|Park, Kun -|
|Allen, Andrew -|
|Seo, Keun -|
|Hamilton, Mary -|
|Abdellrazeq, Gaber -|
|Rihan, Heba -|
|Grimm, Amanda -|
|Davis, William -|
Submitted to: Vaccine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 25, 2011
Publication Date: June 24, 2011
Citation: Park, K.T., Allen, A.J., Bannantine, J.P., Seo, K.S., Hamilton, M.J., Abdellrazeq, G.S., Rihan, H.M., Grimm, A., Davis, W.C. 2011. Evaluation of two mutants of Mycobacterium avium subsp. paratuberculosis as candidates for a live attenuated vaccine for Johne's disease. Vaccine. 29(29-30):4709-4719. Interpretive Summary: The focus of this study was to develop a live vaccine candidate for cattle to prevent Johne’s disease. The strategy was to create a crippled form of the bacterium that causes Johne’s disease, termed Mycobacterium avium subspecies paratuberculosis. In order to cripple the bacterium, a gene was deleted so it can no longer cause disease but it can still temporarily survive in the susceptible host, just long enough to prime a protective immune response to the bacterium. These crippled or mutant forms of the bacterium were not able to survive in macrophages, which are immune cells. The mutants were also tested in cattle and were not found in tissues, but the normal, or wild-type version, was seen in tissues. Yet a strong immune response was observed, indicating the mutant is a great vaccine candidate. This work has impact on control of Johne’s disease through vaccination approaches as well as understanding the biology of the bacterium and how the host responds to the bacterium.
Technical Abstract: Efforts to control Johne’s disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (Map), has been difficult because of a lack of an effective vaccine. To address this problem we examined the potential of targeted gene disruption as a method to develop candidate vaccines with impaired capacity to survive in vitro and in vivo for use as a vaccine. We selected relA and pknG, genes known to be important virulence factors in Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (Mbv), to explore this possibility. Deletion mutants were made in a clinical isolate of Map (K10) and its recombinant strain containing the green fluorescent protein gene (K10-GFP). Comparison of survival in an in vitro assay revealed deletion of either gene reduced survival in monocyte derived macrophages (M Phi) compared to survival of wild type K10. In contrast, survival in vivo was mainly affected by deletion of relA. The bacteria were detected in all 3 wild type infected and 3 of 5 pknG mutant infected calves by tissue culture at 3 months post infection (PI). In contrast, no bacteria were detected in any tissue sample from calves infected with the relA mutant by culture or PCR (p < 0.05). The percentage of Map culture positive tissues (9 different tissue sites from each animal) at the time of necropsy were 48.1 % (28.7 – 68.1, 95 % CI) for wild type, 24.4 % (12.9 – 39.6) for the pknG mutant, and 0 % (0 – 7.87) for the relA mutant infected calves, respectively. Flow cytometric analysis of the immune response to the wild type K10-GFP and the mutant strains showed deletion of either gene did not affect their capacity to elicit a strong proliferative response to soluble antigen extract (SAg) or live Map. Quantitative RT-PCR revealed genes encoding IFN-gamma, IL-17, IL-22, T-bet, RORC, and granulysin were up-regulated in PBMC stimulated with live Map 3 months PI compared to the response of PBMC pre-infection. The findings show deletion of relA alone may be sufficient for development of a vaccine.