Vaccines

The present invention is exemplified by reference to the following examples and figures.

In all figures, adeno-ova (adenovirus vector containing OVA protein) was used as a positive control in first injection. P/B (prime/boost) is a positive control with first injection of Adeno-Ova, and second, boost injection of Ova protein in AS A (AS H in FIG. 6B).

FIG. 1: Siinfekl-specific CD 8 frequency in PBLs 7 days after primary injection with AS A STxB Ova and AS H STxB Ova vaccines.

FIG. 2 Siinfekl-specific CD 8 frequency in PBLs 14 days after primary injection with AS A STxB Ova and AS H STxB Ova vaccines.

FIG. 3 Effector T cell response persistency assessed in PBLs through siinfekl-specific cytokine-producing CD8 T cells at day 15 after primary injection with AS A STxB Ova and AS H STxB Ova vaccines.

FIG. 4 Effector T cell response persistency assessed in PBLs through antigen-specific cytokine-producing CD8 T cells at day 15 after primary injection with AS A STxB Ova and AS H STxB Ova vaccines.

FIG. 5 Effector T cell response assessed by cytotoxic activity detected in vivo15 days after primary injection with AS A STxB Ova and AS H STxB Ova vaccines.

FIG. 6: (A) Siinfekl-specific CD8 frequency in PBLs 47 days after second injection with AS A STxB Ova and AS H STxB Ova vaccines. (B) Kinetics of the Siinfekl-specific CD8 frequency in PBLs from day 0 to day 98.

FIG. 7: Effector T cell response assessed through antigen-specific cytokine-producing CD4 T cells in PBLs 47 days after second injection with AS A and AS H STxB Ova vaccines.

FIG. 8: Effector T cell response assessed through antigen-specific cytokine-producing CD8 T cells in PBLs 47 days after second injection with AS A and AS H STxB Ova vaccines.

FIG. 9: Effector T cell response assessed by Cytotoxic activity detected in vivo 47 days after second Injection with AS A STxB Ova and AS H STxB Ova vaccines.

FIG. 10A: Humoral response 15 days and 40 days post second injection with AS A STxB Ova and AS H STxB Ova vaccines.

FIG. 10B: Anti-Ova memory B cells frequency assessed in spleen 78 days after the second injection of ASH STxB-OVA.

FIG. 11: Siinfekl-specific CD8 frequency in PBLs with AS A, AS F, AS D, AS E, STxB-ova vaccines 13 days post primary injection.

FIG. 12A: Siinfeki-specific CD8 frequency in PBLs with AS A, AS B, AS C, AS G, AS I, and AS H STxB-ova vaccines, 15 days post first injection.

FIG. 12B: Siinfekl-specific CD8 frequency in PBLs with AS A, AS B, AS C, AS G, AS I, and AS H STxB-ova vaccines 6 days post second injection.

FIG. 13: Siinfekl-specific CD8 frequency in PBLs for different doses of STxB-ova vaccines formulated with the same dose of AS H.

FIG. 14: Evaluation of the immune response induced in vivo by STxB-ova with AS J (two doses) or AS K measured in PBLs 14 days after first injection. (A) Siinfeki-specific CD8 frequency. (B) antigen-specific cytokine-producing CD8 frequency. (C) Siinfekl-specific lysis detected in vivo

FIG. 15: Siinfekl-specific CD8 frequency in PBLs with AS L, AS G, AS M STxB-ova vaccines 14 days post 1^stinjection.

FIG. 16: Siinfekl-specific CD8 frequency in PBLs with AS B, AS C, AS K, AS F or AS T STxB ova vaccines 14 days post 1^stinjection.

FIG. 17: Siinfekl-specific CD8 frequency in PBLs with AS B, AS N, AS I STxB-ova vaccines 14 days post 1^stinjection.

FIG. 18: Siinfekl-specific CD8 frequency in PBLs 14 days post 1^stinjection with AS G, AS O, AS P, AS Q STxB-ova vaccines.

FIG. 19: Siinfekl-specific CD8 frequency in PBLs 14 days post 1^stinjection with AS G, AS R, AS S STxB-ova vaccines.

FIG. 20: Humoral response detected 15 days after the second injection performed either 14 or 42 days after the first injection with AS A StxB-ova vaccine.

FIG. 21: Siinfekl-specific CD8 frequency in PBLs 14 days post 1^stinjection with AS G, AS L, AS U, AS V STxB-ova vaccines.

FIG. 22: Siinfekl-specific CD8 frequency in PBLs 14 days post 1^stinjection with ASW1, ASW2-ova vaccines.

EXAMPLES
1. Reagents and Media
1.1 Preparation of Adjuvanted STxB-Ova

STxB coupled to full length Chicken ovalbumin: to allow the chemical coupling of proteins to a defined acceptor site in STxB, a cysteine was added to the C-terminus of the wild-type protein, yielding STxB-Cys. The recombinant mutant STxB-Cys protein was produced as previously described (Haicheur et al.; 2000, J. Immunol. 165, 3301). Endotoxin concentration determined by the Limulus assay test was below 0.5 EU/ml. STxB-ova has been previously described (HAICHEUR et al., 2003, Int. Immunol., 15, 1161-1171) and was kindly provided by Ludger Johannes and Eric Tartour (Curie Institute) .

StxB coupled to full length chicken ovalbumin was formulated in each of the adjuvant systems noted below.

1.2 Galabiose Binding Assay

The Gb3 receptor preferentially recognized by the B subunit of Shiga toxin is a cell surface glycosphingolipid, globotriaosylceramide (Galα1-4Galβ1-4 glucosylceramide), where Gal is Galactose. The method described below is based on that described byTarrago-Trani (Protein Extraction and Purification 38, pp 170-176, 2004), and involves an affinity chromatography on a commercially available galabiose-linked agarose gel (calbiochem). Galabiose (Galα1→4Gal) is the terminal carbohydrate portion of the oligosacharide moiety of Gb3 and is thought to represent the minimal structure recognized by the B subunit of Shiga toxin. This method has been successfully used to purify Shiga toxin directly from E. coli lysate. Therefore it can be assumed that proteins that bind this moiety will bind the Gb3 receptor.

The protein of interest in PBS buffer (500 μl) is mixed with 100 μl of immobilised galabiose resin (Calbiochem) previously equilibrated in the same buffer, and incubated for 30 min to 1 hour at 4° C. on a rotating wheel. After a first centrifugation at 500 rpm for 1 min, the pellet is washed twice with PBS. The bound material is then eluated twice by re-suspending the final pellet in 2×500 μl of 100 mM glycine pH 2.5. Samples corresponding to the flow-through, the pooled washes and the pooled eluates are then analyzed by SDS Page, Coomassie staining and Western blotting. These analytical techniques allow identification of whether the protein is bound to the galabiose, and hence will bind the Gb3 receptor.

1.3—Preparation of Oil in Water Emulsion for Use in Adjuvant Systems.

Preparation of oil in water emulsion followed the protocol as set forth in WO 95/17210. The emulsion contains: 5% Squalene 5% tocopherol 2.0% tween 80; the particle size is 180 nm.

Preparation of Oil in Water Emulsion (2 Fold Concentrate)

Tween 80 was dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100 ml two fold concentrate emulsion 5 g of DL alpha tocopherol and 5 ml of squalene were vortexed until mixed thoroughly. 90 ml of PBS/Tween solution was added and mixed thoroughly. The resulting emulsion was then passed through a syringe and finally microfluidised by using an M110S microfluidics machine. The resulting oil droplets have a size of approximately 180 nm.

1.4—Preparation of Adjuvant Systems.
1.4.1 Adjuvant System A: QS21 and 3D-MPL

A mixture of lipid (such as phosphatidylcholine either from egg-yolk or synthetic) and cholesterol and 3 D-MPL in organic solvent, was dried down under vacuum (or alternatively under a stream of inert gas). An aqueous solution (such as phosphate buffered saline) was then added, and the vessel agitated until all the lipid was in suspension. This suspension was then microfluidised until the liposome size was reduced to about 100 nm, and then sterile filtered through a 0.2 μm filter. Extrusion or sonication could replace this step.

Typically the cholesterol:phosphatidylcholine ratio was 1:4 (w/w), and the aqueous solution was added to give a final cholesterol concentration of 5 to 50 mg/ml.

The liposomes have a defined size of 100 nm and are referred to as SUV (for small unilamelar vesicles). The liposomes by themselves are stable over time and have no fusogenic capacity. Sterile bulk of SUV was added to PBS to reach a final concentration of 10, 20 or 100 μg/ml of 3D-MPL. PBS composition was Na2HPO4: 9 mM; KH2PO4:48 mM; NaCl: 100 mM pH 6.1. QS21 in aqueous solution was added to the SUV. This mixture is referred as DQMPLin. Stx-OVA was then added. Between each addition of component, the intermediate product was stirred for 5 minutes. The pH was checked and adjusted if necessary to 6.1+/−0.1 with NaOH or HCl.

In the experiments described in section 3.1 below, StxB-OVA was at a concentration of 4, 10, 20 or 100 μg/ml and 3D-MPL and QS21 were at a concentration of 10 μg/ml. In these cases, the injection volume of 50 μl corresponded to 0.2-5 μg of STxB-OVA and 0.5 μg of 3D-MPL and QS21. The results for an injection of 0.2 μg of STxB-OVA are shown in FIGS. 1-10. Experiments were also carried out where an injection volume of 50 μl corresponded to 0.5, 1 and 5 μg of STxB-OVA. These experiments gave comparable results to those shown in FIGS. 1 to 10.

In other experiments, StxB-OVA was at a concentration of 20 or 40 μg/ml and 3D-MPL and QS21 were at a concentration of 20 or 100 μg/ml. In these experiments, the injection volume of 25 μl corresponded to 0.5 μg of STXB-OVA and 0.5 μg of 3D-MPL and QS21 (shown in FIGS. 12A and 12B) or 1 μg STxB-OVA and 2.5 μg each 3D-MPL and QS21 (shown in FIGS. 11 and 20)

1.4.2 Adjuvant System B:QS21
1.4.2.1:Adjuvant System B1

The adjuvant was prepared according to the methods used for Adjuvant system A but omitting the 3 D-MPL.

StxB-OVA and QS21 were adjusted at a concentration of 10 or 20 μg/ml.

Injection volumes of 25 or 50 μl corresponded to 0.5 μg of StxB-OVA and 0.5 μg of QS21 (as shown in FIGS. 12A, 12B and 17)

1.4.2.2:Adjuvant system B2

QS21 was diluted at a concentration of 100 μg/ml in PBS pH 6.8 before addition of StxB-OVA to reach a final antigen concentration of 40 μg/ml.

An injection volume of 25 μl corresponded to 1 μg of StxB-OVA and 2.5 μg of QS21 (as shown in FIG. 16)

1.4.3 Adjuvant System C: 3D-MPL
1.4.3.1 :Adjuvant System C1

Sterile bulk of 3D-MPL was diluted at 100 or 200 pg/ml in a sucrose solution at a final concentration of 9.25%. StxB-OVA was added to reach an antigen concentration of 20 or 40 μg/ml.

Injection volume of 25 μl corresponded to 1 μg of StxB-OVA and 5 μg of 3D-MPL (seen in FIG. 16) or 0.5 μg of StxB-OVA and 2.5 μg of 3D-MPL (results not shown, but comparable).

1.4.3.2:Adjuvant System C2

The adjuvant was prepared according to the methods used for Adjuvant system A but omitting the QS21.

StxB-OVA and MPL were adjusted to a concentration of 10 μg/ml.

An injection volume of 50 μl corresponded to 0.5 μg of StxB-OVA and 0.5 μg of MPL.

1.4.4 Adjuvant System D: 3D-MPL and QS21 in an Oil in Water Emulsion

Sterile bulk emulsion prepared as in example 1.3 was added to PBS to reach a final concentration of 250 or 500 μl of emulsion per ml (v/v). 3 D-MPL was then added to reach a final concentration of 50 or 100 μg/ml. QS21 was then added to reach a final concentration of 50 or 100 μg per ml. Between each addition of component, the intermediate product was stirred for 5 minutes. StxB-OVA was then added to reach a final concentration of 10 or 40 μg/ml. Fifteen minutes later, the pH was checked and adjusted if necessary to 6.8+/−0.1 with NaOH or HCl. Injection volume of 25 or 50 μl corresponded to 0.5 or 1 μg of STxB-Ova, 2.5 μg of 3 D-MPL and QS21, 12.5 μl or 25 μ1 of emulsion. An experiment using a 50 μl injection volume is shown in FIG. 11. The experiment using a 25 μl injection volume gave comparable results.

1.4.5 Adjuvant System E: High Dose 3D-MPL and QS21 in an Oil in Water Emulsion.

Sterile bulk emulsion prepared as in example 1.3 was added to PBS to reach a final concentration of 500 μl of emulsion per ml (v/v). 200 μof 3D-MPL and 200 μg QS21 were added. Between each addition of component, the intermediate product was stirred for 5 minutes. StxB-OVA was then added to reach a final concentration of 40 μg/ml.

Fifteen minutes later, the pH was checked and adjusted if necessary to 6.8+/−0.1 with NaOH or HCl.

Injection volume of 25 μl corresponded to 1 μg of STxB-Ova, 5 μg of both immunostimulants and 12.5 μl emulsion.

1.4.6 Adjuvant system F: 3D-MPL and QS21 in an Low Oil in Water Emulsion.

Oil in water emulsion was as in example 1.3 with cholesterol being added to the organic phase to reach a final composition of 1% squalene, 1% tocopherol, 0.4% tween 80, and 0.05% Cholesterol. After formation of the emulsion, 3 D-MPL was then added to reach a final concentration of 100 μg/ml. QS21 was then added to reach a final concentration of 100 μg per ml. Between each addition of component, the intermediate product was stirred for 5 minutes. StxB-OVA was then added to reach a final concentration of 40 μg/ml. Fifteen minutes later, the pH was checked and adjusted if necessary to 6.8+/−0.1 with NaOH or HCl. Injection volume of 25 μl corresponded to 1 μg of STxB-Ova, 2.5 μg of 3 D-MPL and QS21, 2.5 μl emulsion.

1.4.7 Adjuvant System G: CpG2006

Sterile bulk CpG was added to PBS or NaCl 150 mM solution to reach a final concentration of 100 or 200 μg/ml.

StxB-OVA was then added to reach a final concentration of 10 or 20 μg/ml. The CpG used was a 24-mers with the following sequence 5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (Seq ID No. 4). Between each addition of component, the intermediate product was stirred for 5 minutes. The pH was checked and adjusted if necessary to 6.1+/−0.1 with NaOH or HCl.

Injection volume of 50 μl corresponded to 0.5 μg of STxB-Ova and 5 μg of CpG (FIGS. 12A, 12B and 21). Experiments were done with injection volumes of 25 μl (corresponding to 05 μg of STxB-Ova and 5 μg of CpG). Results are not shown but were comparable.

1.4.8 Adjuvant System H: QS21, 3D-MPL and CpG2006

Sterile bulk CpG was added to PBS solution to reach a final concentration of 100 μg/ml. PBS composition was Na₂HPO₄: 9 mM; KH_{2pk PO}₄: 48 mM; NaCl: 100 mM pH 6.1. StxB-OVA was then added to reach a final concentration of 20 μg/ml. Finally, QS21 and 3 D-MPL were added as a premix of sterile bulk SUV containing 3 D-MPL and QS21 referred as DQMPLin to reach final 3D-MPL and QS21 concentrations of 10 μg/ml.

The CpG used was a 24-mers with the following sequence 5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (Seq ID No.4). Between each addition of component, the intermediate product was stirred for 5 minutes. The pH was checked and adjusted if necessary to 6.1+/−0.1 with NaOH or HCl.

Injection volume of 50 μl corresponded to 1 μg of STxB-Ova, 0.5 μg of 3 D-MPL and QS21 and 5 μg of CpG. This formulation was then diluted in a solution of 3D-MPL/QS21 and CpG (at a concentration of 10, 10 and 100 μg/ml respectively) to obtain doses of 0.2, 0.04 and 0.008 μg of StxB-OVA. (these formulations used for experiments shown in FIGS. 1 to 10 and 13) In the experiment shown in FIGS. 12A and 12B, CpG was at a concentration of 100 μg/ml, 3D-MPL and QS21 at a concentration of 10 μg/ml and StxB-OVA at a concentration of 10 μg/ml.

Injection volume of 50 μl corresponded to 0.5 μg of StxB-OVA, 0.5 μg of 3D-MPL and QS21 and 5 μg of CpG.

In one further experiment, CpG was at a concentration of 1000 μg/ml, 3D-MPL and QS21 at a concentration of 100 μg/ml and StxB-OVA at a concentration of 40 μg/ml.

Injection volume of 25 μl corresponded to 1 μg of StxB-OVA, 2.5 μg of 3D-MPL and QS21 and 25 μg of CpG. Results from this experiment are not shown, but are comparable with the results seen with other concentrations of components.

1.4.9 Adjuvant System I: QS21 and CpG2006

Sterile bulk CpG was added to PBS or NaCl 150 mM solution to reach a final concentration of 100 or 200 μg/ml. PBS composition was PO4 10 mM, NaCl 150 mM pH 7.4 or Na2HPO4: 9 mM; KH2PO4: 48 mM; NaCl: 100 mM pH 6.1. StxB-OVA was then added to reach a final concentration of 10 or 20 μg/ml. Finally, QS21 was added as a premix of sterile bulk SUV and QS21 (referred as DQ, prepared as in example 1.3.14) to reach final QS21 concentration of 10 or 20 82 /ml.

Injection volumes of 50 μl corresponded to 0.5 μg of STxB-Ova, 0.5 μg of QS21 and 5 μg of CpG (FIGS. 12A and 12B)

Experiments were also done with injection volumes of 25 μl (corresponding 0.5 μg of STxB-Ova, 0.5 μg of QS21 and 5 μg of CpG). Results are not shown but were comparable.

1.4.10 Adjuvant System J: Incomplete Freunds Adjuvant (IFA)

IFA was obtained from CALBIOCHEM. IFA was emulsified with a volume of antigen using vortex during one minute.

STxB-ova was diluted at 40 μg/ml concentration in PBS pH 6.8 or 7.4 and mixed with 500 μl/ml of IFA either used as such or after a 20-fold dilution in PBS.

Injection volume of 25 μl corresponded to 1 μg of STxB-ova and 12.5 or 0.625 μl of IFA (shown in FIG. 14).

In other experiments, StxB-OVA was diluted at 10 μg/ml in PBS pH 6.8 or 7.4 and mixed with 500 or 250 μl/ml of IFA. Injection volume of 50 μl corresponded to 0.5 μg of StxB-OVA and 12.5 or 25 μl of IFA. These experiments gave comparable results to those shown in FIG. 14.

1.4.11 Adjuvant System K: Oil In Water Emulsion
1.4.11.1 Adjuvant System K1

Sterile bulk emulsion was prepared as in example 1.3 except that 3D-MPL and QS21 were omitted.

Injection volume of 25 μl corresponded to 1 μg of StxB-OVA and 12.5 μl of emulsion. Results are shown as adjuvant system K in FIG. 16.

1.4.11.2 Adjuvant System K2

Sterile bulk emulsion was prepared as in Adjuvant system F except that 3D-MPL and QS21 were omitted.

Injection volume of 25 μl corresponded to 1 μg of StxB-OVA and 2.5 μl of emulsion containing Cholesterol.

Results are not shown, but were comparable to those seen with adjuvant system K1.

1.4.12 Adjuvant system L: Poly I:C

Poly I:C (polyinosinic-polycytidylic acid) is a commercial synthetic mimetic of viral RNA from Amersham. In some experiments, StxB-OVA was diluted in NaCl 150 mM to reach a final concentration of 20 μg/ml. Sterile bulk Poly I:C was then added to reach a final concentration of 20 μg/ml.

Between each addition of component, the intermediate product was stirred for 5 minutes.

Injection volume of 25 μl corresponded to 0.5 μg of STxB-Ova and 0.5 μg of Polyl:C (shown in FIGS. 15 and 21)

In other experiments, StxB-OVA was at a concentration of 10 μg/ml and Poly I:C at a concentration of 20 or 100 μg/ml.

Injection volume of 50 μl corresponded to 0.5 μg StxB-OVA and 1 or 5 μg of Poly I:C.

These experiments gave comparable results to those shown in FIGS. 15 and 21.

1.4.13 Adjuvant System M: CpG5456

StxB-OVA was diluted in NaCl 150 mM to reach a final concentration of 20 μg/ml.

Sterile bulk CpG was then added to reach a final concentration of 200 μg/ml.

The CpG used was a 22-mers with the sequence 5′-TCG ACG TTT TCG GCG CGC GCC G-3′ (CpG 5456). Between each addition of component, the intermediate product was stirred for 5 minutes.

Injection volume of 25 μl corresponded to 0.5 μg of STxB-Ova and 5 μg of CpG.

1.4.14 Adjuvant system N: QS21 and Poly I:C

A mixture of lipid (such as phosphatidylcholine either from egg-yolk or synthetic) and cholesterol in organic solvent, was dried down under vacuum (or alternatively under a stream of inert gas). An aqueous solution (such as phosphate buffered saline) was then added, and the vessel agitated until all the lipid was in suspension. This suspension was then microfluidised until the liposome size was reduced to about 100 nm, and then sterile filtered through a 0.2 μm filter. Extrusion or sonication could replace this step.

Typically the cholesterol:phosphatidylcholine ratio was 1:4 (w/w), and the aqueous solution was then added to give a final cholesterol concentration of 5 to 50 mg/ml. . The liposomes have a defined size of 100 nm and are referred to as SUV (for small unilamelar vesicles). The liposomes by themselves are stable over time and have no fusogenic capacity.

Sterile bulk of SUV was added to PBS to reach a final concentration of 100 μg/ml of MPL. QS21 in aqueous solution was added to the SUV to reach a final QS21 concentration of

100 pg/ml. This mixture of liposome and QS21 is referred as DQ. Sterile bulk Poly I:C (Amersham, as before) was diluted in NaCl 150 mM to reach a final concentration of 20 μg/ml before addition of DQ to reach a final concentration of 20 μg/ml in QS21. StxB-OVA was then added to reach a final concentration of 20 μg/ml. Between each addition of component, the intermediate product was stirred for 5 minutes.

Injection volume of 25 μl corresponded to 0.5 μg of STxB-Ova, 0.5 μg of QS21 and 0.5 μg of Polyl:C.

1.4.15 Adjuvant System O: CpG2006 and Oil in Water Emulsion

Oil in water emulsion was prepared as in example 1.3.

Sterile bulk emulsion was added to PBS to reach a final concentration of 500 μl of emulsion per ml (v/v). CpG was then added to reach a final concentration of 200 μg/ml. Between each addition of component, the intermediate product was stirred for 5 minutes. StxB-OVA was then added to reach a final concentration of 20 μg/ml.

Fifteen minutes later, the pH was checked and adjusted if necessary to 6.8 +/−0.1 with NaOH or HCl.

The CpG used was a 24-mers with the following sequence 5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (Seq ID No.4).

Injection volume of 25 μl corresponded to 0.5 μg of STxB-Ova, 5 μg of CpG and 12.5 μl of emulsion.

1.4.16 Adjuvant System P: CpG2006 and Oil in Water Emulsion

An oil-in-water emulsion was prepared following the recipe published in the instruction booklet contained in Chiron Behring FluAd vaccine.

A citrate buffer was prepared by mixing 36.67 mg of citric acid with 627.4 mg of Na citrate 0.2H2O in 200 ml H2O. Separately, 3.9 g of squalene and 470 mg of Span 85 were mixed under magnetic stirring.

470 mg of Tween 80, was mixed with the citrate buffer. The resulting mixture was added to the squalene/Span 85 mixture and mixed “vigorously” with magnetic stirring. The final volume was 100 ml.

The mixture was then put in the M110S microfluidiser (from Microfluidics) to reduce the size of the oil droplets. A z average mean of 145 nm was obtained with a polydispersity of 0.06. This size was obtained on the Zetasizer 3000HS (from Malvern) using the following technical conditions:

- laser wavelength: 532 nm (Zeta3000HS).
- laser power: 50 mW (Zeta3000HS).
- scattered light detected at 90° (Zeta3000HS).
- temperature: 25° C.,
- duration: automatic determination by the soft,
- number: 3 consecutive measurements,
- z-average diameter: by cumulants analysis

Sterile bulk of the resulting emulsion was added to PBS to reach a final concentration of 500 μl of emulsion per ml (v/v). CpG was then added to reach a final concentration of 200 μg/ml. Between each addition of component, the intermediate product was stirred for 5 minutes. StxB-OVA was then added to reach a final concentration of 20 μg/ml. Fifteen minutes later, the pH was checked and adjusted if necessary to 6.8+/−0.1 with NaOH or HCl.

The CpG used was a 24-mers with the following sequence 5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (Seq ID No.4)

Injection volume of 25 μl corresponded to 0.5 μg of STxB-Ova, 5 μg of CpG and 12.5 μl emulsion.

1.4.17 Adjuvant system Q: CpG2006 and IFA water in oil emulsion IFA, obtained from CALBIOCHEM, was added to PBS to reach a final concentration of 500 μl of emulsion per ml (v/v). CpG was then added to reach a final concentration of 200 μg/ml. Between each addition of component, the intermediate product was stirred for 5 minutes. StxB-OVA was then added to reach a final concentration of 20 μg/ml. Fifteen minutes later, the pH was checked and adjusted if necessary to 7.4+/−0.1 with NaOH or HCl.

The CpG used was a 24-mers with the following sequence 5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (Seq ID No.4)

Injection volume of 25 μl corresponded to 0.5 μg of STxB-Ova and 5 μg of CpG, 12.5 μl emulsion.

1.4.18 Adjuvant System R: CpG2006 and Al(OH)3

Al(OH)₃from Brentag was diluted at final concentration of 1 mg/ml (Al+++) in water for injection. StxB-OVA was adsorbed on Al+++ at a concentration of 20 μg/ml during 30 minutes. CpG was added to reach a concentration of 200 μg/ml and incubated for 30 minutes before addition of NaCl to reach a final concentration of 150 mM. All incubations were performed at room temperature under orbital shacking

The CpG used was a 24-mers with the following sequence 5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (Seq ID No.4)

Injection volume of 25 μl corresponded to 0.5 μg of STxB-Ova, 5 μg of CpG and 25 μg of Al+++.

1.4.19 Adjuvant System S: CpG2006 and AlPO4

AlPO4 from Brentag was diluted at final concentration of 1 mg/ml (Al+++) in water for injection. STxB-OVA was adsorbed on Al+++ at a concentration of 20 μg/ml during 30 minutes. CpG was added to reach a concentration of 200 μg/ml and incubated for 30 minutes before addition of NaCl to reach a final concentration of 150 mM. All incubations were performed at room temperature under orbital shacking The CpG used was a 24-mers with the following sequence 5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (Seq ID No.4)

Injection volume of 25 μl corresponded to 0.5 μg of STxB-Ova, 5 μg of CpG and 25 μg of Al+++.

1.4.20 Adjuvant System T: 3D-MPL and Al(OH)3

Al(OH)3 from Brentag was diluted at a final concentration of 1 mg/ml (Al+++) in water for injection. StxB-OVA was adsorbed on Al+++ at a concentration of 40 or 20 μg/ml during a 30-minute period. 3D-MPL was added to reach a concentration of 100 μg/ml and incubated for 30 minutes before addition of NaCl to reach a final concentration of 150 mM. All incubations were performed at room temperature under orbital shaking

Injection volume of 25 μ/l corresponded to 1 or 0.5 μg of STxB-Ova, 2.5 μg of 3D-MPL and 25 μg of Al+++. Results for 1 μg of STxB-Ova are shown in FIG. 16.

Experiments where 0.5 μg STxB-Ova were injected are not shown, but gave comparable results to that shown in FIG. 16.

1.4.21 Adjuvant System U: TLR2-Ligand

The TLR2 ligand used was a synthetic Pam3CysSerLys4, a bacterial lipopeptide purchased from Microcollections which is known to be TLR2 specific. StxB-OVA was diluted in NaCl 150 mM or in PBS pH 7.4 to reach a final concentration of 10 or 20 μg μg/ml. Sterile bulk Pam3CysSerLys4 was then added to reach a final concentration of 40, 100 and 200 μg/ml. Between each addition of component, the intermediate product was stirred for 5 minutes.

Injection volume of 50 μl corresponded to 0.5 μg of STxB-Ova and 5 or 10 μg of Pam3CysSerLys4. (Results for 5 μg shown in FIG. 21, see section 3.2.9 for discussion of results with other doses of TLR2)

In other experiments, injection volume of 25 μl corresponded to 0.5 μg of StxB-OVA and 1 μg of Pam3CysSerLys4.

1.4.22 Adjuvant System V: TLR7/8 Ligand.

The TLR 7/8 ligand used was an imiquimod derivative known as resiquimod or R-848 (Cayla). R-848 is a low molecular weight compound of the imidazoquinoline family that have potent anti-viral and anti-tumor properties in animal models. The activity of imiquimod is mediated predominantly through the induction of cytokines including IFN-α and IL-12. R-848 is a more potent analogue of imiquimod (Akira, S. and Hemmi, H.; IMMUNOLOGY LETTER, 85, (2003), 85-95). STxB-OVA was diluted in PBS pH 7.4 to reach a final concentration of 10 or 20 μg/ml. Sterile bulk R-848 was then added to reach a final concentration of 20 and 100 μg/ml. Between each addition of component, the intermediate product was stirred for 5 minutes.

Injection volume of 50 μl corresponded to 0.5 μg of STxB-Ova and 1 or 5 μg of R-848.

In other experiment, injection volume of 25 μl corresponded to 0.5 μg of STxB-OVA and 0.5 μg of R-848.

1.4.22 Adjuvant System W: AlPO4.
1.4.22.1 Adjuvant System W1

AlPO4 from Brentag was diluted at final concentration of 0.5 mg/ml (Al+++) in water for injection. STxB-OVA was adsorbed on Al+++ at a concentration of 10 μg/ml during 30 minutes before addition of NaCl to reach a final salt concentration of 150 mM. All incubations were performed at room temperature under orbital shacking

Injection volume of 50 μl corresponded to 0.5 μg of STxB-Ova and 25 μg of Al+++.

1.4.22.2 Adjuvant System W2

AlPO4 from Brentag was diluted in PBS pH 7.4 at final concentration of 0.5 mg/ml (Al+++). STxB-OVA was adsorbed on Al+++ at a concentration of 10 μg/ml during 30 minutes. All incubations were performed at room temperature under orbital shacking

Injection volume of 50 μl corresponded to 0.5 μg of STxB-Ova, 5 μg of CpG and 25 μg of Al+++. Examination by SDS-PAGE as set out in XXXXX indicated that about 70% of the antigen was not adsorbed onto the AlPPO4

1.5 Determination of Level of Adsorbed Antigen in an Antigen/Metal Salt Complex

The formulation of interest is centrifuged for 6 min at 6500 g. A sample of the resulting supernatant is denatured for 5 minutes at 95° C., and loaded onto an SDS-PAGE gel in reducing sample buffer. A sample of the antigen without adjuvant is also loaded. The gel is then run at 200V, 200 mA for 1 hour. The gel is then silverstained according to the Daichi method. Levels of free antigen in the formulation are determined by comparing the sample from the adjuvanted formulation with the antigen without adjuvant. Other techniques that are well known in the art, such as Western blotting, may also be used.

Example 2

Vaccination of C57/B6 mice with vaccines of the invention:

Various formulations as described above were used to vaccinate 6-8 week old C57BL/B6 female mice (10/group). The mice received either one or two injections spaced 14 days apart and were bled during weeks 1, 2, 3 and 8 (for actual bleed days see specific examples) The mice were vaccinated intramuscularly (injection into the left gastrocnemien muscle of a final volume of either 25 μl or 50 μl). The Ovalbumin recombinant adenovirus was injected at a dose varying from 5 10⁷to 10⁸VP.

Ex-vivo PBLs stimulation were performed in complete medium which is RPMI 1640 (Biowitaker) supplemented with 5% FCS (Harlan, Holland), 1 μg/ml of each anti-mouse antibodies CD49d and CD28 (BD, Biosciences), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 μg/ml streptamycin sulfate, 10 units/ml penicillin G sodium (Gibco), 10 μg/ml streptamycin 50 μM B-ME mercaptoethanol and 1000X diluted non-essential amino-acids, all these additives are from Gibco Life technologies. Peptide stimulations were always performed at 37° C., 5% CO2.

2.1 Immunological Assays:
2.1.1 Detection of Antigen-Specific T Cells

Isolation of PBLs and tetramer staining. Blood was taken from retro orbital vein (50 μl per mouse, 10 mice per group) and directly diluted in RPMI + heparin (LEO) medium. PBLs were isolated through a lymphoprep gradient (CEDERLANE). Cells were then washed, counted and finally 1-5 10⁵cells were re-suspended in 50 μl FACS buffer (PBS, FCS1%, 0.002%NaN3) containing CD16/CD32 antibody (BD Biosciences) at 1/50 final concentration (f.c.). After 10 min., 50 μl of the tetramer mix was added to cell suspension. The tetramer mix contains 0.2 μl or 1 μl of siinfekl-H2Kb tetramer-PE from respectively Immunosource or Immunomics Coulter, according to availability.Anti-CD8a-PercP ( 1/100 F.c.) and anti-CD4-APC ( 1/200 F.c.) (BD Biosciences) antibodies were also added in the test. The cells were then left for either 45 minutes at room temperature (for Immunosource tetramer) or 10 minutes at 37° C. (for Immunomics Coulter tetramer) before being washed once and analysed using a FACS Calibur™ with CELLQuest™ software.

2.1.2 Intracellular Cytokine Staining (ICS).

ICS was performed on blood samples taken as described in paragraph 2.1.1. 5 to 10 10⁵PBLs were re-suspended in complete medium supplemented or not with either 1 μg/ml of siinfekl peptide or a pool of 17 15-mer Ova peptides (11 MHC classI-restricted peptides and 6 MHC classII-restricted peptides) present at a concentration of each 1 μg/ml. After 2 hours, 1 μg/ml Brefeldin-A (BD, Biosciences) was added for 16 hours and cells were collected after a total of 18 hours. Cells were washed once and then stained with anti-mouse antibodies all purchased at BD, Biosciences; all further steps were performed on ice. The cells were first incubated for 10 min. in 50 μl of CD16/32 solution ( 1/50 f.c., FACS buffer). 50 μl of T cell surface marker mix was added ( 1/100 CD8a perCp, 1/100 CD4 PE) and the cells were incubated for 20 min. before being washed. Cells were fixed & permeabilised in 200 μl of perm/fix solution (BD, Biosciences), washed once in perm/wash buffer (BD, Biosciences) before being stained at 4° C. with anti IFNg-APC and anti IL2-FITC either for 2 hours or overnight. Data were analysed using a FACS Caliburm with CELLQueSt™ software.

In FIG. 14B, the anti-CD4 antibody was labeled with APC Cy7, the anti-CD8 was labeled with PercP Cy5.5, and an anti-TNFa-PE antibody was included in the cytokine staining step.

2.1.3 Cell Mediated Cytotoxic Activity Detected in vivo (CMC in vivo).

To assess siinfekl-specific cytotoxicity, immunized and control mice were injected with a mixture of targets consisting of 2 differentially CFSE-labeled syngeneic splenocyte and lymphnode populations, loaded or not with 1 nM siinfekl peptide. For the differential labeling, carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes—Palmoski et al. ; 2002, J. Immunol. 168, 4391-4398) was used at a concentration of 0.2 μM or 2.5 μM. Both types of targets were pooled at 1/1 ratio and re-suspended at a concentration of 10⁸targets/ml. 200 μl of target mix were injected per mouse into the tail vein 15 days after 1^stinjection. Cytotoxicity was assessed by FACS^Ranalysis on either draining lymphnode or blood (jugular vein) taken from sacrificed animal at different time points (4,18H or 24H after target injection). The mean percentage lysis of siinfekl-loaded target cells was calculated relative to antigen-negative controls with the following formula:

$lysis % = 100 - (\frac{corrected target (+)}{control target (-)} \times 100)$

$Corrected target += target + \times \frac{(preinj . -)}{(preinj . +)}$

Pre-injected target cells=mix of peptide-pulsed targets (preinj.+) and non-pulsed (preinj.−) targets acquired by FACS before injection in vivo. Corrected target (+)=number of peptide-pulsed targets acquired by FACS after injection in vivo, corrected in order to take into account the number of preinj+cells in the preinjected mix (see above).

2.1.4 Ag Specific Antibody Titer (Individual Analysis of Total IgG): ELISA.

Serological analysis was assessed 15 days and 40 days after second injection. Mice (10 per group) were bled by retro-orbital puncture. Anti-ova total IgG were measured by ELISA. 96 well-plates (NUNC, Immunosorbant plates) were coated with antigen overnight at 4° C. (50 μl per well of ova solution (ova 10 μg/ml, PBS). The plates were then washed in wash buffer (PBS/0.1% Tween 20 (Merck)) and saturated with 100 μl of saturation buffer (PBS/0.1% Tween 20/1% BSA/10% FCS) for 1 hour at 37° C. After 3 further washes in the wash buffer, 100 μl of diluted mouse serum was added and incubated for 90 minutes at 37° C. After another three washes, the plates were incubated for another hour at 37° C. with biotinylated anti-mouse total IgG diluted 1000 times in saturation buffer. After saturation 96w plates were washed again as described above. A solution of streptavidin peroxydase (Amersham) diluted 1000 times in saturation buffer was added, 50 μl per well. The last wash was a 5 steps wash in wash buffer. Finally, 50 μl of TMB (3,3′,5,5′-tetramethylbenzidine in an acidic buffer—concentration of H₂O₂is 0.01%—BIORAD) per well was added and the plates were kept in the dark at room temperature for 10 minutes

To stop the reaction, 50 μl of H₂SO₄0.4N was added per well. The absorbance was read at a wavelength of 450/630 nm by an Elisa plate reader from BIORAD. Results were calculated using the softmax-pro software,

2.1.5 B Cell Elispot

Spleen and bone marrow cells were collected at 78 days after 2^ndinjection and cultured at 37° C. for five days in complete medium supplemented with 3 μg/ml of CpG 2006 and 50 U/ml of rhIL-2 to cause memory B cells to differentiate into antibody-secreting plasma cells. After five days, 96-well filter plates were incubated with ethanol 70% for 10 minutes, washed, and coated with either ovalbumin (50 μg/ml) or an a goat anti-mouse Ig antiserum. They were then saturated with complete medium. Cells were harvested, washed and dispatched on the plates at 2×10⁵cells/well for one hour at 37° C. The plates were then stored overnight at 4° C. The day after, the cells were discarded by washing the plates with PBS Tween 20 0.1%. The wells were then incubated at 37° C. for one hour with an anti-IgG biotynilated antibody diluted in 1/500 PBS, washed and incubated for one hour with extravidin-horseradish peroxidase (4 μg/ml). After a washing step, the spots were revealed by a 10 minute incubation with a solution of amino-ethyl-carbazol (AEC) and H₂O₂and fixed by washing the plates with tap water. Each cell that has secreted IgG or Ova-specific IgG appears as a red spot. The results are expressed as frequency of ova-specific IgG spots per 100 total IgG spots.

3. Results

The results described below show that the efficiency of the STxB system at inducing CD8 responses was dramatically improved by combining it with various adjuvant systems or some of their components.

3.1 Data with Adjuvant Systems A & H
3.1.1 Evaluation of the Primary Response with AS A and AS H

The results obtained show that low dose (0.2 μg) immunization with STxB-ova in the absence of adjuvant does not induce a strong CD8 T cell immune response that can be detected ex-vivo. By contrast, a strong immune response is observed when STXB-OVA is combined with either adjuvant system A or H. Furthermore a clear advantage is demonstrated over the adjuvanted protein.

STxB-ova adjuvanted with adjuvant system A or H is potent at inducing a strong and persistent primary response. It induces high frequency of antigen-specific CD8 T cells (FIG. 1—injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG for AS H. Methods carried out as described in 2.1.1 above, mice were bled at 7 days after 1^stinjection). In addition, FIG. 2 (injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG for AS H. Methods carried out as described in 2.1.1 above, mice were bled at 14 days after 1^stinjection) shows that this siinfekl-specific CD8 response still increases between day 7 and day 14 after injection. This is not observed upon vaccination with the adjuvanted protein, but is rather characteristic of the primary response induced by a live vector such as adenovirus. The primed CD8 T cells are readily differentiated effector T cells, which produce IFNγ whether the stimulation is performed with the immunodominant peptide or a pool of ova peptides (respectively shown in FIGS. 3 and 4, injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG for AS H. Methods carried out as described in 2.1.2 above, mice were bled at 14 days after 1^stinjection). The higher frequency of responder CD8 T cells observed upon restimulation with the peptide pool indicates that the primary CD8 T cell repertoire is not limited to the class 1 immunodominant epitope. In addition, high cytotoxic activity can be detected in vivo only when STxB-ova is adjuvanted (FIG. 5—injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG for AS H. Methods carried out as described in 2.1.3 above at 18 hours following target injection).

Finally the primary response induced by AS H adjuvanted STxB-ova is strongly persistent, as illustrated in FIG. 6B (injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG. methods carried out as described in 2.1.1 above, mice were bled at different time points).

3.1.2 Evaluation of the Secondary Response with AS A and AS H

Combining the STxB toxin delivery system with potent adjuvants also improves amplitude and persistence of the secondary immune response. This is best exemplified by evaluating the response 47 days after the boost. Importantly, the high CD8 response induced by the adjuvanted STxB-OVA is of similar intensity and persistence as that induced by a recombinant adenovirus prime/adjuvanted protein boost strategy (FIG. 6A—injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG for AS H. Methods carried out as described in 2.1.1 above, mice bled 47 days following 2^ndinjection). Regarding effector T-cell population, cytokine-producing T cells are still detected in both CD4 and CD8 T cell compartments (FIGS. 7 and 8—injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG for AS H. Methods carried out as described in 2.1.2 above, mice were bled 47 days following 2^ndinjection, PBLs were stimulated with a pool of ova peptides). Moreover, at this late time point, a cytotoxic activity can still be detected in vivo 4 hours (data not shown), and 24 hours (FIG. 9—injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG for AS H.

Methods carried out as described in 2.1.3 above) after target injection.

The humoral response has been investigated 15 days and 40 days after boost (FIG. 10a—injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG for AS H. Methods carried out as described in 2.1.4 above, results shown through the geomean calculation for each group of 10 mice). In the absence of adjuvant, STxB-ova alone is unable to induce any B cell response. By contrast, equivalent antibody titers are detected whether the adjuvanted protein is coupled to STxB or not at both time points tested.

In FIG. 10B (injections included 0.2 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG. methods carried out as described in 2.1.5 above) the anti-ova memory B cell frequency is shown 78 days post injection. Although the antibody titers detected 15 and 40 days after two injections are equivalent, the quality of the memory B cell response is different as a higher frequency of memory B cells is detected when STxB-ova is adjuvanted as compared to adjuvanted protein. STxB-ova alone is unable to induce memory B cell on its own.

Interestingly, when priming and boost are given 42 days instead of 14 days apart (FIG. 20—injection included 0.5 μg of STXB-OVA and 0.5 μg of 3D-MPL and QS21, methods carried out as in 2.1.4 above), humoral response induced by STxB-OVA AS A is higher than OVA AS A, again suggesting that when combined with adjuvantation, vectorisation may induce a higher frequency of B cell memory cells.

3.1.3 Evaluation of the Immune Response Induced by Low Doses of STxB-OVA Combined with the As H Adjuvant System

FIG. 13 (injections included 0.008, 0.04, 0.2 or 1 μg of STxB-OVA, 0.5 μg of 3D-MPL and QS21, and 5 μg CPG. Methods carried out as described in 2.1.1 above, mice bled 14 days after 1^stinjection) shows that a siinfekl-specific CD8 population can still be detected 14 days after a single injection of doses as low as 8 ng of STxB-ova, corresponding to 4 ng of antigen, formulated in AS H. These results show that the combined use of adjuvant and STxB system could allow a significant reduction of antigen dose without decreasing the induced T cell response.

3.2 Evaluation of the Immune Response Induced by STxB-OVA Combined With Other Adjuvant Systems.

We next wanted to find out whether adjuvant systems other than AS A or AS H could also synergise with the STxB vectorization system.

3.2.1 Evaluation of the Immune Response Following Vaccination with AS A, F, D or E STxB Ova Vaccines.

The evaluation of the primary response clearly indicates that an adjuvanted STxB-ova induces a high frequency of antigen specific TCD8 (FIG. 11—methods carried out as described in 2.1.1 above, mice bled at 13 days after 1^stinjection), whatever the adjuvant system tested. Remarkably, this is seen even with AS D and AS E for which no detectable CD8 response can usually be detected after a single immunization with adjuvanted protein. The adjuvanted STxB-ova strongly primes CD8 T cells which are readily differentiated into cytokine-secreting effector T cells (data not shown).

3.2.2 Evaluation of the Immune Response Induced by STxB-OVA Combined With Individual Components of Adjuvant Systems (3D-MPL-AS C2, QS21-AS B, CpG2006-AS G)

We next evaluated the different component of the previous adjuvant systems in vivo. FIG. 12A (methods carried out as described in 2.1.1 above, mice bled at 15 days after 1^stinjection) shows that the a siinfekl-specific CD8 population can be detected if STxB-ova is adjuvanted with a single immunostimmulant such as QS21 or a TLR9-ligand such as CpG and to a lesser extent with a TLR-4 ligand such as 3 D-MPL (AS C2), this latter immunostimulant been even more efficient when used as higher dose (AS C1) as in FIG. 16. As above, these primed CD8 T cells are readily differentiated cytokine-secreting effector cells (data not shown). The secondary CD8 responses induced by each adjuvant component alone are equivalent, but higher responses are observed when STxB-ova is adjuvanted with a combination of QS21 and at least one TLR ligand (FIG. 12B—methods carried out as described in 2.1.1 above, mice bled at 6 days after 2^ndinjection).

3.2.3 Evaluation of the Immune Response Induced by STxB-OVA Combined with Adjuvant J or Adjuvant K

In contrast to previous published observations, increase of CD8 response is also observed when STxB-OVA is combined with emulsion such as IFA. Formulation with IFA, a water in oil emulsion, increases CD8 responses in a dose dependent manner. Increased frequency of siinfekl-specific CD8 T cells (FIG. 14A) corresponds to improved CD8 effector functions such as cytokine production (FIG. 14B) and cytotoxic activity (FIG. 14C). Similar results are obtained when STxB-ova is combined with an oil in water emulsion

3.2.4 Evaluation of the Immune Response Induced by STxB Ova Combined with Adjuvant System Cl, B, K, F or T

We next evaluated AS T and the different components of adjuvant system F. FIG. 16 shows that when combined to STxB-OVA, each component is able to increase the siinfekl-specific CD8 T response. However, the highest response is observed when the components are associated in the formulation.

3.2.5 Evaluation of the Immune Response Induced by STxB Ova Combined with Adjuvant L, G or M.

FIG. 15 shows that combination of STX-B-OVA with TLR ligands such as poly I:C (TLR3) or CpG sequences (TLR9) representative of categories B and C significantly increases the amplitude of the siinfekl specific CD8 T response.

3.2.6 Evaluation of the Immune Response Induced by STxB Ova Combined with Adjuvant system B, N or I

FIG. 17 shows that CD8 response induced by STxB-OVA is clearly improved when adjuvanted with either QS21 alone or QS21 combined with a TLR3 ligand (poly I:C) or a TLR9 ligand (CpG).

3.2.7 Evaluation of the Immune Response Induced by STxB Ova Combined with Adjuvant System G, 0, P or Q

FIG. 18 shows that the CD8 response induced by STxB-OVA is clearly improved when adjuvanted with either CpG alone or CpG combined with IFA or with different oil-in-water emulsions.

3.2.8 Evaluation of the Immune Response Induced by STxB Ova Combined with Adjuvant System G, R or S

FIG. 19 shows that the CD8 response induced by STX-B-OVA is clearly improved when adjuvanted with either CpG alone or CpG combined with Al(OH)₃or AlPO₄.

3.2.9 Evaluation of the Immune Response Induced by STxB Ova Combined with Adjuvant System G, L, U or V

FIG. 21 shows that, in addition to TLR9 and 3 ligands, combination of STX-B-OVA with TLR2 and TLR7/8 ligands also significantly increases the amplitude of the siinfekl specific CD8 T response. TLR2 ligand was tested at a range of doses from 0.2 to 10 μg. No increase was seen at doses below 5 μg. Interestingly, a reduced response was seen when the dose was increased to 10 μg. This could be explained by the ability of TLR2 ligand to induce regulatory molecules such as IL-10.

3.2.10 Evaluation of the Immune Response Induced by STxB Ova Combined with Adjuvant System W1 or W2.

FIG. 22 shows that the combination of STxB-Ova with AS W1 (which contains aluminium phosphate in a formulation in which the antigen is adsorbed onto the aluminium salt) gives little improvement in the immune response over that seen with unadjuvanted STxB-ova peptide. However, when the composition is formulated such that some of the antigen (in this case about 70%) is not adsorbed onto the aluminium salt, for example by performing the adsorption with aluminium salt dissolved in phosphate buffered saline as is seen in AS W2, then an improvement in immune response is seen over that given by STxB-Ova without adjuvant.

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