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Influenza Virus Hemagglutinin Glycoproteins with Different N-Glycan Patterns Activate Dendritic Cells In Vitro Wen-Chun Liu,a Yu-Li Lin,b Maureen Spearman,c Pei-Yun Cheng,b Michael Butler,c Suh-Chin Wua,d Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwana; Department of Medical Research, National Taiwan University Hospital, Taiwanb; Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canadac; Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwand

ABSTRACT

Influenza virus hemagglutinin (HA) N-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response. Considered essential for controlling innate and adaptive immune responses against influenza virus infections, dendritic cells (DCs) trigger proinflammatory and adaptive immune responses in hosts. In this study, we engineered Chinese hamster ovary (CHO) cell lines expressing recombinant HA from pandemic H1, H5, and H7 influenza viruses. rH1HA, rH5HA, and rH7HA were obtained as wild-type proteins or in the presence of kifunensine (KIF) or further with endo-␤-N-acetylglucosaminidase-treated KIF (KIFⴙE) to generate single-N-acetylglucosamine (GlcNAc) N-glycans consisting of (i) terminally sialylated complex-type N-glycans, (ii) high-mannose-type N-glycans, and (iii) single-GlcNAc-type N-glycans. Our results show that high-mannose-type and single-GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70, and hIL-10 production in human DCs. Significantly higher HLA-DR, CD40, CD83, and CD86 expression levels, as well reduced endocytotic capacity in human DCs, were noted in the high-mannose-type rH1HA and single-GlcNAc-type rH1HA groups than in the complex-type N-glycan rH1HA group. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). The high-mannose-type or single-GlcNAc-type N-glycans of both avian and human HA types are more stimulatory than the complex-type N-glycans. HA-stimulated DC activation was accomplished partially through a mannose receptor(s). These results provide more understanding of the contribution of glycosylation of viral proteins to the immune responses and may have implications for vaccine development. IMPORTANCE

Influenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role in the link between innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5HA, and rH7HA proteins containing distinct N-glycan patterns. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human DCs. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). High-mannose-type and single-GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering mouse and human DC activation and maturation. We believe these results provide some useful information for influenza vaccine development regarding how influenza virus HA proteins with different types of N-glycans activate DCs.

I

nfluenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations (1). Members of the Orthomyxoviridae family, influenza type A and B viruses consist of single-stranded eight-segment negativesense genomic RNA, helical viral ribonucleoprotein (RNP) complexes (RNA segments NP, PB2, PB1, and PA), and four viral envelope proteins (hemagglutinin [HA], neuraminidase [NA], M1 matrix protein, and M2 ion channel protein). Type A influenza viruses have been further classified into 18 HA (H1 to H18) and 11 NA (N1 to N11) serotypes on the basis of the antigenic characteristics of their HA and NA glycoproteins (2–4). The HA glycoprotein, which is the major target of infection-blocking antibodies, exhibits continuous changes in antigenic properties under immune selective pressure (5). Considered the most potent professional antigen-presenting cells, dendritic cells (DCs) link innate and adaptive immunity (6). When they encounter microbial pathogens, endogenous danger signals, or inflammatory mediators, DCs elicit rapid and shortlived innate immune responses before migrating to secondary

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lymphoid organs and enhancing adaptive immunity (7). DCs are also capable of inducing immunotolerance under certain conditions (8). Two major DC subsets are found in mice and humans: (i) myeloid DCs (mDCs; also called conventional DCs) that participate directly in antigen presentation and naive T-cell activation and (ii) plasmacytoid DCs (pDCs) that produce type I interferons (IFNs) in response to viral infections (9, 10). Because of their key role in immune regulation, DCs have been used as immunother-

Received 9 March 2016 Accepted 17 April 2016 Accepted manuscript posted online 20 April 2016 Citation Liu W-C, Lin Y-L, Spearman M, Cheng P-Y, Butler M, Wu S-C. 2016. Influenza virus hemagglutinin glycoproteins with different N-glycan patterns activate dendritic cells in vitro. J Virol 90:6085– 6096. doi:10.1128/JVI.00452-16. Editor: S. Perlman, University of Iowa Address correspondence to Suh-Chin Wu, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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apeutic agents in the development of prophylactic and therapeutic vaccines for cancer and both infectious and immune-related diseases (11, 12). Considered essential for controlling innate and adaptive immune responses against influenza virus infections (13), DCs trigger proinflammatory and adaptive immune responses in hosts (14). Both mDCs and pDCs can be activated by vaccinations with trivalent inactivated or live-attenuated viruses, mainly via Tolllike receptor 7 (TLR7)/type I IFN pathways (15). mDCs and pDCs also comprise different heterologous subsets with unique phenotypes and functions. It has been reported that migratory lungderived and lymph node-derived DCs can be infected by H2N2 (16), pH1N1 (17), and H5N1 influenza viruses (18, 19). The pH1N1 virus induces lower levels of antiviral IFN and proinflammatory tumor necrosis factor alpha (TNF-␣) cytokine expression; it reportedly replicates as efficiently as other seasonal H1N1 and H3N2 viruses in human mDCs (17). Highly pathogenic avian H5N1 viruses can induce productive infections in human mDCs and mouse primary lung DCs (18), whereas H7N9 viruses only result in impaired IFN production in infected human mDCs (20). We previously reported that recombinant HA proteins from H5N1 and pH1N1 influenza viruses are capable of triggering mouse mDC activation and maturation (21). For this study, we used Chinese hamster ovary (CHO) cell expression to obtain rH1HA, rH5HA, and rH7HA proteins consisting of (i) terminally sialylated complex-type N-glycans, (ii) highmannose-type N-glycans, and (iii) single-N-acetylglucosamine (GlcNAc)-type N-glycans. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human mDCs for cytokine production, surface marker expression, and endocytosis. We hope the findings presented here can be used in support of vaccine development by clarifying the relationship between influenza virus HA antigens and mDC activation and maturation. MATERIALS AND METHODS Ethics statement. The human mDCs used in this study were approved by the Research Ethics Committee of National Taiwan University Hospital. Completed informed consent forms were obtained from all donors, who were given detailed descriptions of the research process, including information on blood collection and storage and immune cell isolation. rHA and kifunensine (KIF)-rHA protein expression and purification. Soluble pH1HA (A/Texas/05/2009), H5HA (A/Thailand/KAN-1/ 2004), and H7HA (A/Shanghai/02/2013) proteins were constructed with HA cDNA sequences. Highly productive, stable clones for the expression of these proteins were selected as described previously (22). The HA protein-expressing vector was a modified pISID vector that included pITID (A/Texas/05/2009, pH1HA), pIKID (A/Thailand/KAN-1/2004, H5HA), and pIS2ID (A/Shanghai/02/2013, H7HA), also as previously described (22). Briefly, CHO/dhfr⫺ cells (ATCC CRL-9096) were selected with minimal essential medium alpha without ribonucleosides/deoxyribonucleosides (RNS/dRNS) supplemented with 10% dialyzed fetal bovine serum (FBS) and 200 ␮g/ml Zeocin (Invitrogen) following transfection. Highly productive, stable CHO cell clones were selected after screening 80 to 100 transfected stable clones in CHO/dhfr⫺ cells, with methotrexate (MTX; Sigma) concentrations increased stepwise from 0.02 to 1.00 ␮M. Amplified cell clone culture supernatants were harvested for CHO-rHA protein purification by nickel-chelated resin affinity chromatography (Tosoh), followed by filtering on endotoxin-free columns (Cellufine ET clean; Chisso) to achieve residual endotoxin levels of ⬍1 endotoxin unit (EU)/␮g.

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Endo H digestion of KIF-rHA glycoproteins. Endo-␤-N-acetylglucosaminidase (endo H) was used to cleave high-mannose and some hybrid oligosaccharides from N-linked glycoproteins within the chitobiose core. KIF-rHA protein (H1, H5, or H7) samples (10 or 50 ␮g/ml) were reacted with endo H (New England BioLabs) for 3 h at 37°C. Posttreatment molecular shifts were determined by Coomassie blue staining or Western blotting. Horseradish peroxidase-conjugated anti-His antibodies (Affymetrix) were used for Western blotting characterization. Glycan profile analyses. Purified rHA proteins (H1, H5, and H7) produced in CHO cells were analyzed for glycan structures according to methods established by Royle et al. (23). Samples were subjected to SDSPAGE (Criterion TGX; Bio-Rad) and stained with Coomassie blue. Gel bands were cut into 1-mm3 pieces, frozen overnight at ⫺20°C, washed with acetonitrile and 20 mM sodium bicarbonate (1:1), and dried in a SpeedVac centrifuge. Peptide-N-glycosidase F (PNGase F; Promega) was used overnight at 37°C to remove glycans from proteins within gels. Glycans were removed from gels by sonication with water, desalted with Dowex, filtered through 45-␮m filters, dried with a SpeedVac centrifuge, and labeled with 2-aminobenzamide (2-AB). After the removal of excess 2-AB, samples were subjected to hydrophilic-interaction liquid chromatography (HILIC; X-Bridge amide 3.5-␮m column; Waters) to separate individual glycan structures. The 2-AB glycans were digested with jack bean ␣-mannosidase (Prozyme) prior to another round of HILIC to confirm their structures. A 2-AB-labeled dextran ladder standard was also separated by HILIC and used to generate a fifth-order polynomial to provide glucose unit (GU) values for individual peaks recognized in the glycan samples. GU values were compared to those shown in the NIBRT GlycoBase database. Mouse bone marrow-derived mDC generation. mDCs were generated from the bone marrow of C57BL/6 mice as described previously (21). All procedures involving animals were conducted in accordance with guidelines established by the Laboratory Animal Center of National Tsing Hua University (NTHU). Animal use protocols were reviewed and approved by the NTHU Institutional Animal Care and Use Committee (approval no. 10002). Mice that survived the experiments were sacrificed with carbon dioxide (CO2) to ameliorate suffering. Human mDC generation. DCs were generated from human peripheral blood mononuclear cells (PBMCs) collected from healthy donors as described previously (24). Briefly, PBMCs were isolated by standard Ficoll-Histopaque (Sigma-Aldrich) gradient centrifugation, with low-density fractions from the 42.5-to-50% interface being recovered. CD14⫹ cells were further purified by positive selection with anti-CD14⫹ microbeads in conjunction with the AutoMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Cells (1 ⫻ 106/ml) were cultured in complete RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 25 mM HEPES in 24-well plates with granulocytemacrophage colony-stimulating factor (GM-CSF; 20 ng/ml) and interleukin-4 (IL-4; 20 ng/ml) (both from PeproTech). Fresh medium containing GM-CSF and IL-4 was added every 2 to 3 days. Human monocyte-derived DCs (human mDCs) were routinely collected on day 6 of each culturing cycle for use with rHA, rHA (KIF), or rHA (KIF⫹E) protein. Cytokine production analyses. For mouse mDC cytokine production, TNF-␣ produced by mouse mDCs was detected by intracellular staining as previously described (21). Briefly, DCs were treated with phosphate-buffered saline (PBS), 100 ng/ml lipopolysaccharide (LPS; from Escherichia coli O111:B4; Sigma), and 10 or 50 ␮g/ml rHA (H1, H5, or H7) or KIF-rHA (H1, H5, or H7) with or without endo H for 6 h. Brefeldin A (10 ␮g/ml; BioLegend) was added for the final 4 h. Cells were fixed, permeabilized, stained with a mouse anti-TNF-␣ monoclonal antibody (BioLegend), and analyzed with a flow cytometer and Accuri C6 software (BD Biosciences). To determine human mDC cytokine secretion levels, DC culture supernatants were collected following treatment with PBS, LPS, or rHA for 48 h (IL-12 p40, IL-12 p70, IL-10). Detection was performed with

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FIG 1 Characterization of rHA glycoproteins with different N-glycan patterns. (A) N-glycan patterns for rHA (H1, H5, and H7) proteins. A terminally sialylated complex-type pattern was observed for rHA, a high-mannose-type pattern was observed for KIF-rHA, and a single-GlcNAc-type pattern was observed for KIF-rHA treated with endo H under mild conditions (37°C for 3 h). rHA, rHA (KIF), and rHA (KIF⫹E) molecular weights were determined by Coomassie blue staining (B) or Western blotting (C).

enzyme-linked immunosorbent assay (ELISA) kits (R&D) according to the manufacturer’s instructions. Surface marker expression analyses. Murine mDC maturation was determined in terms of IAb, CD40, and CD86 expression as described in reference 21. Human mDC maturation was examined in terms of HLADR, CD40, CD83, and CD86 expression (24). Immature mouse mDCs derived from C57BL/6 mouse bone marrow were treated with PBS, 100 ng/ml LPS, and 50 ␮g/ml wild-type rHA or KIF-treated rHA (H1, H5, or H7) for 18 h, stained with mouse anti-CD11c, -IAb, -CD40, or -CD86 antibodies (BioLegend), and analyzed by flow cytometry (Accuri C6 software). Immature human mDCs derived from PBMCs collected from healthy donors were treated with PBS, 100 ng/ml LPS, and 10 ␮g/ml wild-type rHA or KIF-treated rHA (H1, H5, or H7) for 48 h, stained with anti-human anti-CD11c, -HLA-DR, -CD40, -CD83, or -CD86 antibodies (eBioscience), and analyzed by flow cytometry (FACSCalibur). mDC endocytotic analyses using dextran-FITC uptake. Stimulated human mDCs were suspended in staining buffer (1% FBS plus 0.01% NaN3 dissolved in PBS) with 200 ␮g/ml dextran-fluorescein isothiocyanate (FITC; Sigma) and incubated in darkness at 4 or 37°C for 1 h, after which cells were washed with cold PBS and analyzed with a FACSCalibur flow cytometer. Antibody blocking assays. Mouse mDCs were treated with 1 ␮g/ml anti-mannose receptor (anti-MR) antibodies (ab64693; Abcam) or antiDectin-1 antibodies (mabg-mdect; InvivoGen) at 37°C overnight to block MRs or Dectin-1 C-type lectin receptors prior to rHA, rHA (KIF), or rHA (KIF⫹E) (10 ␮g/ml) protein stimulation. Intracellular TNF-␣ production was determined by using Accuri C6 flow cytometry and analyzed by Accuri C6 software (BD Biosciences). Percentages of TNF-␣⫹ mDCs were determined. Statistical analyses. All results were analyzed with two-tailed Student t tests (GraphPad Prism v5.03). Statistical significance levels are indicated as follows in all of the figures: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

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RESULTS

Use of KIF and KIFⴙE treatments to characterize rH1HA, rH5HA, and rH7HA proteins. Soluble recombinant HA glycoproteins from pH1N1 (A/Texas/05/2009), H5N1 [A/Thailand/ 1(KAN-1)/2004], and H7N9 (A/Shanghai/02/2013) viruses were expressed from stable CHO cell clones selected from dhfr/MTX amplification, purified by nickel-chelated affinity chromatography, and filtered through endotoxin-free columns to achieve residual endotoxin levels below 1 EU/␮g. To examine the various effects of different N-glycan patterns on DC activation, we also grew the same stable CHO cell clones in medium containing 100 ␮M KIF, a mannosidase inhibitor capable of blocking the highmannose trimming process during complex-type N-glycan biosynthesis. This results in high-mannose (mannose-terminated)type N-glycans (Fig. 1A). KIF-treated rHA proteins (containing high-mannose-type N-glycans) were further treated with endo H (KIF⫹E) under mild conditions (37°C for 3 h), as previously described for HIV-1 envelope glycoprotein glycan analysis (25), to obtain single-GlcNAc-type N-glycans (Fig. 1A). Three N-glycan forms were obtained: a terminally sialylated complex type, a high-mannose type, and a single-GlcNAc type, as determined by SDS-PAGE analyses (Fig. 1B and C). Since fetuin contains both ␣-2,3- and ␣-2,6-linked sialic acids, we also examined rHA protein-binding properties by using different Nglycan patterns. Our results indicate that the fetuin-binding properties of the rH1HA, rH5HA, and rH7HA proteins containing complex-type N-glycans are relatively weak and that those of the KIF⫹E-treated rH5HA and rH7HA proteins are significantly stronger (Fig. 2A to C).

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FIG 2 rHA and KIF-rHA fetuin-binding capabilities (H1, H5, and H7). Shown are fetuin-binding assay results for soluble and recombinant rHA, rHA (KIF), and rHA (KIF⫹E) proteins. Twofold serially diluted H1 (A), H5 (B), and H7 (C) proteins (starting at 10 ␮g/ml and diluted in PBST–Tween 20 buffer containing anti-His antibodies) were added to ELISA plates coated with 100 ␮g/ml bovine fetuin and cultured for 1 h at room temperature prior to the plotting of ELISA binding curves. OD450, optical density at 450 nm.

N-glycan profiling of rH1HA, rH5HA, and rH7HA proteins with and without KIF treatment. N-glycan profiling of rH1HA, rH5HA, and rH7HA proteins with and without KIF treatment was performed by HILIC as described previously (22). The rH1HA, rH5HA, and rH7HA glycoproteins all contained complex-type N-glycans with bi-, tri-, and tetra-antennary structures with one or more terminal sialic acids (Sia1– 4Gal1– 4Man3GlcNAc4 – 6 with or without Fuc). The major glycoforms were as follows: for rH1HA, Sia1Gal2Man3GlcNAc4 with or without Fuc, Man7GlcNAc2 at 25.2% and Sia2Gal2Man3GlcNAc4Fuc at 20.4% (Fig. 3A; Table 1); for rH5HA, Sia1Gal2Man3GlcNAc4Fuc at 26.8% and Sia2Gal2Man3GlcNAc4Fuc at 22.8% (Fig. 4A; Table 1);

and for rH7HA, Sia1–3Gal3Man3GlcNAc5Fuc at 36.6% and Sia1– 4Gal4Man3GlcNAc6Fuc at 27.0% (Fig. 5A; Table 1). Following KIF treatment, rHA (KIF) protein N-glycans were found to be of the high-mannose type (Man5–9GlcNAc2) mixed with much smaller amounts of hybrid glycans (Man4GlcNAc2Fuc). The major glycoforms for rH1HA (KIF) were Man8GlcNAc2 at 24.6% and Man9GlcNAc2 at 45.5% (Fig. 3B; Table 1); for rH5HA (KIF), they were Man8GlcNAc2 at 32.9% and Man9GlcNAc2 at 38.2% (Fig. 4B; Table 1); and for rH7HA (KIF), they were Man7GlcNAc2 at 19.3% and Man8GlcNAc2 at 27.3% (Fig. 5B; Table 1). Mouse mDC activation and maturation by rH1HA, rH5HA, and rH7HA proteins containing different N-glycans. Purified

FIG 3 HILIC results and N-glycan profiles of rH1HA proteins. Purified rH1HA (A) and rH1HA (KIF) (B) proteins were subjected to SDS-PAGE, stained with Coomassie blue, and destained. N-glycans were removed from gels with PNGase F, dried, labeled with 2-AB, and analyzed by HILIC to identify individual glycan structures. A 2-AB-labeled dextran ladder was separated by HILIC and used as a standard to provide GU values for individual peaks in glycan samples from rH1HA proteins. Shown are rH1HA and rH1HA (KIF) HA N-glycan profiles.

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TABLE 1 N-glycan analysis of CHO cell-expressed rHA and rHA (KIF) proteinsa GU value

Glycan profile

rH1HA

rH1HA (KIF)

rH5HA

rH5HA (KIF)

rH7HA

rH7HA (KIF)

4.39 5.39 6.15 6.17 6.71 6.92–7.04 7.06 7.21 7.48 7.55 7.75–7.9 7.93 8.03 8.39 8.55–8.79 8.80 9.11–9.84 9.46 10.05–11.5

M3 M4 A2G1, M5 M5 FA2G1 M6 A2G2, M6 A2G1S FA2G2, FA2G1S1 A2G2S1 M7 A2G2S1, FA2G2S1, M7, FA3G2 A2G2S2 FA2G2S2 M8 FA3G3 FA3G3S1, FA3G3S2, FA3G3S3 M9 FA4G4S1, FA4G4S2, FA4G4S3, FA4G4S4

0 0 7.7 0 ⬍1 0 3.0 1.1 4.7 4.3 0 25.2 2.8 20.4 0 3.5 14.1 0 4.9

0.6 1.3 0 3.4 0 5.6 0 0 0 0 9.8 0 0 0 24.6 0 0 45.5 0

0 0 1.7 0 1.3 0 2.3 2.2 7.0 4.2 0 26.8 2.3 22.8 0 3.9 16.7 0 4.7

0 0.4 0 1.2 0 6.9 0 0 0 0 13.7 0 0 0 32.9 0 0 38.2 0

0 0 2.4 0 0 0 1.1 0 0 5.0 0 6.7 3.0 8.7 0 5.8 36.6 0 27.0

1.3 3.3 0 8.2 0 12.3 0 0 0 0 19.3 0 0 0 27.3 0 0 12.6 0

a

Abbreviations: F, fucose; G, galactose; M, mannose; A2, GlcNAc4Man3; A3, GlcNAc5Man3; S, sialic acid; A4, GlcNAc6Man3.

rHA proteins containing complex-type, high-mannose-type (KIF-treated), or single-GlcNAc-type (KIF⫹E-treated) N-glycans were added to cultured, bone marrow-derived mDCs isolated from mice as described previously (21). Flow cytometry was used to measure intracellular TNF-␣ and IAb (major histocompatibility complex class II), CD40, and CD86 expression levels. Results indicate that mouse mDCs treated with rH1HA (KIF) or rH1HA

(KIF⫹E) protein induced significantly higher levels of TNF-␣, IAb, CD40, and CD86 did than mouse mDCs treated with rH1HA protein (Fig. 6). Similar results were found for mouse mDCs treated with rH7HA (KIF) or rH7HA (KIF⫹E) protein; that is, both groups exhibited increased levels of TNF-␣ and CD40 (Fig. 6). In contrast, mouse mDCs treated with rH5HA (KIF) showed reduced levels of TNF-␣, CD40, and CD86, but those

FIG 4 HILIC results and N-glycan profiles of rH5HA proteins. Purified rH5HA (A) and rH5HA (KIF) (B) proteins were subjected to SDS-PAGE, stained with Coomassie blue, and destained. N-glycans were removed from gels with PNGase F, dried, labeled with 2-AB, and analyzed by HILIC to identify individual glycan structures. A 2-AB-labeled dextran ladder was separated by HILIC and used as a standard to provide GU values for individual peaks recognized in glycan samples from rH5HA proteins. Shown are rH5HA and rH5HA (KIF) HA N-glycan profiles.

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FIG 5 HILIC results and N-glycan profiles of rH7HA proteins. Purified rH7HA (A) and rH7HA (KIF) (B) proteins were subjected to SDS-PAGE, stained with Coomassie blue, and destained. N-glycans were removed from gels with PNGase F, dried, labeled with 2-AB, and analyzed by HILIC to identify individual glycan structures. A 2-AB-labeled dextran ladder was separated by HILIC and used as a standard to provide GU values for individual peaks recognized in glycan samples from rH7HA proteins. Shown are rH7HA and rH7HA (KIF) HA N-glycan profiles.

treated with rH5HA (KIF⫹E) showed TNF-␣ and CD40 levels similar to those of mouse mDCs treated with complex-type N-glycan rH5HA proteins (Fig. 6). Combined, these results indicate that the high-mannose and single-GlcNAc types of N-glycans were more effective in triggering mouse mDC activation and maturation than the complex-type H1HA and H7HA N-glycans. hIL-12 p40, hIL-12 p70, and hIL-10 cytokine production by human mDCs treated with rH1HA, rH5HA, and rH7HA proteins containing different N-glycans. To confirm the capability of high-mannose or single-GlcNAc-type N-glycans to trigger more potent mDC activation than complex-type N-glycans, we obtained human mDCs from CD14⫹ PBMCs collected from healthy donors as described previously (24). These mDCs were treated with 10 or 50 ␮g/ml rHA, rHA (KIF), or rHA (KIF⫹E) protein for 48 h. Culture supernatants were collected and subjected to ELISA to determine hIL-12 p40, hIL-12 p70, and hIL-10 secretion levels. Results show that human mDCs treated with 10 ␮g/ml rH1HA (KIF⫹E) or rH5HA (KIF⫹E) induced significantly higher levels of hIL-12 p40 and hIL-10 production than those treated with rH1HA, rH1HA (KIF), rH5HA, or rH5HA (KIF). For mDCs treated with 10 ␮g/ml rH7HA, the high-tolow order of hIL-12 p40 and hIL-10 secretion levels was rH7HA (KIF) ¡ rH7HA (KIF⫹E) ¡ rH7HA (Fig. 7A and C). At higher treatment doses (50 ␮g/ml), both the rH1HA (KIF) and rH1HA (KIF⫹E) groups exhibited higher hIL-12 p40, hIL-12 p70, and hIL-10 levels than the rH1HA group (Fig. 7D to F). For the rH5HA and rH7HA groups with or without KIF or KIF⫹E treatment, significant differences in induced hIL-12 p40, hIL-12 p70, or hIL-10 production were not observed, with the exception of

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reduced hIL-12 p40 levels in the rH7HA (KIF) group (Fig. 7D to F). Combined, these results suggest that high-mannose-type and single-GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70, and hIL-10 production in human mDCs. Enhancement of human mDC maturation by rH1HA proteins containing different N-glycans. On the basis of our observation that high-mannose and single-GlcNAc types of N-glycans in the rH1HA group induced the most potent hIL12 p40, hIL12 p70, and hIL-10 activation in human mDCs, we attempted to measure specific surface markers for human mDC maturation. According to our results, the expression levels of the HLA-DR, CD40, CD83, and CD86 costimulatory molecules in human mDCs increased significantly in the rH1HA (KIF) and rH1HA (KIF⫹E) groups but not in the rH1HA group (Fig. 8). Costimulatory molecule levels for human mDC maturation in the rH1HA (KIF) and rH1HA (KIF⫹E) groups were found to be in much higher ranges than the positive LPS control group, i.e., approximately 50 to 70% higher than in the rH1HA group (Fig. 8). The results were reproducible with cultured mDCs from another human donor. As the immature mDCs that exert potent endocytic activity for capturing and processing antigens can lose the ability to endocytose and process antigens once they differentiate into immunostimulatory antigen-presenting cells (26), we evaluated the previously reported endocytotic capacity of human mDCs to take up FITC-dextran (27). Our results indicate that human mDCs from two human donors treated with 50 ␮g/ml rH1HA protein exhibited either 52 or 92% uptake of FITC-labeled dextran, compared to 92 or 95% uptake by the PBS control group

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FIG 6 Cytokine production and surface marker expression from murine mDCs treated with rHA proteins containing different N-glycan patterns. (A) Murine mDCs were treated with PBS or 10 ␮g/ml rHA proteins (H1, H5, or H7) or KIF-rHA proteins (H1, H5, or H7), with or without endo H, or with LPS and intracellularly stained with anti-TNF-␣ antibodies. TNF-␣⫹ mDC percentages were determined by flow cytometry. Mouse mDCs treated with PBS, rHA proteins (H1, H5, or H7), KIF-rHA proteins (H1, H5, or H7), with or without endo H, or with LPS were stained with anti-CD11c antibody plus anti-IAb (B), anti-CD40 (C), or anti-CD86 (D) antibodies to determine surface marker expression levels. Results, expressed as mean values ⫾ standard deviations, are from two or more individual experiments. Two-tailed Student t test results: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. PBS was used as a negative control, and LPS was used as a positive control. MFI, mean fluorescence intensity.

(Fig. 9A, D, F, and I). A significant decrease in FITC-dextran uptake was observed in human mDCs treated with either rH1HA (KIF) (7.5 or 18%) or rH1HA (KIF⫹E) (3.9 or 16.9%) (Fig. 9B, C, G, and H). In other words, more significant reductions in endocytotic capacity for human mDC maturation were noted in the high-mannose- and single-GlcNAc-type N-glycan groups than in the complex-type N-glycan group. Altered mouse mDC activation partially through MR(s). Several membrane-associated C-type lectins (e.g., the macrophage MR, macrophage galactose lectin, and DC-SIGN) have been reported to exhibit strong innate immunity to influenza virus infection (28). To address the mechanism(s) leading to altered mDC activation by rHA proteins containing different N-glycans, we used anti-MR or anti-Dectin-1 antibodies to block mouse mDCs prior to rHA, rHA (KIF), and rHA (KIF⫹E) stimulation. Our results indicate that the levels of TNF-␣ production by rH1HA (KIF), rH1HA (KIF⫹E), rH5HA, rH7HA, and rH7HA (KIF) were significantly reduced by treatment with 1 ␮g/ml anti-MR antibody (Fig. 10A) but not by treatment with 1 ␮g/ml anti-Dectin-1 antibody (Fig. 10B). To further clarify whether this effect is HA specific or also occurs with other N-linked glycoproteins, we also used a recombinant erythropoietin (EPO) glycoprotein for mouse mDC activation, and the results showed a relatively low level of TNF-␣ production upon EPO treatment, even after anti-MR or

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anti-Dectin-1 antibody treatment (Fig. 10). Similarly, relatively low levels of TNF-␣ production in mouse mDCs were observed with bovine serum albumin (BSA) and PBS treatments. Taken together, these results reveal that mDC activation was HA specific and occurred partially through MR(s). Overall levels of glycosylation among different HA heterosubtypes. Although the type and complexity of glycan chains attached to a single site are clearly relevant, the overall levels of glycosylation (i.e., the number of potential sites) among different HA heterosubtypes can also affect the stimulatory activity on mouse and human mDCs. Comparing the HA protein sequences of pH1N1 (A/Texas/05/2009), H5N1 [A/Thailand/ 1(KAN-1)/2004], and H7N9 (A/Shanghai/02/2013) virus strains, there are six potential glycosylation sites for pH1N1, six potential glycosylation sites for H5N1, and five potential glycosylation sites for H7N9 (Fig. 11). Therefore, the overall numbers of glycosylation sites are identical in the pH1N1 and H5N1 subtypes but slightly lower in the H7N9 subtype. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). The high-mannose-type or single-GlcNAc-type N-glycans of both avian and human HA types are more stimulatory than the complex-type N-glycans.

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FIG 7 hIL-12 p40, hIL-12 p70, and hIL10 cytokine production by human mDCs treated with rHA protein containing different N-glycan patterns. Human mDCs

were cocultured with 10 ␮g/ml (A to C) or 50 ␮g/ml (D to F) rHA protein or KIF-rHA proteins (H1, H5, or H7) with or without endo H for 48 h. Culture supernatants were harvested for cytokine assessment, including hIL-12p40 (A, D), hIL-12p70 (B, E), and hIL-10 (C, F). Data are expressed as mean values ⫾ standard deviations from one of three independent experiments. Two-tailed Student t test results: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. PBS was used as a negative control, and LPS was used as a positive control.

DISCUSSION

Influenza virus HA N-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response (29). We obtained stable-expression CHO cells to produce rH1HA, rH5HA, and rH7HA proteins containing complex-type and high-mannose-type (KIF-treated) N-glycans (Table 1; Fig. 3 to 5). Highmannose-type N-glycans were further digested with endo H under mild nondenaturing conditions (37°C for 3 h) to obtain single-GlcNAc-type N-glycans as previously described for obtaining single-GlcNAc-type N-glycans from high-mannose HIV-1 gp140 trimers (25). According to our data, the single-GlcNActype N-glycans from rH1HA, rH5HA, and rH7HA following treatment with KIF⫹E all had lower molecular weights (Fig. 1B and C). Further, the purified rH1HA, rH5HA, and rH7HA proteins all had relatively low fetuin-binding intensities, with the exceptions of the H5 and H7 single-GlcNAc forms rH5HA (KIF⫹E) and rH7HA (KIF⫹E) (Fig. 2). However, treatment of mouse and human mDCs with rH5HA (KIF⫹E) and rH7HA (KIF⫹E) did not result in the highest levels of TNF-␣, hIL-12 p40, hIL-12 p70, and hIL-10 secretion (Fig. 6 and 7). This suggests that the fetuinbinding intensity of the cells did not correlate with stronger mDC activation. We previously reported that CHO cells expressing rH5HA proteins exhibited approximately 25-fold lower binding affinities for specific ␣2-3-linked sialic acids than the strong binding affinities of insect cell-expressed rH5HA proteins containing paucimannose-type N-glycans (22). However, the complex-type

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N-glycans of rH5HA proteins produced in CHO cells induced better immunogenicity than insect cell-expressed rH5HA proteins in terms of serum hemagglutination inhibition and neutralization titers, thereby enhancing the protection of mice against lethal H5N1 viral challenges (22). The single-GlcNAc N-glycan forms of rH1HA and rH5HA proteins have also been reported to induce more potent T cells and a more diverse B-cell repertoire, as well as to provide better protective immunity in mice and ferrets (30, 31). Our data indicate that the complex-type N-glycans of rH5HA and rH7HA induced higher TNF-␣, I Ab, CD40, and CD86 expression levels in mouse mDCs than rH1HA complex-type N-glycans. Further, higher levels of hIL-12 p40, hIL-12 p70, and hIL-10 cytokine production and HLA-DR, CD40, CD83, and CD86 expression were observed in human mDCs treated with complex-type N-glycans from rH5HA and rH7HA than in human mDCs treated with complex-type N-glycans from rH1HA. Also, compared to complex-type N-glycans from rH1HA, reduced endocytotic strengths were observed in human mDCs induced by complextype N-glycans from rH5HA and rH7HA, suggesting that they may trigger DC activation and/or maturation more efficiently than complex-type N-glycans from rH1HA. Our previous results suggested that HA alone is responsible for this activation via the TLR4/MyD88 pathway (21). However, HA does not exist as a single entity in vivo and it is unclear if this activation has any relevance during virus infection. Rather than use rHA proteins to stimulate mouse or human mDCs, some researchers have directly

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FIG 8 Expression of KIF-rH1HA-enhanced HLA-DR, CD40, CD83, and CD86 in human mDCs. Human mDC maturation status was evaluated at 48 h posttreatment with 10 ␮g/ml rH1HA, rH1HA (KIF), rH1HA (KIF⫹E), or PBS (negative control [NC]) or 100 ng/ml LPS (positive control [PC]). mDCs were stained with anti-CD11c antibody plus anti-HLA-DR (A), anti-CD40 (B), anti-CD83 (C), or anti-CD86 (D) antibody and analyzed by flow cytometry. Indicated are differences in mean fluorescence intensity (MFI) between PBS, LPS, or rHA treatment (black curves) and a medium-only control (gray curves). MFI values are shown in flow cytometry profiles.

measured mouse or human mDC activation via infections with live influenza viruses, which can trigger several independent activation pathways (TLRs, retinoic acid-inducible gene 1-like receptors, etc.) (17, 20, 32). It was reported that the influenza viruses they used infected human mDCs more efficiently than mouse mDCs but with similar IP-10 gene expression levels. However, they also reported that the H5N1 influenza virus was capable of replicating more efficiently than the pH1N1 virus in human mDCs and that the observed differences in infectivity levels were not due to cellular receptor oligosaccharide links (32). In addition, compared to the efficient induction of antiviral and proinflammatory genes in the mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, the pH1N1 virus (A/Finland/553/2009) induced weak type I IFN (IFN-␣/ ␤), type III IFN (IFN-␭1 to -␭3), CXCL10, and TNF-␣ gene expression in human mDCs, despite its ability to replicate readily in human mDCs (17). Our results indicating similar cytokine production in human mDCs stimulated with rH5HA

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and rH7HA (Fig. 7) differ from those reported by another research group that the H7N9 virus induced significantly lower levels of proinflammatory cytokines (IFN-␣/␤, IFN-␥, IP-10, TNF-␣, and IL-6) than the H5N1 virus, although their replication levels are similar (as well as similar to the H3N2 virus replication level) in human mDCs (20). Membrane-associated C-type lectins on DCs were known to be associated with innate immunity to influenza A virus infection (28). The extents of glycosylation and the HA patterns determine the sensitivity of viruses to lectin-mediated innate defenses. In addition, pandemic pH1N1 and avian H5N1 and H7N9 viruses were resistant to soluble C-type lectin surfactant protein D in host cells, and this resulted in decreased antiviral activities in mice and significant pathology in the mouse lower respiratory tract (33, 34). Another report indicated that the macrophage MR recognized the same spectrum of monosaccharides as the collectins do and served as a major endocytic receptor in the infectious entry of influenza virus into murine

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FIG 9 Effects of rH1HA and KIF-rH1HAs on human mDC endocytotic capacity. Immature human mDCs were stimulated with PBS (negative control [NC]),

LPS (100 ng/ml, positive control [PC]), or 50 ␮g/ml rHA or rHA (KIF) for 24 h and then incubated with dextran-FITC for 1 h at 4°C (gray curves, isotype control) or 37°C (black curves) prior to analysis by flow cytometry. Percentages of dextran-FITC⫹ human mDCs from two donors are shown (A, B, C, D, and E for one donor and E, F, G, H, and I for the other).

FIG 10 Altered mouse mDC activation partially through MR(s). Mouse mDCs were left untreated or treated with 1 ␮g/ml anti-MR antibodies (A) or 1 ␮g/ml anti-Dectin-1 antibodies (B). Mouse mDCs were treated with rHA, rHA (KIF), rHA (KIF⫹E) (10 ␮g/ml), LPS (100 ng/ml), EPO glycoprotein, BSA (10 ␮g/ml), or PBS, and intracellular TNF-␣ production was determined. Percentages of TNF-␣⫹ mDCs (A, B) are indicated. Results are expressed as mean values ⫾ standard deviations from three independent experiments. Student t test results: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

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macrophages (35). Our results indicate that high-mannosetype and single-GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering activation and maturation in both mouse and human mDCs. It is likely that terminally sialylated complex-type N-glycans provide shielding against the activation of pattern recognition receptors (e.g., mannose or another C-type lectin) on mDC surfaces. To further clarify the mechanism(s) leading to altered mDC activation by rHA proteins containing different N-glycans, we used anti-MR or anti-Dectin-1 antibodies to block the mouse mDCs prior to rHA, rHA (KIF), and rHA (KIF⫹E) stimulation. Our results showed that blocking with anti-MR, but not anti-Dectin-1, antibodies resulted in significant decreases in TNF-␣ levels in mouse mDCs treated with rH1HA (KIF), rH1HA (KIF⫹E), rH5HA, rH7HA, and rH7HA (KIF) (Fig. 10). However, these results conflict with one report of mannose-terminated rH5HA protein N-glycans (but not complex-type N-glycans) suppressing CpG-induced IFN-␣ release in pDCs via binding with BDCA-2, a C-type lectin receptor (36). It has also been reported that the mannose moieties of HIV-1 gp120 glycoproteins induced immunosuppressive hIL-10 expression in human mDCs via the DC-SIGN of C-type lectin receptors (37). Additional studies are required to determine whether C-type lectin receptors account for more effective human mDC activation and maturation by high-mannose-type or single-GlcNAc-type N-glycans associated with rHA proteins. Still, we believe our findings provide useful information in support of vaccine develop-

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DC Activation by Influenza Virus Hemagglutinin

FIG 11 Alignment of influenza A virus HA protein sequences. The HA ectodomain sequences of three influenza viruses, A/Texas/05/2009 (pH1N1) (GenBank accession no. ACP41934), A/Thailand/1(KAN-1)/2004 (H5N1) (GenBank accession no. AFF60787), and A/Shanghai/02/2013 (H7N9) (GenBank accession no. AGL44438), were aligned by using the Influenza Virus Resource database. Identical amino acids are represented by dots, and noncorresponding amino acids sites are represented by dashes. The predicted N-linked glycosylation sites (N-X-S/T) are red.

ment regarding how influenza virus HA proteins with different types of N-glycans activate mDCs.

5.

FUNDING INFORMATION This work was funded by Ministry of Science and Technology, Taiwan (MOST) (MOST 104-2321-B-007-006, MOST 105-2321-B-007-005, and MOST 105-2321-B-007-006). This work was funded by National Tsing Hua University (NTHU) (105N742CV8). The funders had no role in study design, data collection and analysis, the decision to publish, or manuscript preparation.

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