Production of human monoclonal antibodies, which inhibit in vitro growth of Plasmodium falciparum

目前还没有研制出有效的疟疾疫苗。用人源单抗进行被动免疫治疗可能是一种有价值的治疗选择。从感染恶性疟原虫患者的淋巴细胞中构建了一个组合免疫球蛋白基因文库，并用于制备人抗恶性疟原虫裂殖子表面蛋白1(MSP-1)C端19 kDa片段(MSP-1)的单抗。在细菌表达系统中产生MSP-LIS特异性Fab片段。FCR3和3D7代表MSP-1_&lt；19&gt；的二态等位基因变异株，Fab片段对恶性疟原虫裂殖子进行免疫荧光染色，表明Fab片段与保守区域有关。为了检验这些Fabs的表位是否被免疫血清识别，还使用了所罗门群岛10名疟疾免疫者的血清或8名淋巴细胞捐赠者的血浆进行了竞争ELISA。与对照血清相比，3份免疫血清和3份供体血浆显示出明显的抑制作用。在其中一个Fab片段P125中，检测了重链和轻链第三互补决定区的单一氨基酸修饰对亲和力的影响。重组PCR用于修饰轻链中的Tyr^；lt；92；或Ile^；；97；；重链中的Val^；；101；或Trp^；没有发现有效的Tyr^&lt；92&gt；和Val^&lt；101&gt；，但证明了11e^&lt；97&gt；用Gly、Leu、Glu、Ala和Ser和Trp^&lt；107&gt；用Arg和Ser替换的可能性。在这些修饰的Fab片段中，与Ile^&lt；97&gt；Leu和Typ^&lt；107&gt；Ser突变的Fab的亲和力略高于原始Fab。修饰后的抗体可用于分析MSP-1和19&gt；的表位结构。

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>.