Progress 06/01/23 to 05/31/24
Outputs
Target Audience:Academic and industrial researchers involved in pathogenic microbiology and vaccinology. Poultry producers Changes/Problems:No changes or problems to report. What opportunities for training and professional development has the project provided?This project has provided invaluable training to the graduate student conducting the majority other work as well as the undergraduate students (non-paid) in the lab. How have the results been disseminated to communities of interest?Our data was disseminated in the following key presentations this past year: Geary S. J. "Mycoplasma gallisepticum Subunit Vaccine Development". Invited speaker. The University of Melbourne Veterinary School (UniMelb), Melbourne, Australia. 6/1/23. Jeremy M. Miller, Rosemary G. Ozyck, Lawrence K. Silbart, Jessica B. Malek, Edan R. Tulman, Steven M. Szczepanek, Steven J. Geary. "Advancements in the Development of a Safe and Efficacious Subunit Vaccine Against Mycoplasma gallisepticum". 7/23 at the International Organization of Mycoplasmolgy Congress (IOM) Osaka, Japan Geary S. J. "Connecting the Dots, Cutting Edge Research and the Field". Keynote speaker. The Third Internatioanl Avian Mycoplasma Conference, Gran Canaria, Spain 7/13/24. What do you plan to do during the next reporting period to accomplish the goals?In the next funding year, we plan to: -Conduct vaccination/challenge experiments to assess the contribution of each of the subunit components individually to more thoroughly assess the contribution of each. -Compare our vaccine to the commercial bacterin MGBac. - We have obtained a licensed salmonella vector from Dr. Roy Curtis and have designed the sequences of the subunits to be inserted into this vector. We will complete and verify our salmonella vectored vaccine construct and assess its efficacy in vaccination/challenge experiments. If successful, this should make our vaccine more affordable for commercial use.
Impacts
What was accomplished under these goals?
We incorporation a total of seven different adjuvants (separately) into our vaccine construct and evaluated their efficacies. We found that different adjuvants resulted in variable levels of protection as determined by viable MG load and pathogenicity based on tracheal thickness. Only the CpG oligodeoxynucleotide (CpG ODN 2007) adjuvant resulted in statistically significant reductions of bacterial recovery and tracheal thickness measures. We conducted extensive immunological assessments to evaluate the contribution of each component of our vaccine. We found that all components were recognized by the chicken immune system and contributed to the overall protection observed. Overall, our subunit vaccine demonstrates the benefit of using verified MG attachment proteins in conjunction with the primary expressed phase variable vlhA proteins to develop an efficacious vaccine that combats MG disease. We also conducted a vaccination/challenge experiment with our vaccine construct and challenged it with a heterologous pathogenic MG strain (VA94). We found that it protected against this strain as well.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2024
Citation:
NPJ Vaccine. Revised manuscript submitted and awaiting final decision.Based on the reviewer's comments it looks very likely to be accepted soon.
Jeremy M. Miller, Rosemary Grace Ozyck, Patrick L. Pagano, Esmeralda F. Hernandez, Megan E. Davis, Anton Q. Karam, Jessica B. Malek, Arlind Mara, Edan R. Tulman, Steven M. Szczepanek, Steven J. Geary*
Corresponding Author:
Correspondence to Steven J. Geary.
Title:
Rationally Designed Mycoplasma gallisepticum Vaccine Using a Recombinant Subunit Approach
Abstract:
Mycoplasma gallisepticum (MG) is an avian respiratory pathogen causing significant global economic losses to the chicken and turkey industries. Currently available vaccines to combat MG are either live-attenuated or bacterins. Although current vaccines provide measures of protective immunity, they still have drawbacks. Live-attenuated MG vaccines have potential for reversion to virulence and some are virulent in young chickens. Bacterins provide incomplete protection. Both live-attenuated vaccine and bacterins present difficulty in distinguishing vaccinated from naturally infected animals. Based on our knowledge of MG adherence and virulence, we rationally designed and developed a subunit vaccine consisting of the primary adhesin GapA, the cytadhesin-related molecule CrmA, and four early-phase-expressed Variable Lipoprotein Hemagglutinins (VlhAs) (3.03, 3.06, 4.07, 5.05) of the virulent strain Rlow. Recombinantly produced proteins were administered at a dose of 50 �g per protein utilizing a prime-boost schedule with three weeks between doses to compare adjuvant formulations in multiple studies. Chickens were challenged with the virulent strain Rlow. Incorporation of different adjuvants resulted in variable levels of protection as determined by viable MG load and pathogenicity based on tracheal thickness. Only the CpG oligodeoxynucleotide (CpG ODN 2007) adjuvant resulted in statistically significant reductions of bacterial recovery and tracheal thickness measures. Overall, our subunit vaccine demonstrates the benefit of using verified MG attachment proteins in conjunction with the primary expressed phase variable vlhA proteins to develop an efficacious vaccine that combats MG disease. Our vaccine maintains a high safety profile with no possibility of reversion to virulence. It is also amenable to rapid modification via the incorporation of variant VlhAs, other virulence factors from emerging strains, or other components of significant avian pathogens, such as Mycoplasma synoviae.
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:Vaccine researchers, both academic and industrial. Changes/Problems:No changes or problems What opportunities for training and professional development has the project provided?This project has provided invaluable training to the graduate student conducting the majority other work as well as the other students (non-paid) in the lab. How have the results been disseminated to communities of interest?Jeremy Miller (Grad student) presented this work at the Colleg of Ag Grad Student Forum in March of 2023. GEARY SJ Invited seminar at the University of Melbourne Veterinary School, Melbourne Australia: "Mycoplasma gallisepticum Subunit Vaccine Development". Geary SJ. "Rationally Designed Mycoplasma gallisepticum Subunit Vaccine". Invited speaker. University of Technology Sydney (UTS), Sydney, Australia. 3/1/23. What do you plan to do during the next reporting period to accomplish the goals?We plan to: Assess the affects of additonal adjuvants. Continue to optimize the vaccine construct, determined to be the most efficatious. Compare our vaccine to the commercial MGBac
Impacts
What was accomplished under these goals?
We have made considerable progress on developing a safe and efficacious subunit vaccine against Mycoplasma gallisepticum. We determined that a dose of 10ug per subunit in conjunction with the adjuvant Addavax was not as efficacious as the commercial vaccine MgBac. We then made considerable improvements to the production of our subunit proteins that resulted in higher purity subunits, more consistent and efficient production, and the ability to increase our dose substantially. Our dose was increased to 50ug per subunit, and we tested the influence of the various components of the vaccine. We tested GapA + CrmA vs VlhA 3.03, 3.06, 4.07, and 5.05 vs all six subunits together. All subunits were adjuvanted with Addavax. We determined that formulations including GapA and CrmA performed the best and trended toward reductions in pathology as determined by reduced tracheal thickness. The GapA + CrmA alone formulation also trended strongly toward reducing bacterial recovery. We rapidly followed up on this by looking at the influence of different adjuvants. To improve our statistical power, we increased our group sizes to 15 birds/group. We chose to move forward with the formulation including all six subunits at a dose of 50ug per subunit. After testing Montanide ISA 78VG, Addavax, and Alum, it was found that Alum was the most promising adjuvant tested. It significantly reduced bacterial recoveries and trended strongly toward reducing pathology. Based on this success, we took an ambitious approach of testing another four adjuvants across two different studies. The first study is evaluating the influence of the adjuvants MPLA (TLR4) and CpG ODNs (TLR21). Combinations of TLR agonists and other adjuvants such as Alum are commonly used to further increase immune responses beyond that of either alone. This is a strategy employed in the human adjuvant systems (e.g. AS04 contains Alum and MPLA). The second TLR agonist study is evaluating the influence of the adjuvants Pam2CSK4 (a synthetic diacylated lipopeptide) and Pam3CSK4 (a triacylated lipopeptide). In humans they are agonists to TLR 2/6 and TLR 2/1 respectively. However, chickens do not have TLR 6 and both synthetic lipopeptides are believed to act through highly similar TLR1/6/10-like molecules. Understanding the specific influence of these TLR agonists in a vaccine context is especially relevant to Mycoplasma infections given that unlike most bacteria, some Mycoplasmas have both diacylated and triacylated lipopeptides. Our studies have all meaningfully contributed to furthering optimization and development of this promising vaccine candidate.
Publications
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