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

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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.