Microparticles as antigen delivery systems for oral vaccination

Many gastro-intestinal infections are still endemic in large parts of the world causing morbidity and mortality among humans and animals. Mucosal surfaces are the major entrance for infectious pathogens and the induction of local adaptive immune responses are required to combat intestinal pathogens. Furthermore, the gastrointestinal system performs a balancing act in making the decision between immunity and oral tolerance, as a discrimination between harmful and beneficial antigens. Without doubt, the development of vaccines constitutes one of the major breakthroughs of human medicine, allowing us to control and prevent numerous infectious diseases in an efficient and cost-effective manner. Especially in developing countries, oral vaccination provides different social and economic advantages (needle-free, non-invasive administration route, patient compliance, less risk of contamination,…). Current oral vaccines, based on killed or attenuated pathogens, provoke an inefficient immune response or maintain the risk of reversion to virulence and the induction of disease in immunocompromised individuals. Subunit vaccines are a safe alternative, however are less efficacious and require adjuvants. A promising strategy for vaccination with safe, biodegradable non-replicating antigen delivery systems has gained increased interest for eliciting cellular and humoral immune responses. However, the low gastric pH and degradation by proteolytic enzymes present in the gastrointestinal tract, are huge obstacles that inhibit the ability to successfully develop new mucosal vaccines. Microparticulate encapsulation strategies can protect the antigen from degradation, increase the concentration of antigen in the vicinity of mucosal tissue for better absorption and selectively target immune inductive sites, namely the gut-associated lymphoid tissue (GALT), Peyer’s patches (PP) and intestinal antigen presenting cells (APCs), including dendritic cells (DCs). Microbial (bacterial toxins, CpG DNA) and synthetic (microspheres, liposomes, PLGA,…) strategies have been investigated. Contemporary developments in the use of delivery systems and adjuvants have an undeniable value in the oral vaccine development, but a better understanding of the unique properties of mucosal surfaces and the vaccination strategies are indispensable. A first part of this thesis describes an introduction on the intestinal immune system expanding on antigen sampling, presentation and epithelial crosstalk in the small intestine. Furthermore, we summarize current registered human oral vaccines and highlight the importance of animal models in the contribution to the translational research into humans. Lastly, we address the rationale and key biological and physicochemical aspects of oral vaccine design and emphasize the use of yeast-derived β-glucan microparticles as an oral vaccine delivery platform. In a second part of this thesis, we performed a layer-by-layer synthesis of hollow yeast shells through electrostatic interactions using ovalbumin (OVA) as model antigen. Zeta-potential measurements and the quantification of antigen loading gave more information about the OVA-yeast shell interaction and its encapsulation efficiency. Additionally, the kinetics of antigen release and processing is investigated via OVA-DQ-loaded yeast particles in pronase solutions and in in vitro cultured murine BMDCs. In a third part of this thesis, we investigated the particle characteristics of β-glucan microparticles and evaluated their potential as an oral antigen delivery vehicle in human intestinal epithelial cell lines and murine models. A first finding is that β-glucan microparticles are of an ideal size of natural fungal pathogens to be recognized and highly efficient internalized by human Caco-2 and HT-29 cells. Internalization of β-glucan microparticles increased with incubation time and was dose-dependent. Secondly, β-glucan microparticles are capable of reaching the murine Peyer’s patches (specialized lymphoid tissue of the small intestine) via the M cell-mediated transcellular pathway and promoted MHCII–mediated presentation of the model antigen ovalbumin. Additionally, β-glucan microparticles induced intestinal secretory-IgA (S-IgA) and secretory component (SC) antibodies and a mixed Th1/Th17 cellular-mediated immune response against the model antigen ovalbumin. The final part of this thesis, focuses on porous antigen-loaded polyelectrolyte microparticles obtained by the spray-drying technique, namely calcium carbonate- and mannitol-based microspheres. The spray-drying method not only enhances the stability and controls the particle size ranges of the dry formulation, it also avoids the cold-chain-constrained settings, which is a very important aspect for vaccination campaigns in third world countries. Stability tests of CP and MP revealed negligible or no antigen loss in gastric and intestinal conditions. Both particle types are successfully internalized by intestinal epithelial cells and subsequently induced MHC class II and co-stimulatory molecules