Tailorable Trimethyl chitosans as adjuvant for intranasal immunization

Abstract

Tailorable Trimethyl Chitosans as Adjuvant for Intranasal Immunization Active vaccination has proven to be the most (cost) effective tool in the fight against infectious diseases. Nowadays, most vaccines are administered via parenteral injection. However, the risk of contaminated needles and need for trained personnel have risen interest in alternative immunization routes. As live attenuated vaccines raise considerable safety issues, (nasal) vaccine development now mainly focuses on purified, well-characterized antigenic proteins. However, these subunit vaccines are generally less immunogenic and need potent adjuvant (system)s to elicit an adequate immune response. The use of muco-adhesive polymers like N,N,N-trimethylchitosan (TMC), a partially quaternized, water-soluble chitosan derivative, can enhance the immune responses against antigens. In this thesis novel synthetic methods are presented to obtain structural variants of TMC that allow tailorability of the degrees of quaternization (DQ) and acetylation (DAc) and exclude the introduction of other alterations such as O-methylation (DOM)and polymer chain scission. Additionally, thiol-moieties are introduced in a controllable manner to yield thiolated TMC. This tailorability of TMC provided the possibility to properly establish structure-activity-relationships in in vitro biological assays and in in vivo (nasal) vaccination studies. On Caco-2, cells O-methyl free TMCs demonstrated a larger decrease in trans-epithelial electrical resistance (TEER) than O-methylated TMCs while TMCs with a high DAc (~50%), were not able to open tight junctions. Also, with increasing DQ, an increase in cytotoxicity (MTT) and membrane permeability (LDH) was observed. On the other hand, TMCs with a high DAc (~50%) demonstrated no cell toxicity (MTT, LDH release) up to a concentration of 10 mg/ml. The extent of lysozyme-catalyzed degradation of TMC was highly dependent on the DAc; polymers with the highest DAc showed the largest decrease in molecular weight. Using Calu-3 cells, XTT and LDH cell viability tests showed a slight reduction in cytotoxicity for thiolated TMCs as compared to the non-thiolated polymers with similar DQs. The adjuvant properties of TMCs as nasal adjuvant in mice, using whole inactivated influenza virus as antigen, were strongly decreased by re-acetylation of TMC, whereas the DQ and DOM hardly affected the adjuvanticity of TMC. The inferior adjuvant effect of TMC-RA over that of TMC might be caused by a slightly lower stability of TMC-RA in the nasal cavity. In vitro experiments with human and murine dendritic cells indicated that the degree of N-acetylation is critical for the adjuvant effect of TMC on human DCs, but not on mice DCs. Finally, a covalently-stabilized polymeric nanocarrier system was introduced based upon the formation of intracellularly degradable disulfides between thiolated TMC (TMC-SH) and (oppositely charged) thiolated hyaluronic acid (HA-SH). Stabilized TMC-S-S-HA nanoparticles showed superior stability in saline solutions compared to non-stabilized particles (composed of non-thiolated polymers), but readily disintegrated when a disulfide reducing agent was introduced. In both the nasal and intradermal immunization study, Ovalbumin-loaded stabilized TMC-S-S-HA particles demonstrated superior immunogenicity compared to non-stabilized particles. Further research is required to investigate the potential applications for these novel TMC(-systems) in nasal vaccin