Kevin Canis

Comparative Efficacy of Neutralizing Antibodies Elicited by Recombinant Hemagglutinin Proteins from Avian H5N1 Influenza Virus

ABSTRACT 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 α2,3-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.

Comparison of Methods for Profiling O-Glycosylation HUMAN PROTEOME ORGANISATION HUMAN DISEASE GLYCOMICS/PROTEOME INITIATIVE MULTI-INSTITUTIONAL STUDY OF IgA1

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiatives activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Comparison of Methods for Profiling O-glycosylation: HUPO Human Disease Glycomics/Proteome Initiative Multi-Institutional Study of IgA1

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiatives activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

The plasma von Willebrand factor O-glycome comprises a surprising variety of structures including ABH antigens and disialosyl motifs.

Summary.  Background: von Willebrand factor (VWF) is a key component for maintenance of normal hemostasis. Its glycan moieties, accounting for about 20% of its molecular weight, have been shown to affect many of its properties. Previous studies reported correlations between VWF secretion, half‐life and the nature or presence of its N‐glycans, and more importantly between VWF plasma level and the type of N‐linked ABH antigens. Despite the presence of 10 predicted O‐glycosylation sites, the O‐glycome remains poorly characterized, impairing the complete elucidation of its influence on VWF functions. So far only a single glycan structure, a disialyl core 1 glycan, has been identified. Objectives: To define an exhaustive profile of the VWF O‐glycan structures to help the understanding of their role in VWF regulation and properties. Methods: Plasma‐derived VWF O‐linked sugars were isolated and analyzed using state‐of‐the‐art mass spectrometry methodologies. Results and conclusions: We provide here a detailed analysis of the human plasma‐derived VWF O‐glycome. Eighteen O‐glycan structures including both core 1 and core 2 structures are now demonstrated to be present on VWF. Amongst the newly determined structures are unusual tetra‐sialylated core 1 O‐glycans and ABH antigen‐containing core 2 O‐glycans. In conjunction with current models explaining VWF activity, knowledge of the complete O‐glycome will facilitate research aimed at providing a better understanding of the influence of glycosylation on VWF functions.

Mapping the N-glycome of human von Willebrand factor.

vWF (von Willebrand factor) is a key component for maintenance of normal haemostasis, acting as the carrier protein of the coagulant Factor VIII and mediating platelet adhesion at sites of vascular injury. There is ample evidence that vWF glycan moieties are crucial determinants of its expression and function. Of particular clinical interest, ABH antigens influence vWF plasma levels according to the blood group of individuals, although the molecular mechanism underlying this phenomenon remains incompletely understood. The present paper reports analyses of the human plasma vWF N-glycan population using advanced MS. Glycomics analyses revealed approximately 100 distinct N-glycan compositions and identified a variety of structural features, including lactosaminic extensions, ABH antigens and sulfated antennae, as well as bisecting and terminal GlcNAc residues. We estimate that some 300 N-glycan structures are carried by human vWF. Glycoproteomics analyses mapped ten of the consensus sites known to carry N-glycans. Glycan populations were found to be distinct, although many structural features were shared across all sites. Notably, the H antigen is not restricted to particular N-glycosylation sites. Also, the Asn(2635) site, previously designated as unoccupied, was found to be highly glycosylated. The delineation of such varied glycan populations in conjunction with current models explaining vWF activity will facilitate research aimed at providing a better understanding of the influence of glycosylation on vWF function.

Mass Spectrometric Analysis of Mutant Mice

Mass spectrometry (MS) has proven to be the preeminent tool for the rapid, high-sensitivity analysis of the primary structure of glycans derived from diverse biological sources including cells, fluids, secretions, tissues, and organs. These analyses are anchored by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) analysis of permethylated derivatives of glycan pools released from the samples, to produce glycomic mass fingerprints. The application of complimentary techniques, such as chemical and enzymatic digestions, GC-MS linkage analysis, and tandem mass spectrometry (MS/MS) utilizing both electrospray (ES) and MALDI-TOF/TOF, together with bioinformatic tools allows the elucidation of incrementally more detailed structural information from the sample(s) of interest. The mouse as a model organism offers many advantages in the study of human biology, health, and disease; it is a mammal, shares 99% genetic homology with humans and its genome supports targeted mutagenesis in specific genes to produce knockouts efficiently and precisely. Glycomic analyses of tissues and organs from mice genetically deficient in one or more glycosylation gene and comparison with data collected from wild-type samples enables the facile identification of changes and perturbations within the glycome. The Consortium for Functional Glycomics (CFG) has been applying such MS-based glycomic analyses to a range of murine tissues from both wild-type and glycosylation-knockout mice in order to provide a repository of structural data for the glycobiology community. In this chapter, we describe in detail the methodologies used to prepare, derivatize, purify, and analyze glycan pools from mouse organs and tissues by MS. We also present a summary of data produced from the CFG systematic structural analysis of wild-type and knockout mouse tissues, together with a detailed example of a glycomic analysis of the Mgat4a knockout mouse.

Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding

The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120SU plays an essential role in virus binding to target CD4+ T-cells and is a major vaccine target. Gp120 has remarkably high levels of N-linked glycosylation and there is considerable evidence that this “glycan shield” can help protect the virus from antibody-mediated neutralization. In recent years, however, it has become clear that gp120 glycosylation can also be included in the targets of recognition by some of the most potent broadly neutralizing antibodies. Knowing the site-specific glycosylation of gp120 can facilitate the rational design of glycopeptide antigens for HIV vaccine development. While most prior studies have focused on glycan analysis of recombinant forms of gp120, here we report the first systematic glycosylation site analysis of gp120 derived from virions produced by infected T lymphoid cells and show that a single site is exclusively substituted with complex glycans. These results should help guide the design of vaccine immunogens.

Glycoengineering approach to half-life extension of recombinant biotherapeutics.

The potential for protein-engineered biotherapeutics is enormous, but pharmacokinetic modulation is a major challenge. Manipulating pharmacokinetics, biodistribution, and bioavailability of small peptide/protein units such as antibody fragments is a major pharmaceutical ambition, illustrated by the many chemical conjugation and recombinant fusion approaches being developed. We describe a recombinant approach that leads to successful incorporation of polysialic acid, PSA for the first time, onto a therapeutically valuable protein. This was achieved by protein engineering of the PSA carrier domain of NCAM onto single-chain Fv antibody fragments (one directed against noninternalizing carcinoembryonic antigen-CEA and one against internalizing human epidermal growth factor receptor-2-HER2). This created novel polysialylated antibody fragments with desired pharmacokinetics. Production was achieved in human embryonic kidney cells engineered to express human polysialyltransferase, and the recombinant, glycosylated product was successfully fractionated by ion-exchange chromatography. Polysialylation was verified by glycosidase digestion and mass spectrometry, which showed the correct glycan structures and PSA chain length similar to that of native NCAM. Binding was demonstrated by ELISA and surface plasmon resonance and on live cells by flow cytometry and confocal immunofluorescence. Unexpectedly, polysialylation inhibited receptor-mediated endocytosis of the anti-HER2 scFv. Recombinant polysialylation led to an estimated 3-fold increase in hydrodynamic radius, comparable to PEGylation, leading to an almost 30-fold increase in blood half-life and a similar increase in blood exposure. This increase in bioavailability led to a 12-fold increase in tumor uptake by 24 h. In summary, recombinant polysialylation of antibody fragments in our system is a novel and feasible approach applicable for pharmacokinetic modulation, and may have wider applications.

Simian Immunodeficiency Virus from the Sooty Mangabey and Rhesus Macaque Is Modified with O-Linked Carbohydrate

ABSTRACT Although stretches of serine and threonine are sometimes sites for O-linked carbohydrate attachment, specific sequence and structural determinants for O-linked attachment remain ill defined. The gp120 envelope protein of SIVmac239 contains a serine-threonine-rich stretch of amino acids at positions 128 to 139. Here we show that lectin protein from jackfruit seed (jacalin), which binds to non- and monosialylated core 1 O-linked carbohydrate, potently inhibited the replication of SIVmac239. Selection of a jacalin-resistant SIVmac239 variant population resulted in virus with specific substitutions within amino acids 128 to 139. Cloned simian immunodeficiency virus (SIV) variants with substitutions in the 128-to-139 region had infectivities equivalent to, or within 1 log unit of, that of SIVmac239 and were resistant to the inhibitory effects of jacalin. Characterization of the SIVmac239 gp120 O-linked glycome showed the presence of core 1 and core 2 O-linked carbohydrate; a 128-to-139-substituted variant gp120 from jacalin-resistant SIV lacked O-linked carbohydrate. Unlike that of SIVmac239, the replication of HIV-1 strain NL4-3 was resistant to inhibition by jacalin. Purified gp120s from four SIVmac and SIVsm strains bound jacalin strongly in an enzyme-linked immunosorbent assay, while nine different HIV-1 gp120s, two SIVcpz gp120s, and 128-to-139-substituted SIVmac239 gp120 did not bind jacalin. The ability or inability to bind jacalin thus correlated with the presence of the serine-threonine-rich stretch in the SIVmac and SIVsm gp120s and the absence of such stretches in the SIVcpz and HIV-1 gp120s. Consistent with sequence predictions, two HIV-2 gp120s bound jacalin, while one did not. These data demonstrate the presence of non- and monosialylated core 1 O-linked carbohydrate on the gp120s of SIVmac and SIVsm and the lack of these modifications on HIV-1 and SIVcpz gp120s.

Gp120 on HIV-1 Virions Lacks O-Linked Carbohydrate

As HIV-1-encoded envelope protein traverses the secretory pathway, it may be modified with N- and O-linked carbohydrate. When the gp120s of HIV-1 NL4-3, HIV-1 YU2, HIV-1 Bal, HIV-1 JRFL, and HIV-1 JRCSF were expressed as secreted proteins, the threonine at consensus position 499 was found to be O-glycosylated. For SIVmac239, the corresponding threonine was also glycosylated when gp120 was recombinantly expressed. Similarly-positioned, highly-conserved threonines in the influenza A virus H1N1 HA1 and H5N1 HA1 envelope proteins were also found to carry O-glycans when expressed as secreted proteins. In all cases, the threonines were modified predominantly with disialylated core 1 glycans, together with related core 1 and core 2 structures. Secreted HIV-1 gp140 was modified to a lesser extent with mainly monosialylated core 1 O-glycans, suggesting that the ectodomain of the gp41 transmembrane component may limit the accessibility of Thr499 to glycosyltransferases. In striking contrast to these findings, gp120 on purified virions of HIV-1 Bal and SIV CP-MAC lacked any detectable O-glycosylation of the C-terminal threonine. Our results indicate the absence of O-linked carbohydrates on Thr499 as it exists on the surface of virions and suggest caution in the interpretation of analyses of post-translational modifications that utilize recombinant forms of envelope protein.