Chimeric flavivirus vectors

Abstract

The invention provides chimeric flavivirus vectors including foreign peptides inserted into the envelope proteins of the vectors and methods of using these vectors.

1 . A method for identifying a site in the envelope protein of a chimeric flavivirus or a genetically attenuated flavivirus that is permissive for insertion of a foreign peptide of a Hepatitis C virus, a Hepatitis B virus, a Hepatitis A virus, or an Influenza virus, said method comprising the steps of:
 (i) introducing a nucleic acid molecule encoding said foreign peptide into a gene encoding a flavivirus envelope protein;   (ii) generating a flavivirus vector comprising an envelope protein encoded by said gene, wherein said envelope protein comprises said foreign peptide; and   (iii) determining whether the flavivirus vector generated in step (ii) is permissive for said insertion.   
     
     
         2 . The method of  claim 1 , wherein said flavivirus vector is a chimeric flavivirus vector comprising a first flavivirus in which a structural protein or proteins has been replaced with a corresponding structural protein or proteins of a second flavivirus. 
     
     
         3 . The method of  claim 2 , wherein said first flavivirus is selected from the group consisting of Japanese encephalitis, Dengue-1, Dengue-2, Dengue-3, Dengue-4, Yellow fever, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, Ilheus, ticke-borne encephalitis, Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses. 
     
     
         4 . The method of  claim 3 , wherein said first flavivirus is Yellow fever virus. 
     
     
         5 . The method of  claim 2 , wherein said second flavivirus is selected from the group consisting of Japanese encephalitis, Dengue-1, Dengue-2, Dengue-3, Dengue-4, Yellow fever, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, Ilheus, ticke-borne encephalitis, Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses. 
     
     
         6 . The method of  claim 5 , wherein said second flavivirus is Japanese encephalitis virus. 
     
     
         7 . The method of  claim 2 , wherein the structural proteins of the first flavivirus that have been replaced with the corresponding structural proteins of the second flavivirus are membrane and envelope proteins. 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein said nucleic acid molecule is introduced into said envelope gene randomly by transposon mutagenesis. 
     
     
         10 . The method of  claim 1 , wherein determination of whether said flavivirus vector generated in step (ii) is permissive for said insertion is carried out by analysis of (a) the infectivity of said flavivirus vector, (b) the stability of the sequence of said foreign peptide upon multiple passages of the vector, (c) the growth properties of said flavivirus vector, or (d) whether the flavivirus vector can be neutralized with antibodies against the envelope protein of said first flavivirus. 
     
     
         11 . The method of  claim 10 , further comprising comparing the analysis of the flavivirus vector with a similar analysis of the flavivirus from which it was derived. 
     
     
         12 . The method of  claim 1 , wherein said genetically attenuated flavivirus is Yellow Fever YF 17D. 
     
     
         13 . A flavivirus vector comprising an envelope protein that comprises a foreign peptide of Hepatitis C virus, Hepatitis B virus, Hepatitis A virus, or Influenza virus. 
     
     
         14 . The flavivirus vector of  claim 13 , wherein said vector is a chimeric flavivirus comprising a first flavivirus in which a structural protein or proteins have been replaced with a corresponding structural protein or proteins of a second flavivirus. 
     
     
         15 . The flavivirus vector of  claim 14 , wherein said first flavivirus is selected from the group consisting of Japanese encephalitis, Dengue-1, Dengue-2, Dengue-3, Dengue-4, Yellow fever, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, Ilheus, ticke-borne encephalitis, Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses. 
     
     
         16 . The flavivirus vector of  claim 14 , wherein said first flavivirus is Yellow fever virus. 
     
     
         17 . The flavivirus vector of  claim 14 , wherein said second flavivirus is selected from the group consisting of Japanese encephalitis, Dengue-1, Dengue-2, Dengue-3, Dengue-4, Yellow fever, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, Ilheus, ticke-borne encephalitis, Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses. 
     
     
         18 . The flavivirus vector of  claim 17 , wherein said second flavivirus is Japanese encephalitis virus. 
     
     
         19 . The flavivirus vector of  claim 14 , wherein the structural proteins of the first flavivirus that have been replaced with the corresponding structural proteins of the second flavivirus are membrane and envelope proteins. 
     
     
         20 . (canceled) 
     
     
         21 . The flavivirus vector of  claim 13 , wherein said vector comprises a genetically attenuated flavivirus. 
     
     
         22 . The flavivirus vector of  claim 21 , wherein said genetically attenuated flavivirus is Yellow Fever YF 17D. 
     
     
         23 . A pharmaceutical composition comprising the flavivirus vector of  claim 13  and a pharmaceutically acceptable carrier or diluent. 
     
     
         24 . A method of delivering a peptide of a Hepatitis C virus, Hepatitis B virus, Hepatitis A virus, or Influenza virus to a patient, said method comprising administering to said patient the composition of  claim 23 . 
     
     
         25 . (canceled) 
     
     
         26 . A nucleic acid molecule comprising the genome of the flavivirus vector of  claim 13  or the complement thereof. 
     
     
         27 . The method of  claim 1 , wherein said foreign peptide is 10-100 amino acids in length or 20-55 amino acids in length. 
     
     
         28 . The method of  claim 1 , wherein said foreign peptide is of a Hepatitis C virus selected from the group consisting of genotypes 1a, 1b, 2a, 2b, 2c, 3a, 4a, 4b, 4c, and 4d. 
     
     
         29 . The method of  claim 1 , wherein said foreign peptide is of a Hepatitis C virus nucleocapsid protein, E1 glycoprotein, or E2 glycoprotein. 
     
     
         30 . The flavivirus vector of  claim 13 , wherein said foreign peptide is 10-100 amino acids in length or 20-55 amino acids in length. 
     
     
         31 . The flavivirus vector of  claim 13 , wherein said foreign peptide is of a Hepatitis C virus is selected from the group consisting of genotype 1a, 1b, 2a, 2b, 2c, 3a, 4a, 4b, 4c, and 4d. 
     
     
         32 . The flavivirus vector of  claim 13 , wherein said foreign peptide is of a Hepatitis C virus nucleocapsid protein, E1 glycoprotein, or E2 glycoprotein. 
     
     
         33 . The flavivirus vector of  claim 13 , wherein said vector comprises said foreign peptide at a single site in the envelope protein or comprises a multiplicity of said foreign peptides at different sites in the envelope protein. 
     
     
         34 . The flavivirus vector of  claim 33 , wherein said foreign peptide(s) contain a single epitope or said foreign peptide(s) contain multiple epitopes. 
     
     
         35 . The flavivirus vector of  claim 34 , wherein said multiple epitopes are from different species or genotypes. 
     
     
         36 . The flavivirus vector of  claim 35 , wherein said different genotypes are selected from the group consisting of Hepatitis C genotypes 1a, 1b, 2a, 2b, 2c, 3a, 4a, 4b, 4c, and 4d. 
     
     
         37 . The flavivirus vector of  claim 34 , wherein said epitope(s) is a B-cell epitope. 
     
     
         38 . The flavivirus vector of  claim 37 , wherein the B-cell epitope is selected from the group consisting of: ETHVTGGNAGRTTAGLVGLLTPGAKQN (SEQ ID NO:29); IQLINTNGSWHINSTALNCNESLNTGW (SEQ ID NO:30); LFYQHKFNSSGCPERLASCR (SEQ ID NO:31); and PSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPL (SEQ ID NO:32). 
     
     
         39 . The flavivirus vector of  claim 34 , wherein said epitope(s) is a T H  cell or a cytotoxic T-lymphocyte (CTL) epitope. 
     
     
         40 . The flavivirus vector of  claim 39 , wherein the T H  cell epitope is IQLINT (SEQ ID NO:33) or said CTL epitope is selected from the group consisting of: STNPKPQR (SEQ ID NO:15); YLLPRRGPRL (SEQ ID NO:16); GPRLGVRAT (SEQ ID NO:17); YPWPLYGNEGCGWAGWLLSP (SEQ ID NO:18); GFADLMGYIPLVGAPL (SEQ ID NO:19); DLMGYIPLV (SEQ ID NO:20); LLALLSCLTV (SEQ ID NO:21); REGNASRCWVAVTPTVATRD (SEQ ID NO:22); STGLIHLHQ (SEQ ID NO:23); LLADARVCSC (SEQ ID NO:24); CWHYPPRPCGI (SEQ ID NO:25); CVIGGVGNNT (SEQ ID NO:26); RRLTDFAQGW (SEQ ID NO:27); and TINYTIFK (SEQ ID NO:28).