Compositions and Methods for the Treatment and Prophylaxis of Multiple Strains and Subtypes of HIV-1

Abstract

A self-adjuvanting immunogenic composition comprising multiple immunogens, each immunogen comprising a lipopeptide cap, a universal T helper sequence and an immunodominant HIV-1 Tat B cell epitope. The immunogen also comprises one or more linker sequences and/or polar charged amino acid sequences. The HIV-1 Tat B cell epitope of each immunogen has an amino acid sequence of V-D-P-Xaa7-L-Xaa9-P-W-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-amide SEQ ID NO: 1, in which the amino acid positions at Xaa7, Xaa9 and Xaa12 are selected from specific amino acid residues choices and in which the amino acid positions at Xaa13-Xaa16 may be absent or specific amino acid residue choices. The lipopeptide is a dipalmitoyl-S-glyceryl-cysteine or a tripalmitoyl-S-glyceryl cysteine or N-acetyl (dipalmitoyl-S-glyceryl cysteine), each with an optional neutral amino acid linker. Optional polar sequences of at least four charged polar amino acids enhance solubility of the immunogen and are located at the carboxy terminal end of the lipopeptide cap, optionally flanked by neutral linker amino acids, or elsewhere in the immunogen. In the composition, each immunogen differs from another immunogen by an amino acid variation at amino acid position Xaa7, Xaa9 or Xaa12 of the immunodominant HIV-1 Tat epitope. Such compositions can induce anti-HIV-1 Tat antibodies with geometric mean titers of greater than 1,000,000 on multiple HIV-1 Tat variants, when employed to immunize a subject, without any extrinsic adjuvant.

1 . A method of making a self-adjuvanting immunogenic composition comprising multiple immunogens, each immunogen comprising:
 (a) an HIV-1 Tat B cell epitope having an amino acid sequence of V-D-P-Xaa7-L-Xaa9-P-W-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16 (SEQ ID NO: 1);
 wherein Xaa7 is selected from the group consisting of R, K, N and S; 
 wherein Xaa9 is selected from the group consisting of E and D; 
 wherein Xaa12 is selected from the group consisting of K and N; 
 wherein Xaa13 is absent or H; 
 wherein Xaa14 is absent or P; 
 wherein Xaa15 is absent or G; and 
 wherein Xaa16 is absent or S; 
   (b) a universal T helper sequence;   (c) a sequence of charged polar amino acids;   (d) a dipalmitoyl-S-glyceryl-cysteine lipopeptide cap; and   wherein each said immunogen differs from another said immunogen in said composition by an amino acid variation at amino acid position Xaa7, Xaa9 or Xaa12 of said HIV-1 Tat epitope,   the method comprising synthesizing and linking together in a single synthesis mixture said immunogen components (a), (b) and (c) using solid phase synthesis methods, and introducing equimolar amounts of variant amino acids at an amino acid position selected from the group consisting of Xaa7, Xaa9 and Xaa12 and combinations thereof; and coupling the lipopeptide cap to the linked components at the N-terminal serine.   
     
     
         2 . The method according to  claim 1 , further comprising:
 (i) synthesizing said immunogens using solid phase synthesis methods commencing with an amidated C-terminal serine;
 continuing the cycles of synthesis towards the N-terminus of the HIV-1 Tat B cell epitope and introducing equimolar amounts of variant amino acids at one or more amino acid positions selected from the group consisting of Xaa7, Xaa9 and Xaa12 in a single synthesis mixture at the appropriate time during synthesis; 
 continuing the cycles of synthesis through the T helper sequence and the sequence of charged polar amino acids to the N-terminus; and 
   (ii) coupling the dipalmitoyl-S-glyceryl-cysteine lipopeptide cap to the immunogen at the N-terminal serine;   wherein for each immunogen, the lipopeptide cap is linked to the sequence of charged polar amino acids, which is linked to the universal T helper sequence, which is linked to the B cell epitope.   
     
     
         3 . The method according to  claim 1 , wherein each sequence of charged polar amino acids is optionally flanked by one or more neutral linker amino acids. 
     
     
         4 . The method according to  claim 3 , wherein said linker is selected from the group consisting of -S-, -S-S-, -G-, and -G-S-. 
     
     
         5 . The method according to  claim 3 , wherein said sequence of charged polar amino acids comprises a sequence of from 4 to 8 amino acids selected individually from the group consisting of K, E, D, and R, which is optionally flanked by said linker amino acids. 
     
     
         6 . The method according to  claim 5 , wherein said sequence of charged polar amino acids is selected from the group consisting of -K-K-K-K-, -S-K-K-K-K-S-, G-K-K-K-K-G, S-K-K-K-K-K-K-S, and G-K-K-K-K-K-K-G (SEQ ID NOs: 65, 64, 70, 69, and 71). 
     
     
         7 . The method according to  claim 3 , wherein said universal T cell helper sequence is selected from the group consisting of:
 (a) Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-Xaa SEQ ID NO: 6, wherein said Xaa is absent or L, with an optional amino acid linker;   (b) Xaa1-K-Xaa2-V-A-A-W-T-L-K-A-A-Xaa3 SEQ ID NO: 46, wherein Xaa1 and Xaa3 are each a D-Alanine and Xaa2 is L-cyclohexylalanine; and   (c) F-N-N-F-T-V-S-F-W-L-R-V-P-K-V-S-A-S-H-L-E- SEQ ID NO: 23.   
     
     
         8 . The method according to  claim 1 , wherein the composition comprises immunogens containing from two to 16 different said variants selected from the group consisting of 
       
         
           
                 
                 
                 
               
                     
                   V-D-P-R-L-E-P-W-K-H-P-G-S, 
                   SEQ ID NO: 15 
                 
                     
                     
                 
                     
                   V-D-P-K-L-E-P-W-K-H-P-G-S, 
                   SEQ ID NO: 16 
                 
                     
                     
                 
                     
                   V-D-P-N-L-E-P-W-K-H-P-G-S, 
                   SEQ ID NO: 18 
                 
                     
                     
                 
                     
                   V-D-P-S-L-E-P-W-K-H-P-G-S, 
                   SEQ ID NO: 17 
                 
                     
                     
                 
                     
                   V-D-P-K-L-E-P-W-N-H-P-G-S, 
                   SEQ ID NO: 21 
                 
                     
                     
                 
                     
                   V-D-P-N-L-E-P-W-N-H-P-G-S, 
                   SEQ ID NO: 19 
                 
                     
                     
                 
                     
                   V-D-P-S-L-E-P-W-N-H-P-G-S, 
                   SEQ ID NO: 22 
                 
                     
                     
                 
                     
                   V-D-P-N-L-D-P-W-N-H-P-G-S, 
                   SEQ ID NO: 20 
                 
                     
                     
                 
                     
                   V-D-P-R-L-D-P-W-K-H-P-G-S, 
                   SEQ ID NO: 32 
                 
                     
                     
                 
                     
                   V-D-P-K-L-D-P-W-K-H-P-G-S, 
                   SEQ ID NO: 33 
                 
                     
                     
                 
                     
                   V-D-P-N-L-D-P-W-K-H-P-G-S, 
                   SEQ ID NO: 34 
                 
                     
                     
                 
                     
                   V-D-P-S-L-D-P-W-K-H-P-G-S, 
                   SEQ ID NO: 35 
                 
                     
                     
                 
                     
                   V-D-P-R-L-E-P-W-N-H-P-G-S, 
                   SEQ ID NO: 36 
                 
                     
                     
                 
                     
                   V-D-P-R-L-D-P-W-N-H-P-G-S, 
                   SEQ ID NO: 37 
                 
                     
                     
                 
                     
                   V-D-P-K-L-D-P-W-N-H-P-G-S, 
                   SEQ ID NO: 38 
                 
                     
                   and 
                     
                 
                     
                     
                 
                     
                   V-D-P-S-L-D-P-W-N-H-P-G-S. 
                   SEQ ID NO: 39 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         9 . The method according to  claim 8 , wherein the composition comprises immunogens containing four different said variants from the group. 
     
     
         10 . The method according to  claim 8 , wherein the composition comprises immunogens containing six different said variants from the group. 
     
     
         11 . The method according to  claim 8 , wherein the composition comprises immunogens containing eight different said variants from the group. 
     
     
         12 . The method according to  claim 8 , wherein the composition comprises immunogens containing twelve different said variants from the group. 
     
     
         13 . The method according to  claim 1 , wherein, in each immunogen, the lipopeptide cap further comprises an amino acid linker sequence of -S-S- residues; and said universal T cell helper sequence is Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L SEQ ID NO: 47 with an amino acid linker of -S- linking it to the B cell epitope. 
     
     
         14 . The method according to  claim 13 , wherein a polar amino acid sequence of -K-K-K-K- (SEQ ID NO: 65) is located between the -S-S- of said linker residues. 
     
     
         15 . A pharmaceutical composition produced by the method of  claim 1 . 
     
     
         16 . A method of making a water-soluble, self-adjuvanting immunogenic composition comprising 16 immunogens, each immunogen comprising:
 (a) a different HIV-1 Tat B cell epitope sequence selected from the group consisting of   
       
         
           
                 
                 
                 
               
                     
                   V-D-P-R-L-E-P-W-K-H-P-G-S, 
                   SEQ ID NO: 15 
                 
                     
                     
                 
                     
                   V-D-P-K-L-E-P-W-K-H-P-G-S, 
                   SEQ ID NO: 16 
                 
                     
                     
                 
                     
                   V-D-P-N-L-E-P-W-K-H-P-G-S, 
                   SEQ ID NO: 18 
                 
                     
                     
                 
                     
                   V-D-P-S-L-E-P-W-K-H-P-G-S, 
                   SEQ ID NO: 17 
                 
                     
                     
                 
                     
                   V-D-P-K-L-E-P-W-N-H-P-G-S, 
                   SEQ ID NO: 21 
                 
                     
                     
                 
                     
                   V-D-P-N-L-E-P-W-N-H-P-G-S, 
                   SEQ ID NO: 19 
                 
                     
                     
                 
                     
                   V-D-P-S-L-E-P-W-N-H-P-G-S, 
                   SEQ ID NO: 22 
                 
                     
                     
                 
                     
                   V-D-P-N-L-D-P-W-N-H-P-G-S, 
                   SEQ ID NO: 20 
                 
                     
                     
                 
                     
                   V-D-P-R-L-D-P-W-K-H-P-G-S, 
                   SEQ ID NO: 32 
                 
                     
                     
                 
                     
                   V-D-P-K-L-D-P-W-K-H-P-G-S, 
                   SEQ ID NO: 33 
                 
                     
                     
                 
                     
                   V-D-P-N-L-D-P-W-K-H-P-G-S, 
                   SEQ ID NO: 34 
                 
                     
                     
                 
                     
                   V-D-P-S-L-D-P-W-K-H-P-G-S, 
                   SEQ ID NO: 35 
                 
                     
                     
                 
                     
                   V-D-P-R-L-E-P-W-N-H-P-G-S, 
                   SEQ ID NO: 36 
                 
                     
                     
                 
                     
                   V-D-P-R-L-D-P-W-N-H-P-G-S, 
                   SEQ ID NO: 37 
                 
                     
                     
                 
                     
                   V-D-P-K-L-D-P-W-N-H-P-G-S, 
                   SEQ ID NO: 38 
                 
                     
                   and 
                     
                 
                     
                     
                 
                     
                   V-D-P-S-L-D-P-W-N-H-P-G-S; 
                   SEQ ID NO: 39 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         (b) a universal T helper sequence; 
         (c) a dipalmitoyl-S-glyceryl-cysteine lipopeptide cap; and 
         (d) a sequence of charged polar amino acids, the method comprising:
 (i) synthesizing said immunogens using solid phase synthesis methods commencing with an amidated C-terminal serine;
 continuing the cycles of synthesis towards the N-terminus of the HIV-1 Tat B cell epitope and introducing equimolar amounts of each variant amino acid for each position at the amino acid positions Xaa7, Xaa9 and Xaa12 in a single synthesis mixture at the appropriate time during synthesis; 
 continuing the cycles of synthesis through the T helper sequence and the sequence of charged polar amino acids to the N-terminus; and 
 
 (ii) coupling the dipalmitoyl-S-glyceryl-cysteine lipopeptide cap to the immunogen at the N-terminal serine; 
 
         wherein for each immunogen, the lipopeptide cap is linked to the sequence of charged polar amino acids, which is linked to the universal T helper sequence, which is linked to the B cell epitope. 
       
     
     
         17 . A pharmaceutical composition produced by the method of  claim 16 . 
     
     
         18 . A self-adjuvanting immunogenic composition comprising multiple immunogens, each immunogen comprising:
 (a) an HIV-1 Tat B cell epitope having an amino acid sequence of V-D-P-Xaa7-L-Xaa9-P-W-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16 (SEQ ID NO: 1);
 wherein Xaa7 is selected from the group consisting of R, K, N and S; 
 wherein Xaa9 is selected from the group consisting of E and D; 
 wherein Xaa12 is selected from the group consisting of K and N; 
 wherein Xaa13 is absent or H; 
 wherein Xaa14 is absent or P; 
 wherein Xaa15 is absent or G; and 
 wherein Xaa16 is absent or S; 
   (b) a universal T helper sequence;   (c) a sequence of charged polar amino acids;   (d) a dipalmitoyl-S-glyceryl-cysteine lipopeptide cap; and   wherein each said immunogen differs from another said immunogen in said composition by an amino acid variation at amino acid position Xaa7, Xaa9 or Xaa12 of said HIV-1 Tat epitope.