AU614971B2 - Hiv polypeptide, its preparation and use in detection of hiv antibodies - Google Patents

Description

1111i.... III~. ~06BL95PE61 zAXMAQjsjbdouWp j6Jnjitk, I 1.5 IAZAXMAflisqldONW1NffIHO.qQDSV 1d OC- 4- 1-25 I6 4- 9u7 1 AU-AI-.17958/88 WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau 0i INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCTf) (21) International Application Number: PCT/F188/00083 (74) Agent: RUSKA CO OY; Runeberginkatu 5, SF- 00,00 Helsinki (Fl).

(22) International Filing Date: 30 May 1988 (30.05.88) (81) Designated States: AT (European patent), AU BE (Eu- (31) Priority Application Number: 872409 ropean patent), CH (European patent), DE (European patent), DK, Fl, FR (European patent), GB (32) Priority Date: 29 May 1987 (29.05.87) (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (Euro- (33) Priority Country: FI pean patent), SU, US.

(71) Applicant (for all designated States except US): LAB- Published SYSTEMS OY [Fl/Fl]; PL 8, SF-00881 Helsinki W~rith international search report.

(72) Inventors;, and Inventorws/Applicants (for US only) :HUHTALA, Marja- JP 23FE '18 Liisa [Fl/Fl]; Kuoretie 5 F, SF-02170 Espoo NARVXNEN, Ale [Fl/Fl]; Kulmakatu 2 C 72, SF- 00170 Helsinki KORKOLAINEN, Mirja fF1/ FI]; Ruusankatu 4 A 7, SF-00250 Helsinki AUSTpiALIAN 2 DEC 19 88 [PATENT

OFFICE

(S4)Title: HIV POLYPE PTIDE, ITS PREPARATION AND USE IN DETECTION OF HIV ANTIBODIES (57) Abstract This invention relates to a new polypeptide the amino acid sequence of which corresponds to the sequence SGKLICTTAVPWNAS. This synthetic env-peptide can be used in immunochemnical detection of HIV-l virus.

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Declarant'sName. Labsystems Oy D eclaran t's N am 0 E B. RICE CO PATENT ATTORNEYS This form is suitable for any type of Patent Application. No ]egalisation required.

WO 88/09339 PCT/F188/00083 1 HIV polypeptide its preparation and use in detection of HIV antibodies.

Field of the invention The present invention relates to immunochemical detection of human immunodeficiency virus type 1 (HIV-1). Especially the invention relates to a new polypeptid, to be used in the detection.

Background of the invention The first case of acquired immunodeficiency syndrome (AIDS) was reported in 1981. Epidemiological studies have shown that the cause of this deadly disease is a human retrovirus, now known as human immunodeficiency virus type 1 (HIV-1) (Popovic M. et al., Science 1984, 224:497-500).

The various proteins of the retrovirus have been described and sequenced by several groups (Myers G. et al., Human Retroviruses and AIDS: A Compilation and analysis of nucleic acid and amino acid sequences, Theoretical and Biophysics Group, Los Alamos, 1987, p. 11-53). In most infected individuals antibodies,-which are reactive with the envelope proteins gpl20 and gp41, are produced.

So far detection of antibodies against HIV-1 has been mainly based on: a) whole virus immunoassays, b) gene technologically produced virus protein immunoassays, or c) immunoblotting by separating the viral proteins first in SDS-PAGE (either gene technologically produced viral proteins or by cocultivation the HIV-virus) and transferring the separated proteins to nitrocellulose sheet and detecting the separated proteins with HIV-1 antibody-positive sera using conventional immunoblotting techniques.

The known assays, however, have several drawbacks: In assays employing the whole virus large quantities of the virus must be cultivated as supply for test reagents.

In spite of safety measures there are dangers associated with the large scale cultivation. There also exists a risk that test reagents prepared with the inactivated virus can WO 881/09339 PCT/F188/00083 2 be contaminated with live virus. Thus, persons who handle the reagents may be subjected to the risk of HIV-1 infection.

Another drawback, which apply also to EIA employing proteins produced by gene technology, is the occurence of false positive test results. These are partly due to presence of cellular components in these preparations. This necessisates the performance of confirmatory tests.

The present enzyme immunoassays do not allow distinctions between HIV type 1 and type 2.

The present assays require expensive laboratory equipment or skillful technical assistance. Thus these assays are not very suitable to be used e.q. in undevelopment countries.

Synthetic peptides representing conserved "immunodominant" epitopes of the viral proteins provide an attractive alternative to virusderived antigens. A plurality of such peptides have been proposed e.g. in US 4629783 (corresponds to WO-86/06414).

General description of the invention The present invention relates to a env-peptide corresponding to the amino acid sequence SGKLICTTAVPWNAS Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-Ala-Val-Prc-Trp-Asn-Ala-Ser), to methods for the production of this peptide, and to methods using this peptide, especially to methods for the detection of antibodies to HIV-1 virus.

It has been found that the peptide is highly sensitive and specifically immunoreactive with HIV-1 infected sera.

The sensitivity and specificity of tests employing this peptide are on the same level as those of tests employing whole virus.

The new peptide can be prepared conviniently by conventional synthetic methods with no infectious risks.

Because of the shortness of the peptide, sequence recombinant technics need not be used.

Immunochemical studies have shown that the invented peptide is an envelope peptide developed from an "immuno- WO 88/09339 PCT/FI88/00083 S3 dominant" epitope region of the transmembrane protein gp41 in HIV-1.

It is obvious that the exact requence can be also modified to some extent in order to obtain immunochemically equivalent sequences.

The env-peptide does not react with antibodies to HIV-2.

Detailed description of the invention In accordance with the present invention a small synthetic env-peptide SGKLICTTAVPWNAS has been synthesized.

This peptide is derived from env-residues 599-613 of the transmembrane protein gp41 of HIV-1. This epitope is located in a hydrophobic domain of gp41 but, nevertheless, it has turned out to be highly immunogenic epitope in eliciting an immune response to HIV-1. Especially it seems to elicit the primary responses at the early stages of the infection and the antibodies formed persist through the different stages of the infection. The peptide is therefore most suitable for the detection of HIV-1 infections.

The detection can be carried out by using any suitable immunochemical technics e.g. enzyme immunoassay (EIA), radioimmunoassay (RIA) or fluoroimmunoassay (FIA).

Especially, the env-peptide can be coupled to a protein carrier e.g. albumin or transferrin. This peptide-conjugate can be immunochemically identified with rabbit antibody to the peptide or with HIV-1 antibody positive human sera. The sensitivity and specificity of this test are the same as those of whole virus enzyme-linked immunosorbent assays or whole virus immunoblottings. The specificity of the reaction I 30 can be confirmed by addition of unconjugated env-peptide to the sample incubation mixture. The test results can be made readable with a naked eye.

In stead of the described env-peptide its immunochemically equivalent homologues can be used. These related peptides can be easily detected by polyclonal or monoclonal antibodies or HIV-1 antibody positive sera directed against the env-peptide. Such a related peptide is e.g. SGKLICTAVP-

WNAS.

WO 88/09339 PCT/FI88/00083 4 The env-peptide can be synthetized by conventional methods starting from the individual amino acids. Such methods are described e.g. by Barany Merrifield in The Peptides, ed. by Gross E. and Meienkoffer Academic Press, New York, 1979, p. 1-284.

In the following the env-peptide, its preparation and its applicability and use in detecting HIV-1 is further exemplified.

In the accompanying drawings Fig. 1 shows the reactivity of the env-peptide EIA in a reference polulation and in patients with HIV-1 infection, Fig. 2 shows reactivity of the env-peptide EIA in different stages of HIV-1 infection, Figs. 3A and 3B show comparison of the sensitivity of the env-peptide EIA with whole virus EIA, and Fig. 4 shows immunoblot analysis of HIV-1 proteins and the env-peptide-BSA-conjugates with different antibody samples.

Synthesis of the env-peptide The env-peptide SGKLICTTAVPWNAS (later EP-peptide) and related peptide SGKLICTAVPWNAS (later RP-peptide) were chemically synthesized on a t-butyloxycarbonyl (BOC)methylbenzyl-cysteine-phenyl-acetamidomethyl (PAM) polystyrene/divinylbenzene resin (Applied Biosystems, Inc.).

Benzyl based side chain protections and the alfa-amino group t-butyloxycarbonyl (t-Boc) protections were used in amino acids (Applied Biosystems). The peptides were cleaved from resin and protecting groups were removed by conventional procedures. The synthetic peptides were purified with reversed phase high performance liquid chromatography. The amino acid sequences were confirmed by automated Edman degradation with a gas-phase sequencer (Model 470A, Applied Biosystems). The purity of the peptides was approximately 99 based on amino acid sequence data.

Coupling of the env-peptide to protein The chemically synthezised EP-peptide and RP-peptide were WO 88/09339 PCT/FI88/00083 coupled to a carrier protein e.g. albumin by using heterobifunctional cross linkers e.g. m-maleimidobenzoic acid Nhydroxysuccinimide ester (MBS) (Liu et al., Biochemistry, 1979, 18:690-697). Briefly, 1 mg of bovine serum albumin (BSA) in 10 mM sodium phosphate (pH 7.2) was incubated with 4 mg of MBS in dimethylformamide for 30 min at 25 Unreacted MBS and solvent were removed on a Sephadex PD-10 (TM) column equilibrated in 50 mM sodium phosphate buffer (pH A 100-molar excess of the peptide relative to the carrier protein was added to the reaction mixture and incubated for an additional 3 hours at 25 °C.

Uncoupled peptide was removed by repeated dialysis.

Use of the peptide in EIA The coupled EP-peptide and RP-peptide (about 1 .pg peptipg BSA in 100 were used for preparation of EIA.

Polystyrene microtitration plates were coated with the EPpeptide or the RP-peptide (5 ug/ml) coupled to BSA, in phosphate-buffered saline (PBS; 10 mM sodium phosphate, 0.1 M NaC1, pH The coated plates were incubated for two hours at 37 "C with 100 Pl of serum specimens (diluted 1:40 in PBS containing 1 BSA and 0.02 Tween 20 and the unbound antibodies were removed by three washes (200 1l) with 0.02 Tween 20. The plates were then reacted for one hour at 37 °C with swine anti-human IgG alkaline phosphatase conjugate (Labsystems Oy) followed by three washes as above and exposure to paranitrophenyl phosphate (Sigma) as substrate. The absorbance values were measured at 405 nm using a microtitration plate reader (Multiskan, Labsystems).

The synthetic peptides were tested for their ability to be specifically recognized by 15 randomly selected HIV-1 antibody-positive sera and by 10 HIV-1 antibody-negative sera using the above described enzyme immunoassay. The results are given in Table 1.

-7 l :ii 1 i II- WO 88/09339 PCT/F188/00083 Table 1. Anti-peptide EIA-titres positive serum samples of 15 HIV-1 antibody- Serum EP-peptide RP-peptide 220 200 200 220 310 600 115 190 520 230 220 370 2000 320 320 210 160 94 100 320 500 110 150 170 170 170 300 1700 300 300 The positivity and negativity of the selected serum samples were studied in two antibody EIA tests, Vironostika HTLV-III (Organon Teknika) and Labsystems whole virus lysate kits, and confirmed by immunoblottning using whole virus antigen strips prepared by Labsystems. The reactivity of the sera with each peptide was defined as the reciprocal of antibody dilution at half-saturating serum dilution. The EP-peptide and RP-peptide reacted with all antibody-positive sera. The positive reactions could be totally inhibited by addition of the EP-peptide to the incubation mixtures (100 U g/ml).

For reference, tests were also conducted with a similarily prepared and coupled unrelated peptide MDIPQTKQ- DLELPKLAG (later UP-peptide). Incubation with the UP-peptide did not affect the reactivity. The UP-peptide gave no reaction with the HIV-1 antibody-positive sera. None of the HIV-1 antibody-negative sera recognized any of the peptides.

e i WO 88/09339 pcT/F]88/00083 7 The specificity of the immunological reaction described above can be confirmed by addition of 100 to 200 Ug/ml of free EP-peptide to the studied serum sample (or blood sample or other body fluid) when incubating the solid support containing the EP-conjugate with the sample.

Clinical studies with the EIA A clinical evaluation of the EP-peptide EIA was carried out with 1000 HIV-1 antibody-negative blood donors and with 144 individuals at various stages of HIV-1 infection being seropositive in immunoblotting and whole virus EIA. Results are given in Figure 1. In order to avoid the effect of variations between the different EIA determinations, the results were calculated and expressed as enzyme immunoassay units (EIU) according to the equation indicated in Figure 1.

Sera of 1000 HIV-1 antibody-negative blood donors gave in the EP-peptide EIA a mean value of 0.9 EIU and a standard deviation SD) of 1.2 EIU. The'cut-off value (4.2 EIU) was adjusted to be two times the mean value plus two standard deviations. None of the blood donor sera gave EIA values above the cut-off value. Of the 144 patients with HIV-1 infection and positive in whole virus EIA and immunoblotting, 143 had values exceeding the cut-off level and 43 values exceeding the measuring range (150 EIU).

Antibody reactivities of different stages of HIV-1 infection were also compared. Results are given in Figure 2.

The EP-peptide EIA detected all 94 asymptomatic patients (ASX), 28 of the 29 patients with LAS, all 8 ARC patients and all 13 AIDS patients studied.

The rate of false-positivity of the EP-peptide EIA was studied in a panel of sera giving easily positive reaction.

The results are given in Table 2.

WO 88/09339 PCT/FI88/00083 8 Patient group Positive in Positive in EP-peptide whole virus EIA EIA* Kidney transplantation 0/20 (100 0/20 (100 Patients under hemodialysis 0/20 (100 0/20 (100 Miscellanous infections 2/50 (96 13/50 (74 Vironostika HTLV-III (Organon) kit was used.

The rate of false-positivity of the EP-peptide assay was 2.2 whereas in the whole virus EIA the rate was 14.4 The sensitivity of the test to detect early infection has been studied prospectively in two patients A and B known to be exposed to HIV-1 by sexual transmission but seronegative both in immunoblotting and in whole virus EIA on initial testing. Results are given in Figure 3A and 3B. The EP-peptide antibodies are detectable clearly, two- to threefold over cutt-off level (patient at the time seroconversion is observed by immunoblotting preciding reaction .in whole virus EIA. In immunoblotting, however, antibodies to gp41 do not appear at the time they are detectable in the peptide EIA.

Purification of human antibodies to the env-peptide The EP-peptide was coupled to AH-sepharose-4B (Pharmacia) via the carboxy terminal cysteine residue using m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS, Sigma) as coupling reagent. Sepharose (100 mg) was first swollen in mmol/l K 2

HPO

4 and 150 mmol/l NaCl buffer pH 7.4 (PBS).

Three per cent MBS solution (50 i in dimethyl formamide (Sigma) was added to 1 ml sepharose suspension which was then stirred gently at room temperature for 30 min. The sepharose suspension was washed twice with PBD and 1 mg peptide was added. After coupling the peptide-sepharose was mixed with 2 ml sepharose-4B (Pharmacia) and used for preparation of the affinity column. A pool (1.5 ml) of HIV-1 antibody-positive sera, all of which were highly immunoreactive in the peptide EIA, was passed through the _i -i i i WO 88/09339 PCT/F188/00083 9 affinity column. After the elution of unbound sera from the column with PBS, it was washed with 500 mmol/l NaCl in mmol/l phosphate buffer (pH Column-bound Ig molecules were eluted with 100 mmol/l Gly-HC1 buffer (pH 2.8) and the acid buffer was changed to PBS using Sephadex (TM, Pharmacia) column.

Immunoblotting using the env-peptide and affinity purified human antibodies to the env-peptide For immunoblotting the EP-peptide (3 g) conjugated to BSA via MBS was run using 10 polyacrylamide gels (SDS-PAGE) under reduction (1 B-mercaptoethanol). After separation, the peptide was transferred to nitrocellulose and tested for immunoreactivity with HIV-l-positive or HIV-l-negative sera, diluted 1:100, or with purified antipeptide Ig fraction diluted 1:5 (5 ug/ml); these dilutions gave equal absorbance values in the EP-peptide EIA. Also, EP-peptide-BSA conjugate was tested for its immunoreactivity with rabbit anti-BSA antiserum (Cappel Laboratories). Bound antibodies were detected by rabbit anti-human IgG-peroxidase conjugate (DAKO) and by swine anti-rabbit Ig-peroxidase conjugate (Liu et al., Biochemistry 1979, 18:690-697).

RIPA using the env-peptide For preparation of radio-immunoprecipitation assays (RIPA) antigen, log phase cultures of HIV-l-infected C10/MT2 cells were used. After starvation 2 x 106 cells were metabolically labelled with 35 S-cysteine (0.5 m Ci) in suspension at 37 OC for 22 h. The cell lysate was prepared by pelleting the cells and washing them in PBS and using a non-ionic detergent containing lysis buffer (0.5 ml) (10 mM Tris-HCl containing 140 mM MgCli, 1 mM dithiotreitol, 1 mM PMSF and 0.5 NP-40 pH For the precipitation assay, 1u of this antigen preparation was used per 15 p 1 of serum sample. The precipitates were collected with protein A sepharose (Pharmacia) and after extensive washing were boiled with SDS-beta-mercaptoethanol (1 and analyzed in SDS-PAGE (12 followed by autoradiography. The results of WO 88/09339 PCT/FI88/00083 the analysis are given in Fig. 4.

Immunofluorescence using the env-peptide HIV-l-infected H9 cells were attached to glass slides coated with poly-L-lysine (Sigma). The cells were fixed with paraformaldehyde (Riedel-de-Haen) in PBS. The fixed cells were permeabilized with 0.05 saponin (Merck) in PBS and treated with rabbit serum in PBS-saponin. Cells were stained with the unfractionated HIV-1 serum pool or with a HIV-l-negative human serum diluted 1:50 or with anti-peptide Ig fraction (23 P g/ml) for 30 min at 37 The cells were washed three times PBS-saponin and the bound antibodies were detected by incubating for 30 min at 37 °C with biotinylated sheep anti-human IgG with fluorescein-streptavidin conjugate (Amersham).

Example of a diagnostic test The following procedure is suitable in diagnostic tests for screening HIV-1 antibody positive sera.

The coupled EP-peptide (about 1 1 g EP-peptide/5 1 g BSA in 100 P 1) and also the UP-peptide are absorbed for instance as small spots to a solid support, for instance to nitrocellulose. The unabsorbed peptide-conjugate is washed off using for instance 50 mM sodium phosphate pH 7.2 (PBS).

Thereafter the solid support is incubatec, about 1 h, in a protein solution, for instance albumin (about 5 mg/100 ml) in 50 mM sodium phosphate, pH 7.2, containing 0.2 Triton- X-100 Thereafter the solid support containing the EPconjugate and the UP-peptide is incubated with the studied serum sample about 1 h. The unbound sample is washed off with PBS. The absorbed antibodies to the EP-peptide conjugate can then be detected by incubating the solid support with anti-human IgG conjugated with an enzyme marker, the presence of which can be determined by addition of the enzyme-substrate. As a marker can be used for instance horse-radish-peroxidase (HRP), which can be determined for instance by adding o-phenylene diamine.

(There are of course numerous other ways to mark the anti- WO 88/09339 PCT/FI88/00083 11 IgG.) The result is determined as positive by visual examination, when the spot containing the EP-conjugate develops a colored reaction while the spot containing the UP-conjugate remains uncolored. The result is determined as negative when the spot containing EP-conjugate do not develope a colored reaction or develops equal color as the spot containing UP-conjugate.

Claims (9)

1. A polypeptide and its immunochemically equivalent homologues characterized in that its amino acid sequence corresponds to the sequence SGKLICTTAVPWNAS.

2. A peptide according to claim 1 characterized in that its sequence is SGKLICTTAVPWNAS.

3. A peptide according to claim 1 characterized in that its sequence is SGKLICTAVPWNAS. S. 4. A method for the detection of antibodies to HIV-1 virus in a sample, in which method a polypeptide, or its immunochemically equivalent homologue, which is capable of forming an imimunochemical complex with antibodies to HIV-1, is incubated with the sample and the immunochemical complex formation is determined, characterized in that the amino acid sequence of the peptide corresponds to the sequence SGKLICTTAVPWNAS. A method according to claim 4 characterized in that the peptide is conjugated to a protein carrier.

6. A method according to claim 4 or 5 characterized in that the specificity of the immunocomplex f,,mation is confirmed by adding the peptide as uncoupled to the incubation mixture.

7. A method according to claim 5 or 6 characterized in that the protein is selected from the group consisting of albumin and transferrin.

8. A method according to claim 7 characterized in that the protein is albumin.

9. A method according to claim 8 characterized in that the albumin is bovine serum albumin. A method according to any of claims 6 to 9 characterized in that the amount of the uncoupled peptide is 100-200 ug/ml of the studied sample.

11. A method according to any of claims 4 to characterized in that the cysteine is bonded to the acid /zU' ~end of the polypeptide. 19 13

12. A method according to claim 11 characterized in that, for the detection, the peptide is attached to a solid surface. DATED this 12th day of April 1991 LABSYSTEMS OY Patent Attorneys for the .0 .eApplicant: RICE CO. 009 *too too0 4 T000