ARS | Publication request: Profiling Host Antibody Responses to Mycobacterium avium subspecies paratuberculosis Infection Using Protein Arrays

Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 14, 2007
Publication Date: November 26, 2007
Citation: Bannantine, J.P., Paustian, M., Waters, W.R., Stabel, J.R., Palmer, M.V., Li, L., Kapur, V. 2007. Profiling Host Antibody Responses to Mycobacterium avium subspecies paratuberculosis Infection Using Protein Arrays. Infection and Immunity. 76(2):739-749.

Interpretive Summary: From the annotation of the Mycobacterium paratuberculosis genome sequence, we are now able to look at all of the genes encoded by this bacterium that causes Johne’s disease. This annotation also gives us some very preliminary information about each gene. We carefully selected a set of 93 genes from this genome to clone and express in Escherichia coli. With the proteins encoded by these 93 genes now in hand, we developed a protein array on nitrocellulose. The protein array was exposed to sera from Johne’s disease cattle and sera from healthy cattle. Dominant antigens emerged from these studies. Also, cross-reactive antigens were identified when the array was exposed to sera from cattle infected with mycobacteria other than M. paratuberculosis. These studies have yielded excellent candidate antigens for use in a subunit vaccine or for development of an antigen-based diagnostic test.

Technical Abstract: Along with the complete genome sequence of Mycobacterium avium subspecies paratuberculosis, technologies are now developed for the construction of protein arrays to detect the presence of antibodies against M. avium subsp paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These include unknown hypothetical proteins, cell surface proteins as well as proteins encoded on large sequence polymorphisms present uniquely in M. avium subsp paratuberculosis. Also included are previously reported or known M. avium subsp paratuberculosis antigens to serve as a frame of reference. The array was exposed to sera from M. avium subsp paratuberculosis immunized mice and a rabbit to identify immunodominant antigens in those laboratory animals. Furthermore, sera from healthy, experimentally infected, and clinical cattle were probed with the array to identify antigens in the context of Johne’s disease stages. Distinct sets of antigens emerged when data were compared between Johne’s disease animals versus that of immunized mice; however, the antibody profile for infected rabbits closely mimicked that of cattle, indicating that this host may serve as a model for the cattle humoral response to M. avium subsp paratuberculosis. Cross-reactive proteins were identified by exposing the array to sera from M. bovis and M. avium-infected cows. These data show a higher degree of cross reactivity with M. avium than with the more distantly related M. bovis. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp paratuberculosis. These results provide a basis for understanding humoral immune responses in hosts exposed to M. avium subsp paratuberculosis as well as open new frontiers in vaccine and diagnostic development.