Tumor therapy in mice by using a tumor antigen linked to modulin peptides from Staphylococcus epidermidis

Vaccine 28 (2010) 7146–7154

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Tumor therapy in mice by using a tumor antigen linked to modulin peptides from Staphylococcus epidermidis Maika Durantez a , Catherine Fayolle b,c , Noelia Casares a , Virginia Belsue a , José I. Riezu-Boj a , Pablo Sarobe a , Jesús Prieto a,d , Francisco Borrás-Cuesta a , Claude Leclerc b,c , Juan J. Lasarte a,∗ a

University of Navarra, Centre for Applied Medical Research (CIMA), Gene Therapy and Hepatology Area, Pamplona 31008, Spain Institut Pasteur, Unité de Régulation Immunitaire et Vaccinologie, Paris F-75015, France INSERM, U883, Paris F-75015, France d CIBERehd, Pamplona, Spain b c

a r t i c l e

i n f o

Article history: Received 5 January 2010 Received in revised form 25 June 2010 Accepted 17 August 2010 Available online 15 September 2010 Keywords: T-cells Dendritic cells Vaccination Peptides Antigen targeting

a b s t r a c t Staphylococcus epidermidis releases a complex of at least four peptides, termed phenol-soluble modulins (PSM), which stimulate macrophages to produce proinflammatory cytokines via activation of TLR2 signalling pathway. We demonstrated that covalent linkage of PSM peptides to an antigen facilitate its capture by dendritic cells and, in combination with different TLR ligands, can favour the in vivo induction of strong and persistent antigen-specific immune responses. Treatment of mice grafted with HPV16-E7expressing tumor cells (TC-1) with poly(I:C) and a peptide containing ␣Mod linked to the H-2Db -restricted cytotoxic T-cell epitope E7(49–57) from HPV16-E7 protein allowed complete tumor regression in 100% of the animals. Surprisingly, this immunomodulatory property of modulin-derived peptides was TLR2 independent and partially dependent upon the EGF-receptor signalling pathway. Our results suggest that alpha or gamma modulin peptides may serve as a suitable antigen carrier for the development of anti-tumoral or anti-viral vaccines. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Recombinant proteins or peptides are generally poor immunogens and do not induce strong T-cell immune responses if they are not combined with appropriate adjuvants, or modified to improve their capture by dendritic cells (DCs). DCs, the professional antigen presenting cells (APCs) of the immune system, process the antigens into peptides and present them on MHC class I and II to T lymphocytes [1]. They orchestrate a repertoire of immune responses from tolerance to self-antigens to resistance to infectious pathogens [2,3] depending upon their maturation status (reviewed in [4]). Thus, it is generally accepted that the efficient activation of T-cell immune responses is dependent on DC maturation triggered by a combination of stimuli derived from microbial products, endogenous danger signals, or signals delivered by antigen-activated T-cells. For these reasons, several vaccination strategies incorporate the use of adjuvants able to trigger maturation of DC, either using ligands for pattern recognition receptors (PRRs) [5–9], agonistic antibodies against costimulatory molecules [9,10] or cytokines [11]. Moreover,

∗ Corresponding author at: Centre for Applied Medical Research, CIMA, Gene Therapy and Hepatology Area, Avenida Pío XII, 55, 31008 Pamplona, Navarra, Spain. Tel.: +34 948 194700; fax: +34 948 194717. E-mail address: [email protected] (J.J. Lasarte). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.08.070

the development of procedures to target antigens to DC in vivo to favour cross-presentation of antigens would greatly improve vaccination protocols [12–17]. Some of the most efficient PRRs-mediated signals for the maturation of DCs are triggered by the interactions of Toll-Like Receptors (TLRs), with their respective ligands (reviewed in [18]). The recognition of ligands by TLRs gives rise to a quick activation of the innate immunity upon the induction of proinflammatory cytokines and the upregulation of costimulatory molecules and thus favours the activation of an efficient adaptive immunity. In 1999, Mehlin et al. identified an inflammatory polypeptide complex from the Grampositive bacteria Staphylococcus epidermidis with proinflammatory activities. This complex was termed phenol-soluble modulins (PSM) and it is composed of at least 4 peptides named modulins PSM␣, PSM␤, PSM␥ and PSM␦ of 22, 44, 25 and 23 amino acids in length, respectively [19,20]. This complex strongly activates the production of TNF-␣, IL-1␤ and IL-6 by monocytes. Since TLR2 transduces the response to phenol-soluble modulin [21], it is likely that PSM binds to TLR2. Thus, we speculated that the production of fusion polypeptides containing the individual PSM peptides linked to an antigen might constitute an efficient strategy to target antigens to TLR2-expressing DC in vivo. In the present study, we demonstrate that synthetic PSM␣ (␣Mod) and PSM␥ (␥Mod) peptides linked to an antigen can favour its capture by antigen presenting cells (APC) and, when injected with an adjuvant, induce

M. Durantez et al. / Vaccine 28 (2010) 7146–7154 Table 1 Synthetic peptides used. Peptide abbreviation

Sequence

␣Mod ␥Mod SIINFEKL OVA(235–264) CF-OVA(235–264) ␣Mod-SIINFEKL SIINFEKL-␣Mod ␣ModScramble-SIINFEKL ISQ-SIINFEKL CF-␣Mod-SIINFEKL ␥Mod-SIINFEKL SIINFEKL-␥Mod CF-␥Mod-SIINFEKL ␣Mod-E7(49–57) E7(49–57) p1073–1081 ␣Mod-1073 ␥Mod-1073

MADVIAKIVEIVKGLIDQFTQK MAADIISTIGDLVKWIIDTVNKFKK SIINFEKL ASGTMSMLVLLPDEVSGLEQLESIINFEKL CF-ASGTMSMLVLLPDEVSGLEQLESIINFEKL MADVIAKIVEIVKGLIDQFTQKSIINFEKL SIINFEKLMADVIAKIVEIVKGLIDQFTQK IGLIIEQIQAMDAKDVFVKTVKSIINFEKL ISQAVHAAHAEINEAGRSIINFEKL CF-MADVIAKIVEIVKGLIDQFTQKSIINFEKL MAADIISTIGDLVKWIIDTVNKFKKSIINFEKL SIINFEKLMAADIISTIGDLVKWIIDTVNKFKK CF-MAADIISTIGDLVKWIIDTVNKFKKSIINFEKL MADVIAKIVEIVKGLIDQFTQKRAHYNIVTF RAHYNIVTF CVNGVCWTV MADVIAKIVEIVKGLIDQFTQKCVNGVCWTV MAADIISTIGDLVKWIIDTVNKFKKCVNGVCWTV

The underlined sequence defines the corresponding cytotoxic T cell epitope within the peptide.

potent CTL responses that can cure mice of a transplantable tumor. Notably, we have found that this ability of modulin-derived peptides was not related to TLR2, but dependent, at least partially upon the epidermal growth factor receptor signalling pathway. 2. Materials and methods 2.1. Mice Female C57BL/6 mice, 6–8 weeks old, from Harlan (Barcelona, Spain), female TLR2-KO mice bred onto a C57BL/6 background, and kindly given by M. Chignard (Pasteur Institute) were housed in appropriated animal care facilities during the experimental period and handled following the international guidelines required for experimentation with animals. 2.2. Peptides Peptides from Table 1, corresponding to: ␣Modulin and ␥Modulin from S. epidermidis, the immunodominant H-2b restricted CTL epitope SIINFEKL (amino acids 257–264 from chicken ovalbumin), peptide OVA(235–264) corresponding to a N-terminus extended version of SIINFEKL, as well as combinations of ␣ and ␥ modulins with SIINFEKL, were purchased from PolyPeptide (Strasbourg, France). The peptide ␣ModScramble-SIINFEKL, containing a scrambled version of the ␣Mod sequence linked to the SIINFEKL epitope, the peptide ISQAVHAAHAEINEAGR-SIINFEKL, containing the T helper epitope OVA (323–339) linked to SIINFEKL epitope (ISQ-SIINFEKL), the peptide RAHYNIVTF corresponding to the HPV16-E7(49–57) H2-Db -restricted epitope [22], the peptide ␣Mod-E7(49–57) (containing ␣Modulin and E7(49–57) epitope), the peptides ␣Mod-1073 and ␥Mod-1073 (containing the HLA-A2restricted cytotoxic T-cell determinant p1073–1081 from hepatitis C virus NS3 protein[23]) as well as the carboxyfluoroscein (CF) labeled peptides from Table 1 were synthesized manually as previously described [24]. Carboxyfluoroscein succinimidyl ester (CFSE; Molecular Probes) was coupled to the N-terminus of the peptides as a final step of peptide synthesis. The purity of the peptides was analyzed by HPLC. The levels of endotoxin were below the limit of detection when tested by Quantitative Chromogenic Limulus Amebocyte Lysate assay (Cambrex, Walkersville, MD, USA). 2.3. Binding assays Splenocytes were isolated from C57BL/6 or TLR2-KO naive mice, fixed with glutaraldehyde solution (0.05% in PBS) for 10 min at

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37 ◦ C. Then, cells were incubated for 30 min at 4 ◦ C with the CFlabeled peptides at a final concentration of 6–8 ␮g/ml. After two washes with PBS, cells were analyzed by flow cytometry. For double staining experiments, cells were previously incubated with antiCD4 (Clone H129.19), -CD8 (Clone 53-6.7), -CD11c (Clone HL-3), -CD19 (Clone 1D3), -F4/80 (Clone GF12) (all from BD-Biosciences, San Diego, CA), or anti-GR-1 (Clone RB6-8C5, eBiosciences, San Diego, CA) antibodies labeled with phycoerythrin (PE), then fixed and incubated with CF-labeled peptides and analyzed by flow cytometry. BMDC or CD11c+ purified dendritic cells from C57BL/6 mice were also used in these binding experiments with CFlabeled peptides as described above. When indicated, 100 ␮g/ml of ␣Mod-SIINFEKL, ␥Mod-SIINFEKL or SIINFEKL peptides, or 7 ␮M of amphiregulin, epiregulin, HB-EGF or ovalbumin were added simultaneously to the CF-labeled peptides to inhibit their binding. 2.4. In vitro analysis of DC maturation BMDC were generated from mouse femur marrow cell cultures as previously described [5]. Non-adherent dendritic cells were harvested at day 7 and cultured in the presence/absence of different stimuli at 37 ◦ C and 5% CO2 . After 48 h of culture, supernatants were harvested and IL-12 (p70) and TNF-␣ were measured by ELISA (BD-Pharmingen), according to manufacturer’s instructions. Alternatively, after 14 h of culture of BMDC with the different stimuli, total RNA was purified, and after DNase treatment, reverse transcription and quantitative real-time PCR for IL-12 (p40) and TNF-␣ was performed as previously described [25] using primers AGATGAAGGAGACAGAGGAG and GGAAAAAGCCAACCAAGCAG for IL-12 and CTTCCAGAACTCCAGGCGGT and GGTTTGCTACGACGACGTGGGC for TNF-␣. mRNA values were represented by the formula: 2Ct , where Ct indicates the difference in threshold cycle between control (␤-actin, amplified using primers CGCGTCCACCCGCGAG and CCTGGTGCCTAGGGCG) and target genes. Expression of DC maturation markers was measured by flow cytometry (BD Biosciences) as described [5]. 2.5. In vitro assays for antigen presentation BMDC were cultured in presence/absence of the indicated stimuli. After 48 h of culture, DC were collected and plated in 96-well plates at different cell concentrations and used as APC in the presence of 105 non-adherent cells/well from OT-1 transgenic mice. After 2 days of culture, cells were pulsed with 0.5 ␮Ci/well of [3 H]Thymidine for 8 h. They were then harvested and radioactivity was measured using a scintillation counter (Topcount, Packard, Meriden, CT). 2.6. In vivo killing and ELISPOT assays C57BL/6 wt or TLR2-KO mice were immunized i.v. with 200 ␮l of a saline solution containing 5 nmol of the indicated peptide mixed with or without poly(I:C) (50 ␮g/mice). In some experiments, peptides were mixed with other TLR ligands as adjuvant. Seven days or 60 days after immunization, CTL activity was measured by an in vivo killing assay as previously described [26]. In some cases, mice were immunized with 50 nmol OVA (2.2 mg/mice) plus poly(I:C) by i.v. route. In some experiments, mice were treated orally with erlotinib (Roche Products Ltd. UK) (100 mg/kg) in 0.5% methylcellulose as indicated. T-cells producing IFN-␥ were enumerated by ELISPOT using a kit from BD Biosciences (San Diego, CA) according to manufacturer instructions culturing 8 × 105 splenocytes in the absence/presence of SIINFEKL (10 ␮M) or E7(49–57) (10 ␮M). The numbers of spots were counted using an automated ELISPOT reader (CTL, Aalen, Germany).

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2.7. Tumor rejection experiments Mice were injected i.v. at day 0 with the indicated immunogen or with saline alone. One week later, mice were challenged s.c. with 105 EG7-OVA cells. For treatment of established tumors, mice received 5 × 105 TC-1 cells and at day 16, once the tumors were around 8 mm in diameter, and at day 23 they received an i.v. injection of saline or the indicated immunogen. Tumor size was monitored twice a week with a calliper. Mice were sacrificed when the mean tumor diameter was greater than 20 mm. Kaplan–Meier plot of mice survival for each immunogen is depicted. 2.8. Statistical analysis Normality was assessed with Shapiro–Wilk W test. Statistical analyses were performed using parametric (Student’s t-test and one-way ANOVA) and non-parametric (Kruskal–Wallis and Mann–Whitney U) tests. For all tests a p value <0.05 was considered statistically significant. Descriptive data for continuous variables are reported as means ± SEM. SPSS 9.0 (SPSS Inc., Chicago, IL, USA) for Windows was used for statistical analysis. 3. Results 3.1. Alpha and gamma derived modulin peptides bind to antigen presenting cells One of the main features allowing an immunogen to induce an efficient immune response is its capacity to be efficiently cap-

tured by APC. We speculated that PSM peptides derived from S. epidermidis may have this capacity, since they activate the TLR2 signalling pathway [21], a molecule expressed at the surface of DC [27]. However, the binding of PSM peptides to TLR2 or to DC has not been yet analyzed. Thus, to address this issue, we synthesized the following CF labeled peptides: CF-␣Mod-SIINFEKL and CF-␥Mod-SIINFEKL, containing respectively the alpha or gamma modulin sequence bound to the CD8+ T-cell epitope SIINFEKL from ovalbumin (OVA(257–264)), and CF-OVA(235–264) peptide, an ovalbumin derived 30-mer peptide containing SIINFEKL at the C-terminus (Table 1). Flow cytometry studies showed that both CF-␣Mod-SIINFEKL and CF-␥Mod-SIINFEKL peptides bind to splenocytes (Fig. 1A and B) as well as to BMDC (Fig. 1C). Indeed, mouse splenocytes incubated with CF-␣Mod-SIINFEKL showed higher fluorescence intensity than splenocytes incubated with the control peptide CF-OVA(235–264). This binding was significantly inhibited by the unlabeled ␣Mod-SIINFEKL peptide, but not by the SIINFEKL peptide (Fig. 1A), suggesting that this peptide interaction with splenocytes requires ␣-modulin. The binding capacity of CF␣Mod-SIINFEKL was also observed with BMDC or freshly isolated splenic CD11c+ cells (Fig. 1C and data not shown respectively). Similar results were found with the CF-␥Mod-SIINFEKL peptide. In this case, two fluorescence peaks, with low and bright fluorescence, were observed. The fluorescence obtained with the CFSE labeled irrelevant peptide, CF-OVA(235–264), was similar to the low fluorescent peak observed with CF-␥Mod-SIINFEKL (Fig. 1B), suggesting that this second peak was due to non-specific binding. Incubation of splenocytes with the unlabeled ␥Mod-SIINFEKL peptide signifi-

Fig. 1. PSM peptides bind to mouse splenocytes and BMDC. Splenocytes (A and B) or BMDC (C) from C57BL/6 mice were fixed with glutaraldehyde and incubated with either CF-␣Mod-SIINFEKL (A and C), CF-␥Mod-SIINFEKL (B and C) or the control peptide CF-OVA(235–264) (A–C). Gray histograms represent non-labeled cells. To study the specificity of binding of modulin peptides, cells were incubated with CFSE labeled peptides in the presence of unlabeled ␣Mod-SIINFEKL (A), ␥Mod-SIINFEKL (B) or SIINFEKL (A and B) peptides. After 30 min at 4 ◦ C, cells were analyzed by flow cytometry. (C) Double staining of splenocytes with CF-␥Mod-SIINFEKL peptide and the indicated PE-labeled antibodies. The bottom histograms of panel C indicate the percentage of positive cells for these surface markers which are stained with the CF-␥Mod-SIINFEKL peptide. Results are representative of three independent experiments.

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Fig. 2. PSM peptides induce maturation of BMDC, improve antigen presentation and induce CTL responses. (A) Expression of mRNA encoding IL-12 (p40) or TNF-␣. C57BL/6 BMDC were cultured for 14 h with LPS (1 ␮g/ml) or the indicated peptides (50 ␮M) and mRNA for IL-12 (p40) and TNF-␣ was measured by real-time PCR. (B) Expression of DC maturation markers. C57BL/6 BMDC were cultured in the presence of the indicated peptides (50 ␮M). After 48 h of culture, cells were analyzed by flow cytometry using specific antibodies. (C) In vitro assay for antigen presentation. BMDC were cultured in the presence of the indicated peptides (10 ␮M). Forty eight hours later, the cells were washed and distributed in 96-well plates at different concentrations and incubated in the presence of 105 T-cells/well from transgenic OT-1 mice. Forty eight hours later, T-cell proliferation was measured by measuring 3 H-thymidine incorporation. * means a p value <0.05. (D) In vivo induction of specific CTL responses. C57BL/6 mice were immunized i.v. with 50 nmol of the indicated peptide. The in vivo induction of SIINFEKL-specific CTL responses was measured by an in vivo killing assay. Results are representative of three independent experiments.

cantly reduced the bright fluorescence. However, incubation with the SIINFEKL peptide did not affect this signal, indicating that the binding of the peptide was due to the presence of the ␥-modulin moiety. Similar to CF-␣Mod-SIINFEKL, CF-␥Mod-SIINFEKL peptide was able to bind to BMDC (Fig. 1C) and also to freshly isolated splenic CD11c+ cells (data not shown). Double stainings were then carried out using the CF-␥ModSIINFEKL peptide and anti-CD4, CD8, CD11c, CD19, F4/80 or GR-1 phycoerythrin-labeled antibodies (Fig. 1D). The CF-␥Mod-SIINFEKL peptide was found to bind to 24.4% of CD4+ cells, 17.8% of CD8+ cells, 62.1% of CD11c+ cells, 52.9% of CD19+ cells, 66.2% of F4/80+ cells and 27.3% of GR-1+ cells. Experiments carried out with the CF-␣Mod-SIINFEKL peptide (Supplementary Fig. 1), and with CF␥Mod-SIINFEKL indicated that both ␣-modulin and ␥-modulin bind to various cell subtypes and in particular to APC, a property that can be exploited to target antigens to these cells. 3.2. ˛-Modulin and -modulin peptides induce maturation of dendritic cells and increase antigen presentation PSM peptides derived from S. epidermidis may activate cytokine production in mononuclear phagocytes [19]. Thus, we examined whether ␣Mod and ␥Mod peptides were able to stimulate BMDC to produce proinflammatory cytokines. When BMDC were cultured in the presence of these peptides for 14 h, ␣- and ␥-modulins were able to increase in a lesser extend than LPS, but significantly, the expression of mRNAs for IL-12 (p40) and TNF-␣ cytokines (Fig. 2A). However, no significant levels of IL-12 or TNF-␣ were detected in the culture supernatant of these cells after 48 h of culture (data not shown).

Incubation of BMDC with ␣Mod increased the expression of CD54, CD86 and MHC class II molecules, whereas ␥Mod was slightly less efficient and promoted only the expression of CD54 and MHC class II molecules (Fig. 2B). These results suggest that modulins might promote antigen-specific immune response by stimulating, in some degree, DC maturation. We then analyzed whether covalent linkage of an antigen to ␣Mod or ␥Mod peptides could facilitate the presentation of this antigen to specific T-cells. BMDC were incubated with 10 ␮M of either ␣Mod-SIINFEKL, ␥Mod-SIINFEKL, OVA(235–264), ␣Mod, ␥Mod or SIINFEKL and used as APC to activate the proliferation of OT-1 transgenic T lymphocytes (carrying a specific T-cell receptor for the SIINFEKL epitope). The highest proliferation of OT-1 T lymphocytes was observed with DC incubated with SIINFEKL alone, which does not require antigen processing and can bind directly to MHC class I molecules at the DC surface. Interestingly, when we pulsed DC with longer peptides (30–33 mer), which require antigen processing, we found that ␣Mod-SIINFEKL and ␥Mod-SIINFEKL peptides stimulated higher OT-1 T-cell proliferation than the OVA(235–264) peptide (p < 0.05) suggesting that modulin moieties have a beneficial effect on antigen presentation (Fig. 2C). 3.3. Immunization with ˛Mod-SIINFEKL or Mod-SIINFEKL peptide in combination with TLR3 or TLR9 ligand induces a cytotoxic T-cell response specific for the SIINFEKL peptide We next tested whether mice immunized with ␣Mod-SIINFEKL or ␥Mod-SIINFEKL peptide in the absence of any exogenously added adjuvant developed OVA-specific T-cell responses. Thus,

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Fig. 3. In vivo induction of CTL responses by modulin-derived peptides and TLR ligands. C57BL/6 were immunized with 5 nmol of the indicated peptide with 50 ␮g of poly(I:C) (A and B) or with the indicated TLR ligand (C): PGN (peptidoglycan, 50 ␮g/mice), poly(I:C), EDA (extra domain A from fibronectin, 50 ␮g/mice), imiquimod or CpG/DOTAP (30 ␮g/60 ␮g). Seven (A and C) or 60 days (B) after immunization, mice were sacrificed and the specific cytotoxic activity against the SIINFEKL peptide was measured by an in vivo killing assay. Results are representative of 2–3 independent experiments.

C57BL/6 mice were immunized twice with 50 nmol/mouse of ␣Mod-SIINFEKL, ␥Mod-SIINFEKL or SIINFEKL in saline. Seven days after the second immunization, we measured the SIINFEKL-specific CTL response by an in vivo killing assay. Although immunization with ␣Mod-SIINFEKL was able to induce a significant CTL activity, as compared to immunization with SIINFEKL or ␥ModSIINFEKL peptide (Fig. 2D), this response was not able to protect mice against a challenge with 5 × 105 EG7-OVA tumor cells (data not shown). We then explored whether immunization with modulinderived peptides, in the presence of different adjuvants, was able to induce efficient CTL priming. C57BL/6 mice were immunized i.v. with 5 nmol of the indicated peptides mixed with 50 ␮g poly(I:C). As shown in Fig. 3A, immunization of mice with ␣ModSIINFEKL, SIINFEKL-␣Mod, ␥Mod-SIINFEKL or SIINFEKL-␥Mod in the presence of poly(I:C) induced strong cytotoxic T-cell responses. However, ␣ModScramble-SIINFEKL (containing a scrambled version of the ␣Mod sequence linked to the SIINFEKL epitope), ISQ-SIINFEKL (containing the T helper epitope OVA(323–339)), SIINFEKL or OVA(235–264) peptides were not capable of activating T-cell responses, as well as the combination of ␣Mod or ␥Mod peptides with SIINFEKL. Similar results were found when we measured the number of IFN-␥ producing cells specific for SIINFEKL peptide by ELISPOT (Supplementary Fig. 2A). The capacity of these immunogens to activate a long-lasting response was also studied. It was found that two months after immunization, mice immunized with either ␣Mod-SIINFEKL or ␥Mod-SIINFEKL peptides still showed significant OVA-specific T-cell responses (Fig. 3B and Supplementary Fig. 2B). The capacity of other TLR ligands, in combination with modulinderived peptides, to enhance activation of a cytotoxic T-cell response against the SIINFEKL peptide was also studied. As shown in Fig. 3C, immunization of mice with modulin-derived peptides together with TLR3 and TLR9 ligands, and to a lesser extent with TLR4 or TLR7 ligands, induced strong CTL responses. Similar results were found when we measured the number of IFN-␥ producing cells specific for SIINFEKL peptide by ELISPOT (not shown).

3.4. Immunization with ˛Mod or Mod derived peptides in combination with poly(I:C) allows tumor rejection We then evaluated the capacity of modulin peptides carrying the OVA CTL epitope to protect mice from challenge with EG7OVA tumor cells. Mice were immunized with 5 nmol of various peptides with poly(I:C) and 7 days after immunization, 5 × 105 EG7OVA cells were injected subcutaneously. Whereas all mice injected with SIINFEKL, poly(I:C) or saline developed tumors, immunization with ␣Mod-SIINFEKL or ␥Mod-SIINFEKL peptide (Fig. 4A) induced 100 and 90% of protection of mice against tumor growth, respectively. To better evaluate the potency of modulin-derived peptides as anti-tumoral vaccines, we synthesized an ␣Mod-E7(49–57) peptide, which contains the cytotoxic T-cell epitope 49–57 from human papilloma virus E7 protein, and studied its therapeutic efficacy in mice grafted with the HPV16-E7-expressing tumor cells TC-1. We found that immunization with ␣Mod-E7(49–57) plus poly(I:C) induced high numbers of IFN-␥ producing cells specific for the E7(49–57) peptide (Fig. 4B). To assay the therapeutic activity of this peptide, C57BL/6 mice were injected s.c. with 5 × 105 TC1 cells and, when tumor mean diameters were around 8 mm (day 16) and 1 week later (day 23), they were treated with saline, ␣Mod-SIINFEKL (as a control peptide), peptide E7(49–57) or ␣Mod-E7(49–57) plus poly(I:C). We found that, whereas treatment with peptide E7(49–57) cured only 36% of mice, 11 out of 11 (100%) animals treated with ␣Mod-E7(49–57) rejected the tumor (Fig. 4C). 3.5. Covalent linkage between modulin peptides and the p1073 cytotoxic T-cell epitope from hepatitis C virus NS3 protein allows the induction of strong T-cell responses against the epitope To extend our findings to another immunogen and to evaluate the potential use of modulin-derived peptides for an anti-viral vaccine, we synthesized modulin peptides linked to a well characterized cytotoxic T-cell determinant from the non-structural protein NS3 from hepatitis C virus, the HLA-A2-restricted NS3 pep-

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Fig. 4. Induction of anti-tumor immunity. (A) C57BL/6 mice were immunized i.v. with 5 nmol of the indicated peptide in combination with 50 ␮g of poly(I:C). Control groups received saline or poly(I:C) alone. Seven days after immunization, 5 × 105 EG7-OVA tumor cells were injected subcutaneously. Mice were sacrificed when at least one of the tumor diameters exceeded 2 cm. Kaplan–Meier plot of mice survival for each treatment is depicted. (B) C57BL/6 were immunized with 5 nmol of the indicated peptide and 50 ␮g of poly(I:C). Seven days later, T-cell responses specific for the peptide E7 (49–57) were assayed by an ELISPOT to measure IFN-␥ producing cells. (C) C57B/6 mice were injected with 5 × 105 TC-1 cells and 16 and 23 days later, mice were treated intravenously with saline, ␣Mod-SIINFEKL plus poly(I:C), E7(49–57) peptide plus poly(I:C) or with ␣Mod-E7(49–57) plus poly(I:C). Tumor size, presented as the average of two perpendicular diameters, was measured at regular intervals. The number of mice free of tumors on day 70 relative to the total number of animals included is indicated. Results are representative of two independent experiments.

tide 1073–1081 [23]. HHD transgenic mice (which express the human HLA-A2 molecule [28]) were immunized with 5 nmol of ␣Mod-1073, ␥Mod-1073 or p1073 in the presence of poly(I:C). Seven days after immunization, responses were analyzed by ELISPOT and in vivo killing assay using target cells pulsed with peptide 1073. As shown in Fig. 5, immunization with ␣Mod or ␥Mod coupled to peptide 1073 allowed the induction of strong T-cell immune responses against the peptide 1073, whereas only low Tcell responses were observed in mice immunized with the peptide 1073 and poly(I:C).

3.6. The capacity of modulin-derived peptides to induce T-cell responses is independent upon the activation of the TLR2 pathway PSM peptides derived from S. epidermidis were shown to activate the TLR2 signalling pathway [21]. Thus, to determine if the immunogenicity of modulin peptide constructs depends upon this pathway, we immunized C57BL/6 wt and TLR2-KO mice with modulin-derived peptides. As expected, immunization of wild type C57BL/6 mice with ␣Mod-SIINFEKL or ␥Mod-SIINFEKL induced strong cytotoxic T responses against target cells incubated with the SIINFEKL peptide. Interestingly, similar responses were observed in mice lacking TLR2 (Fig. 6A and Supplementary Fig. 3), suggesting that the TLR2 pathway is not responsible for the capacity of modulin-derived peptides to favour the induction of T-cell responses against the SIINFEKL epitope. Moreover, binding assays using the CF labeled peptides showed that both CF-␣ModSIINFEKL and CF-␥Mod-SIINFEKL peptides retained the capacity to bind to the surface of TLR2-KO splenocytes (Fig. 6B) suggesting that this binding takes place via a receptor molecule other than TLR2. 3.7. Inhibition of EGF-receptor signalling pathway affects the immunomodulatory activity of modulin-derived peptides

Fig. 5. In vivo induction of T-cell responses against the hepatitis C NS3 peptide p1073 by ␣ or ␥-derived modulin peptide linked to p1073. C57BL/6 were immunized with 5 nmol of the indicated peptide and 50 ␮g of poly(I:C). Seven days later, T-cell responses specific for the peptide p1073 (1073) from hepatitis C NS3 protein were assayed by an ELISPOT for IFN-␥ producing cells (A) and by an in vivo killing assay using target cells pulsed with peptide p1073 (B). Results are representative of two independent experiments.

Umezawa et al. previously demonstrated that ␦-hemolysins are able to inhibit binding of EGF to its receptor [29]. Since the homology of ␦-hemolysins and modulin-derived peptides is very high (see alignment between different modulin-derived peptides and delta lysins on Supplementary Fig. 4), we tested the capacity of various EGF-receptor ligands to inhibit the binding of modulinderived peptides to the surface of splenocytes. We observed that amphiregulin (AR) (Fig. 7A and B), and in a lesser extent, epireg-

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Fig. 6. Activation of CTL responses by modulin-derived peptides in TLR2-KO mice. C57BL/6 wild type or TLR2-KO mice were immunized i.v. with 5 nmol of the indicated peptide and 50 ␮g of poly(I:C). Seven days after, mice were sacrificed and the specific cytotoxic activity against the SIINFEKL peptide induced after immunization with the different peptides (A) was measured by an in vivo killing assay. (B) Binding assays of CF-␣Mod-SIINFEKL or CF-␥Mod-SIINFEKL peptide to splenocytes from TLR2-KO mice. Cells were labeled as in Fig. 1. As control, cells were stained with the labeled control peptide CF-OVA(235–264) or left unlabeled (gray histogram). Results are representative of two independent experiments.

ulin and HB-EGF (data not shown), but not ovalbumin, were able to significantly reduce the binding of both CF-␣Mod-SIINFEKL and CF-␥Mod-SIINFEKL peptides to splenocytes (Fig. 7A and B). This result prompted us to study the in vivo effect of erlotinib, a tyrosine kinase inhibitor of the EGF-R signalling pathway, on the capacity of modulin-derived peptides to induce a CTL response against SIINFEKL. Thus, C57BL/6 mice were immunized on day 0 with ␣Mod-SIINFEKL or ␥Mod-SIINFEKL and poly(I:C) and treated orally with PBS or with erlotinib (100 mg/kg) from days −1 to 4. A group of mice immunized with 50 nmol of ovalbumin and poly(I:C) was also included to discard a potential immunosuppressive activity of erlotinib treatment. Administration of erlotinib did not affect the capacity of OVA administered with poly(I:C) to induce CTL responses (Fig. 7C). In contrast, erlotinib was able to inhibit significantly the in vivo induction of CTL responses by ␣Mod-SIINFEKL or ␥Mod-SIINFEKL.

4. Discussion In the present study, we analyze whether PSM␣ and PSM␥ peptides from S. epidermidis could be used to deliver antigens to APC for optimal induction of T-cell responses. We synthesized hybrid peptides containing PSM␣ or PSM␥ and the well characterized SIINFEKL CTL epitope from ovalbumin and tested their capacity to bind to APC, activate their maturation, improve antigen presentation and induce specific T-cell responses in vivo. Flow cytometry studies carried out with CF-␣Mod-SIINFEKL and CF-␥Mod-SIINFEKL showed that these constructs are able to bind specifically to several splenocyte subpopulations and, in particular to APC, a property that allows antigen targeting to these cells. We also demonstrated that synthetic modulin peptides induced the upregulation of costimulatory molecules, as well as the expression of mRNA for IL-12 and TNF-␣ in BMDC, suggesting that monomeric

Fig. 7. Implication of EGF-R in the induction of CTL responses by modulin-derived peptides. Binding assays of CF-␣Mod-SIINFEKL (A) or CF-␥Mod-SIINFEKL (B) peptide in the presence/absence of the EGF-R ligand amphiregulin or OVA protein. Splenocytes from C57BL/6 mice were fixed with glutaraldehyde and incubated with either CF␣Mod-SIINFEKL or CF-␥Mod-SIINFEKL peptide in the presence or absence of amphiregulin (AR) or OVA protein. Gray histograms represent non-labeled cells. After 30 min of incubation at 4 ◦ C, cells were analyzed by flow cytometry. (C) Effect of erlotinib on the induction of CTL responses. At day 0, C57BL/6 mice were immunized with ␣ModSIINFEKL or ␥Mod-SIINFEKL peptide and poly(I:C) and treated or not by i.g administration of erlotinib at days −1, 0, 1, 2, 3 and 4. As control, a group of mice was immunized with 50 nmol OVA and poly(I:C). Results are representative of three independent experiments.

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modulin-derived peptides may induce some degree of DC maturation. Antigen presentation by DC is a critical step for the induction of an Ag-specific T-cell immune response, and thus, a vaccine candidate should be able to improve this process. We found that proliferation of OT-1 T-cells was enhanced by DC pulsed with ␣Mod-SIINFEKL or ␥Mod-SIINFEKL peptides as compared to the OVA(235–264) peptide. These results suggest that linkage of SIINFEKL to ␣/␥-modulins may favour targeting and/or antigen presentation, leading to an increased number of Kb -SIINFEKL complexes at the surface of DC and thus to an improved proliferation of T lymphocytes. Having shown that ␣/␥-Mod derived peptides are able to bind to the surface of dendritic cells, activate their maturation and improve antigen presentation in vitro, we then tested their capacity to induce in vivo a CTL response against the linked antigen. When mice were immunized with ␣Mod-SIINFEKL in the absence of adjuvant, only a weak OVA-specific immune response was detected. This result suggests that PSM-derived peptides are not capable to induce a complete DC maturation to induce full activation of T-cells. Similar results have been observed with fusion proteins, such as the recently described NK lectin group receptor 1 (DNGR1) conjugated with SIINFEKL [17], or the anti-DEC205 antibody coupled to ovalbumin, able to deliver the Ag to DC but unable to induce their maturation and consequently leading to tolerance [30]. However, combination of these fusion proteins with exogenous maturation stimuli induced strong cellular immune responses against the antigen [17,30]. Immunization of mice with both ␣Mod or ␥Mod derived peptides, in combination with the doublestranded RNA poly(I:C), an activator of TLR3 [31], induced strong T-cell responses specific for SIINFEKL whereas the SIINFEKL peptide, or the OVA(235–264) peptide, injected with poly(I:C) was not able to induce detectable responses. Remarkably, immunization of mice with ␣Mod-SIINFEKL or ␥Mod-SIINFEKL in combination with poly(I:C) was able not only to protect mice against challenge with EG7-OVA tumor cells, but also to induce the eradication of already established tumors. This capacity of modulin-derived peptides to elicit an immune response against a linked CTL epitope, and to reject established tumors was demonstrated with peptide E7(49–57), a well characterized H2-Db cytotoxic T-cell determinant from HPV16-E7 protein [22]. We also confirmed the immunomodulatory activity of modulin-derived peptides using peptide p1073, another well characterized HLA-A2 restricted cytotoxic T-cell determinant from hepatitis C virus NS3 protein. CpG (a TLR9 ligand) and to a lesser extent EDA (TLR4) and imiquimod (TLR7), but not peptidoglycan (TLR2), were also shown to promote CTL responses induced by modulin-derived peptides. We do not have a clear explanation for these differences in synergy between some adjuvants and modulin-derived peptides. Tentatively, it may be speculated that those TLR able to induce production of type I IFN (TLR3, TLR9 and TLR4, reviewed in [32]) are able to synergize with PSM-derived peptides. Indeed, it has been recently described that IFN-␣/␤ signalling is crucial on the ability of TLR3-L and TLR9-L to prime CTLs, although IFN-␣/␤ derived signals alone are insufficient for the induction of fully efficient CTL in the absence of CD4+ T-cell help [33]. Further studies are needed to elucidate the mechanisms responsible for these synergistic effects. It has been described that PSM peptides are TLR2 agonists, however we have found that the immunomodulatory capacity of modulins was not dependent upon signalling through TLR2. Moreover, the binding capacity of modulin-derived peptides was also comparable in splenocytes from normal and TLR2-KO mice. We do not have a clear interpretation for this discrepancy, however, it could be speculated that the peptide complex obtained after hot aqueous phenol extraction from S. epidermidis might contain other modulins responsible for TLR2 signalling. Alternatively, it

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has been suggested that PSM peptides produced by S epidermidis [19] or S. carnosus [20] may form aggregates greater than 10 kD, as occurs for S. aureus ␦-toxin which may form multimeric aggregates with molecular weights in the range of 5000–200,000 kD [34]. These aggregates might elicit a higher degree of cross-linking of their receptors than the PSM-derived synthetic peptides and thus stimulate cytokine production. We found that various ligands for EGF-receptor were able to reduce the binding of modulin-derived peptides to splenocytes. The inhibitory effect of erlotinib, an inhibitor of the EGF-receptor tyrosine kinase, on the capacity of modulin-derived peptides to induce CTL in vivo, may suggest that the activation of EGF-R signalling pathway play an important role in the immunomodulatory properties of these peptides. Further studies are needed to elucidate the fine mechanisms of action of modulins in this process. In summary, the present study has established that covalent linkage of a peptidic antigen to ␣Mod and ␥Mod favours its capture by DC and its presentation to T-cells. Immunization by modulinderived peptides, together with a TLR3 or TLR9 ligand, induces strong and long-lasting anti-tumoral protective and therapeutic immune responses. The immunomodulatory capacity of these modulin-derived peptides is independent upon TLR2 but seems to require, at least partially, the activation of the EGF-R signalling pathway. Thus, ␣/␥-modulin peptides may be considered as suitable antigen carriers for the development of anti-tumor or anti-viral vaccines. Acknowledgements This work was supported by grants from Ministerio de Educación y Ciencia (MEC) (SAF2007-61432) and from Gobierno de Navarra to JJ. Lasarte, from MEC to M Durantez, by “UTE project CIMA” and from the Ligue Nationale Contre le Cancer (Equipe Labellisée 2007) to C. Leclerc. We thank Drs M.A. Avila, C Berasain and J Hernandez for their helpful discussion. We also thank to Elena Ciordia and Eneko Elizalde for excellent animal care. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2010.08.070. References [1] Townsend A, Bodmer H. Antigen recognition by class I-restricted T lymphocytes. Annu Rev Immunol 1989;7:601–24. [2] Steinman RM, Banchereau J. Taking dendritic cells into medicine. Nature 2007;449(7161):419–26. [3] Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol 2003;21:685–711. [4] Reis e Sousa C. Dendritic cells in a mature age. Nat Rev Immunol 2006;6(6):476–83. [5] Lasarte JJ, Casares N, Gorraiz M, Hervas-Stubbs S, Arribillaga L, Mansilla C, et al. The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo. J Immunol 2007;178(2):748–56. [6] Cuadros C, Lopez-Hernandez FJ, Dominguez AL, McClelland M, Lustgarten J. Flagellin fusion proteins as adjuvants or vaccines induce specific immune responses. Infect Immun 2004;72(5):2810–6. [7] Tighe H, Takabayashi K, Schwartz D, Marsden R, Beck L, Corbeil J, et al. Conjugation of protein to immunostimulatory DNA results in a rapid, long-lasting and potent induction of cell-mediated and humoral immunity. Eur J Immunol 2000;30(7):1939–47. [8] LeibundGut-Landmann S, Gross O, Robinson MJ, Osorio F, Slack EC, Tsoni SV, et al. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17. Nat Immunol 2007;8(6):630–8. [9] Ahonen CL, Doxsee CL, McGurran SM, Riter TR, Wade WF, Barth RJ, et al. Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN. J Exp Med 2004;199(6):775–84. [10] Wen T, Bukczynski J, Watts TH. 4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function. J Immunol 2002;168(10):4897–906.

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