Modified gp140 envelope polypeptides of hiv-1 isolates, compositions, stabilized trimeric complexes, and uses thereof

Abstract

This invention provides a modified gp140 envelope polypeptide of an HIV-1 isolate comprising a gp120 polypeptide portion comprising consecutive amino acids and a gp41 ectodomain polypeptide portion comprising consecutive amino acids, said gp41 ectodomain polypeptide portion being modified to comprise isoleucine (I) at an amino acid position equivalent to amino acid position 535; glutamine (Q) at an amino acid position equivalent to amino acid position 543; serine (S) at an amino acid position equivalent to amino acid position 553; lysine (K) at an amino acid position equivalent to amino acid position 567; and arginine (R) at an amino acid position equivalent to amino acid position 588, the amino acid positions being numbered by reference to the HIV-1 isolate KNH1144. This invention also provides nucleic acids encoding such a polypeptide, vectors, host cells, trimeric complexes and compositions thereof. Also provided are antibodies generated against the modified polypeptides and trimeric complexes, and methods of using the modified polypeptides, compositions and trimeric complexes.

1 - 96 . (canceled) 
     
     
         97 . A purified, modified HIV-1 gp140 envelope polypeptide comprising (1) a gp120 polypeptide portion comprising consecutive amino acids and (2) a gp41 ectodomain polypeptide portion comprising consecutive amino acids, said gp41 ectodomain polypeptide portion being heterologous to the gp41 ectodomain polypeptide portion of a KNH1144 HIV-1 isolate and being modified to comprise methionine (M) at an amino acid position equivalent to amino acid position 535 (M535); glutamine (Q) at an amino acid position equivalent to amino acid position 543 (Q543); serine (S) at an amino acid position equivalent to amino acid position 553 (S553); lysine (K) at an amino acid position equivalent to amino acid position 567 (K567); and arginine (R) at an amino acid position equivalent to amino acid position 588 (R588), wherein the amino acid positions are numbered by reference to the KNH1144 HIV-1 isolate. 
     
     
         98 . The purified, modified gp140 envelope polypeptide of  claim 97 , wherein (1) the gp120 polypeptide portion is further modified to comprise a cysteine (C) residue at an amino acid position equivalent to amino acid position 492, numbered by reference to the JR-FL HIV isolate, and (2) the gp41 ectodomain polypeptide portion is modified to comprise a cysteine (C) residue at an amino acid position equivalent to amino acid position 596, numbered by reference to the JR-FL HIV-1 isolate. 
     
     
         99 . The purified, modified HIV-1 gp140 envelope polypeptide of  claim 97  or  claim 98 , wherein the gp41 ectodomain polypeptide portion further comprises a proline (P) at an amino acid position equivalent to amino acid position 559, numbered by reference to the KNH1144 HIV-1 isolate. 
     
     
         100 . The purified, modified HIV-1 gp140 envelope polypeptide of  claim 97  wherein the gp120 or gp41 ectodomain is derived from a HIV-1 isolate of clade A, B, C, D, E, F, G, H, J or O. 
     
     
         101 . The purified, modified HIV-1 gp140 envelope polypeptide of  claim 97 , wherein the HIV-1 isolate is an HIV-1 JR-FL , HIV-1 Ba-L , HIV-1 5768 , HIV-1 DH123 , HIV-1 GUN-1 , HIV-1 89.6 , or HIV-1 HXB2  isolate. 
     
     
         102 . A purified, modified gp160 polypeptide which comprises a consecutive amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8. 
     
     
         103 . A method of producing a stabilized trimeric complex of HIV gp120 and gp41 wherein said gp41 comprises the following mutations numbered by reference to the KNH1144 HIV-1 isolate: X535M, X543Q, X553S, X567K, and X588R, comprising the steps of:
 a) obtaining an expression vector comprising a nucleic acid which encodes a gp120 envelope polypeptide and a gp41 envelope polypeptide;   b) introducing the following mutations into the portion of the nucleic acid which encodes the gp41 envelope polypeptide: X535M, X543Q, X553S, X567K, and X588R;   c) transfecting a cell with the expression vector resulting from step b) under conditions permitting expression of the gp120 envelope polypeptide and the gp41 envelope polypeptide; and   d) purifying the gp120 envelope polypeptides and gp41 envelope polypeptides expressed in step c) under conditions permitting association of gp120 envelope polypeptides and gp41 envelope polypeptides so as to form the stabilized trimeric complex.   
     
     
         104 . A composition comprising the purified, modified polypeptide of any one of  claims 97  to  99 , and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant. 
     
     
         105 . A composition comprising the purified, modified polypeptide of  claim 102  and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant. 
     
     
         106 . A trimeric complex which comprises a noncovalent oligomer of three identical purified, modified HIV-1 gp140 envelope polypeptides of any one of  claims 97  to  99 . 
     
     
         107 . A trimeric complex which comprises a noncovalent oligomer of three identical purified, modified HIV-1 gp140 envelope polypeptides, each gp140 polypeptide comprising a gp120 polypeptide portion and a gp41 ectodomain polypeptide portion derived from the purified, modified gp160 polypeptide of  claim 102 . 
     
     
         108 . A composition comprising the trimeric complex of  claim 106 , a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant. 
     
     
         109 . A composition comprising the trimeric complex of  claim 107 , a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant. 
     
     
         110 . The composition of  claim 108 , further comprising a non-ionic detergent. 
     
     
         111 . The composition of  claim 109 , further comprising a non-ionic detergent.