A high-growth PR8 virus for pandemic vaccine production in ST6-Vero cells

用于在 ST6-Vero 细胞中生产大流行疫苗的高生长 PR8 病毒

• 批准号: 8251012
• 负责人: Pamuk Bilsel
• 金额: $ 22.01万
• 依托单位: FLUGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-27 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8251012
• 关键词: Antigens; Biological Assay; Biological Sciences; Bioreactors; California; Cell Culture Techniques; Cell Density; Cell Line; Cells; Clinical; Dose; Engineering; Generations; Genes; Genome; Goals; Growth; H1N1 vaccine; Hemagglutination; Hemagglutinin; Human; Immune response; Industry; Infection; Influenza; Influenza A Virus, H1N1 Subtype; Investigation; KAI1 gene; Kilogram; Kinetics; MDCK cell; Meridians; Methodology; Methods; Mus; Mutation; National Institute of Allergy and Infectious Disease; Parents; Phase; Plaque Assay; Plasmids; Process; Production; Productivity; Property; Proteins; Puerto Rico; Qualifying; Relative (related person); Reliance; Safety; Sampling; Seeds; Serial Passage; Serum-Free Culture Media; Sialyltransferases; Speed; Suspension Culture; System; Technology; Time; TimeLine; Vaccine Production; Vaccines; Variant; Vero Cells; Vertebral column; Viral; Virus; base; density; egg; experience; flu; immunogenicity; in vivo; influenza outbreak; influenza virus vaccine; influenzavirus; lot production; manufacturing process; method development; non-compliance; novel; novel vaccines; pandemic disease; pandemic influenza; phase 1 study; phase 2 study; positional cloning; receptor; response; scale up; tumor; tumorigenic

DESCRIPTION (provided by applicant): A high-growth PR8 virus for pandemic vaccine production in ST6-Vero cells. In 2009, the traditional means of vaccine manufacturing for the H1N1 influenza pandemic failed to yield sufficient doses during the peak of the crisis. A vaccine production method that can universally accommodate variant strains of virus in much shorter timelines would greaty benefit the interdiction of a growing pandemic threat. Cell-based production systems have been proposed to replace the protracted egg-based processes. Cell culture-based technology is robust, reliable and is a more rapid and efficient alternative to egg-based technology for vaccine producers. For pandemic vaccine production, distinct advantages of cell-based over egg-based vaccine production are speed (12 weeks), capacity (kg scale) and versatility (quick response to new antigens) so that production lots with significantly higher viru yields may be obtained in nearly half of the time. Despite great promise, no cell-based system has been approved for use by the FDA and technical barriers currently exist in the use of cell lines for manufacturing of flu vaccine. These barriers are 1) limited virus production from current cell systems, and 2) reliance on cell lines that spontaneously form tumors in vivo. To overcome these challenging barriers, FluGen Inc. has generated a Vero cell line that stably expresses the human 2,6 sialyltransferase gene I necessary for generation of human-specific influenza receptors (ST6-Vero). ST6-Veros offer greater infection rates with virus isolated from clinical samples than normal Vero cells. ST6-Vero also may be grown to highly packed cell densities in commercial bioreactors thereby providing more host cells for viral replication. The goal of this proposal is to generate a high-growth donor A/Puerto Rico/8/34 (HG-PR8) virus to further enhance viral propagation in FluGen's ST6-Vero cells. The specific aims of the Phase I project are to 1) generate a high-growth PR8 donor virus by serial passaging in ST6-Vero cells; 2) demonstrate increased productivity of ST6-Vero cells by producing HGPR8- H1N1 vaccine virus in scaled-up suspension cultures; and 3) confirm the antigenicity and immunogenicity of the ST6-Vero produced HGPR8-H1N1 pandemic virus by demonstrating protection in mice. Upon completion of these Phase I aims, a HG-PR8/ST6-Vero cell-based vaccine production system will be realized that is capable of yielding extraordinary titers of 109 pfu/ml, and triggering the expected immune response, as evidenced by protection of mice by viral challenge. For Phase II, qualification of the ST6-Vero cell line and method development for scaling up the process of producing H1N1 pandemic vaccine under GMP conditions will be pursued. The intended commercial product is a licensable platform vaccine production system that can rapidly produce pandemic vaccine efficiently and at very high titers. PUBLIC HEALTH RELEVANCE: A high-growth PR8 virus for pandemic vaccine production in ST6-Vero cells. In 2009, the traditional means of vaccine manufacturing in eggs failed to yield sufficient doses during the peak of the H1N1 influenza pandemic crisis. FluGen Inc. proposes to engineer a cell-based vaccine manufacturing system with the highest rate of production in the industry for these Phase I investigations. Our proprietary ST6-Vero cell line stably expresses the human ?2,6 sialyltransferase gene I necessary for generation of human-specific influenza receptors. ST6-Vero cells result in increased virus yields and hence produce greater amounts of hemagglutinin than normal Vero cells. ST6-Vero also may be grown to highly packed cell densities in commercial bioreactors thereby providing more host cells for viral replication. To enhance cell-based productivity even further, we will generate a high-growth A/Puerto Rico/8/34 (HGPR8) donor virus specific for ST6-Vero which will confer high viral growth property in ST6-Vero cells. The combination of the high replication of HGPR8 in highly infectable ST6-Vero cells that may be grown to immense densities will provide a rapid and high yielding vaccine production system.

描述(申请人提供)：一种用于在ST6-Vero细胞中生产大流行疫苗的高增长PR8病毒。2009年，H1N1流感大流行的传统疫苗生产手段在危机高峰期未能生产出足够剂量的疫苗。一种能够在更短的时间内普遍适应不同病毒株的疫苗生产方法，将极大地有利于阻止日益增长的大流行威胁。已经提出了基于细胞的生产系统来取代旷日持久的基于鸡蛋的生产过程。基于细胞培养的技术是强大、可靠的，对于疫苗生产商来说，是一种比基于鸡蛋的技术更快速、更有效的替代技术。对于大流行疫苗的生产，基于细胞的疫苗生产相对于基于鸡蛋的疫苗生产的明显优势是速度(12周)、能力(公斤规模)和通用性(对新抗原的快速反应)，因此可以在近一半的时间内获得显著更高的病毒产量的生产批次。尽管前景看好，但FDA还没有批准使用基于细胞的系统，而且目前在使用细胞系生产流感疫苗方面存在技术障碍。这些障碍是1)当前病毒产量有限 细胞系统，以及2)依赖体内自发形成肿瘤的细胞系。为了克服这些具有挑战性的障碍，FluGen Inc.培育了一种稳定表达人类2，6唾液酸基转移酶基因I的Vero细胞系，该基因是产生人类特异性流感受体(ST6-Vero)所必需的。与正常的Vero细胞相比，ST6-Veros对从临床样本中分离的病毒具有更高的感染率。ST6-Vero也可以在商业生物反应器中培养到高度密集的细胞密度，从而为病毒复制提供更多的宿主细胞。这项提议的目标是产生一种高增长的捐赠者A/波多黎各/8/34(HG-PR8)病毒，以进一步增强病毒在FluGen的ST6-Vero细胞中的繁殖。第一阶段项目的具体目标是1)通过在ST6-Vero细胞中连续传代来产生高增长的PR8供体病毒；2)通过在放大的悬浮培养中生产HGPR8-H1N1疫苗病毒来提高ST6-Vero细胞的生产力；以及3)通过在小鼠身上展示保护作用来证实ST6-Vero产生的HGPR8-H1N1大流行病毒的抗原性和免疫原性。在完成这些第一阶段的目标后，将实现一个基于HG-PR8/ST6-Vero细胞的疫苗生产系统，能够产生109pfu/ml的非凡效价，并引发 预期的免疫反应，如病毒攻击对小鼠的保护。对于第二阶段，将继续进行ST6-Vero细胞系的鉴定和方法开发，以扩大在GMP条件下生产H1N1大流行疫苗的过程。预期的商业产品是一个可获得许可的平台疫苗生产系统，该系统可以快速、高效和非常高滴度地生产大流行疫苗。 公共卫生相关性：一种用于在ST6-Vero细胞中生产大流行疫苗的高增长PR8病毒。2009年，在H1N1流感大流行危机的高峰期，传统的鸡蛋疫苗生产手段未能产生足够的剂量。FluGen Inc.建议为这些第一阶段的研究设计一种基于细胞的疫苗制造系统，该系统具有业界最高的生产率。我们的专利ST6-Vero细胞系稳定表达人类2，6唾液酸基转移酶基因I，这是产生人类特异性流感受体所必需的。ST6-Vero细胞导致病毒产量增加，因此比正常Vero细胞产生更多的血凝素。ST6-Vero也可以在商业生物反应器中培养到高度密集的细胞密度，从而为病毒复制提供更多的宿主细胞。为了进一步提高基于细胞的生产力，我们将产生一种针对ST6-Vero的高增长A/波多黎各/8/34(HGPR8)供体病毒，它将赋予ST6-Vero细胞高病毒生长特性。HGPR8在高度可感染的ST6-Vero细胞中的高复制相结合，可以培养到极高的密度，将提供快速和高产的疫苗生产系统。