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|Title:||Development of a Thermostabilization Platform for Point-of-Care Diagnostics, Food Safety, and Vaccine Deployment|
|Abstract:||Thermostabilization of labile biomolecules is crucial for the advancement of global health. The World Health Organization has identified accessible quality-assured diagnostics, prevention of antibiotic resistance, and universal access to vaccination as three of the most important issues for global health today. One major challenge is that many biomolecules, such as enzymes and vaccines, are thermally unstable and require refrigeration at all times. Maintaining refrigeration is a major logistical problem, since many developing countries and rural areas do not have access to reliable electricity. Moreover, the cost to maintain the cold chain infrastructure presents a substantial financial barrier. Therefore, there is a great need for the thermostabilization of labile biomolecules. This work presents a simple thermostabilization method that uses pullulan and trehalose to encapsulate labile biomolecules within a sugar glass matrix. To address the problem of accessible quality-assured diagnostics, we thermally stabilized molecular biology reagents in ready-to-use mastermixes. Thermally stabilized polymerase chain reaction (PCR) and rolling circle amplification (RCA) mastermixes were shown to be stable for at least 12 weeks at room temperature. This can significantly improve the accessibility of diagnostic tools since, DNA amplification techniques such as PCR and RCA can be used as diagnostic tools to provide rapid detection of diseases with high specificity and sensitivity. To address the problem of antibiotic resistance, this work demonstrated a method to stabilize bacteriophages as a coating on surfaces for food safety applications. Bacteriophages, are viruses that kill bacteria with high specificity while having a low risk of developing antibiotic resistance. The bacteriophages encapsulated in pullulan-trehalose films were able to retain infectivity for up to 3 months at ambient storage conditions. We further optimized the stability of bacteriophage in pullulan-trehalose films performing a four factor, two level design of experiment (DOE). It was shown that vacuum drying and storing the films in a closed container with low-humidity can increased the long-term viability of bacteriophage by over 1000-fold. To address the need for universal immunization, we stabilized enveloped DNA and RNA viral vaccines by drying them in a pullulan and trehalose mixture. The thermally stabilized live-attenuated HSV-2 vaccine retained immunogenicity for at least 2 months when stored at 40 °C. Inactivated influenza vaccine stabilized in pullulan and trehalose retained immunogenicity for at least 3 months at 40 °C. Overall, this thesis presents a versatile thermostabilization method using pullulan and trehalose as stabilizers. This method was used to thermally stabilize molecular biology reagents, bacteriophages, and vaccines. The achievements from this work have the potential to significantly improve global health by creating thermally stable molecular diagnostic kits that are accessible and reliable, by preventing antibiotic resistance with stabilized bacteriophage coatings, and by deploying thermally stable vaccine for universal immunization.|
|Appears in Collections:||Open Access Dissertations and Theses|
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