Brown W C, Zhao S, Logan K S, Grab D J, Rice-Ficht A C
International Laboratory for Research on Animal Diseases, Nairobi, Kenya.
Vet Parasitol. 1995 Mar;57(1-3):189-203. doi: 10.1016/0304-4017(94)03120-l.
Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of reactivity, and proteins separated by anion exchange revealed two patterns of reactivity when selected T cell clones were assayed for stimulation by antigenic fractions. Studies using a continuous-flow electrophoresis apparatus have indicated the feasibility of identifying T cell-stimulatory proteins from parasite membranes as well as from the cytosolic fraction of B. bovis merozoites. The Th cell clones reactive with these different hemoparasites expressed either unrestricted or Th1 cytokine profiles, and were generally characterized by the production of high levels of IFN-gamma. A comprehensive study of T cell and macrophage responses to defined parasite antigens will help elucidate the reasons for vaccine failure or success, and provide clues to the mechanisms of acquired immunity that are needed for vaccine development.
目前用于牛血液寄生虫的疫苗采用减毒活生物体或与抗寄生虫药物同时使用的强毒生物体。尽管这类疫苗可能有效，但对于大多数血液寄生虫而言，针对异源寄生虫分离株攻击感染所产生的获得性抗性机制尚未明确界定。传统上，潜在保护性抗原的选择利用抗体来识别免疫显性蛋白。然而，大量研究表明，高抗体滴度的诱导既不能预测抗原赋予保护性免疫的能力，也与保护作用无关。由于成功的寄生虫已经进化出逃避抗体的策略，因此应采用其他策略来识别保护性免疫原。通过细胞因子的释放，辅助性T细胞1型（Th1）样T细胞和巨噬细胞介导针对许多细胞内寄生虫的保护性免疫，因此很可能在针对牛血液寄生虫的保护性免疫中发挥重要作用。因此，使用针对微小泰勒虫裂殖体或牛巴贝斯虫裂殖子的可溶性或膜抗原的CD4 + T细胞克隆来鉴定在体外引发强烈Th细胞反应的寄生虫抗原。可溶性胞质寄生虫抗原通过凝胶过滤、阴离子交换色谱或羟基磷灰石色谱或它们的组合进行分级分离，然后测试各组分诱导Th细胞克隆增殖的能力。该程序能够鉴定出含有约10 kDa和24 kDa微小泰勒虫蛋白的刺激组分。针对纯化的24 kDa条带产生的抗血清与一种约30 kDa的天然裂殖体蛋白发生反应。用分级分离的牛巴贝斯虫裂殖子可溶性抗原检测牛巴贝斯虫特异性Th细胞克隆，发现至少存在五个不同的抗原表位。当检测选定的T细胞克隆对抗原组分的刺激时，通过凝胶过滤分离的蛋白显示出四种反应模式，通过阴离子交换分离的蛋白显示出两种反应模式。使用连续流动电泳装置的研究表明，从寄生虫膜以及牛巴贝斯虫裂殖子的胞质组分中鉴定T细胞刺激蛋白是可行的。与这些不同血液寄生虫发生反应的Th细胞克隆表达不受限制或Th1细胞因子谱，并且通常以高水平干扰素-γ的产生为特征。对T细胞和巨噬细胞对特定寄生虫抗原的反应进行全面研究将有助于阐明疫苗失败或成功的原因，并为疫苗开发所需的获得性免疫机制提供线索。
