Developing DNA fusion gene vaccines : from pre-clinical models to clinical studies

Abstract

We previously showed that a DNA vaccine encoding a tumour antigen linked to Fragment C (FrC) of tetanus toxin induced protective immunity in vaccinated mice, whereas a DNA vaccine lacking the FrC sequence did not.

Several changes were made to the design of the vaccine in order to activate CD8+ T cell responses.  Firstly the C-terminal domain of FrC was removed and secondly the minimized tumour epitope was placed at the C-terminus of the remaining domain of FrC (DOM).  In order to assess whether this design was applicable to a non-viral tumour antigen we chose an H-2 Kb epitope from CEA, referred to as EAQN.  The sequence encoding this octamer was fused to the C-terminus of domain 1 of FrC (pDOM.EAQN).  This vaccine activated high levels of epitope-specific CTLs whereas vaccines against the whole CEA gene and the CEA gene fused to FrC were unable to induce CTL against EAQN. However the CTL specific for EAQN were unable to lyse tumour cells expressing CEA in vitro suggesting that the tumour cells failed to present the peptide.  Several epitopes have been described from CEA that are restricted by the human class I molecule HLA-A2.  We placed each of these in turn into our vaccine.  All of the vaccines were able to elicit high levels of CD8+ T cells specific for their respective epitopes.  A DNA vaccine encoding a recently discovered epitope generated a CTL capable of lysing tumour cells expressing CEA.

In order to move this novel vaccine design towards a clinical trial, an established epitope from a matrix protein (pp65) of cytomegalovirus was studied.  The sequence encoding this nonamer was fused to the C-terminus of the first domain of FrC (pDOM.NLV).  This vaccine was able to generate high levels of CD8+ T cells specific for this epitope.  These CTL were able to lyse cells that were infected with a modified vaccine Ankara expressing the matrix protein-pp65.  We also tested the vaccine format in human cells.  We transfected dendritic cells with RNA transcribed from the vaccine.  These dendritic cells were potent stimulators of autologous CD8+ T cells and led to 50-fold expansion over 7 days in vitro.  A prime-boost strategy was also developed in this model that combined our DNA vaccine with long peptide vaccination.

Finally we studied immune responses in patients who had received this DNA vaccine in the context of allogeneic stem cell transplantation.

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