Rhoptry proteins (ROPs) are involved in the cell invasion and parasitophorous vacuole (PV) formation and also vital for survival of Toxoplasma gondii (T. gondii) within host cells. ROP8 have a main role during the early phase of infection and can express in tachyzoite and bradyzoite forms. In the present study, we designed a novel multi-epitope DNA vaccine encoding the potential B and T-cell epitopes from ROP8 protein to evaluate the immunity and protective efficacy against acute T. gondii infection in BALB/c mice. For this purpose, several bioinformatics online servers were used. At first, the potential epitopes were selected for T and B cells using immune epitope database (IEDB) and BCPREDS online services. Then, the selected epitopes were fused together by SAPGTP linker. Finally, the physico-chemical features, secondary and tertiary structures, antigenicity, and allergenicity of the peptide were evaluated through different bioinformatics tools. Lastly, the multi-epitope peptide was successfully cloned into pcDNA3.1 expression vector. The DNA vaccine was subcutaneously injected into BALB/c mice and the immune responses of the vaccinated mice and controls were determined. The obtained results revealed that the multi-epitope ROP8 peptide has 183 amino acid residues with average molecular weight (MW) of 18.974 kDa. More than 98 % residues of the peptide were incorporated in favored and allowed regions of the Ramachandran plot. The antigenicity of multi-epitope peptide were estimated 0.8751 and 0.7649 by ANTIGENpro and VaxiJen servers, respectively. BALB/c mice immunized with DNA vaccine showed significantly increased the level of specific anti-T. gondii antibodies (P < 0.05), and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a TH1-type cellular immune response with production of IFN-γ and prolonged survival time, compared with the control groups (P < 0.05). The findings revealed that the multi-epitope ROP8 DNA vaccine induced strong humoral and cellular responses and prolonged the survival time in BALB/c mice, suggesting selection of potential epitopes may be a promising strategy for the design of multi-epitope-based vaccines.

中文翻译：

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用新型多表位ROP8 DNA疫苗对弓形虫进行急性感染，可在小鼠中诱导强烈的B细胞和T细胞反应。

Rhoptry蛋白（ROPs）参与细胞侵袭和寄生虫液泡（PV）的形成，并且对于弓形虫（T. gondii）在宿主细胞内的存活也至关重要。ROP8在感染的早期阶段起主要作用，并且可以以速殖子和缓殖子的形式表达。在本研究中，我们设计了一种新型多表位DNA疫苗，该疫苗编码来自ROP8蛋白的潜在B和T细胞表位，以评估BALB / c小鼠对急性弓形虫感染的免疫力和保护功效。为此，使用了多个生物信息学在线服务器。首先，使用免疫表位数据库（IEDB）和BCPREDS在线服务为T和B细胞选择潜在的表位。然后，将选定的表位通过SAPGTP接头融合在一起。最后是理化特征，二级和三级结构 通过不同的生物信息学工具评估了肽的抗原性和变应原性。最后，将多表位肽成功克隆到pcDNA3.1表达载体中。将DNA疫苗皮下注射到BALB / c小鼠中，并测定了接种的小鼠和对照的免疫应答。获得的结果表明，多表位ROP8肽具有183个氨基酸残基，平均分子量（MW）为18.974kDa。超过98％的肽残基被掺入了Ramachandran图的有利和允许区域。通过ANTIGENpro和VaxiJen服务器分别估计多表位肽的抗原性为0.8751和0.7649。用DNA疫苗免疫的BALB / c小鼠显示特异性抗T水平显着增加。刚地抗体（P <0.05），以及以IgG2a产生为主的混合IgG1 / IgG2a反应。与对照组相比，免疫小鼠还表现出TH1型细胞免疫反应，产生IFN-γ并延长了生存时间（P <0.05）。研究结果表明，多表位ROP8 DNA疫苗可诱导强烈的体液和细胞反应，并延长了BALB / c小鼠的存活时间，这表明选择潜在表位可能是设计基于多表位疫苗的有前途的策略。