Rational design, manufacture and evaluation of respirable TB subunit vaccines Public Deposited

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Last Modified
  • March 21, 2019
Creator
  • Shi, Shuai
    • Affiliation: Eshelman School of Pharmacy
Abstract
  • The purpose of this dissertation is to compare the in vitro immune response of newly designed respirable TB subunit vaccines based on Ag85B/TB10.4 and their different combinations in order to make a recommendation of the best antigen/antigen combination for future animal protection study. The second goal is to develop a particle size comparator to evaluate the batch-to-batch reproducibility of inhalation products. Antigens were expressed and purified as soluble recombinants. The solubility of TB10.4 and TB10.4-Ag85B was dramatically improved by recombinant fusion to the C-terminal of a bacterial protein thioredoxin. A purification method involving three chromatographic steps and one enzymatic treatment was successfully developed to purify soluble TB10.4 and TB10.4-Ag85B to homogeneity. The secondary structure and melting temperature of Ag85B, TB10.4 and TB10.4-Ag85B were characterized by circular dichroism. Purified soluble recombinant antigens were encapsulated into polylactide-co-glycolide (PLGA) microparticles by emulsion/spray-drying. By employing the concept of Quality-by-Design (QbD), the spray-drying parameters were successfully optimized to give the desired particle properties. Particle size was optimized to a target of 3 μm with a single mode and narrow size distributions. A mass median aerodynamic diameter (MMAD) of 3.3μm and a fine particle fraction of 61.5% suggested that these particles are suitable for pulmonary delivery. PLGA microparticles encapsulating different antigens or antigen combination showed highly reproducible physicochemical properties such as size, surface charge and glass transition temperature. Moreover, antigen encapsulation did not compromise its integrity. In vitro evaluation of TB subunit vaccines demonstrated that antigens encapsulated into PLGA microparticles induced much stronger and sustained Ag85B specific MHC II immune response compared to antigen solution formulations. Moreover, our data suggested that antigen blend of Ag85B and TB10.4 induced much better Ag85B response than antigen fusion TB10.4-Ag85B. We also developed a particle size comparator to evaluate the batch-to-batch reproducibility of inhalation products based on orthogonal partial least square analysis (OPLS). It was shown that OPLS coupled with Pareto scaling gave the best performance in terms of consistency with the Product Quality Research Institute (PQRI) working group evaluation. In addition, OPLS-based comparator showed better performance than that based on chi-square ratio statistics proposed by PQRI.
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  • In Copyright
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Eshelman School of Pharmacy."
Advisor
  • Hickey, Anthony J.
Degree granting institution
  • University of North Carolina at Chapel Hill
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Place of publication
  • Chapel Hill, NC
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  • Open access
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