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Blood, Vol. 113, Issue 1, 37-45, January 1, 2009
Enhancement of DNA tumor vaccine efficacy by gene gun–mediated codelivery of threshold amounts of plasmid-encoded helper antigen
Blood Leitner et al.
113: 37
Supplemental materials for: Leitner et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 99.7 KB)
- Table S1. Survival of immunized mice past 10, 20, 25, 30, and 35 days (after challenge) (PDF, 89.7 KB) -
Number of mice shown are from between 2 and 17 independent immunization/challenge experiments. Numbers represent survivors / total number of mice.
- Figure S1. Verification of TCR expression by transfection and on tumor cells (JPG, 76.7 KB)
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(A) BHK-21 cells were transfected with pVax-TCRβ and stained intracellularly with anti-TCR Vβ12 antibody (dotted line: pVax-transfected, solid line: pVax-TCRβ transfected). (B) No surface expression of either TCR chain was detected on transfected cells (shown is staining of pVax-TCRβ transfected BHK-21 cells; dotted line: pVax-transfected, solid line: pVax-TCRβ transfected). (C) Expression of cell surface TCR by MBL-2 tumor cells after staining with anti-TCR Vβ12 antibody (dotted line: isotype control, solid line: Vβ12 mab). (D) MBL-2 cells were stained with monoclonal antibodies against MHC-I (right peak) or MHC-II (left peak) or isotype control antibody (solid peak) and analyzed by flow cytometry demonstrating uniform expression of MHC-I but absence of MHC-II on the lymphoma cells.
- Figure S2 (JPG, 31 KB)
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To test the specificity of the protective immune response, mice (n= 8/group) were immunized with plasmid encoding the TCRβ-chain expressed by MBL-2, EL4 or RMA-cells plus the helper plasmid (6 ng β-gal/shot, solid circle) or empty vaccine vector plus helper plasmid (triangle) and challenged with a syngeneic murine T-cell lymphoma that expresses a different TCR (C6VL).
- Figure S3. Low-dose helper antigen also enhances the efficacy of a non-lymphoma DNA vaccine (JPG, 25.4 KB)
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To rule out that the effect of low-dose helper antigen co-delivered with a tumor antigen was restricted to the lymphoma model used in this study, mice were immunized by gene gun with gold particles carrying 1 µg/shot hgp100-plasmid and 6 ng/shot β-gal plasmid (filled circle), with gp100 and empty pcDNA plasmid (open triangle), empty pcDNA and β-gal plasmid (×), or were not immunized (open circle). The animals received three immunizations at three week intervals and were challenged s.q. 10 days after the last immunization with 1 × 105 B16.F10 melanoma cells. Tumor measurements were stopped when half or more mice in a group required euthanasia.
- Figure S4. Humoral immune responses against helper antigen (JPG, 29 KB)
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The serum titer of β-gal–specific antibodies in mice immunized with 1 µg pVax-TCRβ (or empty pVax) plus low dose (6 ng/shot) pSport-β-gal (or pcDNA) was determined by ELISA (average of 3–4 mice/group with SE). Serum from mice immunized twice with 1 µg/shot pSport-β-gal was used as a positive control. The lack of β-gal–specific antibodies after immunization with low-dose helper plasmid was confirmed with sera from mice immunized with the same plasmids as well as other combinations involving β-gal (not shown).
- Figure S5. Potential impact of the immunization against a self-antigen (TCR) on host T cells (JPG, 40.5 KB)
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Peripheral blood lymphocytes from mice immunized with the different vaccines were obtained 10 days after the last immunization, gated on the CD3+ population and analyzed for changes in the Vβ12+ T-cell subset using Vβ10+ cells as a reference population (mean plus SEM of 4 mice) by flow cytometry.
- Figure S6. Vaccine efficacy is not dependent on epitope spreading (JPG, 41.3 KB)
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Mice were immunized with the TCRβ/β-gal plasmid-carrying gold particles (1 µg and 6 ng/shot, respectively), empty pVax plasmid, pcDNA-gag or were not immunized. Then, mice were challenged either with MBL-2 (as in Figs. 1–3) (n= 7/group) or with EL-4 cells (n= 8/group). The latter express the same TCR as MBL-2, but do not express the muLV-derived gag antigen (EL4(-gag)). Shown is the average tumor size for each group with SEM. Data were analyzed by ANOVA followed by two-sided T test. MBL-2 tumors were significantly (p<0.001) smaller in mice immunized with either pcDNA-gag or pVax-TCRβ/β-gal compared to mice immunized with empty plasmid. In contrast, EL4(-gag) induced tumors were only significantly smaller in mice immunized with pVax-TCRβ/β-gal (p=0.05), but not in mice immunized with pcDNA-gag.
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