WO1991013360A1 - NOUVEAUX PEPTIDES D'HIV p24, ANTIGENES DIAGNOSTIQUES ET METHODE D'IMMUNO-ANALYSE DISCRIMINANTE - Google Patents

NOUVEAUX PEPTIDES D'HIV p24, ANTIGENES DIAGNOSTIQUES ET METHODE D'IMMUNO-ANALYSE DISCRIMINANTE Download PDF

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Publication number
WO1991013360A1
WO1991013360A1 PCT/SE1991/000114 SE9100114W WO9113360A1 WO 1991013360 A1 WO1991013360 A1 WO 1991013360A1 SE 9100114 W SE9100114 W SE 9100114W WO 9113360 A1 WO9113360 A1 WO 9113360A1
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PCT/SE1991/000114
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Jonas Blomberg
Rüdiger Pipkorn
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Replico Medical Aktiebolag
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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

  • New HIV p 24 peptides, diagnostic antigens and discriminative immunoassay method relate to new peptides, to 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 a cluster of epitopes of HIV-1 p24 and to a method of discriminating between a false and true diagnosed HIV-1 p24 protein antibody positive serum sample using an immunoassay.
  • 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
  • EIA enzyme immunoassay
  • 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 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, bacterial growth in buffers etc. It has been observed that a small portion of sera from humans unlikely to be HIV-1 infected contains antibodies against one or a few HIV-1 proteins, mostly derived from the interior gag part of the virus. One example of such a protein is the p24 protein. Such reactivities can give rise to "indeterminate" HIV-1 electroforetic immunoblot (EIB) reactions. They are a constant cause of concern in laboratories responsible for confirmation of HIV-1 antibody screening results. Anti-p24 reactions is a major diagnostic problem in both developed and developing nations. It is costly to run several confirmatory tests on each of these sera. 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.
  • EIB electroforetic immunoblot
  • HIV-1 EIB reactivity is characterised by a reactivity with p24 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 p24 proteins are involved.
  • a single anti-p24 band can be a signal of an early HIV-infection, be due to an HIV-2-infection, be the consequence of a "cryptic" anti-HIV serological reaction (see Ranki AM, Krohn M, Allain JP, et al.: Long latency precedes overt seroconversion in sexually transmitted human immunodeficiency virus infection. Lancet 1987, ii:589-593), or be a false-positive reaction in a person who never encountered HIV.
  • confirmatory tests are Western blotting, inhibition EIA, peptide EIA and radioimmuno precipitation. Usually 3 or 4 confirmatory tests are necessary to discriminate between false and true positive sera.
  • This antigen-antibody complex is detected by an antihuman immunoglobulin G (IgG) antibody conjugated 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, antibodies against almost all of the virus proteins are present. This test is rather sensitive and it permits separation of the immune response to different proteins originating from different parts of a virus organism.
  • IgG immunoglobulin G
  • Inhibition EIA is a technique that discriminates between false and true reactivity by competition 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 antibodies. 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 positive antibodies and not on direct indication, it is associated 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 reactions because the reasons for the immune response are different. 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.
  • 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
  • RIPA radioimmunoprecipitation test
  • the antibodies from the patient serum sample are bound to radioactively labelled HIV-1 proteins.
  • the antigen-antibody complexes are immobilised on protein A beads, and are then solubilised and subjected to electrophoresis in polyacrylamide.
  • 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 advantageous to study env reactivity by RIPA.
  • the disadvantages 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 according to the present invention is simpler for discriminating between true and false HIV-1 anti-p24 reactions. Furthermore, it is safer and in certain respects more reliable than known techniques. It is also fast, and the test is performed in 3-4 h, compared to known techniques, in which up to 24 h are necessary. 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
  • the present invention relates to peptides of the formula
  • the present invention relates to 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 p24.
  • 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 epitopes of HIV-1 p24, optionally coupled to a carrier, is added to the 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.
  • 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 protected by an envelope.
  • the interior parts called gag
  • proteins 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.
  • envelope (env) gp41, gp120, gp160 p protein
  • the numbers indicate the approximate molecular weights of the proteins expressed in kilodaltons.
  • p55 is a precursor of p24 and p17.
  • gp160 is a precursor of gp120 and gp41.
  • an enzyme cleaves the p55 molecule from the p24 molecule.
  • the whole p24 protein constitutes a part of the original p55 protein.
  • p55 is also included in this definition.
  • an epitope or clusters of epitopes of HIV-1 p24 used, is intended to include the same epitope 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 of the HIV-1 p24 protein.
  • X represents a chemical bond or a sequence of at least 4, and preferably 8, particular amino acid residues, which each are chosen from the group consisting of -Thr- and -Ser-.
  • 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 invention.
  • Said amino acid sequence acts as a coupling facilitating spacer, which permits proper bindning to the carrier to which the peptides according to the present invention will be bound during the discrimination method.
  • the sequence X should not be an amino acid sequence which adversely affects the result of the diagnosis method.
  • 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.
  • 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.
  • carrier used herein should be interpreted broadly, and it can be any material which is compatible with and not negatively affects the method according to the present invention, for example resins, microplates, plastic surfaces, gels, matrixes etc.
  • 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.
  • the immuno assay method according to the present invention can be enzyme immunoassay (EIA), radioimmuno assay (RIA), immunoassay involving metal labelling, fluorescence immunoassay (FIA) or an immunoassay in which the peptide is soluble and inhibits another reaction.
  • EIA enzyme immunoassay
  • RIA radioimmuno assay
  • FIA fluorescence immunoassay
  • an immunoassay in which the peptide is soluble and inhibits another reaction can be enzyme immunoassay (EIA), radioimmuno assay (RIA), immunoassay involving metal labelling, fluorescence immunoassay (FIA) or an immunoassay in which the peptide is soluble and inhibits another reaction.
  • FIA fluorescence immunoassay
  • the peptides according to the present invention have ability to bind to antibodies raised against amino acid sequences corresponding to an epitope or clusters of epitopes of HIV-1 p24. Thus, these antibodies are not raised against HIV-1 proteins. It is reasonable to assume that p24 antibody reactions are due either to coincidental or selection controlled antigenic mimicry between the C-terminus part of the p24 amino acid sequence and an unknown non-HIV-epitope. The origin may be autoimmunity or immunization with a retrovirus other than HIV.
  • Figure 1 Distribution of absorbance differences in EIA with synthetic peptides derived from HIV-1 gag.
  • Figure 2. Patterns of reactivity with six long synthetic peptides derived from HIV-1 p24.
  • FIG. 3 Electrophoretic immunoblotting with HIV-1 antigen of one p24 EIB and HIV-1 gag 318-378 reactive human serum (number I, Table 3) before and after threefold absorption "in-well” with solid-phase-bound HIV-1 gag 318- 378 (A), HTLV-I gag 118-140 (B), or wells mock-coated with PBS (C).
  • 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 predetermined cut off value are deemed to be positive.
  • ENVACORE confirmatory HIV-1 antibody competition antibody EIA and the Du Pont Biotech HIV-1 electrophoretic immunoblot (EIB) system. They were used according to the in- structions of the manufacturers.
  • EIB strips were measured with a Bio-Rad 620 Video Densitometer (Richmond, California, USA) (see Blomberg J, Klasse PJ: Quantification of immunoglobulin G on electrophoretic immunoblot strips as a tool for human immunodeficiency virus serodiagnosis. J Clin Microbiol 1988, 26:111-115).
  • Peptide EIA s were performed approximately as described (see Blomberg J, Nilsson I, Andersson M: Viral antibody 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 immunodeficiency 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 ⁇ l of 20 ⁇ l/ml of peptide in PBS-M (PBS: 8.0 g NaCl, 0.2 g
  • HIV-1 hxb2 gag 143-156, 203-218, 260-276 and 335-351 are comprised in the present invention.
  • the amino acid sequences of the peptides are expressed herein with the one letter symbol system in an attempt to in a more simple way show the relation between the longer peptides and the shorter fragments thereof.
  • the peptides were synthesized using Fmoc chemistry on an Applied Biosystems 430A solid phase automatic peptide synthesizer. They were subsequently purified by reversephase HPLC on a C18 column. Their purity was 95% according to an analytical HPLC procedure.
  • the peptides can be used as a diagnostic antigen either solved in the serum sample or bound to a carrier.
  • a diagnostic antigen either solved in the serum sample or bound to a carrier.
  • HIV-1 p24 reactive sera were derived from 785 repeatedly HIV-1 antibody screening test positive sera. These were in their turn derived from around 150 000 sera subjected to HIV-1 antibody screening tests at primary test sites. The distribution of reactivities in the nine sera with p24 bands in HIV-1 EIB can be seen in Table 2.
  • Fig la four p24 positive sera reacted, two strongly, with the N-terminal long peptide HIV-1 gag 133-193, one serum with HIV-1 gag 262-338 and two with the C-terminal HIV-1 gag 318-378 in oxidized form.
  • One serum had a reactivity with gag 133-193 just below the stipulated cut-off absorbance difference of 0.4. None of the p24 positive sera reacted with one of the shorter HIV-1 gag peptides.
  • TP confirmed HIV-1 antibody positive sera.
  • BD HIV-1 seronegative blood donor sera.
  • the peptide number codes are evident from Table 1. Absorption of p24 positive sera with immobilized peptides p24 antibody positive sera were diluted to yield an absorbance increment versus the negative control well of 0.5-1.0 in EIA dilution fluid (3% BSA, 0.2% gelatin in PBS-M). Sera were incubated in wells precoated with p24 peptides for 1 h. The supernatant was then transferred to a neighbouring well, and incubated for another hour. This was repeated another time. A total of three one hour rounds of absorptions were thus made.
  • the long peptides were chosen to optimize serological reactivity with HIV gag antibodies. Long peptides simulate conformational (non-continuous) epitopes of HIV-1 p24 gag particularly well. Such long peptides have to our knowledge never before been used for HIV serology. Thus, the long peptides, especially the HIV-1 gag 133-193 and
  • 318-378 were excellent reagents for both discrimination of true from false HIV-1 antibody reactivity and for detection of both types of reactivity.
  • a combination of several peptides according to the present invention are used as diagnostic antigens.

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Abstract

On décrit des peptides ou leur parties antigéniques et un antigéne diagnostique. Ledit antigène a la capacité de se fixer sur des anticorps qui ont une affinité de liaison avec des composés contenant une séquence d'amino-acides correspondant à un déterminant antigénique ou à des groupes de déterminants antigéniques de p24 HIV-1; l'antigène décrit se compose principalement dudit antigène qui est sélectionné à partir desdits peptides ou de leurs parties antigéniques. On décrit en outre un procédé de discrimination entre un échantillon de sérum d'un sujet correctement diagnostiqué comme étant HIV-positif et un échantillon faussement diagnostiqué comme étant HIV-positif au moyen d'une immuno-analyse utilisant ledit antigène diagnostique.
PCT/SE1991/000114 1990-02-20 1991-02-18 NOUVEAUX PEPTIDES D'HIV p24, ANTIGENES DIAGNOSTIQUES ET METHODE D'IMMUNO-ANALYSE DISCRIMINANTE WO1991013360A1 (fr)

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SE9000595A SE468168B (sv) 1990-02-20 1990-02-20 Hiv-1, p24 peptider, diagnostiska antigener och foerfarande foer saerskiljande diagnostik av preliminaert hiv-1 positiva serumprov
SE9000595-0 1990-02-20

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Cited By (12)

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WO1992022572A1 (fr) * 1991-06-13 1992-12-23 Replico Medical Ab Nouveaux peptides gag et env du vih-1, diagnostic
EP0732339A1 (fr) * 1990-12-14 1996-09-18 BEHRINGWERKE Aktiengesellschaft Peptides de la protéine gag d'HIV, leur procédé de préparation et leur utilisation
US6689879B2 (en) 1998-12-31 2004-02-10 Chiron Corporation Modified HIV Env polypeptides
US6706859B1 (en) 1999-03-04 2004-03-16 Bionor Immuno As HIV peptides, antigens, vaccine compositions, immunoassay kit and a method of detecting antibodies induced by HIV
EP1307130A4 (fr) * 2000-02-04 2005-01-12 Beth Israel Hospital Vaccin contre le virus de l'immunodeficience humaine
US7009037B2 (en) 2000-09-04 2006-03-07 Bionor Immuno As Modified HIV-1gag p17 peptide and immunogenic composition
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
WO2013182660A1 (fr) 2012-06-06 2013-12-12 Bionor Immuno As Vaccin contre le vih
WO2016005508A1 (fr) 2014-07-11 2016-01-14 Bionor Immuno As Procédé permettant de réduire et/ou de retarder les effets pathologiques du virus de l'immunodéficience humaine i (vih) ou de réduire le risque de développer le syndrome d'immunodéficience acquise (sida)
WO2016177833A1 (fr) 2015-05-04 2016-11-10 Bionor Immuno As Schéma posologique pour un vaccin contre le vih

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NATURE, Vol. 336, December 1988, DOUGLAS F. NIXON et al.: "HIV-1 gag-Specific Cytotoxic T Lymphocytes Defined with Recombinant Vaccinia Virus and Synthetic Peptides", see page 484 - page 487. *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0732339A1 (fr) * 1990-12-14 1996-09-18 BEHRINGWERKE Aktiengesellschaft Peptides de la protéine gag d'HIV, leur procédé de préparation et leur utilisation
WO1992022572A1 (fr) * 1991-06-13 1992-12-23 Replico Medical Ab Nouveaux peptides gag et env du vih-1, diagnostic
US6689879B2 (en) 1998-12-31 2004-02-10 Chiron Corporation Modified HIV Env polypeptides
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US7709003B2 (en) 1999-03-04 2010-05-04 Bionor Immuno As Method of producing an HIV-1 immune response
US6706859B1 (en) 1999-03-04 2004-03-16 Bionor Immuno As HIV peptides, antigens, vaccine compositions, immunoassay kit and a method of detecting antibodies induced by HIV
US7709004B2 (en) 1999-03-04 2010-05-04 Bionor Immuno As Method of producing an HIV-1 immune response
US7311915B2 (en) 1999-03-04 2007-12-25 Bionor A/S Method of producing an HIV-1 immune response
EP1307130A4 (fr) * 2000-02-04 2005-01-12 Beth Israel Hospital Vaccin contre le virus de l'immunodeficience humaine
US7009037B2 (en) 2000-09-04 2006-03-07 Bionor Immuno As Modified HIV-1gag p17 peptide and immunogenic composition
US7612168B2 (en) 2000-09-04 2009-11-03 Bionor Immuno As Modified HIV peptides, antigens, compositions, immunoassay kit and a method of detecting antibodies induced by HIV
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
WO2013182660A1 (fr) 2012-06-06 2013-12-12 Bionor Immuno As Vaccin contre le vih
US10335482B2 (en) 2012-06-06 2019-07-02 Bionor Immuno As Method of inducing an anti-HIV-1 immune response comprising administering a C5/TM-GP41 peptide dimer
EP3967323A2 (fr) 2012-06-06 2022-03-16 Bionor Immuno AS Vaccin contre le vih
WO2016005508A1 (fr) 2014-07-11 2016-01-14 Bionor Immuno As Procédé permettant de réduire et/ou de retarder les effets pathologiques du virus de l'immunodéficience humaine i (vih) ou de réduire le risque de développer le syndrome d'immunodéficience acquise (sida)
WO2016177833A1 (fr) 2015-05-04 2016-11-10 Bionor Immuno As Schéma posologique pour un vaccin contre le vih

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SE9000595L (sv) 1991-08-21
SE9000595D0 (sv) 1990-02-20
AU7444891A (en) 1991-09-18

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