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五十川 正記イソガワ マサノリ

所属部署医学研究科ウイルス学分野
職名講師
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Last Updated :2020/06/02

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研究分野

  • ライフサイエンス, ウイルス学

論文

  • 7-Deaza-7-fluoro modification confers on 4'-cyano-nucleosides potent activity against entecavir/adefovir-resistant HBV variants and favorable safety., Sanae Hayashi, Nobuyo Higashi-Kuwata, Debananda Das, Kota Tomaya, Kohei Yamada, Shuko Murakami, David J Venzon, Shin-Ichiro Hattori, Masanori Isogawa, Stefan G Sarafianos, Hiroaki Mitsuya, Yasuhito Tanaka, Antiviral research, 176, 104744 - 104744,   2020年04月, 査読有り, We designed, synthesized and identified a novel nucleoside derivative, 4'-C-cyano-7-deaza-7-fluoro-2'-deoxyadenosine (CdFA), which exerts potent anti-HBV activity (IC50 ~26 nM) with favorable hepatocytotoxicity (CC50 ~56 μM). Southern blot analysis using wild-type HBV (HBVWT)-encoding-plasmid-transfected HepG2 cells revealed that CdFA efficiently suppresses the production of HBVWT (IC50 = 153.7 nM), entecavir (ETV)-resistant HBV carrying L180M/S202G/M204V substitutions (HBVETVR; IC50 = 373.2 nM), and adefovir dipivoxil (ADV)-resistant HBV carrying A181T/N236T substitutions (HBVADVR; IC50=192.6 nM), whereas ETV and ADV were less potent against HBVETVR and HBVADVR (IC50: >1,000 and 4,022.5 nM, respectively). Once-daily peroral administration of CdFA to human-liver-chimeric mice over 14 days (1 mg/kg/day) comparably blocked HBVWT and HBVETVR viremia by 0.7 and 1.2 logs, respectively, without significant changes in body-weight or serum human-albumin levels, although ETV only slightly suppressed HBVETVR viremia (CdFA vs ETV; p = 0.032). Molecular modeling suggested that ETV-TP has good nonpolar interactions with HBVWT reverse transcriptase (RTWT)'s Met204 and Asp205, while CdFA-TP fails to interact with Met204, in line with the relatively inferior activity against HBVWT of CdFA compared to ETV (IC50: 0.026 versus 0.003 nM). In contrast, the 4'-cyano of CdFA-TP forms good nonpolar contacts with RTWT's Leu180 and RTETVR's Met180, while ETV-TP loses interactions with RTETVR's Met180, explaining in part why ETV is less potent against HBVETVR than CdFA. The present results show that CdFA exerts potent activity against HBVWT, HBVETVR and HBVADVR with enhanced safety and that 7-deaza-7-fluoro modification confers potent activity against drug-resistant HBV variants and favorable safety, shedding light to further design more potent and safer anti-HBV nucleoside analogs.
  • Designing a therapeutic hepatitis B vaccine to circumvent immune tolerance, Human Vaccines & Immunotherapeutics,   2019年12月06日, 査読有り
  • The antiviral effects of human microRNA miR-302c-3p against hepatitis B virus infection, Hamada-Tsutsumi S, Naito Y, Sato S, Takaoka A, Kawashima K, Isogawa M, Ochiya T, Tanaka Y, Alimentary Pharmacology and Therapeutics,   2019年, 査読有り
  • Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production, Inoue J, Ninomiya M, Umetsu T, Nakamura T, Kogure T, Kakazu E, Iwata T, Takai S, Sano A, Fukuda M, Watashi K, Isogawa M, Tanaka Y, Shimosegawa T, McNiven M.A, Masamune A, Journal of virology, 93, (15) ,   2019年, 査読有り
  • Type I interferon signaling prevents hepatitis B virus-specific T cell responses by reducing antigen expression., Kawashima K, Isogawa M, Hamada-Tsutsumi S, Baudi I, Saito S, Nakajima A, Tanaka Y, Journal of virology, 92, (23) ,   2018年09月, 査読有り
  • Intrahepatic Cross-Presentation and Hepatocellular Antigen Presentation Play Distinct Roles in the Induction of HBV-Specific CD8+ T Cell Responses., Murata Y, Kawashima K, Sheikh K, Tanaka Y, Isogawa M, Journal of virology, 92, (21) ,   2018年08月, 査読有り
  • A novel association between core mutations in hepatitis B virus genotype F1b and hepatocellular carcinoma in Alaskan Native People., Hayashi S, Khan A, Simons BC, Homan C, Matsui T, Ogawa K, Kawashima K, Murakami S, Takahashi S, Isogawa M, Ikeo K, Mizokami M, McMahon BJ, Tanaka Y, Hepatology (Baltimore, Md.), 69, (1) 19 - 33,   2018年06月, 査読有り
  • Circulating let-7 levels in serum correlate with the severity of hepatic fibrosis in chronic hepatitis C, Matsuura K, Aizawa N, Enomoto H, Nishiguchi S, Toyoda H, Kumada T, Iio E, Ito K, Ogawa S, Isogawa M, Alter H.J, Tanaka Y, Open Forum Infectious Diseases, 5, (11) ,   2018年, 査読有り
  • How are HBV-specific CD8 + T-cell responses induced?, Isogawa M, Murata Y, Kawashima K, Tanaka Y, Future Virology, 13, (12) 825 - 827,   2018年, 査読有り
  • Genome-Wide Association Study Identifies TLL1 Variant Associated With Development of Hepatocellular Carcinoma After Eradication of Hepatitis C Virus Infection, Kentaro Matsuura, Hiromi Sawai, Kazuho Ikeo, Shintaro Ogawa, Etsuko Iio, Masanori Isogawa, Noritomo Shimada, Atsumasa Komori, Hidenori Toyoda, Takashi Kumada, Tadashi Namisaki, Hitoshi Yoshiji, Naoya Sakamoto, Mina Nakagawa, Yasuhiro Asahina, Masayuki Kurosaki, Namiki Izumi, Nobuyuki Enomoto, Atsunori Kusakabe, Eiji Kajiwara, Yoshito Itoh, Tatsuya Ide, Akihiro Tamori, Misako Matsubara, Norifumi Kawada, Ken Shirabe, Eiichi Tomita, Masao Honda, Shuichi Kaneko, Sohji Nishina, Atsushi Suetsugu, Yoichi Hiasa, Hisayoshi Watanabe, Takuya Genda, Isao Sakaida, Shuhei Nishiguchi, Koichi Takaguchi, Eiji Tanaka, Junichi Sugihara, Mitsuo Shimada, Yasuteru Kondo, Yosuke Kawai, Kaname Kojima, Masao Nagasaki, Katsushi Tokunaga, Yasuhito Tanaka, GASTROENTEROLOGY, 152, (6) 1383 - 1394,   2017年05月, 査読有り, BACKGROUND & AIMS: There is still a risk for hepatocellular carcinoma (HCC) development after eradication of hepatitis C virus (HCV) infection with antiviral agents. We investigated genetic factors associated with the development of HCC in patients with a sustained virologic response (SVR) to treatment for chronic HCV infection. METHODS: We obtained genomic DNA from 457 patients in Japan with a SVR to interferon-based treatment for chronic HCV infection from 2007 through 2015. We conducted a genome-wide association study (GWAS), followed by a replication analysis of 79 candidate single nucleotide polymorphisms (SNPs) in an independent set of 486 patients in Japan. The study end point was HCC diagnosis or confirmation of lack of HCC (at follow-up examinations until December 2014 in the GWAS cohort, and until January 2016 in the replication cohort). We collected clinical and laboratory data from all patients. We analyzed expression levels of candidate gene variants in human hepatic stellate cells, rats with steatohepatitis caused by a choline-deficient L-amino acid-defined diet, and a mouse model of liver injury caused by administration of carbon tetrachloride. We also analyzed expression levels in liver tissues of patients with chronic HCV infection with different stages of fibrosis or tumors vs patients without HCV infection (controls). RESULTS: We found a strong association between the SNP rs17047200, located within the intron of the tolloid like 1 gene (TLL1) on chromosome 4, and development of HCC; there was a genome-wide level of significance when the results of the GWAS and replication study were combined (odds ratio, 2.37; P = 2.66 x 10(-8)). Multivariate analysis showed rs17047200 AT/TT to be an independent risk factor for HCC (hazard ratio, 1.78; P = .008), along with male sex, older age, lower level of albumin, advanced stage of hepatic fibrosis, presence of diabetes, and higher post-treatment level of alpha-fetoprotein. Combining the rs17047200 genotype with other factors, we developed prediction models for HCC development in patients with mild or advanced hepatic fibrosis. Levels of TLL1 messenger RNA (mRNA) in human hepatic stellate cells increased with activation. Levels of Tll1 mRNA increased in liver tissues of rodents with hepatic fibrogenesis compared with controls. Levels of TLL1 mRNA increased in liver tissues of patients with progression of fibrosis. Gene expression levels of TLL1 short variants, including isoform 2, were higher in patients with rs17047200 AT/TT. CONCLUSIONS: In a GWAS, we identified the association between the SNP rs17047200, within the intron of TLL1, and development of HCC in patients who achieved an SVR to treatment for chronic HCV infection. We found levels of Tll1/TLL1 mRNA to be increased in rodent models of liver injury and liver tissues of patients with fibrosis, compared with controls. We propose that this SNP might affect splicing of TLL1 mRNA, yielding short variants with high catalytic activity that accelerates hepatic fibrogenesis and carcinogenesis. Further studies are needed to determine how rs17047200 affects TLL1 mRNA levels, splicing, and translation, as well as the prevalence of this variant among other patients with HCC. Tests for the TLL1 SNP might be used to identify patients at risk for HCC after an SVR to treatment of HCV infection.
  • Genome-Wide Association Study Identifies TLL1 Variant Associated With Development of Hepatocellular Carcinoma After Eradication of Hepatitis C Virus Infection., Kentaro Matsuura, Hiromi Sawai, Kazuho Ikeo, Shintaro Ogawa, Etsuko Iio, Masanori Isogawa, Noritomo Shimada, Atsumasa Komori, Hidenori Toyoda, Takashi Kumada, Tadashi Namisaki, Hitoshi Yoshiji, Naoya Sakamoto, Mina Nakagawa, Yasuhiro Asahina, Masayuki Kurosaki, Namiki Izumi, Nobuyuki Enomoto, Atsunori Kusakabe, Eiji Kajiwara, Yoshito Itoh, Tatsuya Ide, Akihiro Tamori, Misako Matsubara, Norifumi Kawada, Ken Shirabe, Eiichi Tomita, Masao Honda, Shuichi Kaneko, Sohji Nishina, Atsushi Suetsugu, Yoichi Hiasa, Hisayoshi Watanabe, Takuya Genda, Isao Sakaida, Shuhei Nishiguchi, Koichi Takaguchi, Eiji Tanaka, Junichi Sugihara, Mitsuo Shimada, Yasuteru Kondo, Yosuke Kawai, Kaname Kojima, Masao Nagasaki, Katsushi Tokunaga, Yasuhito Tanaka, Gastroenterology, 152, (6) 1383 - 1394,   2017年05月, 査読有り
  • A New Class of Hepatitis B and D Virus Entry Inhibitors, Proanthocyanidin and Its Analogs, That Directly Act on the Viral Large Surface Proteins, Senko Tsukuda, Koichi Watashi, Taichi Hojima, Masanori Isogawa, Masashi Iwamoto, Katsumi Omagari, Ryosuke Suzuki, Hideki Aizaki, Soichi Kojima, Masaya Sugiyama, Akiko Saito, Yasuhito Tanaka, Masashi Mizokami, Camille Sureau, Takaji Wakita, HEPATOLOGY, 65, (4) 1104 - 1116,   2017年04月, 査読有り, Introduction of direct-acting antivirals against hepatitis C virus (HCV) has provided a revolutionary improvement in the treatment outcome. In contrast to HCV, however, the strategy for developing new antiviral agents against hepatitis B virus (HBV), especially viral-targeting compounds, is limited because HBV requires only four viral genes for its efficient replication/ infection. Here, we identify an oligomeric flavonoid, proanthocyanidin (PAC) and its analogs, which inhibit HBV entry into host cells by targeting the HBV large surface protein (LHBs). Through cell-based chemical screening, PAC was identified to inhibit HBV infection with little cytotoxic effect. PAC prevented the attachment of the preS1 region in the LHBs to its cellular receptor, sodium taurocholate cotransporting polypeptide (NTCP). PAC was shown to target HBV particles and impair their infectivity, whereas it did not affect the NTCP-mediated bile acid transport activity. Chemical biological techniques demonstrated that PAC directly interacted with the region essential for receptor binding in the preS1 region in the LHBs protein. Importantly, PAC had a pan-genotypic anti-HBV activity and was also effective against a clinically relevant nucleoside analog-resistant HBV isolate. We further showed that PAC augmented the ability of a nucleoside analog, tenofovir, to interrupt HBV spread over time in primary human hepatocytes by cotreatment. Moreover, derivative analysis could identify small molecules that demonstrated more-potent anti-HBV activity over PAC. Conclusion: PAC and its analogs represent a new class of anti-HBV agents that directly target the preS1 region of the HBV large surface protein. These agents could contribute to the development of a potent, well-tolerated, and broadly active inhibitor of HBV infection.
  • Characteristics of escape mutations from occult hepatitis B virus infected patients with hematological malignancies in South Egypt., Elkady A, Iijima S, Aboulfotuh S, Mostafa Ali E, Sayed D, Abdel-Aziz NM, Ali AM, Murakami S, Isogawa M, Tanaka Y, World journal of hepatology, 9, (9) 477 - 486,   2017年03月, 査読有り
  • Molecular epidemiology of co-infection with hepatitis B virus and human immunodeficiency virus (HIV) among adult patients in Harare, Zimbabwe, Ian Baudi, Sayuki Iijima, Nyasha Chin'ombe, Sekesai Mtapuri-Zinyowera, Shuko Murakami, Masanori Isogawa, Atsuko Hachiya, Yasumasa Iwatani, Yasuhito Tanaka, JOURNAL OF MEDICAL VIROLOGY, 89, (2) 257 - 266,   2017年02月, 査読有り, core promoter/precore (BCP/PC) and S regions of HBV, as wel The objective of this study was to determine the prevalence of co-infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV) and the genetic characteristics of both viruses among pre-HIV-treatment patients in Harare, Zimbabwe. This cross-sectional survey involved 176 remnant plasma samples collected from consenting HIV patients (median age 35 [18-74]) between June and September 2014. HBV seromarkers were determined by high-sensitivity chemiluminescence assays. Molecular evolutionary analyses were conducted on the basall as part of the HIV pol region. Of the 176 participants (65.7% female), 19 (10.8%) were positive for HBsAg (median 0.033IU/ml (IQR 0.01-415). The HBsAg incidence was higher in men than women (P=0.009). HBsAg-positive subjects had lower median CD4 counts (P=0.016). HBV DNA was detectable in 12 HBsAg-positive samples (median 3.36 log cp/ml (2.86-4.51), seven being amplified and sequenced. All isolates were subgenotype A1 without HBV drug resistance mutations but each had at least one BCP/PC mutation. PreS deletion mutants and small S antigen variants M133I/T and D144G were identified. Of the 164 HIV isolates successfully genotyped, 163 (99.4%) were HIV-1 subtype C and only one was HIV-1 subtype F1. Sixteen (9.8%) had at least one drug resistance mutation, predominantly non-nucleoside reverse transcriptase inhibitor-related mutations, observed mostly among female participants. This study shows that co-infection with HBV is present among HIV patients enrolling into HIV care in Zimbabwe, suggesting that HBV screening and monitoring programmes be strengthened in this context. J. Med. Virol. 89:257-266, 2017. (c) 2016 Wiley Periodicals, Inc.
  • Erratum: Correction: CD40 Activation Rescues Antiviral CD8+ T Cells from PD-1-Mediated Exhaustion (PLoS pathogens (2013) 9 7 (e1003490)), Isogawa M, Chung J, Murata Y, Kakimi K, Chisari F.V, PLoS pathogens, 13, (5) ,   2017年, 査読有り
  • B型肝炎治療不応例における新規Entecavir耐性変異(rtA186T)の同定, 林 佐奈衣, 村上 周子, 尾曲 克己, 松居 剛志, 飯尾 悦子, 五十川 正記, 渡邊 綱正, 狩野 吉康, 田中 靖人, Nagoya Medical Journal, 55, (1) 35 - 42,   2016年06月, B型肝炎ウイルス(HBV)の持続感染者に対する推奨薬である核酸アナログ(NAs;nucleos(t)ide analogues)は、HBVの逆転写酵素(rt;reverse transcriptase)を阻害しHBV複製を抑制する有用な治療薬だが、多剤耐性株の出現による治療不応が懸念される。本研究ではラミブジン(LAM;lamivudine)からエンテカビル(ETV;entecavir)治療へ切り替え後、viral breakthrough(VBT)を発症したB型慢性肝炎症例に対して、ETV治療前と治療中に含まれる血中HBV遺伝子を解析した結果、VBT時に既知LAM耐性変異(rtL180M/M204V)および新規ETV耐性変異(rtI163V/A186T)を同定した。1.24倍長HBV genotype Ceクローンのポリメラーゼrt領域に対して上述の各変異を挿入し、ETVに対する感受性評価とHBV複製効率を解析した。その結果、I163V/A186T/LAMr、A186T/LAMrは既知ETV耐性株(S202G/LAMr)と同程度のETV耐性能を示した。また、I163V/A186T/LAMr、A186T/LAMrの複製効率は野生株より有意に低値を示したが、163/LAMrと野生株に差はみられなかった。VBT時の患者血清をヒト肝細胞キメラマウスへ感染させ、ETV感受性試験とHBV株含有率の変化を次世代シーケンサーを用いて解析した。その結果、ETV投与前/中/後の全経過でI163V/A186T/LAMrが優勢であり、ETV耐性能を示した。3D docking simulationを用いて逆転写酵素とETVの結合性評価を行った結果、A186T/LAMrはETV結合能が低下する可能性が示唆された。以上より、VBTを発症したETV不応患者より新規変異(rtI163V/A186T)を世界で初めて同定し、このrtA186T変異はETV耐性であることを証明した。(著者抄録)
  • Host Genetic Variants Influencing the Clinical Course of Hepatitis B Virus Infection, Kentaro Matsuura, Masanori Isogawa, Yasuhito Tanaka, JOURNAL OF MEDICAL VIROLOGY, 88, (3) 371 - 379,   2016年03月, 査読有り, The clinical course of hepatitis B virus (HBV) infection greatly differs in individuals. Various viral, host, and environmental factors influence the natural history of HBV infection. Recent genome-wide association studies identified several host genetic factors influencing the clinical course of HBV infection. Genetic variations in HLA class II loci were significantly associated with susceptibility to persistent HBV infection. Other polymorphisms in or near the genes EHMT2, TCF19, and HLA-C, located near HLA class II loci, and UBE2L3 were also associated with persistent HBV infection. Meanwhile, polymorphisms in KIF1B, GRIK1, and STAT4 were associated with HBV-related hepatocellular carcinoma (HCC). Interestingly, HLA class II genetic variations were strongly associated with not only persistent HBV infection, but also disease progression and HBV-related HCC in chronic hepatitis B. Understanding the various genetic factors associated with the clinical course of HBV infection is essential for personalized treatment and surveillance of disease progression and HCC. (C) 2015 Wiley Periodicals, Inc.
  • Novel 4'-modified nucleoside analogs exert antiviral replication against hepatitis B virus with drug resistance mutations, Hayashi S, Murakami S, Omagari K, Matsui T, Isogawa M, Watanabe T, Tanaka Y, Takamatsu Y, Maeda K, Mitsuya H, Maeda K, Kohgo S, Mitsuya H, Matsui T, Karino Y, Kohgo S, Mitsuya H, Acta Hepatologica Japonica, 57, (6) 299 - 301,   2016年, 査読有り
  • [Immune responses to HBV infection]., Isogawa M, Tanaka Y, Nihon rinsho. Japanese journal of clinical medicine, 73 Suppl 9, 397 - 401,   2015年12月, 査読有り
  • Host-virus interactions in hepatitis B virus infection., Guidotti LG, Isogawa M, Chisari FV, Current opinion in immunology, 36, 61 - 66,   2015年10月, 査読有り
  • Characterization of novel entecavir resistance mutations., Hayashi S, Murakami S, Omagari K, Matsui T, Iio E, Isogawa M, Watanabe T, Karino Y, Tanaka Y, Journal of hepatology, 63, (3) 546 - 553,   2015年09月, 査読有り
  • Immunosurveillance of the liver by intravascular effector CD8(+) T cells., Guidotti LG, Inverso D, Sironi L, Di Lucia P, Fioravanti J, Ganzer L, Fiocchi A, Vacca M, Aiolfi R, Sammicheli S, Mainetti M, Cataudella T, Raimondi A, Gonzalez-Aseguinolaza G, Protzer U, Ruggeri ZM, Chisari FV, Isogawa M, Sitia G, Iannacone M, Cell, 161, (3) 486 - 500,   2015年04月, 査読有り
  • Immune responses to HBV infection, Isogawa M, Tanaka Y, Nihon rinsho. Japanese journal of clinical medicine, 73, 397 - 401,   2015年, 査読有り
  • Immunobiology of hepatitis B virus infection., Isogawa M, Tanaka Y, Hepatology research : the official journal of the Japan Society of Hepatology, 45, (2) 179 - 189,   2015年01月, 査読有り
  • Validation of cross-genotype neutralization by hepatitis B virus-specific monoclonal antibodies by in vitro and in vivo infection., Hamada-Tsutsumi S, Iio E, Watanabe T, Murakami S, Isogawa M, Iijima S, Inoue T, Matsunami K, Tajiri K, Ozawa T, Kishi H, Muraguchi A, Joh T, Tanaka Y, PloS one, 10, (2) ,   2015年, 査読有り
  • CD40 activation rescues antiviral CD8⁺ T cells from PD-1-mediated exhaustion., Isogawa M, Chung J, Murata Y, Kakimi K, Chisari FV, PLoS pathogens, 9, (7) ,   2013年, 査読有り
  • Kupffer Cells Hasten Resolution of Liver Immunopathology in Mouse Models of Viral Hepatitis, Giovanni Sitia, Matteo Iannacone, Roberto Aiolfi, Masanori Isogawa, Nico van Rooijen, Cristina Scozzesi, Marco E. Bianchi, Ulrich H. von Andrian, Francis V. Chisari, Luca G. Guidotti, PLOS PATHOGENS, 7, (6) ,   2011年06月, 査読有り, Kupffer cells (KCs) are widely considered important contributors to liver injury during viral hepatitis due to their proinflammatory activity. Herein we utilized hepatitis B virus (HBV)-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1) protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology.
  • Pathogenesis of hepatitis B virus infection,Pathogenèse de l'infection par le virus de l'hépatite B, Chisari F.V, Isogawa M, Wieland S.F, Pathologie Biologie, 58, (4) 258 - 266,   2010年08月, 査読有り
  • Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection, Ken Takahashi, Shinichi Asabe, Stefan Wieland, Urtzi Garaigorta, Pablo Gastaminza, Masanori Isogawa, Francis V. Chisari, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107, (16) 7431 - 7436,   2010年04月, 査読有り, Hepatitis C virus (HCV), a member of the Flaviviridae family, is a single-stranded positive-sense RNA virus that infects > 170 million people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite its ability to block the innate host response in infected hepatocyte cell lines in vitro, HCV induces a strong type 1 interferon (IFN) response in the infected liver. The source of IFN in vivo and how it is induced are currently undefined. Here we report that HCV-infected cells trigger a robust IFN response in plasmacytoid dendritic cells (pDCs) by a mechanism that requires active viral replication, direct cell-cell contact, and Toll-like receptor 7 signaling, and we show that the activated pDC supernatant inhibits HCV infection in an IFN receptor-dependent manner. Importantly, the same events are triggered by HCV subgenomic replicon cells but not by free virus particles, suggesting the existence of a novel cell-cell RNA transfer process whereby HCV-infected cells can activate pDCs to produce IFN without infecting them. These results may explain how HCV induces IFN production in the liver, and they reveal a heretofore unsuspected aspect of the innate host response to viruses that can subvert the classical sensing machinery in the cells they infect, and do not infect or directly activate pDCs.
  • Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment, Karl-Dimiter Bissig, Stefan F. Wieland, Phu Tran, Masanori Isogawa, Tam T. Le, Francis V. Chisari, Inder M. Verma, JOURNAL OF CLINICAL INVESTIGATION, 120, (3) 924 - 930,   2010年03月, 査読有り, A paucity of versatile small animal models of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has been an impediment to both furthering understanding of virus biology and testing antiviral therapies. We recently described a regulatable system for repopulating the liver of immunodeficient mice (specifically mice lacking fumaryl. acetoacetate hydrolase [Fah], recombination activating gene 2 [Rag2], and the gamma-chain of the receptor for IL-2 [Il-2r gamma]) with human hepatocytes. Here we have shown that a high transplantation dose (3 x 10(6) to 5 x 10(6) human hepatocytes/mouse) generates a higher rate of liver chimerism than was previously obtained in these mice, up to 95% human-hepatocyte chimerism. Mice with a high level. of human liver chimerism propagated both HBV and HCV, and the HCV-infected mice were responsive to antiviral treatment. This human liver chimeric mouse model will expand the experimental possibilities for studying HBV and HCV infection, and possibly other human hepatotropic pathogens, and prove useful for antiviral drug testing.
  • Immune effectors required for hepatitis B virus clearance, Priscilla L. Yang, Alana Althage, Josan Chung, Holly Maier, Stefan Wieland, Masanori Isogawa, Francis V. Chisari, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107, (2) 798 - 802,   2010年01月, 査読有り, To better define the mechanism(s) likely responsible for viral clearance during hepatitis B virus (HBV) infection, viral clearance was studied in a panel of immunodeficient mouse strains that were hydrodynamically transfected with a plasmid containing a replication-competent copy of the HBV genome. Neither B cells nor perforin were required to clear the viral DNA transcriptional template from the liver. In contrast, the template persisted for at least 60 days at high levels in NOD/Scid mice and at lower levels in the absence of CD4(+) and CD8(+) T cells, NK cells, Fas, IFN-gamma (IFN-gamma), IFN-alpha/beta receptor (IFN-alpha/beta R1), and TNF receptor 1 (TNFR1), indicating that each of these effectors was required to eliminate the transcriptional template from the liver. Interestingly, viral replication was ultimately terminated in all lineages except the NOD/Scid mice, suggesting the existence of redundant pathways that inhibit HBV replication. Finally, induction of a CD8(+) T cell response in these animals depended on the presence of CD4(+) T cells. These results are consistent with a model in which CD4(+) T cells serve as master regulators of the adaptive immune response to HBV; CD8(+) T cells are the key cellular effectors mediating HBV clearance from the liver, apparently by a Fas-dependent, perforin-independent process in which NK cells, IFN-gamma, TNFR1, and IFN-alpha/beta R play supporting roles. These results provide insight into the complexity of the systems involved in HBV clearance, and they suggest unique directions for analysis of the mechanism(s) responsible for HBV persistence.
  • A virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis C virus infection in vitro., Cheng G, Montero A, Gastaminza P, Whitten-Bauer C, Wieland SF, Isogawa M, Fredericksen B, Selvarajah S, Gallay PA, Ghadiri MR, Chisari FV, Proceedings of the National Academy of Sciences of the United States of America, 105, (8) 3088 - 3093,   2008年02月, 査読有り
  • Platelets prevent IFN-alpha/beta-induced lethal hemorrhage promoting CTL-dependent clearance of lymphocytic choriomeningitis virus., Iannacone M, Sitia G, Isogawa M, Whitmire JK, Marchese P, Chisari FV, Ruggeri ZM, Guidotti LG, Proceedings of the National Academy of Sciences of the United States of America, 105, (2) 629 - 634,   2008年01月, 査読有り
  • PD-1 : PD-L1 interactions contribute to the functional suppression of virus-specific CD8(+) T lymphocytes in the liver, Holly Maier, Masanori Isogawa, Gordon J. Freeman, Francis V. Chisari, JOURNAL OF IMMUNOLOGY, 178, (5) 2714 - 2720,   2007年03月, 査読有り, Mechanisms contributing to the development of chronic viral infections, including chronic hepatitis B virus (HBV) infections, are not well understood. We have shown recently that production of IFN-gamma, an important antiviral cytokine, by HBV-specific CTLs is rapidly induced when they enter the liver of HBV transgenic mice, and then rapidly suppressed, despite the continued presence of Ag. Suppression of IFN-gamma production by the CTLs coincides with the up-regulation of programmed cell death (PD)-1, a cell surface signaling molecule known to inhibit T cell function. To determine whether PD-1 plays a role in the functional suppression of IFN-gamma secretion by CTLs, we treated HBV transgenic mice with blocking Abs specific for PD ligand (PD-L)1, the most widely expressed PD-1 ligand, and adoptively transferred HBV-specific CTLs. Treatment with anti-PD-L1 Abs resulted in a delay in the suppression of IFN-gamma-producing CTLs and a concomitant increase in the absolute number of IFN-gamma-producing CTLs in the liver. These results indicate that PD-1:PD-L1 interactions contribute to the suppression of IFN-gamma secretion observed following Ag recognition in the liver. Blockade of inhibitory pathways such as PD-1:PD-L1 may reverse viral persistence and chronic infection in cases in which the CTL response is suppressed by this mechanism.
  • PD-1:PD-L1 interactions contribute to the functional suppression of virus-specific CD8+ T lymphocytes in the liver., Maier H, Isogawa M, Freeman GJ, Chisari FV, Journal of immunology (Baltimore, Md. : 1950), 178, (5) 2714 - 2720,   2007年03月, 査読有り
  • Persistent hepatitis C virus infection in vitro: Coevolution of virus and host, Jin Zhong, Pablo Gastaminza, Josan Chung, Zania Stamataki, Masanori Isogawa, Guofeng Cheng, Jane A. McKeating, Francis V. Chisari, JOURNAL OF VIROLOGY, 80, (22) 11082 - 11093,   2006年11月, 査読有り, The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host.
  • Platelets mediate cytotoxic T lymphocyte-induced liver damage, M Iannacone, G Sitia, M Isogawa, P Marchese, MG Castro, PR Lowenstein, FV Chisari, ZM Ruggeri, LG Guidotti, NATURE MEDICINE, 11, (11) 1167 - 1169,   2005年11月, 査読有り, We found that platelet depletion reduces intrahepatic accumulation of virus-specific cytotoxic T lymphocytes ( CTLs) and organ damage in mouse models of acute viral hepatitis. Transfusion of normal but not activation-blocked platelets in platelet-depleted mice restored accumulation of CTLs and severity of disease. In contrast, anticoagulant treatment that prevented intrahepatic fibrin deposition without reducing platelet counts did not avert liver injury. Thus, activated platelets contribute to CTL-mediated liver immunopathology independently of procoagulant function.
  • Oscillating CD8(+) T cell effector functions after antigen recognition in the liver, M Isogawa, Y Furuichi, FV Chisari, IMMUNITY, 23, (1) 53 - 63,   2005年07月, 査読有り, When hepatitis B virus (HBV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) are adoptively transferred into HBV transgenic mice, they enter the liver, recognize antigen, secrete interferon gamma (IFN gamma), inhibit viral replication, and kill their target cells, causing hepatitis. In the current study, we examined the impact of antigen recognition on the evolution of the activation phenotype, antiviral effector functions, expansion and contraction kinetics, and compartmentalization of the transferred CTLs. The results reveal that noncytolytic and cytolytic effector functions and expansion-contraction kinetics of the CTLs are regulated asynchronously and in an oscillatory manner as a consequence of antigen recognition in the liver and in association with PD-1 upregulation. We suggest that such oscillations maximize viral clearance and minimize tissue injury during HBV infection and that poor coordination of these events could lead to viral persistence and chronic liver disease.
  • Toll-like receptor signaling inhibits hepatitis B virus replication in vivo, M Isogawa, MD Robek, Y Furuichi, FV Chisari, JOURNAL OF VIROLOGY, 79, (11) 7269 - 7272,   2005年06月, 査読有り, Toll-like receptors (TLR) play a key role in innate immunity. To examine the ability of diverse TLRs to modulate hepatitis B virus (HBV) replication, HBV transgenic mice received a single intravenous injection of ligands specific for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9. All of the ligands except for TLR2 inhibited HBV replication in the liver noncytopathically within 24 It in a α/β interferon-dependent manner. The ability of these TLR ligands to induce antiviral cytokines at the site of HBV replication suggests that TLR activation could represent a powerful and novel therapeutic strategy for the treatment of chronic HBV infection.
  • Immunization with the gene expressing woodchuck hepatitis virus nucleocapsid protein fused to cytotoxic-T-lymphocyte-associated antigen 4 leads to enhanced specific immune responses in mice and woodchucks, M Lu, M Isogawa, Y Xu, G Hilken, JOURNAL OF VIROLOGY, 79, (10) 6368 - 6376,   2005年05月, 査読有り, A number of options are available to modify and improve DNA vaccines. An interesting approach to improve DNA vaccines is to fuse bioactive domains, like cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), to an antigen. Such fusion antigens are expressed in vivo and directed to immune cells by the specific bioactive domain and therefore possess great potential to induce and modulate antigen-specific immune responses. In the present study, we tested this new approach for immunomodulation against hepadnavirus infection in the woodchuck model. Plasmids expressing the nucleocapsid protein (WHcAg) and e antigen (WHeAg) of woodchuck hepatitis virus (WHV) alone or in fusion to the extracellular domain of woodchuck CTLA-4 and CD28 were constructed. Immunizations of mice with plasmids expressing WHcAg or WHeAg led to a specific immunoglobulin G2a (IgG2a) -dominant antibody response. In contrast, fusions of WHcAg to CTLA-4 and CD28 induced a specific antibody response with comparable levels of IgG1 and IgG2a. Furthermore, the specific IgG1 response to WHcAg/WHeAg developed immediately after a single immunization with the CTLA-4-WHcAg fusion. Woodchucks were immunized with plasmids expressing WIIeAg or the CTLA-4-WHcAg fusion and subsequently challenged with WHV. CTLA-4-WHcAg showed an improved efficacy in induction of protective immune responses to WHV. In particular, the anti-WHsAg antibody response developed earlier after challenge in woodchucks that received immunizations with CTLA-4-WHcAg, consistent with the hypothesis that anti-WHs response is dependent on a Th cell response to WHcAg. In conclusion, the use of fusion genes represents a generally applicable strategy to improve DNA vaccination.
  • Differential dynamics of the peripheral and intrahepatic cytotoxic T lymphocyte response to hepatitis B surface antigen, M Isogawa, K Kakimi, H Kamamoto, U Protzer, FV Chisari, VIROLOGY, 333, (2) 293 - 300,   2005年03月, 査読有り, The distribution and dynamics of the cytotoxic T lymphocyte (CTL) response to hepatitis B surface antigen (HBsAg) were studied in mice after intramuscular DNA immunization and after hepatic infection by a recombinant adenovirus that expresses the hepatitis B virus genome (Ad-HBV). CTLs specific for HBsAg accumulate preferentially in the spleen after DNA immunization but are primarily intrahepatic after Ad-HBV infection. The secondary CTL response to Ad-HBV in DNA-primed mice is characterized by rapid depletion of effector CTLs from the spleen, and their expansion in the liver where they cause hepatitis, secrete interferon gamma (IFNgamma), and inhibit HBV gene expression. Suppression of HBsAg synthesis is accompanied by disappearance of intrahepatic IFNgamma-producing CTLs and their reaccumulation in the spleen. The data Suggest a possible explanation for the paucity and functional deficiency of HBV-specific CTLs in the periphery during chronic HBV infection, and that the severity of infection can be worsened by a preexisting CTL response if neutralizing antibody is not also present. (C) 2005 Elsevier Inc. All rights reserved.
  • Mutant hepatitis B virus surface antigens (HBsAg) are immunogenic but may have a changed specificity, Zheng, X, KM Weinberger, R Gehrke, M Isogawa, G Hilken, T Kemper, Y Xu, DL Yang, W Jilg, M Roggendorf, MJ Lu, VIROLOGY, 329, (2) 454 - 464,   2004年11月, 査読有り, Mutant hepatitis B virus with substitutions within the coding region for HBV surface antigen (HBsAg) has been found naturally in chronic carriers. It is therefore important to clarify whether the identified substitutions within the HBsAg have impact on the antigenicity and immunogenicity of HBsAg. A total of nine mutated HBV s-genes with single representative mutations were generated by site-directed mutagenesis and subcloned into an expression vector. The binding of polyclonal and monoclonal antibodies to these mutant HBsAg (mtHBsAg) was tested by immunofluorescence (IF) staining of cells transfected with the expression vectors. The amino acid (aa) substitutions like G145R, F134S, and C147W affected the binding of anti-HBs antibodies to corresponding mtHBsAg to different extents. The impact of aa substitutions G145R and F134S on the immunogenicity was accessed by genetic immunization of mice with vectors expressing middle HBsAg with the corresponding mutations. The immunized mice developed antibodies to recombinant HBsAg containing the HBV preS region and HBsAg-specific cytotoxic T-cell. However, the development of antibody response to wild-type small HBsAg was significantly impaired by the aa substitutions in HBsAg. Based on this fact, we further investigated whether the mtHBsAg with the aa substitution G145R is able to induce mutant-specific antibody responses. Strikingly, serum samples from mice immunized with mtHBsAg with G145R recognized plasma-derived mtHBsAg. Two mouse MAbs specific to mtHBsAg were generated. One MAb recognized mtHBsAg with G145R but not wild type and other mtHBsAg. We conclude that HBsAg with aa substitutions are immunogenic but may have a changed fine specificity. (C) 2004 Elsevier Inc. All rights reserved.
  • MMPs are required for recruitment of antigen-nonspecific mononuclear cells into the liver by CTLs, G Sitia, M Isogawa, M Iannacone, IL Campbell, FV Chisari, LG Guidotti, JOURNAL OF CLINICAL INVESTIGATION, 113, (8) 1158 - 1167,   2004年04月, 査読有り, We recently showed that andgen-nonspecific inflammatory cells are recruited into the liver when hepatitis B virus (HBV)-specific CTLs are injected into HBV transgenic mice, and that this process amplifies the severity of liver disease. We also showed that the severity of CTL-induced liver disease is ameliorated by the depletion of Gr-1(+) cells (Gr-1 is an antigen highly expressed by neutrophils), which, secondarily, abolishes the intrahepatic recruitment of all andgen-nonspecific Gr-1(-) mononuclear cells (NK and NKT cells, T and B lymphocytes, monocytes, macrophages, dendritic cells) despite the strong induction of chemokine gene expression. Those results suggested that in addition to chemokine expression, CTL-induced functions are necessary for mononuclear cell recruitment to occur. We now report that MMPs known to be produced by Gr-1(+) cells are rapidly induced in the livers of CTL-injected mice. The inhibition of MMP activity reduced the intrahepatic recruitment of andgen-nonspecific mononuclear cells and much of the attending liver disease without affecting the migration or antiviral potential of antigen-specific CTLs. The notion that the inhibition of MMP activity is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.
  • MMPs are required for recruitment of antigen-nonspecific mononuclear cells into the liver by CTLs, G Sitia, M Isogawa, M Iannacone, IL Campbell, FV Chisari, LG Guidotti, JOURNAL OF CLINICAL INVESTIGATION, 113, (8) 1158 - 1167,   2004年04月, 査読有り, We recently showed that andgen-nonspecific inflammatory cells are recruited into the liver when hepatitis B virus (HBV)-specific CTLs are injected into HBV transgenic mice, and that this process amplifies the severity of liver disease. We also showed that the severity of CTL-induced liver disease is ameliorated by the depletion of Gr-1(+) cells (Gr-1 is an antigen highly expressed by neutrophils), which, secondarily, abolishes the intrahepatic recruitment of all andgen-nonspecific Gr-1(-) mononuclear cells (NK and NKT cells, T and B lymphocytes, monocytes, macrophages, dendritic cells) despite the strong induction of chemokine gene expression. Those results suggested that in addition to chemokine expression, CTL-induced functions are necessary for mononuclear cell recruitment to occur. We now report that MMPs known to be produced by Gr-1(+) cells are rapidly induced in the livers of CTL-injected mice. The inhibition of MMP activity reduced the intrahepatic recruitment of andgen-nonspecific mononuclear cells and much of the attending liver disease without affecting the migration or antiviral potential of antigen-specific CTLs. The notion that the inhibition of MMP activity is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.
  • Depletion of neutrophils blocks the recruitment of antigen-nonspecific cells into the liver without affecting the antiviral activity of hepatitis B virus-specific cytotoxic T lymphocytes, G Sitia, M Isogawa, K Kakimi, SF Wieland, FV Chisari, LG Guidotti, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 99, (21) 13717 - 13722,   2002年10月, 査読有り, Using transgenic mice that replicate hepatitis B virus (HBV) in their livers, we previously showed that passively transferred HBV-specific cytotoxic T cells (CTLs) recruit antigen-nonspecific lymphomononuclear and polymorphonuclear inflammatory cells that contribute to the pathogenesis of liver disease. This process is chemokine-dependent, because we recently showed that blocking the chemokines CXCL9 and CXCL10 reduces the recruitment of antigen-nonspecific lymphomononuclear cells and the severity of liver disease after CTL injection. In the current study we show that the severity of the CTL-initiated liver disease is also ameliorated by the depletion of neutrophils. Interestingly, depletion of neutrophils does not affect the intrahepatic migration or antiviral activity of CTLs, but it profoundly inhibits the recruitment of all antigen-nonspecific cells into the liver. This effect occurs in face of high intrahepatic levels of chemokine gene expression, suggesting that neutrophil-dependent functions other than chemokine induction are necessary for the recruitment process to occur. The notion that depletion of neutrophils is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.
  • Immunogenicity and tolerogenicity of hepatitis B virus structural and nonstructural proteins: Implications for immunotherapy of persistent viral infections, K Kakimi, M Isogawa, JS Chung, A Sette, FV Chisari, JOURNAL OF VIROLOGY, 76, (17) 8609 - 8620,   2002年09月, 査読有り, Persistent hepatitis B virus (HBV) infection is characterized by a weak and narrowly focused CD8(+) T-cell response to HBV that is thought to reflect the induction of central and/or peripheral tolerance to HBV proteins in neonatal and adult onset infections, respectively. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may lead to viral clearance in chronically infected individuals. The present study was performed to compare the relative immunogenicities and tolerogenicities of HBV structural (envelope [ENV]) and nonstructural (polymerase [POL]) proteins at the CD8(+) cytotoxic T lymphocyte (CTL) level in transgenic mice that replicate HBV in the liver and secrete infectious virus into the blood, thus representing an excellent model of persistent HBV infection. Interestingly, the mice were tolerant to the ENV but not to the POL proteins at the CTL level. Furthermore, the POL-specific CTLs had no impact on HBV replication or liver function in vivo, even though they were readily induced and reached the liver after DNA immunization, reflecting their relatively low avidity and the low level at which the POL protein is expressed by the hepatocyte. Collectively, these results suggest that the factors that make POL less tolerogenic also make POL-specific CTLs relatively inefficient effector cells when they reach the target organ. Immunotherapeutic strategies to control HBV infection by inducing virus-specific CTL responses in chronically infected subjects should be evaluated in light of this observation.
  • Helper T cell determinant peptide contributes to induction of cellular immune responses by peptide vaccines against hepatitis C virus, K Hiranuma, S Tamaki, Y Nishimura, S Kusuki, M Isogawa, G Kim, M Kaito, K Kuribayashi, Y Adachi, Y Yasutomi, JOURNAL OF GENERAL VIROLOGY, 80, (1) 187 - 193,   1999年01月, 査読有り, The capacity of novel subunit vaccines to generate cytotoxic T lymphocytes (CTLs) against hepatitis C virus (HCV) was assessed. BALB/c mice were immunized with peptides based on the CTL and helper T cell (Th) epitopes of the HCV core, with a mixture of CTL and Th peptides (CTL+Th) or with a conjugated Th-CTL peptide, Mice immunized with CTL, CTL+Th and Th-CTL peptides, but not those immunized with Th peptide, developed HCV core CTL epitope-specific effector cells. Cytotoxic activity induced by immunization with Th-CTL was much higher than that induced by immunization with CTL+Th or CTL alone, However, rapid and high cytotoxic activities against HCV core were not only detected after immunization with peptides containing the CTL epitope but also as a result of infection with recombinant vaccinia virus carrying the HCV core gene after immunization with the Th epitope alone. Immunization with peptides containing the Th epitope also elicited spleen cell proliferation. This study demonstrates the capacity of both Th and CTL activated peptide vaccines to elicit CD8(+), MHC class I-restricted CTLs, The capacity of such CTLs to contribute towards a protective and/or pathogenic immune response against HCV can now be assessed in mouse models.

MISC

  • B型肝炎に対する免疫治療の展望, 五十川正記, 田中靖人, 月刊メディカル・サイエンス・ダイジェスト, 44,   2018年07月30日
  • B型肝炎治療 2018 B型肝炎ウイルス感染・複製機構:なぜHBV排除は困難なのか, 五十川正記, 田中靖人, Mebio, 35,   2018年01月
  • 新規エンテカビル耐性株の同定とその特徴, 林 佐奈衣, 村上 周子, 尾曲 克己, 松居 剛志, 飯尾 悦子, 五十川 正記, 渡邊 綱正, 狩野 吉康, 田中 靖人, 肝臓, 56, (Suppl.1) A255 - A255,   2015年04月
  • B型肝炎ウイルスGenotype Fにおける肝細胞癌関連因子の検討, 林佐奈衣, 五十川正記, 村上周子, 飯島沙幸, 堤進, 尾曲克己, 渡邊綱正, 田中靖人, 日本ウイルス学会学術集会プログラム・抄録集, 62nd,   2014年10月31日
  • 初代ヒト肝細胞を用いたB型肝炎ウイルス感染初期の宿主免疫応答とウイルス生活環の解析, 堤進, 渡邊綱正, 村上周子, 飯島沙幸, 林佐奈衣, 尾曲克己, 五十川正記, 田中靖人, 日本ウイルス学会学術集会プログラム・抄録集, 62nd,   2014年10月31日


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