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The Journal of Immunology, 2007, 179, 3371 -3379
Copyright © 2007 by The American Association of Immunologists, Inc.
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B Subunit of Shiga Toxin-Based Vaccines Synergize with {alpha}-Galactosylceramide to Break Tolerance against Self Antigen and Elicit Antiviral Immunity1

Olivier Adotevi2,*,{dagger}, Benoit Vingert2,*, Ludovic Freyburger2,*, Protul Shrikant{ddagger}, Yu-Chun Lone§, Françoise Quintin-Colonna*, Nacilla Haicheur*,{dagger}, Mohamed Amessou, André Herbelin||, Pierre Langlade-Demoyen§, Wolf H. Fridman{dagger},#, François Lemonnier§, Ludger Johannes3,¶ and Eric Tartour3,4,*,{dagger}

* Equipe d’accueil 4054 Université Paris-Descartes, Ecole Nationale Vétérinaire d’Alfort, Paris, France; {dagger} Unité d’Immunologie Biologique, Hopital Européen Georges Pompidou, Assistance Publique des Höpitaux de Paris, Paris, France; {ddagger} Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263; § Unité d’Immunité Cellulaire Antivirale, Département d’Immunologie, Institut Pasteur, Paris, France; Unité Mixte de Recherche 144, Institut Curie/Centre National de la Recherche Scientifique, Traffic and Signaling Laboratory, Paris, France; || Centre National de la Recherche Scientifique Fédération de Recherche 2444, Université Paris 5 René Descartes, Hopital Necker, Paris, France; and # Unité Mixte de Recherche-S872 Centre de Recherche des Cordeliers, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The nontoxic B subunit of Shiga toxin (STxB) targets in vivo Ag to dendritic cells that preferentially express the glycolipid Gb3 receptor. After administration of STxB chemically coupled to OVA (STxB-OVA) or E7, a polypeptide derived from HPV, in mice, we showed that the addition of {alpha}-galactosylceramide ({alpha}-GalCer) resulted in a dramatic improvement of the STxB Ag delivery system, as reflected by the more powerful and longer lasting CD8+ T cell response observed even at very low dose of immunogen (50 ng). This synergy was not found with other adjuvants (CpG, poly(I:C), IFN-{alpha}) also known to promote dendritic cell maturation. With respect to the possible mechanism explaining this synergy, mice immunized with {alpha}-GalCer presented in vivo the OVA257–264/Kb complex more significantly and for longer period than mice vaccinated with STxB alone or mixed with other adjuvants. To test whether this vaccine could break tolerance against self Ag, OVA transgenic mice were immunized with STxB-OVA alone or mixed with {alpha}-GalCer. Although no CTL induction was observed after immunization of OVA transgenic mice with STxB-OVA, tetramer assay clearly detected specific anti-OVA CD8+ T cells in 8 of 11 mice immunized with STxB-OVA combined with {alpha}-GalCer. In addition, vaccination with STxB-OVA and {alpha}-GalCer conferred strong protection against a challenge with vaccinia virus encoding OVA with virus titers in the ovaries reduced by 5 log compared with nonimmunized mice. STxB combined with {alpha}-GalCer therefore appears as a promising vaccine strategy to more successfully establish protective CD8+ T cell memory against intracellular pathogens and tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vaccine delivery systems and adjuvants approved for human use (e.g., aluminum salts, MF59, virosomes) primarily stimulate humoral immune responses (1). However, preclinical studies strongly suggest that successful vaccines against intracellular pathogens (e.g., HIV, Mycobacterium tuberculosis, malaria), but also cancer vaccines, will most likely require both humoral and cell-mediated responses (2, 3, 4, 5, 6). Live attenuated pathogens or whole inactivated organisms have been shown to activate both arms of the immune system in human (7, 8), but these vaccines are difficult to produce, potentially unsafe, or poorly immunogenic because some viral vectors may block different physiological steps of T cell priming (9). Development of subunit vaccines able to mimic or even improve the efficiency of replicative vectors to elicit a robust specific cytotoxic CD8+ T cell response therefore represents an ongoing competitive challenge.

Dendritic cells (DCs)5 have been shown to be the most potent APCs for the induction of primary T cell responses. In humans, clinical studies using healthy recipients also proved the immunogenicity and safety of DCs, and demonstrated that a single injection of a small number of Ag-pulsed DCs is sufficient to rapidly expand T cell immunity for both naive and recall Ags (10). However, generation and ex vivo manipulation of DCs are laborious, and many variables such as the choice of DC lineage, the dosage, and the administration schedule still need to be optimized. Direct Ag targeting to DCs in vivo will therefore offer several advantages. For example, after injection of DCs, only 3–5% reach lymphoid organs and their migration is restricted to the regional lymph nodes (11). In contrast, previous studies showed a broad loading of DCs in various lymphoid organs after directly targeting Ag in vivo to DCs (12).

Initial studies showed that when Ags were specifically targeted to immature DCs in vivo, Ag presentation to CD8+ T cells led to a profound peripheral T cell tolerance (13). From these pioneering studies, it was concluded that efficient priming of CD8+ T cells in vivo will require the delivery of Ag to DCs, followed by optimal presentation of peptides derived from the Ag by HLA-class I molecules (cross-presentation) in combination with a maturation stimulus for DCs. Indeed, several groups demonstrated that when coupled to Ag, various nonreplicative vectors that bind to preferentially expressed DC molecules (e.g., DEC 205, DC-specific ICAM3-grabbing nonintegrin, CD11b) triggered CD8+ T cell responses in mice (14, 15, 16, 17) sometimes associated with Th1-biased CD4+ T cell response and humoral immunity (18, 19, 20, 21). In most cases, addition of adjuvants promoting DC maturation was required to observe immunological responses, although some vectors are endowed with the ability to both target and activate DCs (22, 23).

Shiga toxin from Shigella dysenteriae is composed of an A subunit that mediates toxicity and a B subunit (nontoxic B subunit of Shiga toxin (STxB)), a nontoxic homopentameric protein responsible for toxin binding and internalization into target cells by interacting with the glycolipid Gb3 (24). We have previously shown that STxB efficiently targets exogenous peptides into the MHC class I and II pathways and delivers Ag in vivo directly to DCs, which preferentially expressed the Gb3 receptor (25, 26, 27). STxB-based vaccine induced humoral response (27, 28) and a robust and long-lasting CD8+ T cell response (27, 29). All these results were obtained without the use of adjuvants and may be explained by the ability of STxB to increase costimulatory and MHC class II molecules on DCs (30) and to induce TNF-{alpha} on some cells (31), which could indirectly promote maturation of DCs. However, when myeloid DCs derived from bone marrow were incubated with STxB, no maturation of these cells was observed (29). In addition, because immune response and tumor protection were obtained with doses of STxB conjugate ranging from 50 to 100 µg and required repetitive immunizations, we wondered whether the addition of a DC maturation factor to STxB-based vaccines might improve their efficiency. After screening of conventional adjuvants, we focused on {alpha}-galactosylceramide ({alpha}-GalCer), a glycosphingolipid, also known as KRN7000, originally extracted from marine sponges on the basis of its antitumor properties (32). Unlike common microbial adjuvants, which signal through TLRs, {alpha}-GalCer functions as an Ag presented by CD1d to NKT cells expressing a conserved semi-invariant Ab TCR (33). Activation of NKT by {alpha}-GalCer results in a rapid release of cytokines, followed by NK stimulation and maturation of DCs (34, 35, 36). Previous studies have shown that coadministration of {alpha}-GalCer with various immunogens enhanced the level of Ag-specific CD8+ T cells (35, 36, 37, 38, 39, 40). In the present work, we report a dramatic synergy between {alpha}-GalCer- and STxB-based vaccine leading to potent CD8+ T cell response with the use of very low dose of Ag (50 ng). Vaccines combining STxB and {alpha}-GalCer were efficient to break tolerance against self Ag and elicited antiviral immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female C57BL/6 (H-2b) mice were purchased from Charles River Laboratories. OT-I TCR transgenic (TG) mice specific for the OVA peptide (SIINFEKL) in the context of H-2Kb were provided by C. Leclerc (Institut Pasteur, Paris, France). OVA-TG mice were obtained from P. Shrikant (Roswell Park Cancer Institute, Buffalo, NY) with the permission of M. Jenkins (University of Minnesota, Minneapolis, MN). These mice present the C57BL/6 background and express low levels of the membrane-associated form of chicken OVA protein in most actin-expressing cells (41). Mice were kept under specific pathogen-free conditions and were used between 6 and 8 wk of age, according to institutional guidelines.

Chemical reagents (recombinant proteins, peptides, adjuvant)

Purified chicken OVA (grade V) was purchased from Sigma-Aldrich. Synthetic OVA-derived peptide OVA257–264 (SIINFEKL) and HPV16-E7-derived peptide E749–57 (RAHYNIVTF) or E743–57 (GQAEPDRAHYNIVTF) were obtained from NeoMPS and stored in PBS.

STxB-OVA was obtained by chemical coupling, as previously described (27). Briefly, OVA was first activated via amino groups on lysine side chains using the heterobifunctional cross-linker m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Pierce). Activated OVA was then reacted with STxB-Cys, and the reaction product was purified by gel filtration and immunoaffinity chromatography. STxB-E743–57 was produced using a chemical coupling between the N-bromoacetylated E743–57 peptide and the sulfhydril group of the STxB-Cys recombinant protein, as previously described (29). Removal of contaminating LPS was conducted using Acticlean Etox columns (Biotech-IgG). After purification, endotoxin concentrations determined by the Limulus assay test (BioWhittaker) were <0.5 EU/mg.

The invariant NKT cell ligand {alpha}-GalCer (KRN7000) was kindly supplied by Kirin Brewery.

Poly(I:C) and IFA were purchased from Sigma-Aldrich. CpG oligodeoxynucleotide 1826 was purchased from Proligo.

IFN-{alpha} was provided by I. Gresser (Institut National de la Santé et de la Recherche Médicale, Paris, France).

Cells

The mouse thymoma cell line EL4 (H-2b) was provided by K. Rock (University of Massachusetts Medical School, Worcester, MA) and P. Jeannin (Angers University Hospital, Angers, France).

ELISPOT assay

ELISPOT assay was performed, as previously described (29).

Cytotoxicity assay

Cytotoxicity was assessed on 51Cr-labeled target cells, as previously described (26).

Flow cytometry

To detect anti-OVA257–264/Kb- or anti-E749–57/Db-specific CD8+ T cells, the cells were stained with OVA257–264/Kb or E749–57/Db tetramer, according to the manufacturer’s recommendations (Beckman Coulter Immunomics). Briefly, cells were incubated with PE-labeled tetramer (45 min at 4°C in the dark). After incubation and washes, labeled anti-CD8 mAbs (eBioscience) were used to phenotype the positive tetramer CD8+ T cells. Irrelevant tetramers recognizing a vesicular stomatitis virus (VSV)-derived peptide in the context of Kb or Db molecules were used in each experiment. Naive nonimmunized mice were also included as control for these experiments.

Proliferation assay to detect specific anti-OVA CD4+ T cells

CD4+ T cells were purified from spleen and labeled with CFSE (Molecular Probes), used at 0.5 µM for 30 min at 20°C. Labeling was stopped by repeated washing with ice-cold PBS supplemented with 5% FCS. Cells were then incubated with T cell-depleted splenocytes as APCs pulsed or not with free OVA protein and cocultured for 5 days in AIM V serum-free medium. Proliferation in absence of APC sensitization was subtracted as background from values obtained after OVA pulsing.

Ex vivo cross-presentation assay

Anti-OVA-specific CD8+ T cells derived from OT-1 mice were labeled with CFSE. CD11c+-enriched DCs (106), isolated as described (29) from mice vaccinated with various vaccine formulations, were cocultured with OT-1 cells (5 x 105) for 72 h. Dilution of CFSE detected by FACS analysis was considered to be an indicator of OT-1 cell proliferation after Ag recognition.

Immunization

Mice were immunized with various doses of Ags, as indicated in the legend of the figures. Adjuvants were always used at the same dosage for each injection, as follows: CpG, 50 µg; poly(I:C), 250 µg; IFN-{alpha}, 100,000 IU; {alpha}-GalCer, 2 µg.

Route of injection was s.c. for CpG, poly(I:C), and IFN-{alpha}. {alpha}-GalCer was always injected via the i.p. route. Volume injected via the s.c. or i.p. route was 150–200 µl.

Serological analysis

Serological analysis was performed, as previously described, for measurement of anti-OVA Abs (27). The same protocol was used for anti-STxB Ab detection, except that STxB was coated on flat-bottom microtiter plates instead of OVA.

Antiviral protection experiments

C57BL/6 mice (five mice per group) were injected twice (days 0 and 21) i.p. (200 µl) with different vaccine formulations, and control mice were injected with PBS. Eight days after the last injection, mice were challenged i.p. with 2.5 x 106 PFU recombinant vaccinia virus (rVV, Westerns Reserve strains) expressing either the OVA or the HBx cDNA derived from hepatitis B virus provided by N. Etchart (Institut National de la Santé et de la Recherche Médicale, Lyon, France) and Lone YC (Institut Pasteur, Paris, France), respectively. After 4 days, the ovaries of the mice were harvested and homogenized with a mechanical tissue grinder. The homogenates were clarified by centrifugation at 4000 x g for 10 min; the number of rVV PFU in the resultant supernatant was enumerated by infecting BHK 21 cell monolayers with 10-fold serial dilutions of these fluids; and plaques were counted after 2 days in culture at 37°C in a 5% CO2 environment, as previously described (42).

Statistical analysis

Statistical analysis was performed using the Mann-Whitney U test and the Kruskal-Wallis test. Significance was defined as p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
{alpha}-GalCer increased the efficiency of STxB coupled to Ag to induce specific CTL

A first series of experiments tested the ability of various well-defined adjuvants to increase the efficiency of STxB-OVA to trigger a specific CTL response.

As previously reported (29), when mice were immunized twice with the STxB-OVA conjugate (50 µg) alone, an induction of anti-OVA257–264 CD8+ T cells corresponding to 0.4% of CD8+ T cells was demonstrated (Figs. 1 and 2A). The use of some adjuvants (poly(I:C), CpG, and {alpha}-GalCer) combined with STxB-OVA significantly enhanced the frequency of anti-OVA257–264 CD8+ T cells. However, whereas poly(I:C) and CpG led to a modest increase of anti-OVA257–264 CD8+ T cells detected by specific tetramer not exceeding 1% of total CD8+ T cells, the glycolipid {alpha}-GalCer elicited a dramatic increase of the percentage of specific anti-OVA CTL (Fig. 1). Indeed, after two immunizations with STxB-OVA and {alpha}-GalCer, 4.6% of CD8+ T cells stained positively with OVA257–264/Kb tetramer directly ex vivo without any in vitro restimulation step (Fig. 1). {alpha}-GalCer was administered via the i.p. route because it is much less effective via the s.c. route, whereas CpG and poly(I:C) were administered via the s.c. route in this experiment. However, similar results were observed when these two adjuvants were injected via the i.p. route (data not shown). We therefore focused on analysis of this synergy between STxB and {alpha}-GalCer. Because previous studies reported that repeated administration of {alpha}-GalCer may lead to anergy and Th2 polarization (43, 44) and no difference was observed when {alpha}- GalCer was injected during both the first and second immunizations or only at priming, we only combined this adjuvant and STxB-OVA during the first immunization.


Figure 1
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  		FIGURE 1. Role of adjuvants on the levels of anti-OVA257–264 CD8+ T cells induced by the STxB-OVA conjugate. Mice were s.c. immunized twice (days 0 and 21) with STxB-OVA alone (50 µg = 0.5 nmol) diluted in PBS or combined with various adjuvants (IFA, v/v; IFN-{alpha}, 100,000 IU, days 0, 1, and 2; poly(I:C), 250 µg, days 0 and 21; CpG, 50 µg, days 0 and 21). {alpha}-GalCer (2 µg) was only admixed with STxB-OVA on day 0. The second immunization was performed with STxB-OVA alone. CD8+ T cells from spleen were isolated 7 days after the last immunization and directly stained ex vivo with PE-labeled OVA257–264/Kb tetramer and allophycocyanin-labeled anti-CD8 mAb. Values shown correspond to mean ± SD obtained with specific tetramer after subtracting values from irrelevant tetramer recognizing a VSV-derived peptide in the context of Kb. These results are representative of three experiments with four mice per group.

 

Figure 2
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  		FIGURE 2. {alpha}-GalCer increases the efficiency of STxB to induce long-lasting and functional specific anti-OVA257–264 CD8+ T cells even at very low doses of Ag. Mice were s.c. immunized twice (days 0 and 21) with various doses of STxB-OVA alone (A, left) or combined with {alpha}-GalCer (A, right; C and D) or with free OVA admixed with {alpha}-GalCer (B). As mentioned in Materials and Methods, {alpha}-GalCer was only administered during the first immunization. A–C, CD8+ T cells from spleens were isolated 7 days after the last immunization and directly stained ex vivo with PE-labeled OVA257–264/Kb tetramer and allophycocyanin-labeled anti-CD8 mAb. Results shown are gated on CD8+ T cells. An irrelevant tetramer recognizing a VSV-derived peptide in the context of Kb and an isotype control mAb were included as controls. These results are representative of three experiments with four mice per group. D, Mice were immunized with STxB-OVA (1 µg) mixed with {alpha}-GalCer (2 µg). Left, IFN-{gamma}-producing OVA257–264/Kb-specific CD8+ T cells were detected ex vivo by an IFN-{gamma} ELISPOT assay using EL4 pulsed ({blacksquare}) or not ({square}) with the OVA257–264 peptide (SL8). SFC = spot-forming cells were read using an ELISPOT automated reader system. Right, CTL activity was measured in a standard 4-h 51Cr release assay on the target EL4 cells pulsed with ({blacksquare}) or without ({square}) the OVA257–264 peptide at an E:T ratio of 100:1. Three mice per group were immunized, and these experiments were reproduced two times.

 
When different doses of the STxB-OVA conjugate were mixed with {alpha}-GalCer, this adjuvant was found to significantly induce the anti-OVA257–264 CTL response even at very low doses of Ag (50 ng of STxB-OVA corresponding to 25 ng of equivalent OVA Ag) (Fig. 2A). In contrast, free OVA even at high doses (200 µg) combined with {alpha}-GalCer elicited a low frequency of anti-OVA257–264 CTL not exceeding 1% of total CD8+ T cells (Fig. 2B) compared with 5–7% of anti-OVA257–264-specific CTL when OVA was coupled to STxB and combined with {alpha}-GalCer. In addition, no significant induction of anti-OVA257–264 CD8+ T cells was shown when <50 µg of OVA was added to {alpha}-GalCer or when OVA alone was used for immunization (Fig. 2B). The other adjuvants (IFN-{alpha}, poly(I:C), CpG) did not allow this priming of CTL when a low dose of STxB-OVA conjugate was used reinforcing the significance of the synergy between STxB and {alpha}-GalCer (data not shown).

The specific anti-OVA257–264 CD8+ T cells induced by the combination of {alpha}-GalCer and the STxB-OVA conjugate were long lasting, because 2.49% of CD8+ T cells stained positively with OVA257–264/Kb tetramer directly ex vivo 202 days after the first immunization (Fig. 2C).

Because tetramer analysis does not discriminate between anergic and functional CD8+ T cells, two functional tests, an ELISPOT assay and a cytotoxic assay, on 51Cr-labeled target cells without an in vitro stimulation were performed. As shown in Fig. 2D, after vaccination with STxB-OVA (1 µg) mixed with {alpha}-GalCer, large numbers of CD8+ T cells produced IFN-{gamma} (mean 61/105 cells) when coincubated with OVA257–264 peptide-pulsed EL4 cells (Fig. 2D, left). Similarly, as shown in Fig. 2D (right), spleen cells from mice immunized with STxB-OVA mixed with {alpha}-GalCer efficiently lysed EL4 target cells loaded with the OVA257–264 peptide, whereas no cytotoxicity was observed against EL4 alone.

To check whether these results could be reproduced with another more clinically relevant Ag, mice were immunized twice with STxB coupled to a polypeptide derived from the HPV16-E7 protein (STxB-E743–57) at a low dose (1 µg). A marked induction of anti-E7 CTL detectable ex vivo by the E749–57 Db tetramer (1.12% of CD8+ T cells) was also demonstrated (Fig. 3). These CTL were functional (data not shown). In contrast, at this dosage, only low levels of E7-specific CTL (0.12% of CD8+ T cells) were detected after immunization with STxB-E743–57 alone. The E743–57 polypeptide mixed with {alpha}-GalCer did not prime a CTL response (Fig. 3).


Figure 3
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  		FIGURE 3. {alpha}-GalCer also increases the efficiency of STxB-E7 to elicit anti-E7 CTL. Mice were immunized on days 0 and 21 with STxB-E743–57 (1 µg) alone or mixed with {alpha}-GalCer or with the free polypeptide E743–57 mixed with {alpha}-GalCer. Seven days after the last immunization, CD8+ T cells from spleen were isolated and directly stained with PE-labeled E749–57/Db tetramer. (Cells were previously gated on CD8+ T cells.) An irrelevant tetramer recognizing a lymphocytic choriomeningitis virus-derived peptide in the context of Db was included as controls. These results are representative of two experiments with three mice per group.

 
We then investigated whether the synergy observed between {alpha}-GalCer and STxB was restricted to induction of CTL or could be extended to other immune responses. Mice vaccinated with the STxB-OVA conjugate mixed with {alpha}-GalCer displayed a more potent anti-OVA CD4+ T cell response compared with mice immunized with STxB-OVA alone or free OVA mixed with IFA (Fig. 4A). However, although {alpha}-GalCer increased the anti-OVA IgG2a response compared with free OVA with adjuvant, this humoral response was not enhanced when STxB was admixed with {alpha}-GalCer compared with STxB alone (Fig. 4B).


Figure 4
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  		FIGURE 4. CD4+ T cell and humoral responses after STxB-OVA-based vaccine immunization. A, Mice (n = 3 per group) were immunized twice (days 0 and 21) with STxB-OVA (0.05 nmol) alone or combined with {alpha}-GalCer (2 µg). As control, mice were vaccinated with OVA (0.05 nmol) mixed with IFA. Seven days after the last immunization, CD4+ T cells were purified from spleen and labeled with CFSE. They were then incubated with T cell-depleted splenocytes as APCs pulsed with free OVA protein and cocultured for 5 days in AIM V serum-free medium. Proliferation in the absence of APC sensitization was subtracted as background from values obtained after OVA pulsing. These experiments were reproduced two times. B, One week after the last immunization, serum was collected and anti-OVA IgG2a and IgG1 were measured by ELISA.

 
Analysis of potential mechanisms leading to the synergy between STxB and {alpha}-GalCer

In a previous study, the adjuvant property of {alpha}-GalCer was related to its ability to promote maturation of DCs (35, 36). However, we also confirmed that other adjuvants used in this study (e.g., CpG, poly(I:C)) also activated DCs, but their enhancing effect on the STxB immunogenicity remained modest (Fig. 1 and data not shown).

Because the levels of CD1d may affect NKT cell activation (45), we tested whether STxB could modulate the expression of CD1d. After STxB administration in mice, no significant change was detected in CD1d expression on APCs (DCs and B cells) derived from splenocytes (data not shown).

Because one striking consequence of the synergy between STxB and {alpha}-GalCer is the ability to markedly reduce the efficient dose of STxB vaccine, we analyzed the in vivo presentation of Ag after vaccination. DCs derived from mice immunized with low doses of STxB-OVA (1 µg) combined with {alpha}-GalCer, 7 days earlier, more significantly presented the OVA257–264/Kb complex in vivo than mice vaccinated with STxB alone (Fig. 5). This presentation was observed for both DCs derived from spleen or draining lymph node and persisted for at least 12 days (data not shown). This increased presentation could also be detected when STxB was mixed with other adjuvants, but the levels of Ag presentation appeared to be lower than those observed with {alpha}-GalCer (Fig. 5A and data not shown).


Figure 5
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  		FIGURE 5. Combination of STxB-OVA and {alpha}-GalCer enhances the cross-presentation of OVA by DCs. A, Mice (n = 3 per group) were immunized with STxB-OVA alone (1 µg = 0.01 nmol) or combined with poly(I:C) or with {alpha}-GalCer (2 µg). Seven days after vaccination, CD11c+-enriched DCs were cocultured with CFSE-labeled OT-1 cells for 72 h. These experiments were reproduced three times with similar results. Dot plots were gated on CFSE-labeled CD8+ OT-1 cells. B, Mice were immunized i.p. with STxB coupled to FITC either alone or mixed with {alpha}-GalCer (2 µg). Six hours later, splenocytes were collected and DCs were semipurified. Cells were stained with PerCP-labeled anti-CD11c Abs and analyzed by cytometry. Fl-1 corresponds to gate channel 1 of the cytometer to detect FITC fluorochrome.

 
A possible explanation for this enhanced cross-presentation of STxB-OVA mediated by {alpha}-GalCer was that {alpha}-GalCer increased STxB uptake by DCs. When mice were injected with STxB coupled to FITC, a slight increase of STxB uptake by DCs derived from splenocytes was observed in the presence of {alpha}-GalCer (2.6 vs 1.4% of CD11c+ cells) (Fig. 5B). As expected, DCs were the main cells targeted by STxB in the presence or absence of {alpha}-GalCer.

STxB conjugate mixed with {alpha}-GalCer broke tolerance against self Ags

It is often criticized that exogenous Ags such as OVA and viral proteins do not mimic the clinical situations in which therapeutic vaccines will be developed because most tumor Ags are self Ags and, during chronic infection, tolerance to viral protein is already established. For these indications, a potential vaccine is therefore expected to be endowed with the ability to break tolerance against self Ag. For this purpose, we selected a novel TG mouse model that expresses OVA on the surface of all cells. When these mice were vaccinated with STxB-OVA alone, no anti-OVA257–264 CD8+ T cell induction was observed in either blood or spleen at various times after primary or secondary immunizations (Fig. 6 and data not shown). In contrast, specific tetramer assay detected significant levels of anti-OVA257–264 CD8+ T cells in 8 of 11 mice immunized with STxB-OVA combined with {alpha}-GalCer (Fig. 6). Some of them (4 of 7 vaccinated mice) were functional because they produced IFN-{gamma} using an ELISPOT assay. These specific CD8+ T cells seemed to be induced by the vaccine because they were not present before immunization (data not shown). These anti-OVA-specific CD8+ T cells were essentially found after primary immunizations and rapidly disappeared from the blood and from the spleen. It should be emphasized that this strain of mice expresses membrane OVA in all organs (41) (see Discussion).


Figure 6
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  		FIGURE 6. STxB-OVA combined with {alpha}-GalCer primes anti-OVA257–264 CD8+ T cells in OVA-TG mice. OVA-TG mice were immunized with STxB-OVA (0.1 nmol) combined or not with {alpha}-GalCer (2 µg). Fourteen days later, CD8+ T cells from spleen were isolated and directly stained with PE-labeled OVA257–264/Kb tetramer and allophycocyanin-labeled anti-CD8 mAb. Each square represents values from individual mice and corresponds to results obtained with specific OVA257–264/Kb tetramer after subtracting the values obtained with an irrelevant tetramer recognizing a VSV-derived peptide in the context of Kb. Two series of experiments were performed with similar results.

 
STxB conjugate mixed with {alpha}-GalCer induces antiviral protective immunity

To investigate the clinical relevance of the synergy observed between STxB and {alpha}-GalCer, we challenged mice with rVV encoding OVA (rVV-OVA), 7 days after vaccination, and measured virus titers in the ovaries 5 days later to assess protection. Vaccination of mice with STxB-OVA and {alpha}-GalCer conferred potent protection against rVV-OVA, with virus titers in the ovaries reduced by 5 log (3.92 x 103 PFU) compared with those of mice treated with PBS (8.28 x 108 PFU) (Fig. 7). Immunization with STxB-OVA alone conferred a statistically significant reduction of virus titers in the ovary by >2 log, but this effect was dramatically amplified by the addition of {alpha}-GalCer.


Figure 7
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  		FIGURE 7. STxB-OVA combined with {alpha}-GalCer induces antiviral protective immunity against a challenge with rVV-OVA. Mice (five mice per group) were injected twice (days 0 and 21) i.p. (200 µl) either with STxB B-OVA alone (0.05 nmol = 5 µg), or B-OVA (0.05 nmol) combined with {alpha}-GalCer, or OVA (0.05 nmol) mixed with {alpha}-GalCer, and control mice were injected with PBS. Similarly to the previous experiments, {alpha}-GalCer was not added during the second immunization. Eight days after the last injection, mice were challenged i.p. with 2.5 x 106 PFU rVV-OVA (Westerns Reserve strains) expressing OVA cDNA. After 4 days, ovaries were assayed for rVV titers by plaque assay on BHK 21 cells. Results represent mean of PFU from six mice per group. Values of p were calculated by Student’s t test.

 
To exclude a direct role of {alpha}-GalCer in this antiviral protection, we showed that mice immunized with OVA and {alpha}-GalCer exhibited a slight reduction of infectious virus titers from 8.28 x 108 PFU in PBS-treated mice to 7.73 x 108 PFU corresponding to less than 1 log reduction of virus titers (Fig. 7). The specificity of the protection was documented by challenging STxB-OVA/{alpha}-GalCer-immunized mice, with an irrelevant VV encoding hepatitis Bx protein. No significant reduction of viral load was observed (data not shown).

Intrinsic immunogenicity of STxB

Because intrinsic immunogenicity may raise certain problems, we directly addressed the immunogenicity of this nonlive vector. First, we showed that anti-STxB Abs were elicited after two immunizations with STxB-OVA. The levels of anti-STxB Abs did not appear to differ between the groups immunized in the presence or not of {alpha}-GalCer (Fig. 8A). In a second experiment, we demonstrated that the presence of anti-STxB did not inhibit the subsequent CTL response after repeated immunization, because the frequency of anti-OVA257–264 CD8+ T cells increased from 0.4% after two immunizations to 2.4% after the third immunization (Fig. 8B).


Figure 8
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  		FIGURE 8. Analysis of the immunogenicity of STxB and its influence on CTL response. A, Mice (n = 3 per group) were immunized twice (days 0 and 21) with STxB-OVA (0.5 nmol) alone or combined with {alpha}-GalCer (2 µg). As control, mice were vaccinated with OVA (0.5 nmol) mixed with IFA. One week after the last immunization, serum was collected and anti-STxB IgG were measured by ELISA. B, Mice were immunized once, twice, or three times (days 0, 21, and 28) with STxB-OVA (0.5 nmol). CD8+ T cells from spleens were isolated 7 days after the last immunization and directly stained ex vivo with PE-labeled OVA257–264/Kb tetramer and allophycocyanin-labeled anti-CD8 mAb. An irrelevant tetramer recognizing a VSV-derived peptide in the context of Kb and an isotype control mAb were included as controls of background, which was never above 0.1% of CD8+ T cells. These results are representative of two experiments with three mice per group. C, Mice (n = 4) were preimmunized or not with STxB-E7 (0.2 nmol) (day 0) and then vaccinated with 0.2 nmol of STxB-OVA mixed with {alpha}-GalCer on days 21 and 35. IFN-{gamma}-producing OVA257–264/Kb-specific CD8+ T cells were detected ex vivo by an IFN-{gamma} ELISPOT assay using EL4 pulsed ({blacksquare}) or not ({square}) with the OVA257–264 peptide (SL8). SFC = spot-forming cells were read using an ELISPOT automated reader system.

 
Finally, we showed that when mice were preimmunized with STxB-E7 and then vaccinated with STxB-OVA, the number of anti-OVA-CD8+ T cells measured by ex vivo ELISPOT assay did not differ between the preimmunized and nonpreimmunized groups (Fig. 8C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study demonstrates that STxB-based vaccines combined with {alpha}-GalCer resulted in a dramatic improvement of the STxB Ag delivery system assessed by the more powerful CD8+ T cell response observed even at very low doses of immunogen (50 ng). This vaccine formulation was also efficient to break tolerance against a self Ag and to protect against a VV challenge.

Approaches that target various DC surface endocytic receptors are known to increase the efficiency of Ag presentation and immune response (46). Very small amounts of Ags targeted to DCs via the anti-DEC 205 mAb in combination with a maturation stimulus were capable of inducing specific CD4+ and CD8+ T cell immunity (14). However, most other vaccine formulations that have allowed successful reduction of the Ag dose were only effective on humoral responses (47, 48). The selective ability of {alpha}-GalCer compared with other adjuvants to successfully reduce the STxB dosage for efficient induction of CD8+ T cell response may reflect a synergy between these two components and will be of value to facilitate vaccine production and for preparation of multivalent antigenic vaccines usually limited by the total protein content of the vaccine.

In contrast to replicative vectors, nonlive carrier proteins are often criticized because of their inability to diffuse systemically. We have previously shown that after s.c. administration of STxB-OVA, an immunodominant OVA peptide was presented by DCs from draining lymph node, but also by DCs from spleen, which indicates a systemic diffusion of STxB-Ag conjugate (29). In this study, STxB-OVA conjugate immunization led to significant antiviral protection in peripheral organs such as ovaries after challenge with VV. This result supports the broad systemic distribution and the functionality of the CTL induced by this vaccine and extends previous results about the ability of nonreplicative vectors to induce antiviral immunity (49, 50, 51).

Therapeutic vaccination against tumors or chronic viral infection will need to overcome tolerance against self Ags, because most tumor Ags are also expressed by nontransformed cells, and will need to reactivate anergic T cells associated with Ag persistency in chronic infection (52, 53). Except for a few reports using nonreplicating carriers (12, 50), in most studies, only live vectors and vaccines based on ex vivo pulsed DCs were efficient to trigger cellular immune responses directed against self Ags (54, 55). We demonstrated that when OVA was used to mimic a self Ag in OVA-TG mice, a marked induction of CTL was observed with the STxB-OVA conjugate combined with {alpha}-GalCer. Although the STxB-OVA conjugate efficiently elicited CTL response in wild-type mice, it failed to trigger CD8+ T cells when administered alone in OVA-TG mice, which reinforces the value of the synergy between STxB and {alpha}-GalCer. In contrast to the long-lasting CTL response observed in wild-type mice, it should be noted that the anti-OVA CD8+ T cells elicited in OVA-TG mice rapidly waned, which could be expected in view of the broad expression of the transgene in all organs. Various groups have reported that high Ag load or chronic Ag presentation can be deleterious to immunity and result in the development of dysfunctional memory CD8+ T populations with poor survival characteristics or the deletion of CD8+ T cells by exhaustion (56, 57, 58). Alternatively, these anti-OVA-specific CD8+ T cells may have migrated to peripheral tissues that express OVA. As previously reported, we detected expression of the OVA257–264/Kb complex on DCs of these OVA-TG mice (41) (data not shown).

{alpha}-GalCer coadministered with various immunogens (e.g., proteins, recombinant virus, tumor cells) has already been shown to enhance the level of Ag-specific CD8+ T cell response (35, 36, 37, 38, 39, 40). However, in contrast with the present study, high doses of soluble protein ranging from 100 µg to 5 mg were necessary to prime the CD8+ T cell response (35, 36, 38, 39). Previous studies suggested that the ability of {alpha}-GalCer to promote DC maturation largely accounted for its adjuvant effect (35, 59). However, the synergy observed between STxB and {alpha}-GalCer could not only be related to the {alpha}-GalCer-mediated maturation process assessed by the increase of costimulatory and MHC class II molecules on DCs because other adjuvants, tested in this study, which also induced DC maturation, only moderately enhanced the immunogenicity of the STxB-OVA conjugate. It has to be mentioned that poly(I:C) and CpG may have worked better if they have been formulated in an emulsion, attached to microparticles, or conjugated to the Ag, as already reported (60).

The lack of correlation between conventional markers of DC maturation and the ability of DCs to elicit immunity has already been reported (61, 62). Different combinations of vectors targeting DCs and adjuvants promoting DC maturation also failed to prime CD8+ T cell response (21). DCs are heterogeneous cell populations, and the types of DC maturation induced by adjuvant are not equivalent. It is therefore possible that the combination of vectors targeting different DC subpopulations with various adjuvants induced qualitatively distinct immune responses (63, 64).

Mice immunized with {alpha}-GalCer presented in vivo the OVA257–264/ Kb complex more significantly and for a longer period (>10 days) than mice vaccinated with STxB alone or mixed with other adjuvants tested. A direct relationship between this enhanced presentation and the synergy observed between STxB and {alpha}-GalCer was not formally demonstrated in this study. However, using a similar strategy combining a vector targeting DCs (anti-Dec205 mAb) and a maturation stimulus, Bonifaz et al. (14) found presentation of Ag in the lymph node for up to 15 days after immunization with just 50 ng of Ag, whereas a similar level of presentation was only observed with high doses of free Ag (500 µg). Although some studies have reported that a full differentiation program could be induced in T cells following brief exposure to Ags (65), other studies have also shown that prolonged Ag presentation was required to induce sustained cellular immunity (66, 67). Bachmann et al. (68) recently reported that Ag persistence for <9 days failed to efficiently induce long-lived CD8+ T cell memory.

With respect to the possible extrapolation of these results to humans, it should be noted that STxB recognizes the same glycolipid Gb3 in humans and mice, and that distribution of this receptor is preserved between the two species; {alpha}-GalCer activates human CD1d-restricted invariant NKT cells (69, 70). In addition, administration of {alpha}-GalCer in humans and STxB in preclinical models did not result in any significant toxicity (71, 72, 73).

Injection of {alpha}-GalCer-pulsed mature DCs in humans led to >100-fold expansion of NKT cells and increased cytokine production in most patients (74), whereas administration of free {alpha}-GalCer induced cytokine production only in some patients with high NKT cell counts before treatment (72, 73). Because humans have fewer CD1d-restricted invariant NKT cells than mice, correction of this defect may improve {alpha}-GalCer activity in human. The G-CSF and FMS-like tyrosine kinase 3 ligand chimeric cytokine, progenipoietin-1, has been recently shown to markedly expand the splenic and hepatic NKT cell population and to enhance functional responses to {alpha}-GalCer (75).

Although we did not detect anti-STxB Abs in healthy subjects (data not shown), we cannot exclude that humans may be primed against the Shiga toxin following natural infection. However, the present results showed that prepriming of mice with STxB does not have a negative impact on CTL response induction by STxB-based vaccines.

STxB combined with {alpha}-GalCer therefore appears to be a promising vaccine strategy to more successfully establish protective CD8+ T cell memory against intracellular pathogens and tumors.


   Acknowledgments
 
We thank N. Merillon for technical expert assistance and V. Cerundolo for critically reading the manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors would like to mention that the Institut Curie has decided to set up a biotech company partly based on this technology. This work has not been sponsored by this company and no author of this manuscript will have any responsibility in this company. No material described in this study will be developed and commercialized by this company. However, Ludger Johannes and Eric Tartour have been invited to acquire stocks (5550 Euros) in this company as coinventors of this technology.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Association pour la Recherche sur le Cancer, the Fondation de France, the Canceropole d’Ile de France, Centre d’Investigation Clinique en Biothérapie (AP-HP/INSERM), the European Economic Community "Cancer Immunotherapy," and Pôle de competitivité "Projet Immucan." B.V. is a fellow of the Fondation de France. Back

2 O.A., B.V., and L.F. contributed equally to this work. Back

3 L.J. and E.T. were the principal investigators. Back

4 Address correspondence and reprint requests to Dr. Eric Tartour, Hopital Européen Georges Pompidou, Unité d’Immunologie Biologique, 20 Rue Leblanc 75908 Paris Cedex 15, France. E-mail address: eric.tartour{at}egp.aphp.fr Back

5 Abbreviations used in this paper: DC, dendritic cell; {alpha}-GalCer, {alpha}-galactosylceramide; STxB, nontoxic B subunit of Shiga toxin; TG, transgenic; VSV, vesicular stomatitis virus; VV, vaccinia virus. Back

Received for publication March 2, 2007. Accepted for publication June 24, 2007.


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 Materials and Methods
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