| Group A rotaviruses (ARV) are the single most significant cause of severe dehydrating diarrhea in infants and young children in both developed and developing countries worldwide. In the developing countries, the problem seems even more serious and urgent, an estimated 18 million cases of moderately severe diarrhea and over 600,000 children died of this illness yearly. Because of the high morbidity and mortality associated with rotavirus diarrhea, it is apparently urgent need to develop some more effective and safe rotavirus vaccines to prevent rotavirus infections.In the recent years, adenoviruses have been used to make recombinant genetic engineering vaccines, and the potential and perspective seem to be rather encouraging. In our previous work, we have found that mice immunized either intranasally or orally with the adeno-based rotavirus recombinants could expressing human ARV protein VP6 and VP7, initiating a high level of systemic immune response against rotavirus infection.For designing an acceptable vaccine, safety is always the paramount significance of any other criteria to be considered, it is particularly true when develop a vaccine for use in infants and young children. The present study was carried out based on our previous work, trying to optimize the codons of the recombinant virus aiming to appropriately elevate the expressing level of the encoded human rotavirus protein (VP6 and VP7), the designed titer of the expressed proteins should be the titer that could evoke a sufficient protective immunity, safe and acceptable to children. The optimized codons of the gene VP6 and VP7were artificially synthesized and inserted into the adenovirus backbone plasmid, the plasmid then transfected into 293 cells for generating mature viral particles. Four recombinants were successfully constructed and designated as: rvAdVP6(o), rvAdG1VP7(o), rvAdG2VP7(o) and rvAdG3VP7(o). The expression level of the optimized and wild type VP6 and VP7 gene, as well as the subsequently induced immune responses in mice were identified and evaluated. The main results of the experiments are summarized as follows:1. Improved expression of human rotavirus antigens in the recombinant adenoviruses by codon optimizationIt has been known that codon optimization can raise the expression level of genes of many viral proteins. In the present study, we have artificially synthesized four genes of group A human rotavirus that encode VP6, G1VP7, G2VP7 and G3VP7 according to the human biased codon. The modified genes were transfected into 293 cells using adenovirus vectors and the gene products, the respective proteins were produced. The expression level of each gene was detected by Western Blot. The results showed a remarkable increase of the expression level in comparison with the wild type control. For evaluating the genetic stability of the optimized genes, the four 293 cell lines with non-replicating adenoviruses bearing the genes of VP6, G1VP7, G2VP7 and G3VP7 were continuously propagated up to 20 passages. The results demonstrated a steady stability of the cells in the passage, namely, the recombinant adenoviruses remained genetically stable and kept a potent and high level of expression ability. What is more, the unwanted Replication-Competent Adenoviruses were not found at least within the first 10 passages for all the four recombinants. The above results indicate that our recombinant adenoviruses are potentially applicable for further development and improvement of the adenovirus-vectored vaccines.2. Optimization of the codon strengthened the immune responses in mice model6～8-week female BALB/c mice were randomly grouped and immunized intranasally with 10~8 TCID50 rvAdVP6(o) and rvAdVP6, respectively. After the first immunization, the mice were boosted twice with a 14-day interval. The immunization results were recorded as follows:(a) Both adenoviruses with optimized and wild type genes of RV could generate high-level of serum IgG against rotavirus. However, the serum antibody level induced by rvAdVP6(o) was much higher than that of rvAdVP6.(b) Murine mucosal immunity was induced after intranasal administration of the both optimized and wild type recombinant adenoviruses. whereas the higher-level of sIgG and sIgA was only detected from the lung lavage, lung and intestinal homogenates and feces in rvAdVP6(o) immunized mice.(c) Cell mediated immunity was observed in rvAdVP6(o) administered mice and in rvAdVP6 administered mice. But the rvAdVP6(o) could induce a stronger cell mediated immune response in comparison with the rvAdVP6 as seen in the ELISpot results in which much more spots appeared signifying the higher level of cell mediated immune response.(d) The immunized mice shed significantly lower amount of viral antigens in feces as compared with the control group inoculated with an empty vector rvAdpSC. Also, the viral antigen shed from mice immunized with rvAdVP6(o) was lower than that immunized with rvAdVP6.In summary, the recombinant adenoviruses which encode optimized human rotavirus VP6 proteins (rvAdVP6(o)) could induce stronger immune and protective responses against the challenge of the rotavirus than the wild type(rvAdVP6) at the same immunizing dosage.3. Bio-distribution and persistence of VP6 gene expressed recombinant adenovirus in mice modelFor safe application as the oral vaccine, it is necessary to investigate the bio-distribution of the recombinant adenovirus after oral administration. Seven groups of mice (ten adult female mice per time point) were orally inoculated with 109 TCID50 rvAdVP6(o). Control group was given phosphate buffered saline (PBS) instead. Fourteen kinds of tissues were harvested at each time point (4h, 12h, 1d, 4d, 7d, 14d, 28d), including brain, tongue, trachea, esophagus, lung, liver, spleen, stomach, kidney, large intestine, small intestine, heart, ovary and Peyer's patchs. Immunohistochemistry was used to determine the histological localization of the recombinant adenoviruses and the expression of VP6 protein in the immunized mice. The results showed that the endothelial cells in large intestine were positively stained at 4 hours post-infection. Meanwhile, Real-time PCR was used to quantify recombinant adenoviruses and VP6 gene copies in the different organs of the immunized mice. The result showed that 4 hours after oral vaccination, the recombinant adenoviruses and VP6 gene could be detected in large intestine, stomach, small intestine, esophagus and Peyer's patchs in most of the mice. After12 hours, copy numbers of the recombinant adenoviruses and VP6 gene were reduced significantly. while they could still be detected in large intestine, stomach, small intestine, esophagus, but could not be detected in Peyer's patchs. By day 1, the recombinant adenoviruses and VP6 sequence could only be detected in large intestine and esophagus. And by day 4 and 7, the recombinant adenoviruses could still be detected in large intestine, esophagus and stomach. At day 14, the recombinant adenoviruses could only be detected in esophagus. This result indicated that the recombinant adenoviruses and VP6 gene had widely distributed and durably expressed in vivo.In conclusion, four recombinant adenoviruses encoding the optimized human rotavirus antigens were successfully constructed, the expression of optimized genes significantly increased in comparison with the wild type RV genes. Immune responses in mice induced by optimized recombinant adenovirus also showed a higher level than the wide type ones. The Bio-distribution and persistence were detected upon oral application in mice. The result, therefore, provide an important clue and experimental support for the development of rotavirus genetic engineering vaccine against rotavirus infection. |
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