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The Development of a Process and Quality Control Methods for a Conjugate Vaccine Against Streptococcis Pnuemoniae Serotype1

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The Development of a Process and Quality Control Methods for a Conjugate Vaccine Against Streptococcis Pnuemoniae Serotype1 THE DEVELOPMENT OF A PROCESS AND QUALITY CONTROL METHODS FOR A CONJUGATE VACCINE AGAINST STREPTOCOCCUS PNEUMONIAE SEROTYPE 1 By Cesarina Edmonds – Smith Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY In the Department of Chemistry UNIVERSITY OF CAPE TOWN June 2013 University of Cape Town Supervisors: Associate Professor Neil Ravenscroft Doctor Seanette Wilson The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Declaration I declare that “The development of a process and quality control methods for a conjugate vaccine against Streptococcus pneumoniae serotype 1” is my own work and that all sources that I have used or quoted have been indicated and acknowledged by means of complete references. ________________________________ Cesarina Edmonds-Smith University of Cape Town i Abstract Pneumonia is the leading cause of death in children worldwide and is estimated to kill 1.6 million children every year. Pneumonia affects children and families everywhere, but is most prevalent in sub–Saharan Africa and South–east Asia. Serotype 1 is responsible for up to 20 % of invasive pneumococcal diseases (IPD) in developing countries and has been the cause of several outbreaks in the African meningitis belt. Conjugate vaccines are effective in young children, induce immunological memory and reduce carriage. A conjugate vaccine against 7 serotypes (PCV7) was licensed in 2000 which resulted in a dramatic reduction of IPD. An increase in the number of cases due to non–vaccine serotypes (serotype replacement) led to the recent development and licensure of 10– and 13– valent conjugate vaccines that provide broader coverage. This thesis describes the development of purification and conjugation processes and associated analytical methods for the preparation of a Streptococcus pneumoniae serotype 1 polysaccharide (Pn1) conjugate vaccine. The Pn1 polysaccharide was purified following a two–step process utilising a differential filtration with ethanol. Analytical tests including size analysis, uronic acid composition, O– acetylation and purity (nucleic acids and protein) were optimized and performed on Pn 1 lots. The purified polysaccharide was found to meet World Health Organisation (WHO) specifications. The purified polysaccharide is viscous with a rigid structure that hampers full conjugation reactions and detailed characterisation. Size–reduction was performed and shown to have no impact on the structural integrity of the generated saccharide. The O–acetylated size– reduced polysaccharide was amenable to full nuclear magnetic resonance (NMR) characterisation to confirmUniversity the structural identity of ofCape Pn1 and determine Town the percentage of cell wall polysaccharide (CWPS) and the degree and position of O–acetylation present. Composition analysis was performed using published hydrolysis methods, however, they resulted in low recoveries and therefore alternative microwave assisted conditions were investigated followed by chromatographic separation and analysis. The size–reduced polysaccharide was conjugated to hydrazide–derivatized protein carriers via the polysaccharide carboxyl groups. The conjugates prepared using different activators were evaluated in mice and the immunogenicity data showed that they were non–inferior to two commercially available conjugate vaccines. ii Acknowledgments All the work behind this thesis could not have been possible without the help and support of many people. I would like to thank the following people for their contribution to the preparation of this thesis: Foremost, my principal supervisor Associate Professor Neil Ravenscroft for his patience, guidance, motivation, enthusiasm and expertise in all things vaccine related and for his many hours in front of the NMR machine. To my second supervisor Doctor Seanette Wilson whose insights, valued comment and suggestions have been of great benefit for the improvement of my work and in writing my thesis. Doctor Ebrahim Mohammed for all your help and guidance in and out of the laboratory and everyone else involved in the pneumococcal project at The Biovac Institute especially Dr. Nelius Swart, Dr. Ike James, Shantal Dorasamy, Francisca Theunissen, Margaret Lennon, Rochelle Hendricks, Charlie Nemugumoni, Lizelle Gordon, Daria Kow, Dr. Melinda Scanlen, Charmaine Steggink and Patrick Tippoo. Past and present members of the Bioanalytical and Vaccine Research group including Tanith Curtin, Dr. Meredith Hearshaw and Taigh Anderson. A special thank you to Ms. Astrid Trimmel for her great assistance and friendship and wonderful talks and encouragement. The Biovac Institute and Program for Appropriate Technology for Health (PATH) for the opportunity to work on such an interesting and engaging project and for their financial assistance. The National ResearchUniversity Foundation (NRF) for of their Cape financial assistance. Town And finally, to my family and friends, for their love, support and constant patience during the course of this PhD. iii Contents Declaration ............................................................................................................ i Abstract ........................................................................................................... ii Acknowledgments................................................................................................... iii Contents .......................................................................................................... iv Abbreviations .......................................................................................................... ix Chapter 1. Introduction ............................................................................................ 1 1.1 Early history of Streptococcus pneumoniae .............................................................. 2 1.2 Surface components of Streptococcus pneumoniae ................................................. 3 1.3 Pneumococcal disease ............................................................................................ 4 1.3.1 Non-invasive pneumococcal disease ................................................................. 5 1.3.2 Invasive pneumococcal disease ........................................................................ 5 1.4 Serogroup distribution of Streptococcus pneumoniae in Africa ................................. 6 1.5 Immune response to polysaccharide and conjugate vaccines .................................. 9 1.6 Pneumococcal vaccines ......................................................................................... 13 1.6.1 Whole cell vaccines ......................................................................................... 13 1.6.2 Pneumococcal polysaccharide vaccines .......................................................... 14 1.6.3 Conjugate vaccines ......................................................................................... 16 1.6.3.1 Prevnar7 ....................................................................................................... 17 1.6.3.2 Vaccine trials incorporating more serotypes ................................................. 20 1.6.3.3 Synflorix ....................................................................................................... 22 1.6.3.4 Prevnar13 ..................................................................................................... 25 1.6.4 PneumococcalUniversity vaccines in development of Cape ......................................................... Town 28 1.6.5 Pneumococcal serotype 1 ................................................................................ 29 1.7 Aim and objectives ................................................................................................. 30 Chapter 2. General Methods .................................................................................. 31 2.1 Fermentation of Pn1 ............................................................................................... 31 2.2 Purification of Pn1 .................................................................................................. 32 2.3 Colorimetric assays ................................................................................................ 33 2.3.1 Uronic acid assay ............................................................................................ 34 2.3.2 O-Acetyl Assay ................................................................................................ 35 2.3.3 UV protein assay ............................................................................................. 36 iv 2.3.4 Bradford (Coomassie) protein assay ................................................................ 37 2.3.5 Trinitrobenzenesulfonic acid assay .................................................................. 37 2.4 Immunological Assays ............................................................................................ 39 2.4.1 ELISA .............................................................................................................. 39 2.4.2 Nephelometry .................................................................................................. 40 2.5 Electrophoresis .....................................................................................................
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