Hep B Blog

Hepatitis B Research Review: February

 

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. 

 Summary: This month, researchers in Beijing, China have reported that a therapeutic vaccine composed of polylactic acid microparticles loaded with HBV surface antigen and the mouse STING agonist DMXAA showed efficacy in clearing HBV infection in a mouse model. Researchers from Wuhan, China have reported that SOX2, a transcription factor important for cell proliferation is also a host restriction factor for HBV infection. Also, researchers from the University of Boulder in conjunction with Dr. James Chen’s lab in Dallas have reported the synthesis of two potent cGAS inhibitors.

The incorporation of cationic property and immunopotentiator in poly (lactic acid) microparticles promoted the immune response against chronic hepatitis B – Journal of Controlled Release

This paper from the Chinese Academy of Sciences in Beijing, introduces a microparticle vaccine which may be used to treat chronic HBV infection (CHB). The 1μm diameter microparticle is made from polylactic acid (PLA), which is a biodegradable polymer typically synthesized from plant starch. The microparticle also contains didodecyldimethylammonium bromide (DDAB) which is a double-chain cationic surfactant. This group has previously shown that DDAB may be used as a carrier for the HBV surface protein (HBsAg). DDAB also gives the microparticle a positive charge, which accelerates its phagocytosis into antigen-presenting cells (APCs) and facilitates its escape from lysosomal degradation once in the cell. Additionally, the group loaded microparticles with the mouse STING agonist  5,6-dimethylxanthenone-4-acetic acid (DMXAA). The microparticles were refereed to as DDAB-PLA (DP) and DDAB-PLA-DMXAA (DP-D) respectively. Both types of microparticle were saturated with HBV surface antigen (HBsAg). The microparticles were first tested on mouse bone marrow dendritic cells (BMDCs). Administration of microparticles caused less than a 20% reduction of cell viability in these cultures. BMDCs treated with DP-D microparticles had at least ten-fold more expression of IRF-7 and IFN-β mRNA as measured by RT-qPCR than those treated with HBsAg or DP microparticles alone. Surprisingly, the DP-D microparticle-treated cells also had about twice the expression of these genes compared to the positive control HBsAg + DMXAA, which contained ten times more DMXAA than the microparticles. This indicates that the DP-D microparticles induced the STING pathway with high efficiency due to their bioavailability. Next, the group found that DP-D microparticles induced the highest level of chemokine expression (measured via RT-qPCR) and immune cell recruitment (measured via flow cytometry) at the site of injection in inoculated mice compared with HBsAg alone, HBsAg with aluminum salts (traditional vaccine adjuvant), and DP microparticles. This result shows that the DP-D microparticles induced both an innate immune response and an adaptive immune response in mice. Further, the group showed that BMDCs treated with DP-D microparticles had a high level of maturation, expressing CD40, CD86, and MHCII molecules on their surface as measured by flow cytometry. Finally, the group administered the HBsAg-primed microparticles to mice infected with recombinant HBV (rAAV-1.3HBV virus, serotype ayw). Mice treated with both types of microparticles showed a higher cytokine response as well as a higher titer of anti-HBsAg antibody as measured by ELISA. Mice treated with the DP-D microparticles had the most profound immune cell activation and  fastest clearance of serum HBsAg. The microparticle vaccine introduced in this publication is promising because it is highly efficient in delivering antigen to immune cells. The microparticles are unique in that they contain a small molecule STING agonist inside. This design is clever because this vaccine stimulates the innate immune system by activating STING and the adaptive immune system by displaying HBsAg to APCs. This promotes HBV clearance in a multifaceted approach: immune cells produce cytokines through the STING pathway, T cells recognize and destroy infected cells, and B cells secrete anti-HBsAg antibodies to neutralize newly formed viruses. This publication highlights the versatility of biodegradable microparticle technology in designing unique approaches to combat infection. Micro- and nanoparticle delivery systems represent a promising avenue for future drugs to combat HBV and other viruses.

SOX2 Represses Hepatitis B Virus Replication by Binding to the Viral EnhII/Cp and Inhibiting the Promoter Activation – Viruses
This paper from Wuhan University in China identifies the protein sex determining region Y box 2 (SOX2) as a host factor that restricts HBV replication. SOX2 is a transcription factor critical for cell proliferation and the tumorigenecity of solid tumors. In 2006, expression of SOX2 along with three other transcription factors was shown to convert somatic cells into induced pluripotent stem cells. Overexpression of SOX2 indicates poor prognosis in patients undergoing resection of HCC. In HCC cells, SOX2 has also been found to induce the expression of programmed death ligand-1 (PD-L1), leading to the tumor’s evasion of the host immune system. Previously, it has been demonstrated that HBV infection induces increased expression of SOX2 in hepatocytes. This study demonstrates that SOX2 inhibits HBV replication by binding to the Enhancer II (EnhII) and Core Promoter (Cp) regions of the HBV genome. By binding to the EnhII/Cp region, SOX2 disrupts the transcription of the mRNA species precore, core, polymerase, and pgRNA. This reduction of mRNA transcription results in reduced levels of core-associated DNA, HBV surface antigen (HBsAg), and HBV e antigen (HBeAg). To learn this, the group co-transfected both HepG2 and Huh7 cells with a fixed concentration of  HBV 1.3-mer plasmid DNA alongside variable concentrations of Flag-tagged SOX2 in pcDNA3.1 plasmid DNA. Cells transfected with higher concentrations of SOX2 plasmid DNA showed reduced levels of HBV mRNAs (3.5, 2.4, and 2.1 kb) via Northern blotting. SOX2-transfected cells also showed reduced levels of HBV core-associated DNA via qPCR as well as reduced levels of both HBsAg and HBeAg via ELISA. Next, in order to learn  if SOX2 interacts directly with an HBV promoter, a dual-luciferase reporter assay was implemented. Here, four vectors were used, each containing one of the HBV enhancer and/or promoter sequences (preS1, preS2, EnhⅡ/Cp, and EnhⅠ/Xp) upstream of a firefly luciferase reporter. Each of these firefly luciferase reporter vectors were co-transfected into HepG2 cells alongside variable concentrations of SOX2 plasmid DNA. A plasmid encoding Renilla luciferase was also included at a constant concentration in each transfection as a control for transfection efficiency. While firefly luciferase has an emission of 625 nm (red), Renilla luciferase has an emission of 525 nm (green). Therefore, levels of red fluorescence were used to measure the activity of the HBV enhancer/promoter sequences and levels of green fluorescence were utilized as a control for transfection efficiency. Co-transfection with SOX2 significantly diminished the luciferase activity of the EnhII/Cp reporter only and in a dose-responsive manner, indicating its interaction with that region of the HBV genome. Further, using HBV-producing HepAD38 cells, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) was used to isolate SOX2 protein and then determine what DNA sequence it was bound to. The EnhII/Cp sequence was found to be highly enriched on SOX2 protein. In order to determine which part of the SOX2 protein is required for binding to the EnhII/Cp region, truncated forms of SOX2 were generated in the pcDNA3.1 plasmid. Using the assays described above, it was found that only SOX2 mutants lacking the high mobility group (HMG) domain were unable to bind to the EnhII/Cp region and suppress HBV products. Interestingly, it was found that SOX2 mutants lacking the transcription activation (TA) domain were still able to bind to the EnhII/Cp region. Further, it was demonstrated by Western blot of subcellular fractions and immunofluorescence that SOX2 mutants lacking the HMG domain were unable to enter the nucleus. Finally, studies were performed in an in vivo BALB/c mouse model. Mice were given a hydrodynamic injection of an adeno-associated viral vector conferring HBV (pAAV-HBV1.3) alongside pcDNA3.1 plasmid DNA conferring SOX, SOX2 lacking HMG domain ( SOXΔHMG), or empty vector. Levels of HBsAG and HBeAg in the blood at days two and four were reduced only in mice given the full length SOX2 plasmid. Additionally, mice given the full length SOX2 plasmid had a reduction of 3.5kb HBV mRNA in liver tissues as measured by qPCR and a lower abundance of HBV core antigen (HBcAg) in liver tissues as measured by immunohistochemical staining. This study shows that SOX2 protein, previously shown to be upregulated by HBV, plays an anti-HBV role in the liver. SOX2 is therefore a new host restriction factor of HBV replication. SOX2 may be one protein which contributes to HBV-induced hepatocarcinogenesis, given its role in promoting the transcription of genes involved in cell proliferation. In the future, SOX2 may be utilized for its anti-HBV activity or targeted for the treatment of HCC.

 Discovery of Small Molecule Cyclic GMP-AMP Synthase Inhibitors – The Journal of Organic Chemistry

This paper from the University of Colorado Boulder introduces the development of novel small molecule inhibitors of the protein cyclic GMP-AMP synthase (cGAS). This publication is in conjunction with Dr. James Chen’s laboratory at the University of Texas Southwestern Medical Center in Dallas, Texas. Dr. Chen’s lab discovered cGAS in 2012. cGAS is a cytosolic, double-stranded DNA (dsDNA)-sensing protein. It belongs to the nucleotidyltransferase family of enzymes which transfer nucleoside monophosphates, the substituents of nucleic acids. When cGAS recognizes dsDNA, it synthesizes the cyclic dinucleotide cyclic GMP-AMP (cGAMP). cGAMP acts as a second messenger and activates the stimulator of interferon genes protein (STING). Once activated, STING triggers TBK1- and IKK-mediated activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). In the nucleus, IRF3 and NF-kB induce the expression of type I interferons and other inflammatory cytokines. cGAS is essential for detecting foreign pathogens which contain dsDNA and triggering an innate immune response to clear them. However, excessive or dysfunctional cGAS activity may lead to chronic inflammation and/or autoimmunity. Pharmacologic inhibition of cGAS may provide treatments for diseases including Aicardi-Goutiés syndrome (AGS), lupus erythematosus, and cancer. Current small molecule inhibitors of cGAS are limited by poor specificity and/or cellular activity. In this study, a high throughput virtual screen (HTVS) was utilized to screen about 1.75 million drug-like compounds for activity against the dimer-forming and DNA-binding faces of mouse cGAS (mcGAS). mcGAS was utilized for the in silico screen because the human cGAS (hcGAS)-DNA complex was only recently published. From this virtual screen, ten compounds were further investigated, leading to the selection of one lead compound. This lead was further optimized for greater potency through chemical modifications resulting in the analogues CU-32 and CU-76. The IC50 of both compounds is below 1µM. To test these compounds’ selectivity for cGAS, human monocyte cells THP-1 were either transfected with  interferon-stimulatory DNA (ISD) or infected with Sendai virus (SeV). ISD is a 45-basepair DNA known to activate cGAS, while SeV is a single-stranded RNA (ssRNA) virus known to activate the RIG-I-MAVS pathway; both stimuli are known to result in IRF3 activation and dimerization. Following treatment with both compounds, Western blot of the cells was conducted probing for the formation of IRF3 dimers. In ISD-treated cells, CU-32 and CU-76 inhibited the formation of IRF3 dimers in a dose responsive manner. Neither compound had any effect on IRF3 dimer formation in SeV-infected cells. This result indicates that these inhibitors are selective to cGAS. Using in silico molecular docking studies, the group speculates that these compounds disrupt the interface of the cGAS dimer, allosterically inhibiting dimerization. The discovery of novel cGAS inhibitors is exciting and important for multiple reasons. These compounds, if made commercially available will allow for improved experimentation investigating the cGAS/STING pathway. If these compounds or their derivatives are found to be safe and effective in humans, they may be promising candidates for the treatment of autoimmune disorders or cancer.

 

Meet our guest blogger, David Schad, B.Sc., Junior Research Fellow at the Baruch S. Blumberg Institute studying programmed cell death such as apoptosis and necroptosis in the context of hepatitis B infection under the direction of PI Dr. Roshan Thapa. David also mentors high school students from local area schools as part of an after-school program in the new teaching lab at the PA Biotech Center. His passion is learning, teaching and collaborating with others to conduct research to better understand nature.

 

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