| Budget Amount *help |
¥3,840,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥240,000)
Fiscal Year 2007: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2006: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
An effective vaccine for malaria has not yet been developed. Passive immunotherapy with human monoclonal antibodies may provide a valuable therapeutic alternative. A combinatorial immunoglobulin gene library was constructed from lymphocytes of patients infected with Plasmodium falciparum and screened for the production of human monoclonal antibodies to the C terminal 19-kDa fragment of P. falciparum merozoite surface protein 1 (MSP-1_<19>). MSP-lis-specific Fab fragments were produced in bacterial expression system. Immunofluorescence staining of P. falciparum merozoites by the Fab fragments was demonstrated on FCR3 and 3D7 strains, which were representatives of dimorphic allelic variants in MSP-1_<19>, suggesting the Fab's reativity to a conserved region. To examine whether the epitope for these Fabs was recognized by immune sera, competition ELISA was also performed using sera from ten malaria-immune individuals in the Solomon Islands or plasmas from eight donors of lymphocytes. Three of the immune sera and three of donors' plasmas showed significant inhibition compared with control sera. The effect of single amino acid modifications in the third complementarity-determining regions of the heavy and light chains on affinity was examined in one of the Fab fragments, P125. Recombination PCR was used to modify Tyr^<92> or Ile^<97> in the light chain and Val^<101> or Trp^<107> in the heavy chain. No effective replacements for Tyr^<92> and Val^<101> were found, but possible substitutions of 11e^<97> with Gly, Leu, Glu, Ala and Ser, and of Trp^<107>with Arg and Ser were demonstrated. Of these modified Fab fragments, the affinities of Fabs with Ile^<97>Leu and Typ^<107>Ser mutations were slightly higher than that of the original Fab. The modified antibodies may be applicable to analyze epitope structures of MSP-1_<19>.
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