TH64126B - Vaccines against encephalitis and West Nile virus - Google Patents

Abstract

------02/10/2017------(OCR) Page 1 of 1 page Summary of Invention This invention provides for a weakened flavivirus vaccine, i.e. Japanese encephalitis virus and West Nile virus, as well as methods of manufacture and use of these vaccines. ------------ Current invention refers to the attenuated flavovirus vaccine. activities such as vaccines against Japanese encephalitis virus and West Nile virus, as well as the production and use methods of these vaccines.

Claims (5)

Of the membrane protein of Japanese encephalitis virus or West Nile virus, respectively. Page 2 of 4 Page 1 0. The flavivirus of claim 1, further comprising an attenuating mutation in the outer domain (ectodomain) of the membrane protein 1. The flavivirus of claim 10, wherein the outer domain mutation is in an amino acid selected from 1. 2. The group consisting of amino acids 1 to 5 of the outer domain. The flavivirus of claim 11, wherein the outer domain mutation is a substitution in amino acid 5 of the outer domain 1. 3. The flavivirus of claim 12, wherein the outer domain mutation is a substitution of glutamine with proline 1. 4. The flavivirus of claim 1 or 3, wherein the flavivirus further comprising one or more attenuating mutations in the envelope protein. At one or more residues corresponding to the amino acids of the West Nile virus envelope protein selected from the group consisting of amino acids 107, 138, 176, 177, 244, 264, 280, 316 and 440. 1 5. The flavivirus of claim 14, wherein the flavivirus comprises an attenuating mutation of the envelope protein at residues corresponding to the amino acids of the West Nile virus envelope protein 107, 316 and 440. 1 6. The flavivirus of claim 1 or 3, further comprising an attenuating mutation. into a hydrophobic pocket at the hinge region of the envelope protein of Flavivirus 1. 7. The flavivirus of claim 14, wherein the mutation at the hinge region occurs in an amino acid corresponding to amino acid 204 of the envelope protein of Dengue 1 virus 1. 8. The flavivirus of claim 14, wherein the mutation at the hinge region occurs in one or more of the amino acids at the hinge region corresponding to amino acids 48-61, 127-131 and 196-283 of the envelope protein of Yellow Fever virus 1. 9. The flavivirus of claim 1 or 3, further comprising an attenuating mutation, into the 3' non-coding region of Flavivirus 2. 0. The flavivirus of claim 1 or 3, further comprising an attenuating mutation. Into the capsid protein of the flavivirus Page 3 of 4 Page 2 1. A Japanese encephalitis virus vaccine composition comprising the flavivirus of claim 1 and a pharmaceutically acceptable carrier or diluent. 2 2. A method for producing a Japanese encephalitis virus vaccine comprising an attenuated recombinant chimeric flavivirus, the method comprising introducing mutations into the transmembrane domain of the membrane protein of the chimeric flavivirus comprising a yellow fever virus, wherein the membrane protein and envelope protein of the yellow fever virus are replaced by the membrane protein and envelope protein of the Japanese encephalitis virus, wherein said mutation renders the flavivirus attenuated compared to the corresponding flavivirus without the mutation. and further, wherein the mutation is a substitution of amino acid 60 of membrane protein 2. 3. The method of claim 22, wherein the mutation results in increased genetic stability of the flavivirus compared to the corresponding flavivirus without the mutation 2. 4. The method of claim 22, wherein the mutation results in increased multiplication of the flavivirus compared to the corresponding flavivirus without the mutation 2. 5. The nucleic acid molecule coding for the flavivirus genome of claim 1 or 3, or a complement thereof 2. 6. The method for producing the flavivirus of claim 1 or 3, the method comprising introducing into a cell the nucleic acid molecule corresponding to the flavivirus genome. and the separation of the intracellularly produced flavivirus from the cells or supernatant thereof. 2 7. The method of claim 26, wherein the cells are Vero cells. 2 8. The method of claim 26, wherein the cells are grown in serum-free medium. 2 9. The vaccine composition of claim 21, wherein a mutation in the transmembrane domain in the chimeric flavivirus results in the substitution of an arginine by a cysteine at amino acid position 60 of the membrane protein. 3 0. The chimeric flavivirus comprising a mutant YF-17D yellow fever virus, wherein the membrane protein and the envelope protein of the YF-17D yellow fever virus are replaced by the membrane protein and the envelope protein of the SA14-14-2 strain of Japanese encephalitis virus, and said membrane protein of said SA14-14-2 strain of Japanese encephalitis virus in said chimeric flavivirus. Comprising an arginine substitution at amino acid position 60 of the membrane protein 3. 1. The flavivirus of claim 30, wherein the arginine is replaced by cysteine 3. 2. The vaccine composition comprising the flavivirus of claim 30 or claim 31. 3. The West Nile virus immunological composition comprising the flavivirus of claim 3 and a pharmaceutically acceptable carrier or diluent. 3. A method for producing flavivirus, the method comprising cultivating the flavivirus on virocells, wherein the flavivirus comprises the membrane protein and envelope protein of Japanese encephalitis virus (JEV) strain SA14-14-2, and the membrane protein of said JEV strain SA14-14-2. Including the replacement of arginine with cysteine at amino acid position 60 3 5. The method of claim 34, wherein said flavivirus is a chimeric flavivirus comprising a sequence coding for a capsid protein and a non-structural protein. (non-structural protein) of yellow fever virus and the membrane protein and envelope protein of said JEV strain SA14-14-2, comprising said substitutions. 3 6. The method of claim 35, wherein the yellow fever virus is the YF-17D strain. 3 7. The method of claim 34, wherein said flavivirus is an intact JEV virus strain SA14-14-2, comprising said substitutions. 3 8. The method of claim 34, further comprising collecting said flavivirus from said virocells. 3 9. The method of claim 38, further comprising formulating said flavivirus for administration. ------------ Amended August 23, 2017 1. An attenuated chimeric flavivirus comprising a yellow fever virus, wherein the membrane protein and envelope protein of the yellow fever virus are replaced by the membrane protein and envelope protein of Japanese encephalitis virus, wherein the transmembrane domain of the membrane protein of the chimeric flavivirus comprises a mutation, wherein the mutation replaces 60 amino acids of the Japanese encephalitis virus membrane protein. ---------- 1. Recombinant flavovirus comprising a mutation in the membrane protein. 2. The flavovirus of claim 1, wherein the mutation attenuates the flavovirus. 3. The flavovirus of claim 2, wherein the mutation reduces visceral/hemolytic virulence. 4. The flavovirus of claim 1, wherein the mutation increases the stability of the flavovirus. 5. The flavovirus of claim 1, wherein the mutation results in increased viral multiplication compared to the non-mutated flavovirus. 6. The flavovirus of claim 1, wherein the flavovirus is a chimeric flavovirus. 7. The flavovirus of claim 6, wherein the chimeric flavovirus comprises a capsid and non-structural proteins of a first flavovirus, and a cell membrane and/or envelope protein of a second virus. 8. The flavovirus according to claim 7, wherein the first flavovirus is yellow fever virus. 9. The flavovirus according to claim 8, wherein the yellow fever virus is YF-17D1. 0. The flavovirus according to claim 7, wherein the second flavovirus is Japanese encephalitis virus1. 1. The flavovirus according to claim 7, wherein the second flavovirus is West Nile virus1. 2. The flavovirus according to claim 7, wherein the second flavovirus is selected from the group consisting of dengue virus. Encephalitis virus S. t. Louis, Murray Valley encephalitis virus and tick-borne encephalitis virus 1 3. The flavovirus of claim 12, wherein the dengue viruses are dengue-1, dengue-2, dengue-3, dengue-4 viruses 1 4. The flavovirus of claim 1, wherein the mutation is within the transmembrane domain of the membrane protein 1 5. The flavovirus of claim 14, wherein the mutation is a substitution of one or more amino acids corresponding to amino acids 40-75 of the transmembrane helix within the membrane protein of Japanese encephalitis virus or West Nile virus 1 6. The flavovirus of claim 15, wherein the mutation is a substitution of one or more amino acids corresponding to amino acid 60 of the membrane protein of Japanese encephalitis virus 1 7. The flavovirus of claim 16, wherein the mutation results in a substitution of Arginine with cysteine at amino acid position 60 of membrane protein 1. 8. Flavovirus of claim 15, wherein the mutation is a substitution of one amino acid corresponding to amino acid position 66 of West Nile virus membrane protein 1. 9. Flavovirus of claim 18, wherein the mutation results in a substitution of leucine with proline at amino acid position 66 of membrane protein 2. 0. Flavovirus of claim 15, wherein the mutation occurs in one or more amino acids at positions 60, 61, 62, 63, 64, 65, or 66 of the membrane protein of Japanese encephalitis virus or West Nile virus 2. 1. Flavovirus of claim 1, wherein the mutation is external to the domain of membrane protein 2. 2. Flavovirus of claim 21, wherein the mutation is in a selectable amino acid. 3. The flavovirus of claim 22, wherein the mutation is a substitution of one amino acid, the fifth, outside of domain 2. 4. The flavovirus of claim 23, wherein the mutation is a substitution of glutamine with proline. 5. The flavovirus of claim 1, wherein the flavovirus comprises one or more mutations in the envelope protein at residues corresponding to the envelope protein amino acids of West Nile virus selected from the group consisting of amino acids 107, 138, 176, 177, 224, 264, 280, 316, and 440. 6. The flavovirus of claim 25, wherein the flavovirus comprises one or more mutations in the envelope protein at residues corresponding to the envelope protein amino acids of West Nile virus selected from the group consisting of amino acids 107, 138, 176, 177, 224, 264, 280, 316, and 440. Envelop protein at residues corresponding to West Nile virus envelope protein amino acids 107, 316, and 440. 2 7. Flavovirus of claim 25, wherein the flavovirus comprises mutations in residues corresponding to West Nile virus at position 66 of the membrane protein and at positions 107, 316, and 440 of the envelope protein. 2 8. Flavovirus of claim 1, further comprising mutations appearing in the hydrophobic pocket of the hing region within the membrane protein of flavovirus. 2 9. Flavovirus of claim 28, wherein the mutation appears in the amino acid sequence corresponding to amino acid 204 of the dengue virus envelope protein. 1 3 0. Flavovirus of claim 28, wherein the mutation is within the amino acid region. One or more hyphens corresponding to the yellow fever virus envelope protein amino acids 48-61, 127-131, and 196-283. 3 1. The flavovirus of claim 1 further comprises an attenuating mutation in the 3' non-coding region of the flavovirus. 3 2. The flavovirus of claim 1 further comprises an attenuating mutation in the capsid protein of the flavovirus. 3 3. The vaccine composition comprises the flavovirus of claim 1 and a drug-acceptable vector or diluent. 3 4. The flavovirus used as the vaccine composition of claim 33 induces and produces an immune response in a patient. 3 5. The method of claim 34, where the patient is virus-free. But there is a risk of infection with flavovirus 3. 6. Flavoviruses used as a vaccine component according to claim 34, wherein the patient has been infected with flavovirus 3. 7. Vaccine comprising a recombinant virus, wherein there is a method of vaccine production that induces mutations in the membrane protein of flavovirus 3. 8. Use according to claim 37, wherein the mutation attenuates the flavovirus compared to the corresponding non-mutated flavovirus 3. 9. Use according to claim 37, wherein the mutation stabilizes the non-mutated corresponding flavovirus 4. 0. Use according to claim 37, wherein the mutation causes the multiplication of the non-mutated corresponding flavovirus 4. 1. The use according to claim 37, wherein the flavovirus is a chimeric flavovirus 4. 2. A nucleic acid molecule corresponding to the flavovirus genome according to claim 1 or an equivalent thereof. 4. 3. The flavovirus according to claim 1, wherein the method comprises inducing into cells a nucleic acid molecule corresponding to the flavovirus genome and extracting the flavovirus generated in the cells from those cells or supernatants. 4. 4. The use according to claim 43, wherein the cell is a virocell 4. 5. The use according to claim 43, wherein the cells are cultured in a serum-free liquid culture medium.