Cheng X.,Nanjing Southeast University |
Wang S.,Nanjing Southeast University |
Dai X.,Nanjing Southeast University |
Shi C.,Changchun Institute of Biological Products Co. |
And 5 more authors.
PLoS ONE | Year: 2012
Background: The identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation. Methodology/Principal Findings: First, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 μg (placebo), 10 μg and 20 μg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×105 genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 μg of the HEV p179 produced anti-HEV with titers of 1:104-1:105 and were completely protected from infection. Rabbits vaccinated with 10 μg produced anti-HEV with titers of 1:103-1:104 and were protected from hepatitis, but two out of the five rabbits showed virus shedding. Conclusions/Significance: Rabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected. © 2012 Cheng et al.
PubMed | Changchun Institute of Biological Products Co. and Nanjing Southeast University
Type: | Journal: Antiviral research | Year: 2016
We investigated the immunogenicity difference between hepatitis E vaccine p239 derived from hepatitis E virus (HEV) genotype 1 and vaccine p179 derived from HEV genotype 4; and the presence of genotype-specific neutralizing epitopes. HEV ORF2 recombinant proteins (p166W01, p166Mex, p166US and p166Chn) derived from the four HEV genotypes were used to detect anti-HEV IgGs in sera of mice and humans vaccinated with p179 or p239 and in sera of rhesus monkey challenged with HEV genotype 1 or 4 strains. Then monoclonal antibodies (mAbs) against genotype 1 or 4 ORF2 recombinant proteins were prepared and their immunoreactivity was assessed using ELISA and Western blotting; their neutralizing activity was evaluated by an in vitro PCR-based neutralization assay. The results revealed significant immunogenicity difference between the two vaccines: p239-induced IgGs reacted more strongly against p166W01 and p166Mex than against p166US and p166Chn in mice and humans. By contrast, p179-induced IgGs showed a stronger reactivity against p166US and p166Chn than against p166W01 and p166Mex. This difference has also been observed in the sera of rhesus monkeys challenged with HEV genotype 1 or 4 strains. Moreover, besides the two common neutralizing mAbs 3G1 and 5G5, two genotype-specific neutralizing mAbs, 2B1 and 4C5, were obtained. 2B1 could specifically bind to recombinant proteins derived from genotypes 1 and 2 and neutralized only genotypes 1 and 2 strains, while 4C5 immunoreacted specifically against recombinant proteins derived from genotypes 3 and 4 and neutralized only genotypes 3 and 4 strains. These findings revealed the existence of immunogenicity difference between the p179 and p239 vaccines and demonstrated that this difference could be due to the presence of HEV genotype-specific neutralization epitopes.
Liu Z.,Jilin University |
Liu H.,Jilin University |
Liu L.,Changchun Institute of Biological Products Co. |
Su X.,Jilin University
Microchimica Acta | Year: 2016
We report on a widely applicable approach for protein detection by using triple-helix DNA mediated CuInS2 quantum dot (QD) and graphene oxide (GO) nanocomposite. The CuInS2 QDs were coated with mercaptopropionic acid and then covalently linked to a hairpin aptamer against lysozyme (HLA). Single-stranded DNA (triple helix-forming oligonucleotide; THFO) readily absorbs on the surface of GO via π-stacking interaction, and this results in the formation of THFO-GO. If HLA-CuInS2 QDs are added to the THFO-GO system, the fluorescence of HLA-CuInS2 QDs (at excitation/emission wavelengths of 590/665 nm) is quenched. Lysozyme has a higher affinity for HLA than THFO. Therefore, in the presence of lysozyme, it will bind to the HLA-CuInS2 QD and displace the THFO-GO. This results in the restoration of fluorescence that is related to the concentration of lysozyme. The fluorescence of the QDs is turned on. The calibration plot is linear in the 0.01 to 1.8 ng·mL‾1 concentration range, with a 3 pg·mL‾1 detection limit (at a signal-to-noise ratio of 3). The method was also applied to study the inhibition of lysozyme by IvyEc. In our perception, this method has a wide scope in that it may become applicable to any protein for which an appropriate aptamer is available. [Figure not available: see fulltext.] © 2016 Springer-Verlag Wien
PubMed | Health Science University, Changchun Institute of Biological Products Co., Xi'an Jiaotong University and Reagent p Scientific Research Management Center
Type: Journal Article | Journal: PloS one | Year: 2017
Intranasal vaccination is more potent than parenteral injection for the prevention of influenza. However, because the poor efficiency of antigen uptake across the nasal mucosa is a key issue, immunostimulatory adjuvants are essential for intranasal vaccines. The immunomodulator mannatide or polyactin (PA) has been used for the clinical treatment of impaired immunity in China, but its adjuvant effect on an inactivated trivalent influenza vaccine (ITIV) via intranasal vaccination is unclear. To explore the adjuvant effect of PA, an inactivated trivalent influenza virus with or without PA or MF59 was instilled intranasally once a week in BALB/c mice. Humoral immunity was assessed by both the ELISA and hemagglutination inhibition (HI) methods using antigen-specific antibodies. Splenic lymphocyte proliferation and the IFN- level were measured to evaluate cell-mediated immunity. The post-vaccination serum HI antibody geometric mean titers (GMTs) for the H1N1 and H3N2 strains, antigen-specific serum IgG and IgA GMTs, mucosal SIgA GMT, splenic lymphocyte proliferation, and IFN- were significantly increased in the high-dose PA-adjuvanted vaccine group. The seroconversion rate and the mucosal response for the H3N2 strain were significantly elevated after high-dose PA administration. These adjuvant effects of high-dose PA for the influenza vaccine were comparable with those of the MF59 adjuvant, and abnormal signs or pathological changes were not found in the evaluated organs. In conclusion, PA is a novel mucosal adjuvant for intranasal vaccination with the ITIV that has safe and effective mucosal adjuvanticity in mice and successfully induces both serum and mucosal antibody responses and a cell-mediated response.
Sun L.-J.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2016
Objective To develop a relative fluorescent quantitative PCR method for mitochondrial antiviral signaling protein (MAVS) SYBR Green I in Guinea pigs infected with avian influenza virus (AIV) subtype H5N1, and determine the expression levels of MAVS in lung tissue of Guinea pigs before and after infection. Methods RNA was extracted from the lung tissue of Guinea pigs and reversely transcribed into cDNA, which was 10-fold serially diluted to 9 concentrations (1. 00 E + 10 ∼ 1. 00 E + 02 copies/ μl) for PCR amplification. A standard curve was plotted using β-actin as an internal reference. The amplification efficiencies of two genes were compared, and the method was verified for specificity, reproducibility and sensitivity. The expression levels of MAVS in lung tissue of Guinea pigs before and after challenge with H5N1 AIV. Results The dilution range of cDNA template was 1. 00 E + 09 ∼ 1. 00 E + 04. The difference between slopes of standard curves of MAVS and β-actin genes was less than 0. 1, with a R2 value of 0. 999, while the amplification efficiencies were 100. 2% and 100. 3% respectively. Either of the melting curves of the two genes showed a single peak, and clear target fragments were amplified, no non-specific amplification was observed. Both the coefficients of variation (CV) of Ct values of the two genes were less than 10%. The sensitivity of the developed method was 1. 00 E + 03 copies/μl. The expression level of MAVS in lung tissue of Guinea pigs infected with H5N1 AIV was down-regulated. Conclusion A relative fluorescent quantitative PCR method for MAVS SYBR Green I in Guinea pigs infected with AIV subtype H5N1 was successfully developed, and infection with H5N1 AIV decreased the expression level of MAVS in Guinea pigs.
Cao Y.-F.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013
Objective: To express fusion human insulin-like growth factor-1 (hIGF-1) in prokaryotic cells, and purify and identify the expressed product. Methods: The sequence of natural hIGF-1 gene was subjected to samesense mutation according to the E. coli preferred codons, synthesized amplified by PCR and cloned into vector pET48b(+). The construct recombinant plasmid pET48b-Trx A-hIGF-1 was transformed to E. coli BL21 (DE3) for expression under induction of IPTG. The expressed fusion protein Trx A-hIGF-1 was purified by nickel ion affinity chromatography, digested with enterokinase and identified by SDS-PAGE. Results: Recombinant plasmid pET48b-Trx A-hIGF-1 was constructed correctly as proved by colony PCR and sequencing. The expressed fusion protein, with a relative molecular mass of about 26 000, contained about 40% of total somatic protein and mainly existed in a soluble form, of which the proportion of soluble protein to total target protein was not less than 90%. The fusion protein reached a purity of not less than 90% and a concentration of 0.5 mg/ml, which was digested with enterokinase into hIGF-1 with a relative molecular mass of about 8 000 and Trx A with a relative molecular mass of about 18 000. The hIGF-1 showed specific binding to rabbit anti-hIGF-1 polyclonal antibody. Conclusion: The hIGF-1 fusion protein was successfully expressed, which laid a foundation of study on biological activity and large-scale production of hIGF-1.
Xia Q.-J.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013
Objective: To prepare the immunoglobulin of yolk(IgG) against hepatitis A virus (HAV) and develop a double antibody sandwich ELISA method for HAV antigen content in HA vaccine. Methods: IgY against HAV was prepared by immunizing healthy laying hens with purified HAV, purified by several steps, then determined for protein content by ultraviolet spectrophotometry, for relative molecular mass and purity by reduced SDS-PAGE, and for titer, specificity as well as heat, acid and base stabilities by indirect ELISA. A double antibody sandwich ELISA method was developed using the purified IgY as coating antibody, and HRP-labeled monoclonal antibody against HAV as the secondary antibody, and used for determination of HAV antigen content in HA vaccine during production, and the results were compared with those by commercial ELISA kit. Results: The IgY concentration after purification by affinity chromatography was 3.67 mg/ml. The relative molecular masses heavy and light chains of IgY were 66 000 and 27 000 respectively, while the purity was 96.78%, and the titer was 1:16 000 at most. The purified IgY showed only positive reaction with HAV, while showed no reactions with poliovirus or enterovirus 71 (EV71). Treatment at 25-70°C for 15 min or with Tris-HCl buffer at pH 3.0-10.0 for 2 h showed no significant influence on the titer of IgY. The log of HAV concentration at a range of 15.62-2 000.00 ng/ml was linearly related to the A450 value, with a R2 value of 0.935. The minimum detection limit of the developed ELISA method was 7.81 ng/ml, while the coefficient of relationship of detection result was 0.952 7 to that by commercial kit. Conclusion: The prepared IgY showed high concentration, purity, titer, specificity and stability, by which the developed double antibody sandwich ELISA method might be used for determination of HAV antigen content in HA vaccine during production.
Zhao D.-P.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013
Objective: To evaluate the safety and immunogenicity of influenza A H1N1 virus split vaccine in animals. Methods: Abnormal toxicity test was performed according to the requirements in Chinese Pharmacopoeia (Volume III, 2005 edition). Kunming mice were randomly divided into seven groups, ten for each. The mice in three test groups were injected i. m. with influenza A H1N1 virus split vaccine at hemagglutinin (HA) contents of 15, 30 and 45 μg/0.5 ml, while those in three parallel control groups with seasonal influenza H1N1 virus split vaccine at the corresponding HA contents, and those in negative control group with PBS, respectively. The vaccines were given at a dosage of 0.1 ml for 2 times at an interval of 21 d. Venous blood samples were collected 0, 21, 28, 35 and 42 d after the first immunization, from which sera were separated and determined for hemagglutination inhibition (HI) antibody level. Results: The abnormal toxicity test result met the requirements in Chinese Pharmacopoeia (Volume III, 2005 edition). The serum antibody levels in three test groups 21, 28, 35 and 42 d after the first immunization increased significantly as compared with those in negative control group, which reached a high level on 21 d and a peak value on 28 d (each P < 0.01). However, the antibody levels in test groups 0, 21, 28 and 42 d were higher than those immunized with the control vaccine at the corresponding HA contents (P > 0.05). The serum HI antibody positive rates of mice 21 d after immunization with test vaccine at HA contents of 15, 30 and 45 μg/0.5 ml were 80%, 90% and 90%, while the protection rates were 100%, 90% and 90%, respectively, which showed no significant difference with those in parallel control groups (each P < 0.05). The HI antibody positive rates of mice 28, 35 and 42 d after immunization with 15 and 45 μg/0.5 ml were more than 80%, while that with 30 μg/0.5 ml was slightly low. However, the protection rates of HI antibody in three test groups were 90%-100%. Conclusion: Influenza A H1N1 virus split vaccine showed good safety and immunogenicity in Kunming mice.
He W.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2014
Objective: To study the effect of recombinant endostatin (rhES) on endothelial cells by atomic force microscope (AFM). Methods: The proliferative activities of human umbilical vein endothelial ECV304 cells treated with rhES at various concentrations (0.05-2.4 μg / ml) were determined by MTT assay. ECV304 cells were treated with 0.8 and 2 μg / ml rhES respectively, then observed for overall morphology by AFM, and for local morphology on surface by SPI 3800 New DFM. Results: The rhES inhibited the proliferation of ECV304 cells significantly (P < 0.001), and decreased the thickness of adherent ECV304 cells, both in dose-dependent modes. The surface of cells after treatment with rhES changed from smooth to rough, on which some small processes were formed. However, the surface structure of ECV304 cells after treatment with rhES showed an irregular change. Conclusion: The samples for atomic force microscopy were easy to prepare, while the resolving power was high, indicating that the method was suitable for in situ observation of adherent cells.
Xu J.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2012
Objective: To preliminarily develop a procedure for preparation of interferon gelatin microspheres (IFN-GMS) and investigate their characteristics. Methods: IFN-GMS were prepared, based on which the condition for preparation, such as gelatin concentration, pH value and sodium sulfate concentration, was optimized according to the status of microspheres including homogeneity, formation rate, quantity of debris as well as adhesion. The prepared IFN-GMS were determined for IFNα2b activity, based on which the encapsulation rate was calculated. Results: The optimal temperature, gelatin concentration, pH value and sodium sulfate conentration for preparation of IFN-GMS were 50°C, 40 g/L, 3.4 or 3.6 and 300 g/L respectively. The microspheres were at a mean diameter of 18 μm, more than 70% of which were at diameters of 15-20 μm. The encapsulation rate was more than 85%. Conclusion: The IFN-GMS may be used for preparation of controlled-released injection.