Selection of small inhibitory molecules against filoviruses

描述(由申请人提供)：埃博拉丝状病毒(EBOV)和马尔堡丝状病毒(MARV)在非洲引起周期性的破坏性出血热暴发。由于这些感染导致的高死亡率和这些病毒在人类群体中的高传播性，这组病毒已被列入美国疾病控制和预防中心建立的A类精选制剂名单。目前还没有针对这些致命病毒的抗病毒疗法。虽然最近丝状病毒疫苗的发展看起来很有希望，但对任何疫苗的免疫力都不是立竿见影的。其中一种候选疫苗最近被证明在致命挑战后24小时给予某种程度上有效，这表明接触后预防是可能的。此外，这项研究表明，感染期间病毒载量的减少对有害后果有重大影响。这些发现再次证实了这样一种观点，即即使是暂时降低病毒载量的抗病毒药物，在降低丝状病毒感染引起的死亡率方面也可能非常有效。即使疫苗开发成功，使用抗丝病毒药物作为应对零星暴发的权宜之计也是非常有益的，因为认为在不久的将来将在非洲人口中广泛接种针对这些病毒的疫苗是不切实际的。在这里，我们建议通过选择针对EBOV表面糖蛋白GP1的受体结合域(RBD)的核酸适体来开发这种抗病毒疗法。适配子是专门与蛋白质或小的靶分子结合的寡核苷酸(通常为20-50个核苷酸)。可以选择与蛋白质的精确区域结合并破坏功能的适体。EBOV糖蛋白GP1RBD将成为适配子的极好靶点，因为该蛋白的这一区域位于细胞外，既存在于病毒粒子上，也存在于受感染的细胞上，使适配子能够随时接触到分子。此外，GP1与其受体(S)结合在允许细胞上的适体干扰将有效地中断病毒生命周期，从而减少感染者体内的病毒血症，并减少病毒向他人的传播。在目标1中，我们将选择靶向GP1受体结合域(RBD)并阻止GP1与允许细胞结合的适体。在目标2中，我们将对GP1适配子(S)进行优化，并对其稳定性和靶向亲和力进行表征。然后，我们将测试适体在细胞系和主要靶细胞中阻断EBOV gp依赖转导的有效性。靶向EBOV gp并阻止病毒粒子进入细胞的适配子的开发应该被证明在减少感染者的病毒血症方面非常成功。到目前为止的研究表明，减少病毒血症不仅在提高感染者的存活率方面被证明是有效的，而且可能会减少病毒在人群中的传播，并提供一种暂时控制疫情的手段。公共卫生相关性：丝状病毒、埃博拉病毒和马尔堡病毒的爆发是零星和不可预测的。这些病毒往往是致命的，目前还没有可用的抗病毒治疗方法。由于这些病毒的致命性和在人类群体中快速传播的能力，丝状病毒已被列入生物防御A类名单，使抗病毒开发成为科学家的首要任务。在这里，我们建议选择被称为针对埃博拉病毒糖蛋白的适体的小的合成RNA。适配子与糖蛋白的结合将阻止病毒的感染。一旦检测到暴发，这种新型抗病毒药物可以用来控制和限制病毒的传播。

DESCRIPTION (provided by applicant): The filoviruses, Ebola (EBOV) and Marburg (MARV), cause periodic devastating hemorrhagic fever outbreaks in Africa. Because of the high rates of mortality caused by these infections and high transmissibility of these viruses in the human population, this group of viruses has been placed on the Category A select agent list that has been established by the Centers for Disease Control and Prevention. No anti-viral therapies are currently available against these deadly viruses. While recent developments of filoviral vaccines look promising, immunity to any vaccine is not immediate. One of vaccine candidates was recently shown to be somewhat effective when given 24 hours following lethal challenge suggesting that post exposure prophylaxis is possible. Furthermore, this study demonstrated that reduction in virus load during the infection had a significant impact on deleterious outcomes. These findings re-enforce the idea that antivirals that even transiently reduce virus load may be quite effective at decreasing mortality resulting from filovirus infection. The use of antivirals against filoviruses as a stop-gap measure against sporadic outbreaks will be highly beneficial even if vaccine development is successful as it is unrealistic to believe that wide spread vaccination of African populations against these viruses will occur in the near future. Here, we propose to develop such anti-viral therapy by the selection of nucleic acid aptamers against the receptor binding domain (RBD) of the EBOV surface glycoprotein GP1. Aptamers are oligonucleotides (generally 20-50 bp) that specifically bind to proteins or small target molecules. Aptamers can be selected that bind to a precise region of a protein and disrupt function. The EBOV glycoprotein GP1 RBD will serve as an excellent target for aptamers because this region of the protein resides extracellularly on both virions and infected cells allowing aptamers ready access to the molecule. Furthermore, aptamer interference of GP1 binding to its receptor(s) on permissive cells would effectively interrupt the viral life cycle and thereby reduce viremia within an infected individual and reduce spread of the virus to others. In Aim 1, we will select aptamers that target the GP1 receptor binding domain (RBD) and prevent binding of GP1 to permissive cells. In Aim 2, we will optimize the GP1 aptamer(s) and characterize aptamer stability and target affinity. We will then test the efficacy of the aptamers in blocking EBOV GP dependent transduction in cell lines and primary target cells. Development of aptamers that target EBOV GP and block virion entry into cells should prove highly successful in reducing viremia in the infected individual. Studies to date suggest that reduced viremia will not only prove effective at increasing survival of the infected individual, but will potentially reduce viral spread in a population and provide a means to transiently control outbreaks. PUBLIC HEALTH RELEVANCE: Outbreaks of the filoviruses, Ebola virus and Marburg virus, are sporadic and unpredictable. These viruses are frequently deadly and no current anti-viral treatments are available. Because of the lethality of these viruses and their ability to be rapidly transmitted within a human population, the filoviruses have been placed on the biodefense Category A list making antiviral development a top priority for scientists. Here, we propose to select small, synthetic RNAs called aptamers against the Ebola virus glycoprotein. Binding of the aptamers to the glycoprotein will block infection of the virus. Such novel antivirals could be used to control and limit the spread of the virus once an outbreak is detected.