病毒抗原表达的抑制可提高高滴度HBV携带者小鼠的治疗性疫

StephenW

Gastroenterology. 2020 Jan 28. pii: S0016-5085(20)30125-6. doi: 10.1053/j.gastro.2020.01.032. [Epub ahead of print]
Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-titer HBV Carrier Mice.
Michler T1, Kosinska AD1, Festag J2, Bunse T1, Su J2, Ringelhan M3, Imhof H2, Grimm D4, Steiger K5, Mogler C5, Heikenwalder M6, Michel ML7, Guzman CA8, Milstein S9, Sepp-Lorenzino L9, Knolle P10, Protzer U11.
Author information

1
    Institute of Virology, Technical University of Munich / Helmholtz Zentrum München, Trogerstrasse 30, D-81675 Munich, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany.
2
    Institute of Virology, Technical University of Munich / Helmholtz Zentrum München, Trogerstrasse 30, D-81675 Munich, Germany.
3
    Institute of Virology, Technical University of Munich / Helmholtz Zentrum München, Trogerstrasse 30, D-81675 Munich, Germany; Department of Internal Medicine II, University Hospital rechts der Isar, Technical University of Munich, Ismaninger Str. 22, D-81675 Munich, Germany.
4
    Department of Infectious Diseases/Virology, Heidelberg University Hospital, BioQuant, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany.
5
    Institute of Pathology, Technical University of Munich, Trogerstrasse 18, D-81675 Munich, Germany.
6
    Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany.
7
    Institut Pasteur, 28 rue Dr Roux, 75015 Paris, France.
8
    Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany.
9
    Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
10
    Institute of Molecular Immunology, University Hospital rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany.
11
    Institute of Virology, Technical University of Munich / Helmholtz Zentrum München, Trogerstrasse 30, D-81675 Munich, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany. Electronic address: [email protected].

Abstract
BACKGROUND & AIMS:

Hepatitis B virus (HBV) infection persists because the virus-specific immune response is dysfunctional. Therapeutic vaccines might be used to end immune tolerance to the virus in patients with chronic infection, but these have not been effective in patients. In patients with chronic HBV infection, high levels of virus antigens might prevent induction of HBV-specific immune responses. We investigated whether knocking down expression levels of HBV antigens in liver might increase the efficacy of HBV vaccines in mice.
METHODS:

We performed studies with male C57BL/6 mice that persistently replicate HBV (genotype D, serotype ayw)-either from a transgene or after infection with an adeno-associated virus that transferred an overlength HBV genome-and expressed HB surface antigen (HBsAg) at levels relevant to patients. Small hairpin or small interfering (si)RNAs against the common 3'-end of all HBV transcripts were used to knock-down antigen expression in mouse hepatocytes. siRNAs were chemically stabilized and conjugated to N-acetylgalactosamine to increase liver uptake. Control mice were given either entecavir or non-HBV specific siRNAs and vaccine components. Eight to twelve weeks later, mice were immunized twice with a mixture of adjuvanted HBV S and core antigen, followed by a modified vaccinia virus Ankara vector to induce HBV-specific B- and T-cell responses. Serum and liver samples were collected and analyzed for HBV-specific immune responses, liver damage, and viral parameters.
RESULTS:

In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction), compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies, and increased numbers and functionality of HBV-specific, CD8+ T-cells in mice with low, but not in mice with high levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia following administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells and HBV was eliminated.
CONCLUSIONS:

In mice with high levels of HBV replication, knockdown of HBV antigen expression along with a therapeutic vaccination strategy, but not knockdown alone, increased numbers of effector T cells and eliminated the virus. These findings indicate that high titers of virus antigens reduce the efficacy of therapeutic vaccination. Anti-HBV siRNAs and therapeutic vaccines are each being tested in clinical trials-their combination might cure chronic HBV infection.

Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.
KEYWORDS:

Viral hepatitis; immunization; immunotherapy; siRNA

PMID:
    32001321
DOI:
    10.1053/j.gastro.2020.01.032

StephenW

肠胃病学。 2020年1月28日。pii：S0016-5085（20）30125-6。 Doi：10.1053 / j.gastro.2020.01.032。 [Epub提前发行]
病毒抗原表达的抑制可提高高滴度HBV携带者小鼠的治疗性疫苗效力。
Michler T1，Kosinska AD1，Festag J2，Bunse T1，Su J2，Ringelhan M3，Imhof H2，Grimm D4，Steiger K5，Mogler C5，Heikenwalder M6，Michel ML7，Guzman CA8，Milstein S9，Sepp-Lorenzino L9，Knolle P10 U11。
作者信息

1个
慕尼黑工业大学病毒学研究所/ Helmholtz ZentrumMünchen，Trogerstrasse 30，D-81675慕尼黑，德国;德国感染研究中心（DZIF），慕尼黑，海德堡和汉诺威/不伦瑞克的合作伙伴，德国。
2
慕尼黑工业大学病毒学研究所/ Helmholtz ZentrumMünchen，Trogerstrasse 30，D-81675慕尼黑，德国。
3
慕尼黑工业大学病毒学研究所/ Helmholtz ZentrumMünchen，Trogerstrasse 30，D-81675慕尼黑，德国；慕尼黑工业大学伊萨尔分校伊萨尔分校伊斯曼宁格分校第二医院内科。 22，D-81675慕尼黑，德国。
4
海德堡大学医院，BioQuant传染病/病毒学系，德国海因堡D-69120的Im Neuenheimer Feld 267；德国感染研究中心（DZIF），慕尼黑，海德堡和汉诺威/不伦瑞克的合作伙伴，德国。
5
慕尼黑工业大学病理学研究所，Trogerstrasse 18，D-81675慕尼黑，德国。
6
德国癌症研究中心（DKFZ）慢性炎症和癌症科，德国海德堡D-69120的Im Neuenheimer Feld 242。
7
巴斯德研究所（Pasteur Institut），鲁克斯大街28号，邮政编码75015，巴黎，法国。
8
Inhoffenstr亥姆霍兹感染研究中心疫苗学和应用微生物学系。 7，D-38124不伦瑞克，德国；德国感染研究中心（DZIF），慕尼黑，海德堡和汉诺威/不伦瑞克的合作伙伴，德国。
9
Alnylam Pharmaceuticals，300 Third Street，Cambridge，MA 02142，USA。
10
慕尼黑工业大学伊萨尔大学医院分子免疫学研究所，德国伊斯曼宁格大街22号，D-81675；德国感染研究中心（DZIF），慕尼黑，海德堡和汉诺威/不伦瑞克的合作伙伴，德国。
11
慕尼黑工业大学病毒学研究所/ Helmholtz ZentrumMünchen，Trogerstrasse 30，D-81675慕尼黑，德国;德国感染研究中心（DZIF），慕尼黑，海德堡和汉诺威/不伦瑞克的合作伙伴，德国。电子地址：protzer @ tum .de。

抽象
背景与目的：

乙型肝炎病毒（HBV）感染持续存在，因为该病毒特异性免疫反应功能异常。治疗性疫苗可用于终止慢性感染患者对该病毒的免疫耐受性，但对患者无效。在患有慢性HBV感染的患者中，高水平的病毒抗原可能会阻止HBV特异性免疫反应的诱导。我们调查了敲低肝脏中HBV抗原的表达水平是否可以提高小鼠HBV疫苗的功效。
方法：

我们对雄性C57BL / 6小鼠进行了研究，这些小鼠从转基因或感染腺相关病毒后连续复制HBV（基因型D，血清型ayw），并转移了HBV基因组，并在与患者相关的水平。针对所有HBV转录本共同3'-末端的小发夹或小干扰（si）RNA用于敲除小鼠肝细胞中的抗原表达。 siRNA经过化学稳定处理，并与N-乙酰半乳糖胺缀合以增加肝脏吸收。给予对照小鼠恩替卡韦或非HBV特异性siRNA和疫苗成分。八到十二周后，用佐剂的HBV S和核心抗原的混合物免疫小鼠两次，然后用修饰的牛痘病毒Ankara载体免疫，以诱导HBV特异的B细胞和T细胞反应。收集血清和肝脏样品，并分析HBV特异性免疫反应，肝损伤和病毒参数。
结果：

在两种HBV感染模型中，与给予对照RNA的小鼠相比，表达肝细胞特异性小发夹RNA或经皮下注射siRNA的小鼠的HBV抗原，HBV复制和病毒血症水平降低（减少1-3 log10）。 。疫苗接种可在HBV抗原水平低的小鼠中诱导产生HBV中和抗体，并增加HBV特异性CD8 + T细胞的数量和功能，但在HBV抗原水平高的小鼠中则不。最初具有高滴度HBV和HBV抗原表达敲低的小鼠，但没有因恩替卡韦给药后病毒血症降低的小鼠，消除了发育的多功能HBV特异性CD8 + T细胞和HBV。
结论：在具有高水平HBV复制的小鼠中，击倒HBV抗原表达以及治疗性疫苗接种策略（但不单独击倒）会增加效应T细胞的数量并消除病毒。 这些发现表明高滴度的病毒抗原降低了治疗性疫苗接种的功效。 抗HBV siRNA和治疗性疫苗均在临床试验中进行测试，它们的组合可治愈慢性HBV感染。

版权所有©2020 AGA Institute。 由Elsevier Inc.出版。保留所有权利。
关键字：

病毒性肝炎; 免疫 免疫疗法 siRNA

PMID：
     32001321
DOI：
     10.1053 / j.gastro.2020.01.032