RESUMEN
BACKGROUND: A Sabin strain-based inactivated poliomyelitis vaccine (Sabin-IPV) is the rational option for completely eradicating poliovirus transmission. The neutralizing capacity of Sabin-IPV immune serum to different strains of poliovirus is a key indicator of the clinical protective efficacy of this vaccine. METHODS: Sera collected from 500 infants enrolled in a randomized, blinded, positive control, phase 2 clinical trial were randomly divided into 5 groups: Groups A, B, and C received high, medium, and low doses, respectively, of Sabin-IPV, while groups D and E received trivalent oral polio vaccine and Salk strain-based IPV, respectively, all on the same schedule. Immune sera were collected after the third dose of primary immunization, and tested in cross-neutralization assays against 19 poliovirus strains of all 3 types. RESULTS: All immune sera from all 5 groups interacted with the 19 poliovirus strains with various titers and in a dose-dependent manner. One type 2 immunodeficiency-associated vaccine-derived poliovirus strain was not recognized by these immune sera. CONCLUSIONS: Sabin-IPV vaccine can induce protective antibodies against currently circulating and reference wild poliovirus strains and most vaccine-derived poliovirus strains, with rare exceptions. CLINICAL TRIALS REGISTRATION: NCT01056705.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sueros Inmunes/inmunología , Vacuna Antipolio de Virus Inactivados/inmunología , Vacuna Antipolio Oral/inmunología , Poliovirus/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Relación Dosis-Respuesta Inmunológica , Humanos , Lactante , Pruebas de Neutralización , Poliomielitis/prevención & control , Poliomielitis/transmisión , Poliomielitis/virología , Poliovirus/genética , Vacuna Antipolio de Virus Inactivados/administración & dosificación , Vacuna Antipolio Oral/administración & dosificación , Análisis de Secuencia de ADN , VacunaciónRESUMEN
BACKGROUND: The development of a Sabin strain-based inactivated poliovirus vaccine (Sabin-IPV) is imperative to protecting against vaccine-associated paralytic poliomyelitis in developing countries. METHODS: In this double-blinded, parallel-group, noninferiority trial, eligible infants aged 60-90 days were randomly assigned in a ratio of 1:1 to receive either 3 doses of Sabin-IPV or Salk strain-based IPV (Salk-IPV) at 30-day intervals and a booster at the age of 18 months. Immunogenicity and safety were assessed on the basis of a protocol. RESULTS: Of 1438 infants, 1200 eligible infants were recruited and received either Sabin-IPV or Salk-IPV. From the Sabin-IPV and Salk-IPV groups, 570 and 564 infants, respectively, completed the primary immunization and formed the per-protocol population. The seroconversion rates of the participants who received Sabin-IPV were 100%, 94.9%, and 99.0% (types I, II, and III, respectively), and those of the participants who received Salk-IPV were 94.7%, 91.3%, and 97.9% 1 month after the completion of primary immunization. An anamnestic response for poliovirus types I, II, and III was elicited by a booster in both groups. Except in the case of fever, other adverse events were similar between the 2 groups. CONCLUSIONS: The immune response induced by Sabin-IPV was not inferior to that established with Salk-IPV.
Asunto(s)
Anticuerpos Antivirales/sangre , Poliomielitis/prevención & control , Vacuna Antipolio de Virus Inactivados/administración & dosificación , Vacuna Antipolio de Virus Inactivados/inmunología , Método Doble Ciego , Femenino , Humanos , Esquemas de Inmunización , Lactante , Masculino , Vacuna Antipolio de Virus Inactivados/efectos adversosRESUMEN
OBJECTIVE: The live-attenuated oral polio vaccine (OPV) will be no longer used when wild poliovirus (WPV) eliminating in worldwide, according to GPEI (the Global Polio Eradication Initiative) Reports. It is planning to replace OPV by Sabin-based inactivated poliovirus vaccine (sIPV) in developing countries, with purpose of reducing of the economic burden and maintaining of the appropriate antibody levels in population. It studied serial fractional doses immunized by intradermal injection (ID) in rats, to reduce consume of antigen and financial burden, maintaining sufficient immunogenicity; Methods: Study groups were divided in 4 groups of dose gradient, which were one-tenth (1/10), one-fifth (1/5), one-third (1/3) and one-full dose (1/1), according to the volume of distribution taken from the same batch of vaccine (sIPV). Wistar rats were injected intradermally with the needle and syringe sing the mantoux technique taken once month for 3 times. It was used as positive control that intramuscular inoculation (IM) was injected with one-full dose (1/1) with same batch of sIPV. PBS was used as negative control. Blood samples were collected via tail vein. After 30 d with 3 round of immunization, it analyzed the changes of neutralization antibody titers in the each group by each immunization program end; Results: The results of seroconversion had positive correlation with different doses in ID groups. The higher concentration of D-antigen (D-Ag) could conduct higher seroconversion. Furthermore, different types of viruses had different seroconversion trend. It showed that the geometric mean titers (GMTs) of each fractional-dose ID groups increased by higher concentration of D-Ag, and it got significant lower than the full-dose IM group. At 90th days of immunization, the GMTs for each poliovirus subtypes of fractional doses were almost higher than 1:8, implied that it could be meaning positive seroprotection titer for polio vaccine types, according to WHO suggestion; Conclusions: The fractional dose with one-fifth (1/5) could be used by intradermal injection to prevent poliovirus infection, if there were more human clinical detail research consistent with this findings in rats.
Asunto(s)
Vacuna Antipolio de Virus Inactivados/administración & dosificación , Vacuna Antipolio de Virus Inactivados/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Femenino , Inyecciones Intradérmicas , Masculino , Pruebas de Neutralización , Ratas WistarRESUMEN
BACKGROUND: Current influenza control strategies require an active surveillance system. Pseudotyped viral particles (pp) together with the evaluation of pre-existing immunity in a population might satisfy this requirement. However, the reliability of using pp in neutralizing antibody (nAb) detection are undefined. METHODOLOGY/PRINCIPAL FINDINGS: Pseudotyped particles of A(H1N1)pmd09 (A/California/7/2009) and HPAI H5N1 (A/Anhui/1/2005), as well as their reassortants, were generated. The reliability of using these pp in nAb detection were compared concurrently with the corresponding viruses by a hemagglutination inhibition test, as well as ELISA-, cytopathic effect-, and fluorescence-based microneutralization assays. In the qualitative detection on nAbs, the pp and their corresponding viruses were in complete agreement, with an R2 value equal to or near 1 in two different populations. In the quantitative detection on nAbs, although the geometric mean titers (95% confidence interval) differed between the pp and viruses, no significant difference was observed. Furthermore, humoral immunity against the reassortants was evaluated; our results indicated strong consistency between the nAbs against reassortant pp and those against naïve pp harboring the same hemagglutinin. CONCLUSION/SIGNIFICANCE: The pp displayed high reliability in influenza virus nAb detection. The use of reassortant pp is a safe and convenient strategy for characterizing emerging influenza viruses and surveying the disease burden.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Humana/sangre , Virión/inmunología , Adolescente , Adulto , Animales , Anticuerpos Antivirales/inmunología , Células Cultivadas , Perros , Células HEK293 , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/inmunología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Persona de Mediana Edad , Virus Reordenados/inmunología , Adulto JovenRESUMEN
The World Health Organization has recommended that a Sabin inactivated polio vaccine (IPV) should gradually and synchronously replace oral polio vaccines for routine immunizations because its benefits in eliminating vaccine-associated paralytic poliomyelitis have been reported in different phases of clinical trials. It is also considered important to explore new tetravalent diphtheria, tetanus, and acellular pertussis-Sabin IPV (DTaP-sIPV) candidate vaccines for possible use in developing countries. In this study, the immunogenicity of a combined tetravalent DTaP-sIPV candidate vaccine was investigated in primates by evaluating the neutralizing antibody responses it induced. The dynamic profiles of the antibody responses to each of the separate antigenic components and serotypes of Sabin IPV were determined and their corresponding geometric mean titers were similar to those generated by the tetravalent diphtheria, tetanus, and acellular pertussis-conventional IPV (DTaP-cIPV), the tetravalent diphtheria, tetanus, and acellular pertussis (DTaP), and Sabin IPV vaccines in the control groups. This implies that protective immunogenic effects are conferred by this combined tetravalent formulation.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/inmunología , Vacuna Antipolio de Virus Inactivados/inmunología , Vacuna Antipolio Oral/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antivirales/sangre , Difteria/prevención & control , Macaca mulatta/inmunología , Poliomielitis/prevención & control , Tétanos/prevención & control , Vacunas Combinadas/inmunología , Tos Ferina/prevención & controlRESUMEN
Highly pathogenic avian influenza (HPAI) viruses pose a global pandemic threat, for which rapid large-scale vaccine production technology is critical for prevention and control. Because chickens are highly susceptible to HPAI viruses, the supply of chicken embryos for vaccine production might be depleted during a virus outbreak. Therefore, developing HPAI virus vaccines using other technologies is critical. Meeting vaccine demand using the Vero cell-based fermentation process has been hindered by low stability and yield. In this study, a Vero cell-based HPAI H5N1 vaccine candidate (H5N1/YNVa) with stable high yield was achieved by reassortment of the Vero-adapted (Va) high growth A/Yunnan/1/2005(H3N2) (YNVa) virus with the A/Anhui/1/2005(H5N1) attenuated influenza vaccine strain (H5N1delta) using the 6/2 method. The reassorted H5N1/YNVa vaccine maintained a high hemagglutination (HA) titer of 1024. Furthermore, H5N1/YNVa displayed low pathogenicity and uniform immunogenicity compared to that of the parent virus.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Virus Reordenados/crecimiento & desarrollo , Cultivo de Virus/métodos , Animales , Embrión de Pollo , Chlorocebus aethiops , Femenino , Humanos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Virus Reordenados/inmunología , Virus Reordenados/patogenicidad , Vacunas Atenuadas/inmunología , Células VeroRESUMEN
BACKGROUND: The production of Sabin inactivated poliovirus vaccine (IPV) can reduce biosafety requirements in the posteradication/post-oral poliovirus vaccine (OPV) era. We conducted a phase II, randomized, positive-controlled trial to assess the safety and immunogenicity of Sabin IPV. METHODS: The test groups (A, B, and C) received 3 doses of high, middle, and low D antigen (D Ag) of Sabin IPV at ages 2, 3, and 4 months, respectively. Infants in 2 control groups, group D and group E, received 3 doses of trivalent OPV and conventional IPV (cIPV), respectively, on the same schedule as that of groups A, B, and C. Serum samples were collected before and 30 days after the administration of the third dose. RESULTS: In total, 500 infants were randomly assigned to 5 groups, and 449 infants completed the vaccine series. No serious adverse events were associated with vaccinations. After 3 doses, the seroconversion rates in groups A, B, C, D, and E were 100%, 97.8%, 96.6%, 100%, and 90.1%, respectively, for type 1 poliovirus; 97.7%, 95.7%, 78.7%, 100%, and 90.1%, respectively, for type 2; and 98.8%, 98.9%, 93.3%, 100%, and 97.8%, respectively, for type 3. CONCLUSIONS: Sabin IPV has good safety characteristics. The seroconversion rates for type 1 poliovirus (most appropriate concentration, 15 D Ag units [DU]), type 2 (32 DU), and type 3 (45 DU) Sabin IPV were similar to those of the OPV and cIPV control groups. CLINICAL TRIALS REGISTRATION: NCT01056705.
Asunto(s)
Poliomielitis/inmunología , Vacuna Antipolio de Virus Inactivados/efectos adversos , Vacuna Antipolio de Virus Inactivados/inmunología , Vacuna Antipolio Oral/efectos adversos , Vacuna Antipolio Oral/inmunología , Poliovirus/inmunología , Anticuerpos Antivirales/sangre , Distribución de Chi-Cuadrado , Intervalos de Confianza , Femenino , Humanos , Lactante , Masculino , Vacuna Antipolio de Virus Inactivados/administración & dosificación , Vacuna Antipolio Oral/administración & dosificaciónRESUMEN
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.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/inmunología , Animales , Anticuerpos Antivirales/inmunología , Embrión de Pollo , Chlorocebus aethiops , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Recombinación Genética , Células VeroRESUMEN
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.
Asunto(s)
Adaptación Biológica , Virus de la Influenza B/crecimiento & desarrollo , Animales , Chlorocebus aethiops , Análisis por Conglomerados , Pruebas de Inhibición de Hemaglutinación , Virus de la Influenza B/genética , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia , Pase Seriado , Células Vero , Carga ViralRESUMEN
UNLABELLED: DNA immunization has been used to induce either humoral or cellular immune responses against many antigens, including hepatitis C virus (HCV). In addition, DNA immunizations can be enhanced or modulated at the nucleotide level. Genetic immunizations were examined in BALB/c mice through the use of plasmids and chimeric DNA constructs encoding HCV core proteins and hepatitis B virus (HBV) precore (preC) regions. Plasmids encoding the truncated HCV core induced potent humoral and cellular responses to HCV; pcDNA3.0A-C154 produced a stronger antibody response than pcDNA3.0A-C191 (P < 0.01) and pcDNA3.0A-C69 (P < 0.05). HBV preC enhanced the humoral and cellular immune responses of BALB/c mice to HCV; however, pcDNA3.0A-C69preC resulted in a weak cytotoxic T lymphocyte (CTL) response. In addition, the humoral and cellular immune responses to HCV of groups immunized with pcDNA3.0A-C154preC and pcDNA3.0A-C191preC plasmids were higher than those of groups immunized with pcDNA3.0A-C154 and pcDNA3.0A-C191. In vivo CTL responses verified that mice immunized with preC core fused DNAs showed significantly high specific lysis compared with mice immunized with HCV cores only (P < 0.01). In our study, pcDNA3.0A-C154preC led to the highest immune response among all DNA constructs. CONCLUSION: DNA that encodes truncated HCV core proteins may lead to increased immune responses in vivo, and these responses may be enhanced by HBV preC.
Asunto(s)
Formación de Anticuerpos/efectos de los fármacos , Hepacivirus/inmunología , Virus de la Hepatitis B/inmunología , Inmunidad Celular/efectos de los fármacos , Proteínas del Núcleo Viral/inmunología , Vacunas contra Hepatitis Viral/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica , Hepacivirus/genética , Virus de la Hepatitis B/genética , Hepatitis C/prevención & control , Inmunización/métodos , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Linfocitos T Citotóxicos/efectos de los fármacos , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Vacunas contra Hepatitis Viral/uso terapéuticoRESUMEN
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.
Asunto(s)
Biotecnología/métodos , Vacunas contra la Hepatitis A/metabolismo , Vacunas de Productos Inactivados/metabolismo , Vacunas Virales/metabolismo , Inactivación de Virus , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Cultivo de Virus/métodosRESUMEN
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.
Asunto(s)
Técnicas de Química Analítica/métodos , Vacuna Antipolio Oral/química , Proteínas/análisis , Aminoácidos/metabolismo , Calibración , Fenoles/química , Ácido Tricloroacético/químicaRESUMEN
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.
Asunto(s)
Glicoles de Etileno/farmacología , Vacuna Antipolio de Virus Inactivados/inmunología , Animales , Antiinfecciosos Locales/administración & dosificación , Antiinfecciosos Locales/farmacología , Antiinfecciosos Locales/toxicidad , Antígenos Virales/análisis , Antígenos Virales/inmunología , Peso Corporal/efectos de los fármacos , Chlorocebus aethiops , Estabilidad de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Glicoles de Etileno/administración & dosificación , Glicoles de Etileno/toxicidad , Cobayas , Ratones , Pruebas de Neutralización , Vacuna Antipolio de Virus Inactivados/administración & dosificación , Vacuna Antipolio de Virus Inactivados/toxicidad , Células VeroRESUMEN
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.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos de la Hepatitis C/inmunología , Vacunas de ADN/inmunología , Vacunas contra Hepatitis Viral/inmunología , Animales , Células COS , Chlorocebus aethiops , ADN Recombinante/inmunología , Expresión Génica , Hepatitis B/sangre , Hepatitis B/inmunología , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/genética , Hepatitis C/sangre , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/sangre , Antígenos de la Hepatitis C/genética , Inmunización/métodos , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Vacunas de ADN/genética , Vacunas contra Hepatitis Viral/genéticaRESUMEN
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.
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Hepacivirus/fisiología , Proteínas Estructurales Virales/fisiología , Virión/fisiología , Ensamble de Virus , Animales , Técnica del Anticuerpo Fluorescente , Hepacivirus/inmunología , Inmunización , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Spodoptera , Proteínas Estructurales Virales/inmunologíaRESUMEN
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.
Asunto(s)
Reactores Biológicos , Virus de la Hepatitis A/crecimiento & desarrollo , Cultivo de Virus/métodos , Animales , Antígenos Virales/metabolismo , Chlorocebus aethiops , Microesferas , Células Vero/metabolismo , Células Vero/virología , Replicación ViralRESUMEN
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.