Robert P. de Vries

Characterization of H7N9 influenza A viruses isolated from humans.

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

A General Strategy for the Chemoenzymatic Synthesis of Asymmetrically Branched N-Glycans

Sweet Variety Proteins fold into a great variety of shapes—but, topologically, they always start as a more or less straight line of linked amino acids. In contrast, carbohydrates manifest a range of structures in which the sugar building blocks connect through multiple branch points. Wang et al. (p. 379, published online 26 July; see the Perspective by Kiessling and Kraft) designed a versatile precursor that could be transformed into many different branched glycans with distinct building blocks along each branch. Oligosaccharides synthesized from a versatile common precursor can be used to probe protein-carbohydrate interactions. [Also see Perspective by Kiessling and Kraft] A systematic, efficient means of producing diverse libraries of asymmetrically branched N-glycans is needed to investigate the specificities and biology of glycan-binding proteins. To that end, we describe a core pentasaccharide that at potential branching positions is modified by orthogonal protecting groups to allow selective attachment of specific saccharide moieties by chemical glycosylation. The appendages were selected so that the antenna of the resulting deprotected compounds could be selectively extended by glycosyltransferases to give libraries of asymmetrical multi-antennary glycans. The power of the methodology was demonstrated by the preparation of a series of complex oligosaccharides that were printed as microarrays and screened for binding to lectins and influenza-virus hemagglutinins, which showed that recognition is modulated by presentation of minimal epitopes in the context of complex N-glycans.

Preferential recognition of avian-like receptors in human influenza A H7N9 viruses.

Avian Affinity for H7N9 Structural analyses of the binding of avian origin H7N9 influenza viruses have revealed how the receptor-binding characteristics differentiate between birds and mammals, and studies involving the use of whole viruses have suggested that the virus is acquiring human-type receptor specificity. In contrast, Xu et al. (p. 1230) show that the H7 hemagglutinin strongly retains its specificity for avian-type receptors by using cocrystal structures with receptor analogs and glycan binding analysis with recombinant hemagglutinin against a library of receptor analogs. Thus, current human H7N9 viruses appear to remain poorly adapted to human receptors, and additional mutations will be required to achieve specificity for human-type receptors equivalent to those of human pandemic viruses. The hemagglutinin of 2013 avian-origin H7N9 influenza virus is poorly adapted for efficient human-to-human transmission. The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu226, a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the H7 hemagglutinin reveals negligible binding to humanlike α2-6–linked receptors and strong preference for a subset of avian-like α2-3–linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission.

Recombinant Soluble, Multimeric HA and NA Exhibit Distinctive Types of Protection against Pandemic Swine-Origin 2009 A(H1N1) Influenza Virus Infection in Ferrets

ABSTRACT The emergence and subsequent swift and global spread of the swine-origin influenza virus A(H1N1) in 2009 once again emphasizes the strong need for effective vaccines that can be developed rapidly and applied safely. With this aim, we produced soluble, multimeric forms of the 2009 A(H1N1) HA (sHA3) and NA (sNA4) surface glycoproteins using a virus-free mammalian expression system and evaluated their efficacy as vaccines in ferrets. Immunization twice with 3.75-μg doses of these antigens elicited strong antibody responses, which were adjuvant dependent. Interestingly, coadministration of both antigens strongly enhanced the HA-specific but not the NA-specific responses. Distinct patterns of protection were observed upon challenge inoculation with the homologous H1N1 virus. Whereas vaccination with sHA3 dramatically reduced virus replication (e.g., by lowering pulmonary titers by about 5 log10 units), immunization with sNA4 markedly decreased the clinical effects of infection, such as body weight loss and lung pathology. Clearly, optimal protection was achieved by the combination of the two antigens. Our observations demonstrate the great vaccine potential of multimeric HA and NA ectodomains, as these can be easily, rapidly, flexibly, and safely produced in high quantities. In particular, our study underscores the underrated importance of NA in influenza vaccination, which we found to profoundly and specifically contribute to protection by HA. Its inclusion in a vaccine is likely to reduce the HA dose required and to broaden the protective immunity.

The influenza A virus hemagglutinin glycosylation state affects receptor-binding specificity

In this study we evaluated the receptor-binding properties of recombinant soluble hemagglutinin (HA) trimers (subtype H2 and H7) produced in insect S2 cells, human HEK293T or HEK293S GnTI(-) cells, which produce proteins with paucimannose, complex or high-mannose N-linked glycans, respectively. The results show that HA proteins that only differ in their glycosylation status possess different receptor fine specificities. HEK293T cell-produced HA displayed a very narrow receptor specificity. However, when treated with neuraminidase this HA was able to bind more glycans with similar specificity as HEK293S GnTI(-) cell-produced HA. Insect cell-produced HA demonstrated decreased receptor specificity. As a consequence, differences in HA fine receptor specificities could not be observed with the insect cell-, but were readily detected with the HEK293S GnTI(-) cell-produced HAs.

A Single Immunization with Soluble Recombinant Trimeric Hemagglutinin Protects Chickens against Highly Pathogenic Avian Influenza Virus H5N1

Background The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. In addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. Effective vaccination against HPAI H5N1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission. Methodology/Principal Findings In the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sH53) produced in mammalian cells. The secreted, purified sH53 was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. It was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with H5N1 after a single vaccination. Protected animals did not shed challenge virus as determined by a quantitative RT-PCR on RNA isolated from trachea and cloaca swabs. Also in mice, vaccination with sH53 provided complete protection against challenge with HPAI H5N1. Conclusions/Significance Our results demonstrate that sH53 constitutes an attractive vaccine antigen for protection of chickens and mammals against HPAI H5N1. As these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses.

Only Two Residues Are Responsible for the Dramatic Difference in Receptor Binding between Swine and New Pandemic H1 Hemagglutinin

In view of its critical role in influenza A virus (IAV) tropism and pathogenesis, we evaluated the receptor binding properties of HA proteins of the closely related swine and new pandemic human IAVs. We generated recombinant soluble trimeric H1 ectodomains of several IAVs and analyzed their sialic acid binding properties using fetuin-binding and glycan array analysis. The results show that closely related swine and new pandemic H1 proteins differ dramatically in their ability to bind these receptors. Although new pandemic H1 protein exhibited hardly any binding, swine H1 bound efficiently to a number of α2–6-linked sialyl glycans. The responsible amino acids were identified by analyzing chimeric H1 proteins and by performing systematic site-directed mutagenesis of swine and new pandemic human H1 proteins. The difference was found to map to residues at positions 200 and 227. Although substitution of either residue significantly affected the binding phenotype, substitution of both was found to act synergistically and reverse the phenotype almost completely. Modeling of the T200A and E227A substitutions into the crystal structure of the new pandemic human H1 protein revealed the loss of potential hydrogen bond formation with Gln191, which is part of the 190-loop of the receptor binding site, and with the penultimate galactose, respectively. Thus, a residue not belonging to the receptor binding site may affect the interaction of HA with its receptor. Interestingly, whereas alanine at position 200 is found in most new pandemic human viruses, the residue at position 227 in these viruses is invariably a glutamic acid.

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

ABSTRACT Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA3) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA3 glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH53) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH53 preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man9GlcNAc2 moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man5GlcNAc2 moieties derived from HEK293S GnTI(−) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(−) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.

Influenza A virus entry into cells lacking sialylated N-glycans

Influenza A virus (IAV) enters host cells after attachment of its hemagglutinin (HA) to surface-exposed sialic acid. Sialylated N-linked glycans have been reported to be essential for IAV entry [Chu VC, Whittaker GR (2004) Proc Natl Acad Sci USA 102:18153–18158], thereby implicating the requirement for proteinaceous receptors in IAV entry. Here we show, using different N-acetylglucosaminyl transferase 1 (GnT1)-deficient cells, that N-linked sialosides can mediate, but are not required for, entry of IAV. Entry into GnT1-deficient cells was fully dependent on sialic acid. Although macropinocytic entry appeared to be affected by the absence of sialylated N-glycans, dynamin-dependent entry was not affected at all. However, binding of HA to GnT1-deficient cells and subsequent entry of IAV were reduced by the presence of serum, which could be reversed by back-transfection of a GnT1-encoding plasmid. The inhibitory effect of serum was significantly increased by inhibition of the viral receptor-destroying enzyme neuraminidase (NA). Our results indicate that decoy receptors on soluble serum factors compete with cell surface receptors for binding to HA in the absence of sialylated N-glycans at the cell surface. This competition is particularly disturbed by the additional presence of NA inhibitors, resulting in strongly reduced IAV entry. Our results indicate that the balance between HA and NA is important not only for virion release, but also for entry into cells.

A Human-Infecting H10N8 Influenza Virus Retains a Strong Preference for Avian-type Receptors

Recent avian-origin H10N8 influenza A viruses that have infected humans pose a potential pandemic threat. Alterations in the viral surface glycoprotein, hemagglutinin (HA), typically are required for influenza A viruses to cross the species barrier for adaptation to a new host, but whether H10N8 contains adaptations supporting human infection remains incompletely understood. We investigated whether H10N8 HA can bind human receptors. Sialoside glycan microarray analysis showed that the H10 HA retains a strong preference for avian receptor analogs and negligible binding to human receptor analogs. Crystal structures of H10 HA with avian and human receptor analogs revealed the basis for preferential recognition of avian-like receptors. Furthermore, introduction of mutations into the H10 receptor-binding site (RBS) known to convert other HA subtypes from avian to human receptor specificity failed to switch preference to human receptors. Collectively, these findings suggest that the current H10N8 human isolates are poorly adapted for efficient human-to-human transmission.