MERITS ANDRES (EE)
ZUSINAITE EVA (EE)
UNIV TARTU (EE)
| WO2018138727A1 | 2018-08-02 | |||
| WO2019172982A1 | 2019-09-12 | |||
| WO2019126690A1 | 2019-06-27 |
MARTINEZ, M. ET AL.: "Synonymous virus genome recoding as a tool to impact viral fitness", TRENDS IN MICROBIOLOGY, vol. 24, no. 2, 2016, pages 134 - 147, XP029396444, DOI: 10.1016/j.tim.2015.11.002
QUAX, T. ET AL.: "Codon bias as a means to fine-tune gene expression", MOLECULAR CELL, vol. 59, no. 2, 2015, pages 149 - 161, XP055704582, DOI: 10.1016/j.molcel.2015.05.035
See also references of EP 3866847A4
| CLAIMS 1 . Live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid comprising a partly codon deoptimized Zika viral genome. 2. A recombinant, isolated or substantially purified nucleic acid comprising a partly codon deoptimized Zika viral genome or partly codon deoptimized region thereof. 3. A vector containing the nucleic acid of claim 2. 4. A cell or isolate containing the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the nucleic acid of the claim 2, or the vector of claim 3. 5. A vaccine comprising the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, or the cell or isolate of claim 4. 6. A pharmaceutical preparation comprising the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, or the cell or isolate of claim 4. 7. An immunogenic composition comprising the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, or the cell or isolate of claim 4. 8. A method of (1 ) treating a subject having a natural Zika viral infection, (2) reducing the severity of a natural Zika viral infection in a subject, or (3) preventing a subject from contracting a Zika viral infection naturally, said method comprising the step of administering to the subject: the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 ; the recombinant, isolated or substantially purified nucleic acid of claim 2; the vector of claim 3; the cell or isolate of claim 4; the vaccine of claim 5; the pharmaceutical preparation of claim 6; or the immunogenic composition of claim 7. 9. Use of: the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 ; the recombinant, isolated or substantially purified nucleic acid of claim 2; the vector of claim 3; the cell or isolate of claim 4; the vaccine of claim 5; the pharmaceutical preparation of claim 6; or the immunogenic composition of claim 7, in the preparation of a medicament for (1 ) treating a subject having a natural Zika viral infection, (2) reducing the severity of a natural Zika viral infection in a subject, or (3) preventing a subject from contracting a Zika viral infection naturally. 10. A method of generating a live attenuated Zika virus vaccine, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, or recombinant, isolated or substantially purified nucleic acid comprising a partly codon deoptimized Zika viral genome or partly codon deoptimized region thereof, comprising the step of partly codon deoptimizing a Zika viral genome. 1 1 . A method of preparing a vaccine comprising live attenuated recombinant Zika virus, said method comprising the steps of: (1 ) codon deoptimizing a Zika viral genome to produce a partly codon deoptimized live attenuated Zika virus; and (2) enabling the partly codon deoptimized live attenuated Zika virus to replicate. 12. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized Zika viral genome comprises at least about 200 codon changes compared with wild-type or virulent Zika virus. 13. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized Zika viral genome comprises no more than about 800 codon changes, compared with wild-type or virulent Zika virus. 14. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized Zika viral genome comprises between about 200 and about 800 codon changes, compared with wild-type or virulent Zika virus. 15. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized Zika viral genome comprises a minimum of about 286 codon changes, compared with wild-type or virulent Zika virus. 16. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized Zika viral genome comprises a maximum of about 651 codon changes, compared with wild-type or virulent Zika virus. 17. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized Zika viral genome comprises between about 286 and 651 codon changes in the viral genome, compared with wild-type or virulent Zika virus. 18. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein some or all codon changes of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus are situated immediately next to one another, in sequence. 19. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein some or all codon changes of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus are spaced apart from each other such that they are not situated immediately next to one another, in sequence. 20. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein some codon changes of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus are spaced apart from each other and some of the codon changes are situated immediately next to one another. 21 . The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein codon deoptimization occurs in no less than about a 1700 nucleotide region of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus, and optionally the 1700 nucleotide region is continuous/contiguous or the 1700 nucleotide region is not continuous/not contiguous. 22. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein codon deoptimization occurs in no more than in about a 7900 nucleotide region of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus, and optionally the 7900 nucleotide region is continuous/contiguous or the 7900 nucleotide region is not continuous/not contiguous. 23. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein codon deoptimization occurs in a continuous region of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus with a length of about 1800 to about 3600 nucleotides. 24. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein codon deoptimization results in no less than about an 1800 nucleotide region of the genome compared with wild-type or virulent Zika virus, with no less than about 250 codon changes within that nucleotide region. 25. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein codon deoptimization results in no more than about a 7900 nucleotide region of the genome compared with wild-type or virulent Zika virus, with no more than about 800 codon changes within that nucleotide region. 26. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein about 20-60% of the coding region of the genome is codon deoptimized compared with wild-type or virulent Zika virus, preferably 18-36% of the genome, compared with wild- type or virulent Zika virus. 27. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the non-structural region of the viral genome is codon deoptimized. 28. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein only the non-structural region of the viral genome is codon deoptimized. 29. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein every 3rd or 4th codon is deoptimized along the nonstructural ZIKV coding region. 30. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein any one or more of the genes NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5 are codon deoptimized. 31 . The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein any contiguous genome region from the NS1 to NS5 region corresponding to at least 600 amino acid residues of viral polyprotein is codon deoptimized. 32. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the genes NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5 are codon deoptimized. 33. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the genes NS1 , NS2A, NS2B and NS3 are codon deoptimized. 34. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the gene NS3 is codon deoptimized. 35. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein approximately 700 codon substitutions are made along the entire nonstructural ZIKV coding region compared with wild-type or virulent Zika virus. 36. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimization results in slower polyprotein translation leading to slower replication and, as a result, in attenuation of the virus, compared with wild-type or virulent Zika virus. 37. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein a codon for an amino acid with high codon degeneracy is changed to a synonymous codon that is used least frequently or rarely in the genome of Homo sapiens. 38. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the genome region most rich in codons that can be substituted for rare codon variants is codon deoptimized. 39. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the viral genome prior to codon deoptimization has a very similar nucleotide sequence to a Zika strain associated with microcephaly, preferably Brazilian Zika virus (ZIKV) strain Bel-1819016. 40. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has the deoptimized codons of the NS3 region of vaccine candidate ZIKV-DO-NS3 represented by SEQ ID NO:3, 4, 5 or 10. 41 . The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has about 200 or more of the codon changes of the NS3 region of vaccine candidate ZIKV-DO-NS3 represented by SEQ ID NO:3, 4, 5 or 10. 42. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has the deoptimized codons of the NS1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 regions of the vaccine candidate Z I KV- DO-scattered represented by SEQ ID NO:6, 7 or 1 1 . 43. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has about 200 or more of the codon changes of the NS1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 regions of the vaccine candidate ZIKV- DO-scattered represented by SEQ ID NO:6, 7 or 1 1 . 44. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has the deoptimized codons of the NS1 , NS2A, NS2B and/or NS3 regions of the vaccine candidate ZIKV-DO represented by SEQ ID NO:8, 9 or 12. 45. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has about 200 or more of the codon changes of the NS1 , NS2A, NS2B and/or NS3 regions of the vaccine candidate ZIKV-DO represented by SEQ ID NO:8, 9 or 12. 46. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has the deoptimized codons of the nonstructural region represented by SEQ ID NO:1 or as shown in Figure 1 b. 47. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has 1 or more of the codon changes represented by SEQ ID NO:1 . 48. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has at least 90 percent of the deoptimized codons of the NS3 region of vaccine candidate ZIKV-DO-NS3 represented by SEQ ID NO:3, 4, 5 or 10. 49. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has at least 90 percent of the deoptimized codons of the NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5 regions of the vaccine candidate ZIKV- DO-scattered represented by SEQ ID NO:6 or 7. 50. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the codon deoptimized genome has at least 90 percent of the deoptimized codons of the NS1 , NS2A, NS2B and NS3 regions of the vaccine candidate ZIKV-DO represented by SEQ ID NO:8 or 9. 51 . The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of claim 1 , the recombinant, isolated or substantially purified nucleic acid of claim 2, the vector of claim 3, the cell or isolate of claim 4, the vaccine of claim 5, the pharmaceutical preparation of claim 6, the immunogenic composition of claim 7, the method of claim 8, the use of claim 9, or the method of claim 10 or 1 1 , wherein the vaccine, pharmaceutical preparation or immunogenic composition comprises a delivery system, carrier or aid. |
TECHNICAL FIELD
[0001 ] This invention generally relates to a codon deoptimized Zika virus genome. In particular, embodiments of the invention concern a vaccine comprising live attenuated Zika virus comprising a partly codon deoptimized viral genome, a Zika virus comprising a partly codon deoptimized viral genome, as well as their use in methods of treatment and prevention of viral infection.
BACKGROUND ART
[0002] Zika virus (ZIKV) has very recently emerged as a major human pathogen (Baud D, Gubler DJ, Schaub B, Lanteri MC, Musso D. An update on Zika virus infection. Lancet. 2017 Nov 4;390(10107):2099-2109). It is a mosquito-transmitted member of the Flavivirus genus first isolated in 1947 in Uganda from a rhesus monkey. The first human infection was recorded in 1954, but since then human infections have been reported only rarely. Since 2007 there have been a number of outbreaks in the Pacific of varying severity affecting at least 10 island nations. A particularly explosive outbreak occurred in French Polynesia in 2013 with more than 30,000 cases (Cao-Lormeau VM, Roche C, Teissier A, Robin E, Berry AL, Mallet HP, Sail AA, Musso D. Zika virus, French Polynesia, South pacific, 2013. Emerg Infect Dis. 2014 Jun;20(6):1085-6; Musso D, Nilles EJ, Cao-Lormeau VM. Rapid spread of emerging Zika virus in the Pacific area. Clin Microbiol Infect. 2014 Oct;20(10):O595-6. doi: 10.1 1 1 1/1469- 0691 .12707. Epub 2014 Aug 4). ZIKV subsequently emerged and spread rapidly and extensively in the Americas, starting from 2015 (Zanluca C, Melo VC, Mosimann AL, Santos Gl, Santos CN, Luz K. First report of autochthonous transmission of Zika virus in Brazil. Mem Inst Oswaldo Cruz. 2015 Jun;1 10(4):569-72). ZIKV infections are most commonly asymptomatic. Symptomatic ZIKV infections are generally mild, with fever and rash being the dominant signs (Baud D, Gubler DJ, Schaub B, Lanteri MC, Musso D. An update on Zika virus infection. Lancet. 2017 Nov 4;390(10107):2099-2109).
[0003] ZIKV has emerged as an important human pathogen due to its neurotropism, resulting in an increased incidence of neurological malformation, in particular, microcephaly of the developing foetus and its association with post-infectious Guillain-Barre syndrome (Kleber de Oliveira W, Cortez-Escalante J, De Oliveira WT, do Carmo GM, Henriques CM, Coelho GE, Araiijo de Franga GV. Increase in Reported Prevalence of Microcephaly in Infants Born to Women Living in Areas with Confirmed Zika Virus Transmission During the First Trimester of Pregnancy - Brazil, 2015. MMWR Morb Mortal Wkly Rep. 2016 Mar 1 1 ;65(9):242- 7. doi: 10.15585/mmwr.mm6509e2; Cao-Lormeau VM, Blake A, Mons S, Lastere S, Roche C, Vanhomwegen J, Dub T, Baudouin L, Teissier A, Larre P, Vial AL, Decam C, Choumet V, Halstead SK, Willison HJ, Musset L, Manuguerra JC, Despres P, Fournier E, Mallet HP, Musso D, Fontanet A, Neil J, Ghawche F. Guillain; Barre Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet. 2016 Apr 9;387(10027):1531 -1539. doi: 10.1016/S0140-6736(16)00562-6. Epub 2016 Mar 2). The evidence for a causal link between ZIKV and these neurological manifestations is now very strong.
[0004] There are no licensed vaccines or antivirals available for ZIKV infection.
[0005] Codon usage bias refers to the redundancy of the genetic code, where amino acids are determined by synonymous codons that occur in different organisms at different frequencies. The process of codon optimization, where each amino acid is encoded by the most abundant codon, is frequently exploited to improve gene expression in heterologous systems, a strategy that is used to increase immune responses to antigens. Instead, codon deoptimization (CD), where each or selected number of amino acid residues is encoded by the less abundant codon, is used to decrease gene expression leading to reduced viral protein production while the composition of viral antigens remains the same. The approach can also result in additional virus attenuation by removing/altering of RNA secondary structures of functional importance (Song Y, Gorbatsevych O, Liu Y, Mugavero J, Shen SH, Ward CB, Asare E, Jiang P, Paul A V, Mueller S, Wimmer E. Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes. Proc Natl Acad Sci U S A. 2017 Oct 10;1 14(41 ): E8731 -E8740. doi:10.1073/pnas.17143851 14. Epub 2017 Sep 25). This strategy is successfully used to attenuate replication of human and livestock infecting viruses (Mueller S, Papamichail D, Coleman JR, Skiena S, Wimmer E. Reduction of the rate of poliovirus protein synthesis through large-scale codon deoptimization causes attenuation of viral virulence by lowering specific infectivity. J Virol. 2006 Oct;80(19):9687-96; Stobart CC, Rostad CA, Ke Z, Dillard RS, Hampton CM, Strauss JD, Yi H, Hotard AL, Meng J, Pickles RJ, Sakamoto K, Lee S, Currier MG, Moin SM, Graham BS, Boukhvalova MS, Gilbert BE, Blanco JC, Piedra PA, Wright ER, Moore ML. A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation. Nat Commun. 2016 Dec 21 ; 7:13916. doi: 10.1038/ncomms13916; Diaz-San Segundo F, Medina GN, Ramirez-Medina E, Velazquez-Salinas L, Koster M, Grubman MJ, de los Santos T. Synonymous Deoptimization of Foot-and-Mouth Disease Virus Causes Attenuation In Vivo while Inducing a Strong Neutralizing Antibody Response. J Virol. 2015 Nov 18;90(3):1298-310. doi: 10.1 128/JVI.02167-15. Print 2016 Feb 1 ; Baker SF, Nogales A, Martinez-Sobrido L. Downregulating viral gene expression: codon usage bias manipulation for the generation of novel influenza A virus vaccines. Future Virol. 2015 Jun;10(6):715-730.; Meng J, Lee S, Hotard AL, Moore ML. Refining the balance of attenuation and immunogenicity of respiratory syncytial virus by targeted codon deoptimization of virulence genes. MBio. 2014 Sep 23;5(5):e01704-14. doi: 10.1 128/mBio.01704-14). However, its application for arboviruses, infecting both vertebrate and mosquito host and thus adapted to replication in hosts with different codon usage is less trivial; only few examples are known (Nougairede A, De Fabritus L, Aubry F, Gould EA, Holmes EC, de Lamballerie X. Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells. PLoS Pathog. 2013 Feb;9(2):e1003172. doi: 10.1371 /journal.ppat.1003172. Epub 2013 Feb 21 ; de Fabritus L, Nougairede A, Aubry F, Gould EA, de Lamballerie X. Attenuation of tick- borne encephalitis virus using large-scale random codon re-encoding. PloS Pathog. 2015 Mar 3;1 1 (3):e1004738. doi: 10.1371/journal. ppat.1004738. eCollection 2015 Mar.; de Fabritus L, Nougairede A, Aubry F, Gould EA, de Lamballerie X. Utilisation of ISA Reverse Genetics and Large-Scale Random Codon Re-Encoding to Produce Attenuated Strains of Tick-Borne Encephalitis Virus within Days. PLoS One. 2016 Aug 22;1 1 (8):e0159564. doi: 10.1371 /journal. pone.0159564. eCollection 2016).
[0006] The CD method is one of several massive synonymous mutagenesis methods. Related but non-identical methods utilising different underlying principles for attenuation are codon pair bias deoptimization (Coleman JR, Papamichail D, Skiena S, Futcher B, Wimmer E, Mueller S. Virus attenuation by genome-scale changes in codon pair bias. Science. 2008 Jun 27;320(5884):1784-7. doi: 10.1 126/science.1 155761 ; Le Nouen C, Brock LG, Luongo C, McCarty T, Yang L, Mehedi M, Wimmer E, Mueller S, Collins PL, Buchholz UJ, DiNapoli JM. Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization. Proc Natl Acad Sci U S A. 2014 Sep 9;1 1 1 (36):13169-74. doi: 10.1073/pnas.141 12901 1 1 . Epub 2014 Aug 25; Mueller S, Coleman JR, Papamichail D, Ward CB, Nimnual A, Futcher B, Skiena S, Wimmer E. Live attenuated influenza virus vaccines by computer-aided rational design. Nat Biotechnol. 2010 Jul;28(7):723-6. doi: 10.1038/nbt.1636. Epub 2010 Jun 13) and dinucleotide frequency modification (Atkinson NJ, Witteveldt J, Evans DJ, Simmonds P. The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication. Nucleic Acids Res. 2014 Apr;42(7):4527-45. doi: 10.1093/nar/gku075. Epub 2014 Jan 26.). Usually these two methods are considered different from each other, though the achieved attenuation may or may not actually be the same (Futcher B, Gorbatsevych O, Shen SH, Stauft CB, Song Y, Wang B, Leatherwood J, Gardin J, Yurovsky A, Mueller S, Wimmer E. Reply to Simmonds et al. Codon pair and dinucleotide bias have not been functionally distinguished. Proc Natl Acad Sci U S A. 2015 Jul 14;1 12(28):E3635-6. doi: 10.1073/pnas.15077101 12. Epub 2015 Jun 12; Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P. RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. Elife. 2014 Dec 9;3:e04531 . doi: 10.7554/eLife.04531 ; Simmonds P, Tulloch F, Evans DJ, Ryan MD. Attenuation of dengue (and other RNA viruses) with codon pair recoding can be explained by increased CpG/UpA dinucleotide frequencies. Proc Natl Acad Sci U S A. 2015 Jul 14;1 12(28):E3633-4). Clearly, however, these methods are very different from CD.
SUMMARY OF THE INVENTION
[0007] According to a first embodiment of the present invention, there is provided live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid comprising a partly codon deoptimized Zika viral genome.
[0008] According to a second embodiment of the present invention, there is provided a recombinant, isolated or substantially purified nucleic acid comprising a partly codon deoptimized Zika viral genome or partly codon deoptimized region thereof.
[0009] According to a third embodiment of the present invention, there is provided a vector containing the nucleic acid of the second embodiment.
[0010] According to a fourth embodiment of the present invention, there is provided a cell or isolate containing the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of the first embodiment, the nucleic acid of the second embodiment, or the vector of the third embodiment.
[001 1 ] According to a fifth embodiment of the present invention, there is provided a vaccine comprising the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of the first embodiment, the recombinant, isolated or substantially purified nucleic acid of the second embodiment, the vector of the third embodiment, or the cell or isolate of the fourth embodiment.
[0012] According to a sixth embodiment of the present invention, there is provided a pharmaceutical preparation comprising the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of the first embodiment, the recombinant, isolated or substantially purified nucleic acid of the second embodiment, the vector of the third embodiment, or the cell or isolate of the fourth embodiment.
[0013] According to a seventh embodiment of the present invention, there is provided an immunogenic composition comprising the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of the first embodiment, the recombinant, isolated or substantially purified nucleic acid of the second embodiment, the vector of the third embodiment, or the cell or isolate of the fourth embodiment.
[0014] According to an eighth embodiment of the present invention, there is provided a method of (1 ) treating a subject having a natural Zika viral infection, (2) reducing the severity of a natural Zika viral infection in a subject, or (3) preventing a subject from contracting a Zika viral infection naturally, said method comprising the step of administering to the subject: the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of the first embodiment; the recombinant, isolated or substantially purified nucleic acid of the second embodiment; the vector of the third embodiment; the cell or isolate of the fourth embodiment; the vaccine of the fifth embodiment; the pharmaceutical preparation of the sixth embodiment; or the immunogenic composition of the seventh embodiment.
[0015] According to a ninth embodiment of the present invention, there is provided use of: the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of the first embodiment; the recombinant, isolated or substantially purified nucleic acid of the second embodiment; the vector of the third embodiment; the cell or isolate of the fourth embodiment; the vaccine of the fifth embodiment; the pharmaceutical preparation of the sixth embodiment; or the immunogenic composition of the seventh embodiment, in the preparation of a medicament for (1 ) treating a subject having a natural Zika viral infection, (2) reducing the severity of a natural Zika viral infection in a subject, or (3) preventing a subject from contracting a Zika viral infection naturally.
[0016] According to a tenth embodiment of the present invention, there is provided: a live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of the first embodiment; a recombinant, isolated or substantially purified nucleic acid of the second embodiment; a vector of the third embodiment; a cell or isolate of the fourth embodiment; a vaccine of the fifth embodiment; a pharmaceutical preparation of the sixth embodiment; or an immunogenic composition of the seventh embodiment, for use in (1 ) treating a subject having a natural Zika viral infection, (2) reducing the severity of a natural Zika viral infection in a subject, or (3) preventing a subject from contracting a Zika viral infection naturally.
[0017] According to an eleventh embodiment of the present invention, there is provided a method of generating a live attenuated Zika virus vaccine, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, or recombinant, isolated or substantially purified nucleic acid comprising a partly codon deoptimized Zika viral genome or partly codon deoptimized region thereof, comprising the step of partly codon deoptimizing a Zika viral genome.
[0018] According to a twelfth embodiment of the present invention, there is provided a method of preparing a vaccine comprising live attenuated recombinant Zika virus, said method comprising the steps of: (1 ) codon deoptimizing a Zika viral genome to produce a partly codon deoptimized live attenuated Zika virus; and (2) enabling the partly codon deoptimized live attenuated Zika virus to replicate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 a. Schematic representation of codon deoptimized ZIKV genomes. ZIKV- DO - amino acid codons in NS1 -NS2A-NS2B-NS3 regions are maximally deoptimized; ZIKV- DO-NS3 - amino acid codons in NS3 region are maximally deoptimized; ZIKV-DO-scattered - amino acid codons are deoptimized with 3-4 codon gaps through all the nonstructural ZIKV genome region. In ZIKV-scatter genome every 3 rd -4 th amino acid codon has been deoptimized in favour of rarely used codons. Grey area - ZIKV structural region encompassing C, prM and E (unchanged); white area/s - unchanged; nonstructural region, pink area labelled ‘CD modified’ - codon deoptimized region.
[0020] Figure 1 b. An example of computational codon deoptimization of the nonstructural ZIKV region [SEQ ID NO:1 ], with changes indicated by way of underlining and insertion arrows.
[0021 ] Figure 2. Graph showing the percentage survival of mice infected intracranially with Zika wt virus (MR 766) or Zika vaccine based on clone ZIKV-DO-NS3 over 15 days post infection. The graph shows that the vaccine comprising live-attenuated codon deoptimized Zika virus (based on ZIKV-DO-NS3) did not result in lethal infection in mice as compared to Zika wt virus.
[0022] Figure 3. A. Graph showing the clinical score of mice infected intracranially with Zika wt virus (MR 766) or Zika vaccine based on clone ZIKV-DO-NS3 over 15 days post infection. B. Graph showing the body weight of mice infected intracranially with Zika wt virus (MR 766) or Zika vaccine based on clone ZIKV-DO-NS3 over 15 days post infection. The graphs show that the vaccine comprising live-attenuated codon deoptimized Zika virus based on ZIKV-DO-NS3 did not show signs of disease nor weight loss.
[0023] Figure 4. Graph showing the percentage survival of mice either vaccinated with a Zika vaccine based on clone ZIKV-DO-NS3 or not, and challenged with Zika wt virus (MR 766), over 15 days post infection. The graph shows that vaccinated mice were fully protected from lethal infection with no mortality.
[0024] Figure 5. Graph showing the body weight loss of mice either vaccinated with Zika vaccine based on clone ZIKV-DO-NS3 or not, and challenged with Zika wt virus (MR 766), over 7 days post infection. The graph shows that vaccinated mice were fully protected from lethal infection with no weight loss.
[0025] Figure 6. A. Clinical score criteria used for the graph shown in B. B. Graph showing the clinical score of mice either vaccinated with Zika vaccine based on clone ZIKV-DO-NS3 or not, and challenged with Zika wt virus (MR 766), over 6 days post infection. The graph shows that vaccinated mice were fully protected from lethal infection with no disease signs.
[0026] Figure 7. Graph showing the level (PFU/IFU) of Zika virus in brain tissue of mice either vaccinated with Zika vaccine based on clone ZIKV-DO-NS3 or not, and challenged with Zika wt virus. The graph shows that there was no detectable virus in the brains of vaccinated mice at day 6 after Zika challenge.
[0027] Figure 8. Graph showing that a vaccine based on ZIKV-DO-NS3 that is given subcutaneously to mice can induce a cellular response in the lymph nodes, as compared with a naive non-vaccinated mouse group.
[0028] Figure 9. Graph showing that a vaccine based on ZIKV-DO-NS3 that is given to mice induced a strong ZIKV antibody response, as compared with a naive non-vaccinated mouse group.
[0029] Figure 10. Graph showing a vaccine based on ZIKV-DO-NS3 induced a strong ZIKV neutralising antibody response in mice as compared with a naive non-vaccinated mouse group.
[0030] Figure 1 1 . Graphs showing that a vaccine based on ZIKV-DO-NS3 that is given subcutaneously to mice can induce an immune response (B and T cell response) in the draining lymph nodes compared with a naive non-vaccinated mouse group.
DESCRIPTION OF SEQUENCES
[0031 ] SEQ ID NO:1 . See Figure 1 b. Computational codon deoptimization of a nonstructural ZIKV region, with changes indicated by way of underlining and insertion arrows. SEQ ID NO:1 , below, with changed nucleotides marked in bold and underline. The sequence derives from the ZIKV- DO-scattered vaccine candidate, in particular the ZIKV- DO-scattered NS3 region.
[0032] CAAAGAAGTAAAAAAAGGGGAGACCACGGATGGAGTATACAGAGTAATGACGCGTAGAC
TGCTAGGTTCGACACAAGTTGGTGTAGGAGTTATGCAAGAAGGGGTCTTTCATACTA TGTGGCATGTC
ACAAAAGGTTCCGCGCTGCGTAGCGGTGAAGGTAGACTTGATCCGTACTGGGGAGAT GTAAAGCAGGA
TCTAGTATCATACTGTGGTCCGTGGAAGCTAGATGCGGCCTGGGACGGTCACAGCGA GGTACAGCTCT
TGGCGGTACCCCCCGGAGAAAGAGCGAGGAATATCCAGACTCTACCCGGAATATTTA AAACAAAGGAT
GGTGACATTGGAGCGGTAGCGCTGGATTATCCAGCAGGAACGTCAGGATCTCCGATC CTAGACAAATG
TGGGAGAGTAATAGGACTTTATGGTAATGGGGTCGTAATCAAAAATGGTAGTTATGT TAGTGCGATCA
CCCAAGGTAGGAGGGAAGAAGAAACTCCTGTTGAATGCTTCGAGCCGTCGATGCTGA AAAAGAAGCAG
CTAACGGTCTTAGACTTACATCCTGGAGCGGGGAAAACCCGAAGAGTTCTTCCGGAA ATAGTCCGTGA
AGCGATAAAAACACGTCTCCGTACTGTAATCTTAGCTCCGACCAGGGTTGTAGCTGC TGAAATGGAAG
AGGCCCTTCGTGGGCTTCCAGTACGTTATATGACGACAGCAGTCAATGTAACCCACT CTGGTACAGAA
ATCGTTGACTTAATGTGTCATGCCACCTTTACTTCACGTCTACTACAACCAATCAGA GTTCCCAACTA
TAATCTATATATTATGGATGAAGCCCACTTCACGGATCCCTCAAGTATAGCGGCAAG AGGATATATTT
CAACAAGGGTTGAAATGGGCGAGGCGGCGGCCATCTTCATGACGGCCACGCCACCGG GAACCCGTGAT
GCATTTCCGGATTCCAACTCACCGATTATGGACACGGAAGTGGAAGTTCCAGAGAGA GCGTGGAGCTC
AGGTTTTGATTGGGTAACGGATCATTCGGGAAAAACAGT
[0033] SEQ ID NO:2. ZIKV-wild type nonstructural region nucleotide sequence, with locations of nonstructural regions NS1 to NS5 indicated.
[0034] (NSl ) GATGTGGGGTGCTCGGTGGACTTCTCAAAGAAGGAGACGAGATGCGGTACAGGG
GTGTTCGTCTATAACGACGTTGAAGCCTGGAGGGACAGGTACAAGTACCATCCTGAC TCCCCCCGTAG
ATTGGCAGCAGCAGTCAAGCAAGCCTGGGAAGATGGTATCTGCGGGATCTCCTCTGT TTCAAGAATGG
AAAACATCATGTGGAGATCAGTAGAAGGGGAGCTCAACGCAATCCTGGAAGAGAATG GAGTTCAACTG
ACGGTCGTTGTGGGATCTGTAAAAAACCCCATGTGGAGAGGTCCACAGAGATTGCCC GTGCCTGTGAA
CGAGCTGCCCCACGGCTGGAAGGCTTGGGGGAAATCGTACTTCGTCAGAGCAGCAAA GACAAATAACA
GCTTTGTCGTGGATGGTGACACACTGAAGGAATGCCCACTCAAACATAGAGCATGGA ACAGCTTTCTT
GTGGAGGATCATGGGTTCGGGGTATTTCACACTAGTGTCTGGCTCAAGGTTAGAGAA GATTATTCATT
AGAGTGTGATCCAGCCGTTATTGGAACAGCTGTTAAGGGAAAGGAGGCTGTACACAG TGATCTAGGCT
ACTGGATTGAGAGTGAGAAGAATGACACATGGAGGCTGAAGAGGGCCCATCTGATCG AGATGAAAACA
TGTGAATGGCCAAAGTCCCACACATTGTGGACAGATGGAATAGAAGAGAGTGATCTG ATCATACCCAA
GTCTTTAGCTGGGCCACTCAGCCATCACAATACCAGAGAGGGCTACAGGACCCAAAT GAAAGGGCCAT
GGCACAGTGAAGAGCTTGAAATTCGGTTTGAGGAATGCCCAGGCACTAAGGTCCACG TGGAGGAAACA
TGTGGAACAAGAGGACCATCTCTGAGATCAACCACTGCAAGCGGAAGGGTGATCGAG GAATGGTGCTG
CAGGGAGTGCACAATGCCCCCACTGTCGTTCCGGGCTAAAGATGGCTGTTGGTATGG AATGGAGATAA
GGCCCAGGAAAGAACCAGAAAGCAACTTAGTAAGGTCAATGGTGACTGCA (NS2A) GGATCAACTGAT CACATGGACCACTTCTCCCTTGGAGTGCTTGTGATCCTGCTCATGGTGCAGGAAGGGCTG AAGAAGAG
AATGACCACAAAGATCATCATAAGCACATCAATGGCAGTGCTGGTAGCTATGATCCT GGGAGGATTTT CAATGAGTGACCTGGCTAAGCTTGCAATTTTGATGGGTGCCACCTTCGCGGAAATGAACA CTGGAGGA GATGTAGCTCATCTGGCGCTGATAGCGGCATTCAAAGTCAGACCAGCGTTGCTGGTATCT TTCATCTT CAGAGCTAATTGGACACCCCGTGAAAGCATGCTGCTGGCCTTGGCCTCGTGTCTTTTGCA AACTGCGA TCTCCGCCTTGGAAGGCGACCTGATGGTTCTCATCAATGGTTTTGCTTTGGCCTGGTTGG CAATACGA GCGATGGTTGTTCCACGCACTGATAACATCACCTTGGCAATCCTGGCTGCTCTGACACCA CTGGCCCG GGGCACACTGCTTGTGGCGTGGAGAGCAGGCCTTGCTACTTGCGGGGGGTTTATGCTCCT CTCTCTGA AGGGAAAAGGCAGTGTGAAGAAGAACTTACCATTTGTCATGGCCCTGGGACTAACCGCTG TGAGGCTG GTCGACCCCATCAACGTGGTGGGACTGCTGTTACTCACAAGGAGTGGGAAGCGG (NS2B ) AGCTGGCC CCCTAGCGAAGTACTCACAGCTGTTGGCCTGATATGCGCATTGGCTGGAGGGTTCGCCAA GGCAGATA TAGAGATGGCTGGGCCCATGGCCGCGGTCGGTCTGCTAATTGTCAGTTACGTGGTCTCAG GAAAGAGT GTGGACATGTACATTGAAAGAGCAGGTGACATCACATGGGAAAAAGATGCGGAAGTCACT GGAAACAG TCCCCGGCTCGATGTGGCGCTAGATGAGAGTGGTGATTTCTCCCTGGTGGAGGATGACGG TCCCCCCA TGAGAGAGATCATACTCAAGGTGGTCCTGATGACCATCTGTGGCATGAATCCAATAGCCA TACCCTTT GCAGCTGGAGCGTGGTACGTATACGTGAAGACTGGAAAAAGG (NS3 ) AGTGGTGCTCTATGGGATGTG CCTGCTCCCAAGGAAGTAAAAAAGGGGGAGACCACAGATGGAGTGTACAGAGTAATGACT CGTAGACT GCTAGGTTCAACACAAGTTGGAGTGGGAGTTATGCAAGAGGGGGTCTTTCACACTATGTG GCACGTCA CAAAAGGATCCGCGCTGAGAAGCGGTGAAGGGAGACTTGATCCATACTGGGGAGATGTCA AGCAGGAT CTGGTGTCATACTGTGGTCCATGGAAGCTAGATGCCGCCTGGGACGGGCACAGCGAGGTG CAGCTCTT GGCCGTGCCCCCCGGAGAGAGAGCGAGGAACATCCAGACTCTGCCCGGAATATTTAAGAC AAAGGATG GGGACATTGGAGCGGTTGCGCTGGATTACCCAGCAGGAACTTCAGGATCTCCAATCCTAG ACAAGTGT GGGAGAGTGATAGGACTTTATGGCAATGGGGTCGTGATCAAAAATGGGAGTTATGTTAGT GCCATCAC CCAAGGGAGGAGGGAAGAAGAGACTCCTGTTGAGTGCTTCGAGCCCTCGATGCTGAAGAA GAAGCAGC TAACTGTCTTAGACTTGCATCCTGGAGCTGGGAAAACCAGGAGAGTTCTTCCTGAAATAG TCCGTGAA GCCATAAAAACAAGACTCCGTACTGTGATCTTAGCTCCAACCAGGGTTGTCGCTGCTGAA ATGGAGGA GGCCCTTAGAGGGCTTCCAGTGCGTTATATGACAACAGCAGTCAATGTCACCCACTCTGG AACAGAAA TCGTCGACTTAATGTGCCATGCCACCTTCACTTCACGTCTACTACAGCCAATCAGAGTCC CCAACTAT AATCTGTATATTATGGATGAGGCCCACTTCACAGATCCCTCAAGTATAGCAGCAAGAGGA TACATTTC AACAAGGGTTGAGATGGGCGAGGCGGCTGCCATCTTCATGACCGCCACGCCACCAGGAAC CCGTGACG CATTTCCGGACTCCAACTCACCAATTATGGACACCGAAGTGGAAGTCCCAGAGAGAGCCT GGAGCTCA GGCTTTGATTGGGTGACGGATCATTCTGGAAAAACAGTTTGGTTTGTTCCAAGCGTGAGG AACGGCAA TGAGATCGCAGCTTGTCTGACAAAGGCTGGAAAACGGGTCATACAGCTCAGCAGAAAGAC TTTTGAGA CAGAGTTCCAGAAAACAAAACATCAAGAGTGGGACTTTGTCGTGACAACTGACATTTCAG AGATGGGC GCCAACTTTAAAGCTGACCGTGTCATAGATTCCAGGAGATGCCTAAAGCCGGTCATACTT GATGGCGA GAGAGTCATTCTGGCTGGACCCATGCCTGTCACACATGCCAGCGCTGCCCAGAGGAGGGG GCGCATAG
GCAGGAATCCCAACAAACCTGGAGATGAGTATCTGTATGGAGGTGGGTGCGCAGAGA CTGACGAAGAC CATGCACACTGGCTTGAAGCAAGAATGCTCCTTGACAATATTTACCTCCAAGATGGCCTC ATAGCCTC
GCTCTATCGACCTGAGGCCGACAAAGTAGCAGCCATTGAGGGAGAGTTCAAGCTTAG GACGGAGCAAA
GGAAGACCTTTGTGGAACTCATGAAAAGAGGAGATCTTCCTGTTTGGCTGGCCTATC AGGTTGCATCT
GCCGGAATAACCTACACAGATAGAAGATGGTGCTTTGATGGCACGACCAACAACACC ATAATGGAAGA
TAGTGTGCCGGCAGAGGTGTGGACCAGACACGGAGAGAAAAGAGTGCTCAAACCGAG GTGGATGGACG
CCAGAGTTTGTTCAGATCATGCGGCCCTGAAGTCATTCAAGGAGTTTGCCGCTGGGA AAAGA ( NS 4A )
GGAGCGGCTTTTGGAGTGATGGAAGCCCTGGGAACACTGCCAGGACACATGACAGAG AGATTCCAGGA
AGCCATTGACAACCTCGCTGTGCTCATGCGGGCAGAGACTGGAAGCAGGCCTTACAA AGCCGCGGCGG
CCCAATTGCCGGAGACCCTAGAGACCATAATGCTTTTGGGGTTGCTGGGAACAGTCT CGCTGGGAATC
TTCTTCGTCTTGATGAGGAACAAGGGCATAGGGAAGATGGGCTTTGGAATGGTGACT CTTGGGGCCAG
CGCATGGCTCATGTGGCTCTCGGAAATTGAGCCAGCCAGAATTGCATGTGTCCTCAT TGTTGTGTTCC
TATTGCTGGTGGTGCTCATACCTGAGCCAGAAAAGCAAAGATCTCCCCAGGACAACC AAATGGCAATC
ATCATCATGGTAGCAGTAGGTCTTCTGGGCTTGATTACCGCC ( NS 4 B ) AATGAACTCGGATGGTTGGA
GAGAACAAAGAGTGACCTAAGCCATCTAATGGGAAGGAGAGAGGAGGGGGCAACCAT AGGATTCTCAA
TGGACATTGACCTGCGGCCAGCCTCAGCTTGGGCCATCTATGCTGCCTTGACAACTT TCATTACCCCA
GCCGTCCAACATGCAGTGACCACCTCATACAACAACTACTCCTTAATGGCGATGGCC ACGCAAGCTGG
AGTGTTGTTTGGTATGGGCAAAGGGATGCCATTCTACGCATGGGACTTTGGAGTCCC GCTGCTAATGA
TAGGTTGCTACTCACAATTAACACCCCTGACCCTAATAGTGGCCATCATTTTGCTCG TGGCGCACTAC
ATGTACTTGATCCCAGGGCTGCAGGCAGCAGCTGCGCGTGCTGCCCAGAAGAGAACG GCAGCTGGCAT
CATGAAGAACCCTGTTGTGGATGGAATAGTGGTGACTGACATTGACACAATGACAAT TGACCCCCAAG
TGGAGAAAAAGATGGGACAGGTGCTACTCATAGCAGTAGCCGTCTCCAGCGCCATAC TGTCGCGGACC
GCCTGGGGGTGGGGGGAGGCTGGGGCCCTGATCACAGCCGCAACTTCCACTTTGTGG GAAGGCTCTCC
GAACAAGTACTGGAACTCCTCTACAGCCACTTCACTGTGTAACATTTTTAGGGGAAG TTACTTGGCTG
GAGCTTCTCTAATCTACACAGTAACAAGAAACGCTGGCTTGGTCAAGAGACGT ( NS5 ) GGGGGTGGAA
CAGGAGAGACCCTGGGAGAGAAATGGAAGGCCCGCTTGAACCAGATGTCGGCCCTGG AGTTCTACTCC
TACAAAAAGTCAGGCATCACCGAGGTGTGCAGAGAAGAGGCCCGCCGCGCCCTCAAG GACGGTGTGGC
AACGGGAGGCCATGCTGTGTCCCGAGGAAGTGCAAAGCTGAGATGGTTGGTGGAGCG GGGATACCTGC
AGCCCTATGGAAAGGTCATTGATCTTGGATGTGGCAGAGGGGGCTGGAGTTACTACG CCGCCACCATC
CGCAAAGTTCAAGAAGTGAAAGGATACACAAAAGGAGGCCCTGGTCATGAAGAACCC GTGTTGGTGCA
AAGCTATGGGTGGAACATAGTCCGTCTTAAGAGTGGGGTGGACGTCTTTCATATGGC GGCTGAGCCGT
GTGACACGCTGCTGTGTGACATAGGTGAGTCATCATCTAGTCCTGAAGTGGAAGAAG CACGGACGCTC
AGAGTCCTCTCCATGGTGGGGGATTGGCTTGAAAAAAGACCAGGAGCCTTTTGTATA AAAGTGTTGTG
CCCATACACCAGCACTATGATGGAAACCCTGGAGCGACTGCAGCGTAGGTATGGGGG AGGACTGGTCA
GAGTGCCACTCTCCCGCAACTCTACACATGAGATGTACTGGGTCTCTGGAGCGAAAA GCAACACCATA
AAAAGTGTGTCCACCACGAGCCAGCTCCTCTTGGGGCGCATGGACGGGCCTAGAAGG CCAGTGAAATA
TGAGGAGGATGTGAATCTCGGCTCTGGCACGCGGGCTGTGGTAAGCTGCGCTGAAGC TCCCAACATGA
AGATCATTGGTAACCGCATTGAAAGGATCCGCAGTGAGCACGCGGAAACGTGGTTCT TTGACGAGAAC CACCCATATAGGACATGGGCTTACCATGGAAGCTATGAGGCCCCCACACAAGGGTCAGCG TCCTCTCT
AATAAACGGGGTTGTCAGGCTCCTGTCAAAACCCTGGGATGTGGTGACTGGAGTCAC AGGAATAGCCA TGACCGACACCACACCGTATGGTCAGCAAAGAGTTTTCAAGGAAAAAGTGGACACTAGGG TGCCAGAC CCCCAAGAAGGCACTCGTCAGGTTATGAGCATGGTCTCTTCCTGGTTGTGGAAAGAGCTA GGCAAACA CAAACGGCCACGAGTCTGTACCAAAGAAGAGTTCATCAACAAGGTTCGTAGCAATGCAGC ATTAGGGG CAATATTTGAAGAGGAAAAAGAGTGGAAGACTGCAGTGGAAGCTGTGAACGATCCAAGGT TCTGGGCT CTAGTGGACAAGGAAAGAGAGCACCACCTGAGAGGAGAGTGCCAGAGTTGTGTGTATAAC ATGATGGG AAAAAGAGAAAAGAAACAAGGGGAATTTGGAAAGGCCAAGGGCAGCCGCGCCATCTGGTA TATGTGGC TAGGGGCTAGATTTCTAGAGTTCGAAGCCCTTGGATTCTTGAACGAGGATCACTGGATGG GGAGAGAG AACTCAGGAGGTGGTGTTGAAGGGCTGGGATTACAAAGACTCGGATATGTCCTAGAAGAG ATGAGTCG TATACCAGGAGGAAGGATGTATGCAGATGACACTGCTGGCTGGGACACCCGCATTAGCAG GTTTGATC TGGAGAATGAAGCTCTAATCACCAACCAAATGGAGAAAGGGCACAGGGCCTTGGCATTGG CCATAATC AAGTACACATACCAAAACAAAGTGGTAAAGGTCCTTAGACCAGCTGAAAAAGGGAAAACA GTTATGGA CATTATTTCGAGACAAGACCAAAGGGGGAGCGGACAAGTTGTCACTTACGCTCTTAACAC ATTTACCA ACCTAGTGGTGCAACTCATTCGGAATATGGAGGCTGAGGAAGTTCTAGAGATGCAAGACT TGTGGCTG CTGCGGAGGTCAGAGAAAGTGACCAACTGGTTGCAGAGCAACGGATGGGATAGGCTCAAA CGAATGGC AGTCAGTGGAGATGATTGCGTTGTGAAGCCAATTGATGATAGGTTTGCACATGCCCTCAG GTTCTTGA ATGATATGGGAAAAGTTAGAAAGGACACACAAGAGTGGAAACCCTCAACTGGATGGGACA ACTGGGAA GAAGTTCCGTTTTGCTCCCACCACTTCAACAAGCTCCATCTCAAGGACGGGAGGTCCATT GTGGTTCC CTGCCGCCACCAAGATGAACTGATTGGCCGGGCCCGCGTCTCTCCAGGGGCGGGATGGAG CATCCGGG AGACTGCTTGCCTAGCAAAATCATATGCGCAGATGTGGCAGCTCCTTTATTTCCACAGAA GGGACCTC CGACTGATGGCCAATGCCATTTGTTCATCTGTGCCAGTTGACTGGGTTCCAACTGGGAGA ACTACCTG GTCAATCCATGGAAAGGGAGAATGGATGACCACTGAAGACATGCTTGTGGTGTGGAACAG AGTGTGGA TTGAGGAGAACGACCACATGGAAGACAAGACCCCAGTTACGAAATGGACAGACATTCCCT ATTTGGGA AAAAGGGAAGACTTGTGGTGTGGATCTCTCATAGGGCACAGACCGCGCACCACCTGGGCT GAGAACAT TAAAAACACAGTCAACATGGTGCGCAGGATCATAGGTGATGAAGAAAAGTACATGGACTA CCTATCCA CCCAAGTTCGCTACTTGGGTGAAGAAGGGTCTACACCTGGAGTGCTGTAA (NS5 end)
[0035] SEQ ID NO:3. Vaccine candidate ZIKV-DO-NS3 nonstructural region nucleotide sequence, showing the codon deoptimized NS3 region. The NS3 region of vaccine candidate ZIKV-DO-NS3 has the same nucleotide changes as the NS3 region of vaccine candidate ZIKV-DO. In the deoptimized region changed nucleotides are marked in bold and underline.
(NS3 ) AGTGGTGCGCTCTGGGATGTCCCCGCGCCCAAGGAAGTAAAAAAGGGTGAGACCACGGAT GGC GTCTACCGAGTAATGACCCGTCGACTACTCGGTTCGACGCAAGTAGGCGTCGGCGTAATG CAAGAGGG TGTATTCCACACCATGTGGCATGTAACGAAAGGCTCGGCGCTACGATCCGGTGAAGGTCG ATTGGATC CGTACTGGGGCGATGTAAAGCAAGATCTAGTCTCGTACTGTGGTCCGTGGAAGCTCGATG CCGCCTGG
GACGGTCACTCCGAGGTCCAGTTATTGGCCGTCCCCCCGGGCGAGCGAGCGCGCAAT ATACAAACTCT ACCCGGCATATTCAAGACGAAGGATGGTGACATTGGCGCGGTAGCGCTAGATTACCCGGC GGGCACTT CGGGCTCGCCGATACTCGACAAGTGTGGTCGAGTCATAGGCTTGTATGGTAATGGTGTAG TCATAAAA AATGGTAGTTATGTAAGTGCCATAACCCAAGGTCGCCGCGAAGAAGAGACCCCCGTAGAG TGCTTCGA GCCCTCGATGCTAAAGAAGAAGCAACTCACTGTATTAGACTTGCATCCCGGCGCGGGTAA AACCCGCC GAGTATTGCCCGAAATAGTACGTGAAGCCATAAAAACGCGATTACGTACCGTCATATTAG CGCCGACC CGCGTAGTAGCGGCGGAAATGGAGGAGGCCTTGCGAGGTTTGCCGGTCCGTTATATGACG ACGGCGGT AAATGTAACCCATTCGGGCACGGAAATAGTAGACTTAATGTGCCATGCCACCTTCACCTC GCGTCTCC TCCAGCCGATACGAGTACCCAATTATAATCTATATATTATGGATGAGGCCCATTTCACGG ATCCCTCG AGTATAGCGGCGCGAGGCTACATTTCGACGCGCGTAGAGATGGGTGAGGCGGCGGCCATA TTCATGAC CGCCACGCCGCCGGGCACCCGTGACGCGTTCCCGGACTCGAATTCGCCGATTATGGACAC CGAAGTCG AAGTACCGGAGCGAGCCTGGTCCTCGGGTTTTGATTGGGTCACGGATCATTCGGGCAAAA CGGTATGG TTCGTACCGTCCGTCCGCAATGGTAATGAGATAGCGGCGTGTCTAACGAAGGCGGGCAAA CGGGTAAT ACAGTTATCCCGAAAGACCTTCGAGACGGAGTTCCAAAAAACGAAACATCAAGAGTGGGA CTTCGTAG TCACGACCGACATTTCGGAGATGGGTGCCAATTTCAAAGCGGACCGTGTAATAGATTCGC GCCGATGC CTCAAGCCGGTAATATTGGATGGTGAGCGAGTAATTCTAGCGGGCCCCATGCCCGTAACG CATGCCTC CGCGGCCCAACGCCGCGGTCGCATAGGTCGCAATCCCAATAAACCCGGCGATGAGTATCT ATATGGCG GTGGTTGCGCGGAGACCGACGAAGACCATGCGCATTGGTTGGAAGCGCGAATGTTATTGG ACAATATT TACTTACAAGATGGTTTAATAGCCTCGTTATATCGACCCGAGGCCGACAAAGTAGCGGCC ATTGAGGG CGAGTTCAAGTTGCGCACGGAGCAACGCAAGACCTTCGTCGAATTAATGAAACGAGGCGA TTTGCCCG TATGGCTAGCCTATCAAGTAGCGTCGGCCGGCATAACCTACACGGATCGACGATGGTGCT TCGATGGT ACGACCAATAATACCATAATGGAAGATAGTGTGCCGGCAGAGGTGTGGACCAGACACGGA GAGAAAAG AGTGCTCAAACCGAGGTGGATGGACGCCAGAGTTTGTTCAGATCATGCGGCCCTGAAGTC ATTCAAGG AGTTTGCCGCTGGGAAAAGA (NS3 end)
[0036] SEQ ID NO:4. Vaccine candidate ZIKV-DO-NS3 nonstructural region nucleotide sequence, showing the entire codon deoptimized NS3 region, with deoptimized region shown in underline.
[0037] (NS3 ) AGTGGTGCGCTCTGGGATGTCCCCGCGCCCAAGGAAGTAAAAAAGGGTGAGACC
ACGGATGGCGTCTACCGAGTAATGACCCGTCGACTACTCGGTTCGACGCAAGTAGGC GTCGGCGTAAT
GCAAGAGGGTGTATTCCACACCATGTGGCATGTAACGAAAGGCTCGGCGCTACGATC CGGTGAAGGTC
GATTGGATCCGTACTGGGGCGATGTAAAGCAAGATCTAGTCTCGTACTGTGGTCCGT GGAAGCTCGAT
GCCGCCTGGGACGGTCACTCCGAGGTCCAGTTATTGGCCGTCCCGCCGGGCGAGCGA GCGCGCAATAT
ACAAACTCTACCCGGCATATTCAAGACGAAGGATGGTGACATTGGCGCGGTAGCGCT AGATTACCCGG
CGGGCACTTCGGGCTCGCCGATACTCGACAAGTGTGGTCGAGTCATAGGCTTGTATG GTAATGGTGTA
GTCATAAAAAATGGTAGTTATGTAAGTGCCATAACCCAAGGTCGCCGCGAAGAAGAG ACCCCCGTAGA
GTGCTTCGAGCCCTCGATGCTAAAGAAGAAGCAACTCACTGTATTAGACTTGCATCC CGGCGCGGGTA
AAACCCGCCGAGTATTGCCCGAAATAGTACGTGAAGCCATAAAAACGCGATTACGTA CCGTCATATTA
GCGCCGACCCGCGTAGTAGCGGCGGAAATGGAGGAGGCCTTGCGAGGTTTGCCAGTC CGTTATATGAC GACGGCGGTAAATGTAACCCATTCGGGCACGGAAATAGTAGACTTAATGTGCCATGCCAC CTTCACCT
CGCGTCTCCTCCAGCCGATACGAGTACCCAATTATAATCTATATATTATGGATGAGG CCCATTTCACG GATCCCTCGAGTATAGCGGCGCGAGGCTACATTTCGACGCGCGTAGAGATGGGTGAGGCG GCGGCCAT ATTCATGACCGCCACGCCGCCGGGCACCCGTGACGCGTTCCCGGACTCGAATTCGCCGAT TATGGACA CCGAAGTCGAAGTACCGGAGCGAGCCTGGTCCTCGGGTTTTGATTGGGTCACGGATCATT CGGGCAAA ACGGTATGGTTCGTACCGTCCGTCCGCAATGGTAATGAGATAGCGGCGTGTCTAACGAAG GCGGGCAA ACGGGTAATACAGTTATCCCGAAAGACCTTCGAGACGGAGTTCCAAAAAACGAAACATCA AGAGTGGG ACTTCGTAGTCACGACCGACATTTCGGAGATGGGTGCCAATTTCAAAGCAGACCGTGTAA TAGATTCG CGCCGATGCCTCAAGCCGGTAATATTGGATGGTGAGCGAGTAATTCTAGCGGGCCCCATG CCCGTAAC GCATGCCTCCGCGGCCCAACGCCGCGGTCGCATAGGTCGCAATCCCAATAAACCCGGCGA TGAGTATC TATATGGCGGTGGTTGCGCGGAGACCGACGAAGACCATGCGCATTGGTTGGAAGCGCGAA TGTTATTG GACAATATTTACTTACAAGATGGTTTAATAGCCTCGTTATATCGACCCGAGGCCGACAAA GTAGCGGC CATTGAGGGCGAGTTCAAGTTGCGCACGGAGCAACGCAAGACCTTCGTCGAATTAATGAA ACGAGGCG ATTTGCCCGTATGGCTAGCCTATCAAGTAGCGTCGGCAGGTATAACCTACACGGATCGAC GATGGTGC TTCGATGGTACGACCAATAATACCATAATGGAAGATAGTGTGCCGGCAGAGGTGTGGACC AGACACGG AGAGAAAAGAGTGCTCAAACCGAGGTGGATGGACGCCAGAGTTTGTTCAGATCATGCGGC CCTGAAGT CATTCAAGGAGTTTGCCGCTGGGAAAAGA
[0038] SEQ ID NO:5. Vaccine candidate ZIKV-DO-NS3, with deoptimized region shown in underline, with locations of nonstructural regions indicated. The entire NS3 region of vaccine candidate ZIKV-DO-NS3 is shown together with all flanking nonstructural regions (NS1 to NS5).
[0039] (NSl ) GATGTGGGGTGCTCGGTGGACTTCTCAAAGAAGGAGACGAGATGCGGTACAGGG
GTGTTCGTCTATAACGACGTTGAAGCCTGGAGGGACAGGTACAAGTACCATCCTGAC TCCCCCCGTAG
ATTGGCAGCAGCAGTCAAGCAAGCCTGGGAAGATGGTATCTGCGGGATCTCCTCTGT TTCAAGAATGG
AAAACATCATGTGGAGATCAGTAGAAGGGGAGCTCAACGCAATCCTGGAAGAGAATG GAGTTCAACTG
ACGGTCGTTGTGGGATCTGTAAAAAACCCCATGTGGAGAGGTCCACAGAGATTGCCC GTGCCTGTGAA
CGAGCTGCCCCACGGCTGGAAGGCTTGGGGGAAATCGTACTTCGTCAGAGCAGCAAA GACAAATAACA
GCTTTGTCGTGGATGGTGACACACTGAAGGAATGCCCACTCAAACATAGAGCATGGA ACAGCTTTCTT
GTGGAGGATCATGGGTTCGGGGTATTTCACACTAGTGTCTGGCTCAAGGTTAGAGAA GATTATTCATT
AGAGTGTGATCCAGCCGTTATTGGAACAGCTGTTAAGGGAAAGGAGGCTGTACACAG TGATCTAGGCT
ACTGGATTGAGAGTGAGAAGAATGACACATGGAGGCTGAAGAGGGCCCATCTGATCG AGATGAAAACA
TGTGAATGGCCAAAGTCCCACACATTGTGGACAGATGGAATAGAAGAGAGTGATCTG ATCATACCCAA
GTCTTTAGCTGGGCCACTCAGCCATCACAATACCAGAGAGGGCTACAGGACCCAAAT GAAAGGGCCAT
GGCACAGTGAAGAGCTTGAAATTCGGTTTGAGGAATGCCCAGGCACTAAGGTCCACG TGGAGGAAACA
TGTGGAACAAGAGGACCATCTCTGAGATCAACCACTGCAAGCGGAAGGGTGATCGAG GAATGGTGCTG
CAGGGAGTGCACAATGCCCCCACTGTCGTTCCGGGCTAAAGATGGCTGTTGGTATGG AATGGAGATAA
GGCCCAGGAAAGAACCAGAAAGCAACTTAGTAAGGTCAATGGTGACTGCA (NS2A) GGATCAACTGAT CACATGGACCACTTCTCCCTTGGAGTGCTTGTGATCCTGCTCATGGTGCAGGAAGGGCTG AAGAAGAG
AATGACCACAAAGATCATCATAAGCACATCAATGGCAGTGCTGGTAGCTATGATCCT GGGAGGATTTT CAATGAGTGACCTGGCTAAGCTTGCAATTTTGATGGGTGCCACCTTCGCGGAAATGAACA CTGGAGGA GATGTAGCTCATCTGGCGCTGATAGCGGCATTCAAAGTCAGACCAGCGTTGCTGGTATCT TTCATCTT CAGAGCTAATTGGACACCCCGTGAAAGCATGCTGCTGGCCTTGGCCTCGTGTCTTTTGCA AACTGCGA TCTCCGCCTTGGAAGGCGACCTGATGGTTCTCATCAATGGTTTTGCTTTGGCCTGGTTGG CAATACGA GCGATGGTTGTTCCACGCACTGATAACATCACCTTGGCAATCCTGGCTGCTCTGACACCA CTGGCCCG GGGCACACTGCTTGTGGCGTGGAGAGCAGGCCTTGCTACTTGCGGGGGGTTTATGCTCCT CTCTCTGA AGGGAAAAGGCAGTGTGAAGAAGAACTTACCATTTGTCATGGCCCTGGGACTAACCGCTG TGAGGCTG GTCGACCCCATCAACGTGGTGGGACTGCTGTTACTCACAAGGAGTGGGAAGCGG (NS2B ) AGCTGGCC CCCTAGCGAAGTACTCACAGCTGTTGGCCTGATATGCGCATTGGCTGGAGGGTTCGCCAA GGCAGATA TAGAGATGGCTGGGCCCATGGCCGCGGTCGGTCTGCTAATTGTCAGTTACGTGGTCTCAG GAAAGAGT GTGGACATGTACATTGAAAGAGCAGGTGACATCACATGGGAAAAAGATGCGGAAGTCACT GGAAACAG TCCCCGGCTCGATGTGGCGCTAGATGAGAGTGGTGATTTCTCCCTGGTGGAGGATGACGG TCCCCCCA TGAGAGAGATCATACTCAAGGTGGTCCTGATGACCATCTGTGGCATGAATCCAATAGCCA TACCCTTT GCAGCTGGAGCGTGGTACGTATACGTGAAGACTGGAAAAAGG (NS3 ) AGTGGTGCGCTCTGGGATGTC CCCGCGCCCAAGGAAGTAAAAAAGGGTGAGACCACGGATGGCGTCTACCGAGTAATGACC CGTCGACT ACTCGGTTCGACGCAAGTAGGCGTCGGCGTAATGCAAGAGGGTGTATTCCACACCATGTG GCATGTAA CGAAAGGCTCGGCGCTACGATCCGGTGAAGGTCGATTGGATCCGTACTGGGGCGATGTAA AGCAAGAT CTAGTCTCGTACTGTGGTCCGTGGAAGCTCGATGCCGCCTGGGACGGTCACTCCGAGGTC CAGTTATT GGCCGTCCCGCCGGGCGAGCGAGCGCGCAATATACAAACTCTACCCGGCATATTCAAGAC GAAGGATG GTGACATTGGCGCGGTAGCGCTAGATTACCCGGCGGGCACTTCGGGCTCGCCGATACTCG ACAAGTGT GGTCGAGTCATAGGCTTGTATGGTAATGGTGTAGTCATAAAAAATGGTAGTTATGTAAGT GCCATAAC CCAAGGTCGCCGCGAAGAAGAGACCCCCGTAGAGTGCTTCGAGCCCTCGATGCTAAAGAA GAAGCAAC TCACTGTATTAGACTTGCATCCCGGCGCGGGTAAAACCCGCCGAGTATTGCCCGAAATAG TACGTGAA GCCATAAAAACGCGATTACGTACCGTCATATTAGCGCCGACCCGCGTAGTAGCGGCGGAA ATGGAGGA GGCCTTGCGAGGTTTGCCAGTCCGTTATATGACGACGGCGGTAAATGTAACCCATTCGGG CACGGAAA TAGTAGACTTAATGTGCCATGCCACCTTCACCTCGCGTCTCCTCCAGCCGATACGAGTAC CCAATTAT AATCTATATATTATGGATGAGGCCCATTTCACGGATCCCTCGAGTATAGCGGCGCGAGGC TACATTTC GACGCGCGTAGAGATGGGTGAGGCGGCGGCCATATTCATGACCGCCACGCCGCCGGGCAC CCGTGACG CGTTCCCGGACTCGAATTCGCCGATTATGGACACCGAAGTCGAAGTACCGGAGCGAGCCT GGTCCTCG GGTTTTGATTGGGTCACGGATCATTCGGGCAAAACGGTATGGTTCGTACCGTCCGTCCGC AATGGTAA TGAGATAGCGGCGTGTCTAACGAAGGCGGGCAAACGGGTAATACAGTTATCCCGAAAGAC CTTCGAGA CGGAGTTCCAAAAAACGAAACATCAAGAGTGGGACTTCGTAGTCACGACCGACATTTCGG AGATGGGT GCCAATTTCAAAGCAGACCGTGTAATAGATTCGCGCCGATGCCTCAAGCCGGTAATATTG GATGGTGA GCGAGTAATTCTAGCGGGCCCCATGCCCGTAACGCATGCCTCCGCGGCCCAACGCCGCGG TCGCATAG
GTCGCAATCCCAATAAACCCGGCGATGAGTATCTATATGGCGGTGGTTGCGCGGAGA CCGACGAAGAC CATGCGCATTGGTTGGAAGCGCGAATGTTATTGGACAATATTTACTTACAAGATGGTTTA ATAGCCTC
GTTATATCGACCCGAGGCCGACAAAGTAGCGGCCATTGAGGGCGAGTTCAAGTTGCG CACGGAGCAAC
GCAAGACCTTCGTCGAATTAATGAAACGAGGCGATTTGCCCGTATGGCTAGCCTATC AAGTAGCGTCG
GCAGGTATAACCTACACGGATCGACGATGGTGCTTCGATGGTACGACCAATAATACC ATAATGGAAGA
TAGTGTGCCGGCAGAGGTGTGGACCAGACACGGAGAGAAAAGAGTGCTCAAACCGAG GTGGATGGACG
CCAGAGTTTGTTCAGATCATGCGGCCCTGAAGTCATTCAAGGAGTTTGCCGCTGGGA AAAGA ( NS 4A )
GGAGCGGCTTTTGGAGTGATGGAAGCCCTGGGAACACTGCCAGGACACATGACAGAG AGATTCCAGGA
AGCCATTGACAACCTCGCTGTGCTCATGCGGGCAGAGACTGGAAGCAGGCCTTACAA AGCCGCGGCGG
CCCAATTGCCGGAGACCCTAGAGACCATAATGCTTTTGGGGTTGCTGGGAACAGTCT CGCTGGGAATC
TTCTTCGTCTTGATGAGGAACAAGGGCATAGGGAAGATGGGCTTTGGAATGGTGACT CTTGGGGCCAG
CGCATGGCTCATGTGGCTCTCGGAAATTGAGCCAGCCAGAATTGCATGTGTCCTCAT TGTTGTGTTCC
TATTGCTGGTGGTGCTCATACCTGAGCCAGAAAAGCAAAGATCTCCCCAGGACAACC AAATGGCAATC
ATCATCATGGTAGCAGTAGGTCTTCTGGGCTTGATTACCGCC ( NS 4 B ) AATGAACTCGGATGGTTGGA
GAGAACAAAGAGTGACCTAAGCCATCTAATGGGAAGGAGAGAGGAGGGGGCAACCAT AGGATTCTCAA
TGGACATTGACCTGCGGCCAGCCTCAGCTTGGGCCATCTATGCTGCCTTGACAACTT TCATTACCCCA
GCCGTCCAACATGCAGTGACCACCTCATACAACAACTACTCCTTAATGGCGATGGCC ACGCAAGCTGG
AGTGTTGTTTGGTATGGGCAAAGGGATGCCATTCTACGCATGGGACTTTGGAGTCCC GCTGCTAATGA
TAGGTTGCTACTCACAATTAACACCCCTGACCCTAATAGTGGCCATCATTTTGCTCG TGGCGCACTAC
ATGTACTTGATCCCAGGGCTGCAGGCAGCAGCTGCGCGTGCTGCCCAGAAGAGAACG GCAGCTGGCAT
CATGAAGAACCCTGTTGTGGATGGAATAGTGGTGACTGACATTGACACAATGACAAT TGACCCCCAAG
TGGAGAAAAAGATGGGACAGGTGCTACTCATAGCAGTAGCCGTCTCCAGCGCCATAC TGTCGCGGACC
GCCTGGGGGTGGGGGGAGGCTGGGGCCCTGATCACAGCCGCAACTTCCACTTTGTGG GAAGGCTCTCC
GAACAAGTACTGGAACTCCTCTACAGCCACTTCACTGTGTAACATTTTTAGGGGAAG TTACTTGGCTG
GAGCTTCTCTAATCTACACAGTAACAAGAAACGCTGGCTTGGTCAAGAGACGT ( NS5 ) GGGGGTGGAA
CAGGAGAGACCCTGGGAGAGAAATGGAAGGCCCGCTTGAACCAGATGTCGGCCCTGG AGTTCTACTCC
TACAAAAAGTCAGGCATCACCGAGGTGTGCAGAGAAGAGGCCCGCCGCGCCCTCAAG GACGGTGTGGC
AACGGGAGGCCATGCTGTGTCCCGAGGAAGTGCAAAGCTGAGATGGTTGGTGGAGCG GGGATACCTGC
AGCCCTATGGAAAGGTCATTGATCTTGGATGTGGCAGAGGGGGCTGGAGTTACTACG CCGCCACCATC
CGCAAAGTTCAAGAAGTGAAAGGATACACAAAAGGAGGCCCTGGTCATGAAGAACCC GTGTTGGTGCA
AAGCTATGGGTGGAACATAGTCCGTCTTAAGAGTGGGGTGGACGTCTTTCATATGGC GGCTGAGCCGT
GTGACACGCTGCTGTGTGACATAGGTGAGTCATCATCTAGTCCTGAAGTGGAAGAAG CACGGACGCTC
AGAGTCCTCTCCATGGTGGGGGATTGGCTTGAAAAAAGACCAGGAGCCTTTTGTATA AAAGTGTTGTG
CCCATACACCAGCACTATGATGGAAACCCTGGAGCGACTGCAGCGTAGGTATGGGGG AGGACTGGTCA
GAGTGCCACTCTCCCGCAACTCTACACATGAGATGTACTGGGTCTCTGGAGCGAAAA GCAACACCATA
AAAAGTGTGTCCACCACGAGCCAGCTCCTCTTGGGGCGCATGGACGGGCCTAGAAGG CCAGTGAAATA
TGAGGAGGATGTGAATCTCGGCTCTGGCACGCGGGCTGTGGTAAGCTGCGCTGAAGC TCCCAACATGA
AGATCATTGGTAACCGCATTGAAAGGATCCGCAGTGAGCACGCGGAAACGTGGTTCT TTGACGAGAAC CACCCATATAGGACATGGGCTTACCATGGAAGCTATGAGGCCCCCACACAAGGGTCAGCG TCCTCTCT
AATAAACGGGGTTGTCAGGCTCCTGTCAAAACCCTGGGATGTGGTGACTGGAGTCAC AGGAATAGCCA TGACCGACACCACACCGTATGGTCAGCAAAGAGTTTTCAAGGAAAAAGTGGACACTAGGG TGCCAGAC CCCCAAGAAGGCACTCGTCAGGTTATGAGCATGGTCTCTTCCTGGTTGTGGAAAGAGCTA GGCAAACA CAAACGGCCACGAGTCTGTACCAAAGAAGAGTTCATCAACAAGGTTCGTAGCAATGCAGC ATTAGGGG CAATATTTGAAGAGGAAAAAGAGTGGAAGACTGCAGTGGAAGCTGTGAACGATCCAAGGT TCTGGGCT CTAGTGGACAAGGAAAGAGAGCACCACCTGAGAGGAGAGTGCCAGAGTTGTGTGTATAAC ATGATGGG AAAAAGAGAAAAGAAACAAGGGGAATTTGGAAAGGCCAAGGGCAGCCGCGCCATCTGGTA TATGTGGC TAGGGGCTAGATTTCTAGAGTTCGAAGCCCTTGGATTCTTGAACGAGGATCACTGGATGG GGAGAGAG AACTCAGGAGGTGGTGTTGAAGGGCTGGGATTACAAAGACTCGGATATGTCCTAGAAGAG ATGAGTCG TATACCAGGAGGAAGGATGTATGCAGATGACACTGCTGGCTGGGACACCCGCATTAGCAG GTTTGATC TGGAGAATGAAGCTCTAATCACCAACCAAATGGAGAAAGGGCACAGGGCCTTGGCATTGG CCATAATC AAGTACACATACCAAAACAAAGTGGTAAAGGTCCTTAGACCAGCTGAAAAAGGGAAAACA GTTATGGA CATTATTTCGAGACAAGACCAAAGGGGGAGCGGACAAGTTGTCACTTACGCTCTTAACAC ATTTACCA ACCTAGTGGTGCAACTCATTCGGAATATGGAGGCTGAGGAAGTTCTAGAGATGCAAGACT TGTGGCTG CTGCGGAGGTCAGAGAAAGTGACCAACTGGTTGCAGAGCAACGGATGGGATAGGCTCAAA CGAATGGC AGTCAGTGGAGATGATTGCGTTGTGAAGCCAATTGATGATAGGTTTGCACATGCCCTCAG GTTCTTGA ATGATATGGGAAAAGTTAGAAAGGACACACAAGAGTGGAAACCCTCAACTGGATGGGACA ACTGGGAA GAAGTTCCGTTTTGCTCCCACCACTTCAACAAGCTCCATCTCAAGGACGGGAGGTCCATT GTGGTTCC CTGCCGCCACCAAGATGAACTGATTGGCCGGGCCCGCGTCTCTCCAGGGGCGGGATGGAG CATCCGGG AGACTGCTTGCCTAGCAAAATCATATGCGCAGATGTGGCAGCTCCTTTATTTCCACAGAA GGGACCTC CGACTGATGGCCAATGCCATTTGTTCATCTGTGCCAGTTGACTGGGTTCCAACTGGGAGA ACTACCTG GTCAATCCATGGAAAGGGAGAATGGATGACCACTGAAGACATGCTTGTGGTGTGGAACAG AGTGTGGA TTGAGGAGAACGACCACATGGAAGACAAGACCCCAGTTACGAAATGGACAGACATTCCCT ATTTGGGA AAAAGGGAAGACTTGTGGTGTGGATCTCTCATAGGGCACAGACCGCGCACCACCTGGGCT GAGAACAT TAAAAACACAGTCAACATGGTGCGCAGGATCATAGGTGATGAAGAAAAGTACATGGACTA CCTATCCA CCCAAGTTCGCTACTTGGGTGAAGAAGGGTCTACACCTGGAGTGCTGTAA (NS5 end)
[0040] SEQ ID NO:6. Vaccine candidate ZIKV-DO-scattered entire nonstructural region nucleotide sequence, with locations of nonstructural regions indicated. In the deoptimized region changed nucleotides are marked in bold and underline.
[0041 ] (NSl ) GATGTAGGGTGCTCGGTAGACTTCTCAAAGAAGGAAACGAGATGCGGTACGGGG
GTATTCGTCTATAACGATGTTGAAGCCTGGCGTGACAGGTACAAATACCATCCTGAT TCCCCCCGTCG
ATTGGCAGCAGCGGTCAAGCAAGCGTGGGAAGATGGTATATGCGGGATCTCGTCTGT TTCACGTATGG
AAAACATAATGTGGAGATCGGTAGAAGGGGAGCTAAACGCAATCCTAGAAGAGAATG GTGTTCAACTG
ACGGTAGTTGTAGGATCTGTAAAAAACCCGATGTGGAGAGGTCCGCAGAGATTGCCG GTACCTGTAAA
CGAGCTGCCCCACGGTTGGAAGGCTTGGGGTAAATCGTACTTCGTAAGAGCAGCAAA AACAAATAACT
CGTTTGTCGTGGATGGTGATACACTGAAGGAATGTCCACTCAAACATCGTGCATGGA ACTCGTTTCTT GTAGAGGATCATGGTTTCGGGGTATTTCATACTAGTGTCTGGCTAAAGGTTAGAGAAGAT TATTCGTT AGAGTGTGATCCGGCCGTTATTGGTACAGCTGTTAAAGGAAAGGAGGCGGTACACAGTGA TCTAGGTT ACTGGATTGAAAGTGAGAAGAATGATACATGGAGGCTAAAGAGGGCCCATCTAATCGAGA TGAAAACG TGTGAATGGC CGAAGT CC C AC ACGT T GT GG AC AG AT GGTATAGAAGAGTCGGAT C T GAT C AT ACC GAA GTCTTTAGCGGGGCCACTCAGTCATCACAATACGAGAGAGGGCTATAGGACCCAAATGAA AGGTCCAT GGCACTCGGAAGAGCTTGAAATACGGTTTGAGGAATGTCCAGGCACTAAAGTCCACGTGG AAGAAACA TGTGGTACAAGAGGACCGTCTCTGAGATCGACCACTGCAAGTGGAAGGGTAATCGAGGAA TGGTGTTG CAGGGAGTGCACGATGCCCCCACTATCGTTCCGGGCGAAAGATGGCTGTTGGTATGGTAT GGAGATAC GTCCCAGGAAAGAACCGGAAAGCAACTTAGTACGTTCAATGGTAACTGCA ( NS 2A ) G GAT C GAC T GAT CACATGGATCACTTCTCCCTTGGAGTACTTGTAATCCTGCTCATGGTACAGGAAGGGCTA AAGAAGAG AATGACGACAAAGATCATAATAAGCACATCGATGGCAGTACTGGTAGCTATGATACTGGG AGGATTTT CGATGAGTGACCTAGCTAAGCTTGCGATTTTGATGGGTGCGACCTTCGCGGAAATGAATA CTGGAGGA GATGTAGCGCATCTGGCGCTAATAGCGGCATTTAAAGTCAGACCGGCGTTGCTGGTATCG TTCATCTT CCGUGCTAATTGGACGCCCCGTGAATCGATGCTGCTGGCGTTGGCCTCGTGTCTATTGCA AACTGCGA TATCCGCCTTGGAAGGTGACCTGATGGTACTCATCAATGGTTTTGCGTTGGCCTGGTTAG CAATACGA GCGATGGTAGTTCCACGCACGGATAACATCACGTTGGCAATCCTAGCTGCTCTGACGCCA CTGGCCCG TGGCACACTGCTTGTAGCGTGGAGAGCGGGCCTTGCTACGTGCGGGGGGTTTATGCTACT CTCTCTGA AAGGAAAAGGCAGTGTAAAGAAGAACTTACCGTTTGTCATGGCGCTGGGACTAACGGCTG TAAGGCTG GTCGATCCCATCAACGTAGTAGGACTGCTGTTACTAACAAGGAGTGGGAAACGG ( NS 2 B ) AGCTGGCC GCCTAGCGAAGTACTAACAGCTGTTGGTCTGATATGCGCATTGGCGGGAGGGTTCGCGAA GGCAGATA TAGAAATGGCTGGGCCGATGGCCGCGGTAGGTCTGCTAATAGTCAGTTACGTAGTCTCAG GAAAAAGT GTGGACATGTATATTGAAAGAGCGGGTGACATCACATGGGAAAAAGATGCGGAAGTAACT GGAAACAG TCCGCGGCTCGATGTAGCGCTAGATGAAAGTGGTGATTTTTCCCTGGTAGAGGATGACGG TCCCCCCA TGAGAGAAATCATACTCAAAGTAGTCCTGATGACGATCTGTGGCATGAATCCGATAGCCA TACCGTTT GCAGCTGGTGCGTGGTACGTATACGTAAAGACTGGAAAACGT ( NS 3 ) AGTGGTGCTCTATGGGATGTA CCTGCTCCCAAAGAAGTAAAAAAAGGGGAGACCACGGATGGAGTATACAGAGTAATGACG CGTAGACT GCTAGGTTCGACACAAGTTGGTGTAGGAGTTATGCAAGAAGGGGTCTTTCATACTATGTG GCATGTCA CAAAAGGTTCCGCGCTGCGTAGCGGTGAAGGTAGACTTGATCCGTACTGGGGAGATGTAA AGCAGGAT CTAGTATCATACTGTGGTCCGTGGAAGCTAGATGCGGCCTGGGACGGTCACAGCGAGGTA CAGCTCTT GGCGGTACCCCCCGGAGAAAGAGCGAGGAATATCCAGACTCTACCCGGAATATTTAAAAC AAAGGATG GTGACATTGGAGCGGTAGCGCTGGATTATCCAGCAGGAACGTCAGGATCTCCGATCCTAG ACAAATGT GGGAGAGTAATAGGACTTTATGGTAATGGGGTCGTAATCAAAAATGGTAGTTATGTTAGT GCGATCAC CCAAGGTAGGAGGGAAGAAGAAACTCCTGTTGAATGCTTCGAGCCGTCGATGCTGAAAAA GAAGCAGC TAACGGTCTTAGACTTACATCCTGGAGCGGGGAAAACCCGAAGAGTTCTTCCGGAAATAG TCCGTGAA GCGATAAAAACACGTCTCCGTACTGTAATCTTAGCTCCGACCAGGGTTGTAGCTGCTGAA ATGGAAGA GGCCCTTCGTGGGCTTCCAGTACGTTATATGACGACAGCAGTCAATGTAACCCACTCTGG TACAGAAA
TCGTTGACTTAATGTGTCATGCCACCTTTACTTCACGTCTACTACAACCAATCAGAG TTCCCAACTAT AATCTATATATTATGGATGAAGCCCACTTCACGGATCCCTCAAGTATAGCGGCAAGAGGA TATATTTC
AACAAGGGTTGAAATGGGCGAGGCGGCGGCCATCTTCATGACGGCCACGCCACCGGG AACCCGTGATG
CATTTCCGGATTCCAACTCACCGATTATGGACACGGAAGTGGAAGTTCCAGAGAGAG CGTGGAGCTCA
GGTTTTGATTGGGTAACGGATCATTCGGGAAAAACAGTTTGGTTTGTTCCGAGCGTG AGGAATGGCAA
TGAGATAGCAGCTTGTCTAACAAAGGCTGGTAAACGGGTCATACAACTCAGCAGAAA AACTTTTGAAA
CAGAGTTCCAAAAAACAAAACATCAAGAATGGGACTTTGTTGTTACAACTGACATAT CAGAGATGGGT
GCCAACTTTAAAGCTGACCGTGTCATAGATTCGAGGAGATGCCTAAAGCCGGTCATA CTAGATGGCGA
GCGAGTCATTCTGGCGGGACCCATGCCGGTCACACATGCGAGCGCTGCCCAAAGGAG GGGGCGTATAG
GCAGGAATCCGAACAAACCTGGTGATGAGTATCTATATGGAGGTGGTTGCGCAGAGA CGGACGAAGAC
CATGCGCACTGGCTTGAAGCGAGAATGCTCCTAGACAATATTTATCTCCAAGATGGT CTCATAGCCTC
GCTATATCGACCTGAAGCCGACAAAGTAGCGGCCATTGAGGGTGAGTTCAAGCTAAG GACGGAGCAAC
GTAAGACCTTTGTAGAACTCATGAAAAGAGGTGATCTTCCTGTATGGCTGGCCTATC AAGTTGCATCT
GCGGGAATAACCTATACAGATAGAAGATGGTGTTTTGATGGCACGACGAACAACACC ATAATGGAAGA
TTCGGTGCCGGCAGAAGTGTGGACCAGACATGGAGAGAAACGTGTGCTCAAACCGAG GTGGATGGATG
CCAGAGTTTGTTCAGATCATGCGGCGCTGAAGTCATTTAAGGAGTTTGCGGCTGGGA AAAGA ( NS 4A )
GGTGCGGCTTTTGGTGTAATGGAAGCCCTGGGAACACTGCCGGGACACATGACGGAG AGATTCCAAGA
AGCCATTGATAACCTCGCTGTACTCATGCGGGCGGAGACTGGAAGTAGGCCTTACAA AGCCGCGGCGG
CGCAATTGCCGGAAACCCTAGAGACGATAATGCTTTTAGGGTTGCTGGGTACAGTCT CGCTAGGAATC
TTCTTTGTCTTGATGCGTAACAAGGGCATAGGTAAGATGGGCTTTGGTATGGTGACT CTAGGGGCCAG
CGCGTGGCTCATGTGGCTATCGGAAATTGAACCAGCCAGAATAGCATGTGTCCTAAT TGTTGTATTCC
TATTGCTGGTAGTACTCATACCTGAACCAGAAAAGCAACGTTCTCCCCAGGATAACC AAATGGCAATA
ATCATCATGGTAGCGGTAGGTCTTCTAGGCTTGATTACGGCCU ( NS 4 B ) AATGAACTAGGATGGTTGG
AAAGAACAAAGTCGGACCTAAGCCATCTAATGGGTAGGAGAGAGGAAGGGGCAACCA TAGGTTTCTCA
ATGGATATTGACCTGCGTCCAGCCTCAGCGTGGGCCATCTATGCGGCCTTGACAACG TTCATTACCCC
GGCCGTCCAACATGCGGTGACCACCTCGTACAACAACTATTCCTTAATGGCGATGGC GACGCAAGCTG
GTGTGTTGTTTGGTATGGGTAAAGGGATGCCGTTCTACGCATGGGATTTTGGAGTCC CGCTACTAATG
ATAGGTTGTTACTCACAATTAACGCCCCTGACCCTAATAGTAGCCATCATTTTACTC GTGGCGCATTA
CATGTACTTAATCCCAGGGCTACAGGCAGCAGCGGCGCGTGCTGCGCAGAAGAGAAC GGCGGCTGGCA
TCATGAAAAACCCTGTTGTAGATGGAATAGTAGTGACTGACATAGACACAATGACGA TTGACCCCCAA
GTAGAGAAAAAGATGGGTCAGGTGCTACTAATAGCAGTAGCGGTCTCCAGCGCGATA CTGTCGCGGAC
CGCCTGGGGTTGGGGGGAGGCGGGGGCCCTGATAACAGCCGCAACGTCCACTTTGTG GGAAGGTTCTC
CGAACAAATACTGGAACTCGTCTACAGCCACGTCACTGTGTAATATTTTTAGGGGTA GTTACTTGGCG
GGAGCTTCTCTAATATACACAGTAACGAGAAACGCTGGTTTGGTCAAGCGTCGT ( NS 5 ) GGGGGTGGT
ACAGGAGAGACGCTGGGAGAGAAATGGAAAGCCCGCTTGAATCAGATGTCGGCGCTG GAGTTCTATTC
CTACAAAAAATCAGGCATCACGGAGGTGTGCCGTGAAGAGGCCCGTCGCGCCCTCAA AGACGGTGTGG
CGACGGGAGGCCATGCGGTGTCCCGAGGTAGTGCAAAGCTAAGATGGTTGGTAGAGC GGGGATATCTG
CAGCCCTATGGTAAGGTCATTGATCTAGGATGTGGCCGTGGGGGCTGGTCGTACTAC GCCGCGACCAT CCGCAAAGTACAAGAAGTGAAAGGTTACACAAAAGGTGGCCCTGGTCATGAAGAACCCGT GTTGGTGC AAAGTTATGGGTGGAATATAGTCCGTCTAAAGAGTGGGGTAGACGTCTTTCATATGGCGG CGGAGCCG TGTGATACGCTGCTGTGTGATATAGGTGAGTCGTCATCTAGTCCGGAAGTGGAAGAAGCG CGGACGCT CCGTGTCCTCTCCATGGTAGGGGATTGGCTAGAAAAAAGACCGGGAGCCTTTTGTATAAA AGTATTGT GCCCATATACCAGCACTATGATGGAAACGCTGGAGCGACTACAGCGTAGGTATGGTGGAG GACTGGTA AGAGTGCCACTATCCCGCAACTCGACACATGAGATGTATTGGGTCTCTGGTGCGAAAAGC AATACCAT AAAAAGTGTATCCACCACGTCGCAGCTCCTCTTAGGGCGCATGGATGGGCCTAGACGTCC AGTGAAAT ATGAAGAGGATGTGAATCTAGGCTCTGGCACGCGTGCTGTGGTAAGTTGCGCTGAAGCGC CCAACATG AAAATCATTGGTAATCGCATTGAAAGGATACGCAGTGAGCATGCGGAAACGTGGTTTTTT GACGAGAA TCACCCATATAGGACGTGGGCTTACCATGGTAGCTATGAGGCGCCCACACAAGGTTCAGC GTCCTCGC TAATAAACGGTGTTGTCAGGCTACTGTCAAAACCGTGGGATGTGGTAACTGGAGTCACGG GAATAGCC ATGACGGACACCACACCGTATGGTCAACAAAGAGTTTTTAAGGAAAAAGTAGACACTAGG GTACCAGA CCCCCAAGAAGGTACTCGTCAGGTAATGAGCATGGTATCTTCCTGGTTATGGAAAGAGCT AGGTAAAC ACAAACGTCCACGAGTCTGTACGAAAGAAGAGTTTATCAACAAGGTACGTAGCAATGCGG CATTAGGG GCGATATTTGAAGAAGAAAAAGAGTGGAAAACTGCAGTGGAAGCGGTGAACGATCCGAGG TTCTGGGC GCTAGTGGACAAAGAAAGAGAGCATCACCTGAGAGGTGAGTGCCAGTCGTGTGTGTATAA TATGATGG GAAAACGTGAAAAGAAACAAGGTGAATTTGGAAAAGCCAAGGGCAGCCGTGCCATCTGGT ATATGTGG CTAGGTGCTAGATTTCTAGAATTCGAAGCCCTTGGATTCTTGAATGAGGATCACTGGATG GGTAGAGA GAACTCGGGAGGTGGTGTAGAAGGGCTGGGTTTACAAAGACTAGGATATGTCCTAGAAGA AATGAGTC GTATACCGGGAGGAAGGATGTATGCGGATGACACTGCGGGCTGGGACACGCGCATTAGCC GTTTTGAT CTGGAAAATGAAGCTCTAATAACCAACCAAATGGAAAAAGGGCACAGGGCGTTGGCATTG GCGATAAT CAAGTATACATACCAAAATAAAGTGGTAAAAGTCCTTAGACCGGCTGAAAAAGGTAAAAC AGTTATGG ATATTATTTCGCGTCAAGACCAAAGGGGTAGCGGACAAGTAGTCACTTACGCGCTTAACA CATTTACG AACCTAGTGGTACAACTCATTCGTAATATGGAGGCGGAGGAAGTTCTAGAAATGCAAGAC TTATGGCT GCTGCGTAGGTCAGAGAAAGTAACCAACTGGTTACAGAGCAACGGTTGGGATAGGCTAAA ACGAATGG CGGT CAGT GG AG AT GAT T GTGT T GT G AAGC CGAT T GAT GATCGTT T T GC AC AT GC GC TCAGGTTCTTA AATGATATGGGTAAAGTTAGAAAAGACACACAAGAATGGAAACCCTCGACTGGATGGGAT AACTGGGA AGAAGTACCGTTTTGCTCGCACCACTTCAATAAGCTCCATCTAAAGGACGGGCGTTCCAT TGTGGTAC CCTGCCGCCATCAAGATGAACTAATTGGCCGGGCGCGCGTCTCTCCGGGGGCGGGATGGT CGATCCGG GAGACGGCTTGCCTAGCGAAATCATATGCGCAAATGTGGCAGCTACTTTATTTCCATAGA AGGGACCT ACGACTGATGGCGAATGCCATTTGTTCGTCTGTGCCAGTAGACTGGGTTCCGACTGGGAG AACGACCT GGTCAATACATGGAAAGGGTGAATGGATGACGACTGAAGACATGCTAGTGGTGTGGAATA GAGTGTGG ATAGAGGAGAACGATCACATGGAAGATAAGACCCCAGTAACGAAATGGACGGACATTCCC TATTTAGG AAAAAGGGAAGATTTGTGGTGTGGTTCTCTCATAGGTCACAGACCGCGTACCACCTGGGC GGAGAACA TTAAAAATACAGTCAACATGGTACGCAGGATCATAGGTGATGAAGAAAAATATATGGACT ACCTATCG
ACCCAAGTTCGTTACTTGGGTGAAGAAGGTTCTACACCTGGTGTGCTGTAA [0042] SEQ ID NO:7. Vaccine candidate ZIKV-DO-scattered sequence with deoptimized region shown in underline, with locations of nonstructural regions indicated.
[0043] (NSl ) GATGTAGGGTGCTCGGTAGACTTCTCAAAGAAGGAAACGAGATGCGGTACGGGG
GTATTCGTCTATAACGATGTTGAAGCCTGGCGTGACAGGTACAAATACCATCCTGAT TCCCCCCGTCG
ATTGGCAGCAGCGGTCAAGCAAGCGTGGGAAGATGGTATATGCGGGATCTCGTCTGT TTCACGTATGG
AAAACATAATGTGGAGATCGGTAGAAGGGGAGCTAAACGCAATCCTAGAAGAGAATG GTGTTCAACTG
ACGGTAGTTGTAGGATCTGTAAAAAACCCGATGTGGAGAGGTCCGCAGAGATTGCCG GTACCTGTAAA
CGAGCTGCCCCACGGTTGGAAGGCTTGGGGTAAATCGTACTTCGTAAGAGCAGCAAA AACAAATAACT
CGTTTGTCGTGGATGGTGATACACTGAAGGAATGTCCACTCAAACATCGTGCATGGA ACTCGTTTCTT
GTAGAGGATCATGGTTTCGGGGTATTTCATACTAGTGTCTGGCTAAAGGTTAGAGAA GATTATTCGTT
AGAGTGTGATCCGGCCGTTATTGGTACAGCTGTTAAAGGAAAGGAGGCGGTACACAG TGATCTAGGTT
ACTGGATTGAAAGTGAGAAGAATGATACATGGAGGCTAAAGAGGGCCCATCTAATCG AGATGAAAACG
TGTGAATGGCCGAAGTCCCACACGTTGTGGACAGATGGTATAGAAGAGTCGGATCTG ATCATACCGAA
GTCTTTAGCGGGGCCACTCAGTCATCACAATACGAGAGAGGGCTATAGGACCCAAAT GAAAGGTCCAT
GGCACTCGGAAGAGCTTGAAATACGGTTTGAGGAATGTCCAGGCACTAAAGTCCACG TGGAAGAAACA
TGTGGTACAAGAGGACCGTCTCTGAGATCGACCACTGCAAGTGGAAGGGTAATCGAG GAATGGTGTTG
CAGGGAGTGCACGATGCCCCCACTATCGTTCCGGGCGAAAGATGGCTGTTGGTATGG TATGGAGATAC
GTCCCAGGAAAGAACCGGAAAGCAACTTAGTACGTTCAATGGTAACTGCA (NS2A) GGATCGACTGAT
CACATGGATCACTTCTCCCTTGGAGTACTTGTAATCCTGCTCATGGTACAGGAAGGG CTAAAGAAGAG
AATGACGACAAAGATCATAATAAGCACATCGATGGCAGTACTGGTAGCTATGATACT GGGAGGATTTT
CGATGAGTGACCTAGCTAAGCTTGCGATTTTGATGGGTGCGACCTTCGCGGAAATGA ATACTGGAGGA
GATGTAGCGCATCTGGCGCTAATAGCGGCATTTAAAGTCAGACCGGCGTTGCTGGTA TCGTTCATCTT
CCGTGCTAATTGGACGCCCCGTGAATCGATGCTGCTGGCGTTGGCCTCGTGTCTATT GCAAACTGCGA
TATCCGCCTTGGAAGGTGACCTGATGGTACTCATCAATGGTTTTGCGTTGGCCTGGT TAGCAATACGA
GCGATGGTAGTTCCACGCACGGATAACATCACGTTGGCAATCCTAGCTGCTCTGACG CCACTGGCCCG
TGGCACACTGCTTGTAGCGTGGAGAGCGGGCCTTGCTACGTGCGGGGGGTTTATGCT ACTCTCTCTGA
AAGGAAAAGGCAGTGTAAAGAAGAACTTACCGTTTGTCATGGCGCTGGGACTAACGG CTGTAAGGCTG
GTCGATCCCATCAACGTAGTAGGACTGCTGTTACTAACAAGGAGTGGGAAACGG (NS2B ) AGCTGGCC
GCCTAGCGAAGTACTAACAGCTGTTGGTCTGATATGCGCATTGGCGGGAGGGTTCGC GAAGGCAGATA
TAGAAATGGCTGGGCCGATGGCCGCGGTAGGTCTGCTAATAGTCAGTTACGTAGTCT CAGGAAAAAGT
GTGGACATGTATATTGAAAGAGCGGGTGACATCACATGGGAAAAAGATGCGGAAGTA ACTGGAAACAG
TCCGCGGCTCGATGTAGCGCTAGATGAAAGTGGTGATTTTTCCCTGGTAGAGGATGA CGGTCCCCCCA
TGAGAGAAATCATACTCAAAGTAGTCCTGATGACGATCTGTGGCATGAATCCGATAG CCATACCGTTT
GCAGCTGGTGCGTGGTACGTATACGTAAAGACTGGAAAACGT (NS3 ) AGTGGTGCTCTATGGGATGTA
CCTGCTCCCAAAGAAGTAAAAAAAGGGGAGACCACGGATGGAGTATACAGAGTAATG ACGCGTAGACT
GCTAGGTTCGACACAAGTTGGTGTAGGAGTTATGCAAGAAGGGGTCTTTCATACTAT GTGGCATGTCA
CAAAAGGTTCCGCGCTGCGTAGCGGTGAAGGTAGACTTGATCCGTACTGGGGAGATG TAAAGCAGGAT CTAGTATCATACTGTGGTCCGTGGAAGCTAGATGCGGCCTGGGACGGTCACAGCGAGGTA CAGCTCTT
GGCGGTACCCCCCGGAGAAAGAGCGAGGAATATCCAGACTCTACCCGGAATATTTAA AACAAAGGATG
GTGACATTGGAGCGGTAGCGCTGGATTATCCAGCAGGAACGTCAGGATCTCCGATCC TAGACAAATGT
GGGAGAGTAATAGGACTTTATGGTAATGGGGTCGTAATCAAAAATGGTAGTTATGTT AGTGCGATCAC
CCAAGGTAGGAGGGAAGAAGAAACTCCTGTTGAATGCTTCGAGCCGTCGATGCTGAA AAAGAAGCAGC
TAACGGTCTTAGACTTACATCCTGGAGCGGGGAAAACCCGAAGAGTTCTTCCGGAAA TAGTCCGTGAA
GCGATAAAAACACGTCTCCGTACTGTAATCTTAGCTCCGACCAGGGTTGTAGCTGCT GAAATGGAAGA
GGCCCTTCGTGGGCTTCCAGTACGTTATATGACGACAGCAGTCAATGTAACCCACTC TGGTACAGAAA
TCGTTGACTTAATGTGTCATGCCACCTTTACTTCACGTCTACTACAACCAATCAGAG TTCCCAACTAT
AATCTATATATTATGGATGAAGCCCACTTCACGGATCCCTCAAGTATAGCGGCAAGA GGATATATTTC
AACAAGGGTTGAAATGGGCGAGGCGGCGGCCATCTTCATGACGGCCACGCCACCGGG AACCCGTGATG
CATTTCCG GAT T C C AAC T C AC C GAT T AT GG AC AC G G AAG T G G AAG T T C C AG AG AG AG C G T GG AG C T C A
GGTTTTGATTGGGTAACGGATCATTCGGGAAAAACAGTTTGGTTTGTTCCGAGCGTG AGGAATGGCAA
TGAGATAGCAGCTTGTCTAACAAAGGCTGGTAAACGGGTCATACAACTCAGCAGAAA AACTTTTGAAA
CAGAGT T C C AAAAAAC AAAAC AT C AAGAAT GGG AC TTTGTTGT T AC AAC T G AC AT AT C AGAG AT GGGT
GCCAACTTTAAAGCTGACCGTGTCATAGATTCGAGGAGATGCCTAAAGCCGGTCATA CTAGATGGCGA
GCGAGTCATTCTGGCGGGACCCATGCCGGTCACACATGCGAGCGCTGCCCAAAGGAG GGGGCGTATAG
GCAGGAATCCGAACAAACCTGGTGATGAGTATCTATATGGAGGTGGTTGCGCAGAGA CGGACGAAGAC
CATGCGCACTGGCTTGAAGCGAGAATGCTCCTAGACAATATTTATCTCCAAGATGGT CTCATAGCCTC
GCTATATCGACCTGAAGCCGACAAAGTAGCGGCCATTGAGGGTGAGTTCAAGCTAAG GACGGAGCAAC
GTAAGACCTTTGTAGAACTCATGAAAAGAGGTGATCTTCCTGTATGGCTGGCCTATC AAGTTGCATCT
GCGGGAATAACCTATACAGATAGAAGATGGTGTTTTGATGGCACGACGAACAACACC ATAATGGAAGA
TTCGGTGCCGGCAGAAGTGTGGACCAGACATGGAGAGAAACGTGTGCTCAAACCGAG GTGGATGGATG
CCAGAGTTTGTTCAGATCATGCGGCGCTGAAGTCATTTAAGGAGTTTGCGGCTGGGA AAAGA ( NS 4A )
GGTGCGGCTTTTGGTGTAATGGAAGCCCTGGGAACACTGCCGGGACACATGACGGAG AGATTCCAAGA
AGCCATTGATAACCTCGCTGTACTCATGCGGGCGGAGACTGGAAGTAGGCCTTACAA AGCCGCGGCGG
CGCAATTGCCGGAAACCCTAGAGACGATAATGCTTTTAGGGTTGCTGGGTACAGTCT CGCTAGGAATC
TTCTTTGTCTTGATGCGTAACAAGGGCATAGGTAAGATGGGCTTTGGTATGGTGACT CTAGGGGCCAG
CGCGTGGCTCATGTGGCTATCGGAAATTGAACCAGCCAGAATAGCATGTGTCCTAAT TGTTGTATTCC
TATTGCTGGTAGTACTCATACCTGAACCAGAAAAGCAACGTTCTCCCCAGGATAACC AAATGGCAATA
ATCATCATGGTAGCGGTAGGTCTTCTAGGCTTGATTACGGCC ( NS 4 B ) AATGAACTAGGATGGTTGGA
AAGAACAAAGTCGGACCTAAGCCATCTAATGGGTAGGAGAGAGGAAGGGGCAACCAT AGGTTTCTCAA
TGGATATTGACCTGCGTCCAGCCTCAGCGTGGGCCATCTATGCGGCCTTGACAACGT TCATTACCCCG
GCCGTCCAACATGCGGTGACCACCTCGTACAACAACTATTCCTTAATGGCGATGGCG ACGCAAGCTGG
TGTGTTGTTTGGTATGGGTAAAGGGATGCCGTTCTACGCATGGGATTTTGGAGTCCC GCTACTAATGA
TAGGTTGTTACTCACAATTAACGCCCCTGACCCTAATAGTAGCCATCATTTTACTCG TGGCGCATTAC
ATGTACTTAATCCCAGGGCTACAGGCAGCAGCGGCGCGTGCTGCGCAGAAGAGAACG GCGGCTGGCAT CATGAAAAACCCTGTTGTAGATGGAATAGTAGTGACTGACATAGACACAATGACGATTGA CCCCCAAG
TAGAGAAAAAGATGGGTCAGGTGCTACTAATAGCAGTAGCGGTCTCCAGCGCGATAC TGTCGCGGACC GCCTGGGGTTGGGGGGAGGCGGGGGCCCTGATAACAGCCGCAACGTCCACTTTGTGGGAA GGTTCTCC GAACAAATACTGGAACTCGTCTACAGCCACGTCACTGTGTAATATTTTTAGGGGTAGTTA CTTGGCGG GAGCTTCTCTAATATACACAGTAACGAGAAACGCTGGTTTGGTCAAGCGTCGT ( NS5 ) GGGGGTGGTA CAGGAGAGACGCTGGGAGAGAAATGGAAAGCCCGCTTGAATCAGATGTCGGCGCTGGAGT TCTATTCC TACAAAAAATCAGGCATCACGGAGGTGTGCCGTGAAGAGGCCCGTCGCGCCCTCAAAGAC GGTGTGGC GACGGGAGGCCATGCGGTGTCCCGAGGTAGTGCAAAGCTAAGATGGTTGGTAGAGCGGGG ATATCTGC AGCCCTATGGTAAGGTCATTGATCTAGGATGTGGCCGTGGGGGCTGGTCGTACTACGCCG CGACCATC CGCAAAGTACAAGAAGTGAAAGGTTACACAAAAGGTGGCCCTGGTCATGAAGAACCCGTG TTGGTGCA AAGTTATGGGTGGAATATAGTCCGTCTAAAGAGTGGGGTAGACGTCTTTCATATGGCGGC GGAGCCGT GTGATACGCTGCTGTGTGATATAGGTGAGTCGTCATCTAGTCCGGAAGTGGAAGAAGCGC GGACGCTC CGTGTCCTCTCCATGGTAGGGGATTGGCTAGAAAAAAGACCGGGAGCCTTTTGTATAAAA GTATTGTG CCCATATACCAGCACTATGATGGAAACGCTGGAGCGACTACAGCGTAGGTATGGTGGAGG ACTGGTAA GAGTGCCACTATCCCGCAACTCGACACATGAGATGTATTGGGTCTCTGGTGCGAAAAGCA ATACCATA AAAAGTGTATCCACCACGTCGCAGCTCCTCTTAGGGCGCATGGATGGGCCTAGACGTCCA GTGAAATA TGAAGAGGATGTGAATCTAGGCTCTGGCACGCGTGCTGTGGTAAGTTGCGCTGAAGCGCC CAACATGA AAATCATTGGTAATCGCATTGAAAGGATACGCAGTGAGCATGCGGAAACGTGGTTTTTTG ACGAGAAT CACCCATATAGGACGTGGGCTTACCATGGTAGCTATGAGGCGCCCACACAAGGTTCAGCG TCCTCGCT AATAAACGGTGTTGTCAGGCTACTGTCAAAACCGTGGGATGTGGTAACTGGAGTCACGGG AATAGCCA TGACGGACACCACACCGTATGGTCAACAAAGAGTTTTTAAGGAAAAAGTAGACACTAGGG TACCAGAC CCCCAAGAAGGTACTCGTCAGGTAATGAGCATGGTATCTTCCTGGTTATGGAAAGAGCTA GGTAAACA CAAACGTCCACGAGTCTGTACGAAAGAAGAGTTTATCAACAAGGTACGTAGCAATGCGGC ATTAGGGG CGATATTTGAAGAAGAAAAAGAGTGGAAAACTGCAGTGGAAGCGGTGAACGATCCGAGGT TCTGGGCG CTAGTGGACAAAGAAAGAGAGCATCACCTGAGAGGTGAGTGCCAGTCGTGTGTGTATAAT ATGATGGG AAAACGTGAAAAGAAACAAGGTGAATTTGGAAAAGCCAAGGGCAGCCGTGCCATCTGGTA TATGTGGC TAGGTGCTAGATTTCTAGAATTCGAAGCCCTTGGATTCTTGAATGAGGATCACTGGATGG GTAGAGAG AACTCGGGAGGTGGTGTAGAAGGGCTGGGTTTACAAAGACTAGGATATGTCCTAGAAGAA ATGAGTCG TATACCGGGAGGAAGGATGTATGCGGATGACACTGCGGGCTGGGACACGCGCATTAGCCG TTTTGATC TGGAAAATGAAGCTCTAATAACCAACCAAATGGAAAAAGGGCACAGGGCGTTGGCATTGG CGATAATC AAGTATACATACCAAAATAAAGTGGTAAAAGTCCTTAGACCGGCTGAAAAAGGTAAAACA GTTATGGA TATTATTTCGCGTCAAGACCAAAGGGGTAGCGGACAAGTAGTCACTTACGCGCTTAACAC ATTTACGA ACCTAGTGGTACAACTCATTCGTAATATGGAGGCGGAGGAAGTTCTAGAAATGCAAGACT TATGGCTG CTGCGTAGGTCAGAGAAAGTAACCAACTGGTTACAGAGCAACGGTTGGGATAGGCTAAAA CGAATGGC GGTCAGTGGAGATGATTGTGTTGTGAAGCCGATTGATGATCGTTTTGCACATGCGCTCAG GTTCTTAA ATGATATGGGTAAAGTTAGAAAAGACACACAAGAATGGAAACCCTCGACTGGATGGGATA ACTGGGAA
GAAGTACCGTTTTGCTCGCACCACTTCAATAAGCTCCATCTAAAGGACGGGCGTTCC ATTGTGGTACC CTGCCGCCATCAAGATGAACTAATTGGCCGGGCGCGCGTCTCTCCGGGGGCGGGATGGTC GATCCGGG
AGACGGCTTGCCTAGCGAAATCATATGCGCAAATGTGGCAGCTACTTTATTTCCATA GAAGGGACCTA
CGACTGATGGCGAATGCCATTTGTTCGTCTGTGCCAGTAGACTGGGTTCCGACTGGG AGAACGACCTG
GTCAATACATGGAAAGGGTGAATGGATGACGACTGAAGACATGCTAGTGGTGTGGAA TAGAGTGTGGA
TAGAGGAGAACGATCACATGGAAGATAAGACCCCAGTAACGAAATGGACGGACATTC CCTATTTAGGA
AAAAGGGAAGATTTGTGGTGTGGTTCTCTCATAGGTCACAGACCGCGTACCACCTGG GCGGAGAACAT
TAAAAATACAGTCAACATGGTACGCAGGATCATAGGTGATGAAGAAAAATATATGGA CTACCTATCGA
CCCAAGTTCGTTACTTGGGTGAAGAAGGTTCTACACCTGGTGTGCTGTAA ( NS5 end )
[0044] SEQ ID NO:8. Vaccine candidate ZIKV-DO nonstructural region nucleotide sequence, with locations of nonstructural regions indicated. Only regions NS1 to NS3 are shown. In the deoptimized region changed nucleotides are marked in bold and underline.
[0045] ( NS 1 ) GTCGGTTGTTCGGTAGATTTTTCGAAAAAAGAAACGCGATGTGGTACGGGTGTA TTTGTATATAATGACGTAGAAGCGTGGCGAGACCGATACAAGTATCATCCGGACTCGCCG CGACGATT AGCGGCGGCGGTAAAACAAGCGTGGGAAGACGGTATATGCGGTATATCGTCGGTATCGCG AATGGAAA ATATAATGTGGCGATCGGTAGAAGGTGAGTTAAATGCGATACTAGAAGAGAATGGCGTAC AACTAACG GTAGTAGTCGGCTCGGTAAAAAATCCCATGTGGCGAGGTCCGCAGCGATTGCCCGTCCCC GTCAATGA GCTACCCCATGGTTGGAAGGCGTGGGGTAAATCGTACTTCGTACGAGCGGCGAAGACGAA TAATTCCT TTGTAGTCGATGGTGACACGCTAAAGGAATGCCCGTTAAAACATCGAGCGTGGAATTCCT TTTTGGTC GAGGATCATGGTTTCGGTGTATTCCATACCAGTGTATGGTTAAAGGTACGAGAAGATTAT TCGTTAGA GTGTGATCCGGCCGTAATTGGCACGGCGGTAAAGGGCAAGGAGGCGGTACATAGTGATCT CGGTTACT GG AT T GAG AG T G AG AAG AAT G AC AC GT G GC GC C T AAAG CG CG C C C AT C T AAT AG AG AT G AAAAC GT G T GAATGGCCGAAGTCGCACACGTTGTGGACGGATGGCATAGAAGAGAGTGATCTAATAATA CCCAAGTC GTTAGCGGGTCCGTTATCCCATCATAATACCCGAGAGGGTTACCGCACCCAAATGAAAGG TCCGTGGC ATAGTGAAGAGTTGGAAATTCGGTTCGAGGAATGCCCGGGTACCAAGGTACACGTCGAGG AAACGTGT GGCACGCGAGGCCCGTCGCTACGATCGACCACCGCGTCCGGCCGCGTCATAGAGGAATGG TGCTGCCG CGAGTGCACGATGCCCCCGCTATCGTTCCGGGCGAAAGATGGTTGTTGGTATGGAATGGA GATACGCC CCCGCAAAGAACCGGAATCCAATTTAGTACGCTCGATGGTCACCGCGGGCTCGACCGATC ATATGGAC CATTTCTCGTTG ( NS2A) GGCGTCTTGGTCATACTATTAATGGTCCAAGAAGGTCTAAAGAAGCGAAT GACCACGAAGATAATAATATCCACGTCGATGGCGGTCCTAGTAGCGATGATACTAGGCGG CTTTTCGA TGAGTGACCTAGCGAAGTTGGCGATTTTGATGGGTGCCACCTTCGCGGAAATGAATACCG GCGGCGAT GTAGCGCATCTAGCGCTAATAGCGGCGTTCAAAGTACGACCGGCGTTGCTAGTATCGTTC ATATTCCG AGCGAATTGGACGCCCCGTGAATCCATGCTACTAGCCTTGGCCTCGTGTTTGTTGCAAAC CGCGATAT CGGCCTTGGAAGGTGACCTAATGGTATTAATAAATGGTTTCGCGTTGGCCTGGTTGGCGA TACGAGCG ATGGTAGTACCGCGCACCGATAATATAACCTTGGCGATACTAGCGGCGCTAACGCCGCTA GCCCGGGG TACGCTATTGGTCGCGTGGCGAGCGGGTTTGGCGACCTGCGGTGGTTTTATGTTATTATC GCTAAAGG GCAAAGGTAGTGTCAAGAAGAATTTACCGTTTGTAATGGCCCTAGGCCTCACCGCGGTCC GCCTAGTA GACCCCATAAATGTCGTCGGCCTACTATTATTAACGCGCAGTGGTAAGCGGTCCTGGCCC ( NS 2 B ) CC CTCCGAAGTATTAACGGCGGTAGGTCTAATATGCGCGTTGGCGGGCGGTTTCGCCAAGGC GGATATAG AGATGGCGGGTCCCATGGCCGCGGTAGGTCTACTCATTGTAAGTTACGTCGTATCGGGCA AGAGTGTC GAC AT GT AC AT T GAACGAGC GGGT GAC AT AAC GT GGGAAAAAG AT GC GG AAGT AAC CGGCAATAGT C C CCGGTTAGATGTCGCGCTCGATGAGAGTGGTGATTTCTCGCTAGTCGAGGATGACGGTCC CCCGATGC GAGAGATAATATTAAAGGTCGTACTAATGACCATATGTGGTATGAATCCGATAGCCATAC CCTTCGCG GCGGGCGCGTGGTACGTATACGTCAAGACCGGCAAACGC ( NS 3 ) AGTGGTGCGCTCTGGGATGTCCCC GCGCCCAAGGAAGTAAAAAAGGGTGAGACCACGGATGGCGTCTACCGAGTAATGACCCGT CGACTACT CGGTTCGACGCAAGTAGGCGTCGGCGTAATGCAAGAGGGTGTATTCCACACCATGTGGCA TGTAACGA AAGGCTCGGCGCTACGATCCGGTGAAGGTCGATTGGATCCGTACTGGGGCGATGTAAAGC AAGATCTA GTCTCGTACTGTGGTCCGTGGAAGCTCGATGCCGCCTGGGACGGTCACTCCGAGGTCCAG TTATTGGC CGTCCCCCCGGGCGAGCGAGCGCGCAATATACAAACTCTACCCGGCATATTCAAGACGAA GGATGGTG ACATTGGCGCGGTAGCGCTAGATTACCCGGCGGGCACTTCGGGCTCGCCGATACTCGACA AGTGTGGT CGAGTCATAGGCTTGTATGGTAATGGTGTAGTCATAAAAAATGGTAGTTATGTAAGTGCC ATAACCCA AGGTCGCCGCGAAGAAGAGACCCCCGTAGAGTGCTTCGAGCCCTCGATGCTAAAGAAGAA GCAACTCA CTGTATTAGACTTGCATCCCGGCGCGGGTAAAACCCGCCGAGTATTGCCCGAAATAGTAC GTGAAGCC ATAAAAACGCGATTACGTACCGTCATATTAGCGCCGACCCGCGTAGTAGCGGCGGAAATG GAGGAGGC CTTGCGAGGTTTGC CGGT CC GT T AT AT G AC GACGGC GGT AAAT GT AACC C ATT CGGGCACGGAAAT AG TAGACTTAATGTGCCATGCCACCTTCACCTCGCGTCTCCTCCAGCCGATACGAGTACCCA ATTATAAT CTATATATTATGGATGAGGCCCATTTCACGGATCCCTCGAGTATAGCGGCGCGAGGCTAC ATTTCGAC GCGCGTAGAGATGGGTGAGGCGGCGGCCATATTCATGACCGCCACGCCGCCGGGCACCCG TGACGCGT TCCCGGACTCGAATTCGCCGATTATGGACACCGAAGTCGAAGTACCGGAGCGAGCCTGGT CCTCGGGT TTTGATTGGGTCACGGATCATTCGGGCAAAACGGTATGGTTCGTACCGTCCGTCCGCAAT GGTAATGA GATAGCGGCGTGTCTAACGAAGGCGGGCAAACGGGTAATACAGTTATCCCGAAAGACCTT CGAGACGG AGTTCCAAAAAACGAAACATCAAGAGTGGGACTTCGTAGTCACGACCGACATTTCGGAGA TGGGTGCC AATTTCAAAGCGGACCGTGTAATAGATTCGCGCCGATGCCTCAAGCCGGTAATATTGGAT GGTGAGCG AGTAATTCTAGCGGGCCCCATGCCCGTAACGCATGCCTCCGCGGCCCAACGCCGCGGTCG CATAGGTC GCAATCCCAATAAACCCGGCGATGAGTATCTATATGGCGGTGGTTGCGCGGAGACCGACG AAGACCAT GCGCATTGGTTGGAAGCGCGAATGTTATTGGACAATATTTACTTACAAGATGGTTTAATA GCCTCGTT ATATCGACCCGAGGCCGACAAAGTAGCGGCCATTGAGGGCGAGTTCAAGTTGCGCACGGA GCAACGCA AG AC CT T CGT CG AATTAAT G AAACGAGGCG AT TT GC CCGT AT GGC T AGC CT AT C AAGT AGC GT CGGC C GGCATAACCTACACGGATCGACGATGGTGCTTCGATGGTACGACCAATAATACCATAATG GAAGATAG TGTGCCGGC
[0046] SEQ ID NO:9. Vaccine candidate ZIKV-DO sequence, with the deoptimized region shown in underline, with locations of nonstructural regions NS1 to NS5 indicated and shown in full. [0047] (NSl ) GTCGGTTGTTCGGTAGATTTTTCGAAAAAAGAAACGCGATGTGGTACGGGTGTA
TTTGTATATAATGACGTAGAAGCGTGGCGAGACCGATACAAGTATCATCCGGACTCG CCGCGACGATT AGCGGCGGCGGTAAAACAAGCGTGGGAAGACGGTATATGCGGTATATCGTCGGTATCGCG AATGGAAA ATATAATGTGGCGATCGGTAGAAGGTGAGTTAAATGCGATACTAGAAGAGAATGGCGTAC AACTAACG GTAGTAGTCGGCTCGGTAAAAAATCCCATGTGGCGAGGTCCGCAGCGATTGCCCGTCCCC GTCAATGA GCTACCCCATGGTTGGAAGGCGTGGGGTAAATCGTACTTCGTACGAGCGGCGAAGACGAA TAATTCCT TTGTAGTCGATGGTGACACGCTAAAGGAATGCCCGTTAAAACATCGAGCGTGGAATTCCT TTTTGGTC GAGGATCATGGTTTCGGTGTATTCCATACCAGTGTATGGTTAAAGGTACGAGAAGATTAT TCGTTAGA GTGTGATCCGGCCGTAATTGGCACGGCGGTAAAGGGCAAGGAGGCGGTACATAGTGATCT CGGTTACT GGATTGAGAGTGAGAAGAATGACACGTGGCGCCTAAAGCGCGCCCATCTAATAGAGATGA AAACGTGT GAATGGCCGAAGTCGCACACGTTGTGGACGGATGGCATAGAAGAGAGTGATCTAATAATA CCCAAGTC GTTAGCGGGTCCGTTATCCCATCATAATACCCGAGAGGGTTACCGCACCCAAATGAAAGG TCCGTGGC ATAGTGAAGAGTTGGAAATTCGGTTCGAGGAATGTCCGGGTACCAAGGTACACGTCGAGG AAACGTGT GGCACGCGAGGCCCGTCGCTACGATCGACCACCGCGTCCGGCCGCGTCATAGAGGAATGG TGCTGCCG CGAGTGCACGATGCCCCCGCTATCGTTCCGGGCGAAAGATGGTTGTTGGTATGGAATGGA GATACGCC CCCGCAAAGAACCGGAATCCAATTTAGTACGCTCGATGGTCACCGCGGGCTCGACCGATC ATATGGAC CATTTCTCGTTG (NS2A) GGCGTCTTGGTCATACTATTAATGGTCCAAGAAGGTCTAAAGAAGCGAAT GACCACGAAGATAATAATATCCACGTCGATGGCGGTCCTAGTAGCGATGATACTAGGCGG CTTTTCGA TGAGTGACCTAGCGAAGTTGGCGATTTTGATGGGTGCCACCTTCGCGGAAATGAATACCG GCGGCGAT GTAGCGCATCTAGCGCTAATAGCGGCGTTCAAAGTACGACCGGCGTTGCTAGTATCGTTC ATATTCCG AGCGAATTGGACGCCCCGTGAATCCATGCTACTAGCCTTGGCCTCGTGTTTGTTGCAAAC CGCGATAT CGGCCTTGGAAGGTGACCTAATGGTATTAATAAATGGTTTCGCGTTGGCCTGGTTGGCGA TACGAGCG ATGGTAGTACCGCGCACCGATAATATAACCTTGGCGATACTAGCGGCGCTAACGCCGCTA GCCCGGGG TACGCTATTGGTCGCGTGGCGAGCGGGTTTGGCGACCTGCGGTGGTTTTATGTTATTATC GCTAAAGG GCAAAGGTAGTGTCAAGAAGAATTTACCGTTTGTAATGGCCCTAGGCCTCACCGCGGTCC GCCTAGTA GACCCCATAAATGTCGTCGGCCTACTATTATTAACGCGCAGTGGTAAGCGGTCCTGGCCC (NS2B ) CC CTCCGAAGTATTAACGGCGGTAGGTCTAATATGCGCGTTGGCGGGCGGTTTCGCCAAGGC GGATATAG AGATGGCGGGTCCCATGGCCGCGGTAGGTCTACTCATTGTAAGTTACGTCGTATCGGGCA AGAGTGTC GACATGTACATTGAACGAGCGGGTGACATAACGTGGGAAAAAGATGCGGAAGTAACCGGC AATAGTCC CCGGTTAGATGTCGCGCTCGATGAGAGTGGTGATTTCTCGCTAGTCGAGGATGACGGTCC CCCGATGC GAGAGATAATATTAAAGGTCGTACTAATGACCATATGTGGTATGAATCCGATAGCCATAC CCTTCGCG GCGGGCGCGTGGTACGTATACGTCAAGACCGGCAAACGC (NS3 ) AGTGGTGCGCTCTGGGATGTCCCC GCGCCCAAGGAAGTAAAAAAGGGTGAGACCACGGATGGCGTCTACCGAGTAATGACCCGT CGACTACT CGGTTCGACGCAAGTAGGCGTCGGCGTAATGCAAGAGGGTGTATTCCACACCATGTGGCA TGTAACGA AAGGCTCGGCGCTACGATCCGGTGAAGGTCGATTGGATCCGTACTGGGGCGATGTAAAGC AAGATCTA GTCTCGTACTGTGGTCCGTGGAAGCTCGATGCCGCCTGGGACGGTCACTCCGAGGTCCAG TTATTGGC
CGTCCCGCCGGGCGAGCGAGCGCGCAATATACAAACTCTACCCGGCATATTCAAGAC GAAGGATGGTG ACATTGGCGCGGTAGCGCTAGATTACCCGGCGGGCACTTCGGGCTCGCCGATACTCGACA AGTGTGGT
CGAGTCATAGGCTTGTATGGTAATGGTGTAGTCATAAAAAATGGTAGTTATGTAAGT GCCATAACCCA AGGTCGCCGCGAAGAAGAGACCCCCGTAGAGTGCTTCGAGCCCTCGATGCTAAAGAAGAA GCAACTCA CTGTATTAGACTTGCATCCCGGCGCGGGTAAAACCCGCCGAGTATTGCCCGAAATAGTAC GTGAAGCC ATAAAAACGCGATTACGTACCGTCATATTAGCGCCGACCCGCGTAGTAGCGGCGGAAATG GAGGAGGC CTTGCGAGGTTTGCCGGTCCGTTATATGACGACGGCGGTAAATGTAACCCATTCGGGCAC GGAAATAG TAGACTTAATGTGCCATGCCACCTTCACCTCGCGTCTCCTCCAGCCGATACGAGTACCCA ATTATAAT CTATATATTATGGATGAGGCCCATTTCACGGATCCCTCGAGTATAGCGGCGCGAGGCTAC ATTTCGAC GCGCGTAGAGATGGGTGAGGCGGCGGCCATATTCATGACCGCCACGCCGCCGGGCACCCG TGACGCGT TCCCGGACTCGAATTCGCCGATTATGGACACCGAAGTCGAAGTACCGGAGCGAGCCTGGT CCTCGGGT TTTGATTGGGTCACGGATCATTCGGGCAAAACGGTATGGTTCGTACCGTCCGTCCGCAAT GGTAATGA GATAGCGGCGTGTCTAACGAAGGCGGGCAAACGGGTAATACAGTTATCCCGAAAGACCTT CGAGACGG AGTTCCAAAAAACGAAACATCAAGAGTGGGACTTCGTAGTCACGACCGACATTTCGGAGA TGGGTGCC AATTTCAAAGCGGACCGTGTAATAGATTCGCGCCGATGCCTCAAGCCGGTAATATTGGAT GGTGAGCG AGTAATTCTAGCGGGCCCCATGCCCGTAACGCATGCCTCCGCGGCCCAACGCCGCGGTCG CATAGGTC GCAATCCCAATAAACCCGGCGATGAGTATCTATATGGCGGTGGTTGCGCGGAGACCGACG AAGACCAT GCGCATTGGTTGGAAGCGCGAATGTTATTGGACAATATTTACTTACAAGATGGTTTAATA GCCTCGTT ATATCGACCCGAGGCCGACAAAGTAGCGGCCATTGAGGGCGAGTTCAAGTTGCGCACGGA GCAACGCA AGACCTTCGTCGAATTAATGAAACGAGGCGATTTGCCCGTATGGCTAGCCTATCAAGTAG CGTCGGCA GGTATAACCTACACGGATCGACGATGGTGCTTCGATGGTACGACCAATAATACCATAATG GAAGATAG TGTGCCGGCAGAGGTGTGGACCAGACACGGAGAGAAAAGAGTGCTCAAACCGAGGTGGAT GGACGCCA GAGTTTGTTCAGATCATGCGGCCCTGAAGTCATTCAAGGAGTTTGCCGCTGGGAAAAGA ( NS 4 A) GGA GCGGCTTTTGGAGTGATGGAAGCCCTGGGAACACTGCCAGGACACATGACAGAGAGATTC CAGGAAGC CATTGACAACCTCGCTGTGCTCATGCGGGCAGAGACTGGAAGCAGGCCTTACAAAGCCGC GGCGGCCC AATTGCCGGAGACCCTAGAGACCATAATGCTTTTGGGGTTGCTGGGAACAGTCTCGCTGG GAATCTTC TTCGTCTTGATGAGGAACAAGGGCATAGGGAAGATGGGCTTTGGAATGGTGACTCTTGGG GCCAGCGC ATGGCTCATGTGGCTCTCGGAAATTGAGCCAGCCAGAATTGCATGTGTCCTCATTGTTGT GTTCCTAT TGCTGGTGGTGCTCATACCTGAGCCAGAAAAGCAAAGATCTCCCCAGGACAACCAAATGG CAATCATC ATCATGGTAGCAGTAGGTCTTCTGGGCTTGATTACCGCC ( NS 4 B ) AATGAACTCGGATGGTTGGAGAG AACAAAGAGTGACCTAAGCCATCTAATGGGAAGGAGAGAGGAGGGGGCAACCATAGGATT CTCAATGG ACATTGACCTGCGGCCAGCCTCAGCTTGGGCCATCTATGCTGCCTTGACAACTTTCATTA CCCCAGCC GTCCAACATGCAGTGACCACCTCATACAACAACTACTCCTTAATGGCGATGGCCACGCAA GCTGGAGT GTTGTTTGGTATGGGCAAAGGGATGCCATTCTACGCATGGGACTTTGGAGTCCCGCTGCT AATGATAG GTTGCTACTCACAATTAACACCCCTGACCCTAATAGTGGCCATCATTTTGCTCGTGGCGC ACTACATG TACTTGATCCCAGGGCTGCAGGCAGCAGCTGCGCGTGCTGCCCAGAAGAGAACGGCAGCT GGCATCAT GAAGAACCCTGTTGTGGATGGAATAGTGGTGACTGACATTGACACAATGACAATTGACCC CCAAGTGG
AGAAAAAGATGGGACAGGTGCTACTCATAGCAGTAGCCGTCTCCAGCGCCATACTGT CGCGGACCGCC TGGGGGTGGGGGGAGGCTGGGGCCCTGATCACAGCCGCAACTTCCACTTTGTGGGAAGGC TCTCCGAA
CAAGTACTGGAACTCCTCTACAGCCACTTCACTGTGTAACATTTTTAGGGGAAGTTA CTTGGCTGGAG CTTCTCTAATCTACACAGTAACAAGAAACGCTGGCTTGGTCAAGAGACGT ( NS 5 ) GGGGGT GGAACAG GAGAGACCCTGGGAGAGAAATGGAAGGCCCGCTTGAACCAGATGTCGGCCCTGGAGTTCT ACTCCTAC AAAAAGTCAGGCATCACCGAGGTGTGCAGAGAAGAGGCCCGCCGCGCCCTCAAGGACGGT GTGGCAAC GGGAGGCCATGCTGTGTCCCGAGGAAGTGCAAAGCTGAGATGGTTGGTGGAGCGGGGATA CCTGCAGC CCTATGGAAAGGTCATTGATCTTGGATGTGGCAGAGGGGGCTGGAGTTACTACGCCGCCA CCATCCGC AAAGTTCAAGAAGTGAAAGGATACACAAAAGGAGGCCCTGGTCATGAAGAACCCGTGTTG GTGCAAAG CTATGGGTGGAACATAGTCCGTCTTAAGAGTGGGGTGGACGTCTTTCATATGGCGGCTGA GCCGTGTG ACACGCTGCTGTGTGACATAGGTGAGTCATCATCTAGTCCTGAAGTGGAAGAAGCACGGA CGCTCAGA GTCCTCTCCATGGTGGGGGATTGGCTTGAAAAAAGACCAGGAGCCTTTTGTATAAAAGTG TTGTGCCC ATACACCAGCACTATGATGGAAACCCTGGAGCGACTGCAGCGTAGGTATGGGGGAGGACT GGTCAGAG TGCCACTCTCCCGCAACTCTACACATGAGATGTACTGGGTCTCTGGAGCGAAAAGCAACA CCATAAAA AGTGTGTCCACCACGAGCCAGCTCCTCTTGGGGCGCATGGACGGGCCTAGAAGGCCAGTG AAATATGA GGAGGATGTGAATCTCGGCTCTGGCACGCGGGCTGTGGTAAGCTGCGCTGAAGCTCCCAA CATGAAGA TCATTGGTAACCGCATTGAAAGGATCCGCAGTGAGCACGCGGAAACGTGGTTCTTTGACG AGAACCAC CCATATAGGACATGGGCTTACCATGGAAGCTATGAGGCCCCCACACAAGGGTCAGCGTCC TCTCTAAT AAACGGGGTTGTCAGGCTCCTGTCAAAACCCTGGGATGTGGTGACTGGAGTCACAGGAAT AGCCATGA CCGACACCACACCGTATGGTCAGCAAAGAGTTTTCAAGGAAAAAGTGGACACTAGGGTGC CAGACCCC CAAGAAGGCACTCGTCAGGTTATGAGCATGGTCTCTTCCTGGTTGTGGAAAGAGCTAGGC AAACACAA ACGGCCACGAGTCTGTACCAAAGAAGAGTTCATCAACAAGGTTCGTAGCAATGCAGCATT AGGGGCAA TATTTGAAGAGGAAAAAGAGTGGAAGACTGCAGTGGAAGCTGTGAACGATCCAAGGTTCT GGGCTCTA GTGGACAAGGAAAGAGAGCACCACCTGAGAGGAGAGTGCCAGAGTTGTGTGTATAACATG ATGGGAAA AAGAGAAAAGAAACAAGGGGAATTTGGAAAGGCCAAGGGCAGCCGCGCCATCTGGTATAT GTGGCTAG GGGCTAGATTTCTAGAGTTCGAAGCCCTTGGATTCTTGAACGAGGATCACTGGATGGGGA GAGAGAAC TCAGGAGGTGGTGTTGAAGGGCTGGGATTACAAAGACTCGGATATGTCCTAGAAGAGATG AGTCGTAT ACCAGGAGGAAGGATGTATGCAGATGACACTGCTGGCTGGGACACCCGCATTAGCAGGTT TGATCTGG AGAATGAAGCTCTAATCACCAACCAAATGGAGAAAGGGCACAGGGCCTTGGCATTGGCCA TAATCAAG TACACATACCAAAACAAAGTGGTAAAGGTCCTTAGACCAGCTGAAAAAGGGAAAACAGTT ATGGACAT TATTTCGAGACAAGACCAAAGGGGGAGCGGACAAGTTGTCACTTACGCTCTTAACACATT TACCAACC TAGTGGTGCAACTCATTCGGAATATGGAGGCTGAGGAAGTTCTAGAGATGCAAGACTTGT GGCTGCTG CGGAGGTCAGAGAAAGTGACCAACTGGTTGCAGAGCAACGGATGGGATAGGCTCAAACGA ATGGCAGT CAGTGGAGATGATTGCGTTGTGAAGCCAATTGATGATAGGTTTGCACATGCCCTCAGGTT CTTGAATG ATATGGGAAAAGTTAGAAAGGACACACAAGAGTGGAAACCCTCAACTGGATGGGACAACT GGGAAGAA GTTCCGTTTTGCTCCCACCACTTCAACAAGCTCCATCTCAAGGACGGGAGGTCCATTGTG GTTCCCTG CCGCCACCAAGATGAACTGATTGGCCGGGCCCGCGTCTCTCCAGGGGCGGGATGGAGCAT CCGGGAGA
CTGCTTGCCTAGCAAAATCATATGCGCAGATGTGGCAGCTCCTTTATTTCCACAGAA GGGACCTCCGA CTGATGGCCAATGCCATTTGTTCATCTGTGCCAGTTGACTGGGTTCCAACTGGGAGAACT ACCTGGTC
AATCCATGGAAAGGGAGAATGGATGACCACTGAAGACATGCTTGTGGTGTGGAACAG AGTGTGGATTG AGGAGAACGACCACATGGAAGACAAGACCCCAGTTACGAAATGGACAGACATTCCCTATT TGGGAAAA AGGGAAGACTTGTGGTGTGGATCTCTCATAGGGCACAGACCGCGCACCACCTGGGCTGAG AACATTAA AAACACAGTCAACATGGTGCGCAGGATCATAGGTGATGAAGAAAAGTACATGGACTACCT ATCCACCC AAGTTCGCTACTTGGGTGAAGAAGGGTCTACACCTGGAGTGCTGTAA (NS5 end)
[0048] SEQ ID NO:10. Vaccine candidate ZIKV-DO-NS3, more extensive sequence of flanking regions, with the deoptimized region shown in underline, with positions of key regions indicated.
[0049] AGTTGTTGATCTGTGTGAATCAGACTGCGACAGTTCGAGTTTGAAGCGAAAGCTAGCAA
CAGTATCAACAGGTTTTATTTTGGATTTGGAAACGAGAGTTTCTGGTCATGAAAAAC CCAAAAAAGAA
ATCCGGAGGATTCCGGATTGTCAATATGCTAAAACGCGGAGTAGCCCGTGTGAGCCC CTTTGGGGGCT
TGAAGAGGCTGCCAGCCGGACTTCTGCTGGGTCATGGGCCCATCAGGATGGTCTTGG CGATTCTAGCC
TTTTTGAGATTCACGGCAATCAAGCCATCACTGGGTCTCATCAATAGATGGGGTTCA GTTGGGAAAAA
AGAGGCTATGGAAATAATAAAGAAGTTCAAGAAAGATCTGGCTGCCATGCTGAGAAT AATCAATGCTA
GGAAGGAGAAGAAGAGACGAGGCGCAGATACTAGTGTCGGAATTGTTGGCCTCCTGC TGACCACAGCT
ATGGCAGCGGAGGTCACTAGACGTGGGAGTGCATACTATATGTACTTGGACAGAAAC GATGCTGGGGA
GGCCATATCTTTTCCAACCACATTGGGGATGAATAAGTGTTATATACAGATCATGGA TCTTGGACACA
TGTGTGATGCCACCATGAGCTATGAATGCCCTATGCTGGATGAGGGGGTGGAACCAG ATGACGTCGAT
TGTTGGTGCAACACGACGTCAACTTGGGTTGTGTACGGAACCTGCCATCACAAAAAA GGTGAAGCACG
GAGATCTAGAAGAGCTGTGACGCTCCCCTCCCATTCCACTAGGAAGCTGCAAACGCG GTCGCAAACCT
GGTTGGAATCAAGAGAATACACAAAGCACTTGATTAGAGTCGAAAATTGGATATTCA GGAACCCTGGC
TTCGCGTTAGCAGCAGCTGCCATCGCTTGGCTTTTGGGAAGCTCAACGAGCCAAAAA GTCATATACTT
GGTCATGATACTGCTGATTGCCCCGGCATACAGCATCAGGTGCATAGGAGTCAGCAA TAGGGACTTTG
TGGAAGGTATGTCAGGTGGGACTTGGGTTGATGTTGTCTTGGAACATGGAGGTTGTG TCACCGTAATG
GCACAGGACAAACCGACTGTCGACATAGAGCTGGTTACAACAACAGTCAGCAACATG GCGGAGGTAAG
ATCCTACTGCTATGAGGCATCAATATCAGACATGGCTTCGGACAGCCGCTGCCCAAC ACAAGGTGAAG
CCTACCTTGACAAGCAATCAGACACTCAATATGTCTGCAAAAGAACGTTAGTGGACA GAGGCTGGGGA
AATGGATGTGGACTTTTTGGCAAAGGGAGCCTGGTGACATGCGCTAAGTTTGCATGC TCCAAGAAAAT
GACCGGGAAGAGCATCCAGCCAGAGAATCTGGAGTACCGGATAATGCTGTCAGTTCA TGGCTCCCAGC
ACAGTGGGATGATCGTTAATGACACAGGACATGAAACTGATGAGAATAGAGCGAAAG TTGAGATAACG
CCCAATTCACCAAGAGCCGAAGCCACCCTGGGGGGTTTTGGAAGCCTAGGACTTGAT TGTGAACCGAG
GACAGGCCTTGACTTTTCAGATTTGTATTACTTGACTATGAATAACAAGCACTGGTT GGTTCACAAGG
AGTGGTTCCACGACATTCCATTACCTTGGCACGCTGGGGCAGACACCGGAACTCCAC ACTGGAACAAC
AAAGAAGCACTGGTAGAGTTCAAGGACGCACATGCCAAAAGGCAAACTGTCGTGGTT CTAGGGAGTCA
AGAAGGAGCAGTTCACACGGCCCTTGCTGGAGCTCTGGAGGCTGAGATGGATGGTGC AAAGGGAAGGC
TGTCCTCTGGCCACTTGAAATGTCGCCTGAAAATGGATAAACTTAGATTGAAGGGCG TGTCATACTCC TTGTGTACTGCAGCGTTCACATTCACCAAGATCCCGGCTGAAACACTGCACGGGACAGTC ACAGTGGA
GGTACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCCAGCTCAGATGGCGGTGGA CATGCAAACTC TGACCCCAGTTGGGAGGTTGATAACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACT CTAAGATG ATGCTGGAACTTGATCCACCATTTGGGGACTCTTACATTGTCATAGGAGTCGGGGAGAAG AAGATCAC CCACCACTGGCACAGGAGTGGCAGCACCATTGGAAAAGCATTTGAAGCCACTGTGAGAGG TGCCAAGA GAATGGCAGTCTTGGGAGACACAGCCTGGGACTTTGGATCAGTTGGAGGCGCTCTCAACT CATTGGGC AAGGGCATCCATCAAATTTTTGGAGCAGCTTTCAAATCATTGTTTGGAGGAATGTCCTGG TTCTCACA AATTCTCATTGGAACGTTGCTGATGTGGTTGGGTCTGAACACAAAGAATGGATCTATTTC CCTTATGT GCTTGGCCTTAGGGGGGGTGTTGATCTTCTTATCCACAGCCGTCTCTGCT (NS1 ) GATGTGGGGTGCT CGGTGGACTTCTCAAAGAAGGAGACGAGATGCGGTACAGGGGTGTTCGTCTATAACGACG TTGAAGCC TGGAGGGACAGGTACAAGTACCATCCTGACTCCCCCCGTAGATTGGCAGCAGCAGTCAAG CAAGCCTG GGAAGATGGTATCTGCGGGATCTCCTCTGTTTCAAGAATGGAAAACATCATGTGGAGATC AGTAGAAG GGGAGCTCAACGCAATCCTGGAAGAGAATGGAGTTCAACTGACGGTCGTTGTGGGATCTG TAAAAAAC CCCATGTGGAGAGGTCCACAGAGATTGCCCGTGCCTGTGAACGAGCTGCCCCACGGCTGG AAGGCTTG GGGGAAATCGTACTTCGTCAGAGCAGCAAAGACAAATAACAGCTTTGTCGTGGATGGTGA CACACTGA AGGAATGCCCACTCAAACATAGAGCATGGAACAGCTTTCTTGTGGAGGATCATGGGTTCG GGGTATTT CACACTAGTGTCTGGCTCAAGGTTAGAGAAGATTATTCATTAGAGTGTGATCCAGCCGTT ATTGGAAC AGCTGTTAAGGGAAAGGAGGCTGTACACAGTGATCTAGGCTACTGGATTGAGAGTGAGAA GAATGACA CATGGAGGCTGAAGAGGGCCCATCTGATCGAGATGAAAACATGTGAATGGCCAAAGTCCC ACACATTG TGGACAGATGGAATAGAAGAGAGTGATCTGATCATACCCAAGTCTTTAGCTGGGCCACTC AGCCATCA CAATACCAGAGAGGGCTACAGGACCCAAATGAAAGGGCCATGGCACAGTGAAGAGCTTGA AATTCGGT TTGAGGAATGCCCAGGCACTAAGGTCCACGTGGAGGAAACATGTGGAACAAGAGGACCAT CTCTGAGA TCAACCACTGCAAGCGGAAGGGTGATCGAGGAATGGTGCTGCAGGGAGTGCACAATGCCC CCACTGTC GTTCCGGGCTAAAGATGGCTGTTGGTATGGAATGGAGATAAGGCCCAGGAAAGAACCAGA AAGCAACT TAGTAAGGTCAATGGTGACTGCA (NS2A) GGATCAACTGATCACATGGACCACTTCTCCCTTGGAGTG CTTGTGATCCTGCTCATGGTGCAGGAAGGGCTGAAGAAGAGAATGACCACAAAGATCATC ATAAGCAC ATCAATGGCAGTGCTGGTAGCTATGATCCTGGGAGGATTTTCAATGAGTGACCTGGCTAA GCTTGCAA TTTTGATGGGTGCCACCTTCGCGGAAATGAACACTGGAGGAGATGTAGCTCATCTGGCGC TGATAGCG GCATTCAAAGTCAGACCAGCGTTGCTGGTATCTTTCATCTTCAGAGCTAATTGGACACCC CGTGAAAG CATGCTGCTGGCCTTGGCCTCGTGTCTTTTGCAAACTGCGATCTCCGCCTTGGAAGGCGA CCTGATGG TTCTCATCAATGGTTTTGCTTTGGCCTGGTTGGCAATACGAGCGATGGTTGTTCCACGCA CTGATAAC ATCACCTTGGCAATCCTGGCTGCTCTGACACCACTGGCCCGGGGCACACTGCTTGTGGCG TGGAGAGC AGGCCTTGCTACTTGCGGGGGGTTTATGCTCCTCTCTCTGAAGGGAAAAGGCAGTGTGAA GAAGAACT TACCATTTGTCATGGCCCTGGGACTAACCGCTGTGAGGCTGGTCGACCCCATCAACGTGG TGGGACTG CTGTTACTCACAAGGAGTGGGAAGCGG (NS2B ) AGCTGGCCCCCTAGCGAAGTACTCACAGCTGTTGG CCTGATATGCGCATTGGCTGGAGGGTTCGCCAAGGCAGATATAGAGATGGCTGGGCCCAT GGCCGCGG
TCGGTCTGCTAATTGTCAGTTACGTGGTCTCAGGAAAGAGTGTGGACATGTACATTG AAAGAGCAGGT GACATCACATGGGAAAAAGATGCGGAAGTCACTGGAAACAGTCCCCGGCTCGATGTGGCG CTAGATGA
GAGTGGTGATTTCTCCCTGGTGGAGGATGACGGTCCCCCCATGAGAGAGATCATACT CAAGGTGGTCC TGATGACCATCTGTGGCATGAATCCAATAGCCATACCCTTTGCAGCTGGAGCGTGGTACG TATACGTG AAGACTGGAAAAAGG (NS3 ) AGTGGTGCGCTCTGGGATGTCCCCGCGCCCAAGGAAGTAAAAAAGGGT GAGACCACGGATGGCGTCTACCGAGTAATGACCCGTCGACTACTCGGTTCGACGCAAGTA GGCGTCGG CGTAATGCAAGAGGGTGTATTCCACACCATGTGGCATGTAACGAAAGGCTCGGCGCTACG ATCCGGTG AAGGTCGATTGGATCCGTACTGGGGCGATGTAAAGCAAGATCTAGTCTCGTACTGTGGTC CGTGGAAG CTCGATGCCGCCTGGGACGGTCACTCCGAGGTCCAGTTATTGGCCGTCCCGCCGGGCGAG CGAGCGCG CAATATACAAACTCTACCCGGCATATTCAAGACGAAGGATGGTGACATTGGCGCGGTAGC GCTAGATT ACCCGGCGGGCACTTCGGGCTCGCCGATACTCGACAAGTGTGGTCGAGTCATAGGCTTGT ATGGTAAT GGTGTAGTCATAAAAAATGGTAGTTATGTAAGTGCCATAACCCAAGGTCGCCGCGAAGAA GAGACCCC CGTAGAGTGCTTCGAGCCCTCGATGCTAAAGAAGAAGCAACTCACTGTATTAGACTTGCA TCCCGGCG CGGGTAAAACCCGCCGAGTATTGCCCGAAATAGTACGTGAAGCCATAAAAACGCGATTAC GTACCGTC ATATTAGCGCCGACCCGCGTAGTAGCGGCGGAAATGGAGGAGGCCTTGCGAGGTTTGCCA GTCCGTTA TATGACGACGGCGGTAAATGTAACCCATTCGGGCACGGAAATAGTAGACTTAATGTGCCA TGCCACCT TCACCTCGCGTCTCCTCCAGCCGATACGAGTACCCAATTATAATCTATATATTATGGATG AGGCCCAT TTCACGGATCCCTCGAGTATAGCGGCGCGAGGCTACATTTCGACGCGCGTAGAGATGGGT GAGGCGGC GGCCATATTCATGACCGCCACGCCGCCGGGCACCCGTGACGCGTTCCCGGACTCGAATTC GCCGATTA TGGACACCGAAGTCGAAGTACCGGAGCGAGCCTGGTCCTCGGGTTTTGATTGGGTCACGG ATCATTCG GGCAAAACGGTATGGTTCGTACCGTCCGTCCGCAATGGTAATGAGATAGCGGCGTGTCTA ACGAAGGC GGGCAAACGGGTAATACAGTTATCCCGAAAGACCTTCGAGACGGAGTTCCAAAAAACGAA ACATCAAG AGTGGGACTTCGTAGTCACGACCGACATTTCGGAGATGGGTGCCAATTTCAAAGCAGACC GTGTAATA GATTCGCGCCGATGCCTCAAGCCGGTAATATTGGATGGTGAGCGAGTAATTCTAGCGGGC CCCATGCC CGTAACGCATGCCTCCGCGGCCCAACGCCGCGGTCGCATAGGTCGCAATCCCAATAAACC CGGCGATG AGTATCTATATGGCGGTGGTTGCGCGGAGACCGACGAAGACCATGCGCATTGGTTGGAAG CGCGAATG TTATTGGACAATATTTACTTACAAGATGGTTTAATAGCCTCGTTATATCGACCCGAGGCC GACAAAGT AGCGGCCATTGAGGGCGAGTTCAAGTTGCGCACGGAGCAACGCAAGACCTTCGTCGAATT AATGAAAC GAGGCGATTTGCCCGTATGGCTAGCCTATCAAGTAGCGTCGGCAGGTATAACCTACACGG ATCGACGA TGGTGCTTCGATGGTACGACCAATAATACCATAATGGAAGATAGTGTGCCGGCAGAGGTG TGGACCAG ACACGGAGAGAAAAGAGTGCTCAAACCGAGGTGGATGGACGCCAGAGTTTGTTCAGATCA TGCGGCCC TGAAGTCATTCAAGGAGTTTGCCGCTGGGAAAAGA (NS4A) GGAGCGGCTTTTGGAGTGATGGAAGCC CTGGGAACACTGCCAGGACACATGACAGAGAGATTCCAGGAAGCCATTGACAACCTCGCT GTGCTCAT GCGGGCAGAGACTGGAAGCAGGCCTTACAAAGCCGCGGCGGCCCAATTGCCGGAGACCCT AGAGACCA TAATGCTTTTGGGGTTGCTGGGAACAGTCTCGCTGGGAATCTTCTTCGTCTTGATGAGGA ACAAGGGC ATAGGGAAGATGGGCTTTGGAATGGTGACTCTTGGGGCCAGCGCATGGCTCATGTGGCTC TCGGAAAT TGAGCCAGCCAGAATTGCATGTGTCCTCATTGTTGTGTTCCTATTGCTGGTGGTGCTCAT ACCTGAGC
CAGAAAAGCAAAGATCTCCCCAGGACAACCAAATGGCAATCATCATCATGGTAGCAG TAGGTCTTCTG GGCTTGATTACCGCC ( NS 4 B ) AAT G AAC T C G GAT G GT T G G AG AG AAC AAAG AG T G AC C T AAG C C AT C T AATGGGAAGGAGAGAGGAGGGGGCAACCATAGGATTCTCAATGGACATTGACCTGCGGCC AGCCTCAG CTTGGGCCATCTATGCTGCCTTGACAACTTTCATTACCCCAGCCGTCCAACATGCAGTGA CCACCTCA TACAACAACTACTCCTTAATGGCGATGGCCACGCAAGCTGGAGTGTTGTTTGGTATGGGC AAAGGGAT GCCATTCTACGCATGGGACTTTGGAGTCCCGCTGCTAATGATAGGTTGCTACTCACAATT AACACCCC TGACCCTAATAGTGGCCATCATTTTGCTCGTGGCGCACTACATGTACTTGATCCCAGGGC TGCAGGCA GCAGCTGCGCGTGCTGCCCAGAAGAGAACGGCAGCTGGCATCATGAAGAACCCTGTTGTG GATGGAAT AG T G GT G AC T G AC AT T G AC AC AAT G AC AAT T G AC C C C C AAG T G G AG AAAAAG AT G GG AC AGG T G C T AC TCATAGCAGTAGCCGTCTCCAGCGCCATACTGTCGCGGACCGCCTGGGGGTGGGGGGAGG CTGGGGCC CTGATCACAGCCGCAACTTCCACTTTGTGGGAAGGCTCTCCGAACAAGTACTGGAACTCC TCTACAGC CACTTCACTGTGTAACATTTTTAGGGGAAGTTACTTGGCTGGAGCTTCTCTAATCTACAC AGTAACAA GAAACGCTGGCTTGGTCAAGAGACGT ( NS5 ) GGGGGTGGAACAGGAGAGACCCTGGGAGAGAAATGGA AGGCCCGCTTGAACCAGATGTCGGCCCTGGAGTTCTACTCCTACAAAAAGTCAGGCATCA CCGAGGTG TGCAGAGAAGAGGCCCGCCGCGCCCTCAAGGACGGTGTGGCAACGGGAGGCCATGCTGTG TCCCGAGG AAGTGCAAAGCTGAGATGGTTGGTGGAGCGGGGATACCTGCAGCCCTATGGAAAGGTCAT TGATCTTG GATGTGGCAGAGGGGGCTGGAGTTACTACGCCGCCACCATCCGCAAAGTTCAAGAAGTGA AAGGATAC ACAAAAGGAGGCCCTGGTCATGAAGAACCCGTGTTGGTGCAAAGCTATGGGTGGAACATA GTCCGTCT TAAGAGTGGGGTGGACGTCTTTCATATGGCGGCTGAGCCGTGTGACACGCTGCTGTGTGA CATAGGTG AGTCATCATCTAGTCCTGAAGTGGAAGAAGCACGGACGCTCAGAGTCCTCTCCATGGTGG GGGATTGG CTTGAAAAAAGACCAGGAGCCTTTTGTATAAAAGTGTTGTGCCCATACACCAGCACTATG ATGGAAAC CCTGGAGCGACTGCAGCGTAGGTATGGGGGAGGACTGGTCAGAGTGCCACTCTCCCGCAA CTCTACAC ATGAGATGTACTGGGTCTCTGGAGCGAAAAGCAACACCATAAAAAGTGTGTCCACCACGA GCCAGCTC CTCTTGGGGCGCATGGACGGGCCTAGAAGGCCAGTGAAATATGAGGAGGATGTGAATCTC GGCTCTGG CACGCGGGCTGTGGTAAGCTGCGCTGAAGCTCCCAACATGAAGATCATTGGTAACCGCAT TGAAAGGA TCCGCAGTGAGCACGCGGAAACGTGGTTCTTTGACGAGAACCACCCATATAGGACATGGG CTTACCAT GGAAGCTATGAGGCCCCCACACAAGGGTCAGCGTCCTCTCTAATAAACGGGGTTGTCAGG CTCCTGTC AAAACCCTGGGATGTGGTGACTGGAGTCACAGGAATAGCCATGACCGACACCACACCGTA TGGTCAGC AAAGAGTTTTCAAGGAAAAAGTGGACACTAGGGTGCCAGACCCCCAAGAAGGCACTCGTC AGGTTATG AGCATGGTCTCTTCCTGGTTGTGGAAAGAGCTAGGCAAACACAAACGGCCACGAGTCTGT ACCAAAGA AGAGTTCATCAACAAGGTTCGTAGCAATGCAGCATTAGGGGCAATATTTGAAGAGGAAAA AGAGTGGA AGACTGCAGTGGAAGCTGTGAACGATCCAAGGTTCTGGGCTCTAGTGGACAAGGAAAGAG AGCACCAC CTGAGAGGAGAGTGCCAGAGTTGTGTGTATAACATGATGGGAAAAAGAGAAAAGAAACAA GGGGAATT TGGAAAGGCCAAGGGCAGCCGCGCCATCTGGTATATGTGGCTAGGGGCTAGATTTCTAGA GTTCGAAG CCCTTGGATTCTTGAACGAGGATCACTGGATGGGGAGAGAGAACTCAGGAGGTGGTGTTG AAGGGCTG GGATTACAAAGACTCGGATATGTCCTAGAAGAGATGAGTCGTATACCAGGAGGAAGGATG TATGCAGA TGACACTGCTGGCTGGGACACCCGCATTAGCAGGTTTGATCTGGAGAATGAAGCTCTAAT CACCAACC
AAATGGAGAAAGGGCACAGGGCCTTGGCATTGGCCATAATCAAGTACACATACCAAA ACAAAGTGGTA AAGGTCCTTAGACCAGCTGAAAAAGGGAAAACAGTTATGGACATTATTTCGAGACAAGAC CAAAGGGG
GAGCGGACAAGTTGTCACTTACGCTCTTAACACATTTACCAACCTAGTGGTGCAACT CATTCGGAATA TGGAGGCTGAGGAAGTTCTAGAGATGCAAGACTTGTGGCTGCTGCGGAGGTCAGAGAAAG TGACCAAC TGGTTGCAGAGCAACGGATGGGATAGGCTCAAACGAATGGCAGTCAGTGGAGATGATTGC GTTGTGAA GCCAATTGATGATAGGTTTGCACATGCCCTCAGGTTCTTGAATGATATGGGAAAAGTTAG AAAGGACA CACAAGAGTGGAAACCCTCAACTGGATGGGACAACTGGGAAGAAGTTCCGTTTTGCTCCC ACCACTTC AACAAGCTCCATCTCAAGGACGGGAGGTCCATTGTGGTTCCCTGCCGCCACCAAGATGAA CTGATTGG CCGGGCCCGCGTCTCTCCAGGGGCGGGATGGAGCATCCGGGAGACTGCTTGCCTAGCAAA ATCATATG CGCAGATGTGGCAGCTCCTTTATTTCCACAGAAGGGACCTCCGACTGATGGCCAATGCCA TTTGTTCA TCTGTGCCAGTTGACTGGGTTCCAACTGGGAGAACTACCTGGTCAATCCATGGAAAGGGA GAATGGAT GACCACTGAAGACATGCTTGTGGTGTGGAACAGAGTGTGGATTGAGGAGAACGACCACAT GGAAGACA AGACCCCAGTTACGAAATGGACAGACATTCCCTATTTGGGAAAAAGGGAAGACTTGTGGT GTGGATCT CTCATAGGGCACAGACCGCGCACCACCTGGGCTGAGAACATTAAAAACACAGTCAACATG GTGCGCAG GATCATAGGTGATGAAGAAAAGTACATGGACTACCTATCCACCCAAGTTCGCTACTTGGG TGAAGAAG GGTCTACACCTGGAGTGCTGTAA (NS5 end)
GCACCAATCTTAATGTTGTCAGGCCTGCTAGTCAGCCACAGCTTGGGGAAAGCTGTGCAG CCTGTGAC
CCCCCAGGAGAAGCTGGGAAACCAAGCCTATAGTCAGGCCGAGAACGCCATGGCACG GAAGAAGCCAT
GCTGCCTGTGAGCCCCTCAGAGGATACTGAGTCAAAAAACCCCACGCGCTTGGAGGC GCAGGATGGGA
AAAGAAGGTGGCGACCTTCCCCACCCTTCAATCTGGGGCCTGAACTGGAGATCAGCT GTGGATCCCCA
GAAGAGGGACTAGTGGTTAGAGGAGACCCCCCGGAAAACGCAAAACAGCATATTGAC GCTGGGAAAGA
CCAGAGACTCCATGAGTTTCCACCACGCTGGCCGCCAGGCACAGATCGCCGAACTTC GGCGGCCGGTG
TGGGGAAATCCATGGTTTCT
[0050] SEQ ID NO:1 1. Vaccine candidate ZIKV-DO-scattered, more extensive sequence of flanking regions, with deoptimized region shown in underline, with locations of key regions indicated.
[0051 ] AGTTGTTGATCTGTGTGAATCAGACTGCGACAGTTCGAGTTTGAAGCGAAAGCTAGCAA
CAGTATCAACAGGTTTTATTTTGGATTTGGAAACGAGAGTTTCTGGTCATGAAAAAC CCAAAAAAGAA
ATCCGGAGGATTCCGGATTGTCAATATGCTAAAACGCGGAGTAGCCCGTGTGAGCCC CTTTGGGGGCT
TGAAGAGGCTGCCAGCCGGACTTCTGCTGGGTCATGGGCCCATCAGGATGGTCTTGG CGATTCTAGCC
TTTTTGAGATTCACGGCAATCAAGCCATCACTGGGTCTCATCAATAGATGGGGTTCA GTTGGGAAAAA
AGAGGCTATGGAAATAATAAAGAAGTTCAAGAAAGATCTGGCTGCCATGCTGAGAAT AATCAATGCTA
GGAAGGAGAAGAAGAGACGAGGCGCAGATACTAGTGTCGGAATTGTTGGCCTCCTGC TGACCACAGCT
ATGGCAGCGGAGGTCACTAGACGTGGGAGTGCATACTATATGTACTTGGACAGAAAC GATGCTGGGGA
GGCCATATCTTTTCCAACCACATTGGGGATGAATAAGTGTTATATACAGATCATGGA TCTTGGACACA
TGTGTGATGCCACCATGAGCTATGAATGCCCTATGCTGGATGAGGGGGTGGAACCAG ATGACGTCGAT
TGTTGGTGCAACACGACGTCAACTTGGGTTGTGTACGGAACCTGCCATCACAAAAAA GGTGAAGCACG
GAGATCTAGAAGAGCTGTGACGCTCCCCTCCCATTCCACTAGGAAGCTGCAAACGCG GTCGCAAACCT GGTTGGAATCAAGAGAATACACAAAGCACTTGATTAGAGTCGAAAATTGGATATTCAGGA ACCCTGGC
TTCGCGTTAGCAGCAGCTGCCATCGCTTGGCTTTTGGGAAGCTCAACGAGCCAAAAA GTCATATACTT GGTCATGATACTGCTGATTGCCCCGGCATACAGCATCAGGTGCATAGGAGTCAGCAATAG GGACTTTG TGGAAGGTATGTCAGGTGGGACTTGGGTTGATGTTGTCTTGGAACATGGAGGTTGTGTCA CCGTAATG GCACAGGACAAACCGACTGTCGACATAGAGCTGGTTACAACAACAGTCAGCAACATGGCG GAGGTAAG ATCCTACTGCTATGAGGCATCAATATCAGACATGGCTTCGGACAGCCGCTGCCCAACACA AGGTGAAG CCTACCTTGACAAGCAATCAGACACTCAATATGTCTGCAAAAGAACGTTAGTGGACAGAG GCTGGGGA AATGGATGTGGACTTTTTGGCAAAGGGAGCCTGGTGACATGCGCTAAGTTTGCATGCTCC AAGAAAAT GACCGGGAAGAGCATCCAGCCAGAGAATCTGGAGTACCGGATAATGCTGTCAGTTCATGG CTCCCAGC ACAGTGGGATGATCGTTAATGACACAGGACATGAAACTGATGAGAATAGAGCGAAAGTTG AGATAACG CCCAATTCACCAAGAGCCGAAGCCACCCTGGGGGGTTTTGGAAGCCTAGGACTTGATTGT GAACCGAG GACAGGCCTTGACTTTTCAGATTTGTATTACTTGACTATGAATAACAAGCACTGGTTGGT TCACAAGG AGTGGTTCCACGACATTCCATTACCTTGGCACGCTGGGGCAGACACCGGAACTCCACACT GGAACAAC AAAGAAGCACTGGTAGAGTTCAAGGACGCACATGCCAAAAGGCAAACTGTCGTGGTTCTA GGGAGTCA AGAAGGAGCAGTTCACACGGCCCTTGCTGGAGCTCTGGAGGCTGAGATGGATGGTGCAAA GGGAAGGC TGTCCTCTGGCCACTTGAAATGTCGCCTGAAAATGGATAAACTTAGATTGAAGGGCGTGT CATACTCC TTGTGTACTGCAGCGTTCACATTCACCAAGATCCCGGCTGAAACACTGCACGGGACAGTC ACAGTGGA GGTACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCCAGCTCAGATGGCGGTGGACAT GCAAACTC TGACCCCAGTTGGGAGGTTGATAACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACT CTAAGATG ATGCTGGAACTTGATCCACCATTTGGGGACTCTTACATTGTCATAGGAGTCGGGGAGAAG AAGATCAC CCACCACTGGCACAGGAGTGGCAGCACCATTGGAAAAGCATTTGAAGCCACTGTGAGAGG TGCCAAGA GAATGGCAGTCTTGGGAGACACAGCCTGGGACTTTGGATCAGTTGGAGGCGCTCTCAACT CATTGGGC AAGGGCATCCATCAAATTTTTGGAGCAGCTTTCAAATCATTGTTTGGAGGAATGTCCTGG TTCTCACA AATTCTCATTGGAACGTTGCTGATGTGGTTGGGTCTGAACACAAAGAATGGATCTATTTC CCTTATGT GCTTGGCCTTAGGGGGGGTGTTGATCTTCTTATCCACAGCCGTCTCTGCT (NS1 ) GATGTAGGGTGCT CGGTAGACTTCTCAAAGAAGGAAACGAGATGCGGTACGGGGGTATTCGTCTATAACGATG TTGAAGCC TGGCGTGACAGGTACAAATACCATCCTGATTCCCCCCGTCGATTGGCAGCAGCGGTCAAG CAAGCGTG GGAAGATGGTATATGCGGGATCTCGTCTGTTTCACGTATGGAAAACATAATGTGGAGATC GGTAGAAG GGGAGCTAAACGCAATCCTAGAAGAGAATGGTGTTCAACTGACGGTAGTTGTAGGATCTG TAAAAAAC CCGATGTGGAGAGGTCCGCAGAGATTGCCGGTACCTGTAAACGAGCTGCCCCACGGTTGG AAGGCTTG GGGTAAATCGTACTTCGTAAGAGCAGCAAAAACAAATAACTCGTTTGTCGTGGATGGTGA TACACTGA AGGAATGTCCACTCAAACATCGTGCATGGAACTCGTTTCTTGTAGAGGATCATGGTTTCG GGGTATTT CATACTAGTGTCTGGCTAAAGGTTAGAGAAGATTATTCGTTAGAGTGTGATCCGGCCGTT ATTGGTAC AGCTGTTAAAGGAAAGGAGGCGGTACACAGTGATCTAGGTTACTGGATTGAAAGTGAGAA GAATGATA CATGGAGGCTAAAGAGGGCCCATCTAATCGAGATGAAAACGTGTGAATGGCCGAAGTCCC ACACGTTG TGGACAGATGGTATAGAAGAGTCGGATCTGATCATACCGAAGTCTTTAGCGGGGCCACTC AGTCATCA
CAATACGAGAGAGGGCTATAGGACCCAAATGAAAGGTCCATGGCACTCGGAAGAGCT TGAAATACGGT TTGAGGAATGTCCAGGCACTAAAGTCCACGTGGAAGAAACATGTGGTACAAGAGGACCGT CTCTGAGA
TCGACCACTGCAAGTGGAAGGGTAATCGAGGAATGGTGTTGCAGGGAGTGCACGATG CCCCCACTATC GTTCCGGGCGAAAGATGGCTGTTGGTATGGTATGGAGATACGTCCCAGGAAAGAACCGGA AAGCAACT TAGTACGTTCAATGGTAACTGCA (NS2A) GGATCGACTGATCACATGGATCACTTCTCCCTTGGAGTA CTTGTAATCCTGCTCATGGTACAGGAAGGGCTAAAGAAGAGAATGACGACAAAGATCATA ATAAGCAC ATCGATGGCAGTACTGGTAGCTATGATACTGGGAGGATTTTCGATGAGTGACCTAGCTAA GCTTGCGA TTTTGATGGGTGCGACCTTCGCGGAAATGAATACTGGAGGAGATGTAGCGCATCTGGCGC TAATAGCG GCATTTAAAGTCAGACCGGCGTTGCTGGTATCGTTCATCTTCCGTGCTAATTGGACGCCC CGTGAATC GATGCTGCTGGCGTTGGCCTCGTGTCTATTGCAAACTGCGATATCCGCCTTGGAAGGTGA CCTGATGG TACTCATCAATGGTTTTGCGTTGGCCTGGTTAGCAATACGAGCGATGGTAGTTCCACGCA CGGATAAC ATCACGTTGGCAATCCTAGCTGCTCTGACGCCACTGGCCCGTGGCACACTGCTTGTAGCG TGGAGAGC GGGCCTTGCTACGTGCGGGGGGTTTATGCTACTCTCTCTGAAAGGAAAAGGCAGTGTAAA GAAGAACT TACCGTTTGTCATGGCGCTGGGACTAACGGCTGTAAGGCTGGTCGATCCCATCAACGTAG TAGGACTG CTGTTACTAACAAGGAGTGGGAAACGG (NS2B) AGCTGGCCGCCTAGCGAAGTACTAACAGCTGTTGG TCTGATATGCGCATTGGCGGGAGGGTTCGCGAAGGCAGATATAGAAATGGCTGGGCCGAT GGCCGCGG TAGGTCTGCTAATAGTCAGTTACGTAGTCTCAGGAAAAAGTGTGGACATGTATATTGAAA GAGCGGGT GACATCACATGGGAAAAAGATGCGGAAGTAACTGGAAACAGTCCGCGGCTCGATGTAGCG CTAGATGA AAGTGGTGATTTTTCCCTGGTAGAGGATGACGGTCCCCCCATGAGAGAAATCATACTCAA AGTAGTCC TGATGACGATCTGTGGCATGAATCCGATAGCCATACCGTTTGCAGCTGGTGCGTGGTACG TATACGTA AAGACTGGAAAACGT (NS3 ) AGTGGTGCTCTATGGGATGTACCTGCTCCCAAAGAAGTAAAAAAAGGG GAGACCACGGATGGAGTATACAGAGTAATGACGCGTAGACTGCTAGGTTCGACACAAGTT GGTGTAGG AGTTATGCAAGAAGGGGTCTTTCATACTATGTGGCATGTCACAAAAGGTTCCGCGCTGCG TAGCGGTG AAGGTAGACTTGATCCGTACTGGGGAGATGTAAAGCAGGATCTAGTATCATACTGTGGTC CGTGGAAG CTAGATGCGGCCTGGGACGGTCACAGCGAGGTACAGCTCTTGGCGGTACCCCCCGGAGAA AGAGCGAG GAATATCCAGACTCTACCCGGAATATTTAAAACAAAGGATGGTGACATTGGAGCGGTAGC GCTGGATT ATCCAGCAGGAACGTCAGGATCTCCGATCCTAGACAAATGTGGGAGAGTAATAGGACTTT ATGGTAAT GGGGTCGTAATCAAAAATGGTAGTTATGTTAGTGCGATCACCCAAGGTAGGAGGGAAGAA GAAACTCC TGTTGAATGCTTCGAGCCGTCGATGCTGAAAAAGAAGCAGCTAACGGTCTTAGACTTACA TCCTGGAG CGGGGAAAACCCGAAGAGTTCTTCCGGAAATAGTCCGTGAAGCGATAAAAACACGTCTCC GTACTGTA ATCTTAGCTCCGACCAGGGTTGTAGCTGCTGAAATGGAAGAGGCCCTTCGTGGGCTTCCA GTACGTTA TATGACGACAGCAGTCAATGTAACCCACTCTGGTACAGAAATCGTTGACTTAATGTGTCA TGCCACCT TTACTTCACGTCTACTACAACCAATCAGAGTTCCCAACTATAATCTATATATTATGGATG AAGCCCAC TTCACGGATCCCTCAAGTATAGCGGCAAGAGGATATATTTCAACAAGGGTTGAAATGGGC GAGGCGGC GGCCATCTTCATGACGGCCACGCCACCGGGAACCCGTGATGCATTTCCGGATTCCAACTC ACCGATTA TGGACACGGAAGTGGAAGTTCCAGAGAGAGCGTGGAGCTCAGGTTTTGATTGGGTAACGG ATCATTCG GGAAAAACAGTTTGGTTTGTTCCGAGCGTGAGGAATGGCAATGAGATAGCAGCTTGTCTA ACAAAGGC
TGGTAAACGGGTCATACAACTCAGCAGAAAAACTTTTGAAACAGAGTTCCAAAAAAC AAAACATCAAG AATGGGACTTTGTTGTTACAACTGACATATCAGAGATGGGTGCCAACTTTAAAGCTGACC GTGTCATA
GATTCGAGGAGATGCCTAAAGCCGGTCATACTAGATGGCGAGCGAGTCATTCTGGCG GGACCCATGCC GGTCACACATGCGAGCGCTGCCCAAAGGAGGGGGCGTATAGGCAGGAATCCGAACAAACC TGGTGATG AGTATCTATATGGAGGTGGTTGCGCAGAGACGGACGAAGACCATGCGCACTGGCTTGAAG CGAGAATG CTCCTAGACAATATTTATCTCCAAGATGGTCTCATAGCCTCGCTATATCGACCTGAAGCC GACAAAGT AGCGGCCATTGAGGGTGAGTTCAAGCTAAGGACGGAGCAACGTAAGACCTTTGTAGAACT CATGAAAA GAGGTGATCTTCCTGTATGGCTGGCCTATCAAGTTGCATCTGCGGGAATAACCTATACAG ATAGAAGA TGGTGTTTTGATGGCACGACGAACAACACCATAATGGAAGATTCGGTGCCGGCAGAAGTG TGGACCAG ACATGGAGAGAAACGTGTGCTCAAACCGAGGTGGATGGATGCCAGAGTTTGTTCAGATCA TGCGGCGC TGAAGTCATTTAAGGAGTTTGCGGCTGGGAAAAGA ( NS 4A) GGTGCGGCTTTTGGTGTAATGGAAGCC CTGGGAACACTGCCGGGACACATGACGGAGAGATTCCAAGAAGCCATTGATAACCTCGCT GTACTCAT GCGGGCGGAGACTGGAAGTAGGCCTTACAAAGCCGCGGCGGCGCAATTGCCGGAAACCCT AGAGACGA TAATGCTTTTAGGGTTGCTGGGTACAGTCTCGCTAGGAATCTTCTTTGTCTTGATGCGTA ACAAGGGC ATAGGTAAGATGGGCTTTGGTATGGTGACTCTAGGGGCCAGCGCGTGGCTCATGTGGCTA TCGGAAAT TGAACCAGCCAGAATAGCATGTGTCCTAATTGTTGTATTCCTATTGCTGGTAGTACTCAT ACCTGAAC CAGAAAAGCAACGTTCTCCCCAGGATAACCAAATGGCAATAATCATCATGGTAGCGGTAG GTCTTCTA GGCTTGATTACGGCC ( NS 4 B ) AATGAACTAGGATGGTTGGAAAGAACAAAGTCGGACCTAAGCCATCT AATGGGTAGGAGAGAGGAAGGGGCAACCATAGGTTTCTCAATGGATATTGACCTGCGTCC AGCCTCAG CGTGGGCCATCTATGCGGCCTTGACAACGTTCATTACCCCGGCCGTCCAACATGCGGTGA CCACCTCG TACAACAACTATTCCTTAATGGCGATGGCGACGCAAGCTGGTGTGTTGTTTGGTATGGGT AAAGGGAT GCCGTTCTACGCATGGGATTTTGGAGTCCCGCTACTAATGATAGGTTGTTACTCACAATT AACGCCCC TGACCCTAATAGTAGCCATCATTTTACTCGTGGCGCATTACATGTACTTAATCCCAGGGC TACAGGCA GCAGCGGCGCGTGCTGCGCAGAAGAGAACGGCGGCTGGCATCATGAAAAACCCTGTTGTA GATGGAAT AGTAGTGACTGACATAGACACAATGACGATTGACCCCCAAGTAGAGAAAAAGATGGGTCA GGTGCTAC TAATAGCAGTAGCGGTCTCCAGCGCGATACTGTCGCGGACCGCCTGGGGTTGGGGGGAGG CGGGGGCC CTGATAACAGCCGCAACGTCCACTTTGTGGGAAGGTTCTCCGAACAAATACTGGAACTCG TCTACAGC CACGTCACTGTGTAATATTTTTAGGGGTAGTTACTTGGCGGGAGCTTCTCTAATATACAC AGTAACGA GAAACGCTGGTTTGGTCAAGCGTCGT ( NS 5 ) GGGGGTGGTACAGGAGAGACGCTGGGAGAGAAATGGA AAGCCCGCTTGAATCAGATGTCGGCGCTGGAGTTCTATTCCTACAAAAAATCAGGCATCA CGGAGGTG TGCCGTGAAGAGGCCCGTCGCGCCCTCAAAGACGGTGTGGCGACGGGAGGCCATGCGGTG TCCCGAGG TAGTGCAAAGCTAAGATGGTTGGTAGAGCGGGGATATCTGCAGCCCTATGGTAAGGTCAT TGATCTAG GATGTGGCCGTGGGGGCTGGTCGTACTACGCCGCGACCATCCGCAAAGTACAAGAAGTGA AAGGTTAC ACAAAAGGTGGCCCTGGTCATGAAGAACCCGTGTTGGTGCAAAGTTATGGGTGGAATATA GTCCGTCT AAAGAGTGGGGTAGACGTCTTTCATATGGCGGCGGAGCCGTGTGATACGCTGCTGTGTGA TATAGGTG AGTCGTCATCTAGTCCGGAAGTGGAAGAAGCGCGGACGCTCCGTGTCCTCTCCATGGTAG GGGATTGG CTAGAAAAAAGACCGGGAGCCTTTTGTATAAAAGTATTGTGCCCATATACCAGCACTATG ATGGAAAC
GCTGGAGCGACTACAGCGTAGGTATGGTGGAGGACTGGTAAGAGTGCCACTATCCCG CAACTCGACAC ATGAGATGTATTGGGTCTCTGGTGCGAAAAGCAATACCATAAAAAGTGTATCCACCACGT CGCAGCTC
CTCTTAGGGCGCATGGATGGGCCTAGACGTCCAGTGAAATATGAAGAGGATGTGAAT CTAGGCTCTGG CACGCGTGCTGTGGTAAGTTGCGCTGAAGCGCCCAACATGAAAATCATTGGTAATCGCAT TGAAAGGA TACGCAGTGAGCATGCGGAAACGTGGTTTTTTGACGAGAATCACCCATATAGGACGTGGG CTTACCAT GGTAGCTATGAGGCGCCCACACAAGGTTCAGCGTCCTCGCTAATAAACGGTGTTGTCAGG CTACTGTC AAAACCGTGGGATGTGGTAACTGGAGTCACGGGAATAGCCATGACGGACACCACACCGTA TGGTCAAC AAAGAGTTTTTAAGGAAAAAGTAGACACTAGGGTACCAGACCCCCAAGAAGGTACTCGTC AGGTAATG AGCATGGTATCTTCCTGGTTATGGAAAGAGCTAGGTAAACACAAACGTCCACGAGTCTGT ACGAAAGA AGAGTTTATCAACAAGGTACGTAGCAATGCGGCATTAGGGGCGATATTTGAAGAAGAAAA AGAGTGGA AAACTGCAGTGGAAGCGGTGAACGATCCGAGGTTCTGGGCGCTAGTGGACAAAGAAAGAG AGCATCAC CTGAGAGGTGAGTGCCAGTCGTGTGTGTATAATATGATGGGAAAACGTGAAAAGAAACAA GGTGAATT TGGAAAAGCCAAGGGCAGCCGTGCCATCTGGTATATGTGGCTAGGTGCTAGATTTCTAGA ATTCGAAG CCCTTGGATTCTTGAATGAGGATCACTGGATGGGTAGAGAGAACTCGGGAGGTGGTGTAG AAGGGCTG GGTTTACAAAGACTAGGATATGTCCTAGAAGAAATGAGTCGTATACCGGGAGGAAGGATG TATGCGGA TGACACTGCGGGCTGGGACACGCGCATTAGCCGTTTTGATCTGGAAAATGAAGCTCTAAT AACCAACC AAATGGAAAAAGGGCACAGGGCGTTGGCATTGGCGATAATCAAGTATACATACCAAAATA AAGTGGTA AAAGTCCTTAGACCGGCTGAAAAAGGTAAAACAGTTATGGATATTATTTCGCGTCAAGAC CAAAGGGG TAGCGGACAAGTAGTCACTTACGCGCTTAACACATTTACGAACCTAGTGGTACAACTCAT TCGTAATA TGGAGGCGGAGGAAGTTCTAGAAATGCAAGACTTATGGCTGCTGCGTAGGTCAGAGAAAG TAACCAAC TGGTTACAGAGCAACGGTTGGGATAGGCTAAAACGAATGGCGGTCAGTGGAGATGATTGT GTTGTGAA GCCGATTGATGATCGTTTTGCACATGCGCTCAGGTTCTTAAATGATATGGGTAAAGTTAG AAAAGACA CACAAGAATGGAAACCCTCGACTGGATGGGATAACTGGGAAGAAGTACCGTTTTGCTCGC ACCACTTC AATAAGCTCCATCTAAAGGACGGGCGTTCCATTGTGGTACCCTGCCGCCATCAAGATGAA CTAATTGG CCGGGCGCGCGTCTCTCCGGGGGCGGGATGGTCGATCCGGGAGACGGCTTGCCTAGCGAA ATCATATG CGCAAATGTGGCAGCTACTTTATTTCCATAGAAGGGACCTACGACTGATGGCGAATGCCA TTTGTTCG TCTGTGCCAGTAGACTGGGTTCCGACTGGGAGAACGACCTGGTCAATACATGGAAAGGGT GAATGGAT GACGACTGAAGACATGCTAGTGGTGTGGAATAGAGTGTGGATAGAGGAGAACGATCACAT GGAAGATA AGACCCCAGTAACGAAATGGACGGACATTCCCTATTTAGGAAAAAGGGAAGATTTGTGGT GTGGTTCT CTCATAGGTCACAGACCGCGTACCACCTGGGCGGAGAACATTAAAAATACAGTCAACATG GTACGCAG GATCATAGGTGATGAAGAAAAATATATGGACTACCTATCGACCCAAGTTCGTTACTTGGG TGAAGAAG GTTCTACACCTGGTGTGCTGTAA (NS5 end)
GCACCAATCTTAATGTTGTCAGGCCTGCTAGTCAGCCACAGCTTGGGGAAAGCTGTG CAGCCTGTGAC
CCCCCAGGAGAAGCTGGGAAACCAAGCCTATAGTCAGGCCGAGAACGCCATGGCACG GAAGAAGCCAT
GCTGCCTGTGAGCCCCTCAGAGGATACTGAGTCAAAAAACCCCACGCGCTTGGAGGC GCAGGATGGGA
AAAGAAGGTGGCGACCTTCCCCACCCTTCAATCTGGGGCCTGAACTGGAGATCAGCT GTGGATCCCCA
GAAGAGGGACTAGTGGTTAGAGGAGACCCCCCGGAAAACGCAAAACAGCATATTGAC GCTGGGAAAGA
CCAGAGACTCCATGAGTTTCCACCACGCTGGCCGCCAGGCACAGATCGCCGAACTTC GGCGGCCGGTG TGGGGAAATCCATGGTTTCT
[0052] SEQ ID NO:12. Vaccine candidate ZIKV-DO, more extensive sequence of flanking regions, with deoptimized region shown in underline, with locations of key regions indicated.
[0053] AGTTGTTGATCTGTGTGAATCAGACTGCGACAGTTCGAGTTTGAAGCGAAAGCTAGCAA CAGTATCAACAGGTTTTATTTTGGATTTGGAAACGAGAGTTTCTGGTCATGAAAAACCCA AAAAAGAA ATCCGGAGGATTCCGGATTGTCAATATGCTAAAACGCGGAGTAGCCCGTGTGAGCCCCTT TGGGGGCT TGAAGAGGCTGCCAGCCGGACTTCTGCTGGGTCATGGGCCCATCAGGATGGTCTTGGCGA TTCTAGCC TTTTTGAGATTCACGGCAATCAAGCCATCACTGGGTCTCATCAATAGATGGGGTTCAGTT GGGAAAAA AGAGGCTATGGAAATAATAAAGAAGTTCAAGAAAGATCTGGCTGCCATGCTGAGAATAAT CAATGCTA GGAAGGAGAAGAAGAGACGAGGCGCAGATACTAGTGTCGGAATTGTTGGCCTCCTGCTGA CCACAGCT ATGGCAGCGGAGGTCACTAGACGTGGGAGTGCATACTATATGTACTTGGACAGAAACGAT GCTGGGGA GGCCATATCTTTTCCAACCACATTGGGGATGAATAAGTGTTATATACAGATCATGGATCT TGGACACA TGTGTGATGCCACCATGAGCTATGAATGCCCTATGCTGGATGAGGGGGTGGAACCAGATG ACGTCGAT TGTTGGTGCAACACGACGTCAACTTGGGTTGTGTACGGAACCTGCCATCACAAAAAAGGT GAAGCACG GAGATCTAGAAGAGCTGTGACGCTCCCCTCCCATTCCACTAGGAAGCTGCAAACGCGGTC GCAAACCT GGTTGGAATCAAGAGAATACACAAAGCACTTGATTAGAGTCGAAAATTGGATATTCAGGA ACCCTGGC TTCGCGTTAGCAGCAGCTGCCATCGCTTGGCTTTTGGGAAGCTCAACGAGCCAAAAAGTC ATATACTT GGTCATGATACTGCTGATTGCCCCGGCATACAGCATCAGGTGCATAGGAGTCAGCAATAG GGACTTTG TGGAAGGTATGTCAGGTGGGACTTGGGTTGATGTTGTCTTGGAACATGGAGGTTGTGTCA CCGTAATG GCACAGGACAAACCGACTGTCGACATAGAGCTGGTTACAACAACAGTCAGCAACATGGCG GAGGTAAG ATCCTACTGCTATGAGGCATCAATATCAGACATGGCTTCGGACAGCCGCTGCCCAACACA AGGTGAAG CCTACCTTGACAAGCAATCAGACACTCAATATGTCTGCAAAAGAACGTTAGTGGACAGAG GCTGGGGA AATGGATGTGGACTTTTTGGCAAAGGGAGCCTGGTGACATGCGCTAAGTTTGCATGCTCC AAGAAAAT GACCGGGAAGAGCATCCAGCCAGAGAATCTGGAGTACCGGATAATGCTGTCAGTTCATGG CTCCCAGC ACAGTGGGATGATCGTTAATGACACAGGACATGAAACTGATGAGAATAGAGCGAAAGTTG AGATAACG CCCAATTCACCAAGAGCCGAAGCCACCCTGGGGGGTTTTGGAAGCCTAGGACTTGATTGT GAACCGAG GACAGGCCTTGACTTTTCAGATTTGTATTACTTGACTATGAATAACAAGCACTGGTTGGT TCACAAGG AGTGGTTCCACGACATTCCATTACCTTGGCACGCTGGGGCAGACACCGGAACTCCACACT GGAACAAC AAAGAAGCACTGGTAGAGTTCAAGGACGCACATGCCAAAAGGCAAACTGTCGTGGTTCTA GGGAGTCA AGAAGGAGCAGTTCACACGGCCCTTGCTGGAGCTCTGGAGGCTGAGATGGATGGTGCAAA GGGAAGGC TGTCCTCTGGCCACTTGAAATGTCGCCTGAAAATGGATAAACTTAGATTGAAGGGCGTGT CATACTCC TTGTGTACTGCAGCGTTCACATTCACCAAGATCCCGGCTGAAACACTGCACGGGACAGTC ACAGTGGA GGTACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCCAGCTCAGATGGCGGTGGACAT GCAAACTC TGACCCCAGTTGGGAGGTTGATAACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACT CTAAGATG ATGCTGGAACTTGATCCACCATTTGGGGACTCTTACATTGTCATAGGAGTCGGGGAGAAG AAGATCAC
CCACCACTGGCACAGGAGTGGCAGCACCATTGGAAAAGCATTTGAAGCCACTGTGAG AGGTGCCAAGA GAATGGCAGTCTTGGGAGACACAGCCTGGGACTTTGGATCAGTTGGAGGCGCTCTCAACT CATTGGGC
AAGGGCATCCATCAAATTTTTGGAGCAGCTTTCAAATCATTGTTTGGAGGAATGTCC TGGTTCTCACA AATTCTCATTGGAACGTTGCTGATGTGGTTGGGTCTGAACACAAAGAATGGATCTATTTC CCTTATGT GCTTGGCCTTAGGGGGGGTGTTGATCTTCTTATCCACAGCCGTCTCTGCTGAT (NS1 ) GTCGGTTGTT CGGTAGATTTTTCGAAAAAAGAAACGCGATGTGGTACGGGTGTATTTGTATATAATGACG TAGAAGCG TGGCGAGACCGATACAAGTATCATCCGGACTCGCCGCGACGATTAGCGGCGGCGGTAAAA CAAGCGTG GGAAGACGGTATATGCGGTATATCGTCGGTATCGCGAATGGAAAATATAATGTGGCGATC GGTAGAAG GTGAGTTAAATGCGATACTAGAAGAGAATGGCGTACAACTAACGGTAGTAGTCGGCTCGG TAAAAAAT CCCATGTGGCGAGGTCCGCAGCGATTGCCCGTCCCCGTCAATGAGCTACCCCATGGTTGG AAGGCGTG GGGTAAATCGTACTTCGTACGAGCGGCGAAGACGAATAATTCCTTTGTAGTCGATGGTGA CACGCTAA AGGAATGCCCGTTAAAACATCGAGCGTGGAATTCCTTTTTGGTCGAGGATCATGGTTTCG GTGTATTC CATACCAGTGTATGGTTAAAGGTACGAGAAGATTATTCGTTAGAGTGTGATCCGGCCGTA ATTGGCAC GGCGGTAAAGGGCAAGGAGGCGGTACATAGTGATCTCGGTTACTGGATTGAGAGTGAGAA GAATGACA CGTGGCGCCTAAAGCGCGCCCATCTAATAGAGATGAAAACGTGTGAATGGCCGAAGTCGC ACACGTTG TGGACGGATGGCATAGAAGAGAGTGATCTAATAATACCCAAGTCGTTAGCGGGTCCGTTA TCCCATCA TAATACCCGAGAGGGTTACCGCACCCAAATGAAAGGTCCGTGGCATAGTGAAGAGTTGGA AATTCGGT TCGAGGAATGTCCGGGTACCAAGGTACACGTCGAGGAAACGTGTGGCACGCGAGGCCCGT CGCTACGA TCGACCACCGCGTCCGGCCGCGTCATAGAGGAATGGTGCTGCCGCGAGTGCACGATGCCC CCGCTATC GTTCCGGGCGAAAGATGGTTGTTGGTATGGAATGGAGATACGCCCCCGCAAAGAACCGGA ATCCAATT TAGTACGCTCGATGGTCACCGCGGGCTCGACCGATCATATGGACCATTTCTCGTTG (NS2A) GGCGTC TTGGTCATACTATTAATGGTCCAAGAAGGTCTAAAGAAGCGAATGACCACGAAGATAATA ATATCCAC GTCGATGGCGGTCCTAGTAGCGATGATACTAGGCGGCTTTTCGATGAGTGACCTAGCGAA GTTGGCGA TTTTGATGGGTGCCACCTTCGCGGAAATGAATACCGGCGGCGATGTAGCGCATCTAGCGC TAATAGCG GCGTTCAAAGTACGACCGGCGTTGCTAGTATCGTTCATATTCCGAGCGAATTGGACGCCC CGTGAATC CATGCTACTAGCCTTGGCCTCGTGTTTGTTGCAAACCGCGATATCGGCCTTGGAAGGTGA CCTAATGG TATTAATAAATGGTTTCGCGTTGGCCTGGTTGGCGATACGAGCGATGGTAGTACCGCGCA CCGATAAT ATAACCTTGGCGATACTAGCGGCGCTAACGCCGCTAGCCCGGGGTACGCTATTGGTCGCG TGGCGAGC GGGTTTGGCGACCTGCGGTGGTTTTATGTTATTATCGCTAAAGGGCAAAGGTAGTGTCAA GAAGAATT TACCGTTTGTAATGGCCCTAGGCCTCACCGCGGTCCGCCTAGTAGACCCCATAAATGTCG TCGGCCTA CTATTATTAACGCGCAGTGGTAAGCGGTCCTGGCCC (NS2B ) CCCTCCGAAGTATTAACGGCGGTAGG TCTAATATGCGCGTTGGCGGGCGGTTTCGCCAAGGCGGATATAGAGATGGCGGGTCCCAT GGCCGCGG TAGGTCTACTCATTGTAAGTTACGTCGTATCGGGCAAGAGTGTCGACATGTACATTGAAC GAGCGGGT GACATAACGTGGGAAAAAGATGCGGAAGTAACCGGCAATAGTCCCCGGTTAGATGTCGCG CTCGATGA GAGTGGTGATTTCTCGCTAGTCGAGGATGACGGTCCCCCGATGCGAGAGATAATATTAAA GGTCGTAC TAATGACCATATGTGGTATGAATCCGATAGCCATACCCTTCGCGGCGGGCGCGTGGTACG TATACGTC AAGACCGGCAAACGC (NS3 ) AGTGGTGCGCTCTGGGATGTCCCCGCGCCCAAGGAAGTAAAAAAGGGT
GAGACCACGGATGGCGTCTACCGAGTAATGACCCGTCGACTACTCGGTTCGACGCAA GTAGGCGTCGG CGTAATGCAAGAGGGTGTATTCCACACCATGTGGCATGTAACGAAAGGCTCGGCGCTACG ATCCGGTG
AAGGTCGATTGGATCCGTACTGGGGCGATGTAAAGCAAGATCTAGTCTCGTACTGTG GTCCGTGGAAG CTCGATGCCGCCTGGGACGGTCACTCCGAGGTCCAGTTATTGGCCGTCCCGCCGGGCGAG CGAGCGCG CAATATACAAACTCTACCCGGCATATTCAAGACGAAGGATGGTGACATTGGCGCGGTAGC GCTAGATT ACCCGGCGGGCACTTCGGGCTCGCCGATACTCGACAAGTGTGGTCGAGTCATAGGCTTGT ATGGTAAT GGTGTAGTCATAAAAAATGGTAGTTATGTAAGTGCCATAACCCAAGGTCGCCGCGAAGAA GAGACCCC CGTAGAGTGCTTCGAGCCCTCGATGCTAAAGAAGAAGCAACTCACTGTATTAGACTTGCA TCCCGGCG CGGGTAAAACCCGCCGAGTATTGCCCGAAATAGTACGTGAAGCCATAAAAACGCGATTAC GTACCGTC ATATTAGCGCCGACCCGCGTAGTAGCGGCGGAAATGGAGGAGGCCTTGCGAGGTTTGCCG GTCCGTTA TATGACGACGGCGGTAAATGTAACCCATTCGGGCACGGAAATAGTAGACTTAATGTGCCA TGCCACCT TCACCTCGCGTCTCCTCCAGCCGATACGAGTACCCAATTATAATCTATATATTATGGATG AGGCCCAT TTCACGGATCCCTCGAGTATAGCGGCGCGAGGCTACATTTCGACGCGCGTAGAGATGGGT GAGGCGGC GGCCATATTCATGACCGCCACGCCGCCGGGCACCCGTGACGCGTTCCCGGACTCGAATTC GCCGATTA TGGACACCGAAGTCGAAGTACCGGAGCGAGCCTGGTCCTCGGGTTTTGATTGGGTCACGG ATCATTCG GGCAAAACGGTATGGTTCGTACCGTCCGTCCGCAATGGTAATGAGATAGCGGCGTGTCTA ACGAAGGC GGGCAAACGGGTAATACAGTTATCCCGAAAGACCTTCGAGACGGAGTTCCAAAAAACGAA ACATCAAG AGTGGGACTTCGTAGTCACGACCGACATTTCGGAGATGGGTGCCAATTTCAAAGCGGACC GTGTAATA GATTCGCGCCGATGCCTCAAGCCGGTAATATTGGATGGTGAGCGAGTAATTCTAGCGGGC CCCATGCC CGTAACGCATGCCTCCGCGGCCCAACGCCGCGGTCGCATAGGTCGCAATCCCAATAAACC CGGCGATG AGTATCTATATGGCGGTGGTTGCGCGGAGACCGACGAAGACCATGCGCATTGGTTGGAAG CGCGAATG TTATTGGACAATATTTACTTACAAGATGGTTTAATAGCCTCGTTATATCGACCCGAGGCC GACAAAGT AGCGGCCATTGAGGGCGAGTTCAAGTTGCGCACGGAGCAACGCAAGACCTTCGTCGAATT AATGAAAC GAGGCGATTTGCCCGTATGGCTAGCCTATCAAGTAGCGTCGGCAGGTATAACCTACACGG ATCGACGA TGGTGCTTCGATGGTACGACCAATAATACCATAATGGAAGATAGTGTGCCGGCAGAGGT GT GG AC C AG ACACGGAGAGAAAAGAGTGCTCAAACCGAGGTGGATGGACGCCAGAGTTTGTTCAGATCA TGCGGCCC TGAAGTCATTCAAGGAGTTTGCCGCTGGGAAAAGA ( NS 4 A) GGAGCGGCTTTTGGAGTGATGGAAGCC CTGGGAACACTGCCAGGACACATGACAGAGAGATTCCAGGAAGCCATTGACAACCTCGCT GTGCTCAT GCGGGCAGAGACTGGAAGCAGGCCTTACAAAGCCGCGGCGGCCCAATTGCCGGAGACCCT AGAGACCA TAATGCTTTTGGGGTTGCTGGGAACAGTCTCGCTGGGAATCTTCTTCGTCTTGATGAGGA ACAAGGGC ATAGGGAAGATGGGCTTTGGAATGGTGACTCTTGGGGCCAGCGCATGGCTCATGTGGCTC TCGGAAAT TGAGCCAGCCAGAATTGCATGTGTCCTCATTGTTGTGTTCCTATTGCTGGTGGTGCTCAT ACCTGAGC CAGAAAAGCAAAGATCTCCCCAGGACAACCAAATGGCAATCATCATCATGGTAGCAGTAG GTCTTCTG GGCTTGATTACCGCC ( NS 4 B ) AAT G AAC T C G GAT G GT T G G AG AG AAC AAAG AG T G AC C T AAG C C AT C T AATGGGAAGGAGAGAGGAGGGGGCAACCATAGGATTCTCAATGGACATTGACCTGCGGCC AGCCTCAG CTTGGGCCATCTATGCTGCCTTGACAACTTTCATTACCCCAGCCGTCCAACATGCAGTGA CCACCTCA TACAACAACTACTCCTTAATGGCGATGGCCACGCAAGCTGGAGTGTTGTTTGGTATGGGC AAAGGGAT
GCCATTCTACGCATGGGACTTTGGAGTCCCGCTGCTAATGATAGGTTGCTACTCACA ATTAACACCCC TGACCCTAATAGTGGCCATCATTTTGCTCGTGGCGCACTACATGTACTTGATCCCAGGGC TGCAGGCA
GCAGCTGCGCGTGCTGCCCAGAAGAGAACGGCAGCTGGCATCATGAAGAACCCTGTT GTGGATGGAAT AG T G GT G AC T G AC AT T G AC AC AAT G AC AAT T G AC C C C C AAG T G G AG AAAAAG AT G GG AC AGG T G C T AC TCATAGCAGTAGCCGTCTCCAGCGCCATACTGTCGCGGACCGCCTGGGGGTGGGGGGAGG CTGGGGCC CTGATCACAGCCGCAACTTCCACTTTGTGGGAAGGCTCTCCGAACAAGTACTGGAACTCC TCTACAGC CACTTCACTGTGTAACATTTTTAGGGGAAGTTACTTGGCTGGAGCTTCTCTAATCTACAC AGTAACAA GAAACGCTGGCTTGGTCAAGAGACGT ( NS5 ) GGGGGTGGAACAGGAGAGACCCTGGGAGAGAAATGGA AGGCCCGCTTGAACCAGATGTCGGCCCTGGAGTTCTACTCCTACAAAAAGTCAGGCATCA CCGAGGTG TGCAGAGAAGAGGCCCGCCGCGCCCTCAAGGACGGTGTGGCAACGGGAGGCCATGCTGTG TCCCGAGG AAGTGCAAAGCTGAGATGGTTGGTGGAGCGGGGATACCTGCAGCCCTATGGAAAGGTCAT TGATCTTG GATGTGGCAGAGGGGGCTGGAGTTACTACGCCGCCACCATCCGCAAAGTTCAAGAAGTGA AAGGATAC ACAAAAGGAGGCCCTGGTCATGAAGAACCCGTGTTGGTGCAAAGCTATGGGTGGAACATA GTCCGTCT TAAGAGTGGGGTGGACGTCTTTCATATGGCGGCTGAGCCGTGTGACACGCTGCTGTGTGA CATAGGTG AGTCATCATCTAGTCCTGAAGTGGAAGAAGCACGGACGCTCAGAGTCCTCTCCATGGTGG GGGATTGG CTTGAAAAAAGACCAGGAGCCTTTTGTATAAAAGTGTTGTGCCCATACACCAGCACTATG ATGGAAAC CCTGGAGCGACTGCAGCGTAGGTATGGGGGAGGACTGGTCAGAGTGCCACTCTCCCGCAA CTCTACAC ATGAGATGTACTGGGTCTCTGGAGCGAAAAGCAACACCATAAAAAGTGTGTCCACCACGA GCCAGCTC CTCTTGGGGCGCATGGACGGGCCTAGAAGGCCAGTGAAATATGAGGAGGATGTGAATCTC GGCTCTGG CACGCGGGCTGTGGTAAGCTGCGCTGAAGCTCCCAACATGAAGATCATTGGTAACCGCAT TGAAAGGA TCCGCAGTGAGCACGCGGAAACGTGGTTCTTTGACGAGAACCACCCATATAGGACATGGG CTTACCAT GGAAGCTATGAGGCCCCCACACAAGGGTCAGCGTCCTCTCTAATAAACGGGGTTGTCAGG CTCCTGTC AAAACCCTGGGATGTGGTGACTGGAGTCACAGGAATAGCCATGACCGACACCACACCGTA TGGTCAGC AAAGAGTTTTCAAGGAAAAAGTGGACACTAGGGTGCCAGACCCCCAAGAAGGCACTCGTC AGGTTATG AGCATGGTCTCTTCCTGGTTGTGGAAAGAGCTAGGCAAACACAAACGGCCACGAGTCTGT ACCAAAGA AGAGTTCATCAACAAGGTTCGTAGCAATGCAGCATTAGGGGCAATATTTGAAGAGGAAAA AGAGTGGA AGACTGCAGTGGAAGCTGTGAACGATCCAAGGTTCTGGGCTCTAGTGGACAAGGAAAGAG AGCACCAC CTGAGAGGAGAGTGCCAGAGTTGTGTGTATAACATGATGGGAAAAAGAGAAAAGAAACAA GGGGAATT TGGAAAGGCCAAGGGCAGCCGCGCCATCTGGTATATGTGGCTAGGGGCTAGATTTCTAGA GTTCGAAG CCCTTGGATTCTTGAACGAGGATCACTGGATGGGGAGAGAGAACTCAGGAGGTGGTGTTG AAGGGCTG GGATTACAAAGACTCGGATATGTCCTAGAAGAGATGAGTCGTATACCAGGAGGAAGGATG TATGCAGA TGACACTGCTGGCTGGGACACCCGCATTAGCAGGTTTGATCTGGAGAATGAAGCTCTAAT CACCAACC AAATGGAGAAAGGGCACAGGGCCTTGGCATTGGCCATAATCAAGTACACATACCAAAACA AAGTGGTA AAGGTCCTTAGACCAGCTGAAAAAGGGAAAACAGTTATGGACATTATTTCGAGACAAGAC CAAAGGGG GAGCGGACAAGTTGTCACTTACGCTCTTAACACATTTACCAACCTAGTGGTGCAACTCAT TCGGAATA TGGAGGCTGAGGAAGTTCTAGAGATGCAAGACTTGTGGCTGCTGCGGAGGTCAGAGAAAG TGACCAAC TGGTTGCAGAGCAACGGATGGGATAGGCTCAAACGAATGGCAGTCAGTGGAGATGATTGC GTTGTGAA
GCCAATTGATGATAGGTTTGCACATGCCCTCAGGTTCTTGAATGATATGGGAAAAGT TAGAAAGGACA CACAAGAGTGGAAACCCTCAACTGGATGGGACAACTGGGAAGAAGTTCCGTTTTGCTCCC ACCACTTC
AACAAGCTCCATCTCAAGGACGGGAGGTCCATTGTGGTTCCCTGCCGCCACCAAGAT GAACTGATTGG CCGGGCCCGCGTCTCTCCAGGGGCGGGATGGAGCATCCGGGAGACTGCTTGCCTAGCAAA ATCATATG CGCAGATGTGGCAGCTCCTTTATTTCCACAGAAGGGACCTCCGACTGATGGCCAATGCCA TTTGTTCA TCTGTGCCAGTTGACTGGGTTCCAACTGGGAGAACTACCTGGTCAATCCATGGAAAGGGA GAATGGAT GACCACTGAAGACATGCTTGTGGTGTGGAACAGAGTGTGGATTGAGGAGAACGACCACAT GGAAGACA AGACCCCAGTTACGAAATGGACAGACATTCCCTATTTGGGAAAAAGGGAAGACTTGTGGT GTGGATCT CTCATAGGGCACAGACCGCGCACCACCTGGGCTGAGAACATTAAAAACACAGTCAACATG GTGCGCAG GATCATAGGTGATGAAGAAAAGTACATGGACTACCTATCCACCCAAGTTCGCTACTTGGG TGAAGAAG GGTCTACACCTGGAGTGCTGTAA (NS5 end)
GCACCAATCTTAATGTTGTCAGGCCTGCTAGTCAGCCACAGCTTGGGGAAAGCTGTG CAGCCTGTGAC
CCCCCAGGAGAAGCTGGGAAACCAAGCCTATAGTCAGGCCGAGAACGCCATGGCACG GAAGAAGCCAT
GCTGCCTGTGAGCCCCTCAGAGGATACTGAGTCAAAAAACCCCACGCGCTTGGAGGC GCAGGATGGGA
AAAGAAGGTGGCGACCTTCCCCACCCTTCAATCTGGGGCCTGAACTGGAGATCAGCT GTGGATCCCCA
GAAGAGGGACTAGTGGTTAGAGGAGACCCCCCGGAAAACGCAAAACAGCATATTGAC GCTGGGAAAGA
CCAGAGACTCCATGAGTTTCCACCACGCTGGCCGCCAGGCACAGATCGCCGAACTTC GGCGGCCGGTG
TGGGGAAATCCATGGTTTCT
[0054] Preferred features, embodiments and variations of the invention may be discerned from the following Detailed Description of the invention.
DETAILED DESCRIPTION
[0055] The present inventors have primarily developed a vaccine comprising live- attenuated Zika virus comprising a (partly) codon deoptimized Zika viral genome. Using codon deoptimization (CD) technology, the inventors inserted a number of codon changes in the genome of the virus (wild-type Zika virus), with the objective of decreasing replication efficiency in mammalian cells and rendering the virus attenuated compared to wild-type ZIKV. Using this strategy, some resulting viruses were strongly attenuated but still produced viral proteins to a level comparable to wild-type virus. Thus, using codon deoptimization technology, the inventors were able to generate live attenuated ZIKV vaccine candidates.
[0056] By inserting a substantial number of changes into each vaccine candidate, the chance of reversion to wild-type is negligible, which is a crucial safety feature of the vaccines. This represents a substantial competitive advantage over vaccines with only a small number of mutations. To the best of the inventors’ knowledge, no other ZIKV vaccines have been generated using codon deoptimization technology. [0057] Codon deoptimization in case of Zika virus presumably results in slower polyprotein translation leading to slower replication and, as a result, in attenuation of the virus, compared with wild-type Zika virus. Such vaccine candidates have virtually no risk of deattenuation (the chance of reversion to wild-type is negligible) because of too many substitutions, all of which have, taken alone, minimal effect on virus, have been made in the coding sequence.
[0058] ‘Codon deoptimization’ (CD), as used herein, involves replacing normal codons in the wild-type Zika virus genome with synonymous codons so that the resulting virus proteins are identical to wild-type virus proteins. Moreover, the resulting virus is highly attenuated, but protein function is not compromised.
[0059] By‘live attenuated’ it is meant that the virus demonstrates substantially reduced or preferably no clinical signs of disease when administered to a subject, compared with wild- type Zika virus.
[0060] In some embodiments codon deoptimization results in no less than about 200 codon changes in the viral genome. In some embodiments codon deoptimization results in no more than about 800 codon changes in the viral genome (with the upper limit for substitution being where the virus does not usually grow at all). In some embodiments codon deoptimization results in between about 200 and about 800 codon changes in the viral genome. This 200 to 800 codon change range includes all integers between 200 and 800, including 201 , 202...798 and 799 codon changes. In some embodiments codon deoptimization results in a minimum of about 286 codon changes in the viral genome. In some embodiments codon deoptimization results in a maximum of about 651 codon changes in the viral genome. In some embodiments codon deoptimization results in between about 286 and 651 codon changes in the viral genome. This range includes all integers between 286 and 651 , including 287...650 codon changes. In some embodiments some or all of the codon changes can be situated immediately next to one another, in sequence. In some embodiments some or all of the codon changes can be spaced apart from each other such that they are not situated immediately next to one another, in sequence - E.g. 3 to 4 codon (triplet) spacings. In some embodiments some of the codon changes can be spaced apart from each other and some of the codon changes can be situated immediately next to one another.
[0061 ] In some embodiments codon deoptimization occurs in no less than about a 1700 nucleotide region of the genome. The region can be continuous/contiguous or not. In some embodiments codon deoptimization occurs no more than in about a 7900 nucleotide region of the genome. The region can be continuous/contiguous or not. In some embodiments codon deoptimization occurs in a continuous genome region with a length of about 1800 to about 3600 nucleotides. In some embodiments codon deoptimization results in no less than about an 1800 nucleotide region of the genome, with no less than about 250 codon changes within that nucleotide region. In some embodiments codon deoptimization results in no more than about a 7900 nucleotide region of the genome, with no more than about 800 codon changes within that nucleotide region. In some embodiments about 20-60% of the coding region of the genome is codon deoptimized, preferably 18-36% of the genome, compared to wild-type ZIKV.
[0062] In some embodiments the non-structural region of the viral genome is codon deoptimized. In some embodiments only the non-structural region of the viral genome is codon deoptimized. In some embodiments any one or more of the genes NS1 , 2A, NS2B, NS3, NS4A, NS4B and NS5 are codon deoptimized. In some embodiments any contiguous genome region from the NS1 to NS5 region corresponding to at least 600 amino acid residues of viral polyprotein is codon deoptimized. In some embodiments the genes NS1 , 2A, NS2B, NS3, NS4A, NS4B and NS5 are codon deoptimized. In some embodiments every 3 rd or 4 th codon is deoptimized along the entire nonstructural ZIKV coding region. In some embodiments the genes NS1 , 2A, NS2B and NS3 are codon deoptimized. In some embodiments approximately 700 base changes are made. In some embodiments the gene NS3 is codon deoptimized. In some embodiments about 350 changes base changes are made. In some embodiments approximately 700 codon substitutions are made along the entire nonstructural ZIKV coding region.
[0063] In some embodiments the codon deoptimization results in slower polyprotein translation leading to slower replication and, as a result, in attenuation of the virus. In some embodiments every codon in the wild-type Zika virus genome or region thereof was analyzed in terms of its usage frequency in Homo sapiens, and if the codon was frequent then it was changed in the viral genome to a least frequently used synonymous codon. In some embodiments a codon for an amino acid with codon degeneracy was changed only if the synonymous codons for that amino acid occurred in significantly different frequencies of usage in the genome of Homo sapiens. In some embodiments Asp, and Asn codons of the viral genome are left unchanged. In some embodiments a codon for an amino acid with high codon degeneracy was changed to a synonymous codon that was used least frequently or rarely in the genome of Homo sapiens. In some embodiments a viral region most rich in codons that can be substituted for rare codon variants is codon deoptimized. In some embodiments Leu codons of the viral genome are changed. In some embodiments Leu codons of the viral genome are changed to the rare CUA codon. In some embodiments the viral genome prior to codon deoptimization has a very similar nucleotide sequence to a Zika strain associated with microcephaly. In some embodiments the wild-type Zika viral genome is that of Brazilian Zika virus (ZIKV) strain Bel-1819016. In some embodiments the chance of deattenuation to wild- type Zika is negligible.
[0064] Preferably the codon deoptimized Zika viral genome is generated using codon deoptimization technology.
[0065] In some embodiments the codon deoptimized genome has the deoptimized codons of vaccine candidate ZIKV-DO-NS3 as shown in the NS3 region of SEQ ID NO:3, 4, 5 or 10. In some embodiments the codon deoptimized genome can have about 200 or more of the codon changes of vaccine candidate ZIKV-DO-NS3 shown in SEQ ID NO:3, 4, 5 or 10, including all integers between about 200 and about 350, including 201 , 202...348 and 349 codon changes.
[0066] In some embodiments the codon deoptimized genome has the deoptimized codons of vaccine candidate ZIKV- DO-scattered as shown in SEQ ID NO:6, 7 or 1 1 . In some embodiments the codon deoptimized genome can have about 200 or more of the codon changes of vaccine candidate ZIKV-DO-scattered shown in SEQ ID NO: 6, 7 or 1 1 , including all integers between about 200 and about 700, including 201 , 202...698 and 699 codon changes.
[0067] In some embodiments the codon deoptimized genome has the deoptimized codons of vaccine candidate ZIKV-DO as shown in SEQ ID NO:8, 9 or 12. In some embodiments the codon deoptimized genome can have about 200 or more of the codon changes of vaccine candidate ZIKV-DO-scattered shown in SEQ ID NO: 8, 9 or 12, including all integers between about 200 and about 700, including 201 , 202...698 and 699 codon changes.
[0068] In some embodiments the codon deoptimized genome has the deoptimized codons of the nonstructural region of SEQ ID NO:1 as shown in Figure 1 b. In some embodiments the codon deoptimized genome can have about 1 or more of the codon changes of SEQ ID NO:1 , including all integers between about 1 and about 72, including 2, 3 ... 70 and 71 codon changes.
[0069] The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid comprising a partly codon deoptimized Zika viral genome can be of any suitable form and can be prepared in any suitable way. Likewise, the recombinant, isolated or substantially purified nucleic acid comprising a partly codon deoptimized Zika viral genome or partly codon deoptimized region thereof can be prepared in any suitable way. Such techniques are described elsewhere in this specification (eg. see below), the entire contents of which are incorporated herein by way of cross- reference.
[0070] Likewise, a vaccine, pharmaceutical preparation or immunogenic composition comprising the above can be of any suitable form and can be prepared in any suitable way. Such techniques are described elsewhere in this specification, the entire contents of which are incorporated herein by way of cross- reference.
[0071 ] In addition to a live attenuated recombinant Zika virus vaccine, pharmaceutical preparation or immunogenic composition, the present invention encompasses recombinant Zika virus particles, nucleic acid and genetic vaccines that comprise a partly codon deoptimized Zika viral genome in the form of a nucleic acid. The nucleic acid can be DNA or RNA that is self-replicating/self-amplifying once used for vaccination. The nucleic acid can relate to the Zika viral genome or Zika viral anti-genome. Such techniques are described in the following references, the entire contents of which are incorporated herein by way of cross- reference: Karl Ljungberg & Peter Liljestrom (2015) Self-replicating alphavirus RNA vaccines, Expert Review of Vaccines, 14:2, 177-194, DOI: 10.1586/14760584.2015.965690; Rodriguez- Gascon A, del Pozo-Rodriguez A, Solinis MA (2014) Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles. Int J Nanomedicine. 9: 1833-1843; US 2014/01 12979 A1 .
[0072] The vaccine, pharmaceutical preparation or immunogenic composition can comprise live virus or inactivated virus, provided that it is self-replicating/self-amplifying after vaccination. If inactivated, it can be inactivated in any suitable way (e.g. using high or low temperatures, or chemically).
[0073] The vaccine, pharmaceutical preparation or immunogenic composition can comprise a delivery system or carrier or aid, and these can be of any suitable form and can be prepared in any suitable way. Suitable examples include a plasmid or vector to assist with self-replication/self-amplification, an RNA nanocarrier for RNA delivery, and lipid-based formulations for delivery, including liposomes, nanoemulsions and solid lipid nanoparticles.
[0074] In some embodiments the vaccine can be prepared by way of passing recombinant ZIKV through a filter, such as a 0.22 pm hydrophilic PVDF membrane or hydrophilic Polyethersulfone membrane.
[0075] In some embodiments the vaccine can be stored long term and remain viable at a temperature of between about -20°C and about -80°C. By "long-term" it is meant that the vaccine can remain viable for at least 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 days. In some embodiments it is possible that the vaccine can remain viable for more than 60 days.
[0076] The live attenuated virus can be in the form of an isolate. The isolate may comprise cells, such as mammalian, insect (e.g. mosquito) or other types of cells.
[0077] The method of preventing the subject from contracting a viral infection, treating a subject having a viral infection, or reducing the severity of a viral infection, can be carried out in any suitable way.
[0078] The vaccine, live attenuated virus, pharmaceutical preparation and immunogenic composition (described hereafter as "the compositions") can be administered independently, either systemically or locally, by any method standard in the art, for example, subcutaneously, intravenously, parenterally, intraperitoneally, intradermally, intramuscularly, topically, or nasally.
[0079] The compositions can comprise conventional non-toxic, physiologically or pharmaceutically acceptable ingredients or vehicles suitable for the method of administration and are well known to an individual having ordinary skill in this art. The compositions can, for example, comprise an adjuvant. The adjuvant can be, for example, an aluminium salt (e.g. aluminium hydroxide), monophosphoryl lipid A, or, emulsion of water and oil (e.g. MF59). The term "pharmaceutically acceptable carrier" as used herein is intended to include diluents such as saline and aqueous buffer solutions. The compositions can be in aqueous or lyophilized form.
[0080] A variety of devices are known in the art for delivery of the compositions including, but not limited to, syringe and needle injection, bifurcated needle administration, administration by intradermal patches or pumps, intradermal needle-free jet delivery (intradermal etc), intradermal particle delivery, or aerosol powder delivery.
[0081 ] The compositions can be administered independently one or more times to achieve, maintain or improve upon a desired effect/result. It is well within the skill of an artisan to determine dosage or whether a suitable dosage of the composition comprises a single administered dose or multiple administered doses. An appropriate dosage depends on the subject's health, the induction of immune response and/or prevention of infection caused by the alphavirus, the route of administration and the formulation used. For example, a therapeutically active amount of the compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the composition to elicit a desired response in the subject. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, a subject may be administered a 'booster' vaccination one or two weeks following the initial administration.
[0082] The vector can also be prepared in any suitable way.
[0083] The cell (insect, mammalian or other) or isolate comprising the vector or virus can be prepared in any suitable way.
[0084] Suitable protocols for carrying out one or more of the above-mentioned techniques can be found in "Current Protocols in Molecular Biology", July 2008, JOHN WILEY AND SONS; D. M. WEIR ANDCC BLACKWELL, "Handbook Of Experimental Immunology", vol. I- IV, 1986; JOHN E. COLIGAN, ADA M. KRUISBEEK, DAVID H. MARGULIES, ETHAN M. SHEVACH, WARREN STROBER, "Current Protocols in Immunology", 2001 , JOHN WILEY & SONS; "Immunochemical Methods In Cell And Molecular Biology", 1987, ACADEMIC PRESS; SAMBROOK ET AL., "Molecular Cloning: A Laboratory Manual, 3d ed.,", 2001 , COLD SPRING HARBOR LABORATORY PRESS; "Vaccine Design, Methods and Protocols", Volume 2, Vaccines for Veterinary Diseases, Sunil Thomas in Methods in Molecular Biology (2016); and, "Vaccine Design, Methods and Protocols", Volume 1 : Vaccines for Human Diseases, Sunil Thomas in Methods in Molecular Biology (2016), the entire contents of which are incorporated herein by way of cross-reference.
[0085] Any suitable type of subject can be used. The subject can be any suitable mammal. Mammals include humans, primates, livestock and farm animals (e.g. horses, sheep and pigs), companion animals (e.g. dogs and cats), and laboratory test animals (e.g. rats, mice and rabbits). The subject is preferably human.
[0086] 'Nucleic acid' as used herein includes 'polynucleotide', 'oligonucleotide', and 'nucleic acid molecule', and generally means a polymer of DNA or RNA, which can be single- stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
[0087] As used herein, the term 'recombinant' refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.
[0088] The terms 'isolated' or 'purified' as used herein mean essentially free of association with other biological components/contaminants, e.g., as a naturally occurring protein that has been separated from cellular and other contaminants by the use of antibodies or other methods or as a purification product of a recombinant host cell culture.
[0089] Preferred embodiments of the invention are defined in the following numbered paragraphs:
[0090] 1 . Live attenuated recombinant Zika virus, recombinant Zika virus, recombinant
Zika virus particle or recombinant Zika virus nucleic acid comprising a partly codon deoptimized Zika viral genome.
[0091 ] 2. A recombinant, isolated or substantially purified nucleic acid comprising a partly codon deoptimized Zika viral genome or partly codon deoptimized region thereof.
[0092] 3. A vector containing the nucleic acid of paragraph 2.
[0093] 4. A cell or isolate containing the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of paragraph 1 , the nucleic acid of the paragraph 2, or the vector of paragraph 3.
[0094] 5. A vaccine comprising the live attenuated recombinant Zika virus, recombinant
Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of paragraph 1 , the recombinant, isolated or substantially purified nucleic acid of paragraph 2, the vector of paragraph 3, or the cell or isolate of paragraph 4.
[0095] 6. A pharmaceutical preparation comprising the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of paragraph 1 , the recombinant, isolated or substantially purified nucleic acid of paragraph 2, the vector of paragraph 3, or the cell or isolate of paragraph 4.
[0096] 7. An immunogenic composition comprising the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of paragraph 1 , the recombinant, isolated or substantially purified nucleic acid of paragraph 2, the vector of paragraph 3, or the cell or isolate of paragraph 4.
[0097] 8. A method of (1 ) treating a subject having a natural Zika viral infection, (2) reducing the severity of a natural Zika viral infection in a subject, or (3) preventing a subject from contracting a Zika viral infection naturally, said method comprising the step of administering to the subject:
[0098] the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of paragraph 1 ; the recombinant, isolated or substantially purified nucleic acid of paragraph 2; the vector of paragraph 3; the cell or isolate of paragraph 4; the vaccine of paragraph 5; the pharmaceutical preparation of paragraph 6; or the immunogenic composition of paragraph 7.
[0099] 9. Use of: the live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid of paragraph 1 ; the recombinant, isolated or substantially purified nucleic acid of paragraph 2; the vector of paragraph 3; the cell or isolate of paragraph 4; the vaccine of paragraph 5; the pharmaceutical preparation of paragraph 6; or the immunogenic composition of paragraph 7, in the preparation of a medicament for (1 ) treating a subject having a natural Zika viral infection, (2) reducing the severity of a natural Zika viral infection in a subject, or (3) preventing a subject from contracting a Zika viral infection naturally.
[00100] 10. A method of generating a live attenuated Zika virus vaccine, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, or recombinant, isolated or substantially purified nucleic acid comprising a partly codon deoptimized Zika viral genome or partly codon deoptimized region thereof, comprising the step of partly codon deoptimizing a Zika viral genome.
[00101 ] 1 1 . A method of preparing a vaccine comprising live attenuated recombinant Zika virus, said method comprising the steps of: (1 ) codon deoptimizing a Zika viral genome to produce a partly codon deoptimized live attenuated Zika virus; and (2) enabling the partly codon deoptimized live attenuated Zika virus to replicate.
[00102] 12. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized Zika viral genome comprises at least about 200 codon changes compared with wild-type or virulent Zika virus.
[00103] 13. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized Zika viral genome comprises no more than about 800 codon changes, compared with wild-type or virulent Zika virus.
[00104] 14. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized Zika viral genome comprises between about 200 and about 800 codon changes, compared with wild-type or virulent Zika virus.
[00105] 15. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized Zika viral genome comprises a minimum of about 286 codon changes, compared with wild-type or virulent Zika virus.
[00106] 16. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized Zika viral genome comprises a maximum of about 651 codon changes, compared with wild-type or virulent Zika virus.
[00107] 17. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized Zika viral genome comprises between about 286 and 651 codon changes in the viral genome, compared with wild-type or virulent Zika virus.
[00108] 18. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein some or all codon changes of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus are situated immediately next to one another, in sequence.
[00109] 19. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein some or all codon changes of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus are spaced apart from each other such that they are not situated immediately next to one another, in sequence.
[001 10] 20. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein some codon changes of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus are spaced apart from each other and some of the codon changes are situated immediately next to one another.
[001 1 1 ] 21 . The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein codon deoptimization occurs in no less than about a 1700 nucleotide region of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus, and optionally the 1700 nucleotide region is continuous/contiguous or the 1700 nucleotide region is not continuous/not contiguous.
[001 12] 22. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein codon deoptimization occurs in no more than in about a 7900 nucleotide region of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus, and optionally the 7900 nucleotide region is continuous/contiguous or the 7900 nucleotide region is not continuous/not contiguous.
[001 13] 23. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein codon deoptimization occurs in a continuous region of the codon deoptimized Zika viral genome compared with wild-type or virulent Zika virus with a length of about 1800 to about 3600 nucleotides.
[001 14] 24. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein codon deoptimization results in no less than about an 1800 nucleotide region of the genome compared with wild-type or virulent Zika virus, with no less than about 250 codon changes within that nucleotide region.
[001 15] 25. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein codon deoptimization results in no more than about a 7900 nucleotide region of the genome compared with wild-type or virulent Zika virus, with no more than about 800 codon changes within that nucleotide region.
[001 16] 26. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein about 20-60% of the coding region of the genome is codon deoptimized compared with wild-type or virulent Zika virus, preferably 18-36% of the genome, compared with wild-type or virulent Zika virus.
[001 17] 27. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the non- structural region of the viral genome is codon deoptimized.
[001 18] 28. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein only the non-structural region of the viral genome is codon deoptimized.
[001 19] 29. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein every 3 rd or 4 th codon is deoptimized along the nonstructural ZIKV coding region.
[00120] 30. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein any one or more of the genes NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5 are codon deoptimized.
[00121 ] 31 . The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein any contiguous genome region from the NS1 to NS5 region corresponding to at least 600 amino acid residues of viral polyprotein is codon deoptimized.
[00122] 32. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the genes NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5 are codon deoptimized.
[00123] 33. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the genes NS1 , NS2A, NS2B and NS3 are codon deoptimized.
[00124] 34. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the gene NS3 is codon deoptimized.
[00125] 35. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein approximately 700 codon substitutions are made along the entire nonstructural ZIKV coding region compared with wild-type or virulent Zika virus.
[00126] 36. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimization results in slower polyprotein translation leading to slower replication and, as a result, in attenuation of the virus, compared with wild-type or virulent Zika virus.
[00127] 37. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein a codon for an amino acid with high codon degeneracy is changed to a synonymous codon that is used least frequently or rarely in the genome of Homo sapiens.
[00128] 38. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the genome region most rich in codons that can be substituted for rare codon variants is codon deoptimized.
[00129] 39. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the viral genome prior to codon deoptimization has a very similar nucleotide sequence to a Zika strain associated with microcephaly, preferably Brazilian Zika virus (ZIKV) strain Bel-1819016.
[00130] 40. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has the deoptimized codons of the NS3 region of vaccine candidate ZIKV-DO-NS3 as represented by SEQ ID NO:3, 4, 5 or 10.
[00131 ] 41 . The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has about 200 or more of the codon changes of the NS3 region of vaccine candidate ZIKV-DO-NS3 as represented by SEQ ID NO:3, 4, 5 or 10.
[00132] 42. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has the deoptimized codons of the NS1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 regions of the vaccine candidate ZIKV-DO-scattered as represented by SEQ ID NO:6, 7 or 1 1 .
[00133] 43. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has about 200 or more of the codon changes of the NS1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 regions of the vaccine candidate ZIKV-DO-scattered as represented by SEQ ID NO: 6, 7 or 1 1 .
[00134] 44. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has the deoptimized codons of the NS1 , NS2A, NS2B and/or NS3 regions of the vaccine candidate ZIKV-DO as represented by SEQ ID NO:8, 9 or 12.
[00135] 45. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has about 200 or more of the codon changes of the NS1 , NS2A, NS2B and/or NS3 regions of the vaccine candidate ZIKV-DO as represented by SEQ ID NO:8, 9 or 12.
[00136] 46. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has the deoptimized codons of the nonstructural region as represented by SEQ ID NO:1 or as shown in Figure 1 b.
[00137] 47. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has 1 or more of the codon changes of SEQ ID NO:1 .
[00138] 48. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has at least 90 percent of the deoptimized codons of the NS3 region of vaccine candidate ZIKV-DO-NS3 as represented by SEQ ID NO:3, 4, 5 or 10. (For clarity, at least 90 percent includes 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 and 100 percent.) [00139] 49. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has at least 90 percent of the deoptimized codons of the NS1 ,
NS2A, NS2B, NS3, NS4A, NS4B and NS5 regions of the vaccine candidate ZIKV-DO- scattered as represented by SEQ ID NO :6, 7 or 1 1 .
[00140] 50. The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the codon deoptimized genome has at least 90 percent of the deoptimized codons of the NS1 , NS2A, NS2B and NS3 regions of the vaccine candidate ZIKV-DO as represented by SEQ ID NO:8, 9 or 12.
[00141 ] 51 . The live attenuated recombinant Zika virus, recombinant Zika virus, recombinant Zika virus particle or recombinant Zika virus nucleic acid, the recombinant, isolated or substantially purified nucleic acid, the vector, the cell or isolate, the vaccine, the pharmaceutical preparation, the immunogenic composition, the method, the use, or the method of any one or more of the preceding paragraphs (context permitting), wherein the vaccine, pharmaceutical preparation or immunogenic composition comprises a delivery system, carrier or aid.
[00142] Any of the features described herein can be combined in any combination with any one or more of the other features described herein within the scope of the invention.
EXAMPLES
[00143] Construction of ZIKV vaccine candidates using codon deoptimization technology
[00144] In order to generate a live attenuated Zika vaccine, we first constructed infectious clones of a Brazilian Zika virus (ZIKV) strain— Bel-1819016— which has a very similar sequence to those strains associated with microcephaly. Using the infectious clone and codon deoptimization (CD) technology, we inserted a number of changes in the non-structural (NS1 , NS2A, NS2B, NS3) regions of the virus, with the objective of decreasing replication efficiency in mammalian cells and rendering the virus attenuated compared to wild-type ZIKV. Using this strategy, we generated a panel of clones for further testing. Clones that could be successfully ‘rescued’ were tested for their ability to replicate in mammalian and mosquito cells. The resulting viruses were strongly attenuated but still produced viral proteins to a level comparable to wild-type virus. Thus, using CD technology, we were able to generate a panel of live attenuated ZIKV vaccine candidates.
[00145] Synthetic sequence of Bel-1819016 strain from Brazil (very close to the strains shown to be associated with microcephaly) was used as the initial ZIKV genome. All the changes were made only in the nonstructural part of ZIKV genome to prevent possible adverse effect on structure of viral antigens which may result from altered dynamics of their translation. Three attenuated candidates were constructed:
[00146] 1 . ZIKV-DO with a codon deoptimized NS1 -NS2A-NS2B-NS3 region (see Figure 1 a and SEQ ID:8 and 9). Approximately 3900 bases (36% of the genome) were de-optimized for human cells.
[00147] 2. ZIKV-DO-NS3 with a codon deoptimized NS3 region (see Figure 1 a and SEQ
ID NO: 3, 4 and 5).
[00148] 3. ZIKV-DO-scattered where deoptimized codons are scattered over all the nonstructural genome part from the beginning of NS1 till the end of NS5 (see Figure 1 a and SEQ ID NO:6 and 7).
METHODS
[00149] Process of deoptimization
[00150] In contrast to an optimization process, which can be done using free software or online, there is no publicly available program for CD. Therefore, it was done manually. In case of ZIKV-DO and ZIKV-DO-NS3 every codon in the indicated sequence was analyzed in terms of its usage frequency in Homo sapiens. If the codon was frequent it was manually changed to a synonymous but the less used one. For instance, amino acid Leucine (Leu) can be encoded by six different codons with the following frequencies: UUA - 15%, UUG - 12%, CUU - 12%, CUC - 10%, CUA - 5%, and CUG - 46%. If the Leu codon in the original sequence was represented by highly abundant CUG (46%), it was changed to rare CUA (5%). Some codons were left unchanged: Methionine (Met) and Tryptophan (Trp) as both of them are encoded by only one codon; and, Asparagine (Asn) and Aspartic acid (Asn) as their codons are used at almost the same frequency. Altogether, -700 changes were made in the ZIKV- DO genome and -350 changes in ZIKV-DO-NS3 genome. In the case of ZIKV-DO-scattered, approximately every 3 rd or 4 th codon was deoptimized along the entire nonstructural ZIKV coding region. Again, Met, Trp, Asp, and Asn codons were left unchanged. Approximately 700 substitutions were inserted in the case of ZIKV-DO-scattered.
[00151 ] Rescue of deoptimized ZIKVs
[00152] Deoptimized sequences were purchased as synthetic DNA fragments and were used to replace wildtype (wt) counterparts in the initial pCCI-ZIKV-wt clone using appropriate unique restriction sites. Obtained cDNA clones were verified by restriction analysis and sequencing of deoptimized regions. Plasmid DNAs were amplified using E. coli NEB Turbo strain and purified using Macherey-Nagel Xtra Midi preparation kit. Plasmids were linearized using Agel (BshTI) restriction enzyme and spin-column purified. Capped transcripts, corresponding to viral genome RNAs, were synthesized in vitro with Ambion mMessage- mMachine kit using linearized plasmid DNAs as templates. The quality and integrity of synthesized RNAs was verified by gel-electrophoresis. Obtained in vitro transcription mixtures were used for transfection of Vero E6 cells (derived from African green monkey kidney) by lipofection using Lipofectamine 2000 (Invitrogen) reagent and manufacturer’s protocol. Transfected cells were incubated for 14 days and the cells’ supernatant was then used for infection of new Vero E6 or Ae. albopictus cells C6/36.
[00153] Titration of codon deoptimized ZIKVs
[00154] All codon deoptimized ZIKV vaccine candidates failed to form plaques in Vero E6 cells indicating that they were attenuated. Their titration was therefore performed using A549Npro cells deficient in intracellular immune response. Ordinary plaque forming assay was used for titration with incubation time of 7 days for plaque formation. Infected cells were stained with crystal violet solution and formed plaques counted to obtain viral titers.
[00155] Improved propagation of attenuated ZIKV-DO-NS3 strain with deoptimized NS3 region
[00156] We used the Vero E6 clone for propagation of the virus. TPCK-treated (N-tosyl-L- phenylalanine chloromethyl ketone) trypsin (at 0.5 pg/ml concentration) increased the titer of ZIKV-DO-NS3. The FBS (fetal bovine serum) content in virus growth media could be reduced to 1 % or replaced with 0.2% BSA (bovine serum albumin). ZIKV-DO-NS3 was titrated only on A549NPro cells. The best MOIs (multiplicities of infection) for infection were 0.01 -0.1 pfu/cell.
[00157] Virus was propagated on Vero E6 cells. 100 mm plates, 37°C 5% CO2. Cells were -50-80% confluent at the moment of infection. Low MOI was used (0.01 -0.1 pfu/cell). Cells were washed with PBS (phosphate buffered saline) and infected in 2 ml of serum-free DMEM (Dulbecco’s modified Eagle’s medium) for 2 hours with rocking of the plate every 10-15 minutes; then 8 ml virus growth medium (VGA, DMEM + 0.2% BSA + Pen-Strep + 0.5 pg/ml TPCK) was added (inoculum was not removed). During incubation, plates were gently rocked back and forth 4-5 times every day for the first 5 days to facilitate spread of virus over the plate. Growth media were sampled (approximately 0.5 ml) at Days 7, 10, and 14. Virus titers were determined on A549NPro cells using immuno-plaque assay with anti- ZIKV NS3 rabbit antibody (in house) and IRDye 800CW goat anti-rabbit secondary antibody (LI-COR). Cells were incubated for 96 hours before fixation. Virus titers in samples: Day 7 - 2x10 * 7 pfu/ml; Day 10 - 1 .5x10 * 7 pfu/ml; Day 14 - 5x10 * 7 pfu/ml. The samples were also titrated by classical plaque titration on A549NPro cells (incubation time - 8 days) with the same or similar results.
[00158] RESULTS [00159] Codon deoptimized ZIKV genomes
[00160] Cloning of codon deoptimized ZIKV genomes resulted in three plasmids, ZIKV- DO, ZIKV-DO-NS3 and ZIKV-DO-scattered, whose coding regions are schematically depicted in Fig. 1 a.
[00161 ] A representative computational codon deoptimization is depicted in Fig. 1 b.
[00162] Rescue of attenuated ZIKVs with deoptimized nonstructural regions
[00163] The deoptimized ZIKV vaccine candidates were rescued in Vero E6 cells and passaged 3 times with no significant cytopathic effect for up to 14 days. No protein expression was detected by western blot in Vero E6 cells (except ZIKV-DO-scattered). Subsequently, ZIKV-DO and ZIKV-DO-NS3 were passaged in mosquito Ae. albopictus cells for 7 days. Protein expression (NS3 and Envelope proteins) for ZIKV-DO and ZIKV-DO-NS3 viruses was confirmed in insect cells by western blot analysis. Supernatants collected from both Vero E6 and C6/36 cells were plaque-titrated on A549NPro cells.
[00164] Testing of vaccines in vivo
[00165] To test the vaccines in vivo, we required a suitable immunocompetent mouse model. Most mouse models of ZIKV infection are based on mice with an impaired immune system, making them inappropriate for vaccine studies. We have been successful in generating an immunocompetent C57BL/6 adult mouse model of ZIKV infection, using ZIKV strain MR766. The model is based on intracranial (i.c) infection of adult wild-type mice with 4 x 10 5 PFU ZIKV. Mice show high susceptibility to infection in our model (Figure 2), with all mice dying by d6 p.i. This high susceptibility to infection makes our mouse model particularly suitable for use in vaccine testing. One vaccine candidate, ZIKA-DO-NS3, was initially selected for further testing. In contrast to the 100% mortality seen in mice infected with ZIKV MR766, mice given 4 x 10 5 PFU of the live attenuated ZIKV vaccine based on ZIKA-DO-NS3 showed no mortality. Thus, the vaccine candidate ZIKA-DO-NS3 is extremely safe.
[00166] For the experiments with ZIKV-DO-NS3, there were 5 mice per group and each experiment was repeated 3 times.
[00167] In our mouse model of i.c. ZIKV infection, as seen in Figure 3, mice show prominent signs of disease, which are measured by clinical score and loss of body weight. Clinical score is measured by assessing and scoring a number of clinical signs: every 5% weight loss scores one point; noticeable hesitation in activity scores one point, significant reduction in activity scores 2 points, move only when pushed scores 3 points (select just one of these three movement assessments); rough fur scores 1 point; hunching scores one point; trembling scores one point; standing on hind limbs scores one point. These scores are added together to give a total clinical score. Disease was assessed in mice infected with the vaccine candidate based on ZIKA-DO-NS3 used in Figure 2. Mice infected with 4 x 10 5 PFU ZIKV MR766 showed a dramatic increase in clinical score and weight loss. In contrast, infection of C57BL/6 mice with 4 x 10 5 PFU of the live attenuated ZIKV based on ZIKA-DO-NS3 did not affect clinical score and there was no weight loss. Thus, the vaccine candidate based on ZIKA- DO-NS3 is extremely safe.
[00168] To test vaccine efficacy, as seen in Figure 4, mice were immunised with 2 x 10 4 PFU of the live attenuated vaccine based on ZIKV-DO-NS3 subcutaneously (s.c). Control mice received PBS. We adopted a booster regimen, with s.c immunisation at days 0, 7 and 14. Fourteen days following vaccination, mice were given a lethal i.c challenge with 4 x 10 5 PFU ZIKV. The control (non-vaccinated) mice all died within 6 days of infection. In contrast, there was no mortality in the vaccinated mice. n=5 mice per group.
[00169] To test vaccine efficacy, as seen in Figure 5, mice were immunised with the live attenuated vaccine ZIKV-DO-NS3 subcutaneously (s.c). Control mice received PBS. We adopted a booster regimen, with s.c immunisation at days 0, 7 and 14. Fourteen days following vaccination, mice were given a lethal i.c challenge with 4 x 10 5 PFU ZIKV. The control (non- vaccinated) mice showed substantial loss of body weight from day 3 until death on day 6. In contrast, there was no loss of body weight in the vaccinated mice. n=5 mice per group.
[00170] To test vaccine efficacy, as seen in Figure 6, mice were immunised with the live attenuated vaccine ZIKV-DO-NS3 subcutaneously (s.c). Control mice received PBS. We adopted a booster regimen, with s.c immunisation at days 0, 7 and 14. Fourteen days following vaccination, mice were given a lethal i.c challenge with 4 x 10 5 PFU ZIKV. The control (non- vaccinated) mice showed a dramatic increase in clinical score from day 4 until death on day 6. In contrast, there was no increase in clinical score at any time point in the vaccinated mice. n=5 mice per group.
[00171 ] To test vaccine efficacy, as seen in Figure 7, mice were immunised with the live attenuated vaccine ZIKV-DO-NS3 subcutaneously (s.c). Control mice received PBS. We adopted a booster regimen, with s.c immunisation at days 0, 7 and 14. Fourteen days following vaccination, mice were given a lethal i.c challenge with 4 x 10 5 PFU ZIKV. The control (non- vaccinated mice) showed very high levels of ZIKV virus in brain tissue at 6 days p.i. In contrast, there was no detectable virus in the brains of vaccinated mice at day 6. n=5 mice per group. [00172] We conducted an initial assessment of immunological mechanisms of protection mediated by the vaccine, and the results are shown in Figure 8. C57BL/6 mice were immunized s.c with 2 x 10 4 PFU of the live attenuated vaccine ZIKV-DO-NS3 on days 0, 7 and 14. The mice were killed one week later and the draining lymph nodes were collected for analysis. There was a significant increase in the cellularity of draining lymph nodes in vaccinated mice compared to non-vaccinated mice (n=5 mice per group).
[00173] We conducted an initial assessment of immunological mechanisms of protection mediated by the vaccine, and the results are shown in Figure 9. C57BL/6 mice were immunized s.c with 2 x 10 4 PFU of the live attenuated vaccine ZIKV-DO-NS3 on days 0, 7 and 14. The mice were bled and the ZIKV-specific antibody response assessed. Vaccinated mice mounted a strong antibody response against ZIKV. No ZIKV-specific antibodies were detected in non-vaccinated mice (n=5 mice per group).
[00174] We conducted an initial assessment of immunological mechanisms of protection mediated by the vaccine, and the results are shown in Figure 10. C57BL/6 mice were immunized s.c with 2 x 10 4 PFU of the live attenuated vaccine ZIKV-DO-NS3 on days 0, 7 and 14. The mice were bled and the ZIKV neutralising antibody response assessed. ZIKV-specific neutralising antibodies were induced by the vaccine, as measured using a plaque reduction neutralisation test (PRNT). No ZIKV-specific neutralising antibodies were detected in non- vaccinated mice (n=5 mice per group).
[00175] We conducted an assessment of immunological mechanisms of protection mediated by the vaccine, and the results are shown in Figure 1 1 . We found that s.c inoculation of ZIKV-DO-NS3 induces an immune response in the draining lymph nodes. C57BL/6 mice were immunized s.c with 2 x 10 4 PFU of the live attenuated vaccine ZIKV-DO-NS3 on days 0, 7 and 14. Control mice were given PBS. Vaccinated and control mice were euthanized 6 days after the last immunisation. Draining lymph nodes posterior axillary, bilateral regions were collected. Numbers of CD4+ T cells, CD8+ T cells and B cells were quantitated using flow cytometry. Mice immunized with ZIKV-DO-NS3 mounted a strong T and B cell responses. Weak T and B cell responses were detected in non-vaccinated mice (n=4 mice per group).
[00176] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.
[00177] In the present specification and claims (if any), the word 'comprising' and its derivatives including 'comprises' and 'comprise' include each of the stated integers but does not exclude the inclusion of one or more further integers.
[00178] The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.
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