The tumor-associated antigen disialoganglioside-GD2 is expressed on neuroblastoma and melanoma and is an established target for passive immunotherapy. The aim of this study was to develop an active immunization strategy leading to the induction of a humoral anti-GD2 immune response. However, carbohydrates and glycolipids are T cell-independent antigens (TI) and usually evoke a poor immune response in tumor-bearing hosts.Here, we describe the identification, characterization and in vivo efficacy of cyclic peptides mimicking the structure of glycolipid GD2, i.e. GD2 mimotopes, in order to overcome T-cell independency. First, GD2 peptide mimotopes were identified by biopanning experiments of a phage-display library displaying circular decapeptides against the human/mouse chimeric anti-GD2 antibody (Ab) ch14.18. Thirteen independent phage clones were isolated which bind to ch14.18 with high specificity. Competitive binding of phages expressing GD2 peptide mimotopes to ch14.18 antibody revealed two superior peptide candidates, mimotope A (MA) and and mimotope D (MD), which were subjected to further evaluation. Second, two plasmid DNA minigene vaccines were generated by overlapping PCR encoding for MA and MD, respectively. The plasmids were based on pSecTag2-A also including a kappa leader sequence, a T-cell helper epitope from HIV-1 gp 120 (T1) and a myc-tag. Minigene expression was demonstrated following transfection of COS-7 cells in western-blots and GD2 mimikry was determined in solid phase ELISA experiments. Third, the efficacy of these mimotope DNA vaccines to induce a tumor protective anti-GD2 immune response was tested in the syngeneic NXS2 model of neuroblastoma expressing ganglioside GD2. The DNA vaccination was accomplished with attenuated Salmonella typhimurium (SL 7207) used as an oral vaccine carrier. Only mice receiving the mimotope DNA vaccines revealed a decrease in primary tumor growth by 50% and a dramatic reduction of spontaneous liver metastases with a mean liver weight of 1g in both groups (MA and MD) in contrast to negative controls (3g). Interestingly, mice immunized with KLH conjugated peptide mimotopes A and D revealed an increased rate of s.c. tumor growth and spontaneous liver metastasis with average liver weights of 5 (MA) and 7 (MD), respecively, suggesting the induction of tolerance using this peptide vaccine approach. Finally the highest anti-GD2 humoral immune response was observed in sera of mice from both GD2 mimotope DNA vaccine groups, consistent with the anti-tumor reponse observed in vivo. Based on these data, we belive that GD2 mimotope DNA vaccines may provide a useful strategy for active immunization against neuroblastoma.

Abstract: The disruption of self‐tolerance against neuroblastoma is the ultimate goal of an effective DNA‐vaccine. We demonstrate the induction of protective immunity against syngeneic murine NXS2 neuroblastoma in A/J mice following vaccination with tyrosine hydroxylase (TH)‐derived antigens. Oral gene delivery was accomplished using an attenuated strain of Salmonella typhimurium as a carrier harboring vectors encoding for mouse tyrosine hydroxylase (mTH) antigens. Vaccination was effective in protecting animals from a lethal challenge with wild‐type NXS2 tumor cells. These findings were extended by comparing efficacy of mTH minigene vaccines with a minigene vaccine comprising three novel epitopes isolated fom NXS2 neuroblastoma cells. For this purpose, MHC class I was immunoprecipitated from NXS2 cell lysates, and peptides were eluted and examined in tandem‐mass spectrometry analysis. This led to the identification of three novel natural MHC class I peptide ligands: TEALPVKLI, from ribonucleotide reductase M2; NEYIMSLI, from Ser/Thr protein phosphatase 2A; and FEMVSTLI, of unknown origin. Two minigenes were constructed, one encoding for the three novel epitopes and the second for three known mTH‐derived epitopes with high predicted binding affinity to MHC class I, by cloning them into the mammalian expression vector pCMV‐3FUB. Immunized mice showed a reduction in primary tumor growth and the absence of spontaneous liver metastasis in the majority of animals. Importantly, there was no significant difference between the two minigenes, suggesting that, compared with tumor peptide isolation, mTH epitope prediction is similarly effective for designing efficient DNA‐minigene vaccines. In summary, these findings establish proof of the concept that disruption of self‐tolerance against neuroblastoma‐associated epitopes may be an effective adjuvant therapeutic strategy.

Background Allergen-induced sensitization and airway disease are the results of adverse immune reactions against environmental antigens that may be prevented or inhibited by immune modifying strategies.Objective To investigate the effects of the novel immune response modifier resiquimod (R-848), from the family of imidazol-derivates, in a murine model of allergen-mediated Th2-immune responses and concomitant airway inflammation and airway hyper-reactivity.Methods BALB/c mice were systemically sensitized with ovalbumin (OVA) on days 1 and 14 and challenged with OVA aerosol on days 28 and 29. R-848 was applied intranasally to sensitized animals once prior to the first allergen airway challenge, on day 27.Results A single application of R-848 significantly reduced numbers of eosinophils and lymphocytes in bronchoalveolar lavage fluid and inhibited mucus gland hyperplasia, compared with sensitized and challenged controls. Associated with the decrease in airway inflammation, single intranasal treatment with R-848 abolished the development of airway hyper-reactivity after allergen sensitization and airway challenges. Additionally, Th2-cytokine production in lung tissues from sensitized and R-848-treated animals was reduced, whereas IL-12 and IFN-gamma production was increased, compared with non-treated sensitized mice.Conclusion These data indicate that R-848 effectively inhibits allergen-induced airway inflammation and hyper-reactivity by modulation of increased Th2-immune responses.

BACKGROUND:Transcriptional factors of the signal transducer and activator of transcription (STAT) family play an important role in orchestrating immune reactions. OBJECTIVE:The aim of the current study was to investigate the role of STAT-1 in murine allergen-induced sensitization and development of airway inflammation (AI) and airway hyperreactivity (AHR), cardinal features of bronchial asthma. METHODS:BALB/c mice were systemically sensitized to ovalbumin and challenged with ovalbumin through the airways. Decoy oligonucleotide (ODN) specific for STAT-1 was applied once locally to the airways of sensitized animals before allergen airway challenges. RESULTS:Single application of decoy ODN markedly and significantly reduced numbers of eosinophils and lymphocytes in bronchoalveolar lavage fluids compared with those seen in sensitized and challenged animals receiving mutant control ODN. Associated with this decrease in eosinophilic AI were significantly reduced levels of IL-5 in BAL fluid, of CD40 expression in peribronchial infiltrates, and of vascular cell adhesion molecule 1 expression in vascular endothelial cells, respectively. In addition, development of AHR after allergen sensitization and airway challenges was effectively abolished after local STAT-1 decoy ODN treatment. CONCLUSION:The data indicate that a decoy ODN neutralizing STAT-1 effectively inhibits allergen-induced AI and AHR, probably by attenuating upregulation of costimulatory and adhesion molecules, and suggest a significant role of STAT-1 in asthma pathology.