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(12) Patent Application Publication (10) Pub. No.: US 2015/0037370 A1 Corbeil Et Al US 2015 0037370A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0037370 A1 Corbeil et al. (43) Pub. Date: Feb. 5, 2015 (54) DIATOM-BASEDVACCINES (86). PCT No.: PCT/US2O12/062112 S371 (c)(1), (71) Applicants: The Regents of the University of (2) Date: Apr. 23, 2014 California, Oakland, CA (US); Synaptic Related U.S. Application Data Research, LLC, Baltimore, MD (US) (60) Provisional application No. 61/553,139, filed on Oct. (72) Inventors: Lynette B. Corbeil, San Diego, CA 28, 2011. (US); Mark Hildebrand, La Jolla, CA Publication Classification (US); Roshan Shrestha, San Diego, CA (US); Aubrey Davis, Lakeside, CA (51) Eiko.29s (2006.01) (US) Rachel Schrier, Del Mar, CA CI2N 7/00 (2006.01) (US); George A. Oyler, Lincoln, NE A6139/02 (2006.01) (US); Julian N. Rosenberg, Naugatuck, A61E36/06 (2006.01) CT (US) A6139/02 (2006.01) (52) U.S. Cl. (73) Assignees: SYNAPTIC RESEARCH, LLC, CPC ............... A61K 39/295 (2013.01); A61K 36/06 Baltimore, MD (US): THE REGENTS (2013.01); A61 K39/107 (2013.01); A61 K OF THE UNIVERSITY OF 39/102 (2013.01); C12N 700 (2013.01); A61 K CALIFORNIA, Oakland, CA (US) 2039/523 (2013.01) USPC .................. 424/2011; 424/93.21; 424/261.1; y x- - - 9 (57) ABSTRACT 22) PCT Fled: Oct. 26, 2012 This invention pprovides diatom-based vaccines. Patent Application Publication Feb. 5, 2015 Sheet 1 of 19 US 2015/0037370 A1 83 : RE: Repests 388x ExF8. Patent Application Publication Feb. 5, 2015 Sheet 2 of 19 US 2015/0037370 A1 Fig. 2 Patent Application Publication Feb. 5, 2015 Sheet 3 of 19 US 2015/0037370 A1 '. : 88:838: Patent Application Publication Feb. 5, 2015 Sheet 4 of 19 US 2015/0037370 A1 mine corveiescent tra &888 isix: Fig. 4 Patent Application Publication Feb. 5, 2015 Sheet 5 of 19 US 2015/0037370 A1 88: 3:33 Patent Application Publication Feb. 5, 2015 Sheet 6 of 19 US 2015/0037370 A1 Fig. 6 Patent Application Publication Feb. 5, 2015 Sheet 7 of 19 US 2015/0037370 A1 Patent Application Publication Feb. 5, 2015 Sheet 8 of 19 US 2015/0037370 A1 AGCTIGCGCITICCGAGAACCCCCAA AGCAACGGC'CCAATCAAGAATGATCCGA CAACGGCGAGCATAGCA ACACGTCCGTCTTGGAGTAGAATCATCAGT, GTGGATGAATA CACAGATGAATGACATTAAAAGCATGAACATGTAGAGAGTAGGAGGTAGAGATTGAATG GTAGCATTGCGATGT (GTTTTGGTCAGCAATGATGAGTGGATACCAAATGAGAAAG TTGAATCTCGCCTTTGAGCT CACCCGACGTATTGACCAAAG TAGCCTGACAAAATCC TTGGAGAGACAAGAGGATCAAAGAACCAGGGGGGCGATAACCCAAGCCGTCCAA AGAGGCAAGGAGGAGAAACT CACCCAGTGAGAAGAAGGAAGGCAGGGCGGTGGCG AAAGCAGAGGCAA CGAGGACAGACTCCTGGGGEGAGCAACGAATACCAGAAG GA GAAGTTTAGACACTTGAACCGCTACCTACAATGACAAACTATCCTA: CGATTTTGATGTTC GTTGGTTAGAATTCAA ACTGTAAGTTGGATTGTGAGAAGATCAGAAGTTGAACGAACACA TCTTTCCGATCATTCACCTCCACACTGCAACAACACGG ACTCTCCGCGGCAGCTCTC GTCCCCATICTCTCTCCTGTTCTGGCTCTGACACGACCAAACCAA CGACAAGTTTCACA AAAGGGAGTCCTTTAACGAGATAE GTTTTTTATAAAGAGTCCCAAEAGAAAGACAAATTG ATTCCTCCGTGCAAACGCGCAAATAAACACCACGTCCAAATCCATACTTTCAGAGTA TCCAACAAGTGTTGAAGGACAGGTAGTGAACTAACGTATCTTCCCCCCGACTGGAECCA TCAACAAGGCGAA CAAATCCATCAACCCCAAAAATCGATACCAAACCGATA CAACAAGIGACAAAAAAGCTGAACGAGAAACGAAAAGAAAAAACAAAA AAATTAGAGCAAAAAAACAGACACAAATAC GTAAAACACAACAAECCAGICA CAGGCGGCCGCAAGGCACCCCAGGCTTACACITATGCTTCCGGCCGAAAIG GGGATGAGAGGACCGCGAGATTCAGGAGCAAGGAAGCRAAAAGGAGAAA AAAACACGGAATACCACCGTTGAATATCCCAATGGCATCGTAAAGAACATTGAGG CATCAGICAGGCCAATGACCTATAACCAGACCGTTCAGCTGGATAACGGCCT AAAGACCGAAAGAAAAAAAGCACAAGITATCCGGCCTTACACACIGCC CGCCGAGAAGCCACCGGAATCCGAGGCAAGAAAGACGGTGAGCTGGGAA GGGAEAGGCACCCTTGACACCGTCCATGAGCAAACTGAAACGTCATCGC CGGAGGAAACCACGACGATTCCGGCAGCTACACAATATCGCAAGAGTGGCG GACGGIGAAAACCGGCCIATTCCCAAAGGGTTTATTGAGAAATGTTTCGTC CAGCCAAECCCTGGGTGAGTTTCACCAGTTTGATTTAAACGTGGCCAATAGGACAACTT CCGCCCCCGTTTCACCAGGGCAAATATTAACGCAAGGCGACAAGGTGCTGATGCCG Fig. 8A Patent Application Publication Feb. 5, 2015 Sheet 9 of 19 US 2015/0037370 A1 CGGCGACAGGCACAGCCGTGGAGGCTCCAGTCGGCAGAAGCTAATG AAACAACAGACGCGAGAGGGCAGGGCGGGGCGAAAGATCGGACCGGCACT AAAAGCCAGATAACAGAGCGATGCGCGCGATTTGCGGIATAAGAAATAAC GAATGAACCCGAAGAGCAAAAAGAGGTAGCTATGAAGCAGCGTATACAGTGAC AGGACAGCGACAGCTACAGGCCAAGGCAAATGATGICAAATCTCCGGCTGG AAGCACAACCAGCAGAAGAAGCCCGCGCTGCGTGCCGAACGCTGGAAAGCGGAAAA CAGGAAGGGAGGCGAGGCGCCCGGAGAAATGAACGGCCTTGCGACGAG AACAGGGGCGGGAAAIGCAGAAGGACACCTATAAAAGAGAGAGCCGTATCG CGGTGGAGACAGAGGATATATTGACACGCCCGGGCGACGGATGGGATCCCCC GGCCAGGCACGCGCGICAGAAAAGTCCCCGTGAACTTACCCGGGGGCAA CGGGGAGAAAGCGGCGCATGAGACCACCGATAGGCCAGTGTGCCGGTCCCGTATC GGGGAAGAAGGGCGACCAGCCACCGCGAAAATGACACAAAAACGCCATAACCTGA GTCGGGGAAATAAATGICAGGCTCCCTATACACAGCCAGTCGCAGGCGACCATA GTGACGGATAGIGIGITACAGTATTAGTAGICTGTTTTTTATGCAAAACAATT TAATAATGAAAACATTACGTCTCGITCAGCITICTGTACAAAGGGTT GAATCGCATGCGGTACCGGCGGAAGGIGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG TGCCCAICCTGGTCGAGCIGGACGGCGACCAAACGGCCACAAGTTCAGCGICTCCGGCGA GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCIGAAGTTCATCTGCACCACCGGCA AG TCCCCGTCCCCTGGCCCACCCTCGTGACCACCCTGACC TACGGCCTGCAGTCCTTCAGCC GCTACCCCGACCACATGAAGCAGCACCACCTTCAAGTCCGCCATGCCCGAAGGCACGT CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGGAAG TTCGAGGGCGACACCCTGGTTGAACCGCACGAGCIGAAGGGCATCGACTTCAAGGAGGACG GCAA CATCCI'GGGGCACA AGCI'GGAGTACA ACACAACAGCCACAACGTCTATA ICAI'GGC CGACAAGCAGAAAACOGCACAAGGIGAACTTCAAGACCGCCACA ACACCAGGACGGC AGCGIGCAGCi"CGCCGACCACIACCAGCAGAACACCCCCATCGGCGACGGCCCCGGCGC IGCCCGACAACCACTACCTGAGCACCCAGCCGCCCTGAGCAAAGACCCCAA CGAGAAGCG CCATICACATGGICCIGCTGGAGTTCGIGACCGCCGCCGGGAT"CACTCTCGGCA'GGACGAG CTGACAAGTAAGCGGCCGCATACGGAGGGAACAATGAGCCGTAGCACAATGGTA Fig. 8B Patent Application Publication Feb. 5, 2015 Sheet 10 of 19 US 2015/0037370 A1 CATCGGCAGCTAAGATCCAATGGCAAGGACCAAGTGCGGAACTGTGCAGCA GATCTTAGCGTGAGAGGTATTGTCCTCTGTCAGGAG TAGATAGTAGATGETCTTTTTAAA CTAAAAIGCTAACGTCCGAA. TCC, CATCGCAGCTA ATCCGACACAAAAGACAAAA GCTAGGTATGTGTACTA CATCTCCGTGCTAGAAACACATAGATAGGAAACACACCA: CAATAGTCATTGTAGCTTACTTATACACGCAGCAC TTCCCCTGACTGGCAGAGGC GCATTGAGAAAATCGATCT CAACAAGTTTAG TAGCACCCCTAGACCATACTAAG TCTCCTTCGTCTTTGGTGTAGGCATGTTGGACACAACGAGGTAAAACACAACACAAACAA GTGTCCAGCAAAGTAGTAGCTGCTCCAGTTCTCCCGGGGGATCCACTAGTECTAGAGCGGC CGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTCCCTTAGGAGGG'TAATGCGCGC GGCGTAACAGCCAAGCGCCGGGAAAG ACCGCECACAATCCACA CAACATACGAGCCGGAAGCATAAAGGTAAAGCCTGGGGTGCCTAATGAGTGAGC AACTC ACATTAATGCCTTC CGCT CACTCCCCCCTTCCAGCCGGAAACCGTCGTCCCACCTCC ATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTCCGTATTGGGCGCTCTCCGCTTC CTCGCT CACT CACTCGCT, GCCCCCCCCTTCCGCTCCCCCGACCGCTATCAGCCACCA AAGGCGGTAATACGGTTACCACAGAATCAGGGGATAACGCAGGAAAGAA CATGGAGCAA AAGGCCAGCAA AAGGCCAGGAACCGTAAAAAGGCCGCGTGCTGGCGTCCAAGGC CCGCCCCCCTGACGAGCAT CACAAAAATCGACGCCAAGICAGAGGGGCGAAACCCGACA GGACTATAAAGATACCAGGCGTCCCCCTGGAAGCCCCCG GCGCECTCCTGTCCGA CCCTGCCGCTTACCGGATACCGTCCGCCTTCTCCC, TCGGGAAGCGGGCGC TTC CA, TAGCTCACGCTGTAGGTATCT CACTTCGGTGTAGGTCGTCCCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCGCGCCTATCCGGTAACTATCGCTTGAGCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACGGTAACAGGATTAGCAGAGC GAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGGGGGCCTAACTACGGCTACACTAGA AGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTACCTTCGGAAAAAGAGTGGTA GCTCTTGATCCGGCAAACAA ACCACCGCTGGAGCGGGGTTTTTGGCAAGCAGCA GATTACGCGCAGAAAAAAAGGA CTCAAGAAG ACCTTGATCTTCACGGGGTCTGAC GCTCAGTGGAACGAAAACTCACGTTAAGGGATTGGTCATGAGATTACAAAAAGGATC TCACCAGAICCITTAAAAAAAATGAAGTAAACAACEAAAGTATATATGAGTA AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCECAGC (GATCTGTCTA Fig. 8C Patent Application Publication Feb. 5, 2015 Sheet 11 of 19 US 2015/0037370 A1 TTTCGTCATCCAAGTGCCGACCCCCGCGGTAGAAACTACGATACGGGAGGGCT TACCATCTGGCCCCAGTGCTG CAATGATACCGCGAGACCCACGCT CACCGGCTCCAGATT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCCCAGAACTGGTCCTCCAACTTTATCC GCCICCATCCAGTCATAAG GCCGGGAAGC AGAGAAGAGCGCCAGTAAA GTTTCCCCAACC TriGriCCCA GCACAGCCACGTCC'C' CACCCTCC, CCTTGG AT GGCTICATICAGCTCCGGCCCA ACGATCAAGGCGAGACAEGATCCCCCA GTGGC AAAAAAGCGGTTAGCTCCTTCGGTCCCCGATCGGCAGAAGTAAGTIGGCCGCAGTGT TATCACTCATGGTTAGGCAGCACTGCATAATTCCTTACGTCAGCCATCCGTA AGAG CTTTTCGIGACTGGTGAGTACT CAACCAAGTCACGAGAAAGEGRATGCGGCGACCG AGTIGCTCTTGCCCGGCGT CAAACGGGATAATACCGCGCCACAAGCAGAACTTAAAAG TGCTCATCATGGAAAACGTTCTTCGGGGCGAAAACTCCAAGGATCTEACCGCTGTGAG ATCCAGTCGATGTAACCCACCGTG (CACCCAACTGACCAGCATCTEACTTCACC AGCGTTTCGGGTGAGCAAAAACAGGAAGGCAAAAIGCCGCAAAAAAGGGAATAAGGGCGA CACGGAAA GTGAA, ACCAACCCCiCAAAAGAAGCAATCAGGG TTATTGTCTCATGAGCCGATACAATTGAATGTATTAGAAAAATAAACAAATAGGGGT CCGCGCACATTTCCCCCAAAACGCCACCAAATTCTAACCGIAA; ATTTCTTAAAA' CCCCTTAAATTTTTGTAAAICAGCCATTTTTAACCAAAGGCCCAAATCGGCAAAAC CCITATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGTCCAGTTTGGAACAAGA GT CCACTATTAAAGAACGGGACTCCAACGTCAAAGGGCGAAAAACCGCTACAGGGCGA TGGCCCACTACGTGAACCAT CACCCTAATCAAGTTTGGGGTCGAGGGCCGTAAAGCA CTAAATCGGAACCCAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACG TGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCAGGGCGCTGGCAAGTGTAGC GGTCACGCTGCGCGAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCC CATICGCCATTCAGGCGCGCAACGGGGAAGGGCGATCGGGCGGGCCCTCGCTA TACGCCAGCTGGCGAAAGGGGGATGGCGCAAGGCGATTAAGEGGGTAACGCCAGGGET TTCCCAGTCACGACGGAAAACGACGGCCA GTGAGCGCGCGEAAFACGACCACAAG GCCGAATCGGTACGTACCCCCCCCCCCCCGAGGICGACGCTATCGATA (SEQ ID NO:8) Underlined in FCP promoter and terminator Boid as Gateway B fragment Italic c e GFP Fig. 8D Patent Application Publication Feb. 5, 2015 Sheet 12 of 19 US 2015/0037370 A1 AGCTGCGCTCCGAGAACCCCCAAAGCAACGGCTCCAACAAGAAG ACCGA CAACGGCGAGCATAG CAACACGTCCGT CTTGGAG TAGAATCACAE GTGGGATGAATA CACAGATGAATGACATTAAAAGCATGAACAGTTAGAGAGTAGGAGGAGAGATTGAATG GTAGCATEGCGATGETGETTEGGCAGCATAGAEGAGGGAEACCAAATGATGAAAG TTGAACCGCGTGAGCTCAGCGGACGATGACGAAAGTAGCCGATCAAAAICC TTGGAGAGTACAAGAGGATCAAAGAATCCAGTGGGGGCGATAACTCCAAGCTCGTTCCAA AGAGGCAAGGAGGTAGAAAC CACCCAGTTGA, GAAGAAGGAAGGCAGGGCGGGGCG AAAGCAGAGGCAACGAGGACAGACTTCCTGGGGTTGAGCAACGAATATECCAGAAGGA GAAGTTTAGAGACTGAACCCCACCACAATGACAAAGTACGTACGATTGATGTG GTTGGTTATGAATTCAAACTGAAGGGATTGGAGAAGACAGAAGTGAACGA ACACA TCTCCGATCATICAC CTCCACACGCAA CAACACGGACECCCGCGGCAGGTCC GTCGCCATCTCGTCCTGGTTGGCGGAGACGAGGAAAGCAA
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