Delivery of functional DNA and messenger RNA to mammalian phagocytic cells by recombinant yeast.

Among the different vaccination approaches, DNA/RNA vaccination represents a promising means in particular for the induction of effective cellular immune responses conferred by CD8-positive T lymphocytes. To achieve such immune responses, there is a need for novel delivery systems that allow the introduction of nucleic acids to the cytosol of immune cells. We show, for the first time, the delivery of functional DNA and messenger RNA (mRNA) to mammalian antigen-presenting cells, including murine macrophages and human dendritic cells, using the yeast Saccharomyces cerevisiae as the delivery vehicle. After transfer of the particular nucleic acid, subsequent antigen processing and presentation were demonstrated in a human system. Remarkably, release of DNA/mRNA does not require additional 'helper' proteins such as listeriolysin. In conclusion, the yeast-based system described here is superior to many bacterial and viral systems in terms of efficacy, safety and targeting suggesting 'mycofection' as a promising approach for the development of a novel type of live vaccines.

Human cytomegalovirus protein pp65: an efficient protein carrier system into human dendritic cells.

Protein-based immunogens are usually poor inducers of CD8(+) T cells. To enhance the induction of CD8(+) T cells, one approach is the use of protein immunogens coupled to protein transduction domains (PTDs). These are small cationic peptide sequences that significantly enhance the uptake of fused proteins into dendritic cells (DC) and then mediate their presentation in the context of major histocompatibility complex class I (MHC-I) and MHC-II molecules. One drawback of this system is the high concentrations of PTD-fusion proteins required. Here, we show that proteins fused to the human cytomegalovirus tegument protein pp65 were bound with higher efficiency to DCs than those fused to the described PTDs TatPTD and Penetratin. Furthermore, the fusion of pp65 to proteins led to an enhanced uptake of these proteins by DCs. Once taken up, CD4(+) and CD8(+) memory T cells were strongly stimulated ex vivo demonstrating that pp65 was efficiently processed and presented in the context of both MHC-I and MHC-II. These data make pp65 a promising delivery system to induce cellular immune responses by fused protein vaccines.