N-glycan analysis by CGE-LIF: profiling influenza A virus hemagglutinin N-glycosylation during vaccine production

Glycoproteins, such as monoclonal antibodies as well as recombinant and viral proteins produced in mammalian cell culture play an important role in manufacturing of many biopharmaceuticals. To ensure consisting quality of the corresponding products, glycosylation profiles have to be tightly controlled, as glycosylation affects important properties of the corresponding proteins, including bioactivity and antigenicity. This study describes the establishment of a method for analyzing N-glycosylation patterns of mammalian cell culture-derived influenza A virus glycoproteins used in vaccine manufacturing. It comprises virus purification directly from cell culture supernatant, protein isolation, deglycosylation, and clean-up steps as well as "fingerprint" analysis of N-glycan pools by CGE-LIF, using a capillary DNA-sequencer. Reproducibility studies of CGE-LIF, virus purification, and sample preparation have been performed. For demonstrating its applicability, the method was exemplarily used for monitoring batch-to-batch reproducibility in vaccine production, with respect to the glycosylation pattern of the membrane protein hemagglutinin of influenza A/PR/8/34 (H1N1) virus. This method allows characterization of variations in protein glycosylation patterns, directly by N-glycan "fingerprint" alignment.     

Increased in vivo immunological potency of HB-110, a novel therapeutic HBV DNA vaccine, by electroporation

Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.     

流感病毒糖蛋白糖链的作用和功能研究

流感病毒是近几年的研究热点之一.糖链在流感病毒生活周期中发挥重要作用,例如宿主细胞表面的唾液酸化糖链是病毒侵染细胞时的特异性受体,宿主决定的病毒糖蛋白糖链结构影响病毒的宿主范围和毒力.本文从糖组学角度综述糖链在甲型流感病毒生活周期中的重要作用,着重阐述病毒血凝素糖基化的影响因素及其对病毒宿主范围、毒力的影响和在病毒演化...     

An integrated microfluidic device for influenza and other genetic analyses

An integrated microfluidic device capable of performing a variety of genetic assays has been developed as a step towards building systems for widespread dissemination. The device integrates fluidic and thermal components such as heaters, temperature sensors, and addressable valves to control two nanoliter reactors in series followed by an electrophoretic separation. This combination of components is suitable for a variety of genetic analyses. As an example, we have successfully identified sequence-specific hemagglutinin A subtype for the A/LA/1/87 strain of influenza virus. The device uses a compact design and mass production technologies, making it an attractive platform for a variety of widely disseminated applications.     

Gold immunochromatographic strips for enhanced detection of Avian influenza and Newcastle disease viruses

A new technique that uses gold immunochromatographic strips enhances the detection sensitivity by inducing the clustering of additional gold nanoparticles (AuNPs) around the immunogold particles immobilized on nitrocellulose strips. The additional AuNPs provide an intense signal that can be detected by the naked eye. The AuNPs were synthesized and conjugated to monoclonal antibodies using self-assembly. Other antibodies were immobilized in a defined detection zone on the nitrocellulose membrane. The detection principle is based on a “sandwich” immunoreaction, where gold-labeled antibodies serve as signal vehicles. To improve the sensitivity of the strips, we use a mixture of 1% HAuCl₄ and 10 mmol L⁻¹ NH₂OH·HCl to “enlarge” the gold nanoparticles. The detecting limits of Avian influenza virus (AIV) and Newcastle disease virus (NDV) are significantly increased. Compared with commercial test strips, this method is 100-fold more sensitive. This method is easy to perform and can be carried out on-site in test laboratories.     

Synthesis and specific influenza A virus‐adsorptive functionality of alkyl curdlan sulfate‐coated membrane filter

To develop a biomaterial with an influenza virus‐adsorptive functionality, an alkyl curdlan sulfate was prepared by ionic interaction between a positively charged didodecyldimethyl ammonium bromide and a negatively charged sulfate group of curdlan sulfate, which has potent anti‐HIV activity, and then it was coated on a membrane filter with a 1‐μm pore size by hydrophobic interaction with the long alkyl groups in the curdlan sulfate. The alkyl curdlan sulfate with the degree of alkylation (DOA) of 0.03 (one didodecyldimethyl group/12 sugar residues with 36 hydroxyl or sulfate groups) showed potent anti‐HIV activity in a 50% effective concentration (EC₅₀) as low as 0.87 μg/mL (standard curdlan sulfate EC₅₀ = 0.3 μg/mL), and the activity decreased with increasing DOA. A DOA higher than 0.1 (one didodecyldimethyl group/three sugar residues with nine hydroxyl or sulfate groups) gave no anti‐HIV activity. Although both curdlan sulfate and alkyl curdlan sulfates did not inhibited infection of Madin‐Darby canine kidney cells by influenza viruses, the alkyl curdlan sulfate‐coated membrane filter was found to have a specific adsorptive functionality for influenza A virus in vitro. When 1.6 mg of the alkyl curdlan sulfate with the DOA of 0.03 was coated on a membrane filter (φ13 mm; pore size, 1 μm), three stacked alkyl curdlan sulfate‐coated membrane filters dramatically decreased hemagglutination to 1/4–1/32. However, the membrane filter did not effectively remove on influenza B viruses, and thus a membrane filter without alkyl curdlan sulfate was not effective against influenza viruses. These results can therefore be presumed to demonstrate that the alkyl curdlan sulfate‐coated membrane filter removed influenza A viruses by adsorption between the negatively charged sulfate groups and the positively charged envelope protein of the virus. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

一枝蒿酮酸酰胺衍生物的合成和抗A3, B型流感病毒和 单纯I, II型疱疹病毒活性研究

从新疆一枝蒿中分离得到了单体化合物一枝蒿酮酸, 然后以一枝蒿酮酸和有机胺为原料, 在偶联剂DCC, HOBt/DMAP的作用下, 合成了13种未见文献报道的一枝蒿酮酸酰胺衍生物2a～2m. 所合成的化合物经过IR, 1H NMR, ESI-MS等分析方法进行了表征, 并对化合物2a～2m进行初步的体外抗A3, B型流感病毒和单纯I, II型疱疹病毒活性研究. 初步试验结果表明化合物2a同时具有抗A3, B型流感病毒活性, 而且抗B型流感病毒活性比母体化合物的活性较高. 化合物2d的抗B型流感病毒活性比母体化合物高16倍, 化合物2e同时具有较强的抗单纯I, II型疱疹病毒活性.     

Recognition for avian influenza virus proteins based on support vector machine and linear discriminant analysis

Total 200 properties related to structural characteristics were employed to represent structures of 400 HA coded proteins of influenza virus as training samples. Some recognition models for HA proteins of avian influenza virus (AIV) were developed using support vector machine (SVM) and linear discriminant analysis (LDA). The results obtained from LDA are as follows: the identification accuracy (Ria) for training samples is 99.8% and Ria by leave one out cross validation is 99.5%. Both Ria of 99.8% for training samples and Ria of 99.3% by leave one out cross validation are obtained using SVM model, respectively. External 200 HA proteins of influenza virus were used to validate the external predictive power of the resulting model. The external Ria for them is 95.5% by LDA and 96.5% by SVM, respectively, which shows that HA proteins of AIVs are preferably recognized by SVM and LDA, and the performances by SVM are superior to those by LDA.     

Silver nanoparticles coated graphene electrochemical sensor for the ultrasensitive analysis of avian influenza virus H7

A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify avian influenza virus H7 (AIV H7) by using silver nanoparticle-graphene (AgNPs-G) as trace labels in clinical immunoassays. The device consists of a gold electrode coated with gold nanoparticle-graphene nanocomposites (AuNPs-G), the gold nanoparticle surface of which can be further modified with H7-monoclonal antibodies (MAbs). The immunoassay was performed with H7-polyclonal antibodies (PAbs) that were attached to the AgNPs-G surface (PAb-AgNPs-G). This method of using PAb-AgNPs-G as detection antibodies shows high signal amplification and exhibits a dynamic working range of 1.6 × 10⁻³∼16 ng/mL, with a low detection limit of 1.6 pg/mL at a signal-to-noise ratio of 3σ. In summary, we showed that this novel immunosensor is highly specific and sensitive to AIV H7, and the established assay could potentially be applied to rapidly detect other pathogenic microorganisms.     

Recognition of sialylated poly-N-acetyllactosamine chains on N- and O-linked glycans by human and avian influenza A virus hemagglutinins

Human influenza viruses are proposed to recognize sialic acids (pink diamonds) on glycans extended with poly-LacNAc chains (LacNAc=(yellow circle+blue square)). N- and O-linked glycans were extended with different poly-LacNAc chains with α2-3- and α2-6-linked sialic acids recognized by human and avian influenza viruses, respectively. The specificity of recombinant hemagglutinins (receptors in green) was investigated by using glycan microarray technology.     

Indium-tin-oxide thin film transistor biosensors for label-free detection of avian influenza virus H5N1

As continuous outbreak of avian influenza (AI) has become a threat to human health, economic development and social stability, it is urgently necessary to detect the highly pathogenic avian influenza H5N1 virus quickly. In this study, we fabricated indium-tin-oxide thin-film transistors (ITO TFTs) on a glass substrate for the detecting of AI H5N1. The ITO TFT is fabricated by a one-shadow-mask process in which a channel layer can be simultaneously self-assembled between ITO source/drain electrodes during magnetron sputtering deposition. Monoclonal anti-H5N1 antibodies specific for AI H5N1 virus were covalently immobilized on the ITO channel by (3-glycidoxypropyl)trimethoxysilane. The introduction of target AI H5N1 virus affected the electronic properties of the ITO TFT, which caused a change in the resultant threshold voltage (V_T) and field-effect mobility. The changes of I_D–V_G curves were consistent with an n-type field effect transistor behavior affected by nearby negatively charged AI H5N1 viruses. The transistor based sensor demonstrated high selectivity and stability for AI H5N1 virus sensing. The sensor showed linear response to AI H5N1 in the concentrations range from 5 × 10⁻⁹ g mL⁻¹ to 5 × 10⁻⁶ g mL⁻¹ with a detection limit of 0.8 × 10⁻¹⁰ g mL⁻¹. Moreover, the ITO TFT biosensors can be repeatedly used through the washing processes. With its excellent electric properties and the potential for mass commercial production, ITO TFTs can be promising candidates for the development of label-free biosensors.     

Interdigitated array microelectrode based impedance immunosensor for detection of avian influenza virus H5N1

Continuous outbreaks of avian influenza (AI) in recent years with increasing threat to animals and human health have warranted the urgent need for rapid detection of pathogenic AI viruses. In this study, an impedance immunosensor based on an interdigitated array (IDA) microelectrode was developed as a new application for sensitive, specific and rapid detection of avian influenza virus H5N1. Polyclonal antibodies against AI virus H5N1 surface antigen HA (Hemagglutinin) were oriented on the gold microelectrode surface through protein A. Target H5N1 viruses were then captured by the immobilized antibody, resulting in a change in the impedance of the IDA microelectrode surface. Red blood cells (RBCs) were used as biolabels for further amplification of the binding reaction of the antibody-antigen (virus). The binding of target AI H5N1 onto the antibody-modified IDA microelectrode surface was further confirmed by atomic force microscopy. The impedance immunosensor could detect the target AI H5N1 virus at a titer higher than 10³ EID₅₀/ml (EID₅₀: 50% Egg Infective Dose) within 2 h. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear range for the titer of H5N1 virus was found between 10³ and 10⁷ EID₅₀/ml. Equivalent circuit analysis indicated that the electron transfer resistance of the redox probe [Fe(CN)₆]^3−/4− and the double layer capacitance were responsible for the impedance change due to the protein A modification, antibody immobilization, BSA (bovine serum albumin) blocking, H5N1 viruses binding and RBCs amplification. No significant interference was observed from non-target RNA viruses such as Newcastle disease virus and Infectious Bronchitis disease virus. (The H5N1 used in the study was inactivated virus.)     

Three‐dimensional printed magnetophoretic system for the continuous flow separation of avian influenza H5N1 viruses

As a result of the low concentration of avian influenza viruses in samples for routine screening, the separation and concentration of these viruses are vital for their sensitive detection. We present a novel three‐dimensional printed magnetophoretic system for the continuous flow separation of the viruses using aptamer‐modified magnetic nanoparticles, a magnetophoretic chip, a magnetic field, and a fluidic controller. The magnetic field was designed based on finite element magnetic simulation and developed using neodymium magnets with a maximum intensity of 0.65 T and a gradient of 32 T/m for dragging the nanoparticle–virus complexes. The magnetophoretic chip was designed by SOLIDWORKS and fabricated by a three‐dimensional printer with a magnetophoretic channel for the continuous flow separation of the viruses using phosphate‐buffered saline as carrier flow. The fluidic controller was developed using a microcontroller and peristaltic pumps to inject the carrier flow and the viruses. The trajectory of the virus–nanoparticle complexes was simulated using COMSOL for optimization of the carrier flow and the magnetic field, respectively. The results showed that the H5N1 viruses could be captured, separated, and concentrated using the proposed magnetophoretic system with the separation efficiency up to 88% in a continuous flow separation time of 2 min for a sample volume of 200 μL.