Search

T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies., Graphical abstract Here we developed one reporter gene assay comprising two cell lines, CHO-CD112-CD3 scFv and Jurkat-NFAT-TIGIT, which can be adopted in the bioactivity determination of anti-TIGIT antibody with high accuracy and specificity.Image 1

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV). SFTS is mainly characterized by severe fever with thrombocytopenia and has a high mortality rate. The virus has been found in China, South Korea, and Japan. Effective antiviral drugs or vaccines still have been unavailable. Now, two vaccine manufacturers in China are actively engaged in the development of the vaccine. To promote the development of SFTS vaccines and ensure their effective quality control, we developed national antigen and antibody references. We collaborative calibrated the standards; evaluated the homogeneity and stability of the national SFTS standards. The national SFTS vaccine antigen and antibody references met the Chinese national standards and can be used to standardize quality control for the manufacture of SFTS vaccines. And also can be used into the study the dose-response relationship of SFTS vaccines, determine clinical doses, and evaluate vaccine immunogenicity.

The high global burden of tuberculosis (TB) underscores the urgent need for an effective TB vaccine since the only licensed Bacillus Calmette-Guérin (BCG) vaccine is ineffective in preventing adult pulmonary TB and affords no protection against latent TB infection (LTBI). Herein we investigated the potential of Mycobacterium tuberculosis (Mtb) antigen proteins AEC comprised of Ag85b and ESAT6-CFP10 proteins in conjunction with aluminum (Al) and polyriboinosinic-polyribocytidylic acid (poly-IC) as a novel subunit vaccine against TB. The immunogenicity and protection induced by the adjuvanted vaccine were evaluated in two animal models. Mice vaccinated with AEC/Al/poly-IC exhibited significant antigen-specific humoral immune responses and cell-mediated immunity as determined by immunoassay and multicolor flow cytometric assay, and the protective effect of the vaccine was demonstrated in a guinea pig model of latent Mtb infection. Compared to the control group, the mean pathological scores and bacterial loads in lungs and spleens of AEC/Al/poly-IC-immunized guinea pigs were significantly reduced. These data indicate that the AEC/Al/poly-IC is highly immunogenic in mice and can effectively protect guinea pigs against latent Mtb infection; it may represent a promising candidate vaccine for the control of latent TB.

Sludge gasification for the production of hydrogen-rich gas is a promising technology. In this paper, a pilot study on the hydrogen-rich gas production by sludge gasification using waste heat of blast furnace slag was carried out, and the mass and energy balance of gasification process using waste heat from blast furnace slag were evaluated. The results show that the higher the gasification temperature, the higher the hydrogen content in the gas. When the gasification temperature reaches 880 °C, the hydrogen content in the gas reaches the maximum,35.3%. The technology of sludge gasification combined with waste heat recovery of high furnace slag is feasible. Its efficiency of heat recovery can reach up to 64.35%, and the gasification efficiency and energy consumption ratio can reach to 42.30% and 3.67, respectively.

Adoptive cell therapy using chimeric antigen receptor T (CAR-T) cells, which is a promising cancer immunotherapy strategy, has been developing very rapidly in recent years. CAR-T cells are genetically modified T cells that can specifically recognize tumor specific antigens on the surface of tumor cells, and then effectively kill tumor cells. At present, exciting results are being achieved in clinical applications of CAR-T cells for patients with hematological malignancies. The research and development of CAR-T cells for various targets and for the treatment of solid tumors have become a hot topic worldwide, so an increasing number of investigational new drug applications (INDAs) and new drug applications (NDAs) of CAR-T cell products are expected to be submitted in future. The quality control and nonclinical research of these products are of great significance in ensuring the safety and effectiveness of these products; however, they also present great challenges and difficulties. This article discusses the general principles of and key issues regarding the quality control and nonclinical research of CAR-T cell products based on their product characteristics and on relevant guidelines for gene and cell therapy products. Keywords: Chimeric antigen receptor T cells, Quality control, Nonclinical research, Safety, Efficacy, Clinical trials, Cancer immunotherapy

Biocatalysis has become the main approach to produce cytidine diphosphate choline (CDP-choline), which has been applied for treatment of acute craniocerebral injury and consciousness after brain surgery. However, salt accumulates with the production and inhibits enzyme activity, and eventually reduces yield and product accumulation rate. Our work provided a possible solution to this problem by applying a computational designed halophilic choline kinase. The halotolerant CKI (choline kinase) was designed following a unique strategy considering the most variable residue positions on the protein surface among target enzymes from different sources. The basic and neutral surface residues were replaced with acidic ones. This approach was enlightened by features of natural halophilic enzymes. Mutants in the work represented higher catalytic activities and IC50 (inhibit activity by 50%) at high salt concentrations (over 1200 mM). Furthermore, when the mutant was used in fed-batch production, the CDP-choline accumulation rate doubled comparing with process using wild-type CKI at acetate concentration of over 700 mM. The maximum titer was 151 ± 3.2 mM, the productivity was 5.8 ± 0.1 mM·L−1 h−1, and molar yield to CMP and utilization efficiency of energy were 85.3 and 63.5%. The idea of computational design in our work can also be applied to modify other enzymes in industry, and sheds light on alleviating effect of salt accumulation during industrial manufacturing process.

Long-term and excessive herbicide use has led to some environmental concerns and especially, herbicide resistance evolution in weeds. Here, we confirmed acetolactate synthase (ALS) inhibiting herbicide penoxsulam resistance and cross resistance to acetyl-coenzyme carboxylase (ACCase) inhibiting herbicides (cyhalofop-butyl and metamifop) in a global weed Echinochloa crus-galli population resistant to these herbicides (R). Penoxsulam metabolism study indicated that degradation rate was significantly higher in R than susceptible E. crus-galli population (S). RNA-sequencing revealed that a cytochrome P450 (P450) gene, CYP81A68, expressed higher in R versus S. Rice seedlings overexpressing this CYP81A68 gene are resistant to penoxsulam, cyhalofop-butyl and metamifop, and penoxsulam resistance is due to enhanced metabolism via O-demethylation. Deletion analysis of the CYP81A68 gene promoter identified an efficient region, in which differential methylation of CpG islands occurred between R and S. Collectively, these results demonstrate that upregulation of E. crus-galli CYP81A68 gene endows generalist metabolic resistance to commonly used ALS- and ACCase-inhibiting herbicides in rice fields and epigenetic regulation may play a role in the resistance evolution. This research could contribute to strategies reducing herbicide environmental impacts by judicious selection of alternative herbicide and non-chemical control tactics.

Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide (rhBNP). In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGlo-G1 was constructed by transfecting pGloSensor™ 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGlo-G1 showed high precision with intra-assay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay (paired t test, p = 0.630). All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP. Keywords: RhBNP, cGMP, GloSensor technology, Reporter assay

The management of hand-foot-and-mouth disease(HFMD) epidemic is difficult due to the frequent emergence of non-EV71 and non-CVA16 enteroviruses and some cases testing negative for HFMD-associated causative agents. To clarify the virus spectrum of mild and severe HFMD, a comprehensive virome analysis of 238 samples was performed using next-generation sequencing (NGS). The data revealed total thirteen mammalian- and plant- virus families and diverse viral populations including enteroviruses, common respiratory viruses, diarrhea-related viruses, plant viruses and anelloviruses. A total of 18 viruses from 7 virus families were identified in severe cases, versus 37 viruses from 12 virus families in mild cases. Moreover, complicated mixed-infections of enteroviruses with common respiratory viruses were mainly found in severe cases(P = 0.013), while diarrhea-related viruses were mainly found in mild cases(P 0.001). This study provides the preliminary understanding of viromes both in mild and severe cases, which may benefit the detection of etiologic agents and prevention of HFMD.

Eosinophilic asthma is characterized by the eosinophilic inflammation with the allergen independent activation of Th2 lymphocytes. Since IL-5 plays an important role in the maturation, survival and migration of eosinophils, hence the pathogenesis of eosinophilic asthma, biotherapeutics targeting IL-5/IL-5Rα have been developed and/or marketed, including Mepolizumab, Reslizumab, and Benralizumab. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current mode of action (MOA) based method used in the quality control and stability tests for anti-IL-5 mAbs is anti-proliferation assay, which is tedious with long duration and high variation. We describe here the development and validation of a reporter gene assay (RGA), based on an IL-5-dependent TF-1 cell line variant we established that stably expresses the luciferase reporter under the control of STAT5 response elements. After careful optimization, we demonstrate the excellent specificity, precision, accuracy and linearity of the established RGA. Our study also proves that the assay is superior on precision, sensitivity and assay simplicity to the anti-proliferation assay. The established RGA is also applicable to another anti-IL-5Rα mAb. These results show for the first time that this novel RGA, based on the IL-5-IL-5R-STAT5 pathway, can be a valuable supplement to the anti-proliferation assay and employed in the bioactivity determination of anti-IL-5/anti-IL-5Rα biotherapeutics.

More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FceRIγ effector cell line that stably expresses CD32a-FceRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FceRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FceRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FceRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.

Seasonal inactivated quadrivalent influenza vaccines are currently formulated to include antigens from two strains of influenza A and a strain from each of the two circulating influenza B virus lineages. However, the applicability of the potency assay currently required for the release of vaccines has been hindered due to cross-reactivity between the two B strains. In this study, a reversed-phase high-performance liquid chromatography method previously developed for the separation and quantitative determination of the hemagglutinin content in trivalent influenza vaccine preparations was further extended and found to be adaptable for the assessment of all four hemagglutinin antigens present in quadrivalent influenza vaccines. Vaccines prepared from monovalent bulks and commercial quadrivalent products from the past three vaccination seasons in the Northern Hemisphere were tested with the new method. The results showed excellent resolution of all four hemagglutinins from frequently interfering formulation agents such as surfactants. This method provides a simple approach for fast evaluation of quality and hemagglutinin strain identification in influenza vaccines. It is also the only physicochemical method capable of distinguishing the B strains in quadrivalent influenza vaccines.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by a phlebovirus of the Bunyaviridae family, which is designated as SFTS virus (SFTSV). To our knowledge, no efficient SFTSV vaccine exists. Here, we report the identification of a standard virus strain for the eight major SFTSV strains circulating in China for use in evaluating the SFTSV vaccine. Rabbits were immunized with the SFTSV strains and the cross-neutralization capacities of SFTSV anti-sera were determined in microculture cytopathic effect (CPE)-inhibition assays. The mean cross-neutralization capacity of the eight SFTSV anti-sera ranged from 62.4 to 142.6%, compared to autologous strains. The HB29 strain demonstrated strong cross-reactivity with heterologous antibodies, and 33 serum samples from SFTS patients efficiently neutralized HB29, suggesting its broad cross-reactivity. In addition, HB29 demonstrated good replication in Vero and MRC-5 cells (8.0 and 6.0 lg 50% cell culture-infectious dose/mL, respectively) and significant CPE, which satisfied the requirements for a standard virus strain. The HB29 isolate was proven identical to the reported HB29 strain by DNA sequencing, and showed high homology in the S segments with other SFTSV strains (94.8–99.7%). Our results suggest that HB29 may be the best candidate standard strain for use in SFTS vaccine development in China.

Development of anti-VEGF based biologic agents has been a focus in cancer treatment for the past decades, and several anti-VEGF pharmaceuticals have been already approved for treatment of various medical indications especially in cancer. The first anti-angiogenic agent approved by FDA was bevacizumab (BVZ, trade name Avastin, Genentech/Roche), a humanized anti-VEGF monoclonal antibody. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current method widely used in the lot release and stability test for clinical trial batches of BVZ is anti-proliferation assay using primary human umbilical vein endothelial cells (HUVEC), which is tedious with high assay variations. We describe here the development and preliminary validation of a reporter gene assay (RGA) that is based on an HEK293 cell line stably expressing vascular endothelial growth factor receptor 2 (VEGFR-2), and a luciferase reporter under the control of nuclear factor activated T cell (NFAT) response elements. Our study shows this assay not only to be superior on precision, sensitivity and assay simplicity compared with HUVEC assay, but also applicable to other VEGF-targeted biotherapeutics. These results show for the first time that this new reporter assay, based on the VEGF-VEGFR-NFAT pathway, can be a viable supplement to the HUVEC assay and employed in potency determination of BVZ and other kinds of anti-VEGF antibody-based biotherapeutics.

Complex structure and structure-function relationship of biopharmaceuticals require extensive analytical characterization and appropriate quality control of the products. Despite rapid development of sophisticated physicochemical techniques, biological activity measurement remains the critical role in inferring the high-order structure of biopharmaceuticals. Cell-based biological assays are mostly applied to determine the biological activity of biopharmaceuticals, however, refined biological assays are continually needed to increase their robustness. Reporter gene assays (RGAs) which are mechanism of action (MOA) related, less variable, accurate, precise, and labor-saving are becoming more and more recognized and adopted in the quality control. Here we discuss the importance of bioactivity determination, the strength and weakness of various assay formats with RGAs. We also introduce the mechanism of RGAs, and present a number of examples for RGAs to determine the bioactivity of various biopharmaceuticals, which indicate their extensive use in the screening, characterization, quality control, stability and biosimilarity study. We believe that with the rapid development of biotechnology, new strategies of bioassays based on RGAs will be more widely applied in various fields of biopharmaceuticals.

Up to now, all tested Ebola virus vaccines have been based on the virus strain from the Zaire outbreak in 1976. We aimed to assess the safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine expressing the glycoprotein of the 2014 epidemic strain.We did this randomised, double-blind, placebo-controlled, phase 1 clinical trial at one site in Taizhou County, Jiangsu Province, China. Healthy adults (aged 18-60 years) were sequentially enrolled and randomly assigned (2:1), by computer-generated block randomisation (block size of six), to receive placebo, low-dose adenovirus type-5 vector-based Ebola vaccine, or high-dose vaccine. Randomisation was pre-stratified by dose group. All participants, investigators, and laboratory staff were masked to treatment allocation. The primary safety endpoint was occurrence of solicited adverse reactions within 7 days of vaccination. The primary immunogenicity endpoints were glycoprotein-specific antibody titres and T-cell responses at day 28 after the vaccination. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, number NCT02326194.Between Dec 28, 2014, and Jan 9, 2015, 120 participants were enrolled and randomly assigned to receive placebo (n=40), low-dose vaccine (n=40), or high-dose vaccine. Participants were followed up for 28 days. Overall, 82 (68%) participants reported at least one solicited adverse reaction within 7 days of vaccination (n=19 in the placebo group vs n=27 in the low-dose group vs n=36 in the high-dose group; p=0·0002). The most common reaction was mild pain at the injection site, which was reported in eight (20%) participants in the placebo group, 14 (35%) participants in the low-dose group, and 29 (73%) participants in the high-dose vaccine group (p0·0001). We recorded no statistical differences in other adverse reactions and laboratory tests across groups. Glycoprotein-specific antibody titres were significantly increased in participants in the low-dose and high-dose vaccine groups at both day 14 (geometric mean titre 421·4 [95% CI 249·7-711·3] and 820·5 [598·9-1124·0], respectively; p0·0001) and day 28 (682·7 [424·3-1098·5] and 1305·7 [970·1-1757·2], respectively; p0·0001). T-cell responses peaked at day 14 at a median of 465·0 spot-forming cells (IQR 180·0-1202·5) in participants in the low-dose group and 765·0 cells (400·0-1460·0) in those in the high-dose group. 21 (18%) participants had mild fever (n=9 in the placebo group, n=6 in the low-dose group, and n=6 in the high-dose group). No serious adverse events were recorded.Our findings show that the high-dose vaccine is safe and robustly immunogenic. One shot of the high-dose vaccine could mount glycoprotein-specific humoral and T-cell response against Ebola virus in 14 days.China National Science and Technology, Beijing Institute of Biotechnology, and Tianjin CanSino Biotechnology.

Hispolon was the main antitumor active ingredient in Phellinus sensu lato species. In order to confirm the dual regulating estrogenic ingredient and obtain more effective natural estrogen replacement drugs, hispolon was separated from Phellinus lonicerinus (Bond.) Bond. et sing. Hispolon exhibited significant anti-proliferative effect against estrogen-sensitive ER (+) MCF-7 cells in the absence of estrogen, and exhibits antagonistic effects on 17β-estradiol (E2)-induced MCF-7 cell proliferation when E2 and the different concentrations of hispolon were treated simultaneously. Hispolon also inhibited the proliferation of estrogen-negative ER (−) MDA-MB-231 cells at the concentration of 5.00 × 10− 5 M. The yeast two-hybrid experiments showed that hispolon had strong and non-selective effects on the estrogen receptor (ER) α and ERβ at a concentration of 1.00 × 10− 6 M. The ERβ-binding ability of hispolon was larger than ERα in the concentration range of 1.00 × 10− 9 M and 1.00 × 10− 7 M. Hispolon could increase the body weight coefficient, serum E2 and progesterone contents in immature female mice at dose of 9.10 × 10− 6 mol/kg, and increase coefficient of thymus and spleen in mice. The Gscores of hispolon-ERα and hispolon-ERβ docked complexes were − 7.93 kcal/mol and − 7.79 kcal/mol in docking simulations. Hispolon presented dual regulating estrogenic activities, which showed estrogenic agonist activity at low concentration or lack of endogenous estrogen, and the estrogenic antagonistic effect was stimulated at high concentrations or too much endogenous estrogen. Hispolon could be used for treating the estrogen deficiency-related disease with the benefit of non-toxic to normal cells, good antitumor effects and estrogenic activity.

The detection of variants is one of the important aspects in quality control of recombinant DNA drugs. In this study, a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection is described for the separation of interferon alpha-2a (rhIFN α-2a) from several product related variants. The methodology employed a core-shell C18 column with a linear gradient elution of 0.2% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 1.0mL/min, and the temperature of the column was maintained at 60°C. The method was validated in terms of linearity, sensitivity, intra- and inter-day variations. Compared to the European Pharmacopeia RP-HPLC method of rhIFN α-2a analysis, this new method can separate N-methionylated variant in both drug substance and finished product, and analyze the variants in untreated, oxidized sample and slightly degraded samples more efficiently. In conclusion the method has an improved capability to detect variants in rhIFN α-2a products.

All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.

Trametenolic acid B (TAB), the bioactive component in the Trametes lactinea (Berk.) Pat, was reported to possess cytotoxic activities and thrombin inhibiting effects. This study was performed to investigate the effects of TAB on H(+)/K(+)-ATPase and gastric cancer. The H(+)/K(+)-ATPase inhibitory activity was determined by gastric parietal cells. Compared to the normal control group, TAB (10, 20, 40 and 80 μg/mL) inhibited the H(+)/K(+)-ATPase activity by 15.97, 16.96, 24.86 and 16.25%, respectively. In the study, 36 Kunming mice were randomly divided into six groups: control, model, TAB-L (TAB, 5 mg/kg/day, i.g.), TAB-M (TAB, 20 mg/kg/day, i.g.), TAB-H (TAB, 40 mg/kg/day, i.g.) and omeprazole (OL, 10 mg/kg/day, i.g.). All mice except the control group were administrated with anhydrous alcohol (5.0 mL/kg, i.g.) for induced gastric-ulcer 1h after the 5th day. At the same time, the control mice were given the same volume of physiological saline. After 4h, TAB was evaluated for H(+)/K(+)-ATPase inhibitory activities of ulcerative gaster, gastric ulcer index and ulcer inhibition. In vitro, the anti-proliferation effect of TAB to gastric cancer cell (HGC-27) in acid environment was detected by MTT, and the apoptosis morphological changes were also observed by Hoechst 33258 dye assay. The results indicated that TAB inhibited moderately H(+)/K(+)-ATPase activity in vitro. Compared to the model group, TAB showed anti-ulcer effects in gastric tissue with the dosages of 20 and 5 mg/kg in vivo. Apart from that, TAB could selectively inhibit gastric cancer cell viability and reduce cell apoptosis against HGC-27 cells at low doses in acid environment.

The active components of most meningococcal vaccines are four antigenic serogroup capsular polysaccharides (A, C, Y, W135). The vaccines, monovalent or multivalent mixtures of either free polysaccharides or polysaccharides conjugated to antigenic carrier proteins, may be in liquid or lyophilised formulations, with or without excipients. Acid hydrolysis and chromatographic methods for serogroup quantitation, which were previously optimised and qualified using polysaccharide-based standards and a narrow range of real vaccines, are here challenged with multiple lots of a broad assortment of additional multivalent polysaccharide-based meningococcal vaccine products. Centrifugal filtration successfully removed all interfering lactose excipient without loss of polysaccharides to allow for the determination of Y and W135 serogroups. Replicate operations by three different analysts indicated high method reproducibility. Results indicated some lot-to-lot and product-to-product variations. However, all vaccines were within general specifications for each serogroup polysaccharide, with the exception of all lots of one polysaccharide vaccine - which by these methods were found to be deficient in the serogroup A component only. These robust techniques are very useful for the evaluation of antigen content and consistency of manufacture. The deformulation, hydrolysis and chromatographic methods may be adaptable for the evaluation of other types of polysaccharide-based vaccines.

The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.

The Decade of Vaccines Collaboration and development of the Global Vaccine Action Plan provides a catalyst and unique opportunity for regulators worldwide to develop and propose a global regulatory science agenda for vaccines. Regulatory oversight is critical to allow access to vaccines that are safe, effective, and of assured quality. Methods used by regulators need to constantly evolve so that scientific and technological advances are applied to address challenges such as new products and technologies, and also to provide an increased understanding of benefits and risks of existing products. Regulatory science builds on high-quality basic research, and encompasses at least two broad categories. First, there is laboratory-based regulatory science. Illustrative examples include development of correlates of immunity; or correlates of safety; or of improved product characterization and potency assays. Included in such science would be tools to standardize assays used for regulatory purposes. Second, there is science to develop regulatory processes. Illustrative examples include adaptive clinical trial designs; or tools to analyze the benefit-risk decision-making process of regulators; or novel pharmacovigilance methodologies. Included in such science would be initiatives to standardize regulatory processes (e.g., definitions of terms for adverse events [AEs] following immunization). The aim of a global regulatory science agenda is to transform current national efforts, mainly by well-resourced regulatory agencies, into a coordinated action plan to support global immunization goals. This article provides examples of how regulatory science has, in the past, contributed to improved access to vaccines, and identifies gaps that could be addressed through a global regulatory science agenda. The article also identifies challenges to implementing a regulatory science agenda and proposes strategies and actions to fill these gaps. A global regulatory science agenda will enable regulators, academics, and other stakeholders to converge around transformative actions for innovation in the regulatory process to support global immunization goals.

Background Respiratory infections caused by influenza viruses spread rapidly, resulting in significant annual morbidity and mortality worldwide. Currently, the most effective public health measure against infection is immunisation with an influenza vaccine matching the relevant circulating influenza strains. Although a number of developments in terms of influenza vaccine production, safety and immunogenicity have been reported, limitations in our understanding of vaccine stability still exist. In this report we seek to identify compounds that increase influenza vaccine thermostability. Methods We use plaque inhibition on confluent MDCK cells to identify compounds which inhibit the entry of various seed strain viruses. The effect of these compounds on vaccine thermal lability is evaluated through SRID analysis. The significance of these results is tested by a two-way analysis of variance (ANOVA) method. Results We identify two compounds which selectively inhibit entry of different group I or group II influenza strains through prevention of the neutral-pH to low-pH conformational change of hemagglutinin. Compounds which were able to inhibit virus entry were also able to limit thermally induced potency loss in matched influenza vaccines. Furthermore, we demonstrate that this effect is independent of product formulation or the presence of multiple HA types. Conclusions This work provides further evidence for a link between HA conformational stability in the virus and thermostability of the corresponding vaccine preparation. It also suggests straightforward approaches to improve the stability and predictability of influenza vaccine preparations.

a b s t r a c t Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neu- raminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 g/dose. The SRID assays require the availability of strain- specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neu- raminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS E , a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures.

There is an urgent need for a novel vaccine that is effective against human Enterovirus 71 (EV71) outbreaks. A double-blind, randomized controlled study was to evaluate the safety and immunogenicity of a human EV71 vaccine in healthy adults, children and infants. The vaccine dosages were 200 U and 400 U for children and adults, and 100 U, 200 U and 400 U for infants. Subjects were randomized to receive different dosages of the vaccine or placebo. Adults received intramuscular injection on Days 0, 14 and 28. Children and Infants received on Days 0, 28 and 56. The novel human EV71 inactivated vaccine was well tolerated and highly immunogenic in healthy volunteers, especially in infant populations. For immune response, the seropositive rates (with titers ≥≥1:8) of neutralizing antibody [NTAb] increased to 100% for all dosage groups after the second vaccination. For NTAb seronegative infants before vaccination, after one dose, the NTAb GMTs were 29.7 (95% CI, 13.1–67.2), 10.1 (95% CI, 6.6–15.3), and 27.4 (95% CI, 14.3–52.2) in the 100 U, 200 U, and 400 U vaccine groups, respectively; after two doses, the GMTs were 114.1 (95% CI, 44.5–292.4), 159.5 (95% CI, 49.3–515.3), and 509.0 (95% CI, 181.3–1429.1), respectively. Trial registration: ClinicalTrial.gov identifier: NCT01273246 and NCT01273233 .

A pH- and thermo-sensitive berberine hydrochloride liposome modified by the poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate) was synthesized. The morphology and size were determined by atomic force microscope (AFM) and dynamic light scattering (DLS). The phase transition temperature (Tpm) and phase transition pH (pH*) of the copolymer–liposome were obtained by differential scanning calorimetry (DSC). The results indicate that the size of copolymer–liposome depends on the mass ratios of the copolymer to soya bean lecithin (SPC) and the solution pH value, and the size can reach a maximum value at phase transition pH (pH*). The Tpm and pH* were affected by the mass ratios of the copolymer to SPC and properties of the copolymer. The release of berberine hydrochloride (BH) from the copolymer–liposome was investigated and the maximum release of BH was caused at Tpm and pH* of the copolymer–liposome. The results illustrate that BH released from the liposome is due to the phase transition of the copolymer–liposome, where both Tpm and pH* were acted as switches of the phase transition, and both spacial and temporal releases can be carried out by adjusting the temperature, pH value and release time.

Ethnopharmacological relevance Chinese traditional medicine Rhizoma drynariae (Gusuibu) is widely used for clinically treating osteoporosis and bone non-union. Naringin and its active metabolite naringenin are the main active ingredients of Rhizoma drynariae total flavonoids. Aim of the study The purpose of this paper is to confirm estrogenic and anti-estrogenic activity of naringin and naringenin, and provide the basic data to further study for the dose–effect relationship and the mechanism for Rhizoma drynariae in treatment of osteoporosis and other estrogen deficiency-related diseases. Materials and methods Naringin was extracted from Rhizoma drynariae. Naringin and its metabolin naringenin were tested estrogenic and anti-estrogenic activities through the experiment of cell proliferation and uterus weight gain in mice. Their estrogen-receptor binding abilities were tested by yeast two-hybrid experiment and nuclear receptor cofactor assays (RCAS) experiment, and their possible binding sites for ERβ were performed by computer aided molecular docking technology. Results Naringin and naringenin showed significant effects on the proliferation of estrogen-sensitive ER(+) MCF-7 cells in the absence of estrogen. Induction increased proliferation as the drug concentration, and the strongest proliferation appeared at a concentration of 8.6 × 10−5 M. When estradiol (10−10 M) and the different concentrations of naringin or naringenin were treated at the same time, naringin and naringenin could result in antagonistic effects on estradiol-induced MCF-7 cell proliferation, but they did not significantly affect proliferation of estrogen-insensitive ER(−) MDA-MB-231 cells. Naringin and naringenin exhibited higher binding capacity to estrogen receptor β (ERβ) than estrogen receptor α (ERα) in yeast two-hybrid experiments and nuclear receptor cofactor assays (RCAS) experiment. Docking simulation between naringin/naringenin and ERβ were performed, and the corresponding binding free energies of naringin–receptor and naringenin–receptor docked complexes were −7.95 and −10.45 kcal/mol. Hydrogen bonds were found between naringin and the amino acid residues Lys304 and His308. The oxygen atom (O11) of naringenin formed hydrogen bond to Arg346, and there may be hydrophobic space interactions between phenyl group (C13–C18) of naringenin and the amino acid residues Leu298, Met336, Met340, Phe356, Ile376 and Leu380. Conclusions Naringin and naringenin revealed a double directional adjusting function of estrogenic and anti-estrogenic activities. Both of them showed estrogenic agonist activity at low concentration or lack of endogenous estrogen. On the other hand, they also acted as estrogenic antagonists at high concentrations or too much endogenous estrogen. They produced estrogenic and anti-estrogenic effects primarily through selectively binding with ERβ, which could prevent and treat osteoporosis with the mechanism of estrogenic receptor agitation. This paper confirmed the estrogenic and anti-estrogenic activity of naringin and naringenin, and further studies were still essential to study their dose–effect relationship and the anti-osteoporosis mechanism for Rhizoma drynariae in the treatment of osteoporosis and other estrogen deficiency-related diseases.

The viral cDNA nuclear import is an important requirement for human immunodeficiency virus type 1 (HIV-1) replication in dividing and nondividing cells. Our recent study identified a specific interaction of importin α3 (Impα3) with HIV-1 integrase (IN) and its involvement in viral cDNA nuclear import. In this study, we have performed a more detailed investigation on the molecular mechanism of how HIV-1 IN interacts with Impα3. Our results revealed a reduced interaction between the two IN mutants INKK215,9AA (IN215,9) and INRK263,4AA (IN263,4) with Impα3, while an IN double mutant, IN215,9/263,4, was severely impaired for its Impα3-binding ability, even though it was still found interacting with other cofactors, IN interactor I and Transportin3. Immunostaining and fractionation analysis have shown that YFP-IN215,9/263,4 failed to localize in the nucleus of transfected cells. Also, we found that both major and minor nuclear localization signal binding grooves of Impα3 are involved in interaction with IN. All of these results suggest a cargo protein–import receptor type of interaction. Finally, the effect of IN215,9/263,4 mutations on HIV-1 replication was evaluated, and real-time quantitative PCR analysis showed that, while mutant virus (v215,9/263,4) had a slightly lowered total viral DNA, the 2-long-terminal-repeat DNA, a marker for nuclear import, was greatly reduced during v215,9/263,4 infection in both dividing and nondividing cells. Also, by cell fractionation assay, we found that a significant proportion of viral cDNA was still retained in cytoplasmic fraction of v215,9/263,4-infected cells. Overall, our study provides strong evidence that 211KELQKQITK and 262RRKAK regions of IN C-terminal domain are required for Impα3 interaction and HIV-1 cDNA nuclear import.

For guiding the modification of the lead compound, the DFT (density functional theory) method, with the basis set 6- 31G*, was employed to calculate the molecular geometries and electronic structures of sesquiterpene lactones as aromatase inhibitors. According to the correlation analysis, ELUMO (energy of lowest unoccupied molecular orbital) had positive impact on the inhibition activity. Quantitative structure-activity relationship model based on stepwise multiple binomial regression was established. Docking between sesquiterpene lactones and human aromatase was simulated. The pharmacophore analysis results of the docking complex showed that the external double bond of compound 1 (10-epi-8-deoxycumambrin B) is not the pharmacophore and could be modified to eliminate the cytotoxicity of the molecule.

Neuraminidase-induced immune responses are correlated with protection of humans and animals from influenza. However, the amounts of neuraminidase in influenza vaccines are yet to be standardized. Thus, a simple method capable of quantifying neuraminidase would be desirable. Here we identified two universally conserved sequences in all influenza A and B neuraminidases, one representing a novel finding of nearly 100% conservation near the enzymatically active site. Antibodies generated against the two highly conserved sequences bound to all nine subtypes of influenza A neuraminidase and demonstrated remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Importantly, employing these antibodies for the analyses of vaccines from eight manufacturers using the same vaccine seeds revealed marked variations of neuraminidase levels in addition to considerable differences between lots from the same producer. The reasons for the absence or low level of neuraminidase in vaccine preparations are complex and could be multi-factorial. The antibody-based assays reported here could be of practical value for better vaccine quality control.

The single radial immunodiffusion (SRID) method currently used to determine the hemagglutinin (HA) content of the inactivated influenza vaccines depends on the availability of reference HA antigen and corresponding anti-serum, updated and provided annually by World Health Organization (WHO) collaborative centers. Particularly early in a pandemic outbreak, reference reagents could be the bottleneck in vaccine development and release. Therefore, other reliable tests capable of quantifying HA content could substantially shorten the time needed for vaccine formulation. Here electrophoretic separation of deglycosylated samples in conjunction with densitometry was used to quantify HA contents of H1N1 vaccine at multiple manufacturing sites. We found the overall consistency between the alternative method and traditional SRID was 88-122% in seven lots of vaccine bulks from four subtypes (types) of influenza vaccine, confirming its suitability to quantify HA content. Moreover, we used the alternative method to prepare a national HA antigen reference in China for quality control of 2009 pandemic influenza A (H1N1) vaccines prior to the arrival of the WHO SRID reference standards, subsequently confirming good agreement between both methods. The alternative method for vaccine quantification enabled the Chinese health authority to approve H1N1 vaccine 1 month earlier than otherwise possible.

The fusion peptide is the only universally conserved sequence in the hemagglutinins of all 16 subtypes of influenza A and two genetic lineages of influenza B viruses. Here, peptides selected by bioinformatics approach were modified and conjugated to overcome serious technical hurdles such as the high hydrophobicity and weak immunogenicity of the viral fusion peptides. Antibodies generated against fusion peptides demonstrated remarkable specificity against the viral sequences and robustness of quantitatively analyzing the viral hemagglutinins even under stringent conditions. As quantitatively revealed by antibody-binding experiments, the fusion peptides of diverse hemagglutinins are exposed to the same degree upon unfolding at neutral pH to the physiologically fusogenic state. To our knowledge, this is the first report on the quantitative determination of virtually all influenza vaccines using a single universal antibody.

With a goal of developing a medication for the treatment of MUC1 expressing human cancers, a recombinant heat shock protein 65-MUC1 fusion protein (HSP65-MUC1) between BCG derived heat shock protein 65 (HSP65) and MUC1 derived peptide (MUC1) was developed. To move the HSP65-MUC1 into a phase I clinical trial, a comprehensive non-clinical safety evaluation was conducted. The evaluation comprised of single-dose toxicity and repeat-dose toxicity studies both in mice and rhesus monkeys. The data from the study indicates that the treatment with HSP65-MUC1 is not associated with obvious toxicity in the tested animals. The changes in clinical chemistry and hematology in both the mice and monkeys were considered to be mild because there were no indications of overt toxicity after administering HSP65-MUC1. The data provided here contributed to the approval of initiating a phase I clinical trial with HSP65-MUC1 for the treatment of patients with MUC1-positive breast cancer in China.

To develop CpG oligodeoxynucleotides (CpG ODNs) based therapy for prevention and treatment of severe acute respiratory syndrome (SARS), we selected a novel CpG ODN (BW001), which displays B-type CpG ODN structure feature at the 5' and A-type CpG ODN structure feature at the 3', and tested for its anti-SARS-CoV activity. We found that the supernatants of human PBMCs stimulated by BW001 significantly protected Vero cells from SARS-CoV infection. BW001 could stimulate human PBMCs and pDCs to secrete high level of IFN-alpha and promote human PBMCs and B cells to proliferate. Furthermore, we demonstrated that BW001 could activate CD19+ B cells and CD56+ NK cells in human PBMCs. In addition, BW001 could enhance NK cytotoxicity and IFN-gamma secretion in human PBMCs. Together, BW001 represents a novel type of CpG ODN and may have potential for the development of treatment and prevention for SARS as well as other viral associated diseases.

Using the newly installed SIS receiving system of the 13.7 m telescope of PMO, the CO isotope line pair, (CO)-C-13 and (CO)-O-18 J = 1 - 0, were observed in a search for dense cores in 64 IRAS sources with high color indices or H2O masers. Maps were obtained for some of the sources. The line pair was detected in all the target sources. The emission of (CO)-C-13 J = 1 - 0 is strong showing that the emission regions are dense. The pair lines of the CO isotopes were measured for the first time in at least 60 sources. Antenna temperatures, center velocities and line widths are obtained with Gaussian fitting. Preliminary analyses were made for the emission intensities and line profiles.

The effect of Si overgrowth on the structural and luminescence properties of strained Ge layer grown on Si(1 0 0) is studied. Capping Si leads to the dissolution of Ge island apex and reduced island height. The structural changes in island shape, especially in chemical composition during Si overgrowth have a large effect on the PL properties. The integrated PL intensity of Ge layer increases and there are large blue shifts in peak energies after capping Si. The PL spectra from buried Ge layer are consistent with type-II band alignment in SiGe/Si. We show that the PL properties from buried Ge layer may be tailored by modifying the cap layer growth conditions as well as post-growth annealing. (C) 1999 Elsevier Science B.V. All rights reserved.

Using the 25-m telescope of Shanghai Observatory, Joint Radio Astronomy Laboratory, 19 known maser sources and 20 candidates in H II regions were surveyed for water vapour maser emission at 22.2 GHz. 21 sources were detected, of which two are for the first time. Combining with some previous data, the time variations in the flux and velocity are obtained and discussed. Time variation in the peak flux density is common and is of two types, a change in the intensity of the same peak, and a swapping-over of two peaks. The corresponding IRAS sources were identified and the relation between the maser emission and the infrared parameters discussed. It is shown that maser production rate is more sensitive to the infrared flux intensity than the infrared colour distribution.

The reverse transcriptase (RT) activity assay is widely used in the quality control of biological products including gene therapy products for it can detect all infectious retroviruses in biological starting materials, cell banks and final products. But the lack of reference standard for RT activity results in significant systemic error and incomparability between laboratories. To standardize RT activity assay, this study established a sensitive real time PCR and developed the first national reference standard, which was prepared from a raw material of recombinant moloney murine leukemia virus reverse transcriptase. Our tests indicated that the candidate reference standard met relevant requirements of the national reference standard. The activity value of candidate RT standard was determined as 26.4 U per ampoule by a collaborative calibration among four laboratories, with the within-laboratory geometric coefficients of variation (GCVs) ranging from 2.7% to 9.6% and the between-laboratory GCV values being 9.8%. The thermal accelerated stability test showed that no loss of activity was observed when the candidate RT standard was stored at 4°C, 25°C and 37 °C compared with -20°C even after 9 months. The applicability study showed that the cell bank and viral bank of an adenovirus gene therapy product were positive and the final products were negative. As a conclusion, the candidate preparation was suitable to serve as a Chinese national standard for RT activity assay and can be used in the quality control of gene therapy products.