Study of Japanese encephalitis virus DNA vaccine using the transcutaneous immunization by chitosan complexes

• Main Author: 黃瀚寧
• Other Authors: Chang-Jer Wu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/54062649295141543232

碩士 === 國立臺灣海洋大學 === 食品科學系 === 96 === DNA vaccine represents an innovated novel approach to vaccine research and might potentially circumvent some of the shortcomings of the conventional vaccine. However, the DNA vaccine when applied by intramuscular injections needed a large volume plasmid DNA, and needed some expensive instruments when applied by gene gun. Apparently, the more convenient and efficient delivery strategies are required to obtain high titers of protective immunity in DNA vaccine. Skin is the biggest organ of human that makes cutaneous immunization easy to operate. The epidermis of skin contains the antigen presenting cells and Langerhans cells (LC), which can modulate the immune responses. Transcutaneous immunization, introduction of antigens by topical application to intact skin, has many practical merits compared to injectable routes of administration. In order to ensure the outcome of therapy, the efficiency of gene delivery is very important. In this study, we used cationic chitosan as vehicle for transcutaneous delivery. In vitro transfection efficiency of plasmid chitosan/DNA complexes was analyzed by the expression of green fluorescent protein (GFP) in BHK-21 and Vero cells. The optimal ratio of the plasmid DNA (pGFP-N1) to chitosan for maximal transfection efficiency was N/P ratio = 3 in the BHK-21 cells or N/P ratio = 5 in the Vero cells. Then, the formulation of the chitosan/DNA complexes was applied to the hair-removed dorsal skin of C3H/HeN mice. After 3 days, we could find the green fluorescence protein expression in the hair follicle, epidermal and spleen by fluorescence microscope. In immunization studies, we used the Japanese encephalitis virus (JEV) membrane-envelope gene expressive plasmid (pCJ-3/ME) to test the protective immunity of DNA vaccine/chitosan complexes induced by transcutaneous immunization. The C3H/HeN mice transcutaneously immunized by JEV DNA vaccine/chitosan complexes could induce the specific and protective antibody to against 50xLD50 of JEV (Beijing-1 strain) challenge. These results demonstrate that DNA vaccine cooperate with transcutaneous immunization may hold promise for the prophylaxis of pandemic pathogen.