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The Journal of Immunology, 1999, 163: 322-329.
Copyright © 1999 by The American Association of Immunologists

Genetic Immunization of Mice Against Listeria monocytogenes Using Plasmid DNA Encoding Listeriolysin O1

Kenneth A. Cornell*,{dagger}, H. G. Archie Bouwer*,{ddagger},§, David J. Hinrichs*,{ddagger},§ and Ronald A. Barry2,*,§

* Immunology Research, Veterans Affairs Medical Center, {dagger} INTERLAB, {ddagger} Earle A. Chiles Research Institute, and § Department of Molecular Microbiology and Immunology, Oregon Health Sciences Center, Portland, OR 97207


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The development of protective immunity against many intracellular bacterial pathogens commonly requires sublethal infection with viable forms of the bacteria. Such infection results in the in vivo activation of specific cell-mediated immune responses, and both CD4+ and CD8+ T lymphocytes may function in the induction of this protective immunity. In rodent models of experimental infection with Listeria monocytogenes, the expression of protective immunity can be mediated solely by the immune CD8+ T cell subset. One major target Ag of Listeria-immune CD8+ T cells is the secreted bacterial hemolysin, listeriolysin O (LLO). In an attempt to generate a subunit vaccine in this experimental disease model, eukaryotic plasmid DNA expression vectors containing genes encoding either the wild-type or modified forms of recombinant LLO were generated and used for genetic vaccination of naive mice. Results of these studies indicate that the intramuscular immunization of mice with specifically designed plasmid DNA constructs encoding recombinant forms of LLO stimulates peptide-specific CD8+ immune T cells that exhibit in vitro cytotoxic activity. More importantly, such immunization can provide protective immunity against a subsequent challenge with viable L. monocytogenes, demonstrating that this experimental approach may have direct application in prevention of acute disease caused by intracellular bacterial pathogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The vaccination of experimental animals with plasmid DNA containing genes encoding unique proteins has proven effective in generating both humoral and cellular Ag-specific immune responses. Immunization of various species (ranging from mice to non-human primates) with unique plasmid DNA constructs encoding foreign proteins has resulted in immune responses to Ags derived from a variety of infectious agents, including influenza (1, 2, 3, 4, 5), HIV (6, 7, 8, 9, 10), rabies (11, 12), the hepatitis C and B viruses (13, 14, 15), malaria (16, 17), and mycobacteria (18, 19). Such immunization also has proven effective in inducing protective immunity in several animal models of viral disease (1, 3, 10, 11, 20, 21), as well as in the induction of specific antitumor immune responses (22, 23, 24, 25) and in the down-regulation of the expression of experimental autoimmune encephalomyelitis (26). Collectively, these observations suggest the potential application of this unique methodology to vaccine development and as a means to enhance tumor-specific cellular immunity and control autoimmune responses.

The generation of efficacious vaccines against many intracellular bacterial pathogens has proven problematic, as induction of protective immunity is evident only following recovery from sublethal infection with many microbial pathogens. Historically, experimental infection of mice with Listeria monocytogenes has evolved as the prototypic model for characterizing protective immunity to intracellular bacterial pathogens. Preliminary studies in this disease model have demonstrated that expression of protective immunity is dependent on Ag-specific T lymphocytes, which function via soluble mediators to enhance the bactericidal activity of phagocytic cells (27, 28). While CD4+ immune T cells play a critical role in the development of this immune response, the expression of protective immunity can be mediated solely by CD8+ immune T lymphocytes (29, 30, 31). One of the prominent Ags recognized by these CD8+ T cells is the secreted L. monocytogenes hemolysin, listeriolysin O (LLO)3, a protein which also functions as an essential virulence factor for this bacterial pathogen (32, 33, 34, 35, 36). Experimental studies with inbred BALB/c mice have revealed that a H2-Kd-restricted LLO-derived peptide, designated LLO91–99, is a target of immune CD8+ T cells that are induced following sublethal infection with L. monocytogenes (31, 37). These CD8+ T cells exhibit in vitro cytotoxicity against both LLO91–99-pulsed target cells and Listeria-infected phagocytic cell monolayers, and also provide in vivo protection following systemic challenge with this pathogen (31, 38, 39). In addition, other L. monocytogenes Ags (including the p60 and metalloprotease proteins) also can serve as a source of H2-Kd-restricted peptide epitopes recognized by CD8+ immune CTL recovered from immunized BALB/c mice (36, 38, 39, 40, 41, 42, 43).

To determine whether genetic immunization would be effective in the acute disease model of experimental murine listeriosis, we immunized BALB/c mice with eukaryotic plasmid DNA expression vectors containing either the wild type or modified forms of hly, the gene encoding LLO. Following a series of intramuscular immunizations, the in vivo stimulation of LLO91–99-specific CD8+ CTL was evaluated by in vitro cytotoxicity assays, and the development of protective immunity was assessed following lethal challenge with L. monocytogenes. Results of these studies demonstrated that genetic immunization of BALB/c mice with plasmid DNA constructs encoding the wild-type LLO molecule induced low levels of CTL in vivo, but little or no specific immunity. In contrast, immunization of mice with another plasmid DNA construct, encoding a recombinant LLO molecule containing both a substituted mammalian signal peptide sequence and a mutation resulting in reduced hemolytic activity of LLO, optimally induced LLO91–99-specific CTL activity and provided protective immunity against subsequent challenge with L. monocytogenes. These results demonstrate that genetic immunization with a specifically designed plasmid DNA construct can mimic the Ag-specific immune CTL response observed following sublethal infection with this pathogen. More importantly, these findings suggest that this unique immunization methodology can provide effective protection against an acute bacterial disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacteria

L. monocytogenes 10403 serotype 1 was originally obtained from the American Type Culture Collection (ATCC, Manassas, VA). Virulence has been maintained by repeated passage in BALB/c mice, and the LD50 for these inbred mice is 1 x 104 CFU (36). The generation and characterization of the LO28-W492A mutant strain of L. monocytogenes (kindly provided by Dr. Pascale Cossart, Pasteur Institute, Paris, France) has been described previously (44). Broth cultures of bacteria were established using brain heart infusion (BHI) medium (Difco, Detroit, MI).

Plasmid DNA constructs

The full-length hly gene was PCR amplified from L. monocytogenes DNA template using gene-specific sense (5'-CCCATGAAAAAAATAATGCTAGTTTT-3') and antisense (5'-CAATTATTCGATTGGATTATCTACTTT-3') oligonucleotide primers. The PCR product was cloned into the pCR3 plasmid vector (Invitrogen, Carlsbad, CA) and transformed into competent Escherichia coli TOP10F' cells according to the manufacturer’s instructions. Transformants bearing plasmids containing the complete hly gene in both forward (pLLO6) and reverse (pLLO11) orientations, relative to the strong CMV intermediate-early promoter sequence (PCMV), contained in this plasmid vector, were identified by restriction endonuclease analysis. The mutant hly gene construct (encoding a tryptophan to alanine change at amino acid position 492 of LLO) was PCR amplified from DNA template derived from the LO28-W492A mutant strain of L. monocytogenes and cloned into the pCR3.1 plasmid vector (Invitrogen) and analyzed as described above. hly gene sequences of all of the plasmid constructs were further confirmed by automated sequence analysis using a DNA Dye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).

Chimeric genes containing the signal peptide sequence derived from the gene encoding the murine tissue plasminogen activator protein (tPA) (45) fused to hly were constructed by PCR as follows. Truncated hly wild-type and W492A mutant genes (lacking the bacterial signal peptide sequence) were PCR amplified using a BamHI-containing sense oligonucleotide (5'-TCTGGATCCGATGCATCTGCATTCAATAAAG-3') and the hly antisense primer. The PCR products were cloned into pGEM-T plasmid vector (Promega, Madison, WI) and transformed into competent E. coli JM109 cells. The signal sequence from the tPA gene was PCR amplified from the pTAM2.5-a plasmid (ATCC 63042) using sense (5'-ATGAAGAGAGAGCTGCTGTGTGTACTGC-3') and BamHI-containing antisense (5'-TCTGGATCCTCTTCTGAACCTCCCATGTATT-3') oligonucleotide primers. The tPA signal sequence fragment and the truncated hly gene fragments were separately generated using BamHI/SphI digestion and ligated (Rapid DNA Ligation Kit, Boehringer Mannheim, Indianapolis, IN) to form chimeric gene templates. The chimeric genes were subsequently PCR amplified using the tPA gene sense and hly antisense primers and cloned into PCR3.1 plasmid vector. Following transformation into TOP10F' cells, plasmids containing the chimeric genes in forward and reverse orientations, relative to the PCMV sequence, were identified by restriction endonuclease digestion and confirmed by automated DNA sequence analysis. All plasmid constructs were maintained in the E. coli transformants under ampicillin selection, and large-scale concentrated preparations (3.0–8.5 mg/ml) of these constructs were generated using Plasmid Giga Kits (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions.

Mice and immunizations

Four- to 5-wk-old female BALB/cBkl and BALB/cJ mice were purchased from B&K Universal (Freemont, CA) and The Jackson Laboratory (Bar Harbor, ME), respectively. Mice were provided unrestricted access to food and water. For active immunization with viable L. monocytogenes, 6- to 8-wk-old mice received i.v. injections with 0.05–0.10 LD50 (~300–1000 CFU) in 0.2 ml of PBS. For immunization with the plasmid constructs, 6-to 8-wk-old mice received the first of a series of three i.m. immunizations (via the tibialis anterior muscles) at 3- to 4-wk intervals with ~100–125 µg of plasmid DNA in 50 µl of normal saline. Normal control mice received either no immunization or were immunized with either 0.2 ml of PBS (i.v.) or 50 µl of saline (i.m.).

Cell lines and synthetic peptide reagents

The J774 macrophage/monocyte cell line was maintained in antibiotic-free DMEM (Life Technologies, Grand Island, NY) supplemented with nonessential amino acids (Life Technologies) and 5% FBS (Tissue Culture Biologicals, Tulare, CA). The H2-Kd–transfected RMAS cell line (RMAS-Kd; originally obtained from Dr. Mike Bevan, University of Washington, Seattle, WA) was maintained in antibiotic-free RPMI (Life Technologies) supplemented with 10% FBS and 400 µg/ml Geneticin (Life Technologies). The peptides designated LLO91–99 (GYKDGNEYI) and p60217–225 (KYGVSVQDI) were synthesized at the Portland Veterans Affairs Medical Center with an Applied Biosystems Synergy apparatus using standard Fmoc chemistry. These peptides represent the major epitopes from the L. monocytogenes LLO and p60 proteins, respectively, recognized by protective CD8+ T lymphocytes derived from Listeria-immunized BALB/c mice (40, 41).

Activation of CTL and adoptive transfer of cells

Spleen cells obtained from normal mice, Listeria-immunized mice (at 3–8 wk following sublethal infection), or plasmid DNA-immunized mice (at 22–38 days following the final i.m. injection) were cocultured with peptide-pulsed, irradiated naive spleen cells (as stimulator cells). These stimulator cells were irradiated (3,000 rad from a 137cesium source) and pulsed with either LLO91–99 or p60217–225 for 1–2 h at room temperature (1 x 107 cells/ml, 5 x 10-7 M peptide in RPMI 1640 medium supplemented with 2% FBS). The stimulator cells were washed once then cocultured at 37°C with the donor spleen cell populations (from immunized mice) for 6 days. The cells were cocultured at 5 x 106 total cells per ml (at a donor to stimulator cell ratio of 1:1) in RPMI 1640 medium supplemented with 10% FBS and 23.8 mM sodium bicarbonate, 25 mM HEPES, 1 mM sodium pyruvate, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate (all Sigma, St. Louis, MO), and 30 U/ml recombinant human IL-2 (Tecin, Biological Response Modifiers Program, National Cancer Institute, Frederick, MD). Following culture, cells were washed twice with RPMI 1640 medium (without antibiotic), then used for adoptive transfer studies or in vitro cytotoxicity assays. In adoptive transfer experiments, groups of three to four mice served as recipients for each effector cell population, and each mouse was infused (via the lateral tail vein) with 3 x 107 viable cells in 0.2 ml of RPMI 1640 medium.

In vitro assays of cytotoxic activity

Target cells for the chromium release cytotoxicity assays consisted of chromium labeled, peptide-pulsed RMAS-Kd cells. Approximately 5 x 106 RMAS-Kd target cells were labeled with 250 µCi of Na51CrO4 (NEN Life Science Products, Boston, MA) for 60 min, washed twice, and then pulsed with a 10-9 M concentration of LLO91–99 or p60217–225 for 60 min. The peptide pulsed targets were added in 100 µl volumes to 96-well round-bottom microtiter plates at 1 x 104 cells/well, and effector cells were added in a 100 µl volume at the indicated E:T ratios. Following a 4-h incubation at 37°C, 150 µl of supernatant from each well was collected, relative radioactivity (cpm) determined (Micromedic gamma counter, ICN Micromedic Systems, Huntsville, AL), and the percent lysis calculated as: 100 x (experimental cpm - spontaneous cpm)/(total cpm - spontaneous cpm). Data reported represent means of triplicate wells. Total release was determined following lysis of target cells with 5% Triton X-100 (Bio-Rad, Redmond, CA), and spontaneous release was less than 7% for all experiments.

Target cells for the CFU reduction cytotoxicity assays consisted of Listeria-infected J774 cell monolayers. J774 cells were deposited at 1–2 x 105 cells/well in 24-well tissue-culture plates in 1.0 ml antibiotic-free DMEM with 5% FBS, cultured overnight, and then infected with L. monocytogenes (obtained from a log phase broth culture) at a multiplicity of infection of 2–5. After 60 min, the infected cell monolayers were washed once with sterile PBS, then covered with 0.5 ml of warm (37°C) DMEM supplemented with 5% FBS and 40 µg/ml gentamicin sulfate. Culture-stimulated effector cells were added (at indicated E:T ratios) in 0.5 ml of warm (37°C) DMEM with 5% FBS at 3–4 h after infection of the J774 monolayers. Assays were terminated 4–5 h later, and the number of intracellular bacteria remaining in each well was determined by hypotonic lysis of the J774 cell monolayers with 1.0 ml sterile distilled water, serial dilution of monolayer lysates from each well in PBS, and plating the dilutions on BHI agar (Difco). Following overnight culture at 37°C, the number of bacterial CFU per individual wells was determined. Data provided represent means of triplicate wells, and are calculated as follows: percent CFU reduction = [1 - (CFU in target cell monolayers incubated with effector cells)/(mean CFU in target cell monolayers incubated without effector cells)] x 100. For all experiments, the number of bacteria recovered from wells of Listeria-infected J774 cells cultured in the absence of effector cell populations ranged from 6.64 to 7.48 log10 CFU.

In vivo assay of immune protection

Levels of in vivo protection expressed by immunized mice were determined as previously described (30, 46). Briefly, groups of normal or immunized BALB/c mice received an i.v. injection with ~2 LD50 (~20,000 CFU) of L. monocytogenes in 0.2 ml of PBS, either simultaneous to infusion of culture-stimulated effector cell populations or at 3–4 wk following the final immunization with plasmid DNA. Control groups consisted of normal (nonimmunized) mice and mice previously immunized (4–12 wk earlier) with a sublethal injection of viable L. monocytogenes. Two days following bacterial challenge, spleens were removed from individual mice and homogenized in PBS, and serial dilutions (in PBS) of the homogenates were plated out on BHI agar. Following overnight culture at 37°C, CFU per spleen of individual mice were calculated and the mean level of protection for each group determined. Protection was indicated by reduced numbers of CFU in spleens of immunized mice, and calculated as: log protection = (log10 CFU/spleen of test mice) - (mean log10 CFU/spleen of normal control group).

Statistics

Analyses of the mean (±SEM) determinations for the chromium release and CFU reduction cytotoxicity assays and for the immune protection assays were performed by ANOVA (Tukey test) using the Instat biostatistical computer program (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunization of mice with plasmid DNA encoding LLO

Our initial attempts to induce LLO-specific immunity involved immunization of mice with a plasmid DNA construct containing the hly gene encoding the wild-type form of LLO. Mice were injected with this plasmid construct three times at 3–4 wk intervals. At 4 wk after the final injection, spleen cells obtained from these immunized mice were cocultured with irradiated, LLO91–99-pulsed syngeneic spleen cells obtained from naive donors (as stimulator cells), then used as effector cells in chromium release or CFU reduction cytotoxicity assays. As a positive control, spleen cells obtained from other mice at 5 wk following sublethal infection with L. monocytogenes were cocultured with this same stimulator cell population.

Results of the chromium release assays demonstrated that low, but significant (p < 0.05) levels of LLO91–99-specific cytotoxicity could be detected following culture stimulation of splenic lymphoid cells obtained from pLLO6-immunized mice or Listeria-immunized mice (Fig. 1GoA). In contrast, similar culture stimulation of lymphoid cells obtained from mice immunized with the parent plasmid lacking the hly gene (pCR3) or a plasmid DNA construct containing the hly gene in the reverse orientation (pLLO11) did not stimulate LLO91–99-specific CTL. In addition, none of these effector cell populations expressed any specific cytotoxic activity against target cells pulsed with the heterologous peptide, p60217–225 (Fig. 1GoB).



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  		FIGURE 1. Effector CTL derived from mice immunized with plasmid DNA containing the hly gene exhibit cytotoxic activity against LLO91–99-pulsed target cells. Mice were immunized i.m. three times with either saline, the pCR3 parent plasmid lacking the hly gene, or the plasmid constructs containing hly in the forward (pLLO6) or reverse (pLLO11) orientation (relative to the PCMV sequence), respectively. Spleen cells were obtained from mice at 28 days following the final saline or plasmid DNA immunization or from Listeria-immune mice at 35 days following sublethal infection, cocultured with LLO91–99-pulsed stimulator cells, then used as effector cells (at the indicated E:T cell ratios) against chromium labeled RMAS-Kd cells pulsed with either the LLO91–99 (A) or the p60217–225 (B) synthetic peptides.

 
These same effector cell populations derived from the plasmid DNA- or Listeria-immunized mice also were assessed for the ability to specifically recognize J774 cell monolayers infected with L. monocytogenes. This assay is performed by brief infection of the H2k J774 phagocytic cell line with the bacteria followed by coculture with BALB/c-derived cytotoxic effector cell populations (32, 36, 47). As observed in the chromium release cytotoxicity assays, low, but significant (p < 0.05), levels of cytotoxic activity could be observed following coculture of the infected J774 cell monolayers with culture-stimulated spleen cells derived from mice immunized with the pLLO6 plasmid or with viable L. monocytogenes, but not with the pCR3 or pLLO11 control plasmids (Fig. 2Go).



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  		FIGURE 2. CTL derived from mice immunized with plasmid DNA containing the hly gene recognize Listeria-infected target cells. Culture-stimulated spleen cells obtained from plasmid DNA- or Listeria-immunized mice (as described in Fig. 1Go) were used as effector cell populations (at indicated E:T cell ratios) against Listeria-infected J774 cell monolayers. CTL recognition of Listeria-infected target cells is indicated by reduced CFU recovered from individual wells of the infected cell monolayers (see Materials and Methods for details). The mean (±SEM) log10 CFU observed in wells of Listeria-infected J774 cells cultured in the absence of effector cell populations was 7.01 (±0.04).

 
Protective immunity observed following plasmid immunization of mice

The data described above demonstrate that genetic immunization with the pLLO6 plasmid can prime LLO91–99-specific cytotoxic cells in vivo. To determine whether these cells could mediate in vivo protection, effector CTL derived from either plasmid DNA- or Listeria-immunized mice were evaluated for their capacity to adoptively transfer protection to naive recipients, as determined by subsequent i.v. challenge of these cell recipients with L. monocytogenes.

In three of four experiments conducted, effector CTL derived from mice immunized with the plasmid DNA construct (pLLO6) containing the hly gene in the forward orientation could adoptively transfer low, but rarely significant, levels of protection to naive mice (Fig. 3GoA). CTL derived from Listeria-immunized mice consistently provided greater, and significant (p < 0.05), levels of protection following cell transfer (Fig. 3GoA). Adoptive transfer of similar cell populations derived from mice immunized with the parent plasmid (pCR3) or the plasmid construct containing the hly gene in the reverse orientation (pLLO11) did not provide any protection. Interestingly, the levels of active immunity expressed in mice immunized with either pLLO6 or the control plasmids (pCR3 and pLLO11) were relatively undetectable (Fig. 3GoB), especially as compared with the level of immunity expressed by mice sublethally infected with L. monocytogenes at 6 wk before bacterial challenge.



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  		FIGURE 3. Expression of adoptive and active immunity following immunization of mice with culture-stimulated effector cells, plasmid DNA, or viable L. monocytogenes. A, Spleen cells derived from mice immunized with plasmid DNA or viable Listeria were stimulated in culture (as described in Fig. 1Go) and then infused into normal BALB/c mice. Simultaneous to cell transfer, recipient mice and normal controls received an i.v. challenge with L. monocytogenes. B, At 34 days following the final saline or plasmid DNA immunization, or at 41 days following sublethal infection with L. monocytogenes, immunized mice and normal controls received an i.v. challenge with L. monocytogenes. At 2 days following L. monocytogenes challenge, the number of CFU per spleen of individual mice was determined. Data shown (log protection) represent the difference in mean log10 CFU recovered from spleens of normal control mice (6.69 ± 0.06) and the log10 CFU recovered from spleens of individual mice either infused with the effector cell populations (A) or immunized with saline, plasmid DNA or viable L. monocytogenes (B).

 
Generation of plasmid constructs encoding mutant and/or chimeric forms of the hly gene

The apparent failure to induce active immunity in experimental mice vaccinated with the plasmid DNA construct encoding LLO could be influenced by one or more factors, including 1) the inability of this vaccination methodology to induce adequate protection against a rapidly replicating, antigenically complex bacterial pathogen; 2) the suboptimal in vivo expression of the hly gene product, LLO, following plasmid DNA immunization; and 3) the potential toxicity of LLO for host APC following in vivo expression of this recombinant hemolysin. Therefore, additional plasmid constructs containing mutant or modified hly genes were generated (see Table IGo) for testing in this experimental disease model. One of these plasmid constructs (p492A) contained the hly gene cloned from the LO28-W492A mutant strain of L. monocytogenes, in which a dinucleotide base pair mutation results in an amino acid change (from tryptophan to alanine) in the cholesterol binding region (amino acid position 492) of the LLO molecule (44). This mutant form of the LLO product exhibits a 100- to 1000-fold reduction in hemolytic activity relative to wild-type LLO (Ref. 44 , and data not shown), and thus in vivo expression of this recombinant LLO molecule should be less toxic to the APC. Plasmid DNA constructs encoding chimeric hly genes also were generated by substituting the signal sequence derived from the gene encoding the tPA for the bacterial signal sequence of the wild-type and 492A mutant hly genes, then subcloning these chimeric genes into the pCR3 plasmid vector. Additional plasmid constructs containing these modified genes in the reverse orientations (see Table IGo) were developed and used as immunization controls.


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  		Table I. Description of plasmid DNA constructs

 
Immunization of mice with plasmid constructs encoding mutant and/or chimeric forms of the hly gene

Mice were immunized i.m. three times with the modified plasmid constructs at 3- to 4-wk intervals, and at 21–34 days following the last immunization spleen cells from donor mice were stimulated in vitro with irradiated, LLO91–99-pulsed syngeneic stimulator cells. Following culture, the effector cell populations were evaluated for cytotoxic activity against peptide-pulsed target cells or against Listeria-infected J774 cell monolayers. Data from these experiments demonstrated that effector CTL obtained from mice immunized with either the plasmid construct encoding the 492A mutant form of LLO (p492A) or the plasmid construct encoding the chimeric LLO molecule with the tPA signal sequence substitution (pTpaLLO6) exhibited enhanced cytotoxic activity relative to effector CTL derived from mice immunized with the plasmid construct encoding the wild-type hly gene (pLLO6) (Figs. 4Go and 5). Furthermore, effector CTL derived from mice immunized with the chimeric, mutant hly gene plasmid construct (pTpa492A) consistently exhibited the greatest cytotoxic activity, in some instances exceeding the cytotoxic activity observed using effector CTL derived from Listeria-immunized mice (Fig. 5Go).



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  		FIGURE 4. CTL derived from mice immunized with plasmid DNA containing the mutant or chimeric forms of the hly gene exhibit cytotoxic activity against LLO91–99-pulsed target cells. Mice were immunized with plasmid DNA constructs containing either the wild-type hly gene (pLLO6), the W492A mutant hly gene (p492A), the wild-type hly gene in reverse orientation with respect to the PCMV promoter sequence (pLLO11), the wild-type hly gene with the substituted murine tPA signal sequence (pTpaLLO6), or the W492A mutant hly gene with the substituted murine tPA signal sequence (pTpa492A). Spleen cells obtained from mice at 31 days following the final plasmid DNA immunization or at 38 days following sublethal infection with L. monocytogenes were stimulated in culture, then used as effector cell populations (at indicated E:T ratios) against chromium labeled RMAS-Kd cells pulsed with either LLO91–99 (A) or p60217–225 (B).

 


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  		FIGURE 5. CTL derived from mice immunized with plasmid DNA constructs containing mutant or chimeric hly genes recognize Listeria-infected target cells. Effector CTL derived from plasmid DNA or Listeria-immunized mice (as described in Fig. 4Go) were tested for cytotoxic activity against Listeria-infected J774 cell monolayers. Cytotoxic activity (percent CFU reduction) was determined as indicated in Fig. 2Go. The mean (±SEM) log10 CFU observed in wells of Listeria-infected J774 cells cultured in the absence of effector cell populations was 6.83 (±0.09).

 
The effector CTL populations derived from donor mice immunized with these improved plasmid constructs were further evaluated as to their ability to adoptively transfer protection to naïve syngeneic recipients. In each of four experiments, recipients of effector CTL derived from mice immunized with the plasmid DNA constructs containing the mutant or chimeric hly genes consistently demonstrated protection against L. monocytogenes challenge (Fig. 6GoA). Furthermore, in three of four of these experiments, effector CTL derived from mice immunized with the pTpa492A plasmid (encoding the 492A mutant LLO with the substituted tPA signal sequence) provided optimal adoptive protection to naive recipients (Fig. 6GoA). In contrast, similarly culture-stimulated spleen cells derived from mice immunized with the various control plasmid constructs (pCR3, pLLO11, p492AR, pTpaLLO11, or pTpa492AR) never resulted in effector cell populations exhibiting specific cytotoxicity as measured in vitro or in vivo (Figs. 4Go, 5Go, and 6GoA; other data not shown).



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  		FIGURE 6. Expression of adoptive and active immunity following immunization of mice with the mutant or chimeric hly plasmid DNA constructs. A, CTL populations derived from mice immunized with plasmid DNA or L. monocytogenes (as described in Fig. 4Go) were infused into normal BALB/c mice. Simultaneous to cell transfer, recipient mice and normal control mice received an i.v. challenge with L. monocytogenes, and 2 days later the number of CFU per spleen of individual mice was determined. B, At 37 days following final immunization with plasmid DNA or at 44 days following sublethal infection with L. monocytogenes, immunized mice received an i.v. challenge with L. monocytogenes, and 2 days later the number of CFU per spleen of individual mice was determined. Data shown (log protection) represent the difference in mean log10 CFU recovered from spleens of normal control mice (6.69 ± 0.06) and the log10 CFU recovered from individual spleens of adoptively or actively immunized mice.

 
To assess levels of active immunity following vaccination with the improved plasmid DNA constructs, additional groups of mice were challenged with viable L. monocytogenes at 33–39 days following the last plasmid DNA immunization. Results of these experiments demonstrated that only mice immunized with the pTpa492A plasmid construct (encoding the 492A mutant LLO with the substituted tPA signal sequence) exhibited significant levels (p < 0.05) of active immunity against bacterial challenge (Fig. 6GoB). In contrast, mice immunized with the p492A plasmid (encoding the mutant LLO) or the pTpaLLO6 plasmid (encoding wild-type LLO with the substituted tPA signal sequence) exhibited either little (nonsignificant) or no protection against a lethal challenge with L. monocytogenes. Mice immunized with the pLLO11, p492AR, and pTpaLLO11 control plasmid constructs never exhibited any protection (Fig. 6GoB; other data not shown). Thus, these results demonstrate that immunization of naïve BALB/c mice with a plasmid DNA construct (pTpa492A) containing a mutant, chimeric form of the hly gene not only stimulates LLO91–99-specific CTL, but also provides in vivo protection against subsequent challenge with this intracellular bacterial pathogen.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The evaluation of DNA vaccination in this murine disease model was facilitated by previous studies characterizing the protective immune response to experimental L. monocytogenes infection. The essential role of CD8+ cytotoxic T cells in this protective response, as well as the identification of a major target Ag (LLO) and a H2-Kd-restricted LLO peptide (LLO91–99) recognized by these immune CTL, were critical to the evaluation of genetic immunization in this model of intracellular bacterial pathogenesis. Although our preliminary attempts to induce protective immunity with a plasmid construct (pLLO6) encoding the wild-type form of LLO were largely unsuccessful, they did suggest that LLO91–99-specific CD8+ CTL could be primed in vivo. These initial experimental findings encouraged us to use other recombinant forms of the hly gene to facilitate this immunization event. One of these recombinant genes involved the substitution of the bacterial signal sequence of LLO with an eukaryotic signal sequence derived from the tPA gene (45). This chimeric gene was designed to facilitate enhanced in vivo expression of LLO by eukaryotic host APC populations (i.e., presumably tissue-specific macrophages and/or dendritic cells). A second hly gene was cloned from a mutant strain of L. monocytogenes in which a tryptophan to alanine change at amino acid position 492 (i.e., within the cholesterol-binding region of LLO) dramatically reduces the hemolytic activity of this bacterial toxin (44). This mutant gene was used to attenuate the potential toxicity of the recombinant LLO molecule expressed in vivo. A third recombinant hly gene incorporated both the substituted murine tPA signal sequence and the W492A mutation. Eukaryotic plasmid expression vectors containing each of these three modified hly gene constructs were evaluated as potential DNA vaccines in the induction of LLO-specific immunity. The data reported here demonstrate that immunization of mice with the plasmid DNA construct (pTpa492A) that encodes the less toxic LLO molecule fused to a mammalian signal sequence resulted in optimal in vivo priming of LLO91–99-specific CD8+ CTL and provided good protection against subsequent challenge with L. monocytogenes.

The specificity of this protective immune response was indicated by the ability of CTL derived from mice immunized with the pTpa492A plasmid DNA construct to recognize L. monocytogenes-infected J774 cells and to lyse RMAS-Kd cells pulsed with the LLO91–99, but not the p60217–225, peptide. In contrast, no CTL activity or in vivo protection was observed in mice immunized with plasmids containing any of the hly gene constructs in the reverse orientation (relative to the CMV promoter). This finding suggests that the protective response observed following immunization of experimental mice with the plasmid DNA vaccines is not a function of the innate immune response to nonmethylated CpG motifs present in the bacterial DNA (48, 49), as has been reported previously in the murine model of antilisterial immunity (50). However, as previously suggested (51, 52, 53), the presence of such immunostimulatory CpG motifs may provide an important intracellular signal that facilitates the subsequent induction of this LLO-specific immune response following immunization with the pTpa492A plasmid.

Although the level of protective immunity induced following immunization with the pTpa492A plasmid construct does not approach that observed following sublethal infection with viable L. monocytogenes, it approximates the level of protection observed following immunization of mice with a recombinant strain of Bacillus subtilis expressing LLO (35). Presumably, the protective immune response observed in mice immunized with the pTpa492A plasmid or the recombinant B. subtilis is mediated solely by LLO-specific T cells. In contrast, protective immunity induced following infection with viable L. monocytogenes represents the cellular response to several bacterial Ags, including LLO as well as the p60 and metalloprotease proteins of L. monocytogenes. These latter proteins also function as a source of H2-Kd-restricted peptides recognized by immune CD8+ CTL in the BALB/c murine model of experimental listeriosis (31, 37, 38, 40, 41, 42, 43, 54). In addition, BALB/c mice can be immunized successfully with a L. monocytogenes mutant (designated 92F) containing a disrupted LLO91–99 peptide epitope (a tyrosine to phenylalanine change at amino acid 92, which inactivates this amino acid anchor residue for binding to the Kd MHC class I molecule), further suggesting that other L. monocytogenes Ags can function as targets of the protective immune response (39). Thus, the significant level of protection (i.e., ranging from 1.5 to 2.5 log10 reduction in CFU per spleen) observed in mice immunized with the pTpa492A plasmid DNA construct is rather remarkable, as this response is induced in the absence of bacterial infection and is directed at a single Ag expressed by a rapidly dividing, intracellular bacterial pathogen. The data provided here also demonstrate that the H2-Kd restricted LLO91–99 epitope is a major target of the immune CD8+ cytotoxic T cells induced following immunization with the pTpa492A plasmid. However, whether such immunization also induces the MHC class Ib restricted immune CD8+ T cell response previously described in this murine model of experimental listeriosis (55) remains to be determined.

Recently, Uchijima et al. (56) have reported the successful immunization of mice with plasmid DNA constructs containing an oligonucleotide sequence encoding only the LLO91–99 epitope. Interestingly, they could not stimulate LLO91–99-specific CD8+ CTL nor induce in vivo protection using the wild-type bacterial codon sequence; that is, this immunization was successful only when the LLO91–99 sequence information was provided by substituted codons frequently found in murine genes. Their findings contradict an earlier report (57) describing immunization of BALB/c mice with a bacterial codon-encoded LLO91–99 minigene expressed in a recombinant vaccinia viral vector. The data we report here further demonstrate that in vivo activation of LLO91–99-specific CD8+ CTL and induction of protective immunity can be effected following immunization with plasmid DNA containing the bacterial codon-encoded form of LLO, albeit the expression of this Ag is regulated by a mammalian signal sequence. Thus, our findings demonstrate that the modification of nucleotide sequences to contain mammalian-specific codons need not be a prerequisite for the application of plasmid DNA vaccination in prevention of bacterial disease. In addition, our data demonstrate the nascent development of the LLO91–99-specific CTL responses following immunization with plasmids containing the full-length hly gene. This obviates the need to generate plasmid DNA vectors containing multiple nucleotide sequences encoding several peptides (derived from a single protein Ag) to accommodate the binding motifs of different MHC class I alleles, and thereby suggests the favorable application of this immunization methodology to outbred mammalian populations.

A number of laboratories are conducting studies directed at enhancing the efficacy of plasmid DNA immunization, either via improved plasmid DNA vectors, incorporation of multiple recombinant Ags or peptides, or co-injection of plasmid DNA constructs encoding cytokines that influence the immune response. Using one or more of these approaches, it may be possible to further augment the induction of protective immunity to L. monocytogenes following immunization with plasmid DNA constructs encoding wild-type or modified forms of LLO or other L. monocytogenes proteins. These studies should continue to provide information essential for the eventual application of this unique immunization methodology in the induction of protective immunity to other intracellular bacterial pathogens causing acute or chronic disease in mammalian populations.


   Acknowledgments
 
We thank Drs. Harriet Robinson, Michael Barry, and Stephan Johnston for helpful discussions, and Christian Sinai, Eva Barber, and Cheyenne Welch for technical assistance.


   Footnotes
 
1 This work was supported by the Department of Veterans Affairs and by National Institutes of Health Grants AI23455 and AI40783. Back

2 Address correspondence and reprint requests to Dr. R. Barry, Immunology Research P3-RD4, VA Medical Center, P.O. Box 1034, Portland, OR 97207. E-mail address: Back

3 Abbreviations used in this paper: LLO, listeriolysin O; LLO91–99, peptide corresponding to amino acids 91–99 of LLO; BHI, brain heart infusion; p60217–225, peptide corresponding to amino acids 217–225 of the p60 protein of L. monocytogenes; tPA, murine tissue plasminogen activator protein; PCMV, the CMV strong intermediate-early promoter sequence. Back

Received for publication November 23, 1998. Accepted for publication April 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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