CS258289B1 - Peptide with antigenic determinants' properties and method of its production - Google Patents

Abstract

The present invention relates to a peptide having properties the antigenic determinants of formula (I) H-Met-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-X (I) wherein X is hydroxy or amine, and its immunoadjuvant conjugate substances such as muramyl dipeptide, with carriers of the protein or macromolecular type polymers, especially latex, spheron and polystyrene. The peptide was prepared by synthesis in solid state and it has been shown to him can be used as diagnostics for detection antibodies against the human-causing virus acquired immunodeficiency syndrome (AIDS). The peptide is coming into consideration for production monodeterminant antibodies.

Description

The invention relates to peptides having the properties of the antigenic determinants of formula I

H-Met-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-X (I) wherein X represents a hydroxyl or amine group and its conjugate with muramyl dipeptide-type immunoadjuvants, carriers of the type proteins or macromolecular polymers, in particular latex, spheron and polystyrene, and processes for their preparation.

The compound of the invention is novel and has been shown to possess antigenic determinants of p14 protein, a protein product of the human immunodeficiency virus (HIV) gene of tat. It can be used as a diagnostic tool for detecting antibodies against acquired immunodeficiency virus (AIDS) virus or AIDS-related complex (ARC). Further, it is potentially also useful for the production of monodeterminant antibodies.

Currently, acquired immunodeficiency syndrome (AIDS) is diagnosed by the presence of antibodies against HIV in the blood serum. The assay is performed using the so-called ELISA assay, which is based on the interaction of serum antibodies with the antigen, i.e. the HIV (inactivated detergent) virus, by which the ELISA plates are coated. The virus is obtained by a relatively complex procedure, first it is produced in tissue culture by infected cells, and then the virus is isolated from the tissue medium and further purified several times before it can be used to coat ELISA plates. It is a very complicated and costly work, but most of all highly dangerous, because it is always working with highly infectious material. ELISA plates, even if coated with an inactivated virus preparation, should be considered as potentially infectious and should be handled accordingly. Practically all laboratory AIDS kits manufactured by the world's leading manufacturers are based on the above-mentioned procedure. However, the assay is not entirely specific, giving a small percentage of false positive results, despite intensive purification by some contaminants in tissue culture. Therefore, confirmatory tests based on a different principle, either by immunofluorescence or by Western blotting, should be carried out in positive cases. In 1987, the first safe diagnostic kits are expected to contain only some of its structural proteins, instead of the entire HIV virus, obtained by gene manipulation in a production bacterial or eukaryotic system. Biochrom has already launched a safe diagnostic kit based on synthetic peptides. This should eliminate the need for confirmatory tests, since one defined antigen and not a mixture of the two would be used, as is the case with whole virus. These kits are likely to be highly specific and, in view of the genetic variability of the virus, will, in addition to better diagnosis, also make it possible to determine disease prognosis.

The compound of the invention has been synthesized by the method of the invention, which comprises anchoring a carboxyterminal amino acid on a polystyrene-type polymeric carrier with chloromethyl or benzhydrylamino groups and exposing the peptide chain by sequential condensation. Thereafter, the peptide obtained is acidolytically cleaved from the polymeric support and purified.

The peptide of formula I was prepared by the Merrifield method of solid phase peptide synthesis. The polymeric carrier used was polystyrene sulfurated with divinylbenzene, preferably one percent, with chloromethyl or benzhydrylamine functional groups. N 'are protected ^from amino acid-labile protecting group, preferably a Boc-group, its cleavage in various stages of the synthesis was performed by acidolysis, preferably a mixture of TFA and DCM or a saturated solution of hydrogen chloride in DCM. The side functional groups of the multifunctional amino acids were protected with benzyl type groups. The condensation of protected amino acids was carried out by suitably activated derivatives, preferably symmetrical anhydrides or HOBt esters. Upon completion of the synthesis, the peptide was cleaved from the resin and simultaneously deprotected of the multifunctional amino acids were acidolytically removed, preferably by treatment with liquid HF or TRMSA in admixture with TFA and thianisol. The crude product was purified either by column chromatography on carriers used in peptide chemistry, preferably silica gel, coated with hydrophobic carbon chains or carriers acting as molecular sieves. The peptide was characterized by amino acid analysis and its purity was verified by chromatographic and electrophoretic methods.

The amino acid sequence of the peptide of formula (I) has been derived from the amino acid sequence of the p14 virus protein causing human immunodeficiency (HIV) to include antigenic determinants of said protein. The peptide may serve for diagnostic purposes, as demonstrated by the inventors and for the production of monodeterminant antibodies.

The diagnostic reaction is based on the interaction of the synthetic peptide of the invention with antibodies produced in an organism that has been infected with a virus. The synthetic peptide of formula (I) is intended for the detection of antibodies produced by infected individuals against the viral protein p14, an immunodeficiency virus (HIV) in humans. In practice, for example, an enzyme-linked immunoassay (ELISA) which has been performed as described in U.S. Pat. AO No. 256 960. Table 1 shows the absorbance values for individual positive sera, diluted 1: 100. The sera used were positive when tested in a commercial ELISA, where HIV was used as the antigen and was also verified by Western blotting. The reaction with the control negative serum was at background level and did not exceed 0.1 in absorbance values.

Table 1

Absorbance values of positive sera diluted 1: 100

serum no. 1 2 3 4 5 6 7 8 absorbance 0.49 0.63 0.77 0.79 1.31 0.71 1.46 1.50 serum no. 9 10 11 12 13 14 15 Dec absorbance 1,02 0.93 1.46 0.55 0.79 1.44 1.58

The peptide of the invention can further be used to produce monodeterminant antibodies against the p14 protein directed against an antigenic determinant comprising a peptide by which immunization has been carried out. The inventors have immunized experimental animals, preferably mice or rabbits, with a peptide of formula I, preferably in admixture with muramyldipeptide, covalently bound muramyldipeptide to a peptide of formula I or polymerized glutaraldehyde or active ester method, or after binding to a suitable protein, preferably cow serumalbumin, thyroglobulin or keyhole limpet hemocyanin, using a condensing agent, preferably glutaraldehyde, carbodiimide, bifunctional agents or active esters. Freund's complete adjuvant or muramyldipeptide was used as an immunoadjuvant.

After immunization of mice with a peptide of formula I with a covalently bound muramyldipeptide, it was shown that the mouse hybridoma lines produced antibodies that reacted with the peptide of formula I. In the ELISA, the absorbance ranged from 0.30 to 0.59. Reaction with control negative serum did not exceed 0.1.

The results obtained show that the peptide of the invention can be used as a diagnosis and is useful in the production of monodeterminant antibodies.

The following exemplary embodiment illustrates, but is not limited to, the substance and method of the invention.

Example

2.0 g of polystyrene cross-linked 1% divinylbenzene benzhydrylamine resin was charged to the peptide synthesizer reaction vessel. The synthesis included 12 cycles, consisting of the following steps:

(1) protecting group cleavage, TFA: DCM (1: 1), 1x5 min, 1x30 min;

(2) wash, DCM, 6x1 min;

(3) washing, 2-propanol, 2x1 min;

(4) wash, DCM, 2x1 min;

(5) neutralization, TEAsDCM (1: 9), 1x1 min, 1x3 min;

(6) wash, DCM, 6x1 min;

(7) condensation using preformed symmetrical anhydrides (3-fold excess), to a negative reaction to amino groups;

(8) wash, DCM, 3x1 min.

The following protected amino acids were condensed in each cycle: Boc-Lys (BrZ), Boc-Trp (For), Boc-Pro, Boc-Glu (OBzl), Boc-Leu, Boc-Arg (Tos), Boo-Arg mixed condensation (Tos) and DCC in the reaction vessel, Boc-Pro, Boc-Asp (OBzl), Boc-Val, Boc-Pro, Boc-Glu (OBzl), and Boo-Met. The first synthetic cycle began with step (4). After completion of the synthesis, 4.80 g of peptidyl resin were obtained. The peptide was cleaved from the peptidyl resin (0.5 g) and at the same time deprotected with liquid HF by the low-high method. After removal of HF, the product and resin were filtered off, washed with ethyl acetate and the product extracted into 20% acetic acid and lyophilized. Yield 187 mg. The crude peptide (100 mg) was purified on a column (1 x 100 cm) containing as a stationary phase a polyacrylamide gel, mobile phase 1 acetic acid, flow rate 7 ml / h. Fractions containing a homogeneous product were pooled and lyophilized. The resulting product had an amino acid analysis consistent with theoretical values, the purity of the product was confirmed by analytical HPLC (Rt 11.7 min, mobile phase 50% MeOH, 50% water containing 0.1% TFA), thin layer chromatography (Rf 0.35) in n-butanol: acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield: 74 mg H-Met-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-NH 3

Explanatory notes to abbreviations used in the text

HIV virus causing immunodeficiency in humans

ARC complex of AIDS related diseases

ELISA enzyme immunoassay

HPLC high performance liquid chromatography

TFA trifluoroacetic acid

TFMSA trifluromethanesulfonic acid

HOBt N-hydroxybenztriazole

TEA triethylamine

Boc t-butyloxycarbonyl

This is p-toluenesulfonyl

Bzl benzyl

For formyl

DCC N, N'-dicyclohexylcarbodiimide

HF hydrogen fluoride

Arg arginine

Asp aspartic acid

Glu glutamic acid

Leu leucine

Lys lysine

Met methionine

For proline

Trp tryptophan

Val valin a

Claims (2)

OBJECT OF THE INVENTION A peptide having the properties of antigenic determinants of the general formula I, H-Met-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-X (I) wherein X represents a hydroxyl or amine group and its conjugate with muramyl dipeptide-type immunoadjuvants, type-carriers proteins or macromolecular polymers, especially latex, spheron and polystyrene. 2. A process for the production of a peptide of formula (I) according to claim 1, characterized in that: A carboxyterminal amino acid is anchored to a polystyrene-type polymeric carrier with chloromethyl or benzhydrylamine groups and the peptide chain is exposed by sequential condensation, whereupon the peptide obtained is acidolytically cleaved from the polymeric carrier and purified.