Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
Int J Mol Sci. 2023 Sep 27;24(19):14622. doi: 10.3390/ijms241914622.
Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (0.04-0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72-144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.
病毒样颗粒 (VLPs) 已被提议作为 SARS-CoV-2 疫苗开发的一种有吸引力的工具，既可以作为（1）一种具有高免疫原性和低反应原性的疫苗候选物，也可以作为（2）在功能和中和测定中替代活病毒。尽管已经在 Sf9 昆虫细胞中探索了多种 SARS-CoV-2 VLP 设计，但确保 VLP 可行的一个关键参数是 VLP 刺突产量（即 VLP 中的刺突蛋白含量），这在很大程度上尚未报道。在这项研究中，我们表明，通过表达刺突蛋白与天然冠状病毒膜和/或包膜蛋白结合来生产 SARS-CoV-2 VLP 的常见策略可以形成 VLP，但刺突产量极低（约 0.04-0.08mg/L）。相比之下，将刺突外域融合到流感血凝素（HA）跨膜域和细胞质尾部，并共同表达 M1 可将 VLP 刺突产量提高到约 0.4mg/L。更重要的是，这种产量的增加转化为更高的 VLP 刺突抗原密度（96 个刺突单体/VLP），更接近天然 SARS-CoV-2 病毒（72-144 个刺突单体/病毒）。假型化进一步允许生产功能性的 alpha（B.1.1.7）、beta（B.1.351）、delta（B.1.617.2）和 omicron（B.1.1.529）SARS-CoV-2 VLP，这些 VLP 能够结合靶标 ACE2 受体。最后，我们使用简单的基于细胞的 ELISA 检测试剂盒在生物安全 1 级（BSL-1）进行检测，证明了假型化 VLP 用于测试中和抗体活性的实用性。总的来说，这项研究强调了在实现更高 VLP 刺突产量方面，假型化相对于天然 SARS-CoV-2 VLP 设计的优势，并证明了假型化 VLP 作为替代活病毒在 SARS-CoV-2 变体疫苗和治疗开发中的有用性。
