The Preliminary Study Of Immune Effects Of AD41-Based Rotavirus VP6Recombinant Adenovirus

Group A rotaviruse(Group A Rotavirus, ARV) is the primary pathogenic cause of severe diarrhea in infants and young children under the age of five in the world. There are more than527.000people dying from rotavirus infection, and more than two million children are seriously ill each year worldwide. Considering the serious consequence of rotavirus infection and lack of effective treatment, the WHO has taken the development of rotavirus vaccine as the preferential project.Inoculation of RV vaccine was known as the best method to decrease the rate of RV infection, and oral immunization is the optimal way. The adenovirus vector is the most widely used in gene therapy and recombinant vaccine currently. Adenovirus can be propagated in respiratory tract and intestine, and it can induce general immunity and local mucosal immunity. In addition, adenovirus can be inoculated by oral or intranasal administration, without fever or other effects, and it is safe and reliable when used to inoculate the infant. Therefore, the future of using adenovirus as the vector to prepare the engineering vaccine of rotavirus is bright and challenging. Our group has successfully expressd the antigen of rotavirus using adenovirus vector, and we have done many systemic researches on the genetic engineering vaccine of rotavirus. Our previous work demonstrated that mice immunized intranasally with the Ad5-based rotavirus recombinants could express human ARV protein, initiating a high level of systemic immune response against rotavirus infection. In addition, oral route is recognized as the best immune approach of RV vaccine. The oral administration is the simplest way and there is security risk in intranasal immunization (viruses may invade the brain). Therefore, it is important to explore the recombinant rotavirus vaccine by oral administration.Human adenovirus type41is a member of group F virus, and it is the natural pathogen of gastrointestinal tract. The majority of the whole genome of Ad41is similar to other group adenoviruses except a small number of ORFs, and it has great potential to become the ideal gene transfer vector for oral administration as its tropism to gastrointestinal tract. Our previous work showed that the acid tolerance of Ad41was better than that of Ad5in vitro. Therefore, we can expect a better immune response against rotavirus if model animals are immunized orally with the Ad41--based rotavirus recombinants instead of Ad5-based ones.Recently, a new type of recombinant Ad41vector with El region deleted, and a HAdV-41E1B55K-expressing cell line,293TE7, were established in our lab. Here, we attempted to construct the infectious clone of HAdV-41using this cell line, which we hope would facilitate the virological study of HAdV-41using reverse genetics methods. We also tried to prepare an Ad41-based rotavirus VP6non-replicating recombinant adenovirus which could stably express VP6protein, and explore the feasibility of intestinal adenoviral Ad41as vaccine vector of diarrhea virus, the favorable evidence for Ad41as an oral vaccine vector also will be elucidated in this study.1Construction of an infectious clone of human adenovirus type41Objective:Human adenovirus type41(HAdV-41) is well-known for its non-cultivable property, to construct an infectious clone of HAdV-41for further study of the HAdV-41and Ad41vector system. Methods and Results:Firstly, the DNA fragment, which contained the left and right ends of HAdV-41as well as Kanamycin-resistant gene and pBR322replication origin, was excised from previously construct -ed plasmid pAd41-GFP. Homologous recombination was executed to generate pKAd41by co-transform E.coli BJ5183strain with this fragment and HAdV-41genomic DNA. Virus was rescued from pKAd41-transfected293TE7, a HAdV-41E1B55K-expressing cell line. Genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two Fibers on the virion was confirmed by Western blot. Silver stain confirmed that HAdV-41has a relatively complete protein fraction in the protein level. Immunofluorescence showed that more expression of Hexon could be found in293TE7rather than in293cells after HAdV-41infection. Non-lytic replication of HAdV-41was reserved in293TE7cells since very few progeny viruses were released to the culture medium.293TE7cells were infected with different MOI of HAdV-41respectively, then cells were observed after infection for1d to13d, and the results confirmed that there was no emergence of plaque in the cells. Conclusion:These results illustrated pKAd41was an effective infectious clone, and suggested that pKAd41and293TE7cells combined to be an ideal system for virological study of HAdV-41.2The preliminary study of immune effects of oral administration of Ad41-based rotavirus VP6recombinant adenovirusObjective:To explore the feasibility of adenovirus type41(Ad41) as a vaccine vector for the rotavirus. We prepared and identified the Ad41-based rotavirus VP6non-replicating recombinant adenovirus, and then observed the mucosal, humoral and cellular immune responses and protection in mouse model. Methods and Results:In this research, recombinant Ad41carrying the VP6gene of human rotavirus (rvAd41-VP6(o)) was successfully constructed and identified by Electron microscopy, PCR, RT-PCR and Western-Blot and other methods. Observation by Electron microscope showed that rvAd41-VP6(o) had typical morphological features of icosahedral adenovirus. Genomic integrity of rvAd41-VP6(o) was verified by restriction analysis. PCR and RT-PCR revealed that, there was stable integration and transcription of specific VP6gene in rvAd41-VP6(o), and it had preferable genetic stability. Western blot confirmed that rvAd41-VP6(o) could stably expressed the VP6 protein. Two Fibers on the virion were confirmed by Western blot. One-step growth curve showed that non-lytic replication of HAdV-41was reserved in293TE7cells since very few progeny viruses were released to the culture medium. In our previous work, we found that the tolerance of recombinant adenovirus to the acid in vitro was higher than Ad5-based ones(viral viability:PH=1,20min:Ad4110%, Ad51%; PH=2,30min:Ad41100%, Ad51%). Moreover,6-8-week female BALB/c mice were randomly grouped and immunized intranasally with1010VP rvAd41-VP6(o) and rvAd5-VP6(o), respectively. The results were recorded as follows:after the same dosage oral vaccination of rvAd41-VP6(o) and rvAd5-VP6(o), the serum IgG level against rotavirus induced by rvAd41-VP6(o)(100%,10/10; OD:1.0300±0.1984) was higher than that of rvAd5-VP6(o)(30%,3/10;0.3428±0.2760) after three times of immunization by ELISA detection; The immunized mice shed lower amount of viral antigens in feces as compared with the rvAd5-VP6(o); Compared to rvAd5-VP6(o), rvAd41-VP6(o) induced stronger mucosal immune response by detection of the intestinal IgA level through ELISA; However, the level of cellular immunity did not get statistically significant results by detection of ELISpot and CBA. Conclusion:We have successfully prepared the Ad41-based rotavirus VP6recombinant adenoviruses rvAd41-VP6(o), which have favorable biological properties in vitro; The results of animal experiment suggested that intestinal adenovirus vector Ad41as a vaccine vector for diarrhea virus was more suitable than Ad5.In conclusion, adenovirus HAdV-41infectious clone pKAd41was successfully constructed, and293TE7-HAdV-41system was established, which laid the foundation for further study of the biological traits and function of HAdV-41. This study also successfully prepared Ad41-based rotavirus VP6recombinant adenovirus rvAd41--VP6(o) which could stably express VP6protein, and confirmed that the intestinal adenoviral Ad41is appropriate as gene-transfer vector for intestinal administration and Ad41as a vector for diarrhea virus vaccines was much better than Ad5.