DEVELOPING MULTI-ANTIGEN VACCINE FOR THE TREATMENT OF CHRONIC HBV

项目摘要/摘要 这个SBIR项目的最终目标是开发一种免疫疗法，基于我们的专利病毒样- 水泡(VLV)平台，用于治疗慢性乙肝病毒感染患者。在第一阶段，使用 小鼠慢性乙肝感染模型，我们已经建立了一个概念验证，即用VLV治疗 表达多种乙肝病毒抗原(聚合酶、HBcAg和MHBs)的载体-称为VLV-3xT2a-导联 由于乙肝表面抗原(乙肝表面抗原)的显著丧失，乙肝表面抗原是清除乙肝病毒的替代标志[见 进度报告]。在完成第一阶段的里程碑中，我们展示了(I)诱导多特异性HBVT 细胞，(Ii)对急性攻击性感染的保护，以及(Iii)在小鼠模型中HBs Ag的显著降低 在第二阶段，我们将使用相同的小鼠慢性模型来测试优化的素材- 加强方案，以最大限度地提高清除病毒所需的免疫反应。同时，我们将确定 临床实用的最佳给药途径(肌肉内或皮下注射)。此外, 我们将进行药代动力学、毒性和体内生物分布研究，以符合 FDA对我们IND前会议关于VLV-3xT2A免疫疗法开发的要求的回应。这个 该项目的目标是开发一种免疫疗法，以实现功能性治愈，其特征是 使用新型VLV乙肝病毒持续丢失HBs Ag(有或不有HBs Ag抗体血清转换) 治疗。第二阶段的具体目标如下：目标1：定义、优化和验证主数和升压 治疗方案。这一目标将包括四项任务：制定主要助推战略和测量免疫力 根据评估产生的应答：1)T细胞应答的幅度、特异性和功能性 表型(T细胞细胞因子反应、细胞溶解活性等)和2)抗体反应(即抗体滴度 和抗体特异性)。我们还将确定VLV-3xT2APrime-Boost的顺序和时间 能产生最佳免疫反应的给药。测定T细胞对乙肝病毒抗原的反应 在人类MHC I类和MHC II类分子的背景下来自VLV-3xT2A。比较肌肉内注射与 通过分析幼稚小鼠的CD4+和CD8+T细胞反应的皮下给药途径。评估 AAV介导的慢性乙肝模型VLV-3xT2APRIME-Boost方案的疗效我们会 确定来自幼稚小鼠最强初始增强的免疫反应是否可以清除持久性 通过携带乙肝病毒在小鼠肝脏中启动乙肝病毒复制。目标2：生物分布、药代动力学、 安全性、毒理学和放大研究。这一目标涉及两项任务：进行生物分配和 VLV-3xT2A型药物的药代动力学研究 在小鼠身上进行VLV安全性和毒理学研究。这些结果将构成设计和 由GLP合同实验室进行未来的毒理学研究，根据FDA对IND提交给 在人类身上进行VLV-3xT2a的临床研究。确定扩大VLV生产的条件，并 符合当前良好制造规范(CGMP)标准的工艺步骤，并说明 最佳Prime-Boost VLV疗法的质量和稳定性概况。

PROJECT SUMMARY/ABSTRACT The ultimate objective of this SBIR project is to develop an immunotherapy, based on our patented virus-like- vesicle (VLV) platform, for the treatment of patients infected with chronic hepatitis B virus. In Phase I, using a murine model of chronic HBV infection, we have established a Proof-of-Concept that treatment with a VLV vector expressing multiple HBV antigens (Polymerase, HBcAg and MHBs) - referred to as VLV-3xT2A - leads to a significant loss of hepatitis B surface antigen (HBsAg), a surrogate marker for HBV clearance [see Progress Report]. In fulfilling the Phase I milestones, we demonstrated (i) induction of multi-specific HBV T cells, (ii) protection from acute challenge infection, and (iii) significant reduction in HBsAg in a mouse model of persistent HBV replication.In Phase II, we will use the same murine chronic model to test an optimized prime- boost regimen to maximize the immune responses needed for clearance of the virus. In parallel, we will identify an optimal route of administration (intra-muscular or subcutaneous) that is practical for clinical use. In addition, we will perform pharmacokinetics, toxicity, and in vivo biodistribution studies in line with the feedback from the FDA’s response to our Pre-IND meeting request regarding development of VLV-3xT2A immunotherapy. The goal of this project is to develop an immunotherapy in order to achieve a functional cure, characterized by sustained loss of HBsAg (with or without HBsAg antibody seroconversion) using new VLV-based HBV therapies. The specific aims for Phase II are as follows: Aim 1: Define, optimize and validate prime and boost treatment regimens. This aim will include four tasks: Develop a prime-boost strategy and measure immune responses generated with respect to evaluating: 1) T cell response magnitude, specificity, and functional phenotype (T cell cytokine responses, cytolytic activity, etc.), and 2) antibody responses (i.e., antibody titers and antibody specificity). We will also determine the order and timing of VLV-3xT2A prime-boost administrations that results in the optimum immune response. Determine T cell responses to HBV antigens from VLV-3xT2A in the context of human MHC class I and MHC class II molecules. Compare intramuscular vs. subcutaneous routes of administration by assaying CD4+ and CD8+ T cell responses in naïve mice. Evaluate the efficacy of VLV-3xT2A prime-boost regimen using the AAV-mediated model of chronic HBV. We will determine whether the immune response from the strongest prime-boost in naïve mice can clear persistent HBV replication initiated in the mouse liver by AAV delivery of HBV. Aim 2: Biodistribution, pharmacokinetics, safety, toxicology, and scale-up studies. This aim involves two tasks: Conduct biodistribution and pharmacokinetic studies of VLV-3xT2A using the optimal route of administration determined in Aim 1 and conduct VLV safety and toxicology studies in mice. These results will form the basis for designing and conducting future toxicology studies by a GLP contract lab, per FDA requirements for IND submission to conduct clinical studies on VLV-3xT2A in humans. Identify conditions for scaling up VLV production and process steps amenable to current good manufacturing practice (cGMP) standards and characterize the quality and stability profiles of the optimal prime-boost VLV therapeutics.