Exosome-display as a strategy to enhance the immunogenicity of SARS-CoV-2 vaccines based on adenoviral vectors

摘要：SARS-CoV-2冠状病毒(CoV)的出现突显了我们缺乏准备，以及 强调了在开发试剂、工具、诊断方法方面迅速建设能力的重要性 和治疗SARS-CoV-2和具有大流行潜力的相关Cov。一个直接的目标是生产 一种能够诱导对SARS-CoV-2快速、高水平保护性免疫的疫苗，理想的情况是遵循单一的 中枪了。几种候选疫苗现在已经进入临床试验。然而，更长期的目标是 研究“通用”冠状病毒疫苗、疫苗或主要疫苗的可能性：加强疫苗接种方案， 提供持久和广泛的交叉反应免疫力，以对抗具有高可能性的蝙蝠溢出的冠状病毒。 冠状病毒表面刺突(S)糖蛋白是中和抗体(NAB)和T细胞的主要靶标，是 这是疫苗设计的一个有吸引力的目标。以受体结合域(RBD)为靶点的NAB提供保护， 但通常是特定于菌株的，缺乏广泛性。存在广泛反应的保护性表位 RBD的特征并不是很好。因此，比较全长或截短的疫苗，稳定 S免疫原的变体，可能会为潜在的保护相关因素提供一些线索。另一项重要的 令人担忧的是疾病的增强，已经观察到使用选定疫苗的相关冠状病毒疫苗 全灭活病毒(WIV)疫苗等投放平台。这与Th2有关- 有偏见的免疫反应和克服这个问题，冠状病毒疫苗应该引起在很大程度上偏向Th1 回应。因此，旨在比较不同疫苗诱导的免疫表型的研究 平台，以及S免疫原的不同变体，可以帮助更好地了解 免疫反应在中介保护方面是最佳的，没有感染时的免疫病理风险。 我们将使用三种疫苗开发一种有效的免疫原性、优化的SARS-CoV-2疫苗平台 接近了。(1)首先，我们将以几种不同的形式改造SARS-CoV-2 S，全长免疫原，a 分泌稳定的融合前形式或单独的RBD结构域。(2)其次，我们将扩大或扩大 融合前S对宿主来源的胞外小泡的免疫识别 在体内，通过产生融合-Ag结构，将Ag拴在高度浓缩的蛋白质结构域上 外显体。胞外体是纳米尺寸的电动汽车，被证明在细胞中发挥重要作用：细胞通讯和 免疫反应的调节，由于它们有能力将抗原呈递给T细胞和B细胞。(3)最后，我们将发展 非复制的稀有种腺病毒(Ad)载体疫苗已经建立了快速 临床生产和监管批准，可以在免疫原性损失最小的情况下进行热稳定 并已在人体临床试验中证明是安全的。这项研究将全面评估和表型 SARS-CoV-2疫苗在单针方案中的数量和概况。这些数据将提供有价值的 为后续PRIME：BOOST方案的设计和未来的挑战实验提供信息。

SUMMARY: The emergence of the SARS-CoV-2 coronavirus (CoV) highlighted our lack of preparedness, and has emphasized the importance of rapidly building capacity in the development of reagents, tools, diagnostics and therapeutics for SARS-CoV-2 and related CoVs with pandemic potential. An immediate goal is to produce a vaccine which can elicit rapid, high-level protective immunity against SARS-CoV-2, ideally following a single- shot. Several candidate vaccines have now advanced into clinical trials. However, a longer term goal is to investigate the possibility for a “universal” CoV vaccine, a vaccine or prime:boost vaccination regimen which provides durable and broadly cross-reactive immunity against CoVs with high potential for spillover from bats. The CoV surface spike (S) glycoprotein is a major target for neutralizing antibodies (NAbs) and T cells, and is an attractive target for vaccine design. NAbs which target the receptor binding domain (RBD) confer protection, but are usually strain-specific and lack breadth. The existence of broadly reactive, protective epitopes outside of the RBD are not well-characterized. Therefore, vaccines which compare full length or truncated, stabilized variants of the S immunogen, could shed some light into potential correlates of protection. Another important concern is disease enhancement, which has been observed for related CoV vaccines using selected vaccine delivery platforms such as the whole-inactivated virus (WIV) vaccine. This has been associated with a Th2- biased immune response and to overcome this issue, CoV vaccines should elicit a largely Th1 biased response. Therefore, studies which aim to compare the phenotype of immunity elicited by different vaccine platforms, and to different variants of the S immunogen, could help to better understand which components of the immune response are optimal in mediating protection, without the risk of immunopathology upon infection. We will develop a potently immunogenic, optimized vaccine platform for SARS-CoV-2 using three approaches. (1) Firstly, we will engineer SARS-CoV-2 S in several different forms, a full-length immunogen, a secreted stabilized pre-fusion form or the RBD domain alone. (2) Secondly, we will augment or broaden immune recognition of pre-fusion S by targeting it to host-derived extracellular vesicles (EVs) including exosomes in vivo, by generating fusion-Ag constructs which tether Ag to a protein domain highly enriched in exosomes. Exosomes are nano-sized EVs shown to play important roles in cell:cell communication and in the regulation of immune responses, due to their ability to present Ag to T- and B-cells. (3) Finally, we will develop non-replicating, rare species adenoviral (Ad) vectored vaccines which have established protocols for rapid clinical manufacturing and regulatory approval, can be thermostabilized with minimal losses to immunogenicity and have demonstrated safety in human clinical trials. This study will comprehensively evaluate and phenotype the magnitude and profile of SARS-CoV-2 vaccines in single-shot regimens. These data will provide valuable information for the design of subsequent prime:boost regimens and for challenge experiments in the future.