严重急性呼吸综合征冠状病毒2(SARS-CoV-2)同源三聚体刺突(S)蛋白通过与血管紧张素转换酶2(ACE2)受体结合来介导宿主细胞进入,因此是冠状病毒病19(COVID-19)疫苗中的关键病毒抗原。尽管有COVID-19疫苗,低疫苗覆盖率以及未接种疫苗和免疫功能受损的受试者导致了值得关注的SARS-CoV-2变体的出现。因此,新的和/或更新的疫苗的持续开发对于防止此类新变体是必不可少的。在这项研究中,我们使用昆虫细胞-杆状病毒表达载体系统(IC-BEVS)开发了一种可扩展的生物过程,以生产高质量的S蛋白,融合前构象稳定,纳入基于病毒体的COVID-19候选疫苗。通过探索不同的生物过程工程策略(即,信号肽,杆状病毒转移载体,细胞系,感染策略和制剂缓冲液),我们能够获得~4mg/L的纯化S蛋白,which,据我们所知,是迄今为止使用昆虫细胞获得的最高值。此外,昆虫细胞来源的S蛋白表现出与哺乳动物细胞相似的聚糖加工和储存时的中期稳定性(在-80和4°C下或在5个冻融循环后长达90天)。值得注意的是,S蛋白的抗原性,作为单一抗原或显示在病毒体表面,通过ELISA证实,与ACE2受体结合,泛SARS抗体CR3022和S蛋白上各种表位簇的中和抗体。在4°C下储存1个月的病毒体-S上也保持结合能力。这项工作证明了使用IC-BEVS产生高度糖基化和复杂S蛋白的潜力,在不损害其完整性和抗原性的情况下,纳入基于病毒体的COVID-19候选疫苗。
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and/or updated vaccines is essential for protecting against such new variants. In this study, we developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein, stabilized in its pre-fusion conformation, for inclusion in a virosome-based COVID-19 vaccine candidate. By exploring different bioprocess engineering strategies (i.e., signal peptides, baculovirus transfer vectors, cell lines, infection strategies and formulation buffers), we were able to obtain ~4 mg/L of purified S protein, which, to the best of our knowledge, is the highest value achieved to date using insect cells. In addition, the insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage (up to 90 days at -80 and 4 °C or after 5 freeze-thaw cycles). Noteworthy, antigenicity of S protein, either as single antigen or displayed on the surface of virosomes, was confirmed by ELISA, with binding of ACE2 receptor, pan-SARS antibody CR3022 and neutralizing antibodies to the various epitope clusters on the S protein. Binding capacity was also maintained on virosomes-S stored at 4 °C for 1 month. This work demonstrates the potential of using IC-BEVS to produce the highly glycosylated and complex S protein, without compromising its integrity and antigenicity, to be included in a virosome-based COVID-19 vaccine candidate.