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。正在使用各种方法来开发有效的 RSV 疫苗。RSV 融合 (F) 亚基，尤其是裂解的三聚体融合前 F，是正在开发的最有希望的候选疫苗之一。融合前构象在自然感染过程中引发了大部分中和抗体。然而，这种融合前构象是亚稳态的并且易于转化为融合后构象，从而阻碍了这种构建体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融合前 F 糖蛋白 DS-Cav1，它在动物研究中显示出高免疫原性并诱导中和反应。IE. 需要具有融合前构象的切割三聚体）并提供高蛋白质表达水平。本报告描述了候选疫苗的开发，包括载体设计和细胞培养工艺开发，以应对这些挑战。与双基因载体的共表达相比，单个质粒共转染以表达 DS-Cav1 和弗林蛋白酶（用于 DS-Cav1 切割和激活）表现出更好的蛋白质产物表达和融合前构象。基于这些测量选择顶级克隆。通过接种密度优化和双相低温温度下降进一步提高了蛋白质表达水平。综合努力导致收获时的高产量分批补料生产约为 1,500 毫克/升（或高达 15,000 剂/升）。该过程被放大并证明在 50 L 规模下可重复用于毒性和 I 期临床试验。初步的 I 期数据表明融合前抗原具有良好的疗效（Crank 等，2019）。