Optimization and Advanced Proof-of-Concept Studies of a Listeria-vectored Multi-Antigenic Vaccine against Tuberculosis

项目摘要 结核病（TB）是世界上最重要的疾病之一，并且是针对结核病的安全有效的疫苗。 病原体结核分枝杆菌（Mtb），其比目前仅部分可用的更有效 有效M.牛卡介苗（Bacille Calmette-Guérin，BCG）疫苗是急需的。一般认为 需要一种改进的BCG替代疫苗和一种有效的异源加强疫苗， 与结核病的斗争。该项目的目的是优化和进行先进的概念验证研究 在小动物和非人灵长类动物（NHP）中， 重组减毒单核细胞增生李斯特菌载体疫苗抗结核病。 活减毒重组单核细胞增生李斯特菌（rLm）疫苗提供了优于其他疫苗的主要优点。 加强疫苗的方法，包括佐剂中的蛋白质和病毒载体疫苗，就成本而言， 易于制造、免疫原性和功效。在初步研究中，我们发现了一种改进的 多缺失李斯特菌载体（Lm ΔactA ΔinlB prfA*），并证明表达四个关键基因的rLm疫苗 免疫保护性Mtb蛋白（rLmMtb 4Ag）当用作免疫保护剂时， 在小鼠中针对Mtb气溶胶攻击的初免-加强免疫接种策略中的异源加强疫苗， 豚鼠此外，通过第一代rLm载体递送免疫保护性Mtb蛋白更容易。 比通过重组病毒载体递送或与有效佐剂一起施用更有效。 本申请的目的是优化表达4 Mtb的Lm-载体疫苗的表达 抗原;将其抗原库扩展到六种抗原以提高其效力;并评估优化的 作为一种独立疫苗和作为一种疫苗的安全性、免疫原性和有效性的最终先导rLm疫苗候选物 在小鼠、豚鼠和非人灵长类动物（NHP）中给予BCG致敏动物异源加强疫苗 肺结核模型。我们将通过以下方式实现这一目标：a）优化以下的蛋白表达盒： rLmMtb 4Ag疫苗;系统评价其他新型Mtb抗原的免疫原性和在 小鼠，选择前两种抗原，随后构建rLmMtb 6 Ag先导疫苗候选物; B） 对优化的rLmMtb 6Ag先导候选疫苗进行全面的概念验证研究， 作为单独和异源加强疫苗在小鼠模型中的安全性、免疫原性和有效性 c）对先导rLmMtb 6Ag疫苗作为标准进行选定的概念验证研究， 单独和异源加强疫苗在豚鼠模型中的安全性、免疫原性和疗效 肺结核;以及d）由于Aeras需要NHP中的概念验证以使疫苗进入临床前开发- 评价了主要的rLmMtb 6Ag候选疫苗作为独立疫苗的安全性、免疫原性和 与Aeras、Bioqual和Tulane National Primate合作在NHP肺结核模型中的疗效 研究中心。

PROJECT SUMMARY Tuberculosis (TB) is one of the world's most important diseases, and a safe and effective vaccine against the causative agent Mycobacterium tuberculosis (Mtb) that is more potent than the currently available only partially effective M. bovis strain Bacille Calmette-Guérin (BCG) vaccine is sorely needed. It is generally acknowledged that both an improved replacement vaccine for BCG and a potent heterologous booster vaccine are needed in the fight against TB. The purpose of this project is to optimize and conduct advanced proof-of-concept studies in small animals and non-human primates (NHP) of a second-generation heterologous multiantigenic recombinant attenuated Listeria monocytogenes-vectored vaccine against TB. Live attenuated recombinant Listeria monocytogenes (rLm) vaccines offer major advantages over other approaches to booster vaccines, including protein in adjuvant and virus-vectored vaccines, in terms of cost, ease of manufacture, immunogenicity and efficacy. In preliminary studies, we have identified an improved multi-deletional Listeria vector (Lm ΔactA ΔinlB prfA*) and demonstrated that rLm vaccines expressing four key immunoprotective Mtb proteins (rLmMtb4Ag) substantially augment protective immunity when used as a heterologous booster vaccine in a prime-boost vaccination strategy against Mtb aerosol challenge in mice and guinea pigs. Moreover, delivering the immunoprotective Mtb protein via a first generation rLm vector was more efficacious than delivering it via a recombinant viral vector or administering it with a potent adjuvant. The goal of this application is to optimize expression of an Lm-vectored vaccine expressing 4 Mtb antigens; expand its antigen repertoire to six antigens to increase its potency; and to evaluate the optimized final lead rLm vaccine candidate for safety, immunogenicity and efficacy as a standalone vaccine and as a heterologous booster vaccine to BCG-primed animals in mouse, guinea pig, and non-human primate (NHP) models of pulmonary TB. We shall accomplish this goal by: a) Optimizing the protein expression cassette of rLmMtb4Ag vaccine; systematically evaluating additional novel Mtb antigens for immunogenicity and efficacy in mice, selecting the top two antigens, and subsequently constructing a rLmMtb6Ag lead vaccine candidate; b) Conducting comprehensive proof-of-concept studies of the optimized rLmMtb6Ag lead vaccine candidate for safety, immunogenicity, and efficacy as standalone and heterologous booster vaccine in the mouse model of pulmonary TB; c) Conducting selected proof-of-concept studies of the lead rLmMtb6Ag vaccine as a standa- lone and heterologous booster vaccine for safety, immunogenicity and efficacy in a guinea pig model of pulmonary TB; and d) as Aeras requires proof-of-concept in NHP for a vaccine to enter preclinical develop- ment, evaluating the lead rLmMtb6Ag candidate as a standalone vaccine for safety, immunogenicity and efficacy in a NHP model of pulmonary TB in collaboration with Aeras, Bioqual, and Tulane National Primate Research Centre.