Modulation of monocyte activity by hepatocellular MicroRNA delivery through HBsAg particles: Implications for pathobiology of chronic hepatitis B.

BACKGROUND AND AIMS: HBsAg serves as an important immune-modulatory factor in chronic hepatitis B. One aspect of such modulation may act through monocytes, which are the major Ag-presenting cells taking up HBsAg. There is evidence for the encapsulation of hepatocellular microRNAs (miRNAs) by HBsAg particles, while its pathobiological significance is unclear. Here, we characterized the miRNA profile in patients with chronic hepatitis B and probed their association with liver inflammation. APPROACHES AND RESULTS: We collected plasma from patients that are treatment-naive with chronic hepatitis B (n = 110) and quantified total/HBsAg-enveloped miRNAs by qRT-PCR and plasma cytokines by ELISA. The biological effects of HBsAg-delivered miRNAs in monocytes were evaluated using multiple approaches. The clinical significance of candidate miRNAs and cytokines was corroborated in patients with HBV-associated advanced liver diseases. The plasma miRNA profile showed 2 major clusters, one significantly associated with HBsAg titer and the other correlated with liver inflammation. Among HBsAg-carried miRNAs, miR-939 displayed the most significant correlation with IL-8. Mechanistically, miR-939 in subviral particles enters monocytes and significantly augments IL-8 production through the mitogen-activated protein kinase (MAPK) p38 signaling pathway. Finally, the findings that miR-939 positively correlated with IL-8 level and inflammation/fibrosis stage in the cohort of HBV-associated advanced liver diseases support its causative role in the progression of liver diseases. CONCLUSIONS: HBsAg particles carry hepatocellular miRNAs, including miR-939, which enter monocytes and alter their functional status, such as IL-8 secretion. Our findings demonstrate that the HBsAg-miR-939-IL-8 axis may play a crucial role in HBV-induced hepatic necro-inflammation and the progression of advanced liver diseases.

Chimeric antigen receptors of HBV envelope proteins inhibit hepatitis B surface antigen secretion.

OBJECTIVES: Chronic hepatitis B (CHB) caused by HBV infection greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) plays critical roles in the pathogenesis of CHB. HBsAg loss is the key indicator for cure of CHB, but is rarely achieved by current approved anti-HBV drugs. Therefore, novel anti-HBV strategies are urgently needed to achieve sustained HBsAg loss. DESIGN: We developed multiple chimeric antigen receptors (CARs) based on single-chain variable fragments (scFvs, namely MA18/7-scFv and G12-scFv), respectively, targeting HBV large and small envelope proteins. Their impacts on HBsAg secretion and HBV infection, and the underlying mechanisms, were extensively investigated using various cell culture models and HBV mouse models. RESULTS: After secretory signal peptide mediated translocation into endoplasmic reticulum (ER) and secretory pathway, MA18/7-scFv and CARs blocked HBV infection and virion secretion. G12-scFv preferentially inhibited virion secretion, while both its CAR formats and crystallisable fragment (Fc)-attached versions blocked HBsAg secretion. G12-scFv and G12-CAR arrested HBV envelope proteins mainly in ER and potently inhibited HBV budding. Furthermore, G12-scFv-Fc and G12-CAR-Fc strongly suppressed serum HBsAg up to 130-fold in HBV mouse models. The inhibitory effect lasted for at least 8 weeks when delivered by an adeno-associated virus vector. CONCLUSION: CARs possess direct antiviral activity, besides the well-known application in T-cell therapy. Fc attached G12-scFv and G12-CARs could provide a novel approach for reducing circulating HBsAg.

Dynamic Monitoring of Biomolecular Hydrodynamic Dimensions by Magnetization Motion on Quartz Crystal Microbalance.

Hydrodynamic dimension (HD) is the primary indicator of the size of bioconjugated particles and biomolecules. It is an important parameter in the study of solid-liquid two-phase dynamics. HD dynamic monitoring is crucial for precise and customized medical research as it enables the investigation of the continuous changes in the physicochemical characteristics of biomolecules in response to external stimuli. However, current HD measurements based on Brownian motion, such as dynamic light scattering (DLS), are inadequate for meeting the polydisperse sample demands of dynamic monitoring. In this paper, we propose MMQCM method samples of various types and HD dynamic monitoring. An alternating magnetic field of frequency ωm excites biomolecule-magnetic bead particles (bioMBs) to generate magnetization motion, and the quartz crystal microbalance (QCM) senses this motion to provide HD dynamic monitoring. Specifically, the magnetization motion is modulated onto the thickness-shear oscillation of the QCM at the frequency ωq. By analysis of the frequency spectrum of the QCM output signal, the ratio of the magnitudes of the real and imaginary parts of the components at frequency ωq ± 2ωm is extracted to characterize the particle size. Using the MMQCM approach, we successfully evaluated the size of bioMBs with different biomolecule concentrations. The 30 min HD dynamic monitoring was implemented. An increase of ∼10 nm in size was observed upon biomolecular structural stretching. Subsequently, the size of bioMBs gradually reduced due to the continuous dissociation of biomolecules, with a total reduction of 20∼40 nm. This HD dynamic monitoring demonstrates that the release of biomolecules can be regulated by controlling the duration of magnetic stimulation, providing valuable insights and guidance for controlled drug release in personalized precision medicine.

Super-Aerophilic Biomimetic Cactus for Underwater Dispersed Microbubble Capture, Self-Transport, Coalescence, and Energy Harvesting.

Human ocean activities are inseparable from the supply of energy. The energy contained in the gas-phase components dispersed in seawater is a potential universal energy source for eupelagic or deep-sea equipment. However, the low energy density of bubbles dispersed in water introduces severe challenges to the potential energy harvesting of gas-phase components. Here, a super-aerophilic biomimetic cactus is developed for underwater dispersive microbubble capture and energy harvesting. The bubbles captured by the super-aerophilic biomimetic cactus spines, driven by the surface tension and liquid pressure, undergo automatic transport, coalescence, accumulation, and concentrated release. The formerly unavailable low-density dispersive surface free energy of the bubbles is converted into high-density concentrated gas buoyancy potential energy, thereby providing an energy source for underwater in situ electricity generation. Experiments show a continuous process of microbubble capture by the biomimetic cactus and demonstrate a 22.76-times increase in output power and a 3.56-times enhancement in electrical energy production compared with a conventional bubble energy harvesting device. The output energy density is 3.64 times that of the existing bubble energy generator. This work provides a novel approach for dispersive gas-phase potential energy harvesting in seawater, opening up promising prospects for wide-area in situ energy supply in underwater environments.

5' preS1 Mutations To Prevent Large Envelope Protein Expression from Hepatitis B Virus Genotype A or Genotype D Markedly Increase Polymerase-Envelope Fusion Protein.

Hepatitis B virus (HBV) large (L) envelope protein is translated from 2.4-kb RNA. It contains preS1, preS2, and S domains and is detected in Western blotting as p39 and gp42. The 3.5-kb pregenomic RNA produces core and polymerase (P) proteins. We generated L-minus mutants of a genotype A clone and a genotype D clone from 1.1-mer or 1.3-mer construct, with the former overproducing pregenomic RNA. Surprisingly, mutating a preS1 ATG codon(s) or introducing a nonsense mutation soon afterwards switched secreted p39/gp42 to a p41/p44 doublet, with its amount further increased by a nonsense mutation in the core gene. A further-downstream preS1 nonsense mutation prevented p41/p44 production. Tunicamycin treatment confirmed p44 as the glycosylated form of p41. In this regard, splicing of 3.5-kb RNA to generate a junction at nucleotides (nt) 2447 to 2902 for genotype D enables translation of p43, with the N-terminal 47 residues of P protein fused to the C-terminal 371 residues of L protein. Indeed p41/p44 were detectable by an antibody against the N terminus of P protein and eliminated by a nonsense mutation at the 5' P gene or a point mutation to prevent that splicing. Therefore, lost L (and core) protein expression from the 1.1-mer or 1.3-mer construct markedly increased p41/p44 (p43), the P-L fusion protein. Cotransfection with an expression construct for L/M proteins reversed high extracellular p41/p44 associated with L-minus mutants, suggesting that L protein retains p43 in wild-type HBV to promote its intracellular degradation. Considering that p43 lacks N-terminal preS1 sequence critical for receptor binding, its physiological significance during natural infection and therapeutic potential warrant further investigation. IMPORTANCE The large (L) envelope protein of hepatitis B virus (HBV) is translated from 2.4-kb RNA and detected in Western blotting as p39 and gp42. Polymerase (P) protein is expressed at a low level from 3.5-kb RNA. The major spliced form of 3.5-kb RNA will produce a fusion protein between the first 47 residues of P protein and a short irrelevant sequence, although also at a low level. Another spliced form has the same P protein sequence fused to L protein missing its first 18 residues. We found that some point mutations to eliminate L and core protein expression from overlength HBV DNA constructs converted p39/gp42 to p41/gp44, which turned out to be the P-L fusion protein. Thus, the P-L fusion protein can be expressed at extremely high level when L protein expression is prevented. The underlying mechanism and functional significance of this variant form of L protein warrant further investigation.

Passive Automatic Switch Relying on Laplace Pressure for Efficient Underwater Low-Gas-Flux Bubble Energy Harvesting.

The buoyancy potential energy contained in bubbles released by subsea geological and biological activities represents a possible in situ energy source for underwater sensing and detection equipment. However, the low gas flux of the bubble seepages that exist widely on the seabed introduces severe challenges. Herein, a passive automatic switch relying on Laplace pressure is proposed for efficient energy harvesting from low-gas-flux bubbles. This switch has no moving mechanical parts; it uses the Laplace-pressure difference across a curved gas-liquid interface in a biconical channel as an invisible "microvalve". If there is mechanical equilibrium between the Laplace-pressure difference and the liquid-pressure difference, the microvalve will remain closed and prevent the release of bubbles as they continue to accumulate. After the accumulated gas reaches a threshold value, the microvalve will open automatically, and the gas will be released rapidly, relying on the positive feedback of interface mechanics. Using this device, the gas buoyancy potential energy entering the energy harvesting system per unit time can be increased by a factor of more than 30. Compared with a traditional bubble energy harvesting system without a switch, this system achieves a 19.55-fold increase in output power and a 5.16-fold enhancement in electrical energy production. The potential energy of ultralow flow rate bubbles (as low as 3.97 mL/min) is effectively collected. This work provides a new design philosophy for passive automatic-switching control of gas-liquid two-phase fluids, presenting an effective approach for harvesting of buoyancy potential energy from low-gas-flux bubble seepages. This opens a promising avenue for in situ energy supply for subsea scientific observation networks.

Lost Small Envelope Protein Expression from Naturally Occurring PreS1 Deletion Mutants of Hepatitis B Virus Is Often Accompanied by Increased HBx and Core Protein Expression as Well as Genome Replication.

Hepatitis B virus (HBV) transcribes coterminal mRNAs of 0.7 to 3.5 kb from the 3.2-kb covalently closed circular DNA, with the 2.1-kb RNA being most abundant. The 0.7-kb RNA produces HBx protein, a transcriptional transactivator, while the 3.5-kb pregenomic RNA (pgRNA) drives core and P protein translation as well as genome replication. The large (L) and small (S) envelope proteins are translated from the 2.4-kb and 2.1-kb RNAs, respectively, with the majority of the S protein being secreted as noninfectious subviral particles and detected as hepatitis B surface antigen (HBsAg). pgRNA transcription could inhibit transcription of subgenomic RNAs. The present study characterized naturally occurring in-frame deletions in the 3' preS1 region, which not only codes for L protein but also serves as the promoter for 2.1-kb RNA. The human hepatoma cell line Huh7 was transiently transfected with subgenomic expression constructs for envelope (and HBx) proteins, dimeric constructs, or constructs mimicking covalently closed circular DNA. The results confirmed lost 2.1-kb RNA transcription and HBsAg production from many deletion mutants, accompanied by increases in other (especially 2.4-kb) RNAs, intracellular HBx and core proteins, and replicative DNA but impaired virion and L protein secretion. The highest intracellular L protein levels were achieved by mutants that had residual S protein expression or retained the matrix domain in L protein. Site-directed mutagenesis of a high replicating deletion mutant suggested that increased HBx protein expression and blocked virion secretion both contributed to the high replication phenotype. Our findings could help explain why such deletions are selected at a late stage of chronic HBV infection and how they contribute to viral pathogenesis. IMPORTANCE Expression of hepatitis B e antigen (HBeAg) and overproduction of HBsAg by wild-type HBV are implicated in the induction of immune tolerance to achieve chronic infection. How HBV survives the subsequent immune clearance phase remains incompletely understood. Our previous characterization of core promoter mutations to reduce HBeAg production revealed the ability of the 3.5-kb pgRNA to diminish transcription of coterminal RNAs of 2.4 kb, 2.1 kb, and 0.7 kb. The later stage of chronic HBV infection often selects for in-frame deletions in the preS region. Here, we found that many 3' preS1 deletions prevented transcription of the 2.1-kb RNA for HBsAg production, which was often accompanied by increases in intracellular 3.5-, 0.7-, and especially 2.4-kb RNAs, HBx and core proteins, and replicative DNA but lost virion secretion. These findings established the biological consequences of preS1 deletions, thus shedding light on why they are selected and how they contribute to hepatocarcinogenesis.

Pregnancy-related acute kidney injury at high altitude: a retrospective observational study in a single center.

BACKGROUND: Pregnancy-related acute kidney injury (Pr-AKI) is associated with maternal and fetal morbidity and mortality. There are few studies focusing on Pr-AKI at high altitude in the literature. OBJECTIVES: to investigate the incidence, etiology, clinical features and maternal-fetal outcomes of Pr-AKI in women living at high altitude. METHODS: 6,512 pregnant women attending the Department of Obstetrics & Gynecology at local hospital from January 2015 to December 2018 were screened for Pr-AKI. Patients with serum creatinine above normal range(> 70umol/L) then underwent assessment to confirm the diagnosis of Pr-AKI. AKI was diagnosed and staged based on Kidney Disease Improving Global Outcomes(KDIGO) guideline. Individuals meeting the Pr-AKI criteria were recruited. Their clinical data were recorded and retrospectively analyzed. RESULTS: Pr-AKI was identified in 136/6512(2.09 %) patients. Hypertensive disorders of pregnancy(HDP) was the leading cause of Pr-AKI(35.3 %). 4(2.9 %) women died and the majority(86.1 %) had recovered renal function before discharge. Fetal outcomes were confirmed in 109 deliveries with gestational age ≥ 20 weeks. Pre-term delivery occurred in 30(27.3 %) cases and perinatal deaths in 17(15.5 %). The rate of low birth weight infant(LBWI) and intrauterine growth restriction(IUGR) was 22.0 and 10.9 % respectively. 16(14.5 %) infants were admitted to NICU after birth. Patients with HDP had a higher cesarean rate(56.3 %). More IUGR(25.0 %) and LBWI(37.8 %) were observed in their infants with a higher risk of admission to NICU(22.0 %). High altitude might have an adverse impact on HDP-related Pr-AKI patients with earlier terminated pregnancy and more stillbirth/neonatal death. Logistic regression models indicated that uncontrolled blood pressure, high altitude and advanced AKI were associated with adverse fetal outcomes in HDP-related Pr-AKI patients. CONCLUSIONS: Pr-AKI was not rare in high-altitude regions and caused severe fetal morbidities and mortalities. Uncontrolled blood pressure, high altitude and advanced AKI were all risk factors for adverse fetal outcomes in Pr-AKI patients, especially for those with hypertensive disorders of pregnancy.

A Dynamic Threshold Cancellation Technique for a High-Power Conversion Efficiency CMOS Rectifier.

Power conversion efficiency (PCE) has been one of the key concerns for power management circuits (PMC) due to the low output power of the vibrational energy harvesters. This work reports a dynamic threshold cancellation technique for a high-power conversion efficiency CMOS rectifier. The proposed rectifier consists of two stages, one passive stage with a negative voltage converter, and another stage with an active diode controlled by a threshold cancellation circuit. The former stage conducts the signal full-wave rectification with a voltage drop of 1 mV, whereas the latter reduces the reverse leakage current, consequently enhancing the output power delivered to the ohmic load. As a result, the rectifier can achieve a voltage and power conversion efficiency of over 99% and 90%, respectively, for an input voltage of 0.45 V and for low ohmic loads. The proposed circuit is designed in a standard 130 nm CMOS process and works for an operating frequency range from 800 Hz to 51.2 kHz, which is promising for practical applications.

Interferon-inducible MX2 is a host restriction factor of hepatitis B virus replication.

BACKGROUND & AIMS: Non-cytolytic cure of HBV-infected hepatocytes by cytokines, including type I interferons (IFNs), is of importance for resolving acute and chronic infection. However, as IFNs stimulate hundreds of genes, those most relevant for HBV suppression remain largely unknown. Amongst them are the large myxovirus resistance (Mx) GTPases. Human MX1 (or MxA) is active against many RNA viruses, while MX2 (or MxB) was recently found to restrict HIV-1, HCV, and herpesviruses. Herein, we investigated the anti-HBV activity of MX2. METHODS: The potential anti-HBV activity of MX2 and functional variants were assessed in transfected and HBV-infected hepatoma cells and primary human hepatocytes, employing multiple assays to analyze the synthesis and decay of HBV nucleic acids. The specific roles of MX2 in IFN-α-driven inhibition of HBV transcription and replication were assessed by MX2-specific shRNA interference (RNAi). RESULTS: Both MX2 alone and IFN-α substantially inhibited HBV replication, due to significant deceleration of the synthesis and slight acceleration of the turnover of viral RNA. RNAi knockdown of MX2 significantly reduced the inhibitory effects of IFN-α. Strikingly, MX2 inhibited HBV infection by reducing covalently closed circular DNA (cccDNA), most likely by indirectly impairing the conversion of relaxed circular DNA to cccDNA rather than by destabilizing existing cccDNA. Various mutations affecting the GTPase activity and oligomerization status reduced MX2's anti-HBV activity. CONCLUSION: MX2 is an important IFN-α inducible effector that decreases HBV RNA levels but can also potently inhibit HBV infection by indirectly impairing cccDNA formation. MX2 likely has the potential for therapeutic applications aimed at curing HBV infection by eliminating cccDNA. LAY SUMMARY: This study shows that the protein MX2, which is induced by interferon-α, has important anti-hepatitis B virus (HBV) effector functions. MX2 can reduce the amount of covalently closed circular DNA, which is the form of DNA that HBV uses to maintain viral persistence within hepatocytes. MX2 also reduces HBV RNA levels by downregulating synthesis of viral RNA. MX2 likely represents a novel intrinsic HBV inhibitor that could have therapeutic potential, as well as being useful for improving our understanding of the complex biology of HBV and the antiviral mechanisms of interferon-α.

Immune Complex Vaccination.

Antibody/antigen binding results in immune complexes (IC) that have a variety of regulatory functions. One important feature is the enhanced host immune activation against antigen contained in the complex. ICs play important roles at several critical steps that lead to B and T cell activation, including antigen targeting/retention, facilitated antigen uptake, antigen presenting cell activation and proper balancing of positive and negative stimulatory signals. In both poultry industry and clinical health care, ICs have been used as preventive and therapeutic vaccines. With our deepening understanding of antibody biology, particularly in light of new revelations of regulatory functions of Fc receptors, mechanistically more precise engineering has spearheaded tailored use of this tool for infection control and cancer therapy. IC-based treatment and prophylaxis have been tested to different extents in HBV, HIV and influenza viral infection control and are actively examined as an alternative treatment for several forms of tumor. As a part of this book series, this chapter aims to discuss the mechanistic aspects of IC signaling and their impact on immune cells. We give samples how this old technology has been used by practitioners over the last several decades and suggest potential paths for future development of IC-based immune therapy.

Fluorescence-based biochemical analysis of human hepatitis B virus reverse transcriptase activity.

Although the unique mechanism by which hepatitis B virus (HBV) polymerase primes reverse transcription is now well-characterized, the subsequent elongation process remains poorly understood. Reverse transcriptase (RT)-RNase H sequences from polymerase amino acid 304 (the C-terminal part of spacer domain) to 843 were expressed in Escherichia coli and purified partially. RT elongation activity was investigated using the fluorescent-tagged primer and homopolymeric RNA templates. RT elongation activity depended on both Mg2+ and Mn2+, and had low affinity for purine deoxynucleotides, which may be related with the success of adefovir, tenofovir, and entecavir. However, the polymerization rate was lower than that of human immunodeficiency virus RT. All HBV genotypes displayed similar RT activity, except for genotype B, which demonstrated increased elongation activity.

Biomarkers distinguish HBeAg seroconverted from non-converted individuals in chronic hepatitis B patients treated with a therapeutic vaccine.

Cytokine assays of host immune responses to vaccination can indicate vaccine efficacy. Here we tested the hypothesis that assays of the cytokine status of infected individuals prior to therapeutic vaccination might provide a guide to vaccine therapeutic efficacy. If so, cytokine analysis might be used to select appropriate patients for therapeutic vaccination. Data were obtained from a panel of 14 cytokine/chemokine assays that were done during a phase III clinical trial of HBsAg-HBIG therapeutic vaccine (YIC) treatment of chronic hepatitis B (CHB) patients. Summarized assay results were compared between patients who responded by HBeAg-seroconversion and non-responders. Though no single cytokine or chemokine showed clear correlation with responsiveness, by bio-mathematical analysis with Boolean modelling, the combined results revealed that plasma IL-10, IL-33 and MIP-1α together correlated best with responsiveness. However, the difference between HBeAg seroconverted and non-converted YIC-treated CHB patients was maximized when results of all 14 cytokine/chemokine assays were included and showed a sensitivity around 0.59, and a specificity of 0.8. It suggested that the combined analysis of these elements may be useful to screen appropriate CHB patients for therapeutic vaccination with YIC.

Dosimetric study of GZP6 60 Co high dose rate brachytherapy source.

The purpose of this study was to obtain dosimetric parameters of GZP6 60 Co brachytherapy source number 3. The Geant4 MC code has been used to obtain the dose rate distribution following the American Association of Physicists in Medicine (AAPM) TG-43U1 dosimetric formalism. In the simulation, the source was centered in a 50 cm radius water phantom. The cylindrical ring voxels were 0.1 mm thick for r ≤ 1 cm, 0.5 mm for 1 cm < r ≤ 5 cm, and 1 mm for r > 5 cm. The kerma-dose approximation was performed for r > 0.75 cm to increase the simulation efficiency. Based on the numerical results, the dosimetric datasets were obtained. These results were compared with the available data of the similar 60 Co high dose rate sources and the detailed dosimetric characterization was discussed.

Lower-Order Compensation Chain Threshold-Reduction Technique for Multi-Stage Voltage Multipliers.

This paper presents a novel threshold-compensation technique for multi-stage voltage multipliers employed in low power applications such as passive and autonomous wireless sensing nodes (WSNs) powered by energy harvesters. The proposed threshold-reduction technique enables a topological design methodology which, through an optimum control of the trade-off among transistor conductivity and leakage losses, is aimed at maximizing the voltage conversion efficiency (VCE) for a given ac input signal and physical chip area occupation. The conducted simulations positively assert the validity of the proposed design methodology, emphasizing the exploitable design space yielded by the transistor connection scheme in the voltage multiplier chain. An experimental validation and comparison of threshold-compensation techniques was performed, adopting 2N5247 N-channel junction field effect transistors (JFETs) for the realization of the voltage multiplier prototypes. The attained measurements clearly support the effectiveness of the proposed threshold-reduction approach, which can significantly reduce the chip area occupation for a given target output performance and ac input signal.

Functional characterization of hepatitis B virus core promoter mutants revealed transcriptional interference among co-terminal viral mRNAs.

Hepatitis B virus (HBV) has a 3.2 kb circular DNA genome. It employs four promoters in conjunction with a single polyadenylation signal to generate 3.5, 2.4, 2.1 and 0.7 kb co-terminal RNAs. The 3.5 kb RNA is subdivided into the precore RNA for e-antigen expression and pregenomic RNA for genome replication. When introduced to a genotype A clone, several core promoter mutations markedly enhanced HBV genome replication, but suppressed e-antigen expression through up-regulation of pregenomic RNA at the expense of precore RNA. In this study, we found such mutations also diminished envelope proteins and hepatitis B surface antigen, products of the 2.1 and 2.4 kb subgenomic RNAs. Indeed, Northern blot analysis revealed overall increase in 3.5 kb RNA, but reduction in all subgenomic RNAs. To validate transcriptional interference, we subcloned 1.1×, 0.7× and 0.6× HBV genome, respectively, to a vector with or without a cytomegalovirus (CMV) promoter at the 5' end, so as to produce the pregenomic RNA, 2.4 kb RNA, and 2.1 kb RNA in large excess or not at all. Parallel transfection of the three pairs of constructs into a human hepatoma cell line confirmed the ability of pregenomic RNA to suppress all subgenomic transcripts and established the ability of the 2.4 and 2.1 kb RNAs to suppress the 0.7 kb RNA. Consistent with our findings, pregenomic RNA of the related duck HBV has been reported to interfere with transcription of the subgenomic RNAs. Transcriptional interference might explain why HBV produces so little 0.7 kb RNA and HBx protein despite a strong X promoter.

Novel recombinant hepatitis B virus vectors efficiently deliver protein and RNA encoding genes into primary hepatocytes.

Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection.

Nanotoxicity of tungsten trioxide nanosheets containing oxygen vacancy to human umbilical vein endothelial cells.

Because of the excellent performance in photochemistry, WO3 is increasingly applied in the field of biology and medicine. However, little is known about the mechanism of WO3 cytotoxicity. In this work, WO3 nanosheets with oxygen vacancy are synthesized by solvothermal method, then characterized and added to culture medium of human umbilical vein endothelial cells (HUVECs) with different concentrations. We characterized and analyzed the morphology of nano-WO3 by transmission electron microscopy and calculated the specific data of oxygen vacancy by XPS. It is the first time the effect of WO3-x on cells that WO3-x can cause oxidative stress in HUVEC cells, resulting in DNA damage and thus promoting apoptosis. Transcriptome sequencing is performed on cells treated with low and high concentrations of WO3-x, and a series of key signals affecting cell proliferation and apoptosis are detected in differentially expressed genes, which indicates the research direction of nanotoxicity. The expression levels of key genes are also verified by quantitative PCR after cell treatment with different concentrations of WO3-x. This work fills the gap between the biocompatibility of nano WO3-x materials and molecular cytology and paves the way for investigating the mechanism and risks of oxygen vacancy in cancer therapy.

A bispecific antibody exhibits broad neutralization against SARS-CoV-2 Omicron variants XBB.1.16, BQ.1.1 and sarbecoviruses.

The Omicron subvariants BQ.1.1, XBB.1.5, and XBB.1.16 of SARS-CoV-2 are known for their adeptness at evading immune responses. Here, we isolate a neutralizing antibody, 7F3, with the capacity to neutralize all tested SARS-CoV-2 variants, including BQ.1.1, XBB.1.5, and XBB.1.16. 7F3 targets the receptor-binding motif (RBM) region and exhibits broad binding to a panel of 37 RBD mutant proteins. We develop the IgG-like bispecific antibody G7-Fc using 7F3 and the cross-neutralizing antibody GW01. G7-Fc demonstrates robust neutralizing activity against all 28 tested SARS-CoV-2 variants and sarbecoviruses, providing potent prophylaxis and therapeutic efficacy against XBB.1 infection in both K18-ACE and BALB/c female mice. Cryo-EM structure analysis of the G7-Fc in complex with the Omicron XBB spike (S) trimer reveals a trimer-dimer conformation, with G7-Fc synergistically targeting two distinct RBD epitopes and blocking ACE2 binding. Comparative analysis of 7F3 and LY-CoV1404 epitopes highlights a distinct and highly conserved epitope in the RBM region bound by 7F3, facilitating neutralization of the immune-evasive Omicron variant XBB.1.16. G7-Fc holds promise as a potential prophylactic countermeasure against SARS-CoV-2, particularly against circulating and emerging variants.

Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients: experiences and findings.

BACKGROUND & AIMS: Even though various experimental therapeutic approaches for chronic hepatitis B infection have been reported, few of them have been verified by clinical trials. We have developed an antigen-antibody (HBsAg-HBIG) immunogenic complex therapeutic vaccine candidate with alum as adjuvant (YIC), aimed at breaking immune tolerance to HBV by modulating viral antigen processing and presentation. A double-blind, placebo-controlled, phase II B clinical trial of YIC has been reported previously, and herein we present the results of the phase III clinical trial of 450 patients. METHODS: Twelve doses of either YIC or alum alone as placebo were administered randomly to 450 CHB patients and they were followed for 24weeks after the completion of immunization. The primary end point was HBeAg seroconversion, and the secondary end points were decrease in viral load, improvement of liver function, and histology. RESULTS: In contrast to the previous phase II B trial using six doses of YIC and alum as placebo, six more injections of YIC or alum resulted in a decrease of the HBeAg seroconversion rate from 21.8% to 14.0% in the YIC group, but an increase from 9% to 21.9% in the alum group. Decrease in serum HBV DNA and normalization of liver function were similar in both groups (p>0.05). CONCLUSIONS: Overstimulation with YIC did not increase but decreased its efficacy due to immune fatigue in hosts. An appropriate immunization protocol should be explored and is crucial for therapeutic vaccination. Multiple injections of alum alone could have stimulated potent inflammatory and innate immune responses contributing to its therapeutic efficacy, and needs further investigation.