Escherichia Coli Expressing Recombinant WT1 Protein and Listeriolysin O Stimulate Antigen-Specific T Cells Following Vaccination in C57BL/6 Mice.

The Wilm’s Tumor (WT1) gene is expressed at high levels in most types of leukemia, but also in various types of solid tumors. We tested the efficacy of recombinant Escherichia Coli (E. Coli) expressing Listeriolysin O (LLO) as an antigen delivery vector for tumor specific immunotherapy targeting WT1 gene products. C57BL/6 (B6) mice were immunised with E. Coli expressing a form of WT1 protein truncated of 70 amino acids at the N-Terminus portion, and then challenged subcutaneously with 5X106 WT1-expressing TRAMP-C tumor cells. A significant inhibitory effect on TRAMP-C tumor growth was observed in vivo in vaccinated mice, with no signs of toxicity in tissues such as liver and kidney on histopathlogic examination. Furthermore, splenocytes from immunised mice were stimulated in vitro either with peptides that have been shown to contain major histocompatibility (MHC) class I binding anchor motifs (P126: RMFPNAPYL and P330: CNKRYFKL), or with irradiated TRAMP-C tumor cells. Specific CD8-positive cytotoxic T lymphocytes (CTLs) responses against TRAMP-C tumor cells were seen in cultures stimulated with irradiated tumor cells. However, no CTL activity was found in cultures stimulated with the relevant WT1 peptides that specifically lyse TRAMP-C tumor cells or the leukemia cell line RMA-S cells pulsed with each of the peptides. These results suggest that P126 and P330 might not be naturally processed WT1 epitopes for B6 mice and supports the possibility of other B6 CTLs epitopes. We are currently using a WT1 peptide library to screen immunised animals to look for novel peptides. Immunisation with E. Coli expressing WT1 protein but not LLO did not show any effect on TRAMP-C tumor growth in vivo. In this latter group, western blot analysis revealed WT1 specific antibodies directed against the N-terminus portion of the WT1 protein in the sera of immunised mice. Altogether the results presented here showed that E. Coli expressing WT1 could stimulated antigen-specific T cells dependant on LLO co-expression and suggest possible vaccine strategies for generating CD8+ T cell responses against tumors.