| Group A rotavirus(RV) is the most important and common cause of severe diarrhea in infants, children and infancy animal worldwide. In developing countries, rotavirus may cause an estimated 18 million cases of moderately severe and severe diarrhea and over 870 000 deaths in the young each year.In developed countries, rotavirus may resulted in substantial economical damage. For example, medical expenditure associated with rotavirus may reach 260 000 000$ each year,in the United States.Because of significant morbidity and mortality associated with rotavirus diarrhea, there is an urgent need to develop rotavirus vaccines targeted for use in early infancy.Currently, there are many developing rotavirus candidate vaccines, including attenuated vaccine, inactivated vaccine, VLPs vaccine, subunit vaccine, DNA vaccine, transgenetic plant vaccine, and so on. The immune response of these vaccines varies greatly, so there is no rotavirus vaccine available for rountine immunization at the time being. Because of several rotavirus serotype, multivalent vaccine containing neutralizing antigen is superior to monovalent vaccine. Previously, we have successfully constructed recombinant adenoviruses encoding group A rotavirus protein G1VP7, G2VP7, G3VP7 and VP6 (marked as rvAdG1VP7, rvAdG2VP7, rvAdG3VP7 and rvAdVP6), and the mice immunized with the recombinant adenovirus rvAdG1VP7, rvAdG2VP7 and rvAdG3VP7 could generate specific systemic immune response against rotavirus. RV VP6 protein is RV group-specific antigen, having strong antigenicity and immunogenicity, which can induce protective immunity. Anti-VP6 IgA mAb has been shown to protect mice from RV infection. Heterologous protection can be obtained by intramuscularly injecting VP6 DNA vaccines. Intranasal immunization of mice with VP6 protein and the mucosal adjuvant induces nearly complete protection against murine RV(strain EDIM[epizootic diarrhea of infant mice virus]) shedding for at least 1 year. In order to clarify the feasibility of adenovirus vector-based multivalent genetic engineering vaccine of RV, the present work was carried out on the basis of our previous ones, aiming to investigated the immune responses induced by the adenoviruses expressing VP6 gene of group A rotavirus. The results are following as: 1. Balb/c mice were immunized with rvAdVP6, via intranasal or oral route for 2 times, and serum IgG antibodies and the level of related cytokines were determined afterwards. The results demonstrated that the immunizing with rvAdVP6 intranally or orally could induce strong rotavirus-specific immune responses in mice, including humoral immunity and cell-mediated immunity. However, the results also implied that the primary immune responses triggered by the immunization is probably associated with the route of immunization. Th2-like response is predominant in mice immunized with rvAdVP6 via oral route, while Th1-like response is predominant in mice immunized with rvAdVP6 via intranasal route. RV-specific immune responses induced in the oral immunization group is less stronger than that of the intranasal immunization group.The cause is probably related with the inactivated or /and destroyed effect of recombinant adenovirus in the gastrointestinal tract, resulting the decrease of expression product and the attenuation of immunogenicity. 2. In order to obtain more effective multivalent RV genetic engineering vaccine, RV VP6 gene was optimized in the essay, on the basis of our previous reports, aiming to enhance its immunogenicity and immuno-protective efficacy. According to the improved PCR strategy, we redesign the cDNA sequence of rotavirus VP6 gene and its synthesis is optimized through two different methods. The results have shown that RV VP6 gene is successfully optimized through the gradual splicing method. The optimized RV VP6 gene obviously increased about 20% in (G+C)% compared to wild-type RV VP6 gene. In summary, rvAdVP6 also induced strong RV-specific immune responses, as well as rvAdG1VP7, rvAdG2VP7 and rvAdG3VP7; the optimized RV VP6 gene appeared to be potent in enhancing its immunogenicity and immuno-protective efficacy, because its (G+C)% increased about 20% than that of the wild-type RV VP6 gene. This study, therefore, laid a further strong foundation for the development of novel rotavirus genetic engineering vaccine. |
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