严重急性呼吸系统综合症冠状病毒2(SARS-CoV-2)大流行引起了近代历史上最大的公共卫生危机。许多人预计未来冠状病毒会引入人类群体。因此,了解这些病毒的基本生物学至关重要。在自然感染中,SARS-CoV-2Spike(S)糖蛋白与所有其他病毒蛋白共表达,修改细胞区室以最大化病毒体组装。相比之下,当蛋白质分离表达时,大部分S被降解,就像目前的分子疫苗一样。探索S的成熟途径,我们通过将S融合到tetraspanin蛋白CD81来重定向其成熟。CD81是细胞外囊泡(EV)或外泌体的限定成分。电动汽车是由所有细胞大量产生的,挤压成血液和淋巴,并在细胞和系统之间转移货物(估计每mL血浆1012EV)。电动汽车,像血小板一样,可以在无关的供体之间输血。当将脯氨酸稳定形式的应变DeltaS融合到柔性中时,CD81的大细胞外环,而不是在溶酶体中降解,S被挤出到电动汽车中。含有CD81-S融合物的EV大量产生并且可以分离至高纯度。纯化的CD81::SEV结合ACE2,并且通过低温电子显微镜(EM)观察在个体EV上显示的S。CD81::S-融合EV是无毒的并且在小鼠中引发抗S三聚体和抗RBD抗体应答。该报告显示了最大化病毒糖蛋白组装和释放的设计路径,而不依赖于潜在致病性非结构性病毒蛋白的共表达。
目的:严重急性呼吸道综合症冠状病毒2大流行引起了近期历史上最大的公共卫生危机。为了了解S的成熟途径,我们将S与四跨膜蛋白CD81融合。所得分子分泌在细胞外囊泡中并在小鼠中诱导抗体。这可能是病毒糖蛋白疫苗的一般设计途径。
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic caused the biggest public health crises in recent history. Many expect future coronavirus introductions into the human population. Hence, it is essential to understand the basic biology of these viruses. In natural infection, the SARS-CoV-2 Spike (S) glycoprotein is co-expressed with all other viral proteins, which modify cellular compartments to maximize virion assembly. By comparison, most of S is degraded when the protein is expressed in isolation, as in current molecular vaccines. To probe the maturation pathway of S, we redirected its maturation by fusing S to the tetraspanin protein CD81. CD81 is a defining constituent of extracellular vesicles (EVs) or exosomes. EVs are generated in large numbers by all cells, extruded into blood and lymph, and transfer cargo between cells and systemically (estimated 1012 EVs per mL plasma). EVs, like platelets, can be transfused between unrelated donors. When fusing the proline-stabilized form of strain Delta S into the flexible, large extracellular loop of CD81 rather than being degraded in the lysosome, S was extruded into EVs. CD81-S fusion containing EVs were produced in large numbers and could be isolated to high purity. Purified CD81::S EVs bound ACE2, and S displayed on individual EV was observed by cryogenic electron microscopy (EM). The CD81::S-fusion EVs were non-toxic and elicited an anti-S trimer and anti-RBD antibody response in mice. This report shows a design path to maximize viral glycoprotein assembly and release without relying on the co-expression of potentially pathogenic nonstructural viral proteins.
OBJECTIVE: The severe acute respiratory syndrome coronavirus 2 pandemic caused the biggest public health crises in recent history. To understand the maturation pathway of S, we fused S to the tetraspanin protein CD81. The resulting molecule is secreted in extracellular vesicles and induces antibodies in mice. This may be a general design path for viral glycoprotein vaccines.