Transcutaneous immunization in mice - Wiley Online Library

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Jul 28, 2005 - needles, which is a common practice in remote parts of the world, ..... peptide as antigen, which worked best in the previous trial. In par- allel, we investigated if ..... We are grateful to Ms. C. Klein for expert technical assistance. pSPA7235 ... Nishijima T, Tokura Y, Imokawa G, Seo N, Furukawa F, Takigawa.

Int. J. Cancer: 118, 364–372 (2006) ' 2005 Wiley-Liss, Inc.

Transcutaneous immunization in mice: Induction of T-helper and cytotoxic T lymphocyte responses and protection against human papillomavirus–induced tumors Kerstin Dell1*, Robert Koesters2 and Lutz Gissmann1 1 Deutsches Krebsforschungszentrum, Heidelberg, Germany 2 Abteilung Pathologie, Universit€ atsklinik Heidelberg, Heidelberg, Germany Previous reports have shown that transcutaneous immunization (TCI) with proteins or peptides in combination with adjuvants efficiently induces specific cellular and humoral immune responses. However, depending on the kind of skin pretreatment, induction of cellular immune responses was restricted to generation of either specific cytotoxic T lymphocytes (CTLs) or T-helper (Th) cells. In this study, we induced antigen-specific CTL responses together with the appropriate Th responses by TCI of C57BL/6 mice. We applied ovalbumin protein or an ovalbuminderived fusion peptide containing a CTL and Th epitope together with a combination of cholera toxin (CT) and CpG oligodeoxynucleotide (CpG) onto cold wax–depilated and hydrated bare skin. TCI with the ovalbumin fusion peptide induced more robust CTL and Th responses than that with ovalbumin protein. The fusion peptide in combination with the nontoxic CT derivative CTA1D2D1 and CpG induced an antigen-specific CTL response, albeit less efficiently than in combination with complete CT. Further, we compared the potency of HPV-16 E7 oncoprotein–derived peptides containing single (CTL) or multiple (CTL 1 Th 1 B cell) epitopes to induce effective CTL responses. Strong E7-specific CTL responses were detected only after TCI with the E7 multiepitope peptide. This peptide was also shown to protect mice against tumor growth after challenge with HPV-16 E7-positive tumor cells. TCI with E7 protein and CT/CpG led to formation of an E7specific humoral immune response. ' 2005 Wiley-Liss, Inc. Key words: transcutaneous immunization; human papillomavirus type 16 E7 (HPV-16 E7); CTA1-D2D1; cytotoxic T lymphocyte; T helper cell; ovalbumin; tumor protection

TCI is a novel strategy in modern vaccinology. By this application, blood-borne transmission of diseases through the reuse of needles, which is a common practice in remote parts of the world, can be circumvented and better compliance of vaccinees obtained. Apart from its benefits toward vaccine safety, TCI targets LCs, a dense population of highly accessible APCs within the skin.1 An important feature of vaccination against viral infections and virus-based tumors is the induction of CTLs.2–4 Upon activation by APCs, predominantly DCs, CTLs are able to selectively kill antigen-expressing cells.5 For clearance of certain viral infections, the aid of Th cells for CTL activation is necessary.6 The bias to one of the 2 Th subsets (Th1 > Th2) is a prerequisite for specific CTL induction.7 Generation of specific CTLs against proteinaceous or peptidebased vaccines in combination with adjuvants after TS of the skin is possible.8–10 TS leads to removal of the outermost horny layer of the skin, resulting in enhanced permeability and maturation and activation of LCs via inflammatory cytokines secreted from keratinocytes.11,12 The barrier function of the SC can also be bypassed by hydration of the skin, which leads to swelling of the keratinocytes and pooling of fluid into intercellular spaces. This kind of pretreatment of the skin leads to development of humoral13,14 and Th responses after TCI with proteins or peptides.15–17 Another way of antigen entry through the skin is uptake via noncornified epithelial cells of the HF. Wax depilation induces development of homogenous anagen phase, which is accompanied by a locally increased number of LCs.18,19 In contrast to spontaneous anagen development, depilation-induced anagen phase causes the slight Publication of the International Union Against Cancer

inflammatory effect of plucking,20 which is demonstrated by the upregulation of epidermal ICAM-1,21 thus attracting dendritic epidermal T cells and LCs to the HF area.21 Besides skin pretreatment, successful TCI requires coadministration of an adjuvant to induce both cellular and humoral immune responses.22–24 CT, a member of the bacterial ADP-ribosylating exotoxin family secreted by Vibrio cholerae, is one of the most potent adjuvants in TCI. CT is an 86 kDa heterodimer composed of 2 subunits: the toxic, enzymatically active CTA1 with ADPribosyltransferase activity and the nontoxic pentameric CTB oligomer, which binds to the ubiquitous membrane ganglioside GM1.22 Kahlon et al.8 showed that CT as adjuvant and simultaneous pretreatment of the skin by TS generated strong CTL responses following TCI with an OVA-specific, CTL-restricted epitope peptide. CTA1-DD, a nontoxic 37 kDa fusion polypeptide where CTA1 is linked to a dimer of the IgG/IgM/IgA-binding fragment of Staphylococcus aureus, has so far not been tested in TCI. Unmethylated oligodeoxynucleotides containing CpG motifs activate cells of the innate and adaptive immune system via tolllike receptor-9. CpG DNA is a powerful adjuvant with the capacity to induce preferentially Th1 responses and to support the generation of specific CTLs.25 Major cellular targets are macrophages, DCs26 and LCs.27 CpG efficiently stimulates CTL responses when combined with peptides as antigens and is even superior to incomplete Freund’s adjuvant in s.c. peptide vaccination.28 Applied as adjuvant in TCI, CpG supports the generation of specific CTLs in peptide immunization after TS9 and, in combination with CT and after hydration of the skin, biases the cellular immune response toward a Th1-type response.29 HPV-16 E7 is one of the oncoproteins responsible for the transformation of HPV-16-infected ectocervical epithelial cells, which may lead to development of cervical cancer.30,31 Therapeutic vaccination strategies aim at generating systemic CTL-mediated immune responses that affect local cytotoxicity against virusinfected cells.32 So far, TCI with peptides plus adjuvants elicited either CTL responses after TS 8,12 or Th responses after hydration of the skin.29 However, long-term protection against viral infection after vaccination relies on the generation of CTL memory cells, which are preferentially induced by Th cells.33 To simultaneously induce CTL and Th responses by TCI in C57BL/6 mice, we used OVA Abbreviations: APC, antigen-presenting cell; BCIP/NBT, 5-bromo-4chloro-3-indolyl phosphate/nitroblue tetrazolium; CCPM, corrected counts per minute; CT, cholera toxin; CTL, cytotoxic T lymphocyte; DC, dendritic cell; HF, hair follicle; HPV, human papillomavirus; HRP, horseradish peroxidase; ICAM, intercellular adhesion molecule; i.n., intranasal; IPTG, isopropylthiogalactoside; LC, Langerhans cell; MPBS-T, 5% fatfree milk powder in PBS 1 0.05% Tween; OD, optical density; OVA, ovalbumin; PBMC, peripheral blood mononuclear cell; RT, room temperature; SC, stratum corneum; TCI, transcutaneous immunization; Th, T helper; TS, tape-stripping. *Correspondence to: Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Fax : 149-6221-424932. E-mail: [email protected] Received 8 February 2005; Accepted after revision 25 May 2005 DOI 10.1002/ijc.21360 Published online 28 July 2005 in Wiley InterScience (www.interscience.


protein as well as an OVA fusion peptide comprising a CTL and a Th epitope in combination with CT and CpG as adjuvants. Furthermore, the adjuvanticity of a nontoxic CT derivative was analyzed in combination with the OVA fusion peptide as antigen. We show that TCI after cold wax depilation and hydration of the skin with the OVA fusion peptide in combination with CT and CpG as adjuvants leads to strong antigen-specific CTL and Th responses. Replacement of CT by CTA1-D2D1 results in decreased but still significant CTL immune response. TCI with an HPV-16 E7-based multiepitope peptide encompassing a CTL, Th and B-cell epitope in combination with CT and CpG induces a longlasting immune response, which is capable of protecting mice against repeated challenges of HPV-expressing tumor cells. Material and methods Mice C57BL/6 female mice were obtained from Charles River Wiga breeding laboratories (Sulzfeld, Germany). Mice were maintained under pathogen-free conditions and used at 6–8 weeks of age. Cell lines and culture conditions All cell lines were of C57BL/6 origin (H2b). EL-4 thymoma cells34 and RMA cells,35 a Rauscher virus–induced T lymphoma line, were cultured in RPMI-1640 medium supplemented with 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin and 0.01 mM b-mercaptoethanol (complete medium). OVA-transfected EL-4 (E.G7)34 and HPV-16 E7–transfected RMA cells (2F11)36 were cultured in complete RPMI medium supplemented with 400 lg/ml G418 (GIBCO, Paisley, UK). C3 cells derived from embryonic mouse cells transfected with the HPV-16 genome37 were cultured in complete RPMI medium supplemented with 100 lg/ml kanamycin and 800 lg/ml G418. Peptides, proteins and adjuvants Peptides were synthesized using a fully automatic peptide synthesizer (Multisyntec, Witten, Germany) and evaluated by HPLC. For immunizations, an OVA-specific fusion peptide was generated containing both an MHC class I–restricted CTL and an MHC class II–restricted Th-cell epitope. This fusion peptide (OVA257–280, SIINFEKLTEWTSSNVMEERKIKV) mimics the naturally occurring adjacent sequence order of the CTL and Th epitope (H-2Kb MHC class I epitope, 257SIINFEKL264; I-Ab MHC class II epitope, 38 265TEWTSSNVMEERKIKV280 ). The E7 multiepitope peptide (E744–62, QAEPDRAHYNIVTFCCKCD) comprises 3 different epitopes in an overlapping form: the H-2Db MHC class I epitope 49RAHYNIVTF57, the I-A and I-E MHC class II epitope 48DRAHYNIVTF57 and the linear panspecific B-cell epitope 39 Alternatively, mice were immunized with an E7 44QAEPD48. single epitope peptide (E749–57) encompassing only the E7 CTL sequence (49RAHYNIVTF57). All peptides for immunizations were dissolved in PBS/22% (v/v) DMSO. OVA protein (grade V) was purchased from Sigma (Taufkirchen, Germany). E7 his(6)tagged protein produced in Escherichia coli40 was kindly provided by Dr. P. Sehr (Deutsches Krebsforschungszentrum, Heidelberg, Germany). Proteins were dissolved in PBS/DMSO. For in vitro restimulation, mouse spleen cells were incubated either with the OVA-specific CTL (257SIINFEKL264) or Th (265TEWTSSNVMEERKIKV280) epitope or with the E7-specific CTL (49RAHNIVTF57) epitope peptide. Peptides were dissolved in DMSO at 10 mg/ml and further diluted in PBS. Peptides were used at a working concentration of 1 lg/ml (CTL-restricted peptides) or 400 lg/ml (OVA Th-restricted peptide). As adjuvants for TCI, CT was purchased from Sigma and synthetic phosphorothioate–stabilized oligonucleotides 1826 (CpG, 50 -TCC ATG ACG TTC CTG ACG TT-30 ) were obtained from Eurogentec (Seraing, Belgium). For s.c. immunizations, Titermax Gold Adjuvant (Sigma) was used.


Construction of the pT7-CTA1-D2D1 expression vector Domain D (D1) of protein A from S. aureus was amplified by PCR using the D1 primers 50 -TTCGGCCCCAAAGGCCGATGCGCAACAAAA-30 and 50 -AACTCGAGTTATCCATCAGCTTTCGGTGCTTGAGA-30 and plasmid pSPA723541 as template. A second domain D (D2) of protein A from S. aureus was amplified by PCR using the D2 primers 50 -GGAGCTCAAGCTCCAAAAGCTGAT-30 and 50 -AGGCCTTTGGGGCCTGAGATTCGTTTAATTTT-30 . D1 and D2 PCR products both encode identical D domains at the amino acid level but differ by the presence of unique restriction sites to facilitate cloning. D1 and D2 were dimerized by SfiI digestion and ligated; subsequently, D2D1 dimers were digested with SacI and XhoI and cloned into the vector pCRII (Invitrogen, Karlsruhe, Germany). This vector was designated pCR-D2D1. The A1 subunit of cholera toxin was amplified by PCR using CTA1 primers 50 -GGGAAGCTTCATATGGGTAATGATGATAAGTTATATC-30 and 50 -AAAGAGCTCCCGATGATCTTGGAGCATTC-30 and plasmid pRG-CT42 as template. The PCR product with flanking HindIII and SacI sites was cloned into pCRD2D1 at HindIII/SacI. The resulting fusion gene, CTA1-D2D1, was then cut by NdeI/XhoI and inserted into the prokaryotic expression vector pDEST17 (Invitrogen) at NdeI/SalI. This construct was designated pT7-CTA1-D2D1 because it allows expression of recombinant protein in T7-RNA polymerase expressing E. coli cells, e.g., BL21 star cells (Invitrogen). Expression and purification of the CTA1-D2D1 fusion protein For production of the fusion protein, E. coli BL21 Star cells transformed with pT7-CTA1-D2D1 were grown in 1 3 LB medium supplemented with 100 lg/ml ampicillin. Induction with 1 mM IPTG was performed at OD600 nm 5 0.5. Three hours postinduction, cultures were harvested by centrifugation and solubilized using 6 M guanidine-HCl. After addition of distilled water to allow refolding, the fusion protein was purified by affinity chromatography using IgG-Sepharose (Amersham Pharmacia, Freiburg, Germany) as described.43 Immunizations Two days prior to immunization, mice were shaved on a restricted area of about 200 mm2 of the abdomen and depilated with cold wax (Veet; Reckitt Benckiser, Mannheim, Germany) under isoflurane inhalation anesthesia. On the day of immunization, the shaved skin was hydrated by rubbing with waterdrenched gauze and by allowing water to remain for 10 min. The skin was dried with gauze before applying the vaccine. During the immunization procedure, mice were kept under deep ketamine/ rompun anesthesia to prevent grooming for at least 45 min. Ketamine 10% (75 mg/kg) plus rompun 2% (10 mg/kg) was given by i.p. injection. The vaccine (35 ll) was applied onto the bare skin and left for 30 min before removing with tap water. Mice were transcutaneously immunized either with peptides (OVA257–280 and E749–57, 100 lg/mouse; E744–62, 200 lg/mouse) or with proteins (OVA and E7, 100 lg/mouse) dissolved in PBS/DMSO with or without CT (in PBS, 50 lg/mouse) and CpG (in HPLC-purified water, 100 lg/mouse) as adjuvants. For s.c. immunization, peptides were dissolved in PBS, mixed 1:1 with TM according to the manufacturer’s recommendation and applied into the tail base. Mice were immunized 2 or 3 times in biweekly intervals. Spleens were removed 1 week after the last boost. Blood serum samples for evaluation of specific antibody levels were drawn at the day of splenectomy. Tumor protection Mice were immunized transcutaneously 3 times in biweekly intervals either with 200 lg E744–62 peptide and CT/CpG or with CT/CpG alone or were left untreated. Nine days after the last boost, mice were challenged s.c. into the right flank with 5 3 105 C3 cells in 100 ll PBS under isoflurane anesthesia. Tumor growth



was monitored every 6–7 days. Mice with tumor 200 mm were killed. Blood samples for evaluation of the E7-specific CTL frequency in the PBMC population were taken from the tail vein. 3KEDTA served as anticoagulant (per mouse 100 ll 0.369 M 3KEDTA in water; Sigma). PBMCs were pooled and isolated by Ficoll Paque Plus (Amersham Biosciences, Freiburg, Germany) gradient centrifugation according to the manufacturer’s recommendations. 2

Generation of CTL lines After splenectomy, single cell cultures were prepared. During the first 5 days, 2 3 107 spleen cells were cocultured either with 2 3 106 g-irradiated E.G7 (100 Gy) or with g-irradiated 2F11 cells (200 Gy) in 10 ml a-modified minimum essential medium (Sigma) supplemented with 10% FCS, 4 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.1 mM b-mercaptoethanol (CTL medium). For long-term culture, spleen cell cultures were expanded in 24-well plates and maintained in CTL medium supplemented with 25 mM methyl-a-mannopyranoside (Sigma) and with 5% (vol/vol) supernatant from rat spleen cells treated with concanavalin A as a source of IL-2. Once a week, bulk cultures were restimulated with 1 to 2 3 105 g-irradiated transfectants expressing the appropriate antigens as stimulators and 5 3 106 g-irradiated spleen cells (33 Gy) from naive C57BL/6 mice as syngeneic feeder cells per well. Cultures were grown at 37°C and 7.5% CO2 in a humidified incubator. IL-4 and IFN-c Elispot assays The number of OVA- and E7-specific CTLs and OVA-specific Th1 cells was determined by IFN-g and the number of OVA-specific Th2 cells by IL-4 Elispot assay. E7-specific PBMC CTLs were assayed by IFN-g Elispot. Sterile 96-well MultiScreen HA plates (MAHAS4510; Millipore, Eschborn, Germany) were equilibrated with PBS and coated overnight either with an antimouse IFN-g MAb (0.6 lg/well, clone R4-6A2; Pharmingen, Heidelberg, Germany) or with an antimouse IL-4 antibody (0.6 lg/well, clone 11B11; Pharmingen) at 4°C. The next day, antibodies were removed and plates were washed once with PBS. After blocking with complete RPMI medium for 2 hr at 37°C, splenocytes were seeded in serial 2-fold dilutions whereas only single-well approaches were performed with PBMCs. Cells were left untreated or restimulated either with 0.2 lg/well pokeweed mitogen (Sigma) as a positive control, with 0.025 lg/well OVA257–264 CTL epitope or with E749–57 CTL epitope (only IFN-g Elispot) or with 10 lg/well OVA Th epitope (IFN-g and IL-4 Elispot). After 24 (IFN-g Elispot) or 48 (IL-4 Elispot) hr incubation at 37°C and 7.5% CO2, cells were removed and plates were washed 5 times with PBS 1 0.01% Tween and once with PBS followed by incubation with the appropriate secondary antibodies (biotinylated antimouse IFN-g, 0.2 lg/well, clone XMG1.2, or biotinylated anti-IL-4, 0.3 lg/well, clone BVD6-24G2; both from Pharmingen) for 24 hr at 4°C. Subsequently, plates were washed 4 times with PBS, and 100 ll/well streptavidin alkaline phosphatase (diluted 1:500 in PBS, Pharmingen) were added for 2 hr at RT. After washing the plates 4 times with PBS, 75 ll/well BCIP/NBT (Sigma) were added as substrate. After 1–5 min, the reaction was stopped by rinsing the plates with water. Evaluation was performed by counting the spots in a reader (Autoimmundiagnostika GmbH, Strassberg, Germany) Elispot reader. Numbers of specific spots per 106 cells were determined for each mouse by subtracting the number of spots of the internal negative control (cells incubated with medium alone) from the number of spots after incubation with the appropriate antigen-specific peptide. 51

Cr-release assay The ability of CTLs to lyse antigen-presenting cells was analyzed by 51Cr-release assay after 2 or 3 restimulations in vitro. Assays were performed 5 days after the last in vitro restimulation of splenocytes. Ten thousand target cells (E.G7 or 2F11 or the

appropriate parental cells) labeled with 50 lCi Na251CrO4 (Perkin Elmer, Rodgau, Germany) were coincubated with titrated numbers of effector cells (CTLs) in 200 ll complete medium per well on a 96-well round-bottomed tissue culture plate (Nunc, Wiesbaden, Germany) for 1 hr at 37°C. After for 4 hr incubation at 37°C, 50 ll of supernatant from each well were transferred to a 96-well LumaPlate (Packard, Groningen, the Netherlands) and air-dried overnight at RT. Released radioactivity was measured in a Microbeta counter (Wallac, Turku, Finland). Specific lysis of target cells was calculated according to the formula [(release by CTL – spontaneous release)/(total release – spontaneous release)] 3 100. Spontaneous Cr release was determined using 51Cr-labeled target cells in the absence of effector cells, and total release was determined by adding 2% cell-lysing Triton X-100 (Sigma) to target cells in the absence of effector cells. Sample values of >10% lysis activity were scored positive. Lymphoproliferation assay Functional antigen-specific OVA Th-cell responses were measured by [3H]-thymidine incorporation. After splenectomy, single cell suspensions of spleens (4 3 105/well) were added to a 96-well flat-bottomed tissue culture plate (Corning, Corning, NY) in 0.2 ml CTL medium (without IL-2). Cells were cultured either with medium alone or with the appropriate Th-restricted peptide (10 lg/well). After 72 hr at 37°C and 7.5% CO2, 1 lCi [3H]-thymidine per well was added, and incubation continued for another 18 hr at 37°C. Cellular DNA from each well was transferred onto a printed glass fiber filtermat (Wallac) by a cell harvester (Inotech, Wohlen, Switzerland). Subsequently, scintillator sheets (Meltlex A, Wallac) were melted onto the filtermats. b-Irradiation was measured in the microbeta counter. Results are presented as CCPM after peptide restimulation minus CCPM of the internal medium control. Measurement of antibody responses The presence of anti-E7 antibodies in sera of immunized mice was determined by ELISA. Briefly, 96-well polysorb plastic plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with E. coli purified E7 protein (100 ng/well in PBS) or E744-72 peptide (1500 ng/well). After washing with PBS-T, plates were blocked with MPBS-T for 1 hr at 37°C. Following washing with PBS-T, prediluted sera (in 2-fold dilutions starting from 1:25 after protein coating and 1:5 after peptide coating) were added, and plates were incubated for 1 hr at 37°C. After washing, plates were incubated for 1 hr at 37°C with 1:2,500 diluted HRP-coupled antimouse IgG-specific secondary antibody (100 ll/well in MPBS-T; Dianova, Hamburg, Germany). ABTS (1 mg/ml, Sigma) in 100 mM sodium acetate, 50 mM sodium dihydrogen phosphate-monohydrate (pH 4.2, adjusted with acetic acid) and freshly added 30% H2O2 (0.4 ll/ml) was used as substrate. OD was measured in an ELISA reader at 405 nm after 10 min and 30 min incubation at RT. Titers are presented as the log reciprocal value of the highest serum dilution which reached an OD405 nm above 0.03 [5 cut-off: mean OD values of preimmune sera 1 (3 3 SEM)]. Results TCI with an OVA-specific fusion peptide in the presence of CT and CpG induces antigen-specific CTLs and Th cells Other investigators have reported that, after TS or hydration of the skin, either CTLs or Th-cell responses can be induced.8,12,29,44 Using multiepitope peptides in TCI, we show that both arms of the cellular immune response are induced at the same time without removal of the SC of the skin. To establish TCI, we used an OVA-based fusion peptide containing both an OVA CTL and Th epitope and OVA protein as model vaccines. Prior to application of the vaccine, the abdomen of C57BL/6 mice was depilated and the skin hydrated. In 3 independent experiments (comprising a total of 80 mice), one of which



FIGURE 2 – Proliferation of splenocytes of mice immunized two times transcutaneously either with 100 lg OVA protein or 100 lg OVA257–280 peptide in the presence or absence of CT/CpG (50 lg/ 100 lg). Positive controls were mice immunized with 100 lg OVA protein plus TM s.c., negative controls were mice immunized transcutaneously with CT/CpG alone. Cells were stimulated in vitro with the OVA-restricted Th epitope for 4 days. On day 3, [3H]-thymidine was added. Hatched bars represent the CCPM after peptide restimulation from individual mice minus the CCPM of the internal medium control (median values 6 SEM of each group are shown as black bars).

is shown in Figure 1, similar results were obtained. C57BL/6 mice (2–4/group) received twice either 100 lg OVA257–280 fusion peptide or OVA protein with or without CT and CpG. Mice of the positive control group were immunized s.c. with OVA protein plus Titermax; negative controls received only CT and CpG. Antigenspecific CTL, Th1 and Th2 cells were detected ex vivo by IFN-g (CTL, Th1; Fig. 1a,b) and IL-4 (Th2, Fig. 1c) Elispot analysis after incubation of splenocytes with the OVA-specific CTL- or Th-restricted epitope (see Material and methods). In all 3 readouts, TCI with the OVA257–280 fusion peptide plus CT and CpG resulted in the highest number of specifically activated T lymphocytes, even when compared to the positive control (Fig. 1). TCI with OVA protein plus adjuvants was less efficient. Topical application of the OVA257–280 fusion peptide or complete protein without adjuvants reached only levels equivalent to or slightly above those of the negative controls. In general, the CTL-specific assay gave the highest response, followed by the Th1 and Th2 assays. In a separate experiment, antigen-specific Th-cell proliferation was measured by incorporation of tritiated thymidine under conditions of in vitro peptide restimulation (see Material and methods). In accord with the results obtained by the IFN-g and IL-4 Elispot assays, we found the highest proliferation in the group of mice that received the OVA257–280 fusion peptide plus CT and CpG transcutaneously (Fig. 2), followed by the positive control group (OVA protein plus TM s.c.; Fig. 2).

FIGURE 1 – CTL, Th1 and Th2 responses of mice immunized twice with 100 lg OVA protein 6 CT/CpG (50 lg/100 lg), 100 lg OVA257–280 fusion peptide 6 CT/CpG or CT/CpG alone. With the exception of the positive control group (s.c.), immunizations were performed transcutaneously. Results from IFN-g CTL (a), IFN-g Th1 (b) and IL-4 Th2 (c) Elispot assays are shown. Splenocytes were restimulated with either the OVA CTL-restricted epitope (a) or the OVA Th-restricted epitope peptide (b, c). Each hatched bar represents the spot counts per million cells for each mouse (median values 6 SEM are shown as black bars).

Additional boosting induces OVA-specific functional CTLs also in the presence of the nontoxic CT mutant CTA1-D2D1 Even after repeated restimulations of splenocytes in vitro, we failed to detect biologically active CTLs by 51Cr-release assays after 2 TCIs (data not shown). Therefore, we repeated the experiment by applying 3 immunizations with the OVA257–280 fusion peptide as antigen, which worked best in the previous trial. In parallel, we investigated if CT as adjuvant can be replaced by the nontoxic CT derivative CTA1-D2D1 that was generated analogously to the previously published CTA1-DD.45 The basic difference between CTA1-DD and CTA1-D2D1 consists in the spacing of the 2 D domains. In CTA1-DD, there is a 2–amino acid insertion (PE) which is lacking in both the individual Ig binding domains of CTA1-D2D1 and the native protein A of S. aureus.



FIGURE 3 – Cytolytic activity of CTLs after 3 TCIs of mice (n 5 3/ group) with 100 lg OVA257–280 fusion peptide plus adjuvants as indicated (50 lg CT, 50 lg CTA1D2D1, 100 lg CpG). Splenocytes were assayed after 2 in vitro restimulations by 51Cr-release assay. Cytotoxic activities against OVAexpressing EG7 cells vs. the parental EL-4 cells at indicated effector to target ratios were determined. Mean values 6 SEM of all mice of each group are plotted.

CTA1-D2D1 was expressed in E. coli and purified by IgG column affinity chromatography. The recombinant protein efficiently binds to membrane-bound Ig and becomes internalized by B cells (data not shown). Mice (2–4/group) received 100 lg OVA257–280 peptide plus CT/CpG, or CTA1-D2D1/CpG, or CT, CTA1-D2D1 or CpG alone. Control mice received only CTA1-D2D1/CpG. As shown in Figure 3, the highest antigen-specific cytotoxic activity measured by a 51Cr-release assay was obtained after TCI with the OVA257–280 fusion peptide plus CT/CpG or, somewhat less efficiently, with CTA1-D2D1/CpG, whereas the peptide plus the single adjuvant or the adjuvants alone had no effect.

TCI with an HPV-16 E7 multiepitope peptide in combination with CT and CpG induces functional antigen-specific CTLs To extend our study beyond the model antigen OVA, we immunized C57BL/6 mice transcutaneously 3 times with 2 different peptides encompassing epitopes of the oncoprotein HPV-16 E7. The E749–57 single epitope peptide represents the E7-specific, Dbrestricted CTL epitope, whereas the E744–62 multiepitope peptide contains the same CTL epitope overlapping with a Th epitope39 and a linear B-cell epitope (see Material and Methods). Mice were immunized according to the optimized TCI protocol (3 3 TCI including CT/CpG as adjuvants). Mice received 100 lg E749–57 or 200 lg E744–62 (equimolar to E749–57) plus CT/CpG. Positive controls were immunized s.c. with E744–62 in TM; negative controls received only adjuvants. As shown in Figure 4a, an IFN-g Elispot assay performed ex vivo showed increased numbers of E7-specific CTLs in mice that received E744–62 peptide s.c or by TCI. Within the group transcutaneously immunized with E749–57 CTL epitope plus CT and CpG, only one mouse was positive. No spots were detected in the group immunized transcutaneously with E7 protein plus CT/CpG (data not shown).

The ability of E7 antigen-specific CTLs to lyse E7-expressing target cells (2F11) was analyzsed by 51Cr-release assay. After 2 rounds of in vitro restimulation, mice immunized with E744–62 peptide plus adjuvants either by TCI or s.c. showed significant E7specific cytotoxic activity at an effector to target ratio of 20 (TCI 16%, 23%, 12%; s.c. pooled cells from all 3 mice 42%; data not shown). E7-specific lysis in the TCI group further increased after an additional round of in vitro restimulation to 35%, 52% and 14%, respectively (Fig. 4b). Pooled spleen cells of the 3 mice immunized with the E744–62 peptide plus TM s.c. reached 55% of specific lysis (Fig. 4b). No antigen-specific cytolytic activity was measured in any of the mice immunized with E749–57 peptide plus CT/CpG or in the pooled cells of the negative control TCI group (CT/CpG). TCI with the E7 protein plus adjuvants induces E7-specific serum IgG antibodies When we performed ELISA after coating the plates with E744– 62 peptide, only one mouse of 3 which were immunized 3 times with 200 lg E744–62 peptide plus Titermax responded with a low anti-E7 serum IgG titer (log reciprocal value 5 80, data not shown). None of the mice immunized by E744–62 peptide TCI had measurable serum IgG. Similarly, there was no measurable titer in mice immunized with the E7 protein (3 3 100 lg E7 protein 1 TM s.c., 3 3 100 lg E7 protein 1 CT/CpG by TCI). In contrast, when coating the plates with E7 protein, all E7 protein–immunized mice responded (Fig. 5). TCI with the E7 multiepitope peptide prevents tumor growth of HPV-16-transformed tumor cells in mice To assess if TCI with the E7 multiepitope peptide can prevent tumor growth, C57BL/6 mice were immunized 3 times either with E744–62 peptide plus CT/CpG (n 5 7) or with CT/CpG alone (n 5



FIGURE 5 – HPV-16 E7-specific humoral immune responses after 3 immunizations with either 100 lg E7 protein s.c., 100 lg E7 protein plus CT/CpG (50 lg/100 lg) transcutaneously or CT/CpG alone. Serum IgG antibodies directed against the whole E7 protein were measured by ELISA. Individual E7-specific titers are represented (hatched bars). Black bars illustrate the appropriate means 6 SEM. Serial 1:2 dilutions were performed with a starting dilution of 1:25. Data represent the log reciprocal highest dilution factor of each mouse which reached an OD405nm above the cut-off (5 0.03). The cut-off was determined by mean OD405nm values of preimmune sera 1 3 3 SEM.

(86%) that had been immunized with E744–62 plus CT/CpG were tumor-free (Fig. 6a,c). As shown in Figure 6b, the median size of E7-expressing tumors is negatively correlated with the presence of E7-specific CTLs in the peripheral blood. From these data, we conclude that antitumor activity in vivo results from the development of E7-specific cellular immune responses. Rechallenge of the surviving vaccinated mice (n 5 6) 42 days after the first challenge demonstrated sustained immunity since 5/6 mice remained tumor-free for another 58 days (Fig. 6c). At the day of rechallenge, 4 unvaccinated mice received 5 3 105 C3 cells and developed tumors within 13 days. FIGURE 4 – HPV-16 E7-specific CTL responses of mice after 3 TCIs with either 100 lg E749–57 peptide 1 CT/CpG (50 lg/100 lg), 200 lg E744–62 peptide 1 CT/CpG or CT/CpG alone. The positive control group was immunized s.c. with 200 lg E744–62 plus TM. (a) The number of CTLs was measured by IFN-g Elispot ex vivo after restimulation of splenocytes with E749–57 peptide. (b) E7-specific cytotoxicity of splenocytes was measured after 3 in vitro restimulations. CTLs were cocultured with E7 transfectants (2F11 cells) or the appropriate nontransfectants (RMA cells) at indicated effector to target ratios. Note that median values of the E744–62 TCI group (n 5 3) are shown, whereas cells of the E744–62 TM s.c. group (n 5 3) were pooled.

9) or were left untreated (n 5 9). Nine days after the last boost, mice were challenged s.c. with 5 3 105 HPV-16-transformed (E7positive) C3 cells. Two days prior to and 12 days after challenge (i.e., 7 and 21 days after the last boost), the presence of E7-specific CTLs was determined by IFN-g Elispot assay within groups of pooled PBMCs obtained from living animals. This assay revealed that high numbers of E7-specific CTLs were generated only in mice immunized with E744–62 peptide plus CT/CpG [370 and 260 spots/106 cells at days 7 (data not shown) and 21 after the last boost (Fig. 6b)]. At day 13, all mice in the control groups developed tumors (Fig. 6a,c), which was correlated with an absence of E7-specific CTLs in PBMCs in nontreated mice and a minor number of spots in the CT/CpG group (Fig. 6b). In contrast, 6/7 mice

Discussion The development of needle-free vaccination is a high priority for the World Health Organization.46 In recent years, TCI techniques have been improved, especially by developing formats of application such as skin patches suitable for clinical use in humans. So far, human studies in TCI are restricted to the induction of humoral immune responses (for review, see Glenn et al.47). In animal studies, TCI with epitope-specific peptides in combination with adjuvants generated antigen-specific CTLs and Th cells. Pretreatment of the skin by TS was reported to be critical for the generation of CTLs.8 In our study, we avoided removal of the SC by TS but depilated and hydrated the mouse skin before application of the aqueous antigen/adjuvant solution. Removal of hair by depilation facilitates access of the antigens/adjuvants to the noncornified keratinocytes of the HF. Moreover, depilation-induced anagen phase of HF initiates the recruitment of LCs,19 possibly caused by the enhanced expression of ICAM-1 on keratinocytes.21 Whereas peptide TCI following TS in combination with the strong immunomodulator CT8 or CpG9 as adjuvant induced CTL induction without TS was not successful. Our data confirm that coadministration of an OVA peptide either with CT or with CpG onto depilated and hydrated skin did not evoke strong specific CTL responses, suggesting that the skin barrier is not disrupted by depilation. However, as shown here, pretreatment of the skin by TS is



no longer necessary for induction of robust T-cell responses if both adjuvants (CT and CpG) are combined. TCI in the presence of CT as adjuvant after hydration was reported to generate Th responses.15–17 CT leads to activation of LCs after topical application, indicated by the increased expression of CD11c, ICAM-1 and MHC class II molecules and by morphologic changes such as rounding and loss of dendrites.48 Moreover, CT induces LC migration to draining lymph nodes even after application to intact skin.8,48 In TCI, application of CT favors Th2-cell responses.29 By combining CT and CpG in TCI, the activation/migration of LCs appears to be enhanced and the CT-specific bias toward a Th2-

type response is abandoned in favor of Th1,29 which is confirmed by our results (Fig. 1). Successful vaccination against viral infections depends on the presence of CTL memory cells,33 which can be generated only with the aid of Th cells during priming.33,49 Here, we demonstrate the simultaneous induction of both CTLs and Th cells in TCI using an OVA fusion peptide encompassing both an OVA-specific CTL and a Th-restricted epitope. Mice were immunized transcutaneously either with the OVA fusion peptide or with the complete protein plus CT and CpG as adjuvants. In all experiments, the strongest functional CTL and Th-cell responses were obtained after TCI with the peptide plus adjuvants. In accordance with previously published data,29 the Th response was mostly of the Th1 type. In contrast, others have found that both protein and peptide applied by TCI exerted similar CTL intensities.8 The different results might be explained by the fact that, in contrast to Kahlon et al.,8 we used a 15-fold lesser molar amount of protein than peptide. Another difference is the more rigorous barrier disruption by TS than by the depilation used in our study. A limitation of the use of CT resides in its cell type–associated toxicity. For instance, following i.n. immunization with CT in mice, its B subunit accumulates within olfactory nerve cells, thereby eliciting a high risk of CNS side effects.44,50 In contrast, CTA1-D2D1 binds via the DD dimer specifically to B cells but not to epithelial and nerve cells. When administered i.n. or s.c. CTA1-DD supports the formation of CTL responses51 (our own unpublished observations). Normally, B cells do not reside within the epidermis and thus can be excluded as APCs in a TCI protocol containing CTA1D2D1. Agren et al.52 suggest that the adjuvanticity of the CTA1 subunit is independent of the mode of uptake and thus may exert adjuvant effects even in the absence of B cells. It seems likely that LCs within the epidermis represent the targeted APCs. We evaluated the adjuvant potential of CTA1-D2D1 on CTL induction in TCI in combination with the OVA fusion peptide as model antigen with and without CpG as coadjuvant. Our data show that CTA1D2D1 is active when administered in combination with CpG albeit at a lower level than the cholera holotoxin. Persistent infection with HPV-16 is responsible for >50% of cervical cancers worldwide.53 Therapeutic HPV-16 vaccination strategies aim at the induction of a cell-mediated immune response directed against the HPV-16 E6 and/or E7 oncoproteins, which are constitutively expressed within tumor cells. E7 protein expression in keratinocytes alters expression of genes that influence the host resistance to infection and immune function, leading to peripheral tolerance within the CTL population.30 Tindle30 assumes that successful therapeutic vaccination has to take into account how E7 evades the immune response and has to overcome the E7-tolerogenic status of the patient. One possibility might be to combine the activation of CTLs by E7 with the induction of an inflamma-

FIGURE 6 – Growth of HPV-16 E7-positive tumors in mice after 3 TCIs with 200 lg E744–62 plus CT/CpG (50 lg/100 lg) (n 5 7), CT/ CpG only (n 5 9) or untreated controls (n 5 9). Animals were subsequently challenged s.c. with 5 3 105 C3 cells 9 days after the last boost. Surviving mice were rechallenged 42 days later. (a) Development of C3 tumors. Tumor sizes (mm2) were measured in vivo at day 13 after the first challenge. Results of individual mice are shown. Differences in tumor size between untreated and CT/CpG-treated groups vs. the E744–62 peptide group are highly significant (p < 0.001 in both comparisons by t-test), whereas differences between the 2 control groups are not significant (p 5 0.79). (b) Correlation between E7-specific CTL frequency and median tumor sizes after the first C3 challenge. The number of E7 CTLs was determined in groups of pooled PBMCs by IFN-g Elispot assay 12 days postchallenge (5 21 days after the last boost). In parallel, median tumor sizes (mm2) 6 SEM measured in vivo 13 days postchallenge are shown (described in a). (c) Percentage of tumor-free mice over time starting at the first challenge (day 0) with C3 cells (for details, see a, b). Days of challenges are marked (.). Mice were killed when tumors reached about 200 mm2 Tumor growth was controlled every 6–7 days.



tory environment, which thereby provides signal to turn immature, tolerizing DCs into mature, activating DCs. Vaccine adjuvants, like proinflammatory cytokines (e.g., IFN-a), may support the required danger signals. We used CT and CpG as adjuvants, which are known to be high IFN-a inducers, and immunized mice transcutaneously in the presence of CT and CpG with HPV-16 E7-specific peptides consisting either of a Db-restricted CTL epitope (E749–57) or of a combination of the CTL, a Th and a B-cell epitope (E744–62). Whereas no CTL response after TCI with the E749– 57 single epitope peptide was detected, the E744–62 multiepitope peptide induced strong functional E7-specific CTL responses, as evidenced by positive IFN-g Elispot and 51Cr-release assays and long-term protection against growth of tumor cells (Figs. 4, 6). Fernando et al.33 found that after s.c. immunization, only the E744–62 (not the E749–57) peptide provided long-term protection against tumor challenge and suggested that Th cells induced by E744–62 are responsible for this effect. We actually detected a weak Th response by depleting the CD81 lymphocytes of mice immunized s.c. with the E744–62 peptide (data not shown). Depletion was necessary since the CTL and Th epitopes within the E744–62 peptide are overlapping (E7-specific CD81 T cells recognized both the E749–57 and E744–62 peptides); thus, clear identification of MHC class II–restricted T-cell responses was not feasible by IFN-g Elispot in the presence of both CD41 and CD81 T lymphocytes (E7-specific CD41 T cells recognized only E744– 62). Attempts to measure Th response by lymphoproliferation assay were unsuccessful (data not shown). Yet, it is very likely that a Th response was induced in our experiments since we obtained strong and sustained activity against the growth of HPV16 E7-positive cells that parallels the induction of E7-specific CTLs obtained from peripheral blood of living animals. So far, we

have not performed depletion of T-cell subsets from animals of a tumor experiment; thus, we still miss the final proof of whether long-term protection is accompanied by generation of E7 Th cells and subsequently induced E7-specific memory cells. TCI with E7 protein induced humoral immune responses against E7 protein but at titers much lower than those obtained after s.c. E7 protein immunization. TCI with the E7 multiepitope peptide failed to generate E7 protein- or peptide-specific serum antibody levels. Only one of 3 mice immunized s.c. with E744–62 showed slightly elevated E7 peptide-specific titers (data not shown). None of the sera of mice immunized with E7 protein showed reactivity against E744–62. We conclude that various E7-specific B-cell epitopes are recognized in vivo and that the linear sequence QAEPD within E744–62 represents a minor epitope. TCI against high-risk HPV types might be of benefit in resource-poor areas of the world where >80% of cervical cancers occur32 and where injections often carry the risk of transmission of blood-borne diseases. Future research should aim at further improvement of HPV-specific TCI, including other viral antigens. Our data show that TCI might be a valid alternative to existing vaccination strategies.

Acknowledgements We are grateful to Ms. C. Klein for expert technical assistance. pSPA7235 was kindly provided by Dr. Tim Foster (Trinity College, Dublin, Ireland). pRG-CT was kindly provided by Mr. F.H. Burton (University of Minnesota, Minneapolis, MN).

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