An obstacle to the use of adenovirus as a vaccine carrier is the induction of neutralizing antibodies to hexon, the major protein component of the icosahedral capsid. The Program Project is designed to exploit the fact that human subjects have no preexisting neutralizing antibodies to chimpanzee adenovirus 68 (AdC68) that would limit its efficacy. Nevertheless, subsequent use of AdC68 for booster immunization would be limited by antibodies generated in response to the primary dose. The solution lies in using structural knowledge to design and create a series of novel AdC68 derivatives with modified neutralization epitopes. Three-dimensional crystal structures are known for human adenovirus types 2 and 5 hexons, and the overall virion architecture is known from cryo-electron microscopy. Comparison of all known hexon structures and sequences shows that there is a common molecular fold that provides an accurate and representative model for designing new adenovirus vectors. The hypervariable regions of AdC68 hexon are likely to contain the epitopes recognized by neutralizing antibodies. In this project, molecular modeling will suggest sites where modifications can be made without structural disruption. The AdC68 hexon crystal structure will be determined to confirm and refine the structural predictions. Simultaneously, the AdC68 hexon epitopes responsible for antibody neutralization will be identified experimentally by generating a panel of anti-hexon neutralizing monoclonal antibodies (mAbs). The panel will be used to find neutralization escape mutants by screening AdC68 virions with randomly-introduced mutations in the epitope regions. In addition, random pentapeptide insertions will be made in the hexon hypervariable regions to identify sites that tolerate extensive structural modification. The crystal structures for escape mutant hexons will be solved to refine structural information for each epitope. The combined information will be used to synthesize variants with disrupted epitopes. Finally, """"""""artificial serotypes"""""""" with multiply-disrupted epitopes will be tested against the antibody panel and )olyclonal neutralizing sera to demonstrate their efficacy as booster vaccine carriers.
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