Polat Ceylan, Gülçe İz Sultan, Döşkaya Mert, Can Hüseyin, Caner Ayşe, Değirmenci Aysu, Balcan Erdal, Gürüz Yüksel
Ege University Faculty of Engineering, Department of Bioengineering, Izmir, Turkey.
Mikrobiyol Bul. 2013 Jan;47(1):122-34. doi: 10.5578/mb.4348.
Toxoplasma gondii the causative agent of toxoplasmosis is an obligate intracellular parasite with a wide host range including all warm-blooded animals and birds. T.gondii infection causes congenital toxoplasmosis in newborns and this may lead to fetal anomalies, retinochoroiditis leading to blindness, lethal toxoplasmic encephalitis in immune compromised patients, and organ failure in transplantation patients. The pathogenesis of toxoplasmosis change due to differences in the specific immune response elicited by diverse T.gondii strains. The protective immunity against toxoplasmosis is conferred by cellular immune responses. In the present study, two different strains isolated from Turkey named T.gondii Ankara and Ege were used to evaluate the types of humoral and cellular immune responses elicited by adjuvanted tachyzoite protein vaccines in an animal model. In the study, 6-8 weeks old female BALB/c mice were used and six study groups (each contains three mice) were composed for vaccination. The first and second groups were vaccinated with T.gondii Ankara and Ege (TAnkPE and TEgePE, respectively) tacyhzoite lysates, the third and fourth groups were vaccinated by tacyhzoite lysates adjuvanted with Freund's adjuvant (TAnkPE-Freund; TEgePE-Freund, respectively). The fifth and sixth groups were vaccinated with PBS and Freund's adjuvant as controls. Immunization of the animals was performed two times at three weeks intervals. The serum samples were collected before vaccination and after each vaccination to determine the IgG response by Western blotting, and IgG1 and IgG2a responses by ELISA. To determine the cellular immune response, CD8/CD4 cell ratio, intracellular IFN-g and IL-4 levels were determined in stimulated spleen cells grown in cell culture systems by flow cytometry. Toxoplasma IgG antibodies were only detected in TAnkPE-Freund group. IgG1 and IgG2a responses did not increase in any vaccination groups and there was not any polarization towards IgG1 or IgG2a. There was no significant increase in CD8/CD4 ratio of stimulated spleen cells. IFN-g level was increased in only TAnkPE-Freund vaccination group, however IL-4 levels were increased in TAnkPE-Freund, TEgePE-Freund and TEgePE groups. Our data showed that TAnkPE-Freund vaccine led to increase in IgG and IFN-g responses in BALB/c mice, however, tachyzoite lysate vaccines developed in this study did not induce sufficient protective immune response against toxoplasmosis. Thus, use of specific immunogenic proteins must be taken into consideration in the future vaccine development studies against toxoplasmosis.
弓形虫是弓形虫病的病原体，是一种专性细胞内寄生虫，宿主范围广泛，包括所有温血动物和鸟类。弓形虫感染可导致新生儿先天性弓形虫病，这可能会导致胎儿畸形、导致失明的视网膜脉络膜炎、免疫功能低下患者的致命性弓形虫脑炎以及移植患者的器官衰竭。由于不同弓形虫菌株引发的特异性免疫反应存在差异，弓形虫病的发病机制也会发生变化。针对弓形虫病的保护性免疫是由细胞免疫反应赋予的。在本研究中，使用从土耳其分离出的两种不同菌株，即弓形虫安卡拉株和埃格株，来评估佐剂化速殖子蛋白疫苗在动物模型中引发的体液免疫和细胞免疫反应类型。在该研究中，使用6 - 至8周龄的雌性BALB/c小鼠，并组成六个研究组（每组包含三只小鼠）进行疫苗接种。第一组和第二组分别用弓形虫安卡拉株和埃格株（分别为TAnkPE和TEgePE）的速殖子裂解物进行接种，第三组和第四组分别用佐剂弗氏佐剂的速殖子裂解物进行接种（分别为TAnkPE - Freund；TEgePE - Freund）。第五组和第六组分别接种PBS和弗氏佐剂作为对照。动物免疫分两次进行，间隔三周。在接种前以及每次接种后采集血清样本，通过蛋白质印迹法测定IgG反应，通过酶联免疫吸附测定法测定IgG1和IgG2a反应。为了确定细胞免疫反应，通过流式细胞术在细胞培养系统中培养的刺激脾细胞中测定CD8/CD4细胞比率、细胞内干扰素 - γ和白细胞介素 - 4水平。仅在TAnkPE - Freund组中检测到弓形虫IgG抗体。在任何接种组中，IgG1和IgG2a反应均未增加，并且没有向IgG1或IgG2a的任何极化现象。刺激脾细胞的CD8/CD4比率没有显著增加。仅在TAnkPE - Freund接种组中干扰素 - γ水平升高，然而在TAnkPE - Freund、TEgePE - Freund和TEgePE组中白细胞介素 - 4水平升高。我们的数据表明，TAnkPE - Freund疫苗导致BALB/c小鼠中IgG和干扰素 - γ反应增加，然而，本研究中开发的速殖子裂解物疫苗并未诱导出针对弓形虫病的足够保护性免疫反应。因此，在未来针对弓形虫病的疫苗开发研究中，必须考虑使用特异性免疫原性蛋白。
