To address the need for a safe, efficacious vaccine against SARS-CoV-2 infection with the critical properties of enabling both blocking viral entry into cells and clearing virus from cells already infected, we have developed a bivalent, human adenovirus serotype 5 (hAd5) SARS-CoV-2 S-Fusion + N-ETSD vaccine that is currently in clinical testing. This vaccine uses the next-generation hAd5 [E1-, E2b-, E3-] platform previously used successfully in cancer patients with pre-existing adenovirus immunity, engineered to express both SARS-CoV-2 spike (S) protein modified to improve the generation of neutralizing antibodies to block entry of the virus, and nucleocapsid (N) protein with an Enhanced T cell Stimulation Domain (ETSD) to activate CD4+ and CD8+ T cells to clear the virus and block replication by killing infected cells. The targeting of N to endosomes and lysosomes to enhance CD4+ and CD8+ T-cell responses distinguishes our vaccine. In our previously reported pre-clinical studies we showed that in mice, the hAd5 S-Fusion + N-ETSD vaccine elicits both humoral and T-cell responses that are robust and T helper cell 1 (Th1) dominant. Here we report that the hAd5 S-Fusion + N-ETSD vaccine is recognized by anti-sera and T cells from previously SARS-CoV-2 infected patients, and that the presence of N is vital for T-cell recall. The findings presented herein: i. demonstrate specific recognition of hAd5 S-Fusion + N-ETSD infected cells by plasma antibodies from previously SARS-CoV-2 infected patients, but not antibodies from virus-naive subjects; ii. show enhanced binding of plasma SARS-CoV-2 antibodies from previously infected patients to monocyte-derived dendritic cells (MoDCs) expressing the hAd5 S-Fusion + N-ETSD vaccine as compared to hAd5 S-Fusion alone; iii. reveal N-ETSD localizes to vesicles associated with MHC class II antigen presentation, including endosomes, lysosomes, and autophagosomes in MoDCs; iv. demonstrate endosome/lysosome-targeted N-ETSD elicits higher interferon-gamma T-cell responses than cytoplasm-localized N; and v. N-ETSD alone or in the hAd5 S-Fusion + N-ETSD construct induces both CD4+ and CD8+ T cell memory recall. This recognition of hAd5 S-Fusion + N-ETSD vaccine antigens by T cells from previously SARS-CoV-2 infected patients, together with the ability of this vaccine candidate to elicit de novo immune responses in naive mice suggests that it re-capitulates the natural immune response to SARS-CoV-2 to activate both B and T cells towards viral neutralization and recognition of infected cells, critical for prevention of COVID-19 disease. Intriguingly, our hAd5 S-Fusion + N-ETSD T-cell biased vaccine has the potential to not only provide protection for uninfected individuals, but also to be utilized as a therapeutic for already infected patients to induce rapid clearance of the virus by activating T cells to kill the virus-infected cells, thereby reducing viral replication and lateral transmission.

中文翻译：

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hAd5 S融合+ N-ETSD感染先前感染SARS-CoV-2的患者的自体树突状细胞诱导的Th1优势核仁和穗状抗原特异性CD4 +和CD8 +记忆T细胞召回

为了满足针对SARS-CoV-2感染的安全有效疫苗的需求，该疫苗具有能够阻止病毒进入细胞并从已感染的细胞清除病毒的关键特性，我们开发了一种二价人腺病毒血清型5（hAd5）目前正在临床测试中的SARS-CoV-2 S-Fusion + N-ETSD疫苗。该疫苗使用了下一代hAd5 [E1-，E2b-，E3-]平台，该平台先前已成功用于具有预先存在的腺病毒免疫力的癌症患者，被设计用于表达经修饰的SARS-CoV-2峰值（S）蛋白，以改善产生可阻止病毒进入的中和抗体，以及带有增强T细胞刺激域（ETSD）的核衣壳（N）蛋白，以激活CD4 +和CD8 + T细胞来清除病毒并通过杀死感染的细胞来阻止复制。将N靶向内体和溶酶体以增强CD4 +和CD8 + T细胞反应是我们疫苗的独特之处。在我们先前报道的临床前研究中，我们表明，在小鼠中，hAd5 S-融合+ N-ETSD疫苗引起体液和T细胞反应，两者均很强健，并且T辅助细胞1（Th1）占主导地位。在这里，我们报道hAd5 S融合+ N-ETSD疫苗被先前SARS-CoV-2感染患者的抗血清和T细胞识别，并且N的存在对于T细胞召回至关重要。本文介绍的发现：证明先前被SARS-CoV-2感染患者的血浆抗体对hAd5 S-融合+ N-ETSD感染的细胞有特异性识别，但对未感染病毒的受试者的抗体没有特异性识别；ii。与单独的hAd5 S-Fusion相比，显示先前感染患者的血浆SARS-CoV-2抗体与表达hAd5 S-Fusion + N-ETSD疫苗的单核细胞衍生树突细胞（MoDC）的结合增强；iii。揭示N-ETSD位于与MoDCs中的II类MHC抗原呈递相关的囊泡，包括内体，溶酶体和自噬体；iv。证明以内体/溶酶体为靶标的N-ETSD比定位于细胞质的N引起更高的干扰素-γT细胞反应；v。单独使用N-ETSD或在hAd5 S-Fusion + N-ETSD构建体中诱导N-ETSD诱导CD4 +和CD8 + T细胞记忆回忆。先前受SARS-CoV-2感染的患者的T细胞对hAd5 S融合+ N-ETSD疫苗抗原的识别，连同该疫苗候选物在幼稚小鼠中引发从头免疫反应的能力表明，它重新概括了对SARS-CoV-2的天然免疫反应，从而激活B和T细胞以实现病毒中和和识别感染细胞，这一点至关重要用于预防COVID-19疾病。有趣的是，我们的hAd5 S融合+ N-ETSD T细胞偏向疫苗不仅可以为未感染的个体提供保护，而且还可以用作已感染患者的治疗剂，通过激活T来诱导病毒的快速清除细胞杀死被病毒感染的细胞，从而减少病毒复制和横向传播。