Cannas Angela, Naguleswaran Arunasalam, Müller Norbert, Gottstein Bruno, Hemphill Andrew
Institute of Parasitology, University of Berne, Laenggass-Strasse 122, 3012 Berne, Switzerland.
J Parasitol. 2003 Feb;89(1):44-50. doi: 10.1645/0022-3395(2003)089[0044:RCIONC]2.0.CO;2.
C57BL/6 mice were vaccinated with a bacterially expressed and purified polyhistidine-tagged full-length version of the microneme protein NcMIC3 (recNcMIC3) emulsified in Ribi Adjuvant System (RAS). Subsequently, they were challenged by intraperitoneal inoculation of 2 x 10(6) live Neospora caninum tachyzoites. As controls, groups of mice received phosphate-buffered saline (PBS)-RAS alone (adjuvant control) or were treated with PBS before infection (infection control). The protective effect of vaccination was assessed by Neospora-specific polymerase chain reaction (PCR), immunohistochemical investigation of brain tissue, and serological means (enzyme-linked immunosorbent assay). Assessment by PCR performed on DNA from different organs revealed that in all treatment groups parasite DNA could only be detected in brain tissue. According to the PCR results. the recNcMIC3 vaccine conferred protection to 75% of mice (n = 16 in 2 independent experiments), whereas application of PBS-RAS and of PBS alone resulted in protection of 12.5% and 0% of mice, respectively (n = 16 as above). Mice in the PBS-treated infection control group were affected by evident clinical signs of neosporosis starting on day 6 postinfection (p.i.). Conversely, none of the animals treated with either PBS-RAS or recNcMIC3 exhibited any symptoms until day 21 p.i. Immunohistochemical staining of paraffin-embedded brain tissue sections confirmed the protective effect of recNcMIC3 vaccination. Quantitative Neospora-specific real-time PCR revealed that infection intensities were lower in the brain tissues of recNcMIC3-vaccinated mice compared with PBS-RAS-treated adjuvant control mice. Serological analysis showed that the protective effect observed in recNcMIC3-vaccinated mice was associated with a Th2-type IgG1 antibody response directed against native NcMIC3 and a mixed IgG1-IgG2a antibody response directed against the recombinant antigen itself. Taken together, these results demonstrated that recombinant NcMIC3 vaccine confers a significant protectivity against experimentally induced cerebral neosporosis in mice.
将细菌表达并纯化的带有多聚组氨酸标签的微小膜体蛋白NcMIC3全长版本（recNcMIC3）乳化于Ribi佐剂系统（RAS）中，对C57BL/6小鼠进行免疫接种。随后，通过腹腔接种2×10⁶个活的犬新孢子虫速殖子对其进行攻毒。作为对照，各组小鼠分别单独接受磷酸盐缓冲盐水（PBS）-RAS（佐剂对照）或在感染前用PBS处理（感染对照）。通过新孢子虫特异性聚合酶链反应（PCR）、脑组织免疫组织化学研究和血清学方法（酶联免疫吸附测定）评估免疫接种的保护效果。对来自不同器官的DNA进行PCR检测发现，在所有治疗组中，仅在脑组织中能检测到寄生虫DNA。根据PCR结果，recNcMIC3疫苗使75%的小鼠获得保护（在2个独立实验中n = 16），而单独应用PBS-RAS和PBS分别使12.5%和0%的小鼠获得保护（n = 16，如上所述）。PBS处理的感染对照组小鼠在感染后第6天（p.i.）开始出现明显的新孢子虫病临床症状。相反，用PBS-RAS或recNcMIC3处理的动物在感染后第21天之前均未表现出任何症状。石蜡包埋的脑组织切片免疫组织化学染色证实了recNcMIC3免疫接种的保护效果。定量新孢子虫特异性实时PCR显示，与PBS-RAS处理的佐剂对照小鼠相比，recNcMIC3免疫接种小鼠脑组织中的感染强度更低。血清学分析表明，在recNcMIC3免疫接种小鼠中观察到的保护作用与针对天然NcMIC3的Th2型IgG1抗体反应以及针对重组抗原本身的混合IgG1-IgG2a抗体反应有关。综上所述，这些结果表明重组NcMIC3疫苗对小鼠实验性诱导的脑新孢子虫病具有显著的保护作用。
