CS258288B1 - 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-Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Ile-Arg- -Leu-X (I) wherein X represents a hydroxyl or amino group, and a protein-type conjugate thereof or macromolecular polymers, especially latex, spheron and polystyrene. The peptide was prepared by solid synthesis phase and it has been shown that it can be used as diagnosticians in detecting antibodies against virus causing human syndrome acquired immunodeficiency (AIDS). Peptide it goes further into monodeterminative production antibodies.

Description

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

H-Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Ile-Arg-Leu-X (I), wherein X is a hydroxyl or amine group, and its conjugate with carrier type proteins or macromolecular polymers, especially latex , spheron and polystyrene and a process for their production.

The substance of the invention is novel and has been shown to possess antigenic determinants of the glycoprotein gp 41, a protein product of the env 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 purified in several steps 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 immunofluorescence or Western blotting, should be carried out in positive cases. The first safe diagnostic kits are expected to be launched in 1986, which will contain only some of its structural proteins, instead of the whole HIV virus, obtained by gene manipulation in a production bacterial or eukaryotic system. This should eliminate the need for confirmatory tests, since one defined antigen will be used, not a mixture thereof, 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. Similar results should be achieved using some HIV epitopes, where a similar kit is not yet produced, but a monoclonal antibody set against oligopeptides from the internal p24 viral protein is already on the market, and work on immunogenic peptides synthesized on p24 is expected in the near future. by analyzing the primary sequence of HIV envelope proteins.

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. As polymeric carrier, polystyrene cross-linked with divinylbenzene, preferably one percent, with chloromethyl or benzhydrylamine functional groups was used. The N-alpha-amino groups were protected with an acid-labile protecting group, preferably a Boc group, and its cleavage 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 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 TFMSA 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 peptide of formula (I) has been derived from the amino acid sequence of the glycoprotein gp 41 virus causing human immunodeficiency (HIV) to include antigenic determinants of said glycoprotein. 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 for the detection of antibodies produced by infected individuals against viral glycroprophin gp 41, a virus that causes 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. The reaction with the peptide showed the same absorbance values over time as the antigen prepared from the HIV virus. The reaction with the control negative serum was at background level and did not exceed 0.1 in absorbance values. Positive sera showed an absorbance of 5 to 10 times higher, with occasionally higher values (1.75).

The peptide of the invention can further be used for the production of monodeterminant antibodies to the glycoprotein gp41 directed against an antigenic determinant comprising a peptide by which immunization has been carried out. The inventors have immunized experimental animals, preferably rabbits, with a peptide of formula I, preferably polymerized glutaraldehyde or active ester method, or after binding to a suitable protein, preferably bovine 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 immunoadjuvants.

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.

Following! The exemplary embodiment and the method according to the invention are merely illustrative but not limiting.

Example

2.0 g of benzhydrylamine resin sieved with 1% divinylbenzene was placed in the reaction vessel of the peptide synthesizer. The synthesis involved 11 cycles consisting of the following steps: (1) cleavage of protecting groups, 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, TEA: DCM (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-Leu, Boc-Arg (Tos), condensation by mixing Boc-Arg (Tos) and DCC in the reaction vessel, Boc-Ile, Boc-Ser (Bzl), Boc-Arg (Tos ), condensation as in previous Arg, Boc-Asp (OBzl), Boc-Arg (Tos), condensation as in previous Arg, Boc-Asp (OBzl), Boc-Arg (Tos), condensation as in previous Arg, Boc- Glu (OBzl) and Boc-Gly. The first synthetic cycle began with step (4). After completion of the synthesis, 4.85 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 resins were filtered, washed with ethyl acetate and the product extracted into 20% acetic acid, lyophilized and purified on a column (1 x 100 cm) containing polyacrylamide gel as stationary phase, 1 N acetic acid mobile phase, flow rate 7 ml / h. Fractions containing 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 (R _fc 7.9 min, mobile phase 40 4 MeOH, 60% water containing 0.1 4 TPa), thin layer chromatography <R _f 0 , 35 in n-butanol: acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5,

Yield: 74 mg of H-Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Ile-Arg-Leu-1H-

Explanation 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

TFMSA trifluoromethanesulfonic acid

HOBt N-hydroxybenztriazole

TEA triethylamine

Boc t-butyloxycarbonyl

This is p-toluenesulfonyl

Bzl benzyl

CDD Ν, Ν'-dicyclohexylcarbodiimide

HF hydrogen fluoride

Arg arginine

Asp aspartic acid

Glu glutamic acid

Gly glycine

Ile isoleucine

Leu leuoin

Ser serin

Claims (2)

SUBJECT OF THE INVENTION A peptide having the properties of antigenic determinants of the general formula I, H-Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Ile-Arg-Leu-X (I), wherein X represents a hydroxyl or amine group and its conjugate with carrier type proteins or macromolecular polymers, especially latex, spheron and polystyrene. 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, followed by acidolytic cleavage from the polymeric carrier and purification. .