EP0472659A1 - Anticorps monoclonaux de retrovirus lymphotropes t - Google Patents

Anticorps monoclonaux de retrovirus lymphotropes t

Info

Publication number
EP0472659A1
EP0472659A1 EP90909217A EP90909217A EP0472659A1 EP 0472659 A1 EP0472659 A1 EP 0472659A1 EP 90909217 A EP90909217 A EP 90909217A EP 90909217 A EP90909217 A EP 90909217A EP 0472659 A1 EP0472659 A1 EP 0472659A1
Authority
EP
European Patent Office
Prior art keywords
hiv
antibodies
amino acid
antigen
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP90909217A
Other languages
German (de)
English (en)
Other versions
EP0472659A4 (en
Inventor
Bryan T. Butman
Thomas Michael Venetta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
VENETTA, THOMAS M.
Akzo Nobel NV
Original Assignee
Akzo NV
Akzo Nobel NV
VENETTA Thomas M
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Akzo NV, Akzo Nobel NV, VENETTA Thomas M filed Critical Akzo NV
Publication of EP0472659A1 publication Critical patent/EP0472659A1/fr
Publication of EP0472659A4 publication Critical patent/EP0472659A4/en
Ceased legal-status Critical Current

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Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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 invention relates to monoclonal antibodies, peptides that comprise the epitopes of said monoclonal antibodies and assays utilizing said monoclonal antibodies and said peptides for the detection of T- lymphotropic retroviruses, particularly HIV-1, HIV-2 and SIV.
  • the T-lymphotropic retrovirus family includes among other lentiviruses the simian retrovirus SIV and the human retroviruses HIV-1 (the likely etiologic agent of AIDS) and HIV-2. Although HIV-1 and HIV-2 are related evolutionally, nucleic acid sequence analysis reveals that HIV-2 is more closely related to SIV than it is to HIV-1. Guyader et al. (1987) noted only 42% overall genomi ⁇ sequence identity between the HIV-1 and HIV-2 isolates they compared.
  • HIV-1 and HIV-2 genomes have a typical retroviral configuration comprising LTR's, ⁇ a ⁇ and env regions that encode viral structural proteins, sequences encoding one or more enzyme, including a reverse transcriptase and other ORF's and regulatory elements.
  • the a ⁇ region of HIV-1 encodes a precursor peptide known as p55.
  • p55 is processed to produce among other proteins the major core or capsid protein known as p24.
  • the analogous crag precursor is larger, known as p57, and the major core protein is known as p26.
  • Guyader et al. (1987) found only 58% identity of amino acids between HIV-1 and HIV-2 crag proteins.
  • -_ Even among distant isolates of HIV-1 there is a greater than 90% identity of gag proteins. That and other data support the hypothesis that although HIV-1 and HIV-2 are somewhat related, they are nevertheless distinct retroviral species.
  • HIV-1 and possibly HIV-2 have such an impact on the human immune system, it is desirable, in fact imperative that sensitive, rapid diagnostic assays for detecting presence of HIV be available for population screening, quality control in blood banks, diagnosis, furtherance of our understanding of those viruses to assure the goal of obtaining a vaccine and cure, and the like. Because of ease and convenience, it is preferable that the assays be immunology-based, such as ELISA's, and for reproducibility, specificity and consistency that the reagents be monoclonal antibodies and defined antigenic peptides.
  • p24 antigenemia has been shown to be an early sign of HIV infection (Kessler et al., 1987; Wall et al., 1987) and the observation that clinical progression of AIDS sequelae is associated with reduction in anti-p24 while patients with AIDS can die with high levels of anti-env titers (Coates et al., 1987), it would be advantageous for the assay to be directed to detecting gag products such as p24/p26.
  • Minassian et al. described a monoclonal antibody identified as R1C7 that was raised against HIV-2.
  • R1C7 an anti-capsid antibody (p26) , reacted not only with the three HIV-2 isolates tested, but with the five HIV-l ' isolates and seven SIV isolates that were tested.
  • p26 an anti-capsid antibody
  • R1C7 bound to 55KD and 26KD HIV-2 proteins, to 24KD and 55KD HIV-l proteins and to a 28KD SIV protein.
  • Niedrig et al. developed a panel of 29 monoclonal antibodies to HIV-l.
  • One antibody was directed to pl7 and its precursor p32 whereas the remainder reacted with p24 and some of those also reacted with p55.
  • the pl7 antibody was found to be HIV-l specific.
  • 20 reacted in immunoblots with the corresponding capsid protein (p 6) of HIV-2 and five of those also recognized the corresponding SIV protein, p28.
  • Niedrig et al. make no mention of antibody titer, the efficacy of the antibodies in a antigen capture assay or which of the antibodies bind to p26, p55 or both.
  • several of antibodies reacted with a 22KD protein of unknown function in HIV-2 preparations.
  • the assays use a variety of techniques such as Western blot, enzyme- linked immunosorbent assay (ELISA) or indirect immuno- fluorescent assay and employ either antibodies to whole virus or purified viral antigens, see for example, Gallo et al., U.S. Patent No. 4,520,113; Sarngadharan, et al., (1984); and Robert-Guroff et al. (1982).
  • ELISA enzyme- linked immunosorbent assay
  • indirect immuno- fluorescent assay employ either antibodies to whole virus or purified viral antigens, see for example, Gallo et al., U.S. Patent No. 4,520,113; Sarngadharan, et al., (1984); and Robert-Guroff et al. (1982).
  • the instant invention relates to monoclonal antibodies, the cell lines producing those antibodies, the peptides that comprise the epitopes of those antibodies and assays using those antibodies and peptides for the detection of HIV-l and HIV-2 gene products as well as SIV gene products.
  • the antibodies react with the p24/p26 capsid protein.
  • the nonapeptide that comprises an HIV-l/HIV-2 conserved epitope is disclosed and a capture ELISA using a combination of three monoclonal antibodies that can detect simultaneously HIV-l and HIV-2 is disclosed.
  • Figure 1 Graph depicting reactivity of culture supernatants in capture ELISA. A detailed legend appears in Table 1.
  • FIG. 1 Protein A-purified antibodies were used as probe to separated HIV-2 proteins in immunoblots. Lanes 1 and 2 are positive controls and Lane 3 is a negative control.
  • Figure 4 Diagram of some of the recombinant p24 peptides used to map epitopes.
  • Figure 5 Diagram of four regions of p24 to which various monoclonal antibodies bind.
  • FIG. 6 Photographs of Westerns reacting various monoclonals with blotted gag and gag fragments. Lane 1 in each photo contains whole virus lysate. Lane 5 in each photo is a negative control p24 " plasmid and Lane 6 in each photo is another negative control containing non-HIV-infected MOLT lysate. Figure 7. Graph representing results of ELISA's using sequential overlapping nonapeptides as antigen to determine epitope of 7-D4.
  • Figure 8 Diagram depicting epitope mapping using sequential overlapping nonapeptides as antigen in ELISA.
  • Figure 9 Composition of the regions that comprise the 7-D4 epitope.
  • FIG. 10 Graph of sensitivity of a capture ELISA using two anti-p24 antibodies, 6-C10 and 5-B4, on the solid phase and HIV-l infected MOLT 3 lysate as the antigen.
  • An HRP conjugated human anti-HIV was the reporter.
  • Figure 11 Graph of sensitivity of a capture ELISA using 6-C10 and 5-B4 with and without 7-D4 on the solid phase to detect p26 of HIV-2.
  • Figure 13 Comparison of HIV-l dose response curves between the three antibody capture ELISA and a reverse transcriptase assay.
  • the instant invention relates to monoclonal antibodies and their production, im unoassays and oligopeptides.
  • the methods that were used are known in the art and are discussed only briefly throughout the specification. Suitable methods to practice the invention may be found in Meth Enzymology 121 r (1986) and other available reference materials.
  • Monoclonal antibodies were produced according to established procedures (Kohler & Milstein, 1975) .
  • mice Female BALB/c mice were immunized intraperitoneally repeatedly with lysates of HIV-l infected MOLT 3 cells emulsified in complete Freund's adjuvant (50%) . Sensitized spleen cells were fused with P3X63-Ag8.653 myeloma cells using PEG 1500. Heterokaryons were selected in HAT medium, cloned and screened for reactivity to HIV antigens in a capture ELISA. The IgG fraction of polyclonal human anti-HIV was coated onto wells of icrotiter dishes. HIV-l (produced in MOLT 3 cells) and culture supernatants were added simultaneously to the wells. Bound murine antibodies were detected with an enzyme-labelled anti- mouse Ig antibody. Data representative of the screening is depicted in Figure 1. Designation of the sample numbers is set forth in Table 1.
  • Candidate anti-HIV clones were tested further in Western blots (Towbin et al., 1979). Lysates of HIV- infected MOLT 3 cells were separated through a 12% acrylamide gel under denaturing conditions. The proteins were transferred to nitrocellulose and individual strips were blocked and reacted with the culture supernatants. Bound antibody was detected using an enzyme-labelled goat anti-mouse Ig antibody. Antibodies reacting specifically with p24 were selected ( Figure 2) . Designation of the strips is set forth in Table 2. Table 2
  • 7-D4 recognized a protein of approximately 27,000 molecular weight in lysates of SIV ⁇ c, Epitooe Mapping
  • the amino acids that comprise the p24 epitope of 7- D4 were mapped in the following manner.
  • the gag region and portions of gag were subcloned in an expression vector. Briefly, viral DNA of a bacteriophage (cDNA library HIV-l ⁇ ., clone HAT 3 (Starcich et al., (1986)) was digested with EcoRI and by ligation into the pBR322- derived plasmid pMLB1113 to produce a plasmid identified as clone 29 which contained the EcoRI/SstI gag/pol ORF. Clone 29 was digested with SstI to remove extraneous vector sequences and religated to produce plasmid gag/pol 1.2.
  • This latter plasmid was sonicated, blunt- ended and ligated with EcoRI linkers. The mixture was then digested with EcoRI. ligated into AORF8 (Meissner et al. 1987) and packaged.
  • a AORF8 expression library was generated in E. coli and screened with a human anti- HIV polyclonal antibody and a mouse anti-p24 (HIV-l) monoclonal antibody. The positives were selected, expanded and the expressed peptides were characterized by Western blotting, immunoassay and nucleotide sequencing. The recombinant p24 peptides gag 8, gag 126, gag 107 and gag 141 were expressed in E. coli.
  • clone 29 was used as a template and oligonucleotides corresponding to the 5" and 3' ends of the published sequence were used in a polymerase chain reaction to generate a complete sequence of the gag protein p24.
  • the 5' end contained an EcoRI site and the 3• end contained a IJamHI site.
  • the reaction product was digested with EcoRI and B_amHI and then ligated into PMLB1113.
  • the characterization of the recombinant p24 peptides is presented in Figure 4.
  • the various recombinant p24 peptides were used as antigen in ELISA's and in Western blots to determine whether or not a given monoclonal antibody bound a given peptide.
  • the reactivity pattern of any one monoclonal antibody with the panel of p24 peptides allowed a localization of the recognized epitope to one of four regions as shown in Table 4 and Figures 5 and 6.
  • Synthetic peptides are configured after the epitope sequence and either unmodified or conjugated to carriers are used as antigen.
  • peptides can be conjugated to PPD, tetanus toxoid, KLH or BSA using glutaraldehyde, carbodiimide or N-maleimidobenzoyl hydroxuccinimide ester.
  • Antibodies may be raised n vivo as in mice, goats or other lab animals or in vitro using a system of materials and methods similar to the IVIS of Hana Biologies (Alameda, CA) .
  • Another benefit is that large quantities of the epitope sequence can be produced synthetically or using standard recombinant DNA techniques as described above and the peptides can serve as antigen in immunology-based assays and kits for the detection of circulating antibody or for the detection of circulating antigen in an inhibition type assay.
  • Another benefit relates to improving the assays disclosed herein. Without extending the survey, it is unclear whether the epitope identified in the HIV-l isolate described herein is specific to that isolate and furthermore to the HIV-2 and SIV isolates described herein.
  • the epitope can be engineered, that is substituting one or more amino acids or alternatively derivitizing the epitope, etc., with a view to identifying a related sequence with a greater degree of conservation among a larger variety of HIV isolates or to obtaining a related sequence with a greater degree of reactivity in assays.
  • the nonapeptide analysis apparently identified a discrete linear epitope comprised of amino acids 142- 158 of the HIV-l gag that is conserved in HIV-2 and SIV, it is to be understood that the instant invention relates to monoclonal antibodies, epitopes of said monoclonal antibodies and assays using said antibodies and said peptides that are capable of detecting gag encoded proteins of HIV-l, HIV-2 and SIV.
  • each was used as a capture or HRP- conjugate antibody in a sandwich assay. Briefly, the monoclonal antibody was coated on wells and 10 ⁇ l of disruption buffer added. The antigen samples suspended in detergent buffer or controls in a volume of 100 ⁇ l were added next and incubated at 37 ⁇ C for 90 minutes.
  • Antibodies 5-B4, 6-C10 and 7-E10 worked best as both capture and conjugated antibodies. Maximal signals were obtained with the HRP-human anti-HIV as the conjugate.
  • the sensitivity of the capture ELISA using these three antibodies is less than 10 pg/ml (less than 1 pg/well) of HIV-l p24 antigen and less than 0.5 ng/ml of HIV-2 p26 antigen ( Figure 12) .
  • the sensitivity is found despite the presence of HIV antibodies in the
  • a capture ELISA using the three antibodies 5-B4, 6-C10 and 7-D4 was also compared to a reverse transcriptase assay for the detection of whole virus.
  • the ELISA was 25,000 times more sensitive than the reverse transcriptase assay ( Figure 13) .

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • AIDS & HIV (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des anticorps monoclonaux, les lignées cellulaires produisant ces anticorps, le peptide comprenant les épitopes de ces anticorps, ainsi que des analyses utilisant lesdits anticorps et lesdits peptides pour la détection de produits de gènes du VIH-1 et du VIH-2. Lesdits anticorps réagissent notamment avec la protéine capside p24/p26. L'invention concerne également le nonapeptide comprenant un épitope conservé de VIH-1/VIH-2, ainsi qu'un ELISA de capture utilisant une combinaison de trois anticorps monoclonaux pouvant détecter simultanément le VIH-1 et le VIH-2.
EP19900909217 1989-05-15 1990-05-14 T-lymphotropic retrovirus monoclonal antibodies Ceased EP0472659A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35188289A 1989-05-15 1989-05-15
US351882 1989-05-15

Publications (2)

Publication Number Publication Date
EP0472659A1 true EP0472659A1 (fr) 1992-03-04
EP0472659A4 EP0472659A4 (en) 1992-04-08

Family

ID=23382831

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19900909217 Ceased EP0472659A4 (en) 1989-05-15 1990-05-14 T-lymphotropic retrovirus monoclonal antibodies

Country Status (8)

Country Link
EP (1) EP0472659A4 (fr)
JP (1) JPH04505621A (fr)
KR (1) KR0168447B1 (fr)
AU (1) AU642886B2 (fr)
CA (1) CA2032509A1 (fr)
FI (1) FI915333A0 (fr)
WO (1) WO1990014358A1 (fr)
ZA (1) ZA903492B (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK53291D0 (da) * 1991-03-25 1991-03-25 Carlbiotech Ltd As Smaa peptider og peptidrelaterede stoffer samt farmaceutiske praeparater indeholdende saadanne forbindelser
SE9101863D0 (sv) * 1991-06-13 1991-06-13 Replico Medical Ab Peptider, diagnostiskt antigen, vaccinkomposition och foerfarande foer selektering av hiv-stammar
US6399294B1 (en) * 1994-10-20 2002-06-04 Institut Pasteur Nucleotide sequences of HIV-1 type (or subtype) O retrovirus antigens
FR2777285B1 (fr) * 1998-04-10 2000-05-19 Bio Merieux Ligand peptidique presentant une affinite specifique vis-a-vis de la proteine p24 du retrovirus hiv-1
US6818392B2 (en) 2000-12-06 2004-11-16 Abbott Laboratories Monoclonal antibodies to human immunodeficiency virus and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0345461A2 (fr) * 1988-06-10 1989-12-13 Abbott Laboratories Anticorps monoclonaux de souris contre VIH-IP24 et leur utilisation dans des tests de diagnostic

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4755457A (en) * 1985-02-05 1988-07-05 Robert Guroff Marjorie Method for detecting HTLV-III neutralizing antibodies in sera
US4843011A (en) * 1985-08-01 1989-06-27 Akzo N.V. Monoclonal antibodies for binding HTLV-III proteins, and cell lines for their production
US4888290A (en) * 1987-11-06 1989-12-19 Coulter Corporation Monoclonal antibody specific to HIV antigens
EP0330359A3 (fr) * 1988-02-25 1991-06-05 Bio-Rad Laboratories, Inc. Composition utile pour le diagnostic et le traitement de l'infection par le HIV-I
CA2017021A1 (fr) * 1989-05-22 1990-11-22 Johannes Jacobus Schalken Epreuve de diagnostic pour la detection des anticorps contre les antigenes d'un ou de plusieurs virus
DE4039925A1 (de) * 1990-12-14 1992-06-17 Behringwerke Ag Ausgewaehlte peptide des gruppenspezifischen antigens (gag) von humanem immundefizienzvirus (hiv), ihre herstellung und verwendung

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0345461A2 (fr) * 1988-06-10 1989-12-13 Abbott Laboratories Anticorps monoclonaux de souris contre VIH-IP24 et leur utilisation dans des tests de diagnostic

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9014358A1 *

Also Published As

Publication number Publication date
AU5835590A (en) 1990-12-18
KR0168447B1 (ko) 1999-01-15
CA2032509A1 (fr) 1990-11-16
ZA903492B (en) 1991-02-27
WO1990014358A1 (fr) 1990-11-29
EP0472659A4 (en) 1992-04-08
KR920701243A (ko) 1992-08-11
JPH04505621A (ja) 1992-10-01
AU642886B2 (en) 1993-11-04
FI915333A0 (fi) 1991-11-12

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