序贯免疫策略诱导针对登革病毒保守抗原表位免疫应答的研究

Dengue fever is one of the most prevalent vector-borne infectious diseases in the tropical of subtropical regions of the world. During the last 5 year, the reported dengue fever cases increases logarithmically in China. The rate of its increase has reached number one among all C-type notifiable infectious diseases recorded by the Chinese CDC. Previously, it has been generally agreed that all dengue fever cases in China are imported, but a 2015 epidemiological study demonstrated the endemic circulation of DENV-1 in Guangdong province. .. Currently, only one tetravalent dengue vaccine, which is based on the yellow fever virus vector (YF17D) expressing 4 separate DENV prM and E proteins from each of the 4 dengue serotypes, has been licensed and marketed in selected Asian and Latin America countries. Despite some level of protective immunity, the vaccine failed to protect against infection by all 4 dengue serotypes, and it augmented disease severity in vaccinated young children under the age of 9 years. Thus, developing a more protective and safer vaccine is urgently needed. Recently, we have demonstrated in a phase I human clinical trials that sequential immunization with vaccines containing variant antigens elicited heightened immune responses to conserved epitopes. Therefore, we hypothesize that sequential immunization with recombinant protein vaccines and chimpanzee adenovirus vectored vaccines expressing variant dengue antigens is capable of stimulating greater antibody and T cell responses targeting conserved dengue epitopes. .. To test this hypothesis, we will construct recombinant protein vaccines comprised of the extracellular domain (E80) of dengue virus E protein representing each of the 4 major dengue serotypes. We will also make monovalent, bivalent and tetravalent chimpanzee adenovirus (AdC68) vectored vaccines expressing either 1, or 2, or 4 dengue E80 proteins. We will then compare the antibody and T cell responses among various vaccination strategies with these vaccines in mice. Finally, we will examine whether the most optimal vaccination regimen will confer protection against challenge by each of the 4 dengue serotypes in a rhesus macaque model. These studies will demonstrate whether sequential immunization is more superior to traditional vaccination regimens. It will also reveal whether AdC68 vector can be used effectively to construct novel dengue vaccines.

登革热是全世界流行最广的虫媒传染病之一。该病近5年来我国呈对数型增长,上升速率跃居丙类法定传染病之首。既往病例以输入型为主,如今登革病毒1型（DENV-1）在广东省已呈现本土化流行趋势。即将上市的赛诺菲公司的四价登革热疫苗虽然对部分血清型的DENV有保护作用，但在受试幼童中有明显副作用。所以,亟待研发更安全、有效的登革热疫苗。本课题负责人近期临床研究发现，采用多种疫苗序贯免疫可诱导针对保守抗原表位的免疫应答。.. 据此，本项目拟利用表达DENV-1至-4型E蛋白膜外区的重组蛋白疫苗和黑猩猩腺病毒载体疫苗对小鼠进行序贯免疫，比对该免疫策略和传统免疫策略的优劣,并利用恒河猴DENV感染模型评价疫苗诱导的免疫保护作用。期望该研究能揭示登革热疫苗新型免疫策略的保护机制,同时为进一步开发出以新型腺病毒为载体的登革热疫苗提供依据。

在登革疫苗研发中，如何诱导均衡的、四价保护性免疫应答是最关键的科学问题。既往研究中均期望通过调节4个单价免疫原的比例达到该目的，但效果不理想。本研究通过调整免疫策略，以“序贯免疫”取代传统的“混合免疫”方式，预期通过强烈激活针对“保守性表位”的免疫应答，来产生四价保护性免疫。初期研究以登革病毒包膜蛋白膜外区之EDIII结构域为抗原，发现两种免疫策略均可诱导出针对登革病毒I、II、III、IV型抗原的T细胞反应，以及针对登革病毒I、II、III型的中和抗体反应。相比较之下，“序贯免疫”比“混合免疫”诱导出更强的Th2免疫应答，及较弱的中和抗体反应（王欣等，2018）。在同期进行的拓展性研究中，我们比较了寨卡病毒（与登革病毒同属黄病毒科，病毒结构和基因组成相类似）包膜蛋白膜外区之EDIII结构域和整个膜外区E80（包括三个结构域：EDI、EDII、EDIII）做为备选疫苗的可行性。结果发现，虽然EDIII和E80均能在小鼠和恒河猴中诱导出Th1、Th2反应和中和抗体（Liang et al., 2019; Qu et al., 2019；Yang et al., 2020），但只有E80在恒河猴中产生抗病毒保护性免疫，而EDIII则基本没有效果（Yang et al., 2020）。鉴于这些新发现，我们重新构建了4个表达登革病毒E80的备选疫苗，再次系统地比较了这些免疫原在“序贯免疫”和“混合免疫”在小鼠模型中的差异。初步结果显示，“序贯免疫”和“混合免疫”在诱导Th1和Th2免疫应答上不分伯仲，它们在诱导抗原特异性的结合抗体上也不相上下；至于它们在所诱导出中和抗体的强度和广度中是否有明显差异，尚在研究中。上述研究首次揭示的通过调整免疫策略来促进登革疫苗研发的可行性和局限性，对其它多价疫苗的研发有参考价值。