COMBINED PROPHYLACTIC AND THERAPEUTIC VACCINES

Abstract

The present invention relates to an immunogenic polypeptide comprising (i) a B-cell epitope, (ii) a T-cell epitope, and (iii) a scaffold polypeptide, wherein said scaffold polypeptide is a thioredoxin polypeptide. The present invention further relates to said immunogenic polypeptide for use in medicine and for use in treating and/or preventing inappropriate proliferation of cells and/or infection with an infectious agent, preferably HPV infection, as well as to polypeptides and vectors encoding said immunogenic polypeptide.

Claims

1. An immunogenic polypeptide comprising (i) a B-cell epitope, (ii) a T-cell epitope, and (iii) a scaffold polypeptide, wherein said scaffold polypeptide is a thioredoxin polypeptide.

2. The immunogenic polypeptide of claim 1, wherein at least one of said B-cell epitope and said T-cell epitope is an epitope of an antigen of an infectious agent and/or of a tumor antigen.

3. The immunogenic polypeptide of claim 1, wherein both the B-cell epitope and the T-cell epitope are epitopes of papillomavirus (PV) polypeptides.

4. The immunogenic polypeptide of claim 1, wherein said B-cell epitope and said T-cell epitope are derived from non-identical polypeptides, more preferably are derived from non-homologous polypeptides.

5. The immunogenic polypeptide of claim 1, wherein said T-cell epitope is a peptide derived from an E6 or E7 polypeptide, preferably from an E7 polypeptide.

6. The immunogenic polypeptide of claim 1, wherein said B-cell epitope is a peptide corresponding to amino acids 20 to 38 of the L2 polypeptide of HPV16.

7. The immunogenic polypeptide of claim 1, wherein said immunogenic polypeptide comprises PV L2 N-terminal peptides from at least two, preferably at least four, more preferably at least six, most preferably all HPV genotypes selected from the list consisting of HPV 16, 18, 31, 33, 35, 6, 51, and 59.

8. The immunogenic polypeptide of claim 1, wherein said thioredoxin is a thioredoxin of a thermophilic archaebacterium.

9. The immunogenic polypeptide of claim 1, wherein said immunogenic polypeptide further comprises an oligomerization domain.

10. The immunogenic polypeptide of claim 1, wherein said immunogenic polypeptide comprises the amino acid sequence of SEQ ID NO:3 or a sequence at least 80% identical to said sequence.

11. The immunogenic polypeptide of claim 1, wherein said immunogenic polypeptide comprises the amino acid sequence of SEQ ID NO:4.

12. (canceled)

13. (canceled)

14. A polynucleotide encoding the immunogenic polypeptide according to claim 1, preferably comprising the sequence of SEQ ID NO:33 or a sequence at least 80% identical thereto, more preferably comprising the sequence of SEQ ID NO:34 or a sequence at least 80% identical thereto.

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

21. A composition comprising an anti-PV antibody and/or anti-PV T cells produced or producible by contacting a subject with the immunogenic polypeptide according to claim 1, preferably wherein said subject is non-human.

22. A method of treating and/or preventing inappropriate proliferation of cells and/or infection with an infectious agent, preferably HPV infection, comprising contacting a subject with the immunogenic polypeptide according to claim 1 and, thereby, treating and/or preventing HPV infection.

23. The method of claim 22, wherein said subject is suffering from at least one PV-related lesion.

Description

FIGURE LEGENDS

[0068] FIG. 1: Schematic representation of a preferred embodiment of an immunogenic polypeptide according to the present invention.

[0069] FIG. 2: Number of spot forming units/million cells in the T-cell activation assay of Example 1. A: mice immunized with PfTrx-(OVA257-264)3X-OVX313, B: mice immunized with PfTrx-L2(20-38)8mer-(OVA257-264)3X-OVX313, C: mice immunized with PfTrx-L2(20-38)8mer-OVX313.

[0070] FIG. 3: Anti-HPV16 neutralization titers of mice immunized according to Example 2. Antigens were A: PfTrx-L2(20-38)8mer-OVX313; B: PfTrx-L2(20-38)8mer-(OVA257-264)3X-OVX313).

[0071] FIG. 4: Anti-HPV18 neutralization titers of mice immunized according to Example 3. Antigens were A: PfTrx-L2(20-38)8mer-OVX313; B: PfTrx-L2(20-38)8mer-(OVA257-264)3X-OVX313).

[0072] FIG. 5: Same as FIG. 3, but results from Example 4, i.e. group B was immunized with PfTrx-L2(20-38)8mer-(flankE7(49-57))3X-OVX313).

[0073] FIG. 6: Same as FIG. 4, but results from Example 4, i.e. group B was immunized with PfTrx-L2(20-38)8mer-(flankE7(49-57))3X-OVX313).

[0074] FIG. 7: Tumor growth over time in vaccinated (A) and non-vaccinated (B) mice of Example 6. M1 to M9 indicates numbers of mice.

[0075] FIG. 8: Tumor growth over time in vaccinated (A) and non-vaccinated (B) mice of Example 7. M1 to M12 indicates numbers of mice.

[0076] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1

[0077] 6 to 8 week-old C57BL/6N female mice (6 or 3 per group) were immunized with 20 μg PfTrx-(OVA257-264)3X-OVX313; PfTrx-L2(20-38)8mer-(OVA257-264)3X-OVX313; or PfTrx-L2(20-38)8mer-OVX313 (plus Addavax 50% (v/v) at the base of the tail subcutaneously. 7 days later, splenocytes were obtained, then IFN-gamma Elispot was performed with in vitro stimulation by OVA257-264 peptide. The OVA257-264 peptide is the T-cell epitope from ovalbumin corresponding to amino acids 257-264. OVX313 is the IMX313 oligomerization domain, PfTrx is the thioredoxoin of Pyrococcus furiosus, L2(20-38)8mer is the peptide having the amino acid sequence of SEQ ID NO:1, all as described elsewhere herein. As shown in FIG. 2, the antigen containing both B-cell and T-cell epitopes stimulates an increased anti-OVA T cell response compared to the B-cell epitope only.

EXAMPLE 2

[0078] 6 to 8 week-old BALB/c female mice (10 per group) were intramuscularly immunized 4 times with 2 weeks as immunization interval. The amount of 20 μg different antigens (PfTrx-L2(20-38)8mer-OVX313 or PfTrx-L2(20-38)8mer-(OVA257-264)3X-OVX313) was used with Addavax 50% (v/v) as immune-adjuvant. Sera were collected from mice one month after the last immunization and analyzed against HPV 16 pscudovirions using the L1-PBNA (pseudovirion-based neutralization assay). As shown in FIG. 3, an antigen containing both B-cell and T-cell epitopes produces comparable anti-HPV16 neutralizing antibody titers compared to the antigen with only B cell epitope.

EXAMPLE 3

[0079] 6 to 8 week-old BALB/c female mice (10 per group) were intramuscularly immunized 4 times with 2 weeks as immunization interval. The amount of 20 μg of different antigens (PfTrx-L2(20-38)8mer-OVX313 or PfTrx-L2(20-38)8mer-(OVA257-264)3X-OVX313) was used with Addavax 50% (v/v). Sera collected from mice one month after the last immunization and analyzed against HPV 18 pseudovirions using the L1-PBNA (pseudovirion-based neutralization assay). As shown in FIG. 4, an antigen containing both B-cell and ovalbumin-T-cell epitopes produces comparable anti-HPV18 neutralizing antibody titers compared to the antigen with only B cell epitope.

EXAMPLE 4

[0080] 6 to 8 week-old BALB/c female mice (10 per group) were intramuscularly immunized 4 times with 2 weeks as immunization interval. The amount of 20 μg different antigens (PfTrx-L2(20-38)8mer-OVX313 or PfTrx-L2(20-38)8mer-(flankE749-57)3X-OVX313) was used with Addavax 50% (v/v). Sera collected from mice one month after the last immunization and analyzed against HPV 16 pseudovirions using the L1-PBNA (pseudovirion-based neutralization assay). As shown in FIG. 5, an antigen containing both B-cell and E7-T-cell epitopes produces comparable anti-HPV16 neutralizing antibody titers compared to the antigen with only B cell epitope.

EXAMPLE 5

[0081] The 6 to 8 week-old BALB/c female mice (10 per group) were intramuscularly immunized 4 times with 2 weeks as immunization interval. The amount of 20 μg different antigens (A. PfTrx-L2(20-38)8mer-OVX313; B. PfTrx-L2(20-38)8mer-(flank E749-57)3X-OVX313) was used with Addavax 50% (v/v). Sera collected from mice one month after the last immunization and analyzed against HPV 18 pseudovirions using the L1-PBNA (pseudovirion-based neutralization assay). As shown in FIG. 6, an antigen containing both B-cell and E7-T-cell epitopes produces comparable anti-HPV18 neutralizing antibody titers compared to the antigen with only B cell epitope.

EXAMPLE 6

[0082] 18 C57BL/6N mice were subcutaneously injected with 0.2×10.sup.6 of TC-1 tumor cells (i.e. cells derived from lung epithelium of C57BL/6 mice, transformed with HPV E6, E7 and c-Ha ras) into the right flank. Half of the tumor mice (9 out of 18 mice) were immunized at day 23, 27 and 31 with 20 μg antigen PfTrx-L2(20-38)8mer-(flankE7(49-57))3X-OVX313 (plus Addavax 50% (v/v)) at the base of the tail subcutaneously. The other 9 tumor mice were not vaccinated. Tumor size was measured with a caliper every 3 or 4 days. Mice were sacrificed when the tumor volume was over 1 cm.sup.3 or the tumor diameter was over 1.5 cm. As shown in FIG. 7, vaccinated mice (A) show strongly impeded tumor growth compared to the non-vaccinated controls (B).

EXAMPLE 7

[0083] 24 C57BL/6N mice were subcutaneously injected with 0.2×10.sup.6 of TC-1 tumor cells into the right flank. Half of the tumor mice (12 out of 24 mice) were immunized at day 7 and 12 with 20 μg antigen PfTrx-L2(20-38)8mer-(flankE7(49-57))3X-OVX313 (plus Addavax 50% (v/v)) at the base of the tail subcutaneously. The other 12 tumor mice not vaccinated. Tumor size was measured with a caliper every 3 or 4 days. Mice were sacrificed when the tumor volume was over 1 cm.sup.3 or the tumor diameter was over 1.5 cm. As shown in FIG. 8, vaccinated mice (A) show strongly impeded tumor growth compared to the non-vaccinated controls (B), indicating that two times vaccination is sufficient to induce a strong anti-tumor response.