RESUMO
Amino acid positions recognized by monoclonal antibodies (MAbs) in the influenza A virus nucleoprotein (NP) have been reported. As these residues were scattered in the three-dimensional (3D) structure of NP, no patterns of the architecture of antibody-binding sites could be inferred. Here, we used site-specific mutagenesis and ELISA to screen the amino acids surrounding position 470 recognized by the MAb 3/1 as a linear epitope. Ten amino acid residues involved in the reaction of NP with the MAb 3/1 and the MAb 469/6 were identified. Our data are the first to outline a compact site recognized by MAbs in the 3D structure of the influenza virus NP.
Assuntos
Sítios de Ligação de Anticorpos/genética , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Análise Mutacional de DNA , Vírus da Influenza A/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas do Nucleocapsídeo , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas do Core Viral/químicaRESUMO
BACKGROUND: Approximately 5-20% of chronic myeloid leukemia (CML) patients demonstrate primary resistance or intolerance to imatinib. None of the existing predictive scores gives a good prognosis of TKI efficacy. Gene polymorphisms, expression and microRNAs are known to be involved in the pathogenesis of TKI resistance in CML. The aim of our study is to find new molecular markers of TKI therapy efficacy in CML patients. METHODS: Newly diagnosed patients with Ph+ CML in chronic phase were included in this study. Optimal and non-optimal responses to TKI were estimated according to ELN 2013 recommendation. We performed genotyping of selected polymorphisms in 62 blood samples of CML patients, expression profiling of 33 RNA samples extracted from blood and miRNA profiling of 800 miRNA in 12 blood samples of CML patients. RESULTS: The frequencies of genotypes at the studied loci did not differ between groups of patients with an optimal and non-optimal response to TKI therapy. Analysis of the expression of 34,681 genes revealed 26 differently expressed genes (p < 0.05) in groups of patients with different TKI responses, but differences were very small and were not confirmed by qPCR. Finally, we did not find difference in miRNA expression between the groups. CONCLUSIONS: Using modern high-throughput methods such as whole-exome sequencing, transcriptome and miRNA analysis, we could not find reliable molecular markers for early prediction of TKI efficiency in Ph+ CML patients.
Assuntos
Exoma , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , MicroRNAs/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Transcriptoma , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Genótipo , Humanos , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Prognóstico , Resultado do Tratamento , Adulto JovemRESUMO
We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Vietnam/1203/04 (H5N1) deltaHA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1) virus. The mutants were selected with a panel of eight anti-HA monoclonal antibodies (MAbs), seven to A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and the mutants' HA genes were sequenced. The amino acid changes suggested three MAb groups: four MAbs reacted with the complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, two MAbs reacted with the epitope corresponding to the antigenic site A in H3 HA, and two MAbs displayed unusual behavior: each recognized amino acid changes at two widely separate antigenic sites. Five changes were detected in amino acid residues not previously reported as changed in H5 escape mutants, and four others had substitutions not previously described. The HA antigenic structure differs substantially between A/Vietnam/1203/04 (H5N1) virus and the low-pathogenic A/Mallard/Pennsylvania/10218/84 (H5N2) virus we previously characterized (N. V. Kaverin et al., J. Gen. Virol. 83:2497-2505, 2002). The hemagglutination inhibition reactions of the MAbs with recent highly pathogenic H5N1 viruses were consistent with the antigenic-site amino acid changes but not with clades and subclades based on H5 phylogenetic analysis. These results provide information on the recognition sites of the MAbs widely used to study H5N1 viruses and demonstrate the involvement of the HA antigenic sites in the evolution of highly pathogenic H5N1 viruses, findings that can be critical for characterizing pathogenesis and vaccine design.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Mapeamento de Epitopos/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Mutação de Sentido Incorreto/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Embrião de Galinha , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de ProteínaAssuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Recidiva Local de Neoplasia/diagnóstico , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Citocromo P-450 CYP1B1/genética , Feminino , Seguimentos , Proteínas de Fusão bcr-abl/sangue , Humanos , Fator Regulador 1 de Interferon/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Recidiva Local de Neoplasia/genética , Poli(ADP-Ribose) Polimerases/genética , Polimorfismo de Nucleotídeo Único , Proteínas Quinases/genética , Indução de Remissão/métodos , Suspensão de TratamentoRESUMO
Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies. In the present study five monoclonal antibodies (MAbs) were produced against NP of A/Duck/Novosibirsk/56/05 (H5N1) influenza virus. Two MAbs discerned human and avian influenza strains on ELISA testing. The NP expressed in a prokaryotic system was used for the analysis of site-specific mutants carrying amino acid substitutions in the relevant positions. Amino acid residues in positions 100 and 101 were shown to be recognized by the MAbs. The residue in position 100 is host-specific, and its recognition by the MAb 2E6 may be useful for the differentiation of human and avian viruses. The data are discussed in view of the effects of amino acid substitutions in influenza virus NP affecting both host range and antibody-binding specificity.
Assuntos
Anticorpos Antivirais/imunologia , Epitopos/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas do Core Viral/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Patos , Epitopos/genética , Humanos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas do Nucleocapsídeo , Ligação ProteicaRESUMO
The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.