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In vitro and ex vivo evaluation of a multi-epitope heparinase vaccine for various malignancies.

Tang XD, Guo SL, Wang GZ, Li N, Wu YY, Fang DC, Fan YH, Yang SM - Cancer Sci. (2013)

Bottom Line: The results showed that multi-epitope vaccines Hpa525 + 277 + 405 + 16 and Hpa8 + 310 + 315 + 363 induced higher Hpa-specific lysis of various cancer cells from different tissues in a HLA-A2-restricted and heparanase-specific manner compared with the single epitope vaccines Hpa525, Hpa277, Hpa405, Hpa16, Hpa8, Hpa310, Hpa315 and Hpa363, both in vitro and ex vivo.However, these heparanase-specific CTL did not lyse heparanase-expressing autologous lymphocytes and dendritic cells, which confirms the safety of these multi-epitope vaccines.Therefore, the present study provides theoretical evidence for the use of heparanase multi-epitope vaccines for clinical application.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China; Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China.

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Heparanase specificity and HLA-A2 restriction of cytotoxic T lymphocytes (CTL) generated from different heparanase vaccines in vitro (A) and ex vivo (B). The CTL generated from the HLA-A2-restricted nonapeptide HIVpol(476-484)(ILLEPVHGV) derived from HIV served as the negative peptide control (NP). (A) The cytotoxic activity of CTL induced by heparanase multi-epitope vaccines and single epitope peptides against MCF-7 (A[a, b]), MCF-7/Hpa (A[c, d]), HepG2 (A[e, f]) and HepG2/HLA-A2 cells (A[g,h]) in vitro. (B) The cytotoxic activity of CTL induced by heparanase multi-epitope vaccines and single epitope peptides against MCF-7 (B[a, b]), MCF-7/Hpa (B[c, d]), HepG2 (B[e, f]), and HepG2/HLA-A2 cells (B[g, h]) ex vivo. E/T, effector/target.
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fig04: Heparanase specificity and HLA-A2 restriction of cytotoxic T lymphocytes (CTL) generated from different heparanase vaccines in vitro (A) and ex vivo (B). The CTL generated from the HLA-A2-restricted nonapeptide HIVpol(476-484)(ILLEPVHGV) derived from HIV served as the negative peptide control (NP). (A) The cytotoxic activity of CTL induced by heparanase multi-epitope vaccines and single epitope peptides against MCF-7 (A[a, b]), MCF-7/Hpa (A[c, d]), HepG2 (A[e, f]) and HepG2/HLA-A2 cells (A[g,h]) in vitro. (B) The cytotoxic activity of CTL induced by heparanase multi-epitope vaccines and single epitope peptides against MCF-7 (B[a, b]), MCF-7/Hpa (B[c, d]), HepG2 (B[e, f]), and HepG2/HLA-A2 cells (B[g, h]) ex vivo. E/T, effector/target.

Mentions: To further confirm the heparanase specificity of the CTL in vitro and ex vivo, we used the HLA-A2-positive, heparanase-negative breast cancer cell line MCF-7 (Fig.4). MCF-7 cells were transfected with recombinant, replication-defective adenovirus (Ad-Hpa) encoding a full-length cDNA of human heparanase at a multiplicity of infection of 200 and cultured for 2 days in fresh DMEM medium containing 10% FCS. In our previous study, western blot analysis showed that heparanase protein could be detected in MCF-7/Hpa cells, whereas in MCF-7 cells the expression of heparanase is very weak.19 Heparanase-specific CTL were generated using peptide-pulsed DC from HLA-A2-positive PBMC and DC from the bone marrow of C57BL/6-Tg transgenic mice. After being stimulated three times, the killing effect of CTL induced by Hpa525 + 277 + 405 + 16 and Hpa8 + 310 + 315 + 363 and their corresponding single peptides Hpa525, Hpa277, Hpa405, Hpa16, Hpa8, Hpa310, Hpa315 and Hpa363 were determined at various E/T ratios using a 4-h 51Cr release assay. The results showed that these heparanase-specific CTL could lyse MCF-7/Hpa cells, whereas no obvious lysis of MCF-7 cells was detected even at the highest E/T ratio. Furthermore, compared with the corresponding single heparanase peptides, heparanase multi-epitope vaccines elicited more robust specific lysis of MCF-7/Hpa cells (Fig.4). These results clearly indicate that the CTL specifically targeted heparanase peptides that were presented in the context of HLA-A2 and the multi-epitope vaccines of human heparanase could elicit much more potent killing effects compared with their corresponding single peptides.


In vitro and ex vivo evaluation of a multi-epitope heparinase vaccine for various malignancies.

Tang XD, Guo SL, Wang GZ, Li N, Wu YY, Fang DC, Fan YH, Yang SM - Cancer Sci. (2013)

Heparanase specificity and HLA-A2 restriction of cytotoxic T lymphocytes (CTL) generated from different heparanase vaccines in vitro (A) and ex vivo (B). The CTL generated from the HLA-A2-restricted nonapeptide HIVpol(476-484)(ILLEPVHGV) derived from HIV served as the negative peptide control (NP). (A) The cytotoxic activity of CTL induced by heparanase multi-epitope vaccines and single epitope peptides against MCF-7 (A[a, b]), MCF-7/Hpa (A[c, d]), HepG2 (A[e, f]) and HepG2/HLA-A2 cells (A[g,h]) in vitro. (B) The cytotoxic activity of CTL induced by heparanase multi-epitope vaccines and single epitope peptides against MCF-7 (B[a, b]), MCF-7/Hpa (B[c, d]), HepG2 (B[e, f]), and HepG2/HLA-A2 cells (B[g, h]) ex vivo. E/T, effector/target.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317872&req=5

fig04: Heparanase specificity and HLA-A2 restriction of cytotoxic T lymphocytes (CTL) generated from different heparanase vaccines in vitro (A) and ex vivo (B). The CTL generated from the HLA-A2-restricted nonapeptide HIVpol(476-484)(ILLEPVHGV) derived from HIV served as the negative peptide control (NP). (A) The cytotoxic activity of CTL induced by heparanase multi-epitope vaccines and single epitope peptides against MCF-7 (A[a, b]), MCF-7/Hpa (A[c, d]), HepG2 (A[e, f]) and HepG2/HLA-A2 cells (A[g,h]) in vitro. (B) The cytotoxic activity of CTL induced by heparanase multi-epitope vaccines and single epitope peptides against MCF-7 (B[a, b]), MCF-7/Hpa (B[c, d]), HepG2 (B[e, f]), and HepG2/HLA-A2 cells (B[g, h]) ex vivo. E/T, effector/target.
Mentions: To further confirm the heparanase specificity of the CTL in vitro and ex vivo, we used the HLA-A2-positive, heparanase-negative breast cancer cell line MCF-7 (Fig.4). MCF-7 cells were transfected with recombinant, replication-defective adenovirus (Ad-Hpa) encoding a full-length cDNA of human heparanase at a multiplicity of infection of 200 and cultured for 2 days in fresh DMEM medium containing 10% FCS. In our previous study, western blot analysis showed that heparanase protein could be detected in MCF-7/Hpa cells, whereas in MCF-7 cells the expression of heparanase is very weak.19 Heparanase-specific CTL were generated using peptide-pulsed DC from HLA-A2-positive PBMC and DC from the bone marrow of C57BL/6-Tg transgenic mice. After being stimulated three times, the killing effect of CTL induced by Hpa525 + 277 + 405 + 16 and Hpa8 + 310 + 315 + 363 and their corresponding single peptides Hpa525, Hpa277, Hpa405, Hpa16, Hpa8, Hpa310, Hpa315 and Hpa363 were determined at various E/T ratios using a 4-h 51Cr release assay. The results showed that these heparanase-specific CTL could lyse MCF-7/Hpa cells, whereas no obvious lysis of MCF-7 cells was detected even at the highest E/T ratio. Furthermore, compared with the corresponding single heparanase peptides, heparanase multi-epitope vaccines elicited more robust specific lysis of MCF-7/Hpa cells (Fig.4). These results clearly indicate that the CTL specifically targeted heparanase peptides that were presented in the context of HLA-A2 and the multi-epitope vaccines of human heparanase could elicit much more potent killing effects compared with their corresponding single peptides.

Bottom Line: The results showed that multi-epitope vaccines Hpa525 + 277 + 405 + 16 and Hpa8 + 310 + 315 + 363 induced higher Hpa-specific lysis of various cancer cells from different tissues in a HLA-A2-restricted and heparanase-specific manner compared with the single epitope vaccines Hpa525, Hpa277, Hpa405, Hpa16, Hpa8, Hpa310, Hpa315 and Hpa363, both in vitro and ex vivo.However, these heparanase-specific CTL did not lyse heparanase-expressing autologous lymphocytes and dendritic cells, which confirms the safety of these multi-epitope vaccines.Therefore, the present study provides theoretical evidence for the use of heparanase multi-epitope vaccines for clinical application.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China; Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China.

Show MeSH
Related in: MedlinePlus