Zhu Yuchao, Xu Yanan, Hong Lu, Zhou Chunxue, Chen Jia
Department of Radiology, The Affiliated Hospital of Medical School of Ningbo University, Ningbo, China.
The Ningbo Women and Children's Hospital, Ningbo, China.
Front Microbiol. 2021 Apr 28;12:630682. doi: 10.3389/fmicb.2021.630682. eCollection 2021.
, an obligate intracellular protozoan parasite, can cause infect almost all warm-blooded animals and humans. To evaluate the immunogenicity and protective efficacy of GRA39 (TgGRA39) in mice by using DNA immunization, we constructed a recombinant eukaryotic plasmid pVAX-TgGRA39. The specific immune responses in immunized mice were analyzed by serum antibody and cytokine measurements, lymphocyte proliferation assays and flow cytometry of T lymphocyte subclasses. Also, protective efficacy against acute and chronic infection was assessed by observing the survival time after challenge with the highly virulent RH strain (Genotype I) and counting the number of cyst-forming in brain at 4 weeks post-infection with the cyst-forming PRU strain of (Genotype II), respectively. Our results showed that DNA immunization with pVAX-GRA39 via intramuscular injection three times, at 2-week intervals could elicit humoral and cellular immune response, indicated by enhanced levels of IgG and IgG2a antibodies (a slightly elevated IgG2a to IgG1 ratio), and increased levels of cytokines IFN-γ, IL-2, IL-12, IL-17A, IL-17F, IL-22 and IL-23 and percentages of CD3+ CD4+ CD8- and CD3+ CD8+ CD4- T cells, in contrast to non-immunized mice. The significant increase in the expression levels of IL-6, TGF-β1, IL-1β, and the transcription factor factors RORγt, RORα, and STAT3 involved in the activation and pathway of Th17 and Tc17 cells, were also observed. However, no significant difference was detected in level of IL-4 and IL-10 ( > 0.05). These effective immune responses had mounted protective immunity against infection, with a prolonged survival time (16.80 ± 3.50 days) and reduced cyst numbers (44.5%) in comparison to the control mice. Our data indicated that pVAX-TgGRA39 could induce effective humoral, and Th1-type, Th17, and Tc17 cellular immune responses, and may represent a promising vaccine candidate against both acute and chronic infection.
作为一种专性细胞内原生动物寄生虫，可感染几乎所有温血动物和人类。为了通过DNA免疫评估GRA39（TgGRA39）在小鼠中的免疫原性和保护效果，我们构建了重组真核质粒pVAX-TgGRA39。通过血清抗体和细胞因子检测、淋巴细胞增殖试验以及T淋巴细胞亚群的流式细胞术分析免疫小鼠中的特异性免疫反应。此外，分别通过观察用高毒力RH株（基因型I）攻击后的存活时间以及在感染形成包囊的PRU株（基因型II）4周后计数脑中形成包囊的数量，评估对急性和慢性感染的保护效果。我们的结果表明，每隔2周通过肌肉注射三次pVAX-GRA39进行DNA免疫可引发体液和细胞免疫反应，表现为IgG和IgG2a抗体水平升高（IgG2a与IgG1的比例略有升高），以及细胞因子IFN-γ、IL-2、IL-12、IL-17A、IL-17F、IL-22和IL-23水平升高，CD3+ CD4+ CD8-和CD3+ CD8+ CD4- T细胞百分比增加，与未免疫小鼠相比。还观察到参与Th17和Tc17细胞活化和信号通路的IL-6、TGF-β1、IL-1β以及转录因子RORγt、RORα和STAT3的表达水平显著增加。然而，IL-4和IL-10水平未检测到显著差异（P>0.05）。这些有效的免疫反应对感染产生了保护性免疫，与对照小鼠相比，存活时间延长（16.80±3.50天）且包囊数量减少（44.5%）。我们的数据表明，pVAX-TgGRA39可诱导有效的体液免疫以及Th1型、Th17和Tc17细胞免疫反应，可能是一种针对急性和慢性感染的有前景的疫苗候选物。
