Structural and Immunogenicity Analysis of Reconstructed Ancestral and Consensus P48/45 for cross-species Antimalaria Vaccine

The development of the antimalaria vaccine holds a promising future in malaria control. One of the antimalaria vaccine strategies known as the transmission-blocking vaccine is designed to inhibit the parasite transmission between human and mosquito by targeting parasite gametocyte. Previously, we found that P48/45 that include in the 6-Cysteine protein family shared by Plasmodium sp. We also detected vaccine properties that possess by all human-infecting Plasmodium and could be used as a cross-species antimalaria vaccine. In this study, we investigated the efficacy of P48/45 through the ancestral and consensus reconstruction approach. P48/45 phylogenetic and time tree analysis was done by RAXML and BEAST2. GRASP server and Ugene software were used to reconstructed ancestral and consensus sequences, respectively. The protein structural prediction was done by using inspired and Rosetta program. Each protein characteristic of P48/45 was analyzed by assessing hydrophobicity and Post-Translational Modification sites. Meanwhile, the Epitope sequence for B-cell, T-cell, and MHC was determined using an immunoinformatic approach. Lastly, molecular docking simulation was done to determine native binding interactions of P48/45-P230. The result showed a distinct protein characteristic of ancestral and consensus sequences. The immunogenicity analysis revealed the number of epitopes in the ancestral sequence is greater. The study also found a conserved epitope located in the binding site and consists of specific Post-Translational Modification sites. Hence, our research provides detailed insight into ancestral and consensus P48/45 efficacy.抗疟疾疫苗的研发在疟疾防控领域展现出极具潜力的发展前景。其中一类被称为传播阻断疫苗（transmission-blocking vaccine）的抗疟疾疫苗策略，旨在通过靶向疟原虫配子体，阻断疟原虫在人与蚊之间的传播。此前本团队的研究发现，P48/45属于疟原虫（Plasmodium sp.）共有的6半胱氨酸蛋白家族（6-Cysteine protein family）。同时我们还证实，所有感染人类的疟原虫均携带P48/45的疫苗相关特性，该蛋白可作为跨物种抗疟疾疫苗的候选靶点。本研究通过祖先序列与共有序列重构的方法，探究了P48/45的疫苗效力。研究采用RAXML与BEAST2工具完成了P48/45的系统发育与时间树分析；分别通过GRASP服务器与Ugene软件完成了祖先序列与共有序列的重构。蛋白质结构预测借助Inspired与Rosetta程序完成。本研究通过分析疏水性与翻译后修饰（Post-Translational Modification）位点，对P48/45的各项蛋白特征展开表征。同时，通过免疫信息学方法鉴定了B细胞、T细胞与主要组织相容性复合体（Major Histocompatibility Complex, MHC）的表位序列。最后，通过分子对接模拟实验探究了P48/45与P230的天然结合相互作用。研究结果显示，祖先序列与共有序列的蛋白特性存在显著差异。免疫原性分析表明，祖先序列所包含的表位数量更多。本研究还发现，结合位点处存在一处保守表位，且该表位带有特定的翻译后修饰位点。综上，本研究为解析祖先序列与共有序列形式的P48/45的疫苗效力提供了详尽的研究视角与理论支撑。