Developing ICAM-1 for rhinovirus therapeutics

描述（由申请方提供）：人鼻病毒（HRV）是普通感冒的主要病原体，也是人类最常见的急性感染性疾病。由于大量的HRV血清型，以前的鼻病毒暴露提供的免疫保护很少，使疫苗接种方法无效。在大多数人中，HRV不会引起严重的疾病;然而，在哮喘和免疫功能低下的个体的情况下，HRV感染可导致危及生命的并发症。在102种HRV血清型中，90%使用细胞间粘附分子（ICAM）-1作为宿主受体。仅含有胞外结构域的可溶性ICAM-1在临床试验中显示出一些预防和治疗益处。其抗鼻病毒的作用可能是由于ICAM-1作为诱饵对病毒的中和和破坏。在哺乳动物细胞中生产可溶性ICAM-1的高成本阻碍了ICAM-1用于鼻病毒治疗的进一步开发。ICAM-1含有5个细胞外免疫球蛋白（IG）超家族结构域，结构研究表明只有第一个N-末端结构域（D1）直接参与与鼻病毒的结合。我们假设ICAM-1 D1足以中和和破坏鼻病毒。此外，单独使用D1可以允许在细菌系统中大规模和低成本生产，因为它缺乏任何真核翻译后修饰。迄今为止，单独产生D1的尝试还没有成功，因为D1在没有D2的情况下不会折叠。然而，本文的初步数据表明，能够中和鼻病毒的功能性D1突变体可以被工程化用于细菌中的大规模生产。我们还假设，D1的定向进化对鼻病毒的亲和力更高，诱导病毒解离的效力更大，这将使D1对鼻病毒治疗有效。我们的具体目标是：1.验证工程化ICAM-1 D1在鼻病毒治疗中的功效。a.将通过SPR检查ICAM-1 D1突变体与鼻病毒结合的能力，并使用沉降技术和TEM检查其诱导病毒破坏的能力。B. D1突变体抑制病毒感染的效力将通过PFU的减少来定量。C.我们将构建携带编码增强型绿色荧光蛋白（eGFP）的基因的重组鼻病毒（HRV 14和HRV 16），用于实时监测病毒繁殖，并提供检测ICAM-1效力的灵敏方法。2.通过进化D1以获得更高的亲和力和亲合力，将ICAM-1 D1工程化为更有效的鼻病毒抑制剂。a.使用酵母展示系统，功能性ICAM-1D 1将进一步进行定向进化，以高亲和力结合HRV 3、HRV 14和HRV 16。B.功能性D1突变体将被生产成二聚体、三聚体和四聚体形式，以通过亲合力效应增加其对病毒的亲和力。C.我们将检查D1突变体对病毒的亲和力对其诱导病毒破坏和抑制病毒诱导的细胞病变效应的效力的影响。人类鼻病毒（Human rhinoviruses，HRV）是普通感冒的主要病原体，也是人类最常见的急性传染病。在大多数人中，HRV不会引起严重的疾病;然而，在哮喘和免疫功能低下的个体的情况下，HRV感染可导致危及生命的并发症。在这项研究中，我们建议开发鼻病毒治疗利用宿主受体作为诱饵，将有效地对90%的鼻病毒血清型。

DESCRIPTION (provided by applicant): Human rhinoviruses (HRV) are the major causative agent of the common cold and the most common acute infectious illness in humans. Due to the large number of HRV serotypes, little immunological protection is offered by prior rhinovirus exposure, rendering vaccination approaches ineffective. In most people, HRV does not cause severe disease; however, in the case of asthmatics and immuno-compromised individuals, HRV infection can lead to life threatening complications. Of the 102 HRV serotypes, 90% use intercellular adhesion molecule (ICAM)-1 as a host receptor. Soluble ICAM-1 containing only the extracellular domains has shown some prophylactic and therapeutic benefit in clinical trials. Its effect against rhinovirus was likely due to the neutralization and disruption of the virus by ICAM-1 as a decoy. Further development of ICAM-1 for use in rhinovirus therapeutics has been hampered by the high cost of production of soluble ICAM-1 in mammalian cells. ICAM-1 contains five extracellular immunoglobulin (Ig) superfamily domains, and structural studies have indicated that only the first N-terminal domain (D1) is directly involved in binding to rhinovirus. We hypothesize that ICAM-1 D1 is sufficient to neutralize and disrupt rhinovirus. Moreover, the use of D1 alone may permit large-scale and low-cost production in a bacterial system as it lacks any eukaryotic posttranslational modification. Attempts to produce D1 alone thus far have not been successful, as D1 does not fold without D2. However, preliminary data herein suggest that functional D1 mutants capable of neutralizing rhinovirus can be engineered for large-scale production in bacteria. We also hypothesize that directed evolution of D1 toward higher affinity to the rhinovirus and greater potency for induction of virus dissociation will poise D1 to be effective for rhinovirus therapeutics. Our specific aims are: 1. To validate the efficacy of engineered ICAM-1 D1 in rhinovirus therapeutics. a. ICAM-1 D1 mutants will be examined for their ability to bind to rhinovirus by SPR and to induce virus disruption using a sedimentation technique and TEM. b. The potency of the D1 mutants in inhibiting virus infection will be quantified by a reduction in PFU. c. We will construct recombinant rhinoviruses (HRV14 and HRV16) that carry a gene encoding enhanced green fluorescence protein (eGFP) for real-time monitoring of virus propagation and to provide a sensitive means to test the potency of ICAM-1. 2. To engineer ICAM-1 D1 to be a more potent rhinovirus inhibitor by evolving D1 for higher affinity and avidity. a. Functional ICAM-1 D1 will be further subjected to directed evolution, using a yeast display system, for high affinity binding to HRV3, HRV14, and HRV16. b. Functional D1 mutant will be produced into dimeric, trimeric, and tetrameric forms to increase its affinity to the virus by avidity effect. c. We will examine the effect of the affinity of the D1 mutants to the viruses on their potency to induce viral disruption and to inhibit virus-induced cytopathic effects. PUBLIC HEALTH RELEVANCE Human rhinoviruses (HRV) are the major causative agent of the common cold and the most common acute infectious illness in humans. In most people, HRV does not cause severe disease; however, in the case of asthmatics and immuno-compromised individuals, HRV infection can lead to life threatening complications. In this study we propose to develop rhinovirus therapeutics utilizing host receptor as a decoy that will be effective against 90% of the rhinovirus serotypes.