Start Date: Jul 21, 2011
End Date: Dec 31, 2013
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) two 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 three 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 six 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 three 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 seven 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.