Expression of Avian Influenza Virus (H5N1) Hemagglutinin and Matrix Protein 1 in Pichia pastoris and Evaluation of their Immunogenicity in Mice

The conventional avian influenza vaccines rely on development of neutralizing antibodies against the HA and NA antigens. However, these antigens are highly variable, and hence there is a need for better vaccine candidates which would offer broader protection in animals. The M1 of avian influenza is another major structural protein that has conserved epitopes that are reported to induce CD8+ T cells and can contribute to protection against morbidity and mortality from influenza. Hence in an effort to study the immune response of rM1 either alone or in combination with rHA, the hemagglutinin (HA) and matrix protein (M1) of A/Hatay/2004/H5N1 strain of avian influenza were expressed in Pichia pastoris as his-tagged proteins and purified through Ni-NTA chromatography. The His-tag was removed using TEV protease cleavage site and the immunogenicity of purified rHA and rM1 either alone or in combination was determined in mice. One group of mice was immunized with 5 μg of purified rHA, the other group was immunized with rM1, and a third group of mice were immunized with 5 μg of rHA and rM1. All the animals were boosted twice, once on 28 days postimmunization (dpi) and the second on 42 dpi. The immune response was evaluated by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) assay. The group of mice immunized with rHA and rM1 together showed significantly higher immune response against rHA and rM1 than mice immunized with either HA or M1 antigens. The addition of rM1 with rHA resulted in increased HI titer in animals immunized with both the antigens. These results suggest that the HA and M1 expressed in P. pastoris can be utilized in combination for the development of faster and cost-effective vaccines for circulating and newer strains of avian influenza and would aid in combating the disease in a pandemic situation, in which production time matters greatly.     

Catalytic and biochemical features of a monoclonal antibody heavy chain, JN1-2, raised against a synthetic peptide with a hemagglutinin molecule of influenza virus

It has long been an important issue to produce a catalytic antibody that possesses the ability to lose the infectivity of a bacteria or virus. The monoclonal antibody JN1-2 was generated using a synthetic peptide (TGLRNGITNKVNSVIEKAA) conjugated with human IgG. The peptide sequence includes the conserved region of the hemagglutinin molecule (HA(1) and HA(2) domains), which locates on the envelope of the influenza virus and plays an important role in influenza A virus infection. The monoclonal antibody specifically reacted with the HA2 domain, not only of H2 but also of an H1 strain of the H1N1 subtype (H1 strain). The heavy chain (JN1-2-H) isolated from the parent antibody showed catalytic activity cleaving the above antigenic peptide with very high turnover (kcat = 26 min(-1)), and it could slowly degrade the recombinant HA(2) domain by the catalytic function. Interestingly, the heavy chain exhibited the ability to reduce the infectivity of type A H1N1 but not type B, indicating specificity to type A. This characteristic monoclonal catalytic antibody heavy chain could suppress the infection of the influenza virus in vitro assays.     

Multifunctional nanoparticles as simulants for a gravimetric immunoassay

Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and ¹H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au–tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology.     

Downstream processing of Vero cell-derived human influenza A virus (H1N1) grown in serum-free medium

A downstream processing was examined for Vero cell-derived human influenza virus (H1N1) grown in serum free medium. Vero cell banks were established in serum free medium and characterized according to regulatory requirements. Serum free Vero cells were grown on Cytodex 3 microcarriers in 5L bioreactor and infected with influenza A virus (A/New Caledonia/99/55). The harvests were processed with the sequence of inactivation, clarification, anion exchange chromatography (DEAE FF), Cellufine Sulfate Chromatography (CSC) and size exclusion chromatography (Sepharose 6FF). Host cell DNA (hcDNA) was mainly removed with DEAE FF column and CSC by 40 and 223 fold, respectively. Most of Vero cell proteins were eliminated in CSC and Sepharose 6FF unit operation by about 13 fold. The overall scheme resulted in high recovery of hemagglutinin (HA) activity and the substantial removal of total protein, host protein and DNA. The total protein content and DNA content per 15 μg HA protein in final product was 89 μg and 33 pg, respectively, which complied with regulatory requirements for single strain influenza vaccines. SDS-PAGE analysis and Western blotting confirmed the purity of the final product. In conclusion, the suggested downstream process is suitable for the purification of microcarrier-based cell-derived influenza vaccine.     

Expression,Purification, and Characterization of Hepatitis B Virus Surface Antigens (HBsAg) in Yeast <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

Prevention of the prevalence of HB depends upon the development of efficient diagnostic reagent and preventive vaccine. Pichia pastoris offers many advantages over the other expression systems in the production of recombinant HBsAg. In this study, we reported that the recombinant P. pastoris strains were cultured in shake flasks and then scaled up in a 5.0-l bioreactor: approximately 27 mg/l of the protein and the maximal cell OD at 600 nm of 310 were achieved in the bioreactor. The recombinant HBsAg was purified by three steps of purification procedures. SDS-PAGE showed that the purified recombinant HBsAg constituted only one homogeneous band of ~24 kDa. CsCl density gradient ultracentrifugation assay indicated that the density of the HBsAg was 1.2 mg/ml, which was in agreement with the natural HBsAg, the HBsAg expressed in Saccharomyces cerevisiae and in mammalian cells. Electron microscope observation revealed that the purified recombinant HBsAg was homogeneous 22-nm particles, suggesting the HBsAg expressed in P. pastoris was self-assembled to virus-like structures. Competitive ELISA indicated that P. pastoris-derived HBsAg possessed the excellent immunoreaction with anti-HBsAg. Animal immunization showed that the immunogenicity of P. pastoris-derived HBsAg was superior to that of S. cerevisiae-derived HBsAg. Together, our results demonstrated that the recombinant HBsAg expressed in P. pastoris could provide promising, inexpensive, and large-scale materials for the diagnostic reagent and vaccine to prevent HBV infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Rushi Liu and Qinlu Lin contributed equally to this work.     

禽流感病毒HA蛋白与人受体的分子对接

血凝素(hemagglutinin,HA)是位于禽流感病毒表面的糖蛋白。在病毒感染过程中,HA与禽类宿主细胞表面受体结合,介导病毒膜与宿主核内体膜的融合,在传染过程中发挥关键作用。自然界中的禽流感病毒处于不断演化之中,其HA的禽受体结合位点常常发生氨基酸变异。因此,当HA变异体与人受体结合能力较强时,禽流感病毒往往会发生跨种传播而感染人。为预防禽流感的跨种传播,人们迫切需要发展大规模快速检测或预测HA变异体与人受体结合亲和力的方法,以评估各种新发禽流感病毒的跨种传播能力,提前筛选出有潜在危险的病毒株。针对此问题,本研究以H7N9亚型的HA蛋白H7为研究对象,发展了一种运用分子对接的计算方法,预测HA变异体与人受体的结合亲和力。该方法的计算结果表明,H7与人受体的结合亲和力普遍弱于有较强传染人能力的H1,说明H7N9亚型病毒的跨种传播能力普遍较弱;但是,计算分析也揭示,部分新发的H7N9毒株的HA有强的人受体结合亲和力,提示在自然演化过程中,H7N9病毒有可能演化出具有较强的感染人能力的新毒株,这与2013年禽流感疫情的实际发生情况相一致。因此,本文所发展的计算方法可用于快速预测新发禽流感病毒HA与人受体的结合亲和力,为新发禽流感病毒的跨种传播风险评估提供理论依据。     

Development of recombinant protein-based influenza vaccine. Expression and affinity purification of H1N1 influenza virus neuraminidase

The influenza virus surface glycoprotein antigen neuraminidase (NA) is a crucial viral enzyme with many potential medical applications; therefore, the development of efficient upstream and downstream processing strategy for the expression and purification of NA is of high importance. In the present work the NA gene from the H1N1 influenza virus strain A/Beijing/262/95 was cloned from viral RNA and expressed in expresSF+ insect cells using the baculovirus expression vector system (BVES). A limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify the recombinant H1N1 neuraminidase. Affinity-ligand design was based on mimicking the interactions of the lock-and-key (LAK) motif (Phe-Gly-Gln), a common structural moiety found in the subunit interface of glutathione S-transferase I (GST I), and plays an important structural role in subunit-subunit recognition. Solid-phase combinatorial chemistry was used to synthesize 13 variants of the lock-and-key lead ligand (Phe-Trz-X, where X was selected alpha-amino acid) using the 1,3,5-triazine moiety (Trz) as the scaffold for assembly. One immobilized ligand, bearing phenylalanine and isoleucine linked on the chlorotriazine ring (Phe-Trz-Ile), displayed high affinity for NA. Absorption equilibrium and molecular modeling studies were carried out to provide a detailed picture of Phe-Trz-Ile interaction with NA. This LAK-mimetic affinity adsorbent was exploited in the development of a facile purification protocol for NA, which led to 335-fold purification in a single-step. The present purification procedure is the most efficient reported so far for recombinant NA.     

Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in <Emphasis Type="Italic">Bombyx mori</Emphasis> Chrysalides

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.     

Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin

BACKGROUND: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure-function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. RESULTS: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l(-1) from Escherichia coli. Chemical glycosylation with glycosyl-beta-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. CONCLUSIONS: Erythropoietin expressed in E. coli bearing specific Asn-->Cys mutations at natural glycosylation sites can be glycosylated using beta-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins.     

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.     

Synthesis of Four Pentacyclic Triterpene–Sialylglycopeptide Conjugates and Their Affinity Assays with Hemagglutinin

Influenza outbreaks pose a serious threat to human health. Hemagglutinin (HA) is an important target for influenza virus entry inhibitors. In this study, we synthesized four pentacyclic triterpene conjugates with a sialylglycopeptide scaffold through the Cu(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) and prepared affinity assays of these conjugates with two HAs, namely H1N1 (A/WSN/1933) and H5N1 (A/Hong Kong/483/97), respectively. With a dissociation constant (K_D) of 6.89 μM, SCT-Asn-betulinic acid exhibited the strongest affinity with the H1N1 protein. Furthermore, with a K_D value of 9.10 μM, SCT-Asn-oleanolic acid exhibited the strongest affinity with the H5N1 protein. The conjugates considerably enhanced antiviral activity, which indicates that pentacyclic triterpenes can be used as a ligand to improve the anti-influenza ability of the sialylglycopeptide molecule by acting on the HA protein.     

Functional Expression of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Protective Antigen in <Emphasis Type="Italic">E. coli</Emphasis>

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.     

Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

А protocol for the efficient and selective recovery of human ferritin heavy chain (FTH1) expressed intracellularly in Hansenula polymorpha was developed. It was based on electropermeabilisation and an increase in the cell wall porosity by pulsed electric field (PEF) treatment and subsequent incubation with a low concentration of a lytic enzyme. Irreversible plasma membrane permeabilisation was induced by applying rectangular electric pulses in the flow mode. The electrical treatment itself did not cause the release of the recombinant protein but induced the sensitisation of H. polymorpha cells to the lytic enzyme. Consequently, the subsequent incubation of the permeabilised cells with lyticase led to the recovery of approximately 90% of the recombinant protein, with a purification factor of 1.8. A similar efficiency was obtained by using the industrial lytic enzyme Glucanex. The released FTH1 appears in the form of an oligomer with a molecular mass of approximately 480 kDa, which is able to bind iron. The possibility for scaling the proposed protocol is discussed.     

Immunogenicity of Japanese Encephalitis Virus Envelope Protein by Hyphantria cunea Nuclear Polyhedrosis Virus Vector in Guinea Pig

Japanese encephalitis virus (JEV) is an important pathogen causing febrile syndrome, encephalitis, and death. Envelop (E) glycoprotein is the major target of inducing neutralizing antibodies and protective immunity in host. In this study, E glycoprotein of JEV was expressed in Spodoptera frugiperd 9 cells as a fusion protein containing a gX signal sequence of pseudorabies virus. This purified HcE recombinant protein was evaluated for their immunogenicity and protective efficacy in guinea pig. The survival rates of guinea pig immunized with HcE protein was significantly increased over that of JE vaccine. This result indicates helpful information for developing a subunit vaccine against JEV.     

检测禽流感H5亚型病毒的阻抗型免疫研究

研制了一种可用于H5亚型禽流感病毒快速检测的阻抗型免疫传感器。通过蛋白A将H5N1表面抗原血凝素(HA)的单克隆抗体固定于金叉指阵列微电极表面,并与待测溶液中的目标抗原H5N1进行免疫反应。在[Fe(CN)6]3"/4"溶液中进行电化学阻抗谱扫描,表征电极的表面修饰及抗原捕获过程。当H5N1病毒浓度在21～26 HA unit/50μL范围时,其浓度的对数值与叉指阵列微电极的电子传递阻抗的变化值呈线性关系,相关系数为0.9885;检出限为20 HA unit/50μL,检测时间为1 h。此传感器特异性好,灵敏度高,可以重复使用,在病原微生物快速检测领域具有良好的应用前景。     

High Levels of Expression of Fibroblast Growth Factor 21 in Transgenic Tobacco (<Emphasis Type="Italic">Nicotiana benthamiana)</Emphasis>

Fibroblast growth factor-21 (FGF21) is a hepatic hormone that plays a critical role in metabolism, stimulating fatty acid oxidation in the liver and glucose uptake in adipose tissue. In this study, we produced tobacco plants expressing human recombinant FGF21 (hFGF21) via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the human FGF21 gene fused with green florescence protein and a histidine tag. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the hFGF21 gene was correctly translated in tobacco plants. Seven days after agroinfection, the recombinant hFGF21 had accumulated to levels as high as 450 μg g⁻¹ fresh weight in leaves of agroinfected plants. The recombinant hFGF21 was purified from plant tissues by Ni–NTA affinity chromatography, and the purified hFGF21 stimulated glucose uptake in 3T3/L1 cells. This indicated that the recombinant hFGF21 expressed via the PVX viral vector in N. benthamiana was biologically active.     

Differential receptor binding affinities of influenza hemagglutinins on glycan arrays

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.     

Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting‐out

In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine‐tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni‐chelated Sepharose Fast‐Flow affinity column, ammonium sulfate salting‐out and Sephadex G‐75 size‐exclusion column in turn. The purity analysis by SDS–PAGE, high‐performance size‐exclusion and reversed‐phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti‐angiogenic activity under the effective range of 5.0–25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%. Copyright © 2013 John Wiley & Sons, Ltd.     

单管高灵敏度等温扩增技术快速检测甲型H1N1流感病毒

建立了单管逆转录环介导等温扩增法(RT-LAMP)快速检测甲型H1N1流感病毒的方法.针对甲型H1N1流感病毒的M基因和HA基因的保守区,设计了两组特异性引物,分别用于筛选甲型流感病毒及鉴定甲型H1N1流感病毒.对反应体系中的关键因素进行优化,反应结果可直接通过浊度或者SYBR Green Ⅰ荧光进行判定.本方法最低可...