Sharma Shailendra Kumar, de Val Natalia, Bale Shridhar, Guenaga Javier, Tran Karen, Feng Yu, Dubrovskaya Viktoriya, Ward Andrew B, Wyatt Richard T
IAVI Neutralizing Antibody Center at the Scripps Research Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Cell Rep. 2015 Apr 28;11(4):539-50. doi: 10.1016/j.celrep.2015.03.047. Epub 2015 Apr 16.
Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, the conformationally fixed HIV-1 envelope glycoprotein (Env) trimers in the pre-fusion state (SOSIP) display molecular homogeneity and structural integrity at relatively high levels of resolution. However, the SOSIPs necessitate full Env precursor cleavage, which requires endogenous furin overexpression. Here, we developed an alternative strategy using flexible peptide covalent linkage of Env subdomains to produce soluble, homogeneous, and cleavage-independent Env mimics, called native flexibly linked (NFL) trimers, as vaccine candidates. This simplified design avoids the need for furin co-expression and, in one case, antibody affinity purification to accelerate trimer scale-up for preclinical and clinical applications. We have successfully translated the NFL design to multiple HIV-1 subtypes, establishing the potential to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses.
病毒糖蛋白通过pH激活或受体结合激活介导病毒进入，并且以亚稳态的融合前状态存在，这种状态可通过定向理性设计来稳定。正如最近报道的那样，处于融合前状态的构象固定的HIV-1包膜糖蛋白（Env）三聚体（SOSIP）在相对较高的分辨率下显示出分子同质性和结构完整性。然而，SOSIP需要完全切割Env前体，这需要内源性弗林蛋白酶的过表达。在这里，我们开发了一种替代策略，利用Env亚结构域的柔性肽共价连接来产生可溶性、均一且不依赖切割的Env模拟物，称为天然柔性连接（NFL）三聚体，作为疫苗候选物。这种简化的设计避免了弗林蛋白酶共表达的需要，并且在一种情况下，避免了抗体亲和纯化，以加速三聚体的扩大生产用于临床前和临床应用。我们已经成功地将NFL设计转化为多种HIV-1亚型，确立了其成为生产用于HIV-1或其他病毒的类似天然、有序Env三聚体的通用方法的潜力。
