No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. Ming-Zong Lai (Institute of Molecular Biology, Academia Sinica, Taiwan) for comments on the manuscript. The authors thank Hsilin Cheng and Ting-Fang Wang (Institute of Molecular Biology, Academia Sinica, Taiwan) for acquiring the TOF/TOF mass data.
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Figure legends
Figure 1. Construction, production and identification of rE7m and rlipo-E7m.
(a) The alignment of the amino acid sequence of wild-type HPV16 E7 (Accession number: NP_041326) and the inactive E7 (E7m). Every mutant site of E7m is highlighted. The DNA sequence of the E7m gene was derived using codon usage of E. coli and fully synthesized using the assembly PCR method. The PCR product was cloned into the pET22b vector for rE7m expression and into the modified pET22b vector with a lipid signal peptide for rlipo-E7m expression. The recombinant proteins contained an additional HHHHHH sequence (HisTag) at the C-terminus and were expressed under the control of the T7 promoter. (b) The rlipo-E7m and rE7m protein purification process was monitored using 15% reducing SDS-PAGE followed stained by Coomassie Blue staining and immunoblotting using anti-HisTag antibodies. The recombinant rlipo-E7m was expressed in E. coli strains C43 (DE3). Lane 1, rE7m expression after IPTG induction; lane 2, protein expression in the absence of IPTG induction; lane 3, extracted fraction of rE7m; lane 4, purified recombinant rE7m. Lanes 5-8 show immunoblotting to monitor the rE7m purification process, and the samples in these lanes are the
same as those in lanes 1-4, respectively. Lane 9, rlipo-E7m expression after IPTG induction; lane 10, protein expression in the absence of IPTG induction;
lane 11, purified rlipo-E7m. Lanes 12-14 show immunoblotting to monitor the rlipo-E7m purification process, and the samples in these lanes are the same as those in lanes 9-11, respectively. The non-lipidated form of rE7m was expressed in E. coli BL21 (DE3) star strain. The arrows indicate the electrophoretic positions of rE7m or rlipo-E7m in the gels or blots. (c) Intact rlipo-E7m was analyzed by LC/MS. LC/MS analysis revealed the presence of five major post-translational modifications of rlipo-E7m. Molecular masses (in daltons) were determined using a maximum entropy algorithm. The five major peaks displayed masses of 15384.5, 15400, 15412.5, 15426 and 15439. (d) N-terminal rlipo-E7m fragments were obtained and identified after digestion of rlipo-E7m with trypsin. The digested sample was analyzed on a Waters®
MALDI micro MX™ mass spectrometer. The MALDI-TOF MS spectra revealed the existence of five peaks with m/z values of 1452, 1466, and 1480, 1492 and 1506.
Figure 2. Bone marrow-derived dendritic cells (BM-DCs) are activated by
rlipo-E7m through TLR2. (a) Splenocytes were isolated from wild-type mice and plated at a density of 2×105 cells/well in 96-well plates. The cells were incubated with various concentrations of LPS (0–1000 g/ml), Pam3 (0–1000
g/ml), rlipo-E7m (0–100 g/ml) or rE7m (0–1000 g/ml) for 48 hrs. During the final 24 hrs, 1 Ci of [3H]-thymidine was added to each well to measure DNA synthesis. The data are represented as the mean ± SD of triplicate samples. (b) BM-DCs from wild-type mice were cultured in medium supplemented with LPS (0.01 g/ml), rE7m (10 g/ml) or rlipo-D1E3 (10g/ml) in the presence or absence of polymyxin B (20g/ml). After a 24-hr incubation, the expression of the dendritic cell surface markers CD40 and CD86 was analyzed using flow cytometry. Experiments were performed in triplicate and the mean fluorescence intensity (MFI) of cells cultured in medium alone was defined as 100%. (c) For the cytokine secretion studies, BM-DCs were cultured in medium supplemented with LPS (0.01 g/ml), Pam3 (0.15 g/ml), rE7m (0.1-10g/ml) or rlipo-E7m (0.1-10g/ml) in the presence or absence of polymyxin B (20g/ml). After a 24-hr incubation, the supernatants were harvested and analyzed for TNF- and IL-12 (p40) production by ELISA. The data are presented as the mean±SD from three independent experiments. (d) BM-DCs from wild-type, TLR1-knockout (TLR1-KO), TLR2-KO or TLR6-KO
mice were cultured either in medium alone or in medium supplemented with LPS (0.01 g/ml), Pam3 (0.15 g/ml), rE7m (10g/ml), or rlipo-E7m (10g/ml).
After a 24-hr incubation, the supernatants were harvested and analyzed for TNF- production by ELISA. The data are represented as the mean±SD of triplicate samples.
Figure 3. Enhancement of anti-E7m IgG antibody titers and induction of a Th1-biased immune response after administration of rlipo-E7m (a) C57BL/6 mice (n=5) were immunized twice by subcutaneous injection of 30 μg of rE7m in PBS or of 30μg of rlipo-E7m in PBS at two-week intervals. Sera were collected, and IgG or (b) IgG2b/IgG1 responses against rE7m were evaluated by ELISA. The IgG2b/IgG1 ratios in both groups are shown in the insert. (c) Mice were injected subcutaneously of 30 g of rE7m or rlipoE7m twice at two-week intervals. Seven days after the second immunization, splenocytes were isolated and stimulated with rE7m (10 g/ml) for 4-5 days. The supernatants were collected, and the levels of IFN-or IL-5 were measured by ELISA. Data are expressed as means+SD of samples (n=5).
Figure 4. Immunization with rlipoE7m induces higher levels of E7-specific cytotoxic T lymphocyte activity. (a) Splenocytes (2x105 cells/well) from immunized mice were incubated with or without 10 g/ml of RAHYNIVTF (RAH) peptide for 48 hrs in an anti-IFN--coated 96-well ELISPOT plate. The IFN--secreting spots were measured using an ELISPOT reader. Data are expressed as means+SD of six animals per group. (b) The RAH-specific CD8+ T cells were detected by tetramer staining and analyzed by flow cytometry. The percentage of tetramer-positive cells among the CD8+ T cells is indicated within each panel. The numbers of TetraRAH-tetramer +/CD8+
cells in total CD8 cells (1 x105) from three experiments were showed. Bar graphs indicating the mean percentage of RAH tetramer+/CD8+ cells among the CD8+ cells. Data are expressed as the mean + SD. * P < 0.05. (c) To assess the cytotoxic effect of rlipo-E7m immunization on tumor cells, a standard chromium-release assay was performed. Isolated splenocytes (effector cells) were stimulated with RAH (10 g/ml) for 7 days in the presence of rIL-2 (10 unit/ml). The target cells (5x103/ well) were incubated with different ratio of effector cells (1:25, 1:50, 1:100). Specific lysis (%) was calculated as follows: 100 x (Sample releasecpm – Spontaneous releasecpm)/(Maximum
releasecpm – Spontaneous releasecpm).
Figure 5. Immunization with rlipo-E7m induces a strong anti-tumor effect. (a) Mice were immunized twice by subcutaneous injection of rE7m/rlipo-E7m (10
g) or PBS at one-week intervals. Seven days after the final immunization, mice were subcutaneously injected with 2x105 TC-1 cells in a total volume of 200 l subcutaneously. (b) Mice were subcutaneously inoculated with TC-1 cells (2x105/mouse). After 7 days, tumor-bearing mice (5-10 mice per group) were injected once with rE7m/rlipo/rE7m (30 g/mouse) or PBS. The tumor volume was shown as length x width x width/2 (mm3). Data are expressed as means+SEM. (c) Three groups (E7m/CD4, E7m/CD8 and rlipo-E7m/Rat IgG) of mice were injected of anti-CD4, anti-CD8 and control antibodies, respectively, one day before the injection of TC-1 cells. These groups and two additional groups (rlipo-E7m and PBS) were injected once with rlipo-E7m (10 g/mouse) or PBS seven days after inoculated with TC-1 cells (2x105/mouse). The tumor volume was shown as length x width x width/2 (mm3). Data are expressed as means+SEM.