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dc.contributor.advisorMiddaugh, Charles Russell
dc.contributor.authorIyer Gowrishankara, Vidyashankara
dc.date.accessioned2014-07-28T02:10:18Z
dc.date.available2014-07-28T02:10:18Z
dc.date.issued2012-12-31
dc.date.submitted2012
dc.identifier.otherhttp://dissertations.umi.com/ku:12464
dc.identifier.urihttp://hdl.handle.net/1808/14832
dc.description.abstractThe objective of this project is to explore of the use of the empirical phase diagram approach in the formulation development of several forms of vaccines that exist today such as recombinant based subunit vaccines and the traditional live attenuated viral vaccines. The chapters in this dissertation describe the formulation development of three vaccines. The first is a monovalent recombinant-based subunit vaccine targeted against that 2009 pandemic "swine" influenza (Chapter 1). The second is a trivalent vaccine consisting of three recombinantly produced proteins from S. pneumonia (Chapter 2). The third is a trivalent live viral canine vaccine targeted against canine distemper, parainfluenza and kennel cough caused by canine adenovirus type 2 (Chapter 3). The first aim in each chapter is the biophysical characterization of the individual biological entities. This was accomplished by initially by fully characterizing each of the antigens using multiple analytical techniques such as intrinsic tryptophan fluorescence and circular dichroism spectroscopy and static light scattering experiments among others. The ideal preformulation conditions were identified using the empirical phase diagram technique. Influenza is a prevalent, highly contagious and sometimes fatal respiratory disease. Vaccination provides an effective approach to control the disease, but because of frequent changes in the structure of the major surface proteins, there is great need for a technology that permits rapid preparation of new forms of the vaccine each year in sufficient quantities. Recently, using a safe, simple, time- and cost-effective plant viral vector-based transient expression system, the hemagglutinin antigen of H1N1 influenza A strain (HAC1), an H1N1 influenza vaccine candidate, has been produced in Nicotiana benthamiana plants. As a step towards the generation of a commercially viable subunit influenza vaccine, we developed HAC1 formulations in the presence and absence of an aluminum salt adjuvant (Alhydrogel®), analyzed their properties, and assessed immunogenicity in an animal model. Biophysical properties of HAC1 were evaluated using several spectroscopic and light scattering techniques as a function of pH and temperature combined with data analysis using an empirical phase diagram approach. Excipients that were potent stabilizers of the recombinant protein were identified using intrinsic fluorescence spectroscopy. The adsorptive capacity and thermal stability of the protein on the surface of Alhydrogel® were then examined in the presence and absence of selected stabilizers using UV absorbance after centrifugation and intrinsic fluorescence spectroscopy, respectively. Immunogenicity studies conducted in mice demonstrated that the highest level of serum immune responses (hemagglutination-inhibiting antibody titers), with a 100% seropositive rates, were induced by HAC1 in the presence of Alhydrogel®, and this response was elicited regardless of the solution conditions of the formulation. The preformulation of a trivalent recombinant protein based vaccine candidate for protection against Streptococcus pneumoniae is described both in the presence and absence of aluminum salt adjuvants. The biophysical properties of the three protein-based antigens, fragments of PsaA, StkP and PcsB, were studied using several spectroscopic and light scattering techniques. An empirical phase diagram was constructed to assess the overall conformational stability of the three antigens as a function of pH and temperatures. A variety of excipients were screened based on their ability to stabilize each antigen using intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. Sorbitol, sucrose, and trehalose stabilized three proteins in solution. The addition of manganese also showed a drastic increase in the thermal stability of PsaA in solution. The adsorption and desorption processes of each of the antigens to aluminum salt adjuvants were evaluated and the stability of the adsorbed proteins were then assessed using intrinsic fluorescence spectroscopy and FTIR spectroscopy. All three proteins showed good adsorption to Alhydrogel. PsaA was destabilized when adsorbed onto Alhydrogel and adding sodium phosphate showed a stabilizing effect. PcsB was found to be stabilized when adsorbed to Alhydrogel, and no destabilizing or stabilizing effects were seen in the case of StkP. The empirical phase diagram based approach was utilized to rapidly identify stabilizing buffer conditions and excipients for potential use in a complex trivalent live-virus veterinary vaccine. The attenuated versions of canine distemper virus (CDV), canine parainfluenza virus (CPI) and the canine adenovirus type 2 (CAV2) were purified separately to enable study of the physical stability of the individual viruses. The CDV and CPI viruses were purified using centrifugation and/or sucrose gradient techniques, and CAV2 virus was isolated using ammonium sulfate precipitation. The integrity of the three viruses was confirmed by transmission electron microscopy and purity was assessed by SDS-PAGE. The structural properties and conformational stability of the three viruses were then studied using various spectroscopic and light scattering techniques. The data obtained from the various methods were then used to construct an empirical phase diagram to characterize the physical stability of the individual viruses as a function of pH and temperature. Potential stabilizers were then identified for each virus using an assay based on the aggregation state of the viral particles and the conformational state of the viral proteins. Sucrose and sorbitol were demonstrated to be promising common stabilizers for all three live attenuated viruses. In conclusion, the empirical phase diagram approach was used to successfully formulate three vaccines in solution and while adsorbed on to the surface of aluminum salt adjuvants. The EPD proves to be a robust tool that can aid immensely in the early stages of formulation.
dc.format.extent150 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
dc.subjectPharmaceutical sciences
dc.subjectAdjuvants
dc.subjectAntigen
dc.subjectDevelopment
dc.subjectEmpirical phase diagram
dc.subjectFormulation
dc.subjectVaccine
dc.titlePreformulation Development of a monovalent recombinant-based subunit vaccine, a multivalent recombinant-based subunit vaccine and a multivalent live attenuated viral vaccine.
dc.typeDissertation
dc.contributor.cmtememberVolkin, David B
dc.contributor.cmtememberLaurence, Jennifer
dc.contributor.cmtememberGehrke, Stevin
dc.contributor.cmtememberDetamore, Micheal
dc.thesis.degreeDisciplineBioengineering
dc.thesis.degreeLevelPh.D.
kusw.embargo.termsEmbargo in effect until Dec. 31, 2014
dc.rights.accessrightsopenAccess


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