Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells.
Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells.
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DOI：
10.1038/s41541-018-0071-7
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发表时间：
2018
期刊：
影响因子：
9.2
通讯作者：
Draper SJ
中科院分区：
文献类型：
--
作者：
Jin J;Tarrant RD;Bolam EJ;Angell-Manning P;Soegaard M;Pattinson DJ;Dulal P;Silk SE;Marshall JM;Dabbs RA;Nugent FL;Barrett JR;Hjerrild KA;Poulsen L;Jørgensen T;Brenner T;Baleanu IN;Parracho HM;Tahiri-Alaoui A;Whale G;Moyle S;Payne RO;Minassian AM;Higgins MK;Detmers FJ;Lawrie AM;Douglas AD;Smith R;de Jongh WA;Berrie E;Ashfield R;Draper SJ
恶性疟原虫网织红细胞结合蛋白同源物5（PfRH 5）是一种领先的无性血液阶段疟疾疫苗候选者。在临床试验的准备中，使用ExpreS 2平台（基于黑腹果蝇S2稳定细胞系系统）在cGMP下产生称为“RH5.1”的全长PfRH 5蛋白疫苗作为可溶性产物。在开发高产单克隆S2细胞系后，在cGMP活动前生产主细胞库。使用C-标签亲和色谱法处理培养物上清液，然后进行尺寸排阻色谱法和病毒减少过滤。整个过程产生>400 mg高纯度RH5.1蛋白。QC检测显示MCB和RH5.1产品符合所有规定的验收标准，包括无菌性、纯度和鉴别。RH5.1疫苗产品在−80 °C下储存，可稳定储存18个月以上。在佐剂系统AS 01 B中配制后对蛋白质的表征表明，RH5.1在临床疫苗施用所需的时间范围内是稳定的，并且在添加RH5.1后对AS 01 B的脂质体制剂没有可辨别的影响。随后的小鼠免疫证实了RH5.1/AS 01 B疫苗具有免疫原性，并且在体外可诱导针对血液期恶性疟原虫的功能性生长抑制抗体。RH5.1/AS 01 B被认为适用于人类，并已进入I/IIa期临床试验。我们的数据支持未来使用果蝇S2细胞和C-tag平台技术，以实现符合cGMP的其他新型和“难以表达”的重组蛋白疫苗的生物制造。一种针对血液阶段疟疾的候选疫苗克服了先前的障碍进入临床试验。PfRH 5蛋白是恶性疟原虫血液阶段感染的重要促进剂，也是疫苗策略的有希望的靶点。不幸的是，以免疫原性的、临床上可行的方式生产蛋白质的努力遇到了困难。在这里，由英国詹纳研究所的西蒙德雷珀领导的研究人员使用果蝇表达系统生产了超过400毫克的高纯度蛋白质。该疫苗与免疫增强佐剂一起配制，在体外试验中，该疫苗在小鼠中引发了对血液阶段恶性疟原虫具有抑制作用的抗体。PfRH 5候选疫苗及其佐剂已被批准在英国进行临床试验，作者希望所使用的表达系统可能有利于其他“困难”蛋白的表达。
Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a leading asexual blood-stage vaccine candidate for malaria. In preparation for clinical trials, a full-length PfRH5 protein vaccine called “RH5.1” was produced as a soluble product under cGMP using the ExpreS2 platform (based on a Drosophila melanogaster S2 stable cell line system). Following development of a high-producing monoclonal S2 cell line, a master cell bank was produced prior to the cGMP campaign. Culture supernatants were processed using C-tag affinity chromatography followed by size exclusion chromatography and virus-reduction filtration. The overall process yielded >400 mg highly pure RH5.1 protein. QC testing showed the MCB and the RH5.1 product met all specified acceptance criteria including those for sterility, purity, and identity. The RH5.1 vaccine product was stored at −80 °C and is stable for over 18 months. Characterization of the protein following formulation in the adjuvant system AS01B showed that RH5.1 is stable in the timeframe needed for clinical vaccine administration, and that there was no discernible impact on the liposomal formulation of AS01B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01B vaccine was immunogenic and could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01B was judged suitable for use in humans and has since progressed to phase I/IIa clinical trial. Our data support the future use of the Drosophila S2 cell and C-tag platform technologies to enable cGMP-compliant biomanufacture of other novel and “difficult-to-express” recombinant protein-based vaccines. A vaccine candidate for blood-stage malaria has overcome previous hurdles to enter clinical trials. The protein PfRH5 is an essential blood-stage infection facilitator of malarial parasite Plasmodium falciparum, and a promising target for vaccine strategies. Unfortunately, efforts to produce the protein in an immunogenic, clinically-viable way have been met with difficulty. Here, researchers led by Simon Draper, from the UK’s Jenner Institute, used a fruit fly expression system to produce over 400 mg of high-purity protein. Formulated with an immunity-boosting adjuvant, the vaccine elicited antibodies in mice that proved inhibitory to blood-stage P. falciparum during in vitro assays. The PfRH5 vaccine candidate and its adjuvant have been approved for a clinical trial in the UK, and the authors hope that the expression system used may be beneficial in the expression of other ‘difficult’ proteins.
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