Department of Molecular Microbiology and Immunology, Saint Louis University, Saint Louis, United States.
Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, United States.
Elife. 2023 Sep 19;12:RP87555. doi: 10.7554/eLife.87555.
A hallmark of dengue virus (DENV) pathogenesis is the potential for antibody-dependent enhancement, which is associated with deadly DENV secondary infection, complicates the identification of correlates of protection, and negatively impacts the safety and efficacy of DENV vaccines. Antibody-dependent enhancement is linked to antibodies targeting the fusion loop (FL) motif of the envelope protein, which is completely conserved in mosquito-borne flaviviruses and required for viral entry and fusion. In the current study, we utilized saturation mutagenesis and directed evolution to engineer a functional variant with a mutated FL (D2-FL), which is not neutralized by FL-targeting monoclonal antibodies. The FL mutations were combined with our previously evolved prM cleavage site to create a mature version of D2-FL (D2-FLM), which evades both prM- and FL-Abs but retains sensitivity to other type-specific and quaternary cross-reactive (CR) Abs. CR serum from heterotypic (DENV4)-infected non-human primates (NHP) showed lower neutralization titers against D2-FL and D2-FLM than isogenic wildtype DENV2 while similar neutralization titers were observed in serum from homotypic (DENV2)-infected NHP. We propose D2-FL and D2-FLM as valuable tools to delineate CR Ab subtypes in serum as well as an exciting platform for safer live-attenuated DENV vaccines suitable for naïve individuals and children.
登革热病毒（DENV）发病机制的一个标志是抗体依赖性增强作用的潜力，这与致命的 DENV 二次感染有关，使保护相关因素的鉴定复杂化，并对 DENV 疫苗的安全性和有效性产生负面影响。抗体依赖性增强作用与针对包膜蛋白融合环（FL）基序的抗体有关，该基序在蚊媒黄病毒中完全保守，是病毒进入和融合所必需的。在本研究中，我们利用饱和诱变和定向进化工程技术，构建了一种具有突变 FL（D2-FL）的功能性变体，该变体不受针对 FL 的单克隆抗体中和。将 FL 突变与我们之前进化的 prM 切割位点结合，产生了成熟的 D2-FL（D2-FLM），它逃避了 prM 和 FL-Abs 的识别，但仍对其他型特异性和四元交叉反应（CR）Abs 敏感。来自异型（DENV4）感染的非人类灵长类动物（NHP）的 CR 血清对 D2-FL 和 D2-FLM 的中和效价低于同源野生型 DENV2，而在同型（DENV2）感染的 NHP 血清中观察到类似的中和效价。我们提出 D2-FL 和 D2-FLM 是在血清中描绘 CR Ab 亚型的有价值的工具，也是一种安全的减毒 DENV 疫苗的激动人心的平台，适合于初治个体和儿童。
