Hagenaars Niels, Mastrobattista Enrico, Verheul Rolf J, Mooren Imke, Glansbeek Harrie L, Heldens Jacco G M, van den Bosch Han, Jiskoot Wim
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Pharm Res. 2009 Jun;26(6):1353-64. doi: 10.1007/s11095-009-9845-y. Epub 2009 Feb 18.
The purpose of this study was the development and physicochemical and immunological characterization of intranasal (i.n.) vaccine formulations of whole inactivated influenza virus (WIV) coated with N,N,N-trimethyl chitosan (TMC).
Synthesized TMCs with a degree of quarternization of 15% (TMC15) or 37% (TMC37) were tested in vitro for their ability to decrease the transepithelial resistance (TEER) of an epithelial cell monolayer. TMC15- and TMC37-coated WIV (TMC15-WIV and TMC37-WIV) were characterized by zeta potential measurements, dynamic light scattering, electron microscopy and gel permeation chromatography. Mice were vaccinated i.n. with selected vaccine formulations and immunogenicity was determined by measuring serum hemagglutination inhibition (HI) and serum IgG, IgG1 and IgG2a/c titers. Also a pulse-chase study with TMCs in solution administered i.n. 2 h prior to WIV was performed. Protective efficacy of vaccination was determined by an aerosol virus challenge.
TMC37 induced a reversible decrease in TEER, suggesting the opening of tight junctions, whereas TMC15 did not affect TEER. Simple mixing of (negatively charged) WIV with TMC15 or TMC37 resulted in positively charged particles with TMCs being partially bound. Intranasal immunization with TMC37-WIV or TMC15-WIV induced stronger HI, IgG, IgG1 and IgG2a/c titers than WIV alone. TMC37-WIV induced the highest immune responses. Both TMC15-WIV and TMC37-WIV provided protection against challenge, whereas WIV alone was not protective. Intranasal administration of TMC prior to WIV did not result in significant immune responses, indicating that the immunostimulatory effect of TMC is primarily based on improved i.n. delivery of WIV.
Coating of WIV with TMC is a simple procedure to improve the delivery and immunogenicity of i.n. administered WIV and may enable effective i.n. vaccination against influenza.
本研究旨在开发用N，N，N-三甲基壳聚糖（TMC）包被的全灭活流感病毒（WIV）鼻内（i.n.）疫苗制剂，并对其进行物理化学和免疫学表征。
测试了季铵化程度为15%（TMC15）或37%（TMC37）的合成TMCs在体外降低上皮细胞单层跨上皮电阻（TEER）的能力。通过ζ电位测量、动态光散射、电子显微镜和凝胶渗透色谱对TMC15包被的WIV（TMC15-WIV）和TMC37包被的WIV（TMC37-WIV）进行表征。用选定的疫苗制剂对小鼠进行鼻内接种，并通过测量血清血凝抑制（HI）以及血清IgG、IgG1和IgG2a/c滴度来确定免疫原性。还进行了一项脉冲追踪研究，即在接种WIV前2小时经鼻内给予溶液中的TMCs。通过气溶胶病毒攻击来确定疫苗接种的保护效果。
TMC37导致TEER可逆性降低，提示紧密连接开放，而TMC15不影响TEER。（带负电荷的）WIV与TMC15或TMC37简单混合产生带正电荷的颗粒，TMCs部分结合。用TMC37-WIV或TMC15-WIV进行鼻内免疫诱导的HI、IgG、IgG1和IgG2a/c滴度比单独使用WIV更强。TMC37-WIV诱导的免疫反应最强。TMC15-WIV和TMC37-WIV均提供了针对攻击的保护，而单独的WIV没有保护作用。在接种WIV前经鼻内给予TMC未产生显著的免疫反应，表明TMC的免疫刺激作用主要基于改善WIV的鼻内递送。
用TMC包被WIV是一种简单的方法，可改善鼻内给予的WIV的递送和免疫原性，并可能实现有效的鼻内流感疫苗接种。
