Superior protective immunity against murine listeriosis by combined vaccination with CpG DNA and recombinant Salmonella enterica serovar typhimurium

Abstract

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.

FIG. 1.

Kinetics of p60-specific CD8 T-cell development in spleens of BALB/c mice after vaccination with either Salmonella strain SB824(pHR241) or CPG and the p60_217-225 peptide (for designations of the vaccine groups, see Table 1). MHC tetramer staining and FACS analysis were performed on days −7, 0, 7, 14, and 28 of the immunization schedule. Means and standard deviations of 10 mice per vaccination group are indicated.

FIG. 2.

Kinetics of p60-specific CD8 T-cell development in spleens of BALB/c mice after combined vaccination with Salmonella SB824(pHR241) and CPG with p60_217-225 (for designations of the vaccine groups, see Table 1). MHC tetramer staining and FACS analysis were performed on days −7, 0, 7, 14, and 28 of the immunization schedule. Means and standard deviations of 10 mice per vaccination group are indicated.

FIG. 3.

Protective capacity of p60-specific CD8 T cells induced by different vaccination protocols (for designation of vaccine groups see Table 1). (A to F) On days 7, 14, or 28 mice were intravenously challenged with a lethal dose of L. monocytogenes. Three days later, the bacterial loads of spleens with Listeria were determined. The results are expressed as the mean log₁₀ CFU ± the standard deviations of 10 mice per time point and vaccination group. An asterisk indicates a value that differs significantly from that of the respective nonimmunized group of mice (*, P < 0.01).

FIG. 4.

Absolute numbers of p60_217-225-tetramer-positive central memory (T_CMC), effector memory (T_EMC), and effector (T_EC) CD8 T cells per 10⁶ splenocytes on days 7, 14, and 28 of the immunization schedule (for designation of vaccine groups see Table 1). (A to F) Cells were stained for expression of CD127 and CD62L. Bars show means and standard deviations of 10 mice per time point and vaccination group. Asterisks indicate values that differ significantly from the corresponding values obtained from mice of group S and C-S, respectively (*, P < 0.01).