Epitope-Independent Purification of Native-Like Envelope Trimers from Diverse HIV-1 Isolates

Abstract

Soluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising native-like (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed SOSIP, into truncated Env sequences. However, the abundances of NL trimeric conformers vary among Envs, necessitating purification by monoclonal antibodies (MAbs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography (HIC), and size exclusion chromatography (SEC) to isolate NL trimers from nonnative Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native PAGE, SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (>95% pure), fully cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative-stain electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in nonnative Envs. Screened for antigenicity, some Envs, like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing MAb affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to MAbs for isolation of NL Env trimers. IMPORTANCEThe production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heterogeneous Env conformers, even with the addition of stabilizing mutations, have presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While MAbs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, MAb affinity chromatography can be expensive, the necessary MAb may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs. T he envelope glycoprotein (Env) on the virus surface is the sole target of HIV-1 neutralizing antibodies (NAbs). Many vaccine strategies involve the use of Env-...

Changes in Structure and Antigenicity of HIV-1 Env Trimers Resulting from Removal of a Conserved CD4 Binding Site-Proximal Glycan

“…To examine the effects of N197 glycan removal within a native trimer, the N197Q mutation was introduced into the BG505 SOSIP construct, and both WT and N197Q trimers were transiently expressed in HEK293F cells. The two Env trimers were purified by a combination of lectin affinity chromatography, anion exchange chromatography, HIC, and a final SEC step to generate pure trimer preparations (89). The SEC elution profiles of the BG505 SOSIP.664 WT and the N197Q mutant showed the expected trimer peaks with identical elution volumes (Fig.…”

“…The flowthrough from the DEAE column was collected, buffer exchanged into a solution containing 2 M (NH4) 2 SO 4 and 0.1 M NaH 2 PO 4 (pH 7.0) (buffer A), and loaded onto a proPac HIC-10 column (Thermo Scientific) equilibrated with buffer A. Trimeric Env was eluted from the hydrophobic interaction chromatography (HIC) column with a linear gradient from 0 to 100% buffer B (0.1 M NaH 2 PO 4 , pH 7.0). Subsequent purification of the Env trimer was done by gel filtration using a HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated with 1ϫ phosphate-buffered saline (PBS) (pH 7.4) (1 mM EDTA, 0.02% NaN 3 ) (89). Fractions (500 l each) were collected and analyzed by using SDS-PAGE and blue native PAGE (BN-PAGE).…”

cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV‐1 envelope glycoprotein vaccine candidate

“…BG505 and several other genotypes of SOSIP trimer can be successfully purified using 2G12, but some cannot due to contamination by a subset of non‐native pseudo‐trimers that are not removed by an SEC column (Ringe et al, ). Other chromatographic procedures may be helpful here (Verkerke et al, ). However, our preferred option is to use a conformationally selective bNAb such as PGT145 to positively select only the native‐like trimer population (de Taeye et al, ; Pugach et al, ; Ringe et al, ).…”

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

“…Additional studies in this area are justified. In this context, Verkerke et al have described an alternative way to purify SOSIP-stabilized gp140s that does not rely on any antibody-affinity columns (57). The use of this epitope-independent method, compared to a method using a bNAb selection column(s), could allow the identification of any as-yet-unseen effects on the resulting glycosylation profile.…”