're in this phase. And this is some taken from the hepatitis B research network where we enroll patients that we thought were in the immune tolerant phase, put them on an anticoagulant and also add on capillated interferon. As you would expect, the DNA drop, and when you stop the treatment, the DNA shoot up. But look at the liver enzymes. They start off normal. They have a little bit of a rise during the interferon treatment, but when treatment was stopped, when the virus DNA was climbing, there was no flare in ALT, which is really in contrast to most other situations when you see the DNA rise from two log to eight log, you would expect an ALT increase. Thus, these patients do behave very differently. Similarly, in the hepatitis B research network led by our co-moderator, Kimmy, we look at the immunological response. And yes, if you look at a key value in terms of immune response as measured by the LPR response, there was no statistically significant value in the immune-tolerant patients compared to the immune-active phases. But we always argue statistical significance versus clinical significance, because you can see that the height of the bar here is very, very different. And it is because we have a small number of patients in this group compared to these other groups. And you do wonder if we have similar numbers, whether that will be a key value that would bridge statistical significance. So I'm sure that Antonio is gonna talk more about this, but you know what is in a name? Everyone has some proposed different names. And we do recognize that initial designation, although we put a word immune, is really based on clinical phenotyping, high DNA, normal liver enzyme, minimal liver inflammation, but not based on immunological studies. But as I've shown you, for these patients, when you withdraw treatment, despite a rapid rebound of DNA, there's no ALT flare, suggesting that their response to the virus is different. And even though in Qmesome study, statistically, this immune response was present and not statistically different, they do seem to be weaker. And I would look forward to hearing my co-panelists and share their opinion, which I'm sure will spark a debate later on. Thank you. Thank you so much, Anna, for that introduction and perspective. So the next speaker, as Anna has already introduced, would be Dr. Antonio Bertoletti from the Duke National University of Singapore Medical School in Singapore, presenting late at night from Singapore. Antonio. Thank you. Thank you for the introduction. Thank you for inviting me. And thanks for Anna to really set up the historical point of view. So I'm Antonio Bertoletti. I'm working at Duke-NUS Medical School in Singapore. These are my... Oh, sorry. Yeah. These are my disclosure that are not in any case related to this talk. And let me start with the sort of definition of immunotolerance. So let's say for an immunology, immunotolerance, as I... As is written here, is a state of unresponsive of the immune system to substance or tissue that normally elicit an immune response. I would say also to viruses that normally elicit an immune response. But I would say the historical, instead the HPV nomenclature, say that this is a phase of chronic hepatitis B characterized by active HPV replication and normality of transaminase. Now, the real problem is whether these two definition can actually really coincide in patients with chronic hepatitis B. So in a way, I would say that it is true that in patients with chronic hepatitis B, we have general defect of HPV adaptive immunity. So patients with chronic hepatitis B do have defects on adaptive immunity, on T-cells, on S-specific B-cells. So there are defects of HPV-specific humoral and cellular immune response if we compare them with patients that have resolved acute hepatitis. The real question is whether this, let's say immune defects are really present in selected phase of HPV infection. A little bit what Anna was before saying. And basically whether it is true that during the phase of immune tolerance, there are less HPV-specific T and B-cell response. Let me start it from, let's say, an analysis of the HPV-specific B-cell response. Some analysis that we have done in the past where we use particular reagents that now I don't want to go to the details, but let's say reagents that are going to bind specifically to the B-cells. These reagents are fluorochrome and you can basically quantify both S-antigen-specific B-cells and core-specific B-cells as is shown in these slides. So now, if we are going to quantify the S-specific B-cells in patients with chronic hepatitis B as indicated here, we can find them and they are actually at higher frequency than what we found in patients with acute and resolved hepatitis. Now the question is whether these B-cells are differentially present in the different phases of the disease. And here you can pretty much see that the frequency of the S-specific B-cells is identical among the different phases of the disease. I mean, there is also the point that we say we didn't find any correlation with S-antigen, but this is a point that we say maybe we can discuss and is not related to this talk. Therefore, there are basically no quantitative difference of S-specific B-cells in the different phases of chronic hepatitis B. What about their function? So we use the same reagents to basically analyze the function of the B-cells. We took the T-cells, we basically sort them, and we have done a sort of early spot measuring the production of the antibody by the B-cells. So the first point, which is indicated here on the right, is that actually the core-specific B-cells present in all the patients with chronic hepatitis are actually functional, but we know that patients with chronic hepatitis B do make antibodies against core. And the big difference was actually that instead the S-specific B-cell present in chronic patients indicated here, they were not able or produced very, very little quantity of antibodies if we compare with the S-specific B-cells that were present in vaccinated. Now, again, the other question is there were differences in the different phases of the chronic HBV infection about the function of the B-cells? Not really. Here, the different columns indicate the different patients in the different phases of chronic hepatitis B. And you can see that they were pretty much identical. So again, we can find no functional difference of S-specific B-cells in different phases of chronic hepatitis B. So now let's, and therefore, in a way, for first conclusion is we cannot find any sign of B-cell immunotolerant in selected phases of chronic hepatitis B. But let's go to the T-cells. And I would say first is whether, I would say the problem is whether the transaminases can be really correlated with the quantity and their function of the HBV-specific T-cells, because this is a little bit what is the usual idea. In reality, if we quantify antigen-specific T-cells, HBV-specific T-cells with different methods, and here I'm just showing data from an old paper where we quantify the cell directly as vivo with tetramer, what we find, what the T-cell quantities is really more associated with HBV control and not in the damage. So it was correlated with low quantity of HBV DNA, but there was no correlation with the quantity of transaminases. And we find pretty much similar point of view in, let's say, similar results also when we are analyzing the intrapartic T-cells. So we find that in patients with chronic hepatitis B with perfect normal transaminases, we can actually find high frequency of HBV-specific T-cells. So it's not really the quantity of HBV-specific T-cells is not really, cannot really be quantified by transaminases, and instead is proportional to HBV control. Sorry. Now let's go a little bit more in the detail and try really to analyze the profile of the HBV-specific T-cells and those of the general T-cells in patients that were really selected during the phase of immunotolerance and during the phase of chronic active hepatitis. Here, different to what Anna have shown before, we choose patients that, however, have different age. And this is a very important, let's say, characteristic. So the immunotolerant here, they're really all young patients. The chronic active, they were adults. So we analyze general T-cell response. We didn't find evidence, no evidence of any sort of T helper to alternate balance. We didn't find any alter in production, let's say classical symptoms of sort of immunotolerance. And also we didn't find in the general T-cells, these are general T-cells present in the circulation of the patient. We didn't find any frequency of high expression of PD-1, TIN-3. So all the exhaustion markers. And then we analyze the antigen specificity cell, the frequency of antigen specificity cells. In black, there are the immunotolerant, but young immunotolerant. And in white here is represented the chronic active hepatitis. But these were all patients with age at least of 30 years. And again, here you can see that actually the frequency of the antigen specificity cells was a little bit higher in the immunotolerant patients. So in reality, what we were detecting here is actually a little bit the opposite of what we thought. There were more antigen specificity cells during this phase of immunotolerance, but however, really link with the age of the patients. Now, how can be, let's say, because of this difference in HPV specificity cell frequency and function. So the first point that I think is important to remember is that in reality, the transaminase can actually block the function of the T-cells. So when you have a patient with high level of transaminase, their T-cells are not really feeling very well because they lack an enzyme that is arginine that is important for the function of the T-cells. This is coming from old data, I would say of Francis Ari that already show some of this data. And now we have done other data in the peripheral blood. But more importantly, there was also this paper of a group of Malamaini that actually demonstrated again, that also in the intrapartic T-cells, the function of the T-cells was really altered in the presence of transaminases. And again, the problem is that the transaminases in reality reduce the functionality of the T-cells. We also have done another big study to again, quantify and try to understand better why we found more cells actually in the immunotolerant phase, in the young immunotolerant phase. And we actually have analyzed a large quantity of patients. And what we found that actually was definitely that, for example, if we quantify the frequency of HBV-specific T-cells, and this was data that are coming from more than 180 patients where we analyze T-cell response for S, corporeal and X. And you can see that actually, particularly for S-specific T-cells, the quantity of S-specific T-cells was higher at the young age. And it starts to decline with the age. Again, showing that the duration of infection can impact on HBV-specific T-cell quantity. So again, therefore, these two variable, arginase and duration of infection, can clearly explain why HBV-specific T-cells appear more conserved in the chronic hepatitis B patients at young age. And most of them, they were actually labeled as immunotolerant. Now, briefly, but what is really the case of this different profile of transaminase? And this have been already somehow said already by Anna. The fact is that when we have high level of transaminases, we have a large infiltration of inflammatory cells. And this was well demonstrated, again, by a recent paper of Qiumin Zhang, where they analyzed with multiplex dimensional imaging mass cytometry, the phenotype and the quantity of the cells that were present in the intraparty. And they find that there was an association between transaminases level and quantity of macrophage myeloid cells, basically inflammatory cells. Why we find this mainly in the adult? Well, again, here the answer is somehow easy. Unfortunately, with age, we have an increase of our, let's say, inflammatory response. Something that, for example, we have seen even recently during the COVID-19 pandemic, where we can see that actually the patients that were young, and particularly the kids, do not have a rarely severe COVID-19. And again, the pro-inflammatory response really changed during the lifetime. And therefore, it's more likely that we have inflammatory reaction during, let's say, the adult time. So therefore, conclusion number two, there are really no sign of T cell deletion, of lack of functionality in the immunotolerant phase. But however, there are definitely low inflammatory events in this phase. There is another aspect that I want just to touch, because I think Matteo Iannacone that will speak later will basically go much more in the details here. And there is a fact that in the absence of inflammation, the T cells are being induced, probably only by the fact that the antigen is presented by the hepatocytes. Because there is no inflammation, there is no large destruction of the hepatocytes. The contrary, when there is inflammation, the T cell can be actually induced and activated by dendritic cells. Did we find some of these difference that are actually altering the function of the T cells? Yes, we did. Actually, if we are taking the T cells of classical immunotolerant, we can see that they are much more responding to interleukin-2. They need interleukin-2 to, let's say, proliferate. Something that we don't find in the T cells of the immune active that are looking more like classical exhausted T cells. And this data have been actually further confirmed by a nice study of Evan Newell that have analyzed with large panel of tetramer many antigen-specific T cells. And they actually find that the HBV-specific T cells present in the immunotolerant patient, they were actually phenotypically different. They were looking more as a sort of naive-like T cells. So therefore, in a way, there are differences in function and phenotype of T cell present in the immunotolerant phase of chronic hepatitis B. Whether these differences are sufficient to define some of the chronic patient completely immunotolerant, we can discuss it later. I don't think so. But what I think is really the big problem in general in chronic hepatitis B is that we need to better characterize the quantity and the quality of immune response in these patients. In reality, at the moment, again, selection and clinical management of patients with chronic HBV infection rely exclusively on the assessment of virological and biological markers. And in reality, these cannot measure the great heterogeneity that I show you here that is present in patients with chronic hepatitis B, the heterogeneity of HBV-specific immunity. I wrote an article recently about that. And what I think we really need is actually rapid assay that can really measure precisely the level of antigen-specific T cell response in our patients with chronic hepatitis and really define better what is their immunological profile. Thank you very much for your attention. And if there are questions later, I will be happy to answer them. Bye. Thank you so much, Antonio. I think I'm sure there'll be many questions in the audience. If you want to post the questions in the chat box, please feel free to do so. It's now my pleasure to introduce Professor Simon Ling, my fellow Canadian from the University of Toronto and a clinician investigator at the Toronto Sick Kids Research Institute. And he's going to talk about management of immunotolerant hepatitis B in children. So Simon, it's all yours. Thanks very much, Carla, for the introduction and for the opportunity to talk today. I'm just getting the slides. Okay, hopefully you can see the correct view there. This is my one disclosure and learning objectives for this 15 minutes, I'm ready to try to take a quick tour through these controversies. First of all, some of the limitations in trying to decide on a clinical definition of immune tolerance. Some of the evidence around the impact such as it is of immune tolerant hep B on children's health. And then we'll talk a bit about goals of treatment if we were to consider treating the immune tolerant phase, look at some of the conflicting available evidence of treatment efficacy and just finish up with a list of knowledge gaps where some more research might help us with our clinical decision making. So to begin, maybe with stating the obvious, the prevalence of immune tolerant chronic hep B in children really depends on the clinical definition used, and that of course determines what patients we're talking about and who we may be considering in treatment decisions. First of all, let's begin with ALT. If we look at a large group of children here from the HPRN Pediatric Cohort Study, 274 children, the immune tolerance should fit into this bottom right quadrant of the graph where there is high viral load on the x-axis and normal ALT on the y-axis where ALT is expressed in upper limits of normal. Perhaps surprisingly compared to traditional understanding of immune tolerance in children, very few of these dots based on the baseline data of these children appear in that quadrant and far more, the majority, the vast majority are in this chronic hepatitis area. Exactly how many and what proportion fall in the immune tolerance will depend of course on where you set your upper limit of normal. So if we take on the right of this bar diagram, the site-specific upper limits of normal many labs at this time, we're still using an upper limit of normal of 40 or 45 for ALT, then just over a third of our patients will be classified as immune tolerant. If we use what I believe to be the true normal, updated upper limit of normal based on several studies now in pediatrics trying to remove other causes of mild elevation of ALT, specifically NAFLD of course, then that number, that proportion drops down to just over 10%. What about biopsy data? Biopsy data is difficult to come by in pediatric hepatitis B in general and specifically in the immune tolerance where there's little need for biopsy clinically. Most of the data does relate to clinically acquired biopsies. This data again comes from the HVRN pediatric centers and here we have the ISHAC inflammation on biopsy on the x-axis and ALT on the y-axis. The upper limit of normal is about one and a half logs, which I've marked there in the red line. So there's a very small number of patients here that have normal ALT, but with some mild degree of inflammation showing on those biopsies. Some more recent data from China based on one of their treatment trials for immune tolerant children with hepatitis B, in which 69 children were enrolled here. They all had to have an ALT below 60 and the median values were 45 in the treatment group and 48 in the control group. Again, you can see that these biopsies were mostly with some degree of inflammation on the Shoya scale here. So the majority of these biopsies do show some degree of inflammation. So how you define this clinically will determine how many and which patients we're talking about. What are the consequences of hepatitis B infection in the immune tolerant phase in children? Well, there are a number of cohorts that have been studied longitudinally over the years. This again is the HPRN Pediatric Cohort Outcomes data. If we look at those hard outcomes of death from liver disease, cirrhosis, and HCC, you can see that these are really vanishingly rare in this pediatric cohort. And this is all children, not just those with immune tolerant. So 360 children followed on average for just over four years, almost 1,500 person years of follow-up, and only one incident case of cirrhosis during that time. The single death was nothing to do with liver disease. So we are not seeing those significant hard outcomes. The question is, are we seeing slowly progressive liver fibrosis, for example, that we might be better to identify and treat? And there we have very little data available. We do not yet have longitudinal elastography studies, although we do have some cross-sectional elastography and biopsy studies. Interestingly, if you begin with immune tolerant chronic hepatitis B, you will often remain with immune tolerant chronic hepatitis B over a prolonged period of follow-up during childhood. Here's the HBRN data comparing baseline with last visit about four years later. And you can see most of these children are again immune tolerant at their last visit. There's a little movement in and out along the way, just this small proportion who at last visit are in the chronic hepatitis phase, and some that were taken off into a treatment trial. Similar data from this Italian study, 69 children followed for, again, a median of just over four years. The immune tolerant group are marked in green at baseline here in that bottom right quadrant again, and stay green in the last follow-up data in the right-hand graph, and you can see the vast majority of the green markers remain in that immune tolerant phase, with just maybe one or two creeping into the immune active or into the inactive phases. So the impact of immune tolerant chronic hepatitis B during childhood years seems to be limited. The question then, if we are considering what we should do with these children, and whether we should or should not be treating them, what goals of treatment might we have? Here's four and some checkboxes, and let's see if we can tick off any of these as good reasons to be considering treatment of children with immune tolerant hepatitis B. So first of all, the prevention of cirrhosis. Well, I have shown the HBRN data. Cirrhosis in childhood is very rarely found. There is a small proportion of patients that are found to be cirrhotic during childhood, but really the cirrhosis picks up during the 20s and 30s. And so our question really would be whether treatment during childhood might prevent some of these early adult cirrhotic events from happening. We would have to treat a lot of children to prevent cirrhosis during childhood. And based on some of that biopsy data that we have, we know that cirrhosis can be present very early. In fact, in one of the elastography studies recently published from China, the two cirrhotic children in 140 children studied, both of them were in the first two years of life. So we'd have to treat a lot of children. We'd have to start very early if we were aiming to prevent cirrhosis during childhood. What about the prevention of hepatocellular carcinoma? Well, again, these are very rare events, tragic when they happen, but very rare events during childhood. And again, this incident starts to pick up in early adulthood into middle adulthood. Again, we would have to treat a lot of children to prevent HCC during childhood. Two questions really arise here. One of which is whether treatment early in childhood might have some impact on the biology of carcinogenicity and change the risk of HCC for many years to come into adulthood. That I think is an unanswered question, although there's reasons to think that might be true. And secondly, is the regional variation here. We know that in sub-Saharan Africa, for example, HCC in adolescence is more common than in North America. So maybe there needs to be some regionally specific considerations for that. Can we improve quality of life and reduce the stigmatization? We have some data from the HPRN looking at quality of life for children with chronic hepatitis B. This, again, is all come as not just immune tolerant. And we see that from 244 children studied that the health-related quality of life was generally similar to children without hepatitis B. There were certain domains where quality of life was reduced. Interestingly, the degree of reduction in those certain domains was more pronounced as ALT became higher. And therefore, one can assume, therefore, that the immune tolerance would have less of this mild impact on their quality of life. So again, based just on quality of life, not a strong goal for expanding treatment to the immune tolerance here. Of note, stigmatization is probably not measured well by the current health-related quality of life scores. I'm not aware of data from children looking specifically at stigmatization related to hepatitis B. This is a big issue in the clinic. A lot of teens, in particular, find this problem. So we need to wait for some data on this before we know whether that's a good goal for treatment. And finally, what about reducing transmission? I think here, vaccination is really our primary approach to reducing transmission of hepatitis B. But there is a group of teens who are beginning sexual activity, men with sex with men, and people who inject drugs in their teenage years. And these are high-risk groups that we have really very little data on. The data on this really starts from 18 years and above. So question mark for that. And just as a place marker, I'll mention pregnancy here. Obviously, a bigger topic not to be discussed in this session, but an important potential for reducing transmission by treating hepatitis B that will be e-antigen positive and potentially with normal ALT late in pregnancy. So then let's turn to the treatment. The story about treatment for immune-tolerant hepatitis B in children really starts with this publication in 2006 from King's College Hospital in London, looking at a pilot study of 23 children, mean age of 10 years, with mostly normal ALTs, treated with lamivudine initially for three weeks, and then combination therapy, lamivudine and interferon, for a further 10 months. And the results were quite striking from this small group of patients. Only 2% showed e-seroconversion, but a full 17% lost S antigen and became NTS positive, which prompted interest in further studies. Two multicenter studies I'll show. This first one, again, led by the King's College group, but a multicenter international study treating initially for eight weeks with either lamivudine or entecavir, and then combination of lamivudine or entecavir with interferon for a further 56 weeks, and then some follow-up after that. And patients were randomized into either that treatment group or an untreated follow-up group. This PEG-interferon monotherapy group was discontinued early, and only three patients went into that group. Unfortunately, the study, as you can see, the results were disappointing, and only one patient met the S loss, S-seroconversion endpoint. Similarly, in a North American multicenter study from the HVRN, this is the pediatric IT trial from the HVRN, where entecavir was given for eight weeks and then combination therapy with PEG-interferon up to week 48, and then the combined primary endpoint was measured at week 96. And unfortunately, again, only two out of 60 patients included in this study met the primary endpoint of E loss and DNA below 1,000. So disappointing results. There is, however, this study from China, which took a slightly different approach, again, randomized to either a control group, untreated, or a treatment group. And within this study, patients began with interferon, and after a period of treatment of 12 weeks on interferon, if the viral load had not fallen significantly, so it was less than a two log fall in viral load, then lamivudine was added to the end of the trial at week 96 when outcomes were evaluated. If there was a bigger fall in DNA, then interferon monotherapy was continued to week 72 and stopped In fact, only one patient entered that arm. So really, all of these randomized patients went down the combined therapy arm. And this study, again, showed impressive S-antigen seroconversion results here, 22% at week 96. The authors of this study modeled their likely response to antiviral therapy against ALT and produced this interesting model graph, which we can see that inclusion of these patients between a true upper limit of normal of ALT, which from the pediatric studies is around 30, up to the inclusion level for this study of 60, we're perhaps seeing more of a response, more likelihood of a response in these patients with mild elevation of ALT compared to those with truly normal ALT. So conclusions then, first of all, immune-tolerant hep B accounts really for a small minority of children, at least in North America, at any given time. Cirrhosis and HCC are very rare outcomes in childhood, efficacy and cost-effectiveness of treatment to prevent these outcomes during childhood is certainly unclear, as are the potential to make a difference to these outcomes later in adulthood. A note that HCC does differ in Africa, and we may need to think about this regionally. Efficacy of treatment to prevent other outcomes is unclear, but I would say at least for quality of life and horizontal transmission in North America, we're likely to have low impact here if we just are treating to achieve that goal, with a reminder about the importance of pregnancy. Treatment trials of combination therapy have had conflicting results, as I've shown there, two studies were negative. We don't have data looking at long-term viral suppression with a nucleotide analog throughout childhood to see whether that changes the liver status as these children transition into young adulthood. Those studies, of course, would be very challenging to do over such a prolonged duration. And so finally, recommendations have therefore been to not treat children with immune-tolerant hepatitis B, and what might change that recommendation? Well, I think if we had evidence of advancing fibrosis in the immune-tolerant phase through childhood, that would make us think again. If there was evidence that early childhood treatment changed the long-term cancer outlook for these patients into adulthood, that would also be impactful. Perhaps some evidence from specific subgroups that we can make a difference either with transition in high-risk teens or by reducing stigmatization. And of course, finally, the availability of new therapies with truly high cure rates would make a big difference to all of what we're discussing today. So thank you for your attention, and I'll be happy to be available for questions during the question time. Thank you, Simon. A great presentation. And finally, it's my pleasure to introduce our last speaker, Professor Mark Thurse, who is a professor of hepatology in the Faculty of Medicine in Imperial College London. He's also a senior member of the ESL Governing Board, our sister organization. So Mark is going to talk about controversies in management of adults with immune-tolerant hepatitis B. Please, Mark, go ahead. Thanks very much indeed. Many thanks for the invitation to participate. So let's kick off by trying to identify what the controversies are here. And they mirror clearly a number of the issues that were raised by Simon just now in paediatric hepatology and also by our previous speakers who dealt with the immunology. So is it really immune-tolerant? How can we tell? And will the patient need a biopsy? Is immune-tolerant really benign, as we were originally led to believe by Howard Thomas and colleagues? Should we be treating? And if so, what should we treat? And then as Simon set me up a little bit, we should probably discuss what treatment to use and how to manage pregnant women who've got immune-tolerant hepatitis B. And then perhaps something for a bit of a debate really is, does the risk of transmission justify use of treatment and in what context? So this original definition of immune tolerance, patients who were E antigen positive, chronic hepatitis B, six months of substantial positivity, therefore normal transaminases, high viral load, and we'd tag onto the end here, absence of histological injury. I suppose we could add this to the list of controversies that is why are the guidelines of the three major societies different in many ways when dealing with this particular group of patients. Amongst the differences that I'd quite like to highlight is the level of ALT, which was previously highlighted for pediatrics, but I think is also relevant for adult patients. I think 40 is probably relatively high. The level of HBV DNA, and then obviously the need for biopsy and indications for treatment all differ slightly. And when you're sat in front of our individual patient, we need to bear that in mind and make a decision that's actually for the benefit of the individual. So this is just highlighting the gap between what's considered to be, if you like, serological or clinical immune tolerance versus histological immune tolerance. Amongst those in this study with serological immune tolerance, then actually a reasonable proportion had some form of histological injury. In this graph, I've just highlighted the fibrosis stage, but similar for inflammatory grade in this group of patients. Many had more inflammation visible to the pathologist than was apparent to the biochemist. And the predictors here of who had histological disease, if you like, was those whose age was over 35, whose ALT values were over 35 for a male, 25 for a female, and whose albumin was lower than the normal limits. And the consequences following these patients up for up to 180 months or more, and this is a Korean study, showed actually those patients who had histological injury despite being clinically immune tolerant, they progressed. And one of the problems, I think, with some of these studies, of course, is they assume that we only look at the group of patients on one occasion, whereas in reality, of course, we look more frequently than that. One other question I have, and Simon referred to this earlier, about African populations. So this is one of our studies, a cohort study that went on in a couple of villages in Gambia, West Africa, looking at who's immune tolerant. So actually, in the light blue here, in 1974, at the inception of this cohort, and most of these are children at the time that they were recruited, only a third were young adults. Okay, at that time, there was 30% or thereabouts of immune tolerant. Moving forward to our most recent survey of this group of patients, actually only 2% were immune tolerant. And actually, the majority of patients who had been followed up had actually become either inactive carriers or had indeed resolved the infection completely, had become surface negative. Having said that, of course, in this population, the onset of complications from hepatitis B, such as hepatocellular carcinoma, starts in early adulthood at about the age of 30. Okay, so back to focusing on immune tolerant, is it benign? And let me deal with this in three different ways. First, what's the impact on the hepatocytes, the generation of viral variants, and then clinical progression of the disease. So this is from Bill Mason's group in collaboration with Patrick Kennedy and colleagues in the UK, looking at integration of hepatitis B into the host genome. And this is specifically looking at those patients who were classified as immune tolerant. And what you can see, rather randomly spread across the whole genome, apart from the white chromosome, for some reason, and maybe just because it's small, there were multiple integration sites in the majority of patients. So something biologically important is already going on during this stage of the infection. Furthermore, those integrations are also associated with clonal proliferation of cells. So that suggests that these cells have some survival advantage, that they acquire through the hepatitis B, the clone sizes are really quite large, and the numbers of clones are similar, whether you're immune tolerant or in more advanced, if you like, stages of the disease. So biologically, your ALT and your HPV DNA might be telling you one thing, but if you dig deeper down into the biology, something else is going on. What about then the emergence of hepatitis B variants? So it's not surprising, of course. We know that the replication of hepatitis B is not entirely accurate, and therefore, inevitably, there will be variants that arise, and particularly when you've got high levels of virus, which implies high levels of replication. But what I would draw your attention to here is that variants of hepatitis B, including deletions and variations in the surface antigen, which are associated with immune escape, and also in the reverse transcriptase polymerase open reading frames, that there are variants that arise there which have been associated with resistance to antiviral therapy with nucleoside or nucleotide analogs, as well as variants associated with more aggressive disease. So clearly, the longer that these are allowed, that this phase of high replication is allowed to go on, the more variants will potentially arise. So what about disease prognosis? What happens over, let's say, in this case, a 10-year period in immune-tolerant patients, and here, compared with patients who've got immune-active disease who've been treated? And it looks a little worrying in this study, where those with immune-tolerant disease have disease progression. So on the left is a cumulative incidence of hepatocellular carcinoma. On the right, cumulative incidence of death or transplantation. And you can see a significantly higher rate in patients who were classified as immune-tolerant. So that's a cause for concern, and it may make you rush towards your prescription pad. But we need to take that into context because there are several other similar studies in this area. And this is a meta-analysis by Lee and colleagues published fairly recently. And this suggests, overall, that the incidence of, choosing here, hepatocellular carcinoma as an endpoint isn't significantly different between the two groups. So, and that's reassuring, perhaps, but it also begs the question, why are these outcomes different? Firstly, a number of these studies use different ALT cutoffs, and I tried to highlight earlier on that that's a potential issue. Secondly, HPV DNA levels are different or defined differently in different cohorts. And also there's an issue about HPV DNA and ALT sampling frequency. So in clinical practice, we're doing these fairly frequently. In cohort studies, sometimes they're just done at baseline. So if you do decide, well, you know, we want to treat, what are your justifications? And actually, these are exactly the same as Simon presented earlier, is to, obviously, to prevent disease progression to cirrhosis, hepatocellular carcinoma, to prevent transmission. I'll deal with the prevention of mother-to-child transmission in just a moment. And then there's, obviously, there's that other form of transmission. What's been interesting in a country like the UK where the majority of patients with hepatitis B are from immigrant communities or be often second generation, there has been very little transmission. It's not an issue. However, every now and again, we get cases, and we had a series of young women in the not too distant past presenting with acute hepatitis B and eventually traced back to a rather amorous young 17-year-old who was eventually persuaded in order to prevent further transmission to take some nucleoside analogs. And then perhaps there's an argument for treating based on the WHO elimination strategy. And if you choose to treat, then how? Finite therapy, of course, is based around the use of pegylated interferon alpha. And in pretty much all the guidelines, immune-tolerant hepatitis B is a contraindication to the use of pegylated interferon alpha. And I would find it very difficult to have that conversation with a patient. It's not something that I would contemplate. So we're really based really on using indefinite therapy strategies using nucleoside or nucleotide analogs. And this is, I suppose, is a sort of landmark study in this area. It was sponsored by Gilead using tenofovir or tenofovir in combination with emtricitabine. So a nucleoside and a nucleotide analog in combination. And when you look at this graph, it looks really encouraging. So these are patients with immune-tolerant hepatitis B and you see something like six and a half log of reduction in viral load by 48 to 64 weeks. And that to me looks encouraging. However, bearing in mind that a lot of these patients started with a viral load that was substantially higher than seven logs. And actually, this is the reality of the situation, which even in the dual therapy group, actually you've got less than 80% of patients who've achieved undetectable viral load. And we don't spend too much time these days talking about emergence of resistance and of course resistance to tenofovir is incredibly rare, but that's because we don't treat very many patients with immune-tolerant hepatitis B. And what's perhaps worrying from this study is that about 9% of the patients across both of those groups had variants in conserved sites that would not normally vary in the polymerase and reverse transcriptase genes, suggesting that there is quite a significant risk that those patients would develop antiviral-resistant varying mutations. So changing the topic again now to control or prevention of mother-to-child transmission. So we know that perinatal transmission results in roughly 90% chronicity and the rates of chronicity in the child has been responsible for around 40% of transmission in Asia, probably 10% in sub-Saharan Africa, but we do have some tools to prevent this. And the mothers of course, who are potentially the source of that transmission are those high viral loads immune-tolerant women. So first of all, we have birth dose vaccine, then there is the birth dose hyperimmune immunoglobulin, and more recently, the use of antiviral therapy, so treatment in highly infectious mothers. And this was a key study here, the PAN study published about seven years ago, showing, and it's quite interesting looking at per protocol, which I'll look at again in just a second with another study. In the per protocol analysis, those mothers who were treated in the third trimester of pregnancy with tenofovir had no transmission, but of course, not everybody sticks to the protocol. Further development of this, which I think is interesting is this exploration by Segal and colleagues in Cambodia about the need for the hyperimmune immunoglobulin, which is both expensive and also rather difficult to access in many parts of the world. So here they considered tenofovir eligibility in the mothers to be antigen positivity or an HPV DNA over 200,000 international units. And what's really interesting here is first of all, the duration, the risk of transmission versus the duration of tenofovir use. So for those who'd had four weeks of treatment, there was no transmission. Furthermore, in those who'd had four weeks of at least four weeks of tenofovir treatment, even if they didn't get HPV, there was no transmission. So that is a serious contender going forward for use in certainly low and middle income countries. The other question about this is, well, what happens after the child is born? And probably worth reminding you that there was no increase in fetal abnormalities in either of these studies. But after the child was born, around 40% decided to continue treatment. We don't have the follow-up of that. Of those 60% who stopped, there were some flares, but none of the flares of hepatitis were particularly serious was anecdotally, we've seen actually some seroconversions, both e-antigen and then even a surface antigen seroconversion after withdrawal postpartum. So with that, I will just finish with a summary. So first of all, to say and underline what other people have said, which is a diagnosis of immune tolerant HPV remains a little challenging. Can we be sure in an individual patient? The natural history is probably benign, but it doesn't get us around the fact that these patients need regular surveillance. If you are going to consider treatment, then consider using two nucleoside analogs. So use tenofovir and intecovir because of that risk of antiviral resistance. But this is a relatively evidence-free zone. And then I suppose in that last section, I'm just advocating for increased use of tenofovir in immune tolerant mothers at the time of birth. And with that, thank you very much for your attention. And I'm happy to answer some questions. That's a great presentation, Mark. So I would ask all the speakers and panelists to turn on their screen. And this is an opportunity for the panelists to ask each other questions. And maybe if you would like to address some of the themes that were mentioned in the chat box, some of the questions, for example, Antonio, I'll start it off. The whole arginine story and ALT, there was a comment about what about other non-HPV related conditions as a factor. And what came to mind for me was something like non-alcoholic fatty liver disease and ALT flares affecting T cell function. I mean, I don't have to discover anything that, you know, there is no question that arginine is important for T cell function. And if you have a high level of transaminase, you have a dysfunctional T cell. Then what does it mean dysfunctional T cells? You know, also here, it's not means that you became completely immunodepressed. It means that the T cells are working a little bit less. And we can see this for sure in vitro. And I would say, I'm back to data. I would say, these are data that are coming back from initial analysis. I would say of the serum of the patient done by Frank Cesari in the past. And then I would say biopsies of the patients with chronic hepatitis B done by Mala Maligni, where she was showing very well that in general, the T cells in the presence of transaminases are dysfunctional, but again, slightly dysfunctional. It's not a complete block of the function. Then again, you know, maybe no, I leave it to others to answer different questions. There are four open questions in the chat box. Perhaps that would be something I could read out for others to respond to if that's okay. Yeah. One for you, Mark, from Dr. Sowa, at what HPV DNA level would you consider new treatment in inactive HPV mothers pregnant beneficial? I think Mark is frozen on the screen. His video is frozen. Now he's back. Now he's back. Okay. Did you get that question? Sorry, Carla, I lost connection for a minute. Oh, no problem. It's a question about what viral load level would you consider nucleoside analog treatment in pregnancy inactive HPV mothers beneficial? Yeah, so for antiviral therapy, it would be around 200,000 international units per mL. So there's two reasons for that. One, it's made its way into the guidelines, but we know that the rate of transmission with mothers whose viral load is less than that is negligible or non-existent. The other thing is, and those who are treated, if a viral load comes down to those sorts of levels, they don't transmit, so it protects. So the level in the guidelines is good. And Antonio, I believe you are actively typing, but you may answer online if you would like, about from Dr. Nomov, but given the effect of raised ALT and duration of infection and HPV-specific T cell functionality, what would be the optimal subpopulation where therapeutic vaccines might be better chance for functional cure or surface antigen loss? Yeah, okay. Thank you for this question. I mean, what I really would like to point out here, which is really how can we distinguish the patients that needs different treatment? First, I would say it's important that we start to understand what kind of treatment. If we have a treatment that basically is designed to boost antigen-specific immunity and not only inhibit viral replication, I do sincerely think that we need, again, to have now a better idea of the real HPV, of the status of HPV-specific immunity, which cannot, again, be only derived from the transaminases and the ALT. And this, I think, is, in general, a problem of how we treat patients with chronic hepatitis B. Then, in general, I can answer to Nicola and say, well, you know, I do think that in general, if you are, let's say, not 50 years old, but you are 25, 30 years old, you are still a little bit more HPV-specific T cells. And perhaps this is better. It's also better because probably if you have not a lot of fibrosis, your T cells that you're going to induce will reach hepatocytes much better. This, again, is something that has been very well seen by data in mouse model, where the fibrosis is actually blocking the access of T cells to the infected hepatocyte. But in general, I'm really thinking that as a, let's say, field, we need now to develop better analysis of the general HPV immunity in patients with different, let's say, analysis. I don't know, there is Adam Gehring have just done also fantastic analysis of all the different analysis that can be done. I personally think we should do for something simple that can really be used in the clinic and not go too much on difficult assay that are important for research, but not then for clinical analysis. But I do think this is something that we need to do. Actually, Antonio, would you be able to think about people who have less of a viral load or less of inactive carrier or whatever? I mean, is that something that you could consider for people who have less chance of bad outcome, but lower barrier to overcome? Would you think of it that way? I'm just wondering if Dr. Naumov's question might be addressing that kind of... No, but yes, Cameron. I mean, you know, as I said, as you know, the quantity of virus is somehow inversely proportional to the level of HBV immunity, unrelated to age. So if you say, oh, perhaps vaccine therapy in these patients that have already very low level of S antigen, very low level of HBV DNA might work better. Yeah, probably, yes. But again, I'm studying really patients that have similar level of S antigen and HBV. I can see huge differences on the immunity. I do think this is a variable that we need now to analyze. I think Dr. Lamar. Yeah, Antonio, so I'm wondering what to treat these patients with. So it looks like we have... The immunologist can now provide us with better phenotyping tools. So we can then, you know, hopefully, yeah. But I'm not quite sure what to treat them with. So therapeutic vaccination has not had a great outcome so far. Probably needs something to work with it. And you've also shown us that the B cells are a bit lazy, frankly. So probably not a group of people to go for. I don't know how the B cells are lazy. I think they're just overwhelmed, just to be... Sorry, I... No, no, Mark, yes. On the other hand, I mean, for example, now we, everybody, I mean, we are, there is a lot of hope for the reduction of S antigen. And somehow the aim of the reduction of S antigen has always been set with the idea that he has to recover this immune response against HBV. Maybe that could be a little bit true, but we need also to analyze, again, the patients that have something to recover. If we are treating, let's say, reducing S antigen in that patients that do not have anything anymore, we cannot do anything. And personally, I don't think that we will leave one single golden bullet, one single treatment. We are going towards, let's say, a combination of strategies that will have to be analyzed better, but in association with virological, clinical, and immunological parameters. To define the population that can respond to the different therapies. And I guess, Matteo, you might think that maybe those agonist or antagonist approach may be of use if they don't have a really bad, negative, adverse off-target effects. I'm sorry, Kyungmi, I was answering a question from Frank, actually, sorry. Can you repeat it? Oh, I think we were discussing, like, first of all, who, you know, would we treat? And if so, what do we treat with? I think was sort of the question, that that's the direction the questions were going. Yeah, yeah, yeah. No, so- And Antonio said it should be a combination. And so I was simply asking for your thoughts about the immune modulatory effects. Yeah, well, I mean, of course, this is already done in mouse models. So, I mean, we have to see where this applies to immune-tolerant patients. But if they are any informative at all, I think that the sooner, the better, if you want to reinvigorate the CDAT cells. So, I mean, more children and young adults would be my, in terms of reinvigorating CDAT cells, this is just from a T-cell biology point of view. Then, of course, there are other issues to take into consideration. But if one wants to really have the maximum effect on T-cells, I think that I would rather see young adults more than, and possibly with agonistic co-stimulation rather than blockade of checkpoint inhibition to me. Mark, and maybe you would like to answer two of the questions in the chat as well about FibroScan and about tenofovir for perinatal flares. Yeah, no, that's why I put my hand up, indeed. So, first of all, I think, I mean, so, look, we know that we can get a better handle on the stage of disease, both in probably adults and in kids by doing biopsy. But let's be realistic. This isn't feasible, nor is it, frankly, ethical to be biopsying people at multiple time points. So, the current tools that we're using is HPV DNA and ALT, and we've shown quite clearly they're not great. So, yes, FibroScan is, I think, is a very sensible idea to be doing annually. It's completely non-invasive. And at least in Europe, it's relatively cheap. And that's what we're doing with our patients. So, I would definitely recommend that. The second question about evidence of using tenofovir to prevent perinatal disease flare rather than transmission. Actually, I don't think I'm aware of any data, but clearly, if there is any data, Anna would know. Anna, any comment? Sorry, I missed the last part. What should I know? The question, Anna, is, is there any evidence that tenofovir is effective to prevent perinatal disease flare in HPV, as opposed to, obviously, we know it's effective to prevent mother-to-child transmission. But obviously, some women after pregnancy have a disease flare. And does tenofovir prevent that? Well, I'm not aware of any formal study. So, people have oftentimes said that postpartum, you might have a flare, but it's actually not as common as we think. And since you don't know which patient is gonna have a flare, you actually can't prevent unless you put every pregnant woman on tenofovir. So, I think that we should monitor. And if the liver enzymes do go up and associate a high virus level, you'll then be treating rather than preventing. Because to prevent would mean that we need to put everyone on treatment since we don't know who is gonna develop a flare. Right, exactly, yeah. I have a question for Simon in the last couple of minutes. Just curious, for children, for pediatrics, I don't know as much, but are you treating mostly with entecovir versus tenofovir because of the bone development, metabolic bone issues? We are treating mostly with entecovir. I think it probably varies a little by center. We happen to be in the entecovir trial. So, we sort of stuck with that afterwards. But I think in general, most pediatricians probably using entecovir. What age? The TAF pediatric study is well advanced. It's not reported yet. It's not published yet. So, we'll see what that shows. And I suspect that that will become a drug for the future for children as well. Right. If I could ask for the group. So, there is a, I think, growing sentiments about we should treat everybody who has a detectable virus, chronic hepatitis B. I'm getting a sense from, certainly from Simon, that for children, he doesn't see the benefit of that, at least during that time. I wonder what the group generally thinks about that. Is there a way I can put you guys on the spot and say yay or nay? Yes, no, or maybe? Maybe Dr. Third, because you're on the somehow top left of my screen. Okay. The randomizer, fine. Thank you. Okay. So, I don't believe in treating everybody. I think if it's focused around transmission possibilities, then clearly everybody around the infected person should be vaccinated and educated to avoid transmission. It turns out, I mean, we did some modeling work and published it recently on the cost-effectiveness of the treat-all strategy. And it's not cost-effective. And also, you're giving someone, we know it's relatively safe, a drug, but actually you're giving someone a drug and committing them to a lot of follow-up and they may not need it because most immune-tolerant young people will clear spontaneously. Thank you. Simon, just to reiterate what you said. Yeah, I don't think we're there yet. I think we need more data, particularly about long-term implications. There's a really nice study from Meiwei Chang's group in Taiwan looking at prediction of severe fibrosis in 38-year-old adults from S antigen levels in teenagers. So, it might be that with more data, we can build a story that there are predictors in teenage years of groups that we should be putting on treatment, but we're not there yet. Mateo, your thoughts? Yeah, I mean, in terms of clinical considerations, obviously I refer to Mark and Simon, of course, but I mean, again, from a T cell biology point of view, I think that if you want to have a chance of improving the T cell response, I think it would be still very hard, but probably easier in young adults with HP antigen positive infection rather than later on. Also in terms of the number of cells as shown by Antonio that are there, and also, I think, in terms of their dysfunction, I think they should be more prone to immunotherapeutic interventions. Thank you. Antonio? I mean, to me, it's back to the point of say, yes, we need more data, but we need more quality of data. And I honestly think it's time that we cannot decide everything only based on transaminases and virology. And at that point, we can actually say, okay, this guy needs a treatment, or no, this guy is going to probably control spontaneously. Clearly, we need to study a lot. We need to do longitudinal analysis, but I don't see why we should not really do that. And not only really relying on virological and transaminases to categorize the patients, which I don't think is correct. And let me chime in. The final word from Anna. Yeah, to the immunologists. We would love for you guys to come up with an assay that we can actually order clinically that would tell us this patient, despite all these clinical phenotype is ready, the immune response is prime, go ahead and treat. But I think we all agree that we're not going to be recommending treat all of those because some of them you can keep doing it and it's not doing anything. So we look forward to a blood test, not a liver biopsy, to give us the guidance. I agree 100%, Anna. And this is our fault as well, because we are doing analysis that are too complicated. And I think we need to go to something that is easier. And I agree, but we are working on it. So with everyone agreeing, Dr. Coffin, would you like to close the meeting? Well, I would just like to thank everybody for a wonderful webinar, some really interesting talks and discussion. This will be available in liver learning afterwards for people to look at. And I think the speakers are also welcome for follow-up email questions as well for any of the people participating. So thank you again for participating.

immune tolerance

chronic hepatitis B

clinical implications

controversies

immune response

diagnostic tools

treatment decisions

patients

research

natural history

treatment strategies