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Characterization and specificity of newly defined protective antigens of Plasmodium berghei XAT

新定义的伯氏疟原虫 XAT 保护性抗原的表征和特异性

基本信息

批准号：
12670240
负责人：
KOBAYASHI Fumie
金额：
$ 2.18万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670240/
关键词：
malaria protective antigen vaccine Plasmodium berghei

项目摘要

Plasmodium berghei XAT is an irradiation induced attenuated variant of the lethal strain, P. berghei NK65. It has been suggested that Th1 type antibody, IgG2a, in hyperimmune sera suppressed the parasitemia, indicating that antibody-mediated immunity is important in P. berghei XAT infection. However, antigens which are involved in the protective immunity have not yet been identified. In the present study, we established hybridomas producing protective monoclonal antibodies against P. berghei XAT infection. The characterization, stage specificity and localization of the protective antigens were examined. Spleen cells from mice immunized with Plasmodium berghei XAT were fused with P3U1 myeloma cells. Seven hybridomas secreting monoclonal antibodies (mAb) which reacted strongly with P. berghei XAT were recloned. The clones were expanded in the form of ascites fluids. The isotypes of 7 mAbs were found to be IgMs (5 clones) and IgG1s (2 clones) and only mAbs identified as IgG1s were protective in passive transfer experiments. The other anti-P. berghei XAT mAbs were not protective on passive transfer. Protective mAbs showed high levels of antibody titer to P. berghei XAT and low levels to P. berghei NK65 in ELISA. The molecular weights of the recognized antigens are 150, 160 kDa as determined by Western blotting under reducing and nonreducing conditions. Indirect immunofluorescent antibody tests showed that the target antigens were synthesized only in the schizont stage. The detailed localization of the protective antigens was conferred by the immunoelectron microscopy. The antigens were localized in the parasitophorous vacuole space and in the clefts in the infected erythrocyte cytoplasm. To know the mechanisms by which the antigens recognized by these mAbs are involved in the protection would provide an important information for the development of a blood-stage malarial vaccine.

伯氏疟原虫 XAT 是致命菌株伯氏疟原虫 NK65 的辐射诱导减毒变种。有人提出，超免疫血清中的 Th1 型抗体 IgG2a 抑制了寄生虫血症，表明抗体介导的免疫在伯氏疟原虫 XAT 感染中很重要。然而，尚未鉴定出参与保护性免疫的抗原。在本研究中，我们建立了产生针对伯氏疟原虫 XAT 感染的保护性单克隆抗体的杂交瘤。检查了保护性抗原的特征、阶段特异性和定位。用伯氏疟原虫 XAT 免疫小鼠的脾细胞与 P3U1 骨髓瘤细胞融合。重新克隆了7个分泌与伯氏疟原虫XAT强烈反应的单克隆抗体(mAb)的杂交瘤。克隆以腹水的形式扩增。发现 7 种 mAb 的同种型为 IgM（5 个克隆）和 IgG1（2 个克隆），并且只有被鉴定为 IgG1 的 mAb 在被动转移实验中具有保护性。另一个反P。 berghei XAT mAb 对被动转移没有保护作用。在 ELISA 中，保护性单克隆抗体显示出针对伯氏疟原虫 XAT 的高水平抗体滴度，以及针对伯氏疟原虫 NK65 的低水平抗体滴度。在还原和非还原条件下通过蛋白质印迹测定，所识别的抗原的分子量为150、160 kDa。间接免疫荧光抗体测试表明，目标抗原仅在裂殖体阶段合成。保护性抗原的详细定位通过免疫电子显微镜获得。抗原定位于寄生液泡空间和受感染红细胞细胞质的裂隙中。了解这些单克隆抗体识别的抗原参与保护的机制将为血期疟疾疫苗的开发提供重要信息。