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.

总结 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了小分子和多糖的免疫原性。然而，在这方面， 破伤风类毒素（TTxd）仅占TTxd疫苗中蛋白质的20-70%， 含有数百种“非预期/污染物”梭菌蛋白。在TTxd之前通常需要进行纯化 使用结合疫苗载体。TT用甲醛解毒，使用超过30天的孵育， 阻断了一个赖氨酸亚群，该亚群不能用于与抗原缀合。医学合作者 威斯康星州学院已经设计了具有8个独立突变的全长无毒性破伤风毒素（M8 TT 减少催化、移位和结合功能。FinaBio（FinaBio）建议开发 并在专有工程E.大肠杆菌菌株，具有独特的氧化环境。这 一种菌株已成功地用于生产几克/升的CRM 197，一种突变形式的白喉 已成功用作重组表达的疫苗蛋白载体的毒素。这项建议 利用重组DNA技术、生物技术、生物化学和免疫学方法， 并测试下一代破伤风结合疫苗平台的免疫效力。 阶段I的具体目标是：亚克隆和规模化生产并纯化>95%的> 1 g/L的M8 TT 并测试M8 TT相对于常规TTxd的免疫学性质以产生M8 TT的缀合物。 流感嗜血杆菌亚型B多核糖醇磷酸糖PRP与M8 TT和TTxd结合，以确定 如果PRP-TTxd是比PRP-TTxd更有效PRP和TT缀合疫苗。如果成功，第二阶段 研究将优化M8 TT生产工艺至50 L生产规模，并表征产品， 临床前试验中的安全性和有效性。此外，M8 TT作为疫苗载体蛋白的效用将进一步被研究。 探索了额外的抗原，包括小分子和其他多糖。最终，一个上级 破伤风疫苗蛋白将通过用新的， 现代化、经济、有效、安全的TT结合疫苗平台。