Progress 05/15/07 to 01/14/09
Outputs
OUTPUTS: Activities revolving around experimentation towards the goals of the project were the main output of this work. These activities included cloning and development of an inducible expression vector system to be used in Flavobacterium. These activities also included methods development for the introduction of E. coli derived plasmid material in to Flavobacterium, and attempts to overcome the native Flavobacterium restriction barrier. PARTICIPANTS: PARTICIPANTS AT VPI Vito G. DelVecchio, Ph.D.,. Principal Investigator: Dr. DelVecchio was the PI on this project. He oversaw the scientific strategy of the project and was integral to the implementation of planned activities, resolution of unexpected outcomes and implementation of new strategies as problems and issues arose. Tim Alefantis, Ph.D., M.B.A.: Dr. Alefantis directed the day-to-day scientific activities of the project and troubleshooting of project problems. He also coordinated efforts and discussions with outside collaborators. Clarissa Dake, Laboratory Technician: Ms. Dake performed day-to-day scientific activities including molecular biology and microbiology experiments. Ashley Kolinovsky, Laboratory Technician: Ms. Kolinovsky performed day-to-day scientific activities including molecular biology and microbiology experiments. Jessica Trichillo, Laboratory Manager: Ms. Trichillo performed day-to-day scientific activities including strain culturing, molecular biology and microbiology. PARTICIPANTS AT THE UNIVERSITY OF IDAHO Ken Cain, Ph.D.: Dr. Cain provided several strains of pathogenic Flavobacterium psychrophilum for use in this project. He also sponsored IACUC application and approval for proposed Phase I animal studies and collaborative support for the project. Dr. Cain was slated to perform animal studies under this project, however the project did not progress to the expected point at which animal studies would be performed. PARTICIPANTS AT THE UNIVERSITY OF MICHIGAN Mark McBride, Ph.D.: Dr. McBride provided the pCP29 plasmid for use in this project along with advice on general Flavobacterium genetics and molecular biology. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: One single major problem caused a delay and significant impact on the course of the project. F. psychrophilum contains a restriction modification system that was incompatible with E. coli derived plasmids. Since VPI performed all molecular biological work in E. coli it was critical to overcome the restriction barrier and introduce E. coli derived plasmids into F. psychrophilum. However, although significant efforts were undertaken, and utilized all remaining time and funds available, VPI was not able to successfully introduce plasmid into several F. psychrophilum strains. This halted all subsequent work on the proposed tasks. Although work never progressed to bacterial inactivation strategies, VPI also intended to use its improbed bacterial inactivation platform called the Gene-Mediated Inactivation Vaccine (GeMI-Vax) platform. Both the intended inactivation platform and GeMI-Vax are methods of killing bacteria by expression of proteins from a plasmid and, as such are inherently similar. VPI had moved other non-USDA projects to GeMI-Vax, which VPI felt was superior to the proposed method, and wanted to align the USDA project with its overall vaccine strategy.
Impacts
The objective of this research was to develop an inactivated bacterial vaccine to prevent Flavobacterium psychrophilum induced disease. VPI established ties with Dr. Kenneth Cain of the University of Idaho to provide F. psychrophilum and perform immunogenicity and efficacy studies in trout. Three strains of F. psychrophilum were received from Dr. Cain. Then development of an inducible promoter system was initiated. VPI requested and received the pCP29 E. coli - F. psychrophilum shuttle plasmid from Dr. Mark McBride. In order to give the project the best chance of success, VPI planned to utilize its Gene-Mediated Inactivated Vaccine (GeMI-Vax) strategy for genetic inactivation of F. psychrophilum. However, use of this vaccine platform, and any platform based on expression of a killing protein, required strict and tight control of an inducible promoter system to prevent premature expression of the killing protein. VPI designed and constructed a putative IPTG inducible expression system (in pCP29) for F. psychrophilum using a strong Flavobacterium promoter (atpD gene promoter) with an inserted lac protein binding site. VPI cloned the putative IPTG inducible promoter into the pCP29 plasmid (then called pCP29-atpD) and also made two additional constructs with green fluorescent protein cloned behind the atpD promoter and the same construct also containing the lacIq gene behind the pCP29 ORF1 constitutive promoter. Completion of these constructs marked the first known attempt to produce an IPTG inducible plasmid system for Flavobacterium which could be used by many investigators for research purposes. VPI then attempted to use these constructs in functional analyses in F.p. by introduction of the E. coli derived plasmids into F.p. F. psychrophilum contains an incompatible restriction modification system with E. coli and VPI spent a great deal of effort was put forth trying to develop an electroporation procedure in order to facilitate transformation. However, none of these experiments were successful and absorbed a great deal of project time. As a final strategy conjugation was attempted with the assistance of Dr. McBride. Unfortunately, after multiple attempts, use of additional Flavobacterium strains and advice received from Dr. McBride, this process was also unsuccessful. Most of these experiments were performed during the Phase I requested extension period. During the conjugation studies VPI knew that much more work would be needed to overcome the restriction barrier and that it was unlikely animal studies would be possible in Phase I. Thus, VPI decided to postpone the Phase I immunogenicity and efficacy studies. After informing Dr. Cain of our decision Dr. Cain terminated our collaboration and requested VPI to return the FP53 and FP120 strains which VPI agreed to and did. VPI did have full intentions on collaborating with Dr. Cain in animal immunogenicity and efficacy studies once the restriction barrier issue was resolved and F. psychrophilum vaccine was produced. At that point the requested NCE was on the verge of expiring and VPI had exhausted the remaining Phase I funds working on a resolution to the restriction barrier issue.
Publications
- No publications reported this period
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