Abstract
Strains of Listeria monocytogenes (LM) that are deficient in the virulence factor listeriolysin O (LLO) are highly attenuated and are thought not to elicit protective immunity. This failure has been attributed to the inability of the bacterium to enter the host cell cytosol and access MHC class I Ag processing machinery. We reexamined this issue using recombinant strains of LM that are deficient in LLO but express an additional CD8 T cell epitope derived from lymphocytic choriomeningitis virus. After infection with LLO-deficient strains, we find sizable priming of epitope-specific CD8 T cells and the development of a functional memory cell population. Mice primed with the LLO-deficient LM strain are equally resistant against high-dose challenge with virulent LM as mice primed with wild-type virulent bacteria and also resist heterologous challenge with lymphocytic choriomeningitis virus. Interestingly, priming with a low dose of LLO-deficient LM, which occurred in environment of reduced inflammation (IFN-γ), allowed rapid amplification of Ag-specific CD8 T cells by booster immunization, despite an undetectable primary response. We conclude that the generation of protective immunity by LLO-deficient strains of LM does in fact occur and that this highly attenuated LM strain may be a useful platform for vaccine delivery.
Memory CD8 T cells are essential for resistance to repeat infections with many bacterial and viral infections, and the generation of these cells is an important goal of vaccination. Critical to the effective design of vaccines is an understanding of the important aspects of host–pathogen interactions that lead to CD8 T cell activation, expansion, contraction, and finally, the development of a long-lived functional memory CD8 T cell pool that provides protective immunity against subsequent infections (1, 2, 3).
Listeria monocytogenes (LM)5 is a Gram-positive, facultative intracellular bacterium that has been extensively studied as a model of cell-mediated immunity (4, 5, 6). Infection of humans and animals, which often occurs upon ingestion of contaminated food, can lead to serious disease particularly in immunocompromised individuals (7). The bacteria are initially phagocytosed by macrophages and hepatocytes and thereby enclosed within host cell vacuoles. Subsequent secretion by LM of the hemolysin listeriolysin O (LLO) promotes escape from the vacuole compartment and release into the cytosol. Cytosolic growth and cell-to-cell spread of the organism allows infection of neighboring cells to occur while maintaining a protective intracellular niche (6).
Due to its intracellular lifestyle, immunity to repeat LM infection is dependent on CD8 T cells and not Abs. Sublethal infection with wild-type (WT) LM generates robust CD8 T cell responses, long-term memory, and protective CD8 T cell-mediated clearance upon secondary challenge (4). Infection with some attenuated strains of LM, such as those that cannot perform cell-to-cell spread or that cannot multiply within the cytosol, also leads to robust expansion of epitope-specific CD8 T cells and development of protective memory populations (8, 9, 10). However, several studies concluded that LM strains deficient in LLO, which cannot escape from the phagosome compartment, were avirulent and unable to elicit protective immunity (5, 11, 12). Although the reason for this is unknown, one implication is that reaching the cytosolic compartment and host MHC class I Ag-processing machinery is critical to the development of functional CD8 T cell memory.
In this report, we make the unexpected discovery that LLO-deficient LM are capable of inducing robust CD8 T cell priming against endogenous and recombinant (r) Ags which leads to the development of a functional, long-lived memory cell population. Upon secondary challenge with a high dose of virulent LM or after lymphocytic choriomeningitis virus (LCMV) infection, LLO-deficient LM memory mice are able to provide protective immunity similarly to WT-infected controls. Lowering the dose of LLO-deficient LM infection by 100- to 1000-fold results in priming of undetectable numbers of Ag-specific CD8 T cells; however, the vaccinated mice were able to mount an enhanced response to early (day 7 post primary infection) booster LM immunization and generate increased numbers of memory CD8 T cells. These results demonstrate that cytosolic entry is not required for the development of protective immune responses and may indicate the usefulness of highly attenuated strains of bacteria in vaccine design.
Materials and Methods
Mice and infections
BALB/c mice (6–10 wk old) were obtained from the National Cancer Institute (Frederick, MD). All animal experiments followed approved Institutional Animal Care and Use Committee protocols. The virulent LM strains that express LCMV-derived NP118-126 epitope in secreted (s) form XFL303 or HSL236 (WT LM-NPs) or nonsecreted (ns) form XFL304 (WT LM-NPns) (13), the LLO-deficient DP-L2161 (14), and the LLO-deficient NP118-126-expressing strains, LLO-deficicient LM-NPs, and LLO-deficient LM-NPns were grown, injected i.v., and quantified as described (15). The Armstrong strain of LCMV (LCMV-Arm; 2 × 105 PFU per mouse i.p.) was used as described (9).
Construction of LLO-deficient LM-NPs and LLO-deficient LM-NPns
LLO-deficient LM were constructed by allelic exchange as described previously (16). Vector pKSV7 (17) with a temperature-sensitive origin of replication, containing 500 bp of sequence upstream of the hly gene and 500 bp of sequence within the hly gene (a gift from D. Portnoy, University of California, Berkeley, CA), was introduced into the genome of competent WT LM-NPs and WT LM-NPns strains by electroporation. Colonies were selected at permissive temperature (30°C) on brain/heart infusion agar plates supplemented with chloramphenicol (10 mg/ml). Transformants were then grown on brain-heart infusion plates with chloramphenicol at nonpermissive temperature (41°C) to select for chromosomal integration of plasmids. Isolated clones were screened for plasmid excision and in-frame deletion of the hly gene by being streptomycin and kanamycin resistant, chloramphenicol sensitive, non-β-hemolytic on blood agar, and by mice surviving an infection of >109 organisms in the absence of any detectable LLO91-99-specific CD8+ T cell response. Equivalent growth curves also were measured in vitro between the parent strains (LM-NPs and LM-NPns) and the LLO-deficient strains.
Abs, peptides, and in vivo CD8 T cell depletion
The following mAbs were used: αIFN-γ (clone XMG1.2), αCD8 (53-6.7), anti-Thy1.2 (53-2.1), anti-TNF (MP6-XT22), anti-CD127 (A7R34), anti-CD27 (LG.7F9), anti-CD62L (MEL-14), anti-IL-2 (JES6-5H4), and rat IgG2a and IgG2b isotype controls (eBR2a, KLH/G2b-1-2). Defined LM LLO91-99 and p60217-225 or LCMV NP118-126 CD8 T cell epitopes were used as described (18). In vivo CD8 T cell depletion was performed by i.p injection of mAb 2.43 (0.25 mg per mouse) (19) on days −2 and −1 relative to LCMV challenge as described previously (15).
Quantification of Ag-specific CD8+ T cell responses and IFN-γ ELISA
The magnitude of the epitope-specific CD8 T cell response was determined by intracellular cytokine staining for IFN-γ or MHC class I tetramer staining as described (9, 20). The total number of epitope-specific CD8 T cells per spleen was calculated from the percentage of IFN-γ+CD8+ T cells, the percentage of CD8 T cells in each sample, and the total number of cells per spleen. IFN-γ concentrations in the serum were determined with an IFN-γ ELISA kit (BD OptEIA; BD Biosciences) according to the manufacturer’s recommendations.
In vivo cytotoxicity assay
Target cells were prepared by incubating equal numbers of naive BALB/c splenocytes with or without NP118-126 peptide (1 μM) for 1 h. After washing without peptide and with peptide, fractions were labeled at 37°C for 12 min with 0.25 μM CFSE (low) and 2.5 μM CFSE (high), respectively. Cells were washed, combined, and ∼10 × 106 total cells transferred into naive, WT LM-NPs, or LLO-deficient LM-NPs-immune mice i.v. The percentage killing was calculated using the following formula: 100 − ((percentage peptide pulsed in immune/percentage unpulsed in immune)/(percentage peptide pulsed in naive/percentage unpulsed in uninfected) × 100) (21).
Determination of CFU and PFU
On the indicated days after infection, portions of the spleen and liver were placed in a 0.2% IGEPAL (Sigma-Aldrich) solution. Organs were homogenized, and serial dilutions were plated onto tryptic soy agar containing 50 μg/ml streptomycin. Bacterial colonies were counted following plate incubation for ∼24 h at 37°C. The limit of detection (LOD) was 100 organisms per tissue. LCMV titers in homogenates of spleen were determined by plaque assay on VERO cells as described (9).
Results
LLO-deficient LM-NPs prime robust CD8 T cell responses
Previous work has shown that infection with LLO-deficient strains of LM fails to elicit protective immunity (5, 11, 12). However, it is unknown whether LLO-deficient strains cannot prime CD8 T cell responses, due to the inability of LM to reach the cytosol of the cell and Ag-processing machinery, or whether priming occurs and functional memory cells fail to develop. To examine whether LLO-deficient bacteria can elicit primary CD8 T cell responses, rLM strains expressing either a secreted (s) or nonsecreted (ns) LLO-deficient epitope from the nucleoprotein of LCMV (NP118-126) were used. LM-NPs and LM-NPns strains (13) were constructed by integration of an Ag cassette into the bacterial genome between the lecithinase operon and the lactate dehydrogenase operon. The Ag cassette contains the NP118-126 epitope fused to dihydrofolate reductase and includes a signal sequence in the case of LM-NPs to ensure efficient secretion. LM-NPs and LM-NPns strains were then engineered to have an in-frame deletion in the hly gene resulting in LLO-deficient LM-NPs and LLO-LM-NPns strains (Fig. 1⇓A). This deletion removes the LLO91-99 epitope recognized by CD8 T cells and prevents the production of functional molecules of LLO. LLO-deficient strains of LM are found only within host-cell vacuoles and are therefore incapable of intracellular growth (22). The deletion resulted in an avirulent strain of bacteria with an unknown LD50 that is >109 organisms.
Ag-specific CD8 T cell priming after LLO-deficient LM immunization. A, Construction of LLO-deficient LM strain. B, BALB/c (H-2d) mice were infected on day 0 i.v. with virulent LM expressing as a secreted fusion protein the LCMV-derived NP118-126 epitope (WT LM-NPs; 1 × 103; 0.1 LD50) or with LLO-deficient NP118-126 expressing LM (LLO− LM-NPs; 2 × 108). B, Bacterial numbers in the spleens and livers on day 3 postchallenge. C, The frequency of CD8 T cells that are NP118-126 specific is shown from representative mice at day 7 after infection as determined by peptide-MHC class I tetramers. Splenocytes from naive mice were used as staining control. D, The percentage of IFN-γ+/CD8+ T cells from the spleen of representative mice in the presence (upper numbers) or absence (lower numbers) of peptide(s) stimulation. E, Total number per spleen of LLO91-99-, NP118-126-, and p60217-225-specific CD8 T cells at day 7 after infection. Data are presented as mean + SD for three mice per group. One of four similar experiments is shown. Line represents the LOD.
Infection of BALB/c mice with either 103 CFU of virulent WT strain LM-NPs or with 200,000-fold more CFU of LLO-deficient LM-NPs (2 × 108), resulted in similar numbers of bacteria in the spleen and liver 3 days after infection (Fig. 1⇑B). Comparable clearance also was observed 7 days after infection (data not shown). At these doses of infection, similar priming of NP118-126-specific CD8 T cells 7 days after infection could be easily measured in both groups of immunized mice by either tetramer staining (Fig. 1⇑C) or staining for IFN-γ after short-term in vitro incubation with NP118-126 peptide (Fig. 1⇑D). Although similar frequencies and total numbers of p60217-225-specific CD8 T cells also were detected in both groups, as expected, no priming of LLO91-99-specific CD8 T cells could be measured after infection with LLO-deficient LM-NPs (Fig. 1⇑, D and E). In addition, phenotypic analysis of NP118-126-specific CD8 T cells showed similar low expression of CD127, CD62L, and low IL-2 production at day 7 after WT or LLO-deficient LM infections, suggesting that both immunizations elicited Ag-specific effector CD8 T cells (data not shown). Therefore, despite the highly attenuated nature of LLO-deficient bacteria and the inability of the LM to access the cytosolic Ag-processing machinery, it is possible to achieve similar total numbers of Ag-specific CD8 T cells as seen after WT LM infection at the peak of the primary response against both endogenous (p60217-225) and engineered (NP118-126) epitopes (Fig. 1⇑E).
LLO-deficient LM elicit CD8 T cells against nsAg
Infection of mice with virulent rLM strains expressing NP118-126 as either s or ns fusion protein primes NP118-126-specific CD8 T cell responses that differ by 3- to 4-fold (13). This difference has been attributed to the inability of bacterial nsAgs to efficiently enter the endogenous MHC class I presentation pathway, which involves degradation of proteins by the proteasome and transport into the endoplasmic reticulum. Because LLO-deficient LM cannot enter the cytosol, all bacterial Ags derived from infection must be processed through an exogenous MHC class I processing pathway. Although it was already clear that priming against the p60217-225s epitope was similar between LLO-containing and LLO-deficient LM, we generated LLO-deficient LM-NPns bacteria where the NP118-126 epitope was now expressed as an ns fusion protein (13). We then compared infection with LLO-deficient LM-NPs and LLO-deficient LM-NPns to assess whether a 3- to 4-fold difference in the number of NP118-126-specific CD8 T cells could still be detected in a situation where all Ags were restricted to exogenous Ag-processing pathways.
After infection of mice with either WT or LLO-deficient strains, we found that priming against NP118-126 was similar regardless of whether LM contained LLO (Fig. 2⇓). Infection with WT or LLO-deficient strains that secrete NP118-126 resulted in 4-fold more Ag-specific CD8 T cells, compared with ns strains. Priming against p60217-225 also was similar between LLO-deficient LM-NPs (Fig. 2⇓A) and LLO-deficient LM-NPns (Fig. 2⇓B). This suggests that an additional level of Ag processing exists that distinguishes bacterial nsAgs from sAgs irrespective of the intracellular compartment of the organisms.
Recombinant LLO-deficient LM priming of CD8 T cells specific for nsAg. BALB/c mice were infected with WT (3 × 103) and LLO-deficient LM (3 × 109) that are engineered to express NP118-126 epitope either in LM-NPs (A) or LM-NPns (B) forms. Total number per spleen of LLO91-99-, NP118-126-, and p60217-225-specific CD8 T cells at day 7 after infection. Data are presented as mean ± SD for three mice per group. All of the mice survived without detectable bacteria on day 7 after infection.
Kinetics of the CD8 T cell response after infection with LLO-deficient LM
Because previous reports have described the failure of immunization with LLO-deficient LM to provide protective immunity, we sought to follow the kinetics of Ag-specific CD8 T cell expansion and contraction after primary infection (Fig. 3⇓). Both WT and LLO-deficient bacteria generated NP118-126-specific CD8 T cells that expanded in number until day 7. This was followed by a period of contraction and memory generation that was indistinguishable between the two strains examined. Therefore, despite the 6-log difference in initial bacterial challenge, mice infected with either WT or LLO-deficient strains go through similar programs of expansion and contraction phases, and still maintain ∼105 NP118-126-specific CD8 T cells per spleen 30 days after infection.
Kinetics of Ag-specific CD8 T cell responses after WT and LLO-deficient LM infections. BALB/c mice were infected with WT (3 × 103) and LLO-deficient (3 × 109) LM on day 0 and total number of LLO91-99- and NP118-126-specific CD8 T cells were determined by intracellular IFN-γ staining on indicated days after infection. Data are presented as mean ± SD of three mice per group.
Phenotypic and functional characteristics of memory cells after LLO-deficient LM infection
We further examined the phenotypic and functional markers of NP118-126-specific memory CD8 T cells 30 days after infection with WT or LLO-deficient LM-NPs (Figs. 4⇓ and 5⇓). Regardless of the type of infection, similar frequencies of NP118-126-specific CD8 T cells were detected at this time point (Fig. 4⇓A) and the Ag-specific CD8 T cells were found to be largely CD127+, CD27high, and CD62Lhigh (Fig. 4⇓B), consistent with a central memory phenotype (23, 24, 25). Upon stimulation with NP118-126 peptide, most of the IFN-γ producing NP118-126-specific CD8 T cells in both groups produced TNF and a similar albeit lower frequency also made IL-2. Therefore, by both phenotypic and functional measures, NP118-126-specific memory CD8 T cells generated by infection with LLO-deficient LM-NPs are indistinguishable from memory cells generated by virulent LM-NPs infection.
Phenotypic and functional markers of memory CD8 T cells after WT and LLO-deficient LM infections. BALB/c mice were infected with WT (1 × 103) and LLO-deficient (5 × 108) LM and NP118-126-specific CD8 T cells were analyzed for phenotypic (CD127, CD27, CD62L) and functional (TNF, IL-2) markers 30 days post initial challenge. A, The frequency of NP118-126-specific CD8 T cells in the spleen was determined by intracellular IFN-γ staining after short term ex vivo incubation with peptide. Total number of NP118-126-specific cells in both group of mice was similar (∼0.6 × 105 per spleen). B, IFN-γ producing CD8 T cells (gated as shown in A were stained for the expression of indicated markers. Numbers inside flow profiles represent the frequency of cells that were scored as positive for particular marker. Representative profiles are shown. Similar data were obtained from three mice per group.
In vivo cytotoxicity of memory CD8 T cells after WT and LLO-deficient LM challenges. Naive and immune mice infected with either WT or LLO-deficient LM-NPs (30 days after infection) received equal numbers (1 × 107/mouse) of naive splenocytes that were previously incubated in the absence or in the presence of NP118-126 peptide and labeled with low (0.25 μM; w/o peptide incubation) or high (2.5 μM; w/peptide incubation) concentrations of CFSE. A, Six hours after transfer, the splenocytes from all groups of mice were analyzed and the percent of cytotoxicity was determined. Numbers inside panels indicate the percentage cytotoxicity. B, Percentage cytotoxicity. Each symbol represents the value obtained from an individual animal. One of two similar experiments is shown.
We also examined the in vivo cytotoxic capabilities of NP118-126-specific memory cells and found that 6 h after transfer of peptide-coated CFSE-labeled splenocytes, ∼50% of Ag-expressing targets in both types of memory hosts were destroyed, compared with Ag-negative controls (Fig. 5⇑). These data demonstrate that infection with either virulent or LLO-deficient LM results in robust priming of Ag-specific cytolytic memory CD8 T cells that phenotypically and functionally resemble central memory CD8 T cells.
LLO-deficient LM immunization results in robust protective immunity
We next asked whether the memory CD8 T cells generated by infection with LLO-deficient LM would be able to respond and provide protective immunity upon challenge with a high dose (∼10 LD50) of virulent LM (Fig. 6⇓A) or upon heterologous LCMV challenge (Fig. 7⇓A).
Secondary infection of WT or LLO-deficient LM immune mice. A, Experimental design. BALB/c mice were infected with non-NP118-126-expressing WT or LLO-deficient LM (1 × 103 or 2 × 109, respectively), or infected with NP118-126-expressing LLO-deficient strain of LM with 2 × 109 (Hi LLO-deficient LM-NPs) or 100-fold lower (2 × 107; Lo LLO-deficient LM-NPs) doses on day 0. Two months after primary immunizations all groups of mice were infected for the second time with WT LM-NPs (2 × 105; 10 LD50). B, CD8 T cell memory levels in indicated groups of mice at the time of secondary challenge. Total number per spleen of LLO91-99-, NP118-126-, and p60217-225-specific CD8 T cells at day 65 after infection. Data are presented as mean ± SD for three mice per group. nd, Not detectable. C, Frequency of LLO91-99-, NP118-126-, and p60217-225-specific CD8 T cells from representative mice at day 5 post secondary challenge (d65 + 5). The frequency of IFN-γ+/CD8+ T cells responding to the indicated peptide stimulation is shown. D, Total number per spleen of Ag-specific CD8 T cells before (d65) and after secondary challenge (d65 + 5). Data are presented as mean ± SD for three mice per group. E, Bacterial numbers in the spleen and liver 3 days after secondary challenge. Naive (nonimmune) mice were used as controls. All of the naive mice died after d3 (∗). Percentages inside panels indicate the frequency of reduction in CFU detected on day 3 post secondary challenge or the percentages of mice analyzed that showed detectable levels of infection.
LCMV challenge of LLO-deficient LM-NPs-immune mice. A, Experimental design. BALB/c mice were infected with 2 × 108 of LLO-deficient LM-NPs on day 0. Some of the LM-immune mice were depleted of CD8 T cells before LCMV challenge. An additional group of nonimmune (naive) mice were used as controls. B, LCMV titers in the spleen 3 days post LCMV challenge. Data are presented as mean ± SD for three mice per group. Percentages inside panel indicate the frequency of reduction in PFU detected on day 3 post LCMV infection. Line represents the LOD. C, Frequency of NP118-126-specific CD8 T cells in the spleen from representative mice at day 5 post viral infection. The frequency of IFN-γ+/CD8+ T cells responding to the NP118-126 peptide stimulation is shown. D, Total number per spleen of NP118-126-specific CD8 T cells before secondary challenge (d30) and after secondary challenge (d30 + 5). Data are presented as mean + SD for three mice per group.
We first infected mice with either WT (1 × 103) or LLO-deficient LM that do not express the NP118-126 epitope (2 × 109) or high (2 × 109) or low (100-fold less; 2 × 107) doses of LLO-deficient LM-NPs. The total number of LLO91-99, NP118-126, and p60217-225-specific memory CD8 T cells was measured 65 days after infection (Fig. 6⇑B). All mice were then challenged with 2 × 105 CFU of virulent LM-NPs (∼10 LD50 for naive mice), and the secondary expansion of Ag-specific CD8 T cells was measured as well as the clearance of LM from the spleen and liver. We found substantial expansion of memory CD8 T cell populations in all immunized mice 5 days after challenge with virulent LM as well as the development of new primary responses in the cases where new Ags were introduced (Fig. 6⇑, C and D). Surprisingly, we also found strong protective immunity by all previously infected mice, compared with a naive control group (Fig. 6⇑E). Additionally, one mouse from the WT LM group and two mice from the high LLO-deficient LM-NPs group had undetectable levels of bacteria remaining at day 3 after infection. Even primary infection with the LLO-deficient LM strain that does not express either the LLO91-99 or the NP118-126 epitopes resulted in strong protective immunity. Importantly, mice primed with 100-fold less LLO-deficient LM-NPs, generated Ag-specific memory CD8 T cells that provided protection against high-dose virulent LM rechallenge (Fig. 6⇑E). Finally, primary immunization with LLO-deficient LM that expresses NP118-126ns generated detectable numbers of memory NP118-126-specific CD8 T cells that were able to provide protection (>99.9% reduction in bacterial numbers) after high-dose virulent LM secondary challenge (data not shown). Thus, infection with LLO-deficient LM can generate memory CD8 T cell populations that are able to provide robust protective immunity against challenge with virulent bacteria.
We next asked whether NP118-126-specific memory CD8 T cells evoked after primary LLO-deficient LM-NPs immunization can provide protection against viral (LCMV) challenge. As shown in Fig. 7⇑, >99% reduction in PFU was observed in the spleen on day 3 post LCMV challenge of LLO-deficient LM-NPs immune mice, compared with naive controls. Importantly, protection is mediated by CD8 T cells because in vivo CD8 T cell depletion of LLO-deficient LM-NPs immune mice before LCMV challenge made those mice equally susceptible to LCMV infection as naive, LCMV-infected controls (Fig. 7⇑B). In addition, memory NP118-126-specific CD8 T cells were able to undergo Ag-driven proliferation in response to LCMV challenge, resulting in higher numbers of Ag-specific CD8 T cells than were found in naive mice given the same infection (Fig. 7⇑, C and D). Taken together, these results suggest that LLO-deficient strains of LM can be used to generate CD8 T cell responses that can provide protection against heterologous infections.
CD8 T cell priming with very low dose of LLO-deficient LM
After primary infections with either 1 × 103 WT LM-NPs or with high (2 × 108 or 2 × 109) doses of LLO-deficient LM-NPs, similar numbers of NP118-126-specific CD8 T cells were found at the peak of the expansion and at the memory phases of the CD8 T cell responses. In addition, both virulent and LLO-deficient LM infections stimulated an identical rate of effector to memory CD8 T cell conversion (Figs. 3⇑ and 4⇑ and data not shown). Finally, the duration of infection did not differ between mice immunized with virulent and LLO-deficient LM (Fig. 1⇑) suggesting that 5- to 6-log-higher numbers of LLO-deficient LM injected at day 0 were needed to prime similar CD8 T cell responses, compared with WT LM-NPs infection. Recent experiments have shown that inflammation controls the rate of effector to memory CD8 T cell conversion in vivo, suggesting that lowering the extent of inflammation early after infection might facilitate accelerated generation of memory CD8 T cells (26).
Therefore, to formally test the idea that immunization with highly attenuated LLO-deficient LM might prime CD8 T cells in an environment of reduced inflammation and accelerate the generation of memory-like CD8 T cells, naive BALB/c mice were infected either with high (2 × 108) or 100-fold lower dose (2 × 106) of LLO-deficient LM-NPs on day 0 (Fig. 8⇓A). The high-dose infection evoked substantial NP118-126 CD8 T cell expansion (Fig. 8⇓B), and those cells were CD127low and failed to produce IL-2 after peptide stimulation (effector CD8 T cells; data not shown). Only one of three mice infected with 100-fold lower dose of LLO-deficient LM-NPs showed detectable NP118-126-specific CD8 T cells (none of the mice had measurable p60217-225-specific CD8 T cell response; data not shown) on day 7 after infection, suggesting highly inefficient priming of CD8 T cell compartment. Nevertheless, both sets of immunized mice, together with naive controls (naive), were boosted with WT LM-NPs on day 7 after primary immunization. As expected (26, 27), effector NP118-126-specific CD8 T cells from high-dose LLO-deficient LM-NPs immunized mice did not expand by day 5 after booster challenge, and their memory CD8 T cell numbers (day 35 after secondary challenge) were similar to the control (nonboosted) group of immune mice (Fig. 8⇓, C and D). Surprisingly, substantial expansion of NP118-126-specific CD8 T cells after boost was observed in mice initially immunized with a low number of LLO-deficient LM-NPs. More importantly, those mice contained higher-memory CD8 T cell numbers at day 35, compared with the other two experimental groups (Fig. 8⇓, C and D). Taken together, these results suggest that a low dose of LLO-deficient LM infection might prime an undetectable, but real, number of Ag-specific CD8 T cells that are able to respond to earlier booster immunization. To explore whether Ag-specific CD8 T cells were indeed primed in a low inflammation environment, the level of IFN-γ in the serum of mice infected with either a high- or low-dose of LLO-deficient LM-NPs was examined 24 h after immunization (Fig. 9⇓). After a high-dose infection, IFN-γ in the range of 35–65 pg/0.1 ml of serum was detected. However, after low-dose infection, IFN-γ could not be detected, supporting the idea that this type of immunization primes CD8 T cells in a low inflammation environment. Future experiments will address the feasibility of low-dose infection as an approach to accelerate CD8 T cell memory generation.
Secondary infection early after primary challenge with different doses of LLO-deficient LM. A, Experimental design. BALB/c mice were infected with 2 × 108 (High) or 2 × 106 (Low) doses of LLO-deficient LM-NPs. Week after primary challenge both groups of mice and additional group of naive mice were infected with WT LM-NPs (1 × 104; ∼1LD50) and NP118-126-specific CD8 T cells were determined in the spleen at indicated days after infection. B, Total number of LLO91-99-, and NP118-126-specific CD8 T cells at day 7 after infection. The response of individual mice is shown. One of three mice infected with low dose of LLO-deficient LM had detectable levels of NP118-126-specific CD8 T cells. Line represents the LOD. C, The percentage of IFN-γ+/CD8+ T cells from representative mice in the presence (upper numbers) or absence (lower numbers) of peptide(s) stimulation. D, Total number per spleen of NP118-126-specific CD8 T cells at indicated days after infection. Data are presented as mean ± SD for three mice per group per time point.
IFN-γ serum concentrations after primary challenge with different doses of LLO-deficient LM. A, Experimental design. BALB/c mice were infected with 2 × 108 (high) or 2 × 106 (low) doses of LLO-deficient LM-NPs and IFN-γ concentrations in the serum were determined 24 h later. B, The concentration of IFN-γ in serum of control (naive) and infected mice. Each symbol represents the value obtained from an individual animal. Line represents the LOD.
Discussion
Previous investigators examining infection with LLO-deficient LM have concluded that cytosolic entry is necessary for the development of protective immunity (5, 11, 12). Support for this conclusion also has been provided by the lower IL-12 and TNF production by the host during LLO-deficient LM infection, suggesting that infection with virulent and attenuated strains generate fundamentally different responses by the host (28). Interestingly, microarray evidence shows that cytosolic entry by LM induces a completely different genetic profile by host cells (29). Given these conclusions in the literature, our results showing the strong development of protective immunity by LLO-deficient LM-NPs bacteria are surprising and warrant a reexamination of this topic.
We first examined the primary response of CD8 T cells after infection with a high dose of LLO-deficient LM-NPs and compared this to the response against WT LM (Fig. 1⇑). Although previous work has focused on the failure of LLO-deficient strains to generate protective immune response, there has not been a clear demonstration of the ability of this attenuated LM to activate CD8 T cell expansion and development into effector cells. We found similar priming of NP118-126- and p60217-225-specific CD8 T cells after infection with 0.1 LD50 of virulent LM or a 5-log-higher dosage of infection with LLO-deficient LM-NPs (Fig. 1⇑). These two doses of infection resulted in similar bacterial loads both early after infection and at day 7. Due to the vacuole localization of LLO-deficient LM, NP118-126 and p60217-225 Ags are likely to be processed through exogenous MHC class I processing mechanisms. Exogenous Ag have been shown to be capable of playing a major role in CD8 T cell priming, be it through cross-priming by professional APCs or another mechanism (30). Our data showing ∼10-fold lower numbers of NP118-126-specific CD8 T cells after LLO-deficient LM-NPns, compared with infection with LLO-deficient LM-NPs, would suggest that there are additional levels of processing that are affected by the secretion of bacterial Ags even when contained within a membrane-bound organelle.
The effector cells stimulated by LLO-deficient LM contracted in number with similar kinetics to WT LM-infected mice (Fig. 3⇑) and developed a long-lasting memory cell population that phenotypically and functionally resembled central memory T cells. The functional competency of NP118-126 memory CD8 T cells (Figs. 4⇑ and 5⇑), demonstrated by cytokine production and cytotoxic killing in response to peptide-coated targets, is in contrast with infection with heat-killed LM, which creates a substantial memory cell compartment that is unable to acquire effector functions (31).
Although we still detect >99% clearance of challenge LM infection after primary immunization with the reduced dose of LLO-deficient LM-NPs (107 CFU), it is 2–3 logs less efficient than the high-dose immunization (109 CFU). It is likely that very high doses of LLO-deficient LM are required to induce priming and protective immunity given the extremely attenuated nature of the bacteria and its rapid clearance from the host (12). This may, in part, explain why our results differ from one report that focused on a dose of infection not surpassing 107 organisms and only examined strains of bacteria that were not engineered to express additional CD8 T cell epitopes (12). Although strong protective immunity was observed without the NP118-126 epitope at a high dose of immunization (Fig. 6⇑E; LLO-deficient LM), we did not perform a titration of the infecting dose. Therefore, it is possible that the addition of NP118-126 allows for protective immunity to occur at lower dosages of immunization with LLO-deficient LM. In another report, when LLO-deficient LM failed to generate protective immunity, the experimental design was quite different from ours, with the secondary challenge occurring only 12–16 h after the primary infection (11). Because this does not allow time for the primary CD8 T cell response to develop and memory to form, this approach does not really address the vaccine potential capacity of a primary LLO-deficient LM immunization.
The degree of CD8 T cell-mediated protection after secondary infection depends on the quality and quantity of memory CD8 T cells (1, 8). Therefore, increasing the representation of these cells in vivo remains an important goal in successful vaccine designs. Classical prime-boost protocols where primary memory CD8 T cells expansion after booster immunizations leads to increased numbers of secondary memory are often used to achieve sufficient representation of memory CD8 T cells. Because generation of primary memory CD8 T cells takes time (32), exploring modalities to accelerate development of these cells might lead to rapid amplification of Ag-specific CD8 T cell memory. Recently, we have shown that priming of naive CD8 T cells in an environment of low inflammation (IFN-γ) accelerates the generation of CD8 T cells with phenotypic and functional (i.e., ability to respond to booster challenge) characteristics of memory CD8 T cells (data not shown and Ref. 26, 33). In this study, infection with a very low dose of highly attenuated LLO-deficient LM did not elicit measurable numbers of Ag-specific CD8 T cells in the spleen (LOD was 5000 cells per spleen) but did result in a substantially better expansion of Ag-specific CD8 T cells after early booster immunization with LM, compared with nonimmunized mice given the same challenge (Fig. 8⇑). Interestingly, due to their apparent ability to enter a second round of Ag-driven proliferation after secondary challenge, the numbers of memory CD8 T cells were increased in low-dose LLO-deficient LM-primed, compared with control, groups of mice given the same booster immunization. In addition, IFN-γ is not detectable in vivo early after infection with a low dose of LLO-deficient LM (Fig. 9⇑). This suggests that, because of the inability of LLO-deficient LM to efficiently multiply in vivo, inflammation is reduced at the time when naive CD8 T cells are primed by this immunization. Importantly, these data suggest that LLO-deficient LM might be used in a prime-boost strategy even at doses that are 10,000-fold or more below the LD50.
Taken together, immunization with LLO-deficient strains of LM generate functional memory CD8 T cell responses that can provide protection against bacterial or viral infections. These results may be useful in vaccine design, because we show that use of highly attenuated strains of LM can generate robust protective immunity against encounter with virulent organisms. Finally, LLO-deficient bacteria can be engineered to express additional CD8 T cell epitopes, raising the possibility of generating protection against multiple bacteria or viruses with a single live, attenuated dose of LM.
Acknowledgments
We thank Rebecca Podyminogin for technical assistance.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported by National Institutes of Health Grants R0IAI42767, R0IAI46653, and R0IAI50073 (to J.T.H.), T32AI07511 (to S.E.H.), and American Cancer Society Grant Seed Grant IRG-77-004-28 (to V.P.B.).
↵2 S.E.H. and V.P.B. contributed equally to this work.
↵3 Current address: Department of Laboratory Medicine and Pathology, University of Minnesota Medical Center, Center for Immunology, Minneapolis, MN 55454.
↵4 Address correspondence and reprint requests to Dr. John T. Harty, Department of Microbiology, University of Iowa, 3-501 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242. E-mail address: john-harty{at}uiowa.edu
↵5 Abbreviations used in this paper: LM, Listeria monocytogenes; LCMV, lymphocytic choriomeningitis virus; s, secreted; ns, nonsecreted; WT, wild type; LOD, limit of detection.
- Received March 14, 2006.
- Accepted June 23, 2006.
- Copyright © 2006 by The American Association of Immunologists
References
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