SUPPLEMENTARY DATA

Results

Therecombinant Lmdd-MPFG strain was stable in vivo after 20 generations

The in vivo stability of the Lmdd-MPFG strain was examinedaccording tothe number of viable bacteria at every five passages, by plating the bacteria on both selective and nonselective media. We observed no significant differences (P=0.1085) in colony forming unit (CFU) counts after 20 generations by plating under each condition, suggesting that the recombinant bacteria are extremely stable, with no loss or modification of the transgene during sequential replications in vivo (SupplementaryFigure S1a). The expression of the segments was retained by the bacteria recovered from the spleens 3 days post-injection after every 5 passages, showing the in-vivo stability and the capacity to retain the chromosomal transgeneof the strains (SupplementaryFigure S1b).

Materials and methods

Western blot

The culture supernatants from which Listeria strains grew were trichloroacetic acid (TCA) precipitated and resuspended in 0.1 N NaOH + 2% SDS as described. Blots were stained with an anti-flag monoclonal antibody (Zymed, San Diego, CA) and alkaline phosphatase-labeled anti-rabbit goat antibody (Kirkegaard and Perry, Gaithersburg, MD) was used for detection of bound anti-flag antibody as recommended by the manufacturer. Blots were developed with Super Signal WestDura chemiluminescent substrate (Pierce, Rockford, IL) and visualized using a CCD camera.

In vivo stability studies and expression of Lmdd-MPFG

The stability of the recombinant L. monocytogenes integrants was determined after 20passages in-vivo as described by Peters et al(Peters and Paterson, 2003). Briefly, a dose of 5×107 CFU of Lmdd-MPFG was injected intravenously (i.v.) into 6–8 week old mice. Single cell suspensions were prepared from the spleens three days after the injection and plated on Brain-Heart Infusion (BHI) plates in the absence of D-alanine to select for Lmdd-MPFG growth. Following every five passages,25 colonies were tested for growth on BHI plates containing 100 μg/ml D-alanine. At the end of the stability test, one colony recovered was tested for MPFG expression by Western-blot with anti-flag antibody.

Detection of anti-MPFG Abs by enzyme-linked immunosorbent assay (ELISA)

Sera were collected at week fourfollowing the last vaccination and assayed for antibodies specific to HBc-Ag and MPFG fusion peptides by ELISA. For detection of anti-HBc Abs,anELISA kit from Diagnostic Automation Incorporation (Calabasas, CA) was used according to the instructions. The ELISA-Peptide assay was used to detect MPFG fusion peptides HBx52-60, HBx140-148, AFP158-166 and MAGE271-279. Briefly, ninety-six-well plates were coated with different MPFG peptides overnight at 4°C and then blocked by incubation with 10 mg/ml BSA. Mouse sera were diluted 1:10 in PBS and applied to duplicate wells, followed by goat anti–mouse IgG horseradish peroxidase (HRP) conjugates (Sigma, St. Louis, MO) as the secondary Ab at a dilution of 1:5000. ABTS 1 Component Microwell Substrate (BioFX Laboratories, Owings Mills, MD) was added subsequently and the assay results wereanalyzed at OD405.

In vivo depletion of CD4 or CD8 T lymphocyte subsets

Immunized mice were injected with 500 µg i.p. rat IgG (Abcam, Cambridge, MA), anti-CD4 (clone GK1.5), and/or anti-CD8 (clone GK 2.43) on day-3 and day-1 prior to challenge with Lmdd-MPFG. The depletion was verified 24 h following mAbs injection by analyzing the CD8 and CD4 T cell populations in the blood. The in-vivo cytotoxicity assay was assessed following depletion by flow cytometric (FC) analysis of spleen.

T-cell proliferation assay

Responses to the six peptides were assessed in vitro by measuring proliferation after antigen exposure. Splenocytes were established from immunized mice in culture at 5×105 cells/well in flat-bottom 96-well plates and stimulated with 5 µg/mL, 20 µg/mL, or 50 µg/mL peptides in the presence of IL-2 (20 U/mL) (Protech, London, UK). Splenocytes were also established without peptide or with 5 µg/mL Con A. Then 200 µl final culture volumes were incubated at 37°C for 5 days and pulsed with the addition of [3H] thymidine at 0.4 µCi/well 16 h before harvesting. Proliferation was measured using a Wallac 1450 scintillation counter. Results were expressed using the stimulation index (SI), which is the ratio of counts per min (CPM) from peripheral blood mononuclear cells (PBMCs) exposed to peptide stimulation to the CPM from PBMC cultured in medium only.

IFN-γ ELISA

Splenocytes from immunized mice (three per group) were cultured in 24-well plates at 5×106 cells/well in-vitro in the presence of 5 µg/L of the variouspeptides and IL-2 (20 U/mL) in 1 mL of complete RPMI medium. Samples from supernatants were collected on day 3 and tested for the presence of IFN-γ using a mouse IFN-γ ELISA kit (BD Biosciences) according to the manufacturer’s recommendations.

Cell staining and flow cytometry

Splenocytes harvested 5 days after the last immunization were stimulated with 20 µg/mL various peptides in the presence of Golgiplug and then stained with PE-Cy5–conjugated mAb against CD8 (clone 53-6.7; eBioscience) and PE-conjugated IFN-γ (clone XMG1.2; eBioscience).Treg cell staining, followed the isolation of TIL populations from the tumor-bearing mice wasas follows: cells were stained with CD4-FITC, CD25-APC, CD4-PE, anti-GITR-FITC, anti-CD62L-PE, anti-CD127-PE, anti-CCR7-PE, anti-TGF-β-PE, anti-CD152-PE (CTLA-4), anti-CCR4-PE, and anti-IL-10-PE and assayed on a FACScan flow cytometer (Becton Dickinson). Antibodies and their respective isotypes, used as a negative control for surface and intracellular staining, were all purchased from BD PharMingen. The mouse regulatory T cell staining kit(eBioscience) was used for intracellular cell staining for Foxp3 according to the instructions.

Cytotoxicity assay

For the measurement of cytolytic activity in vivo, target cells were pooled with splenocytes from naive mice and pulsed with 20 µg/mL HBx140-148 peptide and labeled with 6 μM CFSE (CFSEhigh) (Molecular Probes) fluorescence intensity. Uncoated splenocytes (control target cells) were labeled with 0.3 μM low CFSE (CFSElow) fluorescence intensity. The mixed target cells (2107), at a ratio of 1:1, were adoptively transferred into the immunized mice. At 24 h after injection of the CFSE-labeled targets cells, spleens were removed and the ratio of CFSElow to CFSEhigh cells was determined by flow cytometry. The percentage of cytotoxicity was determined as follows: (1-CFSEhigh/CFSElow) ×100%. Due to the limited amounts of TILs, the cytotoxic activity of TILs and MPFG-specific CTL against various targets was assayed by LDH release assays, as described previously (Chen et al., 2009).

Interferon-γ (IFN-γ), IL-4, and IL-10 ELISPOT assay

Detection of antigen-specific IFN-γ-secreting T cells from TILs in response to peptide stimulation was performed using ELISPOT kits (BioSource International, Camarillo, CA). Briefly, 50 µl of cell suspensions from immunized or tumor burden mice at 107 cells/ml were added to each well and incubated in the presence of 5 µg/ml various peptides/stimulatorsplus IL-2 (5 U/ml) overnight at 37°C. Splenocytes without peptide or with 2.5 µg/ml Concanavalin A (Con A) were used as control. For the IL-4 and IL-10 Elispot assays, splenocytes from naive or immunized mice were plated with 1ng/ml PMA and 0.5 ng/ml Inomycine (Sigma-Aldrich). IFN-γ, IL-4 and IL-10 spots in the wells were then developed according to the manuscript. Results were expressed as spot-forming units (SFU)/106 cells after subtracting background spots.

Isolation of tumor infiltrating lymphocytes (TIL)

At selected time-points after treatment, tumors were removed from mice and single-cell suspensions were prepared by enzymatic digestion. Resected tumors were weighed, minced into small (1–2 mm3) pieces with a scalpel, and immersed in 10 mL of digestion mixture [5% FBS in RPMI 1640, 0.5 mg/ml collagenase A (Roche Diagnostic), 0.2 mg/ml hyaluronidase, type V (Sigma-Aldrich), and 0.02 mg/ml DNase I (Sigma-Aldrich)] per 0.25 g of tumor tissue. The resulting cell suspensions were filtered sequentially through 70- and 40-µm cell strainers (BD Falcon) and washed with 5% FBS in RPMI 1640. Red blood cells (RBC) were lysed by brief incubation in 0.15 M ammonium chloride solution, and cell debris was removed by centrifugation using a Lympholyte-M gradient as recommended by the manufacturer (Cedarlane).

Immunohistochemistry

Immunoperoxidase staining of CD4+ or foxP3 T cells was done on 4-μm sections of formalin-fixed paraffin-imbedded tumors. Sections were deparaffinized and subjected to heat-induced antigen retrieval using DAKO's target retrieval solution for 25 min. Following endogenous peroxidase removal using 3% hydrogen peroxide in methanol, the samples were incubated 2×15 min with avidin-biotin blocking reagent (Vector, Burlingame, CA) and protein-blocking reagent (DAKO, Carpinteria, CA) for 15 min. Slides were incubated with a 1:80 dilution of primary antibody (anti-CD4 or Foxp3) or isotype control (BD PharMingen, San Diego, CA) overnight at 4°C. Immunodetection was done using a secondary biotinylated-polyclonal anti-Rat IgG (Sigma, St. Louis, MO). Diaminobenzidine/hydrogen peroxidase was used as the chromogen substrate. Serial sections were stained by routine H&E.

Supplementary Figure legends

Figure S1. In vivo stability and expression studies of Lmdd-MPFG strain. A, In vivo stability was examined by immunizing mice with 5×107 CFU of the p1565-MPFG strain intravenously in the tail vein. The CFUs were determined in the homogenized spleens after 24, 48, and 72 h. Viable CFUs were determined after plating on both selective and nonselective media. No colonies were recovered at the time points of 72 h. Columns, mean number of CFU from each mouse; bars, SD. B, Expression of MPFG fusion protein by Lmdd-MPFG by passaging in vivo. Protein extracts were prepared by precipitating culture supernatants with 10% trichloroacetic acid (TCA) at 4°C. The Western blot represents the expression of MPFG in the total proteins secreted by the Lmdd-MPFG strains. The blot was stained with a 1:1,000 dilution of anti-flag antibodies.

Figure S2. Frequency of total CD8+ and CD4+ cells in the PBL following the indicated antibody treatment.500 µg rat IgG, anti-CD4, or anti-CD8 (clone GK 2.43) were injected intraperitoneally (i.p.) to immunized mice on day-3 and day-1 prior to challenge with Lmdd-MPFG. The CD8 and CD4 T cell populations in the blood 24 hours after mAbs injection were analyzed byflow cytometry to verify complete depletion.

FigureS3. Detection of Th2/antibody mediated responses. Splenocytes from immunized or control mice were harvested and cultured in the presence of PMA and Inomycine. A, HBc-specific Abs elicited in HLA-A2 Tg mice were measured by enzyme-linkedimmunosorbent assay (ELISA).B,The number of cytokine-secreting cells for IL-4, and IL-10 was determined as described in the Supplementary Materials and Methods.