刚地弓形虫 RHΔompdcΔuprt 活疫苗突变株可在小鼠和猫体内诱导强烈的保护性免疫，抵抗弓形虫病。

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats.

机构信息

School of Basic Medical Sciences and Forensic Medicine, Hangzhou Medical College, Hangzhou, Zhejiang, China.

Engineering Research Center of Novel Vaccine of Zhejiang Province, Hangzhou Medical College, Hangzhou, China.

出版信息

Infect Dis Poverty. 2023 Jun 15;12(1):60. doi: 10.1186/s40249-023-01109-9.

DOI:10.1186/s40249-023-01109-9

PMID:37322556

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10266960/

Abstract

BACKGROUND

Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats.

METHODS

First, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel-Cox) test, Student's t test and one-way ANOVA.

RESULTS

The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8 T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80 strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%).

CONCLUSIONS

The avirulent RHΔompdcΔuprt strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.

摘要

背景

刚地弓形虫是一种专性细胞内顶复门寄生虫，可引起人畜共患的弓形虫病。开发有效的抗弓形虫疫苗来控制弓形虫病至关重要，本研究旨在探索一种活减毒疫苗在小鼠和猫中的免疫保护作用。

方法

首先，通过 CRISPR-Cas9 系统敲除弓形虫的 ompdc 和 uprt 基因。然后，评估该突变株的细胞内增殖和毒力。随后，检测该突变株在小鼠和猫中诱导的免疫反应，包括抗体滴度、细胞因子水平和 T 淋巴细胞亚群。最后，通过用不同株的速殖子或猫的 ME49 株的包囊对小鼠进行攻毒，评估免疫保护作用。此外，为了发现针对弓形虫病的有效免疫因素，进行了被动免疫。使用 GraphPad Prism 软件进行对数秩（Mantel-Cox）检验、学生 t 检验和单因素方差分析。

结果

通过 CRISPR-Cas9 系统构建了 RHΔompdcΔuprt。与野生型株相比，突变株的增殖明显减少（P<0.05）。此外，该突变株在小鼠（BALB/c 和 BALB/c-nu）和猫模型中表现出毒力减弱。值得注意的是，在 RHΔompdcΔuprt 注射小鼠的组织中发现的病理变化有限。此外，与未免疫组相比，突变株可诱导小鼠产生高水平的 IgG（IgG1 和 IgG2a）抗体和细胞因子（IFN-γ、IL-4、IL-10、IL-2 和 IL-12）（P<0.05）。值得注意的是，所有 RHΔompdcΔuprt 疫苗接种的小鼠均能耐受 RHΔku80 和 ME49 和 WH6 株的致死性攻击而存活。与未感染的小鼠相比，免疫血清和脾细胞，特别是 CD8 T 细胞，可显著延长（P<0.05）感染 RHΔku80 株的小鼠的存活时间。此外，与未免疫的猫相比，用突变株免疫的猫产生高水平的抗体和细胞因子（P<0.05），并显著减少粪便中卵囊的脱落数量（95.3%）。