CS272926B1 - Peptides with properties of hpv-virus protein's antigen determinant and method of their production - Google Patents

Abstract

The invention concerns peptides with antigen determinant properties of H-Met-Gly-Thr-Pro-Phe-Ser-Pro-Val-Thr-Pro-Ala-Leu-Pro-Thr-Gly-Pro-Val-NH2 and their conjugates with biologically inactive carriers of the type of proteins or macromolecular polymers, particularly latex, spheron and polystyrene or with active membranes. The peptides were prepared by synthesis in the solid phase and it has been shown that they can be used as diagnostics for detection of antibodies against human papillomaviruses of type 6 and 11.

Description

The invention relates to a peptide having the properties of an antigenic determinant of formula (I)

H-Met-Gly-Thr-Pro-Phe-Ser-Pro-Val-Thr-Pro-Ala-Leu-Pro-Thr-Gly-Pro-Val-NH2 (I) and its conjugate with protein or macromolecular polymer type carriers , in particular latex, spheron and polystyrene or activated membranes, and processes for their preparation.

The compounds of the invention are novel and have been shown to possess antigenic determinants of the protein encoded by the L-2 region of the HPV-6 and HPV-11 genomes. The peptides may be useful as diagnostic agents for the detection of antibodies in persons infected with these HPV types.

Various serological techniques have been used in the past to demonstrate immune responses to HPV, such as an immunoaggregation assay (Almeida JD, and P. Goffe, 1965, Lancet ii, 1205-1207) by agar gel diffusion (Cubie HA, 1972, J. Hyg. 70 677-690), complement fixation (Genver J, 1971, Acta Dermato-venerol 51, 365-373), radioimmunoassay (Hefton JM, M. Eisinger 1977, J. Gen. Virol. 38, 179-182) and neutralization text (Kienzler JL et al. 1983, Br. J. Dermatol. 108, 665-672). Since HPV cannot be cultivated in vitro, and therefore it is not possible to use classical methods for the preparation of viral antigens on tissue cultures to produce an antigen, the only source of antigens was until recently natural viral lesions. However, the possibilities of obtaining preparative amounts of viral antigens are limited. Some HPV-induced lesions, such as conoyloma accuminatum, contain very few viral portions and only viral nucleic acids are detectable in some HPV-associated malignant tumors.

These drawbacks are solved by the method according to the invention, which comprises using a synthetic peptide with the properties of the antigenic determinant of the protein encoded by the L-2 region of the genorau to detect antibodies raised following HPV 6 and HPV 11 infection.

The compounds according to the invention have been synthesized by the method according to the invention, which comprises anchoring a carboxyterminal amino acid on a polystyrene-type polymer carrier with chloromethyl or benzhydrylamine groups and exposing the peptide chain by sequential condensation of the individual amino acids. Thereafter, the peptide obtained is acidolytically cleaved from the polymeric support and purified.

The peptides of formula (I) were prepared by the Merrifield method of solid phase peptide synthesis. As polymeric carrier, polystyrene crosslinked 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 at each stage of the synthesis was performed 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 the protecting groups of the multifunctional amino acids were removed by acidolysis, preferably by treatment with liquid HP or TPMSA in admixture with TFA and thioanisole. The crude product was purified by either column chromatography on carriers used in peptide chemistry, preferably silica gel, coated with hydrophobic carbon chains or carriers acting as molecular sieves. The peptides were characterized by an amino acid

Their analysis and their purity were verified by chromatographic and electrophoretic methods.

The amino acid sequence of peptide I was derived from the L-2 region of the HPV 6 genome, which is homologous to HPV 11, and also includes the antigenic determinant of the L-2 genome protein. The peptides may serve for diagnostic purposes, as demonstrated by the inventors, and for the production of monodeterminant antibodies.

The basis of the diagnostic reaction is to form a complex of the synthetic peptides of the invention with antibodies produced in an organism which has been infected with HPV 6 or

CS 272 926 Bl

HPV 11. For example, an enzyme-linked immunoassay (ELISA) may be used in practice. The synthetic peptide was conjugated to a proteinaceous carrier (e.g. BSA) with glutaraldehyde and the sera were applied after binding the antigen to a microtiter ELISA plate, rinsing with buffer and saturation of vacant sites with 1% BSA solution. After incubation and washing with buffer, peroxidase labeled porcine anti-human IgG antibody was added. When antibodies to the selected HPV 6 or HPV 11 antigen deterrent were present in the serum to be tested, peroxidase-labeled antibody bound to the antigen-lique antibody complex. The amount of bound antibodies is proportional to their concentration in the serum tested. Detection of peroxidase activity is accomplished by oxidation of o-phenylenediamine with oxygen released from the peroxide present in the substrate solution. Absorbance values for positive samples ranged from O.L. 1.2 AV to 2.612 A.V. (74 samples tested), the values for negative sera did not exceed 0.5 A.V. (250 determinations).

The results obtained show that the peptides of the invention can be used as diagnostics for the detection of specific antibodies and are suitable for the production of monodeterminant antibodies.

The following examples and methods of the present invention are illustrative and not limiting.

Example 1

100 mg of benzhydrylamine copoly (styrene-1% divinylbenzene) resin (amino group content 0.4 meg / g) was placed in a flow reactor (1 ml internal volume). The synthesis was performed on a manually operated self-synthesizer synthesizer, author's certificate No. 262081, continuous by the flow method, see author's certificate No. 255089. 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, to a weakly acidic effluent, (controlled by a moistened pH paper); (3) neutralization, 5% N-ethyl-N, N-diisopropylamine in chloroform, 3 min; (4) washing, DMF, to neutral effluent;

(5) condensation: protected amino acid, 0.4 meg in DCM (or DCM: DMF, 4: 1 if necessary), add HOBt, 0.4 meg in DMF, add DCC, 0.4 meg in DCM, 30 min at 20 ° C , filter, N, N'-dicyclohexylurea, washed with DCM, DCM evaporated under vacuum (T 25 ° C) diluted in DMF, filtrated, washed with DMF, DMF supplement to a concentration of 0.2M, injected into a flow reactor, to react _doi: ineutrální response to ninhy * · toil; (6) wash, DCM, 5 min. The flow rate during the flushing cycles was about 20-30 ml / min, the inert gas overpressure was 20-50 kPa. The synthesis was terminated by cleavage of the gOC protecting group from the last condensed amino acid (step (1)) and the 1-resin peptides were washed with DCM and methanol and dried with a stream of nitrogen. The crude peptide was obtained after cleavage of peptydyl resin with liquid hydrogen fluoride. After removal of HF, the product and resins were suspended in ethyl acetate, filtered, washed with ethyl acetate, and the product extracted into 20% acetic acid and lyophilized. Yield 49.5 mg. The crude peptide was purified on a column (2.6 x 13 cm) containing hydrophobic carbon chain coated silica gel as a stationary phase, 50% methanol mobile phase, 50% water containing 0.1% TFA. 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 HPCL (Rt 3.3 min, mobile phase 60% MeOH, 40% water containing 0.1% TFA), thin layer chromatography (Rf 0.21 in n-butanol system): acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5- Yield 18.2 mg H-Met-Gly-Thr-Pro-Phe-Ser-Pro-Val-Thr-Pro-Ala- Leu-Pro-Thr-Gly-Pro-Val-NH2.

Example 2

To a solution of 26 mg (0.37 µM) of BSA in 2.6 ml of saline was added

6.3 mg (3.7 / UM - 10 molar excess) of peptide in 1.3 ml saline. 0.39 was slowly added dropwise to the mixture with vigorous stirring at room temperature

CS 272 926 B1 ml of 1% glutaraldehyde solution and the mixture was left at room temperature in the dark for 16 hours. Then the solution was transferred to the dialyzed intestine and dialyzed for 4 hours against a 0.5 M solution of ethanolamine pH 7.2 and overnight against a saline solution with 0.05% sodium azide. Protein concentration was determined in the conjugate solution and stored at -20 ° C after filling.

Example 3

The polystyrene microtiter plate was incubated with 100 µl of a synthetic peptide-BSA conjugate solution in carbonate buffer (0.015 M NagGO 4, 0.03 M NaHCO 3,

0.003 M NaN (pH = 9.6) 16 h at 37 ° C (antigen concentration 0.0625 / Ug / well). Saturation of free spots in the well was performed by incubation with 100 µl of a 1% solution of cow's serum albumin in carbonate buffer for 1 h at 20 ° C. The wells were rinsed with phosphate buffer (0.034 M KaCL, 0.002 µl KOI, 0.001 M KHgPO ^ 0.006 M Na ₂ HPO ₄ , 2 HgO, pH = 7-2) with the addition of O.1 nonionic detergent (Triton X-100). Then, 100 µL of the examined serum diluted with phosphate buffer pH = 7.2 was added to the wells, with 0.1% addition of nonionic detergent (Triton X-100) and enriched with 10% bovine serum (BS). After incubation (20 ° C, 60 min) and rinsing with phosphate buffer pH 7.2 with 0.1% detergent, 100 µl of peroxidase-labeled porcine anti-human immunoglobulin G antibody in phosphate buffer pH = 7.2, with 0.1% nonionic detergent (Triton) was added to the wells. X-100) and enriched with 10% BS. After incubation (20 ° C, 60 min) and washed 5 times with phosphate buffer, once with citrate buffer, peroxidase activity was detected by adding 100 µL of a 0.04% o-phenylenediamine solution in citrate buffer (0.066 M Na 2 HPO 2, 2H ₂ O and 0.034 M). citric acid solution (pH = 5 · 0) and enriched with 0.006% HgOg.

The reaction was terminated by the addition of 100 µl of 2 M sulfuric acid and the color evaluated quantitatively at 492 nm. The peptide with the properties of the antigenic determinants of the protein encoded by the L-2 region of the HPV-6 and 11 genome was used to test more than 400 human sera. While less than 15% of sera from healthy individuals showed the presence of specific antibodies, most sera (52%) from patients with condyloma accuminatum (disease caused by papillomaviruses type 6 and 11) reacted specifically with the antigen. The preparation of the synthetic peptide of the invention allows the introduction of the type-specific serological assay necessary to study the role of papillomaviruses in the pathogenesis of human diseases.

Explanation of abbreviations used in the text:

HPV human papillomavirus ELISA $ enzymoimmunological test BSA bovine serum albumin FROM. optical density HPLC high performance liquid chromatography TEA trifluoroacetic acid DCM dichloromethane MeOH methanol TEMSA Trigluoromethanesulfonic acid HOBt N-hydroxybenzotriazole TEA triethylamine Bac t-butyloxycarbonyl It with p-toluenesulfonyl Bzl benzyl For formyl

Claims (2)

Peptides having the properties of the antigenic determinants of the HPV protein of formula I H-Met-Gly-Thr-Pro-Phe-Ser-Pro-Val-Thr-Pro-Ala-Leu-Pro-Thr-Gly-Pro-Val-NH2 (I) and their conjugates with biologically inactive protein-type carriers; or macromolecular polymers, especially latex, spheron and polystyrene or with activated membranes. 2. A process for the production of peptides of formula (I) according to claim 1, characterized in that a carboxyterminal amino acid is anchored to the polystyrene-type polymeric carrier with chloromethyl or henzhydrylamine groups and the peptide chain is subjected to sequential condensation. purifies and binds to macromolecules! a proteinaceous carrier, preferably bovine serum albumin, using glutaraldehyde or soluble carbodiimide.