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JOURNAL OF VIROLOGY, July 2008, p. 6200–6208 0022-538X/08/$08.00⫹0 doi:10.1128/JVI.00187-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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Comparative Efficacy of Neutralizing Antibodies Elicited by Recombinant Hemagglutinin Proteins from Avian H5N1 Influenza Virus䌤 Chih-Jen Wei,1† Ling Xu,1† Wing-Pui Kong,1 Wei Shi,1 Kevin Canis,2 James Stevens,3‡ Zhi-Yong Yang,1 Anne Dell,2 Stuart M. Haslam,2 Ian A. Wilson,3 and Gary J. Nabel1* Vaccine Research Center, NIAID, National Institutes of Health, Bldg. 40, Room 4502, MSC-3005, 40 Convent Drive, Bethesda, Maryland 20892-30051; Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, United Kingdom2; and Department of Molecular Biology & Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, BCC206, La Jolla, California 920373 Received 25 January 2008/Accepted 1 April 2008

of H5N1 avian influenza virus are strongly associated with exposure to infected domestic fowl (21). Effective vaccination is a critical tool that supports public health efforts to reduce influenza virus morbidity and mortality. Each year, the World Health Organization selects three influenza virus strains as targets for inactivated vaccine development. While the trivalent inactivated influenza virus vaccines currently used in the United States are manufactured using embryonated eggs, it will be difficult to rapidly scale up this technology for the mass production of vaccine in the event of a potential pandemic (18). Recently, a new cell culturebased approach for influenza virus vaccine development, involving the production of influenza virus in cell culture followed by virus inactivation and purification, has been proposed and tested (1). While offering advantages over egg-based approaches, e.g., cell culture technology can be scaled up in shorter periods of time, cell culture-based approaches for H5N1 manufacture still require the production of a potentially hazardous virus (1). It has been demonstrated that protection provided by the trivalent influenza virus vaccine is mediated primarily by antihemagglutinin (HA) neutralizing antibodies. Thus, a recombinant protein-based approach utilizing purified HA proteins expressed in different mammalian systems offers another alternative for influenza virus vaccine development. This platform provides advantages over current approaches, including welldescribed technologies for mass production and reduced biohazards during manufacturing. Various prototypes produced

Since 1889, at least five influenza virus pandemics have occurred, the most catastrophic of which was the Spanish influenza of 1918, which resulted in 20 to 50 million deaths worldwide (4, 8). Today, an average of about 200,000 influenza virus-related hospitalizations and about 36,000 influenza virusrelated deaths occur in a typical winter-seasonal epidemic in the United States (14). First appearing in 1997, the highly pathogenic avian influenza H5N1 virus continues to spread globally (19). The current global outbreak of H5N1 avian influenza virus among domestic and wild birds, and its potential adaptation to humans, has accelerated influenza H5N1 virus research and pandemic preparedness. More than 300 cases of human H5N1 influenza virus infection had been confirmed. Of these cases, nearly 200 individuals have died as a consequence of infection (22). Although a few instances of human-to-human H5N1 influenza virus transmission have been documented, the current H5N1 virus has not yet acquired the ability to spread efficiently within the human population, and most human cases

* Corresponding author. Mailing address: Vaccine Research Center, NIAID, National Institutes of Health, Bldg. 40, Room 4502, MSC3005, 40 Convent Drive, Bethesda, MD 20892-3005. Phone: (301) 496-1852. Fax: (301) 480-0274. E-mail: [email protected]. † These authors contributed equally to this work. ‡ Present address: Molecular Virology and Vaccines Branch, Influenza Division, NCIRD, CCID, Centers for Disease Control and Prevention, 1600 Clifton Rd., Mail stop G-16, Atlanta, GA 30333. 䌤 Published ahead of print on 16 April 2008. 6200

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Although the human transmission of avian H5N1 virus remains low, the prevalence of this highly pathogenic infection in avian species underscores the need for a preventive vaccine that can be made without eggs. Here, we systematically analyze various forms of recombinant hemagglutinin (HA) protein for their potential efficacy as vaccines. Monomeric, trimeric, and oligomeric H5N1 HA proteins were expressed and purified from either insect or mammalian cells. The immunogenicity of different recombinant HA proteins was evaluated by measuring the neutralizing antibody response. Neutralizing antibodies to H5N1 HA were readily generated in mice immunized with the recombinant HA proteins, but they varied in potency depending on their multimeric nature and cell source. Among the HA proteins, a high-molecular-weight oligomer elicited the strongest antibody response, followed by the trimer; the monomer showed minimal efficacy. The coexpression of another viral surface protein, neuraminidase, did not affect the immunogenicity of the HA oligomer, as expected from the immunogenicity of trimers produced from insect cells. As anticipated, HA expressed in mammalian cells without NA retained the terminal sialic acid residues and failed to bind ␣2,3-linked sialic acid receptors. Taken together, these results suggest that recombinant HA proteins as individual or oligomeric trimers can elicit potent neutralizing antibody responses to avian H5N1 influenza viruses.

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in a baculovirus-insect cell expression system have proven safe and effective in clinical studies for both H1N1 and H3N2 influenza viruses (7, 10, 11, 15–17). In this study, we systematically tested various recombinant HA proteins as alternatives to egg-based vaccine candidates against influenza virus infection. H5N1 HA proteins were expressed and purified from either insect or mammalian cells. The immunogenicity of different recombinant HA proteins was evaluated by antibody neutralization. The data suggest that stable, trimeric viral spikes serve as the optimal protein immunogens to elicit neutralizing antibodies against H5N1 isolates, an approach that may be applicable to seasonal influenza and other viruses. MATERIALS AND METHODS

reader (Wyatt Technology, Santa Barbara, CA). Additionally, mammalian cellexpressed HAs were produced by the cotransfection of a 1/10 ratio (wt/wt) of NA(KAN-1) or NA(New Caledonia/99) expression vector for the glycan array analysis, the mass spectrometry (MS) analysis of HA N-glycan composition, or the NA-coexpressed HA proteins that also were used for immunization. The molecular weights of the HA oligomer, trimer, or monomer proteins were determined by density gradient sedimentation as previously described (9). Vaccination. Female BALB/c mice (6 to 8 weeks old; Jackson Laboratories) were immunized intramuscularly with 20 ␮g of inactivated influenza virus subvirion vaccine [rgA/Vietnam/1203/2004 (H5N1); Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH) or 20 ␮g of purified protein in 50 ␮l of phosphate-buffered saline (PBS) (pH 7.4) and mixed with 50 ␮l of Ribi adjuvant (Sigma, St. Louis, MO) in PBS, pH 7.4, as recommended, at weeks 0 and 3. Blood was collected 14 days after each immunization, and serum was isolated. Animal experiments were conducted in full compliance with all relevant federal regulations and NIH guidelines. ELISA and isotyping of anti-HA antibodies. The mouse anti-HA immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) titers were measured by a previously described method (23). Purified trimeric HA was used to coat the plate, and anti-HA antibodies were detected by peroxidaseconjugated goat anti-mouse IgG and IgM antibody (Jackson ImmunoResearch, West Grove, PA). The subclasses of anti-HA antibodies also were determined by ELISA using antibodies to IgA, IgG1, IgG2a, IgG2b, IgG3, and IgM (Calbiochem, Gibbstown, NJ). Production of pseudotyped lentiviral vectors and measurement of neutralizing antibodies. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described (6, 24). Glycan array analysis. The glycan microarray analysis of the HA proteins was performed as previously described (24). MS of HA N-glycans. The HA N-glycans were prepared for MS analysis as previously described (5). Briefly, purified HA glycoproteins were reduced, carboxymethylated, and digested with L-1-tosylamido-2-phenylmethyl chloromethyl ketone (TPCK) bovine pancreas trypsin (EC 3.4.21.4). The N-glycans were enzymatically released by digestion with PNGase F (EC 3.5.1.52; Roche Molecular Biochemicals) and purified by reverse-phase C18 Sep-Pak (Waters Corp.) chromatography. Prior to MS analyses, the released N-glycans were permethylated and purified using a reverse-phase C18 Sep-Pak (Waters Corp.). Matrixassisted laser desorption ionization–time of flight (MALDI-TOF) data were acquired on a Voyager-DE STR mass spectrometer (PerSeptive Biosystems, Framingham, MA) in the reflectron mode with delayed extraction. Permethylated samples were dissolved in 10 ␮l of 80% (vol/vol) methanol in water, and 1 ␮l of dissolved sample was premixed with 1 ␮l of matrix (10 mg/ml 2,5-dihydroxybenzoic acid [DHB] in 80% [vol/vol] aqueous methanol) before being loaded onto a metal plate. MALDI-TOF/TOF experiments were performed on a 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA) operated in the reflectron positive ion mode.

RESULTS Expression, purification, and characterization of HA proteins. To evaluate the efficacy of recombinant HA proteins as potential vaccine candidates, cDNAs encoding the ectodomain of HA (A/Thailand/KAN-1/2004) were cloned into a baculovirus transfer vector, pAcGP67A, or a mammalian expression vector, CMV/R 8␬B (6), to allow the efficient secretion of HA proteins (Fig. 1A). The multibasic protease cleavage site PQRERRRKKRG between HA1 and HA2 was mutated to PQRETRG to reduce the efficiency of processing. To stabilize the trimeric conformation of HA proteins, a bacteriophage-trimerizing foldon sequence was engineered into the constructs, and a His tag was introduced at the COOH terminus for purification purposes (12). A thrombin cleavage site was inserted between the HA and foldon sequence to ensure the cleavage of the foldon and His tag, if necessary. After the generation of baculovirus vectors expressing HA proteins, the expression of HA proteins was carried out by either infecting Hi5 cells or transfecting 293F cells with mammalian expression vectors. The expression of secreted

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Genes and expression vectors. Based on H3 numbering (20), a cDNA corresponding to residues 11 to 500 of the HA from A/Thailand/KAN-1/2004 (KAN-1; GenBank accession no. AAS65615) was synthesized using human-preferred codons as described previously (6) by using GeneArt (Regensburg, Germany). This construct terminates at the bromelain cleavage site (12). Alternatively, the 14 amino acids (EISGVKLESIGIYQ) between the bromelain cleavage site and the transmembrane domain of HA were inserted to produce the ⌬TM construct. The original viral protease cleavage site PQRERRRKKRG was changed to PQRETRG in order to retain the uncleaved and unprocessed proteins. The purified protein contains additional residues at the C terminus (ISGRLVPRGS PGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH), in which the thrombin cleavage site is in italics, the bacteriophage T4 fibritin foldon trimerization sequence is underlined, and the His tag is in boldface (12). The inserts were cloned into the cytomegalovirus/human T-cell leukemia virus type 1 repetitive sequence (CMV/R) 8␬B expression vector for efficient expression in mammalian cells (6) or into the baculovirus transfer vector pAcGP67A (BD Biosciences, Bedford, MA). Genes for NA(KAN-1)(H5N1) and NA(New Caledonia/99)(H1N1) (GenBank accession nos. AY555150 and AJ518092, respectively) also were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) and were cloned into the expression vector CMV/R 8␬B. Baculovirus production. HA proteins were produced by the cotransfection of baculovirus transfer vector with BaculoGold-linearized baculovirus DNA (BD Biosciences, Bedford, MA) into Spodoptera frugiperda (Sf9) cells (Invitrogen, Carlsbad, CA) using the BaculoGold transfection buffer set (BD Biosciences, Bedford, MA) and subsequently was amplified in the same cells according to the manufacturer’s instructions. Protein expression and purification. Plasmids expressing a secreted HA were transfected into the human embryonic kidney cell line 293F using 293fectin (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. 293F cells were cultured in Freestyle 293 expression medium (Invitrogen, Carlsbad, CA), and supernatant was collected 72 to 96 h posttransfection and cleared by centrifugation and filtration. HA proteins were purified as previously described (6), with minor modifications. Briefly, HA was recovered from the cell supernatant by metal affinity chromatography using Ni Sepharose high-performance resin (GE Healthcare, Piscataway, NJ). Fractions containing HA were combined and subjected to ion-exchange chromatography using a MonoQ HR10/10 column (GE Healthcare, Piscataway, NJ). HA oligomers, trimers, and monomers then were separated by gel filtration chromatography using a Hi-Load 16/60 Superdex 200-pg column (GE Healthcare, Piscataway, NJ). To remove the foldon sequence and His tag, HA proteins were subjected to thrombin digestion (EMD Chemicals, Inc., San Diego, CA) at 3 U/mg at 4°C overnight. Insect-expressed HA proteins (KAN-1) were purified as previously described (12). Trichoplusia ni (Hi5) cells were infected at a multiplicity of infection of 10 and cultured in Express Five serum-free medium (Invitrogen, Carlsbad, CA). The cell culture was maintained at 27°C with gentle shaking. The culture suspension was collected 96 h after infection, and the HA proteins were purified using the same method as that described for mammalian cell-expressed proteins, except that, after using the MonoQ column, HA protein was left overnight at 4°C to precipitate ferritin (12). HA protein from A/Vietnam/1203/2004 (VN1203) was purified as previously described (12). The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using a mouse monoclonal anti-Penta His antibody (Qiagen, Hilden, Germany), mouse monoclonal anti-HA antibody 10D10 (24), or a rabbit polyclonal anti-HA antibody (Immune Technology, New York, NY). Protein purity also was examined by dynamic light scattering using a DynaPro plate

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proteins was first confirmed by Western blotting using antiHis tag or anti-HA antibodies. The secreted HA proteins then were purified using a nickel affinity and MonoQ anionexchange column, followed by using a Superdex200 gel filtration column to separate HA oligomer, trimer, and monomer. In Hi5 cells, HA was expressed as two major species, a high-molecular-weight oligomer and an uncleaved trimer (Fig. 1B). The molecular sizes of insect-expressed HA oligomer and trimer were estimated to be 1,321 and 214 kDa, respectively, as determined by density gradient sedimentation. When the foldon sequence was removed by thrombin digestion, the majority of the HA proteins appeared as a cleaved trimer, and a small fraction of cleaved monomer also was present (Fig. 1B). The expression of insect-ex-

pressed HA also was confirmed by SDS-PAGE (Fig. 1C) and by Western blot analysis using an antibody against HA, and this revealed a slightly lower molecular size after the cleavage of the trimerization motif and His tag (Fig. 1C). Its removal was confirmed by Western blotting using an antiHis tag antibody (Fig. 1C). The mammalian cell-expressed HA also appeared as a high-molecular-mass oligomer and trimer (Fig. 1B) with molecular masses of 1,394 and 222 kDa, respectively, as determined by density gradient sedimentation. In contrast to the insect-produced protein, the peak size of the trimer showed a higher molecular mass due to the more extensive glycosylation in mammalian cells. In addition, unlike its insect-expressed counterpart, the highmolecular-mass oligomer remained intact after thrombin

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FIG. 1. H5 HA expression vectors, biochemical purification, and characterization of insect- and mammalian cell-expressed HA proteins. (A) Vectors encoding H5 HA (A/Thailand/KAN-1/2004) with a mammalian codon preference were prepared in the mammalian expression vector CMV/R 8␬B or the baculovirus transfer vector pAcGP67A. The cleavage site between HA1 and HA2 was mutated in order to obtain full-length proteins. The clones were engineered to contain a trimeric foldon sequence and hexa-His tag (see Materials and Methods) at the COOH terminus, which could be removed by thrombin digestion. These vectors were introduced into the 293F renal epithelial cells or Hi5 cells, as described in Materials and Methods, to generate recombinant protein that was purified in monomeric, trimeric, or oligomeric forms for immunization studies. (B) The HA proteins purified from insect or mammalian cells existed as a high-molecular-mass oligomer (⬎700 kDa) and trimer (uncleaved trimer; ⬎200 kDa) after gel filtration (red line). After thrombin digestion, HA protein eluted as a trimer (cleaved trimer) and a small fraction of monomer (cleaved monomer) also could be detected (black line). The figures present superimposed elution profiles of insect- or mammalian cell-expressed HA proteins overlaid with calibration standards (blue lines). (C) Insect or mammalian cell-derived proteins purified in the oligomer, trimer, and cleaved trimer forms were analyzed by SDS-PAGE (left). Uncleaved trimer and cleaved trimer also were analyzed by Western blotting using a polyclonal anti-HA antibody (middle), and the removal of the foldon domain and His tag from the trimer was confirmed by the decrease in the size of the HA band and by using an anti-His tag antibody (right). HA-F, uncleaved HA with a foldon trimerization domain and His tag.

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cleavage, although trimeric and monomeric species were detected (Fig. 1B). The expression of HA proteins of the expected size also was confirmed by SDS-PAGE and Western blot analysis (Fig. 1C). For subsequent immunogenicity studies, only the peak fractions of each species were collected in 2-ml aliquots. An analysis of these fractions by dynamic light scattering confirmed that each immunogen was of ⬎97% homogeneity (data not shown). Humoral immune responses elicited by insect-expressed recombinant HAs. Humoral immunity in mice was evaluated by vaccination with different forms of HA expressed in insect cells. Mice were immunized with 20 ␮g of oligomers, trimers, or monomers in Ribi adjuvant twice at an interval of 3 weeks. Antisera from the immunized animals were collected 14 days after the second immunization and analyzed for neutralization activity using a previously described HA/neuraminidase (NA)-

pseudotyped, lentiviral vector assay (24). To analyze the ability of insect-expressed HA to induce neutralizing antibodies, HA/ NA-pseudotyped reporters were incubated with antisera from immunized mice, and the neutralizing antibody activity was measured using a luciferase assay. Sera from animals immunized with insect-expressed oligomer and uncleaved trimer inhibited pseudovirus entry effectively, indicating the presence of neutralizing antibodies to H5 HA (Fig. 2A). The thrombindigested trimer elicited only a modest level of neutralizing antibody against KAN-1 HA (Fig. 2A), although a cleaved trimer from a closely related strain (VN1203) elicited levels comparable to those observed with uncleaved trimer (Fig. 2A). These differences likely were related to the relative stability of the cleaved HA trimer of these two strains when mixed with adjuvant. The total HA antibodies were measured by ELISA (Fig. 2D) and used to determine the ratio of neutralizing to

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FIG. 2. Ability of the trimeric HAs from mammalian or insect cells to elicit neutralizing antibodies: higher neutralizing antibody responses were elicited by oligomers than by trimers, and the titers increased with a repeat immunization. (A) The neutralization by antisera from five mice immunized with insect-expressed HAs prepared from insect cells was assessed by the incubation of mouse sera with HA/NA-pseudotyped lentiviral reporter vectors encoding luciferase. The percent neutralization was calculated by the reduction of luciferase activity relative to the values achieved in the absence of sera. Among the insect cell-expressed proteins, oligomer, uncleaved trimer, and cleaved trimer from the VN1203 strain all elicited potent neutralizing antibodies against KAN-1 HA/NA pseudovirus, while the cleaved trimer induced only modest neutralizing activity. (B) Mammalian high-molecular-mass oligomers induced the highest titer of neutralizing antibody response, followed by cleaved trimer, uncleaved trimer, and cleaved monomer. Statistically significant differences were observed between oligomer and trimer (P ⬍ 0.0001), oligomer and cleaved trimer (P ⬍ 0.0001), and oligomer and cleaved monomer (P ⬍ 0.0001) forms. (C) Mice received a single injection of mammalian cell-expressed oligomer or a secondary boost, as indicated, 3 weeks after the initial injection. The neutralizing antibody responses were measured in serum samples collected 14 days after each vaccination using the HA/NA-pseudotyped lentivirus reporter assay. All animals were immunized with 20 ␮g/injection of HA protein in 50 ␮l PBS and an equal volume of Ribi as the adjuvant. (D) Total HA antibodies in mice immunized with insect- or mammalian cell-expressed HA proteins were measured by ELISA. (E) Antisera from mice immunized with insect- or mammalian cell-expressed HA proteins were characterized for IgG1, IgG2a, and IgM responses. Similarly to mice immunized with inactivated H5N1 subvirion vaccine (inact. H5 vaccine), both insect- and mammalian cell-expressed oligomeric and trimeric HA induced the highest levels of IgG2a, followed by IgG1. Naı¨ve mouse serum was used as a negative control. IgG3 and IgA also were tested but were negative for all sera (not shown).

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nary core fucosylated structures. The sialylation of the complex glycans is a predominant feature, with fully sialylated bi-, tri-, and tetra-antennary structures being observed at m/z 2966, 3776, and 4586, respectively. The spectra derived from the N-glycans of HA coexpressed with NA(KAN-1)(H5N1) again showed compositions consistent with those of bi-, tri-, and tetra-antennary core fucosylated structures (m/z 1835 to 3503; Hex3HexNAc4Fuc-NeuAc1Hex7HexNAc6Fuc) (Fig. 4). However, the sialylation of these glycans has been greatly reduced, as clearly observed by a comparison of the core fucosylated tetra-antennary glycans. Without the coexpression of NA, core fucosylated tetra-antennary glycans with 1, 2, 3, and 4 sialic acid residues were observed in abundance at m/z 3503, 3864, 4225, and 4586, respectively (Fig. 4A, Table 1). The coexpression of NA caused a loss of the signals at m/z 3864, 4225, and 4586 and a concurrent increase in the abundance of the nonsialylated core fucosylated tetra-antennary glycans at m/z 3142 (Fig. 4A, Table 1). Similarly, a dramatic reduction in HA N-glycan sialylation was observed by the coexpression of NA (New Caledonia/99)(H1N1) (Fig. 4C, Table 1). Given that the mammalian cell-expressed oligomers elicited the strongest neutralizing antibody response, we tested whether the inclusion of NA during protein expression would affect its immunogenicity. In the construct that terminates at the bromelain cleavage site, the inclusion of NA during protein expression reduced the ability of HA to elicit antibodies that neutralize HA/NA-pseudotyped virus (Fig. 5A). Since these proteins appeared similarly on purification after gel filtration, this difference most likely was due to their stability in the Ribi adjuvant. In particular, the coexpression of NA with the bromelain site HA construct was noted to reduce protein precipitation. In contrast, the inclusion of NA in the ⌬TM construct, which has 14 additional amino acids between the bromelain cleavage site and the transmembrane domain, did not affect the neutralizing antibody response (Fig. 5B), suggesting that the additional sequence stabilized the protein in the presence of adjuvant. The rationale for testing the ⌬TM construct was that it contained more of the HA protein ectodomain and might, therefore, represent a more native protein with additional determinants as an immunogen. However, the ⌬TM construct primarily formed oligomers that lacked trimers, suggesting that the inclusion of the additional amino acids destabilized the trimeric form of the protein. Taking these results together, the coexpression of NA is not required to elicit neutralizing antibodies by transmembrane-deleted, stabilized oligomers of the HA protein, suggesting that this form of HA serves as a preferred immunogen. DISCUSSION Although egg-based vaccines have been used to combat seasonal flu, the lengthy production cycles and limited manufacturing capacity of egg-based vaccines are not conducive to facilitating a rapid response during a potential influenza virus pandemic. Several clinical studies have shown that recombinant HA-based vaccines purified from baculovirus expression systems are safe and effective against H1N1 and H3N2 influenza viruses (7, 10, 11, 15–17). The protein-based approach represents an attractive alternative to egg-based technology,

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total binding antibodies. Among the insect cell-expressed proteins, uncleaved trimer elicited the highest percentage of neutralizing antibodies, followed by oligomer and cleaved trimer. Isotypes of antibodies elicited by insect-expressed oligomer and trimer were mostly IgG1 and IgG2a and were similar whether the immunogen was generated in mammalian or insect cells (Fig. 2E). Comparison of the immunogenicity of mammalian cell-expressed oligomers, trimers, and monomers. Mammalian cellexpressed HA oligomers, trimers, and monomers were analyzed for their ability to elicit neutralizing antibody against H5 HA. Vaccination in mice was performed similarly, and antisera were collected and analyzed for their neutralizing activities. Neutralizing antibody titers were significantly higher for animals immunized with oligomers (Fig. 2B). Cleaved trimers also elicited a neutralizing antibody response, although the titer was lower than that of the oligomers in this lentiviral neutralization assay (Fig. 2B). While uncleaved trimers also induced neutralizing antibody, no detectable antibody responses were found in the animals immunized with cleaved monomers (Fig. 2B). When the ratio of neutralizing to total HA binding antibodies (Fig. 2D) was calculated, antisera from mice immunized with mammalian cell-expressed oligomer showed the highest percentage of neutralizing antibodies, suggesting that these complexes better preserve the physiologic trimeric spike structure. The ratios of neutralizing to total HA binding antibodies in mice immunized with uncleaved and cleaved trimer were ⬃70% lower than those of mice immunized with oligomer. While a single dose of mammalian cell-expressed oligomers elicited only modest levels of neutralizing antibody, as shown by the lentiviral neutralization assay, neutralizing titers against H5 (KAN-1) pseudovirus were enhanced substantially after a secondary boost 3 weeks after the initial injection (Fig. 2C). Like insect-expressed HA, mammalian cell-expressed oligomer and trimer injected with Ribi adjuvant elicited antibodies of IgG1 and IgG2a subclasses that were similar to the antibodies elicited by the inactivated influenza H5N1 subvirion vaccine (Fig. 2E). NA is not required for HA-elicited neutralizing antibody responses. NA has been shown to play a role in viral release from cells (2). This viral enzyme cleaves terminal sialic acid residues from carbohydrate moieties on the surfaces of infected cells and, therefore, promotes the release of progeny viruses (2). NA also cleaves sialic acid residues from HA, thereby preventing the aggregation of viruses (2). HA proteins made with or without NA coexpression behaved differently in the glycan array binding analysis (Fig. 3). HA trimers expressed without NA showed no prominent binding to any of the glycans tested (Fig. 3A). In contrast, mammalian cell-expressed HA trimers that coexpressed NA preferentially bound to ␣2,3-linked sialic acid oligosaccharides (Fig. 3B). The effect of the coexpression of NA on HA glycosylation was investigated by MS analysis (Fig. 4, Table 1). The N-linked glycans from HA (Fig. 4A) and HA coexpressed with NA(KAN-1)(H5N1) (Fig. 4B) or NA(New Caledonia/99)(H1N1) (Fig. 4C) were purified, derivatized, and subjected to MALDITOF (MS). The spectra derived from HA without the coexpression of NA showed predominantly complex type N-glycans (m/z 1835 to 4586; Hex3HexNAc4Fuc-NeuAc4Hex7HexNAc6 Fuc) (Fig. 4), which is consistent with bi-, tri-, and tetra-anten-

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since it uses HA proteins as antigens and does not require the production of potentially dangerous live virus. Also, with this approach, vaccine production is not limited by the supply of eggs and can be easily scaled up in a good-manufacturingpractices facility. Recently, a recombinant HA vaccine against avian H5N1 influenza virus has demonstrated tolerability in humans (16). However, this vaccine only induced protective neutralizing antibody titers in 50% of the subjects receiving the highest dose (two doses of 90 ␮g vaccine). Since it has been previously reported that recombinant HA proteins expressed in insect cells tend to form monomers (13), the suboptimal immunogenicity of this H5 HA vaccine may be due in part to recombinant HA protein not being presented in its native trimeric conformation. In this study, we cloned the ectodomain of HA from an H5N1 virus (KAN-1) and expressed the HA proteins in mammalian or insect cells. HA proteins initially were purified using

a nickel affinity column followed by anion-exchange and gel filtration chromatography. The entire purification process can be completed in 2 to 3 days, and protein production can easily be scaled up. In both Hi5 insect cells and 293F mammalian cells, HA proteins were expressed as high-molecular-weight oligomers and stabilized trimers, demonstrating that the trimerizing foldon sequence indeed prevented the HA from dissociating into monomers. Upon the removal of the foldon sequence by thrombin digestion, only trimers and monomers were present in the insect-expressed proteins, whereas in the mammalian cell-expressed proteins only a small portion of monomer was observed after the removal of the foldon sequence. The discrepancy may be due to the different glycosylation states of proteins derived from insect cells versus proteins produced in mammalian cells, although it is certainly not conclusive. We then evaluated the immunogenicity of these different

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FIG. 3. Glycan array analysis of the specificity of HA trimers expressed with or without NA. Glycan microarray analysis of H5 HA proteins expressed without (A) or with (B) the coexpression of NA. Glycans with related linkages are grouped by color and include selected glycoproteins (orange), predominantly ␣2,3 sialosides (yellow) or ␣2,6 sialosides (green), ␣2,8 ligands (blue), and others (purple). This analysis was performed by the Core H of the Consortium for Functional Genomics at Emory University.

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TABLE 1. Assignments of major molecular ions observed in MALDI spectra of permethylated N-glycans from HA trimers expressed with or without NA (m/z) for: HA/NA (KAN-1)

HA/NA (New Caledonia)

Hex5HexNAc2 Hex3HexNAc4Fuc1 Hex4HexNAc4Fuc1 Hex3HexNAc5Fuc1 Hex5HexNAc4Fuc1 Hex4HexNAc5Fuc1 Hex5HexNAc5Fuc1 Hex5HexNAc4Fuc1NeuAc1 Hex5HexNAc5Fuc1 Hex5HexNAc6Fuc1 Hex5HexNAc5Fuc1NeuAc1 Hex5HexNAc6Fuc1 Hex5HexNAc4Fuc1NeuAc2 Hex6HexNAc5Fuc1NeuAc1 Hex7HexNAc6Fuc1 Hex7HexNAc6Fuc2 Hex7HexNAc7Fuc1 Hex6HexNAc5Fuc1NeuAc2 Hex7HexNAc6Fuc1NeuAc1 Hex7HexNAc6Fuc2NeuAc1 Hex6HexNAc5Fuc1NeuAc3 Hex7HexNAc6Fuc1NeuAc2 Hex7HexNAc6Fuc2NeuAc2 Hex7HexNAc6Fuc1NeuAc3 Hex7HexNAc6Fuc1NeuAc4

1,580 1,835 2,040 2,081 2,244 2,285 2,489 2,605 2,693 2,734 2,851 2,938 2,966 3,054 3,142 3,316 3,387 3,415 3,503 3,677 3,776 3,864 4,038 4,225 4,586

1,580 1,835 2,040 2,081 2,244 2,285 2,489 NDa 2,693 2,734 ND 2,938 ND ND 3,142 3,316 3,387 ND 3,503 ND ND ND ND ND ND

1,580 1,835 2,040 2,081 2,244 2,285 2,489 ND 2,693 2,734 ND 2,938 ND ND 3,142 3,316 3,387 ND 3,503 ND ND ND ND ND ND

a

ND, not determined.

forms of HA derived from either insect or mammalian cells using an HA/NA-pseudotyped lentiviral system (24). In this assay, the neutralization activity can be determined easily by measuring the ability of antisera from mice immunized with recombinant HA proteins to inhibit pseudovirus entry. It has been shown that this pseudotype inhibition assay correlates highly with traditional microneutralization and hemagglutination inhibition assays (6, 24) and can be easily performed in a conventional biosafety level 2 laboratory with biosafety level 3 practices. Among the proteins produced from mammalian cells, high-molecular-weight oligomers elicited the highest titers of neutralizing antibody, followed by the cleaved trimers and uncleaved trimers. Cleaved monomers failed to induce significant neutralizing antibodies against H5N1 virus, even though anti-H5 antibodies were detected by ELISA. This may be due to the preferential induction of antibodies against epitopes present in the monomeric form and not in the trimer, similarly to that observed with human immunodeficiency virus type 1 gp120 monomers and trimers (reviewed in reference 3). It also is possible that the monomeric form is less immunogenic than the trimer/oligomer forms of the same protein. In a separate study, antisera from animals immunized with mammalian

FIG. 5. HA-elicited antibody response is independent of NA coexpression in oligomers from transmembrane-deleted, but not bromelain site-truncated, HA. Neutralizing antibody titers in sera from mice immunized with HA proteins produced with or without the coexpression of NA were examined. (A) In the bromelain site construct, the inclusion of NA [bromelain site oligomer (⫹NA)] reduced HA’s ability to induce neutralizing antibodies compared to that of the bromelain site construct containing no NA [bromelain site oligomer (⫺NA)]. (B) In ⌬TM oligomers, coexpression with NA [oligomer (⌬TM, ⫹NA)] did not alter the immunogenicity of the HA proteins compared to ⌬TM oligomer expression without NA [oligomer (⌬TM, ⫺NA)].

cell-expressed oligomers or cleaved trimers were examined, and their ability to elicit neutralizing antibodies against different H5N1 strain pseudoviruses was similar (unpublished data), though we cannot exclude the possibility of differences in their fine specificity. It should be noted that, although Ribi adjuvant does not contain any denaturants or reducing agents, its effect on the stability and conformation of HA proteins is unknown. We attempted to analyze this effect biochemically but were unable to extract HA proteins from the lipid-rich components of this adjuvant. Although NA plays an essential role in viral replication and infection, the trimming of terminal sialic acid from the HA proteins by NA did not affect the immunogenicity of recombinant HA oligomers. However, the addition of NA did prevent the precipitation of purified protein and facilitated the production of the HA oligomers (data not shown). The removal of terminal sialic acids by NA appeared to be important for the receptor binding of HA. Glycan binding analyses of HA expressed in the insect cell, which lacks sialic acids, have revealed a similar ␣2-3 specificity (12) to the NA-coexpression mammalian HA protein, which bound to ␣2,3-linked sialic acid oligosaccharides. These findings are consistent with the observation that insect-produced, stabilized trimers elicited substantial levels of neutralizing antibodies (Fig. 2A) despite the lack of the sialylation of HA in this cell type. Although previous studies have shown that recombinant HA proteins derived from insect cells elicit immune responses (7, 10, 11, 15–17), our data provide evidence that oligomeric or trimeric HA produced in mammalian cells are comparable or slightly better in eliciting neutralizing antibodies against avian H5N1 virus. Further testing will be required to determine

FIG. 4. MALDI-TOF mass spectra of permethylated N-glycans from HA trimers expressed with or without NA. Shown are profiles of N-glycans from HA (A), HA coexpressed with NA isolated from the KAN-1 strain (B), or HA coexpressed with NA isolated from the New Caledonia (NC) strain (C) from the 50% (vol/vol) acetonitrile fraction from C18 Sep-Paks. All molecular ions are [M ⫹ Na]⫹. Structural assignments are based on monosaccharide composition, MALDI-TOF/TOF (MS/MS) analysis, and knowledge of N-glycan biosynthetic pathways.

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whether other adjuvants, such as alum, QS-21, or MF-59, can improve the immunogenicity of recombinant HA proteins. Not only could the potency of these adjuvants differ but also their effects on the stability of the trimer may vary. These results eventually will require validation with the most active and manufacturable forms in human clinical trials. Nonetheless, our data demonstrate that recombinant mammalian cell- or insect-expressed trimeric HA proteins represent a promising approach to the development of vaccines relevant to seasonal and pandemic influenza virus. ACKNOWLEDGMENTS

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We thank Ati Tislerics and Hamani Henderson for manuscript preparation, Brenda Hartman and Michael Cichanowski for the preparation of figures, and members of the G.J.N. laboratory for helpful advice and discussions. We acknowledge The Consortium for Functional Glycomics, funded by the NIGMS GM62116, and David F. Smith, Emory University School of Medicine, Atlanta, GA, for the glycan array analysis. We thank Jacob Lebowitz for performing the sedimentation equilibrium measurements. Monovalent influenza virus subvirion vaccine, rgA/Vietnam/1203/2004 (H5N1), NR-4143, was obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH. This research was supported by the Intramural Research Program, Vaccine Research Center, NIAID, NIH, and by the Biotechnology and Biological Sciences Research Council (BBSRC) of the Wellcome Trust (A.D. and S.M.H.). A.D. was supported as a BBSRC Professorial Research Fellow.

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