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
  • 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
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention is directed to novel peptides which inhibit HIV-induced fusion and syncytia formation and, thus, inhibit one of the major cytopathological effects of HIV.
• the peptides mimic the amino terminus of the transmembrane protein of HIV virus.
• the invention is demonstrated by way of examples in which the efficacy of such inhibitory peptides is measured by an in vitro assay system.
• enveloped viruses The entry of enveloped viruses into mammalian cells is thought to occur by a process of membrane fusion mediated by viral glycoproteins.
• the paramyxoviruses deliver their nucleocapsids into the cytoplasm of the infected cells by fusion of the viral membrane with the plasma membrane.
• orthomyxoviruses, rhabdoviruses, alphaviruses, and certain retroviruses have been demonstrated to enter the cell via coated pits which are internalized and become part of the endosomal compartment (White and Helenius, 1980 Proc.
• Virology 110:243-247 Following acidification of the endocytic vesicles, the fusion activity of specific viral glycoproteins is activated and causes fusion between the viral membrane and that of the endosomal vesicle membrane, resulting in the release of the nucleocapsids into the cytoplasm.
• HA hemagglutinin
• F protein of Sendai virus a paramyxovirus
• the fusion activity, but not the receptor-binding activity, of the influenza hemagglutinin requires activation by proteolytic cleavage (by a host-specific enzyme) of an inactive precursor (HA o ) into two subunits (Lazarowitz and Choppin, 1975, Virology 68L:444-454).
• a similar cleavage is required to activate the F protein of Sendai virus (F o ) yeilding two disulfide-linked subunits F 1,2 (Scheid and Choppin, 1974, Virology 57:475-490; Huang et al., 1981, Virology 110:243- 247).
• Cleavage of HA o or F o exposes hydrophobic amino termini on the HA 2 and F 1 subunits, which are thought to interact with the lipid bilayer of the target cell membrane and mediate fusion (Garten et al., 1981 Virology 115:361- 374; Richardson et al., 1980, Virology 105.205-222).
• fusion peptide to glutamic acid residues either abolished fusion or increased the pH threshold and decreased the efficiency of fusion (Gething et al., 1986, J. Cell Biol.
• Sendai F 1 were reported to be potent inhibitors of infectivity of each virus (Richardson et al., 1980, Virology
• fusion-peptide hypothesis for viral induced fusion.
• Hull et al. describe a measles virus mutant that is resistant to fusion inhibitory oligopeptides. Sequencing of the F glycoprotein revealed three amino acid changes, none of which were located within the hydrophobic NH 2 -terminal "fusion-peptide" region. The findings reported by Hull et al. suggest that the positioning of the F 1 amino terminus within a functional F 1,2 conformation, and not within thecell membrane, is the mediator of the fusion process.
• HIV Human immunodeficiency virus
• AIDS acquired immune deficiency syndrome
• This glycoprotein is synthesized as a precursor of 160 Kd and is subsequently processed into two proteins, gp120 and gp41.
• the gp120 portion has been shown to bind directly tothe cellular CD4 receptor molecule, hence producing HIV's tropism for host cells displaying the CD4 surface protein; whereas. gp41 serves to anchor the envelope glycoprotein complex in the viral membrane.
• a prominent feature in the cytopathology of HIV infection is the formation of multinucleated syncytia (Barre-Sinoussi et al., 1983,
• Vaccine trials are currently underway in an attempt to control the spread of the virus among the population.
• antiviral agents such as AZT (3'-azido-2',3'-dideoxythymidine), which interfere with the viral polymerase activity and, therefore, viral replication.
• Novel peptide sequences capable of inhibiting HIV-induced cell fusion are described.
• the peptides of the invention mimic regions within a hydrophobic domain located at the amino terminus of the transmembrane protein of the HIV envelope.
• Such peptides have amino acid sequences that are homologous to this region found in various strains of HIV including but not limited to the amino terminus of gp41 of HIV-1 and the amino terminus of gp40 of HIV-2.
• the peptides are at least two amino acids in length and can be used for the inhibition of HIV-induced fusion and syncytia formation which is a major cytopathological effect of HIV-infection in vivo.
• oligopeptides of the invention are demonstrated herein by way of examples in which the oligopeptides AVG and AVGIGA inhibited HIV-induced syncytia formation as measured by an in vitro assay using recombinant virus expressing glycoproteins of the BHIO strain of HIV, and host cells that constitutively express the CD4 target antigen.
• A (alanine), R (arginine), N (asparagine), D (aspartic acid), C (cysteine), Q (glutamine), E (glutami ⁇ acid), G
• Figure 1 Imunoprecipitation, using AIDS patient antisera, of envelope glycoproteins in recombinant (Wenv) and wild type (w.t.) vaccinia virus-infected 3 H-leucine labeled HeLa T4 cells at 3, 6, and 10 hours post infection.
• Figure 2. SDS polyaery1amide gel of 35 S-methionine labeled HeLa T4 cells at 12 hours post infection: (a) wild- type (w.t.) immunoprecipitate with patient antisera; (b) Wenv immun ⁇ precipitate with patient antisera; (c) w.t. cell lysate; (d) Wenv cell lysate.
• FIG. 3 HeLa T4 cells infected with 1 p.f.u/cell Wenv at 7 hours post infection: (a) no peptide; (b) 8mM peptide AVGIGA; (c) 8mM peptide AVGAIG; and (d) 8mM peptide AVG.
• Figure 4. Quantitation of peptide inhibition. HeLa T4 cells were infected at an m.o.i. of 0.1 with Wenv in the presence of various concentrations of each peptide and quantitated for syncytia at 20 hours post infection.
• Novel peptide sequences are described which are capable of inhibiting HIV-induced cell fusion and cytopathic syncytia formation.
• the invention is based, in part, on the discovery that peptides which are substantially homologous to the hydrophobic domain of the amino terminus of the transmembrane protein of HIV are effective inhibitors of HIV-induced fusion of infected cells.
• the peptides of the invention have amino acid sequences that correspond to a region within a hydrophobic domain comprising approximately 21 to 40 amino acids located at the amino terminus of the transmembrane protein of the
• HIV envelope glycoprotein for example, the first 21 to 30 amino acids of gp41 HIV-1, or the first 21-37 amino acids of gp40 of HIV-2.
• the complete genomic sequence of HIV-1 has been previously reported (Wain Hobson et al., 1985, Cell
• the peptides of the invention may comprise oligopeptides (e.g., at least two amino acid residues), polypeptides (e.g., more than 10 amino acid residues) or proteins (e.g., polypeptides having a molecular weight of approximately 10 Kd or about 100 amino acid residues in length) which contain such sequences.
• oligopeptides e.g., at least two amino acid residues
• polypeptides e.g., more than 10 amino acid residues
• proteins e.g., polypeptides having a molecular weight of approximately 10 Kd or about 100 amino acid residues in length
• Z and X when present, each comprises one or more amino acid residues, a hydrophobic group (e.g., carboxybenzoxyl, dansyl or t-butyloxycarbonyl), or a cross reactive group (e.g. alkylating agent).
• m and n each comprises an integer of at least O.
• Z and X when present, each comprises one or more amino acid residues, a hydrophobic group (e.g., carboxybenzoxyl, dansyl or t-butyloxycarbonyl), or a cross reactive group (e.g. alkylating agent).
• m and n each comprises an integer of at least 0. G
• Z and X when present, each comprises one or more amino acid residues, a hydrophobic group (e.g., carboxybenzoxyl, dansyl or t-butyloxycarbonyl), or a cross reactive group (e.g. alkylating agent).
• m and n each comprises an integer of at least 0.
• Amino acids in boldface type may be inserted between amino acid residues at positions indicated above by * (when present) or substituted for the amino acid residue indicated directly above within any of the peptide sequences shown.
• the peptides of the invention are synthesized to resemble the amino-terminal region of the HIV transmembrane protein, variations in the amino acid sequence, the steric configuration, the type of covalent bond which links the amino acid residues, and/or addition of groups to the amino- or carboxy-terminal residues are within the scope of the invention.
• the peptides of the invention may include altered sequences to accomodate strain-to-strain variations among different HIV isolates as well as conservative alterations which result in a silent change thus producing a functionally equivalent peptide.
• the peptides depicted in Tables I through VI may be altered by various changes such as insertions, deletions and substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use.
• conservative substitutions would involve the substitution of one or more amino acids within the peptide sequences shown in Tables I through VI with another amino acid having similar polarity and hydrophobicity/hydrophilicity characteristics resulting in a silent alteration and a functionally equivalent molecule.
• Such conservative substitutions incude are not limited to substitutions within the following groups of amino acids: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine; and methionine, norleucine.
• the steric configuration of the peptides may be altered in order to increase the inhibitory activity of the peptides.
• the use of a D-isomer rather than the L-isomer at the amino terminus of the peptide and/or opposite configurations of the first two amino acids of the peptide may provide for an increased inhibitory function.
• Such variations in the steric configuration may enhance inhibitory activity by protecting the peptide from proteolysis or by affecting the affinity of the peptide for its binding site.
• Additional amino acids or other chemical groups may be added to either the amino or carboxy terminus of the peptides in order to alter or enhance their activity.
• hydrophobic groups such as carbobenzoxyl, dansyl or t-butyloxycarbonyl
• t-butyloxycarbonyl may also increase inhibitory activity.
• groups such as carbobenzoxyl or dansyl may have less effect, or may result in a decrease in inhibitory activity.
• peptides derivatized by the addition of chemically reactive groups that allow for the formation of covalent bonds between the peptides of the invention and their binding sites may be prepared. Such peptides would form covalent bonds with their binding sites and bind irreversibly to their target.
• the peptides may be derivatized with an alkylating agent such as chloromethylketone (Powers, 1980, in Methods in Enzymology, Jakoby and Wilkek, eds, Acad. Press, N.Y., Vol. 46.197-208).
• alkylating agent such as chloromethylketone (Powers, 1980, in Methods in Enzymology, Jakoby and Wilkek, eds, Acad. Press, N.Y., Vol. 46.197-208).
• Such derivatives are essentially alkylating agents that can react with and modify histidine, serine and sulfhydryl residues thus allowing the formation of covalent bonds between the inhibitory peptide
• the peptides of the invention may be synthesized or prepared by any technique known in the art. Short peptides can be synthesized on a solid support or in solution in accordance with conventional techniques. Commercially available automated synthesizers may be conveniently used. See, for example, Merrifield, 1969, Adv. Enzymol. Relat. Areas Mol. Bio. 32:221-296; Tarn et al., 1983, J. Am. Chem. Soc. 105:6442; Konig and Geiger, 1970, Chem. Ber. 103:788-789 and 2034-2040).
• nucleotide coding sequences for the peptides of the invention may be cloned and expressed using techniques well known in the art. See, for example, Maniatus et al.. Molecular Cloning, A Laboratory Manual, CSH, Cold Spring Harbor Laboratory, 1982.
• peptide analogs may be synthesized which comprise the sequences shown and described supra in which the amino acid residues are linked one to the other by non-peptide covalent bonds.
• Such covalent linkages can be made using reactions well known in the art involving the amino and/or carboxyl groups of the amino acids as well as any other reactive group,
• non-peptide (or non-amide) linkages which can be used to synthesize these peptide analogos of the invention include but are not limited to imino, ester, hydrazide, semicarbazide, and azo bonds to name but a few.
• the inhibitory activity of the peptides can be measured and the functional equivalency and/or increased efficacy of the altered peptides can readily be tested using the in vitro assay system described in Section 6.2 herein. Accordingly, the efficacy of any peptide of the invention may be assessed by its relative ability to inhibit the formation of syncytia in vitro using a cell line that constitutively expresses the CD4 receptor molecule and a recombinant vaccinia virus that expresses the HIV env glycoprotein. These recombinant viruses induce the formation of syncytia which are morphologically indistinguishable from those formed by authentic HIV. The ability of the peptides or altered peptides of the invention to inhibit syncytia formation in this assay system is indicative of the inhibitory activity of these peptides against authentic HIV-induced fusion.
• the assay system described herein which utilizes a recombinant vaccinia virus that expresses the HIV glycoprotein offers several advantages for characterizing the peptides of the invention and for screening antiviral agents which may be useful against HIV-infection.
• the efficacy of inhibitory and/or antiviral compounds can be assessed without risking exposure to authentic HIV and thus eliminates the possibility of contracting AIDS.
• the recombinant vaccinia virus described in the Example of Section 6.2 was constructed using the IHD strain of vaccinia.
• This vaccinia strain unlike the WR strain which has commonly been used to construct recombinants demonstrates increased levels of expression of the foreign gene inserted into the virus, and has a decreased cytopathic effect.
• the recombinant vaccinia can be used to infect the target test cells and results in a greatly increased level of surface expression of the HIV envelope glycoprotein at shorter times after infection.
• synchronous expression in culture is achieved.
• the decreased cytopathic effect of the IHD vaccinia strain may reduce the risks for the technicians who must handle the recombinants and run the assay system making the assay system described herein very desirable for evaluation, assessment characterization and screening of inhibitory and/or antiviral compounds.
• the properties of this vaccinia virus also makes the recombinant virus exemplified herein a desirable candidate for potential use in vaccines.
• the inhibitory peptides of the invention may be used in vivo to prevent the formation of syncytia and, thus, inhibit the progression of HIV infection within an exposed patient.
• Effective doses of the peptides of the invention formulated in suitable pharmacological carriers may be administered by any appropriate route including but not limited to injection (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, etc.), by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and vaginal epithelial linings, nasopharyngeal mucosa, intestinal mucosa, etc); etc.
• injection e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, etc.
• epithelial or mucocutaneous linings e.g., oral mucosa, rectal and vaginal epithelial linings, nasopharyngeal mucosa, intestinal mucos
• the peptides may be mixed in any suitable pharmacological carrier, linked to a carrier molecule and/or incorporated into liposomes, microcapsules, and controlled release preparations prior to administration in vivo.
• the peptides of the invention may also be used to identify different strains of HIV.
• the peptides of the invention will exert an inhibitory effect on
• HIV strains that have transmembrane proteins which share the sequence specificity of the peptide.
• an HIV strain with a very divergent transmembrane sequence may not be inhibited. Therefore, the peptides having known sequences could be used in an in vitro assay to quickly determine whether a particular strain of HIV isolated from a patient is a new variant. Accordingly, a cell line which constitutively expresses the CD4 receptor, such as the HeLa
• T4 cell line of the Examples described in Section 6.2 infra would be used as the target cells in vitro.
• HIV isolated from a patient can be used to infect the CD4 positive cell line in vitro in the presence of a peptide of the invention which is specific for a particular strain of HIV. Failure of the peptide to inhibit virus-induced fusion would indicate that the HIV isolate is a different strain.
• the human immunodeficiency virus (HIV-1) envelope glycoprotein is essential for virus entry and the formation of multinucleated giant cells by cell fusion, the major virus-induced cytopathic effect.
• the subsections below describe (a) oligopeptides which inhibit HIV-induced fusion; and (b) an in vitro assay system devised to determine the effects of potential fusion inhibitors.
• a recombinant vaccinia virus expressing the envelope glycoprotein of the BH-10 strain of HIV was generated and used to infect a HeLa cell line (HeLa T4) which constitutively expresses the CD4 receptor molecule.
• Syncytium induction in this system was observed as early as 4 hours post-infection and continued until the entire monolayer was fused.
• the N-terminus of the gp41 subunit of the HIV envelope protein is very hydrophobic, and we considered it likely that it may be involved in virus-induced cell fusion.
• a hexapeptide which was identical in amino acid sequence to the N-terminus of gp41 of the BH-10 isolate was found to almost completely prevent virus-induced cell fusion.
• oligopeptides which are homologous to the fusion peptide of HIV inhibit virus- induced cytopathology.
• OLIGOPEPTIDE INHIBITORS Three synthetic peptides were made with varying degrees of homology to the N-terminal amino acids of gp41 from different HIV strains. The first peptide:
• Peptides were synthesized by the solid phase procedure using an Applied Biosystems model 430A instrument. The completed peptides were removed from the resin and purified by high performance liquid chromatography.
• insertion vectors were used to insert the foreign gene into vaccinia virus via homologous recombination in vivo (Mackett et al.,
• these insertion plasmids contain an isolated vaccinia virus promoter including the transcriptional initiation site and 200-300 base pairs of upstream DNA, with several unique restriction endonuclease cleavage sites positioned downstream from the transcriptional start point; both sides of the promoter and restriction sites are flanked by nonessential vaccinia virus
• TK gene TK- mutants are obtained which can be identified by their ability to be grown on TK- host cells in the presence of BUdR.
• Recombinants may be further distinguished from wild type parental virus by screening of plaques with a labeled DNA probe specific for the foreign gene, immunological analysis using antiserum specific for the foreign protein and 125 I-labeled protein A from S. aureus or a second antibody, or color screening by use of an insertion vector containing the ⁇ -galactosidase gene of E. coli.
• recombinant plaques expressing ⁇ -galactosidase activity become blue.
• recombinant virus stocks were prepared in any of a variety of possible host cell types.
• a plasmid insertion vector was used to construct the recombinant vaccinia viruses described in more detail below.
• the envelope gene was obtained from plasmid pBH10 and inserted into the vaccinia recombinant plasmid vector pSC11.
• the plasmid pBH-10 (Ratner et al., 1985, Nature 313:277-284) contains a DNA clone of the unintegrated HTLV-III genome in the Sst I site of plasmid pSP65.
• the entire envelope gene was subcloned by digesting pBH10 with Sall and XhoI followed by gel isolation of the 3.1 kilobase pair fragment. This fragment was ligated to the SalI digested plasmid vector pGem-3 (Promega Biotech, Madison, WI). Orientation of the env gene was determined by restriction analysis.
• Plasmids were recircularized with T4 ligase and used to transform E. coli HB101 cells . Plasmids from individual colonies were sequenced by the dideoxy method to determine that 56 nucleotides remained upstream of the translation initiation site of the env gene. DNA fragments containing the entire env gene were obtained by cleavage with EcoRI and PstI, after which blunt ends were created using the Klenow fragment of DNA polymerase and deoxyribonucleoside triphosphate. These fragments were inserted into the Smal site of pSCll containing the ⁇ -galactosidase gene
• TK-143 cells were infected with the IHD strain of vaccinia at an m.o.i. of 0.1 p.f.u./cell. At 2 h.p.i., cells were transfected with a calcium phosphate precipitate of 5 ⁇ g pVenv-1 and 15 ⁇ g of salmon sperm DNA/ml of Hepes-buffered saline.
• Stocks of TK ⁇ vaccinia virus were prepared in TK-143 cells (Smith and
• TK-143 cells were infected with 100-200 p.f.u. of TK- vaccinia virus in the presence of BUdR (25 ⁇ g/ml).
• BUdR 25 ⁇ g/ml
• the monolayers were overlayed with 1% low melting agarose containing 300 ⁇ g/ml 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (Xgal).
• Xgal 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside
• blue plaques were picked and further purified by two additional rounds of plaque purification. Plaque-purified virus was grown in CV-1 cells and purified as described by Joklik
• HeLa T4 HeLa T4 that constitutively expresses the CD4 receptor molecule to test whether or not the recombinant vaccinia virus was capable of inducing cell fusion
• OLIGOPEPTIDES INHIBIT HIV- INDUCED SYNCYTIUM FORMATION
• the peptides synthesized in Section 6.1 were tested for their ability to inhibit syncytium formation in HeLa T4 cells infected with a recombinant vaccinia virus that expresses the glycoprotein of the BH10 strain of HIV.
• the greatest degree of inhibition was observed with peptide AVGIGA (derived from the transmembrane protein of the BH10 strain of HIV), while peptide AVGAIG (derived from the transmembrane protein of the WMJ strain of HIV) showed a lesser inhibitory effect.
• Peptide AVG exhibited an inhibitory effect that was between that observed for peptide AVGIGA and peptide AVGAIG (FIG. 4).
• the I 50 (concentration at which 50% syncytium are inhibited) for peptides AVGIGA and AVG were both about 0.75 mM and the I 50 for peptide AVGAIG was about 2.5 mM.
• peptide AVGIGA was about 2.5-fold more effective than peptide AVGAIG at inhibiting cell fusion.
• concentration tested (8 mM) peptide AVGIGA was slightly more effective than both peptide and AVGAIG peptide AVG.
• FIG. 5 No differences were observed in the level of env gene products in the presence or absence of peptide indicating that the inhibitory effects of these peptides do not reside at the level of protein synthesis or proteolytic processing of gp160.
• T4 cells were infected with Wenvl and analyzed for surface expression by indirect surface immunofluorescence (FIG. 3).
• the relative fluorescence intensities of cells treated with high concentrations of each peptide were the same as cells that received no peptide, indicating that syncytium inhibition was not due to a block in transport of the glycoprotein to the cell surface.

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