title: 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.
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: Hagenaars
authors.forename: Rolf J
authors.initials: RJ
authors.lastname: Verheul
authors.forename: Imke
authors.initials: I
authors.lastname: Mooren
authors.forename: Pascal H J L F
authors.initials: PH
authors.lastname: de Jong
authors.forename: Enrico
authors.initials: E
authors.lastname: Mastrobattista
authors.forename: Harrie L
authors.initials: HL
authors.lastname: Glansbeek
authors.forename: Jacco G M
authors.initials: JG
authors.lastname: Heldens
authors.forename: Han
authors.initials: H
authors.lastname: van den Bosch
authors.forename: Wim E
authors.initials: WE
authors.lastname: Hennink
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
mesh_terms: D000107:Acetylation; D000276:Adjuvants, Immunologic / Q000008:administration & dosage / Q000737:chemistry; D000281:Administration, Intranasal; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002626:Chemistry, Pharmaceutical; D048271:Chitosan / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D004195:Disease Models, Animal; D004339:Drug Compounding; D005260:Female; D007115:Immunization Schedule; D007071:Immunoglobulin A, Secretory / Q000378:metabolism; D007074:Immunoglobulin G / Q000097:blood; D009980:Influenza A virus / Q000276:immunology*; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D017817:Nasal Lavage Fluid / Q000276:immunology; D009976:Orthomyxoviridae Infections / Q000276:immunology / Q000517:prevention & control* / Q000821:virology; D013329:Structure-Activity Relationship; D013997:Time Factors; D015164:Vaccines, Inactivated / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D000914:Antibodies, Viral; D007071:Immunoglobulin A, Secretory; D007074:Immunoglobulin G; D007252:Influenza Vaccines; C118071:N-trimethyl chitosan chloride; D015164:Vaccines, Inactivated; D048271:Chitosan