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Unité de Biologie des Régulations Immunitaires and
Unité de Biochimie Cellulaire, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1129, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies, we have demonstrated that an invasive and nonreplicative vector, the detoxified adenylate cyclase of Bordetella pertussis (CyaA)3 can deliver CTL epitopes into the cytosol of eukaryotic cells. Moreover, purified CyaA toxins carrying a CTL epitope from the nucleoprotein of lymphocytic choriomeningitis virus were shown to stimulate strong specific CTL responses mediated by class I-restricted CD8+ T cells and were able to induce protection against a lethal intracerebral infection with this virus (7, 8). This opens up the possibility of using this molecule to develop cancer vaccines.
In the present study, using an OVA-expressing tumor mouse model, we show that recombinant CyaA molecules carrying a CD8+ OVA epitope cannot only elicit CTL responses and protect mice against the graft of lethal dose of tumor cells but can also cause a regression of established tumors in an Ag-specific manner, thus establishing the potential of these molecules to develop antitumor therapeutic immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EL4 is a C57BL/6 thymoma, and E.G7 is a chicken egg OVA-transfected subclone of EL4 (9). The C57BL/6-derived murine melanoma B16 and the OVA-transfected B16, MO5, were kindly provided by L. Rosthein and L. Sigal (University of Massachusetts, Worcester, MA). P815 (H-2d) and RDM4 (H-2k) cells were used to assay the MHC class I restriction of effector cells. Cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5.10-5 M of 2-ME. E.G7 cells were maintained in medium containing G418 at 0.4 mg/ml. MO5 cells were grown in medium containing 2 mg/ml of G418 and 60 µg/ml of hygromycin B.
Peptide and recombinant adenylate cyclases
The OVA synthetic peptide SIINFEKL (amino acids 257–264, H-2b-restricted) was synthesized by Neosystem (Strasbourg, France) and stored in PBS.
CyaA-OVA is a recombinant CyaA toxin resulting from the insertion of the amino acid sequence (one letter code) ASIINFEKLGT (the amino acid sequence of the OVA epitope is underlined) between Arg224 and Ala225 of CyaA. This protein is invasive and displays full catalytic activity. It was produced in the BLR strain of Escherichia coli (Novagen, R&D Systems, Abington, U.K.) harboring the plasmid pCACT-ova21 (10), which carries both the cyaC gene, required for the activation of the pro-CyaA toxin, and the modified cyaA gene, under the transcriptional control of the lac UV5 promoter. CyaA-OVA E5 is a derivative of CyaA-OVA, which is catalytically inactive (but still invasive) as a result of the insertion of the dipeptide Leu-Gln between codons 188 and 189 of CyaA-OVA. In addition, a Pro residue was inserted between Arg224 of CyaA and the peptide insert ASIINFEKLGT. Details of the construction and plasmid sequences can be provided upon request. The toxins, which were overproduced in the E. coli BLR strain, accumulated as inclusion bodies. After solubilization in 8 M urea and 20 mM HEPES-sodium (pH 7.5), they were purified to >95% homogeneity (as judged by SDS-gel analysis, data not shown) by two sequential chromatographies on DEAE-Sepharose and phenyl-Sepharose as described previously (7). Toxin concentrations were determined spectrophotometrically from the absorption at 280 nm using a molecular extinction coefficient of 142,000 M-1 · cm-1.
Immunization of mice for CTL assay and prevention of tumor growth
Female C57BL/6 mice (aged 6–8 wk) from Iffa Credo (L’Arbresle, France) were immunized i.p. on days 0, 21, and 42 with 50 µg of purified recombinant adenylate cyclase toxins mixed with 1 mg of aluminum hydroxide in PBS. Control groups were injected i.p. with either PBS or wild-type (wt) CyaA mixed with 1 mg of aluminum hydroxide in PBS.
Cytotoxicity assay
Spleen cells were removed from immunized mice at 7 days after the last injection. The spleens from three or four mice in each treatment group were pooled. Single-cell suspensions of spleen cells (2.5 x 107 cells) from in vivo-primed mice were cocultured with 106 irradiated (15,000 rad) E.G7 cells. After 5–6 days in culture, effector cells were harvested and cultured with 104 51Cr-labeled targets in round-bottom microwells (200 µl) at the indicated E:T ratio. For peptide sensitization of target cells, 50 µM of peptide was added to the target cells during the labeling period. In some cases, effector cells were depleted of CD4+ or CD8+ T cells by incubation with anti-CD4 or anti-CD8 mAb, followed by separation with immunomagnetic beads before the CTL assay as described previously (7). After 4 h at 37°C, 100 µl of supernatant from duplicate cultures was collected and counted, and the percentage of specific release was calculated as described previously (7). Results are the mean of duplicate cultures. The SD of duplicate wells was always <15–20% of the specific 51Cr release.
To determine the Kb specificity of the cytotoxic response, the effector cells were tested for CTL activity on 51Cr-labeled P815 (H-2d) or RDM4 (H-2k) cells incubated either with medium alone or with the OVA p257–264 peptide.
In vivo tumor protection studies
C57BL/6 mice were immunized as described with the indicated Ags. At 7 days after the last immunization (day 0), the mice were injected s.c. on the right flank with 2 x 104 MO5 tumor cells that had been washed three times in PBS, in a 0.2 ml volume. To evaluate whether the antitumor immunity was OVA-specific, other groups of mice immunized with CyaA-OVA or CyaA-OVA E5 mixed with 1 mg of aluminum hydroxide in PBS were challenged with 2 x 104 nontransfected B16 tumor cells. As controls, PBS and wt CyaA-injected mice were also inoculated with the tumor cells. All experiments included six or eight mice per group and were repeated at least three times.
In separate experiments, immunized and nonimmunized mice were treated i.p. on days 3, 10, 20, 24, and 32 after tumor inoculation with the recombinant toxins (50 µg) in aluminum hydroxide.
Mice were followed for tumor growth and survival. Tumor growth
was assessed by measuring the diameter of the tumor in centimeters
(recorded as the average of two perpendicular diameter measurements).
Mice with tumors of 
3.0 cm were killed and scored as "not
surviving". The survival of mice was recorded as the percentage of
mice surviving after the tumor graft.
Treatment of established MO5 and E.G7 tumors
Mice were injected s.c. on the right flank with 2 x 104 MO5 or 2 x 104 E.G7 tumor cells washed three times in PBS. Next, mice were treated with a total of five i.p. injections of CyaA-OVA or CyaA-OVA E5 (50 µg mixed with 1 mg of aluminum hydroxide in PBS) on days 1, 7, 14, 21, and 32 postchallenge. Control groups received PBS or wt CyaA following the same protocol. The mice were then followed for tumor growth and survival.
Statistical analysis
Statistical difference was determined using the method of Kaplan and Meier (11). Statistical significance was determined at the <0.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We constructed two recombinant CyaA toxins carrying the CD8+ T cell epitope from OVA (amino acid sequence 257–264) genetically inserted into the middle of the catalytic domain. This sequence corresponds to the main CD8+ T cell (Kb-restricted) epitope derived from OVA when this protein was artificially introduced into or expressed by mammalian cells (8). In CyaA-OVA, the peptide ASIINFEKLGT (one letter code, the OVA sequence is underlined) was inserted between Arg224 and Ala225 of CyaA. This toxin is catalytically active and fully invasive (i.e., it translocates into eukaryotic cells as efficiently as the wt CyaA). In CyaA-OVA E5, the peptide inserted between Arg224 and Ala225 was PASIINFEKLGT (an additional Pro residue was inserted at the N terminus of the epitope to facilitate construction). This toxin carries an additional insert of the dipeptide Leu-Gln between residues D188 and I189, a modification that completely abolishes the adenylate cyclase activity. Both toxins were produced in E. coli and purified to homogeneity by previously described procedures (7).
Immunization of mice with recombinant adenylate cyclase toxins carrying OVA epitope elicits CD8+ T cell responses
We first analyzed the capacity of the recombinant CyaA toxins
carrying the OVA epitope to trigger specific CTL responses (Fig. 1
). C57BL/6 mice were inoculated i.p. on days 0, 21, and
42 with 50 µg of CyaA-OVA or CyaA-OVA E5 in the presence of alum. On
day 49, CTL activity was determined after a secondary in vitro
stimulation of splenocytes of immunized mice with irradiated E.G7
cells. Cells from mice injected with PBS (Fig. 1a) and from
mice injected with the wt CyaA (Fig. 1b) were unable to lyse
E.G7 target cells or EL4 cells loaded with the OVA p257–264 peptide.
In contrast, effector cells from mice primed with CyaA-OVA (Fig. 1c) or CyaA-OVA E5 (Fig. 1d) were able to lyse
E.G7 target cells or EL4 cells loaded with the OVA p257–264 peptide.
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To characterize the cytolytic activity of the effector cells from
CyaA-OVA- or CyaA-OVA E5-immunized mice, we depleted the lymphocyte
population of either CD8+ or CD4+ T cells. As
shown in Fig. 2a, the CTL response induced by
CyaA-OVA E5 was strongly increased by removing CD4+ T
cells, whereas this response was totally abolished by removing
CD8+ T cells. Similar results were found for the CTL
response induced by CyaA-OVA (data not shown).
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b
for CyaA-OVA E5 and data not shown for CyaA-OVA). Hence, immunization
with CyaA-OVA or CyaA-OVA E5 induced MHC class I-restricted
peptide-specific CD8+ cytotoxic T cells. Immunization of mice with CyaA-OVA or CyaA-OVA E5 induces protective immunity to MO5 tumor challenge
To analyze the capacity of the recombinant adenylate cyclases carrying the OVA CTL epitope to protect mice against the graft of MO5 melanoma cells transfected with the OVA gene, mice were immunized i.p. on days 0, 21, and 42 with 50 µg of purified recombinant CyaA-OVA or CyaA-OVA E5 or wt CyaA mixed with 1 mg of aluminum hydroxide. At 7 days after the last immunization, mice were injected s.c. with 2 x 104 MO5 cells (>10 times the lethal dose to 50% of the animals tested). As an additional control, naive mice (PBS with alum) were also inoculated with the tumor cells.
All mice grafted with tumor cells were monitored for tumor growth,
which was recorded as the average tumor diameter in centimeters (Fig. 3a). When MO5 cells were injected s.c. into
C57BL/6 mice injected with PBS or wt CyaA, tumors grew progressively.
Because some animals began to die at 1 mo after the tumor graft, tumor
growth was not recorded beyond 30 days. In contrast to the PBS- and wt
CyaA-immunized mice, no tumor growth was detected in mice immunized
with CyaA-OVA or CyaA-OVA E5 at 30 days after the tumor graft (Fig. 3a).
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b also shows that immunization with CyaA-OVA or
CyaA-OVA E5 increased mouse survival. Indeed, at 
100 days after the
tumor graft, only 8% of mice of the PBS group and 4% of the wt CyaA
group had survived. In contrast, 27% and 56% of the CyaA-OVA- or
CyaA-OVA E5-immunized mice, respectively, had survived. These protected
animals were still alive 6 mo later (data not shown). Injection of an
equivalent dose of wt CyaA had no effect on tumor growth or survival in
comparison with control animals (PBS with alum)
(p = 0.43). In contrast, the tumor growth and
mortality of the CyaA-OVA- or CyaA-OVA E5-immunized groups were
significantly reduced compared with the group of mice injected with wt
CyaA (p < 0.0001).
As immunization with CyaA-OVA or CyaA-OVA E5 induced only partial
protection against the tumor graft, we tested whether additional
injections of CyaA-OVA or CyaA-OVA E5 molecules administered after the
tumor graft would result in better protection of the immunized animals.
As shown in Fig. 4, a and b, these
additional injections did not confer a survival advantage to mice
grafted with MO5 cells. Combined treatment using three injections of
CyaA-OVA before the tumor challenge followed by five additional
injections was no more effective than three injections alone
(p = 0.12 between Figs. 3b and
4b). Although the survival of the group that had been
vaccinated and treated after the challenge with CyaA-OVA E5 (Fig. 4b) was higher than the survival of the group that had been
vaccinated with CyaA-OVA E5 alone (Fig. 3b), the level of
protection achieved with the combined treatment was only slightly
higher than that obtained with only three injections
(p = 0.043 between Figs. 3b and
4b).
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c) and died following the
tumor graft (Fig. 4d). Thus, the protective antitumor
immunity induced by CyaA-OVA or CyaA-OVA E5 is selectively directed
against tumor cells that express OVA. Therapeutic antitumor immunity induced by recombinant adenylate cyclases carrying the OVA epitope
In considering the clinical application of a tumor therapeutic
strategy, it is clearly more relevant to start immunization when the
animals already bear tumors. Thus, in the next experiment, 2 x
104 tumor cells were implanted under the skin of naive
animals, and these mice were treated with the recombinant CyaA toxins
starting 1 day later. Next, animals received four i.p. injections
(spaced 1 wk apart) of PBS, wt CyaA, CyaA-OVA, or CyaA-OVA E5 (Fig. 5); their survival was followed. Mice that were in the
PBS group or treated with wt CyaA had a median survival time of 45
days. In contrast, mice treated with CyaA-OVA had a significantly
longer median survival (p = 0.0018); 1 of the
15 treated animals was still alive at day 100, when the experiment was
stopped. A better therapeutic effect was observed in the group
treated with CyaA-OVA E5 (p < 0.0001), with 6
of 15 animals surviving at day 100. It must be noted that when the
treatment with CyaA-OVA E5 was started at 7 days after the graft of
tumor cells, treated mice did not survive longer than control animals
(data not shown).
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, mice immunized with
CyaA-OVA or CyaA-OVA E5 showed significantly increased survival
(p < 0.001) compared with animals that
received either PBS or wt CyaA. Indeed, 40% and 50% of animals
immunized with CyaA-OVA and CyaA-OVA E5, respectively, were capable of
rejecting tumor cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several approaches to develop efficient therapeutic antitumoral vaccines have been reported (4, 5, 6, 12, 13), but there are always safety concerns when using live vaccines. To our knowledge, this study represents the first demonstration that a nonreplicative and nontoxic vector carrying a single CTL epitope can stimulate efficient protective and therapeutic immunity. Recently, the anthrax toxin lethal factor (LF) has been used to stimulate a peptide-specific CTL response in mice. In particular, the OVA (257–264) CTL epitope carried by the anthrax toxin was shown to be presented in association with MHC class I molecules both in vitro and in vivo. However, the capacity of the recombinant anthrax toxin to stimulate an antitumor immunity has not been reported (14, 15).
Two main conclusions can been drawn from this study: 1)
detoxified adenylate cyclase carrying the OVA CTL epitope (CyaA-OVA E5)
elicits Ag-specific cytotoxic responses that are effective in
protecting mice from a lethal tumor challenge, and 2) immunization with
CyaA-OVA E5 also leads to a marked suppression of established growing
tumors. Although protection was not complete, therapeutic efficacy
might be further improved by the codelivery of cytokines such as IL-12
(16, 17), IL-2, IFN-
, TNF-
, IL-4, IL-6, and
granulocyte-macrophage CSF (18, 19) with CyaA-OVA E5.
With the identification of minimal peptides derived from tumor-associated Ags (20, 21, 22) and point-mutated fragments from oncogenes that bind to MHC class I molecules (23, 24), it now becomes feasible to utilize strategies with recombinant CyaA carrying different tumor CTL epitopes. This approach may represent a significant potential for use in immunotherapy of cancers.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Claude Leclerc, Unité de Biologie des Régulations Immunitaires, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail address:
3 Abbreviations used in this paper: CyaA, adenylate cyclase toxin of Bordetella pertussis; wt, wild type.
Received for publication October 28, 1998. Accepted for publication December 23, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, granulocyte-macrophage colony-stimulating factor, and -interferon gene or admixed with conventional adjuvants. Cancer Res. 54:182.This article has been cited by other articles:
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T. Basar, P. Guermonprez, M. Rojas, and C. Leclerc Delivery of CD8+ T-Cell Epitopes into Major Histocompatibility Complex Class I Antigen Presentation Pathway by Bordetella pertussis Adenylate Cyclase: Delineation of Cell Invasive Structures and Permissive Insertion Sites Infect. Immun., January 1, 2000; 68(1): 247 - 256. [Abstract] [Full Text] [PDF]
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