Location: Ruminant Diseases and Immunology Research
Project Number: 5030-32000-236-002-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2022
End Date: Jul 31, 2024
1. Identify virulence determinants of Mycoplasma mycoides. 2. Following identification of virulence determinants, utilize synthetic genome and other approaches to engineer Mycoplasma mycoides cluster agents for enhancing the understanding of disease pathogenesis and for use as potential vaccines. 3. Determine the role of surface lipoproteins for vaccine enhancement of disease in Mycoplasma mycoides subsp. mycoides small colony (Mmmsc).
Obj 1. a) Mmmsc mutants will be generated by transposon mutagenesis targeting the genes of the pathway operon involved in transport and utilization of glycerol. All mutants will be screened for their abilities to produce H2O2 and their cytotoxic capabilities in-vitro when grown in glycerol. Those found to be deficient in these activities will be used to challenge adult cows, with wild-type virulent progenitor Mmmsc serving as the positive control. If H2O2 is determined to be an actual virulence factor, further analysis will be performed. b) Comparative genomic analysis using archived and newly obtained Mmmsc field strains to identify virulence determinants suitable for deletion or as vaccine targets. c) Virulence determinants will also be identified by RNAseq experiments over a two week period of exposure to Mmmsc. This will also provide us with additional host response data critical for vaccine design. Obj 2. a) Use the JCVI (J. Craig Venter Institute) synthetic genome approach on Mmmsc. We plan to engineer Mmmsc using genome transplantation technology once we have identified virulence targets for deletion. b) Subunit vaccine constructs using identified virulence determinants, assessing various quantities and combinations, with various adjuvant formulations Obj 3. Cows will be vaccinated with a preparation of lipid associated membrane proteins (LAMPs) from Mmmsc, delipidated LAMPs (dLAMPs) or sterile saline, following a prime-boost regimen (dose, number of booster shots, timing of shots, adjuvants, and route of administration will all be empirically optimized). Serum will be collected prior to vaccination, two days and two weeks after each vaccination to determine antigen-specific antibody titers and cytokine profiles. After vaccination, cows will be challenged with virulent Mmmsc. Mycoplasma load will be determined, tissues will be harvested for histopathology, and blood will be analyzed for antibody/cytokine profiles. Continuous data (i.e. cytokine concentrations in serum) will be analyzed using parametric statistical analyses, whereas discontinuous data will be analyzed using non-parametric tests. Groups of two will be analyzed with a t-test and groups of 3 or more will be analyzed by ANOVA with an appropriate post- hoc test. Power analysis on small pilot studies will be used to help determine the appropriate number of animals for each experiment based on the variance observed.