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

柏氏疟原虫XAT是辐照诱导致死菌株柏氏疟原虫NK65的减毒变种。提示高免疫血清中Th1型抗体IgG2a抑制寄生虫血症，提示抗体介导的免疫在伯氏假体XAT感染中起重要作用。然而，参与保护性免疫的抗原尚未被确定。在本研究中，我们建立了产生抗伯氏假体XAT感染的保护性单克隆抗体的杂交瘤。检测了保护性抗原的特征、阶段特异性和定位。用贝格氏疟原虫XAT免疫小鼠脾细胞与P3U1骨髓瘤细胞融合。我们克隆了7株能与柏氏假单抗发生强烈反应的单克隆抗体杂交瘤。克隆体以腹水的形式膨胀。7个单抗的同型分别为IgMs（5个克隆）和IgG1s（2个克隆），只有IgG1s的单抗在被动转移实验中具有保护作用。另一个反p。berghei XAT单抗对被动转移无保护作用。ELISA结果显示，保护性单抗对柏氏假体XAT抗体效价高，对柏氏假体NK65抗体效价低。在还原和非还原条件下，经Western blotting测定所识别抗原的分子量分别为150、160 kDa。间接免疫荧光抗体试验表明，目标抗原仅在分裂体阶段合成。保护性抗原的详细定位是通过免疫电子显微镜确定的。抗原定位于寄生物液泡间隙和感染红细胞胞质间隙。了解这些单克隆抗体识别的抗原参与保护的机制将为开发血期疟疾疫苗提供重要信息。

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.