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
• • 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
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the invention relates generally to immunoassays in which antibodies specific for human immunodeficiency virus type 1 (HIV-1) are detected.
• HIV-1 human immunodeficiency virus type 1
• HIV-1 is the proposed causative agent of Acquired Immune Deficiency Syndrome (AIDS) .
• Individuals infected by HIV-1 produce serum antibodies which bind various viral proteins, particularly the core, poly erase, and envelope proteins (Allan et al., 1985, Science 228:1091; Chang et al., 1985, Science 228:93, and 1985, Nature 315:151; Crowl et al., 1985, Cell 41:979; Veronese et al., 1985, Science 229:1402, and 1986, Science 231:1289). Immunoassays which detect these antibodies are used to diagnose HIV-1 infection and to screen donated blood for HIV-1 contamination.
• ELISAs enzyme-linked im unosorbant assays
• RIAs radioimmunoassays
• Whole viral lysates which contain both HIV and non-HIV proteins, and purified viral components, including recombinant proteins and synthetic peptides, have been used as antibody adsorbants.
• Western blots have been used to detect antibodies which recognize HIV proteins that have been separated by size.
• the invention provides diagnostic and detection methods which can be used to distinguish between persons who are infected with HIV-1 and persons who have been vaccinated against HIV-1 with peptides corresponding to all or a portion of the principal neutralizing determinant (PND) , and also offers a more sensitive and less expensive diagnostic method, e.g., for detecting seroconversion.
• PND principal neutralizing determinant
• the invention features a method of diagnosing HIV-1 infection in a human which includes contacting a biological sample from the human with a first antigen which is capable of forming immune complexes with antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes production in an human of HIV-neutralizing antibodies, formation of immune complexes with the first antigen being diagnostic of an HIV infection in the individual.
• 'neutralization* refers to the ability of the antibody to inhibit HIV infection of cells by cell-free virions, or fusion of infected and uninfected cells, or both; 'immune complex' refers to a non-covalently bound antibody/antigen pair; 'specific for' and 'reacting with' mean capable of specifically binding to an antigen; an antibody that is 'substantially incapable of forming immune complexes' means -that there will be less than a three-fold difference between the binding of the antibody to a specified antigen and the binding of normal human serum to the same antigen.
• the biological sample may be a bodily fluid, e.g., serum; and the first antigen is immobilized on a solid support, although any standard immunoassay format can be used.
• the method may further include the steps of contacting the sample with a second antigen which is immobilized on a second solid support, wherein the second antigen is the immunogen.
• the first antigen is HIV-1 gpl60 envelope protein which lacks a portion of the principal neutralizing determinant (PND) or a portion of the PND to which neutralizing antibodies can be raised, i.e., a neutralizing portion thereof.
• PND principal neutralizing determinant
• the 'principal neutralizing determinant' encompasses the region of gpl60 located between amino acids 297 and 331, inclusive, according to the number convention of Ratner et al., 1985, Nature 313:277, hereby incorporated by reference.
• this protein is gpl60 ⁇ 135.
• gpl60 ⁇ 135 means any gpl60 protein that lacks the PND; gpl60 ⁇ 135 can be expressed from a recombinant gene that has been engineered to delete the DNA coding for the PND or a portion of the PND to which neutralizing anitbodies are raised, or may be a protein which has been chemically altered to lack the PND.
• the second antigen includes the PND of gpi ⁇ O; more preferably, it includes intact HIV-1 gpl60 envelope protein (i.e., which contains the principal neutralizing determinant), e.g., gpl ⁇ OMN, or a fragment thereof or a synthetic peptide having an amino acid sequence corresponding to that of gpl60, any of which may be the immunogen.
• intact HIV-1 gpl60 envelope protein i.e., which contains the principal neutralizing determinant
• gpl ⁇ OMN a fragment thereof or a synthetic peptide having an amino acid sequence corresponding to that of gpl60, any of which may be the immunogen.
• the second antigen is a protein fragment or a synthetic peptide having an amino acid sequence corresponding to that of the PND of gpl60, e.g., the PND of gpl ⁇ OMN (i.e., gpl60 from the MN isolate) or a neutralizing portion thereof; most preferably, the second antigen is a combination of gpl ⁇ OMN and the PND of the immunogen.
• an 'immunogen' is an antigen which causes the production of neutralizing antibodies by the immune system of a mammal, e.g., a rabbit on a human.
• the detection method in which the antigen is gpl60 ⁇ 135 results in detection of HIV-1-specific antibodies which recognize regions of gpl ⁇ o lying outside the PND
• the detection method in which the antigen is gpl60 results in detection of antibodies specific for any portion of the gpi ⁇ O molecule, and will include the detection of PND-specific antibodies.
• a biological sample from a person infected with HIV-1 would include antibodies detectable by both methods, whereas a sample from a person who had been immunized against HIV-1 with PND peptides but who is not infected with the virus, would include antibodies detectable only in the latter method, which employs either the complete gpl60 molecule, i.e., that contains the PND, or PND peptides, or a combination of the two.
• an amino acid sequence 'corresponding to' or 'derived from' means the amino acid sequence is identical to all or a portion of another amino acid sequence.
• 'MN' refers to the MN prototype of HIV or a viral variant of the MN prototype; the MN prototype virus is defined by the amino acid sub-sequence within the PND region of the gpl ⁇ O envelope protein R-I-H-I-G-P-G-R-A-F-Y; MN viral variants are herein defined as variants which exhibit complete amino acid sequence homology at residues I-G-P-G-R, and at least 36% homology with the remaining 6 amino acids of the MN sequence given above.
• the method further includes contacting the sample with an agent (e.g., an enzymatically or radioactively labeled component) capable of binding to the first and second adsorbed antibodies, and detecting the bound agent as an indication of the presence in the sample of first and second antibodies; for example, the agent may be protein A or a labeled antibody capable of binding to the antibodies.
• an agent e.g., an enzymatically or radioactively labeled component
• the agent may be protein A or a labeled antibody capable of binding to the antibodies.
• the invention also features an HIV-1 envelope in which the principal neutralizing determinant, i.e., the region lying between amino acid residues 297-331, inclusive, or a region encompassing all or part of the PND, or a neutralizing portion of the PND has been deleted.
• this protein is gpl60 ⁇ 135, and may be immobilized on a solid support.
• the invention also features a. it for detecting the presence of HIV-1 antibodies in a human biological sample, which includes a first antigen capable of forming immune complexes with antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes production in a human of HIV-neutralizing antibodies, and an agent capable of reacting with first and second antibodies.
• the first antigen is immobilized on a solid support; preferably, it is HIV-I gpl60 which contains a deletion of the PND region lying between residues 297 and 331, or a portion of the PND to which neutralizing antibodies are raised; most preferably, the first antigen is gpl60 ⁇ 135.
• the detection agent may be labeled and may be, for example, protein A or a labeled anti-human immunoglobulin, e.g., a labeled Fc-region- specific antibody.
• the kit may further include the second antigen immobilized on a support; preferably, the second antigen is a peptide having an amino acid sequence corresponding to the amino acid sequence of an immunogen which causes production of HIV neutralizing antibodies, i.e., the HIV-1 PND sequence from the MN variant; more preferably, gpl60 is immobilized on the second support, and the peptides have sequences corresponding to the sequence of the PND of the MN PND.
• the invention features a method of detecting HIV-1 infection in a person, which includes contacting an antibody-containing biological sample from the person with HIV-1 gpl ⁇ OMN, formation of immune complexes between the sample antibodies and gpl60MN being diagnostic of HIV infection.
• the use of gpi60 containing the PND sequence of the MN isolate provides for a more sensitive assay and allows for the earlier detection of seroconversion than can be obtained with other gpl60 PND sequences, e.g., IIIB.
• the first antigen is immobilized on a solid support.
• the antigen may further include a peptide having an amino acid sequence corresponding to an HIV-1 gpl60 PND or a neutralizing portion of the PND.
• a synthetic peptide corresponding to all or a portion of the PND are added to assays based on a gpl60 adsorbant, the amount of gpl60 used in the assay can be reduced without a loss in assay sensitivity.
• the invention also features a kit for detecting HIV- 1 antibodies in a human biological sample, which contains immobilized HIV-1 gpl ⁇ OMN, and an agent capable of reacting with an antibody.
• the sample is serum and the agent is a labeled molecule.
• the invention also features a kit for diagnosing the presence of HIV-1 antibodies in a human biological sample which includes combined immobilized gpl60 and a peptide having an amino acid sequence corresponding to that of the MN PND or a portion thereof, and an agent capable of reacting with the antibodies.
• Methods of the invention are useful for distinguishing between HIV-1 infection and immunization, and are inexpensive and safe; for example, recombinant protein components, such as full-length gpl ⁇ O, which are expensive to produce, may be supplemented with synthetic peptides having the same antigenic sites. Methods of the invention thus reduce expense without sacrificing sensitivity, and enable early detection of seroconversion.
• Drawings Fig. 1 is an ELISA in which gpl60 ⁇ 135 and gpl ⁇ OIIIB
• gpl ⁇ O from the IIIB isolate are compared as antigens for a panel of HIV positive human sera.
• Fig. 2 is an ELISA in which the response of the gpl ⁇ O ⁇ 135-based assay was compared to the response of the gpl ⁇ OIIIB-based assay to various dilutions of a single serum.
• Fig. 3 is an ELISA in which PND-based peptides were used.
• Fig. 4 is an ELISA in which the antigen consisted of synthetic peptides in combination with gpl ⁇ OIIIB.
• Fig. 5 is a Western blot in which gpl60 ⁇ 135 and gpl ⁇ OIIIB were probed with normal human serum (NHS) , human HIV positive (+) serum, or anti-RP174C serum.
• NHS normal human serum
• gpl60 ⁇ 135 and gpl ⁇ OIIIB were probed with normal human serum (NHS) , human HIV positive (+) serum, or anti-RP174C serum.
• the invention provides a method of detecting and distinguishing between antibodies which recognize different regions of the HIV-1 envelope protein gpl ⁇ O, e.g., the principal neutralizing determinant vs. non-PND regions, without a significant loss of sensitivity for the antibodies. This is accomplished by using, as an adsorbant, a recombinant envelope protein from which the amino acids corresponding to only one determinant are deleted; as a result, antibodies directed against the non-deleted determinant(s) are detected, whereas those directed against the deleted determinant are not detected. Preparation of recombinant ⁇ pl ⁇ OIIIB- gp!60 ⁇ 135.
• gpl60 ⁇ 135 is a gpl ⁇ O polypeptide which contains a deletion of the PND region.
• gpl60 ⁇ 135 is used herein as an example of a gpl60 polypeptide having a deletion which renders the protein incapable of reacting with an antibody to the PND; the deletion may encompass one or more amino acids between residues 297 and 331, inclusive.
• the preparation of gpl60 ⁇ 135 was described by Javaherian et al., 1989, Proc. Nat. Aca. Sci. 86:6768.
• gpl60 ⁇ 135 was derived from the gpl ⁇ OIIIB envelope gene cloned into the baculovirus transfer vector pAc ⁇ lO (Rusche et al, 1987, supra) .
• any MN polypeptide or fragment, or any MN hybrid which retains the PND of the MN isolate may be used, e.g., gpl ⁇ OMN.
• the gp 60MN/IIIB hybrid was constructed by direct replacement of the Bglll fragment of the MN envelope. This resulted in a hybrid IIIB/MN envelope gene having an MN PND and contained in a IIIB gpl ⁇ O framework. Antibodies generated to the resulting PND region of the hybrid molecule are specific for MN.
• This hybrid envelope gene contained in the baculovirus transfer vector pAc ⁇ lO was then used to generate recombinant baculovirus, as described in Rusche et al., 1987, supra.
• Synthetic peptides corresponding to the principal neutralizing determinant were prepared by the Merrifield solid-phase synthesis procedure (Merrifield, 1963, J. Amer. Chem. Soc. 85:2149). Peptides were purified by reverse- phase HPLC and characterized by amino acid composition analysis, according to procedures well-known in the art. The designation of the peptides, the isolate to which they correspond, and their sequences are as follows: RP135 (IIIB) NNTRKSIRIQRGPGRAFVTIGKIGC RP174C (IIIB) CNNTRKSIRIQRGPGRAFVTIGKIGC RP1 2 (MN) YNKRKRIHIGPGRAFYTTKNIIGC
• RP70C (MN) INCTRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAHCNIS
• Peptides RP174c and RP70c each contain an intramolecular disulfide bond between the two cysteine residues.
• a method for creating such a bond is described in Zhang et al., 1988, Biochemistry 27:3785. The existence of these bonds was confirmed by mass spectrometric analysis. Detection of Antibodies
• the adsorbed antibodies are detected by any of several well-known methods, one example of which is an enzyme-linked i munosorbant assay (ELISA) .
• ELISA enzyme-linked i munosorbant assay
• the antigen (gpl60 ⁇ 135, gpl ⁇ OIIIB, gpl60MN/IIIB, synthetic peptides, or a combination of these reagents) is diluted in suitable buffer such as a sodium carbonate buffer (pH 9.6) to a concentration of >0.125 ug/ml (preferably 1 ug/ml) and 50 ul is added to each well of a polystyrene 96-well microtiter plate (Immunodynamics, Chantilly, VA) .
• suitable buffer such as a sodium carbonate buffer (pH 9.6) to a concentration of >0.125 ug/ml (preferably 1 ug/ml) and 50 ul is added to each well of a polystyrene 96-well microtiter plate (Immunodynamics, Chantilly, VA) .
• the wells are emptied, refilled with a blocking solution such as 10 mg/ml bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.02% sodium azide, and allowed to incubate :> 60 min at RT.
• BSA bovine serum albumin
• PBS-T phosphate-buffered saline
• Serum samples to be tested for the presence of anti-gpl ⁇ O antibodies are diluted in PBS-T and added to the plate (50 ul per well) . Several serum dilutions may be selected.
• a labeled antibody-detecting agent solution is added to the wells and allowed to incubate for 30-90 min at RT.
• agents which may be used are goat anti- human IgG:horseradish peroxidase conjugate, goat anti-human IgG:alkaline phosphatase conjugate, Staphylococcal protein A:horseradish peroxidase conjugate, or any conventional conjugate.
• the wells are washed three times with PBS-T and treated with a suitable substrate solution such as the horseradish peroxidase substrate 3,3*-azino-bis(3-ethyl benzthiazoline- 6-sulfonic acid) (ABTS) and hydrogen peroxide.
• ABTS horseradish peroxidase substrate 3,3*-azino-bis(3-ethyl benzthiazoline- 6-sulfonic acid)
• the solutions are allowed to incubate 15-20 min and the absorbance of the solution in each well (at 410 nm for ABTS) is determined. The absorbance of each solution is proportional to the amount of antibody adsorbed by the antigen.
• the data demonstrates that immunoassays which employ gpl60 ⁇ 135 and gpl ⁇ OIIIB are equally effective for detecting anti-HIV antibodies in all of the human serum samples tested.
• a single HIV-positive serum was tested at varying dilutions using the above procedure. Dilutions ranged from 1:1 to 1:8192.
• the response of the gpl60 ⁇ 135-based assay was identical to the response of the gpl60IIIB-based assay at each dilution, within experimental error.
• Antisera were prepared according to conventional methods by immunizing the designated animal with the corresponding peptide or protein. Each serum was tested in immunoassays using each of four different antigens: RP135, RP142, gpl60 ⁇ 135 and gpl ⁇ OIIIB. Antigens were coated at 1 ug/ml in carbonate buffer, and bound antibodies were detected using goat anti-human IgG:horseradish peroxidase conjugate. The resulting absorbance values are shown in Fig. 3.
• Antisera from the guinea pigs immunized with RP174c produced significant absorbance when RP135 and gpl ⁇ OIIIB were used as antigens, but produced no response when gpl60 ⁇ 135 or RP142 (a peptide from the MN PND) were used.
• Antiserum elicited by gpl60 ⁇ 135 or gpl60IIIB produced a similar response when gpl60 ⁇ 135 of gpl ⁇ OIIIB were used as antigens.
• Table 1 shows that the response of the assay when gpl60MN/IIIB was used as the antigen was greater. Table 1 also shows that the response of the gpl60MN/IIIB-based assay to the normal human serum pool was also greater than the. response of the gpl60IIIB-based assay, but if these background values are subtracted, the response to the HIV-positive serum pool remains greater when gpl60MN/IIIB is used as the antigen, particularly at lower concentrations of gpl ⁇ O. PND Peptides in Combination with gpl ⁇ O as Antigen
• Synthetic peptide RP142 corresponding to the principal neutralizing determinant of the MN isolate, was used in combination with gpl ⁇ OIIIB as a antigen.
• Wells of a microtiter plate were coated with varying concentrations of gpl ⁇ OIIIB (in carbonate buffer) , either with or without peptide RP142.
• the response of each assay to an HIV- positive human serum is shown in Fig. 4.
• the data in Fig. 4 shows that a combination of the synthetic peptide and gpl60 results in higher sensitivity in the ELISA and allows for use of lower concentrations of gpl ⁇ O than when gpl ⁇ O is used alone as an antigen.
• Gpl60 ⁇ 135 and gpl ⁇ OIIIB were individually subjected to SDS polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters (Towbin et al., 1979, Proc. Nat. Aca. Sci. 76:4350). Blots were treated with an HIV-positive human serum sample, antisera raised against RP174c in guinea pigs, and normal human serum. Adsorbed antibodies were visualized by treatment of the blots with goat anti-human IgG:horseradish peroxidase conjugate or goat anti-mouse IgG:horseradish peroxidase conjugate, followed by a diaminobenzidine solution and hydrogen peroxide.
• Fig. 5 shows that the HIV positive human serum recognized both of the blotted gpl ⁇ Os, whereas the anti-RPl74c serum recognized gpl60IIIB, but not gpl60 ⁇ 135. Normal human serum did not recognize either protein

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Molecular Biology (AREA)
• Virology (AREA)
• Immunology (AREA)
• Engineering & Computer Science (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Organic Chemistry (AREA)
• Urology & Nephrology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Microbiology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
• Biophysics (AREA)
• AIDS & HIV (AREA)
• Gastroenterology & Hepatology (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=24135683

Family Applications (1)

Country Status (4)

Families Citing this family (2)

Family Cites Families (3)

• 1991
  • 1991-06-07 WO PCT/US1991/004021 patent/WO1991019011A1/en not_active Ceased
  • 1991-06-07 JP JP91511485A patent/JPH05507559A/ja active Pending
  • 1991-06-07 CA CA 2082806 patent/CA2082806A1/en not_active Abandoned
  • 1991-06-07 EP EP91912258A patent/EP0532673A1/en not_active Withdrawn

Also Published As

Similar Documents

Legal Events