Materials and methods for producing cleaved, HIV-1 envelope glycoprotein trimers
Expression vectors and mammalian cell lines containing them are described that enable the recombinant production of HIV-1 envelope proteins, including SOSIP modified gp140 trimers capable of inducing broadly neutralizing antibodies.
1. An expression vector comprising a first nucleic acid sequence encoding an HIV Env glycoprotein and a second nucleic acid sequence encoding a furin protease, wherein (i) expression of the nucleic acid sequence encoding the Env glycoprotein is controlled by a first constitutive promoter and the expression of the nucleic acid sequence encoding the furin protease is controlled by a second constitutive promoter, (ii) the first constitutive promoter is more potent than the second constitutive promoter, and (iii) the expression vector integrates into the genome of a mammalian cell.
2. The expression vector of claim 1 , wherein the Env glycoprotein comprises a gp120 subunit and a gp41 subunit, wherein (i) the gp120 subunit and the gp41 subunit are linked by an inter-subunit disulfide bond, and (ii) the isoleucine at position 559 of the gp41 subunit is substituted with proline.
3. The expression vector of claim 1 , wherein the Env glycoprotein forms the cleaved trimer BG505 SOSIP.664.
4. The expression vector of claim 1 , wherein the Env glycoprotein forms the cleaved trimer B41 SOSIP.664.
5. The expression vector of claim 1 , wherein the first constitutive promoter is a CMV promoter.
6. The expression vector of claim 1 , wherein the second constitutive promoter is an EF1 alpha promoter.
7. The expression vector of claim 1 , wherein the first nucleic acid sequence further comprises a synthetic poly A sequence.
8. The expression vector of claim 1 , wherein the expression vector comprises a DNA plasmid.
9. The expression vector of claim 8 , wherein the DNA plasmid comprises SEQ ID NO: 1.
10. The expression vector of claim 1 , wherein the expression vector integrates into the genome of a mammalian cell via Flp-mediated recombination.
11. An isolated mammalian cell comprising the expression vector of claim 1 .
12. The isolated mammalian cell of claim 11 , which is a 293T cell.
13. The isolated mammalian cell of claim 11 , which is a CHO cell.
14. A method of generating a mammalian cell line that expresses cleaved HIV Env glycoproteins, the method comprising:
(a) transfecting one or more mammalian cells with the expression vector of claim 1 under conditions wherein the expression vector integrates into the genome of the one or more mammalian cells;
(b) selecting one or more mammalian cells comprising the expression vector integrated into the genome thereof and expressing cleaved HIV Env glycoproteins; and
(c) propagating the selected mammalian cells indefinitely, thereby generating a mammalian cell line that expresses cleaved HIV Env glycoproteins.
15. A method of producing cleaved Env glycoproteins, wherein the method comprises (i) culturing the isolated mammalian cell of claim 11 under conditions to express the first and second nucleic acid sequences to provide HIV Env glycoproteins and furin proteases, and (ii) allowing the furin protease to cleave the Env glycoproteins to provide cleaved Env glycoproteins.
16. The method of claim 15 , which further comprises isolating and purifying the cleaved Env glycoproteins.