|ABDELLRAZEQ, GABER - Washington State University|
|ELNAGGAR, MAHMOUD - Washington State University|
|PARK, KUNTAEK - Seoul National University|
|HULUBEI, VICTORIA - Washington State University|
|FRY, LINDSAY - US Department Of Agriculture (USDA)|
|DAVIS, WILLIAM - Washington State University|
|SOUZA, CLEVERSON - Washington State University|
|BACKER, BRIAN - Washington State University|
|KHALIEL, SAMY - Washington State University|
Submitted to: Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/31/2018
Publication Date: 6/26/2018
Citation: Abdellrazeq, G.S., Elnaggar, M.M., Bannantine, J.P., Park, K.T., Souza, C.D., Backer, B., Hulubei, V., Fry, L.M., Khaliel, S.A., Torky, H.A., Schneider, D.A., Davis, W.C. 2018. A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria. Veterinary Research. 49:53. https://doi.org/10.1186/s13567-018-0549-3.
Interpretive Summary: This communication describes a novel method to evaluate vaccine candidates prior to conducting expensive animal trials for testing the usefulness of the vaccine to promote pathogen killing. We describe development of an ex vivo (outside the animal) method for assessing the bacteriocidal activity of immune cells (specifically, effector memory T cells) rapidly growing in response to stimulation with antigen processed by dendritic cells. Macrophages infected with Mycobacterium avium paratuberculosis were used as target cells. To help demonstrate killing of the mycobacteria, effector cells were overlaid with infected target cells to assist in the killing. Following 6 hr of incubation, effector cells were washed away and the macrophage processed to determine the extent of killing mediated by the effectors cells. A unique aspect of this study is that killed vs live bacteria were determined by quantitative PCR using a single copy probe. As described, propidium monoazide was used to differentiate live and dead bacteria. PMA intercalates into the DNA of dead bacteria and blocks PCR activity allowing for enumeration of live bacteria. In addition to demonstration of killing activity, we demonstrate a major component of the immune memory response is directed toward a major membrane protein on the bacteria. The results from this study show a method is now available to assess the potential efficacy of candidate vaccines ex vivo before expensive testing is conducted in animals.
Technical Abstract: A major impediment to the development of a vaccine for Mycobacterium avium paratuberculosis (Map) and other mycobacterial pathogens has been the lack of methods to fully evaluate the efficacy of candidate vaccines before testing in field trials. Testing of the proliferative response to candidate live attenuated and peptide-based vaccines ex vivo has not yielded correlative data predictive of efficacy in vivo. To address this problem, we developed a three-cell assay platform consisting of blood dendritic cell (bDC)/monocyte-derived dendritic cell (MoDC)/monocyte-derived macrophage (MoMphi) for use in the presentation of candidate vaccine antigens to autologous lymphocytes from control and vaccinated animals. A flow cytometric assay was developed to assess the proliferative response to antigenic peptides presented by MoDC pulsed with candidate vaccine antigens. A 6 hour killing assay was developed and used to demonstrate cytotoxic effector function of T cells proliferating in response to peptides presented by MoDC. Effector T cells were able to kill Map present within MoMphi. As suggested by these results, the unique co-culture platform has potential for use in assessing efficacy of candidate vaccines to Map and other pathogens ex vivo.