This study aimed to test the efficacy of a DNA vaccine encompassing GRA35, GRA42, and GRA43 in inducing protective immunity against the highly virulent T. gondii RH strain (type I) and the brain cyst-forming PRU strain (type II).
The eukaryotic plasmids pVAX-GRA35, pVAX-GRA42, and pVAX-GRA43 were constructed and formulated into two- or three-gene cocktail DNA vaccines. The indirect immunofluorescence assay (IFA) was used to analyze their expression and immunogenicity. Mice were immunized with a single-gene, two-genes, or multicomponent eukaryotic plasmid, intramuscularly. We assessed antibody levels, cytotoxic T-cell (CTL) responses, cytokines, and lymphocyte surface markers by using flow cytometry. Additionally, mouse survival and cyst numbers in the brain of mice challenged 1 to 2 months postvaccination were determined.
Specific humoral and cellular immune responses were elicited in mice immunized with single-, two-, or three-gene cocktail DNA vaccine, as indicated by significant increases in serum antibody concentrations of total IgG, IgG2a/IgG1 ratio, cytokine levels (IFN-γ, IL-2, IL-12, IL-4, and IL-10), lymphocyte proliferation, lymphocyte populations (CD4+ and CD8+ T lymphocytes), CTL activities, and survival, as well as decreased brain cysts, in comparison with control mice. Moreover, compared with pVAX-GRA35 + pVAX-GRA42, pVAX-GRA42 + pVAX-GRA43, or pVAX-GRA35 + pVAX-GRA43, multicomponent DNA vaccine with three genes (pVAX-GRA35 + pVAX-GRA42 + pVAX-GRA43) induced the higher humoral and cellular immune responses, including serum antibody concentrations, cytokine levels, lymphocyte proliferation, lymphocyte populations, CTL activities and survival, resulting in prolonged survival time and reduced brain cyst loads. Furthermore, mice immunized with pVAX-GRA35 + pVAX-GRA42, pVAX-GRA42 + pVAX-GRA43, or pVAX-GRA35 + pVAX-GRA43 showed greater Th1 immune responses and protective efficacy than the single-gene-vaccinated groups.
These results demonstrate that TgGRA35, TgGRA42, or TgGRA43 are vaccine candidates against T. gondii infection, and the three-gene DNA vaccine cocktail conferred the strongest protection against T. gondii infection.
■本研究旨在测试包含GRA35,GRA42和GRA43的DNA疫苗在诱导针对高毒力弓形虫RH菌株(I型)和脑囊肿形成PRU菌株(II型)的保护性免疫中的功效。
■构建真核质粒pVAX-GRA35、pVAX-GRA42和pVAX-GRA43,并将其配制成两基因或三基因混合物DNA疫苗。采用间接免疫荧光法(IFA)分析其表达和免疫原性。用单基因免疫小鼠,两个基因,或多组分真核质粒,肌内注射.我们评估了抗体水平,细胞毒性T细胞(CTL)反应,细胞因子,用流式细胞仪检测淋巴细胞表面标志物。此外,确定了接种疫苗后1至2个月受到攻击的小鼠的小鼠存活率和大脑中的囊肿数量。
■在单次免疫的小鼠中引起特异性体液和细胞免疫应答,two-,或者三基因鸡尾酒DNA疫苗,如总IgG的血清抗体浓度显着增加所示,IgG2a/IgG1比值,细胞因子水平(IFN-γ,IL-2、IL-12、IL-4和IL-10),淋巴细胞增殖,淋巴细胞群(CD4+和CD8+T淋巴细胞),CTL活动,和生存,以及减少的脑囊肿,与对照小鼠相比。此外,与pVAX-GRA35+pVAX-GRA42,pVAX-GRA42+pVAX-GRA43或pVAX-GRA43相比,具有三个基因(pVAX-GRA35+pVAX-GRA42+pVAX-GRA43)的多组分DNA疫苗诱导更高的体液和细胞免疫应答,包括血清抗体浓度,细胞因子水平,淋巴细胞增殖,淋巴细胞群,CTL活动和生存,导致延长的生存时间和减少的脑囊肿负荷。此外,用pVAX-GRA35+pVAX-GRA42、pVAX-GRA42+pVAX-GRA43或pVAX-GRA35+pVAX-GRA43免疫的小鼠比单基因接种组显示更大的Th1免疫应答和保护功效。
■这些结果表明,TgGRA35、TgGRA42或TgGRA43是针对弓形虫感染的候选疫苗,三基因DNA疫苗混合物赋予了对弓形虫感染的最强保护。