Development of Rotavirus Vaccine Using Adenoviral Vector
Date Issued
2005
Date
2005
Author(s)
Su, Chien-Wen
DOI
zh-TW
Abstract
Rotavirus infections are the most important cause of severe, even life-threatening dehydrating diarrhea in infants and children. Because of the significant disease burden, vaccines against rotavirus are urgently needed.
The viral particle is composed of three layers: inner, middle, and outer capsids. V6 protein is the major component of the middle-layer capsid and is highly antigenic and immunogenic. It is also more conserved than outer capsid proteins. Either full-length or the cytoplasmic domain of VP6 can elicit protective antibody response. In addition, NSP4 protein is a nonstructural transmembrane protein important for viral assembly and is suggested as an enterotoxin. The cytoplasmic domain (amino acid 86-175) include the region that cause diarrhea. Therefore, antibody against NSP4 may effectively reduce the diarrhea symptom. Because of the higher conservation and the importance for viral structure and function, vaccines against VP6 and NSP4 were developed in this research.
Adenoviral vector systems were utilized for a variety of animal virus vaccine, and considered to be effective. In this study, we used the adenoviral vector to develop rotavirus vaccine. VP6, VP6197-397, NSP4, and NSP486-175 gene fragments were recovered from rotavirus, then cloned into pShuttle-CMV vector. The vector was used to transform bacteria BJ5183 which carried adenoviral genome to generate recombinant adenoviral DNA through homologous recombination. The recombinant adenoviral DNA was then transfected into 293A cell to generate recombinant adenoviruses carrying each gene fragment. Four recombinant adenoviruses carrying VP6, VP6197-397, NSP4, and NSP486-175 gene were established. Target protein expression in 293A cells could be detected by indirect immunofluorescence assay. In order to get pure viral progeny, three serial plaque purification procedures were carried out. After large scale amplification of recombinant adenoviruses, double CsCl gradient banding and dialysis were performed to purify viral particles. The virus titer was determined, and the mice were immunized with 1×109 pfu viral particles twice via oral or intranasal administration routes. The mice were challenged with rotavirus and the antibody responses were analyzed.
In order to prepare antigens to detect specific antibody, the Sf-9 cell were infected with recombinant baculovirus to express VP6 protein. On the other hand, the NSP486-175 were expressed by E.coli system and purified by Ni2+ column, for detection of the anti-NSP486-175 antibody. The ELISA results showed that immunization of mouse with VP6 recombinant adenoviruses via either oral or intranasal route could induce anti-VP6 IgG antibody response. Low level IgA antibody could also be detected in mouse serum. This study showed that using adenoviral vector carrying VP6 gene could induce antibody response in mice. There is potential for the development of rotavirus vaccine.
Subjects
腺病毒載體
輪狀病毒
VP6
NSP4
疫苗
Adenoviral Vector
Vaccine
Rotavirus
SDGs
Type
other