RESUMO
Vaccine-induced memory B cell responses to evolving viruses like influenza A involve activation of pre-existing immunity and generation of new responses. To define the contribution of these two types of responses, we analyzed the response to H7N9 vaccination in H7N9-naive adults. We performed comprehensive comparisons at the single-cell level of the kinetics, Ig repertoire, and activation phenotype of established pre-existing memory B cells recognizing conserved epitopes and the newly generated memory B cells directed toward H7 strain-specific epitopes. The recall response to conserved epitopes on H7 HA involved a transient expansion of memory B cells with little observed adaptation. However, the B cell response to newly encountered epitopes was phenotypically distinct and generated a sustained memory population that evolved and affinity matured months after vaccination. These findings establish clear differences between newly generated and pre-existing memory B cells, highlighting the challenges in achieving long-lasting, broad protection against an ever-evolving virus.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Adulto , Anticorpos Antivirais/metabolismo , Formação de Anticorpos , Células Cultivadas , Epitopos/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Memória Imunológica , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores de Antígenos de Linfócitos B/genética , Análise de Célula Única , Vacinação , Adulto JovemRESUMO
Induction of an antibody response capable of recognizing highly diverse strains is a major obstacle to the development of vaccines for viruses such as HIV and influenza. Here, we report the dynamics of B cell expansion and evolution at the single-cell level after vaccination with a replication-competent adenovirus type 4 recombinant virus expressing influenza H5 hemagglutinin. Fluorescent H1 or H5 probes were used to quantitate and isolate peripheral blood B cells and their antigen receptors. We observed increases in H5-specific antibody somatic hypermutation and potency for several months beyond the period of active viral replication that was not detectable at the serum level. Individual broad and potent antibodies could be isolated, including one stem-specific antibody that is part of a new multidonor class. These results demonstrate prolonged evolution of the B cell response for months after vaccination and should be considered in efforts to evaluate or boost vaccine-induced immunity.
Assuntos
Adenoviridae/genética , Linfócitos B/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Adenoviridae/imunologia , Administração Oral , Adolescente , Adulto , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Feminino , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunogenicidade da Vacina , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Hipermutação Somática de Imunoglobulina/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Replicação Viral/imunologia , Adulto JovemRESUMO
Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.
Assuntos
Actinas/metabolismo , Movimento Celular/genética , Genoma Humano/genética , MicroRNAs/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , Transdução de Sinais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/patologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.