Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9 West Watkins Mill Rd., Gaithersburg, MD, 20878, USA.
Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9 West Watkins Mill Rd., Gaithersburg, MD, 20878, USA.
J Biotechnol. 2021 Jan 10;325:261-270. doi: 10.1016/j.jbiotec.2020.10.014. Epub 2020 Oct 15.
Respiratory syncytial virus (RSV) is a highly contagious virus causing severe infection in infants and the elderly. Various approaches are being used to develop an effective RSV vaccine. The RSV fusion (F) subunit, particularly the cleaved trimeric pre-fusion F, is one of the most promising vaccine candidates under development. The pre-fusion conformation elicits the majority of neutralizing antibodies during natural infection. However, this pre-fusion conformation is metastable and prone to conversion to a post-fusion conformation, thus hindering the potential of this construct as a vaccine antigen. The Vaccine Research Center (VRC) at the National Institutes of Health (NIH) designed a structurally stabilized pre-fusion F glycoprotein, DS-Cav1, that showed high immunogenicity and induced a neutralizing response in animal studies. To advance this candidate to clinical manufacturing, a production process that maintained product quality (i.e. a cleaved trimer with pre-fusion conformation) and delivered high protein expression levels was required. This report describes the development of the vaccine candidate including vector design and cell culture process development to meet these challenges. Co-transfection of individual plasmids to express DS-Cav1 and furin (for DS-Cav1 cleavage and activation) demonstrated a superior protein product expression and pre-fusion conformation compared to co-expression with a double gene vector. A top clone was selected based on these measurements. Protein expression levels were further increased by seeding density optimization and a biphasic hypothermia temperature downshift. The combined efforts led to a high-yield fed-batch production of approximately 1,500 mg/L (or up to 15,000 doses per liter) at harvest. The process was scaled up and demonstrated to be reproducible at 50 L-scale for toxicity and Phase I clinical trial use. Preliminary phase I data indicate the pre-fusion antigen has a promising efficacy (Crank et al., 2019).
呼吸道合胞病毒（RSV）是一种高度传染性病毒，可导致婴儿和老年人严重感染。目前正在采用各种方法来开发有效的 RSV 疫苗。RSV 融合（F）亚基，特别是切割的三聚体前融合 F，是正在开发的最有前途的疫苗候选物之一。前融合构象在自然感染过程中引发了大多数中和抗体。然而，这种前融合构象是亚稳定的，容易转化为融合后构象，从而阻碍了该结构作为疫苗抗原的潜力。美国国立卫生研究院（NIH）的疫苗研究中心（VRC）设计了一种结构稳定的前融合 F 糖蛋白 DS-Cav1，该蛋白在动物研究中表现出高免疫原性，并诱导了中和反应。为了将该候选物推进到临床生产，需要开发一种生产工艺，该工艺能够保持产品质量（即具有前融合构象的切割三聚体）并实现高蛋白表达水平。本报告介绍了该疫苗候选物的开发，包括载体设计和细胞培养工艺开发，以应对这些挑战。单独转染质粒表达 DS-Cav1 和弗林（用于 DS-Cav1 切割和激活），与共表达双基因载体相比，显示出更高的蛋白产物表达和前融合构象。根据这些测量结果，选择了一个最佳克隆。通过优化接种密度和两相低温降温进一步提高了蛋白表达水平。通过组合努力，实现了约 1500mg/L（或每升高达 15000 剂）的高产量补料分批生产。该工艺已放大，并在 50L 规模上证明可用于毒性和 I 期临床试验。初步 I 期数据表明，前融合抗原具有良好的疗效（Crank 等人，2019）。
