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）的出现凸显了我们缺乏准备， 强调了在开发试剂、工具和诊断方法方面迅速建设能力的重要性， 以及SARS-CoV-2和具有大流行潜力的相关CoV的治疗剂。一个直接的目标是生产 一种疫苗，可以引发快速，高水平的保护性免疫，对SARS-CoV-2，理想的是在一个单一的， 枪了几种候选疫苗现已进入临床试验阶段。然而，更长远的目标是 调查“通用”CoV疫苗的可能性，疫苗或初免：加强疫苗接种方案， 提供持久和广泛的交叉反应免疫力，以对抗蝙蝠溢出的可能性很高的冠状病毒。 CoV表面刺突（S）糖蛋白是中和抗体（NAb）和T细胞的主要靶点， 一个有吸引力的疫苗设计目标。靶向受体结合结构域（RBD）的NAb提供保护， 但通常是菌株特异性的并且缺乏广度。广泛反应性保护性表位的存在， 的RBD没有很好的特点。因此，比较全长或截短的疫苗， S免疫原的变体，可以揭示一些保护的潜在相关性。另一个重要 令人担忧的是疾病增强，这在使用选定疫苗的相关CoV疫苗中已观察到 递送平台，例如全灭活病毒（WIV）疫苗。这与Th 2相关， 为了克服这个问题，CoV疫苗应该引起很大程度上偏向于Th 1的免疫应答， 反应因此，旨在比较不同疫苗引起的免疫表型的研究 平台，以及S免疫原的不同变体，可以帮助更好地了解 免疫应答在介导保护中是最佳的，而没有感染后免疫病理学的风险。 我们将开发一个有效的免疫原性，优化的疫苗平台，为SARS-CoV-2使用三个 接近。(1)首先，我们将设计几种不同形式的SARS-CoV-2S，全长免疫原， 分泌的稳定的融合前形式或单独的RBD结构域。(2)第二，我们会增加或扩大 通过将融合前S靶向宿主来源的细胞外囊泡（EV）来免疫识别融合前S， 通过产生融合-Ag构建体，其将Ag拴系到高度富集的蛋白质结构域， 外来体外泌体是纳米尺寸的EV，显示出在细胞：细胞通信中和在细胞内环境中发挥重要作用。 由于它们将Ag呈递给T细胞和B细胞的能力，它们调节免疫应答。(3)最后，我们将开发 非复制型、稀有物种腺病毒（Ad）载体疫苗，其已经建立了快速免疫的方案， 临床生产和监管批准，可以热稳定，免疫原性损失最小 并且已经在人体临床试验中证明了安全性。本研究将全面评估和表型 SARS-CoV-2疫苗在单次注射方案中的规模和特征。这些数据将提供有价值的 为后续预充：加强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.