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Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers.

TitleInfluences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers.
Publication TypeJournal Article
Year of Publication2015
AuthorsRinge RP, Yasmeen A, Ozorowski G, Go EP, Pritchard LK, Guttman M, Ketas TA, Cottrell CA, Wilson IA, Sanders RW, Cupo A, Crispin M, Lee KK, Desaire H, Ward AB, Klasse PJ, Moore JP
JournalJ Virol
Volume89
Issue23
Pagination12189-210
Date Published2015 Dec
ISSN1098-5514
KeywordsAntibodies, Monoclonal, Calorimetry, Differential Scanning, Chromatography, Affinity, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, env Gene Products, Human Immunodeficiency Virus, Enzyme-Linked Immunosorbent Assay, HIV-1, Mass Spectrometry, Microscopy, Electron, Neutralization Tests, Protein Engineering, Protein Multimerization, Protein Structure, Tertiary, Recombinant Proteins, Surface Plasmon Resonance
Abstract

UNLABELLED: We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components.

IMPORTANCE: Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.

DOI10.1128/JVI.01768-15
Alternate JournalJ. Virol.
PubMed ID26311893
PubMed Central IDPMC4645310
Grant ListR01 AI094797 / AI / NIAID NIH HHS / United States
1UM1 AI100663 / AI / NIAID NIH HHS / United States
UM1 AI100663 / AI / NIAID NIH HHS / United States
R21 AI112389 / AI / NIAID NIH HHS / United States
R37 AI036082 / AI / NIAID NIH HHS / United States
R37 AI36082 / AI / NIAID NIH HHS / United States
P01 AI110657 / AI / NIAID NIH HHS / United States
P01 AI082362 / AI / NIAID NIH HHS / United States