Modified envelope protein of human immunodeficiency virus and use thereof

1 . A recombinant protein, which comprises gp120 and gp41 ectodomain (gp41ECTO) derived from human immunodeficiency virus (HIV), wherein the gp120 is located between β27 and α8 of the gp41ECTO;

and the recombinant protein comprises: α6, α7, β27 of gp41ECTO; gp120; α8, α9 of gp41ECTO, from its N-terminal to C-terminal;

and wherein:

(i) the gp41ECTO comprises a substitution of 1-12 consecutive amino acids in a linkage region between β27 and α8 with gp120; and wherein the linkage region corresponds to amino acid positions 607-618 of a gp160 sequence of isolate HXB2; or

ii) the gp120 is inserted between adjacent amino acids in a linkage region between β27 and α8 of gp41ECTO; and wherein the linkage region corresponds to amino acid positions 607-618 of a gp160 sequence of isolate HXB2.

2 . The recombinant protein according to claim 1 , wherein the gp41ECTO comprises a substitution of 7 consecutive amino acids in a region corresponding to amino acid positions 610-616 of the gp160 sequence of isolate HXB2 with gp120.

3 . The recombinant protein according to claim 1 ,

wherein the recombinant protein comprises a disulfide bond between amino acid positions corresponding to positions 501 and 605 of a gp160 sequence of isolate HXB2.

4 . The recombinant protein according to claim 1 , wherein the N-terminal and/or C-terminal of the gp120 is linked to the gp41ECTO via a direct linkage or a peptide linker.

5 . The recombinant protein according to claim 1 , which further possesses one or more of the following features:

(1) the gp41ECTO comprises the following amino acid substitution: 1559P;

(2) the gp120 comprises the following amino acid substitution: T332N;

(3) the gp120 comprises the following amino acid substitutions: E64K and H66R;

(4) the gp120 comprises the following amino acid substitution: A316W;

(5) the N-linked glycosylation site (PNGS) near the CD4bs epitope of the gp120 is replaced to prevent glycosylation; wherein the PNGS is selected from the group consisting of N276, N301, N360, N463;

(6) the gp120 comprises an internal disulfide bond between I201C and A433C;

(7) the recombinant protein comprises a disulfide bond between E49C and L555C;

the numbering of the above positions is according to the numbering in gp160 of HIV-1 isolate HXB2.

6 . The recombinant protein according to claim 1 , wherein the gp41ECTO and gp120 are derived from the same or different HIV-1 strains.

7 . The recombinant protein according to claim 1 , comprising an amino acid sequence selected from:

(1) an amino acid sequence consisting of amino acid residues at positions 40 to 651 of the sequence set forth in SEQ ID NO: 1;

(2) an amino acid sequence consisting of amino acid residues at positions 40 to 652 of the sequence set forth in SEQ ID NO:2;

(3) an amino acid sequence consisting of amino acid residues at positions 40 to 651 of the sequence set forth in SEQ ID NO:3;

(4) an amino acid sequence consisting of amino acid residues at positions 40 to 652 of the sequence set forth in SEQ ID NO:4;

(5) an amino acid sequence consisting of amino acid residues at positions 40 to 665 of the sequence set forth in SEQ ID NO:5;

(6) an amino acid sequence consisting of amino acid residues at positions 40 to 678 of the sequence set forth in SEQ ID NO:6;

(7) an amino acid sequence consisting of amino acid residues at positions 40 to 661 of the sequence set forth in SEQ ID NO:7;

(8) an amino acid sequence consisting of amino acid residues at positions 40 to 666 of the sequence set forth in SEQ ID NO:8;

(9) an amino acid sequence consisting of amino acid residues at positions 40 to 671 of the sequence set forth in SEQ ID NO:9;

(10) an amino acid sequence consisting of amino acid residues at positions 40 to 676 of the sequence set forth in SEQ ID NO:10;

(11) an amino acid sequence consisting of amino acid residues at positions 36 to 607 of the sequence set forth in SEQ ID NO:11;

(12) an amino acid sequence consisting of amino acid residues at positions 36 to 646 of the sequence set forth in SEQ ID NO:12;

(13) an amino acid sequence consisting of amino acid residues at positions 36 to 648 of the sequence set forth in SEQ ID NO:13;

(14) an amino acid sequence consisting of amino acid residues at positions 36 to 638 of the sequence set forth in SEQ ID NO:14;

(15) an amino acid sequence consisting of amino acid residues at positions 36 to 639 of the sequence set forth in SEQ ID NO:15;

(16) an amino acid sequence consisting of amino acid residues at positions 36 to 836 of the sequence set forth in SEQ ID NO:16;

(17) an amino acid sequence consisting of amino acid residues at positions 36 to 673 of the sequence set forth in SEQ ID NO:30;

(18) an amino acid sequence consisting of amino acid residues at positions 36 to 678 of the sequence set forth in SEQ ID NO:31;

(19) an amino acid sequence consisting of amino acid residues at positions 36 to 683 of the sequence set forth in SEQ ID NO:32;

(20) an amino acid sequence consisting of amino acid residues at positions 36 to 678 of the sequence set forth in SEQ ID NO:33;

(21) an amino acid sequence consisting of amino acid residues at positions 36 to 688 of the sequence set forth in SEQ ID NO:34;

(22) an amino acid sequence consisting of amino acid residues at positions 36 to 670 of the sequence set forth in SEQ ID NO:35;

(23) an amino acid sequence consisting of amino acid residues at positions 36 to 676 of the sequence set forth in SEQ ID NO:36;

(24) an amino acid sequence consisting of amino acid residues at positions 36 to 680 of the sequence set forth in SEQ ID NO:37;

(25) an amino acid sequence consisting of amino acid residues at positions 36 to 673 of the sequence set forth in SEQ ID NO:38;

(26) an amino acid sequence consisting of amino acid residues at positions 36 to 678 of the sequence set forth in SEQ ID NO:39; or

(27) an amino acid sequence consisting of amino acid residues at positions 36 to 683 of the sequence set forth in SEQ ID NO:40.

8 . The recombinant protein according to claim 1 ,

wherein the recombinant protein comprises a signal peptide and/or a Kozak consensus sequence at its N-terminal; and/or

the recombinant protein comprises a tag sequence at its C-terminal.

9 . A fusion protein, which comprises the recombinant protein according to claim 1 , and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal.

10 . The fusion protein according to claim 9 , comprising an amino acid sequence selected from:

(1) an amino acid sequence consisting of amino acid residues at positions 33 to 836 of the sequence set forth in SEQ ID NO: 16; or

(2) an amino acid sequence consisting of amino acid residues at positions 34 to 837 of the sequence set forth in SEQ ID NO:17.

11 . A multimer comprising a plurality of monomers, wherein each monomer is independently the recombinant protein according to claim 1 or a fusion protein comprising the recombinant protein and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal.

12 . An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding:

(i) the recombinant protein according to claim 1 ,

(ii) a fusion protein comprising the recombinant protein of (i) and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal, or

(iii) a multimer comprising a plurality of monomers, wherein each monomer is independently selected from the recombinant protein of (i) or the fusion protein of (ii).

13 . A vector, which comprises the isolated nucleic acid molecule according to claim 3 .

14 . An isolated host cell which comprises the isolated nucleic acid molecule according to claim 12 or a vector comprising the isolated nucleic acid molecule.

15 . A particle, displaying on its surface:

(i) the recombinant protein according to claim 1 ,

(ii) a fusion protein comprising the recombinant protein of (i) and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal, or

(iii) a multimer comprising a plurality of monomers, wherein each monomer is independently selected from the recombinant protein of (i) or the fusion protein of (ii);

and wherein the particle is a liposome or a nanoparticle.

16 . A pseudoviral particle, comprising on its surface:

(i) the recombinant protein according to claim 1 ,

(ii) a fusion protein comprising the recombinant protein of (i) and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal, or

(iii) a multimer comprising a plurality of monomers, wherein each monomer is independently selected from the recombinant protein of (i) or the fusion protein of (ii).

17 . A packaging system for producing a pseudoviral particle, which comprises: (i) an expression vector comprising the nucleic acid molecule according to claim 12 , and (ii) a packaging vector.

18 . A modified HIV virus, which expresses a fusion protein as its envelope protein; the fusion protein comprising the recombinant protein of claim 1 and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal.

19 . An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the genome of the modified HIV virus according to claim 18 .

20 . A vector, which comprises the isolated nucleic acid molecule according to claim 19 .

21 . A composition, which comprises any one of (i)-(ix):

(i) the recombinant protein according to claim 1 ;

(ii) a fusion protein comprising the recombinant protein of (i) and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal;

(iii) a multimer comprising a plurality of monomers, wherein each monomer is independently selected from the recombinant protein of (i) or the fusion protein of (ii);

(iv) an isolated nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding any one of (i)-(iii);

(v) a particle displaying on its surface any one of (i)-(iii);

(vi) a pseudoviral particle displaying on its surface any one of (i)-(iii);

(vii) a packaging system comprising (a) an expression vector comprising the nucleic acid molecule of (iv), and (b) a packaging vector;

(viii) a modified HIV virus expressing the fusion protein of (ii) as its envelope protein;

(ix) an isolated nucleic acid molecule or vector comprising a nucleotide sequence encoding the genome of the modified HIV virus of (viii).

22 . The composition according to claim 21 , wherein the composition is an immunogenic composition or a vaccine.

23 . The composition according to claim 21 , which comprises an antiretroviral agent selected from a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, and a fusion protein inhibitor.

24 . A method for inducing an immune response against HIV in a subject or for preventing and/or treating an HIV infection in a subject, which comprises administering to a subject in need thereof an immunologically effective amount of any one of (i)-(x):

(i) the recombinant protein according to claim 1 ;

(ii) a fusion protein comprising the recombinant protein of (i) and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal;

(iii) a multimer comprising a plurality of monomers, wherein each monomer is independently selected from the recombinant protein of (i) or the fusion protein of (ii);

(iv) an isolated nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding any one of (i)-(iii);

(v) a particle displaying on its surface any one of (i)-(iii);

(vi) a pseudoviral particle displaying on its surface any one of (i)-(iii);

(vii) a packaging system comprising (a) an expression vector comprising the nucleic acid molecule of (iv), and (b) a packaging vector;

(viii) a modified HIV virus expressing the fusion protein of (ii) as its envelope protein;

(ix) an isolated nucleic acid molecule or vector comprising a nucleotide sequence encoding the genome of the modified HIV virus of (viii); or

(x) a composition comprising any of the foregoing.

25 . The recombinant protein according to claim 1 , wherein the gp120 is located between amino acid positions corresponding to positions 609 and 610 of the gp160 sequence of isolate HXB2; or, the gp120 is located between amino acid positions corresponding to positions 616 and 617 of the gp160 sequence of isolate HXB2.

26 . The recombinant protein according to claim 1 , wherein:

(i) the gp120 is a modified gp120 that has a furin cleavage site containing a mutation to prevent being cleaved compared to a natural gp120, or the furin cleavage site is deleted in the modified gp120 compared to the natural gp120;

(ii) the gp120 is a modified gp120 that has C-terminal truncation of 1-11 amino acids compared with a natural gp120, or that has a deletion of 1-11 consecutive amino acids in a region corresponding to amino acid positions 501-511 of a gp160 sequence of isolate HXB2; or

(iii) the gp120 is a natural gp120.

27 . The recombinant protein of claim 4 , wherein the peptide linker is (GmS)n, wherein m is an integer selected from 1-4, and n is an integer selected from 1-3.

28 . The fusion protein of claim 9 , wherein the transmembrane region and intracellular region sequences of gp41 and the gp41ECTO in the recombinant protein are derived from the same HIV-1 strain.

29 . The method of claim 24 , wherein the subject is a human.