Molecularly cloned acquired immunodeficiency syndrome polypeptides and their methods of use

Abstract

Diagnostic product and vaccine for Acquired Immuno-deficiency Syndrome (AIDS) and methods for making and using same, wherein viral polypeptide sequences from an AIDS associated retrovirus are expressed directly or as a fusion polypeptide in a prokaryotic or mammalian cell expression host to produce a diagnostic product which specifically binds complementary antibody produced by individuals afflicted with AIDS or a vaccine against AIDS which confers resistance to infection by AIDS associated retrovirus. The reverse transcriptase of an AIDS associated retrovirus is used separately or in a whole cell assay to identify compounds which selectively inhibit retroviral reverse transcriptase.

1 . A composition comprising a predetermined polypeptide encoded by the genome of an AIDS associated retrovirus which composition is essentially free of other naturally occurring AIDS associated polypeptides or human proteins from cells for which the AID-associated retrovirus is naturally infective, said predetermined polypeptide sequence having at least one antigenic determinant capable of specifically binding complementary antibody.  
     
     
         2 . The composition of  claim 1  wherein said predetermined polypeptide sequence is a sequence encoded by the gag region of the genome of an AIDS associated retrovirus.  
     
     
         3 . The composition of  claim 1  wherein said predetermined polypeptide sequence is a fusion of the AIDS associated retroviral polypeptide with a viral or bacterial polypeptide.  
     
     
         4 . The composition of  claim 1  wherein said predetermined polypeptide sequence is derived from a sequence encoded by the env region of the genome of an AIDS associated retrovirus.  
     
     
         5 . The composition of  claim 1  wherein said predetermined polypeptide sequence is from gp-41, gp-65 or gp-120.  
     
     
         6 . The composition of  claim 1  wherein a normally glycosylated predetermined polypeptide sequence of an AIDS associated retrovirus is not glycosylated.  
     
     
         7 . The composition of  claim 1  wherein said predetermined polypeptide sequence contains methionine or formylemethionine at the N-terminus.  
     
     
         8 . A process comprising isolating a predetermined Polypeptide sequence of an AIDS associated retrovirus from a prokaryotic or mammalian recombinant expression host containing DNA sequence encoding said predetermined polypeptide sequence, wherein the isolated predetermined polypeptide sequence contains at least one antigenic determinant capable of specifically binding complementary antibody.  
     
     
         9 . The polypeptide product made by the process of  claim 8 .  
     
     
         10 . A composition comprising a predetermined fragment of a predetermined polypeptide sequence of an AIDS associated retrovirus, said fragment containing at least one antigenic determinant capable of specifically binding complementary antibody.  
     
     
         11 . The composition of  claim 10  essentially free of other AIDS associated viral polypeptides.  
     
     
         12 . The composition of  claim 10  wherein said polypeptide fragment is the N-terminal sequence of a predetermined polypeptide sequence of an AIDS associated retrovirus.  
     
     
         13 . The composition of  claim 10  wherein said polypeptide fragment is the C-terminal sequence of a predetermined polypeptide sequence of an AIDS associated retrovirus.  
     
     
         14 . The composition of  claim 13  wherein said polypeptide fragment is a fragment of the AIDS associated gp120 env protein.  
     
     
         15 . The composition of  claim 10  wherein said polypeptide fragment is from a sequence encoded by the gag region of the genome of an AIDS associated retrovirus.  
     
     
         16 . The composition of  claim 10  wherein said polypeptide fragment is from p-24.  
     
     
         17 . The composition of  claim 10  wherein said polypeptide fragment is from a sequence encoded by the env region of the genome of an AIDS associated retrovirus.  
     
     
         18 . The composition of  claim 10  wherein said polypeptide fragment is from gp-41, gp-65 or p120.  
     
     
         19 . A fusion polypeptide comprising (a) a first predetermined polypeptide sequence or fragment thereof, of an AIDS associated retrovirus having at least one antigenic determinant capable of specifically binding complementary antibody and (b) a second polypeptide sequence which is not immunologically cross-reactive with antibodies normally present in a biologically derived sample—which is to be assayed for the presence of said complementary antibody.  
     
     
         20 . The fusion polypeptide of  claim 19  wherein said predetermined polypeptide sequence is from a sequence encoded by the gag region of the genome of an AIDS associated retrovirus.  
     
     
         21 . The fusion polypeptide of  claim 19  wherein said predetermined polypeptide sequence is from p-24.  
     
     
         22 . The fusion polypeptide of  claim 19  wherein said predetermined polypeptide sequence is from a sequence encoded by the env region of a genome of an AIDS associated retrovirus.  
     
     
         23 . The fusion polypeptide of  claim 19  wherein said predetermined polypeptide sequence is from gp-41, gp-65 or gp-120.  
     
     
         24 . The fusion polypeptide of  claim 19  wherein the first and second polypeptide sequences are fused.  
     
     
         25 . The fusion polypeptide of  claim 24  wherein said fusion is by a peptide bond.  
     
     
         26 . The fusion polypeptide of  claim 19  wherein the amino terminus of the first polypeptide sequence is fused to the carboxyl terminus of the second polypeptide sequence.  
     
     
         27 . A composition comprising a variant polypeptide sequence, of an AIDS associated retrovirus containing at least one antigenic determinant capable of specifically binding complementary antibody to an AIDS associated retrovirus.  
     
     
         28 . The composition of  claim 27  wherein said variant polypeptide sequence is an insertion, deletion or substitution of an amino acid residue of a polypeptide sequence of an AIDS associated retrovirus.  
     
     
         29 . The composition of  claim 27  wherein said variant polypeptide sequence is a labeled or bound derivative of a polypeptide sequence of an AIDS associated retrovirus.  
     
     
         30 . A diagnostic test kit comprising the composition of claims  1 ,  10 ,  19  or  27 .  
     
     
         31 . The composition of claims  1 ,  10 ,  19  or  27  which is immobilized on a solid phase.  
     
     
         32 . The composition of claims  1 ,  10 ,  19  or  27  which is labeled with a detectable marker.  
     
     
         33 . A vaccine comprising the composition of claims  1 ,  10 ,  19  or  27 , wherein said polypeptide is capable of inducing the production of neutralizing antibodies which confer resistance to infection by AIDS associated retrovirus.  
     
     
         34 . The vaccine of  claim 33  wherein the AIDS-associated polypeptide is a fusion with a second polypeptide and said second Polypeptide does not normally induce antibodies which cross-react with proteins which are naturally occurring in the subject such vaccine is directed to.  
     
     
         35 . The vaccine of  claim 34  wherein the second polypeptide is a trpLE fusion.  
     
     
         36 . The vaccine of  claim 33  including a pharmaceutically acceptable vehicle.  
     
     
         37 . A method of vaccination against AIDS comprising the administration of the vaccine of  claim 33  at a dosage level which is effective in raising antibodies in a human subject.  
     
     
         38 . The DNA sequence encoding the composition of  claim 19  or  27 .  
     
     
         39 . The DNA sequence encoding the fusion polypeptide of claims  1  or  10  wherein said DNA sequence is essentially free of DNA complementary to the naturally occurring flanking RNA sequences.  
     
     
         40 . A replicable prokaryotic or mammalian cell expression vector containing the DNA sequence of claims  38  or  39  which is capable of transforming a host cell to expression of an AIDS associated polypeptide.  
     
     
         41 . A prokaryotic or mammalian cell culture containing the expression vector of  claim 40 .  
     
     
         42 . A method for the detection of antibody contained in a test sample comprising: 
 a) contacting said sample with the composition of claims  31  or  32  to bind the diagnostic product with complementary antibody in said sample; and    b) detecting the amount bound or unbound detectable marker.    
     
     
         43 . The method of  claim 42  wherein the test sample is urine, saliva or a blood fluid.  
     
     
         44 . A reverse transcriptase of an AIDS associated retrovirus which is essentially free of other AIDS associated retrovirus proteins.  
     
     
         45 . A cell-free DNA sequence encoding the reverse transcriptase of  claim 44 .  
     
     
         46 . A replicable expression vector containing the DNA sequence of  claim 45 , said vector being capable of expressing reverse transcriptase when contained within a transformed cell.  
     
     
         47 . A eukaryotic cell culture containing the expression vector of  claim 46 .  
     
     
         48 . The culture of  claim 47  wherein the cells are those of a mammalian cell line.  
     
     
         49 . An assay for identifying compounds which inhibit reverse transcriptase of an AIDS associated retrovirus comprising: 
 a) reacting the reverse transcriptase of  claim 44  with a compound which is suspected to be capable of inhibiting said reverse transcriptase; and    b) measuring the level of reverse transcriptase activity of the reaction mixture of step (a).    
     
     
         50 . The assay of  claim 49  wherein the reverse transcriptase is presen in a transformed recombinant mammalian host cell.  
     
     
         51 . A composition comprising an AIDS-associated retroviral E′ polypeptide, which composition is free of proteins from AIDS-associated retrovirus infected cells that are not encoded by the AIDS-associated retrovirus and which is free of infectious AIDS-associated retroviral virions.  
     
     
         52 . The composition of  claim 51  wherein the E′ polypeptide has a relative molecular weight on SDS PAGE of about 28,000 or about 26,500.  
     
     
         53 . The composition of  claim 51  wherein the E′ polypeptide has the amino acid sequence of the E′ polypeptide of FIG. 2 or its naturally-occurring alleles.  
     
     
         54 . A composition comprising an E′ polypeptide which is other than the E′ polypeptide of FIG. 2 or its naturally-occurring alleles but which is immunologically cross-reactive with the E′ polypeptide of FIG. 2 or its naturally-occurring alleles.  
     
     
         55 . The composition of  claim 51  which is a vaccine.  
     
     
         56 . The composition of  claim 51  which is sterile, comprises a pharmaceutically-acceptable carrier, and contains the E′ polypeptide in an amount sufficient upon administration to an animal to elicit the formation of antibodies to the E′ polypeptide.  
     
     
         57 . The composition of  claim 51  which further contains known amounts of other predetermined AIDS-associated retroviral polypeptides.  
     
     
         58 . A diagnostic test kit for AIDS-associated retroviral infection comprising the composition of  claim 51 .  
     
     
         59 . The composition of  claim 51  wherein the E′ polypeptide is labelled with a detectable marker.  
     
     
         60 . The composition of  claim 59  wherein the detectable marker is an enzyme, radioisotope or fluorescent substituent.  
     
     
         61 . The composition of  claim 58  wherein the test kit comprises an immobilized antibody capable of binding the E′ polypeptide, said antibody being in a container separate from the E′ polypeptide.  
     
     
         62 . An antibody capable of binding an AIDS-associated retroviral E′ polypeptide which is free of antibody capable of binding any other AIDS-associated retroviral-encoded polypeptide and which is free of bound E′ polypeptide.  
     
     
         63 . The antibody of  claim 62  which is immobilized.  
     
     
         64 . The antibody of  claim 63  which is immobilized by adsorption to a polyolefin or to antibody capable of binding the constant region of the antibody of  claim 63 .  
     
     
         65 . A method for the early determination of the presence of an AIDS-associated retroviral infection in a test subject comprising 
 (a) obtaining a sample from the test subject;    (b) detecting the presence in the test sample of a polypeptide encoded by the RNA of the AIDS-associated retrovirus which is other than a polypeptide component of the purified AIDS-associated retrovirus virion.    
     
     
         66 . The method of  claim 65  wherein the detected polypeptide is an E′ polypeptide.  
     
     
         67 . The method of  claim 65  further comprising additionally detecting the presence of a predetermined polypeptide component of the AIDS-associated retrovirus virion.  
     
     
         68 . Nucleic acid encoding an AIDS-associated retroviral E′ polypeptide free of flanking proviral sequences encoding other complete AIDS-associated retroviral polypeptides.  
     
     
         69 . A replicable vector comprising the nucleic acid of  claim 68 .  
     
     
         70 . The nucleic acid of  claim 68  which is labelled with a detectable substituent.