SARS-CoV Spike protein epitopes mapping with RNA Q-beta displayed peptides: A vaccine candidate.

We propose to investigate a novel epitope mapping approach of SARS-CoV S proteins, using RNA phage Qβdisplayed peptides, and to evaluate the potential of these engineered hybrid phages as antibody neutralizingdeterminants critically important in the development of an efficacious vaccine candidate. Among the proteinscoded by the SARS-CoV genome, S protein mediates its cell entry and induces neutralizing antibodies. Thesepivotal functions are achieved by key amino acid peptide(s) through their exposed position on the virus surface.Phage Qβ is a small positive strand RNA virus with a 4.2 kb genome encoding 4 proteins. These are coat protein(Cp), maturation (or A2) protein, read-through or minor coat protein (or A1), and the RNA-dependent RNApolymerase or RNA replicase (RdRp) protein. As an RNA virus, phage Qβ possesses a key feature for its rapidevolution and adaptation: the RdRp protein that does not have proofreading activity during replication, resultingin higher mutation rates which simulate in vitro evolution and affinity-maturation processes. The A1 protein hasrecently been successfully used for fusion and display of randomized peptides in our laboratory, which isimportant because of its number and position on the phage surface. These fundamental concepts of RNA displaywill be exploited to investigate the following three specific aims:1. Design, construct, express and characterize hybrid phages Qβ exposing peptide of S protein epitopeson the exterior surface. Potential S protein continuous and discontinuous (chimeric) epitopes will be localizedand checked for exposition on A1 protein, using a combination of three computer bioinformatics software. Theidentified epitopes will be designed as oligonucleotides and cloned by fusion to the end of A1 minor coat proteingene for the novel RNA display system. These constructs will be characterized and expressed for hybrid phages(phagotopes) production. For any testing and selection of variant phages, we will use anti-S Abs.2. Randomize and optimize the major epitopes of S protein. A novel biopanning method will be developedfor selecting the appropriate randomized phages mimicking S protein epitopes (mimotopes) against the existingSARS-CoV and S protein mono/polyclonal antibodies. The selected mimotope(s) will be easily optimized andevolved through at least ten rounds of biopanning. The determinants of the randomized mimotopes pool willalso be classified by antibody type, and studied for any potential affinity to other viral cellular receptors differentfrom the natural SARS-CoV. Non-selected hybrid phages will be analyzed in correlation with the antibodies.3. Stabilize and initiate evaluation of the potential binding and neutralization of variant phages to SARS-CoV antibodies. We hypothesize that epitope peptide flanked by paired cysteines can be stabilized on thesurface of the mimotopes and/or phagotopes. The prepared hybrid phages will be analyzed and evaluated fortheir potential binding to all SARS-CoV antibodies and/or sera. The antibodies will be tested and classified foraffinity with the phagotopes and/or mimotopes in comparison to the wild type SARS-CoV or pseudotype model.