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1.
Virus Res ; 112(1-2): 95-9, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16022903

RESUMO

Linear immunogenic peptides corresponding to amino acid sequences from the NS1 non-structural protein from tick-borne encephalitis virus (strain Sophyin) were predicted using established algorithms and synthesized. Of the 12 peptides predicted, 11 were able to induce peptide-specific antibodies in BALB/c mice but only 1 of these 11 was able to induce antibodies, which reacted with the native protein in a radio-immune precipitation assay. This peptide corresponds to amino acids 37--55, and forms one of the predicted structurally conserved alpha helices of the virus NS1 protein. It was able to protect 60% of animals against lethal challenge with the homologous highly pathogenic tick-borne encephalitis virus strain, and adoptive transfer experiments indicated the involvement of the antibodies induced by this peptide in its protective activity in mice.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/prevenção & controle , Peptídeos/síntese química , Peptídeos/imunologia , Proteínas não Estruturais Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Modelos Animais de Doenças , Encefalite Transmitida por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/mortalidade , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , Proteínas não Estruturais Virais/química , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
2.
BMC Microbiol ; 5: 45, 2005 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-16076390

RESUMO

BACKGROUND: Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. RESULTS: The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. CONCLUSION: Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/imunologia , Vacinas de DNA , Vaccinia virus/imunologia , Proteínas não Estruturais Virais/imunologia , Vacinas Virais , Animais , Bactérias/genética , Bactérias/imunologia , Citomegalovirus/imunologia , Humanos , Imunização Secundária , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Vacinas de DNA/toxicidade , Proteínas não Estruturais Virais/toxicidade , Vacinas Virais/toxicidade
4.
Biochemistry (Mosc) ; 65(6): 640-50, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10887281

RESUMO

Contacts of phosphate groups at positions -12, -15, and -18 in relation to the transcription initiation site in the non-template strand of lac UV5 promoter with lysines or histidines of E. coli RNA polymerase in the open complex model were studied. A number of synthetic oligonucleotides from the -10-area of the non-template strand containing activated 5'-terminal phosphate group were cross-linked with holo- or core-enzyme of RNA polymerase. 5'-N-Hydroxybenzotriazole phosphodiesters of oligonucleotides were used as phosphate activated derivatives. They are capable of phosphorylating amino groups of lysines and histidines in the enzyme molecule that are brought into proximity with activated phosphate in the complex, resulting in the formation of a covalent bond between the oligonucleotide and the protein. The analysis of the products of cross-linking allowed the protein subunit and the amino acid residue taking part in the formation of the covalent bond for each oligonucleotide to be identified. It was found that all oligonucleotides from the non-template strand of promoter in the complex with the holo-enzyme are bound with the sigma70-subunit. When analyzing the products of partial cleavage of the complexes cross-linked at cysteines and methionines using SDS-PAGE, it was shown that phosphate at position -12 made contacts with His180 or His242 of the sigma70-subunit, the reactive amino acid residue being located between the first and second conservative regions. Phosphate at position -15 is located near lysines from two different areas--between Met413 and Met456 (regions 2.3 and 2.4) and between Met470 and Met507 (region 3.1). Phosphate at position -18 makes preferential contacts with a lysine situated between Met470 and Met507 (region 3.1). Based on the analysis of contacts of phosphate groups and the structure of the isolated sigma70-subunit established previously, a scheme of the mutual arrangement of the oligonucleotide and the sigma70-subunit possessed by the holo-enzyme has been proposed.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Óperon Lac , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Sequência de Bases , Reagentes de Ligações Cruzadas/química , Hidrólise , Sondas Moleculares , Oligonucleotídeos/química , Ligação Proteica
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