Improve the safety of an efficacious live-attenuated HIV-1 vaccine through unnatu

描述（由申请人提供）：迫切需要一种安全有效的人类免疫缺陷病毒1型（HIV-1）疫苗来遏制全球艾滋病流行，但仍然难以捉摸。在迄今为止开发和测试的所有HIV-1疫苗形式中，nef缺失的减毒活HIV-1疫苗（nef-LAV）在HIV-1感染的猴免疫缺陷病毒/恒河猴模型中提供最佳的HIV-1获得保护;然而，其安全性问题排除了其临床应用。在过去的几年里，我们在内布拉斯加大学林肯分校开发了一种非天然氨基酸（UAA）介导的空白密码子抑制方法来控制HIV-1蛋白的生物合成。我们的方法需要通过空白密码子（不编码天然蛋白质氨基酸的密码子，如无义三联体密码子和移码密码子）抑制来操纵必需蛋白质的生物合成，所述空白密码子由自然界中未发现的UAA精确控制。我们将该技术应用于HIV-1疫苗的研究，通过将几组编码UAA的空白密码子引入病毒基因组中来控制HIV-1的复制。我们成功地证明了我们可以在体外打开和关闭HIV-1复制。我们假设，我们的新策略允许减毒活HIV-1仅在自然界中未发现的三种人工调控元件（新型<$nef-LAV）存在下复制，这将大大提高传统<$nef-LAV的安全性，同时仍然保持其对HIV-1获得的良好疗效。因此，我们建议用三个具体目标来检验这一假设。具体目标1，控制新型<$nef-LAV的体外复制，其中我们将应用并进一步优化空白密码子抑制策略，并将抑制tRNA-aaRS对掺入HIV-1基因组中，通过补充或去除UAA来控制HIV-1在哺乳动物细胞中的体外复制。具体目标2，以控制新的<$nef-LAV体内复制，其中我们将使用人源化- BLT小鼠比较和对比在具体目标1中已经通过体外多循环复制测试的新的<$nef-LAV与常规<$nef-LAV的体内复制能力和动力学。Specific Aim 2的结果将用于进一步优化Specific Aim 1中的新型nef-LAV病毒设计和开发。具体目标3，新型<$nef-LAV体内保护，其中我们将使用hu-BLT小鼠比较新型<$nef-LAV与常规<$nef-LAV对HIV-1直肠攻击的保护率。由于我们的策略可以通过在HIV-1上提供额外的安全锁来大大提高<$nef-LAV的安全性，因此它使修改后的<$nef-LAV在临床试验中实用。此外，在本研究中开发和进一步优化的方法/工具可以应用于针对其他病原性病毒或细菌的疫苗的生成，因此预计将对疫苗研究产生更广泛的影响。

DESCRIPTION (provided by applicant): A safe and effective human immunodeficiency virus type one (HIV-1) vaccine is urgently needed to stem worldwide AIDS pandemic, but still remains elusive. Among all the HIV-1 vaccine modalities developed and tested thus far, nef-deleted live-attenuated HIV-1 vaccines (¿nef-LAV) confer the best protection against HIV-1 acquisition in simian immunodeficiency virus/rhesus macaque model of HIV-1 infection; however, its safety concerns preclude its clinical use. We at University of Nebraska-Lincoln in the past years have developed an unnatural amino acid (UAA)-mediated blank codon suppression approach to control HIV-1 protein biosynthesis. Our approach entails the manipulation of essential protein biosynthesis through blank codon (codons that do not encode a natural proteinogenic amino acid, such as nonsense triplet codon and frameshift codon) suppression that is precisely controlled by UAA that is not found in nature. We applied this technology to HIV-1 vaccine research by introducing several sets of blank codons coding for UAA into virus genome to control HIV-1 replication. We successfully demonstrated that we could turn HIV-1 replication on and off in vitro. We hypothesize that our novel strategy to allow live-attenuated HIV-1 to replicate only in the presence of the three artificial regulatory elements that are not found in nature (novel ¿nef-LAV) will greatly improve the safety of conventional ¿nef-LAV while still maintains its good efficacy against HIV-1 acquisition. We therefore propose to test this hypothesis with three specific aims. Specific Aim 1, to control the replication of novel ¿nef-LAV in vitro, of which we will apply and further optimize blank codon suppression strategy and incorporate suppression tRNA-aaRS pair into the HIV-1 genome to control HIV-1 replication by supplementing or withdrawing UAA in mammalian cells in vitro. Specific Aim 2, to control novel ¿nef-LAV replication in vivo, of which we will compare and contrast the in vivo replication capacity and kinetics of the novel ¿nef-LAV that has passed multi-cycle replication tests in vitro in the Specific Aim 1 with conventional ¿nef-LAV using humanized- BLT mice. The results of the Specific Aim 2 will be used to further optimize the novel ¿nef-LAV virus design and development in the Specific Aim 1. Specific Aim 3, novel ¿nef-LAV in vivo protection, of which we will compare the protection rate of novel ¿nef-LAV vs. conventional ¿nef-LAV against HIV-1 rectal challenge using hu-BLT mice. Since our strategy can greatly improves the safety of ¿nef-LAV by providing additional safety locks on HIV-1, it makes the modified ¿nef-LAV practical in clinical trial. In addition, the approaches/tools that are developed and further optimized in this research can be applied to the generation of vaccines against other pathogenic viruses or bacteria, and therefore is expected to exert broader impact on vaccine research.