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 破伤风和白喉毒素是预防疾病的高效疫苗。作为“载体蛋白”， Tetanius和白喉毒素增强了小分子和多糖的免疫原性。然而， Tetanius毒素（TTXD）仅代表TTXD疫苗和TTXD疫苗中蛋白质的20-70％ 包含数百种“非意外/污染物”梭蛋白。在TTXD之前通常需要纯化 使用共轭疫苗载体。用甲醛对TT进行排毒，使用30天以上的孵育 阻断无法与抗原结合的子集赖氨酸。医疗合作者 威斯康星大学已经设计了一个全长的毒性破伤风毒素（M8TT），并具有8个独立突变 减少催化，易位和结合功能。在这里，Fina Biosoltions（Finabio）提出的提案 并在具有独特的氧化环境的专有工程大肠杆菌菌株中生产M8TT。这 菌株已成功地产生多克/l量的CRM197，一种突变形式的白喉形式 已成功用作重组表达的疫苗蛋白载体的毒素。这个建议 使用重组DNA技术，生物技术，生物化学和免疫学方法来生产 并测试下一代结合破伤风疫苗平台的免疫学效力。 第一阶段的具体目的是：亚克隆和扩大生产和净化> 1 g/l的M8TT> 95％ 纯度;并测试M8TT与常规TTXD的免疫学特性，以产生共轭物 血友病影响亚型B多核糖磷酸糖PRP与M8TT和TTXD结合 如果PRP-TTXD比PRP-TTXD是对PRP和TT的偶联疫苗。如果成功，第二阶段 研究将将M8TT制造过程优化为50升生产量表，并表征 临床前试验的安全性和效率。此外，M8TT作为疫苗载体蛋白的实用性将进一步 用其他抗原（包括小分子和其他多糖）探索。最终，上级 破伤风疫苗蛋白将通过推进一项使用新的，新的，一种新的工业技术来商业化 现代化，经济，有效和安全的共轭TT疫苗平台。