Methods and compositions for impairing multiplication of HIV-1

Abstract

A composition which elicits antibodies to multiple known variants of Tat protein of HIV-1 of both the B and non-B clades contains the peptide R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 SEQ ID NO: 23, and preferably an additional at least two variants of a peptide or polypeptide of the formula: R1-Asp-Pro-Y 7 -Leu-X 9 -Pro-Trp-Z 12 -R2 (SEQ ID NO: 8). In this composition, at least one of the two variants contains Arg at Y 7 and Lys at Z 12 , and in at least a second of the two variants Y 7 is Asn and Z 12 is Asn. Vaccinal and pharmaceutical compositions can contain one or more such peptides associated with carrier proteins, associated in multiple antigenic peptides or as part of recombinant proteins. Diagnostic compositions and uses are described for assessing the immune status of vaccinated patients.

1 . A composition comprising at least two variants of a peptide or polypeptide of the formula 
 R1-Asp-Pro-Y 7 -Leu-X 9 -Pro-Trp-Z 12 -R2 SEQ ID NO: 8,    wherein Y 7  is selected from the group consisting of Arg, Lys, Ser and Asn;    wherein X 9  is selected from the group consisting of Glu and Asp;    wherein Z 12  is selected from the group consisting of Lys and Asn;    wherein R1 is selected from the group consisting of hydrogen, a lower alkyl, a lower alkanoyl, and a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl;    wherein R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of one or up to about 5 additional amino acids, optionally substituted with an amide, and    wherein in at least one of said two variants Y 7  is Arg and Z 12  is Lys, and in at least a second of said two variants Y 7  is Asn and Z 12  is Asn.    
     
     
         2 . The composition according to  claim 1  wherein R1 is Val, resulting in the sequence Val-Asp-Pro-Y 7 -Leu-X 9 -Pro-Trp-Z 12  SEQ ID NO: 37.  
     
     
         3 . The composition according to  claim 1  wherein R1 is X 2 -Pro-Val, wherein X 2  is selected from the group consisting of Glu and Asp, resulting in the sequence X 2 -Pro-Val-Asp-Pro-Y 7 -Leu-X 9 -Pro-Trp-Z 12  SEQ ID NO: 38, optionally substituted with a lower alkyl or lower alkanoyl.  
     
     
         4 . The composition according to  claim 1  wherein R2 is -His-Pro-Gly-Ser-SEQ ID NO:16 optionally substituted with an amide.  
     
     
         5 . The composition according to  claim 1 , wherein said variant sequences are selected from the group consisting of 
 R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2    R1-Asp-Pro-Arg-Leu-Asp-Pro-Trp-Lys-R2    R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Asn-R2    R1-Asp-Pro-Arg-Leu-Asp-Pro-Trp-Asn-R2    R1-Asp-Lys-Arg-Leu-Glu-Pro-Trp-Lys-R2    R1-Asp-Lys-Arg-Leu-Asp-Pro-Trp-Lys-R2    R1-Asp-Lys-Arg-Leu-Glu-Pro-Trp-Asn-R2    R1-Asp-Lys-Arg-Leu-Asp-Pro-Trp-Asn-R2    R1-Asp-Ser-Arg-Leu-Glu-Pro-Trp-Lys-R2    R1-Asp-Ser-Arg-Leu-Asp-Pro-Trp-Lys-R2    R1-Asp-Ser-Arg-Leu-Glu-Pro-Trp-Asn-R2    R1-Asp-Ser-Arg-Leu-Asp-Pro-Trp-Asn-R2    R1-Asp-Asn-Arg-Leu-Glu-Pro-Trp-Lys-R2    R1-Asp-Asn-Arg-Leu-Asp-Pro-Trp-Lys-R2    R1-Asp-Asn-Arg-Leu-Glu-Pro-Trp-Asn-R2, and    R1-Asp-Asn-Arg-Leu-Asp-Pro-Trp-Asn-R2.    
     
     
         6 . The composition according to  claim 1 , comprising 
 R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2 SEQ ID NO:17, and    R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 SEQ ID NO:18    and one or more sequences selected from the group consisting of:    R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-R2 SEQ ID NO:19    R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Asn-R2 SEQ ID NO:22    R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-R2 SEQ ID NO:20    R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2 SEQ ID NO:24    R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-R2 SEQ ID NO:21; and    R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 SEQ ID NO:23.    
     
     
         7 . The composition according to  claim 1 , comprising at least seven of said variant amino acid sequences of SEQ ID NO: 8.  
     
     
         8 . The composition according to  claim 6 , comprising all eight of said sequences.  
     
     
         9 . The composition according to  claim 1 , further comprising: at least one peptide or polypeptide of the formula: 
 R3-Lys-X 42 -Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-R4 SEQ ID NO: 5,    wherein X 42  is selected from the group consisting of Gly or Ala;    wherein R3 is selected from the group consisting of hydrogen, a lower alkyl, a lower alkanoyl, and a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl;    wherein R4 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of one or up to about 5 additional amino acids excluding the basic amino acids -Lys-Arg-Arg-, optionally substituted with an amide.    
     
     
         10 . The composition according to  claim 1  wherein said peptides or polypeptides are produced recombinantly or synthetically.  
     
     
         11 . The composition according to  claim 1  wherein one or more of said peptides is expressed as a synthetic peptide coupled to a carrier protein.  
     
     
         12 . The composition according to  claim 1  wherein one or more of said peptides is expressed as a multiple antigenic peptide, optionally coupled to a carrier protein.  
     
     
         13 . The composition according to  claim 1  wherein one or more of said peptides is expressed within a recombinantly produced protein, optionally fused in frame with a carrier protein.  
     
     
         14 . The composition according to  claim 13 , wherein said carrier protein is selected from the group consisting of an  E. coli  DnaK protein, a GST protein, a mycobacterial heat shock protein 70, a diphtheria toxoid, a tetanus toxoid, a galactokinase, an ubiquitin, an α-mating factor, a β-galactosidase, and an influenza NS-1 protein.  
     
     
         15 . A pharmaceutical composition comprising a composition of  claim 1 , a pharmaceutically acceptable carrier and an optional adjuvant.  
     
     
         16 . A method for inducing anti-HIV-1 Tat antibodies, said method comprising the steps of: 
 exposing a subject to an effective amount of composition of  claim 1 , which actively induces antibodies that react with HIV-1 Tat proteins from different strains or subtypes of HIV-1.    
     
     
         17 . An in vitro method for determining the presence and or titer of antibodies induced by immunization to a Tat immunogen comprising: 
 (A) contacting a biological sample from an immunized subject with a peptide or polypeptide composition of  claim 1  bound to a solid support;    (B) washing said support to eliminate any material from said biological sample which is not bound to said sequences;    (C) contacting said support with a reagent associated with a detectable label, wherein said reagent detects binding between said sequences on said solid support and antibody in said biological sample, and wherein said label produces a detectable signal.    
     
     
         18 . An antibody composition comprising at least two antibodies, each antibody binding to a different peptide or polypeptide of the composition of  claim 1 .  
     
     
         19 . The antibody composition according to  claim 18 , wherein each antibody is selected from the group consisting of an isolated polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, an antibody produced by screening phage displays, a single chain antibody, and an antibody fragment.  
     
     
         20 . The antibody composition according to  claim 18 , comprising an antibody that binds to an epitope of R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 or R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2.  
     
     
         21 . The antibody composition according to  claim 18 , comprising an antibody which specifically binds to an epitope having the amino acid sequence Lys-X 42 -Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- SEQ ID NO: 10, wherein X 42  is selected from the group consisting of Gly and Ala.  
     
     
         22 . A pharmaceutical composition comprising an antibody or antibody composition of  claim 18 , and a pharmaceutically acceptable carrier and an optional adjuvant.  
     
     
         23 . A method for reducing the viral levels of HIV-1, said method comprising the steps of administering to a human an antibody or antibody composition of  claim 18 .  
     
     
         24 . A synthetic gene comprising a nucleic acid sequence encoding sequentially the peptides or polypeptides of the composition of  claim 1 .  
     
     
         25 . The synthetic gene according to  claim 24  wherein each nucleic acid sequence encoding an amino acid sequence is separated by a spacer sequence.  
     
     
         26 . A pharmaceutical composition comprising a synthetic gene of  claim 24 , a pharmaceutically acceptable carrier and an optional adjuvant.  
     
     
         27 . A synthetic molecule comprising the synthetic gene of  claim 24 , operatively linked to regulatory nucleic acid sequences, which direct and control expression of the product of said synthetic gene in a host cell.  
     
     
         28 . A mammalian host cell containing a synthetic gene of  claim 24.