Chromosomal deletions and the foreign antigen

cassette insertion were confirmed by PCR sequencing. The final foreign antigen cassette is shown graphically in Figure 1. Gel electrophoresis and Western blotting to nitrocellulose was performed using standard methods. A commercially available rabbit polyclonal antibody to E. coli alkaline phosphatase (Abcam, Cambridge, MA, USA) was used with a goat anti-rabbit peroxidase secondary antibody (KPL, Stem Cell Compound Library manufacturer Gaithersburg, MD, USA) and a chemiluminescent substrate (LumiGlo; KPL). Bacterial cultures were grown for approximately 16 hr in trypticase soy broth (TSB). J774A.1 murine macrophage monolayers (ATCC, Manassas, VA, USA) in 24-well plates were infected at a multiplicity of infection (MOI) of 20:1, and gentamicin exclusion assays for intracellular survival were performed as previously described (21). L929 murine fibroblast monolayers (ATCC) were infected with L. monocytogenes (MOI 1:50) and plaques measured five days later (22). Animal experiments were reviewed and approved by the IACUC at Massachusetts General Hospital and 8–12 week female BALB/c mice from Charles River Laboratories (Wilmington, MA, USA) were used for all

experiments. L. monocytogenes strains were grown Protease Inhibitor Library in vitro overnight in TSB containing streptomycin (100 μg/mL). Cultures were pelleted, washed once with normal saline and resuspended in sterile normal saline. Serial 10-fold dilutions were made and groups of six mice were injected intraperitoneally (i.p.) in a 300 μL volume. In addition to the vaccine strains expressing the influenza antigen, three groups

of control animals received either the wild type, the BMB72, parent strain or the BMB54 parent strain. Amisulpride Animal health was assessed several times daily and the median lethal dose (LD50) was calculated (23). For visceral persistence studies, mice were inoculated once i.p. with 0.1 LD50 of either the wild type (WT), BMB54, or BMB72. Mice were sacrificed at days 1, 3, 7, and 11, and the spleens and livers homogenized for one minute in 2 mL buffered saline, serially diluted and plated in triplicate on TSB plates. For ELISpot studies mice received approximately 0.1 LD50 of relevant strains and were sacrificed seven days later. Murine spleens were pooled by vaccine strain (three animals/group), processed with mesh strainers and red cells were lysed using ammonium chloride buffer. ELISpot experiments were performed using a pair of monoclonal antibodies (one biotinylated) directed against mouse interferon (IFN)-γ (Pierce, Rockford, IL, USA). Plates were then washed and developed with streptavidin–alkaline phosphatase conjugate and nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad, Hercules, CA, USA). The lectin control stimulus for murine ELISpot studies was concanavalin A.