Polyclonal Fab phage display libraries with a high percentage of diverse clones to Cryptosporidiumparvum glycoproteins

Abstract

The protozoan parasite Cryptosporidium parvum is regarded as a major public health problem world-wide, especially for immunocompromised individuals. Although no effective therapy is presently available, specific immune responses prevent or terminate cryptosporidiosis and passively administered antibodies have been found to reduce the severity of infection. Therefore, as an immunotherapeutic approach against cryptosporidiosis, we set out to develop C. parvum-specific polyclonal antibody libraries, standardised, perpetual mixtures of polyclonal antibodies, for which the genes are available. A combinatorial Fab phage display library was generated from the antibody variable region gene repertoire of mice immunised with C. parvum surface and apical complex glycoproteins which are believed to be involved in mediating C. parvum attachment and invasion. The variable region genes used to construct this starting library were shown to be diverse by nucleotide sequencing. The library was subjected to one round of antigen selection on C. parvum glycoproteins or a C. parvum oocyst/sporozoite preparation. The two selected libraries showed specific reactivity to the glycoproteins as well as to the oocyst/sporozoite preparation, with 50–73% antigen-reactive members. Fingerprint analysis of individual clones from the two antigen-selected libraries showed high diversity, confirming the polyclonality of the selected libraries. Furthermore, immunoblot analysis on the oocyst/sporozoite and glycoprotein preparations with selected library phage showed reactivity to multiple bands, indicating diversity at the antigen level. These C. parvum-specific polyclonal Fab phage display libraries will be converted to libraries of polyclonal full-length antibodies by mass transfer of the selected heavy and light chain variable region gene pairs to a mammalian expression vector. Such polyclonal antibody libraries would be expected to mediate effector functions and provide optimal passive immunity against cryptosporidiosis.