WO2008133759A2 - Antiviral peptides - Google Patents

Antiviral peptides Download PDF

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Publication number
WO2008133759A2
WO2008133759A2 PCT/US2008/000452 US2008000452W WO2008133759A2 WO 2008133759 A2 WO2008133759 A2 WO 2008133759A2 US 2008000452 W US2008000452 W US 2008000452W WO 2008133759 A2 WO2008133759 A2 WO 2008133759A2
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WO
WIPO (PCT)
Prior art keywords
seq
xaa
peptide
xaai
absent
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Application number
PCT/US2008/000452
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French (fr)
Other versions
WO2008133759A3 (en
Inventor
Francis V. Chisari
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The Scripps Research Institute
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Publication of WO2008133759A2 publication Critical patent/WO2008133759A2/en
Publication of WO2008133759A3 publication Critical patent/WO2008133759A3/en

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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

  • Viral diseases can be difficult to treat because viruses enter mammalian cells, where they perform many of their functions, including transcription and translation of viral proteins, as well as replication of the viral genome. Thus, viruses are protected not only from the host's immune system, but also from medicines administered to the host, as the viral infection progresses.
  • the invention relates to peptides that can prevent or treat infection by human immunodeficiency virus and members of the Flaviviridae family.
  • the invention also relates to peptides that can inactivate these viruses before they enter a cell.
  • the invention is based on the discovery of certain antiviral peptides some of which have an amphipathic alpha-helical structure and particular amino acid compositions.
  • many of the present peptides inactivate viruses that are free in solution, thereby preventing viral infection of mammalian cells.
  • the present peptides are derived from and/or are related the Hepatitis C polyprotein, but are capable of inactivating flaviviruses, paramyxoviruses, and human immunodeficiency virus (HIV).
  • the invention involves the discovery that an amphipathic alpha-helical peptide (SEQ ID NO: 43) derived from the membrane anchor domain of the Hepatitis C virus (HCV) NS5A protein is virocidal for HCV, i.e. it is capable of inactivating HCV, at nanomolar concentrations in vitro.
  • the peptide prevents de novo HCV infection and suppresses ongoing infection by inactivating both extracellular and intracellular infectious particles.
  • the peptide inhibits infection by selected flaviviruses, paramyxoviruses, and HIV.
  • the peptide is also nontoxic in vitro and in vivo at doses at least 100-fold higher than required for antiviral activity. Its amphipathic alpha-helical structure is necessary but not sufficient for its virocidal activity, which depends on its amino acid composition but not its primary sequence or its chirality.
  • the invention provides an isolated peptide of 14 to 50 D- or L- amino acids in length comprising an amino acid sequence of formula I: Xaa i -Xaa 2 -Xaa 3 -W-L-Xaa 6 -Xaa 7 -Xaa 8 -W-Xaa i O -W-Xaa 1 2 -Xaai 3 -Xaa 14 -
  • Xaai 5 -Xaai 6 -Xaai 7 -Xaa 18 - Xaai9-Xaa 20 -Xaa 2 rXaa 2 2 (SEQ ID NO: 163), wherein: Xaai is serine (S) or absent; Xaa 2 is glycine (G) or absent; Xaa 3 is serine (S), aspartic acid (D) or threonine (T); W is tryptophan; L is leucine; Xaa 6 is arginine (R), aspartic acid (D), lysine (K), tryptophan (W) or glutamic acid (E); Xaa 7 is aspartic acid (D), arginine (R), glutamic acid (E), alanine (A), isoleucine (I) or lysine (K); Xaa 8 is isoleucine (I) or valine (V); Xaaio is as
  • Xaai 8 - Xaai 9 -Xaa 20 -Xaa 2 i-Xaa 22 (SEQ ID NO: 164) , wherein: Xaai, Xaa 2 , Xaa 21 and Xaa 22 are absent; S is serine; W is tryptophan; L is leucine; Xaa ⁇ 5 is arginine (R), aspartic acid (D), glutamic acid (E) or lysine (K); Xaa 7 is arginine (R), aspartic acid (D) or lysine (K); I is isoleucine; W is tryptophan; Xaaio is aspartic acid (D), arginine (R), or lysine (K); C is cysteine; Xaa ⁇ is lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D); V is valine; Xaai 7 is serine
  • Xaai 8 -Xaai 9 -Xaa 20 -Xaa 2 i-Xaa 22 (SEQ ID NO: 178), wherein: Xaai, Xaa 2 , Xaa 21 and Xaa 22 are absent; S is serine; W is tryptophan; L is leucine; R is arginine; I is isoleucine; C is cysteine; V is valine; Xaa 7 is aspartic acid (D), arginine (R), or lysine (K); Xaaio is arginine (R) or lysine (K); Xaa ]4 is lysine (K), glutamic acid (E) or aspartic acid (D); Xaa 17 is serine (S) or absent; Xaai 8 is arginine (R), lysine (K) or absent; Xaai 9 is phenylalanine (F) or absent; and
  • a peptide of the invention comprises an amino acid sequence of formula IV:
  • Xaai8-Xaa 19 -Xaa 20 -Xaa 2 i-Xaa 22 (SEQ ID NO: 184), wherein: Xaai, Xaa 2 , Xaa 21 and Xaa 22 are absent; S is serine; W is tryptophan; L is leucine; I is isoleucine; C is cysteine; V is valine;; and Xaa ⁇ Xaa- 7 , Xaaio and Xaai4 are arginine (R) or lysine (K); Xaa ⁇ is serine (S) or absent; Xaai 8 is arginine (R), lysine (K) or absent; Xaai 9 is phenylalanine (F) or absent; and Xaa 20 is arginine (R), lysine (K) or absent.
  • the invention provides isolated peptide of 14 to 50 D- or L- amino acids in length
  • SLVECIWDWIDRLWS SEQ ID NO: 101
  • LVECIWD WIDRLWS SEQ ID NO: 102
  • a peptide of the invention comprises an amino acid sequence of formula VI: Xaai -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 -Xaa 6 -L- V-Xaa 9 -C-I-W-
  • Xaai 3 -W-I-Xaa, 6 -Xaa I7 -L-W-S- Xaa 2 i-Xaa 22 (SEQ ID NO: 596), wherein: Xaa ⁇ Xaa 2 , Xaa 2 t and Xaa 22 are absent; Xaa 3 is lysine (K), glutamic acid (E), aspartic acid (D), arginine (R) or absent; Xaa 4 is phenylalanine (F) or absent; Xaa 5 is aspartic acid (D), arginine (R), lysine (K) or absent; Xaa ⁇ $ is serine (S) or absent; L is leucine; V is valine; Xaa 9 is lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D); C is cysteine; I is isoleucine; W is tryptophan; Xa
  • a peptide of the invention comprises an amino acid sequence of formula VIII:
  • the isolated peptide comprises an amino acid sequence selected from the group consisting of:
  • SWRLDIWDWICESVLDFK (SEQ ID NO: 1 19), DWLRIIWDWVCSVVSDFK (SEQ ID NO: 123), SWLWEVWDWVLHVLSDFK (SEQ ID NO: 124),
  • TWLRAIWDWVCTALTDFK (SEQ ID NO: 125), SWLRDVWDWVCTVLSDFK (SEQ ID NO: 126), SWLRDIWDWISEVLSDFK (SEQ ID NO: 127), SWLDRIWRWICKVLSRFE (SEQ ID NO: 128), SWLDDIWDWICEVLSDFE (SEQ ID NO: 129),
  • SWLRRIWRWICKVLSRFK (SEQ ID NO: 130), SWLKEIWEWICDVLSEFR (SEQ ID NO: 131 ), SWLKDIWDWICEVLSDFR (SEQ ID NO: 132), SWLKDIWDWICEVLSDFK (SEQ ID NO: 133), SWLREIWEWICDVLSEFK (SEQ ID NO: 134), SWLREIWEWICEVLSEFK (SEQ ID NO: 135), SWLDRIWRWICKVLSRFE (SEQ ID NO: 136), SWLDDIWDWICEVLSDFE (SEQ ID NO: 137), SWLRRIWRWICKVLSRFK (SEQ ID NO : 138), '
  • SWLRDIWDWIREVLSDFK (SEQ ID NO: 139), SWLRDIWDWIEEVLSDFK (SEQ ID NO: 140), SGSWLRDIWDWICEVLSDFK (SEQ ID N0:141), GSWLRDIWDWICEVLSDFK (SEQ ID NO: 142), SWLRDIWDWICEVLSDFKT (SEQ ID NO: 143),
  • SWLRDIWDWICEVLSDFKTW (SEQ ID NO: 144), SWLEKIWKWICRVLSKFD (SEQ ID NO: 165), SWLRKIWKWICEVLSDFK (SEQ ID NO: 166), SWLRDIWDWICKVLSKFK (SEQ ID NO: 167), SWLRRIWRWICEVLSDFK (SEQ ID NO: 168),
  • SWLRDIWDWICRVLSRFK (SEQ ID NO: 169), SWLRRIWDWICRVLSDFK (SEQ ID NO: 170), SWLRKIWDWICKVLSDFK (SEQ ID NO: 171), SWLRRIWDWICEVLSRFK (SEQ ID NO: 172), SWLRKIWDWICEVLSKFK (SEQ ID NO: 173),
  • SWLRDIWRWICRVLSDFK (SEQ ID NO: 174), SWLRDIWKWICKVLSDFK (SEQ ID NO: 175), SWLDRIWDWICRVLSRFK (SEQ ID NO: 176), SWLRDIWDWICKVLSKFK (SEQ ID NO: 177), SWLRDIWRWICKVLSRFK (SEQ ID NO: 179),
  • SWLRDIWKWICKVLSKFK (SEQ ID NO: 180), SWLRKIWKWICEVLSKFK (SEQ ID NO: 181), SWLRRIWRWICEVLSRFK (SEQ ID NO: 182), SWLRRIWRWICDVLSRFK (SEQ ID NO: 183), SWLRKIWKWICKVLSKFK (SEQ ID NO: 185), SWLRRIWRWICRVLSRFK (SEQ ID NO: 186), SWLRRIWRWICRVLSRFR (SEQ ID NO: 187), SWLKKIWKWICKVLSKFK (SEQ ID NO: 188), SWRLDIWDWICESVLDF (SEQ ID NO: 189),
  • SWRLDIWDWICESVLD (SEQ ID NO: 190), SWRLDIWDWICESVL (SEQ ID NO: 191), SWRLDIWDWICESV (SEQ ID NO: 192), DWLRIIWDWVCSVVSDF (SEQ ID NO: 193), DWLRIIWDWVCSVVSD (SEQ ID NO: 194),
  • DWLRIIWDWVCSVVS (SEQ ID NO: 195), DWLRIIWDWVCSVV (SEQ ID NO: 196), SWLWEVWDWVLHVLSDF (SEQ ID NO: 197), SWLWEVWDWVLHVLSD (SEQ ID NO: 198), SWLWEVWDWVLHVLS (SEQ ID NO: 199),
  • SWLWEVWDWVLHVL (SEQ ID NO: 200), TWLRAIWDWVCTALTDF (SEQ ID NO: 201), TWLRAIWDWVCTALTD (SEQ ID NO: 202), TWLRAIWDWVCTALT (SEQ ID NO: 203), TWLRAIWDWVCTAL (SEQ ID NO: 204),
  • SWLRDVWDWVCTVLSDF (SEQ ID NO: 205), SWLRDVWDWVCTVLSD (SEQ ID NO: 206), SWLRDVWDWVCTVLS (SEQ ID NO: 207), SWLRDVWDWVCTVL (SEQ ID NO: 208), SWLRDIWDWISEVLSDF (SEQ ID NO: 209),
  • SWLRDIWDWISEVLSD (SEQ ID NO: 210), SWLRDIWDWISEVLS (SEQ ID NO: 21 1), SWLRDIWDWISEVL (SEQ ID NO: 212), SWLDRIWRWICKVLSRF (SEQ ID NO: 213), SWLDRIWRWICKVLSR (SEQ ID NO: 214), SWLDRIWRWICKVLS (SEQ ID NO: 215), SWLDRIWRWICKVL (SEQ ID NO: 216), SWLDDIWDWICEVLSDF (SEQ ID NO: 217), SWLDDIWDWICEVLSD (SEQ ID NO: 218),
  • SWLDDIWDWICEVLS (SEQ ID NO: 219), SWLDDIWDWICEVL (SEQ ID NO: 220), SWLRRIWRWICKVLSRF (SEQ ID NO:221), SWLRRIWRWICKVLSR (SEQ ID NO: 222), SWLRRIWRWICKVLS (SEQ ID NO: 223),
  • SWLRRIWRWICKVL (SEQ ID NO: 224), SWLKEIWEWICDVLSEF (SEQ ID NO: 225), SWLKEIWEWICDVLSE (SEQ ID NO: 226), SWLKEIWEWICDVLS (SEQ ID NO: 227), SWLKEIWEWICDVL (SEQ ID NO: 228),
  • SWLKDIWDWICEVLSDF (SEQ ID NO: 229), SWLKDIWDWICEVLSD (SEQ ID NO: 230), SWLKDIWDWICEVLS (SEQ ID NO: 231), SWLKDIWDWICEVL (SEQ ID NO: 232), SWLKDIWDWICEVLSDF (SEQ ID NO: 233),
  • SWLKDIWDWICEVLSD (SEQ ID NO: 234), SWLKDIWDWICEVLS (SEQ ID NO: 235), SWLKDIWDWICEVL (SEQ ID NO: 236), SWLREIWEWICDVLSEF (SEQ ID NO: 237), SWLREIWEWICDVLSE (SEQ ID NO: 238),
  • SWLREIWEWICDVLS (SEQ ID NO: 239), SWLREIWEWICDVL (SEQ ID NO: 240), SWLREIWEWICEVLSEF (SEQ ID NO: 591), SWLREIWEWICEVLSE (SEQ ID NO: 592), SWLREIWEWICEVLS (SEQ ID NO: 593), SWLREIWEWICEVL (SEQ ID NO: 594), SWLDRIWRWICKVLSRF (SEQ ID NO: 241), SWLDRIWRWICKVLSR (SEQ ID NO: 242), SWLDRIWRWICKVLS (SEQ ID NO: 243),
  • SWLDRIWRWICKVL SEQ ID NO: 244
  • SWLDDIWDWICEVLSDF SEQ ID NO: 245
  • SWLDDIWDWICEVLSD SEQ ID NO: 246
  • SWLDDIWDWICEVLS SEQ ID NO: 247
  • SWLDDIWDWICEVL SEQ ID NO: 248
  • SWLRRIWRWICKVLSRF (SEQ ID NO: 249), SWLRRIWRWICKVLSR (SEQ ID NO: 250), SWLRRIWRWICKVLS (SEQ ID NO: 251), SWLRRIWRWICKVL (SEQ ID NO: 252), SWLRDIWDWIREVLSDF (SEQ ID NO: 253),
  • SWLRDIWDWIREVLSD (SEQ ID NO: 254), SWLRDIWDWIREVLS (SEQ ID NO: 255), SWLRDIWDWIREVL (SEQ ID NO: 256), SWLRDIWDWIEEVLSDF (SEQ ID NO: 257), SWLRDIWDWIEEVLSD (SEQ ID NO: 258),
  • SWLRDIWDWIEEVLS (SEQ ID NO: 259), SWLRDIWDWIEEVL (SEQ ID NO: 260), SGSWLRDIWDWICEVLSDF (SEQ ID NO: 261), SGSWLRDIWDWICEVLSD (SEQ ID NO: 262), SGSWLRDIWDWICEVLS (SEQ ID NO: 263),
  • SGSWLRDIWDWICEVL (SEQ ID NO: 264), GSWLRDIWDWICEVLSDF (SEQ ID NO: 265), GSWLRDIWDWICEVLSD (SEQ ID NO: 266), GSWLRDIWDWICEVLS (SEQ ID NO: 267), GSWLRDIWDWICEVL (SEQ ID NO: 268), SWLRDIWDWICEVLSDFK (SEQ ID NO: 269), SWLRDIWDWICEVLSDF (SEQ ID NO: 270), SWLRDIWDWICEVLSD (SEQ ID NO: 271), SWLRDIWDWICEVLS (SEQ ID NO: 272),
  • SWLRDIWDWICEVLSDFKT (SEQ ID NO: 273), SWLRDIWDWICEVLSDFK (SEQ ID NO: 274), SWLRDIWDWICEVLSDF (SEQ ID NO: 275), SWLRDIWDWICEVLSD (SEQ ID NO: 276), KFDLVSECIWDWIDLRWS (SEQ ID NO: 277),
  • VSECIWDWIDLRWS (SEQ ID NO: 281), KFDSVVSCVWDWIIRLWD (SEQ ID NO: 282),
  • VVSCVWDWIIRLWD SEQ ID NO: 286)
  • KFDSLVHLVWDWVEWLWS SEQ ID NO: 288)
  • FDSLVHLVWDWVEWLWS (SEQ ID NO: 289), DSLVHLVWDWVEWLWS (SEQ ID NO:290), SLVHLVWDWVEWLWS (SEQ ID NO: 291), LVHLVWDWVEWLWS (SEQ ID NO: 292), KFDTLATCVWDWIARLWT (SEQ ID NO: 293),
  • LATCVWDWIARLWT (SEQ ID NO: 297), KFDSLVTCVWDWVDRLWS (SEQ ID NO: 298),
  • SLVTCVWDWVDRLWS (SEQ ID NO: 301), LVTCVWDWVDRLWS (SEQ ID NO: 302),
  • SLVESIWDWIDRLWS (SEQ ID NO: 306)
  • LVESIWDWIDRLWS (SEQ ID NO: 307)
  • DSLVECIWDWIDKLWS (SEQ ID NO: 330), SLVECIWDWIDKLWS (SEQ ID NO: 331),
  • DSLVECIWDWIDKLWS (SEQ ID NO: 335), SLVECIWDWIDKLWS (SEQ ID NO: 336),
  • LVECIWDWIDKLWS (SEQ ID NO: 337), KFESLVDCIWEWIERLWS (SEQ ID NO: 338), FESLVDCIWEWIERLWS (SEQ ID NO: 339), ESLVDCIWEWIERLWS (SEQ ID NO: 340), SLVDCIWEWIERLWS (SEQ ID NO: 341),
  • DSLVECIWDWIDDLWS (SEQ ID NO: 355), SLVECIWDWIDDLWS (SEQ ID NO: 356),
  • FRSLVKCIWRWIRRLWS (SEQ ID NO: 359), RSLVKCIWRWIRRLWS (SEQ ID NO: 360),
  • FDSLVERIWDWIDRLWS (SEQ ID NO: 364), DSLVERIWDWIDRLWS (SEQ ID NO: 365),
  • FDSLVEEIWDWIDRLWS (SEQ ID NO: 369), DSLVEEIWDWIDRLWS (SEQ ID NO : 370),
  • SLVEEIWDWIDRLWS (SEQ ID NO: 371), LVEEIWDWIDRLWS (SEQ ID NO: 372), KFDSLVECIWDWIDRLWSGS (SEQ ID NO: 373), FDSLVECIWDWIDRLWSGS (SEQ ID NO: 374), DSLVECIWDWIDRLWSGS (SEQ ID NO: 375),
  • SLVECIWDWIDRLWSGS (SEQ ID NO: 376), LVECIWDWIDRLWSGS (SEQ ID NO: 377), KFDSLVECIWDWIDRLWSG (SEQ ID NO: 378), FDSLVECIWDWIDRLWSG (SEQ ID NO: 379), DSLVECIWDWIDRLWSG (SEQ ID NO: 380),
  • KFDSLVECIWDWIDRLWS (SEQ ID NO: 384), FDSLVECIWDWIDRLWS (SEQ ID NO: 385), DSLVECIWDWIDRLWS (SEQ ID NO: 386), SLVECIWDWIDRLWS (SEQ ID NO: 387), WTKFDSLVECIWDWIDRLWS (SEQ ID NO: 388), TKFDSLVECIWDWIDRLWS (SEQ ID NO: 389),
  • KFDSLVECIWDWIDRLWS (SEQ ID NO: 390), FDSLVECIWDWIDRLWS (SEQ ID NO: 391), DSLVECIWDWIDRLWS (SEQ ID NO: 392), SWLEKIWKWICRVLSKF (SEQ ID NO: 393), SWLEKIWKWICRVLSK (SEQ ID NO: 394),
  • SWLEKIWKWICRVLS SEQ ID NO: 395), SWLEKIWKWICRVL (SEQ ID NO: 396), SWLRKIWKWICEVLSDF (SEQ ID NO: 397), SWLRKIWKWICEVLSD (SEQ ID NO: 398), SWLRKIWKWICEVLS (SEQ ID NO: 399),
  • SWLRKIWKWICEVL SEQ ID NO: 400
  • SWLRDIWDWICKVLSKF SEQ ID NO: 401
  • SWLRDIWDWICKVLSK SEQ ID NO: 402
  • SWLRDIWDWICKVLS SEQ ID NO: 403
  • SWLRDIWDWICKVL SEQ ID NO: 404
  • SWLRRIWRWICEVLSDF SEQ ID NO: 405
  • SWLRRIWRWICEVLSD SEQ ID NO: 406
  • SWLRRIWRWICEVLS SEQ ID NO: 407
  • SWLRRJWRWICEVL SEQ ID NO: 408
  • SWLRDIWDWICRVLSRF SEQ ID NO : 409
  • SWLRDIWDWICRVLSR (SEQ ID NO: 410), SWLRDIWDWICRVLS (SEQ ID NO: 411), SWLRDIWDWICRVL (SEQ ID NO: 412), SWLRRIWDWICRVLSDF (SEQ ID NO: 413), SWLRRIWDWICRVLSD (SEQ ID NO: 414), SWLRRIWDWICRVLS (SEQ ID NO: 415), SWLRRIWDWICRVL (SEQ ID NO: 416), SWLRKIWDWICKVLSDF (SEQ ID NO: 417), SWLRKIWDWICKVLSD (SEQ ID NO: 418),
  • SWLRKIWDWICKVLS SEQ ID NO: 419
  • SWLRKIWDWICKVL SEQ ID NO: 420
  • SWLRRIWDWICEVLSRF SEQ ID NO: 421
  • SWLRRIWDWICEVLSR SEQ ID NO: 422
  • SWLRRIWDWICEVLS SEQ ID NO: 423
  • SWLRRIWDWICEVL (SEQ ID NO: 424), SWLRKIWDWICEVLSKF (SEQ ID NO: 425), SWLRKIWDWICEVLSK (SEQ ID NO: 426), SWLRKIWDWICEVLS (SEQ ID NO: 427), SWLRKIWDWICEVL (SEQ ID NO: 428),
  • SWLRDIWRWICRVLSDF SEQ ID NO: 429), SWLRDIWRWICRVLSD (SEQ ID NO: 430), SWLRDIWRWICRVLS (SEQ ID NO: 431), SWLRDIWRWICRVL (SEQ ID NO: 432), SWLRDIWKWICKVLSDF (SEQ ID NO: 433),
  • SWLRDIWKWICKVLSD (SEQ ID NO: 434), SWLRDIWKWICKVLS (SEQ ID NO: 435), SWLRDIWKWICKVL (SEQ ID NO: 436), SWLDRIWDWICRVLSRF (SEQ ID NO: 437), SWLDRIWDWICRVLSR (SEQ ID NO: 438),
  • SWLDRIWDWICRVLS (SEQ ID NO: 439), SWLDRIWDWICRVL (SEQ ID NO: 440), SWLRDIWDWICKVLSKF (SEQ ID NO: 441), SWLRDIWDWICKVLSK (SEQ ID NO: 442), SWLRDIWDWICKVLS (SEQ ID NO: 443), SWLRDIWDWICKVL (SEQ ID NO: 444), SWLRDIWRWICKVLSRF (SEQ ID NO: 445), SWLRDIWRWICKVLSR (SEQ ID NO: 446), SWLRDIWRWICKVLS (SEQ ID NO: 447),
  • SWLRDIWRWICKVL (SEQ ID NO: 448), SWLRDIWKWICKVLSKF (SEQ ID NO: 449), SWLRDIWKWICKVLSK (SEQ ID NO: 450), SWLRDIWKWICKVLS (SEQ ID NO: 451), SWLRDIWKWICKVL (SEQ ID NO : 452),
  • SWLRKIWKWICEVLSKF (SEQ ID NO: 453), SWLRKIWKWICEVLSK (SEQ ID NO: 454), SWLRKIWKWICEVLS (SEQ ID NO: 455), SWLRKIWKWICEVL (SEQ ID NO: 456), SWLRRIWRWICEVLSRF (SEQ ID NO: 457),
  • SWLRRIWRWICEVLSR (SEQ ID NO: 458), SWLRRIWRWICEVLS (SEQ ID NO: 459), SWLRRIWRWICEVL (SEQ ID NO: 460), SWLRRIWRWICDVLSRF (SEQ ID NO: 461), SWLRRIWRWICDVLSR (SEQ ID NO: 462),
  • SWLRRIWRWICDVLS SEQ ID NO: 463
  • SWLRRIWRWICDVL SEQ ID NO: 464
  • SWLRKIWKWICKVLSKF SEQ ID NO: 465
  • SWLRKIWKWICKVLSK SEQ ID NO: 466
  • SWLRKIWKWICKVLS SEQ ID NO: 467
  • SWLRKIWKWICKVL SEQ ID NO: 468
  • SWLRRIWRWICRVLSRF SEQ ID NO: 469
  • SWLRRIWRWICRVLSR SEQ ID NO: 470
  • SWLRRIWRWICRVLS SEQ ID NO: 471
  • SWLRRIWRWICRVL SEQ ID NO: 472
  • SWLRRIWRWICRVLSRF SEQ ID NO: 473
  • SWLRRIWRWICRVLSR SEQ ID NO: 474
  • SWLRRIWRWICRVLS SEQ ID NO: 475
  • SWLRRIWRWICRVL SEQ ID NO: 476
  • SWLKKIWKWICKVLSKF (SEQ ID NO: 477), SWLKKIWKWICKVLSK (SEQ ID NO: 478), SWLKKIWKWICKVLS (SEQ ID NO: 479), SWLKKIWKWICKVL (SEQ ID NO: 480), DFKSLVRCIWKWIKELWS (SEQ ID NO: 481),
  • LVECIWKWIKRLWS (SEQ ID NO: 490), KFKSLVKCIWDWIDRLWS (SEQ ID NO: 491),
  • LVKCIWDWIDRLWS (SEQ ID NO: 495), KFDSLVECIWRWIRRLWS (SEQ ID NO: 496),
  • LVECIWRWIRRLWS (SEQ ID NO: 500), KFRSLVRCIWDWIDRLWS (SEQ ID NO: 501), FRSLVRCIWDWIDRLWS (SEQ ID NO: 502), RSLVRCIWDWIDRLWS (SEQ ID NO: 503), SLVRCIWDWIDRLWS (SEQ ID NO: 504), LVRCIWDWIDRLWS (SEQ ID NO: 505),
  • FKSLVECIWDWIKRLWS (SEQ ID NO: 522)
  • RSLVRCIWDWIRDLWS (SEQ ID NO: 538), SLVRCIWDWIRDLWS (SEQ ID NO: 539),
  • KSLVKCIWDWIDRLWS SEQ ID NO: 543
  • SLVKCIWDWIDRLWS SEQ ID NO: 544
  • KSLVKCIWKWIDRLWS SEQ ID NO: 553
  • SLVKCIWKWIDRLWS SEQ ID NO: 554.
  • SLVDCIWRWIRRLWS (SEQ ID NO: 569), LVDCIWRWIRRLWS (SEQ ID NO: 570), KFKSLVKCIWKWIKRLWS (SEQ ID NO: 571), FKSLVKCIWKWIKRLWS (SEQ ID NO: 572), KSLVKCIWKWIKRLWS (SEQ ID NO: 573),
  • SLVKCIWKWIKRLWS (SEQ ID NO: 574), LVKCIWKWIKRLWS (SEQ ID NO: 575), KFRSLVRCIWRWIRRLWS (SEQ ID NO: 576), FRSLVRCIWRWIRRLWS (SEQ ID NO: 577), RSLVRCIWRWIRRLWS (SEQ ID NO: 578),
  • SLVRCIWRWIRRLWS (SEQ ID NO: 584), LVRCIWRWIRRLWS (SEQ ID NO: 585), KFKSLVKCIWKWIKKLWS (SEQ ID NO: 586), FKSLVKCIWKWIKKLWS (SEQ ID NO: 587), KSLVKCIWKWIKKLWS (SEQ ID NO: 588), SLVKCIWKWIKKLWS (SEQ ID NO: 589), and LVKCIWKWIKKLWS (SEQ ID NO: 590).
  • the isolated peptide of the invention consists of any one of the above amino acid sequences, in particular, SEQ ID NO: 119, 123 to 144, 165 to 177; 179 to 183; 185 to 594.
  • a peptide of the invention has viricidal activity.
  • a peptide of the invention is 18 to 40 D- or L-amino acids in- length, 18 to 30 D- or L-amino acids in-length, or 18 to 22 D- or L-amino acids in- length.
  • a peptide of the invention includes D-amino acids.
  • a peptide of the invention includes L-amino acids.
  • the peptide includes a dansyl moiety.
  • the peptide has an EC 5O of about 3 ⁇ M or less; about 2 ⁇ M or less; about 1 ⁇ M or less; about 500 nM or less; about 400 nM or less; or about 300 nM.
  • the peptides are active against HIV, the hepatitis C virus or Flaviviruses such as the West Nile virus or the Dengue virus.
  • the invention provides a pharmaceutical composition that includes any peptide of the invention described herein.
  • the pharmaceutical composition is a microbicide or a vaginal cream.
  • the pharmaceutical composition includes an antiviral agent.
  • the antiviral agent is a protease inhibitor, a polymerase inhibitor, an integrase inhibitor, an entry inhibitor, an assembly/secretion inhibitor, a translation inhibitor or an immunostimulant.
  • the antiviral agent is ⁇ - interferon, pegylated interferon, ribavirin, amantadine, rimantadine, pleconaril, acyclovir, zidovudine, lamivudine, Indenavir (Merck), telaprivir (Vertex), Tenofivir (Gilead), R1626 (Roche), GS-9137 (Gilead), Fuzeon (Roche, Trimeris), Celgosivir (Migenix), VGX-410c (VGX pharmaceuticals), 0 IMO-2125 (Idera pharmaceuticals) or a combination thereof.
  • the invention provides an article of manufacture that includes a vessel for collecting a body fluid and any peptide of the invention described herein.
  • the vessel is a collection bag, tube, capillary tube or syringe.
  • the vessel is evacuated.
  • the vessel further includes a biological stabilizer.
  • the peptide is attached or adsorbed onto the vessel so that the peptide is retained in the vessel after materials placed therein have been removed. When attached or adsorbed onto the vessel, the peptide is still able to inhibit viral infection.
  • the invention provides a composition that includes a sample from the body of a mammal and any peptide of the invention described herein.
  • the composition also includes a biological stabilizer.
  • the sample is a blood product.
  • the blood product is plasma, platelet, leukocytes or stem cell.
  • the biological stabilizer is an anti-coagulant, preservative, protease inhibitor, or any combination thereof.
  • the anti-coagulant is citrate, ethylene diamine tetraacetic acid, heparin, oxalate, fluoride or any combination thereof.
  • the preservative is boric acid, sodium formate and sodium borate.
  • the protease inhibitor is dipeptidyl peptidase IV. In some embodiments, the peptide and/or stabilizer is freeze dried.
  • the invention provides (1) a method for inactivating a virus that involves contacting the virus with a peptide or composition of the invention, (2) method for preventing or treating infection of a mammalian cell by a virus that involves contacting the mammalian cell with with a peptide or composition of the invention, or (3) a method for preventing or treating a viral infection in a mammal that involves administering to the mammal an effective amount of with a peptide or composition of the invention.
  • the virus is a Flavivirus, an Hepacivirus or a human immunodeficiency virus.
  • the virus is a Hepatitis C virus, West Nile virus, or Dengue virus.
  • the mammalian cell is a human cell.
  • the mammal is a human.
  • the peptide is administered. topically or systematically.
  • the invention provides (1) a method for inactivating a human immunodeficiency virus, (2) a method for preventing or treating infection of a mammalian cell by a human immunodeficiency virus, and (3) a method for preventing or treating infection of a mammal by a human immunodeficiency virus. These methods involve contacting the virus or mammalian cell, or administering to the mammal, a peptide of the invention or a composition comprising a peptide of the invention.
  • a peptide that can be used is 14 to 50 D- or L-amino acids in length and comprises an alpha-helical structure, and wherein the polar amino acids are located on the same face of the alpha helical structure, and the nonpolar amino acids are located on the other face of the alpha-helical structure.
  • the polar amino acids are located on the same face of the alpha- helical structure and all the non-polar amino acids are located on the other face of the alpha-helical structure resulting in perfect amphipathicity. Perfect amphipathicity can be determined using a helical wheel as known in the art.
  • a peptide that can be used in these methods of the invention includes nonpolar amino acids selected from the group consisting of alanine, valine, leucine, methionine, isoleucine, phenylalanine, and tryptophan.
  • the polar amino acids are selected from the group consisting of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine, hydroxylysine, ornithine, serine and threonine.
  • the cysteine residue in the peptide is located at a position N-terminal to a serine on the peptide.
  • the cycteine is located four positions N-terminal to the serine on the peptide. In some embodiments, the cysteine is located at position 11 relative to the N-terminus of the peptide. In some embodiments, amino acids 16 and 18 relative to the N-terminus of the peptide are charged, in particular, amino acids 16 and 18 are charged positive and negative, positive and positive, or negative and positive, respectively.
  • a peptide that can be used in a method of the invention is 14 to 40 D- or L-amino acids in-length, 14 to 30 D- or L-amino acids in-length, 14 to 25 D- or L-amino acids in-length, or 14 to 18 D- or L-amino acids in-length.
  • the peptide is 14 amino acids in length; the amino acids are arginine, cysteine, glutamate, serine, valine, two aspartates, two leucines, two isoleucines and three tryptophan residues; and the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure.
  • the peptide is 15 amino acids in length; the amino acids are arginine, cysteine, glutamate, two serines, valine, two aspartates, two leucines, two isoleucines and three tryptophan residues; and the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha- helical structure.
  • the peptide is 16 amino acids in length; the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines and three tryptophan residues; and the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure.
  • the peptide is 17 amino acids in length; the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines, three tryptophan and a phenylalanine; and the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure.
  • the peptide is 18 amino acids in length; the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines, three tryptophan, a phenylalanine and a lysine; and the arginine, cysteine, glutamate, serine, aspartate and lysine residues are located on the same face of the alpha-helical structure.
  • a peptide that can be used in a method of the invention is composed of D amino acids. In other embodiments, it is composed of L amino acids. In some embodiments, it further comprises a dansyl moiety.
  • a peptide that can be used in a method of the invention comprises the amino acid sequence:
  • QIVGGVYLLPRRGPRLGV (SEQ ID NO: 4), QPGYPWPLYGNEGCGWAG (SEQ ID NO: 5), LYGNEGCGWAGWLLSPRG (SEQ ID NO: 6), GWAGWLLSPRGSRPSWGP (SEQ ID NO: 7), IFLLALLSCLTVPASAYQ (SEQ ID NO: 8),
  • DAILHTPGCVPCVREGNA (SEQ ID NO: 9), LPTTQLRRHIDLLVGSAT (SEQ ID NO: 10), RHIDLLVGSATLCSALYV (SEQ ID NO: 11), GSATLCSALYVGDLCGSV (SEQ ID NO: 12), ALYVGDLCGSVFLVGQLF (SEQ ID NO: 13),
  • IMDMIAGAHWGVLAGIAY SEQ ID NO: 14
  • HINSTALNCNESLNTGWL SEQ ID NO: 15
  • NCNESLNTGWLAGLFYQH SEQ ID NO: 16
  • LASCRRLTDFAQGWGPIS SEQ ID NO: 17
  • TDFAQGWGPISYANGSGL SEQ ID NO: 18
  • GPISYANGSGLDERPYCW SEQ ID NO: 19
  • GSGLDERPYCWHYPPRPC SEQ ID NO: 20
  • WMNSTGFTKVCGAPPCVI SEQ ID NO: 21
  • PCVIGGVGNNTLLCPTDC SEQ ID NO: 22
  • MYVGGVEHRLEAACNWTR SEQ ID NO: 23
  • YLYGVGSSIASWAIKWEY SEQ ID NO: 24
  • SIASWAIKWEYVVLLFLL SEQ ID NO: 25
  • KWEYVVLLFLLLADARVC SEQ ID NO: 26
  • WMMLLISQAEAALENLVI SEQ ID NO: 27
  • GAVYAFYGMWPLLLLLLA SEQ ID NO: 28
  • GMWPLLLLLLALPQRAYA SEQ ID NO: 29
  • TLVFDITKLLLAIFGPLW SEQ ID NO: 30
  • VSTATQTFLATCIN SEQ ID NO: 31
  • ATQTFLATCINGVCWTVY SEQ ID NO: 32
  • DSSVLCECYDAGCAWYEL SEQ ID NO: 33
  • AYMNTPGLPVCQDHLEFW SEQ ID NO: 34
  • LEFWEGVFTGLTHIDAHF SEQ ID NO: 35
  • HPITKYIMTCMSADLEVV SEQ ID NO: 36
  • VTSTWVLVGGVLAAL SEQ ID NO: 37
  • WVLVGGVLAALAAYCLST (SEQ ID NO: 38), LAALAAYCLSTGCVV (SEQ ID NO: 39), EVFWAKHMWNFISGIQYL (SEQ ID NO: 40), MWNFISGIQYLAGLSTLP (SEQ ID NO: 41), PAILSPGALVVGVVCAAI (SEQ ID NO: 42),
  • SWLRDIWDWICEVLSDFK (SEQ ID NO: 43), DWICEVLSDFKTWLKAKL (SEQ ID NO: 44), YVSGMTTDNLKCPCQIPS (SEQ ID NO: 45), SSGADTEDVVCCSMS (SEQ ID NO: 46), DTEDVVCCSMSYSW (SEQ ID NO: 47),
  • SSGADTEDVVCCSMSYSW (SEQ ID NO: 48), DVVCCSMSYSWTGAL (SEQ ID NO: 49), TVTESDIRTEEAIYQCCD (SEQ ID NO: 50), GNTLTCYIKARAACRAAG (SEQ ID NO: 51), RAAGLQDCTMLVCGDDLV (SEQ ID NO: 52),
  • CTMLVCGDDLVVICESAG (SEQ ID NO: 53), DDLVVICESAGVQEDAAS (SEQ ID NO: 54), LELITSCSSNVSVAHDGA (SEQ ID NO: 55), HTPVNSWLGNIIMFAPTL (SEQ ID NO: 56), APTLWARMILMTHFFSVL (SEQ ID NO: 57), DQLEQALNCEIYGACYSI (SEQ ID NO: 58), GVPPLRAWRHRARSVRAR (SEQ ID NO: 59), WRHRARSVRARLLSRGGR (SEQ ID NO: 60), GWFTAGYSGGDIYHSVSH (SEQ ID NO: 61),
  • LYGNEGLGWAGWLLSPRG SEQ ID NO:62
  • IFLLALLSCITVPVSAAQ SEQ ID NO:63
  • IFLLALLSCLTIPASAYE SEQ ID NO:64
  • MSATFCSALYVGDLCGGV SEQ ID NO:65
  • GAAALCSAMYVGDLCGSV SEQ ID NO:66
  • ALYVGDLCGGVMLAAQVF (SEQ ID NO:67), AMYVGDLCGSVFLVAQLF (SEQ ID NO:68), IIDIVSGAHWGVMFGLAY (SEQ ID NO:69), VVDMVAGAHWGVLAGLAY (SEQ ID NO:70), VDVQYMYGLSPAITKYVV (SEQ ID N0:71),
  • YLYGIGSAVVSFAIKWEY (SEQ ID NO:72), WMLILLGQAEAALEKLVV (SEQ ID NO:73), WMMLLIAQAEAALENLVV (SEQ ID NO:74), GVVFDITKWLLALLGPAY (SEQ ID NO:75), ELIFTITKILLAILGPLM (SEQ ID NO:76),
  • VSQSFLGTTISGVLWTVY (SEQ ID NO:77), ATQSFLATCVNGVCWTVY (SEQ ID NO:78), SWLRDVWDWVCTILTDFK (SEQ ID NO:79), SWLRDVWDWICTVLTDFK (SEQ ID NO:80), DWVCTILTDFKNWLTSKL (SEQ ID N0:81),
  • DWICTVLTDFKTWLQSKL (SEQ ID NO: 82), ASEDVYCCSMSYTWT (SEQ ID NO:83), EDDTTVCCSMSYSW (SEQ ID NO:84), CTMLVCGDDLVVICESAG (SEQ ID NO:85), PTMLVCG DDLVVISESQG (SEQ ID NO:86), SWLRPIWPWICEVLSDFK (SEQ ID NO: 91), SWLRDIWDWICEVL (SEQ ID NO: 92), SWLRDIWDWICEVLS (SEQ ID NO: 93), SWLRDIWDWICEVLSD (SEQ ID NO: 94),
  • SWLRDIWDWICEVLSDF (SEQ ID NO: 95), KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), SIWRDWVDLICEFLSDWK (SEQ ID NO: 97), KWLCRIWSWISDVLDDFE (SEQ ID NO: 98), FDSLVECIWDWIDRLWS (SEQ ID NO: 99),
  • DSLVECIWDWIDRLWS (SEQ ID NO: 100), SLVECIWDWIDRLWS (SEQ ID NO: 101), or LVECIWDWIDRLWS (SEQ ID NO: 102).
  • a peptide that can be used in a method of the invention consists of the amino acid sequence of any of SEQ ID NO: 4-86 and 91- 102. In some embodiments, a peptide that can be used in a method of the invention has an EC 50 of about 3 ⁇ M or less, about 2 ⁇ M or less, about 1 ⁇ M or less, about 500 nM or less, about 400 nM or less, about 300 nM.
  • the invention provides for an isolated peptide of 14 to 50 D- or L-amino acids in-length, having an amphipathic ⁇ -helical structure and antiviral activity against HIV and/or a virus of the Flaviviridae family.
  • the peptide has a sequence comprising any one of formulae IX-XIII:
  • Xaaj, Xaa 4 , Xaa 5 , Xaa 8 , Xaan, Xaaj 2 , Xaai 5 , Xaai 6 and Xaai 8 are separately each a polar amino acid; and Xaa 2 , Xaa 3 , Xaa ⁇ , Xaa 7 , Xaa9, Xaaio, Xaan, Xaai 4 , and Xaaj 7 are separately each a nonpolar amino acid.
  • the invention provides a fusion peptide formed by attaching a 14 amino acid peptide (the N-terminal peptide) to the N-terminus of a peptide of any of formulae IX to XIII.
  • the 14 amino acid N-terminal peptide has the structure: Rx-Ry-Ry-Rx-Ry-Ry-Rx-Rx-Ry-Ry-Rx-Rx-Ry-Ry-Rx (SEQ ID NO: 1 17), wherein each Rx is separately a polar amino acid, and each Ry is separately a nonpolar amino acid.
  • the invention provides a fusion peptide formed by attaching a 12 amino acid peptide (the C-terminal peptide) to the C-terminus of a peptide of formula XIII.
  • the resulting fusion peptide has the structure of formulae XIV:
  • Xaa 23 , Xaa 26 , Xaa 29 , and Xaa 3 o are separately each a polar amino acid
  • Xaa 2 , Xaa 3 , Xaa ⁇ , Xaa 7 , Xaa 9 , Xaaio, Xaan, Xaai 4 , Xaan, Xaa 20 , Xaa 2 i, Xaa 24 , Xaa 25 , Xaa 27 , and Xaa 28 are separately each a nonpolar amino acid.
  • the invention provides a fusion peptide having a sequence that corresponds to the 14 amino acid N-terminal peptide of SEQ ID NO: 1 17 attached by a peptide bond to the N-terminus of a peptide of formula XIV.
  • a peptide of the invention is a peptide comprising at least 14 contiguous amino acids of any of the above described peptides.
  • the invention provides for the use of a peptide of formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIV in the preparation of a medicament for preventing and/or treating infection in a mammal by a human immunodeficiency virus, Hepatitis C virus, West Nile virus, or Dengue virus.
  • the invention provides for the use of a peptide of formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIV in the preparation of a medicament for preventing and/or treating infection in a mammal by a Flavivirus or an Hepacivirus.
  • FIG. 1 illustrates the location of the peptides with respect to the HCV polyprotein genotype Ia (H77 isolate, having SEQ ID NO:1) and the corresponding anti-HCV activity. Thirteen of the peptides tested inhibited infectivity by 90% or more.
  • FIG. 2 illustrate that peptide 1 having the amino acid sequence SWLRDIWDWICEVLSDFK (SEQ ID NO:43) with either L- or D-amino acids (A) prevents initiation of HCV infection when added to the virus before cells are exposed to virus; (B) terminates ongoing HCV infection; (C) inhibits HCV infection in growth-arrested Huh-7 cells; (D) enters the cell and (E) inhibits intracellular HCV particle infectivity.
  • the EC 50 of peptide 1 is approximately 300 nM (F and G), as shown by two separate experiments.
  • FIG. 3 is a bar graph showing inhibition of HCV infection of Huh-7.5.1 cells by various synthetic peptides, of which peptide 1 (SEQ ID NO:43) was the most effective inhibitor of viral infection.
  • FIG. 4A-G are data showing the inhibitory characteristic and stability of peptide 1 (SEQ ID NO:43). Both the L- and D- isomers of peptide 1 are highly inhibitory (A). Peptide 1 acts on the virus (B) and is virocidal for HCV (C) by disrupting HCV virions (D). Both the L- and D-isomers are effective at permeabilizing membranes (E). The D-form of peptide 1 displays enhanced serum stability (F) and a slightly lower IC 50 (G).
  • FIG. 5A-D are results showing the effective concentration (EC 50 ) and toxicity (LC 50 ) of the L- and D-forms of peptide 1 (SEQ ID NO:43).
  • FIG. 5 A shows the percent infected Huh-7.5.1 cells as a function of concentration of the L- form of peptide 1, illustrating that the EC 50 of the L-form of peptide 1 is 0.6 micromolar.
  • FIG. 5B shows the percent infected Huh-7.5.1 cells as a function of concentration of the D-form of peptide 1 (with D- instead of L-amino acids), illustrating that the EC 50 of the D-form of peptide 1 is 1.0 micromolar.
  • FIG. 5 A shows the percent infected Huh-7.5.1 cells as a function of concentration of the L- form of peptide 1, illustrating that the EC 50 of the L-form of peptide 1 is 0.6 micromolar.
  • FIG. 5B shows the percent infected Huh-7.5.1 cells as
  • FIG. 5C shows the percent viable Huh-7 cells as a function of concentration of the L-form (square symbols) and D-form (diamond symbols) of peptide 1, illustrating that the LC 50 of the L-form of peptide 1 is 46 micromolar and the LC 50 of the D-form of peptide 1 is 87 micromolar.
  • FIG. 5D shows the percent viable Huh-7.5.1 cells as a function of concentration of the L-form (square symbols) and D-form (diamond symbols) of peptide 1, illustrating that the LC 50 of the L-form of peptide 1 is 64 micromolar and the LC 5O of the D-form of peptide 1 is 105 micromolar.
  • FIG. 6A-E illustrate the amphipathic ⁇ -helical nature of peptide 1 (SEQ ID NO:43).
  • Helical wheel diagram of peptide 1 shows that the amino acid distribution results in a hydrophilic (or polar) face and a hydrophobic (or non-polar) face (FIG. 6A).
  • Circular dichroism results show the ⁇ -helical structure of the L- and D- isomers of peptide 1 (FIG. 6B), the effect of dansylation on the ⁇ -helical structure of the L- and D-isomers of peptide 1 (FIG. 6C), and the ⁇ -helical structures of variants of peptide 1 having C-terminal truncations (FIG.
  • FIG. 7 illustrate the liposome-release assays results obtained for various truncation variants of peptide 1 (SEQ ID NO:43). The sequences of these truncated peptides are provided in Table 9.
  • FIG. 8 is a graph showing that peptide 1 (SEQ ID NO:43; referred to as #7208 in this Figure) does not block vesicular stomatitis virus (VSV) infection. Additional studies indicate that peptide 1 does not block infection by influenza virus, vaccinia virus, Borna disease virus, lymphocytic choriomeningitis virus or adenovirus.
  • VSV vesicular stomatitis virus
  • FIG. 9 is a graph showing that peptide 2022 (peptide 1) with sequence SWLRDIWDWICEVLSDFK (SEQ ID NO:43) and peptide 2013 having the sequence S WLRDIWD WICEVL (SEQ ID NO:92) inhibit essentially 100 % of Dengue viral infection as detected by ELISA.
  • FIG. 1 OA-D are graphs showing the percent inhibition of Dengue viral infection for various peptides.
  • FIG. 1 OA-D are graphs showing the percent inhibition of Dengue viral infection for various peptides.
  • FIG. 1 OA shows dose-dependent inhibition of Dengue viral infection by peptide 2022 (peptide 1 ; SEQ ID NO:43), peptide 2013 (SEQ ID NO:92), and peptide 2017 (SEQ ID NO: 107), as detected by FACS analysis of cells intracellularly stained for Dengue viral antigens.
  • peptide 2022 peptide 1
  • SEQ ID NO:92 peptide 2013
  • peptide 2017 SEQ ID NO: 107
  • OB-D also show inhibition of Dengue viral infection by peptide variants and demonstrates the inhibitory effect amphipathic peptides that are variants of peptide 1 as detected by intracellular FACS staining. These data show that variants and homologues of peptide 1 retain inhibitory activity so long as the amphipathicity of the peptide structure is maintained. Note that peptide 1 (SEQ ID NO:43) is called peptide L-7208 in FIG. lOB-D. FIG.
  • FIG. 11 depicts results illustrating the ability of peptide 1 (SEQ ID NO:43; called 2022 in this figure) and peptide 2012 (S WLRDIWD WICEVLSD, SEQ ID NO:94) to inhibit West Nile Viral (WNV) infection when incubated with infectious virus prior to adding the virus to Huh-7 cells.
  • WNV West Nile Viral
  • FIG. 12 graphically illustrates that both the L- and D- forms of peptide 1 (i.e. L-2022 [SWLRDIWDWICEVLSDFK] (SEQ ID NO:43) and D-2022 (SEQ ID NO:43 with D-amino acids) inhibit HIV-I infection at concentrations between 1.25 to 5.0 micromolar.
  • L-2022 [SWLRDIWDWICEVLSDFK] SEQ ID NO:43
  • D-2022 SEQ ID NO:43 with D-amino acids
  • peptide 2018 DIWD WICEVLSDFK
  • N-terminally truncated 14-mer version of peptide 1 has similar antiviral activity
  • peptide 2054 SWLRDIWD WICEV
  • a C-terminally truncated 13-mer analog of peptide 1 is slightly less active.
  • peptide 2015 (S WLRDIWDWI) (SEQ ID NO: 105), a C-terminally truncated 10-mer version of peptide 1 has no activity; nor does peptide 6938 (LYGNEGCGWAGWLLSPRG) (SEQ ID NO:6), an 18-mer derived from the core protein of HCV.
  • FIG. 13A-B graphically illustrates that the L-7208 HS peptide (SIWRDWVDLICEFLSDWK, SEQ ID NO:97), is almost as active as the highly active L-7208 SEQ ID NO:43 peptide (also called the L-2022 peptide or "peptide 1").
  • the L-7208 HS peptide has the same amino acid composition as the L-7208 peptide, but the hydrophobic amino acids in the L-7208 HS peptide (SEQ ID NO:97) have been scrambled, thereby changing the amino acid sequence but maintaining its amphipathicity.
  • the L-7208 HS peptide (SEQ ID NO:97), the L-7208 peptide (SEQ ID NO:43) and the D-7208 peptide (SEQ ID NO:43 with D- amino acids instead of L-amino acids) all inhibit essentially 100 % HIV infection at concentrations of about 20 micromolar.
  • Another peptide (called 3229, SWRLDIWDWICESVLDFK, SEQ ID NO: 119) whose amino acids were exchanged to reduce its amphipathicity, but which has the same amino acid composition as the L-7208 (SEQ ID NO:43) peptide, exhibited little if any activity.
  • FIG. 14A-C illustrates that the peptides destabilize the HIV-I virions extracellularly.
  • FIG. 14A illustrates that large amounts of free HIV-I capsids are released from infectious virions after treatment of HIV-I preparations with peptide 1 (designated here as L-7208 (SEQ ID NO:43)), indicating that the virions were lysed by the peptide.
  • L-7208 SEQ ID NO:43
  • essentially no HIV-I capsid was released after HIV-I virions were treated with DMSO or with control peptide 6938 (LYGNEGCGWAGWLLSPRG) (SEQ ID NO:6).
  • the amount of virus-associated capsid was reduced in the L-7208 (SEQ ID NO:43)-treated virus (FIG. 14B) but not in the peptide 6938
  • FIG. 14C shows the percent of HIV-I capsid internalized into cells after treatment with DMSO and 5 or 10 micromolar of peptides 6938 (SEQ ID NO:6) or L-7208 (SEQ ID NO:43). While essentially 100% of control amounts of HIV-I capsid were internalized when cells were treated with 5 or 10 micromolar 6938 (SEQ ID NO:6), HIV-I capsid internalization was inhibited up to 10-fold by treatment with peptide L-7208 (SEQ ID NO:43).
  • FIG. 15 is a bar graph illustrating that peptides with amphipathic structures similar to the amphipathic structure of peptide L-7208 (SEQ ID NO:43) are also strongly inhibitory of HIV infection.
  • amphipathic peptides with strong anti- HIV activity include peptide 3222 (SEQ ID NO: 127), peptide 3226 (SEQ ID NO: 128), peptide 3228 (SEQ ID NO: 130), peptide L-7208 2D to 2 Pro (SEQ ID NO:91), and L-7208 HS with hydrophilic amino acids scrambled (KWLCRIWSWISDVLDDFE, SEQ ID NO:98).
  • FIG. 16A-D Peptide 1 (SEQ ID NO:43; amphipathic "viracide”) neutralizes not only cell-free HIV, but also cell-associated and internalized HIV.
  • SEQ ID NO:43 amphipathic "viracide”
  • CD4 + T- lymphocytes, macrophages or DC O.lxlO 6 cells
  • NL4.3 BaL 1 ng of p24
  • Wild-type Peptide 1 or its non-amphipathic variant (SEQ ID NO: 1 19; 5 ⁇ M) were added together with virus to CD4 + T cells, macrophages and DC (panels 1 to 3), to HIV -pulsed DC prior to T cell incubation (panel 4), or added 3 days post-infection to T cells (panel 5). Supernatants were collected after different days and viral replication was monitored by p24 ELISA. Error bars represent standard errors of duplicates. These experiments are representative of three independent experiments using three different donors.
  • B 293T cells transfected with NL4.3 or NL4.3 BaL for 24 h were treated with or without peptide (5 ⁇ M) for 1 h at 37°C and washed to remove peptides.
  • TZM cells Twenty-four hours post-peptide treatment, infectivity of 293T-released viruses was scored on TZM cells. Infection was measured 48 h post-infection by ⁇ -galactosidase activity. Error bars represent standard errors of duplicates. These experiments are representative of two independent experiments.
  • C TZM cells were exposed to pNL4.3- ⁇ Env viruses (1 ng of p24) pseudotyped with gpl ⁇ O NL4.3 (X4), gpl ⁇ O BaL (R5) or VSVG with or without peptide (5 ⁇ M). Infection was measured 48 h post-infection by ⁇ - galactosidase activity. Error bars represent standard errors of duplicates. Data are expressed in % of infection.
  • TZM cells were either pretreated with peptide (5 ⁇ M) for 1 , 2, 4 and 8 h, washed extensively to remove peptides and then exposed to NL4.3 (1 ng of p24), or cells were first exposed to virus and peptide was added 1, 2, 4 and 8 h later. Infection was measured 48 h post-infection by ⁇ -galactosidase activity. Error bars represent standard errors of duplicates.
  • FIG. 17A-E Peptide 1 (SEQ ID NO:43; amphipathic "viracide”) destroys the integrity of both the membrane and capsid core of HIV.
  • SEQ ID NO:43 amphipathic "viracide”
  • A Purified NL4.3 virus (20 ng of p24 in PBS) was incubated with or without Peptide 1 (5 ⁇ M) for 30 min at 37°C and loaded over a 20-70% sucrose gradient. Each collected fraction of the gradient was analyzed for capsid (p24 ELISA and immunoblot), RT (by exoRT assay) and gp41 content (by immunoblot). The density of each fraction (g/cm 3 ) shown in the lower two panels, was determined by measuring the refractive index.
  • TZM cells (500,000) were exposed to 1 ng of p24 of NL4.3 for 1 h at 4°C, washed extensively to remove unbound virus and lysed.
  • virus internalization cells were exposed to virus for 2 h at 37 0 C, washed, trypsinized to remove attached virus and lysed. Amounts of attached and internalized virus were determined by p24 ELISA in cell lysates. Error bars represent the standard errors of duplicates. Data are expressed in percentage of attachment or internalization.
  • (C) Same as (A) except that virus was treated with decreasing concentrations of Peptide 1 for 30 min at 37°C (top left panel), with 5 ⁇ M of Peptide 1 for 15, 30 and 60 min at 37°C (bottom left panel), for 30 min at 4, 25 or 37°C (top right panel), and for 30 min at pH 8, 7, 6 and 5 (bottom right panel). Gradient fractions were analyzed for HIV capsid content by p24 ELISA.
  • D Same as (A) except that virus was treated either with Peptide 1 or with its non- amphipathic variant (SEQ ID NO:119).
  • (E) Same as (A) except that virus was first trypsinized for 15 min at 37°C, incubated in 10% FCS to neutralize trypsin, microfuged for 90 min at 4°C, resuspended and immediately loaded over a sucrose gradient for virus integrity evaluation by p24 ELISA.
  • FIG. 18A-F Peptide 1 (SEQ ID NO:43; amphipathic "viracide”) inhibits both HIV genital epithelial transmigration and LC/DC transmission of HIV.
  • SEQ ID NO:43 amphipathic "viracide”
  • Either cell-free or cell-associated HIV was added to the apical surface of primary genital epithelial cells (PGEC) for 8 h at 37°C, and amounts of transcytosed viruses were quantified by p24 ELISA of the lower chamber corresponding to the basal PGEC surface.
  • Peptide 1 SEQ ID NO:43
  • SEQ ID NO:43 the effect of Peptide 1 (SEQ ID NO:43) on HIV transmigration, it (5 ⁇ M) was added just after addition of the virus to PGEC and its effects were compared with its non-amphipathic variant (SEQ ID NO: 119). Results are expressed in % of p24 of the original inoculum. Error bars represent standard errors of duplicates. Results are representative of 4 independent experiments using PGEC derived from 4 donors.
  • B PGEC were treated twice daily with 200 ⁇ M of Peptide 1 (SEQ ID NO:43) or 0.01% saponin for a week. No washes were performed in order to maintain a continuous exposure of cells to peptides.
  • FIG. 18C-F epidermal sheets were infected with HIV NL4.3-BaL-eGFP (100 ng p24) and directly incubated with 10 ⁇ M Peptide 1 (SEQ ID NO:43) or DMSO control. After 3 days, epidermal sheets were removed and 200,000 CCR5 + Jurkat cells were added for an additional 4 days.
  • Migrated DC/LC epidermal cells day 3) and samples of the co-cultures (day 5 and 7) were analyzed for Green Fluorescent Protein (GFP) expression by FACS.
  • C HIV infection of migrated DC/LC is depicted as percentage of total cells. Error bars represent standard errors of duplicates
  • D The co-cultures were further analyzed for infection at day 7 by FACS. The percentage of infected cells is depicted.
  • E The donor variability at day 5 is depicted. Error bars represent standard errors of duplicates.
  • the invention relates to peptides that can prevent or treat viral infection or that can inactivate a virus before it enters a cell.
  • the invention involves the discovery that certain peptides derived from the hepatitis C viral polyprotein, e.g. those having sequences set forth in SEQ ID NO: 4-61, can prevent or treat infection of a mammal by viruses of several types, including viruses in the Flaviviridae family, measles, respiratory syncytial virus (RSV) and HIV, as well as inactivate such viruses.
  • viruses of several types including viruses in the Flaviviridae family, measles, respiratory syncytial virus (RSV) and HIV, as well as inactivate such viruses.
  • RSV respiratory syncytial virus
  • the invention also involves the discovery that several peptides from the HCV polyprotein (SEQ ID NO:1) are highly effective at inhibiting HIV infection as well as infection of a virus in the Flaviviridae family.
  • the invention involves the discovery that "peptide 1" (SEQ ID NO:43), derived from the membrane anchor domain of NS5A (NS5A-1975), was particularly potent against HIV, hepatitis C virus, measles, RSV and Flaviviruses such as Dengue virus and West Nile virus. For example, 20 ⁇ M of peptide 1 completely inhibited HIV infection and concentrations as low as 0.3 ⁇ M were strongly inhibitory of HIV infection.
  • the present peptides are effective at preventing or treating infection by a variety of HIV strains, including those that use CXCR4 and CCR5 as coreceptors.
  • Peptides of the invention include, for example, those having sequences set out in SEQ ID NO: 4-86, 91-102, 119, 123-144, 165-177, 179-183 and 185-594 and peptides of about 8 to about 50 amino acids that are capable of forming an ⁇ - helical structure and can inhibit viral infection in a mammalian cell.
  • the invention provides an antiviral peptide or combinations of antiviral peptides, various compositions and combinations containing such antiviral peptide(s), and a method for inhibiting viral infection in a mammalian cell that utilizes such peptide(s).
  • the invention also provides an article of manufacture containing such antiviral peptide(s).
  • peptides from the hepatitis C viral polyprotein of the hepatitis C genotype Ia exhibit strong inhibition of HIV infection.
  • the HCV polyprotein sequence from which the peptides were originally obtained has sequence SEQ ID NO: 1 and can be found in the NCBI database as accession number NP 671491 (gi: 22129793).
  • the amino acid sequence of this HCV polyprotein (SEQ ID NO:1) is as follows.
  • AGVGIYLLPN R Another example of an HCV polyprotein amino acid sequence that may serve as a source of related peptides can be found in the NCBI database as accession number BAB32872 (gi: 13122262). See ncbi.nlm.nih.gov; Kato et al. J. Med. Virol. 64: 334-339 (2001). This HCV was isolated from a fulminant hepatitis patient, and its amino acid sequence (SEQ ID NO:2) is as follows.
  • HCV polyprotein amino acid sequence that may served a source of related peptides can be found in the NCBI database as accession number Q9WMX2 (gi: 68565847). See ncbi.nlm.nih.gov. This sequence was obtained from the Conl isolate of HCV.
  • the amino acid sequence (SEQ ID NO:3) is the following.
  • LALPPRAYAM DREMAASCGG AVFVGLILLT LSPHYKLFLA 841 RLIWWLQYFI TRAEAHLQVW IPPLNVRGGR DAVILLTCAI
  • HCV polyprotein sequences are available and can serve a source of other anti -viral peptides.
  • Taiwan isolate of hepatitis C virus is available in the NCBI database at accession number P29846 (gi: 266821). See ncbi.nlm.nih.gov.
  • the HCV polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins.
  • the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is affected by two viral proteases.
  • the first one cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3- NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites.
  • the NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components.
  • NS3 protease The complex formation of the NS3 protease with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites.
  • the NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities.
  • NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
  • the HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication.
  • the first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein (C. Lin et al., J. Virol. 68, 8147-8157 (1994)).
  • HCV has three structural proteins, the N-terminal nucleocapsid protein (termed “core”) and two envelope glycoproteins, "El” (also known as E) and “E2” (also known as E2/NS1). See, Houghton et al. (1991) Hepatology 14:381-388, for a discussion of HCV proteins, including El and E2.
  • the El protein is detected as a 32-35 kDa species and is converted into a single endo H-sensitive band of approximately 18 Kda.
  • E2 displays a complex pattern upon immunoprecipitation consistent with the generation of multiple species (Grakoui et al. (1993) J Virol. 67:1385-1395; Tomei et al. (1993) J. Virol.
  • HCV envelope glycoproteins El and E2 form a stable complex that is co- immunoprecipitable (Grakoui et al. (1993) J. Virol. 67:1385-1395; Lanford et al. (1993) Virology 197:225-235; Ralston et al. (1993) J. Virol. 67:6753-6761).
  • Antiviral Peptides One aspect of the invention is an antiviral peptide.
  • An antiviral peptide is a peptide that can prevent or treat infection of HIV, measles, RSV or a virus of the Flaviviridae family, herein a peptide inhibitor or a peptide of the invention.
  • HIV Human Immunodeficiency Virus, a virus that causes immunodeficiency by attacking CD4+ cells in the body.
  • Measles also known as rubeola, is a disease caused by a virus, specifically a paramyxovirus of the genus Morbillivirus. Measles is spread through respiration (contact with fluids from an infected person's nose and mouth, either directly or through aerosol transmission), and is highly contagious — 90% of people without immunity sharing a house with an infected person will catch it.
  • Respiratory syncytial virus is a spherical or pleomorphic enveloped virus (100-350 nm) with single-stranded, negative sense linear RNA.
  • RSV Respiratory syncytial virus
  • groups A and B Two major groups of strains of human RSV exist, groups A and B.
  • the strains of group A predominate.
  • the invention is directed to treating and preventing both strains of
  • RSV causes cold-like symptoms in adults and older children. However, it can cause serious problems in young babies, including pneumonia and severe breathing problems. In rare cases it can lead to death.
  • diverse types of HIV can effectively be inactivated or infection by diverse types of HIV can be treated and/or prevented because evidence provided herein indicates that the present peptides disrupt or lyse viruses without injuring mammalian cells.
  • the process by which the present peptides inhibit HIV infection is not particularly strain, clade or type specific.
  • the present peptides exhibit activity against a variety of HIV types.
  • one aspect of the invention is directed to treating and inhibiting infection by any HIV clade, type, subtype or strain.
  • the invention is also directed to inactivating HIV of any clade, type, subtype or strain.
  • the HIV that can be inactivated, treated or inhibited by the present compositions or methods is generally HIV-I or HIV-2, preferably HIV-I .
  • the HIV that can be inactivated, treated or inhibited according to the invention is generally of clade A, B, C or D. for example HIV-I
  • the inventive compositions and methods are also useful for treating and preventing infections by the HIV-I.
  • group M or O subtypes and sub-subtypes.
  • the members of group M are broken out into nine equidistant phylogenetic subtypes. These are labeled Al, A2, B, C, D, Fl, F2, G, H, J and K.
  • the sequences within any one-subtype or sub-subtype share more genetically than they do with sequences from other subtypes throughout their genomes.
  • a Flaviviridae is a spherical, enveloped virus having a linear, single- stranded RNA genome of positive polarity.
  • the family Flaviviridae includes the genera Flavivirus, Hepacivirus and Pestivirus.
  • the invention contemplates treatment of Flaviviridae infections, including infections caused by any virus from any of the genera Flavivirus, Hepacivirus and Pestivirus, as well as viruses of the unassigned genera of Flaviviridae.
  • the present peptides can be used to treat infections caused by the following viruses of the Flavivirus genus: Tick-borne encephalitis, Central European encephalitis, Far Eastern encephalitis, Rio Bravo, Japanese encephalitis, Kunjin, Murray Valley encephalitis, St Louis encephalitis, West Nile encephalitis, Tyulenly, Ntaya, Kenya S, Dengue type 1, Dengue type 2, Dengue type 3, Dengue type 4, Modoc, and Yellow Fever.
  • the present peptides can be used to treat infections caused by the following viruses of the Pestivirus genus: Bovine viral diarrhea virus 1, Bovine viral diarrhea virus 2, Hog cholera (classical swine fever virus), and Border disease virus.
  • the present peptides can be used to treat infections caused by hepatitis C virus, which is classified in the Hepacivirus genus.
  • Viruses of the unassigned genera of Flaviviridae whose infections can also be treated with the peptides of the invention include: GB virus- A, GB virus-B and GB virus-C.
  • GB virus- A GB virus- A
  • GB virus-B GB virus-C
  • an appropriate dosage for such a peptide methods known in the art, including, without limitation, those described herein can be used.
  • Viral infection in the presence or absence of a peptide of the invention can be evaluated, for example, by determining intracellular viral RNA levels, detection of viral protein or the number of viral foci by immunoassays using antibody against viral proteins as described herein. Viral inactivation can also be evaluated using these methods as described herein.
  • the antiviral activity of a peptide can also be determined using the liposome release assay as exemplified herein.
  • a peptide has antiviral activity if can prevent or reduce viral infection or inactivate the virus by any amount, for example, by 2 fold or more than 2 fold.
  • a peptide of the invention can prevent or reduce viral infection by 2-5 fold, 5-10 fold, or more than 10 fold.
  • peptides listed in Table 3 can inhibit viral infection by more than ten-fold, including, for example, peptides with SEQ ID NO:6, 8, 12, 13, 14, 24, 27, 30, 32, 43, 44, 47, 48 and 53.
  • Other peptides listed in Table 3 can inhibit viral infection by five-fold to ten-fold, including peptides with SEQ ID NO:21, 23, 28 and 37.
  • the remainder of the peptides inhibit viral infection by at least two-fold and some of the remaining peptides inhibit viral infection by up to about five-fold.
  • These peptides exhibit such inhibition of viral infection at concentrations of nanomolar and low micromolar levels.
  • a peptide of the invention is a polymer of ⁇ -amino acids linked by amide bonds between the ⁇ -amino and ⁇ -carboxyl groups.
  • amino acid refers to an ⁇ -amino acid.
  • the amino acids included in the peptides of the invention can be L-amino acids or D-amino acids.
  • the amino acids used in the peptides of the invention can be naturally-occurring and non-naturally occurring amino acids.
  • a peptide of the present invention can be made from genetically encoded amino acids, naturally occurring non-genetically encoded amino acids, or synthetic amino acids.
  • the amino acid notations used herein for the twenty genetically encoded L-amino acids and some examples of non-encoded amino acids are provided in Table 1.
  • a peptide of the invention will include at least 8 to about 50 amino acid residues, usually about 14 to 40 amino acids, more usually fewer than about 35 or fewer than about 25 amino acids in length.
  • a peptide of the invention will be as small as possible, while still maintaining substantially all of the activity of a larger peptide.
  • a peptide of the invention may be 8, 9, 10, 1 1, 12 or 13 amino acids in length.
  • the length of the peptide selected by one of skilled in the art may relate to the stability and/or sequence of the peptide.
  • peptide 1 exhibits optimal antiviral activity when it has about 18 amino acids, and truncations from the C-terminal end do not eliminate its antiviral activity, until five or so amino acids are deleted. Nonetheless, peptides with sequences different from SEQ ID NO:43 may exhibit optimal activity when they are longer than 18 amino acids or shorter than 13 amino acids. This may be due to sequence differences that stabilize or modify the secondary structure of the peptide.
  • the peptides can be derivatized with agents that enhance the stability and activity of the peptides.
  • peptides can be modified by attachment of a dansyl moiety or by incorporation of non-naturally occurring amino acids so as to improve the activity and/or conformation stability of the peptides.
  • Use of non- natural amino acids and dansyl moieties can also confer resistance to protease cleavage. It may also be desirable in certain instances to join two or more peptides together in one peptide structure.
  • peptides from other HCV strains exhibit excellent anti-viral activity including: genotype IB (SWLRDVWD WICTVLTDFK, SEQ ID NO:80); genotype 2A (S WLRDV WDWVCTILTDFK, SEQ ID NO:79); genotype 3A (DWLRIIWDWVCSVVSDFK, SEQ ID NO: 123); genotype 4A (SWLWEVWDWVLHVLSDFK, SEQ ID NO: 124); genotype 5 A (TWLRAIWDWVCTALTDFK, SEQ ID NO: 125); and genotype 6A (SWLRDVWDWVCTVLSDFK, SEQ ID NO: 126) all exhibit anti-viral activity.
  • genotype IB SWLRDVWD WICTVLTDFK, SEQ ID NO:80
  • genotype 2A S WLRDV WDWVCTILTDFK, SEQ ID NO:79
  • genotype 3A DWLRIIWDWVCSVVSDFK, SEQ ID NO: 123
  • genotype 4A S
  • the invention is also directed to peptidomimetics of the antiviral peptides of the invention.
  • a peptidomimetic is a peptide analog, such as those commonly used in the pharmaceutical industry as non-peptide drugs, that has properties analogous to those of the template peptide.
  • Advantages of peptide mimetics over natural peptide embodiments may include more economical production, greater chemical stability, altered specificity and enhanced pharmacological properties such as half- life, absorption, potency and efficacy.
  • the amino acid residues of a peptide of the invention can form an amphipathic ⁇ -helical structure in solution.
  • ⁇ -helix refers to a right-handed coiled conformation.
  • An ⁇ -helix has 3.6 amino acid residues per turn.
  • Certain amino acid residues tend to contribute to the formation of ⁇ -helical structures in polypeptides, for example, alanine, cysteine, leucine, methionine, glutamate, glutamine, histidine and lysine.
  • the inventive peptides are ⁇ -helical in aqueous solution.
  • the aqueous solution can, for example, have a physiological pH, and/or physiological salts.
  • the amphipathic ⁇ -helical structures of the present peptides are detected at moderate temperatures, such as at about 4 °C to about 50 °C, or at about room temperature to about body temperature.
  • the peptides ⁇ -helical structure under physiological temperatures and physiological pH values.
  • An ⁇ -helical structure can be detected using methods known in the art including, without limitation, circular dichroism spectroscopy (CD), nuclear magnetic resonance (NMR), crystal structure determination and optical rotary dispersion (ORD).
  • CD circular dichroism spectroscopy
  • NMR nuclear magnetic resonance
  • ORD optical rotary dispersion
  • amphipathic means that the ⁇ -helical peptides have a hydrophilic (or polar) face and a hydrophobic (or non-polar) face, wherein such a "face” refers to a longitudinal surface of the peptide.
  • a helical wheel is apparent when an ⁇ -helical peptide is viewed down its longitudinal axis (e.g. as shown in FIG.
  • one side of the helical wheel that circles this longitudinal axis is composed of hydrophilic (or polar) residues and the other side of the helical wheel is composed of hydrophobic (or nonpolar) residues.
  • hydrophilic or polar
  • hydrophobic or nonpolar residues
  • the hydrophilic and hydrophobic faces of the ⁇ -helix can therefore be identified based on the nature of the amino acids present.
  • the hydrophilic face of an ⁇ -helix will consist of a larger number of hydrophilic, charged and/or polar amino acids than is present on the hydrophobic face.
  • the hydrophobic face of an amphipathic ⁇ -helix consists of hydrophobic and/or non-polar amino acids that facilitate insertion into lipid bilayers.
  • the hydrophobic face may have one or more hydrophilic or polar amino acid as long as a sufficient number of non-polar amino acids are present that enable membrane insertion.
  • the hydrophilic face of the ⁇ -helix consists of charged or otherwise polar amino acids, while the hydrophobic face of the ⁇ -helix consists of non-polar amino acid residues.
  • the hydrophilic face of the ⁇ -helix consists of charged or otherwise polar amino acids, while the hydrophobic face of the ⁇ -helix consists of non-polar amino acid residues. See for example, the helical wheel of the peptide 1 (SEQ ID NO:43), which is shown in FIG. 6A.
  • Whether any given peptide sequence has a sufficient number of non-polar amino acids to enable membrane insertion can be determined using methods that are well known in the art, including without limitation, methods involving liposomal dye release described in the examples herein.
  • whether a peptide has an amphipathic ⁇ -helical structure can be determined using software available on the internet such as http://cti.itc.virginia.edu/ ⁇ cmg/Demo/wheel/wheelApp.html (last visited Aug. 15, 2006) and http://www.bioinf.man.ac.uk/ ⁇ gibson/ HelixDraw/helixdraw.html (last visited Aug. 15, 2006).
  • a schematic diagram illustrating the amphipathic ⁇ -helical structure of the peptide of SEQ ID NO: 43 is shown in FIG. 6A.
  • peptides of the invention can be found in Table 3.
  • Other peptides of the invention include those peptides having conservative amino acid substitutions compared to those shown in Table 3.
  • Peptides of the invention also include those having amino acid compositions that resemble the peptides shown in Table 3. These include peptides that have sequences of SEQ ID NO: 96, 97 and 98, which are shown in Table 11. These sequences correspond to the reverse variant of SEQ ID NO: 43 or they constitute a "scrambled" variant of SEQ ID NO: 43.
  • a retro or reverse variant of a peptide such as SEQ ID NO 43 will have an amino acid composition that resembles that of the original peptide (SEQ ID NO: 43), but the amino acid sequence will be the reverse of that of the original peptide.
  • the scrambled variant of a peptide such as SEQ ID NO: 43 will also have an amino acid composition that resembles the original peptide (SEQ ID NO: 43), but the order of the amino acid will be scrambled or mixed up without altering the relative positions of the hydrophobic and hydrophilic residues.
  • a peptide that is a "hydrophobic scrambled" variant of SEQ ID NO: 43 will have the same amino acid composition as that of SEQ ID NO: 43.
  • hydrophobic amino acid residues will be altered without altering the relative positions of hydrophobic and hydrophilic residues within the sequence such that the amphipathicity of the variant peptide resembles that of the original peptide.
  • a "hydrophilic scrambled" variant of SEQ ID NO: 43 will have the same amino acid composition as that of SEQ ID NO: 43, but the order of the hydrophilic amino acid residues will be altered without altering the relative positions of hydrophobic and hydrophilic residues within the sequence such that the amphipathicity of the variant peptide resembles that of the original peptide.
  • the term “scrambling” or “scrambled,” with respect to a hydrophilic (polar) amino acid, is used to indicate that while the positions of each hydrophilic (polar) amino acid are held constant, any other hydrophilic (polar) amino acid can be placed at that position.
  • the term “scrambling” or “scrambled,” with respect to a hydrophobic (nonpolar) amino acid is used to indicate that while the positions of each hydrophobic (nonpolar) amino acid are held constant, any other hydrophobic (nonpolar) amino acid can be placed at that position.
  • a peptide of the invention will have an amino acid sequence that is identical to the sequences shown in Table 3, as well as variants of such sequences.
  • Such variants can result from one or more amino acid truncations, conservative substitutions, scrambling of just the hydrophilic amino acids, scrambling of just the hydrophobic residues within a sequence, scrambling of both hydrophilic and hydrophobic amino acids, replacement of naturally occurring amino acids with non- naturally occurring amino acids or other modifications such as dansylation.
  • conservative substitutions scrambling of just the hydrophilic amino acids
  • scrambling of just the hydrophobic residues within a sequence scrambling of both hydrophilic and hydrophobic amino acids
  • replacement of naturally occurring amino acids with non- naturally occurring amino acids or other modifications such as dansylation Such variant peptides are further described in the next section.
  • the invention embraces numerous peptide homologues and variants.
  • genotype 1 B S WLRDVWD WICTVLTDFK, SEQ ID NO:80
  • genotype 2A SWLRDVWD WVCTILTDFK, SEQ ID NO:79
  • genotype 3A D WLRIIWD WVCSVVSDFK, SEQ ID NO: 123
  • genotype 4A SWLWEVWDWVLHVLSDFK, SEQ ID NO: 124
  • genotype 5 A TWLRAI WD WVCTALTDFK, SEQ ID NO: 125
  • genotype 6A S WLRDVWD WVCTVLSDFK, SEQ ID NO: 126) all exhibit antiviral activity.
  • invention is also directed to peptide homologues of the active peptides disclosed herein.
  • a peptide homologue is a peptidyl sequence from an HCV isolate other than the H77 isolate having SEQ ID NO:1.
  • a peptide of the invention can be a homologue of a peptide with an amino acid sequence of any of SEQ ID NO:4-61.
  • one peptide homologue of the invention has SEQ ID NO:62, which is a homologue of peptide SEQ ID NO:6.
  • LYGNEGLGWAGWLLSPRG (SEQ ID NO:62).
  • the sequence of peptide inhibitor SEQ ID NO:62 is found in HCV polyprotein sequences SEQ ID NO:2 and 3.
  • Another peptide inhibitor homologue of the invention has SEQ ID NO: 63 or 64, which are homologues of peptide SEQ ID NO:8.
  • IFLLALLSCITVPVSAAQ SEQ ID NO:63;
  • Another peptide inhibitor homologue of the invention has SEQ ID NO: 65 or 66, which are homologues of peptide SEQ ID NO: 12.
  • sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
  • Another peptide inhibitor homologue of the invention has SEQ ID NO:67 or
  • ALYVGDLCGGVMLAAQVF SEQ ID NO:67
  • AMYVGDLCGSVFLVAQLF SEQ ID NO:68
  • Another peptide inhibitor homologue of the invention has SEQ ID NO: 69 or 70, which are homologues of peptide SEQ ID NO: 14.
  • IIDIVSGAHWGVMFGLAY SEQ ID NO:69
  • VVDMVAGAHWGVLAGLAY SEQ ID NO:70
  • the sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
  • Another peptide inhibitor homologue of the invention has SEQ ID NO:71 or 72, which are homologues of peptide SEQ ID NO:24.
  • VDVQYMYGLSPAITKYVV (SEQ ID NO:71)
  • YLYGIGSAVVSFAIKWEY (SEQ ID NO:72)
  • Another peptide inhibitor homologue of the invention has SEQ ID NO: 73 or 74, which are homologues of peptide SEQ ID NO:27.
  • WMLILLGQAEAALEKLVV (SEQ ID NO:73)
  • WMMLLIAQAEAALENLVV (SEQ ID NO:74)
  • sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
  • Another peptide inhibitor homologue of the invention has SEQ ID NO: 75 or
  • GVVFDITKWLLALLGPAY SEQ ID NO:75
  • ELIFTITKILLAILGPLM SEQ ID NO:76
  • the peptide inhibitor homologue has SEQ ID NO:77 or 78, which are homologues of peptide SEQ ID NO:32.
  • VSQSFLGTTISGVLWTVY SEQ ID NO:77
  • ATQSFLATCVNGVCWTVY SEQ ID NO:78.
  • the sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
  • the peptide inhibitor homologue has SEQ ID NO: 79 or 80, which are homologues of peptide SEQ ID NO:43.
  • SWLRDVWDWVCTILTDFK (SEQ ID NO:79); SWLRDVWDWICTVLTDFK (SEQ ID NO:80).
  • the peptide inhibitor homologue has SEQ ID NO: 81 or 82, which are homologues of peptide SEQ ID NO:44.
  • DWVCTILTDFKNWLTSKL SEQ ID NO:81
  • DWICTVLTDFKTWLQSKL SEQ ID NO:82.
  • the sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
  • the peptide inhibitor homologue has SEQ ID NO:83 or 84, which are homologues of peptide SEQ ID NO:47.
  • ASEDVYCCSMSYTWT (SEQ ID NO:83); EDDTTVCCSMSYSW (SEQ ID NO:84).
  • the peptide inhibitor homologue has SEQ ID NO:85 or 86, which are homologues of peptide SEQ ID NO:53.
  • CTMLVCGDDLVVICESAG SEQ ID NO:85
  • PTMLVCG DDLVVISESQG SEQ ID NO:86.
  • the sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
  • a peptide variant is any peptide having an amino acid sequence that is not identical to a segment in the polyprotein sequence of a HCV isolate.
  • a peptide of the invention can have a variant sequence that results from conservative amino acid substitutions.
  • Amino acids that are substitutable for each other generally reside within similar classes or subclasses. As known to one of skill in the art, amino acids can be placed into different classes depending primarily upon the chemical and physical properties of the amino acid side chain. For example, some amino acids are generally considered to be hydrophilic or polar amino acids and others are considered to be hydrophobic or nonpolar amino acids.
  • Polar amino acids include amino acids having acidic, basic or hydrophilic side chains and nonpolar amino acids include amino acids having aromatic or hydrophobic side chains. Nonpolar amino acids may be further subdivided to include, among others, aliphatic amino acids.
  • the definitions of the classes of amino acids as used herein are as follows.
  • Nonpolar Amino Acid refers to an amino acid having a side chain that is uncharged at physiological pH, that is not polar and that is generally repelled by aqueous solution.
  • Examples of genetically encoded hydrophobic amino acids include Ala, He, Leu, Met, Trp, Tyr and VaI.
  • Examples of non-genetically encoded nonpolar amino acids include t-BuA, Cha and NIe.
  • Aromatic Amino Acid refers to a nonpolar amino acid having a side chain containing at least one ring having a conjugated ⁇ -electron system (aromatic group).
  • aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others.
  • substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others.
  • Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan.
  • Non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, a-2-thienylalanine, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.
  • Aliphatic Amino Acid refers to a nonpolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain.
  • Examples of genetically encoded aliphatic amino acids include Ala, Leu, VaI and He.
  • Examples of non-encoded aliphatic amino acids include NIe.
  • Polar Amino Acid refers to a hydrophilic amino acid having a side chain that is charged or uncharged at physiological pH and that has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • Polar amino acids are generally hydrophilic, meaning that they have an amino acid having a side chain that is attracted by aqueous solution.
  • genetically encoded polar amino acids include asparagine, cysteine, glutamine, lysine and serine.
  • non-genetically encoded polar amino acids include citrulline, homocysteine, N-acetyl lysine and methionine sulfoxide.
  • Acidic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
  • Basic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of greater than 7.
  • Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion.
  • genetically encoded basic amino acids include arginine, lysine and histidine.
  • non-genetically encoded basic amino acids include amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.
  • Ionizable Amino Acid refers to an amino acid that can be charged at a physiological pH.
  • Such ionizable amino acids include acidic and basic amino acids, for example, D-aspartic acid, D-glutamic acid, D-histidine, D-arginine, D-lysine, D- hydroxylysine, D-ornithine, L-aspartic acid, L-glutamic acid, L-histidine, L-arginine, L-lysine, L-hydroxylysine or L-ornithine.
  • tyrosine has both a nonpolar aromatic ring and a polar hydroxyl group.
  • tyrosine has several characteristics that could be described as nonpolar, aromatic and polar.
  • the nonpolar ring is dominant and so tyrosine is generally considered to be hydrophobic.
  • cysteine in addition to being able to form disulfide linkages, cysteine also has nonpolar character.
  • cysteine can be used to confer hydrophobicity or nonpolarity to a peptide.
  • hydrophilic or polar amino acids contemplated by the present invention include, for example, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine, hydroxylysine, ornithine, serine, threonine, and structurally related amino acids.
  • the polar amino is an ionizable amino acid such as arginine, aspartic acid, glutamic acid, histidine, hydroxylysine, lysine, or ornithine.
  • hydrophobic or nonpolar amino acid residues examples include, for example, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tryptophan, tyrosine and the like.
  • amino acid sequence of a peptide can be modified so as to result in a peptide variant that includes the substitution of at least one amino acid residue in the peptide for another amino acid residue, including substitutions that utilize the D rather than L form.
  • One or more of the residues of the peptide can be exchanged for another, to alter, enhance or preserve the biological activity of the peptide.
  • Such a variant can have, for example, at least about 10% of the biological activity of the corresponding non-variant peptide.
  • Conservative amino acid substitutions are often utilized, i.e., substitutions of amino acids with similar chemical and physical properties, as described above.
  • conservative amino acids substitutions involve exchanging aspartic acid for glutamic acid; exchanging lysine for arginine or histidine; exchanging one nonpolar amino acid (alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine) for another; and exchanging one polar amino acid (aspartic acid, asparagine, glutamic acid, glutamine, glycine, serine, threonine, etc.) for another.
  • substitutions are introduced, the variants can be tested to confirm or determine their levels of biological activity.
  • the peptides of the invention can have a sequence that includes any one of formulae IX-XIII:
  • Xaai, Xaa 4 , Xaa 5 , Xaa 8 , Xaan, Xaaj 2 , Xaai 5 , Xaai 6 and Xaai 8 are polar amino acids;
  • Xaa 2 , Xaa 3 , Xaa ⁇ , Xaa 7 , Xaa 9 , Xaaio, Xaai 3 , Xaa ⁇ , and Xaa ⁇ are nonpolar amino acids.
  • the present peptides can have additional peptidyl sequences at either the N-terminus or the C-terminus.
  • the invention provides a fusion peptide formed by attaching a 14 amino acid peptide (the N-terminal peptide) to the N-terminus of a peptide of any of formulae IX to XIII.
  • the 14 amino acid N-terminal peptide has the structure: Rx-Ry-Ry-Rx-Ry- Ry-Rx-Rx-Ry-Ry-Rx-Rx-Ry-Rx (SEQ ID NO: 1 17), wherein each Rx is separately a polar amino acid, and each Ry is separately a nonpolar amino acid.
  • the invention also provides a fusion peptide formed by attaching a 12 amino acid peptide (the C-terminal peptide) to the C-terminus of a peptide of formula XIII.
  • the resulting fusion peptide has the structure of formulae XIV:
  • Xaai, Xaa ⁇ Xaa 5 , Xaa 8 , Xaan, Xaai 2 , Xaa ] 5 , Xaa )6 , Xaais, Xaai 9 , Xaa 22 , Xaa 23 , Xaa 26 , Xaa 29 , and Xaa 30 are separately each a polar amino acid;
  • Xaa 2 , Xaa 3 , Xaa ⁇ , Xaa 7 , Xaa 9 , Xaaio, Xaan, Xaai 4 , Xaai 7 , Xaa 2 o, Xaa 2 i, Xaa 24 , Xaa 25 , Xaa 27 , and Xaa 28 are separately each a nonpolar amino acid.
  • the invention also provides a fusion peptide having a sequence that corresponds to the 14 amino acid N-terminal peptide of SEQ ID NO: 117 attached by a peptide bond to the N-terminus of a peptide of formula XIV.
  • a peptide of the invention is a peptide comprising at least 14 contiguous amino acids of any of the above described peptides.
  • a peptide variant can also result from "scrambling" of the hydrophilic and/or hydrophobic residues within a sequence as long as the amphipathic ⁇ -helical secondary structure of the peptide in solution is maintained.
  • an "isolated" peptide is a peptide that exists apart from its native environment and is therefore not a product of nature.
  • An isolated peptide may exist in a purified form or may exist in a non-native environment such as, for example, in a cell or in a composition with a solvent that may contain other active or inactive ingredients.
  • an "isolated" peptide free of at least some of sequences that naturally flank the peptide (i.e., sequences located at the N-terminal and C-terminal ends of the peptide) in the protein from which the peptide was originally derived.
  • a “purified” peptide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a purified peptide preparation is at least 50 %, at least 60 %, at least 70 %, at least 80 % or at least 90 % by weight peptide. Purity can be determined using methods known in the art, including, without limitation, methods utilizing chromatography or polyacrylamide gel electrophoresis.
  • the present peptides or variants thereof can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by enzyme catalyzed peptide synthesis or with the aid of recombinant DNA technology.
  • Solid phase peptide synthetic method is an established and widely used method, which is described in references such as the following: Stewart et al.. Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc. 85_ 2149 (1963); Meienhofer in "Hormonal Proteins and Peptides," ed.; CH.
  • Peptides of the invention can be cyclic peptides so long as they retain antiviral activity.
  • Such cyclic peptides are generated from linear peptides typically by covalently joining the amino terminus to the terminal carboxylate. To insure that only the termini are joined amino and carboxylate side chains can be protected with commercially available protecting groups.
  • one of skill in the art may choose to cyclize peptide side chains to one of the amino or carboxylate termini, or to another amino acid side chain. In this case, protecting groups can again be used to guide the cyclization reaction as desired.
  • Cyclization of peptides can be performed using available procedures. For example, cyclization can be performed in dimethylformamide at a peptide concentration of 1-5 mM using a mixture of benzotriazole-1-yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP, Novabiochem) (5 eq. with respect to crude peptide) and N,N-diisopropylethylamine (DIEA, Fisher) (40 eq.). The amount of DIEA is adjusted to achieve an apparent pH 9-10. The reaction can be followed by any convenient means, for example, by MALDI-MS and/or HPLC.
  • N-acyl derivatives of an amino group of the peptide or peptide variants may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide.
  • O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N-acylation and O- acylation may be carried out together, if desired.
  • Salts of carboxyl groups of a peptide or peptide variant of the invention may be prepared in the usual manner by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide
  • a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate
  • an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • Acid addition salts of the peptide or variant peptide, or of amino residues of the peptide or variant peptide may be prepared by contacting the peptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid.
  • Esters of carboxyl groups of the peptides may also be prepared by any of the usual methods known in the art.
  • Peptides of the invention can be employed to prevent, treat or otherwise ameliorate infection by any human immunodeficiency virus (HIV), measles virus, or respiratory syncytial virus as well, as any virus from the Flaviviridae family.
  • HIV human immunodeficiency virus
  • peptides of the invention can be used to inactivate any of these viruses, in vivo, in vitro or ex vivo.
  • a peptide of the invention can be used to prevent, treat or otherwise ameliorate infection by any HIV, measles or RSV virus and any virus from the Flaviviridae family, as well as prevent, treat or otherwise ameliorate the associated disease conditions.
  • the present peptides can be used as a therapeutic agent to prevent sexual transmission of HIV, to treat and inhibit HIV disease progression, to inhibit HIV multiplication, to reduce, HIV viral load, to promote CD4+ responses against HIV, to promote CD8+ responses against HIV, to cure HIV infection, to increase the safety of blood and blood products used in transfusions, and to increase the safety of clinical laboratory samples.
  • the peptides of the invention can also be used to inactivate viruses in bodily fluid samples and tissue samples, and to inhibit or prevent infection amongst people who must handle these samples.
  • a peptide of the invention can be employed to prevent, treat or otherwise ameliorate infection by a virus of the Flaviviridae family, which includes, without limitation, viruses in the genera Flavivirus, Pestivirus, and Hepacivirus, as described above.
  • Flavivirus genus include viruses that cause Tick- borne encephalitis, Central European encephalitis, Far Eastern encephalitis, Rio Bravo, Japanese encephalitis, Kunjin, Murray Valley encephalitis, St Louis encephalitis, West Nile encephalitis, Tyulenly, Ntaya, Kenya S, Dengue type 1 , Dengue type 2, Dengue type 3, Dengue type 4, Modoc, and Yellow Fever.
  • Pestivirus genus include Bovine viral diarrhea virus 1, Bovine viral diarrhea virus 2, Hog cholera (classical swine fever virus), and Border disease virus.
  • the Hepacivirus genus include hepatitis C virus. Additional members of the Flaviviridae family include the unassigned GB virus-A, GB virus-B, and GB virus- C. Members of the Flaviviridae family of viruses are known to cause a variety of diseases including, for example, Dengue fever, Hepatitis C infection, Japanese encephalitis, Kyasanur Forest disease, Murray Valley encephalitis, St. Louis encephalitis, Tick-borne encephalitis, West Nile encephalitis and Yellow fever.
  • a peptide of the invention can be used to prevent, treat or otherwise ameliorate infection by a member of the Flaviviridae family of viruses and its associated disease conditions.
  • examples of various applications of the invention include, without limitation, use as a therapeutic for patients with Dengue fever, Dengue hemorrhagic fever, Dengue shock syndrome, Japanese aencephalitis, Kyasanur forest disease, Murray Valley encephalitis, St. Louis Encephalitis, Tick- borne meningoencephalitis, Chronic hepatitis C infection, to prevent graft infection during liver transplantation, to prevent sexual transmission, to increase the safety of blood and blood product used in transfusions, and to increased safety of clinical laboratory samples.
  • the invention provides a method for preventing, inhibiting or otherwise ameliorating viral infection of mammalian cell, such as a human cell, or a method for preventing, inhibiting, treating or otherwise ameliorating acute or chronic infection of a mammal such as a human by a human immunodeficiency virus, measles virus, respiratory syncytial virus or a virus of the Flaviviridae family.
  • preventing is intended to include the administration of a peptide of the invention to a mammal such as a human who could be or has been exposed to a human immunodeficiency virus for purposes of inhibiting infection.
  • the mammal who could be exposed to HIV includes, without limitation, someone present in an area where these viruses are prevalent or commonly transmitted, e.g., Africa, Southeast Asia, China, South Asia, Australia, India, the United States, Russia, as well as Central and South American countries.
  • the mammal who could be exposed to HIV also includes someone who has been a recipient of donated body tissue or fluids, for example, a recipient of blood or one or more of its components such as plasma, platelets, or stem cells; and medical, clinical or dental personnel who handle body tissues and fluids.
  • a mammal who has been exposed to HIV includes, without limitation, someone who has had contact with the body tissue or fluid, e.g. blood, of an infected person or otherwise have come in contact with HIV.
  • preventing is intended to include the administration of a peptide of the invention to a mammal such as a human who could be or has been exposed to a member of the Flaviviridae family to inhibit infection by a virus of the Flaviviridae family.
  • the mammal who could be exposed to a virus of the Flaviviridae family includes, without limitation, someone present in an area where these viruses are prevalent or common, e.g. the tropics, Southeast Asia and the Far East, South Asia, Australia and Papua New guinea, the United States, Russia, Africa, as well as Central and South American countries.
  • the mammal who could be exposed to a virus of the Flaviviridae family also includes someone who has been bitten by a deer or forest tick or a mosquito; a recipient of donated body tissue or fluids, for example, a recipient of blood or one or more of its components such as plasma, platelets, or stem cells; and medical, clinical or dental personnel who handle body tissues and fluids.
  • a mammal who has been exposed to a virus of the Flaviviridae family also includes someone who has been bitten by a deer or forest tick or a mosquito; a recipient of donated body tissue or fluids, for example, a recipient of blood or one or more of its components such as plasma, platelets, or stem cells; and medical, clinical or dental personnel who handle body tissues and fluids.
  • Flaviviridae family include, without limitation, someone who has had contact with the body tissue or fluid, e.g. blood, of an infected person or otherwise have come in contact with HCV or any other virus of the Flaviviridae family.
  • preventing is intended to include the administration of a peptide of the invention to a mammal such as a human who could be or has been exposed to a measles or respiratory syncytial virus to inhibit infection by measles or RSV.
  • Treatment of, or treating HIV infection, measles infection, RSV infection or infection by a virus of the Flaviviridae family is intended to include a reduction of the viral load or the alleviation of or diminishment of at least one symptom typically associated with the infection.
  • the treatment also includes alleviation or diminishment of more than one symptom.
  • the treatment cures, e.g., substantially inhibits viral infection and/or eliminates the symptoms associated with the infection.
  • Symptoms or manifestations of viral exposure or infection are specific for the particular infection, and these are known in the art.
  • HIV infection Early symptoms of HIV infection include flu-like symptoms within three to six weeks after exposure to the virus. This illness, called Acute HIV Syndrome, may include fever, headache, tiredness, nausea, diarrhea and enlarged lymph nodes.
  • Dengue fever and dengue hemorrhagic fever are caused by one of four Flavivirus serotypes. Symptoms of these conditions include sudden onset of fever, severe headache, joint and muscular pains and rashes, as well as high fever, thrombocytopenia and haemoconcentration. Clinical indications of also include high fever, petechial rash with thrombocytopenia and leucopenia, and haemorrhagic tendency. Symptoms of Japanese aencephalitis include fever, headache, neck rigidity, cachexia, hemiparesis, convulsions and heightened body temperature.
  • Japanese encephalitis can be diagnosed by detection of antibodies in serum and cerebrospinal fluid.
  • Symptoms of Kyasanur forest disease include high fever, headache, haemorrhages from nasal cavity and throat, and vomiting.
  • Symptoms of St. Louis encephalitis include fever, headache, neck stiffness, stupor, disorientation, coma, tremors, occasional convulsions and spastic paralysis.
  • Symptoms of Murray Valley encephalitis include fever, seizures, nausea and diarrhea in children, and headaches, lethargy and confusion in adults.
  • Symptoms of West Nile virus infection include flu-like symptoms, malaise, fever, anorexia, nausea, vomiting, eye pain, headache, myalgia, rash and lymphadenopathy, as well as encephalitis (inflammation of the brain) and meningitis (inflammation of the lining of the brain and spinal cord), meningismus, temporary blindness, seizures and coma.
  • West Nile infection can be diagnosed using ELISA to detect antibodies in the blood or cerebrospinal fluids.
  • Symptoms of Yellow fever include fever, muscle aches, headache, backache, a red tongue, flushed face, red eyes, hemorrhage from the gastrointestinal tract, bloody vomit, jaundice, liver failure, kidney insufficiency with proteinuria, hypotension, dehydration, delirium, seizure and coma.
  • Symptoms of hepatitis C infection include, without limitation, inflammation of the liver, decreased appetite, fatigue, abdominal pain, jaundice, flu-like symptoms, itching, muscle pain, joint pain, intermittent low-grade fevers, sleep disturbances, nausea, dyspepsia, cognitive changes, depression headaches and mood changes.
  • HCV infection could also be diagnosed by detecting antibodies to the virus, detecting liver inflammation by biopsy, liver cirrhosis, portal hypertension, thyroiditis, cryoglobulinemia and glomerulonephritis.
  • HCV infection could be diagnosed.
  • diagnosis of exposure or infection or identification of one who is at risk of exposure to HCV could be based on medical history, abnormal liver enzymes or liver function tests during routine blood testing.
  • infection by a member of the Flaviviridae family can be diagnosed using ELISA for detecting viral antigens or anti-viral antibodies, immunofluorescence for detecting viral antigens, polymerase chain reaction (PCR) for detecting viral nucleic acids and the like.
  • Symptoms of measles include a fever for at least three days, as well as cough, a runny nose, and conjunctivitis.
  • the fever may reach up to 40° Celsius (104° Fahrenheit).
  • Koplik's spots seen inside the mouth are pathognomonic
  • measles rash is classically described as a generalized, maculopapular, erythematous rash that begins several days after the fever starts. It starts on the head before spreading to cover most of the body, often causing itching. The rash is said to "stain,” changing color from red to dark brown, before disappearing.
  • RSV causes respiratory tract infections in patients of all ages. It is the major cause of lower respiratory tract infection during infancy and childhood. Symptoms include recurrent wheezing and asthma may develop among individuals who suffered severe RSV infection during the first few months of life.
  • Methods of preventing, treating or otherwise ameliorating acute or chronic viral infection include contacting the cell with an effective amount of a peptide of the invention or administering to a mammal such as a human a therapeutically effective amount of a peptide of the present invention.
  • Methods of inactivating the virus include contacting the virus with an effective amount of the peptide of the invention..
  • a peptide of the invention can be administered in a variety of ways.
  • Routes of administration include, without limitation, oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, vaginal, dermal, transdermal (topical), transmucosal, intrathoracic, intrapulmonary and intranasal (respiratory) routes.
  • the means of administration may be by injection, using a pump or any other appropriate mechanism.
  • a peptide of the invention may be administered in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the peptides of the invention may be essentially continuous over a pre-selected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • the dosage to be administered to a mammal may be any amount appropriate to reduce or prevent viral infection or to treat at least one symptom associated with the viral infection.
  • Some factors that determine appropriate dosages are well known to those of ordinary skill in the art and may be addressed with routine experimentation. For example, determination of the physicochemical, toxicological and pharmacokinetic properties may be made using standard chemical and biological assays and through the use of mathematical modeling techniques known in the chemical, pharmacological and toxicological arts. The therapeutic utility and dosing regimen may be extrapolated from the results of such techniques and through the use of appropriate pharmacokinetic and/or pharmacodynamic models. Other factors will depend on individual patient parameters including age, physical condition, size, weight, the condition being treated, the severity of the condition, and any concurrent treatment.
  • the dosage will also depend on the peptide(s) chosen and whether prevention or treatment is to be achieved, and if the peptide is chemically modified. Such factors can be readily determined by the clinician employing viral infection models such as the HCV cell culture/JFH-1 infection model described herein, or other animal models or test systems that are available in the art.
  • a peptide of the invention, a variant thereof or a combination thereof may be administered as single or divided dosages, for example, of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages may provide beneficial results.
  • the absolute weight of a given peptide included in a unit dose can vary widely.
  • the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.
  • Daily doses of the peptides of the invention can vary as well.
  • Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day.
  • a peptide of the invention may be used alone or in combination with a second medicament.
  • the second medicament can be a known antiviral agent such as, for example, an interferon-based therapeutic or another type of antiviral medicament such as ribavirin.
  • the second medicament can be an anticancer, antibacterial, or antiviral agent.
  • the antiviral agent may act at any step in the life cycle of the virus from initial attachment and entry to egress.
  • the added antiviral agent may interfere with attachment, fusion, entry, trafficking, translation, viral polyprotein processing, viral genome replication, viral particle assembly, egress or budding.
  • the antiviral agent may be an attachment inhibitor, entry inhibitor, a fusion inhibitor, a trafficking inhibitor, a replication inhibitor, a translation inhibitor, a protein processing inhibitor, an egress inhibitor, in essence an inhibitor of any viral function.
  • the effective amount of the second medicament will follow the recommendations of the second medicament manufacturer, the judgment of the attending physician and will be guided by the protocols and administrative factors for amounts and dosing as indicated in the PHYSICIAN'S DESK REFERENCE.
  • the effectiveness of the method of treatment can be assessed by monitoring the patient for signs or symptoms of the viral infection as discussed above, as well as determining the presence and/or amount of virus present in the blood, e.g. the viral load, using methods known in the art including, without limitation, polymerase chain reaction and transcription mediated amplification.
  • the invention provides a pharmaceutical composition comprising a peptide of the invention.
  • a peptide of the invention is synthesized or otherwise obtained, purified as necessary or desired and then lyophilized and stabilized.
  • the peptide can then be adjusted to the appropriate concentration and then combined with other agent(s) or pharmaceutically acceptable carrier(s).
  • pharmaceutically acceptable it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • compositions containing a therapeutic peptide of the invention can be prepared by procedures known in the art using well-known and readily available ingredients.
  • the peptide can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like.
  • excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives.
  • Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone.
  • Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate.
  • Agents for retarding dissolution can also be included such as paraffin.
  • Resorption accelerators such as quaternary ammonium compounds can also be included.
  • Surface active agents such as cetyl alcohol and glycerol monostearate can be included.
  • Adsorptive carriers such as kaolin and bentonite can be added.
  • Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added.
  • the compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They may also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.
  • a peptide may be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum.
  • the active peptide may also be presented as a bolus, electuary or paste.
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts including the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • the total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.
  • Tablets or caplets containing the peptides of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate.
  • Caplets and tablets can also include inactive ingredients such as cellulose, pre-gelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like.
  • Hard or soft gelatin capsules containing at least one peptide of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil.
  • inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like
  • liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil.
  • enteric-coated caplets or tablets containing one or more peptides of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.
  • Orally administered therapeutic peptide of the invention can also be formulated for sustained release.
  • a peptide of the invention can be coated, micro-encapsulated (see WO 94/ 07529, and U.S. Patent No.4,962,091), or otherwise placed within a sustained delivery device.
  • a sustained-release formulation can be designed to release the active peptide, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time.
  • Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, draining devices and the like.
  • a therapeutic peptide of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes.
  • a pharmaceutical formulation of a therapeutic peptide of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.
  • a therapeutic peptide may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers.
  • preservatives can be added to help maintain the shelve life of the dosage form.
  • the active peptides and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active peptides and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water
  • formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol,” polyglycols and polyethylene glycols, C ⁇ -C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol,” isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.
  • organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol,” poly
  • an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings.
  • Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and ⁇ -tocopherol and its derivatives can be added.
  • the peptides are formulated as a microbicide, which is administered topically or to mucosal surfaces such as the vagina, the rectum, eyes, nose and the mouth.
  • the therapeutic agents may be formulated as is known in the art for direct application to a target area.
  • Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap.
  • a peptide of the invention can be formulated as a vaginal cream or a microbicide to be applied topically.
  • Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols.
  • the therapeutic peptides of the invention can be delivered via patches or bandages for dermal administration.
  • the peptide can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer.
  • an adhesive polymer such as polyacrylate or acrylate/vinyl acetate copolymer.
  • the backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • the active peptides can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051,842.
  • a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01 % to 95 % of the total weight of the formulation, and typically 0.1-85 % by weight.
  • Drops such as eye drops or nose drops, may be formulated with one or more of the therapeutic peptides in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents.
  • Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
  • the therapeutic peptide may further be formulated for topical administration in the mouth or throat.
  • the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier.
  • the pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art.
  • pharmaceutically acceptable carriers such as physiologically buffered saline solutions and water.
  • diluents such as phosphate buffered saline solutions pH 7.0-8.0.
  • the peptides of the invention can also be administered to the respiratory tract.
  • the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention.
  • dosage forms comprise an amount of at least one of the agents of the invention effective to treat or prevent the clinical symptoms of the viral infection. Any statistically significant attenuation of one or more symptoms of the infection that has been treated pursuant to the method of the present invention is considered to be a treatment of such infection within the scope of the invention.
  • the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator, or a metered-dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds., pp. 197-224, Butterworths, London, England, 1984).
  • MDI pressurized metered dose inhaler
  • the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds., pp. 197-224
  • Therapeutic peptides of the present invention can also be administered in an aqueous solution when administered in an aerosol or inhaled form.
  • other aerosol pharmaceutical formulations may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/mL and about 100 mg/mL of one or more of the peptides of the present invention specific for the indication or disease to be treated.
  • Dry aerosol in the form of finely divided solid peptide or nucleic acid particles that are not dissolved or suspended in a liquid are also useful in the practice of the present invention.
  • Peptides of the present invention may be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 ⁇ m, alternatively between 2 and 3 ⁇ m.
  • Finely divided particles may be prepared by pulverization and screen filtration using techniques well known in the art.
  • the particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder.
  • the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units.
  • the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.
  • the therapeutic peptides of the invention are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifiuoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Nebulizers include, but are not limited to, those described in U.S. Patent Nos.
  • Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co., (Valencia, CA).
  • the therapeutic agent may also be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler.
  • atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).
  • a therapeutic peptide of the invention may also be used in combination with one or more known therapeutic agents, for example, a pain reliever; an antiviral agent such as an anti-HBV, anti-HCV (HCV inhibitor, HCV protease inhibitor) or an anti-herpetic agent; an antibacterial agent; an anti-cancer agent; an antiinflammatory agent; an antihistamine; a bronchodilator and appropriate combinations thereof, whether for the conditions described or some other condition.
  • a pain reliever an antiviral agent such as an anti-HBV, anti-HCV (HCV inhibitor, HCV protease inhibitor) or an anti-herpetic agent
  • an antibacterial agent such as an anti-cancer agent
  • an antiinflammatory agent such as an antihistamine, a bronchodilator and appropriate combinations thereof, whether for the conditions described or some other condition.
  • the invention provides an article of manufacture that includes a pharmaceutical composition containing a peptide of the invention for controlling microbial infections.
  • Such articles may be a useful device such as a vaginal ring, a condom, a bandage or a similar device.
  • the device holds a therapeutically effective amount of a pharmaceutical composition for controlling viral infections.
  • the device may be packaged in a kit along with instructions for using the pharmaceutical composition for control of the infection.
  • the pharmaceutical composition includes at least one peptide of the present invention, in a therapeutically effective amount such that viral infection is controlled.
  • An article of manufacture may also be a vessel or filtration unit that can be used for collection, processing or storage of a biological sample containing a peptide of the invention.
  • a vessel may be evacuated.
  • Vessels include, without limitation, a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter.
  • the filtration unit can be part of another device, for example, a catheter for collection of biological fluids.
  • the peptides of the invention can also be adsorbed onto or covalently attached to the article of manufacture, for example, a vessel or filtration unit.
  • the peptides of the invention can be in filtration units integrated into biological collection catheters and vials, or added to collection vessels to remove or inactivate viral particles that may be present in the biological samples collected, thereby preventing transmission of the disease.
  • the invention also provides a composition comprising a peptide of the invention and one or more clinically useful agents such as a biological stabilizer.
  • Biological stabilizer includes, without limitation, an anticoagulant, a preservative and a protease inhibitor.
  • Anticoagulants include, without limitation, oxalate, ethylene diamine tetraacetic acid, citrate and heparin.
  • Preservatives include, without limitation, boric acid, sodium formate and sodium borate.
  • Protease inhibitors include inhibitors of dipeptidyl peptidase IV.
  • compositions comprising a peptide of the invention and a biological stabilizer may be included in a collection vessel such as a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter or any other container or vessel used for the collection, processing or storage of a biological samples.
  • the invention also provides a composition comprising a peptide of the invention and a biological sample such as blood, semen or other body fluids that is to be analyzed in a laboratory or introduced into a recipient mammal.
  • a peptide of the invention can be mixed with blood prior to laboratory processing and/or transfusions.
  • the peptides of the invention can be included in physiological media used to store and transport biological tissues, including transplantation tissues.
  • biological tissues including transplantation tissues.
  • liver, heart, kidney and other tissues can be bathed in media containing the present peptides to inhibit viral transmission to transplant recipients.
  • the invention is further illustrated by the following non-limiting Examples.
  • Example 1 Methods Cell culture and reagents. Huh-7 and Huh-7.5.1 cells were described in
  • HeLa, Hep2, LLC- MK2, MRC-5, MA- 104, Vero and MDBK cells were obtained from the American Type Culture Collection. All cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS), 10 mM Hepes buffer, 100 units/ml penicillin, 100 mg/ml streptomycin and 100 ⁇ M non-essential amino acids (Invitrogen) at 5% CO 2 . Peptide synthesis.
  • Huh-7 cells were fixed with 4 % paraformaldehyde, and stained with previously determined optimal dilutions of these antibodies followed by incubation with a 1 : 1000 dilution of Alexa555-conjugated secondary antibodies to human or rabbit IgG (Molecular Probes, Eugene, OR), respectively.
  • Cell nuclei were stained with Hoechst dye or determined by differential interference contrast (DIC).
  • DIC differential interference contrast
  • MTT cytotoxicity assay The cytotoxic effect of peptides was measured in an MTT cytotoxicity assay according to the manufacturer's instructions (MTT assay kit, Cat# 30-1010K, ATCC, Manassas, VA). In brief, serial 2-fold dilutions of peptide in 5 % dextrose H 2 O containing 0.5% DMSO or DMSO alone were added to 5000-10,000 cells in a 96 well plate. After 72 hours at 37 0 C, 1/10 volume of MTT solution (5mg/mL in PBS) was added to each well and the plates were returned to the incubator.
  • MTT assay kit Cat# 30-1010K, ATCC, Manassas, VA.
  • HRP horse radish peroxidase
  • Pierce horse radish peroxidase
  • Assay controls included (a) wells that were coated with 1 ⁇ g of dansylated D-peptide 1 and incubated with rabbit polyclonal anti-dansyl antibody (1 :400 dilution) (Molecular Probes, Eugene, OR) followed by HRP-conjugated goat anti-rabbit IgG (1 :25,000 dilution) (Pierce, Rockford, IL); (b) wells coated with 1 ⁇ g influenza hemagglutinin (HA) peptide (Sigma, St. Louis, MO) and incubated with mouse monoclonal anti- HA antibody (1 :600 dilution) (Sigma, St. Louis, MO) followed by HRP-conjugated goat anti-mouse IgG (1 :25,000 dilution) (Pierce, Rockford, IL).
  • HA hemagglutinin
  • Velocity sedimentation ultracentrifugation The sedimentation velocity of native and peptide-treated infectious HCV particles was examined by rate zonal ultracentrifugation in continuous sucrose gradients as previously described. Briefly, 100 ⁇ L samples were layered onto 5 mL preformed 10 to 50 % continuous sucrose gradients and spun for 1 hour at 200,000 x g in a SW60.Ti rotor at 4 °C. After centrifugation, 12 fractions (400 ⁇ L each) were collected from the top and analyzed for virus infectivity and HCV RNA content as described above.
  • Huh-7 cells were infected by JFH-I at a multiplicity of infection (moi) of 0.01. Ten days later, infected cells were washed 3x with PBS, trypsinized, and resuspended in complete culture medium at a concentration of 1x10 5 cells/mL. The cells were lysed by four freeze-thaw cycles in dry ice and a 37 0 C water bath and centrifuged for 2 minutes at 14,000 rpm to remove cell debris. The supernatant was assayed for infectivity in a standard titration assay as described above.
  • moi multiplicity of infection
  • virus infections To determine if selected peptides inhibit other virus infections, varying concentrations of each peptide or DMSO were added to virus stocks of predetermined infectivity (1-10 5 ffu or TCID 5 o/mL), incubated at 37 0 C for at least 1 hour and then added to susceptible cells, unless specified. After 2-4 days of infection, the cultures were assessed by comparative assessment of cytopathic effect (CPE) or by immunostaining or immunoassay with antibody against the corresponding viral protein as described below.
  • CPE cytopathic effect
  • HBV Since HBV is not infectious in vitro, we examined the impact of peptides on HBs antigenicity by quantitative ELISA analysis as described in Guidotti et al., J Virol 69, 6158-69 (1995) and on HBV DNA content by quantitative PCR analysis as described in Thimme et al., J Virol 11, 68-76 (2003).
  • the Example describes the identification of anti-viral peptides of the invention.
  • a peptide library of 441 overlapping peptides covering the complete hepatitis C viral polyprotein of hepatitis C genotype 1 a (H77) (SEQ ID NO: 1 ) was prepared and tested for inhibition of hepatitis C viral infection using a cell culture model of hepatitis C viral infection described in a related application (U.S. Ser. No. 11/541,488).
  • the peptides were about 18 amino acids in length with 1 1 overlapping amino acids.
  • the peptide library was made by NIH AIDS Research and Reference Reagent Program (Cat # 7620, Lot # 1).
  • the peptide library was screened by an HCV assay.
  • the peptides were reconstituted in 100 % DMSO at a final concentration 10 mg/mL, and stored in -20 0 C.
  • the peptide stock solution was diluted 1 :200 to a final concentration approximately 20 ⁇ M in complete DMEM growth medium containing 50 focus forming units (ffu) of HCV.
  • the virus-peptide mixture was transferred to Huh-7.5.1 cells at a density of 8000 cells per well in a 96-well plate. After adsorption for 4 hours at 37 °C, the inoculum was removed.
  • the cells were washed 2 times, overlaid with 120 ⁇ L fresh growth medium and incubated at 37 0 C. After 3 days of culture, the cells were fixed with paraformaldehyde and immunostained with antibody against HCV nonstructural protein NS5A. The numbers of HCV foci were counted under fluorescent microscopy and the result is expressed as percentage (%) of control foci detected in cells inoculated with virus and 0.5 % DMSO, but without peptides.
  • HCV infection was profoundly inhibited (90-100 %) by peptides with SEQ ID NO:6, 8, 12, 13, 14, 24, 27, 30, 32, 43, 44, 47, 48 and 53. No evidence of toxicity was detected when Huh-7.5.1 cells were incubated with these peptides.
  • these peptides can be used in antiviral compositions and methods for inhibiting HCV infection. Peptides that inhibited infection by more than 90 % were selected for further analysis.
  • Validation of the antiviral activity of these inhibitory peptides was performed by comparing the ability of highly (>95 %) purified preparations of each peptide (20 ⁇ M) to inhibit the expansion of HCV RNA in Huh-7.5.1 cells 72 hours after infection (moi 0.1) relative to the solvent control (0.5 % DMSO).
  • the intracellular HCV RNA content of peptide-treated and untreated cells was quantified by real time RT-QPCR.
  • the peptide stock solution and DMSO solvent were diluted 1 : 100 and mixed with an equal volume of stock viral supernatant to yield a final peptide concentration of 18 ⁇ M and a final DMSO concentration of 0.5%.
  • the virus-peptide and virus-DMSO mixtures were then used to infect Huh-7.5.1 cells at a multiplicity of infection (moi) of 0.1. After 3 days incubation at 37 °C, cells were washed, lysed and total cellular RNA was isolated.
  • moi multiplicity of infection
  • Reverse transcription and quantitative real-time PCR (RT-QPCR) was performed as described in Cheng et al., Proc Natl Acad Sci USA 103, 8499-504 (2006).
  • HCV and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) transcript levels were determined relative to a standard curve comprised of serial dilutions of plasmid containing the HCV JFH-I cDNA or human GAPDH gene.
  • the relative HCV RNA content in infected cells was determined after normalization to cellular GAPDH mRNA levels.
  • the detection limit of the RT-QPCR protocol is approximately 1 copy of HCV RNA per 1000 cells.
  • the HCV RNA transcript level was measured by real time RT-QPCR with the primers S'-TCTGCGGAACCGGTGAGTA-S' (sense, SEQ ID NO: 89) and 5'- TC AGGC AGTACC AC AAGGC-3' (antisense, SEQ ID NO: 90)', and normalized to cellular GAPDH levels. Inhibitory activity was detected by comparing the normalized intracellular HCV RNA levels of the peptide-treated and solvent-treated inocula. Results are summarized in the following table.
  • peptide 1 (SEQ ID NO:43) derived from the N- terminus of NS5A was the most effective peptide inhibitor of HCV and inhibited viral expansion by more than 4 orders of magnitude.
  • Peptide 1 contains residues 3- 21 of the amphipathic alpha helical N-terminal membrane anchor domain of the HCV NS5A protein.
  • peptides having SEQ ID NO:6, 8, 12, 13, 14, 24, 27, 30, 32, 44, 47, 48 and 53 also strongly inhibited HCV infection as measured using a cell culture model of hepatitis C viral infection described in a related application (U.S. Ser. No. 1 1/541,488). Other peptides exhibited good inhibition of HCV infection. These HCV-derived synthetic peptides that were effective inhibitors were from both structural and non-structural regions of the HCV polyprotein.
  • peptide #1 SEQ ID NO:43
  • the antiviral activity of a series of N-terminal and C-terminal truncations of peptide 1 was analyzed using the focus reduction assay and by measuring the reduction in intracellular HCV RNA as described.
  • the solvent system was a H 2 O and acetonitrile solvent system with a linear gradient of 5 % to 70 % for 30 minutes.
  • Mass spectral analysis was performed by PE Sciex API-100 mass spectrometer. This confirmed the molecular masses of the synthesized peptides.
  • mg peptide per mL (A 280 x DF x MW) / e, where A 280 was the actual absorbance of the solution at 280 nm in a 1-cm cell, DF was the dilution factor, MW was the molecular weight of the peptide and e was the molar extinction coefficient of each chromophore at 280 nm.
  • Results summarized in the following table, show that the peptides having C- terminal truncations of 1 to 4 amino acid residues retained antiviral activity. Removal of as few as 2 amino acids from the N-terminus destroyed antiviral activity.
  • Huh-7.5.1 cells were inoculated with HCV (moi 0.1) and peptide 1 (18 ⁇ M) or 0.5 % DMSO as a control. After adsorption for 4 hours at 37 0 C, the virus-peptide and virus-DMSO inocula were removed, the cells were washed 2 times, overlaid with 120 ⁇ L fresh growth medium and incubated at 37 °C. At the indicated time points, total cellular RNA was isolated and the HCV RNA level was measured. As shown in FIG.
  • peptide 1 can terminate an ongoing HCV infection, it was added to infected cells 3 days after virus inoculation, when 10 % of the cells were HCV E2 positive, and it was maintained in the culture medium and replenished every time the cells were split. More specifically, Huh-7 cells were infected with HCV (moi 0.1). On day 3 postinfection, when approximately 10 % of the cells were infected (HCV E2 positive by immunostaining), peptide 1 (18 ⁇ M) or 0.5 % DMSO were added and the cells were incubated at 37 °C and split 1 : 6 every 3-4 days when reaching confluency.
  • peptide 1 can suppress infection in nondividing cells with established infection
  • the D-form of peptide 1 and DMSO were added to growth- arrested and highly differentiated Huh-7 cells (see Sainz et al., J. Virol. 80,10253-7 (2006)) 15 days after infection, when >90 % of the cells were HCV E2-positive (not shown). More specifically, Huh-7 cells were treated with 1 % DMSO for 10 days to induce differentiation and growth arrest at which time they were infected by HCV at moi of 0.01. Fifteen days after infection, when >90 % cells were HCV E2-positive, the L- and D-isomers of peptide 1 (18 ⁇ M) were added in complete growth medium.
  • the culture medium was replaced every day with medium containing fresh peptide.
  • total cellular RNA was isolated and HCV RNA content was measured.
  • the infected cells were treated with recombinant human IFN ⁇ (PBL biomedical Laboratories, Piscataway, NJ) at 100 U/mL that was replenished every day as above.
  • the D-form of peptide 1 suppressed intracellular HCV RNA to the same extent as IFN ⁇ by day 5 and it was even more inhibitory (>95 %) than IFN ⁇ on days 15 and 20 of treatment.
  • peptide 1 might be able to inactivate HCV intracellularly.
  • peptide 1 was evaluated to determine whether it can enter cells and destabilize intracellular virus particles.
  • a fluorescent form of peptide 1 containing a dansyl group at its N-terminus was diluted in complete culture medium to a final concentration of 18 ⁇ M and incubated with Huh-7 cells for 4 hours at 37 0 C before being washed 5 times with PBS.
  • Treated cells were then fixed by 4 % paraformaldehyde, and immunostained with rabbit polyclonal antibody against the dansyl group (Molecular Probes, Eugene, OR) and analyzed by confocal fluorescence microscopy.
  • a fluorescent D-form of peptide 1 containing a dansyl group at its N-terminus efficiently enters Huh-7 cells, accumulating in granular structures in the cytoplasm.
  • the number of intracellular infectious HCV particles was reduced by 3-fold (FIG. 2E), without any change in intracellular HCV RNA (not shown).
  • Huh-7 cells that had been infected with JFH-I at an moi 0.01 for 10 days were washed 4 times, and treated with D-isomers of the peptide 1 (18 ⁇ M) or DMSO (0.5%). After 6 hours incubation, intracellular HCV infectivity, extracellular HCV infectivity and cellular HCV RNA content were determined. The results suggest that, in addition to its extracellular virocidal activity, peptide 1 can enter cells and inactivate intracellular virus, albeit less efficiently, without blocking HCV replication.
  • EC 50 median effective concentration
  • RT-QPCR real time quantitative polymerase chain reaction
  • Inhibitory activity was quantified by comparing the amount of cell-associated HCV RNA in cells exposed to the virus-peptide inocula versus the virus-DMSO control.
  • the results indicate that peptide 1 (and peptide 2, which overlaps with peptide 1) significantly blocks viral binding/attachment/uptake while none of other peptides are active at this level.
  • peptide #1 was added to the cells at different times relative to the time of addition of the inoculum.
  • the L-isomer of peptide 1 was added to the virus or to the cells at different times relative to inoculation according to the following protocol.
  • Huh-7.5.1 cells were seeded at 8000 cells per well in a 96-well plate and the next day they were infected with 800 ffu/well of HCV.
  • Peptide was added at a final concentration of 18 ⁇ M under the following conditions: (1) pre-inoculation: peptide was added to cells for 4 h at 37° C followed by washing 4 times with growth medium before virus infection; (2) co-inoculation: peptide was added to cells together with virus for 4 hours at which time the cells were washed as above and replenished with complete media without peptide; (3) post ⁇ inoculation: cells were infected for 4 hours at which point the virus was removed and the peptide was added and left on the cells for the duration of the experiment without washing. At 24 and 72 hours postinfection, cells were lysed and the HCV RNA level was measured by real time RT-QPCR and normalized to cellular GAPDH levels.
  • peptide 1 was strongly inhibitory if it was added to the cells together with the virus; much less inhibitory if added to the cells 4 hours after infection; and entirely noninhibitory if added to the cells for 4 hours and removed before the virus was added.
  • an HCV virocidal assay was performed in which viral supernatant was pretreated with the L- or D-isomers or DMSO for 1 hour before the HCV RNA content and infectivity remaining in the supernatant were measured.
  • a peptide has virocidal activity, it was diluted in complete growth medium containing 5x10 5 ffu/ml of HCV to a final concentration of 18 ⁇ M and incubated at 37°C for varying lengths of time at which point the virus-peptide mixture was analyzed for total HCV RNA and infectivity and compared with a comparably prepared virus-DMSO control.
  • HCV RNA content was measured by real time RT-QPCR, and normalized to the level of GAPDH RNA that was added to the RNA samples to control for RNA extraction efficiency.
  • HCV infectivity was measured by diluting 25 ⁇ L of each sample in growth medium 200- fold (i.e. below the inhibitory concentration of the diluted peptide) and residual infectivity was determined by incubating the diluted samples with Huh-7.5.1 cells and counting the number of HCV E2 -positive foci 3 days later.
  • the peptides reduced the total HCV RNA content and infectivity by 100-fold and more than 10,000-fold, respectively, within 1 hour of incubation in contrast to the DMSO control.
  • peptide 1 is virocidal to HCV.
  • the sedimentation velocity of untreated, peptide-treated and DMSO-treated virus particles was measured by rate zonal ultracentrifugation in continuous sucrose gradients. Briefly, the L- and D- isomers of peptide 1 (18 ⁇ M) were added to separate aliquots of viral supernatant containing 1x10 5 ffu/mL of HCV and incubated for 4 hours at 37 °C.
  • a third aliquot of virus was incubated with 0.5 % DMSO as a control. After incubation, 100 ⁇ L samples were analyzed by velocity sedimentation ultracentrifuge as described in the Methods. Twelve fractions (400 ⁇ L each) were collected from the top and analyzed for virus infectivity and HCV RNA content. As shown in FIG. 4D, the DMSO control samples displayed a unique peak of viral RNA which was virtually abolished by the L- and D- isomers. As expected, the infectivity of all fractions was abolished as well.
  • peptide 1 is derived from the membrane anchor domain of NS5A which is predicted to be an in-plane amphipathic alpha helix, it may destabilize and disrupt the HCV virion by permeabilizing its envelope.
  • a liposome dye release assay was conducted. The liposome dye release assay involved incubating cholesterol-phospholipid liposomes encapsulating a fluorescent dye with peptides or DMSO and then measuring fluorescence release.
  • Liposomes (Large Unilamellar Vesicles, LUV) were prepared as follows. A mixture containing 28 mg of total lipids (12 mM) composed of 10 parts 1- Palmitoyl-2-Oleoyl-5/?-Glycero-3-Phosphocholine (POPC), 1 1 parts 1,2- Dipalmitoyl-sw-Glycero-3-Phosphocholine (DPPC), 1 part 1 -Palmitoyl-2-Oleoyl-sfl- Glycero-3-[Phospho-L-Serine] (POPS), and 6 parts cholesterol (Avanti Polar Lipids, Inc., Alabaster, AL) was dissolved in 1 mL chloroform, 1 mL ether, and 2 mL sulforhodamine B (100 mM in 10 mM Hepes, pH 7.2; SulfoB, Molecular Probes, Eugene, OR).
  • POPC Palmitoyl-2-Ole
  • the mixture was sonicated at 4 0 C in a Branson 2210 waterbath sonicator (Danbury, CT) for 10 minutes. After removing organic solvents in a Buchi Rotavapor R-114 (Labortechnik AG, Flawil, Switzerland), the lipids were resuspended in another 2 mL of SulfoB and the resultant lipid vesicles were sized by repeated extrusion through a stack of 0.8, 0.4, and 0.2 ⁇ m polycarbonate membrane filters using a Mini-Extruder (Avanti Polar Lipids, Inc., Alabaster, AL). The SulfoB-loaded liposomes were separated from unentrapped SufloB on a Sephadex G-25 column. Dye release assays were performed in an Aminco-Bowman Series 2 Luminescence Spectrometer (Thermo Electron Corporation, Waltham, MA).
  • % SulfoB released 100 * (F - F 0 )/(F m - F 0 )
  • F is the fluorescence intensity observed in the presence of the peptides
  • F 0 is the basal fluorescence intensity before addition of peptide
  • F 100 is the fluorescence intensity corresponding to 100 % SulfoB release obtained by the addition of 25 ⁇ L of 10 % Triton X-100.
  • the L- and D-isomers of peptide 1 (10 ⁇ M) instantly permeabilized 70 % of the liposomes while DMSO was inactive. The effect was dose-dependent down to 160 nM, at which point 20 % of the liposomes were permeabilized (not shown).
  • Example 6 The L- and D-forms of Peptide 1 Inhibits HCV Infection at Noncytotoxic Concentrations in Vitro and in Vivo Peptides composed of L-amino acids are susceptible to proteolysis, which could shorten their half-life and, thus, their biological activity.
  • peptide 1 was synthesized using all D-amino acids, purified to > 95 % homogeneity, and its antiviral activity and serum stability were compared with a similarly pure preparation of the L-type version of peptide #1. Both L- and D-type peptides were diluted 1 : 100 in complete growth medium (10 % FBS) and mixed with an equal volume of viral supernatant.
  • the diluted peptide was incubated at 37 °C for 1 hour, 2 hours and 4 hours before mixing with viral supernatant.
  • the HCV RNA transcript level was measured by real time RT-QPCR and normalized to cellular GAPDH levels. The results (FIG.
  • IC 50 the peptide concentration that inhibits HCV infection by 50%
  • LC 50 50 % lethal concentrations of Peptide 1
  • IC 50 the peptide concentration that inhibits HCV infection by 50%
  • >95% pure peptide stock solution 3.6 mM in DMSO
  • An aliquot of peptide from each dilution was then diluted 1 : 100 in complete growth medium and mixed with an equal volume of virus supernatant.
  • the % inhibition of HCV infection was calculated by comparing the intracellular HCV RNA content in cells infected with virus between peptide treatment and DMSO control. Peptide cytotoxic activity was measured by MTT cytotoxicity assay according to the manufacturer's instructions (ATCC Cat# 30-101 OK) and described in the Methods. The peptide concentration that reduced 50 % of the OD reading was designated the LC 5 O-
  • both the L- and D-isomers of peptide 1 were highly inhibitory, displaying IC 50 values of 0.79 ⁇ M and 0.32 ⁇ M, respectively, while their LC 50 values were approximately 100-300-fold higher.
  • the slightly lower (IC 5 o) of the D-isomer may reflect its increased serum-stability since the antiviral activity of the L-isomer was reduced by approximately 2 logs after 1 hour of preincubation in serum while the D-isomer was fully active after at least 24 hours of incubation (FIG. 4G).
  • Peptide 1 also displayed a favorable toxicity profile in vivo since both the L- and D-isomers were entirely nontoxic when administered intravenously at doses as high as 0.5 mg per 25 gm C57B1/6 mouse administered at weekly intervals for 3 consecutive weeks. See Table 6 below. Importantly, peptide 1 was not immunogenic after repeated intravenous administration as antibodies directed against peptide 1 were not detected in the serum of those mice as shown in the following Table 7.
  • Peptide cytotoxicity was measured by MTT cytotoxicity assay based on the protocol provided in the ATCC MTT assay kit (Cat# 30-1010K). In brief, 5000- 10,000 cells were seeded per well in a 96 well plate. Following overnight growth, 100 ⁇ L fresh medium plus 20 ⁇ L of 2-fold serially diluted peptide was added. Media without peptides was added to at least 3 wells as untreated controls. The cells were then incubated for 72 hours at 37 0 C, 5 % CO 2 . After this incubation, 1/10 volume of MTT solution (5 ⁇ g/mL in PBS) was added to each well, and the cells were returned to the incubator.
  • MTT cytotoxicity assay based on the protocol provided in the ATCC MTT assay kit (Cat# 30-1010K). In brief, 5000- 10,000 cells were seeded per well in a 96 well plate. Following overnight growth, 100 ⁇ L fresh medium plus 20 ⁇ L of 2-fold serially diluted peptide was added. Media without
  • Fresh human blood (treated with EDTA) was centrifuged 100Og for 10 minutes to remove the supernatant and buffy coat. The red blood cells were then washed twice in PBS, and resuspended to a final concentration of 8 % with and without 16 % FBS. Serial 2-fold dilutions of peptide were prepared in 60 ⁇ L PBS in a 96- well microtiter plate, and 60 ⁇ L of the suspended human red blood cells with and without FBS were added. The plates were incubated for lhour at 37 0 C. After this incubation 120 ⁇ L PBS was added to each well and the plates were centrifuged at 100Og for 5mins.
  • mice BALB/c mice, 7 weeks old, about 23 g
  • mice were each injected with 92 ⁇ g L-type peptide 1 ( ⁇ 4 mg/kg) in 200 ⁇ L PBS (spun 14,000 rpm for 3 minutes before injection).
  • each of three mice was given 200 ⁇ L PBS containing 5 % DMSO.
  • the mice were monitored for acute toxicity during the first 3 hours after injection. Results are summarized in the following table.
  • mice were then weighed on days 0, 3, 5, 7 and 10. Peptide-injected mice gained weight at the same rate as the controls.
  • the secondary structure of peptide 1 (SEQ ID NO:43) was analyzed using the tool of helical Wheel Applet available online at cti.itc.virginia.edu/ ⁇ cmg/Demo/wheel/wheelApp.html (last visited Aug. 15, 2006).
  • the resulting helical wheel (FIG. 6A) shows that peptide 1 is amphipathic, having both hydrophobic and hydrophilic faces.
  • the secondary structure of peptide 1 was also analyzed using circular dichroism (CD) spectroscopy using an Aviv model 62DS CD spectrometer (Aviv Associates Inc., Lakewood, N.J.).
  • CD circular dichroism
  • the CD spectra of peptides were measured at 25 0 C using a 1 mm path-length cell. Three scans per sample were performed over the wavelength range of 190 to 260 nm in 10 mM potassium phosphate buffer, pH 7.0. Data were collected at 0.1 nm interval with a scan rate of 60 nm/min and is given in mean molar ellipticity [q].
  • the peptide concentrations were 50 ⁇ M.
  • peptide 1 To determine whether the antiviral activity of peptide 1 is dependent on its primary amino acid sequence, four derivative peptides from peptide 1 were synthesized to a purity > 95 %.
  • the four derivatives having the same composition of amino acids included (1) the reversed the sequence of peptide 1 (also called retro- peptide); (2) scrambled hydrophobic amino acids; (3) scrambled hydrophilic amino acids; and (4) a derivative in which the aspartic acid residues (D) were replaced with proline residues (P).
  • the antiviral activity of the peptides was examined by HCV focus reduction assay at three peptide concentrations: 18 ⁇ M, 6 ⁇ M and 2 ⁇ M, as described above.
  • Peptide 1 for example, derived from the membrane anchor domain of NS5A (NS5A-1975) was highly potent as a single dose of this peptide completely blocked HCV infection with an EC 5 o of 289 nJVl without evidence of cytotoxicity. The antiviral effect was evident for at least 1 1 days post infection. The peptide was most active when it was added to the cells together with the virus. Preincubation of the peptide with virus significantly reduced viral attachment and infectivity, suggesting that the antiviral activity of NS5A-1975 interacts directly with the virus and destabilizes it.
  • the D- amino acid form of the peptide is fully active, and the D- and L-forms of the peptide display amphipathic ⁇ -helical structure in solution and induce permeabilization of artificial liposomes. Comparative analysis of the antiviral activity, alpha-helicity and membranolytic activity (not shown) of a series of C- and N-terminal truncation mutants (peptide 3-13) were performed. All peptides were synthesized with a purity >95% and their antiviral activities were compared by determining their IC50 values.
  • the helicity of the peptides was determined by circular dichroism (CD) analysis using an Aviv model 62DS circular dichroism (CD) spectrometer (Aviv Associates Inc., Lakewood, N.J.)- The spectra were measured at 25 0 C using a 1 mm path- length cell. Three scans per sample were performed over a wavelength range from 190 to 260 nm in 10 mM potassium phosphate buffer (pH 7.0) with or without 50 % Trifluoroethanol (TFE). Data were collected at 0.1 nm interval with a scan rate of 60 nm/min and is given in mean molar ellipticity [ ⁇ ]. The peptide concentrations were 50 ⁇ M. The percentage of helicity of a peptide was calculated as described in Chen et al., Biochemistry 13, 3350-9 (1974)). Results are shown in the table below
  • SWLRDIWDWICEVLSDFK (SEQIDNO ⁇ S) L-isomer 0.79 37.2 2 SWLRDIWDWICEVLSDFK (SEQ ID NO: 43) D-isomer 0.34 39.0 b. Length Series
  • SGSWLRDIWDWICEVLSDFK (SEQ ID NO: 141) extend N-, 2 a.a 1.7 39.8
  • SWLRDIWDWICEVLSDFKT (SEQIDNO: 143) extend C-, 1 a.a 1.7 39.8
  • SWLRDIWDWICEVLSDFKTW (SEQIDNO: 144) extend C-, 2 a.a 0.51 38.0 c. Ampliipathicity Series
  • VLDLIYSLHKQINRGLKKIVL (SEQIDNO: 147) BVDV analogue >36 31.6 d.
  • a retro-peptide has the reverse sequence of the parental peptide.
  • the prototype strains of HCV genotype 3a, 4a, 5a, and 6a selected were K3A (GenBank accession D28917), EG.ED43 (Yl 1604), ZA.SA13 (AF064490), and HK.6a33 (AY859526), respectively.
  • the 16-mer SWLRDIWD WICEVLSD (SEQ ID NO: 94) (peptide 3 in Table 12) retains full antiviral activity, alpha-helical and membranolytic properties (Table 12b).
  • the antiviral activity of the N- and C-terminally truncated peptides correlated with their membrane permeability activity and amphipathic ⁇ -helical structure.
  • the simultaneous loss of all three activities in the truncation mutants suggests that they are probably functionally linked to the ability of the peptide to inhibit HCV infection.
  • peptide 1 is strongly amphipathic.
  • Table 12c the antiviral activity of these peptides was reduced 5- to >30- fold.
  • VSV vesicular stomatitis virus
  • the cells were washed 2 times, overlaid with 120 ⁇ L fresh growth medium and incubated at 37 °C for 3 days.
  • VSV and HCV infections were assessed by viral cytopathic effect (CPE) and immunostaining with antibody against HCV E2 protein, respectively.
  • Virucidal activity To determine if peptide 1 has virocidal activity against VSV, peptide 1 was diluted in a complete growth medium containing 2 x 105 pfu (ffu)/mL VSV or HCV to a final concentration of 18 ⁇ M. The virus-peptide mixture was then incubated for 4 hours at 37 °C. The VSV and HCV viral titer were then determined by serial dilution and assessed by viral cytopathic effect (CPE) and immunostaining with antibody against HCV E2 protein, respectively. The result (FIG. 8) indicates that peptide 1 does not block VSV infection and has no virocidal activity against VSV. Further experiments indicate that peptide 1 does not block infection by influenza virus, vaccinia virus, Borna disease virus, lymphocytic choriomeningitis virus or adenovirus (data not shown).
  • CPE viral cytopathic effect
  • Vero cells (80,000 cells/well/ml) were seeded for 24 h pre-infection in 24-well plates. Cells were exposed to Dengue- 2 (derived from Vero cells) in the presence of increasing concentration of peptide (or DMSO as control). Viruses and peptide were not removed (cells were not washed) throughout the incubation. Infection was analyzed after 5 days using ELISA that measured the amounts of Dengue-2 capsid released in the supernatant of infected Vero cells.
  • Fluorescent Foci Assay Vero cells were seeded for 24 h pre-infection in 96- well plates. Cells were exposed to Dengue-2 in the presence of increasing concentrations of peptide (or DMSO as control). Viruses and peptide were washed away 2 h post-infection. Supernatants were collected every 3 days post-infection and added to fresh Vero cells for fluorescent foci assay. Newly infected Vero cells were fixed with 4% formaldehyde after 3 days. Cells were then stained with Dengue Env antibodies followed by Alexa-fluor dye conjugated secondary antibodies. Foci were counted using a fluorescent microscope.
  • Dengue infection was inhibited by the present peptides in a dose-dependent manner. Essentially 100% inhibition of Dengue viral infection was observed at concentrations of 20 ⁇ M (FIG. 9).
  • Intracellular FACS Assay Vero cells were seeded for 24 h pre-infection in 6-well plates. Cells were exposed to Dengue-2 in the presence of increasing concentrations of peptide (or DMSO as control). Viruses and peptide were washed away 2 h post-infection. Cells were taken for intracellular staining 3 days post- infection. Cells were stained with appropriate isotype control, Dengue Env, Dengue capsid or tubulin antibodies. Cells were analyzed by FACS.
  • Results when using peptide concentrations of 20 ⁇ M are shown in Table 15. Results for 1.25 to 20 ⁇ M are summarized in the graph shown in FIG. 10A-B.
  • FIG. 1 OB-D show that strong inhibition of Dengue viral infection is correlated with the amphipathicity of the peptide structure, rather than the precise amino acid sequence of the peptide.
  • peptide 1 also called 2022 and L-7208, SEQ ID NO:43
  • peptide 3222 SEQ ID NO: 127
  • peptide 3226 SEQ ID NO: 128
  • peptide 3228 SEQ ID NO: 130
  • peptide L-7208 2D to 2 Pro SEQ ID NO:91
  • L-7208 HS with hydrophobic amino acids scrambled SEQ ID NO:97.
  • the present peptides inhibit Dengue viral infection in a dose-dependent manner. Essentially 100% inhibition of Dengue viral infection was observed at concentrations of 20 ⁇ M (FIG. 1 OA-B).
  • Example 13 Peptide 1 has Strong Antiviral Activity Against West Nile Viral Infection
  • peptide 1 and peptide 2012 SEQ ID NO: 94
  • WNV West Nile Virus
  • A549 cells were infected with 10 2 to 10 5 PFU/mL WNV (New York strain) in the presence of 0.5 % DMSO, peptide 1 or peptide 2012, where a final concentration of peptide was 18 ⁇ M in 0.5 % DMSO. After 3 days of incubation at 37 °C, the cells were fixed and subjected to immunoperoxidase staining to detect WNV protein. Results (FIG.
  • Example 14 Peptide 1 and Variants Thereof Inhibit HIV Infection This Example illustrates that the peptides of the invention can inhibit infection by human immunodeficiency viruses. Materials and Methods
  • Viruses were generated by liposome-mediated transfection of 293T cells using Genejuice (Novagen). Viral supernatants were harvested 48 h post- transfection and filtered through a 0.2- ⁇ M pore size filter to remove cellular debris. Viral inoculum was standardized by p24 (HIV-I capsid) enzyme-linked immunosorbent assay (PerkinElmer Life Sciences).
  • TZM-bl reporter cells are CD4+ CXCR4+ CCR5+ HeLa cells, which contain LacZ gene driven by the HIV-I LTR (promoter). Upon infection, HIV-I Tat protein is produced and activates the HIV-I LTR. TZM-bl cells (80,000 cells/well/ml) were seeded for 24 h pre-infection in 24-well plates.

Abstract

The present application is directed to antiviral peptides, methods of using these peptides to prevent or inhibit infections by a human immunodeficiency virus or a virus from the Flaviviridae family, and pharmaceutical compositions and combinations, as well as articles of manufacture comprising these peptides.

Description

ANTIVIRAL PEPTIDES
This application claims priority to U.S. Provisional Application Serial Numbers 60/879,727, filed January 10, 2007, and 60/967,783, filed September 7, 2007, the contents of which are specifically incorporated herein by reference in their entireties.
This application is also related to U.S. Application Ser. No. 11/541,488, filed September 29, 2006 and PCT Application Ser. No. PCT/US2006/038420, filed September 29, 2006, both of which claim priority to the filing dates of U.S. Provisional Application Ser. Nos. 60/840,328 (filed August 25, 2006) and
60/722,502 (filed September 29, 2005), the contents of all of which are specifically incorporated herein by reference in their entireties. This application is also related to U.S. Provisional Application Serial No. 60/967,812, filed September 7, 2007, the contents of which are specifically incorporated herein by reference in its entirety.
GOVERNMENT FUNDING
The invention described herein was made with United States Government support under Grant Number CAl 08304 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
Viral diseases can be difficult to treat because viruses enter mammalian cells, where they perform many of their functions, including transcription and translation of viral proteins, as well as replication of the viral genome. Thus, viruses are protected not only from the host's immune system, but also from medicines administered to the host, as the viral infection progresses.
Few effective anti- viral agents are currently available and most of those are effective against only a small subset of viruses. Therefore, agents for treating and preventing viral infections are needed. SUMMARY OF THE INVENTION
The invention relates to peptides that can prevent or treat infection by human immunodeficiency virus and members of the Flaviviridae family. The invention also relates to peptides that can inactivate these viruses before they enter a cell. The invention is based on the discovery of certain antiviral peptides some of which have an amphipathic alpha-helical structure and particular amino acid compositions. Surprisingly, many of the present peptides inactivate viruses that are free in solution, thereby preventing viral infection of mammalian cells. Also surprising is that the present peptides are derived from and/or are related the Hepatitis C polyprotein, but are capable of inactivating flaviviruses, paramyxoviruses, and human immunodeficiency virus (HIV).
The invention involves the discovery that an amphipathic alpha-helical peptide (SEQ ID NO: 43) derived from the membrane anchor domain of the Hepatitis C virus (HCV) NS5A protein is virocidal for HCV, i.e. it is capable of inactivating HCV, at nanomolar concentrations in vitro. The peptide prevents de novo HCV infection and suppresses ongoing infection by inactivating both extracellular and intracellular infectious particles. In addition to HCV, the peptide inhibits infection by selected flaviviruses, paramyxoviruses, and HIV. The peptide is also nontoxic in vitro and in vivo at doses at least 100-fold higher than required for antiviral activity. Its amphipathic alpha-helical structure is necessary but not sufficient for its virocidal activity, which depends on its amino acid composition but not its primary sequence or its chirality.
In one aspect, the invention provides an isolated peptide of 14 to 50 D- or L- amino acids in length comprising an amino acid sequence of formula I: Xaa i -Xaa2-Xaa3-W-L-Xaa6-Xaa7-Xaa8-W-Xaa i O-W-Xaa 12-Xaai 3-Xaa 14-
Xaai5-Xaai6-Xaai7-Xaa18- Xaai9-Xaa20-Xaa2rXaa22 (SEQ ID NO: 163), wherein: Xaai is serine (S) or absent; Xaa2 is glycine (G) or absent; Xaa3 is serine (S), aspartic acid (D) or threonine (T); W is tryptophan; L is leucine; Xaa6 is arginine (R), aspartic acid (D), lysine (K), tryptophan (W) or glutamic acid (E); Xaa7 is aspartic acid (D), arginine (R), glutamic acid (E), alanine (A), isoleucine (I) or lysine (K); Xaa8 is isoleucine (I) or valine (V); Xaaio is aspartic acid (D), arginine (R), glutamic acid (E) or lysine (K); Xaa^ is isoleucine (I) or valine (V); Xaa^ is cysteine (C), glutamic acid (E), leucine (L), serine (S) or arginine (R); Xaa14 is glutamic acid (E), lysine (K), aspartic acid (D), threonine (T), histidine (H), serine (S) or arginine (R); Xaais is valine (V), alanine (A) or serine; Xaai6 is leucine (L) or valine (V); Xaaπ is serine (S), threonine (T), leucine (L) or absent; Xaai8 is aspartic acid (D), arginine (R), glutamic acid (E), lysine (K) or absent; Xaa19 is phenylalanine or absent; Xaa20 is lysine (K), glutamic acid (E), arginine (R), aspartic acid (D) or absent; Xaa2i is threonine (T) or absent; and Xaa22 is tryptophan (W) or absent; and wherein the amino acid sequence of formula I is not: SWLRDIWDWICEVLSDFK (SEQ ID NO: 43); SWLRDIWD WICEVLSDF (SEQ ID NO: 95), SWLRDIWD WICEVLSD (SEQ ID NO: 94), SWLRDIWD WICEVLS (SEQ ID NO: 93), or SWLRDIWD WICEVL (SEQ ID NO: 92). In some embodiments, an isolated peptide of the invention comprises an amino acid sequence of formula II:
XaarXaa2-S-W-L-Xaa6-Xaa7-I-W-Xaaio-W-I-C-Xaa,4-V-L-Xaai7-
Xaai8- Xaai9-Xaa20-Xaa2i-Xaa22 (SEQ ID NO: 164), wherein: Xaai, Xaa2, Xaa21 and Xaa22 are absent; S is serine; W is tryptophan; L is leucine; Xaa<5 is arginine (R), aspartic acid (D), glutamic acid (E) or lysine (K); Xaa7 is arginine (R), aspartic acid (D) or lysine (K); I is isoleucine; W is tryptophan; Xaaio is aspartic acid (D), arginine (R), or lysine (K); C is cysteine; Xaaπ is lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D); V is valine; Xaai7 is serine (S) or absent; Xaa18 is aspartic acid (D), arginine (R), lysine (K) or absent; Xaai9 is phenylalanine (F) or absent; and Xaa2o is lysine (K), glutamic acid (E), aspartic acid (D), arginine (R) or absent. In some embodiments, an isolated peptide of the invention comprises an amino acid sequence of formula III:
Xaa,-Xaa2-S-W-L-R-Xaa7-I-W-Xaai0-W-I-C-Xaai4-V-L- Xaa17-
Xaai8-Xaai9-Xaa20-Xaa2i-Xaa22 (SEQ ID NO: 178), wherein: Xaai, Xaa2, Xaa21 and Xaa22 are absent; S is serine; W is tryptophan; L is leucine; R is arginine; I is isoleucine; C is cysteine; V is valine; Xaa7 is aspartic acid (D), arginine (R), or lysine (K); Xaaio is arginine (R) or lysine (K); Xaa]4 is lysine (K), glutamic acid (E) or aspartic acid (D); Xaa17 is serine (S) or absent; Xaai8 is arginine (R), lysine (K) or absent; Xaai9 is phenylalanine (F) or absent; and Xaa20 is lysine (K) or absent.
In some embodiments, a peptide of the invention comprises an amino acid sequence of formula IV:
Xaai-Xaa2-S-W-L-Xaa6-Xaa7-I-W-Xaa10-W-I-C-Xaa14-V-L-Xaa,7-
Xaai8-Xaa19-Xaa20-Xaa2i-Xaa22 (SEQ ID NO: 184), wherein: Xaai, Xaa2, Xaa21 and Xaa22 are absent; S is serine; W is tryptophan; L is leucine; I is isoleucine; C is cysteine; V is valine;; and Xaa^ Xaa-7, Xaaio and Xaai4 are arginine (R) or lysine (K); Xaaπ is serine (S) or absent; Xaai8 is arginine (R), lysine (K) or absent; Xaai9 is phenylalanine (F) or absent; and Xaa20 is arginine (R), lysine (K) or absent. In another aspect, the invention provides isolated peptide of 14 to 50 D- or L- amino acids in length comprising an amino acid sequence of formula V:
Xaai-Xaa2-Xaa3- Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa1o-Xaai i-W-Xaa^ -W-Xaai5-Xaa16-Xaa)7- L-W-Xaa20-Xaa21-Xaa22 (SEQ ID NO: 595), wherein Xaai is tryptophan (W) or absent; Xaa2 is threonine (T) or absent; Xaa3 is lysine (K), glutamic acid (E), arginine (R), aspartic acid (D) or absent; Xaa4 is phenylalanine or absent; Xaa5 is aspartic acid (D), arginine (R), glutamic acid (E), lysine (K) or absent; Xaa6 is serine (S), threonine (T), leucine (L) or absent; Xaa7 is leucine (L) or valine (V); Xaa8 is valine (V), alanine (A) or serine; Xaa9 is glutamic acid (E), lysine (K), aspartic acid (D), threonine (T), histidine (H), serine (S) or arginine (R); Xaaio is cysteine (C), glutamic acid (E), leucine (L), serine (S) or arginine (R); Xaai i is isoleucine (I) or valine (V); W is tryptophan; Xaa13 is aspartic acid (D), arginine (R), glutamic acid (E) or lysine (K); Xaai5 is isoleucine (I) or valine (V); Xaai6 is aspartic acid (D), arginine (R), glutamic acid (E), alanine (A), isoleucine (I) or lysine (K); Xaa^ is arginine (R), aspartic acid (D), lysine (K), tryptophan (W) or glutamic acid (E); L is leucine; Xaa2o is serine (S), aspartic acid (D) or threonine (T); Xaa2i is glycine (G) or absent; and Xaa22 is serine (S) or absent; and wherein the amino acid sequence of formula V is not: KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), FDSLVECIWD WIDRLWS (SEQ ID NO: 99), DSLVECIWDWIDRLWS (SEQ ID NO: 100),
SLVECIWDWIDRLWS (SEQ ID NO: 101), or LVECIWD WIDRLWS (SEQ ID NO: 102).
In some embodiments, a peptide of the invention comprises an amino acid sequence of formula VI: Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L- V-Xaa9-C-I-W-
Xaai3-W-I-Xaa,6-XaaI7-L-W-S- Xaa2i-Xaa22 (SEQ ID NO: 596), wherein: Xaa^ Xaa2, Xaa2 t and Xaa22 are absent; Xaa3 is lysine (K), glutamic acid (E), aspartic acid (D), arginine (R) or absent; Xaa4 is phenylalanine (F) or absent; Xaa5 is aspartic acid (D), arginine (R), lysine (K) or absent; Xaa<$ is serine (S) or absent; L is leucine; V is valine; Xaa9 is lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D); C is cysteine; I is isoleucine; W is tryptophan; Xaa^ is aspartic acid (D), arginine (R), or lysine (K); Xaaj6 is arginine (R), aspartic acid (D) or lysine (K); Xaaπ is arginine (R), aspartic acid (D), glutamic acid (E) or lysine (K); and S is serine. In some embodiments, a peptide of the invention comprises an amino acid sequence of formula VII:
Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L- V-Xaa9-C-I- W- Xaa,3-W-I-Xaa,6-R-L-W-S-Xaa21-Xaa22 (SEQ ID NO: 597), wherein: Xaai, Xaa2, Xaa2i and Xaa22 are absent; Xaa3 is lysine (K) or absent; Xaa4 is phenylalanine (F) or absent; Xaa5 is arginine (R) or lysine (K), or absent; Xaa$ is serine (S) or absent; L is leucine; V is valine; Xaa9 is lysine (K), glutamic acid (E) or aspartic acid (D); C is cysteine; I is isoleucine; W is tryptophan; Xaa^ is arginine (R) or lysine (K); Xaai6 is aspartic acid (D), arginine (R), or lysine (K); R is arginine; and S is serine.
In some embodiments, a peptide of the invention comprises an amino acid sequence of formula VIII:
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-XaacrC-I-W- Xaai3-W-I-Xaai6-Xaa17-L-W-S-Xaa2,-Xaa22 (SEQ ID NO: 598), wherein: Xaaj, Xaa2, Xaa21 and Xaa22 are absent; Xaa3 is arginine (R), lysine (K) or absent; Xaa^t is phenylalanine (F) or absent; Xaa5 is arginine (R), lysine (K) or absent; Xaa6 is serine (S) or absent; L is leucine; V is valine; Xaa9, Xaaj3> Xaai6 and Xaaπ are arginine (R) or lysine (K); C is cysteine; I is isoleucine; W is tryptophan; and S is serine.
In some embodiments of the invention, the isolated peptide comprises an amino acid sequence selected from the group consisting of:
SWRLDIWDWICESVLDFK (SEQ ID NO: 1 19), DWLRIIWDWVCSVVSDFK (SEQ ID NO: 123), SWLWEVWDWVLHVLSDFK (SEQ ID NO: 124),
TWLRAIWDWVCTALTDFK (SEQ ID NO: 125), SWLRDVWDWVCTVLSDFK (SEQ ID NO: 126), SWLRDIWDWISEVLSDFK (SEQ ID NO: 127), SWLDRIWRWICKVLSRFE (SEQ ID NO: 128), SWLDDIWDWICEVLSDFE (SEQ ID NO: 129),
SWLRRIWRWICKVLSRFK (SEQ ID NO: 130), SWLKEIWEWICDVLSEFR (SEQ ID NO: 131 ), SWLKDIWDWICEVLSDFR (SEQ ID NO: 132), SWLKDIWDWICEVLSDFK (SEQ ID NO: 133), SWLREIWEWICDVLSEFK (SEQ ID NO: 134), SWLREIWEWICEVLSEFK (SEQ ID NO: 135), SWLDRIWRWICKVLSRFE (SEQ ID NO: 136), SWLDDIWDWICEVLSDFE (SEQ ID NO: 137), SWLRRIWRWICKVLSRFK (SEQ ID NO : 138), '
SWLRDIWDWIREVLSDFK (SEQ ID NO: 139), SWLRDIWDWIEEVLSDFK (SEQ ID NO: 140), SGSWLRDIWDWICEVLSDFK (SEQ ID N0:141), GSWLRDIWDWICEVLSDFK (SEQ ID NO: 142), SWLRDIWDWICEVLSDFKT (SEQ ID NO: 143),
SWLRDIWDWICEVLSDFKTW (SEQ ID NO: 144), SWLEKIWKWICRVLSKFD (SEQ ID NO: 165), SWLRKIWKWICEVLSDFK (SEQ ID NO: 166), SWLRDIWDWICKVLSKFK (SEQ ID NO: 167), SWLRRIWRWICEVLSDFK (SEQ ID NO: 168),
SWLRDIWDWICRVLSRFK (SEQ ID NO: 169), SWLRRIWDWICRVLSDFK (SEQ ID NO: 170), SWLRKIWDWICKVLSDFK (SEQ ID NO: 171), SWLRRIWDWICEVLSRFK (SEQ ID NO: 172), SWLRKIWDWICEVLSKFK (SEQ ID NO: 173),
SWLRDIWRWICRVLSDFK (SEQ ID NO: 174), SWLRDIWKWICKVLSDFK (SEQ ID NO: 175), SWLDRIWDWICRVLSRFK (SEQ ID NO: 176), SWLRDIWDWICKVLSKFK (SEQ ID NO: 177), SWLRDIWRWICKVLSRFK (SEQ ID NO: 179),
SWLRDIWKWICKVLSKFK (SEQ ID NO: 180), SWLRKIWKWICEVLSKFK (SEQ ID NO: 181), SWLRRIWRWICEVLSRFK (SEQ ID NO: 182), SWLRRIWRWICDVLSRFK (SEQ ID NO: 183), SWLRKIWKWICKVLSKFK (SEQ ID NO: 185), SWLRRIWRWICRVLSRFK (SEQ ID NO: 186), SWLRRIWRWICRVLSRFR (SEQ ID NO: 187), SWLKKIWKWICKVLSKFK (SEQ ID NO: 188), SWRLDIWDWICESVLDF (SEQ ID NO: 189),
SWRLDIWDWICESVLD (SEQ ID NO: 190), SWRLDIWDWICESVL (SEQ ID NO: 191), SWRLDIWDWICESV (SEQ ID NO: 192), DWLRIIWDWVCSVVSDF (SEQ ID NO: 193), DWLRIIWDWVCSVVSD (SEQ ID NO: 194),
DWLRIIWDWVCSVVS (SEQ ID NO: 195), DWLRIIWDWVCSVV (SEQ ID NO: 196), SWLWEVWDWVLHVLSDF (SEQ ID NO: 197), SWLWEVWDWVLHVLSD (SEQ ID NO: 198), SWLWEVWDWVLHVLS (SEQ ID NO: 199),
SWLWEVWDWVLHVL (SEQ ID NO: 200), TWLRAIWDWVCTALTDF (SEQ ID NO: 201), TWLRAIWDWVCTALTD (SEQ ID NO: 202), TWLRAIWDWVCTALT (SEQ ID NO: 203), TWLRAIWDWVCTAL (SEQ ID NO: 204),
SWLRDVWDWVCTVLSDF (SEQ ID NO: 205), SWLRDVWDWVCTVLSD (SEQ ID NO: 206), SWLRDVWDWVCTVLS (SEQ ID NO: 207), SWLRDVWDWVCTVL (SEQ ID NO: 208), SWLRDIWDWISEVLSDF (SEQ ID NO: 209),
SWLRDIWDWISEVLSD (SEQ ID NO: 210), SWLRDIWDWISEVLS (SEQ ID NO: 21 1), SWLRDIWDWISEVL (SEQ ID NO: 212), SWLDRIWRWICKVLSRF (SEQ ID NO: 213), SWLDRIWRWICKVLSR (SEQ ID NO: 214), SWLDRIWRWICKVLS (SEQ ID NO: 215), SWLDRIWRWICKVL (SEQ ID NO: 216), SWLDDIWDWICEVLSDF (SEQ ID NO: 217), SWLDDIWDWICEVLSD (SEQ ID NO: 218),
SWLDDIWDWICEVLS (SEQ ID NO: 219), SWLDDIWDWICEVL (SEQ ID NO: 220), SWLRRIWRWICKVLSRF (SEQ ID NO:221), SWLRRIWRWICKVLSR (SEQ ID NO: 222), SWLRRIWRWICKVLS (SEQ ID NO: 223),
SWLRRIWRWICKVL (SEQ ID NO: 224), SWLKEIWEWICDVLSEF (SEQ ID NO: 225), SWLKEIWEWICDVLSE (SEQ ID NO: 226), SWLKEIWEWICDVLS (SEQ ID NO: 227), SWLKEIWEWICDVL (SEQ ID NO: 228),
SWLKDIWDWICEVLSDF (SEQ ID NO: 229), SWLKDIWDWICEVLSD (SEQ ID NO: 230), SWLKDIWDWICEVLS (SEQ ID NO: 231), SWLKDIWDWICEVL (SEQ ID NO: 232), SWLKDIWDWICEVLSDF (SEQ ID NO: 233),
SWLKDIWDWICEVLSD (SEQ ID NO: 234), SWLKDIWDWICEVLS (SEQ ID NO: 235), SWLKDIWDWICEVL (SEQ ID NO: 236), SWLREIWEWICDVLSEF (SEQ ID NO: 237), SWLREIWEWICDVLSE (SEQ ID NO: 238),
SWLREIWEWICDVLS (SEQ ID NO: 239), SWLREIWEWICDVL (SEQ ID NO: 240), SWLREIWEWICEVLSEF (SEQ ID NO: 591), SWLREIWEWICEVLSE (SEQ ID NO: 592), SWLREIWEWICEVLS (SEQ ID NO: 593), SWLREIWEWICEVL (SEQ ID NO: 594), SWLDRIWRWICKVLSRF (SEQ ID NO: 241), SWLDRIWRWICKVLSR (SEQ ID NO: 242), SWLDRIWRWICKVLS (SEQ ID NO: 243),
SWLDRIWRWICKVL (SEQ ID NO: 244), SWLDDIWDWICEVLSDF (SEQ ID NO: 245), SWLDDIWDWICEVLSD (SEQ ID NO: 246), SWLDDIWDWICEVLS (SEQ ID NO: 247), SWLDDIWDWICEVL (SEQ ID NO: 248),
SWLRRIWRWICKVLSRF (SEQ ID NO: 249), SWLRRIWRWICKVLSR (SEQ ID NO: 250), SWLRRIWRWICKVLS (SEQ ID NO: 251), SWLRRIWRWICKVL (SEQ ID NO: 252), SWLRDIWDWIREVLSDF (SEQ ID NO: 253),
SWLRDIWDWIREVLSD (SEQ ID NO: 254), SWLRDIWDWIREVLS (SEQ ID NO: 255), SWLRDIWDWIREVL (SEQ ID NO: 256), SWLRDIWDWIEEVLSDF (SEQ ID NO: 257), SWLRDIWDWIEEVLSD (SEQ ID NO: 258),
SWLRDIWDWIEEVLS (SEQ ID NO: 259), SWLRDIWDWIEEVL (SEQ ID NO: 260), SGSWLRDIWDWICEVLSDF (SEQ ID NO: 261), SGSWLRDIWDWICEVLSD (SEQ ID NO: 262), SGSWLRDIWDWICEVLS (SEQ ID NO: 263),
SGSWLRDIWDWICEVL (SEQ ID NO: 264), GSWLRDIWDWICEVLSDF (SEQ ID NO: 265), GSWLRDIWDWICEVLSD (SEQ ID NO: 266), GSWLRDIWDWICEVLS (SEQ ID NO: 267), GSWLRDIWDWICEVL (SEQ ID NO: 268), SWLRDIWDWICEVLSDFK (SEQ ID NO: 269), SWLRDIWDWICEVLSDF (SEQ ID NO: 270), SWLRDIWDWICEVLSD (SEQ ID NO: 271), SWLRDIWDWICEVLS (SEQ ID NO: 272),
SWLRDIWDWICEVLSDFKT (SEQ ID NO: 273), SWLRDIWDWICEVLSDFK (SEQ ID NO: 274), SWLRDIWDWICEVLSDF (SEQ ID NO: 275), SWLRDIWDWICEVLSD (SEQ ID NO: 276), KFDLVSECIWDWIDLRWS (SEQ ID NO: 277),
FDLVSECIWDWIDLRWS (SEQ ID NO: 278),
DLVSECIWDWIDLRWS (SEQ ID NO: 279),
LVSECIWDWIDLRWS (SEQ ID NO: 280),
VSECIWDWIDLRWS (SEQ ID NO: 281), KFDSVVSCVWDWIIRLWD (SEQ ID NO: 282),
FDSVVSCVWDWIIRLWD (SEQ ID NO: 283),
DSVVSCVWDWIIRLWD (SEQ ID NO: 284),
SVVSCVWDWIIRLWD (SEQ ID NO: 285),
VVSCVWDWIIRLWD (SEQ ID NO: 286), KFDSLVHLVWDWVEWLWS (SEQ ID NO: 288),
FDSLVHLVWDWVEWLWS (SEQ ID NO: 289), DSLVHLVWDWVEWLWS (SEQ ID NO:290), SLVHLVWDWVEWLWS (SEQ ID NO: 291), LVHLVWDWVEWLWS (SEQ ID NO: 292), KFDTLATCVWDWIARLWT (SEQ ID NO: 293),
FDTLATCVWDWIARLWT (SEQ ID NO: 294),
DTLATCVWDWIARLWT (SEQ ID NO: 295),
TLATCVWDWIARLWT (SEQ ID NO: 296),
LATCVWDWIARLWT (SEQ ID NO: 297), KFDSLVTCVWDWVDRLWS (SEQ ID NO: 298),
FDSLVTCVWDWVDRLWS (SEQ ID NO: 299),
DSLVTCVWDWVDRLWS (SEQ ID NO: 300),
SLVTCVWDWVDRLWS (SEQ ID NO: 301), LVTCVWDWVDRLWS (SEQ ID NO: 302),
KFDSLVESIWDWIDRLWS (SEQ ID NO: 303),
FDSLVESIWDWIDRLWS (SEQ ID NO: 304),
DSLVESIWDWIDRLWS (SEQ ID NO: 305),
SLVESIWDWIDRLWS (SEQ ID NO: 306), LVESIWDWIDRLWS (SEQ ID NO: 307),
EFRSLVKCIWRWIRDLWS (SEQ ID NO: 308),
FRSLVKCIWRWIRDLWS (SEQ ID NO: 309),
RSLVKCIWRWIRDLWS (SEQ ID NO: 310),
SLVKCIWRWIRDLWS (SEQ ID NO: 31 1), LVKCIWRWIRDLWS (SEQ ID NO : 312),
EFDSLVECIWDWIDDLWS (SEQ ID NO: 313),
FDSLVECIWDWIDDLWS (SEQ ID NO: 314),
DSLVECIWDWIDDLWS (SEQ ID NO: 315),
SLVECIWDWIDDLWS (SEQ ID NO: 316), LVECIWDWIDDLWS (SEQ ID NO: 317),
KFRSLVKCIWRWIRRLWS (SEQ ID NO: 318),
FRSLVKCIWRWIRRLWS (SEQ ID NO: 319),
RSLVKCIWRWIRRLWS (SEQ ID NO: 320),
SLVKCIWRWIRRLWS (SEQ ID NO: 321), LVKCIWRWIRRLWS (SEQ ID NO: 322),
RFESLVDCIWEWIEKLWS (SEQ ID NO: 323),
FESLVDCIWEWIEKLWS (SEQ ID NO: 324),
ESLVDCIWEWIEKLWS (SEQ ID NO: 325),
SLVDCIWEWIEKLWS (SEQ ID NO: 326), LVDCIWEWIEKLWS (SEQ ID NO: 327),
RFDSLVECIWDWIDKLWS (SEQ ID NO: 328),
FDSLVECIWDWIDKLWS (SEQ ID NO: 329),
DSLVECIWDWIDKLWS (SEQ ID NO: 330), SLVECIWDWIDKLWS (SEQ ID NO: 331),
LVECIWDWIDKLWS (SEQ ID NO: 332),
KFDSLVECIWDWIDKLWS (SEQ ID NO: 333),
FDSLVECIWDWIDKLWS (SEQ ID NO: 334),
DSLVECIWDWIDKLWS (SEQ ID NO: 335), SLVECIWDWIDKLWS (SEQ ID NO: 336),
LVECIWDWIDKLWS (SEQ ID NO: 337), KFESLVDCIWEWIERLWS (SEQ ID NO: 338), FESLVDCIWEWIERLWS (SEQ ID NO: 339), ESLVDCIWEWIERLWS (SEQ ID NO: 340), SLVDCIWEWIERLWS (SEQ ID NO: 341),
LVDCIWEWIERLWS (SEQ ID NO: 342),
KFESLVECIWEWIERLWS (SEQ ID NO: 343),
FESLVECIWEWIERLWS (SEQ ID NO: 344),
ESLVECIWEWIERLWS (SEQ ID NO: 345), SLVECIWEWIERLWS (SEQ ID NO: 346),
LVECIWEWIERLWS (SEQ ID NO: 347),
EFRSLVKCIWRWIRDLWS (SEQ ID NO: 348),
FRSLVKCIWRWIRDLWS (SEQ ID NO: 349),
RSLVKCIWRWIRDLWS (SEQ ID NO: 350), SLVKCIWRWIRDLWS (SEQ ID NO: 351),
LVKCIWRWIRDLWS (SEQ ID NO: 352),
EFDSLVECIWDWIDDLWS (SEQ ID NO: 353),
FDSLVECIWDWIDDLWS (SEQ ID NO: 354),
DSLVECIWDWIDDLWS (SEQ ID NO: 355), SLVECIWDWIDDLWS (SEQ ID NO: 356),
LVECIWDWIDDLWS (SEQ ID NO: 357),
KFRSLVKCIWRWIRRLWS (SEQ ID NO: 358),
FRSLVKCIWRWIRRLWS (SEQ ID NO: 359), RSLVKCIWRWIRRLWS (SEQ ID NO: 360),
SLVKCIWRWIRRLWS (SEQ ID NO: 361),
LVKCIWRWIRRLWS (SEQ ID NO: 362),
KFDSLVERIWDWIDRLWS (SEQ ID NO: 363),
FDSLVERIWDWIDRLWS (SEQ ID NO: 364), DSLVERIWDWIDRLWS (SEQ ID NO: 365),
SLVERIWDWIDRLWS (SEQ ID NO: 366),
LVERIWDWIDRLWS (SEQ ID NO: 367),
KFDSLVEEIWDWIDRLWS (SEQ ID NO: 368),
FDSLVEEIWDWIDRLWS (SEQ ID NO: 369), DSLVEEIWDWIDRLWS (SEQ ID NO : 370),
SLVEEIWDWIDRLWS (SEQ ID NO: 371), LVEEIWDWIDRLWS (SEQ ID NO: 372), KFDSLVECIWDWIDRLWSGS (SEQ ID NO: 373), FDSLVECIWDWIDRLWSGS (SEQ ID NO: 374), DSLVECIWDWIDRLWSGS (SEQ ID NO: 375),
SLVECIWDWIDRLWSGS (SEQ ID NO: 376), LVECIWDWIDRLWSGS (SEQ ID NO: 377), KFDSLVECIWDWIDRLWSG (SEQ ID NO: 378), FDSLVECIWDWIDRLWSG (SEQ ID NO: 379), DSLVECIWDWIDRLWSG (SEQ ID NO: 380),
SLVECIWDWIDRLWSG (SEQ ID NO: 381),
LVECIWDWIDRLWSG (SEQ ID NO: 382),
TKFDSLVECIWDWIDRLWS (SEQ ID NO: 383),
KFDSLVECIWDWIDRLWS (SEQ ID NO: 384), FDSLVECIWDWIDRLWS (SEQ ID NO: 385), DSLVECIWDWIDRLWS (SEQ ID NO: 386), SLVECIWDWIDRLWS (SEQ ID NO: 387), WTKFDSLVECIWDWIDRLWS (SEQ ID NO: 388), TKFDSLVECIWDWIDRLWS (SEQ ID NO: 389),
KFDSLVECIWDWIDRLWS (SEQ ID NO: 390), FDSLVECIWDWIDRLWS (SEQ ID NO: 391), DSLVECIWDWIDRLWS (SEQ ID NO: 392), SWLEKIWKWICRVLSKF (SEQ ID NO: 393), SWLEKIWKWICRVLSK (SEQ ID NO: 394),
SWLEKIWKWICRVLS (SEQ ID NO: 395), SWLEKIWKWICRVL (SEQ ID NO: 396), SWLRKIWKWICEVLSDF (SEQ ID NO: 397), SWLRKIWKWICEVLSD (SEQ ID NO: 398), SWLRKIWKWICEVLS (SEQ ID NO: 399),
SWLRKIWKWICEVL (SEQ ID NO: 400), SWLRDIWDWICKVLSKF (SEQ ID NO: 401), SWLRDIWDWICKVLSK (SEQ ID NO: 402), SWLRDIWDWICKVLS (SEQ ID NO: 403), SWLRDIWDWICKVL (SEQ ID NO: 404),
SWLRRIWRWICEVLSDF (SEQ ID NO: 405), SWLRRIWRWICEVLSD (SEQ ID NO: 406), SWLRRIWRWICEVLS (SEQ ID NO: 407), SWLRRJWRWICEVL (SEQ ID NO: 408), SWLRDIWDWICRVLSRF (SEQ ID NO : 409),
SWLRDIWDWICRVLSR (SEQ ID NO: 410), SWLRDIWDWICRVLS (SEQ ID NO: 411), SWLRDIWDWICRVL (SEQ ID NO: 412), SWLRRIWDWICRVLSDF (SEQ ID NO: 413), SWLRRIWDWICRVLSD (SEQ ID NO: 414), SWLRRIWDWICRVLS (SEQ ID NO: 415), SWLRRIWDWICRVL (SEQ ID NO: 416), SWLRKIWDWICKVLSDF (SEQ ID NO: 417), SWLRKIWDWICKVLSD (SEQ ID NO: 418),
SWLRKIWDWICKVLS (SEQ ID NO: 419), SWLRKIWDWICKVL (SEQ ID NO: 420), SWLRRIWDWICEVLSRF (SEQ ID NO: 421), SWLRRIWDWICEVLSR (SEQ ID NO: 422), SWLRRIWDWICEVLS (SEQ ID NO: 423),
SWLRRIWDWICEVL (SEQ ID NO: 424), SWLRKIWDWICEVLSKF (SEQ ID NO: 425), SWLRKIWDWICEVLSK (SEQ ID NO: 426), SWLRKIWDWICEVLS (SEQ ID NO: 427), SWLRKIWDWICEVL (SEQ ID NO: 428),
SWLRDIWRWICRVLSDF (SEQ ID NO: 429), SWLRDIWRWICRVLSD (SEQ ID NO: 430), SWLRDIWRWICRVLS (SEQ ID NO: 431), SWLRDIWRWICRVL (SEQ ID NO: 432), SWLRDIWKWICKVLSDF (SEQ ID NO: 433),
SWLRDIWKWICKVLSD (SEQ ID NO: 434), SWLRDIWKWICKVLS (SEQ ID NO: 435), SWLRDIWKWICKVL (SEQ ID NO: 436), SWLDRIWDWICRVLSRF (SEQ ID NO: 437), SWLDRIWDWICRVLSR (SEQ ID NO: 438),
SWLDRIWDWICRVLS (SEQ ID NO: 439), SWLDRIWDWICRVL (SEQ ID NO: 440), SWLRDIWDWICKVLSKF (SEQ ID NO: 441), SWLRDIWDWICKVLSK (SEQ ID NO: 442), SWLRDIWDWICKVLS (SEQ ID NO: 443), SWLRDIWDWICKVL (SEQ ID NO: 444), SWLRDIWRWICKVLSRF (SEQ ID NO: 445), SWLRDIWRWICKVLSR (SEQ ID NO: 446), SWLRDIWRWICKVLS (SEQ ID NO: 447),
SWLRDIWRWICKVL (SEQ ID NO: 448), SWLRDIWKWICKVLSKF (SEQ ID NO: 449), SWLRDIWKWICKVLSK (SEQ ID NO: 450), SWLRDIWKWICKVLS (SEQ ID NO: 451), SWLRDIWKWICKVL (SEQ ID NO : 452),
SWLRKIWKWICEVLSKF (SEQ ID NO: 453), SWLRKIWKWICEVLSK (SEQ ID NO: 454), SWLRKIWKWICEVLS (SEQ ID NO: 455), SWLRKIWKWICEVL (SEQ ID NO: 456), SWLRRIWRWICEVLSRF (SEQ ID NO: 457),
SWLRRIWRWICEVLSR (SEQ ID NO: 458), SWLRRIWRWICEVLS (SEQ ID NO: 459), SWLRRIWRWICEVL (SEQ ID NO: 460), SWLRRIWRWICDVLSRF (SEQ ID NO: 461), SWLRRIWRWICDVLSR (SEQ ID NO: 462),
SWLRRIWRWICDVLS (SEQ ID NO: 463), SWLRRIWRWICDVL (SEQ ID NO: 464), SWLRKIWKWICKVLSKF (SEQ ID NO: 465), SWLRKIWKWICKVLSK (SEQ ID NO: 466), SWLRKIWKWICKVLS (SEQ ID NO: 467),
SWLRKIWKWICKVL (SEQ ID NO: 468), SWLRRIWRWICRVLSRF (SEQ ID NO: 469), SWLRRIWRWICRVLSR (SEQ ID NO: 470), SWLRRIWRWICRVLS (SEQ ID NO: 471), SWLRRIWRWICRVL (SEQ ID NO: 472), SWLRRIWRWICRVLSRF (SEQ ID NO: 473), SWLRRIWRWICRVLSR (SEQ ID NO: 474), SWLRRIWRWICRVLS (SEQ ID NO: 475), SWLRRIWRWICRVL (SEQ ID NO: 476),
SWLKKIWKWICKVLSKF (SEQ ID NO: 477), SWLKKIWKWICKVLSK (SEQ ID NO: 478), SWLKKIWKWICKVLS (SEQ ID NO: 479), SWLKKIWKWICKVL (SEQ ID NO: 480), DFKSLVRCIWKWIKELWS (SEQ ID NO: 481),
FKSLVRCIWKWIKELWS (SEQ ID NO: 482),
KSLVRCIWKWIKELWS (SEQ ID NO: 483),
SLVRCIWKWIKELWS (SEQ ID NO: 484),
LVRCIWKWIKELWS (SEQ ID NO: 485), KFDSLVECIWKWIKRLWS (SEQ ID NO: 486),
FDSLVECIWKWIKRLWS (SEQ ID NO: 487),
DSLVECIWKWIKRLWS (SEQ ID NO: 488),
SLVECIWKWIKRLWS (SEQ ID NO: 489),
LVECIWKWIKRLWS (SEQ ID NO: 490), KFKSLVKCIWDWIDRLWS (SEQ ID NO: 491),
FKSLVKCIWDWIDRLWS (SEQ ID NO: 492),
KSLVKCIWDWIDRLWS (SEQ ID NO: 493),
SLVKCIWDWIDRLWS (SEQ ID NO: 494),
LVKCIWDWIDRLWS (SEQ ID NO: 495), KFDSLVECIWRWIRRLWS (SEQ ID NO: 496),
FDSLVECIWRWIRRLWS (SEQ ID NO: 497),
DSLVECIWRWIRRLWS (SEQ ID NO: 498),
SLVECIWRWIRRLWS (SEQ ID NO: 499),
LVECIWRWIRRLWS (SEQ ID NO: 500), KFRSLVRCIWDWIDRLWS (SEQ ID NO: 501), FRSLVRCIWDWIDRLWS (SEQ ID NO: 502), RSLVRCIWDWIDRLWS (SEQ ID NO: 503), SLVRCIWDWIDRLWS (SEQ ID NO: 504), LVRCIWDWIDRLWS (SEQ ID NO: 505),
KFDSLVRCIWDWIRRLWS (SEQ ID NO: 506),
FDSLVRCIWDWIRRLWS (SEQ ID NO: 507),
DSLVRCIWDWIRRLWS (SEQ ID NO: 508),
SLVRCIWDWIRRLWS (SEQ ID NO: 509), LVRCIWDWIRRLWS (SEQ ID NO: 510),
KFDSLVKCIWDWIKRLWS (SEQ ID NO: 511),
FDSLVKCIWDWIKRLWS (SEQ ID NO: 512),
DSLVKCIWDWIKRLWS (SEQ ID NO: 513),
SLVKCIWDWIKRLWS (SEQ ID NO: 514), LVKCIWDWIKRLWS (SEQ ID NO: 515),
KFRSLVECIWDWIRRLWS (SEQ ID NO: 516),
FRSLVECIWDWIRRLWS (SEQ ID NO: 517),
RSLVECIWDWIRRLWS (SEQ ID NO: 518),
SLVECIWDWIRRLWS (SEQ ID NO: 519), LVECIWDWIRRLWS (SEQ ID NO: 520),
KFKSLVECIWDWIKRLWS (SEQ ID NO: 521),
FKSLVECIWDWIKRLWS (SEQ ID NO: 522),
KSLVECIWDWIKRLWS (SEQ ID NO: 523),
SLVECIWDWIKRLWS (SEQ ID NO: 524), LVECIWDWIKRLWS (SEQ ID NO: 525),
KFDSLVRCIWRWIDRLWS (SEQ ID NO: 526),
FDSLVRCIWRWIDRLWS (SEQ ID NO: 527),
DSLVRCIWRWIDRLWS (SEQ ID NO: 528),
SLVRCIWRWIDRLWS (SEQ ID NO: 529), LVRCIWRWIDRLWS (SEQ ID NO: 530),
KFDSLVKCIWKWIDRLWS (SEQ ID NO: 531),
FDSLVKCIWKWIDRLWS (SEQ ID NO: 532),
DSLVKCIWKWIDRLWS (SEQ ID NO: 533), SLVKCIWKWIDRLWS (SEQ ID NO: 534),
LVKCIWKWIDRLWS (SEQ ID NO: 535),
KFRSLVRCIWDWIRDLWS (SEQ ID NO: 536),
FRSLVRCIWDWIRDLWS (SEQ ID NO: 537),
RSLVRCIWDWIRDLWS (SEQ ID NO: 538), SLVRCIWDWIRDLWS (SEQ ID NO: 539),
LVRCIWDWIRDLWS (SEQ ID NO: 540),
KFKSLVKCIWDWIDRLWS (SEQ ID NO: 541),
FKSLVKCIWDWIDRLWS (SEQ ID NO: 542),
KSLVKCIWDWIDRLWS (SEQ ID NO: 543), SLVKCIWDWIDRLWS (SEQ ID NO: 544),
LVKCIWDWIDRLWS (SEQ ID NO: 545),
KFRSLVKCIWRWIDRLWS (SEQ ID NO: 546),
FRSLVKCIWRWIDRLWS (SEQ ID NO: 547),
RSLVKCIWRWIDRLWS (SEQ ID NO: 548), SLVKCIWRWIDRLWS (SEQ ID NO: 549),
LVKCIWRWIDRLWS (SEQ ID NO: 550),
KFKSLVKCIWKWIDRLWS (SEQ ID NO: 551),
FKSLVKCIWKWIDRLWS (SEQ ID NO: 552),
KSLVKCIWKWIDRLWS (SEQ ID NO: 553), SLVKCIWKWIDRLWS (SEQ ID NO: 554),
LVKCIWKWIDRLWS (SEQ ID NO: 555),
KFKSLVECIWKWIKRLWS (SEQ ID NO: 556),
FKSLVECIWKWIKRLWS (SEQ ID NO: 557),
KSLVECIWKWIKRLWS (SEQ ID NO: 558), SLVECIWKWIKRLWS (SEQ ID NO: 559),
LVECIWKWIKRLWS (SEQ ID NO: 560),
KFRSLVECIWRWIRRLWS (SEQ ID NO: 561),
FRSLVECIWRWIRRLWS (SEQ ID NO: 562), RSLVECIWRWIRRLWS (SEQ ID NO: 563),
SLVECIWRWIRRLWS (SEQ ID NO: 564),
LVECIWRWIRRLWS (SEQ ID NO: 565),
KFRSLVDCIWRWIRRLWS (SEQ ID NO: 566),
FRSLVDCIWRWIRRLWS (SEQ ID NO: 567), RSLVDCIWRWIRRLWS (SEQ ID NO: 568),
SLVDCIWRWIRRLWS (SEQ ID NO: 569), LVDCIWRWIRRLWS (SEQ ID NO: 570), KFKSLVKCIWKWIKRLWS (SEQ ID NO: 571), FKSLVKCIWKWIKRLWS (SEQ ID NO: 572), KSLVKCIWKWIKRLWS (SEQ ID NO: 573),
SLVKCIWKWIKRLWS (SEQ ID NO: 574), LVKCIWKWIKRLWS (SEQ ID NO: 575), KFRSLVRCIWRWIRRLWS (SEQ ID NO: 576), FRSLVRCIWRWIRRLWS (SEQ ID NO: 577), RSLVRCIWRWIRRLWS (SEQ ID NO: 578),
SLVRCIWRWIRRLWS (SEQ ID NO: 579),
LVRCIWRWIRRLWS (SEQ ID NO: 580),
RFRSLVRCIWRWIRRLWS (SEQ ID NO: 581),
FRSLVRCIWRWIRRLWS (SEQ ID NO: 582), RSLVRCIWRWIRRLWS (SEQ ID NO: 583),
SLVRCIWRWIRRLWS (SEQ ID NO: 584), LVRCIWRWIRRLWS (SEQ ID NO: 585), KFKSLVKCIWKWIKKLWS (SEQ ID NO: 586), FKSLVKCIWKWIKKLWS (SEQ ID NO: 587), KSLVKCIWKWIKKLWS (SEQ ID NO: 588), SLVKCIWKWIKKLWS (SEQ ID NO: 589), and LVKCIWKWIKKLWS (SEQ ID NO: 590).
In another embodiment of the invention, the isolated peptide of the invention consists of any one of the above amino acid sequences, in particular, SEQ ID NO: 119, 123 to 144, 165 to 177; 179 to 183; 185 to 594.
In some embodiments, a peptide of the invention has viricidal activity. In some embodiments, a peptide of the invention is 18 to 40 D- or L-amino acids in- length, 18 to 30 D- or L-amino acids in-length, or 18 to 22 D- or L-amino acids in- length. In some embodiments, a peptide of the invention includes D-amino acids. In other embodiments, a peptide of the invention includes L-amino acids. In some embodiments, the peptide includes a dansyl moiety. In some embodiments, the peptide has an EC5O of about 3 μM or less; about 2 μM or less; about 1 μM or less; about 500 nM or less; about 400 nM or less; or about 300 nM. In some embodiments, the peptides are active against HIV, the hepatitis C virus or Flaviviruses such as the West Nile virus or the Dengue virus.
In another aspect, the invention provides a pharmaceutical composition that includes any peptide of the invention described herein. In some embodiments, the pharmaceutical composition is a microbicide or a vaginal cream. In some embodiments, the pharmaceutical composition includes an antiviral agent. In some embodiments, the antiviral agent is a protease inhibitor, a polymerase inhibitor, an integrase inhibitor, an entry inhibitor, an assembly/secretion inhibitor, a translation inhibitor or an immunostimulant. In some embodiments, the antiviral agent is α- interferon, pegylated interferon, ribavirin, amantadine, rimantadine, pleconaril, acyclovir, zidovudine, lamivudine, Indenavir (Merck), telaprivir (Vertex), Tenofivir (Gilead), R1626 (Roche), GS-9137 (Gilead), Fuzeon (Roche, Trimeris), Celgosivir (Migenix), VGX-410c (VGX pharmaceuticals), 0 IMO-2125 (Idera pharmaceuticals) or a combination thereof. In another aspect, the invention provides an article of manufacture that includes a vessel for collecting a body fluid and any peptide of the invention described herein. In some embodiments, the vessel is a collection bag, tube, capillary tube or syringe. In some embodiments, the vessel is evacuated. In some embodiments, the vessel further includes a biological stabilizer. In some embodiments, the peptide is attached or adsorbed onto the vessel so that the peptide is retained in the vessel after materials placed therein have been removed. When attached or adsorbed onto the vessel, the peptide is still able to inhibit viral infection. In another aspect, the invention provides a composition that includes a sample from the body of a mammal and any peptide of the invention described herein. In some embodiments, the composition also includes a biological stabilizer. In some embodiments, the sample is a blood product. In some embodiments, the blood product is plasma, platelet, leukocytes or stem cell. In the compositions or articles of the invention, the biological stabilizer is an anti-coagulant, preservative, protease inhibitor, or any combination thereof. In some embodiments, the anti-coagulant is citrate, ethylene diamine tetraacetic acid, heparin, oxalate, fluoride or any combination thereof. In some embodiments, the preservative is boric acid, sodium formate and sodium borate. In some embodiments, the protease inhibitor is dipeptidyl peptidase IV. In some embodiments, the peptide and/or stabilizer is freeze dried.
In another aspect, the invention provides (1) a method for inactivating a virus that involves contacting the virus with a peptide or composition of the invention, (2) method for preventing or treating infection of a mammalian cell by a virus that involves contacting the mammalian cell with with a peptide or composition of the invention, or (3) a method for preventing or treating a viral infection in a mammal that involves administering to the mammal an effective amount of with a peptide or composition of the invention. In some embodiments of the methods of the invention, the virus is a Flavivirus, an Hepacivirus or a human immunodeficiency virus. In some embodiments, the virus is a Hepatitis C virus, West Nile virus, or Dengue virus. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammal is a human. In some embodiments, the peptide is administered. topically or systematically.
In some embodiments, the invention provides (1) a method for inactivating a human immunodeficiency virus, (2) a method for preventing or treating infection of a mammalian cell by a human immunodeficiency virus, and (3) a method for preventing or treating infection of a mammal by a human immunodeficiency virus. These methods involve contacting the virus or mammalian cell, or administering to the mammal, a peptide of the invention or a composition comprising a peptide of the invention.
In some embodiments, a peptide that can be used is 14 to 50 D- or L-amino acids in length and comprises an alpha-helical structure, and wherein the polar amino acids are located on the same face of the alpha helical structure, and the nonpolar amino acids are located on the other face of the alpha-helical structure. For example, all the polar amino acids are located on the same face of the alpha- helical structure and all the non-polar amino acids are located on the other face of the alpha-helical structure resulting in perfect amphipathicity. Perfect amphipathicity can be determined using a helical wheel as known in the art.
In some embodiments, a peptide that can be used in these methods of the invention includes nonpolar amino acids selected from the group consisting of alanine, valine, leucine, methionine, isoleucine, phenylalanine, and tryptophan. In some embodiments, the polar amino acids are selected from the group consisting of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine, hydroxylysine, ornithine, serine and threonine. In some embodiments, the cysteine residue in the peptide is located at a position N-terminal to a serine on the peptide. In some embodiments, the cycteine is located four positions N-terminal to the serine on the peptide. In some embodiments, the cysteine is located at position 11 relative to the N-terminus of the peptide. In some embodiments, amino acids 16 and 18 relative to the N-terminus of the peptide are charged, in particular, amino acids 16 and 18 are charged positive and negative, positive and positive, or negative and positive, respectively.
In some embodiments, a peptide that can be used in a method of the invention is 14 to 40 D- or L-amino acids in-length, 14 to 30 D- or L-amino acids in-length, 14 to 25 D- or L-amino acids in-length, or 14 to 18 D- or L-amino acids in-length. In some embodiments, the peptide is 14 amino acids in length; the amino acids are arginine, cysteine, glutamate, serine, valine, two aspartates, two leucines, two isoleucines and three tryptophan residues; and the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure. In other embodiments, the peptide is 15 amino acids in length; the amino acids are arginine, cysteine, glutamate, two serines, valine, two aspartates, two leucines, two isoleucines and three tryptophan residues; and the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha- helical structure. In other embodiments, the peptide is 16 amino acids in length; the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines and three tryptophan residues; and the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure. In some embodiments, the peptide is 17 amino acids in length; the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines, three tryptophan and a phenylalanine; and the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure. In some embodiments, the peptide is 18 amino acids in length; the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines, three tryptophan, a phenylalanine and a lysine; and the arginine, cysteine, glutamate, serine, aspartate and lysine residues are located on the same face of the alpha-helical structure. In some embodiments, a peptide that can be used in a method of the invention is composed of D amino acids. In other embodiments, it is composed of L amino acids. In some embodiments, it further comprises a dansyl moiety.
In some embodiments, a peptide that can be used in a method of the invention comprises the amino acid sequence:
QIVGGVYLLPRRGPRLGV (SEQ ID NO: 4), QPGYPWPLYGNEGCGWAG (SEQ ID NO: 5), LYGNEGCGWAGWLLSPRG (SEQ ID NO: 6), GWAGWLLSPRGSRPSWGP (SEQ ID NO: 7), IFLLALLSCLTVPASAYQ (SEQ ID NO: 8),
DAILHTPGCVPCVREGNA (SEQ ID NO: 9), LPTTQLRRHIDLLVGSAT (SEQ ID NO: 10), RHIDLLVGSATLCSALYV (SEQ ID NO: 11), GSATLCSALYVGDLCGSV (SEQ ID NO: 12), ALYVGDLCGSVFLVGQLF (SEQ ID NO: 13),
IMDMIAGAHWGVLAGIAY (SEQ ID NO: 14), HINSTALNCNESLNTGWL (SEQ ID NO: 15), NCNESLNTGWLAGLFYQH (SEQ ID NO: 16), LASCRRLTDFAQGWGPIS (SEQ ID NO: 17), TDFAQGWGPISYANGSGL (SEQ ID NO: 18),
GPISYANGSGLDERPYCW (SEQ ID NO: 19), GSGLDERPYCWHYPPRPC (SEQ ID NO: 20), WMNSTGFTKVCGAPPCVI (SEQ ID NO: 21), PCVIGGVGNNTLLCPTDC (SEQ ID NO: 22), MYVGGVEHRLEAACNWTR (SEQ ID NO: 23),
YLYGVGSSIASWAIKWEY (SEQ ID NO: 24), SIASWAIKWEYVVLLFLL (SEQ ID NO: 25), KWEYVVLLFLLLADARVC (SEQ ID NO: 26), WMMLLISQAEAALENLVI (SEQ ID NO: 27), GAVYAFYGMWPLLLLLLA (SEQ ID NO: 28), GMWPLLLLLLALPQRAYA (SEQ ID NO: 29), TLVFDITKLLLAIFGPLW (SEQ ID NO: 30), VSTATQTFLATCIN (SEQ ID NO: 31), ATQTFLATCINGVCWTVY (SEQ ID NO: 32),
DSSVLCECYDAGCAWYEL (SEQ ID NO: 33), AYMNTPGLPVCQDHLEFW (SEQ ID NO: 34), LEFWEGVFTGLTHIDAHF (SEQ ID NO: 35), HPITKYIMTCMSADLEVV (SEQ ID NO: 36), VTSTWVLVGGVLAAL (SEQ ID NO: 37),
WVLVGGVLAALAAYCLST (SEQ ID NO: 38), LAALAAYCLSTGCVV (SEQ ID NO: 39), EVFWAKHMWNFISGIQYL (SEQ ID NO: 40), MWNFISGIQYLAGLSTLP (SEQ ID NO: 41), PAILSPGALVVGVVCAAI (SEQ ID NO: 42),
SWLRDIWDWICEVLSDFK (SEQ ID NO: 43), DWICEVLSDFKTWLKAKL (SEQ ID NO: 44), YVSGMTTDNLKCPCQIPS (SEQ ID NO: 45), SSGADTEDVVCCSMS (SEQ ID NO: 46), DTEDVVCCSMSYSW (SEQ ID NO: 47),
SSGADTEDVVCCSMSYSW (SEQ ID NO: 48), DVVCCSMSYSWTGAL (SEQ ID NO: 49), TVTESDIRTEEAIYQCCD (SEQ ID NO: 50), GNTLTCYIKARAACRAAG (SEQ ID NO: 51), RAAGLQDCTMLVCGDDLV (SEQ ID NO: 52),
CTMLVCGDDLVVICESAG (SEQ ID NO: 53), DDLVVICESAGVQEDAAS (SEQ ID NO: 54), LELITSCSSNVSVAHDGA (SEQ ID NO: 55), HTPVNSWLGNIIMFAPTL (SEQ ID NO: 56), APTLWARMILMTHFFSVL (SEQ ID NO: 57), DQLEQALNCEIYGACYSI (SEQ ID NO: 58), GVPPLRAWRHRARSVRAR (SEQ ID NO: 59), WRHRARSVRARLLSRGGR (SEQ ID NO: 60), GWFTAGYSGGDIYHSVSH (SEQ ID NO: 61),
LYGNEGLGWAGWLLSPRG (SEQ ID NO:62), IFLLALLSCITVPVSAAQ (SEQ ID NO:63), IFLLALLSCLTIPASAYE (SEQ ID NO:64), MSATFCSALYVGDLCGGV (SEQ ID NO:65), GAAALCSAMYVGDLCGSV (SEQ ID NO:66),
ALYVGDLCGGVMLAAQVF (SEQ ID NO:67), AMYVGDLCGSVFLVAQLF (SEQ ID NO:68), IIDIVSGAHWGVMFGLAY (SEQ ID NO:69), VVDMVAGAHWGVLAGLAY (SEQ ID NO:70), VDVQYMYGLSPAITKYVV (SEQ ID N0:71),
YLYGIGSAVVSFAIKWEY (SEQ ID NO:72), WMLILLGQAEAALEKLVV (SEQ ID NO:73), WMMLLIAQAEAALENLVV (SEQ ID NO:74), GVVFDITKWLLALLGPAY (SEQ ID NO:75), ELIFTITKILLAILGPLM (SEQ ID NO:76),
VSQSFLGTTISGVLWTVY (SEQ ID NO:77), ATQSFLATCVNGVCWTVY (SEQ ID NO:78), SWLRDVWDWVCTILTDFK (SEQ ID NO:79), SWLRDVWDWICTVLTDFK (SEQ ID NO:80), DWVCTILTDFKNWLTSKL (SEQ ID N0:81),
DWICTVLTDFKTWLQSKL (SEQ ID NO: 82), ASEDVYCCSMSYTWT (SEQ ID NO:83), EDDTTVCCSMSYSW (SEQ ID NO:84), CTMLVCGDDLVVICESAG (SEQ ID NO:85), PTMLVCG DDLVVISESQG (SEQ ID NO:86), SWLRPIWPWICEVLSDFK (SEQ ID NO: 91), SWLRDIWDWICEVL (SEQ ID NO: 92), SWLRDIWDWICEVLS (SEQ ID NO: 93), SWLRDIWDWICEVLSD (SEQ ID NO: 94),
SWLRDIWDWICEVLSDF (SEQ ID NO: 95), KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), SIWRDWVDLICEFLSDWK (SEQ ID NO: 97), KWLCRIWSWISDVLDDFE (SEQ ID NO: 98), FDSLVECIWDWIDRLWS (SEQ ID NO: 99),
DSLVECIWDWIDRLWS (SEQ ID NO: 100), SLVECIWDWIDRLWS (SEQ ID NO: 101), or LVECIWDWIDRLWS (SEQ ID NO: 102).
In some embodiments, a peptide that can be used in a method of the invention consists of the amino acid sequence of any of SEQ ID NO: 4-86 and 91- 102. In some embodiments, a peptide that can be used in a method of the invention has an EC50 of about 3 μM or less, about 2 μM or less, about 1 μM or less, about 500 nM or less, about 400 nM or less, about 300 nM.
In another aspect, the invention provides for an isolated peptide of 14 to 50 D- or L-amino acids in-length, having an amphipathic α-helical structure and antiviral activity against HIV and/or a virus of the Flaviviridae family.
In one embodiment, the peptide has a sequence comprising any one of formulae IX-XIII:
Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8- IX Xaa9-Xaaio-Xaan-Xaai2-Xaai3-Xaai4 (SEQ ID NO: 112)
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9- X
XaaiO-Xaai i-Xaai2-Xaa,3-Xaai4- Xaa,5 (SEQ ID NO: 113)
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaaio- XI
Xaai i-Xaai2-Xaai3-Xaai4- Xaa,5-Xaa16 (SEQ ID NO: 114) Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaai0-Xaai i- XII Xaai2-Xaai3-Xaai4- Xaai5-Xaa,6-Xaa) 7 (SEQ ID NO: 1 15)
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaaio-Xaan- XIII
Xaa12-Xaa13-XaaH- Xaai5-Xaai6-Xaai7-Xaa,8 (SEQ ID NO: 1 16)
wherein: Xaaj, Xaa4, Xaa5, Xaa8, Xaan, Xaaj2, Xaai5, Xaai6 and Xaai8 are separately each a polar amino acid; and Xaa2, Xaa3, Xaaό, Xaa7, Xaa9, Xaaio, Xaan, Xaai4, and Xaaj7 are separately each a nonpolar amino acid.
In another embodiment, the invention provides a fusion peptide formed by attaching a 14 amino acid peptide (the N-terminal peptide) to the N-terminus of a peptide of any of formulae IX to XIII. The 14 amino acid N-terminal peptide has the structure: Rx-Ry-Ry-Rx-Ry-Ry-Rx-Rx-Ry-Ry-Rx-Rx-Ry-Rx (SEQ ID NO: 1 17), wherein each Rx is separately a polar amino acid, and each Ry is separately a nonpolar amino acid.
In another embodiment, the invention provides a fusion peptide formed by attaching a 12 amino acid peptide (the C-terminal peptide) to the C-terminus of a peptide of formula XIII. The resulting fusion peptide has the structure of formulae XIV:
Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaai o- Xaaii-Xaai2-Xaai3-Xaai4- Xaai 5-Xaai6-Xaai7-Xaai8 -Xaai 9-Xaa20-Xaa2i- Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa3o (SEQ ID NO: 1 18), wherein: Xaai, Xaa4, Xaa5, Xaa8, Xaan, Xaai2, Xaa15, Xaai6, Xaa!8, Xaaig, Xaa22,
Xaa23, Xaa26, Xaa29, and Xaa3o are separately each a polar amino acid; and
Xaa2, Xaa3, Xaaό, Xaa7, Xaa9, Xaaio, Xaan, Xaai4, Xaan, Xaa20, Xaa2i, Xaa24, Xaa25, Xaa27, and Xaa28 are separately each a nonpolar amino acid.
In some embodiments, the invention provides a fusion peptide having a sequence that corresponds to the 14 amino acid N-terminal peptide of SEQ ID NO: 1 17 attached by a peptide bond to the N-terminus of a peptide of formula XIV. In another embodiment, a peptide of the invention is a peptide comprising at least 14 contiguous amino acids of any of the above described peptides.
In another aspect, the invention provides for the use of a peptide of formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIV in the preparation of a medicament for preventing and/or treating infection in a mammal by a human immunodeficiency virus, Hepatitis C virus, West Nile virus, or Dengue virus.
In another aspect, the invention provides for the use of a peptide of formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIV in the preparation of a medicament for preventing and/or treating infection in a mammal by a Flavivirus or an Hepacivirus.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
DESCRIPTION OF THE FIGURES FIG. 1 illustrates the location of the peptides with respect to the HCV polyprotein genotype Ia (H77 isolate, having SEQ ID NO:1) and the corresponding anti-HCV activity. Thirteen of the peptides tested inhibited infectivity by 90% or more.
FIG. 2 illustrate that peptide 1 having the amino acid sequence SWLRDIWDWICEVLSDFK (SEQ ID NO:43) with either L- or D-amino acids (A) prevents initiation of HCV infection when added to the virus before cells are exposed to virus; (B) terminates ongoing HCV infection; (C) inhibits HCV infection in growth-arrested Huh-7 cells; (D) enters the cell and (E) inhibits intracellular HCV particle infectivity. The EC50 of peptide 1 is approximately 300 nM (F and G), as shown by two separate experiments.
FIG. 3 is a bar graph showing inhibition of HCV infection of Huh-7.5.1 cells by various synthetic peptides, of which peptide 1 (SEQ ID NO:43) was the most effective inhibitor of viral infection. FIG. 4A-G are data showing the inhibitory characteristic and stability of peptide 1 (SEQ ID NO:43). Both the L- and D- isomers of peptide 1 are highly inhibitory (A). Peptide 1 acts on the virus (B) and is virocidal for HCV (C) by disrupting HCV virions (D). Both the L- and D-isomers are effective at permeabilizing membranes (E). The D-form of peptide 1 displays enhanced serum stability (F) and a slightly lower IC50 (G).
FIG. 5A-D are results showing the effective concentration (EC50) and toxicity (LC50) of the L- and D-forms of peptide 1 (SEQ ID NO:43). FIG. 5 A shows the percent infected Huh-7.5.1 cells as a function of concentration of the L- form of peptide 1, illustrating that the EC50 of the L-form of peptide 1 is 0.6 micromolar. FIG. 5B shows the percent infected Huh-7.5.1 cells as a function of concentration of the D-form of peptide 1 (with D- instead of L-amino acids), illustrating that the EC50 of the D-form of peptide 1 is 1.0 micromolar. FIG. 5C shows the percent viable Huh-7 cells as a function of concentration of the L-form (square symbols) and D-form (diamond symbols) of peptide 1, illustrating that the LC50 of the L-form of peptide 1 is 46 micromolar and the LC50 of the D-form of peptide 1 is 87 micromolar. FIG. 5D shows the percent viable Huh-7.5.1 cells as a function of concentration of the L-form (square symbols) and D-form (diamond symbols) of peptide 1, illustrating that the LC50 of the L-form of peptide 1 is 64 micromolar and the LC5O of the D-form of peptide 1 is 105 micromolar.
FIG. 6A-E illustrate the amphipathic α-helical nature of peptide 1 (SEQ ID NO:43). Helical wheel diagram of peptide 1 shows that the amino acid distribution results in a hydrophilic (or polar) face and a hydrophobic (or non-polar) face (FIG. 6A). Circular dichroism results show the α-helical structure of the L- and D- isomers of peptide 1 (FIG. 6B), the effect of dansylation on the α-helical structure of the L- and D-isomers of peptide 1 (FIG. 6C), and the α-helical structures of variants of peptide 1 having C-terminal truncations (FIG. 6D) and N-terminal truncations (FIG. 6E). The sequences of these truncated peptides are provided in Table 9. FIG. 7 illustrate the liposome-release assays results obtained for various truncation variants of peptide 1 (SEQ ID NO:43). The sequences of these truncated peptides are provided in Table 9.
FIG. 8 is a graph showing that peptide 1 (SEQ ID NO:43; referred to as #7208 in this Figure) does not block vesicular stomatitis virus (VSV) infection. Additional studies indicate that peptide 1 does not block infection by influenza virus, vaccinia virus, Borna disease virus, lymphocytic choriomeningitis virus or adenovirus.
FIG. 9 is a graph showing that peptide 2022 (peptide 1) with sequence SWLRDIWDWICEVLSDFK (SEQ ID NO:43) and peptide 2013 having the sequence S WLRDIWD WICEVL (SEQ ID NO:92) inhibit essentially 100 % of Dengue viral infection as detected by ELISA. Peptide 2017 having the sequence LRDIWDWICEVLSDFK (SEQ ID NO: 107) had slightly less activity, inhibiting Dengue viral infection by about 84 %. FIG. 1 OA-D are graphs showing the percent inhibition of Dengue viral infection for various peptides. FIG. 1 OA shows dose-dependent inhibition of Dengue viral infection by peptide 2022 (peptide 1 ; SEQ ID NO:43), peptide 2013 (SEQ ID NO:92), and peptide 2017 (SEQ ID NO: 107), as detected by FACS analysis of cells intracellularly stained for Dengue viral antigens. As shown, at concentrations of 20 μM almost 100 % of Dengue viral infection was inhibited by peptide 2022 (peptide 1) and peptide 2013, as detected by FACS. Peptide 2017 at 20 μM had slightly less activity, inhibiting Dengue viral infection by about 80 %. FIG. 1 OB-D also show inhibition of Dengue viral infection by peptide variants and demonstrates the inhibitory effect amphipathic peptides that are variants of peptide 1 as detected by intracellular FACS staining. These data show that variants and homologues of peptide 1 retain inhibitory activity so long as the amphipathicity of the peptide structure is maintained. Note that peptide 1 (SEQ ID NO:43) is called peptide L-7208 in FIG. lOB-D. FIG. 11 depicts results illustrating the ability of peptide 1 (SEQ ID NO:43; called 2022 in this figure) and peptide 2012 (S WLRDIWD WICEVLSD, SEQ ID NO:94) to inhibit West Nile Viral (WNV) infection when incubated with infectious virus prior to adding the virus to Huh-7 cells. In this figure, between 102 and 105 PFU of WNV were preincubated with either DMSO (first column) or peptide 1 (18 micromolar) (third column) or peptide 2012 (fifth column) before being added to Huh-7 cells in a microtiter plate. The seventh column displays uninfected cells. Five (5) days later, the wells were stained with a WNV-specific antibody. As shown, viral antigen was easily detectable throughout the monolayers that had been incubated with DMSO-treated virus. In contrast, there were only 4-5 foci of stained cells in the cultures that were inoculated with 105 PFU of peptide-treated virus, and no evidence of infection in cultures that were inoculated with lower doses of virus..
FIG. 12 graphically illustrates that both the L- and D- forms of peptide 1 (i.e. L-2022 [SWLRDIWDWICEVLSDFK] (SEQ ID NO:43) and D-2022 (SEQ ID NO:43 with D-amino acids) inhibit HIV-I infection at concentrations between 1.25 to 5.0 micromolar. It also illustrates that peptide 2018 (DIWD WICEVLSDFK) (SEQ ID NO: 108), an N-terminally truncated 14-mer version of peptide 1 has similar antiviral activity, and that peptide 2054 (SWLRDIWD WICEV) (SEQ ID NO: 103), a C-terminally truncated 13-mer analog of peptide 1 is slightly less active. In contrast, peptide 2015 (S WLRDIWDWI) (SEQ ID NO: 105), a C-terminally truncated 10-mer version of peptide 1 has no activity; nor does peptide 6938 (LYGNEGCGWAGWLLSPRG) (SEQ ID NO:6), an 18-mer derived from the core protein of HCV.
FIG. 13A-B graphically illustrates that the L-7208 HS peptide (SIWRDWVDLICEFLSDWK, SEQ ID NO:97), is almost as active as the highly active L-7208 SEQ ID NO:43 peptide (also called the L-2022 peptide or "peptide 1"). The L-7208 HS peptide has the same amino acid composition as the L-7208 peptide, but the hydrophobic amino acids in the L-7208 HS peptide (SEQ ID NO:97) have been scrambled, thereby changing the amino acid sequence but maintaining its amphipathicity. Thus, the L-7208 HS peptide (SEQ ID NO:97), the L-7208 peptide (SEQ ID NO:43) and the D-7208 peptide (SEQ ID NO:43 with D- amino acids instead of L-amino acids) all inhibit essentially 100 % HIV infection at concentrations of about 20 micromolar. Another peptide (called 3229, SWRLDIWDWICESVLDFK, SEQ ID NO: 119) whose amino acids were exchanged to reduce its amphipathicity, but which has the same amino acid composition as the L-7208 (SEQ ID NO:43) peptide, exhibited little if any activity. These data indicate that the amphipathicity of the peptide is important for activity, but the exact sequence of the peptide is not critical. Results for HIV R9BaL from 293 T cells are shown in FIG. 13 A. Results for HIV R9BaL from CEM T cells are shown in FIG. 13B. -
FIG. 14A-C illustrates that the peptides destabilize the HIV-I virions extracellularly. For example, FIG. 14A illustrates that large amounts of free HIV-I capsids are released from infectious virions after treatment of HIV-I preparations with peptide 1 (designated here as L-7208 (SEQ ID NO:43)), indicating that the virions were lysed by the peptide. In contrast, essentially no HIV-I capsid was released after HIV-I virions were treated with DMSO or with control peptide 6938 (LYGNEGCGWAGWLLSPRG) (SEQ ID NO:6). In keeping with these observations, the amount of virus-associated capsid was reduced in the L-7208 (SEQ ID NO:43)-treated virus (FIG. 14B) but not in the peptide 6938
(LYGNEGCGWAGWLLSPRG, SEQ ID NO:6) treated specimens. Finally, FIG. 14C shows the percent of HIV-I capsid internalized into cells after treatment with DMSO and 5 or 10 micromolar of peptides 6938 (SEQ ID NO:6) or L-7208 (SEQ ID NO:43). While essentially 100% of control amounts of HIV-I capsid were internalized when cells were treated with 5 or 10 micromolar 6938 (SEQ ID NO:6), HIV-I capsid internalization was inhibited up to 10-fold by treatment with peptide L-7208 (SEQ ID NO:43).
FIG. 15 is a bar graph illustrating that peptides with amphipathic structures similar to the amphipathic structure of peptide L-7208 (SEQ ID NO:43) are also strongly inhibitory of HIV infection. Thus, amphipathic peptides with strong anti- HIV activity include peptide 3222 (SEQ ID NO: 127), peptide 3226 (SEQ ID NO: 128), peptide 3228 (SEQ ID NO: 130), peptide L-7208 2D to 2 Pro (SEQ ID NO:91), and L-7208 HS with hydrophilic amino acids scrambled (KWLCRIWSWISDVLDDFE, SEQ ID NO:98).
FIG. 16A-D. Peptide 1 (SEQ ID NO:43; amphipathic "viracide") neutralizes not only cell-free HIV, but also cell-associated and internalized HIV. (A) CD4+ T- lymphocytes, macrophages or DC (O.lxlO6 cells) were exposed to NL4.3 BaL (1 ng of p24) for 1 day, washed three times with medium, and cultured in a flat bottom 96-well plate. Wild-type Peptide 1 or its non-amphipathic variant (SEQ ID NO: 1 19; 5 μM) were added together with virus to CD4+ T cells, macrophages and DC (panels 1 to 3), to HIV -pulsed DC prior to T cell incubation (panel 4), or added 3 days post-infection to T cells (panel 5). Supernatants were collected after different days and viral replication was monitored by p24 ELISA. Error bars represent standard errors of duplicates. These experiments are representative of three independent experiments using three different donors. (B) 293T cells transfected with NL4.3 or NL4.3 BaL for 24 h were treated with or without peptide (5 μM) for 1 h at 37°C and washed to remove peptides. Twenty-four hours post-peptide treatment, infectivity of 293T-released viruses was scored on TZM cells. Infection was measured 48 h post-infection by β-galactosidase activity. Error bars represent standard errors of duplicates. These experiments are representative of two independent experiments. (C) TZM cells were exposed to pNL4.3-ΔEnv viruses (1 ng of p24) pseudotyped with gplόO NL4.3 (X4), gplόO BaL (R5) or VSVG with or without peptide (5 μM). Infection was measured 48 h post-infection by β- galactosidase activity. Error bars represent standard errors of duplicates. Data are expressed in % of infection. (D) TZM cells were either pretreated with peptide (5 μM) for 1 , 2, 4 and 8 h, washed extensively to remove peptides and then exposed to NL4.3 (1 ng of p24), or cells were first exposed to virus and peptide was added 1, 2, 4 and 8 h later. Infection was measured 48 h post-infection by β-galactosidase activity. Error bars represent standard errors of duplicates.
FIG. 17A-E. Peptide 1 (SEQ ID NO:43; amphipathic "viracide") destroys the integrity of both the membrane and capsid core of HIV. (A) Purified NL4.3 virus (20 ng of p24 in PBS) was incubated with or without Peptide 1 (5 μM) for 30 min at 37°C and loaded over a 20-70% sucrose gradient. Each collected fraction of the gradient was analyzed for capsid (p24 ELISA and immunoblot), RT (by exoRT assay) and gp41 content (by immunoblot). The density of each fraction (g/cm3) shown in the lower two panels, was determined by measuring the refractive index. (B) For virus attachment, TZM cells (500,000) were exposed to 1 ng of p24 of NL4.3 for 1 h at 4°C, washed extensively to remove unbound virus and lysed. For virus internalization, cells were exposed to virus for 2 h at 370C, washed, trypsinized to remove attached virus and lysed. Amounts of attached and internalized virus were determined by p24 ELISA in cell lysates. Error bars represent the standard errors of duplicates. Data are expressed in percentage of attachment or internalization. These experiments are representative of two independent experiments. (C) Same as (A) except that virus was treated with decreasing concentrations of Peptide 1 for 30 min at 37°C (top left panel), with 5 μM of Peptide 1 for 15, 30 and 60 min at 37°C (bottom left panel), for 30 min at 4, 25 or 37°C (top right panel), and for 30 min at pH 8, 7, 6 and 5 (bottom right panel). Gradient fractions were analyzed for HIV capsid content by p24 ELISA. (D) Same as (A) except that virus was treated either with Peptide 1 or with its non- amphipathic variant (SEQ ID NO:119). (E) Same as (A) except that virus was first trypsinized for 15 min at 37°C, incubated in 10% FCS to neutralize trypsin, microfuged for 90 min at 4°C, resuspended and immediately loaded over a sucrose gradient for virus integrity evaluation by p24 ELISA.
FIG. 18A-F. Peptide 1 (SEQ ID NO:43; amphipathic "viracide") inhibits both HIV genital epithelial transmigration and LC/DC transmission of HIV. (A) Either cell-free or cell-associated HIV was added to the apical surface of primary genital epithelial cells (PGEC) for 8 h at 37°C, and amounts of transcytosed viruses were quantified by p24 ELISA of the lower chamber corresponding to the basal PGEC surface. To determine the effect of Peptide 1 (SEQ ID NO:43) on HIV transmigration, it (5 μM) was added just after addition of the virus to PGEC and its effects were compared with its non-amphipathic variant (SEQ ID NO: 119). Results are expressed in % of p24 of the original inoculum. Error bars represent standard errors of duplicates. Results are representative of 4 independent experiments using PGEC derived from 4 donors. (B) PGEC were treated twice daily with 200 μM of Peptide 1 (SEQ ID NO:43) or 0.01% saponin for a week. No washes were performed in order to maintain a continuous exposure of cells to peptides. After overnight incubation, CellQuanti-MTTTM reagent was added and cell viability was quantified by OD 570 nm reading. For FIG. 18C-F, epidermal sheets were infected with HIV NL4.3-BaL-eGFP (100 ng p24) and directly incubated with 10 μM Peptide 1 (SEQ ID NO:43) or DMSO control. After 3 days, epidermal sheets were removed and 200,000 CCR5+ Jurkat cells were added for an additional 4 days.
Migrated DC/LC epidermal cells (day 3) and samples of the co-cultures (day 5 and 7) were analyzed for Green Fluorescent Protein (GFP) expression by FACS. (C) HIV infection of migrated DC/LC is depicted as percentage of total cells. Error bars represent standard errors of duplicates (D) The co-cultures were further analyzed for infection at day 7 by FACS. The percentage of infected cells is depicted. (E) The donor variability at day 5 is depicted. Error bars represent standard errors of duplicates. (F) DC (50,000) were exposed to HIV-I NL4.3-eGFP (X4), NL4.3-BaL- eGFP (R5) or to NL4.3ΔEnv-eGFP pseudotyped with NL4.3 gpl60 env (25 ng p24) together with 10 μM Peptide 1 or DMSO control for 2 h at 37°C. Cells were washed 3 times, activated CD4+ T cells were added for 3 days and GFP expression was measured by FACS. Error bars represent standard errors of duplicates. These results are representative of 3 independent experiments.
DETAILED DESCRIPTION OF THE INVENTION The invention relates to peptides that can prevent or treat viral infection or that can inactivate a virus before it enters a cell. The invention involves the discovery that certain peptides derived from the hepatitis C viral polyprotein, e.g. those having sequences set forth in SEQ ID NO: 4-61, can prevent or treat infection of a mammal by viruses of several types, including viruses in the Flaviviridae family, measles, respiratory syncytial virus (RSV) and HIV, as well as inactivate such viruses. The invention also involves the discovery that several peptides from the HCV polyprotein (SEQ ID NO:1) are highly effective at inhibiting HIV infection as well as infection of a virus in the Flaviviridae family. In addition, the invention involves the discovery that "peptide 1" (SEQ ID NO:43), derived from the membrane anchor domain of NS5A (NS5A-1975), was particularly potent against HIV, hepatitis C virus, measles, RSV and Flaviviruses such as Dengue virus and West Nile virus. For example, 20 μM of peptide 1 completely inhibited HIV infection and concentrations as low as 0.3 μM were strongly inhibitory of HIV infection.
Moreover, the present peptides are effective at preventing or treating infection by a variety of HIV strains, including those that use CXCR4 and CCR5 as coreceptors. Peptides of the invention include, for example, those having sequences set out in SEQ ID NO: 4-86, 91-102, 119, 123-144, 165-177, 179-183 and 185-594 and peptides of about 8 to about 50 amino acids that are capable of forming an α- helical structure and can inhibit viral infection in a mammalian cell. The invention provides an antiviral peptide or combinations of antiviral peptides, various compositions and combinations containing such antiviral peptide(s), and a method for inhibiting viral infection in a mammalian cell that utilizes such peptide(s). The invention also provides an article of manufacture containing such antiviral peptide(s).
Peptides Obtained from Hepatitis C Virus According to the invention, peptides from the hepatitis C viral polyprotein of the hepatitis C genotype Ia (H77) as well as hepatitis genotypes IB, 2 A, 4 A, 5 A and 6 A exhibit strong inhibition of HIV infection. Thus, for example, the HCV polyprotein sequence from which the peptides were originally obtained has sequence SEQ ID NO: 1 and can be found in the NCBI database as accession number NP 671491 (gi: 22129793). The amino acid sequence of this HCV polyprotein (SEQ ID NO:1) is as follows.
1 MSTNPKPQRK TKRNTNRRPQ DVKFPGGGQI VGGVYLLPRR
41 GPRLGVRATR KTSERSQPRG RRQPIPKARR PEGRTWAQPG 81 YPWPLYGNEG CGWAGWLLSP RGSRPSWGPT DPRRRSRNLG
121 KVIDTLTCGF ADLMGYIPLV GAPLGGAARA LAHGVRVLED
161 GVNYATGNLP GCSFSIFLLA LLSCLTVPAS AYQVRNSSGL
201 YHVTNDCPNS SIVYEAADAI LHTPGCVPCV REGNASRCWV
241 AVTPTVATRD GKLPTTQLRR HIDLLVGSAT LCSALYVGDL 281 CGSVFLVGQL FTFSPRRHWT TQDCNCSIYP GHITGHRMAW
321 DMMMNWSPTA ALVVAQLLRI PQAIMDMIAG AHWGVLAGIA
361 YFSMVGNWAK VLVVLLLFAG VDAETHVTGG SAGRTTAGLV
401 GLLTPGAKQN IQLINTNGSW HINSTALNCN ESLNTGWLAG
441 LFYQHKFNSS GCPERLASCR RLTDFAQGWG PISYANGSGL 481 DERPYCWHYP PRPCGIVPAK SVCGPVYCFT PSPVVVGTTD
521 RSGAPTYSWG ANDTDVFVLN NTRPPLGNWF GCTWMNSTGF
561 TKVCGAPPCV IGGVGNNTLL CPTDCFRKHP EATYSRCGSG
601 PWITPRCMVD YPYRLWHYPC TINYTIFKVR MYVGGVEHRL
641 EAACNWTRGE RCDLEDRDRS ELSPLLLSTT QWQVLPCSFT 681 TLPALSTGLI HLHQNIVDVQ YLYGVGSSIA SWAIKWEYVV
721 LLFLLLADAR VCSCLWMMLL ISQAEAALEN LVILNAASLA
761 GTHGLVSFLV FFCFAWYLKG RWVPGAVYAF YGMWPLLLLL
801 LALPQRAYAL DTEVAASCGG VVLVGLMALT LSPYYKRYIS 841 WCMWWLQYFL TRVEAQLHVW VPPLNVRGGR DAVILLMCVV 881 HPTLVFDITK LLLAIFGPLW ILQASLLKVP YFVRVQGLLR
921 ICALARKIAG GHYVQMAIIK LGALTGTYVY NHLTPLRDWA
961 HNGLRDLAVA VEPVVFSRME TKLITWGADT AACGDIINGL
1001 PVSARRGQEI LLGPADGMVS KGWRLLAPIT AYAQQTRGLL
1041 GCIITSLTGR DKNQVEGEVQ IVSTATQTFL ATCINGVCWT 1081 VYHGAGTRTI ASPKGPVIQM YTNVDQDLVG WPAPQGSRSL
1121 TPCTCGSSDL YLVTRHADVI PVRRRGDSRG SLLSPRPISY
1161 LKGSSGGPLL CPAGHAVGLF RAAVCTRGVA KAVDFIPVEN
1201 LETTMRSPVF TDNSSPPAVP QSFQVAHLHA PTGSGKSTKV
1241 PAAYAAQGYK VLVLNPSVAA TLGFGAYMSK AHGVDPNIRT 1281 GVRTITTGSP ITYSTYGKFL ADGGCSGGAY DIIICDECHS 1321 TDATSILGIG TVLDQAETAG ARLVVLATAT PPGSVTVSHP
1361 NIEEVALSTT GEIPFYGKAI PLEVIKGGRH LIFCHSKKKC
1401 DELAAKLVAL GINAVAYYRG LDVSVIPTSG DVVVVSTDAL
1441 MTGFTGDFDS VIDCNTCVTQ TVDFSLDPTF TIETTTLPQD
1481 AVSRTQRRGR TGRGKPGIYR FVAPGERPSG MFDSSVLCEC
1521 YDAGCAWYEL TPAETTVRLR AYMNTPGLPV CQDHLEFWEG
1561 VFTGLTHIDA HFLSQTKQSG ENFPYLVAYQ ATVCARAQAP
1601 PPSWDQMWKC LIRLKPTLHG PTPLLYRLGA VQNEVTLTHP
1641 ITKYIMTCMS ADLEVVTSTW VLVGGVLAAL AAYCLSTGCV
1681 VIVGRIVLSG KPAIIPDREV LYQEFDEMEE CSQHLPYIEQ
1721 GMMLAEQFKQ KALGLLQTAS RQAEVITPAV QTNWQKLEVF
1761 WAKHMWNFIS GIQYLAGLST LPGNPAIASL MAFTAAVTSP
1801 LTTGQTLLFN ILGGWVAAQL AAPGAATAFV GAGLAGAAIG
1841 SVGLGKVLVD ILAGYGAGVA GALVAFKIMS GEVPSTEDLV
1881 NLLPAILSPG ALVVGVVCAA ILRRHVGPGE GAVQWMNRLI
1921 AFASRGNHVS PTHYVPESDA AARVTAILSS LTVTQLLRRL
1961 HQWISSECTT PCSGSWLRDI WDWICEVLSD FKTWLKAKLM
2001 PQLPGIPFVS CQRGYRGVWR GDGIMHTRCH CGAEITGHVK
2041 NGTMRIVGPR TCRNMWSGTF PINAYTTGPC TPLPAPNYKF
2081 ALWRVSAEEY VEIRRVGDFH YVSGMTTDNL KCPCQIPSPE
2121 FFTELDGVRL HRFAPPCKPL LREEVSFRVG LHEYPVGSQL
2161 PCEPEPDVAV LTSMLTDPSH ITAEAAGRRL ARGSPPSMAS
2201 SSASQLSAPS LKATCTANHD SPDAELIEAN LLWRQEMGGN
2241 ITRVESENKV VILDSFDPLV AEEDEREVSV PAEILRKSRR
2281 FARALPVWAR PDYNPPLVET WKKPDYEPPV VHGCPLPPPR
2321 SPPVPPPRKK RTVVLTESTL STALAELATK SFGSSSTSGI
2361 TGDNTTTSSE PAPSGCPPDS DVESYSSMPP LEGEPGDPDL
2401 SDGSWSTVSS GADTEDVVCC SMSYSWTGAL VTPCAAEEQK
2441 LPINALSNSL LRHHNLVYST TSRSACQRQK KVTFDRLQVL
2481 DSHYQDVLKE VKAAASKVKA NLLSVEEACS LTPPHSAKSK
2521 FGYGAKDVRC HARKAVAHIN SVWKDLLEDS VTPIDTTIMA
2561 KNEVFCVQPE KGGRKPARLI VFPDLGVRVC EKMALYDVVS
2601 KLPLAVMGSS YGFQYSPGQR VEFLVQAWKS KKTPMGFSYD
2641 TRCFDSTVTE SDIRTEEAIY QCCDLDPQAR VAIKSLTERL
2681 YVGGPLTNSR GENCGYRRCR ASGVLTTSCG NTLTCYIKAR
2721 AACRAAGLQD CTMLVCGDDL VVICESAGVQ EDAASLRAFT
2761 EAMTRYSAPP GDPPQPEYDL ELITSCSSNV SVAHDGAGKR
2801 VYYLTRDPTT PLARAAWETA RHTPVNSWLG NIIMFAPTLW
2841 ARMILMTHFF SVLIARDQLE QALNCEIYGA CYSIEPLDLP
2881 PIIQRLHGLS AFSLHSYSPG EINRVAACLR KLGVPPLRAW
2921 RHRARSVRAR LLSRGGRAAI CGKYLFNWAV RTKLKLTPIA
2961 AAGRLDLSGW FTAGYSGGDI YHSVSHARPR WFWFCLLLLA
3001 AGVGIYLLPN R Another example of an HCV polyprotein amino acid sequence that may serve as a source of related peptides can be found in the NCBI database as accession number BAB32872 (gi: 13122262). See ncbi.nlm.nih.gov; Kato et al. J. Med. Virol. 64: 334-339 (2001). This HCV was isolated from a fulminant hepatitis patient, and its amino acid sequence (SEQ ID NO:2) is as follows.
1 MSTNPKPQRK TKRNTNRRPE DVKFPGGGQI VGGVYLLPRR
41 GPRLGVRTTR KTSERSQPRG RRQPIPKDRR STGKAWGKPG
81 RPWPLYGNEG LGWAGWLLSP RGSRPSWGPT DPRHRSRNVG
121 KVIDTLTCGF ADLMGYIPVV GAPLSGAARA VAHGVRVLED
161 GVNYATGNLP GFPFSIFLLA LLSCITVPVS AAQVKNTSSS
201 YMVTNDCSND SITWQLEAAV LHVPGCVPCE RVGNTSRCWV
241 PVSPNMAVRQ PGALTQGLRT HIDMVVMSAT FCSALYVGDL
281 CGGVMLAAQV FIVSPQYHWF VQECNCSIYP GTITGHRMAW
321 DMMMNWSPTA TMILAYVMRV PEVIIDIVSG AHWGVMFGLA
361 YFSMQGAWAK VIVILLLAAG VDAGTTTVGG AVARSTNVIA
401 GVFSHGPQQN IQLINTNGSW HINRTALNCN DSLNTGFLAA
441 LFYTNRFNSS GCPGRLSACR NIEAFRIGWG TLQYEDNVTN
481 PEDMRPYCWH YPPKPCGVVP ARSVCGPVYC FTPSPVVVGT
521 TDRRGVPTYT WGENETDVFL LNSTRPPQGS WFGCTWMNST
561 GFTKTCGAPP CRTRADFNAS TDLLCPTDCF RKHPDATYIK
601 CGSGPWLTPK CLVHYPYRLW HYPCTVNFTI FKIRMYVGGV
641 EHRLTAACNF TRGDRCDLED RDRSQLSPLL HSTTEWAILP
681 CTYSDLPALS TGLLHLHQNI VDVQYMYGLS PAITKYVVRW
721 EWVVLLFLLL ADARVCACLW MLILLGQAEA ALEKLVVLHA
761 ASAANCHGLL YFAIFFVAAW HIRGRVVPLT TYCLTGLWPF
801 CLLLMALPRQ AYAYDAPVHG QIGVGLLILI TLFTLTPGYK
841 TLLGQCLWWL CYLLTLGEAM IQEWVPPMQV RGGRDGIAWA
881 VTIFCPGVVF DITKWLLALL GPAYLLRAAL THVPYFVRAH
921 ALIRVCALVK QLAGGRYVQV ALLALGRWTG TYIYDHLTPM
961 SDWAASGLRD LAVAVEPIIF SPMEKKVIVW GAETAACGDI
1001 LHGLPVSARL GQEILLGPAD GYTSKGWKLL APITAYAQQT
1041 RGLLGAIVVS MTGRDRTEQA GEVQILSTVS QSFLGTTISG
1081 VLWTVYHGAG NKTLAGLRGP VTQMYSSAEG DLVGWPSPPG
1121 TKSLEPCKCG AVDLYLVTRN ADVIPARRRG DKRGALLSPR
1161 PISTLKGSSG GPVLCPRGHV VGLFRAAVCS RGVAKSIDFI
1201 PVETLDVVTR SPTFSDNSTP PAVPQTYQVG YLHAPTGSGK
1241 STKVPVAYAA QGYKVLVLNP SVAATLGFGA YLSKAHGINP
1281 NIRTGVRTVM TGEAITYSTY GKFLADGGCA SGAYDIIICD
1321 ECHAVDATSI LGIGTVLDQA ETAGVRLTVL ATATPPGSVT
1361 TPHPDIEEVG LGREGEIPFY GRAIPLSCIK GGRHLIFCHS
1401 KKKCDELAAA LRGMGLNAVA YYRGLDVSII PAQGDVVVVA 1441 TDALMTGYTG DFDSVIDCNV AVTQAVDFSL DPTFTITTQT
1481 VPQDAVSRSQ RRGRTGRGRQ GTYRYVSTGE RASGMFDSVV
1521 LCECYDAGAA WYDLTPAETT VRLRAYFNTP GLPVCQDHLE
1561 FWEAVFTGLT HIDAHFLSQT KQAGENFAYL VAYQATVCAR
1601 AKAPPPSWDA MWKCLARLKP TLAGPTPLLY RLGPITNEVT
1641 LTHPGTKYIA TCMQADLEVM TSTWVLAGGV LAAVAAYCLA
1681 TGCVSIIGRL HVNQRVVVAP DKEVLYEAFD EMEECASRAA
1721 LIEEGQRIAE MLKSKIQGLL QQASKQAQDI QPAMQASWPK
1761 VEQFWARHMW NFISGIQYLA GLSTLPGNPA VASMMAFSAA
1801 LTSPLSTSTT ILLNIMGGWL ASQIAPPAGA TGFVVSGLVG
1841 AAVGSIGLGK VLVDILAGYG AGISGALVAF KIMSGEKPSM
1881 EDVINLLPGI LSPGALVVGV ICAAILRRHV GPGEGAVQWM
1921 NRLIAFASRG NHVAPTHYVT ESDASQRVTQ LLGSLTITSL
1961 LRRLHNWITE DCPIPCSGSW LRDVWDWVCT ILTDFKNWLT
2001 SKLFPKLPGL PFISCQKGYK GVWAGTGIMT TRCPCGANIS
2041 GNVRLGSMRI TGPKTCMNTW QGTFPINCYT EGQCAPKPPT
2081 NYKTAIWRVA ASEYAEVTQH GSYSYVTGLT TDNLKIPCQL
2121 PSPEFFSWVD GVQIHRFAPT PKPFFRDEVS FCVGLNSYAV
2161 GSQLPCEPEP DADVLRSMLT DPPHITAETA ARRLARGSPP
2201 SEASSSVSQL SAPSLRATCT THSNTYDVDM VDANLLMEGG
2241 VAQTEPESRV PVLDFLEPMA EEESDLEPSI PSECMLPRSG
2281 FPRALPAWAR PDYNPPLVES WRRPDYQPPT VAGCALPPPK
2321 KAPTPPPRRR RTVGLSESTI SEALQQLAIK TFGQPPSSGD
2361 AGSSTGAGAA ESGGPTSPGE PAPSETGSAS SMPPLEGEPG
2401 DPDLESDQVE LQPPPQGGGV APGSGSGSWS TCSEEDDTTV
2441 CCSMSYSWTG ALITPCSPEE EKLPINPLSN SLLRYHNKVY
2481 CTTSKSASQR AKKVTFDRTQ VLDAHYDSVL KDIKLAASKV
2521 SARLLTLEEA CQLTPPHSAR SKYGFGAKEV RSLSGRAVNH
2561 IKSVWKDLLE DPQTPIPTTI MAKNEVFCVD PAKGGKKPAR
2601 LIVYPDLGVR VCEKMALYDI TQKLPQAVMG ASYGFQYSPA
2641 QRVEYLLKAW AEKKDPMGFS YDTRCFDSTV TERDIRTEES
2681 IYQACSLPEE ARTAIHSLTE RLYVGGPMFN SKGQTCGYRR
2721 CRASGVLTTS MGNTITCYVK ALAACKAAGI VAPTMLVCGD
2761 DLVVISESQG TEEDERNLRA FTEAMTRYSA PPGDPPRPEY
2801 DLELITSCSS NVSVALGPRG RRRYYLTRDP TTPLARAAWE
2841 TVRHSPINSW LGNIIQYAPT IWVRMVLMTH FFSILMVQDT
2881 LDQNLNFEMY GSVYSVNPLD LPAIIERLHG LDAFSMHTYS
2921 HHELTRVASA LRKLGAPPLR VWKSRARAVR ASLISRGGKA
2961 AVCGRYLFNW AVKTKLKLTP LPEARLLDLS SWFTVGAGGG
3001 DIFHSVSRAR PRSLLFGLLL LFVGVGLFLL PAR
Another example of an HCV polyprotein amino acid sequence that may served a source of related peptides can be found in the NCBI database as accession number Q9WMX2 (gi: 68565847). See ncbi.nlm.nih.gov. This sequence was obtained from the Conl isolate of HCV. The amino acid sequence (SEQ ID NO:3) is the following.
1 MSTNPKPQRK TKRNTNRRPQ DVKFPGGGQI VGGVYLLPRR 41 GPRLGVRATR KTSERSQPRG RRQPIPKARQ PEGRAWAQPG
81 YPWPLYGNEG LGWAGWLLSP RGSRPSWGPT DPRRRSRNLG
121 KVIDTLTCGF ADLMGYIPLV GAPLGGAARA LAHGVRVLED
161 GVNYATGNLP GCSFSIFLLA LLSCLTIPAS AYEVRNVSGV
201 YHVTNDCSNA SIVYEAADMI MHTPGCVPCV RENNSSRCWV 241 ALTPTLAARN ASVPTTTIRR HVDLLVGAAA LCSAMYVGDL
281 CGSVFLVAQL FTFSPRRHET VQDCNCSIYP GHVTGHRMAW
321 DMMMNWSPTA ALVVSQLLRI PQAVVDMVAG AHWGVLAGLA
361 YYSMVGNWAK VLIVMLLFAG VDGGTYVTGG TMAKNTLGIT
401 SLFSPGSSQK IQLVNTNGSW HINRTALNCN DSLNTGFLAA 441 LFYVHKFNSS GCPERMASCS PIDAFAQGWG PITYNESHSS
481 DQRPYCWHYA PRPCGIVPAA QVCGPVYCFT PSPVVVGTTD
521 RFGVPTYSWG ENETDVLLLN NTRPPQGNWF GCTWMNSTGF
561 TKTCGGPPCN IGGIGNKTLT CPTDCFRKHP EATYTKCGSG 601 PWLTPRCLVH YPYRLWHYPC TVNFTIFKVR MYVGGVEHRL 641 EAACNWTRGE RCNLEDRDRS ELSPLLLSTT EWQVLPCSFT
681 TLPALSTGLI HLHQNVVDVQ YLYGIGSAVV SFAIKWEYVL
721 LLFLLLADAR VCACLWMMLL IAQAEAALEN LVVLNAASVA
761 GAHGILSFLV FFCAAWYIKG RLVPGAAYAL YGVWPLLLLL
801 LALPPRAYAM DREMAASCGG AVFVGLILLT LSPHYKLFLA 841 RLIWWLQYFI TRAEAHLQVW IPPLNVRGGR DAVILLTCAI
881 HPELIFTITK ILLAILGPLM VLQAGITKVP YFVRAHGLIR
921 ACMLVRKVAG GHYVQMALMK LAALTGTYVY DHLTPLRDWA
961 HAGLRDLAVA VEPVVFSDME TKVITWGADT AACGDIILGL
1001 PVSARRGREI HLGPADSLEG QGWRLLAPIT AYSQQTRGLL 1041 GCIITSLTGR DRNQVEGEVQ VVSTATQSFL ATCVNGVCWT
1081 VYHGAGSKTL AGPKGPITQM YTNVDQDLVG WQAPPGARSL
1121 TPCTCGSSDL YLVTRHADVI PVRRRGDSRG SLLSPRPVSY
1161 LKGSSGGPLL CPSGHAVGIF RAAVCTRGVA KAVDFVPVES
1201 METTMRSPVF TDNSSPPAVP QTFQVAHLHA PTGSGKSTKV 1241 PAAYAAQGYK VLVLNPSVAA TLGFGAYMSK AHGIDPNIRT
1281 GVRTITTGAP ITYSTYGKFL ADGGCSGGAY DIIICDECHS
1321 TDSTTILGIG TVLDQAETAG ARLVVLATAT PPGSVTVPHP
1361 NIEEVALSST GEIPFYGKAI PIETIKGGRH LIFCHSKKKC
1401 DELAAKLSGL GLNAVAYYRG LDVSVIPTSG DVIVVATDAL 1441 MTGFTGDFDS VIDCNTCVTQ TVDFSLDPTF TIETTTVPQD
1481 AVSRSQRRGR TGRGRMGIYR FVTPGERPSG MFDSSVLCEC
1521 YDAGCAWYEL TPAETSVRLR AYLNTPGLPV CQDHLEFWES 1561 VFTGLTHIDA HFLSQTKQAG DNFPYLVAYQ ATVCARAQAP
1601 PPSWDQMWKC LIRLKPTLHG PTPLLYRLGA VQNEVTTTHP
1641 ITKYIMACMS ADLEVVTSTW VLVGGVLAAL AAYCLTTGSV
1681 VIVGRIILSG KPAIIPDREV LYREFDEMEE CASHLPYIEQ
1721 GMQLAEQFKQ KAIGLLQTAT KQAEAAAPVV ESKWRTLEAF
1761 WAKHMWNFIS GIQYLAGLST LPGNPAIASL MAFTASITSP
1801 LTTQHTLLFN ILGGWVAAQL APPSAASAFV GAGIAGAAVG
1841 SIGLGKVLVD ILAGYGAGVA GALVAFKVMS GEMPSTEDLV
1881 NLLPAILSPG ALVVGVVCAA ILRRHVGPGE GAVQWMNRLI
1921 AFASRGNHVS PTHYVPESDA AARVTQILSS LTITQLLKRL
1961 HQWINEDCST PCSGSWLRDV WDWICTVLTD FKTWLQSKLL
2001 PRLPGVPFFS CQRGYKGVWR GDGIMQTTCP CGAQITGHVK
2041 NGSMRIVGPR TCSNTWHGTF PINAYTTGPC TPSPAPNYSR
2081 ALWRVAAEEY VEVTRVGDFH YVTGMTTDNV KCPCQVPAPE
2121 FFTEVDGVRL HRYAPACKPL LREEVTFLVG LNQYLVGSQL
2161 PCEPEPDVAV LTSMLTDPSH ITAETAKRRL ARGSPPSLAS
2201 SSASQLSAPS LKATCTTRHD SPDADLIEAN LLWRQEMGGN
2241 ITRVESENKV VILDSFEPLQ AEEDEREVSV PAEILRRSRK
2281 FPRAMPIWAR PDYNPPLLES WKDPDYVPPV VHGCPLPPAK
2321 APPIPPPRRK RTVVLSESTV SSALAELATK TFGSSESSAV
2361 DSGTATASPD QPSDDGDAGS DVESYSSMPP LEGEPGDPDL
2401 SDGSWSTVSE EASEDVVCCS MSYTWTGALI TPCAAEETKL
2441 PINALSNSLL RHHNLVYATT SRSASLRQKK VTFDRLQVLD
2481 DHYRDVLKEM KAKASTVKAK LLSVEEACKL TPPHSARSKF
2521 GYGAKDVRNL SSKAVNHIRS VWKDLLEDTE TPIDTTIMAK
2561 NEVFCVQPEK GGRKPARLIV FPDLGVRVCE KMALYDVVST
2601 LPQAVMGSSY GFQYSPGQRV EFLVNAWKAK KCPMGFAYDT
2641 RCFDSTVTEN DIRVEESIYQ CCDLAPEARQ AIRSLTERLY
2681 IGGPLTNSKG QNCGYRRCRA SGVLTTSCGN TLTCYLKAAA
2721 ACRAAKLQDC TMLVCGDDLV VICESAGTQE DEASLRAFTE
2761 AMTRYSAPPG DPPKPEYDLE LITSCSSNVS VAHDASGKRV
2801 YYLTRDPTTP LARAAWETAR HTPVNSWLGN IIMYAPTLWA
2841 RMILMTHFFS ILLAQEQLEK ALDCQIYGAC YSIEPLDLPQ
2881 IIQRLHGLSA FSLHSYSPGE INRVASCLRK LGVPPLRVWR
2921 HRARSVRARL LSQGGRAATC GKYLFNWAVR TKLKLTPIPA
2961 ASQLDLSSWF VAGYSGGDIY HSLSRARPRW FMWCLLLLSV
3001 GVGIYLLPNR
Additional examples of HCV polyprotein sequences are available and can serve a source of other anti -viral peptides. For example a Taiwan isolate of hepatitis C virus is available in the NCBI database at accession number P29846 (gi: 266821). See ncbi.nlm.nih.gov. In infected cells, the HCV polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. The generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is affected by two viral proteases. The first one, as yet poorly characterized, cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3- NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protease with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
The HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication. The first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein (C. Lin et al., J. Virol. 68, 8147-8157 (1994)).
HCV has three structural proteins, the N-terminal nucleocapsid protein (termed "core") and two envelope glycoproteins, "El" (also known as E) and "E2" (also known as E2/NS1). See, Houghton et al. (1991) Hepatology 14:381-388, for a discussion of HCV proteins, including El and E2. The El protein is detected as a 32-35 kDa species and is converted into a single endo H-sensitive band of approximately 18 Kda. By contrast, E2 displays a complex pattern upon immunoprecipitation consistent with the generation of multiple species (Grakoui et al. (1993) J Virol. 67:1385-1395; Tomei et al. (1993) J. Virol. 67:4017-4026). The HCV envelope glycoproteins El and E2 form a stable complex that is co- immunoprecipitable (Grakoui et al. (1993) J. Virol. 67:1385-1395; Lanford et al. (1993) Virology 197:225-235; Ralston et al. (1993) J. Virol. 67:6753-6761).
Antiviral Peptides One aspect of the invention is an antiviral peptide. An antiviral peptide is a peptide that can prevent or treat infection of HIV, measles, RSV or a virus of the Flaviviridae family, herein a peptide inhibitor or a peptide of the invention.
"HIV" is Human Immunodeficiency Virus, a virus that causes immunodeficiency by attacking CD4+ cells in the body. The term "HIV," as used herein, includes any HIV, including all groups and subtypes (clades) of HIV-I and HIV-2. In some embodiments, the HIV is HIV-I .
Measles, also known as rubeola, is a disease caused by a virus, specifically a paramyxovirus of the genus Morbillivirus. Measles is spread through respiration (contact with fluids from an infected person's nose and mouth, either directly or through aerosol transmission), and is highly contagious — 90% of people without immunity sharing a house with an infected person will catch it.
Respiratory syncytial virus (RSV) is a spherical or pleomorphic enveloped virus (100-350 nm) with single-stranded, negative sense linear RNA. Two major groups of strains of human RSV exist, groups A and B. The strains of group A predominate. The invention is directed to treating and preventing both strains of
RSV. RSV causes cold-like symptoms in adults and older children. However, it can cause serious problems in young babies, including pneumonia and severe breathing problems. In rare cases it can lead to death.
According to the invention, diverse types of HIV can effectively be inactivated or infection by diverse types of HIV can be treated and/or prevented because evidence provided herein indicates that the present peptides disrupt or lyse viruses without injuring mammalian cells. Accordingly, the process by which the present peptides inhibit HIV infection is not particularly strain, clade or type specific. Thus, the present peptides exhibit activity against a variety of HIV types. Thus, one aspect of the invention is directed to treating and inhibiting infection by any HIV clade, type, subtype or strain. The invention is also directed to inactivating HIV of any clade, type, subtype or strain. Guidance on the different clades can be found in HIV Sequence Compendium 2002 Kuiken C, Foley B, Freed E, Hahn B, Marx P, McCutchan F, Mellors J, Wolinsky S, and Korber B, editors. Published by the Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, LA-UR number 03-3564, incorporated herein by reference. In particular, consensus sequence data for HIV-I clades A, B, C and D and a number of isolates can be found on pages 490 to 550; consensus sequence data for HIV-2 clades A, B, C and D and a number of isolates can be found one pages 554 to 578.
The HIV that can be inactivated, treated or inhibited by the present compositions or methods is generally HIV-I or HIV-2, preferably HIV-I . The HIV that can be inactivated, treated or inhibited according to the invention is generally of clade A, B, C or D. for example HIV-I However, the inventive compositions and methods are also useful for treating and preventing infections by the HIV-I. group M or O subtypes and sub-subtypes. The members of group M are broken out into nine equidistant phylogenetic subtypes. These are labeled Al, A2, B, C, D, Fl, F2, G, H, J and K. The sequences within any one-subtype or sub-subtype share more genetically than they do with sequences from other subtypes throughout their genomes.
A Flaviviridae is a spherical, enveloped virus having a linear, single- stranded RNA genome of positive polarity. The family Flaviviridae includes the genera Flavivirus, Hepacivirus and Pestivirus. The invention contemplates treatment of Flaviviridae infections, including infections caused by any virus from any of the genera Flavivirus, Hepacivirus and Pestivirus, as well as viruses of the unassigned genera of Flaviviridae. For example, the present peptides can be used to treat infections caused by the following viruses of the Flavivirus genus: Tick-borne encephalitis, Central European encephalitis, Far Eastern encephalitis, Rio Bravo, Japanese encephalitis, Kunjin, Murray Valley encephalitis, St Louis encephalitis, West Nile encephalitis, Tyulenly, Ntaya, Uganda S, Dengue type 1, Dengue type 2, Dengue type 3, Dengue type 4, Modoc, and Yellow Fever. Moreover, the present peptides can be used to treat infections caused by the following viruses of the Pestivirus genus: Bovine viral diarrhea virus 1, Bovine viral diarrhea virus 2, Hog cholera (classical swine fever virus), and Border disease virus. In addition, the present peptides can be used to treat infections caused by hepatitis C virus, which is classified in the Hepacivirus genus. Viruses of the unassigned genera of Flaviviridae, whose infections can also be treated with the peptides of the invention include: GB virus- A, GB virus-B and GB virus-C. To determine the level of antiviral activity a peptide has against a particular type of HIV, measles, RSV or member of the Flaviviridae family, and an appropriate dosage for such a peptide, methods known in the art, including, without limitation, those described herein can be used. Viral infection in the presence or absence of a peptide of the invention can be evaluated, for example, by determining intracellular viral RNA levels, detection of viral protein or the number of viral foci by immunoassays using antibody against viral proteins as described herein. Viral inactivation can also be evaluated using these methods as described herein. The antiviral activity of a peptide can also be determined using the liposome release assay as exemplified herein. A peptide has antiviral activity if can prevent or reduce viral infection or inactivate the virus by any amount, for example, by 2 fold or more than 2 fold. For example, a peptide of the invention can prevent or reduce viral infection by 2-5 fold, 5-10 fold, or more than 10 fold. As illustrated hereinbelow, many of the peptides listed in Table 3 can inhibit viral infection by more than ten-fold, including, for example, peptides with SEQ ID NO:6, 8, 12, 13, 14, 24, 27, 30, 32, 43, 44, 47, 48 and 53. Other peptides listed in Table 3 can inhibit viral infection by five-fold to ten-fold, including peptides with SEQ ID NO:21, 23, 28 and 37. The remainder of the peptides inhibit viral infection by at least two-fold and some of the remaining peptides inhibit viral infection by up to about five-fold. These peptides exhibit such inhibition of viral infection at concentrations of nanomolar and low micromolar levels.
A peptide of the invention is a polymer of α-amino acids linked by amide bonds between the α-amino and α-carboxyl groups. Thus, the term "amino acid," as used herein, refers to an α-amino acid. The amino acids included in the peptides of the invention can be L-amino acids or D-amino acids. Moreover, the amino acids used in the peptides of the invention can be naturally-occurring and non-naturally occurring amino acids. Thus, a peptide of the present invention can be made from genetically encoded amino acids, naturally occurring non-genetically encoded amino acids, or synthetic amino acids. The amino acid notations used herein for the twenty genetically encoded L-amino acids and some examples of non-encoded amino acids are provided in Table 1.
Table 1
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
A peptide of the invention will include at least 8 to about 50 amino acid residues, usually about 14 to 40 amino acids, more usually fewer than about 35 or fewer than about 25 amino acids in length. A peptide of the invention will be as small as possible, while still maintaining substantially all of the activity of a larger peptide. Thus, a peptide of the invention may be 8, 9, 10, 1 1, 12 or 13 amino acids in length. Moreover, the length of the peptide selected by one of skilled in the art may relate to the stability and/or sequence of the peptide. Thus, for example, while peptide 1 (SEQ ID NO:43) exhibits optimal antiviral activity when it has about 18 amino acids, and truncations from the C-terminal end do not eliminate its antiviral activity, until five or so amino acids are deleted. Nonetheless, peptides with sequences different from SEQ ID NO:43 may exhibit optimal activity when they are longer than 18 amino acids or shorter than 13 amino acids. This may be due to sequence differences that stabilize or modify the secondary structure of the peptide. In addition, the peptides can be derivatized with agents that enhance the stability and activity of the peptides. For example, peptides can be modified by attachment of a dansyl moiety or by incorporation of non-naturally occurring amino acids so as to improve the activity and/or conformation stability of the peptides. Use of non- natural amino acids and dansyl moieties can also confer resistance to protease cleavage. It may also be desirable in certain instances to join two or more peptides together in one peptide structure.
Moreover, peptides from other HCV strains exhibit excellent anti-viral activity including: genotype IB (SWLRDVWD WICTVLTDFK, SEQ ID NO:80); genotype 2A (S WLRDV WDWVCTILTDFK, SEQ ID NO:79); genotype 3A (DWLRIIWDWVCSVVSDFK, SEQ ID NO: 123); genotype 4A (SWLWEVWDWVLHVLSDFK, SEQ ID NO: 124); genotype 5 A (TWLRAIWDWVCTALTDFK, SEQ ID NO: 125); and genotype 6A (SWLRDVWDWVCTVLSDFK, SEQ ID NO: 126) all exhibit anti-viral activity. The invention is also directed to peptidomimetics of the antiviral peptides of the invention. Peptidomimetics are structurally similar to peptides having peptide bonds, but have one or more peptide linkages optionally replaced by a linkage such as, -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, by methods known in the art. Thus, a peptidomimetic is a peptide analog, such as those commonly used in the pharmaceutical industry as non-peptide drugs, that has properties analogous to those of the template peptide. (Fauchere, J., Adv. Drug Res., 15: 29 (1986) and Evans et al., J. Med. Chem., 30:1229 (1987)). Advantages of peptide mimetics over natural peptide embodiments may include more economical production, greater chemical stability, altered specificity and enhanced pharmacological properties such as half- life, absorption, potency and efficacy.
In some embodiments, the amino acid residues of a peptide of the invention can form an amphipathic α-helical structure in solution. The term "α-helix" refers to a right-handed coiled conformation. In a polypeptide, the α-helical structure results from hydrogen bonding between the backbone N-H group of one amino acid and the backbone C=O group of an amino acid four residues earlier. An α-helix has 3.6 amino acid residues per turn. Certain amino acid residues tend to contribute to the formation of α-helical structures in polypeptides, for example, alanine, cysteine, leucine, methionine, glutamate, glutamine, histidine and lysine.
However, formation of an α-helix also depends upon the solution, pH and temperature in which a peptide resides. Thus, according to the invention, the inventive peptides are α-helical in aqueous solution. The aqueous solution can, for example, have a physiological pH, and/or physiological salts. In general, the amphipathic α-helical structures of the present peptides are detected at moderate temperatures, such as at about 4 °C to about 50 °C, or at about room temperature to about body temperature. Thus, for example, the peptides α-helical structure under physiological temperatures and physiological pH values.
An α-helical structure can be detected using methods known in the art including, without limitation, circular dichroism spectroscopy (CD), nuclear magnetic resonance (NMR), crystal structure determination and optical rotary dispersion (ORD). As used herein, the phrase "amphipathic" means that the α-helical peptides have a hydrophilic (or polar) face and a hydrophobic (or non-polar) face, wherein such a "face" refers to a longitudinal surface of the peptide. A helical wheel is apparent when an α-helical peptide is viewed down its longitudinal axis (e.g. as shown in FIG. 6A), one side of the helical wheel that circles this longitudinal axis is composed of hydrophilic (or polar) residues and the other side of the helical wheel is composed of hydrophobic (or nonpolar) residues. Thus, when the peptides of the invention lie on a hydrophilic surface, the hydrophilic face of the peptide will tend to be in contact with the hydrophilic surface. One the other hand, when confronted with a hydrophobic surface, the hydrophobic face of the peptides of the invention will tend to be in contact with the hydrophobic surface.
In an amphipathic α-helical peptide, the hydrophilic and hydrophobic faces of the α-helix can therefore be identified based on the nature of the amino acids present. The hydrophilic face of an α-helix will consist of a larger number of hydrophilic, charged and/or polar amino acids than is present on the hydrophobic face. The hydrophobic face of an amphipathic α-helix consists of hydrophobic and/or non-polar amino acids that facilitate insertion into lipid bilayers. The hydrophobic face may have one or more hydrophilic or polar amino acid as long as a sufficient number of non-polar amino acids are present that enable membrane insertion. In general, a majority of the amino acid residues on the hydrophilic face of the α-helix are charged or otherwise polar amino acids, while a majority of the amino acid residues on the hydrophobic face of the α-helix are non-polar amino acids. Thus in many embodiments, the hydrophilic face of the α-helix consists of charged or otherwise polar amino acids, while the hydrophobic face of the α-helix consists of non-polar amino acid residues. See for example, the helical wheel of the peptide 1 (SEQ ID NO:43), which is shown in FIG. 6A.
Whether any given peptide sequence has a sufficient number of non-polar amino acids to enable membrane insertion can be determined using methods that are well known in the art, including without limitation, methods involving liposomal dye release described in the examples herein. In addition, whether a peptide has an amphipathic α-helical structure can be determined using software available on the internet such as http://cti.itc.virginia.edu/~cmg/Demo/wheel/wheelApp.html (last visited Aug. 15, 2006) and http://www.bioinf.man.ac.uk/~gibson/ HelixDraw/helixdraw.html (last visited Aug. 15, 2006). A schematic diagram illustrating the amphipathic α-helical structure of the peptide of SEQ ID NO: 43 is shown in FIG. 6A.
Examples of peptides of the invention can be found in Table 3. Other peptides of the invention include those peptides having conservative amino acid substitutions compared to those shown in Table 3. Peptides of the invention also include those having amino acid compositions that resemble the peptides shown in Table 3. These include peptides that have sequences of SEQ ID NO: 96, 97 and 98, which are shown in Table 11. These sequences correspond to the reverse variant of SEQ ID NO: 43 or they constitute a "scrambled" variant of SEQ ID NO: 43. A retro or reverse variant of a peptide such as SEQ ID NO 43 will have an amino acid composition that resembles that of the original peptide (SEQ ID NO: 43), but the amino acid sequence will be the reverse of that of the original peptide. The scrambled variant of a peptide such as SEQ ID NO: 43 will also have an amino acid composition that resembles the original peptide (SEQ ID NO: 43), but the order of the amino acid will be scrambled or mixed up without altering the relative positions of the hydrophobic and hydrophilic residues. Thus, a peptide that is a "hydrophobic scrambled" variant of SEQ ID NO: 43 will have the same amino acid composition as that of SEQ ID NO: 43. However, the order of the hydrophobic amino acid residues will be altered without altering the relative positions of hydrophobic and hydrophilic residues within the sequence such that the amphipathicity of the variant peptide resembles that of the original peptide. Similarly, a "hydrophilic scrambled" variant of SEQ ID NO: 43 will have the same amino acid composition as that of SEQ ID NO: 43, but the order of the hydrophilic amino acid residues will be altered without altering the relative positions of hydrophobic and hydrophilic residues within the sequence such that the amphipathicity of the variant peptide resembles that of the original peptide. In general, the term "scrambling" or "scrambled," with respect to a hydrophilic (polar) amino acid, is used to indicate that while the positions of each hydrophilic (polar) amino acid are held constant, any other hydrophilic (polar) amino acid can be placed at that position. Similarly, the term "scrambling" or "scrambled," with respect to a hydrophobic (nonpolar) amino acid, is used to indicate that while the positions of each hydrophobic (nonpolar) amino acid are held constant, any other hydrophobic (nonpolar) amino acid can be placed at that position. Thus, a peptide of the invention will have an amino acid sequence that is identical to the sequences shown in Table 3, as well as variants of such sequences. Such variants can result from one or more amino acid truncations, conservative substitutions, scrambling of just the hydrophilic amino acids, scrambling of just the hydrophobic residues within a sequence, scrambling of both hydrophilic and hydrophobic amino acids, replacement of naturally occurring amino acids with non- naturally occurring amino acids or other modifications such as dansylation. Such variant peptides are further described in the next section.
Peptide Homologues and Variants
The invention embraces numerous peptide homologues and variants. For example, while the present anti-viral peptides were originally isolated from hepatitis C genotype 1 a (H77), similar peptides from other HCV strains exhibit excellent anti-viral activity including: genotype 1 B (S WLRDVWD WICTVLTDFK, SEQ ID NO:80); genotype 2A (SWLRDVWD WVCTILTDFK, SEQ ID NO:79); genotype 3A (D WLRIIWD WVCSVVSDFK, SEQ ID NO: 123); genotype 4A (SWLWEVWDWVLHVLSDFK, SEQ ID NO: 124); genotype 5 A (TWLRAI WD WVCTALTDFK, SEQ ID NO: 125); and genotype 6A (S WLRDVWD WVCTVLSDFK, SEQ ID NO: 126) all exhibit antiviral activity.
Thus, invention is also directed to peptide homologues of the active peptides disclosed herein. A peptide homologue is a peptidyl sequence from an HCV isolate other than the H77 isolate having SEQ ID NO:1. Thus, a peptide of the invention can be a homologue of a peptide with an amino acid sequence of any of SEQ ID NO:4-61. Thus, for example, one peptide homologue of the invention has SEQ ID NO:62, which is a homologue of peptide SEQ ID NO:6.
LYGNEGLGWAGWLLSPRG (SEQ ID NO:62). The sequence of peptide inhibitor SEQ ID NO:62 is found in HCV polyprotein sequences SEQ ID NO:2 and 3.
Another peptide inhibitor homologue of the invention has SEQ ID NO: 63 or 64, which are homologues of peptide SEQ ID NO:8. IFLLALLSCITVPVSAAQ (SEQ ID NO:63);
IFLLALLSCLTIPASAYE (SEQ ID NO:64).
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
Another peptide inhibitor homologue of the invention has SEQ ID NO: 65 or 66, which are homologues of peptide SEQ ID NO: 12.
MSATFCSALYVGDLCGGV (SEQ ID NO:65) GAAALCSAMYVGDLCGSV (SEQ ID NO:66)
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3. Another peptide inhibitor homologue of the invention has SEQ ID NO:67 or
68, which are homologues of peptide SEQ ID NO: 13.
ALYVGDLCGGVMLAAQVF (SEQ ID NO:67) AMYVGDLCGSVFLVAQLF (SEQ ID NO:68)
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
Another peptide inhibitor homologue of the invention has SEQ ID NO: 69 or 70, which are homologues of peptide SEQ ID NO: 14.
IIDIVSGAHWGVMFGLAY (SEQ ID NO:69) VVDMVAGAHWGVLAGLAY (SEQ ID NO:70) The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
Another peptide inhibitor homologue of the invention has SEQ ID NO:71 or 72, which are homologues of peptide SEQ ID NO:24.
VDVQYMYGLSPAITKYVV (SEQ ID NO:71) YLYGIGSAVVSFAIKWEY (SEQ ID NO:72)
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
Another peptide inhibitor homologue of the invention has SEQ ID NO: 73 or 74, which are homologues of peptide SEQ ID NO:27.
WMLILLGQAEAALEKLVV (SEQ ID NO:73) WMMLLIAQAEAALENLVV (SEQ ID NO:74)
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3. Another peptide inhibitor homologue of the invention has SEQ ID NO: 75 or
76, which are homologues of peptide SEQ ID NO:30.
GVVFDITKWLLALLGPAY (SEQ ID NO:75); ELIFTITKILLAILGPLM (SEQ ID NO:76).
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
In another embodiment, the peptide inhibitor homologue has SEQ ID NO:77 or 78, which are homologues of peptide SEQ ID NO:32.
VSQSFLGTTISGVLWTVY (SEQ ID NO:77); ATQSFLATCVNGVCWTVY (SEQ ID NO:78). The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
In another embodiment, the peptide inhibitor homologue has SEQ ID NO: 79 or 80, which are homologues of peptide SEQ ID NO:43.
SWLRDVWDWVCTILTDFK (SEQ ID NO:79); SWLRDVWDWICTVLTDFK (SEQ ID NO:80).
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
In another embodiment, the peptide inhibitor homologue has SEQ ID NO: 81 or 82, which are homologues of peptide SEQ ID NO:44. DWVCTILTDFKNWLTSKL (SEQ ID NO:81); DWICTVLTDFKTWLQSKL (SEQ ID NO:82).
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3. In another embodiment, the peptide inhibitor homologue has SEQ ID NO:83 or 84, which are homologues of peptide SEQ ID NO:47.
ASEDVYCCSMSYTWT (SEQ ID NO:83); EDDTTVCCSMSYSW (SEQ ID NO:84).
The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
In another embodiment, the peptide inhibitor homologue has SEQ ID NO:85 or 86, which are homologues of peptide SEQ ID NO:53.
CTMLVCGDDLVVICESAG (SEQ ID NO:85); PTMLVCG DDLVVISESQG (SEQ ID NO:86). The sequences of these peptide inhibitors are found in HCV polyprotein sequences SEQ ID NO:2 and 3.
A peptide variant is any peptide having an amino acid sequence that is not identical to a segment in the polyprotein sequence of a HCV isolate. Thus, a peptide of the invention can have a variant sequence that results from conservative amino acid substitutions. Amino acids that are substitutable for each other generally reside within similar classes or subclasses. As known to one of skill in the art, amino acids can be placed into different classes depending primarily upon the chemical and physical properties of the amino acid side chain. For example, some amino acids are generally considered to be hydrophilic or polar amino acids and others are considered to be hydrophobic or nonpolar amino acids. Polar amino acids include amino acids having acidic, basic or hydrophilic side chains and nonpolar amino acids include amino acids having aromatic or hydrophobic side chains. Nonpolar amino acids may be further subdivided to include, among others, aliphatic amino acids. The definitions of the classes of amino acids as used herein are as follows.
"Nonpolar Amino Acid" refers to an amino acid having a side chain that is uncharged at physiological pH, that is not polar and that is generally repelled by aqueous solution. Examples of genetically encoded hydrophobic amino acids include Ala, He, Leu, Met, Trp, Tyr and VaI. Examples of non-genetically encoded nonpolar amino acids include t-BuA, Cha and NIe.
"Aromatic Amino Acid" refers to a nonpolar amino acid having a side chain containing at least one ring having a conjugated δ-electron system (aromatic group). The aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others. Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan. Commonly encountered non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, a-2-thienylalanine, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.
"Aliphatic Amino Acid" refers to a nonpolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids include Ala, Leu, VaI and He. Examples of non-encoded aliphatic amino acids include NIe.
"Polar Amino Acid" refers to a hydrophilic amino acid having a side chain that is charged or uncharged at physiological pH and that has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids are generally hydrophilic, meaning that they have an amino acid having a side chain that is attracted by aqueous solution. Examples of genetically encoded polar amino acids include asparagine, cysteine, glutamine, lysine and serine. Examples of non-genetically encoded polar amino acids include citrulline, homocysteine, N-acetyl lysine and methionine sulfoxide. "Acidic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
"Basic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Examples of genetically encoded basic amino acids include arginine, lysine and histidine. Examples of non-genetically encoded basic amino acids include amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine. "Ionizable Amino Acid" refers to an amino acid that can be charged at a physiological pH. Such ionizable amino acids include acidic and basic amino acids, for example, D-aspartic acid, D-glutamic acid, D-histidine, D-arginine, D-lysine, D- hydroxylysine, D-ornithine, L-aspartic acid, L-glutamic acid, L-histidine, L-arginine, L-lysine, L-hydroxylysine or L-ornithine.
As will be appreciated by those having skill in the art, the above classifications are not absolute. Several amino acids exhibit more than one characteristic property, and can therefore be included in more than one category. For example, tyrosine has both a nonpolar aromatic ring and a polar hydroxyl group. Thus, tyrosine has several characteristics that could be described as nonpolar, aromatic and polar. However, the nonpolar ring is dominant and so tyrosine is generally considered to be hydrophobic. Similarly, in addition to being able to form disulfide linkages, cysteine also has nonpolar character. Thus, while not strictly classified as a hydrophobic or nonpolar amino acid, in many instances cysteine can be used to confer hydrophobicity or nonpolarity to a peptide.
The classifications of the above-described genetically encoded and non- encoded amino acids are summarized in Table 2, below. It is to be understood that Table 2 is for illustrative purposes only and does not purport to be an exhaustive list of amino acid residues that may comprise the peptides and peptide analogues described herein. Other amino acid residues that are useful for making the peptides described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein. Another source of amino acid residues is provided by the website of RSP Amino Acids Analogues, Inc. (www.amino-acids.com). Amino acids not specifically mentioned herein can be conveniently classified into the above- described categories on the basis of known behavior and/or their characteristic chemical and/or physical properties as compared with amino acids specifically identified.
TABLE 2
Figure imgf000064_0001
Figure imgf000065_0001
In some embodiments, hydrophilic or polar amino acids contemplated by the present invention include, for example, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine, hydroxylysine, ornithine, serine, threonine, and structurally related amino acids. In one embodiment the polar amino is an ionizable amino acid such as arginine, aspartic acid, glutamic acid, histidine, hydroxylysine, lysine, or ornithine.
Examples of hydrophobic or nonpolar amino acid residues that can be utilized include, for example, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tryptophan, tyrosine and the like.
In addition, the amino acid sequence of a peptide can be modified so as to result in a peptide variant that includes the substitution of at least one amino acid residue in the peptide for another amino acid residue, including substitutions that utilize the D rather than L form. One or more of the residues of the peptide can be exchanged for another, to alter, enhance or preserve the biological activity of the peptide. Such a variant can have, for example, at least about 10% of the biological activity of the corresponding non-variant peptide. Conservative amino acid substitutions are often utilized, i.e., substitutions of amino acids with similar chemical and physical properties, as described above. Hence, for example, conservative amino acids substitutions involve exchanging aspartic acid for glutamic acid; exchanging lysine for arginine or histidine; exchanging one nonpolar amino acid (alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine) for another; and exchanging one polar amino acid (aspartic acid, asparagine, glutamic acid, glutamine, glycine, serine, threonine, etc.) for another. When substitutions are introduced, the variants can be tested to confirm or determine their levels of biological activity.
For example, in some embodiments, the peptides of the invention can have a sequence that includes any one of formulae IX-XIII:
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8- IX
Xaa9-Xaa10-Xaa11-Xaai2-Xaai3-Xaai4 (SEQ ID NO: 1 12)
Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9- X XaaiO-Xaan-Xaa12-Xaa,3-Xaai4- Xaai5 (SEQ ID NO: 1 13)
Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaai 0- XI
Xaai ,-Xaa12-Xaai3-Xaai4- Xaai5-Xaai6 (SEQ ID NO: 114) Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaaio-Xaan- XII
Xaai2-Xaai3-Xaa14- Xaa]5-Xaai6-Xaai7 (SEQ ID NO: 115)
Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaai O-Xaai i - XIII
Xaa,2-Xaai3-Xaai4- Xaai5-Xaai6-Xaai7-Xaai8 (SEQ ID NO: 116) wherein:
Xaai, Xaa4, Xaa5, Xaa8, Xaan, Xaaj2, Xaai5, Xaai 6 and Xaai8 are polar amino acids; and
Xaa2, Xaa3, Xaaβ, Xaa7, Xaa9, Xaaio, Xaai3, Xaaπ, and Xaa^ are nonpolar amino acids. In other embodiments, the present peptides can have additional peptidyl sequences at either the N-terminus or the C-terminus. Thus, for example, the invention provides a fusion peptide formed by attaching a 14 amino acid peptide (the N-terminal peptide) to the N-terminus of a peptide of any of formulae IX to XIII. The 14 amino acid N-terminal peptide has the structure: Rx-Ry-Ry-Rx-Ry- Ry-Rx-Rx-Ry-Ry-Rx-Rx-Ry-Rx (SEQ ID NO: 1 17), wherein each Rx is separately a polar amino acid, and each Ry is separately a nonpolar amino acid.
The invention also provides a fusion peptide formed by attaching a 12 amino acid peptide (the C-terminal peptide) to the C-terminus of a peptide of formula XIII. The resulting fusion peptide has the structure of formulae XIV:
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaaio-Xaa1i- Xaa12-Xaa13-Xaai4- Xaai5-Xaai6-Xaai7-Xaai8 -Xaai9-Xaa2o-Xaa2i-Xaa22- Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa3o (SEQ ID NO: 118), XIV wherein:
Xaai, Xaa^ Xaa5, Xaa8, Xaan, Xaai2, Xaa] 5, Xaa)6, Xaais, Xaai9, Xaa22, Xaa23, Xaa26, Xaa29, and Xaa30 are separately each a polar amino acid; and
Xaa2, Xaa3, Xaaό, Xaa7, Xaa9, Xaaio, Xaan, Xaai4, Xaai7, Xaa2o, Xaa2i, Xaa24, Xaa25, Xaa27, and Xaa28 are separately each a nonpolar amino acid. The invention also provides a fusion peptide having a sequence that corresponds to the 14 amino acid N-terminal peptide of SEQ ID NO: 117 attached by a peptide bond to the N-terminus of a peptide of formula XIV.
In another embodiment, a peptide of the invention is a peptide comprising at least 14 contiguous amino acids of any of the above described peptides. A peptide variant can also result from "scrambling" of the hydrophilic and/or hydrophobic residues within a sequence as long as the amphipathic α-helical secondary structure of the peptide in solution is maintained.
Methods of Making a Peptide of the Invention In the context of the present invention, an "isolated" peptide is a peptide that exists apart from its native environment and is therefore not a product of nature. An isolated peptide may exist in a purified form or may exist in a non-native environment such as, for example, in a cell or in a composition with a solvent that may contain other active or inactive ingredients. In one embodiment, an "isolated" peptide free of at least some of sequences that naturally flank the peptide (i.e., sequences located at the N-terminal and C-terminal ends of the peptide) in the protein from which the peptide was originally derived. A "purified" peptide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Thus, a purified peptide preparation is at least 50 %, at least 60 %, at least 70 %, at least 80 % or at least 90 % by weight peptide. Purity can be determined using methods known in the art, including, without limitation, methods utilizing chromatography or polyacrylamide gel electrophoresis.
The present peptides or variants thereof, can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by enzyme catalyzed peptide synthesis or with the aid of recombinant DNA technology. Solid phase peptide synthetic method is an established and widely used method, which is described in references such as the following: Stewart et al.. Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc. 85_ 2149 (1963); Meienhofer in "Hormonal Proteins and Peptides," ed.; CH. Li, Vol.2 (Academic Press, 1973), pp.48-267; and Bavaay and Merrifield, "The Peptides," eds. E. Gross and F. Meienhofer, Vol.2 (Academic Press, 1980) pp.3-285. These peptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; ligand affinity chromatography; or crystallization or precipitation from non-polar solvent or nonpolar/polar solvent mixtures. Purification by crystallization or precipitation is preferred.
Peptides of the invention can be cyclic peptides so long as they retain antiviral activity. Such cyclic peptides are generated from linear peptides typically by covalently joining the amino terminus to the terminal carboxylate. To insure that only the termini are joined amino and carboxylate side chains can be protected with commercially available protecting groups. In some embodiments, one of skill in the art may choose to cyclize peptide side chains to one of the amino or carboxylate termini, or to another amino acid side chain. In this case, protecting groups can again be used to guide the cyclization reaction as desired.
Cyclization of peptides can be performed using available procedures. For example, cyclization can be performed in dimethylformamide at a peptide concentration of 1-5 mM using a mixture of benzotriazole-1-yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP, Novabiochem) (5 eq. with respect to crude peptide) and N,N-diisopropylethylamine (DIEA, Fisher) (40 eq.). The amount of DIEA is adjusted to achieve an apparent pH 9-10. The reaction can be followed by any convenient means, for example, by MALDI-MS and/or HPLC.
N-acyl derivatives of an amino group of the peptide or peptide variants may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N-acylation and O- acylation may be carried out together, if desired. Salts of carboxyl groups of a peptide or peptide variant of the invention may be prepared in the usual manner by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
Acid addition salts of the peptide or variant peptide, or of amino residues of the peptide or variant peptide, may be prepared by contacting the peptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the peptides may also be prepared by any of the usual methods known in the art.
Methods of Use Peptides of the invention can be employed to prevent, treat or otherwise ameliorate infection by any human immunodeficiency virus (HIV), measles virus, or respiratory syncytial virus as well, as any virus from the Flaviviridae family. In addition, peptides of the invention can be used to inactivate any of these viruses, in vivo, in vitro or ex vivo. A peptide of the invention can be used to prevent, treat or otherwise ameliorate infection by any HIV, measles or RSV virus and any virus from the Flaviviridae family, as well as prevent, treat or otherwise ameliorate the associated disease conditions. Thus, the present peptides can be used as a therapeutic agent to prevent sexual transmission of HIV, to treat and inhibit HIV disease progression, to inhibit HIV multiplication, to reduce, HIV viral load, to promote CD4+ responses against HIV, to promote CD8+ responses against HIV, to cure HIV infection, to increase the safety of blood and blood products used in transfusions, and to increase the safety of clinical laboratory samples. The peptides of the invention can also be used to inactivate viruses in bodily fluid samples and tissue samples, and to inhibit or prevent infection amongst people who must handle these samples.
Similarly, a peptide of the invention can be employed to prevent, treat or otherwise ameliorate infection by a virus of the Flaviviridae family, which includes, without limitation, viruses in the genera Flavivirus, Pestivirus, and Hepacivirus, as described above. Members of the Flavivirus genus include viruses that cause Tick- borne encephalitis, Central European encephalitis, Far Eastern encephalitis, Rio Bravo, Japanese encephalitis, Kunjin, Murray Valley encephalitis, St Louis encephalitis, West Nile encephalitis, Tyulenly, Ntaya, Uganda S, Dengue type 1 , Dengue type 2, Dengue type 3, Dengue type 4, Modoc, and Yellow Fever. Members of the Pestivirus genus include Bovine viral diarrhea virus 1, Bovine viral diarrhea virus 2, Hog cholera (classical swine fever virus), and Border disease virus. The Hepacivirus genus include hepatitis C virus. Additional members of the Flaviviridae family include the unassigned GB virus-A, GB virus-B, and GB virus- C. Members of the Flaviviridae family of viruses are known to cause a variety of diseases including, for example, Dengue fever, Hepatitis C infection, Japanese encephalitis, Kyasanur Forest disease, Murray Valley encephalitis, St. Louis encephalitis, Tick-borne encephalitis, West Nile encephalitis and Yellow fever.
A peptide of the invention can be used to prevent, treat or otherwise ameliorate infection by a member of the Flaviviridae family of viruses and its associated disease conditions. Thus, examples of various applications of the invention include, without limitation, use as a therapeutic for patients with Dengue fever, Dengue hemorrhagic fever, Dengue shock syndrome, Japanese aencephalitis, Kyasanur forest disease, Murray Valley encephalitis, St. Louis Encephalitis, Tick- borne meningoencephalitis, Chronic hepatitis C infection, to prevent graft infection during liver transplantation, to prevent sexual transmission, to increase the safety of blood and blood product used in transfusions, and to increased safety of clinical laboratory samples.
In one embodiment, the invention provides a method for preventing, inhibiting or otherwise ameliorating viral infection of mammalian cell, such as a human cell, or a method for preventing, inhibiting, treating or otherwise ameliorating acute or chronic infection of a mammal such as a human by a human immunodeficiency virus, measles virus, respiratory syncytial virus or a virus of the Flaviviridae family.
As used herein "preventing" is intended to include the administration of a peptide of the invention to a mammal such as a human who could be or has been exposed to a human immunodeficiency virus for purposes of inhibiting infection. The mammal who could be exposed to HIV includes, without limitation, someone present in an area where these viruses are prevalent or commonly transmitted, e.g., Africa, Southeast Asia, China, South Asia, Australia, India, the United States, Russia, as well as Central and South American countries. The mammal who could be exposed to HIV also includes someone who has been a recipient of donated body tissue or fluids, for example, a recipient of blood or one or more of its components such as plasma, platelets, or stem cells; and medical, clinical or dental personnel who handle body tissues and fluids. A mammal who has been exposed to HIV includes, without limitation, someone who has had contact with the body tissue or fluid, e.g. blood, of an infected person or otherwise have come in contact with HIV.
In addition, "preventing" is intended to include the administration of a peptide of the invention to a mammal such as a human who could be or has been exposed to a member of the Flaviviridae family to inhibit infection by a virus of the Flaviviridae family. The mammal who could be exposed to a virus of the Flaviviridae family includes, without limitation, someone present in an area where these viruses are prevalent or common, e.g. the tropics, Southeast Asia and the Far East, South Asia, Australia and Papua New guinea, the United States, Russia, Africa, as well as Central and South American countries. The mammal who could be exposed to a virus of the Flaviviridae family also includes someone who has been bitten by a deer or forest tick or a mosquito; a recipient of donated body tissue or fluids, for example, a recipient of blood or one or more of its components such as plasma, platelets, or stem cells; and medical, clinical or dental personnel who handle body tissues and fluids. A mammal who has been exposed to a virus of the
Flaviviridae family include, without limitation, someone who has had contact with the body tissue or fluid, e.g. blood, of an infected person or otherwise have come in contact with HCV or any other virus of the Flaviviridae family.
In addition, "preventing" is intended to include the administration of a peptide of the invention to a mammal such as a human who could be or has been exposed to a measles or respiratory syncytial virus to inhibit infection by measles or RSV.
Treatment of, or treating HIV infection, measles infection, RSV infection or infection by a virus of the Flaviviridae family is intended to include a reduction of the viral load or the alleviation of or diminishment of at least one symptom typically associated with the infection. The treatment also includes alleviation or diminishment of more than one symptom. Ideally, the treatment cures, e.g., substantially inhibits viral infection and/or eliminates the symptoms associated with the infection.
Symptoms or manifestations of viral exposure or infection are specific for the particular infection, and these are known in the art.
Early symptoms of HIV infection include flu-like symptoms within three to six weeks after exposure to the virus. This illness, called Acute HIV Syndrome, may include fever, headache, tiredness, nausea, diarrhea and enlarged lymph nodes.
These symptoms usually disappear within a week to a month and are often mistaken for another viral infection. During this acute period, the quantity of the virus in the body will be high and it spreads to different parts, particularly the lymphoid tissue. At this stage, the infected person is more likely to pass on the infection to others. The viral quantity then drops as the body's immune system launches an orchestrated fight. More persistent or severe symptoms may not surface for several years, even a decade or more, after HIV first enters the body in adults, or within two years in children born with the virus. This period of "asymptomatic" infection varies from individual to individual. Some people may begin to have symptoms as soon as a few months, while others may be symptom-free for more than 10 years. However, during the "asymptomatic" period, the virus will be actively multiplying, infecting, and killing cells of the immune system.
Regarding symptoms associated with infection by a member of the Flaviviridae family, Dengue fever and dengue hemorrhagic fever, for example, is caused by one of four Flavivirus serotypes. Symptoms of these conditions include sudden onset of fever, severe headache, joint and muscular pains and rashes, as well as high fever, thrombocytopenia and haemoconcentration. Clinical indications of also include high fever, petechial rash with thrombocytopenia and leucopenia, and haemorrhagic tendency. Symptoms of Japanese aencephalitis include fever, headache, neck rigidity, cachexia, hemiparesis, convulsions and heightened body temperature. Japanese encephalitis can be diagnosed by detection of antibodies in serum and cerebrospinal fluid. Symptoms of Kyasanur forest disease include high fever, headache, haemorrhages from nasal cavity and throat, and vomiting. Symptoms of St. Louis encephalitis include fever, headache, neck stiffness, stupor, disorientation, coma, tremors, occasional convulsions and spastic paralysis. Symptoms of Murray Valley encephalitis include fever, seizures, nausea and diarrhea in children, and headaches, lethargy and confusion in adults. Symptoms of West Nile virus infection include flu-like symptoms, malaise, fever, anorexia, nausea, vomiting, eye pain, headache, myalgia, rash and lymphadenopathy, as well as encephalitis (inflammation of the brain) and meningitis (inflammation of the lining of the brain and spinal cord), meningismus, temporary blindness, seizures and coma. West Nile infection can be diagnosed using ELISA to detect antibodies in the blood or cerebrospinal fluids. Symptoms of Yellow fever include fever, muscle aches, headache, backache, a red tongue, flushed face, red eyes, hemorrhage from the gastrointestinal tract, bloody vomit, jaundice, liver failure, kidney insufficiency with proteinuria, hypotension, dehydration, delirium, seizure and coma. Symptoms of hepatitis C infection include, without limitation, inflammation of the liver, decreased appetite, fatigue, abdominal pain, jaundice, flu-like symptoms, itching, muscle pain, joint pain, intermittent low-grade fevers, sleep disturbances, nausea, dyspepsia, cognitive changes, depression headaches and mood changes. HCV infection could also be diagnosed by detecting antibodies to the virus, detecting liver inflammation by biopsy, liver cirrhosis, portal hypertension, thyroiditis, cryoglobulinemia and glomerulonephritis. In addition HCV infection could be diagnosed. In addition, diagnosis of exposure or infection or identification of one who is at risk of exposure to HCV could be based on medical history, abnormal liver enzymes or liver function tests during routine blood testing. Generally, infection by a member of the Flaviviridae family can be diagnosed using ELISA for detecting viral antigens or anti-viral antibodies, immunofluorescence for detecting viral antigens, polymerase chain reaction (PCR) for detecting viral nucleic acids and the like.
Symptoms of measles include a fever for at least three days, as well as cough, a runny nose, and conjunctivitis. The fever may reach up to 40° Celsius (104° Fahrenheit). Koplik's spots seen inside the mouth are pathognomonic
(diagnostic) for measles but are not always present, even in real cases of measles, because they are transient and may disappear within a day of arising. The characteristic measles rash is classically described as a generalized, maculopapular, erythematous rash that begins several days after the fever starts. It starts on the head before spreading to cover most of the body, often causing itching. The rash is said to "stain," changing color from red to dark brown, before disappearing.
RSV causes respiratory tract infections in patients of all ages. It is the major cause of lower respiratory tract infection during infancy and childhood. Symptoms include recurrent wheezing and asthma may develop among individuals who suffered severe RSV infection during the first few months of life.
Methods of preventing, treating or otherwise ameliorating acute or chronic viral infection include contacting the cell with an effective amount of a peptide of the invention or administering to a mammal such as a human a therapeutically effective amount of a peptide of the present invention. Methods of inactivating the virus include contacting the virus with an effective amount of the peptide of the invention..
A peptide of the invention can be administered in a variety of ways. Routes of administration include, without limitation, oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, vaginal, dermal, transdermal (topical), transmucosal, intrathoracic, intrapulmonary and intranasal (respiratory) routes. The means of administration may be by injection, using a pump or any other appropriate mechanism.
A peptide of the invention may be administered in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the peptides of the invention may be essentially continuous over a pre-selected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
The dosage to be administered to a mammal may be any amount appropriate to reduce or prevent viral infection or to treat at least one symptom associated with the viral infection. Some factors that determine appropriate dosages are well known to those of ordinary skill in the art and may be addressed with routine experimentation. For example, determination of the physicochemical, toxicological and pharmacokinetic properties may be made using standard chemical and biological assays and through the use of mathematical modeling techniques known in the chemical, pharmacological and toxicological arts. The therapeutic utility and dosing regimen may be extrapolated from the results of such techniques and through the use of appropriate pharmacokinetic and/or pharmacodynamic models. Other factors will depend on individual patient parameters including age, physical condition, size, weight, the condition being treated, the severity of the condition, and any concurrent treatment. The dosage will also depend on the peptide(s) chosen and whether prevention or treatment is to be achieved, and if the peptide is chemically modified. Such factors can be readily determined by the clinician employing viral infection models such as the HCV cell culture/JFH-1 infection model described herein, or other animal models or test systems that are available in the art.
The precise amount to be administered to a patient will be the responsibility of the attendant physician. However, to achieve the desired effect(s), a peptide of the invention, a variant thereof or a combination thereof, may be administered as single or divided dosages, for example, of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages may provide beneficial results. The absolute weight of a given peptide included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least one peptide of the invention, or a plurality of peptides specific for a particular cell type can be administered. Alternatively, the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g. Daily doses of the peptides of the invention can vary as well. Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day. A peptide of the invention may be used alone or in combination with a second medicament. The second medicament can be a known antiviral agent such as, for example, an interferon-based therapeutic or another type of antiviral medicament such as ribavirin. The second medicament can be an anticancer, antibacterial, or antiviral agent. The antiviral agent may act at any step in the life cycle of the virus from initial attachment and entry to egress. Thus, the added antiviral agent may interfere with attachment, fusion, entry, trafficking, translation, viral polyprotein processing, viral genome replication, viral particle assembly, egress or budding. Stated another way, the antiviral agent may be an attachment inhibitor, entry inhibitor, a fusion inhibitor, a trafficking inhibitor, a replication inhibitor, a translation inhibitor, a protein processing inhibitor, an egress inhibitor, in essence an inhibitor of any viral function. The effective amount of the second medicament will follow the recommendations of the second medicament manufacturer, the judgment of the attending physician and will be guided by the protocols and administrative factors for amounts and dosing as indicated in the PHYSICIAN'S DESK REFERENCE.
The effectiveness of the method of treatment can be assessed by monitoring the patient for signs or symptoms of the viral infection as discussed above, as well as determining the presence and/or amount of virus present in the blood, e.g. the viral load, using methods known in the art including, without limitation, polymerase chain reaction and transcription mediated amplification.
Pharmaceutical Compositions In one embodiment, the invention provides a pharmaceutical composition comprising a peptide of the invention. To prepare such a pharmaceutical composition, a peptide of the invention is synthesized or otherwise obtained, purified as necessary or desired and then lyophilized and stabilized. The peptide can then be adjusted to the appropriate concentration and then combined with other agent(s) or pharmaceutically acceptable carrier(s). By "pharmaceutically acceptable" it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
Pharmaceutical formulations containing a therapeutic peptide of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the peptide can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone. Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution can also be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds can also be included. Surface active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They may also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.
For oral administration, a peptide may be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. The active peptide may also be presented as a bolus, electuary or paste. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts including the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.
Tablets or caplets containing the peptides of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pre-gelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatin capsules containing at least one peptide of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated caplets or tablets containing one or more peptides of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.
Orally administered therapeutic peptide of the invention can also be formulated for sustained release. In this case, a peptide of the invention can be coated, micro-encapsulated (see WO 94/ 07529, and U.S. Patent No.4,962,091), or otherwise placed within a sustained delivery device. A sustained-release formulation can be designed to release the active peptide, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, draining devices and the like.
A therapeutic peptide of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes. A pharmaceutical formulation of a therapeutic peptide of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.
Thus, a therapeutic peptide may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form. The active peptides and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active peptides and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
These formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol," polyglycols and polyethylene glycols, C \ -C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol," isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes. It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol and its derivatives can be added. In some embodiments the peptides are formulated as a microbicide, which is administered topically or to mucosal surfaces such as the vagina, the rectum, eyes, nose and the mouth. For topical administration, the therapeutic agents may be formulated as is known in the art for direct application to a target area. Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap. Thus, in one embodiment, a peptide of the invention can be formulated as a vaginal cream or a microbicide to be applied topically. Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus, the therapeutic peptides of the invention can be delivered via patches or bandages for dermal administration. Alternatively, the peptide can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. The backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns.
Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The active peptides can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01 % to 95 % of the total weight of the formulation, and typically 0.1-85 % by weight. Drops, such as eye drops or nose drops, may be formulated with one or more of the therapeutic peptides in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
The therapeutic peptide may further be formulated for topical administration in the mouth or throat. For example, the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier.
The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0.
The peptides of the invention can also be administered to the respiratory tract. Thus, the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention. In general, such dosage forms comprise an amount of at least one of the agents of the invention effective to treat or prevent the clinical symptoms of the viral infection. Any statistically significant attenuation of one or more symptoms of the infection that has been treated pursuant to the method of the present invention is considered to be a treatment of such infection within the scope of the invention.
Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator, or a metered-dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds., pp. 197-224, Butterworths, London, England, 1984).
Therapeutic peptides of the present invention can also be administered in an aqueous solution when administered in an aerosol or inhaled form. Thus, other aerosol pharmaceutical formulations may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/mL and about 100 mg/mL of one or more of the peptides of the present invention specific for the indication or disease to be treated. Dry aerosol in the form of finely divided solid peptide or nucleic acid particles that are not dissolved or suspended in a liquid are also useful in the practice of the present invention. Peptides of the present invention may be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 μm, alternatively between 2 and 3μm. Finely divided particles may be prepared by pulverization and screen filtration using techniques well known in the art. The particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.
For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic peptides of the invention are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifiuoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Nebulizers include, but are not limited to, those described in U.S. Patent Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co., (Valencia, CA). For intra-nasal administration, the therapeutic agent may also be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker). A therapeutic peptide of the invention may also be used in combination with one or more known therapeutic agents, for example, a pain reliever; an antiviral agent such as an anti-HBV, anti-HCV (HCV inhibitor, HCV protease inhibitor) or an anti-herpetic agent; an antibacterial agent; an anti-cancer agent; an antiinflammatory agent; an antihistamine; a bronchodilator and appropriate combinations thereof, whether for the conditions described or some other condition.
Miscellaneous Compositions and Articles of Manufacture
In one embodiment, the invention provides an article of manufacture that includes a pharmaceutical composition containing a peptide of the invention for controlling microbial infections. Such articles may be a useful device such as a vaginal ring, a condom, a bandage or a similar device. The device holds a therapeutically effective amount of a pharmaceutical composition for controlling viral infections. The device may be packaged in a kit along with instructions for using the pharmaceutical composition for control of the infection. The pharmaceutical composition includes at least one peptide of the present invention, in a therapeutically effective amount such that viral infection is controlled.
An article of manufacture may also be a vessel or filtration unit that can be used for collection, processing or storage of a biological sample containing a peptide of the invention. A vessel may be evacuated. Vessels include, without limitation, a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter. The filtration unit can be part of another device, for example, a catheter for collection of biological fluids. Moreover, the peptides of the invention can also be adsorbed onto or covalently attached to the article of manufacture, for example, a vessel or filtration unit. Thus, when material in the article of manufacture is decanted therefrom or passed through the article of manufacture, the material will not retain substantial amounts of the peptide. However, adsorption or covalent attachment of the peptide to the article of manufacture kills viruses or prevents their transmission, thereby helping to control viral infection. Thus, for example, the peptides of the invention can be in filtration units integrated into biological collection catheters and vials, or added to collection vessels to remove or inactivate viral particles that may be present in the biological samples collected, thereby preventing transmission of the disease.
The invention also provides a composition comprising a peptide of the invention and one or more clinically useful agents such as a biological stabilizer. Biological stabilizer includes, without limitation, an anticoagulant, a preservative and a protease inhibitor. Anticoagulants include, without limitation, oxalate, ethylene diamine tetraacetic acid, citrate and heparin. Preservatives include, without limitation, boric acid, sodium formate and sodium borate. Protease inhibitors include inhibitors of dipeptidyl peptidase IV. Compositions comprising a peptide of the invention and a biological stabilizer may be included in a collection vessel such as a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter or any other container or vessel used for the collection, processing or storage of a biological samples. The invention also provides a composition comprising a peptide of the invention and a biological sample such as blood, semen or other body fluids that is to be analyzed in a laboratory or introduced into a recipient mammal. For example, a peptide of the invention can be mixed with blood prior to laboratory processing and/or transfusions. In another embodiment, the peptides of the invention can be included in physiological media used to store and transport biological tissues, including transplantation tissues. Thus, for example, liver, heart, kidney and other tissues can be bathed in media containing the present peptides to inhibit viral transmission to transplant recipients. The invention is further illustrated by the following non-limiting Examples.
EXAMPLES
Example 1: Methods Cell culture and reagents. Huh-7 and Huh-7.5.1 cells were described in
Zhong, J. et al., Proc Natl Acad Sci USA 102, 9294-9 (2005). HeLa, Hep2, LLC- MK2, MRC-5, MA- 104, Vero and MDBK cells were obtained from the American Type Culture Collection. All cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS), 10 mM Hepes buffer, 100 units/ml penicillin, 100 mg/ml streptomycin and 100 μM non-essential amino acids (Invitrogen) at 5% CO2. Peptide synthesis. Highly purified peptides (>95% purity) were synthesized with either L- or D-amino acids using fluorenylmethoxycarbonyl (Fmoc) chemistry by A & A Labs, LLC (San Diego, CA), Mimotopes Pty Ltd (Clayton Victoria, Australia), or at The Scripps Research Institute. HCV production and infection. The infectious genotype 2a HCV clone JFHl
(see Kato, T. et al., J Med Virol 64, 334-9 (2001)) was used in this study. The JFH-I plasmid construct used for stock virus production, the cell culture conditions used for JFH-I infection, and the analytical procedures used to detect virus production were described in Zhong, J. et al., Proc Natl Acad Sci USA 102, 9294-9 (2005). Indirect immunofluorescence. Intracellular staining of proteins or peptides was performed as described by Zhong, J. et al., Proc Natl Acad Sci USA 102, 9294-9 (2005). The recombinant human monoclonal (IgGl) anti-E2 antibody was provided by D. Burton (see Zhong, J. et al., Proc Natl Acad Sci USA 102, 9294-9 (2005)), a rabbit polyclonal antibody against NS5A was provided by M. Houghton (see Zhong, J. et al., Proc Natl Acad Sci USA 102, 9294-9 (2005)), and rabbit anti- dansyl polyclonal antibody was purchased from Molecular Probes (Eugene, OR) and diluted 1 :400 for use. Huh-7 cells were fixed with 4 % paraformaldehyde, and stained with previously determined optimal dilutions of these antibodies followed by incubation with a 1 : 1000 dilution of Alexa555-conjugated secondary antibodies to human or rabbit IgG (Molecular Probes, Eugene, OR), respectively. Cell nuclei were stained with Hoechst dye or determined by differential interference contrast (DIC). Cellular localization of the proteins or peptides was analyzed on a Radiance 2100 Rainbow laser scanning confocal microscope (LSCM) (Bio-Rad, Hercules, CA).
MTT cytotoxicity assay. The cytotoxic effect of peptides was measured in an MTT cytotoxicity assay according to the manufacturer's instructions (MTT assay kit, Cat# 30-1010K, ATCC, Manassas, VA). In brief, serial 2-fold dilutions of peptide in 5 % dextrose H2O containing 0.5% DMSO or DMSO alone were added to 5000-10,000 cells in a 96 well plate. After 72 hours at 37 0C, 1/10 volume of MTT solution (5mg/mL in PBS) was added to each well and the plates were returned to the incubator. Two hours later, the media was removed, 150 μL DMSO was added to dissolve the purple formazan precipitate, and the plate was shaken at 150rpm for 10 minutes before the absorbance was read at 570nm. The peptide concentration that reduced the OD reading by 50 % was designated the 50 % lethal concentration (LC50).
In vivo cytotoxicity analysis. To examine the cytotoxic potential of peptide 1 in mice, pure peptide (> 95%) was dissolved in DMSO at a concentration of 50-100 mg/mL, which was then diluted in endotoxin-free 5% dextrose/H2O and 200 μL of peptide solution or DMSO (5 %) control were injected intravenously (tail vein) into groups of 7-9 week old C57BL/6J mice that were weighed before injection and on days 1, 2, 4 and 6 and 8 after injection. Selected animals were injected three times with 0.5 mg of the D-isomer at weekly intervals and monitored every other day for one week after each injection. In vivo immunogenicity analysis. To determine if peptide 1 is immunogenic, sera from mice that were injected three times intravenously with 0.5 mg of the D- isomer at weekly intervals were tested for anti-peptide 1 antibody using a peptide- specific ELISA. Briefly, 1 μg of D-peptide 1 or PBS was coated overnight at 4 0C onto wells of flat bottom ImmunoPlates (Nalge Nunc Intl, Rochester, NY), washed three times with PBS containing 0.05 % Tween 20 (wash buffer); then blocked overnight at 4 0C with 5 % nonfat dry milk containing 10 % FBS; washed 4 times followed by addition of serial 2-fold dilutions of mouse serum starting at 1 :10 dilution and incubated for 1 hour at room temperature. Wells were then washed 10 times and incubated with a secondary horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG (1 :25000 dilution) (Pierce, Rockford, IL) for 1 hour at room temperature, washed 10 times and developed with tetramethylbenzidine (Pierce) according to the manufacturer's directions and the optical density was measured at 650 run and the absorbance of pepti de-coated and PBS-coated control wells were compared and sera whose absorbance were greater than 2-fold higher in peptide- coated wells than PBS-coated wells were considered positive. Assay controls included (a) wells that were coated with 1 μg of dansylated D-peptide 1 and incubated with rabbit polyclonal anti-dansyl antibody (1 :400 dilution) (Molecular Probes, Eugene, OR) followed by HRP-conjugated goat anti-rabbit IgG (1 :25,000 dilution) (Pierce, Rockford, IL); (b) wells coated with 1 μg influenza hemagglutinin (HA) peptide (Sigma, St. Louis, MO) and incubated with mouse monoclonal anti- HA antibody (1 :600 dilution) (Sigma, St. Louis, MO) followed by HRP-conjugated goat anti-mouse IgG (1 :25,000 dilution) (Pierce, Rockford, IL).
Velocity sedimentation ultracentrifugation. The sedimentation velocity of native and peptide-treated infectious HCV particles was examined by rate zonal ultracentrifugation in continuous sucrose gradients as previously described. Briefly, 100 μL samples were layered onto 5 mL preformed 10 to 50 % continuous sucrose gradients and spun for 1 hour at 200,000 x g in a SW60.Ti rotor at 4 °C. After centrifugation, 12 fractions (400 μL each) were collected from the top and analyzed for virus infectivity and HCV RNA content as described above.
Intracellular HCV infectivity determination. Huh-7 cells were infected by JFH-I at a multiplicity of infection (moi) of 0.01. Ten days later, infected cells were washed 3x with PBS, trypsinized, and resuspended in complete culture medium at a concentration of 1x105 cells/mL. The cells were lysed by four freeze-thaw cycles in dry ice and a 37 0C water bath and centrifuged for 2 minutes at 14,000 rpm to remove cell debris. The supernatant was assayed for infectivity in a standard titration assay as described above.
Other virus infections. To determine if selected peptides inhibit other virus infections, varying concentrations of each peptide or DMSO were added to virus stocks of predetermined infectivity (1-105 ffu or TCID5o/mL), incubated at 370C for at least 1 hour and then added to susceptible cells, unless specified. After 2-4 days of infection, the cultures were assessed by comparative assessment of cytopathic effect (CPE) or by immunostaining or immunoassay with antibody against the corresponding viral protein as described below. Since HBV is not infectious in vitro, we examined the impact of peptides on HBs antigenicity by quantitative ELISA analysis as described in Guidotti et al., J Virol 69, 6158-69 (1995) and on HBV DNA content by quantitative PCR analysis as described in Thimme et al., J Virol 11, 68-76 (2003).
Example 2: HCV Peptides Inhibit Hepatitis C Viral Infection
The Example describes the identification of anti-viral peptides of the invention.
A peptide library of 441 overlapping peptides covering the complete hepatitis C viral polyprotein of hepatitis C genotype 1 a (H77) (SEQ ID NO: 1 ) was prepared and tested for inhibition of hepatitis C viral infection using a cell culture model of hepatitis C viral infection described in a related application (U.S. Ser. No. 11/541,488). The peptides were about 18 amino acids in length with 1 1 overlapping amino acids. The peptide library was made by NIH AIDS Research and Reference Reagent Program (Cat # 7620, Lot # 1).
To identify peptides that display antiviral activity against HCV infection, the peptide library was screened by an HCV assay. The peptides were reconstituted in 100 % DMSO at a final concentration 10 mg/mL, and stored in -20 0C. The peptide stock solution was diluted 1 :200 to a final concentration approximately 20 μM in complete DMEM growth medium containing 50 focus forming units (ffu) of HCV. The virus-peptide mixture was transferred to Huh-7.5.1 cells at a density of 8000 cells per well in a 96-well plate. After adsorption for 4 hours at 37 °C, the inoculum was removed. The cells were washed 2 times, overlaid with 120 μL fresh growth medium and incubated at 37 0C. After 3 days of culture, the cells were fixed with paraformaldehyde and immunostained with antibody against HCV nonstructural protein NS5A. The numbers of HCV foci were counted under fluorescent microscopy and the result is expressed as percentage (%) of control foci detected in cells inoculated with virus and 0.5 % DMSO, but without peptides.
The results of these assays are shown in FIG. 1 and the following table.
Table 3: Inhibition of HCV Infection
No. Peptide Sequence % of Fold >10- 5-10 2-5 SEQ
Mock Inhibition fold fold fold ID
NO:
6930 QIVGGVYLLPRRGPRLGV 45.2 2.2 * 4
6937 QPGYPWPLYGNEGCGWAG 50.0 2.0 * 5
6938 LYGNEGCGWAGWLLSPRG 2.4 42.0 *** 6
6939 GWAGWLLSPRGSRPSWGP 45.2 2.2 * 7
6951 IFLLALLSCLTVPASAYQ 2.4 42.0 *** 8
6957 DAILHTPGCVPCVREGNA 21.4 4.7 * 9
6962 LPTTQLRRHIDLLVGSAT 38.1 2.6 * 10
6963 RHIDLLVGSATLCSALYV 31.0 3.2 * 1 1
6964 GSATLCSALYVGDLCGSV 1.0 100.0 *** 12
6965 ALYVGDLCGSVFLVGQLF 1.0 100.0 *** 13
6975 IMDMIAGAHWGVLAGIAY 2.4 42.0 *** 14
6986 HINSTALNCNESLNTGWL 40.5 2.5 * 15
6987 NCNESLNTGWLAGLFYQH 35.7 2.8 * 16
6991 LASCRRLTDFAQGWGPIS 35.7 2.8 * 17
6992 TDFAQGWGPISYANGSGL 31.0 3.2 * 18
6993 GPISYANGSGLDERPYCW 23.8 4.2 * 19
6994 GSGLDERPYCWHYPPRPC 33.3 3.0 * 20
7005 WMNSTGFTKVCGAPPCVI 16.7 6.0 ** 21
7007 PCVIGGVGNNTLLCPTDC 33.3 3.0 * 22
7016 MYVGGVEHRLEAACNWTR 16.7 6.0 ** 23
7026 YLYGVGSSIASWAIKWEY 2.4 42.0 *** 24
7027 SIASWAIKWEYVVLLFLL 40.5 2.5 * 25 No. Peptide Sequence % of Fold >10- 5-10 2-5 SEQ
Mock Inhibition fold fold fold ID
NO:
7028 KWEYVVLLFLLLADARVC 47.6 2.1 * 26
7031 WMMLLISQAEAALENL Vl 4.8 21.0 *** 27
7038 GAVYAFYGMWPLLLLLLA 19.0 5.3 ** 28
7039 GMWPLLLLLLALPQRAYA 31.0 3.2 * 29
7052 TLVFDITKLLLAIFGPLW 1.0 100.0 *** 30
7725 VSTATQTFLATCIN 40.5 2.5 * 31
7078 ATQTFLATCINGVCWTVY 2.4 42.0 *** 32
7142 DSSVLCECYDAGCAWYEL 40.5 2.5 * 33
7146 AYMNTPGLPVCQDHLEFW 40.5 2.5 * 34
7148 LEFWEGVFTGLTHIDAHF 33.3 3.0 * 35
7160 HPITKYIMTCMSADLEVV 38.1 2.6 * 36
7729 j TST WVLVGGVLAAL 1 1.9 8.4 ** 37
7163 WVLVGGVLAALAAYCLST 26.2 3.8 * 38
7730 LAALAAYCLSTGCVV 21.4 4.7 * 39
7177 EVFWAKHMWNFISGIQYL 23.8 4.2 * 40
7178 MWNFISGIQYLAGLSTLP 42.9 2.3 * 41
7195 PAILSPGALVVGVVCAAI 42.9 2.3 * 42
7208 SWLRDIWDWICEVLSDFK 1.0 100.0 *** 43
7209 DWICEVLSDFKTWLKAKL 2.4 42.0 *** 44
7226 YVSGMTTDNLKCPCQIPS 38.1 2.6 * 45
7740 SSGADTEDVVCCSMS 42.9 2.3 * 46
7741 DTEDVVCCSMSYSW 2.4 42.0 *** 47
7270 SSGADTEDVVCCSMSYSW 4.5 22.0 *** 48
7742 DVVCCSMSYSWTGAL 23.8 4.2 * 49
7304 TVTESDIRTEEAIYQCCD 35.7 2.8 * 50
7313 GNTLTCYIKARAACRAAG 45.2 2.2 * 51
7315 RAAGLQDCTMLVCGDDLV 50.0 2.0 * 52
7316 CTMLVCGDDLVVICESAG 1.0 100.0 *** 53
7317 DDLVVICESAGVQEDAAS 26.2 3.8 * 54
7323 LELITSCSSNVSVAHDGA 42.9 2.3 * 55
7329 HTPVNSWLGNIIMFAPTL 47.6 2.1 * 56
7331 APTLWARMILMTHFFSVL 45.2 2.2 * 57
7334 DQLEQALNCEIYGACYSI 28.6 3.5 * 58
7342 GVPPLRAWRHRARSVRAR 50.0 2.0 * 59
7343 WRHRARSVRARLLSRGGR 47.6 2.1 * 60
7350 GWFTAGYSGGDIYHSVSH 42.9 2.3 * 61
Total 14 4 41
Of the 441 peptides, 382 had no effect on HCV infection or blocked it by less than 20 % (not shown in Table 3). Forty-one peptides slightly inhibited HCV infection by about 2- to 5 -fold. Four peptides inhibited HCV infection by about 5- to 10-fold. Fourteen peptides inhibited HCV infection by more than 10-fold. In particular, HCV infection was profoundly inhibited (90-100 %) by peptides with SEQ ID NO:6, 8, 12, 13, 14, 24, 27, 30, 32, 43, 44, 47, 48 and 53. No evidence of toxicity was detected when Huh-7.5.1 cells were incubated with these peptides. These results identify peptide inhibitors that may modify or inhibit one or more steps in the viral life cycle. Moreover, according to the invention, these peptides can be used in antiviral compositions and methods for inhibiting HCV infection. Peptides that inhibited infection by more than 90 % were selected for further analysis.
Validation of the antiviral activity of these inhibitory peptides was performed by comparing the ability of highly (>95 %) purified preparations of each peptide (20 μM) to inhibit the expansion of HCV RNA in Huh-7.5.1 cells 72 hours after infection (moi 0.1) relative to the solvent control (0.5 % DMSO). To quantify the inhibitory effect of peptides on HCV infection, the intracellular HCV RNA content of peptide-treated and untreated cells was quantified by real time RT-QPCR. The peptide stock solution and DMSO solvent were diluted 1 : 100 and mixed with an equal volume of stock viral supernatant to yield a final peptide concentration of 18 μM and a final DMSO concentration of 0.5%. The virus-peptide and virus-DMSO mixtures were then used to infect Huh-7.5.1 cells at a multiplicity of infection (moi) of 0.1. After 3 days incubation at 37 °C, cells were washed, lysed and total cellular RNA was isolated.
Total cellular RNA was isolated by the guanidine thiocyanate method using standard protocols after adding 5 μg of yeast tRNA per 500 μL sample as a carrier. Reverse transcription and quantitative real-time PCR (RT-QPCR) was performed as described in Cheng et al., Proc Natl Acad Sci USA 103, 8499-504 (2006). HCV and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) transcript levels were determined relative to a standard curve comprised of serial dilutions of plasmid containing the HCV JFH-I cDNA or human GAPDH gene. The relative HCV RNA content in infected cells was determined after normalization to cellular GAPDH mRNA levels. The detection limit of the RT-QPCR protocol is approximately 1 copy of HCV RNA per 1000 cells.
The HCV RNA transcript level was measured by real time RT-QPCR with the primers S'-TCTGCGGAACCGGTGAGTA-S' (sense, SEQ ID NO: 89) and 5'- TC AGGC AGTACC AC AAGGC-3' (antisense, SEQ ID NO: 90)', and normalized to cellular GAPDH levels. Inhibitory activity was detected by comparing the normalized intracellular HCV RNA levels of the peptide-treated and solvent-treated inocula. Results are summarized in the following table.
Table 4: Inhibitory Peptide Hierarchy
Figure imgf000094_0001
Figure imgf000095_0001
Based on the hierarchy of infectivity, the most active peptides were reassigned numerical designators to reflect their position in the hierarchy of infectivity as shown in the above Table. Thus, peptide 1 (SEQ ID NO:43) derived from the N- terminus of NS5A was the most effective peptide inhibitor of HCV and inhibited viral expansion by more than 4 orders of magnitude. Peptide 1 contains residues 3- 21 of the amphipathic alpha helical N-terminal membrane anchor domain of the HCV NS5A protein.
However, peptides having SEQ ID NO:6, 8, 12, 13, 14, 24, 27, 30, 32, 44, 47, 48 and 53 also strongly inhibited HCV infection as measured using a cell culture model of hepatitis C viral infection described in a related application (U.S. Ser. No. 1 1/541,488). Other peptides exhibited good inhibition of HCV infection. These HCV-derived synthetic peptides that were effective inhibitors were from both structural and non-structural regions of the HCV polyprotein.
Example 3: Analyses of N- and C-terminal Truncated Peptide 1
To define the antiviral action of peptide #1 (SEQ ID NO:43), the antiviral activity of a series of N-terminal and C-terminal truncations of peptide 1 was analyzed using the focus reduction assay and by measuring the reduction in intracellular HCV RNA as described.
Highly purified peptides (>95% purity) were used for these studies. All peptides were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry on pre-loaded wang resin by A & A Labs, LLC (San Diego, CA). The peptides were synthesized on the Symphony multiple peptide synthesizer (Protein Technologies Inc, Tucson, AZ). The crude peptides were then purified and analyzed by reverse- phase Gilson HPLC system (Gilson, Inc. Middleton, WI). The column used was Cl 8 column (Grace Vydac, Hesperia, California) with bead size 20 mm and length 250 mm. The solvent system was a H2O and acetonitrile solvent system with a linear gradient of 5 % to 70 % for 30 minutes. Mass spectral analysis was performed by PE Sciex API-100 mass spectrometer. This confirmed the molecular masses of the synthesized peptides. Peptide concentration was determined using the extinction coefficient of the chromophore residues (Tryptophan or Tyrosine), where Tryptophan = 5560 AU/mmole/mL and Tyrosine 1200 = AU/mmole/mL.
Calculations were made using the formula: mg peptide per mL = (A280 x DF x MW) / e, where A280 was the actual absorbance of the solution at 280 nm in a 1-cm cell, DF was the dilution factor, MW was the molecular weight of the peptide and e was the molar extinction coefficient of each chromophore at 280 nm. Results, summarized in the following table, show that the peptides having C- terminal truncations of 1 to 4 amino acid residues retained antiviral activity. Removal of as few as 2 amino acids from the N-terminus destroyed antiviral activity.
Table 5: Anti-HCV Activity of Truncated Variants of Peptide 1
Fold Fold
Working Focus Inhibition Inhibition
Peptides
Figure imgf000096_0001
ConcenReducon HCV on HCV
IU tration tion (72h) RNA RNA
(24h) (72h)
SWLRDIWDWICEVLSDFK 43
19.5μM 0 47697.9 301779.7
SWLRDIWDWICEVLSD 94
18.4μM 0 9852.6 234207.1 SWLRDIWDWICEVL 23.6μM 0 20274.7 23717
SWLRDIWDWICE 104
21.4μM 107 0.8 0.5
SWLRDIWDWI 105
25.6μM 65 0.8 0.6
SWLRDIWD 106
24.7μM 58 1.3 1.0
LRDIWDWICEVLSDFK 107
18.6μM 125 0.7 0.6
DIWDWICEVLSDFK 108
27.1μM 125 1.0 0.7
WDWICEVLSDFK 109
24.7μM 38 2.2 1.4
WICEVLSDFK 1 10
27.5μM 45 1.1 1.0
CEVLSDFK 1 1 1 NA 53 1.1 0.7
Mock 51
Example 4: Peptide Prevents Initiation of HCV Infection and Suppresses
Established Infection Based on its virocidal activity the following experiment was conducted to determine whether peptide 1 can completely and permanently inhibit the establishment of HCV infection. Huh-7.5.1 cells were inoculated with HCV (moi 0.1) and peptide 1 (18 μM) or 0.5 % DMSO as a control. After adsorption for 4 hours at 37 0C, the virus-peptide and virus-DMSO inocula were removed, the cells were washed 2 times, overlaid with 120 μL fresh growth medium and incubated at 37 °C. At the indicated time points, total cellular RNA was isolated and the HCV RNA level was measured. As shown in FIG. 2A, in contrast to the virus spread observed in control cultures, intracellular viral RNA progressively decreased and ultimately disappeared (fewer than 1 copy per 1000 cells) in cells infected with virus that was incubated with peptide 1 prior to inoculation, and it remained undetectable for at least 11 days. These results suggest that, by virtue of its virocidal activity, peptide 1 prevents initiation and spread of HCV infection.
To determine if peptide 1 can terminate an ongoing HCV infection, it was added to infected cells 3 days after virus inoculation, when 10 % of the cells were HCV E2 positive, and it was maintained in the culture medium and replenished every time the cells were split. More specifically, Huh-7 cells were infected with HCV (moi 0.1). On day 3 postinfection, when approximately 10 % of the cells were infected (HCV E2 positive by immunostaining), peptide 1 (18 μM) or 0.5 % DMSO were added and the cells were incubated at 37 °C and split 1 : 6 every 3-4 days when reaching confluency. The peptide and DMSO were replenished every time the cells were split at which point total cellular RNA was isolated and the HCV RNA transcript level was measured. As shown in Figure 2B, while intracellular HCV RNA increased in DMSO-treated cells, both the L- and D-forms of peptide 1 (SEQ ID NO:43) abruptly halted viral expansion and viral RNA gradually decreased to less than 1 copy every 1000 cells by day 45. Importantly, HCV RNA remained negative for at least 15 days after peptide withdrawal (not shown). These results suggest that peptide 1 not only prevents HCV infection by destroying the virus and blocking cell-to-cell spread, it can also terminate ongoing HCV infection in a dividing cell culture.
To determine if peptide 1 can suppress infection in nondividing cells with established infection, the D-form of peptide 1 and DMSO were added to growth- arrested and highly differentiated Huh-7 cells (see Sainz et al., J. Virol. 80,10253-7 (2006)) 15 days after infection, when >90 % of the cells were HCV E2-positive (not shown). More specifically, Huh-7 cells were treated with 1 % DMSO for 10 days to induce differentiation and growth arrest at which time they were infected by HCV at moi of 0.01. Fifteen days after infection, when >90 % cells were HCV E2-positive, the L- and D-isomers of peptide 1 (18 μM) were added in complete growth medium. The culture medium was replaced every day with medium containing fresh peptide. At the indicated time points, total cellular RNA was isolated and HCV RNA content was measured. For comparison, the infected cells were treated with recombinant human IFNα (PBL biomedical Laboratories, Piscataway, NJ) at 100 U/mL that was replenished every day as above. As shown in Figure 2C, the D-form of peptide 1 suppressed intracellular HCV RNA to the same extent as IFNα by day 5 and it was even more inhibitory (>95 %) than IFNα on days 15 and 20 of treatment. These results indicate that peptide 1 can potently suppress persistent HCV infection in growth-arrested cells.
These results also suggest that in addition to its extracellular virocidal activity, peptide 1 might be able to inactivate HCV intracellularly. To test this hypothesis, peptide 1 was evaluated to determine whether it can enter cells and destabilize intracellular virus particles. To determine if the peptide actually enters the cells, a fluorescent form of peptide 1 containing a dansyl group at its N-terminus was diluted in complete culture medium to a final concentration of 18 μM and incubated with Huh-7 cells for 4 hours at 37 0C before being washed 5 times with PBS. Treated cells were then fixed by 4 % paraformaldehyde, and immunostained with rabbit polyclonal antibody against the dansyl group (Molecular Probes, Eugene, OR) and analyzed by confocal fluorescence microscopy. As shown in FIG. 2D, a fluorescent D-form of peptide 1 containing a dansyl group at its N-terminus efficiently enters Huh-7 cells, accumulating in granular structures in the cytoplasm. Importantly, when infected cells were incubated with the D-isomer of peptide 1 for 6 hours, the number of intracellular infectious HCV particles was reduced by 3-fold (FIG. 2E), without any change in intracellular HCV RNA (not shown). Huh-7 cells that had been infected with JFH-I at an moi 0.01 for 10 days were washed 4 times, and treated with D-isomers of the peptide 1 (18 μM) or DMSO (0.5%). After 6 hours incubation, intracellular HCV infectivity, extracellular HCV infectivity and cellular HCV RNA content were determined. The results suggest that, in addition to its extracellular virocidal activity, peptide 1 can enter cells and inactivate intracellular virus, albeit less efficiently, without blocking HCV replication. To determine the median effective concentration (EC50) of peptide #1, peptide stock solution (3.6 mM in DMSO) was serially 2-fold diluted in DMSO. An aliquot of peptide from each dilution was then diluted 1 :100 in complete growth medium and mixed with equal volume of virus supernatant. The virus-peptide mixture was then used to infect Huh-7.5.1 cells (MOI = 0.1). After adsorption for 4 hours at 37 0C, the virus-peptide inoculum was removed. The cells were washed 2 times, overlaid with 120 μL fresh growth medium and incubated at 37 °C for 3 days. Cells were lysed and subjected to RNA analysis. The HCV RNA transcript level was measured by real time RT-QPCR and normalized to cellular GAPDH levels. The inhibition of HCV infection was calculated by comparing the intracellular HCV RNA transcript between the peptide treatment and solvent control. The results (FIG. 2F-G) show that the EC50 of peptide #1 is approximately 300 nM under these conditions.
Example 5: Determination of the Mechanism of Antiviral Activity of
Peptide U 1 (SEQ ID NO:43)
To define the mechanism of antiviral activity of peptide #1, its ability to prevent the binding/attachment/uptake by cells of viral RNA in an infectious inoculum cells was examined. Huh-7.5.1 cells were seeded at 8000 cells per well in a 96-well plate. Sixteen hours later, the cells were incubated with HCV at MOI = 0.1 in the presence or absence of peptide at a concentration of 18 μM. After adsorption for 4 hours at 37 °C, the virus-peptide inoculum was removed. The cells were washed 2 times, lysed and subjected to RNA analysis. The HCV RNA transcript level was measured by real time quantitative polymerase chain reaction (RT-QPCR) assay and normalized to cellular GAPDH levels. Inhibitory activity was quantified by comparing the amount of cell-associated HCV RNA in cells exposed to the virus-peptide inocula versus the virus-DMSO control. The results (FIG. 3) indicate that peptide 1 (and peptide 2, which overlaps with peptide 1) significantly blocks viral binding/attachment/uptake while none of other peptides are active at this level.
To further define the mechanism of action, peptide #1 was added to the cells at different times relative to the time of addition of the inoculum. To determine the mechanism of action, that is, to determine if the inhibitory peptides act at the level of the virus or the cell or both, the L-isomer of peptide 1 was added to the virus or to the cells at different times relative to inoculation according to the following protocol. Huh-7.5.1 cells were seeded at 8000 cells per well in a 96-well plate and the next day they were infected with 800 ffu/well of HCV. Peptide was added at a final concentration of 18 μM under the following conditions: (1) pre-inoculation: peptide was added to cells for 4 h at 37° C followed by washing 4 times with growth medium before virus infection; (2) co-inoculation: peptide was added to cells together with virus for 4 hours at which time the cells were washed as above and replenished with complete media without peptide; (3) post^inoculation: cells were infected for 4 hours at which point the virus was removed and the peptide was added and left on the cells for the duration of the experiment without washing. At 24 and 72 hours postinfection, cells were lysed and the HCV RNA level was measured by real time RT-QPCR and normalized to cellular GAPDH levels.
As shown in FIG. 4B, peptide 1 was strongly inhibitory if it was added to the cells together with the virus; much less inhibitory if added to the cells 4 hours after infection; and entirely noninhibitory if added to the cells for 4 hours and removed before the virus was added. These results suggest that peptide 1 either blocks a very early step in the HCV life cycle or it is virocidal for HCV.
To distinguish between these alternatives, an HCV virocidal assay was performed in which viral supernatant was pretreated with the L- or D-isomers or DMSO for 1 hour before the HCV RNA content and infectivity remaining in the supernatant were measured. To determine if a peptide has virocidal activity, it was diluted in complete growth medium containing 5x105 ffu/ml of HCV to a final concentration of 18 μM and incubated at 37°C for varying lengths of time at which point the virus-peptide mixture was analyzed for total HCV RNA and infectivity and compared with a comparably prepared virus-DMSO control. HCV RNA content was measured by real time RT-QPCR, and normalized to the level of GAPDH RNA that was added to the RNA samples to control for RNA extraction efficiency. HCV infectivity was measured by diluting 25 μL of each sample in growth medium 200- fold (i.e. below the inhibitory concentration of the diluted peptide) and residual infectivity was determined by incubating the diluted samples with Huh-7.5.1 cells and counting the number of HCV E2 -positive foci 3 days later.
As illustrated in FIG. 4C, the peptides reduced the total HCV RNA content and infectivity by 100-fold and more than 10,000-fold, respectively, within 1 hour of incubation in contrast to the DMSO control. These results suggest that peptide 1 is virocidal to HCV. To confirm this interpretation, the sedimentation velocity of untreated, peptide-treated and DMSO-treated virus particles was measured by rate zonal ultracentrifugation in continuous sucrose gradients. Briefly, the L- and D- isomers of peptide 1 (18 μM) were added to separate aliquots of viral supernatant containing 1x105 ffu/mL of HCV and incubated for 4 hours at 37 °C. A third aliquot of virus was incubated with 0.5 % DMSO as a control. After incubation, 100 μL samples were analyzed by velocity sedimentation ultracentrifuge as described in the Methods. Twelve fractions (400 μL each) were collected from the top and analyzed for virus infectivity and HCV RNA content. As shown in FIG. 4D, the DMSO control samples displayed a unique peak of viral RNA which was virtually abolished by the L- and D- isomers. As expected, the infectivity of all fractions was abolished as well. Collectively, these results strongly suggest that peptide disrupts the structural integrity of HCV; i.e. it is virocidal for HCV. Since peptide 1 is derived from the membrane anchor domain of NS5A which is predicted to be an in-plane amphipathic alpha helix, it may destabilize and disrupt the HCV virion by permeabilizing its envelope. To test this hypothesis, a liposome dye release assay was conducted. The liposome dye release assay involved incubating cholesterol-phospholipid liposomes encapsulating a fluorescent dye with peptides or DMSO and then measuring fluorescence release.
Liposomes (Large Unilamellar Vesicles, LUV) were prepared as follows. A mixture containing 28 mg of total lipids (12 mM) composed of 10 parts 1- Palmitoyl-2-Oleoyl-5/?-Glycero-3-Phosphocholine (POPC), 1 1 parts 1,2- Dipalmitoyl-sw-Glycero-3-Phosphocholine (DPPC), 1 part 1 -Palmitoyl-2-Oleoyl-sfl- Glycero-3-[Phospho-L-Serine] (POPS), and 6 parts cholesterol (Avanti Polar Lipids, Inc., Alabaster, AL) was dissolved in 1 mL chloroform, 1 mL ether, and 2 mL sulforhodamine B (100 mM in 10 mM Hepes, pH 7.2; SulfoB, Molecular Probes, Eugene, OR). The mixture was sonicated at 4 0C in a Branson 2210 waterbath sonicator (Danbury, CT) for 10 minutes. After removing organic solvents in a Buchi Rotavapor R-114 (Labortechnik AG, Flawil, Switzerland), the lipids were resuspended in another 2 mL of SulfoB and the resultant lipid vesicles were sized by repeated extrusion through a stack of 0.8, 0.4, and 0.2 μm polycarbonate membrane filters using a Mini-Extruder (Avanti Polar Lipids, Inc., Alabaster, AL). The SulfoB-loaded liposomes were separated from unentrapped SufloB on a Sephadex G-25 column. Dye release assays were performed in an Aminco-Bowman Series 2 Luminescence Spectrometer (Thermo Electron Corporation, Waltham, MA).
Twelve microliters of liposomes were diluted to a final concentration of 120 μM in 1 mL Hepes buffer in a stirred cuvette at room temperature. The samples were excited at a wavelength of 535 nm, and emission was monitored at 585 nm. After 60 seconds equilibration, peptides (10 μL) were added to the cuvette starting at a concentration of 10 μM and the kinetics of membrane disruption was monitored by the increase in SulfoB fluorescence. The percentage of SulfoB released by the addition of peptides was calculated by the equation % SulfoB released = 100 * (F - F0)/(Fm - F0) where F is the fluorescence intensity observed in the presence of the peptides, F0 is the basal fluorescence intensity before addition of peptide, and F100 is the fluorescence intensity corresponding to 100 % SulfoB release obtained by the addition of 25 μL of 10 % Triton X-100. As shown in FIG. 4E, the L- and D-isomers of peptide 1 (10 μM) instantly permeabilized 70 % of the liposomes while DMSO was inactive. The effect was dose-dependent down to 160 nM, at which point 20 % of the liposomes were permeabilized (not shown). These results suggest that the virocidal effect of peptide
1 reflects its ability to permeabilize cholesterol-rich phospholipid membranes. Example 6: The L- and D-forms of Peptide 1 Inhibits HCV Infection at Noncytotoxic Concentrations in Vitro and in Vivo Peptides composed of L-amino acids are susceptible to proteolysis, which could shorten their half-life and, thus, their biological activity. To examine this possibility and to determine if specific peptide-viral protein interactions mediate antiviral activity, peptide 1 was synthesized using all D-amino acids, purified to > 95 % homogeneity, and its antiviral activity and serum stability were compared with a similarly pure preparation of the L-type version of peptide #1. Both L- and D-type peptides were diluted 1 : 100 in complete growth medium (10 % FBS) and mixed with an equal volume of viral supernatant.
In addition, to compare the serum stability of the L- and D-type peptides, the diluted peptide was incubated at 37 °C for 1 hour, 2 hours and 4 hours before mixing with viral supernatant. The virus-peptide mixture was then used to infect Huh-7.5.1 cells (MOI = 0.1). After adsorption for 4 hours at 37 °C, the virus- peptide inoculum was removed. The cells were washed 2 times, overlaid with 120 μL fresh growth medium and incubated at 37 °C for 3 days. Cells were lysed and subjected to RNA analysis. The HCV RNA transcript level was measured by real time RT-QPCR and normalized to cellular GAPDH levels. The results (FIG. 4F) show that whereas approximately 95 % of the antiviral activity of the L-peptide was lost within 1 hour in 10 % FBS at 37 °C, the D-peptide was entirely stable for at least 4 hours under the same conditions. Thus, in addition to low immunogenicity and possible oral bioavailability, peptides composed of D-amino acids have the potential therapeutic advantage of enhanced serum stability.
The 50 % inhibitory (IC50) and 50 % lethal (LC50) concentrations of Peptide 1 were tested. To determine the IC50 (the peptide concentration that inhibits HCV infection by 50%) of peptide 1, >95% pure peptide stock solution (3.6 mM in DMSO) was serially 2-fold diluted in DMSO. An aliquot of peptide from each dilution was then diluted 1 : 100 in complete growth medium and mixed with an equal volume of virus supernatant. The virus-peptide mixture was then used to infect Huh-7.5.1 cells (moi = 0.1) and incubated at 37 °C for 3 days. The % inhibition of HCV infection was calculated by comparing the intracellular HCV RNA content in cells infected with virus between peptide treatment and DMSO control. Peptide cytotoxic activity was measured by MTT cytotoxicity assay according to the manufacturer's instructions (ATCC Cat# 30-101 OK) and described in the Methods. The peptide concentration that reduced 50 % of the OD reading was designated the LC5O-
As shown in FIG. 4 A, both the L- and D-isomers of peptide 1 were highly inhibitory, displaying IC50 values of 0.79 μM and 0.32 μM, respectively, while their LC50 values were approximately 100-300-fold higher. The slightly lower (IC5o) of the D-isomer may reflect its increased serum-stability since the antiviral activity of the L-isomer was reduced by approximately 2 logs after 1 hour of preincubation in serum while the D-isomer was fully active after at least 24 hours of incubation (FIG. 4G). Peptide 1 also displayed a favorable toxicity profile in vivo since both the L- and D-isomers were entirely nontoxic when administered intravenously at doses as high as 0.5 mg per 25 gm C57B1/6 mouse administered at weekly intervals for 3 consecutive weeks. See Table 6 below. Importantly, peptide 1 was not immunogenic after repeated intravenous administration as antibodies directed against peptide 1 were not detected in the serum of those mice as shown in the following Table 7.
Table 6 Mouse Cytotoxicity of Peptide I
Pre-
Compounds day 1 day 2 day 4 day 6 day 8 day 10 day 12 day 14 day 16 day 18 day 20 day 22 inject
DMSO* 23.5 22 22.3 23.1 24 24.3(2nd) 24.7 24.7 25.1 25.3(3rd) 25.6 25.7 26.8
DMSO 28.1 28.3 28.4 28.6 28.5 28.1
DMSO 26.9 26.9 27.2 27.4 27.7 27.7
DMSO 25.7 25.7 25.6 26.1 26.8 27.4
L-Peptide I 1 l .Omg 23.3 23.3 23.4 23.6 23.5 23.6
L-Peptide 1 , l .Omg 25.4 23.4 23.7 24.8 25.4 25.9
L-Peptide 1 , l .Omg 24.0 died
L-Peptide 1 , l .Omg 23.8 died
L-Peptide 1 0.5mg 23.1 23.2 23.9 24.1 24.8 25.1
L-Peptide 1 0.5mg 23.9 24.2 24.7 24.7 24.9 25.3
L-Peptide 1 0.5mg 27.6 27.8 28.5 28.9 29.4 29.3
D-Peptide 1 , l .Omg 26.5 24.5 died
D-Peptide 1 , l .Omg 26.3 23.3 died
D-Peptide 1 , l .Omg 26.4 24.1 died
D-Peptide I , l .Omg 28.7 26.0 died
D-Peptide 1
0.5mg 24.8 23.2 23.1 23.1 23.7 24.3
D-Peptide 1
0.5mg* 24.5 23.1 24.6 25.4 25.4 25.3(2nd) 25.1 25.4 25 25.3(3rd) 25.2 25.4 25.5
D-Peptide 1
0.5mg* 25.2 23.9 25.4 25.5 25.6- 25.7(2nd) 25.9 26.1 27.1 27.5(3rd) 27.6 28.3 28.6
D-Peptide 1
0.5mg* 24.8 23.2 23.1 23.1 23.7 24.3(2nd) 24.8 25.2 24.7 25.4(3rd) 25.8 25.9 26.1
* Animals were injected three times with 0.5 mgs of the D-isomer or control DMSO at weekly intervals and monitored every other day
Table 7 In vivo immunogenicity of D-Peptide 1
Serum D-Peptide 1 #1 D-Peptide 1 #2 D-Peptide 1 #3 HA peptide HBV core dansyl-Peptide
DMSO Pre- bleed dilution Pre- bleed Pre- bleed Pre- bleed control control 1 control dansyl-
D- D- D- D-
HA HBV
Coating Peptide PBS Peptide PBS Peptide PBS Peptide PBS D- pRς peptide PBb core 1 1 1 1 Peptide ™ύ 1
1 :10 0.526 0.497 0.354 0.345 0.429 0.437 0.314 0.293 1.1305 0.0869 0.5761 0.0386 1.2921 0.118
1 :20 0.401 0.399 0.278 0.271 0.389 0.394 0.251 0.261 1 :40 0.285 0.271 0.159 0.163 0.251 0.265 0.176 0.180 1 :80 0.177 0.177 0.104 0.108 0.158 0.159 0.107 0.108
Figure imgf000107_0001
Figure imgf000107_0002
Serum D-Peptide 1 #1 D-Peptide 1 #2 D-Peptide 1 #3 HA peptide HBV core dansyl-Peptide
DMSO 3rd bleed dilution 3rd bleed 3rd bleed 3rd bleed control control 1 control dansyl-
D- D- D- D-
HBV
Coating Peptide PBS Peptide PBS Peptide PBS Peptide PBS PBS pe Hp?tiHde PBS core Peptide 1 1 1 1
1
1 :10 0.302 0.274 0.365 0.342 0.268 0.254 0.351 0.385 0.9347 0.0784 0.5815 0.0414 1.0994 0.116
1 :20 0.259 0.247 0.292 0.304 0.271 0.290 0.306 0.320 1 :40 0.177 0.182 0.199 0.198 0.181 0.191 0.189 0.207 1 :80 0.114 0.118 0.134 0.129 0.116 0.122 0.126 0.131
Example 7: Peptide Toxicity
Peptide cytotoxicity was measured by MTT cytotoxicity assay based on the protocol provided in the ATCC MTT assay kit (Cat# 30-1010K). In brief, 5000- 10,000 cells were seeded per well in a 96 well plate. Following overnight growth, 100 μL fresh medium plus 20 μL of 2-fold serially diluted peptide was added. Media without peptides was added to at least 3 wells as untreated controls. The cells were then incubated for 72 hours at 37 0C, 5 % CO2. After this incubation, 1/10 volume of MTT solution (5 μg/mL in PBS) was added to each well, and the cells were returned to the incubator. Two hours later, the medium was removed, 150 μL DMSO was added to dissolve the purple precipitate formazan, and the plate was shaken at 150 rpm for 10 minutes. Absorbance at 570nm less background at 670 nm is a reliable measure of cell death. Cytotoxicity (LD50) of individual peptides was defined as the peptide concentration that caused 50 % cell death. The results (FIG. 5A) show that the LC50 values of the L- and D-forms of peptide 1 are virtually identical (3.8 and 3.7 μM, respectively, without FBS; and 26.7 and 36.8 μM with FBS).
Fresh human blood (treated with EDTA) was centrifuged 100Og for 10 minutes to remove the supernatant and buffy coat. The red blood cells were then washed twice in PBS, and resuspended to a final concentration of 8 % with and without 16 % FBS. Serial 2-fold dilutions of peptide were prepared in 60 μL PBS in a 96- well microtiter plate, and 60 μL of the suspended human red blood cells with and without FBS were added. The plates were incubated for lhour at 37 0C. After this incubation 120 μL PBS was added to each well and the plates were centrifuged at 100Og for 5mins. Aliquots of 100 μL of supernatant were transferred to a new 96-well microtiter plate. Hemoglobin release is monitored using microplate ELISA reader by measuring the absorbance at 414 nm. In the plate, zero and 100 % hemolysis are determined in PBS and 0.1 % Triton X-100, respectively. Percent hemolysis as determined according to the formula: [(A4I401H in the peptide solution - A414Pm in PBS) /(A4140n, in 0.1 % Triton X-100 - A4Mmn in PBS)] x 100. The results (FIG. 5B) indicate that the LC5O values of the L- and D-peptides against human red blood cells, when tested in the presence of serum, were similar to each other and similar to their LC50 against hepatocyte cell lines in vitro. Importantly, the LC50 values against both cell types is consistently 50 to 100-fold higher than the EC50 values for each peptide.
As a preliminary measurement of the in vivo cytotoxicity of the peptides 1, 2 and 3 (see Table 4) a group of three mice (BALB/c mice, 7 weeks old, about 23 g) were each injected with 92 μg L-type peptide 1 ( ~ 4 mg/kg) in 200 μL PBS (spun 14,000 rpm for 3 minutes before injection). In the control group, each of three mice was given 200 μL PBS containing 5 % DMSO. The mice were monitored for acute toxicity during the first 3 hours after injection. Results are summarized in the following table.
Table 8: Peptides 1, 2 and 3 are Nontoxic in C57BL/6 Mice
Figure imgf000110_0001
No change in appearance, activity or behavior was observed. The mice were then weighed on days 0, 3, 5, 7 and 10. Peptide-injected mice gained weight at the same rate as the controls.
Example 8: Physical Properties of Peptide 1 Correlate with its Antiviral Activity
The secondary structure of peptide 1 (SEQ ID NO:43) was analyzed using the tool of helical Wheel Applet available online at cti.itc.virginia.edu/~cmg/Demo/wheel/wheelApp.html (last visited Aug. 15, 2006). The resulting helical wheel (FIG. 6A) shows that peptide 1 is amphipathic, having both hydrophobic and hydrophilic faces.
The secondary structure of peptide 1 was also analyzed using circular dichroism (CD) spectroscopy using an Aviv model 62DS CD spectrometer (Aviv Associates Inc., Lakewood, N.J.). The CD spectra of peptides were measured at 25 0C using a 1 mm path-length cell. Three scans per sample were performed over the wavelength range of 190 to 260 nm in 10 mM potassium phosphate buffer, pH 7.0. Data were collected at 0.1 nm interval with a scan rate of 60 nm/min and is given in mean molar ellipticity [q]. The peptide concentrations were 50 μM. Spectra highly characteristic of amphotropic α-helices were observed for the L and D form of peptide 1 (FIG. 6B). In addition, dansylation enhances the amphotropic α-helical structure of peptide 1 (FIG. 6C). Thus, the peptides of the invention can have dansyl moieties covalently attached thereto. The secondary structures of various truncated derivatives of peptide 1 (Table
9) were analyzed using CD spectroscopy. Results indicate that a deletion of 2 or 4 amino acids from the C-terminus of peptide 1 did not eliminate the α-helical structure of the peptide (FIG. 6D). In contrast, deletion of 2 amino acids from the N-terminus of peptide 1 did eliminate the α-helical structure of the peptide (FIG. 6E).
The anti-HCV activity of these truncated variants of peptide 1 were also determined. Results in the table below indicate that the antiviral activity of peptide 1 (L-form) correlates with its α-helical structure.
Figure imgf000112_0001
Example 9: Liposome-Dye Release Assay
The peptides in Table 9 were also tested using the liposome release assay as discussed above. Results (FIG. 7) indicate that the antiviral activity of the various derivatives of peptide 1 correlates with the ability to cause liposome dye release. Thus, the antiviral activity of peptide 1 correlates with the α-helical structure and liposome dye release as summarized in the following table.
Table 10: Structure/function Relationship of Peptide 1 and Truncations Thereof
Figure imgf000113_0001
Figure imgf000113_0002
Example 10: Structure-activity Analyses of the Antiviral Activity of
Peptide 1
To determine whether the antiviral activity of peptide 1 is dependent on its primary amino acid sequence, four derivative peptides from peptide 1 were synthesized to a purity > 95 %. The four derivatives having the same composition of amino acids included (1) the reversed the sequence of peptide 1 (also called retro- peptide); (2) scrambled hydrophobic amino acids; (3) scrambled hydrophilic amino acids; and (4) a derivative in which the aspartic acid residues (D) were replaced with proline residues (P). The antiviral activity of the peptides was examined by HCV focus reduction assay at three peptide concentrations: 18 μM, 6 μM and 2 μM, as described above.
Results, which are summarized in the following table shows that the antiviral activity of peptide 1 correlates with the α-helical structure, but not with the primary amino acid sequence. Table 11: Antiviral Activity of Scrambled Derivatives of Peptide 1
Figure imgf000114_0001
In sum, by screening a synthetic HCV peptide library, 13 peptides were identified that could inhibit HCV infection efficiently. Peptide 1 , for example, derived from the membrane anchor domain of NS5A (NS5A-1975) was highly potent as a single dose of this peptide completely blocked HCV infection with an EC5o of 289 nJVl without evidence of cytotoxicity. The antiviral effect was evident for at least 1 1 days post infection. The peptide was most active when it was added to the cells together with the virus. Preincubation of the peptide with virus significantly reduced viral attachment and infectivity, suggesting that the antiviral activity of NS5A-1975 interacts directly with the virus and destabilizes it. The D- amino acid form of the peptide is fully active, and the D- and L-forms of the peptide display amphipathic α-helical structure in solution and induce permeabilization of artificial liposomes. Comparative analysis of the antiviral activity, alpha-helicity and membranolytic activity (not shown) of a series of C- and N-terminal truncation mutants (peptide 3-13) were performed. All peptides were synthesized with a purity >95% and their antiviral activities were compared by determining their IC50 values. The helicity of the peptides was determined by circular dichroism (CD) analysis using an Aviv model 62DS circular dichroism (CD) spectrometer (Aviv Associates Inc., Lakewood, N.J.)- The spectra were measured at 25 0C using a 1 mm path- length cell. Three scans per sample were performed over a wavelength range from 190 to 260 nm in 10 mM potassium phosphate buffer (pH 7.0) with or without 50 % Trifluoroethanol (TFE). Data were collected at 0.1 nm interval with a scan rate of 60 nm/min and is given in mean molar ellipticity [θ]. The peptide concentrations were 50 μM. The percentage of helicity of a peptide was calculated as described in Chen et al., Biochemistry 13, 3350-9 (1974)). Results are shown in the table below
Table 12 Structure-activity Analyses of the Antiviral Activity of Peptide 1
Table 1 Structure-activity analyses (SARs) of Viracide
No. Peptides Description IC50 (μM) Helicity (%) a. Viracide
1 SWLRDIWDWICEVLSDFK (SEQIDNO^S) L-isomer 0.79 37.2 2 SWLRDIWDWICEVLSDFK (SEQ ID NO: 43) D-isomer 0.34 39.0 b. Length Series
3 SWLRDIWDWICEVLSD (SEQ ID NO: 94) ΔC-, 2a.a 0.98 37.1
4 SWLRDIWDWICEVL (SEQ ID NO: 92) ΔC-, 4a.a 11.3 34.2
5 SWLRDIWDWICEV(SEQIDNO: 103) ΔC-, 5a.a >27 O
6 SWLRDIWDWICE (SEQ ID NO: 104) ΔC-, 6a.a >27 O
7 SWLRDIWDWI (SEQ ID NO: 105) ΔC-, 8a.a >27 O
8 SWLRDIWD (SEQ ID NO: 106) ΔC-, lOa.a >27 O
9 LRDIWDWICEVLSDFK (SEQ ID NO: 107) ΔN-, 2a.a >27 4.4
10 DIWDWICEVLSDFK(SEQIDNO: 108) ΔN-, 4a.a >27 2.2
11 WDWICEVLSDFK (SEQ ID NO: 109) ΔN-, 6a.a >27 O
12 WICEVLSDFK (SEQ ID NO: 110) ΔN-, 8a.a >27 O
13 CEVLSDFK(SEQ1DNO: 111) ΔN-, lOa.a >27 O
14 SGSWLRDIWDWICEVLSDFK (SEQ ID NO: 141) extend N-, 2 a.a 1.7 39.8
15 GSWLRDIWDWICEVLSDFK (SEQIDNO: 142) extend N-, 1 a.a 0.51 38.0
16 SWLRDIWDWICEVLSDFKT (SEQIDNO: 143) extend C-, 1 a.a 1.7 39.8
17 SWLRDIWDWICEVLSDFKTW (SEQIDNO: 144) extend C-, 2 a.a 0.51 38.0 c. Ampliipathicity Series
18 SWRLIDWDWICEVLSDFK (SEQIDNO: 145) less amphipathic 4.0 22.6
19 SWRLDIWDWICESVLDFK (SEQIDNO: 117) less amphipathic >30 21.5
20 GIGKFLHSAKKFGKAFVGEIMNS (SEQlDNO: 146) Magainin 2 >27 21.7
21 DWLKAFYDKVAEKLKEAF (SEQIDNO: 120) Apo 18A >28 48.0
22 VLDLIYSLHKQINRGLKKIVL (SEQIDNO: 147) BVDV analogue >36 31.6 d. Primary Sequence Series
23 KFDSLVECIWDWIDRLWS (SEQ ID NO: 96) L-Retro 0.85 40.7
24 KFDSLVECIWDWIDRLWS (SEQ ID NO: 96) D-Retro 0.48 ND
25 KWLCRIWSWISDVLDDFE (SEQ ID NO: 98) Hydrophilic scrambled 0.50 38.1
26 SrWRDWVDLICEFLSDWK. (SEQ ID NO: 97) Hydrophobic scrambled 0.40 37.0 e. Genotype Series
27 SWLRDVWDWICTVLTDFK (SEQ ID NO: 80) HCV 1 b analogue 3.9 50.0
28 SWLRDVWDWVCTILIDFK (SEQ ID NO: 79) HCV 2a analogue 2.1 30.2
29 DWLRπWDWVCSVVSDFK (SEQ ID NO: 123) HCV 3a analogue 0.55 42.4
30 SWLWEVWDWVLHVLSDFK (SEQ ID NO: 124) HCV 4a analogue 7.0 39.3
31 TWLRAIWDWVCIALTDFK (SEQ ID NO: 125) HCV 5a analogue 7.1 42.0
32 SWLRDVWDWVCTVLSDFK (SEQ ID NO: 126) HCV 6a analogue 3.5 36.7
/ Cysteine Series
33 SWLRDIWDWISEVLSDFK (SEQ ID NO: 127) C to S 13.5 31.4
34 SWLRDIWDWIREVLSDFK (SEQ ID NO: 139) C to R 12.5 35.7
35 SWLRDIWDWIEEVLSDFK (SEQ ID NO: 140) C to E 13.0 31.9
A retro-peptide has the reverse sequence of the parental peptide. The prototype strains of HCV genotype 3a, 4a, 5a, and 6a selected were K3A (GenBank accession D28917), EG.ED43 (Yl 1604), ZA.SA13 (AF064490), and HK.6a33 (AY859526), respectively.
The 16-mer SWLRDIWD WICEVLSD (SEQ ID NO: 94) (peptide 3 in Table 12) retains full antiviral activity, alpha-helical and membranolytic properties (Table 12b). The antiviral activity of the N- and C-terminally truncated peptides correlated with their membrane permeability activity and amphipathic α-helical structure. The simultaneous loss of all three activities in the truncation mutants suggests that they are probably functionally linked to the ability of the peptide to inhibit HCV infection.
As illustrated in the helical wheel diagram shown in FIG. 6A and B, peptide 1 is strongly amphipathic. To determine if amphipathicity is necessary for its antiviral activity, we tested two variants whose amphipathicity was reduced by swapping the positions of its amino acids (underlined in Table 12c) while maintaining its amino acid composition and alpha helicity (peptide 18 and 19). As shown in Table 12c, the antiviral activity of these peptides was reduced 5- to >30- fold. These results contrast with the retention of antiviral activity in the variants whose hydrophobic or hydrophilic amino acids were selectively scrambled while retaining their amphipathicity (Table 12d, peptide 25 and 26). Collectively, these results suggest that amphipathicity is necessary for antiviral activity. It is not sufficient, however, since three previously well-characterized amphipathic alpha- helical peptides such as Maganin 2 (Bechinger et al., Protein Sci 2, 2077-84 (1993)), apolipoprotein 18A (Chung et al., J Biol Chem 260, 10256-62 (1985)), and the peptide 1 analogue from BVDV (Sapay et al., Biochemistry 45, 2221-33 (2006)) had no antiviral activity against HCV even at high concentrations (Table 12c, peptide 20-22). To determine if primary amino acid sequence is required for antiviral activity, a series of peptides that have the same amino acid composition as the parental peptides but different primary sequences was tested. As shown in Table 12d, analogues of peptide 1 containing either reversed (retro-) (peptide 23 and 24) or scrambled sequences (peptide 25 and 26) that maintained amphipathicity and alpha-helicity were fully active. These results indicate that the antiviral activity of the peptide is independent of its primary amino acid sequence as long as its amphipathic alpha-helical structure is maintained.
To determine if the antiviral activity is strictly dependent on amino acid composition, we compared the activity of several analogues of peptide 1 from six HCV genotypes whose amino acid sequence and composition are not strictly conserved. As shown in Table 12e, the antiviral activity of the peptides was found to vary over a 10-fold range with genotypes Ia and 3a (peptide 28 and 29) being most active and genotypes 4a and 5a (peptide 30 and 31) least active. These results imply that the amino acid composition of the peptide appears to influence the efficiency of its antiviral activity. It is noteworthy that the cysteine residue in peptide 1 , which could lead to disulfide bond formation between peptide monomers, is essential for antiviral activity (Table 12f).
Example 11: Effect of Peptides on VSV Infection
To determine whether the antiviral activity of peptide #1 is specific for HCV, similar experiments were conducted on other enveloped viruses, e.g. vesicular stomatitis virus (VSV). Two assays were used to test the antiviral activity of peptide #1 against VSV. Blockade of infection. To examine if peptide 1 blocks VSV infection, peptide 1 at final concentration 18 μM and VSV from 1 to 10,000 pfu/mL were concurrently added to Huh-7 cells. In parallel, peptide and HCV (10,000 ffu/mL) were added to cells as control. After adsorption for 4 hours at 37 °C, the virus- peptide inoculum was removed. The cells were washed 2 times, overlaid with 120 μL fresh growth medium and incubated at 37 °C for 3 days. VSV and HCV infections were assessed by viral cytopathic effect (CPE) and immunostaining with antibody against HCV E2 protein, respectively.
Virucidal activity. To determine if peptide 1 has virocidal activity against VSV, peptide 1 was diluted in a complete growth medium containing 2 x 105 pfu (ffu)/mL VSV or HCV to a final concentration of 18 μM. The virus-peptide mixture was then incubated for 4 hours at 37 °C. The VSV and HCV viral titer were then determined by serial dilution and assessed by viral cytopathic effect (CPE) and immunostaining with antibody against HCV E2 protein, respectively. The result (FIG. 8) indicates that peptide 1 does not block VSV infection and has no virocidal activity against VSV. Further experiments indicate that peptide 1 does not block infection by influenza virus, vaccinia virus, Borna disease virus, lymphocytic choriomeningitis virus or adenovirus (data not shown).
Example 12: Effect of Peptides on Dengue-2 Infection
The following experiments were performed to determine which peptides inhibited Dengue-2 viral infection. Peptides tested are shown in the table below.
Table 13
Enzyme-linked Immunosorbent Assay. Vero cells (80,000 cells/well/ml) were seeded for 24 h pre-infection in 24-well plates. Cells were exposed to Dengue- 2 (derived from Vero cells) in the presence of increasing concentration of peptide (or DMSO as control). Viruses and peptide were not removed (cells were not washed) throughout the incubation. Infection was analyzed after 5 days using ELISA that measured the amounts of Dengue-2 capsid released in the supernatant of infected Vero cells.
Fluorescent Foci Assay: Vero cells were seeded for 24 h pre-infection in 96- well plates. Cells were exposed to Dengue-2 in the presence of increasing concentrations of peptide (or DMSO as control). Viruses and peptide were washed away 2 h post-infection. Supernatants were collected every 3 days post-infection and added to fresh Vero cells for fluorescent foci assay. Newly infected Vero cells were fixed with 4% formaldehyde after 3 days. Cells were then stained with Dengue Env antibodies followed by Alexa-fluor dye conjugated secondary antibodies. Foci were counted using a fluorescent microscope.
Results are summarized in the following table and in FIG. 9.
Table 14: Inhibition of Dengue Infection as Detected by ELISA
Figure imgf000122_0001
As illustrated in Table 14 and FIG. 9, Dengue infection was inhibited by the present peptides in a dose-dependent manner. Essentially 100% inhibition of Dengue viral infection was observed at concentrations of 20 μM (FIG. 9).
Intracellular FACS Assay: Vero cells were seeded for 24 h pre-infection in 6-well plates. Cells were exposed to Dengue-2 in the presence of increasing concentrations of peptide (or DMSO as control). Viruses and peptide were washed away 2 h post-infection. Cells were taken for intracellular staining 3 days post- infection. Cells were stained with appropriate isotype control, Dengue Env, Dengue capsid or tubulin antibodies. Cells were analyzed by FACS.
Results when using peptide concentrations of 20 μM are shown in Table 15. Results for 1.25 to 20 μM are summarized in the graph shown in FIG. 10A-B. FIG. 1 OB-D show that strong inhibition of Dengue viral infection is correlated with the amphipathicity of the peptide structure, rather than the precise amino acid sequence of the peptide. Thus, following peptides are highly inhibitory of Dengue viral infection: peptide 1 (also called 2022 and L-7208, SEQ ID NO:43), peptide 3222 (SEQ ID NO: 127), peptide 3226 (SEQ ID NO: 128), peptide 3228 (SEQ ID NO: 130), peptide L-7208 2D to 2 Pro (SEQ ID NO:91), and L-7208 HS with hydrophobic amino acids scrambled (SEQ ID NO:97).
Table 15: Inhibition of Dengue Infection as Detected by FACS
* PI -Propidium iodide staining for measuring dead cells; N/A - non-applicable
Figure imgf000124_0001
As shown in Table 15 and FIG. 10A-B, the present peptides inhibit Dengue viral infection in a dose-dependent manner. Essentially 100% inhibition of Dengue viral infection was observed at concentrations of 20 μM (FIG. 1 OA-B).
Fluorescent Foci Assay. Vero cells were seeded for 24 hours pre-infection in 96-well plates. Cells were exposed to Dengue-2 in the presence of increasing concentrations of peptide (or DMSO as control). Viruses and peptide were washed away 2 hours post-infection. Supernatants were collected every 3 days postinfection and added to fresh Vero cells for fluorescent foci assay. Newly infected Vero cells were fixed with 4 % formaldehyde after 3 days. Cells were then stained with antibodies directed to the Dengue Envelop protein followed by Alexa-fluor dye conjugated secondary antibodies. Foci were counted using a fluorescent microscope. The results (not shown) confirm that the present peptides strongly inhibit Dengue viral infection. Essentially 100% inhibition of Dengue viral infection was observed at concentrations of 20 μM.
Example 13: Peptide 1 has Strong Antiviral Activity Against West Nile Viral Infection
In this study, the activity of peptide 1 and peptide 2012 (SEQ ID NO: 94) against the West Nile Virus (WNV), a Flavivirus, was examined. A549 cells were infected with 102 to 105 PFU/mL WNV (New York strain) in the presence of 0.5 % DMSO, peptide 1 or peptide 2012, where a final concentration of peptide was 18 μM in 0.5 % DMSO. After 3 days of incubation at 37 °C, the cells were fixed and subjected to immunoperoxidase staining to detect WNV protein. Results (FIG. 11) show that the cell monolayer with 105 PFU/mL treated with DMSO was almost completely destroyed, and all the cells in the lower titer wells expressed WNV protein. In contrast, the monolayers in the peptide-treated cells were intact, and little or no WNV protein was detected. In particular, the WNV protein staining intensity was the same as the uninfected negative control wells, irrespective of the dose of the viral inoculum. These results demonstrate that peptide 1 (SEQ ID NO:43) and peptide 2012 (SEQ ID NO:94) have strong antiviral activity against WNV infection.
Example 14: Peptide 1 and Variants Thereof Inhibit HIV Infection This Example illustrates that the peptides of the invention can inhibit infection by human immunodeficiency viruses. Materials and Methods
Viruses were generated by liposome-mediated transfection of 293T cells using Genejuice (Novagen). Viral supernatants were harvested 48 h post- transfection and filtered through a 0.2-μM pore size filter to remove cellular debris. Viral inoculum was standardized by p24 (HIV-I capsid) enzyme-linked immunosorbent assay (PerkinElmer Life Sciences).
TZM-bl reporter cells are CD4+ CXCR4+ CCR5+ HeLa cells, which contain LacZ gene driven by the HIV-I LTR (promoter). Upon infection, HIV-I Tat protein is produced and activates the HIV-I LTR. TZM-bl cells (80,000 cells/well/ml) were seeded for 24 h pre-infection in 24-well plates.
The following peptides were tested for inhibition of HIV infection.
Table 16: Peptides tested for Anti-HIV Activity
Peptide No. Sequence SEQ ID NO:
6938 LYGNEGCGWAGWLLSPRG 6
2015 SWLRDIWDWI 105
2054 SWLRDIWDWICEV 103
2018 DIWDWICEVLSDFK 108
L-2022 (L-7208) SWLRDIWDWICEVLSDFK 43
D-2022 (D-7208) SWLRDIWDWICEVLSDFK NA
L-7208 HS SIWRDWVDLICEFLSDWK 97
T 7908 Q1 f2D → 2 Pro) SWLRPIWPWICEVLSDFK
3222 SWLRDIWDWISEVLSDFK 127
3226 SWLDRIWRWICKVLSRFE 128
3227 SWLDDIWDWICEVLSDFE 129
3228 SWLRRIWRWICKVLSRFK 130
3229 SWRLDIWDWICESVLDFK 119 3244 DWLKAFYDKVAEKLKEAF 120
3310 DIWDWICEV 121
3311 RDIWDWICEV 122
Note that the L-2022 peptide (also called "peptide 1" and L-7208) has the same sequence as the D-2022 peptide (also called D-7208) but the L-2022 peptide is composed of L-amino acids while the D-2022 peptide is composed of D-amino acids. In addition, the L-7208 HS peptide (SEQ ID NO:97) and the 3229 peptide (SEQ ID NO:119) have the same amino acid composition as the L-7208 peptide (SEQ ID NO:43). However, while the L-7208 HS peptide has an amphipathic structure, the 3229 peptide does not. The 3310 peptide (SEQ ID NO:121) and the 331 1 peptide (SEQ ID NO: 122) contain the central nine and ten amino acid sequences, respectively, of the highly active peptide L-2022 (SEQ ID NO:43), but they are so short that they have minimal activity. The 3244 and 6938 peptides were used as controls.
Cells were exposed to HIV-I in the presence of increasing concentrations of peptide, using DMSO without peptide as control. The peptide was added to the virus and then immediately added to target cells. While initial experiments utilized the HIV clade B CCR5 virus (JR-CSF), later experiments were performed on other HIV strains, including recombinant HIV strains containing an NL4-3 (CXCR4) backbone and envelope (GP 120) proteins derived from either R9BaL, ADA or YU2 (all of which display CCR5 tropism).
Viruses and peptides were washed away 2 h post-infection. Infected cells were analyzed by beta-galactosidase activity 48 h post-infection. For beta- galactosidase activity, infected TZM-bl cells were washed twice with 1 ml of phosphate-buffered saline and lysed in 100 niM potassium phosphate, pH 7.8, containing 0.2% Triton X-IOO. Plates were stored at -80 °C for 16 h and thawed on ice, and 20 μl of lysate was transferred to a 96- well plate for detecting beta- galactosidase activity. Galacton-Star substrate (Applied Biosystems, Bedford, MA) was diluted 1 :50 in the reaction buffer diluent (100 mM sodium phosphate pH 7.5, 1 mM MgCl2, 5% Sapphire-IITM enhancer) to make the reaction buffer. 100 μl of reaction buffer was added to 20 μl of lysate, and the light emission was measured over 1 s in a microplate luminometer after 30 min. Results
The peptides of the invention are effective inhibitors of HIV infection. As shown in FIG. 12, the 2054, 2018, L-2022 and D-2022 peptides with SEQ ID NOs: 103, 108 and 43, respectively, exhibited essentially 100% inhibition of HIV infection when present at concentrations of 20 micromolar. Even lower concentrations of 2018, L-2022 and D-2022 peptides (SEQ ID NOs: 108 and 43) were also highly effective. In particular, the 2018 and both isomers of the 2022 peptide inhibited 90-100% of HIV infection at 5 micromolar concentrations. The D-isomer of the 2022 (SEQ ID NO:43) peptide remained highly active at concentrations as low as 1.25 micromolar (FIG. 12). FIG. 13 further illustrates the efficacy of the present peptides. In particular,
FIG. 13 shows that at 20 micromolar concentrations, peptide L-7208 HS (SIWRDWVDLICEFLSDWK, SEQ ID NO:97), is as active as the highly active SEQID NO:43 peptide. The L-7208 HS (SEQ ID NO:97) peptide has the same amino acid composition as the highly active SEQ ID NO: 43 L-2022 peptide, but the hydrophobic amino acids have been scrambled in the L-7208 HS peptide so that while it retains its amphipathicity, it has a different sequence than the SEQ ID NO: 43 L-2022 peptide. These data indicate that while the amphipathicity of the peptide is important for activity, the exact sequence of the peptide is not critical. The same results were observed with HIV clade B CCR5 virus (JR-CSF), and recombinant HIV strains containing an NL4-3 (CXCR4) backbone and envelope (GPl 20) proteins derived from either R9BaL, ADA or YU2 (all of which display CCR5 tropism). Moreover, the present peptides are highly effective at inhibiting infection of different cell types. Inhibition of HIV R9BaL infection in 293 T cells is shown in FIG. 13 A, and similar results for HIV R9BaL infection of CEM T cells are shown in FIG. 13B.
The present peptides disrupt HIV-I and release the viral contents into the medium. Thus, FIG. 14A illustrates that large amounts of HIV-I capsid were released from virions after treatment of HIV-I with peptide L-7208 (SEQ ID NO:43), suggesting that the peptide disrupted the virion extracellularly. In contrast, little or no HIV-I capsid was released after treatment with DMSO or with peptide 6938 (SEQ ID NO:6) (FIG. 14A) which served as negative controls.
In keeping with this observation, significantly less viral-associated HIV-I capsid was observed after treatment with peptide L-7208 (SEQ ID NO:43) than after treatment with DMSO or with peptide 6938 (SEQ ID NO:6) (FIG. 14B). Finally, FIG. 14C shows the percent of HIV-I capsid internalized into cells after the virions were treated with DMSO and 5 or 10 micromolar of peptides 6938 (SEQ ID NO:6) or L-7208 (SEQ ID NO:43). While essentially 100% of control amounts of HIV-I capsid were internalized when the virions were treated with 5 or 10 micromolar peptide 6938 (SEQ ID NO:6) before infection, HIV-I capsid internalization was inhibited up to 10-fold by treatment with peptide L-7208 (SEQ ID NO:43).
FIG. 15 shows that peptides with amphipathic structures similar to the amphipathis structure of peptide L-7208 (SEQ ID NO:43) are also strongly inhibitory of HIV infection. Thus, amphipathic peptides with strong anti-HIV activity include peptide 3222 (SEQ ID NO: 127), peptide 3226 (SEQ ID NO: 128), peptide 3228 (SEQ ID NO: 130), peptide L-7208 2D to 2 Pro (SEQ ID NO:91), and L-7208 HS with hydrophilic amino acids scrambled (KWLCRIWSWISDVLDDFE, SEQ ID NO:98). Therefore, as observed for HCV and Dengue, the antiviral activity of the present peptides towards HIV is dependent on the amphipathicity of the peptide. Furthermore, when HIV infectivity was inhibited, HIV capsids were released into the supernatant, indicating that the peptide disrupts or lyses the HIV virion. Therefore, these peptides, which were derived from hepatitis C viral genome, are highly effective anti-HIV agents and can readily inhibit HIV infection.
Example 15: Antiviral Specificity and Spectrum of Peptide 1
To determine if the antiviral activity of peptide 1 is specific to HCV, antiviral activity against a large series of other viruses listed in Table 17 was examined as follows.
Varying concentrations of peptide or DMSO were added to virus stocks of predetermined infectivity (1-105 ffu or TCID50/mL) using 2-fold serial dilutions starting from 18-20 μM. The virus-peptide and virus-DMSO mixtures were incubated at 370C for at least 1 hour before addition to susceptible cells unless specified. In parallel, peptide and HCV (10,000 ffu/mL) were added to Huh-7.5.1 cells as a positive control for antiviral activity. After 2-4 days of infection, the cultures were assessed either by detection of a cytopathic effect (CPE) or by immunostaining with antibody against the corresponding viral protein, respectively and described in Methods. HCV infections were carried out with JFH-I (genotype 2a) and with chimeric viruses containing the structural region of prototype isolates from genotypes Ia (H77), Ib (conl) and an additional genotype 2a molecular clone (J6CF). To produce the chimeric HCV genomes a recombinant PCR approach was used. The J6CF-, H77, and Con 1 -JFH-I chimeric HCV genomes are generated as described in Pietschmann et al., Proc Natl Acad Sci USA 103, 7408-13 (2006) by replacing the corresponding JFH-I core-NS2 region in pUC-vJFH by the corresponding sequences from J6CF, H77 and Conl. Infectious JFH-I and chimeric viruses were produced by transfection of in vitro synthesized genomic HCV RNA into Huh-7.5.1 cells and virus stocks containing 104-105 ffu/niL were prepared as described in Zhong et al, Proc Natl Acad Sci USA 102, 9294-9 (2005).
Since HBV is not infectious in vitro, we examined the impact of peptides on the antigenicity of HBV by quantitative ELISA analysis and on HBV DNA content by quantitative PCR analysis as described as described above.
For Dengue-2 infection, Vero cells, obtained from Christopher Aiken, Vanderbilt University School of Medicine, Nashville, TN, were seeded for 24 hours pre-infection in 6-well plates. Cells were incubated with Dengue-2, provided by Richard Kinney, CDC, Atlanta, GA, in the presence of increasing concentrations of peptides (DMSO as control). Viruses and peptide were washed away 4 hours postinfection. Cells were taken for intracellular FACS staining 3 days post-infection. Cells were stained with either appropriate isotype control (BD biosciences Pharmingen, La Jolla, CA) or Dengue Envelope-specific antibody (Chemicon, Temecula, CA), and analyzed by intracellular FACS assay (FACSort, BD biosciences, San Jose, CA). Dengue-2 virus intracellular FACS assay (IFSA) set up by S. Selvarajah and P. Gallay, unpublished data.
For HIV-I infection, wild-type pNL4.3, provided by Malcolm Martin through the NIH AIDS Research and Reference Reagent Program, or infectious molecular clones derived from pNL4.3 in which the envelope gene has been replaced with envelope genes of BaL and JR-CSF (Bobardt et al., J Virol 81, 295- 405 (2007) was used. The molecular clones were used to generate infectious HIV by liposome-mediated transfection of 293T cells using Genejuice (Novagen, San Diego, CA). TZM-bl reporter cells are CD4+ CXCR4+ CCR5+ HeLa cells, which contain LacZ gene driven by the HIV-I LTR promoter (contributed by John C. Kappes, Xiaoyun Wu, and Tranzyme Inc, and obtained through the NIH AIDS Research and Reference Reagent Program). Cells were exposed to HIV-I in the presence of increasing concentration of peptides (DMSO as control). Viruses and peptides were washed away 2 hours post-infection. Infected cells were analyzed by beta-galactosidase activity 48 hours post-infection as described by Chatteηi et al., J Biol Chem 280, 40293-300 (2005).
For measles infection, the measles virus wild-type strain (WTF) was grown on BJAB cells and used to infect Vero cells expressing human SLAM-receptor at moi 0.3 in the presence or absence of peptides or DMSO. All reagents were provided by T.B.H. Geijtenbeek, VU University Medical Center, Amsterdam, Netherlands. At 48 hours post-infection, WTF infection was measured by staining the cells with anti-MV H antibodies (CVl, CV4) (Chemicon, Temecula, CA), followed by staining with the goat anti-mouse IgG-FITC antibody as described by de Witte et al, J Virol 80, 3477-86 (2006).
For West Nile virus infection, serial 2-fold dilutions of peptide 1 were added to a stock of WNV-NY diluted to Ix 105 pfu. Virus-peptide mixtures were immediately added to Huh-7 cells at an moi = 1 and incubated for 48 hours at 37 0C. Infected cells were visualized by immunostaining with an anti-WNV polyclonal antibody as described by Fredericksen et al., J Virol 80, 2913-23 (2006)
Other viruses studied include HSV-I strain F (ATCC Cat# VR-733), bovine viral diarrhea virus strain NADL (VR-534), human coronavirus strain 229E (VR- 740), human coxsackievirus B5 strain Faulkner (VR- 185), human respiratory syncytial virus strain Long (VR-26), human rhinovirus 14 strain 1059 (VR-284), human Rotavirus strain WISC2 (VR-2417), and vesicular stomatitis virus strain Indiana Lab (VR- 1238). These viruses were purchased from the American Type Culture Collection and cultured in Huh-7, MDCK (ATCC Cat# CCL-34), MRC-5 (CCL-171), HeLa (CCL-2), Hep-2 (CCL-23), HeLa, MA- 104 (CRL-2378), and Huh-7 cells, respectively, as recommended by the supplier or using Huh-7 cells when the cells are permissive. Recombinant adenovirus subtype 5 expressing GFP driven by CMV promoter was provided by U. Protzer, University of Cologne Sprinzl et al., J Virol 75, 5108-18 (2001), and used to infect human HEK293 cells (CRL- 1573). Recombinant vaccinia virus was provided by B. Moss, NIAID (Thimme et al., J Virol 77, 68-76 (2003) and used to infect Huh-7 cells. Mouse- adapted influenza A/WSN/33 was provided by Adolfo Garcia-Sastre (Falcon et al., J Gen Virol 86, 2817-21 (2005) (The Mount Sinai School of Medicine, New York, NY). LCMV (Armstrong) and BDV (He80) as well as Vero cells were provided by Juan Carlos de Ia Torre (see Clemente et al., J Virol 81, 5968-77 (2007) and Buchmeier et al., Virology 113, 73-85 (1981)) (The Department of Immunology, TSRI, La Jolla, CA). All the three viruses were used to infect the Vero cells. To determine if selected peptides inhibit the virus infections, serial 2-fold diluted peptides (from 18-20 μM) or DMSO were added to virus stocks of predetermined infectivity (1-105 ffu or TCID50/mL), incubated at 37 0C for 1 hour and then added to susceptible cells for 2-4 days at which time the cultures were assessed by comparative assessment of cytopathic effect (CPE) (BVDV, Coxsackie virus, HSVl, coronavirus, influenza virus, rhinovirus, rotavirus, RSV, vaccina virus and VSV) or by immunostaining with antibody against the corresponding viral protein (Borna (see Buchmeier et al., Virology 113, 73-85 (1981)), LCMV (see Clemente et al., J Virol 81, 5968-77 (2007)), adenovirus (see Spinzl et al., J Virol 75, 5108-18 (2001)), respectively.
Results indicate that at a concentration of 18 μM, peptide 1 had no significant effect on the infectivity of adenovirus 5, Borna disease virus, bovine viral diarrhea virus, coronavirus, coxsackie virus, herpes simplex virus 1 , influenza A virus, lymphocytic choriomeningitis virus, rhinovirus, rotavirus, vaccinia virus, or vesicular stomatitis virus, or on the antigenicity and DNA content of hepatitis B virus.
In contrast, the peptide strongly inhibited the infectivity of other human Flaviviridae members, including West Nile virus and dengue 2 virus. Surprisingly, peptide 1 strongly inhibited infectivity of the paramyxoviruses, measles and respiratory syncytial virus, as well as the human immunodeficiency virus- 1. The IC50 values of peptide 1 against these viruses are presented in Table 17 below.
Table 17 Antiviral specificity and spectrum of Viracide
Virus Enveloped Genome IC50 (μM)
HCV (JFH-I) genotype 2a yes RNA + 0.6
HCV (H77 envelope) genotype Ia yes RNA + 3.9
HCV (Conl envelope) genotype Ib yes RNA + 1.6
HCV (J6CF envelope) genotype 2a yes RNA + 1.1
Dengue Virus yes RNA + 2.0
West Nile Virus yes RNA + 4.5
Measles Virus yes RNA + 2.7
Respiratory Syncytial Virus yes RNA - 4.5
Human Immunodeficiency Virus yes RNA + 1.3
Adenovirus no DNA >18
Borna Disease Virus y J es RNA - >18
Bovine Viral Diarrhea Virus y jes RNA + >10
Coronavirus 229E yes RNA + >18
Coxsackie Virus no RNA + >18
Hepatitis B Virus yes DNA >18
Herpes Simplex Virus 1 yes DNA >10
Influenza Virus y jes RNA - >18
Lymphocytic Choriomeningitis Virus yes RNA - >18
Rhinovirus no RNA + >18
Rotavirus WISC2 no RNA + >18
Vaccinia Virus yes DNA >18
Vesicular Stomatitis Virus yes RNA - >18
Example 16: Peptide 1 Has Potent Antiviral Activity Against HIV
The ability of the D-isomer of peptide 1 to block HIV infection together with a panel of host and viral peptides that also have amphipathic structures were examined.
Methods
Cells. Immature DC were cultured as described in Geijtenbeek et al., Cell 100, 587-97 (2000). In short, human blood monocytes were isolated from buffy coats by use of a Ficoll gradient and a subsequent CD 14 selection step using a MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Purified monocytes were differentiated into immature DC in the presence of 500 U/ml of interleukin-4 (IL-4) and 800 U/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Schering-Plough, Brussels, Belgium). On day 6, the phenotype of the cultured DC was confirmed by flow cytometry. DC express high levels of MHC class I and II, CDl Ib, CDl Ic, ICAM-I and low levels of CD80, CD83 and CD86. PBMC were isolated from buffy coats, and activated with phytohemagglutinin (3μg/mL). On day 3, cells were washed and cultured with IL-2 (100 U/ml). CD4+ T-lymphocytes and monocyte-derived macrophages were isolated and characterized as described previously17. TZM-bl cells express CD4, CXCR4 and CCR5 and contain an integrated lacZ gene driven by the HIV-I LTR (Wei et al. Antimicrob Agents chemother 46, 1896-1905 (2002)). Primary genital epithelial cells (PGEC) were provided by B. Kahn of the Department of Obstetrics and Gynecology at Scripps Clinic. By rotating cotton swabs against the vaginal walls, several million cells were collected per individual. Cells were immediately placed in sterile PBS, held at 4 "C, and transported to the laboratory. After centrifugation (300 g for 5 min), the cell pellet was digested in 1 mg/mL of collagenase-dispase (Roche Molecular Biochemicals) containing 1 mg/mL of DNase (Sigma) and 0.15 mg/mL of N-alpha-p-tosyl-L-lysine chloromethyl ketone (Sigma) for 1 hour at 37 °C. The digest was centrifuged (1,000 g for 20 min) and resuspended in 250 mg/mL of PBS/BSA. After additional centrifugation, the pellet was resuspended in 5 mg/mL of PBS/BSA and loaded onto a 50 % Percoll gradient. PGEC were then isolated from contaminating by FACS cell sorting as previously described in Bobardt et al., J Virol 81 , 395-405 (2007) and propagated into collagen type I- coated T-25 flasks in DMEM Fl 2 medium containing 10 % FCS and epithelial cell growth supplement (100 μg/ml) (Sigma). PGEC were passaged less than three times prior to use in order to maintain their original features. Viruses. 293T cells were transfected with proviral plasmid (9 μg) and VSV- G envelope plasmid (1 μg). At day 2, VSV-G pseudotyped viruses were harvested and used to acutely infect Jurkat cells. Two days post-infection, viruses were harvested and p24 content was measured by ELISA (Perkin Elmer Life Sciences). The following proviral constructs were used, wild-type pNL4.3 (X4), pNL4.3-BaL (R5) in which wild-type NL4.3 envelope was switched for the R5 BaL envelope, the pNL4.3-ΔEnv, which lacks gpl60, the pNL4.3-eGFP (X4) and the pNL4.3-BaL- eGFP (R5), which encode the GFP gene instead of the Nef gene. Primary HIV-I, HIV-I drug-resistant, HIV-2, SIV and SHIV viruses were obtained through the NIH AIDS Research and Reference Reagent Program and amplified in PHA/IL-2 stimulated PBMC.
Infections. TZM cells (100,000 cells/mL) were exposed to HIV (1 ng of p24) for 4 hours in the presence or absence of Peptide 1, washed, and infection was measured 48 h post-infection by β-galactosidase activity. Without washing, TZM cytoxicity is observed at > 100 μM of peptide 1 (data not shown). DC, CD4+ T- lymphocytes or macrophages (0.1 x 106 cells) were exposed to virus (1 ng of p24) for 1 day, washed three times with medium, and cultured in a flat bottom 96-well plate. Supernatants were collected after different days and viral replication was monitored by p24 ELISA.
Peptide. As discussed above, peptide 1 composed of D-amino acids exhibits prolonged anti-HIV activities in serum than peptide 1 composed of L-amino acids.
Interestingly, peptide 1 composed of L-amino acids is not degraded in serum (i.e. after 4 h at 37°C), but instead is tightly bound (interaction maintained in an SDS- gel) to unknown seric molecules (data not shown). This tight association may result in the attenuated anti-HIV activity of the L-amino acids peptide in serum overtime.
Thus, in the following studies, exclusively the D form of peptide one is used.
Peptides were disolved in DMSO and subsequently diluted in RPMI or DMEM in the absence of serum. Transmigration assay. PGEC were seeded onto the upper face of collagen I- and fibronectin-coated 12-mm-diameter, 3-μm-pore-size polycarbonate membrane transwells at a density of 105 cells/well, and cultured until formation of tight junctions is achieved. The inserts were fed every 2 days. The monolayer on the filter effectively divides the well into an apical compartment and a basolateral compartment. The integrity of each cell monolayer was measured using an epithelial volt/ohm meter (Millipore). To ensure the integrity of the PGEC barrier, the elevated transpithelial electrical resistance of each cell monolayer was monitored and the paracellular passage of the extracellular marker inulin was measured by the permeability coefficient as measured by diffusion assay using C- carboxylated inulin (molecular weight 5000, Sigma) in the upper chamber as described in Bobardt et al., J Virol 81, 395-405 (2007). After verifying the integrity of the monolayer on the Transwell filters, PGEC were exposed to HIV (added to the upper chamber) and release of HIV into the basal chamber was monitored by measuring amounts of viral capsid by p24 ELISA. Specifically, cell-free HIV (10 ng of p24 of NL4.3ΔEnv pseudotyped with or without NL4.3 gpl60, or cell- associated virus (incubated with 104 CCR5+ Jurkat cells/1 OOμL) were added to the upper chamber for 8 h at 37°C in the presence or absence of 5 μM of peptide 1. Amounts of transcytosed viruses were quantified by p24 ELISA in the lower chamber corresponding to the basal surface.
Ex vivo infection and transmission. Healthy skin was obtained from plastic surgery and cut into 3 mm thick slices using a dermatome within 3 h after surgery. The split skin was incubated with 1 mg/ml Dispase II (Roche Diagnostics, Penzberg, Germany) in complete IMDM (Iscoves Modified Dulbecco's Medium (IMDM), 10% FCS and 10 μg/ml gentamycine) at 4°C for 18 h. Dermal and epidermal tissues were separated mechanically and cut into pieces of 1 cm2. The epidermal sheets were floated on 200 μL IMDM in a 24-well plate, the epidermal site upwards. Subsequently, the epidermal sheets were infected with HIV NL4.3-
BaL-eGFP (100 ng p24) by pipetting the virus underneath the sheets into the medium. Either 10 μM of Peptide 1 or the appropriate volume of the DMSO control solution was added in a total volume of 500 μL. After 2 h, 1.5 mL complete IMDM was added and the sheets were cultured for 3 days. The epidermal sheets were removed and 200,000 CCR5+ Jurkat cells were added for an additional 4 days. Migrated epidermal cells (day 3) and samples of the co-cultures (day 5 and 7) were harvested, fixed in 4% paraformaldehyde/PBS and analysed for GFP expression using flow cytometry. To determine the phenotype of the infected migrated cells, the cells were double-stained with the LC marker Langerin. All infected cells were Langerin positive (data not shown). No changes were observed after addition of peptide 1 concerning LC migration, LC maturation and LC viability as determined by flow cytometry (data not shown).
DC transmission assay. DC were plated at 50.000 cells/well in 96-well v- bottom plate. Cells were incubated with HIV NL4.3-eGFP (XA), NL4.3-BaL-eGFP (R5) or the single round NL4.3ΔEnv-eGFP-pseudotyped with NL4.3 gpl60 (XA) (25 ng p24) for 2 h at 37°C. Cells were washed 3 times with warm medium and PHA/IL-2-activated CD4+ T cells (200,000) were added. Cells were cultured in a flat bottom 96-well plate, harvested after 3 days, fixed in 4% paraformaldehyde/PBS and GFP expression was measured by FACS.
Cytotoxicity. PGEC were plated in clear bottom 96-well plates. Cells were serial diluted from 55,000 cells to 25 cells in 100 μL complete DMEM. Fifteen μL of the CellQuanti-MTTTM reagent (Gentaur Belgium) was added and cells were incubated for 4 h at 37°C. Then 100 μL of the solubilization solution was added and the plate was shaken for 1 h at room temperature. The OD 570 run was measured on a Molecular Devices SpectraMax 384 Plus reader. A linear relationship was observed between OD 570 nm and the cell number. The detection limit was estimated to be 950 cells from the blank control. To determine the cytotoxicity of peptides on PGEC, 55,000 cells were plated per 80 μL well in clear bottom 96-well plates. Cells were treated twice daily with 200 μM of Peptide 1 or 0.01% saponin for a week. No washes were performed in order to maintain a continuous exposure of cells to peptides.
Virus velocity sedimentation assay. Viruses were microcentrifuged for 90 min at 4°C to remove free capsid, resuspended in PBS, exposed to peptide under various conditions and loaded over a 20-70% sucrose gradient. After ultracentrifugation at 20,000 rpm for 24 h in a SW41 T rotor, fractions were collected and tested for their content in viral proteins. HIV-I capsid was detected either by p24 ELISA or by immunoblot using anti-capsid IgG obtained through the AIDS Research and Reference Program. HIV-I gp41 was detected by immunoblot using anti-gp41 IgG obtained through the AIDS Research and Reference Program. RT was detected by exoRT assay as described previously20. Briefly, aliquots (10 μl) of each fraction were mixed with RT reaction cocktail (20 μl). The reaction mixtures were incubated at 37°C for 2 h, spotted on DE81 filters, and washed twice in 2x SSC (Ix SSC is 0.15 M NaCl, 0.015 M sodium citrate) and once in 95% ethanol, and 3H was quantified by liquid scintillation counting. The density of each sucrose gradient fraction was determined by measuring the refractive index as described previously21.
Attachment and internalization assay. Attachment and internalization assays were conducted as described in Bobardt et al., J Virol 81 :395-405 (2007). For virus attachment, TZM cells (500,000) were exposed to 1 ng of p24 of virus for 1 h at 4°C, washed extensively to remove unbound virus and lysed. Under these conditions, no virus is internalized into cells. For virus internalization, TZM cells (500,000) were exposed to 1 ng of virus for 2 h at 37°C, washed, trypsinized to remove attached virus and lysed. Amounts of attached and internalized virus were determined by p24 ELISA in cell lysates.
Results
Results are summarized in the following table.
Figure imgf000140_0001
The results in Table 18(a) were obtained as follows. TZM cells were exposed to HIV-I NL4.3 in the presence of increasing amounts of amphipathic peptides derived either from host or viral proteins. Viruses and peptides were washed away after 4 h. Infection was measured 48 h post-infection by β-galactosidase activity. Table 18B. Antiviral Spectrum of Peptide 1.
Figure imgf000141_0001
For Table 18(b), target cells were exposed to viruses (HIV-I, HIV-2, SIV, SHIV, . Adeno-GFP or VSV-GFP) in the presence of increasing amounts of wild-type peptide 1 (SWLRDIWDWICEVLSDFK, SEQ ID NO: 43) or its non-amphipathic variant (SWRLOIΨOWICESVLDFK, SEQ ID NO: 119), which lacks antiviral activity. TZM infection was scored by β-galactosidase activity (HIV and SIV) or GFP content (VSV-GFP), whereas 293 infection was scored by GFP content (Adeno-GFP). Results are representative of three independent experiments. Results (triplicate) are expressed in concentration (μM) of peptide required to inhibit 50% (IC50) or 90% (IC90) of virus infectivity.
Results indicate that most of the host-derived peptides, including hecate, cynthaurin, piscidin, PGLa, latarcin-1, buforin 2, LL-37, did not affect HIV infection while those derived from apolipoprotein A-I, dermaseptin, and melittin were mildly antiviral at high concentrations (Table 18a). Similarly, most virus- derived peptides, such as Semliki Forest virus replicase protein- 1, influenza virus M2 protein, influenza virus hemagglutinin, HIV-I Vpr, and the feline immunodeficiency virus glycoprotein, did not affect HIV infection (Table 18a). In contrast, amphipathic peptides derived from HIV gp41 and HCV NS5A (Peptide 1), previously shown to inhibit HCV infection, efficiently blocked HIV infection (Table 18a). The amphipathic peptide derived from HIV gp41 corresponds to the FDA- approved T20 peptide that blocks HIV infection by binding to gp41 and interfering with fusion (see Kilby et al., Nat. Med. 4, 1302-07 (1998)). In contrast, peptide 1 does not correspond to any sequence of the HIV genome and, thus, represents a novel anti-HIV agent. Peptide 1 blocked all HIV-I isolates from different clades with various co- receptor usages (CCR5 or CXCR4 usage) (Table 18b), drug-resistant viruses (Table 18b), and other lentiviruses (HIV-2, SIV and SHIV) (Table 18b) at low micromolar or submicromolar concentrations. However, it did not block the infectivity of adenovirus or vesicular stomatitis virus (VSV) (Table 18b), suggesting that peptide 1 selectively neutralizes HIV.
Peptide 1 blocked HIV infection of the three major in vivo targets of HIV: CD4+ T-lymphocytes, macrophages and DC (FIG. 16a, panel 1-3). Remarkably, it also blocked HIV infection and transmission of a DC-T cell co-culture (FIG. 16a, panel 4). Similarly, HIV-transfected 293T cells, briefly treated with the peptide and washed, produced only non-infectious particles (FIG. 16b), suggesting that Peptide 1 can neutralize viruses on the surface (budding particles) as well as within cells (assembling particles). Moreover, peptide 1 can block an established infection, since it abrogates HIV replication even if it is added 3 days post-infection (FIG. 16a, panel 5).
Peptide 1 does not abrogate HIV infection by interfering with gpl20 or gp41, since it inhibits infectivity of VSV-G-pseudotyped HIV (FIG. 16c). To address the antiviral function in more detail, peptide 1 was added either before or after addition of HIV to cells (FIG. 16d). When cells were pre-incubated with peptide 1 and washed before adding virus, no inhibitory effect is observed (FIG. 16d, left panel). In contrast, addition of peptide 1 to the virus 2 hours before it is added to the cells (not shown) or 2 hours afterwards with maintenance of the peptide in the culture thereafter, prevents HIV infection (FIG. 16d, right panel), suggesting that Peptide 1 acts on the virus rather than on cells. As discussed above, peptide 1 is virocidal for HCV, likely by destabilizing it at the level of the membrane. Therefore, the impact of peptide 1 on the structural integrity of HIV was analyzed. Untreated viruses sediment at a density of 1.16 g/cm3 as demonstrated by the presence of viral capsid, gp41 and RT (FIG. 17a, left panel). In contrast, all viral components, including membrane-associated gp41, relocated to the top of the gradient upon peptide treatment (FIG. 17a, right panel), suggesting that peptide 1 destroys the integrity of viral particles. Supporting this hypothesis, peptide 1 prevents viral attachment to and internalization into cells (FIG. 17b). Presumably, peptide 1 creates holes in the membrane that destabilize the physical linkage between the small end of the conical capsid core and the viral envelope, called the core-envelope linkage (CEL). The core undergoes disassembly in the absence of the CEL. Peptide 1 -induced holes could promote virus swelling or shrinkage in response to an osmotic imbalance between the interior and the exterior of the virus. However, Peptide 1 destroyed HIV even in an isotonic buffer, which mimics the solute concentration of the intraviral milieu (data not shown). Even submicromolar concentrations (0.6 μM) of peptide 1 suffice to destabilize HIV (FIG. 17c). The antiviral effect is not temperature-dependent, since peptide 1 destabilizes HIV integrity at 4, 25 and 37°C (FIG. 17c). The antiviral effect is rapid, since HIV is destroyed within 15 min (FIG. 17c), and it is active under acidic conditions (FIG. 17c), suggesting that peptide 1 could be active in the acidic milieu of the female genital tract if it were used as a microbicide. A non- amphipathic variant of peptide 1 does not destroy HIV (FIG. 17d), supporting the notion that, as previously shown for HCV, the amphipathicity of Peptide 1 is crucial for its antiviral activity (Table 18b). Membrane association of the full length HCV NS5A protein is restricted to a subset of intracellular membranes, suggesting the existence of specific receptors for its anchor. A recent study reported that trypsin treatment of cellular membranes abolished the binding of an extended peptide representing the complete NS5A membrane anchor (see Cho et al. J Virol. 2007). The authors suggested that a protein mediates NS5 A binding to its target membrane in the endoplasmic reticulum. To determine if peptide 1 interacts with HIV membrane proteins, experiments were conducted to determine if preincubation of HIV with trypsin would reduce its susceptibility to destruction by peptide 1. Importantly, trypsin treatment of the virus did not prevent peptide 1 from destabilizing HIV (FIG. 17e), suggesting that it does not require trypsin-sensitive molecules on the virus for its virocidal activity. Thus, peptide 1 uses either trypsin-resistant proteineous receptors or nonproteineous receptors (e.g. lipids) to bind HIV. The resistance of VSV, and many other viruses that are insensitive to peptide 1 , may reflect either of these alternatives. Since HCV and HIV bud from lipid rafts, whereas VSV does not, it is likely that the protein and/or lipid composition of the VSV membrane differs from that of HIV. The enrichment of a specific proteinaceous or lipid receptor for the NS5A anchor domain in the HIV membrane would explain why it has no effect on VSV, and most importantly, is not toxic for host cells. An effective anti-HIV agent should interfere with the three different mechanisms involved in HIV transmission: epithelial transmigration, DC-mediated transmission and infection of mucosal target cells. To examine the antiviral effect of peptide 1 on HIV transcytosis, an in vitro assay that mimics HIV transmigration through primary genital epithelial cells (PGEC) was developed. HIV efficiently transmigrated, in contrast to a virus lacking gplόO (FIG. 18a), suggesting that the viral glycoprotein is required for transcytosis and that the PGEC layer does not allow nonspecific transmigration of viral particles. Importantly, peptide 1 prevented both cell-free and cell-associated HIV transmigration (FIG. 18a). Peptide 1 was not cytolytic for PGEC at peptide doses at least 10- to 100-fold higher than required for antiviral activity (FIG. 18b). Thus, peptide 1 prevents HIV transmigration without interfering with epithelial integrity.
Langerhans cells (LC), a DC subset present in the epidermal and epithelial tissues, play an important role in the transmission of HIV from mucosal sites to T cells residing in lymphoid tissues. Therefore, we used an ex vivo transmission model to mimic LC -mediated transmission of HIV. Epidermal sheets were infected with HIV in presence or absence of peptide 1. After three days, migrated LC were either analyzed for direct infection or for their ability to transmit HIV to T cells. The peptide blocked both LC infection (FIG. 18c) and HIV transmission to T cells (FIG. 18 d and e). Thus, peptide 1 effectively prevents the transmission of HIV by LC ex vivo.
Monocyte-derived DC were used to determine the role of the subepithelial DC subset. Transmission of HIV from DC to T cells can occur dependently or independently of infection of the DC. Importantly, peptide 1 blocked both HIV transmission pathways (FIG. 18f). Moreover, it blocked the transmission of the replication-defective virus, demonstrating that peptide 1 affects captured virus in DC (FIG. 18f). Thus, peptide 1 interferes with HIV transmission at three different levels: PGEC transmigration, DC/LC-mediated T cell transmission and direct DC/LC infection. Thus, peptide 1 is an attractive antiviral agent for the treatment and prevention of HIV. Its rapid intra- and extracellular virocidal mode of action, stability at low pH, and ability to prevent transepithelial transmigration and cell-to- cell transmission makes it especially attractive as a candidate microbicide. By targeting the lipid composition of HIV membranes, peptide 1 is not likely to select for escape variants and, if used in combination with agents that do select for resistance variants, peptide 1 is likely to prevent their spread. Thus, peptide 1 appears to represent the prototype of a new generation of antiviral agents that have promise for the treatment and prevention of HIV.
Example 17: Additional Peptides of the Invention
Additional peptides of the invention and their antiviral activity are shown in the following table. The EC50 and EC90 values were determined as above.
Figure imgf000146_0001
Figure imgf000147_0001
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All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.
The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an antibody" includes a plurality (for example, a solution of antibodies or a series of antibody preparations) of such antibodies, and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

WHAT IS CLAIMED IS:
1. An isolated peptide of 14 to 50 D- or L- amino acids in length comprising an amino acid sequence of formula I:
Xaai-Xaa2-Xaa3-W-L-Xaa6-Xaa7-Xaa8-W-Xaaio-W-Xaai2-Xaai3-Xaa14- Xaai5-Xaai6-Xaaπ-Xaai8- Xaai9-Xaa20-Xaa2i-Xaa22 (SEQ ID NO: 163), wherein:
Xaai is serine (S) or absent;
Xaa2 is glycine (G) or absent;
Xaa3 is serine (S), aspartic acid (D) or threonine (T);
W is tryptophan; L is leucine;
Xaaβ is arginine (R), aspartic acid (D), lysine (K), tryptophan (W) or glutamic acid (E);
Xaa7 is aspartic acid (D), arginine (R), glutamic acid (E), alanine (A), isoleucine (I) or lysine (K);
Xaa8 is isoleucine (I) or valine (V);
Xaaio is aspartic acid (D), arginine (R), glutamic acid (E) or lysine (K);
Xaai2 is isoleucine (I) or valine (V);
Xaaπ is cysteine (C), glutamic acid (E), leucine (L), serine (S) or arginine
(R);
Xaaj4 is glutamic acid (E), lysine (K), aspartic acid (D), threonine (T), histidine (H), serine (S) or arginine (R);
Xaa15 is valine (V), alanine (A) or serine (S);
Xaaj6 is leucine (L) or valine (V);
Xaaπ is serine (S), threonine (T), leucine (L) or absent;
Xaai8 is aspartic acid (D), arginine (R), glutamic acid (E), lysine (K) or absent;
Xaai9 is phenylalanine or absent;
Xaa20 is lysine (K), glutamic acid (E), arginine (R), aspartic acid (D) or absent; Xaa2i is threonine (T) or absent; and Xaa22 is tryptophan (W) or absent; and wherein the amino acid sequence of formula I is not:
SWLRDIWDWICEVLSDFK (SEQ ID NO: 43);
SWLRDIWDWICEVLSDF (SEQ ID NO: 95),
SWLRDIWDWICEVLSD (SEQ ID NO: 94),
SWLRDIWDWICEVLS (SEQ ID NO: 93), or
SWLRDIWDWICEVL (SEQ ID NO: 92).
2. The isolated peptide of claim 1, which comprises an amino acid sequence selected from the group consisting of:
SWRLDIWDWICESVLDFK (SEQ ID NO: 119), DWLRIIWDWVCSVVSDFK (SEQ ID NO: 123), SWLWEVWDWVLHVLSDFK (SEQ ID NO: 124), TWLRAIWDWVCTALTDFK (SEQ ID NO: 125), SWLRDVWDWVCTVLSDFK (SEQ ID NO: 126), SWLRDIWDWISEVLSDFK (SEQ ID NO: 127), SWLDRIWRWICKVLSRFE (SEQ ID NO: 128), SWLDDIWDWICEVLSDFE (SEQ ID NO: 129), SWLRRIWRWICKVLSRFK (SEQ ID NO: 130), SWLKEIWEWICDVLSEFR (SEQ ID NO: 131), SWLKDIWDWICEVLSDFR (SEQ ID NO: 132), SWLKDIWDWICEVLSDFK (SEQ ID NO: 133), SWLREIWEWICDVLSEFK (SEQ ID NO: 134), SWLREIWEWICEVLSEFK (SEQ ID NO: 135), SWLDRIWRWICKVLSRFE (SEQ ID NO: 136), SWLDDIWDWICEVLSDFE (SEQ ID NO: 137), SWLRRIWRWICKVLSRFK (SEQ ID NO: 138), SWLRDIWDWIREVLSDFK (SEQ ID NO: 139), SWLRDIWDWIEEVLSDFK (SEQ ID NO: 140), SGSWLRDIWDWICEVLSDFK (SEQ ID NO: 141), GSWLRDIWDWICEVLSDFK (SEQ ID NO: 142), SWLRDIWDWICEVLSDFKT (SEQ ID NO: 143), SWLRDIWDWICEVLSDFKTW (SEQ ID NO: 144), SWRLDIWDWICESVLDF (SEQ ID NO: 189), SWRLDIWDWICESVLD (SEQ ID NO: 190), SWRLDIWDWICESVL (SEQ ID NO: 191), SWRLDIWDWICESV (SEQ ID NO: 192), DWLRIIWDWVCSVVSDF (SEQ ID NO: 193), DWLRIIWDWVCSVVSD (SEQ ID NO: 194), DWLRIIWDWVCSVVS (SEQ ID NO: 195), DWLRIIWDWVCSVV (SEQ ID NO: 196), SWLWEVWDWVLHVLSDF (SEQ ID NO: 197), SWLWEVWDWVLHVLSD (SEQ ID NO: 198), SWLWEVWDWVLHVLS (SEQ ID NO: 199), SWLWEVWDWVLHVL (SEQ ID NO: 200), TWLRAIWDWVCTALTDF (SEQ ID NO: 201), TWLRAIWDWVCTALTD (SEQ ID NO: 202), TWLRAIWDWVCTALT (SEQ ID NO: 203), TWLRAIWDWVCTAL (SEQ ID NO: 204), SWLRDVWDWVCTVLSDF (SEQ ID NO: 205), SWLRDVWDWVCTVLSD (SEQ ID NO: 206), SWLRDVWDWVCTVLS (SEQ ID NO: 207), SWLRDVWDWVCTVL (SEQ ID NO: 208), SWLRDIWDWISEVLSDF (SEQ ID NO: 209), SWLRDIWDWISEVLSD (SEQ ID NO: 210), SWLRDIWDWISEVLS (SEQ ID NO: 211), SWLRDIWDWISEVL (SEQ ID NO: 212), SWLDRIWRWICKVLSRF (SEQ ID NO: 213), SWLDRIWRWICKVLSR (SEQ ID NO: 214), SWLDRIWRWICKVLS (SEQ ID NO: 215), SWLDRIWRWICKVL (SEQ ID NO: 216), SWLDDIWDWICEVLSDF (SEQ ID NO: 217), SWLDDIWDWICEVLSD (SEQ ID NO: 218), SWLDDIWDWICEVLS (SEQ ID NO: 219), SWLDDIWDWICEVL (SEQ ID NO: 220), SWLRRIWRWICKVLSRF (SEQ ID NO:221), SWLRRIWRWICKVLSR (SEQ ID NO: 222), SWLRRIWRWICKVLS (SEQ ID NO: 223), SWLRRIWRWICKVL (SEQ ID NO: 224), SWLKEIWEWICDVLSEF (SEQ ID NO: 225), SWLKEIWEWICDVLSE (SEQ ID NO: 226), SWLKEIWEWICDVLS (SEQ ID NO: 227), SWLKEIWEWICDVL (SEQ ID NO: 228), SWLKDIWDWICEVLSDF (SEQ ID NO: 229), SWLKDIWDWICEVLSD (SEQ ID NO: 230), SWLKDIWDWICEVLS (SEQ ID NO: 231), SWLKDIWDWICEVL (SEQ ID NO: 232), SWLKDIWDWICEVLSDF (SEQ ID NO: 233), SWLKDIWDWICEVLSD (SEQ ID NO: 234), SWLKDIWDWICEVLS (SEQ ID NO: 235), SWLKDIWDWICEVL (SEQ ID NO: 236), SWLREIWEWICDVLSEF (SEQ ID NO: 237), SWLREIWEWICDVLSE (SEQ ID NO: 238), SWLREIWEWICDVLS (SEQ ID NO: 239), SWLREIWEWICDVL (SEQ ID NO: 240), SWLREIWEWICEVLSEF (SEQ ID NO: 591), SWLREIWEWICEVLSE (SEQ ID NO: 592), SWLREIWEWICEVLS (SEQ ID NO: 593), SWLREIWEWICEVL (SEQ ID NO: 594), SWLDRIWRWICKVLSRF (SEQ ID NO: 241), SWLDRIWRWICKVLSR (SEQ ID NO: 242), SWLDRIWRWICKVLS (SEQ ID NO: 243), SWLDRIWRWICKVL (SEQ ID NO: 244), SWLDDIWDWICEVLSDF (SEQ ID NO: 245), SWLDDIWDWICEVLSD (SEQ ID NO: 246), SWLDDIWDWICEVLS (SEQ ID NO: 247), SWLDDIWDWICEVL (SEQ ID NO: 248), SWLRRIWRWICKVLSRF (SEQ ID NO: 249), SWLRRIWRWICKVLSR (SEQ ID NO: 250), SWLRRIWRWICKVLS (SEQ ID NO: 251), SWLRRIWRWICKVL (SEQ ID NO: 252), SWLRDIWDWIREVLSDF (SEQ ID NO: 253), SWLRDIWDWIREVLSD (SEQ ID NO: 254), SWLRDIWDWIREVLS (SEQ ID NO: 255), SWLRDIWDWIREVL (SEQ ID NO: 256), SWLRDIWDWIEEVLSDF (SEQ ID NO: 257), SWLRDIWDWIEEVLSD (SEQ ID NO: 258), SWLRDIWDWIEEVLS (SEQ ID NO: 259), SWLRDIWDWIEEVL (SEQ ID NO: 260), SGSWLRDIWDWICEVLSDF (SEQ ID NO: 261), SGSWLRDIWDWICEVLSD (SEQ ID NO: 262), SGSWLRDIWDWICEVLS (SEQ ID NO: 263), SGSWLRDIWDWICEVL (SEQ ID NO: 264), GSWLRDIWDWICEVLSDF (SEQ ID NO: 265), GSWLRDIWDWICEVLSD (SEQ ID NO: 266), GSWLRDIWDWICEVLS (SEQ ID NO: 267), GSWLRDIWDWICEVL (SEQ ID NO: 268), SWLRDIWDWICEVLSDFK (SEQ ID NO: 269), SWLRDIWDWICEVLSDF (SEQ ID NO: 270), SWLRDIWDWICEVLSD (SEQ ID NO: 271), SWLRDIWDWICEVLS (SEQ ID NO: 272), SWLRDIWDWICEVLSDFKT (SEQ ID NO: 273), SWLRDIWDWICEVLSDFK (SEQ ID NO: 274), SWLRDIWDWICEVLSDF (SEQ ID NO: 275), and SWLRDIWDWICEVLSD (SEQ ID NO: 276).
3. The isolated peptide of claim 1, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 19, 123-144, 189-276 and 591- 594.
4. The isolated peptide of claim 1, which comprises an amino acid sequence of formula II:
Xaai-Xaa2-S-W-L-Xaa6-Xaa7-I-W-Xaai0-W-I-C-Xaa14-V-L-Xaai7-
Xaa!8- Xaai9-Xaa20-Xaa2i-Xaa22 (SEQ ID NO: 164), wherein:
Xaai, Xaa2, Xaa21 and Xaa22 are absent;
S is serine; W is tryptophan; L is leucine;
Xaa^ is arginine (R), aspartic acid (D), glutamic acid (E) or lysine (K);
Xaa7 is arginine (R), aspartic acid (D) or lysine (K);
I is isoleucine; W is tryptophan;
Xaaio is aspartic acid (D), arginine (R), or lysine (K);
C is cysteine; Xaai4 is lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D); V is valine;
Xaaπ is serine (S) or absent
Xaai8 is aspartic acid (D), arginine (R), lysine (K) or absent; Xaai9 is phenylalanine (F) or absent; and
Xaa2o is lysine (K), glutamic acid (E), aspartic acid (D), arginine (R) or absent.
5. The isolated peptide of claim 4, which comprises an amino acid sequence selected from the group consisting:
SWLEKIWKWICRVLSKFD (SEQ ID NO: 165);
SWLRKIWKWICEVLSDFK (SEQ ID NO: 166);
SWLRDIWDWICKVLSKFK (SEQ ID NO: 167);
SWLRRIWRWICEVLSDFK (SEQ ID NO: 168);
SWLRDIWDWICRVLSRFK (SEQ ID NO: 169);
SWLRRIWDWICRVLSDFK (SEQ ID NO: 170);
SWLRKIWDWICKVLSDFK (SEQ ID NO: 171);
SWLRRIWDWICEVLSRFK (SEQ ID NO: 172);
SWLRKIWDWICEVLSKFK (SEQ ID NO: 173);
SWLRDIWRWICRVLSDFK (SEQ ID NO: 174);
SWLRDIWKWICKVLSDFK (SEQ ID NO: 175);
SWLDRIWDWICRVLSRFK (SEQ ID NO: 176);
SWLRDIWDWICKVLSKFK (SEQ ID NO: 177);
SWLEKIWKWICRVLSKF (SEQ ID NO: 393);
SWLEKIWKWICRVLSK (SEQ ID NO: 394);
SWLEKIWKWICRVLS (SEQ ID NO: 395);
SWLEKIWKWICRVL (SEQ ID NO: 396);
SWLRKIWKWICEVLSDF (SEQ ID NO: 397);
SWLRKIWKWICEVLSD (SEQ ID NO: 398); SWLRKIWKWICEVLS (SEQ ID NO: 399); SWLRKIWKWICEVL (SEQ ID NO: 400); SWLRDIWDWICKVLSKF (SEQ ID NO: 401); SWLRDIWDWICKVLSK (SEQ ID NO: 402); SWLRDIWDWICKVLS (SEQ ID NO: 403); SWLRDIWDWICKVL (SEQ ID NO: 404); SWLRRIWRWICEVLSDF (SEQ ID NO: 405); SWLRRIWRWICEVLSD (SEQ ID NO: 406); SWLRRIWRWICEVLS (SEQ ID NO: 407); SWLRRIWRWICEVL (SEQ ID NO: 408); SWLRDIWDWICRVLSRF (SEQ ID NO: 409); SWLRDIWDWICRVLSR (SEQ ID NO: 410); SWLRDIWDWICRVLS (SEQ ID NO: 41 1); SWLRDIWDWICRVL (SEQ ID NO: 412); SWLRRIWDWICRVLSDF (SEQ ID NO: 413); SWLRRIWDWICRVLSD (SEQ ID NO: 414); SWLRRIWDWICRVLS (SEQ ID NO: 415); SWLRRIWDWICRVL (SEQ ID NO: 416); SWLRKIWDWICKVLSDF (SEQ ID NO: 417); SWLRKIWDWICKVLSD (SEQ ID NO: 418); SWLRKIWDWICKVLS (SEQ ID NO: 419); SWLRKIWDWICKVL (SEQ ID NO: 420); SWLRRIWDWICEVLSRF (SEQ ID NO: 421); SWLRRIWDWICEVLSR (SEQ ID NO: 422); SWLRRIWDWICEVLS (SEQ ID NO: 423); SWLRRIWDWICEVL (SEQ ID NO: 424); SWLRKIWDWICEVLSKF (SEQ ID NO: 425); SWLRKIWDWICEVLSK (SEQ ID NO: 426); SWLRKIWDWICEVLS (SEQ ID NO: 427); SWLRKIWDWICEVL (SEQ ID NO: 428); SWLRDIWRWICRVLSDF (SEQ ID NO: 429); SWLRDIWRWICRVLSD (SEQ ID NO: 430); SWLRDIWRWICRVLS (SEQ ID NO: 431); SWLRDIWRWICRVL (SEQ ID NO: 432); SWLRDIWKWICKVLSDF (SEQ ID NO: 433); SWLRDIWKWICKVLSD (SEQ ID NO: 434); SWLRDIWKWICKVLS (SEQ ID NO: 435); SWLRDIWKWICKVL (SEQ ID NO: 436); SWLDRIWDWICRVLSRF (SEQ ID NO: 437); SWLDRIWDWICRVLSR (SEQ ID NO: 438); SWLDRIWDWICRVLS (SEQ ID NO: 439); SWLDRIWDWICRVL (SEQ ID NO: 440); SWLRDIWDWICKVLSKF (SEQ ID NO: 441); SWLRDIWDWICKVLSK (SEQ ID NO: 442); SWLRDIWDWICKVLS (SEQ ID NO: 443); and SWLRDIWDWICKVL (SEQ ID NO: 444).
6. The isolated peptide of claim 4, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 165-177 and 393-444.
7. The isolated peptide of claim 4, which comprises an amino acid sequence of formula III:
Xaa,-Xaa2-S-W-L-R-Xaa7-I-W-Xaa10-W-I-C-Xaai4-V-L-Xaa17-
Xaai8-Xaai9-Xaa20-Xaa2i-Xaa22 (SEQ ID NO: 178), wherein:
Xaai, Xaa2, Xaa2i and Xaa22 are absent;
S is serine; W is tryptophan; L is leucine; R is arginine; I is isoleucine; C is cysteine; V is valine; Xaa7 is aspartic acid (D), arginine (R), or lysine (K);
Xaaio is arginine (R) or lysine (K);
Xaai4 is lysine (K), glutamic acid (E) or aspartic acid (D);
Xaaπ is serine (S) or absent;
Xaai8 is arginine (R), lysine (K) or absent;
Xaai9 is phenylalanine (F) or absent; and
Xaa20 is lysine (K) or absent.
8. The isolated peptide of claim 7, which comprises an amino acid sequence selected from the group consisting of:
SWLRDIWRWICKVLSRFK (SEQ ID NO: 179);
SWLRDIWKWICKVLSKFK (SEQ ID NO: 180);
SWLRKIWKWICEVLSKFK (SEQ ID NO: 181);
SWLRRIWRWICEVLSRFK (SEQ ID NO: 182);
SWLRRIWRWICDVLSRFK (SEQ ID NO: 183);
SWLRDIWRWICKVLSRF (SEQ ID NO: 445);
SWLRDIWRWICKVLSR (SEQ ID NO: 446);
SWLRDIWRWICKVLS (SEQ ID NO: 447);
SWLRDIWRWICKVL (SEQ ID NO: 448);
SWLRDIWKWICKVLSKF (SEQ ID NO: 449);
SWLRDIWKWICKVLSK (SEQ ID NO: 450);
SWLRDIWKWICKVLS (SEQ ID NO: 451);
SWLRDIWKWICKVL (SEQ ID NO: 452);
SWLRKIWKWICEVLSKF (SEQ ID NO: 453);
SWLRKIWKWICEVLSK (SEQ ID NO: 454);
SWLRKIWKWICEVLS (SEQ ID NO: 455);
SWLRKIWKWICEVL (SEQ ID NO: 456);
SWLRRIWRWICEVLSRF (SEQ ID NO: 457);
SWLRRIWRWICEVLSR (SEQ ID NO: 458); SWLRRIWRWICEVLS (SEQ ID NO: 459); SWLRRIWRWICEVL (SEQ ID NO: 460); SWLRRIWRWICDVLSRF (SEQ ID NO: 461); SWLRRIWRWICDVLSR (SEQ ID NO: 462); SWLRRIWRWICDVLS (SEQ ID NO: 463); and SWLRRIWRWICDVL (SEQ ID NO: 464).
9. The isolated peptide of claim 7, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 179-183 and 445-464.
10. The isolated peptide of claim 4, which comprises an amino acid sequence of formula IV:
Xaai-Xaa2-S-W-L-Xaa6-Xaa7-I-W-Xaai0-W-I-C-Xaai4-V-L-Xaai7-
Xaai8-Xaai9-Xaa20-Xaa2i-Xaa22 (SEQ ID NO: 184), wherein:
Xaai, Xaa2, Xaa2j and Xaa22 are absent;
S is serine; W is tryptophan; L is leucine; I is isoleucine; C is cysteine; V is valine;
Xaa6j Xaa7, Xaaio and Xaa]4 are arginine (R) or lysine (K);
Xaaπ is serine (S) or absent;
Xaais is arginine (R), lysine (K) or absent;
Xaai9 is phenylalanine (F) or absent; and
Xaa20 is arginine (R), lysine (K) or absent.
11. The isolated peptide of claim 10, which comprises an amino acid sequence selected from the group consisting of:
SWLRKIWKWICKVLSKFK (SEQ ID NO: 185); SWLRRIWRWICRVLSRFK (SEQ ID NO: 186); SWLRRIWRWICRVLSRFR (SEQ ID NO: 187); SWLKKIWKWICKVLSKFK (SEQ ID NO: 188); SWLRKIWKWICKVLSKF (SEQ ID NO: 465); SWLRKIWKWICKVLSK (SEQ ID NO: 466); SWLRKIWKWICKVLS (SEQ ID NO: 467); SWLRKIWKWICKVL (SEQ ID NO: 468); SWLRRIWRWICRVLSRF (SEQ ID NO: 469); SWLRRIWRWICRVLSR (SEQ ID NO: 470); SWLRRIWRWICRVLS (SEQ ID NO: 471); SWLRRIWRWICRVL (SEQ ID NO: 472); SWLRRIWRWICRVLSRF (SEQ ID NO: 473); SWLRRIWRWICRVLSR (SEQ ID NO: 474); SWLRRIWRWICRVLS (SEQ ID NO: 475); SWLRRIWRWICRVL (SEQ ID NO: 476); SWLKKIWKWICKVLSKF (SEQ ID NO: 477); SWLKKIWKWICKVLSK (SEQ ID NO: 478); SWLKKIWKWICKVLS (SEQ ID NO: 479); and SWLKKIWKWICKVL (SEQ ID NO: 480).
12. The isolated peptide of claim 10, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 185-188 and 465-480.
13. The isolated peptide of claims 1, which has viricidal activity.
14. The isolated peptide of claim 1, which is 18 to 40 D- or L-amino acids in- length.
15. The isolated peptide of claim 1, which is 18 to 30 D- or L-amino acids in- length.
16. The isolated peptide of claim 1, which is 18 to 22 D- or L-amino acids in- length.
17. A pharmaceutical composition comprising the isolated peptide of claim 1.
18. The pharmaceutical composition of claim 17, which is a microbicide.
19. The pharmaceutical composition of claim 17, which is a vaginal cream.
20. The pharmaceutical composition of claim 17, further comprising an antiviral agent.
21. The pharmaceutical composition of claim 20, wherein the antiviral agent is a protease inhibitor, a polymerase inhibitor, an integrase inhibitor, an entry inhibitor, an assembly/secretion inhibitor, a translation inhibitor, an immunostimulant or any combination thereof.
22. The pharmaceutical composition of claim 20, wherein the antiviral agent is α-interferon, pegylated interferon, ribavirin, amantadine, rimantadine, pleconaril, acyclovir, zidovudine, lamivudine, Indenavir (Merck), telaprivir (Vertex), Tenofivir (Gilead), Rl 626 (Roche), GS-9137 (Gilead), Fuzeon (Roche, Trimeris), Celgosivir (Migenix), VGX-410c (VGX pharmaceuticals), 0 IMO-2125 (Idera pharmaceuticals), or any combination thereof.
23. An article of manufacture comprising a vessel for collecting a body fluid and a peptide of claim 1.
24. The article of claim 23, wherein the vessel is a collection bag, tube, capillary tube or syringe.
25. The article of claim 23, wherein the vessel is evacuated.
26. The article of claim 23, further comprising a biological stabilizer.
27. The article of claim 26, wherein the stabilizer is an anti-coagulant, preservative, protease inhibitor, or any combination thereof.
28. The article of claim 27, wherein the anti-coagulant is citrate, ethylene diamine tetraacetic acid, heparin, oxalate, fluoride or any combination thereof.
29. The article of claim 27, wherein the preservative is boric acid, sodium formate and sodium borate.
30. The article of claim 27, wherein the protease inhibitor is dipeptidyl peptidase IV.
31. The article of claim 26, wherein the peptide and/or stabilizer is freeze dried.
32. A composition comprising a sample from the body of a mammal and a peptide of claim 1.
33. The composition of claim 32, further comprising a biological stabilizer.
34. The composition of claim 33, wherein the stabilizer is an anti-coagulant, a preservative, a protease inhibitor, or any combination thereof.
35. The composition of claim 34, wherein the anticoagulant is citrate, ethylene diamine tetraacetic acid, heparin, oxalate, fluoride or any combination thereof.
36. The composition of claim 34, wherein the preservative is boric acid, sodium formate and sodium borate.
37. The composition of claim 34, wherein the protease inhibitor is dipeptidyl peptidase IV.
38. The composition of claim 32, wherein the sample is a blood product.
39. The composition of claim 38, wherein the blood product is plasma, platelet, leukocytes or stem cell.
40. A method for inactivating a virus comprising contacting the virus with a peptide of claim 1.
41. The method of claim 40, wherein the virus is a Flavivirus, an Hepacivirus or a human immunodeficiency virus.
42. A method for preventing or treating infection of a mammalian cell by a virus comprising contacting the mammalian cell with a peptide of claim 1.
43. The method of claim 42, wherein the virus is a Flavivirus, an Hepacivirus or a human immunodeficiency virus.
44. The method of claim 42, wherein the virus is Hepatitis C virus, West Nile virus, Dengue virus or human immunodeficiency virus.
45. The method of claim 42, wherein the mammalian cell is a human cell.
46. A method for preventing or treating a viral infection in a mammal comprising administering to the mammal an effective amount of a peptide of claim 1.
47. The method of claim 46, wherein the mammal is a human.
48. The method of claim 46, wherein the virus is a Flavivirus, an Hepacivirus or a human immunodeficiency virus.
49. The method of claim 46, wherein the virus is Hepatitis C virus, West Nile virus, Dengue virus or human immunodeficiency virus.
50. The method of claim 46, wherein the peptide is administered topically or systematically.
51. A method for inactivating a human immunodeficiency virus comprising contacting the virus with a peptide of 14 to 50 D- or L-amino acids in length, wherein the peptide comprises an alpha-helical structure, and wherein all the polar amino acids are located on the same face of the alpha helical structure, and all the nonpolar amino acids are located on the other face of the alpha-helical structure.
52. The method of claim 51, wherein the nonpolar amino acids are selected from the group consisting of alanine, valine, leucine, methionine, isoleucine, phenylalanine, and tryptophan.
53. The method of claim 51 , wherein the polar amino acids are selected from the group consisting of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine, hydroxylysine, ornithine, serine and threonine.
54. The method of claim 53, wherein the cysteine is located at a position N- terminal to a serine on the peptide.
55. The method of claim 53, wherein the cycteine is located four positions N- terminal to the serine on the peptide.
56. The method of claim 53, wherein the cysteine is located at position 11 relative to the N-terminus of the peptide.
57. The method of claim 51, wherein amino acids 16 and 18 relative to the N- terminus of the peptide are charged, and wherein amino acids 16 and 18 are charged positive and negative, positive and positive, or negative and positive, respectively.
58. The method of claim 51, wherein the peptide is 14 to 40 D- or L-amino acids in-length.
59. The method of claim 51, wherein the peptide is 14 to 30 D- or L-amino acids in-length.
60. The method of claim 51, wherein the peptide is 14 to 25 D- or L-amino acids in-length.
61. The method of claim 51, wherein the peptide is 14 to 18 D- or L-amino acids in-length.
62. The method of claim 61, wherein (a) the peptide is 14 amino acids in length; (b) the amino acids are arginine, cysteine, glutamate, serine, valine, two aspartates, two leucines, two isoleucines and three tryptophan residues; and (c) the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure.
63. The method of claim 62, wherein the amino acid sequence of the peptide is:
SWLRDIWDWICEVL (SEQ ID NO: 92), or LVECIWDWIDRLWS (SEQ ID NO: 102).
64. The method of claim 61, wherein (a) the peptide is 15 amino acids in length; (b) the amino acids are arginine, cysteine, glutamate, two serines, valine, two . aspartates, two leucines, two isoleucines and three tryptophan residues; and (c) the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure.
65. The method of claim 64, wherein the amino acid sequence of the peptide is:
SWLRDIWDWICEVLS (SEQ ID NO: 93), or SLVECIWDWIDRLWS (SEQ ID NO: 101).
66. The method of claim 61, wherein (a) the peptide is 16 amino acids in length; (b) the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines and three tryptophan residues; and (c) the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure.
67. The method of claim 66, wherein the amino acid sequence of the peptide is:
SWLRDIWDWICEVLSD (SEQ ID NO: 94), or DSLVECIWDWIDRLWS (SEQ ID NO: 100).
68. The method of claim 61, wherein (a) the peptide is 17 amino acids in length; (b) the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines, three tryptophan and a phenylalanine; and (c) the arginine, cysteine, glutamate, serine and aspartate residues are located on the same face of the alpha-helical structure.
69. The method of claim 68, wherein the amino acid sequence of the peptide is:
SWLRDIWDWICEVLSDF (SEQ ID NO: 95), or FDSLVECIWDWIDRLWS (SEQ ID NO: 99).
70. The method of claim 61, wherein (a) the peptide is 18 amino acids in length;(b) the amino acids are arginine, cysteine, glutamate, two serines, valine, three aspartates, two leucines, two isoleucines, three tryptophan, a phenylalanine and a lysine; and (c) the arginine, cysteine, glutamate, serine, aspartate and lysine residues are located on the same face of the alpha-helical structure.
71. The method of claim 70, wherein the amino acid sequence of the peptide is:
SWLRDIWDWICEVLSDFK (SEQ ID NO: 43), KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), SIWRDWVDLICEFLSDWK (SEQ ID NO: 97) or KWLCRIWSWISDVLDDFE (SEQ ID NO: 98).
72. The method of claim 51, wherein the EC50 of the peptide is about 3 μM or less.
73. The method of claim 51, wherein the EC50 of the peptide is about 2 μM or less.
74. The method of claim 51, wherein the EC50 of the peptide is about 1 μM or less.
75. The method of claim 51 , wherein the EC50 of the peptide is about 500 nM or less.
76. The method of claim 51 , wherein the EC50 of the peptide is about 400 nM or less.
77. The method of claim 51 , wherein the EC50 of the peptide is about 300 nM.
78. The method of claim 51 , wherein the peptide comprises an amino acid sequence selected from the group consisting of:
SWLRPIWPWICEVLSDFK (SEQ ID NO: 91), SWLRDIWDWICEVL (SEQ ID NO: 92), SWLRDIWDWICEVLS (SEQ ID NO: 93), SWLRDIWDWICEVLSD (SEQ ID NO: 94), SWLRDIWDWICEVLSDF (SEQ ID NO: 95), KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), SIWRDWVDLICEFLSDWK (SEQ ID NO: 97), KWLCRIWSWISDVLDDFE (SEQ ID NO: 98), FDSLVECIWDWIDRLWS (SEQ ID NO: 99), DSLVECIWDWIDRLWS (SEQ ID NO: 100), SLVECIWDWIDRLWS (SEQ ID NO: 101), and LVECIWDWIDRLWS (SEQ ID NO: 102).
79. The method of claim 51 , wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 91-102.
80. The method of claim 51 , wherein the peptide comprises an amino acid sequence selected from the group consisting of:
QIVGGVYLLPRRGPRLGV (SEQ ID NO: 4), QPGYPWPLYGNEGCGWAG (SEQ ID NO: 5), LYGNEGCGWAGWLLSPRG (SEQ ID NO: 6), GWAGWLLSPRGSRPSWGP (SEQ ID NO: 7), IFLLALLSCLTVPASAYQ (SEQ ID NO: 8), DAILHTPGCVPCVREGNA (SEQ ID NO: 9), LPTTQLRRHIDLLVGSAT (SEQ ID NO: 10), RHIDLLVGSATLCSALYV (SEQ ID NO: 11), GSATLCSALYVGDLCGSV (SEQ ID NO: 12), ALYVGDLCGSVFLVGQLF (SEQ ID NO: 13), IMDMIAGAHWGVLAGIAY (SEQ ID NO: 14), HINSTALNCNESLNTGWL (SEQ ID NO: 15), NCNESLNTGWLAGLFYQH (SEQ ID NO: 16), LASCRRLTDFAQGWGPIS (SEQ ID NO: 17), TDFAQGWGPISYANGSGL (SEQ ID NO: 18), GPISYANGSGLDERPYCW (SEQ ID NO: 19), GSGLDERPYCWHYPPRPC (SEQ ID NO: 20), WMNSTGFTKVCGAPPCVI (SEQ ID NO: 21), PCVIGGVGNNTLLCPTDC (SEQ ID NO: 22), MYVGGVEHRLEAACNWTR (SEQ ID NO: 23), YLYGVGSSIASWAIKWEY (SEQ ID NO: 24), SIASWAIKWEYVVLLFLL (SEQ ID NO: 25), KWEYVVLLFLLLADARVC (SEQ ID NO: 26), WMMLLISQAEAALENLVI (SEQ ID NO: 27), GAVYAFYGMWPLLLLLLA (SEQ ID NO: 28), GMWPLLLLLLALPQRAYA (SEQ ID NO: 29), TLVFDITKLLLAIFGPLW (SEQ ID NO: 30), VSTATQTFLATCIN (SEQ ID NO: 31), ATQTFLATCINGVCWTVY (SEQ ID NO: 32), DSSVLCECYDAGCAWYEL (SEQ ID NO: 33), AYMNTPGLPVCQDHLEFW (SEQ ID NO: 34), LEFWEGVFTGLTHIDAHF (SEQ ID NO: 35), HPITKYIMTCMSADLEVV (SEQ ID NO: 36), VTSTWVLVGGVLAAL (SEQ ID NO: 37), WVLVGGVLAALAAYCLST (SEQ ID NO: 38), LAALAAYCLSTGCVV (SEQ ID NO: 39), EVFWAKHMWNFISGIQYL (SEQ ID NO: 40), MWNFISGIQYLAGLSTLP (SEQ ID NO: 41), PAILSPGALVVGVVCAAI (SEQ ID NO: 42), SWLRDIWDWICEVLSDFK (SEQ ID NO: 43), DWICEVLSDFKTWLKAKL (SEQ ID NO: 44), YVSGMTTDNLKCPCQIPS (SEQ ID NO: 45), SSGADTEDVVCCSMS (SEQ ID NO: 46), DTEDVVCCSMSYSW (SEQ ID NO: 47), SSGADTEDVVCCSMSYSW (SEQ ID NO: 48), DVVCCSMSYSWTGAL (SEQ ID NO: 49), TVTESDIRTEEAIYQCCD (SEQ ID NO: 50), GNTLTCYIKARAACRAAG (SEQ ID NO: 51), RAAGLQDCTMLVCGDDLV (SEQ ID NO: 52), CTMLVCGDDLVVICESAG (SEQ ID NO: 53), DDLVVICESAGVQEDAAS (SEQ ID NO: 54), LELITSCSSNVSVAHDGA (SEQ ID NO: 55), HTPVNSWLGNIIMFAPTL (SEQ ID NO: 56), APTLWARMILMTHFFSVL (SEQ ID NO: 57), DQLEQALNCEIYGACYSI (SEQ ID NO: 58), GVPPLRAWRHRARSVRAR (SEQ ID NO: 59), WRHRARSVRARLLSRGGR (SEQ ID NO: 60), GWFTAGYSGGDIYHSVSH (SEQ ID NO: 61), LYGNEGLGWAGWLLSPRG (SEQ ID NO:62), IFLLALLSCITVPVSAAQ (SEQ ID NO:63), IFLLALLSCLTIPASAYE (SEQ ID NO:64), MSATFCSALYVGDLCGGV (SEQ ID NO:65), GAAALCSAMYVGDLCGSV (SEQ ID NO:66), ALYVGDLCGGVMLAAQVF (SEQ ID NO:67), AMYVGDLCGSVFLVAQLF (SEQ ID NO:68), IIDIVSGAHWGVMFGLAY (SEQ ID NO:69), VVDMVAGAHWGVLAGLAY (SEQ ID NO:70), VDVQYMYGLSPAITKYVV (SEQ ID NO:71), YLYGIGSAVVSFAIKWEY (SEQ ID NO:72), WMLILLGQAEAALEKLVV (SEQ ID NO:73), WMMLLIAQAEAALENLVV (SEQ ID NO:74), GVVFDITKWLLALLGPAY (SEQ ID NO:75), ELIFTITKILLAILGPLM (SEQ ID NO:76), VSQSFLGTTISGVLWTVY (SEQ ID NO:77), ATQSFLATCVNGVCWTVY (SEQ ID NO:78), SWLRDVWDWVCTILTDFK (SEQ ID NO:79), SWLRDVWDWICTVLTDFK (SEQ ID NO:80), DWVCTILTDFKNWLTSKL (SEQ ID NO:81), DWICTVLTDFKTWLQSKL (SEQ ID NO:82), ASEDVYCCSMSYTWT (SEQ ID NO:83), EDDTTVCCSMSYSW (SEQ ID NO: 84), CTMLVCGDDLVVICESAG (SEQ ID NO:85), and PTMLVCG DDLVVISESQG (SEQ ID NO:86).
81. The method of claim 51 , wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4-86.
82. The method of claim 51 , wherein the peptide comprises an amino acid sequence selected from the group consisting of:
LYGNEGCGWAGWLLSPRG (SEQ ID NO: 6), IFLLALLSCLTVPASAYQ (SEQ ID NO: 8), GSATLCSALYVGDLCGSV (SEQ ID NO: 12), ALYVGDLCGSVFLVGQLF (SEQ ID NO: 13), IMDMIAGAHWGVLAGIAY (SEQ ID NO: 14), WMNSTGFTKVCGAPPCVI (SEQ ID NO: 21), MYVGGVEHRLEAACNWTR (SEQ ID NO: 23), YLYGVGSSIASWAIKWEY (SEQ ID NO: 24), WMMLLISQAEAALENLVI (SEQ ID NO: 27), GAVYAFYGMWPLLLLLLA (SEQ ID NO: 28), TLVFDITKLLLAIFGPLW (SEQ ID NO: 30), ATQTFLATCINGVCWTVY (SEQ ID NO: 32), VTSTWVLVGGVLAAL (SEQ ID NO: 37), DWICEVLSDFKTWLKAKL (SEQ ID NO: 44), DTEDVVQCSMSYSW (SEQ ID NO: 47), SSGADTEDVVCCSMSYSW (SEQ ID NO: 48), and CTMLVCGDDLVVICESAG (SEQ ID NO: 53).
83. The method of claim 51 , wherein the peptide consists of an amino acid sequence selected from the group consisting of 6, 8, 12, 13, 14, 21, 23, 24, 27, 28, 30, 32, 37, 44, 47, 48 and 53.
84. The method of claim 51, wherein each of the amino acids in the peptide is a D-amino acid.
85. The method of claim 51, wherein each of the amino acids in the peptide is an L-amino acid.
86. The method of claim 51 , wherein the peptide further comprises a dansyl moiety.
87. A method for preventing or treating infection of a mammalian cell by a human immunodeficiency virus comprising contacting the cell with a peptide of 14 to 50 D- or L-amino acids in length, wherein the peptide comprises an alpha-helical structure, and wherein the polar amino acids are located on the same face of the alpha helical structure, and the nonpolar amino acids are located on the other face of the alpha-helical structure.
88. The method of claim 87, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4-86 and 91-102.
89. The method of claim 87, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4-86 and 91-102.
90. A method for preventing or treating infection of a mammal by a human immunodeficiency virus comprising administering to the mammal a peptide of 14 to 50 D- or L-amino acids in length, wherein the peptide comprises an alpha-helical structure, and wherein the polar amino acids are located on the same face of the alpha helical structure, and the nonpolar amino acids are located on the other face of the alpha-helical structure.
91. The method of claim 90, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4-86 and 91-102.
92. The method of claim 90, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4-86 and 91-102.
93. An isolated peptide of 14 to 50 D- or L- amino acids in length comprising an amino acid sequence of formula V:
Xaai-Xaa2-Xaa3- Xaa^Xaas-Xaaό-Xaav-Xaag-Xaag-Xaaio-Xaai i-W-Xaao -W-Xaa,5-Xaa,6-Xaai7- L-W-Xaa20-Xaa2i-Xaa22 (SEQ ID NO: 595), wherein
Xaai is tryptophan (W) or absent;
Xaa2 is threonine (T) or absent;
Xaa3 is lysine (K), glutamic acid (E), arginine (R), aspartic acid (D) or absent;
Xaa4 is phenylalanine or absent;
Xaa5 is aspartic acid (D), arginine (R), glutamic acid (E), lysine (K) or absent;
Xaa6 is serine (S), threonine (T), leucine (L) or absent;
Xaa7 is leucine (L) or valine (V);
Xaa8 is valine (V), alanine (A) or serine;
Xaa9 is glutamic acid (E), lysine (K), aspartic acid (D), threonine (T), histidine (H), serine (S) or arginine (R);
Xaaio is cysteine (C), glutamic acid (E), leucine (L), serine (S) or arginine
(R);
Xaaπ is isoleucine (I) or valine (V);
W is tryptophan;
Xaao is aspartic acid (D), arginine (R), glutamic acid (E) or lysine (K);
Xaai5 is isoleucine (I) or valine (V);
Xaai6 is aspartic acid (D), arginine (R), glutamic acid (E), alanine (A), isoleucine (I) or lysine (K);
Xaaπ is arginine (R), aspartic acid (D), lysine (K), tryptophan (W) or glutamic acid (E);
L is leucine; Xaa20 is serine (S), aspartic acid (D) or threonine (T); Xaa2i is glycine (G) or absent; and Xaa22 is serine (S) or absent; and wherein the amino acid sequence of formula V is not:
KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), FDSLVECIWDWIDRLWS (SEQ ID NO: 99), DSLVECIWDWIDRLWS (SEQ ID NO: 100), SLVECIWDWIDRLWS (SEQ ID NO: 101), or LVECIWDWIDRLWS (SEQ ID NO: 102).
94. The peptide of claim 93, which comprises an amino acid sequence selected from the group consisting of:
KFDLVSECIWDWIDLRWS (SEQ ID NO: 277), FDLVSECIWDWIDLRWS (SEQ ID NO: 278), DLVSECIWDWIDLRWS (SEQ ID NO: 279), LVSECIWDWIDLRWS (SEQ ID NO: 280), VSECIWDWIDLRWS (SEQ ID NO: 281), KFDSVVSCVWDWIIRLWD (SEQ ID NO: 282), FDSVVSCVWDWIIRLWD (SEQ ID NO: 283), DSVVSCVWDWIIRLWD (SEQ ID NO: 284), SVVSCVWDWIIRLWD (SEQ ID NO: 285), VVSCVWDWIIRLWD (SEQ ID NO: 286), KFDSLVHLVWDWVEWLWS (SEQ ID NO: 288), FDSLVHLVWDWVEWLWS (SEQ ID NO: 289), DSLVHLVWDWVEWLWS (SEQ ID NO:290), SLVHLVWDWVEWLWS (SEQ ID NO: 291), LVHLVWDWVEWLWS (SEQ ID NO: 292), KFDTLATCVWDWIARLWT (SEQ ID NO: 293), FDTLATCVWDWIARLWT (SEQ ID NO: 294), DTLATCVWDWIARLWT (SEQ ID NO: 295), TLATCVWDWIARLWT (SEQ ID NO: 296), LATCVWDWIARLWT (SEQ ID NO: 297), KFDSLVTCVWDWVDRLWS (SEQ ID NO: 298), FDSLVTCVWDWVDRLWS (SEQ ID NO: 299), DSLVTCVWDWVDRLWS (SEQ ID NO: 300), SLVTCVWDWVDRLWS (SEQ ID NO: 301), LVTCVWDWVDRLWS (SEQ ID NO: 302), KFDSLVESIWDWIDRLWS (SEQ ID NO: 303), FDSLVESIWDWIDRLWS (SEQ ID NO: 304), DSLVESIWDWIDRLWS (SEQ ID NO: 305), SLVESIWDWIDRLWS (SEQ ID NO: 306), LVESIWDWIDRLWS (SEQ ID NO: 307), EFRSLVKCIWRWIRDLWS (SEQ ID NO: 308), FRSLVKCIWRWIRDLWS (SEQ ID NO: 309), RSLVKCIWRWIRDLWS (SEQ ID NO: 310), SLVKCIWRWIRDLWS (SEQ ID NO: 311), LVKCIWRWIRDLWS (SEQ ID NO: 312), EFDSLVECIWDWIDDLWS (SEQ ID NO: 313), FDSLVECIWDWIDDLWS (SEQ ID NO: 314), DSLVECIWDWIDDLWS (SEQ ID NO: 315), SLVECIWDWIDDLWS (SEQ ID NO: 316), LVECIWDWIDDLWS (SEQ ID NO: 317), KFRSLVKCIWRWIRRLWS (SEQ ID NO: 318), FRSLVKCIWRWIRRLWS (SEQ ID NO: 319), RSLVKCIWRWIRRLWS (SEQ ID NO: 320), SLVKCIWRWIRRLWS (SEQ ID NO: 321), LVKCIWRWIRRLWS (SEQ ID NO: 322), RFESLVDCIWEWIEKLWS (SEQ ID NO: 323), FESLVDCIWEWIEKLWS (SEQ ID NO: 324), ESLVDCIWEWIEKLWS (SEQ ID NO: 325), SLVDCIWEWIEKLWS (SEQ ID NO: 326), LVDCIWEWIEKLWS (SEQ ID NO: 327), RFDSLVECIWDWIDKLWS (SEQ ID NO: 328), FDSLVECIWDWIDKLWS (SEQ ID NO: 329), DSLVECIWDWIDKLWS (SEQ ID NO: 330), SLVECIWDWIDKLWS (SEQ ID NO: 331), LVECIWDWIDKLWS (SEQ ID NO: 332), KFDSLVECIWDWIDKLWS (SEQ ID NO: 333), FDSLVECIWDWIDKLWS (SEQ ID NO: 334), DSLVECIWDWIDKLWS (SEQ ID NO: 335), SLVECIWDWIDKLWS (SEQ ID NO: 336), LVECIWDWIDKLWS (SEQ ID NO: 337), KFESLVDCIWEWIERLWS (SEQ ID NO: 338), FESLVDCIWEWIERLWS (SEQ ID NO: 339), ESLVDCIWEWIERLWS (SEQ ID NO: 340), SLVDCIWEWIERLWS (SEQ ID NO: 341), LVDCIWEWIERLWS (SEQ ID NO: 342), KFESLVECIWEWIERLWS (SEQ ID NO: 343), FESLVECIWEWIERLWS (SEQ ID NO: 344), ESLVECIWEWIERLWS (SEQ ID NO: 345), SLVECIWEWIERLWS (SEQ ID NO: 346), LVECIWEWIERLWS (SEQ ID NO: 347), EFRSLVKCIWRWIRDLWS (SEQ ID NO: 348), FRSLVKCIWRWIRDLWS (SEQ ID NO: 349), RSLVKCIWRWIRDLWS (SEQ ID NO: 350), SLVKCIWRWIRDLWS (SEQ ID NO: 351), LVKCIWRWIRDLWS (SEQ ID NO: 352), EFDSLVECIWDWIDDLWS (SEQ ID NO: 353), FDSLVECIWDWIDDLWS (SEQ ID NO: 354), DSLVECIWDWIDDLWS (SEQ ID NO: 355), SLVECIWDWIDDLWS (SEQ ID NO: 356), LVECIWDWIDDLWS (SEQ ID NO: 357), KFRSLVKCIWRWIRRLWS (SEQ ID NO: 358), FRSLVKCIWRWIRRLWS (SEQ ID NO: 359), RSLVKCIWRWIRRLWS (SEQ ID NO: 360), SLVKCIWRWIRRLWS (SEQ ID NO: 361), LVKCIWRWIRRLWS (SEQ ID NO: 362), KFDSLVERIWDWIDRLWS (SEQ ID NO: 363), FDSLVERIWDWIDRLWS (SEQ ID NO: 364), DSLVERIWDWIDRLWS (SEQ ID NO: 365), SLVERIWDWIDRLWS (SEQ ID NO: 366), LVERIWDWIDRLWS (SEQ ID NO: 367), KFDSLVEEIWDWIDRLWS (SEQ ID NO: 368), FDSLVEEIWDWIDRLWS (SEQ ID NO: 369), DSLVEEIWDWIDRLWS (SEQ ID NO: 370), SLVEEIWDWIDRLWS (SEQ ID NO: 371), LVEEIWDWIDRLWS (SEQ ID NO: 372), KFDSLVECIWDWIDRLWSGS (SEQ ID NO: 373), FDSLVECIWDWIDRLWSGS (SEQ ID NO: 374), DSLVECIWDWIDRLWSGS (SEQ ID NO: 375), SLVECIWDWIDRLWSGS (SEQ ID NO: 376), LVECIWDWIDRLWSGS (SEQ ID NO: 377), KFDSLVECIWDWIDRLWSG (SEQ ID NO: 378), FDSLVECIWDWIDRLWSG (SEQ ID NO: 379), DSLVECIWDWIDRLWSG (SEQ ID NO: 380), SLVECIWDWIDRLWSG (SEQ ID NO: 381), LVECIWDWIDRLWSG (SEQ ID NO: 382), TKFDSLVECIWDWIDRLWS (SEQ ID NO: 383), KFDSLVECIWDWIDRLWS (SEQ ID NO: 384), FDSLVECIWDWIDRLWS (SEQ ID NO: 385), DSLVECIWDWIDRLWS (SEQ ID NO: 386), SLVECIWDWIDRLWS (SEQ ID NO: 387), WTKFDSLVECIWDWIDRLWS (SEQ ID NO: 388), TKFDSLVECIWDWIDRLWS (SEQ ID NO: 389), KFDSLVECIWDWIDRLWS (SEQ ID NO: 390), FDSLVECIWDWIDRLWS (SEQ ID NO: 391), and DSLVECIWDWIDRLWS (SEQ ID NO: 392).
95. The peptide of claim 93, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 277-392.
96. The isolated peptide of claim 93, which comprises an amino acid sequence of formula VI:
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W- Xaa13-W-I-Xaa,6-Xaa17-L-W-S- Xaa2rXaa22 (SEQ ID NO: 596), wherein:
Xaai, Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is lysine (K), glutamic acid (E), aspartic acid (D), arginine (R) or absent;
Xaa4 is phenylalanine (F) or absent;
Xaa5 is aspartic acid (D), arginine (R), lysine (K) or absent; Xaaό is serine (S) or absent L is leucine; V is valine;
Xaag is lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D); C is cysteine; I is isoleucine; W is tryptophan; Xaaπ is aspartic acid (D), arginine (R), or lysine (K); Xaai6 is arginine (R), aspartic acid (D) or lysine (K);
Xaai7 is arginine (R), aspartic acid (D), glutamic acid (E) or lysine (K); and
S is serine.
97. The peptide of claim 96, which comprises an amino acid sequence selected from the group consisting of:
DFKSLVRCIWKWIKELWS (SEQ ID NO: 481);
FKSLVRCIWKWIKELWS (SEQ ID NO: 482);
KSLVRCIWKWIKELWS (SEQ ID NO: 483);
SLVRCIWKWIKELWS (SEQ ID NO: 484);
LVRCIWKWIKELWS (SEQ ID NO: 485);
KFDSLVECIWKWIKRLWS (SEQ ID NO: 486);
FDSLVECIWKWIKRLWS (SEQ ID NO: 487);
DSLVECIWKWIKRLWS (SEQ ID NO: 488);
SLVECIWKWIKRLWS (SEQ ID NO: 489);
LVECIWKWIKRLWS (SEQ ID NO: 490);
KFKSLVKCIWDWIDRLWS (SEQ ID NO: 491);
FKSLVKCIWDWIDRLWS (SEQ ID NO: 492);
KSLVKCIWDWIDRLWS (SEQ ID NO: 493);
SLVKCIWDWIDRLWS (SEQ ID NO: 494);
LVKCIWDWIDRLWS (SEQ ID NO: 495);
KFDSLVECIWRWIRRLWS (SEQ ID NO: 496);
FDSLVECIWRWIRRLWS (SEQ ID NO: 497);
DSLVECIWRWIRRLWS (SEQ ID NO: 498);
SLVECIWRWIRRLWS (SEQ ID NO: 499);
LVECIWRWIRRLWS (SEQ ID NO: 500);
KFRSLVRCIWDWIDRLWS (SEQ ID NO: 501);
FRSLVRCIWDWIDRLWS (SEQ ID NO: 502);
RSLVRCIWDWIDRLWS (SEQ ID NO: 503); SLVRCIWDWIDRLWS (SEQ ID NO: 504);
LVRCIWDWIDRLWS (SEQ ID NO: 505);
KFDSLVRCIWDWIRRLWS (SEQ ID NO: 506);
FDSLVRCIWDWIRRLWS (SEQ ID NO: 507);
DSLVRCIWDWIRRLWS (SEQ ID NO: 508);
SLVRCIWDWIRRLWS (SEQ ID NO: 509);
LVRCIWDWIRRLWS (SEQ ID NO: 510);
KFDSLVKCIWDWIKRLWS (SEQ ID NO: 511);
FDSLVKCIWDWIKRLWS (SEQ ID NO: 512);
DSLVKCIWDWIKRLWS (SEQ ID NO: 513);
SLVKCIWDWIKRLWS (SEQ ID NO: 514);
LVKCIWDWIKRLWS (SEQ ID NO: 515);
KFRSLVECIWDWIRRLWS (SEQ ID NO: 516);
FRSLVECIWDWIRRLWS (SEQ ID NO: 517);
RSLVECIWDWIRRLWS (SEQ ID NO: 518);
SLVECIWDWIRRLWS (SEQ ID NO: 519);
LVECIWDWIRRLWS (SEQ ID NO: 520);
KFKSLVECIWDWIKRLWS (SEQ ID NO: 521);
FKSLVECIWDWIKRLWS (SEQ ID NO: 522);
KSLVECIWDWIKRLWS (SEQ ID NO: 523);
SLVECIWDWIKRLWS (SEQ ID NO: 524);
LVECIWDWIKRLWS (SEQ ID NO: 525);
KFDSLVRCIWRWIDRLWS (SEQ ID NO: 526);
FDSLVRCIWRWIDRLWS (SEQ ID NO: 527);
DSLVRCIWRWIDRLWS (SEQ ID NO: 528);
SLVRCIWRWIDRLWS (SEQ ID NO: 529);
LVRCIWRWIDRLWS (SEQ ID NO: 530);
KFDSLVKCIWKWIDRLWS (SEQ ID NO: 531);
FDSLVKCIWKWIDRLWS (SEQ ID NO: 532); DSLVKCIWKWIDRLWS (SEQ ID NO: 533);
SLVKCIWKWIDRLWS (SEQ ID NO: 534);
LVKCIWKWIDRLWS (SEQ ID NO: 535);
KFRSLVRCIWDWIRDLWS (SEQ ID NO: 536);
FRSLVRCIWDWIRDLWS .(SEQ ID NO: 537);
RSLVRCIWDWIRDLWS (SEQ ID NO: 538);
SLVRCIWDWIRDLWS (SEQ ID NO: 539);
LVRCIWDWIRDLWS (SEQ ID NO: 540);
KFKSLVKCIWDWIDRLWS (SEQ ID NO: 541);
FKSLVKCIWDWIDRLWS (SEQ ID NO: 542);
KSLVKCIWDWIDRLWS (SEQ ID NO: 543);
SLVKCIWDWIDRLWS (SEQ ID NO: 544); and
LVKCIWDWIDRLWS (SEQ ID NO: 545).
98. The peptide of claim 96, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 481 to 545.
99. The isolated peptide of claim 96, which comprises an amino acid sequence of formula VII:
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W- Xaa13-W-I-Xaa,6-R-L-W-S-Xaa2,-Xaa22 (SEQ ID NO: 597), wherein:
Xaai, Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is lysine (K) or absent;
Xaa4 is phenylalanine (F) or absent;
Xaa5 is arginine (R) or lysine (K), or absent
Xaaβ is serine (S) or absent;
L is leucine; V is valine;
Xaa9 is lysine (K), glutamic acid (E) or aspartic acid (D); C is cysteine; I is isoleucine; W is tryptophan;
Xaai3 is arginine (R) or lysine (K),
Xaai6 is aspartic acid (D), arginine (R), or lysine (K); and
R is arginine; S is serine.
100. The peptide of claim 99, which comprises an amino acid sequence selected from the group consisting of:
KFRSLVKCIWRWIDRLWS (SEQ ID NO: 546); FRSLVKCIWRWIDRLWS (SEQ ID NO: 547); RSLVKCIWRWIDRLWS (SEQ ID NO: 548); SLVKCIWRWIDRLWS (SEQ ID NO: 549); LVKCIWRWIDRLWS (SEQ ID NO: 550); KFKSLVKCIWKWIDRLWS (SEQ ID NO: 551); FKSLVKCIWKWIDRLWS (SEQ ID NO: 552); KSLVKCIWKWIDRLWS (SEQ ID NO: 553); SLVKCIWKWIDRLWS (SEQ ID NO: 554); LVKCIWKWIDRLWS (SEQ ID NO: 555); KFKSLVECIWKWIKRLWS (SEQ ID NO: 556); FKSLVECIWKWIKRLWS (SEQ ID NO: 557); KSLVECIWKWIKRLWS (SEQ ID NO: 558); SLVECIWKWIKRLWS (SEQ ID NO: 559); LVECIWKWIKRLWS (SEQ ID NO: 560); KFRSLVECIWRWIRRLWS (SEQ ID NO: 561); FRSLVECIWRWIRRLWS (SEQ ID NO: 562); RSLVECIWRWIRRLWS (SEQ ID NO: 563); SLVECIWRWIRRLWS (SEQ ID NO: 564); LVECIWRWIRRLWS (SEQ ID NO: 565); KFRSLVDCIWRWIRRLWS (SEQ ID NO: 566). FRSLVDCIWRWIRRLWS (SEQ ID NO: 567). RSLVDCIWRWIRRLWS (SEQ ID NO: 568).
SLVDCIWRWIRRLWS (SEQ ID NO: 569).
LVDCIWRWIRRLWS (SEQ ID NO: 570).
101. The peptide of claim 99, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 546 to 570.
102. The isolated peptide of claim 96, which comprises an amino acid sequence of formula VIII:
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W- Xaai3-W-I-Xaa16-Xaa,7-L-W-S-Xaa2,-Xaa22 (SEQ ID NO: 598), wherein:
Xaa1 ? Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is arginine (R), lysine (K) or absent;
Xaa4 is phenylalanine (F) or absent;
Xaa5 is arginine (R), lysine (K) or absent;
Xaa<5 is serine (S) or absent;
L is leucine; V is valine;
Xaap, Xaai3i Xaai6 and Xaai7 are arginine (R) or lysine (K); and
C is cysteine; I is isoleucine; W is tryptophan; S is serine.
103. The peptide of claim 102, which comprises an amino acid sequence selected from the group consisting of:
KFKSLVKCIWKWIKRLWS (SEQ ID NO: 571); FKSLVKCIWKWIKRLWS (SEQ ID NO: 572); KSLVKCIWKWIKRLWS (SEQ ID NO: 573); SLVKCIWKWIKRLWS (SEQ ID NO: 574); LVKCIWKWIKRLWS (SEQ ID NO: 575); KFRSLVRCIWRWIRRLWS (SEQ ID NO: 576); FRSLVRCIWRWIRRLWS (SEQ ID NO: 577); RSLVRCIWRWIRRLWS (SEQ ID NO: 578); SLVRCIWRWIRRLWS (SEQ ID NO: 579); LVRCIWRWIRRLWS (SEQ ID NO: 580); RFRSLVRCIWRWIRRLWS (SEQ ID NO: 581); FRSLVRCIWRWIRRLWS (SEQ ID NO: 582); RSLVRCIWRWIRRLWS (SEQ ID NO: 583); SLVRCIWRWIRRLWS (SEQ ID NO: 584); LVRCIWRWIRRLWS (SEQ ID NO: 585); KFKSLVKCIWKWIKKLWS (SEQ ID NO: 586). FKSLVKCIWKWIKKLWS (SEQ ID NO: 587). KSLVKCIWKWIKKLWS (SEQ ID NO: 588). SLVKCIWKWIKKLWS (SEQ ID NO: 589). LVKCIWKWIKKLWS (SEQ ID NO: 590).
104. The peptide of claim 102, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 571-590.
105. A method for inactivating a human immunodeficiency virus comprising contacting the virus with a peptide of formula V, VI, VII or VIII:
(V) XaapXaa2-Xaa3- Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa1o-
Xaai i-W-Xaai3-W-Xaai5-Xaai6-Xaai7- L-W-Xaa20-Xaa2i-
Xaa22 (SEQ ID NO: 595), wherein
Xaai is tryptophan (W) or absent;
Xaa2 is threonine (T) or absent;
Xaa3 is lysine (K), glutamic acid (E), arginine (R), aspartic acid (D) or absent;
Xaa4 is phenylalanine or absent; Xaa5 is aspartic acid (D), arginine (R), glutamic acid (E), lysine (K) or absent;
Xaaό is serine (S), threonine (T), leucine (L) or absent;
Xaa7 is leucine (L) or valine (V);
Xaa8 is valine (V), alanine (A) or serine;
Xaa9 is glutamic acid (E), lysine (K), aspartic acid (D), threonine (T), histidine (H), serine (S) or arginine (R);
Xaaio is cysteine (C), glutamic acid (E), leucine (L), serine (S) or arginine (R);
Xaai i is isoleucine (I) or valine (V);
W is tryptophan;
Xaai3 is aspartic acid (D), arginine (R), glutamic acid (E) or lysine (K);
Xaai5 is isoleucine (I) or valine (V);
Xaa16 is aspartic acid (D), arginine (R), glutamic acid (E), alanine (A), isoleucine (I) or lysine (K);
Xaai7 is arginine (R), aspartic acid (D), lysine (K), tryptophan (W) or glutamic acid (E);
L is leucine;
Xaa20 is serine (S), aspartic acid (D) or threonine (T);
Xaa2i is glycine (G) or absent; and
Xaa22 is serine (S) or absent; and wherein the amino acid sequence of formula V is not:
KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), FDSLVECIWDWIDRLWS (SEQ ID NO: 99), DSLVECIWDWIDRLWS (SEQ ID NO: 100), SLVECIWDWIDRLWS (SEQ ID NO: 101), or LVECIWDWIDRLWS (SEQ ID NO: 102); (VI) Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W-Xaai3-W-I-
Xaa,6-Xaa,7-L-W-S- Xaa2,-Xaa22 (SEQ ID NO: 596), wherein:
Xaai, Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is lysine (K), glutamic acid (E), aspartic acid (D), arginine (R) or absent;
Xaa4 is phenylalanine (F) or absent;
Xaa5 is aspartic acid (D), arginine (R), lysine (K) or absent;
Xaaό is serine (S) or absent
L is leucine; V is valine;
Xaa9 is lysine (K), arginine (R), glutamic acid (E) or aspartic acid
(D);
C is cysteine; I is isoleucine; W is tryptophan; Xaa13 is aspartic acid (D), arginine (R), or lysine (K); Xaai6 is arginine (R), aspartic acid (D) or lysine (K); Xaai 7 is arginine (R), aspartic acid (D), glutamic acid (E) or lysine (K); and S is serine;
(VII) Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W-Xaai3-
W-I-Xaai6-R-L-W-S-Xaa2i-Xaa22 (SEQ ID NO: 597), wherein:
Xaai, Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is lysine (K) or absent;
Xaa_i is phenylalanine (F) or absent;
Xaa5 is arginine (R) or lysine (K), or absent
Xaaό is serine (S) or absent;
L is leucine; V is valine;
Xaa9 is lysine (K), glutamic acid (E) or aspartic acid (D);
C is cysteine; I is isoleucine; W is tryptophan; Xaaπ is arginine (R) or lysine (K),
Xaai6 is aspartic acid (D), arginine (R), or lysine (K); and
R is arginine; S is serine;
(VIII) Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W-
Xaa13-W-I-Xaa16-XaaI 7-L-W-S-Xaa2,-Xaa22 (SEQ ID NO: 598), wherein:
Xaai, Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is arginine (R), lysine (K) or absent;
Xaa4 is phenylalanine (F) or absent;
Xaa5 is arginine (R), lysine (K) or absent;
Xaaβ is serine (S) or absent;
L is leucine; V is valine;
Xaa9, Xaai3; Xaai6 and Xaaπ are arginine (R) or lysine (K); and
C is cysteine; I is isoleucine; W is tryptophan; S is serine.
106. The method of claim 105, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 277-392 and 481 to 590.
107. The method of claim 105, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 277-392 and 481 to 590.
108. A method for preventing or treating infection of a mammalian cell by a human immunodeficiency virus comprising contacting the cell with a peptide of formula V, VI, VII or VIII:
(V) Xaai-Xaa2-Xaa3- Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaai0-
Xaai i -W-Xaai 3-W-Xaai 5-Xaai 6-Xaai 7- L-W-Xaa20-Xaa2i - Xaa22 (SEQ ID NO: 595), wherein
Xaai is tryptophan (W) or absent;
Xaa2 is threonine (T) or absent;
Xaa3 is lysine (K), glutamic acid (E), arginine (R), aspartic acid (D) or absent;
Xaa4 is phenylalanine or absent;
Xaa5 is aspartic acid (D), arginine (R), glutamic acid (E), lysine (K) or absent;
Xaaδ is serine (S), threonine (T), leucine (L) or absent;
Xaa7 is leucine (L) or valine (V);
Xaa8 is valine (V), alanine (A) or serine;
Xaa9 is glutamic acid (E), lysine (K), aspartic acid (D), threonine (T), histidine (H), serine (S) or arginine (R);
Xaaio is cysteine (C), glutamic acid (E), leucine (L), serine (S) or arginine (R);
Xaai i is isoleucine (I) or valine (V);
W is tryptophan;
Xaai3 is aspartic acid (D), arginine (R), glutamic acid (E) or lysine (K);
Xaai 5 is isoleucine (I) or valine (V);
Xaai6 is aspartic acid (D), arginine (R), glutamic acid (E), alanine (A), isoleucine (I) or lysine (K);
Xaaπ is arginine (R), aspartic acid (D), lysine (K), tryptophan (W) or glutamic acid (E);
L is leucine;
Xaa2o is serine (S), aspartic acid (D) or threonine (T);
Xaa2i is glycine (G) or absent; and
Xaa22 is serine (S) or absent; and wherein the amino acid sequence of formula V is not:
KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), FDSLVECIWDWIDRLWS (SEQ ID NO: 99), DSLVECIWDWIDRLWS (SEQ ID NO: 100), SLVECIWDWIDRLWS (SEQ ID NO: 101), or LVECIWDWIDRLWS (SEQ ID NO: 102);
(VI) Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W-Xaai3-W-I-
Xaa,6-Xaa17-L-W-S- Xaa21-Xaa22 (SEQ ID NO: 596), wherein:
Xaal 5 Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is lysine (K), glutamic acid (E), aspartic acid (D), arginine (R) or absent;
Xaazj is phenylalanine (F) or absent;
Xaa5 is aspartic acid (D), arginine (R), lysine (K) or absent;
Xaa^ is serine (S) or absent
L is leucine; V is valine;
Xaa9 is lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D);
C is cysteine; I is isoleucine; W is tryptophan;
Xaaπ is aspartic acid (D), arginine (R), or lysine (K);
Xaa16 is arginine (R), aspartic acid (D) or lysine (K);
Xaaπ is arginine (R), aspartic acid (D), glutamic acid (E) or lysine (K); and S is serine;
(VII) Xaai-XaarXaarXaa^Xaas-Xaae-L-V-Xaag-C-I-W-Xaaπ-
W-I-Xaa16-R-L-W-S-Xaa21-Xaa22 (SEQ ID NO: 597), wherein:
Xaai, Xaa2, Xaa2i and Xaa22 are absent; Xaa3 is lysine (K) or absent; Xaβ4 is phenylalanine (F) or absent;
Xaa5 is arginine (R) or lysine (K), or absent
Xaaό is serine (S) or absent;
L is leucine; V is valine;
Xaa9 is lysine (K), glutamic acid (E) or aspartic acid (D);
C is cysteine; I is isoleucine; W is tryptophan;
Xaaπ is arginine (R) or lysine (K),
Xaai6 is aspartic acid (D), arginine (R), or lysine (K); and
R is arginine; S is serine;
(VIII) Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W-
Xaai3-W-I-Xaai6-Xaai7-L-W-S-Xaa21-Xaa22 (SEQ ID NO: 598), wherein:
Xaai, Xaa2, Xaa2j and Xaa22 are absent;
Xaa3 is arginine (R), lysine (K) or absent;
Xaa4 is phenylalanine (F) or absent;
Xaa5 is arginine (R), lysine (K) or absent;
Xaa6 is serine (S) or absent;
L is leucine; V is valine;
Xaa9, Xaai3 Xaai6 and Xaa17 are arginine (R) or lysine (K); and
C is cysteine; I is isoleucine; W is tryptophan; S is serine.
109. The method of claim 108, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 277-392 and 481 to 590.
110. The method of claim 108, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 277-392 and 481 to 590.
1 11. A method for preventing or treating infection of a mammal by a human immunodeficiency virus comprising administering to the mammal a peptide of formula V, VI, VII or VIII:
(V) Xaai-Xaa2-Xaa3- Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaaio-
Xaai i-W-Xaai3-W-Xaai5-Xaai6-Xaai7- L-W-Xaa2o-Xaa2i-
Xaa22 (SEQ ID NO: 595), wherein
Xaai is tryptophan (W) or absent;
Xaa2 is threonine (T) or absent;
Xaa3 is lysine (K), glutamic acid (E), arginine (R), aspartic acid (D) or absent;
Xaa4 is phenylalanine or absent;
Xaa5 is aspartic acid (D), arginine (R), glutamic acid (E), lysine (K) or absent;
Xaa6 is serine (S), threonine (T), leucine (L) or absent;
Xaa7 is leucine (L) or valine (V);
Xaa8 is valine (V), alanine (A) or serine;
Xaa9 is glutamic acid (E), lysine (K), aspartic acid (D), threonine (T), histidine (H), serine (S) or arginine (R);
Xaaio is cysteine (C), glutamic acid (E), leucine (L), serine (S) or arginine (R);
Xaaπ is isoleucine (I) or valine (V);
W is tryptophan;
Xaaπ is aspartic acid (D), arginine (R), glutamic acid (E) or lysine (K);
Xaais is isoleucine (I) or valine (V);
Xaai6 is aspartic acid (D), arginine (R), glutamic acid (E), alanine (A), isoleucine (I) or lysine (K); Xaaπ is arginine (R), aspartic acid (D), lysine (K), tryptophan (W) or glutamic acid (E); L is leucine;
Xaa20 is serine (S), aspartic acid (D) or threonine (T); Xaa2i is glycine (G) or absent; and Xaa22 is serine (S) or absent; and wherein the amino acid sequence of formula V is not:
KFDSLVECIWDWIDRLWS (SEQ ID NO: 96), FDSLVECIWDWIDRLWS (SEQ ID NO: 99), DSLVECIWDWIDRLWS (SEQ ID NO: 100), SLVECIWDWIDRLWS (SEQ ID NO: 101), or LVECIWDWIDRLWS (SEQ ID NO: 102);
(VI) Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W-Xaai3-W-I-
Xaai6-Xaa,7-L-W-S- Xaa2,-Xaa22 (SEQ ID NO: 596), wherein:
Xaai, Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is lysine (K), glutamic acid (E), aspartic acid (D), arginine (R) or absent;
Xaa* is phenylalanine (F) or absent;
Xaa5 is aspartic acid (D), arginine (R), lysine (K) or absent;
XasLβ is serine (S) or absent
L is leucine; V is valine;
Xaa9 is lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D);
C is cysteine; I is isoleucine; W is tryptophan;
Xaao is aspartic acid (D), arginine (R), or lysine (K);
Xaai6 is arginine (R), aspartic acid (D) or lysine (K); Xaaπ is arginine (R), aspartic acid (D), glutamic acid (E) or lysine (K); and S is serine;
(VII) Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-Xaa9-C-I-W-Xaa,3-
W-I-Xaai6-R-L-W-S-Xaa2,-Xaa22 (SEQ ID NO: 597), wherein:
Xaal5 Xaa2, Xaa21 and Xaa22 are absent;
Xaa3 is lysine (K) or absent;
Xaa_i is phenylalanine (F) or absent;
Xaa5 is arginine (R) or lysine (K), or absent
Xaaό is serine (S) or absent;
L is leucine; V is valine;
Xaa9 is lysine (K), glutamic acid (E) or aspartic acid (D);
C is cysteine; I is isoleucine; W is tryptophan;
Xaa13 is arginine (R) or lysine (K),
Xaaiό is aspartic acid (D), arginine (R), or lysine (K); and
R is arginine; S is serine;
(VIII) Xaa, -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L- V-Xaa9-C-I-W- Xaa13-W-I-Xaai6-Xaai7-L-W-S-Xaa21-Xaa22 (SEQ ID NO: 598), wherein:
Xaaj, Xaa2, Xaa2i and Xaa22 are absent;
Xaa3 is arginine (R), lysine (K) or absent;
Xaa_t is phenylalanine (F) or absent;
Xaa5 is arginine (R), lysine (K) or absent;
Xaaβ is serine (S) or absent;
L is leucine; V is valine;
Xaa9, Xaai3 Xaai6 and Xaaπ are arginine (R) or lysine (K); and
C is cysteine; I is isoleucine; W is tryptophan; S is serine.
112. The method of claim 111, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 277-392 and 481 to 590.
1 13. The method of claim 111, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 277-392 and 481 to 590.
1 14. Use of a peptide of formulae I, II, III, IV, V, VI, VII, or VIII in the preparation of a medicament for preventing and/or treating infection in a mammal by a human immunodeficiency virus, Hepatitis C virus, West Nile virus, or Dengue virus.
1 15. Use of a peptide of formulae I, II, III, IV, V, VI, VII, or VIII in the preparation of a medicament for preventing and/or treating infection in a mammal by a Flavivirus or an Hepacivirus.
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WO2009014615A2 (en) * 2007-07-19 2009-01-29 The Board Of Trustees Of The Leland Stanford Junior University Amphipathic alpha-helical peptide compositions as antiviral agents
RU2526799C2 (en) * 2012-09-06 2014-08-27 Виталий Александрович Шестаков Pharmaceutical composition and method for preparing antiviral fractions (antivirus-c)
JP2018531880A (en) * 2015-06-25 2018-11-01 ナンヤン テクノロジカル ユニヴァーシティー Broad spectrum anti-infective peptide
WO2022220749A1 (en) * 2021-04-13 2022-10-20 Nanyang Technological University Anti-viral peptide compositions and methods to improve biological activity thereof
WO2022232275A1 (en) * 2021-04-27 2022-11-03 Oak Crest Institute Of Science Materials and methods for the prevention and treatment of viral diseases
WO2024054009A1 (en) * 2022-09-08 2024-03-14 대한뉴팜㈜ Novel antiviral peptide

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WO2009014615A2 (en) * 2007-07-19 2009-01-29 The Board Of Trustees Of The Leland Stanford Junior University Amphipathic alpha-helical peptide compositions as antiviral agents
WO2009014615A3 (en) * 2007-07-19 2009-12-03 The Board Of Trustees Of The Leland Stanford Junior University Amphipathic alpha-helical peptide compositions as antiviral agents
US8728793B2 (en) 2007-07-19 2014-05-20 The Board Of Trustees Of The Leland Stanford Junior University Amphipathic alpha-helical peptide compositions as antiviral agents
RU2526799C2 (en) * 2012-09-06 2014-08-27 Виталий Александрович Шестаков Pharmaceutical composition and method for preparing antiviral fractions (antivirus-c)
JP2018531880A (en) * 2015-06-25 2018-11-01 ナンヤン テクノロジカル ユニヴァーシティー Broad spectrum anti-infective peptide
EP3313862A4 (en) * 2015-06-25 2019-01-23 Nanyang Technological University Broad-spectrum anti-infective peptides
US10351604B2 (en) 2015-06-25 2019-07-16 Nanyang Technological University Broad-spectrum anti-infective peptides
US11866460B2 (en) 2015-06-25 2024-01-09 Nanyang Technological University Broad-spectrum anti-infective peptides
WO2022220749A1 (en) * 2021-04-13 2022-10-20 Nanyang Technological University Anti-viral peptide compositions and methods to improve biological activity thereof
WO2022232275A1 (en) * 2021-04-27 2022-11-03 Oak Crest Institute Of Science Materials and methods for the prevention and treatment of viral diseases
WO2024054009A1 (en) * 2022-09-08 2024-03-14 대한뉴팜㈜ Novel antiviral peptide

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