Millipore HA cellulose ester (MAHAS4510) was used for IgA ELISpot

assays and Immobilon P (MAIPS4510) membrane-bottom plates were used for IFN-γ ELISpot assays. For IgA ELISpots, the antigens used were recombinant nucleoprotein, recombinant listeriolysin, and sonicated WT listerial antigen. Antibodies in cultured lymphocyte supernatants were also harvested for soluble vaccine-specific immunoglobulins by ELISA, as previously described (25), an assay also known as the ALS assay (30). For IFN-γ ELISpots, control wells included phytohemagglutinin (PHA) and “CEF”, a commercially available standard peptide pool including 32 CMV, EBV and influenza virus peptides, 8–12 BGB324 supplier amino acids in length (AnaSpec, San Jose, CA, USA). Test peptides included the same three influenza peptide pools and the listeriolysin O (LLO) (25) peptide pool described above. The complex whole listerial antigen was also used in IFN-γ ELISpot studies. Spots were counted by an automated reader (Immunospot; CTL, Shaker

Heights, OH, USA). Low level spot counts in unstimulated medium-only control wells were subtracted from the test wells. The IFN-γ ELISpot results are presented as mean values of duplicate wells per condition as spot-forming Trichostatin A cells (SFC)/106 PBMC. A positive response for an individual was defined as more than two-fold greater than baseline results for that antigen and over 100 SFC/106 PBMC (31, 32). Because IFN-γ responses did not appear related to the oral dose given, results were also analyzed as a whole by organism given, comparing pre-immune with peak values. Serum samples were studied by ELISA to quantify IgG and IgA directed against sonicated listerial antigens, recombinant his-tagged listeriolysin and Influenza A nucleoprotein over time. Antigens were suspended in PBS and used to coat Nunc-Immuno Maxisorp 96-well plates (Nalge Nunc International, Roskilde, Denmark). Assays were performed as described (9) and read on a Vmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Endpoint dilutions are reported as the highest dilution at which a serum sample was ≥0.14 OD units at 405 nm, an arbitrarily chosen cutoff value. Four-fold or greater increases in endpoint titer were considered a positive result. The differences in geometric means between groups were compared statistically with the Mann–Whitney PLEKHB2 test. Both vaccine strains were demonstrated by sequencing to contain the expected deletions and heterologous fusion antigen. The introduction of the attenuating mutations ΔactA/plcB and ΔactA/inlB did not significantly alter growth kinetics in TSB broth as measured by optical density, nor did the incorporation of the “empty” integration vector, pPL2. Introduction of the foreign antigen fusion cassette did moderately alter growth kinetics; both the rate of growth and the final density of growth were slightly depressed (OD600nm∼1.5 to 2.0 vs. ∼2.3 to 2.7).