Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

Characterization of a Novel Capsid Assembly Modulator for the Treatment of Chronic Hepatitis B Virus Infection.

The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs ("antiviral intensification"). Here, we describe GS-SBA-1, a capsid assembly modulator (CAM) belonging to class CAM-E, that demonstrates potent inhibition of extracellular HBV DNA in vitro (EC50 [50% effective concentration] = 19 nM) in HBV-infected primary human hepatocytes (PHHs) as well as in vivo in an HBV-infected immunodeficient mouse model. GS-SBA-1 has comparable activities across HBV genotypes and nucleos(t)ide-resistant mutants in HBV-infected PHHs. In addition, GS-SBA-1 demonstrated in vitro additivity in combination with tenofovir alafenamide (TAF). The administration of GS-SBA-1 to PHHs at the time of infection prevents covalently closed circular DNA (cccDNA) formation and, hence, decreases HBV RNA and antigen levels (EC50 = 80 to 200 nM). Furthermore, GS-SBA-1 prevents the production of extracellular HBV RNA-containing viral particles in vitro. Collectively, these data demonstrate that GS-SBA-1 is a potent CAM that has the potential to enhance viral suppression in combination with an NA.

Expanded profiling of Remdesivir as a broad-spectrum antiviral and low potential for interaction with other medications in vitro.

Remdesivir (GS-5734; VEKLURY) is a single diastereomer monophosphoramidate prodrug of an adenosine analog (GS-441524). Remdesivir is taken up by target cells and metabolized in multiple steps to form the active nucleoside triphosphate (GS-443902), which acts as a potent inhibitor of viral RNA-dependent RNA polymerases. Remdesivir and GS-441524 have antiviral activity against multiple RNA viruses. Here, we expand the evaluation of remdesivir's antiviral activity to members of the families Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae. Using cell-based assays, we show that remdesivir can inhibit infection of flaviviruses (such as dengue 1-4, West Nile, yellow fever, Zika viruses), picornaviruses (such as enterovirus and rhinovirus), and filoviruses (such as various Ebola, Marburg, and Sudan virus isolates, including novel geographic isolates), but is ineffective or is significantly less effective against orthomyxoviruses (influenza A and B viruses), or hepadnaviruses B, D, and E. In addition, remdesivir shows no antagonistic effect when combined with favipiravir, another broadly acting antiviral nucleoside analog, and has minimal interaction with a panel of concomitant medications. Our data further support remdesivir as a broad-spectrum antiviral agent that has the potential to address multiple unmet medical needs, including those related to antiviral pandemic preparedness.

A Combined Model of SARIMA and Prophet Models in Forecasting AIDS Incidence in Henan Province, China.

Acquired immune deficiency syndrome (AIDS) is a serious public health problem. This study aims to establish a combined model of seasonal autoregressive integrated moving average (SARIMA) and Prophet models based on an L1-norm to predict the incidence of AIDS in Henan province, China. The monthly incidences of AIDS in Henan province from 2012 to 2020 were obtained from the Health Commission of Henan Province. A SARIMA model, a Prophet model, and two combined models were adopted to fit the monthly incidence of AIDS using the data from January 2012 to December 2019. The data from January 2020 to December 2020 was used to verify. The mean square error (MSE), mean absolute error (MAE), and mean absolute percentage error (MAPE) were used to compare the prediction effect among the models. The results showed that the monthly incidence fluctuated from 0.05 to 0.50 per 100,000 individuals, and the monthly incidence of AIDS had a certain periodicity in Henan province. In addition, the prediction effect of the Prophet model was better than SARIMA model, the combined model was better than the single models, and the combined model based on the L1-norm had the best effect values (MSE = 0.0056, MAE = 0.0553, MAPE = 43.5337). This indicated that, compared with the L2-norm, the L1-norm improved the prediction accuracy of the combined model. The combined model of SARIMA and Prophet based on the L1-norm is a suitable method to predict the incidence of AIDS in Henan. Our findings can provide theoretical evidence for the government to formulate policies regarding AIDS prevention.

Targeting lipid biosynthesis pathways for hepatitis B virus cure.

Chronic hepatitis B virus (HBV) infection is characterized by the presence of high circulating levels of non-infectious lipoprotein-like HBV surface antigen (HBsAg) particles thought to contribute to chronic immune dysfunction in patients. Lipid and metabolomic analysis of humanized livers from immunodeficient chimeric mice (uPA/SCID) revealed that HBV infection dysregulates several lipid metabolic pathways. Small molecule inhibitors of lipid biosynthetic pathway enzymes acetyl-CoA carboxylase (ACC), fatty acid synthase, and subtilisin kexin isozyme-1/site-1 protease in HBV-infected HepG2-NTCP cells demonstrated potent and selective reduction of extracellular HBsAg. However, a liver-targeted ACC inhibitor did not show antiviral activity in HBV-infected liver chimeric mice, despite evidence of on-target engagement. Our study suggests that while HBsAg production may be dependent on hepatic de novo lipogenesis in vitro, this may be overcome by extrahepatic sources (such as lipolysis or diet) in vivo. Thus, a combination of agents targeting more than one lipid metabolic pathway may be necessary to reduce HBsAg levels in patients with chronic HBV infection.

Spatio-Temporal Analysis of Influenza-Like Illness and Prediction of Incidence in High-Risk Regions in the United States from 2011 to 2020.

About 8% of the Americans contract influenza during an average season according to the Centers for Disease Control and Prevention in the United States. It is necessary to strengthen the early warning for influenza and the prediction of public health. In this study, Spatial autocorrelation analysis and spatial scanning analysis were used to identify the spatiotemporal patterns of influenza-like illness (ILI) prevalence in the United States, during the 2011-2020 transmission seasons. A seasonal autoregressive integrated moving average (SARIMA) model was constructed to predict the influenza incidence of high-risk states. We found the highest incidence of ILI was mainly concentrated in the states of Louisiana, District of Columbia and Virginia. Mississippi was a high-risk state with a higher influenza incidence, and exhibited a high-high cluster with neighboring states. A SARIMA (1, 0, 0) (1, 1, 0)52 model was suitable for forecasting the ILI incidence of Mississippi. The relative errors between actual values and predicted values indicated that the predicted values matched the actual values well. Influenza is still an important health problem in the United States. The spread of ILI varies by season and geographical region. The peak season of influenza was the winter and spring, and the states with higher influenza rates are concentrated in the southeast. Increased surveillance in high-risk states could help control the spread of the influenza.

M6A Classification Combined With Tumor Microenvironment Immune Characteristics Analysis of Bladder Cancer.

BACKGROUND: Studies have shown that N6-methyl adenosine (m6A) plays an important role in cancer progression; however, the underlying mechanism of m6A modification in tumor microenvironment (TME) cell infiltration of bladder cancer remains unclear. This study aimed to investigate the role of m6A modification in TME cell infiltration of bladder cancer. METHODS: The RNA expression profile and clinical data of bladder cancer were obtained from The Cancer Genome Atlas and Gene Expression Omnibus. We assessed the m6A modification patterns of 664 bladder cancer samples based on 20 m6A regulators through unsupervised clustering analysis and systematically linked m6A modification patterns to TME cell infiltration characteristics. Gene ontology and gene set variation analyses were conducted to analyze the underlying mechanism based on the assessment of m6A methylation regulators. Principal component analysis was used to construct the m6A score to quantify m6A modification patterns of bladder cancer. RESULTS: The genetic and expression alterations in m6A regulators were highly heterogeneous between normal and bladder tissues. Three m6A modification patterns were identified. The cell infiltration characteristics were highly consistent with the three immune phenotypes, including immune rejection, immune inflammation, and immune desert. The biological functions of three m6A modification patterns were different. Cox regression analyses revealed that the m6A score was an independent signature with patient prognosis (HR = 1.198, 95% CI: 1.031-1.390). Patients with a low-m6A score were characterized by increased tumor mutation burden, PD-L1 expression, and poorer survival. Patients in the low-m6A score group also showed significant immune responses and clinical benefits in the CTLA-4 immunotherapy cohort (p =0.0069). CONCLUSIONS: The m6A methylation modification was related to the formation of TME heterogeneity and complexity. Assessing the m6A modification pattern of individual bladder cancer will improve the understanding of TME infiltration characteristics.

Functional analysis of murine aryl hydrocarbon (AH) receptors defective in nuclear import: impact on AH receptor degradation and gene regulation.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is also a substrate for the 26S proteasome. However, the subcellular location of the degradation events or the requirement for nuclear transport has not been resolved. To gain insight into both ligand-dependent and independent degradation of the AHR, studies were designed to evaluate the relationship between AHR localization, stability, and gene regulation in a defined cell culture model system. The strategy of these studies was to generate stable cell lines expressing murine AHR proteins that were defective in nuclear import and then to assess the location of the AHR, the time course of AHR degradation, and the level of induction of endogenous CYP1A1 protein after exposure to 2,3,7,8-tetrachlorodibezo-p-dioxin (TCDD), geldanamycin (GA), or the protease inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal (MG-132). Mutation within the putative nuclear localization sequence (NLS) resulted in AHR mutants that were severely defective in nuclear import as evaluated by immunocytochemical staining after exposure to TCDD, GA, or MG-132. Importantly, the NLS mutants exhibited identical levels of degradation along a similar time course as wild-type AHR after exposure to TCDD or GA when stably expressed in either murine hepatoma cells (Hepa-1) or hamster lung cells (E36). In contrast, the NLS mutants were severely defective in ligand-mediated induction of CYP1A1 expression. These findings imply that the proteolytic machinery present in the cytoplasmic compartment is sufficient to degrade the AHR and that nuclear translocation, binding with ARNT, or DNA binding are not necessary for efficient degradation of the AHR.

Ligand-dependent and independent modulation of aryl hydrocarbon receptor localization, degradation, and gene regulation.

Changes in the concentration or subcellular location of the key proteins involved in signal transduction pathways have been shown to impact gene regulation. Studies were designed to evaluate the relationship between aryl hydrocarbon receptor (AHR) localization, stability, and gene regulation in a defined system where the endogenous AHR protein could be evaluated. The findings indicate that treatment of cells with geldanamycin (GA) or MG-132 (an inhibitor of the 26S proteasome) results in nuclear translocation of the endogenous AHR in both human HepG2 and murine Hepa-1 cells without induction of endogenous CYP1A1 protein. Exposure to GA resulted in the degradation of AHR by >90% in the nucleus via the 26S proteasome. Importantly, the reduced level of AHR resulted in a 50% reduction in the maximal level of CYP1A1 induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In all treatments the concentration of the AHR nuclear translocator (ARNT) protein was unchanged and had no impact on the localization of the AHR. Thus, ligand-independent translocation of the AHR to the nucleus was not sufficient to induce CYP1A1 in the absence of ligand, but reductions in the level of the endogenous AHR protein pool shifted the dose-response curve for TCDD to the right.