Almdni, Sabir M.S. (2013) Recombinant antibodies against Clostridium difficile Toxin A. PhD thesis. http://theses.gla.ac.uk/4727/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Recombinant antibodies against Clostridium difficile Toxin A College of Medical Veterinary and Life Sciences School of Life Sciences February 2013 Sabir M.Shakir Almdni Supervisor: Dr R. Aitken A thesis submitted for the degree of Doctor of Philosophy Abstract Clostridium difficile is a major cause of nosocomial intestinal infection. The pathogen possesses two potent toxins, Toxin A and Toxin B, both of which contribute to diarrhoea, intestinal inflammation and tissue damage. Antibiotics are effective against the disease, however around 20 % of patients on treatment relapse after the termination of antibiotic therapy. The binding of Toxin A to a receptor on human intestinal epithelial cells initiates disease: this is considered the starting point from which the toxin elicits its effect. One feature of the carboxy-terminal domain of Toxin A is the presence of repeating units of amino acids that form a series of binding sites able to recognise disaccharides and trisaccharides on glycolipid and glycoprotein receptor molecules. Antibody response against the toxin can protect against C. difficile disease and efforts to generate vaccines have focused upon the carboxy-terminal, receptor binding domain. The aims of this project were to use phage display to isolate recombinant antibodies against those features of the carboxy-terminal domain of Toxin A thought to be responsible for receptor-binding and to assess if the antibodies were capable protecting against the action of Toxin A. Using published crystallographic data that has shown the interaction of Toxin A and trisaccharide, a region of about 113 amino acids from the carboxy- terminal region of Toxin A was expressed as a fusion to maltose-binding protein. The MBP fusion protein was expressed, purified on amylose resin, and characterised. The fusion protein was then used to isolate single chain antibodies from the Tomlinson libraries of scFvs, a synthetically diversified 2 phage display library of single scaffold human antibodies. Conventional bio- panning methods were used in which the MBP fusion protein was bound to a plastic surface and the phage display libraries were pre-mixed with native MBP to inhibit the isolation of anti-MBP antibodies. Progressive enrichment of scFvs through 3 rounds of selection was observed. Those scFvs that showed strongest reaction against the target protein in ELISA but failed to react with native MBP were sequenced, expressed as soluble antibodies and purified on nickel chelating columns. While the resulting panel of scFvs showed similarities of sequence, none were identical. All were reactive with native, full-length Toxin A and appeared to bind to conformational (nonlinear) epitopes. Cross-reaction with Toxin B from C. difficile was also evident. A panel of truncation mutants were generated from the MBP fusion protein and using these in ELISA with the scFvs, reactivity appeared to be directed to features of a long repeat sequence of Toxin A. To assay whether the isolated scFvs possessed biological activity of significance, in vitro and in vivo protection assays were established. For experiments in vitro, the action of Toxin A upon cultured Vero cells was studied. Native Toxin A triggered a conversion of the cells from stellate to rounded morphology. When cells were exposed to 100 ng of toxin, this effect was evident within 60 minutes; at a 10-fold lower dose, the minimum quantity to which a response was detectable, virtually all cells had undergone rounding within 2 h. When individual scFvs were mixed with 10 ng of Toxin A prior to addition to Vero cells, there was a consistent delay in cytopathic activity that extended to 5 h. In this assay, the percentage of cells that had retained their stellate morphology 5 h post-challenge was dependent on the scFv used. To quantify the potency of this neutralising activity, the amount of each scFv required to achieve 50% 3 protection during a 2 h challenge period was established. This revealed 3.5-fold difference between the most and the least effefctive scFv. The most potent scFv was used in an in vivo assay in which Toxin A was administered to the ligated intestinal loops of rats. Again, protective activity was evident. Overall, phage display technology enabled the assembly of a panel of scFv antibodies against the putative receptor binding site in the carboxy-terminal domain of Toxin A from C. difficile. The scFvs were able to protect against the cytopathic activity of Toxin A in vitro and in vivo and proposals are made about how these observations could be taken forward in a model of C. difficile infection that best mimics the human disease. 4 Table of Contents Chapter One Introduction ................................................................ 17 1.1 Overview ......................................................................... 17 1.2 Pathogenesis .................................................................... 19 1.3 Epidemiology .................................................................... 28 1.4 Risk factor ....................................................................... 29 1.4.1 Toxin function ............................................................. 30 1.4.2 Toxin expression regulation ............................................. 32 1.5 Strains of importance .......................................................... 34 1.6 Diagnosis ......................................................................... 39 1.7 Treatment and therapeutics .................................................. 41 1.7.1 Antibiotic therapy ......................................................... 42 1.7.2 Non-antibiotic therapy ................................................... 45 1.8 Antibodies ....................................................................... 51 1.9 Phage display .................................................................... 55 1.9.1 Antibody formats .......................................................... 57 1.9.2 Affinity maturation ....................................................... 58 1.9.3 Antibody libraries ......................................................... 59 1.9.4 Naïve libraries ............................................................. 61 1.9.5 Immune libraries .......................................................... 62 1.9.6 Synthetic libraries ......................................................... 62 1.9.7 The choice of phage coat protein ....................................... 63 1.9.8 Some limitations of phage display ...................................... 64 1.9.9 Selection in antibody phage display .................................... 65 1.9.10 Screening of phage displayed antibodies and antibody expression 67 1.10 Overall aims of the project ................................................... 68 Chapter Two Protein expression and purification .................................... 70 2.1 Introduction ..................................................................... 70 2.1.1 Toxin A binding to its receptor .......................................... 70 2.1.2 General strategy ........................................................... 71 2.1.3 Maltose-binding protein .................................................. 71 2.1.4 Aims for the experiments ................................................ 72 2.2 Materials and methods ......................................................... 73 2.2.1 Construction, expression and purification of maltose-binding protein (MBP) fusions ................................................................ 73 2.2.1.1 Confirmation of sequence .......................................... 73 2.2.1.2 Primer design for putative binding site of TcdA-MBP fusion .. 74 2.2.1.3 Polymerase chain reaction (PCR) .................................. 75 2.2.1.4 Preparation of pCG806 and target insert ......................... 77 2.2.1.5 Ligation and transformation ........................................ 79 2.2.1.6 Analysis of transformants ........................................... 80 2.2.1.7 Sequencing ............................................................ 81 2.2.1.8 Bacterial growth, induction and expression of the native MBP and fusions proteins ............................................................... 81 2.2.1.9 Purification of the proteins using affinity chromatography ... 81 2.2.1.10 SDS-PAGE analysis of purified proteins ........................... 82 2.2.1.11 Electroblotting of the purified proteins .......................... 83 2.2.1.12 Protein assay ......................................................... 84 2.2.2 Construction of truncation mutants from the putative binding site of TcdA-MBP fusion .................................................................. 84 2.2.2.1 Construction of MBP-SR1, MBP-LR, MBP-SR2 and MBP-SR3