Location: Infectious Bacterial Diseases Research2012 Annual Report
1a. Objectives (from AD-416):
Proposed objectives for research: 1) Evaluate cloned MAP proteins as potential vaccine candidates. 2) Evaluate genomic DNA clones as potential vaccine candidates. The use of pathogen-specific proteins in the development of new vaccines has gained interest in recent years. Subunit vaccines are favorable because they fail to induce granulomatous lesions at the site of injection that are noted with the heat-killed whole cell commercial vaccine that is currently used in the US. In addition, subunit vaccines have the potential to avoid interference with diagnostic tests that are commonly associated with use of a whole cell vaccine. Although sequencing of the genome of MAP strain K-10 was completed in 2004, only a small percentage of the over 4300 genes or gene products have been fully characterized. Data from the genome project will greatly facilitate a directed approach to identify antigens that can be used as vaccine candidates. Currently, we have available in our laboratory over 600 cloned proteins that can be evaluated in a directed fashion as potential immunogens. The proposed research will identify and characterize cloned proteins, singularly and in pools (>2), for efficacy in protection against MAP challenge in mouse and calf models. Host responses to vaccination will be assessed to provide insight into protective responses that mediate infection in the challenge models. Using expression library immunization, large numbers of bacterial genes can be evaluated to determine if they offer protection against infection. Expression library immunization proceeds through successive rounds of immunization with each round reducing the number of clones to ultimately lead to a single or select group of protective sequences. Ideally, one protective sequence will be identified but the identification of multiple protective coding regions is more likely and may yield enhanced protection over a singular clone. Previously, we demonstrated that pools consisting of 1500 cloned MAP genes were protective against MAP infection in a mouse model (Huntley et al., 2005). Further reduction within pools to 108 genes also resulted in strong protection against MAP infection (Huntley et al., 2005). The proposed research will identify and characterize cloned genes, singularly and in pools, for efficacy in protection against MAP challenge in mouse and calf models. Host responses to vaccination will be assessed to provide insight into protective responses that mediate infection in the challenge models.
1b. Approach (from AD-416):
Objective 1: Six- to 8-week old male Balb/c mice (Bar Harbor, ME) will be housed in a temperature and humidity controlled room in BSL-2 level containment with free access to water and standard mouse chow during the studies. The NADC Animal Care and Use Committee will approve all animal procedures in the studies. Mice will be randomly assigned to treatment groups (n = 10) consisting of: 1) Control noninfected (no vaccine, no MAP); 2) Control infected (no vaccine, MAP); 3) Adjuvant/ infected (Sigma adjuvant system); 4) 74F construct/infected; 5) MAP proteins/infected. Each mouse will receive 100 micrograms of protein subcutaneously, and will then be boosted with the same protein(s) 2 weeks after the initial immunization. Two weeks following the booster administration, mice will be inoculated intraperitoneally with live, virulent MAP (NADC strain 167; 108 cfu). A polyprotein construct, Map74F, has shown efficacy in reducing MAP infection in the mouse and goat model (Chen et al., 2007) and will be used as a positive control in our studies (Cornell University). Control noninfected and Control infected mice will receive sterile PBS to simulate vaccination. After 3 months of infection, mice will be anesthetized by inhalation of isofluorane and decapitated with a guillotine. Tissues (spleen, liver, mesenteric lymph node, ileum) will be removed and processed for culture on Herrold’s egg yolk medium (Becton-Dickinson). At 12 weeks of incubation viable MAP recovered from tissues will be enumerated. Portions of each spleen will be macerated for isolation of splenocytes. Splenocytes will be cultured with medium control (NS), Con A (10 micrograms/ml), MPS (10 micrograms/ml), and the appropriate protein(s) used as the vaccine. Secretion of cytokines such as IL-4, IL10, IL-12, IL-23 and IFN-gamma will be assessed on culture supernatants after 24 hr of incubation. Flow cytometric analyses will be performed after 6 days of incubation to evaluate effects of immunization on CD4, CD8, gamma delta TCR subpopulations, as well as monocytes and B cell populations. A profile of activation markers such as CD25, CD45RO, CD44, CD62L and MHCII on T cell subsets will also be determined. Objective 2: Similar to above except treatment groups will consist of: 1) Control noninfected (no vaccine, no MAP); 2) Control infected (no vaccine, MAP); 3) Vaccination with MAP gene clones (singular or pools; vaccine, MAP). Each mouse will receive 50 micrograms of pooled DNA intramuscularly, and then boosted with the same clone pool preparation 3 weeks later. Two weeks following the booster administration, mice will be inoculated intraperitoneally with live, virulent MAP (NADC clinical strain 167; 108 bacteria per mouse). Control noninfected and Control infected mice will receive sterile PBS as a sham inoculation. DNA candidates will be selected based on the 7 best clone pools from previous research in our laboratory (Huntley et al, 2005). Selection criteria will be that clones are present in at least two of the seven clone pools. The clones meeting these criteria will be reassigned to new clone pools consisting of 10 pools of 50 clones each.
3. Progress Report:
This collaborative research project encompasses measures to ensure animal health through the control and management of the disease by improving knowledge of host immunity during infection. The primary goals of the project have been modified from the directed approach of using uncharacterized recombinant Mycobacterium avium subspecies paratuberculosis (MAP) proteins to immunize mice as outlined above to a randomized, discovery approach. The new approach involves the optimization and implementation of new protocols for the isolation and characterization of membrane preparations of MAP. Briefly, 3 different isolates of MAP (K-10, 167 and 509) were grown up in liquid medium to log phase, harvested, and sonicated for recovery of outer membranes. Cell envelope and membrane protein complexes of MAP have been characterized by blue native gel and 2D gel electrophoresis. The major protein complexes identified on 2D gels will be excised and their composition identified using mass spectrophotometry. Concurrently, experiments have been outlined to determine the antigenicity of major protein complexes of MAP by performing Western blot analyses with anti-sera from cattle with Johne’s disease. Once antigenic proteins are identified, proteins will be pooled to make up a cocktail of 3-4 proteins for immunization of mice as described in the original protocol for this study. This will allow us to assess the protection of the host animal (mice) against challenge with live MAP with novel, antigenic MAP proteins. These MAP proteins will be selected from the outer membranes of MAP so are more likely to have been seen by the animal during a normal infection and, thus, make more likely candidates for a vaccine.