Type A influenza viruses are contagious and even life-threatening if left untreated. So far, no broadly protective vaccine is available due to rapid antigenic changes and emergence of new subtypes of influenza virus. In this study, we exploited bioinformatics tools in order to design a subunit chimeric vaccine from the antigenic and highly conserved regions of HA and M2 proteins of H7N9 subtype of influenza virus. We used mucosal adjuvant candidates, including CTxB, STxB, ASP-1, and LTB to stimulate mucosal immunity and analyzed the combination of HA2, M2e, and the adjuvant. Furthermore, to improve the antigen function and to maintain their three-dimensional structure, 12 different linkers including six rigid linkers and six flexible linkers were used. The 3D structure model was generated using a combination of homology and ab initio modeling methods and the molecular dynamics of the model were analyzed, either. Analysis of different adjuvants showed that using CtxB as an adjuvant, results in higher overall vaccine stability and higher half-life among four adjuvant candidates. Fusion of antigens and the CTxB in the form of M2e-linker-CTxB-linker-HA2 has the most stability and half life compared to other combination forms. Furthermore, the KPKPKP rigid linker showed the best result for this candidate vaccine among 12 analyzed linkers. The changes in the vaccine 3D structure made by linker insertion found to be negligible, however, although small, the linker insertion between the antigens causes the structure to change slightly. Eventually, using predictive tools such as Ellipro, NetMHCpan I and II, CD4episcore, CTLpred, BepiPred and other epitope analyzing tools, we analyzed the conformational and linear epitopes of the vaccine. The solubility, proteasome cleavage sites, peptidase and potential chemical cutters, codon optimization, post translational modification were also carried out on the final vaccine. It is concluded that M2e-Linker-CTxB-Linker-HA2 combination of chimeric vaccine retains its 3D structure and antigenicity when KPKPKP used as linker and CTxB used as adjuvant.

中文翻译：

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基于HA2，M2e和CTxB设计流感病毒嵌合亚单位疫苗：一项生物信息学研究

如果不及时治疗，A型流感病毒具有传染性，甚至危及生命。迄今为止，由于快速的抗原变化和流感病毒新亚型的出现，没有广泛的保护性疫苗可用。在这项研究中，我们利用生物信息学工具从流感病毒的H7N9亚型HA和M2蛋白的抗原性和高度保守区域设计亚单位嵌合疫苗。我们使用包括CTxB，STxB，ASP-1和LTB在内的粘膜佐剂候选物刺激粘膜免疫，并分析了HA2，M2e和佐剂的组​​合。此外，为了改善抗原功能并维持其三维结构，使用了12种不同的接头，包括6个刚性接头和6个柔性接头。使用同源性和从头算建模方法的组合生成3D结构模型，并分析模型的分子动力学。对不同佐剂的分析表明，使用CtxB作为佐剂，可在四种佐剂中提高疫苗的整体稳定性和半衰期。与其他组合形式相比，M2e-接头-CTxB-接头-HA2形式的抗原与CTxB融合具有最大的稳定性和半衰期。此外，在12个分析的接头中，KPKPKP刚性接头显示了该候选疫苗的最佳结果。发现通过接头插入引起的疫苗3D结构的改变可以忽略不计，但是，尽管很小，但是抗原之间的接头插入导致结构略微改变。最终，使用Ellipro等预测工具，NetMHCpan I和II，CD4episcore，CTLpred，BepiPred和其他表位分析工具，我们分析了疫苗的构象和线性表位。还对最终疫苗进行了溶解度，蛋白酶体切割位点，肽酶和潜在的化学切割剂，密码子优化，翻译后修饰。结论是，当KPKPKP用作接头而CTxB用作佐剂时，嵌合疫苗的M2e-接头-CTxB-接头-HA2组合保留了其3D结构和抗原性。