A menu of mutations was developed that is useful in fine-tuning the attenuation and growth characteristics of dengue virus vaccines.

The application generally relates to the attenuation of a RNA virus or of a clone thereof and involves the alteration of sequence space, more particularly the reduction, of mutational robustness of said RNA virus or clone. The means of the application are more particularly dedicated to the attenuation of an infectious RNA virus or clone, for the production of immunogenic composition or vaccine. More particularly, the means of the application involve the replacement of codon(s) by different codon(s), which is(are) selected to differ by only one nucleotide from a codon STOP, more particularly by different but synonymous codon(s), which is(are) selected to differ by only one nucleotide from a codon STOP.

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 provides for modified Flavivirus such as a modified dengue virus type 1, 2, 3, 4, a combination of these, or a tetravalent combination of these. The modification according to various aspects of the invention results in reduced viral protein expression compared to a parent virus, wherein the reduction in expression is the result of recoding one or more regions of the virus. For example, the prM, or envelope (E) region can be recoded. In various embodiments one or more regions are recoded by reducing the codon pair bias or codon usage bias of the protein-encoding sequence. These modified Flaviviruses are used as vaccine compositions to provide a protective immune response.

Modified genomes of an organism comprising at least one coding sequence comprising at least one mutation, wherein the mutation generates an underrepresented sequence that is underrepresented in the unmodified genome of the organism are provided. Organism and cells comprising the modified genomes of the invention, pharmaceutical compositions comprising the organisms and cells of the invention, as well as methods of making the modified genomes are also provided.

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.

Embodiments herein report compositions, uses and manufacturing of dengue virus constructs and live attenuated dengue viruses. Some embodiments concern a composition that includes, but is not limited to, a tetravalent dengue virus composition. In certain embodiments, compositions can include constructs of one or more serotypes of dengue virus, such as dengue-1 (DEN-1) virus, dengue-2 (DEN-2) virus, dengue-3 (DEN-3) or dengue-4 (DEN-4) virus constructs. In other embodiments, constructs disclosed herein can be combined in a composition to generate a vaccine against more one or more dengue virus constructs that may or may not be subsequently passaged in mammalian cells.

The application relates to the attenuation of a flavivirus, such as a West Nile Virus (WNV), Zika virus (ZIKV), Japanese Encephalitis Virus (JEV), Dengue Virus (DV), in particular DV4, or Usutu virus. The application notably provides a live and attenuated flavivirus, such as a live and attenuated WNV or ZIKV, comprising a mutated M protein. Said mutated M protein comprises or consists of a sequence, wherein at least the amino acids at positions 36 and 43 in said sequence are mutated. The application also provides embodiments deriving from said live and attenuated flavivirus, such as a WNV or ZIKV, such as nucleic acids, cells, cDNA clones, immunogenic compositions as well as uses and methods.

Embodiments herein report compositions, uses and manufacturing of dengue virus constructs and live attenuated dengue viruses. Some embodiments concern a composition that includes, but is not limited to, a tetravalent dengue virus composition. In certain embodiments, compositions can include constructs of one or more serotypes of dengue virus, such as dengue-1 (DEN-1) virus, dengue-2 (DEN-2) virus, dengue-3 (DEN-3) or dengue-4 (DEN-4) virus constructs. In other embodiments, constructs disclosed herein can be combined in a composition to generate a vaccine against more one or more dengue virus constructs that may or may not be subsequently passaged in mammalian cells.

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.