Molecular characterization and humoral immunity of bovine rotavirus VP4, VP6, and VP7
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The studies described in this thesis include the production and characterization of three bovine rotavirus structural proteins, VP4, VP6 and VP7, which are very important in eliciting protective immune responses in convalescent animals. To understand these proteins at a molecular level, and the role of individual proteins in immunity, rotavirus VP4, VP6 and VP7 proteins were expressed by recombinant vaccinia virus and baculovirus. The evaluation of the immunogenicity of the recombinant proteins may aid in the development of more effective subunit vaccines to prevent and control rotavirus infection. The recombinant forms of VP4, VP6 and VP7 expressed by recombinant vaccinia virus and baculovirus were analyzed by immunoprecipitations and western blotting. These in vitro characterizations revealed that the different recombinant forms of VP4 are similar in size and antigenicity to authentic BRV VP4. I have shown that: (1) the recombinant VP4 protein produced in mammalian cells or insect cells had the same molecular weight as the authentic protein; (2) the trypsin cleavage patterns of recombinant VP4 were similar to the pattern produced by trypsin treatment of authentic VP4, and; (3) recombinant VP4 was stable in BSC-1 and Sf9 cells, while recombinant VP4 expressed by baculovirus was not stable during post-translational processing. The reason for this instability has been found. There is a proteolytic agent in infected Sf9 cells which cleaves the VP4 protein. The characterization of recombinant VP6 demonstrated that its configuration closely mimicked the authentic VP6 protein. It was shown that: (1) the recombinant VP6 protein produced in mammalian cells or insect cells had the same molecular weight and antigenicity as authentic BRV VP6; (2) recombinant VP6 is not expressed on the cell surface but in the cytoplasm of infected cells, and; (3) recombinant VP6 protein could be assembled into single shelled particles in vitro in a fashion identical to authentic VP6 protein. Characterization of recombinant VP7 produced by vaccinia virus revealed that it was similar to authentic VP7. However, VP7 expressed by recombinant baculovirus was similar to the native form of the primary translation product but differed in its post-translational modification. The differences are: (1) the molecular weight of VP7 expressed by recombinant baculovirus was lower than the molecular weight of authentic VP7 or VP7 expressed by recombinant vaccinia virus, and; (2) VP7 expressed by recombinant baculovirus was secreted into the medium, which is different from VP7 expressed by rotavirus and recombinant vaccinia virus. However. when tunicamycin was used to inhibit glycosylation, the VP7 expressed by recombinant baculovirus was of the same molecular weight as VP7 expressed by rotavirus or recombinant vaccinia virus and none were secreted into the medium. This also suggested that VP7 expressed by recombinant vaccinia virus and baculovirus were decorated with N-linked oligosaccharides, as is authentic VP7. The antigenicity of recombinant VP4, VP6 and VP7 and their ability to induce protective immune responses were analyzed by immunizing mice with live recombinant vaccinia virus or recombinant proteins produced by baculovirus mixed with adjuvant. ELISA results indicate that antibodies raised in mice which recognize rotavirus after two immunizations. Antibodies in the milk were also analyzed by ELISA. The results indicate that vaccination elicits high lacteal Ab titers. We also examined whether antibodies raised in vaccinated mice reacted with individual rotavirus proteins. The ELISA and immunoprecipitation results indicate that antibody induced by the recombinant viruses were specific to the BRV protein expressed by the specific vaccine viruses. Protection of neonates from rotavirus infection was tested by challenging mice suckling from vaccinated dams. The results indicate that protection from rotavirus diarrhea occurred in neonates nursed by dams immunized with recombinant vaccinia virus expressing VP7, a mixture of recombinant vaccinia viruses expressing VP4, VP6 and VP7, recombinant baculovirus expressing VP7 and a mixture of recombinant baculoviruses expressing VP4, VP6 and VP7, while protection was not conferred to offspring suckling from dams immunized with recombinant baculovirus expressing VP6, wild type vaccinia virus, placebo and sentinel. In the vaccinia virus system, two promoters were used, one was a vaccinia virus 7.5 K gene promoter, the other was a vaccinia virus consensus late gene promoter. The results indicate that the consensus late gene promoter expresses significantly higher levels of protein than the 7.5 K promoter. However, the increase in the level of protein expression is dependent on the gene being expressed. For example, the consensus late gene promoter produced > 16 times the quantity of VP6 protein when compared to that produced by the 7.5 K promoter, while gene 4 (VP4) and 8 (VP7) expressed only 4-8 times the quantity of protein. Thus, both the gene and the promoter appear to influence the level of expression. In conclusion, these studies characterized recombinant BRV VP4, VP6 and VP7 proteins expressed by vaccinia virus and baculovirus and defined the immunogenicity of these proteins in dam vaccination studies. These studies may lead to the development of more effective vaccines to deliver rotavirus antigens and to prevent rotavirus infection.