设计治疗性乙型肝炎疫苗以规避免疫耐受。

StephenW

Hum Vaccin Immunother. 2019 Dec 6:1-18. doi: 10.1080/21645515.2019.1689745. [Epub ahead of print]
Designing a therapeutic hepatitis B vaccine to circumvent immune tolerance.
Whitacre DC1,2, Peters CJ1,2, Sureau C3, Nio K4, Li F5, Su L5, Jones JE1, Isogawa M6, Sallberg M7, Frelin L7, Peterson DL8, Milich DR1,2.
Author information

1
    Department of Immunology, VLP Biotech, Inc., JLABS San Diego, San Diego, CA, USA.
2
    Department of Immunology, Vaccine Research Institute of San Diego, San Diego, CA, USA.
3
    Molecular Virology Laboratory, Institut National de la Transfusion Sanguine (INTS), Paris, France.
4
    Graduate School of Medicine, Department of Gastroenterology, Kanazawa University, Kanazawa, Ishikawa, Japan.
5
    Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
6
    Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
7
    Department of Laboratory Medicine, Division of Clinical Microbiology, F68, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockhold, Sweden.
8
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.

Abstract

An effective prophylactic hepatitis B virus (HBV) vaccine has long been available but is ineffective for chronic infection. The primary cause of chronic hepatitis B (CHB) and greatest impediment for a therapeutic vaccine is the direct and indirect effects of immune tolerance to HBV antigens. The resulting defective CD4+/CD8+ T cell response, poor cytokine production, insufficient neutralizing antibody (nAb) and poor response to HBsAg vaccination characterize CHB infection. The objective of this study was to develop virus-like-particles (VLPs) that elicit nAb to prevent viral spread and prime CD4+/CD8+ T cells to eradicate intracellular HBV. Eight neutralizing B cell epitopes from the envelope PreS1 region were consolidated onto a species-variant of the HBV core protein, the woodchuck hepatitis core antigen (WHcAg). PreS1-specific B cell epitopes were chosen because of preferential expression on HBV virions. Because WHcAg and HBcAg are not crossreactive at the B cell level and only partially cross-reactive at the CD4+/CD8+ T cell level, CD4+ T cells specific for WHcAg-unique T cell sites can provide cognate T-B cell help for anti-PreS1 Ab production that is not curtailed by immune tolerance. Immunization of immune tolerant HBV transgenic (Tg) mice with PreS1-WHc VLPs elicited levels of high titer anti-PreS1 nAbs equivalent to wildtype mice. Passive transfer of PreS1 nAbs into human-liver chimeric mice prevented acute infection and cleared serum HBV from mice previously infected with HBV in a model of CHB. At the T cell level, PreS1-WHc VLPs and hybrid WHcAg/HBcAg DNA immunogens elicited HBcAg-specific CD4+ Th and CD8+ CTL responses.
KEYWORDS:

Hbv; immune tolerance; neutralizing antibody; pres1; therapeutic vaccine

PMID:
    31809638
DOI:
    10.1080/21645515.2019.1689745

StephenW

嗡嗡声疫苗免疫。 2019十二月6：1-18。 doi：10.1080 / 21645515.2019.1689745。 [Epub提前发布]
设计治疗性乙型肝炎疫苗以规避免疫耐受。
Whitacre DC1,2，Peters CJ1,2，Sureau C3，Nio K4，Li F5，Su L5，Jones JE1，Isogawa M6，Sallberg M7，Frelin L7，Peterson DL8，Milich DR1,2。
作者信息

1个
    VLP Biotech，Inc.免疫学系，美国加利福尼亚州圣地亚哥JLABS圣地亚哥。
2
    美国加州圣地亚哥圣地亚哥疫苗研究所免疫学系。
3
    法国巴黎国家输血血研所（INTS）分子病毒学实验室。
4
    日本石川县金泽市金泽大学消化科医学研究生院。
5
    美国北卡罗来纳大学教堂山分校医学院微生物学和免疫学系Lineberger综合癌症中心，美国北卡罗来纳州。
6
    名古屋市立大学医学研究科病毒与肝病学系，日本名古屋。
7
    瑞典斯托克霍尔德卡罗林斯卡大学附属医院卡罗林斯卡大学卡罗林斯卡研究所F68临床微生物学实验室医学科。
8
    弗吉尼亚联邦大学生物化学与分子生物学系，美国弗吉尼亚州里士满。

抽象

有效的预防性乙型肝炎病毒（HBV）疫苗早已可用，但对慢性感染无效。慢性乙型肝炎（CHB）的主要原因和治疗性疫苗的最大障碍是对HBV抗原的免疫耐受的直接和间接作用。导致的CD4 + / CD8 + T细胞缺陷，细胞因子产生不良，中和抗体（nAb）不足以及对HBsAg疫苗接种的不良反应是CHB感染的特征。这项研究的目的是开发能诱导nAb阻止病毒传播并引发CD4 + / CD8 + T细胞根除细胞内HBV的病毒样颗粒（VLP）。来自包膜PreS1区域的八个中和B细胞表位被整合到HBV核心蛋白物种的变异中，该蛋白是土拨鼠肝炎核心抗原（WHcAg）。选择PreS1特异的B细胞表位是因为在HBV病毒粒子上优先表达。因为WHcAg和HBcAg在B细胞水平上没有交叉反应，而在CD4 + / CD8 + T细胞水平上只有部分交叉反应，所以特异于WHcAg独特T细胞位点的CD4 + T细胞可以为抗PreS1 Ab产生提供相关的TB细胞帮助。不能被免疫耐受所限制。用PreS1-WHc VLP免疫免疫耐受的HBV转基因（Tg）小鼠会引起与野生型小鼠相当的高滴度抗PreS1 nAbs水平。将PreS1 nAbs被动转移到人肝嵌合小鼠中可预防急性感染，并可从以前在CHB模型中感染过HBV的小鼠中清除血清HBV。在T细胞水平，PreS1-WHc VLP和WHcAg / HBcAg杂合DNA免疫原引发了HBcAg特异性CD4 + Th和CD8 + CTL反应。
关键字：

乙肝病毒免疫耐受中和抗体； pres1;治疗性疫苗

PMID：
    31809638
DOI：
    10.1080 / 21645515.2019.1689745

StephenW

https://www.tandfonline.com/doi/ ... 645515.2019.1689745

齐欢畅

不看好

StephenW

Discussion

Immune tolerance to an infectious agent such as the HBV is not a binary event (tolerance vs no tolerance), but rather represents complex host-viral interactions. Because immune tolerance is clonal, all viral antigens and their constituent epitopes are not equally tolerogenic nor are all cell types equally susceptible to tolerance induction. For example, the secreted HBeAg is more tolerogenic than the cellular HBcAg,69 and one HBeAg-specific epitope is more tolerogenic than another epitope on the same HBeAg.70,71 Similarly, one would expect different degrees of T cell tolerance to epitopes on the PreS1, PreS2, and HBsAg envelope antigens because T cell recognition is distinct between these envelope regions.55 At the cellular level, Th cells are often more susceptible to tolerance induction than B cells or CTL.72 These phenomenon represent forms of “split tolerance”. Immune tolerance preferentially occurs in high avidity clones and is mediated by deletional and non-deletional mechanisms. Therefore, low avidity T cells that have escaped deletion most likely constitute the bulk of the T cell repertoire during chronic HBV infection and low avidity T cells are more prone to negative regulation (i.e., checkpoint inhibitors, regulatory T cells, metabolic dysfunction, and clonal exhaustion). Other hosts and viral factors such as age of infection, immune status, antigen load, secreted or cytosolic antigen and phase of infection affect immune tolerance as well.9,10 For vaccine immunotherapy to be effective it must target either non-tolerant clones or non-deleted clones in which tolerance can be reversed or circumvented. Therefore, we targeted PreS1-specific B cells and HBcAg-specific CTL effector cells and bypassed the more tolerant prone Th cells by substituting heterospecific, WHcAg-specific Th cells for HBV-specific Th cells. Although B cell defects have been defined during chronic HBV infection,6 low-level antibodies to most viral antigens are present during the active phases of infection often masked in immune complexes.66 Further, HBc/HBeAg-specific CTL can be detected in the periphery and the liver during chronic infection,73,74 however, similar to B cell antibody production, CTL function is suboptimal and not sufficient to mediate viral clearance. It is anticipated that both of these nascent effector pathways can be enhanced by providing more efficient Th cell function using the WHcAg platform to present neutralizing, PreS1-specific B cell epitopes and HBcAg-specific CTL epitopes.

Accomplishments of the current study include:

    Defined eight PreS1 B cell epitopes (highly conserved amongst HBV genotypes).

    Inserted each of the eight PreS1 B cell epitopes onto WHcAg carrier VLPs.

    Consolidated all eight PreS1 epitopes onto two PreS1-WHc VLPs (VLP-1.6 and VLP-1.9).

    Demonstrated that PreS1-WHc VLPs elicited PreS1-specific Ab that recognizes native HBV virions and multiple L/M/S-HBsAg particles of both major serotypes (ad/ay).

    Demonstrated that six PreS1-WHc VLPs elicited nAbs specific for N-terminal, central and C-terminal B cell domains of the PreS1 region, however, the central B cell domain elicited superior neutralizing Ab. A previous study of PreS1-HBc VLPs concluded that anti-PreS1 Abs specific for the N- and C-terminus were not viral neutralizing.61 This suggests that the fine specificity of the B cell response to the PreS1 region is variable and can be influenced by the choice of HBcAg or WHcAg carrier.

    Demonstrated that PreS1-WHc VLPs circumvented immune tolerance and elicited equivalent levels of nAb to the PreS1 region in wildtype, HBV-Tg, HBc/HBeAg-Tg, and PreS1-Tg mice lineages.

    Demonstrated that PreS1-WHc VLPs primed crossreactive, HBcAg-specific CD4+/CD8+ T cells as well as WHcAg-heterospecific CD4+/CD8+ T cells.

    Demonstrated that in human-liver chimeric mice passive transfer of PreS1 nAbs prevented an acute HBV infection.

    Demonstrated that in human-liver chimeric mice previously infected with HBV (model of chronic infection), PreS1 nAbs cleared serum HBV and may arrest HBV spread in the liver possibly reducing cccDNA levels.16

    Produced hybrid WHcAg/HBcAg particles as DNA constructs, which elicited HBcAg-specific CD8+ CTL.

The relative scarcity of PreS1 antigen relative to the major HBsAg is a limiting factor for anti-PreS1 nAb production during a natural HBV infection. The capacity of the highly immunogenic WHcAg carrier to display multiple PreS1 neutralizing B cell epitopes overcomes this limitation. For example, 240 copies of each of the eight PreS1 B cell epitopes are displayed per PreS1-WHc VLP. A combined PreS1-WHc VLP-1.6/VLP-1.9 vaccine formulated in an adjuvant suitable for human use given in a prime/boost protocol with an optimized WHcAg/HBcAg DNA construct would represent a strong candidate therapeutic HBV vaccine. This vaccine would be capable of circumventing immune tolerance and eliciting multiple PreS1 nAb specificities as well as HBcAg-specific CD8+ CTL to target intracellular HBV DNA including cccDNA. Although a PreS1-WHc VLP prime – hybrid WHcAg/HBcAg DNA boost regimen could be given as a monotherapy, combination with an antiviral agent would enhance efficacy by reducing viral load. Inserting multiple neutralizing B cell PreS1 epitopes will mitigate the possibility of nAb escape mutants, which may be problematic when treating an established HBV infection. Bacterial production of PreS1-WHc VLPs together with a WHcAg/HBcAg DNA immunogen would be cost-efficient and compatible with any antiviral treatment for maximum efficacy. The ultimate goal is to produce both PreS1-WHc VLPs and WHcAg-HBcAg hybrid VLP DNA constructs for the dual purposes of eliciting PreS1-specific nAbs and HBcAg-specific CTL. The current study was limited primarily to evaluating the PreS1-specific humoral response in immune tolerant HBV-Tg mice and in human-liver chimeric mice infected with HBV. Future studies will be necessary to evaluate the ability of the WHcAg-HBcAg hybrid DNA constructs produced herein to circumvent HBcAg-specific CTL immune tolerance.

Although therapeutic HBV vaccines have not been successful to date, a number of experimental vaccines show promise in animal models or in the clinic,74,75 especially PreS-containing candidates.76,77 However, most experimental vaccines rely exclusively on the use of HBV-derived antigens, subject to the direct and indirect effects of immune tolerance, unlike the current PreS1-WHc VLP technology. An alternative approach for bypassing immune tolerant, dysfunctional endogenous T cells is the adoptive transfer of “engineered” HBV-specific T cells, which are demonstrating efficacy in animal models and in one clinical trial.78–80 In a recent study, a single adoptive transfer of transfected HBV-specific T cells demonstrated effective reduction in HBV DNA without severe liver disease in an animal model. However, achieving long-term control of HBV infection required the combined use of the PreS1 peptide viral entry-inhibitor Myrcludex B.16,78 We argue that regardless of the efficacy of any and all antiviral therapies, the induction of nAbs should be included to prevent the spread of HBV to uninfected hepatocytes, which may indeed be a requirement for the complete cure of CHB infection.

In addition to use as a therapeutic HBV vaccine, other possible applications for PreS1-WHc VLPs include: Use as a preventative vaccine in low-to-nonresponders to the conventional HBsAg vaccine; vaccination of pregnant HBV+ carrier mothers in order to provide passive transfer of PreS1-specific neutralizing Abs to block transmission during and after birth; an immunotherapy for chronic HDV infection; and prior to immunosuppressive therapy, vaccination of HBV+ liver transplant recipients in order to prevent infection of the new liver.

StephenW

讨论区

对诸如HBV的传染原的免疫耐受性不是二元事件（耐受性vs无耐受性），而是代表复杂的宿主-病毒相互作用。由于免疫耐受是克隆性的，因此所有病毒抗原及其组成表位均不具有相同的致耐受性，也不是所有细胞类型都同样容易诱导耐受。例如，分泌的HBeAg比细胞HBcAg更具有耐受性，69，一个HBeAg特异性表位比同一个HBeAg上的另一个表位具有更大的耐受性。70,71同样，人们会期望T细胞对HBeAg的表位具有不同程度的耐受性PreS1，PreS2和HBsAg包膜抗原是因为在这些包膜区域之间T细胞识别是不同的。 55在细胞水平上，Th细胞通常比B细胞或CTL更易诱导耐受。72这些现象代表“分裂耐受”的形式。免疫耐受优先发生在高亲和力克隆中，并由缺失和非缺失机制介导。因此，在慢性HBV感染期间，逃避缺失的低亲和力T细胞很可能构成T细胞库的大部分，而低亲和力T细胞更容易产生负调节作用（即检查点抑制剂，调节性T细胞，代谢功能障碍和克隆）精疲力尽）。其他宿主和病毒因素（如感染年龄，免疫状态，抗原负荷，分泌性或胞质抗原和感染阶段）也会影响免疫耐受性。 9,10为使疫苗免疫治疗有效，它必须靶向可逆转或规避耐受性的非耐性克隆或非缺失克隆。因此，我们靶向PreS1特异性B细胞和HBcAg特异性CTL效应细胞，并通过用异种特异性WHcAg特异性Th细胞替代HBV特异性Th细胞来绕开了耐受性更强的Th细胞。尽管在慢性HBV感染期间已经定义了B细胞缺陷，但在感染的活跃阶段通常会被免疫复合物掩盖，存在针对大多数病毒抗原的6种低水平抗体。 66此外，在慢性感染期间可以在外周和肝脏中检测到HBc / HBeAg特异性CTL，73,74然而，与B细胞抗体产生相似，CTL功能次优，不足以介导病毒清除。预期可以通过使用WHcAg平台提供更有效的Th细胞功能来呈现中和的PreS1特异性B细胞表位和HBcAg特异性CTL表位，从而增强这两个新生效应途径。

本研究的成就包括：

定义了八个PreS1 B细胞表位（在HBV基因型中高度保守）。

将八个PreS1 B细胞表位分别插入WHcAg载体VLP。

将所有八个PreS1表位整合到两个PreS1-WHc VLP（VLP-1.6和VLP-1.9）上。

证明PreS1-WHc VLP引发了PreS1特异性抗体，该抗体可识别天然HBV病毒体和两种主要血清型（ad / ay）的多个L / M / S-HBsAg颗粒。

证明了六个PreS1-WHc VLP引发了对PreS1区域的N端，中央和C端B细胞结构域具有特异性的nAb，但是，中央B细胞结构域引发了中和性的Ab。对PreS1-HBc VLP的先前研究得出的结论是，对N和C端特异的抗PreS1 Abs不能中和病毒。 61这表明B细胞对PreS1区域的精细特异性是可变的，并且会受到HBcAg或WHcAg载体选择的影响。

在野生型，HBV-Tg，HBc / HBeAg-Tg和PreS1-Tg小鼠谱系中，证明PreS1-WHc VLP绕过了免疫耐受，并引起了与PreS1区相当的nAb水平。

证明PreS1-WHc VLP引发了交叉反应性，HBcAg特异性CD4 + / CD8 + T细胞以及WHcAg异种CD4 + / CD8 + T细胞。

证明在人肝嵌合小鼠中被动转移PreS1 nAb可预防急性HBV感染。

证明在先前感染了HBV（慢性感染模型）的人肝嵌合小鼠中，PreS1 nAbs清除了血清HBV并可能阻止HBV在肝脏中的传播，可能会降低cccDNA的水平。 16

产生杂交的WHcAg / HBcAg颗粒作为DNA构建体，引发HBcAg特异性CD8 + CTL。

PreS1抗原相对于主要HBsAg的相对稀缺是自然HBV感染期间抗PreS1 nAb产生的限制因素。具有高度免疫原性的WHcAg载体显示多个PreS1中和B细胞表位的能力克服了这一限制。例如，每个PreS1-WHc VLP显示八个PreS1 B细胞表位中每一个的240个拷贝。在适合人类使用的佐剂中配制成的PreS1-WHc VLP-1.6 / VLP-1.9组合疫苗，在初免/加强方案中与优化的WHcAg / HBcAg DNA构建体一起使用，将代表一种强大的候选治疗性HBV疫苗。该疫苗将能够规避免疫耐受并引发多种PreS1 nAb特异性以及HBcAg特异性CD8 + CTL，以靶向细胞内HBV DNA（包括cccDNA）。尽管可以将PreS1-WHc VLP初免-混合WHcAg / HBcAg DNA增强方案作为单药治疗，但与抗病毒药联用可通过降低病毒载量来提高疗效。插入多个中和的B细胞PreS1表位将减轻nAb逃逸突变体的可能性，这在治疗已建立的HBV感染时可能会出现问题。 PreS1-WHc VLP的细菌生产以及WHcAg / HBcAg DNA免疫原将具有成本效益，并且可以与任何抗病毒治疗兼容，以实现最大功效。最终目标是同时产生PreS1-WHc VLP和WHcAg-HBcAg杂合VLP DNA构建体，以达到引发PreS1特异性nAb和HBcAg特异性CTL的双重目的。目前的研究主要限于评估免疫耐受的HBV-Tg小鼠和感染HBV的人肝嵌合小鼠的PreS1特异性体液反应。未来的研究对于评估本文生产的WHcAg-HBcAg杂交DNA构建体规避HBcAg特异性CTL免疫耐受的能力将是必要的。

尽管迄今为止治疗性乙肝病毒疫苗尚未成功，但许多实验性疫苗在动物模型或临床研究中显示出希望，74,75尤其是含PreS的候选药物。76,77然而，大多数实验性疫苗完全依赖于乙肝病毒的使用抗原，受到免疫耐受的直接和间接影响，这与当前的PreS1-WHc VLP技术不同。绕过免疫耐受，功能失调的内源性T细胞的另一种方法是过继转移“工程化”的HBV特异性T细胞，这在动物模型和一项临床试验中均证明了其有效性。78-80在最近的一项研究中，单个过继性在动物模型中，转染的HBV特异性T细胞的转移证明HBV DNA有效降低，而没有严重的肝脏疾病。但是，要实现对HBV感染的长期控制，需要结合使用PreS1肽病毒进入抑制剂Myrcludex B.16,78。我们认为，无论任何抗病毒治疗的有效性如何，nAb的诱导均应包括在内。防止HBV传播到未感染的肝细胞，这确实可能是彻底治愈CHB感染所必需的。

除用作治疗性HBV疫苗外，PreS1-WHc VLP的其他可能用途还包括：在对常规HBsAg疫苗低应答或无应答的情况下用作预防性疫苗；为怀孕的HBV +携带者母亲接种疫苗，以提供PreS1特异性中和抗体的被动转移，以阻止出生期间和出生后的传播；慢性HDV感染的免疫疗法；在免疫抑制治疗之前，应接种HBV +肝移植受者疫苗，以防止新肝感染。