This invention provides an attenuated virus comprising a modified viral genome engineered to containing multiple nucleotide substitutions that reduce the codon pair bias of a virus protein encoding sequence relative to a first host while the codon pair bias relative to a second host is not substantially reduced. In another embodiment, the invention provides an attenuated virus comprising modified viral genome engineered to containing multiple nucleotide substitutions that reduce the codon pair bias of a virus protein-encoding sequence relative to a first host and a second host. The attenuated virus may be used in a vaccine composition for inducing a protective immune response in a subject. The invention also provides a method of synthesizing the attenuated virus. Further, this invention further provides a method for preventing a subject from becoming afflicted with a virus-associated disease comprising administering to the subject a prophylactically effective dose of a vaccine composition comprising the attenuated virus.

The instant invention discloses a closed-system, serum-free, microcarrier-based up-stream process to produce dengue virus. The process comprises a closed-system cell expansion comprising cell passages taking place in closed-system containers. These cell expansions represent a closed process, with replacement media, a cell-detachment agent, and quench medium added though sterile, weldable tubing (a closed-system environment) to eliminate all open aseptic processing following the initial vial thaw of the adherent cells. The viral production takes place in a closed-system bioreactor in adherent cell culture grown on microcarriers providing sufficient cell mass to support the production of dengue virus.

The invention provides flavivirus vaccines and methods of making and using these vaccines.

The present disclosure relates to attenuated Zika viruses and vaccines, attenuated chimeric Zika viruses and vaccines, and to multivalent immunogenic compositions comprising Zika vaccines and vaccines to other flaviviruses. The chimeric Zika viruses includes a first nucleotide sequence encoding at least one structural protein from a Zika virus (ZIKV), a second nucleotide sequence encoding at least one nonstructural protein from a first flavivirus, and a third nucleotide sequence of a 3 untranslated region from a second flavivirus. The multivalent immunogenic compositions comprise an attenuated ZIKV vaccine or an attenuated chimeric ZIKV vaccine (or their combination) together with one or more of a first attenuated virus that is immunogenic against dengue serotype 1, a second attenuated virus that is immunogenic against dengue serotype 2, a third attenuated virus that is immunogenic against dengue serotype 3, and a fourth attenuated virus that is immunogenic against dengue serotype 4. The present disclosure further relates to methods of inducing immune responses, as well as preventing or treating ZIKV infections, and in certain embodiments, the combined prevention or treatment of ZIKV and another flavivirus, e.g., dengue virus.

The subject application provides a genetically modified recombinant facultative intracellular invasive bacterial pathogen (RFIIBP) or a recombinant attenuated Salmonella vaccine (RASV) strains encoding Zika virus (ZIKV) antigens. These strains exhibit high immunogenicity, complete safety, and attenuation, and are unable to persist or be shed in an infective or viable form. These RFIIBPs and RASVs also exhibit regulated delayed attenuation in vivo, regulated delayed in vivo synthesis of protective ZIKV antigens. Methods of inducing mucosal, systemic and cellular immunities in hosts are also provided and antibodies produced using the disclosed RFIIBPs and RASVs can comprise neutralizing antibodies against ZIKV that do not crossreact with Dengue virus (DENV).

The present invention relates to a genomic sequences encoding for an attenuated mutant Zika virus. The inventors have introduced some specific substitutions at very specific positions in the epidemic genomic sequence for restoring some fixation sites for miR-4279 that were originally present in the endemic genomic sequence. Moreover the inventors have additionally introduced mutation leading to the abrogation of the N-glycosylation site on the E protein which will prevent the generation of auto-antibodies responsible for Guillain-Barre syndrome. The inventors have produced additional mutations of the virus that result to a dramatic reduction of the cytopathic effects without affecting the capacity to produce high titers of virus. In particular the present invention relates to a genomic sequence characterized by the sequence represented by SEQ ID NO:1 wherein at least one site of fixation for miR-4279 is restored.

The present invention provides methods for large-scale flaviviral vaccine production and manufacture. The methods provided herein are specifically contemplated for large-scale production and manufacture of live, attenuated flaviviral vaccines such as live, attenuated, dengue virus vaccines. Further, the methods provided herein pertain to formulation of live, attenuated, monovalent, divalent, trivalent, or tetravalent viral vaccine products.

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 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.

The invention provides flavivirus vaccines and methods of making and using these vaccines.

This invention provides an attenuated virus comprising a modified viral genome engineered to containing multiple nucleotide substitutions that reduce the codon pair bias of a virus protein encoding sequence relative to a first host while the codon pair bias relative to a second host is not substantially reduced. In another embodiment, the invention provides an attenuated virus comprising modified viral genome engineered to containing multiple nucleotide substitutions that reduce the codon pair bias of a virus protein-encoding sequence relative to a first host and a second host. The attenuated virus may be used in a vaccine composition for inducing a protective immune response in a subject. The invention also provides a method of synthesizing the attenuated virus. Further, this invention further provides a method for preventing a subject from becoming afflicted with a virus-associated disease comprising administering to the subject a prophylactically effective dose of a vaccine composition comprising the attenuated virus.