US010023845B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 , 023 ,845 B2 Wimmer et al . ( 45 ) Date of Patent: Jul. 17, 2018 ( 54 ) METHODS OF MAKING MODIFIED VIRAL ( 58 ) Field of Classification Search GENOMES None See application file for complete search history . (71 ) Applicant: The Research Foundation for The State University of New York , Albany , (56 ) References Cited NY (US ) U . S . PATENT DOCUMENTS ( 72 ) Inventors: Eckard Wimmer , East Setauket , NY 6 ,696 ,289 B1 2 /2004 Bae et al. (US ) ; Steve Skiena , Setauket , NY (US ) ; 2004 / 0097439 A95 /2004 Nicolas et al . Steffen Mueller , Kings Point, NY (US ) ; 2004 /0209241 A1 10 / 2004 Hermanson et al . Bruce Futcher , Stony Brook , NY (US ) ; 2008 /0118530 A1 5 / 2008 Kew et al . 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C12N 7700 ( 2013 .01 ) ; A61K 39/ 13 (57 ) ABSTRACT ( 2013 . 01 ) ; A61K 39 / 145 (2013 . 01 ) ; COOK This invention provides an attenuated virus which comprises 14 /005 ( 2013 .01 ) ; A61K 2039 /5254 ( 2013 .01 ) ; a modified viral genome containing nucleotide substitutions C12N 2720 /12361 (2013 .01 ) ; C12N engineered in multiple locations in the genome , wherein the 2740 / 15061 ( 2013 .01 ) ; CI2N 2760 / 16061 substitutions introduce synonymous deoptimized codons ( 2013 .01 ) ; C12N 2760 / 16121 ( 2013 .01 ) ; CI2N into the genome . The instant attenuated virus may be used in 2760 / 16134 ( 2013 .01 ) ; CI2N 2760 / 16162 a vaccine composition for inducing a protective immune ( 2013 .01 ) ; C12N 2770 /20061 (2013 . 01 ) ; CI2N response in a subject. The invention also provides a method 2770 /24161 (2013 .01 ) ; C12N 2770 / 32061 of synthesizing the instant attenuated virus. Further, this ( 2013 .01 ) ; C12N 2770 / 32621 ( 2013 .01 ) ; C12N invention further provides a method for preventing a subject 2770 / 32634 ( 2013 . 01 ) ; C12N 2770 / 32661 from becoming afflicted with a virus -associated disease (2013 .01 ) ; C12N 2770 / 32662 (2013 .01 ) ; C12N comprising administering to the subject a prophylactically 2770 / 32671 ( 2013 .01 ) ; C12N 2770 / 32721 effective dose of a vaccine composition comprising the ( 2013 .01 ) ; C12N 2770 / 32761 ( 2013 .01 ) ; C12N instant attenuated virus . 2770 / 32762 ( 2013 .01 ) 25 Claims, 18 Drawing Sheets US 10 ,023 ,845 B2 Page 2 ( 56 ) References Cited Cis -Replicating Element in the Coding Sequence of 2 Apro ” , J . Virol . ( 2001 ) , vol. 75 : 22 , pp . 10979 - 10990 . Girard , S . et al. , “ Poliovirus Induces Apoptosis in the Mouse Central OTHER PUBLICATIONS Nervous System ” , J . Virol. ( 1999 ) , vol. 73 : 7 , pp . 6066 -6072 . 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