Characterization of immune responses induced by plasmids encoding bovine rotavirus antigens in the murine model

by Suradhat, Sanipa

DNA immunization effectively induces humoral and cell-mediated irnmunity to numerous infectious diseases. To investigate the potential use of DNA-based vaccine for induction of bovine rotavirus (BRV) specific immune responses, I characterized the immune responses induced by plasmids encoding 2 important neutralizing antigens, VP4 and VP7, in a murine model. lmmunizationwith plasmids encoding VP4 protein did not induced detectable BRV-specific antibody responses in mice. However, it induced BRV-specific cell-mediated immune responses characterized by an increased number of BRV-specific cytokine secreting cells in the spleens of imrnunized mice. In addition, the immunized animais were primed for both antibody and cellular immune responses following BRV boost. The priming effect was lasted, for at least 9 months, after plasmid imrnunization. However, plasmids encoding VP4 did not boost the BRVspecific immune responses in the BRV-primed mice. The results from this study demonstrated that although immunization with plasmids encoding VP4 did not induce detectable BRV-specific antibody response, the immune responses induced by plasmid encoding VP4, alone or following BRV boost, were biased toward Th1 -like responses. This conclusion was based on the findings that the spleens of immunized mice contained predominantly BRV-specific 1FN-y and lgG2a secreting cells and that CD4' cells were the major source of IFN-y production in the spleens. The BRV-VP7 is also considered an important antigen for inducing protective immunity against rotaviruses. However, this study indicated that the VP7, either by Ïtself or in the context of viral particle, was poorly immunogenic in mice. Prelirninary results indicatedthat plasmids encoding VP7 were less efficient for induction of immune responses than the plasmids encoding VP4. This project was then explored several approachesto enhance the immunogenicity of the VP7 protein when expressed by the plasmid, utilizing information gathered from different areas including molecular biology, virology, and immunology. These approaches included: i) enhancing the level of gene expression by modifying the expression cassette of the plasmids; ii) changing the cellular localization of the plasmid-expressed protein; iii) CO-administration of plasmids encoding VP4 and VP7; iv) creating plasmids encoding a deletion mutant of VP7; and v) construction of plasmids encoding chimeric proteins of VP7 and complement C3d or bovine herpes virus-1 glycoprotein D (BHV-1 gD). The results from these studies demonstrated that cellular localization and nature of the VP7 antigen couid greatly influence the imrnunogenicity of the plasrnid-expressed antigen and the pattern of immune responses in immunized mice. Plasrnids encoding membrane-boundVP7 induced the best BRV-specific immune responses, foliowing BRV boost or in BRV-primedmice, when compared with the authentic and the secretory versions. In addition, the plasmid encoding a deletion mutant, truncated VP7, induced predominantly BRV-specific IL-4 production, instead of the predominantly IFN-y production previously observed following plasrnid irnmunization. Strategies that had previously been shown to enhance the immunogenicity of protein antigens were also examined. However, different outcornes were observed when these strategies were applied to plasmid immunization. First, addition of the C3d gene into the expression cassette which encoded antigen-C3d chimeras inhibited the induction of both humoral and cell-rnediated, antigen-specific immune responses. Secondly, CO-expression of the gD molecule as a gD-VP7 chimera enhanced the level iii of BRV-specific antibody responses following viral boost or in the BRV-primed mice. These results with the gD protein suggested an aiternative approach for improving the imrnunogenicityof poorly imrnunogenic antigens. However, findings in this study also suggested that the gD protein rnay have biological activities that could play a significant imrnunomodulatory role in immunized mice. In conclusion, none of the plasmids used in this study induced BRV-specific antibody responses following a pnmary immunization. However, immunologicai activity was evident following BRV administration, either pricjr to or following plasmid immunization. Each plasmid induced a distinct pattern of immune responses in both naive and BRV-pnmed mice. These investigations demonstrated specific ways that DNA immunization could be modified to modulate the outcome of BRV-specific immune responses-