Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.登革病毒（Dengue virus, DENV）是引发登革热（dengue fever）与登革出血热（dengue hemorrhagic fever）的致病原。该病毒在全球120余个国家呈地方性流行，每年造成超过3.5亿人次感染。登革病毒疫苗研发颇具挑战，原因在于需同时诱导针对四种抗原性各异的登革病毒血清型的保护效力，且有研究证据表明，在特定条件下，疫苗接种可能因针对该病毒的特异性免疫反应而加重疾病病情。尽管多款减毒活四价登革病毒疫苗已展现出部分保护效果，但诱导针对全部四种血清型的均衡保护性免疫仍存在较大难度。相较于全病毒制剂，本研究正探索基于登革病毒E蛋白的颗粒亚单位疫苗的研发潜力，该蛋白展示于通过非润湿模板颗粒复制（Particle Replication in Non-wetting Template, PRINT）技术精准制备的纳米颗粒表面。本文报道了一款登革病毒2型（DENV2）纳米颗粒疫苗候选株的免疫原性研究：研究中将登革病毒2型E蛋白的胞外域构建为分泌型重组蛋白（secreted recombinant protein, sRecE），经纯化后吸附于不同尺寸与形态的聚乳酸-羟基乙酸共聚物（poly (lactic-co-glycolic acid), PLGA）纳米颗粒表面。实验结果显示，无需额外添加佐剂，经PRINT纳米颗粒吸附的sRecE即可诱导出较单纯可溶性sRecE蛋白更高的IgG滴度（IgG titers），以及更强效的DENV2特异性中和抗体应答（neutralizing antibody response）。抗原转运（antigen trafficking）相关研究表明，PRINT纳米颗粒展示的sRecE可通过构建抗原储库（antigen depot），延长抗原在引流淋巴结（draining lymph nodes）中的生物利用度。本研究结果证实，PRINT纳米颗粒是一种极具前景的递送平台，可用于制备针对登革病毒、寨卡病毒（Zika）等黄病毒属（flaviviruses）病毒的亚单位疫苗。