Journal articles recommended by our Emerging Scholars Scientific and Medical Advisors
Each month, one of our emerging scholars in the field of hepatitis B basic and clinical research recommends one or two current scholarly article(s) in the field.
PICK OF THE MONTH - August 2024
For August, our presenting emerging scholar is Barbara Testoni, PhD.
Yu Y, Kass MA, Zhang M, Youssef N, Freije CA, Brock KP, Aguado LC, Seifert LL, Venkittu S, Hong X, Shlomai A, de Jong YP, Marks DS, Rice CM, Schneider WM. Deep mutational scanning of hepatitis B virus reveals a mechanism for cis-preferential reverse transcription. Cell. 2024 May 23;187(11):2735-2745.e12. doi: 10.1016/j.cell.2024.04.008. Epub 2024 May 8. PMID: 38723628; PMCID: PMC11127778. [website link]
Deep mutational scanning of hepatitis B virus reveals a mechanism for cis-preferential reverse transcription
Yu et al. previously described a method to initiate the replication of HBV using synthetic RNA, thus eliminating contaminating background signals from input virus or plasmid DNA (Yu et al. DOI: 10.1126/sciadv.adg6265). Here, they combine this approach with deep mutational scanning to assess the fitness of thousands of HBV variants in a cell culture assay. This assay measures the frequency that an HBV pgRNA is reverse transcribed to full- length minus-strand HBV DNA.
The results help clarifying the mechanisms of HBV Pol translation and cis-preference for pgRNA. With a very elegant combination of biochemical and functional experiments, the authors demonstrated that Pol translation involves leaky ribosome scanning and that the unstructured C terminus of HBV Pol encodes a ribosome-stalling motif that enforces Pol’s cis preference for pgRNA. Next step will be to determine why cis preference is important for the virus life cycle, which will require testing mutants in a more natural setting that includes robust virus amplification and spread.
PICK OF THE MONTH - March 2024
For March, our presenting emerging scholar is Mohsin Khan, MSc, PhD.
Tsukuda S, Harris JM, Magri A, Balfe P, Siddiqui A, Wing PA, et al. (2024) PLoS Pathog 20(1): e1011917. https://doi.org/10.1371/journal.ppat.1011917. [website link]
The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome
The RNA methylation (m6A modification) is the most abundant modification in the eukaryotic system. After it was first identified in the early 1970s, researchers have always been interested in identifying the role of m6A in multiple physiological events. However, due to the intricacies and complexities of the RNA molecule, and the unavailability of a robust high-throughput analysis platform, the overall growth in the field was incremental. The development of RNA-seq technology and its recent advancement have not just revived the m6A-related research field but transformed it to a greater extent. With the advent of RNA-seq technology, the m6A modification and its role in various physiological events, including viral pathogenesis, have been extensively investigated. In the year 2018, the presence of m6A modification in HBV-transcripts was identified for the first time, and it was discovered that the stem-loop structure of HBV transcripts is m6A modified, which has a significant role in viral replication.
Researchers at the University of Oxford have now identified that ALKBH5, a demethylase that removes m6A from RNA, can regulate the HBV transcriptome under hypoxia. It was observed that under hypoxic conditions, HBV transcriptome showed an increased abundance of viral transcripts, and this abundance is dependent on m6A modification. The team also noted that during hypoxia, ALKBH5 is induced, and inhibition of ALKBH5 via RNA-silencing can significantly reduce HBV pre-genomic RNA. ALKBH5 was not only seen to affect HBV transcripts but also found to regulate the stability of HIF-alpha, a cellular sensor that is stimulated under low-oxygen conditions.
In summary, this report provided detailed information about how m6A demethylase can contribute to cellular adaptation to hypoxic stress. Although this investigation deciphered the ALKBH5-hypoxia axis in the context of HBV infection, this mechanism may be more widely applicable to other hepatotropic pathogens.
PICK OF THE MONTH - January 2024
For January, our presenting emerging scholar is Lena Allweiss, PhD.
Giraud G, Rodà M, Huchon P, et al. Nucleic Acids Res. Published online December 19, 2023. doi:10.1093/nar/gkad1200. [website link]
G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes.
When HBV infects hepatocytes, the viral DNA is transported into the cell´s nucleus and it converted into a small viral chromosome, called an episome or “covalently closed circular DNA” (cccDNA). This cccDNA is a very stable molecule, difficult to target with antiviral drugs and is therefore believed to be one of the main reasons for viral persistence. From the cccDNA, viral RNAs are transcribed leading to the production of viral proteins and new progeny virus. We currently do not completely understand the generation of cccDNA, its stability and degradation processes, or the regulation of its transcription. By conducting research and expanding our knowledge on these processes, we hope to find new druggable targets or strategies to eliminate cccDNA or shut off cccDNA transcription in order to cure HBV infection.
The study by Giraud and colleagues describes a new mechanism of transcriptional regulation of the cccDNA. The cccDNA was shown to contain secondary structures called G-quadruplexes, which promoted liquid-liquid phase separation (i.e. the creation of membrane-less organelles in the nucleus) and with it enhanced viral transcription. G-quadruplexes could therefore present a new druggable target.
PICK OF THE MONTH - November 2023
For November, our presenting emerging scholar is Yumeng Zhang, MD.
Zhang Y, Bourgine M, Wan Y, et al. [published online ahead of print, 2023 Oct 11]. J Hepatol. 2023;S0168-8278(23)05146-2. [website link]
Therapeutic vaccination with lentiviral vector in HBV-persistent mice and two inactive HBsAg carriers
A interesting study recently published in Journal of Hepatology introduces a novel approach in combating chronic hepatitis B virus (HBV) infection. Researchers developed a lentiviral-vectored therapeutic vaccine and evaluated its effectiveness in murine models and two inactive HBsAg carriers. Among the various proteins tested, the large HBsAg (LHBs) encoded by the lentiviral vector (LV-LHBs) emerged as the most promising candidate. In murine models, LV-LHBs showed exceptional potential by eliciting robust antigen-specific T cells and significantly reducing serum HBsAg levels along with the viral load. Remarkably, a substantial portion of HBV-persistent mice vaccinated with LV-LHBs achieved a sustained loss of serum HBsAg and a significant reduction in HBV-positive hepatocytes within the liver. Moreover, in human participants, specifically in two inactive HBsAg carriers, vaccination with LV-LHBs displayed encouraging results. One patient experienced a considerable increase in LHBs-specific T cells, while the other exhibited a detectable but weaker response. Notably, both patients demonstrated sustained reductions in HBsAg levels, indicating a positive therapeutic outcome. The study's success in expanding HBV-specific T cells, reducing HBV-positive hepatocytes, and lowering serum HBsAg levels showcases the promising potential of the lentiviral-vectored therapeutic vaccine in the treatment of chronic HBV infection, offering hope for improved therapies in the future, particularly therapeutic vaccination.
PICK OF THE MONTH - November 2023
For November, our presenting emerging scholar is Huan Yan, MD, PhD.
Key Factors for "Fishing" NTCP as a Functional Receptor for HBV and HDV
Huan Y, Chunli W. PMID: 36851726 PMCID: PMC9959848 DOI: 10.3390/v15020512 [website link]
The eLife paper in 2012 reported a discovery identifying Sodium Taurocholate Co-transporting Polypeptide (NTCP) as a functional HBV receptor. This important study has been well recognized as a landmark in the HBV field, which opened the door for many HBV and HDV infection-related studies. In this review, the authors retrospected their bumpy and exciting journey for identifying this long-sought bona fide HBV receptor and summarized the crucial factors for our successful receptor hunting by comparing the efforts in catching a particular fish. This review may also provide helpful insight into the pitfalls and critical factors in identifying a protein target by peptide or protein baits through cross-linking and immunoprecipitation.
PICK OF THE MONTH - August 2023
Discovery of a first-in-class orally available HBV cccDNA inhibitor
Wang L, Zhu Q, Zhang JD, Zhang Y, Ni X, Xiang K, Jiang J, Li B, Yu Y, Hu H, Zhang M, Wu W, Zeng J, Yan Z, Dai J, Sun K, Zhang X, Chen D, Feng S, Sach-Peltason L, Young JAT, Gao L. J Hepatol. 2023 Apr;78(4):742-753. doi: 10.1016/j.jhep.2022.12.014. PMID: 36587899 [website link]
The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure.
PICK OF THE MONTH - July 2023
For July, our presenting emerging scholar is Barbara Testoni, PhD.
Discovery of a first-in-class orally available HBV cccDNA inhibitor
Wang L, Zhu Q, Zhang JD, Zhang Y, Ni X, Xiang K, Jiang J, Li B, Yu Y, Hu H, Zhang M, Wu W, Zeng J, Yan Z, Dai J, Sun K, Zhang X, Chen D, Feng S, Sach-Peltason L, Young JAT, Gao L. J Hepatol. 2023 Apr;78(4):742-753. doi: 10.1016/j.jhep.2022.12.014. PMID: 36587899 [website link]
As a result of the persistence of the viral minichromosome in the infected liver, i.e. covalently closed circular (ccc) DNA, viral relapse is almost universal when nucleos(t)ide analogue (NA) treatment is stopped prior to HBsAg loss. So far, no compounds in the new drug development pipelines have demonstrated a significant effect on cccDNA levels. Given the complexity of cccDNA biology and its key role in viral persistence, the discovery of orally available antiviral agents that could deplete the intrahepatic cccDNA pool would change the HBV cure paradigm.
Here, Wang et al. report the discovery of a small molecule, ccc_R08, that decreases the HBV cccDNA reservoir in preclinical relevant study models, including HBV-infected primary human hepatocytes, the HBV circle mouse model and the uPA-SCID humanized liver mouse model. Besides the antiviral effects on HBV antigen secretion and viral replication, the compound demonstrated its ability to decrease the cccDNA reservoir in the liver. This is the first time that a small molecule has been shown to act on the already established cccDNA pool, with a preferential effect on “transcriptionally active” molecules.
However, the exact mode of action of the compound remains to be determined. Indeed, the integrated analysis of host transcriptomics and viral RNA expression and replication suggested that the anti-HBV effect of ccc_R08 is most likely mediated by the regulation of a host gene regulatory network. Using an elegant forward and reverse pharmacology approach, the authors identified putative effectors of the pharmacological effect of ccc_R08, including CHEK1, CHEK2, TOP2A, and ATM, but also novel candidates to be explored by future studies, such as transcription factors, druggable proteins, and signal transduction proteins.
Altogether, this study not only represents the proof-of-concept that targeting the established liver cccDNA pool is possible, but also provides the ground to generate new knowledge on the cellular networks involved in the persistence and regulation of the HBV minichromosome. Further functional investigations of these candidates may reveal new avenues for the identification of molecular targets involved in cccDNA depletion.
PICK OF THE MONTH - June 2023
For June, our presenting emerging scholar is Tung-Hung Su, MD, PHD.
A multicenter randomized-controlled trial of nucleos(t)ide analogue cessation in HBeAg-negative chronic hepatitis B
Bömmel F, Stein K, Heyne R, Petersen J, Buggisch P, Berg C, Zeuzem S, Stallmach A, Sprinzl M, Schott E, Pathil-Warth A, Arnim U, Keitel V, Lohmeyer J, Simon KG, Trautwein C, Trein A, Hüppe D, Cornberg M, Lammert F, Ingiliz P, Zachoval R, Hinrichsen H, Zipprich A, Klinker H, Schulze Zur Wiesch J, Schmiedeknecht A, Brosteanu O, Berg T. DOI: 10.1016/j.jhep.2022.12.018. PMID: 37062574 [website link]
The article discusses the STOP-NUC that aimed to assess the possibility of achieving a functional cure for chronic hepatitis B by discontinuing long-term nucleos(t)ide analogues (NUCs) treatment. NUCs are the standard treatment for chronic hepatitis B, but they rarely result in a functional cure, defined as the loss of hepatitis B surface antigen (HBsAg). The trial involved 166 HBeAg-negative patients without advanced fibrosis who had been on continuous NUC treatment for at least four years at baseline. The patients were randomized into two groups: stopped NUC treatment (Arm A) while the other continued treatment (Arm B) for a 96-week observation period.
The results showed that the group that discontinued NUC treatment (Arm A) had a significantly higher rate of HBsAg loss (10.1%) compared to the group that continued treatment (0% in Arm B). Patients with baseline HBsAg levels below 1,000 IU/ml had a greater chance of HBsAg loss. No serious adverse events related to treatment cessation were reported. The results of the STOP-NUC trial provide evidence supporting the concept of stopping NUC treatment as a potential therapeutic option to induce functional cure in certain patients with chronic hepatitis B. However, adopting the stopping NUC strategy should have a close monitoring schedule and protocol for retreatment in case of a severe flare. Further investigation into other ethnicities should be conducted.
PICK OF THE MONTH - April 2023
For April, our presenting emerging scholar is Julie Lucifora, PhD, HDR. She has selected two articles.
A 3-Year Course Of Bulevirtide Monotherapy May Cure Hdv Infection In Cirrhotics
Anolli MP, Degasperi E, Allweiss L, Sangiovanni A, Maggioni M, Scholtes C, Oberhardt V, Neumann-Haefelin C, Dandri M, Zoulim F, Lampertico P. J Hepatol. A 3-Year Course Of Bulevirtide Monotherapy May Cure Hdv Infection In Cirrhotics. 2023 Jan 3:S0168-8278(22)03475-4. doi: 10.1016/j.jhep.2022.12.023. Online ahead of print. PMID: 36931396 [website link]
Chronic Hepatitis Delta (CHD) leads to the most severe form of viral hepatitis and until recently patients had very limited therapeutic options. Bulevertide is the first specific drug against hepatitis delta virus (HDV) that was conditionally approved for the treatment of CHD in 2020 in Europe. It was designed to block HDV entry and thereby propagation within the liver of infected patients. In this case report, the authors described the first case of CHD cure in a patient with severe clinical conditions, including compensated cirrhosis and portal hypertension, receiving Bulevertide monotherapy. HDV viral load declined rapidly and became negative 28 weeks after beginning of the treatment. After 3 years, the treatment was stopped and HDV viral load remained undetectable for at least 72 weeks. All markers of liver function were normalized during or after treatment. This article raises hopes for patients with CHD.
Improved hepatitis delta virus genome characterization by single molecule full-length genome sequencing combined with VIRiONT pipeline
Charre C, Regue H, Dény P, Josset L, Chemin I, Zoulim F, Scholtes C. Improved hepatitis delta virus genome characterization by single molecule full-length genome sequencing combined with VIRiONT pipeline. J Med Virol. 2023 Mar;95(3):e28634. doi: 10.1002/jmv.28634. PMID: 36879535 [website link]
Hepatitis Delta Virus (HDV) is a small virus with a high genetic variability. Differences in HDV genome sequences may lead to differences in replication, propagation, pathogenesis and response to treatment. It is therefore very important to be able to properly assess HDV genome sequences variations in patients. So far, this was not routinely performed because sequencing approaches available were challenged by the features of the HDV genome. In this article, the authors developed a workflow to amplify, sequence, and analyze the whole HDV genome in a single fragment from patient’s sera. They made available online for free the bioinformatic tools necessary to analyze data. This new approach, that can be implemented in medical centers, will help to better characterize circulating HDV genomes among the population and allow correlation with disease outcome and response to treatments.
PICK OF THE MONTH - March 2023
For March, our presenting emerging scholar is Angel Yen-Chun Liu, MD.
Wen-Juei Jeng, George V Papatheodoridis, Anna S F Lok. Hepatitis B. Lancet 2023 Feb 9; S0140-6736(22)01468-4. doi: 10.1016/S0140-6736(22)01468-4.
https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(22)01468-4.pdf
Impact: The article generally and intact summarize the epidemiology, virology, pathophysiology, prevention, diagnosis, natural history, management as well as new therapies in development of hepatitis B. It can provide the effective information to understand the hepatitis B infection, especially from informative and exquisite figures.
PICKS OF THE MONTH - January 2023
For January our presenting emerging scholars are Julie Dang, PhD, MPH and Mohsin Khan, MSc, PhD.
Pham TN, Le DH, Dao DV, Phan LT, Pham TT, Nguyen TB, Mize GW, Gish RG, Lee WM, Trang A, Le AN. Establishing baseline framework for hepatitis B virus micro-elimination in Ho Chi Minh City, Vietnam–A community-based seroprevalence study. The Lancet Regional Health-Western Pacific. 2023 Jan 1;30:100620.
https://www.thelancet.com/journals/lanwpc/article/PIIS2666-6065(22)00235-8/fulltext
Impact: This is the first population-based community HBV study to estimate the prevalence of HBV statuses in Vietnam. Use of three sero-marker screening for HBV enabled researchers to distinguish among those who were: HBV susceptible population (negative all three seromarkers), chronic carriers (HBsAg(+)), HBV exposure with immune control (anti-HBc total (+), with or without anti-HBs), and HBV vaccination (anti-HBs(+) without HBsAg). Researchers also found wide variations in HBsAg (+) and HBV vaccination rates between districts, risk factors, and socio-economic statuses. This study demonstrated the feasibility of conducting a large-scale comprehensive HBV screening and access to care program in an HBV endemic country that involved collaboration among diverse stakeholders including government, health care institutions, community organizations, and private sector.
Chu JYK, Chuang YC, Tsai KN, et al. Autophagic membranes participate in hepatitis B virus nucleocapsid assembly, precore and core protein trafficking, and viral release. Proc Natl Acad Sci U S A. 2022;119(30):e2201927119.
https://www.pnas.org/doi/10.1073/pnas.2201927119
It is widely known that Hepatitis B virus (HBV) replication is regulated by autophagy. This investigation, led by Prof. Jing-hsiung James Ou at UCLA, has precisely examined and characterized how autophagic membranes participate in the HBV life cycle. Autophagy is a process in which the cells eliminate their organelles and macromolecules. Under normal circumstances, autophagy ensures homeostasis and proper recycling of cellular biomaterials. However, the signaling events of autophagy are often altered during stress, inflammation, and infection. Interestingly many pathogens, especially viruses, have evolved with successful strategies to hijack this cellular process to support their replication. HBV is one among those viruses that are successful invaders and play with autophagic machinery efficiently.
Autophagy, at the cellular level, starts with the formation of isolation membranes that are called phagophores (PP). PP are crescent structures that subsequently expand to form complete double-membrane vesicles called autophagosomes (APS). The APS can fuse with either lysosome or MVBs (multivesicular bodies) to form autolysosomes or amphisomes respectively.
In this investigation, it was observed that the PP and APS, purified from the cells that contained replicating HBV, are highly enriched with core particles. These PP- and APS-associated core particles contain hypo-phosphorylated core protein. The PP-associated core particles have mostly double-stranded HBV DNA, while APS-associated core particles are rich in both, single- and double-stranded DNA. With some additional experiments, it was further confirmed that APS-associated core particles are nucleocapsids that contain viral pgRNA (progenome RNA) and/or DNA. It was also observed that the association of HBV core with PP and APS is independent of pre-core protein. Interestingly, the ectopically expressed core protein did not localize to APS. These observations suggest that the cellular trafficking of individually expressed core protein does not entirely mimic the trafficking of core protein originating from active HBV replication. Possibly, the packaging of pgRNA is a cooperative event that is required for the association of core to APS.
Additionally, it was noted that HBV could induce the formation of amphisome, and disruption of this event suppresses HBV release from the cells. It signifies that although HBV induces autophagy via PP and APS formation, it does not promote autolysosome formation. In other words, HBV induces only early events of autophagy and utilizes PP and APS for packaging and replication purposes. However, it directs a major pool of APS towards amphisome formation and does not promote the degradative stage of autophagy to ensure the successful release of mature HBV.
In summary, this report provided detailed information about the intracellular trafficking of HBV proteins and the relation between autophagy and HBV replication. This will be very helpful for the development of novel anti-HBV drugs that target the autophagic pathway.
PICK OF THE MONTH - December 2022
For December, our presenting emerging scholar is Peter Block, MD, MSc.
Yuen M-F, Lim S-G, Plesniak R, et al. Efficacy and safety of bepirovirsen in chronic hepatitis B infection. N Engl J Med 2022; 387:1957-68.
https://pubmed.ncbi.nlm.nih.gov/36346079/
The search for an HBV cure has been an area of active investigation for decades. While the backbone of our current treatment – nucleotide/nucleoside analogues (NAs) – can suppress the virus, they rarely eradicate it. The scientific community has therefore sought to develop novel therapeutics with curative effects. The article highlighted here describes the clinical efficacy of bepirovirsen, a new antiviral agent that may bring us closer to this goal.
Bepirovirsen is an antisense RNA-based drug delivered that impairs HBV replication by targeting viral messenger RNA in the infected hepatocyte. This study evaluated its antiviral activity through a phase 2b clinical trial in patients with CHB either receiving (or not receiving) NA therapy. Study participants were randomized into different treatment arms, where they received varying durations of therapy with bepirovirsen and then were followed for 24 weeks after completion of treatment.
The major takeaway is that a significant minority of study participants treated with bepirovirsen achieved a functional cure from CHB, defined by the sustained clearance of both HBV DNA and HBV surface antigen (HBsAg). While this primary endpoint was achieved in only 6% of the total study population, rates of response were higher in certain subgroups. Specifically, upwards of 16-25% of patients with low pre-treatment levels of HBsAg attained clearance of both HBV and HBsAg after receiving bepirovirsen. These rates of functional cure are much higher than what is typically seen in those receiving conventional monotherapy with NAs.
Overall, the study reports promising clinical data on an emerging treatment option for chronic hepatitis B. Future studies on bepirovirsen will be monitored closely by the HBV community, as such studies should clarify the durability of bepirovirsen’s antiviral activity, safety profile, and long-term outcomes.
PICKS OF THE MONTH - November 2022
For November, our presenting emerging scholar is Lena Allweiss, PhD. She has selected two articles.
Smc5/6 silences episomal transcription by a three-step function. Abdul F, Diman A, Baechler B, Ramakrishnan D, Kornyeyev D, Beran RK, Fletcher SP, Strubin M. Nat Struct Mol Biol. 2022 Sep;29(9):922-931. doi: 10.1038/s41594-022-00829-0
https://pubmed.ncbi.nlm.nih.gov/36097294/
The chromosomal maintenance complex SMC5/6 is a restriction factor against HBV because it blocks transcription from its episomal viral genome. For an active infection to be established, HBV relies on its regulatory protein HBx to specifically degrade this complex and relieve its transcriptional suppression. This publication describes the mechanism by which SMC5/6 silences viral transcription from the HBV genome - a mechanism that might hold true for the restriction of other DNA virus such as HIV and HPV. Elucidating this process will not only generate basic knowledge about the interaction of viruses with their hosts but might also assist with the design of antiviral drugs targeting HBx.
Conversion of hepatitis B virus relaxed circular to covalently closed circular DNA is supported in murine cells. Wei L, Cafiero TR, Tseng A, Gertje HP, Berneshawi A, Crossland NA, Ploss A. JHEP Rep. 2022 Jul 9;4(9):100534. doi: 10.1016/j.jhepr.2022.100534.
https://pubmed.ncbi.nlm.nih.gov/36035363/
Mice are often used to study viral infections and test novel antiviral treatments. In the case of HBV, however, mice cannot simply be used for these purposes since HBV does not naturally infect mice. Scientists are currently trying to develop a genetically modified mouse model that is engineered to express missing factors or eliminate inhibitory factors necessary to establish an HBV infection in these mice. In this publication, the authors show that a crucial step in HBV infection, the conversion of incoming viral relaxed circular DNA to covalently closed circular DNA, does take place in mouse hepatocytes, thus ruling out this step as a potential block for infection establishment. This knowledge is important because it will help create such a mouse model for HBV research, a model in which this step does not need to be modified.