RESUMO
Bivalent COVID-19 vaccines that contain BA.1 or BA.4/BA.5 have been introduced worldwide in response to pandemic waves of Omicron subvariants. This prospective cohort study was aimed to compare neutralizing antibodies (Nabs) against Omicron subvariants (BA.1, BA.5, BQ.1.1, BN.1, and XBB.1) before and 3-4 weeks after bivalent booster by the types of SARS-CoV-2 variants in prior infections and bivalent vaccine formulations. A total of 21 participants were included. Prior BA.1/BA.2-infected, and BA.5-infected participants showed significantly higher geometric mean titers of Nab compared to SARS-CoV-2-non-infected participants after bivalent booster (BA.1, 8156 vs. 4861 vs. 1636; BA.5, 6515 vs. 4861 vs. 915; BQ.1.1, 697 vs. 628 vs. 115; BN.1, 1402 vs. 1289 vs. 490; XBB.1, 434 vs. 355 vs. 144). When compared by bivalent vaccine formulations, Nab titers against studied subvariants after bivalent booster did not differ between BA.1 and BA.4/BA.5 bivalent vaccine (BA.1, 4886 vs. 5285; BA.5, 3320 vs. 4118; BQ.1.1, 311 vs. 572; BN.1, 1028 vs. 1095; XBB.1, 262 vs. 362). Both BA.1 and BA.4/BA.5 bivalent vaccines are immunogenic and provide enhanced neutralizing activities against Omicron subvariants. However, even after the bivalent booster, neutralizing activities against the later Omicron strains (BQ.1.1, BN.1, and XBB.1) would be insufficient to provide protection.
RESUMO
Selenoprotein is associated with a variety of serious diseases, including infectious diseases, neurodegenerative disorders, cancer and cardiovascular disease. The aim of this study was to produce a new transgenic (Tg) rat expressing human selenoprotein M (SelM) in order to examine the protective function of the antioxidant status in vivo. To achieve this, a new lineage of Tg rats was produced by the microinjection of pCMV/GFP-hSelM constructs into a fertilized rat egg. Several conclusions can be drawn based on the results of the present study. The human SelM gene was successfully expressed at both the transcription and protein levels in the CMV/GFP-hSelM Tg rats. This Tg rat showed a different enzyme activity for the antioxidant protein in the various tissues examined. In response to the 2,2'-azobiz(2-amidinopropane) dihydrochloride (AAPH) injection, the Tg rats showed a lower level of antioxidant and H2O2 concentration as the activity of the antioxidant enzyme was maintained at a higher level in the Tg rats than in the non-Tg rats. Also, the neutrophil-to-lymphocyte ratio was significantly increased in this Tg rat, even though the level of corticosterone remained unchanged in both genotypes. Thus, the results of this study demonstrated that the CMV/GFP-hSelM Tg rat can serve as an animal model for the maintenance of a high level of antioxidant status and can be used to study the biological function of selenoprotein in infectious diseases, cardiovascular disease and cancer.
Assuntos
Antioxidantes/metabolismo , Expressão Gênica , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Leucócitos/citologia , Selenoproteínas/genética , Superóxido Dismutase/metabolismo , Animais , Animais Geneticamente Modificados , Corticosterona/metabolismo , Citomegalovirus , Modelos Animais de Doenças , Eritrócitos/enzimologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peróxido de Hidrogênio/sangue , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Selenoproteínas/metabolismoRESUMO
Transportation can cause stress to laboratory animals and alter physiological characteristics that may confound experimental results. The authors investigated stress-related effects of 3-4 h of transportation by truck in two strains of mice (C57BL/6, which are known to be aggressive, and ICR, which are less aggressive). Transported mice had sufficient space and access to water, though temperature in the truck was lower than what is usually recommended. Transportation affected the following parameters in both strains of mice: (i) serum corticosterone concentrations, (ii) expression of the chaperone proteins Hsp70 and Grp78 in various tissues and (iii) concentrations of serological enzymes that are associated with liver disease. These parameters also differed substantially between the two strains of mice.
Assuntos
Criação de Animais Domésticos , Temperatura Baixa/efeitos adversos , Meio Ambiente , Estresse Psicológico/sangue , Animais , Comportamento Animal , Corticosterona/sangue , Chaperona BiP do Retículo Endoplasmático , Enzimas/sangue , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Chaperonas Moleculares/metabolismo , Estresse Psicológico/fisiopatologia , Fatores de Tempo , Meios de TransporteRESUMO
Nucleic acid amplification techniques are used frequently for rapid diagnosis of viral diseases. In this study, a real-time polymerase chain reaction protocol that uses primers specific for the viral VP4 gene and the commercial SYBR Green reagent were evaluated for the quantitative measurement of human rotavirus (HRV) RNA in human stool specimens. SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. The assay resulted in a sensitive and reproducible detection of targets ranging from low (<10(2)rotavirus cDNA copies/reaction) to high numbers (>10(6)rotavirus cDNA copies/reaction). No cross-reaction was found with crude cell culture stocks of coxsackievirus, echovirus, poliovirus, hepatitis A virus and adenovirus. Analysis with the HRV cDNA standard demonstrated high reproducibility with a coefficient of variation (CV) of 0.2-0.9%. Daily performance among three different laboratories showed a CV no greater than 8%, indicating an intermediate level of variation. These results demonstrate the feasibility of this method for quantitative analysis of human rotavirus in clinical samples.
Assuntos
RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Rotavirus/virologia , Rotavirus/isolamento & purificação , Análise de Variância , Benzotiazóis , Proteínas do Capsídeo/genética , Criança , Pré-Escolar , Diaminas , Fezes/virologia , Humanos , Lactente , Variações Dependentes do Observador , Compostos Orgânicos/metabolismo , Quinolinas , RNA Viral/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Rotavirus/genética , Infecções por Rotavirus/diagnóstico , Sensibilidade e Especificidade , Temperatura de TransiçãoRESUMO
Pertussis toxin (PTx) is an essential component of the acellular pertussis (aP) vaccine. However, because PTx in its native form is considered too toxic for human vaccine use, it must be inactivated into a stable, nontoxic form by treatment with chemical detoxifying agents or by genetic modification. Therefore, testing for the residual PTx in the aP vaccine is a major quality control step for vaccine manufacturers and regulatory authorities. The histamine sensitization test is currently the standard safety test method for all aP vaccines, regardless of the vaccine formula or the detoxification process, except for those with genetically modified PTx. However, test result variability and ethical concerns regarding animal use necessitate an alternative method. In vitro assays based on the biochemical properties of PTx have been considered as potential alternatives to the histamine sensitization test. In this study, the suitability of assays based on the ADP-ribosyltransferase and carbohydrate binding activities of PTx was assessed for PTx after treatment with formaldehyde, glutaraldehyde or both denaturants in sequence. The results indicated a distinctive pattern of the biochemical activities depending on the detoxification methods and storage conditions. These results suggest that although a more careful study is needed, these in vitro biochemical assays can be considered potential alternatives to the histamine sensitization test, as they might provide more specific safety information of aP vaccines.
Assuntos
Carboidratos/química , Toxina Pertussis/química , Toxina Pertussis/toxicidade , Vacina contra Coqueluche/química , Vacina contra Coqueluche/toxicidade , Coqueluche/prevenção & controle , ADP Ribose Transferases/química , ADP Ribose Transferases/imunologia , ADP Ribose Transferases/toxicidade , Animais , Bioensaio , Carboidratos/imunologia , Histamina/imunologia , Humanos , Toxina Pertussis/imunologia , Vacina contra Coqueluche/imunologia , Controle de Qualidade , Vacinas Acelulares/química , Vacinas Acelulares/imunologia , Vacinas Acelulares/toxicidadeRESUMO
The histamine sensitization test is a widely used method for measuring the residual toxicity of pertussis toxin in acellular pertussis vaccines. Although it has been used as a routine assay for decades, the current protocols are difficult to standardize because the test results vary considerably and are based on several factors, including mouse strain, age and sex. In this study, we observed that mice of strains CD1, ddY and C57/BL6 were sufficiently sensitive to pertussis toxin among six mice strains tested and that aged male mice were more sensitive to pertussis toxin than younger or female mice. Using this animal model, we showed pertussis toxin dose-dependent responses in the two histamine sensitization test protocols based on either lethal end-point determination or mouse rectal temperature measurement. Sensitivity to pertussis toxin was further enhanced by the addition of lipopolysaccharide in both methods. With these improvements, pertussis toxin activity can be estimated more accurately and reproducibly using a reduced number of animals.
Assuntos
Bioensaio/métodos , Histamina/toxicidade , Toxina Pertussis/toxicidade , Vacina contra Coqueluche/efeitos adversos , Tecnologia Farmacêutica/métodos , Coqueluche/prevenção & controle , Fatores Etários , Animais , Bioensaio/normas , Temperatura Corporal , Feminino , Masculino , Camundongos , Toxina Pertussis/análise , Vacina contra Coqueluche/imunologia , Análise de Sobrevida , Tecnologia Farmacêutica/normas , Vacinas Acelulares/efeitos adversos , Vacinas Acelulares/imunologiaRESUMO
Recently, the genetic modification of mesenchymal stem cells (MSCs) has led to increased differentiation potential. For the therapeutic application of genetically modified MSCs, it is crucial to evaluate their characteristics and safety. In this study, we investigated the effects of bone morphogenetic protein 2 (BMP2) gene transfer on the characteristics and biodistribution of human MSCs. Lentiviral-mediated BMP2 transduction to MSCs enhanced osteocyte differentiation and decreased adipocyte differentiation. Although there is no significant difference in cell proliferation capacity, MSCs transduced BMP2 proliferate somewhat higher than nontransduced or GFP transduced MSCs. No significant changes were observed in surface antigen expression in genetically modified MSCs. In vivo transplantation of lentiviral-mediated BMP2 gene transferred MSCs to nude mice did not result in tumor formation. To evaluate the biodistribution of genetically modified cells, MSCs carrying BMP2 were injected into the tail vein of femur fractured mice. The introduced MSCs were detected in the spleen, testis and fractured femur 28 days post-implantation. These findings suggest that diverse safety tests for genetically modified MSCs should be considered, particularly when a lentivirus mediated gene transfer method is used.
Assuntos
Proteína Morfogenética Óssea 2/genética , Diferenciação Celular , Vetores Genéticos , Lentivirus/genética , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Adipócitos/citologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fraturas do Fêmur/cirurgia , Citometria de Fluxo , Expressão Gênica , Humanos , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Camundongos , Camundongos Nus , Osteoblastos/citologia , Osteogênese/fisiologia , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução GenéticaRESUMO
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/genéticaRESUMO
Tau is a neuronal phosphoprotein responsible for the formation of the neurofibrillary tangles in Alzheimer's disease. To characterize the changes in global gene expression in the brain of transgenic mice that overexpress human Tau23 protein in response to the increase of Tau23 phosphorylation, total RNA extracted from the hippocampus of 12-month-old transgenic and wild-type mice was converted to cDNA, labeled with biotin and hybridized to oligonucleotide microarrays. The microarray results were confirmed by real-time RT-PCR and Western blotting method. It was determined that 43 genes were up-regulated and 8 genes were down-regulated by Tau23 in transgenic mice compared to controls, based on the arbitrary difference in the 2-fold change. Among the up-regulated transcripts, those encoding for transporter and oxidoreductase were dramatically over-represented, followed by those related to regulatory molecule, cytoskeletal protein, signaling molecule, and extracellular matrix protein. Genes encoding for transcription factor, regulatory molecule, miscellaneous function, and chaperone were significantly reduced in the down-regulated group. The major genes in the up-regulated categories included Ecrg4, Folr1, Defb11, Aqp1 and Soctdc1. The major genes in the down-regulated categories were Ncor1, Gpm6a, and Hspd1. These results indicate that the microarray analysis identifies several gene functional groups and individual genes that respond to a sustained increase in Tau23 phosphorylation levels in the brain of transgenic mice. In addition, the results suggest the microarray test is a useful tool for increased understanding of the role of Tau23 protein in regulating neurodegenerative disorders.
Assuntos
Encéfalo/fisiologia , Regulação da Expressão Gênica , Camundongos Transgênicos , Fosfopiruvato Hidratase , Proteínas tau , Animais , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Biopharmaceutical products produced from cell cultures have a potential for viral contamination from cell sources or from adventitious introduction during production. The objective of this study was to assess viral clearance in the production of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs). We selected Japanese encephalitis virus (JEV), bovine viral diarrhea virus (BVDV), and minute virus of mice (MVM) as relevant viruses to achieve the aim of this study. A downstream process for the production of purified HPV-16 L1 VLPs consisted of detergent lysis of harvested cells, sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. The capacity of each purification/treatment step to clear viruses was expressed as reduction factor by measuring the difference in log virus infectivity of sample pools before and after each process. As a result, detergent treatment (0.5% v/v, Nonidet P-40/phosphate-buffered saline) was effective for inactivating enveloped viruses such as JEV and BVDV, but no significant reduction (< 1.0 log(10)) was observed in the non-enveloped MVM. The CsCl equilibrium density centrifugation was fairly effective for separating all three relevant adventitious viruses with different CsCl buoyant density from that of HPV-16 L1 VLPs (JEV, BVDV, and MVM = 4.30, 3.10, > or = 4.40 log(10) reductions). Given the study conditions we used, overall cumulative reduction factors for clearance of JEV, BVDV, and MVM were > or = 10.50, > or = 9.20, and > or = 6.40 log(10) in 150 ml of starting cell cultures, respectively.
Assuntos
Papillomavirus Humano 16/isolamento & purificação , Insetos/virologia , Animais , Técnicas de Cultura de Células , DNA Viral/isolamento & purificação , Papillomavirus Humano 16/crescimento & desenvolvimento , RNA Viral/isolamento & purificaçãoRESUMO
Insect cell culture has greatly increased in part due to the widespread use of insect virus-based vectors for efficient expression of foreign proteins. Insect cells such as Sf9 cells are susceptible to arboviruses which may pose a safety concern by adventitious introduction during the production process. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived biotechnological products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses that are known to be infectious in insect cells. Here we report the development of a quantitative assay for JEV RNA using real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay was performed using LightCycler and RNA amplification kit SYBR Green I. The JEV specific primer was selected from the 3' untranslated region, and the expected band size was 323 base pairs (bp). The sensitivity of the assay was calculated to be approximately 15 TCID(50)per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. JEV clearance was determined during the purification process of rHPV-16 L1 VLPs by CsCl equilibrium density centrifugation. The comparative results obtained by real-time RT-PCR assay for JEV and infectivity titrations suggested that the real-time RT-PCR assay could have an additive effect on the interpretation and evaluation of virus clearance, especially during the virus removal process.