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Gene Gun-Mediated DNA Vaccination Induces Antitumor Immunity Against Human Papillomavirus Type 16 E7-Expressing Murine Tumor Metastases in the Liver and Lungs

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Gene Gun-Mediated DNA Vaccination Induces Antitumor Immunity Against Human Papillomavirus Type 16 E7-Expressing Murine Tumor Metastases in the Liver and Lungs Gene Therapy (1999) 6, 1972–1981 1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt Gene gun-mediated DNA vaccination induces antitumor immunity against human papillomavirus type 16 E7-expressing murine tumor metastases in the liver and lungs C-H Chen1,2,HJi3, KW Suh4,5, MA Choti4, DM Pardoll1 and T-C Wu1,3,6,7 Departments of 1Oncology, 3Pathology, 4Surgery, 6Obstetrics and Gynecology, and 7Molecular Microbiology and Immunology, The Johns Hopkins Medical Institutions, Baltimore, Maryland, USA; 2Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan; and 5Department of Surgery, School of Medicine, Ajou University, South Korea DNA vaccination has emerged as an attractive approach assess the E7-specific T cell-mediated and humoral for tumor immunotherapy. The aim of this study was to immunity. Mice vaccinated with Sig/E7/LAMP-1 DNA gen- evaluate the potency of DNA vaccines in preventing and erated the strongest E7-specific CTL activities, the highest treating the liver and lung metastases of a human papil- numbers of E7-specific CD8+ cell precursors and the high- lomavirus-16 (HPV-16) E7-expressing murine tumor (TC- est titers of E7-specific antibodies. While both E7 DNA and 1). We used the gene gun method to vaccinate C57BL/6 Sig/E7/LAMP-1 DNA generated potent antitumor immunity mice intradermally with DNA vaccines containing the HPV- in the liver and lung metastases models, the Sig/E7/LAMP- 16 E7 gene, the E7 gene linked to the sorting signals of 1 DNA was more potent under stringent conditions. DNA the lysosome-associated membrane protein-1 (Sig/E7/ vaccination with E7-expressing plasmids was effective in LAMP-1), or the ‘empty’ plasmid vector. The in vivo anti- controlling liver and lung metastases of an E7-expressing tumor immunity was analyzed in both tumor prevention and murine tumor. Our data suggest that antigen-specific DNA tumor regression experiments. In addition, cytotoxic T lym- vaccination can potentially be applied to control liver and phocyte (CTL) assays, enzyme-linked immunospot assay lung metastases of tumors with defined tumor-specific and enzyme-linked immunoabsorbent assay were used to antigens. Keywords: DNA vaccine; metastasis; human papillomaviruses; LAMP-1; antitumor; E7 Introduction autoimmunity, more flexible in deciding the amount of antigen administered and the methods of antigen presen- Metastasis is a common cause of death in many cancer tation to the immune system, more reproducible from patients. Conventional therapies, such as surgery, chemo- patient to patient and has the potential to correlate clini- therapy and radiotherapy, have limited success in con- cal outcome to a specific immune response.2 Therefore, trolling cancer metastases. The ideal cancer therapy antigen-specific cancer immunotherapy represents a should act systemically in addition to its local antitumor desirable approach for controlling tumors. effect. In this regard, immunotherapy is especially In the past few years, several reports have shown that attractive because it has the potency to eradicate systemic antigen-specific cancer vaccines, in the form of cell-based tumors in multiple sites in the body and the specificity vaccines, protein/peptide-based vaccines and viral vec- to discriminate between neoplastic and non-neoplastic tor-based vaccines, are effective for the treatment of met- 1 cells. astases.1 However, production of the autologous tumor Tumor-specific antigens, when efficiently presented by + cell-based vaccines is labor intensive and technically chal- antigen presenting cells (APCs) to both CD8 cytotoxic T 3 + lenging. The application of peptide-based vaccines is lymphocytes (CTLs) and CD4 helper T cells, are capable limited by major histocompatibility complex (MHC) of inducing potent T cell-mediated immunity – the most restriction and the necessity of defining the CTL epitopes. crucial component of antitumor immunity. There are sev- The disadvantages of viral vector-based vaccines include eral additional advantages of using an antigen-specific unnecessary immune responses to vectors and other cancer vaccine. It is less likely to generate nonspecific potential virus-associated toxicity. In contrast, naked plasmid DNA is safe, low immunogenically and can be repeatedly administered. DNA vaccines can be easily Correspondence: T-C Wu, Department of Pathology, The Johns Hopkins prepared on a large scale with high purity and are highly Hospital, 600 North Wolfe Street, Baltimore, Maryland 21205, USA stable relative to proteins and other biologic polymers Received 28 June 1999; accepted 31 August 1999 (for review, see Refs 4–6). DNA vaccination for cancer metastases C-H Chen et al 1973 Figure 1 Schematic diagram of wild-type E7 and Sig/E7/LAMP-1 DNA constructs. The wild-type HPV-16 E7 gene and the chimeric Sig/E7/LAMP-1 gene were cloned into the BamHI cloning sites of the pCMVneoBam expression vector downstream of the cytomegalovirus promoter as described in Materials and methods. We chose human papilliomavirus-16 (HPV-16) E7 as a model antigen for vaccine development because HPVs, particularly HPV-16, are associated with most cervical cancers. The HPV oncogenic proteins, E6 and E7, are important in the induction and maintenance of cellular transformation and are co-expressed in most HPV-con- taining cervical cancers. Therefore, vaccines or immuno- therapies targeting E7 and/or E6 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. We have previously linked the sorting signals of the lysosome-associated membrane protein-1 (LAMP-1) to the HPV-16 E7 antigen, creating a chimera, Sig/E7/LAMP-1 (see Figure 1). We found that mice vac- cinated with Sig/E7/LAMP-1-containing recombinant vaccinia virus can generate strong antitumor immunity against HPV-16 E7-expressing tumors in subcutaneous,7 lung metastasis8 and hepatic metastasis models (Chen et al, unpublished data). However, immune responses to the vaccinia vaccine vector can limit the effectiveness of re- vaccination with vaccinia virus.9 The use of naked DNA vaccines can circument this problem. We used liver and lung metastasis models in the cur- rent study because besides regional lymph nodes, the liver and lungs are the most common sites of distant metastases of cervical cancers.10 In addition, when developing clinical applications for the treatment of com- mon solid malignancies, demonstrating efficacy of ther- apy within visceral sites such as the liver or lung is important.11 Therefore, the aim of this study was to test whether DNA vaccines encoding HPV-16 E7 could induce protective immunity against HPV-16 E7-express- ing murine tumor metastases in the liver or lungs. Results Figure 2 CTL response in C57BL/6 mice immunized with various recom- Vaccination with Sig/E7/LAMP-1 DNA generates potent binant DNA vaccines. C57BL/6 mice were immunized with empty plas- E7-specific CD8+ T cell-mediated immune response mid, E7 DNA or Sig/E7/LAMP-1 DNA via gene gun. Splenocytes were + harvested 2 weeks after the last DNA vaccination and stimulated in vitro CD8 T lymphocytes are one of the most crucial compo- b nents of the antitumor effect.12 CTL assay and enzyme with E7-specific H-2D CTL epitope and interleukin-2 for 6 days. A stan- dard 4 h chromium release assay was performed against MC57G (H-2b) linked immunospot (ELISPOT) assays were used to cells pulsed with E7 CTL peptide. Target cell-specific lysis was determined determine the E7-specific T cell-mediated immunity gen- as described in Materials and methods. Results shown here are from one erated by DNA vaccine. For the standard chromium representative experiment of two performed. DNA vaccination for cancer metastases C-H Chen et al 1974 Figure 3 E7-specific CD8+ T cell precursors in C57BL/6 mice immunized with various recombinant DNA vaccines using ELISPOT assays. C57BL/6 mice were immunized with empty plasmid, E7 DNA or Sig/E7/LAMP-1 DNA via gene gun. Splenocytes were harvested 2 weeks after the last DNA vaccination. The number of IFN-␥ producing E7-specific CD8+ T cell precursors was determined using the ELISPOT assay (see text for the detailed method). (a) The spot numbers were the mean of triplicates ± s.e. in each vaccinated group. (b) Representative ELISPOT pictures in each vaccinated group. Results shown here are from one representative experiment of two performed. release cytotoxicity assay, mice vaccinated with assay indicated that Sig/E7/LASMP-1 DNA-vaccinated Sig/E7/LAMP-1 DNA generated the highest E7-specific mice generated the highest CD8+ T cell-mediated immunity. CTL activity, while mice vaccinated with E7 DNA gener- ated only slightly higher E7-specific CTL activity than the Vaccination with Sig/E7/LAMP-1 DNA generates the mice vaccinated with the empty plasmid (Figure 2). The highest T helper-type I cytokine responses ELISPOT assay is a sensitive function asaay for IFN-␥ To determine the cytokine profiles generated by E7 DNA production by individual cell and can be applied to quan- and Sig/E7/LAMP-1 DNA, splenocytes from vaccinated tify antigen-specific CD8+ T cell precursors.13 We used the mice were stimulated in vitro with E7 protein for 72 h. ELISPOT assay to determine the precursor frequencies of The supernatants were harvested and assayed for the E7-specific CD8+ T cells in each vaccination group. presence of IFN-␥ and IL-4. As shown in Figure 4, the Around 120 E7-specific CD8+ T cells were detected per highest IFN-␥ level was found in the mice vaccinated 106 spenocytes derived from the Sig/E7/LAMP-1 DNA- with Sig/E7/LAMP-1 DNA. Vaccination with E7 DNA vaccinated mice, but only 10–20/106 in E7 DNA-vacci- generated the second highest IFN-␥ release. IL-4 could nated mice. We did not detect E7-specific IFN-␥ secreting not be detected in the supernatant of any vaccinated CD8+ T cells in control of empty plasmid-vaccinated mice group.
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