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Abstract
The goal of this research was to develop new vehicles that deliver protein antigens into the cytoplasm of antigen presenting cells (APCs) leading to cytotoxic T lymphocyte (CTL) activation. Particles that encapsulate protein and target APCs have been prepared. They are stable at extracellular pH but degrade in the acidic environment of endosomes after phagocytosis. This degradation destabilizes the endosomal membrane releasing the protein antigen into the cytoplasm of APCs, where it is processed along the pathway that leads to CTL activation.
Chapter 1 discusses the concept of using subunit vaccines as alternatives to traditional immunization strategies, with particular attention being paid to protein-based vaccines. A summary of protein-based vaccine delivery vehicles is given, as well as an explanation of the general goals of this doctoral research.
Initial investigation into the design and synthesis of a novel acid-degradable crosslinker and pH-sensitive hydrogels is described in Chapter 2. Chapter 3 expands upon this work by presenting the synthesis of a new crosslinker and the preparation of acid-degradable, protein antigen-loaded particles. pH-Dependent protein release from the delivery vehicle is demonstrated and evaluation of the system's vaccine potential by in vitro experiments is discussed. Chapter 4 outlines studies into the optimization of the crosslinker structure and particle properties and more in depth in vitro experiments are performed. Additionally, in vivo studies demonstrate that particles containing a model antigen stimulate protective immunity in mice, supporting the potential use of the particles as protein-based vaccines.
In Chapter 5, receptor ligands that aid in vaccine delivery are incorporated into the particles. Active targeting to APCs is explored by addition of mannose residues to the particles. Additionally, incorporation of immunostimulatory CpG oligonucleotides into the particles is described. In vitro studies demonstrate that CpG particles activate APCs to express costimulatory molecules and secrete cytokines, which leads to enhanced vaccine efficacy in vivo.
The preparation of pH-sensitive polyureas, polyurethanes and polyamidoamines is presented in Chapter 6 and Chapter 7. These polymers contain acid-degradable linkages embedded in the backbone and are stable at pH 7.4 but degrade rapidly into small molecules at pH 5.0. Initial particle formation using these polymers is described.
