Advertisement

 

 

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

Epitope-Independent Purification of Native-Like Envelope Trimers from Diverse HIV-1 Isolates.
Author Information (click to view)

Verkerke HP, Williams JA, Guttman M, Simonich CA, Liang Y, Filipavicius M, Hu SL, Overbaugh J, Lee KK,


Verkerke HP, Williams JA, Guttman M, Simonich CA, Liang Y, Filipavicius M, Hu SL, Overbaugh J, Lee KK, (click to view)

Verkerke HP, Williams JA, Guttman M, Simonich CA, Liang Y, Filipavicius M, Hu SL, Overbaugh J, Lee KK,

Advertisement
Share on FacebookTweet about this on TwitterShare on LinkedIn

Journal of virology 2016 Sep 2990(20) 9471-82 doi 10.1128/JVI.01351-16

Abstract
UNLABELLED
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.

IMPORTANCE
The 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.

Submit a Comment

Your email address will not be published. Required fields are marked *

three × five =

[ HIDE/SHOW ]