WO1987003883A2 - Use of oligopeptides in the treatment of viral infections - Google Patents
Use of oligopeptides in the treatment of viral infections Download PDFInfo
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- WO1987003883A2 WO1987003883A2 PCT/EP1986/000759 EP8600759W WO8703883A2 WO 1987003883 A2 WO1987003883 A2 WO 1987003883A2 EP 8600759 W EP8600759 W EP 8600759W WO 8703883 A2 WO8703883 A2 WO 8703883A2
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- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 37
- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 37
- 208000036142 Viral infection Diseases 0.000 title claims 4
- 230000009385 viral infection Effects 0.000 title claims 4
- 238000011282 treatment Methods 0.000 title description 6
- 241000700605 Viruses Species 0.000 claims abstract description 20
- 102000005962 receptors Human genes 0.000 claims abstract description 11
- 108020003175 receptors Proteins 0.000 claims abstract description 11
- 230000001413 cellular effect Effects 0.000 claims abstract description 5
- 230000035515 penetration Effects 0.000 claims abstract description 4
- 150000001413 amino acids Chemical class 0.000 claims description 22
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 10
- 102000014914 Carrier Proteins Human genes 0.000 claims description 9
- 108010078791 Carrier Proteins Proteins 0.000 claims description 9
- -1 aromatic amino acid Chemical class 0.000 claims description 8
- 241000709664 Picornaviridae Species 0.000 claims description 6
- 238000002649 immunization Methods 0.000 claims description 6
- 238000011321 prophylaxis Methods 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- 125000001931 aliphatic group Chemical group 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000000271 synthetic detergent Substances 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- 125000001424 substituent group Chemical group 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 29
- 235000001014 amino acid Nutrition 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 7
- 239000000539 dimer Substances 0.000 description 7
- 238000006386 neutralization reaction Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 208000000474 Poliomyelitis Diseases 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 241000709661 Enterovirus Species 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000002788 anti-peptide Effects 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 229910001868 water Inorganic materials 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- 241000430519 Human rhinovirus sp. Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 229940073584 methylene chloride Drugs 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102100023321 Ceruloplasmin Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 229910020939 NaC104 Inorganic materials 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 201000009240 nasopharyngitis Diseases 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000709643 Echovirus E9 Species 0.000 description 1
- 101710146739 Enterotoxin Proteins 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001068027 Homo sapiens Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Proteins 0.000 description 1
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 102100034464 Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 102100021696 Syncytin-1 Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 238000000333 X-ray scattering Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000002850 nasal mucosa Anatomy 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
- C12N2770/32011—Picornaviridae
- C12N2770/32711—Rhinovirus
- C12N2770/32722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Oligopeptides having variable length and sequences from the receptor-binding region of viruses which use specific, cellular receptors for the penetration of host cells.
Description
USE OF OLIGOPEPTIDES IN THE TREATMENT OF VIRAL SNFECTtONS
Although the common cold is a usually harmless illness, secondary infections can nevertheless have serious consequences. Common cold can be caused by a number of different viruses and microorganisms whereby rhino viruses play an important role. It is known that a protective immunity against a particular serotype can be induced by live or inactivated viruses. The greatest problem in the development of a vaccine against rhino viruses is the large number (ca. 120) of serotypes, as the immunity induced in this way is serotype specific. In recent years the possibility has been discovered of obtaining antibodies against parts of the antigenic protein which are normally not recognised by the immune system by employing short synthetic peptides optionally linked to carrier proteins.
Vaccines comprising such peptides for example against foot and mouth disease (FED) and E. coli enterotoxin are already in an advanced stage of development.
Oligopeptides having variable lengths with sequences from the receptorbinding regions of viruses which employ a specific cellular receptor for penetration of the host cell induce, optionally after coupling to a suitable carrier protein, on local or systemic application neutralising antibodies, which on infection by the natural antigen (=complete virus particle) are not increasingly formed. The three-dimensional structure of Rhino 14 and Polio 1 virus particles has been clarified by X-ray scattering. Regions have been identified which are binding sites for cellular receptors and which consist of deep tucks or "canyons" on the surface of the virus particle. It has been stated in the literature that these receptor-binding sites are not accessible to anti-bodies for steric reasons.
It has however become apparent that antibodies obtained using oligopeptides do in fact neutralise the virus particles, i.e. that they are suitable for the treatment or prophylaxis of infections caused by such viruses. In a particular embodiment according to the invention oligopeptides are used which have partial sequences from the VP1 and VP3 envelope proteins of Rhino 14 which lie at the base of the tuck and are in part also conserved in other Picorna viruses.
Oligopeptides having these sequences have induced antibodies in test animals, which were positive in an in vitro neutralisation test.
Preferably oligopeptides are used having sequences from the receptorbinding region of picorna viruses PVPR and having the general formula
NH2-X1-5-Tyr-X6-Pro-Gly-Ala-X7-X8-Pro-X9-14-COOH
(His) (Ile) (Lys)
(Val) wherein X1 14 stand independently for any amino acid whereby one or more of the amino acids X1-5 or X9-14 may be replaced by a direct bond.
Particularly suitable according to the invention are oligopeptides with conserved sequences from the capsid proteins VP3 and VP1 having the formula
PVP3
NH2-X"1-5-Tyr-X"6-Pro-Pro-GLy-Ala-X"7-X"8-Pro-X"9-11-Cys-COOH
(Ile)
(Val) PVP3 wherein X'1 stands for a basic amino acid such as Lys, His or Arg X'2 stands for an aliphatic or aromatic amino acid such as Leu, Val, Ile, Ala, Tyr, Phe or Trp, X'3 stands for any amino acid, X'4 stands for an aliphatic amino acid such as Leu, Val, Ile, Ala, Thr, Tyr, or Ser, X'5 stands for Ala, Ser, Thr,
Val, Ile or Leu, X'6 stands for Thr, Ala, Ile, Ser, Leu or Val and X'7-X' stand independently for any amino acid,
or having the formula PVP1
NH2-X"1-X"2-Gln-X"4-Met.Tyr-Val-Pro-Gly-Ala-Pro-X"-Pro-X"9-X"10-Cyc-COOH wherein X"1 and X"2 are the same or different and each represent any amino acid, stands for Ala, Ile, Tyr, Val, Leu, Phe or Trp, X"8 and X"9 are the same or different and each represent a charged amino acid such as Lys, His, Arg,
Asp, Asn, Glu or Gln and X". stands for any amino acid whereby one or more of the amino acids defined by X'1 to X'5, X'g to X'1l or X"1, X"2, X"g or X"10 may be replaced by a direct bond.
Especially suitable according to the invention are oligopeptides with partial acid sequences from the receptor-binding region of the Rhino-14 virus and having the formula
NH2-Val-Val-Gln-Ala-Met-Tyr-Val-Pro-Pro-Gly-Ala-Pro-Asn-Pro-Lys-Glu-Cyc-COOH or PVPla
NH2-Lys-Leu-Ile-Leu-Ala-Tyr-Thr-Pro-Pro-Gly-Ala-Arg-Gly-Pro-Gln-Asp-Cyc-COOH
PVP3a.
The oligopeptides according to the invention may be prepared e.g. by conventional solid phase peptide synthesis preferably on automatic or semiautomatic peptide synthesizers. The amino acids in protected form are successively coupled. Following introduction of the final amino acid the oligopeptide is cleaved from the resin and deprotected. The final product is purified and analysed in conventional manner, e.g. employing high pressure liquid chromatography.
These oligopeptides can also be used in dimeric or polymeric form. A monomeric oligopeptide containing a Cys can for example be oxidised into the dimer.
Oligopeptides of variable lengths with sequences from the receptor-binding regions of viruses can be chemically coupled to carrier proteins, e.g. keyholelimpet hemocyamin (KLH) or BSA, to form immunogenic conjugates. For immunisation in humans the carrier must be physiologically acceptable. Preferably e.g.
carriers will be used which themselves act as vaccines e.g. tetanus toxoid and/ or diptheria toxoid.
The oligopeptides according to the invention are particularly useful for immunisation in particular against rhino viruses. This can be achieved by administering an effective amount of an oligopeptide according to the invention as such or in dimeric or polymeric form, or linked to a physiologically acceptable carrier protein.
Prophylactic protection can for example be induced by repeated local application to the mucous membrane of the nose. Prophylaxis can also be achieved e.g. by intramuscular application e.g. of 100 ug to 1 mg of the oligopeptide in question. The oligopeptides can however also be used for the therapeutic treatment of existing infections.
In particular oligopeptides are employed with partial amino acid sequences from picorna virus envelope proteins which are involved in the binding of virus particles on cellular receptors. The peptides contain sequences from the capsid proteins VP1 and VP3 of Rhino virus type 14. Parts of these peptides are however conserved for various other picorna viruses such as Rhino, Polio or FMD and can therefore induce neutralising antibodies against other Rhino-Serotypes and Polio and FMD-viruses or other Picorna viruses.
The oligopeptides according to the invention can for example be employed with pharmaceutically acceptable diluents or carriers and e.g. additionally with a natural or synthetic detergent for local e.g. nasal application.
Therapeutic preparations can contain one or more oligopeptides or dimers, polymers or conjugates thereof in the form of cocktails. They can also contain other antigens in physical admixture. Preparations for nasal administration can contain e.g. 0.1 to 0.5 mg/ml of oligopeptide.
The following examples illustrate the invention without in any way limiting the scope thereof. Temperatures are given in degrees centigrade.
EXAMPLE 1 : Preparation of the heptadecapeptide
H2N-Val-Val-Gln-Ala-Met-Tyr-Val-Pro-Pro-Gly-Ala-Pro-Asn-Pro Lys-Glu-Cys-COOH (PVPla)
The peptide is prepared stepwise employing solid phase-peptide synthesis on a p-alkoxybenzylalcohol polystyrene resin (1% DVB cross-linked).
The coupling of the first 16 acids proceeds in each case via the FMOC-amino acid derivative whereby the following amino acids are provided with a side chain protecting group as indicated) - Cys (acetamidomethyl-) - Glu (y-tert.-butylester) - Lys (e-amino-BOC) - Tyr (tert.-butylether)-.
a) Coupling of Val to H-16-p-Alkoxybenzylalcoholresin
The following reagents were reacted in a semi-automatic peptide synthesizer (SP 640 - Labortec) for 90 min. at 200C in DMF as a solvent:
1. eq. -H-16-p-alkoxybenzylalcohol-resin
3. eq. BOC-Val-OH
3. eq. hydroxybenzotriazole (HOBT)
3.3. eq. dicyclohexylcarbodiimide (DUCT)
All excess reactants and side products are removed by filtration and washing.
The preceding coupling steps are carried out in an analogous manner.
b) Separation of peptide from resin with simultaneous removal of acid labile
protecting groups
The peptide coupled resin is shaken for 90 min. at 20"C with 55% trifluoroacetic acid in methylenechloride. The peptide is separated from the resin by filtration and the residual resin washed through twice with 20% trifluoroacetic acid in methylenechloride and once with methylenechloride alone. 10% anisol is added to the separation solutions as t-butyl cation scavenger. The peptide filtrate is evaporated at 350C and the residue reprecipitated from ether, washed three times with ether and dried in vacuum.
c) Removal of the -Cys(acetamidomethyl)-protecting group
The peptide is dissolved in 80% aq. trifluoroacetic acid, kept free of oxygen with nitrogen gas and reduced to Cys-peptide mercaptide with 1.2 eq.
Hg(II)-acetate for 15 min. at 00. Liberation of peptide from mercaptide is carried out using H2S. The resulting difficultly soluble Hg sulfide is completely removed by filtration and washed through with 80% acetic acid ( 2- free). The peptide filtrate is evaporated at 35C and directly lyophilised from water (02-free).
d) Preparative HPLC-purification of the peptide
Carrier : RP C 18, 75-20 microns 300 A (silica gel)
Buffers : A2.8 g NaC104
2 ml H3PO4+H2O 2 litre solution
B 2.8 g NaC104
2 ml H3P04
1200 ml acetonitrile + H20 2 litre solution.
Gradient system 100% A, 0.1% TFA - 60 min. - 100% B (0.1% TFA/CH3CN 40/60)
Pure fractions are combined and lyophilised. The pure peptide is then freed from the peptide-TFA-salt form by strongly basic ion-exchange (acetate-form).
The resu-lting peptide had a MW of 1800.14 glmol.
Aminoacid analysis.
Val 2.88 (3.00) Glu 1.95(2.00) Ala 1.98(2.00)
Met 0.84 (4.00) Gly 0.98(1.00) Tyr 0.92(1.00)
Pro 3.90 (4.00) Asp 0.96(1.00) Lys 1.00(1.00)
Cys 0.93 (1.00)
Other peptides according to the invention and in particular PVP3a can be prepared in an analogous manner.
EXAMPLE 2 : Neutralisation of Rhino 14 viruses with antibodies
against synthetic Peptides 1. Peptides employed
PVPla and PVP3a as hereinbefore illustrated.
2. Chemical coupling of the peptides to KLH.
To a solution of 10 mg of KLH in 0.9 ml of sodium phosphate buffer are added 0.1 ml 50mM 3-maleicimidobenzoic acid-N-hydroxy-succinimide ester (MBS) in dimethylformamide and the mixture stirred for 30 minutes at room temperature. A further 0.05 ml of MBS solution are then added and stirring continued for a further 30 minutes. The cloudiness is centrifuged off and the clear solution chromatographed at 4" over a Sephadex G25-column (elution with phosphate buffer, pH 7.5). The eluate is measured photometrically and the fractions having the highest protein content combined.
10 mg of peptide are dissolved at pH 6.5 in 1 ml of 0.1M phosphate buffer/ lmM ethylenediaminetetracetate (EDTA) (optionally with up to 20% acetonitrile).
1 ml of the peptide solution and lml of the reacted KLH-solution are combined, the pH value ajusted to 8.5 and the solution saturated with nitrogen. The mixture is incubated for 2 hours at room temperature and then dialysed against deficient PBS.
3. Immunisation of test animals
The conjugate solution obtained e.g. as described under 2 above is adjusted with deficient PBS to a protein concentration of 0.4 mg/ml. This solution is mixed 1:1 with complete Freund's adjuvant and injected intradermally into New
Zealand White Rabbits (lml per rabbit). After 10-14 days the animals are injected a second time subcutaneously with the same amount of conjugate mixed this time with incomplete Freund's adjuvant. After 7 and 14 days serum is taken from the animals for antibody purifaction. The antibody level against the peptide is determined by an ELISA.
4. Purification of the antibodies 4.1 Preparation of the affinity column.
5 ml of Affigel-102 are washed with 300 ml of cold distilled water, equilibrated with 0.1M phosphate buffer and sucked off. l00mg of maleicimido-B- alanyl-succinimide ester are dissolved in methanol/dimethylformamide (2/1) and the gel is then suspended therein and shaken for 2 hours at room temperature.
The gel is then washed on a suction filter with PBS and resuspended in a solution of lOmg peptide in 10ml of O.1M phosphate (pH6.5) with 0.ism EDTA. The pH value is adjusted to 8.0 with 1N NaOH, the suspension shaken for 1 hour at
R.T. and then washed alternately with PBS and 0.1M glycine HCI/0.15M NaCl (pH 2.8) (total 2 litres). The gel is stored suspended in PBS with 0.03% sodium azide at 4 .
4.2 Affinity chromatoqraphy
5m1 of anti-serum, diluted with an equal volume of PBS are very slowly applied to a small column (2,5-3m1 prepared with a gel as described under 4.1) equilibrated with PBS. The column is washed through with PBS until no further protein was detectable in the eluate (extinction at 280nm = O). The bound antibodies are eluted with 0.1M glycine HC1/0.15M NaCl (pH 2.8), whereby each fraction is added to 25u 2M Tris per ml of fraction volume. The fractions containing antibody are determined photometrically (extinction at 280nm), combined and dialysed against PBS.
5. Neutralisation test
In cell-culture microtiter plates 0.1 ml of various purified antipeptide antisera (prepared e.g. as described under 4.2) prediluted (1:2) in a combination of Eagles MEM with 2% FCS, 40nM Mg C12 and 1% penicillin/Streptomycin (Medium
A) are diluted out in half rows with the same medium. Controls contain either a 1 : 10 pre-diluted neutralising rabbit antiserum against complete virus particles (positive control) or medium A alone (virus control). To each well is added 0.05ml of viral broth (4.5 x 105 pfu/ml; pre-diluted 1:10 with medium A) and incubated for 1 hour at 340 in 5% humid C02-atmosphere. 0.5ml of a freshly prepared HELA-Ohio cell suspension (105 cell/ml) are then added to each well and incubation continued for 48 hours under the same conditions.
Medium is removed by shaking off and the cells remaining in the wells stained with crystal violet (2.5 g crystal violet, dissolved in 16.5ml of ethanol and 80ml of formamide and filled to 260ml with dist. water). Anti-PVPla- and anti-PVP3a-antibodies neutralised Rhino 14 infection up to an end-dilution of 1 : 32. At the same time the cells in the virus control wells are completely lysed (no staining). Antibodies against peptides having completely different structures prepared in the same manner did not neutralise the infection.
EXAMPLE 3
Neutralisation of various serotypes of human rhino virus with antibodies against synthetic peptides
The preparation of the antibodies and the neutralisation test are carried out as described in Example 2. Various serotypes of human rhino viruses, polio 1 and 2 viruses, echo 9 virus and Coxcackie B virus are employed in the neutralisation test. The results of which are given in the following table.
Activity Serotype (rhino) or virus tested (dilution factor)
none la,2,4,7,8,13,15,18,19,20,21,22,25,33,38,45,47,
62,75,85,Polio 1, Polio 2, Echo 9,Coxsackie B 3
weak 9,10,16,40,45,68,71
1:8)
medium 17,24,26,28,32,36,51,58,72 (1:8-1:32)
strong 3,5,6,14,23,27,35,37,48,50,55,64
1:32)
The viruses underlined belong to a group of rhino viruses which use a different receptor for cell penetration.
From these results it can be seen that ca. 60% of the serotypes investigated are neutralised, 25% equally well or better and 33% less strongly than
Rhino 14.
This clearly shows that the antibodies neutralise, and can therefore protect against a large group of human rhino virus serotypes.
EXAMPLE 4
Local intranasal immunisation with a dimeric oligopeptide in detergent solution 1. Preparation of the dimer of PVP3a
To a solution of 4 mg of PVP3a oligopeptide in 2 ml of sterile 0.1M sodium phosphate buffer (pH = 8.0) are added 20ul of 30% H202 with stirring at R.T.
which is continued for 2 hours. By this time more than 98% of the free sulfhydryl groups are oxidised (Ellmann's reagent). In order to facilitate the taking up of the dimer through the nasal epithelium, Triton x-100, a non-ionic detergent is added at a concentration of 0.1%.
2. Intranasal immunisation
The dimer preparation thus obtained is diluted to a concentration of 0.2mg/ml with PBS containing 0.1% Triton x-100. Each of two rabbits receives in total 1 ml (0.2mg). 0.5 ml in each nostril, applied with a plastic syringe (without needle).
Two control animals receive the same amount of 0.1% Triton x-100 in PBS without peptide dimer. This treatment is repeated every 6 to 8 days and blood samples taken every 6 to 8 days to prepare serum for determination of antipeptide antibody content. Three weeks following commencement of the treatment a large increase in anti-peptide antibodies is seen in rabbits immunised with peptide dimer.
3. Neutralisation test
With the help of an affinity column the anti-peptide antibodies contained in the serum are purified (cf. Example 1.4) and show the same results as given in the table of Example 3.
Claims
WE CLAIM
1. Oligopeptides having variable length and sequences from the receptor-binding region of viruses which use specific, cellular receptors for the penetration of host cells.
2. An oligopeptide according to Claim 1 having the formula
NH2-X1-5-Tyr-X6-Pro-Pro-Gly-Ala-X7-X8-Pro-X9-14-COOH
(His) (Ile) (Lys)
(Val) wherein X1 14 stand independently for any amino acid whereby one or more of the amino acids X1-5 or X9 14 may be replaced by a direct bond.
3. An oligopeptide according to Claim 2 having the formula PVP3
NH2-X"1-5-Tyr-X"6-Pro-Pro-Gly-Ala-X"7-X"8-Pro-X"9-11-cyc-COOH
(Ile)
(Val) PVP3 or PVP1
NH2-X"1 -X"2-Cln-X "4-Met-Tyr-Val-Pro-Pro-Gly-Ala-Pro-X" 8-Pro-X"9-X"10-Cys-C00H
(His) (Lys) (Lys) wherein X'1 stands for a basic amino acid, X'2 stands for an aliphatic or aromatic amino acid, X'4 stands for an aliphatic amino acid, X'5 stands for
Ala, Ser, Thr, Val, Ile or Leu, X'6 stands for Thr, Ala, Ile, Ser, Leu or Val and X'3 and X"7-X"11 independently stand for any amino acid;
and X"4 stands for
Ala, Ile, Tyr, Val, Leu, Phe or Trp, X"4 stands for Ala, Ile, Tyr, Val, Leu,
Phe or Trp, X"8 and X"9 are the same or different and each represent a charged amino acid and X"1, X"2 and X"10 are the same or different and each represent any amino acid, whereby one or more of the amino acids defined by X'1 to X'5,
X"9 to X"11 or X"1, X"2, X"9 or X"10 may be replace by a direct bond.
4. An oligopeptide according to Claim 3 wherein X'1 stands for Lys, His or
Arg, X'2 stands for Leu, Val, Ile, Ala, Tyr, Phe or Trp, X'4 stands for Leu,
Val, Ile, Ala, Thr, Tyr or Ser, X"8 and X" are the same or different and each represent Lys, His, Arg, Asp, Asn, Glu or Gln and the remaining substituents are as defined in Claim 3, whereby one or more of the amino acids defined by X1 to X'5, X'g to X'll or X"1, X"2, X" or X''1O 10 may be replaced by a direct bond.
5. An oligopeptide according to Claim 3 selected from
NH2-Val-Val-Gln-Ala-Met-Tyr-Val-Pro-Pro-Gly-Ala-Pro-Asn-Pro-Lys-Glu-Cyc-COOH or PVPla
NH2-Lys-Leu-Ile-Leu-Ala-Tyr-Thr-Pro-Pro-Gly-Ala-Arg-Gly-Pro-Gln-Asp-Cyc-COOH
PVP3a.
6. An oligopeptide according to any one of Claims 1 to 5 in dimeric or polymeric form.
7. An oligopeptide'according to any one of Claims 1 to 5 conjugated to a carrier protein.
8. A pharmaceutical composition comprising an oligopeptide according to any one of Claims 1 to 6 optionally conjugated to a physiologically acceptable carrier protein together with a pharmaceutically acceptable diluent or carrier.
9. A pharmaceutical composition according to Claim 8 comprising an oligopeptide according to any one of Claims 1 to 6 optionally conjugated to a physiologically acceptable carrier protein together with a pharmaceutically acceptable, natural or synthetic detergent in a form suitable for nasal administration.
10. An oligopeptide according to any one of Claims 1 to 6 optionally conjugated to a physiologically acceptable carrier protein for use in the therapeutic or prophylactic treatment of viral infections.
11. An oligopeptide according to-any one of Claims 1 to 6 optionally conjugated to a physiologically acceptable carrier protein for use in immunisation against viral infections.
12. A oligopeptide for use according to Claim 10 or 11 whereby the viral infections are caused by rhino and other picorna viruses.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP87500583A JPS63502034A (en) | 1985-12-23 | 1986-12-17 | Use of oligopeptides in the treatment of viral infections |
| DK437987A DK437987D0 (en) | 1985-12-23 | 1987-08-21 | oligopeptides |
| KR870700766A KR880700819A (en) | 1985-12-23 | 1987-08-22 | How to use oligopeptides to treat a virus infection |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0372985A AT385413B (en) | 1985-12-23 | 1985-12-23 | METHOD FOR PRODUCING NEUTRALIZING ANTIBODIES |
| ATA3729/85 | 1985-12-23 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1987003883A2 true WO1987003883A2 (en) | 1987-07-02 |
| WO1987003883A3 WO1987003883A3 (en) | 1987-11-05 |
Family
ID=3554592
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1986/000759 WO1987003883A2 (en) | 1985-12-23 | 1986-12-17 | Use of oligopeptides in the treatment of viral infections |
Country Status (9)
| Country | Link |
|---|---|
| JP (1) | JPS63502034A (en) |
| AT (1) | AT385413B (en) |
| AU (1) | AU6848687A (en) |
| DK (1) | DK437987D0 (en) |
| IL (1) | IL81058A0 (en) |
| NZ (1) | NZ218760A (en) |
| PH (1) | PH25351A (en) |
| WO (1) | WO1987003883A2 (en) |
| ZA (1) | ZA869678B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0142387A1 (en) * | 1983-08-26 | 1985-05-22 | Anda Biologicals | Process for the preparation of vaccines specific for LH and HCG and process for their detection |
-
1985
- 1985-12-23 AT AT0372985A patent/AT385413B/en not_active IP Right Cessation
-
1986
- 1986-12-17 WO PCT/EP1986/000759 patent/WO1987003883A2/en unknown
- 1986-12-17 JP JP87500583A patent/JPS63502034A/en active Pending
- 1986-12-17 AU AU68486/87A patent/AU6848687A/en not_active Abandoned
- 1986-12-22 NZ NZ218760A patent/NZ218760A/en unknown
- 1986-12-22 PH PH34642A patent/PH25351A/en unknown
- 1986-12-22 IL IL81058A patent/IL81058A0/en unknown
- 1986-12-23 ZA ZA869678A patent/ZA869678B/en unknown
-
1987
- 1987-08-21 DK DK437987A patent/DK437987D0/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| NZ218760A (en) | 1991-06-25 |
| ATA372985A (en) | 1987-09-15 |
| DK437987A (en) | 1987-08-21 |
| AT385413B (en) | 1988-03-25 |
| ZA869678B (en) | 1988-08-31 |
| PH25351A (en) | 1991-05-13 |
| JPS63502034A (en) | 1988-08-11 |
| WO1987003883A3 (en) | 1987-11-05 |
| IL81058A0 (en) | 1987-03-31 |
| AU6848687A (en) | 1987-07-15 |
| DK437987D0 (en) | 1987-08-21 |
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