Identification Of Mimotopes By Screening Of A Bacterially-displayed Random Peptide Library And Its Application In Vaccine Development

Bacterial surface display systems, as an alternative system of phage display, have wide range of biotechnological and industrial applications, in which live bacterial vaccine development is the most promising and attractive. To date, a diverse variety of well-known vaccine candidates (an immunogenic peptide or protein) or mimotopes screened from phage displayed peptide library have been successfully displayed on bacterial surface and have induced higher antibody response to the native antigen. Nevertheless, numerous studies have demonstrated that the insertion of heterologous epitopes into loops of bacterial proteins would potentially change the environment of epitopes and inevitably disturb its structure integrity, which usually leads to the failure of Abs response against the native antigen. Since epitope mapping has also been accomplished by bacterial peptide library, bacteria displayed mimotopes screened from random library should be directly used as recombinant live vaccines.Firstly, a monoclonal antibody against the HBV preS protein and a commercially available bacterial peptide library were involved as a model system to practice this process. After five successive rounds of biopanning, bacterial clones interacting with the monoclonal antibody 3B9 were greatly enriched. The R-RG-Y motif was identified as mimotopes of HBV preS protein at 135-140 residues from sequence analysis. Strong responses against HBV preS protein were obtained after mice were immunized by i.p injection of live bacteria without any adjuvant. These results imply that bacterially-displayed mimotopes screened from peptide library could be directly used as live vaccine in case of emergency infectious diseases.To overcome the limitation of conventional biopanning, FACS was subsequently introduced to epitope screening from FliTrx?peptide library. Epitopes of themonoclonal antibody were successfully identified from the constructed model library or FliTrx?peptide library. Due to more complexity of polyclonal antibody over monoclonal antibody, a subtractive FACS strategy was established, which constitute the foundation for epitope screening from patient serum in the further study.To complement the existing constrained libraries, a novel bacterial random peptide library, with 5x10 primary bacterial colonies, was constructed using the ?-domain of IgA protease as an anchoring motif. The DNA sequence analysis showed that the library had sufficient complexity and diversity. This non-constrained system provides an alternative tool for further epitope screening.In conclusion, bacterially-displayed mimotopes screened from bacterial peptide library could be directly used as live vaccines, which proved to be a less expensive, yet more effective and rapid route for vaccine development than the conventional methods. By this strategy, we could also obtain mimotopes for the unrecognized or previously unknown antigens from patient serum. In this context, it is of great significance to develop the effective vaccines for such emerging infectious diseases whose pathogen is still unknown.