Recent cases of avian influenza H5N1 and the swine-origin 2009 H1N1 have caused a great concern that a global disaster like the 1918 influenza pandemic may occur again. Viral transmission begins with a critical interaction between hemagglutinin (HA) glycoprotein, which is on the viral coat of influenza, and sialic acid (SA) containing glycans, which are on the host cell surface. To elucidate the role of HA glycosylation in this important interaction, various defined HA glycoforms were prepared, and their binding affinity and specificity were studied by using a synthetic SA microarray. Truncation of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. The contribution of each monosaccharide and sulfate group within SA ligand structures to HA binding energy was quantitatively dissected. It was found that the sulfate group adds nearly 100-fold (2.04 kcal/mol) in binding energy to fully glycosylated HA, and so does the biantennary glycan to the monoglycosylated HA glycoform. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. Thus, removal of structurally nonessential glycans on viral surface glycoproteins may be a very effective and general approach for vaccine design against influenza and other human viruses.flu vaccine ͉ glycan binding ͉ glycosylation T he highly pathogenic H5N1 and the 2009 swine-origin influenza A (H1N1) viruses have caused global outbreaks and raised a great concern that further changes in the viruses may occur to bring about a deadly pandemic (1, 2). Important contributions to our understanding of influenza infections have come from the studies on hemagglutinin (HA), a viral coat glycoprotein that binds to specific sialylated glycan receptors in the respiratory tract, allowing the virus to enter the cell (3-6). To cross the species barrier and infect the human population, avian HA must change its receptorbinding preference from a terminally sialylated glycan that contains ␣2,3 (avian)-linked to ␣2,6 (human)-linked sialic acid motifs (7), and this switch could occur through only two mutations, as in the 1918 pandemic (8). Understanding the factors that affect influenza binding to glycan receptors is thus critical for developing methods to control any future crossover influenza strains that have pandemic potential.HA is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem region (3). Both regions carry N-linked oligosaccharides (9), which affect the functional properties of HA (10, 11). Among different subtypes of influenza A viruses, there is extensive variation in the glycosylation sites of the head region, whereas the stem oligosaccharides are more conserved and required for fusion activity (11). Glycans near antigenic peptide epitopes interfere with antibody recognition (12), and glycans near the proteolytic ...

Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.endoglycosidase | Fc glycosylation | glycoengineered antibodies | homogeneous antibodies | sugar oxazoline

Vaccination is the most effective measure at preventing influenza virus infections. However, current seasonal influenza vaccines are only protective against closely matched circulating strains. Even with extensive monitoring and annual reformulation our efforts remain one step behind the rapidly evolving virus, often resulting in mismatches and low vaccine effectiveness. Fortunately, many next-generation influenza vaccines are currently in development, utilizing an array of innovative techniques to shorten production time and increase the breadth of protection. This review summarizes the production methods of current vaccines, recent advances that have been made in influenza vaccine research, and highlights potential challenges that are yet to be overcome. Special emphasis is put on the potential role of glycoengineering in influenza vaccine development, and the advantages of removing the glycan shield on influenza surface antigens to increase vaccine immunogenicity. The potential for future development of these novel influenza vaccine candidates is discussed from an industry perspective.

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HA mg ) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HA mg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HA fg ) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HA mg vaccine activated more diverse B-cell repertoires than the HA fg vaccine and produced antibodies with cross-strain binding ability. In summary, the HA mg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.glycoprotein engineering | broadly neutralizing antibody H

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Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2␣ subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCEThe OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant. Orf virus (ORFV), a member of the Parapoxvirus genus and the Poxviridae family, is the causative agent of contagious ecthyma in sheep, goats, and other ruminants. The disease is characterized by the development of pustular lesions around the nostrils and mouth with a high incidence rate and a low mortality rate in healthy adult animals. In contrast, infection in immunosuppressed animals or in lambs may be fatal (1). ORFV is also of concern as a source of zoonotic infection because it can cause cutaneous lesions in humans in contact with infected animals. Persistent infection with ORFV can be observed in goats and sheep, and while the severity of lesions is reduced compared with that seen in primary infection, this persistence suggests that the virus is able to evade host immunity (2-4). In line with this observation, ORFV has been shown to encode several proteins that modulate the host response to infection. These include viral homologues of ovine cytokines, such as vascular endothelial growth factor, interleukin-10 (IL-10), and a granulocyte-macrophage colony-stimulating factor (GM-CSF)-inhibiting protein, as well as an apoptosis inhibitor (5-7). ORFV also antagonizes interferon (IFN) signaling, and this is done by the product...

Termination detection is a fundamental problem in distributed computing. Many algorithms have been proposed, but only the Chandrasekaran and Venkatesan ( C V ) algorithm [l]is known to be optimal in worst-case message complexity. This optimal algorithm, however, has several undesirable properties. First, it always requires M' + 2 * IEl+ n -1 control messages, whether it is worst case o r best case, where M' is the number of basic messages issued b y the underlying computation after the algorithm starts, IEl is the number of channels in the system, and n is the number of processes. Second, ita worst-case detection delay is O(M'). In a message-intensive computation, that might not be tolerable. Third, the maximum amount of space needed b y each process is O(M'), a quantity not known at compile time, making it necessary t o use the more expensive dynamic memory allocation. Last, it works only for FIFO channels.The purpose of this paper is to remedy these drawbacks, while keeping its strength. W e propose an algorithm that requires M' + 2(n -1) control messages in the word case, but much fewer on the average, and in the best case, it uses only 2(n -1) control messages, no matter how large M' is. The worst-case detection delay is O(n), as compared to the CValgorithm's O(M'); and the space Complexity is e(n) for each process, a compile-time known quantity. The algorithm works equally well for both FIFO and Non-FIFO channels.

BackgroundThe third and fourth heart sound (S3 and S4) are two abnormal heart sound components which are proved to be indicators of heart failure during diastolic period. The combination of using diastolic heart sounds with the standard ECG as a measurement of ventricular dysfunction may improve the noninvasive diagnosis and early detection of myocardial ischemia.MethodsIn this paper, an adaptive method based on time-frequency analysis is proposed to detect the presence of S3 and S4. Heart sound signals during diastolic periods were analyzed with Hilbert-Huang Transform (HHT). A discrete plot of maximal instantaneous frequency and its amplitude was generated and clustered. S3 and S4 were recognized by the clustered points, and performance of the method was further enhanced by period definition and iteration tracking.ResultsUsing the proposed method, S3 and S4 could be detected adaptively in a same method. 90.3% of heart sound cycles with S3 were detected using our method, 9.6% were missed, and 9.6% were false positive. 94% of S4 were detected using our method, 5.5% were missed, and 16% were false positive.ConclusionsThe proposed method is adaptive for detecting low-amplitude and low-frequency S3 and S4 simultaneously compared with previous detection methods, which would be practical in primary care.