| BackgroundProtein transduction is an emerging technology that could best be described as the internalization of chemical compounds, peptides, nucleic acids or full-length proteins into cells in a receptor- and transporter-independent manner when conjugated with a so-called protein transduction domains (PTDs). This process relies on the inherent property of PTDs of being able to penetrate the cell membrane.Protein transduction was first reported a decade ago by Green and Frankel who indendently demonstrated that the Tat protein from HIV-1 virus was able to enter cells when added to the surrounding media. Subsequently, several other proteins with transducing capabilities have been identified, including Drosophila hemeotic transcription factor ANTP (encoded by the antennapedia gene) and the herpes simplex virus type I (HSV-1)VP22.It has been demonstrated that protein transduction not only could deliver pharmacologically relevant compounds and proteins into cells, but also could improve the outcome of cancer gene therapy when fused with certain therapeutic genes coding for tumor suppressor genes, pro-apoptotic molecules or prodrug activating enzymes. More recently, it has been reported that PTDs have the capability of introduing antigens into MHC I-dependent antigen presentation pathway and could enhancing the anti-tumor immunity induced by peptide or nucleic acid vaccines.However, the relationship between the transducing capabilities of PTDs and the enhancement of antitumor immunity elicited by immunization of antigens fusion with PTDs. AimEvaluating the antitumor immunity provoked by loading dendritic cells with nucleic acid vaccines coding hTERT 1540 epitope fusion with HIV-1 Tat-PTD or PTD4 delivered by recombinant replication-defective adenoviruses, determining whether the transducing capabilities of PTDs could affect the efficacy of nucleic acid vaccines. Methods and Results1. The eukaryotic expression cassettes of I540-PTD and I540-PTD4 were designed based on the amino acid sequence of the epitope of hTERT 1540, HIV-1 Tat-PTD and PTD4. The coding sequences were obtained by adding kozak sequence after back-translation using human preferred codons. Then the whole expression cassettes were constructed by phosphorylation and annealing of 6 oligonucleotides chemically synthesized, and ligating into pSP72 in turns. The resulting plasmids pSP72-I540-PTD and pSP72-I540-PTD4 were verified by restriction enzyme digestion and DNA sequencing.2. The I540-PTD and I540-PTD4 expression cassettes were subcloned into the shuttle plasmid pDC315 and both the pDC315-I540-PTD and pDC315-I540-PTD4 yielded a fragment of 121bp just as we predicted after digested with EcoRI. Ten days after co-transfecting HEK293 cells with pDC315-I540-PTD and pDC315-I540-PTD4 with genomic plasmid pBHGloxdeltaE13Cre respectively, typical cytopathic effects were seen. The lysates containing recombinant replication-defective adenoviruses Ad-I540-PTD and Ad-I540-PTD4 were obtained by three consecutive free-thaw cycles after harvesting the HEK293 cells. The infectious titers of Ad-I540-PTD and Ad-I540-PTD4 are 8.9x109 pfu/ml and 6.3x109pfu/ml, 5.8x109 pfu/ml and 5.4x109 pfu/ml respectively determined by end-point dilution assay and plaque assay. Then Ad-I540-PTD and Ad-I540-PTD4 were confirmed by amplifying the 167bp products by PCR with specific primers using recombinant adenoviruses DNA as templates. Immunohistochemical analysis using anti-His tag monoclonalantibody demonstrated that the U2OS infected with Ad-I540-PTD and Ad-I540-PTD4 are both strong positive stained.3. PBMC were isolated from healthy HLA-A*0201+ donors'buffy coats by FicoLL-Hypaque density gradient centrifugation. Dendritic cells were generated from PBMC in the presence of GM-CSF and IL-4. Typical mature morphological features could be seen after activiating the dendritic cells by TNF-a.4. The stimulating index(SI) of autologous T cells were determined by MTT assays by co-culture with PBMC, dendritic cells and dendritic cells infected with Ad-I540-PTD or Ad-I540-PTD4. It was observed that PBMC and uninfected dendritic cells could only stimulate the proliferation of autologous T cells moderately, but the Sis were significantly increased when using dendritic cells post-infection with Ad-I540-PTD or Ad-I540-PTD4(P<0.01) and reaches 11.75±0.26 and 12.17±0.46 when the dendritic cells to T cells ratio was 1:10. However, there was no significant difference between the groups of dendritic cells infected with Ad-I540-PTD or Ad-I540-PTD4 (P>0.05).5. TNF-a released by CTLs primed by PBMC, dendritic cells, dendritic cells transduced with Ad-I540-PTD or Ad-I540-PTD4 were determined by ELISA using HLA-A*0201+ MCF-7 cells and hTERT- HLA-A*0201+ U2OS cells as target cells at E:T ratio 50:1. Spontaneous secretion of TNF-a by CTLs could be detected, especially the CTLs primed by dendritic cells transduced with Ad-I540-PTD or Ad-I540-PTD4(P<0.01). And when stimulated with U2OS cells, the levels of TNF-a released by CTLs primed by dendritic cells transduced with Ad-I540-PTD or Ad-I540-PTD4 reached 35.6±2.9 pg/ml and 31.9±3.1 pg/ml, significantly lower than that of being stimulated by MCF-7, which were 80.9±3.8 pg/ml and 84.3±3.5 pg/ml respectively(PO.Ol).6. Specific lysis were assessed by chromium release assay using MCF-7 as target cells. CTLs generated from Ad-I540-PTD and Ad-I540-PTD4 transduced-dendritic cells exhibited higher lysis rales than that generated from PBMC or non-transduced dendritic cel!s(P<0.01), and the lysis rates increased with the E:T ratios. At an E:T ratio of 100:1, the specific lysis rates of CTLs generated from Ad-I540-PTD and Ad-I540-PTD4 transduced-dendritic cells reached 75.7±4.1% and 79.3±3.7% respectively, but there was no significantdifference between the two groups. Conclusion1. We have successfully designed and constructed the recombinant replication-defective adenoviruses Ad-I540-PTD and Ad-I540-PTD4 harboring I540-PTD and I540-PTD4 expression cassette respectively.2. Both Ad-I540-PTD and Ad-I540-PTD4 transduced dendritic cells could give rise to potent anti-telomerase-specific CTL response in vitro, implying that protein transduction domains such as HIV-1 TAT-PTD and PTD4 could be applicable to introduce antigens into MHC I-dependent antigen presentation pathway and could enhancing the anti-tumor immunity induced by fusion gene vaccines.3. Though the protein transduction activity of PTD4 is about 30-fold of that of HIV-1 TAT-PTD, the anti-tumor immunity elicited by dendritic cells transduced with Ad-I540-PTD and Ad-I540-PTD4 have no significant difference, demonstrated that the protein transduction activities of PTDs do not correlated with the levels of antigen-specific CTLs generated by fusion gene vaccines. |
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