Recombinant Flavivirus Vaccines

• US 20080175862A1
• Filed: 04/24/2006
• Published: 07/24/2008
• Est. Priority Date: 04/24/2005
• Status: Active Grant

• Current Assignee: Sanofi Pasteur Biologics, LLC (Sanofi )
• Original Assignee: Acambis, Inc. (Sanofi )
• Inventors: Pugachev, Konstantin V., Monath, Thomas P., Guirakhoo, Farshad
• Granted Patent: US 8,124,398 B2
• Time in Patent Office: Days
• US Class Current: 424/205.1
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2710/16234 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/24122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2770/24134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/24162 by genetic engineering
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides recombinant flavivirus vaccines that can be used in the prevention and treatment of flavivirus infection. The vaccines of the invention contain recombinant flaviviruses including attenuating mutations.

Claims

1. A recombinant flavivirus comprising a mutation that provides a small decrease in viscerotropism to an already attenuated flavivirus.

2. The recombinant flavivirus of claim 1, wherein the flavivirus is a yellow fever virus.

3. The recombinant flavivirus of claim 1, wherein the flavivirus is a chimeric flavivirus.

4. The recombinant flavivirus of claim 3, wherein the chimeric flavivirus comprises the capsid and non-structural proteins of a first flavivirus and the membrane and envelope proteins of a second flavivirus.

5. The recombinant flavivirus of claim 4, wherein the first flavivirus is a yellow fever virus.

6. The recombinant flavivirus of claim 5, wherein the yellow fever virus is YF17D.

7. The recombinant flavivirus of claim 4, wherein the second flavivirus is selected from the group consisting of Japanese encephalitis, dengue-1, dengue-2, dengue-3, dengue-4, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, Ilheus,

Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Alkhurma, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses.

8. The recombinant flavivirus of claim 7, wherein the second flavivirus is a West Nile virus.

9. The recombinant flavivirus of claim 8, wherein the West Nile virus comprises substitutions in envelope amino acids 107, 316, and 440.

10. The recombinant flavivirus of claim 1, wherein the mutation comprises a deletion.

11. The recombinant flavivirus of claim 1, wherein the mutation comprises a substitution.

12. The recombinant flavivirus of claim 1, wherein the mutation is in the 3′

untranslated region of the recombinant flavivirus and comprises less than 30 nucleotides.

13. The recombinant flavivirus of claim 12, wherein the mutation destabilizes a stem structure in the 3′

untranslated region of the virus, or possible alternative elements of predicted secondary structure or overall structure.

14. The recombinant flavivirus of claim 13, wherein the stem structure is in a non-conserved region of the 3′

untranslated region of the virus.

15. The recombinant flavivirus of claim 14, wherein the mutation is selected from the group consisting of d7, dA, dB, dC, and dD.

16. The recombinant flavivirus of claim 12, wherein the mutation comprises one or more nucleotides of conserved sequence 2 (CS2).

17. The recombinant flavivirus of claim 16, wherein the mutation is CS2 d5 or CS2 d16.

18. The recombinant flavivirus of claim 1, wherein the mutation is in the capsid sequence of the recombinant flavivirus.

19. The recombinant flavivirus of claim 18, wherein the mutation comprises a deletion of 1-3 amino acids of the capsid protein.

20. The recombinant flavivirus of claim 19, wherein the mutation comprises a deletion of amino acids in Helix I of the capsid protein.

21. The recombinant flavivirus of claim 20, wherein the mutation is C2.

22. The recombinant flavivirus of claim 1, wherein the mutation comprises a substitution of an envelope amino acid.

23. The recombinant flavivirus of claim 22, wherein the mutation comprises a substitution in an amino acid selected from the group consisting of envelope amino acids 138, 176, 177, 244, 264, and 280, or combinations thereof.

24. The recombinant flavivirus of claim 23, wherein the mutation comprises substitutions in a combination of envelope amino acids selected from the group consisting of 176, 177, and 280;

176, 177, 244, 264, and 280; and

138, 176, 177, and 280.

25. The recombinant flavivirus in claim 9, wherein the mutation comprises a substitution in an amino acid selected from the group consisting of envelope amino acids 138, 176, 177, 244, 264, and 280, or combinations thereof.

26. The recombinant flavivirus of claim 1, wherein the flavivirus comprises a mutation in two or more of the 3′

untranslated region of the virus, the capsid protein, and the envelope protein.

27. The recombinant flavivirus of claim 26, wherein the combinations of different types of mutations are C2+d7, C2+dB, C2+dD, or C2+E#5.

28. The recombinant flavivirus of claim 1, wherein the flavivirus further comprises a mutation in the hinge region of the envelope protein of the flavivirus.

29. The recombinant flavivirus of claim 1, wherein the flavivirus further comprises a mutation in the membrane protein of the flavivirus.

30. The recombinant flavivirus of claim 13, wherein the mutant flavivirus is adapted to a cell culture substrate, resulting in spontaneous modification of the mutation (e.g., deletion), or in a second site mutation, which does not affect attenuation in vivo.

31. The recombinant flavivirus of claim 30, wherein the spontaneous modification increases the original 5-nucleotide deletion in dC mutant to 24 nucleotides.

32. A method of preventing or treating flavivirus infection in a subject, the method comprising administering to the subject a vaccine comprising the recombinant flavivirus of claim 1.

33. A pharmaceutical composition comprising the recombinant flavivirus of claim 1.

34. A nucleic acid molecule comprising the genome of the recombinant flavivirus of claim 1.

35. A method of attenuating a flavivirus vaccine candidate, the method comprising introducing into the flavivirus vaccine candidate a mutation that reduces the viscerotropism of the flavivirus candidate.