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

描述（由申请方提供）：一种用于在ST 6-Vero细胞中生产大流行性疫苗的高生长PR 8病毒。 2009年，H1N1流感大流行的传统疫苗生产方式未能在危机高峰期生产足够的剂量。一种能够在更短的时间内普遍适应变异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过程。基于细胞培养的技术是强大的，可靠的，是一个更快速，更有效的替代鸡蛋为基础的疫苗生产技术。对于大流行疫苗生产，基于细胞的疫苗生产相对于基于蛋的疫苗生产的明显优势在于速度（12周）、容量（千克规模）和多功能性（对新抗原的快速反应），因此可以在近一半的时间内获得具有显著更高维鲁产量的生产批次。尽管前景广阔，但FDA尚未批准使用基于细胞的系统，目前在使用细胞系制造流感疫苗方面存在技术障碍。这些障碍是：1）从电流中产生的有限病毒 细胞系统，和2）依赖于在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.已经产生了稳定表达产生人特异性流感受体所必需的人2，6唾液酸转移酶基因I的Vero细胞系（ST 6-Vero）。与正常Vero细胞相比，ST 6-Veros对从临床样品中分离的病毒的感染率更高。ST 6-Vero也可以在商业生物反应器中生长至高度堆积的细胞密度，从而为病毒复制提供更多的宿主细胞。本提案的目的是生成高生长供体A/波多黎各/8/34（HG-PR 8）病毒，以进一步增强FluGen的ST 6-Vero细胞中的病毒繁殖。 I期项目的具体目标是：1）通过在ST 6-Vero细胞中连续传代产生高生长的PR 8供体病毒; 2）通过在按比例放大的悬浮培养物中产生HGPR 8- H1N1疫苗病毒来证明ST 6-Vero细胞的生产力增加; 3）通过在小鼠中证明保护作用来证实ST 6-Vero产生的HGPR 8-H1N1大流行性病毒的抗原性和免疫原性。在完成这些I期目标后，将实现基于HG-PR 8/ST 6-Vero细胞的疫苗生产系统，该系统能够产生109 pfu/ml的非凡滴度，并触发免疫反应。 预期的免疫应答，如通过病毒攻击保护小鼠所证明的。在第二阶段，将对ST 6-Vero细胞系进行鉴定，并开发方法，以扩大在GMP条件下生产H1N1大流行性流感疫苗的工艺。预期的商业产品是一种可获得许可的平台疫苗生产系统，可以快速有效地生产高滴度的大流行疫苗。 公共卫生相关性：用于在ST 6-Vero细胞中生产大流行疫苗的高生长PR 8病毒。2009年，在H1N1流感大流行危机的高峰期，鸡蛋中生产疫苗的传统方法未能产生足够的剂量。FluGen Inc.建议设计一个基于细胞的疫苗生产系统，该系统在这些I期研究中具有行业内最高的生产率。我们专有的ST 6-Vero细胞系稳定表达人类？产生人特异性流感受体所必需的2，6唾液酸转移酶基因I。ST 6-Vero细胞导致病毒产量增加，因此产生比正常Vero细胞更大量的血凝素。ST 6-Vero也可以在商业生物反应器中生长至高度堆积的细胞密度，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率，我们将产生对ST 6-Vero特异性的高生长A/波多黎各/8/34（HGPR 8）供体病毒，其将在ST 6-Vero细胞中赋予高病毒生长特性。HGPR 8在高度可感染的ST 6-Vero细胞中的高复制的组合可以生长到巨大的密度，这将提供快速和高产的疫苗生产系统。

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.