Detection of HIV-1 DNA

Abstract

The determination of the nucleotide sequence of HTLV-III DNA; identification, isolation and expression of HTLV-III sequences which encode immunoreactive polypeptides by recombinant,DNA methods and production of viral RNA are disclosed. Such polypeptides can be employed in immunoassays to detect HTLV-III.

1 . Immunoreactive HTLV-III polypeptide expressed by cells transformed with a recombinant vector containing HTLV-III cDNA.  
     
     
         2 . A polypeptide of  claim 1  wherein said HTLV-III cDNA encodes an env gene sequence.  
     
     
         3 . A polypeptide of  claim 2  which is immunoreactive with sera of patients with acquired immunodeficiency syndrome or sera containing antibodies to HTLV-III.  
     
     
         4 . A polypeptide of  claim 1  wherein said HTLV-III cDNA encodes an env-lor gene sequence.  
     
     
         5 . A polypeptide of  claim 4  which is immunoreactive with sera of patients with acquired immunodeficiency syndrome or sera containing antibodies to HTLV-III.  
     
     
         6 . A polypeptide of  claim 1  wherein said HTLV-III cDNA is an EcoRI restriction fragment.  
     
     
         7 . A polypeptide of  claim 6  which is immunoreactive with sera of patients with acquired immunodeficiency syndrome or sera containing antibodies to HTLV-III.  
     
     
         8 . Isolated HTLV-III envelope polypeptide.  
     
     
         9 . Isolated HTLV-III polypeptide encoded by an env-lor gene sequence.  
     
     
         10 . Isolated cDNA encoding an HTLV-III gene.  
     
     
         11 . cDNA of  claim 10  encoding the HTLV-III env gene.  
     
     
         12 . cDNA of  claim 10  encoding the HTLV-III env-lor gene sequence.  
     
     
         13 . cDNA of  claim 10  encoding an EcoRI restriction fragment of HITLV-III cDNA which encodes a polypeptide immunoreactive with sera of patients with acquired immunodeficiency syndrome or sera containing antibodies to HTLV-III.  
     
     
         14 . Isolated cDNA encoding for an HTLV-III polypeptide which is immunoreactive.  
     
     
         15 . Isolated cDNA of  claim 14  encoding for an envelope polypeptide which is immunoreactive with sera of patients with acquired immunodeficiency syndrome or sera containing antibodies to HTLV-III.  
     
     
         16 . Isolated cDNA of  claim 14  which is an EcoRI restriction fragment.  
     
     
         17 . A DNA probe comprising a DNA sequence which is essentially homologous to a portion of the HTLV-III genome unique to the virus.  
     
     
         18 . A DNA probe of  claim 17  wherein the DNA sequence is essentially homologous to a portion of the HTLV III genome which encodes a polypeptide immunoreactive with sera of AIDS patents.  
     
     
         19 . A hybrid protein comprising an HTLV-III polypeptide linked to at least one other polypeptide.  
     
     
         20 . A hybrid protein of  claim 19  comprising an HTLV-III polypeptide linked to an indicator polypeptide.  
     
     
         21 . A hybrid protein of  claim 20  wherein said indicator polypeptide comprises beta-galactosidase.  
     
     
         22 . An isolated RNA transcript of the env gene of HTLV-III.  
     
     
         23 . An isolated RNA transcript of  claim 22  having a label which emits a detectable signal.  
     
     
         24 . An isolated RNA transcript of  claim 23  wherein said label comprises a radioisotope.  
     
     
         25 . A recombinant vector containing HTLV-III DNA capable of expression upon insertion into host cells.  
     
     
         26 . OmpA vector containing HTLV-III cDNA.  
     
     
         27 . pMR 100 vector containing HTLV-III cDNA.  
     
     
         28 . A method of producing HTLV-III polypeptide, comprising the steps of: 
 a. cleaving HTLV-III cDNA to produce cDNA fragments;    b. inserting the cDNA fragments into an expression vector to form a recombinant vector;    c. transforming an appropriate host cell with the recombinant vector; and    d. culturing the transformed host cell under conditions sufficient for expression of the polypeptide encoded for by the inserted HTLV-III cDNA.    
     
     
         29 . A method of  claim 28  wherein the cleaving step comprises digesting the HTLV-III cDNA with restriction endonucleases to produce restriction fragments of cDNA.  
     
     
         30 . A method of  claim 28  wherein the cleaving step comprises shearing the HTLV-III cDNA to produce cDNA fragments.  
     
     
         31 . A method of producing HTLV-III envelope polypeptide, comprising the steps of: 
 a. cleaving HTLV-III genomic cDNA with the restriction endonuclease Sstl;    b. digesting the cleaved cDNA with restriction endonucleases sufficient to generate restriction fragments which encompass at least a portion of the env gene;    c. isolating the restriction fragments;    d. producing DNA fragments of about 200-500 base pairs in length from the restriction fragments;    e. isolating the DNA fragments of about 200-500 base pairs;    f. inserting the isolated fragments into the open reading frame expression vector pMR100 for production of hybrid proteins comprising an env gene product and beta-galactosidase;    g. transforming lac z  E. coli  cells with the vector;    h. plating the transformed cells on MacConkey agar plates, maintaining the plates under conditions sufficient for the formation of colonies and selecting cell colonies exhibiting a red color;    i. culturing transformed cells from the selected colonies under conditions which allow expression of the hybrid protein;    j. obtaining cellular protein from the cultured transformed cells;    k. separating the cellular protein obtained;    l. contacting the separated protein with sera from AIDS patients to identify protein which is immunoreactive with the sera; and    m. isolating the immunoreactive protein.    
     
     
         32 . A fusion protein produced by the method of  claim 31 .  
     
     
         33 . A method of  claim 31 , further comprising the step of separating the env gene expression product from the remainder of the hybrid protein.  
     
     
         34 . A HTLV-III envelope polypeptide produced by the method of  claim 33 .  
     
     
         35 . Antibody specifically reactive with HTLV-III envelope polypeptide.  
     
     
         36 . An antibody of  claim 35  which is monoclonal.  
     
     
         37 . Anibody specifically reactive with HTLV-III polypeptide produced by recombinant DNA techniques.  
     
     
         38 . An antibody of  claim 37  which is monoclonal.  
     
     
         39 . An immunoassay for the detection of HTLV-III employing antibody which reacts specifically with HTLV-III polypeptide produced by recombinant DNA techniques.  
     
     
         40 . An immunoassay for the detection of HTLV-III employing antibody which reacts specifically with HTLV-III envelope polypeptide.  
     
     
         41 . An immunoassay of  claim 40  wherein said antibody is monoclonal.  
     
     
         42 . A sandwich type immunoradiometric assay for the detection of HTLC III employing an immobilized antibody which reacts with HTLV-III polypeptide and a soluble antibody which reacts with HTLV-III polypeptide.  
     
     
         43 . An assay kit comprising an antibody which reacts specifically with HTLV-III polypeptide bound to a solid phase and a labeled soluble antibody which reacts specifically with HTLV-III polypeptide.  
     
     
         44 . A method of detecting antibodies against HTLV-III in a bodily fluid comprising the steps of: 
 a. contacting an immunoadsorbent comprising an HTLV-III polypeptide bound to a solid phase with a bodily fluid until antibodies against HTLV-III polypeptide in the bodily fluid bind the solid phase polypeptide;    b. separating the immunoadsorbent from the bodily fluid;    c. contacting the immunoadsorbent with a labeled HTLV-III polypeptide or labeled antibody against human immunoglobulin; and    d. determining the amount of labeled polypeptide bound to immunoadsorbent as an indication of antibody to HTLV-III.    
     
     
         45 . A kit for determining the presence of antibody against HTLV-III in a bodily fluid comprising: 
 a. an immunoadsorbent comprising a HTLV-III polypeptide bound to a solid phase; and    b. labeled HLTV-III polypeptide or a labeled antibody against human immunoglobulin.    
     
     
         46 . A method of detecting HTLV-III nucleic acid in a bodily fluid comprising the steps of: 
 a. adsorbing the nucleic acid in a bodily fluid onto an adsorbent;    b. denaturing the adsorbed nucleic acid;    c. contacting the adsorbed nucleic acid with a HTLV-III DNA or RNA probe; and    d. determining if the probe hybridizes with the adsorbed nucleic acid.    
     
     
         47 . A method of  claim 46  wherein the bodily fluid is a cell lysate.  
     
     
         48 . A hybridoma cell line which produces antibody specifically reactive with HTLV-III envelope polypeptide.