Selection of small inhibitory molecules against filoviruses

描述（由申请人提供）：丝状病毒，埃博拉病毒（EBOV）和马尔堡病毒（MARV），在非洲引起周期性的破坏性出血热暴发。由于这些感染引起的高死亡率以及这些病毒在人群中的高传播性，这组病毒已被列入疾病控制和预防中心建立的A类精选病原体名单。目前还没有针对这些致命病毒的抗病毒疗法。虽然丝状病毒疫苗的最新进展看起来很有希望，但对任何疫苗的免疫都不是立即产生的。一种候选疫苗最近被证明在致命攻击后24小时接种有一定效果，这表明暴露后预防是可能的。此外，该研究表明，感染期间病毒载量的减少对有害结果有显著影响。这些发现再次证实了这样一种观点，即即使是暂时减少病毒载量的抗病毒药物也可能相当有效地降低丝状病毒感染造成的死亡率。即使疫苗开发取得成功，使用针对丝状病毒的抗病毒药物作为应对零星暴发的权宜之计也将非常有益，因为相信在不久的将来会在非洲人口中广泛接种这些病毒疫苗是不现实的。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治疗。适配体是一种寡核苷酸（通常为20-50 bp），可以特异性地与蛋白质或小靶分子结合。可以选择与蛋白质的精确区域结合并破坏其功能的适体。EBOV糖蛋白GP1 RBD将作为适体体的极好靶标，因为该蛋白的这一区域位于细胞外的病毒粒子和受感染细胞上，允许适体体随时进入分子。此外，适体干扰GP1与其受体在允许细胞上的结合将有效地中断病毒的生命周期，从而减少受感染个体内的病毒血症，减少病毒向他人的传播。在Aim 1中，我们将选择靶向GP1受体结合域（RBD）并阻止GP1与许可细胞结合的适体。在目标2中，我们将优化GP1适配体，并表征适配体的稳定性和靶标亲和力。然后，我们将测试适体在细胞系和原代靶细胞中阻断EBOV GP依赖转导的功效。针对EBOV GP并阻断病毒粒子进入细胞的适体的开发，在减少感染个体的病毒血症方面应该是非常成功的。迄今为止的研究表明，减少病毒血症不仅可以有效地提高受感染个体的存活率，而且还可能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暴发是散发的和不可预测的。这些病毒通常是致命的，目前还没有抗病毒的治疗方法。由于这些病毒的致命性及其在人群中迅速传播的能力，丝状病毒已被列入生物防御a类名单，使抗病毒药物的开发成为科学家的首要任务。在这里，我们建议选择小的，合成的rna称为适体抗埃博拉病毒糖蛋白。与糖蛋白结合的适体将阻止病毒的感染。一旦发现疫情爆发，这种新型抗病毒药物可用于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.