登革病毒血清特异型人源中和抗体对抗体依赖性感染增强(ADE)的抑制及分子机制

Dengue virus has four serotypes. Primary infection of dengue virus induces life-long protection against the same serotype. Secondary infection of a different serotype is the major risk factor for dengue hemorrhagic fever. Human antibody responses are dominated by non- and weakly-neutralizing antibodies, which are cross-reactive with multiple virus serotypes. These antibodies mediate viral infection of monocytes, causing antibody-dependent enhancement (ADE). Previous studies demonstrate: 1) not all neutralizing antibodies are able to inhibit ADE; 2) whether a neutralizing antibody can inhibit ADE depends on its ability to compete with the immunodominant non- and weakly-neutralizing antibodies for virus binding. It is generally recognized that serotype-specific neutralizing antibodies are the major component of the protective immune response against dengue virus. Here we found that human serotype-specific neutralizing antibodies target various epitopes, and a portion of these epitopes do not overlap with those of non- and weakly-neutralizing antibodies. Based on these preliminary results, we hypothesize that: 1) not all serotype-specific neutralizing antibodies are able to inhibit ADE; 2) besides competing with non- and weakly-neutralizing antibodies for virus binding, serotype-specific neutralizing antibodies can inhibit ADE by alternative mechanisms. This study aims to evaluate the ability of serotype-specific neutralizing antibodies in ADE inhibition. Furthermore, by a combination of fluorescent-labeled virus, confocal microscopy and cryo-electron microscopy, the molecular mechanism of ADE inhibition will be studied. The anticipated result will improve our understanding of ADE inhibition by dengue virus neutralizing antibodies, which could have significant impacts on vaccine and therapeutic antibody development.

登革病毒有四种血清型，初次感染病毒对同血清型终生免疫，二次感染不同血清型是登革出血热的主要风险因素。人体免疫应答产生大量与多血清型病毒交叉反应的非中和、弱中和抗体，介导病毒感染单核细胞，引起抗体依赖性感染增强（ADE）。研究发现：1）中和抗体并不都能抑制ADE；2）一种抗体是否抑制ADE取决于其能否与非中和、弱中和抗体竞争结合病毒。一般认为血清特异型中和抗体对登革病毒感染起主要保护作用。申请人前期研究发现人源血清特异型中和抗体抗原表位多样，且部分不与非中和、弱中和抗体重合。据此我们提出假说：1）血清特异型中和抗体并不都能抑制ADE；2）除了与非中和、弱中和抗体竞争结合病毒，存在其他抑制ADE的机制。本课题将验证血清特异型中和抗体抑制ADE的功能，结合荧光标记病毒、共聚焦显微镜和冷冻电镜等方法分析其分子机制。预期结果将提高对中和抗体抑制ADE的认识，对疫苗和治疗性抗体的开发产生重要影响。

登革病毒有四种血清型，初次感染病毒对同血清型终生免疫，二次感染不同血清型是登革出血热的主要风险因素。人体免疫应答产生大量与多血清型病毒交叉反应的非中和、弱中和抗体，介导病毒感染单核细胞，引起抗体依赖性感染增强（ADE）。研究发现：1）中和抗体并不都能抑制ADE；2）一种抗体是否抑制ADE取决于其能否与非中和、弱中和抗体竞争结合病毒。一般认为血清特异型中和抗体对登革病毒感染起主要保护作用。申请人前期研究发现人源血清特异型中和抗体抗原表位多样，且部分不与非中和、弱中和抗体重合。近期研究报道，靶向包膜蛋白结构域III非免疫优势的抗体具有开发成治疗性抗体的潜力。本课题围绕一株靶向非免疫优势表位的中和抗体，结合结构分析与分子动力学模拟，预测能够提高其与多种血清型病毒抗原形成新的相互作用（如盐桥）的突变。由于登革热病毒不同血清型间序列区别较大，模拟结果发现对于不同的血清型，突变体将与不同抗原形成不同的新相互作用。实验结果证实了所设计突变能够提高抗体与多种血清型登革热病毒重组抗原的亲和力。已知中和抗体在亚中和浓度下能引起ADE，本研究所设计的抗体可能具有更狭窄的引起ADE的浓度窗口，具有一定的应用潜力。进一步研究将着重于提高抗体与登革热血清四型病毒的结合能力，实现对全部四种血清型登革热病毒的结合与中和。这可能将依赖于基于机器学习的蛋白设计与高通量筛选新方法。

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