CS277003B6 - Peptides with antigenic determinant properties of the HIV-1 env gene product - Google Patents

Abstract

The solution relates to peptides with the properties of antigenic determinants of the protein product of the HIV-1 env gene of the general formula I. H-X-Asn-Y-Z (I) where X denotes Gly-Glu-Arg-Asp-Arg-Asp-Arg- -Ser-Ile-Arg-Leu-Val and Y denotes Gly-Ser-Leu (Ia) or X denotes Cys-Val-Pro-Thr-Asp-Pro and Y denotes Pro-Gln-Glu-Val-Val (lb) or X denotes Ile-Asn-Cys-Thr-Arg-Pro- -Asn-Asn (lc) and Y denotes Thr-Arg-Lys-Ser-Ile-Arg-Ile or X denotes Gly-Gly-Gly-Asp-Met-Arg-Asp and Y denotes Trp-Arg-Ser-Glu-Leu-Tyr (Id) or X denotes Val-Pro-Trp-Asn-Ala-Ser-Trp- -Ser (Ie) and Y denotes Lys -Ser-Leu-Glu-Gln-Ile and Z represents a hydroxyl or amino group, and their conjugates with immunoadjuvant substances of the muryl dipeptide type, with carriers of the protein type or macromolecular polymers, for example latex, spheron and polystyrene or with some activated membranes. The peptides were prepared by solid phase synthesis and have been shown to be useful as diagnostics in the detection of antibodies against the virus causing acquired immunodeficiency syndrome in humans. Alternatively, a conjugate of any of the peptides with a protein carrier can be used to prepare monodeterminant antibodies against gp 160 HIV-1.

Description

Peptides with the properties of antigenic determinants of the protein product of the HIV-1 env gene

Field of technology

The invention relates to peptides with properties of antigenic determinants of the protein product of the HIV-1 env gene.

Prior art

The peptides according to the invention are new and have shown, inter alia, the properties of the antigenic determinants of the glycoprotein gp160, a protein product of the env gene of the HIV-1 virus causing immunodeficiency syndrome (AIDS) in humans.

The essence of the invention

The invention relates to novel peptides with the properties of antigenic determinants of the protein product of the env gene of the HIV-1 virus of the general formula I

HX-Asn-YZ (I) where X is Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Ile-Arg-Leu-Val and Y is Fly-Ser-Leu (Ia), or X is Gys -Val-Pro-Thr-Asp-Pro and Y denotes Pro-Gln-Glu-Val-Val (Ib), or X denotes Ule-Asn-Cys-Thr-Arg-Pro-Asn-Asn and Y denotes Thr-Arg -Lys-Ser-Ile-Arg-Ile (Ic), or X denotes Gly-Gly-Gly-Asp-Met-Arg-Asp and Y denotes Trp-Arg-Ser-Glu-Leu-Tyr (Id), or X denotes Val-Pro-Trp-Asn-Ala-Ser-Trp-Ser and Y denotes Lys-Ser-Leu-Glu-Gln-Ile (le) and Z denotes a hydroxyl or amine group, and their conjugates with muramyl dipeptide-type immunoadjuvants, with carriers of the type of proteins or wet-molecular polymers, for example latex, spheron and polystyrene, or with some activated membranes.

The peptides of formula I were prepared by the Merrifield solid phase peptide synthesis method. Divinylbenzene crosslinked polystyrene, preferably one percent, with p-methylbenzhydrylamine or chloromethyl functional groups was used as the polymeric support. The N-amino groups were protected by an acid-labile protecting group, preferably a Boc group, its cleavage in the individual stages of the synthesis was carried out acidolytically, preferably with a mixture of TFA and DCM or HCl in DCM, U.S. Pat. The side functional groups of the multifunctional amino acids were protected with benzyl-type groups. Condensations of protected amino acids were performed with appropriately activated derivatives, preferably HOBt esters. Condensations were accelerated in an ultrasonic bath as described in MS. copyright certificate No. 271 584. Termination

CS 277003 B6 2 reaction was monitored by color change of acid-base indicator, preferably bromophenol blue according to MS. No. 268,230. Upon completion of the synthesis, the peptide was cleaved from the resin and the protecting groups of the multifunctional amino acids were removed acidolytically, preferably by the action of liquid HF. The crude product was purified either by column chromatography on supports used in peptide chemistry, preferably carriers acting as molecular sieves. The peptides were characterized by amino acid analysis and their purity was verified by chromatographic and electrophoretic methods.

The amino acid sequence of the peptides of formula I was derived from the amino acid sequence of the gp160 glycoprotein of the virus causing HIV-1 immunodeficiency in humans to include the artigenic determinants of said protein.

The basis of the diagnostic reaction, the implementation of which is explained in more detail, for example, Czechoslovakia. No. 258,288, is the interaction of the synthetic peptides of the invention with antibodies produced in an organism that has been infected with a virus. The synthetic peptides of formula I are intended for the detection of antibodies produced by infected individuals against the viral glycoprotein gp160 of HIV-1. In practical use, for example, an enzyme-linked immunosorbent assay (ELISA) can be advantageously used, which has been carried out by the inventors as already described in U.S. Pat. author's certificate No. 256 960. Absorbance values for individual positive sera, diluted in a ratio of 1. 20 to 100, were significantly higher compared to negative sera. The sera used were positive when tested in a commercial ELISA assay using HIV-1 lysate as antigen and also verified by Western Blot. The reaction with the control negative serum was at the background level and did not exceed the average of 0.15 in absorbance values.

The peptides of the invention could further be used to produce monodeterminant antibodies to the gp160 glycoprotein, whether polyclonal or monoclonal, directed against an antigenic determinant comprising the peptide to be immunized. The inventors immunized experimental animals, preferably mice or rabbits, with peptides of formula I, preferably in admixture with muramyl dipeptide, covalently linked muramyl dipeptide to peptides of formula I or peptides polymerized by glutaraldehyde or active ester method or after binding to a suitable protein, preferably bovine serum albumin, thyroglobulin or hemocyanin from keyhole limpet, using a condensing agent, preferably glutaraldehyde, carbodiimide, bifunctional reagents or active esters. The inventors used Freund's complete adjuvant or muramyl dipeptide as the immunoadjuvant.

Detection of antibodies was performed by an ELISA assay with a peptide of formula I anchored on a plastic polystyrene plate. The portable procedure of the ELISA test is described in MS. Author's Certificate no. 256 960. Using the appropriate anti-rabbit or anti-mouse IgG labeled with an enzyme, preferably a peroxidase or alkaline phosphatase, the resulting sera gave positive reaction up to a dilution of 10 ^"4-10".

Exemplary embodiments

Example 1

400 mg of p-methylbenzhydrylamine copoly (styrene-1% divinylbenzene) resin (MBHA, amino group content 0.4 meg / g) was placed in a flow reactor (internal volume 10 ml). The synthesis was performed on a manually operated synthesizer of our own design (see Czechoslovak author's certificate No. 262 081) by a continuous flow method (Czechoslovak author's certificate No. 255 089). The N- << -amino groups were protected by a Boc group, the side chains of the multifunctional amino acids were protected by benzyl-type groups. Arg p-toluenesulfone and protected amino acids were activated for HOBt ester condensation. One synthetic cycle included the following steps:

1. cleavage of Boc protecting groups, 40% TFA in DCM, 30 min (3 min wash, 27 min at rest),

2. washing, DCM into weakly acidic effluent (controlled with moistened pH paper),

3. neutralization, 5% N-ethyl-N, N-diisopropylamine in chloroform, 3 min,

4. wash, DMF, to neutral effluent,

5. condensation, protected amino acid, 0.8 meq in DCM (or DCM: DMF, 4: 1, if necessary), add HOBt, 0.8 meq in DMF, add DCC, 0.8 meq in DCM, 30 min at 20 ° C, filter. N, N-dicyclohexylurea, wash with DCM, evaporate with DCM under reduced pressure (t <25 ° C), dilute with DMF. filter, wash with DMF, make up to 0.2 M with DMF, add a few drops of a 0.001 M solution of bromophenol blue in DMF (70 mg / 100 ml), inject into a flow reactor, allow to react in an ultrasonic bath until the acid-base indicator changes from blue to yellow colors,

6. wash, DMF, 5min.

The flow rate during the washing cycles was about 20 to 30 ml / min, the inert gas overpressure 20 to 50 kPa. The synthesis was terminated by cleavage of the Boc protecting group from the last amino acid (step (1)) and the peptisyl resin was washed with DCM, methanol and dried under a stream of nitrogen. Yield 920 mg. The crude peptide was obtained after cleavage of the peptidyl resin with liquid hydrogen fluoride. After removal of HF, the product and resin were suspended in ethyl acetate, filtered, washed with ethyl acetate and the product extracted into 20% acetic acid and lyophilized. Yield 330 mg. The crude peptide was purified on a column (2.6 x 100 cm) containing a polysmide biogel as stationary phase. Fractions containing homogeneous product were pooled and lyophilized. The resulting product had an amino acid analysis identical to the theoretical values, the purity of the product was confirmed by analytical HPLC (Rt 4.5 min, mobile phase 50% MeOH, 50% water containing 0.1% TFA), thin layer chromatography (n-butanol system : acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield:

mg H-Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Ile-Arg-Leu-Val-Asn-Gly-Ser-Leu-NH ₂

Example 2

Following the procedure described in Example 1, peptide Ib was synthesized. A p-methylbenzyl group was used to protect the cysteine side group. Yield of peptidyl resin 820 mg, yield of crude product 262 mg. The resulting product had an amino acid analysis identical to the theoretical values, the purity of the product was confirmed by analytical HPLC (Rt 4.5 min, mobile phase 40% MeOH, 60% water containing 0.1% TFA), thin layer chromatography (n-butanol system: acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield:

132.5 mg H-Cys-Val-Pro-ThrAsp-Pro-Asn-Pro-Gln-Glu-Val-Val-hn ₂

Example 3

Following the procedure described in Example 1, peptide Ic was synthesized. A p-methylbenzyl group was used to protect the cysteine side group. Yield of peptide-resin 1 005 mg, yield of crude product 346 mg. The resulting product had an amino acid analysis identical to the theoretical values. The purity of the product was confirmed by analytical HPLC (Rt 6.0 min, mobile phase 45% MeOH, 55% water containing 0.1% TFA), thin layer chromatography (n-butanol: acetic acid: water -4: 1: 1 ) and paper electrophoresis at pH 2.5.

Yield:

201 mg H-Ile-Asn-Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-HN ₂

Example 4

Following the procedure described in Example 1, peptide Id was synthesized. A formyl group was used to protect the side function of tryptophan. Yield of peptidyl resin 945 mg, yield of crude product 401 mg. The resulting product had an amino acid analysis identical to the theoretical values, the purity of the product was confirmed by analytical HPLC (Rt 7.5 min, mobile phase 45% MeOH, 55% water containing 0.1% TFA), thin layer chromatography (n-butanol system: acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield:.

199 mg H-Gly-Gly-Gly-Asp-Met-Arg-Asp-Asn-Trp-Arg-Ser-Glu-Leu-Tyr-NH ₂

Example 5

Following the procedure described in Example 1, peptide Ie was synthesized. A formyl group was used to protect the side function of tryptophan. Yield of peptidyl resin 840 mg, yield of crude product 368 mg. The resulting product had an amino acid analysis identical to the theoretical values, the purity of the product was confirmed by analytical HPLC / Rt 7.5 min, mobile phase 55% MeOH, 45% water containing 0.1% TFA), thin layer chromatography (n-butanol system: acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield:

94.0 mg H-Val-Pro-Trp-Asn-Ala-Ser-Trp-Ser-Asn-Lys-Ser-Leu-Glu-Gln-Ile-NH ₂

Example 6

Following the procedure described in Example 1, peptide Ia was synthesized. Polystyrene crosslinked with 1% divinylbenzene functionalized with chloromethyl groups (Cl content 0.8 meq / g) was used as polymer carrier. The first protected amino acid Boc-Leu was attached to the support by esterification with KF. Yield of peptidyl resin 940 mg, yield of crude product 298 mg. The resulting product had an amino acid analysis identical to the theoretical values, the purity of the product was confirmed by analytical HPLC (Rt 8.5 min, mobile phase 45% MeOH, 45% water containing 0.1% TFA), thin layer chromatography (n-butanol system : acetic acid: water - 4: 1: 1) and electrophase on paper at pH 2.5.

Yield:

104 mg H-Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Ile-Arg-Leu-Val-Asn-Gly-Ser-Leu-OH

Example 7

To a solution of 10 mg of peptide Ia in 10 ml of HCl (pH 3.0) was added a solution of 10 mg of ECD in 10 ml of HCl (pH 3.0) with stirring at 0 ° C, the mixture was left for 15 min at 0 ° C, then a solution of 10 mg KLH in 0.1 M carbonate buffer was added so that the resulting pH was neutral. The mixture was incubated for 2 h at 20 ° C, after which time a solution of 10 mg MDP in PBS buffer pH 7.2 was added with stirring, and the mixture was incubated for 16 h at 8 ° C. The reaction mixture was then dialyzed against PBS, aliquoted into individual immunization doses and stored at -20 ° C. Amino acid analysis of the conjugate revealed that an average of 30 peptide molecules bound per molecule of protein carrier. A characteristic scattering of molecular weights from 5,000 to 30,000 was observed in gel permeation chromatography

Example 8

Peptides Ia to Ie of the invention were coated on wells of polystyrene ELISA plates. Appropriately diluted sera (in this case 1:20) were then pipetted into the wells with a diluent solution containing a blocking indifferent protein, usually bovine serum albumin at a concentration of 2 to 5% by weight. After incubation for 15 minutes at 37 ° C, serum samples were aspirated from the wells, washed 4 times with phosphate buffer pH 7.2 with 0.05% by weight, a nonionic detergent such as Tween 20. An appropriately diluted porcine conjugate solution was then pipetted into the well. horseradish peroxidase anti-human γ-globulin. Incubation was for 15 minutes at 37 ° C. The wells were then washed 4 times and a solution of a chromogen, such as o-phenylenediamine, was pipetted into them. The color reaction was induced in 5 to 7 minutes. The reaction was stopped by the addition of 2N Η $ 80 ₄ and the color intensity was read at 490 nm. Negative sera from blood donors and positive sera for the presence of antibodies to HIV 1 have been used, verified by tests such as Wellcome and Western blot).

Absorbance (490 nm) Negative sera Positive serum β β * »» · »« »« ·· »« * <» β β «β« · Β β β β ® β »β. « Peptide la 0.107 0.079 0.615 0.524 0.102 0.062 0.676 0.489 lb 0.080 0.160 0.811 0.850 0.079 0.157 0.725 0.784 Ic 0.086 0.157 0.903 0.574 0.097 0.174 0.880 0.588 Id 0.104 0.211 0.905 0.666 0.106 0.170 0.934 0.779 Ie 0.288 0.092 0.886 0.923 0.214 0.097 0.928 0.927

Industrial applicability

The peptides of the invention have the properties of the intigenic determinants of the glycoprotein gp160, a protein product of the eno gene of the HIV-1 virus, which causes immunodeficiency syndrome (AIDS) in humans. In addition, they may well serve as suitable diagnostics for the detection of antibodies to HIV and, in addition, are useful for the production of monodeterminant antibodies.

Explanations of abbreviations used in the text:

HIV virus causing immunodeficiency in humans ARC complex of AIDS-related diseases AIDS syndrome acquired immunodeficiency

ELISA enzyme immunoassay

HPLC high performance liquid chromatography

TFA trifluoroacetic acid

DCM dichloromethane

DMF N, N-dimethylformamide

HOBt N-hydroxybenzotriazole

Boc t-butyloxycarbonyl

Tos p-toluenesulfonyl

Bzl benzyl

For formyl

DIEA ethyl-diisopropylamine

MeOh methanol

DCC N, N-dicyclohexylcarbodiimide

HF hydrogen fluoride

Arg arginine

Asp aspartic acid

Ί

CS 277003 Β6

Asn Cys Gin Gly Glu lie igG Leu Lys Met Pro Ser Thr Trp Val ECD asparagine cysteine glutamine glycine glutamic acid. isoleucine immunoglobulin G leucine lysine methionine proline serine threonine tryptophan val in N-ethyl-N- (3-dimethylaminopropyl) carbodiimide hydrochloride KLH MDP PBS hemocyanin keyhole limpetui murymyldipeptide phosphoric buffer.

PATENT CLAIMS

Claims (1)

Peptides with the properties of antigenic determinants of the protein product of the env gene of the HIV-1 virus, of general formula I. H-X-Asn-Y-Z (I) where X and Y denotes Gly-Glu-Arg-Asp-Asp-Arg-Ser-Ile-Arg-Leu-Val indicates Gly-Ser-Leu (la), or X and Y denotes Cys-Val-Pro-Thr-Asp-Pro denotes Pro-Gln-Glu-Val-Val (lb), or X and Y denotes Ile-Asn-Cys-Thr-Arg-Pro-Asn-Asn denotes Thr-Arg-Lys-Ser-Ile-Arg-Ile (Ic), or X and Y denotes Gly-Gly-Gly-Asp-Met-Arg-Asp denotes Trp-Arg-Ser-Glu-Leu-Tyr (Id), or X and Y means Val-Ori-Trp-Asn-Ala-Ser-Trp-Ser means Lys-Ser-Leu-Glu-Gln-Ile (le), and Z denotes a hydroxyl or amine group, and their conjugates with muramyl dipeptide-type immunoadjuvants with carriers of the protein or macromolecular polymers, for example latex, spheron and polystyrene, or with certain activated membranes.