WO1991008227A1 - NEW HIV-1 p17 PEPTIDES, DIAGNOSTIC ANTIGENS AND IMMUNOASSAY METHOD - Google Patents

NEW HIV-1 p17 PEPTIDES, DIAGNOSTIC ANTIGENS AND IMMUNOASSAY METHOD Download PDF

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Publication number
WO1991008227A1
WO1991008227A1 PCT/SE1990/000756 SE9000756W WO9108227A1 WO 1991008227 A1 WO1991008227 A1 WO 1991008227A1 SE 9000756 W SE9000756 W SE 9000756W WO 9108227 A1 WO9108227 A1 WO 9108227A1
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ala
ser
gln
asn
gly
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PCT/SE1990/000756
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French (fr)
Inventor
Jonas Blomberg
Rüdiger Pipkorn
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Replico Medical Aktiebolag
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Priority claimed from SE8903985A external-priority patent/SE8903985L/en
Application filed by Replico Medical Aktiebolag filed Critical Replico Medical Aktiebolag
Publication of WO1991008227A1 publication Critical patent/WO1991008227A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to new peptides, to a diagnostic antigen having the ability of binding to anti ⁇ bodies which have a binding affinity for compounds con ⁇ taining an amino acid sequence corresponding to an epitope or a cluster of epitopes of HIV-1 pl7 and to a method of discriminating between a false and true diagnosed HIV-1 positive serum sample using an immunoassay.
  • a standard HIV-1 antibody screening test usually based on an enzyme immunoassay (EIA)
  • EIA enzyme immunoassay
  • the sample is considered positive, i.e. the subject is probably infected with HIV-1.
  • confirmatory tests are performed, because the serum sample might be positive even though the subject is not HIV-1 infected.
  • HIV-1 antibodies are present, i.e. the individual is HIV-1 infected (true HIV-1 seropositivity)
  • HIV antibodies are present, but the person is not HIV infected
  • antibodies against contaminating components for example cellular proteins, are present if the virus used for the assay has been produced by eukaryotic cells, or bacterial proteins if the protein used for the assay is a recombinant protein produced by bacteria
  • the test has technical deficiencies, which lead to a positive reaction, for example insufficient blocking, bac ⁇ terial growth in buffers etc.
  • HIV-1 EIB reactivity is characterised by a reactivity with pl7 protein with or without its precursor p55 in the gag part, but with no other HIV-1 protein. Most likely a false HIV-1 antibody test is obtained when tests based on whole virus are used, and in particular all tests in which pl7 pro ⁇ teins are involved.
  • the most common confirmatory tests are Western blot ⁇ ting, inhibition EIA, peptide EIA and radioimmuno precipi ⁇ tation. Usually 3 or 4 confirmatory tests are necessary to discriminate between false and true positive sera.
  • This antigen-antibody complex is detec ⁇ ted by an antihuman immunoglobulin G (IgG) antibody conju ⁇ gated to an enzyme, that in presence of a substrate will produce a colored band on the strips. If a full (normal) HIV-1 seropositivity according to a) above exists, anti ⁇ bodies against almost all of the virus proteins are pre ⁇ sent. This test is rather sensitive and it permits sepa- ration of the immune response to different proteins origi ⁇ nating from different parts of a virus organism.
  • IgG immunoglobulin G
  • Inhibition EIA is a technique that discriminates between false and true reactivity by compe ⁇ tition of labelled true positive antibodies and the serum sample from the patient for binding to HIV-1 bound to a solid phase. If the patient serum sample inhibits the labelled antibodies it is clear that they bind to the same sites on the HIV-1 proteins and that the antibodies in the patient serum sample bind as strong as the probed anti ⁇ bodies. If the reaction is false, none of those conditions are funfilled.
  • Peptide EIA is a technique that further separates the immune response to single epitopes. By this technique it is possible to discriminate between true and false reac ⁇ tions because the reasons of the immuno response are dif- ferent. It is possible to use peptide EIA to indicate that antibodies raised against env proteins are present. Peptide EIA is a more sensitive technique than Western blot in indicating env antibodies. However, at present the use of this technique is somewhat restricted because the test may fail if there are slight variations in peptide sequence of the virus like in certain patients from Africa.
  • RIPA radioimmunoprecipitation test
  • the anti ⁇ bodies from the patient serum sample are bound to radio- actively labelled HIV-1 proteins.
  • the antigen-antibody complexes are immobilised on protein A beads, and are then solubilised and subjected to electrophoresis in polyacryl- amide.
  • This test requires high affinity of the antibodies and it is rather sensitive. It can discriminate fairly well between true and false reactions. It is particularly advantegeous to study env reactivity by RIPA.
  • the disad ⁇ vantages with this test is that it is rather expensive, time consuming and laborious. Further, there is an aspect of hazard in working with radioactive material.
  • the method gives an additional safety margin, because it positively identifies the false positive reactions. To sum up, it is possible to select additional not HIV-1 infected individuals from a group of individuals which have been diagnosed as possibly HIV-1 positive, compared to any hitherto known method.
  • the present invention relates to a pep ⁇ tide of the formula a) H-X-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Asn-Gln-Val-Ser- -Gln-Asn-Tyr-X-Z, b) H-X-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Ser-Gln-Val-Ser- -Gln-Asn-Tyr-X-Z, c) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Asn-Ser-Gln-Val-Ser- -Gln-Asn-Tyr-X-Z, d) H-X-Ala-Ala-Ala-Ala-Ala-Gly-Thr-Gly-Asn-Ser-Ser-Gln-Val-Ser- -Gl
  • the present invention relates to a diagnostic antigen having the ability of binding to anti ⁇ bodies which have a binding affinity for compounds con- taining an amino acid sequence corresponding to an epitope or clusters of epitopes of HIV-1 pl7.
  • the present invention relates to a method of discriminating between a false and true diagnosed HIV-1 positive serum sample from a subject, characterised in that at least one diagnostic antigen having the ability of binding to antibodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or clusters of epi- topes of HIV-1 pl7, optionally coupled to a carrier, is • added to the serum sample, and that possible antigen/an- tibody complexes formed are detected using an immunoassay, whereby the discrimination is established based upon that said serum sample is false HIV-1 positive if said .comple- xes are detected, and true HIV-1 positive if said com ⁇ plexes are not detected.
  • said diagnostic antigen is a diagnostic antigen according to the invention.
  • HIV-1 HIV-1
  • HTLV-1 and HTLV-2 do not induce AIDS, but AIDS-related diseases, for example T-cell leukemia.
  • the present invention is therefore concerned with HIV-1 and amino acid sequences unique for the HIV-1 proteins are contemplated.
  • the interior parts, for example the nucleocapsid or the core containing the RNA, of the HIV-1 viruses are pro ⁇ tected by an envelope.
  • the interior parts, called gag (group-specific antigen) and the envelope, called env are built up of different proteins. These proteins, i.e. the precursors, are predominant in the beginning of the virus life cycle, while others are predominant in later phases.
  • Table 1 lists major genes and gene products of HIV-1.
  • p55 is a precursor of p24 and/or pl7.
  • gpl60 is a precursor of gpl20 and gp41.
  • an enzyme cleaves the p55 molecule from the pi7 molecule.
  • the whole pl7 protein constitutes a part of the original p55 protein.
  • p55 is also included in this defi- nition.
  • an epitope or clusters of epitopes of HIV-1 pl7 used, is intended to include the same epi ⁇ tope or epitopes of HIV-1 p55 "as well.
  • the peptides according to the present invention have ability to react with antibodies raised against an amino acid sequence corresponding to or closely related to an epitope or clusters of epitopes in the position 118-132, i.e. the C-terminal position, of the HIV-1 pl7 protein in the interior of the HIV-1 virus.
  • the peptides according to the invention basically comprises said 15 C-terminal amino acids of the pl7 protein, i.e.- Ala-Ala-Ala-Asp-Thr-Gly- -His-Ser-Asn-Gln-Val-Ser-Gln-Asn-Tyr-, optionally with one or a few amino acids replaced by other amino acids, especially in the corresponding position 123 to 127.
  • This C-terminal portion of HIV-1 pl7 is dissimilar to the amino acid sequences of all other retroviruses. Further, as indicated above, the peptides according to the present invention optionally have 1-5 amino acid residues inserted between the corresponding pl7 positions 121 and 122. All these new peptides and/or parts thereof react in a uniquely specific way with the above mentioned non HIV-1 antibodies.
  • X represents a chemical bond, the amino acid sequence -Pro- -Ile-Val-Gln-Asn-Ile-Gln-Gly- or a sequence of at least 4, and preferably 8, particular amino acid residues, which each are chosen from the group consisting of -Thir- and
  • X is an amino acid sequence, it can be located either in the C- or N-terminus of the peptides but not in both ends at the same time. If it is for example located in the C-terminus, X in the N-terminus corresponds to a chemical bond and vice versa. However, X can be a bond at both ends of the peptide according to the present inven ⁇ tion. Said amino acid sequence acts as a coupling facili ⁇ tating spacer, which permits proper bindning to the car ⁇ rier to which the peptides according to the present inven- tion will be bound during the discrimination method.
  • the sequence -Pro-Ile-Val-Gln-Asn-Ile-Gln-Gly- corresponds to the sequence in position 133-140 in the HIV-1 p24 protein and is particularly suitable as a coupling sequence.
  • the sequence X should not be an amino acid sequence which adversely affects the result of the diagnosis method. Accordingly, it should not have a too high charge or be too hydrophobic and it should not disturb the conformation of the peptides.
  • the amino acids threonine and serine also fulfill these requirements particularily well, and any one of the amino acids in said sequence X can be threonine or serine, with the proviso that X is not the p24 derived sequence containing the 8 amino acids above.
  • the number of amino acids in this spacer sequence should be at least 4, but in a preferred embodiment according to the present invention 8 amino acids are used.
  • polypeptides according to the present invention can be bound via the amino acid sequence X to a carrier by physical/chemical interaction, as for example covalent binding, ionic binding, hydrogen binding or hydrophobic binding.
  • covalent binding are esther, ether and disulfide binding.
  • carrier used herein should be inter ⁇ preted broadly, and it can be any material which is compa ⁇ tible with and not negatively affects the method according to the present invention, for example resins, microplates, plastic surfaces, gels, matrixes etc.
  • epipe used herein means antigenic or immunogenic determinant and relates to a specific part of a structure of an antigen inducing an immuno response, and the produced antibodies are directed against this part.
  • the peptides according to the pre ⁇ sent invention have ability to bind to antibodies raised against amino acid sequences corresponding to an epitope or clusters of epitopes of HIV-1 pl7.
  • these anti ⁇ bodies are not raised against HIV-1 proteins.
  • pl7 antibody reactions are due either to coincidental or selection controlled antigenic mimicry between the C-terminus part of the pi7 amino acid sequence and an unknown non-HIV-epitope.
  • An autoimmune origin appears to be possible. From a general retroviral perspective, the C-ter ⁇ minal portion of the membrane anchoring protein of gag and its surrounding sequences, is the most variable portion of gag. Its functions are unknown.
  • HIV-1 has neither. This emphasizes the uniqueness of the C-terminus of HIV-1. Further, the existence of a low frequency of persons unlikely to be HIV-1 infected having antibodies to this sequence which is dissimilar to other retrovirus sequences indicates that the C- erminus of pi7 contains at least one epitope which mimics a non-retroviral epitope.
  • the C-terminus of HIV-1 pl7 has point mutated into a similarity with at least two non-HIV-1 antigens (thymosin alpha 1 and an unknown one), or by a recombinatorial event involving a cellular sequence containing both could have been transduced into an HIV-1 predecessor after the diver- gence of HIV-1 and HIV-2/SIVmac-smm.
  • the pl7 antibodies would be auto-antibodies.
  • Figure 1 sho s the distribution of absorbance diffe ⁇ rences between the samples and a defined limit in a whole virus based HIV-1 antibody screening test (Organon Viro- nostica) of ten pl7 reactive sera.
  • FIG 2 shows the distribution of absorbance diffe ⁇ rences in EIA with synthetic peptides derived from HIV-1 gag of a) ten pl7 reactive sera, b) 19 confirmed HIV-1 antibody positive sera and c) ten HIV seronegative blood donors.
  • Figure 3 shows electrophoretic immunoblotting (EIB) with HIV-1 antigen of three (A-C) pi7 reactive human sera ("false HIV pl7 pos sera”) after absorption over night with resin-bound HIV-1 gag 118-140 (P) or with resin without any peptide (R).
  • EIB electrophoretic immunoblotting
  • Figure 4 shows the distribution of anti-pl7 reacti ⁇ vity measured (in arbitrary units) by reflectance densito- metry of electrophoretic immunoblot strips with HIV-1 antigen after absorption over night with resin-bound HTLV-I gag 111-130 or with resin-bound HIV-1 gag 118-140.
  • EXPERIMENTS Identification of HIV-1 p!7 reactive sera
  • a standard whole virus HIV-1 antibody screening test preferably the Organon Vironostica-anti-HIV (Organon Technica) is performed. Serum samples are allowed to react with whole HIV-1 virus bound to a solid phase. Possible binding of antibodies to the different parts of the virus is detected by measuring the absorbance of the sample. Samples showing an absorbance above a predeter- mined cut off value are deemed to be positive.
  • Fig. 1 The distribution of the differences between the absorbance of the sample and the cut-off value obtained with the Organon whole virus HIV-1 antibody assay are depicted in Fig. 1. The average absorbance difference was 0.118.
  • a comparison test performed with commercial HIV-1 antibody tests no serum reacted in the Abbott HIV-1 EIA, which employs a recombinant antigen including both a portion of gag and env. None of the 10 sera reacted in the
  • EIB strips were measured with a Bio-Rad 620 Video Densitometer (Richmond, California, USA) (see Blomberg J, Klasse PJ: Quantification of immunoglobulin G on electro ⁇ phoretic immunoblot strips as a tool for human immuno ⁇ deficiency virus serodiagnosis. J Clin Microbiol 1988, 26:111-115).
  • Peptide EIA s were performed approximately as descri- bed (see Blomberg J, Nilsson I, Andersson M: Viral anti ⁇ body screening system that uses a standardized single dilution immunoglobulin G enzyme immunoassay with multiple antigens. J Clin Microbiol 1983, 17:1081-1091 and Klasse PJ, Pipkorn R, Blomberg J: Presence of antibodies to a putatively immunosuppressive part of human immunodeficien ⁇ cy virus (HIV) envelope glycoprotein gp41 is strongly associated with health among HIV-positive subjects. Proc Natl Acad Sci USA 1988, 85:5225-5229).
  • HIV human immunodeficien ⁇ cy virus
  • Peptide D is a peptide according to the present invention.
  • Peptide E is entirely contained within peptide D.
  • a previously recognized similarity between thymosin alpha-1 and HIV-1 pl7 is present in the sequence of peptides B and C (see Naylor PH, Naylor CW, Badamchian M, et al.: Human immuno ⁇ deficiency virus contains an epitope immunoreactive with thymosin alphal and the 30-amino acid synthetic pi7 group specific antigen HGP-30. Proc Natl Acad Sci USA 1987,
  • the carboxy terminus of pl7 is at residue 132, a tyrosine.
  • the peptides were synthesized using Fmoc chemistry on an Applied Biosystems 430A solid phase automatic peptide synthesizer. They were subsequently purified by reverse- phase HPLC on a C18 column. Their purity was 95% according to an analytical HPLC procedure.
  • the peptides -bound to a resin carrier were synthesized on a PAM resin. After de ⁇ blocking with concentrated trifluoroacetic acid for 1 h the unprotected peptides remained covalently coupled to the resin through their carboxy terminus.
  • the amino acid sequence was determined by the degradation method according to Edman.
  • the peptides can be used as a diagnostic antigen either solved in the serum sample or bound to a carrier.
  • Method 1 The diagnostic antigen according to the present invention, i.e. the peptide D, can be used in a peptide EIA, wherein it is bound to a micro titer plate serving as a carrier.
  • a micro titer plate serving as a carrier.
  • at least two peptides corresponding to one or more epitopes of HIV-1 pl7 is used, of which one is a peptide according to the present invention, for example peptide D and peptide B and/or C above.
  • Each serum sample is added to the wells and possible binding of antibodies present in the serum to the antigen occurs.
  • the pattern obtained after the measurement of the absorbance shows accurately or very likely if the serum is true or false positive.
  • a combination of reactivity for the different pepti ⁇ des used is considered for determination of false or true positive serum samples. This evaluation can either be done manually or with a micro processor. If only peptide D reacts, the serum is doubtless false positive, i.e. the patient is not HIV-1 infected. If only peptide C reacts, the serum is doubtless true positive. Further, the serum sample is true positive when peptide B and C reacts weakly, but likely false positive when peptide B and D reacts weakly. However, certain rare combinations of reactivity are impossible to interpret in an accurate way, but most of the pl7 reactive sera are possible to determine in a much more reliable way than hitherto known using the method according to the present invention.
  • Fig. 2 shows an example of reactivity for pl7 posi- tive sera using the five ab ⁇ vementioned synthetic pl7 derived peptides A-E compared to confirmed HIV-1 antibody positive sera and negative sera.
  • Fig. 2a The distribution of IgG reactivities of pl7 reactive sera on the five pl7 derived peptides is shown in Fig. 2a. Only two peptides, A and D, yielded absorbance differences exceeding 0.3. This distribution was clearly different from the reactivity patterns of the confirmed HIV-1 anti ⁇ body positive sera (Fig. 2b) and the sera from HlV-nega- tive Swedish blood donors (Fig. 2c). The confirmed posi- tive sera reacted primarily with peptides B and C, and to a lesser extent with peptides A, D and E. The blood donor sera reacted only with peptide A. Method 2
  • the serum sample is absorbed in at least two different tubes containing one type of peptide in each.
  • One of the peptides is the diagnostic antigen according to the present invention, i.e. peptide D, while the other or the others is (are) reference peptide(s). All of the peptides used are bound to a solid phase, for example resin beads.
  • peptide F i.e. the HTLV-1 peptide.
  • Fig. 4 This figure shows, on the left side, where the lines start, the initial intensity of pl7 band in EIB for both HTLV-1 and HIV-1 before absorption, and the right end points of the lines represent the band intensity for peptide D from HIV-1 after absorption.
  • the HTLV-1 band intensity is unchanged after absorption with HTLV-I peptide.
  • the lines represent the degree of the intensity reduction of the pl7 band before and after absorption, and shows that pl7 antibodies are absorbed away from the serum sample.
  • the peptide according to the present invention is used as such, i.e. not bound to any carrier.
  • Antibodies reactive with pl7 in EIB have been found in a serum sample.
  • the peptide D is added to the serum sample or a dilution of the serum sample.
  • a referencs peptide with a sequence unrelated to that of peptide D is added to another portion of the serum sample or a dilution of the serum sample. If the binding of antibodies to pl7 in.EIB is inhibited in the presence of peptide D, but not in the presence of the reference peptide, the serum sample is false-positive.
  • immunoassay methods can be used according to the present invention, for example radioimmuno assay (RIA), fluorescence immuno assay (FIA) and an immuno assay involving metal labelling.
  • RIA radioimmuno assay
  • FFA fluorescence immuno assay
  • metal labelling an immuno assay involving metal labelling.

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Abstract

Peptides of the formulae (a, b, c, d, e, f, g, h, i or j), or antigenic parts thereof, wherein one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating amino acid sequence and Z represents -NH2 or -OH, a diagnostic antigen selected from said peptides and a method of discriminating between a false and true diagnosed HIV-1 positive serum sample, are described.

Description

NEW HIV-1 P17 PEPTIDES, DIAGNOSTIC ANTIGENS AND IMMUNOASSAY METHOD
The present invention relates to new peptides, to a diagnostic antigen having the ability of binding to anti¬ bodies which have a binding affinity for compounds con¬ taining an amino acid sequence corresponding to an epitope or a cluster of epitopes of HIV-1 pl7 and to a method of discriminating between a false and true diagnosed HIV-1 positive serum sample using an immunoassay. Background art
For diagnosis of HIV-1 infection of individuals a standard HIV-1 antibody screening test, usually based on an enzyme immunoassay (EIA), is initially performed with a serum sample from the subject. If a certain cut off value is exceeded in the test result, the sample is considered positive, i.e. the subject is probably infected with HIV-1. For confirmation of this preliminary diagnosis, confirmatory tests are performed, because the serum sample might be positive even though the subject is not HIV-1 infected.
The reasons to a positive result in the tests could be the following; a) HIV-1 antibodies are present, i.e. the individual is HIV-1 infected (true HIV-1 seropositivity) b) HIV antibodies are present, but the person is not HIV infected c) antibodies against contaminating components, for example cellular proteins, are present if the virus used for the assay has been produced by eukaryotic cells, or bacterial proteins if the protein used for the assay is a recombinant protein produced by bacteria d) the test has technical deficiencies, which lead to a positive reaction, for example insufficient blocking, bac¬ terial growth in buffers etc. It has been observed that a small portion of sera from humans unlikely to be HIV-1 infected contains anti¬ bodies against one or a few HIV-1 proteins, mostly derived from the interior gag part of the virus. Such reactivities can give rise to "indeterminate" HIV-1 electroforetic im- munoblot (EIB) reactions. They are a constant cause of concern in laboratories responsible for confirmation of HIV-1 antibody screening results. The elucidation of the biological reason for these reactions and the development of tests facilitating the discrimination of "true" from "false" HIV-1 antibodies then are important objects for research.
One of the most common types of false-positive HIV-1 EIB reactivity is characterised by a reactivity with pl7 protein with or without its precursor p55 in the gag part, but with no other HIV-1 protein. Most likely a false HIV-1 antibody test is obtained when tests based on whole virus are used, and in particular all tests in which pl7 pro¬ teins are involved. The most common confirmatory tests are Western blot¬ ting, inhibition EIA, peptide EIA and radioimmuno precipi¬ tation. Usually 3 or 4 confirmatory tests are necessary to discriminate between false and true positive sera.
In Western blotting purified whole HIV-1 virus is fractionated by molecular weight using electroforesis on a polyacrylamide gel. The separated HIV-1 proteins, which function as viral antigens, are then transferred from the gel via electroforetic blotting to a nitrocellulose mem¬ brane. Alternatively, recombinant proteins or synthetic peptides corresponding to HIV-1 proteins can be used as antigens. The blot is cut into strips which are then reac¬ ted with test and control serum or plasma samples. During incubation, if anti-HIV-1 antibodies are present in the sample, they will bind to the viral antigens on the nitro- cellulose strips. This antigen-antibody complex is detec¬ ted by an antihuman immunoglobulin G (IgG) antibody conju¬ gated to an enzyme, that in presence of a substrate will produce a colored band on the strips. If a full (normal) HIV-1 seropositivity according to a) above exists, anti¬ bodies against almost all of the virus proteins are pre¬ sent. This test is rather sensitive and it permits sepa- ration of the immune response to different proteins origi¬ nating from different parts of a virus organism.
Reactions against both gag (gag = p24, pl7, p55) proteins from the interior parts of the virus and env (env = gp41, gpl60, gpl20) proteins from the envelope of the virus are required for this test. The risk for such a pattern to arise randomly is very small. If a patient has antibodies against a few single virus proteins, the un¬ reliability is more pronounced. Then it is necessary to distinguish an incomplete but true immune response against HIV-1 from an antibody reaction against another protein with ability to induce the same antibodies as a HIV-1 protein (false positivity). Reactions with pl7> p24 and p55 are most common of the gag reactions, wherein pl7 reactions are most common and p55 are least common. The appearance and the intensity of the different bands in EIB are dependent on if the infection is recent. Consequently, it is impossible to be absolutely sure if a single gag band is true or false using only a Western blot. This is a considerable disadvantage of the method. Inhibition EIA (competition EIA) is a technique that discriminates between false and true reactivity by compe¬ tition of labelled true positive antibodies and the serum sample from the patient for binding to HIV-1 bound to a solid phase. If the patient serum sample inhibits the labelled antibodies it is clear that they bind to the same sites on the HIV-1 proteins and that the antibodies in the patient serum sample bind as strong as the probed anti¬ bodies. If the reaction is false, none of those conditions are funfilled. However, as this test is a negative test, i.e. it is based on non-reactivity of possibly false posi¬ tive antibodies and not on direct indication, it is asso¬ ciated with a certain unreliability. Peptide EIA is a technique that further separates the immune response to single epitopes. By this technique it is possible to discriminate between true and false reac¬ tions because the reasons of the immuno response are dif- ferent. It is possible to use peptide EIA to indicate that antibodies raised against env proteins are present. Peptide EIA is a more sensitive technique than Western blot in indicating env antibodies. However, at present the use of this technique is somewhat restricted because the test may fail if there are slight variations in peptide sequence of the virus like in certain patients from Africa.
In the radioimmunoprecipitation test, RIPA, the anti¬ bodies from the patient serum sample are bound to radio- actively labelled HIV-1 proteins. The antigen-antibody complexes are immobilised on protein A beads, and are then solubilised and subjected to electrophoresis in polyacryl- amide. This test requires high affinity of the antibodies and it is rather sensitive. It can discriminate fairly well between true and false reactions. It is particularly advantegeous to study env reactivity by RIPA. The disad¬ vantages with this test is that it is rather expensive, time consuming and laborious. Further, there is an aspect of hazard in working with radioactive material. Consequently there is a great demand for a method of reliable, fast, simple and cheap discrimination between false and true HIV-1 antibody reactions, especially for developing countries, where false seropositivity is a large problem. This demand is satisfied by the present invention. Compared to the known technique the method according to the present invention is simpler for discriminating bet¬ ween true and false HIV-1 gag reactions. Furthermore, it is much safer and more reliable than known techniques. It is also fast, and the test is performed in a 3-4 h, com¬ pared to known techniques, in which up to 24 h are neces¬ sary. Finally, it should be at least ten times cheaper per test than a Western blot and much cheaper than the other conventional confirmatory tests. In combination with the conventional methods, the method gives an additional safety margin, because it positively identifies the false positive reactions. To sum up, it is possible to select additional not HIV-1 infected individuals from a group of individuals which have been diagnosed as possibly HIV-1 positive, compared to any hitherto known method. Description of the invention In one aspect the present invention relates to a pep¬ tide of the formula a) H-X-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Asn-Gln-Val-Ser- -Gln-Asn-Tyr-X-Z, b) H-X-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Ser-Gln-Val-Ser- -Gln-Asn-Tyr-X-Z, c) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Asn-Ser-Gln-Val-Ser- -Gln-Asn-Tyr-X-Z, d) H-X-Ala-Ala-Ala-Ala-Ala-Gly-Thr-Gly-Asn-Ser-Ser-Gln- -Val-Ser-Gln-Asn-Tyr-X-Z, e) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Arg-Gly-Asx-Ser-Ser- -Gln-Val-Ser-Gln-Asn-Tyr-X-Z, f) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Ser-Ser-Lys-Val-Ser- -Gln-Asn-Tyr-X-Z, g) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Ser-Ser-Gln-Val-Ser- -Gln-Asn-Tyr-X-Z, h) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Gly-Ser-Gln-Val-Ser-
-Gln-Asn-Tyr-X-Z, i) H-X-Ala-Ala-Ala-Ala-Gln-Gln-Ala-Ala-Ala-Thr-Lys-Asn-
-Ser-Ser-Ser-Val-Ser-Gln-Asn-Tyr-X-Z, or j ) H-X-Gln-Ala-Ala-Ala-Asp-Ala-Gly-Asn-Asn-Ser-Gln-Val-
-Ser-Gln-Asn-Tyr-X-Z, in which X represents a chemical bond or a coupling- facilitating amino acid sequence, and Z represents -NH_ or -OH. In another aspect the present invention relates to a diagnostic antigen having the ability of binding to anti¬ bodies which have a binding affinity for compounds con- taining an amino acid sequence corresponding to an epitope or clusters of epitopes of HIV-1 pl7.
In yet another aspect the present invention relates to a method of discriminating between a false and true diagnosed HIV-1 positive serum sample from a subject, characterised in that at least one diagnostic antigen having the ability of binding to antibodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or clusters of epi- topes of HIV-1 pl7, optionally coupled to a carrier, is •added to the serum sample, and that possible antigen/an- tibody complexes formed are detected using an immunoassay, whereby the discrimination is established based upon that said serum sample is false HIV-1 positive if said .comple- xes are detected, and true HIV-1 positive if said com¬ plexes are not detected.
In a preferred embodiment of the invention said diagnostic antigen is a diagnostic antigen according to the invention. Other characteristics and features of the present invention appears from the attached claims.
Among the AIDS-inducing retro viruses so far known, the most common in the world is HIV-1, while the partially structurally different HIV-2 is mainly restricted to West Africa. The retro viruses HTLV-1 and HTLV-2 do not induce AIDS, but AIDS-related diseases, for example T-cell leukemia. The present invention is therefore concerned with HIV-1 and amino acid sequences unique for the HIV-1 proteins are contemplated. The interior parts, for example the nucleocapsid or the core containing the RNA, of the HIV-1 viruses are pro¬ tected by an envelope. The interior parts, called gag (group-specific antigen) and the envelope, called env, are built up of different proteins. These proteins, i.e. the precursors, are predominant in the beginning of the virus life cycle, while others are predominant in later phases. Table 1 lists major genes and gene products of HIV-1.
TABLE 1 Gene Gene products/proteins group-specific antigen/core
(gag) pl7, p24, p55 poly erase (pol) p31, p51, p66 envelope (env) gp41, gpl20, gpl60
p = protein gp = glycoprotein
The numbers indicate the approximate molecular weights of the proteins expressed in kilodaltons. p55 is a precursor of p24 and/or pl7. gpl60 is a precursor of gpl20 and gp41.
At a certain stage of the virus life cycle, an enzyme cleaves the p55 molecule from the pi7 molecule. Thus, the whole pl7 protein constitutes a part of the original p55 protein. As the present invention relates to peptides with the ability to react with antibodies induced by an amino acid sequence corresponding to an epitope or clusters of epitopes of HIV-1 pl7, p55 is also included in this defi- nition. Thus, throughout the present specification and. claims, the expression "an epitope or clusters of epitopes of HIV-1 pl7" used, is intended to include the same epi¬ tope or epitopes of HIV-1 p55 "as well.
The peptides according to the present invention have ability to react with antibodies raised against an amino acid sequence corresponding to or closely related to an epitope or clusters of epitopes in the position 118-132, i.e. the C-terminal position, of the HIV-1 pl7 protein in the interior of the HIV-1 virus. The peptides according to the invention basically comprises said 15 C-terminal amino acids of the pl7 protein, i.e.- Ala-Ala-Ala-Asp-Thr-Gly- -His-Ser-Asn-Gln-Val-Ser-Gln-Asn-Tyr-, optionally with one or a few amino acids replaced by other amino acids, especially in the corresponding position 123 to 127. This C-terminal portion of HIV-1 pl7 is dissimilar to the amino acid sequences of all other retroviruses. Further, as indicated above, the peptides according to the present invention optionally have 1-5 amino acid residues inserted between the corresponding pl7 positions 121 and 122. All these new peptides and/or parts thereof react in a uniquely specific way with the above mentioned non HIV-1 antibodies.
In the peptides according to the present invention, X represents a chemical bond, the amino acid sequence -Pro- -Ile-Val-Gln-Asn-Ile-Gln-Gly- or a sequence of at least 4, and preferably 8, particular amino acid residues, which each are chosen from the group consisting of -Thir- and
-Ser-. When X is an amino acid sequence, it can be located either in the C- or N-terminus of the peptides but not in both ends at the same time. If it is for example located in the C-terminus, X in the N-terminus corresponds to a chemical bond and vice versa. However, X can be a bond at both ends of the peptide according to the present inven¬ tion. Said amino acid sequence acts as a coupling facili¬ tating spacer, which permits proper bindning to the car¬ rier to which the peptides according to the present inven- tion will be bound during the discrimination method. The sequence -Pro-Ile-Val-Gln-Asn-Ile-Gln-Gly- corresponds to the sequence in position 133-140 in the HIV-1 p24 protein and is particularly suitable as a coupling sequence. The sequence X should not be an amino acid sequence which adversely affects the result of the diagnosis method. Accordingly, it should not have a too high charge or be too hydrophobic and it should not disturb the conformation of the peptides. The amino acids threonine and serine also fulfill these requirements particularily well, and any one of the amino acids in said sequence X can be threonine or serine, with the proviso that X is not the p24 derived sequence containing the 8 amino acids above. The number of amino acids in this spacer sequence should be at least 4, but in a preferred embodiment according to the present invention 8 amino acids are used.
The polypeptides according to the present invention can be bound via the amino acid sequence X to a carrier by physical/chemical interaction, as for example covalent binding, ionic binding, hydrogen binding or hydrophobic binding. Examples of covalent binding are esther, ether and disulfide binding. The expression "carrier" used herein should be inter¬ preted broadly, and it can be any material which is compa¬ tible with and not negatively affects the method according to the present invention, for example resins, microplates, plastic surfaces, gels, matrixes etc. The expression "epitope" used herein means antigenic or immunogenic determinant and relates to a specific part of a structure of an antigen inducing an immuno response, and the produced antibodies are directed against this part. As stated above, the peptides according to the pre¬ sent invention have ability to bind to antibodies raised against amino acid sequences corresponding to an epitope or clusters of epitopes of HIV-1 pl7. Thus, these anti¬ bodies are not raised against HIV-1 proteins. It is reasonable to assume that pl7 antibody reactions are due either to coincidental or selection controlled antigenic mimicry between the C-terminus part of the pi7 amino acid sequence and an unknown non-HIV-epitope. An autoimmune origin appears to be possible. From a general retroviral perspective, the C-ter¬ minal portion of the membrane anchoring protein of gag and its surrounding sequences, is the most variable portion of gag. Its functions are unknown. Features of this region are the sequence motifs -Pro-Pro-Pro-Tyr and -Pro-Thr-Ala- -Pro- which are recognizable in a majority of retrovi- ruses. The latter, but not the former, motif is present in HIV-2 and many, but not all, SIV viruses. HIV-1 has neither. This emphasizes the uniqueness of the C-terminus of HIV-1. Further, the existence of a low frequency of persons unlikely to be HIV-1 infected having antibodies to this sequence which is dissimilar to other retrovirus sequences indicates that the C- erminus of pi7 contains at least one epitope which mimics a non-retroviral epitope. It is of interest that the sequence responsible for many of the false positive pl7 antibody reactions located at about gag 120-130 neighbours a region of pl7 which is similar to thy osin alpha 1 (see Naylor PH, Naylor CW, Badamchian M, et al.: Human immunodeficiency virus con¬ tains an epitope immunoreactive with thymosin alphal and the 30-amino acid synthetic pl7 groupspecific antigen HPG- 30. Proc Natl Acad Sci USA 1987, 84:2951-2955). Either, then, the C-terminus of HIV-1 pl7 has point mutated into a similarity with at least two non-HIV-1 antigens (thymosin alpha 1 and an unknown one), or by a recombinatorial event involving a cellular sequence containing both could have been transduced into an HIV-1 predecessor after the diver- gence of HIV-1 and HIV-2/SIVmac-smm. In at least the lat¬ ter case, the pl7 antibodies would be auto-antibodies. Other examples of autoantibodies cross-reacting with retroviral gag sequences are known (see Query CC, Keene JD: A human autoimmune protein associated with Ul RNA contains a region of homology that is cross-reactive with retroviral p30 gag antigen. Cell 1987, 51:211-220). Brief description of the Drawings
Figure 1 sho s the distribution of absorbance diffe¬ rences between the samples and a defined limit in a whole virus based HIV-1 antibody screening test (Organon Viro- nostica) of ten pl7 reactive sera.
Figure 2 shows the distribution of absorbance diffe¬ rences in EIA with synthetic peptides derived from HIV-1 gag of a) ten pl7 reactive sera, b) 19 confirmed HIV-1 antibody positive sera and c) ten HIV seronegative blood donors. Figure 3 shows electrophoretic immunoblotting (EIB) with HIV-1 antigen of three (A-C) pi7 reactive human sera ("false HIV pl7 pos sera") after absorption over night with resin-bound HIV-1 gag 118-140 (P) or with resin without any peptide (R).
Figure 4 shows the distribution of anti-pl7 reacti¬ vity measured (in arbitrary units) by reflectance densito- metry of electrophoretic immunoblot strips with HIV-1 antigen after absorption over night with resin-bound HTLV-I gag 111-130 or with resin-bound HIV-1 gag 118-140. EXPERIMENTS Identification of HIV-1 p!7 reactive sera
First a standard whole virus HIV-1 antibody screening test, preferably the Organon Vironostica-anti-HIV (Organon Technica), is performed. Serum samples are allowed to react with whole HIV-1 virus bound to a solid phase. Possible binding of antibodies to the different parts of the virus is detected by measuring the absorbance of the sample. Samples showing an absorbance above a predeter- mined cut off value are deemed to be positive.
In the present case about 150 000 healthy persons in the region of Southern Sweden were tested, and 785 were regarded positive. Blood donors, pregnant women and heterosexuals worrying about HIV, but essentially without risk behaviour, provided the great majority of these samples.
Subsequently these 785 positive sera were subjected to a confirmatory electroimmuno blotting (EIB) assay, i.e. a Western blot (described above). Ten of these 785 samples showed a single pl7 pattern of serological activity. Among the persons donating the 10 sera were 6 women and 4 men. The ages were 20, 21, 22, 22, 27, 31, 33 and 33. The ages were unknown for two persons. The reasons for making the HIV antibody test stated on the request form were: blood donor 2, heterosexual without stated risk behaviour 5, pregnant woman 2, mental hospital patient 1. The persons were not contacted themselves, because it was not neces- sary to trouble them at this stage of the investigation concerning the nature of the false-positive HIV serological reactions.
The distribution of the differences between the absorbance of the sample and the cut-off value obtained with the Organon whole virus HIV-1 antibody assay are depicted in Fig. 1. The average absorbance difference was 0.118. In a comparison test performed with commercial HIV-1 antibody tests, no serum reacted in the Abbott HIV-1 EIA, which employs a recombinant antigen including both a portion of gag and env. None of the 10 sera reacted in the
»
Abbott ENVACORE competition EIA. Neither did any of them react in the Pasteur ELAVIA-2 and DuPont HIV-2 EIB.
The two above mentioned commercial assays EIA and EIB were used according to the instructions of the manufac¬ turers. Immunoassays
EIB strips were measured with a Bio-Rad 620 Video Densitometer (Richmond, California, USA) (see Blomberg J, Klasse PJ: Quantification of immunoglobulin G on electro¬ phoretic immunoblot strips as a tool for human immuno¬ deficiency virus serodiagnosis. J Clin Microbiol 1988, 26:111-115).
Peptide EIA:s were performed approximately as descri- bed (see Blomberg J, Nilsson I, Andersson M: Viral anti¬ body screening system that uses a standardized single dilution immunoglobulin G enzyme immunoassay with multiple antigens. J Clin Microbiol 1983, 17:1081-1091 and Klasse PJ, Pipkorn R, Blomberg J: Presence of antibodies to a putatively immunosuppressive part of human immunodeficien¬ cy virus (HIV) envelope glycoprotein gp41 is strongly associated with health among HIV-positive subjects. Proc Natl Acad Sci USA 1988, 85:5225-5229). Briefly, 100 μl of 20 μl/ml of peptide in PBS-M (PBS: 8.0 g NaCl, 0.2 g KH2P04, 1.4 g Na2HP04«2H20, 0.2 g KC1 per liter) (PBS-M: PBS with 10 μl/ml of sodium merthiolate) was allowed to bind first for 2-6 h at room temperature, then overnight at 4°C. 200 μl of blocking solution (4% bovine serum albumin (BSA)/0.2% gelatin/0.05% Tween 20) were added, allowed to bind for 2-6 h at room temperature, then over night at 4°C, and frozen. At the day of use, the plates were thawed and washed three times in washing fluid
(freshly made 0.05% Tween 20 in PBS). 100 μl of appropria¬ tely diluted control sera and test sera diluted 1/50 in diluent I (3% BSA, 0.2% gelatin, 0.05% Tween 20 in PBS-M) were then added and the plates were incubated with shaking at room temperature for 1 h. After another wash, 100 μl biotinylated affinity purified goat anti-human IgG (Sigma) diluted 1/1500 in diluent II (0.2% gelatin in PBS-M) were added, and incubated with shaking for 1 h at room tempera¬ ture. After washing, 100 μl avidin-peroxidase (Sigma) diluted 1/300 in diluent II were added, and the plates were incubated with shaking for 1 h at room temperature. The plates were washed thoroughly and beaten dry. 100 μl substrate solution (20 mg o-phenylenediamine + 10 μl 30% H«0« in color buffer, made fresh each day) (Color buffer: 34.7 M citric acid, 66.7 mM Na2HP0., pH 5.0), was added, and the plates were incubated in the dark at room temperature for 30 min and read at 450 nm in a microplate photometer. Synthetic peptides The following peptides were used in the experiments: A HIV-1 gag 1-20
-Met-Gly-Ala-Arg-Ala-Ser-Val-Leu-Ser-Gly-Gly-Glu-Leu-Asp- -Arg-Trp-Glu-Lys-Ile-Arg- B HIV-1 £ 2 88-109 -Val-His-Gln-Arg-Ile-Glu-Ile-Lys-Asp-Thr-Lys-Glu-Ala-Leu- -Asp-Lys-Ile-Glu-Glu-Glu-Gln-Asn- C HIV-1 gag 101-122
-Leu-Asp-Lys-Ile-Glu-Glu-Glu-Gln-Asn-Lys-Ser-Lys-Lys-Lys- -Ala-Gln-Gln-Ala-Ala-Ala-Asp-Thr- D HIV-1 gag 118-140
-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Asn-Gln-Val-Ser-Gln-Val- Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Asn-Ile-Gln-Gly- E HIV-1 gag 127-140
-Gln-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Asn-Ile-Gln-Gly- F HTLV-I gag 111-130
-Pro-Asp-Ser-Asp-Pro-Gln-Ile-Pro-Pro-Pro-Tyr-Val-Glu-Pro- Thr-Ala-Pro-Gln-Val-Leu-
The sequences of peptides B, C and D overlap. Peptide D is a peptide according to the present invention. Peptide E is entirely contained within peptide D. A previously recognized similarity between thymosin alpha-1 and HIV-1 pl7 is present in the sequence of peptides B and C (see Naylor PH, Naylor CW, Badamchian M, et al.: Human immuno¬ deficiency virus contains an epitope immunoreactive with thymosin alphal and the 30-amino acid synthetic pi7 group specific antigen HGP-30. Proc Natl Acad Sci USA 1987,
84:2951-2955). The carboxy terminus of pl7 is at residue 132, a tyrosine.
The peptides were synthesized using Fmoc chemistry on an Applied Biosystems 430A solid phase automatic peptide synthesizer. They were subsequently purified by reverse- phase HPLC on a C18 column. Their purity was 95% according to an analytical HPLC procedure. The peptides -bound to a resin carrier were synthesized on a PAM resin. After de¬ blocking with concentrated trifluoroacetic acid for 1 h the unprotected peptides remained covalently coupled to the resin through their carboxy terminus. The amino acid sequence was determined by the degradation method according to Edman.
According to the invention, the peptides can be used as a diagnostic antigen either solved in the serum sample or bound to a carrier. Hereinafter, in methods 1-3, some modes of using the diagnostic antigen according to the present invention will be described. Method 1 The diagnostic antigen according to the present invention, i.e. the peptide D, can be used in a peptide EIA, wherein it is bound to a micro titer plate serving as a carrier. In one or more wells of the micro titer plate at least two peptides corresponding to one or more epitopes of HIV-1 pl7 is used, of which one is a peptide according to the present invention, for example peptide D and peptide B and/or C above. Each serum sample is added to the wells and possible binding of antibodies present in the serum to the antigen occurs. The pattern obtained after the measurement of the absorbance, shows accurately or very likely if the serum is true or false positive. A combination of reactivity for the different pepti¬ des used is considered for determination of false or true positive serum samples. This evaluation can either be done manually or with a micro processor. If only peptide D reacts, the serum is doubtless false positive, i.e. the patient is not HIV-1 infected. If only peptide C reacts, the serum is doubtless true positive. Further, the serum sample is true positive when peptide B and C reacts weakly, but likely false positive when peptide B and D reacts weakly. However, certain rare combinations of reactivity are impossible to interpret in an accurate way, but most of the pl7 reactive sera are possible to determine in a much more reliable way than hitherto known using the method according to the present invention.
Fig. 2 shows an example of reactivity for pl7 posi- tive sera using the five abαvementioned synthetic pl7 derived peptides A-E compared to confirmed HIV-1 antibody positive sera and negative sera.
The distribution of IgG reactivities of pl7 reactive sera on the five pl7 derived peptides is shown in Fig. 2a. Only two peptides, A and D, yielded absorbance differences exceeding 0.3. This distribution was clearly different from the reactivity patterns of the confirmed HIV-1 anti¬ body positive sera (Fig. 2b) and the sera from HlV-nega- tive Swedish blood donors (Fig. 2c). The confirmed posi- tive sera reacted primarily with peptides B and C, and to a lesser extent with peptides A, D and E. The blood donor sera reacted only with peptide A. Method 2
In this method the serum sample is absorbed in at least two different tubes containing one type of peptide in each. One of the peptides is the diagnostic antigen according to the present invention, i.e. peptide D, while the other or the others is (are) reference peptide(s). All of the peptides used are bound to a solid phase, for example resin beads.
Eight of the ten sera with the pl7 reactivity pattern were incubated with 10 mg phosforamidite resin beads carrying either peptide D or F or beads without peptide. The sera had first been diluted with a half volume in EIA dilution fluid (3% BSA, 0.2% gelatin in PBS-M). The amount of peptide used in each tube was approximately 5 mg, as approximately half of the weight of the peptide-coupled beads was calculated to be due to peptide. On a molar basis, this should greatly exceed the amount of antibody in the diluted serum. After 18 h of absorption the super¬ natant was directly incubated with EIB strips, and analy- zed according to the standard EIB protocol. In six of the eight sera the anti-pl7 activity could be totally or nearly totally absorbed out by resin-bound peptide D.
In no serum it could be absorbed out by peptide F, i.e. the HTLV-1 peptide. This appears from Fig. 4. This figure shows, on the left side, where the lines start, the initial intensity of pl7 band in EIB for both HTLV-1 and HIV-1 before absorption, and the right end points of the lines represent the band intensity for peptide D from HIV-1 after absorption. The HTLV-1 band intensity is unchanged after absorption with HTLV-I peptide. Thus, the lines represent the degree of the intensity reduction of the pl7 band before and after absorption, and shows that pl7 antibodies are absorbed away from the serum sample. Thus, all or the great majority of the EIB-reactive anti- pl7 antibodies of these pl7 reactive sera must have been directed to one or several narrowly clustered epitope(s) within the pl7 portion of HIV-1 gag 118-140, i.e. peptide D according to the present invention. Method 3
In a third method, an inhibition method, the peptide according to the present invention is used as such, i.e. not bound to any carrier. Antibodies reactive with pl7 in EIB have been found in a serum sample. The peptide D is added to the serum sample or a dilution of the serum sample. A referencs peptide with a sequence unrelated to that of peptide D is added to another portion of the serum sample or a dilution of the serum sample. If the binding of antibodies to pl7 in.EIB is inhibited in the presence of peptide D, but not in the presence of the reference peptide, the serum sample is false-positive. Other immunoassay methods can be used according to the present invention, for example radioimmuno assay (RIA), fluorescence immuno assay (FIA) and an immuno assay involving metal labelling. The possibilities of using the diagnostic antigen according to the present invention in connection with said assay methods are obvious for a skilled in the art.
The difference in "peptide reactivity spectrum" demonstrated here is useful for discrimination of true from false-positive HIV-1 pl7 EIB reactions. A cross- reaction due to a coincidental cross-reactivity is likely to occur on only one epitope. The antibodies raised towards the virus most likely will react with several epitopes on the protein.

Claims

1. Peptides of the formula a) H-X-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Asn-Gln-Val-Ser- Gln-Asn-Tyr-X-Z, b) H-X-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Ser-Gln-Val-Ser- Gln-Asn-Tyr-X-Z, c) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Asn-Ser-Gln-Val-Ser- Gln-Asn-Tyr-X-Z,
"d) H-X-Ala- la-Ala-Ala-Ala-Gly-Thr-Gly-Asn-Ser-Ser-Gln-
Val-Ser-Gln-Asn-Tyr-X-Z, e) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Arg-Gly-Asn-Ser-Ser- • Gln-Val-Ser-Gln-Asn-Tyr-X-Z, f) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Ser-Ser-Lys-Val-Ser- Gln-Asn-Tyr-X-Z, g) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Ser-Ser-Gln-Val-Ser-
Gln-Asn-Tyr-X-Z, h) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Gly-Ser-Gln-Val-Ser- Gln-Asn-Tyr-X-Z, i) H-X-Ala-Ala-Ala-Ala-Gln-Gln-Ala-Ala-Ala-Thr-Lys-Asn-
Ser-Ser-Ser-Val-Ser-Gln-Asn-Tyr-X-Z, or j ) H-X-Gln-Ala-Ala-Ala-Asp-Ala-Gly-Asn-Asn-Ser-Gln-Val- Ser-Gln-Asn-Tyr-X-Z, in which X represents a chemical bond or an coupling- facilitating amino acid sequence and Z represents -NH„ or -OH.
2. Peptide according to claim 1, wherein the coupling-facilitating amino acid sequence represents at least 4, preferably 8, amino acid residues, which each is chosen from the group consisting of -Thr- and -Ser-, or the amino acid sequence -Pro-Ile-Val-Gln-Ans-Ile-Gln-Gly-.
3. A diagnostic antigen having the ability of binding to antibodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or clusters of epitopes of HIV-1 pl7, c h a r a c t e r i s e d in that it mainly consists of an antigen, which antigen is selected from peptides comprising the amino acid sequence a) H-X-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Asn-Gln-Val-Ser- Gln-Asn-Tyr-X-Z, b) H-X-Ala-Ala-Ala-Asp-Thr-Gly-His-Ser-Ser-Gln-Val-Ser- Gln-Asn-Tyr-X-Z, c) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Asn-Ser-Gln-Val-Ser- Gln-Asn-Tyr-X-Z, d) H-X-Ala-Ala-Ala-Ala-Ala-Gly-Thr-Gly-Asn-Ser-Ser-Gln- Val-Ser-Gln-Asn-Tyr-X-Z, e) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Arg-Gly-Asn-Ser-Ser- Gln-Val-Ser-Gln-Asn-Tyr-X-Z, f) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Ser-Ser-Lys-Val-Ser- Gln-Asn-Tyr-X-Z, g) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Ser-Ser-Gln-Val-Ser- Gln-Asn-Tyr-X-Z, h) H-X-Ala-Ala-Ala-Asp-Thr-Gly-Asn-Gly-Ser-Gln-Val-Ser-
Gln-Asn-Tyr-X-Z, i) H-X-Ala-Ala-Ala-Ala-Gln-Gln-Ala-Ala-Ala-Thr-Lys-Asn- Ser-Ser-Ser-Val-Ser-Gln-Asn-Tyr-X-Z, or j ) H-X-Gln-Ala-Ala-Ala-Asp-Ala-Gly-Asn-Asn-Ser-Gln-Val-
Ser-Gln-Asn-Tyr-X-Z, or antigenic parts thereof, wherein one X represents a chemical bond and the other X represents a chemical bond or a coupling-facilitating amino acid sequence and Z represents -NH2 or -OH.
4. A method of discriminating between a false and true diagnosed HIV-1 positive'serum sample, which first has been diagnosed as positive in a standard HIV-1 antibody screening EIA test and then shown to exhibit a pl7 pattern of serological activity in an electrophoretic immunoblot assay, c h a r a c t e r i s e d in that at least one diagnostic antigen having the ability of binding to antibodies which have a binding affinity for compounds containing an amino acid sequence corresponding to an epitope or clusters of epitopes of HIV-1 pl7, optionally coupled to a carrier, is added to said serum sample, and that possible antigen/antibody complexes formed are detected using an immunoassay, whereby the discrimination is established based upon that said serum sample is false HIV-1 positive if said complexes are detected, and true HIV-1 positive if said complexes are not detected.
5. A method according to claim 4, c h a r a c t e ¬ r i s e in that the immunoassay for detection of said antibody/antigen complex is an enzyme immuno assay (EIA), radio immunoassay (RIA), immunoassay involving metal labelling, fluorescense immunoassay (FIA) or an immunoassay in which the peptide is soluble and inhibits another reaction.
6. A method according to claim 4, c h a r a c t e ¬ r i s e d in that the carrier is a resin, microplate, plastic surface, gel or matrix.
PCT/SE1990/000756 1989-11-27 1990-11-22 NEW HIV-1 p17 PEPTIDES, DIAGNOSTIC ANTIGENS AND IMMUNOASSAY METHOD WO1991008227A1 (en)

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SE8903985-3 1989-11-27
SE9000356A SE9000356L (en) 1989-11-27 1990-02-01 Peptide(s) corresp. to HIV-1 P17 C-terminal - used for discriminating between a false and true diagnosed HIV-1 positive serum sample
SE9000356-7 1990-02-01

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WO2009043155A2 (en) * 2007-10-03 2009-04-09 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Distinct hiv-1 gag and env epitopes of hla alleles associated with differential susceptibility to hiv-1 infection
WO2009043155A3 (en) * 2007-10-03 2009-05-28 Ca Minister Health & Welfare Distinct hiv-1 gag and env epitopes of hla alleles associated with differential susceptibility to hiv-1 infection

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