Immunological characterization of potential protective proteins of Mycobacterium avium subspecies Paratuberculosis
Abstract
Mycobacterium avium subsp. paratuberculosis ( M. paratuberculosis), the causative agent of bovine paratuberculosis, is very closely related to M. avium subsp. avium ( M. avium). A new generation paratuberculosis vaccine that can confer better protection without causing any adverse reactions will greatly aid in the control of this disease in cattle. One approach to developing such a vaccine is to utilize bacterial, viral or DNA vaccine vectors known to stimulate robust cell-mediate immunity (CMI) for expression and delivery of protective protein(s) of M. paratuberculosis. In this study, we used Brucella abortus RB51 (RB51), replication-defective human adenovirus (HAd) and plasmid DNA (pSecTag) as vaccine vectors to deliver selected potential protective proteins of M. paratuberculosis. Based on review of the published literature, we selected four potential protective protein candidates of M. paratuberculosis: Fibronectin attachment protein (FAP), Antigen 85B (85B), Superoxide dismutase (SOD), and 35 kDa Major membrane protein (MMP). We tested the ability of the generated recombinant vaccines to induce antigen-specific immune responses and protection against M. avium TMC724 challenge in C57BL/6 or BALB/c mice. We first tested the ability of strain RB51 to stably express the selected proteins. Consistent expression of antigen 85B and SOD, but not FAP and MMP, was detected in the respective recombinant RB51 strains. Mice vaccinated with the recombinant RB51 strain expressing antigen 85B (RB51/85B) developed antigen-specific Th1 type CMI, but not antibodies, as determined by the production of interferon-γ, but not IL-5 and IL-4, by the in vitro stimulated splenocytes. Mice vaccinated with the recombinant RB51 strain expressing SOD (RB51/SOD) developed antigen-specific IgG2c, but not IgG1, antibodies, suggesting the development of a Th1 type immune response. However, no SOD-specific T cell responses were detected in these mice. Mice immunized with RB51/85B, but not RB51/SOD, developed protection against M. avium challenge as revealed by a significant decrease in bacterial load in their spleen. We generated two recombinant HAd that expressed antigen 85B (HAd/85B) and SOD (HAd/SOD) upon transduction of the host cells. Mice inoculated with HAd/85B and HAd/SOD developed robust antigen-specific Th1 type of immune response as determined by the IgG subisotype analyses and production of interferon-γ, but not IL-5 and IL-4, cytokines by the in vitro stimulated splenocytes. However, mice vaccinated with either HAd/85B or HAd/SOD did not exhibit increased resistance against the M. avium challenge. The protective potential of FAP and MMP were tested by constructing DNA vaccines using the eukaryotic expression plasmid pSecTag2. Immunization of BALB/c mice with the DNA vaccines expressing FAP (pSecTag/FAP) and MMP (pSecTag/MMP) induced a mixed Th1 and Th2 type of immune response and did not confer protection against M. avium challenge. Overall, these results suggest that RB51 and HAd are suitable vectors for inducing Th1 type immune responses against M. paratuberculosis proteins. Our results also indicate that not all proteins of M. paratuberculosis can be stably expressed in strain RB51 and not all expressed proteins can elicit similar extent of antigen-specific T cell responses. Nonetheless, the results of these studies support further testing of RB51/85B as a potential vaccine candidate against Johne's disease in the target ruminant hosts.
Degree
Ph.D.
Advisors
Vemulapalli, Purdue University.
Subject Area
Veterinary services
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