An Epitope-Substituted DNA Vaccine Improves Safety and Immunogenicity against Dengue Virus Type 2

Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope (E) protein of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP) mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT)-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.

登革病毒（Dengue virus, DENV）是一种全球性流行病原，可分为4个血清型（DENV1~4）。针对登革病毒包膜（E）蛋白的交叉反应性非中和抗体，可结合细胞表面的Fcγ受体（Fcγ receptors, FcγR），进而通过抗体依赖增强效应（antibody-dependent enhancement, ADE）加重异型血清型病毒的感染。鉴定并修饰增强型表位或可缓解登革病毒感染的增强效应。本研究对针对登革病毒结构域I~II的交叉反应性单克隆抗体DB21-6与DB39-2进行了表征；这类抗体在体外与体内均仅表现出微弱的中和活性，却能强效增强登革病毒的感染。此外，这两株增强型单克隆抗体DB21-6与DB39-2可与登革热患者感染后血清中的抗体产生竞争结合。研究通过噬菌体展示技术、结构预测以及病毒样颗粒（virus-like particle, VLP）突变体定位，鉴定了这两株增强型单克隆抗体的表位：DB21-6的反应残基为N8、R9、V12与E13，而DB39-2的反应残基为N8、R9与E13。对N8位点进行替换，可在维持病毒样颗粒分泌能力的同时，降低DB21-6与DB39-2的结合活性。相较于野生型（wild-type, WT）免疫血清，N8位点替换（N8R）DNA疫苗诱导的免疫血清在体外与体内均表现出更强的中和活性与保护效果。此外，使用N8R免疫血清处理可降低AG129小鼠的感染致死增强效应。上述研究结果表明，对增强型表位进行鉴定与替换，可作为开发安全型登革疫苗的全新策略。