Search Results (17)

Anthrax remains a formidable bioterrorism threat for which new, optimized and thermostable vaccines are needed. We previously demonstrated that five immunizations of rabbits with a multiple-antigenic-peptide (MAP) vaccine in either Freund’s adjuvant or human-use adjuvants can elicit antibody (Ab) against the loop-neutralizing determinant (LND), a cryptic neutralizing epitope in the 2β2-2β3 loop of protective antigen from Bacillus anthracis (B. anthracis), which mediates complete protection of rabbits from inhalation spore challenge with the B. anthracis Ames strain. To develop a more immunogenic vaccine, we molecularly constructed a virus-like particle (VLP) vaccine, comprising the Woodchuck hepatitis core antigen capsid (WHcAg) displaying 240 copies of the LND epitope on each nanoparticle. Initial studies showed that the LND-VLP was immunogenic in rabbits following two immunizations, and passive transfer of the rabbit sera into A/J mice conferred complete protection from aerosol challenge with B. anthracis. Further optimization of the vaccine revealed that the lyophilized LND-VLP vaccine was capable of eliciting highly protective levels of neutralizing antibody with two immunizations, and in some rabbits, a single immunization, using human-use adjuvants. A lyophilized LND-VLP nanoparticle vaccine may be an effective stand-alone vaccine or may complement PA-based vaccines as a future pre- or post-exposure vaccine for anthrax. Full article

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV’s evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus–host interaction of this unique group of animal viroid-like agents in relation to HDV. Full article

Orthohepadnavirus causes chronic hepatitis in a broad range of mammals, including primates, cats, woodchucks, and bats. Hepatitis B virus (HBV) X protein inhibits type-I interferon (IFN) signaling, thereby promoting HBV escape from the human innate immune system and establishing persistent infection. However, whether X proteins of Orthohepadnavirus viruses in other species display a similar inhibitory activity remains unknown. Here, we investigated the anti-IFN activity of 17 Orthohepadnavirus X proteins derived from various hosts. We observed conserved activity of Orthohepadnavirus X proteins in inhibiting TIR-domain-containing adaptor protein inducing IFN-β (TRIF)-mediated IFN-β signaling pathway through TRIF degradation. X proteins from domestic cat hepadnavirus (DCH), a novel member of Orthohepadnavirus, inhibited mitochondrial antiviral signaling protein (MAVS)-mediated IFNβ signaling pathway comparable with HBV X. These results indicate that inhibition of IFN signaling is conserved in Orthohepadnavirus X proteins. Full article

During the emergence of infectious diseases, evaluating the efficacy of newly developed vaccines requires antigen proteins. Available methods enhance antigen protein productivity; however, structural modifications may occur. Therefore, we aimed to construct a novel transient overexpression vector capable of rapidly producing large quantities of antigenic proteins in mammalian cell lines. This involved expanding beyond the exclusive use of the human cytomegalovirus (CMV) promoter, and was achieved by incorporating a transcriptional enhancer (CMV enhancer), a translational enhancer (woodchuck hepatitis virus post-transcriptional regulatory element), and a promoter based on the CMV promoter. Twenty novel transient expression vectors were constructed, with the vector containing the human elongation factor 1-alpha (EF-1a) promoter showing the highest efficiency in expressing foreign proteins. This vector exhibited an approximately 27-fold higher expression of enhanced green fluorescent protein than the control vector containing only the CMV promoter. It also expressed the highest level of severe acute respiratory syndrome coronavirus 2 receptor-binding domain protein. These observations possibly result from the simultaneous enhancement of the transcriptional activity of the CMV promoter and the human EF-1a promoter by the CMV enhancer. Additionally, the synergistic effect between the CMV and human EF-1a promoters likely contributed to the further enhancement of protein expression. Full article

Hepatitis B virus (HBV) remains a dominant cause of hepatocellular carcinoma (HCC). Recently, it was shown that HBV and woodchuck hepatitis virus (WHV) integrate into the hepatocyte genome minutes after invasion. Retrotransposons and transposable sequences were frequent sites of the initial insertions, suggesting a mechanism for spontaneous HBV DNA dispersal throughout the hepatocyte genome. Several somatic genes were also identified as early insertional targets in infected hepatocytes and woodchuck livers. Head-to-tail joints (HTJs) dominated amongst fusions, indicating their creation by non-homologous end-joining (NHEJ). Their formation coincided with the robust oxidative damage of hepatocyte DNA. This was associated with the activation of poly(ADP-ribose) polymerase 1 (PARP1)-mediated dsDNA repair, as reflected by the augmented transcription of PARP1 and XRCC1; the PARP1 binding partner OGG1, a responder to oxidative DNA damage; and increased activity of NAD⁺, a marker of PARP1 activation, and HO1, an indicator of cell oxidative stress. The engagement of the PARP1-mediated NHEJ repair pathway explains the HTJ format of the initial merges. The findings show that HBV and WHV are immediate inducers of oxidative DNA damage and hijack dsDNA repair to integrate into the hepatocyte genome, and through this mechanism, they may initiate pro-oncogenic processes. Tracking initial integrations may uncover early markers of HCC and help to explain HBV-associated oncogenesis. Full article

Infection with hepatitis B virus (HBV) is responsible for the increasing global hepatitis burden, with an estimated 296 million people being carriers and living with the risk of developing chronic liver disease and cancer. While the current treatment options for chronic hepatitis B (CHB), including oral nucleos(t)ide analogs and systemic interferon-alpha, are deemed suboptimal, the path to finding an ultimate cure for this viral disease is rather challenging. The lack of suitable laboratory animal models that support HBV infection and associated liver disease progression is one of the major hurdles in antiviral drug development. For more than four decades, experimental infection of the Eastern woodchuck with woodchuck hepatitis virus has been applied for studying the immunopathogenesis of HBV and developing new antiviral therapeutics against CHB. There are several advantages to this animal model that are beneficial for performing both basic and translational HBV research. Previous review articles have focused on the value of this animal model in regard to HBV replication, pathogenesis, and immune response. In this article, we review studies of drug development and preclinical evaluation of direct-acting antivirals, immunomodulators, therapeutic vaccines, and inhibitors of viral entry, gene expression, and antigen release in the woodchuck model of CHB since 2014 until today and discuss their significance for clinical trials in patients. Full article

Chronic hepatitis induced by hepatitis B virus (HBV) infection is a serious public health problem, leading to hepatic cirrhosis and liver cancer. Although the currently approved medications can reliably decrease the virus load and prevent the development of hepatic diseases, they fail to induce durable off-drug control of HBV replication in the majority of patients. The roots of Isatis indigotica Fortune ex Lindl., a traditional Chinese medicine, were frequently used for the prevention of viral disease in China. In the present study, (−)-lariciresinol ((−)-LRSL), isolated from the roots of Isatis indigotica Fortune ex Lindl., was found to inhibit HBV DNA replication of both wild-type and nucleos(t)ide analogues (NUCs)-resistant strains in vitro. Mechanism studies revealed that (−)-LRSL could block RNA production after treatment, followed by viral proteins, and then viral particles and DNA. Promoter reporter assays and RNA decaying dynamic experiments indicated that (−)-LRSL mediated HBV RNA reduction was mainly due to transcriptional inhibition rather than degradation. Moreover, (−)-LRSL in a dose-dependent manner also inhibited other animal hepadnaviruses, including woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Combining the analysis of RNA-seq, we further found that the decrease in HBV transcriptional activity by (−)-LRSL may be related to hepatocyte nuclear factor 1α (HNF1α). Taken together, (−)-LRSL represents a novel chemical entity that inhibits HBV replication by regulating HNF1α mediated HBV transcription, which may provide a new perspective for HBV therapeutics. Full article

Current treatment options for patients infected with hepatitis B virus (HBV) are suboptimal, because the approved drugs rarely induce cure due to the persistence of the viral DNA genome in the nucleus of infected hepatocytes, and are associated with either severe side effects (pegylated interferon-alpha) or require life-long administration (nucleos(t)ide analogs). We report here the evaluation of the safety and therapeutic efficacy of a novel, humanized antibody (hzVSF) in the woodchuck model of HBV infection. hzVSF has been shown to act as a viral entry inhibitor, most likely by suppressing vimentin-mediated endocytosis of virions. Targeting the increased vimentin expression on liver cells by hzVSF after infection with HBV or woodchuck hepatitis virus (WHV) was demonstrated initially. Thereafter, hzVSF safety was assessed in eight woodchucks naïve for WHV infection. Antiviral efficacy of hzVSF was evaluated subsequently in 24 chronic WHV carrier woodchucks by monotreatment with three ascending doses and in combination with tenofovir alafenamide fumarate (TAF). Consistent with the proposed blocking of WHV reinfection, intravenous hzVSF administration for 12 weeks resulted in a modest but transient reduction of viral replication and associated liver inflammation. In combination with oral TAF dosing, the antiviral effect of hzVSF was enhanced and sustained in half of the woodchucks with an antibody response to viral proteins. Thus, hzVSF safely but modestly alters chronic WHV infection in woodchucks; however, as a combination partner to TAF, its antiviral efficacy is markedly increased. The results of this preclinical study support future evaluation of this novel anti-HBV drug in patients. Full article

Hepatocellular carcinoma (HCC) is an important cause of cancer death worldwide, and hepatitis B virus (HBV) infection is a major etiology, particularly in the Asia-Pacific region. Lack of sensitive biomarkers for early diagnosis of HCC and lack of effective therapeutics for patients with advanced HCC are the main reasons for high HCC mortality; these clinical needs are linked to the molecular heterogeneity of hepatocarcinogenesis. Animal models are the basis of preclinical and translational research in HBV-related HCC (HBV-HCC). Recent advances in methodology have allowed the development of several animal models to address various aspects of chronic liver disease, including HCC, which HBV causes in humans. Currently, multiple HBV-HCC animal models, including conventional, hydrodynamics-transfection-based, viral vector-mediated transgenic, and xenograft mice models, as well as the hepadnavirus-infected tree shrew and woodchuck models, are available. This review provides an overview of molecular mechanisms and animal models of HBV-HCC. Additionally, the metastatic tumor antigen 1 (MTA1), a cancer-promoting molecule, was introduced as an example to address the importance of a suitable animal model for studying HBV-related hepatocarcinogenesis. Full article

As current interventions for chronic hepatitis B (CHB) rarely induce cure, more effective drugs are needed. Short-term treatment of woodchucks with the novel immunomodulator AIC649, a parapoxvirus-based stimulator of toll-like receptor 9 dependent and independent pathways, has been shown to reduce viral DNA and surface antigen via a unique, biphasic response pattern. The present study evaluated long-term AIC649 treatment in combination with Entecavir for potency and safety in woodchucks. AIC649 monotreatment induced modest reductions in serum viral DNA and surface and e antigens that were associated with the same biphasic response pattern previously observed. Entecavir monotreatment reduced transiently viremia but not antigenemia, while AIC649/Entecavir combination treatment mediated superior viral control. Undetectability of viral antigens and elicitation of antibodies in AIC649/Entecavir-treated woodchucks correlated with the expression of interferons and suppression of viral replication in liver. Combination treatment was well tolerated, and liver enzyme elevations were minor and transient. It was concluded that the AIC649-mediated effects were most likely based on an improvement and/or reconstitution of antiviral immune responses that are typically deficient in CHB. As a combination partner to Entecavir, the antiviral efficacy of AIC649 was markedly enhanced. This preclinical study supports future evaluation of AIC649 for treatment of human CHB. Full article

Clinical diagnosis of hepatocellular carcinoma (HCC) relies heavily on radiological imaging. However, information pertaining to liver cancer treatment such as the proliferation status is lacking. Imaging tumor proliferation can be valuable in patient management. This study investigated ¹⁸F-labeled clofarabine ([¹⁸F]CFA) targeting deoxycytidine kinase (dCK) for PET imaging of dCK-dependent proliferation in HCC. Since clinical PET scans showed a high liver background uptake of [¹⁸F]CFA, the aim of this study was to reduce this liver background uptake. A clinically relevant animal model of spontaneously developed HCC in the woodchucks was used for imaging experiments. Several modifiers were tested and compared with the baseline PET scan: Forodesine, probenecid, and cold clofarabine, all applied before the hot [¹⁸F]CFA injection to evaluate the reduction in liver background uptake. Application of forodesine before hot [¹⁸F]CFA injection did not reduce the background uptake. Instead, it increased the background by 11.6–36.3%. Application of probenecid also increased the liver background uptake by 16.6–32.1%. Cold CFA application did reduce the liver background uptake of [¹⁸F]CFA, comparing to the baseline scan. Combining cold CFA with [¹⁸F]CFA for PET imaging of liver cancers is a promising strategy, worthy of further clinical evaluation. Full article

The multikinase inhibitor sorafenib is the only standard first-line therapy for hepatocellular carcinoma (HCC). Here, we report the dose-dependent effects of sorafenib on the immune response, which is related to nuclear factor of activated T cells 1 (NFAT1) activity. In vitro and in vivo experiments were performed with low and high doses of sorafenib using human T cells and spontaneous developed woodchuck HCC models. In vitro studies demonstrated that following exposure to a high dose of sorafenib the baseline activity of NFAT1 in T cells was significantly increased. In a parallel event, high dose sorafenib resulted in a significant decrease in T cell proliferation and increased the proportion of PD-1 expressing CD8+ T cells with NFAT1 activation. In the in vivo model, smaller tumors were detected in the low-dose sorafenib treated group compared to the placebo and high-dose treated groups. The low-dose sorafenib group showed a significant tumor growth delay with significantly more CD3+ cells in tumor. This study demonstrates that sorafenib has immunomodulatory effects in a dose- and time-dependent manner. Higher dose of sorafenib treatment was associated with immunosuppressive action. This observed effect of sorafenib should be taken into consideration in the selection of optimum starting dose for future trials. Full article

In the last two centuries, since the dawn of modern geology, heavy minerals have been used to investigate sediment provenance and for many other scientific or practical applications. Not always, however, with the correct approach. Difficulties are diverse, not just technical and related to the identification of tiny grains, but also procedural and conceptual. Even the definition of “heavy minerals” is elusive, and possibly impossible. Sampling is critical. In many environments (e.g., beaches), both absolute and relative heavy mineral abundances invariably increase or decrease locally to different degrees owing to hydraulic-sorting processes, so that samples close to "neutral composition" are hard to obtain. Several widely shared opinions are misleading. Choosing a narrow size-window for analysis leads to increased bias, not to increased accuracy or precision. Only point-counting provides real volume percentages, whereas grain-counting distorts results in favor of smaller minerals. This paper also briefly reviews the heavy mineral associations typically found in diverse plate-tectonic settings. A mineralogical assemblage, however, only reproduces the mineralogy of source rocks, which does not correlate univocally with the geodynamic setting in which those source rocks were formed and assembled. Moreover, it is affected by environmental bias, and by diagenetic bias on top in the case of ancient sandstones. One fruitful way to extract information on both provenance and sedimentological processes is to look for anomalies in mineralogical–textural relationships (e.g., denser minerals bigger than lower-density minerals; harder minerals better rounded than softer minerals; less durable minerals increasing with stratal age and stratigraphic depth). To minimize mistakes, it is necessary to invariably combine heavy mineral investigations with the petrographic analysis of bulk sand. Analysis of thin sections allows us to see also those source rocks that do not shed significant amounts of heavy minerals, such as limestone or granite, and helps us to assess heavy mineral concentration, the “outer” message carrying the key to decipher the “inner message” contained in the heavy mineral suite. The task becomes thorny indeed when dealing with samples with strong diagenetic overprint, which is, unfortunately, the case of most ancient sandstones. Diagenesis is the Moloch that devours all grains that are not chemically resistant, leaving a meager residue difficult or even impossible to interpret when diagenetic effects accumulate through multiple sedimentary cycles. We have conceived this friendly little handbook to help the student facing these problems, hoping that it may serve the purpose. Full article

In the hydrodynamic injection (HI) BALB/c mouse model with the overlength viral genome, we have found that woodchuck hepatitis virus (WHV) could persist for a prolonged period of time (up to 45 weeks), while hepatitis B virus (HBV) was mostly cleared at week four. In this study, we constructed a series of chimeric genomes based on HBV and WHV, in which the individual sequences of a 1.3-fold overlength HBV genome in pBS-HBV1.3 were replaced by their counterparts from WHV. After HI with the WHV-HBV chimeric constructs in BALB/c mice, serum viral antigen, viral DNA (vDNA), and intrahepatic viral antigen expression were analyzed to evaluate the persistence of the chimeric genomes. Interestingly, we found that HI with three chimeric WHV-HBV genomes resulted in persistent antigenemia in mice. All of the persistent chimeric genomes contained the preC region and the part of the C region encoding the N-terminal 1–145 amino acids of the WHV genome. These results indicated that the preC region and the N-terminal part of the C region of the WHV genome may play a role in the persistent antigenemia. The chimeric WHV-HBV genomes were able to stably express viral antigens in the liver and could be further used to express hepadnaviral antigens to study their pathogenic potential. Full article

Hepatitis delta virus (HDV) is a defective RNA virus that has an absolute requirement for a virus belonging to the hepadnaviridae family like hepatitis B virus (HBV) for its replication and formation of new virions. HDV infection is usually associated with a worsening of HBV-induced liver pathogenesis, which leads to more frequent cirrhosis, increased risk of hepatocellular carcinoma (HCC), and fulminant hepatitis. Importantly, no selective therapies are available for HDV infection. The mainstay of treatment for HDV infection is pegylated interferon alpha; however, response rates to this therapy are poor. A better knowledge of HDV–host cell interaction will help with the identification of novel therapeutic targets, which are urgently needed. Animal models like hepadnavirus-infected chimpanzees or the eastern woodchuck have been of great value for the characterization of HDV chronic infection. Recently, more practical animal models in which to perform a deeper study of host virus interactions and to evaluate new therapeutic strategies have been developed. Therefore, the main focus of this review is to discuss the current knowledge about HDV host interactions obtained from cell culture and animal models. Full article