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1.
PLoS Negl Trop Dis ; 7(5): e2243, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23738026

RESUMO

BACKGROUND: We have developed a manufacturing strategy that can improve the safety and genetic stability of recombinant live-attenuated chimeric dengue vaccine (DENVax) viruses. These viruses, containing the pre-membrane (prM) and envelope (E) genes of dengue serotypes 1-4 in the replicative background of the attenuated dengue-2 PDK-53 vaccine virus candidate, were manufactured under cGMP. METHODOLOGY/PRINCIPAL FINDINGS: After deriving vaccine viruses from RNA-transfected Vero cells, six plaque-purified viruses for each serotype were produced. The plaque-purified strains were then analyzed to select one stock for generation of the master seed. Full genetic and phenotypic characterizations of the master virus seeds were conducted to ensure these viruses retained the previously identified attenuating determinants and phenotypes of the vaccine viruses. We also assessed vector competence of the vaccine viruses in sympatric (Thai) Aedes aegypti mosquito vectors. CONCLUSION/SIGNIFICANCE: All four serotypes of master vaccine seeds retained the previously defined safety features, including all three major genetic loci of attenuation, small plaques, temperature sensitivity in mammalian cells, reduced replication in mosquito cell cultures, and reduced neurovirulence in new-born mice. In addition, the candidate vaccine viruses demonstrated greatly reduced infection and dissemination in Aedes aegypti mosquitoes, and are not likely to be transmissible by these mosquitoes. This manufacturing strategy has successfully been used to produce the candidate tetravalent vaccine, which is currently being tested in human clinical trials in the United States, Central and South America, and Asia.


Assuntos
Vacinas contra Dengue/genética , Vacinas contra Dengue/imunologia , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Aedes , Animais , Animais Recém-Nascidos , Linhagem Celular , Dengue/patologia , Dengue/virologia , Vacinas contra Dengue/efeitos adversos , Vacinas contra Dengue/normas , Modelos Animais de Doenças , Feminino , Instabilidade Genômica , Camundongos , Camundongos Endogâmicos ICR , Controle de Qualidade , Tecnologia Farmacêutica/métodos , Temperatura , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/normas , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/normas , Ensaio de Placa Viral , Virulência , Replicação Viral/efeitos da radiação
2.
Vaccine ; 30(8): 1513-20, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22178727

RESUMO

Formulations of chimeric dengue vaccine (DENVax) viruses containing the pre-membrane (prM) and envelope (E) genes of serotypes 1-4 expressed in the context of the attenuated DENV-2 PDK-53 genome were tested for safety, immunogenicity and efficacy in interferon receptor knock-out mice (AG129). Monovalent formulations were safe and elicited robust neutralizing antibody responses to the homologous virus and only limited cross-reactivity to other serotypes. A single dose of monovalent DENVax-1, -2, or -3 vaccine provided eighty or greater percent protection against both wild-type (wt) DENV-1 (Mochizuki strain) and DENV-2 (New Guinea C strain) challenge viruses. A single dose of monovalent DENVax-4 also provided complete protection against wt DENV-1 challenge and significantly increased the survival times after challenge with wt DENV-2. In studies using tetravalent mixtures, DENVax ratios were identified that: (i) caused limited viremia, (ii) induced serotype-specific neutralizing antibodies to all four DENV serotypes with different hierarchies, and (iii) conferred full protection against clinical signs of disease following challenge with either wt DENV-1 or DENV-2 viruses. Overall, these data highlight the immunogenic profile of DENVax, a novel candidate tetravalent dengue vaccine and the advantage of sharing a common attenuated genomic backbone among the DENVax monovalent vaccines that confer protection against homologous or heterologous virus challenge.


Assuntos
Vacinas contra Dengue/efeitos adversos , Vacinas contra Dengue/imunologia , Interferons/deficiência , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Temperatura Corporal , Peso Corporal , Dengue/mortalidade , Dengue/patologia , Dengue/prevenção & controle , Vacinas contra Dengue/administração & dosagem , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Análise de Sobrevida , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Carga Viral , Viremia/prevenção & controle
3.
Vaccine ; 29(43): 7456-62, 2011 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-21803103

RESUMO

Thermal stability is important for the manufacture, distribution and administration of vaccines, especially in tropical developing countries, where particularly adverse field conditions exist. Current live-attenuated flavivirus vaccines exhibit relatively poor liquid stability in clinical settings, and clinicians are instructed to discard the yellow fever vaccine 1h after reconstitution. We have identified novel combinations of excipients that greatly enhance the thermal stability of live-attenuated DEN-2 PDK-53-based flavivirus vaccine candidates. Liquid formulations comprising a sugar, albumin and a pluronic polymer minimized the loss of flavivirus infectious titer to less than 0.5 log(10)pfu after storage for at least 8h at 37°C, 7 days at room temperature or at least 11 weeks at 4°C. Additionally, these formulations prevented reduction of viral infectivity after two freeze-thaw cycles of virus. Formulated candidate vaccines were readily lyophilized and reconstituted with minimal loss of viral titers. In mice, the formulations were safe and did not hinder the ability of the vaccine virus to generate a potent, protective immune response. These formulations provided significantly greater liquid-phase stability than has been reported previously for other flavivirus vaccine formulations. The enhanced thermal stability provided by the formulations described here will facilitate the effective distribution of flavivirus vaccines worldwide.


Assuntos
Vacinas contra Dengue , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Vacinas Virais , Vírus do Nilo Ocidental/imunologia , Vacina contra Febre Amarela , Albuminas , Animais , Química Farmacêutica , Chlorocebus aethiops , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Camundongos , Testes de Neutralização , Polímeros , Estabilidade Proteica , Temperatura , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Vacina contra Febre Amarela/administração & dosagem , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologia
4.
Am J Trop Med Hyg ; 84(6): 978-87, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21633037

RESUMO

Three tetravalent formulations of chimeric dengue (DENVax) viruses containing the pre-membrane and envelope genes of serotypes 1-4 expressed by the attenuated DENV-2 PDK-53 genome were tested for safety, immunogenicity, and efficacy in cynomolgus macaques (Macaca fascicularis). Subcutaneous injection of the DENVax formulations was well-tolerated. Low levels of viremia of only one of the four vaccine viruses were detected yet virus neutralizing antibody titers were induced against all four dengue virus serotypes after one or two administrations of vaccine. All animals immunized with the high-dose formulation were protected from viremia, and all immunized animals were completely protected from DENV-3 and DENV-4 challenge. A lower dose of DENVax formulation partially protected animals from DENV-1 or DENV-2 challenge. In contrast, all control animals developed high levels of viremia for multiple days after challenge with DENV 1-4. This study highlights the immunogenicity and efficacy of the tetravalent DENVax formulations in nonhuman primates.


Assuntos
Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Análise de Variância , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Células Cultivadas , Chlorocebus aethiops , Dengue/imunologia , Imunidade Celular , Macaca fascicularis , Testes de Neutralização , RNA Viral/sangue , Vacinação , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/imunologia , Células Vero , Viremia/prevenção & controle
5.
Virology ; 406(2): 328-35, 2010 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-20708768

RESUMO

The FG extended loop in domain III of the dengue virus type 2 (DENV2) envelope protein is postulated to be a molecular determinant for host cell infectivity. To determine the contribution of the FG loop to virus infectivity, an infectious cDNA clone of DENV2 was manipulated by deleting amino acids in the loop (VEPGΔ) to mimic tick-borne flaviviruses or by substituting these AAs with RGD or RGDK/S to mimic motifs present in other mosquito-borne flaviviruses. We found the FG loop to be dispensable for infection of C6/36 cells but critical for infection of Aedes aegypti mosquito midguts and mammalian cells. All the FG loop mutants were able to bind to and enter mammalian cells but replication of VEPGΔ in Vero cells at 37 °C was delayed until acquisition of secondary mutations. Reduced binding of DENV2 type-specific monoclonal antibody 3H5 to mutant viruses confirmed the FG loop motif as its target epitope.


Assuntos
Aedes/virologia , Vírus da Dengue/fisiologia , Dengue/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus da Dengue/química , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Células Hep G2 , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Células Vero , Proteínas do Envelope Viral/genética , Replicação Viral
6.
Virology ; 396(2): 305-15, 2010 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-19913272

RESUMO

The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.


Assuntos
Vírus da Dengue/fisiologia , Fusão de Membrana/fisiologia , Proteínas Virais de Fusão/fisiologia , Aedes/virologia , Animais , Antígenos Virais/imunologia , Linhagem Celular , Chlorocebus aethiops , Dengue/virologia , Vírus da Dengue/genética , Feminino , Mutagênese Sítio-Dirigida , Transfecção , Células Vero , Proteínas Virais de Fusão/genética , Replicação Viral/genética , Replicação Viral/fisiologia
7.
J Virol ; 79(12): 7300-10, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15919884

RESUMO

Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers. Two D2/WN chimeras (D2/WN-E2 and -V2) engineered in the backbone of the PDK53-E and -V viruses retained all of the PDK-53 vaccine characteristic phenotypic markers of attenuation and were immunogenic in mice and protected mice from a high-dose 10(7) PFU challenge with wild-type WN NY99 virus. This report further supports application of the genetic background of the D2 PDK-53 virus as a carrier for development of live-attenuated, chimeric flavivirus vaccines in general and the development of a chimeric D2/WN vaccine virus against WN disease in particular.


Assuntos
Vírus da Dengue/imunologia , Recombinação Genética , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Febre do Nilo Ocidental/prevenção & controle , Vírus do Nilo Ocidental/imunologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Humanos , Imunização , Camundongos , Dados de Sequência Molecular , Fenótipo , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/patogenicidade
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