击倒病毒抗原表达可提高高滴度HBV小鼠的治疗性疫苗效力

StephenW

Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-titer HBV Carrier Mice
Thomas Michler1,10, ∗
, Anna D. Kosinska1,10, ∗
, Julia Festag1
, Till Bunse1,10
, Jinpeng Su1
, Marc Ringelhan1,2
, Hortenzia Imhof1
, Dirk Grimm3,10
, Katja Steiger4
, Carolin Mogler4
, Mathias Heikenwalder5
, Marie-Louise Michel6
, Carlos A. Guzman7,10
, Stuart Milstein8
, Laura Sepp-Lorenzino8
, Percy Knolle9,10
, Ulrike Protzer1,10, ∗, 'Correspondence information about the author Ulrike ProtzerEmail 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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Publication History
Published online: January 28, 2020Accepted: January 20, 2020Received in revised form: December 28, 2019Received: June 5, 2019

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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.
Keywords:
Viral hepatitis, immunization, siRNA, immunotherapy

StephenW

击倒病毒抗原表达可提高高滴度HBV小鼠的治疗性疫苗效力
托马斯·米切勒1,10，*
，Anna D.Kosinska1,10，*
朱莉娅·菲斯塔格（Julia Festag1）
，直到Bunse1,10
苏金鹏1
，马克·林格汉1,2
，Hortenzia Imhof1
，德克·格林3,10
，Katja Steiger4
，Carolin Mogler4
，Mathias Heikenwalder5
，玛丽·路易丝·米歇尔6
，卡洛斯·古兹曼（Carlos A. Guzman）7,10
，斯图尔特·米尔斯坦8
劳拉·塞普（Laura Sepp-Lorenzino）8
，珀西·诺勒9,10
，，Ulrike Protzer1,10，∗，'有关作者Ulrike Protzer的通讯信息给作者发送电子邮件Ulrike Protzer给作者电子邮件Ulrike Protzer
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DOI：https：//doi.org/10.1053/j.gastro.2020.01.032
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出版历史
在线发布：2020年1月28日接受：2020年1月20日收到修订版：2019年12月28日收到：2019年6月5日

抽象
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图形概要

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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失调。治疗性疫苗可用于终止慢性感染患者对病毒的免疫耐受性，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，这些小鼠从转基因中或感染腺相关病毒后一直复制HBV（基因型D，血清型ayw），并转移了超长的HBV基因组，并在HBV处表达HB表面抗原（HBsAg）。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。 8至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特异性的B细胞和T细胞应答。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却不。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。
关键字：
病毒性肝炎，免疫，siRNA，免疫疗法