The current coronavirus disease 2019 (COVID-19) pandemic was the result of the rapid transmission of a highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is no efficacious vaccine or therapeutic. Toward the development of a vaccine, here we expressed and evaluated as potential candidates four versions of the spike (S) protein using an insect cell expression system: receptor binding domain (RBD), S1 subunit, the wild-type S ectodomain (S-WT), and the prefusion trimer-stabilized form (S-2P). We showed that RBD appears as a monomer in solution, whereas S1, S-WT, and S-2P associate as homotrimers with substantial glycosylation. Cryo-electron microscopy analyses suggested that S-2P assumes an identical trimer conformation as the similarly engineered S protein expressed in 293 mammalian cells but with reduced glycosylation. Overall, the four proteins confer excellent antigenicity with convalescent COVID-19 patient sera in enzyme-linked immunosorbent assay (ELISA), yet show distinct reactivities in immunoblotting. RBD, S-WT and S-2P, but not S1, induce high neutralization titres (>3-log) in mice after a three-round immunization regimen. The high immunogenicity of S-2P could be maintained at the lowest dose (1 μg) with the inclusion of an aluminium adjuvant. Higher doses (20 μg) of S-2P can elicit high neutralization titres in non-human primates that exceed 40-times the mean titres measured in convalescent COVID-19 subjects. Our results suggest that the prefusion trimer-stabilized SARS-CoV-2 S-protein from insect cells may offer a potential candidate strategy for the development of a recombinant COVID-19 vaccine.

当前的2019年冠状病毒疾病（COVID-19）大流行是高致病性冠状病毒，严重的急性呼吸综合征冠状病毒2（SARS-CoV-2）快速传播的结果，目前尚无有效的疫苗或治疗剂。为了开发疫苗，在这里我们使用昆虫细胞表达系统表达并评估了穗蛋白（S）蛋白质的四个版本作为潜在候选者：受体结合域（RBD），S1亚基，野生型S胞外域（S- WT）和融合前三聚体稳定形式（S-2P）。我们显示，RBD在溶液中以单体形式出现，而S1，S-WT和S-2P以均三聚体的形式伴有大量糖基化。低温电子显微镜分析表明，S-2P假定的三聚体构象与在293个哺乳动物细胞中表达的类似工程化的S蛋白相同，但糖基化程度降低。总体而言，这四种蛋白质在酶联免疫吸附测定（ELISA）中与恢复期的COVID-19患者血清具有优异的抗原性，但在免疫印迹中却表现出不同的反应性。三轮免疫方案后，RBD，S-WT和S-2P而非S1在小鼠中诱导高中和效价（> 3-log）。含有铝佐剂可以将S-2P的高免疫原性维持在最低剂量（1μg）。较高剂量（20μg）的S-2P可以在非人灵长类动物中引起高中和滴度，其超过恢复期COVID-19受试者中平均滴度的40倍。