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1.
APMIS ; 128(11): 593-602, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32870528

RESUMO

Induction of broad Th1 cellular immune responses and cytokines is crucial characteristics for vaccines against intracellular infections such as hepatitis C virus (HCV). Plants (especially oilseed tissues) and plant-immunomodulators (like oil bodies) offer cost-effective and scalable possibilities for the production of immunologically relevant and safe vaccine antigens and adjuvants, respectively. Herein, we provide data of the murine immunization by transgenic canola oilseed-derived HCV core protein (HCVcp) soluble extract (TSE) and Escherichia coli- derived rHCVcp in combination with Canola oil bodies (oil) compared to that of the Freund's (FA) adjuvant. Mice immunized by TSE+ oil developed both strong humeral (IgG) and Th1-biased cellular responses, manifested by high levels of IFN-γ and lower IgG1/IgG2a ratio and IL-4 secretion. Results of the intracellular cytokine staining indicated that TSE+ oil immunization in mice triggered both CD4+ and CD8+ T cells to release IFN-γ, while CD4+ cells were mostly triggered when FA was used. Analyses by qRT-PCR indicated that a combination of rHCVcp/TSE with oil body induced high levels of IL-10 cytokines compared to that of the FA adjuvant. These characteristics are important properties for the design of an HCV vaccine candidate and indicate the potential of Canola-derived antigen and oil bodies in addressing these concerns.


Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/prevenção & controle , Proteínas Recombinantes/administração & dosagem , Células Th1/efeitos dos fármacos , Proteínas do Core Viral/administração & dosagem , Vacinas contra Hepatite Viral/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C Crônica/imunologia , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Imunidade Celular/efeitos dos fármacos , Imunoglobulina G/biossíntese , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Óleo de Brassica napus/administração & dosagem , Óleo de Brassica napus/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Células Th1/virologia , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/imunologia , Vacinas contra Hepatite Viral/biossíntese
2.
Mol Med Rep ; 19(2): 1016-1023, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569131

RESUMO

Hepatitis C virus (HCV) infection remains a major public health issue despite the introduction of several direct­acting antiviral agents (DAAs), with some 185 million individuals infected with HCV worldwide. There is an urgent need for an effective prophylactic HCV vaccine. In the present study, we constructed genetic vaccines based on novel recombinant adeno­associated viral (rAAV) vectors (AAV2/8 or AAV2/rh32.33) that express the envelope glycoprotein E2 from the HCV genotype 1b. Expression of HCV E2 protein in 293 cells was confirmed by western blot analysis. rAAV2/8.HCV E2 vaccine or rAAV2/rh32.33.HCV E2 vaccine was intramuscularly injected into C57BL/6 mice. HCV E2­specific antigen was produced, and long­lasting specific antibody responses remained detectable XVI weeks following immunization. In addition, the rAAV2/rh32.33 vaccine induced higher antigen­specific antibody levels than the rAAV2/8 vaccine or AAV plasmid. Moreover, both AAV vaccines induced neutralizing antibodies against HCV genotypes 1a and 1b. Finally, it is worth mentioning that neutralizing antibody levels directed against AAV2/rh32.33 were lower than those against AAV2/8 in both mouse and human serum. These results demonstrate that AAV vectors, especially the AAVrh32.33, have particularly favorable immunogenicity for development into an effective HCV vaccine.


Assuntos
Anticorpos Neutralizantes/biossíntese , Dependovirus/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/biossíntese , Hepatite C Crônica/prevenção & controle , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Adulto , Animais , Anticorpos Neutralizantes/sangue , Dependovirus/genética , Feminino , Vetores Genéticos/química , Vetores Genéticos/imunologia , Células HEK293 , Hepacivirus/genética , Anticorpos Anti-Hepatite C/sangue , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Soros Imunes/química , Imunidade Humoral/efeitos dos fármacos , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/biossíntese , Vacinas de DNA/genética , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/genética
3.
Iran Biomed J ; 23(1): 57-67, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30056690

RESUMO

Background: Hepatitis C virus (HCV) is a blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was to design and prepare a specific mRNA, without 5' cap and poly (A) tail transcribed in vitro capable of coding core protein and also to determine its functionality. Methods: Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of 5ʹ and 3ʹ untranslated regions of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) sequences of eIF4G related to human dendritic cells (DCs), and the Core gene of HCV. To design the modified mRNA, the 5' cap and poly (A) tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA (IVT-mRNA) and the expressions of green fluorescent protein (GFP), and Core genes were determined by microscopic examination and Western blotting assay. Results: Cell transfection results showed that despite the absence of 5' cap and poly (A) tail, the structure of the mRNA was stable. Moreover, the successful expressions of GFP and Core genes were achieved. Conclusion: Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV.


Assuntos
Hepacivirus/genética , Proteínas do Core Viral/genética , Vacinas contra Hepatite Viral/biossíntese , Células Dendríticas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Monócitos/citologia , Conformação de Ácido Nucleico , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Termodinâmica , Transcrição Genética
4.
J Virol ; 91(1)2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27795422

RESUMO

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE: A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.


Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/biossíntese , Hepatite C/prevenção & controle , Proteínas Recombinantes de Fusão/biossíntese , Proteínas do Envelope Viral/biossíntese , Vacinas contra Hepatite Viral/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/química , Antígenos Virais/química , Antígenos Virais/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Cromatografia em Agarose/métodos , Reações Cruzadas , Epitopos/química , Epitopos/imunologia , Hepacivirus/química , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/química , Humanos , Soros Imunes/química , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Camundongos , Testes de Neutralização , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Vacinação , Vacinas Sintéticas , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/biossíntese
6.
Vaccine ; 32(32): 4039-50, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-24892250

RESUMO

The hepatitis E virus (HEV) vaccine, Hecolin(®), was licensed in China for the prevention of HEV infection and HEV-related diseases with demonstrated safety and efficacy [1,2]. The vaccine is composed of a truncated HEV capsid protein, p239, as the sole antigen encoded by open reading frame 2 and produced using Escherichia coli platform. The production of this virus-like particle (VLP) form of the antigen was successfully scaled up 50-fold from a bench scale to a manufacturing scale. Product consistency was demonstrated using a combination of biophysical, biochemical and immunochemical methods, which revealed comparable antigen characteristics among different batches. Particle size of the nanometer scale particulate antigen and presence of key epitopes on the particle surface are two prerequisites for an efficacious VLP-based vaccine. The particle size was monitored by several different methods, which showed diameters between 20 and 30nm for the p239 particles. The thermal stability and aggregation propensity of the antigen were assessed using differential scanning calorimetry and cloud point assay under heat stress conditions. Key epitopes on the particulate antigen were analyzed using a panel of murine anti-HEV monoclonal antibodies (mAbs). The immuno reactivity to the mAbs among the different antigen lots was highly consistent when analyzed quantitatively using a surface plasmon resonance technique. Using a sandwich ELISA to probe the integrity of two different epitopes in the antigen, the specific antigenicity of multiple batches was assessed to demonstrate consistency in these critical product attributes. Overall, our findings showed that the antigen production process is robust and scalable during the manufacturing of Hecolin(®).


Assuntos
Proteínas do Capsídeo/imunologia , Vírus da Hepatite E , Vacinas Sintéticas/biossíntese , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas contra Hepatite Viral/biossíntese , Animais , Anticorpos Monoclonais Murinos/imunologia , Antígenos Virais/imunologia , Epitopos/imunologia , Feminino , Camundongos Endogâmicos BALB C , Peso Molecular , Tamanho da Partícula , Vacinas Sintéticas/normas , Vacinas de Partículas Semelhantes a Vírus/normas , Vacinas contra Hepatite Viral/normas
7.
Nat Med ; 19(7): 869-78, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23836237

RESUMO

Despite major advances in the understanding and treatment of hepatitis C, a preventive vaccine remains elusive. The marked genetic diversity and multiple mechanisms of persistence of hepatitis C virus, combined with the relatively poor immune response of the infected host against the virus, are major barriers. The lack of robust and convenient model systems further hampers the effort to develop an effective vaccine. Advances in our understanding of virus-host interactions and protective immunity in hepatitis C virus infection provide an important roadmap to develop potent and broadly directed vaccine candidates targeting both humoral and cellular immune responses. Multiple approaches to generating and testing viral immunogens have met with variable success. Several candidates have advanced to clinical trials based on promising results in chimpanzees. The ultimate path to a successful preventive vaccine requires comprehensive evaluations of all aspects of protective immunity, innovative application of state-of-the-art vaccine technology and properly designed vaccine trials that can affirm definitive endpoints of efficacy.


Assuntos
Descoberta de Drogas/tendências , Hepacivirus/imunologia , Vacinas contra Hepatite Viral/síntese química , Animais , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepacivirus/fisiologia , Hepatite C/prevenção & controle , Hepatite C/terapia , Humanos , Imunidade/genética , Imunidade/fisiologia , Modelos Biológicos , Pan troglodytes , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/uso terapêutico
8.
Vaccine ; 28(5): 1234-40, 2010 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-19941986

RESUMO

Recombinant viruses expressing foreign antigens may provide a convenient vaccine vector capable of inducing preventative immunity. In this study, we explored the capacity of highly attenuated Coxsackievirus B3 (CVB3) to act as a recombinant vector to deliver foreign genes into experimental animals for the purpose of vaccination. The infectious cDNA of highly attenuated CVB3, YYFF, which has been reported previously (Vaccine 27:1974), was used to construct a recombinant YYFF cDNA (YYFF-HCV) by inserting a truncated form of hepatitis C virus (HCV) envelope protein E2 as an antigenic marker immediately upstream from the gene encoding the VP4 capsid protein. In YYFF-HCV-infected HeLa cells, HCV E2 expression was confirmed by immunoblotting and fluorescence microscopy. YYFF-HCV induced the production of antibodies and the cytotoxic T-lymphocyte (CTL) response to HCV E2 in the inoculated mice. Moreover, YYFF-HCV induced no inflammation in the virus-immunized mice. These results demonstrate that recombinant CVB3 expressing a foreign gene can act as a live vaccine vector capable of inducing humoral and cell-mediated immune responses directed against a foreign protein.


Assuntos
Enterovirus Humano B/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Formação de Anticorpos/imunologia , Células COS , Chlorocebus aethiops , Enterovirus Humano B/genética , Enterovirus Humano B/metabolismo , Expressão Gênica , Células HeLa , Hepacivirus/genética , Anticorpos Anti-Hepatite C/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/metabolismo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/genética
9.
World J Gastroenterol ; 13(34): 4630-5, 2007 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-17729420

RESUMO

AIM: To investigate the hotspots, direction, and the time course of evolution of hepatitis A virus in the process of consecutive cell culture passage in human KMB17 diploid cells. METHODS: Wild type hepatitis A virus H2w was serially propagated in KMB17 cells until passage 30, and the full-length genomes of H2w and its six chosen progenies were determined by directly sequencing RT-PCR products amplified from viral genomic RNA. Alignment comparison of sequences from H2w with its six progenies and phylogenetic analysis of the whole VP1 region from H2w, progenies of H2w, and other cell culture adapted hepatitis A virus were then carried out to obtain data on the molecular evolution of hepatitis A virus in the process of consecutive passage in KMB17 cells. RESULTS: Most of the mutations occurred by passage 5 and several hotspots related to adaptation of the virus during cell growth were observed. After that stage, few additional mutations occurred through the remaining duration of passage in KMB17 cells except for mutation in the virulence determinants, which occurred in the vicinity of passage 15. The phylogenetic analysis of the whole VP1 region suggested that the progenies of H2w evolved closely to other cell culture adapted hepatitis A virus, i.e. MBB, L-A-1, other than its progenitor H2w. CONCLUSION: Hepatitis A virus served as a useful model for studying molecular evolution of viruses in a given environment. The information obtained in this study may provide assistance in cultivating the next generation of a seed virus for live hepatitis A vaccine production.


Assuntos
Proliferação de Células , Diploide , Evolução Molecular , Vírus da Hepatite A/genética , Pulmão/virologia , Mutação , RNA Viral , Vacinas contra Hepatite Viral/genética , Linhagem Celular , Análise Mutacional de DNA , Bases de Dados Genéticas , Vírus da Hepatite A/crescimento & desenvolvimento , Vírus da Hepatite A/imunologia , Vírus da Hepatite A/patogenicidade , Humanos , Pulmão/citologia , Pulmão/embriologia , Filogenia , Fatores de Tempo , Vacinas contra Hepatite Viral/biossíntese , Fatores de Virulência/genética , Replicação Viral
10.
Vaccine ; 23(22): 2893-901, 2005 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-15780738

RESUMO

It was evaluated its antigenicity, immunogenicity and efficacy of a candidate recombinant hepatitis E virus (HEV) vaccine, referred hitherto as HEV 239 vaccine. The vaccine peptide has a 26 amino acids extension from the N terminal of another peptide, E2, of the HEV capsid protein, which has been shown to protect monkeys against HEV infection previously. The vaccine peptide is similar as E2 in that: first, the vaccine peptide migrates predominantly as dimer in SDS-PAGE and it is dissociated into monomers by heating; second, its dimeric form of which predominantly recognized by HEV reactive human serum; and third, it shows the same pattern of reaction as E2 with a panel of eight monoclonal antibodies that had been raised against E2. In contrast to E2, the vaccine peptide aggregates to form particles of 13 nm mean radius, and consequently, it is more than 240 times more immunogenic than E2. Using alum as adjuvant, immunizing dose determined in mice was 80-250 ng for the vaccine and >60 microg for E2. Rhesus monkeys twice vaccinated with a 10 microg or a 20 microg formulation of this vaccine showed essentially the same antibody response, whereas the response to a 5 microg formulation was delayed but reached similar antibody levels. All the three vaccine formulations afford complete protection against infection with 10(4) genome equivalent dose of the homologous genotype 1 virus. At higher virus dose of 10(7), the same vaccine formulation partially protected against the infection and completely protected against hepatitis. The efficacy of the vaccine was essentially the same for the homologous genotype 1 virus and heterologous genotype 4 virus.


Assuntos
Hepatite E/prevenção & controle , Vacinas contra Hepatite Viral/imunologia , Animais , Eletroforese em Gel de Poliacrilamida , Hepatite E/imunologia , Macaca mulatta , Vacinas contra Hepatite Viral/biossíntese
11.
Sheng Wu Gong Cheng Xue Bao ; 20(2): 262-8, 2004 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-15969119

RESUMO

An E. coli expressed recombinant antigen NE2 was reported to aggregate into homo-oligomer, and can induce protective antibodies on rhesus monkey, but its immunogenicty was much weak after being purified. In this study, three N-terminal extension mutant of NE2 were expressed in E. coli, one of which named HEV 239 was found to aggregate into particle. HEV 239 antigen had good reactivity with sera of hepatitis E patients. The reactivity of HEV 239 against neutralization monoclonal antibody 8C11 was similar as NE2 antigen, while the reactivity of it against another neutralization monoclonal antibody 8H3 is much better than NE2 antigen, which indicated better antigenicity of HEV 239 than NE2. The diameter of purified HEV 239 particulate antigen was between 15 nm to 30 nm. The ED50 of immunization of HEV 239 particle adsorbed by aluminum adjuvant to BALB/c mice was between 0.08 microg to 0.25 microg. In contrast, the seraconversion rate of mice immunized by NE2 antigen adsorbed by aluminium adjuvant was only 25% on 60 microg vaccination. These results suggested that HEV 239 antigen particle has better immunogenicity as well as antigenicity than those of NE2 antigen, so it is a better vaccine candidate against HEV.


Assuntos
Vírus da Hepatite E/genética , Proteínas do Nucleocapsídeo/biossíntese , Proteínas do Nucleocapsídeo/imunologia , Proteínas Recombinantes/biossíntese , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Vetores Genéticos/genética , Antígenos de Hepatite/imunologia , Vírus da Hepatite E/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/imunologia
12.
Arch Virol ; 145(12): 2557-73, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11205105

RESUMO

Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis with over 180 million cases worldwide. Vaccine development for HCV has been difficult. Presently, the virus cannot be grown in tissue culture and there is no vaccine or effective therapy against this virus. In this research, we describe the development of an experimental plant-derived subunit vaccine against HCV. A tobamoviral vector was engineered to encode a consensus sequence of hypervariable region 1 (HVR1), a potential neutralizing epitope of HCV, genetically fused to the C-terminal of the B subunit of cholera toxin (CTB). This epitope was selected from among the amino acid sequences of HVR1 "mimotopes" previously derived by phage display technology. The nucleotide sequence encoding this epitope was designed utilizing optimal plant codons. This mimotope is capable of inducing cross-neutralizing antibodies against different variants of the virus. Plants infected with recombinant tobacco mosaic virus (TMV) engineered to express the HVR1/CTB chimeric protein, contained intact TMV particles and produced the HVR1 consensus peptide fused to the functionally active, pentameric B subunit of cholera toxin. Plant-derived HVR1/CTB reacted with HVR1-specific monoclonal antibodies and immune sera from individuals infected with virus from four of the major genotypes of HCV. Intranasal immunization of mice with a crude plant extract containing the recombinant HVR1/CTB protein elicited both anti-CTB serum antibody and anti-HVR1 serum antibody which specifically bound to HCV virus-like particles. Using plant-virus transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant-derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccines.


Assuntos
Hepacivirus/genética , Proteínas de Plantas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Vacinas contra Hepatite Viral/biossíntese , Administração Intranasal , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Feminino , Vetores Genéticos , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas de Plantas/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus do Mosaico do Tabaco/genética , Vacinação , Vacinas de Subunidades/biossíntese , Vacinas de Subunidades/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia
13.
Vopr Virusol ; 40(5): 215-8, 1995.
Artigo em Russo | MEDLINE | ID: mdl-8659176

RESUMO

Modernization of the protocol and optimization of the technology of preparation of inactivated hepatitis A vaccine helped prepare the experimental lots of inactivated vaccine meeting all the requirements to such preparations and the WHO recommendations.


Assuntos
Hepatite A/prevenção & controle , Hepatovirus/imunologia , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/imunologia , Animais , Células Cultivadas , Chlorocebus aethiops
14.
J Hepatol ; 18 Suppl 2: S20-6, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8182268

RESUMO

The development of the formalin-inactivated hepatitis A vaccine, VAQTA, culminates nearly two decades of the basic science studies of VAQTA in hepatitis A virology at the MRL. The master seed virus for production of VAQTA is derived from the F'(P18) variant of the strain CR326F which has been studied in human clinical trials and shown to the highly attenuated. The antigen is highly purified to make possible the consistency and thoroughness of its inactivation by formalin. Phase I clinical studies of VAQTA were initiated in 1989 and have progressed since that time to the recent Phase III clinical trials which demonstrated efficacy of a single dose of the vaccine in preventing clinical hepatitis A disease in pediatric populations in Monroe, NY.


Assuntos
Hepatovirus/imunologia , Vacinas contra Hepatite Viral/biossíntese , Animais , Formaldeído , Vacinas contra Hepatite A , Humanos , Vacinas de Produtos Inativados/biossíntese
15.
Cytotechnology ; 9(1-3): 173-87, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1283686

RESUMO

Microcarrier culture was investigated for the propagation of attenuated hepatitis A vaccine in the anchorage-dependent human fibroblast cell line, MRC-5. Cells were cultivated at 37 degrees C for one to two weeks, while virus accumulation was performed at 32 degrees C over 21 to 28 days. The major development focus for the microcarrier process was the difference between the cell and virus growth phases. Virus antigen yields, growth kinetics, and cell layer/bead morphology were each examined and compared for both the microcarrier and stationary T-flask cultures. Overall, cell densities of 4-5 x 10(6) cells/ml at 5-10 milligrams beads were readily attained and could be maintained in the absence of infection at either 37 degrees C or 32 degrees C. Upon virus inoculation, however, substantial cell density decreases were observed as well as 2.5 to 10-fold lower per cell and per unit surface area antigen yields as compared to stationary cultures. The advantages as well as the problems presented by the microcarrier approach will be discussed.


Assuntos
Biotecnologia/instrumentação , Dextranos , Hepatovirus/crescimento & desenvolvimento , Microesferas , Antígenos Virais/análise , Adesão Celular/fisiologia , Contagem de Células , Divisão Celular/fisiologia , Linhagem Celular , Fluoresceínas , Vacinas contra Hepatite A , Temperatura Alta , Humanos , Reprodutibilidade dos Testes , Vacinas Atenuadas/biossíntese , Vacinas contra Hepatite Viral/biossíntese
16.
Vaccine ; 10 Suppl 1: S99-101, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1335671

RESUMO

The isolation and adaptation of hepatitis A virus to cell culture opened the way to the development of vaccines. Based on experience with inactivated poliovaccines, a similar approach was chosen for the development of an inactivated hepatitis A vaccine. Strain HM175, adapted to MRC-5 human diploid cells, was used as the virus strain. Vaccine production starts with growth and multiplication of the seed virus in MRC-5 cells. The harvests are clarified, purified and concentrated. Inactivation by formaldehyde is carried out on a pool of purified harvests. Close control of all process parameters results in consistent production of completely inactivated and highly immunogenic vaccine lots. Quality control testing is based on the general requirements for biologicals of WHO and National Control Authorities. Tests have been developed and validated to show the purity of the cell substrate used for each production cycle, the quality of the virus harvest, the adequacy of the purification and inactivation processes, and the conformity to stringent specifications for purity, safety and potency of the final bulk vaccine filled in final containers. The vaccine is characterized by adequate identity tests, by its reaction with polyclonal and monoclonal antibodies, by its immunogenicity in laboratory animals and by the detailed study of the immune response in primates and human volunteers. The final result of the development of adequate production and testing methods, confirmed by extensive characterization studies, is the availability of a consistent, safe and potent hepatitis A vaccine.


Assuntos
Hepatovirus/imunologia , Vacinas contra Hepatite Viral , Linhagem Celular , Anticorpos Anti-Hepatite A , Vacinas contra Hepatite A , Anticorpos Anti-Hepatite/biossíntese , Hepatovirus/crescimento & desenvolvimento , Hepatovirus/isolamento & purificação , Humanos , Controle de Qualidade , Vacinas de Produtos Inativados/biossíntese , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/normas , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/imunologia , Vacinas contra Hepatite Viral/normas , Replicação Viral
17.
J Hosp Infect ; 18 Suppl A: 164-9, 1991 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1679780

RESUMO

Vaccination is one of the major preventive measures against infectious diseases. With the exception of the hepatitis B vaccine, the vaccines in use today are produced from the infectious agents themselves, either by attenuation or inactivation. Although these products have been successful in controlling many diseases, there are several reasons why efforts are being made to improve their quality. In addition there are some infectious diseases for which vaccines are not available because the causal agents cannot be grown in sufficient quantities. New approaches will be required to obtain effective vaccines against these diseases. In this paper, these approaches to the design of new vaccines are described using hepatitis B, rabies and foot-and-mouth disease as examples.


Assuntos
Vacinas Sintéticas/biossíntese , Vacinas Virais/biossíntese , Animais , Mapeamento Cromossômico , Febre Aftosa/prevenção & controle , Hepatite B/prevenção & controle , Humanos , Vacinas Antirrábicas/biossíntese , Vacinas Antirrábicas/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/normas , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/genética , Vacinas Virais/genética , Vacinas Virais/normas
20.
Tsitologiia ; 32(7): 736-40, 1990.
Artigo em Russo | MEDLINE | ID: mdl-2175469

RESUMO

The karyological analysis of the cell line 4647 used for production of a killed vaccine to Hepatitis A virus was run in the 98th, 107th, 117th and 127th passages by the routine and C, G, and Ag methods of differential chromosome staining. A considerable balancing of the chromosome composition at 107-127 passage levels is shown. The cells of line 4647 present a significant heterogeneity, as to the number of chromosomes, and do not belong to any distinct modal class. The modal number of chromosomes ranged from 61 to 66 and from 121 to 125 for hyperdiploid and polyploid cells, respectively. The stable modal class of cells was established in the tetrasomic region, when culturing in the medium containing 10% CS from the 107th passage, and in the medium containing 10% FBS from the 117th passage, which conforms to one of the WHO requirements asserted to the substrate cells.


Assuntos
Rim/ultraestrutura , Animais , Linhagem Celular , Células Cultivadas/ultraestrutura , Chlorocebus aethiops , Bandeamento Cromossômico/métodos , Meios de Cultura , Marcadores Genéticos , Hepatovirus/imunologia , Cariotipagem , Fatores de Tempo , Vacinas contra Hepatite Viral/biossíntese
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