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

Abstract

HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

“…Hence, there are no indications that SOSIP trimer expression in CHO cells is problematic from the antigenicity perspective. This assessment is consistent with reports that producer cell variation has only a subtle influence on the glycosylation of native-like trimers (48)(49)(50).…”

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

“…Purification can also be problematic. These translational difficulties reflect the extensive glycosylation of HIV‐1 Env, with glycans comprising half the mass, and also the presence of nine intramolecular disulfide bonds in the gp120 subunit and another in gp41 (Behrens & Crispin, ; Go et al, ; Ward & Wilson, ). The processing state of the glycans has proven to be particularly sensitive to the precise quaternary structure of the Env complex (Behrens & Crispin, ; Pritchard et al, ).…”

The expanding role of mass spectrometry in the field of vaccine development

“…(ii) To identify the variables that impact Env's glycosylation profile at sites with divergent glycosylation. The study concluded that the processing of many glycosylation sites on recombinant Envs are not affected by the expression system …”