Tyrosine Hydroxylase-Based DNA-Vaccination Is Effective Against Murine Neuroblastoma H.N. Lode, MD, 1,2 * U. Pertl, MD, 1 R. Xiang, MD, PhD 1 , G. Gaedicke, MD, 2 and R.A. Reisfeld, PhD 1 Background. The disruption of self-toler- ance against neuroblastoma is the ultimate goal of an effective DNA-vaccine. Procedure. Here we demonstrate the induction of a protective immunity against syngeneic murine NXS2 neu- roblastoma in A/J mice, following vaccination with tyrosine hydroxylase (TH) derived anti- gens. Oral gene delivery was accomplished us- ing an attenuated strain of Salmonella typhimu- rium as a carrier harboring vectors encoding for mTH antigens. Results. Vaccination was effec- tive in protecting animals from a lethal chal- lenge with wild-type NXS2 tumor cells. Con- clusions. These results provide the first evidence of the TH self antigen being recog- nized by T-cells and demonstrate that a TH- based DNA vaccine is a potentially useful im- munotherapeutic strategy for neuroblastoma. Med. Pediatr. Oncol. 35:641–646, 2000. © 2000 Wiley-Liss, Inc. Key words: tyrosine hydroxylase; DNA vaccines; immunotherapy; neuroblastoma INTRODUCTION The effective treatment of Stage 4 neuroblastoma is one of the major challenges in pediatric oncology, since its outcome remains poor, even after high-dose chemo- therapy and autologous bone marrow or stem cell trans- plantation. The development of effective alternative strategies appears to be the only realistic way to further improve outcome of this neoplasm. A new era for cancer immunotherapy emerged in the last decade based on the recognition that some tumors encode tumor rejection antigens, which are capable of inducing protective immunity, particularly through the cellular arm of the immune system where CD8 + cyto- toxic T cells (CTL) seem best equipped to recognize tumor cells as foreign and eradicate them. Several strat- egies focusing on CD8 + T cells include tumor cells trans- duced with cytokine genes, dendritic cells pulsed with tumor antigens or tumor cells transfected with costimu- latory molecules to deliver the antigen-specific signal concomitant with a second costimulatory signal to CD8 + T cells [1]. In fact, CD8 + T cells recognize peptides presented by MHC class I molecules. These peptides are usually derived from cytosolic proteins, such as tyrosine hydroxylase, which are degraded in 20S proteasomes and translocated to the endoplasmic reticulum (ER) by trans- porters associated with antigen processing (TAP1 and TAP2) before they associate into complexes with MHC Class I molecules [2], which are transported to the cell membrane and recognized by CD8 + T cells [3]. DNA vaccines are based on the discovery that immu- nization with “naked” DNA leads to strong and persistent cell-mediated and humoral immune responses to antigens encoded by plasmid DNA [4–6]. The design of DNA vaccines is highly flexible, as more than one immunogen can be expressed in a plasmid together with additional sequences that serve as immunomodulators. Further- more, outside the inserts the plasmid contains transcrip- tional control elements for expression of vaccine inserts in eukaryotic cells [7,8]. The unmethylated CpG motifs of plasmid DNA provide T-helper immunostimulatory activity [8] that mobilizes an immune response against the DNA-expressed immunogen and stimulates mono- cytes and macrophages to produce such cytokines as IL12, TNF- and IFN-/ that, in turn, act on NK cells to induce lytic activity and INF- secretion [9]. Novel ways of enhancing the efficacy of DNA vaccines include growing the plasmid encoding DNA in a nonreplicating strain of Salmonella that can be applied as an oral vac- cine. Thus, the live, attenuated bacteria transport the DNA through the gastrointestinal tract and then through the M cells which cover the Peyer’s patches of the gut. From there, the attenuated bacteria enter APCs (macro- phages and DCs), where they die, because of their mu- tation, liberating multiple copies of the DNA inside the phagocytes [9–11]. Indeed, attenuated bacteria may pro- vide a “danger signal” [12,13] and stimulate the innate 1 The Scripps Research Institute, Department of Immunology, La Jolla, California, USA 2 Charite ´ Children’s Hospital, Department of Pediatrics, Berlin, Ger- many Grant sponsor: NIH; Grant number: CA42508. *Correspondence to: Holger N. Lode, M.D., Assistant Professor, Charite ´ Children’s Hospital, Dept. of Pediatrics, Augustenburgerplatz 1, 13353 Berlin, Germany. E-mail: holger.lode@charite.de Medical and Pediatric Oncology 35:641–646 (2000) © 2000 Wiley-Liss, Inc.