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@#Objective To evaluate the passage stability of H5N1(NIBRG-14) influenza virus vaccine strain in MDCK cells(sMDCK)of serum-free suspension culture.Methods H5N1(NIBRG-14) influenza virus working-bank vaccine strains were passed 15 consecutive times in sMDCK cells.The 8-segment nucleotide sequences(HA,NA,M,NP,NS,PA, PB1 and PB2 genes) of the main-bank,working-bank,virus of P1,P2,P3,P5,P10 and P15 generations were detected for genetic stability by second and first generation sequencing.The stability of amino acid sequences of hemagglutinin(HA)and neuraminidase(NA),the main antigens of the working-bank,P5 and P15 generation viruses,were evaluated by using peptide coverage as indicators;Influenza vaccine was prepared with working-bank,P5 and P15 generation viruses,with which the female BALB/c mice were immunized i.m.with 10 in each group,15 μg HA per mouse,and boosted 28 d later at the same dosage and route.At 28,42 and 56 d after the primary immunization,the mice were detected for the titer of neutralizing antibody in serum to evaluate the stability of immunogenicity.Results No segment insertion or deletion was detected in each generation of influenza virus,and the nucleotide sequence was completely consistent with the main-bank;Single nucleotide polymorphism(SNP) mutations did not occur in the main-bank,working-bank,P1,P2,P3,P5 and P10 generations of viruses,while the possibility of SNP mutation showed in many gene loci of P15 generation virus,with heterozygous SNP accounting for 91.62%.The coverage rate of HA and NA protein peptides of P5 and P15 generation viruses ranged from96.7% to 100%.There was no significant difference in serum neutralizing antibody titer of mice in the working-bank,P5 and P15 groups(H=2.253,2.029 and 1.408,P=0.324,0.363 and 0.495,respectively) at 28,42 and 56 d after the first immunization.Conclusion H5N1(NIBRG-14) influenza virus vaccine strain has good genetic stability in sMDCK cells,which is expected to be used in the production of sMDCK cell matrix pandemic influenza vaccine.
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Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.
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@#ObjectiveTo analyze the protein components in the bulks of H5N1 inactivated influenza virus vaccine(MDCK cells),providing experimental basis for the improvement of the quality control method and the development of the vaccine.MethodsThe H5N1 influenza virus strain was inoculated into MDCK cells. After culturing the virus for 48 h,the virus liquid was harvested,and the original liquid sample was obtained by clarification and ultrafiltration concentration,β-propiolactone inactivation,and two-step chromatography purification with Capto Q and Sephorase 4FF. Morphology of virus particles in the sample was analyzed by transmission electron microscopy,while hemagglutinin identification of the virus bulk by single radial immunodiffusion(SRID)assay,purity by high performance liquid chromatography,and protein electrophoresis by gradient SDS-PAGE. The protein components contained in the samples were analyzed by liquid chromatography-mass spectrometry(LC/MS)combined with secondary mass spectrometry sequence determination of the recovered protein polypeptides.ResultsSpherical H5N1 influenza virus particles of 80 ～ 120 nm were observed under transmission electron microscope,showing the typical shape of influenza virus. The identification test showed that the antigenicity of the virus bulks was consistent with the virus strain. The gradient SDS-PAGE analysis showed that the virus bulk had two bands. LC/MS mass spectrometry analysis showed that H5N1 influenza virus protein was the main component in the bulk samples,including NP,M1,HA,NA and other proteins of H5N1 influenza virus.ConclusionThe protein composition of the bulk during the preparation of H5N1 influenza virus inactivated vaccine was analyzed,which provided a reference for the development and quality control of this type of vaccine.
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Objective To screen a Madin-Darby canine kidney (MDCK) cell line for H5N1 influ-enza virus isolation and to evaluate its safety in vaccine production. Methods MDCK cells were cloned by the method of limiting dilution. Hemagglutination test was used to screen MDCK cells that were suitable for H5N1 influenza virus production. Tests for analyzing the characteristics, extraneous agents, endogenous agents and tumorigenicity of MDCK cells were performed according to Chinese Pharmacopeia Volume Ⅲ. Results A total of 108 MDCK cell lines were obtained and three of them were selected after hemagglutina-tion test. G1 cells were chosen following further screening with tumorigenicity test and receptor abundance analysis. The average number of chromosomes of the MDCK-G1 cells was 78±4. No bacteria, fungi or myco-plasma contamination was detected. In experimental group, each nude mouse was injected with 1×107/ml viable cells to observe their tumorigenicity. Twelve weeks after cell injection, no node was found at injection sites or in gross anatomy. There was no significant difference between the experimental and negative control groups. The result of the tumorigenicity test was negative. No node formation was found after injecting nude mice with cell lysate or cellular DNA collected from equivalent amount of cells. It was indicated that the MDCK-G1 cells were of low-oncogenic potential. Conclusions The MDCK-G1 cell line could be used as a substrate to produce H5N1 influenza virus vaccine.
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OBJECTIVE@#To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus (H5N1).@*METHODS@#Crude extracts of Andrographis paniculata, Curcuma longa (C. longa), Gynostemma pentaphyllum, Kaempferia parviflora (K. parviflora), and Psidium guajava obtained by both water and ethanol extractions were investigated for their cytotoxicity in the Madin-Darby canine kidney cells. Thereafter, they were investigated in vitro for antiviral activity and cytokine response upon H5N1 virus infection.@*RESULTS@#The results revealed that both water and ethanol extracts of all the five studied plants showed significant antiviral activity against H5N1 virus. Among these plants, C. longa and K. parviflora showed strong anti-H5N1 activity. Thus, they were selected for further studies on their cytokine response upon virus infection. It was found that ethanol and water crude extracts of C. longa and K. parviflora induced significant upregulation of TNF-α and IFN-β mRNA expressions, suggesting their roles in the inhibition of H5N1 virus replication.@*CONCLUSIONS@#To the best of the authors' knowledge, this study is among the earliest reports to illustrate the antiviral property of these Asian medicinal plants against the highly pathogenic avian H5N1 influenza virus. The results of this study shed light on alternative therapeutic sources for treatment of H5N1 influenza virus infection in the future.
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Objective To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus (H5N1). Methods Crude extracts of Andrographis paniculata, Curcuma longa (C. longa), Gynostemma pentaphyllum, Kaempferia parviflora (K. parviflora), and Psidium guajava obtained by both water and ethanol extractions were investigated for their cytotoxicity in the Madin–Darby canine kidney (MDCK) cells. Thereafter, they were investigated in vitro for antiviral activity and cytokine response upon H5N1 virus infection. Results The results revealed that both water and ethanol extracts of all the five studied plants showed significant antiviral activity against H5N1 virus. Among these plants, C. longa and K. parviflora showed strong anti-H5N1 activity. Thus, they were selected for further studies on their cytokine response upon virus infection. It was found that ethanol and water crude extracts of C. longa and K. parviflora induced significant upregulation of TNF-α and IFN-β mRNA expressions, suggesting their roles in the inhibition of H5N1 virus replication. Conclusions To the best of the authors’ knowledge, this study is among the earliest reports to illustrate the antiviral property of these Asian medicinal plants against the highly pathogenic avian H5N1 influenza virus. The results of this study shed light on alternative therapeutic sources for treatment of H5N1 influenza virus infection in the future.
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Background: A/H5N1 influenza virus spreads from birds to humans and cause influenza diseases with high mortality rate. Vaccination is the most effective way to protect communities from pandemic, reduce morbidity and mortality. The study of creating A/H5N1 influenza vaccines in conformity with Vietnam was the urgent need. Institute of Vaccine\u2019s Achievement (IVAC) studied production of inactivated influenza vaccine for human on egg-grown from reassortants NIBRG-14. Objectives: In order to produce experimentally A/H5N1 influenza vaccine for human in accordance with WHO requirements and set up a viable process for production of the vaccines. Subjects and method: 10 days embryonated eggs and NIBRG-14 strains were served to the study with LAL method to check endotoxin, Kijehdal method to test total protein. Results: IVAC had produced successfully 5 lots of absorbed vaccine A/H5N1 (FLUVAC) using NIBRG-14 strains and embryonated eggs. Initially, production and quality control processes had been set up at IVAC by applying the recommendations of WHO. Conclusion: The success of the study was a basis of the approval of the government to establish a influenza vaccine manufacturing facilities.
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Humanos , Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Vacunas contra la Influenza , HuevosRESUMEN
The M and NP genes of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA,and cloned into pMD 18-T vector respectively.The expression plasmid containing the M gene (pHM6-m) or the NP gene (pHM6-np) was then constructed by inserting the M or NP gene into the pHM6 eukaryote expression vector; the constructed plasmid was then sequenced.32 BALB/c mice (6-week-old) were divided into four groups at random.Three groups of BALB/c mice were inoculated one time the intramuscular route with either 30 μg of plasmid pHM6-m,30 μg of plasmid pHM6-np or the mixture of plasmid pHM6-m (15 μg ) and pHM6-np(15 μg) respectively.A additional group of mice were injected with 100 μ1 PBS as controls.Two weeks later,all mice were challenged with homologous H5N1 avian influenza virus,and observed in the following 12 days.The survival rates of mice in the pHM6-m group,the pHM6-np group and mixed plasmids group were 62.5% ,25.0% and 50.0%,respectively.Results showed that effective protection could be provided by either pHM6-m or pHM6-np,but pHM6-m provided a better protective effect than pHM6-np.