The present invention relates to attenuated, immunogenic West Nile virus chimeras built on a dengue virus backbone for the production of immunogenic, live, attenuated West Nile virus vaccines.

The invention is related to a dengue virus or chimeric dengue virus that contains a mutation in the 3 untranslated region (3-UTR) comprising a ?30 mutation that removes the TL-2 homologous structure in each of the dengue virus serotypes 1, 2, 3, and 4, and nucleotides additional to the ?30 mutation deleted from the 3-UTR that removes sequence in the 5 direction as far as the 5 boundary of the TL-3 homologous structure in each of the dengue virus serotypes 1, 2, 3, and 4, or a replacement of the 3-UTR of a dengue virus of a first serotype with the 3-UTR of a dengue virus of a second serotype, optionally containing the ?30 mutation and nucleotides additional to the ?30 mutation deleted from the 3-UTR; and immunogenic compositions, methods of inducing an immune response, and methods of producing a dengue virus or chimeric dengue virus.

Nucleic acid molecules comprising a coding sequence with at least one codon substituted to a synonymous codon, a modified form of a virus comprising the nucleic acid molecules of the invention, and methods for producing these nucleic acid molecules, and viruses, are provided.

Attenuated viruses and methods of designing them are disclosed. In one embodiment, there is disclosed an attenuated form of a virulent virus comprising an RNA encoding a viral protein or a nucleic acid sequence transcribable to said RNA, wherein the folding energy or structure of the RNA is changed at positions of evolutionarily conserved RNA structures with respect to that of said RNA encoding said viral protein in the virulent virus so as to bring about attenuation of the virus.

Methods of producing a pathogen with reduced replicative fitness are disclosed, as are attenuated pathogens produced using the methods. In particular examples, the method includes deoptimizing one or more codons in a coding sequence, thereby reducing the replicative fitness of the pathogen. Methods of using the attenuated pathogens as immunogenic compositions are also disclosed.

The present invention relates to deoptimized Yellow Fever viruses and their uses for the treatment of various forms of malignant tumors, and as vaccines against Yellow Fever. The method of the present invention is particularly useful for the treatment of malignant tumors in various organs, such as: breast, skin, colon, bronchial passage, epithelial lining of the gastrointestinal, upper respiratory and genito-urinary tracts, liver, prostate and the brain.

Methods of producing a pathogen with reduced replicative fitness are disclosed, as are attenuated pathogens produced using the methods. In particular examples, the method includes deoptimizing one or more codons in a coding sequence, thereby reducing the replicative fitness of the pathogen. Methods of using the attenuated pathogens as immunogenic compositions are also disclosed.

The invention provides a recombinant West Nile virus, in which the amino acid sequence of envelope E protein is genetically modified to attenuation, and its RNA genome is inserted with a foreign gene fragment. The engineered E gene contains the mutation of five amino acids for reducing its neural virulence to the central nervous system; the integrated foreign gene between E and S1 gene makes a new chimerical virus. Thus, the present invention provides the application of this attenuated West Nile virus as a vaccine in preventive medicine and the application of the RNA-viral vector as a novel gene-drug in the pharmaceutical industry. The newly attenuated virus may fill the gap of no live-attenuated vaccine to the West Nile virus epidemic. The attenuated and recombinant virus can be used as an RNA oncolytic virus to target solid tumors, especially neural tumors, for cancer therapy with higher safety.

The invention is related to a dengue virus or chimeric dengue virus that contains a mutation in the 3 untranslated region (3-UTR) comprising a ?30 mutation that removes the TL-2 homologous structure in each of the dengue virus serotypes 1, 2, 3, and 4, and nucleotides additional to the ?30 mutation deleted from the 3-UTR that removes sequence in the 5 direction as far as the 5 boundary of the TL-3 homologous structure in each of the dengue virus serotypes 1, 2, 3, and 4, or a replacement of the 3-UTR of a dengue virus of a first serotype with the 3-UTR of a dengue virus of a second serotype, optionally containing the ?30 mutation and nucleotides additional to the ?30 mutation deleted from the 3-UTR; and immunogenic compositions, methods of inducing an immune response, and methods of producing a dengue virus or chimeric dengue virus.

Provided is a vaccine combination against multiple dengue virus serotypes and preparation thereof. The vaccine combination includes a first vaccine and a second vaccine, wherein the first vaccine includes a live-attenuated dengue virus and a live-attenuated chimeric dengue virus, and the second vaccine includes a plurality type of recombinant flagellin and envelope domain III fusion proteins, wherein an envelope domain III of each type of the recombinant flagellin and envelope domain III fusion proteins is derived from a different dengue virus serotype. Also provided is a method of preventing or treating viral infection by multiple dengue virus serotypes in a subject using the vaccine combination, including the steps of administering the first and then the second vaccines at a time interval of about 1-5 weeks.