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Anti-tumor Immunotherapy via Antigen Delivery from a Live Attenuated Genetically Engineered Pseudomonas aeruginosa Type III Secretion System-Based Vector
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doi:10.1016/j.ymthe.2006.06.011
Anti-tumor Immunotherapy via Antigen Delivery from a Live Attenuated Genetically Engineered Pseudomonas aeruginosa Type III Secretion System-Based Vector Olivier Epaulard,1,* Bertrand Toussaint,1,* Lauriane Quenee,1 Madiha Derouazi,1 Nabil Bosco,2 Christian Villiers,2 Rozenn Le Berre,3 Benoit Guery,3 Didier Filopon,1 Laurence Crombez,1,4 Patrice N. Marche,2 and Benoit Polack1,y 1
Groupe de Recherche et d’Etude du Processus Inflammatoire, Universite´ J. Fourier EA2938, Hopital de Grenoble, BP 217, 38043 Grenoble Cedex 09, France 2 Laboratoire d’Immunochimie, INSERM U548, CEA Grenoble DRDC/ICH, 17 Rue des Martyrs, 38054 Grenoble Cedex 09, France 3 Laboratoire de Recherche en Pathologie Infectieuse, EA2689 Faculte´ de Me´decine, 59045 Lille Cedex, France 4 Protein’eXpert SA, 15, Rue des Martyrs, 38027 Grenoble, France *These authors contributed equally to this work.
y
To whom correspondence and reprint requests should be addressed at the Groupe de Recherche et d’Etude du Processus Inflammatoire, Universite´ J. Fourier EA2938, Laboratoire d’Enzymologie, De´partement de Biologie et de Pathologie de la Cellule, CHU de Grenoble, BP 217, 38043 Grenoble Cedex 09, France. Fax: +33 4 76 76 56 08. E-mail: [email protected].
Immunotherapy requiring an efficient T lymphocyte response is initiated by antigen delivery to antigenpresenting cells. Several studies have assessed the efficiency of various antigen loading procedures, including microbial vectors. Here a live strain of Pseudomonas aeruginosa was engineered to translocate a recombinant antigenic protein into mammalian cells via the type III secretion system, a bacterial device translocating effector proteins into host cells. Optimization of the vector included virulence attenuation and determination of the N-terminal sequence allowing translocation of fused antigens into cells. In vitro delivery of an ovalbumin fragment by the bacterial vector into dendritic cells induced the activation of ovalbumin-specific CD8+ T lymphocytes. Mice injected with the ovalbumin-delivering vector developed ovalbumin-specific CD8+ T lymphocytes and were resistant to a subsequent challenge with an ovalbumin-expressing melanoma. Moreover, in a curative assay, injection of the vaccine vector 5 and 12 days after tumor implantation led to a complete cure in five of six animals. These results highlight the utility of type III secretion system-based vectors for anti-tumor immunotherapy. Key Words: cancer, immunotherapy, vaccine, type III secretion system, Pseudomonas aeruginosa, dendritic cell
INTRODUCTION The objective of most anti-tumor vaccine strategies is the generation of tumor-specific cytotoxic T lymphocytes (CTLs). This is possible through major histocompatibility complex class I (MHC-I)-restricted presentation of antigen by dendritic cells (DCs) [1]. These cells are powerful antigenpresenting cells (APCs) and play a central role in generating and directing immune response. MHC-I-restricted presentation occurs preferentially for antigens present in the cytoplasm, in contrast to antigens processed after phagocytosis and presented by MHC-II molecules. Different methods of antigen delivery to DCs have been used to obtain MHC-Irestricted antigen presentation, including antigen gene delivery with microbial vectors [1]. Recently, the potential of immunotherapy using antigen delivery by live attenuated
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bacterial vectors has been assessed [2–4]. We previously demonstrated the feasibility of using Pseudomonas aeruginosa as a vector for active enzyme delivery to mammalian cells using type III secretion system (TTSS), a bacterial device translocating effector proteins into the eukaryotic cytoplasm [5]. We hypothesized that a TTSS-based antigen delivering Pseudomonas may induce a MHC-I-restricted presentation by DCs. Here we used P. aeruginosa to deliver a tumor antigen to APCs and generate a CTL response against tumor cells in vivo.
RESULTS Engineering of the Bacterial Vector For the secretion of the Yersinia TTSS toxins YopE and YopH, the N-terminal 15 and 17 amino acids (aa),
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respectively, are sufficient [6]; however, a similar domain has not been discovered at the N-termini of TTSS toxins from other bacterial pathogens [7]. Therefore we determined the region of the P. aeruginosa TTSS toxin exoenzyme S (ExoS) required for the secretion and translocation of an ExoS-fused antigen by generating protein fusions between the N-terminal 129, 96, 54, or 17 aa of ExoS and three reporter proteins that are not normally secreted by the TTSS: P. aeruginosa inhibitor of vertebrate lysozyme (IVY), P. putida catechol 2,3-dioxygenase (CDO), and green fluorescent protein (GFP). We transferred the plasmids into P. aeruginosa strain CHA and assessed the secretion of the fusion proteins after TTSS induction by calcium depletion, a condition known to trigger TTSS [7]. We observed high secretion levels when each of the reporter proteins was fused with the Nterminal 54 aa of ExoS (Figs. 1A and 1B). With the Nterminal ExoS fragments of 129 or 96 aa, IVY but not CDO (Figs. 1A and 1B) or GFP (data not shown) was efficiently secreted. Reporter proteins fused with the Nterminal 17 aa of ExoS were secreted only at low levels (Figs. 1A and 1B). To determine if this N-terminus of ExoS
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was also required for translocation into mammalian cells, we incubated CHA strains expressing different ExoS–CDO fusions with PLB-985 cells. When fused to the N-terminal 54 aa of ExoS, cytoplasmic delivery of CDO was optimal (Fig. 1C). The TTSS toxins ExoS and exoenzyme T (ExoT) are involved in P. aeruginosa pathogenesis [7]. To lessen CHA cytotoxicity, we developed an attenuated CHA-OST mutant (ExoS, ExoT). We assessed the viability of bone-marrow-derived myeloid DCs from C57BL/6 mice 24 h after a 1-h exposure to either CHA or CHA-OST. With the parental strain 50% of the DCs were killed, whereas the survival rate was 95% in the presence of CHA-OST (data not shown). To control fusion protein expression in P. aeruginosa, we cloned the exsA gene encoding the ExoS transcriptional activator [7] under the control of an isopropyl-h-dthiogalactopyranoside (IPTG)-inducible promoter. To assess the efficiency of conditional TTSS control by ExsA, we expressed a fusion gene encoding the N-terminal 54 aa of ExoS and the C-terminus of ovalbumin along with the exsA gene from pS54-Ova ExsAi in strain CHA-OST
FIG. 1. Type III secretion system analysis. (A) Secretion of ExoS–IVY. SDS–PAGE of culture supernatants of CHA strains transformed with pS(129, 96, 54, or 17)IVY and grown in the absence () or presence (+) of EGTA to induce TTSS activation is shown. Theoretical molecular weights of the fusion proteins are 30.7 kDa for ExoS129–IVY, 27.1 kDa for ExoS96–IVY, 23.1 kDa for ExoS54–IVY, and 18.8 kDa for ExoS17–IVY. The arrows indicate the positions of the fusion proteins. (B) Secretion of ExoS–CDO. CHA strains transformed with pS(129, 96, 54, or 17)-CDO were grown in the presence of EGTA. The CDO activity in the culture supernatant was measured with a spectrophotometric assay. Each point represents the average of three independent experiments. Error bars represent a 95% CI. (C) Translocation of ExoS–CDO. CHA strains transformed with pS(129, 96, 54, or 17)-CDO were incubated with PLB-985 cells for 1 h. CDO activity in PLB-985 cells and in the bacteria was measured with a spectrophotometric assay. The results are expressed as the ratio (PLB-985 activity/bacteria activity) 100. Each point represents the average of three independent experiments. Error bars represent a 95% CI. (D) Western blot of pellets (P) and supernatants (SN) from cultures of CHA-OST S54-Ova, CHA-OST S0-Ova, and CHA-BD S54-Ova grown without supplementation, with IPTG, or with both IPTG and EGTA. ExoS–Ova fusion proteins were detected with an anti-ovalbumin polyclonal antibody.
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S54-Ova. Addition of IPTG to the culture resulted in an increase in the intrabacterial level of S54-Ova, and we observed high-level secretion of S54-Ova only upon calcium depletion (Fig. 1D). As a control for antigen delivery, we elaborated secretion- or translocation-negative strains. First, we transferred pS54-Ova ExsAi in mutant CHA-BD lacking TTSS structure proteins PopB and PopD. Deletion of popB and popD allows protein secretion (Fig. 1D) but not translocation [8]. In addition, we elaborated pS0-Ova ExsAi (lacking ExoS N-terminal sequence) and transferred it in mutant CHA-OST. As expected, S0-Ova was produced in CHA-OST but was not secreted (Fig. 1D). Subsequently, we evaluated antigen delivery to DCs in vitro and in vivo with four strains: (i) CHA-OST S54Ova (secreting and translocating the antigen by TTSS), (ii) CHA-OST S0-Ova (not secreting nor translocating the antigen), (iii) CHA-BD S54-Ova (secreting but not translocating the antigen, and (iv) CHA-OST S54-GFP (secreting and translocating GFP as control antigen). In Vitro Evaluation of the Vector We evaluated whether exposure of DCs to P. aeruginosa delivering the fusion protein allows DC maturation and activation. We induced the expression of S54-Ova in the CHA-OST strain by IPTG and incubated bacteria for 1 h with DCs from C57BL/6 (MHC-I H-2Kb) mice. Within 24 h more than 90% of DCs were activated (Fig. 2A). We then evaluated if exposure to the vaccine vector induces antigen presentation by DCs to specific CD8+ T lymphocytes. We incubated DCs from C57BL/6 mice for 3 h with each of the four strains after IPTG preinduction of the TTSS. We then harvested the DCs and incubated them with B3Z hybridoma cells having a T cell receptor (TCR) specific to the ovalbumin 257–264 peptide (SIINFEKL)
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presented by MHC-I H-2Kb. TCR-mediated B3Z activation was obtained after incubation with DCs that had been exposed to CHA-OST S54-Ova (Fig. 2B). In contrast, we observed no activation after exposure of DCs to phosphate-buffered saline (PBS), CHA-OST S0-Ova, CHA-BD S54-Ova, or CHA-OST S54-GFP (Fig. 2B). The results demonstrated that specific antigen delivery via the P. aeruginosa TTSS was well suited to MHC-I presentation. In Vivo Evaluation of the Vector To assess the ability of the bacterial vector to raise specific CD8+ T lymphocytes in vivo, we injected C57BL/6 mice subcutaneously with 5 106 bacteria at days 1 and 8 and harvested splenocytes at day 13. Staining with anti-CD8+ and H-2Kb-ovalbumin257–264 (SIINFEKL) tetramer demonstrated that up to 1.2% of the CD8+ T lymphocytes were specific for SIINFEKL in mice injected with CHA-OST S54-Ova compared to 0.3% of the CD8+ T lymphocytes in mice injected with one of the other strains (Fig. 3A). We also evaluated (by ELISA and by Western-blot) the anti-ovalbumin antibody response 30 days after vaccination with strains CHA-OST S54-Ova, CHA-OST S54-GFP, and CHA-BD S54-Ova. We detected only a weak antiovalbumin antibody titer (1/90) in mice injected with CHA-OST S54-Ova and CHA-BD S54-Ova; mice injected with CHA-OST S54-GFP did not harbor detectable antiovalbumin antibodies (data not shown). We used the mouse melanoma cell line B16-OVA, which expresses chicken egg ovalbumin, to evaluate the efficacy of the anti-tumor response induced by the four P. aeruginosa strains described above. Injection of B16OVA cells into syngeneic C57BL/6 mice results in rapid tumor development [3]. Here, we performed a prophylactic and a curative anti-B16-OVA assay.
FIG. 2. In vitro DC activation and peptide presentation to specific lymphocytes. (A) FACS analysis of C57BL/6 DC activation at 24 h after a 1-h incubation with or without CHA-OST. The dot plots are representative of two repeated experiments. (B) B3Z hybridoma activation by C57BL/6 DCs previously incubated with various bacterial strains as indicated. The B3Z T cell receptor is specific to the ovalbumin peptide SIINFEKL presented by murine MHC-I H2Kb. B3Z activation was assessed with a h-galactosidase enzymatic assay (absorbance at 570 nm). The vaccine vector (CHA-OST S54-Ova) delivering the ovalbumin fragment by TTSS was compared with three control vectors: CHA-OST S0-Ova (not secreting nor translocating ovalbumin), CHA-BD S54-Ova (secreting but not translocating ovalbumin), and CHA-OST S54-GFP (secreting and translocating GFP as control antigen). The data were obtained from three independent experiments in triplicate. The error bars represent a 95% CI.
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FIG. 3. In vivo activity of P. aeruginosa vector. The vaccine vector (CHA-OST S54-Ova) was compared with the same controls as in Fig. 2B. (A) Quantification of specific anti-ovalbumin peptide SIINFEKL CD8+ T lymphocytes among freshly harvested splenocytes in C57BL/6 mice injected with different bacterial strains at 13 and 5 days before analysis. The cells were quantitated by flow cytometry using anti-CD8–FITC and H-2Kb ovalbumin257–264 (SIINFEKL) tetramer–PE. Dead cells were excluded from analysis after viability assessment using propidium iodide staining. The dot plots are representative of repeated experiments in different animals. (B and C) Prophylactic anti-tumor assay. Subcutaneous tumor challenge was performed with B16-OVA (at day 0) in C57BL/6 mice injected 14 and 7 days before with different bacterial vectors: (5) CHA-OST S54-Ova, n = 8; (o) CHA-OST S0-Ova, n = 6; (z) CHA-BD S54-Ova, n = 7; (X) CHA-OST S54-GFP, n = 8; (.) PBS, n = 8. Animals were sacrificed when larger diameter exceeded 1 cm. (B) Tumor onset; (C) survival. (D) Tumor weight in a curative anti-tumor assay. C57BL/6 mice received an intravenous tumor challenge with B16-OVA at day 1 and were injected with either CHA-OST S54-Ova or CHA-OST S54-GFP at days 5 and 12. Animals were sacrificed at day 16 and metastatic intrathoracic tumor (if any) was weighed. (5) CHA-OST S54-Ova; (X) CHA-OST S54-GFP.
For the prophylactic assay, we injected C57BL/6 mice subcutaneously with either 5 106 bacteria or PBS at both 14 and 7 days before subcutaneous injection of 2 105 B16-OVA cells. Only one of eight mice injected with CHA-OST S54-Ova developed a tumor (at day 55 after injection of B16-OVA cells) (Figs. 3B and 3C). All mice injected with PBS, CHA-OST S0-Ova, or CHA-OST S54GFP developed a tumor within 3 weeks after injection of B16-OVA cells (Figs. 3B and 3C). Mice injected with CHABD S54-Ova had a slightly delayed kinetics of tumor formation (Figs. 3B and 3C). For the curative assay, we injected C57BL/6 mice intravenously with 105 B16-OVA cells at day 1 and subcutaneously injected them with either 5 106 CHA-
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OST S54-GFP or 5 106 CHA-OST S54-Ova at days 5 and 12. We sacrificed the mice at day 16 and harvested intrathoracic metastatic tumors (if any). Six of the six CHA-OST S54-GFP-treated mice (mean tumor weight 98.3 F 34.3 mg), in contrast with 1/6 CHA-OST S54-Ovatreated mice (tumor weight 50 mg), harbored intrathoracic tumor ( P = 0.0192) (Fig. 3D).
DISCUSSION Two of the main difficulties in inducing a specific antitumor response are correct delivery of the targeted antigen and generation of an appropriate activation of APCs. Numerous animal and human studies have suc-
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cessfully loaded DCs ex vivo with protein or peptide, but this requires isolation, in vitro manipulation, and subsequent reinjection of DCs [1]. Injection of antigenic protein alone often results in poor immune system activation and induces only a weak CTL response. Fusion of the antigen with a protein having internalization properties such as Tat from human immunodeficiency virus [9] or ExoA from P. aeruginosa [10] may ensure a cytoplasmic delivery of antigenic protein to APCs, thus inducing an MHC-I-restricted presentation. However, this may not result in APC maturation and activation, which are necessary for generation of a CTL response. A maturation stimulus is required to induce the effectors of immune response, and injection of immature antigenloaded DCs may result in tolerance rather than immunization [11]. Activation occurs through several pathways providing DCs necessary signals to elicit cytokine release. This adjuvant effect, also called a bdanger signalQ [12], can be initiated by engagement of one or more of the toll-like receptors (TLRs), which recognize conserved molecular microbial motifs such as lipopolysaccharide and flagella. Vectors for anti-tumor immunotherapy need to deliver antigen to the MHC-I presentation pathway and to exhibit TLR ligands. The TTSS-based Salmonella vector, by providing both requirements, resulted in protective immunity in a viral infection model [4]. Here the effectiveness of a live attenuated TTSS-based vector for anti-tumor prophylactic and curative immunotherapy was demonstrated for the first time. Antigen delivery by the TTSS was demonstrated in vitro and in vivo by the efficient anti-tumor TCD8+ immune reaction induced by CHA-OST S54-Ova compared with a strain deficient in protein secretion and translocation (CHA-OST S0-Ova) or one deficient in protein translocation (CHA-BD S54-Ova). For the two latter strains, antigen may have been delivered to APCs by phagocytosis or pinocytosis but not processed for MHCI-restricted presentation. Considering the results we obtained, it is highly probable that the following events occur in vivo after vector injection: antigen delivery to antigen-presenting cells (among them mDC) and Toll-like receptor-mediated activation of these cells, subsequent activation and proliferation of antigen-specific CD8+ (and CD4+) T lymphocytes, and cytotoxicity performed by CD8+ T lymphocytes toward antigen-expressing tumor cells. Participation of anti-tumor antibodies in the tumor elimination process is unlikely, as we detected only low titers of anti-ovalbumin antibodies after vector injection. The strengths of the vector described here are its simple administration route and its flexibility in translocating a variety of proteins. The subcutaneous injection of a live antigen-delivering bacteria may allow the immune system to develop a more complete immune response toward the antigen than the reaction obtained with ex vivo-manipulated DCs. The S54-Ova fusion protein has a molecular weight of 19.9 kDa, but larger proteins were also correctly
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secreted and translocated. This property will allow immunization protocols with entire antigenic proteins, which provide panel of putative epitopes at once. This offers the possibility of a wide usage in vaccination, bypassing the limitation associated with the use of short peptides as antigens, which are restricted to particular MHC-I alleles. Clinical phase I trials using live attenuated bacteria have been conducted and these vectors may be acceptable for human therapy [13,14]. Therefore they may become an important tool for the activation of specific CD8+ T lymphocytes in association with existing therapies for cancer patients.
MATERIAL
AND
METHODS
Bacterial Strains The CHA-OST (lacking TTSS toxins ExoS and ExoT) and CHA-BD (lacking TTSS structural proteins PopB and PopD) mutants were generated from the P. aeruginosa strain CHA [15] by Cre/lox-based mutagenesis [16]. Plasmid Construction The ExoS promoter and various lengths of the 5Vend of the ExoS coding sequence were amplified by PCR from CHA DNA. The genes of the P. aeruginosa 16.9-kDa IVY, P. putida 35.1-kDa CDO, 27.1-kDa GFP from pMN406 [17], and C-terminal end of chicken ovalbumin (encoding residues 286–387) were amplified by PCR and cloned in-frame with exoS sequences to generate the series pS(129, 96, 54, or 17)-IVY, -CDO, or -GFP and pS54-GFP and pS54-Ova. The CHA exsA gene (transcriptional activator of the ExoS promoter [7]) was cloned under the control of the IPTGinducible ptac promoter in pTTQ18 [18]. The inducible exsA gene was transferred to pUCP20 to obtain pExsAi. S54-Ova and S54-GFP gene fusions were each cloned in pExsAi to obtain respectively pS54-Ova ExsAi and pS54-GFP ExsAi. pS0-Ova ExsAi was generated by removing the ExoS coding region from pS54-Ova ExsAi, allowing the expression of the ovalbumin C-terminal fragment alone. Plasmids pS54-Ova ExsAi, pS0-Ova ExsAi, and pS54-GFP ExsAi were transferred to the CHA mutants to obtain CHA-OST S54-Ova, CHA-OST S54-GFP, CHA-OST S0-Ova, and CHA-BD S54-Ova. Mammalian Cells PLB-985 is a precursor cell line of human neutrophil polymorphonuclear leukocytes [19]. DCs were generated from the bone marrow of C57BL/6 mice [20]. The murine T cell hybridoma B3Z is specific for the ovalbumin peptide SIINFEKL (residues 257–264) in the context of H-2Kb and harbors the h-galactosidase gene under the control of the IL2 promoter [21]. B16OVA is a melanoma cell line from C57BL/6 mice constitutively expressing ovalbumin [22]. TTSS Analysis Secretion assay. CHA was transformed with each of the plasmids from the series pS(129, 96, 54, or 17)-IVY, -CDO, or -GFP. Resulting strains were grown in Luria–Bertani (LB) medium containing 300 mg/L carbenicillin, 5 mM EGTA, and 20 mM MgCl2 (calcium depletion induced by EGTA triggers P. aeruginosa TTSS activation and secretion of TTSS effectors in culture medium) [7]. Supernatants were analyzed for the presence of secreted fusion protein. ExoS–IVY was detected by SDS–PAGE of the supernatant. ExoS–CDO was quantified by adding 15 mM catechol to the supernatant and measuring the absorbance of 2-hydroxymuconic semialdehyde at 375 nm. ExoS–GFP was quantified by measuring fluorescence in the supernatant. Translocation assay. PLB-985 cells were incubated for 1 h at a multiplicity of infection (m.o.i.) of 5 with CHA transformed with pS(129, 96, 54, or 17)-CDO. After centrifugation at 400g, the pellet (containing the PLB-985) was washed in PBS and lysed on ice with 0.1% Triton X-100 in PBS and the supernatant (containing the bacteria) was sonicated. CDO
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activity was assessed in both fractions and results were expressed as (CDO activity in PLB-985/CDO activity in bacteria) 100.
Infectieuse de Langue Franc¸aise, France, and by a grant from the Faculte´ de Me´decine, Universite´ Joseph Fourier, Grenoble, France.
Western blot. The strains CHA-OST S54-Ova, CHA-BD S54-Ova, and CHAOST S0-Ova were grown in LB containing 300 mg/L carbenicillin alone or with either IPTG or IPTG, MgCl2, and EGTA. After centrifugation, the pellet and supernatant were analyzed by Western blot using an antichicken ovalbumin antibody (Sigma, St. Louis, MO, USA).
RECEIVED FOR PUBLICATION JANUARY 27, 2006; REVISED MAY 1, 2006; ACCEPTED JUNE 23, 2006.
DC Mortality and Activation Assay The CHA-OST S54-Ova strain was grown in LB medium supplemented with IPTG for 1 h and added to DCs at an m.o.i. of 5. After 1 h DCs were washed and maintained for 24 h in culture medium supplemented with 200 mg/L gentamycin prior to FACS analysis. DC death was assessed by propidium iodide staining, DC phenotype by staining with anti-CD11c antibody (e-Biosciences, Atlanta, GA, USA), and DC activation by staining with anti-Iab antibody (BD Biosciences Pharmingen, San Jose, CA, USA). Antigen Presentation Assay C57BL/6 DCs were seeded in flat-bottom 96-well plates at 1 105 cells/well. Bacteria were grown in LB medium supplemented with 300 mg/L carbenicillin and 0.5 mM IPTG and added at an m.o.i. of 5. Supernatant was discarded after 3 h, and B3Z cells were added for 16 h at 1 105 cells/ well in medium supplemented with 250 mg/L gentamycin. After centrifugation, h-galactosidase activity was determined as previously described [3]. In Vivo Immunization Female C57BL/6 mice were purchased from Janvier SA (Le Genest-SaintIsle, France) and used at 6–8 weeks of age. Experiments were approved by the Universite´ J. Fourier Committee for Animal Experimentation. Bacteria were grown in LB containing 300 mg/L carbenicillin and 0.5 mM IPTG and resuspended in PBS before a 100-Al subcutaneous injection into the right flank. Tetramer analysis. Mice were injected with different CHA strains at days 1 and 8 and were sacrificed at day 13. Flow cytometry was performed on freshly harvested splenocytes using anti-CD8-APC antibody and H-2Kbovalbumin257–264 (SIINFEKL) tetramer according to the manufacturer’s instructions (Immunomics, Beckman Coulter, Fullerton, CA, USA). Antibody analysis. Mice were injected subcutaneously in the right flank with different CHA strains at days 1 and 8, and sera were harvested at day 30. For the Western blot analysis, we performed SDS–PAGE with purified ovalbumin (Calbiochem, EMD Bioscience, La Jolla, CA, USA). For the ELISA, we coated wells with purified ovalbumin. Different serum dilutions (from 1/30 to 1/7290) were tested. In both experiments, revelation was performed using anti-mouse immunoglobulin antibody (Amersham Life Science, Little Chalfont, England). Tumor challenge. For the prophylactic assay, mice were injected subcutaneously in the right flank with different CHA strains 14 and 7 days before the injection of 2 105 B16-OVA cells in 100 Al PBS in the left flank. Mice were sacrificed when the tumor diameter reached 1 cm. For the curative assay, mice were injected intravenously with 105 B16-OVA cells in 50 Al PBS at day 1, vaccinated with different CHA strains at days 5 and 12, and sacrificed at day 16 for the evaluation of intrathoracic metastatic tumors.
ACKNOWLEDGMENTS We thank Georges Vassaux (Molecular Oncology Unit, Cancer Research UK Clinical Centre, UK) for the gifts of the B3Z and B16-OVA cell lines, Michael Niederweis (Friedrich-Alexander University, Nqrnberg, Germany) for the gift of pMN406, and David Hacker for critical review of the manuscript. O. Epaulard was supported by a grant from the Colle`ge des Professeurs de Pathologie
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Anti-tumor Immunotherapy via Antigen Delivery from a Live Attenuated Genetically Engineered Pseudomonas aeruginosa Type III Secretion System-Based Vector Olivier Epaulard,1,* Bertrand Toussaint,1,* Lauriane Quenee,1 Madiha Derouazi,1 Nabil Bosco,2 Christian Villiers,2 Rozenn Le Berre,3 Benoit Guery,3 Didier Filopon,1 Laurence Crombez,1,4 Patrice N. Marche,2 and Benoit Polack1,y 1
Groupe de Recherche et d’Etude du Processus Inflammatoire, Universite´ J. Fourier EA2938, Hopital de Grenoble, BP 217, 38043 Grenoble Cedex 09, France 2 Laboratoire d’Immunochimie, INSERM U548, CEA Grenoble DRDC/ICH, 17 Rue des Martyrs, 38054 Grenoble Cedex 09, France 3 Laboratoire de Recherche en Pathologie Infectieuse, EA2689 Faculte´ de Me´decine, 59045 Lille Cedex, France 4 Protein’eXpert SA, 15, Rue des Martyrs, 38027 Grenoble, France *These authors contributed equally to this work.
y
To whom correspondence and reprint requests should be addressed at the Groupe de Recherche et d’Etude du Processus Inflammatoire, Universite´ J. Fourier EA2938, Laboratoire d’Enzymologie, De´partement de Biologie et de Pathologie de la Cellule, CHU de Grenoble, BP 217, 38043 Grenoble Cedex 09, France. Fax: +33 4 76 76 56 08. E-mail: [email protected].
Immunotherapy requiring an efficient T lymphocyte response is initiated by antigen delivery to antigenpresenting cells. Several studies have assessed the efficiency of various antigen loading procedures, including microbial vectors. Here a live strain of Pseudomonas aeruginosa was engineered to translocate a recombinant antigenic protein into mammalian cells via the type III secretion system, a bacterial device translocating effector proteins into host cells. Optimization of the vector included virulence attenuation and determination of the N-terminal sequence allowing translocation of fused antigens into cells. In vitro delivery of an ovalbumin fragment by the bacterial vector into dendritic cells induced the activation of ovalbumin-specific CD8+ T lymphocytes. Mice injected with the ovalbumin-delivering vector developed ovalbumin-specific CD8+ T lymphocytes and were resistant to a subsequent challenge with an ovalbumin-expressing melanoma. Moreover, in a curative assay, injection of the vaccine vector 5 and 12 days after tumor implantation led to a complete cure in five of six animals. These results highlight the utility of type III secretion system-based vectors for anti-tumor immunotherapy. Key Words: cancer, immunotherapy, vaccine, type III secretion system, Pseudomonas aeruginosa, dendritic cell
INTRODUCTION The objective of most anti-tumor vaccine strategies is the generation of tumor-specific cytotoxic T lymphocytes (CTLs). This is possible through major histocompatibility complex class I (MHC-I)-restricted presentation of antigen by dendritic cells (DCs) [1]. These cells are powerful antigenpresenting cells (APCs) and play a central role in generating and directing immune response. MHC-I-restricted presentation occurs preferentially for antigens present in the cytoplasm, in contrast to antigens processed after phagocytosis and presented by MHC-II molecules. Different methods of antigen delivery to DCs have been used to obtain MHC-Irestricted antigen presentation, including antigen gene delivery with microbial vectors [1]. Recently, the potential of immunotherapy using antigen delivery by live attenuated
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bacterial vectors has been assessed [2–4]. We previously demonstrated the feasibility of using Pseudomonas aeruginosa as a vector for active enzyme delivery to mammalian cells using type III secretion system (TTSS), a bacterial device translocating effector proteins into the eukaryotic cytoplasm [5]. We hypothesized that a TTSS-based antigen delivering Pseudomonas may induce a MHC-I-restricted presentation by DCs. Here we used P. aeruginosa to deliver a tumor antigen to APCs and generate a CTL response against tumor cells in vivo.
RESULTS Engineering of the Bacterial Vector For the secretion of the Yersinia TTSS toxins YopE and YopH, the N-terminal 15 and 17 amino acids (aa),
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respectively, are sufficient [6]; however, a similar domain has not been discovered at the N-termini of TTSS toxins from other bacterial pathogens [7]. Therefore we determined the region of the P. aeruginosa TTSS toxin exoenzyme S (ExoS) required for the secretion and translocation of an ExoS-fused antigen by generating protein fusions between the N-terminal 129, 96, 54, or 17 aa of ExoS and three reporter proteins that are not normally secreted by the TTSS: P. aeruginosa inhibitor of vertebrate lysozyme (IVY), P. putida catechol 2,3-dioxygenase (CDO), and green fluorescent protein (GFP). We transferred the plasmids into P. aeruginosa strain CHA and assessed the secretion of the fusion proteins after TTSS induction by calcium depletion, a condition known to trigger TTSS [7]. We observed high secretion levels when each of the reporter proteins was fused with the Nterminal 54 aa of ExoS (Figs. 1A and 1B). With the Nterminal ExoS fragments of 129 or 96 aa, IVY but not CDO (Figs. 1A and 1B) or GFP (data not shown) was efficiently secreted. Reporter proteins fused with the Nterminal 17 aa of ExoS were secreted only at low levels (Figs. 1A and 1B). To determine if this N-terminus of ExoS
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was also required for translocation into mammalian cells, we incubated CHA strains expressing different ExoS–CDO fusions with PLB-985 cells. When fused to the N-terminal 54 aa of ExoS, cytoplasmic delivery of CDO was optimal (Fig. 1C). The TTSS toxins ExoS and exoenzyme T (ExoT) are involved in P. aeruginosa pathogenesis [7]. To lessen CHA cytotoxicity, we developed an attenuated CHA-OST mutant (ExoS, ExoT). We assessed the viability of bone-marrow-derived myeloid DCs from C57BL/6 mice 24 h after a 1-h exposure to either CHA or CHA-OST. With the parental strain 50% of the DCs were killed, whereas the survival rate was 95% in the presence of CHA-OST (data not shown). To control fusion protein expression in P. aeruginosa, we cloned the exsA gene encoding the ExoS transcriptional activator [7] under the control of an isopropyl-h-dthiogalactopyranoside (IPTG)-inducible promoter. To assess the efficiency of conditional TTSS control by ExsA, we expressed a fusion gene encoding the N-terminal 54 aa of ExoS and the C-terminus of ovalbumin along with the exsA gene from pS54-Ova ExsAi in strain CHA-OST
FIG. 1. Type III secretion system analysis. (A) Secretion of ExoS–IVY. SDS–PAGE of culture supernatants of CHA strains transformed with pS(129, 96, 54, or 17)IVY and grown in the absence () or presence (+) of EGTA to induce TTSS activation is shown. Theoretical molecular weights of the fusion proteins are 30.7 kDa for ExoS129–IVY, 27.1 kDa for ExoS96–IVY, 23.1 kDa for ExoS54–IVY, and 18.8 kDa for ExoS17–IVY. The arrows indicate the positions of the fusion proteins. (B) Secretion of ExoS–CDO. CHA strains transformed with pS(129, 96, 54, or 17)-CDO were grown in the presence of EGTA. The CDO activity in the culture supernatant was measured with a spectrophotometric assay. Each point represents the average of three independent experiments. Error bars represent a 95% CI. (C) Translocation of ExoS–CDO. CHA strains transformed with pS(129, 96, 54, or 17)-CDO were incubated with PLB-985 cells for 1 h. CDO activity in PLB-985 cells and in the bacteria was measured with a spectrophotometric assay. The results are expressed as the ratio (PLB-985 activity/bacteria activity) 100. Each point represents the average of three independent experiments. Error bars represent a 95% CI. (D) Western blot of pellets (P) and supernatants (SN) from cultures of CHA-OST S54-Ova, CHA-OST S0-Ova, and CHA-BD S54-Ova grown without supplementation, with IPTG, or with both IPTG and EGTA. ExoS–Ova fusion proteins were detected with an anti-ovalbumin polyclonal antibody.
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S54-Ova. Addition of IPTG to the culture resulted in an increase in the intrabacterial level of S54-Ova, and we observed high-level secretion of S54-Ova only upon calcium depletion (Fig. 1D). As a control for antigen delivery, we elaborated secretion- or translocation-negative strains. First, we transferred pS54-Ova ExsAi in mutant CHA-BD lacking TTSS structure proteins PopB and PopD. Deletion of popB and popD allows protein secretion (Fig. 1D) but not translocation [8]. In addition, we elaborated pS0-Ova ExsAi (lacking ExoS N-terminal sequence) and transferred it in mutant CHA-OST. As expected, S0-Ova was produced in CHA-OST but was not secreted (Fig. 1D). Subsequently, we evaluated antigen delivery to DCs in vitro and in vivo with four strains: (i) CHA-OST S54Ova (secreting and translocating the antigen by TTSS), (ii) CHA-OST S0-Ova (not secreting nor translocating the antigen), (iii) CHA-BD S54-Ova (secreting but not translocating the antigen, and (iv) CHA-OST S54-GFP (secreting and translocating GFP as control antigen). In Vitro Evaluation of the Vector We evaluated whether exposure of DCs to P. aeruginosa delivering the fusion protein allows DC maturation and activation. We induced the expression of S54-Ova in the CHA-OST strain by IPTG and incubated bacteria for 1 h with DCs from C57BL/6 (MHC-I H-2Kb) mice. Within 24 h more than 90% of DCs were activated (Fig. 2A). We then evaluated if exposure to the vaccine vector induces antigen presentation by DCs to specific CD8+ T lymphocytes. We incubated DCs from C57BL/6 mice for 3 h with each of the four strains after IPTG preinduction of the TTSS. We then harvested the DCs and incubated them with B3Z hybridoma cells having a T cell receptor (TCR) specific to the ovalbumin 257–264 peptide (SIINFEKL)
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presented by MHC-I H-2Kb. TCR-mediated B3Z activation was obtained after incubation with DCs that had been exposed to CHA-OST S54-Ova (Fig. 2B). In contrast, we observed no activation after exposure of DCs to phosphate-buffered saline (PBS), CHA-OST S0-Ova, CHA-BD S54-Ova, or CHA-OST S54-GFP (Fig. 2B). The results demonstrated that specific antigen delivery via the P. aeruginosa TTSS was well suited to MHC-I presentation. In Vivo Evaluation of the Vector To assess the ability of the bacterial vector to raise specific CD8+ T lymphocytes in vivo, we injected C57BL/6 mice subcutaneously with 5 106 bacteria at days 1 and 8 and harvested splenocytes at day 13. Staining with anti-CD8+ and H-2Kb-ovalbumin257–264 (SIINFEKL) tetramer demonstrated that up to 1.2% of the CD8+ T lymphocytes were specific for SIINFEKL in mice injected with CHA-OST S54-Ova compared to 0.3% of the CD8+ T lymphocytes in mice injected with one of the other strains (Fig. 3A). We also evaluated (by ELISA and by Western-blot) the anti-ovalbumin antibody response 30 days after vaccination with strains CHA-OST S54-Ova, CHA-OST S54-GFP, and CHA-BD S54-Ova. We detected only a weak antiovalbumin antibody titer (1/90) in mice injected with CHA-OST S54-Ova and CHA-BD S54-Ova; mice injected with CHA-OST S54-GFP did not harbor detectable antiovalbumin antibodies (data not shown). We used the mouse melanoma cell line B16-OVA, which expresses chicken egg ovalbumin, to evaluate the efficacy of the anti-tumor response induced by the four P. aeruginosa strains described above. Injection of B16OVA cells into syngeneic C57BL/6 mice results in rapid tumor development [3]. Here, we performed a prophylactic and a curative anti-B16-OVA assay.
FIG. 2. In vitro DC activation and peptide presentation to specific lymphocytes. (A) FACS analysis of C57BL/6 DC activation at 24 h after a 1-h incubation with or without CHA-OST. The dot plots are representative of two repeated experiments. (B) B3Z hybridoma activation by C57BL/6 DCs previously incubated with various bacterial strains as indicated. The B3Z T cell receptor is specific to the ovalbumin peptide SIINFEKL presented by murine MHC-I H2Kb. B3Z activation was assessed with a h-galactosidase enzymatic assay (absorbance at 570 nm). The vaccine vector (CHA-OST S54-Ova) delivering the ovalbumin fragment by TTSS was compared with three control vectors: CHA-OST S0-Ova (not secreting nor translocating ovalbumin), CHA-BD S54-Ova (secreting but not translocating ovalbumin), and CHA-OST S54-GFP (secreting and translocating GFP as control antigen). The data were obtained from three independent experiments in triplicate. The error bars represent a 95% CI.
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FIG. 3. In vivo activity of P. aeruginosa vector. The vaccine vector (CHA-OST S54-Ova) was compared with the same controls as in Fig. 2B. (A) Quantification of specific anti-ovalbumin peptide SIINFEKL CD8+ T lymphocytes among freshly harvested splenocytes in C57BL/6 mice injected with different bacterial strains at 13 and 5 days before analysis. The cells were quantitated by flow cytometry using anti-CD8–FITC and H-2Kb ovalbumin257–264 (SIINFEKL) tetramer–PE. Dead cells were excluded from analysis after viability assessment using propidium iodide staining. The dot plots are representative of repeated experiments in different animals. (B and C) Prophylactic anti-tumor assay. Subcutaneous tumor challenge was performed with B16-OVA (at day 0) in C57BL/6 mice injected 14 and 7 days before with different bacterial vectors: (5) CHA-OST S54-Ova, n = 8; (o) CHA-OST S0-Ova, n = 6; (z) CHA-BD S54-Ova, n = 7; (X) CHA-OST S54-GFP, n = 8; (.) PBS, n = 8. Animals were sacrificed when larger diameter exceeded 1 cm. (B) Tumor onset; (C) survival. (D) Tumor weight in a curative anti-tumor assay. C57BL/6 mice received an intravenous tumor challenge with B16-OVA at day 1 and were injected with either CHA-OST S54-Ova or CHA-OST S54-GFP at days 5 and 12. Animals were sacrificed at day 16 and metastatic intrathoracic tumor (if any) was weighed. (5) CHA-OST S54-Ova; (X) CHA-OST S54-GFP.
For the prophylactic assay, we injected C57BL/6 mice subcutaneously with either 5 106 bacteria or PBS at both 14 and 7 days before subcutaneous injection of 2 105 B16-OVA cells. Only one of eight mice injected with CHA-OST S54-Ova developed a tumor (at day 55 after injection of B16-OVA cells) (Figs. 3B and 3C). All mice injected with PBS, CHA-OST S0-Ova, or CHA-OST S54GFP developed a tumor within 3 weeks after injection of B16-OVA cells (Figs. 3B and 3C). Mice injected with CHABD S54-Ova had a slightly delayed kinetics of tumor formation (Figs. 3B and 3C). For the curative assay, we injected C57BL/6 mice intravenously with 105 B16-OVA cells at day 1 and subcutaneously injected them with either 5 106 CHA-
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OST S54-GFP or 5 106 CHA-OST S54-Ova at days 5 and 12. We sacrificed the mice at day 16 and harvested intrathoracic metastatic tumors (if any). Six of the six CHA-OST S54-GFP-treated mice (mean tumor weight 98.3 F 34.3 mg), in contrast with 1/6 CHA-OST S54-Ovatreated mice (tumor weight 50 mg), harbored intrathoracic tumor ( P = 0.0192) (Fig. 3D).
DISCUSSION Two of the main difficulties in inducing a specific antitumor response are correct delivery of the targeted antigen and generation of an appropriate activation of APCs. Numerous animal and human studies have suc-
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cessfully loaded DCs ex vivo with protein or peptide, but this requires isolation, in vitro manipulation, and subsequent reinjection of DCs [1]. Injection of antigenic protein alone often results in poor immune system activation and induces only a weak CTL response. Fusion of the antigen with a protein having internalization properties such as Tat from human immunodeficiency virus [9] or ExoA from P. aeruginosa [10] may ensure a cytoplasmic delivery of antigenic protein to APCs, thus inducing an MHC-I-restricted presentation. However, this may not result in APC maturation and activation, which are necessary for generation of a CTL response. A maturation stimulus is required to induce the effectors of immune response, and injection of immature antigenloaded DCs may result in tolerance rather than immunization [11]. Activation occurs through several pathways providing DCs necessary signals to elicit cytokine release. This adjuvant effect, also called a bdanger signalQ [12], can be initiated by engagement of one or more of the toll-like receptors (TLRs), which recognize conserved molecular microbial motifs such as lipopolysaccharide and flagella. Vectors for anti-tumor immunotherapy need to deliver antigen to the MHC-I presentation pathway and to exhibit TLR ligands. The TTSS-based Salmonella vector, by providing both requirements, resulted in protective immunity in a viral infection model [4]. Here the effectiveness of a live attenuated TTSS-based vector for anti-tumor prophylactic and curative immunotherapy was demonstrated for the first time. Antigen delivery by the TTSS was demonstrated in vitro and in vivo by the efficient anti-tumor TCD8+ immune reaction induced by CHA-OST S54-Ova compared with a strain deficient in protein secretion and translocation (CHA-OST S0-Ova) or one deficient in protein translocation (CHA-BD S54-Ova). For the two latter strains, antigen may have been delivered to APCs by phagocytosis or pinocytosis but not processed for MHCI-restricted presentation. Considering the results we obtained, it is highly probable that the following events occur in vivo after vector injection: antigen delivery to antigen-presenting cells (among them mDC) and Toll-like receptor-mediated activation of these cells, subsequent activation and proliferation of antigen-specific CD8+ (and CD4+) T lymphocytes, and cytotoxicity performed by CD8+ T lymphocytes toward antigen-expressing tumor cells. Participation of anti-tumor antibodies in the tumor elimination process is unlikely, as we detected only low titers of anti-ovalbumin antibodies after vector injection. The strengths of the vector described here are its simple administration route and its flexibility in translocating a variety of proteins. The subcutaneous injection of a live antigen-delivering bacteria may allow the immune system to develop a more complete immune response toward the antigen than the reaction obtained with ex vivo-manipulated DCs. The S54-Ova fusion protein has a molecular weight of 19.9 kDa, but larger proteins were also correctly
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secreted and translocated. This property will allow immunization protocols with entire antigenic proteins, which provide panel of putative epitopes at once. This offers the possibility of a wide usage in vaccination, bypassing the limitation associated with the use of short peptides as antigens, which are restricted to particular MHC-I alleles. Clinical phase I trials using live attenuated bacteria have been conducted and these vectors may be acceptable for human therapy [13,14]. Therefore they may become an important tool for the activation of specific CD8+ T lymphocytes in association with existing therapies for cancer patients.
MATERIAL
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METHODS
Bacterial Strains The CHA-OST (lacking TTSS toxins ExoS and ExoT) and CHA-BD (lacking TTSS structural proteins PopB and PopD) mutants were generated from the P. aeruginosa strain CHA [15] by Cre/lox-based mutagenesis [16]. Plasmid Construction The ExoS promoter and various lengths of the 5Vend of the ExoS coding sequence were amplified by PCR from CHA DNA. The genes of the P. aeruginosa 16.9-kDa IVY, P. putida 35.1-kDa CDO, 27.1-kDa GFP from pMN406 [17], and C-terminal end of chicken ovalbumin (encoding residues 286–387) were amplified by PCR and cloned in-frame with exoS sequences to generate the series pS(129, 96, 54, or 17)-IVY, -CDO, or -GFP and pS54-GFP and pS54-Ova. The CHA exsA gene (transcriptional activator of the ExoS promoter [7]) was cloned under the control of the IPTGinducible ptac promoter in pTTQ18 [18]. The inducible exsA gene was transferred to pUCP20 to obtain pExsAi. S54-Ova and S54-GFP gene fusions were each cloned in pExsAi to obtain respectively pS54-Ova ExsAi and pS54-GFP ExsAi. pS0-Ova ExsAi was generated by removing the ExoS coding region from pS54-Ova ExsAi, allowing the expression of the ovalbumin C-terminal fragment alone. Plasmids pS54-Ova ExsAi, pS0-Ova ExsAi, and pS54-GFP ExsAi were transferred to the CHA mutants to obtain CHA-OST S54-Ova, CHA-OST S54-GFP, CHA-OST S0-Ova, and CHA-BD S54-Ova. Mammalian Cells PLB-985 is a precursor cell line of human neutrophil polymorphonuclear leukocytes [19]. DCs were generated from the bone marrow of C57BL/6 mice [20]. The murine T cell hybridoma B3Z is specific for the ovalbumin peptide SIINFEKL (residues 257–264) in the context of H-2Kb and harbors the h-galactosidase gene under the control of the IL2 promoter [21]. B16OVA is a melanoma cell line from C57BL/6 mice constitutively expressing ovalbumin [22]. TTSS Analysis Secretion assay. CHA was transformed with each of the plasmids from the series pS(129, 96, 54, or 17)-IVY, -CDO, or -GFP. Resulting strains were grown in Luria–Bertani (LB) medium containing 300 mg/L carbenicillin, 5 mM EGTA, and 20 mM MgCl2 (calcium depletion induced by EGTA triggers P. aeruginosa TTSS activation and secretion of TTSS effectors in culture medium) [7]. Supernatants were analyzed for the presence of secreted fusion protein. ExoS–IVY was detected by SDS–PAGE of the supernatant. ExoS–CDO was quantified by adding 15 mM catechol to the supernatant and measuring the absorbance of 2-hydroxymuconic semialdehyde at 375 nm. ExoS–GFP was quantified by measuring fluorescence in the supernatant. Translocation assay. PLB-985 cells were incubated for 1 h at a multiplicity of infection (m.o.i.) of 5 with CHA transformed with pS(129, 96, 54, or 17)-CDO. After centrifugation at 400g, the pellet (containing the PLB-985) was washed in PBS and lysed on ice with 0.1% Triton X-100 in PBS and the supernatant (containing the bacteria) was sonicated. CDO
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activity was assessed in both fractions and results were expressed as (CDO activity in PLB-985/CDO activity in bacteria) 100.
Infectieuse de Langue Franc¸aise, France, and by a grant from the Faculte´ de Me´decine, Universite´ Joseph Fourier, Grenoble, France.
Western blot. The strains CHA-OST S54-Ova, CHA-BD S54-Ova, and CHAOST S0-Ova were grown in LB containing 300 mg/L carbenicillin alone or with either IPTG or IPTG, MgCl2, and EGTA. After centrifugation, the pellet and supernatant were analyzed by Western blot using an antichicken ovalbumin antibody (Sigma, St. Louis, MO, USA).
RECEIVED FOR PUBLICATION JANUARY 27, 2006; REVISED MAY 1, 2006; ACCEPTED JUNE 23, 2006.
DC Mortality and Activation Assay The CHA-OST S54-Ova strain was grown in LB medium supplemented with IPTG for 1 h and added to DCs at an m.o.i. of 5. After 1 h DCs were washed and maintained for 24 h in culture medium supplemented with 200 mg/L gentamycin prior to FACS analysis. DC death was assessed by propidium iodide staining, DC phenotype by staining with anti-CD11c antibody (e-Biosciences, Atlanta, GA, USA), and DC activation by staining with anti-Iab antibody (BD Biosciences Pharmingen, San Jose, CA, USA). Antigen Presentation Assay C57BL/6 DCs were seeded in flat-bottom 96-well plates at 1 105 cells/well. Bacteria were grown in LB medium supplemented with 300 mg/L carbenicillin and 0.5 mM IPTG and added at an m.o.i. of 5. Supernatant was discarded after 3 h, and B3Z cells were added for 16 h at 1 105 cells/ well in medium supplemented with 250 mg/L gentamycin. After centrifugation, h-galactosidase activity was determined as previously described [3]. In Vivo Immunization Female C57BL/6 mice were purchased from Janvier SA (Le Genest-SaintIsle, France) and used at 6–8 weeks of age. Experiments were approved by the Universite´ J. Fourier Committee for Animal Experimentation. Bacteria were grown in LB containing 300 mg/L carbenicillin and 0.5 mM IPTG and resuspended in PBS before a 100-Al subcutaneous injection into the right flank. Tetramer analysis. Mice were injected with different CHA strains at days 1 and 8 and were sacrificed at day 13. Flow cytometry was performed on freshly harvested splenocytes using anti-CD8-APC antibody and H-2Kbovalbumin257–264 (SIINFEKL) tetramer according to the manufacturer’s instructions (Immunomics, Beckman Coulter, Fullerton, CA, USA). Antibody analysis. Mice were injected subcutaneously in the right flank with different CHA strains at days 1 and 8, and sera were harvested at day 30. For the Western blot analysis, we performed SDS–PAGE with purified ovalbumin (Calbiochem, EMD Bioscience, La Jolla, CA, USA). For the ELISA, we coated wells with purified ovalbumin. Different serum dilutions (from 1/30 to 1/7290) were tested. In both experiments, revelation was performed using anti-mouse immunoglobulin antibody (Amersham Life Science, Little Chalfont, England). Tumor challenge. For the prophylactic assay, mice were injected subcutaneously in the right flank with different CHA strains 14 and 7 days before the injection of 2 105 B16-OVA cells in 100 Al PBS in the left flank. Mice were sacrificed when the tumor diameter reached 1 cm. For the curative assay, mice were injected intravenously with 105 B16-OVA cells in 50 Al PBS at day 1, vaccinated with different CHA strains at days 5 and 12, and sacrificed at day 16 for the evaluation of intrathoracic metastatic tumors.
ACKNOWLEDGMENTS We thank Georges Vassaux (Molecular Oncology Unit, Cancer Research UK Clinical Centre, UK) for the gifts of the B3Z and B16-OVA cell lines, Michael Niederweis (Friedrich-Alexander University, Nqrnberg, Germany) for the gift of pMN406, and David Hacker for critical review of the manuscript. O. Epaulard was supported by a grant from the Colle`ge des Professeurs de Pathologie
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