Preparations And Characterization Of Recombinant Human Monoclonal Antibody Fab Fragment Specific For Cryptosporidium

Cryptosporidium, which belongs to enteric protozoan parasites of the phylum Apicomplexa, causes cryptosporidiosis in animals and humans. The disease was characterized by diarrhea. Unfortunately, effective therapies to this important opportunistic pathogen have not been developed. Previous researches indicated that Hyperimmune bovine colostrum (HBC), immune serum and monoclonal antibodies specific for surface antigens of sporozoite or merozoite could reduce oocyst shedding and alleviate clinical signs of disease in both animals and humans. Those data proved the efficacy of passive antibody immunotherapy for Cryptosporidium infection. Therefore human antibodies specific for Cryptosporidium should be prepared for passive immunotherapy.A combinatorial human immunoglobulin gene library was constructed from theperipheral lymphocytes of a donor who was orally immunized with oocyst of C.parvum. The library was screened for the production of Fab antibody fragments by using recombinant 40/15 glycoprotein of C.parvum, which located on the surface of sporozoite and was related to pathopoiesis of C.parvum. When 1.01 × 10⁶ clones were screened by colony blotting and enzyme-linked immunsorbent assay, 23 clones showed positive signal. In the second screening by IFA, only two clones, designated Agp40-l and Agp40-2, reacted specifically with the recombinant and native protein of C.parvum oocyst. The heavy and light chain genes of two clones were sequenced and analyzed with the IgBlast program. For the heavy chain genes of Agp40-l, the nearest germline of the V-segment was VH3-30 (92% homology). The closest V-segment germline of the light chain gene was A20 (95% homology). For the heavy chain genes of Agp40-2, the nearest germline of the V-segment was VH3-30 (92% homology). The closest V-segment germline of the light chain gene was 012 (97% homology). The affinity of Agp40-1/Agp40-2 to the recombinant protein was analyzed by surface plasmon resonance assay; the dissociation constants were 5.25 × 10^-9 (M) and 1.47 × 10^-9(M), respectively. Preincubation of oocyst or sporozoites with these Fabs significantly inhibited oocysts from infecting Caco-2 cells and blocked sporozoites'invasion to Caco-2 cells in vitro.These results demonstrated the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibody from human immunized with C.parvum oocysts. It indicated that these human monoclonal antibody fragments should be helpful not only for blocking Cryptosporidium to invade host cells, but also for the development of immunoprophylaxis against cryptosporidiosis.