THERAPEUTIC VACCINE FOR TREATING OR PREVENTING MERKEL CELL POLYOMA VIRUS-ASSOCIATED TUMORS

Abstract

The invention relates to a therapeutic vaccine useful against tumors exhibiting as signature the Large T antigen (LT) of the Merkel Cell PolyomaVirus (MCPyV). Additionally, the therapeutic vaccine may be also useful for preventing tumors in healthy individuals infected with MCPyV. The therapeutic vaccine involves a type-3 secretion system (T3SS) bacterial vector able to deliver a polypeptide comprising LT epitopes to antigen presenting cells (APCs), such as a truncated form of LT. The invention also relates to a fusion protein comprising a truncated form of LT.

Claims

1. A fusion protein which comprises from its N-terminal end to its C-terminal end and fused in frame: at least one secretion peptide signal able to direct said fusion protein to the type 3 secretion system of a bacterial vector, when said fusion protein is in a bacterial vector owning a type 3 secretion system, and one truncated form of the Large T (LT) antigen of the Merkel Cell Polyoma Virus (MCPyV), which has an amino acid sequence having at least 80% identity with one of the amino acid sequences shown in SEQ ID NO: 1 which starts with the amino acid in any one of positions 1 to 5 and ends with the amino acid in any one of the positions 210 to 469.

2. The fusion protein according to claim 1, wherein the truncated form of the Large T antigen of the Merkel Cell Polyoma Virus has: an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1 which starts with the amino acid in position 2 and ends with the amino acid in position 215 and provided that said truncated form of the Large T antigen of the Merkel Cell Polyoma Virus does not bind to the human retinoblastoma protein, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1 which starts with the amino acid in position 2 and ends with the amino acid in position 270, and provided that said truncated form of the Large T antigen of the Merkel Cell Polyoma Virus does not bind to the human retinoblastoma protein, or one of the amino acid sequences shown in SEQ ID NO: 1 which starts with the amino acid in any one of positions 1 to 5, advantageously in position 2, and ends with the amino acid in any one of positions 210 to 215, so that said truncated form of the Large T antigen of the Merkel Cell Polyoma Virus does not bind to the retinoblastoma protein, or one of the amino acid sequences shown in SEQ ID NO: 1 which starts with the amino acid in any one of positions 1 to 5, advantageously in position 2, and ends with the amino acid in any one of positions 216 to 270, which is further mutated in the human retinoblastoma protein binding site located from positions 212 to 216 in SEQ ID NO: 1, so that said truncated form of the Large T antigen of the Merkel Cell Polyoma Virus does not bind to the human retinoblastoma protein.

3. The fusion protein according to claim 2, wherein the truncated form of the Large T antigen of the Merkel Cell Polyoma Virus has one of amino acid sequences shown in SEQ ID NO: 1 which starts with the amino acid in any one of positions 1 to 5, advantageously in position 2, and ends with the amino acid in position 215.

4. A fusion protein according to claim 2, wherein the truncated form of the Large T antigen of the Merkel Cell Polyoma Virus has: one of the amino acid sequences shown in SEQ ID NO: 1 which starts with the amino acid in any one of positions 1 to 5, advantageously in position 2, and ends with the amino acid in any one of positions 250 to 260, advantageously in position 259, which is further mutated in the human retinoblastoma protein binding site located from positions 212 to 216 in SEQ ID NO: 1, so that said truncated form of the Large T antigen of the Merkel Cell Polyoma Virus does not bind to the human retinoblastoma protein; or an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 1 which starts with the amino acid in position 2 and ends with the amino acid in position 259 and provided that said truncated form of the Large T antigen of the Merkel Cell Polyoma Virus does not bind to the human retinoblastoma protein.

5. The fusion protein according to claim 2, wherein the fusion protein is mutated in the human retinoblastoma protein binding site in position 216 in SEQ ID NO: 1.

6. The fusion protein according to claim 5, wherein the mutation in position 216 in SEQ ID NO: 1 is a substitution mutation replacing the Glu residue by a Lys residue (E216.fwdarw.K).

7. The fusion protein according to claim 1, wherein the secretion peptide signal that is able to direct said fusion protein to the type 3 secretion system of a bacterial vector is the N-terminal moiety of the Pseudomonas exoS gene product and has one of the amino acid sequences shown in SEQ ID NO: 2 which starts with the amino acid in position 1 and ends with the amino acid in any one of positions 15 to 129, advantageously ends with the amino acid in any one of positions 15 to 70, advantageously ends with the amino acid in position 54.

8. The fusion protein according to claim 1, wherein it further comprises a Pan-HLA-DR-binding epitope.

9. A bacterial vector owning a type 3 secretion system which is able to express, secrete and transfer, preferably translocate, into mammalian cells, the fusion protein as defined in claim 1.

10. The bacterial vector according to claim 9, wherein said bacterial vector is a bacterium, and in particular an attenuated bacterium.

11. The bacterial vector according to the claim 10, wherein the said bacterium comprises an expression cassette encoding a fusion protein inserted into the chromosome of the bacterium or inserted into a plasmid, the fusion protein comprising from its N-terminal end to its C-terminal end and fused in frame: at least one secretion peptide signal able to direct said fusion protein to the type 3 secretion system of a bacterial vector, when said fusion protein is in a bacterial vector owning a type 3 secretion system, and one truncated form of the Large T (LT) antigen of the Merkel Cell Polyoma Virus (MCPyV), which has an amino acid sequence having at least 80% identity with one of the amino acid sequences shown in SEQ ID NO: 1 which starts with the amino acid in any one of positions 1 to 5 and ends with the amino acid in any one of the positions 210 to 469.

12. The bacterial vector according to claim 10, wherein the bacterium belongs to the genus of Pseudomonas, in particular wherein the bacterium belongs to the Pseudomonas aeruginosa species or to the Pseudomonas syringuae species.

13. The bacterial vector according to claim 10, which is unable to express at least one of the products chosen among the exoS, exoT, exoU and exoY gene products and NDK cytotoxin, preferably which is unable to express at least the exoS, exoT and exoU gene products.

14. The bacterial vector according to any one of the claims 10 to 13, which comprises an expression cassette, wherein the nucleotide sequence encoding a fusion protein comprising from its N-terminal end to its C-terminal end and fused in frame: at least one secretion peptide signal able to direct said fusion protein to the type 3 secretion system of a bacterial vector, when said fusion protein is in a bacterial vector owning a type 3 secretion system, and one truncated form of the Large T (LT) antigen of the Merkel Cell Polyoma Virus one truncated form of the large T (LT) antigen of the Merkel Cell Polyoma Virus (MCPyV), which has an amino acid sequence having at least 80% identity with one of the amino acid sequences shown in SEQ ID NO: 1 which starts with the amino acid in any one of positions 1 to 5 and ends with the amino acid in any one of the positions 210 to 469 is placed under the control of the exoS promoter.

15. The bacterial vector according to claim 9, for use in a method of preventing or combating MCPyV infection, in particular by promoting a CD8+ immune response against MCPyV-infected cells.

16. The bacterial vector for use according to claim 15, wherein the CD8+ immune response is cytotoxic.

17. The bacterial vector for use according to claim 15, wherein the immune response is against cells (a) expressing whole or part of the MCPyV LT antigen and (b) in the genome of which is integrated an MCPyV nucleotide sequence encoding said whole or part of the LT antigen.

18. The bacterial vector for use according to claim 15, wherein the method of combating or preventing MCPyV infection prevents the onset of a tumoral disorder in patients infected with MCPyV.

19. The bacterial vector for use according to claim 15, wherein the method of combating or preventing MCPyV infection reduces the growth or propagation of a tumor in a patient suffering from a tumoral disorder characterized by the presence of tumoral cells expressing the LT antigen of MCPyV.

20. The bacterial vector according to claim 9, for use as a medicament, preferably for use as a medicament in the treatment of cancer.

Description

FIGURES

[0174] FIG. 1 is a schematic representation of plasmid pEAI-S54 described in Wang et al, 2012.

[0175] FIG. 2 shows a Western blot analysis of the supernatant of an LT.sup.VAX culture, using an LT-specific monoclonal antibody.

[0176] FIG. 3 shows the IFNg release (pg/ml) by NP68-specific CD8 T-cells co-cultivated with DCs previously incubated with LT.sup.VAX, as measured by an ELISA assay.

[0177] FIG. 4 shows the IFNg release (pg/ml) by SIINFEKL-specific CD8 T-cells co-cultivated with B16 LT tumor cells, as measured by an ELISA assay.

[0178] FIG. 5 shows the tumor growth in B16 LT tumor cell-bearing mice treated with LT.sup.VAX.

EXPERIMENTALS

Materials and Methods

DNA Cloning

[0179] DNA sequence that encodes the LT protein of Merkel Cell Polyomavirus (MCPyV) as shown in SEQ ID NO: 1 was optimized in silico for expression in Pseudomonas aeruginosa. The DNA sequence that encodes for the LT truncated form LT(2-215) is shown in SEQ ID NO: 22. The DNA sequence that encodes for the LT truncated and mutated form LT(2-259,E216->K) is shown in SEQ ID NO: 7. These sequences were cloned in the monocistronic pEAI-S54-PADRE plasmid (Wang et al, 2012; FIG. 1) downstream the nucleotide PADRE sequence (SEQ ID NO: 9). This generated the plasmids pEAI-S54-PADRE-LT(2-215) and pEAI-S54-PADRE-LT(2-259, E216->K) able to express the fusion protein S54-PADRE-LT(2-215) and S54-PADRE-LT(2-259, E216->K), respectively.

[0180] DNA sequence (SEQ ID NO: 7) that encodes the amino acid sequence (2-259, E216->K) was also cloned in the same monocistronic pEAI-S54-PADRE plasmid downstream the nucleotide PADRE sequence (SEQ ID NO: 9) together with the sequence encoding the NP68 peptide (as shown in SEQ ID NO: 10). This generated the plasmid pEAI-S54-PADRE-LT(2-259, E216->K)-NP68 able to express the fusion protein S54-PADRE-LT(2-259,E216->K)-NP68. The NP68 peptide (SEQ ID NO 5) which corresponds to amino acids 362-378 of the nucleoprotein (NP) protein of the Influenza virus strain A/Nt/60/68, was used as a tag for monitoring the immune response against the expression product.

[0181] Cloned sequence was verified by DNA sequencing.

[0182] Plasmids pEAI-S54-PADRE-LT(2-215) and pEAI-S54-PADRE-LT(2-259, E216-K) have been used for the secretion assay. These plasmids encode for the fusion protein S54-PADRE-LT(2-215) and S54-PADRE-LT(2-259, E216->K) respectively.

[0183] The plasmid pEAI-S54-PADRE-LT(2-259, E216->K)-NP68 has been used for the generation of LT.sup.VAX as this plasmid permitted the in vitro validation of antigen delivery thanks to the presence of NP68. This plasmid encodes for the fusion protein S54-PADRE-LT(2-259,E216->K)-NP68.

[0184] For the generation of the B16 LT tumor cells, the LT-IRES-GFP plasmid was constructed to express under the control of the EF1a promoter, (i) the LT(1-259, E216->K) tagged in C-ter by the ovalbumine peptide (257-264) SIINFEKL (SEQ ID NO: 6) and (ii) the Green Fluorescent Protein (GFP). In the pCDNA3.1 plasmid (Life Technologies) devoid of the CMV promoter, the following elements were inserted from 5 to 3: the EF1a promoter (SEQ ID NO: 25), the DNA sequence encoding LT(1-259, E216->K) (SEQ ID NO: 23), the DNA sequence encoding the SIINFEKL peptide, an Internal Ribosomal Entry Site (IRES; SEQ ID NO: 24) and the DNA sequence encoding GFP (SEQ ID NO: 21) in order to obtain the LT-IRES-GFP plasmid. The SIINFEKL peptide was used as a tag for monitoring the immune response against the B16 LT cell line.

Generation of LT.SUP.VAX

[0185] The LT.sup.VAX bacterial vector was generated by transforming the P. aeruginosa, attenuated strain CHA-OST (Epaulard et al, 2006) with the plasmids pEAI-S54-PADRE-LT(2-215), pEAI-S54-PADRE-LT(2-259, E-216->K) or the plasmid pEAI-S54-PADRE-LT(2-259, E216->K)-NP68. Briefly, the CHA-OST strain was incubated in Luria Bertani broth (Miller) at 37? C., 225 rpm agitation for 16 hours. 2 ml of bacterial culture were resuspended in 300 mM sucrose solution (Euromedex). After 2 washes, 1/10 of the bacterial population was incubated for 20 minutes at 4? C. with 100 ng of plasmid DNA. Electroporation was performed at 1.8 kV for 5 milli seconds. After a 1 hour incubation, at 37? C., 225 rpm in SOC (Super Optimal Catabolite) medium (Life Technologies), bacteria were plated on Pseudomonas isolation agar (PiA) medium (Gibco) supplemented with 300 ?g/ml carbenicillin (Sigma).

Culture of LT.SUP.VAX

[0186] LT.sup.VAX was incubated at 37? C. with 300 rpm agitation, in a chemically defined medium based on the glucose minimal medium (M9) supplemented with magnesium and calcium, named MM9 medium (Le Gou?llec et al, 2013). Precisely, MM9 contains: extract of synthetic yeast without tryptophan 4 g/L (Sigma); FeSO.sub.4 0.4 g/L (Sigma); glucose 2.5 g/L (Euromedex); glycerol 1% (Euromedex); Citric acid 0.36 g/L (Euromedex); M9 Salts Medium 5? (Sigma).

[0187] The expression of the fusion protein S54-PADRE-LT(2-215), S54-PADRE-LT(2-259, E216-K) S54-PADRE-LT(2-259, E216->K)-NP68 and T3SS was induced by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) and the T3SS function was activated by Ca2+ chelation with ethylene glycol tetraacetic acid (EGTA). Briefly, LT.sup.VAX was then diluted at Optical Density (OD.sub.600) of 0.2 in MM9 medium supplemented with 300 ?g/mL carbenicillin, 1.6 mM IPTG, 5 mM EGTA and 20 mM MgCl.sub.2 (all from Euromedex), and incubated at 37? C. with 300 rpm agitation until OD.sub.600 1.6 was reached. LT.sup.VAX was then resuspended in MM9 medium, ready for in vivo or in vitro use.

Protein Secretion Assay

[0188] The LT.sup.VAX culture was centrifuged at 13 000 g for 10 minutes. In order to precipitate proteins, the supernatant was incubated with 20% (v/v) trichloroacetic acid at 4? C. for 10 minutes, then centrifuged at 15000 g, 4? C. for 5 minutes and washed twice with acetone (Sigma), centrifuging at 15000 g for 5 minutes after each wash. Proteins were resuspended in 80 ?l of denaturation buffer (0.5 M Tris-HCL, 0.6 M DTT, 10% SDS, 0.012% bromophenol blue, 15% glycerol) and migrated in SDS-PAGE in a 10% polyacrylamide gel (Ready Gels Precast Gel, Biorad) following manufacturer's instructions.

Western Blot Analysis

[0189] Proteins were loaded in 12% acrylamide gel (Promega), and run in running buffer (Promega). Protein transfer was performed at 120 V for 60 minutes with the precast system (Promega). Anti-LT antibodies (2000-fold dilution, purchased from Santa Cruz Biotech; ref: sc-136172) and anti-mouse horse radish peroxidase secondary antibody (10.sup.4 dilution, from Dutscher) were used.

Cell Lines and Primary Cells

[0190] B16F0 cells (ATCC-CRL-6322) and B16LT tumor cells were cultured in DMEM medium, supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 U/ml), 1 mM glutamine, and 1 mM sodium pyruvate (all items from Life Technologies).

[0191] Primary dendritic cells (DCs) were obtained as follows: bone marrow from tibia and femurs of C57BL/6 mice was flushed and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES buffer, 50 ?g/ml gentamicin (all from Life Technologies) and 50 ?M 2-Mercaptoethanol (Sigma-Aldrich). Red blood cells were lysed by resuspending pelleted cells in Tris-ammonium chloride (Life Technologies) for 2 minutes. Cells were then resuspended in culture medium and cultured for 9 days at 10.sup.6 cells/ml in six-well plate, at 37? C. and 7% CO.sub.2, in culture medium supplemented with 200 ng/ml recombinant human Flt3 ligand (Amgen) for monocyte differentiation into DCs.

[0192] Activated, anti-NP68 T-cells were obtained from splenocytes of F5 transgenic mice (Mamalaki et al, 1993). Spleen was collected aseptically and splenocytes were cultured in DMEM supplemented with 6% FBS, 2 mM L-glutamine, 10 mM HEPES buffer, 50 ?M 2-Mercaptoethanol, 10% recombinant IL-2 and 10 nM NP68 peptide (ProteoGenix). After 5 days of culture, cells were harvested and cultured for two additional days in culture medium, in the absence of peptide NP68.

[0193] Activated, anti-ovalbumine peptide SIINFEKL T-cells were obtained from splenocytes of OT1 transgenic mice (Hogquist et al, 1994; Clarke et al, 2000). Spleen was collected aseptically and splenocytes were cultured in DMEM supplemented with 6% FBS, 2 mM L-glutamine, 10 mM HEPES buffer, 50 ?M 2-Mercaptoethanol and 10 nM SIINFEKL peptide (ProteoGenix). After 5-day culture, cells were harvested and cultured for two additional days in culture medium, in the absence of peptide SIINFEKL.

In Vitro Validation of LT.SUP.VAX .Antigen Delivery

[0194] Primary dendritic cells (DCs) were seeded in 96-well plate at 10.sup.5 cells/well in 100 ?l of medium. DCs were co-incubated with LT.sup.VAX or BacVac? vector (Pseudomonas strain CHA-OST transformed with the pEAI-S54-PADRE plasmid) at multiplicity of infection of 0.1. After 1 hour, bacteria were removed by performing two washes with DC culture medium, and incubating DCs twice (30 minutes at each time) in medium enriched with gentamycin (Life Technologies). Activated, anti-NP68 T-cells were co-incubated with DCs at ratio 1:1 and incubated at 37? C., 7% CO.sub.2. Co-culture supernatants were collected 16 hours later. IFNg levels measured in the co-culture supernatants were quantified by ELISA (ReD systems) following manufacturer's instructions.

Generation of the B16 LT Tumor Cell Line

[0195] B16F0 tumor cells were transfected with the LT-IRES-GFP plasmid, using the jetPEI? transfection reagent (PolyPlus) following manufacturer's instructions to give B16F0 tumor cells expressing LT(1-259, E216->K) (now called B16 LT tumor cells). Stable plasmid integration in the eukaryotic cell genome was obtained by incubating B16 LT tumor cells in medium supplemented with 10% FBS and 0.1 g/L G418 (Sigma). Surviving, GFP-positive tumor cell clones were isolated.

Validation of the B16 LT GFP-Positive Cells for Use in the Tumor Model

[0196] B16 LT, GFP-positive tumor cells (and B16F0 tumor cells) were respectively co-seeded with activated, SIINFEKL-specific T-cells, at ratio 1:1 and co-incubated at 37? C., 7% CO.sub.2. Co-culture supernatants were collected 16 hours later. IFNg levels were quantified by ELISA (ReD systems) following manufacturer's instructions.

Tumor Challenge Experiment

[0197] Female C57BL/6j mice were purchased from Janvier S A (Le Genest-Saint-Isle, France) and kept under pathogen-free conditions in the animal facility of the University Joseph Fourier (Grenoble, France). Experiments were approved by the Animal Experiment Committee of the Region and were performed in accordance with institutional and national guidelines. 2?10.sup.5 B16 LT tumor cells were resuspended in PBS and administered subcutaneously into the flank of 6-8 week old mice. Mice were monitored for tumor appearance every 24 hours. LT.sup.VAX injection was performed subcutaneously (5*10.sup.5 bacteria for the first two injections, then 10.sup.6 for the following injections) when tumor became palpable (5-7 days post inoculation) and then every three-four days throughout the experiment. Groups of 6 mice per condition were used. Tumor size was measured by using a caliper. Two measures were performed.

Results

LT.SUP.VAX .Efficiently Secretes Fusion Proteins Comprising the Truncated Form of LT (2-215) or the Truncated Form of LT(2-259, E216?K)

[0198] LT.sup.VAX is an immunotherapy product based on the BacVac? technology (Epaulard 2006). LT.sup.VAX was obtained by transforming a BacVac? vector (CHA-OST strain of P. aeruginosa) with a plasmid encoding a fusion product of (i) the P. aeruginosa ExoS peptide (1-54), also called the S54 peptide, and (ii) a mutated, truncated form of the LT antigen of MCPyV, hereinafter called LT (2-259, E216.fwdarw.K). The E216.fwdarw.K mutation was introduced to inactivate RBS so that the LT product (in particular the fusion protein) encoded by the plasmid is substantially non-oncogenic. The encoding sequence was optimized for expression in P. aeruginosa. Alternatively, LT.sup.VAX was obtained by transforming the BacVac? vector with a plasmid encoding a fusion product of (i) the S54 peptide, and (ii) a mutated, truncated form of the LT antigen of MCPyV, hereinafter called LT (2-215) This truncation generates an RBS so that the LT product (in particular a fusion protein) encoded by the plasmid is substantially non-oncogenic. The encoding sequence was optimized for expression in P. aeruginosa.

[0199] LT.sup.VAX were cultured and incubated with (i) IPTG to express the exsA gene product which in turn activates the exoS promoter; and (ii) EGTA, to trigger delivery of the fusion protein S54-PADRE-LT(2-259, E216->K) or S54-PADRE-LT(2-215) via the T3SS. As shown in FIG. 2, Western blot analysis of the supernatant of an LT.sup.VAX culture, using an LT-specific monoclonal antibody, demonstrates that the fusion protein comprising the LT (2-259, E216.fwdarw.K) and the LT(2-215) were efficiently expressed and secreted via the T3SS, thanks to the S54 peptide. Secretion of the fusion protein comprising the LT (2-259, E216.fwdarw.K) and the LT(2-215) were not observed in the control test (BacVac? transformed with the pEAI-S54-PADRE plasmid).

[0200] Moreover, secretion tests as described above have also been performed with the following fusion proteins: [0201] S54-LT(2-817) which corresponds to a fusion protein comprising the S54 peptide and the native form of the LT antigen of the MCPyV having the amino acid sequence shown in SEQ ID NO: 13 which starts to the amino acid in position 2 and ends with the amino acid in position 817. The nucleic sequence shown in SEQ ID NO: 14 which has been optimized for expression in P. aeruginosa has been used for the construction of said fusion protein. The nucleic sequence shown in SEQ ID NO: 8 which encodes for the S54 peptide has also been used for the construction of said fusion protein. [0202] S54-PADRE-sT(2-186) which corresponds to a fusion protein comprising the S54 peptide, the PADRE epitope and the native form of the Small T (sT) antigen of the MCPyV having the amino acid sequence shown in SEQ ID NO: 15 which starts to the amino acid in position 2 and ends with the amino acid in position 186. The nucleic sequence shown in SEQ ID NO: 16 which has been optimized for expression in P. aeruginosa has been used for the construction of said fusion protein. The nucleic sequence shown in SEQ ID NO: 8 which encodes for the S54 peptide and the nucleic sequence SEQ ID NO: 9 which encodes for the PADRE epitope have also been used for the construction of said fusion protein. [0203] S54-PADRE-sT(81-186) which corresponds to a fusion protein comprising the S54 peptide, the PADRE epitope and the sT truncated form having the amino acid sequence shown in SEQ ID NO: 15 which starts to the amino acid in position 81 and ends with the amino acid in position 186. The nucleic sequence shown in SEQ ID NO: 17 which has been optimized for expression in P. aeruginosa has been used for the construction of said fusion protein. The nucleic sequence shown in SEQ ID NO: 8 which encodes for the S54 peptide and the nucleic sequence SEQ ID NO: 9 which encodes for the PADRE epitope have also been used for the construction of said fusion protein. [0204] S54-PADRE-sT(2-141) which corresponds to a fusion protein comprising the S54 peptide, the PADRE epitope and the sT truncated form having the amino acid sequence shown in SEQ ID NO: 15 which starts to the amino acid in position 2 and ends with the amino acid in position 141. The nucleic sequence shown in SEQ ID NO: 18 which has been optimized for expression in P. aeruginosa has been used for the construction of said fusion protein. The nucleic sequence shown in SEQ ID NO: 8 which encodes for the S54 peptide and the nucleic sequence SEQ ID NO: 9 which encodes for the PADRE epitope have also been used for the construction of said fusion protein. [0205] S54-PADRE-sT(81-141) which corresponds to a fusion protein comprising the S54 peptide, the PADRE epitope and the sT truncated form having the amino acid sequence shown in SEQ ID NO: 15 which starts to the amino acid in position 81 and ends with the amino acid in position 141. The nucleic sequence shown in SEQ ID NO: 19 which has been optimized for expression in P. aeruginosa has been used for the construction of said fusion protein. The nucleic sequence shown in SEQ ID NO: 8 which encodes for the S54 peptide and the nucleic sequence SEQ ID NO: 9 which encodes for the PADRE epitope have also been used for the construction of said fusion protein.

[0206] The Small T (sT) antigen is an oncoprotein expressed by MCPyV. The first 79.sup.th amino acid of sT antigen are identical to the first 79.sup.th amino acid of LT antigen.

[0207] Said fusion proteins (S54-LT(2-817)), (S54-PADRE-sT(2-186)), (S54-PADRE-sT(81-186)), (S54-PADRE-sT(2-141)), (S54-PADRE-sT(81-141)) are expressed by the bacterial vector LT.sup.VAX but none of them secrete and transfer outside of the bacterial vector despite the presence of the secretion peptide signal S54.

[0208] These experiments demonstrate that it is not easy to predict whether a fusion protein can be secreted and transferred, preferably translocated, by a bacterial vector owning a type 3 secretion system.

DCs that have Received LT (259, E216.fwdarw.K) Thanks to the Vector LT.sup.VAX are Efficient for LT Presentation to T-Cells

[0209] LT.sup.VAX delivers the fusion protein comprising the LT truncated form (2-259, E216.fwdarw.K) antigen tagged by the NP68 peptide. This peptide is frequently used in immunology research to sensitively trace the immune response. This is possible by using T-cells derived from F5 transgenic mice, which have high frequency of NP68-specific T-cells. This tool is particularly useful as there is no immunomonitoring tool specific for LT available on the market.

[0210] As shown in FIG. 3, IFNg release is significantly higher when activated NP68-specific T-cells were co-cultivated with DCs previously co-cultivated with LT.sup.VAX compared to the negative control (NP68-specific T-cells co-cultivated with DCs previously co-cultivated with BacVac?). This reveals that the fusion protein comprising the LT truncated form (2-259, E216.fwdarw.K) as delivered by LT.sup.VAX to DCs was efficiently processed for presentation to CD8 T-cells.

B16 LT Tumor Cells are Efficient for LT Presentation to T-Cells

[0211] The LT-IRES-GFP plasmid used for the generation of the B16 LT tumor cells expresses an mRNA encoding two distinct proteins: (i) LT (2-259, E216.fwdarw.K) tagged by the SIINFEKL peptide and (ii) GFP. The plasmid also expresses the gene conferring resistance to geneticin. Following the isolation of cells that stably integrated the plasmid in the genome by prolonged treatment with geneticin, GFP-positive clones were isolated.

[0212] The SIINFEKL peptide was tagged to LT (2-259, E216.fwdarw.K) in order to confirm that LT (2-259, E216.fwdarw.K) is expressed and processed by the B16 LT tumor cells for presentation to T-cells. Consistently with this hypothesis, IFNg was released by activated, SIINFEKL-specific CD8 T-cells, when co-incubated with B16 LT tumor cells (FIG. 4). Instead, no IFNg was detected when the same T-cells were co-incubated with the parental tumor cell line (B16F0). This demonstrates that the B16 LT tumor cells properly express LT (2-259, E216.fwdarw.K) and processed it for presentation to CD8 T-cells.

LT.SUP.VAX .is Efficient for Tumor Growth Control

[0213] The efficacy of LT.sup.VAX was demonstrated in an in vivo tumor challenge experiment: B16 LT tumor cells were implanted in mice, and LT.sup.VAX treatment was performed when the tumor became palpable, and then repeated every three-four days throughout the experiment. LT.sup.VAX significantly reduced the tumoral growth (p<0.5, T test), with a reduction of the tumor size of approximately 75% relative to tumor-bearing mice treated with the BacVac? control vector (delivering no antigen), as measured 19 days post tumor implantation (FIG. 5).

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