We have developed a new vaccination strategy by using the Salmonella type III secretion system (T3SS) to translocate heterologous antigens into the cytosol of host cells. This leads to an efficient antigen-specific CD8 T cell induction. Recently, we have demonstrated the use of Salmonella's T3SS for the immunoprophylaxis of a solid tumor. The murine fibrosarcoma WEHI 164 was transfected with the DNA sequence encoding the MHC class I-peptide p60(217-225) from Listeria monocytogenes. In the present study, we used this tumor model to investigate the potential of vaccination with recombinant Salmonella in a therapeutic setting. BALB/c mice were subcutaneously challenged with WEHI-p60 cells. Simultaneously or 4 days later, these mice received either an orogastric or intravenous immunization with Salmonella translocating p60. Interestingly, 71-80% of the intravenously and 50-52% of the orogastrically immunized mice showed a complete tumor regression after 14 days. In addition, the distribution of tetramer-positive p60(217-225)-specific CD8 T cell subpopulations in blood and tumor tissue was analyzed. Co-staining with CD62L and CD127 revealed that the frequencies of p60(217-225)-specific effector and effector memory CD8 T cells in blood and in fibrosarcoma tissue were related to the kinetics of tumor regression. In summary, our study demonstrates that therapeutic vaccination with Salmonella leads to efficient induction of tumor-invading effector CD8 T cells that may result in significant tumor regression.

Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) fromListeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.Protein antigens are recognized by T cells as short peptide fragments bound either to major histocompatibility class (MHC) I or to MHC class II molecules on the surface of antigen-presenting cells (APCs). The location of antigens in distinct intracellular compartments of APCs influences their proteolytic processing as well as access to MHC molecules (16, 39). Classically, peptides generated in the cytosol (e.g., derived from viral proteins) by proteasomal degradation are bound to MHC class I molecules after transport across the endoplasmic reticulum membrane by the transporters associated with antigen processing (TAP) (90). Subsequently, MHC class I molecules present these endogenous antigenic peptides to CD8 T cells. In contrast, exogenous antigens (e.g., antigens derived from engulfed bacteria and soluble antigens) are directed into the endosomal/lysosomal pathway for degradation (63). In late endosomal compartments, degraded protein fragments interact with MHC class II molecules and are further trimmed into peptides for presentation to CD4 T cells.However, the synchrony of this system has been challenged by biochemical and functional studies of professional and nonprofessional APCs. Epitopes derived from a diverse ...

Preexisting antivector immunity can severely compromise the ability of Salmonella enterica serovar Typhimurium live vaccines to induce protective CD8 T-cell frequencies after type III secretion system-mediated heterologous protein translocation in orally immunized mice. To circumvent this problem, we injected CpG DNA admixed to the immunodominant p60 217-225 peptide from Listeria monocytogenes subcutaneously into BALB/c mice and coadministered a p60-translocating Salmonella strain by the orogastric route. The distribution of tetramer-positive p60 217-225 -specific effector and memory CD8 T cells was analyzed by costaining of lymphocytes with CD62L and CD127. In contrast to the single oral application of recombinant Salmonella or single immunization with CpG and p60, in the spleens from mice immunized with a combination of both vaccine types a significantly higher level of p60-specific CD8 T cells with a predominance of the effector memory T-cell subset was detected. In vivo protection studies revealed that this CD8 T-cell population conferred sterile protective immunity against a lethal infection with L. monocytogenes. However, p60-specific central memory CD8 T cells induced by single vaccination with CpG and p60 were not able confer effective protection against rapidly replicating intracellular Listeria. In conclusion, we provide compelling evidence that the combination of Salmonella type III-mediated antigen delivery and CpG immunization is an attractive novel vaccination strategy to modulate CD8 differentiation patterns toward distinct antigen-specific T-cell subsets with favorable protective capacities.The type III secretion system (T3SS) of Salmonella enterica serovar Typhimurium can be used to target heterologous antigens directly into the cytosol of antigen-presenting cells (30,32,33). Our laboratory has reported that the single oral immunization of mice with a recombinant Salmonella strain expressing the translocated Yersinia outer protein E (YopE) fused to the immunodominant antigen p60 from Listeria monocytogenes results in the efficient induction of p60-specific CD8 T cells (33). In further experiments, we explored the possibility to induce enhanced levels of antigen-specific CD8 T cells by oral boost immunization using the same recombinant serovar Typhimurium strain (38). We demonstrated that the rapid clearance of the Salmonella vaccine carrier due to anti-Salmonella vector immunity after the second immunization prevents a significant elevation of T-lymphocyte numbers (38). In a more recent study, we showed that heterologous prime-boost immunizations using attenuated serovar Typhimurium and serovar Dublin strains for foreign antigen delivery can be used to bypass anti-Salmonella immunity resulting in enhanced antigen-specific CD8 T-cell induction (40). However, the translation of this heterologous prime-boost immunization approach from mice to man is not trivial due to the lack of S. enterica vaccine strains with different O antigens approved for use in humans.An alternative vaccination strategy to ...