Development of a RPLC-UV method for monitoring uncleaved HIV-1 envelope glycoprotein

Abstract

One of the HIV-1 vaccine design efforts has focused on developing a recombinant HIV-1 trimeric envelope glycoprotein (Env) as an immunogen to induce broadly neutralizing antibodies. A native-like immunogen, the BG505.DS.SOSIP.664 gp140 (Env) construct has been well-characterized as a vaccine candidate. This vaccine candidate comprises of three identical gp120 and truncated gp41 subunits that form into a trimer of heterodimers. During production, recombinant Env is expressed as a gp140 precursor polypeptide in which a furin cleavable site is engineered to generate a heterodimer of gp120 and gp41 subunits. Each heterodimer is connected by an intermolecular disulfide bond, and three heterodimers form into a trimer. Furin cleavage is an important factor to mimic native-like HIV-1 Env conformations and is needed to help induce an immune response. Therefore, it is critical to monitor cleavage for ensuring functionality of the Env vaccine product. In this paper, a new RPLC-UV method coupled with reduction was developed to routinely determine the percentage of uncleaved gp140 relative to the cleaved gp120 and gp41 subunits. Baseline separation was achieved among the gp120, gp41 and uncleaved gp140 peaks, thus enabling relative quantification of uncleaved gp140. Overall, this RPLC-UV approach has been successfully applied to support Env vaccine candidate developments.

Fig. 1

Scheme of uncleaved and cleaved gp140 heterodimers. After reduction with DTT, cleaved gp140 dissociates into the gp120 and gp41 subunits while uncleaved gp140 cannot due to the hexa-arginine residue linker holding the two subunits together.

Fig. 2

RPLC-UV chromatograms of Env under (a) non-reduced and (b) reduced conditions.

Fig. 3

RPLC-UV chromatograms of Env under reduced conditions after being spiked with varying amounts of furin. Env sample was incubated with 0 μL (blue), 0.2 units (orange), 0.4 units (green), 1 unit (black), 2 units (red), and 10 units (purple) of furin. The teal trace was a negative control of 10 units of furin without Env.

Fig. 4

RPLC-UV application for two different HIV-1 Env constructs: profiles of Env trimer constructs (a) BG505.DS.SOSIP.664 and (b) ConC-FP8v2 RnS-3mut-2G-SOSIP.664 after reduction by DTT.