RESUMO
Recombinant baculovirus (BV) expression systems are widely applied in the production of viral capsid proteins and virus-like particles (VLPs) for use as immunogens and vaccine candidates. Traditional density gradient purification of VLPs does not enable complete elimination of BV-derived impurities, including live viruses, envelope glycoprotein gp64 and baculoviral DNA. We used an additional purification system based on ionic strength to purify norovirus (NoV) GII-4 capsid-derived VLPs. The anion exchange chromatography purification led to highly purified VLPs free from BV impurities with intact morphology. In addition, highly purified VLPs induced strong NoV-specific antibody responses in BALB/c mice. Here, we describe a method for NoV VLP purification and several methods for determining their purity, including quantitative PCR for BV DNA detection.
Assuntos
Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Cromatografia por Troca Iônica/métodos , Norovirus/imunologia , Vacinas Virais/imunologia , Animais , Baculoviridae/genética , Proteínas do Capsídeo/isolamento & purificação , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Norovirus/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagemRESUMO
Noroviruses are, after rotaviruses, the second most common causative agents of acute gastroenteritis in young children. We studied norovirus genotypes in faecal specimens collected from Finnish children followed-up prospectively in rotavirus vaccine trials. Almost 5000 faecal specimens collected from cases of acute gastroenteritis were examined using reverse transcriptase-PCR. A total of 1172 cases (25% of all acute gastroenteritis) were associated with noroviruses. Of these, 96% were genogroup GII. GII.4 was the most common genotype (46%) throughout the study period but the proportion of this genotype varied in different norovirus epidemic seasons. Additional norovirus genotypes detected were: GII.7 (15%), GII.3 (14%), GII.1 (9%), GII.b (7%), GII.2 (3%), and GI.3 (2%). GII.4 dominated during the following years: 1998-1999 (75%), 2002-2003 (88%) and 2006-2007 (98%) while recombinant genotype GII.b was dominant between 2003 and 2004 (83%). In conclusion, genotypes GII.4 and GIIb have emerged as predominant norovirus genotypes in endemic gastroenteritis affecting young infants and children in Finland.
Assuntos
Infecções por Caliciviridae/virologia , Fezes/virologia , Gastroenterite/virologia , Norovirus/genética , Infecções por Caliciviridae/epidemiologia , Capsídeo , Proteínas do Capsídeo/genética , Pré-Escolar , Surtos de Doenças , Finlândia/epidemiologia , Gastroenterite/epidemiologia , Genótipo , Humanos , Incidência , Lactente , Norovirus/classificação , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estações do Ano , Análise de Sequência de RNARESUMO
Noroviruses (NoVs) are the second most common cause of viral gastroenteritis after rotavirus in children. NoV genotype GII-4 has emerged as the major type not only in outbreaks of NoV gastroenteritis but also endemic gastroenteritis among infants and young children worldwide. Using baculovirus-insect cell system virus-like particles (VLPs) of NoV genotype GII-4 and an uncommon genotype GII-12 were produced. These VLPs were used in enzyme-linked immunosorbent assays (ELISA) for detection of NoV-specific immunoglobulin G (IgG) and IgA antibodies in 492 serum specimens from Finnish children 0-14 years of age collected between 2006 and 2008. NoV IgG antibody prevalence was 47.3% in the age group 7-23 months and increased up to 91.2% after the age of 5 years. Avidity of NoV IgG antibodies was low in the primary infections while high avidity antibodies were detected in the recurrent infections of the older children. In GII-4 infections, the homologous antibody response to GII-4 VLPs was stronger than to GII-12 VLPs but cross-reactivity between GII-4 and GII-12 was observed. Binding of GII-4 VLPs to a putative carbohydrate antigen receptor H-type 3 could be blocked by sera from children not infected with NoV during a waterborne outbreak of acute gastroenteritis. Therefore, protection against NoV infection correlated with strong blocking activity.
Assuntos
Anticorpos Antivirais/sangue , Infecções por Caliciviridae/epidemiologia , Norovirus/imunologia , Doença Aguda , Adolescente , Fatores Etários , Afinidade de Anticorpos/imunologia , Infecções por Caliciviridae/virologia , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Finlândia/epidemiologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Genótipo , Humanos , Lactente , Recém-Nascido , Prevalência , Estudos SoroepidemiológicosRESUMO
Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis worldwide. NoV GII-4 VP1 protein was expressed in a recombinant baculovirus system using Sf9 insect cells. Several methods for purification and concentration of virus-like particles (VLPs) were evaluated. Electron microscopy (EM) and histo-blood group antigen (HBGA) binding assays showed that repeated sucrose gradient purification followed by ultrafiltration resulted in intact VLPs with excellent binding to H type 3 antigens. VLPs were stable for at least 12 months at 4°C, and up to 7 days at ambient temperature. These findings indicate that this method yielded stable and high-quality VLPs.
Assuntos
Proteínas do Capsídeo/química , Norovirus/metabolismo , Animais , Antígenos de Grupos Sanguíneos/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Temperatura Alta , Insetos , Microscopia Eletrônica , Norovirus/química , Ligação Proteica , Estabilidade Proteica , SacaroseRESUMO
This randomized, double-blind, crossover study in two parts compared tolerability to high doses of formoterol (Oxis Turbuhaler) with that of high doses of terbutaline (Bricanyl Turbuhaler). After Holter monitoring at home, 12 patients were treated with 4+4+4 doses of formoterol Turbuhaler, 6 microg x dose(-1), (total daily metered dose 72 microg) or 4+4+4 doses of terbutaline Turbuhaler, 0.5 mg x dose(-1) (daily dose 6 mg) given in the morning, after lunch and in the evening, for 3 consecutive days. After a one week washout period at home, patients received the alternative treatment. Thereafter, 15 other patients received 8+6+6 doses of formoterol Turbuhaler (total daily metered dose 120 microg) or 8+6+6 doses of terbutaline Turbuhaler (daily dose 10 mg). Pulse, cardiac frequency, blood pressure, serum potassium, electrocardiogram and forced expiratory volume in one second (FEV1) were registered at regular intervals and Holter monitoring was applied during all 4 treatment days. Terbutaline 6 mg showed significantly greater systemic effects than formoterol 72 microg on pulse, blood pressure, cardiac frequency and QTc (QT interval corrected for heart rate). Terbutaline 10 mg had significantly greater effects than formoterol 120 microg on serum potassium levels, pulse, cardiac frequency and QTc. No differences in FEV1 levels were found. Both drugs were safe and generally well tolerated on both dose levels. In conclusion, high doses of formoterol Turbuhaler over 3 days were generally safe and well tolerated. Daily doses of 6 mg and 10 mg terbutaline Turbuhaler were systemically more potent than 72 microg and 120 microg formoterol, respectively. The safety margin thus appears to be wide if patients happen to use extra doses of formoterol in addition to those prescribed for regular use.
Assuntos
Agonistas Adrenérgicos beta/efeitos adversos , Asma/tratamento farmacológico , Etanolaminas/efeitos adversos , Terbutalina/efeitos adversos , Administração por Inalação , Agonistas Adrenérgicos beta/administração & dosagem , Adulto , Idoso , Análise de Variância , Asma/fisiopatologia , Determinação da Pressão Arterial , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Eletrocardiografia , Etanolaminas/administração & dosagem , Feminino , Fumarato de Formoterol , Humanos , Masculino , Pessoa de Meia-Idade , Nebulizadores e Vaporizadores , Potássio/sangue , Testes de Função Respiratória , Terbutalina/administração & dosagem , Resultado do TratamentoRESUMO
BACKGROUND: Baroreflex sensitivity (BRS) is depressed in conditions associated with high sympathetic nerve activity in proportion to circulating noradrenaline (NA) levels. Despite the prognostic importance of measurements of BRS in patients, there is little information on how high NA levels affect arterial baroreflex function. AIM: To understand better the role of NA in cardiovascular homeostasis. METHODS: We gave incremental intravenous NA infusions (at 50 and 100 ng/kg/min) to 12 healthy young men. We measured RR intervals and photoplethysmographic arterial pressures and estimated BRS with cross-spectral and sequence methods during metronome-guided respiration at 0.25 Hz. RESULTS: The high NA infusion rate significantly increased respiratory-frequency (0.15-0.40 Hz) RR interval spectral power and decreased low-frequency (0.04-0.15 Hz) systolic pressure spectral power compared with baseline levels (P < 0.05 for both). Cross-spectral BRS increased from an average (+/- SD) baseline level of 17.3+/-6.6 to 34.1+/-20.8 ms/mmHg at the high NA infusion rate (P < 0.05). Sequence BRS values did not increase significantly during NA infusions. The percentage of sequences with parallel changes in systolic pressures and RR intervals decreased progressively from a baseline level of 16.0+/-12.9 to 10.1+/-7.4 during the low NA infusion rate and to 6.2+/-6.2% during the high rate (P < 0.05 and 0.01, respectively). CONCLUSIONS: Increases in circulating NA to high physiological levels do not depress BRS but interfere with the close baroreflex-mediated coupling that is usually present between arterial pressure and heart rate.