Mutant CyaA polypeptides and polypeptide derivatives suitable for the delivery of immunogenic molecules into a cell

Abstract

The invention relates to mutant CyaA/E570Q+K860 polypeptides suitable for use as proteinaceous vectors for delivering one or more molecules of interest into a cell, in particular into a cell expressing the CD11b receptor. The invention further relates to polypeptide derivatives suitable for eliciting an immune response in a host. The invention is more particularly directed to polypeptides derived from an adenylate cyclase protein (CyaA) either under the form of a toxin or of a toxoid, which are mutant polypeptides. The mutant polypeptides are capable of retaining the binding activity of native CyaA to a target cell and preferably of also retaining the translocating activity of native CyaA through its N-terminal domain into target cells and furthermore have a pore-forming activity which is reduced or suppressed as compared to that of the native CyaA toxin.

Claims

1. A method for preparing a polypeptide mutant of the Bordetella pertussis CyaA protein comprising: a) substituting the glutamic acid residue at position 570 of SEQ ID NO: 1 with another amino acid residue selected from the group consisting of Gln, Asn, Met, Thr, Ser, Gly, Arg, Lys, Val, Leu, Cys, Ile, and Asp; and b) substituting the lysine residue at position 860 of SEQ ID NO: 1 with another amino acid residue selected from the group consisting of Gln, Asn, Met, Thr, Ser, Gly, Arg, Val, Leu, Cys, and Ile, to generate the polypeptide mutant of the Bordetella pertussis CyaA protein.

2. The method of claim 1, wherein the polypeptide mutant of the Bordetella pertussis CyaA protein is combined with one or more molecules of interest for eliciting an immune response comprising an amino acid sequence of a poliovirus antigen, an HIV virus antigen, an influenza virus antigen, a choriomeningitis virus antigen, a tumor antigen, or comprising or consisting of an amino acid sequence of any of these antigens which comprises at least one epitope.

3. The method of claim 2, wherein the molecule of interest for eliciting an immune response is inserted into a permissive site of the polypeptide mutant of the Bordetella pertussis CyaA protein, thereby preserving the capacity of said polypeptide to translocate its N-terminal adenylate cyclase enzyme domain into target cells.

4. The method of claim 2, wherein each of said amino acid sequences for eliciting an immune response is covalently or non-covalently coupled to an amino acid residue of said polypeptide mutant of the Bordetella pertussis CyaA protein.

5. The method of claim 1, wherein the adenylate cyclase activity of the polypeptide mutant of the Bordetella pertussis CyaA protein in cells is partly or totally suppressed as compared to that of the Bordetella pertussis CyaA toxin.

6. The method of claim 5, wherein the polypeptide mutant of the Bordetella pertussis CyaA protein has essentially lost its adenylate cyclase enzyme activity.

7. The method of claim 5, wherein said partial or total suppression of adenylate cyclase activity is achieved by insertion of a dipeptide between the amino acid residues at positions 188 and 189 of SEQ ID NO: 1.

8. The method of claim 1, further comprising inserting an LQ or GS dipeptide between the amino acid residues at positions 188 and 189 of SEQ ID NO: 1.

9. The method of claim 1, comprising: a) substituting the glutamic acid residue at position 570 of SEQ ID NO: 1 with a Gln amino acid residue; and b) substituting the lysine residue at position 860 of SEQ ID NO: 1 with an Arg amino acid residue, to generate the polypeptide mutant of the Bordetella pertussis CyaA protein.

Description

FIGURES

(1) FIG. 1. Substitutions in the pore-forming and acylation domains synergize in decreasing the specific hemolytic activity of CyaA. (A) Sheep erythrocytes (510.sup.8/ml) in TNC buffer were incubated with 5 g/ml of enzymatically active CyaA proteins at 37 C. After 30 min, aliquots of cells suspensions were washed repeatedly to remove unbound CyaA and used to determine the amount of cell-associated and cell-invasive AC activity. Hemolytic activity was measured after 5 hours of incubation as the amount of released hemoglobin by photometric determination (A.sub.541). Activity of intact CyaA was taken as 100%. (B) Erythrocytes were incubated as above with the indicated concentrations of the enzymatically active CyaA-derived proteins for 30 min, washed, and the amount of cell-associated AC enzyme activity was determined. (C) The reduced cell binding activity of proteins with the K860R substitution was compensated for by increasing their concentration from 5 g/ml to 25 g/ml. Activities of CyaA/233OVA (CyaA/OVA) in the presence were taken as 100% value. The results represent average values from at least three independent experiments performed in duplicates. The asterisks indicate statistically significant differences (**, p<0.001) from activities of CyaA (FIG. 1A) or CyaA/OVA (FIG. 1C).

(2) FIG. 2. CyaA/OVA/E570Q+K860R binds and translocates into CD11b.sup.+ monocytes. (A) J774A.1 cells (10.sup.6/ml) were incubated in D-MEM for 30 min at 4 C. with 2.5 g/ml of CyaA, washed repeatedly, and the amount of cell-associated AC activity was determined in cell lyzates. To block the CD11b/CD18 receptor, cells were incubated for 30 min with the CD11b-specific antibody M1/70 (Exbio, Czech Republic) at a final concentration of 10 g/ml prior to addition of CyaA (**, p<0.001). (B) The AC domain translocation capacity of constructs was assessed as the capacity to penetrate cells and convert cytosolic ATP to cAMP. J774A.1 cells were incubated with CyaA constructs for 30 minutes at 37 C. and the amounts of cAMP accumulated in cell lyzates were determined (41). As a control, the CD11b/CD18 receptor was blocked with the anti-CD11b antibody M1/70 as above. Membrane penetration of CD11b/CD18-bound and endocytosed toxin was controlled by using the doubly mutated CyaA/E570K+E581P variant, which is intact for receptor binding but fails to translocate the AC domain across cell membrane and elevate cytosolic cAMP concentrations (Vojtova-Vodolanova et al., 2009). (C) J774A.1 cells were loaded with the K.sup.+ sensitive fluorescent probe PBFI/AM at 9.5 M final external concentration and 25 C. for 45 min in the presence of Pluronic F-127 [0.05% (w/w)]. Cells were washed in HBSS before 3 g ml.sup.1 of the indicated toxins were added. Fluorescence intensity ratio of PBFI (excitation wavelength 340, emission wavelengths 450 and 510 nm) reflecting the intracellular K.sup.+ concentration was recorded every 15 s. The right scale shows intracellular [K.sup.+] values derived from calibration experiments (see Experimental procedures). No cell lysis, assessed as lactate dehydrogenase release, was observed within the time interval of the assay. Results representative of three independent determinations performed in duplicates are shown.

(3) FIG. 3. E570Q+K860R toxoid does not permeabilize J774A.1 cells. (A) Lysis of J774A.1 cells was determined as the amount of lactate dehydrogenase released from 10.sup.5 cells upon 3 h of incubation with 3, 10 and 30 g ml.sup.1 of the indicated protein at 37% in DMEM without serum. The results represent the average of values obtained in two experiments performed in duplicates. (B) Whole-cell patch-clamp measurements were performed on single J774A.1 cells at room temperature exposed to 1 or 10 g/ml of CyaA/233OVA/AC.sup. or CyaA/233OVA/E570Q+K860R/AC.sup. proteins as described in Materials and Methods. The shown curves are representative of six determinations in 3 independent experiments.

(4) FIG. 4. Toxoid with E570Q+K860R substitutions delivers the OVA T-cell epitope for presentation by IVIHC class I molecules and induction of CD8.sup.+ CTLs. (A) BMDC (310.sup.5 cells/well) used as APCs were incubated in the presence of indicated concentrations (0 to 60 nM) of the toxoids harboring the OVA epitope or with mock CyaNAC.sup.. Upon co-culture for 24 hours with B3Z T cells (110.sup.5 cells/well), IL-2 secretion by the stimulated B3Z cells was determined by the CTLL proliferation method. Results are expressed as cpm of incorporated [.sup.3H]thymidine (cpm in the presence of toxoidcpm in the absence of toxoid) SD and are representative of five independent experiments. (B) Analysis of the induction of OVA (SIINFEKL)-specific (SEQ ID NO:12) CTL responses by in vivo killing assay. On day 0, mice received 50 g i.v. of mock AC.sup. or OVA/AC.sup. toxoids and on day 7, they were i.v. injected with a mixture (1:1) of OVA (SIINFEKL) (SEQ ID NO:12) peptide-loaded CFSE.sup.high and unloaded CFSE.sup.low splenocytes. The number of CFSE-positive cells remaining in the spleen after 20 h was determined by FACS analysis, as documented for one representative in vivo killing assay in the upper panel assembly of plots, where percentages of cells in the gates are indicated. The lower panel shows pooled results of in vivo killing assays for three independent experiments. Statistical significance was determined by the Student t test (p=0.75 for OVA/AC.sup. vs. OVA/E570Q+K860R/AC.sup.).

(5) FIG. 5. Model of CyaA action on the membrane. (A) The model predicts an equilibrium between two conformers of CyaA in solution, each of them inserting into cell membrane in different a conformation. One would yield a monomeric CyaA translocation precursor, delivery of the AC domain into cytosol and concomitant influx of calcium ions into cells. The conformer would insert as pore precursor oligomerizing into a CyaA pore. (B) The synergic effect of the E570Q and K860R substitutions would selectively block the capacity of CyaA pore precursors to oligomerize into a pore, while the capacity of translocation precursors to deliver the AC domain across membrane would remain unaffected.

(6) FIG. 6. Amino acid sequence of the Bordetella pertussis CyaA toxin (SEQ ID No 1)

(7) FIG. 7. Amino acid sequence of the Bordetella pertussis CyaA/E570Q+K860R mutant (SEQ ID No 2)

(8) FIG. 8. Amino acid sequence of the Bordetella pertussis CyaA/E570Q+K860R/AC.sup. mutant (SEQ ID No 3)

(9) FIG. 9. Amino acid sequence of the Bordetella pertussis CyaA/233OVA/E570Q+K860R/AC.sup. mutant (SEQ ID No 4)

(10) FIG. 10. Plasmid encoding the CyaA/E570Q+K860R/AC.sup. mutant (QR-AC.sup.).

(11) FIG. 11A-C. DNA sequence of the QR-AC-plasmid encoding the CyaA/E570Q+K860R/AC.sup. mutant (SEQ ID No 5).

(12) FIG. 12. Plasmid encoding the CyaA/233OVA/E570Q+K860R/AC.sup. mutant (OVA-QR-AC.sup.).

(13) FIG. 13A-C. DNA sequence of OVA-QR-AC-plasmid encoding the CyaA/233OVA/E570Q+K860R/AC-mutant (SEQ ID No 6).

(14) FIG. 14. Amino acid sequence of the Bordetella parapertussis CyaA toxin (accession number CAB76450, SEQ ID No 7)

(15) FIG. 15. Amino acid sequence of the Bordetella hinzii CyaA toxin (accession number AAY57201, SEQ ID No 8)

(16) FIG. 16. Amino acid sequence of the Bordetella bronchiseptica CyaA toxin (accession number CAA85481, SEQ ID No 9)

EXAMPLES

Adenylate Cyclase Toxin Translocates Across Target Cell Membrane without Forming a Pore

(17) Materials and Methods

(18) Construction, Production and Purification of CyaA Proteins.

(19) The modifications yielding CyaNAC.sup., CyaA/233OVA, CyaA/E570Q and CyaA/K860R constructs were previously described (13, 20, 21) and were introduced into CyaA/233OVA/AC.sup. individually or in combination. The CyaA-derived proteins were produced in E. coli XL-1 Blue and purified close to homogeneity as previously described (29). During the hydrophobic chromatography, the resin with bound toxin was repeatedly washed with 60% isopropanol (30) to reduce the endotoxin content of CyaA samples below 100 IU/mg of protein, as determined by the LAL assay QCL-1000 (Cambrex).

(20) An Escherichia coli XL1-Blue strain (Stratagene) containing the QR-AC.sup. plasmid (FIG. 10) which encodes the CyaA/E570Q+K860R/AC.sup. mutant was deposited on Mar. 18, 2009 at the CNCM (Collection Nationale de Cultures de Microorganismes, France) under the accession number CNCM 1-4136 (FIG. 10). The DNA sequence of the QR-AC.sup. plasmid (SEQ ID No 5) is disclosed in FIG. 11.

(21) An Escherichia coli XL1-Blue strain (Stratagene) containing the OVA-QR-AC.sup. plasmid (FIG. 12) which encodes the CyaA/233OVA/E570Q+K860R/AC.sup. mutant was deposited on Mar. 18, 2009 at the CNCM (Collection Nationale de Cultures de Microorganismes, France) under the accession number CNCM 1-4137. The DNA sequence of the OVA-QR-AC.sup. plasmid (SEQ ID No 6) is disclosed in FIG. 13.

(22) Cell Binding and Hemolytic Activities on Sheep Erythrocytes.

(23) 510.sup.8 washed sheep erythrocytes in 50 mM Tris pH 7.4, 150 mM NaCl and 2 mM CaCl.sub.2 (TNC buffer) were incubated at 37 C. with 5 g/ml of CyaA proteins and cell binding, cell-invasive AC and hemolytic activities of CyaA were determined as described in detail previously (13). Significance of differences in activity values was analyzed using a one-way analysis of variance (ANOVA) with Bonferroni post-test (SigmaStat v. 3.11, Systat, San Jose, Calif.).

(24) Macrophage Binding, Elevation of cAMP and Cell Lysis Capacities of CyaA.

(25) J774.AI murine monocytes/macrophages (ATCC, number TIB-67) were cultured at 37 C. in a humidified air/CO.sub.2 (19:1) atmosphere in RPMI medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 IU ml.sup.1), streptomycin (100 g ml.sup.1) and amphotericin B (250 ng ml.sup.1). Prior to assays, RPMI was replaced with Dulbecco's modified Eagle's medium (DMEM) (1.9 mM Ca2.sup.+) without FCS and the cells were allowed to rest in DMEM for 1 h at 37 C. in a humidified 5% CO.sub.2 atmosphere (8). J774A.1 macrophages (10.sup.6) were incubated in D-MEM with 2.5 g/ml of CyaA variants for 30 min at 4 C., prior to removal of unbound toxin by three washes in D-MEM. Cells were lyzed with 0.1% Triton X-100 for determination of cell-bound AC activity. For intracellular cAMP assays, 10.sup.5 cells were incubated with CyaA for 30 minutes in D-MEM with 100 M IBMX (3-isobutyl-1-methylxanthin), the reaction was stopped by addition of 0.2% Tween-20 in 100 mM HCl, samples were boiled for 15 min at 100 C., neutralized by addition of 150 mM unbuffered imidazol and cAMP was measured as described (29). To block the CD11b/CD18 receptor, cells were preincubated for 30 min on ice with the CD11b-specific blocking MAb M1/70 (Exbio, Czech Republic) at a final concentration of 10 g/ml prior to addition of CyaA. Toxin-induced lysis of J774A.1 cells was determined using the CytoTox 96 kit assay (Promega) as the amount of lactate dehydrogenase released from 10.sup.5 cells in 3 hours of incubation with 10 g/ml of the appropriate protein at 37 C. in D-MEM as described (8). Significance of differences in activity values was analyzed as above.

(26) Patch Clamp Measurements.

(27) Whole-cell patch-clamp measurements were performed on J774A.1 cells bathing in HBSS (140 mM NaCl, 5 mM KCl, 2 mM CaCl.sub.2, 3 mM MgCl.sub.2, 10 mM Hepes-Na, 50 mM glucose; pH 7.4). Fire-polished glass micropipettes with outer diameter of about 3 m were filled with a solution of 125 mM potassium gluconate, 15 mM KCl, 0.5 mM CaCl.sub.2, 1 mM MgCl.sub.2, 5 mM EGTA, 10 mM HEPES-KOH pH 7.2. The resulting resistances of the microelectrodes were 3 to 5 M Cells were clamped at 40 mV, the data were filtered at 1 kHz and digitized at 2 kHz using Axopatch 200A amplifier, Digidata 1320A digitizer and PClamp-9 software package (Axon Instruments, Foster City, Calif.).

(28) Determination of Decrease of Cytosolic K.sup.+ Concentration.

(29) Cells grown on grass coverslips were washed in HBSS and loaded with 9.5 M PFBI acetoxymethyl ester (PBFI/AM, Molecular Probes, Eugene, Oreg., USA) for 30 min at 25 C. in the presence of 0.05% (w/w) Pluronic F-127 (Sigma, St. Louis, Mo.), in the dark. Ratiometric measurement was performed at 25 C. using a FluoroMax-3 spectrofluorimeter (Jobin Yvon Horriba, France) equipped with DataMax software. Fluorescence intensity of PBFI (excitation wavelength 340, emission wavelengths 450 and 510 nm) was recorded every 15 s and the integration time for each wavelength was 3 s. The ratio of 450/510 nm wavelengths is shown. The observed area of coverslip mounted in the 1 cm cuvette was about 10 mm.sup.2, corresponding to approximately 10.sup.4 cells. Calibration experiments were performed in 50 mM HEPES, pH 7.2, with varying concentrations of Potassium Acetate (10, 30, 60 or 140 mM) and Sodium Acetate (135, 115, 85 or 5 mM), respectively, on cells permeabilized for 30 min with 3 pM valinomycin or nigericin. Final intensity ratio (450/510 nm) is shown on right vertical axis of the plots.

(30) Mice and Cell Lines.

(31) Female C57BL/6 obtained from Charles River Laboratories were kept under specific pathogen-free conditions and manipulated according to institutional guidelines. CTLL-2 cells were obtained from ATCC. B3Z, a CD8.sup.+ specific T cell hybridoma specific for the Kb restricted OVA (SIINFEKL) epitope (SEQ ID NO: 12) was provided by N. Shastri (University of California, Berkeley) and maintained in the presence of 1 mg/ml G418 and 400 g/ml hygromycin B in complete RPMI 1640 medium (Invitrogen Life Technologies) with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 g/ml streptomycin, and 510.sup.5 M 2-ME.

(32) Antigen Presentation Studies.

(33) Bone Marrow Dendritic Cells (BMDC, 310.sup.5 per well) used as APCs were incubated in the presence of various concentrations (0 to 60 nM) of the recombinant CyaNOVA/AC.sup. carrying the OVA (SIINFEKL) epitope (SEQ ID NO: 12) or mock CyaA/AC.sup. and cocultured for 24 hours with B3Z T cells (110.sup.5 per well), selectively recognizing the OVA SIINFEKL/H-2K.sup.b (SEQ ID NO: 12) MHC class I complexes. After 18 h of culture, supernatants were frozen for at least 2 h at 80 C. The amount of IL-2 produced by the stimulated B3Z cells was then determined by the CTLL proliferation method. Briefly, 10.sup.4 cells of the IL-2-dependent CTLL line per well were cultured with 100 l of supernatant in 200 l of final volume. Twenty-four hours later, [.sup.3H]-thymidine (50 Ci/well) was added and cells were harvested 6 h later with an automated cell harvester. Incorporated [.sup.3H]-thymidine was detected by scintillation counting. Each point was done in duplicate and the experiment was repeated five times. Results are expressed in cpm of incorporated [.sup.3H]-thymidine (cpm in the presence of toxoidcpm in the absence of toxoid).

(34) In Vivo Killing Assay.

(35) For CTL priming, mice were immunized by i.v. injection with 50 g of recombinant CyaA/OVA/AC.sup. carrying the OVA (SIINFEKL) epitope (SEQ ID NO: 12) or mock CyaNAC.sup.. Seven days after immunization, naive syngenic splenocytes were pulsed with OVA (SIINFEKL) peptide (SEQ ID NO: 12) (10 g/ml) (30 min, 37 C.), washed extensively and labeled with a high concentration (1.25 M) of carboxyfluoroscein succinimidyl ester (CFSE; Molecular Probes, Eugene, Oreg.). The nonpulsed control population was labeled with a low concentration (0.125 M) of CFSE. CFSE.sup.high- and CFSE.sup.low-labeled cells were mixed in a 1:1 ratio (510.sup.6 cells of each population) and injected i.v. into mice. Spleen cells were collected 20 h after, washed and resuspended in FACS buffer (PBS supplemented with 1% BSA and 0.1% NaN.sub.3). The number of CFSE-positive cells remaining in the spleen after 20 h was determined by FACS. The percentage of specific lysis was calculated as follows: percent specific lysis=100 [100(% CFSE.sup.high immunized mice/% CFSE.sup.low immunized mice)/(% CFSE.sup.high naive mouser/% CFSE.sup.low naive mouse)].

(36) Statistical Analysis:

(37) Significance of differences in values was analyzed using a one-way analysis of variance (ANOVA) with Bonferroni post-test (SigmaStat v. 3.11, Systat, San Jose, Calif.).

(38) Results

(39) Combined Elimination of Negatively Charged Glutamate 570 and of Acylated Lysine 860 Ablates Cell-Permeabilizing Capacity of CyaA.

(40) The working model of CyaA action predicts that CyaA can be modified to lose its pore-forming (hemolytic) activity while preserving the capacity to deliver the AC domain into cytosol of target cells. To test this hypothesis, the inventors sought to produce CyaA constructs exhibiting as low hemolytic and cytolytic activities as possible, building on previous observation that the capacity of CyaA/AC.sup. toxoids to lyze cells can be modulated both up or down by substitutions within the pore-forming domain (8, 12-14, 18). To enable assessment of target cell penetration also for the CyaA/AC.sup. toxoids, the inventors derived such mutants from a CyaA/233OVA toxin that was previously tagged by insertion of the SIINFEKL peptide (SEQ 1D NO: 12) from ovalbumin (OVA). This CyaA variant was chosen as the insertion of reporter Kb-restricted CD8.sup.+ T-cell epitope at residue 233 does not affect the AC activity and allows to quantify translocation of the OVA/AC enzyme into cells as elevation of cytosolic cAMP. More importantly, presence of the OVA epitope allows to assess also the capacity of enzymatically inactive CyaA/233OVA/AC.sup. toxoids to deliver their OVA/AC.sup. domain into cytosol of CD11b.sup.+ antigen presenting cells (APC), as this enables proteasome processing and cell surface presentation of the OVA epitope on MHC Class I glycoproteins that can be determined as stimulation of OVA-specific CD8.sup.+ T cells, both in vitro and in vivo (20).

(41) To generate CyaA/AC.sup. toxoids possibly lacking the cytolytic activity, the inventors combined the E570Q and K860R substitutions previously shown to reduce the specific hemolytic activity of CyaA on sheep erythrocytes, with the E570Q substitution having been found to reduce also the cytolytic activity of the CyaA/AC.sup. on CD11b.sup.+ J774A.1 monocytes (8, 13). These substitutions were engineered into CyaA/233OVA/AC.sup. individually and in combination and the specific hemolytic and cytolytic activities of resulting toxoids were compared using sheep erythrocytes as model CD11b.sup. target and J774A.1 as model CD11b.sup.+ target in parallel (Table I). In agreement with results obtained previously with toxoids lacking the OVA epitope (4, 8, 13, 21), under the used conditions the OVA/AC.sup. toxoids carrying individually the E570Q and K860R substitutions exhibited respectively a two-fold reduced (558) and nil (11) relative hemolytic activity on erythrocytes and the relative cytolytic activity of the E570Q toxoid towards CD11b-expressing J774A.1 cells was also reduced (3710), as compared to OVA/AC.sup.. In turn, as expected from results obtained with an enzymatically active K860R construct, despite the low hemolytic activity on CD11b.sup. erythrocytes, the K860R toxoid exhibited only a slightly reduced relative cytolytic activity on CD11b.sup.+ J774A.1 cells (7222%), confirming that the structural defect caused by the K860R substitution was rescued by interaction with the CD11b/CD18 receptor (4). Nevertheless, when combined with E570Q, the K860R substitution exhibited a clear synergic effect in reducing the relative cytolytic activity of the E570Q+K860R construct towards J774A.1 cells down to 147%.

(42) TABLE-US-00001 TABLE I Cytolytic activities of OVA/AC.sup. and derivatives on sheep erythrocytes and J774A.1 macrophages. Lysis of Lysis of erythrocytes J774A.1 cells Protein (% of AC.sup.).sup.a (% of AC.sup.).sup.b AC.sup. 100 5 100 10 OVA/AC.sup. 93 4 93 12 OVA/E570Q/AC.sup. 55 8** 37 10** OVA/K860R/AC.sup. 1 1** 72 22** OVA-L247Q-AC.sup. 97 3 41 9 OVA/E570Q+K860R/AC.sup. 1 1** 14 7** OVA-E570Q-L247Q-AC.sup. 50 12 40 11 OVA-K860R-L247Q-AC.sup. 1 1 45 11 OVA-E570Q-K860R-L247Q-AC.sup. 0 1 16 10 Table Legend .sup.aLysis of sheep erythrocytes was determined after 4.5 hours as the amount of hemoglobin released upon incubation of 5 10.sup.8 RBC at 37 C. in the presence of 2 mM Ca.sup.2+ with 5 g/ml of the given protein (31). The hemolytic activity of CyaA/AC.sup. was taken as 100% activity. The results represent the average of values obtained in four independent experiments performed in duplicates S.D with two different protein preparations. .sup.bLysis of J774A.1 cells was determined as the amount of released lactate dehydrogenase from 10.sup.5 cells upon 3 hours of cell incubation with 10 g/ml of the appropriate protein at 37 C. in D-MEM. J774A.1 cell lysis by CyaA/AC.sup. was taken as 100%. The results represent the average of values obtained in four separate experiments performed in duplicates S.D with two different protein preparations (*p < 0.05; **p < 0.001).

(43) To enable quantification of capacity of the E570Q+K860R construct to deliver the AC domain into cytosol of cells, the E570Q and K860R substitutions were transferred into enzymatically active constructs derived from CyaA/233OVA (CyaA/OVA). These were produced and purified in the same way as the AC.sup. toxoids (not shown) and characterized for cell binding, hemolytic and AC translocation capacities on sheep erythrocytes. As shown in FIG. 1A and expected from results with toxins lacking the OVA epitope (4, 13, 21), the E570Q substitution had no impact on erythrocyte binding or the capacity of CyaA/OVA to deliver the AC domain into erythrocyte cytosol and selectively reduced only its relative hemolytic activity. As further expected (4), the K860R substitution significantly reduced the capacity of CyaA/OVA to bind and penetrate erythrocytes, causing a sharp reduction of the relative hemolytic and cell-invasive AC activities of the E570Q and E570Q+K860R mutants on erythrocytes.

(44) It has to be noted, that the hemolytic activity of CyaA is a highly cooperative function of the amount of cell-associated CyaA (Hill number >3), suggesting that CyaA oligomerization is a prerequisite for pore formation (22). Therefore, to assess the impact of combined E570Q+K860R substitutions on the hemolytic activity, the loss of erythrocyte-binding capacity of the K860R constructs had to be compensated by increasing their concentration in the assay to 25 g/ml (5 g/ml for intact toxin), in order to achieve binding of equal amounts of all proteins to erythrocytes, as shown in FIG. 1G. Under these conditions the combination of E5700 and K860R substitutions exhibited a clear synergy in further reducing by a factor of two the already impaired hemolytic activities of constructs carrying the E5700 (50%) and K860R substitutions (30%) individually, as shown in FIG. 2C. This suggests that combination of the two substitutions affected the specific cell-permeabilizing capacity of CyaA.

(45) Pore-Forming Activity of CyaA is Dispensable for Membrane Translocation of the AC Domain.

(46) In contrast to impact of the K860R substitution on toxin activity on erythrocytes, both the E570Q and K860R substitutions were previously found to have no effect on the capacity of CyaA to bind and penetrate J774A.1 monocytes expressing the CD11b/CD18 receptor (4, 8). Moreover, as documented in FIG. 2, when the two substitutions were combined in the same toxin molecule, the CyaA/OVA/E570Q+K860R construct exhibited an equal capacity to bind J774A.1 cells (FIG. 2A) and to deliver the AC domain into their cytosol to elevate cytosolic cAMP concentrations (FIG. 2B), as did intact CyaA. At the same time, however, the doubly mutated E570Q+K860R toxoid exhibited an about seven-fold reduced (147%) relative cytolytic capacity on these cells (cf. Table I). As shown in FIG. 2C, when compared with intact CyaA, the singly mutated E570Q and the doubly mutated E570Q+K860R constructs were importantly impaired in the capacity to elicit decrease of intracellular concentration of potassium ions ([K.sup.+]i) in toxin-treated J774A.1 cells. While no cell lysis was detected over 20 min by the assay for release of lactate dehydrogenase, the [K.sup.+]i of J774A.1 cells exposed to 3 g ml.sup.1 of intact CyaA decreased from 140 mM to well below 30 mM already in 10 min upon toxin addition. In turn, when the same amounts of the ER570QiK860R constructs were used (3 g ml.sup.1), the [K.sup.+]i levels in cells did not decrease below 100 mM (FIG. 2C). Indeed, efflux of potassium from cells was previously shown to be the hallmark of insertion of the CyaA pore precursors into cell membrane (32). This suggested that the combination of E570Q and K860R substitutions selectively impaired only the capacity of the toxoid to permeabilize J774A.1 cells and not its capacity to translocate the AC domain across cell membrane.

(47) This conclusion was further supported by an importantly reduced relative cytolytic activity of the corresponding E570Q/AC and E570Q+K860R/AC toxoids, as discussed above (Table I) and documented in detail in FIG. 3A. The doubly mutated E570Q+K860R toxoid at 3 g ml.sup.1 was essentially unable to provoke any detectable release of lactate dehydrogenase from J774A.1 cells in 3 h of incubation, while 20% of cells lysed in the presence of equal amounts of intact toxoid.

(48) To test this, the inventors analyzed the cell-permeabilizing capacity of the E570Q+K860R construct in single whole cell patch-clamp experiments. Here again the AC.sup. toxoids had to be used, in order to avoid the massive ruffling of J774A.1 cells provoked by toxin-generated cAMP (23). As shown in FIG. 3A by a representative recording of ion currents across the membrane of patch-clamped single J774A.1 cells exposed to 1 g/ml of CyaA/OVA/AC.sup., upon an initial lag of about 3 minutes the J774A.1 cells were progressively and massively permeabilized by CyaA/OVA/AC.sup. and the currents across cell membrane reached 3,000 pA within 10 minutes. In contrast, as shown in FIG. 3B, exposure to the CyaA/OVA/E570Q+K860R/AC.sup. reproducibly caused only a transient and minimal initial permeabilization of the cells, with currents across cell membrane not exceeding 200 pA and returning close to zero within 10 minutes after toxoid addition. Quite similar picture was observed when toxoid concentrations were elevated to 10 g ml.sup.1, which was the concentration used for comparisons of relative cytolytic activity of toxoids summarized in Table I. The 10-fold increase of concentration from 1 to 10 g ml.sup.1 resulted in about twofold increase of the currents produced across cell membrane by OVA/AC, while essentially no enhancement of cell permeabilization was observed even at the increased concentration of OVA/E570Q+K860R/AC (note the expanded scale of y-axis for measurements at 10 g ml.sup.1). The shown recordings were representative of at least six determinations from 3 independent experiments and demonstrate that the combination of the E570Q and K860R substitutions had a major impact on the capacity of the toxoid to permeabilize the membrane of J774A.1 cells. Given that the enzymatically active version of the same construct was fully capable to translocate the AC domain into J774A.1 cells (cf. FIG. 2B), these results strongly suggest that the cell-permeabilizing (pore-forming) activity of CyaA was not required for AC domain translocation across cellular membrane.

(49) Membrane-Permeabilizing Activity of CyaA is Dispensable for Delivery of Passenger Antigens to the Cytosolic MHC Class I Pathway.

(50) Since the assay for cytosolic cAMP could not be used for assessment of cell penetration capacity of the AC.sup. toxoids, the surrogate assay for their capacity to deliver the reporter OVA epitope to the cytosolic processing site of the MHC class I antigen presentation pathway was used (7, 24). Towards this end, the inventors determined the capacity of C57BL/6 mouse bone marrow-derived dendritic cells (BMDCs), loaded with the toxoids, to stimulate IL-2 release by B3Z T cells that selectively recognize the complex of K.sup.b MHC class molecules with the SIINFEKL (OVA) peptide (SEQ ID NO:12) on APCs. It has, indeed, been previously shown that the capacity of CyaA/AC.sup. toxoids to translocate the AC domain across the cytoplasmic membrane into cytosol of BMDCs is essential for the capacity of the toxoids to promote presentation of the delivered OVA epitope in complex with the H-2Kb MHC class I molecules. This, in turn, is essential for specific in vitro stimulation of cytotoxic T cells to occur (29). Nevertheless, it was important to confirm here that delivery of the OVA epitope for proteasome processing and subsequent presentation was due to AC domain translocation into cytosol of BMDCs across their cytoplasmic membrane, and was not due to sampling of the added antigen by fluid phase uptake, or endocytosis. For this purpose, a doubly mutated non-translocating OVA/E570K+E581 P/AC.sup. toxoid variant was used, which bears a combination of charge-reversing and a-helix-breaking substitutions of glutamates 570 and 581 in the transmembrane domain of CyaA (33). This construct was previously found to exhibit a full capacity to bind CD11b/CD18-expressing cells (cf. FIG. 2A), while its capacity to translocate the AC domain across target cell membrane was ablated by the combination of E570K and E581 P substitutions (cf. FIG. 2B).

(51) As shown in FIG. 4A, the B3Z hybridoma cells were effectively stimulated upon co-incubation with BMDCs loaded with the OVA/E570Q/AC and OVA/E570Q+K860R/AC toxoids. In contrast, no B3Z stimulation was observed upon co-incubation with BMDCs loaded with the OVA/E570K+E581P/AC toxoid defective in AC domain translocation across cell membrane. Moreover, the OVA/E570Q/AC.sup. and OVA/E570Q+K860R/AC.sup. toxoids induced stimulation of the B3Z lymphocytes by APCs in vitro with as high efficiency as intact OVA/AC.sup. toxoid. These results confirm that the E570Q+K860R double mutant was fully capable to translocate its AC domain into BMDC cytosol for processing and presentation of the OVA epitope by K.sup.b MHC class I molecules, while being essentially unable to permeabilize the J774A.1 cells. These results suggest that the cell-permeabilizing (pore-forming) activity of CyaA was neither required for AC domain translocation across cellular membrane, nor did it play any role in the capacity of CyaA to deliver passenger epitopes into APC cytosol.

(52) To corroborate the observed in vitro antigen delivery capacity of the non-cytolytic toxoids, the inventors assessed their in vivo capacity to prime OVA-specific cytotoxic CD8.sup.+ T lymphocytes (CTL). 50 g of the various OVA-toxoids were injected intravenously into C57BL/6 mice and one week later the OVA-specific CTL responses were assessed in immunized mice by an in vivo killing assay. C57BL/6 mice received i.v. injection of a mixture (1:1) of OVA (SIINFEKL) (SEQ ID NO: 12) peptide-loaded CFSE.sup.high and unloaded CFSE.sup.low splenocytes, followed one day later by FACS analysis of CFSE-labeled cells. As shown in FIG. 4B, immunization of mice with the mock toxoid did not induce any SIINFEKL-specific (SEQ ID NO:12) in vivo CTL activity. In turn, immunization with the E570Q+K860R toxoid induced the same OVA-specific in vivo CTL killing response as the unmutated toxoid used as positive control, with the slight difference in the values of mean response to the intact and doubly mutated toxoids not being statistically significant (p=0.065). These results show that the cell-permeabilizing activity of CyaA was dispensable for the in vivo capacity of the CyaA/233OVA/AC.sup. toxoids to deliver an AC-inserted passenger antigen into cytosol of APCs.

DISCUSSION

(53) The inventors demonstrate here that translocation of the AC domain of CyaA across the membrane of CD11b/CD18 receptor-expressing myeloid target cells does not depend on the capacity of the toxin to form pores and permeabilize the cellular membrane.

(54) As summarized in the model proposed in FIG. 5, the inventors have previously reported that balance between the two activities of CyaA can be shifted by mutations or alternative acylation of CyaA. Enhancement of the pore-forming (hemolytic) activity at the expense of the capacity to deliver AC into cells was, indeed, observed upon lysine substitutions of glutamates 509, 516 and 581 (13, 18), or upon blocking of AC translocation by the 3D1 monoclonal antibody (MAb) (25). In turn, a shift in the opposite direction was observed for the recombinant r-Ec-CyaA, acylated in E. coli by palmitoleyl (C16:1) residues, as compared to the native (C16:0) palmitoylated Bp-CyaA produced by B. pertussis. The r-Ec-CyaA was found to exhibit about four-fold reduced hemolytic activity and about ten-fold lower pore-forming activity in planar lipid bilayers than Bp-CyaA (12), while both CyaA forms were equally active in penetrating cellular membrane and translocating the AC domain into erythrocytes (17, 26). Moreover, recently the CyaA/E570Q construct was found to exhibit a full capacity to deliver the AC domain into both erythrocytes and J774A.1 macrophages, while exhibiting reduced hemolytic activity and lower specific pore-forming capacity in planar lipid bilayers than intact CyaA, with the CyaA/E570Q/AC.sup. toxoid exhibiting a two-fold reduced cytolytic activity on J774A.1 cells (8, 13).

(55) Despite the above mentioned and the many mutant CyaAs that the inventors characterized, the question remained whether formation of a membrane pore by CyaA is required for translocation of the AC domain across the membrane of CD11b-expressing cells. It is worth mentioning that, based on previous comparisons of haemolytic potency of the intact r-Ec-CyaA with that of the native Bp-CyaA purified from B. pertussis, the specific haemolytic activity of the here described r-Ec-CyaA/OVA/E570Q+K860R/AC toxoid on sheep erythrocytes would be reduced by about three orders of magnitude. The residual specific cytolytic activity of r-Ec-CyaA/OVA/E570Q+K860R/AC on CD11b-expressing cells would then be estimated to be about 50-fold lower than that of Bp-CyaA, while the specific capacity of both proteins to deliver the AC domain into these cells would be the same (using intact r-Ec-CyaA as 100% invasive AC activity standard for comparisons). The here described CyaA/233OVA/E570Q+K860R mutant is the first construct with an importantly reduced capacity to permeabilize cells that remains fully capable of translocating the AC domain across cellular membrane. This shows that on its way to cell cytosol the translocating AC domain can bypass the cation-selective pore formed by CyaA.

(56) The mode and path of AC domain translocation across cellular membrane, however, remain to be defined in more detail. Given the differing effects of substitutions of glutamates 509, 516, 570 and 581 on the pore-forming and AC delivery activities of CyaA (8, 13, 18), where the balance between the two activities can be almost entirely shifted in either direction by specific substitutions, the amphipathic helices harboring these glutamate residues appear to be involved in both activities of CyaA in an alternative manner. This is supported by the effect of combined E509K+E516K substitution, which yields a hyper-hemolytic CyaA unable to deliver the AC domain into cells (8, 18), while the here described E570Q+K860R combination yields the opposite, an essentially non-cytolytic CyaA that is fully competent to translocate the AC domain into J774A.1 cells (CD11b.sup.+).

(57) These observations further corroborate the proposed model that the two membrane activities of CyaA would depend on different conformers inserting into membrane, one yielding translocation of the AC domain by toxin monomers and the other leading to formation of oligomeric CyaA pores (13, 18). It is proposed that the transmembrane segments harbouring the critical glutamate residues 509, 516, 570 and 581 can participate in formation of an oligomeric and cation-selective cytolytic pore only if the membrane-inserted pore precursor conformer has the AC domain located outside the cell. In the AC translocating conformer, the same transmembrane segments would adopt a different conformation in the membrane, being potentially obtruded and prevented from entering CyaA oligomers by the polypeptide linking the C-terminal end of the AC domain to transmembrane segments. Support for this interpretation can be deduced from results obtained by Gray and co-workers (25). These authors showed that deletion of the AC domain together with the segment linking it to the pore-forming domain (up to residue 489), or binding of the 3D1 antibody that blocks membrane translocation of the terminal AC domain segment located between residues 373 and 399, importantly enhances the pore-forming (haemolytic) activity of the toxin. This is likely to be due to imposing a conformation on the transmembrane segments of CyaA that is favourable for formation of the otigomeric pores. It remains to be defined what CyaA segments outside of the pore-forming domain are involved in AC domain translocation across membrane. Given the requirement for its structural integrity (27), the large RTX repeat domain (residues 1006 to 1706) is likely to be taking part in AC translocation into cells. It would be sized enough (700 residues) to form a hydrophilic translocation interface within cellular membrane that might allow passage of an unfolded AC domain across the membrane without a concomitant formation of a real cell-permeabilizing pore. Alternatively, CyaA might promote formation of inverted nonlamellar (inverted hexagonal phase) lipid structures (28), which might potentially take part in a well sealed protein-lipid interface through which the AC domain could slide into cell cytosol.

(58) CyaA was, indeed, previously shown to promote formation of inverted non-lamellar (inverted hexagonal phase) lipid structures (28). These might potentially take part in formation of a well-sealed protein-lipid interface, thus allowing translocation of the AC domain across membrane in the absence of cell permeabilization. Formation of non-lamellar lipid structures is favoured in cholesterol-enriched lipid rafts and CyaA was, indeed, recently found to mobilize into rafts in complex with its receptor CD11b/CD18. Moreover, the inventors have recently shown that AC domain translocation across membrane is accomplished only upon relocation of CyaA into rafts (L., Bumba, J., Masin, R., Fiser, and P., Sebo, submitted). Intriguingly, translocation of the catalytic subunit of diphtheria toxin (DT) across the cell cytoplasmic membrane was also previously found to occur without detectable cell permeabilization, when DT was pulsed into cells by low pH upon binding to a truncated GPI-anchored DT receptor (34). The authors did not examine whether the GPI-anchored DT receptor localized into lipid rafts, but this is highly likely. It is, hence, tempting to speculate that the specific lipid composition of the raft membrane may support translocation of different protein toxins into target cells without the need for formation of a true protein conducting pore permeabilizing the cell.

(59) Last not least, a practical discovery reported herein is that the CyaA/E570Q+K860R/AC.sup. toxoid with the much reduced cell-permeabilizing (cytolytic) activity, remains fully active in antigen delivery into CD11b.sup.+ APCs. This is of importance in the light of its potential use as enhanced safety profile tool for delivery of tumor-specific antigens in second generation of CyaNAC.sup.-derived vaccines for immunotherapy of cancer.

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