YopE specific CD8+ T cells provide protection against systemic and mucosal Yersinia pseudotuberculosis infection

Prior studies indicated that CD8+ T cells responding to a surrogate single antigen expressed by Y. pseudotuberculosis, ovalbumin, were insufficient to protect against yersiniosis. Herein we tested the hypothesis that CD8+ T cells reactive to the natural Yersinia antigen YopE would be more effective at providing mucosal protection. We first confirmed that immunization with the attenuated ksgA- strain of Y. pseudotuberculosis generated YopE-specific CD8+ T cells. These T cells were protective against challenge with virulent Listeria monocytogenes expressing secreted YopE. Mice immunized with an attenuated L. monocytogenes YopE+ strain generated large numbers of functional YopE-specific CD8+ T cells, and initially controlled a systemic challenge with virulent Y. pseudotuberculosis, yet eventually succumbed to yersiniosis. Mice vaccinated with a YopE peptide and cholera toxin vaccine generated robust T cell responses, providing protection to 60% of the mice challenged mucosally but failed to show complete protection against systemic infection with virulent Y. pseudotuberculosis. These studies demonstrate that vaccination with recombinant YopE vaccines can generate YopE-specific CD8+ T cells, that can provide significant mucosal protection but these cells are insufficient to provide sterilizing immunity against systemic Y. pseudotuberculosis infection. Our studies have implications for Yersinia vaccine development studies.


Introduction
Three Yersinia species cause disease in humans: Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis [1]. Today, in the United States, Y. pestis infections occur mainly by inoculations from fleas carried by wild rodents [2], the last major plague outbreak in the US occurred in 1924 [3]. Ingestion of contaminated food or water is the source of enteric yersiniosis, caused by Y. enterocolitica or Y. pseudotuberculosis with swine being the most common source. This disease is characterized by fever, gastroenteritis and mesenteric lymphadenitis [4]. Albeit there are differences in routes of infection and disease severity, all Yersinia species are known to disseminate from lymphoid tissues to systemic organs. Many studies have demonstrated the ability of a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 these pathogens to subvert host immune responses and the cellular death pathways that control bacterial replication leading to fulminant disease.
CD8 + T cells are known to be essential for the immune response against viruses; however, they also contribute as a line of defense against intracellular bacterial pathogens. Naïve CD8 + T cells detect infected cells by recognition of peptide antigens presented by the major histocompatibility complex (MHC) class I molecule on the surface of the cells [5]. Most of the peptide antigens presented by MHC class I molecules come from cytosolic proteins, for this reason pathogens or microorganisms that introduce antigens to the cytosol are subject to CD8 + T cell surveillance [6]. Along these lines, bacterial pathogens such as Salmonella spp., Shigella spp., Vibrio cholerae, and Yersinia use a virulence-associated molecular machine called a type III secretion system (T3SS) to directly inject or translocate bacterial toxins from the bacteria to the host-cell cytosol [7]. Using this mechanism, pathogenic Yersinia introduces multiple virulence factors known as Yersinia outer proteins (Yops) into the host cell. Yersinia spp. express six secreted Yops, these are: YopE, YopJ, YopH, YopM, YopO and YopT. These are known to disrupt cellular signaling pathways leading to changes in cytokine production and blockage of phagocytosis [8].
Since Yops are delivered directly to the cytosol by the T3SS, it is reasonable to expect CD8 + T cells might respond to Yop-derived antigens. In the last five years, multiple studies have shown that the T3SS effector protein YopE contains a dominant CD8 + T cell epitope specifically recognized by CD8 + T cells isolated from Y. pestis or Y. pseudotuberculosis infected or immunized mice [9][10][11][12]. YopE, a Rho GTPase-activation protein (GAP), is characterized as a contact-dependent cytotoxin, responsible for inhibition of phagocytic processes by disruption of the actin cytoskeleton [13]. YopE amino acids 69 to 77 (YopE 69-77 ) have been shown to be a dominant epitope recognized by CD8 + T cells in C57BL/6 mice and immunization with peptides containing the epitope confers significant protection from lethal pulmonary challenge with Y. pestis [10].
A recent publication by Zhang et al. showed that effector CD8 + T cells were generated in response to Y. pseudotuberculosis YopE 69-77 . Moreover, that the YopE 69-77 specific CD8 + T cells produced IFN-γ and TNF-α 14 days after infection with a YopE GAP mutant and that these cells were positive for KLRG1, a marker associated with the memory phenotype [14]. The latter report in combination with our own [9,11], suggests that vaccination with an attenuated version of Y. pseudotuberculosis induces a protective antigen-specific CD8 + T-cell response against the wildtype infection. Herein, we test the hypothesis that CD8 + T cells specific to the natural Yersinia antigen YopE would be effective at protecting the host against mucosal Y. pseudotuberculosis infections. We show that mice immunization with Listeria monocytogenes expressing YopE or cholera toxin in addition to a YopE 69-77 peptide resulted in the development of varying degrees of YopE-specific CD8 + T cell protection against Y. pseudotuberculosis systemic or mucosal infection. These data demonstrate that cholera toxin in combination with YopE 69-77 is sufficient to generate partial protection (!60%) to Y. pseudotuberculosis after oral challenge and significantly extend the survival of systemically infected mice.
were housed in specific-pathogen-free conditions in isolator cages with soft bedding. Mice were given free access to food, standard mouse chow, and water throughout the course of the experiment. Animals were cared for by department of laboratory animal staff. During experimental procedures animals were monitored by laboratory staff twice a day and there were no unexpected deaths during this study. Animals in survival studies were considered morbibund if they lost 20% of their body weight and were subsequently euthanized by isoflurane overdose. Animal suffering was minimized by providing free access to food and water, animals had soft bedding for the duration of the experiments and all procedures were done under general anesthesia, 3% isoflurane and oxygen.
Bacterial strains (Table 1) The Yersinia pseudotuberculosis YPIII and isogenic ksgAstrain have been described and used by us previously [15,16]. The ksgAstrain contains a kanamycin-resistance-encoding transposon inserted into the ksgA gene. The attenuated L. monocytogenes ΔactA ΔplcB strain in the 10403S background [17], was used as a vaccine vector in these studies. L. monocytogenes expressing a secretable YopE-OVA fusion protein was constructed as previously described by Lauer et al. by inserting the YopE fragment in-between a sequence encoding the actA promoter and the first 100 amino acids of the ActA protein and a sequence encoding OVA 254-265 in the L. monocytogenes integration vector pPL2 [18]. The construct was stably integrated at the tRNA Arg locus of the bacterial chromosome of the attenuated Listeria monocytogenes ΔactA ΔplcB strain, as described previously [19].

Flow cytometry
For spleen single cell suspensions the tissue was gently dissociated with a syringe plunger through Falcon1 70 μm Cell Strainer (BD Biosciences, San Jose CA). After lysing the erythrocytes with PharmLyse (BD Biosciences, San Jose CA), approximately 1 × 10 6 cells were blocked with FcBlock (clone 2.4G2) for 15 min on ice. Then cells were stained with antibodies (1:1000 dilution) to

Cellular stimulation and intracellular cytokine staining
T-cell stimulation and intracellular cytokine staining was done essentially as we have previously described [20]. Splenocytes from animals in the indicated treatment groups were harvested and stimulated in vitro with 1μg of OVA 257-264 peptide, YopE 69-77 peptide, or the saline vehicle as a control for 24 hours. As a positive control cells were non-specifically stimulated with plate-bound anti-CD3e (1μg/well) and costimulation was provided by treatment with 500ng/well anti-CD28. Cells were then stained with fluorochrome-conjugated antibodies against CD3e, CD8, and IFNγ. Data was collected and analyzed on a FACSCalibur. Briefly, lymphocytes were identified by light scattering and CD3+ cells were gated to facilitate the analysis of IFNγ expressing CD8+ cells.
In vivo inoculations, immunizations, survival and weight change assays Y. pseudotuberculosis strains were grown as follows, after 2 days growth on LB agar plates, single colonies were inoculated into 2 mL LB broth and incubated o/n at 26˚C with rotation.
Overnight cultures were then diluted to the desired concentration in PBS and used for mouse challenges as described below. L. monocytogenes strains were grown overnight in brain-heart infusion broth at 30˚C without rotation, and diluted in PBS to the desired concentration and delivered intravenously. Mice were then visually inspected for signs of illness including lethargy and piloerection. For immunization studies, mice were allowed to rest for the indicated amount of time prior to challenge with the indicated bacteria. For most experiments, mice were challenged sixty days after immunization to allow time for the primary immune response to resolve and memory to be established. Mouse morbidity, weight change and mortality were recorded over a four to six-week time course. Mucosal immunization: on days 0, 7, and 21, mice were anesthetized with isoflurane and immunized intranasally with 10 μg YopE 69-77 peptide mixed with 1μg of Cholera toxin (CT; List Biological Laboratories Inc., Campbell, CA) in 15 μL of PBS. Control mice received CT alone. On day 44, mice were challenged as indicated.

Detection of Ag presentation by LacZ T cell hybridoma assay
2.0 X 10 5 macrophages were incubated with L. monocytogenes expressing the ActA-YopE-OVA fusions, control bacteria expressing secreted OVA, the parental OVA-negative strain, or synthetic SIINFEKL peptide (i.e. OVA 257-264 ) at the indicated concentrations for 12 hours. B3Z T cell hybridomas were co-incubated with the infected macrophages for 18 hours. B3Z responses were analyzed using a LacZ T cell hybridoma assay. After incubation, PBS was used to wash the plates and LacZ buffer and substrate (Roche) was added and cells were further incubated for up to 4 hours. LacZ enzymatic activity was measured by changes in the absorbance of a colorimetric substrate at 570 nm using a BioTek Synergy H4 plate reader.

Data and statistical analysis
Flow cytometry data was analyzed with FlowJo (Tree Star, Ashland OR). Graphing and statistical analysis was performed with Prism (GraphPad Software, La Jolla CA). Kaplan Meier method was used for survival curves and significance calculated using the log-rank test. The nonparametric Mann-Whitney U test was used to determine statistical differences between groups of animals.

Results
Systemic immunization with attenuated Y. pseudotuberculosis generates YopE 69-77 -specific CD8 + T cells A protective immune responses to virulent Y. pseudotuberculosis can be generated by immunization with an attenuated Y. pseudotuberculosis strain lacking the 16s rRNA methyltransferase KsgA, and that protection required CD8 + T cells [9]. To determine if immunization with ksgA -Y. pseudotuberculosis stimulated YopE 69-77 -specific CD8 + T cells, we evaluated the frequency of K b YopE 69-77 tetramer-positive CD8 + T cells at day 8 post-intravenous inoculation with 200 CFU of ksgAbacteria. Ten percent of spleen CD8 + T cells from ksgAimmunized animals showed positive staining for the K b YopE 69-77 tetramer, in contrast to the <2% of spleen CD8 + T cells from naïve mice (Fig 1A, 1B, 1D and 1E). To test if the amount of YopE translocated into host cells limited CD8 + T-cell responses, we also tested a yopK mutant. A Y. pseudotuberculosis strain lacking the T3SS protein YopK is attenuated for virulence and hypertranslocates Yops into host cells [22][23][24]. We found that the frequency of K b YopE 69-77 tetramer + CD8 + T cells in spleens was unaffected by the absence of YopK (Fig 1C and 1E). These results affirm that mice exposed to attenuated Y. pseudotuberculosis via the systemic route generate splenic T cells against the YopE 69-77 epitope.
YopE 69-77 -specific CD8 + T cells generated by attenuated Y. pseudotuberculosis infection are sufficient to protect against virulent rYopE-expressing Listeria monocytogenes We exploited the fact that CD8 + T cells are critical for protecting hosts against the cytosolic pathogen L. monocytogenes, and engineered this pathogen to express and secrete YopE. We inserted either full-length YopE or YopE 64-82 downstream of the first 100 amino acids of the Listeria protein ActA, to allow for secretion of the fusion protein, and upstream of a C-terminal OVA 254-265 tag, to allow for detection of antigen delivery/processing/presentation (Fig 2A). Macrophages were infected with the different L. monocytogenes strains and then incubated with B3Z cells, a T cell hybridoma that is activated by the recognition of H-2K b in association with OVA 257-264 peptide. Both ActA-YopE-OVA and ActA-YopE 64-82 -OVA were capable of being processed and presented by infected macrophages to stimulate B3Z cells equivalently or better than L. monocytogenes expressing OVA from a chromosome-integrated ( Fig 2B) or plasmid-borne allele (Fig 2C). These results demonstrate that insertion of YopE into the ActA-OVA fusion protein does not interfere with antigen processing and presentation to responding T cell hybridomas, and suggests that the YopE portion of the fusion will be correctly processed and presented. Initially we immunized mice IV with 200 CFU of the ksgA mutant or the ksgA yopE double mutant and once memory was established sixty days later, we tested susceptibility to challenge with 5.0 x 10 5 CFU of the wild type parental L. monocytogenes strain, 1043s (Fig 3A), or L. monocytogenes ActAYopE 1-219 (Fig 3B). The ksgAstrain provides no protection against virulent L. monocytogenes challenge. Subsequently, to determine if infection with the ksgAmutant conferred YopE-mediated protection against infection, we immunized C57BL/6 mice with 200 CFU of the Y. pseudotuberculosis ksgA-strain or the attenuated L. monocytogenes ΔactA ΔplcB mutant and 60 days after, mice were challenged with a low dose of 5 x 10 5 CFU (Fig 3C and  3E) or a high dose of 2.0 x 10 6 CFU (Fig 3D and 3F) of virulent L. monocytogenes expressing the ActAYopE 1-219 fusion protein . No significant weight loss or decrease survival was observed between the Y. pseudotuberculosis ksgA-and L. monocytogenes ΔactA ΔplcB immunized mice after low dose L. monocytogenes ActAYopE 1-219 challenge, however,~20% weight loss and 60% survival was observed 1 week after infection in the control mice (Fig 3C and 3D). When mice were challenged with the high dose of L. monocytogenes ActAYopE 1-219 a maximum of  /group A, B). For multiple group comparisons Dunn's multiple-comparison post-test was used for weight change differences at selected time points: *, P 0.05, **, P 0.01, ***, P 0.001, ****, P 0.0001. Log rank Mantel-Cox test was used for survival curves. and 3D). These results show that the host response generated against Y. pseudotuberculosis ksgA-was sufficient to protect against virulent L. monocytogenes in a YopE-specific manner.
Immunization with attenuated L. monocytogenes expressing an YopE epitope mediates CD8+ T-cell protection against lethal Y. pseudotuberculosis challenge To test the requirement for CD8 + T cells for protection against Yersinia following L. monocytogenes immunization, we immunized mice with 5.5 X 10 7 CFU L. monocytogenes ActAYopE 1-219 OVA IV, and then allowed the animals to rest for 60 days. Mice were then treated IP with 200μg of a CD8 depleting antibody or an isotype control antibody two days prior to oral challenge with 9 X 10 8 CFU of Y. pseudotuberculosis YPIII and then every four days throughout the course of the experiment. As shown in Fig 6A, protection following L. monocytogenes immunization required CD8+ T-cells. All animals treated with CD8 neutralizing antibodies succumbed to challenge with Y. pseudotuberculosis within 10 days whereas 70% of animals treated with an isotype control antibody survived the 20 day experiment (p = 0.0001). To confirm effective CD8 T-cell depletion, spleens were harvested from an animal treated with the isotype control antibody and the depleting antibodies prior to challenge with Yersinia. Single cell suspensions were stained with fluorescent antibodies for CD45, CD4, and CD8 an then analyzed by flow cytometry. CD45+ cells were gated on and then CD4 and CD8 expression was analyzed. Treatment with the anti-CD8 antibody eliminated CD8+ cells from the spleen (Fig 6B).

Prior exposure to YopE confers partial protection against lethal Y. pseudotuberculosis oral and systemic infection
To test if induction of functional YopE-specific CD8 + T cells protected against virulent Y. pseudotuberculosis, we immunized mice with either attenuated YopE-expressing bacteria (listed in figure legend (Fig 7A and 7B, Fig 7A.1 and 7B.1) or cholera toxin plus YopE 69-77 peptide. Sixty days after immunization, mice were challenged with the mouse virulent Y. pseudotuberculosis strain YPIII/pIB1. Mice challenged via oral gavage, showed partial protection when immunized with any of the YopE expressing L. monocytogenes strains or the attenuated Y. pseudotuberculosis ΔksgA strain (Fig 7A and 7A.1). Interestingly, mice challenged intravenously showed significant resistance to infection reflected in a prolonged mean day to death after being immunized with all YopE expressing L. monocytogenes strains or the attenuated Y. pseudotuberculosis ΔksgA strain (Fig 7B and 7B.1). Also, a substantial increase in survival was observed in mice immunized with cholera toxin plus the YopE 69-77 peptide (Fig 7C  and 7C.1). These results suggest that a single immunization containing the immunodominant epitope of YopE can confer a partially protective CD8 + T cell driven host response against infection resulting in a delayed mean time to death.

Discussion
In this study, we report that systemic immunization with a live-attenuated strain of Y. pseudotuberculosis generates YopEspecific CD8 + T cells. Immunization of mice with the Y. pseudotuberculosis live-attenuated ksgA-strain was sufficient to protect against challenge with virulent L. monocytogenes expressing YopE. Although not directly tested in this study, this protection was presumably dependent on YopE-specific CD8+ T-cells. Moreover, immunization with live-attenuated L. monocytogenes expressing YopE also results in the generation of YopE-specific CD8 + T cells and confers partial protection against lethal Y. pseudotuberculosis oral and systemic infection. In the case of immunization with L. monocytogenes expressing YopE, we show protection requires YopE-specific CD8+ T-cells. Our results indicate that Yersinia outer proteins can stimulate protective CD8 + T cell mediated immunity. Understanding the basis of immunity to Y. pseudotuberculosis is an important step in development of effective plague vaccines since Y. pestis is very closely related to Y. pseudotuberculosis [25].
The majority of work developing vaccines against Yersinia has focused on vaccines against Y. pestis to protect against plague. These vaccines have largely targeted the TTSS protein LcrV and the F1 protein [26]. Antibodies against these proteins provide significant protection against Y. pestis infection in a variety of animal models of plague [27][28][29]. A F1-LcrV fusion protein, F1V, is currently in late-stage development as a subunit vaccine against plague. In addition, to subunit vaccines targeting F1 and LcrV, a variety of approaches and vectors expressing these antigens provide protection against Y. pestis [26,[30][31][32][33]. Consistent with data presented in this study, we showed years ago that a live-attenuated Y. pestis vaccine provided significant mucosal and systemic protection against high-titer Y. pestis challenge [34]. Even though significant efforts and progress has been made in the preclinical development of the F1V vaccine, this vaccine has potential limitations. The F1 protein is not essential for virulence and natural and engineered strains of Y. pestis lacking F1 are virulent [35]. Therefore, identification of additional protective antigens or epitopes that can be included in future plague vaccine formulations is warrented. Although antibodies can provide protection against Y. pestis, it is now clear that T-cell mediated responses can contribute significantly to vaccine mediated protection. YopE is an attractive T-cell antigen since YopE is required for virulence and it generates very strong CD8 + T-cell responses.
Yersinia species are predominantly extracellular pathogens in vivo with many of their Yopmediated virulence mechanisms targeting phagocytes by interfering with phagocytosis [8]. However, we and others provide evidence that immune responses generally associated with intracellular pathogens can be effective against Yersinia [9-12, 14, 29]. In this study, we took advantage of a recipricol heterologous vaccine/challenge system using Listeria and Yersinia. This allowed us to focus on the contributions of YopE-mediated immunity and in the case of immunizations with Yersinia expressing YopE and challenge with YopE-expressing Listeria we have a system where immunity is dependent on CD8+ T-cells. Altogether, data presented in this study suggests that CD8+ T-cells provide some protection against oral challenge with Yersinia and significantly delay the mean day to death in an intraveneous challenge model.
We recently demonstrated that mice immunized with live-attenuated Listeria expressing OVA were significantly protected from challenge with a virulent Y. pseudotuberculosis strain expressing a YopE-OVA fusion [11]. Protection required ova-specific CD8 + T-cells and perforin. These data were consistent with our earlier studies showing a general requirement for CD8 + T-cells and perforin to protect against Yersinia pseudotuberculosis [9]. This study suggested that CD8 + T-cells killed Yersinia-associated antigen presenting cells (APC) in a perforin-dependent manner. Bystander phagocytes can then engulf Yersinia-associated APCs without interference from the anti-phagocytic Yops. Others have presented evidence that perforin is despensible for CD8+ T-cell-mediated protection from Y. pestis but CD8 T-cell derived TNF-alpha and IFN-γ are critical [36]. It has become clear that cellular immunity to Yersinia, including CD8 + T-cell-mediated immunity, can provide significant protection against infection. Smiley and co-workers defined an immunodominant epitope in YopE, YopE 69-77 , that leads to robust generation of a functional CD8 + T-cell response capable of protecting mice against challenge with Y. pestis [37]. Consistent with the data presented in the current study, Bliska and co-workers demonstrated that YopE-specific CD8 + T-cells provide varying levels of protection against challenge with Yersinia [12]. We extend these studies by showing that immunization with the live-attenuated ksgA mutant induces the expansion of YopE-specific CD8 + T-cells that can provide significant protection against a systemic lethal heterologous challenge with Listeria monocytogenes expressing YopE. These data demonstrate that the protection is YopE-mediated as vaccinated control animals succumb to challenge with the parental L. monocytogenes strain and protection requires CD8+ T-cells. Consistent with findings observed with mucosal immunization with the immunodominant YopE epitope and Cholera toxin and subsequent Y. pestis challenge [10], mucosal immunization with YopE significantly protects against virulent mucosal challenge with Y. pseudotuberculosis (this work and [14]). Furthermore, vaccinated animals treated with an antibody that depletes CD8+ T-cells succumb to infection whereas animals treated with an isotype control antibody are protected strongly suggesting CD8+ T-cells are responsible for protection.
Although not directly tested in this study, we have previously shown that perforin expression by CD8 + T-cells is critical for controlling Yersinia pseudotuberculosis infection [9,11] but others have not seen a requirement for perforin in protecting animals from Y. pestis [36]. Taken together, our work and that of others strongly suggest that the cytolytic functions of CD8 + T-cells in addition to their ability to produce IFN-γ and TNF-alpha are critical to control Yersinia infection. These observations could impact the design of future vaccines targeting Y. pestis and other Yersinia species.