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.
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. ? ? ?
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