H7N9, a novel strain of avian origin influenza was the first recorded incidence where a human was transited by a N9 type influenza virus. Effective vaccination against influenza A (H7N9) is a major concern, since it has emerged as a life threatening viral pathogen. Here, an in silico reverse vaccinology strategy was adopted to design a unique chimeric subunit vaccine against avian influenza A (H7N9). Induction of humoral and cell-mediated immunity is the prime concerned characteristics for a peptide vaccine candidate, hence both T cell and B cell immunity of viral proteins were screened. Antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach were adopted to generate the most antigenic epitopes of avian influenza A (H7N9) proteome. Further, a novel subunit vaccine was designed by the combination of highly immunogenic epitopes along with suitable adjuvant and linkers. Physicochemical properties and secondary structure of the designed vaccine were assessed to ensure its thermostability, hydrophilicity, theoretical PI and structural behavior. Homology modeling, refinement and validation of the designed vaccine allowed to construct a three dimensional structure of the predicted vaccine, further employed to molecular docking analysis with different MHC molecules and human immune TLR8 receptor present on lymphocyte cells. Moreover, disulfide engineering was employed to lessen the high mobility region of the designed vaccine in order to extend its stability. Furthermore, we investigated the molecular dynamic simulation of the modeled subunit vaccine and TLR8 complexed molecule to strengthen our prediction. Finally, the suggested vaccine was reverse transcribed and adapted for E. coli strain K12 prior to insertion within pET28a(+) vector for checking translational potency and microbial expression.

32 33 H7N9, a novel strain of avian origin influenza was the first recorded incidence where a human was 34 transited by a N9 type influenza virus. Effective vaccination against influenza A (H7N9) is a major 35 concern, since it has emerged as a life threatening viral pathogen. Here, an in silico reverse 36 vaccinology strategy was adopted to design a unique chimeric subunit vaccine against avian 37 influenza A (H7N9). Induction of humoral and cell-mediated immunity is the prime concerned 38 characteristics for a peptide vaccine candidate, hence both T cell and B cell immunity of viral 39 proteins were screened. Antigenicity testing, transmembrane topology screening, allergenicity and 40 toxicity assessment, population coverage analysis and molecular docking approach were adopted 41 to generate the most antigenic epitopes of avian influenza A (H7N9) proteome. Further, a novel 42 subunit vaccine was designed by the combination of highly immunogenic epitopes along with 43 suitable adjuvant and linkers. Physicochemical properties and secondary structure of the designed 44 vaccine were assessed to ensure its thermostability, hydrophilicity, theoretical PI and 45 structural behavior. Homology modeling, refinement and validation of the designed vaccine 46 allowed to construct a three dimensional structure of the predicted vaccine, further employed to 47 molecular docking analysis with different MHC molecules and human immune TLR8 receptor 48 present on lymphocyte cells. Moreover, disulfide engineering was employed to lessen the high 49 mobility region of the designed vaccine in order to extend its stability. Furthermore, we 50 investigated the molecular dynamic simulation of the modeled subunit vaccine and TLR8 51 complexed molecule to strengthen our prediction. Finally, the suggested vaccine was reverse 52 transcribed and adapted for E. coli strain K12 prior to insertion within pET28a(+) vector for 53 checking translational potency and microbial expression. 54 55