Brown W C, Shkap V, Zhu D, McGuire T C, Tuo W, McElwain T F, Palmer G H
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164, USA.
Infect Immun. 1998 Nov;66(11):5406-13. doi: 10.1128/IAI.66.11.5406-5413.1998.
Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.
针对边缘无形体这种埃立克体病原体的保护性免疫被假定为需要诱导针对外膜蛋白表位的免疫球蛋白G2（IgG2）抗体，并协同激活巨噬细胞以进行吞噬和杀伤。在本研究中，对用边缘无形体佛罗里达菌株的纯化外膜免疫的犊牛的细胞介导免疫反应进行了表征，包括IgG同种型转换的诱导。重要的是，随后显示这些犊牛在用佛罗里达菌株进行实验性攻击时受到保护，并且产生最高IgG2滴度的犊牛完全免受感染。免疫后获得的外周血单核细胞（PBMC）对整个边缘无形体匀浆和纯化外膜均有强烈增殖反应，并且这种反应性一直持续到攻击时。反应细胞显示为CD4(+) T细胞，培养2至4周的CD4(+) T细胞系也对边缘无形体有特异性增殖，并产生高滴度的γ干扰素。辅助性T细胞反应包括对保守表位的识别，因为PBMC增殖受到同源佛罗里达菌株、四种遗传上不同的边缘无形体菌株和绵羊无形体的刺激。在受保护的犊牛中刺激PBMC反应的外膜蛋白包括主要表面蛋白（MSP）MSP-1、MSP-2和MSP-3，先前已证明它们可诱导对感染的部分保护作用。这些研究首次证明，在用外膜进行保护性免疫以抵抗边缘无形体攻击的牛中存在有效的辅助性T细胞反应，并鉴定出三种可被免疫T细胞识别的MSP。这些实验为随后鉴定MSP-1、MSP-2和MSP-3上的辅助性T细胞表位提供了基础，这些表位是引发由蛋白质或核酸免疫引起的回忆性抗体和效应T细胞反应所必需的。
