击倒病毒抗原表达可提高高滴度乙型肝炎病毒载体小鼠的治

StephenW

Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-Titer Hepatitis B Virus Carrier Mice [url=]Thomas Michler[/url]1,2,∗
,  [url=]Anna D. Kosinska[/url]1,2,∗
,  [url=]Julia Festag[/url]1
,  [url=]Till Bunse[/url]1,2
,  [url=]Jinpeng Su[/url]1
,  [url=]Marc Ringelhan[/url]1,3
,  [url=]Hortenzia Imhof[/url]1
,  [url=]Dirk Grimm[/url]2,4
,  [url=]Katja Steiger[/url]5
,  [url=]Carolin Mogler[/url]5
,  [url=]Mathias Heikenwalder[/url]6
,  [url=]Marie-Louise Michel[/url]7
,  [url=]Carlos A. Guzman[/url]2,8
,  [url=]Stuart Milstein[/url]9
,  [url=]Laura Sepp-Lorenzino[/url]9
,  [url=]Percy Knolle[/url]2,10
,  [url=]Ulrike Protzer[/url]1,2,∗,[url=]Correspondence information about the author  Ulrike Protzer[/url]Email the author  Ulrike ProtzerEmail the author  Ulrike Protzer






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DOI: https://doi.org/10.1053/j.gastro.2020.01.032 |

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Background & AimsHepatitis 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 so far. 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.


MethodsWe 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 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 12 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.


ResultsIn 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 after administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells, and HBV was eliminated.


ConclusionsIn 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.



Keywords:                [url=https://www.gastrojournal.org/action/doSearch?searchType=quick&occurrences=all&ltrlSrch=true&searchScope=fullSite&searchText=Viral Hepatitis&code=ygast-site]Viral Hepatitis[/url], Immunization, siRNA, Immunotherapy

StephenW

击倒病毒抗原表达可提高高滴度乙型肝炎病毒载体小鼠的治疗性疫苗效力
托马斯·米歇勒1,2，*
，Anna D. Kosinska1,2，*
朱莉娅·菲斯塔格（Julia Festag1）
，直到Bunse1,2
苏金鹏1
，马克·林格汉1,3
，Hortenzia Imhof1
，德克·格林2,4
，Katja Steiger5
卡罗琳·莫格勒5
，Mathias Heikenwalder6
，玛丽·路易丝·米歇尔7
，卡洛斯·古兹曼2.8
，斯图尔特·米尔斯坦9
劳拉·塞普（Laura Sepp-Lorenzino）9
，珀西·诺勒2,10
，Ulrike Protzer1,2，*，'有关作者Ulrike Protzer的通讯信息给作者发送电子邮件Ulrike Protzer电子邮件给作者Ulrike Protzer
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DOI：https：//doi.org/10.1053/j.gastro.2020.01.032 |
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背景与目标

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，但到目前为止，这些疫苗尚未奏效。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腺病毒感染后转移了HBV（基因型D，血清型ayw），该病毒转移了超长的HBV基因组，并以与耐心。针对所有HBV转录本共同3'-末端的小发夹或小干扰（si）RNA被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。对照小鼠接受恩替卡韦或非HBV特异的siRNA和疫苗成分。 8到12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抗原水平，HBV复制和病毒血症水平降低（1-3 log10降低）。接种疫苗可在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然。最初具有高滴度HBV和HBV抗原表达敲低的小鼠，但服用恩替卡韦后病毒血症降低的小鼠没有，发展了多功能的HBV特异性CD8 + T细胞，并消除了HBV。
结论

在具有高水平HBV复制的小鼠中，HBV抗原表达的敲除以及一种治疗性接种策略（但不是单独敲除）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。
关键字：
病毒性肝炎，免疫，siRNA，免疫治疗