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Impacts ImmunogenicityGlycosylation MarkedlyN-Hemagglutinin

Influenza Vaccine Design: Viral Based−Toward Animal Cell Culture

Udo Reichl, Erdmann Rapp and Bernd LepeniesJulia Hütter, Jana V. Rödig, Dirk Höper, Peter H. Seeberger,

http://www.jimmunol.org/content/190/1/220doi: 10.4049/jimmunol.1201060December 2012;

2013; 190:220-230; Prepublished online 5J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Toward Animal Cell Culture–Based Influenza VaccineDesign: Viral Hemagglutinin N-Glycosylation MarkedlyImpacts Immunogenicity

Julia Hutter,*,†,1 Jana V. Rodig,‡,1 Dirk Hoper,x Peter H. Seeberger,*,† Udo Reichl,‡,{

Erdmann Rapp,‡ and Bernd Lepenies*,†

The glycoproteins hemagglutinin (HA) and neuraminidase are the major determinants of host range and tissue tropism of the

influenza virus. HA is the most abundant protein in the virus particle membrane and represents the basis of most influenza vaccines.

It has been reported that influenza virus HA N-glycosylation markedly depends on the host cell line used for virus production.

However, little is known about how differential glycosylation affects immunogenicity of the viral proteins. This is of importance for

virus propagation in chicken eggs as well as for innovative influenza vaccine production in mammalian cell lines. In this study, we

investigated the impact of the differential N-glycosylation patterns of two influenza A virus PR/8/34 (H1N1) variants on immu-

nogenicity. Madin–Darby canine kidney cell–derived and Vero cell–derived glycovariants were analyzed for immunogenicity in

a TCR-HA transgenic mouse model. Next-generation pyrosequencing validated the congruence of the potential HA N-glycosylation

sites as well as the presence of the HA peptide recognized by the TCR-HA transgenic T cells. We show that differential HA N-

glycosylation markedly affected T cell activation and cytokine production in vitro and moderately influenced IL-2 production in vivo.

Cocultivation assays indicated that the difference in immunogenicity was mediated by CD11c+ dendritic cells. Native virus degly-

cosylation by endo- and exoglycosidases dramatically reduced cytokine production by splenocytes in vitro and markedly decreased

HA-specific Ab production in vivo. In conclusion, this study indicates a crucial importance of HA N-glycosylation for immunoge-

nicity. Our findings have implications for cell line–based influenza vaccine design. The Journal of Immunology, 2013, 190: 220–230.

Besides ideal hygiene measures, the only known efficientprotection from influenza infection is annual vaccinationbecause the virus regularly undergoes antigenic change

due to gene reassortment (antigenic shift) or point mutations (anti-

genic drift) (1). Strategies for enhancing influenza vaccine protec-tion are intensively investigated. One possibility is to use knownepitope targets for eliciting neutralizing Abs (2, 3). A focus is thedevelopment of vaccines that stimulate the production of Abs ca-pable of neutralizing multiple influenza subtypes (4). Another op-tion is to optimize vaccine uptake into APCs by targeting dendriticcells (DCs) or by increasing Ag immunogenicity (5).The major Ags of influenza virus are the envelope glycoproteins

hemagglutinin (HA) (6) and neuraminidase (7, 8). HA is highlyabundant in the virus particle and is able to induce strong and pro-tective immune responses. As for other glycoproteins, the qualitycharacteristics of HA such as activity (9), antigenicity (9–11),binding avidity (12), and receptor-binding specificity (13) stronglydepend on macro- and microheterogeneity of its N-glycosylation.HA antigenicity is strongly influenced by the HA amino acid se-quence with certain epitopes of HA being highly immunogenic (14,15). Although the impact of HA N-glycosylation on influenza virusbinding to host cell sialosides has been investigated in detail (16–19), less is known about its influence on immunogenicity such asrecognition by and activation of APCs or subsequent T cell priming.In mice, recent studies focused on the impact of influenza virus

N-glycan structures on the interaction between the virus and theimmune system. For instance, it was demonstrated that increasedinfluenza virus glycosylation resulted in decreased virulence,which was partly mediated by surfactant protein D (SP-D)–in-duced virus clearance from the lung (20). In addition to SP-D, themacrophage mannose receptor on airway macrophages contrib-uted to HA glycan recognition (21, 22). Mannose-rich glycans onthe globular head of HA impacted virus sensitivity to neutral-ization by a mannose-specific lectin in mouse lung fluids (23).However, in a DNA-based influenza H5 HA vaccine study, threepotential glycosylation sites of H5 from the influenza A H5N1virus did not substantially influence Ab responses (24).

*Department of Biomolecular Systems, Max Planck Institute of Colloids and Inter-faces, 14476 Potsdam, Germany; †Department of Biology, Chemistry, and Pharmacy,Institute of Chemistry and Biochemistry, Free University of Berlin, 14195 Berlin,Germany; ‡Max Planck Institute for Dynamics of Complex Technical Systems,39106 Magdeburg, Germany; xFriedrich-Loeffler-Institut, 17493 Greifswald–InselRiems, Germany; and {Chair of Bioprocess Engineering, Otto von Guericke Univer-sity, 39106 Magdeburg, Germany

1J.H. and J.V.R. contributed equally to this work.

Received for publication April 10, 2012. Accepted for publication October 30, 2012.

This work was supported by the Max Planck Society, German Federal Ministry ofEducation and Research Grant 0315446 (to B.L.), funding from the EuropeanUnion’s Seventh Framework Programme (FP7-Health-F5-2011) under grant agreement278535 “HighGlycan” (to E.R.), and Collaborative Research Centre Grant 765 (toP.H.S. and B.L.).

The sequences presented in this article have been submitted to the Global Initiativeon Sharing All Influenza Data EpiFlu database (http://www.gisaid.org) under accessionnumber EPI351614.

Address correspondence and reprint requests to Dr. Erdmann Rapp or Dr. BerndLepenies, Max Planck Institute for Dynamics of Complex Technical Systems, Sand-torstrasse 1, 39106 Magdeburg, Germany (E.R.) or Department of BiomolecularSystems, Max Planck Institute of Colloids and Interfaces, Am Muhlenberg 1,14476 Potsdam, Germany (B.L.). E-mail addresses: rapp@mpi-magdeburg.mpg.de (E.R.) and Bernd.Lepenies@mpikg.mpg.de (B.L.)

The online version of this article contains supplemental material.

Abbreviations used in this article: CGE-LIF, capillary gel electrophoresis with laser-induced fluorescence detection; CLR, C-type lectin receptor; DC, dendritic cell; GS,genome sequencer; HA, hemagglutinin; HAI, hemagglutination inhibition; MDCK,Madin–Darby canine kidney; MGL, macrophage galactose-type lectin; MMR, mac-rophage mannose receptor; M-variant, Madin–Darby canine kidney cell–derived in-fluenza virus; b-PL, b-propiolactone; RFU, relative fluorescence unit; SP-D,surfactant protein D; V-variant, Vero cell–derived influenza virus.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

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Little is known about the impact of influenza virusN-glycosylationon immunogenicity in humans. It was reported that HA N-glyco-sylation may mask antigenic epitopes, which can prevent binding ofHA by neutralizing Abs or the recognition of HA epitopes by CD4+

T cells (9, 25). A recent study showed that oligosaccharides presenton the influenza virus HA are a target for recognition by innateimmune proteins of the collectin and pentraxin superfamilies (26).Thus, although there are some indications for recognition of in-fluenza virus HA N-glycans by pattern recognition receptors of theinnate immune system, the impact of differentially glycosylatedHA on T cell activation remains to be determined.To date, vaccine production processes of manufacturers and de-

velopers such as Baxter, GlaxoSmithKline, Kaketsuken, Novartis,and Sanofi Pasteur range from traditional embryonated egg-basedto more recently established cell culture–based processes usingadherent Vero and Madin–Darby canine kidney (MDCK) cells orsuspension cells such as EBx, MDCK, and PER.C6 cells. SomeMDCK and Vero cell–derived vaccines have already been licensed(27–31). The MDCK cell culture–based processes that have beenlicensed so far are the subunit vaccines Optaflu and Celtura (Novartis)as well as a split vaccine (Solvay Pharmaceuticals/Abbott) (27). Theadvantages of cell culture–based processes are the independencefrom egg supply and the possibility of rapid process adjustmentto better match supply with vaccine demand during pandemics.Moreover, cell culture–derived vaccines bear no risk of ana-phylactic reactions caused by egg proteins.Previously, we have shown production cell line–specific HA N-

glycosylation patterns (32–34). Because it is unclear whether andto which extent HA N-glycosylation impacts immunogenicity, inthis study we investigated the effect of different cell culture–de-rived influenza A virus PR/8/34 (H1N1) glycovariants on T cellactivation. Two adherent cell lines, MDCK and Vero, were se-lected for influenza virus production, as these are so far the onlyones having passed all clinical trials. In a TCR transgenic mousemodel, we show that differential HA N-glycosylation markedlyaffected T cell activation and effector cytokine production in vitroand moderately influenced IL-2 production in vivo. Next-generationpyrosequencing validated the congruence of potential HA N-gly-cosylation sites as well as the presence of the HA peptide recog-nized by the TCR of the transgenic T cells. The difference in HA-specific T cell activation induced by the glycovariants in vitro wasmediated by CD11c+ DCs. Native virus deglycosylation by a mix-ture of endo- and exoglycosidases dramatically reduced cytokineproduction by splenocytes, indicating the crucial importance of HAN-glycosylation for immunogenicity. In vivo relevance of degly-cosylation was demonstrated by a marked reduction of HA-specificAb levels upon immunization of mice with the deglycosylated viruspreparations. Thus, our findings may have implications for opti-mized chicken egg influenza vaccine production as well as for in-novative cell line–based influenza vaccine design.

Materials and MethodsVirus production

Adherent MDCK (no. 841211903; European Collection of Cell Cultures,Salisbury, U.K.) and Vero (no. 88020401; European Collection of CellCultures) cells were cultivated in closed roller bottles at 37˚C until con-fluence. Virus was produced using MDCK or Vero cell–adapted influenzaA virus PR/8/34 (H1N1, Amp. 3138; Robert Koch Institute, Berlin, Ger-many) virus seeds as published before (32, 34). Virus-containing super-natant was harvested and cleared 96 h after infection by step gradientcentrifugation (100 3 g for 20 min, 4,000 3 g for 35 min, 10,000 3 g for45 min) and inactivated using b-propiolactone (b-PL) (12, 35). Virus isola-tion was performed at an average of 70,7143 g (31,000 rpm, type 70Ti rotor;Beckman Coulter, Brea, CA) for 90 min at 4˚C. The virus-containing pelletwas washed in ∼32 ml 100 mM Tris (pH 7) and finally resuspended in100 mM Tris (pH 7). Virus preparations were stored at 280˚C.

Native deglycosylation

An aliquot of the MDCK and Vero cell–adapted influenza A virus PR/8/34(H1N1) was natively deglycosylated in solution. All buffers and enzymesused for deglycosylation were purchased from Sigma-Aldrich (Steinheim,Germany) unless otherwise stated. After ultracentrifugation, the viruspellet was resuspended in 160 ml virus infection medium, and 6.7 mlprotease inhibitor (403, no. 11777700; Roche, Mannheim, Germany),50 ml reaction buffer (no. R9150), 10 ml endoglycosidase F2 (no. E0639),10 ml endoglycosidase F3 (no. E2264), and 10 ml a-galactosidase (no.G8507) were added and the mixture was shaken at 450 rpm and 37˚C for24 h in the dark. Then, 10 ml reaction buffer (no. R9025) and 10 mlendoglycosidase F1 (no. E9762) were added and shaking was continued at450 rpm at 37˚C for 24 h in the dark. In the following, 10 ml reactionbuffer (no. R0266), 10 ml a-mannosidase (no. M7257), 10 ml a-neur-aminidase (no. N8271), 10 ml b-N-acetylglucosaminidase (no. A6805), 20U b-galactosidase (no. G5160), 2 ml a-galactosidase, and 2 ml endogly-cosidase F3 were added and the mixture was again shaken at 450 rpm and37˚C for 24 h in the dark. As before, the virus was isolated by ultracen-trifugation at 31,000 rpm and 4˚C for 90 min. The pelleted virus wasresuspended in 100 mM Tris (pH 7) and stored at 280˚C. Total virusprotein was quantified by bicinchoninic acid assay (Thermo Scientific,Rockford, IL). Additionally, protein concentrations were determined bya 10% nonreducing SDS-PAGE (data not shown). Band intensities wereanalyzed using the open source imaging software ImageJ 1.45 (36).

N-glycosylation pattern analysis

N-glycosylation pattern analysis was performed based on a method de-veloped by Laroy et al. (37). For analysis, samples were prepared as de-scribed previously (33, 38) with some optimizations and modifications.These comprise the substitution of 20 mM NaHCO3 (aqueous) with 50 mMNH4HCO3 (aqueous) during enzymatic in gel deglycosylation and N-glycan extraction as well as the reduction of the final sample volume forpeptide-N-glycosidase F digestion to 60 ml, which resulted in an increasedenzyme concentration. Furthermore, labeled samples were desalted andexcess label was removed by hydrophilic interaction liquid chromatog-raphy as described before (39).

Finally, HA N-glycosylation patterns were analyzed as described before(32–34, 38) using an ABI Prism 3100-Avant genetic analyzer (Applied Bio-systems), allowing multiplex capillary gel electrophoresis with laser-inducedfluorescence detection (xCGE-LIF). In an HA N-glycosylation fingerprint, onepeak corresponds to at least one distinct N-glycan structure. Overlays of N-glycosylation fingerprints allowed a direct comparison of different N-glycanpools. All data were processed as published previously (38, 39).

Pyrosequencing analysis

The DNA for full-length genome sequencing using the Roche/454 genomesequencer (GS) FLX (Roche) was prepared according to the protocol ofHoper et al. (40). The equimolar pool of PCR amplicons was fragmentedaccording to the manufacturer’s instructions for preparation of shotgunsequencing libraries. The fragmented DNA was then converted to a GSFLX Titanium Library with the SPRIworks Fragment Library System II(Beckman Coulter, Krefeld, Germany) using a SPRIworks Fragment Li-brary kit II (Beckman Coulter) and a GS FLX Titanium Rapid Librarymolecular identifier adaptor (Roche). The resulting library was quantifiedwith the aid of the KAPA Library Quant Roche 454 Titanium UniversalKitsystem (Kapa Biosystems, Cape Town, South Africa) on a Bio-RadCFX96 real-time PCR system (Bio-Rad, Munchen, Germany). The li-braries were then clonally amplified in the emulsion PCR system with 0.08copies per bead. Finally, the amplified library was sequenced with the GSFLX using Titanium chemistry and the appropriate instrument run proto-col. Raw sequencing data were analyzed with the GS FLX software suite(version 2.5.3; Roche). During sequence assembly, primer sequences weretrimmed off the raw data according to the protocol (40). The HA sequenceis deposited in the Global Initiative on Sharing All Influenza Data EpiFludatabase (http://www.gisaid.org) with the accession number EPI351614.For quasispecies analyses, a mapping of the raw sequencing reads alongthe reference sequence using the GS FLX reference mapper software(version 2.5.3; Roche) was performed.

Ethics statement

Animal experiments were performed in strict accordance with the Germanregulations of the Society for Laboratory Animal Science and the EuropeanHealth Law of the Federation of Laboratory Animal Science Associations.The protocol was approved by the Landesamt fur Gesundheit und Soziales(Berlin, Germany; permit no. G0259/11). All efforts were made to mini-mize suffering.

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Animals

Mice were kept in the animal facility of the Federal Institute for RiskAssessment under specific pathogen-free conditions. TCR-HA transgenicmice were kindly provided from Dr. W. Hansen (Essen, Germany). Thesetransgenic mice express a TCRab specific for the peptide 110–120 fromHA of influenza A virus A/PR/8/34 (H1N1) presented by I-Ed MHC classII molecules (41). BALB/c mice were bred at the Max Planck Institute forInfection Biology (Berlin, Germany).

Whole spleen cell stimulation

Spleens were removed, cells were flushed out, and RBCs were lysed byaddition of ammonium chloride. For Ag-specific T cell stimulation, 23 105

spleen cells from TCR-HA transgenic mice or, as control, from BALB/cwild-type mice were stimulated with different protein concentrations of thefully glycosylated or deglycosylated influenza A virus PR/8/34 (H1N1)MDCK cell–derived (M-variant) or Vero cell–derived influenza virus (V-variant) (0.01, 0.1, and 1 mg/ml). HA110–120 peptide (0.1, 1, and 10 mg/ml)was used as positive control, and 10 mg/ml OVA323–339 peptide (Anaspec,Seraing, Belgium) was used as negative control. After 48 h, cells wereanalyzed for the expression of T cell activation markers (CD69, CD25) byflow cytometry. A portion of cells was first incubated with anti-CD16/32(BD Biosciences) at 4˚C for 30 min, washed, and then stained with cellmarker–specific Abs at 4˚C for 30 min. Data were acquired on a FACS-Canto II flow cytometer (BD Biosciences) and analyzed with the FlowJoanalysis software (Tree Star, Ashland, OR). The cytokine concentrations ofIL-2, IFN-g, and IL-4 in cell supernatants were quantified by ELISA(PeproTech, Hamburg, Germany) according to the manufacturer’s protocol.

LPS contamination in the b-PL–inactivated virus preparations waschecked using a gel clot Limulus amebocyte lysate kit (Lonza, Cologne,Germany) to rule out potential contaminations. The endotoxin detectionlevel of this assay was 0.06 endotoxin unit/ml. The virus preparations werenegative for all concentrations used in the cell stimulation assay.

Stimulation studies with isolated spleen cell subsets

CD11c+ and CD19+ cells were isolated from spleen of BALB/c mice byMACS using CD11c or CD19 microbeads (Miltenyi Biotec, BergischGladbach, Germany) according to the manufacturer’s instructions. Theeluate of the CD11c+ MACS was applied on a second column to enhancecell purity. In the end, a purity of ∼80% was achieved (data not shown).TCR-HA transgenic T cells were purified from spleen by negative se-lection using the MACS Pan T cell isolation kit II (Miltenyi Biotec)according to the manufacturer’s protocol. T cell purity was .95% (datanot shown).

For Ag-specific T cell stimulation, 1 3 104 CD11c+ cells were firstpulsed at 37˚C for 1 h with the Ags mentioned above. In the next step, 1 3105 purified TCR-HAT cells were added and cells were incubated at 37˚Cfor 48 h. T cell activation was analyzed as described before.

In vivo studies

To analyze the immune stimulatory effect of HA N-glycosylation in vivo,female 6- to 8-wk-old BALB/c wild-type mice were immunized i.p. with10 mg fully glycosylated or deglycosylated M- and V-variant or with PBSas control. A boost immunization with the same amount of Ag was done onday 14. On day 28, CD4+ spleen T cells were analyzed for IL-2 and IFN-gproduction by ELISPOT (Abs were purchased from BD Biosciences) afterrestimulation with 20 mg/ml HA110–120 peptide for 18 h. A freshly pre-pared 3-amino-9-ethylcarbazole substrate solution (Sigma-Aldrich) wasused for development. Spots were recorded and analyzed using Bioreader5000 Pro-E (BioSys, Karben, Germany). HA-specific IgG/IgM Ab levelsin sera were measured by ELISA on days 14 and 28. Plates were coatedwith 10 ng/well recombinant influenza A virus PR/8/34 H1N1 HA (SinoBiological, Beijing, China). Sera were also tested for HA inhibition (HAI)activity as previously published (42).

For further analysis of the immunogenic properties of the M- and V-variant, TCR-HA transgenic T cells (purified by MACS) were adoptivelytransferred into female BALB/c wild-typemice. Before injection, cells werelabeled with the cell proliferation dye eFluor 670 (eBioscience). On day 0,1.5 3 107 cells in 150 ml PBS were adoptively transferred by i.v. injectionin the lateral tail vein. Mice were immunized i.p. with 50 mg M- or V-variant on the following day. On day 5, CD4+eFluor 670+ spleen cells wereanalyzed for proliferation by flow cytometry. Furthermore, cytokine pro-duction of spleen cells was measured by ELISPOT as described earlier.

Statistical analysis

Statistical analyses were performed with Student t test. An unpaired t testwas used for analyzing data within one experiment. A paired t test was

used when results were compared across different experiments. All sta-tistical analyses were performed with the Prism software (GraphPadSoftware).

ResultsGenomic composition of Ags

To verify the presence of the HA110–120 peptide from influenza Avirus PR/8/34 (H1N1), the MDCK and Vero cell–adapted virusseeds were sequenced by next-generation pyrosequencing. Thesequence for the HA110–120 peptide was detected for both virusseeds and it was not altered in any detected subpopulation. Becausepyrosequencing allows for the characterization of the quasispeciescomposition, it was also applied to check for homogeneity of virusseeds. As published before (34), the MDCK cell–adapted virus seedwas homogeneous for HA composition (i.e., no other HA sequenceswere detected), indicating that the seed only consists of onepopulation. The Vero cell–adapted virus seed (32) had the sameconsensus sequence as did the MDCK cell–adapted virus seed.Pyrosequencing revealed a subpopulation of 40% with a deletionof isoleucine at position 338 (I338–) and a subpopulation of 11%with a substitution of valine by methionine at position 459 (V459M).Furthermore, a silent point mutation at position 364 (G364G) waspresent in a subpopulation of 12% (Table I). Both the deletionI338– and the substitution V459M are located in the stem regionof the HA molecule within the fusion subdomains (43). The de-letion I338– is located only a few residues before the HA1–HA2cleavage site whereas the substitution V459M is located within thebig a helix of the HA2 subunit. No potential N-glycosylation sitewas altered by virus adaptation from MDCK to Vero cells, neitherin the HA (Table I) (34) nor in the neuraminidase molecule (datanot shown). These results confirm that the altered amino acids inMDCK and Vero cell–derived HA did not affect HA N-glycanrecognition by host immune cells.

Impact of N-glycosylation on immunogenicity

Strict host cell specificity of HA N-glycosylation was confirmed. Asreported previously (33), HA N-glycosylation from MDCK andVero cell–derived virus differs significantly (Fig. 1Ai, 1Bi). Whereasthe M-variant HA of influenza A virus PR/8/34 (H1N1) containstri- and tetra-antennary as well as bisecting N-acetylglucosaminestructures, the V-variant HA exhibits fewer N-glycan structuresand mainly with a low molecular mass. Furthermore, the V-variantHA carries terminal b-galactose and high-mannose structures,whereas the M-variant HA displays terminal a- and b-galactoseand no high-mannose glycan structures (Fig. 1Ai, 1Bi, and Ref.33). To analyze whether this difference in the HA N-glycosylationpatterns influences T cell stimulation, TCR-HA transgenic spleencells were incubated with b-PL–inactivated preparations of theinfluenza A virus PR/8/34 (H1N1) M- or V-variant. T cells fromTCR-HA transgenic mice have a TCRab specific for the HA110–120

peptide presented by I-Ed MHC class II molecules (41). Therefore,it was possible to investigate specifically how CD4+ T cell stim-ulation was affected by differential HA N-glycosylation. Flowcytometry revealed that a higher frequency of splenic T cellsexpressed the activation marker CD69 when stimulated with theV-variant (Fig. 2A). This cell surface glycoprotein is upregulatedvery early during lymphoid activation and represents a costimula-tory molecule involved in lymphocyte proliferation (44). The ob-served difference in CD69 expression on T cells stimulated withthe M- or V-variant was statistically significant when the results ofthree independent experiments were combined (Fig. 2B). In con-trast, there was no significant difference in the expression of theT cell activation marker CD25 (data not shown). Next, we analyzedcytokine production by splenocytes to investigate whether the in-

222 HEMAGGLUTININ N-GLYCOSYLATION IN IMMUNOGENICITY

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fluenza A virus PR/8/34 (H1N1) M- and V-variant induced distinctcytokine profiles. Consistent with the CD69 expression analysis,IL-2 was produced at a significantly higher level by splenocytesincubated with the V-variant than cells incubated with the M-variant (Fig. 3A). Thus, HA N-glycosylation of the V-variant hada marked impact on IL-2 production. For IFN-g, the same tendencyas for IL-2 was observed, albeit to a lesser extent, whereas nodifference was observed for IL-4 (Fig. 3B, 3C). These findingssuggest that the initiated T cell response is markedly influencedby differential HA N-glycosylation.Next, we investigated the kinetics of T cell activation by the two

glycovariants. The difference in CD69 expression was alreadydetectable after 24 h but no longer after 72 h (Supplemental Fig. 1).The production of IL-2 did not yet differ after 24 h (SupplementalFig. 2A). However, splenocytes stimulated with the V-variantproduced still significantly higher IL-2 levels after 72 h com-pared with stimulation with the M-variant (Supplemental Fig. 2B,2C). This indicates that the influenza A virus PR/8/34 (H1N1) V-

variant promotes a faster T cell activation than does the M-variantand thus enhances T cell proliferation.

Differential T cell activation is mediated by CD11c+ DCs

To identify the APC population responsible for the distinct stim-ulatory effect of the influenza A virus PR/8/34 (H1N1) M- and V-variant, CD11c+ cells were separated from spleen, incubated withthe M- or V-variant, and cocultivated with TCR-HA transgenicT cells. The same effects in CD69 expression (Fig. 4A, Supple-mental Fig. 3), IL-2 production (Fig. 4B), as well as IL-4 and IFN-g production (Supplemental Fig. 4) were observed as in the wholespleen cell assay. This finding indicates that CD11c+ DCs playa role in the recognition of the two glycovariants, in CD4+ T cellactivation, and in acquisition of T cell effector functions. We alsoinvestigated whether other subsets of spleen cells contributed todifferential T cell stimulation. However, cocultivation of TCR-HAtransgenic T cells with other spleen cell subsets led to no or veryweak T cell activation (data not shown). Thus, the differences in

Table I. Quasispecies’ composition of Vero cell–adapted influenza A virus PR/8/34 (H1N1) HA

Segment Coded Protein Base Pair Substitution Amino Acid SubstitutionVero Cell–AdaptedVirus Seed (%) (32)

4 HA CAT 1011 — I 338 — 40A 1092 T G 364 G 12G 1375 A V 459 M 11

The deletion I338– is located in the HA1 molecule, only a few amino acids before the HA1–HA2 cleavage site betweenamino acid 344(R)–345(G). The silent point mutation at G364G and the substitution V459M are located in the HA2 molecule.The deletion I338– as well as the substitution are both located in the stem region of the HA molecule.

FIGURE 1. HA N-glycosylation finger-

prints of MDCK (A) and Vero (B) cell–de-

rived influenza virus A/PR/8/34 (H1N1).

For N-glycosylation analysis, influenza vi-

rus proteins were separated by a nonreduc-

ing SDS-PAGE. HA protein bands were

excised and N-glycans obtained from di-

gestion with peptide-N-glycosidase F were

fluorescently labeled. N-glycan patterns of

MDCK (A) and Vero cell–derived HA (B)

were obtained by analyzing HA N-glycan

pools by multiplex CGE-LIF. RFU are

plotted over the normalized migration time

(tmig) in base pairs. One peak represents at

least one distinct N-glycan structure and

tmig increases with the size of the N-glycan

structure. Shifted overlays (i, ii) and direct

overlays (iii) of fully N-glycosylated (i) and

native deglycosylated HA (ii) show efficient

but not complete deglycosylation (note the

different scale in i and ii). Glycoanalysis

indicated that at least ∼90% of HA N-gly-

can structures were cut off. New, truncated

glycan structures detected after deglycosy-

lation on the MDCK (Aii) or Vero (Bii) cell–

derived HA are marked with an asterisk.

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T cell activation induced by the influenza virus glycovariants wereindeed mediated by CD11c+ DCs.

Deglycosylation of the influenza virus HA variantsdramatically affects immunogenicity

To further investigate the effect of HA N-glycosylation on T cellstimulation, the MDCK and Vero cell–derived glycovariants ofinfluenza A virus PR/8/34 (H1N1) were natively deglycosylatedusing a variety of endo- and exoglycosidases. HA bands wereshifted to lower molecular masses in the SDS-PAGE, indicating

successful deglycosylation (data not shown). Bands of fully gly-cosylated HA monomers were found at ∼70 kDa (MDCK-derivedjust above and Vero-derived just below). In contrast, deglycosy-lated preparations exhibited a more diffuse and broader band patternjust below the fully glycosylated band reaching to the nucleopro-tein at ∼55 kDa (data not shown). N-glycosylation pattern analysisconfirmed that both variants were deglycosylated to most parts(Fig. 1Aii–iii, 1Bii–iii). The significant reduction of signal in-tensity from ∼450–500 relative fluorescence units (RFU) to ,50RFU confirmed efficient protein deglycosylation. Although no

FIGURE 2. Increased CD69 expression by splenic T cells upon stimulation with the influenza A virus PR/8/34 (H1N1) V-variant. The frequency of CD4+

CD69+ TCR-HA transgenic splenocytes stimulated with the M- or V-variant was determined by flow cytometry. Cells were gated on CD4+ cells.

Recombinant HA110–120 peptide and OVA323–339 were used as positive and specificity control, respectively. (A) Histoplots showing data from one ex-

periment representative of three independent experiments (duplicates each). (B) Bar diagram summarizing the results from three independent whole spleen

cell assays. Dashed line indicates background frequency of CD4+CD69+ cells. Upon stimulation with the V-variant, a higher frequency of TCR-HA

transgenic splenic T cells expressed the activation marker CD69. Data are expressed as means 6 SEM. *p , 0.05, **p , 0.01 for MDCK versus Vero (by

paired Student t test). ns, Not significant.

FIGURE 3. Cytokine production

by spleen cells stimulated with the

influenza A virus PR/8/34 (H1N1)

glycovariants. Levels of the cyto-

kines IL-2 (A), IL-4 (B), and IFN-g

(C) were measured by ELISA in the

supernatants of stimulated TCR-HA

spleen cells after 48 h (in triplicates).

As control peptide, OVA323–339 pep-

tide was used. Significantly higher IL-

2 levels were produced by splenocytes

stimulated with the V-variant, indicat-

ing the role of HA N-glycosylation for

T cell proliferation. Data are rep-

resentative of four independent

experiments and are expressed as

means 6 SEM. *p , 0.05, **p ,0.01 for MDCK versus Vero at all

concentrations (by unpaired Student

t test). ns, Not significant.

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complete deglycosylation was achieved, glycan analysis showeda reduction of N-glycosylation by at least a factor of 10. Moreover,glycan analysis suggested that Vero cell–derived N-glycan structureswere removed more efficiently (Fig. 1Bii) than MDCK cell–derivedstructures (Fig. 1Aii) (maximum RFUVero , maximum RFUMDCK).Interestingly, deglycosylation resulted in multiple truncated glycanstructures of lower molecular mass on the Vero cell–derived HA(marked with an asterisk in Fig. 1Bii). In contrast, on the MDCKcell–derived HA only one shorter glycan structure was detectedafter deglycosylation (marked with an asterisk in Fig. 1Aii).To investigate whether HA deglycosylation affected immuno-

genicity, again TCR-HA transgenic splenocytes were stimulatedwith the deglycosylated influenza virus variants (Fig. 5). Degly-cosylation led to a dramatically reduced T cell activation as mea-sured by the frequency of CD4+ T cells expressing CD69 (Fig.5A) and cytokine production (Fig. 5B–D) after stimulation withthe V-variant. For splenocyte stimulation with the M-variant, thesame tendency was observed, albeit to a lower extent (Fig. 5). Thismight be due to the fact that HA deglycosylation of the M-variantreduced the HA N-glycosylation level by ∼90% but withoutmodifying most N-glycan structures. In contrast, N-glycans of theV-variant HA were truncated during deglycosylation, leading tomultiple new glycan structures in addition to the reduced level ofglycosylation (Fig. 1Bii). However, for both glycovariants of in-fluenza A virus PR/8/34 (H1N1) a significantly diminished fre-quency of CD4+CD69+ T cells and decreased levels of IL-2, IL-4,and IFN-g were observed upon deglycosylation, particularly atlower protein concentrations (Fig. 5). Interestingly, the reductionof the T cell proliferation cytokine IL-2 was more pronouncedcompared with the effector cytokines IL-4 and IFN-g. This findingsuggests that the N-glycosylation pattern of the V-variant may leadto a faster recognition and virus uptake by APCs and influencesthe kinetics of T cell activation rather than changing the quality ofthe initiated immune response.To confirm the crucial role of CD11c+ DCs, we analyzed the

effect of the HA deglycosylation in the CD11c+/TCR-HA T cellcocultivation assay. Consistent with the results in the whole spleencell assay, IL-2, IFN-g, and IL-4 levels were dramatically reduced

after HA deglycosylation (Supplemental Fig. 4). The reduction incytokine levels was even more pronounced than in the wholespleen cell assay. This finding further confirmed the crucial role ofCD11c+ DCs for HA N-glycan recognition and T cell activation.In conclusion, these observations clearly indicate that HA N-gly-cosylation of the influenza A virus PR/8/34 (H1N1) has a markedimpact on virus immunogenicity.

In vivo studies

To investigate the immune stimulatory effect of HA N-glycosylationin vivo, wild-type mice were immunized with the fully glycosylatedor deglycosylated M- or V-variant. On days 14 and 28, HA-specificAb levels in sera were analyzed by ELISA. Consistent with thein vitro results, anti-HA Ab levels were dramatically reduced in seraof mice immunized with the deglycosylated preparations comparedwith mice immunized with the fully glycosylated M- or V-variant(Fig. 6A, left). An HAI assay performed with serum samples col-lected on day 28 confirmed these results (Fig. 6A, right). Accord-ingly, the frequency of IFN-g–producing CD4+ T cells was slightlylower for spleen cells from mice immunized with the deglycosy-lated virus preparations upon restimulation with the HA110–120

peptide (data not shown). The number of IFN-g–producing CD4+

T cells in spleens of mice immunized with the V-variant was highercompared with the M-variant although values did not reach statis-tical significance (data not shown). Thus, the crucial effect of HAN-glycosylation was demonstrated by a dramatic reduction of HA-specific Ab levels in mice immunized with the deglycosylated virusvariants. Interestingly, HA-specific Ab levels between the M- andV-variant differed on day 14 when the M-variant induced signifi-cantly higher Ab levels (Fig. 6A, left). This effect was still observedon day 28 when sera of immunized mice were tested for hemag-glutination inhibition activity by HAI assay (Fig. 6A, right). Thisfinding might suggest that the glycosylation pattern of the M-variantinduces a bias toward a humoral immune response whereas the V-variant promotes cellular immune responses as described before.To address the role of HA N-glycosylation on T cell prolifer-

ation and cytokine production further in vivo, TCR-HA transgenicT cells were labeled with the cell proliferation dye eFluor 670 and

FIGURE 4. Differential T cell activation by the two influenza A virus PR/8/34 (H1N1) glycovariants is mediated by CD11c+ DCs. MACS-purified

splenic CD11c+ DCs were pulsed with varying concentrations of the M- or V-variant, HA110–120 peptide (positive control), or OVA323–339 peptide

(specificity control). Pulsed DCs were cocultivated with purified TCR-HA transgenic T cells for 48 h. (A) Flow cytometry histoplots showing the frequency

of CD4+CD69+ cells in one experiment representative of three independent experiments (duplicates each). Cells were gated on CD4+ cells. The frequency

of T cells expressing CD69 was increased when DCs were pulsed with the V-variant. (B) Bar diagram showing the IL-2 production after cocultivation of

pulsed CD11c+ DCs with TCR-HA transgenic T cells. Data are representative of three independent experiments (triplicates each) and are expressed as

means 6 SEM. *p , 0.05, ***p , 0.001 for MDCK versus Vero at all concentrations (by unpaired Student t test). ns, Not significant.

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were adoptively transferred into BALB/c wild-type mice. This allowsfor analysis of the fate of HA110–120 peptide–specific T cells innatural conditions. Subsequently, mice were immunized with theb-PL–inactivated virus glycovariants and the proliferation of thetransferred T cells was analyzed 5 d after adoptive T cell transfer(Fig. 6B, 6C). Furthermore, spleen cells were restimulated withHA110–120 peptide and the frequency of cytokine-producing cells wasdetermined using ELISPOT assay (Fig. 6D). Although proliferationof T cells was similar after immunization with the two glycovariants(Fig. 6C), the frequency of IL-2–producing splenocytes was in-creased in mice immunized with the V-variant (Fig. 6D). In con-clusion, the findings observed in the TCR-HA transgenic system andupon immunization of wild-type mice indicate that differential HAN-glycosylation impacts immunogenicity in vitro and in vivo.

DiscussionThis study showed that differences in influenza HA N-glycosyla-tion caused by virus production in different host cell lines havea marked impact on immunogenicity in vitro and in vivo. Fur-thermore, this study emphasizes the advantage of combining next-generation pyrosequencing, high-throughput multiplex CGE-LIF–based HA N-glycosylation pattern analysis, and immunogenicityassays to identify optimal (i.e., highly immunogenic) HA N-gly-cosylation patterns or single N-glycan structures for targeted in-fluenza vaccine design.In this study, we analyzed b-PL–inactivated influenza A virus

PR/8/34 (H1N1) glycovariants of two host cell lines, MDCK andVero, for their HA N-glycosylation pattern and immunogenicity.First and foremost, these cell lines were selected because they areeither already licensed for human influenza vaccine manufacturingor have at least successfully passed clinical trial III studies. Al-though we demonstrate the impact of HA N-glycosylation onimmunogenicity for only two cell lines, screening for highly im-

munogenic N-glycosylation patterns can easily be extended toa variety of other virus expression systems by using the platformpresented in this study. In previous studies, we showed that thehost cell line indeed influences HA N-glycosylation patterns (32–34). Whereas the influenza A virus PR/8/34 (H1N1) M-variantabundantly displays larger, tri- or tetra-antennary, a- and b-ga-lactose–terminated N-glycan structures, the V-variant carriessmaller, exclusively b-galactose–terminated glycan structures andalso some high-mannose N-glycans (33).Whole spleen cell stimulation using a TCR-HA transgenic

system revealed that HA N-glycosylation significantly affectedearly T cell activation and proliferation in vitro. In this assay, theV-variant induced a markedly higher frequency of CD4+ T cellsexpressing the activation marker CD69 than did the M-variant.Moreover, splenocyte stimulation with the V-variant led to sig-nificantly increased IL-2 levels. Interestingly, the production ofthe effector cytokines IFN-g and IL-4 was less affected than IL-2release. IL-2 is a cytokine that is typically produced by Th0 cells,but also by Th1 cells, after Ag stimulation and promotes T cellgrowth, differentiation, and survival (45). That the change in IL-2production was more pronounced than levels of Th1/Th2 effectorcytokines suggests that the V-variant HA N-glycosylation patternsfacilitate faster recognition and virus uptake by APCs. In ten-dency, the V-variant also induced a higher production of the Th1cytokine IFN-g by splenocytes. This finding might be of impor-tance because it is known that induction of a potent Th1 immuneresponse is essential for viral clearance (46).Furthermore, we demonstrated in DC/T cell cocultivation assays

that the difference in T cell priming based on differential HA N-glycosylation was mediated by CD11c+ DCs. As a bridge toadaptive immunity, DCs transport Ags from the site of infection tothe regional lymph nodes or to the spleen where they activate Ag-specific CD4+ T cells and CD8+ T cells by cross-presentation (47).

FIGURE 5. Deglycosylation leads

to a dramatically reduced T cell ac-

tivation in vitro. The influenza A

virus PR/8/34 (H1N1) glycovariants

were natively deglycosylated with

a mixture of endo- and exoglycosi-

dases. TCR-HA transgenic spleno-

cytes were stimulated with the virus

preparations for 48 h. (A) Bar dia-

gram demonstrates the frequency of

CD69+ T cells stimulated with gly-

cosylated versus deglycosylated M-

or V-variant (duplicates each). A

marked reduction in the frequency

of CD4+ T cells expressing the ac-

tivation marker CD69 was observed

for the deglycosylated virus prepa-

rations. Upon deglycosylation, a dra-

matic decrease was also observed for

levels of the cytokines IL-2 (B), IL-4

(C), and IFN-g (D), particularly for

the deglycosylated V-variant. Data

are representative of three indepen-

dent experiments (triplicates each).

Data are expressed as means6 SEM.

*p , 0.05, **p , 0.01, ***p ,0.001, ****p , 0.0001 for glycosy-

lated versus deglycosylated virus

preparations at all concentrations (by

unpaired Student t test). ns, Not sig-

nificant.

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CD4+ T cells generally lead to optimal activation of CD8+ T cellsand promote maturation of Ab-producing B cells (48). In thisstudy, the crucial role of DCs in recognition of the glycovariantswas further confirmed by DC/T cell cocultivation assays withdeglycosylated virus preparations where T cell activation wasalmost completely abrogated.

A number of DC subsets are involved in innate and adaptiveimmunity and thus might be responsible for the different effect onT cell activation by the two influenza A virus PR/8/34 (H1N1)glycovariants. A previous study indicated that CD103+ DCs andCD11bhigh DCs uptake influenza virus particles in the lung andtransport them to the draining mediastinal lymph nodes (49, 50).

FIGURE 6. Immunogenicity of the influenza A virus PR/8/34 (H1N1) glycovariants in vivo. (A) 5 BALB/c wild-type mice per group were prime boost

immunized i.p. with 10 mg fully glycosylated or deglycosylated M- or V-variant on day 0 and on day 14. Left, HA-specific Ab levels in sera were analyzed

by ELISA on days 14 and 28 (triplicates each). Right, HAI activity was tested by HAI assay on day 28 (each symbol represents one mouse). Data are

expressed as means 6 SEM. **p , 0.01, ***p , 0.001, ****p , 0.0001 for glycosylated versus deglycosylated M-variant; ˚p , 0.05, ˚˚p , 0.01 for

glycosylated versus deglycosylated V-variant; #p , 0.05 for glycosylated M- versus V-variant (by unpaired Student t test). Values of p for HAI activity for

glycosylated versus deglycosylated V-variant could not be determined because all HAI values of mice immunized with the deglycosylated V-variant were

null. (B) TCR-HA transgenic spleen T cells were purified by MACS and were labeled with the cell proliferation dye eFluor 670. Subsequently, labeled cells

were adoptively transferred into BALB/c wild-type mice by i.v. injection (day 0) and mice were immunized with 50 mg M- or V-variant or with PBS (day

1). On day 5, spleen cell proliferation and activation were analyzed. For analysis by flow cytometry, cells were gated on eFluor 670+CD4+ cells. (C) The

diagram shows the frequency of transferred cells that had proliferated. A summary of three independent experiments with three to four mice is presented

(each symbol represents one mouse). (D) Splenocytes were restimulated with HA110–120 peptide and the frequency of IL-2–producing cells was analyzed by

ELISPOT (triplicates each). The diagram shows the number of IL-2–producing cells normalized to the number of adoptively transferred cells based on flow

cytometry data. Data are expressed as means 6 SEM.

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CD8a+ DCs were reported to play a crucial role in CTL primingduring influenza infection (51). However, this might rather berelevant for influenza infections of mice, as only a very limitednumber of CTL epitopes within HA are known in humans (52).Recently, a study using a human challenge model revealed thatmemory CD4+ T cell populations correlated with less severe ill-ness (53). Thus, further experiments with human DC populationsand PBMCs are needed to determine the relevance of HA N-glycosylation on influenza virus immunogenicity in humans.Additionally, DCs are known to express high levels of pattern

recognition receptors, particularly C-type lectin receptors (CLRs)(54). Our finding that in vitro the influenza A virus PR/8/34(H1N1) V-variant exhibits a more immunogenic N-glycosylationpattern than does the M-variant suggests differential glycan rec-ognition by DCs. Because some of the V-variant N-glycans are ofthe high-mannose type (33), mannose-rich HA N-glycan structuresmay be recognized by CLRs expressed by splenic CD11c+ DCs.This is in accordance with studies elegantly demonstrating theinvolvement of the macrophage mannose receptor (MMR) inmacrophage infection by the influenza virus (21, 55). In a recentstudy, the macrophage galactose-type lectin (MGL) was identifiedas a second receptor besides MMR for virus recognition by hostcells (56). Binding of influenza virus to the CLRs MMR and MGLwas independent of sialic acid and was mediated by Ca2+-de-pendent recognition of viral glycans by the carbohydrate recog-nition domains of MMR and MGL (56). An additional lectinreported to play a role in influenza virus clearance from bodyfluids is SP-D that may be involved in glycan recognition (20, 22,57). An alternative mechanism that may also account for differ-ential immunogenicity of the influenza virus glycovariants ismasking of antigenic epitopes by HA N-glycosylation (9, 25). Inall of these studies, except for one (9), live influenza virus wasused for the experiments. However, because glycan-bindingreceptors on the APC surface may contribute to virus endocyto-sis, but may also serve as first attachment receptors for virus entry(51), we used b-PL–inactivated virus preparations in this study todecipher the role of N-glycosylation for immunogenicity. In a re-cent study published during revision of this manuscript, immu-nogenicity of influenza HA N-glycosylation was investigated byusing HA recombinantly expressed in HEK or in insect cells (58).The authors showed that the N-glycan structure impacted HAI Abtiters in chicken and mice.In this study, we used natively deglycosylated virus prepara-

tions of two glycovariants and showed in vitro that T cell acti-vation significantly diminished compared with fully glycosylatedHA. This effect was observed for both variants as seen in a markedlydecreased expression of CD69 as well as in reduced cytokine levels,albeit more pronounced for the V-variant. Glycoanalysis revealedthat both influenza Avirus PR/8/34 (H1N1) glycovariants exhibiteda markedly reduced N-glycosylation (by at least 90%), but were notcompletely deglycosylated. Although similar N-glycan structureswere still present on the deglycosylated MDCK cell–derivedHA, new, truncated glycan structures occurred on the deglyco-sylated Vero cell–derived HA. Because T cell activation wasalmost completely abrogated upon stimulation with the degly-cosylated V-variant, the removed terminal carbohydrate moie-ties such as b-galactose or high-mannose structures seem to becrucial for the immune stimulatory effect. Overall, deglycosyla-tion had a dramatic effect on T cell activation, thus confirming theessential role of influenza virus HA N-glycosylation for immu-nogenicity.The differential effect of the M- and V-variant on T cell priming

observed in vitro was also relevant in vivo. Consistent with thein vitro results, HA-specific Ab levels were dramatically reduced in

serum of mice immunized with the deglycosylated virus prepa-rations. These findings revealed that HA N-glycosylation is alsocrucial for the initiation of the humoral immune response. Anal-ysis of CD4+ T cell responses by restimulation of splenic T cellswith the HA110–120 peptide also showed that native virus degly-cosylation led to a reduced number of IFN-g–producing CD4+

T cells. In contrast, CD8+ T cell responses were generally verylow (data not shown). Interestingly, 14 d after the prime immu-nization, mice immunized with the M-variant had higher HA-specific Ab levels than mice immunized with the V-variant. HAinhibition activity was still significantly higher upon immuniza-tion with the M-variant on day 28. This might reflect a differentialinfluence of HA N-glycosylation on humoral and cellular im-mune responses. This finding is in accordance with a recent studyshowing that HA carrying complex glycan structures or onlysingle N-acetylglucosamine residues induces higher HAI Ab titersthan does HA carrying high-mannose glycan structures (58).To address T cell proliferation and cytokine production further

in vivo, we adoptively transferred TCR-HA transgenic T cells intowild-type mice and immunized them with the M- or V-variant. Inthis set-up, a higher frequency of transferred TCR-HA transgenicT cells produced IL-2 when mice were immunized with the V-variant. Overall, these findings confirm the in vitro results inthat the glycans of the V-variant led to a faster activation of T cellsin vivo than did the ones of theM-variant. In contrast, theM-variantHA N-glycosylation pattern showed a stronger effect on the hu-moral immune response. The effects of HA N-glycosylation onimmunogenicity in vivo are remarkable, but its impact on vaccinepotency will need to be addressed more in detail in further stud-ies. The focus of this study was to demonstrate the utility of theplatform presented and to provide evidence that HA N-glycosyl-ation potentially affects influenza vaccine efficacy.Without doubt, there is urgent need for new production pro-

cesses of potent influenza vaccines, as the classical influenzavaccine production in embryonated chicken eggs exhibits somedrawbacks such as a long production time, dependence on eggsupply, and the risk of anaphylactic reactions caused by eggproteins. Because production upscale in a timely fashion is a keychallenge of today’s influenza vaccine manufacturing, particularlyduring pandemics, cell culture–based influenza vaccines indeedrepresent an efficient alternative. Our findings have importantimplications for cell-based influenza vaccine design. The platformpresented in this study allows for a rapid and easy screening of N-glycosylation patterns and correlates them with immunogenicity.Thus, appropriate host cell lines can be selected for virus produc-tion that may even be glyco-designed for optimal N-glycosylationpatterns by genetic engineering approaches (59).

AcknowledgmentsWe thank Uwe Vogel and Moctezuma Reimann for expert technical assis-

tance and Prof. Dr. Thomas Schuler from the Institute for Immunology of

the Charite Berlin for fruitful discussions. We also thank Dr. Boris Hundt

from IDT Biologika (Dessau, Germany) for constructive collaboration and

kind support as well as Sandra Meißner and Dr. Ralf Durrwald from IDT

Biologika for performing the HAI assays. We are also very grateful to Dr.

Wiebke Hansen, University Hospital Essen, for providing the TCR-HA

transgenic mice.

DisclosuresThe authors have no financial conflicts of interest.

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