A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: Effects on glycosylation and antigenicity

José A González-Feliciano; Pearl Akamine; Coral M Capó-Vélez; Manuel Delgado-Vélez; Vincent Dussupt; Shelly J Krebs; Valerie Wojna; Victoria R Polonis; Abel Baerga-Ortiz; José A Lasalde-Dominicci

doi:10.1371/journal.pone.0231679

. 2020 Jun 19;15(6):e0231679. doi: 10.1371/journal.pone.0231679

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: Effects on glycosylation and antigenicity

José A González-Feliciano ¹, Pearl Akamine ¹, Coral M Capó-Vélez ¹, Manuel Delgado-Vélez ¹, Vincent Dussupt ^2,³, Shelly J Krebs ^2,³, Valerie Wojna ⁴, Victoria R Polonis ², Abel Baerga-Ortiz ^1,^5,^*, José A Lasalde-Dominicci ^1,^6,^*

Editor: Juan Pablo Jaworski⁷

¹Molecular Sciences Research Center Inc., University of Puerto Rico, San Juan, Puerto Rico

²U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America

³Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, United States of America

⁴Division of Neurology, Internal Medicine Department and NeuroHIV Research Program, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico

⁵Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico

⁶Department of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico

⁷Consejo Nacional de Investigaciones Cientificas y Tecnicas, ARGENTINA

Competing Interests: The authors have declared that no competing interests exist.

^✉

* E-mail: abel.baerga@upr.edu (ABO); jose.lasalde@upr.edu (JALD)

Roles

José A González-Feliciano: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft

Pearl Akamine: Conceptualization, Data curation, Formal analysis, Writing – original draft

Coral M Capó-Vélez: Conceptualization, Data curation, Writing – original draft, Writing – review & editing

Manuel Delgado-Vélez: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing – review & editing

Vincent Dussupt: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing

Shelly J Krebs: Conceptualization, Data curation, Investigation, Resources, Writing – review & editing

Valerie Wojna: Formal analysis, Methodology, Resources, Validation, Writing – review & editing

Victoria R Polonis: Data curation, Investigation, Resources, Supervision, Writing – original draft, Writing – review & editing

Abel Baerga-Ortiz: Conceptualization, Data curation, Project administration, Supervision, Writing – original draft, Writing – review & editing

José A Lasalde-Dominicci: Funding acquisition, Software, Supervision, Writing – review & editing

Juan Pablo Jaworski: Editor

PMCID: PMC7304579 PMID: 32559193

Abstract

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5–5.5 and 6.0–7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.

Introduction

With 35 million infected individuals worldwide, the human immunodeficiency virus (HIV) remains a major global health concern. Most of the current efforts toward a protective vaccine against HIV center on the envelope glycoprotein (Env), the only protein displayed on the virus surface [1–5]. Env is a two-protein system composed of a monomeric gp120 that binds non-covalently to the trimeric, membrane-spanning gp41 [6,7]. Env-based vaccines are notoriously difficult to produce because of their hydrophobic membrane-proximal regions and their extensive glycosylation [8]. Despite these production hurdles, monomeric gp120 has been produced in large amounts and has been thoroughly tested in numerous vaccine trials, including the landmark RV144 vaccine trial in Thailand [9–11].

After the discovery of patient-derived broadly neutralizing antibodies (bNAbs), which provide cross-clade protection through specific recognition of the viral Env, it was clear that the monomeric gp120 did not contain all the relevant epitopes to elicit broad neutralization, and thus, longer Env constructs that better resemble viral spikes would be needed [12–14]. To satisfy the growing need for longer and more structurally relevant Env constructs, two new families of Env designs have been developed: 1) uncleaved trimers arising from the elimination of the protease cleavage site that divides gp120 and gp41. These ‘uncleaved trimers’ consist of a single chain Env with a sequence that stops short of the membrane-proximal region. Env glycoproteins developed in this category include the uncleaved C97ZA012-gp140 [5], CN54gp140 [15], and CO6980v0c22 gp145 [16]. This family of immunogens has been produced in large scale and tested in rodents and non-human primates. Additionally, some of these constructs are currently in Phase I clinical trials. 2) The native-like trimers consisting of gp120 and gp41 genetically fused either by an engineered disulfide bond or by a flexible peptide linker. HIV-1 Env glycoproteins developed in this category include the SOSIP trimers [4,17,18], NFL trimers [19], and the UFO constructs [20]. Native trimers, particularly BG505 SOSIP, have been characterized structurally and conformationally, and are also currently being tested for safety and preliminary efficacy in patients [21–25].

The extensive glycosylation on these trimeric versions of Env (both uncleaved and native-like) remains a major limitation toward their high-yield production. Env contains approximately 27 sites for N-linked glycosylation, each of which could be occupied by either an oligomannose or a complex glycan, imposing tremendous strain on the protein trafficking machinery of the expression host [3]. It has been generally understood that for an Env vaccine to be effective, its glycan shield should resemble that of the infectious virus [26]. Thus, a number of analytical strategies have been developed to measure the precise chemical nature and distribution of the glycans in Env from viral and recombinant sources [27,28].

The glycosylation of recombinant Env vaccines can be affected by the choice of expression system. This is exemplified in Raska et al. where it is shown that the types of glycans displayed on recombinant gp120 vary depending on the expression system [28]. This observation was confirmed by Yu et al., who reported that the gp120 produced in CHO-K1 cells is substantially more acidic than the same protein produced in 293F cells due to a higher level of decoration with sialic acid, although the effect of this difference on antibody binding was not entirely clear [29]. Similar glycosylation studies have been performed for native-like trimers, particularly for the SOSIP versions produced in CHO-K1 or in HEK293 cells, revealing minimal differences in the complex vs. mannose glycan distribution between the two hosts [30]. From these detailed glycosylation studies it was concluded that the gp120 portion of the SOSIP trimer contains more high-mannose glycans than the gp41 portion, and that those trimers made in CHO-K1 cells contain a higher level of complex glycosylation than the cleaved trimers produced in 293 cells [31]. Such detailed glycosylation studies have not been carried out for any of the other native-like trimers (NFL or UFO), or for the uncleaved Env constructs.

In this work, we compared the overall glycosylation and antigenicity of the uncleaved CO6980v0c22, a subtype C gp145, produced in CHO-K1 and Expi293F (HEK 293-derived cells). This specific Env construct is currently undergoing clinical testing for safety and immunogenicity in uninfected healthy adults in the United States (ClinicalTrials.gov). Our results show considerable differences in the gp145 glycosylation pattern depending on the cell host. These differences in glycosylation, however, do not seem to greatly affect the binding affinity of bNAbs or reactivity against antibodies from HIV-infected patients.

Materials and methods

Antibodies and HIV-1 immunogens

All bNAbs were obtained from the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The HIV-1 CO6980v0c22 was produced by transient transfection of Expi293F cells and purified by a Galanthus nivalis lectin (GNL) affinity column followed by Q-sepharose chromatography. The CHO-K1-produced gp145 was purified following an identical protocol as previously described[16]. Briefly, the culture supernatant was clarified by centrifugation and concentrated by tangential-flow filtration followed by GNL affinity and Q-Sepharose fast flow. The protein was then further concentrated, buffer exchanged into phosphate-buffered saline (PBS), and sterile filtered. Aliquots obtained at 1 mg/mL in phosphate-buffered saline from Advanced Bioscience Laboratories (ABL Inc.) and the U.S. Military HIV Research Program (MHRP), respectively.

Glycan analysis by MALDI-ToF mass spectrometry

Enzymatic release of N-linked glycans from HIV-1 gp145 was carried out following the method of Küster et al. [32] with minor modifications. Briefly, excised bands containing 20 μg of gp145 were incubated with 5000 units of PNGase F (NEB) for 24 hrs. at 37°C [32]. For the analysis of oligosaccharides containing sialic acid, 20 μg of gp145 in solution were incubated with 50 units of α2–3,6,8,9 NEUA (NEB) for 24 hrs. at 37°C, prior to PNGase F digestion. Subsequently, glycans were extracted into LC-MS water in an ultrasonic bath. The extracted glycans were then dried in a vacuum concentrator and dissolved with water. For MALDI-ToF analysis, glycans were co-crystallized with a solution of 50% acetonitrile/50% 20 mM ammonium citrate containing 134 mM of 2′,4′,6′-Trihydroxyacetophenone monohydrate (Sigma-Aldrich). MALDI-ToF MS analysis of the N-glycans was performed in the reflector positive and negative ion mode using a 4800 Plus MALDI ToF/ToF Analyzer (AB Sciex) that was calibrated externally using the Calmix 5 Opti-ToF High-Resolution TIS Calibration Insert (AB Sciex). N-glycan assignments were carried out using GlycoWorkbench software and confirmed by MALDI-ToF post-source decay spectra (S2 Fig) [33].

Peptide mass fingerprinting

The in-gel tryptic digestion of the HIV-1 gp145 protein was performed following the method of Shevchenko et al. with minor modifications [34]. Briefly, 20 μg of gp145 were separated in a 10% Mini-Protean TGX Gel (BioRad). The SDS-PAGE gel was then stained for 1 hr. with Coomassie Brilliant Blue R-250 dye and destained overnight in 50% methanol and 10% acetic acid. Subsequently, the gel band corresponding to the molecular weight of gp145 was excised and destained in 25 mM ammonium bicarbonate diluted in 50% acetonitrile at 37°C. Then, samples were digested overnight at 37°C with PNGase F (NEB). After the N-linked glycans were removed, gel pieces were reduced for 40 min at 37°C with 10 mM dithiothreitol (MilliporeSigma) diluted in 50 mM ammonium bicarbonate (Fluka). Next, alkylation was performed in the dark with 55 mM iodoacetamide diluted in 50 mM ammonium bicarbonate at room temperature (RT). The samples were then digested with 100 μL of proteomic grade trypsin (17 ng/μL) in 10 mM ammonium bicarbonate containing 10% (vol/vol) acetonitrile at 37°C overnight. The peptides were extracted with 200 μL of extraction buffer [1:2 (vol/vol) 5% formic acid/acetonitrile] for 15 min at 37°C. The obtained peptides were dried in a speed vacuum concentrator and resuspended in 40 μL 5% formic acid (Honeywell). To analyze the samples by MS, a mixture of peptides and the MALDI matrix solution [1:1 (vol/vol) sample/72 mM 4-Chloro—α-cyanocinnnamic acid, 70% acetonitrile, and 1.5% formic acid] was spotted onto the MALDI plate insert. MALDI-ToF MS data were acquired in the reflector positive ion mode using a 4800 Plus MALDI ToF/ToF Analyzer (AB Sciex) that was externally calibrated using the Calmix 5 Opti-ToF High-Resolution TIS Calibration Insert (AB Sciex). The mMass—Open Source Mass Spectrometry Tool (Version 5.5.0) was used for spectra and sequence coverage analysis.

Capillary isoelectric focusing

The gp145 produced in CHO-K1 and Expi293F cells was prepared by mixing 25 μg in 100 μL IEF solution containing 0.8% pH 2–9 Servalyte, 4.22/9.46 pI markers, 4.5 M urea, and 0.35% methylcellulose (MC). The samples were vortexed for 10 s and centrifuged at 14,000 rpm for 10 min at RT. Subsequently, 80 μL of the spun sample was transferred to a 2 mL vial containing a 300 μL vial insert. IEF separation of gp145 immunogens was carried out using an imaging cIEF technique on an iCE3^™ Analyzer (ProteinSimple). The PrinCE autosampler temperature was set at 10°C and the separation was performed in a 50 mm, 100 μm (I.D.) fluorocarbon-coated capillary cartridge (cIEF cartridge fluorocarbon-coated). The solutions containing 0.1 M NaOH in 0.1% MC and 0.08 M phosphoric acid in 0.1% MC were used as the catholyte and anolyte, respectively. The instrumental settings for the gp145 immunogens were as follows: prefocus 1 min at 1.5 kV and focus at 3 kV for 7.0 min. The absorbance at 280 nm was monitored using a whole column ultraviolet absorption detector. The resulting electropherograms were calibrated using the iCE3 CFR control software V4.1 (ProteinSimple) and plotted using GraphPad Prism (Version 6.07).

Binding kinetics of HIV-1 gp145 to 2G12 and PG16 bNAbs

The binding kinetics of gp145 immunogens to 2G12 and PG16 bNAbs were measured using the Octet QK^e BLI system (Pall ForteBio Corp). The 2G12 and PG16 bNAbs in 0.5X Kinetics Buffer (PBS with 0.02% Tween and 0.05 mg/mL BSA) were loaded onto an anti-Human IgG Fc Capture sensor (Pall ForteBio Corp). Binding kinetics were determined using serial dilutions of CHO-K1-produced and Expi293F-produced gp145. The BLI method for 2G12 was as follows: 2G12 (0.2 μg/mL) load: 500 s at 1200 rpm; baseline: 200 s at 1200 rpm; association: gp145 serial dilutions 100–3.125 nM in 0.5X Kinetics Buffer 900 s at 1200 rpm and dissociation: 1000 s at 1200 rpm. The BLI method for PG16 was as follows: PG16 (0.357 μg/mL) load: 600 s at 1200 rpm; baseline: 200 s at 1200 rpm; association: gp145 serial dilutions 125–31.25 nM in 0.5 X Kinetics Buffer 900 s at 1200 rpm and dissociation: 1000 s at 1200 rpm. To correct for the instrument drift, a reference well containing only 0.5X Kinetics Buffer was monitored in parallel and used for background subtraction. Curve fitting analysis was performed using the global fitting and 1:1 binding model. The mean of k_a (rate of association), k_d (rate of dissociation), and K_D (dissociation constant) were calculated using the ForteBio Analysis 8.2 program.

Binding kinetics for CD4, b6, VRC01 [35], 2G12, PG9, PG16, 2158, 447-52D, PGT121, and 4E10 antibodies were measured using the Octet Red 96 system with the following assay orientation: Ligand: biotinylated gp145; Analyte: HIV-1 antibody. The biotinylated CO6980v0c22 Env was immobilized on streptavidin biosensors at a single concentration of 10 μg/mL for 250 s, to reach ~50% saturation of the biosensor. After reaching baseline, sensors were dipped into six, two-fold dilutions of the monoclonal antibodies at various concentrations for association. Sensors with analyte-ligand complexes were then moved into kinetics buffer to measure dissociation rates. Association and dissociation rates were measured in real-time and were calculated using the ForteBio Analysis software, which fit the observed global binding curves to a 1:1 binding model. Non-specific binding of the monoclonal antibody to the sensor and/or a buffer only reference was subtracted from all curves. Rate constants were calculated using at least three different concentrations of analyte, to achieve an X²<3 and R²>0.90.

Enzyme-linked immunosorbent assay (ELISA)

To determine whether differences in glycosylation affect antibody recognition, we investigated the immunoreactivity of IgG from plasma of HIV-positive patients toward CHO-K1-produced and Expi293F-produced gp145. Twenty samples (15 HIV-seropositive and 5 control women; 1/10,000 dilution) were obtained from the repository of the Hispanic/Latino Longitudinal HIV-seropositive women and incubated for two hours on Nunc-Immuno MicroWell plates (Sigma-Aldrich) coated with gp145 produced in either CHO-K1 or Expi293F cells (final concentration 0.5 μg/mL). IgG antibodies were detected with an anti-human HRP-conjugated antibody (1/5000 dilution) and absorbance read at 490 nm. Each sample was run in triplicate and values were normalized against the blank. A Two-Way ANOVA with a Dunnett’s multiple comparison test was used to assess statistical differences. Values with a p < 0.05 were considered significant.

Participants’ description

Blood samples of 20 participants, 15 HIV-seropositive and 5 control women, were obtained from the repository of the Hispanic/Latino Longitudinal HIV-seropositive women cohort (20 plasma samples) (IRB protocol 1330107). The inclusion criteria included consenting adults with or without HIV infection and without active systemic infections. All participants consented to have samples stored in the cohort repository for future related studies toward the understanding of HIV infection mechanisms and future treatment modalities. Characteristics of the participants are described in Table 1. The HIV-seropositive group was further divided into those who received no antiretroviral treatment (ART, n = 4), used older ARTs (from 2006–2012, n = 7), and those who used newer ART (≥2012, n = 4).

Table 1. Participant characteristics.

	Controls (n = 5)	HIV-seropositive (n = 15)
Age (years¹)	29 (25, 40)	39 (24, 58)
CD4 cells/mm³	--	371(18, 1007)
HIV RNA copies/mL (log)	--	2,63 (1.70, 5.50) 2 ND², 2 missing
old ART³ combinations (2006–2012, n = 7)	--	nelfinavir, lamivudine, zidovudine, saquinavir, abacavir, atazanavir
new ART³ combinations (≥2012, n = 4)	--	raltegravir, emtricitabine, tenofovir, etravirine

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¹median(range),

²ND = no detectable,

³ART = antiretroviral treatment

Results

Confirmation of HIV-1 gp145 protein identity

The identity of CO6980v0c22 gp145 produced in CHO-K1 and Expi293F cells was confirmed by peptide mass fingerprinting (Fig 1A and 1B). Briefly, the excised protein gel band corresponding to gp145 (S1 Fig) was first PNGase F-digested and then trypsin-digested. The deglycosylated peptides were analyzed by MALDI-ToF. MS results showed an identical distribution of trypsin-cleaved peptides in a mass range of 800–3000 Daltons for HIV-1 gp145 from both cell lines. Furthermore, sequence coverage was similar for gp145 produced in CHO-K1 and Expi293F cells, 53% and 50%, respectively. Collectively, these results confirm that the same protein is being encoded and made by both CHO-K1 and Expi293F cell lines.

Differences in the glycosylation profiles of CHO-K1- and Expi293F-produced HIV-1 gp145

The distribution of N-linked glycans in gp145 produced in CHO-K1 and Expi293F cells was analyzed by MALDI-ToF following PNGase-catalyzed glycan release. The identity of specific glycans was confirmed by post-source decay (PSD) (S2 Fig). MS analysis in reflector positive ion mode revealed a similar distribution of oligomannose-type glycans for gp145 from both cell hosts (Fig 2A and 2C, S1 Table). Specifically, an oligomannose population of Man_5-9GlcNac₂ with peaks at m/z 1256, 1418, 1580, 1742, and 1904 ([M + Na]⁺ ions), respectively, was observed. Furthermore, a higher ratio of mannose to complex N-glycans was seen for the protein produced in either cell line. Bi- and tri-antennary complex N-glycans with core fucose (peaks at m/z 1809 and 2174) were observed in both CHO-K1- and Expi293F-produced gp145.

Fig 2 — The recombinant gp145 (20 μg) produced in (**A-B**) CHO-K1 and (**C-D**) Expi293F cells were incubated overnight with (B and D) and without (A and C) Neuraminidase A (NEUA) at 37°C. Then, gp145 proteins were resolved by SDS-PAGE and the 145 kDa bands were excised. N-glycans were released with PNGase F and were co-crystallized with 2',4',6'-Trihydroxyacetophenone monohydrate (THAP) ionization matrix. The x-axis represents the mass-to charge ratio (m/z) value in Daltons and the y-axis shows the relative abundance (arbitrary units) of the ions. The released glycans were analyzed using the MALDI-ToF reflector positive mode. Predicted glycan structures of the masses shown in the upper panel spectrum are the same as those shown in the spectrum on the lower panel.

The presence of sialylated N-glycans was determined following hydrolysis of sialic acid residues by neuraminidase A (NEUA). Analysis of the released glycans was performed in both reflector positive and reflector negative ion mode (Figs 2 and 3). The data collected from the reflector positive mode revealed the presence of bi-, tri-, and tetra-antennary complex N-glycans with core fucose (peaks at m/z 1808, 2174, and 2538) for gp145 produced in both cell hosts (Fig 2B–2D, S2 Table). A percentage increase in the relative intensity of these glycans in the NEUA-treated samples (in comparison to the untreated samples) suggests that these complex glycans are sialylated. The presence of sialylated glycans was confirmed by the detection of sialylated bi-, tri-, and tetra-antennary complex N-glycans with core fucose (peaks at m/z 1929, 2075, 2390, 2440, and 2754) of the untreated sample in the negative reflector mode (Fig 3A and 3C). When gp145 was treated with NEUA, the signal for these sialylated N-glycans disappeared (Fig 3B and 3D). In order to compare the relative levels of sialylation in CHO-K1- and Expi293F-produced gp145, we plotted the magnitude of neutral glycan increase upon treatment with NEUA for each of the glycan signals (Fig 4). Our results showed that the magnitude of the increase (fold change) is higher for gp145 produced in CHO-K1 cells than for the one produced in Expi 293F. Taken together, these results suggest that CHO-K1 cells produce a gp145 that is more highly sialylated than the one produced in Expi293F.

Fig 3 — The recombinant gp145 (20 μg) produced in (**A-B**) CHO-K1 and (**C-D**) Expi293F cells were incubated overnight with (B and D) and without (A and C) Neuraminidase A (NEUA) at 37°C. Then, gp145 proteins were resolved by SDS-PAGE and the 145 kDa bands were excised. N-glycans were released with PNGase F and were co-crystallized with 2',4',6'-Trihydroxyacetophenone monohydrate (THAP) ionization matrix. The x-axis represents the mass-to charge ratio (m/z) value in Daltons and the y-axis shows the relative abundance (arbitrary units) of the ions. The released glycans were analyzed using the MALDI-ToF reflector negative mode. Predicted glycan structures of the masses shown in the upper panel spectrum are the same as those shown in the spectrum on the lower panel.

Fig 4 — Fold change increase for the composition of acidic N-glycans after the addition of Neuraminidase A (NEUA). To calculate the fold change of glycans obtained by MALDI-ToF analysis, the relative abundance of glycans from NEUA-treated samples was normalized against an untreated sample (NEUA+/NEUA-). The relative abundance of a given glycan was determined by an in-house program that adds the ion counts of the user-designated mass and the related isotopic peaks that are plus-or-minus 1, 2, 3 and 4 mass units, then divides the given glycan total by the sum of the ion counts for all glycans under consideration. In this figure, the glycans with the following masses were considered: 1298.5, 1460.5, 1622.6, 1663.6, 1809.5, 2174.7, and 2539.9 (the principal peaks are seen in Fig 2).

Distribution of charge variants

The observed high sialic acid content in the CHO-K1-produced gp145 was expected to result in a lower isoelectric point (pI). To confirm these expected differences in pI, we measured charge heterogeneity using imaged capillary isoelectric focusing (cIEF) in the presence and absence of NEUA (Fig 5). The pI distribution for the CHO-K1-produced gp145 ranged between 4.3–6.2, whereas that of the Expi293F-produced ranged between 5.4–7.8 (Fig 5A). The observed pI distribution for gp145 produced in both cell hosts was more acidic than the theoretical pI of 8.7 calculated from the amino acid sequence of CO6980v0c22 gp145, however, it is clear that the CHO-K1-produced gp145 is more acidic than the Expi293F-produced gp145. This observation is consistent with a more highly sialylated glycoprotein. As expected, treatment with NEUA shifted the pI of gp145 produced in either cell line to a range of 7.5–9.0 (Fig 5B).

Fig 5 — Electropherograms from 3 consecutive injections of CHO-K1- (blue lines) and Expi293F-produced (black dash lines) gp145 with (B) and without (A) neuraminidase. The prefocusing was performed for 1 min at 1.5 kV and the focusing for 3.0 kV at 4.5 min. Capillary IEF sample solutions contained 0.7 μg/μL of gp145, 5 M urea, pI markers 4.22/9.46 and servalyte 2–9 carrier ampholytes.

Recognition of HIV gp145 by the broadly neutralizing antibodies, 2G12 and PG16

Next, we determined the possible effect of host-dependent glycosylation on the recognition of gp145 by broadly neutralizing antibodies (bNAbs), specifically 2G12 [36–40] and PG16 [41]. These antibodies were chosen because their epitope specificities are known to be the high-mannose cluster on the glycan shield and the glycosylation sites in the V1V2V3 loops of the HIV-1 envelope, respectively [42]. Binding was evaluated via biolayer interferometry (BLI) using the FortéBio Octet QK^e in two different configurations: we either captured the corresponding bNAb onto an anti-Fc surface or we immobilized biotin-labeled gp145 on a streptavidin surface.

Using the Fc capture system to immobilize the bNAb, we did not detect any significant differences in binding of CHO-K1-derived and Expi293F-derived gp145 to 2G12 (1.6 nM vs. 1.2 nM; Fig 6A and 6B and Table 2) and PG16 (6.6 nM vs. 2.8 nM; Fig 6C and 6D and Table 2).

Fig 6 — (**A-D**) Octet QK^e sensorgrams generated by binding of serial of dilutions of gp145 produced in CHO-K1 and Expi293F cells to 2G12 and PG16 bNAbs loaded onto anti-human IgG FC capture (AHC) biosensors. Tables show the k_a (rate of association), k_d (rate of dissociation) and K_D (dissociation constant) values for the interactions of HIV-1 gp145 to (**A-B**) 2G12 and (**C-D**) PG16 bNAbs. Black lines represent the 1:1 global fit curve.

Table 2. Binding of soluble CO6980v0c22 gp145 to Fc-captured bNAbs 2G12 and PG16.

Antibody	Expression Host	k_a (1/Ms)	k_d (1/s)	K_D (nM)
2G12	CHO-K1	1.25E+05	2.23E-04	1.62 ± 0.01
2G12	Expi293F	9.09E+04	1.11E-04	1.2 ± 0.2
PG16	CHO-K1	1.67E+04	1.06E-04	7 ± 3
PG16	Expi293F	2.09E+04	5.97E-05	3 ± 1

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Binding kinetics were obtained using the Octet QKe system. Broadly neutralizing antibodies were captured on an anti-human IgG Fc sensor and dipped into different dilutions of gp145. Association/dissociation rates were analyzed using the ForteBio analysis software. Values represent mean ± standard deviation (STD) from three independent experiments

However, differences were observed in the binding of 2G12 and PG16 when the CHO- and Expi293F-derived gp145s were immobilized onto the sensor (Table 3). These apparent discrepancies in binding values depending on the assay orientation are not uncommon [43]. When bNAbs are captured on an anti-human Fc sensor, the antibody molecules are oriented in the same fashion exposing the antigen binding region in an optimal and uniform manner. Thus, it is not surprising that higher affinities were obtained when the antibodies were captured, than when the antigen is immobilized in a random orientation [43].

Table 3. Binding of bNAbs 2G12 and PG16 to immobilized CO6980v0c22 gp145.

Antibody	Expression Host	k_a (1/Ms)	k_d (1/s)	K_D (nM)
2G12	CHO-K1	4.23E+04	2.23E-02	527 ± 19.3
2G12	Expi293F	4.52E+05	2.98E-02	66.0 ± 3.1
PG16	CHO-K1	1.85E+04	2.62E-03	142 ± 3.7
PG16	Expi293F	3.26E+04	2.05E-03	62.8 ± 0.3

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Binding kinetics were obtained using the Octet Red 96. The CO6980v0c22 gp145 was biotin-labeled and captured on a streptavidin sensor. The resulting sensor was dipped into different dilutions of broadly neutralizing antibodies and association/dissociation rates were analyzed using the ForteBio analysis software. Values represent mean ± KD error.

We also measured the binding affinity between CHO-K1- and Expi293F-derived gp145 to other HIV-1 bNAbs and no differences in binding affinity were observed (S3 Table).

Reactivity of gp145 against HIV+ and HIV- patient plasma

To assess how differences in glycosylation and charge distribution translate to immunoreactivity, we measured the magnitude of binding antibodies from patient’s plasma to gp145 produced in either CHO-K1 or Expi293F cells. Our results showed that the plasma of HIV+ individuals reacted significantly over the background against gp145 (Fig 7A), whereas the plasma of uninfected individuals did not react (Fig 7B). Despite the fact that subtype B is the dominant subtype in the Caribbean region [44], plasma from HIV-infected Puerto Ricans showed substantial cross-reactivity with this subtype C immunogen. This cross-reactivity was not surprising since it had been reported by the group of Zolla-Pazner, where they found that individuals infected with clade B virus made antibodies that reacted with Clade C immunogens[45]. Still, no differences in reactivity were observed between gp145 produced in CHO-K1 and Expi293F, indicating that differences in glycosylation do not affect the recognition by HIV infected patient polyclonal antibodies (Fig 7A and 7B).

Fig 7 — The recombinant protein gp145, produced in either CHO-K1 or Expi293F cells, was tested for its potential to bind antibodies found in HIV+ patients using an ELISA assay. (A) Plasma samples from 16 HIV+ patients, P1-P16, (1:10,000 dilution) were incubated in 96-well plates coated with gp145 samples produced in two different cell lines (0.5 μg/mL per well). Binding was detected with an anti-human IgG HRP-conjugated antibody. Absorbance was read at 490 nm and standardized against the blank. Values significantly different from the blank (represented by asterisks) were considered reactive. Statistical significance was calculated using a Two-Way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (B) HIV+ samples were compared with HIV uninfected samples using One-Way ANOVA. Significant differences (represented by asterisks) were found between the two groups but not between immunogens. ****p<0.001.

Discussion

The HIV envelope glycoprotein (Env) is the most promising candidate for the development of HIV vaccines to date. The short versions of Env, all variants of the gp120, have given mixed results in Phase III efficacy trials, thus highlighting the need for developing new and more complex Env constructs that can elicit a robust immune response. In this paper, we describe efforts to develop a uncleaved gp145 from strain CO6980v0c22, whose amino acid sequence includes the whole gp120 plus a substantial portion of the gp41. This construct was produced using two different expression hosts, and the differences between them were assessed.

The effect of the choice of expression host on the glycosylation and charge distribution for the monomeric gp120 has been explored by others [28,29]. Studies on the glycosylation of A244 and MN gp120 concluded that both proteins are more highly sialylated when expressed in CHO-K1 cells rather than in Expi293F and that this difference in sialylation has some effect on antibody recognition [29]. Similarly, studies on the glycosylation of the consensus B gp120 showed that there are substantial differences in glycosylation depending on the expression host, with Jurkat cells being the host with the most mannose glycosylation [28].

The effect of expression host on glycosylation has been even more closely scrutinized for the production of the native-like SOSIP trimers [30,31]. A detailed study on the glycosylation of BG505.664 SOSIP trimers revealed that, while the gp120 portion of the trimer is mostly decorated with oligomannose glycans, the gp41 portion contains mostly complex glycans, with those made in CHO-K1 cells containing a higher sialic acid content than the cleaved trimers produced in 293 cells [31]. That same study concluded that the uncleaved versions of the glycoprotein contained more complex glycans than the native-like trimeric versions of the corresponding glycoprotein, although this was only reported for gp140 made in 293 cells [31]. Others have reported a similar occurrences of the uncleaved trimer containing a higher proportion of complex glycans [46,47].

In this study, we compared the glycosylation of uncleaved gp145 trimers made in CHO-K1 and Expi293F cells. Our findings that CHO-K1 cells are able to incorporate a larger proportion of sialylated complex glycans is consistent with previous findings [29,31]. These differences in glycosylation did not greatly affect protein recognition by the glycan-specific antibodies, 2G12 and PG16, nor did they affect reactivity against patient-derived antibodies. It was expected that the 2G12 antibody would bind to both CHO- and EXPI293F-produced gp145 with similar affinity, since its epitope is a high-mannose glycan that is likely to be the same in both cell types [48]. Similarly, we did not expect differences in immunogen recognition by PG16 since its epitope has been reported to be conformational and affected by N-glycosylation, but not sensitive to specific monosaccharide units [49]. The group of Binley reported differences in Env recognition by the glycan-dependent bNAb PG9 that were attributed to the linkage of the sialic acid, which is α-2,6 in 293 cells and α-2,3 in CHO-K1 cells [50]. However, we do not see the same sialic linkage-dependent differences in the binding of PG16, probably a reflection that the epitopes for these two antibodies are overlapping but different.

While there is no clear notion of an optimal glycosylation state for HIV immunogens, recent evidence has revealed that virions contain more complex glycosylation than previously thought [30]. It is generally understood that the sialylation of protein therapeutics extends their pharmacological half-life [51]. However, it is not known whether this increase in the half-life of the glycoprotein would affect vaccine efficacy. Still, the glycosylation of recombinant HIV Env vaccines remains an important feature related to their production yield, quality, and stability. Clearly, the choice of expression host affects glycosylation of the resulting protein in dramatic ways that impact its net charge. Future work should attempt to delineate the relationship between specific glycan content and host immune response. Also, experiments should be performed to identify protein production bottlenecks caused by glycosylation in mammalian cell systems. Overall, glycosylation is an important consideration in the design of new immunogens and in the development of methods for quality analysis.

Supporting information

S1 Fig. SDS-PAGE analysis of HIV-1 gp145 produced in CHO-K1 and Expi293F cells.

The recombinant gp145 immunogens were incubated overnight with (+, lanes 3 and 5) and without (-, lanes 2 and 4) Neuraminidase A, and then resolved on 10% SDS-PAGE gels.

(TIF)

Click here for additional data file.^{(486.8KB, tif)}

S2 Fig. MALDI-Post-source decay (PSD) spectra of N-linked glycans derived from CHO-K1 gp145.

The presence of Man5GlcNAc2 (A), Man6GlcNAc2 (B), Man7GlcNAc2 (C), Man8GlcNAc2 (D), Gal2Man3GlcNAc4Fuc1 (E) and Man9GlcNAc2 (F) was confirmed by MALDI-PSD. The x-axis represents the mass-to charge ratio (m/z) value in Daltons and the y-axis shows the relative abundance (arbitrary units) of the ions.

(TIF)

Click here for additional data file.^{(1.8MB, tif)}

S1 Table. N-glycans composition of C06980v0c22 gp145 using MALDI-ToF in reflector positive ion mode.

All the peaks obtained in the MALDI-ToF were compared against the list previously reported by Doores et al., 2010. Some of them were selected randomly for confirmation by MS/MS analysis.

(DOCX)

Click here for additional data file.^{(151KB, docx)}

S2 Table. N-glycans composition of C06980v0c22 gp145 using MALDI-ToF in reflector negative ion mode.

(DOCX)

Click here for additional data file.^{(84.8KB, docx)}

S3 Table. Binding of CO6980v0c22 gp145 to HIV-1 antibodies.

Binding kinetic results were obtained using the Octet Red 96 system. Biotinylated gp145 was immobilized on streptavidin sensors, dipped into two-fold dilutions of the monoclonal antibodies and association/dissociation rates were analyzed using the Octet Molecular Interaction System software. *All CHO-K1 antigenicity results were previously reported by Wieczorek et al. 2015 (16).

(DOCX)

Click here for additional data file.^{(21.3KB, docx)}

Acknowledgments

The authors also thank Advanced Biosciences Laboratories (ABL), Inc. for providing the CO6980v0c22 gp145 reference material and Ms. Elaine Rodríguez for technical support and sample retrieval. A special note of thanks goes to Ms. Nandini Sane from the NIH Division of AIDS providing critical feedback. Also, the authors acknowledge WHO-UNAIDS and the NIH AIDS Reagent Program for providing bNAbs for the current study. Specifically, 1) the VRC01 reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (VRC01), from Dr. John Mascola (cat# 12033); 2) the 2G12 reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (2G12) from Polymun Scientific; 3) the PG16 reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (PG16) from IAVI.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by the National Institutes of Health (NIH) grant R01AI122935 to JAL-D and AB-O. This work was also supported by a cooperative agreement (W81XWH-07-2-0067) between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense (DoD). JAL-D wants to acknowledge the US Civilian Research and Development Foundation (CRDF) for providing the necessary funds (funding agreement # OISE-9531011-NIH) to establish the Clinical Bioreagent Center (CBC). Additional support from NIH grants U54NS43011, S11NS046278, and U54MD007587 to VW enabled the patient sample repository. The authors also acknowledge the Puerto Rico Science, Technology & Research Trust, for financing the acquisition of key instruments.

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PLoS One. doi: 10.1371/journal.pone.0231679.r001

Decision Letter 0

Juan Pablo Jaworski

14 Apr 2020

PONE-D-20-07724

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: effects on glycosylation and antigenicity

PLOS ONE

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Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the manuscript presented by Gonzalez-Feliciano et al, the authors study the glycosylation pattern of the HIV surface protein gp145 in its uncleaved form utilizing two mammalian cell lines, CHO-K1 and Expi293F. The antigen is the primary target for vaccine development against HIV. The authors present literature supporting the notion that different expression systems differ in the way gp145 is decorated, having a potential impact in the generation of and recognition by bNAb. The authors found relevant differences regarding the nature of the glycans in gp145 produced in the two cells lines using suitable technology. They also assess the impact on antigenicity given by glycan differences using bnMAb and polyclonal sera from infected patients. Overall this is an interesting and exciting work that is well written and the subject is of relevance to the field. However, in this reviewer's modest opinion, the manuscript needs minor and major revisions to be considered for publication.

Minor revisions

Figure 2C. Plot shows peak at 1742. The number above the peak reads 17423.57. Please fix the decimal point.

Figures 1, 2, 3 and 4. Please clarify what unit m/z is.

Populations?

Font/size of figure 3 B differ from the other panels in the figure.

Background noise figure 3B and D?

In the figures 3 C there is a peak at ~2800 that is not identified that has a relative intensity comparable with some that are identify and is a potential difference with 3 A. Could you please provide details on why this can be omitted?

Line 288. Please define unit “au”.

Line 289-291. Here you refer to the peaks that you included in the analysis. I) the figure show 7 peaks that were analyzed whereas in the text you mentioned 12. I would like to know why is this difference. Also, the numbers mentioned in the text do not match the number in the figure 2. For example, in figure 2 the peak reads as 1257.4, in all four panels, but in the text you refer to this peak as 1256. Please, these discrepancies need to be fixed.

Line 290. “12987.5” I think is 1298.75.

Line 295. You first refer to isoelectric point and then pI. I suggest to add “(pI)” after “isoelectric point” in this line.

Comparison of bNAb. Here you show that immobilization of the antibody does not show differences in the recognition of gp145 produced by any of the cell lines. Whereas when you use gp145 attached to the surface you do see differences. You justify these differences by the way the antigen is presented to the antibody. Even though I do believe these data need to be published, I would suggest that is shown as supplementary data, so it does not affect the interpretation of the of the other, more solid, result.

Line 358-359: “…even when stratifying for antiretroviral treatment status.” I do not find this stratification anywhere in the figure 7 or anywhere else in the manuscript. Please, either add the missing data or remove this statement.

Figure 7. Please, add what is the meaning of the asterixis in the figure.

I would like if you could add to the discussion what would be the importance of the protein presenting more glycans bearing sialic acid. You used approaches specifically to differentiate these from other glycans but do not mention why they are important in this context neither discuss it. Please, I suggest that you add this information since it would help to put the importance of these differences in context.

Major

In the analysis of the antibody binding you make significant inferences from the data without running any statistical method. I think it is important to show by some statistical method that these differences in binding are significant or not and then based on that discuss the results.

Line 354-355 “Despite the fact that subtype B is the dominant subtype in the Caribbean region…” The sera used to assess reactivity (figure 7), were all from patients infected with subtype B? Or since this is the main subtype in the Caribbean this is an assumption? If available, this information needs to be added to the manuscript. In any way, I would like to see some deeper discussion on how this may affect your results. If there is more literature on this, please cite and discuss. As you acknowledge, subtype mismatch may affect the way these sera recognize the antigen. In other viruses, these mismatches between subtypes may go from none recognition to partial or fairly good recognition. I guess what you are showing belongs to the later. Do you think that heterologous sera may recognize better or worse subtle differences in the glycosylation of gp145?

Reviewer #2: This paper compares the antigenicity and glycan profiles of a clade C gp145 expressed either in CHO-K1 and Expi cells. The authors found the gp145’s to differ in isoelectric point and sialic acid incorporation, but that this did not translate into major antigenic differences in terms of 2G12, PG16 or HIV+ plasma binding. They conclude that there are sizeable differences in glycosylation depending on host cell, but that it is unclear how these differences will translate into vaccine efficacy.

The paper is technically really good. However, the question comes with the samples. It’s not clear why the clade C strain was chosen. Nor is it clear why it was generated as an uncleaved gp145, especially considering the now substantial evidence for better folded forms of trimer ectodomain that would be more authentic representations of the surface spikes from the clade C virus they chose. Uncleaved gp140 is not compact and glycans tend to be more processed compared to native spikes, because of the fewer constraints on glycan processing. It would be even better if they were truly native trimers, as expressed in membranes. The use of a non-native form of gp140 inevitably reduces the power of the findings.

The authors initially did not find any notable differences in binding by 2G12 and PG16. However, in Table 3, line 334, they observed “differences” (lower binding?) without explaining what they mean. For example, do they normalize PG16 binding to that of 2G12? Or do they compare the patterns in the two orientations? Or do they mean that the capture methods lead to different outcomes that may not reflect antibody binding differences? For 2G12, this is not surprising since this antibody recognizes an invariant high mannose epitope that is unlikely to be affected by producer cells. For PG16, the lack of change may in part trace to the fact that CHO cells tend to add alpha 2,3 sialic acids, whereas PG16 prefers the 2,6 sialic acids more commonly found in human production (PMID: 29718999), so PG16 is ultimately ambivalent or even slightly averse to the CHO cell 2,3 glycans. There is in fact a slight loss of PG16 binding to CHO cells, as compared to the control using 2G12 as a control arbiter for binding. Overall, I am not sure the data in S3 Table and elsewhere (Table 2, 3 and other kinetic data) for the Octet work are not different for the two gp145’s. A lot of tabulated Kds could be plotted to check for antibody-specific patterns. KD’s are not massively different, but the degree of difference seems to vary per antibody which may be worth capturing. Otherwise this would not fully investigate the patterns that are justified by the effort in running all these affinity tests.

The two gp145 preps were also purified differently. It is unclear what the method for the CHO version was as it refers to another paper (a brief description would help). A major question is whether these gp145s are monomers or oligomeric forms or various? These different forms will bear different glycans so this is another problem leading to variability, in addition to using uncleaved gp145.

line 70: Native trimers here are actually “near native” and should be referred to as such. While they are a closer match to native, membrane trimers than uncleaved gp145, there are several differences.

line 79: Re: a tremendous strain on the protein trafficking machinery, the other side of this point is that the glycans play a key structural role in folding. Removing some glycans can decrease expression of trimers. So there is more of a trade off with glycans being present.

line 92: There have been some prior analyses on uncleaved gp140. A google search of “uncleaved gp140 glycans” revealed a few articles, including (PMID: 26051934, PMID: 26018173), and there have been a few by Go/Desaire. Uncleaved gp140 glycans tend to be more processed according to (PMID: 26051934), consistent with their less compact, non-native conformation.

line 311: The subtitle heading would be better reversed to show that it is the antibodies binding to the gp145 and not the other way around.

**********

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Reviewer #1: Yes: Lucas M. Ferreri

Reviewer #2: No

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PLoS One. 2020 Jun 19;15(6):e0231679. doi: 10.1371/journal.pone.0231679.r002

Author response to Decision Letter 0

2 May 2020

Response to Reviewer #1:

Thank you for taking the time to read our manuscript thoroughly and for pointing out the technical suitability of the biochemical methods employed to address the question of glycan composition and antigenicity. We have accepted all of your minor revisions and have amended the manuscript to reflect that. Below is a point-by-point response to your comments:

“Minor revisions

Figure 2C. Plot shows peak at 1742. The number above the peak reads 17423.57. Please fix the decimal point.”

We have fixed this mistake in Figure 2C . Thank you for pointing it out.

“Figures 1, 2, 3 and 4. Please clarify what unit m/z is.”

The legends now include an explanation of what the x- and y- axes represent. The lableing of the x-axis of MALDI-ToF spectra as “m/z” is consistent with the published literature of the field.

“Font/size of figure 3 B differ from the other panels in the figure.”

The figure has been amended.

“Background noise figure 3B and D?”

Figure 3 present the MALDI-ToF spectra in the negative mode for glycans that were either fully sialylated (3A and 3C) or enzymatically desialylated (3B and 3D). Because desialylated glycans lose their negative charge their signal is dampened in the negative mode, hence the apparent increase in the noise.

“In the figures 3 C there is a peak at ~2800 that is not identified that has a relative intensity comparable with some that are identify and is a potential difference with 3 A. Could you please provide details on why this can be omitted?”

Thank you for pointing this out. The 2806 signal corresponds to the tetra-antenary sialylated glycans. We have now identified the signal in figure 3C. The signal for this glycan is not one that we detect consistently. Thus, we would not be comfortable drawing conclusions from the presence or absence of this speficic signal at in the current data.

“Line 288. Please define unit “au”.”

Has been changed to “mass units” and it is now in Line 295

“Line 289-291. Here you refer to the peaks that you included in the analysis. I) the figure show 7 peaks that were analyzed whereas in the text you mentioned 12. I would like to know why is this difference. Also, the numbers mentioned in the text do not match the number in the figure 2. For example, in figure 2 the peak reads as 1257.4, in all four panels, but in the text you refer to this peak as 1256. Please, these discrepancies need to be fixed.”

The figures have been changed and the text has been amended.

“Line 290. “12987.5” I think is 1298.75.”

The text has been amended and it is now Line 295

“Line 295. You first refer to isoelectric point and then pI. I suggest to add “(pI)” after “isoelectric point” in this line.”

The text has been amended.

“Comparison of bNAb. Here you show that immobilization of the antibody does not show differences in the recognition of gp145 produced by any of the cell lines. Whereas when you use gp145 attached to the surface you do see differences. You justify these differences by the way the antigen is presented to the antibody. Even though I do believe these data need to be published, I would suggest that is shown as supplementary data, so it does not affect the interpretation of the of the other, more solid, result.”

The reviewer rightly points out the major discrepancy between the two methods of immobilizing molecules for a biosensor binding study. When the antibody is captured on an anti-Fc sensor, all of the antibody molecules are captured in the same orientation. Thus, the resulting sensor surface is fully competent and the data resulting from it should be more reliable. Thus, our interpretation and conclusion that differences in glycosylation and more specifically, sialyation, do not greatly impact antibody binding, are based on the measurements obtained with the captured antibody. We had many discussions internally on whether the binding data obtained with the covalently immobilized gp145 would be more appropriately placed in the supplementary section, but we did not want it to be perceived like we were hiding conflicting results. However, we would be happy to move that table 3 and binding data from the immobilized gp145 to the supplementary section, if that is the reviewer’s recommendation.

“Line 358-359: “…even when stratifying for antiretroviral treatment status.” I do not find this stratification anywhere in the figure 7 or anywhere else in the manuscript. Please, either add the missing data or remove this statement.”

The phrase “…even when stratifying for antiretroviral treatment status.” Has been deleted and is now in Line 368.

“Figure 7. Please, add what is the meaning of the asterixis in the figure.”

We have changed the figure legen to included the statistical test applied to determine significance, and the p-value assigned to each group of asterisks in Lines 376-382.

“I would like if you could add to the discussion what would be the importance of the protein presenting more glycans bearing sialic acid. You used approaches specifically to differentiate these from other glycans but do not mention why they are important in this context neither discuss it. Please, I suggest that you add this information since it would help to put the importance of these differences in context.”

In the world of injectable protein therapeutics a higher proportion of sialic acid is generally thought to contribute to a longer pharmacological half-life once injected, which is typically desirable for most protein therapeutics. This paradigm could be different in the context of vaccines, since a longer half-life is not necessarily correlated with immunogenicity. Thus, the impact of glycosylation on the eventual efficacy of an immunogen is an interesting question that remains somewhat unexplored. We have added a sentence and a new reference (Werner et al., 2007). We have added a sentence on this topic in Lines 429-431.

Major revisions

“In the analysis of the antibody binding you make significant inferences from the data without running any statistical method. I think it is important to show by some statistical method that these differences in binding are significant or not and then based on that discuss the results.”

The following line was added to the figure legend in Lines 378-382:

“Statistical significance was calculated using a Two-Way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. HIV+ samples were compared with HIV uninfected samples using One-Way ANOVA. Significant differences (represented by asterisks) were found between the two groups but not between immunogens. ****p<0.001”

“Line 354-355 “Despite the fact that subtype B is the dominant subtype in the Caribbean region…” The sera used to assess reactivity (figure 7), were all from patients infected with subtype B? Or since this is the main subtype in the Caribbean this is an assumption? If available, this information needs to be added to the manuscript.”

Thank you for the question, since this point was not made clear in the manuscript. The predominant subtype in Puerto Rico (and the rest of the Caribbean) is subtype B, and therefore, we assume that all of our patient sera analyzed are subtype B, although we cannot say that with any measure of scientific certainty. A change has been made in the text to reflect the fact that we do not know the subtype of HIV present in patient serum. Some lines of text have been added to the results section in Lines 363-365.

“In any way, I would like to see some deeper discussion on how this may affect your results. If there is more literature on this, please cite and discuss. As you acknowledge, subtype mismatch may affect the way these sera recognize the antigen. In other viruses, these mismatches between subtypes may go from none recognition to partial or fairly good recognition. I guess what you are showing belongs to the later. Do you think that heterologous sera may recognize better or worse subtle differences in the glycosylation of gp145?”

Some years ago, the group of Susan Zolla-Pazner at NYU examined the sera of both individuals infected with HIV subtype B and immunized with Env from a subtype B strain (Verrier et al. 2000). The IgG’s from both of these groups exposed to subtype B, were found to bind to peptides and recombinant Env’s from different clades. They especially cross-reacted with clade C peptides. Thus, it was not entirely surprising to see this robust cross-clade reactivity in our patient serum samples. We have included a sentence on this previous work by Zolla-Pazner in Lines 363-365.

Although this has not been thoroughly explored, we expected that at least some of the cross-reactive IgGs would recognize the glycans and we also expected that the difference in total negative charge between the two proteins would be large enough to affect IgG recognition. However, we did not see that. IgGs from Puerto Rico patients bound to both gp145 made in CHO (highly sialylated and negative) and gp145 made in 293 (less sialylated and less negative).

Response to Reviewer #2:

Thank you for taking the time to read our manuscript thoroughly and for providing good explanations for our antibody binding results. We appreciate your observation of the technical suitability of the biochemical methods employed in our work to address the question of glycan composition and antigenicity. Below is a point-by-point response to your comments:

“It’s not clear why the clade C strain was chosen. Nor is it clear why it was generated as an uncleaved gp145, especially considering the now substantial evidence for better folded forms of trimer ectodomain that would be more authentic representations of the surface spikes from the clade C virus they chose. Uncleaved gp140 is not compact and glycans tend to be more processed compared to native spikes, because of the fewer constraints on glycan processing. It would be even better if they were truly native trimers, as expressed in membranes. The use of a non-native form of gp140 inevitably reduces the power of the findings.”

The reviewer rightly notices that although HIV infections in Puerto Rico and the rest of the Caribbean are of clade B strains, our group is leading efforts to produce a vaccine based on a clade C strain. The production of clade C vaccine was an assignment from the NIH under grant R01AI122935, the aims of which included the optimization of titer and yield for a CHO cell line expressing the CO6980v0c22 gp145, originally identified in a Tanzanian strain. This immunogen had previously shown to be notoriously difficult to produce in large amounts with diminishing returns on scale-up. Our group has been able to increase the scale and the titers for this problematic construct through the systematic optimization of media conditions. We understand that the NIH is leading efforts to produce numerous vaccines of different clades and CO6980v0c22 happens to be the one that we were assigned or encouraged to work with.

We understand the reviewer’s comment on developing Env versions that are considered to be more physiological or native-like than the uncleaved trimers. However, it is difficult to predict what an effective and protective HIV vaccine will look like. While it is true that the SOSIP trimers have been successfully built to closely resemble the native trimers, their performance in Phase I clinical trials has not yet been reported. They could very well perform as designed, but this remains to be tested. At the present time, there are numerous other types of cleaved, uncleaved and single-domain constructs that are very much still under consideration and undergoing Phase I vaccine trials. The NFL trimers have shown good promise and so have vaccines templated on the external outer domain (eOD) of gp120 and on the fusion peptide (FP) of gp41.

Currently, one of the most anticipated clinical trials (NCT03060629), which is sponsored by Janssen Vaccines, involves the immunization of human participants with the Mosaic uncleaved gp140 trimer as a boost immunogen. While we do not know the results from this Phase 2b trial, it is fair to say that uncleaved trimers are very much under consideration and our work is a step toward understanding glycosylation and sialylation in this family of Env constructs.

Our CO6980v0c22 gp145 is slightly longer than the Mosaic gp140 as it includes the MPER region which is a known epitope for broad neutralization. This construct has been shown to form trimers and also dimers, in a similar proportion as Env constructs that are considered to be more physiological (Wieczorek et al., 2015). Furthermore, the CO6980v0c22 gp145 is currently undergoing Phase I clinical trials (NCT03382418) for safety and immunogenicity and the trial results should become available soon.

While we agree with the reviewer that there are multiple HIV Env versions out there, it is still too early to discard or dismiss any of them, especially while they are undergoing clinical testing.

“The authors initially did not find any notable differences in binding by 2G12 and PG16. However, in Table 3, line 334, they observed “differences” (lower binding?) without explaining what they mean. For example, do they normalize PG16 binding to that of 2G12? Or do they compare the patterns in the two orientations? Or do they mean that the capture methods lead to different outcomes that may not reflect antibody binding differences?”

The last question posed by the reviewer reflects our thinking. When antibodies are uniformly captured on the sensor, we do not see differences in binding between CHO and 293 cells. But when the gp145 is covalently attached via lysine amides, then we see differences in binding. These differences could be due to the mode attachment. In covalent linkage, the proteins are coupled onto a negatively charged surface. Since the gp145 CHO-K1 is a more negative protein than the gp145 made in 293, it will attach differently via the covalent method. Thus, it is our thinking that the data obtained with the sensor generated via antibody capture is, at least in this case, more reliable.

“For 2G12, this is not surprising since this antibody recognizes an invariant high mannose epitope that is unlikely to be affected by producer cells. For PG16, the lack of change may in part trace to the fact that CHO cells tend to add alpha 2,3 sialic acids, whereas PG16 prefers the 2,6 sialic acids more commonly found in human production (PMID: 29718999), so PG16 is ultimately ambivalent or even slightly averse to the CHO cell 2,3 glycans. There is in fact a slight loss of PG16 binding to CHO cells, as compared to the control using 2G12 as a control arbiter for binding.”

We were unaware of this preference of PG16 for the 2,6-linked sialic acid and its selection against the 2,3-linked sialic acid. Our discussion was based on the findings of Doores and Burton from 2010 (PMID 20686044) that both PG9 and PG16 recognize overlapping conformational epitopes that are affected by N-glycosylation perhaps indirectly, but are not sensitive to specific monosaccharide units. However, a more recent report by Crooks et al., 2018 concludes that the binding of PG9 could be enhanced by hypersialylation in human cells. No mention is made in that report about bNAb PG16. A note has been added to the discussion in Lines 418-426.

“Overall, I am not sure the data in S3 Table and elsewhere (Table 2, 3 and other kinetic data) for the Octet work are not different for the two gp145’s. A lot of tabulated Kds could be plotted to check for antibody-specific patterns. KD’s are not massively different, but the degree of difference seems to vary per antibody which may be worth capturing. Otherwise this would not fully investigate the patterns that are justified by the effort in running all these affinity tests.”

We totally agree with this interpretation of the binding data. We do not think that the binding of glycan-specific antibodies is any different for both of these immunogens, despite their differences in complex glycosylation.

“The two gp145 preps were also purified differently. It is unclear what the method for the CHO version was as it refers to another paper (a brief description would help). A major question is whether these gp145s are monomers or oligomeric forms or various? These different forms will bear different glycans so this is another problem leading to variability, in addition to using uncleaved gp145.”

Both immunogens were purified through a GNL lectin affinity column followed by Q-sepharose chromatography, which resulted in the production of a mixture of oligomeric forms that was not further resolved. We have now included a brief description of the purification of gp145 from CHO-K1 in the Methods section in Lines 108-112.

Regarding the possible differences in glycosylation for each oligomeric form, in the past our group has analyzed the glycosylation of trimers compared with monomers of different Env constructs, and have observed no differences. These analyses have been done as part of contract work for a manufacturing organization and we have not received permission to publish that data. Our explanation for this lack of difference in glycosylation between trimers and monomers is that the sub-units are in dynamic equilibrium between the different oligomeric states, causing a homogenization of the glycosylated forms.

“line 70: Native trimers here are actually “near native” and should be referred to as such. While they are a closer match to native, membrane trimers than uncleaved gp145, there are several differences.”

We have substituted the word “native” with “native-like”

“line 79: Re: a tremendous strain on the protein trafficking machinery, the other side of this point is that the glycans play a key structural role in folding. Removing some glycans can decrease expression of trimers. So there is more of a trade off with glycans being present.”

Thank you for pointing this out. While extensive glycosylation poses challenges to the efficient production of glycoproteins, it is essential for protein function. It has been shown that the complete removal of Env glycans from pseudovirions eliminates infectivity (Binley et al., 2010; Ref #17 in our manuscript). Thus glycans are important for function and thus must be present on any vaccine candidate that resembles the infective virus.

We have followed the statement quoted by the reviewer with the following statements: “It has been generally understood that for an Env vaccine to be effective, its glycan shield should resemble that of the infectious virus. Thus, a number of analytical strategies have been developed to measure the precise chemical nature and distribution of the glycans in Env from viral and recombinant sources.” in Lines 80-83.

“line 92: There have been some prior analyses on uncleaved gp140. A google search of “uncleaved gp140 glycans” revealed a few articles, including (PMID: 26051934, PMID: 26018173), and there have been a few by Go/Desaire. Uncleaved gp140 glycans tend to be more processed according to (PMID: 26051934), consistent with their less compact, non-native conformation.”

We have acknowledged prior work on the glycosylation of uncleaved gp140 vs. their native-like version, in which authors showed that uncleaved gp140 is decorated with a higher proportion of complex glycans than the native-like (Pritchard et al., 2015; PMID 26051934), which the reviewer has also identified. We have now added additional references suggested by the reviewer in support of the observation that uncleaved trimers contain more mannose glycans compared to their SOSIP or their membrane-embedded versions, respectively (Ringe et al., 2015 PMID 26311893 and Go et al., 2015 PMID 26018173). They are cited in Lines 412-413. Thank you for the suggestion.

“line 311: The subtitle heading would be better reversed to show that it is the antibodies binding to the gp145 and not the other way around.”

We have changed the subheading to “Recognition of gp145 by broadly neutralizing antibodies 2G12 and PG16”. Thank you for the suggestion.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0231679.r003

Decision Letter 1

Juan Pablo Jaworski

15 May 2020

PONE-D-20-07724R1

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: effects on glycosylation and antigenicity

PLOS ONE

Dear Dr. Baerga-Ortiz,

We would appreciate receiving your revised manuscript by Jun 29 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
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We look forward to receiving your revised manuscript.

Kind regards,

Juan Pablo Jaworski, D.V.M., M.Sc., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Dear authors

Revise your manuscript highlighting strengths and limitations of your current study as suggested by Reviewer 2.

Best,

Dr Jaworski

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have addressed all minor and major comments thoroughly. This reviewer thinks that manuscript should be accepted with no further modifications.

Reviewer #2: The author’s explanation of why they used an uncleaved clade C gp145 is satisfactory. These reasons should be stated clearly in the paper to provide proper context to anchor a reasons as to why the work was done - that the product is in a clinical trial so it is useful to know this information. As it stood, there was no explanation for the choice of strain and format. I think the point that it is in clinical trial should even be stated in the abstract, as I really think that will increase reader’s interest which might otherwise be lost due to the uncleaved platform. I don’t believe that uncleaved gp145 is a useful format, regardless of the clinical trial. At least from the perspective of inducing neutralizing antibodies, uncleaved gp140, like gp120 monomer have by now been well-established in dozens of studies (antigenicity and immunogenicity) as poor choices. It may be in both of the clinical trials mentioned in the response letter that neutralizing antibodies are not the end goal and that cellular or non-neutralizing antibodies are instead the goals. If this is the case, however, evaluating the constructs with PG9 and 2G12 is not crucial and they could be assessed with other antibodies that don’t neutralize, like V3.

The statement that gp120 doesn’t work line 61 and “longer” constructs are needed including MPER is a bit vague. This is not about counting epitopes and including them, but more about conformation. It makes more sense to state that forms that better resemble viral spikes are preferable. Uncleaved gp140 is not really much of an improvement on gp120 as it consists of loose gp120 protomers held together by hydrophobic gp41, as shown by EM.

Overall, I would think that some acknowledgement that gp145 is not a cutting-edge platform for inducing neutralizing antibodies should be added somewhere, as otherwise the paper ignores much of the advances in vaccines in preclinical work in recent years. Uncleaved gp145 may have been a reasonable choice in say 2005, but not really in 2020.

All this said, given that this product is in trial justifies the work, but the link to a trial is needed to be stated prominently to justify it.

Regarding the point in the response letter that uncleaved trimers contain more mannose than SOSIP or membrane versions (PMID 26311893 and 26018173), this statement is at odds with the work of Pritchard mentioned directly above this in the letter where there are more complex glycans on uncleaved gp140 (PMID 26051934). This may be just a typo that they meant complex not high mannose glycans. I think this is correct in the main text.

It is good that some previous papers on studying glycans on uncleaved gp140 have been cited in the discussion, but these also need to be covered in the introduction. Glycopeptide mass spec has been done by Crispin, Desaire and others on uncleaved gp140s and I think that is a key part of the introduction missing. Acknowledging this is OK, because the work here is still justified by comparing the uncleaved trimer production from two cell lines, one of which is FDA approved (CHO) and that the gp140 is in clinical trial. So, I am happy that these references were added, but they need to be said earlier to provide context.

Line 94: the uncleaved gp140 is partly trimer, but also a mix of dimer and other not purified, so I think you need to drop the trimer on line 94.

**********

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Reviewer #2: No

PLoS One. 2020 Jun 19;15(6):e0231679. doi: 10.1371/journal.pone.0231679.r004

Author response to Decision Letter 1

29 May 2020

Response to Reviewer #1: Thank you for your input, which made this manuscript better and more polished.

Response to Reviewer #2: We greatly appreciate your insightful comments on the suitability of an uncleaved gp145 as a protective immunogen. Your feedback has given us the chance to reflect on our efforts to produce viral glycoproteins and to discuss possible avenues for the production of protein-based vaccines against SARS-CoV-2.

“The author’s explanation of why they used an uncleaved clade C gp145 is satisfactory. These reasons should be stated clearly in the paper to provide proper context to anchor a reasons as to why the work was done - that the product is in a clinical trial so it is useful to know this information. As it stood, there was no explanation for the choice of strain and format. I think the point that it is in clinical trial should even be stated in the abstract, as I really think that will increase reader’s interest which might otherwise be lost due to the uncleaved platform.”

We have included a sentence in the abstract and in the introduction to mention the on-going clinical trial, as suggested by the reviewer. And we do agree with the reviewer that the fact CO6980v0c22 is being tested clinically, makes it more relevant to the average reader. However, the objective of our manuscript is to compare biochemical properties for this immunogen expressed in CHO and 293 cells. We are not making any claims of efficacy or immunogenicity for this specific construct. The observations reported by us would still be valid if the gp145 immunogen did not elicit the desired neutralizing humoral response in humans, and could still be useful to researchers expressing other viral glycoproteins for purposes other than vaccine development.

“I don’t believe that uncleaved gp145 is a useful format, regardless of the clinical trial. At least from the perspective of inducing neutralizing antibodies, uncleaved gp140, like gp120 monomer have by now been well-established in dozens of studies (antigenicity and immunogenicity) as poor choices. It may be in both of the clinical trials mentioned in the response letter that neutralizing antibodies are not the end goal and that cellular or non-neutralizing antibodies are instead the goals. If this is the case, however, evaluating the constructs with PG9 and 2G12 is not crucial and they could be assessed with other antibodies that don’t neutralize, like V3.”

The description of the clinical trial (https://clinicaltrials.gov/ct2/show/NCT03382418) clearly states that among the secondary outcomes that will be measured are the response rates and levels of CD8+ and CD4+ T cells, and the response rates and levels of neutralizing antibodies. While the results of these measurements are not presently available, we have chosen not to speculate on what these results might eventually be. We do understand that the reviewer feels strongly that this vaccine is unlikely to yield a neutralizing response. Our manuscript, however, does not make any claims of efficacy or protection, as we focus on the differences in the chemical properties of the immunogen which may result from the choice of expression host.

“The statement that gp120 doesn’t work line 61 and “longer” constructs are needed including MPER is a bit vague. This is not about counting epitopes and including them, but more about conformation. It makes more sense to state that forms that better resemble viral spikes are preferable. Uncleaved gp140 is not really much of an improvement on gp120 as it consists of loose gp120 protomers held together by hydrophobic gp41, as shown by EM.”

Line 61: We do not disagree with this observation and have deleted the phrase: “to preserve known bNAb epitopes within the membrane proximal external region (MPER)” and inserted “that better resemble viral spikes”.

“Overall, I would think that some acknowledgement that gp145 is not a cutting-edge platform for inducing neutralizing antibodies should be added somewhere, as otherwise the paper ignores much of the advances in vaccines in preclinical work in recent years. Uncleaved gp145 may have been a reasonable choice in say 2005, but not really in 2020. All this said, given that this product is in trial justifies the work, but the link to a trial is needed to be stated prominently to justify it.”

We have included a sentence in the abstract and another one in the introduction to mention the on-going clinical trial, as suggested by the reviewer.

“Regarding the point in the response letter that uncleaved trimers contain more mannose than SOSIP or membrane versions (PMID 26311893 and 26018173), this statement is at odds with the work of Pritchard mentioned directly above this in the letter where there are more complex glycans on uncleaved gp140 (PMID 26051934). This may be just a typo that they meant complex not high mannose glycans. I think this is correct in the main text.”

Yes. The reviewer is correct. There was a mistake in the previous response letter. However, as noted by the reviewer, the manuscript contains the correctly referenced finding that SOSIP or membrane versions of Env, contain a higher proportion of mannose glycans than the uncleaved gp140.

“It is good that some previous papers on studying glycans on uncleaved gp140 have been cited in the discussion, but these also need to be covered in the introduction. Glycopeptide mass spec has been done by Crispin, Desaire and others on uncleaved gp140s and I think that is a key part of the introduction missing. Acknowledging this is OK, because the work here is still justified by comparing the uncleaved trimer production from two cell lines, one of which is FDA approved (CHO) and that the gp140 is in clinical trial. So, I am happy that these references were added, but they need to be said earlier to provide context.”

We have included in the introduction (line 93) the following sentence: “From these detailed glycosylation studies it was concluded that the gp120 portion of the SOSIP trimer contains more high-mannose glycans than the gp41 portion, and that those trimers made in CHO-K1 cells contain a higher level of complex glycosylation than the cleaved trimers produced in 293 cells [45].

“Line 94: the uncleaved gp140 is partly trimer, but also a mix of dimer and other not purified, so I think you need to drop the trimer on line 94.”

The word “trimer” has been substituted for “Env constructs”.

Attachment

Submitted filename: Response-Letter-3-PONE-D-20-07724.doc

Click here for additional data file.^{(3MB, doc)}

PLoS One. doi: 10.1371/journal.pone.0231679.r005

Decision Letter 2

Juan Pablo Jaworski

5 Jun 2020

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: effects on glycosylation and antigenicity

PONE-D-20-07724R2

Dear Dr. Baerga-Ortiz,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0231679.r006

Acceptance letter

Juan Pablo Jaworski

10 Jun 2020

PONE-D-20-07724R2

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: effects on glycosylation and antigenicity

Dear Dr. Baerga-Ortiz:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. SDS-PAGE analysis of HIV-1 gp145 produced in CHO-K1 and Expi293F cells.

The recombinant gp145 immunogens were incubated overnight with (+, lanes 3 and 5) and without (-, lanes 2 and 4) Neuraminidase A, and then resolved on 10% SDS-PAGE gels.

(TIF)

Click here for additional data file.^{(486.8KB, tif)}

S2 Fig. MALDI-Post-source decay (PSD) spectra of N-linked glycans derived from CHO-K1 gp145.

(TIF)

Click here for additional data file.^{(1.8MB, tif)}

S1 Table. N-glycans composition of C06980v0c22 gp145 using MALDI-ToF in reflector positive ion mode.

All the peaks obtained in the MALDI-ToF were compared against the list previously reported by Doores et al., 2010. Some of them were selected randomly for confirmation by MS/MS analysis.

(DOCX)

Click here for additional data file.^{(151KB, docx)}

S2 Table. N-glycans composition of C06980v0c22 gp145 using MALDI-ToF in reflector negative ion mode.

(DOCX)

Click here for additional data file.^{(84.8KB, docx)}

S3 Table. Binding of CO6980v0c22 gp145 to HIV-1 antibodies.

(DOCX)

Click here for additional data file.^{(21.3KB, docx)}

Attachment

Submitted filename: Response-Letter-PONE-D-20-07724.doc

Click here for additional data file.^{(3MB, doc)}

Attachment

Submitted filename: Response-Letter-3-PONE-D-20-07724.doc

Click here for additional data file.^{(3MB, doc)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: Effects on glycosylation and antigenicity

José A González-Feliciano

Pearl Akamine

Coral M Capó-Vélez

Manuel Delgado-Vélez

Vincent Dussupt

Shelly J Krebs

Valerie Wojna

Victoria R Polonis

Abel Baerga-Ortiz

José A Lasalde-Dominicci

Roles

Abstract

Introduction

Materials and methods

Antibodies and HIV-1 immunogens

Glycan analysis by MALDI-ToF mass spectrometry

Peptide mass fingerprinting

Capillary isoelectric focusing

Binding kinetics of HIV-1 gp145 to 2G12 and PG16 bNAbs

Enzyme-linked immunosorbent assay (ELISA)

Participants’ description

Table 1. Participant characteristics.

Results

Confirmation of HIV-1 gp145 protein identity

Fig 1. Peptide mass fingerprinting (PMF) analysis of HIV-1 gp145 protein.

Differences in the glycosylation profiles of CHO-K1- and Expi293F-produced HIV-1 gp145

Fig 2. Positive ion MALDI-ToF analysis of N-glycans released from HIV-1 gp145.

Fig 3. Negative ion MALDI-ToF analysis of acidic N-glycans released from HIV-1 gp145.

Fig 4. Abundance of sialylated N-glycans of gp145 produced in CHO-K1 and Expi293F cells.

Distribution of charge variants

Fig 5. The impact of sialylation on HIV-1 gp145 isoelectric point.

Recognition of HIV gp145 by the broadly neutralizing antibodies, 2G12 and PG16

Fig 6. Binding of 2G12 and PG16 bNAbs to HIV-1 gp145.

Table 2. Binding of soluble CO6980v0c22 gp145 to Fc-captured bNAbs 2G12 and PG16.

Table 3. Binding of bNAbs 2G12 and PG16 to immobilized CO6980v0c22 gp145.

Reactivity of gp145 against HIV+ and HIV- patient plasma

Fig 7. IgG binding from HIV+ patient plasma to gp145 produced in CHO-K1 and Expi293F cells.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Juan Pablo Jaworski

Roles

Author response to Decision Letter 0

Decision Letter 1

Juan Pablo Jaworski

Roles

Author response to Decision Letter 1

Decision Letter 2

Juan Pablo Jaworski

Roles

Acceptance letter

Juan Pablo Jaworski

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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