[Preparation of vero cell-adapted influenza H5N1 virus strain by genetic reassortment].

OBJECTIVE: To prepare Vero cell-adapted influenza H5N1 virus strain by Genetic Reassortment and produce influenza H5N1 vaccine using Vero cell as a substrate. METHODS: Embryonated specific pathogen-free (SPF) hen's eggs and Vero cells were co-infected with Vero cell-adapted influenza virus A/Yunnan/1/2005 Va(H3N2) and A/Anhui/1/2005 (H5N1) via reverse genetics. The reassortant was screened with goat antibody against strain A/Yunnan/1/2005 Va(H3N2) and identified for subtype by hemagglutination-inhibition (HI) assays and gene analysis of HA and NA. RESULTS: A Vero cell-adapted influenza H5N1 virus strain was obtained, and there was no significant difference in serum antibody titers of monovalent inactivated vaccine reassorted before and after (F = 0.857, P > 0.05). CONCLUSION: The Vero cell-adapted influenza virus of epidemic strain may be reassortment between Vero cell-adapted and epidemic strains.

Screening of a high growth influenza B virus strain in Vero cells.

Due to the insufficient supply of embryonated chicken eggs, the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus. The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines. However, most of the influenza viruses can not grow well in Vero cells. To develop a new influenza vaccine with Vero cells as a substrate, the virus needs to adapt to this cell substrate to maintain high growth characteristics. By serial passages in Vero cells, the B/Yunnan/2/2005va (B) strain was successfully adapted to Vero cells, with the hemagglutination titer (HAT) of the virus reaching 1:512. The high growth characteristic of this strain is stable up to 21 passages. The strain was identified by hemagglutination inhibition (HAI) test and sequencing respectively; the HA1 gene sequence of the virus was cloned and analyzed. The screening and establishment of high growth B virus provides an important tool for influenza vaccine production in Vero cells.

[Effective inactivatian test of inactivated hepatitis A vaccine using integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction].

OBJECTIVE: To establish an quick, sensitive and specific assay for effective inactivatian test of inactivated hepatitis A vaccine. METHODS: effective inactivatian test of inactivated hepatitis A vaccine were carried out using integrated cell culture/strand-specific RT-PCR (ICC/strand-specific RT-PCR) assay compared with traditional ELISA and nest RT-PCR assay. RESULTS: all the samples were infectious negative detecting by both ICC/ strand-specific RT-PCR and ELISA assay,while some samples appeared false positive detecting by nest RT-PCR. CONCLUSION: ICC/strand-specific RT-PCR assay is a novel, rapid, sensitive and reliable method for effective inactivatian test of inactivated hepatitis A vaccine. Shorting detection period largely, this assay may be used as an alternative method for routine inactivated hepatitis A vaccines test.

[Research on an improved Lowry method to determine content of protein in Sabin IPV].

OBJECTIVE: To establish a method for the content determination of protein in Sabin IPV. METHODS: Using lowry method combined with being precipitated by trichloroacetic acid to determine the content of protein in Sabin IPV. Changing different conditions to optimize the experiment to establish a improved lowry method. And the sample recovery test was also conducted. RESULTS: The method can exclude the interference of free aminoacid, phenols and some other additives. The calibration curve was in good linearity of protein within the range of 2.5 microg/ml-40 Microg/ml, r = 0.9998. Under the best conditions, the mean recovery was 95.32%, the CV in a batch and between batches were both < 10%. CONCLUSION: The method can be used to determine the micro content of protein in vaccines.

[Effect of 2-phenoxyethanol on potency of Sabin inactivated poliomyelitis vaccine and its safety].

OBJECTIVE: To investigate the effect of 2-phenoxyethanol on potency of Sabin inactivated poliomyelitis vaccine (IPV). METHODS: Sabin IPV samples containing 5 mg or 7 mg 2-phenoxyethanol each dosage respectively were placed separately at 4 degrees C, 37 degrees C for 2 days and 7 days. D-antigen contents were tested with ELISA method. Then neutralizing antibodies in mice and guinea pigs were detected. The safety experiment was performed according to unusual toxicity test of China requirement for biological product. RESULTS: After addition of 2-phenoxyethanol, the I, II, and III D-antigen contents of Sabin IPV did not change. The antibody levels in mice and guinea pigs were not different between experimental group and control group. Animals were safe during observation period. CONCLUSION: 2-Phenoxyethanol had no effect on potency and safety of Sabin IPV. It can be used as antiseptic for Sabin IPV.

[Construction of bicistronic vector and its application to combined DNA vaccine].

BACKGROUND: To study preparation of polyvalent DNA vaccine and the control of multiple gene expression. METHODS: A bicistronic vector pcDNA3.0BA was constructed from pcDNA3.0. HCV PC154 gene and HBV preS2S gene were inserted into this vector to form bicistronic expression construct pcDNA3.0BAPC154S2S and monocistronic expression construct pcDNA3.0BAPC154 or pcDNA3.0BAS2S. These plasmids were transiently expressed in COS-7 cells and injected into muscles of BALB/c mice. RESULTS: pcDNA3.0BA contains two cistronic units, which can co-express two kinds of genes, with the first immunogen gene and the second gene serving as additional immunogen or as modulator for the immune responses. HBV surface Ag and HCV core Ag were coexpressed in vitro. The antibody responses and lymphoproliferation to antigens were similar between bicistronic and monocistronic expression construct in mice. CONCLUSION: pcDNA3.0BA is a novel vector, which can coexpress two proteins and elicit polyvalent immune responses.

Expression and self-assembly of HCV structural proteins into virus-like particles and their immunogenicity.

BACKGROUND: The synthesis of virus-like particles (VLPs) provides an important tool to determine the structural requirements for viral particle assembly and virus-host interactions. Our purpose was to express simultaneously all three structural proteins of hepatitis C virus (HCV) in insect cells to investigate the proteins assembly into VLPs and the immunogenicity of these particles. METHODS: HCV gene sequences encoding the structural proteins C, E1, and E2 were amplified with PCR, and recombinant baculoviruses were constructed using recombinant DNA techniques. The expression of HCV structural proteins in insect cells was analyzed by immunofluorescence and SDS-PAGE. The interaction of expressed structural proteins was investigated by immunoprecipitation and immunoblotting. The VLPs in the insect cells were visualized by electron microscopy (EM). VLPs were then purified by sucrose gradient centrifugation and used to immunize BALB/c mice. Antibodies against HCV were tested for in mouse serum samples by an ELISA assay. RESULTS: The recombinant baculoviruses reBV/C and reBV/E1-E2 were constructed successfully. Insect cells co-infected with reBV/C and reBV/E1-E2 expressed HCV C, E1, and E2 proteins with the expected molecular weights of 20kD, 35kD, and 66kD, respectively. The results of immunoprecipitation and immunoblotting assays revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating association of the three structural proteins. Electron microscopy of insect cells co-infected with reBV/C and reBV/E1-E2 demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from serum samples or hepatic tissue samples of HCV infected humans. The VLPs were partially purified. Antibodies to HCV were detectable in the serum of mice immunized with VLPs. CONCLUSION: HCV structural proteins simultaneously expressed in insect cells can interact with each other and assemble into HCV-like particles, which are shown to be immunogenic in mice.

A novel process for production of hepatitis A virus in Vero cells grown on microcarriers in bioreactor.

AIM: To develop a novel process for production of HAV in Vero cells grown on microcarriers in a bioreactor. METHODS: Vero cells infected with HAV strain W were seeded at an initial density of 1 x 10(5) cells/mL into a 7-L bioreactor containing Cytodex-I microcarriers. During the stage of cell proliferation, the following conditions were applied: pH 7.2 +/- 0.2, temperature 37 +/- 0.2 degrees, dissolved oxygen 40% of air saturation and agitation rate 40 r/min. After the stage of virus culture started, the culture conditions were altered to pH 7.2 +/- 0.2, temperature 35 +/- 0.2 degrees, dissolved oxygen 25% of air saturation, agitation rate 50 r/min and perfusion of fresh medium at a flux of 20 mL/h. During the course of fermentation, cell density, HAV antigen titre, glucose, lactate and ammonia levels were monitored. A control experiment using conventional static culture was conducted in the T150 flask. RESULTS: After a 28-d cultivation, cell density increased to 14.0 x 10(6) cells/mL in the bioreactor, 5.6 x 10(9) viable cells and 4,000 mL virus suspension with a titre of 1:64 were harvested. The viral antigen output per cell unit in the bioreactor was 3-fold higher than that in the T150 flask. Meanwhile the metabolic mode of Vero cells did not change after the infection with HAV strain W. CONCLUSION: The process for production of HAV in Vero cells grown on microcarriers in a bioreactor is a novel, efficient and practical way to obtain virus antigen for vaccine purpose. This approach produces more cells and HAV antigen than the conventional static culture. With further improvement, it is possible to be used for the production of hepatitis A vaccine.

No requirement of HCV 5'NCR for HCV-like particles assembly in insect cells.

AIM: To express all three HCV structural proteins in the presence or absence of HCV 5'NCR to investigate the requirement of 5'NCR for the assembly of HCV-like particles in insect cells. METHODS: HCV structural protein encoding sequences CE1E2 and 5'NCR-CE1E2 were amplified with PCR. Recombinant baculovirus were constructed with recombinant DNA techniques. HCV structural proteins expressed in insect cells were analyzed by immunofluorescence and SDS-PAGE. Immunoprecipitation experiment of insect cell lysates with anti-E2 monoclonal antibody (MAb) was carried out and the immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotting with anti-C, anti-E2 MAbs and HCV positive serum. The virus-like particles in insect cells were visualized by electron microscopy (EM). The HCV-like particles were purified by sucrose gradient centrifugation and identified by EM and immune aggregation EM. RESULTS: The recombinant baculovirus reBV/CE1E2 containing HCV C, E1, E2 genes and reBV/CS containing the same structural protein genes plus 5'NCR were constructed. The insect cells infected with either reBV/CE1E2 or reBV/CS expressed HCV C, E1 and E2 proteins with a molecular weight of 20 kD, 35 kD and 66 kD respectively. The results of immunoprecipitation and the immunoblotting revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating the interaction of HCV structural proteins expressed in insect cells. Electron microscopy of insect cells infected with reBV/CE1E2 or reBV/CS demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from sera or hepatic tissues of HCV infected humans. The HCV-like particles were partially purified by sucrose gradient centrifugation, and the purified VLPs showed immuno-reactivity with anti-HCV antibodies. CONCLUSION: HCV 5'NCR is not required for the assembly of HCV-like particles in insect cells, HCV core and envelope proteins are sufficient for viral particle formation.