Screening And Identification Of Mimotopes Of Highly Pathogenic H5N1 Avian Influenza A Virus HA

Avian Influenza (AI) is one of syndrome disease of birds caused by influenza A virus in avian. At present, highly pathogenic avian influenza (HPAI) is listed in A infectious diseases by World Organization for Animal Health (OIE). The disease broke out in many countries of the world, and resulted in a serious economic loss. At the same time, it threatens the public health. The HA is the major surface antigen of influenza virus, which can induce the production of neutralizing antibodies to neutralize the infectivity of the virus. And there are five antigenic sites mapped on the HA. Antigenic changes of HA play an important role in influenza virus variation. We found four monoclonal antibodies which neutralized 34 strains of the H5N1 virus in our antibody panel. It suggested that there's a highly conservative epitope in H5N1 virus. Thus it gives us a clue to research the epitope of HA and epitope-based vaccines, and a approach of overcoming the H5N1 avian influenza virus.In the paper, McAb 8H5 was used to screen for the mimotops from Ph.D.-12TM phage display peptide library. 11 positive phage clones were obtained: 122, 123, 124, 125, 126, 128, 129, 130, 131, 132, 133. The homologous sequence of the HA protein was not found.To assay the affinity of these peptides and McAb 8H5, and the immune response, we design a recombinant protein of corresponding peptide and 239 protein, including single-copy peptide and double-copy peptides. The results of indirect ELISA and competitive ELISA showed high affinity of McAb 8H5 and recombinant proteins, including 239 -123, 239-125, 239-D122 and 239-D124. Then, peptide 123 and 125 as an example, were used to study the affection of copy numbers to the activity of peptide displayed on HBV virus-like particle. 2, 3, 4, 5 copies peptide of 123 and 125 maintained the high affinity to McAb 8H5, except the single copy. Meanwhile, we immunized BALB/c mice with HBV virus-like particle recombinant protein. The immuno- fluorescence assay demonstrated that antibodies specific for HA were induced, and the titer of antibodies raised quickly. It showed the feasibility of mimotops as HA epitope vaccine. Further more, the other 10 peptides were designed for double copies peptide displayed on HBV virus-like particles. Another four high affinity recombinant protein to McAb 8H5 were obtained, including HBc-D122, HBc-D124, HBc-D128 and HBc-D129. There was also a high titer of antibody after immunization. Thus, we chose the HBV VLP as a peptide displaying vector for epitope vaccine.The further research on the peptides results of Dot blot and Western blot showed that the peptide from the linear peptide library should also form a specifical conformation; we called it"conformation-dependent". Meanwhile, the analysis of homologous character of peptide mimotopes and the analysis of evolutional tree of peptide mimotopes, indicated that 122 and 125, 128 and 124, 123 and 129 were the same type of peptide respectively.In conclusion, through the assay of recombinant proteins and the analysis of peptides, we try to found the basis for the research on mimotopes of highly pathogenic H5N1 avian influenza A virus HA.