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

伯氏疟原虫XAT是致死株伯氏疟原虫NK 65的辐射诱导减毒变体。结果表明，Th1型抗体IgG2a可抑制伯氏疟原虫XAT感染，提示抗体介导的免疫在伯氏疟原虫XAT感染中起重要作用。然而，涉及保护性免疫的抗原尚未被鉴定。在本研究中，我们建立了杂交瘤生产保护性单克隆抗体抗伯氏疟原虫XAT感染。检测了保护性抗原的特性、阶段特异性和定位。将伯氏疟原虫XAT免疫小鼠的脾细胞与P3U1骨髓瘤细胞融合。获得7株分泌抗伯氏疟原虫XAT单克隆抗体的杂交瘤细胞。以腹水的形式扩增克隆。7株单抗的同种型为IgM（5个克隆）和IgG1（2个克隆），只有IgG1单抗在被动转移实验中具有保护性。其他抗伯氏疟原虫XAT mAb对被动转移无保护作用。在ELISA中，保护性mAb显示对伯氏疟原虫XAT的高水平抗体滴度和对伯氏疟原虫NK65的低水平抗体滴度。通过还原和非还原条件下的Western印迹法测定，识别的抗原的分子量为150、160 kDa。间接免疫荧光抗体试验表明，靶抗原仅在单核期合成。免疫电镜观察保护性抗原的详细定位。抗原定位于寄生虫空泡空间和感染红细胞胞质的裂隙中。了解这些单克隆抗体识别的抗原参与保护作用的机制，将为疟疾血液期疫苗的研制提供重要信息。

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.