Alphavirus Replicon Vaccines against Botulinum Neurotox*

描述（由申请人提供）： 拟开展研究的目的是构建针对所有7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价和临床前动物研究;进行针对BoNT（血清型A和B）的二价疫苗的生产规模扩大和GMP制造，准备并提交研究性新药（IND）申请，并进行I期临床试验。BoNT通过气溶胶途径具有高度致命性，是一种可信和重大的生物恐怖主义威胁。现有的研究疫苗有明显的缺点。我们提出了一种新的方法，使用一个繁殖缺陷，单循环，RNA复制子载体系统，来自委内瑞拉马脑炎（VEE）病毒的减毒株，产生病毒样复制子颗粒（VRP）表达的羧基末端的一半的重链（HC）的所有血清型的BoNT。初步的动物研究已经证明了这种方法的安全性和可行性，因为单价和二价VRP疫苗都诱导了针对表达的BoNT Hc的中和抗体应答，这赋予了针对同源毒素攻击的长期保护。该项目将包括七个具体目标。(1)构建表达所有七种BoNT血清型的Hc片段的VRP疫苗。(2)对这些疫苗进行体外表征。(3)在小鼠攻毒模型中评价临床前安全性、免疫原性和有效性。(4)对选择用于临床评价的二价（血清型A和B）BoNT Hc片段VRP疫苗的GMP合规性生产工艺进行规模化开发。(5)进行该疫苗的GMP生产。(6)进行基准临床前研究并准备IND。（7）进行1期临床试验。为了实现这些目标，将BoNT Hc的人类密码子优化基因插入VEE复制子载体中。使用排除复制子/辅助重组的新型DNA辅助系统，复制子RNA将被包装到具有赋予树突状细胞向性的VEE糖蛋白外壳的VRP中。将使用已建立的动物模型评价安全性、免疫原性和有效性。将根据HIV VRP疫苗的既往经验进行工艺放大、GMP生产、临床前和临床试验。这些研究的结果将为将来全面的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.