DNA vaccination with gp96peptide fusion proteins induces protection against an intracellular bacterial pathogen
Ulrike K. Rapp1 and
Stefan H. Kaufmann1
1 Max Planck Institute for Infection Biology, Schumannstrasse 2122, 10117 Berlin, Germany
Correspondence to: S. H. E. Kaufmann; E-mail: kaufmann{at}mpiib-berlin.mpg.de
Transmitting editor: S. Akira
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Abstract
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Effective vaccination using in vitro peptide-loaded heat-shock proteins (HSP), tumor-derived HSP and HSP fusion proteins has been shown in viral, parasite and tumor model systems. We demonstrate protective DNA vaccination using gp96peptide fusion proteins against the intracellular bacterial pathogen Listeria monocytogenes in a mouse model. In contrast to previous studies using pathogen-derived HSP as vaccine vehicles, we used recombinant endogenous (Mus musculus) gp96 (GRP94) as a carrier for immunodominant listerial peptides. Analyses of the cellular immune response revealed profound epitope-specific IFN-
and cytotoxic T cell responses. Our findings suggest a predominantly MHC I-restricted T cell response to DNA vaccination with gp96 fusion proteins in the model employed. Most importantly, DNA vaccination induced protection against an otherwise lethal dose of L. monocytogenes.
Keywords: CD8 T cell, gp96, heat-shock protein, Listeria monocytogenes, protective immunity
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Introduction
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Heat-shock proteins (HSP) are a family of proteins expressed in response to cellular stress. They chaperone nascent proteins and prevent accumulation of aggregated or mis-folded proteins (13). In stressed cells, HSP associate with a large pool of peptides, many of them with antigenic potential, as they are likely to be derived from proteins related to the cause of cell stress (47). gp96 belongs to a subgroup of HSP described as glucose-regulated proteins (GRP), which are located in the endoplasmic reticulum (ER) (8). The GRP play an essential role in multiple signaling pathways associated with cell stress, amongst these the unfolded protein response pathway in the ER and the inflammatory response (9,10). Expression of GRP is tissue specific and regulated primarily at the transcriptional level (9,10).
HSP have been exploited as vaccine carriers in parasite, virus and tumor model systems (5,11,12). These vaccination studies have confirmed a natural adjuvant function of HSP (1316), as well as induction of an adoptive immune response against peptides associated with the HSP carrier (47). Immunization with HSPpeptide complexes or HSP fusion proteins has been shown to induce cytotoxic T lymphocyte (CTL) and IFN-
responses directed at the peptide or fusion partner independently of CD4+ T cell help (47). These unique properties indicate preferred involvement of the MHC I antigen-processing pathway (47). However, the mechanisms responsible for these effects remain largely elusive. Recent studies directed at elucidating the cellular uptake and processing of HSP suggest that HSP complexes do not undergo lysosomal processing, but co-localize with MHC I molecules in the early endosome (1719). This could explain the unusual presentation of HSP-associated peptides by MHC I molecules, a processing pathway usually reserved for cytosolic, as opposed to endosomal, antigens.
After establishing the efficacy of HSP as vaccine vehicles, questions regarding the precise mechanism of their action and their role in immunology regained increasing interest. Several aspects of the relevance of HSP in the induction of a peptide-specific cellular response have been the focus of recent investigations, including cellular uptake, processing and signaling (1719). However, we are far from understanding the processes responsible for the unique efficacy of HSP as vaccine carrier molecules and the interactions of HSP with other molecules necessary to define their role in the induction of the observed cellular immune responses.
Experimental infection of mice with Listeria monocytogenes is widely used as a model for analyzing T cell-mediated anti-bacterial protection. This intracellular pathogen egresses into the cytosol and antigens are presented primarily via the MHC I pathway. As a result, MHC I molecules and CD8+ T cells are critical mediators of protection in addition to CD4+ T cells (20,21). In BALB/c mice, classical MHC Ia molecules play the major role in antigen restriction of protective immunity, while in C57Bl/6 mice the non-classical MHC Ib (H-2M3) molecules are central to restriction (20,21). Dominant L. monocytogenes epitopes of MHC Ia have been defined: p60217225 and listeriolysin (LLO)9199 (20,21).
Previous vaccine formulations based on HSP fusion proteins have used recombinant, pathogen-derived HSP60 or HSP70 as vaccine carriers (1113). HSP were loaded in vitro with antigenic peptides or HSP gene fusions were constructed and purified recombinant fusion proteins were used for vaccination (1113). HSP fusion proteins are chimeric N- or C-terminal gene fusions of HSP and the polypeptide of interest (11). We used DNA vaccines comprising recombinant endogenous (Mus musculus) gp96 (GRP94) protein fused to immunodominant peptide antigens LLO9199 and p60217225 of L. monocytogenes. We show for the first time successful DNA vaccination against a bacterial pathogen with HSPpeptide fusion proteins, using an endogenous, ER-derived GRP as carrier.
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Methods
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Mice
All experiments were performed using 8- to 12-week-old BALB/c female mice. Mice were obtained from our breeding facilities at the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin (Berlin, Germany). Naive mice and mice vaccinated with empty vector DNA were used as negative controls. Mice vaccinated with 1 x 103 viable L. monocytogenes EGD were used as positive controls. An additional group was immunized with gp96 DNA devoid of antigenic peptide. For survival studies, 10 mice were used per group; for all other assays, three mice were used per group and time point.
Peptides
Peptide design was based on the original protein sequence. LLO9199: GYKDGNEYI; LLO-elongated: VTNVPPRK GYKDGNEYI VVEKK; p60217225: KYGVSVQDI; p60 elongated: WALSV KYGVSVQDI MSWNN. Elongation of peptides was based on an estimation of those amino acids after which proteasomal cleavage is most likely to occur. Nonamer peptide sequences were elongated just enough to assure that potential proteasomal cleavage would not affect the immunodominant nonamer sequence. The amino acid sequence for the elongated peptides is based on the respective protein sequence.
Construction of vectors expressing fusion protein
gp96 fusion proteins were constructed by site-specific mutagenesis (Stratagene, La Jolla, CA). Each antigenic peptide was fused directly to the C-terminus of the gp96 protein. Fusion proteins were then cloned into the pCI expression vector (Invitrogen, Carlsbad, CA). All five constructs were sequenced and DNA for vaccination was purified from overnight bacterial cultures using Endofree GigaPrep Kits (Qiagen, Valencia, CA).
Naked DNA vaccination
DNA vaccination was performed 3 times at 3-week intervals. Mice were anaesthetized with 200300 µl i.p. Avertin and the hind legs of mice were shaved. DNA (50 µg) was applied intramuscularly into each left and right hind leg of BALB/c female mice.
Depletion of CD4+ and CD8+ T cells
CD4+ and/or CD8+ T cells were depleted 3 times at 7-day intervals, beginning 2 days prior to the second boost. Antibodies (anti-CD8
clone #169; anti-CD4 clone #191) were administered at a dose of 200 µg in a volume of 200 µl by i.p. injection.
Determination of CD8+ T cell response
ELISPOT
Spleens from vaccinated mice were prepared to a single cell suspension and were re-stimulated in vitro with 106 M peptide for 2 days. P815 cells were used as antigen presenting cells (APC) and were pulsed with 106 M peptide for 2 h prior to application in the assay. All tissue cultures were performed in RPMI medium (Biochrom, Berlin, Germany), supplemented with 10% FCS, 1 mM L-glutamine, 100 µg/µl penicillin and 100 U/µl streptomycin (all Biochrom). APC were added at 1 x 105/well to 96-well ELISPOT plates (Millipore, Molsheim, France) together with 1 x 1051 x 106 spleen cells. Plates were incubated at 37°C and 7% CO2 for 2224 h. We used the IFN-
mAb R4-6A2 (rat anti-mouse) and XMG-1.2 (biotin rat anti-mouse), and Sigma Fast substrate tablets (Sigma, Steinheim, Germany) for detection of IFN-
-specific spots. Each value presents the mean of triplicate values.
CTL assay
Spleens from three mice were prepared to a single-cell suspension and were cultured at 4 x 106 spleen cells/ml together with 3 x 106/ml (3000 rad) syngeneic stimulator cells. The stimulator cells had been pulsed at 37°C with 106 M peptide for 1 h. The cells were incubated in a 25-cm2 flask (Falcon, Heidelberg, Germany) at 37°C and 7% CO2 for 5 days. P815 cells were used as targets and were re-stimulated with 106 M peptide and 100 µCi 51Cr for 2 h prior to the assay. Targets were washed twice and added at 1 x 104 targets per well to a round-bottom 96-well tissue culture plate (Falcon) together with responder cells at different E:T ratios. After 34 h of incubation, 25 µl of supernatant was collected and 51Cr activity was determined. The percentage of peptide specific lysis was calculated as: (experimental value x spontaneous release)/(maximum release spontaneous release) x 100. Each value presents the mean of triplicate values.
Protection assay
Mice were challenged with 5 x 104 L. monocytogenes EGD (5 x LD50) 3 weeks after the second boost (63 days after the first vaccination). Survival was monitored for 10 days. Ten mice were used per group.
Statistical analysis
Statistical analysis for IFN-
ELISPOTs was performed using an unpaired Students t-test, with P < 0.05 indicating significant differences (GraphPad Prism 3.0). Statistical analyses for protection assays were performed using the log-rank test, with P < 0.05 indicating significant differences (GraphPad Prism 3.0).
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Results
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Construction and administration of gp96peptide antigen complexes
Consistent with studies focusing on in vitro loading of antigenic peptides onto various HSP, we found an average stable peptide-binding efficiency <10% (data not shown) (12,22). In order to optimize the construction of HSPpeptide complexes with a predictable 1:1 stoichiometry of HSP:fusion partner we constructed DNA encoding HSP fusion proteins (11). This DNA construct encodes a gp96 fusion protein with the antigenic peptide fused directly to the C-terminus of recombinant M. musculus gp96 (GRP94) by site-specific mutagenesis. We then cloned the gp96 fusion protein into the eukaryotic expression vector pCI. We tested four DNA constructs expressing different gp96 fusion proteins, using two immunodominant L. monocytogenes epitopes, LLO and p60, as fusion partners (20): LLO9199 (referred to as LLO-99 in the following), LLO82104 (referred to as LLOe), p60217225 (referred to as p60-217) and p60212230 (referred to as p60e). Since the cellular processing of HSP complexes has not been firmly elucidated, we constructed elongated versions of the originally described nonamer epitopes LLO9199 and p60217225, such that proteasome processing would be less likely to destroy the original peptide epitope sequence. Naked DNA vaccine constructs encoding gp96 fusion proteins were applied intramuscularly 3 times in total, with an interval of 3 weeks between each vaccination.
Peptide-specific IFN-
response induced by DNA vaccination with gp96 fusion proteins
We first analyzed peptide-specific IFN-
responses in vaccinated mice using a standard ELISPOT assay between day 7 and 28 after the second boost (Fig. 1). Spleen cells were re-stimulated with peptide for 2 days and used as effector cells. P815 and J774 cells were pulsed with peptide for 2 h before the assay and used as APC. Naive control, pCI vector control and gp96 alone induced negligible IFN-
responses. In contrast, on day 14 after the second boost, all gp96 fusion proteins induced significant IFN-
responses when re- stimulated either with heat-killed Listeria (HKL) or homologous peptide antigen, as compared to negative controls (P < 0.05). gp96p60e and gp96p60-217 induced equal IFN-
responses when re-stimulated with HKL or homologous antigen, which were both significantly higher than negative controls (P < 0.05). In the case of gp96LLOe and gp96LLO-99, both constructs induced IFN-
responses which were significantly higher than negative controls when re-stimulated with either HKL or homologous antigen (P < 0.05). However, gp96LLOe induced a significantly higher response than gp96LLO-99 when re-stimulated with homologous antigen (P < 0.05). The IFN-
response on day 21 after the second boost was markedly lower than on day 14 (data not shown).
Peptide-specific CTL response induced by DNA vaccination with gp96 fusion proteins
Second, we analyzed the induction of a peptide-specific CTL response between days 7 and 28 after the second boost. Spleen cells were re-stimulated with peptide in the presence of irradiated, naive spleen cells as APC for 5 days and used as effector cells. Peptide-specific CTL activity was measured in a standard 51Cr-release assay using peptide-pulsed P815 cells as targets (Fig. 2). Spleen cells from mice vaccinated with gp96 alone did not induce a measurable CTL response. The CTL response induced by the gp96p60e and gp96LLO-99 constructs on day 14 after the second boost was profound and comparable to the CTL response induced by L. monocytogenes infection. In contrast, the gp96p60-217 and the gp96LLOe constructs induced only marginal peptide specific lysis not exceeding the lysis observed for the control groups. The CTL responses measured on day 21 after the second boost did not exceed the lysis observed for the control groups (data not shown).


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Fig. 2. Induction of a peptide specific CTL response in DNA vaccinated mice. CTL response 14 days after second boost. (A) P815 restimulated with LLO peptides. (B) P815 restimulated with p60 peptides (abbreviations are as described in Fig. 1). For reasons of comprehensibility, the figures show specific target cell lysis only at E/T ratio 20:1, although killing was measured over a range of ratios from 60:1 to 2:1 at 3-fold dilutions. Representative results from one of two experiments with similar results are shown.
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Protection against lethal L. monocytogenes by DNA vaccination with gp96 fusion proteins
We next determined whether HSP-based DNA vaccination was able to confer protective immunity against an otherwise lethal dose of L. monocytogenes (Fig. 3). All four gp96 fusion protein constructs were tested. As expected, the gp96 alone and the pCI vector control did not protect against lethal challenge with L. monocytogenes. The gp96p60e construct induced complete and the gp96LLO-99 construct induced almost complete protection against L. monocytogenes, and in both cases protection was significant (P < 0.05). The protection induced by gp96LLO-99 was significantly higher than the negative control (P < 0.05), and there was no significant difference between the gp96LLO-99 construct and the positive control (P > 0.05). Although the gp96LLOe construct afforded 50% protection, this was not significantly higher than the protection induced by the negative control (P > 0.05). The gp96p60e construct induced equal protection as compared to the positive control and significantly higher protection than the negative control (P < 0.05). However, there was no significant difference between the protection induced by the gp96p60-217 construct and the negative control (P > 0.05). Interestingly, the same two HSP fusion protein constructs for which we observed the most efficient induction of peptide-specific CTL (see Fig. 2) were able to induce statistically significant protection against a normally lethal dose of L. monocytogenes.


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Fig. 3. Protection against L.monocytogenes by DNA vaccination. Mice were challenged with 5 x 104 L.monocytogenes i.v. 3 weeks after the second boost (abbreviations are as described in Fig. 1). Survival was monitored for 10 days. The pCI vector control and the gp96 alone induced significantly lower protection as compared to the positive control (vaccination with viable L.monocytogenes) (P < 0.05). (A) gp96-LLO constructs. The protection induced by gp96-LLO-99 was significantly higher than negative controls (P < 0.05) and there was no significant difference between the gp96-LLO-99 construct and the positive control (P > 0.05). Though the gp96-LLOe construct afforded 50% protection, this was not significantly higher as compared to the negative control (P > 0.05). (B) gp96-p60 constructs. The gp96-p60e construct induced equal protection as compared to the positive control, and significantly higher protection than the negative control (P < 0.05). However, there was no significant difference between the protection induced by the gp96-p60-217 construct and the negative control (P > 0.05). Ten mice were used per group. Representative results from one of two experiments with similar results are shown.
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Which T cell population is responsible for the observed cellular response and the protective immunity?
Finally, we wanted to define the T cell population critical for the cellular response to vaccination. To this end, we depleted CD4+, CD8+ or both CD4+ and CD8+ T cells using the appropriate mAb before the second boost. Results from CTL assays performed between days 7 and 28 after the second boost, as well as survival studies, revealed that the CTL response and protective immunity were mediated primarily by CD8+ T cells and largely independent of CD4+ T cells (see Fig. 4, which shows day 14 results). As expected, there was no significant difference between the protection afforded by the L. monocytogenes vaccinated positive control and the gp96LLO-99 construct in non-depleted mice (P > 0.05). Also, there was no significant difference between mice vaccinated with gp96LLO-99 and depleted of CD4+ T cells, and non-depleted mice (P > 0.05), and both groups showed a significantly higher survival than the negative control (P < 0.05). However, the protection induced in mice immunized with gp96LLO-99 and depleted of either CD8+ T cells or CD4+ plus CD8+ T cells was significantly reduced as compared to the survival observed for the non-depleted mice (P < 0.05). Vaccination with gp96 alone did not induce significant protection as compared to the negative control (P > 0.05).



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Fig. 4. Phenotype of T-cell population critical for DNA vaccine induced immunity. CD4+, CD8+, or both CD4+ and CD8+ T cells were depleted using the appropriate mAb before the second boost. (A) CTL assay at day 14 after second boost, using P815 restimulated with LLO peptide. For reasons of comprehensibility, the figures show specific target cell lysis only at E/T ratio 20:1, although killing was measured over a range of ratios from 60:1 to 2:1 at 3-fold dilutions. Representative results from one of two experiments with similar results are shown. (B) Protection assay with gp96-LLO constructs. There was no significant difference between the protection afforded by the positive control and the gp96-LLO-99 construct in non-depleted mice (P > 0.05). Similarly, protection in mice vaccinated with gp96-LLO-99 and depleted of CD4+ T cells did not significantly differ from non-depleted mice (P > 0.05), and both groups showed a significantly higher survival than the negative control (P < 0.05). However, the protection induced in mice vaccinated with gp96-LLO-99 and depleted of either CD8+ T cells, or CD4+ plus CD8+ T cells was significantly lower than the survival observed for non-depleted mice (P < 0.05). Protection in CD8+ T cell, or the CD4+ plus CD8+ T cell depleted mice did not differ from the negative control (P > 0.05). Vaccination with gp96 alone also failed to afford significant protection as compared to the negative control (P > 0.05). (C) Protection assay with gp96-p60 constructs. Protection in the positive control and the gp96-p60-e vaccinated non-depleted mice was equally high (P > 0.05). Protection in mice vaccinated with gp96-p60-e and depleted of CD4+ T cells, and that in non-depleted mice, did not differ significantly (P > 0.05), and both groups showed a significantly higher survival than the negative control (P < 0.05). However, the protection in mice vaccinated with gp96-p60-e and depleted of either CD8+ T cells, or CD4+ plus CD8+ T cells, was significantly reduced as compared to non-depleted mice (P < 0.05). Neither the CD8+ T cell nor the CD4+ plus CD8+ T cell depleted mice differed from the negative control (P > 0.05). Protective immune responses were determined as described in the legend to Fig. 3. (LLO-99:no depletion, LLO-99--4:depletion of CD4+, LLO-99--8:depletion of CD8+, LLO-99-4+8:depletion of both CD4+ plus CD8+ T cells, abbreviations are the same for gp96-p60e construct). For the CTL assay, three mice per group were used; for the protection assay, 10 mice per group were used (see Methods). Representative results from one of two experiments with similar results are shown.
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With regard to the protection afforded by the gp96p60e construct, there was no significant difference between the protection afforded by the L. monocytogenes-vaccinated positive control and the gp96p60e construct in non-depleted mice (P > 0.05). Protection in mice vaccinated with gp96p60e and depleted of CD4+ T cells, and non-depleted mice did not differ (P > 0.05), and both groups showed a significantly higher survival than the negative control (P < 0.05). However, the protection induced in mice vaccinated with gp96p60e and depleted of either CD8+ T cells or CD4+ plus CD8+ T cells was significantly reduced as compared to the non-depleted mice (P < 0.05). Thus, both for gp96LLO and for gp96p60 vaccination, depletion of CD8+ T cells abrogated protection, while depletion of CD4+ T cells did not do so. We conclude that DNA constructs encoding gp96peptide fusion proteins are capable of inducing potent CD8+ T cell responses, which protect against an intracellular bacterial pathogen.
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Discussion
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Previous studies in virus and parasite model systems have focused on the use of pathogen-derived HSP as vaccine carriers (1115), most frequently cytosolic bacterial HSP60 or HSP70 (1115). In vitro loaded HSPpeptide complexes or HSP fusion proteins were applied s.c. or intradermally as purified recombinant proteins (1115). Alternatively, recombinant HSP fusion proteins were applied intramuscularly as naked DNA (15). The basic rationale behind the use of HSP as vaccine vehicles is based primarily on two observations: (i) the ability of HSP to cross-prime and to induce dominant activation of the MHC I restricted CD8+ T cells (47), and (ii) the strong general immunostimulatory quality of pathogen-derived HSP (1116,22). The application of endogenous HSP was avoided initially because of the anticipated risk of inducing unwanted autoimmune responses to the HSP (23). However, studies have shown that probably due to the high amount of sequence homology between endogenous and pathogen-derived HSP, the induced cellular response is similar (23). We chose endogenous gp96 because we aimed to use the HSP not as an antigen, but solely as a carrier for the fused listerial antigen. The ability of HSP to cross-prime and activate the MHC I antigen-presentation pathway reserved usually for cytosolic, as opposed to endosomal, antigens makes them particularly interesting vaccine vehicles to combat intracellular pathogens, where the induction of a potent CD8+ T cell response is essential (24). Because HSP shuttle antigens towards the MHC I antigen-presentation pathway, we expected that the use of HSP as a vaccine vehicle would promote CD8 T cell responses against the dominant listerial epitopes. Bacterial HSP may contain CpG motifs that stimulate immune responses, thus providing adjuvant activity. We reasoned that if we use murine, endogenous gp96 as a carrier for our assays, the gp96 carrier would be largely ignored by the immune system and we would thus focus the immune response on the antigenic listerial peptides.
In our study we observed induction of a profound peptide-specific IFN-
response and peptide-specific CTL activity for two gp96 fusion protein constructs: gp96LLO9199 and gp96p60212230. The two other constructs tested were able to induce a peptide specific IFN-
response, but negligible peptide-specific CTL lysis. Interestingly, those two constructs, which induced a significant CTL response, also induced efficient protection against challenge with an otherwise lethal dose of L. monocytogenes. Our findings argue against antigen-specific IFN-
production as a robust correlate of protection in experimental listeriosis and point to CTL responses as the more reliable surrogate.
We cannot explain at this point what caused the superior efficacy of the gp96LLO9199 and gp96p60212230 constructs. However, we assume that the reason lies in the processing of the gp96 constructs in the cell and the integrity of the resulting immunodominant peptide epitope. Proteasome cleavage site prediction programs and analysis of immunoproteasome cleavage motifs by MALDI and mass spectrometry may prove helpful in understanding the intracellular processing and efficacy of individual HSP fusion proteins (25). Analyses of the T cell populations responsible for the observed IFN-
and CTL responses are consistent with the assumption that protective immunity induced by HSP-based DNA vaccination is mediated largely by CD8+ T cells. This is in agreement with previously published data by others in parasite and viral model systems (48,1115). However, we cannot formally exclude a contribution of CD4+ T cells to the immune response we observed. Also, we did not analyze the contribution of CD4+ T cells during the priming phase (20,21). However, the use of dominant listerial MHC I epitopes and the lack of listerial MHC II epitopes in our system make a major contribution of Listeria-specific CD4+ T cells unlikely.
We observed no measurable IFN-
and CTL response to immunization with gp96 DNA without peptide. This indicates a low immunostimulatory potential of endogenous gp96 at the dose applied and implies lack of autoimmune reactivity to the vaccine carrier employed. We conclude that endogenous gp96 served solely as a carrier molecule for the protein or peptide associated with it and was responsible, albeit by a mechanism not yet fully understood, for shuttling the associated peptides into the MHC Ia presentation pathway.
For studies using purified proteins, in particular, it remains to be verified how far the observed effects can be ascribed to the HSP complexes themselves, contaminating endotoxin or concanavalin A. Questions regarding endotoxin contamination of the DNA vaccine are legitimate, although the DNA was purified using an Endofree plasmid purification kit. However, we do not assume eventual endotoxin contamination to be responsible for the observed cellular immune response for two reasons. (i) The fusion proteins are generated de novo in the cell after transfection with naked DNA. (ii) Eventual endotoxin contamination should have induced a more significant unspecific immune response to immunization with all constructs equally and the gp96-minus peptide construct in particular.
We describe here efficacious antibacterial protection afforded by a naked DNA vaccine encoding gp96peptide fusion proteins. Protection was accompanied by profound antigen-specific IFN-
secretion and CTL activation. CD8+ T cells were shown to be crucial for all three activities: protection, IFN-
and CTL. Are the protection and the memory responses to L. monocytogenes dependent on CD8+ or CD4+ T cells, or both? This question has been approached in a series of recent studies (2632). Most investigations revealed CD4+ T cells to be essential during the priming, but not during the recall response to infection (3336). In addition, Harty and co-workers have shown that there is no requirement for the co-stimulatory ligand CD40L in exogenous antigen presentation to CD8+ T cells in L. monocytogenes infection (26). In our study, we depleted CD4+ T cells or CD8+ T cells directly before the second booster immunization. Hence, CD4+ T cells could have participated in the priming of CD8+ T cells; yet CD4+ T cells were not essential for recall and protective responses in our system. Given a contribution of CD4+ T cells to the host response, the question arises as to their specificity. Because we used dominant MHC I epitopes of LLO and p60, our constructs were devoid of MHC II epitopes of listerial origin. Thus, CD4+ T cells could only be stimulated by MHC II epitopes derived from the carrier, gp96. Further studies need to resolve the question whether Listeria-specific CD8+ T cells received help from gp96-specific CD4+ T cells.
Our results demonstrate that DNA vaccination with gp96 fusion proteins induces a potent listerial antigen-specific CD8+ T cell response and confers protection against an otherwise lethal challenge with L. monocytogenes. Although many vaccines based on naked DNA or bacterial carriers have been tested in the Listeria model, they have not provided the same degree of protective immunity against lethal challenge (32,3739). These observations suggest that DNA vaccination with gp96 fusion proteins provides a promising strategy for achieving protective immunity against intracellular pathogens. More importantly, the fact that the efficacy of HSP-based vaccines is at least partially independent of CD4+ T cells implies an intriguing potential for application in patients with immune deficiencies. To further analyze the degree of independence of CD4+ T cells in HSP vaccination, their role during priming with HSPantigen constructs should be determined in the future. Further studies directed at elucidating the precise physiological and pathophysiological role of HSP, and the regulatory and signaling circuits involved in the cellular response to HSP-based vaccination, may pave the way for the application of HSP as vaccines against intracellular pathogens.
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Acknowledgements
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The original plasmid expressing murine gp96 was a generous gift from Dr Z. Li (University of Connecticut School of Medicine). We thank Professor Dr P. Kloetzel and Dr A. Sijts for their advice regarding the elongation of LLO and p60 nonamer epitopes. We also thank M. Koch for sharing his expertise with construction of plasmids and the technicians from Dr P. Seilers lab (P. Mann, E. Heinemann and T. Tharmalingam) as well as M. Miyamoto for their assistance with animal handling. We thank Dr B. Raupach for assistance with statistics, and Professor Dr W. Goebel and Dr A. Galmiche for critical reading of the manuscript. This work is supported by the DFG priority program Novel Vaccination Strategies (KA 573/4).
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Abbreviations
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CTLcytotoxic T lymphocyte
ERendoplasmic reticulum
GRPglucose-regulated protein
HKLheat-killed Listeria
HSPheat-shock protein
LLOlisteriolysin
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