&&ReWrAp:HEADERFOOTER:0:ReWrAp&&

Date of Award

1998

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Dr. Mark R. McDermott

Abstract

Despite recent interest in developing novel microparticle (MP)-based antigen delivery systems for mucosal vaccination, existing MP formulations possess a number of liabilities potentially precluding their widespread use. Nevertheless, there is sufficient evidence to suggest that if immunogenically stable vaccine material can be incorporated into biocompatible/biodegradable MPs and delivered to various mucosal surfaces, this may be an effective way to elicit protective systemic and mucosal immunity. To overcome difficulties associated with existing MP formulations, the present work describes the development of a novel polymergrafted starch MP system which is capable of entrapping a wide variety of soluble antigens under mild conditions and which will elicit efficacious immune responses following intragastric (i.g.) or intranasal (i.n.) administration. The novel MP delivery technology was developed using starch, a well-studied, biologically acceptable and non-toxic material when given orally or parenterally. Antigen-containing starch MP were subsequently grafted with a hydrophobic silicone [3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane, TS-PDMS] which, it was hypothesized, would protect microentrapped antigen from the deleterious environment found in the gastrointestinal (GI) tract, facilitate MP uptake by M cells overlying mucosae-associated lymphoid tissues (MALT) and/or act as an adjuvant or immunopotentiator. Moreover, following selective transport into mucosal inductive sites, antigen-containing MPs should stimulate the generation of robust antigen-specific disseminating mucosal and circulating humoral immune responses. In the work reported here, it was demonstrated that antigen-containing TS-PDMS-grafted MPs could be fabricated under mild conditions, allowing for the successful entrapment of a variety of protein and peptide antigens without any demonstrable loss in immunogenicity. Furthermore, it demonstrated that under acidic conditions, protein release from MPs was retarded when MPs were grafted with TS-PDMS. Although protein release from TS-PDMS-grafted MPs compared to ungrafted MPs was hindered, microentrapped protein was still released from TS-PDMS-grafted MPs, thus demonstrating that the MP composition allowed for entrapped proteins to readily learn from the MP matrix. Overall, these observations suggested that TS-PDMS-grafted MPs could serve as an efficacious MP-based mucosal vaccine delivery system. To be considered an attractive alternative to current mucosal MP vaccine delivery vehicles, TS-PDMS-grafted MPs should incite both local mucosal and systemic humoral immunity following mucosal administration of low doses of microentrapped antigen. The studies in this report clearly demonstrated that oral immunization with very low doses of TS-PDMS-grafted MPs simulated both systemic and mucosal humoral immune responses. Indeed, the adjuvanticity of TS-PDMS-grafted MPs seems to have arisen via a unique physicochemical relationship occurring between protein antigen and silicone in the starch matrix. This led to a predominantly Th2-type immune response following oral MP administration of relatively small amounts of microentrapped antigen. Furthermore, serum antibody titres were augmented after an oral or systemic boost, suggesting that the initial immunization protocol stimulated serum antibody memory capability, an advantage when developing successful mucosal immunization strategies. Surprisingly, systemic antigen challenge failed to boost antigen-specific sera igA titres following i.g. immunization with microentrapped or soluble antigen. These results suggest Peyer's patch (PP)-stimulated igA lymphocytes migrate to mucosal lymphoid compartments following i.g. MP administration, while antigen-specific PP-stimulated IgC plasmacytes have the propensity to migrate to both mucosal and systemic lymphoid compartments. In addition to stimulating specific serum antibody responses, i.e. immunization with TS-PDMS-grafted or ungrafted MPs resulted in specific sIgA responses in the gut; this is in contrast to soluble antigen which was incapable of inciting specific intestinal immunity following i.g. administration. Thus, compared to soluble antigen, TS-PDMS-grafted MPs have immunopotentiating activity when delivered orally. The studies outlined in this work strongly suggest that i.g. administration of TS-PDMS-grafted MPs stimulated mucosal immunity via PP. Following i.g. immunization with a low dose of antigen-containing MP, but not soluble antigen, specific proliferation of PP cells was observed. Lymphocyte proliferation was subsequently observed in mesenteric lymph node (MLN) and splenic tissue. In contrast, antigen-specific lymphocyte proliferation was not observed in gut lamina propria (LP) lymphocytes following i.g. immunization with MPs, thus suggesting that MP-induced immunity was incited by MP uptake and processing solely by PP. It was shown also that i.n. immunization with low doses of microentrapped, but not soluble, antigen evoked robust circulating specific IgG responses, indicating that TS-PDMS-grafted MPs could enhance the immunogenicity of an i.n. administered soluble antigen. Indeed, i.n. immunization with microentrapped protein stimulated greater levels of specific sera IgG than was observed after i.g. MP administration with equal amounts of microentrapped antigen. However, unlike i.g. TS-PDMS-grafted MP immunization, antigen specific IgA was not detected in local mucosal secretions or sera following i.n. immunization with microentrapped antigen, thereby suggesting that i.n. immunization expresses unique immune responses compared to those evoked after comparable i.g. immunization protocols. Although that nasal-associated lymphoid tissue (NALT) is considered to be the equivalent of Waldeyer's ring in humans, the exact process of generating immune responses when NALT is exposed to antigen is not clear. The present work describes the development of a novel, rapid and precide method for isolating NALT from mice to study its immune function and cell populations. Following the development of this precise NALT isolation technique, the pathway of i.n. administered TS-PDMS-grafted MPs was examined. It was demonstrated that i.n. immunization with low doses of microentrapped, but not soluble protein, evoked robust circulating IgG responses via NALT cell activation. Numerous antigen-specific spot-forming cells (SFCs) were observed in the NALT and later the posterior cervical lymph nodes (pCLN) and spleen (SPL), confirming the selective drainage of NALT cells exclusively to the pCLN following i.n. administration of a particulate antigen. Although B cell activity was observed in the pCLN following i.n. MP administration, no specific IgA was detected in any lymphoid tissue examined or in nasal secretions following i.n. immunization with TS-PDMS-grafted MPs. This suggested that either pCLN in mice are not intermediate in evoking sIgA responses in the nasopharynx or that TS-PDMS-grafted MPs are incapable of stimulating this arm of the murine immune system. However, since it was previously demonstrated that i.g. immunization with comparable doses of TS-PDMS-grafted MPs evoked intestinal IgA responses, the inability to detect antigen-specific IgA in nasal secretions probably reflects differences between murine NALT and PP and not a unique inability of TS-PDMS-grafted MPs to evoke secretory immunity in the nose. Thus, the present work describes the successful development of a novel polymer-grafted starch MP system which is capable of entrapping a wide variety of soluble antigens under mild conditions and which can elicit efficacious immune responses both orally and nasally, via MALT activation.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS