SE457353B - PERTUSSISTOXIN ANTIGEN APPLIES IMMUNAL ANALYSIS TEST RATE, VACCINE COMPOSITION AND INTRADERMAL SKIN TEST COMPOSITION WITH THE SAME ANTIGEN - Google Patents

Description

457 10 15 20 25 ao 35 353 2 fimbrial hemagglutinin as antigen. J. Infect. Haze. vol 146: 741-745), or sonicated B. pertussis bacteria (see, e.g., Goodman, YE, Wort, AJ and Jackson, FL 1981. Epzyme-linked immunosorbent assay for detection of pertussis immunoglobulin A in nasopharyngeal secretions as an indicator of recent infection- J. Clin. Microbiol. vol. 13: 286-292, and Viljanen, MK, Ruuskanen, O., Granberg, C. and Salmi, TT 1982. Serological diagnosis of pertussis: IgM , IgA and IgG antibodies against Bordetella pertussis measured by enzyme-linked immunosorbent assay. Scand.

J. Infect. Haze. 14: 112-117).

As is well known in the art, the vaccines are vol. against pertussis currently used in the United States and many other countries based on inactivated Bordetella pertussis bacteria. M. Pittman suggested in 1979 that whooping cough be mediated by an exotoxin (pertussis toxin) in 1979. the harmful effects and prolonged immunity of whooping cough. A hypothesis. Reef. Infect. Haze. vol. 1: 401-412) (see Pittman, M. Pertussis toxin: The cause of and in Japan acellular vaccines are currently used which include inactivated pertussis toxin.

Recently, the nucleotide sequence of pertussis toxin was published (Locht, C. and Keith, J. M., l98 §. Pertussis Toxin Gene: Nucledtide Sequence and Genetic Organization, Science, vol. 232, pp. 1258-1264). In this article, the authors suggest, among other things, that synthetic oligopeptides that include protective epitopes will also be useful in the development of a new generation of vaccines, but the article neither describes nor suggests such epitopes.

Another recently published article on pertussis toxin genes is: Nicosia, A., Perugini, M., Franzini, C., Casagli, MC, Borri, MG, Antoni, G., Almoni, M., Neri, P., Ratti, G., and Rappuoli, R., 1986. Cloning and sequencing of the pertussis toxin genes: Operon structure and gene duplication. Proc. 10 15 20 25 30 35 457 353 3 Natl. Acad. Sci. USA, vol. 83, 4631-4635. publication states, among other things, that "Manipulation of this toxin gene using genetic engineering could be a way of producing large amounts of detoxified protein".

This is only a suggestion and no manipulated toxin has been described.

Yet another publication in this field is: Engström, O., Rodmalm, K., Jörnvall, H., Lundquist, G., Kálmán, M., Simonscits, A., Bartfai, T., Löfdahl, S., and Askelöf, P., 1986. Characterization of the N-terminal structure of pertussis toxin subunit Sl and hybridization of oligodeoxyribonucleotide probes with Bordetella pertussis DNA fragment, FEMS Micro- biology Letters, vol. 36, 219-223. This article also presents suggestions, namely "The gene can also be introduced into other organisms for the production of toxin. Sequencing of the gene would allow the synthesis of peptides corresponding to the antigen epitopes of the toxin and consequently allow the development of a synthetic pertussis vaccine." However, the antigenic epitopes of pertussis toxin have not been identified, synthesized or tested.

In the case of intradermal skin test compositions, such compositions for testing the immunity to pertussis have not hitherto been described in the prior art. Disclosure of the Invention The present invention is based on a 17 amino acid polypeptide, namely NH 2 -X1-Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr-Ser- -Arg-Arg-Ser -Val-Ala-Ser-X2-COY where X1 and X2 each represent a possible coupling-facilitating amino acid residue (in the party of the description indicated as amino acid coupling unit), and Y represents -OH or -NH2.

Examples of suitable amino acid linkers are -Lys- and -Cys-. These coupling units facilitate coupling of a carrier, such as bovine serum albumin, to the polypeptide.

This polypeptide has biologically interesting properties, including its ability to react with antibodies from convalescent serum from pertussis patients.

The polypeptide has been synthesized in accordance with per se known solid state techniques.

As is well known in the art, a peptide is synthesized either in acid form (COOH) or in amide form (CONH2) depending on available, resin-bound starting amino acid. In one aspect of the invention there is provided an artificially produced pertussis toxin antigen which reacts with antibodies induced by native pertussis toxin and which comprises at least one peptide sequence selected from the group consisting of the polypeptide NH2-X1-Gln-Thr-Arg-Ala -Asn-Pro-Asn ~ Pro-Tyr-Thr-Ser- -Arg-Arg-Ser ~ Val-Ala-Ser-X2-COY 1 where X and X2 each-represent an optional amino acid coupling unit, and Y represents : OH or p-NH2; and peptide epitopes contained therein.

Because the polypeptide is capable of reacting with antibodies induced by native pertussis toxin, it is per se an antigen, and consequently it is capable of inducing antibodies which react with native toxin in animals. Because the polypeptide is an antigen, it is likely to contain shorter peptide sequences that act as epitopes. The pertussis toxin antigen, which reacts with antibodies induced by native pertussis toxin, does not necessarily comprise more than one such peptide epitope together with a carrier, although it preferably comprises several such epitopes. The term "artificially produced pertussis toxin antigen", as used in this specification and the appended claims, is intended to include pertussis toxin antigens which have been artificially produced, i.e., produced by human effort and not through natural causes that are independent of human participation. Although the polypeptide constituting or forming part of the pertussis toxin antigen of the invention has been chemically synthesized, the polypeptide and peptide epitopes contained therein may be prepared using any other technique, e.g., degradation, cloning, etc., and the term "artificially produced" is intended to cover products made by any such technology.

The word "comprising" in the specification and claims and the expression "consisting essentially of" in the claims indicate that something is included, but that this something does not necessarily constitute the only thing included.

In particular, it should be understood that "antigens which react with antibodies induced by native pertussis toxin and which comprise at least one peptide sequence selected from the group consisting of the polypeptide and peptide epitopes contained therein" are intended to include antigens in which the selected peptide sequence substantially consists of the polypeptide is the only component; antigens in which at least the polypeptide is present together with one or more carriers to which it is linked; antigens in which at least one peptide epitope contained in the polypeptide is present together with one or more carriers to which it or they are linked; antigens in which the polypeptide is present together with at least one peptide epitope contained in the polypeptide and antigens in which at least the polypeptide and at least one peptide epitope contained in the polypeptide are present together with the carrier or carriers to which they are linked. The word "carrier" should be construed broadly, and the carrier can be anything to which the peptide in question can be attached by physical / chemical interaction, such as covalent bonding, ionic bonding, hydrogen bonding or hydrophobic bonding. Examples of such carriers are mineral carriers, eg aluminum hydroxide, calcium phosphate, etc., plastic surfaces such as microplates, spheres, etc., lipids, liposomes, carbohydrates, amino acids, peptides and proteins.

In another aspect of the invention, there is provided a diagnostic immunoassay kit for determining antibodies in a biological fluid sample, which antibodies are induced by native pertussis toxin. As a diagnostic antigen, the test kit comprises at least one antigen selected from the artificially produced antigens, which react with antibodies induced by native pertussis toxin. Depending on the immunoassay used to determine antibodies induced by native pertussis toxin, the test kit may comprise other suitable reagents, such as a carrier to which the diagnostic antigen is linked, a positive standard serum sample, a negative standard serum sample, an enzyme conjugate, such as alkaline phosphatase or peroxidase, substrates for the enzyme conjugate, such as paranitrophenyl phosphate, agar or agarose gel, radiolabeled antigen, buffer solutions and / or washing solutions. Optionally, all reagents in the test kit are in separate, sealed test tubes or vials labeled with specific labels.

The biological fluid sample is preferably a nasopharyngeal secretion sample, saliva sample, blood sample or serum sample from an animal, eg a human.

Examples of immunoassays in which the test kit of the invention can be used are ELISA (enzyme-linked immunosorbent assay), Immunodiffusion, Radioimmunoassay (RIA) and Immunoelectrophoresis (IE). When ELISA (enzyme-linked immunosorbent assay) is used, the test kit according to the invention comprises a) a diagnostic antigen according to the invention b) optionally a carrier for said diagnostic antigen c) optionally a positive standard serum sample d) optionally a negative standard serum sample e) an enzyme conjugate f) optionally a substrate for said enzyme conjugate g) optionally buffer solution (s), and h) optionally washing solution (s).

When immunodiffusion or immunoelectrophoresis (IE) is used, the test kit according to the invention comprises the same as for ELISA, with the exception of ingredients e) and f). Instead, a gel such as agar or agarose gel is needed, but such a gel is usually not included in the test kit because it is widely available.

When radioimmunoassay (RIA) is used, the test kit of the invention comprises the same as for ELISA, with the exception of ingredients e) and f), which are replaced by radiolabeled antigen. Optionally, a solution for precipitating radiolabeled antigen bound to antibodies, such as trichloroacetic acid, or secondary antibodies may also be included in the test kit.

In a further aspect of the invention there is provided a vaccine composition comprising as an immunizing component at least one antigen selected from the artificially produced pertussis toxin antigens which react with antibodies induced by native pertussis toxin, preferably in an amount which is effective in protecting an individual against the disease pertussis, and a nontoxic, pharmaceutically acceptable carrier and / or diluent. The carrier is a carrier as defined above, and the diluent may be a conventional diluent used in this field, such as physiological saline. The vaccine composition of the invention may further comprise an antigen adjuvant in an amount which, together with the amount of said antigen, is effective in protecting an individual against the disease pertussis. Examples of commonly used adjuvants in vaccines for humans are so-called mineral carriers, eg phosphate or hydroxide of calcium or aluminum, on which the antigen in question is adsorbed. An example of a vaccine adjuvant commonly used in veterinary medicine is Freund's complete adjuvant. The vaccine composition may further comprise buffer (s) and / or one or more preservatives as needed, and suitable buffers and preservatives have been described in, for example, the US Pharmacopoeia.

In yet another aspect of the invention there is provided an intradermal skin test composition comprising at least one antigen selected from the artificially prepared pertussis toxin antigens of the invention which react with antibodies induced by native pertussis toxin, in an amount effective to produce an immunological skin reaction to a specific antibody titer in an individual, and a nontoxic, pharmaceutically acceptable carrier and / or diluent. The skin test composition of the invention may further comprise buffer (s) and / or one or more preservatives, as needed.

Useful carriers, diluents, buffers and preservatives have been defined above.

SYNTHESIS OF THE POLYPEPTIDE Solid phase synthesis (Merrifield) of the polypeptide has been performed in a conventional manner, by coupling the amino acids to each other, forming peptide bonds, starting with a solid phase (resin) to which the C-terminus of the first amino acid is coupled, terminal is connected to the N-terminus of the first amino acid, etc. Finally, the constructed peptide is released from the solid phase. Specifically, the following peptide was synthesized: NH 2 -Lys-Gln-Thr-Arg-Ala-Asn-Pro-Asn ~ Pro-Tyr-Thr-Ser- -Arg-Arg-Ser-Val-Ala -Ser-COOH Synthesis of the peptide fragments was performed using a Biosearch Sam Two (Biosearch, Inc. California USA) peptide synthesis machine, using version 2.38 of Biosearch's peptide synthesis automation software for t-BOC amino acids. Standard coupling constants (default) were used for 1 g of starting material (degree of substitution = 0.33 meq / g).

The resin used to synthesize the peptide was a standard Merrifield resin. The Appropriately Protected AlTlinOSYfOIfla Val '2 Boc-Ala-OH (Boc-L-Alanine) Boc-Arg (Tos) ) Boc-Asn-OH (Boc-L-Asparagine) Boc-Lys (CL-Z) -OH (Boc-eo-Chloro-Benzyl-Boc-Pro-OH (Boc-L-Proline) oxycarbonyl-L-Lysine Boc -Ser (Bzl) -OH Boc-Thr (Bzl) -OH (Boc-O ~ Benzyl-L-Serine) (Boc ~ O ~ Benzyl-L-Threonine) Boc-Val-OH (Boc-L-Valine) Boc -Tyr (Cl2-B21) -OH (Boé-oz, s-Dichlorobenzyl1 ~ L-Tyrosine) In terms of details regarding amino acid concentration, solvent volumes, reaction times, etc., the manufacturer's (Biosearch) recommendations were followed. 457 553 10 Specifically used the following chemicals are listed below for the synthesis, cleavage and purification of the synthetic peptides.

SUBSTANCE Acetylimidazole Dimethylformamide Diisopropylethylamine Hydroxybenzyltriazole Methylene chloride Ninhydrin Potassium hydroxide t-Boc amino acids Hydrogen fluoride Resorcinol Dimethylsulfide Hydroxybenzyltriazole Trifluoroacetic acid Acetonitrile Acetonitrile p.a.

Double distilled from 1 g / l ninhydrin, then 5 g / l KOH. The first 10% distillate was discarded. p.a.

HPLC p.a. p.a. p.a. (synthesis) sequanal (HPLC) HPLC p.a.

SUPPLIER Aldrich Fluka Aldrich Aldrich Fisons (UK) Aldrich Bachem AGA x Aldrich Aldrich Rathburn Merck Fluka The completed, resin-bound peptide was removed from the synthesis chamber and washed with methanol, and dried to constant weight under vacuum. Digestion of the Peptide from the Resin Carrier Aliquots (0.59) of the washed and dried resin bound peptide were transferred to a cylindrical (PTFE) polytetrafluoroethylene chamber containing 1 g of resorcinol, 6.5 ml of dimethyl sulfide and a PTFE-coated magnet. The chamber was cooled to -80 ° C in a dry ice-ethanol bath. Hydrofluoric acid (HF) (final volume 10 ml) was then released into the chamber, which was then kept at 0 ° C for 120 minutes on a magnetic stirrer. The acid was then removed using a water aspirator. The chamber was again cooled to -80 ° C and 10 ml of HF was admitted into the chamber, which was placed over a magnetic stirrer at 0 ° C for 45 minutes. Then the acid was removed again using a water aspirator and the remaining mixture was transferred to a sintered glass filter. The resin / peptide mixture was then washed with 100 ml of 10% by volume acetic acid and then with 50 ml of diethyl ether. The acetic acid wash was also washed with 50 ml of diethyl ether in a separatory funnel, and the ether phase was removed from the aqueous phase.

The combined ether washes and acetic acid wash were then evaporated and lyophilized, respectively, and the peptide product was recovered. The peptide was recovered only from the aqueous phase. Purification of the synthetic peptide The various components of the crude synthesis product were dissolved by applying them to a high-pressure chromatography column with reverse phase C-18 silica, using acetonitrile / H 2 O (0.1 vol% trifluoroacetic acid). (PFA)) as the mobile phase, 20 pg-100 mg of 20 (0.1% TFA), centrifuged and injected into a 1 ml loop the crude peptide was dissolved in up to 1 ml of H on a Rheodyne (California, USA HPLC injector The solution was pumped through a pre-packed l0x500 mm Oligosil "reverse-phase" C-18 column (Scandinavian Genetec, Sweden) using a 2152 gradient controller and two 2150 HPLC pumps (LKB, Sweden). Gradient 457 353 10 15 20 25 30 35 12 was linear from 0 to 100% acetonitrile after 60 min.

The flow rate was 2 ml / min. The elution of the synthesis product was monitored at 206-238 nm using a variable wavelength absorption LKB 2131 absorption monitor. Fractions were collected by hand and purified again if necessary by lyophilizing the relevant fractions, redissolving and repeating the chromatographic procedure. The final product was lyophilized and stored in glass tubes at -20 ° C.

PREPARATION OF THE PEPTIAN ANTIGEN FOR IMMUNOUS ANALYSIS The peptide synthesized above was coupled to a carrier, i.e. bovine serum albumin (BSA) in the following manner to form a diagnostic antigen (coating antigen), which was used in ELISA. 1 mg of peptide and 3.6 mg of BSA are dissolved in 2.0 ml of phosphate buffered saline (PBS), pH 7.4.

To this solution was added 30 microliters of a 2.5% (w / v) aqueous glutardialdehyde solution.

The reaction mixture was incubated at room temperature (20-25 ° C) for 1 hour, stopped by adding 0.5 ml of a 5 M aqueous ethanolamine solution and then dialyzed against one liter of PBS at + 4 ° C for 4 hours, exchanging the dialysis fluid after 30 min and after 2 h.

The contents of the dialysis bag are then gel filtered through a column (2.5 x 60 cm) containing Sephacryl (DS-300 gel (Pharmacia, Uppsala, Sweden) equilibrated with PBS, 3.0 ml fractions are collected.

The fractions containing coupled material were pooled and the pooled material was used in ELISA as a coating antigen and added directly, without any prior dilution, to the microplate.

ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA) The following is a general description of ELISA.

The conjugate and the dilution of the conjugate, as well as the time that elapsed before the microplates were read, can vary between the different analyzes. The differences are given according to the general description. ëëê ¥ ħ_ëE§§ElYElN§_êY_§Elêê EQUIPMENT: Microtiter plates (Dynatech, mod. no. l29B).

Antigen for coating microplates (eg peptides and proteins).

Coating Buffer Phosphate Buffered Physiological Saline (PBS) Incubation Buffer: PBS + 0.05 vol% Tween 20 Aqueous Liquid: 0.9% NaCl + 0.05% Tween 20 Serum (eg human and animal reference and test sera) Conjugate (t eg alkaline phosphatase-conjugated porcine anti-human IgG antibodies (Orion Diagnostica) and goat anti-rabbit Ig antibodies (Sigma).

Enzyme substrate: p-nitrophenylphosphate tablets (Sigma), 1 mg / ml substrate buffer (see below) Substrate buffer: 1M aqueous diethanolamine, pH 9.8, + 0.5 mM MgCl2 + 0.02% NaN Pipettes and test tubes PERFORMANCE: 1. Coating of microplates. 3.

Microplates were incubated with 0.1 ml of antigen in PBS at room temperature (20-25 ° C) overnight. Incubation with bovine serum albumin (BSA) and serum.

The plates were washed four times and then incubated with 0.1 ml of 1% (w / v) BSA in PBS at 37 ° C for 1 hour. After washing, 0.1 ml of serum was suitably diluted in incubation buffer to the wells and plate (plates). ) was incubated at room temperature for 1 h. 3. Incubation with conjugate.

After washing, 0.1 ml of conjugate was suitably diluted in incubation buffer to the wells and the plate (s) were incubated at room temperature for 2 hours. After washing, 0.1 ml of enzyme substrate was added. the solution to the wells. The plates were kept at room temperature and the absorbance at 405 nm was read after the time given below.

SPECIFIC DESCRIPTION OF ELISA ENZYME ~ LINKED IMMUNOSORBENT ASSAYS: Enzyme-linked immunosorbent assay (ELISA) for measuring the reaction between the synthetic peptide and antibodies specifically induced by immunization with native pertussis or anticoagulant (rabbit) human).

Each assay was performed in triplicate.

The peptide used as the coating antigen in the ELISA was first treated as described under "Preparation of peptide antigen for immunoassay".

The serum samples were from a rabbit hyperimmunized with highly purified pertussis toxin (Statens Bakteriologiska Laboratorium, Sweden), and from a convalescent pertussis patient (human), obtained from the Statens Bakteriologiska Laboratorium (SBL) in Sweden. The human serum sample is so selected and adjusted that in ELISA with native toxin as antigen gives an absorbance at -405 nm of about 1.0 after 1 hour of incubation.

Both sera were used in a 1/500 dilution. The conjugate used was an alkaline phosphatase-goat anti-rabbit Ig conjugate (Sigma) diluted 1/500 or an alkaline phosphatase-pig anti-human IgG conjugate (Orion Diagnostica) diluted 1/100.

The plates were read at the times indicated below in an automatic ELISA reading device (Titertek, Multiscan). 457 353 The following results were obtained:, In plates read absorbance at Serum É conjugate after 405 nm rabbit goat anti-rabbit at 3 min 1.21 É ¿human 2 pig anti-human É 20 min 1.11 As shown by the The above results bind the synthetic peptide strongly to antibodies induced by native pertussis toxin in convalescent serum samples from a pertussis patient.

Thus, the synthetic peptide acts as an antigen.

Claims (10)

457 353 10 15 20 25 30 35 16 PATENT REQUIREMENTS 1. Artificially produced pertussis toxin antigen which reacts with antibodies induced by native pertussis toxin, characterized in that it consists essentially of at least one peptide sequence selected from the group consisting of the polypeptide -X1-Gln-Thr-Arg- Ala-Asn-Pro-Asn-Pro-Tyr-Thr-Ser-NH2 -Arg-Arg-Ser-Val-Ala-Ser-X2-COY where X1 and X2 each represent an optional coupling-facilitating amino acid residue, and Y represents - OH or -NH 2; and peptide epitopes contained therein. A diagnostic immunoassay kit for the determination of antibodies induced by native pertussis toxin in a sample of biological fluid, characterized in that it comprises as a diagnostic antigen at least one antigen selected from artificially produced antigens which react with antibodies induced of native pertussis toxin and which consist essentially of at least one peptide sequence selected from the group consisting of the polypeptide NH2-X1-Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr-Ser- -Arg- Arg-Ser-Val-Ala-Ser-X2-COY 1 and X2 each represent an optional linker- where X facilitates amino acid residue, and Y represents -OH or -NH2; and peptide epitopes contained therein. A diagnostic immunoassay test kit according to claim 2, characterized in that it further comprises a carrier to which the diagnostic antigen is linked, a positive standard serum sample, a negative standard serum sample, and optionally buffer solution (s) and / or wash solution (s). A diagnostic immunoassay kit according to claim 3, characterized in that it further comprises agar or agarose gel, or radiolabeled antigen, or an enzyme conjugate and optionally a substrate for the enzyme conjugate. Vaccine composition, characterized in that it comprises as an immunizing component at least one antigen selected from artificially produced pertussis toxin antigens, which react with antibodies induced by native pertussis toxin and which consist essentially of at least one peptide sequence selected from the group consisting of the polypeptide NH2-X1-Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr-Ser- -Arg-Arg-Ser-Val-A1a-Ser-X2-COY where X1 represents facilitation amino acid residue, and Y represents and X2 each represents an optional cup -OH or -NH2; and peptide epitopes contained therein, together with a nontoxic, pharmaceutically acceptable carrier and / or diluent. Vaccine composition according to claim 5, characterized by cystoxin antigen (s) in an amount effective for comprising said pertussis to protect an individual against the disease pertussis. Vaccine composition according to claim 5, characterized in that an antigenic adjuvant in an amount which together with it further comprises the amount of said pertussis toxin antigen (s) is effective to protect an individual against the disease whooping cough. Vaccine composition according to claim 5, 6 or 7, characterized in that it further comprises buffer (s) and / or one or more preservatives. An intradermal skin test composition, characterized in that it consists essentially of at least one antigen in an amount effective to elicit an immunological skin response to a specific antibody titer in an individual, the antigen being selected from arterially prepared pertussis toxins. antigens which react with antibodies induced by native pertussis toxin and which comprise at least one peptide sequence selected from the group consisting of the polypeptide NH2-X1-Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr- Ser- -Arg-Arg-Ser-Va1-Ala-Ser-X2-COY where X1 and X2 each represent an optional coupling-facilitating amino acid residue, and Y represents 1 -OH or -NH2; and peptide epitopes contained therein, together with a nontoxic, pharmaceutically acceptable carrier and / or diluent. An intradermal skin test composition according to claim 9, characterized in that it further comprises buffer (s) and / or one or more preservatives.