Copyright by Irnela Bajrovic 2020 The Dissertation Committee for Irnela Bajrovic Certifies that this is the approved version of the following Dissertation: Development and Characterization of Thermostable Thin Films as a Novel Vaccine Dosage Form Committee: Maria A. Croyle, Supervisor Hugh D. Smyth Debadyuti Ghosh Jennifer A. Maynard Charles R. Middaugh For my parents Džemal and Rahima, and my sister, Minela without whom none of this would have been possible. Acknowledgements I joined Dr. Maria Cryole’s lab in 2014 as an undergraduate student hoping to gain some research experience and learn about vaccine delivery. Little did I know that one semester would turn into six years and two degrees that have forever changed my life for the better. First and foremost, I would like to thank Dr. Croyle for giving me the guidance and support to learn and grow as a scientist. I will treasure the time we spent together and everything I learned from her. I would also like to thank my committee members Dr. Hugh Smyth, Dr. Debadyuti Ghosh, Dr. Jennifer Maynard, and Dr. Charles Middaugh for their guidance, support, and encouragement. Special thanks to Stephen Schafer who served as great sounding board and brainstorm partner. To past and present Croyle lab members Dr. Kristina Jonsson-Schmunk, Trang Doan, and Matthew Le, I also say thank you. Your advice and support contributed to the completion of the projects in this document and I am grateful for the friendships we have built. I would also like to thank my family for always encouraging me and reminding me that I am capable of anything I set my mind to. My wonderful parents, Rahima and Džemal, have sacrificed so much to give my sister and I a better life. I hope to honor their sacrifice with this document and a steadfast devotion to science. To my sister Minela, who taught me confidence and ambition, I am eternally indebted. She is my forever role model and I hope to make her proud. And to the friends that are like family, Lauren Cardenas, Zainab Shahid, Shehnaz Haqqani, Saad Dawoodi, Melissa Soto, and Ahlam Qerqez a special thanks for reminding me of the strength I have within. And finally, I would like to thank Karen Stewart, for opening up her home to me while I was preparing this document and providing me with a comfortable and quiet space to focus on my work. I will never forget her generosity. iv Abstract Development and Characterization of Thermostable Thin Films as a Novel Vaccine Dosage Form Irnela Bajrovic, PhD The University of Texas at Austin, 2020 Supervisor: Maria A. Croyle Thermostabilization of vaccines can significantly simplify vaccine storage and distribution processes, eliminating the need for cold-chain maintenance, and resulting in global access to life-saving vaccines. Despite this benefit, all approved vaccines for use by the Food and Drug Administration must be refrigerated for long term storage in order to guarantee potency. The first study described in this thesis demonstrated that formulation of live adenovirus in the novel thin film matrix protects the virus from degradation at 4°C and 20°C for a minimum of three months, as well as 14 days at 37°C and 5 days at 40°C. The film matrix protected virus through 16 freeze-thaw cycles as well. As formulations prepared with surfactant outperformed those without it, the second study was designed to characterize and evaluate the intermolecular interactions between the surfactant and adenovirus capsids. in order to better understand the surfactants contribution to stability. The data suggested that surfactant stabilizes adenovirus by preventing aggregation of capsids via electrostatic and hydrophobic interactions. Additionally, the other formulation components in our multi-component preparation mitigates the interactions between adenovirus and the surfactant without interfering with stability. Lastly, the principles of surfactant stabilization were applied to the identification of alternative excipients for stabilization v of a virus with different properties from adenovirus, H1N1 influenza. The third study evaluated the ability of the thin film platform to induce an immune response and the impact of a natural adjuvants on the cytokine response and bioavailability of the vaccine dose. A preliminary screen demonstrated that vaccination with the thin film platform resulted in a stronger humoral response following mucosal vaccination than with traditional intramuscular vaccination. Additionally, the optimized formulation improved bioavailability of the viral dose across human buccal explants. Further characterization of the immune response also revealed that sublingual routes induced a strong TH1 polarized immune response which resulted in greater protective efficacy than intramuscular immunization. Taken together, these studies identified a novel thin film platform capable of stabilizing adenovirus at ambient temperatures, provide key insights into viral stabilization in the novel thin film platform, and illustrate the utility of the thin film as mucosal vaccine dosage form. vi Development and Characterization of Thermostable Thin Films as a Novel Vaccine Dosage Form by Irnela Bajrovic Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin December 2020 vii Table of Contents Chapter 1: Breaking the Cold Chain for Viral Vaccines: A Move Toward Needle Free Delivery .......................................................................................................................................... 1 1.1 Physical Properties of Viruses ............................................................................................... 5 1.2 Chemical and Environmental Stressors which Impact Virus Stability ............................... 10 1.3 Principles of Formulation Design ....................................................................................... 13 1.4 Liquid Formulations for Viral Stabilization ........................................................................ 15 1.5 Solid Formulations for Vaccine Stabilization ..................................................................... 20 1.6 Spray-Drying ....................................................................................................................... 24 1.7 Film Formation for Vaccine Stabilization ........................................................................... 26 1.8 General Overview: Immune Response to Virus Based Vaccines ....................................... 31 1.9 Administration of Vaccines: The Injectables ...................................................................... 34 1.10 Intranasal Vaccine Delivery .............................................................................................. 38 1.11 Oral Vaccine Delivery ....................................................................................................... 41 1.11.1 Administration of Vaccines: Sublingual and Buccal Delivery ................................... 43 1.12 Objectives .......................................................................................................................... 51 1.13 References ......................................................................................................................... 55 Chapter 2: Novel Technology for Storage and Distribution of Live Vaccines and Other Biological Medicines at Ambient Temperature ....................................................................... 74 2.1 Introduction ......................................................................................................................... 74 2.2 Materials and Methods ........................................................................................................ 79 2.2.1 Materials ....................................................................................................................... 79 2.2.2 Adenovirus Production and Purification ...................................................................... 80 2.2.3 Formulation Screening ................................................................................................. 80 2.2.4 Rate of Release Analysis .............................................................................................. 81 2.2.5 Young’s Modulus and Percent Elongation ................................................................... 81 2.2.6 Viscosity Measurements ............................................................................................... 82 2.2.7 Short-Term Stability ..................................................................................................... 82 2.2.8 Freeze-Thaw Studies .................................................................................................... 83 2.2.9 Scanning Electron Microscopy ..................................................................................... 83 2.2.10 Differential Scanning Calorimetry ............................................................................. 83 2.2.11 X-ray Powder Diffraction ........................................................................................... 83 2.2.12 Fourier Transform Infrared Spectroscopy .................................................................. 84 2.2.13 Statistical Analysis ..................................................................................................... 84 viii 2.3 Results ................................................................................................................................