[CANCER RESEARCH 64, 400 – 405, January 1, 2004] Enhancement of DNA Vaccine Potency by Coadministration of a Tumor Antigen Gene and DNA Encoding Serine Protease Inhibitor-6 Tae Woo Kim, 1 Chien-Fu Hung, 1 David A. K. Boyd, 1 Liangmei He, 1 Cheng-Tao Lin, 1 Dion Kaiserman, 5 Phillip I. Bird, 5 and T-C. Wu 1,2,3,4 Departments of 1 Pathology, 2 Oncology, 3 Obstetrics and Gynecology, and 4 Molecular Microbiology and Immunology, The Johns Hopkins Medical Institutions, Baltimore, Maryland, and 5 Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia ABSTRACT Serine protease inhibitor 6 (SPI-6), also called Serpinb9, inhibits gran- zyme B and thus may provide a method for delaying apoptotic cell death in dendritic cells. We have previously enhanced DNA vaccine potency by targeting antigen to MHC antigen presentation pathways, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, domain II of Pseudomonas aeruginosa exotoxin A, or the sorting signal of the lysosome-associated membrane protein type 1. In this study, we ex- plored intradermal coadministration of DNA encoding SPI-6 with DNA constructs encoding human papillomavirus type 16 E7 linked to these intracellular targeting molecules for its ability to generate E7-specific CD8 T-cell immune responses and E7-specific antitumor effects. This combination of strategies resulted in significantly increased E7-specific CD8 T-cell and CD4 Th1-cell responses, enhanced tumor treatment ability, and stronger tumor protection when compared with vaccination without SPI-6. Among these targeting strategies tested, mice vaccinated with Sig/E7/lysosome-associated membrane protein type 1 mixed with SPI-6 showed the greatest fold increase in E7-specific CD8 T cells (5-fold). Vaccination with a nonfunctional mutant of SPI-6 did not result in immune enhancement, indicating that enhancement was dependent on the antiapoptotic function of SPI-6. Our results suggest that DNA vaccines combining strategies that enhance MHC class I and II antigen processing with SPI-6 have potential clinical implications for control of viral infection and neoplasia. INTRODUCTION DNA vaccines provide a means of administering antigen to the immune system and initiating cell-mediated immune responses. Intra- dermal administration of DNA vaccines via gene gun has proven to be the most effective delivery technique because it allows for the direct delivery of DNA encoding antigen of interest into dendritic cells (DCs; Ref. 1), the most potent professional antigen-presenting cells. DCs process the antigen and travel to draining lymph nodes where antigenic epitopes are presented to naı ¨ve CD8+ and CD4+ T cells, stimulating them to develop into CTLs and T-helper cells, respec- tively (2). The ability of the gene gun to directly target DCs with DNA vaccine constructs has allowed us to evaluate strategies for improving DNA vaccine potency by enhancing antigen processing and presen- tation by DCs. We have previously enhanced MHC class I and MHC class II antigen processing by DCs via several intracellular targeting strategies designed to route human papillomavirus type 16 (HPV-16) E7 antigen to desired subcellular compartments. These strategies include linking DNA encoding E7 antigen to DNA encoding Mycobacterium tuber- culosis heat shock protein 70 (3), calreticulin (4), domain II of Pseudomonas aeruginosa exotin A (5), or the sorting signal of the lysosome-associated membrane protein 1 (LAMP-1; Ref. 6). We have previously shown that mice vaccinated with any of these strategies linked to E7 display enhanced E7-specific CD8+ T-cell responses and antitumor effects when compared with mice vaccinated with wild-type E7 DNA alone. We have recently explored the use of antiapoptotic proteins to enhance DNA vaccine potency by prolonging DC life (7). DCs have a limited life span that hinders their long-term ability to prime anti- gen-specific T cells (8). A principal contributor to the short life of DCs is CTL-induced apoptosis. CTLs are programmed to recognize antigens and kill the cells expressing them. Because DCs express MHC I:antigen peptide complexes, newly primed CTLs sometimes kill the very DCs that activated them (9). Interrupting CTL-induced apoptosis and thereby prolonging the survival of DCs may facilitate the priming of antigen-specific CD8 + T cells and increase cell- mediated immune responses. To this end, we investigated the capacity of the serine protease inhibitor (SPI-6; Ref. 10) to delay CTL-induced DC death. CTLs can secrete granzyme B (GrB) and perforin (11), which act in concert as part of granule-mediated apoptosis, the dominant pathway of CTL- induced apoptosis (for review, see Ref. 12). The serine protease inhibitors (serpins) represent a potential solution to the problem of CTL suicide (13, 14) and CTL-induced DC apoptosis by inactivating GrB (for reviews of serpin classification and regulation, see Refs. 15, 16). Medema et al. (9) demonstrated that maturing DCs naturally express SPI-6 to inhibit GrB-triggered apoptosis and showed that overexpression of SPI-6 provides DCs with a powerful mechanism for defense against cytotoxic T cells. On the basis of these findings, we hypothesized that coadministra- tion of an SPI-6 expression vector with various DNA vaccines would prolong DC life, enhancing antigen presentation by DCs and, there- fore, the immune response. Our data indicated that coadministering DNA encoding SPI-6 with DNA encoding intracellular targeting molecules linked to E7 prolongs DC life by delaying apoptosis of DCs and enhances cell-mediated E7-specific immune responses and anti- tumor effects to a greater extent than either of these strategies alone. These results suggest that a vaccination strategy combining intracel- lular targeting with SPI-6 to prolong DC life may additionally en- hance DNA vaccine potency and may have important future clinical applications. MATERIALS AND METHODS Plasmid DNA Constructs and DNA Preparation. The generation of pcDNA3-E7 (3), pcDNA3-calreticulin /E7 (4), pcDNA3- domain II of Pseudo- monas aeruginosa exotoxin A/E7 (5), pcDNA3-E7/heat shock protein 70 (3), and pcDNA3-Sig/E7/LAMP-1 (6) has been described previously. For gener- ation of pcDNA3-SPI-6, SPI-6 was first amplified with PCR using mouse cDNA as the template and a set of primers, 5'-cccgaattcatgaatactctgtct- gaagga-3' and 5'-ttggatcctggagatgagaacctgccaca-3'. The amplified product was then cloned into the EcoRI/BamHI sites of the pcDNA3 vector. To generate the inactive mutant SPI-6 (mtSPI-6) containing the P14 muta- Received 5/23/03; revised 8/28/03; accepted 10/16/03. Grant support: National Cancer Institute, the Cancer Research Institute, the American Cancer Society (to T-C. W.), and the National Health and Medical Research Council, Australia (to P. I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Drs. Kim and Hung contributed equally to this work. Requests for reprints: Dr. T-C. Wu, Department of Pathology, The Johns Hopkins University School of Medicine, Richard Ross Research Building, Room 512H, 720 Rutland Avenue, Baltimore, MD 21205. Phone: (410) 614-3899; Fax: (410) 287-4295; E-mail: wutc@jhmi.edu. 400 Research. on January 22, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from