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1.
Vet Immunol Immunopathol ; 140(3-4): 244-51, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21288576

RESUMO

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.


Assuntos
Vacinas Protozoárias/administração & dosagem , Theileria parva/imunologia , Theileriose/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Protozoários/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Bovinos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Interferon gama/biossíntese , Ativação Linfocitária , Proteínas de Membrana/administração & dosagem , Proteínas Recombinantes , Linfócitos T Citotóxicos/imunologia , Theileria parva/patogenicidade , Theileriose/imunologia , Vacinas de DNA/administração & dosagem
2.
Proc Natl Acad Sci U S A ; 103(9): 3286-91, 2006 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-16492763

RESUMO

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Linfócitos T Citotóxicos/imunologia , Theileria parva/imunologia , Theileriose/imunologia , Animais , Bovinos , Linhagem Celular , Theileriose/parasitologia , Theileriose/patologia , Vacinação
3.
Microbiol Immunol ; 47(9): 639-51, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14584611

RESUMO

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3-9-16, that recognized a linear epitope located at the N-terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Malpha) whose 3-9-16 epitope is hidden, and an N-terminal-exposed epitope-positive form (Mbeta). The Mbeta protein accounted for about 25-30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mbeta antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mbeta antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb-dependent complement-mediated cytolysis. Similarly, the Mbeta antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mbeta) of which exposes the 3-9-16 epitope located in the N-terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Malpha), probably through its intimate association with the Golgi area and/or the cell membrane.


Assuntos
Anticorpos Monoclonais/imunologia , Complexo de Golgi/virologia , Vírus da Raiva/imunologia , Proteínas da Matriz Viral/análise , Proteínas da Matriz Viral/imunologia , Vírion/química , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Linhagem Celular , Membrana Celular/virologia , Proteínas do Sistema Complemento/imunologia , Cricetinae , Epitopos , Glicoproteínas/análise , Conformação Proteica , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Proteínas do Envelope Viral/análise , Proteínas da Matriz Viral/química
4.
Hepatogastroenterology ; 50(51): 817-20, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12828092

RESUMO

BACKGROUND/AIMS: Possible short-term interferon therapy was investigated in chronic hepatitis C patients with genotype 2a or 2b and low viral-loads. Furthermore, initial changes of hepatic C virus RNA levels in early phase interferon therapy, and the number of pretreatment mutated clones at hypervariable region-1 were determined in order to upgrade interferon therapy efficacy prediction rates. METHODOLOGY: Study subjects were 31 patients with histologically proven chronic hepatitis C, having less than 1 Meq/mL of hepatic C virus RNA levels. Daily dose was defined as 9 MU of interferon; patients with genotype lb were treated for 26 weeks, while those with genotype 2a or 2b were treated for 16 weeks. RESULTS: Sustained response rates showed no difference in efficacy between the 2 groups (66.7% vs. 62.5%). Response rates based on the number of hypervariable region-1 clones indicated that the fewer the number of mutated clones, more significant was the increase in efficacy. Efficacy as hepatic C virus RNA in early phase treatment showed no difference in response rates between negative and positive groups at any time point from day 1. CONCLUSIONS: In a low viral-load group, the number of hypervariable region-1 clones was a critical factor influencing interferon therapy efficacy. Thus, 16-week interferon therapy was effective and economical.


Assuntos
Antivirais/administração & dosagem , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/administração & dosagem , Carga Viral , Adulto , Idoso , Biópsia , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Humanos , Interferon alfa-2 , Interferon-alfa/efeitos adversos , Fígado/patologia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Mutação/genética , RNA Viral/sangue , Proteínas Recombinantes , Resultado do Tratamento , Proteínas do Envelope Viral/genética , Proteínas Virais/genética
5.
J Gen Virol ; 83(Pt 12): 3035-3043, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12466480

RESUMO

The structural changes of the nominal phosphoprotein (P) of rabies virus using a monoclonal antibody, mAb #402-13, was investigated. This mAb recognized a linear epitope that was mapped roughly to a C-terminal region of the P protein, ranging from aa 256 to 297. The P gene products were detected by the mAb in immunoblot assays, the products of which were produced either in BHK-21 cells or in Escherichia coli cells. The mAb, however, detected very low levels of P gene products in immunoprecipitation assays. The mAb recognized the nucleocapsid (NC)-associated P proteins but recognized free P protein and free N-P complex produced in the infected cells much less efficiently. When the P proteins were released from the NC, however, they were no longer recognized by the mAb. Similar results were obtained from BHK-21 cells co-transfected with P and N cDNAs. Furthermore, studies with C-terminally truncated P protein mutants revealed that the NC-binding ability of the P protein was dependent on the presence of the C-terminal epitope region. From these results, it is thought that the 402-13 epitope region is concealed when the P protein is present in a free form or free N-P complex but is exposed when it is associated with the NC. The C-terminal epitope region seemed to be essential for the P protein to be associated with the NC but not for the formation of free N-P complexes with newly synthesized N protein.


Assuntos
Mapeamento de Epitopos , Epitopos/química , Nucleocapsídeo/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Vírus da Raiva/metabolismo , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Linhagem Celular , Cricetinae , Chaperonas Moleculares , Proteínas do Nucleocapsídeo , Fosfoproteínas/genética , Conformação Proteica , Vírus da Raiva/imunologia , Relação Estrutura-Atividade , Proteínas Estruturais Virais/genética
6.
Microbiol Immunol ; 46(7): 449-61, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12222931

RESUMO

We investigated a virus-neutralizing conformational epitope of the rabies virus glycoprotein (G) that is recognized by an anti-G monoclonal antibody (mAb; #1-46-12) and shared by most of the laboratory strains of the virus. To investigate the epitope structure, we isolated escape mutants from the HEP-Flury virus (wild-type; wt) after repeated passages in culture in the presence of the mAb. Immunofluorescence studies indicated that the mutants could be classified into two groups; the Group I lacked the epitope, while Group II preserved the epitope. The latter was dominant under the passage conditions, since Group I disappeared during the continuous passages. G proteins showed different electrophoretic mobilities; G protein of Group I migrated at the same rate as wt G protein, while that of Group II migrated at a slower rate, which was shown to be due to acquisition of an additional oligosaccharide side chain. Nucleotide sequencing of the G gene strongly suggested that amino acid substitutions at Thr-36 by Pro and Ser-39 by Thr of the G protein are responsible for the escape mutations of Groups I and II, respectively. The latter is a unique mutation of the rabies virus that allows the G protein to be glycosylated additionally at Asn-37, a potential glycosylation site that is not glycosylated in the parent virus, in preserving the epitope-positive conformation. These results suggest that to keep the 1-46-12 epitope structure is of greater survival advantage for the virus to escape the neutralization than to destroy it, which could be achieved by acquiring an additional oligosaccharide chain at Asn-37.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Epitopos/imunologia , Glicoproteínas/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Sequência de Bases , Células Cultivadas , Eletroforese em Gel de Poliacrilamida/métodos , Epitopos/química , Evolução Molecular , Imunofluorescência , Glicoproteínas/química , Glicoproteínas/genética , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Testes de Neutralização , Conformação Proteica , Raiva/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
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