Publications by authors named "Michinori Kohara"

163 Publications

Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies.

mSphere 2021 05 12;6(3). Epub 2021 May 12.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

Antibody detection is crucial for monitoring host immune responses to specific pathogen antigens (Ags) and evaluating vaccine efficacies. The luciferase immunoprecipitation system (LIPS) was developed for sensitive detection of Ag-specific antibodies in sera from various species. In this study, we describe NanoLIPS, an improved LIPS assay based on NanoLuciferase (NLuc), and employ the assay for monitoring antibody responses following influenza virus infection or vaccination. We generated recombinant influenza virus hemagglutinin (HA) proteins tagged with N-terminal (N-NLuc-HA) or C-terminal (C-NLuc-HA) NLuc reporters. NLuc-HA yielded an at least 20-fold higher signal-to-noise ratio than did a LIPS assay employing a recombinant HA- luciferase (GLuc) fusion protein. NanoLIPS-based detection of anti-HA antibodies yielded highly reproducible results with a broad dynamic range. The levels of antibodies against C-NLuc-HA generated by mice vaccinated with recombinant vaccinia virus DIs strain expressing an influenza virus HA protein (rDIs-HA) was significantly correlated with the protective effect elicited by the rDIs-HA vaccine. C-NLuc-HA underwent glycosylation with native conformations and assembly to form a trimeric structure and was detected by monoclonal antibodies that detect conformational epitopes present on the globular head or stalk domain of HA. Therefore, NanoLIPS is applicable for evaluating vaccine efficacy. We also showed that C-NLuc-HA is applicable for detection of HA-specific antibodies in sera from various experimental species, including mouse, cynomolgus macaque, and tree shrew. Thus, NanoLIPS-based detection of HA offers a simple and high-sensitivity method that detects native conformational epitopes and can be used in various experimental animal models. Influenza virus HA-specific antibodies can be detected via the hemagglutination inhibition (HI) assay, the neutralization (NT) assay, and the enzyme-linked immunosorbent assay (ELISA). However, these assays have some drawbacks, including narrow dynamic range and the requirement for large amounts of sera. As an alternative to an ELISA-based method, luciferase immunoprecipitation system (LIPS) was developed. We focused on NanoLuciferase (NLuc), which has a small size, higher intensity, and longer stability. In this study, we developed a technically feasible and highly sensitive method for detecting influenza virus-specific antibodies using a NLuc-tagged recombinant HA protein produced in mammalian cells. HA with a C-terminal NLuc extension (C-NLuc-HA) was glycosylated and formed trimeric complexes when expressed in mammalian cells. Furthermore, C-NLuc-HA was recognized not only by monoclonal antibodies that bind to the globular head domain but also by those that bind to the stalk domain. We also demonstrated that the data obtained by this assay correlate with the protection of an experimental vaccine in animal models.
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http://dx.doi.org/10.1128/mSphere.01342-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125058PMC
May 2021

Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models.

Nat Commun 2021 05 11;12(1):2654. Epub 2021 May 11.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.

Most anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules.
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http://dx.doi.org/10.1038/s41467-021-22964-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113231PMC
May 2021

Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition.

J Control Release 2021 02 14;330:61-71. Epub 2020 Dec 14.

Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan. Electronic address:

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.
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http://dx.doi.org/10.1016/j.jconrel.2020.12.013DOI Listing
February 2021

Structure-guided discovery approach identifies potential lead compounds targeting M of SARS-CoV-2.

Virusdisease 2020 Nov 11:1-5. Epub 2020 Nov 11.

Virology Unit, Viral Hepatitis Laboratory, Institut Pasteur du Maroc 1, Place Louis Pasteur, 20360 Casablanca, Morocco.

The ongoing coronavirus disease 19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become fatal for the world with affected population crossing over 25 million in more than 217 countries, consequently declared a global pandemic by the World Health Organization. Unfortunately, neither specific prophylactic or therapeutic drugs nor vaccines are available. To address the unmet medical needs, we explored a strategy identifying new compounds targeting the main protease (M) of SARS-CoV-2. Targeting the SARS-CoV-2 M crystal structure (PDB ID: 6LU7) a combination of in silico screening, molecular docking, and dynamic approaches, a set of 5000 compounds of the ZINC database were screened. As a result, we identified and ranked the top 20 compounds based on the scores of ligand-interaction, their drug-likeness properties, and their predicted antiviral efficacies. The prominent drug-like and potent inhibitory compounds are 2-[2-(2-aminoacetyl) aminoacetyl] amino-3-(4-hydroxyphenyl)-propanamide (ZINC000004762511), 6'-fluoroaristeromycin (ZINC000001483267) and cyclo (L-histidyl-L-histidyl) (ZINC000005116916) scaffolds. Further in vitro and in vivo validations are required to demonstrate anti-SARS-CoV-2 activities.
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http://dx.doi.org/10.1007/s13337-020-00627-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7656896PMC
November 2020

Nanobodies: an unexplored opportunity to combat COVID-19.

J Biomol Struct Dyn 2020 Nov 10:1-3. Epub 2020 Nov 10.

Virology Unit, Viral Hepatitis Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco.

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2). This virus is capable of human-to-human transmission, and is spreading rapidly round the globe, with markedly high fatality rates. Unfortunately, there are neither vaccines nor specific therapies available to combat it, and the developments of such approaches depend on pursuing multiple avenues in biomedical science. Accordingly, in this paper we highlight one such avenue-nanobodies-for potential utility in therapeutic and diagnostic interventions to combat COVID-19.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2020.1845801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678357PMC
November 2020

Cell-penetrating peptide-mediated cell entry of H5N1 highly pathogenic avian influenza virus.

Sci Rep 2020 10 22;10(1):18008. Epub 2020 Oct 22.

Molecular Medical Research Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan.

H5N1 highly pathogenic avian influenza virus (HPAIV) poses a huge threat to public health and the global economy. These viruses cause systemic infection in poultry and accidental human infection leads to severe pneumonia, associated with high mortality rates. The hemagglutinin (HA) of H5N1 HPAIV possesses multiple basic amino acids, as in the sequence RERRRKKR at the cleavage site; however, the role of this motif is not fully understood. Here, we showed that a 33-amino acid long peptide derived from HA of H5N1 HPAIV (HA314-46) has the potential to penetrate various cells and lung tissue through a sialic acid-independent endocytotic pathway. Mutant peptide analyses revealed that the cysteine residue at position 318 and multiple basic amino acids were essential for the cell-penetrating activity. Moreover, reassortant viruses possessing H5 HA could enter sialic acid-deficient cells, and virus internalisation was facilitated by cleavage with recombinant furin. Thus, our findings demonstrate that the HA314-46 motif exhibits cell-penetrating activity through a sialic acid-independent cell entry mechanism.
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http://dx.doi.org/10.1038/s41598-020-74604-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582914PMC
October 2020

Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA.

Virus Res 2020 12 10;290:198191. Epub 2020 Oct 10.

Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan. Electronic address:

Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.
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http://dx.doi.org/10.1016/j.virusres.2020.198191DOI Listing
December 2020

Coronavirus disease 2019-Historical context, virology, pathogenesis, immunotherapy, and vaccine development.

Hum Vaccin Immunother 2020 12 5;16(12):2992-3000. Epub 2020 Aug 5.

Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University , Kagoshima, Japan.

The current Coronavirus Disease 2019 (COVID-19) pandemic is causing great alarm around the world. The pathogen for COVID-19 - severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - is the seventh known coronavirus to cause pneumonia in humans. While much remains unknown about SARS-CoV-2, physicians and researchers have begun to publish relevant findings, and much evidence is available on coronaviruses previously circulating in human and animal populations. In this review, we situate COVID-19 in its context as a transboundary viral disease, and provide a comprehensive discussion focused on the discovery, spread, virology, pathogenesis, and clinical features of this disease, its causative coronaviral pathogen, and approaches to combating the disease through immunotherapies and other treatments and vaccine development. An epidemiological survey revealed a potentially large number of asymptomatic SARS-CoV-2 carriers within the population, which may hamper efforts against COVID-19. Finally, we emphasize that vaccines against SARS-CoV-2, which may be developed by 2021, will be essential for prevention of COVID-19.
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http://dx.doi.org/10.1080/21645515.2020.1787068DOI Listing
December 2020

Pathological and genetic aspects of spontaneous mammary gland tumor in Tupaia belangeri (tree shrew).

PLoS One 2020 18;15(5):e0233232. Epub 2020 May 18.

Joint Faculty of Veterinary Medicine, Transboundary Animal Diseases Centre, Kagoshima University, Kagoshima, Japan.

Mammary gland cancer is the most common cancer occurring in women globally. Incidences of this cancer in Japan are on the increase. Annually, more than 70,000 new cases are recorded in Japan and about 1.7 million in the world. Many cases are still difficult to cure completely, and animal models are required for the characterization of the biology, therapeutic strategy, and preventive measures for spontaneous mammary tumor. The mouse model used currently has some limitations owing to structural differences between mouse and human mammary glands. Tupaia belangeri (tree shrew), which belongs to the Tupaiidae family, shows relatively high genetic homology and structural similarity to human mammary glands. Here, we characterized the spontaneous mammary tumors in 61 female tree shrews of different ages. The incidence rate was 24.6% (15/61), and the rate of simultaneous or metachronous multiplex tumors was 60% (9/15). From the incidence pattern, some cases seemed to be of familial mammary gland tumor, as the offspring of female tree shrews No. 3 and 9 and male tree shrew No. 11 showed a high incidence rate, of 73.3% (11/15). Average incidence age for tumor development was 2 years and 3 months, and the earliest was 10 months. Histochemical analysis indicated that spontaneous mammary gland tumors in the tree shrew show the features of intraductal papillary adenomas (22 cases), except 2 tubulopapillary carcinoma cases (No. 75 and 131). All the cases were positive for the progesterone receptor, whereas 91.3% were positive for the estrogen receptor, and 4.3% were HER-2 positive. We have also confirmed the expression of nectin-4 in some mammary tumor cells. Additionally, we subjected tree shrews to cytodiagnosis or X-ray CT. Thus, the findings of this study highlight the potential of the tree shrew as a valuable new animal model for mammary gland tumor study.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7233572PMC
September 2020

Targeting Host Innate and Adaptive Immunity to Achieve the Functional Cure of Chronic Hepatitis B.

Vaccines (Basel) 2020 May 11;8(2). Epub 2020 May 11.

Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890-0065, Japan.

Despite the availability of an effective preventive vaccine for hepatitis B virus (HBV) for over 38 years, chronic HBV (CHB) infection remains a global health burden with around 257 million patients. The ideal treatment goal for CHB infection would be to achieve complete cure; however, current therapies such as peg-interferon and nucleos(t)ide analogs are unable to achieve the functional cure, the newly set target for HBV chronic infection. Considering the fact functional cure has been accepted as an endpoint in the treatment of chronic hepatitis B by scientific committee, the development of alternative therapeutic strategies is urgently needed to functionally cure CHB infection. A promising target for future therapeutic strategies is immune modulation to restore dysfunctional HBV-specific immunity. In this review, we provide an overview of the progress in alternative therapeutic strategies, including immune-based therapeutic approaches that enhance host innate and adaptive immunity to achieve and increase the functional cure from CHB infection.
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http://dx.doi.org/10.3390/vaccines8020216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349973PMC
May 2020

A Novel Cytological Model of B-Cell/Macrophage Biphenotypic Cell Hodgkin Lymphoma in -Transgenic Mice.

Cancers (Basel) 2020 Jan 14;12(1). Epub 2020 Jan 14.

Department of Diagnostic Pathology, Fujita Health University School of Medicine, Aichi 470-1192, Japan.

Hodgkin lymphoma (HL) is one of the most difficult neoplasms in terms of cytopathological research owing to the lack of established cytological murine models. Although HL is believed to be of lymphoid germinal center B-cell origin, HL cells exhibit unique biphenotypic characteristics of B cells and macrophages. B-cell/macrophage biphenotypic cells have also been identified in the spleen of Lyn-deficient mice. Moreover, Lyn-targeting germinal center-associated nuclear protein (GANP)-transgenic mice ( mice) spontaneously develop a lymphoid tumor. We aimed to investigate whether the lymphoid tumor developed in mice exhibit biphenotypic characteristics of B cells/macrophages that correspond to human HL. Here, we demonstrated GANP overexpression in human HL cells and found that it may regulate transdifferentiation between B cells and macrophages. We also demonstrated that tumors were comparable with B-cell/macrophage biphenotypic Hodgkinoid lymphomas. The tumor cells expressed macrophage-related F4/80, CD68, and CD204 as well as cytoplasmic B220 and µ-/κ-chains; in addition, these cells exhibited phagocytic activity. These cells also expressed transcripts of ; ; and the cytokines monocyte chemoattractant protein (MCP)-1, MCP-5, RANTES, tumor necrosis factor-α and thrombopoietin associated with macrophages as well as granulocyte/macrophage colony-stimulating factor, interleukin (IL)-4, IL-10, IL-12, and IL-13. - mice represent a novel cytological model for the study of cytopathological etiology and oncogenesis of HL.
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http://dx.doi.org/10.3390/cancers12010204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017268PMC
January 2020

Structure-activity relationships of fluorene compounds inhibiting HCV variants.

Antiviral Res 2020 02 17;174:104678. Epub 2019 Dec 17.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, 37673, South Korea; Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, 37673, South Korea. Electronic address:

Approximately 71 million people suffer from hepatitis C virus (HCV) infection worldwide. Persistent HCV infection causes liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, resulting in approximately 400,000 deaths annually. Effective direct-acting antiviral agents (DAAs) have been developed and are currently used for HCV treatment targeting the following three proteins: NS3/4A proteinase that cleaves the HCV polyprotein into various functional proteins, RNA-dependent RNA polymerase (designated as NS5B), and NS5A, which is required for the formation of double membrane vesicles serving as RNA replication organelles. At least one compound inhibiting NS5A is included in current HCV treatment regimens due to the high efficacy and low toxicity of drugs targeting NS5A. Here we report fluorene compounds showing strong inhibitory effects on GT 1b and 3a of HCV. Moreover, some compounds were effective against resistance-associated variants to DAAs. The structure-activity relationships of the compounds were analyzed. Furthermore, we investigated the molecular bases of the inhibitory activities of some compounds by the molecular docking method.
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http://dx.doi.org/10.1016/j.antiviral.2019.104678DOI Listing
February 2020

Intranasal vaccination with HBs and HBc protein combined with carboxyl vinyl polymer induces strong neutralizing antibody, anti-HBs IgA, and IFNG response.

Biochem Biophys Res Commun 2019 11 30;520(1):86-92. Epub 2019 Sep 30.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan. Electronic address:

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis, which is a major public health concern worldwide. Immunization methods incorporating hepatitis B surface-small (HBs-S) antigen and hepatitis B core antigen (HBc) have been proposed as candidate therapeutic vaccines, but the elimination of existing HBV infection remains a challenge. To enhance the efficacy of HBs and HBc vaccination, we investigated HBs-large (HBs-L) as an immunogen, and carboxyl vinyl polymer (CVP) as an excipient. HBs-S or HBs-L, in combination with HBc antigen, was administered subcutaneously (without CVP) or intranasally (with or without CVP) for the evaluation of immune response in the tree shrew, which is considered to be a suitable small animal model of HBV infection. Immunization with HBs-L antigen by either route induced a rapid IgG response. Intranasal immunization with HBs-S or HBs-L and HBc formulated with CVP strongly induced neutralizing antibody activity, IgA response, and HBc-specific expression of the interferon gamma-encoding gene. These data indicated the potential of HBs-L and HBc intranasal immunization with CVP, not only as a therapeutic vaccine, but also as a prophylactic vaccine candidate.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.072DOI Listing
November 2019

Construction of complete Tupaia belangeri transcriptome database by whole-genome and comprehensive RNA sequencing.

Sci Rep 2019 08 26;9(1):12372. Epub 2019 Aug 26.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan.

The northern tree shrew (Tupaia belangeri) possesses high potential as an animal model of human diseases and biology, given its genetic similarity to primates. Although genetic information on the tree shrew has already been published, some of the entire coding sequences (CDSs) of tree shrew genes remained incomplete, and the reliability of these CDSs remained difficult to determine. To improve the determination of tree shrew CDSs, we performed sequencing of the whole-genome, mRNA, and total RNA and integrated the resulting data. Additionally, we established criteria for the selection of reliable CDSs and annotated these sequences by comparison to the human transcriptome, resulting in the identification of complete CDSs for 12,612 tree shrew genes and yielding a more accurate tree shrew genome database (TupaiaBase: http://tupaiabase.org ). Transcriptome profiles in hepatitis B virus infected tree shrew livers were analyzed for validation. Gene ontology analysis showed enriched transcriptional regulation at 1 day post-infection, namely in the "type I interferon signaling pathway". Moreover, a negative regulator of type I interferon, SOCS3, was induced. This work, which provides a tree shrew CDS database based on genomic DNA and RNA sequencing, is expected to serve as a powerful tool for further development of the tree shrew model.
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http://dx.doi.org/10.1038/s41598-019-48867-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710255PMC
August 2019

Basal expression of interferon regulatory factor 1 drives intrinsic hepatocyte resistance to multiple RNA viruses.

Nat Microbiol 2019 07 15;4(7):1096-1104. Epub 2019 Apr 15.

UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA.

Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance. Less attention has been paid to how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RIG-I-like receptor and Toll-like receptor signalling to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show that constitutively expressed IRF1 acts independently of mitochondrial antiviral signalling (MAVS) protein, IRF3 and signal transducer and activator of transcription 1 (STAT1)-dependent signalling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains the basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses, dengue virus and Zika virus. Our findings reveal an unappreciated layer of hepatocyte-intrinsic immunity to these positive-strand RNA viruses and identify previously unrecognized antiviral effector genes.
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http://dx.doi.org/10.1038/s41564-019-0425-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588457PMC
July 2019

Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation.

J Biol Chem 2019 04 12;294(15):5759-5773. Epub 2019 Feb 12.

From the Transboundary Animal Diseases Centre, Kagoshima University, Kagoshima-City 890-8580, Japan; Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima-City 890-8580, Japan. Electronic address:

Hepatitis C virus (HCV) establishes a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HCV life cycle is closely associated with host factors that promote or restrict viral replication, the characterization of which could help to identify potential therapeutic targets. To this end, here we performed a genome-wide microarray analysis and identified ribonucleotide reductase M2 (RRM2) as a cellular factor essential for HCV replication. We found that RRM2 is up-regulated in response to HCV infection in quiescent hepatocytes from humanized chimeric mouse livers. To elucidate the molecular basis of RRM2 expression in HCV-infected cells, we used HCV-infected hepatocytes from chimeric mice and hepatoma cells infected with the HCV strain JFH1. Both models exhibited increased mRNA and protein expression levels. Moreover, siRNA-mediated silencing of suppressed HCV replication and infection. Of note, RRM2 and RNA polymerase nonstructural protein 5B (NS5B) partially co-localized in cells and co-immunoprecipitated, suggesting that they might interact. knockdown reduced NS5B expression, which depended on the protein degradation pathway, as RNA levels did not decrease and NS5B protein stability correlated with RRM2 protein levels. We also found that silencing decreased levels of hPLIC1 (human homolog 1 of protein linking integrin-associated protein and cytoskeleton), a ubiquitin-like protein that interacts with NS5B and promotes its degradation. This finding suggests that there is a dynamic interplay between RRM2 and the NS5B-hPLIC1 complex that has an important function in HCV replication. Together, these results identify a role of host RRM2 in viral RNA replication.
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http://dx.doi.org/10.1074/jbc.RA118.004397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463693PMC
April 2019

Avian H5N1 influenza virus infection causes severe pneumonia in the Northern tree shrew (Tupaia belangeri).

Virology 2019 03 19;529:101-110. Epub 2019 Jan 19.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. Electronic address:

Avian-origin influenza viruses like H5N1 and H7N9 often cause severe symptoms with high mortality in humans. Animal models are useful for clarification of the mechanisms of pathogenicity of these infections. In this study, to expand the potential utility of the Northern tree shrew (Tupaia belangeri) for influenza virus infection, we assessed the pathogenicity of H5N1 and H7N9 avian influenza viruses in tupaia. Infectious virus was detected continuously from nasal, oral, tracheal, and conjunctival swab samples in the animals infected with these viruses. H5N1 influenza virus infection of tupaia caused severe diffuse pneumonia with fever and weight loss. In contrast, H7N9 influenza virus infection caused focal pneumonia. The severity of pneumonia was correlated with proinflammatory cytokine transcript levels. These results indicated that tupaia can be another suitable animal model for avian influenza virus research.
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http://dx.doi.org/10.1016/j.virol.2019.01.015DOI Listing
March 2019

MicroRNA-451a in extracellular, blood-resident vesicles attenuates macrophage and dendritic cell responses to influenza whole-virus vaccine.

J Biol Chem 2018 11 3;293(48):18585-18600. Epub 2018 Oct 3.

From the Department of Immunology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556,

The innate immune system is important for the efficacy of vaccines, but excessive innate immune responses can cause adverse reactions after vaccination. Extracellular vesicles (EVs) are enriched in the blood and can deliver functional RNAs, such as microRNAs (miRNAs), to recipient cells, thereby mediating intercellular communication. However, the role of EVs in controlling the innate immune responses to vaccines has not been fully elucidated. Here, we found that miR-451a is abundant in human serum EVs and that its presence in blood-circulating EVs affects the innate immune responses of macrophages and dendritic cells to inactivated whole-virus vaccines (WV) against influenza. miR-451a in human serum EVs was stable for a week in healthy subjects, and its levels gradually fluctuated over several months. miR-451a within serum EVs was internalized into serum-cultured macrophages and dendritic cells and reduced endogenous 14-3-3ζ protein levels and decreased the expression of type I IFN and interleukin 6 in response to WV stimulation. miR-451a levels in blood-circulating EVs were positively correlated with intracellular miR-451a levels in mouse splenic CD11c cells and inversely correlated with the innate immune response to inactivated WV These findings suggest that miR-451a in circulating EVs is internalized into recipient cells and that this internalization results in an attenuation of the innate immune response to WV. Moreover, a microarray analysis identified several other miRNAs that affect the macrophage response to inactivated WV. Our results reveal that miRNAs in circulating EVs significantly modify the responses of macrophages and dendritic cells to inactivated WV.
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http://dx.doi.org/10.1074/jbc.RA118.003862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290151PMC
November 2018

A Highly Bioactive Lys-Deficient IFN Leads to a Site-Specific Di-PEGylated IFN with Equivalent Bioactivity to That of Unmodified IFN-α2b.

ACS Synth Biol 2018 11 26;7(11):2537-2546. Epub 2018 Oct 26.

Department of Electrical Engineering and Bioscience , Waseda University , Shinjuku, Tokyo , 169-8050 , Japan.

Although conjugation with polyethylene glycol (PEGylation) improves the pharmacokinetics of therapeutic proteins, it drastically decreases their bioactivity. Site-specific PEGylation counters the reduction in bioactivity, but developing PEGylated proteins with equivalent bioactivity to that of their unmodified counterparts remains challenging. This study aimed to generate PEGylated proteins with equivalent bioactivity to that of unmodified counterparts. Using interferon (IFN) as a model protein, a highly bioactive Lys-deficient protein variant generated using our unique directed evolution methods enables the design of a site-specific di-PEGylated protein. Antiviral activity of our di-PEGylated IFN was similar to that of unmodified IFN-α2b. The di-PEGylated IFN exhibited 3.0-fold greater antiviral activity than that of a commercial PEGylated IFN. Moreover, our di-PEGylated IFN showed higher in vitro and in vivo stability than those of unmodified IFN-α2b. Hence, we propose that highly bioactive Lys-deficient proteins solve the limitation of conventional PEGylation with respect to the reduction in bioactivity of PEGylated proteins.
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http://dx.doi.org/10.1021/acssynbio.8b00188DOI Listing
November 2018

Experimental in vitro and in vivo systems for studying the innate immune response during dengue virus infections.

Arch Virol 2018 Jul 8;163(7):1717-1726. Epub 2018 Mar 8.

Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan.

Dengue is the most prevalent arboviral disease in humans and leads to significant morbidity and socioeconomic burden in tropical and subtropical areas. Dengue is caused by infection with any of the four closely related serotypes of dengue virus (DENV1-4) and usually manifests as a mild febrile illness, but may develop into fatal dengue hemorrhagic fever and shock syndrome. There are no specific antiviral therapies against dengue because understanding of DENV biology is limited. A tetravalent chimeric dengue vaccine, Dengvaxia, has finally been licensed for use, but its efficacy was significantly lower against DENV-2 infections and in dengue-naïve individuals. The identification of mechanisms underlying the interactions between DENV and immune responses will help to determine efficient therapeutic and preventive options. It has been well established how the innate immune system responds to DENV infection and how DENV overcomes innate antiviral defenses, however further progress in this field remains hampered by the absence of appropriate experimental dengue models. Herein, we review the available in vitro and in vivo approaches to study the innate immune responses to DENV.
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http://dx.doi.org/10.1007/s00705-018-3784-zDOI Listing
July 2018

Hepatitis C Virus: Viral Quasispecies and Genotypes.

Int J Mol Sci 2017 Dec 22;19(1). Epub 2017 Dec 22.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute, 2-1-6 Kamikitazawa, Setagaya-Ku 156-8506, Japan.

Hepatitis C virus (HCV) mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs), which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.
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http://dx.doi.org/10.3390/ijms19010023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795974PMC
December 2017

Broad-spectrum antiviral agents: secreted phospholipase A targets viral envelope lipid bilayers derived from the endoplasmic reticulum membrane.

Sci Rep 2017 Nov 21;7(1):15931. Epub 2017 Nov 21.

Department of Vaccine and Drug Development, Graduate School of Health Sciences, Kobe University, Kobe, 650-0047, Japan.

Hepatitis C virus (HCV), dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the family Flaviviridae. Their viral particles have the envelope composed of viral proteins and a lipid bilayer acquired from budding through the endoplasmic reticulum (ER). The phospholipid content of the ER membrane differs from that of the plasma membrane (PM). The phospholipase A (PLA) superfamily consists of a large number of members that specifically catalyse the hydrolysis of phospholipids at a particular position. Here we show that the CM-II isoform of secreted PLA obtained from Naja mossambica mossambica snake venom (CM-II-sPLA) possesses potent virucidal (neutralising) activity against HCV, DENV and JEV, with 50% inhibitory concentrations (IC) of 0.036, 0.31 and 1.34 ng/ml, respectively. In contrast, the IC values of CM-II-sPLA against viruses that bud through the PM (Sindbis virus, influenza virus and Sendai virus) or trans-Golgi network (TGN) (herpes simplex virus) were >10,000 ng/ml. Moreover, the 50% cytotoxic (CC) and haemolytic (HC) concentrations of CM-II-sPLA were >10,000 ng/ml, implying that CM-II-sPLA did not significantly damage the PM. These results suggest that CM-II-sPLA and its derivatives are good candidates for the development of broad-spectrum antiviral drugs that target viral envelope lipid bilayers derived from the ER membrane.
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http://dx.doi.org/10.1038/s41598-017-16130-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698466PMC
November 2017

Investigating the hepatitis B virus life cycle using engineered reporter hepatitis B viruses.

Cancer Sci 2018 Jan 7;109(1):241-249. Epub 2017 Dec 7.

Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan.

Chronic infection with hepatitis B virus (HBV) increases the risk of developing fibrosis, cirrhosis or hepatocellular carcinoma. Current therapies are limited to type-I interferons and/or nucleos(t)ide analogues; however, these are only partially effective. The development of novel anti-HBV agents for new treatment strategies has been hampered by the lack of a suitable system that allows the in vitro replication of HBV. Studies of virus infection/replication at the molecular level using wild-type HBV are labor-intensive and time-consuming. To overcome these problems, we previously constructed a recombinant reporter HBV bearing the NanoLuc gene and showed its usefulness in identifying factors that affect HBV proliferation. Because this system mimics the early stage of the HBV life cycle faithfully, we conducted a quantitative analysis of HBV infectivity to several human hepatocyte cell lines as well as the effect of dimethyl sulfoxide and HBV protein X on the early stage of HBV proliferation using this system. Furthermore, we developed a system to produce a reporter HBV expressing a pol gene. These reporter HBV may provide an opportunity to enhance our understanding of the HBV life cycle and aid strategies for the development of new anti-HBV agents.
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http://dx.doi.org/10.1111/cas.13440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765299PMC
January 2018

Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticle-associated toxicity leading to the safe and efficacious decrease in the hepatitis B virus.

J Control Release 2017 Nov 3;266:216-225. Epub 2017 Oct 3.

Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo 060-0812, Japan. Electronic address:

Lipid nanoparticles (LNPs) are the leading technology for delivering short interfering RNA (siRNA) in vivo. While numerous attempts to improve the efficiency of siRNA delivery have been reported, only a few studies of the mechanism of LNP-mediated toxicity and attempts to develop safe LNPs in vivo have been reported, in spite of the significance of such systems, in the light of treatment and clinical applications. We herein report on the elucidation of the mechanism of hepatotoxicity following the intravenous injection of a high dose of hepatotropic LNPs. The LNPs accumulated in liver sinusoidal endothelial cells (LSECs), resulting in their activation and the induction of several cytokines related to neutrophils, followed by neutrophilic inflammation. To circumvent this toxic cascade, the LNPs were modified with a hepatocyte-specific ligand, N-acetyl-d-galactosamine (GalNAc), which resulted in a substantial improvement of hepatocyte-specificity and in a dramatic reduction in toxicity. Moreover, modification of the GalNAc-LNPs with polyethyleneglycol abrogated the LNP-associated toxicity without any detectable loss of gene silencing activity in hepatocytes. Finally, we observed that a single injection of the LNPs resulted in a significant reduction of hepatitis B virus (HBV) genomic DNA and their antigens without any sign of toxicity in chimeric mice with humanized livers that had been persistently infected with HBV. These lines of the fact suggest that the newly designed siRNA-loaded LNPs promise to be a useful technology for the treatment of liver diseases.
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http://dx.doi.org/10.1016/j.jconrel.2017.09.044DOI Listing
November 2017

Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys.

Sci Rep 2017 09 20;7(1):12017. Epub 2017 Sep 20.

Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

Highly pathogenic avian influenza virus (HPAIV) is a serious threat not only to domestic fowls but also to humans. Vaccines inducing long-lasting immunity against HPAIV are required. In the present study, we generated recombinant measles virus (MV) expressing the hemagglutinin protein of HPAIV without the multibasic site necessary for its pathogenicity in chickens using the backbone of an MV vaccine strain (rMV-Ed-H5HA) or a wild-type MV-derived mutant (rMV-HL-Vko-H5HA). We examined protective efficacy of the candidate vaccines in the monkey infection model by the challenge with a HPAIV (H5N1). Cynomolgus monkeys inoculated with the candidate vaccines produced both anti-H5 HA and anti-MV antibodies. They recovered earlier from influenza symptoms than unvaccinated monkeys after the challenge with the HPAIV strain. Chest radiography and histopathological analyses confirmed less severe pneumonia in the vaccinated monkeys. Vaccination tended to suppress viral shedding and reduced the interleukin-6 levels in the lungs. Furthermore, the vaccination with rMV-Ed-H5HA of monkeys with pre-existing anti-MV immunity induced the production of anti-H5 HA antibodies. These results suggest that both candidate vaccines effectively reduce disease severity in naïve hosts, and that rMV-Ed-H5HA is a particularly good candidate vaccine against HPAIV infection.
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http://dx.doi.org/10.1038/s41598-017-08326-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607339PMC
September 2017

Oxidative Stress and Immune Responses During Hepatitis C Virus Infection in Tupaia belangeri.

Sci Rep 2017 08 29;7(1):9848. Epub 2017 Aug 29.

Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. To address the molecular basis of HCV pathogenesis using tupaias (Tupaia belangeri), we characterized host responses upon HCV infection. Adult tupaias were infected with HCV genotypes 1a, 1b, 2a, or 4a. Viral RNA, alanine aminotransferase, anti-HCV core and anti-nonstructural protein NS3 antibody titres, reactive oxygen species (ROS), and anti-3β-hydroxysterol-Δ24reductase (DHCR24) antibody levels were measured at 2-week intervals from 0 to 41 weeks postinfection. All HCV genotypes established infections and showed intermittent HCV propagation. Moreover, all tupaias produced anti-core and anti-NS3 antibodies. ROS levels in sera and livers were significantly increased, resulting in induction of DHCR24 antibody production. Similarly, lymphocytic infiltration, disturbance of hepatic cords, and initiation of fibrosis were observed in livers from HCV-infected tupaias. Intrahepatic levels of Toll-like receptors 3, 7, and 8 were significantly increased in all HCV-infected tupaias. However, interferon-β was only significantly upregulated in HCV1a- and HCV2a-infected tupaias, accompanied by downregulation of sodium taurocholate cotransporting polypeptide. Thus, our findings showed that humoral and innate immune responses to HCV infection, ROS induction, and subsequent increases in DHCR24 auto-antibody production occurred in our tupaia model, providing novel insights into understanding HCV pathogenesis.
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http://dx.doi.org/10.1038/s41598-017-10329-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575003PMC
August 2017

Safety, Tolerability, and Preliminary Efficacy of the Anti-Fibrotic Small Molecule PRI-724, a CBP/β-Catenin Inhibitor, in Patients with Hepatitis C Virus-related Cirrhosis: A Single-Center, Open-Label, Dose Escalation Phase 1 Trial.

EBioMedicine 2017 Sep 19;23:79-87. Epub 2017 Aug 19.

The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Chiba, Japan.

Background: There is currently no anti-fibrotic drug therapy available to treat hepatitis C virus (HCV) cirrhosis. The aim of this study was to assess the safety, tolerability, and anti-fibrotic effect of PRI-724, a small-molecule modulator of Wnt signaling, in patients with HCV cirrhosis.

Methods: In this single-center, open-label, phase 1 trial, we sequentially enrolled patients with HCV cirrhosis who were classified as Child-Pugh (CP) class A or B. PRI-724 was administered as a continuous intravenous infusion of 10, 40, or 160mg/m/day for six cycles of 1week on and 1week off. The primary endpoints were frequency and severity of adverse events. The secondary endpoint was efficacy of PRI-724 in treating cirrhosis based on CP score and liver biopsy. This study is registered with ClinicalTrials.gov (no. NCT02195440).

Findings: Between Sept 3, 2014 and May 2, 2016, 14 patients were enrolled: CP class A:B, 6:8; median age, 62 (range: 43 to 74) years; male:female, 10:4. Twelve of the 14 patients completed six cycles of treatment; one was withdrawn from the study due to possible study drug-related liver injury (grade 3) in the 160mg/m/day dose cohort and one withdrew for personal reasons. Serious adverse events occurred in three patients [21% (3/14)], one of which was possibly related to PRI-724. The most common adverse events were nausea [29% (4/14)] and fatigue [21% (3/14)]. After PRI-724 administration, the CP scores worsened by 1 point in two patients in the 10mg/m/day cohort, improved in three patients at 1, 1, and 2 points in the 40mg/m/day cohort, and improved in one patient by 3 points in the 160mg/m/day cohort. The histology activity index scores of the liver tissue improved in two patients and exacerbated in two patients in the 10mg/m/day cohort, and improved in one patient in the 40mg/m/day cohort.

Interpretation: This study showed that administration of 10 or 40mg/m/day intravenous PRI-724 over 12weeks was well-tolerated by patients with HCV cirrhosis; however, liver injury as a possible related serious adverse event was observed in the 160mg/m/day cohort.

Funding Source: AMED.
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http://dx.doi.org/10.1016/j.ebiom.2017.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605374PMC
September 2017

Efficient siRNA Delivery by Lipid Nanoparticles Modified with a Nonstandard Macrocyclic Peptide for EpCAM-Targeting.

Mol Pharm 2017 10 25;14(10):3290-3298. Epub 2017 Aug 25.

Faculty of Pharmaceutical Sciences, Hokkaido University , Hokkaido 060-0812, Japan.

The development of a specific, effective method for the delivery of therapeutics including small molecules and nucleic acids to tumor tissue remains to be solved. Numerous types of lipid nanoparticles (LNPs) have been developed in attempts to achieve this goal. However, LNPs are probably not taken up by target cells because cancer-targeting LNPs are typically modified with poly(ethylene glycol) (PEG), which inhibits the cellular uptake of LNPs, to passively accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. It would clearly be important to develop a LNP with both a prolonged circulation and cancer-specific efficient uptake for use in an innovative nanodrug delivery system. Herein, we assessed the effect of nonstandard macrocyclic peptides against the epithelial cell adhesion molecule (EpCAM) Epi-1, which was discovered by a random nonstandard peptides integrated discovery (RaPID) system, on the cellular uptake and therapeutics delivery of LNPs. A liposomal siRNA delivery system (MEND) was modified with an Epi-1 lipid-derivative (EpCAM-targeting MEND; ET-MEND). The resulting ET-MEND showed a more than 27-fold increase in cellular uptake in EpCAM-positive cell lines. In the case of negative cells, cellular uptake and the efficiency of the ET-MEND for delivering therapeutics were comparable with those of nonmodified MEND. In addition, when systemically injected, the ET-MEND successfully inhibited gene expression in the tumor tissue at a dose of 0.5 mg siRNA/kg without any obvious toxicity. These results suggest that a combination of a specific peptide ligand can be used to identify a RaPID system and that the use of such a MEND for liposomal drug delivery has the potential for use in developing a system for the efficacious delivery of pharmaceuticals to various cancer cells.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00362DOI Listing
October 2017

Interferon-β response is impaired by hepatitis B virus infection in Tupaia belangeri.

Virus Res 2017 06 25;237:47-57. Epub 2017 May 25.

Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan; Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan. Electronic address:

To date, the chimpanzee has been used as the natural infection model for hepatitis B virus (HBV). However, as this model is very costly and difficult to use because of ethical and animal welfare issues, we aimed to establish the tupaia (Tupaia belangeri) as a new model for HBV infection and characterized its intrahepatic innate immune response upon HBV infection. First, we compared the propagation of HBV genotypes A2 and C in vivo in tupaia hepatocytes. At 8-10days post infection (dpi), the level of HBV-A2 propagation in the tupaia liver was found to be higher than that of HBV-C. Abnormal architecture of liver cell cords and mitotic figures were also observed at 8 dpi with HBV-A2. Moreover, we found that HBV-A2 established chronic infection in some tupaias. We then aimed to characterize the intrahepatic innate immune response in this model. First, we infected six tupaias with HBV-A2 (strains JP1 and JP4). At 28 dpi, intrahepatic HBV-DNA and serum hepatitis B surface antigens (HBsAg) were detected in all tupaias. The levels of interferon (IFN)-β were found to be significantly suppressed in the three tupaias infected with HBV A2_JP4, while no significant change was observed in the three infected with HBV A2_JP1. Expression of toll-like receptor (TLR) 1 was suppressed, while that of TLR3 and TLR9 were induced, in HBV A2_JP1-infected tupaias. Expression of TLR8 was induced in all tupaias. Next, we infected nine tupaias with HBV-A2 (JP1, JP2, and JP4), and characterized the infected animals after 31 weeks. Serum HBsAg levels were detected at 31 weeks post-infection (wpi) and IFN-β was found to be significantly suppressed in all tupaias. TLR3 was not induced, except in tupaia #93 and #96. Suppression of TLR9 was observed in all tupaias, except tupaia #93. Also, we investigated the expression levels of cyclic GMP-AMP synthase, which was found to be induced in all tupaias at 28 dpi and in four tupaias at 31 wpi. Additionally, we evaluated the expression levels of sodium-taurocholate cotransporting polypeptide, which was found to be suppressed during chronic HBV infection. Thus, the tupaia infection model of HBV clearly indicated the suppression of IFN-β at 31 wpi, which might have contributed to the establishment of chronic HBV infection.
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http://dx.doi.org/10.1016/j.virusres.2017.05.013DOI Listing
June 2017
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