title: Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies.
abstract: Two phage display antibody libraries (Tomlinson I and J) were screened against the whole oocysts of Cryptosporidium parvum to select for scFv (single chain variable fragment) antibodies. Three scFv antibodies were selected that bound to C. parvum oocysts as determined by monoclonal phage ELISA. DNA sequencing revealed that clone A11 lacked the majority of its V (H) chain. Clone B10 had a stop codon in the first framework region of the V (H) chain. We changed this stop codon to Gly by site-directed mutagenesis, and designated the variant mutB10. Clone B9 had a complete scFv gene with no internal stop codons. These antibody genes were individually subcloned into the pET-20b expression vector for soluble scFv antibody production. C. parvum infectivity was determined by infection of HCT-8 tissue culture monolayers and quantified by the foci detection method. By incubating C. parvum oocysts with individual scFv antibodies for 1 h at 37 degrees C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependent manner. At the highest soluble scFv concentration tested (4 nmol), the mean number of infectious foci was reduced by 82%, 73% and 94% for the A11, B9 and mutB10 scFv, respectively. This inhibition of oocyst infectivity was abolished when the scFvs were exposed to boiling water. The results showed that the 3 selected scFvs bound to C. parvum oocysts, and their ability to neutralize infectivity may have potential therapeutic potential against cryptosporidiosis.
authors.affiliation: Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Pokorny
authors.forename: Jeanine I
authors.initials: JI
authors.lastname: Boulter-Bitzer
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Hall
authors.forename: Jack T
authors.initials: JT
authors.lastname: Trevors
authors.forename: Hung
authors.initials: H
authors.lastname: Lee
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D018388:Codon, Terminator; D016785:Cryptosporidium parvum / Q000276:immunology* / Q000472:pathogenicity; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D033661:Oocysts / Q000276:immunology; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000913:Antibodies, Protozoan; D018388:Codon, Terminator; D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins