Recombinant E. coli efficiently delivers antigen and maturation signals to human dendritic cells: Presentation of MART1 to CD8⁺ T cells

Abstract

The generation of tumour-specific cytotoxic T-lymphocyte (CTL) responses is the primary focus in the design of immunotherapeutic cancer vaccines. We have recently demonstrated generation of ovalbumin (OVA)-specific CTLs and tumour-protection in a murine tumour model using vaccination with dendritic cells (DCs) pulsed with E. coli expressing listeriolysin O (LLO) and OVA as a model antigen. In this system paraformaldehyde fixation of E. coli/LLO provided an additional safety feature without compromising vaccine efficacy. We therefore reasoned that paraformaldehyde-fixed recombinant E. coli expressing LLO would be an efficient vehicle for the delivery of human tumour antigens to human DCs. In the present study, we demonstrate that fixed E. coli expressing LLO are taken up efficiently by human monocytederived DCs (MoDCs) with minimal toxicity. As a consequence of the interaction with bacteria, human DCs undergo marked phenotypic and functional maturation. Furthermore, we show that fixed E. coli/LLO expressing the well-characterised human melanoma antigen, MART1, efficiently deliver the HLA-A2-restricted MART1 27-35 epitope for processing and presentation on human MoDCs, suggesting the potential of this system as a novel strategy for human tumour immunotherapy. © 2003 Wiley-Liss, Inc. Key words: dendritic cell; E. coli; tumour immunotherapyThe generation of tumour-specific cytotoxic T-lymphocyte (CTL) responses is the primary focus in the design of immunotherapeutic cancer vaccines. It is generally accepted that the initiation of effective antitumour immune responses relies upon antigen-presenting cells (APCs), most notably dendritic cells (DCs), priming CTLs and T helper cells, by processing antigen and presenting it on major histocompatibility (MHC) class I and class II molecules, respectively. DCs are unique in their potent ability to prime naïve T-cell responses, 1,2 and DCs loaded with tumour antigens show increasing potential as vaccine candidates in animal models 3,4 and early-phase clinical trials. [5][6][7][8][9] The generation of appropriate immune responses, and therefore successful vaccination, is highly dependent on the development of effective methods of antigen delivery to DCs. The ideal antigen delivery system should combine efficient processing and presentation of tumour antigens to CD8 ϩ T cells, along with potent DC activation in order to deliver the costimulatory signals necessary for T-cell priming. Preparations of tumour-specific antigens are desirable as less well defined whole tumour cell preparations or lysates contain selfantigens and may result in the induction autoimmunity. In addition, it is desirable to deliver the whole antigen to enable presentation of multiple epitopes without prior knowledge of the patient's HLA haplotype or the nature of the peptide epitopes.A number of vaccine strategies have already been shown to induce potent CTL responses, including infection with recombinant viruses, and DNA or RNA transfection. However, there are safety concerns with the expression of whole a...

Listeriolysin O Expressed in a Bacterial Vaccine Suppresses CD4+CD25high Regulatory T Cell Function In Vivo

“…Using the model tumor Ag OVA, we have demonstrated that E. coli LLO OVA is a powerful tool for successful antitumor vaccination, through its ability to generate high-avidity CTL and Treg functional defect leading to the rejection of tumor. Recombinant E. coli have already been described as protein delivery vectors for professional phagocytic cells (6,20,21) and presentation of the OVA epitope on MHC class I by E. coli LLO/OVA has been shown to be orders of magnitude more efficient than E. coli OVA strains (6,20). Therefore, the difference in efficacy of the two vaccines may be attributed to a difference in efficiency of MHC class I presentation on APCs.…”

“…Through the pore-forming action of LLO, the cytoplasmic contents of the bacteria can then escape into the cytosol and thereby be processed by the proteasome. In vitro, this LLO-mediated process has been shown to improve MHC class I presentation of the OVA H2-K b -restricted epitope SIINFEKL by mouse macrophages (20), mouse bone-marrow-derived dendritic cells (BMDCs) (6), the HLA-A2-restricted MART1 [27][28][29][30][31][32][33][34][35] epitope (21), and the immunodominant epitope of the influenza matrix protein (22) by human monocytes-derived DCs. In vivo, s.c. injection of E. coli LLO expressing the model chicken OVA Ag (E. coli LLO/OVA) has been shown to trigger a very strong antitumor response against the highly aggressive B16/ OVA melanoma cell line (6) (B16-OVA).…”

Potential therapeutic applications of recombinant, invasive E. coli

“…28 E. coli coexpressing LLO and a model antigen (truncated chicken ovalbumin, OVA) were incubated with the cells, taken up and presentation of the OVA epitope on MHC class I was observed. 26,27 In addition, an antitumour effect was observed when these bacteria were used in a vaccination protocol.…”

“…27 Similar in vitro data were obtained when human DCs were used to present MART-1 antigen epitopes. 28 In the present communication, we have used an invasive E. coli to deliver therapeutically relevant molecules to mouse and human nonprofessional phagocytic cells. We demonstrate that protein delivery is much more efficient than gene delivery.…”

Listeriolysin O as a strong immunogenic molecule for the development of new anti-tumor vaccines

“…Additionally, the use of a typical human melanoma antigen (MART1) instead of OVA in the vaccine efficiently delivered the MART127-35 antigen epitope for processing and presentation by human MoDCs. 117 The anti-tumor efficacy of the paraformaldehydefixed E. coli vaccine is maintained, and this vaccine is significantly less harmful to humans. Similarly, another research team illustrated that an LLO-based E. coli vaccine could induce a strong immune response against a WT1-expressing leukemia tumor in vivo through enhanced CTL activity.…”