Dual role of CpG as immune modulator and physical crosslinker in ovalbumin loaded N-trimethyl chitosan (TMC) nanoparticles for nasal vaccination.

Nasal vaccination is a promising, but challenging vaccination strategy. Poor absorption by the nasal epithelium and failure to break nasal tolerance are regarded as important reasons for poor efficacy of nasally applied vaccines. Formulation of the antigen into mucoadhesive nanoparticles, made of N-trimethyl chitosan (TMC) crosslinked with tripolyphosphate (TPP), has been shown to overcome these obstacles. However, although nasally administered antigen loaded TMC/TPP nanoparticles induce a strong humoral response, antibody subtyping indicates a Th2 bias. To design a nasal antigen delivery system capable of inducing stronger Th1 type responses, TPP as a crosslinking agent was replaced by unmethylated CpG DNA, a TLR-9 ligand and a potent inducer of Th1 responses, to prepare ovalbumin (OVA) loaded TMC nanoparticles (TMC/CpG/OVA). Several physicochemical characteristics of TMC/CpG/OVA (size, zetapotential, loading efficiency and antigen release profile) were assessed and compared to TMC nanoparticles prepared by crosslinking with TPP (TMC/TPP/OVA). Mice were nasally administered TMC/TPP/OVA and TMC/CpG/OVA after which antibody responses in serum and nasal washes were assessed and T-cell activation in the spleens determined. TMC/CpG/OVA showed similar physical properties as TMC/TPP/OVA in terms of particle size (380 nm), zetapotential (+21 mV) and antigen release characteristics. Nasal administration of TMC/CpG/OVA and TMC/TPP/OVA to mice resulted in comparable serum IgG levels (ca. 1000 fold higher than those induced by unadjuvanted OVA) and local secretory IgA levels. Moreover, TMC/CpG/OVA induced a 10 fold higher IgG2a response than TMC/TPP/OVA and enhanced the number of OVA specific IFN-gamma-producing T-cells in the spleen. In conclusion, OVA loaded TMC nanoparticles, containing CpG as adjuvant and crosslinker, are capable of provoking strong humoral as well as Th1 type cellular immune responses after nasal vaccination.