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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 p60217-225 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 p60217-225-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 circumvent the obstacle of preexisting antivector immunity might be the combined administration of the above-mentioned Salmonella live vaccine and a synthetic deoxycytidyl-deoxyguanosine (CpG) oligonucleotide mixed with the p60217-225 antigenic peptide. CpG oligonucleotides contain unmethylated CG motifs similar to those found in bacterial DNA that trigger Toll-like receptor 9 (TLR9) in the vertebrate immune system (12, 45, 48). Multiple studies have conclusively demonstrated that CpG oligonucleotides activate innate, humoral, and cellular immune responses (19, 20, 22, 23, 24, 47). Further investigations revealed that CpG DNA admixed to proteinaceous antigens are efficient adjuvants for vaccine-induced CD8 T-cell responses in mice and humans (11, 23, 42).
In the present study, we injected CpG DNA combined with the immunodominant p60217-225 peptide subcutaneously into mice and administered a p60-translocating Salmonella strain orally at different time points. Our results show rapid and consistent CD8 T-cell responses in vivo that confer superior protection against listeriosis compared to single oral immunization with recombinant Salmonella, highlighting the potential of this novel vaccination protocol to enhance antigen-specific CD8 T-cell responses.
(This research was conducted by C. Berchtold in partial fulfillment of the requirements for a Ph.D. from Ludwig Maximilians-University Munich, Munich, Germany, 2009.)
Previously, the construction of plasmid pHR241 has been outlined in detail (33). Briefly, this derivative of pWSK29 is a low-copy-number expression vector and bears the genetic information for translocated chimeric YopE1-138/p60130-477/M45 under expression control of the lac promoter, which is constitutively active in Salmonella. The above-described plasmid was transformed into S. enterica serovar Typhimurium strain SB824 by electroporation. Strain SB824 (32) was engineered by introducing the sptP::kan mutant allele from strain SB237 (18) into the ΔaroA strain SL3261 (13) by P22HTint transduction. Serovar Typhimurium was grown in Luria-Bertani medium supplemented with 0.3 M NaCl (pH 7.4) to allow expression of components and targets of the T3SS encoded by the Salmonella pathogenicity island 1, which mediates Salmonella invasion of host cells (10). L. monocytogenes strain 10403s was used for challenge experiments in vaccinated mice (4).
Specific-pathogen-free female BALB/c mice (6 to 8 weeks old) were purchased from Harlan-Winkelmann (Horst, The Netherlands). For the experiments, animals were housed in groups of five mice under standard barrier conditions in individually ventilated cages (Tecniplast, Buguggiate, Italy). Water and food were withdrawn for 4 h before groups of mice were orally immunized with 5 × 108 CFU of the Salmonella vaccine strain by using round-bottom gavage needles. Thereafter, drinking water ad libitum was offered immediately, and food was provided at 2 h postimmunization. Each experiment was performed at least twice with similar results. Animal experiments were approved by the German authorities and performed according to the legal requirements.
The CpG oligonucleotide 1826 was synthesized on a phosphothioate backbone by Coley Pharmaceutical Group (Wellesley, MA). The oligonucleotide contains two CpG motifs (underlined) with the sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′. Peptide p60217-225 with the sequence KYGVSVQDI was synthesized by Jerini Peptide Technologies (Berlin, Germany). At indicated immunization time points, 100 μg of CpG ODN 1826 combined with 50 μg of p60217-225 were injected subcutaneously into the flanks of the mice.
The generation of tetrameric H2-Kd/p60217-225 complexes has been outlined in detail (6). Briefly, recombinant H2-Kd heavy chain and β2-microglobulin were expressed as insoluble inclusion bodies in Escherichia coli and were further purified. The H2-Kd heavy-chain molecule was mutated to remove the transmembrane and cytosolic domain and to add a specific biotinylation site at the C terminus. Purified proteins were refolded in vitro in the presence of high concentrations of synthetic peptides (Biosythan, Berlin, Germany) to form stable and soluble major histocompatibility complex (MHC)/peptide complexes. Complexes were specifically biotinylated in vitro by adding the enzyme BirA, d-biotin, and ATP. After further purification, biotinylated MHC/peptide complexes were multimerized with streptavidin-PE (Molecular Probes, Eugene, OR). Tetrameric complexes were purified by gel filtration and stored at 2 to 5 mg/ml at 4°C in phosphate-buffered saline (pH 8.0) containing 0.02% sodium azide, 1 μg of pepstatin/ml, 1 μg of leupeptin/ml, and 0.5 ml of EDTA.
At the indicated time points, spleens were removed from animals, harvested by dissociation through a wire mesh, and subsequently resuspended in RP10+, which consists of RPMI 1640 supplemented with 10% fetal calf serum, l-glutamine, HEPES (pH 7.5), 2-mercaptoethanol, penicillin (100 U/ml), streptomycin (100 μg/ml), and gentamicin (50 μg/ml).
The p60217-225-specific T-cell populations were detected with phycoerythrin (PE)-conjugated, tetrameric MHC-I/peptide complexes and concurrently stained for other surface molecules using directly conjugated monoclonal antibodies as described previously (6, 7). Briefly, cells were incubated with ethidium bromide monoazide for live/dead discrimination in fluorescence-activated cell sorting (FACS)-staining buffer (phosphate-buffered saline [pH 7.45], 0.5% bovine serum albumin, and 0.02% sodium azide). Subsequently, cells were stained with the above mentioned MHC-I tetramer and surface markers for 1 h. The following monoclonal antibodies were used: anti-CD8α (clone 53-5.8; Pharmingen, Heidelberg, Germany), anti-CD62L (clone Mel-14; Pharmingen), and anti-CD127 (clone A7R34; eBioscience, San Diego, CA) antibodies. Cells were resuspended in staining buffer and fixed in 1% paraformaldehyde/phosphate-buffered saline (pH 7.45). The data were acquired on a FACSCanto (BD Biosciences, San Jose, CA) and further analyzed with FlowJo software (TreeStar, Ashland, OR).
On days 7, 14, and 28 of the immunization schedule (Table (Table1)1) mice were intravenously challenged with 104 CFU of log-phase L. monocytogenes strain 10403s in 0.1 ml of phosphate-buffered saline. Three days after the challenge, CFU were determined by plating serial dilutions of spleen homogenates on brain heart infusion agar. Colonies were enumerated after 24 to 48 h of incubation. Colony counts were corrected for dilution and averaged to yield CFU per organ. The level of protection was calculated as the log10 difference of the bacterial counts from naive control and immunized mice. Each experiment was performed at least twice with similar results.
The statistical significance of the results was checked with the nonparametric Mann-Whitney U test at the 0.01 significance level. All tests were performed by using the SPSS software (SPSS, Chicago, IL).
Our laboratory has used the p60 protein of L. monocytogenes as a model antigen for the construction of hybrid YopE proteins to be delivered by the Salmonella-T3SS (33). After invasion of host cells and the escape from the phagosome, Listeria constitutively secretes the murein hydrolase p60 (31). Subsequently, p60 is directed to the MHC-I antigen processing pathway, leading to the presentation of antigen-derived peptides to CD8 T cells (27). Analysis of T cells from Listeria-infected BALB/c mice revealed that the immunodominant listerial nonamer peptide p60217-225 is presented to cytotoxic CD8 T lymphocytes in the context of the H2-Kd MHC-I molecule (27, 28). The previously described low-copy-number plasmid pHR241 bears the genetic information for a YopE/p60 hybrid protein (33). The N-terminal 138 amino acids of YopE containing the secretion and translocation domains (36, 41) were fused to p60130-477. Constitutive expression of the respective gene fusion led to the production of a hybrid protein that was shown to be translocated into the cytosol of macrophages by serovar Typhimurium (33).
In a first set of experiments, we compared the efficacy of a single orogastric immunization with the attenuated Salmonella strain SB824(pHR241) to a subcutaneous vaccination with the p60217-225 peptide and CpG for priming of p60-specific CD8 T cells. Spleens from vaccinated BALB/c mice were removed on days 7, 14, and 28 after a single or the last immunization, respectively, and the relative numbers of H2-Kd/p60217-225-tetramer-positive CD8 T cells were assessed. The designation of the vaccination groups and the corresponding immunization schedules are shown in Table Table1.1. In Fig. Fig.11 it is demonstrated that in spleens from mice of group C (single application of CpG with p60217-225 on day 0) the highest level of p60-specific CD8 T cells (6.27% ± 1.28%) was detected as early as 7 days after immunization, followed by a gradual decline of antigen-specific T-cell frequencies on days 14 and 28. In contrast (Fig. (Fig.1),1), a boost immunization protocol applied to mice from vaccination group C-C (application of CpG with p60217-225 on days −7 and 0) led to a prolonged level of more than 5% p60-specific CD8 T cells over a time period of at least 14 days (maximum on day 14; 7.14% ± 1.47%). Single orogastric immunization with SB824(pHR241) expressing translocated YopE/p60 (vaccination group S) resulted in a pronounced p60-specific splenic CD8 T-cell response on day 14 (13.10% ± 1.57%), thereafter descending to a level of ca. 5% on day 28 (Fig. (Fig.1).1). For respective dot plots, please see Fig. S1A to C in the supplemental material.
In summary, subcutaneous immunization with CpG and p60217-225 was used to induce a rapid antigen-specific CD8 T-cell response within 7 days. High frequencies of CD8 T cells in spleens of mice were observed for a minimum of 2 weeks after a boost vaccination with CpG. In contrast, immunization with a recombinant Salmonella strain led to a slower development, albeit a higher frequency of p60-specific CD8 T cells.
In further experiments, we wanted to determine whether the combined application of attenuated Salmonella strain SB824(pHR241) and CpG with p60217-225 results in enhanced p60-specific CD8 T-cell priming. In Fig. Fig.22 it is shown that a classical prime-boost immunization protocol applied to mice from group S-C (vaccination with Salmonella on day −7 and with CpG and p60217-225 on day 0) led to very high levels of p60-specific CD8 T cells (24.0% ± 1.45%) on day 14. However, 14 days later (day 28) the T-cell frequency declined to ca. 5%. Surprisingly, a single concomitant application of SB824(pHR241) and CpG with p60217-225 on day 0 (group S/C) resulted in a steady increase of p60-specific CD8 T-cell numbers from day 7 to day 28 (Fig. (Fig.2).2). Spleens from mice revealed a peak value of 29.5% ± 1.37% antigen-specific T cells on the latter day. In contrast, relatively constant CD8 T-cell frequencies ranging from 5 to 10% between day 0 and day 28 were observed in mice from vaccination group C-S (application of CpG and p60217-225 on day −7 and Salmonella on day 0; Fig. Fig.2).2). For respective dot plots, please see Fig. S1D to F in the supplemental material.
Taken together, these results clearly demonstrate that the combination of the attenuated Salmonella strain SB824(pHR241) and CpG with p60217-225 for vaccination purposes improved antigen-specific CD8 T-cell priming in mice. Especially, the simultaneous application of both vaccines appeared to be useful for the induction of high level splenic CD8 T-cell numbers.
We wanted to investigate whether the amounts of p60217-225-specific CD8 T cells induced by our different vaccination protocols (Table (Table1)1) are sufficient to protect mice against a lethal challenge infection with L. monocytogenes. Therefore, on days 7, 14, and 28 of the immunization schedule, animals were intravenously challenged with 104 CFU of wild-type Listeria. Viable bacterial cells were determined in spleens 3 days after the challenge (Fig. 3A to F). Spleens of nonimmunized mice (group NI) revealed Listeria numbers ranging from 105 to 106 CFU per organ. Despite the presence of p60-specific CD8 T cells in spleens of mice from vaccination groups C and C-C (see Fig. S1A and B in the supplemental material), no significant protection against listeriosis was detected on day 28 (Fig. 3A and B). However, as reported by our laboratory (33), mice immunized with Salmonella strain SB824(pHR241) showed a pronounced reduction of listerial colonization in their spleens (100 to 1,000 CFU) 14 and 28 days after vaccination (Fig. (Fig.3C).3C). Strikingly, the high frequencies of p60-specific T cells in group S-C (day 14) and group S/C (day 28) correlated with full protection against the lethal Listeria infection (Fig. 3D and E). In fact, 9 of 10 mice from both vaccination groups revealed sterile immunity at these time points. In spleens from mice of group C-S (Fig. (Fig.3F)3F) a similar level of protection was observed compared to mice from group S (Fig. (Fig.3C3C).
Interestingly, p60217-225-specific CD8 T cells generated by application of CpG and p60217-225 were not able to confer protection against listeriosis, whereas protective immunity was mediated by p60217-225-specific CD8 T cells after immunization with SB824(pHR241) in combination with CpG-p60217-225. What are the underlying mechanisms of this phenomenon?
Antigen-specific T cells can be divided at least into three subsets (35). Effector CD8 T cells (TEC) are characterized by an immediate effector function, a poor proliferative activity, and a limited in vivo survival. In contrast, memory T cells persist for extended periods due to an antigen-independent homeostatic turnover. Central memory T cells (TCMC) reside preferentially in lymphoid organs and lack immediate effector functions (26), whereas effector memory T cells (TEMC) migrate mainly into nonlymphoid tissue and elicit immediate effector functions on antigen reencounter. Recent results indicate that interleukin-7 receptor α-chain (CD127) surface expression is a marker for long-living memory T cells (14). The combination of surface staining for CD127 and L-selectin (CD62L) further distinguishes between TCMC (CD127high/CD62Lhigh) and TEMC (CD127high/CD62Llow), allowing us to distinguish TEC (CD127low/CD62Llow) from memory T cells at early time points in the in vivo immune responses.
Recent data for the Listeria infection model provided strong evidence that TEMC are the main players in conferring protection against murine listeriosis (15). To determine differentiation patterns into the above mentioned distinct p60-specific CD8 T-cell subsets induced by our immunization strategies, spleens from mice were removed 7, 14, and 28 days after vaccination. Figure S2 in the supplemental material shows representative distributions of TCMC, TEMC, and TEC among p60-specific CD8 T cells, whereas in Fig. 4A to F the absolute numbers of these antigen-specific CD8 T-cell subpopulations per 106 splenocytes are given. Spleens from mice of groups C and C-C revealed TCMC as the predominant p60-specific CD8 T-cell subset (>94%) at all time points (see Fig. S2 in the supplemental material and Fig. 4A and B). Thus, vaccination with CpG and p60217-225 resulted mainly in the induction of splenic antigen-specific CD8 T cells lacking immediate effector function. In contrast, costaining of p60217-225-specific lymphocytes with CD62L and CD127 demonstrated that on days 14 and 28 all three CD8 T-cell subpopulations were present in spleens from mice of immunization groups S and C-S (see Fig. S2 in the supplemental material and Fig. 4C and F). At these two time points, <45% of p60-specific CD8 T cells belonged to the TCMC phenotype. An even more pronounced shift to TEMC and TEC subpopulations was observed in vaccination groups S-C and S/C, with up to 55% TEMC and 13 to 25% TEC in these groups on day 28 (see Fig. S2 in the supplemental material and Fig. 4D and E).
These data clearly show that the combined application of recombinant Salmonella and CpG with p60217-225 is an attractive vaccination strategy to mount an antigen-specific CD8 T-cell response biased toward the induction of lymphocytes with an immediate effector phenotype.
CD8 T cells play a pivotal role in the host defense against viruses, intracellular bacteria, and tumors. However, the induction of potent CD8 T-cell responses to fight microbes or cancer remains a major challenge in vaccine development. Live attenuated bacteria (e.g., S. enterica, L. monocytogenes, and mycobacteria) have been widely used as vaccine carriers for foreign antigen display to induce cell-mediated immunity (9, 21, 37). In the past, the T3SS of S. enterica serovar Typhimurium became an attractive tool for heterologous protein delivery directly into the cytosol of macrophages and dendritic cells, resulting in vigorous antigen-specific CD8 T-cell priming and the induction of protective immunity against viruses, bacteria, and tumors (29, 30, 32, 34). More recent studies from our laboratory revealed that preexisting antivector immunity can severely compromise the ability of Salmonella live vaccines to induce protective CD8 T-cell frequencies in orally immunized mice (38, 39, 40).
An alternative potent immunization strategy to improve T-cell responses is the use of CpG oligonucleotides that are recognized by the innate immune system via TLR9 (1, 12). Stimulation of TLR9 by CpG oligonucleotides leads to strong immune activation and can enhance immune responses to coadministered peptide antigens (23). Indeed, CpG oligonucleotides are one of the most potent adjuvants known for the induction of CD8 T-cell responses in mice (5, 8, 11, 46, 49) and humans (42).
In the present study, we applied a novel vaccination strategy to mice by combining Salmonella T3SS-mediated antigen delivery with subcutaneous CpG immunization. After single oral administration of live recombinant Salmonella, it took 14 days to induce significant p60-specific CD8 T-cell numbers in spleens of mice (Fig. (Fig.1).1). This relatively slow T-cell development was clearly improved by single vaccination with CpG and p60217-225, leading to a markedly faster antigen-specific CD8 T-cell response within 7 days (Fig. (Fig.1).1). Combined application of Salmonella and CpG vaccination did not further enhance the speed of T-cell priming, but clearly resulted in significantly higher frequencies of p60-specific CD8 T cells (Fig. (Fig.2).2). Remarkably, ca. 25 to 30% of all splenic CD8 T cells were p60-tetramer positive in mice of vaccination groups S-C and S/C, respectively (Fig. (Fig.2).2). As a consequence of this extremely efficient CD8 T-cell induction, we observed a sterile protective immunity against listeriosis (Fig. 3D and E). However, not only the quantity of p60-specific CD8 T cells but also the biological functionality of these lymphocytes had a major impact on the protective capacity.
Three distinct subsets of antigen-experienced CD8 T cells have been identified by using a recently described marker combination (CD127 and CD62L) (14, 17). Short-living effector T cells (TEC) can be distinguished from two long-living subsets, known as central memory (TCMC) and effector memory (TEMC) T cells. Effector and memory T cells display diversity with respect to their effector functions, homing potential, and proliferative capacity (35). TEC dominate the expansion phase, migrate to peripheral organs, and display immediate effector function. Thus, TEC are the principal mediators of protection early during primary infection. TEMC preferentially home to peripheral tissues and respond to antigen encounters with immediate effector function but poor numeric expansion (15). In contrast, TCMC home to lymphoid organs and can vigorously expand upon antigen reencounter, therefore potentially assigned to the crucial T-cell population that confers long-lasting protective immunity against infection (16). However, proliferative capacity does not necessarily correlate with protection, since TCMC-likeCD8 T cells induced by vaccination with heat-killed L. monocytogenes reveal vigorous proliferation and expansion after challenge with live Listeria but do not protect against listeriosis, as determined by clearance of the bacteria (25). These findings were supported by a recent study demonstrating that TEMC are more effective than TCMC in conferring protection in the murine Listeria infection model (15). Our results are perfectly in line with these published data. In spleens from mice of vaccination groups S-C (day 14) and S/C (day 28), TEMC were detected as the predominant subset of p60-tetramer-positive CD8 T cells (Fig. 4D and E). These mice revealed sterile immunity against the lethal Listeria challenge (Fig. 3D and E). In contrast, >94% of splenic CD8 T cells from mice vaccinated solely with CpG-p60217-225 (immunization groups C and C-C) belonged to the TCMC subset, and significant protection was not observed (Fig. 4A and B). Interestingly, Bachmann et al. could show that antigen-specific TCMC can confer protection against the exposure with lymphocytic choriomeningitis virus but not against the infection with vaccinia virus (2, 3). In contrast, TEMC protected against both types of viruses. A plausible explanation for this observation is the fact that lymphocytic choriomeningitis virus replicates rather slowly in lymphoid organs, thus giving TCMC time and a suitable environment to develop into effector cells. On the other hand, vaccinia virus replicates rapidly in peripheral organs. In this particular case, protection relies mostly on immediate effector functions, and the functional transition from TCMC to TEMC or TEC cannot occur quickly enough.
In our experimental setup, an important question is why vaccination with CpG-p60217-225 gives rise to TCMC as the predominant p60-specific CD8 T-cell subset in spleens from mice, whereas the combined application of Salmonella strain SB824(pHR241) and CpG-p60217-225 results in a pronounced shift to the induction of the TEMC subset. Different models for T-cell subset diversification have been proposed (44), which differ mainly in the time point during priming and clonal expansion (prior, during, or beyond the first cell division) when differentiation programs are induced. In a recently published elegant study, single-cell adoptive-transfer technology allowed the authors to demonstrate that an individual precursor cell still bears the full plasticity to develop into a plethora of different T-cell subsets (43). This observation targets the shaping of T-cell subset differentiation toward factors that are still operative beyond the first cell division. Obviously, the vaccination strategies introduced in our study modulate differentiation patterns toward distinct antigen-specific CD8 T-cell subsets. Further experiments are needed to identify major factors influencing subset diversification since this knowledge will have important implications for vaccine development. Also, T-cell subset transfer or depletion of particular T-cell subsets in vaccinated mice might be helpful to clearly assign the protection to the TEMC or the TEC subset. This will be an important task for future studies.
We thank Dirk H. Busch (Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich) for providing p60217-225-specific tetrameric MHC-I complexes.
C. Berchtold, J. Heeseman, S. Endres, and C. Bourquin were supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 1202, “Oligonucleotides in Cell Biology and Therapy”). H. Rüssmann was supported by the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Neue Vakzinierungsstrategien, grant RU 838/1-3). B. Köhn, S. Jellbauer, and H. Rüssmann were supported by the Deutsche Forschungsgemeinschaft (grant RU 838/2-1). C. Bourquin and S. Emdres were supported by the Deutsche Forschungsgemeinschaft (grants SFB-TR 36 and En 169/7-2), by a research professorship of LMUexcellent, by the excellence cluster CIPSM 114, and by BayImmuNet.
Editor: A. Camilli
Published ahead of print on 21 September 2009.
†Supplemental material for this article may be found at http://iai.asm.org/.