Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: 1) 1,685 C. pecorum ORF genetic immunization constructs have been designed, oligonucleotides synthesized, and high-quality vaccine constructs produced with 100% success rate were completed in November 2006. 2) An optimized biolistic (gene gun) vaccination modality was developed for vaccination with pools of ORF vaccine constructs. To enhance Chlamydia-protective Th1 immunity, both subunit genes encoding E. coli lethal toxin (LT) were included as genetic adjuvant in all vaccinations used for expression library screening in the A/J mouse model. 3) In the first round of ELI, a library of linear expression elements (LEE) of 1,685 PCR-amplified fragments of all putative ORFs of the C. pecorum genome was constructed. Pools of 8 LEEs each were arranged in a 18x12 rectangular matrix, and were combined into 18 vertical and 12 horizontal groups. Per vaccine group, 10 female, 6-week-old A/J mice were immunized by gene gun on days 0, 3, 6, 20, and 34. All mice were intranasally challenged with 4x108 C. pecorum strain E58 organisms 4 weeks later and sacrificed after 10 days. C. pecorum lung loads were quantified by 23S rRNA FRET PCR, and a protection score was calculated relative to unprotected naive mice (0 percent protection) and optimally protected mice that had received a low-dose live C. pecorum inoculum 4 weeks prior to the high-dose challenge infection (live-vaccinated, 100 percent protection). This round identified several protective pools and allowed reduction of the original pool of 1,053 C. pecorum vaccine candidate genes (a total of 1,685 partial or full gene constructs for vaccination) to 150 genes (a total of 270 partial or full gene constructs). 8) The second round of C. pecorum ELI with 150 candidate genes identified in August 2007 a pool of 55 C. pecorum vaccine candidate genes (90 ORF fragments) among the 270 gene constructs. 4) In the third round, completed in March 2008, these 55 genes were tested in a 8x7 matrix arrangement of pools of 7 or genes. Disappointingly, none of these pools mediated full protection, while several pools mediated partial protection with low variance of the data. Further analyses suggested that this low protection was associated with a Th2 shift of the immune response. This type of immunity is non-protective against chlamydial infection. We attribute this immune shift to an inadequate immunization approach, with potential over-vaccination and insufficient antigen removal that would result in persistent antigenic stimulus leading to Th2-biased immunity. 10) In a series of experiments that are in progress, we use a proven protective gene of C. pneumoniae in a C. pneumoniae - A/J and C57BL/6J mouse challenge model to identify an optimal vaccination modality for low-complexity or single antigen genetic immunization. The preliminary data suggest that either very low genetic antigen doses or a mixed modality genetic vaccine priming followed by protein antigen boost will yield highly protective vaccination outcomes. Once these optimization experiments are completed (anticipated for April 2009), we will use the optimal approach to identify the definitive C. pecorum vaccine antigens within the pool of 55 candidates. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: 1) Analyses of experimental results suggested that low protection observed during vaccination with low-complexity pools or single genes was associated with a Th2 shift of the immune response. This type of immunity is non-protective against chlamydial infection. We attribute this immune shift to an inadequate immunization approach, with potential over-vaccination and insufficient antigen removal that would result in persistent antigenic stimulus leading to Th2-biased immunity. 2) In a series of experiments that are in progress, we use a proven protective gene of C. pneumoniae in a C. pneumoniae - A/J and C57BL/6J mouse challenge model to identify an optimal vaccination modality for low-complexity or single antigen genetic immunization. The preliminary data suggest that either very low genetic antigen doses or a mixed modality genetic vaccine priming followed by protein antigen boost will yield highly protective vaccination outcomes. Once these optimization experiments are completed (anticipated for April 2009), we will use the optimal approach to identify the definitive C. pecorum vaccine antigens within the pool of 55 candidates.
Impacts
1) Out of the total of 1,053 C. pecorum genes (a total of 1,685 partial or full gene constructs for vaccination)a pool of 55 C. pecorum vaccine candidate genes (90 ORF fragments) were identified. 2) Findings during this investigation indicate the need for re-evaluation of genetic immunization in general, and specifically biolistic immunization by the gene gun. It appears that improved technology and stability of genetic vaccine constructs has resulted in long persistence of expression plasmids at the anatomical vaccination site, resulting in a shift towards Th2 immunity that is not protectiuve against chlamydiae and other intracellular bacteria. Ongoing investigations funded from non-CSREES sources allow continuation of the project to optimize genetic immunization against chlamydiae and other intracellular pathogens. Conclusive results from this investigation are anticipated for mid-2009. This will allow identification of the definitive C. pecorum vaccine candidate genes among the present 55 candidates. 3) It is anticipated that identification of 2-3 optimal C. pecorum vaccine candidate proteins will allow combination with the already known 3 best protective proteins for C. abortus to formulate and test a highly effective vaccine aganist chlamydial infections in cattle. 4) It is further anticipated that the knowledge about mechanisms that lead to Th2 immunity after genetic immunization will allow effective manipulation of teh immune response obtained by gteentic immunization. Thsi will have a major impact on the overall field of genetic immunization.
Publications
- 1) Li, Y., Borovkov, A., A. Loskutov, S. Ahluwalia, C. Wang, D. Gao, K. F. Sykes, and B. Kaltenboeck. Identification of Chlamydophila pecorum vaccine candidate genes by expression library immunization. Proceedings of the 88th Annual Meeting of the Conference of Research Workers in Animal Disease. Chicago, Illinois, December 2007: Abstract 17.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Preliminary results were reported at the Conference of Research Workers in Animal Disease in Chicago, December 2007.
PARTICIPANTS: Yihang Li, Graduate Research Assistant, Auburn University, Auburn, AL Dongya Gao, Research Assistant III, Auburn University, Auburn, AL Bernhard Kaltenboeck, Professor, Principal Investigator, Auburn University, Auburn, AL Kathryn F. Sykes, Assistant Professor, Biodesign Institute, Arizona State University, Tempe, AZ Alexandre Borovkov, Research Assistant Professor, Biodesign Institute, Arizona State University, Tempe, AZ Andrey Loskutov, Research Assistant IV, Biodesign Institute, Arizona State University, Tempe, AZ
TARGET AUDIENCES: Researchers in Veterinary Medicine and Animal Protection.
PROJECT MODIFICATIONS: The modifications in the screening approach are driven by the need to search for stochastic combinations of 2 or more partially protective genes, since single genes do not mediate sufficeint protection.
Impacts
Beginning in December 2006, we initiated expression library immunization (ELI) in a murine respiratory disease model to identify candidates for a subunit vaccine against C. pecorum. A library of linear expression elements (LEE) of 1,685 PCR-amplified fragments of all putative ORFs of the C. pecorum genome was constructed. For selection of vaccine candidates, pools of 8 LEEs each were arranged in a 18x12 rectangular matrix, and the pools were combined into 18 vertical and 12 horizontal groups. Per such vaccine group, 10 female, 6-week-old A/J mice were immunized by gene gun vaccination on days 0, 3, 6, 20, and 34. All mice were intranasally challenged with 3.6x108 C. pecorum strain E58 organisms 4 weeks later and sacrificed after 10 days. C. pecorum lung loads were quantified by 23S rRNA FRET PCR, and a protection score was calculated relative to unprotected naive mice (0 percent protection) and optimally protected mice that had received a low-dose live C. pecorum
inoculum 4 weeks prior to the high-dose challenge infection (live-vaccinated, 100 percent protection). Intersections between several highly protective vertical and horizontal vaccine groups were further dissected in a second round of ELI in an 18x9 rectangular matrix of single genes. Only weak protection was found in round 2. This suggests that single individual C. pecorum genes are not highly protective, instead only the combination of partially protective genes mediates subunit vaccine protection comparable to that observed in live-vaccinated mice. An experiment to identify the best combination of such partially protective genes is currently in progress.
Publications
- An abstract of the report of preliminary results is available in the Proceeding of the 2007 Conference of Research Workers in Animal Disease in Chicago.
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Progress 01/01/06 to 12/31/06
Outputs
The starting date of the project is October 1, 2006, with the actual account established in January 2007. Basic organizational procedures for the project have been done until December 31, 2006. The measures include detail planning of sampling methodology on the animal and in the laboratory. Further evaluation of sampling will be performed between 1/2007-4/2007, and actual routine sampling in bi-weekly intervals will be started in May 2007. At that time cows will become exposed to heat stress, and conditions for stress-induced immunosupprossion and chlamydial mastitis will be optimal.
Impacts
Identification of C. pecorum protective antigens is required for a vaccine that will generate immunity against both species of Chlamydia routinely found in ruminants, i.e. C. pecorum and C. abortus. C. abortus protective antigens have already been identified. Once protective antigens of C. pecorum are known, proteins of both species will be produced and incorporated into an experimental vaccine that elicits Thi-biased immunity using standard methodology. Preliminary data with a whole chlamydia organism vaccine indicate that Th1-biased vaccination against chlamydiae will reduce milk somatic cell counts in dairy cows, and will potentially improve fertility.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
A large stock of highly pure C. pecorum elementary bodies, sufficient for all challenge infections, was generated. The mouse lung disease model was retested for identification of an optimal inbred mouse strain. In collaboration on an NIH-funded project on Chlamydia taxogenomics, DNA of C. pecorum type strain E58 had been purified earlier, and the genome was sequenced from this DNA by G. Myers at The Institute for Genomic Research, Rockville, MD. After the annotated genome became available in January 2005, our primer design programs designed PCR oligonucleotides to amplify all genomic coding regions as 1,637 overlapping ORFs of 1,000 bp each. A C. pecorum mammalian expression library for vaccine screening of all ORFs is underway. Purified C. pecorum DNA serves as template for PCRs, and 15-base single stranded overhangs are created at both 5-prime and 3-prime ends to accommodate the annealing of promoter and terminator DNA products. These linear expression elements will
be strategically pooled into 90 inoculating groups, each comprised of 40 ORFs, for biolistic delivery into mice. To favor stimulation of Th1-biased immunity, which is known to be important in the protective type of anti-chlamdial immune response, two plasmids encoding E. coli heat-labile enterotoxin subunits A (LTA) and B (LTB) will be included in the antigen inocula. In preliminary experiments, gene gun delivery with mutant, non-toxic LTA and LTB drove predominantly an antigen-specific strongly Th1-biased antibody response. We are currently confirming this effect with Chlamydia gene immunization in A/J mice and optimizing the C. pecorum intranasal challenge dose in an A/J challenge model. We anticipate starting the ELI screen of the whole C. pecorum genome in early 2006.
Impacts
Identification of C. pecorum protective antigens is required for a vaccine that will generate immunity against both species of Chlamydia routinely found in ruminants, i.e. C. pecorum and C. abortus. C. abortus protective antigens have already been identified. Once protective antigens of C. pecorum are known, proteins of both species will be produced and incorporated into an experimental vaccine that elicits Thi-biased immunity using standard methodology. Preliminary data with a whole chlamydia organism vaccine indicate that Th1-biased vaccination against chlamydiae will reduce milk somatic cell counts in dairy cows, and will potentially improve fertility.
Publications
- No publications reported this period
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