NZ218760A

NZ218760A - Oligopeptides useful for immunisation against viruses especially rhino viruses

Info

Publication number: NZ218760A
Application number: NZ218760A
Authority: NZ
Inventors: Gudrun Werner; Joseph Willis Mccray
Original assignee: Sandoz Ltd
Priority date: 1985-12-23
Filing date: 1986-12-22
Publication date: 1991-06-25
Also published as: IL81058A0; WO1987003883A2; DK437987D0; DK437987A; JPS63502034A; ATA372985A; PH25351A; AU6848687A; ZA869678B; WO1987003883A3; AT385413B

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £18760 21876C o o Priority V * . - " ^N. *• ci . .v P.O. J*umat. No: .. . ~22DECm Vv *?.«' MO No.: Date: NEW ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION OLIGOPEPTIDES AND THEIR USE IN THE TREATMENT OF VIRAL INFECTIONS j-fWc, SANDOZ LTD., 35 Lichtstrasse, CH-4002, Basle, Switzerland; a Swiss Body Corporate hereby declare the invention for which 4- / we pray that a patent may be granted to Dae./us, and the method by which it is to be performed, to be particularly described in and by the following statement: - - 1 - -s 2187 6 C Case 900-9446 OLIGOPEPTIDES AND THEIR USE IN THE TREATMENT OF VIRAL INFECTIONS 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 (FMD) and E. coli enterotoxin are already in an advanced stage of development. Oligopeptides having variable lengths with sequences from the receptor-binding 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 VP! 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 receptor-binding region of picorna viruses PVPR and having the general formula 2187C0 -2- NH_-X. 5-Tyr-Xg-Pro-Pro-Gly-Ala-X7-X8-Pro-Xg ..-COOH (His) (He) (Lys) (Val) wherein X^_^ stand independently for any amino acid whereby one or more of the amino acids X^_^ or Xg ^ may be replaced by a direct bond. Particularly suitable according to the invention are oligopeptides with conserved sequences from the capsid proteins VP3 and VPl having the formula PVP3 NVX' 1 _,--Tyr-X1 g-Pro-Pro-Gly-Al a-X • ?-X' g-Pro-X' g-1 ] -Cys-COOH (He) (Val) PVP3 wherein X'^ stands for a basic amino acid such as Lys, His or ArgjX^ stands for an aliphatic or aromatic amino acid such as Leu, Val, lie, Ala, Tyr, Phe or Trp, X'^ stands for any amino acid, X1^ stands for an aliphatic amino acid such as Leu, Val, He, Ala, Thr, Tyr, or Ser, X'^ stands for Ala, Ser, Thr, Val, He or Leu, X'g stands for Thr, Ala, lie, Ser, Leu or Val and X'^-X'^ stand independently for any amino acid, or having the formula PVP1 NH.-X".-X1' -Gln-X".-Met-Tyr-Val-Pro-Pro-Gly-Ala-Pro-X" -Pro-X"Q-X",n-Cys-C00H 212 4 (His) (Lys) (Lys)pVpj wherein X"^ and X"^ are the same or different and each represent any amino acid, X"^ stands for Ala, He, Tyr, Val, Leu, Phe or Trp, X"g and X"g are the same or different and each represent a charged amino acid such as Lys, His, Arg, Asp, Asn, Glu or Gin and X"^Q stands for any amino acid whereby one or more of the amino acids defined by X'^ to X',., X'g to X'^ or X"^, X'^, X"g or X"^ 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 NH^-Val -Val-G1n-Ala-Met-Tyr-Val-Pro-Pro-Gly-Ala-Pro-Asn-Pro-Lys-Glu-Cys-COOH or PVPla NH^-Lys-Leu-Ile-Leu-Ala-Tyr-Thr-Pro-Pro-Gly-Ala-Arg-Gly-Pro-Gln-Asp-Cysgfififi^ L PVP3a., !Jn 1 NOV 1989 •f 218760 o o -3- 900-9446 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 mono- I meric oligopeptide containing a Cys can for example be oxidised into the dimer. -< - v- -y j Oligopeptides of variable lengths with sequences from the receptor-binding regions of viruses can be chemically coupled to carrier proteins, e.g. keyhole- limpet 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 VPl 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. O 218760 -4- 900-9446 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. 2 187 6 0 -5- 900-9446 EXAMPLE 1 : Preparation of the heptadecapeptide H^N-Val-Val-61n-Ala-Met-Tyr-Val -Pro-Pro-Gly-Al a-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.-butyl ester)- - 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 20°C 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 (DCCI) 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 rain, 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 35°C and the residue reprecipitated from ether, washed three times with ether and dried in vacuum. 218760 -6- 900-9446 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(ID-acetate for 15 min. at 0°. Liberation of peptide from mercaptide is carried out using H^S. The resulting difficultly soluble Hg sulfide is completely removed by filtration and washed through with 80% acetic acid free). The peptide filtrate is evaporated at 35° and directly lyophilised from water ^-free). d) Preparative HPLC-purification of the peptide Carrier : RP C 18, 15-20 microns 300 A (silica gel) Buffers : A 2.8 g NaClO^ 2 ml HgPO^ + H20 2 litre solution B 2.8 g NaC104 2 ml H3P04 1200 ml acetonitrile + ^0 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 resulting peptide had a MW of 1800.14 g/mol. 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. 218760 _7_ 900-9446 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/ ImM ethylenediaminetetracetate (EDTA) (optionally with up to 20% acetonitrile). 1 ml of the peptide solution and 1ml 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 (1ml per rabbit). After 10-14 days the animals are injected a second time subcutaneous!y 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. lOOmg of maleicimido-pal anyl-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 % m "I % o 218760 -8- 900-9446 solution of lOmg peptide in 10ml of 0.1M phosphate (pH6.5) with O.lmM EDTA. The pH value is adjusted to 8.0 with IN NaOH, the suspension shaken for 1 hour at R.T. and then washed alternately with PBS and 0.1M glycine HC1/0.15H NaCl (pH 2.8) (total 2 litres). The gel is stored suspended in PBS with 0.03% sodium azide at 4°. 4.2 Affinity chromatography 5ml of anti-serum, diluted with an equal volume of PBS are very slowly applied to a small column (2,5-3ml 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 =0). The bound antibodies are eluted with 0.1M glycine HC1/0.15M NaCl (pH 2.8), whereby each i: /' fraction is added to 25u 2M Tris per ml of fraction volume. The fractions v 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 Cl^ 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 10^ pfu/ml; pre-diluted 1:10 with medium A) and incubated for 1 hour at 34° in 5% humid C0?-atmosphere. 0.5ml of 5 ^ a freshly prepared HELA-Ohio cell suspension (10 cell/ml) are then added to each well and incubation continued for 48 hours under the same conditions. w 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-diluticn 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. 218760 -9- 900-9446 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, 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% ^02 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%. weak 62,75,85,Polio 1, Polio 2, Echo 9,Coxsackie B 3 9,10,16,40,45,68,71 ( 1:8) medi um 17,24,26,28,32,36,51,58,72 (1:8-1:32) strong ( 1:32) 3,5,6,14,23,27,35,37,48,50,55,64 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 218760 -10- 900-9446 O w'
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 anti-peptide 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. O 216760 -ii- WHAT HE CLAIM IS: X- Synthetic 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 NH -X1_5-Tyr-Xg-Pro-Pro-Gly-Ala-X7-X -Pro-Xg14~C00H (His) (lie) (Lys) (Val) wherein X^ ^ stand independently for any amino acid whereby one or more of the amino acids X^_^ or Xg_-|4 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-pr°-pr°-G1y-A1 a_x' 7"X 'g-Pro-X' gl 1 -Cys-COOH (He) (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) PVP1 wherein X'^ 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',- stands for Ala, Ser, Thr, Val, lie or Leu, X'g stands for Thr, Ala, He, Ser, Leu or Val and X'g and X'^-X'^ independently stand for any amino acid; and X"4 stands for Ala, lie, Tyr, Val, Leu, Phe or Trp, X"g and X"g are the same or different and each represent a charged amino acid and X"i, X"2 and X"io are the same or different and each represent any amino acid, whereby one or more of the amino acids defined by X'j to X's, X'g to X'n or X"i, X"2» X"g or X"io may be replaced by a direct bond. v ^ 218760 -12-
4. An oligopeptide according to Claim 3 wherein X'^ stands for Lys, His or Arg, X'2 stands for Leu, Val, He, Ala, Tyr, Phe or Trp, X'^ stands for Leu, Val, lie, Ala, Thr, Tyr or Ser, X"g and X"g are the same or different and each represent Lys, His, Arg, Asp, Asn, Glu or Gin and the remaining substituents are as defined in Claim 3, whereby one or more of the amino acids defined by X'.| to X'5, X'g to X'-j-j or X".|, X"2, X"g or X"1Q 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-GIy-Ala-Pro-Asn-Pro-Lys-Glu-Cys-COOH or PVPla NH^Lys-Leu-Ile-Leu-Ala-Tyr-Thr-Pro-Pro-Gly-Ala-Arg-Gly-Pro-Gln-Asp-Cys-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 pharmaceutical^ 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
12. A oligopeptide for use according to Claim 10 or 11 whereby the viral infections. if - f NOV 1989 infections are caused by rhino and other picorna viruses. DATED THIS / A. PARK* SON PER AGENTS FORTrlEAPPLiCANTS </div>