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1.
J Biosci Bioeng ; 117(3): 366-71, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24140131

RESUMO

Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease that caused significant mortality in children, but no vaccine is available yet. EV71 virus-like particle (VLP) is the empty capsid consisting of viral structural proteins but can elicit potent immune responses, rendering VLP a promising EV71 vaccine candidate. To evaluate whether VLP remains stable after long-term storage, which is crucial for advancing the VLP vaccine to the clinical setting, we evaluated the effects of NaCl concentration, buffers and temperatures on the VLP stability. We first validated the use of dynamic light scattering (DLS) for measuring the hydrodynamic diameter (≈30-35 nm) of VLP, which was close to the VLP diameter (≈25-27 nm) as measured by transmission electron microscopy (TEM). Using these techniques, we found that EV71 VLP remained stable for 5 months in sodium phosphate (NaPi) buffers with various NaCl concentrations. EV71 VLP also remained morphologically stable in NaPi, citrate and TE(+) buffers for 5 months, yet the enzyme-linked immunosorbent assay (ELISA) revealed that the VLP stored in citrate and TE(+) buffers partially lost the immunogenicity after 5 months. In contrast, the VLP stored in the NaPi buffer at 4°C remained stable macroscopically and microscopically for 5 months, as judged from the DLS, TEM and ELISA. The VLP stored at 25°C and 37°C also retained stability for 1 month, which would obviate the need of a cold chain during the shipping. These data altogether proved the stability of EV71 VLP and suggested that the VLP is amenable to bioprocessing and storage.


Assuntos
Enterovirus , Vacinas de Partículas Semelhantes a Vírus/química , Proteínas Estruturais Virais/química , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Insetos , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Estabilidade Proteica , Cloreto de Sódio/farmacologia , Vacinas Virais/química
2.
Vaccine ; 30(7): 1305-12, 2012 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-22214888

RESUMO

Enterovirus type 71 (EV71) is a virulent form of enteroviruses causing hospitalizations for children less than three years of age. Currently there are no anti-viral therapies or vaccines available for EV71. Due to the high risk of poliomyelitis-like paralysis and fatal encephalitis, an effective vaccine to EV71 could potentially prevent virus-induced morbidity and mortality. In this study, we first tested a potential EV71 vaccine candidate based on virus-like particles (VLP). We vaccinated macaque monkeys to validate the immunogenicity of the VLP vaccine to EV71. We detected the VLP or EV71-specific antibodies, neutralization titers, ELISPOT, and T cell response to find their immune responses to EV71. When the VLP vaccine adjuvanted with alum was given to macaque monkeys, these monkeys developed both specific humoral and cellular immune responses to EV71. Despite lower neutralizing antibodies to EV71 were found in sera of VLP-immunized monkeys than monkeys vaccinated with inactivated EV71, VLP-based vaccine generated a memory immune response to EV71. Hence, VLP-based EV71 vaccine is a potential vaccine against EV71 infection.


Assuntos
Anticorpos Neutralizantes/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Pré-Escolar , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Humanos , Imunidade Celular , Imunidade Humoral , Memória Imunológica , Macaca , Vacinação , Vacinas de Produtos Inativados , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas Virais/genética
3.
Vaccine ; 28(43): 6951-7, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20797455

RESUMO

To develop the enterovirus 71 (EV71) vaccine, we previously constructed a recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under p10 promoter) proteins, which caused P1 cleavage by 3CD protease and self-assembly of virus-like particles (VLPs) in Sf-9 cells. Assuming that reducing the 3CD expression can alleviate the competition with P1 expression and elevate the VLPs yield, hereby we constructed Bac-P1-C3CD and Bac-P1-I3CD expressing 3CD under weaker CMV and IE-1 promoters, respectively. Western blot and ELISA analyses revealed that Bac-P1-C3CD and Bac-P1-I3CD led to the VLPs release into the supernatant and enhanced the extracellular VLPs yield in Sf-9 cells, but gave poor VLPs production in High Five™ (Hi-5) cells. By optimizing the process parameters including host cells, cell density, culture mode and dissolved oxygen (DO), the best extracellular VLPs yield was achieved by infecting Sf-9 cells (4 × 10(6)cells/mL) cultured in the bioreactor (DO=30%) with Bac-P1-C3CD, which approached ≈64.3mg/L and represented a ≈43-fold increase over the yield (1.5mg/L) attained using the old process (Bac-P1-3CD infection of Sf-9 cells in the spinner flasks). The resultant VLPs not only resembled the VLPs produced from Bac-P1-3CD infection in density, size and shape, but also induced potent antibody responses in mouse models. The antibodies neutralized EV71 strains of homologous and heterologous genogroups, implicating the potential of the VLPs to confer cross-protection for the prevention of future epidemics. Altogether, Bac-P1-C3CD and the bioprocess render mass production more economical, obviate the need for cell lysis and hold promise for future industrial vaccine production.


Assuntos
Técnicas de Cultura de Células , Enterovirus Humano A/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Anticorpos Antivirais/sangue , Baculoviridae/genética , Reatores Biológicos , Contagem de Células , Linhagem Celular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Spodoptera/citologia , Cultura de Vírus/métodos
4.
J Med Chem ; 53(16): 5929-41, 2010 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-20681538

RESUMO

A series of pyrrole-indolin-2-ones were synthesized, and their inhibition profile for Aurora kinases was studied. The potent compound 33 with phenylsulfonamido at the C-5 position and a carboxyethyl group at the C-3' position selectively inhibited Aurora A over Aurora B with IC50 values of 12 and 156 nM, respectively. Replacement of the carboxyl group with an amino group led to compound 47, which retained the activity for Aurora B and lost activity for Aurora A (IC50=2.19 microM). Computation modeling was used to address the different inhibition profiles of 33 and 47. Compounds 47 and 36 (the ethyl ester analogue of 33) inhibited the proliferation of HCT-116 and HT-29 cells and suppressed levels of the phosphorylated substrates of Aurora A and Aurora B in the Western blots.


Assuntos
Antineoplásicos/síntese química , Indóis/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirróis/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Aurora Quinase B , Aurora Quinases , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Células HT29 , Células HeLa , Histonas/metabolismo , Humanos , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Fosforilação , Ligação Proteica , Pirróis/química , Pirróis/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
5.
J Med Chem ; 52(9): 2716-23, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19419203

RESUMO

Hepatitis C virus nonstructural protein 3 (HCV NS3) helicase is believed to be essential for viral replication and has become an attractive target for the development of antiviral drugs. A fluorescence resonant energy transfer helicase assay was established for fast screening of putative inhibitors selected from virtual screening using the program DOCK. Soluble blue HT (1) was first identified as a novel HCV helicase inhibitor. Crystal structure of the NS3 helicase in complex with soluble blue HT shows that the inhibitor bears a significantly higher binding affinity mainly through a 4-sulfonatophenylaminophenyl group, and this is consistent with the activity assay. Subsequently, fragment-based searches were utilized to identify triphenylmethane derivatives for more potent inhibitors. Lead optimization resulted in a 3-bromo-4-hydroxyl substituted derivative 12 with an EC(50) value of 2.72 microM to Ava.5/Huh-7 cells and a lower cytotoxicity to parental Huh-7 cells (CC(50) = 10.5 microM), and it indeed suppressed HCV replication in the HCV replicon cells. Therefore, these inhibitors with structural novelty may serve as a useful scaffold for the discovery of new HCV NS3 helicase inhibitors.


Assuntos
Descoberta de Drogas , Hepacivirus/enzimologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Compostos de Tritil/química , Compostos de Tritil/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Hepacivirus/efeitos dos fármacos , Modelos Moleculares , Replicon/efeitos dos fármacos , Software , Proteínas não Estruturais Virais/química
6.
Hepatology ; 40(6): 1415-20, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15565627

RESUMO

Neonatal immunization with hepatitis B (HB) vaccine is highly effective; however, more needs to be learned about the duration of protection and indications for boosters. We measured antibody to HB core antigen (anti-HBc), HB surface antigen (HBsAg), and pre- and postbooster titers of HBsAg antibody (anti-HBs) 15 years after primary neonatal immunization with plasma-derived HB vaccines in 2 cohorts of 15-year-old children. Group A consisted of 78 children who were born to HB e antigen-positive HBsAg carrier mothers and had developed protective levels of anti-HBs antibodies (> or =10 mIU/mL) following HB immunization. Group B consisted of 113 apparently healthy children whose anti-HBs titers after vaccination were unknown. Anti-HBs was undetectable (antibody titer <10 mIU/mL) in 29.9% in group A and 62.4% in group B (P < .001). Anti-HBc was detected in 33.3 % in group A and 4.4 % in group B (P < .001). After a single booster dose of HB vaccine, 2.7% in group A and 3.3% in group B remained anti-HBs-negative. A blunted serological response was noted in approximately 20% in both groups. One HBsAg carrier was detected in group A (1.3%) and 4 in group B (3.5%). Fifteen years after neonatal immunization with plasma-derived HB vaccine, a large proportion of children exhibited waning immunity. This poses the risk of breakthrough infection. A single booster augmented the serological response to the vaccine in most but not all subjects. In conclusion, our findings suggest that one or more booster immunizations are needed in seronegative subjects by at least 15 years following neonatal immunization with plasma-derived HB vaccine.


Assuntos
Vacinas contra Hepatite B/imunologia , Hepatite B/prevenção & controle , Imunização Secundária , Adolescente , Adulto , Feminino , Hepatite B/epidemiologia , Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Humanos , Recém-Nascido , Masculino , Mães , Fatores de Risco , Falha de Tratamento
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