Genetically detoxified tetanus toxin for use in vaccines

用于疫苗的基因解毒破伤风毒素

• 批准号: 10006309
• 负责人: Andrew Lees
• 金额: $ 28.91万
• 依托单位: FINA BIOSOLUTIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-20 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006309
• 关键词: Antibody Formation; Antigens; Bacterial Toxins; Binding; Biochemistry; Biotechnology; Carrier Proteins; Catalysis; Chemicals; Clostridium tetani; Conjugate Vaccines; Diphtheria Toxin; Diphtheria Toxoid; Disease; Disulfides; Economics; Endotoxins; Engineering; Environment; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Fermentation; Formaldehyde; Generations; Genetic Engineering; Haemophilus influenzae; Haemophilus influenzae type b; Immunize; Immunologic Tests; Immunologics; Industrialization; Length; Lysine; Methods; Modernization; Molecular Analysis; Molecular Conformation; Mus; Mutate; Mutation; Organism; Phase; Polysaccharides; Production; Property; Proteins; Protocols documentation; Recombinants; Safety; Small Business Innovation Research Grant; Sugar Phosphates; System; Technology; Tetanus; Tetanus Toxin; Tetanus Toxoid; Tetanus Vaccine; Toxin; Toxoids; Vaccines; Wisconsin; base; cost; cross reacting material 197; disulfide bond; immunogenicity; in vivo; innovation; inorganic phosphate; manufacturability; manufacturing process; medical schools; next generation; phase 2 study; polyribitol phosphate; preclinical study; preclinical trial; prevent; receptor binding; scale up; small molecule; success

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.

概括 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增强小分子和多糖的免疫原性。然而， 破伤风类毒素 (TTxd) 仅占 TTxd 疫苗中蛋白质的 20-70%，并且 TTxd 疫苗 含有数百种“非预期/污染”梭菌蛋白。 TTxd 之前通常需要纯化 使用结合疫苗载体。 TT 经过 30 多天的培养，用甲醛进行了解毒， 阻断不能用于与抗原缀合的赖氨酸子集。医学界的合作者 威斯康星大学设计了一种全长无毒破伤风毒素 (M8TT)，具有 8 个独立突变 减少催化、易位和结合功能。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有工程大肠杆菌菌株中生产 M8TT。这 该菌株已成功用于生产数克/升量的 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。这个提议 采用重组DNA技术、生物技术、生物化学和免疫学方法来生产 并测试下一代破伤风结合疫苗平台的免疫效力。 第一阶段的具体目标是：亚克隆和扩大生产规模，并以 >95% 纯化 > 1 g/L 的 M8TT 纯度;并测试 M8TT 与传统 TTxd 的免疫学特性，以产生 流感嗜血杆菌 b 亚型聚核糖醇磷酸糖 PRP 缀合 M8TT 和 TTxd 进行测定 如果 PRP-TTxd 是比 PRP-TTxd 更有效的 PRP 和 TT 结合疫苗。如果成功的话，第二阶段 研究将优化 M8TT 制造工艺至 50 L 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效用也将进一步得到加强。 使用其他抗原进行探索，包括小分子和其他多糖。最终成为一名优秀的 破伤风疫苗蛋白将通过利用新的、有 50 年历史的工业技术来推进，从而实现商业化 现代化、经济、有效、安全的结合TT疫苗平台。