Dataset of selection and identification of mimotopes of the variant surface glycoprotein of Trypanosoma evansi RoTat1.2 isolate by PhD-12 peptide phage display library panning against monoclonal antibodies

By employing two murine monoclonal antibodies (mAbs), designated as 2E11 (IgG1) and 1C2 (igG1), we have selected and identified the peptide mimotopes of T. evansi RoTat 1.2 variant surface glycoprotein (VSG) [File 1]. The mAbs-selected mimotopes were then used to predict the conformational B epitopes on VSG. First, we produced by the hybridoma technique the mAbs against the T. evansi RoTat 1.2 lysate antigens. The 2E11 mAb was used to immunoprecipitate the target antigen in the parasite lysate, which was then identified to be the VSG by mass spectrometry. Both 2E11 and 1C2 mAbs reacted with the VSG in the Western blots. Further, the biotinylated mAbs were used in three rounds of panning to select the peptide mimotopes out of the phage-displayed 12-mer random peptides library (PhD-12; New England Biolabs, USA) [File 1-4; Fig. 1-2]. The peptide-displaying phage clones specifically bound to the selector mAb in the phage- capture ELISA [File 4 table 6-7]. The peptide coding regions of the selected phages were sequenced [File 4 table 4-5] and the related sequences were blast searched in the non-redundant protein database at https://blast.ncbi.nlm.nih.gov/ [File 5-13]. By using 3D Pepitope algorithms accessed at http://pepitope.tau.ac.il/, the mimotope sequences predicted the conformational B epitopes on T. brucei IlTat1.24 VSG (PDB id: 2VSG_A or 2VSG_B) [File 14-28; Fig. 3-4]. T. brucei IlTat1.24 2VSG_A and 2VSG_B showed homology to T. evansi RoTat1.2 VSGs of various isolates [File 29-35]. Data generated from our laboratory experiments and by application of bioinformatics tools are presented for the B epitope mapping of the VSG of an Indian isolate of Trypanosoma evansi RoTat1.2.
Four different peptide mimotope sequences (deduced amino acid sequences) were obtained from 11 sequences of 2E11-binder clones (File 4 table 4]. The consensus mimotope [HWKAVNWLKPWT] was represented by 8 identical sequences. Whereas, five distinct mimotope sequences were obtained from 14 1C2-binder clones. While the consensus sequence [WHWDANRWWTYR] was represented by 9 identical sequences [File 4 table 5]. The conformational B epitopes were predicted by Pepsurf alignment of 2E11 and 1C2 mAb- selected peptide mimotopes against T. brucei IlTat1.24 VSG (PDB ID: 2VSG_A) using 3D Pepitope algorithms [accessed at: (http://pepitope.tau.ac.il/)] [File 14-28; Fig. 3-4]. The best cluster identified by three 2E11-selected mimotopes consisted of 14 AA [File 24], whereas that identified by two 1C2-selected mimotopes consisted of 17 AA [File 26]. Of these AA in the best clusters, nine AA, i.e., T40, P259, T262, V264, T266, E314, A315, W317 and N318 were found to be shared or common. The conformational B cell epitopes on 2VSG_A predicted by the DiscoTope tool at http://www.iedb.org/ shared some AA positions in both 2E11 and 1C2-selected mimotopes [Fig. 4 & File 36]. These data could be useful for diagnostics, vaccine design, and research in trypanosome biology.

通过采用两种小鼠单克隆抗体（mAbs），分别命名为2E11（IgG1）和1C2（IgG1），我们已筛选并鉴定了锥虫埃万西（Trypanosoma evansi）RoTat 1.2变异表面糖蛋白（VSG）[文件1]的肽模拟物。随后，利用这些mAbs筛选出的模拟物预测了VSG上的构象性B表位。首先，我们通过杂交瘤技术制备了对锥虫埃万西RoTat 1.2裂解抗原的mAbs。2E11 mAb被用于从寄生虫裂解物中免疫沉淀目标抗原，经质谱鉴定确定为VSG。2E11和1C2 mAbs在Western blot中均与VSG发生反应。进一步地，生物素化的mAbs在三次洗选过程中用于从噬菌体展示的12-mer随机肽库（PhD-12；美国新英格兰生物实验室）[文件1-4；图1-2]中筛选肽模拟物。展示肽的噬菌体克隆特异性地结合到选择mAb上，在噬菌体捕获ELISA中表现出反应[文件4表6-7]。所筛选噬菌体的肽编码区域经测序[文件4表4-5]，相关序列在非冗余蛋白质数据库https://blast.ncbi.nlm.nih.gov/中进行BLAST搜索[文件5-13]。通过访问http://pepitope.tau.ac.il/的3D Pepitope算法，预测了锥虫布鲁氏（Trypanosoma brucei）IlTat1.24 VSG（PDB id: 2VSG_A或2VSG_B）上的构象性B表位[文件14-28；图3-4]。锥虫布鲁氏IlTat1.24的2VSG_A和2VSG_B与各种分离株的锥虫埃万西RoTat1.2 VSGs具有同源性[文件29-35]。本研究室实验生成数据和通过生物信息学工具的应用，展示了印度分离株锥虫埃万西RoTat1.2 VSG的B表位定位。从11个2E11结合克隆的11个序列中获得了四种不同的肽模拟物序列（推导的氨基酸序列）。由8个相同序列表示的共识模拟物[HWKAVNWLKPWT]。而来自14个1C2结合克隆的14个序列中获得了五种不同的模拟物序列。共识序列[WHWDANRWWTYR]由9个相同序列表示[文件4表5]。通过3D Pepitope算法[访问：http://pepitope.tau.ac.il/]对2E11和1C2 mAb筛选的肽模拟物与锥虫布鲁氏IlTat1.24 VSG（PDB ID: 2VSG_A）进行Pepsurf比对，预测了构象性B表位。由三个2E11选择的模拟物识别出的最佳簇由14个氨基酸（AA）组成[文件24]，而由两个1C2选择的模拟物识别出的最佳簇由17个AA组成[文件26]。在这些AA中，九个AA，即T40、P259、T262、V264、T266、E314、A315、W317和N318被发现是共有或共享的。通过DiscoTope工具在http://www.iedb.org/上预测的2VSG_A上的构象性B细胞表位与2E11和1C2选择的模拟物中的一些氨基酸位置共享[图4 & 文件36]。这些数据可用于诊断、疫苗设计和锥虫生物学研究。