RESUMEN
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.
Asunto(s)
Exoma , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , MicroARNs/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Transcriptoma , Adulto , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Femenino , Genotipo , Humanos , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Pronóstico , Resultado del Tratamiento , Adulto JovenAsunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Recurrencia Local de Neoplasia/diagnóstico , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Citocromo P-450 CYP1B1/genética , Femenino , Estudios de Seguimiento , Proteínas de Fusión bcr-abl/sangre , Humanos , Factor 1 Regulador del Interferón/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Recurrencia Local de Neoplasia/genética , Poli(ADP-Ribosa) Polimerasas/genética , Polimorfismo de Nucleótido Simple , Proteínas Quinasas/genética , Inducción de Remisión/métodos , Privación de TratamientoRESUMEN
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.
Asunto(s)
Sitios de Unión de Anticuerpos/genética , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunología , Análisis Mutacional de ADN , Virus de la Influenza A/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de la Nucleocápside , Conformación Proteica , Proteínas de Unión al ARN/química , Proteínas del Núcleo Viral/químicaRESUMEN
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.
Asunto(s)
Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Proteínas de Unión al ARN/inmunología , Proteínas del Núcleo Viral/inmunología , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Patos , Epítopos/genética , Humanos , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Gripe Humana/virología , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Proteínas de la Nucleocápside , Unión ProteicaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Epítopos de Linfocito B/inmunología , Proteínas de Unión al ARN/inmunología , Proteínas del Núcleo Viral/inmunología , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de la Nucleocápside , Estructura Terciaria de ProteínaRESUMEN
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.
