Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
mBio. 2024 Sep 11;15(9):e0192224. doi: 10.1128/mbio.01922-24. Epub 2024 Aug 14.
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 10 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.
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.
严重急性呼吸系统综合症冠状病毒 2（SARS-CoV-2）大流行是最近历史上最大的公共卫生危机。许多人预计未来冠状病毒会进入人类群体。因此，了解这些病毒的基本生物学特性至关重要。在自然感染中，SARS-CoV-2 刺突（S）糖蛋白与所有其他病毒蛋白共同表达，这些蛋白修饰细胞区室以最大限度地组装病毒粒子。相比之下，当蛋白在孤立表达时，大多数 S 被降解，例如当前的分子疫苗。为了探究 S 的成熟途径，我们通过将 S 融合到四跨膜蛋白 CD81 上来重新定向其成熟。CD81 是细胞外囊泡（EVs）或外泌体的重要组成部分。所有细胞都会大量产生 EVs，将其挤出到血液和淋巴中，并在细胞之间和全身系统（估计每毫升血浆中有 10 个 EV）中转移货物。EVs 可以在无关供体之间输注，就像血小板一样。当将稳定脯氨酸的 Delta S 融合到 CD81 的灵活的大细胞外环中，而不是在溶酶体中降解时，S 被挤出到 EVs 中。大量产生含有 CD81-S 融合的 EVs，并可以分离到高纯度。纯化的 CD81::S EVs 结合 ACE2，通过低温电子显微镜（EM）观察到单个 EV 上显示的 S。CD81::S 融合 EVs 无毒，并在小鼠中引发针对 S 三聚体和 RBD 抗体的反应。本报告展示了一种设计路径，可以在不依赖潜在致病性非结构病毒蛋白共表达的情况下最大限度地组装和释放病毒糖蛋白。
严重急性呼吸系统综合症冠状病毒 2 大流行是最近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 融合到四跨膜蛋白 CD81 上。由此产生的分子在细胞外囊泡中分泌，并在小鼠中诱导抗体。这可能是一种通用的病毒糖蛋白疫苗设计途径。
