In Pennsylvania, the Hepatitis B Foundation is collaborating with various stakeholders to launch a State Viral Hepatitis Elimination Plan! Join us as we document our process from start to finish!
This month’s installment focuses on speaking with state legislators to ask for funding to support elimination.
In October 2019, the Hepatitis B Foundation had the opportunity to 
speak with Andrew Vaillant, Ph.D., Chief Scientific Officer at Replicor at the annual International HBV Meeting in Melbourne, Australia. Dr. Vaillant gave us an inside look at REP 2139 – their drug candidate developed for the treatment of chronic hepatitis B and HBV/HDV coinfection. REP 2139 is a nucleic acid polymer that removes surface antigen (HBsAg) and as part of combination therapy, has achieved functional cure for chronic HBV (sustained HBsAg loss) and sustained clearance of HDV infection from the blood in early phase II proof of concept clinical trials it has completed to date. REP 2139 is currently in phase II of clinical trials. Below is Dr. Vaillant’s response to a series of questions we posed to him.
1. Replicor’s drug candidate REP 2139 is a nucleic acid polymer (NAP) for the treatment of chronic hepatitis B. Can you explain the mechanism for this drug and how it works?
REP 2139 is a polymer built from the building blocks the body uses to store genetic material in the body (nucleic acids). These building blocks are linked together in a unique pattern to form nucleic acid polymers (or NAPs for short) and in the case of REP 2139, use only naturally occurring nucleic acids and modifications to prevent it being recognized as a foreign molecule. As a result, REP 2139 is very well tolerated and safe in clinical trials.
In HBV infection, the most abundant viral antigen in the blood is the hepatitis B surface antigen (HBsAg) which plays an important role in preventing immune control of HBV. Circulating HBsAg is almost entirely in the form of non-infectious HBV subviral particles (SVP) which are produced independently from viral replication, making this viral antigen difficult to target with approved therapies. REP 2139 naturally enters liver cells (hepatocytes) and blocks the assembly of SVP in any hepatocyte producing SVPs. This mechanism effectively blocks the replenishment of HBsAg in the blood and also reduces HBsAg inside these hepatocytes. The overall antiviral effect of REP 2139 is to allow the body to clear HBsAg in order to reduce or remove the inhibition of immune control caused by this viral antigen.
2. REP 2165 is also mentioned as a drug candidate. Can you explain the difference between REP 2139 and REP 2165.
REP 2165 is a close cousin of REP 2139 being examined for potential use in future therapy with more frequent dosing to improve HBsAg response in selected cases and was proven to be as effective as REP 2139 in this study. More information about REP 2165 can be found under question 6.
3. Can you share the latest results from phase 2 trials? How is REP 2139 administered to patients, and for what duration of time? What kind of side effects can patients expect with REP 2139?
In our latest trials, side effects have been limited to mild effects from pegylated interferon (pegIFN). REP 2139 is currently given in a formulation (REP 2139-Mg) which results in little to no side effects during administration. REP 2139 is currently administered once every week for 48 weeks by intravenous infusion in combination with other antiviral agents. REP 2139-Mg is expected to be as effective with a once weekly injection under the skin (subcutaneous injection) which will be used in future trials.
4. Is REP 2139 equally effective in HBeAg positive and negative patients?
REP 2139 is effective in HBeAg positive and in HBeAg negative patients in multiple genotypes. As REP 2139 targets a host protein involved in SVP formation and not the virus or SVP directly, its antiviral effects are expected to be similar in all HBV genotypes and may also be effective in the presence of other co-infections with HBV such as HCV and HIV.
5. Can REP 2139 be safely used in patients with cirrhosis?
Another of the remarkable features of NAP based therapy is the high rate of flares in liver transaminases during therapy (occurring in almost all participants in the REP 401 study). Patients with these flares had no symptoms or any negative impact on their liver function.
Continually expanding evidence in the field tells us that during treatment of HBV, these flares are signs of elimination of HBV infection from the liver and are not accompanied by changes in liver function. These same features appear to hold true for transaminase flares during NAP therapy and, when occurring in the absence of HBsAg in the blood, are highly correlated with functional cure in our clinical trials. The ability of cirrhotic patients to tolerate these flares will be tested in future trials and we are encouraged by recent results (produced by a different group) with pegIFN in HBV / HDV co-infected patients showing that host mediated transaminase flares may also be well tolerated in cirrhotic patients.
6. Do you anticipate combination therapy will be needed? Will combination therapy include immune modulators like pegylated interferon and/or treatment with antivirals?
Replicor’s latest REP 401 study is the first in the field to feature triple combination therapy: Tenofovir disoproxil fumarate (TDF), pegIFN and either REP 2139-Mg or REP 2165-Mg. REP 2165 is a close cousin of REP 2139 being examined for potential use in future therapy with more frequent dosing to improve HBsAg response in selected cases and was proven to be as effective as REP 2139 in this study. In addition to the excellent control of HBV DNA with TDF exposure, this triple combination therapy for 48 weeks led to meaningful HBsAg decline (greater than a 10-fold reduction from baseline) in 90 % of participants, HBsAg clearance to very low levels similar to HBsAg “negative” in the qualitative test used in the United States (< 0.05 IU/mL) and HBsAg seroconversion (often with very high titers of anti-HBs antibodies) in 60% of participants. After removal of all treatment (including TDF), a 48-week follow-up yielded very encouraging results: 89% had normal liver function, 56% had reduced liver inflammation, 39% had stable virologic control and an additional 39% had functional cure with HBsAg seroconversion. These results illustrate the effectiveness of combining potent HBsAg reduction with immunotherapy but also suggest that direct acting antivirals such as TDF and entecavir may also contribute to establishing functional cure in a combination setting.
7. Surface antigen loss is key to people living with chronic HBV. Do you believe REP 2139 can provide a functional cure for chronic HBV?
In an early clinical study using NAPs alone, HBsAg clearance by itself resulted in virologic control (low level infection with normal liver infection no longer requiring therapy under current guidelines ) or functional cure (complete control of HBV DNA and HBsAg) persisting after removal of all therapy only in a small proportion of patients but stable throughout a 5 year follow-up. Importantly, HBsAg clearance with REP 2139 in a subsequent study led not only to a dramatic improvement in the activity of various immunotherapies (including pegIFN) but to virologic control occurring in a larger proportion of patients after removal of therapy persisting throughout more than 2 years of follow-up. As a result of these early studies, Replicor believes that the best approach to achieving functional cure of HBV infection is to simultaneously combine potent HBsAg reduction using REP 2139 with immunotherapy to restore effective and long-lasting immune control.
8. Which countries do you anticipate phase 3 trials to occur? Do you anticipate trials in the U.S?
Replicor believes that the combination of therapy with NUCs such as TDF and ETV, pegIFN and REP 2139-Mg will be the first available therapy to offer patients a real chance of eliminating the need for therapy and establishing functional control of their HBV infection and normalizing their liver function. Work is ongoing to start a phase II US trial in collaboration with the Aids Clinical Trials Group as soon as possible. We are also planning to assess other immunotherapies, the effectiveness of which we believe will be similarly improved with HBsAg clearance as we have demonstrated for pegIFN.
9. With regard to hepatitis delta, is there a difference in the mechanism for how it works?
REP 2139 is also potently active against HDV infection and is able to rapidly eliminate HDV RNA, normalize liver function and reverse the liver inflammation associated with HBV / HDV co-infection. The completed follow-up results from our long term follow-up study of co-infected participants treated with REP 2139 and pegIFN show complete control of HDV infection at 3.5 years follow-up in the absence of all therapy in a large proportion of patients. In many patients this control of HDV infection was associated with functional cure of HBV and in some patients with virologic control of HBV. This potent effect against HDV infection is assumed to be driven not only from the effect of REP 2139 on SVP (which also forms the envelope of the HDV virus) but on the ability of REP 2139 to interact with different forms of the hepatitis delta antigen protein essential for HDV replication and assembly.
Thank you to Dr. Vaillant for taking the time to talk to us about REP 2139. The results look promising! We look forward to learning more from continuing and new trials with REP 2139, used alone and in combination with antivirals and immune modulators. We know the hepatitis B virus is challenging, but those living with chronic HBV look forward to a day when there are therapies resulting in a durable loss of surface antigen and sustained viral suppression in a reasonable, finite amount of time.
For most people, Valentine’s Day is a day full of love, but for those living with hepatitis B, it can be filled with dread and anticipation. Perhaps you haven’t told your significant other that you have been 
diagnosed with hepatitis B, or maybe you are spending this year alone because you are scared to begin a relationship. This year, instead of focusing on others, take Valentine’s Day to love yourself – and your liver!
Taking Care of Your Liver
Find a Knowledgeable Provider (and be sure to see them regularly!):
Most people who are diagnosed with hepatitis B lead long, healthy lives. The key is proper care and monitoring by a trained healthcare provider. If you do not yet have a healthcare provider who is regularly monitoring your diagnosis, you can search our physician directory to find one near you. You can also search the World Hepatitis Alliance’s member list to find local resources and organizations who can help you identify a provider in your area.
It is always a good idea to conduct your own research as well! Look into what your provider specializes in, as some may be more knowledgeable about the infection than others. Ideally, it would be best to regularly see a hepatologist – someone who specializes directly in diseases of the liver. However, due to finances and other constraints, this may not be an option for everyone. Seeing any doctor is extremely important, but if you only have access to a provider who is not as experienced in hepatitis B, make sure that they are performing the correct tests to monitor the health of your liver. At each follow-up appointment, your doctor should: check your liver enzymes (ALT, AST), perform a physical exam of the liver, and any other blood tests they might feel is needed to determine the stage of the infection and the health of the liver. Sometimes, the doctor will also perform an ultrasound of the liver to get a better picture of what is going on. You can find some questions that are important to ask your doctor here.
Watch What You Consume:
When people are first diagnosed with hepatitis B, they may feel fine and may not consider making small changes in their daily lives. The truth is that your diet plays a large role in the health of your liver! Everything that enters your body is filtered through your liver. This makes adopting healthy habits essential to keeping the liver in good shape. A standard rule of liver disease is to avoid alcohol – even small amounts – and maintain a steady diet of fruits and vegetables. Foods that are high in fat, salt, and sugar content can lead to weight gain, which puts a strain on your liver. Beware of what you are drinking as well! Drinks like juices and sodas might seem like healthier options, but often contain high amounts of sugar. Diet sodas may lack sugar but have other additives which may have other health implications. Opt for flavored water or seltzer to satisfy a sweet craving instead! If healthier beverage options are not readily available, see if any coffee is available. Studies have shown that drinking coffee can lower one’s risk of developing liver damage and liver cancer – just be sure to watch how much sugar and creamer you put in it! Other diseases of the liver, such as fatty liver, can also increase your risk of liver damage and liver cancer, so it is extremely important to be aware of the risks and what you are consuming.
Those living with hepatitis B should also be aware of aflatoxins. Aflatoxins – which can cause liver cancer – are natural toxins that are 
produced by a mold that grows on crops like corn, peanuts, and tree nuts. Aflatoxins are more common in warm, humid parts of the world, such as African countries and areas with tropical climates. Before eating any grains and nuts, check for any signs of mold. If the food appears to be moldy, do not consume it. The World Health Organization also recommends buying grains and nuts as fresh as possible to minimize the risk of aflatoxin exposure. The fresher the food is, the less time it has been in storage, which is where aflatoxins commonly grow.
Be Mindful of Your Stress Levels:
Living with hepatitis B can be a big stressor, especially for those who may face stigma and discrimination. Research shows that stress can negatively impact liver health. Take some time to find ways that might relieve your stress, such as meditation, listening to music. Being social can also be a stress reliever for some, so try spending more time with your trusted friends and family members. Exercise is also a great stress reliever and it has the benefit of helping you maintain a healthy weight!
If you are celebrating Valentine’s Day with your partner or if you are in a new relationship, remember that hepatitis B is preventable and cannot be transmitted casually! Holding hands, kissing, or sharing utensils or food made by someone who is living with hepatitis B will not spread the infection. Hepatitis B is a vaccine-preventable disease so make sure that they have completed their hep B vaccine series. If they are not protected from hepatitis B, be sure to practice safe sex (use a condom) to prevent transmission.
Welcome to the Hepatitis B Research Review! This monthly blog shares recent scientific findings with members of Baruch S. Blumberg Institute (BSBI) labs and the hepatitis B (HBV) community. Technical articles concerning HBV, Hepatocellular Carcinoma, and STING protein will be highlighted as well as scientific breakthroughs in cancer, immunology, and virology. For each article, a brief synopsis reporting key points is provided as the BSBI does not enjoy the luxury of a library subscription. The hope is to disseminate relevant articles across our labs and the hep B community.
This paper from the University of Duisburg-Essen in Germany shows that hepatocytes infected with HBV exhibit innate immune signaling via the pattern precognition receptor (PRR) Toll-Like Receptor 2 (TLR2). The adaptive immune response to HBV infection is well characterized and is broken into phases based on serological testing of antibodies produced against the virus. However, whether HBV infection triggers an innate immune response has remained controversial, with the long-held belief being that HBV evades the innate immune system as a “stealth virus”. Contrary to this view, studies of acute HBV infection in patients have indicated an early, innate immune response to HBV characterized by a natural killer (NK) cell response. Toll-like receptors (TLRs) are a class of membrane-bound receptor proteins which play a key role in innate immunity by recognizing foreign pathogens and activating inflammatory signaling cascades. A previous publication from this group has demonstrated that primary human hepatocytes (PHHs) can be stimulated through the TLR proteins TLR1-9. In this paper, PHHs from human donors were infected with HBV ex vivo. Then, expression of the innate immune cytokines Interleukin 1 Beta (IL1B), Interleukin 6 (IL6), and Tumor Necrosis Factor Alpha (TNFα) were measured by quantitative, reverse-transcription polymerase chain reaction (qRT-PCR). HBV-infected PHHs showed greatly increased expression of these genes at three hours after infection compared to mock-infected and not treated PHHs. Additionally, immunocytochemical staining revealed translocation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) to the nuclei of HBV-infected PHHs, indicating a cytokine response. Next, to characterize the innate immune response caused by HBV infection, a DNA microarray was used. Here, PHHs were either infected with HBV or treated with a known TLR ligand such as Pam3Cys (TLR2 agonist) or poly(I:C) (TLR3 agonist). Then, RNA was extracted from the cells and converted through a complementary DNA (cDNA) intermediate into biotin-labeled anti-sense RNA (aRNA) which was then hybridized to a Human Genome U219 Array Plate. This plate, coated with over 530,000 DNA probes representing over 20,000 human genes served as a scaffold for complementary base-pair binding of the aRNAs derived from the cells. Once bound to the microarray, the biotin-labeled aRNAs were detected by staining with streptavidin phycoerythrin, resulting in a fluorescent signal wherever complementary base-paring occurred. This microarray analysis revealed which specific inflammatory genes were up-regulated in the PHHs by each stimuli. Gene expression signals which were induced by HBV infection were compared with those induced by the TLR agonists. The gene expression profile of HBV-infected PHHs was most similar to that of PHHs treated with the TLR2 agonist Pam3Cys. This data indicates that HBV infection induces a TLR2-like innate immune response. Importantly, no expression of interferon-stimulated genes (ISGs) was detected in the microarray analysis. Finally, PHHs were pre-treated with neutralizing antibodies against TLR2 (nABTLR2) prior to infection with HBV. HBV-mediated induction of IL1B, IL6 and TNF was significantly reduced by nABTLR2 pre-treatment and conversely, HBV replication was increased. In summary, this paper shows that PHHs exhibited an innate immune response to HBV infection via the TLR2 pathway. The group suggests that this response is one of the body’s first steps leading to HBV clearance. Furthermore, in the discussion section the group indicates that the HBV surface antigen (HBsAg) is likely the protein component of HBV which activates TLR2 upon infection. This finding may help in the development of strategies to cure chronic HBV infection.
This paper from Wuhan University in China reports that HBV infection can increase the expression of Programmed Death Ligand 1 (PD-L1) on the surface of infected hepatocytes, allowing them to escape destruction by the adaptive immune system. PD-L1 is the binding partner of Programmed Death 1 (PD-1), an immune checkpoint protein on the surface of T cells. The expression of PD-L1 on cell surfaces allows for their recognition by circulating T cells as part of the body and not an outside threat. This interaction is important for the prevention of autoimmune disorders in which the immune system attacks healthy cells of the body. However, PD-L1 is commonly over-expressed in a number of cancers and is a hallmark of especially aggressive cancers. PD-L1 expression on cancer cells allows them to neutralize T cells which specifically target them. This is one example of an “immune-escape” strategy exhibited by cancers. Accordingly, PD-L1 and PD-1 are the target of a number of FDA approved immunotherapies for cancer including the PD-L1 inhibitors Tecentriq, Bavencio, and Imfinzi and the PD-1 inhibitors Keytruda, Opdivo, and Libtayo. These drugs are some of the first in their class in that they are not small molecules, but are recombinant, monoclonal antibodies. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor which is mutated or deleted in many human cancers. PTEN is a phosphatase, a protein which dephosphorylates other molecules. This group has previously shown that PTEN plays a role in antiviral innate immunity. Therefore, they wanted to see if PTEN also regulates the adaptive immune response in the context of HBV infection. First, they used immunohistochemical staining of patient liver tissues to compare the levels of PTEN and PDL-1 in patients chronically infected with HBV vs healthy controls. There was a reduced staining of PTEN and a heightened staining of PD-L1 in chronic HBV tissues compared to controls. The group then found a similar correlation using immunofluorescence, qPCR and Western blotting of HepG2 cells vs HepG2.2.15 (HBV-producing) cells. They also transfected HepG2 cells and infected mice via hydrodynamic injection with an HBV-containing vector (pHBV1.3) or an empty vector control (pUC18) and then performed qPCR and/or Western blotting. In all systems, HBV infection/production induced a reduction of PTEN and an increase in PD-L1 expression. Then, in order to elucidate this phenomenon further, a PTEN-expressing plasmid was transfected into HepG2.2.15 cells, which resulted in a reduction in PD-L1 mRNA and protein. Conversely, PTEN knockdown in HepG2.2.15 cells resulted in a two-fold increase in PD-L1 mRNA and protein expression. These results show that HBV inhibits PTEN expression which in turn causes up-regulation of PD-L1. Next, the group transfected HepG2 and Huh7 cells with a number of constructs conferring individual HBV proteins. They found that HBV X protein (HBx) and HBV polymerase (HBp) reduced PTEN expression more than any other HBV protein components. Next, the group analyzed how HBV production in hepatocytes affected human T cells grown in co-culture. Jurkat T cells were co-cultured with either HepG2 or HepG2.2.15 cells and then analyzed by flow cytometry. Jurkat T cells grown alongside the HBV-producing HepG2.2.15 cells had a higher incidence of apoptosis, a higher expression of PD-1, and less Interleukin-2 (IL-2) secretion than those grown alongside HepG2 cells. This result indicates that HBV-infected hepatocytes suppress local T cell responses by PD-L1/PD-1 signaling. Finally, the group used a mouse model of HBV infection to show that PTEN over-expression promotes HBV clearance in vivo. This paper shows that PD-L1, a highly studied drug target implicated in the immune-escape of cancers is also up-regulated by HBV infection. Furthermore, the HBV proteins responsible for this up-regulation are HBx and HBp. This finding may help in the development of immunotherapies to treat chronic HBV infection. Perhaps FDA approved PD-L1 or PD-1 inhibitors may be used in conjunction with interferon alpha treatment or HBV antivirals to boost the immune response against HBV-infected hepatocytes.
This paper from National Tsing Hua University in Hsinchu, Taiwan reports the design and testing of nanoparticles which selectively confer immunogene therapy to hepatocellular carcenoma (HCC) cells. Nanoparticles are very small (1-1000nm) particles which have become an attractive novel drug candidate in recent years. The use of nanoparticles as medicine would enable the customizable delivery of DNA, RNA, or protein payloads to cells. The novel nanoparticles presented here deliver both a small interfering RNA (siRNA) against the Programmed Death Ligand 1 (PD-L1) gene as well as a plasmid DNA (pDNA) encoding the cytokine Interleukin 2 (IL-2). The strategy behind the nanoparticles’ design is to both inhibit an immunosuppressive gene (PD-L1) and up-regulate an immunostimmulatory gene (IL-2) in tumor cells. Delivery of such genes to tumor cells would make them more vulnerable to destruction by circulating cytotoxic T cells (CD8+ T cells). This type of approach is needed, because many advanced tumors create an immunosuppressive tumor micro-environment (TME) rendering many cancer treatments ineffective. The nanoparticles presented here are referred to as tumor-targeted lipid dendrimer-calcium phosphate (TT-LDCP) nanoparticles. The nanoparticles consist of a core of calcium phosphate, thymine-capped polyamidomine (PAMAM) dendrimers, siRNA, and pDNA. This core is coated with an inner lipid called DOPA and outer leaflet lipids DOPC, DOTAP, and DSPE-PEG. The nanoparticle is then tagged with SP94 (SFSIIHTPILPL), a polypeptide which selectively binds to HCC cells but not healthy hepatocytes. Dendrimers are repeatedly-branching molecules which exhibit a sphere-like shape. PAMAMs are the most well-characterized class of dendrimers, consisting of branching amide and amine groups. The calcium phosphate and PAMAM dendrimers in the core of the TT-LDCP nanoparticle promote endosomal escape of the nucleic acid payload. Additionally, this group shows that the PAMAM dendrimers in TT-LDCP nanoparticles also activate the STING pathway. The group showed that STING was activated by treating mouse HCC cells HCA-1 with complete nanoparticles or those lacking the dendrimers. Cells treated with complete nanoparticles showed, by Western blot a higher level of both TBK1 and IRF3 phosphorylation than those treated with incomplete nanoparticles. Those cells treated with complete nanoparticles also displayed heightened transcription of the STING-triggered proinflammatory genes Ifnb,Ccl5, and Cxcl10 as measured by qPCR. Furthermore, the group showed that treatment using their nanoparticles of mice bearing orthotopic HCC implants resulted in dendritic cell maturation in those animals, regardless of the identity of the genes delivered. These results indicate that the dendrimers used in the TT-LDCP nanoparticles not only serve for efficient delivery of nucleic acids, but also as adjuvants that stimulate the STING pathway and activate tumor-infiltrating dendritic cells. This publication gives a glimpse into what future therapies for cancer may look like. The nanoparticle designed by this group is unique in that it has multiple functionalities: selectively targeting HCC cells, inhibiting PD-L1 expression, inducing IL-2 expression, and activating the STING pathway. Such a complex design is bound to require fine tuning before it can become a medicine. But a multi-target immunotherapeutic such as this may be exactly what is needed to help the body fight against aggressive, immunosupressive tumors.
Lay Summary:
This month, the innate immune system was the focus of HBV research. Scientists hope to find how the innate immune system interacts with HBV during viral infection and proliferation. Doing so will shed light on host factors which lead to chronic infection and inform antiviral strategies. Notably, this month a human protein, MX2 was found to have potent anti-HBV activity by preventing cccDNA formation. Also, a microRNA encoded by HBV called HBV-miR-3 was found to activate the human innate immune system to limit HBV replication. This month, a paper studying woodchuck hepatitis virus (WHV) traked activation of the innate immune system as well as he adaptive immune system in an acute infection model. Also this month, concerning hepatocellular carcenoma (HCC), the alternative splicing of mRNA in tumors was found to vary in HCC patients based upon their risk factor (HBV, HCV, or alcohol). Finally, a review was published this month concerning STING, an innate immune protein which is not activated by HBV infection but which may prove a valuable tool for cancer treatment.
Meet our guest blogger, David Schad, B.Sc., Junior Research Fellow at the Baruch S. Blumberg Institute studying programmed cell death such as 
