Delivery of class I and class II MHC-restricted T-cell epitopes of listeriolysin of Listeria monocytogenes by attenuated Salmonella

Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215–226) or class I (LLO 91–99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated ΔaroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215–226 epitope, indicating that LLO 215–226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunication with the living, chromosomally integrated Salmonella construct carrying LLO 215–226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91–99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91–99 inserts: 91–99 (normal flagellin amino acids as flanking residues); KK91–99KK (Lys-Lys flanking residues); and AAA91–99AAA (Ala-Ala-Ala flanking residues). Spleen cells from mice immunized with the KK91–99KK flagellin chimeric Salmonella strain showed the greater CTL response indicating that Lys-Lys flanking residues are associated with enhanced immunogenicity, possibly by facilitating the proteolytic excision of the epitope in the endosomal compartment of antigen-presenting cells. A statistically significant decrease in the number of hepatic and/or splenic bacteria was achieved after Listeria challenge in mice immunized with Salmonella constructs expressing either MHC class II LLO 215–226 or class I LLO KK91–99KK flagellin chimera, showing that the immunogenicity of these constructs is associated with an epitope-specific increased resistance to infection.