CS258290B1 - Peptide with antigenic determinant properties and method for its production - Google Patents

Abstract

The solution relates to peptides with the properties of antigenic determinants of the general formula I H-Gly-Gln-Met-Arg-Glu-Pro-Arg-Gly-Ser-Asp-Ile-Ala-X (D , where X denotes a hydroxyl or amino group, and its conjugate with immunoadjuvant substances of the muramyl dipeptide type, carriers of the protein type or macromolecular polymers, in particular latex, spheron and polystyrene. The peptide was prepared by solid-phase synthesis and it was demonstrated that it can be used as a diagnostic agent in the detection of antibodies against the virus causing acquired immunodeficiency syndrome (AIDS) in humans. The peptide is also considered for the production of monodeterminant antibodies.

Description

The invention relates to a peptide having the properties of antigenic determinants of formula X

H-Gly-Gln-Met-Arg-Glu-Pro-Arg-Gly-Ser-Asp-Ile-Ala-X (I) wherein X is a hydroxyl or amine group and its conjugates with muramyl dipeptide immunoadjuvants, with carriers protein 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 the properties of the antigenic determinants of the p24 protein, the protein product of the gag virus gene that causes immunodeficiency (HIV) in humans. 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 in a relatively complex manner, first produced in tissue culture by infected cells, and then the virus is isolated from the tissue medium and further buffered several times before it can be used to coat ELISA plates. It is a very complicated and expensive work, but it is also very 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 as an antigen, instead of the entire HIV virus, obtained by the gene manipulation method in a production bacterial or eukaryotic system. Biochrom has already launched a diagnostic kit based on synthetic peptides. This should eliminate the need for confirmatory tests since one defined antigen and not a mixture thereof would be used, as is the case with whole virus. These kits are likely to be highly specific and will allow, in addition to better diagnosis, to determine disease prognosis due to the genetic variability of the virus

The compound of the present invention has been synthesized by the method of the present invention which comprises anchoring a carboxyterminal amino acid onto a polystyrene-type polymeric carrier with chloromethyl or benzhydrylamine 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. As polymeric carrier, polystyrene cross-linked with divinylbenzene, preferably one percent, with chloromethyl or benzhydrylamine functional groups was used. The N 2 -amino groups were protected with acid-labile protecting groups, preferably a Boc group, its cleavage at each stage of the synthesis was carried out acidolytically, preferably with a mixture of TFA and DCM or a saturated solution of hydrogen chloride in DCM. The side 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 at the same time the protecting groups of the multifunctional amino acids were removed acidolytically, preferably by treatment with liquid HF or TFM5A in admixture with TFA and thioanisole. 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 peptides of formula I was derived from the amino acid sequence of the HIV p24 protein 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 peptides of the invention with antibodies produced in an organism infected with a virus. The synthetic peptide of formula (I) is for the detection of antibodies produced by infected individuals against the viral protein p24 of HIV. 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 0.53 0.86 0.62 0.88 1.24 0.59 1.85 1.67 serum no. 9 10 11 12 13 14 15 Dec 16 absorbance 0.96 0.72 1.13 0.53 0.70 1.52 0.73 1.47 Peptide according to the invention is possible further use for production monodeterminantních antibodies

against a p24 protein directed against an antigenic determinant that includes a peptide that has been immunized. The inventors have immunized test animals preferably rabbits with a peptide of formula I, preferably in admixture with muramyldipeptide, covalently bound muramyldipeptide to a peptide of formula I or a peptide of formula I polymerized with glutaraldehyde or active ester method, or after binding to a suitable protein, preferably cow serumalbumin, thyroglobulin or hemocyanin to keyhole limpet, using a condensing agent, preferably glutaraldehyde, carbodiimide, bifunctional agents or active esters. Freund's complete adjuvant or muramyldipeptide was used as an immunoadjuvant. Sera of rabbits immunized with a peptide of formula I with a covalently bound muramyldipeptide showed a positive reaction in an ELISA assay using HIV as the antigen. Absorbance values ranged from 0.52 to 0.71 at a serum dilution of 1: 1,000, the absorbance value with the control negative serum was less than 0.1.

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

The following exemplary embodiment illustrates, but does not limit, the substance and method of the invention.

Example 1 a d

2.0 g of benzhydrylamine polystyrene resin cross-linked with 1-divinylbenzene was charged to the reaction vessel of the peptide synthesizer. The synthesis included 12 cycles consisting of the following steps ‘(1) cleavage of the protecting groups, TFA: DCM (1: 1), 1x5 min, 1x30 min?

(2) wash, DCM, 6x1 min;

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

51 (? $ 10 (4) washes, DCM, 2x1 min;

(5) neutralization, TEA: DCM (1: 9), 1x1 min, 1x3 min?

(6) piercing, DCM, 6x1 min;

(7) condensation using preformed symmetrical anhydrides (triple excess), to the negative reaction of the no amino group?

(8) wash, DCM, 3x1 min.

The following protected amino acids were condensed in each cycle: Boc-Ala, Boc-Ile, Boc-Asp (OBzl), Boc-Cer (Bzl), Boc-Gly, Boc-Arg (Tos), Boc-Arg blending condensation ( Tos) o DCC in reaction vessel, Boc-Pro, Boc-Glu (OBzl), Boc-Arg (Tos), condensation as in the previous Boc-Arg (Tos), Boc-Met, Boc-Gln and Boc-Gly. The first synthetic cycle began with step (4).

After completion of the synthesis, 4.21 g of peptidyl resin was obtained. The peptide was cleaved from the peptidyl-resin (C, 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 217 mg. The crude peptide (100 mg) was purified on a column (1 x 100 cm) containing polyacrylamide gel as a stationary phase, mobile phase 1 N 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 6.4 min, mobile phase 50% methanol, 50% water containing 0.1% TFA), thin layer chromatography (Rf 0.42) (η-butanol: acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield: 63 mg II-Gly-Cln-Met-Arg-Glu-Pro-Arg-Gly-Ser-Asp-Ile-Ala

Explanations of abbreviations used in the text

HIV virus causing! immunodeficiency in humans

ABC complex of diseases related to AIDS

AIDS syndrome acquired immunodeficiency

ELISA enzyme immunoassay

HPLC high performance liquid chromatography

TFA trifluoroacetic acid

DCM dichloromethane

TFMSA trifluoromethanesulfonic acid

IIOBt U-hydroxybenztriazole

TEA triethylamine

Boc t-butyloxycarbonyl

Tos p-toluenesulfones1

Bzl benzyl

DCC N, N-Dicyclohexylcarbodiimide

HF hydrogen fluoride

Ala alanin

Arg arginine

Asp aspartic acid

Gin glutamine

Glu glutamic acid

Gly glycine

Ile isoleucine

Met methionine

For proline

Ser serin

Claims (2)

A peptide having the properties of antigenic determinants of Formula I: H-Gly-Gln-Met-Arg-Glu-Pro-Arg-Gly-Ser-Asp-Ile-Ala-X (I) wherein X is a hydroxyl or amine group and its conjugate with muramyl dipeptide-type intunoadjuvants, protein-type carriers or macromolecular polymers, in particular spherone and polystyrene latex. 2. A process according to claim 1, wherein a carboxyterminal amino acid is anchored to the polystyrene-type polymeric carrier with chloromethyl or benzhydrylamine groups and the peptide chain is subjected to sequential condensation, whereupon the peptide obtained is acidolytically cleaved from the polymeric carrier and purified.