AU627701B2 - Hiv peptides, artificial hiv antigens and immunoassay kits - Google Patents

Description

I- 71

PCT

OPI DATE 23/05/89 AOJP DATE 29/06/89 APPLN. ID 26242 88 PCT NUMBER PCT/SE88/00570 INTERNATIONAL APPLICATION PUBLISHED UNDER THE VAl INI .u (51) International Patent Classification 4 (11) International Publication Number: WO 89/ 03844 C07K 7/04, G01ON 33/569 Al A61K 39/21 (43) International Publication Date: 5 May 1989 (05.05.89) (21) Internationa) Application Number: PCT/SE88/00570 (74) Agent: AWAPATENT AB; Box 5117, S-200 71 Malm6

(SE).

(22) International Filing Date: 27 October 1988 (27.10.88) (81) Designated States: AT (European patent), AU, BE (Eu- (31) Priority Application Number: 8704185-1 ropean patent), CH (European patent), DE (European patent), DK, FI, FR (European patent), GB (32) Priority Date: 28 October 1987 (28.10.87) (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), NO, SE (33) Priority Country: SE (European patent), US.

(71) Applicant (for all designated States except US): FER- Published RING AB [SE/SE]; Soldattorpvagen 5, S-216 13 Mal- With international search report.

m6 (SE).

(72) Inventors; and Inventors/Applicants (for US only) TROJNAR, Jerzy [SE/SE]; Sten6regatan 36, S-230 44 Vintrie (SE).

WAHREN, Brita [SE/SE]; Fritiofsvigen 10, S-182 64 Djursholm RUDEN, Ulla [SE/SE]; Vidfaresvig 11, S-191 77 Sollentuha (SE).

62 (54) Title: HIV EPTIDES, ARTIFICIAL HIV ANTIGENS AND IMMUNOASSAY KITS (57) Abstract An artificial peptide having an amino acid sequence which corresponds to a naturally occurring amino acid sequence of an HIV comprising an epitope and which further has two cysteine residues located on each side of said epitope, and further having a sulphur bridge between said two cysteine residues, which has been formed by a chemical oxidation step, is described. Furthermore, an artificial antigen which reacts with antibodies induced by an HIV is described. Said antigen mainly consists of an artificial peptide according to the invention. Additionally, a method of detecting antibodies induced by an HIV in a sample of body fluid, wherein said sample is subjected to an immunoassay, especially ELISA, and wherein an artificial antigen according to the invention is used as a diagnostic antigen, is described. A diagnostic immunoassay kit for said method is also described. Finally, a vaccine composition comprising, as an immunizing component, at least one antigen according to the invention, is described.

'WO 89/03844 PCT/SE88/00570 i HIV peptides, artificial HIV antigens and immunoassay kits.

The present invention relates to artificial peptides having an amino acid sequence which corresponds to a naturally occurring amino acid sequence of a HIV comprising an epitope and which further has two cysteine residues located on each side of said epitope, and further having a sulphur bridge between said two cysteine residues which has been formed by a chemical oxidation step. The invention also relates to artificial antigens, which react with antibodies induced by a HIV, a method of detecting antibodies induced by a HIV in a sample of body fluid, a diagnostic immunoassay kit for said method, and'a vaccine composition comprising, as an immunizing component, at least one antigen of the invention.

Background and Prior Art The acquired immunodeficiency syndrome (AIDS) is a sexually transmitted disease that can also be transmitted through contaminated blood or blood products.

It is caused by human immunodeficiency virus (HIV, previously called HTLV-III) which infects and is latently harboured in T4-lymphocytes and monocytes (Wong-Staal, F. and Gallo, Human T-lymphotropic retroviruses.

Nature 317:395-403, 1985). The chronically HIV-infected individual may in turn transmit HIV, most often by sexual contact. During infection, the immune response deteriorates and AIDS develops in 50-70% of the cases.

The fate for the remainder of infected persons is not yet known. When AIDS develops it is lethal and characterised by opportunistic infection, Kaposi sarcoma, other tumors and/or neurological disease.

To diagnose an HIV infection, virus is isolated or the antibody response is measured. Virus isolation is successful in 30-50% of asymptomatic HIV infected i 1 WO 89/03844 PCT/SE88/00570 2 persons, and in 90-100% of patients who developed AIDS. The antibody response is composed of immunoglobulins directed to the various structural and enzymatic components of HIV. They include the virus envelope proteins glycoprotein (gp)120, the transmembrane protein gp41 and their precursor gpl60, the interior structural group antigens p24, 17 and 7/9 and their precursor p 55, the enzymes reverse transcriptase (RT) p65/51 and endonuclease p32 and protease. Antibodies to proteins of the regulatory regions of the HIV genome also develop. The correct identification of an HIV-infected person depends on the type(s) of immunoglobulin (Ig) he produces and on the correct composition of antigens used in the immunoassay.

One common way to establish a diagnosis through antibody detection is to screen serum samples by enzyme- -linked immunosorbent assay (ELISA) (Sarngadharan, M.G., Popovic, Bruch, Schupbach, J. and Gallo, R.C.: Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) inithe serum of patients with AIDS.

Science 224:506-508, 1984; Schtpbach, Haller, 0., Vogt, Ltthy, Joller, Oelz, Popovic, M., Sarngadharan, M.G. and Gallo, R.C.:-Antibodies to HTLV-III in Swiss patients with AIDS and pre-AIDS and in groups at risk for AIDS. The New Engl. J. Med.

312:265-270, 1985). Wells of microplates coated with viral antigens are reacted with the serum samples under investigation, washed, and antihumar Ig added.

The latter reagent is labelled with an enzyme. After washing, the enzyme labelled antihuman Ig remains only if specific antiviral Ig was present in the serum sample. It is visualized by addition of a substrate for the enzyme and the color reaction quantified in a spectrophotometer. To verify this positive ELISA reaction, the serum sample is then added to e.g. Western blots which contain electrophoretically separated virus subcomponents. An immunoreaction on the Western blot will show whether Ig is reactive with the bands charac- "WO 89/0.3844 PCT/SE88/00570 3 teristic of viral subcomponents. Usually two different and characteristic bands are required to establish a definite diagnosis of HIV antibody.

The reliability of HIV antibody detection is dependent on the reagents of the ELISA plate. Lysates of infected cells may contain cellular contaminants, causing false positive serological reactions (Saag, M.S.

and Britz, Asymptomatic blood donor with a false positive HTLV-III western blot. The New Engl. J. Med.

314:118, 1985, Martin, Burger, Caouette, S. and Goldstein, Importance of confirmatory tests after strongly positive HTLV-III screening tests.

The New Engl. J. Med. 314:1577, 1986). Viral lysates usually have an over-representation of gag proteins and less envelope proteins which may cause false negative reactions. In addition to native viral lysates, recombinant-produced polypeptides have been produced (Ghrayeb, Kato. McKinney, Huang, J.J., Chanda, Ho. Sarngadharan, Chang, T.W. and Chang, Human T-cell lymphotropic virus type III (HTLV-III) core antigens: synthesis in Escherichia coli and immunoreactivity with human sera.

DNA 5:93-99, 1986; Chang, Kato, McKinney, Chanda, Barone, Wong-Staal, Gallo, R.C. and Chang, Detection of antibodies to human T-cell lymphotropic virus-III (HTLV-III) with an immunoassay employing a recombinant Escheria coli-derived viral antigenic peptide. Bio/Technology 3:905-909, 1985). The specificity and sensitivity of these assays are good, but not 100% (Chang, T.W. et al. ibid; Deinhardt, Eberle, J. and Gtrtler, Sensitivity and specificity of eight commercial and one recombinant anti-HIV ELISA tests. Lancet: 40, 1987).

The envelope protein gpl20 varies between isola- t tes (Wong-Staal, F. et al, ibid; Weiss, Clapham, Weber, Dalgleish, Lasky, L.A. and I Berman, Variable and conserved neutralization antigens of human immunodeficiency virus. Nature WO 89/03844 PCT/SE88/00570 4 324:572-575, 1986) and consequently peptides from them are not usually recommendable as ELISA antigens.

Antibodies to gp41 have instead been considered the most sensitive diagnostic criterion of HIV infection (Sarngadharan, et al, ibid; Schipbach, et al, ibid). gp41 contains regions that are well conserved between various isolates. Antibodies to p24 disappear with progessive disease (Lange, Paul, D.A., Huisman, de Wolf, van der Berg, Coutinho, Danner, van der Noordaa, J. and Goudsmit, Persistent HIV antigenaemia and decline of HIV core antibodies associated with transition to AIDS.

British. Med. J. 293:1459-1461, 1986) but are the ones which appear early. Regarding known antibody responses, a combination of measuring anti p24 and anti gp41 should therefore be the most reliable for all circumstances.

Performing Western blot is time consuming and expensive. To produce reliable antigens for use in ELISA seems prefercble. No single assay has so far obtained 100% specificity and sensitivity (Deinhardt, et al, ibid; Lange, et al, ibid).

Recently, Gnann, et al (Journal of Virology, Aug. 1987, p. 2639-2641) identified a 12-amino-acid peptide derived from gp41 of HIV, that was recognized by 100% of the sera from HIV-infected sera. The described peptide contained two cystein residues, and the authors stated that they provide evidence that disulfide bond formation plays a key role in the antigenic conformation of the epitope. However, no such evidence was presented. The authors assumed that the peptide had cyclized spontaneously, and they attempted to prove it by reducing the alleged disulfide bond, but they failed. Instead they synthesized another 12-amino-acid peptide in which they had substituted serine for one of the cysteines, an" when this was tested it reacted with only 2 of 22 HIV-positive test sera. The authors 'i WO 89/03844 PCT/SE88/00570 conclude that the presence of both cysteine residues is essential for the antigenic conformation of the epitope, possibly via formation of disulfide bonds.

They have not proved that they had a disulfide bond in the investigated peptide, what they have done is to show that cysteine is better than serine in the specified position of said peptide. The existence of two cysteines in a peptide is not equivalent to a cyclic structure. The cyclization demands a defined chemical step, namely oxidation, and the result can be method dependent. If spontaneous cyclization occures it is often incomplete and accompanied by polymerization.

In primary HIV disease, antibody tests may be negative during a period (Marlink, Allan, J.S., McLane, Essex, Anderson, K.C. and Groopman, Low sensitivity of ELISA testing in early HIV Infection. The New Engl. J. Med. 315:1549, 1986).

The development of a single rapid, sensitive and specific assay for detecting antibodies to HIV, also at an early stage of infection, is most desirable.

Description of the invention The present invention provides i.a. a rapid, sensitive and specific assay for detection of antibodies induced by a HIV.

In one aspect of the invention there is provided an artificial peptide having an amino acid sequence which corresponds to a naturally occurring amino acid sequence of a HIV comprising an epitope and which further has two cysteine residues located on each side of said epitope, and further having a sulphur bridge between said two cysteine residues which has been formed by a chemical oxidation step. It is believed that this stabilization of the peptide by a sulphur bridge between two cysteine residues is responsible I for the useful properties of the peptide, such as an enhancement of the antibody binding activity, as well as the chemical stability of the final product.

if WO 89/03844 PCT/SE88/00570 6 The artificial peptide includes at least two cysteine residues, which are cyclized to form a sulphur bridge. The two cysteine residues which are linked together may have one or more amino acid residues comprising an epitope between themselves, such as 2 to 10 residues, e.g. 5 residues. If the artificial peptide according to the invention includes more than two cysteine residues, still only one sulphur bridge between two cysteine residues is formed by a chemical oxidation step.

ST- ti i an artificial peptide, which is chosen from the group consisting of the peptide having the modified amino acid sequence H-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr- -Ala-Val-Pro-Trp-Asn-Ala-Ser-NH 2 and peptides having a shorter.sequence thereof including the modified sequence -Cys-Ser-Gly-Lys-Leu-Ile-Cys-; I I the peptide having the modified amino acid sequence H-Lys-Ala-Cys-Tyr-Ala-Pro-Pro-Ile-Ser-Gly-Gln-Ile-Arg- -Cys-Ser-Ser-Asn-NH 2

J

and peptides having a shorter sequence thereof including the modified sequence -Cys-Tyr-Ala-Pro-Pro-Ile-Ser-Gly-Gln-Ile-Arg-Cys-; 1 the peptide having the modified amino acid sequence H-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly- -Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-NH 2 and peptides having a shorter sequence thereof including the modified sequence WO 89/03844 PCT/SE88/00570 4 7 -Cys-Ser-Gly-Lys-Leu-Ile-Cys-; the peptide having the modified amino acid sequence H-Cys-Ser-Gly--Lys-Leu-Ile-Cys-Thr-Thr-Ala-NH2 52 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ser-Gly-Lys-Leu-Ile-Cys-; I I the peptide having the modified amino acid sequence H-Asn-Ser-Trp-Gly-Cys--Ala-Phe-Arg-Gln-Val-Cys-His-Thr- -Thr-Val-Pro-Trp-Val.-Asn-Asp-Ser-NH 2 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ala--Phe-Arg-Gln-Val -Cyls-; the peptide having the modified amino acid sequence H-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val -Cys-His-Thr-Thr- -Va 1-NH 2 and peptides having a shorter sequence thereof including the modified sequence.

-Cys-Ala-Phe-Arg-Glu-Val-Cys-; the peptide having the modified amino acid sequence H-Tyr--Leu-Gl n-Asp-Cl n-Al a-Arg-Leu-As n-Ser-Trp-Gl y- -Cys-Ala-Phe-Arg-Gln-Val1-Cys-NH 2 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ala-Phe-Arg--Gln-Val-Cys-, and the peptide having the modified amino acid sequence H-Tyr-Leu-Gln-Asp-Gln-Ala-Arg-Leu-Asn-Sef-Trp-Gly- -Cys-Al a-Phe-Arg-Gln-Val -Cys-His-Thr-Thr-Val -NH I CIU- -CI3~1-~-It WO 89/03844 PCT/SE88/00570 8 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ala-Phe-Arg-Gln-Val-Cys-.

At present, the most preferred artificial peptide according to the invention is H-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr- I I -Ala-Val-Pro-Trp-Asn-Ala-Ser-NH 2 In another aspect of the invention there is provided an artificial antigen which reacts with antibodies induced by a HIV, which antigen mainly consists of an artifical peptide having an amino acid sequence which corresponds to a naturally occurring amino acid sequence of a -HIV comprising an epitope and which further has two cysteine residues located on each side of said epitope, and further having a sulphur bridge between said two cysteine residues which has been formed by a chemical oxidation step.

In the specification and claims the expression "antigen mainly consists of an artificial peptide" indicates that the ability of the antigen to react with antibodies derives from the artificial peptide.

In a preferred embodiment of this aspect of the invention there is provided an artificial antigen which reacts-with antibodies induced by a HIV, which antigen mainly consists of a preferred artificial peptide according to the invention, exemplified above.

The artificial antigens according to the invention can be immobilized or coupled to a carrier, such as mineral carriers, e.g. aluminium hydroxide, calcium phosphate, etc., plastic surfaces, e.g. microplates, beads, etc., proteins, such as bovine serum albumin or an immunizing component, such as keyhole limpet haemocyanin.

Even though the artificial antigens according to j the invention sofar only have been used as diagnostic iri "WO 89/03844 PCT/SE88/00570 9 antigens to detect antibodies induced by a HIV, in a sample of body fluid, it is believed that they can be used as immunizing components in vaccine compositions against a HIV. When a peptide (Example I) of the invention (without carrier) was injected into apes, antibodies directed against the C-terminal portion of the peptide were elicited.

Thus, a further aspect of the invention provides a vaccine composition, which comprises as an immunizing component, at least one antigen selected from artificial antigens according to the invention, together with a nontoxic pharmaceutically acceptable carrier and/or diluent.

In yet another aspect of the invention there is provided a method of detecting antibodies induced by a HIV in a sample of body fluid, wherein said sample is subjected to an immunoassay and an artificial antigen according to the invention is used as a diagnostic antigen. The sample of body fluid can be urine, saliva, tear fluid, milk, serum or blood.

The immunoassay in which the artificial antigens according to the invention can be used as diagnostic antigens is any immunoassay of choice, such as enzymelinked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunodiffusion or immunoelectrophoreses

(IE).

In a preferred embodiment of this aspect of the invention ELISA is used as the immunoassay of choice.

In a further aspect of the invention there is provided a diagnostic immunoassay kit for the detection of antibodies induced by a HIV in a sample of body fluid, wherein an artificial antigen according to the invention is included as a diagnostic antigen. Depending on the immunoassay to be used, the kit will comprise other items, such as a positive standard serum sample, a nega- I tive standard serum sample, in the case of ELISA an enzyme conjugate and optionally a substrate for the o.

enzyme conjugate, and also optionally buffer solution(s) WO 89/03844 PCT/SE88/00570 and/or washing solution(s). Optionally all the reagents in the kit are contained in separate sealed test tubes or vialL marked with specific labels.

Synthesis of the artificial peptides of the invention The artificial peptides of the invention can be produced by any known method of producing a linear peptide sequence, such as cloning, degradation, coupling of one amino acid residue to the next one in liquid phase or coupling the amino acids to one another starting with a solid phase (resin) to which the C-terminal of the first amino acid is coupled, whereupon the C-terminal of the next amino acid is coupled to the N-terminal of the first amino acid, etc., finally releasing the built-up peptide from the solid phase (so-called Merrifield synthesis). Once the appropriate linear peptide sequence is ready, it is subjected to a chemical oxidation step in order to cyclize two cysteine residues thereof, whereby a sulphur bridge is formed between the cysteine residues.

General description of synthesis Tn the examples below, all peptides were synthesized on an Applied Biosystems 430A Peptide Synthesizer using a double coupling program with termination step after the second coupling. The resin used was of 4-methylbenzhydrylamine type with theoretical loading of 0.66 meq/g (Peptides International, Louisville, KY, USA).

The final product of the synthesis was dried in vacuo over night. After drying the peptide-resin was suspended in methanol (70 ml) and saturated with ammonia. The mixture was placed in pressurized steel vessel and left over night with magnetic stirring. The resin was then separated by filtration, washed several times with methanol and thoroughly dried in vacuo. The peptide was then cleaved from the resin by treatment with liquid hydrogen fluorid in the presence of anisole and ethyl-methyl-sulfide as scavangers (HF:anisole:EMS 10:05:05). After removal of hydrogen fluoride by evaporation the residue "WO 89/03844 PCT/SE88/60570 11 was suspended in ethyl acetate (100 ml) and filtered.

The solid was washed on filter with additional ethyl acetate (3x100 ml) and the cleaved peptide extracted with acetic acid (100 ml). The extract was promptly diluted to the volume of 2 dm 3 with 20% acetic acid in methanol and treated with 0.1 M solution of iodine in methanol until the faint brown colour persisted.

Then the Dowex 1x8 ion exchanger in acetate form was added (3 g) (Bio-Rad, Richmond, CA) and the mixture was filtered. The filtrate was evaporated and the residue freeze-dried from 1% acetic acid in water. The product was then purified by reversed phase liquid chromatography on a column filled with Vydac 20-25 p (Separation Group, CA) in a suitable system containing acetonitrile in 0.1% trifluoroacetic acid water solution. The samples collected from the column were analyzed by analytical HPLC Varian 5500 (Sunnyvale, CA) equipped with Bondapak C18 column (Millipore, Milford, Mass.). Fractions containing pure substance were pooled, the solvent was evaporated and the product freeze-dried from 1% acetic acid in water. The final HPLC analysis was performed on ready product and the structure of the peptide was confirmed by amino acid analysis and FAB-MS (Fast atom bombardment spectrometry).

All amino acids used during the synthesis were protected with tert-butoxycarbonyl group at a-aminofunction. The side chain protections used were as follows: Ser(BZL), Thr(BZL), Tyr(2-BrCbz), Lys(2-ClCbz), Orn(Cbz), Asp(BZL), Glu(BZL), Arg(Tos), Cys(Mob).

Amino acid derivatives were delivered by Bachem AG, Switzerland.

EXAMPLE I (The sequence corresponds to a sequence found in HIV-1- -gp-41) H-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-

SI

-Ala-Val-Pro-Trp-Asn-Ala-Ser-NH 2.

7 i i WO 89/03844 PCT/SE88/00570 12 The peptide was prepared according to the general description of synthesis. Purity 99.9% according to HPLC (36% acetonitrile in 0.1% trifluoroacetic acid, retention time 6.79 min at 2 ml/min; detection at 223 nm).

The structure was confirmed by amino acid analysis and by FAB-MS.

EXAMPLE II (The sequence corresponds to a sequence found in HIV-1modified by insertion of the cysteine residue located closer to the C-terminal.) H-Lys-Ala-Cys-Tyr-Ala-Pro-Pro-Ile-Ser-Gly-Gln-Ile-Arg- -Cys-Ser-Ser-Asn-NH 2 The peptide was prepared according to the general description of synthesis.

The structure was confirmed by amino acid analysis and by FAB-MS.

EXAMPLE III (The sequence corresponds to a sequence found in HIV-1- -gp-41) H-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-GlnGln-Gn-Leu-Leu-Gly- -Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-NH 2 I I The peptide was prepared according to the general description of synthesis. The structure was confirmed by amino acid analysis and by FAB-MS.

EXAMPLE IV (The sequence corresponds to a sequence found in HIV-1- -gp-41) H-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-Ala-NH 2 The peptide was prepared according to the general description of synthesis. The structure was confirmed by amino acid analysis and by FAB-MS.

EXAMPLE V (Reference) (The sequence corresponds to a sequence found in HIV-1- -gp-41) Ii P;1 -i WO 89/03844 PCT/SE88/00570 13 H-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-Ala-Val-Pro-Trp-Asn- -Ala-Ser-NH 2 The peptide was prepared according to the general description of synthesis, with the exception of the oxidation step with iodine.

Purity >98.5%(HPCL).

The structure was confirmed by amino acid analysis and by FAB-MS.

EXAMPLE VI (Reference) (The sequence corresponds to a sequence found in HIV-1- -gp-41) H-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr- -Ala-Val-Pro-Trp-Asn-Ala-Ser-NH 2 The peptide was prepared according to the general description of synthesis, with the exception of the oxidation step with iodine.

The structure was confirmed by amino acid analysis and by FAB-MS.

EXAMPLE VII (The sequence corresponds to a sequence found in HIV-2- -gp41) H-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-His-Thr-Thr- -Val-Pro-Trp-Val-Asn-Asp-Ser-NH 2 The peptide was prepared according to the general description of synthesis. The structure was confirmed by amino acid analysis and by FAB-MS.

EXAMPLE VIII (The sequence corresponds to a sequence found in HIV-2 transmembrane) H-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-His-Thr-Thr- -Val-NH 2 The peptide was prepared according to the general description of synthesis. The structure was confirmed by amino acid analysis and by FAB-MS.

WO 89/03844 PCT/SE88/00570 14 EXAMPLE IX (The sequence corresponds to a sequence found in HIV-2 transmembrane) H-Tyr-Leu-Gln-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly- -Cys-Ala-Phe-Arg-Gln-Val-Cys-NH2 1 I The peptide was prepared according to the general description of synthesis. The structure was confirmed by amino acid analysis and by FAB-MS.

EXAMPLE X (The sequence corresponds to a sequence found in HIV-2 transmembrane) H-Tyr-Leu-Gln-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly- -Cys-Ala-Phe-Arg-Gln-Val-Cys-His-Thr-Thr-Val-NH2 1I The peptide was prepared according to the general description of synthesis. The structure was confirmed by amino acid analysis and by FAB-MS.

Detection of antibodies induced by HIV in blood samples For the detection of antibodies induced by HIV in blood samples use was made of ELISA. (Engvall, E.

and Perlmann, Enzyme-linked immunosorbent assay (ELISA). III Quantification of specific antiboides by enzyme labelled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109.129-135,1972).

Materials: Plates: Nunc 96 F, Roskilde, Denmark Conjugate: HRPO rabbit anit-human Ig, Dakopatts, Glostrup, J Denmark Buffers: Carbonate buffer: 0.05 M sodium carbonate buffer, pH approx. Buffer A: Phosphate buffered saline (PBS) without Ca and Mg and with 0.5% bovine serum albumin (BSA) and 0.05% Tween 20% fetal calf serum (FCS). Can be stored at 4 0 C for 1-3 days.

V i y OPD buffer: 0.0347 M citric acid, 0.0667 M 1 I we I V )89/03844 PCT/SE88/00570 Na 2

HPO

4 pH 5.5. Can be stored 1 month. OPD tablets, Dakopatts, Glostrup, Denmark.

Washing solution: 0.9% NaC1 with 0.05% Tween Methods: Coating: A solution of the coating antigen (test peptide), 5 pg to 20 pg per ml, is made in carbonate buffer. 100 pl of the solution is added to each well of stripes or of 96-well microplates. The adsorption takes place over night, 18 hrs, at room temperature.

The coated plates can be stored with their contents in 4 0 C until use.

Serum assay: 1. Empty and wash the plate 4 times with washing solution.

2. Add 100 pl of serum diluted 1:100 in buffer A.

3. Add 100 pl of just buffer A to one well; this well serves as a blank.

4. Incubate 60 min at 37 0 C. (Dilute the conjugate during this period, see 6).

Wash 4 times with washing solution.

Empty.

6. Add 100 pl of a conjugate, e.g. HRPO rabbit anti-human IgG,IgA,IgM diluted 1:15 000 in buffer A (for instance pl added to 0.75 ml, dilute this 1:100).

7. Incubate at 37 0 C for 30 min. (Prepare substrate solution during this period, see 9).

8. Wash 4 times with washing solution.

Empty.

9. Add 100 pl OPD-substrate (6 ml OPD buffer. Use a clean vessel.

15-20 min are needed to dissolve the tablets. When dissolved, add 4 pl H202/ ml).

Li_ WO 89/03844 PCT/SE88/00570 16 Incubate 30 min at toom temperature.

Stop the reaction with 100 pl 2.5 M

H

2 SO4' 11. Read plate at 490 nm. Negative values should be below 0.250.

Using the above described materials and methods the following materials were tested as diagnostic antigens (coating antigens) for the detection of antibodies induced by HIV in serum samples from infected patients: A. A recombinant protein gp 160 (expressed in Baculo- -virus in insect cells). Received from Repligen Corp.

Cambridge, Mass., USA.

B. A recombinant polypeptide p 121 (with conserved region of gp 41, cloned in E. coli) received from Repligen Corp. Cambridge, Mass., USA.

C. A synthetic peptide described in Example V (Reference) of this specification (only one cysteine residue, no cyclization).

D. An artificial peptide according to the invention described in Example I of this specification.

E. An artificial peptide according to the invention described in Example II of this specification.

Serum samples from HIV infected human patients and from non-infected humans were tested.

Their serological status had been confirmed by Western blot.

The results were as follows: HIV antibody ELISA Test Sensitivity Specificity Number of material tested persons A. 97.7 100 B. 97.5 75 87 C. 93.9 90.7 370 t D. 100 99.9 2225 E. 100 100 Lj_ I t vC, WO 89/03844 PCT/SE88/00570 17 From the above results it is evident that the tested artificial peptides according to the invention and exhibits both excellent sensitivity and excellent specificity.

In the same way the following additional artificial peptides according to the invention were tested, namely F. An artificial peptide described in Example III of this specification, G. An artificial peptide described in Example IV of this specification, H. An artificial peptide described in Example VIII of this specification, I. An artificial peptide described in Example IX of this specification, and J. An artificial peptide described in Example X of this specification.

The results were as follows: HIV antibody ELISA Test Sensitivity Specificity Number of material tested persons F. 92.1 (100) G. 84.2 (100) 31 H. 100 98.7 180 I. 100 98.7 180 J. 100 90 21 Even though these peptides are not as good as the peptides D. and E. they show a good reactivity with antibodies induced by HIV.

Comparison of seroreactivity between a cyclic and a linear peptide Using the above described materials and methods (ELISA) the cyclic peptide of Example I and the corresponding li,.ar peptide of Example VI were tested at different serum dilutions.

The results were as follows: I WO 89/03844 PCT/SE8//00570 HIV antibody ELISA HIV seropositive, n=66 Amount of peptide Serum dilution Number of positive reactions with peptide cyclic linear Number of serum dilutions having highest absorbancy values cyclic linear pg pg 1/20 1/100 1/500 1/20 1/100 1/500 1/20 1/100 1/500 66 61 27 154 66 65 36 166 66 58 23 147 467 66 60 19 145 66 61 13 139 66 59 19 144 428 118 166 129 pg Total number of reactions 413 175 HIV seroneative, n=22 pg pg 20 pg 1/20 1/20 1/20 2 11 Total number of reactions 8 14 The results of the above comparison illustrate the advantage of the cyclic peptide as compared to the corresponding linear peptide. Both peptides give positive results with all sera at the lowest serum dilution.

However, the reactions decrease rapidly with the linear peptide, while the cyclic peptide is capable of keeping its configuration for positive antibody reactivity (see the above Table, columns 3 and This is a necessary characteristic to be able to detect with certainty sera having low antibody titres.

W, 3 89/03844 PCT/SE88/00570 19 A direct comparison of the absorbancy values in ELISA shows that the cyclic peptide is by far superior to the linear one (see the above Table, columns 5 and 6).

The relationship is the reverse when it comes to the antibody-negative sera. The specificity is 100% for both peptides, but the background values are higher for the linear peptide. This indicates advantages in both specific and non-specific reactions for the cyclic peptide.

Claims (10)

1. An artificial peptide having an amino acid se- quence which corresponds to a naturally occurring amino acid sequence of a HIV comprising an epitope and which further has two cysteine residues located on each side of said epitope, c h a r a c t e r i s e d in that the peptide has a sulphur bridge between said two cys- teine residues which has been formed by a chemical oxi- dation step and that it is chosen from the group con- sisting of the peptide having the modified amino acid sequence H-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr- -Ala-Val-Pro-Trp-Asn-Ala-Ser-NH 2 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ser-Gly-Lys-Leu-Ile-Cys-; the peptide having the modified amino acid sequence H-Lys-Ala-Cys-Tyr-Ala-Pro--Pro-Ile-Ser-Gly-G1ln-Ile-Arg- -Cys-Ser-Ser-Asn-NH 2 and peptides having a shorter sequence thereof including the modified sequence -Cys-Tyr-Ala-Pro-Pro-Ile-Ser-Gly-Gln-Ile-Arg-Cys-; the peptide having the modified amino acid sequence H-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gn-Leu-Leu-Gly- -Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-NH 2 and peptides having a shorter sequence thereof including the modified sequence T SUSTITUTE HEET ~N ~'SUB~TmJT~ Z3ET The wecis~P~n (i~X~PCT/ SE 8 8 /0 0 5 7 0 PCT it, ;Imc 2 1 19 -1 -Cys--Ser-Gly-Lys-Leu-Ile-C~ the peptide having the modified amino acid sequence H-C~s-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-Ala-NH 2 and peptides having a shorter sequence thereof including the modified sequence I-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val -Cys-His-Thr- -Thr-Val-Pro-Trp-Val -Asn--Asp-Ser-NH 2 and peptides having a shorter sequence thereof including the modified sequence -C s--Ala-Phe-Arg-Gln-Val-Cys-; the peptide having the modified amino acid sequence H-Ser-Trp-Gly-Cys-A1 a-Phe-Arg-Gln-Val -Cys-His-Thr-Thr- -Va 1-NH 2 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ala-Phe-Arg-Glu-Val-Cy's-; L. the peptide having the modified amino acid sequence H-Tyr-Leu-Gln-Asp-Gln-Ala-Arg--Leu-Asn-Ser-Trp-Gly- -Cys--Al a-Phe-Arg--Gln-Va1 -Cys-NH 1_ 2 and peptides having a shorter sequence thereof including the modified sequence -C s-Ala--Phe-Arg-Gln-Val-C's-, and the peptide having the modified amino acid sequence H-Tyr--Leu-Gln-Asp-Gln-Ala-Arg-Leu--Asn-ser-Trp-Gly- -Cys-Ala--Phe-Arg-Gln-Val-Cv s-His-Thr-Thr-Val-NH2 TSUBSTiTUTE SHEET t c The Swcdish Pccni C~fr PCT/ SE 8 8 0 0 5 7 0 PCT Inerno iOnm :uiian 1990 -01- 1 2 22 and peptides having a shorter sequence thereof including the modified sequence -C s-Ala-Phe-Arg-Gln-Val-Cys-.

2. An artificial antigen which reacts with anti- bodies induced by a HIV, c h a r a c t e r i s e d in that the antigen mainly consists of an artificial peptide having an amino acid sequence which corresponds to a naturally occurring amino acid sequence of a HIV comprising an epitope and which further has two cysteine residues located on each side of said epitope, and further having a sulphur bridge between said two cysteine resi- dues which has been formed by a chemical oxidation step, which antigen mainly consists of an artificial peptide chosen from the group consisting of the peptide having the modified amino acid sequence H-Gly-Ile-Trp-Gly-C s-Ser-Gly-Lys-Leu-Ile-C s-Thr-Thr- -Ala-Val-Pro-Trp-Asn-Ala-Ser-NH 2 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ser-Gly-Lys-Leu-Ile-Cys-; the peptide having the modified amino acid sequence H-Lys-Ala-C s-Tyr-Ala-Pro-Pro-Ile-Ser-Gly-Gln-Ile-Arg- -Cys-Ser-Ser-Asn-NH 2 -i and peptides having a shorter sequence thereof including the modified sequence -Cys-Tyr-Ala-Pro-Pro-Ile-Ser-Gly-Gln-Ile-Arg-Cys-; the peptide having the modified amino acid sequence H-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-rln-Leu-Leu-Gly- -Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-L s-NH 2 2 i i 4 II: SUBSTITUTE SHEET The d~ ~n c~kePCT/ SE 8 8 /00 570 '1990 -01- 1 2 23 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ser-Gly--Lys-Leu-Ile-Cys-; the peptide having the modified amino acid sequence H-C ys-Ser-Gl y-Lys-Leu-Il e-C s-Thr-Thr-Al a-NH 2 and pep-,ides having a shorter sequence thereof including the modified sequence -Cy L-Ser-Gly--Lys-Leu-Ile-C' T-; the peptide having the modified amino acid sequence H-Asn-Ser-Trp-Gly-Cys--Ala-Phe-Arg-Gln-Val-C y s-His-Thr- -Thr-Val-Pro-Trp-Val-Asn-Asp-Ser-NH2 and peptides having a shorter sequence thereof including the modified sequence -Cys-. la-Phe-Arg-Gln-Val-Cys-; the peptide having the modified amino acid sequence H-Ser-Trp-Gly-C 7s-Ala-Phe-Arg-Gl n-Val--Cys-His--Thr-Thr- -Val-NH 2 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ala--Phe-Arg-Glu-Val-Cys-; the peptide having the modified amino acid sequence H-Tyr-Leu-Gl n-Asp-Cl n-Al a-Arg-Leu-As n-Ser-Trp-Gl y- -Cys-Ala-Phe-Arg-G-ln-Val-Cys-NH2 L 2 and peptides having a shot sequence thereof including the modified sequence -Cys-Ala-Phe-Arg-Gln-Val-C and the peptide having the modified amino acid sequence I--Tyr-Leu-Gn-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gl y- -Cys--Ala-Phe--Arg-Gln-Val -Cys-His-Thr-Thr-Val-N., I 0< SUBSTITUTE SHEET i i I II "l I "1 III 24 and peptides having a shorter sequence thereof including the modified sequence -Cys-Ala-Phe-Arg-Gln-Val-Cys-.

3. An artificial antigen according to claim 2, characterised in that it has been immobilized or coupled to a carrier.

4. A method of detecting antibodies induced by a HIV in a sample of body fluid, wherein said sample is subjected to an immunoassay, characterised in that an artificial antigen according to any one of claims 2 and 3 is used as a diagnostic antigen.

A method according to claim 4, wherein said sample is subjected to enzyme-linked immunosorbent assay (ELISA) characterised in that an artificial antigen according to any one of claims 2 Jnd 3 is used as a diagnostic coating antigen.

6. A diagnostic immunoassay kit for the detection of antibodies induced by a HIV in a sample of body fluid, characterised in that it comprises as a diagnostic antigen an *2 artificial antigen according to any one of claims 2 and 3.

7. A diagnostic immunoassay kit according to claim 6, characterised in that it additionally comprises a positive standard serum sample, a negative standard serum sample, an enzyme conjugate, optionally a substrate for the enzyme conjugate, and optionally buffer solution(s) and/or washing solution(s).

8. An artificial peptide according to claim 1 substantially as hereinbefore described with reference to any one of the examples.

9. An artificial antigen according to claim 2 substantially as hereinbefore described with reference to Example XI. A method according to claim 4 substantially as hereinbefore described with reference to Example XI. DATED: 15 January 1992 PHILLIPS ORMONDE FITZPATRICK Patent Attorneys For: W ~iS FERRING AB xc T ;fj^ r p I r4 l-Y i i L119P 71 INTERNATIONAL SEARCH REPORT International Application No PCT/SE88/00570 1. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols apply, indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC 4 C 07 K 7/04, G 01 N 33/569, A 61 K 39/21 II. FIELDS SEARCHED Minimum Documentation Searched Classification System Classification Symbols IPC 4 A 61 K 39/21; C 07 K 7/00, /08,

/10; G 01 N 33/569 US Cl 436:510; 424:89; Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched SE, NO, DK, FI classes as above III. DOCUMENTS CONSIDERED TO BE RELEVANT' Category I Citation of Document, with indication, where appropriate, of the relevant passages 1 Relevant to Claim No. X Journal of Virology, Vol. 61, No. 8, Aug. 1987, p. 2639-2641, J. W. Gnann JR. et al.: "Fine Mapping of an Immunodominant Domain in the Transmembrane Glycoprotein of Human Immunodeficiency Virus", see the whole document X WO, Al. 8706005 (ORTHO PHARMACEUTICAL CORPORATION) 1-9 8 October 1987 see page 10, lines 15-30, page 27, lines 26-33 and page 41 EP, 0261224 JP, 63502904 A WO, Al, 8606414 (GENETIC SYSTEMS CORPORATION) 6 November 1986 see in particular page 13 and ulaims 29 and 32 Nature, vol. 329, 17 September 1987, p. 248-250, E. Norrby et al: "Discrimination between anti- bodies to HIV and to related retroviruses using site-directed serology", Special categories of cited documents: 'a later document published after the International filing date documen definin the general state the art whichs not or priority date and not in conflict with the application but document defining the general tate of the art which s not cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the International document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document whic' may throw doubts on priority claim(s) or involve an inventive step which is cited to eatablish the publication date of another document particular relevanc the claimed nvention citation or other special reason (as apecified) cannot be considered to Involve an Inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 1989 -02- 0 8

1989-02-03 International Searching Authority Signature of Auth rlted Offlc Swedish Patent Office r 'f Carl Olof Gus afsson Form PCT/ISA/210 (second sheet) (January 1985) r i r 'lilt International Application No. PCT/SE88/00570 IlI. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category I Citation of Document, with indication, where appropriate, of tte relevant passages Relevant to Claim No Y Y Y A A,P A see in particular fig. 1 and page 249, last paragraph page 250, first paragraph The Journal of Immunology, vol. 137, No. 9, 1 November 1986, p. 2937-2944, J. S. McDougal et al: "Binding of the human retrovirus HTLV-III/LAV/ARV/ /HIV to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and potential for idiotypic mimicry", see page 2941, "DICUSSION" J. gen. Virol. (1987), Vol. 68, p. 2239-224, G. Winkler et al: "Characterization of a Disulphide Bridge-stabilized Antigenic Domain of Tick-borne Encephalitis Virus Structural Glycoprotein", see in particular pages 2239 and 2244 National Library of Medicine, File Medline accession no. 86073671, GR Dreesman et al: "Synthetic hepatitis B surface antigen peptide vaccine", Adv. Exp. Med. Biol. 1985; 185:129-37 see the whole document EP, A2, 0 247 557 HOFFMANN-LA ROCHE CO.) 2 December 1987 see the whole document EP, A2, 0 214 709 (UNITED BIOMEDICAL INC.) 18 March 1987 see the whole document 1-9 1-9 1 1-9 2 Form PCTISAI210 (extra sheet) (January 195)