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

描述（由申请人提供）：一种在ST6-Vero细胞中生产大流行性疫苗的高生长PR8病毒。2009年，在危机最严重的时候，H1N1流感大流行的传统疫苗生产手段未能生产出足够的剂量。一种能够在短得多的时间内普遍适应病毒变异株的疫苗生产方法，将极大地有利于阻止日益严重的大流行威胁。基于细胞的生产系统已被提议取代旷日持久的基于鸡蛋的生产过程。对疫苗生产商来说，基于细胞培养的技术稳健、可靠，是一种比基于鸡蛋的技术更快速、更有效的替代方法。就大流行性流感疫苗生产而言，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在于速度（12周）、容量（公斤级）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病毒产量的生产批次。尽管前景光明，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制造流感疫苗方面存在技术障碍。这些屏障限制了当前病毒的产生

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.