DataSheet_2_Screening Novel Vaccine Candidates for Leishmania Donovani by Combining Differential Proteomics and Immunoinformatics Analysis.docx

Visceral leishmaniasis (VL), also known as kala-azar, is the most dangerous form of leishmaniasis. Currently no effective vaccine is available for clinical use. Since the pathogenicity of different Leishmania strains is inconsistent, the differentially expressed proteins in Leishmania strains may play an important role as virulence factors in pathogenesis. Therefore, effective vaccine candidate targets may exist in the differentially expressed proteins. In this study, we used differential proteomics analysis to find the differentially expressed proteins in two Leishmania donovani strains, and combined with immunoinformatics analysis to find new vaccine candidates. The differentially expressed proteins from L. DD8 (low virulent) and L. 9044 (virulent) strains were analyzed by LC-MS/MS, and preliminarily screened by antigenicity, allergenicity and homology evaluation. The binding peptides of MHC II, IFN-γ and MHC I from differentially expressed proteins were then predicted and calculated for the second screening. IFN-γ/IL-10 ratios and conserved domain prediction were performed to choose more desirable differentially expressed proteins. Finally, the 3D structures of three vaccine candidate proteins were produced and submitted for molecular dynamics simulation and molecular docking interaction with TLR4/MD2. The results showed that 396 differentially expressed proteins were identified by LC-MS/MS, and 155 differentially expressed proteins were selected through antigenicity, allergenicity and homology evaluation. Finally, 16 proteins whose percentages of MHC II, IFN-γ and MHC I binding peptides were greater than those of control groups (TSA, LmSTI1, LeIF, Leish-111f) were considered to be suitable vaccine candidates. Among the 16 candidates, amino acid permease, amastin-like protein and the hypothetical protein (XP_003865405.1) simultaneously had the large ratios of IFN-γ/IL-10 and high percentages of MHC II, IFN-γ and MHC I, which should be focused on. In conclusion, our comprehensive work provided a methodological basis to screen new vaccine candidates for a better intervention against VL and associated diseases.

内脏利什曼病（VL），亦称黑热病，为利什曼病中最危险的形式。目前尚无有效的疫苗可供临床应用。鉴于不同利什曼菌菌株的致病性存在差异，利什曼菌菌株中差异表达的蛋白质可能在发病机制中扮演重要的致病因子角色。因此，在差异表达的蛋白质中可能存在有效的疫苗候选靶点。在本研究中，我们运用差异蛋白质组学分析，以寻找两种利什曼原虫道诺凡尼菌株中的差异表达蛋白，并结合免疫信息学分析以发现新的疫苗候选物。对来自低毒力菌株L. DD8和高毒力菌株L. 9044的差异表达蛋白进行了LC-MS/MS分析，并通过抗原性、致敏性和同源性评估进行初步筛选。随后，对差异表达蛋白的MHC II、IFN-γ和MHC I结合肽进行了预测和计算，以进行第二轮筛选。通过IFN-γ/IL-10比率分析和保守结构域预测，选择了更具吸引力的差异表达蛋白。最终，制备了三种疫苗候选蛋白的三维结构，并提交进行分子动力学模拟以及与TLR4/MD2的分子对接相互作用。结果显示，通过LC-MS/MS鉴定出396个差异表达蛋白，通过抗原性、致敏性和同源性评估筛选出155个差异表达蛋白。最终，16个MHC II、IFN-γ和MHC I结合肽百分比高于对照组（TSA、LmSTI1、LeIF、Leish-111f）的蛋白被认为适合作为疫苗候选物。在这16个候选物中，氨基酸渗透酶、类似amastin蛋白和假设蛋白（XP_003865405.1）同时具有大比率的IFN-γ/IL-10和高百分比的MHC II、IFN-γ和MHC I，应予以重点关注。总之，我们的综合研究为筛选新的疫苗候选物，以更好地干预VL及其相关疾病提供了方法论基础。