Bovine respiratory coronavirus (BRCoV) is a group 2a coronavirus expressing both hemagglutinin-esterase and spike (S) envelope glycoproteins. The S glycoprotein is a primary coronavirus virulence factor responsible for both receptor specificity and membrane fusion-mediated entry into host cells. In addition, the S glycoprotein serves as a major antigen targeted by both the cellular and humoral immune responses and, as such, is an important target for antibody-facilitated virus neutralization. The objective of this research was the design of a safe and effective vaccine against BRCoV using a prime-boost vaccination approach. This method utilized an initial DNA vaccine encoding either the soluble portion of the spike glycoprotein, or the soluble portion of the spike glycoprotein fused in-frame to bovine CD154, administered intramuscularly. The initial priming was followed 14 days later by vaccination with purified immunogenic extracellular portion of S glycoprotein alone or this portion fused in-frame to the soluble portion of the bovine CD40 ligand (CD40L; CD154). The bovine CD40L was included to enhance the immunogenicity of the S glycoprotein and elicit protective immune response against BRCoV infection. Both of the recombinant proteins were expressed in insect Sf9 cells via recombinant baculovirus expression and purified using affinity chromatography. The efficacy of these vaccine approaches in eliciting neutralizing antibody responses, preventing virus replication and spread and the onset of respiratory disease in cattle was then investigated in animal experimental infections. An ELISA was developed and utilized to screen 129 cattle for animals that did not have appreciable antibody titers to BRCoV. In addition, BRCoV-specific serum was obtained from one cow immunized with commercially available vaccine and high-titer anti BRCoV S-specific serum was obtained by immunization of rabbits with the S-CD154-fusion protein. As expected, animals responded to vaccination with the soluble portion of spike. Furthermore, fusion of CD154 to the soluble portion of the spike glycoprotein resulted in a pronounced increase in circulating and neutralizing serum antibody specific for the BRCoV spike glycoprotein.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-06262012-091949 |
| Date | 05 July 2012 |
| Creators | Lum, Genevieve Elizabeth |
| Contributors | Garcia, Matthew, Ph.D, Riggs, Laura M., DVM, Ph.D, Chowdhury, Shafiqul, DVM, Ph.D, Enright, Frederick, DVM, Ph.D, Kousoulas, Konstantin G., Ph. D, Bondioli, Kenneth R, Ph.D |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-06262012-091949/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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