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Link to original content: https://pubmed.ncbi.nlm.nih.gov/31941770/
Glycan Profiles of gp120 Protein Vaccines from Four Major HIV-1 Subtypes Produced from Different Host Cell Lines under Non-GMP or GMP Conditions - PubMed Skip to main page content
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. 2020 Mar 17;94(7):e01968-19.
doi: 10.1128/JVI.01968-19. Print 2020 Mar 17.

Glycan Profiles of gp120 Protein Vaccines from Four Major HIV-1 Subtypes Produced from Different Host Cell Lines under Non-GMP or GMP Conditions

Shixia Wang  1   2 , Yegor Voronin  2 , Peng Zhao  3 , Mayumi Ishihara  3 , Nickita Mehta  3 , Mindy Porterfield  3 , Yuxin Chen  1 , Christopher Bartley  4 , Guangnan Hu  1 , Dong Han  1 , Lance Wells  3 , Michael Tiemeyer  3 , Shan Lu  5
Affiliations

Glycan Profiles of gp120 Protein Vaccines from Four Major HIV-1 Subtypes Produced from Different Host Cell Lines under Non-GMP or GMP Conditions

Shixia Wang et al. J Virol. .

Abstract

Envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is an important target for the development of an HIV vaccine. Extensive glycosylation of Env is an important feature that both protects the virus from antibody responses and serves as a target for some highly potent broadly neutralizing antibodies. Therefore, analysis of glycans on recombinant Env proteins is highly significant. Here, we present glycosylation profiles of recombinant gp120 proteins from four major clades of HIV-1 (A, B, C, and AE), produced either as research-grade material in 293 and CHO cells or as two independent lots of clinical material under good manufacturing practice (GMP) conditions. Almost all potential N-linked glycosylation sites were at least partially occupied in all proteins. The occupancy rates were largely consistent among proteins produced under different conditions, although a few sites showed substantial variability even between the two GMP lots. Our data confirmed previous studies in the field, showing an abundance of oligomannose on Env protein, with 40 to 50% of glycans being Man5 to Man9 on all four proteins under all production conditions. Overall, the differences in occupancy and glycan forms among different Env subtypes produced under different conditions were less dramatic than anticipated, and antigenicity analysis with a panel of six monoclonal antibodies, including antibodies that recognize glycan forms, showed that all four gp120s maintained their antibody-binding profiles. Such findings have major implications for the final production of a clinical HIV vaccine with Env glycoprotein components.IMPORTANCE HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations.

Keywords: Env protein; HIV-1; glycosylation; gp120; vaccine.

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Figures

FIG 1
FIG 1
(A) Sequences of the gp120 proteins belonging to clade A (92UG037.8), clade B (JRFL), clade C (93MW965.26), and clade AE (consensus), aligned to the reference strain HXB2. Identical amino acids are shown as dots, gaps are indicated with dashes, and numbers correspond to the HXB2 sequence. Variable regions V1 to V5 of gp120 are indicated above the sequences. PNGSs predicted on the basis of the consensus glycosylation sequence are shown in red and marked with stars above the sequences. (B) Summary of glycosylation site distributions among the four gp120 proteins. N indicates the presence of a PNGS in the sequence.
FIG 2
FIG 2
(A) Representative MS spectra of glycans identified on research-grade gp120 proteins produced in CHO cells (top) and 293F cells (bottom). The gp120 protein from clade B (strain JR-FL) is shown here as a representative example. Glycan forms corresponding to the most abundant peaks are shown using the standard glycan icons. MS peaks corresponding to oligomannose and complex glycan forms are indicated. (B) Quantitative comparison of relative proportions of various glycan forms (in percent) on CHO- and 293F-derived gp120-B proteins. Glycan structures found at levels greater than 10% of total glycans are highlighted in gray.
FIG 3
FIG 3
(A) PNGS occupancy analysis of gp120 proteins expressed in 293F cells (top row) and CHO cells (bottom row). The levels of glycan occupancy at each PNGS are shown for each of the gp120 proteins (clade A, B, C, or AE), as indicated above each panel. Green and purple bars indicate the proportions of oligomannose glycans and complex glycans, respectively, and gray bars indicate that the site was not occupied by a glycan. ND indicates that peptides were not detected. (B) Differences in glycan occupancy between the 293F- and CHO-produced proteins at each PNGS for each of the gp120 proteins. Percentages of oligomannose and complex glycans were compared at each site, and the differences were plotted based on which protein had larger amounts of that glycan. For example, at position N187 in clade A protein, the 293F-derived protein had 25 percentage points more complex glycans than the CHO-derived protein, while the CHO-derived protein had 18 percentage points more oligomannose glycans than the 293F-derived protein.
FIG 4
FIG 4
MS spectra of glycans released from four gp120 proteins (clades A, B, C, and AE) produced under GMP conditions. Glycan forms corresponding to the most abundant peaks are shown, using the standard glycan icons. MS peaks corresponding to oligomannose and complex glycan forms are indicated.
FIG 5
FIG 5
Quantification of the types of glycan forms released from two independent lots (lot 1 and lot 2) of gp120 proteins (clades A, B, C, and AE) produced under GMP conditions. Numbers indicate the percentages of the corresponding glycan forms or structural classes, color coded as Man5 (M5), Man6 (M6), Man7 (M7), Man8 (M8), Man9 (M9), paucimannose (Pauci) (Man3 and Man4), hybrid, or complex.
FIG 6
FIG 6
Analysis of sialylation (left) and core fucosylation (right) modifications of complex glycans of each gp120 protein (clade A, B, C, or AE) produced in two different GMP lots. Only complex glycans were included in the analysis. 1 and 2 under the bars of the graph indicate lot 1 and lot 2 GMP-grade gp120 proteins, respectively. A, B, C, and AE indicate gp120 proteins from clades A, B, C, and AE, respectively.
FIG 7
FIG 7
(A) PNGS occupancy analysis of GMP-grade gp120 proteins. The levels of glycan occupancy at each PNGS are shown for each of the gp120 proteins (clade A, B, C, or AE), as indicated above each panel. Green and purple bars indicate the proportions of oligomannose glycans and complex glycans, respectively, and gray bars indicate that the site was not occupied by a glycan. ND indicates that peptides were not detected. (B) Differences in glycan occupancy in gp120 proteins produced in CHO cells under research-grade conditions and under GMP conditions at each PNGS. The plots were generated as described for Fig. 3B.
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