CS258289B1 - Peptide with antigenic determinant properties and method for its production - Google Patents

Abstract

The solution relates to a peptide with 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), where X denotes a hydroxyl or amino group, and its conjugate with immunoadjuvant substances of the muramyl dipeptide type, with 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 shown 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 suitable for the production of monodeterminant antibodies.

Description

The invention relates to peptides with antigenic determinant properties of the general formula I

H-Met-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-X (I), where X represents a hydroxyl or amino group, and its conjugates with immunoadjuvant substances of the muramyl dipeptide type, with carriers of the protein type or macromolecular polymers, in particular latex, spheron and polystyrene, and a method for their production.

The substance according to the invention is new and has been shown to possess the properties of antigenic determinants of the pl4 protein, which is a protein product of the tat gene of the human immunodeficiency virus (HIV). It may serve as a suitable diagnostic for the detection of antibodies against the virus causing acquired immunodeficiency syndrome (AIDS) or AIDS-related disease complex (ARC). It is also potentially useful for the production of monodeterminant antibodies.

Currently, acquired immunodeficiency syndrome (AIDS) is diagnosed by the presence of antibodies against the HIV virus in the blood serum. The determination is carried out using the so-called ELISA test, which is based on the interaction of antibodies in the serum with the antigen, i.e. the HIV virus (inactivated detergent), with 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 in several stages before it can be used to coat the ELISA plates. This is a very complex and expensive work, but above all highly dangerous, because the work is done all the time with highly infectious material. ELISA plates, even if they are coated with an inactivated virus preparation, must be considered as potentially infectious material and must be handled accordingly. Practically all laboratory kits for the examination of AIDS, produced by leading world manufacturers, are based on the above-mentioned procedure. However, the test is not entirely specific, it gives a certain small percentage of false positive results, caused despite intensive purification by some contaminating components from tissue culture. Therefore, it is necessary to perform confirmation tests based on a different principle in positive cases, either immunofluorescence or the so-called Western blot method. In 1987, the first safe diagnostic kits are expected to be launched on the market, which instead of the entire HIV virus will contain only some of its structural proteins as antigens, obtained by the method of gene manipulation in a production bacterial or eukaryotic system. The company Biochrom has already launched a safe diagnostic kit based on synthetic peptides. This should eliminate the need for confirmation tests, since one defined antigen would be used and not a mixture of them, as is the case when using the entire virus. These kits will probably be highly specific and, given the genetic variability of the virus, will probably also allow for better diagnostics and, in addition to determining the prognosis of the disease.

The substance according to the invention was synthesized by the method according to the invention, the essence of which consists in anchoring a carboxy-terminal amino acid to a polystyrene-type polymer carrier with chloromethyl or benzhydrylamine groups and exposing a peptide chain by gradual condensation. The obtained peptide is then acidolytically cleaved from the polymer carrier and purified.

The peptide of general formula I was prepared by the Merrifield method of solid-phase peptide synthesis. Polystyrene sulfurized with divinylbenzene, preferably one percent, with chloromethyl or benzhydrylamine functional groups was used as a polymer carrier. The N' ^of the -amino groups were protected by an acid-labile protecting group, preferably a Boc group, its cleavage in individual stages 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 functional groups of the multi-functional amino acids were protected by benzyl-type groups. Condensations of the protected amino acids were carried out with suitably activated derivatives, preferably with symmetrical anhydrides or HOBt esters. After the synthesis was completed, the peptide was cleaved from the resin and at the same time the protecting groups of the multi-functional amino acids were removed acidolytically, preferably by the action of liquid HF or TRMSA in a mixture with TFA and thianisole. The crude product was purified either by column chromatography on supports used in peptide chemistry, preferably silica gel coated with hydrophobic carbon chains or supports functioning 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 general formula I was derived from the amino acid sequence of the p14 protein of the human immunodeficiency virus (HIV) to include the antigenic determinants of said protein. The peptide can serve for diagnostic purposes, as demonstrated by the inventors, and for the production of monodeterminant antibodies.

The basis of the diagnostic reaction is the interaction of the synthetic peptide according to the invention with antibodies formed in an organism that has been infected with a virus. The synthetic peptide of the general formula I is intended for the detection of antibodies produced by infected individuals against the viral protein p14, a virus that causes human immunodeficiency (HIV). In practical use, it is advantageous to use, for example, an enzyme-linked immunosorbent assay (ELISA), which was performed by the authors of the invention as described in Czechoslovak Patent Application No. 256,960. Table 1 shows the absorbance values for individual positive sera, diluted in a ratio of 1:100. The sera used were positive when tested in a commercial ELISA test, where HIV was used as the antigen, and were also verified by the Western blot method. The reaction with the control negative serum was at the background level and the absorbance values did not exceed the limit of 0.1.

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 absorbance 1.02 0.93 1.46 0.55 0.79 1.44 1.58

The peptide according to the invention can further be used for the production of monodeterminant antibodies against the p14 protein, directed against the antigenic determinant, which includes the peptide with which the immunization was carried out. The authors of the invention carried out the immunization of experimental animals, preferably mice or rabbits, with a peptide of general formula I, preferably in a mixture with muramyldipeptide, covalently bound muramyldipeptide to the peptide of general formula I or polymerized glutaraldehyde or by the active ester method or after binding to a suitable protein, preferably bovine serum albumin, thyroglobulin or keyhole limpet hemocyanin, using a condensing agent, preferably glutaraldehyde, carbodiimide, bifunctional agents or using active esters. As an immunoadjuvant, Freund's complete adjuvant or muramyldipeptide was used.

After immunization of mice with the peptide of general formula I with covalently bound muramyl dipeptide, it was demonstrated that mouse hybridoma lines produced antibodies that reacted with the peptide of general formula I. In the ELISA test, the absorbance ranged from 0.30 to 0.59. The reaction with the control negative serum did not exceed the limit of 0.1.

The obtained results show that the peptide according to the invention can be used as a diagnostic and is considered for the production of monodeterminant antibodies.

The following embodiment example only illustrates the substance and method of the invention, but does not limit it in any way.

Example

2.0 g of benzhydrylamine polystyrene resin crosslinked with 1% divinylbenzene was placed in the reaction vessel of the peptide synthesizer. The synthesis involved 12 cycles, consisting of the following steps:

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

(2) washing, DCM, 6x1 min;

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

(4) washing, DCM, 2x1 min;

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

(6) washing, DCM, 6x1 min;

(7) condensation using pre-prepared symmetrical anhydrides (3-fold excess), to a negative reaction on the amino groups;

(8) washing, DCM, 3x1 min.

In individual cycles, the following protected amino acids were condensed: Boc-Lys(BrZ), Boc-Trp(For), Boc-Pro, Boc-Glu(OBzl), Boc-Leu, Boc-Arg(Tos), condensation by mixing Boo-Arg(Tos) and DCC in a 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 the synthesis was completed, 4.80 g of peptidyl resin was obtained. The peptide was cleaved from the peptidyl resin (0.5 g) and the protecting groups were simultaneously removed using 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 was extracted into 20% acetic acid and lyophilized. Yield 187 mg. The crude peptide (100 mg) was purified on a column (1x100 cm) containing a polyacrylamide gel as the stationary phase, IN acetic acid as the mobile phase, flow rate 7 ml/hour. Fractions containing a homogeneous product were combined and lyophilized. The resulting product had an amino acid analysis consistent with the 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 the system n-butanol:acetic acid:water - 4:1:1) and paper electrophoresis at pH 2.5.

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

Explanations of abbreviations used in the text

HIV virus causing human immunodeficiency

ARC AIDS-related disease complex

ELISA enzyme immunoassay

HPLC high performance liquid chromatography

TFA trifluoroacetic acid

TFMSA trifluoromethanesulfonic acid

HOBt N-hydroxybenzotriazole

TEA triethylamine

Boc t-butyloxycarbonyl

Tos 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 valine and

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.