Alphavirus Replicon Vaccines against Botulinum Neurotox*

描述(由申请人提供)： 拟议研究的目标是构建针对所有七种血清型肉毒杆菌神经毒素(BoNT)的甲病毒复制子颗粒疫苗；对这些疫苗进行体外评估和临床前动物研究；进行生产放大和GMP生产针对BoNT(A型和B型)的双价疫苗；准备和提交研究性新药(IND)申请，并进行I期临床试验。通过气雾剂路线，BONT是高度致命的，是一个可信和重大的生物恐怖威胁。现有的研究疫苗存在重大缺陷。我们提出了一种新的方法，利用来自委内瑞拉马脑炎(VEE)病毒减毒株的繁殖缺陷、单循环RNA复制子载体系统，生产表达所有血清型BONT重链(HC)的羧基末端一半的病毒样复制子颗粒(VRP)。初步的动物研究已经证明了这种方法的安全性和可行性，因为单价和双价VRP疫苗都能诱导针对所表达的bot HC的中和抗体反应，从而对同源毒素的攻击提供长期保护。该项目将包括七个具体目标。(1)构建表达7种BONT血清型HC片段的VRP疫苗。(2)对这些疫苗进行体外鉴定。(3)在小鼠攻击模型中评价其临床前安全性、免疫原性和有效性。(4)规模化开发符合GMP的双价(A型和B型)BoT HC片段VRP疫苗的生产工艺，选择用于临床评估。(5)进行该疫苗的GMP生产。(6)开展基准临床前研究并准备IND。(7)进行一期临床试验。为了达到这些目的，人类密码子优化的BONT HC基因将被插入到VEE复制子载体中。使用一种新的DNA辅助系统，阻止复制子/辅助重组，复制子RNA将被包装成具有VEE糖蛋白涂层的VRP，赋予树突状细胞趋向性。安全性、免疫原性和有效性将使用已建立的动物模型进行评估。将根据以前艾滋病毒VRP疫苗的经验进行工艺放大、GMP制造、临床前和临床测试。这些研究结果将为未来全面的7价BoNT HC片段VRP疫苗的临床试验提供基础。

DESCRIPTION (provided by applicant): The objectives of the proposed research are to construct alphavirus replicon particle vaccines directed against all seven serotypes of botulinum neurotoxin (BoNT); perform in vitro evaluations and preclinical animal studies of these vaccines; perform production scale-up and GMP manufacture of a bivalent vaccine against BoNT (serotypes A and B), prepare and submit an Investigational New Drug (IND) application, and perform a phase I clinical trial. BoNT is highly lethal by the aerosol route and is a credible and significant bioterrorist threat. Existing investigational vaccines have significant shortcomings. We propose a novel approach, using a propagation-defective, single cycle, RNA replicon vector system, derived from an attenuated strain of Venezuelan equine encephalitis (VEE) virus, to produce virus-like replicon particles (VRP) expressing the carboxy-terminal half of the heavy chain (Hc) of all serotypes of BoNT. Preliminary animal studies have demonstrated the safety and feasibility of this approach, as both monovalent and bivalent VRP vaccines induced neutralizing antibody responses against the expressed BoNT Hc that conferred long-term protection against challenge with homologous toxin. The project will include seven specific aims. (1) Construct VRP vaccines expressing Hc fragments of all seven BoNT serotypes. (2) Perform in vitro characterization of these vaccines. (3) Evaluate preclinical safety, immunogenicity and efficacy in murine challenge models. (4) Perform scale-up development of processes for GMP-compliant manufacture of a bivalent (serotypes A and B) BoNT Hc fragment VRP vaccine selected for clinical evaluation. (5) Perform GMP manufacture of this vaccine. (6) Perform benchmark preclinical studies and prepare an IND. (7) Conduct a phase 1 clinical trial. To achieve these aims, human codon-optimized genes for BoNT Hc will be inserted into VEE replicon vectors. Using a novel DNA helper system that precludes replicon/helper recombination, the replicon RNAs will be packaged into VRP with a VEE glycoprotein coat that confers dendritic cell tropism. Safety, immunogenicity and efficacy will be evaluated using established animal models. Process scale-up, GMP manufacture, preclinical and clinical testing will be performed based on previous experience with a VRP vaccine for HIV. Results of these studies will provide the basis for future clinical trials of a comprehensive 7-valent BoNT Hc fragment VRP vaccine.