WO2017031280A1 - Fusion proteins that include dengue antigens and methods of use - Google Patents

Fusion proteins that include dengue antigens and methods of use Download PDF

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Publication number
WO2017031280A1
WO2017031280A1 PCT/US2016/047496 US2016047496W WO2017031280A1 WO 2017031280 A1 WO2017031280 A1 WO 2017031280A1 US 2016047496 W US2016047496 W US 2016047496W WO 2017031280 A1 WO2017031280 A1 WO 2017031280A1
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domain
dengue
flagellin
amino acid
loop
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PCT/US2016/047496
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French (fr)
Inventor
Langzhou Song
Hong Li
Xiangyu Liu
Ge LIU
Lynda TUSSEY
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Vaxinnate Corporation
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Publication of WO2017031280A1 publication Critical patent/WO2017031280A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Dengue infection is a major public health problem in tropical areas of the world. About 2.5 billion people are at risk for Dengue infection and about 50 million cases of dengue infection and hundreds of thousands of cases of dengue hemorrhagic fever (DHF) occur in the tropics each year, including Mexico, the Caribbean and parts of Asia and the South Pacific (Gubler, D.J., Ann Acad Med Singapore 27: 227-34 (1998); Guzman, M.G., et al, Nat. Rev. Microbiol. S/S7-S16 (2010)). Dengue viruses are transmitted by peridomestic Aedes spp.
  • Dengue a flavivirus
  • Dengue 1 also referred to as "Denl”
  • Dengue 2 also referred to as “Den2”
  • Dengue 3 also referred to as “Den3”
  • Dengue 4 also referred to as "Den3”
  • a fifth serotype was reported (Dengue 5) (Normile, D., Science 342(6157):415 (2013)).
  • DHF Dengue hemorrhagic fever
  • DSS dengue shock syndrome
  • DHF may be the result of an antibody dependent enhancement (ADE) where non- neutralizing antibodies induced by the primary DEN infection form virus-antibody complexes in secondary infections that are ingested by macrophages through Fc receptors and, thus, enhance virus infection (Kliks, S.C., et al, Am. J. Trop. Med. Hyg. ⁇ 0:444-451 (1989)). DHF occurs primarily in children and can be fatal. Human antibodies to non-neutralizing epitopes in the envelope (E) protein and the membrane (M) protein of Dengue viruses are thought to be highly cross reactive and may cause the ADE (Dejnirattisai, W., et al, Science 7:328(5979):745-748 (2010)).
  • ADE antibody dependent enhancement
  • compositions that employ tetravalent live Dengue flaviviruses to prevent Dengue infection may be limiting due to interference and imbalanced immune response among the serotypes.
  • Compositions that include inactivated flavivirus may result in limited
  • the present invention relates to compositions and fusion proteins that include Dengue antigens and flagellin.
  • the compositions and fusion proteins can be employed in methods to stimulate immune responses that ameliorates or diminishes disease or illness consequent to infection by the Dengue virus.
  • the immune response stimulated can be a protective immune response.
  • the invention is a composition comprising at least four fusion proteins, each of which activates a Toll-like Receptor 5, and includes flagellin and Dengue viral antigens.
  • the first fusion protein of the composition includes a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin.
  • the second fusion protein of the composition includes a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin.
  • the third fusion protein of the composition includes a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin.
  • the fourth fusion protein of the composition includes a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin; and a domain I and a domain II of the Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
  • the invention is a method of stimulating an immune response in a subject by administering a composition comprising at least four fusion proteins, each of which activates a Toll-like Receptor 5, and includes flagellin and Dengue viral antigens.
  • the first fusion protein of the composition includes a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin.
  • the second fusion protein of the composition includes a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin.
  • the third fusion protein of the composition includes a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin.
  • the fourth fusion protein of the composition includes a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin; and a domain I and a domain II of the Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
  • compositions and methods described herein have advantages over current methods of preventing Dengue infection.
  • the compositions include at least four fusion proteins of Dengue antigens from the four serotypes fused to flagellin in a similar configuration that can be readily produced recombinantly, thereby optimizing manufacturing and access to compositions for preventing Dengue infection that can be readily available for use in relatively short-term immunization procotols.
  • FIG. 1 depicts loops in domain 3 for S. typhimurium FliC (SEQ ID NO: 1) based on a known crystal structure and predicted loops in S. typhimurium FljB (SEQ ID NO: 2).
  • FIG. 2 depicts predicted insertion sites in domains 0, 1, 2 and 3 of S. typhimurium FljB (SEQ ID NO: 2) for fusion with antigens, including insertion sites in a loop of domain 3.
  • Domain 0 is predicted at amino acid residues 1-46 and amino acid residues 465-506; Domain 1 is predicted at amino acid residues 47-176 and amino acid residues 415-464; Domain 2 is predicted at amino acid residues 177-190 and amino acid residues 292-414; and Domain 3 is predicted at amino acid residues 191-291.
  • the amino acid number of boundaries between domains 0, 1, 2 and 3 and insertion sites are also indicated.
  • FIG. 3 depicts the structure of flagellin, domains 0, 1, 2 and 3 and insertion sites in flagellin (SEQ ID NO: 2) for generation of fusion proteins that include antigens. Reference to amino acid numbers are made to (SEQ ID NO: 2).
  • FIG. 4A depicts the Dengue envelope ectodomain homodimer (Zhang et al., Nat. Struct. Biol. 70(11):907-912 (2003)). The three domains of the Dengue envelope protein, EI (dark grey), EII (light grey) and EIII (black) are indicated. A stem region connects the stably folded ectodomain portion of the envelope protein with the C-terminal transmembrane (TM) anchor region of the envelope protein.
  • TM transmembrane
  • FIG. 4B depicts the conformation of E (envelope protein) in the mature virus particle and in solution above the fusion pH.
  • the junction between the EI (Domain I of the Dengue viral envelope protein) and EII (Domain II of the Dengue viral envelope protein) domains is structurally complex and includes several peptide strands, depicted by the circle.
  • the linking region extending the C-terminus of EIII to form 80E+ is indicated by arrows.
  • FIG. 4C generally depicts the linear conformation of 80E, 80E+ and 85E Dengue envelope protein antigen components of the fusion proteins of the invention.
  • Domain I EI
  • domain II EII
  • domain III EIII
  • junction loop JL
  • linking region LR
  • portion of a stem region ST.
  • amino acid residues of the envelope protein are with reference to SEQ ID NO: 89.
  • FIG. 5 depicts the amino acid sequence of the fusion protein BV179, E395.STF2ng2 (Dengue 1 strain 16007). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 8)
  • FIG. 6 depicts the amino acid sequence of the fusion protein BV251, STF2ng2 D3.E395 (Dengue 1 strain 16007). 80E D3Ins format. Dotted line, MT leading sequence;
  • FIG. 7 depicts the amino acid sequence of the fusion protein BV255
  • FIG. 8 depicts the amino acid sequence of the fusion protein BV262, PRM16/E400.STF2ng2 (Dengue 1 strain 16007).
  • 80E+ N-term format PRM16 is a leading sequence from PrM and 80E+(l-400) is a longer version of 80E.
  • FIG. 9 depicts the amino acid sequence of the fusion protein BV265, STF2ng2 D3.E400 (Dengue 1 strain 16007).
  • 80E+(L,l-400, SEQ ID NO: 93) includes amino acids residues of the 80E Dengue protein of SEQ ID NO: 41. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 12)
  • FIG. 10 depicts the amino acid sequence of the fusion protein VI 54
  • FIG. 11 depicts the amino acid sequence of the fusion protein BV155 E395.STF2ng2 (Dengue 2 strain 16681). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 14)
  • FIG. 12 depicts the amino acid sequence of the fusion protein BV237 E395.STF2ng2 with linker (Dengue 2 strain 16681). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 15)
  • FIG. 13 depicts the amino acid sequence of the fusion protein BV242
  • STF2ng2D3Ins.E394 (a.a. 2-395 of SEQ ID NO: 42, Dengue 2 strain 16681. 80E D3Ins format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 16)
  • FIG. 14 depicts the amino acid sequence of the fusion protein BV244 E399 (a.a. 2- 400 of SEQ ID NO: 94).STF2ng2 (Dengue 2 strain 16681). 80E+ N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 17)
  • FIG. 15 depicts the amino acid sequence of the fusion protein BV163
  • STF2ng2.3GS.E393 (Dengue 3 strain Portugal 24/94). 80E C-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 18)
  • FIG. 16 depicts the amino acid sequence of the fusion protein BV170 E393.STF2ng2 (Dengue 3 strain Guatemala 24/94). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 19)
  • FIG. 17 depicts the amino acid sequence of the fusion protein BV219 E393.STF2ng2 (Dengue 3 strain Pah881/88). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 20)
  • FIG. 18 depicts the amino acid sequence of the fusion protein BV256
  • FIG. 19 depicts the amino acid sequence of the fusion protein BV272 E398.STF2ng2 (Dengue 3 strain Pah881/88). 80E+ N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 22)
  • FIG. 20 depicts the amino acid sequence of the fusion protein BV164 STF2ng2.3GS. E395 (Dengue 4 strain 341750 (TVP360)). 80E C-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
  • FIG. 21 depicts the amino acid sequence of the fusion protein BV171
  • FIG. 22 depicts the amino acid sequence of the fusion protein BV243 E399.STF2ng2 (a.a.. 2-400 of SEQ ID NO: 96, Dengue 4 strain 341750 (TVP360)).
  • 80E+ N-term format Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag.
  • FIG. 23 depicts the amino acid sequence of the fusion protein B V267
  • PRM16.E400.STF2ng2 (Dengue 4 strain 341750 (TVP360)). 80E+ N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 26)
  • FIG. 24 depicts the amino acid sequence of the fusion protein BV261
  • EIEII.STF2ng2D3.EIII (Dengue 1 strain 16007). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
  • FIG. 25 depicts the amino acid sequence of the fusion protein BV258
  • EIEII.STF2ng2.5GS.D3.EIII (Dengue 1 strain 16007). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 28)
  • FIG. 26 depicts the amino acid sequence of the fusion protein B V270
  • EIEII.5.GS.STF2ng2D3.EIII.3GS (Dengue 1 strain 16007). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 29)
  • FIG. 27 depicts the amino acid sequence of the fusion protein BV239
  • EIEII.STF2ng2.D3.EIII (Dengue 2 strain 16681). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
  • FIG. 28 depicts the amino acid sequence of the fusion protein 8 BV263
  • FIG. 29 depicts the amino acid sequence of the fusion protein BV241 EIII.STF2ng2D3.EIEH (Dengue 2 strain 16681). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 31) [0043]
  • FIG. 29 depicts the amino acid sequence of the fusion protein BV241 EIII.STF2ng2D3.EIEH (Dengue 2 strain 16681). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
  • FIG. 30 depicts the amino acid sequence of the fusion protein BV264
  • EIEII.STF2ng2D3..EIII Dengue 3 strain Pah881/88. 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 33)
  • FIG. 31 depicts the amino acid sequence of the fusion protein BV268
  • EIEII.5GS.STF2ng2D3.EIII Dengue 3 strain Pah881/88. 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Wavy underline, flexible linker. (SEQ ID NO: 34)
  • FIG. 32 depicts the amino acid sequence of the fusion protein BV269
  • EIEII.5GS.STF2ng2D3.EIII.3GS (Dengue 3 strain Pah881/88). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 35)
  • FIG. 33 depicts the amino acid sequence of the fusion protein BV227
  • EIEII.STF2ng2D3.EIII Dengue 4 strain 341750 (TVP360)). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 36)
  • FIG. 34 depicts the amino acid sequence of the fusion protein BV254
  • EIEII.5GC.STF2ng2R3.EIII Dengue 4 strain 341750 (TVP360)). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 37)
  • FIG. 35 depicts the amino acid sequence of the fusion protein BV257
  • FIG. 36 depicts the amino acid sequence of the fusion protein BV316 EIEII.5GS.STF2ng2D3.EIII.3GS(Den4). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker.
  • SEQ ID NO: 38 [0050]
  • FIG. 36 depicts the amino acid sequence of the fusion protein BV316 EIEII.5GS.STF2ng2D3.EIII.3GS(Den4). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Wavy underline, flexible linker.
  • SEQ ID NO: 39
  • FIG. 37 depicts the amino acid sequence of the fusion protein BV293B EIEII.
  • FIG. 38 depicts the amino acid sequence of Dengue 1 NH 16007 80E: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 41)
  • FIG. 39 depicts the amino acid sequence of Dengue 2 NH 16681 80E: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 42)
  • FIG. 40 depicts the amino acid sequence of Dengue 3 Pah881/88 80E: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 43)
  • FIG. 41 depicts the amino acid sequence of DEN4 341750 80E: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 44)
  • FIG. 42 depicts the amino acid sequence of DEN1 Hawaii 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 45)
  • FIG. 43 depicts the amino acid sequence of DEN2 Thailand 16681 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 46)
  • FIG. 44 depicts the amino acid sequence of DEN2 New Guinea C 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 47)
  • FIG. 45 depicts the amino acid sequence of depicts the amino acid sequence of DEN3 CH53489 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted.
  • SEQ ID NO: 48 depicts the amino acid sequence of DEN3SIA 24/94 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted.
  • FIG. 47 depicts the amino acid sequence of DEN4 H241 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 50)
  • FIG. 48 depicts the amino acid sequence of DEN4 61NIID 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted.
  • FIG. 49 depicts the amino acid sequence of EI (Dengue 1, NH 16007): Junction loop, grey highlighted (SEQ ID NO: 52)
  • FIG. 50 depicts the amino acid sequence of EI (Dengue 2, NH 16681): Junction loop, grey highlighted (SEQ ID NO: 56)
  • FIG. 51 depicts the amino acid sequence of EI (Dengue 3, Pah881/88): Junction loop, grey highlighted (SEQ ID NO: 60)
  • FIG. 52 depicts the amino acid sequence of EI (Dengue 4, 341750): Junction loop, grey highlighted (SEQ ID NO: 64)
  • FIG. 53 depicts the amino acid sequence of EI (Dengue 1, Hawaii): Junction loop, grey highlighted (SEQ ID NO: 68)
  • FIG. 54 depicts the amino acid sequence of EI (Dengue 2, Thailand 16681): Junction loop, grey highlighted (SEQ ID NO: 71)
  • FIG. 55 depicts the amino acid sequence of EI (Dengue 2, New Guinea C): Junction loop, grey highlighted (SEQ ID NO: 74)
  • FIG. 56 depicts the amino acid sequence of EI (Dengue 3, CH53489): Junction loop, grey highlighted (SEQ ID NO: 77)
  • FIG. 57 depicts the amino acid sequence of EI (Dengue 3, Portugal 24/94): Junction loop, grey highlighted (SEQ ID NO: 80)
  • FIG. 58 depicts the amino acid sequence of EI (Dengue 4, H241): Junction loop, grey highlighted (SEQ ID NO: 83)
  • FIG. 59 depicts the amino acid sequence of EI (Dengue 4, 61NIID): Junction loop, grey highlighted (SEQ ID NO: 86)
  • FIG. 60 depicts the amino acid sequence of 80 E+ (Dengue 1 NH 16007) Junction loop, grey highlighted (SEQ ID NO: 93)
  • FIG. 61 depicts immunogenicity of monovalent DENV1 fusion proteins in various formats in mice.
  • Groups of 5 BALB/c mice were immunized S.C. with 8 ⁇ g of BV270 (Split, EIEII.5GS.STF2ng2D3.EIII.3GS), BV255 (N-term 80E fusion, E399.3GS.STF2ng2), or BV271 (N-term 85E, E426.STF2ng2) on days 0, 21, and 42.
  • Sera were prepared on days 56 and subjected to FRNT test against strain West Pac 74. Data are shown as FRNT50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
  • GTT geometric mean titers
  • FIG. 62 depicts immunogenicity of monovalent DENV1 fusion protein in split formats, BV270B (strain 16007) and BV293 (strain PU0359) in mice.
  • Groups of 8 BALB/c mice were immunized S.C. with the indicated doses of BV270B or BV293B on days 0, 21, and 42.
  • Sera were prepared on days 56 and subjected to FRNT test against strain West Pac 74.
  • Data are shown as FRNT 50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
  • FIG. 63 depicts immunogenicity of monovalent DENV2 fusion proteins of different formats in mice.
  • Groups of 5 BALB/c mice were immunized S,C, with the 1 ⁇ g or 10 ⁇ g of BV237, BV239, BV241, or BV242 on days 0, 21, and 42.
  • Sera were prepared on days 63 and subjected to FRNT test against strain SI 6803.
  • Data are shown as FRNT 50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
  • FIG. 64 depicts immunogenicity of monovalent DENV2 in Split format with no linker, one linker and two linkers in mice.
  • Groups of 6 BALB/c mice were immunized S.C. with indicated doses of BV239, BV257, or BV263 on days 0, 21, and 42.
  • Sera were prepared on days 56 and subjected to FRNT test against strain SI 6803.
  • Data are shown as FRNT 50 titers of individual mice with geometric mean titers (GMT) in numbers.
  • FIG. 65 depicts immunogenicity of monovalent DENV3 fusion proteins in split (BV269B) and N-term (BV272B) formats in mice.
  • Groups of 5 BALB/c mice were immunized S.C. with indicated doses of BV269B or BV272B on days 0, 14, and 28.
  • Sera were prepared on days 42 and subjected to FRNT test against strain CH54389.
  • Data are shown as FRNT50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
  • FIG. 66 depicts immunogenicity of monovalent DENV4 fusion proteins in split (BV316) and N-term (BV243B) formats in mice.
  • Groups of 6 BALB/c mice were immunized S.C. with indicated doses of BV316 or BV243B on days 0, 14, and 28.
  • Sera were prepared on days 42 and subjected to FRNT test against strain TVP 360.
  • Data are shown as FRNT 50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
  • FIG. 67 depicts the efficacy of TDV formulations in the DENV2/NHP model.
  • FIGs. 68 and 69 depict cytokine (IL-6) induction in mice following immunization with flagellin/Dengue viral envelope protein antigen fusion proteins.
  • FIG. 70 generally depicts 80E Dengue protein antigens fused to flagellin in split format and N-terminal fusion formats. Junction loop (JL).
  • amino acid residues of the envelope protein in a fusion protein are with reference to SEQ ID NO: 41.
  • FIG. 71 generally depicts 80E+ Dengue protein antigens fused to flagellin in split format and N-terminal fusion formats. Junction loop (JL), linking region (LR).
  • amino acid residues of the envelope protein in a fusion protein are with reference to SEQ ID NO: 93.
  • FIG. 72 generally depicts 85E Dengue protein antigens fused to flagellin in split format and N-terminal fusion formats. Junction loop (JL), linking region (LR), portion of the stem region (ST).
  • amino acid residues of the envelope protein in a fusion protein are with reference to SEQ ID NO: 89.
  • the invention is a composition comprising at least four fusion proteins, each of which activates a Toll-like Receptor 5, and includes flagellin and Dengue viral antigens.
  • the first fusion protein of the composition includes a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin.
  • the second fusion protein of the composition includes a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin.
  • the third fusion protein of the composition includes a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin.
  • the fourth fusion protein of the composition includes a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin; and a domain I and a domain II of the Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
  • Fusion protein refers to a protein generated from at least two distinct components, a flagellin or a portion of a flagellin and a Dengue antigen or a portion of a Dengue antigen.
  • the flagellin and Dengue antigen can be linked by covalently or noncovalently.
  • Fusion proteins of the invention can be designated by components of the fusion proteins separated by a ".” or
  • “EIEII.STF2ng2.D3Ins-EIII” refers to a fusion protein of a fljB/STF2 flagellin that has been altered to delete at least one putative glycosylation sites (“ng") (STF2ng2, for example SEQ ID NO: 108) fused at its amino-terminal amino acid to domains I and II (" ⁇ ") of the envelope protein of a Dengue antigen and fusion of domain III of the envelope protein of the Dengue antigen (" ⁇ ") to a loop of domain 3 of flagellin
  • the flagellin in the fusion proteins of the invention can be an S. typhimurium flagellin (UniProt accession number P06179 or P52616), such as a S. typhimurium flagellin selected from the group consisting of (SEQ ID NOS: 1 and 2); an E. coli flagellin (UniProt accession number A0PCV8), such as, for example, (SEQ ID NO: 3); a P.
  • aeruginosa flagellin (UniProt accession number P72151), such as (SEQ ID NO: 4); an Aquifex aeolicus VF5 flagellin (UniProt accession number 067803), such as (SEQ ID NO: 5); a Helicobacter pylori J99 Flagellin A (UniProt accession number P0A052), such as (SEQ ID NO: 6); and a Legionella pneumophila flagellin (UniProt accession number Q48824), such as (SEQ ID NO: 7).
  • the flagellin component of the first fusion protein, second fusion protein, third fusion protein and fourth fusion protein can be similar or distinct from each other.
  • Similar in reference to the flagellin component of the fusion proteins of the invention, means that the flagellin of one or more of the four fusion proteins resembles one or more of the other fusion proteins.
  • the first and the second fusion proteins can include a S. typhimurium flagellin, which would be similar flagellin components of the fusion proteins.
  • “Distinct,” in reference to the flagellin component of the fusion proteins of the invention means that the flagellin of one or more of the four fusion proteins is not the same as one or more of the other fusion proteins.
  • a first protein can include a S. typhimurium flagellin and a second protein can include an E. coli flagellin, which would be distinct flagellin components of the fusion proteins.
  • Domains I and II of the Dengue viral envelope antigen are fused to the amino-terminus of a flagellin.
  • the Dengue viral envelope antigen of domains I and II can be fused to the most terminal amino acid residue of the amino- terminus of flagellin.
  • the junction between domains I and II of the Dengue viral envelope protein is structurally complex and includes several peptide strands, depicted by the circle in FIG. 4B. To form a tertiary structure, the Dengue E protein traverses the EI and EII domains multiple times before entering the EIII domain.
  • Fusion proteins of the invention include domains I and II in a sequence that mimics the sequence of the domains in the Dengue viral envelope protein as I/II/I/II/I (see, for example, FIGs. 4A, 4C and 70-72), which is fused to the amino-terminus of flagellin, with or without a linker, as described infra.
  • Exemplary domains I of Den 1, Den 2, Den 3 and Den 4 are SEQ ID NOS: 52, 56, 60 and 64
  • domains II of Den 1, Den 2, Den 3 and Den 4 are SEQ ID NOS: 53, 57, 61 and 65.
  • a loop of domain 3 of flagellin refers to a stretch of amino acids within domain 3 of flagellin that is, itself, devoid of secondary structures (e.g., B- sheets, a-helices), yet flanks adjacent stretches of amino acids in domain 3 that include secondary structures, such as B-sheets, a-helices. Loops of domain 3 in flagellin can be about 2, about 3, about 4, about 5, about 6, about 7 and between about 5 to about 30 amino acids in length.
  • Flagellin (FljB) from Salmonella typhimurium is depicted in (SEQ ID NO: 2).
  • Domain 3 of Salmonella typhimurium flagellin is between amino acid residue 191 and amino acid residue 291 of (SEQ ID NO: 2).
  • Flagellin from E. coli (UniProt accession number
  • A0PCV8 is depicted in (SEQ ID NO: 3). Domain 3 of £ coli flagellin of (SEQ ID NO: 3) is predicted between amino acid residue 191 and amino acid residue 283 of (SEQ ID NO. 3). P. aeruginosa flagellin (UniProt accession number P72151) is depicted in (SEQ ID NO: 4) with domain 3 predicted. Flagellin from Aquifex aeolicus VF5 (UniProt accession number 067803) is depicted in (SEQ ID NO: 5). Domain 3 of Aquifex aeolicus flagellin is predicted between amino acid residue 197 and amino acid residue 302 of (SEQ ID NO. 5). The flagellin A from
  • Helicobacter pylori J99 (UniProt accession number P0A052) is depicted in SEQ ID NO: 6. Domain 3 of Helicobacter pylori J99 of (SEQ ID NO: 6) is predicted between amino acid residue 189 and amino acid residue 283 of (SEQ ID NO: 6). The flagellin from Legionella pneumophila (UniProt accession number Q48824) is depicted in (SEQ ID NO: 7). Domain 3 of Legionella pneumophila flagellin of (SEQ ID NO: 7) is predicted between amino acid residue 189 and amino acid residue 283 of (SEQ ID NO: 7).
  • X-ray crystallography of Salmonella typhimurium FliC flagellin shows that domain 3 of flagellin includes 6 loops (FIG. 1).
  • the loops in domain 3 of Salmonella typhimurium flagellin are from amino acid residues 211 to 212 (loop 1); amino acid residues 217 to 219 (loop 2); amino acid residues 223 to 229 (loop 3); amino acid residues 237 to 242 (loop 4); amino acid residues 250 to 255 (loop 5) and amino acid residues 259 to 275 (loop 6).
  • FIG. 2 depicts the amino acid sequence of Salmonella typhimurium FljB flagellin (SEQ ID NO: 2), boundaries of domain 0, 1, 2 and 3, and potential sites of fusion of Dengue viral antigens in loops of domain 3 of the flagellin.
  • the site of fusion of a Dengue viral envelope antigen domain III
  • D3I-il also referred to as "D3Ins-il”
  • the site of fusion of a Dengue envelope viral antigen is D3I-ol (also referred to as "D3Ins-ol”) between amino acid residues 277 and 278 of (SEQ ID NO: 2) in loop 6 of domain 3 (see FIG. 1).
  • the Dengue viral envelope antigen (domain III) can be fused to domain 3 of flagellin to generate constructs referred to as D3I-ol and D3I-il .
  • D3I-ol refers to insertion into a loop of domain 3 (Domain 3 Insertion) on the outer (o) or concave surface of flagellin, such as loop 6 of (SEQ ID NO: 2) or (SEQ ID NO: 1).
  • D3I-il refers to a loop of domain 3 on the inner (i) or convex surface of flagellin, such as loop 3 of (SEQ ID NO: 2) and (SEQ ID NO: 1).
  • the tertiary structure of flagellin and portions of flagellin that would be considered concave and convex surfaces of flagellin are described, for example, by Samatey, et al, Nature ⁇ 70:331-337 (2001).
  • D3Ins means insertion of a Dengue antigen (domain III of the Dengue envelope protein) into a loop of domain 3 of flagellin.
  • Insertion of a Dengue antigen into a loop of domain 3 of flagellin refers to fusion of the Dengue antigen to the loop of domain 3 of flagellin by formation of a peptide bond.
  • Exemplary fusion proteins of the invention are SEQ ID NOS: 93, 94, 95 and 96.
  • Exemplary Domain III of Den 1 (SEQ ID NO: 54), Den2 (SEQ ID NO: 58), Den3 (SEQ ID NO: 62), and Den4 (SEQ ID NO: 66) can be fused to at least one loop of domain 3 of flagellin.
  • Fusion of the Dengue viral envelope antigen (domain III) to a loop of domain 3 of flagellin essentially retains domain 3 of flagellin in its tertiary structure.
  • the phrase "essentially retains domain 3 of flagellin in its tertiary structure,” as used herein, refers to maintenance of the tertiary structure of domain 3 of flagellin, which can be assessed by well-established in vivo and in vitro assays described herein that are known to one of ordinary skill in the art, including the ability of flagellin to activate TLR5 and to assess protective immunity.
  • AA Primary amino acid sequence
  • PROF sec Secondary structure prediction where "H” stands for oc-Helix and "E” stands for ⁇ -strand
  • the predicted loops in domain 3 of (SEQ ID NO: 1) are indicated in boxes below.
  • the secondary structures adjacent to the predicted loops are essentially similar to the known secondary structures for Salmonella typhimurium flagellin (FliC, Protein Data Bank ID (PDB): lUCU) (Yonekura, K., etal, Nature, 424: 643-650 (2003)).
  • the predicted secondary structure of FliC (S. typhimurium, (SEQ ID NO: 1)) is substantially similar to the known high resolution atomic model determined by combination of X-ray crystallography and electron microscopy (lUCU, Yonekura, K., et al. Nature, 424: 643- 650 (2003)).
  • the substantial similarity between the predicted secondary structure and the known secondary structure of S. typhimurium FliC (SEQ ID NO: 1) indicates that secondary structures predicted employing CLUSTALW and PUD adjacent to loops of domain 3 of other flagellins, including S. typhimurium FljB (SEQ ID NO: 2), can be employed to select insertion sites that correspond to known loops in domain 3 in other flagellin, such as of S. typhimurium FliC.
  • Protein Data Bank ID: lUCU, (SEQ ID NO: 1) is depicted below. Predicted loops in domain 3 are indicated by boxed text.
  • typhimurium FljB (SEQ ID NO: 2) was performed using the multiple sequence alignment tool CLUSTALW and secondary structure prediction by PUD with PROF sec: structure prediction where "H” stands for alpha-helix and "E” stands for beta-strand (Thompson, J.D., et al., Nucleic Acids Res.22: 4673-4680 (1994); Rost, B., et al., Proteins 19: 55-72 (1994)).
  • the sequence alignment below showed about 74.75% identity of amino acid residues denoted with (*) and about 10.26% difference with about 7.30% strongly similar (:) and about 7.69% (.) weakly similar amino acid residues. Domain boundaries of DO, Dl, D2 and Dl are underlined differently and three D3 insertion sites were marked.
  • typhimurium FljB (SEQ ID NO: 2) that correspond to known loop regions of Domain 3 of S. typhimurium FliC (SEQ ID NO: 1) for points of insertion of Dengue viral envelope protein antigens (domain III) to generate fusion proteins of the inventions.
  • Predicted loops in domain 3 are indicated by boxed text.
  • An insertion site in a predicted loop in domain 3 of S. typhimurium FljB, referred to herein as "D3I-ol," for fusion of a Dengue viral envelope antigen (domain III) is between amino acid residues G277 and A278 of (SEQ ID NO: 2).
  • the insertion site D3I-ol is between predicted ⁇ -strands VTLA (SEQ ID NO: 109), which is amino acid residues 263-266 of (SEQ ID NO: 2), and VVS, which is amino acid residues 293-295 of (SEQ ID NO: 2).
  • D3I- il is between residues T259 and D260 of (SEQ ID NO: 2).
  • the insertion site D3I-il is between ⁇ -strands EVNVA (SEQ ID NO: 110), which corresponds to amino acid residues 254- 258 of SEQ ID NO: 2, and VTLA (SEQ ID NO: 109), which corresponds to amino acid residues 263-266 of SEQ ID NO: 2.
  • An additional insertion site in a third predicted loop in domain 3 of S. typhimurium FljB, referred to herein as "D3I-sl,” is between residue G244 and A245 of (SEQ ID NO: 2).
  • the insertion site of D3I-sl is between ⁇ -strands KYFVTIGG (SEQ ID NO: 111), which corresponds to amino acid residues 234-241 of (SEQ ID NO: 2), and EVNVA (SEQ ID NO: 110), which corresponds to amino acid residues 254-258 of (SEQ ID NO: 2).
  • the secondary structures adjacent to the predicted loops of S. typhimurium FliC are substantially similar to the predicted secondary structures adjacent to loops of domain 3 of S. typhimurium FljB and are depicted in FIG. 1.
  • fusion of the Dengue viral envelope protein antigen can be about 2 to about 10 amino acids towards the carboxy- or amino-terminus of flagellin from the designated insertion site, based on the proximity of the adjacent secondary structural elements.
  • the D3I-ol site can be from amino acid residues G268 through D289 of (SEQ ID NO: 2), which does not invade adjacent ⁇ -strands, and are predicted at VTLA (SEQ ID NO: 109) at amino acid residues 263-266 of SEQ ID NO: 2 and VVS at amino acid residues 293- 295 of (SEQ ID NO: 2).
  • the D3I-il site can only accommodate a shift of 1 or 2 amino acids towards the carboxy -terminus of flagellin of (SEQ ID NO: 2) at amino acid residues D260 or G261 of (SEQ ID NO: 2) before disrupting the neighboring ⁇ -strands, which are predicted at EVNVA (SEQ ID NO: 110) at amino acid residues 254-258 of (SEQ ID NO: 2) and VTLA (SEQ ID NO: 109) at amino acid residues 263-266 of (SEQ ID NO: 2).
  • E. coli FliC flagellin SEQ ID NO: 3
  • Vseudomonas aeruginosa PAOl ' flagellin SEQ ID NO: 4
  • E. coli FliC primary amino acid sequence SEQ ID NO: 3
  • the secondary structures were assigned either directly from known structure of lUCU for Samonella typhimurium FliC. (SEQ ID NO: 1) or by prediction using PHD.
  • coli flagellin SEQ ID NO: 3
  • Pseudomonas aeruginosa PAOl flagellin SEQ ID NO: 4 were assigned using the PHD program. Designations depicted below are AA: Primary amino acid sequence; PROF sec: Secondary structure prediction where "H” stands for oc-Helix and “E” stands for beta-strand (Rost, B., et al., Proteins 19: 55-72 (1994)).
  • NQTTQNVLSLLR (SEQ ID NO : 3)
  • E.coli FliC primary amino acid sequence (SEQ ID NO: 3) was initially aligned with the primary amino acid sequence of Salmonella typhimurium FliC (SEQ ID NO: 1).
  • ECFlic QAGTSVLAQANQTTQNVLSLLR (SEQ ID NO : 3)
  • Sequence alignment of Salmonella typhimurium FliC (STFlic, Protein Data Bank ID: lUCU, (SEQ ID NO: 1) and Pseudomonas aeruginosa P AO 1 flagellin (SaFlixl, (SEQ ID NO: 4) was performed by using the multiple sequence alignment tool CLUSTALW and secondary structure prediction by PHD with PROF sec: denoting secondary structure prediction where "H” stands for alpha-helix and "E” stands for beta-strand (Thompson, J.D., et al., Nucleic Acids Res. 22: 4673-4680 (1994); Rost, B., et al., Proteins 19: 55-72 (1994)).
  • the sequence alignment showed about 36.24% identity of amino acid residues denoted with (*) and about 30.10% difference with about 20.59% strongly similar (:) and about 13.07%) (.) weakly similar amino acid residues.
  • the insertion site referred to herein as "D3I-ol," in a loop of domain 3 of S.
  • typhimurium FljB for fusion to a Dengue viral envelope protein antigen (domain III) is between predicted ⁇ -strands VTLA (SEQ ID NO: 109), which is amino acid residues 263-266 of (SEQ ID NO: 2), and VVS, which is amino acid residues 293-295 of (SEQ ID NO: 2).
  • the insertion site, referred to herein as "D3I-il,” in a loop of domain 3 of flagellin is between ⁇ -strands EVNVA (SEQ ID NO: 110), which corresponds to amino acid residues 254- 258 of (SEQ ID NO: 2), and VTLA (SEQ ID NO: 109), which corresponds to amino acid residues 263-266 of (SEQ ID NO: 2).
  • the insertion site in a loop of domain 3 of flagellin, is between ⁇ -strands KYFVTIGG (SEQ ID NO: 111), which corresponds to amino acid residues 234-241 of SEQ ID NO: 2, and EVNVA (SEQ ID NO: 110), which corresponds to amino acid residues 254-258 of SEQ ID NO: 2 in FljB S. typhimurium, (SEQ ID NO: 2)).
  • the D3I-ol, D3I-il and D3I-sl insertion sites predicted for S. typhimurium FljB, (SEQ ID NO: 2) were loop conformations in the predicted secondary structures of E. coli (SEQ ID NO: 3) and P.
  • aeruginosa (SEQ ID NO: 4) flagellin. Therefore, with reference to the loops in domain 3 of flagellin (SEQ ID NO: 2), predicted loops in domain 3 and insertion sites, such as D3I-il and D3I-ol, can be selected for E. coli (SEQ ID NO: 3) a d P. aeruginosa (SEQ ID NO: 4) flagellin.
  • Insertion sites in loops in domain 3 for fusion to Dengue viral envelope protein antigens (domain III) in E. coli FliC flagellin can include an D3I-ol site between G274 and A275 of (SEQ ID NO: 3) in the loop between ⁇ -strand ITF (amino acid residues 267-269 of (SEQ ID NO: 3) and ⁇ -strand VLTANI (SEQ ID NO: 112), amino acid residues 284-289 of (SEQ ID NO: 3).
  • the D3I-il site is between A257 and D258 of SEQ ID NO: 3 in the loop between ⁇ -strand ELAKLAIKL (SEQ ID NO: 113), amino acid residues 248- 256 of (SEQ ID NO: 3) and IEYK (SEQ ID NO: 114), amino acid residues 262-265 of (SEQ ID NO: 3).
  • the D3I-sl site was selected between S240 and G241 of (SEQ ID NO: 3) in the loop between ⁇ -strand KV (residues 238-239 of (SEQ ID NO: 3)) and ⁇ -strand SID (residues 243-245 of (SEQ ID NO: 3)).
  • the D3I-ol site was selected between Q272 and D273 in the loop between ⁇ -strand TV SLA (SEQ ID NO: 115), amino acid residues 262-266 of (SEQ ID NO: 4) and ⁇ -strand LGITASI (SEQ ID NO: 1 16), amino acid residues 285-291 of (SEQ ID NO: 4).
  • the D3I-il site was between S260 and N261 of (SEQ ID NO: 4) in the loop between ⁇ -strand SLNFDVTVG (SEQ ID NO: 117), amino acid residues 251-259 of (SEQ ID NO: 4) and TVSLA (SEQ ID NO: 118), amino acid residues 262-266 of (SEQ ID NO: 4).
  • the D3I-sl site was selected between S245 and G246 of (SEQ ID NO: 4) in the loop between ⁇ -strand TVFT (SEQ ID NO: 119), residues 238-241 of (SEQ ID NO: 4) and ⁇ -strand GVT (residues 246-248 of (SEQ ID NO: 4)).
  • the point of fusion between the flagellin component and Dengue viral envelope protein antigen (also referred to herein as "Dengue antigen") components of fusion proteins of the invention results in a sequence of unique amino acids.
  • Dengue antigen also referred to herein as "Dengue antigen”
  • fusion proteins employed in methods of the invention to treat humans if this unique sequence of amino acids at the juncture of the fusion of the flagellin component and the Dengue viral envelope protein antigen shares homology with a known human protein, the fusion protein has the potential to elicit an unwanted immune response to the human protein or a portion of the human protein.
  • the sequence of unique amino acids that would be created by fusion of the Dengue viral envelope protein antigen (domain III) to flagellin is assessed for its potential ability to elicit an unwanted immune response.
  • a probe of about 10 to about 12 amino acids in length, which includes the flagellin/Dengue antigen point of fusion is used to probe a database of known human genome sequences. If homology is identified for a stretch of amino acids greater than about 5 amino acids, then the point of fusion sequence is modified with an amino acid substitution of, for example, 1, 2, 3, 4, 5, or 6 amino acids to decrease the homology.
  • the order of preferred amino acids for use in the modification is serine, threonine, alanine and glycine. Generally, a single amino acid substitution is sufficient to modify the homology.
  • the site of insertion of a Dengue viral envelope protein antigen (domain III) in a loop of domain 3 of flagellin can be 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, amino acids towards the carboxy-domain 2 or an amino-domain 2 of flagellin from the insertion sites.
  • a loop of domain 3 to which the Dengue antigen is fused is between amino acid residues 277 and 278 (e.g., D3I-ol) (FIG. 2).
  • fusion of the antigen can occur between amino acid 266 and 282 of (SEQ ID NO: 2).
  • a loop of domain 3 to which the antigen is fused is between amino acid residues 259 to 260 (D3I-il) or amino acid residues 260 to 261.
  • the insertion site in a loop of domain 3 of flagellin in the D3I-il fusion protein of (SEQ ID NO: 2) is between amino acid residues 190 to 191 or between amino acid residues 291 to 292.
  • about 2 to about 4 amino acid residues in the loop of domain 3 can be deleted prior to fusion with the Dengue viral envelope protein antigen (domain III).
  • domain III Dengue viral envelope protein antigen
  • the flagellin for use in the fusion proteins is a flagellin that includes at least one member selected from the group consisting of (SEQ ID NOS: 1-7).
  • the antigen is fused between amino acid residue 191 and amino acid residue 285 of (SEQ ID NO: 1), which is within a loop of domain 3 of flagellin.
  • At least a portion refers to a Dengue viral envelope protein antigen, flagellin or stem region of a Dengue viral envelope protein that is less than or the entirety of the Dengue viral envelope protein antigen, flagellin or stem region of the Dengue viral envelope protein.
  • a portion refers to any part of the Dengue viral envelope protein antigen that is less than the entirety of the Dengue viral envelope protein antigen.
  • Fusion proteins of the invention can include an amino acid linker between at least one of an amino-terminus or a carboxy -terminus of the Dengue antigen and the loop of domain 3 of the flagellin or the amino-terminus of flagellin.
  • the linker such as (SEQ ID NOS: 97-105) is between the carboxy -terminal amino acid of domain I in the domains I/II antigen and the amino-terminus of flagellin.
  • the linker such as (SEQ ID NOS: 97-105) is between the carboxy -terminal amino acid of the domain III Dengue antigen and a loop of domain 3 of flagellin.
  • fusion proteins of the invention include two linkers, specifically, one linker, such as (SEQ ID NOS: 97-105), between the carboxy -terminal amino acid of domain I in the domains I/II antigen and the amino-terminus of flagellin and another linker, such as (SEQ ID NOS: 97-105), between the carboxy-terminal amino acid of the domain III Dengue antigen and a loop of domain 3 of flagellin.
  • linker such as (SEQ ID NOS: 97-105)
  • the linker is between the carboxy-terminal amino acid of the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin, or the carboxy-terminal amino acid of the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin, or the carboxy-terminal amino acid of the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin, or the carboxy-terminal amino acid of the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
  • the fusion proteins of the composition of the invention include a linker, such as an amino acid linker, between the carboxy-terminal amino acid of the domain I of the Dengue 1 viral antigen and the amino-terminal amino acid of the first flagellin, or the carboxy-terminal amino acid of the domain I of the Dengue 2 viral antigen and the amino- terminal amino acid of the second flagellin, or the carboxy-terminal amino acid of the domain I of the Dengue 3 viral antigen and the amino-terminal amino acid of the third flagellin, or the carboxy-terminal amino acid of domain I of the Dengue 4 viral antigen and the amino-terminal amino acid of the fourth flagellin.
  • a linker such as an amino acid linker
  • the fusion proteins of the invention include a linker, such as an amino acid linker, between the carboxy-terminal amino acid of the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin, the carboxy-terminal amino acid of the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin, the carboxy-terminal amino acid of the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin and the carboxy-terminal amino acid of the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin in combination with a linker between the carboxy-terminal amino acid of the domain I of the Dengue 1 viral antigen and the amino-terminal amino acid of the first flagellin, the carboxy- terminal amino acid of the domain I of the Dengue 2 viral antigen and the amino-terminal amino acid of the second flagellin, the carboxy-terminal amino acid of the domain I of the Den
  • the amino acid linker can be between about 2 to about 20 amino acids in length, such as about 2, about 4, about 6, about 8 or about 10 amino acids in length.
  • Preferred amino acid residues would include amino acid residues without side chains or amino acid residues with small side chains, such as glycine, alanine or serine, including combinations of glycine, serine and alanine.
  • Amino acid linkers are devoid of secondary structures, such as a-helices and B- sheets. Amino acid linkers that include amino acids without side chains and devoid of secondary structures are referred to herein as "flexible linkers.”
  • amino acid linkers can include 2, 4 or 6 negatively charged amino acid residues, such as aspartic acid or glutamic acid. Exemplary amino acid linkers are described, for example, in PCT/US2012/000099 (WO
  • Exemplary amino acid residues are unit repeats of glycine and serine residues, such as GSGS (SEQ ID NO: 97), GSGSGS (SEQ ID NO: 98), GSGSGSGS (SEQ ID NO: 99), GSGSGSGSGSGS (SEQ ID NO: 100), GSGSGSGSGSGSGS (SEQ ID NO: 101), GSGSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 102), GSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 103), GSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 104),
  • the linker can include amino acid residues that are native to the naturally occurring Dengue viral envelope protein.
  • the linker that includes amino acid residues that are native to the naturally occurring Dengue viral envelope protein are in a linking region of the Dengue viral envelope protein.
  • a "linking region,” as used herein, means an amino acid residue or peptide that is adjacent to domain III and outside a stem region of a naturally occurring ectodomain of Dengue viral envelope protein.
  • the linking region can be 4 amino acids in length, or 5 amino acids in length, or 6 amino acids in length or 7 amino acids in length or 8 amino acids in length or 9 amino acids in length or 10 amino acids in length.
  • linking regions examples include SSIGK (SEQ ID NO: 106) for Dengue 1, or SSIGQ (SEQ ID NO: 107) for Dengue 2, or SSIGK (SEQ ID NO: 106) for Dengue 3, or SSIGK (SEQ ID NO: 107) for Dengue 4.
  • a linking region can be fused to the carboxy-terminus of domain III of the Dengue antigen that is fused to a loop of domain 3 of flagellin. Fusion proteins that include domains I, II and III of the Dengue envelope protein antigens without a linking region and without at least a portion of a stem region are referred to herein as "80E" fusion proteins that include "80E” Dengue antigens (FIG. 4C), such as SEQ ID NO: 41.
  • Fusion proteins that include an 80E Dengue antigen and a linking region, such as SEQ ID NOS: 93, 94, 95 and 96, are referred to herein as "80E+" fusion proteins that include "80E+” Dengue antigens (FIG. 4C), such as SEQ ID NO: 93.
  • a linking region of the Dengue antigen is indicated by an arrow and, with reference to SEQ ID NO: 89, for example, is 5 amino acid residues in length.
  • Fusion proteins that include (i) domains I and II and (ii) domain III that includes a linking region and at least a portion of a stem region of a Dengue viral envelope protein antigen, are referred to herein as "85E” fusion proteins that include "85E” Dengue antigens (FIG. 4C), such as SEQ ID NO: 89.
  • the 80E, 80E+ and 85E Dengue antigens can be fused to flagellin in split formats, N-term format and C-term format.
  • Split format as used herein with reference to a fusion protein that includes a Dengue antigen (80E, 80E+, 85E) and a flagellin, means that domains I and II of the Dengue viral envelope protein are fused to the amino-terminus of flagellin and the domain III of the Dengue viral envelope protein (with or without a linking region in 80E and 80E+ formats, respectively, and with a linking region and at least a portion of a stem region in 85E format) is fused to at least one loop of domain 3 of the same flagellin.
  • the ectodomain has been "split" into domains I/II and III for fusion to two distinct sites (amino-terminus and loop of domain 3, respectively) of flagellin, as depicted in FIGs. 70- 72.
  • Exemplary split format fusion proteins include SEQ ID NOs: 11, 17, 22, 25, 26 and 27-40.
  • N-term fusion format (also referred to herein as "N-term format), as used herein with reference to a fusion protein that includes a Dengue antigen (80E, 80E+, 85E+) and a flagellin, means that domains I, II and III (with or without a linking region in 80E and 80E+ formats, respectively, and with a linking region and at least a portion of a stem region in 85E format) are collectively fused to the flagellin at one site of the flagellin, specifically the amino- terminus of the flagellin, as depicted in FIGs. 70-72.
  • N-term formats of fusion proteins include SEQ ID NOs: 8, 14, 15, 19, 20 and 21.
  • C-term fusion format (also referred to herein as "C-term format), as used herein with reference to a fusion protein that includes a Dengue antigen (80E, 80E+, 85E+) and a flagellin, means that domains I, II and III (with or without a linking region in 80E and 80E+ formats, respectively, and with a linking region and at least a portion of a stem region in 85E format) are collectively fused to the flagellin at one site of the flagellin, specifically the carboxy- terminus of the flagellin.
  • the fusion proteins of the invention can further include at least one junction loop peptide of a Dengue virus between at least one member selected from the group consisting of the domain I of the Dengue 1 viral antigen and the amino-terminus of the first flagellin; the domain I of the Dengue 2 viral antigen and the amino-terminus of the second flagellin; the domain I of the Dengue 3 viral antigen and the amino-terminus of the third flagellin; the domain I of the Dengue 4 viral antigen and the amino-terminus of the fourth flagellin; the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin; the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin; the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin; and the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
  • a junction loop peptide is between each domain I of each of the four Dengue serotype antigens (Denl, Den2, Den3 and Den4) and the amino terminus of each flagellin component of the four fusion proteins in combination with a junction loop peptide between domain III of each of the four Dengue serotype antigens (Denl, Den2, Den3 and Den4) and the loop of the domain 3 of each flagellin component of the four fusion proteins, wherein the domain III of each of the four Dengue serotype antigens is fused to the junction loop peptide at the carboxy -terminal amino acid residue, such as exemplary fusion proteins as set forth in SEQ ID NOS: 27-38.
  • junction loop refers to a single strand domain boundary between the EIEII domains and the EIII domain.
  • a junction loop is located at about amino acid position 295 for DENVl, 2 and 4 of SEQ ID NOs: 93, 94 and 96 or amino acid position 293 for DENV3 of SEQ ID NO: 95.
  • the junction loop is essentially in the middle of an about 7 amino acid peptide, such as DKLTLKG (SEQ ID NO: 55), depicted as a dashed circle in FIG. 4B and shown in grey highlighted text in FIGs. 49-60).
  • Exemplary overlapping 7 amino acid residues for Dengue 1 are DKLTLKG (SEQ ID NO: 55), Dengue 2 are DKLQLKG (SEQ ID NO: 59), Dengue 3 are DKLELKG (SEQ ID NO: 63), Dengue 4 are EKLRTKG (SEQ ID NO: 67).
  • At least one junction loop peptide can be fused to at least one member selected from the group consisting of the carboxy-terminal amino acid of domain I in the combined EIEII domain of the Dengue antigen for fusion to the amino-terminus of flagellin, and the amino- terminal amino acid of domain III of the Dengue antigen for fusion to a loop of domain 3 of flagellin.
  • a composition of the fusion proteins of the invention are more immunogenic when the EIEII domain of the Dengue antigens is fused to the amino-terminus of flagellin with the junction loop in combination with a flexible linker, such as an amino acid linker of repeat units of glycine and serine (GS n ), in combination with fusion of a junction loop to the amino-terminal amino acid of the EIII domain of the Dengue viral envelope protein antigen (domain III) fused to a loop of domain 3 of flagellin and a flexible linker, such as an amino acid linker of repeat units of glycine and serine (GSn).
  • a flexible linker such as an amino acid linker of repeat units of glycine and serine
  • compositions and methods of the invention can further include at least one Toll-like Receptor (TLR) agonist selected from the group consisting of a Toll-like Receptor 1 agonist, a Toll-like Receptor 2 agonist, a Toll-like Receptor 3 agonist, a Toll-like Receptor 4 agonist, a Toll-like Receptor 5 agonist, a Toll-like Receptor 6 agonist, a Toll-like Receptor 7 agonist, a Toll-like Receptor 8 agonist, a Toll-like Receptor 9 agonist, a Toll-like Receptor 10 agonist, a Toll-like Receptor 11 agonist, a T Toll-like Receptor 12 agonist and a Toll-like Receptorl3 agonist.
  • TLR Toll-like Receptor
  • the Toll-like Receptor agonist included in the composition is a TLR 4 agonist, such as a lipopolysaccharide, or
  • the TLR agonist is at least one member selected from the group consisting of a TLR 3 agonist, a TLR 7 agonist and a TLR 8 agonist.
  • a TLR signaling pathway is an intracellular signal transduction pathway employed by a particular TLR that can be activated by a TLR ligand or a TLR agonist.
  • Common intracellular pathways are employed by TLRs and include, for example, NF- ⁇ , Jun N- terminal kinase and mitogen-activated protein kinase.
  • the Toll-like Receptor agonist in the compositions and methods described herein can include at least one member selected from the group consisting of a TLR1 agonist, a TLR2 agonist (e.g., Pam3Cys, Pam2Cys, bacterial lipoprotein), a TLR3 agonist (e.g., dsRNA), a TLR4 agonist (e.g., bacterial lipopolysaccharide), a TLR5 agonist (e.g., a flagellin), a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist (e.g., unmethylated DNA motifs), TLR10 agonist, a TLR11 agonist and a TLR12 agonist.
  • a TLR1 agonist e.g., Pam3Cys, Pam2Cys, bacterial lipoprotein
  • TLR3 agonist e.g., dsRNA
  • TLR4 agonist
  • the invention is a method of stimulating an immune response in a subject by administering a composition comprising at least four fusion proteins, each of which activates a Toll-like Receptor 5, and includes flagellin and Dengue viral antigens.
  • the first fusion protein of the composition includes a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin.
  • the second fusion protein of the composition includes a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin.
  • the third fusion protein of the composition includes a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin.
  • the fourth fusion protein of the composition includes a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin; and a domain I and a domain II of the Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
  • the methods of the invention can further include the administration of an adjuvant.
  • the adjuvant is a saponin (see, for example, Skene, CD., et al, Methods 40:53-59 (2006) and Rajput, Z.I., et al. J. Zhejiang Univ. Sci. 8(3): 153-161 (2007).
  • Stimulating an immune response refers to the generation of antibodies and/or T-cells to the Dengue viral envelope protein antigen fused to flagellin in the fusion proteins described herein. Stimulating an immune response in a subject can include the production of humoral and/or cellular immune responses that are reactive against the Dengue antigen. Stimulation of an immune response in a subject by administration of the compositions and fusion proteins described herein may provide protective immunity to disease or illness consequent to exposure to the Dengue virus. Fusion proteins of the invention may neutralize Dengue virus, which can be assessed employing well-known techniques, such as those described herein.
  • Neutralization of the Dengue virus in in vitro assays is believed to correlate with the presence of antibodies to the Dengue antigens in the fusion proteins that neutralize the Dengue virus for use in compositions to treat a subject, including use as a vaccine composition to provide protective immunity against disease consequent to Dengue infection.
  • compositions of the invention for use in methods to stimulate immune responses in subjects can be evaluated for the ability to stimulate an immune response in a subject using well-established methods.
  • Exemplary methods to determine whether the compositions of the invention stimulate an immune response in a subject include measuring the production of antibodies specific to the Dengue antigen (e.g., IgG antibodies) by a suitable technique such as, ELISA assays; the potential to induce antibody-dependent enhancement (ADE) of a secondary infection; macrophage-like assays; neutralization assessed by using the Plaque Reduction Neutralization Test (PRNT 50 ); and the ability to generate serum antibodies in non-human models (e.g., mice, rabbits, monkeys) (Messer, et al., PLOS Negl. Trop. Des. 6:el486 (2012)).
  • compositions of the invention that include a fusion protein comprising Dengue antigens as described herein that result in production of antibodies to the protein to thereby cause a subject to be essentially disease-free (not develop viremia) by an otherwise challenge dose of a viral protein or neutralize the Dengue virus or ameliorate disease and illness consequent to exposure to the Dengue virus.
  • Techniques to determine an optimum challenge dose of the Dengue virus are known to one of skill in the art (see, for example, Putnak, et al., J. Inf. Des. 174: 1176-1184 (1996)).
  • Exemplary techniques for determining an optimum challenge dose can include administration of varying doses of virus and a determination of the percent of subjects that do not develop viremia following administration of various challenge doses of virus.
  • Assessment of stimulation of protective immunity can also be made by employing assays that assess the ability of the antibodies produced in response to the fusion proteins to neutralize binding of the Dengue virus to a host cell. It is believed that inhibition of Dengue virus infectivity is indicative of the ability of antibodies, formed from the compositions and by the methods of the invention, to neutralize the binding sites of the naturally Dengue virus ("neutralization of Dengue virus") and, thereby, prevent infection of the host cell as a
  • Inhibition or neutralization of the Dengue viruses is believed to correlate with an ability of an immune response to protect against a Dengue virus or infection, or disease from the Dengue virus.
  • Fusion proteins of the invention can be generated recombinantly or by chemical conjugation using well-established techniques.
  • Chemical conjugation can include conjugation by a reactive group, such as a thiol group (e.g., a cysteine residue) or by derivatization of a primary (e.g., an amino-terminal) or secondary (e.g., lysine) group.
  • a recombinant fusion protein can be generated by operably linking a nucleic acid sequence encoding a flagellin, or a portion of a flagellin that includes a domain 3, to a nucleic acid sequence encoding a Dengue antigen.
  • fusion proteins of the invention can include Dengue antigens that consist essentially of domain III of a Dengue envelope protein or consist of domain III of the Dengue envelope protein fused to at least one loop, such as one, two, three, or four loops, of domain 3 of flagellin in combination with a second Dengue antigen that consists essentially of domains I and II of a Dengue envelope protein or consists of domains I and II of a second Dengue antigen fused to the amino-terminus of the flagellin.
  • Fusion proteins of the invention can be made employing routine molecular biological techniques, as described herein.
  • Host cells can be transfected with nucleic acids encoding fusion proteins of the invention.
  • the host cells can be eukaryotic or prokaryotic host cells. Suitable prokaryotic host cells include E. coli, B. subtilis and Pseudomonas fluorescens.
  • Fusion proteins made in eukaryotic host cells can include a flagellin of the fusion proteins of the invention that is modified from its corresponding native flagellin to delete at least one putative glycosylation site in the nucleic acid sequence encoding the flagellin.
  • the putative glycosylation site that is deleted can include an N-glycosylation site.
  • a flagellin that has been made recombinantly to delete at least one putative glycosylation site is referred to as "ng," for example, STF2ng.
  • at least one putative glycosylation site is mutated.
  • the "N" residue in the putative glycosylation site such as N-X-Serine or N-X-Tyrosine or N-X-Cysteine is mutated to glutamine when fusion proteins are generated recombinantly in eukaryotic host cells.
  • the "N” at amino acid residues 19, 194, 216, 293, 457 and 476 of SEQ ID NO: 108 is mutated to glutamine and the "N" at amino acid residue 101 of SEQ ID NO: 108 is mutated to aspartic acid.
  • the eukaryotic host cells employed in the methods of the invention can include a Saccharomyces eukaryotic host cell, an insect eukaryotic host cell (e.g., at least one member selected from the group consisting of a Baculovirus infected insect cell, such as Spodoptera frugiperda (Sf9) or Trichhoplusia ni (High5) cells; and a Drosophila insect cell, such as Dmel2 cells), a fungal eukaryotic host cell, a parasite eukaryotic host cell (e.g., a Leishmania tarentolae eukaryotic host cell), CHO cells, yeast cells (e.g., Pichia) and a Kluyveromyces lactis host cell.
  • Saccharomyces eukaryotic host cell e.g., at least one member selected from the group consisting of a Baculovirus infected insect cell, such as Spodoptera frugiperda (Sf9)
  • fusion proteins of the invention are made in a Drosophila eukaryotic host cell. In another embodiment, fusion proteins of the invention are made in a Baculovirus eukaryotic host cell. A fusion protein made in Drosophila is identified by the designation "DR.” A fusion protein made in Baculovirus are identified by the designation "BV.”
  • Suitable eukaryotic host cells and vectors can also include plant cells (e.g., tomato; chloroplast; mono- and dicotyledonous plant cells; Arabidopsis thaliana; Hordeum vulgare; Zea mays; potato, such as Solarium tuberosum; carrot, such as Daucus carota L ; and tobacco, such as Nicotiana tabacum, Nicotiana benthamiana (Gils, M., et al, Plant BiotechnolJ.
  • plant cells e.g., tomato; chloroplast; mono- and dicotyledonous plant cells; Arabidopsis thaliana; Hordeum vulgare; Zea mays; potato, such as Solarium tuberosum; carrot, such as Daucus carota L ; and tobacco, such as Nicotiana tabacum, Nicotiana benthamiana (Gils, M., et al, Plant BiotechnolJ.
  • the fusion proteins of the invention can be purified and characterized employing well-known methods (e.g., gel chromatography, cation exchange chromatography, SDS-PAGE), as described herein.
  • the invention includes a protein, polypeptide or peptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%), at least about 95%, at least about 98%> and at least about 99% sequence identity to the fusion proteins of the invention.
  • the length of the protein or nucleic acid encoding can be aligned for comparison purposes is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%), at least about 99% of the length of the reference sequence, for example, the nucleic acid sequence of a Dengue antigen or flagellin that includes domain 3 or fusion protein of the invention of a fusion protein.
  • the default parameters of the respective programs e.g., BLASTN; available at the Internet site for the National Center for Biotechnology Information
  • the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.
  • Another mathematical algorithm employed for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, California) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis, et al, Comput. Appl. Biosci., 10: 3-5 (1994) and FASTA described in Pearson, et al, (Proc. Natl. Acad. Sci USA, 85: 2444-2448 (1988), the teachings of which are hereby incorporated by reference in its entirety).
  • the percent identity between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, California) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
  • the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, California), using a gap weight of 50 and a length weight of 3.
  • the nucleic acid sequence encoding a Dengue envelope protein antigen (EI/EII, EII, EI/II/III) or fusion proteins of the invention and polypeptides of the invention can include nucleic acid sequences that hybridize to nucleic acid sequences or complements of nucleic acid sequences of the invention, for example, the nucleic acid sequence of a flagellin or Dengue antigen employed in the fusion proteins of the invention of a fusion protein of the invention under selective hybridization conditions (e.g., highly stringent hybridization conditions).
  • selective hybridization conditions e.g., highly stringent hybridization conditions.
  • hybridizes under high stringency or “hybridizes under very high stringency conditions,” describe conditions for hybridization and washing of the nucleic acid sequences.
  • Guidance for performing hybridization reactions which can include aqueous and nonaqueous methods, can be found in Aubusel, F.M., et al, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (2001).
  • High stringency conditions are, for example, relatively low salt and/or high temperature conditions. High stringency is provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C. High stringency conditions allow for limited numbers of mismatches between the two sequences. In order to achieve less stringent conditions, the salt concentration may be increased and/or the temperature may be decreased.
  • Medium stringency conditions are achieved at a salt concentration of about 0.1 to 0.25 M NaCl and a temperature of about 37°C to about 55°C, while low stringency conditions are achieved at a salt concentration of about 0.15 M to about 0.9 M NaCl, and a temperature ranging from about 20°C to about 55°C.
  • Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel et al, Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., Units 2.8-2.11, 3.18-3.19 and 4-64.9, (1997).
  • a "subject,” as used herein, can be a mammal, such as a primate or rodent (e.g., rat, mouse). In a particular embodiment, the subject is a human.
  • an "effective amount,” when referring to the amount of a composition and fusion protein of the invention, refers to that amount or dose of the composition and fusion protein, that, when administered to the subject is an amount sufficient for therapeutic efficacy (e.g., an amount sufficient to stimulate an immune response in the subject).
  • the compositions and fusion proteins of the invention can be administered in a single dose or in multiple doses.
  • compositions and fusion proteins of the invention can be accomplished by the administration of the compositions and fusion proteins of the invention by enteral or parenteral means.
  • the route of administration is by oral ingestion (e.g., drink, tablet, capsule form) or intramuscular injection of the composition and fusion protein.
  • Other routes of administration as also encompassed by the present invention including intravenous, intradermal, intraarterial, intraperitoneal, or subcutaneous routes, and nasal administration. Suppositories or transdermal patches can also be employed.
  • compositions and fusion proteins of the invention can be administered ex vivo to a subject's autologous dendritic cells. Following exposure of the dendritic cells to the composition and fusion protein of the invention, the dendritic cells can be administered to the subject.
  • compositions and fusion proteins of the invention can be administered alone or can be coadministered to the patient. Coadminstration is meant to include simultaneous or sequential administration of the composition, fusion protein or polypeptide of the invention individually or in combination. Where the composition and fusion protein are administered individually, the mode of administration can be conducted sufficiently close in time to each other (for example, administration of the composition close in time to administration of the fusion protein) so that the effects on stimulating an immune response in a subject are maximal. It is also envisioned that multiple routes of administration (e.g., intramuscular, oral, transdermal) can be used to administer the compositions and fusion proteins of the invention.
  • routes of administration e.g., intramuscular, oral, transdermal
  • compositions and fusion proteins of the invention can be administered alone or as admixtures with conventional excipients, for example, pharmaceutically, or physiologically, acceptable organic, or inorganic carrier substances suitable for enteral or parenteral application which do not deleteriously react with the extract.
  • suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrolidine.
  • compositions and fusion proteins of the invention can be administered by is oral administration, such as a drink, intramuscular or intraperitoneal injection or intranasal delivery.
  • compositions and fusion proteins alone, or when combined with an admixture can be administered in a single or in more than one dose over a period of time to confer the desired effect (e.g., alleviate prevent viral infection, to alleviate symptoms of virus infection, such as influenza or flaviviral infection).
  • compositions and fusion proteins are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories.
  • carriers for parenteral administration include aqueous solutions of dextrose, saline, pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil, polyoxyethylene-block polymers, and the like.
  • Ampules are convenient unit dosages.
  • the compositions, fusion proteins or polypeptides can also be incorporated into liposomes or administered via transdermal pumps or patches.
  • compositions and fusion proteins of the invention can be administered to a subject on a support that presents the compositions and fusion proteins of the invention to the immune system of the subject to generate an immune response in the subject.
  • the presentation of the compositions and fusion proteins of the invention would preferably include exposure of antigenic portions of the viral protein to generate antibodies.
  • the fusion proteins of the invention are in close physical proximity to one another on the support.
  • the fusion proteins of the invention can be attached to the support by covalent or noncovalent attachment.
  • the support is biocompatible. "Biocompatible,” as used herein, means that the support does not generate an immune response in the subject (e.g., the production of antibodies).
  • the support can be a biodegradable substrate carrier, such as a polymer bead or a liposome.
  • the support can further include alum or other suitable adjuvants.
  • the support can be a virus (e.g., adenovirus, poxvirus, alphavirus), bacteria (e.g., Salmonella) or a nucleic acid (e.g., plasmid DNA, CpG).
  • the dosage and frequency (single or multiple doses) administered to a subject can vary depending upon a variety of factors, including prior exposure to the Dengue virus, the duration of viral infection, prior treatment of the viral infection, the route of administration of the composition or fusion protein; size, age, sex, health, body weight, body mass index, and diet of the subject; nature and extent of symptoms of viral exposure, viral infection and the particular viral responsible for the infection (e.g., Dengue virus infection), or treatment or infection of another antigen, such as a Dengue antigen, kind of concurrent treatment, complications from the viral exposure, viral infection or exposure or other health-related problems.
  • Other therapeutic regimens or agents can be used in conjunction with the methods and compositions and fusion proteins of the present invention.
  • compositions and fusion proteins can be accompanied by other viral therapeutics or use of agents to treat the symptoms of a condition associated with or consequent to exposure to the antigen, such as influenza infection.
  • Adjustment and manipulation of established dosages are well within the ability of those skilled in the art.
  • compositions, fusion proteins and polypeptides of the invention can be administered to a subject on a presenting carrier.
  • a presenting carrier means any composition that presents the compositions, fusion proteins and polypeptides of the invention to the immune system of the subject to generate an immune response in the subject.
  • the presentation of the compositions, fusion proteins and polypeptides of the invention would preferably include exposure of antigenic portions of the viral protein to generate antibodies.
  • polypeptides of the invention are in close physical proximity to one another on the presenting carrier.
  • the compositions, fusion proteins and polypeptides of the invention can be attached to the presenting carrier by covalent or noncovalent attachment.
  • the presenting carrier is biocompatible. "Biocompatible," as used herein, means that the presenting carrier does not generate an immune response in the subject (e.g., the production of antibodies).
  • the presenting carrier can be a biodegradable substrate presenting carrier, such as a polymer bead or a liposome.
  • the presenting carrier can further include alum or other suitable adjuvants.
  • the presenting carrier can be a virus (e.g., adenovirus, poxvirus, alphavirus), bacteria (e.g., Salmonella) or a nucleic acid (e.g., plasmid DNA).
  • a virus e.g., adenovirus, poxvirus, alphavirus
  • bacteria e.g., Salmonella
  • a nucleic acid e.g., plasmid DNA
  • compositions and methods of the invention can further include a carrier.
  • Carrier refers to a molecule (e.g., protein, peptide) that can enhance stimulation of a protective immune response. Carriers can be physically attached (e.g., linked by recombinant technology, peptide synthesis, chemical conjugation or chemical reaction) to a composition (e.g., a protein portion of a naturally occurring viral hemagglutinin) or admixed with the composition.
  • a composition e.g., a protein portion of a naturally occurring viral hemagglutinin
  • Carriers for use in the methods and compositions described herein can include, for example, at least one member selected from the group consisting of Tetanus toxoid (TT), Vibrio cholerae toxoid, Diphtheria toxoid (DT), a cross-reactive mutant (CRM) of diphtheria toxoid, E. coli enterotoxin, E.
  • TT Tetanus toxoid
  • Vibrio cholerae toxoid Vibrio cholerae toxoid
  • DT Diphtheria toxoid
  • CCM cross-reactive mutant
  • LTB heat labile enterotoxin
  • TVB Tobacco mosaic virus
  • RV Tobacco mosaic virus
  • RV protein Rabies virus envelope protein
  • Thy thyroglobulin
  • HSP 60 Kda Keyhole limpet hemocyamin
  • KLH Keyhole limpet hemocyamin
  • ESAT-6 early secreted antigen tuberculosis-6
  • Flaviviruses including Dengue viruses, are small, enveloped viruses with icosahedral capsids.
  • the flavivirus genome is a single-stranded positive-sense RNA (about 11 kb) that is directly translated by the host cell machinery following infection.
  • the viral genome is translated as a single polypeptide that undergoes co- and post-translational cleavage by viral and cellular enzymes to generate three structural proteins of the flavivirus (the capsid (C), the membrane (M) and the envelope (E) proteins); and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Weaver, et al., Annu Rev Microbiol 990:44-649 (2004)).
  • the viral capsid is composed of the C-protein, while both the M- and envelope proteins are located on the envelope surface of the virion (Weaver, S.C., et al, Nat. Rev. Microbiol. 70:789-801 (2004); , Chambers et al, Annu Rev. Microbiol. 44: 649-688 (1990)).
  • the flavivirus envelope protein plays a role in virus assembly. These proteins form a protective shell around the virus, which serves as a cage for the genetic material inside, sheltering the virus until it is released inside a host cell. While simple viruses consist of only a protein shell and genetic information, more complex viruses, such as flaviviruses, also contain a lipid bilayer between the protein shell and viral genome. A flavivirus can enter a host cell when the viral envelope protein binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host- cell membranes.
  • the envelope of a flavivirus may function as a receptor binding protein and to facilitate fusion of the virus and host cell membrane.
  • the envelope protein is a determinant of host range, cell tropism, virulence and elicits neutralizing antibodies during the immune response (Roehrig, Adv Virus Res 59: 141-175 (2003)).
  • the envelope protein is responsible for fusing the virus and host membranes (Chu, et al, J. Virol 75:10543-10555
  • envelope proteins form homodimers on the outer surface of the virus particles (Rey, et al, Nature 375:291-298); Kuhn, et al, Cell 108:111-125
  • Each envelope protein monomer folds into three structural domains (domains I, II and III) predominantly composed of ⁇ -strands.
  • Domain I (also referred to herein as “I” or “DI”) is centrally located in the structure and has an N-glycosylation site in glycosylated envelope proteins.
  • Domain II (also referred to herein as “II” or “DII”) of the envelope protein promotes dimerization and has a fusion loop that inserts into the target host membrane during the pH-dependent fusion of the virus (Modis, et al, Nature ⁇ 27:313-319 (2004); Bressanelli, et al, EMBO J 23:728-738 (2004)).
  • Domain III (also referred to herein as “III” or “Dili”) is at the carboxy-terminus of the envelope protein. Domain III is also referred to as "domain B” in earlier antigenic mapping studies. Domain III has several epitopes that can elicit virus-neutralizing antibodies (Roehrig, Adv Virus Res 59:141-175
  • Domain I of the Dengue 2 flavivirus envelope protein corresponds to amino acids 1-52, 132-193 and 280-296 of (SEQ ID NO: 42); domain II corresponds to amino acids 53-131 and 194-279 of (SEQ ID NO: 42); and domain III corresponds to amino acids 297-495 of (SEQ ID NO: 42) (Modis, Y., et al, Nature ⁇ 27:313-319 (2004)).
  • the location of domains I, II and III of other flavivirus e.g., West Nile vims, Japanese encephalitis, Dengue 1 virus, Dengue 3 virus and Dengue 4 virus
  • the location of domains I, II and III of other flavivirus is based on homology of the Tick-borne encephalitis envelope protein domains and the Dengue 2 envelope protein domains.
  • flavivirus proteins in particular, flaviviruses other than Tick-borne encephalitis flavivirus envelope proteins and Dengue 2 flavivirus envelope proteins, are based on homology to domains in the Tick-borne encephalitis flavivirus envelope protein and the Dengue 2 flavivirus envelope protein.
  • the domain III of the envelope protein of the DEN flavivirus encodes the majority of the flavivirus type-specific contiguous critical/dominant neutralizing epitopes (Roehring, J.T., Adv. Virus Res. 59: 141 (2003)), including the four DEN (DEN1, DEN2, DEN3, DEN4) viruses. Flavivirus envelope proteins are highly homologous. Exemplary envelope protein sequences are shown in (SEQ ID NOS: 41-44 and 89-96).
  • the ectodomain of the Dengue envelope (E) protein includes three domains that have been identified immunologically (Roehrig, J.T., et al, Virology 5:246(2):317-328 (1998)) and by X-ray crystallographically (Modis, Y., et al, Nature 22:427(6972):313-319 (2004), Modis, Y., et al, J. Virol. 76(24): 13097-13100 (2002)).
  • Domain I also referred to as "EI”
  • domain II also referred to as " ⁇ " is the dimerization domain and contains the fusion peptide.
  • Domain III (also referred to as " ⁇ ") is about 100 amino acids in length and contains the putative receptor-binding and the majority of the type-specific neutralizing epitopes (Modis, Y., et al, Nature 22:427(6972):313-319 (2004), Modis, Y., et al, J. Virol.
  • Elll-reactive antibodies produced by mice immunized with virus and boosted with recombinant E protein are largely serotype-specific and do not neutralize all of the genotypes within a given serotype (Shrestha B., et al, PLoS Pathog Apr;6(4):el 000823 (2010)).
  • the role of antibodies to EI/EII is less clear, as they tend to be more cross-reactive and less potent in neutralization (Goncalvez A.P., et al, J Virol 2004 Dec;78(23): 12910-8 (2004)).
  • the synthetic genes encoding the Dengue envelope protein (80E, 80E+ or 85E) were codon-optimized for Baculovirus expression (DNA2.0 Inc., Menlo Park, CA) and cloned into pAcSG2TM (BD Biosciences) vector.
  • Either the entire 80E+ (Dengue 1, 2 and 4, 1-399, SEQ ID NOS: 93, 94, 96; Dengue 3, 1-397 SEQ ID NO: 95) was used to fuse to the N-terminus of full- length sequence of Salmonella typhimurium fljB (flagellin phase 2, STF2), or the "split format" was employed in which EIEII (Dengue 1, 2 and 4, 1-296 SEQ ID NO: 93, 94, 96; Dengue 3, 1- 294, SEQ ID NO: 95) was fused to the amino-terminus of flagellin and EIII (Dengue 1, 2 and 4, 290-399 of SEQ ID NOS: 93, 94, 96 and Dengue 3, 288-398, SEQ ID NO: 95) was fused to a loop of domain 3 of flagellin.
  • EIEII Dengue 1, 2 and 4, 1-296 SEQ ID NO: 93, 94, 96; Dengue 3, 1- 294, SEQ ID NO:
  • the EIEII domain was linked to the N-terminus of flagellin and remaining EIII domain was inserted in domain 3 of flagellin.
  • the EIEII and EIII domains contain an overlapping 7 amino acids.
  • Flexible linkers (GS) 5 (GSGSGSGSGS (SEQ ID NO: 100) was utilized to connect 80% E to flagellin and (GS) 3 was used at the C-terminus of EIII domain.
  • the recombinant baculoviruses were generated by co-transfecting Sf9 cells with the recombinant plasmids and BD BaculoGoldTM DNA following standard Baculovirus Expression protocol (Invitrogen, Carlsbard, CA).
  • the envelope sequences used in the vaccine constructs are: Dengue 1, PU0359 (Genbank accession code: AAN32784); Dengue 2, Thailand 16681, (Genbank accession code: NP 739583); Dengue 3, Pah881/88, (Genbank accession code:
  • the secreted recombinant vaccine candidates were captured by an affinity column.
  • the envelope protein (E) specific antibody 4G2 (ATCC, Manassas, VA) was coupled to the NHS-activated Sepharose 4 Fast Flow Resin (GE) following the manufacture's protocol.
  • the column was washed and equilibrated.
  • the loaded fusion proteins were captured and eluded.
  • the protein was further purified by size exclusion chromatography (10/24 GL, GE).
  • the final formulation buffer is PBS.
  • mice Female BALB/c mice (6-8 weeks old) were purchased from Charles River Laboratories (Wilmington, Massachusetts) and maintained at the AAALAC-accredited animal facility of Princeton University (Princeton New Jersey). Fusion proteins were formulated in DPBS (2.67 mM KC1, 1.57 mM KH2P04, 137.93 NaCl, & 8.06 mM Na2HP04.7H20, pH 7.2) prior to animal studies. Groups of 5-8 mice were immunized subcutaneously (s.c.) on days 0, 21, and 42, and bled on days 56 or 63. Serum samples were stored at -70°C until use.
  • Virus neutralizing antibodies of immune sera were measured by 50% focus reduction neutralization test (FRNT50) following a procedure similar to that previously described (Liu, G, et al., Clinical Vaccine and Immunology, 2015, 22 (5): 516-525). Briefly, sera were heat inactivated at 56 °C for 30 min, serially 2-fold diluted, and subsequently co-incubated with 30-60 PFU of DENV at 37 °C for 1 hr. The mixtures were added to Vero cells in 24-well or 96-well plates and incubated for 1 hr and subsequently incubated in 1% methylcellulose/EMEM containing 2% FBS and antibiotics.
  • FRNT50 50% focus reduction neutralization test
  • FRNT50 test was performed in 96-well plates (micro- FRNT50) for mouse sera. Virus only and medium only controls were included with each dilution series. After 2-5 day incubation at 37 °C, the monolayers on the plates were fixed and blocked in I-Block solution. Infection foci were reacted with flavivirus group specific monoclonal antibody (4G2) and HRP-conjugated goat anti-mouse IgG, and visualized with True Blue substrate. Foci were counted and FRNT50 titers were calculated by Probit analysis using BioStat 2009 software (AnalystSoft Inc.). If the sample was negative for the first dilution (1 : 10 or 1 :20), the FRNT50 titer was assigned one-half (1 ⁇ 2) of the first dilution (5 or 10).
  • EXAMPLE 1 Split format was selected for DENV1 based on comparison of immunogenicity and production yield. [00205] The immunogenicity of DENVl fusion proteins in Split (BV270,
  • BV270 which is based on the E sequence of strain 16007, was produced at an extremely low yield
  • another Split DENVl fusion protein based on strain PU0359 (BV293) of DENVl was generated.
  • BV293 was expressed and purified at a higher yield compared to BV270.
  • BV27B and BV293B are similarly immunogenic in mice.
  • B V293B can be produced at a higher yield. Therefore, BV293B (Split format) was selected for further development of a composition that included DENVl to stimulate an immune response, in particular a protective immune response.
  • EXAMPLE 2 DENV2 fusion proteins in split format (BV239) elicited superior neutralizing antibody titers compared to other fusion proteins in N-term 80E candidate (BV237), D3Ins 80E (BV242) or alternative Split (BV241) formats.
  • BV241 (alternative spilt format" fuses domain III to the amino- terminus of flagellin and domains I and II to a loop of domain 3 of flagellin,
  • EIII.STF2ng2D3.EIEH EIII.STF2ng2D3.EIEH
  • BV237 N-term 80E, E395.3GS.STF2ng2
  • BV242 D3Ins 80E, STF2ng2D3. E395
  • BV239 elicited robust neutralizing antibodies in a dose-dependent manner with a FRNT 50 titer of 1940 at 10 ⁇ g.
  • DENV2 fusion proteins in other formats, including an N-terminal, 85E+ and alternative split formats did not induce significant neutralizing titers. Therefore, the split format was selected as for use in a fusion protein that included DENV2.
  • EXAMPLE 3 DENV2 fusion proteins in split format with two linkers (BV263) elicited superior neutralizing antibody titers compared to those without linker (BV239) or with a linker (BV257).
  • the immunogenicity of DENV2 Split fusion proteins of BV239 (no linker,
  • EIEII.STF2ng2D3.EIII BV257 (one amino acid GS n linker between the carboxy -terminal amino acid of domain II of the dengue antigen and the amino-terminal amino acid of flagellin, EIEII.5GS.STF2ng2D3.EIII) and BV263 (EIEII.5GS.STF2ng2D3.EIII.3GS, a (GS) n linker at the carboxy -terminal amino acid of domain II of DenV2 and the amino-terminal amino acid of flagellin and the amino-terminal amino acid of domain III of DENV2 where fused to an amino acid of a loop of domain 3 of flagellin was compared in mice. As shown in FIG.
  • BV263 elicited the highest neutralizing antibody titers in a dose-dependent manner with a FRNT 50 titer of 2032 at 5 ⁇ g. A significant titer was also seen at the 1 ⁇ g dose level. Therefore, split fusion proteins with two amino acid linkers, such as a fusion protein of BV263, were selected for fusion proteins that include DENV2 antigens and flagellin.
  • the amino acid linkers are units of glycine and serine (GS) n .
  • One of the amino acid linkers is at the carboxy-terminal amino acid of domain II of DenV2 and the amino-terminal amino acid of flagellin and the other amino acid linker is at the amino-terminal amino acid of domain III of DENV2 where fused to an amino acid of a loop of domain 3 of flagellin.
  • This format is referred herein as "2 linkers.”
  • EXAMPLE 4 Two DENV3 fusion proteins in either Split (with 2 linkers, BV269B) or N-term 80E+ (BV272B) format elicited moderate neutralizing antibody titers.
  • BV272B (EIEII.5GS.STF2ng2D3.EIII.3GS) and BV272B (E398.STF2ng2) at 1-9 ⁇ g was compared in mice.
  • FIG. 65 both elicited robust neutralizing antibody titers at a dose as low as 1 ⁇ g, BV272B may be more immunogenic as evidenced by about 2.3 to about 3.4 fold higher titers, when delivered as a monovalent vaccine.
  • production of BV272B is more difficult than BV269B.
  • the latter is also highly immunogenic in a TDV formulation (Table 1). Therefore, the Split fusion protein B V269B was selected for the further development of TDV formulations.
  • EXAMPLE 5 Two DENV4 fusion proteins in either split (with 2 linkers, BV316) or N-term 80E+ (BV243B) format elicited moderate neutralizing antibody titers.
  • mice EIEII.5GS.STF2ng2D3.EIII.3GS
  • BV243B E399.STF2ng2
  • both fusion proteins elicited robust neutralizing antibody titers, ranging from about 20 to about 202 for BV243B and about 45 to about 254 for BV316, respectively. Although the trend of titers was slightly higher for BV316, the difference is not significant. Both fusion proteins appear to be similarly immunogenic when delivered as a monovalent composition.
  • TDV Three tetravalent dengue (TDV) compositions each consisting of 4 fusion proteins elicited moderate to high levels of neutralizing antibodies.
  • mice The immunogenicity of three tetravalent formulations was examined in mice. Groups of 6-8 BALB/c mice were immunized S.C. with the indicated TDV formulations listed in Table 1 or monovalent fusion proteins on days 0, 21, and 42. Sera were collected on day 56 and subjected to FRNT50 test and expressed as GMTs. As shown in Table 1, all three TDV formulations elicited significant levels of neutralizing antibodies. A suitable TDV formulation is expected to induce balanced neutralizing antibody titers against the 4 dengue serotypes to minimize the possibility of causing a potential ADE. Among the three formulations, TDV1 appears to induce more balanced titers to all 4 serotypes (about 320 to about 1660). Therefore, a TDV formulations consisting of 4 Split fusion proteins was further evaluated in a non-human primate animal model described below.
  • EXAMPLE 7 Two tetravalent dengue vaccine (TDV) formulations reduced the mean days of viremia by 2 days in non-human primates.
  • mice Consistent with low neutralizing antibody titers, animals showed breakthrough viremia indicating that the TDV formulations may need to be altered or augmented to provide sufficient protection against viremia.
  • the mean day of viremia in the TDV96 and TDV36 groups was reduced by 2 days (FIG. 67). It is possible that the decreased potency may be a consequence of current recombinant production methods that result in suboptimal folding of the antigen or the flagellin that reduces TLR5 activation by the flagellin and decreased antigen presentation.
  • a tetravalent dengue composition which includes 80E+ immunogens of 4 DENV serotypes fused to flagellin, may potentially serve as an improved subunit dengue vaccine.
  • the target populations include adults and children living in the endemic countries as well as travelers and military personnel.
  • Example 8 Fusion proteins induced cytokine IL-6 in mice following primary immunization.
  • mice Groups of five mice were immunized with the indicated doses of fusion proteins (FIGs. 68 and 69). Sera were harvested 2-3 hours later and evaluated for IL-6 production. Mice receiving the buffer PBS alone were included as negative controls. Geometric mean IL-6 levels (pg/ml of sera) are shown above each data set. The serotype of Dengue construct evaluated is provided at the top of each dataset. The construct designation (e.g. BV 270B or BV263B is provided either on the X axis (FIG. 68) or below the serotyped designation (FIG. 69). The production lot evaluated (e.g. R007 or R003) is provided in FIG. 69.
  • the construct designation e.g. BV 270B or BV263B is provided either on the X axis (FIG. 68) or below the serotyped designation (FIG. 69).
  • the production lot evaluated e.g. R007 or R003 is provided in FIG. 69
  • Fusion proteins that include DENV1 , DENV2, DENV3 and DENV4 antigens induced significant levels of IL-6 levels (FIGs. 68 and 69). Therefore, the flagellin/dengue antigen fusion proteins can activate TLR5 pathway signaling and are TLR5 agonists.

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Abstract

Compositions include fusion proteins of four serotypes of the Dengue viral envelope protein. Domains I and II of the Dengue viral envelope proteins are fused to the amino-terminus of flagellin and domain III of the Dengue viral envelope protein is fused to at least one loop of domain 3 of flagellin. Compositions can further include adjuvants and Toll-like Receptor agonists. The compositions can be employed in methods of stimulating an immune response, in particular an immune response that ameliorates illness or disease consequent to exposure to a Dengue virus.

Description

FUSION PROTEINS THAT INCLUDE DENGUE ANTIGENS AND METHODS OF USE
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 62/207,635, filed on August 20, 2015. The entire teachings of the above application are incorporated herein by reference.
INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE
[0002] This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:
a) File name: 37101064001 seqlisting.txt; created August 18, 2016, 391 KB in size.
GOVERNMENT SUPPORT
[0003] This invention was made with government support under 5R01 All 05206 from NIH Institute: National Institute of Allergy and Infectious Diseases (NIAID). The government has certain rights in the invention.
BACKGROUND
[0004] Dengue infection is a major public health problem in tropical areas of the world. About 2.5 billion people are at risk for Dengue infection and about 50 million cases of dengue infection and hundreds of thousands of cases of dengue hemorrhagic fever (DHF) occur in the tropics each year, including Mexico, the Caribbean and parts of Asia and the South Pacific (Gubler, D.J., Ann Acad Med Singapore 27: 227-34 (1998); Guzman, M.G., et al, Nat. Rev. Microbiol. S/S7-S16 (2010)). Dengue viruses are transmitted by peridomestic Aedes spp.
mosquitoes, which inhabit the tropics, allowing endemicity of Dengue Fever (DF) in these areas.
[0005] Dengue (DEN), a flavivirus, is transmitted by the bite of a mosquito infected with a Dengue virus. Based on the analysis of the envelope protein of the Dengue virus there are at least four serologically and genetically distinct viruses termed Dengue 1 (also referred to as "Denl"), Dengue 2 (also referred to as "Den2"), Dengue 3 (also referred to as "Den3") and Dengue 4 (also referred to as "Den3"). In 2013 a fifth serotype was reported (Dengue 5) (Normile, D., Science 342(6157):415 (2013)).
[0006] Infection by one virus serotype causes DF, a febrile illness, which is not normally life-threatening, and leads to life-long protective immunity against the infecting DEN
serotype/virus. However, individuals that are infected by one serotype/virus remain susceptible to infection by the other three DEN serotypes/viruses. Subsequent infection by one of the other DEN serotypes/viruses can lead to Dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), which are life-threatening diseases (Murphy, B.R., et al., Annu Rev Immunol 29:587-619 (2011)).
[0007] DHF may be the result of an antibody dependent enhancement (ADE) where non- neutralizing antibodies induced by the primary DEN infection form virus-antibody complexes in secondary infections that are ingested by macrophages through Fc receptors and, thus, enhance virus infection (Kliks, S.C., et al, Am. J. Trop. Med. Hyg. ¥0:444-451 (1989)). DHF occurs primarily in children and can be fatal. Human antibodies to non-neutralizing epitopes in the envelope (E) protein and the membrane (M) protein of Dengue viruses are thought to be highly cross reactive and may cause the ADE (Dejnirattisai, W., et al, Science 7:328(5979):745-748 (2010)).
[0008] Compositions that employ tetravalent live Dengue flaviviruses to prevent Dengue infection may be limiting due to interference and imbalanced immune response among the serotypes. Compositions that include inactivated flavivirus may result in limited
immunogenicity and the need for multiple doses, which is difficult in topical areas. Recently, a recombinant dengue vaccine (DENGVAXIA®) has been approved for use in humans age 9-45. However, the immunization regimen is lengthy, requiring immunizations at 0, 6 and 12 months, and is suboptimal in young children not previously exposed (i.e., naive) to the dengue virus (Hadinegoro, et al, N. Engl. J. Med. 373 : 1195-1206 (2015)). Thus, in view of the complexity and the disease burden of Dengue viral infection, there is a need to develop new compositions for use in the prevention and treatment of disease in subjects consequent to Dengue infection.
SUMMARY OF THE INVENTION
[0009] The present invention relates to compositions and fusion proteins that include Dengue antigens and flagellin. The compositions and fusion proteins can be employed in methods to stimulate immune responses that ameliorates or diminishes disease or illness consequent to infection by the Dengue virus. The immune response stimulated can be a protective immune response.
[0010] In an embodiment, the invention is a composition comprising at least four fusion proteins, each of which activates a Toll-like Receptor 5, and includes flagellin and Dengue viral antigens. The first fusion protein of the composition includes a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin. The second fusion protein of the composition includes a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin. The third fusion protein of the composition includes a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin. The fourth fusion protein of the composition includes a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin; and a domain I and a domain II of the Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
[0011] In another embodiment, the invention is a method of stimulating an immune response in a subject by administering a composition comprising at least four fusion proteins, each of which activates a Toll-like Receptor 5, and includes flagellin and Dengue viral antigens. The first fusion protein of the composition includes a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin. The second fusion protein of the composition includes a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin. The third fusion protein of the composition includes a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin. The fourth fusion protein of the composition includes a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin; and a domain I and a domain II of the Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
[0012] The compositions and methods described herein have advantages over current methods of preventing Dengue infection. For example, the compositions include at least four fusion proteins of Dengue antigens from the four serotypes fused to flagellin in a similar configuration that can be readily produced recombinantly, thereby optimizing manufacturing and access to compositions for preventing Dengue infection that can be readily available for use in relatively short-term immunization procotols.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 depicts loops in domain 3 for S. typhimurium FliC (SEQ ID NO: 1) based on a known crystal structure and predicted loops in S. typhimurium FljB (SEQ ID NO: 2).
[0014] FIG. 2 depicts predicted insertion sites in domains 0, 1, 2 and 3 of S. typhimurium FljB (SEQ ID NO: 2) for fusion with antigens, including insertion sites in a loop of domain 3. Domain 0 is predicted at amino acid residues 1-46 and amino acid residues 465-506; Domain 1 is predicted at amino acid residues 47-176 and amino acid residues 415-464; Domain 2 is predicted at amino acid residues 177-190 and amino acid residues 292-414; and Domain 3 is predicted at amino acid residues 191-291. The amino acid number of boundaries between domains 0, 1, 2 and 3 and insertion sites are also indicated.
[0015] FIG. 3 depicts the structure of flagellin, domains 0, 1, 2 and 3 and insertion sites in flagellin (SEQ ID NO: 2) for generation of fusion proteins that include antigens. Reference to amino acid numbers are made to (SEQ ID NO: 2).
[0016] FIG. 4A depicts the Dengue envelope ectodomain homodimer (Zhang et al., Nat. Struct. Biol. 70(11):907-912 (2003)). The three domains of the Dengue envelope protein, EI (dark grey), EII (light grey) and EIII (black) are indicated. A stem region connects the stably folded ectodomain portion of the envelope protein with the C-terminal transmembrane (TM) anchor region of the envelope protein.
[0017] FIG. 4B depicts the conformation of E (envelope protein) in the mature virus particle and in solution above the fusion pH. The junction between the EI (Domain I of the Dengue viral envelope protein) and EII (Domain II of the Dengue viral envelope protein) domains is structurally complex and includes several peptide strands, depicted by the circle. The linking region extending the C-terminus of EIII to form 80E+ is indicated by arrows.
[0018] FIG. 4C generally depicts the linear conformation of 80E, 80E+ and 85E Dengue envelope protein antigen components of the fusion proteins of the invention. Domain I (EI), domain II (EII), domain III (EIII), junction loop (JL), linking region (LR), portion of a stem region (ST). In an embodiment, amino acid residues of the envelope protein are with reference to SEQ ID NO: 89.
[0019] FIG. 5 depicts the amino acid sequence of the fusion protein BV179, E395.STF2ng2 (Dengue 1 strain 16007). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 8)
[0020] FIG. 6 depicts the amino acid sequence of the fusion protein BV251, STF2ng2 D3.E395 (Dengue 1 strain 16007). 80E D3Ins format. Dotted line, MT leading sequence;
Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 9)
[0021] FIG. 7 depicts the amino acid sequence of the fusion protein BV255,
E395.3GS.STF2ng2 (Dengue 1 strain 16007). 80E N-term format with linker. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag; Wavy underline, flexible linker. (SEQ ID NO: 10) [0022] FIG. 8 depicts the amino acid sequence of the fusion protein BV262, PRM16/E400.STF2ng2 (Dengue 1 strain 16007). 80E+ N-term format. PRM16 is a leading sequence from PrM and 80E+(l-400) is a longer version of 80E. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO:
11)
[0023] FIG. 9 depicts the amino acid sequence of the fusion protein BV265, STF2ng2 D3.E400 (Dengue 1 strain 16007). 80E+ D3Ins format. 80E+(L,l-400, SEQ ID NO: 93) includes amino acids residues of the 80E Dengue protein of SEQ ID NO: 41. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 12)
[0024] FIG. 10 depicts the amino acid sequence of the fusion protein VI 54
STF2ng2.3GS.E395 (Dengue 2 strain 16681). 80E C-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 13)
[0025] FIG. 11 depicts the amino acid sequence of the fusion protein BV155 E395.STF2ng2 (Dengue 2 strain 16681). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 14)
[0026] FIG. 12 depicts the amino acid sequence of the fusion protein BV237 E395.STF2ng2 with linker (Dengue 2 strain 16681). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 15)
[0027] FIG. 13 depicts the amino acid sequence of the fusion protein BV242
STF2ng2D3Ins.E394 (a.a. 2-395 of SEQ ID NO: 42, Dengue 2 strain 16681. 80E D3Ins format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 16)
[0028] FIG. 14 depicts the amino acid sequence of the fusion protein BV244 E399 (a.a. 2- 400 of SEQ ID NO: 94).STF2ng2 (Dengue 2 strain 16681). 80E+ N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 17)
[0029] FIG. 15 depicts the amino acid sequence of the fusion protein BV163
STF2ng2.3GS.E393 (Dengue 3 strain Nicaragua 24/94). 80E C-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 18)
[0030] FIG. 16 depicts the amino acid sequence of the fusion protein BV170 E393.STF2ng2 (Dengue 3 strain Nicaragua 24/94). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 19)
[0031] FIG. 17 depicts the amino acid sequence of the fusion protein BV219 E393.STF2ng2 (Dengue 3 strain Pah881/88). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 20)
[0032] FIG. 18 depicts the amino acid sequence of the fusion protein BV256
E393.3GS.STF2ng2 (Dengue 3 strain Pah881/88). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag; Wavy underline, flexible linker. SEQ ID NO: 21
[0033] FIG. 19 depicts the amino acid sequence of the fusion protein BV272 E398.STF2ng2 (Dengue 3 strain Pah881/88). 80E+ N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 22)
[0034] FIG. 20 depicts the amino acid sequence of the fusion protein BV164 STF2ng2.3GS. E395 (Dengue 4 strain 341750 (TVP360)). 80E C-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 23)
[0035] FIG. 21 depicts the amino acid sequence of the fusion protein BV171
80E395.STF2ng2 (Dengue 4 strain 341750 (TVP360)). 80E N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 24) [0036] FIG. 22 depicts the amino acid sequence of the fusion protein BV243 E399.STF2ng2 (a.a.. 2-400 of SEQ ID NO: 96, Dengue 4 strain 341750 (TVP360)). 80E+ N-term format.
Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 25)
[0037] FIG. 23 depicts the amino acid sequence of the fusion protein B V267
PRM16.E400.STF2ng2 (Dengue 4 strain 341750 (TVP360)). 80E+ N-term format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 26)
[0038] FIG. 24 depicts the amino acid sequence of the fusion protein BV261
EIEII.STF2ng2D3.EIII (Dengue 1 strain 16007). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 27)
[0039] FIG. 25 depicts the amino acid sequence of the fusion protein BV258
EIEII.STF2ng2.5GS.D3.EIII (Dengue 1 strain 16007). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 28)
[0040] FIG. 26 depicts the amino acid sequence of the fusion protein B V270
EIEII.5.GS.STF2ng2D3.EIII.3GS (Dengue 1 strain 16007). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 29)
[0041] FIG. 27 depicts the amino acid sequence of the fusion protein BV239
EIEII.STF2ng2.D3.EIII (Dengue 2 strain 16681). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 30)
[0042] FIG. 28 depicts the amino acid sequence of the fusion protein 8 BV263
EIEII.5GS.STF2ng2D3.EIII.3GS (Dengue 2 strain 16681). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 31) [0043] FIG. 29 depicts the amino acid sequence of the fusion protein BV241 EIII.STF2ng2D3.EIEH (Dengue 2 strain 16681). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with
glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 32)
[0044] FIG. 30 depicts the amino acid sequence of the fusion protein BV264
EIEII.STF2ng2D3..EIII (Dengue 3 strain Pah881/88). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 33)
[0045] FIG. 31 depicts the amino acid sequence of the fusion protein BV268
EIEII.5GS.STF2ng2D3.EIII (Dengue 3 strain Pah881/88). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Wavy underline, flexible linker. (SEQ ID NO: 34)
[0046] FIG. 32 depicts the amino acid sequence of the fusion protein BV269
EIEII.5GS.STF2ng2D3.EIII.3GS (Dengue 3 strain Pah881/88). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 35)
[0047] FIG. 33 depicts the amino acid sequence of the fusion protein BV227
EIEII.STF2ng2D3.EIII (Dengue 4 strain 341750 (TVP360)). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Grey highlight, junction loop; Underline, 6XHis tag. (SEQ ID NO: 36)
[0048] FIG. 34 depicts the amino acid sequence of the fusion protein BV254
EIEII.5GC.STF2ng2R3.EIII (Dengue 4 strain 341750 (TVP360)). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 37)
[0049] FIG. 35 depicts the amino acid sequence of the fusion protein BV257
EIEII.5GS.STF2ng2. D3.EIII (Den2)— from BV239. 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 38) [0050] FIG. 36 depicts the amino acid sequence of the fusion protein BV316 EIEII.5GS.STF2ng2D3.EIII.3GS(Den4). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Wavy underline, flexible linker. (SEQ ID NO: 39)
[0051] FIG. 37 depicts the amino acid sequence of the fusion protein BV293B EIEII.
5GS.STF2. D3.EIII.3GS (Denl PUO). 80E+ split format. Dotted line, MT leading sequence; Double underline, Dengue Envelope protein; No underline, flagellin with glycosylation site knock out; Underline, 6XHis tag; Grey highlight, junction loop; Wavy underline, flexible linker. (SEQ ID NO: 40)
[0052] FIG. 38 depicts the amino acid sequence of Dengue 1 NH 16007 80E: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 41)
[0053] FIG. 39 depicts the amino acid sequence of Dengue 2 NH 16681 80E: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 42)
[0054] FIG. 40 depicts the amino acid sequence of Dengue 3 Pah881/88 80E: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 43)
[0055] FIG. 41 depicts the amino acid sequence of DEN4 341750 80E: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 44)
[0056] FIG. 42 depicts the amino acid sequence of DEN1 Hawaii 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 45)
[0057] FIG. 43 depicts the amino acid sequence of DEN2 Thailand 16681 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 46)
[0058] FIG. 44 depicts the amino acid sequence of DEN2 New Guinea C 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 47)
[0059] FIG. 45 depicts the amino acid sequence of depicts the amino acid sequence of DEN3 CH53489 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 48) [0060] FIG. 46 depicts the amino acid sequence of DEN3 Nicaragua 24/94 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted.
(SEQ ID NO: 49)
[0061] FIG. 47 depicts the amino acid sequence of DEN4 H241 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted. (SEQ ID NO: 50)
[0062] FIG. 48 depicts the amino acid sequence of DEN4 61NIID 80E+: EI, single underlined; EII, double underlined; EIII, wavy underlined. Junction loop, grey highlighted.
(SEQ ID NO: 51)
[0063] FIG. 49 depicts the amino acid sequence of EI (Dengue 1, NH 16007): Junction loop, grey highlighted (SEQ ID NO: 52)
[0064] FIG. 50 depicts the amino acid sequence of EI (Dengue 2, NH 16681): Junction loop, grey highlighted (SEQ ID NO: 56)
[0065] FIG. 51 depicts the amino acid sequence of EI (Dengue 3, Pah881/88): Junction loop, grey highlighted (SEQ ID NO: 60)
[0066] FIG. 52 depicts the amino acid sequence of EI (Dengue 4, 341750): Junction loop, grey highlighted (SEQ ID NO: 64)
[0067] FIG. 53 depicts the amino acid sequence of EI (Dengue 1, Hawaii): Junction loop, grey highlighted (SEQ ID NO: 68)
[0068] FIG. 54 depicts the amino acid sequence of EI (Dengue 2, Thailand 16681): Junction loop, grey highlighted (SEQ ID NO: 71)
[0069] FIG. 55 depicts the amino acid sequence of EI (Dengue 2, New Guinea C): Junction loop, grey highlighted (SEQ ID NO: 74)
[0070] FIG. 56 depicts the amino acid sequence of EI (Dengue 3, CH53489): Junction loop, grey highlighted (SEQ ID NO: 77)
[0071] FIG. 57 depicts the amino acid sequence of EI (Dengue 3, Nicaragua 24/94): Junction loop, grey highlighted (SEQ ID NO: 80)
[0072] FIG. 58 depicts the amino acid sequence of EI (Dengue 4, H241): Junction loop, grey highlighted (SEQ ID NO: 83)
[0073] FIG. 59 depicts the amino acid sequence of EI (Dengue 4, 61NIID): Junction loop, grey highlighted (SEQ ID NO: 86)
[0074] FIG. 60 depicts the amino acid sequence of 80 E+ (Dengue 1 NH 16007) Junction loop, grey highlighted (SEQ ID NO: 93)
[0075] FIG. 61 depicts immunogenicity of monovalent DENV1 fusion proteins in various formats in mice. Groups of 5 BALB/c mice were immunized S.C. with 8 μg of BV270 (Split, EIEII.5GS.STF2ng2D3.EIII.3GS), BV255 (N-term 80E fusion, E399.3GS.STF2ng2), or BV271 (N-term 85E, E426.STF2ng2) on days 0, 21, and 42. Sera were prepared on days 56 and subjected to FRNT test against strain West Pac 74. Data are shown as FRNT50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
[0076] FIG. 62 depicts immunogenicity of monovalent DENV1 fusion protein in split formats, BV270B (strain 16007) and BV293 (strain PU0359) in mice. Groups of 8 BALB/c mice were immunized S.C. with the indicated doses of BV270B or BV293B on days 0, 21, and 42. Sera were prepared on days 56 and subjected to FRNT test against strain West Pac 74. Data are shown as FRNT50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
[0077] FIG. 63 depicts immunogenicity of monovalent DENV2 fusion proteins of different formats in mice. Groups of 5 BALB/c mice were immunized S,C, with the 1 μg or 10 μg of BV237, BV239, BV241, or BV242 on days 0, 21, and 42. Sera were prepared on days 63 and subjected to FRNT test against strain SI 6803. Data are shown as FRNT50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
[0078] FIG. 64 depicts immunogenicity of monovalent DENV2 in Split format with no linker, one linker and two linkers in mice. Groups of 6 BALB/c mice were immunized S.C. with indicated doses of BV239, BV257, or BV263 on days 0, 21, and 42. Sera were prepared on days 56 and subjected to FRNT test against strain SI 6803. Data are shown as FRNT50 titers of individual mice with geometric mean titers (GMT) in numbers.
[0079] FIG. 65 depicts immunogenicity of monovalent DENV3 fusion proteins in split (BV269B) and N-term (BV272B) formats in mice. Groups of 5 BALB/c mice were immunized S.C. with indicated doses of BV269B or BV272B on days 0, 14, and 28. Sera were prepared on days 42 and subjected to FRNT test against strain CH54389. Data are shown as FRNT50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
[0080] FIG. 66 depicts immunogenicity of monovalent DENV4 fusion proteins in split (BV316) and N-term (BV243B) formats in mice. Groups of 6 BALB/c mice were immunized S.C. with indicated doses of BV316 or BV243B on days 0, 14, and 28. Sera were prepared on days 42 and subjected to FRNT test against strain TVP 360. Data are shown as FRNT50 titers of individual mice with geometric mean titers (GMT) in numbers above groups.
[0081] FIG. 67 depicts the efficacy of TDV formulations in the DENV2/NHP model.
Groups of 4 rhesus macaques were immunized I.M. with three TDV formulations on days 0, 28, and 56, and challenged with DENV2 virus (104 PFU of S16803, S.C.) on day 117. Sera were collected on days 117-131 and subjected to plaque assay. Data are shown as viremic days of individual animals with mean days of viremia in numbers above groups. *, p < 0.05 in Fisher's exact test.
[0082] FIGs. 68 and 69 depict cytokine (IL-6) induction in mice following immunization with flagellin/Dengue viral envelope protein antigen fusion proteins.
[0083] FIG. 70 generally depicts 80E Dengue protein antigens fused to flagellin in split format and N-terminal fusion formats. Junction loop (JL). In an embodiment, amino acid residues of the envelope protein in a fusion protein are with reference to SEQ ID NO: 41.
[0084] FIG. 71 generally depicts 80E+ Dengue protein antigens fused to flagellin in split format and N-terminal fusion formats. Junction loop (JL), linking region (LR). In an embodiment, amino acid residues of the envelope protein in a fusion protein are with reference to SEQ ID NO: 93.
[0085] FIG. 72 generally depicts 85E Dengue protein antigens fused to flagellin in split format and N-terminal fusion formats. Junction loop (JL), linking region (LR), portion of the stem region (ST). In an embodiment, amino acid residues of the envelope protein in a fusion protein are with reference to SEQ ID NO: 89.
DETAILED DESCRIPTION OF THE INVENTION
[0086] The features and other details of the invention, either as steps of the invention or as combinations of parts of the invention, will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention.
[0087] In an embodiment, the invention is a composition comprising at least four fusion proteins, each of which activates a Toll-like Receptor 5, and includes flagellin and Dengue viral antigens. The first fusion protein of the composition includes a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin. The second fusion protein of the composition includes a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin. The third fusion protein of the composition includes a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin. The fourth fusion protein of the composition includes a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin; and a domain I and a domain II of the Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
[0088] "Fusion protein," as used herein, refers to a protein generated from at least two distinct components, a flagellin or a portion of a flagellin and a Dengue antigen or a portion of a Dengue antigen. The flagellin and Dengue antigen can be linked by covalently or noncovalently.
[0089] Fusion proteins of the invention can be designated by components of the fusion proteins separated by a "." or For example, "EIEII.STF2ng2.D3Ins-EIII" refers to a fusion protein of a fljB/STF2 flagellin that has been altered to delete at least one putative glycosylation sites ("ng") (STF2ng2, for example SEQ ID NO: 108) fused at its amino-terminal amino acid to domains I and II ("ΕΙΕΠ") of the envelope protein of a Dengue antigen and fusion of domain III of the envelope protein of the Dengue antigen ("ΕΠΙ") to a loop of domain 3 of flagellin
("D3Ins").
[0090] The flagellin in the fusion proteins of the invention can be an S. typhimurium flagellin (UniProt accession number P06179 or P52616), such as a S. typhimurium flagellin selected from the group consisting of (SEQ ID NOS: 1 and 2); an E. coli flagellin (UniProt accession number A0PCV8), such as, for example, (SEQ ID NO: 3); a P. aeruginosa flagellin (UniProt accession number P72151), such as (SEQ ID NO: 4); an Aquifex aeolicus VF5 flagellin (UniProt accession number 067803), such as (SEQ ID NO: 5); a Helicobacter pylori J99 Flagellin A (UniProt accession number P0A052), such as (SEQ ID NO: 6); and a Legionella pneumophila flagellin (UniProt accession number Q48824), such as (SEQ ID NO: 7).
[0091] The flagellin component of the first fusion protein, second fusion protein, third fusion protein and fourth fusion protein can be similar or distinct from each other. "Similar," in reference to the flagellin component of the fusion proteins of the invention, means that the flagellin of one or more of the four fusion proteins resembles one or more of the other fusion proteins. For example, the first and the second fusion proteins can include a S. typhimurium flagellin, which would be similar flagellin components of the fusion proteins. "Distinct," in reference to the flagellin component of the fusion proteins of the invention, means that the flagellin of one or more of the four fusion proteins is not the same as one or more of the other fusion proteins. For example, a first protein can include a S. typhimurium flagellin and a second protein can include an E. coli flagellin, which would be distinct flagellin components of the fusion proteins.
[0092] In an embodiment, Domains I and II of the Dengue viral envelope antigen (Den 1, Den 2, Den 3, Den 4) are fused to the amino-terminus of a flagellin. The Dengue viral envelope antigen of domains I and II can be fused to the most terminal amino acid residue of the amino- terminus of flagellin. The junction between domains I and II of the Dengue viral envelope protein is structurally complex and includes several peptide strands, depicted by the circle in FIG. 4B. To form a tertiary structure, the Dengue E protein traverses the EI and EII domains multiple times before entering the EIII domain. Fusion proteins of the invention include domains I and II in a sequence that mimics the sequence of the domains in the Dengue viral envelope protein as I/II/I/II/I (see, for example, FIGs. 4A, 4C and 70-72), which is fused to the amino-terminus of flagellin, with or without a linker, as described infra.
[0093] Exemplary domains I of Den 1, Den 2, Den 3 and Den 4 are SEQ ID NOS: 52, 56, 60 and 64, domains II of Den 1, Den 2, Den 3 and Den 4 are SEQ ID NOS: 53, 57, 61 and 65.
[0094] The phrase "a loop of domain 3 of flagellin," as used herein, refers to a stretch of amino acids within domain 3 of flagellin that is, itself, devoid of secondary structures (e.g., B- sheets, a-helices), yet flanks adjacent stretches of amino acids in domain 3 that include secondary structures, such as B-sheets, a-helices. Loops of domain 3 in flagellin can be about 2, about 3, about 4, about 5, about 6, about 7 and between about 5 to about 30 amino acids in length.
[0095] Flagellin (FljB) from Salmonella typhimurium is depicted in (SEQ ID NO: 2).
Domain 3 of Salmonella typhimurium flagellin is between amino acid residue 191 and amino acid residue 291 of (SEQ ID NO: 2). Flagellin from E. coli (UniProt accession number
A0PCV8) is depicted in (SEQ ID NO: 3). Domain 3 of £ coli flagellin of (SEQ ID NO: 3) is predicted between amino acid residue 191 and amino acid residue 283 of (SEQ ID NO. 3). P. aeruginosa flagellin (UniProt accession number P72151) is depicted in (SEQ ID NO: 4) with domain 3 predicted. Flagellin from Aquifex aeolicus VF5 (UniProt accession number 067803) is depicted in (SEQ ID NO: 5). Domain 3 of Aquifex aeolicus flagellin is predicted between amino acid residue 197 and amino acid residue 302 of (SEQ ID NO. 5). The flagellin A from
Helicobacter pylori J99 (UniProt accession number P0A052) is depicted in SEQ ID NO: 6. Domain 3 of Helicobacter pylori J99 of (SEQ ID NO: 6) is predicted between amino acid residue 189 and amino acid residue 283 of (SEQ ID NO: 6). The flagellin from Legionella pneumophila (UniProt accession number Q48824) is depicted in (SEQ ID NO: 7). Domain 3 of Legionella pneumophila flagellin of (SEQ ID NO: 7) is predicted between amino acid residue 189 and amino acid residue 283 of (SEQ ID NO: 7).
[0096] X-ray crystallography of Salmonella typhimurium FliC flagellin (SEQ ID NO: 1) shows that domain 3 of flagellin includes 6 loops (FIG. 1). The loops in domain 3 of Salmonella typhimurium flagellin (SEQ ID NO: 1) are from amino acid residues 211 to 212 (loop 1); amino acid residues 217 to 219 (loop 2); amino acid residues 223 to 229 (loop 3); amino acid residues 237 to 242 (loop 4); amino acid residues 250 to 255 (loop 5) and amino acid residues 259 to 275 (loop 6). [0097] FIG. 1 identifies the predicted loops (gray) in domain 3 of Salmonella typhimurium FljB flagellin (SEQ ID NO: 2) and compares the location in the sequence to the loops in domain 3 of Salmonella typhimurium FliC flagellin (SEQ ID NO: 1). Darkly shaded arrows depict secondary structures in domain 3 of S. typhimurium FliC, such as β-sheets and a-helices.
[0098] FIG. 2 depicts the amino acid sequence of Salmonella typhimurium FljB flagellin (SEQ ID NO: 2), boundaries of domain 0, 1, 2 and 3, and potential sites of fusion of Dengue viral antigens in loops of domain 3 of the flagellin. In an embodiment, the site of fusion of a Dengue viral envelope antigen (domain III) is D3I-il (also referred to as "D3Ins-il") between amino acid residues 259 and 260 of (SEQ ID NO: 2) in loop 5 of domain 3 (see FIG. 1). In another embodiment, the site of fusion of a Dengue envelope viral antigen (domain III) is D3I-ol (also referred to as "D3Ins-ol") between amino acid residues 277 and 278 of (SEQ ID NO: 2) in loop 6 of domain 3 (see FIG. 1).
[0099] The Dengue viral envelope antigen (domain III) can be fused to domain 3 of flagellin to generate constructs referred to as D3I-ol and D3I-il . "D3I-ol," as used herein, refers to insertion into a loop of domain 3 (Domain 3 Insertion) on the outer (o) or concave surface of flagellin, such as loop 6 of (SEQ ID NO: 2) or (SEQ ID NO: 1). "D3I-il," as used herein, refers to a loop of domain 3 on the inner (i) or convex surface of flagellin, such as loop 3 of (SEQ ID NO: 2) and (SEQ ID NO: 1). The tertiary structure of flagellin and portions of flagellin that would be considered concave and convex surfaces of flagellin are described, for example, by Samatey, et al, Nature ¥70:331-337 (2001).
[00100] The designation "c," with respect to an insertion of an antigen into a loop of domain 3 of flagellin (D3I-cl), refers to a "side-way" portion of domain 3 (FIG. 3).
[00101] The designation "s," with respect to an insertion of an antigen into a loop of domain 3 of flagellin (D3I-sl), means the "tip" of domain 3 of flagellin (FIG. 3).
[00102] The designation "D3Ins," as used herein, means insertion of a Dengue antigen (domain III of the Dengue envelope protein) into a loop of domain 3 of flagellin.
[00103] Insertion of a Dengue antigen into a loop of domain 3 of flagellin refers to fusion of the Dengue antigen to the loop of domain 3 of flagellin by formation of a peptide bond.
[00104] Exemplary fusion proteins of the invention are SEQ ID NOS: 93, 94, 95 and 96. Exemplary Domain III of Den 1 (SEQ ID NO: 54), Den2 (SEQ ID NO: 58), Den3 (SEQ ID NO: 62), and Den4 (SEQ ID NO: 66) can be fused to at least one loop of domain 3 of flagellin.
[00105] Fusion of the Dengue viral envelope antigen (domain III) to a loop of domain 3 of flagellin essentially retains domain 3 of flagellin in its tertiary structure. The phrase "essentially retains domain 3 of flagellin in its tertiary structure," as used herein, refers to maintenance of the tertiary structure of domain 3 of flagellin, which can be assessed by well-established in vivo and in vitro assays described herein that are known to one of ordinary skill in the art, including the ability of flagellin to activate TLR5 and to assess protective immunity.
[00106] The crystal structure of domain 3 of Salmonella typhimurium flagellin (FliC, Protein Data Bank ID (PDB): lUCU) has been reported (Yonekura, K., et al, Nature, 424: 643-650 (2003)) and includes the identification of 6 loops in domain 3 of (SEQ ID NO: 1). Secondary structure prediction of Salmonella typhimurium flagellin FliC using PUD is depicted below and, consistent with the known tertiary structure, predicts 6 loops in domain 3. Designations below are AA: Primary amino acid sequence; PROF sec: Secondary structure prediction where "H" stands for oc-Helix and "E" stands for β-strand (Rost, B., et al., Proteins, 19: 55-72 (1994)). The predicted loops in domain 3 of (SEQ ID NO: 1) are indicated in boxes below. The secondary structures adjacent to the predicted loops are essentially similar to the known secondary structures for Salmonella typhimurium flagellin (FliC, Protein Data Bank ID (PDB): lUCU) (Yonekura, K., etal, Nature, 424: 643-650 (2003)).
[00107] The predicted secondary structure of FliC (S. typhimurium, (SEQ ID NO: 1)) is substantially similar to the known high resolution atomic model determined by combination of X-ray crystallography and electron microscopy (lUCU, Yonekura, K., et al. Nature, 424: 643- 650 (2003)). The substantial similarity between the predicted secondary structure and the known secondary structure of S. typhimurium FliC (SEQ ID NO: 1) indicates that secondary structures predicted employing CLUSTALW and PUD adjacent to loops of domain 3 of other flagellins, including S. typhimurium FljB (SEQ ID NO: 2), can be employed to select insertion sites that correspond to known loops in domain 3 in other flagellin, such as of S. typhimurium FliC.
[00108] Secondary structure prediction using PUD of Flagellin FliC (Salmonella
typhimurium, Protein Data Bank ID: lUCU, (SEQ ID NO: 1) is depicted below. Predicted loops in domain 3 are indicated by boxed text.
.........1.........2.........3.........4.........5.........6
AA AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGL PROF sec EEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHHH
.........7.........8.........9.........10........11........12
AA TQASRNANDGISIAQTTEGALNEI NNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLN
PROF sec HHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH
.........13........14........15........16........17........18
AA EIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKV PROF sec HHHHHHH EEEEE EEEEEE EEEEEEEEEE EEEEEEEEEEE
.........19........20........21... , ....22........23... , ....24
AA SDTAATVTGYADTTIALDNSTFKASATGLG|G^QKIpG^LKFpDTTGKY|YAKVTVT|GGTG|
PROF sec EEEE EEEEE EEE EEE EEEEEEEEEEE
.........25... , ....26........27 ....28........29........30
sec .........31........32........33........34........35........36
TGTASWKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSI SI TTKYTADDGTSKTAL EEEE EEEE EEE EEEE EE EEE
.........37........38........39........40........41........42
NKLGGADGKTEWSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDT EE EEEE EEE HHHHHHHHHHHHHHHHHHHH
.........43........44........45........46........47........48
LRSDLGAVQNRFNSAITNLGNTWNLTSVRSRIEDSDYATEVSNMSRAQILQQAGTSVLA HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHH
.........49...
QANQVPQNVLSLLR (SEQ ID NO: 1)
HH HHHHHHH (predicted secondary structure)
[00109] Secondary structure prediction using PHD of Flagellin FljB (S. typhimurium, (SEQ ID NO: 2). Predicted loops in domain 3 are indicated by boxed text.
.........1.........2.........3.........4.........5.........6
AA MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRI SAKDDAAGQAIANRFTANIKG
PROF_sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
.........7.........8.........9.........10........11........12
AA LTQASRNANDGI SIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
.........13........14........15........16........17........18
AA NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD PROF_sec HHHHHHHH EEEE EEEEE EEEEEEEEE EEEEEEEEEEEEE
.........19........20........21........22... , ....23... , ....24
AA VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTN|G^SVT|GGA1VKF|DADNNKY|FVTIG
PROF_sec EEE EEEEEEE EEEEE EEE EEEEE EEEEEEE
.... , ....25........26... , ....27. .28.. .29........30
AA GFT|GADAAKN|GDYEVNV1ATDGT|VTL|AAGATKTTMPAGATTKT|EVQELKDTPAWSADAKN
PROF_sec E EEEEE EEEE EEE
.........31........32........33........34........35........36
AA ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS PROF_sec EEEE EEEEEE EE EEE EEE
.........37........38........39........40........41........42
AA YTAADGTTKTAANQLGGVDGKTEWTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL PROF sec EEEEEEEEE EEEE EE HHHHHHHH
.........43........44........45........46........47........48
AA QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHH
.........49........50 .
AA QILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO : 2)
PROF_sec HHHHHHHHHHHHHH HHHHHHH (predicted secondary structure)
[00110] The predictions of loops in domain 3 employing PHD compared to known loops identified by X-ray crystallography show that the PHD computer program can be employed to predict amino acid sequences and loops for sites of insertion of antigens within domain 3 of a flagellin. [00111] Sequence alignment between S. typhimurium FliC (SEQ ID NO: 1) and S.
typhimurium FljB (SEQ ID NO: 2) was performed using the multiple sequence alignment tool CLUSTALW and secondary structure prediction by PUD with PROF sec: structure prediction where "H" stands for alpha-helix and "E" stands for beta-strand (Thompson, J.D., et al., Nucleic Acids Res.22: 4673-4680 (1994); Rost, B., et al., Proteins 19: 55-72 (1994)). The sequence alignment below showed about 74.75% identity of amino acid residues denoted with (*) and about 10.26% difference with about 7.30% strongly similar (:) and about 7.69% (.) weakly similar amino acid residues. Domain boundaries of DO, Dl, D2 and Dl are underlined differently and three D3 insertion sites were marked.
[00112] The objective of this alignment was to predict loop regions in Domain 3 of S.
typhimurium FljB (SEQ ID NO: 2) that correspond to known loop regions of Domain 3 of S. typhimurium FliC (SEQ ID NO: 1) for points of insertion of Dengue viral envelope protein antigens (domain III) to generate fusion proteins of the inventions.
[00113] Primary amino acid sequence alignment between FliC (PDB: lUCU (Yonekura, K., et al., Nature, 424: 643-650 (2003)), (SEQ ID NO: 1) and FljB (SEQ ID NO: 2) of Salmonella typhimurium indicated that the two flagellins shared highly conserved DO (domain 0) and Dl (domain 1) domains, but varied in the D2 (domain 2) and D3 (domain 3) domains. However, secondary structure prediction, using PHD (Rost, B., et al., Proteins 19: 55-72 (1994)), showed that both D2 and D3 in the FliC flagellin FliC (SEQ ID NO: 1) and FljB flagellin (SEQ ID NO: 2) of Salmonella typhimurium share similar secondary structures, despite the differences in primary amino acid sequence.
[00114] Sequence alignment and secondary structure prediction using CLUSTALW and PHD of FliC (SEQ ID NO: 1) and FljB (SEQ ID NO: 2) of & typhimurium is depicted below.
Predicted loops in domain 3 are indicated by boxed text.
10 20 30 40 50 60
I I I I I I
FliCxxO MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRI SAKDDAAGQAI NRFTANIKG
lUCU HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH
PROF_sec EEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
FlijBxl MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAG^
PROF_sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
*************************************************
70 80 90 100 110 120
I I I I I I
FliCxxO LTQASRNANDGI SIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL
lUCU HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH
PROF_sec HHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
FlijBxl LTOA^RNANDGJ^SIA^
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
************************************************************
130 140 150 160 170 180
I I I I I I FliCxxO NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYK lUCU HHHHHHHHHH EEEEEE EEEEEE E
PROF_sec HHHHHHHH EEEEE EEEEEE EEEEEEEEEE EEEEEEEEEEE
FlijBxl EIDRVSG^TOjyjGV^
PROF_sec HHHHHHHH EEEE EEEEE EEEEEEEEE EEEEEEEEEEEEE
************************************************* - **** - *
190 200 210 220 230 240
VSDTAATVTGYADT TIALDNSTFKASATGLG|GT|DQKI|DGD|LKF^)DTTGKY|YAKVT
EEEEE EEEE EEEE HHHH HHHHH EEEE EEE EEEEEE EEEE EEEEE EEE EEE EEEEEE
VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGT |G^SVT|GGA1VKF|DADNNKY|FVTIG
EEE EEEEEEE EEEEE EEE EEEEE EEEEEEE
250 260 270 280 290 300
FliCxxO -VT|GG-TGKD|GYYEVSV1DKTNGE1VTL|AGGATSPLTGGLPATAT|EDVKNVQVANADLTEAK
lUCU EEEEEE EEE EEE EEEEE HHHHH
PROF_sec EEEE E EEEEEEE EEEEEEEE EEEE
FlijBxl GFT|GADAAKN]GDYEVNV1A-TDG^TL|AAG
PROF sec EEEEE EEEE EEE
*-* *** * *-* **** ***
D3I site
310 320 330 340 350 360
AALTAAGVTGTAS WKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKD-GSISINTT
HHHHHH EE EEEEEEE EEEEEEEEEE EEEE EEE
EEE E EEE EEEE EEE EEEE
NALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTT
EEEE EEEEEE EE EEE EEE
370 380 390 400 410 420
FliCxxO KYTADDGTSKTALNKLGGADGKTEWSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENP
lUCU EEE EEEEEE EEEE HHHH HH
PROF_sec EE EEEEE EEEE EEE HHHHHHH
Flij Bxl ^ ^^^J- -^ ^^Q^^i PS^ 'YY' IDGKTYJsTASKAAGHDFKAQPELAEAAAKTTENP
PROF_sec EEEEEEEEE EEEE EE ' ' HHHHHHH
*** ***-*** *-*** *******-* **** **** **-*****-****** *****
430 440 450 460 470 480
I I I I I I
LQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSVRSRIEDSDYATEVSNMSR HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHH LOjg^gAALAOVDALR^
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHH
490 500
FliCxxO AQILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 1)
lUCU HHHHHHHHHHHHHHHHHHHHHHHH (empirical structure)
PROF sec HHHHHHHHHHHHHHH HHHHHHH (predicted secondary structure)
FlijBxl AQILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 2)
PROF sec HHHHHHHHHHHHHHH HHHHHHH (predicted secondary structure)
***************************
[00115] An insertion site in a predicted loop in domain 3 of S. typhimurium FljB, referred to herein as "D3I-ol," for fusion of a Dengue viral envelope antigen (domain III) is between amino acid residues G277 and A278 of (SEQ ID NO: 2). The insertion site D3I-ol is between predicted β-strands VTLA (SEQ ID NO: 109), which is amino acid residues 263-266 of (SEQ ID NO: 2), and VVS, which is amino acid residues 293-295 of (SEQ ID NO: 2). Another insertion site in a second predicted loop in domain 3 of S. typhimurium FljB, referred to herein as "D3I- il," is between residues T259 and D260 of (SEQ ID NO: 2). The insertion site D3I-il is between β-strands EVNVA (SEQ ID NO: 110), which corresponds to amino acid residues 254- 258 of SEQ ID NO: 2, and VTLA (SEQ ID NO: 109), which corresponds to amino acid residues 263-266 of SEQ ID NO: 2. An additional insertion site in a third predicted loop in domain 3 of S. typhimurium FljB, referred to herein as "D3I-sl," is between residue G244 and A245 of (SEQ ID NO: 2). The insertion site of D3I-sl is between β-strands KYFVTIGG (SEQ ID NO: 111), which corresponds to amino acid residues 234-241 of (SEQ ID NO: 2), and EVNVA (SEQ ID NO: 110), which corresponds to amino acid residues 254-258 of (SEQ ID NO: 2). The secondary structures adjacent to the predicted loops of S. typhimurium FliC are substantially similar to the predicted secondary structures adjacent to loops of domain 3 of S. typhimurium FljB and are depicted in FIG. 1.
[00116] In an embodiment, fusion of the Dengue viral envelope protein antigen (domain III), can be about 2 to about 10 amino acids towards the carboxy- or amino-terminus of flagellin from the designated insertion site, based on the proximity of the adjacent secondary structural elements. For example, the D3I-ol site can be from amino acid residues G268 through D289 of (SEQ ID NO: 2), which does not invade adjacent β-strands, and are predicted at VTLA (SEQ ID NO: 109) at amino acid residues 263-266 of SEQ ID NO: 2 and VVS at amino acid residues 293- 295 of (SEQ ID NO: 2). In addition, for example, the D3I-il site can only accommodate a shift of 1 or 2 amino acids towards the carboxy -terminus of flagellin of (SEQ ID NO: 2) at amino acid residues D260 or G261 of (SEQ ID NO: 2) before disrupting the neighboring β-strands, which are predicted at EVNVA (SEQ ID NO: 110) at amino acid residues 254-258 of (SEQ ID NO: 2) and VTLA (SEQ ID NO: 109) at amino acid residues 263-266 of (SEQ ID NO: 2).
[00117] Based on predicted secondary structure analysis, loops of domain 3 of E. coli FliC flagellin (SEQ ID NO: 3) and Vseudomonas aeruginosa PAOl 'flagellin (SEQ ID NO: 4) can be predicted. Based on this analysis, E. coli FliC primary amino acid sequence (SEQ ID NO: 3) was aligned with the primary amino acid sequence of Samonella typhimurium FliC (SEQ ID NO: 1). The secondary structures were assigned either directly from known structure of lUCU for Samonella typhimurium FliC. (SEQ ID NO: 1) or by prediction using PHD. The secondary structures of E. coli flagellin (SEQ ID NO: 3) or Pseudomonas aeruginosa PAOl flagellin (SEQ ID NO: 4) were assigned using the PHD program. Designations depicted below are AA: Primary amino acid sequence; PROF sec: Secondary structure prediction where "H" stands for oc-Helix and "E" stands for beta-strand (Rost, B., et al., Proteins 19: 55-72 (1994)).
[00118] Secondary structure prediction using PHD of Flagellin FliC (E. coli, (SEQ ID NO: 3)) is depicted below. Predicted loops in domain 3 are in boxed text.
.........1.........2.........3.........4.........5.........6
MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTANIKG EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
.........7.........8.........9.........10........11........12
LTQASRNANDGI SVAQTTEGALNEINNNLQRIRELSVQATNGTNSDSDLSSIQAEITQRL HHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
.........13........14........15........16........17........18
EEIDRVSEQTQFNGVKVLAENNEMKIQVGANDGETITINLAKIDAKTLGLDGFNIDGAQK HHHHHHHH EEEEE EEEEE EEEEEEEEE EEEEEEEEEE
.........19........20........21........22........23........24
ATGSDLISKFKATGTDNYDVGGKTYTWVESGAVP^ANKP^FVSA|AD|GSLTTSSDTKV|S|
EEEEEE EEEEE EEEEEEE EEEEEE EEEEEE EE
.........25........26........27........28........29........30
|GES|IDATELAKLAIKL|ADKGS|IEYKGITF[TNNTGAELDANG|KGVLTANIDGQDVQFTIDS
EEE EEEEEEEEE EEEE EEE EEEEEE EEEEEE
.........31........32........33........34........35........36
NAPTGAGATITTDTAVYKNSAGQFTTTKVENKAATLSDLDLNAAKKTGSTLWNGATYNV EEE EEEEE EEEEE EEE EEEEEEEEEEEE
.........37........38........39........40........41........42
SADGKTVTDTTPGAPKVMYLSKSEGGSPILVNEDAAKSLQSTTNPLETIDKALAKVDNLR E EEE EEEEE EEE HHHHHHHHHHHHHHHHHHHHH
.........43........44........45........46........47........48
SDLGAVQNRFDSAITNLGNTVNNLSSARSRIEDADYATEVSNMSRAQILQQAGTSVLAQA HHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHH
.........49.
NQTTQNVLSLLR (SEQ ID NO : 3)
HHHHHHH (predicted secondary structure)
[00119] Secondary structure prediction using PFID of flagellin (P. aeruginosa PAOl, (SEQ ID NO: 4)) is depicted below. Predicted loops in domain 3 are indicated by boxed text.
.........1.........2.........3.........4.........5.........6
MALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQI SNRLSNQI SG EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
.........7.........8.........9.........10........11........12
LNVATRNANDGI SLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQ HHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
.........13........14........15........16........17........18
AELTRI SDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAG HHHHHHHH EEEEE EEEE EEEEEEEEE EEE EEEEE
.........19........20........21........22... , ....23... , ....24
AA TVASVAGTATASGIASGTVNLVGGGQVK IAIAAGDSA|KA1IAEKM|DGA|I PNL|SARART|VF PROF_sec EEEEE EEEEEEEEEE EEEEE EEEEEE EEE
.... , ....25........26... , ....21........28........29........30
AA
PROF_sec E EEE EEEEEEEEE EEEEE EEEEEEE EEEEE
.........31........32........33........34........35........36
AA SATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAK WAAGTAATTTIVTGYVQLNSP PROF_sec EE EE EEEEEEEE EEEE EEEE EEEEEEEEEE
.........37........38........39........40........41........42
AA TAYSVSGTGTQASQVFGNASAAQKSSVASVDI STADGAQNAIAWDNALAAIDAQRADLG
PROF_sec EEEEE EEEE EE EE HHHHHHHHHHHHHHHHHHHHHHHHHH
.........43........44........45........46........47........48
AA AVQNRFK TIDNLTNI SENATNARSRIKDTDFAAETAALSK QVLQQAGTAILAQANQLP
PROF_sec HHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHH
.........4
AA QAVLSLLR (SEQ ID NO : 4)
PROF_sec HHHHHHH (predicted secondary structure)
[00120] The E.coli FliC primary amino acid sequence (SEQ ID NO: 3) was initially aligned with the primary amino acid sequence of Salmonella typhimurium FliC (SEQ ID NO: 1).
Sequence alignment was performed employing the multiple sequence alignment tool
CLUSTALW and secondary structure prediction by PHD with PROF sec: denoting secondary structure prediction where "H" stands for alpha-helix and "E" stands for β-strand (Thompson, J.D., et al., Nucleic Acids Res.22: 4673-4680 (1994); Rost, B., et al., Proteins 19: 55-72 (1994)). The sequence alignment showed about 53.98% identity of amino acid residues denoted with (*) and about 21.91% difference with about 12.95%> strongly similar (:) and about 11.16%) (.) weakly similar amino acid residues. The predicted secondary structure of E. coli FliC (ECFlic, (SEQ ID NO: 3) was substantially similar to that of S. typhimurium FliC (STFliC, Protein Data Bank ID: lUCU (Yonekura, K., et al., Nature 424:643-650 (2003)) , (SEQ ID NO: 1) despite differences in primary amino acid sequences.
[00121] Sequence alignment and secondary structure prediction using CLUSTALW and PHD of S. typhimurium FliC (SEQ ID NO: 1) and E. coli FliC (SEQ ID NO: 3) to select loops in domain 3 for fusion to Dengue viral envelope protein antigens (domain III) is depicted below. The predicted loops in domain 3 are indicated by boxed text.
10 20 30 40 50 60
STFlic MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRI SAKDDAAGQAIANRFTANIKG
lUCU HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH
PROF_sec EEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
ECFlic MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRI SAKDDAAGQAIANRFTANIKG
PROF_sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
*********************** - * - *********************************
70 80 90 100 110 120 LTQASRNANDGI SIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH HHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH LTQASRNANDGI SVAQTTEGALNEINNNLQRIRELSVQATNGTNSDSDLSSIQAEITQRL HHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
130 140 150 160 170 180
NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQ--QK HHHHHHHHHH EEEEEE EEEEEE
HHHHHHHH EEEEE EEEEEE EEEEEEEEEE EEEEEEEEEEE
EEIDRVSEQTQFNGVKVLAENNEMKIQVGANDGETITINLAKIDAKTLGLDGFNIDGAQK EEIDRVSEQTQFNGVKVLAENNEMKIQVGANDGETITINLAKIDAKTLGLDGFNIDGAQK HHHHHHHH EEEEE EEEEE EEEEEEEEE EEEEEEEEEE
. ****** *********** . *********** *-* . *****
190 200 210 220 230 240
YKVSDTAATVTGYADTTIALDNSTFKASAT-GLG|GT|DQKI|DGD|LKF|DDTTGKY1YAKVTVT EEEEEE EEEEEEEE HHHH HH HHH EEEE EEE EEEEEE
EEEE EEEEE EEE EEE EEEEEEEE
ATGSDLISKFKATGTDNYDVGGKTYTWVESGAVP^ANKP^FVSA|AD|GSLTTSSDTKV|S| EEEEEE EEEEE EEEEEEE EEEEEE EEEEEE EE
250 260 270 280 290 300
STFlic |G-GTGKD|GYYEVsv[bKTN-G|EVTL|AGGATSPLTGGLPATAT|EDVKNVQVANADLTEAKAA lUCU EEEEEE EEE EEE EEEEE HHHHHHH
PROF_sec EE E EEEEEEE EEEEEEEE EEEEEE
ECFlic |GES|IDATELAKLAIKL|ADKGS|IEYKGITFTNNTG—AELDANG|KGVLTANIDGQDVQFT
PROF_sec EEE EEEEEEEEE EEEE EEE EEEEEE EEEEE
* .... .. * . * . . ** * . * * ** * . . .
D3I site
310 320 330 340 350 360
LTAAGVTGTASWKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDG
HHHH EEEEEEEEE EEEEEEEEEE EEEE EEE
E EEEE EEEE EEE EEEE EE
IDSNAPTGAGATIT TDTAVYKNSAGQFTTTKVENKAATLS DLDLNAAKKTGSTL
E EEE EEEEE EEEEE EEE EEE
370 380 390 400 410 420
TSKTALNKLGGADGKTEWSIGG- -KTYAASKAEGHNFKAQPDLAEAAATTTENPLQKID EEE EEEEEE EEEE HHHH HH HHH
EEEEE EEEE EEE HHHHHHH HHH
WNGATYNVS-ADGKTVTDTTPGAPKVMYLSKSEGGSPILVNEDAAKSLQSTTNPLETID WNGATYNVS ADGKTVTDTTPGAPKVMYLSKSEGGSPILVNEDAAKSLQSTTNPLETID EEEEEEEEEE EEE EEEEE EEE HHHHHHHHHHH
430 440 450 460 470 480
AALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQ HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH KALAKVDNLRSDLGAVQNRFDSAITNLGNTVNNLSSARSRIEDADYATEVSNMSRAQILQ HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHHHHH
490 500
STFl QAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 1)
lUCU HHHHHHHHHHHHHHHHHHH (empirical structure) PROF_sec HHHHHHHHHH HHHHHHH (predicted secondary structure)
ECFlic QAGTSVLAQANQTTQNVLSLLR (SEQ ID NO : 3)
PROF_sec HHHHHHHHHH HHHHHHH (predicted secondary structure)
************ ********
[00122] Sequence alignment of Salmonella typhimurium FliC (STFlic, Protein Data Bank ID: lUCU, (SEQ ID NO: 1) and Pseudomonas aeruginosa P AO 1 flagellin (SaFlixl, (SEQ ID NO: 4)) was performed by using the multiple sequence alignment tool CLUSTALW and secondary structure prediction by PHD with PROF sec: denoting secondary structure prediction where "H" stands for alpha-helix and "E" stands for beta-strand (Thompson, J.D., et al., Nucleic Acids Res. 22: 4673-4680 (1994); Rost, B., et al., Proteins 19: 55-72 (1994)). The sequence alignment showed about 36.24% identity of amino acid residues denoted with (*) and about 30.10% difference with about 20.59% strongly similar (:) and about 13.07%) (.) weakly similar amino acid residues.
[00123] Sequence alignment and secondary structure prediction using CLUSTALW and PHD of S. typhimurium FliC (SEQ ID NO: 1) and P. aeruginosa P AO 1 flagellin (SEQ ID NO: 4) to select loops in domain 3 of the flagellin for fusion to Dengue viral envelope protein antigens (domain III) is depicted below. The predicted loops in domain 3 of flagellin are indicated by boxed text.
10 20 30 40 50 60
STFlic MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG
lUCU HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH
PROF_sec EEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
SaFlixl MALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQI SNRLSNQI SG
PROF sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
.*** ** ** *** * .**- -* *********** *-**.
70 90 100 110 120
LTQASRNANDGI SIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH HHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH LNVATRNANDGI SLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQ HHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
* - ******** - *** - **** . * ***-*-**-**** -**.
130 140 150 160 170 180
NEIDRVSGQTQFNGVKVLAQDN-TLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKY HHHHHHHHHH E EEEEE EEEEEE
HHHHHHHH EEEEE EEEEEE EEEEEEEEEE EEEEEEEEEEE
AELTRI SDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAG HHHHHHHH EEEEE EEEE EEEEEEEEE EEE EEEEE
* - * * * * * - * .***-* ***** *.
190 200 210 220 230 240
KVSDTA- -ATVTGYADTTIALDN STFKASATGLG|G^DQKI|DGD|LKF|DDTTGKY|YA
EEEEEE EEEEEEEE H HHH HHHHH EEEE EEE EEE
EEEE EEEEE EEE EEE EEE
TVASVAGTATASGIASGTVNLVGGGQVK IAlAAGDSA|KA|IAEKM|DGA|I PNL|SARART|VF EEEEE EEEEEEEEEE EEEEE EEEEEE EEE * * - * *
Prim. cons . 2V222AGTAT22G2A22T22L22GGQVK2222A2222222222K2DG2222222222222
Figure imgf000027_0001
PROF_sec
SaFlixl
PROF_sec
D3I site
310 320 330 340 350 360
STFlic KAALTAAGVTGTASWKMSYTDNNGKTIDGGLAVKVG- -DDYYSATQNKDGSISINTTKY
lUCU HHHHHHH EEEEEEEEE EEEEEEEEEE EEEE EEE
PROF_sec EEE EEEE EEEE EEE EEEE E
SaFlixl KGVLTITSATGEN VKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNWAAGTAATTT
PROF sec EEEEEEE EE EEEEEEEE EEEE EEEE E
370 380 390 400 410 420
STFlic TADDGTSKTALNKLGGADGKTEWSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQ
FliCxxO TADDGTSKTALNKLGGADGKTEWSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQ
lUCU EEE EEEEEE EEEE HHHH HHHH
PROF_sec E EEEEE EEEE EEE HHHHHHHHH
SaFlixl IVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSS VASVDISTADGAQNAIA
PROF_sec EEEEEEEEE EEEEE EEEE EE EE HHHHHHHHH
430 440 450 460 470 480
KIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQ HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHH WDNALAAIDAQRADLGAVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQ HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHH
490 500
STFlic ILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 1)
lUCU HHHHHHHHHHHHHHHHHHHHHH (empirical structure)
PROF sec HHHHHHHHHHHHH HHHHHHH (predicted secondary
structure)
SaFlixl VLQQAGTAILAQANQLPQAVLSLLR (SEQ ID NO: 4)
PROF sec HHHHHHHHHHHHH HHHHHHH (predicted secondary structure)
[00124] The insertion site, referred to herein as "D3I-ol," in a loop of domain 3 of S.
typhimurium FljB (SEQ ID NO: 2) for fusion to a Dengue viral envelope protein antigen (domain III) is between predicted β-strands VTLA (SEQ ID NO: 109), which is amino acid residues 263-266 of (SEQ ID NO: 2), and VVS, which is amino acid residues 293-295 of (SEQ ID NO: 2). The insertion site, referred to herein as "D3I-il," in a loop of domain 3 of flagellin is between β-strands EVNVA (SEQ ID NO: 110), which corresponds to amino acid residues 254- 258 of (SEQ ID NO: 2), and VTLA (SEQ ID NO: 109), which corresponds to amino acid residues 263-266 of (SEQ ID NO: 2). The insertion site, referred to herein as "D3I-sl," in a loop of domain 3 of flagellin, is between β-strands KYFVTIGG (SEQ ID NO: 111), which corresponds to amino acid residues 234-241 of SEQ ID NO: 2, and EVNVA (SEQ ID NO: 110), which corresponds to amino acid residues 254-258 of SEQ ID NO: 2 in FljB S. typhimurium, (SEQ ID NO: 2)). The D3I-ol, D3I-il and D3I-sl insertion sites predicted for S. typhimurium FljB, (SEQ ID NO: 2) were loop conformations in the predicted secondary structures of E. coli (SEQ ID NO: 3) and P. aeruginosa (SEQ ID NO: 4) flagellin. Therefore, with reference to the loops in domain 3 of flagellin (SEQ ID NO: 2), predicted loops in domain 3 and insertion sites, such as D3I-il and D3I-ol, can be selected for E. coli (SEQ ID NO: 3) a d P. aeruginosa (SEQ ID NO: 4) flagellin.
[00125] Insertion sites in loops in domain 3 for fusion to Dengue viral envelope protein antigens (domain III) in E. coli FliC flagellin (SEQ ID NO: 3), can include an D3I-ol site between G274 and A275 of (SEQ ID NO: 3) in the loop between β -strand ITF (amino acid residues 267-269 of (SEQ ID NO: 3) and β -strand VLTANI (SEQ ID NO: 112), amino acid residues 284-289 of (SEQ ID NO: 3). The D3I-il site is between A257 and D258 of SEQ ID NO: 3 in the loop between β -strand ELAKLAIKL (SEQ ID NO: 113), amino acid residues 248- 256 of (SEQ ID NO: 3) and IEYK (SEQ ID NO: 114), amino acid residues 262-265 of (SEQ ID NO: 3). The D3I-sl site was selected between S240 and G241 of (SEQ ID NO: 3) in the loop between β-strand KV (residues 238-239 of (SEQ ID NO: 3)) and β-strand SID (residues 243-245 of (SEQ ID NO: 3)).
[00126] For P. aeruginosa flagellin (PAOl, (SEQ ID NO: 4)), the D3I-ol site was selected between Q272 and D273 in the loop between β -strand TV SLA (SEQ ID NO: 115), amino acid residues 262-266 of (SEQ ID NO: 4) and β -strand LGITASI (SEQ ID NO: 1 16), amino acid residues 285-291 of (SEQ ID NO: 4). The D3I-il site was between S260 and N261 of (SEQ ID NO: 4) in the loop between β -strand SLNFDVTVG (SEQ ID NO: 117), amino acid residues 251-259 of (SEQ ID NO: 4) and TVSLA (SEQ ID NO: 118), amino acid residues 262-266 of (SEQ ID NO: 4). The D3I-sl site was selected between S245 and G246 of (SEQ ID NO: 4) in the loop between β-strand TVFT (SEQ ID NO: 119), residues 238-241 of (SEQ ID NO: 4) and β-strand GVT (residues 246-248 of (SEQ ID NO: 4)).
[00127] The point of fusion between the flagellin component and Dengue viral envelope protein antigen (also referred to herein as "Dengue antigen") components of fusion proteins of the invention results in a sequence of unique amino acids. For fusion proteins employed in methods of the invention to treat humans, if this unique sequence of amino acids at the juncture of the fusion of the flagellin component and the Dengue viral envelope protein antigen shares homology with a known human protein, the fusion protein has the potential to elicit an unwanted immune response to the human protein or a portion of the human protein. Upon selection of an insertion site in flagellin, the loop of domain 3 or the amino-terminus of flagellin, the sequence of unique amino acids that would be created by fusion of the Dengue viral envelope protein antigen (domain III) to flagellin is assessed for its potential ability to elicit an unwanted immune response. In the evaluation, a probe of about 10 to about 12 amino acids in length, which includes the flagellin/Dengue antigen point of fusion is used to probe a database of known human genome sequences. If homology is identified for a stretch of amino acids greater than about 5 amino acids, then the point of fusion sequence is modified with an amino acid substitution of, for example, 1, 2, 3, 4, 5, or 6 amino acids to decrease the homology. The order of preferred amino acids for use in the modification is serine, threonine, alanine and glycine. Generally, a single amino acid substitution is sufficient to modify the homology.
[00128] In another embodiment, the site of insertion of a Dengue viral envelope protein antigen (domain III) in a loop of domain 3 of flagellin can be 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, amino acids towards the carboxy-domain 2 or an amino-domain 2 of flagellin from the insertion sites. For example, with reference to (SEQ ID NO: 2), a loop of domain 3 to which the Dengue antigen is fused is between amino acid residues 277 and 278 (e.g., D3I-ol) (FIG. 2). Alternatively, fusion of the antigen can occur between amino acid 266 and 282 of (SEQ ID NO: 2).
[00129] Likewise, with reference to (SEQ ID NO: 2), a loop of domain 3 to which the antigen is fused is between amino acid residues 259 to 260 (D3I-il) or amino acid residues 260 to 261. The insertion site in a loop of domain 3 of flagellin in the D3I-il fusion protein of (SEQ ID NO: 2) is between amino acid residues 190 to 191 or between amino acid residues 291 to 292.
[00130] In another embodiment, about 2 to about 4 amino acid residues in the loop of domain 3 can be deleted prior to fusion with the Dengue viral envelope protein antigen (domain III). The deletions would be designed so that adjacent secondary structures in the flagellin would not be disrupted.
[00131] In a preferred embodiment, the flagellin for use in the fusion proteins is a flagellin that includes at least one member selected from the group consisting of (SEQ ID NOS: 1-7). The antigen is fused between amino acid residue 191 and amino acid residue 285 of (SEQ ID NO: 1), which is within a loop of domain 3 of flagellin.
[00132] "At least a portion," as used herein, refers to a Dengue viral envelope protein antigen, flagellin or stem region of a Dengue viral envelope protein that is less than or the entirety of the Dengue viral envelope protein antigen, flagellin or stem region of the Dengue viral envelope protein. "A portion," as used herein with reference to a Dengue viral envelope protein antigen, including domain I or domain II or domain III, refers to any part of the Dengue viral envelope protein antigen that is less than the entirety of the Dengue viral envelope protein antigen.
[00133] The phrase, "causes the ectodomain to essentially retain its tertiary structure," as used herein, refers to maintenance of the tertiary structure of the ectodomain of the Dengue antigen (domains I, II or III) to thereby mimic the tertiary structure of the respective domain in the naturally occurring Dengue viral antigen, which can be assessed by the ability to generate a sufficient immune response to stimulate a protective immune response in a subject in vivo or viral neutralization in in vitro assays, as described herein.
[00134] Fusion proteins of the invention can include an amino acid linker between at least one of an amino-terminus or a carboxy -terminus of the Dengue antigen and the loop of domain 3 of the flagellin or the amino-terminus of flagellin. In a particular embodiment, the linker, such as (SEQ ID NOS: 97-105), is between the carboxy -terminal amino acid of domain I in the domains I/II antigen and the amino-terminus of flagellin. In another embodiment, the linker, such as (SEQ ID NOS: 97-105), is between the carboxy -terminal amino acid of the domain III Dengue antigen and a loop of domain 3 of flagellin. In an additional embodiment, fusion proteins of the invention include two linkers, specifically, one linker, such as (SEQ ID NOS: 97-105), between the carboxy -terminal amino acid of domain I in the domains I/II antigen and the amino-terminus of flagellin and another linker, such as (SEQ ID NOS: 97-105), between the carboxy-terminal amino acid of the domain III Dengue antigen and a loop of domain 3 of flagellin.
[00135] An additional embodiment, the linker is between the carboxy-terminal amino acid of the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin, or the carboxy-terminal amino acid of the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin, or the carboxy-terminal amino acid of the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin, or the carboxy-terminal amino acid of the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
[00136] In another embodiment, the fusion proteins of the composition of the invention include a linker, such as an amino acid linker, between the carboxy-terminal amino acid of the domain I of the Dengue 1 viral antigen and the amino-terminal amino acid of the first flagellin, or the carboxy-terminal amino acid of the domain I of the Dengue 2 viral antigen and the amino- terminal amino acid of the second flagellin, or the carboxy-terminal amino acid of the domain I of the Dengue 3 viral antigen and the amino-terminal amino acid of the third flagellin, or the carboxy-terminal amino acid of domain I of the Dengue 4 viral antigen and the amino-terminal amino acid of the fourth flagellin. [00137] In a particular embodiment, the fusion proteins of the invention include a linker, such as an amino acid linker, between the carboxy-terminal amino acid of the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin, the carboxy-terminal amino acid of the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin, the carboxy-terminal amino acid of the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin and the carboxy-terminal amino acid of the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin in combination with a linker between the carboxy-terminal amino acid of the domain I of the Dengue 1 viral antigen and the amino-terminal amino acid of the first flagellin, the carboxy- terminal amino acid of the domain I of the Dengue 2 viral antigen and the amino-terminal amino acid of the second flagellin, the carboxy-terminal amino acid of the domain I of the Dengue 3 viral antigen and the amino-terminal amino acid of the third flagellin, and the carboxy-terminal amino acid of domain I of the Dengue 4 viral antigen and the amino-terminal amino acid of the fourth flagellin.
[00138] The amino acid linker can be between about 2 to about 20 amino acids in length, such as about 2, about 4, about 6, about 8 or about 10 amino acids in length. Preferred amino acid residues would include amino acid residues without side chains or amino acid residues with small side chains, such as glycine, alanine or serine, including combinations of glycine, serine and alanine. Amino acid linkers are devoid of secondary structures, such as a-helices and B- sheets. Amino acid linkers that include amino acids without side chains and devoid of secondary structures are referred to herein as "flexible linkers." In an embodiment, amino acid linkers can include 2, 4 or 6 negatively charged amino acid residues, such as aspartic acid or glutamic acid. Exemplary amino acid linkers are described, for example, in PCT/US2012/000099 (WO
2012/115715), by Song, L. et al, and PCT/US2012/000367 (WO 2013/066365), by Song, L., et al. It is believed that negatively charged amino acids at or adjacent to (about 2, 4, 6, 8 or 10 amino acids) the amino- or carboxy -terminus of the Dengue antigen at the site of fusion to a loop of domain 3 or the amino-terminus of flagellin reduce undesirable intramolecular interactions between the negatively charged flagellin and a positively charged Dengue antigen thereby tethering the Dengue antigen at the site of fusion. Exemplary amino acid residues are unit repeats of glycine and serine residues, such as GSGS (SEQ ID NO: 97), GSGSGS (SEQ ID NO: 98), GSGSGSGS (SEQ ID NO: 99), GSGSGSGSGS (SEQ ID NO: 100), GSGSGSGSGSGS (SEQ ID NO: 101), GSGSGSGSGSGSGS (SEQ ID NO: 102), GSGSGSGSGSGSGSGS (SEQ ID NO: 103), GSGSGSGSGSGSGSGSGS (SEQ ID NO: 104),
GSGSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 105). [00139] The linker can include amino acid residues that are native to the naturally occurring Dengue viral envelope protein. In an embodiment, the linker that includes amino acid residues that are native to the naturally occurring Dengue viral envelope protein are in a linking region of the Dengue viral envelope protein. A "linking region," as used herein, means an amino acid residue or peptide that is adjacent to domain III and outside a stem region of a naturally occurring ectodomain of Dengue viral envelope protein. The linking region can be 4 amino acids in length, or 5 amino acids in length, or 6 amino acids in length or 7 amino acids in length or 8 amino acids in length or 9 amino acids in length or 10 amino acids in length. Examples of linking regions include SSIGK (SEQ ID NO: 106) for Dengue 1, or SSIGQ (SEQ ID NO: 107) for Dengue 2, or SSIGK (SEQ ID NO: 106) for Dengue 3, or SSIGK (SEQ ID NO: 107) for Dengue 4. A linking region can be fused to the carboxy-terminus of domain III of the Dengue antigen that is fused to a loop of domain 3 of flagellin. Fusion proteins that include domains I, II and III of the Dengue envelope protein antigens without a linking region and without at least a portion of a stem region are referred to herein as "80E" fusion proteins that include "80E" Dengue antigens (FIG. 4C), such as SEQ ID NO: 41.
[00140] Fusion proteins that include an 80E Dengue antigen and a linking region, such as SEQ ID NOS: 93, 94, 95 and 96, are referred to herein as "80E+" fusion proteins that include "80E+" Dengue antigens (FIG. 4C), such as SEQ ID NO: 93. For example, as shown in FIG. 4B, a linking region of the Dengue antigen is indicated by an arrow and, with reference to SEQ ID NO: 89, for example, is 5 amino acid residues in length.
[00141] Fusion proteins that include (i) domains I and II and (ii) domain III that includes a linking region and at least a portion of a stem region of a Dengue viral envelope protein antigen, are referred to herein as "85E" fusion proteins that include "85E" Dengue antigens (FIG. 4C), such as SEQ ID NO: 89.
[00142] The 80E, 80E+ and 85E Dengue antigens can be fused to flagellin in split formats, N-term format and C-term format.
[00143] "Split format," as used herein with reference to a fusion protein that includes a Dengue antigen (80E, 80E+, 85E) and a flagellin, means that domains I and II of the Dengue viral envelope protein are fused to the amino-terminus of flagellin and the domain III of the Dengue viral envelope protein (with or without a linking region in 80E and 80E+ formats, respectively, and with a linking region and at least a portion of a stem region in 85E format) is fused to at least one loop of domain 3 of the same flagellin. In other words, the ectodomain has been "split" into domains I/II and III for fusion to two distinct sites (amino-terminus and loop of domain 3, respectively) of flagellin, as depicted in FIGs. 70- 72. Exemplary split format fusion proteins include SEQ ID NOs: 11, 17, 22, 25, 26 and 27-40. [00144] "N-term" fusion format (also referred to herein as "N-term format), as used herein with reference to a fusion protein that includes a Dengue antigen (80E, 80E+, 85E+) and a flagellin, means that domains I, II and III (with or without a linking region in 80E and 80E+ formats, respectively, and with a linking region and at least a portion of a stem region in 85E format) are collectively fused to the flagellin at one site of the flagellin, specifically the amino- terminus of the flagellin, as depicted in FIGs. 70-72. N-term formats of fusion proteins include SEQ ID NOs: 8, 14, 15, 19, 20 and 21.
[00145] "C-term" fusion format (also referred to herein as "C-term format), as used herein with reference to a fusion protein that includes a Dengue antigen (80E, 80E+, 85E+) and a flagellin, means that domains I, II and III (with or without a linking region in 80E and 80E+ formats, respectively, and with a linking region and at least a portion of a stem region in 85E format) are collectively fused to the flagellin at one site of the flagellin, specifically the carboxy- terminus of the flagellin.
[00146] In a further embodiment, the fusion proteins of the invention, including fusion proteins that include a linking region, can further include at least one junction loop peptide of a Dengue virus between at least one member selected from the group consisting of the domain I of the Dengue 1 viral antigen and the amino-terminus of the first flagellin; the domain I of the Dengue 2 viral antigen and the amino-terminus of the second flagellin; the domain I of the Dengue 3 viral antigen and the amino-terminus of the third flagellin; the domain I of the Dengue 4 viral antigen and the amino-terminus of the fourth flagellin; the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin; the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin; the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin; and the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
[00147] In a particular embodiment, a junction loop peptide is between each domain I of each of the four Dengue serotype antigens (Denl, Den2, Den3 and Den4) and the amino terminus of each flagellin component of the four fusion proteins in combination with a junction loop peptide between domain III of each of the four Dengue serotype antigens (Denl, Den2, Den3 and Den4) and the loop of the domain 3 of each flagellin component of the four fusion proteins, wherein the domain III of each of the four Dengue serotype antigens is fused to the junction loop peptide at the carboxy -terminal amino acid residue, such as exemplary fusion proteins as set forth in SEQ ID NOS: 27-38.
[00148] A "junction loop," as used herein, refers to a single strand domain boundary between the EIEII domains and the EIII domain. For example, a junction loop is located at about amino acid position 295 for DENVl, 2 and 4 of SEQ ID NOs: 93, 94 and 96 or amino acid position 293 for DENV3 of SEQ ID NO: 95. The junction loop is essentially in the middle of an about 7 amino acid peptide, such as DKLTLKG (SEQ ID NO: 55), depicted as a dashed circle in FIG. 4B and shown in grey highlighted text in FIGs. 49-60). Exemplary overlapping 7 amino acid residues (the "junction loop") for Dengue 1 are DKLTLKG (SEQ ID NO: 55), Dengue 2 are DKLQLKG (SEQ ID NO: 59), Dengue 3 are DKLELKG (SEQ ID NO: 63), Dengue 4 are EKLRTKG (SEQ ID NO: 67).
[00149] At least one junction loop peptide can be fused to at least one member selected from the group consisting of the carboxy-terminal amino acid of domain I in the combined EIEII domain of the Dengue antigen for fusion to the amino-terminus of flagellin, and the amino- terminal amino acid of domain III of the Dengue antigen for fusion to a loop of domain 3 of flagellin. As described herein, a composition of the fusion proteins of the invention are more immunogenic when the EIEII domain of the Dengue antigens is fused to the amino-terminus of flagellin with the junction loop in combination with a flexible linker, such as an amino acid linker of repeat units of glycine and serine (GSn), in combination with fusion of a junction loop to the amino-terminal amino acid of the EIII domain of the Dengue viral envelope protein antigen (domain III) fused to a loop of domain 3 of flagellin and a flexible linker, such as an amino acid linker of repeat units of glycine and serine (GSn).
[00150] Compositions and methods of the invention can further include at least one Toll-like Receptor (TLR) agonist selected from the group consisting of a Toll-like Receptor 1 agonist, a Toll-like Receptor 2 agonist, a Toll-like Receptor 3 agonist, a Toll-like Receptor 4 agonist, a Toll-like Receptor 5 agonist, a Toll-like Receptor 6 agonist, a Toll-like Receptor 7 agonist, a Toll-like Receptor 8 agonist, a Toll-like Receptor 9 agonist, a Toll-like Receptor 10 agonist, a Toll-like Receptor 11 agonist, a T Toll-like Receptor 12 agonist and a Toll-like Receptorl3 agonist. In particular embodiments, the Toll-like Receptor agonist included in the composition is a TLR 4 agonist, such as a lipopolysaccharide, or a TLR 9 agonist, such as CpG
oligodeoxynucleotides or a TLR 3 agonist, or a TLR 7 agonist or a TLR 8 agonist. In another embodiment, the TLR agonist is at least one member selected from the group consisting of a TLR 3 agonist, a TLR 7 agonist and a TLR 8 agonist.
[00151] "Agonist," as used herein in referring to a TLR, means a molecule that activates a TLR signaling pathway. A TLR signaling pathway is an intracellular signal transduction pathway employed by a particular TLR that can be activated by a TLR ligand or a TLR agonist. Common intracellular pathways are employed by TLRs and include, for example, NF-κΒ, Jun N- terminal kinase and mitogen-activated protein kinase. The Toll-like Receptor agonist in the compositions and methods described herein can include at least one member selected from the group consisting of a TLR1 agonist, a TLR2 agonist (e.g., Pam3Cys, Pam2Cys, bacterial lipoprotein), a TLR3 agonist (e.g., dsRNA), a TLR4 agonist (e.g., bacterial lipopolysaccharide), a TLR5 agonist (e.g., a flagellin), a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist (e.g., unmethylated DNA motifs), TLR10 agonist, a TLR11 agonist and a TLR12 agonist.
[00152] In another embodiment, the invention is a method of stimulating an immune response in a subject by administering a composition comprising at least four fusion proteins, each of which activates a Toll-like Receptor 5, and includes flagellin and Dengue viral antigens. The first fusion protein of the composition includes a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin. The second fusion protein of the composition includes a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin. The third fusion protein of the composition includes a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin. The fourth fusion protein of the composition includes a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin; and a domain I and a domain II of the Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
[00153] The methods of the invention can further include the administration of an adjuvant. In a particular embodiment, the adjuvant is a saponin (see, for example, Skene, CD., et al, Methods 40:53-59 (2006) and Rajput, Z.I., et al. J. Zhejiang Univ. Sci. 8(3): 153-161 (2007).
[00154] "Stimulating an immune response," as used herein, refers to the generation of antibodies and/or T-cells to the Dengue viral envelope protein antigen fused to flagellin in the fusion proteins described herein. Stimulating an immune response in a subject can include the production of humoral and/or cellular immune responses that are reactive against the Dengue antigen. Stimulation of an immune response in a subject by administration of the compositions and fusion proteins described herein may provide protective immunity to disease or illness consequent to exposure to the Dengue virus. Fusion proteins of the invention may neutralize Dengue virus, which can be assessed employing well-known techniques, such as those described herein. Neutralization of the Dengue virus in in vitro assays is believed to correlate with the presence of antibodies to the Dengue antigens in the fusion proteins that neutralize the Dengue virus for use in compositions to treat a subject, including use as a vaccine composition to provide protective immunity against disease consequent to Dengue infection.
[00155] The compositions of the invention for use in methods to stimulate immune responses in subjects, can be evaluated for the ability to stimulate an immune response in a subject using well-established methods. Exemplary methods to determine whether the compositions of the invention stimulate an immune response in a subject, include measuring the production of antibodies specific to the Dengue antigen (e.g., IgG antibodies) by a suitable technique such as, ELISA assays; the potential to induce antibody-dependent enhancement (ADE) of a secondary infection; macrophage-like assays; neutralization assessed by using the Plaque Reduction Neutralization Test (PRNT50); and the ability to generate serum antibodies in non-human models (e.g., mice, rabbits, monkeys) (Messer, et al., PLOS Negl. Trop. Des. 6:el486 (2012)).
[00156] "Stimulates a protective immune response," as used herein, means administration of the compositions of the invention that include a fusion protein comprising Dengue antigens as described herein that result in production of antibodies to the protein to thereby cause a subject to be essentially disease-free (not develop viremia) by an otherwise challenge dose of a viral protein or neutralize the Dengue virus or ameliorate disease and illness consequent to exposure to the Dengue virus. Techniques to determine an optimum challenge dose of the Dengue virus are known to one of skill in the art (see, for example, Putnak, et al., J. Inf. Des. 174: 1176-1184 (1996)). Exemplary techniques for determining an optimum challenge dose can include administration of varying doses of virus and a determination of the percent of subjects that do not develop viremia following administration of various challenge doses of virus.
[00157] Assessment of stimulation of protective immunity can also be made by employing assays that assess the ability of the antibodies produced in response to the fusion proteins to neutralize binding of the Dengue virus to a host cell. It is believed that inhibition of Dengue virus infectivity is indicative of the ability of antibodies, formed from the compositions and by the methods of the invention, to neutralize the binding sites of the naturally Dengue virus ("neutralization of Dengue virus") and, thereby, prevent infection of the host cell as a
consequence of stimulating a protective immune response. Inhibition or neutralization of the Dengue viruses is believed to correlate with an ability of an immune response to protect against a Dengue virus or infection, or disease from the Dengue virus.
[00158] Fusion proteins of the invention can be generated recombinantly or by chemical conjugation using well-established techniques. Chemical conjugation can include conjugation by a reactive group, such as a thiol group (e.g., a cysteine residue) or by derivatization of a primary (e.g., an amino-terminal) or secondary (e.g., lysine) group. A recombinant fusion protein can be generated by operably linking a nucleic acid sequence encoding a flagellin, or a portion of a flagellin that includes a domain 3, to a nucleic acid sequence encoding a Dengue antigen. In an embodiment, fusion proteins of the invention can include Dengue antigens that consist essentially of domain III of a Dengue envelope protein or consist of domain III of the Dengue envelope protein fused to at least one loop, such as one, two, three, or four loops, of domain 3 of flagellin in combination with a second Dengue antigen that consists essentially of domains I and II of a Dengue envelope protein or consists of domains I and II of a second Dengue antigen fused to the amino-terminus of the flagellin.
[00159] Fusion proteins of the invention can be made employing routine molecular biological techniques, as described herein. Host cells can be transfected with nucleic acids encoding fusion proteins of the invention. The host cells can be eukaryotic or prokaryotic host cells. Suitable prokaryotic host cells include E. coli, B. subtilis and Pseudomonas fluorescens.
[00160] Fusion proteins made in eukaryotic host cells can include a flagellin of the fusion proteins of the invention that is modified from its corresponding native flagellin to delete at least one putative glycosylation site in the nucleic acid sequence encoding the flagellin. The putative glycosylation site that is deleted can include an N-glycosylation site. A flagellin that has been made recombinantly to delete at least one putative glycosylation site is referred to as "ng," for example, STF2ng. In an embodiment, at least one putative glycosylation site is mutated. For example, the "N" residue in the putative glycosylation site, such as N-X-Serine or N-X-Tyrosine or N-X-Cysteine is mutated to glutamine when fusion proteins are generated recombinantly in eukaryotic host cells. The "N" at amino acid residues 19, 194, 216, 293, 457 and 476 of SEQ ID NO: 108 is mutated to glutamine and the "N" at amino acid residue 101 of SEQ ID NO: 108 is mutated to aspartic acid.
[00161] The eukaryotic host cells employed in the methods of the invention can include a Saccharomyces eukaryotic host cell, an insect eukaryotic host cell (e.g., at least one member selected from the group consisting of a Baculovirus infected insect cell, such as Spodoptera frugiperda (Sf9) or Trichhoplusia ni (High5) cells; and a Drosophila insect cell, such as Dmel2 cells), a fungal eukaryotic host cell, a parasite eukaryotic host cell (e.g., a Leishmania tarentolae eukaryotic host cell), CHO cells, yeast cells (e.g., Pichia) and a Kluyveromyces lactis host cell. In an embodiment, fusion proteins of the invention are made in a Drosophila eukaryotic host cell. In another embodiment, fusion proteins of the invention are made in a Baculovirus eukaryotic host cell. A fusion protein made in Drosophila is identified by the designation "DR." A fusion protein made in Baculovirus are identified by the designation "BV."
[00162] Suitable eukaryotic host cells and vectors can also include plant cells (e.g., tomato; chloroplast; mono- and dicotyledonous plant cells; Arabidopsis thaliana; Hordeum vulgare; Zea mays; potato, such as Solarium tuberosum; carrot, such as Daucus carota L ; and tobacco, such as Nicotiana tabacum, Nicotiana benthamiana (Gils, M., et al, Plant BiotechnolJ. 3:613-20 (2005); He, D.M., et al, Colloids Surf B Biointerfaces, (2006); Huang, Z., et al, Vaccine 79:2163-71 (2001); Khandelwal, A, et al, Virology. 305:207-15 (2003); Marquet-Blouin, E., et al, Plant Mol Biol 51 :459-69 (2003); Sudarshana, M.R., et al. Plant BiotechnolJ. :551-9 (2006); Varsani, A., et al., Virus Res, 120:91-6 (2006); Kamarajugadda S., et al, Expert Rev Vaccines 5:839-49 (2006); Koya V, et al, Infect Immun. 73:8266-74 (2005); Zhang, X., et al, Plant BiotechnolJ. 4:419-32 (2006)).
[00163] The fusion proteins of the invention can be purified and characterized employing well-known methods (e.g., gel chromatography, cation exchange chromatography, SDS-PAGE), as described herein.
[00164] In an additional embodiment, the invention includes a protein, polypeptide or peptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%), at least about 95%, at least about 98%> and at least about 99% sequence identity to the fusion proteins of the invention.
[00165] The percent identity of two amino acid sequences (or two nucleic acid sequences) can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The amino acid sequence or nucleic acid sequences at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100). The length of the protein or nucleic acid encoding can be aligned for comparison purposes is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%), at least about 99% of the length of the reference sequence, for example, the nucleic acid sequence of a Dengue antigen or flagellin that includes domain 3 or fusion protein of the invention of a fusion protein.
[00166] The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al. (Proc. Natl. Acad. Sci. USA, 90:5873- 5877 (1993), the teachings of which are hereby incorporated by reference in its entirety). Such an algorithm is incorporated into the BLASTN and BLASTX programs as described, for example, in Schaffer et al. (Nucleic Acids Res., 29:2994-3005 (2001) and Oehmen, C. S. et al, Bioinformatics 29 (6): 797-798 (2013). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN; available at the Internet site for the National Center for Biotechnology Information) can be used. In one embodiment, the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.
[00167] Another mathematical algorithm employed for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, California) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis, et al, Comput. Appl. Biosci., 10: 3-5 (1994) and FASTA described in Pearson, et al, (Proc. Natl. Acad. Sci USA, 85: 2444-2448 (1988), the teachings of which are hereby incorporated by reference in its entirety).
[00168] The percent identity between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, California) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, California), using a gap weight of 50 and a length weight of 3.
[00169] The nucleic acid sequence encoding a Dengue envelope protein antigen (EI/EII, EII, EI/II/III) or fusion proteins of the invention and polypeptides of the invention can include nucleic acid sequences that hybridize to nucleic acid sequences or complements of nucleic acid sequences of the invention, for example, the nucleic acid sequence of a flagellin or Dengue antigen employed in the fusion proteins of the invention of a fusion protein of the invention under selective hybridization conditions (e.g., highly stringent hybridization conditions). As used herein, the terms "hybridizes under low stringency," "hybridizes under medium
stringency," "hybridizes under high stringency," or "hybridizes under very high stringency conditions," describe conditions for hybridization and washing of the nucleic acid sequences. Guidance for performing hybridization reactions, which can include aqueous and nonaqueous methods, can be found in Aubusel, F.M., et al, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (2001).
[00170] For applications that require high selectivity, relatively high stringency conditions to form hybrids can be employed. In solutions used for some membrane based hybridizations, addition of an organic solvent, such as formamide, allows the reaction to occur at a lower temperature. High stringency conditions are, for example, relatively low salt and/or high temperature conditions. High stringency is provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C. High stringency conditions allow for limited numbers of mismatches between the two sequences. In order to achieve less stringent conditions, the salt concentration may be increased and/or the temperature may be decreased. Medium stringency conditions are achieved at a salt concentration of about 0.1 to 0.25 M NaCl and a temperature of about 37°C to about 55°C, while low stringency conditions are achieved at a salt concentration of about 0.15 M to about 0.9 M NaCl, and a temperature ranging from about 20°C to about 55°C. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel et al, Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., Units 2.8-2.11, 3.18-3.19 and 4-64.9, (1997).
[00171] A "subject," as used herein, can be a mammal, such as a primate or rodent (e.g., rat, mouse). In a particular embodiment, the subject is a human.
[00172] An "effective amount," when referring to the amount of a composition and fusion protein of the invention, refers to that amount or dose of the composition and fusion protein, that, when administered to the subject is an amount sufficient for therapeutic efficacy (e.g., an amount sufficient to stimulate an immune response in the subject). The compositions and fusion proteins of the invention can be administered in a single dose or in multiple doses.
[00173] The methods of the present invention can be accomplished by the administration of the compositions and fusion proteins of the invention by enteral or parenteral means.
Specifically, the route of administration is by oral ingestion (e.g., drink, tablet, capsule form) or intramuscular injection of the composition and fusion protein. Other routes of administration as also encompassed by the present invention including intravenous, intradermal, intraarterial, intraperitoneal, or subcutaneous routes, and nasal administration. Suppositories or transdermal patches can also be employed.
[00174] The compositions and fusion proteins of the invention can be administered ex vivo to a subject's autologous dendritic cells. Following exposure of the dendritic cells to the composition and fusion protein of the invention, the dendritic cells can be administered to the subject.
[00175] The compositions and fusion proteins of the invention can be administered alone or can be coadministered to the patient. Coadminstration is meant to include simultaneous or sequential administration of the composition, fusion protein or polypeptide of the invention individually or in combination. Where the composition and fusion protein are administered individually, the mode of administration can be conducted sufficiently close in time to each other (for example, administration of the composition close in time to administration of the fusion protein) so that the effects on stimulating an immune response in a subject are maximal. It is also envisioned that multiple routes of administration (e.g., intramuscular, oral, transdermal) can be used to administer the compositions and fusion proteins of the invention.
[00176] The compositions and fusion proteins of the invention can be administered alone or as admixtures with conventional excipients, for example, pharmaceutically, or physiologically, acceptable organic, or inorganic carrier substances suitable for enteral or parenteral application which do not deleteriously react with the extract. Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrolidine. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the compositions, fusion proteins or polypeptides of the invention. The preparations can also be combined, when desired, with other active substances to reduce metabolic degradation. The compositions and fusion proteins of the invention can be administered by is oral administration, such as a drink, intramuscular or intraperitoneal injection or intranasal delivery. The compositions and fusion proteins alone, or when combined with an admixture, can be administered in a single or in more than one dose over a period of time to confer the desired effect (e.g., alleviate prevent viral infection, to alleviate symptoms of virus infection, such as influenza or flaviviral infection).
[00177] When parenteral application is needed or desired, particularly suitable admixtures for the compositions and fusion proteins are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. In particular, carriers for parenteral administration include aqueous solutions of dextrose, saline, pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil, polyoxyethylene-block polymers, and the like. Ampules are convenient unit dosages. The compositions, fusion proteins or polypeptides can also be incorporated into liposomes or administered via transdermal pumps or patches. Pharmaceutical admixtures suitable for use in the present invention are well-known to those of skill in the art and are described, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, PA) and WO 96/05309 the teachings of which are hereby incorporated by reference.
[00178] The compositions and fusion proteins of the invention can be administered to a subject on a support that presents the compositions and fusion proteins of the invention to the immune system of the subject to generate an immune response in the subject. The presentation of the compositions and fusion proteins of the invention would preferably include exposure of antigenic portions of the viral protein to generate antibodies. The fusion proteins of the invention are in close physical proximity to one another on the support. The fusion proteins of the invention can be attached to the support by covalent or noncovalent attachment. Preferably, the support is biocompatible. "Biocompatible," as used herein, means that the support does not generate an immune response in the subject (e.g., the production of antibodies). The support can be a biodegradable substrate carrier, such as a polymer bead or a liposome. The support can further include alum or other suitable adjuvants. The support can be a virus (e.g., adenovirus, poxvirus, alphavirus), bacteria (e.g., Salmonella) or a nucleic acid (e.g., plasmid DNA, CpG).
[00179] The dosage and frequency (single or multiple doses) administered to a subject can vary depending upon a variety of factors, including prior exposure to the Dengue virus, the duration of viral infection, prior treatment of the viral infection, the route of administration of the composition or fusion protein; size, age, sex, health, body weight, body mass index, and diet of the subject; nature and extent of symptoms of viral exposure, viral infection and the particular viral responsible for the infection (e.g., Dengue virus infection), or treatment or infection of another antigen, such as a Dengue antigen, kind of concurrent treatment, complications from the viral exposure, viral infection or exposure or other health-related problems. Other therapeutic regimens or agents can be used in conjunction with the methods and compositions and fusion proteins of the present invention. For example, the administration of the compositions and fusion proteins can be accompanied by other viral therapeutics or use of agents to treat the symptoms of a condition associated with or consequent to exposure to the antigen, such as influenza infection. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art.
[00180] The compositions, fusion proteins and polypeptides of the invention can be administered to a subject on a presenting carrier. "Presenting carrier," as used herein, means any composition that presents the compositions, fusion proteins and polypeptides of the invention to the immune system of the subject to generate an immune response in the subject. The presentation of the compositions, fusion proteins and polypeptides of the invention would preferably include exposure of antigenic portions of the viral protein to generate antibodies. The components (e.g., PAMP and a viral protein) of the compositions, fusion proteins and
polypeptides of the invention are in close physical proximity to one another on the presenting carrier. The compositions, fusion proteins and polypeptides of the invention can be attached to the presenting carrier by covalent or noncovalent attachment. Preferably, the presenting carrier is biocompatible. "Biocompatible," as used herein, means that the presenting carrier does not generate an immune response in the subject (e.g., the production of antibodies). The presenting carrier can be a biodegradable substrate presenting carrier, such as a polymer bead or a liposome. The presenting carrier can further include alum or other suitable adjuvants. The presenting carrier can be a virus (e.g., adenovirus, poxvirus, alphavirus), bacteria (e.g., Salmonella) or a nucleic acid (e.g., plasmid DNA). Compositions and fusion proteins of the invention can be administered by use of viral vectors.
[00181] The compositions and methods of the invention can further include a carrier.
"Carrier," as used herein, refers to a molecule (e.g., protein, peptide) that can enhance stimulation of a protective immune response. Carriers can be physically attached (e.g., linked by recombinant technology, peptide synthesis, chemical conjugation or chemical reaction) to a composition (e.g., a protein portion of a naturally occurring viral hemagglutinin) or admixed with the composition.
[00182] Carriers for use in the methods and compositions described herein can include, for example, at least one member selected from the group consisting of Tetanus toxoid (TT), Vibrio cholerae toxoid, Diphtheria toxoid (DT), a cross-reactive mutant (CRM) of diphtheria toxoid, E. coli enterotoxin, E. coli B subunit of heat labile enterotoxin (LTB), Tobacco mosaic virus (TMV) coat protein, protein Rabies virus (RV) envelope protein (glycoprotein), thyroglobulin (Thy), heat shock protein HSP 60 Kda, Keyhole limpet hemocyamin (KLH), an early secreted antigen tuberculosis-6 (ESAT-6), exotoxin A, choleragenoid, hepatitis B core antigen, and the outer membrane protein complex of N. meningiditis (OMPC) (see, for example, Schneerson, R., et al, Prog Clin Biol Res 47:77-94 (1980); Schneerson, R., et al, JExp Med 152:361-76 (1980); Chu, C, et al, Infect Immun 40: 245-56 (1983); Anderson, P., Infect Immun 39:233-238 (1983); Anderson, P., et al, J Clin Invest 76:52-59 (1985); Fenwick, B.W., et al, 54: 583-586 (1986); Que, J.U., et al Infect Immun 5(5:2645-9 (1988); Que, J.U., et al Infect Immun 5(5:2645-9 (1988); (Que, J.U., et al Infect Immun 5(5:2645-9 (1988); Murray, K., et al, Biol Chem 380:211- 283 (1999); Fingerut, E., et al, Vet Immunol Immunopathol 772:253-263 (2006); and Granoff, D.M., et al, Vaccine 77:Suppl 1 :S46-51 (1993)).
[00183] A description of example embodiments of the invention follows.
[00184] Flaviviruses, including Dengue viruses, are small, enveloped viruses with icosahedral capsids. The flavivirus genome is a single-stranded positive-sense RNA (about 11 kb) that is directly translated by the host cell machinery following infection. The viral genome is translated as a single polypeptide that undergoes co- and post-translational cleavage by viral and cellular enzymes to generate three structural proteins of the flavivirus (the capsid (C), the membrane (M) and the envelope (E) proteins); and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Weaver, et al., Annu Rev Microbiol 990:44-649 (2004)). The viral capsid is composed of the C-protein, while both the M- and envelope proteins are located on the envelope surface of the virion (Weaver, S.C., et al, Nat. Rev. Microbiol. 70:789-801 (2004); , Chambers et al, Annu Rev. Microbiol. 44: 649-688 (1990)).
[00185] The flavivirus envelope protein plays a role in virus assembly. These proteins form a protective shell around the virus, which serves as a cage for the genetic material inside, sheltering the virus until it is released inside a host cell. While simple viruses consist of only a protein shell and genetic information, more complex viruses, such as flaviviruses, also contain a lipid bilayer between the protein shell and viral genome. A flavivirus can enter a host cell when the viral envelope protein binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host- cell membranes.
[00186] The envelope of a flavivirus may function as a receptor binding protein and to facilitate fusion of the virus and host cell membrane. As a receptor binding protein, the envelope protein is a determinant of host range, cell tropism, virulence and elicits neutralizing antibodies during the immune response (Roehrig, Adv Virus Res 59: 141-175 (2003)). The envelope protein is responsible for fusing the virus and host membranes (Chu, et al, J. Virol 75:10543-10555
(2004) ; Heinz, et al, Adv Virus Res 59:63-97 (2003); Chu, et al, J. Gen Virol 86:405-412
(2005) ). Crystallographic structures of the Tick-borne encephalitis virus envelope protein and the Dengue-2 (Den 2) virus envelope protein have been determined (Rey, et al, Nature 375:291- 298(1995); Modis, et al, Proc Natl Acad Sci USA 700:6986-6991(2003)). Envelope proteins of flaviviruses have common structural (domains I, II and III) and functional features (receptor binding of virus and host cell and fusion functions) and are class II fusion glycoproteins (Lescar et al, Cell 105: 137-148 (2001)).
[00187] In the pre-fusion conformation, envelope proteins form homodimers on the outer surface of the virus particles (Rey, et al, Nature 375:291-298); Kuhn, et al, Cell 108:111-125
(2002) ; Mukhopadhyay, et al, Science 302:248 (2003)). Each envelope protein monomer folds into three structural domains (domains I, II and III) predominantly composed of β-strands.
Domain I (also referred to herein as "I" or "DI") is centrally located in the structure and has an N-glycosylation site in glycosylated envelope proteins. Domain II (also referred to herein as "II" or "DII") of the envelope protein promotes dimerization and has a fusion loop that inserts into the target host membrane during the pH-dependent fusion of the virus (Modis, et al, Nature ¥27:313-319 (2004); Bressanelli, et al, EMBO J 23:728-738 (2004)). Domain III (also referred to herein as "III" or "Dili") is at the carboxy-terminus of the envelope protein. Domain III is also referred to as "domain B" in earlier antigenic mapping studies. Domain III has several epitopes that can elicit virus-neutralizing antibodies (Roehrig, Adv Virus Res 59:141-175
(2003) ).
[00188] The crystal structure of domains I, II and III of the envelope protein from the Tick- borne encephalitis flavivirus and the Dengue 2 flavivirus has been determined (Rey, F.A., et al., Nature 375:291-298 (1995); Modis, Y., et al, Nature ¥27:313-319 (2004), respectively).
Domain I of the Dengue 2 flavivirus envelope protein corresponds to amino acids 1-52, 132-193 and 280-296 of (SEQ ID NO: 42); domain II corresponds to amino acids 53-131 and 194-279 of (SEQ ID NO: 42); and domain III corresponds to amino acids 297-495 of (SEQ ID NO: 42) (Modis, Y., et al, Nature ¥27:313-319 (2004)). The location of domains I, II and III of other flavivirus (e.g., West Nile vims, Japanese encephalitis, Dengue 1 virus, Dengue 3 virus and Dengue 4 virus) is based on homology of the Tick-borne encephalitis envelope protein domains and the Dengue 2 envelope protein domains. Thus, reference herein to domains of flavivirus proteins, in particular, flaviviruses other than Tick-borne encephalitis flavivirus envelope proteins and Dengue 2 flavivirus envelope proteins, are based on homology to domains in the Tick-borne encephalitis flavivirus envelope protein and the Dengue 2 flavivirus envelope protein.
[00189] The domain III of the envelope protein of the DEN flavivirus encodes the majority of the flavivirus type-specific contiguous critical/dominant neutralizing epitopes (Roehring, J.T., Adv. Virus Res. 59: 141 (2003)), including the four DEN (DEN1, DEN2, DEN3, DEN4) viruses. Flavivirus envelope proteins are highly homologous. Exemplary envelope protein sequences are shown in (SEQ ID NOS: 41-44 and 89-96).
[00190] The ectodomain of the Dengue envelope (E) protein includes three domains that have been identified immunologically (Roehrig, J.T., et al, Virology 5:246(2):317-328 (1998)) and by X-ray crystallographically (Modis, Y., et al, Nature 22:427(6972):313-319 (2004), Modis, Y., et al, J. Virol. 76(24): 13097-13100 (2002)). Domain I (also referred to as "EI") is the central domain and domain II (also referred to as "ΕΠ") is the dimerization domain and contains the fusion peptide. Domain III (also referred to as "ΕΙΠ") is about 100 amino acids in length and contains the putative receptor-binding and the majority of the type-specific neutralizing epitopes (Modis, Y., et al, Nature 22:427(6972):313-319 (2004), Modis, Y., et al, J. Virol.
76(24): 13097-13100 (2002)). (See FIG. 1) Neutralizing antibodies against DENV that bind EIII are speculated to provide passive prophylaxis in rodents (Gromowski G.D., et al, J. Virol.
82(17):8828-37 (2008); Kaufman B.M., et al, Am J Trop Med Hyg Mar; 36(2): 427-34 (1987); Shrestha B., et al, PLoS Pathog Apr;6(4):el 000823 (2010); Sukupolvi-Petty S., et al, J. Virol. Dec;81(23): 12816-26 (2007); Goncalvez A.P., et al, J Virol 2004 Dec;78(23): 12910-8 (2004)). Elll-reactive antibodies produced by mice immunized with virus and boosted with recombinant E protein are largely serotype-specific and do not neutralize all of the genotypes within a given serotype (Shrestha B., et al, PLoS Pathog Apr;6(4):el 000823 (2010)). The role of antibodies to EI/EII is less clear, as they tend to be more cross-reactive and less potent in neutralization (Goncalvez A.P., et al, J Virol 2004 Dec;78(23): 12910-8 (2004)).
[00191] The crystal structure of 5H2 and DENV4 E protein shows the antibody prevents prefusion to postfusion transition, thus, preventing the viral membrane fusion. Fusion proteins that include flagellin and two copies of the EIII generated in E. coli elicited moderate levels of neutralizing antibodies and only provided partial protection against viremia in a nonhuman primate model (Liu, G., et al, Clinical Vaccine and Immunology 22(5):516-525 (2015)). [00192] EXPERIMENTAL PROCEDURES
[00193] Evaluation of in vivo TLR5 activity in mice.
[00194] Groups of 5 female BALB/c mice were immunized subcutaneously at various doses of the fusion proteins. Sera was collected 2-3 hours later and measured for IL-6 levels (pg/ml serum) with a cytokine bead array kit (BD Biosciences).
[00195] Cloning of recombinant Dengue envelope genes.
[00196] The synthetic genes encoding the Dengue envelope protein (80E, 80E+ or 85E) were codon-optimized for Baculovirus expression (DNA2.0 Inc., Menlo Park, CA) and cloned into pAcSG2™ (BD Biosciences) vector. Either the entire 80E+ (Dengue 1, 2 and 4, 1-399, SEQ ID NOS: 93, 94, 96; Dengue 3, 1-397 SEQ ID NO: 95) was used to fuse to the N-terminus of full- length sequence of Salmonella typhimurium fljB (flagellin phase 2, STF2), or the "split format" was employed in which EIEII (Dengue 1, 2 and 4, 1-296 SEQ ID NO: 93, 94, 96; Dengue 3, 1- 294, SEQ ID NO: 95) was fused to the amino-terminus of flagellin and EIII (Dengue 1, 2 and 4, 290-399 of SEQ ID NOS: 93, 94, 96 and Dengue 3, 288-398, SEQ ID NO: 95) was fused to a loop of domain 3 of flagellin. The EIEII domain was linked to the N-terminus of flagellin and remaining EIII domain was inserted in domain 3 of flagellin. The EIEII and EIII domains contain an overlapping 7 amino acids. Flexible linkers (GS)5 (GSGSGSGSGS (SEQ ID NO: 100) was utilized to connect 80% E to flagellin and (GS)3 was used at the C-terminus of EIII domain. The recombinant baculoviruses were generated by co-transfecting Sf9 cells with the recombinant plasmids and BD BaculoGold™ DNA following standard Baculovirus Expression protocol (Invitrogen, Carlsbard, CA). The envelope sequences used in the vaccine constructs are: Dengue 1, PU0359 (Genbank accession code: AAN32784); Dengue 2, Thailand 16681, (Genbank accession code: NP 739583); Dengue 3, Pah881/88, (Genbank accession code:
AAK18606); Dengue 4, 341750, (Genbank accession code: ADA00410).
[00197] Expression and purification of E-flagellin fusion proteins.
[00198] The secreted recombinant vaccine candidates were captured by an affinity column. The envelope protein (E) specific antibody 4G2 (ATCC, Manassas, VA) was coupled to the NHS-activated Sepharose 4 Fast Flow Resin (GE) following the manufacture's protocol. The column was washed and equilibrated. The loaded fusion proteins were captured and eluded. The protein was further purified by size exclusion chromatography (10/24 GL, GE). The final formulation buffer is PBS.
[00199] Evaluation of immunogenicity studies in mice.
[00200] All mouse studies were approved by the Institutional Animal Care and Use
Committee (IACUC) at the Princeton University according to NIH guidelines and IACUC- approved protocols. Female BALB/c mice (6-8 weeks old) were purchased from Charles River Laboratories (Wilmington, Massachusetts) and maintained at the AAALAC-accredited animal facility of Princeton University (Princeton New Jersey). Fusion proteins were formulated in DPBS (2.67 mM KC1, 1.57 mM KH2P04, 137.93 NaCl, & 8.06 mM Na2HP04.7H20, pH 7.2) prior to animal studies. Groups of 5-8 mice were immunized subcutaneously (s.c.) on days 0, 21, and 42, and bled on days 56 or 63. Serum samples were stored at -70°C until use.
[00201] Evaluation of immunogenicity and efficacy in non-human primate study.
[00202] Immunogenicity and efficacy of two TDV formulations were evaluated using an established DENV-2/rhesus macaque infection model (Putnak, et al, J. Inf. Dis. 174: 1176-1184 (1996)). In a DENV/non-human primate model, fusion protein efficacy is measured by reduction in the number of viremic days and/or viremic animals. Groups of four flavivirus antibody negative rhesus macaques (3-5 kg, Covance, Alice, TX) were anesthetized by i.m. injection of a mixture of ketamine (1 lmg/kg) and ACE (0.55 mg per Kg), and immunized i.m. with TDV at a high (96 μg), medium (36 μg) or a low (12 μg) dose on days 0, 28, and 56. On day 117, the monkeys were challenged s.c. with DENV-2 (strain S16803, 104 PFU/animal in 0.5 ml PBS). Blood was collected using Vacutainer venous serum separator tubes (SST, BD) on the days of each immunization and 2 and 4 weeks after each immunization (5 ml each time) and daily on days 117-131. Serum samples were prepared following centrifugation and stored at -70 °C until use.
[00203] Measurement of serum antibodies.
[00204] Virus neutralizing antibodies of immune sera were measured by 50% focus reduction neutralization test (FRNT50) following a procedure similar to that previously described (Liu, G, et al., Clinical Vaccine and Immunology, 2015, 22 (5): 516-525). Briefly, sera were heat inactivated at 56 °C for 30 min, serially 2-fold diluted, and subsequently co-incubated with 30-60 PFU of DENV at 37 °C for 1 hr. The mixtures were added to Vero cells in 24-well or 96-well plates and incubated for 1 hr and subsequently incubated in 1% methylcellulose/EMEM containing 2% FBS and antibiotics. FRNT50 test was performed in 96-well plates (micro- FRNT50) for mouse sera. Virus only and medium only controls were included with each dilution series. After 2-5 day incubation at 37 °C, the monolayers on the plates were fixed and blocked in I-Block solution. Infection foci were reacted with flavivirus group specific monoclonal antibody (4G2) and HRP-conjugated goat anti-mouse IgG, and visualized with True Blue substrate. Foci were counted and FRNT50 titers were calculated by Probit analysis using BioStat 2009 software (AnalystSoft Inc.). If the sample was negative for the first dilution (1 : 10 or 1 :20), the FRNT50 titer was assigned one-half (½) of the first dilution (5 or 10).
EXAMPLE 1 : Split format was selected for DENV1 based on comparison of immunogenicity and production yield. [00205] The immunogenicity of DENVl fusion proteins in Split (BV270,
EIEII.5GS.STF2ng2D3.EIII.3GS), N-term 80E (BV255. E395.3GS.STF2ng2) or N-term 85E (BV271, E426.3GS.STF2ng2) fusion. After three immunizations (3 weeks apart) with 8 μg of each fusion protein, the Split fusion protein elicited greater then about 20-fold higher FRNT50 titers (GMT = 423) than the N-term 80E fusion (GMT = 18) or N-term 85E fusion (GMT = 14) (FIG. 61). Therefore, the Split fusion protein format was selected for DENVl .
[00206] Two DENVl fusion proteins in split formats with a (GS)n linker at the carboxy- terminal amino acid of domain II of DenVl and the amino-terminal amino acid of flagellin and the amino-terminal amino acid of domain III of DENVl where fused to an amino acid of a loop of domain 3 of flagellin (BV293B, BV270B) elicited moderate neutralizing antibody titers. Since BV270, which is based on the E sequence of strain 16007, was produced at an extremely low yield, another Split DENVl fusion protein based on strain PU0359 (BV293) of DENVl was generated. BV293 was expressed and purified at a higher yield compared to BV270. The immunogenicity of DENVl fusion protein BV270B (strain 16007 without His-tag) and BV293B (strain PU0159 without His-tag) was compared at three dose levels in mice. As shown in FIG. 62, BV293B elicited slightly higher neutralizing antibody titers compared to BV270B.
Generally, overall, BV27B and BV293B are similarly immunogenic in mice. However, B V293B can be produced at a higher yield. Therefore, BV293B (Split format) was selected for further development of a composition that included DENVl to stimulate an immune response, in particular a protective immune response.
EXAMPLE 2: DENV2 fusion proteins in split format (BV239) elicited superior neutralizing antibody titers compared to other fusion proteins in N-term 80E candidate (BV237), D3Ins 80E (BV242) or alternative Split (BV241) formats.
[00207] The immunogenicity of DENV2 fusion protein BV239 (Split,
EIEII.STF2ng2D3.EIII), BV241 ("alternative spilt format" fuses domain III to the amino- terminus of flagellin and domains I and II to a loop of domain 3 of flagellin,
EIII.STF2ng2D3.EIEH), BV237 (N-term 80E, E395.3GS.STF2ng2) and BV242 (D3Ins 80E, STF2ng2D3. E395) was compared at 2 dose levels in mice. As shown in FIG. 63, BV239 elicited robust neutralizing antibodies in a dose-dependent manner with a FRNT50 titer of 1940 at 10 μg. In contrast, DENV2 fusion proteins in other formats, including an N-terminal, 85E+ and alternative split formats did not induce significant neutralizing titers. Therefore, the split format was selected as for use in a fusion protein that included DENV2.
EXAMPLE 3 : DENV2 fusion proteins in split format with two linkers (BV263) elicited superior neutralizing antibody titers compared to those without linker (BV239) or with a linker (BV257). [00208] The immunogenicity of DENV2 Split fusion proteins of BV239 (no linker,
EIEII.STF2ng2D3.EIII), BV257 (one amino acid GSn linker between the carboxy -terminal amino acid of domain II of the dengue antigen and the amino-terminal amino acid of flagellin, EIEII.5GS.STF2ng2D3.EIII) and BV263 (EIEII.5GS.STF2ng2D3.EIII.3GS, a (GS)n linker at the carboxy -terminal amino acid of domain II of DenV2 and the amino-terminal amino acid of flagellin and the amino-terminal amino acid of domain III of DENV2 where fused to an amino acid of a loop of domain 3 of flagellin was compared in mice. As shown in FIG. 64, BV263 elicited the highest neutralizing antibody titers in a dose-dependent manner with a FRNT50 titer of 2032 at 5 μg. A significant titer was also seen at the 1 μg dose level. Therefore, split fusion proteins with two amino acid linkers, such as a fusion protein of BV263, were selected for fusion proteins that include DENV2 antigens and flagellin. The amino acid linkers are units of glycine and serine (GS)n. One of the amino acid linkers is at the carboxy-terminal amino acid of domain II of DenV2 and the amino-terminal amino acid of flagellin and the other amino acid linker is at the amino-terminal amino acid of domain III of DENV2 where fused to an amino acid of a loop of domain 3 of flagellin. This format is referred herein as "2 linkers."
EXAMPLE 4: Two DENV3 fusion proteins in either Split (with 2 linkers, BV269B) or N-term 80E+ (BV272B) format elicited moderate neutralizing antibody titers.
[00209] The immunogenicity of DENV3 fusion proteins BV269B
(EIEII.5GS.STF2ng2D3.EIII.3GS) and BV272B (E398.STF2ng2) at 1-9 μg was compared in mice. As shown in FIG. 65, both elicited robust neutralizing antibody titers at a dose as low as 1 μg, BV272B may be more immunogenic as evidenced by about 2.3 to about 3.4 fold higher titers, when delivered as a monovalent vaccine. However, production of BV272B is more difficult than BV269B. The latter is also highly immunogenic in a TDV formulation (Table 1). Therefore, the Split fusion protein B V269B was selected for the further development of TDV formulations.
EXAMPLE 5: Two DENV4 fusion proteins in either split (with 2 linkers, BV316) or N-term 80E+ (BV243B) format elicited moderate neutralizing antibody titers.
[00210] The immunogenicity of DENV4 fusion proteins BV316.
(EIEII.5GS.STF2ng2D3.EIII.3GS) and BV243B (E399.STF2ng2) at 1-10 μg was compared in mice. As shown in FIG. 66, both fusion proteins elicited robust neutralizing antibody titers, ranging from about 20 to about 202 for BV243B and about 45 to about 254 for BV316, respectively. Although the trend of titers was slightly higher for BV316, the difference is not significant. Both fusion proteins appear to be similarly immunogenic when delivered as a monovalent composition. EXAMPLE 6: Three tetravalent dengue (TDV) compositions each consisting of 4 fusion proteins elicited moderate to high levels of neutralizing antibodies.
[00211] The immunogenicity of three tetravalent formulations was examined in mice. Groups of 6-8 BALB/c mice were immunized S.C. with the indicated TDV formulations listed in Table 1 or monovalent fusion proteins on days 0, 21, and 42. Sera were collected on day 56 and subjected to FRNT50 test and expressed as GMTs. As shown in Table 1, all three TDV formulations elicited significant levels of neutralizing antibodies. A suitable TDV formulation is expected to induce balanced neutralizing antibody titers against the 4 dengue serotypes to minimize the possibility of causing a potential ADE. Among the three formulations, TDV1 appears to induce more balanced titers to all 4 serotypes (about 320 to about 1660). Therefore, a TDV formulations consisting of 4 Split fusion proteins was further evaluated in a non-human primate animal model described below.
Table 1. Immunogenicity of TDV formulations in mice
Figure imgf000050_0001
EXAMPLE 7. Two tetravalent dengue vaccine (TDV) formulations reduced the mean days of viremia by 2 days in non-human primates.
[00212] The immunogenicity and efficacy of three TDV formulations listed in Table 2 were evaluated in an established DENV2/NFIP model. Three TDV formulations consisting of 24 meg each (TDV96), 9 meg each (TDV36), or 3 meg each (TDV 12). As shown in Table 2, all three TDV formulations elicit low neutralizing antibody titers with higher titers of DENV-2 (about 10 - about 70). Seventy five to 100% monkeys immunized with TDV96 and TDV36 are seroconverted (FRNT50 >10). Two months after the last immunization, all the monkeys were challenged with DENV2 virus and sera were collected for 14 consecutive days to measure the viremia and virus titers. Consistent with low neutralizing antibody titers, animals showed breakthrough viremia indicating that the TDV formulations may need to be altered or augmented to provide sufficient protection against viremia. The mean day of viremia in the TDV96 and TDV36 groups was reduced by 2 days (FIG. 67). It is possible that the decreased potency may be a consequence of current recombinant production methods that result in suboptimal folding of the antigen or the flagellin that reduces TLR5 activation by the flagellin and decreased antigen presentation.
Table 2. Geometric mean neutralizing antibody titers (GMT) of Day 117 sera from monkeys following immunizations (days 0, 18, 56) with three TDV formulations.
Table 2
Figure imgf000051_0001
[00213] Groups of 4 rhesus macaques were immunized I.M. with the indicated TDV formulations or monovalent candidates on days 0, 28, and 56. Sera were collected on day 84 and subjected to FRNT50 test and expressed as GMTs.
[00214] A tetravalent dengue composition, which includes 80E+ immunogens of 4 DENV serotypes fused to flagellin, may potentially serve as an improved subunit dengue vaccine. The target populations include adults and children living in the endemic countries as well as travelers and military personnel.
[00215] Example 8. Fusion proteins induced cytokine IL-6 in mice following primary immunization.
[00216] Groups of five mice were immunized with the indicated doses of fusion proteins (FIGs. 68 and 69). Sera were harvested 2-3 hours later and evaluated for IL-6 production. Mice receiving the buffer PBS alone were included as negative controls. Geometric mean IL-6 levels (pg/ml of sera) are shown above each data set. The serotype of Dengue construct evaluated is provided at the top of each dataset. The construct designation (e.g. BV 270B or BV263B is provided either on the X axis (FIG. 68) or below the serotyped designation (FIG. 69). The production lot evaluated (e.g. R007 or R003) is provided in FIG. 69. [00217] Fusion proteins that include DENV1 , DENV2, DENV3 and DENV4 antigens induced significant levels of IL-6 levels (FIGs. 68 and 69). Therefore, the flagellin/dengue antigen fusion proteins can activate TLR5 pathway signaling and are TLR5 agonists.
[00218] The teachings of all patents, published applications and references cited herein, including U.S. Patent Nos: 8,420,102; 8,574,588; 8,932,605 and 8,932,598, are incorporated by reference in their entirety.
[00219] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS s claimed is:
A composition comprising:
a) a first fusion protein that activates Toll-like Receptor 5, the first fusion protein including:
i) a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and
ii) a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin;
b) a second fusion protein that activates Toll-like Receptor 5, the second fusion protein including:
i) a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and
ii) a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin;
c) a third fusion protein that activates Toll-like Receptor 5, the third fusion protein including:
i) a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and
ii) a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin; and
d) a fourth fusion protein that activates Toll-like Receptor 5, the fourth fusion protein including:
i) a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin: and
ii) a domain I and a domain II of the second Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
The composition of Claim 1, further including a linking region between at least one of: a) the carboxy-terminal amino acid of the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin,
b) the carboxy-terminal amino acid of the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin, c) the carboxy-terminal amino acid of the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin; and
d) the carboxy-terminal amino acid of the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
The composition of Claims 1 or 2, further including a linker between at least one of: a) the carboxy-terminal amino acid of the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin,
b) the carboxy-terminal amino acid of the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin,
c) the carboxy-terminal amino acid of the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin; and
d) the carboxy-terminal amino acid of the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
The composition of Claim 3, wherein the linker is an amino acid linker.
The composition of Claim 1, further including a linker between at least one of:
a) the carboxy-terminal amino acid of the domain I of the Dengue 1 viral antigen and the amino-terminal amino acid of the first flagellin,
b) the carboxy-terminal amino acid of the domain I of the Dengue 2 viral antigen and the amino-terminal amino acid of the second flagellin,
c) the carboxy-terminal amino acid of the domain I of the Dengue 3 viral antigen and the amino-terminal amino acid of the third flagellin, and
d) the carboxy-terminal amino acid of domain I of the Dengue 4 viral antigen and the amino-terminal amino acid of the fourth flagellin.
The composition of Claim 5, wherein the linker is an amino acid linker.
The composition of Claim 1, further including an adjuvant.
The composition of Claim 1, further including at least one Toll-like Receptor agonist selected from the group consisting of a Toll-like Receptor 1 agonist, a Toll-like Receptor 2 agonist, a Toll-like Receptor 3 agonist, a Toll-like Receptor 4 agonist, a Toll-like Receptor 5 agonist, a Toll-like Receptor 6 agonist, a Toll-like Receptor 7 agonist, a Tolllike Receptor 8 agonist, a Toll-like Receptor 9 agonist, a Toll-like Receptor 10 agonist, a Toll-like Receptor 11 agonist, a Toll-like Receptor 12 agonist and a Toll-like Receptor 13 agonist.
9. The composition of Claim 8, wherein the Toll-like Receptor agonist is the Toll-like
Receptor 4 agonist.
10. The composition of Claim 9, wherein the Toll-like Receptor 4 agonist is a
lipopolysaccharide.
11. The composition of Claim 10, wherein the Toll-like Receptor agonist is the Toll-like Receptor 9 agonist.
12. The composition of Claim 11, wherein the Toll-like Receptor 9 agonist is CpG
oligodeoxynucleotides.
13. The composition of Claim 8, wherein the Toll-like Receptor agonist is at least one
member selected from the group consisting of a Toll-like Receptor 3 agonist, a Toll-like Receptor 7 agonist and a Toll-like Receptor 8 agonist.
14. The composition of Claim 1, further including at least one junction loop peptide between at least one member selected from the group consisting of:
a) the domain I of the Dengue 1 viral antigen and the amino-terminus of the first flagellin;
b) the domain I of the Dengue 2 viral antigen and the amino-terminus of the second flagellin;
c) the domain I of the Dengue 3 viral antigen and the amino-terminus of the third flagellin;
d) the domain I of the Dengue 4 viral antigen and the amino-terminus of the fourth flagellin;
e) the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin;
f) the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin;
g) the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin; and
h) the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
15. A method of stimulating an immune response to a Dengue flavivirus antigen in a subject, comprising the step of administering to the subject a composition that includes:
a) a first fusion protein that activates Toll-like Receptor 5, the first fusion protein including:
i) a domain III of a Dengue 1 viral antigen fused to a loop of domain 3 of a first flagellin; and
ii) a domain I and a domain II of the Dengue 1 viral antigen fused to the amino-terminus of the first flagellin;
b) a second fusion protein that activates Toll-like Receptor 5, the second fusion protein including:
i) a domain III of a Dengue 2 viral antigen fused to a loop of domain 3 of a second flagellin; and
ii) a domain I and a domain II of the Dengue 2 viral antigen fused to the amino-terminus of the second flagellin;
c) a third fusion protein that activates Toll-like Receptor 5, the third fusion protein including:
i) a domain III of a Dengue 3 viral antigen fused to a loop of domain 3 of a third flagellin; and
ii) a domain I and a domain II of the Dengue 3 viral antigen fused to the amino-terminus of the third flagellin; and
d) a fourth fusion protein that activates Toll-like Receptor 5, the fourth fusion
protein including:
i) a domain III of a Dengue 4 viral antigen fused to a loop of domain 3 of a fourth flagellin: and
ii) a domain I and a domain II of the second Dengue 4 viral antigen fused to the amino-terminus of the fourth flagellin.
16. The method of Claim 15, wherein the composition administered to the subject further includes a linker region between at least one of:
a) the carboxy-terminal amino acid of the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin,
b) the carboxy-terminal amino acid of the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin,
c) the carboxy-terminal amino acid of the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin; and d) the carboxy-terminal amino acid of the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
17. The method of Claims 14 or 15, wherein the composition administered to the subject further includes a linker between at least one of:
a) the carboxy-terminal amino acid of the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin,
b) the carboxy-terminal amino acid of the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin,
c) the carboxy-terminal amino acid of the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin; and
d) the carboxy-terminal amino acid of the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
18. The method of Claim 17, wherein the linker is an amino acid linker.
19. The method of Claim 15, wherein the composition administered to the subject further includes a linker between at least one of:
a) the carboxy-terminal amino acid of the domain I of the Dengue 1 viral antigen and the amino-terminal amino acid of the first flagellin,
b) the carboxy-terminal amino acid of the domain I of the Dengue 2 viral antigen and the amino-terminal amino acid of the second flagellin,
c) the carboxy-terminal amino acid of the domain I of the Dengue 3 viral antigen and the amino-terminal amino acid of the third flagellin, and
d) the carboxy-terminal amino acid of domain I of the Dengue 4 viral antigen and the amino-terminal amino acid of the fourth flagellin.
20. The method of Claim 19, wherein the linker is an amino acid linker.
21. The method of Claim 15, further including the step of administering an adjuvant to the subject.
22. The method of Claim 15, wherein the composition administered to the subject further includes at least one Toll-like Receptor agonist selected from the group consisting of a Toll-like Receptor 1 agonist, a Toll-like Receptor 2 agonist, a Toll-like Receptor 3 agonist, a Toll-like Receptor 4 agonist, a Toll-like Receptor 5 agonist, a Toll-like Receptor 6 agonist, a Toll-like Receptor 7 agonist, a Toll-like Receptor 8 agonist, a Toll- like Receptor 9 agonist, a Toll-like Receptor 10 agonist, a Toll-like Receptor 1 1 agonist, a T Toll-like Receptor 12 agonist and a Toll-like Receptorl3 agonist.
23. The method of Claim 22, wherein the Toll-like Receptor agonist is the Toll-like Receptor 4 agonist.
24. The method of Claim 23, wherein the Toll-like Receptor 4 agonist is a
lipopolysaccharide.
25. The method of Claim 22, wherein the Toll-like Receptor agonist is the Toll-like Receptor 9 agonist.
26. The method of Claim 25, wherein the Toll-like Receptor 9 agonist is CpG
oligodeoxynucleotides.
27. The method of Claim 22, wherein the Toll-like Receptor agonist is at least one member selected from the group consisting of a Toll-like Receptor 3 agonist, a Toll-like Receptor 7 agonist and a Toll-like Receptor 8 agonist.
28. The method of Claim 15, wherein the composition administered to the subject further includes at least one junction loop peptide between at least one of the following:
a) the domain I of the Dengue 1 viral antigen and the amino-terminus of the first flagellin;
b) the domain I of the Dengue 2 viral antigen and the amino-terminus of the second flagellin;
c) the domain I of the Dengue 3 viral antigen and the amino-terminus of the third flagellin;
d) the domain I of the Dengue 4 viral antigen and the amino-terminus of the fourth flagellin;
e) the domain III of the Dengue 1 viral antigen and the loop of domain 3 of the first flagellin;
f) the domain III of the Dengue 2 viral antigen and the loop of domain 3 of the second flagellin;
g) the domain III of the Dengue 3 viral antigen and the loop of domain 3 of the third flagellin; and
h) the domain III of the Dengue 4 viral antigen and the loop of domain 3 of the fourth flagellin.
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