Moghadamizad Zeinab, Dalimi Abdolhossein, Pirestani Majid, Ghafarifar Fatemeh
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. 2025 Jul;204:107567. doi: 10.1016/j.micpath.2025.107567. Epub 2025 Apr 10.
This study designed and evaluated a multi-epitope DNA vaccine targeting Toxoplasma gondii immunodominant antigens-ROP5, ROP7, and SAG1-to assess its protective efficacy against acute and chronic toxoplasmosis in BALB/c mice. A bioengineered multi-epitope vaccine construct (MEVC) was synthesized by integrating computationally predicted B- and T-cell epitopes using SAPGTP linkers to ensure conformational stability and epitope accessibility. In silico analyses confirmed the MEVC's antigenicity (VaxiJen score: 0.96), non-allergenicity, solubility (GRAVY index: 0.45), and physicochemical stability (instability index: 32.14; aliphatic index: 78.3), supporting its suitability for immunization. The codon-optimized sequence (753 bp; 253 amino acids) was cloned into the pcDNA3.1(+) plasmid and amplified in Escherichia coli TOP10 cells. Thirty-six female BALB/c mice (6-8 weeks) were divided into three groups (n = 12/group) and immunized intramuscularly with 100 μg MEVC, empty vector, or phosphate-buffered saline (PBS) at weeks 0, 2, and 4. Post-immunization, mice were challenged with acute (2 × 10 RH strain tachyzoites, intraperitoneal) or chronic (10 PRU strain cysts, oral) infection. Molecular docking simulations demonstrated high-affinity binding of the MEVC to murine toll-like receptor 4 via hydrogen bonds and hydrophobic interactions, suggesting adjuvant-like immunogenicity. In vitro expression in HEK-293 cells confirmed protein synthesis, with Western blot detecting a 26 kDa immunoreactive band. MEVC-immunized mice exhibited significantly elevated anti-Toxoplasma IgG titers (1:12,800), dominated by IgG2a isotypes (P < 0.05), and robust IFN-γ production, indicative of Th1-polarized immunity. IL-4 levels remained low, confirming minimal Th2 skewing. Vaccination reduced cerebral cyst burden by 76 % (P < 0.01) in chronic infection, yet survival post-acute challenge extended only two days compared to controls. These results demonstrate partial protection against toxoplasmosis, with the MEVC eliciting cellular and humoral responses effective against chronic infection but limited efficacy in acute settings.
本研究设计并评估了一种靶向弓形虫免疫显性抗原ROP5、ROP7和SAG1的多表位DNA疫苗，以评估其对BALB/c小鼠急性和慢性弓形虫病的保护效果。通过使用SAPGTP接头整合经计算预测的B细胞和T细胞表位，合成了一种生物工程多表位疫苗构建体（MEVC），以确保构象稳定性和表位可及性。计算机模拟分析证实了MEVC的抗原性（VaxiJen评分：0.96）、无致敏性、溶解性（亲水性氨基酸平均指数：0.45）和物理化学稳定性（不稳定指数：32.14；脂肪族指数：78.3），支持其适用于免疫接种。将密码子优化序列（753 bp；253个氨基酸）克隆到pcDNA3.1(+)质粒中，并在大肠杆菌TOP10细胞中扩增。36只6 - 8周龄雌性BALB/c小鼠分为三组（每组n = 12），在第0、2和4周通过肌肉注射100μg MEVC、空载体或磷酸盐缓冲盐水（PBS）进行免疫。免疫后，小鼠接受急性（腹腔注射2×10个RH株速殖子）或慢性（口服10个PRU株包囊）感染攻击。分子对接模拟显示MEVC通过氢键和疏水相互作用与小鼠Toll样受体4具有高亲和力结合，表明具有佐剂样免疫原性。在HEK - 293细胞中的体外表达证实了蛋白质合成，蛋白质印迹检测到一条26 kDa的免疫反应条带。用MEVC免疫的小鼠表现出抗弓形虫IgG滴度显著升高（1:12,800），以IgG2a亚型为主（P < 0.05），并且产生强大的IFN -γ，表明为Th1极化免疫。IL - 4水平保持较低，证实Th2偏向最小。在慢性感染中，疫苗接种使脑包囊负担降低了76%（P < 0.01），但与对照组相比，急性攻击后的存活时间仅延长了两天。这些结果表明该疫苗对弓形虫病有部分保护作用，MEVC引发的细胞和体液反应对慢性感染有效，但在急性情况下疗效有限。
