理想型的DNA疫苗設計是能夠誘導適合該抗原的免疫反應。在傳統免疫學的研究上，抗原在細胞中呈現的位置常會決定免疫反應的走向。在細胞外所呈現的抗原主要被體液型免疫反應所清除，細胞內的抗原主要被細胞型免疫反應所清除。基於此原則，若將分泌型的DNA疫苗表現在細胞內部也許會增加其細胞免疫反應以提高DNA疫苗的效用。故比較分泌型和在細胞內部表現的DNA疫苗對於研究理想型DNA疫苗設計會是一個可行的研究方式。於是，我們實驗室便構築了表現在細胞外部和內部的N’-neu及C’-neu (Sec-N’-neu, Cyt-N’-neu, Sec-C’-neu, Cyt-C’-neu)，利用這四種DNA疫苗觀察來深入探討這個發展DNA疫苗的新議題。
在腫瘤大小測量及生存曲線中的結果發現，這四種DNA疫苗都有顯著的效果，並且抗原在細胞內部呈現的DNA疫苗治療效果會比抗原在細胞外部呈現更佳。我們利用西方點漬法偵測小鼠中是否含有anti-neu的抗體，結果發現只有在分泌型的DNA疫苗組別中才有抗體的產生。於是，利用免疫組織染色觀察所引起的免疫反應，發現抗原呈現在細胞內部的DNA疫苗會使較多的CD8的淋巴球滲入，但抗原呈現在細胞外部的DNA疫苗卻是使較多的CD4淋巴球滲入。更進一步在動物體內分別去除不同的免疫球細胞群，發現在去除CD8淋巴球時，各組都有顯著的影響，這顯示CD8淋巴球在各組DNA疫苗的療效都扮演了重要的角色。而在去除CD4的免疫細胞群時，我們發現只有抗原在細胞內部的DNA疫苗的療效有降低，但對於抗原在細胞外部的DNA疫苗卻反而還有增加療效的現象。由於CD4淋巴球中含有會抑制免疫反應的調節 T細胞 (CD4+CD25+ T 細胞)，所以我們認為分泌型的DNA疫苗也許和調節T細胞有關。於是，我們便分析施打疫苗後的小鼠其CD4+CD25+ T 細胞數量。結果發現，分泌型DNA疫苗的調節T細胞在數量上有上升的情形，而這樣的情形在抗原表現在細胞內部的組別並未發生。
由以上結果我們可以確認：在細胞內部表現的DNA疫苗比分泌型的DNA還要好，而我們認為這可能和分泌型的DNA疫苗會活化調節T細胞有關。

The ultimate objective of rational DNA vaccine design is the induction of pathogen-appropriate immunity. We wanted to find a rational DNA vaccine design to modify secretive form N’-neu DNA vaccine for increasing its therapeutic efficacy or lowering its side effect. The initial cellular events and interactions that occur following DNA immunization are likely to be key to determining the character and magnitude of the resulting immune response. Therefore, the comparison of secretive form DNA vaccine and cytoplasmic DNA vaccine is a practicable approach for studying rational DNA vaccine design. Then, we constructed cytoplasmic N’-neu, secretive C’-neu, and cytoplasmic C’-neu DNA vaccine for studying this issue.

In our results, we found that all of these neu DNA vaccines had the therapeutic efficacy in this animal model based on the tumor volume measurement and the survival curve, and the cytoplasmic DNA vaccines even had better therapeutic efficacy than secretive form. We also detected the anti-neu antibody in serum of the mice treated with secretive form DNA vaccine. The immunohistochemistry studies showed that the significant infiltration of CD4+ T cells and CD8+ T cells into tumors were found in the mice that were treated with all of these DNA vaccine therapies. And we found that the numbers of the CD4+T cells activated by secretive form DNA vaccines are more than by cytoplasmic DNA vaccines. In contrast, the numbers of the CD8+T cells activated by secretive form DNA vaccines are fewer than by cytoplasmic DNA vaccine. Further, we wanted to investigate the role of the CD4+ T cells and CD8+ T cells by depletion Ab from hybridoma. The results showed that when we depleted CD8+ T cells, the therapeutic efficacy of the DNA vaccines is lost. Therefore, CD8+ T cell played an essential role in all of these DNA vaccine therapies. But when we depleted CD4+ T cells, we found that the therapeutic efficacy of secretive form DNA vaccine and cytoplasmic DNA vaccine are different. Combination of secretive form DNA vaccines and the antibody for depletion CD4+ T cells even increases the therapeutic efficacy. These results may be correlated with the number of the CD4+CD25+ T cells. Then we analyzed the number of the CD4+CD25+ T cells in the lymph node. The results showed that the secretive form DNA vaccine increased the number of the CD4+CD25+ T cells, but the cytoplasmic DNA vaccine didn’t.

In conclusion, the results indicated that the cytoplasmic DNA vaccine had better therapeutic efficacy than the secretive form did. And we suggested that it may be correlated with the CD4+CD25 +T cells.

Alegre, M.L., Noel, P.J., Eisfelder, B.J., Chuang, E., Clark, M.R., Reiner, S.L., Thompson, C.B. Regulation of surface and intracellular expression of CTLA4 on mouse T cells. J. Immunol. 157:4762-4770. 1996.

Cederbom, L., H. Hall, F. Ivars. 2000. CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells. Eur. J. Immunol. 30:1538. 2000.

Chen SA, Tsai MH, Wu FT, Hsiang A, Chen YL, Lei HY, Tzai TS, Leung HW, Jin YT, Hsieh CL, Hwang LH, Lai MD. Induction of antitumor immunity with combination of HER2/neu DNA vaccine and interleukin 2 gene-modified tumor vaccine. Clin Cancer Res. 6 :4381-8. 2000.

Chen, Y., D. Hu, D. J. Eling, J. Robbins, T. J. Kipps. DNA vaccines encoding full-length or truncated neu induce protective immunity against neu-expressing mammary tumors. Cancer Res. 58:1965. 1998.

Christianson, T. A., J. K. Doherty, Y. J. Lin, E. E. Ramsey, R. Holmes, E. J. Keenan, G. M. Clinton. NH2-terminally truncated HER-2/neu protein: relationship with shedding of the extracellular domain and with prognostic factors in breast cancer. Cancer Res. 58:5123. 1998.

Disis, M., E. Calenoff, G. McLaughlin, A. E. Murphy, W. Chen, B. Groner, M. Jeschke, N. Lydon, E. McGlynn, R. B. Livingston. Existent T cell and antibody immunity to Her-2/neu protein in patients with breast cancer. Cancer Res. 54:16. 1994.

Disis, M. L., S. M. Pupa, J. R. Gralow, R. Dittadi, S. Menard, M. A. Cheever. High-titer Her-2/neu protein-specific antibody can be detected in patients with early-stage breast cancer. J. Clin. Oncol. 15:3363. 1997

Donnelly J. J., Ulmer J. B., Shiver J. W., Margaret A. L. DNA Vaccines.Annu. Rev. Immunol., 15: 617-648. 1997.

Ewer, M. S., H. R. Gibbs, J. Swafford, R. S. Benjamin. Cardiotoxicity in patients receiving Trastuzumab (Herceptin): primary toxicity, synergistic or sequential stress, or surveillance artifact?. Semin. Oncol. 26:(Suppl. 12):96. 1999.

Feldman, J.. Immunological enhancement: a study of blocking antibodies. Adv. Immunol. 15:167. 1972.

Fendly, B. M., C. Kotts, W. L. T. Wong, I. Figari, W. Harel, L. Staib, M. E. Carver, D. Vetterlein, M. S. Mitchell, H. M. Shephard. Successful immunization of rhesus monkeys with the extracellular domain of p185HER2: a potential approach to human breast cancer. Vaccine Res. 2:129. 1993.

Garza, K. M., S. M. Chan, R. Suri, L. T. Nguyen, B. Odermatt, S. P. Schoenberger, P. S. Ohashi. Role of antigen-presenting cells in mediating tolerance and autoimmunity. J. Exp. Med. 191:2021. 2000.

Godfrey, V., Wilkinson, J., Rinchik, E. & Russell, L. Fatal lymphoreticular disease in the scurfy (sf) mouse requires T cells that mature in a sf thymic environment: potential model for thymic education. Proc. Natl. Acad. Sci. USA 88, 5528-5532 . 1991.

Grant, E. P., M. T. Michalek, A. L. Goldberg, K. L. Rock. Rate of antigen degradation by the ubiquitin-proteasome pathway influences MHC class I presentation. J. Immunol. 155:3750. 1995.

Hurwitz, E., I. Stancovski, M. Sela, Y. Yarden. Suppression and promotion of tumor growth by monoclonal antibodies to ErbB-2 differentially correlate with cellular uptake. Proc. Natl. Acad. Sci. USA 92:3353. 1995.

Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol. 1 :337-42. 2003

Khattri R, Kasprowicz D, Cox T, Mortrud M, Appleby MW, Brunkow ME, Ziegler SF, Ramsdell F. The amount of scurfin protein determines peripheral T cell number and responsiveness. J Immunol. 167 :6312-20. 2001.

Krummel, M.F., Allison, J.P. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182:459-465. 1995.

Lofts, F. J., W. J. Gullick. c-erbB2 amplification and overexpression in human tumors. Cancer Treat. Res. 61:161. 1992.

Mahnke K, Guo M, Lee S. The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments. J Cell Biol. 151: 673-684. 2000.

Mahnke K, Qian Y, Knop J, Enk AH. Induction of CD4+/CD25+ regulatory T cells by targeting of antigens to immature dendritic cells. Blood. 101(12):4862-9. 2003.

Nishimura, T., K. Iwakabe, M. Sekimoto, Y. Ohmi, T. Yahata, M. Nakui, T. Sato, S. Habu, H. Tashiro, M. Sato, A. Ohta. Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo. J. Exp. Med. 190:617. 1999.

Qin, Z., G. Richter, T. Schuler, S. Ibe, X. Cao, T. Blankenstein. B cells inhibit induction of T cell-dependent tumor immunity. Nat. Med. 4:627. 1998.

Pegram, M., D. Slamon. Biological rationale for HER2/neu (c-erbB2) as a target for monoclonal antibody therapy. Oncology 27:13. 2001.

Peoples, G. E., P. S. Goedegebuure, R. Smith, D. C. Linehan, I. Yoshino, T. J. Eberlein. Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same Her-2/neu-derived peptide. Proc. Natl. Acad. Sci. USA 92:432. 1995.

Piechocki MP, Pilon SA, Wei WZ. Complementary Antitumor Immunity Induced by Plasmid DNA Encoding Secreted and Cytoplasmic Human ErbB-2. J. Immunol. 167: 3367-3374. 2001.

Piechocki MP, Pilon SA, Wei WZ. Quantitative measurement of anti-ErbB-2 antibody by flow cytometry and ELISA. J Immunol Methods. 1;259(1-2):33-42. 2002.

Pilon SA, Piechocki MP, Wei WZ. Vaccination with cytoplasmic ErbB-2 DNA protects mice from mammary tumor growth without anti-ErbB-2 antibody. J Immunol. 15;167(6):3201-6. 2001.

Read, S., V. Malmstrom, F. Powrie. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295. 2000.

Rush C, Mitchell T, Garside P. Efficient priming of CD4+ and CD8+ T cells by DNA vaccination depends on appropriate targeting of sufficient levels of immunologically relevant antigen to appropriate processing pathways. J. Immunol. 167: 3367-3374. 2002

Rovero, S., A. Amici, E. DiCarlo, R. Bei, P. Nanni, E. Quaglino, P. Porcedda, K. Boggio, A. Smorlesi, P. L. Lollini. Inhibition of carcinogenesis by DNA vaccination. J. Immunol. 165:5133. 2000.

Seddon B, Mason D. Peripheral autoantigen induces regulatory T cells that prevent autoimmunity. J Exp Med. 189: 877-882. 1999.

Shevach, E.M. CD4+ CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2, 389-400. 2002.

Steitz J, Bruck J, Lenz J, Knop J, Tuting T. Depletion of CD25(+) CD4(+) T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon alpha-induced, CD8(+) T-cell-dependent immune defense of B16 melanoma. Cancer Res. 61 :8643-6. 2001.

Suri-Payer, E., Amar, A.Z., Thornton, A.M., Shevach, E.M. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J. Immunol. 160:1212-1218. 1998.

Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, J. Shimizu, S. Sakaguchi. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10 :1969. 1998.

Thornton AM, Shevach EM. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J Immunol. 164: 183-190. 2000.

Tighe H., Corr M., Roman M., Raz E. Gene vaccination: plasmid DNA is more than just a blueprint. Immunol. Today, 19: 89-97. 1998.

Toellner, K., S. A. Luther, D. M. Sze, R. K. Choy, D. R. Taylor, I. C. M. MacLennan, H. Acha-Orbea. T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching. J. Exp. Med. 187:1193. 1998.

Tokuda, Y., M. Ohta, Y. Suzuki, M. Kubota, T. Tajima. Clinical development of trastuzumab in breast cancer. Breast Cancer 8:93. 2001.

V. Renard, L. Sonderbye, K. Ebbehoj, P. B. Rasmussen, K. Gregorius, T. Gottschalk, S. Mouritsen, A. Gautam, and D. R. Leach. HER-2 DNA and Protein Vaccines Containing Potent Th Cell Epitopes Induce Distinct Protective and Therapeutic Antitumor Responses in HER-2 Transgenic Mice. J. Immunol. 171: 1588 - 1595. 2003.

Walker, R. A., W. J. Gullick, J. M. Varley. An evaluation of immunoreactivity for c-erbB-2 protein as a marker of poor short-term prognosis in breast cancer. Br. J. Cancer 60:426. 1989.

Wright, C., B. Angus, S. Nicholson, J. R. Sainsbury, J. Cairns, W. J. Gullick, P. Kelly, A. L. Harris, C. H. Horne. Expression of c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer. Cancer Res. 49:2087. 1989.

Xingmin Zhang, Leonid Izikson, Liming Liu and Howard L. Weiner. Activation of CD25+CD4+ Regulatory T Cells by Oral Antigen Administration. J. Immunol. 167: 4245 – 4253. 2001.

Yu, D., M. C. Hung. Overexpression of ErbB2 in cancer and ErbB2-targeting strategies. Oncogene 19:6115. 2001.

蔡明寰. 膀胱癌之 (I) 基因治療 及 (II) 抗藥性 之研究。國立成功大學醫學院生物化學研究所碩士論文, 2000。

http://www.doh.gov.tw/statistic/data/死因摘要/91年/表22.xls