A Therapeutic Vaccine for Chronic Hepatitis B

描述（由申请方提供）：本提案的目的是评估使用重组病毒样颗粒（VLP）引发中和抗体并引发与B型肝炎病毒（HBV）抗原反应的CD 4 +/CD 8 + T细胞作为慢性HBV感染的候选免疫治疗剂的可行性。为此目的，将代表HBV包膜Pre-S（1）和（2）抗原的选择的明确定义的中和B细胞表位插入到HBV核心蛋白的物种变体上，即土拨鼠肝炎核心抗原（WHcAg）。选择Pre-S B细胞表位是因为它们在HBV病毒粒子上优先表达。我们先前已经开发了WHcAg作为许多异源B和T细胞表位的VLP疫苗载体，并且在该特定应用中的使用将允许规避存在于慢性感染患者中的T细胞免疫耐受。 WHO估计超过4亿个体慢性感染HBV，并且大约20-40%将发展严重并发症，例如肝硬化、肝衰竭和肝细胞癌。尽管安全有效的HBV预防性疫苗已经存在了20多年，但HBV感染仍然是一个主要的健康问题（每年超过5000万例HBV感染），并且不存在有效的慢性感染治疗方法。抗病毒药物如拉米夫定、阿德福韦、恩替卡韦和聚乙二醇化干扰素-α改善了慢性HBV的治疗选择，但是，由于停药后HBV复制的重新激活，它们的疗效仍然有限。由于慢性HBV感染是一种免疫介导的疾病，并且通过骨髓移植对HBV的过继免疫转移已经实现了慢性HBV感染的消退，因此建议将基于疫苗的免疫治疗作为可能的单药治疗或与抗病毒药物的联合治疗。为此，已经使用HBV包膜抗原（即，HBsAg、HBsAg- PreS（2）和含有亚病毒颗粒的HBsAg-PreS（2）-PreS（1））单独或组合作为佐剂中的蛋白质或作为DNA构建体递送，所有这些都具有相当令人失望的结果。 解释慢性HBV携带者中T细胞应答缺陷的最重要因素是免疫耐受。为了克服HBV慢性携带者免疫耐受的障碍，我们选择构建由插入到WHcAg上的HBsAg Pre-S中和B细胞表位组成的杂合VLP，其与HBcAg约66-68%同源。WHcAg和HBcAg在B细胞水平上不具有交叉反应性，并且对于我们的目的同样重要的是，基于独特的WHcAg特异性T细胞位点和共享的WHcAg/HBcAg特异性T细胞位点，WHcAg和HBcAg之间保守的WHcAg区域在CD 4 + T细胞水平上仅具有部分交叉反应性。因此，对WHcAg独特T细胞位点特异性的CD 4 + T细胞将为抗PreS抗体的产生提供同源T-B细胞帮助，并且不会因对HBcAg特异性T细胞位点的免疫耐受而减少。事实上，在对HBcAg耐受的HBcAg-Tg小鼠中的初步研究中，用杂合WHcAg-PreS VLP免疫在野生型和HBcAg-Tg小鼠中产生等同的高滴度抗PreS抗体。 用HBcAg的密切相关物种变体免疫的另一个优点是WHcAg内的CD 4+和可能的CD 8 + T细胞位点与HBcAg内的位点仅略有不同（即，1或2个氨基酸）可以引发所谓的“旁观者”T细胞帮助，并且由于更高的亲合力T细胞交叉反应性，甚至还可以破坏HBV患者的HBcAg特异性T细胞的T细胞耐受性。从历史上看，打破T细胞对自身抗原的耐受性已经通过用密切相关的物种变体蛋白免疫来完成（诱导实验性自身免疫性关节炎、糖尿病、脑炎等）。 具体而言，在目的1中，我们提出将3个Ag-PreS（1）和1个Ag-PreS（2）中和B细胞表位插入WHcAg VLP载体上，并基于组装、产率、稳定性和免疫原性优化构建体。在目标2中，将在HBV复制的转基因（Tg）小鼠模型中评价基于VLP的候选疫苗的治疗效果，其中HBV在肝细胞中的复制水平与慢性HBV患者感染肝脏中的复制水平相当，而无细胞病理学证据。杂合WHcAg-PreS VLP候选物具有通过至少3种方式降低HBV载量的潜力：（1）抗PreS抗体应通过免疫复合物清除血清HBV;（2）WHc/HBcAg交叉反应性CD 4 + T细胞可通过炎性细胞因子产生减少HBV在肝脏中的复制;和（3）WHc/HBcAg交叉反应性CD 8 + T细胞可通过细胞因子产生以及通过直接肝细胞溶解来减少肝脏中的病毒复制。将在HBV-Tg小鼠模型中监测WHcAg-PreS VLP候选物免疫通过所有这些机制降低HBV载量的能力（目的2）。 预计这两种技术的组合，即生产杂交VLP（VLP Biotech）和慢性HBV感染小鼠模型（TSRI）的能力，将使我们能够在两年的时间内生产和测试至少4种杂交WHcAg-PreS VLP的治疗效果。

DESCRIPTION (provided by applicant): The objective of this proposal is to assess the feasibility of using recombinant virus-like-particles (VLPs) to elicit neutralizing antibodies and prime CD4+/CD8+ T cells reactive with hepatitis B viral (HBV) antigens as candidate immunotherapeutics for chronic HBV infection. For this purpose, selected well- defined neutralizing B cell epitopes representing HBV envelope Pre-S (1) and (2) antigens are being inserted onto a species variant of the HBV core protein, namely the woodchuck hepatitis core antigen (WHcAg). Pre-S B cell epitopes were chosen because of their preferential expression on HBV virions. We have previously developed the WHcAg as a VLP vaccine carrier for a number of heterologous B and T cell epitopes and use in this particular application will permit circumvention of the T cell immune tolerance that exists in chronically infected patients. The WHO estimates that more than 400 million individuals are chronically infected with HBV and approximately 20-40% will develop serious complication such as cirrhosis, liver failure and hepatocellular carcinoma. Although a safe and efficacious preventative vaccine for HBV has been available for over 20 years, HBV infections continue (with more than 50 million HBV infections per year) to be a major health problem and no effective treatments for chronic infection exist. Antiviral drugs such as lamivudine, adefovir, entecavir and pegylated interferon-alpha have improved the therapeutic options for chronic HBV, but, their efficacy remains limited due to reactivation of HBV replication upon drug withdrawal. Because chronic HBV infection is an immune-mediated disease and adoptive transfer of immunity to HBV through bone marrow transplantation has achieved resolution of chronic HBV infection, vaccine-based immunotherapy has been suggested as a possible monotherapy or as a combination therapy with antiviral drugs. To this end a number of clinical trials have been conducted using the HBV envelope antigens (i.e., HBsAg, HBsAg- PreS(2) and HBsAg-PreS(2)-PreS(1) containing subviral particles) either singly or in combination delivered as proteins in adjuvant or as DNA constructs, all with rather disappointing results. The most important factor to explain the defective T cell responses to the HBsAgs in chronic HBV carriers is immune tolerance. To circumvent the obstacle of immune tolerance in HBV chronic carriers, we have chosen to construct hybrid VLPs consisting of HBsAg Pre-S neutralizing B cell epitopes inserted onto the WHcAg, which is approximately 66-68% homologous with the HBcAg. The WHcAg and the HBcAg are not crossreactive at the B cell level and, just as importantly for our purposes, are only partially crossreactive at the CD4+ T cell level based on unique WHcAg-specific T cell sites and shared WHcAg/HBcAg-specific T cell sites in regions of the WHcAg that are conserved between WHcAg and HBcAg. Therefore, CD4+ T cells specific for WHcAg-unique T cell sites will provide cognate T-B cell help for anti-PreS antibody production and will not be curtailed by immune tolerance to HBcAg-specific T cell sites. In fact, in preliminary studies in HBcAg-Tg mice, which are tolerant to HBcAg, immunization with hybrid WHcAg-PreS VLPs elicits equivalent high titer anti-PreS antibodies in wildtype and HBcAg-Tg mice. Another advantage of immunizing with a closely related species variant of the HBcAg is that CD4+ and possibly CD8+ T cell sites within the WHcAg that differ only marginally from the sites within the HBcAg (i.e., 1 or 2 amino acids) may elicit so-called "bystander" T cell help and may also even break T cell tolerance of the HBV patient's HBcAg-specific T cells by virtue of higher avidity T cell crossreactivity. Historically, breaking T cell tolerance to self antigens has been accomplished by immunization with closely related species variant proteins (induction of experimental autoimmune arthritis, diabetes, encephalitis etc.). Specifically, in Aim 1 we propose to insert 3 HBsAg-PreS(1) and 1 HBsAg-PreS(2) neutralizing B cell epitopes onto the WHcAg VLP carrier and optimize the constructs based on assembly, yield, stability and immunogenicity. In Aim 2 the therapeutic efficacy of the VLP-based vaccine candidates will be evaluated in a transgenic (Tg) mouse model of HBV replication, in which HBV replicates in hepatocytes at levels comparable to that in the infected livers of chronic HBV patients without evidence of cytopathology. The hybrid WHcAg-PreS VLP candidates have the potential to reduce HBV load in at least 3 ways: (1) anti-PreS antibodies should clear serum HBV through immune complexes; (2) WHc/HBcAg-crossreactive CD4+ T cells may reduce HBV replication in the liver through inflammatory cytokine production; and (3) WHc/HBcAg-crossreactive CD8+ T cells may reduce viral replication in the liver through cytokine production as well as through direct hepatocyte cytolysis. The ability of immunization with the WHcAg-PreS VLP candidates to reduce HBV load through all of these mechanisms will be monitored in the HBV-Tg mouse model (Aim 2). It is anticipated that the combination of these two technologies, the ability to produce hybrid-VLPs (VLP Biotech) and the murine model of chronic HBV infection (TSRI), will enable us to produce and test the therapeutic efficacy of at least 4 hybrid WHcAg-PreS VLPs in a two-year timeframe.