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Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies

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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°C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependant 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.

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Abbreviations

rAb:

Recombinant antibody

scFv:

Single chain variable fragment antibody

VH :

Variable region of the heavy chain

VL :

Variable region of the light chain

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Acknowledgements

Our research on Cryptosporidium was supported by the Canadian Water Network (CWN) Center of Excellence, Natural Sciences and Engineering Research Council (NSERC) of Canada, and Ontario Ministry of Agriculture and Food. We thank the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NSAID, NIH for oocysts of C. parvum MD strain, provided by Dr. Saul Tzipori. We would also like to thank Sara Mohr of the University of Guelph for supplying the anti-cholera toxin subunit B scFv. The Tomlinson libraries were provided by the Medical Research Council Laboratory of Molecular Biology (now Geneservive Ltd.) (Cambridge, UK).

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Authors and Affiliations

  1. Department of Environmental Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1

    Nicholas J. Pokorny, Jeanine I. Boulter-Bitzer, J. Christopher Hall, Jack T. Trevors & Hung Lee

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  1. Nicholas J. Pokorny
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  2. Jeanine I. Boulter-Bitzer
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  3. J. Christopher Hall
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  4. Jack T. Trevors
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  5. Hung Lee
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Correspondence to Jack T. Trevors or Hung Lee.

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Pokorny, N.J., Boulter-Bitzer, J.I., Hall, J.C. et al. Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies. Antonie van Leeuwenhoek 94, 353–364 (2008). https://doi.org/10.1007/s10482-008-9252-0

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