Relationship between structure and adjuvanticity of N,N,N-trimethyl chitosan (TMC) structural variants in a nasal influenza vaccine

Abstract

The aim of this study was to assess the influence of structural properties of N,N,N-trimethyl chitosan (TMC) on its adjuvanticity. Therefore, TMCs with varying degrees of quaternization (DQ, 22–86%), O-methylation (DOM, 0–76%) and acetylation (DAc 9–54%) were formulated with whole inactivated influenza virus (WIV). The formulations were characterized physicochemically and evaluated for their immunogenicity in an intranasal (i.n.) vaccination/challenge study in mice.

Simple mixing of the TMCs with WIV at a 1:1 (w/w) ratio resulted in comparable positively charged nanoparticles, indicating coating of WIV with TMC. The amount of free TMC in solution was comparable for all TMC-WIV formulations. After i.n. immunization of mice with WIV and TMC-WIV on days 0 and 21, all TMC-WIV formulations induced stronger total IgG, IgG1 and IgG2a/c responses than WIV alone, except WIV formulated with reacetylated TMC with a DAc of 54% and a DQ of 44% (TMC-RA44). No significant differences in antibody titers were observed for TMCs that varied in DQ or DOM, indicating that these structural characteristics play a minor role in their adjuvant properties. TMC with a DQ of 56% (TMC56) formulated with WIV at a ratio of 5:1 (w/w) resulted in significantly lower IgG2a/c:IgG1 ratios compared to TMC56 mixed in ratios of 0.2:1 and 1:1, implying a shift towards a Th2 type immune response. Challenge of vaccinated mice with aerosolized virus demonstrated protection for all TMC-WIV formulations with the exception of TMC-RA44-WIV.

In conclusion, formulating WIV with TMCs strongly enhances the immunogenicity and induces protection against viral challenge in mice after i.n. vaccination. The adjuvant properties of TMCs as i.n. adjuvant are strongly decreased by reacetylation of TMC, whereas the DQ and DOM hardly affect the adjuvanticity of TMC.

Introduction

Intranasal (i.n.) vaccination offers several advantages over the intramuscular (i.m.) route, like simple, needle-free administration without the need for trained personnel, potentially less adverse effects and the induction of local mucosal immune responses [1]. On the other hand, vaccines administered via the i.n. route generally induce low systemic immune responses when compared to i.m. administration likely due to mucociliary clearance and low antigen uptake. Mucoadhesive polymers have been used to increase the immunogenicity of i.n. vaccines by increasing nasal residence time and enhancing antigen presentation [1].

Chitosan, a polysaccharide that is obtained by deacetylation of the natural polymer chitin, has mucoadhesive properties and showed promising results as an adjuvant in nasal vaccines [2], [3], [4], [5]. However, the unfavorable pH-dependent solubility and charge density led to the synthesis of its quaternized derivative N,N,N-trimethyl chitosan (TMC) (Scheme 1), which is well soluble in aqueous solution at neutral pH.

TMC is traditionally synthesized by reaction of chitosan with excess iodomethane in strong alkaline conditions with N-methyl-2-pyrrolidone (NMP) as solvent and the degree of quaternization (DQ) can be varied by varying the number of reaction steps [6]. Besides N-methylation this synthesis method also introduces substantial O-methylation on the hydroxyl groups located at the C-3 and C-6 of the glucosamine unit. The degree of O-methylation (DOM) increases up to 80–90% with increasing DQ [7], [8]. Recently, O-methyl free TMC was synthesized using a novel two-step synthesis procedure, allowing good control of the DQ without altering other structural properties. Both DQ and DOM were found to influence toxicity and transepithelial electrical resistance (TEER), an indicator for opening of tight junctions, in a Caco-2 cell model. A higher DQ leads to more toxicity and a stronger TEER effect, with a maximum effect on TEER at a DQ above 60%. Furthermore, O-methyl free TMC has a much stronger effect on TEER than O-methylated TMC (TMC-OM) and shows more in vitro cell toxicity [8]. Another characteristic of TMCs is the degree of N-acetylation (DAc). Partial reacetylation of TMC (from 17 to 54%) decreased the in vitro cell toxicity and effect on TEER but increased the enzymatic degradability of TMC by lysozyme [9].

Little is known about the relationship between the structural characteristics and adjuvant properties of TMCs in vivo. For TMC-OM solutions in i.n. vaccination with ovalbumin an optimal DQ of 40% was reported although differences were small [10]. Previously, whole inactivated influenza virus (WIV) vaccine was formulated with TMC-OM with a DQ of 15% or 37%. This resulted in positively charged nanoparticles with partially bound TMC-OM. These particles had an intact viral ultrastructure. Strong, protective immune responses were induced after i.n. vaccination [11]. No significant differences were observed between the two different TMC-OMs. Most likely, TMC exerts its adjuvant effect by an improved antigen delivery, through an increased nasal residence time and/or enhanced uptake through the epithelium and by antigen presenting cells.

Besides differences in DQ, the TMC-OMs used in these studies also differed in DOM and, likely, polymer molecular weight. So, the individual contributions of DQ, DAc and DOM on the adjuvant effect of TMC are unknown.

In the present study we investigated for i.n. administered WIV the adjuvant properties of O-methyl free TMCs with varying DQs and reacetylated O-methyl free TMC in comparison to conventional TMC-OMs with similar DQ. The TMC-WIV vaccines were physicochemically characterized and the immunogenicity and protectivity of the vaccines were assessed in a murine challenge model. Additionally, the influence of TMC:WIV ratio on the quality and quantity of humoral immune responses was investigated.