The primary goal of this Phase I study was to assess the safety and bioactivity of tumor lysate-pulsed dendritic cell (DC) vaccination to treat patients with glioblastoma multiforme and anaplastic astrocytoma. Adverse events, survival, and cytotoxicity against autologous tumor and tumor-associated antigens were measured. Fourteen patients were thrice vaccinated 2 weeks apart with autologous DCs pulsed with tumor lysate. Peripheral blood mononuclear cells were differentiated into phenotypically and functionally confirmed DCs. Vaccination with tumor lysatepulsed DCs was safe, and no evidence of autoimmune disease was noted. Ten patients were tested for the development of cytotoxicity through a quantitative PCR-based assay. Six of 10 patients demonstrated robust systemic cytotoxicity as demonstrated by IFN-␥ expression by peripheral blood mononuclear cells in response to tumor lysate after vaccination. Using HLA-restricted tetramer staining, we identified a significant expansion in CD8؉ antigen-specific T-cell clones against one or more of tumorassociated antigens MAGE-1, gp100, and HER-2 after DC vaccination in four of nine patients. A significant CD8؉ T-cell infiltrate was noted intratumorally in three of six patients who underwent reoperation. The median survival for patients with recurrent glioblastoma multiforme in this study (n ‫؍‬ 8) was 133 weeks. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous tumor lysate-pulsed DC vaccine for patients with malignant glioma. We demonstrate for the first time the ability of an active immunotherapy strategy to generate antigenspecific cytotoxicity in brain tumor patients.

DNA vaccines were introduced less than a decade ago but have already been applied to a wide range of infectious and malignant diseases. Here we review the current understanding of the mechanisms underlying the activities of these new vaccines. We focus on recent strategies designed to enhance their function including the use of immunostimulatory (CpG) sequences, dendritic cells (DC), costimulatory molecules and cytokine-and chemokine-adjuvants. Although genetic vaccines have been significantly improved, they may not be sufficiently immunogenic for the therapeutic vaccination of patients with infectious diseases or cancer in clinical trials. One promising approach aimed at dramatically increasing the immunogenicity of genetic vaccines involves making them 'selfreplicating'. This can be accomplished by using a gene encoding RNA replicase, a polyprotein derived from alphaviruses, such as Sindbis virus. Replicase-containing RNA vectors are significantly more immunogenic than conventional plasmids, immunizing mice at doses as low as 0.1 μg of nucleic acid injected once intramuscularly. Cells transfected with 'self-replicating' vectors briefly produce large amounts of antigen before undergoing apoptotic death. This death is a likely result of requisite double-stranded (ds) RNA intermediates, which also have been shown to super-activate DC. Thus, the enhanced immunogenicity of 'self-replicating' genetic vaccines may be a result of the production of pro-inflammatory dsRNA, which mimics an RNA-virus infection of host cells.

'Naked' nucleic acid vaccines are potentially useful candidates for the treatment of patients with cancer, but their clinical efficacy has yet to be demonstrated. We sought to enhance the immunogenicity of a nucleic acid vaccine by making it 'self-replicating'. We accomplished this by using a gene encoding an RNA replicase polyprotein derived from the Semliki forest virus, in combination with a model antigen. A single intramuscular injection of a self-replicating RNA immunogen elicited antigen-specific antibody and CD8+ T-cell responses at doses as low as 0.1 microg. Pre-immunization with a self-replicating RNA vector protected mice from tumor challenge, and therapeutic immunization prolonged the survival of mice with established tumors. The self-replicating RNA vectors did not mediate the production of substantially more model antigen than a conventional DNA vaccine did in vitro. However, the enhanced efficacy in vivo correlated with a caspase-dependent apoptotic death in transfected cells. This death facilitated the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. Naked, non-infectious, self-replicating RNA may be an excellent candidate for the development of new cancer vaccines.

Cancer vaccines targeting 'self' antigens that are expressed at consistently high levels by tumor cells are potentially useful in immunotherapy, but immunological tolerance may block their function. Here, we describe a novel, naked DNA vaccine encoding an alphavirus replicon (self-replicating mRNA) and the self/tumor antigen tyrosinase-related protein-1. Unlike conventional DNA vaccines, this vaccine can break tolerance and provide immunity to melanoma. The vaccine mediates production of double-stranded RNA, as evidenced by the autophosphorylation of protein kinase R. Double-stranded RNA is critical to vaccine function because both the immunogenicity and the antitumor activity of the vaccine are blocked in mice deficient for the RNase L enzyme, a key component of the 2′,5′-linked oligoadenylate synthetase antiviral pathway involved in double-stranded RNA recognition. This study shows for the first time that alphaviral replicon-encoding DNA vaccines activate innate immune pathways known to drive antiviral immune responses, and points the way to strategies for improving the efficacy of immunization with naked DNA.Vaccine vectors based on recombinant viruses have been used for many years, but the delivery of target antigens can be accompanied by unwanted side effects. First, preexisting antibodies can neutralize the recombinant virus before it is able to deliver its payload. Second, structural proteins from the virus can dominate T-and B-cell-mediated immune responses, diverting immunity away from the target immunogen 1,2 . Hence, there is a critical need to develop vaccine vectors that are not only highly immunogenic, but also antigenically simple.The simplest of all recombinant vectors, naked plasmid DNA vaccines, have successfully been used in animal models to induce immune responses to many pathogens and model antigens. DNA vaccines are easy to produce, inexpensive and safe 3 , but for some applications insufficiently immunogenic. A variety of attempts have been made to improve DNA vaccines 4 , including the production of pro-apoptotic proteins 5 . Despite these advances, the poor immunogenicity of plasmid DNA remains apparent when attempting to elicit immunity to weak immunogens, such as non-mutated 'self' tumor-associated antigens that are recognized by anti-tumor T cells. One promising new strategy to improve naked DNA vaccines is to express the target antigen under the control of an alphaviral replicase 6,7 with the premise of using the ability of alphavirus to produce large amounts of viral mRNA (refs. 8 ,9 ). In alphavirus-derived DNA and RNA vaccines, the encoded alphaviral replicase-enzyme complex amplifies self-replicating RNA (replicon). In model systems, replicon containing nucleic acid vaccines display therapeutic efficacy at doses several logs lower than those required by conventional DNA vaccines 10-12 . In the current study, we examined whether a replicase-based DNA vaccine encoding a nonmutated self-antigen could be used to break tolerance and prevent B16 melanoma, a goal not previously ...