Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation.

For a virus-like particle (VLP) to serve as a delivery platform, the VLP must be able to release its cargo in response to a trigger. Here, we use a chemical biology approach to destabilize a self-assembling capsid for a subsequent triggered disassembly. We redesigned the dimeric hepatitis B virus (HBV) capsid protein (Cp) with two differentially addressable cysteines, C150 for reversibly crosslinking the capsid and C124 to react with a destabilizing moiety. The resulting construct, Cp150-V124C, assembles into icosahedral, 120-dimer VLPs that spontaneously crosslink via the C-terminal C150, leaving C124 buried at a dimer-dimer interface. The VLP is driven into a metastable state when C124 is reacted with the bulky fluorophore, maleimidyl BoDIPY-FL. The resulting VLP is stable until exposed to modest, physiologically relevant concentrations of reducing agent. We observe dissociation with FRET relaxation of polarization, size exclusion chromatography, and resistive-pulse sensing. Dissociation is slow, minutes to hours, with a characteristic lag phase. Mathematical modeling based on the presence of a nucleation step predicts disassembly dynamics that are consistent with experimental observations. VLPs transfected into hepatoma cells show similar dissociation behavior. These results suggest a generalizable strategy for designing a VLP that can release its contents in an environmentally responsive reaction.

Inside and outside of virus-like particles HBc and HBc/4M2e: A comprehensive study of the structure.

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.

Multiscale Modeling of Hepatitis B Virus Capsid Assembly and Its Dimorphism.

Hepatitis B virus (HBV) is an endemic, chronic virus that leads to 800000 deaths per year. Central to the HBV lifecycle, the viral core has a protein capsid assembled from many copies of a single protein. The capsid protein adopts different (quasi-equivalent) conformations to form icosahedral capsids containing 180 or 240 proteins: T = 3 or T = 4, respectively, in Caspar-Klug nomenclature. HBV capsid assembly has become an important target for recently developed antivirals; nonetheless, the assembly pathways and mechanisms that control HBV dimorphism remain unclear. We describe computer simulations of the HBV assembly, using a coarse-grained model that has parameters learned from all-atom molecular dynamics simulations of a complete HBV capsid and yet is computationally tractable. Dynamical simulations with the resulting model reproduce experimental observations of HBV assembly pathways and products. By constructing Markov state models and employing transition path theory, we identify pathways leading to T = 3, T = 4, and other experimentally observed capsid morphologies. The analysis shows that capsid polymorphism is promoted by the low HBV capsid bending modulus, where the key factors controlling polymorphism are the conformational energy landscape and protein-protein binding affinities.

Conserved Lysine Residues of Hepatitis B Virus Core Protein Are Not Required for Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection. We found that the two lysine residues were dispensable for all intracellular steps of HBV replication. In particular, all mutants were competent to form the covalently closed circular DNA (cccDNA) via the intracellular amplification pathway, indicating that K7 and K96, or any PTMs of these residues, were not essential for nucleocapsid uncoating, a prerequisite for cccDNA formation. Furthermore, we found that K7A and K7R mutations did not affect de novo cccDNA formation and RNA transcription during infection, indicating that K7 or any PTMs of this residue were dispensable for HBV infection. In addition, we demonstrated that the HBc K7 coding sequence (AAA), as part of the HBV polyadenylation signal UAUAAA, was indispensable for viral RNA production, implicating this cis requirement at the RNA level, instead of any function of HBc-K7, likely constrains the identity of the 7th residue of HBc. In conclusion, our results provided novel insights regarding the roles of lysine residues on HBc, and their coding sequences, in the HBV life cycle. IMPORTANCE Hepatitis B virus (HBV) infection remains a public health burden that affects 296 million individuals worldwide. HBV core protein (HBc) is involved in almost all steps in the HBV life cycle. There are two conserved lysine residues on HBc. Here, we found that neither of them is essential for HBV intracellular replication, including the formation of covalently closed circular DNA (cccDNA), the molecular basis for establishing and sustaining the HBV infection. However, K96 is critical for virion morphogenesis, while the K7 coding sequence, but not HBc-K7 itself, is indispensable, as part of the RNA polyadenylation signal, for HBV RNA production from cccDNA. Our results provide novel insights regarding the role of the conserved lysine residues on HBc, and their coding sequences, in viral replication, and should facilitate the development of antiviral drugs against the HBV capsid protein.

In-Plane, In-Series Nanopores with Circular Cross Sections for Resistive-Pulse Sensing.

Resistive-pulse sensing with solid-state nanopores is a sensitive, label-free technique for analyzing single molecules in solution. To add functionality to resistive-pulse measurements, direct coupling of the nanopores to other pores and nanoscale fluidic elements, e.g., reactors, separators, and filters, in the same device is an important next step. One approach is monolithic fabrication of the fluidic elements in the plane of the substrate, but methods to generate pores with circular cross sections are needed to improve sensing performance with in-plane devices. Here, we report a fabrication method that directly patterns nanopores with circular cross sections in series and in plane with the substrate. A focused ion beam instrument is used to mill a lamella in a nanochannel and, subsequently, bore a nanopore through the lamella. The diameter and geometry of the nanopore are controlled by the current and dose of the ion beam and by the tilt angle and thickness of the lamella. We fabricated devices with vertical and tilted lamellae and nanopores with diameters from 40 to 90 nm in cylindrical and conical geometries. To test device performance, we conducted resistive-pulse measurements of hepatitis B virus capsids. Current pulses from T = 3 capsids (∼31 nm diameter) and T = 4 capsids (∼35 nm diameter) were well resolved and exhibited relative pulse amplitudes (Δi/i) up to 5 times higher than data obtained on nanopores with rectangular cross sections. For smaller pore diameters (<45 nm), which approach the diameters of the capsids, a dramatic increase in the pulse amplitude was observed for both T = 3 and T = 4 capsids. Two and three pores fabricated in series further improved the resolution between the relative pulse amplitude distributions for the T = 3 and T = 4 capsids by up to 2-fold.

Hysteresis in Hepatitis B Virus (HBV) Requires Assembly of Near-Perfect Capsids.

The hepatitis B virus (HBV) must release its contents to initiate infection, making capsid disassembly critical to the viral life cycle. Capsid assembly proceeds through a cascade of weak interactions between copies of capsid protein (Cp) to yield uniform particles. However, there is a hysteresis to capsid dissociation that allows capsids to persist under conditions where they could not assemble. In this study, we have sought to define the basis of hysteresis by examining urea-induced dissociation of in vitro-assembled HBV capsids. In general, capsid samples show a mixture of two pools, differentiated by stability. Labile capsid dissociation corresponds to an ∼5 µM pseudocritical concentration of assembly (pcc), the same as that observed in assembly reactions. Dissociation of the stable pool corresponds to a subfemtomolar pcc, indicative of hysteresis. The fraction of stable capsids in an assembly reaction increases with the integrity of the Cp preparation and when association is performed at a higher ionic strength, which modifies the Cp conformation. Labile complexes are more prevalent when assembly conditions yield many kinetically trapped (incomplete and overgrown) products. Cp isolated from stable capsids reassembles into a mixture of stable and labile capsids. These results suggest that hysteresis arises from an ideal capsid lattice, even when some of the substituents in that lattice have defects. Consistent with structural studies that show a subtle difference between Cp dimers and Cp in capsid, we propose that hysteresis arises when HBV capsids undergo a lattice-dependent structural transition.

Silicon Nanowires Length and Numbers Dependence on Sensitivity of the Field-Effect Transistor Sensor for Hepatitis B Virus Surface Antigen Detection.

Silicon nanowire field effect transistor (NWFET) sensors have been demonstrated to have high sensitivity, are label free, and offer specific detection. This study explored the effect of nanowire dimensions on sensors' sensitivity. We used sidewall spacer etching to fabricate polycrystalline silicon NWFET sensors. This method does not require expensive nanoscale exposure systems and reduces fabrication costs. We designed transistor sensors with nanowires of various lengths and numbers. Hepatitis B surface antigen (HBsAg) was used as the sensing target to explore the relationships of nanowire length and number with biomolecule detection. The experimental results revealed that the sensor with a 3 µm nanowire exhibited high sensitivity in detecting low concentrations of HBsAg. However, the sensor reached saturation when the biomolecule concentration exceeded 800 fg/mL. Sensors with 1.6 and 5 µm nanowires exhibited favorable linear sensing ranges at concentrations from 800 ag/mL to 800 pg/mL. The results regarding the number of nanowires revealed that the use of few nanowires in transistor sensors increases sensitivity. The results demonstrate the effects of nanowire dimensions on the silicon NWFET biosensors.

Multiple epitopes of hepatitis B virus surface antigen targeted by human plasma-derived immunoglobulins coincide with clinically observed escape mutations.

Hepatitis B immune globulin (HBIG) is a human plasma-derived immunoglobulin G concentrate that contains a high titer of neutralizing antibodies (anti-HBs) to the hepatitis B virus (HBV) surface antigen (HBsAg). HBIG is known to be highly effective in treating HBV infections, however, a more systematic characterization of the antibody binding sites on HBsAg and their correlation with emerging "escape" mutations in HBsAg was lacking. By using anti-HBs antibodies from HBIG lots to screen random peptide phage display libraries, we identified five clusters of peptides that corresponded to five distinct anti-HBs binding sites on the HBsAg. Three sites, Site II (C121-C124), Site III (M133-P135), and Site IV (T140-G145), were mapped within the "a" determinant, while the two other sites, Site I (Q101-M103) and Site V (I152-S154), were outside the "a" determinant. We then tested in binding assays HBsAg peptides containing clinically relevant mutations previously reported within these sites, such as Y134S, P142S, and G145R, and observed a significant reduction in anti-HBs binding activity to the mutated sites, suggesting a mechanism the virus may use to avoid HBIG-mediated neutralization. The current HBIG treatment could be improved by supplementing it with site-specific neutralizing monoclonal antibodies that target these mutations for control of HBV infections.

Disassembly of Single Virus Capsids Monitored in Real Time with Multicycle Resistive-Pulse Sensing.

Virus assembly and disassembly are critical steps in the virus lifecycle; however, virus disassembly is much less well understood than assembly. For hepatitis B virus (HBV) capsids, disassembly of the virus capsid in the presence of guanidine hydrochloride (GuHCl) exhibits strong hysteresis that requires additional chemical energy to initiate disassembly and disrupt the capsid structure. To study disassembly of HBV capsids, we mixed T = 4 HBV capsids with 1.0-3.0 M GuHCl, monitored the reaction over time by randomly selecting particles, and measured their size with resistive-pulse sensing. Particles were cycled forward and backward multiple times to increase the observation time and likelihood of observing a disassembly event. The four-pore device used for resistive-pulse sensing produces four current pulses for each particle during translocation that improves tracking and identification of single particles and increases the precision of particle-size measurements when pulses are averaged. We studied disassembly at GuHCl concentrations below and above denaturing conditions of the dimer, the fundamental unit of HBV capsid assembly. As expected, capsids showed little disassembly at low GuHCl concentrations (e.g., 1.0 M GuHCl), whereas at higher GuHCl concentrations (≥1.5 M), capsids exhibited disassembly, sometimes as a complex series of events. In all cases, disassembly was an accelerating process, where capsids catastrophically disassembled within a few 100 ms of reaching critical stability; disassembly rates reached tens of dimers per second just before capsids fell apart. Some disassembly events exhibited metastable intermediates that appeared to lose one or more trimers of dimers in a stepwise fashion.

Characterization of a G-quadruplex from hepatitis B virus and its stabilization by binding TMPyP4, BRACO19 and PhenDC3.

Specific guanine rich nucleic acid sequences can form non-canonical structures, like the four stranded G-quadruplex (GQ). We studied the GQ-forming sequence (named HepB) found in the genome of the hepatitis B virus. Fluorescence-, infrared- and CD-spectroscopy were used. HepB shows a hybrid form in presence of K+, but Na+, Li+, and Rb+ induce parallel structure. Higher concentrations of metal ions increase the unfolding temperature, which was explained by a short thermodynamic calculation. Temperature stability of the GQ structure was determined for all these ions. Na+ has stronger stabilizing effect on HepB than K+, which is highly unusual. The transition temperatures were 56.6, 53.8, 58.5 and 54.4 °C for Na+, K+, Li+, and Rb+ respectively. Binding constants for Na+ and K+ were 10.2 mM and 7.1 mM respectively. Study of three ligands designed in cancer research for GQ targeting (TMPyP4, BRACO19 and PhenDC3) showed unequivocally their binding to HepB. Binding was proven by the increased stability of the bound form. The stabilization was higher than 20 °C for TMPyP4 and PhenDC3, while it was considerably lower for BRACO19. These results might have medical importance in the fight against the hepatitis B virus.

Binding of a Pocket Factor to Hepatitis B Virus Capsids Changes the Rotamer Conformation of Phenylalanine 97.

(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.

Human hepatitis B virus-derived virus-like particle as a drug and DNA delivery carrier.

The controlled release of medications using nanoparticle-based drug delivery carriers is a promising method to increase the efficacy of pharmacotherapy and gene therapy. One critical issue that needs to be overcome with these drug delivery carriers is their target specificity. We focused on the cell tropism of a virus to solve this issue, i.e., we attempted to apply hepatitis B virus-like particle (HBV-VLP) as a novel hepatic cell-selective carrier for medication and DNA. To prepare HBV-VLP, 293T cells were transfected with expression plasmids carrying HBV envelope surface proteins, large envelope protein (L), and small envelope protein (S). After 72 h post-transfection, VLP-containing culture supernatants were harvested, and HBV-VLP was labeled with red fluorescent dye (DiI) and was purified by sucrose gradient ultracentrifugation. An anticancer drugs (geldanamycin or doxorubicin) and GFP-expressing plasmid DNA were incorporated into HBV-VLP, and medication- and plasmid DNA-loaded VLPs were prepared. We evaluated their delivery capabilities into hepatocytes, other organ-derived cells, and hepatocytes expressing sodium taurocholate cotransporting polypeptide (NTCP), which functions as the cellular receptor for HBV by binding to HBV L protein. HBV-VLP selectively delivered both anticancer drugs and plasmid DNA not into HepG2, Huh7, and other organ cells but into HepG2 cells expressing NTCP. In summary, we developed a novel delivery nanocarrier using HBV-VLP that could be used as a hepatitis selective drug- and DNA-carrier for cancer treatment and gene therapy.

All-atom molecular dynamics study of hepatitis B virus containing pregenome RNA in solution.

Immature hepatitis B virus (HBV) captures nucleotides in its capsid for reverse transcription. The nucleotides and nucleotide analog drugs, which are triphosphorylated and negatively charged in the cell, approach the capsid via diffusion and are absorbed into it. In this study, we performed a long-time molecular dynamics calculation of the entire HBV capsid containing pregenome RNA to investigate the interactions between the capsid and negatively charged substances. Electric field analysis demonstrated that negatively charged substances can approach the HBV capsid by thermal motion, avoiding spikes. The substances then migrate all over the floor of the HBV capsid. Finally, they find pores through which they can pass through the HBV capsid shell. Free energy profiles were calculated along these pores for small ions to understand their permeability through the pores. Anions (Cl-) showed higher free energy barriers than cations (Na+ and K+) through all pores, and the permeation rate of Cl- was eight times slower than that of K+ or Na+. Furthermore, the ions were more stable in the capsid than in the bulk water. Thus, the HBV capsid exerts ion selectivity for uptake and provides an environment for ions, such as nucleotides and nucleotide analog drugs, to be stabilized within the capsid.

In silico analysis of mutations associated with occult hepatitis B virus (HBV) infection in South Africa.

Occult hepatitis B virus (OBI) infection is defined by the presence of viral DNA in the liver and/or serum in absence of hepatitis B surface antigen (HBsAg). While multiple studies have identified mutations that are associated with OBI, only a small portion of these mutations have been functionally characterized in vitro. Using complementary in silico approaches, the effects of OBI-associated amino acid mutations on HBV protein function in HBV/HIV-positive ART-naïve South Africans were evaluated. Two OBI-associated mutations in the PreS1 region, one in the PreS2 region, and seven in the surface region of subgenotype A1 sequences were identified as deleterious. In subgenotype A2 sequences, 11 OBI-associated mutations in the PreS1 region, seven in the PreS2 region, and 31 in the surface region were identified as deleterious. In the polymerase region, 14 OBI-associated mutations in subgenotype A1 and 71 OBI-associated mutations in subgenotype A2 were identified as deleterious. This study utilized in silico approaches to characterize the likely impact of OBI-associated mutations on viral function, thereby identifying and prioritizing candidates and reducing the significant cost associated with functional studies that are essential for mechanistic studies of the OBI phenotype.

Binding of Nanoparticles Harboring Recombinant Large Surface Protein of Hepatitis B Virus to Scavenger Receptor Class B Type 1.

(1) Background: As nanoparticles containing the hepatitis B virus (HBV) large (L) surface protein produced in yeast are expected to be useful as a carrier for targeting hepatocytes, they are also referred to as bio-nanocapsules (BNCs). However, a definitive cell membrane receptor for BNC binding has not yet been identified. (2) Methods: By utilizing fluorescence-labeled BNCs, we examined BNC binding to the scavenger receptor class B type 1 (SR-B1) expressed in HEK293T cells. (3) Results: Analyses employing SR-B1 siRNA and expression of SR-B1 fused with a green fluorescent protein (SR-B1-GFP) indicated that BNCs bind to SR-B1. As mutagenesis induced in the SR-B1 extracellular domain abrogates or attenuates BNC binding and endocytosis via SR-B1 in HEK293T cells, it was suggested that the ligand-binding site of SR-B1 is similar or close among high-density lipoprotein (HDL), silica, liposomes, and BNCs. On the other hand, L protein was suggested to attenuate an interaction between phospholipids and SR-B1. (4) Conclusions: SR-B1 can function as a receptor for binding and endocytosis of BNCs in HEK293T cells. Being expressed various types of cells, it is suggested that functions as a receptor for BNCs not only in HEK293T cells but also in other types of cells.

Design, synthesis and evaluation of heteroaryldihydropyrimidine analogues bearing spiro ring as hepatitis B virus capsid protein inhibitors.

GLS4, a potent antiviral drug candidate, has been widely studied and entered into phase II clinical trials. Nevertheless, the therapeutic application of GLS4 is limited due to poor water solubility, short half-life, and low bioavailability. In order to improve the hydrophilicity and pharmacokinetic (PK) properties of GLS4, herein, we retained the dominant fragments, and used a scaffold hopping strategy to replace the easily metabolized morpholine ring of GLS4 with diverse sizes of spiro rings consisting of hydrogen bond donor and acceptor substituents. Potent in vitroanti-HBV activity and low cytotoxicity were observed for compound 4r (EC50 = 0.20 ± 0.00 µM, CC50 > 87.03 µM), which was more potent than the positive control lamivudine (EC50 = 0.37 ± 0.04 µM, CC50 > 100.00 µM) in this assay and was about a quarter as effective as GLS4 (EC50 = 0.045 ± 0.01 µM, CC50 > 99.20 µM). Preliminary structure-activity relationship (SAR) analysis and molecular docking studies were carried out to explore potential interactions and binding mode between compounds and target protein. In terms of the physicochemical properties, 4r was predicted to be consistent with the rule-of-five, which means 4r may have favourable absorption and permeation. Finally, ADMET and PK characteristics of 4r and GLS4 were predicted to be comparable in most aspects, implying that the two compounds may have similar profiles in vivo.

High prevalence of tryptophan-truncated S quasispecies in treatment-naïve chronic hepatitis B patients.

Hepatitis B virus surface antigen (HBsAg) encoded by the S gene is highly expressed during the replication cycle of hepatitis B virus (HBV). However, the frequent usage of tryptophan in HBsAg, which leads to a high cost of biosynthesis, is inconsistent with the high expression level of this protein. Tryptophan-truncated mutation of HBsAg, that is, a tryptophan to stop codon mutation resulting in truncated HBsAg, might help to maintain its high expression with lower biosynthetic cost. We aimed to investigate the prevalence of tryptophan-truncated S quasispecies in treatment-naïve patients with chronic hepatitis B (CHB) by applying CirSeq as well as a site-by-site algorithm developed by us to identify variants at extremely low frequencies in the carboxyl terminus of HBsAg. A total of 730 mutations were identified in 27 patients with CHB, varying from seven to 56 mutations per sample. The number of synonymous mutations was much higher than that of nonsynonymous mutations in the reverse transcriptase (RT) coding region and vice versa in the S coding region, implying that the evolutionary constraints on the RT and S genes might be different. We showed that 25 (92.6 %) of 27 patients had at least one S-truncated mutation, most of which were derived from tryptophan, indicating a high prevalence of tryptophan-truncated S mutations in treatment-naïve patients with CHB. In terms of the RT gene, 21 (77.8 %) patients had pre-existing drug-resistant mutations, while no truncated mutations were detected. Our findings that tryptophan-truncated S quasispecies and drug-resistant RT mutants were highly prevalent in treatment-naïve patients with CHB provide new insights into the composition of the HBV population, which might help optimize the treatment and management of patients with CHB.

A Hepatitis B Virus-Derived Peptide Can Inhibit Infection of Human Lung Cells with SARS-CoV-2 in a Type-1 Interferon-Dependent Manner.

The current COVID-19 pandemic has highlighted the urgent need to develop effective therapeutic strategies. We evaluated the in vitro antiviral effect against SARS-CoV-2 of a hepatitis B virus (HBV) hexamer peptide, Poly6, which is capable of eliciting an antiviral effect against human immunodeficiency virus -1 (HIV-1), as a novel HIV-1 integrase inhibitor, and a strong anticancer immune response in an IFN-I-dependent manner, as a novel potential adjuvant in anticancer immunotherapy. Here, we report that Poly6 exerts an anti-SARS-CoV-2 effect, with an estimated 50% inhibitory concentration of 2.617 µM, in the human bronchial epithelial cell line, Calu-3 but not in Vero-E6 cells, which are deficient in type 1 interferon (IFN-I) signaling. We proved via assays based on mRNA profiles, inhibitors, or blocking antibodies that Poly6 can exert an anti-SARS-CoV-2 effect in an IFN-I-dependent manner. We also found that Poly6 inhibits IL-6 production enhanced by SARS-CoV-2 in infected Calu-3 cells at both the transcription and the translation levels, mediated via IL-10 induction in an IFN-I-dependent manner. These results indicate the feasibility of Poly6 as an IFN-I-inducing COVID-19 drug with potent antiviral and anti-inflammatory activities.

Novel Biomarkers of Hepatitis B Virus and Their Use in Chronic Hepatitis B Patient Management.

Even though an approved vaccine for hepatitis B virus (HBV) is available and widely used, over 257 million individuals worldwide are living with chronic hepatitis B (CHB) who require monitoring of treatment response, viral activity, and disease progression to reduce their risk of HBV-related liver disease. There is currently a lack of predictive markers to guide clinical management and to allow treatment cessation with reduced risk of viral reactivation. Novel HBV biomarkers are in development in an effort to improve the management of people living with CHB, to predict disease outcomes of CHB, and further understand the natural history of HBV. This review focuses on novel HBV biomarkers and their use in the clinical setting, including the description of and methodology for quantification of serum HBV RNA, hepatitis B core-related antigen (HBcrAg), quantitative hepatitis B surface antigen (qHBsAg), including ultrasensitive HBsAg detection, quantitative anti-hepatitis B core antigen (qAHBc), and detection of HBV nucleic acid-related antigen (HBV-NRAg). The utility of these biomarkers in treatment-naïve and treated CHB patients in several clinical situations is further discussed. Novel HBV biomarkers have been observed to provide critical clinical information and show promise for improving patient management and our understanding of the natural history of HBV.