一种通过计算机设计的、编码刚地弓形虫ROP21和ROP29的多表位DNA疫苗对BALB/c小鼠急性和慢性弓形虫病的免疫原性

Immunogenicity of an in-silico designed multi-epitope DNA vaccine encoding ROP21 and ROP29 of Toxoplasma gondii against both acute and chronic toxoplasmosis in BALB/c mice.

作者信息

Karimipour-Saryazdi Amir, Ghaffarifar Fatemeh, Dalimi Abdolhossein, Foroutan Masoud, Sadraei Javid, Horton John

机构信息

Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Department of Basic Medical Sciences, Faculty of Medicine, Abadan University of Medical Sciences, Abadan, Iran.

出版信息

Microb Pathog. 2025 Mar;200:107321. doi: 10.1016/j.micpath.2025.107321. Epub 2025 Jan 22.

DOI:10.1016/j.micpath.2025.107321

PMID:39855488

Abstract

BACKGROUND

Toxoplasma infections are highly prevalent worldwide and can cause serious complications in immunocompromised individuals and lead to congenital infections in neonates. Despite ongoing efforts to develop T. gondii vaccines, none have been developed. A potential target, the ROP21 protein catalyzes the phosphorylation of additional protein substrates and ROP29 is critical for chronic Toxoplasma gondii infection. In this study, recombinant plasmid with epitopes of ROP21 and ROP29 were used as DNA vaccine.

METHODS

An immunoinformatics approach was employed to design a multi-epitope DNA vaccine encoding T. gondii ROP21 and ROP29. The bioinformatic outputs supported the immunogenic and non-allergic nature of multi-epitope vaccine. The recombinant plasmid was transfected in HEK cells. Thereafter, the protective effect of the DNA vaccine was evaluated in BALB/c mice by way of vaccination and challenge of these mice with acute RH and chronic PRU strains of T. gondii, respectively. The immunological responses of the control and vaccinated groups were assessed using survival time, lymphocyte proliferation assays, cytokine and antibody measurements, and determination of the parasite load in the spleen with real-time PCR.

FINDINGS

Multiple epitope (ROP21 and ROP29) DNA immunization stimulated cellular and humoral immune reactions in BALB/c mice and lengthened their life following challenge. Multiple epitope proteins increased significantly the total IgG antibody concentrations, triggered increased IFN-γ cytokine production, increased the mixed IgG1/IgG2a response with a predominance of IgG2a, prolonged the survival duration and reduced the number of brain cysts. The in vivo findings indicated that the multi-epitope DNA vaccine elicited significant production of IgG antibodies (122.16 ng/ml) as well as IFN-γ (12.37 pg/ml) (p < 0.05). Moreover, a significantly reduced parasite-burden (CT: 35.62) and prolonged survival time (14 days) were observed in the immunized groups compared to the controls (p < 0.05). Low IL-4 (5.63 pg/ml) values were detected in vaccinated mice compared to the PBS control (p > 0.05).

CONCLUSIONS

We found that multiepitope protein vaccination could provide more protective immunity against chronic and acute toxoplasmosis infection compared to control.

摘要

背景

弓形虫感染在全球范围内高度流行，可在免疫功能低下的个体中引起严重并发症，并导致新生儿先天性感染。尽管一直在努力开发弓形虫疫苗，但尚未有疫苗问世。一个潜在的靶点是，ROP21蛋白催化其他蛋白质底物的磷酸化，而ROP29对慢性弓形虫感染至关重要。在本研究中，将带有ROP21和ROP29表位的重组质粒用作DNA疫苗。

方法

采用免疫信息学方法设计一种编码弓形虫ROP21和ROP29的多表位DNA疫苗。生物信息学结果支持了多表位疫苗的免疫原性和非过敏性。将重组质粒转染到HEK细胞中。此后，通过分别用急性RH株和慢性PRU株弓形虫对BALB/c小鼠进行接种和攻击，评估DNA疫苗的保护作用。使用生存时间、淋巴细胞增殖试验、细胞因子和抗体测量以及通过实时PCR测定脾脏中的寄生虫载量，评估对照组和接种组的免疫反应。

结果

多表位（ROP21和ROP29）DNA免疫在BALB/c小鼠中刺激了细胞免疫和体液免疫反应，并在攻击后延长了它们的寿命。多表位蛋白显著提高了总IgG抗体浓度，引发了IFN-γ细胞因子产生增加，增加了以IgG2a为主的混合IgG1/IgG2a反应，延长了生存时间并减少了脑囊肿数量。体内研究结果表明，多表位DNA疫苗引发了显著的IgG抗体（122.16 ng/ml）以及IFN-γ（12.37 pg/ml）产生（p < 0.05）。此外，与对照组相比，免疫组的寄生虫负荷显著降低（CT：35.62），生存时间延长（14天）（p < 0.05）。与PBS对照组相比，接种疫苗的小鼠中检测到低水平的IL-4（5.63 pg/ml）（p > 0.05）。