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The Journal of Immunology, 1999, 163: 4510-4518.
Copyright © 1999 by The American Association of Immunologists

Effective DNA Vaccination Against Listeriosis by Prime/Boost Inoculation with the Gene Gun1

Joachim Fensterle*,{dagger}, Leander Grode*, Jürgen Hess*,{dagger} and Stefan H. E. Kaufmann2,*,{dagger}

* Department of Immunology, Max-Planck-Institute for Infection Biology, Berlin, Germany; and {dagger} Department of Immunology, University Clinics of Ulm, Ulm, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protective immunity against Listeria monocytogenes strongly depends on CD8+ T lymphocytes, and both IFN-{gamma} secretion and target cell killing are considered relevant to protection. We analyzed whether we could induce a protective type 1 immune response by DNA vaccination with the gene gun using plasmids encoding for two immunodominant listerial Ags, listeriolysin and p60. To induce a Th1 response, we 1) coprecipitated a plasmid encoding for GM-CSF, 2) employed a prime/boost vaccination schedule with a 45-day interval, and 3) coinjected oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. DNA immunization of BALB/c mice with plasmids encoding for listeriolysin (pChly) and p60 (pCiap) efficiently induced MHC class I-restricted, Ag-specific CD8+ T cells that produced IFN-{gamma}. Coinjection of CpG-ODN significantly increased the frequency of specific IFN-{gamma}-secreting T cells. Although pChly induced specific CD8+ T cells expressing CTL activity, it failed to stimulate CD4+ T cells. Only pCiap induced significant CD4+ T cell and humoral responses, which were predominantly of Th2 type. Vaccination with either plasmid induced protective immunity against listerial challenge, and coinjection of CpG ODN improved vaccine efficacy in some situations. This study demonstrates the feasibility of gene gun administration of plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Listeria monocytogenes, a Gram-positive facultative intracellular bacterium, serves as a useful model microorganism for studying protective immunity against intracellular pathogens (1). Successful immunization strategies against L. monocytogenes and several other intracellular bacteria need to induce a profound T lymphocyte response with emphasis on CD8+ CTL (2, 3, 4, 5, 6, 7), an area in which conventional subunit vaccination strategies often fail (8). Sublethal infection with live L. monocytogenes induces protective immunity against subsequent challenge infection (9). Several studies have demonstrated that CD8+ T cells are induced with specificity against two immunodominant Ags, listeriolysin (Hly)3 and p60 (10, 11, 12). Adoptive transfer experiments have shown that these Ag-specific CD8+ T cells protect against L. monocytogenes (13, 14). More recently, protection against L. monocytogenes was induced by different vaccination approaches, including immunization with recombinant vaccinia virus (15), recombinant attenuated Salmonella typhimurium as protein (16, 17, 18) or DNA carrier (19), and recombinant attenuated Bacillus anthracis strains (20).

The recently developed technique of DNA vaccination represents a type of subunit vaccination strategy, stimulating the immune response to a defined Ag, excluding the possibility of nonspecific components induced by a bacterial carrier (21). Delivery of naked DNA into the muscle or via the gene gun effects Ab formation as well as both MHC class I- and MHC class II-restricted T cell responses (22, 23, 24). Moreover, protection or at least partial protection has been induced by naked DNA vaccination in different animal models against viral pathogens including HIV (25, 26, 27, 28), hepatitis B virus (29), and influenza virus (22), as well as intracellular microbial pathogens such as Leishmania major (30) and Mycobacterium tuberculosis (31, 32).

Although gene gun administration bears several advantages over i.m. injection (33), the former approach seems to favor Th2 cell/B cell responses (34, 35, 36, 37). As Th1 responses are generally considered to be of major importance for control of intracellular pathogens, DNA vaccination approaches against these organisms have to date neglected the gene gun (38). The present experiments were designed to determine the feasibility of naked DNA vaccination by gene gun administration against the paragon intracellular pathogen, L. monocytogenes. We constructed two different plasmid vectors with the genes coding for the dominant listerial Ags, Hly (pChly) and p60 (pCiap), under the control of a CMV promotor. We show that prime/boost vaccination with both constructs by gene gun administration induced potent CD8+ T cell responses specific for the known H-2Kd-restricted dominant epitopes of Hly (Hly 91–99) and p60 (p60 216–225) in BALB/c mice. Coinjection of pChly or pCiap together with oligodeoxynucleotides (ODN) containing CpG motifs (CPG ODN), which have been identified as the main source of adjuvant activity of bacterial DNA (39), significantly increased the level of specific IFN-{gamma}-secreting T cells compared with coinjection with control ODN. Most importantly, both constructs protected against challenge infection with L. monocytogenes. Thus, prime/boost administration of naked DNA with the gene gun is a potent vaccination scheme in the model of experimental listeriosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and cell lines

Female BALB/c mice (6–8 wk old) were bred and kept under specific pathogen-free conditions in the animal facilities of the University of Ulm (Ulm, Germany) and of the Bundesgesundheitsamt für Veterinärmedizin und Verbraucherschutz Berlin. P815 mastocytoma cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (Life Technologies, Karlsruhe, Germany) supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 U/ml), and 2-ME. This medium is termed as RP10 medium. The transfected P815 cell line expressing Hly, PHem3.3 (13), was a generous gift of John Harty (University of Iowa, Iowa City, IA).

Plasmid DNA and ODN

Plasmid construction followed standard techniques (40). Briefly, the plasmid pChly (Fig. 1GoA) was constructed by PCR, amplifying the hly gene of L. monocytogenes without the bacterial signal sequence using a XhoI site containing forward primer and the XbaI site containing reverse primer. The PCR fragment was digested and integrated in the XhoI and XbaI sites of the eukaryotic expression plasmid pCI (Promega, Madison, WI). For the construction of pCiap (Fig. 1GoB), the p60 gene was amplified without the bacterial signal sequence by PCR using an EcoRI site containing forward primer and a XbaI site containing reverse primer. The PCR fragment was digested and integrated into the corresponding sites of the pCI plasmid. The plasmid pCMV-GM-CSF contains the gene encoding GM-CSF under the control of a CMV promotor and was a generous gift of Jörg Reimann (University of Ulm). Plasmid DNA was amplified in Escherichia coli XL1 Blue and purified using the Qiagen Endotoxin Free Plasmid Purification kit (Qiagen, Hilden, Germany). The precipitation of DNA on 1.6 µm gold particles (Bio-Rad Laboratories, Hercules, CA) was performed according to the manufacturer using 1 µg plasmid pChly, pCiap, or pCI coprecipitated with 0.8 µg plasmid pCMV-GM-CSF on 0.5 mg gold. For coprecipitation of pChly, pCiap, and pCMV-GM-CSF on 0.5 mg gold, 0.7 µg of each plasmid was used. The phosphothiate-modified ODN described by Chu and colleagues (41), ODN 1760, containing a CpG motif and ODN 1908, devoid of a CpG motif, were dissolved in PBS in a final concentration of 0.5 µg/ml. ODN were synthesized by Interactiva Biotechnology (Ulm, Germany).



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  		FIGURE 1. Maps of the Hly-encoding plasmid pChly (A) and the p60-encoding plasmid pCiap (B) used for DNA vaccination. The p60-encoding iap gene or the Hly-encoding hly gene of L. monocytogenes was amplified by PCR, excluding the signal sequence and integrated in the eukaryotic expression vector pCI.

 
Peptides

The listeriolysin H-2Kd epitope Hly 91–99 (42) and the p60 H-2Kd epitope p60 217–225 (12) were synthesized by Jerini Biotools GmbH (Berlin, Germany) and stored as stock solutions in PBS at 1 mg/ml.

Immunization

Gene gun inoculations were performed using the Bio-Rad hand-held helium-pulsed gene gun. For immunizations, two nonoverlapping shots per mouse were performed into freshly shaven abdominal skin at discharge pressures of 200 psi using 0.5 mg 1.6 µm DNA-coated gold particles per shot. Subsequently, 10 µg of the CpG-containing ODN 1760 or of the control ODN 1908 was injected intradermally at the site of particle bombardment. The immunization procedure was repeated after 45 days.

Protein purification

L. monocytogenes was grown to an OD (600 nm) of 1. The bacteria were harvested by centrifugation at 3000 x g. The supernatant was concentrated 50-fold using the MiniKros tangential flow membrane (membraPure, Bodenheim, Germany) with a 30-kDa cutoff, and further purified on a Mono-S ion-exchange column (Pharmacia, Piscataway, NJ). The flow through was collected and analyzed for the presence of p60 and Hly. The proteins were detected by SDS-PAGE immunoblotting with an anti-Hly mAb H14-3 (43) and with a polyclonal rabbit anti-p60 serum (17).

SDS-PAGE with immunoblotting

The protein concentration of the flow through was 4 mg/ml. The protein mix was adjusted to 2 µg/µl total protein. Following the manufacturer’s instructions, 20 µg were separated on 10% SDS-PAGE (Bio-Rad). The separated proteins were electrophoretically transferred to Hybond ECL nitrocellulose membrane (Amersham-Pharmacia, Little Chalfont, U.K.) and blocked overnight with PBS containing 1% BSA. The membrane was washed in PBS-Tween 0.05%, and pieces of each lane were incubated with pooled serum diluted 1/150 in PBS for 2 h. After the subsequent wash, membrane pieces were incubated with HRP-coupled anti-mouse IgG (1/20,000; PharMingen, San Diego, CA) for 1 h. The Western blot was developed with the enhanced chemiluminescence kit (Amersham-Pharmacia).

Cytokine ELISPOT assay

The frequency of IFN-{gamma}-secreting T lymphocytes specific for the L. monocytogenes H-2Kd epitopes Hly 91–99 and p60 217–225 was determined by ELISPOT (44). Briefly, 96-well nitrocellulose plates (Millititer HA; Millipore, Bedford, MA) were coated with 5 µg/ml of the anti-mouse IFN-{gamma} mAb R4 (PharMingen) in 100 µl of carbonate buffer, pH 9.6. After overnight incubation at 4°C, plates were washed twice with PBS and blocked for 2 h at 37°C with 100 µl of 1% BSA in PBS. Splenocytes (105) from vaccinated mice were added in 100 µl RP10 per well. P815 cells were coated with 1 µg/ml of Hly 91–99 or p60 217–225 at 37°C for 1 h and subsequently washed twice with RP10. Coated or uncoated P815 cells (105) were added to splenocytes in 100 µl of RP10 and after 20–22-h incubation at 37°C, 5% CO2 in the presence of 30 U/ml IL-2, plates were washed 10 times with 0.05% Tween 20 in PBS (washing buffer). To detect IFN-{gamma} spots, 0.25 µg/ml biotinylated anti-mouse IFN-{gamma} mAb XMB1.2 (PharMingen) in 100 µl wash buffer was added and incubated at 37°C for 2 h. Plates were washed 10 times in wash buffer and incubated for 1 h at 37°C in the presence of 100 µl of a 1/20,000 dilution of alkaline phosphatase-coupled streptavidin (PharMingen). After five washes, spots of IFN-{gamma}-secreting cells were visualized by adding 50 µl of the ready to use substrate BCIP/NBT (Sigma, St. Louis, MO) dissolved in water. The reaction was stopped after 15 min at 37°C by several washes with distilled water. After drying, spots were counted under a dissecting microscope at 3-fold magnification. The frequency of peptide-specific T cells is expressed as the number of IFN-{gamma}-secreting cells per 106 splenocytes.

For detecting Hly- or p60-specific MHC class II-restricted T cells, a slightly modified protocol was used. Ninety-six-well nitrocellulose plates were coated with 2 µg/ml of the IL-4-specific mAb BVD4-1D11 (PharMingen) in 100 µl coating buffer, and the IL-4-secreting cells were detected using 0.15 µg/ml of the biotinylated mAb BVD6-24G2 (PharMingen) in 100 µl wash buffer. Plates for IFN-{gamma} detection were prepared as described above. To induce specific cytokine secretion in CD4+ T cells, 4 x 105 spleen cells/well were incubated in 200 µl RP10 without the addition of IL-2 in the presence or absence of 10 µg heat-denatured, Mono-S-purified p60 or Hly. Incubation times and the detection followed the ELISPOT protocol for CD8+ T cells.

In vitro restimulation of CTL and CTL assay

Spleens from three individual animals 3 wk after the first immunization or 2 wk after prime/boost immunization were pooled and 2 x 106/ml splenocytes were restimulated in the presence of 2 x 105/ml {gamma}-irradiated (20,000 rad) PHem3.3 and 10% Con A supernatant in RP10 medium. After 5 days of culture at 37°C, 5% CO2, lymphocytes were harvested and tested for cytotoxicity in a standard 4-h Cr51 release assay. Briefly, the target cells PHem3.3 or P815 with or without the addition of 1 µg/ml Hly 91–99 were pulsed with 51Cr for 1.5 h. After two washes, 2 x 103 target cells in a volume of 100 µl RP10 were added to 100 µl splenocytes at various E:T ratios. Spontaneous release and total release samples were prepared by adding the targets to wells containing only medium or 1 M HCl, respectively. After 4 h, 100 µl of supernatant was collected and counted in a gamma counter. Percent specific release was calculated as 100 x ((experimental release - spontaneous release)/(total release - spontaneous release)).

Ab ELISA

Following the time schedule of ELISPOT analysis, sera from vaccinated mice were collected and kept at -20°C. For ELISA, 96-well NUNC Maxisorb plates (Nalgene-NUNC, Naperville, IL) were incubated at 4°C overnight with 100 µl 50x concentrated L. monocytogenes EGD supernatant diluted 1/2 in 0.5 M carbonate buffer (pH 9.6). After two washes with PBS-Tween 0.05%, plates were blocked at 37°C for 2 h with 150 µl 1% BSA in PBS. Pooled sera from three mice per group were added starting at a dilution of 1/50 and serially diluted 1/2 in 0.5% BSA in PBS-Tween (dilution buffer). As a control, a 1/800 dilution of the anti-Hly mAb H14-3 (1 mg/ml) was included. For the analysis of isotypes, serum was diluted 1/200 in dilution buffer. After 1.5 h at 37°C, plates were washed three times, and 100 µl alkaline phosphatase-coupled goat anti-mouse IgG, IgG1, or IgG2a diluted 1/1000 in dilution buffer was added. Plates were incubated at 37°C for 1 h and subsequently washed four times. The colorimetric assay was developed with 50 µl p-nitrophenyl phosphate (Sigma) in diethanolamine buffer, pH 9. The OD at 405 nm (OD405) was determined with a SpectraMax 250 ELISA reader (MWG Biotech, Ebersberg, Germany) after 20 min. Each assay was performed in triplicate, and data represent means ± SD of three values.

Protection against bacterial challenge

Three weeks after prime boost vaccination, mice were challenged with 104 L. monocytogenes EGD i.v. Five days after challenge, spleens were dissected and homogenized in PBS. Serial dilutions were plated on tryptic soya agar plates, and after incubation at 37°C, overnight colonies were counted. Data were analyzed using a two-tailed, unpaired t test. Differences between data sets were termed as significant if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene gun vaccination with pChly or pCiap induces IFN-{gamma}-producing T cells with specificity for the dominant MHC class I epitopes

In the first set of experiments, we ascertained whether gene gun immunization with pChly or pCiap induces a specific MHC class I-restricted CD8+ T cell response in BALB/c mice. To analyze the CD8+ T cell response, frequencies of peptide-specific T cells were determined in an ELISPOT assay. Three weeks after one immunization, pChly induced a significant T cell response to the major H-2Kd-restricted epitope Hly 91–99 (Fig. 2GoA). Coinjection of CpG ODN resulted in a 1.8-fold increase in the frequency of specific T cells. In contrast, mice immunized once with the pCiap plasmid showed no significant response against the H-2Kd-restricted epitope p60 217–225 (Fig. 2GoB). Also, the additional injection of a CpG ODN had no effect.



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  		FIGURE 2. Frequencies of Ag-specific IFN-{gamma}-producing CD8+ T cells 3 wk after prime and 5 days after boost vaccination. pChly (A and C) and pCiap (B and D) together with the CpG ODN 1760 (+CpG) or the control ODN 1908 (-CpG) were used for two vaccinations with a 45-day interval. Frequencies of epitope-specific MHC class I-restricted CD8+ T cells were determined using IFN-{gamma} ELISPOT assay. Five days after immunization, immune splenocytes were incubated in the presence of peptide-pulsed (filled bars) or nonpulsed (open bars) P815 cells. Peptide-induced IFN-{gamma} secretion by single cells was visualized and quantified; bars represent the mean ± SD of one assay performed in triplicate.

 
In the second part of the experiment, we tested whether booster immunization could increase the frequency of Ag-specific T cells. In pilot experiments, a 14-day prime/boost interval failed to induce detectable levels of specific CD8+ T cells (data not shown). We therefore extended the interval to 45 days. Booster immunization with pChly induced a moderate increase in T cell frequencies without coinjection of CpG ODN (Fig. 2GoC). In contrast, coinjection of CpG ODN resulted in a pronounced booster effect with a 4-fold increase in the frequency of IFN-{gamma} producers as compared with mice immunized with pChly alone. The second immunization with pCiap also caused a marked increase in T cell frequency (Fig. 2GoD), and treatment with CpG ODN did not further elevate this frequency.

All of the DNA preparations used to date were coprecipitated with pCMV-GM-CSF to enhance activation of dendritic cells (DC) (45). In the next set of experiments, the use of the plasmid pCMV-GM-CSF encoding for GM-CSF was addressed. Gene gun-mediated DNA vaccination with pCMV-GM-CSF coprecipitated with pChly moderately increased the frequency of specific CD8+ T cells (Fig. 3Go), and was therefore included in all subsequent experiments.



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  		FIGURE 3. Frequencies of Ag-specific IFN-{gamma}-producing CD8+ T cells after prime/boost vaccination with pChly in the presence or absence of pCMV-GM-CSF. Five days after prime/boost vaccination at a 45-day interval, frequencies of peptide-specific IFN-{gamma}-secreting CD8+ T cells were analyzed in an ELISPOT assay. Splenocytes from three mice per group were restimulated individually in the presence (filled bars) or absence (open bars) of Hly 91–99. Peptide-induced IFN-{gamma} secretion was visualized and quantified. Bars represent means and SD of the frequencies of specific T cells from three individual mice per group.

 
Following i.m. DNA vaccination, it has been shown that DNA is expressed over a significant time span (22, 46, 47). In additional experiments, we therefore asked whether gene gun vaccination effected a long lasting T cell response. Eleven weeks after the booster immunization, significant levels of IFN-{gamma}-secreting T cells were not detected for both vaccination schedules (Fig. 4Go). In contrast, 1 wk after sublethal infection with L. monocytogenes as a control, mice showed the expected Ag-specific IFN-{gamma} response (Fig. 4Go).



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  		FIGURE 4. Frequencies of Ag-specific IFN-{gamma}-producing T cells 11 wk after prime/boost vaccination. pChly (A) and pCiap (B) together with the CpG-containing ODN 1760 (+CpG) or the control ODN 1908 (-CpG) were used for two vaccinations in a 45-day interval. Eleven weeks after the boost, or 1 wk after sublethal (2 x 103 i.v.) L. monocytogenes EGD infection as a control, immune splenocytes were incubated with peptide-pulsed (filled bars) or nonpulsed P815 cells (open bars). Peptide-induced IFN-{gamma} secretion by a single cell was visualized and quantified; bars represent the mean ± SD of one assay performed in triplicate.

 
These data determine that gene gun administration of naked DNA induces most potent IFN-{gamma} T cell responses when 1) CpG ODN are coadministered and 2) the vaccination encompasses a prime/boost schedule.

DNA vaccination with pChly induces Hly CTL responses to the dominant MHC class I epitope

We next addressed the question as to whether the vaccination schedule delineated in the last series of experiments was capable of inducing specific CTL activity. Three weeks after the prime and 2 wk after the booster immunization, splenocytes were restimulated in vitro with the Hly-transfected cell line PHem3.3 and cytotoxicity was assessed (Fig. 5Go). As expected from the low frequencies of IFN-{gamma} producers, no cytotoxic activity was found after single immunization. However, after the booster immunization, CTL specific for PHem3.3- or Hly 91–99-pulsed P815 were induced. In contrast to the differential T cell frequencies observed in the ELISPOT experiments, the CTL assay revealed only small differences between mice coinjected with CpG ODN or with control ODN.



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  		FIGURE 5. Hly-specific CTL responses in mice after single or prime/boost vaccination. Mice were immunized with pChly together with ODN 1760 (+CpG) or ODN 1908 (-CpG) or the control plasmid pCI +CpG and boosted after 45 days. Three weeks after the first or second immunization, pooled spleen cells of three mice were tested for cytotoxicity in a standard chromium release assay 5 days after restimulation in vitro with the hly-transfected cell line PHem3.3. Lysis was assayed against the Hly transfectant PHem3.3 ({blacksquare}), H2-Kd-restricted peptide Hly 91–99-pulsed P815 cells ({blacktriangleup}), or P815 cells alone ({triangleup}). Spontaneous release was <10% of total release, and the significance level ((3 x SD of spontaneous release)/(total release - spontaneous release)) was <5% in all experiments.

 
DNA vaccination with pCiap, but not with pChly, induces significant CD4+ T cell responses

Potent induction of CD8+ T cells as observed in our experiments is generally paralleled by IFN-{gamma}-secreting CD4+ T cells. We therefore analyzed the type of the Hly- or p60-specific MHC class II-restricted T cell response induced by both constructs. Two weeks after primary immunization, neither construct induced significant numbers of specific CD4+ T cells in an ELISPOT assay using heat-inactivated, Mono-S-purified, Hly and p60 (data not shown). Booster immunization with pCiap resulted in a significant induction of CD4+ T cells, mainly of the Th2 type (Fig. 6Go, A and B). Interestingly, CpG coinjection did not influence the magnitude of specific CD4+ T cells secreting IL-4 (Fig. 6GoB), but only mice coinjected with CpG ODN showed relevant levels of Ag-specific IFN-{gamma}-secreting T cells (Fig. 6GoA). In contrast to the profound CD4+ T cell response induced by pCiap, pChly failed to induce significant levels of specific CD4+ T cells (Fig. 6Go, C and D). Thus, using gene gun DNA vaccination, a profound CD8+ T cell response can be induced in the presence of specific Th2-biased CD4+ T cells and even in the absence of a measurable CD4+ T cell response.



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  		FIGURE 6. p60- or Hly-specific CD4+ T cell responses induced by prime/boost gene gun DNA vaccination with pCiap or pChly. Five days after the boost, pooled splenocytes from three mice per group were incubated in the presence (filled bars) or absence (open bars) of Mono-S-purified, heat-denatured Hly and p60. IFN-{gamma} and IL-4 secretion of splenocytes from mice vaccinated with pCiap (A and B) or pChly (C and D) was quantified. Bars represent the mean ± SD of one assay performed in triplicate. The induction of IL-4 secretion by pCiap ± CpG ODN was highly significant compared with pCI (p < 0.001, two-tailed unpaired t test), as was the induction of IFN-{gamma} secretion by pCiap + CpG ODN compared with pCI (p < 0.001). pChly ± CpG ODN could neither induce significant IL-4 nor IFN-{gamma} production compared with pCI (p > 0.05).

 
DNA vaccination with pCiap, but not with pChly, induces a specific Ab response

Having analyzed the specific CD4+ T cell response induced by pChly and pCiap, we examined the Ab responses by Western blot analysis using Mono-S-purified Hly and p60 (Fig. 7GoA). Only pCiap induced specific IgG Abs, whereas pChly failed to do so. This finding was further confirmed in an ELISA using 50x concentrated L. monocytogenes supernatant as Ag (Fig. 7GoB). Starting from serum dilutions of 1/50, Abs to pChly or pCI were not detectable, whereas the anti-Hly mAb gave a positive signal. In contrast, pCiap induced high levels of Abs with a detectable signal up to a serum dilution of 1/6400. Mice coinjected with CpG ODN showed only marginal differences in Ab titers compared with mice coinjected with control ODN. Ab isotypes induced by pCiap (Fig. 7GoC) were predominantly IgG1, and the coinjection of CpG ODN did not result in measurable titers of IgG2a Abs at serum dilutions of 1/200. Only a slight decrease of IgG1 Abs may be contributed to CpG ODN coinjection. Thus, the Ab data confirm the predominant induction of Th2 CD4+ T cells by pCiap and the absence of significant CD4+ T cell stimulation by pChly using gene gun vaccination.



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  		FIGURE 7. p60- or Hly-specific Ab response induced by prime/boost vaccination with pCiap or pChly. Five days after prime/boost vaccination, serum was taken and pooled sera from three mice per group were analyzed for the presence of specific IgG Abs. A, Western blot analysis of sera diluted 1/150 using Mono-S-purified Hly and p60, as described in Materials and Methods. Only pCiap ± CpG ODN induced detectable levels of specific IgG Abs. B, ELISA with 1/2 serial dilutions of the sera beginning at 1:50 using 50x concentrated L. monocytogenes EGD supernatant as Ag; bars represent the mean ± SD of one assay performed in triplicate. pCiap ± CpG induced similar levels of IgG-specific Abs up to serum dilutions of 1/6400. pChly ± CpG ODN or pCI failed to induce a specific Ab response, whereas the anti-Hly control mAb diluted 1/800 gave a positive signal. C, Isotype analysis of sera from mice vaccinated with pCiap. Sera were diluted 1/200, and the OD405 using alkaline phosphatase-coupled anti-mouse IgG1 (black bars) or IgG2a (gray bars) as secondary Ab was determined; bars represent the mean ± SD of one assay performed in triplicate.

 
DNA vaccination with pChly and pCiap induces protection against L. monocytogenes infection

In the last set of experiments, we attempted to define the optimum vaccination schedules for protection against challenge infection with L. monocytogenes. Three weeks after booster immunization, mice were infected with 104 L. monocytogenes EGD i.v. A minimum delay of 14 days between the last vaccination and infection was necessary to avoid nonspecific protection induced by CpG ODN alone (48, 49). Five days after challenge, mice were sacrificed and splenic bacterial load was determined (Fig. 8Go). Mice vaccinated with pChly or pCiap alone or mice vaccinated with both plasmids at the same time were significantly protected compared with the animals immunized with the control plasmid and CpG ODN. Coadministration of CpG ODN slightly, but significantly, enhanced vaccine efficiency of pChly and resulted in sterile elimination of the pathogen. In the groups vaccinated with pChly and pCiap together, CpG ODN coadministration did not further improve protection significantly. However, vaccination with both plasmids already induced superior protection over vaccination with pChly or pCiap alone. These experiments demonstrate the feasibility of gene gun administration for efficacious DNA vaccination against intracellular bacteria. Moreover, they support the notion that CpG ODN improve protective responses stimulated by suboptimal vaccination protocols.



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  		FIGURE 8. Protection of mice from L. monocytogenes challenge by DNA vaccination with pChly or pCiap. Four or five female BALB/c mice per group were prime/boost vaccinated in a 45-day interval with pChly, pCiap, pCI, or pChly coprecipitated with pCiap together with ODN 1760 (+CpG) or ODN 1908 (-CpG). Three weeks after the booster immunization, mice were challenged with 104 L. monocytogenes EGD (LD50). Five days after challenge, the mice were killed and the bacterial load in spleen was determined. Each dot represents the bacterial load of an individual mouse, and bars represent the means. Protection was highly significant (p < 0.0002, two-tailed unpaired t test) for mice immunized with pChly or pCiap compared with naive mice. The small reduction observed using the control plasmid with ODN 1760 is statistically not significant (p > 0.05). Coinjection of ODN 1760 with pChly significantly reduced bacterial load (p < 0.0001) compared with coinjection with the control ODN 1908, whereas CpG coadministration did not reduce bacterial load significantly (p > 0.05) in the case of immunization with both pChly and pCiap.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although vaccination remains an important control measure for infectious diseases, for numerous pathogens efficient vaccines are not available yet (50). The methodology of naked DNA vaccination has provided a new strategy toward efficacious control of pathogens that have evaded control by traditional vaccination strategies. In animal models, this novel strategy has already shown great promise as a preventive measure against bacterial, parasitic, and viral infections (21).

In the mouse model, CD8+ T cells are pivotal in the control of infection with L. monocytogenes due to the capacity of this organism to egress from the phagosome into the cytosol (1). We have taken advantage of this well-characterized infection model to analyze the feasibility of gene gun administration of naked DNA to induce an antibacterial T cell response. In this work, we describe a vaccination schedule by which gene gun administration of naked DNA constructs encoding dominant Ags of L. monocytogenes induces protective immunity against listerial challenge infection.

It is known that the mode of application of naked DNA has a marked influence on the quality of the immune response (34, 35, 36, 37). Currently, two methods of administration are used most widely: direct injection into the muscle or gene gun administration into the dermis (21). Both techniques can induce efficient B and T cell responses. However, i.m. injection almost exclusively stimulates a type 1 response, whereas gene gun application tends toward stimulating type 2 responses (36). Type 1 responses are promoted by IL-12 and characterized by marked IFN-{gamma} secretion and induction of CTL, whereas IL-4 is predominantly produced during type 2 responses. To effectively combat diseases caused by intracellular pathogens including L. monocytogenes and M. tuberculosis, induction of a type 1 response is critical (1). Consequently, most of the previous work on DNA vaccines against intracellular bacteria employed the i.m. route of DNA administration (38). In our work, we addressed the question as to whether protective responses can be achieved by gene gun administration, which has several advantages over i.m. administration, including greater experimental reproducibility and the requirement for lower amounts of DNA. Although in principle the low amount of DNA required for gene gun vaccination is advantageous, the paucity of CpG motifs may be too low for efficient type 1 responses to be induced. We therefore analyzed effects of different parameters by gene gun application: 1) Coadministration of a GM-CSF-expressing plasmid; 2) coadministration of CpG ODN; and 3) prime/boost immunization schedule at an interval of 45 days.

GM-CSF is an important determinant of DC viability (51, 52) and activation (53), and enhances Ag presentation by these cells (54, 55). In the case of particle-mediated vaccination, direct transformation of DC seems to be essential for vaccine efficiency (56, 57, 58). Most importantly, in almost all studies, the use of GM-CSF has been found to improve the immune response upon DNA vaccination (45, 59, 60, 61). Consistent with these findings, coprecipitation of a GM-CSF-expressing plasmid enhanced the frequency of CD8+ T cells specific for Hly 91–99 and was therefore included in all of our experiments. Note that we coprecipitated this plasmid with the Ag-expressing plasmid to achieve expression in the same cell, because the DC rapidly migrate from the skin to lymph nodes.

Recently, CpG motifs were identified as the main adjuvant activity of bacterial DNA (39, 62, 63). These nonmethylated CpG motifs in bacterial DNA shift immune responses toward the type 1 pole (41, 63, 64, 65). Thus, administration of ODN-comprising CpG motifs has been shown to cure persistent Leishmania major infection (66) and to induce some nonspecific resistance against listeriosis (48, 49). Elimination of these motifs from plasmids used for DNA vaccination markedly reduces the efficiency of i.m. DNA vaccination, but has almost no effect on the outcome of gene gun administration (63, 64). Generally, 100-fold lower amounts of DNA are administered by the ballistic route as compared with i.m. injection (33). We speculated that the amount of accessible, extracellular CpG motifs after DNA administration by gene gun was too low to exert type 1-promoting activity. We therefore coinjected higher concentrations of synthetic ODN encompassing the CpG motif. Indeed, coinjection of CpG ODN with pChly significantly increased the numbers of specific CD8+ T cells in the absence of measurable CD4+ T cell and Ab responses. Coinjection of CpG ODN with pCiap had no influence on specific CD8+ T cell frequencies. However, successful induction of specific IFN-{gamma}-secreting CD4+ T cells with pCiap was dependent on coinjection of CpG ODN. We conclude that CpG ODN administration together with gene gun vaccination does not influence the magnitude of the type 2 response, but is important for the parallel induction of a significant Th1 response. Interestingly, existence of a prominent type 2 response did not interfere with the protective efficacy of gene gun vaccination with pCiap. To our knowledge, this is the first report describing significant improvement by CpG motifs of the immune response induced by gene gun vaccination. The use of such a protocol could be of particular interest for vaccines against pathogens, like Helicobacter pylori, which are controlled by a balanced Th1/Th2 response (67).

In pilot experiments performed with gene gun immunization of pChly, prime/boost intervals of 14 days did not induce significant levels of CTL. Only few attempts have been published to determine vaccination schedules that allow type 1 T cell induction by this application method. Yet, preliminary evidence indicates an important influence of repeated immunizations and the interval between the prime and boost (68, 69). Apparently, shorter intervals favor type 2 responses, whereas longer intervals tend toward type 1 polarization (68). Consistent with this, we find efficacious protection against listeriosis by gene gun DNA administration employing an interval of 45 days between the prime and the boost immunization. In contrast, shorter intervals (14 days) or single immunization failed to stimulate a measurable type 1 T cell response.

During revision of this manuscript, Cornell et al. published that successful DNA vaccination against L. monocytogenes via the i.m. route is only possible if a nonhemolytic form of Hly is used (70). In contrast, vaccination with the hemolytically active Hly failed to induce protection. At first sight, our success with gene gun vaccination using a construct encoding for hemolytic Hly contradicts these findings. This apparent discrepancy, however, can be explained as follows. The i.m. administration of DNA primarily transfects muscle cells leading to cross-priming (21). In contrast, the gene gun is thought to induce an immune response by directly transfected DC (58). We assume that the hemolytic activity of Hly abolishes efficient cross-priming, whereas priming by directly transfected DC is less affected. This speculation is supported by experiments using recombinant Salmonella as carriers for DNA constructs encoding hemolytically active Hly (19), in which professional APC are the primary targets of transfection. However, it is remarkable that, in our experiments, gene gun administration of pChly mounted a CD8+ T cell response in the absence of significant levels of CD4+ T cells. Hly is known to interfere with MHC class II presentation in vitro (71, 72), and hardly any anti-Hly Abs can be found during L. monocytogenes infection in vivo (73). Yet Hly-specific CD4+ T cells can be detected during natural infection of BALB/c mice (74). In our assay system, we can exclude interference of Hly with MHC class II presentation, because the biological activity of Hly was destroyed by heat treatment of the Ag preparation. CD8+ T cell activation in the absence of CD4+ T cells has been described in several models. Of particular interest is the stimulation of Listeria-specific CD8+ T cells in L. monocytogenes-infected MHC class II knockout mice lacking conventional CD4+ T cells (75). Yet we do not want to rule out the existence of Hly-specific CD4+ T cells below the detection limit of our ELISPOT assay, and therefore the possible role of CD4+ T cells in the induction of Hly-specific CD8+ T cells after gene gun vaccination needs to be elucidated in more detail.

Despite the general belief that gene gun injection preferentially induces type 2/humoral immune responses, our work demonstrates the feasibility of this vaccination regime for protective immune response in which type 1 T cells are required. It is rewarding that the most convenient way of DNA administration proved to be an efficacious vaccine, provided a prime/boost schedule was employed.


   Acknowledgments
 
We thank Drs. Jörg Reimann and Reinhold Schirmbeck for providing us the plasmid and for helpful discussion, Caitlin McCoull for secretarial assistance, and Michael Rolph and Jens Zerrahn for critically reading the manuscript.


   Footnotes
 
1 J.F. was supported by the Graduiertenkolleg für Biomolekulare Medizin. S. H. E. U. acknowledges financial support from the Fonds der Chemischen Industrie. Back

2 Address correspondence and reprint requests to Dr. S. H. E. Kaufmann, Max-Planck-Institute of Infection Biology, Monbijoustrasse 2, D-10117 Berlin, Germany. E-mail address: Back

3 Abbreviations used in this paper: Hly, listeriolysin; CpG motifs, unmethylated cytidine-guanosine dinucleotide containing motifs; DC, dendritic cell; ODN, oligodeoxynucleotide. Back

Received for publication March 18, 1999. Accepted for publication August 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Genetic Immunization Elicits Antigen-Specific Protective Immune Responses and Decreases Disease Severity in Trypanosoma cruzi Infection
Infect. Immun., October 1, 2002; 70(10): 5547 - 5555.
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A. Tawab, J. Fields, E. Chao, and R. J. Kurlander
Recombinant lemA without adjuvant induces extensive expansion of H2-M3-restricted CD8 effectors, which can suppress primary listeriosis in mice
Int. Immunol., February 1, 2002; 14(2): 225 - 232.
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B. I. Loehr, R. Pontarollo, R. Rankin, L. Latimer, P. Willson, L. A. Babiuk, and S. van Drunen Littel-van den Hurk
Priming by DNA immunization augments T-cell responses induced by modified live bovine herpesvirus vaccine
J. Gen. Virol., December 1, 2001; 82(12): 3035 - 3043.
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U. Zugel, A.-M. Sponaas, J. Neckermann, B. Schoel, and S. H. E. Kaufmann
gp96-Peptide Vaccination of Mice against Intracellular Bacteria
Infect. Immun., June 1, 2001; 69(6): 4164 - 4167.
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T. Yamada, H. Uchiyama, T. Nagata, M. Uchijima, T. Suda, K. Chida, H. Nakamura, and Y. Koide
Protective Cytotoxic T Lymphocyte Responses Induced by DNA Immunization against Immunodominant and Subdominant Epitopes of Listeria monocytogenes Are Noncompetitive
Infect. Immun., May 1, 2001; 69(5): 3427 - 3430.
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T. Ohashi, S. Hanabuchi, H. Kato, H. Tateno, F. Takemura, T. Tsukahara, Y. Koya, A. Hasegawa, T. Masuda, and M. Kannagi
Prevention of Adult T-Cell Leukemia-Like Lymphoproliferative Disease in Rats by Adoptively Transferred T Cells from a Donor Immunized with Human T-Cell Leukemia Virus Type 1 Tax-Coding DNA Vaccine
J. Virol., October 15, 2000; 74(20): 9610 - 9616.
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A. Tanghe, O. Denis, B. Lambrecht, V. Motte, T. van den Berg, and K. Huygen
Tuberculosis DNA Vaccine Encoding Ag85A Is Immunogenic and Protective When Administered by Intramuscular Needle Injection but Not by Epidermal Gene Gun Bombardment
Infect. Immun., July 1, 2000; 68(7): 3854 - 3860.
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