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Pre-existing anti-Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis
Microbes and Infection 9 (2007) 1447e1453 www.elsevier.com/locate/micinf
Original article
Pre-existing anti-Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis Victo`ria E. Sevil Dome`nech, Klaus Panthel, Katrin M. Meinel, Sebastian E. Winter, Holger Ru¨ssmann* Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians-University Munich, Pettenkoferstrasse 9a, 80336 Munich, Bavaria, Germany Received 2 May 2007; accepted 30 July 2007 Available online 7 August 2007
Abstract Our laboratory has focused its research on the use of the type III secretion system of Salmonella enterica serovar Typhimurium to translocate heterologous antigens directly into the cytosol of antigen-presenting cells. We have previously reported that the single oral immunization of mice with a recombinant Salmonella aroA/sptP mutant strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes in a type III-mediated fashion results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal Listeria challenge infection. In the present study, we determined whether pre-existing anti-Salmonella vector immunity influences the induction of p60-specific CD8 T cells and modulates protective immunity against listeriosis after oral vaccination with recombinant Salmonella. After single oral immunization, the Salmonella aroA/sptP double mutant strain was found to colonize spleens of mice for 21 days. In contrast, the period of colonization was significantly shortened to 6 days due to anti-Salmonella vector immunity after second oral immunization. The latter scenario led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific T lymphocytes elicited after single oral immunization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Salmonella enterica serovar Typhimurium; Type III secretion system; CD8 T cell; Listeriosis; Anti-vector immunity; Protective Immunity
1. Introduction In the past, attenuated Salmonella strains have received much attention for their potential as homologous and heterologous antigen delivery systems for oral immunization [1,2]. The type III secretion system (T3SS) of Salmonella enterica serovar Typhimurium can be used to target foreign proteins Abbreviations: CFU, colony-forming units; Elispot, enzyme-linked immunospot; IFN-g, interferon-g; MHC, major histocompatibility complex; T3SS, type III secretion system; YopE, Yersinia outer protein E. * Corresponding author. Tel.: þ49 89 5160 5280; fax: þ49 89 5160 5223. E-mail address: [email protected] (H. Ru¨ssmann). 1286-4579/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2007.07.010
directly into the cytosol of antigen-presenting cells [3,4]. Our laboratory has previously 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 p60specific CD8 T cells and confers protection against a lethal wild-type Listeria challenge infection [5]. In a more recent study, we explored the possibility to induce enhanced levels of antigen-specific CD8 T cells by oral boost immunizations of mice with recombinant Salmonella expressing translocated YopE/p60 [6]. We found that the ability of the Salmonella vaccine strain to colonize the intestine, mesenteric lymph nodes,
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and spleen was markedly impaired after boost immunizations. Salmonella was rapidly cleared from these organs within 7 days after the second oral application. However, this was not attributed to the presence of existing p60-specific CD8 T cells but probably to serovar-specific anti-lipopolysaccharide antibodies directed against the vector itself. Further results revealed that frequencies of p60-specific CD8 T cells could not be increased after a second or third oral application [6]. We speculated that the observed rapid clearance of the Salmonella vaccine carrier after boost immunizations prevented a significant elevation of T lymphocyte numbers. In the present study, we investigated the impact of preexisting anti-Salmonella vector immunity on p60-specific CD8 T-cell priming and on protection against murine listeriosis. 2. Materials and methods 2.1. Plasmids, bacterial strains, and growth conditions Previously, the construction of plasmid pHR241 has been outlined in detail [5]. Briefly, this derivative of pWSK29 is a low-copy-number expression vector and bears the genetic information for translocated chimeric YopE1e138/p60130e477/ M45 under expression control of the lac promoter, which is constitutively active in Salmonella. The hybrid protein was M45 epitope-tagged at its C-terminus. This tag (MDRSRDRLPPFETETRIL) is derived from the E4e6/7 protein of adenovirus [5]. The above described plasmid was transformed into Salmonella enterica serovar Typhimurium strain SB824 by electroporation. Strain SB824 [3] was engineered by introducing the sptP::kan mutant allele from strain SB237 [7] into the DaroA strain SL3261 [8] by P22HTint transduction. Serovar Typhimurium was grown in LuriaeBertani medium supplemented with 0.3 M NaCl, pH 7.0, to allow expression of components and targets of the T3SS encoded by the Salmonella pathogenicity island 1 which mediates Salmonella invasion of host cells [9]. L. monocytogenes strain 10403s [10] was used for challenge experiments in Salmonella-vaccinated mice. 2.2. Oral immunization of mice with recombinant Salmonella Specific-pathogen-free female BALB/c mice, 6e8 weeks old, were purchased from HarlaneWinkelmann (Borchen, Germany). For the experiments, animals were housed in groups of five mice under standard barrier conditions in individually ventilated cages (Tecniplast, Buguggiate, Italy) equipped with steel grid floors and autoclaved filter paper. Water and food were withdrawn for 4 h before groups of mice were orally immunized with 5 108 colony-forming units (CFU) of the respective recombinant Salmonella vaccine strain by using round bottom gavage needles. Thereafter, drinking water ad libitum was offered immediately and food 2 h post immunization. Prime and boost immunizations were separated by a period of 30 days. 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.
2.3. Analysis of Salmonella loads in intestines, Peyer’s patches, mesenteric lymph nodes and spleens At the indicated time points post immunization, mice were euthanized by CO2 asphyxiation, and samples from the intestinal tract, Peyer’s patches, mesenteric lymph nodes, and spleens were removed for analysis. Intestinal contents from the cecum were weighed before resuspending them in 500 ml of 4 C phosphate-buffered saline, pH 7.4. The numbers of CFU per 1000 mg from intestinal content were determined by plating appropriate dilutions on MacConkey agar plates containing kanamycin at 50 mg/ml. It is noteworthy that pHR241 was remarkably stable in vivo, with w98% of the bacterial population retaining the plasmid 14 days after infection (data not shown). To analyze colonization, three Peyer’s patches and three mesenteric lymph nodes per animal and the spleen were removed aseptically. Spleens were weighed before homogenizing them in 4 C phosphate-buffered saline, pH 7.4, containing 0.5% Tergitol and 0.5% bovine serum albumin. The numbers of CFU per spleen, from three Peyer’s patches, or from three mesenteric lymph nodes, respectively, were determined by plating appropriate dilutions on MacConkey agar plates containing kanamycin at 50 mg/ml. 2.4. Elispot assay Thirty days after immunization with SB824 (pHR241) mice were sacrificed and splenocytes were analyzed directly ex vivo for epitope-specific CD8 T cell responses using an interferongamma (IFN-g) enzyme-linked immunospot (Elispot) assay as described previously [11]. Assays were performed in nitrocellulose-backed 96-well microtiter plates (Nunc, Wiesbaden, Germany) coated with a rat anti-mouse IFN-g monoclonal antibody (clone RMMG-1; Biosource, Camarillo, USA). Unseparated splenocytes (6 105 per well) were stimulated for 6 h in round-bottomed microtiter plates in the presence of a 106 M concentration of the CD8 T cell epitope p60217e225. Subsequently, activated cells (4 105 per well) were transferred to Elispot plates and incubated overnight. Elispot plates were developed with a biotin-labeled rat anti-mouse IFN-g monoclonal antibody (clone XMG1.2; Pharmingen, San Diego, USA), horseradish peroxidaseestreptavidin conjugate (Dianova, Hamburg, Germany), and aminoethylcarbazole dye solution. The frequency of antigen-specific cells was calculated as the number of spots per splenocyte seeded. The specificity and sensitivity of the Elispot assay were controlled with IFN-g-secreting CD8 T-cell lines specific for p60217e225. Recovery of seeded CD8 T cells was >90%. 2.5. In vivo protection assay Thirty days after immunization with SB824 (pHR241), mice were challenged intravenously with 104 CFU of logphase L. monocytogenes strain 10403s in 0.2 ml of phosphate-buffered saline. Three days after the challenge, CFU were determined by plating serial dilutions of spleen
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homogenates on PALCAM Listeria selective agar (Merck, Darmstadt, Germany). Colonies were enumerated after 48 h of incubation. Colony counts were corrected for dilution and averaged to yield CFU per organ. Positive control mice received a sublethal intraperitoneal dose of 103 CFU of Listeria 60 days before the challenge infection. The level of protection was calculated as the log10 difference of the bacterial count from immunized mice and na€ıve control mice. Each experiment was performed at least twice with similar results. 2.6. Statistical analysis The statistical significance of the results was checked with the nonparametric Tukey multiple comparison test at the 0.001 significance level. All tests were performed using WINKS statistical analysis software (Texasoft, Cedar Hill, USA). 3. Results 3.1. Time course of colonization and persistence of recombinant Salmonella strains in Peyer’s patches and spleens after single oral immunization of mice In a recently published study, we orally immunized BALB/c mice with the attenuated serovar Typhimurium aroA/sptP mutant strains SB824 or SB824 (pHR241) expressing chimeric YopE1e138/p60130e477/M45, and the kinetics of colonization and persistence of the bacteria in vivo were investigated [6]. The time course of colonization was determined by counting the numbers of viable bacteria, as CFU, in the cecum, in mesenteric lymph nodes, and in spleens on days 2, 4, and 7 after oral immunization [6]. We demonstrated that 2 days after oral immunization, both strains colonized the intestine but were not detectable in mesenteric lymph nodes and spleens. The time course of colonization of both strains, SB824 and SB824 (pHR241) was progressive, with dissemination of the bacteria into mesenteric lymph nodes on day 4 post immunization, and into spleens on day 7 post immunization, respectively. In the current study, we determined the time course of colonization and persistence of SB824 and SB824 (pHR241) on days 7, 14, 21, and 28 after single oral immunization. We decided to count the numbers of viable bacteria, as CFU, in Peyer’s patches and spleens of immunized mice. As shown in Fig. 1, SB824 and SB824 (pHR241) were recovered from both organs 7 days after oral application (mean standard deviation, 5.0 4.1 102 CFU of SB824 and 4.1 3.9 102 CFU of SB824 (pHR241) in Peyer’s patches; 5.5 3.7 102 CFU of SB824 and 5.4 3.7 102 CFU of SB824 (pHR241) in spleens). On day 14 post administration, both bacterial strains were still present in Peyer’s patches and spleens (2.0 1.2 101 CFU of SB824 and 1.6 1.6 101 CFU of SB824 (pHR241) in Peyer’s patches; 3.8 2.3 101 CFU of SB824 and 4.1 3.5 101 CFU of SB824 (pHR241) in spleens). However, the numbers of bacteria were significantly reduced as compared to day 7 after inoculation. On day 21 post immunization, only few numbers of SB824 and SB824 (pHR241) colonized spleens (1.0 0.6 101 CFU and
Fig. 1. Time course of colonization and persistence of recombinant Salmonella strains in Peyer’s patches (PP) and spleens. BALB/c mice received a single oral immunization of either serovar Typhimurium aroA/sptP mutant strain SB824 (open bars) or SB824 (pHR241) expressing translocated YopE/p60/ M45 (filled bars). Seven, 14, 21, and 28 days later, mice were sacrificed and the numbers of bacteria present in the respective organ were determined as CFU. The standard deviations of 15 individual mice per group are indicated. Comparing same time points, values for SB824 and SB824 (pHR241) do not differ significantly (P > 0.001).
1.3 0. 3 101 CFU, respectively), whereas both strains could not be isolated from Peyer’s patches at this time point. By day 28, all mice had cleared both recombinant strains. As previously reported [6], the colonization profiles of SB824 and SB824 (pHR241) did not differ significantly, indicating that the plasmid-mediated expression of hybrid YopE/ p60/M45 did not diminish Salmonella’s ability to persist in the host. Taken together, we found that both attenuated vaccine strains were detectable for 21 days in spleens of orally immunized mice. 3.2. Time course of colonization and persistence of Salmonella in mouse tissues after prime immunization with SB824 and boost immunization with SB824 (pHR241) As previously demonstrated [6], the ability of Salmonella to colonize the intestine, mesenteric lymph nodes, and spleen was markedly impaired after prime-boost immunizations with SB824 (pHR241) expressing translocated YopE/p60/ M45 probably due to anti-vector immunity. To determine the exact time point of bacterial clearance after boost application, we decided to orally prime-immunize BALB/c mice with SB824 (Fig. 2, immunization group A). Thirty days later, mice were boost-immunized with SB824 (pHR241). Subsequently, in this group of mice, colonization and persistence of the latter strain in cecum, Peyer’s patches, mesenteric lymph nodes, and in spleens were investigated on days 31, 34, 36, and 37. Results from the respective experiments are shown in Fig. 3. Bacteria were able to colonize all of the above mentioned tissues on day 1 post boost immunization. Four and 6 days after the second bacterial inoculation (days 34 and 36), SB824 (pHR241) was still present in all organs. As previously shown, [6] on day 37 bacteria could not be isolated from mice. Thus, SB824 (pHR241) was only able to colonize and persist in organs from mice for 6 days after prior exposure to SB824.
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Fig. 2. Schedule of immunization, mouse tissue sampling, and Elispot assay. Two groups of mice were orally prime-immunized on day 0 with either SB824 (immunization group A) or SB824 (pHR241) (immunization group B). On day 30, mice from group A were boost-immunized with SB824 (pHR241) and 1, 4, 6, and 7 days later, samples from cecum, Peyer’s patches, mesenteric lymph nodes, and spleens were collected and analyzed for the presence of viable Salmonella. On day 60 (immunization group A) or on day 30 (immunization group B), frequencies of p60-specific CD8 T cells were determined in mice from groups A and B by Elispot assays.
3.3. Impact of anti-Salmonella vector immunity on p60-specific CD8 T-cell priming Our laboratory has gained much experience with the use of the p60 protein of L. monocytogenes as a model antigen to be delivered by the Salmonella-T3SS [5]. After invasion of host cells and the escape from the phagosome, Listeria constitutively secretes the murine hydrolase p60 [12]. Subsequently, p60 is directed to the major histocompatibility complex (MHC) class I antigen processing pathway, leading to presentation of antigen-derived peptides to CD8 T cells [13]. Analysis of T cells from Listeria-infected BALB/c mice revealed that the immunodominant listerial nonamer peptide p60217e225 is presented to cytotoxic CD8 T lymphocytes in the context of the H2-Kd MHC class I molecule [13,14]. Previously, we have demonstrated that SB824 (pHR241) is capable of translocating YopE/p60/M45 directly into the cytosol
Fig. 3. Time course of colonization and persistence of Salmonella after boost immunization. BALB/c mice were prime-immunized with SB824 and boostimmunized with SB824 (pHR241) 30 days later (see also Fig. 2, immunization group A). On days 31, 34, 36, and 37 samples from cecum, Peyer’s patches (PP), mesenteric lymph nodes (MLN), and spleens were collected and the numbers of bacteria present in the respective organ were determined as CFU. The standard deviations of 15 individual mice per group are indicated.
of macrophages cells via the T3SS [5]. Single oral immunization of mice with this recombinant Salmonella strain led to the induction of a p60-specific CD8 T-cell response and animal protection against a virulent L. monocytogenes challenge [5]. In the next set of experiments, we wanted to answer the question whether a shortened colonization period of the Salmonella vaccine carrier due to pre-existing vector immunity is sufficient to induce p60217e225-specific CD8 T cells. Therefore, two groups of BALB/c mice were prime-immunized on day 0 with either SB824 (Fig. 2, immunization group A) or SB824 (pHR241) (Fig. 2, immunization group B). Thirty days later, mice from group A received a boost immunization with SB824 (pHR241). On day 60 (immunization group A) or on day 30 (immunization group B), mice were sacrificed and Elispot assays were performed to determine the frequencies of p60217e225-specific CD8 T cells. T-cell frequencies were calculated as the number of IFN-g spots generated per 105 spleen cells in the presence of the corresponding synthetic peptide. In Fig. 4 it is demonstrated that mice from group A revealed moderate numbers of IFN-g-producing cells reactive with p60217e225 (4.4 4.7 101 per 105 splenocytes). In comparison, the frequency of p60-specific CD8 T cells (2.1 1.0 102 per 105 splenocytes) was significantly higher (P < 0.001) in mice from group B. Thus, in our immunization setting anti-Salmonella vector immunity influences antigenspecific T-cell priming. 3.4. Impact of anti-Salmonella vector immunity on vaccine-induced protective immunity In further experiments, we investigated whether the amount of p60217e225-specific CD8 T cells induced after encountering
Fig. 4. Influence of pre-existing anti-Salmonella vector immunity on p60-specific CD8 T-cell priming in spleens of mice. Two groups of mice were orally prime-immunized on day 0 with either SB824 (immunization group A) or SB824 (pHR241) (immunization group B) (see also Fig. 2). On day 30, mice from group A were boost-immunized with SB824 (pHR241). On day 60 (immunization group A) or on day 30 (immunization group B), the amount of p60-specific CD8 T cells was determined by Elispot assays. The frequencies of cells reactive with p60217e225 are shown as the number of reactive cells per 105 splenocytes. The standard deviations of triplicate cultures from 15 individual mice per group are indicated. The asterisk indicates values of mice from group A that differ significantly (P < 0.001) from that of mice from group B.
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anti-Salmonella vector immunity (Fig. 2, immunization group A) is sufficient to protect mice against a lethal challenge infection with L. monocytogenes. Thirty days after immunization with SB824 (pHR241) (day 60 in immunization group A and day 30 in immunization group B), animals were intravenously challenged with 104 CFU of wild-type Listeria. CFU were determined in spleens 3 days after the challenge. Spleens of uninfected mice were colonized with 4.0 1.9 105 CFU of Listeria (Fig. 5). In contrast, in spleens of mice that had received a sublethal intraperitoneal dose of 103 CFU of Listeria 60 days before the challenge infection, 2.2 0.9 102 CFU were detected. All mice from the latter group were able to clear L. monocytogenes from spleens 10 days after the challenge and, thus, were totally protected against the lethal infection (data not shown). The same level of protection was observed for mice from immunization group B (long-term antigen display for 21 days). These animals also revealed a pronounced reduction of listerial colonization in their spleens (2.6 0.7 102 CFU). Interestingly, the relatively low amount of p60217e225-specific CD8 T cells found in mice from immunization group A (short-term antigen display for 6 days) was not able to confer protection against listeriosis, because spleens of these animals were colonized with 3.4 2.1 105 CFU of Listeria. 4. Discussion During its interaction with host cells, S. enterica serovar Typhimurium employs a T3SS for cytosolic targeting of
Fig. 5. Influence of pre-existing anti-Salmonella vector immunity on the induction of protective immunity against listeriosis. Positive control mice received a sublethal intraperitoneal dose of 103 CFU of Listeria 60 days before the challenge infection. Negative control mice were uninfected. Two groups of mice were orally prime-immunized on day 0 with either SB824 (immunization group A) or SB824 (pHR241) (immunization group B) (see also Fig. 2). On day 30, mice from group A were boost-immunized with SB824 (pHR241). On day 60 (immunization group A) or on day 30 (immunization group B) mice were intravenously challenged with 104 CFU of L. monocytogenes 10403s. The bacterial load of spleens with Listeria was determined 3 days later. Results are expressed as the mean log10 CFU standard deviation of 15 individual mice per group. Asterisks indicate values that differ significantly from that of the negative control group (P < 0.001).
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virulence factors [15]. This protein translocation system is a useful tool for heterologous antigen delivery by attenuated Salmonella vaccine carrier strains [4]. Previously, we used YopE from Y. enterocolitica as a carrier molecule for Salmonella T3SS-dependent cytosolic delivery of the immunodominant CD8 T-cell antigen p60 from L. monocytogenes [5]. After single oral immunization of mice with recombinant Salmonella it was shown that translocation of hybrid YopE/p60 led to p60-specific antigen presentation and CD8 T-cell priming. This vaccination strategy resulted in protection against a lethal wild-type Listeria challenge infection. In the present study, we modulated the period and kinetic of colonization with recombinant Salmonella by taking advantage of pre-existing anti-vector immunity induced after prime immunization [6]. We wanted to answer the question as to whether a shortened colonization period of mice with an attenuated vaccine strain is sufficient to induce a protective antigenspecific CD8 T-cell response. In a first set of experiments, the colonization and persistence profiles of serovar Typhimurium aroA/sptP mutant strains SB824 and SB824 (pHR241) expressing chimeric YopE/p60/M45 were determined after single oral immunization. We found that both strains were detectable in spleens of mice for 21 days. Dunstan et al. demonstrated that an aroA S. enterica serovar Typhimurium mutant reached a maximum burden of 103 CFU in spleens on day 14 post immunization [16]. On day 21 after inoculation the bacterial load in spleens declined to approximately 102 CFU [16]. At both time points, the aroA/sptP double mutant strain used in our study revealed bacterial recovery rates that were 10 times lower. A single mutation of the sptP gene in serovar Typhimurium has been shown to lead to an impaired ability to colonize the spleen of orally infected mice [7]. Thus, the additional mutation of the sptP gene in SB824 results in further attenuation. In contrast to the long-term colonization of SB824 (pHR241) over 21 days after prime immunization, this strain was only able to colonize and persist in animals organs for exactly 6 days after prior exposure of mice to SB824. The reduced colonization period due to anti-vector immunity after boost immunization led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific cytotoxic T lymphocytes elicited after long-term colonization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis. CD8 T cells are stimulated when peptides from endogenously processed antigens derived from viruses, intracellular bacteria or tumors are presented on MHC class I-restricted molecules [17]. Antigen presentation to CD8 T cells usually occurs during the first few days of acute viral or bacterial infection [18,19]. In fact, several studies demonstrated that na€ıve CD8 T cells require only a brief antigen-dependent instructional period before entering an antigen-independent program of proliferation and differentiation. In vitro data showed that less than 3 h of antigenic stimulation is sufficient to induce multiple rounds of division, and 24 h of stimulation can lead to full differentiation into effector CD8 T cells [19e21]. In respect to an in vivo L. monocytogenes infection that had been
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terminated by ampicillin treatment 24 h post bacterial inoculation, unimpaired development of effector CD8 T cell populations, as well as protective immunity was observed [18]. A more recent study suggests that shortening the infectious period of Listeria to 24e48 h does not alter expansion and size of the primary CD8 T-cell response but diminishes the capacity of T lymphocytes to differentiate into memory cells [22]. A possible explanation for these obvious differences in T-cell priming induced by Listeria and the recombinant Salmonella SB824 (pHR241) mutant strain might be the particular intracellular life style in antigen-presenting cells exhibited by these two bacterial species. Whereas L. mononcytogenes escapes from the phagosome and proliferates in the cytosol of infected cells, serovar Typhimurium replicates inside the phagosome that poses a hostile and changing environment characterized by poor nutrient content, progressive decrease of the pH, and delivery of antibacterial peptides and lysosomal enzymes [23]. In fact, the general paradigm of differentiation, expansion, and contraction of CD8 T cells derived from the Listeria infection model has been challenged by a recent study conducted by Luu and colleagues [24]. It was demonstrated that CD8 T cells induced by recombinant serovar Typhimurium expressing the model antigen ovalbumin undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. The delay in CD8 T-cell priming in this experimental setting might be related to Salmonella’s phagosomal life style. The same group has reported previously, that another intracellular bacterium, Mycobacterium bovis (bacillus CalmetteeGuerin), which also resides within phagosomes, also induces delayed CD8 T-cell induction [25]. We are aware that anti-Salmonella vector immunity not only influences the colonization period of the vaccine strain but also has an impact on Salmonella’s ability to replicate and express the heterologous p60 antigen. Surprisingly, we have recently demonstrated that the Salmonella vaccine strain revealed a more rapid invasion and systemic dissemination after the first boost immunization [6]. A possible explanation for this phenomenon might be the recruitment of chemokine receptor CX3CR1-positive lamina propria dendritic cells after the first oral application. This cell type has been shown to sample luminal antigens as well as entero-invasive pathogens [26], thus contributing to a faster dissemination but also to a more rapid elimination of bacteria after second immunization. To elucidate the influence of the colonization period on CD8 T-cell priming under conditions lacking anti-Salmonella vector immunity, we have orally immunized na€ıve mice with SB824 (pHR241) and terminated the immunization by intraperitoneal injection of the fluorquinolone ciprofloxacin (45 mg/kg per day) on day 6 (data not shown). By day 8, viable Salmonella could not be detected in spleens, Peyer’s patches and the cecum. However, 30 days after vaccination no significant differences in p60-specific CD8 T-cell frequencies were observed as compared to mice that were pre-immunized with the vector strain (data not shown). As expected, the former group of mice was not protected against a lethal Listeria challenge infection. These data confirm that longer than 6 days of bacterial presence is required to elicit a protective
T-cell response. In future experiments, we plan to determine the duration of vaccine carrier persistence that is necessary to induce protective CD8 T-cell immunity. There are conflicting reports concerning the impact of prior vector priming on the immunogenicity of recombinant Salmonella-based vaccines. Some data indicated that prior exposure to Salmonella enhanced antibody responses to a foreign antigen delivered orally by Salmonella [27,28]. These findings were contradicted by studies reporting that prior exposure to Salmonella can dramatically reduce serum antibody responses to a foreign antigen [29e31]. Our data demonstrate that preexisting Salmonella immunity to a homologous serovar compromises the ability of a live Salmonella vector to induce protective antigen-specific CD8 T-cell frequencies. However, this handicap might be overcome by using a heterologous Salmonella serovar for second immunization.
Acknowledgements H.R. was supported by the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm ‘‘Neue Vakzinierungsstrategien‘‘, grant RU 838/1-3).
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V.E. Sevil Dome`nech et al. / Microbes and Infection 9 (2007) 1447e1453 [13] E.G. Pamer, J.T. Harty, M.J. Bevan, Precise prediction of a dominant class-I restricted epitope of Listeria monocytogenes, Nature 353 (1991) 852e855. [14] E.G. Pamer, Direct sequence identification and kinetic analysis of an MHC class I-restricted Listeria monocytogenes CTL epitope, J. Immunol. 152 (1994) 686e694. [15] J.E. Gala´n, H. Wolf-Watz, Protein delivery into eukaryotic cells by type III secretion machines, Nature 444 (2006) 567e573. [16] S.J. Dunstan, C.P. Simmons, R.A. Strugnell, Comparison of the abilities of different attenuated Salmonella typhimurium strains to elicit humoral immune responses against a heterologous antigen, Infect. Immun. 66 (1998) 732e740. [17] M.J. Bevan, Antigen presentation to cytotoxic T lymphocytes in vivo, J. Exp. Med. 182 (1995) 639e641. [18] R. Mercado, S. Vijh, S.E. Allen, K. Kerksieck, I.M. Pilip, E.G. Pamer, Early programming of T cell populations responding to bacterial infection, J. Immunol. 165 (2000) 6833e6839. [19] M.J. van Stipdonk, E.E. Lemmens, S.P. Schoenberger, Na€ıve CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation, Nat. Immunol. 2 (2001) 415e422. [20] S.M. Kaech, R. Ahmed, Memory CD8þT cell differentiation: initial antigen encounter triggers a developmental program in na€ıve cells, Nat. Immunol. 2 (2001) 415e422. [21] P. Wong, E.G. Pamer, Cutting edge: antigen-independent CD8 T cell proliferation, J. Immunol. 166 (2001) 5864e5868. [22] M.A. Williams, M.J. Bevan, Shortening the infectious period does not alter expansion of CD8 T cells but diminishes their capacity to differentiate into memory cells, J. Immunol. 173 (2004) 6694e6702. [23] B.D. Jones, S. Falkow, Salmonellosis: host immune responses and bacterial virulence determinants, Annu. Rev. Immunol. 14 (1996) 533e561.
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[24] R.A. Luu, K. Gurnani, R. Dudani, R. Kammara, H. van Faassen, J.-C. Sirard, L. Krishnan, S. Sad, Delayed expansion and contraction of CD8þT cell responses during infection with virulent Salmonella typhimurium, J. Immunol. 177 (2006) 1516e1525. [25] R. Dudani, Y. Chapdelaine, H.H. Faassen, D.K. Smith, H. Shen, L. Krishnan, S. Sad, Multiple mechanisms compensate to enhance tumor-protective CD8þT cell response in the long-term despite poor CD8þT cell priming initially: comparison between an acute versus a chronic intracellular bacterium expressing a model antigen, J. Immunol. 168 (2002) 5737e5745. [26] J.H. Niess, S. Brand, X. Gu, L. Landsman, S. Jung, B.A. McCormick, J.M. Vyas, M. Boes, H.L. Ploegh, J.G. Fox, D.R. Littman, H.-C. Reinecker, CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance, Science 307 (2005) 254e258. [27] J.X. Bao, J.D. Clements, Prior immunologic experience potentiates the subsequent antibody response when Salmonella strains are used as vaccine carriers, Infect. Immun. 59 (1991) 3841e3845. [28] B.L. Whittle, N.K. Verma, The immune response to a B-cell epitope delivered by Salmonella is enhanced by prior immunological experience, Vaccine 15 (1997) 1737e1740. [29] S.R. Attridge, R. Davies, J.T. LaBrooy, Oral delivery of foreign antigens by attenuated Salmonella: consequences of prior exposure to the vector strain, Vaccine 15 (1997) 155e162. [30] M. Roberts, A. Bacon, J. Li, S. Chatfield, Prior immunity to homologous and heterologous Salmonella serotypes suppresses local and systemic anti-fragment C antibody responses and protection from tetanus toxin in mice immunized with Salmonella strains expressing fragment C, Infect. Immun. 67 (1999) 3810e3815. [31] C.J. Vindurampulle, S.R. Attridge, Impact on vector priming on the immunogenicity of recombinant Salmonella vaccines, Infect. Immun. 71 (2003) 287e297.
Original article
Pre-existing anti-Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis Victo`ria E. Sevil Dome`nech, Klaus Panthel, Katrin M. Meinel, Sebastian E. Winter, Holger Ru¨ssmann* Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians-University Munich, Pettenkoferstrasse 9a, 80336 Munich, Bavaria, Germany Received 2 May 2007; accepted 30 July 2007 Available online 7 August 2007
Abstract Our laboratory has focused its research on the use of the type III secretion system of Salmonella enterica serovar Typhimurium to translocate heterologous antigens directly into the cytosol of antigen-presenting cells. We have previously reported that the single oral immunization of mice with a recombinant Salmonella aroA/sptP mutant strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes in a type III-mediated fashion results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal Listeria challenge infection. In the present study, we determined whether pre-existing anti-Salmonella vector immunity influences the induction of p60-specific CD8 T cells and modulates protective immunity against listeriosis after oral vaccination with recombinant Salmonella. After single oral immunization, the Salmonella aroA/sptP double mutant strain was found to colonize spleens of mice for 21 days. In contrast, the period of colonization was significantly shortened to 6 days due to anti-Salmonella vector immunity after second oral immunization. The latter scenario led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific T lymphocytes elicited after single oral immunization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Salmonella enterica serovar Typhimurium; Type III secretion system; CD8 T cell; Listeriosis; Anti-vector immunity; Protective Immunity
1. Introduction In the past, attenuated Salmonella strains have received much attention for their potential as homologous and heterologous antigen delivery systems for oral immunization [1,2]. The type III secretion system (T3SS) of Salmonella enterica serovar Typhimurium can be used to target foreign proteins Abbreviations: CFU, colony-forming units; Elispot, enzyme-linked immunospot; IFN-g, interferon-g; MHC, major histocompatibility complex; T3SS, type III secretion system; YopE, Yersinia outer protein E. * Corresponding author. Tel.: þ49 89 5160 5280; fax: þ49 89 5160 5223. E-mail address: [email protected] (H. Ru¨ssmann). 1286-4579/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2007.07.010
directly into the cytosol of antigen-presenting cells [3,4]. Our laboratory has previously 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 p60specific CD8 T cells and confers protection against a lethal wild-type Listeria challenge infection [5]. In a more recent study, we explored the possibility to induce enhanced levels of antigen-specific CD8 T cells by oral boost immunizations of mice with recombinant Salmonella expressing translocated YopE/p60 [6]. We found that the ability of the Salmonella vaccine strain to colonize the intestine, mesenteric lymph nodes,
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and spleen was markedly impaired after boost immunizations. Salmonella was rapidly cleared from these organs within 7 days after the second oral application. However, this was not attributed to the presence of existing p60-specific CD8 T cells but probably to serovar-specific anti-lipopolysaccharide antibodies directed against the vector itself. Further results revealed that frequencies of p60-specific CD8 T cells could not be increased after a second or third oral application [6]. We speculated that the observed rapid clearance of the Salmonella vaccine carrier after boost immunizations prevented a significant elevation of T lymphocyte numbers. In the present study, we investigated the impact of preexisting anti-Salmonella vector immunity on p60-specific CD8 T-cell priming and on protection against murine listeriosis. 2. Materials and methods 2.1. Plasmids, bacterial strains, and growth conditions Previously, the construction of plasmid pHR241 has been outlined in detail [5]. Briefly, this derivative of pWSK29 is a low-copy-number expression vector and bears the genetic information for translocated chimeric YopE1e138/p60130e477/ M45 under expression control of the lac promoter, which is constitutively active in Salmonella. The hybrid protein was M45 epitope-tagged at its C-terminus. This tag (MDRSRDRLPPFETETRIL) is derived from the E4e6/7 protein of adenovirus [5]. The above described plasmid was transformed into Salmonella enterica serovar Typhimurium strain SB824 by electroporation. Strain SB824 [3] was engineered by introducing the sptP::kan mutant allele from strain SB237 [7] into the DaroA strain SL3261 [8] by P22HTint transduction. Serovar Typhimurium was grown in LuriaeBertani medium supplemented with 0.3 M NaCl, pH 7.0, to allow expression of components and targets of the T3SS encoded by the Salmonella pathogenicity island 1 which mediates Salmonella invasion of host cells [9]. L. monocytogenes strain 10403s [10] was used for challenge experiments in Salmonella-vaccinated mice. 2.2. Oral immunization of mice with recombinant Salmonella Specific-pathogen-free female BALB/c mice, 6e8 weeks old, were purchased from HarlaneWinkelmann (Borchen, Germany). For the experiments, animals were housed in groups of five mice under standard barrier conditions in individually ventilated cages (Tecniplast, Buguggiate, Italy) equipped with steel grid floors and autoclaved filter paper. Water and food were withdrawn for 4 h before groups of mice were orally immunized with 5 108 colony-forming units (CFU) of the respective recombinant Salmonella vaccine strain by using round bottom gavage needles. Thereafter, drinking water ad libitum was offered immediately and food 2 h post immunization. Prime and boost immunizations were separated by a period of 30 days. 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.
2.3. Analysis of Salmonella loads in intestines, Peyer’s patches, mesenteric lymph nodes and spleens At the indicated time points post immunization, mice were euthanized by CO2 asphyxiation, and samples from the intestinal tract, Peyer’s patches, mesenteric lymph nodes, and spleens were removed for analysis. Intestinal contents from the cecum were weighed before resuspending them in 500 ml of 4 C phosphate-buffered saline, pH 7.4. The numbers of CFU per 1000 mg from intestinal content were determined by plating appropriate dilutions on MacConkey agar plates containing kanamycin at 50 mg/ml. It is noteworthy that pHR241 was remarkably stable in vivo, with w98% of the bacterial population retaining the plasmid 14 days after infection (data not shown). To analyze colonization, three Peyer’s patches and three mesenteric lymph nodes per animal and the spleen were removed aseptically. Spleens were weighed before homogenizing them in 4 C phosphate-buffered saline, pH 7.4, containing 0.5% Tergitol and 0.5% bovine serum albumin. The numbers of CFU per spleen, from three Peyer’s patches, or from three mesenteric lymph nodes, respectively, were determined by plating appropriate dilutions on MacConkey agar plates containing kanamycin at 50 mg/ml. 2.4. Elispot assay Thirty days after immunization with SB824 (pHR241) mice were sacrificed and splenocytes were analyzed directly ex vivo for epitope-specific CD8 T cell responses using an interferongamma (IFN-g) enzyme-linked immunospot (Elispot) assay as described previously [11]. Assays were performed in nitrocellulose-backed 96-well microtiter plates (Nunc, Wiesbaden, Germany) coated with a rat anti-mouse IFN-g monoclonal antibody (clone RMMG-1; Biosource, Camarillo, USA). Unseparated splenocytes (6 105 per well) were stimulated for 6 h in round-bottomed microtiter plates in the presence of a 106 M concentration of the CD8 T cell epitope p60217e225. Subsequently, activated cells (4 105 per well) were transferred to Elispot plates and incubated overnight. Elispot plates were developed with a biotin-labeled rat anti-mouse IFN-g monoclonal antibody (clone XMG1.2; Pharmingen, San Diego, USA), horseradish peroxidaseestreptavidin conjugate (Dianova, Hamburg, Germany), and aminoethylcarbazole dye solution. The frequency of antigen-specific cells was calculated as the number of spots per splenocyte seeded. The specificity and sensitivity of the Elispot assay were controlled with IFN-g-secreting CD8 T-cell lines specific for p60217e225. Recovery of seeded CD8 T cells was >90%. 2.5. In vivo protection assay Thirty days after immunization with SB824 (pHR241), mice were challenged intravenously with 104 CFU of logphase L. monocytogenes strain 10403s in 0.2 ml of phosphate-buffered saline. Three days after the challenge, CFU were determined by plating serial dilutions of spleen
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homogenates on PALCAM Listeria selective agar (Merck, Darmstadt, Germany). Colonies were enumerated after 48 h of incubation. Colony counts were corrected for dilution and averaged to yield CFU per organ. Positive control mice received a sublethal intraperitoneal dose of 103 CFU of Listeria 60 days before the challenge infection. The level of protection was calculated as the log10 difference of the bacterial count from immunized mice and na€ıve control mice. Each experiment was performed at least twice with similar results. 2.6. Statistical analysis The statistical significance of the results was checked with the nonparametric Tukey multiple comparison test at the 0.001 significance level. All tests were performed using WINKS statistical analysis software (Texasoft, Cedar Hill, USA). 3. Results 3.1. Time course of colonization and persistence of recombinant Salmonella strains in Peyer’s patches and spleens after single oral immunization of mice In a recently published study, we orally immunized BALB/c mice with the attenuated serovar Typhimurium aroA/sptP mutant strains SB824 or SB824 (pHR241) expressing chimeric YopE1e138/p60130e477/M45, and the kinetics of colonization and persistence of the bacteria in vivo were investigated [6]. The time course of colonization was determined by counting the numbers of viable bacteria, as CFU, in the cecum, in mesenteric lymph nodes, and in spleens on days 2, 4, and 7 after oral immunization [6]. We demonstrated that 2 days after oral immunization, both strains colonized the intestine but were not detectable in mesenteric lymph nodes and spleens. The time course of colonization of both strains, SB824 and SB824 (pHR241) was progressive, with dissemination of the bacteria into mesenteric lymph nodes on day 4 post immunization, and into spleens on day 7 post immunization, respectively. In the current study, we determined the time course of colonization and persistence of SB824 and SB824 (pHR241) on days 7, 14, 21, and 28 after single oral immunization. We decided to count the numbers of viable bacteria, as CFU, in Peyer’s patches and spleens of immunized mice. As shown in Fig. 1, SB824 and SB824 (pHR241) were recovered from both organs 7 days after oral application (mean standard deviation, 5.0 4.1 102 CFU of SB824 and 4.1 3.9 102 CFU of SB824 (pHR241) in Peyer’s patches; 5.5 3.7 102 CFU of SB824 and 5.4 3.7 102 CFU of SB824 (pHR241) in spleens). On day 14 post administration, both bacterial strains were still present in Peyer’s patches and spleens (2.0 1.2 101 CFU of SB824 and 1.6 1.6 101 CFU of SB824 (pHR241) in Peyer’s patches; 3.8 2.3 101 CFU of SB824 and 4.1 3.5 101 CFU of SB824 (pHR241) in spleens). However, the numbers of bacteria were significantly reduced as compared to day 7 after inoculation. On day 21 post immunization, only few numbers of SB824 and SB824 (pHR241) colonized spleens (1.0 0.6 101 CFU and
Fig. 1. Time course of colonization and persistence of recombinant Salmonella strains in Peyer’s patches (PP) and spleens. BALB/c mice received a single oral immunization of either serovar Typhimurium aroA/sptP mutant strain SB824 (open bars) or SB824 (pHR241) expressing translocated YopE/p60/ M45 (filled bars). Seven, 14, 21, and 28 days later, mice were sacrificed and the numbers of bacteria present in the respective organ were determined as CFU. The standard deviations of 15 individual mice per group are indicated. Comparing same time points, values for SB824 and SB824 (pHR241) do not differ significantly (P > 0.001).
1.3 0. 3 101 CFU, respectively), whereas both strains could not be isolated from Peyer’s patches at this time point. By day 28, all mice had cleared both recombinant strains. As previously reported [6], the colonization profiles of SB824 and SB824 (pHR241) did not differ significantly, indicating that the plasmid-mediated expression of hybrid YopE/ p60/M45 did not diminish Salmonella’s ability to persist in the host. Taken together, we found that both attenuated vaccine strains were detectable for 21 days in spleens of orally immunized mice. 3.2. Time course of colonization and persistence of Salmonella in mouse tissues after prime immunization with SB824 and boost immunization with SB824 (pHR241) As previously demonstrated [6], the ability of Salmonella to colonize the intestine, mesenteric lymph nodes, and spleen was markedly impaired after prime-boost immunizations with SB824 (pHR241) expressing translocated YopE/p60/ M45 probably due to anti-vector immunity. To determine the exact time point of bacterial clearance after boost application, we decided to orally prime-immunize BALB/c mice with SB824 (Fig. 2, immunization group A). Thirty days later, mice were boost-immunized with SB824 (pHR241). Subsequently, in this group of mice, colonization and persistence of the latter strain in cecum, Peyer’s patches, mesenteric lymph nodes, and in spleens were investigated on days 31, 34, 36, and 37. Results from the respective experiments are shown in Fig. 3. Bacteria were able to colonize all of the above mentioned tissues on day 1 post boost immunization. Four and 6 days after the second bacterial inoculation (days 34 and 36), SB824 (pHR241) was still present in all organs. As previously shown, [6] on day 37 bacteria could not be isolated from mice. Thus, SB824 (pHR241) was only able to colonize and persist in organs from mice for 6 days after prior exposure to SB824.
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Fig. 2. Schedule of immunization, mouse tissue sampling, and Elispot assay. Two groups of mice were orally prime-immunized on day 0 with either SB824 (immunization group A) or SB824 (pHR241) (immunization group B). On day 30, mice from group A were boost-immunized with SB824 (pHR241) and 1, 4, 6, and 7 days later, samples from cecum, Peyer’s patches, mesenteric lymph nodes, and spleens were collected and analyzed for the presence of viable Salmonella. On day 60 (immunization group A) or on day 30 (immunization group B), frequencies of p60-specific CD8 T cells were determined in mice from groups A and B by Elispot assays.
3.3. Impact of anti-Salmonella vector immunity on p60-specific CD8 T-cell priming Our laboratory has gained much experience with the use of the p60 protein of L. monocytogenes as a model antigen to be delivered by the Salmonella-T3SS [5]. After invasion of host cells and the escape from the phagosome, Listeria constitutively secretes the murine hydrolase p60 [12]. Subsequently, p60 is directed to the major histocompatibility complex (MHC) class I antigen processing pathway, leading to presentation of antigen-derived peptides to CD8 T cells [13]. Analysis of T cells from Listeria-infected BALB/c mice revealed that the immunodominant listerial nonamer peptide p60217e225 is presented to cytotoxic CD8 T lymphocytes in the context of the H2-Kd MHC class I molecule [13,14]. Previously, we have demonstrated that SB824 (pHR241) is capable of translocating YopE/p60/M45 directly into the cytosol
Fig. 3. Time course of colonization and persistence of Salmonella after boost immunization. BALB/c mice were prime-immunized with SB824 and boostimmunized with SB824 (pHR241) 30 days later (see also Fig. 2, immunization group A). On days 31, 34, 36, and 37 samples from cecum, Peyer’s patches (PP), mesenteric lymph nodes (MLN), and spleens were collected and the numbers of bacteria present in the respective organ were determined as CFU. The standard deviations of 15 individual mice per group are indicated.
of macrophages cells via the T3SS [5]. Single oral immunization of mice with this recombinant Salmonella strain led to the induction of a p60-specific CD8 T-cell response and animal protection against a virulent L. monocytogenes challenge [5]. In the next set of experiments, we wanted to answer the question whether a shortened colonization period of the Salmonella vaccine carrier due to pre-existing vector immunity is sufficient to induce p60217e225-specific CD8 T cells. Therefore, two groups of BALB/c mice were prime-immunized on day 0 with either SB824 (Fig. 2, immunization group A) or SB824 (pHR241) (Fig. 2, immunization group B). Thirty days later, mice from group A received a boost immunization with SB824 (pHR241). On day 60 (immunization group A) or on day 30 (immunization group B), mice were sacrificed and Elispot assays were performed to determine the frequencies of p60217e225-specific CD8 T cells. T-cell frequencies were calculated as the number of IFN-g spots generated per 105 spleen cells in the presence of the corresponding synthetic peptide. In Fig. 4 it is demonstrated that mice from group A revealed moderate numbers of IFN-g-producing cells reactive with p60217e225 (4.4 4.7 101 per 105 splenocytes). In comparison, the frequency of p60-specific CD8 T cells (2.1 1.0 102 per 105 splenocytes) was significantly higher (P < 0.001) in mice from group B. Thus, in our immunization setting anti-Salmonella vector immunity influences antigenspecific T-cell priming. 3.4. Impact of anti-Salmonella vector immunity on vaccine-induced protective immunity In further experiments, we investigated whether the amount of p60217e225-specific CD8 T cells induced after encountering
Fig. 4. Influence of pre-existing anti-Salmonella vector immunity on p60-specific CD8 T-cell priming in spleens of mice. Two groups of mice were orally prime-immunized on day 0 with either SB824 (immunization group A) or SB824 (pHR241) (immunization group B) (see also Fig. 2). On day 30, mice from group A were boost-immunized with SB824 (pHR241). On day 60 (immunization group A) or on day 30 (immunization group B), the amount of p60-specific CD8 T cells was determined by Elispot assays. The frequencies of cells reactive with p60217e225 are shown as the number of reactive cells per 105 splenocytes. The standard deviations of triplicate cultures from 15 individual mice per group are indicated. The asterisk indicates values of mice from group A that differ significantly (P < 0.001) from that of mice from group B.
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anti-Salmonella vector immunity (Fig. 2, immunization group A) is sufficient to protect mice against a lethal challenge infection with L. monocytogenes. Thirty days after immunization with SB824 (pHR241) (day 60 in immunization group A and day 30 in immunization group B), animals were intravenously challenged with 104 CFU of wild-type Listeria. CFU were determined in spleens 3 days after the challenge. Spleens of uninfected mice were colonized with 4.0 1.9 105 CFU of Listeria (Fig. 5). In contrast, in spleens of mice that had received a sublethal intraperitoneal dose of 103 CFU of Listeria 60 days before the challenge infection, 2.2 0.9 102 CFU were detected. All mice from the latter group were able to clear L. monocytogenes from spleens 10 days after the challenge and, thus, were totally protected against the lethal infection (data not shown). The same level of protection was observed for mice from immunization group B (long-term antigen display for 21 days). These animals also revealed a pronounced reduction of listerial colonization in their spleens (2.6 0.7 102 CFU). Interestingly, the relatively low amount of p60217e225-specific CD8 T cells found in mice from immunization group A (short-term antigen display for 6 days) was not able to confer protection against listeriosis, because spleens of these animals were colonized with 3.4 2.1 105 CFU of Listeria. 4. Discussion During its interaction with host cells, S. enterica serovar Typhimurium employs a T3SS for cytosolic targeting of
Fig. 5. Influence of pre-existing anti-Salmonella vector immunity on the induction of protective immunity against listeriosis. Positive control mice received a sublethal intraperitoneal dose of 103 CFU of Listeria 60 days before the challenge infection. Negative control mice were uninfected. Two groups of mice were orally prime-immunized on day 0 with either SB824 (immunization group A) or SB824 (pHR241) (immunization group B) (see also Fig. 2). On day 30, mice from group A were boost-immunized with SB824 (pHR241). On day 60 (immunization group A) or on day 30 (immunization group B) mice were intravenously challenged with 104 CFU of L. monocytogenes 10403s. The bacterial load of spleens with Listeria was determined 3 days later. Results are expressed as the mean log10 CFU standard deviation of 15 individual mice per group. Asterisks indicate values that differ significantly from that of the negative control group (P < 0.001).
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virulence factors [15]. This protein translocation system is a useful tool for heterologous antigen delivery by attenuated Salmonella vaccine carrier strains [4]. Previously, we used YopE from Y. enterocolitica as a carrier molecule for Salmonella T3SS-dependent cytosolic delivery of the immunodominant CD8 T-cell antigen p60 from L. monocytogenes [5]. After single oral immunization of mice with recombinant Salmonella it was shown that translocation of hybrid YopE/p60 led to p60-specific antigen presentation and CD8 T-cell priming. This vaccination strategy resulted in protection against a lethal wild-type Listeria challenge infection. In the present study, we modulated the period and kinetic of colonization with recombinant Salmonella by taking advantage of pre-existing anti-vector immunity induced after prime immunization [6]. We wanted to answer the question as to whether a shortened colonization period of mice with an attenuated vaccine strain is sufficient to induce a protective antigenspecific CD8 T-cell response. In a first set of experiments, the colonization and persistence profiles of serovar Typhimurium aroA/sptP mutant strains SB824 and SB824 (pHR241) expressing chimeric YopE/p60/M45 were determined after single oral immunization. We found that both strains were detectable in spleens of mice for 21 days. Dunstan et al. demonstrated that an aroA S. enterica serovar Typhimurium mutant reached a maximum burden of 103 CFU in spleens on day 14 post immunization [16]. On day 21 after inoculation the bacterial load in spleens declined to approximately 102 CFU [16]. At both time points, the aroA/sptP double mutant strain used in our study revealed bacterial recovery rates that were 10 times lower. A single mutation of the sptP gene in serovar Typhimurium has been shown to lead to an impaired ability to colonize the spleen of orally infected mice [7]. Thus, the additional mutation of the sptP gene in SB824 results in further attenuation. In contrast to the long-term colonization of SB824 (pHR241) over 21 days after prime immunization, this strain was only able to colonize and persist in animals organs for exactly 6 days after prior exposure of mice to SB824. The reduced colonization period due to anti-vector immunity after boost immunization led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific cytotoxic T lymphocytes elicited after long-term colonization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis. CD8 T cells are stimulated when peptides from endogenously processed antigens derived from viruses, intracellular bacteria or tumors are presented on MHC class I-restricted molecules [17]. Antigen presentation to CD8 T cells usually occurs during the first few days of acute viral or bacterial infection [18,19]. In fact, several studies demonstrated that na€ıve CD8 T cells require only a brief antigen-dependent instructional period before entering an antigen-independent program of proliferation and differentiation. In vitro data showed that less than 3 h of antigenic stimulation is sufficient to induce multiple rounds of division, and 24 h of stimulation can lead to full differentiation into effector CD8 T cells [19e21]. In respect to an in vivo L. monocytogenes infection that had been
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terminated by ampicillin treatment 24 h post bacterial inoculation, unimpaired development of effector CD8 T cell populations, as well as protective immunity was observed [18]. A more recent study suggests that shortening the infectious period of Listeria to 24e48 h does not alter expansion and size of the primary CD8 T-cell response but diminishes the capacity of T lymphocytes to differentiate into memory cells [22]. A possible explanation for these obvious differences in T-cell priming induced by Listeria and the recombinant Salmonella SB824 (pHR241) mutant strain might be the particular intracellular life style in antigen-presenting cells exhibited by these two bacterial species. Whereas L. mononcytogenes escapes from the phagosome and proliferates in the cytosol of infected cells, serovar Typhimurium replicates inside the phagosome that poses a hostile and changing environment characterized by poor nutrient content, progressive decrease of the pH, and delivery of antibacterial peptides and lysosomal enzymes [23]. In fact, the general paradigm of differentiation, expansion, and contraction of CD8 T cells derived from the Listeria infection model has been challenged by a recent study conducted by Luu and colleagues [24]. It was demonstrated that CD8 T cells induced by recombinant serovar Typhimurium expressing the model antigen ovalbumin undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. The delay in CD8 T-cell priming in this experimental setting might be related to Salmonella’s phagosomal life style. The same group has reported previously, that another intracellular bacterium, Mycobacterium bovis (bacillus CalmetteeGuerin), which also resides within phagosomes, also induces delayed CD8 T-cell induction [25]. We are aware that anti-Salmonella vector immunity not only influences the colonization period of the vaccine strain but also has an impact on Salmonella’s ability to replicate and express the heterologous p60 antigen. Surprisingly, we have recently demonstrated that the Salmonella vaccine strain revealed a more rapid invasion and systemic dissemination after the first boost immunization [6]. A possible explanation for this phenomenon might be the recruitment of chemokine receptor CX3CR1-positive lamina propria dendritic cells after the first oral application. This cell type has been shown to sample luminal antigens as well as entero-invasive pathogens [26], thus contributing to a faster dissemination but also to a more rapid elimination of bacteria after second immunization. To elucidate the influence of the colonization period on CD8 T-cell priming under conditions lacking anti-Salmonella vector immunity, we have orally immunized na€ıve mice with SB824 (pHR241) and terminated the immunization by intraperitoneal injection of the fluorquinolone ciprofloxacin (45 mg/kg per day) on day 6 (data not shown). By day 8, viable Salmonella could not be detected in spleens, Peyer’s patches and the cecum. However, 30 days after vaccination no significant differences in p60-specific CD8 T-cell frequencies were observed as compared to mice that were pre-immunized with the vector strain (data not shown). As expected, the former group of mice was not protected against a lethal Listeria challenge infection. These data confirm that longer than 6 days of bacterial presence is required to elicit a protective
T-cell response. In future experiments, we plan to determine the duration of vaccine carrier persistence that is necessary to induce protective CD8 T-cell immunity. There are conflicting reports concerning the impact of prior vector priming on the immunogenicity of recombinant Salmonella-based vaccines. Some data indicated that prior exposure to Salmonella enhanced antibody responses to a foreign antigen delivered orally by Salmonella [27,28]. These findings were contradicted by studies reporting that prior exposure to Salmonella can dramatically reduce serum antibody responses to a foreign antigen [29e31]. Our data demonstrate that preexisting Salmonella immunity to a homologous serovar compromises the ability of a live Salmonella vector to induce protective antigen-specific CD8 T-cell frequencies. However, this handicap might be overcome by using a heterologous Salmonella serovar for second immunization.
Acknowledgements H.R. was supported by the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm ‘‘Neue Vakzinierungsstrategien‘‘, grant RU 838/1-3).
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