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首页> 外文会议>Conference on vaccine technology VI >ACCELERATED MASS PRODUCTION OF INFLUENZA VIRUS SEED STOCKS IN HEK-293 SUSPENSION CELL CULTURES BY REVERSE GENETICS
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ACCELERATED MASS PRODUCTION OF INFLUENZA VIRUS SEED STOCKS IN HEK-293 SUSPENSION CELL CULTURES BY REVERSE GENETICS

机译：逆向遗传学加速了HEK-293悬浮细胞培养物中流感病毒种子股的批量生产

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Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Viral Vaccine (CW) as reported by the 2015 WHO Informal Consultation report titled "Influenza Vaccine Response during the Start of a Pandemic". In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza A/Puerto Rico/8/34 H1N1 strain, and advances in the large-scale transfection of suspension cultured HEK-293 cells. Transfection in shake flasks was performed using 1ug of total plasmid and 1×10~6 cells/mL. The supernatant was harvested after 48 hpt and used to infect a new shake flasks at 1×10~6 cells/mL for virus amplification. 3-L bioreactor was inoculated and transfected at 1×10~6 cells/mL with 1ug of total plasmid and harvested after 48hpt and the virus generated was amplified in shake flask. Quantification by TCID50, SRID, Dot-blot and TRPS were performed as well as characterization by TEM and HA and NA sequencing. Small-scale transfection in shake flasks generated 1.5×10~5 IVP/mL after 48 hpt and 1×10~7 IVP/mL after 96 hpi. For large-scale experiment a 3-L controlled stirred tank bioreactor resulted in supernatant (P0) virus titer of 5×10~4 IVP/mL and 2.8×10~7 IVP/mL after only one amplification (P1) in HEK-293 suspension cells. We demonstrate the efficent generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CW for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Thus, this innovative approach is better suited to rationally design and mass produce the CW within timelines dictated by pandemic situations and produce effective responsiveness than previous methodology.
机译：尽管对基于鸡蛋的流感疫苗产生的能力和替代技术进行了重大进展，但对候选病毒疫苗（CW）的候选病毒疫苗（CW）有限，仍有限制为2015年的非正式磋商报告所需的时间受到限制“流感疫苗反应在大流行开始期间”。在以前的工作中，我们已经表明，HEK-293悬浮液和血清培养基中的细胞培养是流感候选疫苗的细胞培养制造的有效生产平台。本报告中，利用流感A / Puerto Rico / 8/34 H1N1菌株的逆遗传学的重组DNA技术，以及悬浮培养HEK-293细胞的大规模转染的进展。使用1ug总质粒和1×10〜6个细胞/ ml进行摇烧烧瓶中的转染。在48个HPT后收获上清液，并用于在1×10〜6个细胞/ ml下感染用于病毒扩增的新摇瓶。将3-L生物反应器接种并在1×10〜6个细胞/ mL中转染，在1UG的总质粒中，在48HPT后收获，并且在摇瓶中扩增生物的病毒。通过TCID50，SRID，点印迹和TRP的定量以及TEM和HA和NA测序表征。在98 HPT后，在48 HPT后产生1.5×10〜5 IVP / mL的摇瓶中的小尺寸转染，96 HPI后1×10〜7 IVP / mL。对于大型实验，3-L控制搅拌釜生物反应器导致上清液（P0）病毒滴度为5×10〜4 IVP / mL，仅在HEK-293中仅一次扩增（P1）后2.8×10〜7 IVP / ml悬浮细胞。我们证明了在两个顺序步骤（转染/救援和感染/生产）的受控生物反应器条件下PR8骨架重新置于H1N1的效果产生。从HA和Na大流行序列开始，这种方法可以在两周内为流感疫苗制造提供CW。因此，这种创新的方法更适合理性设计，并且群众在大流行情况决定的时间表内产生CW，并产生比以前的方法的有效响应性。

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