Expression of hiv polypeptides and production of virus-like particles

Abstract

The present invention relates to the efficient expression of HIV polypeptides in a variety of cell types, including, but not limited to, mammalian, insect, and plant cells. Synthetic expression cassettes encoding the HIV Gag-containing polypeptides are described, as are uses of the expression cassettes in applications including DNA immunization, generation of packaging cell lines, and production of Env-, tat- or Gag-containing proteins. The invention provides methods of producing Virus-Like Particles (VLPs), as well as, uses of the VLPs including, but not limited to, vehicles for the presentation of antigens and stimulation of immune response in subjects to whom the VLPs are administered.

1 . An expression cassette comprising a first polynucleotide sequence and a second polynucleotide sequence, wherein the first polynucleotide sequence encodes an HIV Gag polypeptide and has at least 90% sequence identity to SEQ ID NO:20, and the second polynucleotide sequence encodes an HIV Env polypeptide. 
     
     
         2 . The expression cassette of  claim 1 , wherein the first polynucleotide sequence that encodes the HIV Gag polypeptide has at least 90% sequence identity to SEQ ID NO:9. 
     
     
         3 . The expression cassette of  claim 1 , wherein the first polynucleotide sequence that encodes the HIV Gag polypeptide has at least 90% sequence identity to SEQ ID NO:4. 
     
     
         4 . The expression cassette of  claim 1 , wherein the second polynucleotide sequence that encodes the HIV Env polypeptide has at least 90% sequence identity to SEQ ID NO:71 or to SEQ ID NO:72. 
     
     
         5 . The expression cassette of  claim 1 , wherein the first polynucleotide sequence encodes an HIV Gag polypeptide of a first HIV strain and the second polynucleotide sequence encodes an HIV Env polypeptide of a second HIV strain that is different from the first HIV strain. 
     
     
         6 . The expression cassette of  claim 1 , further comprising a third polynucleotide sequence that encodes an HIV protease polypeptide. 
     
     
         7 . The expression cassette of  claim 6 , wherein the third polynucleotide sequence that encodes the HIV protease polypeptide has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:78, and SEQ ID NO:79. 
     
     
         8 . The expression cassette of  claim 6 , wherein the first polynucleotide sequence encodes an HIV Gag polypeptide of a first HIV strain and the third polynucleotide sequence encodes an HIV protease polypeptide of a third HIV strain that is different from the first HIV strain. 
     
     
         9 . The expression cassette of  claim 8 , wherein the second polynucleotide sequence encodes an HIV Env polypeptide of a second HIV strain, and wherein the first, second, and third HIV strains are different. 
     
     
         10 . A cell comprising an expression cassette of  claim 1 , wherein the first and second polynucleotide sequences are operably linked to control elements compatible with expression of the HIV Gag polypeptide and the HIV Env polypeptide. 
     
     
         11 . A method for producing a chimeric HIV polypeptide, the method comprising incubating mammalian cells comprising the expression cassette of  claim 1  under conditions suitable for expressing the HIV Gag polypeptide and the HIV Env polypeptide, wherein the HIV Gag polypeptide and the HIV Env polypeptide are expressed. 
     
     
         12 . A gene delivery vector comprising the expression cassette of  claim 1 , wherein the first and second polynucleotide sequences are operably linked to control elements compatible with expression of the HIV Gag polypeptide and the HIV Env polypeptide. 
     
     
         13 . A packaging cell line comprising host cells transfected with the gene delivery vector of  claim 12 . 
     
     
         14 . The packaging cell line of  claim 13 , wherein the host cells are mammalian cells. 
     
     
         15 . The packaging cell line of  claim 14 , wherein the host cells are selected from the group consisting of baby hamster kidney (BHK) cells, African green monkey kidney (VERO) cells, human fibrosarcoma (HT1080) cells, human embryonic kidney (HEK293) cells, human rhabdomyoscarcoma (RD) cells, African green monkey kidney fibroblast (COS-7) cells, Chinese hamster ovary (CHO) cells, mouse myeloma (SB20) cells, and human cervical cancer (HeLa) cells. 
     
     
         16 . A method of raising an immune response to a chimeric HIV polypeptide, the method comprising administering to a subject the gene delivery vector of  claim 12 . 
     
     
         17 . A vaccine composition comprising the gene delivery vector of  claim 12 . 
     
     
         18 . The vaccine composition of  claim 17 , wherein the gene delivery vector is a viral vector derived from the pox family of viruses. 
     
     
         19 . The vaccine composition of  claim 18 , wherein the gene delivery vector is a canarypox vector. 
     
     
         20 . The vaccine composition of  claim 17 , further comprising an adjuvant. 
     
     
         21 . An expression cassette, comprising a first polynucleotide sequence that encodes an HIV Gag polypeptide, a second polynucleotide sequence encodes an HIV Env polypeptide, and a third polynucleotide that encodes an HIV protease polypeptide. 
     
     
         22 . The expression cassette of  claim 21 , wherein the HIV Gag polypeptide, the HIV Env polypeptide, and the HIV protease polypeptide are from different HIV strains. 
     
     
         23 . The expression cassette of  claim 21 , wherein the first polynucleotide sequence that encodes the HIV Gag polypeptide has at least 90% sequence identity to SEQ ID NO:20. 
     
     
         24 . The expression cassette of  claim 23 , wherein the first polynucleotide sequence that encodes the HIV Gag polypeptide has at least 90% sequence identity to SEQ ID NO:9. 
     
     
         25 . The expression cassette of  claim 23 , wherein the first polynucleotide sequence that encodes the HIV Gag polypeptide has at least 90% sequence identity to SEQ ID NO:4. 
     
     
         26 . The expression cassette of  claim 21 , wherein the second polynucleotide sequence that encodes the HIV Env polypeptide has at least 90% sequence identity to SEQ ID NO:71 or to SEQ ID NO:72. 
     
     
         27 . The expression cassette of  claim 21 , wherein the third polynucleotide sequence that encodes the HIV protease polypeptide has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:78, and SEQ ID NO:79. 
     
     
         28 . A gene delivery vector comprising the expression cassette of  claim 21 , wherein the first, second, and third polynucleotide sequences are operably linked to control elements compatible with expression of the HIV Gag polypeptide, the HIV Env polypeptide, and the HIV protease polypeptide. 
     
     
         29 . A gene delivery vector comprising the expression cassette of  claim 21  in a viral vector derived from the pox family of viruses. 
     
     
         30 . A vaccine composition comprising the gene delivery vector of  claim 29 . 
     
     
         31 . The vaccine composition of  claim 30 , wherein the gene delivery vector is a viral vector derived from the pox family of viruses. 
     
     
         32 . The vaccine composition of  claim 31 , wherein the gene delivery vector is a canarypox vector. 
     
     
         33 . The vaccine composition of  claim 30 , further comprising an adjuvant.