Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Microb Pathog. 2021 Jun;155:104925. doi: 10.1016/j.micpath.2021.104925. Epub 2021 Apr 29.
Current study deals with a novel multi-epitope vaccine designed in silico and its confirmation experiments for potential efficacy in BALB/c mice. Major histocompatibility complex (MHC)-binding and B-cell binding epitopes of five Toxoplasma antigens (SAG1, ROP16, GRA12, MIC4 and M2AP) were predicted. Selected epitopes were fused together using SAPGTP linker, and antigenicity, allergenicity, physico-chemical features, secondary and tertiary structures and validations were all performed via bioinformatics servers. Then, vaccine construct was cloned into pLEXSY-neo 2.1 vector. After Leishmania tarentolae transfection, live recombinant and wild parasites were subcutaneously injected into 6-8 week female BALB/c mice and immune responses were measured. Results showed that the peptide possessed 282 amino acid residues with average molecular weight of 28.06 kDa. About 90% of the peptide residues were incorporated in favored and allowed regions of the Ramachandran plot. Vaccinated mice showed remarkably elevated levels of specific antibodies (P < 0.05) with predominance of IgG2a production. Also, a Th immune response with production of IFN-γ and relatively increased survival rate against intraperitoneal challenge with RH strain was demonstrated in immunized mice than control groups (P < 0.05). Also, very low levels of IL-4 were demonstrated, which showed statistically significant association with controls (P < 0.05). The findings clarified that multi-epitope vaccine expressed in Leishmania tarentolae induced significant immune responses against acute toxoplasmosis.
本研究涉及一种新型多表位疫苗的设计及其在 BALB/c 小鼠中的潜在疗效的验证实验。预测了五种弓形虫抗原（SAG1、ROP16、GRA12、MIC4 和 M2AP）的主要组织相容性复合体（MHC）结合和 B 细胞结合表位。选择的表位使用 SAPGTP 接头融合在一起，并通过生物信息学服务器进行抗原性、变应原性、物理化学特性、二级和三级结构以及验证。然后，疫苗构建体被克隆到 pLEXSY-neo 2.1 载体中。在 Leishmania tarentolae 转染后，活重组和野生寄生虫被皮下注射到 6-8 周龄雌性 BALB/c 小鼠中，并测量免疫反应。结果表明，该肽具有 282 个氨基酸残基，平均分子量为 28.06 kDa。大约 90%的肽残基位于 Ramachandran 图谱的有利和允许区域。与对照组相比，接种疫苗的小鼠表现出显著升高的特异性抗体水平（P < 0.05），以 IgG2a 产生为主。此外，在免疫小鼠中还证明了 Th 免疫反应，产生 IFN-γ，并且相对于 RH 株的腹腔内攻毒，存活率相对增加（P < 0.05）。此外，还证明了非常低水平的 IL-4，与对照组相比具有统计学显著相关性（P < 0.05）。这些发现表明，在 Leishmania tarentolae 中表达的多表位疫苗诱导了针对急性弓形体病的显著免疫反应。
