Characterization and autoproteolytic activity of N[superscript pro] of bovine viral diarrhea virus

http://www.worldcat.org/oclc/4155684

DNA vaccination and recombinant protein expression to identify immunogenic and protective genes from M avium subsp paratuberculosis

Paratuberculosis (Johne\u27s disease) is a chronic granulomatous infection of cattle, caused by the intracellular bacterium Mycobacterium avium subsp. paratuberculosis (referred to hereafter as M. paratuberculosis). Currently available vaccines are not effective at preventing infection or transmission of the bacterium to other animals. The objectives of this dissertation were to develop and test DNA vaccines that prevent M. paratuberculosis infection, to examine the host immune response induced by protective DNA vaccines, and to identify immunogenic proteins from M. paratuberculosis. In an effort to identify protective M. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones. Eleven clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging with live, virulent M. paratuberculosis. Four clone pools demonstrated a significant reduction (P \u3c 0.05) of M. paratuberculosis infection in mice when compared to other clone pools and non-vaccinated, infected control mice. In a second study, one of the protective clone pools was partitioned into 10 new clone arrays of 108 clones each and an immunization experiment was performed to identify protective clone arrays. When groups of mice were immunized with clone array DNA via gene gun, four clone arrays provided significant protection (P \u3c 0.05) from M. paratuberculosis challenge. Comparison of the nucleotide sequences from protective clone arrays identified 26 sequences that may have contributed to the protection observed in these mice. Additionally, protective clone arrays were found to induce IL-10 and IL-12 cytokine production, which indicated induction of both TH1 and TH2 immune responses. In a final experiment, the efficacy of the M. paratuberculosis 19 kDa lipoprotein was evaluated as an immunomodulator in cattle with Johne\u27s disease. Control, non-infected cattle did not produce antibodies or cell-mediated immune responses against the 19 kDa protein. However, the 19 kDa protein induced cellular immune responses (IFN-gamma production) in subclinically infected cattle and induced humoral immune responses (19 kDa-specific antibody production) in clinically infected cattle. Taken together, these three studies have identified a number of M. paratuberculosis antigens that may aid in the development of vaccines to prevent Johne\u27s disease