The Interview Mystery unraveled: Age-dependent immunity against hepatitis B virus Hepatitis B is a global epidemic. An estimated 240 million people are chronically infected with hepatitis B virus (HBV) worldwide. These chronic carriers have a higher risk of developing cirrhosis and hepatocellular carcinoma, constituting a great burden on the health system, especially in Asian countries. Prof. Ding-Shinn Chen and Prof. Pei-Jer Chen, renowned HBV experts in Taiwan, have devoted their entire careers to fighting HBV. As hepatitis specialists, they have both conducted several studies aimed at unraveling the mystery surrounding HBV. One of their studies, published in 2015, provides insight into the relationship between the gut microbiota and liver immunity against HBV. In this interview, the two professors share the results of their study and experience in the war against HBV. Why do you hypothesize that immunity against HBV is associated with the gut microbiota? After being infected with HBV, the host may develop acute or chronic hepatitis. Ninety-five percent of adult-acquired infected patients are able to eliminate the virus, leading to spontaneous clearance. However, the immune systems of neonates and children are not mature enough to wipe out the virus. More than 90% of infected neonates and 30% of children aged 1–5 years fail to resolve HBV and develop chronic infections. This age-dependent immunity is well known, but the underlying mechanism remains poorly understood. The hosts appear to develop immunity against HBV after birth as their immune systems gradually mature. So, what “teaches” our immunity against HBV? Unlike other organs, the liver is supplied by both the hepatic artery and the portal vein, accounting for 25% and 75% of the total liver blood flow, respectively. The portal vein collects blood from the gastrointestinal tracts and transports the absorbed substances to the liver. After birth, the neonate starts feeding, and the gut microbiota gradually starts to grow. Because humans also gradually develop liver immunity against HBV after birth, we speculate that the gut microbiota might play a role in liver immunity against HBV. What is the primary finding of this study? In this study, adult mice (12 weeks old) could clear HBV after transfection, but their younger counterparts (6 weeks old) remained HBV-positive 26 weeks after transfection. Adult mice lost the ability to clear HBV after elimination of the gut microbiota with antibiotics, which supports our hypothesis that the microbiota plays a crucial role in liver immunity against HBV. Furthermore, young mice with a loss-of-function Toll-like receptor 4 (TLR4) mutation exhibited rapid HBV clearance, which suggests that the TLR4 pathway may play a role in immune tolerance of HBV. What was the greatest difficulty in conducting this study? The main obstacle in proving our hypothesis was building a good experimental model. Generally, mice are the best animal models for studies of human immunological functions. However, because the host range of HBV is very narrow, mice are not susceptible to it. This has been the greatest limitation for HBV studies in the past. We spent nearly ten years building the hydrodynamic transfection mouse model, which is able to mimic features of HBV infection observed in humans, including age-dependent chronicity. It was not until we developed this system that could we answer our question. In recent years, many countries around the globe have used this animal model to conduct HBV studies, especially China, where HBV is an extremely critical public health issue. What is the next step of this research? We want to know exactly which molecules released from the gut microbiota stimulate the immune system and which cells in the liver help to build up immunity against HBV. If we can answer these questions, we may be able to find a way to stimulate liver immunity and finally win the war against HBV. Many HBV carriers are now being treated with antiviral agents such as entecavir. In HBV carriers without cirrhosis, the National Health Insurance only covers the expenditure of these agents during the first three years of treatment. However, some patients develop acute exacerbation or even hepatic failure after discontinuation of these agents. Can we predict the risk of acute exacerbation after drug discontinuation? Acute exacerbation after drug discontinuation is predictable. The presence of hepatitis B e antigen and a high hepatitis B surface antigen concentration are predictors of relapse. However, the difficulty lies in the timing of discontinuation. If the carrier’s liver function improves after a three-year treatment, is it safe to stop the treatment? There are many criteria for drug discontinuation. Some suggest that treatment should be continued until the hepatitis B surface antigen disappears. However, this is not a practical goal for Asians, who require more time before the hepatitis B surface antigen disappears due to early infection. Others suggest life-long treatment. Nevertheless, HBV is an infectious disease, and we still hope to eliminate the virus one day. Since the development of direct antiviral agents, curing HCV is no longer a dream. Will a cure for HBV be just around the corner? What are the difficulties in conducting these studies? The medications for HBV and hepatitis C virus (HCV) are similar, and both are oral agents that inhibit viral replication. The HCV antiviral agent inhibits RNA polymerase and has a high cure rate. In comparison, the antiviral agent for HBV targets reverse transcriptase with a fair control rate. HBV is likely to reemerge in the blood and may induce devastating acute hepatitis once the antiviral agent is discontinued. What is the main difference between the two viruses? HCV is an RNA virus. Once it stops replicating, there is no reservoir in our body. In contrast, HBV belongs to the family of DNA viruses. It hides in liver cell nuclei and will integrate into our liver DNA. Our immune system has difficulty eliminating it. The mechanism by which cells recognize foreign DNA remains poorly understood. If we can better understand how cells recognize foreign DNA, we will be able to develop techniques to help the body clear DNA viruses. What are the new strategies for treating HBV? Many new strategies have been proposed, including immune therapy, CRISPR/Cas9 gene editing and viral entry blockade. CRISPR/Cas9, which is now gaining much attention, directly edits the HBV ccc-form DNA in hepatic cells. However, all of these strategies face some difficulties. Some are now undergoing clinical trials, but the initial results have not met expectations. All of the current anti-HBV agents in clinical use act on reverse transcriptase. In fact, all of these agents were initially developed for the treatment of human immunodeficiency virus (HIV). Most of their anti-HBV properties were serendipitously discovered when treating patients with both HIV and HBV infections. Many researchers are devoted to developing new anti-HBV agents. However, HBV is a very clever and tricky virus. There are still many challenges ahead! What are the strategies of the WHO (World Health Organization) for controlling the viral hepatitis epidemic? The WHO proposed Sustainable Development Goals (SDGs) for viral hepatitis in 2015. The SDGs are composed of three global targets, including reducing new chronic viral hepatitis infections by 90%, reducing the number of deaths due to viral hepatitis by 65% and providing proper treatment to 80% of eligible persons with chronic hepatitis infections by 2030. In fact, viral hepatitis has long been neglected by the WHO, which mainly focused on HIV, malaria and tuberculosis. However, the number of deaths caused by viral hepatitis, especially in Asian countries, is higher than that of the above three infectious diseases. It was wise for the WHO to propose the SGDs for viral hepatitis. Because almost all countries follow the health policies proposed by the WHO, this will make it easier to raise the awareness on a global scale. What are the other major difficulties in preventing HBV on a global scale? First, there are still no medications to cure chronic HBV infection. Second, the availability of screening tools is still low in developing countries with limited medical resources. Third, in some countries, especially China, those who are infected with HBV still suffer from social stigma. On the other hand, although the vertical transmission of HBV can be prevented by hepatitis B immunoglobulins and the HBV vaccine, if the carrier mother has a very high viral load, there is still a 10% chance that her neonates will become carriers even with the above measures. How can the 10% vertical transmission be prevented? Prof. MH Chang, a renowned pediatric doctor at National Taiwan University, has conducted many studies on this topic. Based on what was known about the prevention of HIV vertical transmission, anti-HBV agents were administered during the last trimester of pregnancy. The initial study results are quite promising. We hope vertical transmission cases in Taiwan can be eradicated one day. How did you persuade the government to adopt a universal neonatal HBV vaccination in the 1980s? Did you face any difficulties in promoting the policy to the general population? HBV was already a serious medical problem in Taiwan in the 1970s. There were no effective medications or vaccines back then, and many people died miserably every year. As a result, when HBV vaccines first came out in the 1980s, many Taiwanese experts tried very hard to persuade the government to implement mass vaccinations. Although it was too late to treat patients who already had HBV, we hoped that vaccinations could save the next generation. Luckily, in the early 1980s, Taiwan’s economy began to boom. The government was convinced and was able to spend more money on infrastructure and health care. They invited experts from different disciplines to discuss policies. With increasing evidence suggesting that HBV was a serious problem, my mentor, Professor Juei-Low Sung, called for neonatal vaccinations in the meetings. Finally, the government started the nationwide neonatal vaccination policy in 1984. In the early years after implementing neonatal vaccination, the general population and even some physicians did not know the importance of HBV vaccinations. Some doctors even opposed the policy. They criticized the policy makers for exposing Taiwan’s neonates to unknown risks for nothing but their own fame. This affected the public opinion and caused much trouble in implementing the policy at that time. As a result, the government held an international symposium, inviting both international HBV experts and local experts, as well as local doctors, who opposed the policy. After days of discussion, most were persuaded, and the policy was successfully promoted. The hepatitis C virus (HCV) is another critical public health issue in Taiwan. What are the past, present and future strategies for HCV control in Taiwan? HCV was first identified in 1989. After its discovery, we immediately conducted epidemiological studies in 1990 and found that HCV was a killer in Taiwan second only to HBV. HCV is a unique virus. A high percentage of the infected persons will develop chronic infections. Most do not exhibit any symptoms after being infected. Over time, these chronic infected persons may develop cirrhosis and hepatocellular carcinoma 3-4 decades later. The importance of HCV has been neglected for a long time. However, the prevalence of HCV infection in adults is approximately 2% to 4% in Taiwan. Among infected adults, 70% to 80% (approximately 600,000 people) develop chronic infections. Prevention of HCV is quite simple and includes the use of disposable needles and syringes, the sterilization of surgical instruments, and the screening of donated blood for the presence of hepatitis viruses. Using these measures, the number of new cases of infection decreased drastically. However, what can we do for chronic HCV-infected persons? Two to three decades ago, interferon was the only treatment for HCV and had high relapse rates after discontinuation, and the cure rate was less than 5%. As a result, in early 1990, we at the National Taiwan University Hospital launched studies on HCV treatment and found that combining ribavirin with interferon resulted in a higher cure rate (approximately 50%). Later, by using long-acting interferons, the cure rate of this combination therapy increased to 70-75%. However, the combination of these two medication was not perfect. The cure rate was still low, approximately 25% to 55%, in white and black populations due to the host genotype. In addition, the side effects of these two drugs limited their usage. Interferon causes hematopoietic suppression, flu-like symptoms, depression and hair loss, and ribavirin causes hemolytic anemia. Many patients could not tolerate these side effects and were thus unable to receive proper treatment. Therefore, new agents were developed. A 90-95% cure rate was achieved with the so-called direct-acting antivirals (DAAs) within only 3-6 months of treatment. However, the drugs were terribly expensive. Initially, a 3- to 6-month-course of treatment cost approximately 94,000 US dollars. The patients could barely afford the drugs without health insurance, which sparked much criticism. HCV is still an important health issue in Taiwan. We hope that these new drugs will be covered by the National Health Insurance (NHI) to ensure appropriate treatment for these patients. However, finances are still the key issue. The Taiwanese government has already negotiated with some pharmaceutical companies, and the new anti-HCV DAAs will be covered by the NHI in 2017. As a result of many people’s efforts, the death rates caused by hepatocellular carcinoma and cirrhosis in Taiwan have gradually declined. Doctors and researchers are responsible for providing high-quality study results to convince our government to enact policies. The hepatitis D virus (HDV) is quite mysterious. How is HDV treated and prevented? The hepatitis D virus (HDV) is quite special. It requires the envelope of HBV to form its viral particle. If there is no HBV envelope, HDV cannot complete its life cycle and therefore will not survive. If we can prevent HBV transmission, HDV will no longer be a problem. However, once infected with HDV, it is difficult to eradicate the virus. Chronic HDV carriers may develop cirrhosis and fulminant hepatitis. At present, there is no effective medication for HDV infection. Prolonged administration of interferon may have an effect, but only a minority of patients are able to clear the virus. The prevalence of HDV infection in asymptomatic HBV carriers in Taiwan was approximately 4% according to our study 20 years ago. No similar study has been conducted in recent years, but the prevalence today may be lower. However, in intravenous drug users, the prevalence of HDV infection remains high; therefore, we should still pay attention to HDV. As HDV is easy to prevent and its prevalence in HBV carriers is low, the number of studies of HDV and the development of new drugs targeting HDV worldwide have decreased. The hepatitis E virus (HEV) is associated with a high mortality rate among pregnant women. What are the reasons for this? This deadly phenomenon is only observed during the third trimester of pregnancy. There are some hypotheses, including viral replication in the placenta and hormonal changes during pregnancy, but none of them have been validated. HEV can infect many other animals, including monkeys, deer and hogs. However, high mortality rates during the third trimester have not been observed in experimental studies in monkeys. We still do not known the mechanism responsible for this. HEV is transmitted through the fecal-oral route. As a result, public and personal hygiene is of great importance. Furthermore, an HEV vaccine has recently been developed in China, with good effectiveness according to the clinical trial data. In addition, the hepatitis A virus (HAV) is another hepatitis virus transmitted through the fecal-oral route. Less than 3% of the young population in Taipei currently has antibodies against HAV. Although the hepatitis A vaccination is effective, the price of the vaccine decreases the willingness of some people to get vaccinated. However, I still advocate that all health care providers without hepatitis A antibodies should receive the vaccination. Doctors and nurses are at high risk because they are likely to be infected through contact with hepatitis A patients, especially when bathing or changing the diapers of infected children. Any suggestions for medical students and young doctors? Dream and don’t give up! As a doctor, your mission is to improve people’s health. Try your best to take care of your patients or contribute by taking part in public health policies. You must follow your dreams, keep up your enthusiasm and never give up!   War against HBV Featured Research The gut microbiota may play a key role in age-dependent HBV clearance Hepatitis B virus (HBV) is one of the most common infectious diseases worldwide. According to the World Health Organization, more than 2 billion people are infected with HBV, and 240 million people are chronic carriers. These chronic carriers have a higher risk of developing cirrhosis and hepatocellular carcinoma than the general population, placing a great burden on the health care system. A unique feature of HBV infection in humans is that viral clearance depends heavily on the age of exposure. Ninety-five percent of adult-acquired infections lead to spontaneous clearance, whereas more than 90% of exposed neonates and 30% of children aged 1–5 years fail to resolve HBV and develop chronic infections. It is postulated that “liver tolerance” and “immune immaturity” to HBV result in high viral persistence in the early stage of life but that the maturation of liver immunity later in life allows for HBV clearance. However, this maturation process has not been clarified. The liver has a unique blood supply system, with one-fourth of the blood supply coming from the hepatic artery and three-fourths of the blood supply coming from the portal vein, which collects blood from the gastrointestinal tract. As a result, it is plausible to speculate that the signal stimulating liver immunity may come from the gastrointestinal tract. Who is sending out the messages? In recent years, growing evidence has revealed that the gut microbiota may play a key role in immune system development. Although the liver is not in direct contact with these commensals, constant exposure to microbe-derived metabolites through the gut-liver axis may shape liver immunity. In neonates, the gut microbiota will gradually develop after oral intake and will become stable 2 to 3 years later, which is compatible with the development of liver immunity against HBV. In January 2015, Professor Chen and his team published their study, which was mainly about the relationship between the gut microbiota and liver immunity against HBV, in the Proceedings of the National Academy of Sciences of the United States of America (PNAS). In this study, mice were transfected with HBV and studied. Adult mice (12 weeks old) cleared HBV within 6 weeks after transfection, while their young counterparts (6 weeks old) remained HBV-positive 26 weeks after transfection. In addition, antibiotic-induced sterilization of the gut microbiota at a young age prevented adult mice from rapidly clearing HBV. Possible molecular mechanisms of clearing HBV were elucidated by using mice with specific gene mutations. Based on the above results, gut microbiota development may be associated with age-dependent HBV clearance. This relationship may guide new treatments aimed at helping neonates eradicate HBV. Reference Han-Hsuan Chou, Wei-Hung Chien, Li-Ling Wu, Chi-Hung Cheng, Chen-Han Chung, Jau-Haw Horng, Yen-Hsuan Ni, Hong-Tai Tseng, Dafei Wu, Xuemei Lu, Hurng-Yi Wang, Pei-Jer Chen, and Ding-Shinn Chen. Age-related immune clearance of hepatitis B virus infection requires the establishment of gut microbiota. PNAS 2015 112 (7) 2175-2180; published ahead of print February 2, 2015, DOI:10.1073/pnas.1424775112 Professor Hurng-Yi Wang Graduate Institute of Clinical Medicine, College of Medicine Institute of Ecology and Evolutionary Biology hurngyi@ntu.edu.tw Professor Pei-Jer Chen Graduate Institute of Clinical Medicine, College of Medicine Hepatitis Research Center, National Taiwan University Hospital peijerchen@ntu.edu.tw Professor Ding-Shinn Chen Department of Internal Medicine, College of Medicine Hepatitis Research Center, National Taiwan University Hospital chends@ntu.edu.tw