Efficacy of recombinant adenovirus expressing a fusion gene from GM-CSF and Epstein-Barr virus LMP2A in a mouse tumor model

Figures & data

Figure 1. A. Electrophoresis results for GM-CSF and LMP2A. Lane 1 = DNA DL2000 Marker; lanes 2 and 3 = GM-CSF gene; lane 4 = DNA DL2000 Marker; lane 5 = LMP2A gene. B. Electrophoresis results for the fusion gene GC2A. Lane 1 = DNA DL2000 Marker; lanes 2 and 3 = GC2A genes. C. Identification of pAdTrack-CMV-GC2A. Lane 1 = enzyme analysis after Bg1II and EcoRV digest; lane 2 = DNA DL2000 Marker; lane 3 = pAdTrack-CMV-GC2A. D. Restriction enzyme digestion for pAd-GC2A analysis and identification. Lane 1 = DNA DL15000 Marker; lane 2 = PacI enzyme digestion of recombinant vector.

Figure 2. Delection of the fusion gene GM-CSF and LMP2A expression in recombinant adenovirus. A. The 293 cells transfected by recombinant adenovirus vectors (200 ×,56 h). B. Western blotting analysis of expressed (Lane 1 = western blotting with GM-CSF of Adenovirus 5 control; lane 2 = western blotting with GM-CSF of vAd-GC2A; lane 3 = Protein Markers; lane 4 = western blotting with LMP2A of Adenovirus 5 control; lane 5 = western blotting with LMP2A of vAd-GC2A; lane 6 = protein markers).

Figure 3. Specific IgG antibody response of mice immunized with recombinant adenovirus by intraperitoneal injection. Recombinant adenovirus maximum dose was 5 × 10⁸ virus particles/100 μl, recombinant adenovirus secondary dose was 5 × 10⁷ virus particles/100 μl, and PBS control dose was 100 μl. The recombinant adenovirus maximum dose exerted the most proliferative effect, antibody titers peaked on week 7.

Table 1. Specific CTL killing activity of immunized mice (%).

Groups	E/T = 60:1	E/T = 40:1	E/T = 20:1	E/T = 10:1
PBS control	22.01 ± 1.04	24.30 ± 2.50	22.06 ± 2.12	22.28 ± 1.62
adenovirus 5 control	29.09 ± 1.14	32.40 ± 3.10	25.08 ± 1.06	23.98 ± 1.56
adenovirus 5+ plasmid	28.93 ± 1.21	31.78 ± 1.42	27.92 ± 1.93	24.90 ± 1.29
adenovirus 5+ LMP2A	31.23 ± 1.34	35.35 ± 1.25	32.92 ± 1.25	29.38 ± 1.56
adenovirus 5+ hGM-CSF	43.06 ± 1.18	49.32 ± 1.37	46.38 ± 1.26	41.04 ± 2.46
recombinant adenovirus	64.16 ± 1.08	66.70 ± 6.90	57.08 ± 2.73	58.93 ± 2.24

Note: Compared with the PBS control group,

* P ≤ 0.05, compared with the PBS control group,

** P ≤ 0.01.

Figure 4. Tumor size. The adenovirus vaccine group was injected with 2 × 10⁸ plaque-forming unit recombinant adenovirus, other groups were injected with 2 × 10⁸ plaque-forming unit (adenovirus 5+ LMP2A), (adenovirus 5+ hGM-CSF), (adenovirus 5+ plasmid) (soluble in 250 μL PBS)/mouse, respectively. The adenovirus 5 control group was injected with 2 × 10⁸ plaque-forming unit adenovirus 5 (soluble in 250 μL PBS)/mouse, the PBS control group was injected with 250 μL PBS/mouse.

Table 2. Subcutaneous tumor model results (P < 0.01).

Group	Number	Tumor size (mm^3)	Inhibitionrate(means ± SD)
Adenovirus vaccine	5	306.23 ± 108.2mm^Citation3	0.76 ± 0.10
Adenovirus 5+ LMP2A	5	1149.26 ± 102 mm^3	11.39 ± 0.09
Adenovirus 5+ hGM-CSF	5	927.34 ± 113 mm^3	28.50 ± 0.11
Adenovirus 5+ plasmid	5	1206.18 ± 109 mm^3	0.07 ± 0.10
Adenovirus 5 control	5	1274.0 ± 115 mm^3	0 ± 0.09
PBS control	5	1297.0 ± 103 mm^3	0.000

Note: Results for two groups of BALB/c mouse tumor models.

** Compared with the control group (P ≤ 0.01).

Figure 5. Immunohistochemical staining of tumor tissue in groups. (A) Recombinant adenovirus 5 group. (B)(Adenovirus 5+ LMP2A) group. (C) (Adenovirus 5+ hGM-CSF) group. (D) (Adenovirus 5+ plasmid) group. (E) Adenovirus 5. (F) PBS. The tumor cells in the recombinant adenovirus 5 group compared with the control group, the CD4 cells were more, and in a large number of CD4 in tumor tissue; Adenovirus 5(LMP2A) and Adenovirus 5 (hGM-CSF) group can also be seen with a certain extent part of tumor tissue necrosis and some of CD4 cells, but CD4 cells were less than recombinant adenovirus 5 group, there is no significant difference in the staining results on adenovirus 5 and (adenovirus 5+ plasmid) group. PBS had no tumor necrosis and CD4 cells.

Figure 6. The recombinant diagram of pAdTrack-CMV and pAdEasy-1.

Liu MT , Chang YT , Chen SC , Chuang YC , Chen YR , Lin CS , Chen JY . Epstein-Barr virus latent membrane protein 1 represses p53-mediated DNA repair and transcriptional activity. Oncogene. 2005;24:2635-42. doi:10.1038/sj.onc.1208319. PMID:15829976

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