Screen of MN glycan mutant supernatants for improvements to bN-mAb binding.

<p>A FIA was used to identify 293 GnTI^- expressed MN-rgp120 glycan variants exhibiting improved bN-mAb binding profiles as compared to the wildtype MN sequence expressed in GnTI^- (MN358). Recombinant gp120s were expressed in GnTI^- 293 cells via transient transfection, and transfection supernatants were normalized to contain ~2μg/mL. MN-rgp120 variants were captured onto 96 well black plates using a 1μg/mL concentration of mouse monoclonal antibody to an N-terminal gD tag. Binding curves to the VRCO1 bN-mAb, which binds a conformation dependent epitope in the CD4 binding site, were used to assay for maintenance of overall secondary and tertiary structure. All screening assays were performed in duplicate. MN-rgp120 glycan variants were assayed for improved antigenicity to a panel of glycan dependent bN-mAbs to be considered for further analysis.</p

Modification of N-linked glycosylation sites in MN- and A244-rgp120.

<p>(<b>A</b>) The A244-rgp120 or MN-rgp120 sequences were analyzed for the presence of highly conserved glycans known to be important for bN-mAb binding within the C2-C3 domains. Glycosylation sites are represented as either black (present in RV144 immunogen) or grey (absent in original RV144 immunogen) structures. (<b>B</b>) A ribbon diagram depicts the 3-dimensional arrangement of the N289, N301, N332, and N334 PNGS. The structure is based on crystal structure of the BG505 SOSIP.664 gp140 trimer (in gold) bound to the PGT122 bN-mAb (in grey) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196370#pone.0196370.ref041" target="_blank">41</a>]. The N301 and N332 glycan structures immobilized by the PGT122 antibody are indicated in green, while the asparagine residues at the base of relevant PNGS are indicated in red. (<b>C</b>) Site directed mutagenesis was used to create MN- or A244-rgp120 variants introducing one or more of the indicated PNGS. A summary of the PNGS variant constructs assayed is shown. The constructs with identical number and location of PNGS to the RV144 rgp120 immunogens are marked with an asterisk.</p

Binding of MN glycan variants to extended panel of bN-mAbs.

<p>The MN-rgp120 variants MN358 and MN1320 expressed in GnTI^- cells were compared to MN_GNE for improved binding to an array of bN-mAbs. Recombinant gp120s were expressed in GnTI^- 293 cells via transient transfection. Purified MN_GNE or transfection supernatants were normalized to contain 4μg/mL rgp120 and captured using 2μg/mL of mouse monoclonal antibody 34.1, then assayed by FIA for bN-mAb binding. Results are reported in μg/mL as (EC50), the concentration of antibody required for a half-maximal binding, measured in Relative Fluorescence Units (RFU) on a titration-binding curve. Values are reported as ≥2.5μg/mL if titration curves did not plateau or if mean EC50 was ≥2.5μg/mL. Binding curves to bN-mAbs were performed in quadruplicate. The rp120 constructs exhibiting statistically significant differences in EC50 values (p<0.05) from the MN_GNE are noted in bold. Human IgGK polyclonal antibody was used as a negative control and purified goat polyclonal antibody raised against rgp120 (PB94) was used as a coating control.</p

Endo H digest and immunoblot of A244 gp120 glycan variants.

<p>A244-rgp120s containing either N332 or N334 based PNGS were expressed in either CHO-S (lanes 1–4) or HEK 293 GnTI^- cells (lanes 5–8) via transient transfection. Purified protein was subjected to Endo H or mock digest (digest buffer alone), and analyzed for mobility on 4–12% reducing SDS-PAGE gels. Immunoblots were probed with the mouse monoclonal 34.1 that binds a conformation independent epitope in the N-terminal gD tag of all expressed proteins, and visualized with goat-anti-mouse HRP conjugated polyclonal sera.</p

Endo H digest and immunoblot of MN gp120 glycan variants.

<p>Wildtype MN rgp120 expressed in CHO GnTI^- cells, or MN glycoform mutants with single, double, or triple glycan additions were expressed in GnTI^- cells via transient transfection. Purified MN_GNE or GnTI^- cells supernatants containing expressed rgp120 were immunoprecipitated via a monoclonal antibody to an N-terminal gD tag bound to protein-G coated beads. Immunoprecipiated rgp120 variants were subjected to Endo H or mock digest (digest buffer alone), and analyzed for mobility on 4–12% reducing SDS-PAGE gels. Immunoblots were probed with the mouse monoclonal 34.1 and visualized with goat-anti-mouse HRP-conjugated polyclonal sera.</p

SDS-PAGE analysis of A244_N332 rgp120 HIV produced in 5F MGAT1^- CHO and CHO-S cells treated with PNGase or EndoH.

<p>Enzymes and buffers were purchased from (New England Biolabs, Ipswich, MA). Purified protein was denatured and reduced then incubated overnight at 37°C with or without glycosidase. Protein was resolved (2 μg/lane) on 4–12% SDS-PAGE gel and stained with Simply Blue. Plus (+) indicates enzyme treatment, minus indicates untreated.</p

The timeline for development of stable MGAT1^- CHO cell lines expressing HIV-1 rgp120.

<p>Leading clones expressing 0.2–0.4 g/L in shake flasks under standard laboratory conditions were selected in less than two months. Production was subsequently increased to levels of g/L rgp120 production with minimal feed optimization.</p

Analysis of A244_N332-rgp120 secreted from stable MGAT1^- CHO cell lines.

<p>Six stable MGAT1^- CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. <b>(A)</b> Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. <b>(B)</b> Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1^- CHO cell lines. (<b>C)</b> SDS PAGE of rgp120 produced by the 5F MGAT1^- CHO cell line. Supernatant samples (10 <i>μ</i>l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).</p

Primary identification of high producer MGAT1^- CHO lines expressing A244_N332 rgp120 by immunofluorescent labeling.

<p><b>(A)</b> G418 selected colonies visible in a single 35mm well illuminated with white light at 6 days. <b>(B)</b> The same single 35mm well illuminated with 490 nm wavelength light. Colonies actively secreting rgp120 have a green “halo” visible at 525 nm. <b>(C)</b> Relative mean exterior fluorescence of halo for more than 10,000 colonies imaged by the ClonePix2 plotted by rank. The top ranking 0.1% of colonies (44) were robotically picked and cultured. The six clones expressing 0.2–0.4 g/L at day 56 are shown in red.</p

Binding of bN-mAbs to A244-rgp120 produced in normal and A244_N332-rgp120 produced in MGAT1^- CHO cell lines.

<p>A244_N332-rgp120 was purified from the stable clone 5F MGAT1^- CHO cell line (closed circles) or from the MGAT1^- CHO cells (open circles) transiently transfected with the UCSC 1331 plasmid. A244-rgp120 expressed and purified from transiently transfected CHO-S cells (open squares). Antibody binding was measured by a fluorescent immunoassay (FIA).</p