Wu Qinli, Zhang Zhongao, Chu Hongkun, Xia Bing, Li Weiqi, Ding Jianzu, Ding Haojie, Zheng Bin, Gao Meng, Wang Youru, El Shanawany Eman E, Tan Feng, Ye Huayue, Zhuo Xunhui, Lu Shaohong
School of Basic Medical Sciences and Forensic Medicine, Hangzhou Medical College, Hangzhou, 310013, China.
Engineering Research Center of Novel Vaccine of Zhejiang Province, Hangzhou Medical College, Hangzhou, China.
Infect Dis Poverty. 2025 Jun 23;14(1):55. doi: 10.1186/s40249-025-01332-6.
Toxoplasma gondii, an intracellular parasitic protozoan, which infects almost all warm-blooded animals, including humans, causes toxoplasmosis. However, we lack effective drugs and vaccines to control toxoplasmosis, representing a clinical challenge. Therefore, safe and effective vaccines are urgently needed. In this study, a self-replicating mRNA vaccine comprising four T. gondii antigens: ROP18, TGME49_237490, TGME49_268230, and MIC13, named 4x-mRNA-LNP (lipid nanoparticle), was developed, and its protective efficacy was evaluated in mice.
The expression of this vaccine in eukaryotic Human embryonic kidney 293 T (HEK-293 T) cells and mouse myoblast (C2C12) cells were analyzed, followed by enzyme-linked immunosorbent assay (ELISA) evaluation of the elicited humoral immune response. Subsequently, the vaccine-triggered immune responses in mice were detected, including antibody titers, T lymphocyte subsets, and cytokine levels. Finally, its immunoprotective effects were evaluated after challenging mice with T. gondii PRU oocysts or tachyzoites of different strains and analyzing the pathological changes, parasite loads, and mouse survival time. Western blotting and ELISA confirmed the successful eukaryotic expression and immunogenicity of 4x-mRNA, respectively. Statistical analyses, including the log-rank (Mantel-Cox) test, Student's t-test, and one-way ANOVA, were performed using GraphPad Prism software.
Mice vaccinated with 4x-mRNA-LNP generated higher levels of IgG1 and IgG2a antibodies (P < 0.05) and cytokines (IL-2, IL-4, IL-10, IL-12, IFN-γ) (P < 0.05) compared with the control group. The high specific IgG titer was maintained for at least 10 weeks after the last vaccination. The proportion of CD3CD4 T cells and CD3CD8 T cells also increased significantly (P < 0.05), along with increased spleen cell proliferation in 4x-mRNA-LNP-vaccinated mice. Notably, limited pathological changes and < 10 fg of parasites/mg were found in the immunized mice tissues post-pathogen challenge. During observation for 30 days, 4x-mRNA-LNP-immunized mice survived significantly longer under challenge with lethal doses of RH, ME49, or WH6 tachyzoites (survival rates = 60%, 80%, and 60%, respectively). Following PRU oocyst challenge, vaccinated mice had notably decreased cyst burdens (72.5%, P < 0.05) compared with control mice.
The 4x-mRNA-LNP vaccine triggered effective long-term antibody levels in mice, thus representing a promising candidate to further develop anti-toxoplasmosis vaccines.
刚地弓形虫是一种细胞内寄生的原生动物，可感染包括人类在内的几乎所有温血动物，引发弓形虫病。然而，我们缺乏有效的药物和疫苗来控制弓形虫病，这是一个临床挑战。因此，迫切需要安全有效的疫苗。在本研究中，开发了一种包含四种刚地弓形虫抗原（ROP18、TGME49_237490、TGME49_268230和MIC13）的自我复制mRNA疫苗，命名为4x-mRNA-LNP（脂质纳米颗粒），并在小鼠中评估了其保护效果。
分析该疫苗在真核人类胚胎肾293T（HEK-293T）细胞和小鼠成肌细胞（C2C12）细胞中的表达，随后通过酶联免疫吸附测定（ELISA）评估引发的体液免疫反应。随后，检测疫苗在小鼠中引发的免疫反应，包括抗体滴度、T淋巴细胞亚群和细胞因子水平。最后，在用刚地弓形虫PRU卵囊或不同菌株的速殖子攻击小鼠并分析病理变化、寄生虫载量和小鼠存活时间后，评估其免疫保护作用。蛋白质免疫印迹法和ELISA分别证实了4x-mRNA在真核细胞中的成功表达和免疫原性。使用GraphPad Prism软件进行统计分析，包括对数秩（Mantel-Cox）检验、学生t检验和单因素方差分析。
与对照组相比，接种4x-mRNA-LNP的小鼠产生了更高水平的IgG1和IgG2a抗体（P<0.05）以及细胞因子（IL-2、IL-4、IL-10、IL-12、IFN-γ）（P<0.05）。最后一次接种后，高特异性IgG滴度至少维持了10周。接种4x-mRNA-LNP的小鼠中，CD3CD4 T细胞和CD3CD8 T细胞的比例也显著增加（P<0.05），同时脾细胞增殖增加。值得注意的是，在病原体攻击后，免疫小鼠组织中发现的病理变化有限，且每毫克组织中的寄生虫量<10 fg。在观察期30天内，用致死剂量的RH、ME49或WH6速殖子攻击时，接种4x-mRNA-LNP的小鼠存活时间显著延长（存活率分别为60%、80%和60%）。在用PRU卵囊攻击后，与对照小鼠相比，接种疫苗的小鼠的包囊负担显著降低（72.5%，P<0.05）。
4x-mRNA-LNP疫苗在小鼠中引发了有效的长期抗体水平，因此是进一步开发抗弓形虫病疫苗的有希望的候选者。
