Chao Bian

The PB2 E627K mutation contributes to the high polymerase activity and enhanced replication of H7N9 influenza virus

Human infection by H7N9 influenza virus was first identified in China in March 2013. As of 12 August 2013, a total of 135 documented cases with 44 fatalities had been reported. Genetic and laboratory analyses of the novel H7N9 viruses isolated from patients indicate that these viruses possess several polymerase gene mutations previously associated with human adaptation and potential pandemic capabilities. However, the function of these mutations in the emergence and pathogenicity of the viruses is not well known. In this study, we demonstrate that the PB2 E627K mutation, which occurs in over 70u200a% of the H7N9 patient isolates, promotes the replication of H7N9 virus by enhancing PB2 polymerase activity and enhances virulence in mice. Our results show the PB2 E627K mutation has played an important role in this H7N9 influenza outbreak and in the pathogenicity of the H7N9 virus.

PEDV ORF3 encodes an ion channel protein and regulates virus production

Several studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV‐infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity.

Broad Humoral and Cellular Immunity Elicited by a Bivalent DNA Vaccine Encoding HA and NP Genes from an H5N1 Virus

Influenza A virus is highly variable and a major viral respiratory pathogen that can cause severe illness in humans. Therefore it is important to induce a sufficient immune response specific to current strains and to heterosubtypic viruses with vaccines. In this study, we developed a dual-promoter-based bivalent DNA vaccine that encodes both hemagglutinin (HA) and nucleoprotein (NP) proteins from a highly pathogenic A/Chicken/Henan/12/2004 (H5N1) virus. Our results show that the expression levels of HA and NP genes from the dual-promoter plasmid are similar to those seen when they are expressed individually in independent plasmids. When the bivalent DNA vaccine was inoculated via intramuscular injection and in vivo electroporation, high levels of both humoral and cellular immune responses were elicited against homologous H5N1 virus and heterosubtypic H9N2 virus. Furthermore, no obvious antigenic competition was observed between HA and NP proteins in the dual-promoter-based bivalent vaccine compared to monovalent vaccines. Our data suggest that a combination of influenza surface and internal viral genes in a dual-promoter-expressing plasmid may provide a new approach for developing a DNA vaccine that may protect not only specifically against a currently circulating strain, but also may cross-protect broadly against new heterosubtypic viruses.

Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

n Abstractn n Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.n n

Novel sinomenine derivative 1032 improves immune suppression in experimental autoimmune encephalomyelitis

Sinomenine (SIN) is an alkaloid isolated from the Chinese medicinal plant Sinomenium acutum. It is widely used as an immunosuppressive drug for treating autoimmune diseases. Due to its poor efficiency, the large-dose treatment presents some side effects and limits its further applications. In this study, we used chemical modification to improve the therapeutic effect of SIN in vitro and in vivo. A new derivative of sinomenine, named 1032, demonstrates significantly improved immunosuppressive activity over that of its parent natural compound (SIN). In an experimental autoimmune encephalomyelitis (EAE) model, 1032 significantly reduced encephalitogenic T cell responses and induced amelioration of EAE, which outcome was related to its selective inhibitory effect on the production of IL-17. By contrast, SIN treatment only led to a moderate alleviation of EAE severity and the expression level of IL-17 was not significantly reduced. Furthermore, 1032 exhibited suppression of Th17, but not Treg, cell differentiation, a result probably related to its inhibitory effect on IkappaB-alpha degradation as well as on IL-6 and TNF-alpha secretion in BMDCs. We speculate that 1032 as a novel anti-inflammatory agent may target DC to block IL-6 production, which in turn would terminate Th17 cell development. Thus, SIN derivative 1032 presents considerable potential in new drug development for treating autoimmune and inflammatory disease.

Immune Protection Induced on Day 10 Following Administration of the 2009 A/H1N1 Pandemic Influenza Vaccine

Background The 2009 swine-origin influenza virus (S-OIV) H1N1 pandemic has caused more than 18,000 deaths worldwide. Vaccines against the 2009 A/H1N1 influenza virus are useful for preventing infection and controlling the pandemic. The kinetics of the immune response following vaccination with the 2009 A/H1N1 influenza vaccine need further investigation. Methodology/Principal Findings 58 volunteers were vaccinated with a 2009 A/H1N1 pandemic influenza monovalent split-virus vaccine (15 µg, single-dose). The sera were collected before Day 0 (pre-vaccination) and on Days 3, 5, 10, 14, 21, 30, 45 and 60 post vaccination. Specific antibody responses induced by the vaccination were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). After administration of the 2009 A/H1N1 influenza vaccine, specific and protective antibody response with a major subtype of IgG was sufficiently developed as early as Day 10 (seroprotection rate: 93%). This specific antibody response could maintain for at least 60 days without significant reduction. Antibody response induced by the 2009 A/H1N1 influenza vaccine could not render protection against seasonal H1N1 influenza (seroconversion rate: 3% on Day 21). However, volunteers with higher pre-existing seasonal influenza antibody levels (pre-vaccination HI titer ≥1∶40, Group 1) more easily developed a strong antibody protection effect against the 2009 A/H1N1 influenza vaccine as compared with those showing lower pre-existing seasonal influenza antibody levels (pre-vaccination HI titer <1∶40, Group 2). The titer of the specific antibody against the 2009 A/H1N1 influenza was much higher in Group 1 (geometric mean titer: 146 on Day 21) than that in Group 2 (geometric mean titer: 70 on Day 21). Conclusions/Significance Recipients could gain sufficient protection as early as 10 days after vaccine administration. The protection could last at least 60 days. Individuals with a stronger pre-existing seasonal influenza antibody response may have a relatively higher potential for developing a stronger humoral immune response after vaccination with the 2009 A/H1N1 pandemic influenza vaccine.

Conserved amino acids W423 and N424 in receptor-binding domain of SARS-CoV are potential targets for therapeutic monoclonal antibody.

n Abstractn n The receptor-binding domain (RBD) on spike protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is the main region interacting with the viral receptor-ACE2 and is a useful target for induction of neutralizing antibodies against SARS-CoV infection. Here we generated two monoclonal antibodies (mAbs), targeting RBD, with marked virus neutralizing activity. The mAbs recognize a new conformational epitope which consists of several discontinuous peptides (aa. 343–367, 373–390 and 411–428) and is spatially located neighboring the receptor-binding motif (RPM) region of the RBD. Importantly, W423 and N424 residues are essential for mAb recognition and are highly conserved among 107 different strains of SARS, indicating that the residues are the most critical in the epitope which is a novel potential target for therapeutic mAbs. A human–mouse chimeric antibody, based upon the original murine mAb, was also constructed and shown to possess good neutralizing activity and high affinity.n n

Synthetic peptides derived from SARS coronavirus S protein with diagnostic and therapeutic potential

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS‐CoV) is an important viral structural protein. Based on bioinformatics analysis, 10 antigenic peptides derived from the S protein sequence were selected and synthesized. The antigenicity and immunoreactivity of all the peptides were tested in vivo and in vitro. Four peptides (P6, P8, P9 and P10) which contain B cell epitopes of the S protein were identified, and P8 peptide was confirmed in vivo to have a potential in serological diagnosis. By using a syncytia formation model, we tested the neutralization ability of all 10 peptides and their corresponding antibodies. It is interesting to find that P8 and P9 peptides inhibited syncytia formation, suggesting that the P8 and P9 spanning regions may provide a good target for anti‐SARS‐CoV drug design. Our data suggest that we have identified peptides derived from the S protein of SARS‐CoV, which are useful for SARS treatment and diagnosis.

Copper nano-clusters prepared by one-step electrodeposition and its application on nitrate sensing

This paper describes the fabrication and characterization of copper nano-clusters prepared by a simple one-step electrodeposition process on platinum microelectrode, and the application for nitrate determination. The one-step electrodepostion process was performed by chronoamperometry scan in acidic copper sulphate electrolyte directly. The SEM and electrochemical examination showed that the morphologies and microstructures of deposited copper layers can be precisely controlled by using different deposition voltages. It was found that the copper layer is porous when the deposition voltage is higher than -500 mV, and this porous layer has a larger effective surface area compared with the corresponding smooth flat copper layer deposited under voltage less than -300 mV. Under the optimized deposition voltage, copper clusters constructed by uniform nanoparticles with an average diameter of about 100 nm can be obtained. The mechanism of electrodeposition process for this method was also speculated. The copper layers deposited under different voltages are used in a series of tests in order to evaluate their performance for nitrate sensing. The experimental results reveal that the microelectrode modified by fixed potential deposition under -700 mV had a higher sensitivity of 39.31 μA/mmolL−1 for nitrate detection within the concentration ranging from 0.1 mmolL−1 to 4.0 mmolL−1.

Development of retinol-binding protein 4 immunocolloidal gold fast test strip using high-sensitivity monoclonal antibodies generated by DNA immunization

DNA immunization is an efficient method for high-affinity monoclonal antibody generation. Here, we describe the generation of several high-quality monoclonal antibodies (mAbs) against retinol-binding protein 4 (RBP4), an important marker for kidney abnormality and dysfunction, with a combination method of DNA priming and protein boost. The mAbs generated could bind to RBP4 with high sensitivity and using these mAbs, an immunocolloidal gold fast test strip was constructed. The strip can give a result in <5 min and is very sensitive with a detection limit of about 1 ng/ml. A small-scale clinical test revealed that the result of this strip was well in accordance with that of an enzyme-labeled immunosorbent assay kit currently available on the market. Consequently, it could be useful for more convenient and faster RBP4 determination in the clinic.