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Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.
- Source :
-
Retrovirology [Retrovirology] 2014 Apr 25; Vol. 11, pp. 33. Date of Electronic Publication: 2014 Apr 25. - Publication Year :
- 2014
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Abstract
- Background: Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized.<br />Results: We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145.<br />Conclusions: The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.
- Subjects :
- Animals
Antibodies, Neutralizing genetics
Antibodies, Neutralizing immunology
Antigens, Viral immunology
CHO Cells
Cell Line
Cricetulus
Furin genetics
Furin immunology
Gene Expression immunology
Glycoproteins immunology
HEK293 Cells
HIV Antibodies genetics
HIV Antibodies immunology
HIV Envelope Protein gp120 biosynthesis
HIV Envelope Protein gp120 genetics
HIV Envelope Protein gp120 immunology
HIV Envelope Protein gp41 biosynthesis
HIV Envelope Protein gp41 genetics
HIV-1 genetics
HIV-1 immunology
Humans
Protein Multimerization
Recombinant Proteins genetics
Recombinant Proteins immunology
Vaccines biosynthesis
Vaccines immunology
env Gene Products, Human Immunodeficiency Virus genetics
env Gene Products, Human Immunodeficiency Virus immunology
Antigens, Viral genetics
Gene Expression genetics
Glycoproteins genetics
HIV-1 metabolism
Vaccines genetics
env Gene Products, Human Immunodeficiency Virus biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 1742-4690
- Volume :
- 11
- Database :
- MEDLINE
- Journal :
- Retrovirology
- Publication Type :
- Academic Journal
- Accession number :
- 24767177
- Full Text :
- https://doi.org/10.1186/1742-4690-11-33