RESUMO
We have previously reported on a universal human influenza A vaccine, based on the external domain of the transmembrane viral M2-protein (M2e) [Nature Medicine 5 (1999) 1119]. M2-protein is scarcely present on the virus but is abundantly expressed on virus-infected cells. The external domain, M2e, is 23-amino acids long and as such weakly immunogenic. But when presented on an appropriate carrier, such as hepatitis B virus core (HBc) particles, it induces a high titer antibody response that in mice effectively protects against a potentially lethal influenza infection. The advantage of M2e as an antigen is the conservation of its sequence that has hardly changed since the first influenza virus was isolated in 1933, despite numerous epidemics and several pandemics. Various constructs, e.g. M2e fused at the N-terminus of the HBc subunit or inserted in the immuno-dominant loop, were evaluated as a vaccine. They conferred full protection when administered together with an adjuvant. Several adjuvants were tested in conjunction with intraperitoneal vaccine administration, while the non-toxic enterotoxin mutant LT(R192G) was used for intranasal vaccination. Appropriate combinations of vaccine construct and adjuvant allowed to obtain anti-M2e IgG2a serum titers above 10,000, and this provided complete protection.
Assuntos
Vírus da Influenza A/imunologia , Vacinas contra Influenza , Influenza Humana/prevenção & controle , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Sequência Conservada , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Humanos , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-alpha-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of alpha-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.
Assuntos
Manosidases/química , Manosidases/metabolismo , Pichia/genética , Pichia/metabolismo , Polissacarídeos/biossíntese , Trichoderma/enzimologia , Trichoderma/genética , Animais , Sequência de Bases , Primers do DNA/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Técnicas In Vitro , Manosidases/genética , Neuraminidase/química , Neuraminidase/genética , Oligopeptídeos , Polissacarídeos/química , Engenharia de Proteínas , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genéticaRESUMO
Current influenza vaccines require repeated administration for long-term protection. Failure to develop broad-spectrum vaccines may be attributed to the chronic presentation of hypervariable, immunodominant epitopes displayed on the viral surface that keep the immune response somewhat fixed and limited by suppression of broadly neutralizing, low-titered antibodies. To test this hypothesis, we have attempted to dampen the immunogenicity of variable epitopes and potential immunodominant domains of the A/Victoria/3/75 (H3N2) neuraminidase by site-directed mutagenesis. The results suggest that the neuraminidase structure is extremely flexible, since many substitution combinations were tolerated, and constitute proof-of-principle that the antigenicity of this protein can be modulated to a large extent. However, mice immunized with neuraminidase mutants containing multiple amino acid substitutions showed a reduced protection rate against heterologous virus in comparison with the reference groups.
Assuntos
Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Vacinas Sintéticas/imunologia , Animais , Reações Cruzadas , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , SpodopteraRESUMO
The head domain of recombinant neuraminidase of A/Victoria/3/75 influenza virus was produced in a secreted form in the methylotrophic yeast Pichia pastoris using the P. pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-mating-factor signal sequence. Cultures in shake flasks provided expression levels of approximately 2.5-3 mg/l. Recombinant neuraminidase was purified from the culture medium to over 99% homogeneity. Although P. pastoris-secreted products are believed to carry shorter N-linked carbohydrate side chains than glycoproteins of S. cerevisiae, secreted neuraminidase was hyperglycosylated, with N-glycans of the high-mannose type containing up to 30-40 mannose residues. N-glycans were phosphorylated and only partially sensitive to alpha-mannosidase treatment. Balb/c mice immunized three times with 2 microg purified recombinant neuraminidase were 50% protected against a lethal challenge of mouse-adapted homologous virus; removal of glycosylation at the top of neuraminidase resulted in improved protection. The results provide a system for the production of an effective recombinant influenza vaccine that can easily be scaled up.
Assuntos
Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Sintéticas/imunologia , Animais , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/biossíntese , Neuraminidase/química , Pichia/genética , Vacinas Sintéticas/biossínteseRESUMO
The complete nucleotide sequence of the influenza A/PR/8/34 nucleoprotein gene was determined after cloning for dsDNA copy in pBR322. The nucleoprotein gene is 1517 nucleotides long of which 1446 nucleotides code for 482 amino acids. The calculated amino acid composition is in good agreement with those published for influenza A nucleoprotein genes. The amino acid sequence of the nucleoprotein contains clusters of basic amino acids and proline, a property shared with other nucleic-acid-associated proteins like Semliki forest virus nucleocapsid protein, VP1 protein of polyoma virus and Simian virus 40, and the core antigen of hepatitis B virus. The described nucleoprotein structure brings the number of sequenced genes of influenza A/PR/8/34 to five out of eight genes.
Assuntos
Genes Virais , Genes , Vírus da Influenza A/genética , Nucleoproteínas/genética , Sequência de Bases , Clonagem Molecular , Códon , Enzimas de Restrição do DNA , DNA Recombinante/análise , Hibridização de Ácido Nucleico , Plasmídeos , RNA Viral/genética , DNA Polimerase Dirigida por RNARESUMO
Crystals of bacteriophage MS2 have been obtained by slowly cooling a 1% virus solution from 23 degrees C to 0 degrees C in the presence of poly(ethylene glycol) 6000. The crystals were colorless, needle-like, anisotropic and very fragile. Electron microscopic observation of the crystals revealed a two-dimensional lattice of particles with RNA phage morphology and dimensions. Preliminary X-ray examination of the crystals confirmed their viral nature.
Assuntos
Colífagos/análise , Cristalização , Escherichia coli/análise , Microscopia Eletrônica , RNA Viral/análise , Solubilidade , Difração de Raios XRESUMO
Bacteriophage MS2 RNA is 3,569 nucleotides long. The nucleotide sequence has been established for the third and last gene, which codes for the replicase protein. A secondary structure model has also been proposed. Biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis. As the sequences for the other regions of this RNA have been published already, the complete, primary chemical structure of a viral genome has now been established.
Assuntos
Colífagos/análise , RNA Viral/análise , Proteínas Virais , Sequência de Bases , Códon , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Viral/metabolismoRESUMO
32P-Labeled MS2 RNA was partially digested with ribonuclease T1 (guanyloribonuclease; ribonucleate 3'-guanylo-oligonucleotidohydrolase; EC 3.1.4.8) or with epilson-carboxymethyl-lysine-41 pancreatic ribonuclease A (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase; EC 3.1.4.22). A series of overlapping fragments was obtained which allowed the reconstruction of a 361-nucleotide-long 3'-terminal sequence. A unique reading frame could be deduced, which indicated that the replicase gene ends with a U-A-G termination signal and is followed by a 174-nucleotide-long untranslated segment.
