Towards an intranasal influenza vaccine based on whole inactivated influenza virus with N,N,N-trimethyl chitosan as adjuvant

Abstract

Vaccination provides the best form of protection against seasonal and pandemic influenza. Intranasal (i.n.) vaccination against influenza poses several advantages to an intramuscular injection, such as simple, noninvasive administration and the potential induction of mucosal immune responses in addition to systemic ones. The aim of in this thesis is the rational design of an inactivated i.n. influenza vaccine. Initially, four commonly used, nonadjuvanted influenza vaccines were tested to determine the optimal antigen formulation for i.n. administration. From these studies, whole inactivated virus (WIV) was selected as the best candidate for further optimization as an i.n. inactivated influenza vaccine. Next, WIV was formulated with N,N,N-trimethylchitosan (TMC), a water-soluble, cationic, mucoadhesive polymer. The addition of TMC, which is partially bound to the WIV particles and partially present in solution, strongly enhanced the immunogenicity of the vaccines and induced complete protection after i.n. vaccination in mice. Additionally, the relationship between structure and adjuvanticity of TMC structural variants in an i.n. WIV-based vaccine was investigated. Furthermore, the influence of TMC on the nasal residence time, antigen distribution in the nasal cavity and local toxicity was assessed. The increased adjuvanticity of TMC-WIV vaccines could not be explained by a prolonged nasal residence time, but likely the interaction between WIV and the mucosal surfaces is enhanced by formulation with TMC. Minimal local toxicity was observed. Next, a study was performed to clarify why reacetylated TMC(TMCRA)did not work as an adjuvant, in contrast to all other TMC structural variants. It appeared that TMCRA did not enhance the antigen uptake by epithelial cells as much as the other TMCs. Interestingly, initial studies on dendritic cells (DCs) suggest that TMCRA does act as an immunostimulant on human DCs but not as pronounced on murine DCs. Finally, the colloidal stability of these TMC-WIV vaccines was studied and revealed that stable colloidal formulations of positively charged, TMC-coated WIV particles can be obtained with all TMC variants. However, the TMC:WIV w/w ratio; the salt concentration and the type of TMC influence the stability of the formed TMC-WIV formulations. Freeze drying these formulations seems feasible for further enhancement of the colloidal stability. Altogether, the research in this thesis resulted in the rational design of an i.n. influenza vaccine, based on WIV with TMC as an adjuvant, that shows promising results for further development.