Genetically detoxified tetanus toxin for use in vaccines

摘要 破伤风和白喉类毒素是预防疾病的高效疫苗。作为“载体蛋白”， 破伤风和白喉类毒素可增强小分子和多糖的免疫原性。然而， 破伤风类毒素(TTxd)只占TTxd疫苗和TTxd疫苗中蛋白质的20%-70% 含有数百种“非预期的/受污染的”梭状芽胞杆菌蛋白质。在TTxd之前通常需要提纯 使用结合疫苗载体。TT用甲醛解毒，使用超过30天的孵化时间， 阻断一个不能与抗原结合的子集赖氨酸。医疗中心的合作者 威斯康星大学已经设计出一种具有8个独立突变的全长无毒破伤风毒素(M8TT) 减少催化、转位和结合功能。在这里，菲纳生物解决方案(FinaBio)建议开发 并在具有独特氧化环境的专利工程大肠杆菌菌株中制造M8TT。这 该菌株已成功用于生产多克/L剂量的CRM197，白喉的一种突变形式 已成功用作重组表达疫苗蛋白载体的毒素。这项建议 使用重组DNA技术、生物技术、生物化学和免疫学方法生产 并测试这一新一代结合破伤风疫苗平台的免疫效力。 第一阶段的具体目标是：亚克隆和规模化生产，提纯M8TT；1g/L at&gt；95% 纯度；并检测M8TT与常规TTxd的免疫学性质，以产生M8TT的结合物 结合M8TT和TTxd的b型流感嗜血杆菌多聚核糖醇磷酸糖 如果PRP-TTxd对PRP和TT是比PRP-TTxd更有效的结合疫苗。如果成功，则进入第二阶段 研究将M8TT的制造工艺优化到50 L的生产规模，并对产品进行表征 临床前试验的安全性和有效性。此外，M8TT作为疫苗载体蛋白的用途还将进一步 探索额外的抗原，包括小分子和其他多糖。归根结底，一个上级 破伤风疫苗蛋白将通过推进一项已有50年历史的工业技术实现商业化， 现代化、经济、高效、安全的TT结合疫苗平台。

Summary Tetanus and diphtheria toxoids are highly effective vaccines for preventing diseases. As “carrier proteins”, tetanus and diphtheria toxoids enhance the immunogenicity of small molecules and polysaccharides. However, tetanus toxoid (TTxd) represents only 20-70% of the protein in the TTxd vaccine and the TTxd vaccine contains hundreds of ‘un-intended/contaminant’ clostridial proteins. Purification is often needed prior to TTxd use a conjugate vaccine carrier. TT is detoxified with formaldehyde, using an over 30-day incubation that blocks a subset lysines that cannot then be used for conjugation with antigens. Collaborators at the Medical College of Wisconsin have engineered a full-length, atoxic tetanus toxin (M8TT) with 8 independent mutations reducing catalysis, translocation, and binding functions. Here, Fina Biosolutions (FinaBio) proposes to develop and manufacture M8TT in a proprietary engineered E. coli strain that has a unique oxidative environment. This strain has been used successfully to produce multi-grams/L amounts of CRM197, a mutated form of diphtheria toxin that has been successfully used as a recombinantly expressed vaccine protein carrier. This proposal uses recombinant DNA technology, biotechnology, biochemistry and immunological approaches to produce and test the immunological potency of this next generation conjugate tetanus vaccine platform. The Specific Aims for Phase I are to: subclone and scale up production and purify > 1 g/L of M8TT at >95% purity; and to test the immunological properties of M8TT versus conventional TTxd to produce a conjugate of Hemophilus influenzae subtype b polyribitol phosphate sugar PRP conjugated to M8TT and TTxd to determine if PRP-TTxd is a more potent conjugate vaccine to PRP and TT than PRP-TTxd. If successful, Phase II studies will optimize the M8TT manufacturing process to 50 L production scale and characterize the product for safety and efficacy in pre-clinical trials. In addition, the utility of M8TT as a vaccine carrier protein will be further explored with additional antigens, including small molecules and other polysaccharides. Ultimately, a superior tetanus vaccine protein will be commercialized by advancing a 50-year old industrial technology with a new, modernized, economic, effective, and safe conjugate TT vaccine platform.