Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
PLoS Negl Trop Dis. 2012;6(5):e1636. doi: 10.1371/journal.pntd.0001636. Epub 2012 May 1.
Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control.
METHODOLOGY/PRINCIPAL FINDINGS: We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials.
CONCLUSIONS/SIGNIFICANCE: We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.
登革热病毒（DENV）是世界热带和亚热带地区的重大公共卫生威胁。针对病毒包膜（E）蛋白的治疗性抗体代表了一种有前途的疾病控制免疫疗法。
方法/主要发现：我们针对登革热病毒 2 型（DENV-2）的 E 蛋白产生了 17 种新型的小鼠单克隆抗体（mAb），这些 mAb 与 E 蛋白具有高反应性。我们进一步使用 DENV-2 的重组 E 蛋白结构域 I-II（E-DI-II）和 III（E-DIII）对 mAb 进行了剖析。通过噬斑减少中和试验（PRNT）和用致死剂量的 DENV-2 进行的小鼠保护试验，我们鉴定了 4 种针对 DENV-2 感染具有高中和活性的血清型特异性 mAb。在这 4 种 mAb 中，针对 E-DIII 的 DB32-6 是针对多种 DENV-2 株系的最强中和 mAb。通过噬菌体展示和病毒样颗粒（VLPs），我们发现 E-DIII A 链中的残基 K310 是 mAb DB32-6 结合 E-DIII 的关键。我们成功地将 DB32-6 转化为人源化版本，保留了中和 DENV-2 的效力，并且不会增强病毒感染。DB32-6 在两种小鼠模型中，甚至在暴露后试验中，对不同株系的 DENV-2 引起的死亡率均显示出治疗效果。
结论/意义：我们使用新型表位作图策略，通过将噬菌体展示与 VLPs 相结合，鉴定了具有强中和活性的重要 A 链表位。这项研究引入了潜在的治疗性抗体，这些抗体可能能够提供针对多种 DENV-2 感染的广泛保护，而不会在人类中增强其活性。
