Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies

Abstract

BackgroundRecombinant 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.ResultsWe 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.ConclusionsThe 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.

A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene

“…Hence, the subtype A BG505 SOSIP.664 trimers are not vulnerable to V3 clipping ( Fig. 1D) (49). Production of non-V3-clipped B41 SOSIP.664 trimers in low-serum medium.…”

“…To facilitate the production of B41 SOSIP.664 trimers on a larger scale, we made stable 293T and CHO lines using procedures previously described for the corresponding BG505 trimers (49). We then attempted to adapt these lines to growth in media containing lower serum concentrations, and we succeeded with the CHO line (see Materials and Methods).…”

“…We have described elsewhere how to make stable CHO and 293T cell lines that express fully cleaved BG505 SOSIP.664 trimers (49). We used the same method to make lines that produce B41 SOSIP.664 trimers.…”

“…Western blotting of SDS-PAGE gels was carried out as described previously, using the anti-gp120 monoclonal antibody (MAb) ARP3119 (NIBSC Reagent Repository, United Kingdom) or a pool of serum derived from individuals infected with subtype B HIV-1 strains (HIV-Ig) (49). ARP3119 recognizes a conserved, linear epitope (residues EDIISLW) in the C1 region of gp120 and hence detects the 70-kDa fragment that is produced when V3-clipped gp120 is fractionated on a reducing SDS-PAGE gel (the other, 50-kDa, fragment does not contain the ARP3119 epitope).…”

Antigenicity-defined conformations of an extremely neutralization-resistant HIV-1 envelope spike

“…Two such preparations-a soluble Env trimer, known as "SOSIP.664," stabilized by several modifications (12,35), and a detergent-solubilized clade B Env with the CT deleted (JR-FL EnvΔCT) (36)-appear to present substantial technical barriers to large-scale, clinical-grade production (37,38). Purification of full-length, membrane-bound CH120.6 Env in large quantities will be difficult also.…”