| Bordetella pertussis is the etiological agent of 'whooping cough', an acute respiratory disease responsible for 400,000 deaths annually. Passive immunity is an ideal approach to protect infants (under 4 months) and the immune-compromised, who cannot generate an immune response, against pertussis. In this study, 288 individual clones were grown from a previously constructed single chain variable fragment (scFv) antibody library against B. pertussis using M13 phage display. The clones were screened for the ability to bind to B. pertussis cells, filamentous haemagglutinin (FHA), and pertactin (PRN) in enzyme-linked immunosorbent assays (ELISA). Based on the binding affinities, the clones were put into 8 groups. The scFv DNA inserts from the 288 clones were digested with BstOI and 18 unique restriction patterns, named Types 1 to 18, were found. Four of the scFv (T1, 5, 7, and 18) displayed the ability to bind to FHA and PRN. Also, Types 1, 5, and 7 inhibited the binding of B. pertussis to HEp-2 cells. Soluble scFv proteins were isolated from Types 1, 5, and 7 clones and shown to be functional in ELISA. Maltose-binding protein (MBP) fusions composed of three different regions of FHA (heparin binding domain, carbohydrate recognition domain, and the RGD triplet motif) and a region of PRN (the RGD triplet motif) were constructed to map the binding epitopes of those scFv. The binding epitope could not be resolved since the FHA-RGD fusion displayed an affinity to IgG molecules. A murine passive protection model was developed in which T1, 5, and 7 were found to protect mice against infection. A Streptococcus gordonii clone secreting T5 scFv was constructed. This clone was able to produce functional scFv that recognized B. pertussis cells in an ELISA. The three scFv antibodies and the recombinant S. gordonii clone produced in this thesis are good candidates for future studies in the development of a live mucosal vaccine that can be used in passive immunity against pertussis. |
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