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3.
Proc Natl Acad Sci U S A ; 113(20): 5682-7, 2016 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-27114505

RESUMO

The αß T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/µm(2) This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.


Assuntos
Antígenos CD4/química , Antígeno HLA-A24/química , Cadeias HLA-DRB1/química , Sítios de Ligação , Antígenos CD4/metabolismo , Células HEK293 , Antígeno HLA-A24/metabolismo , Cadeias HLA-DRB1/metabolismo , Humanos , Proteínas Ligantes de Maltose/química , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Ressonância de Plasmônio de Superfície
5.
Proc Natl Acad Sci U S A ; 109(33): 13353-8, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22826228

RESUMO

Human leukocyte antigen HLA-B alleles have better protective activity against HIV-1 than HLA-A alleles, possibly due to differences in HLA-restricted HIV-1-specific CD8+ cytotoxic T lymphocyte (CTL) function, but the mechanism is unknown. HIV-1 negative regulatory factor (Nef) mediates down-regulation of surface expression of class I HLA (HLA-I) and may therefore impair immune recognition by CTL. Because of sequence differences in the cytoplasmic domains, HLA-A and -B are down-regulated by Nef but HLA-C and -E are not affected. However, the latter are expressed at low levels and are not of major importance in the CTL responses to HIV-1. Here, we compared the role of the cytoplasmic domains of HLA-A and -B in Nef-mediated escape from CTL. We found HLA-B cytoplasmic domains were more resistant to Nef-mediated down-regulation than HLA-A cytoplasmic domains and demonstrated that these differences affect CTL recognition of virus-infected cells in vitro. We propose that the relative resistance to Nef-mediated down-regulation by the cytoplasmic domains of HLA-B compared with HLA-A contributes to the better control of HIV-1 infection associated with HLA-B-restricted CTLs.


Assuntos
Regulação para Baixo/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Alelos , Sequência de Aminoácidos , Linhagem Celular , Citoplasma/imunologia , Epitopos/imunologia , Infecções por HIV/virologia , Antígenos HLA-A/química , Antígenos HLA-A/genética , Antígenos HLA-B/química , Antígenos HLA-B/genética , Antígeno HLA-B7/imunologia , Antígenos HLA-C/química , Antígenos HLA-C/imunologia , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Estrutura Terciária de Proteína , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
6.
J Infect Dis ; 209(9): 1354-61, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24415790

RESUMO

BACKGROUND: Antibodies play a major role in the protection against influenza virus in human. However, the antibody level is usually short-lived and the cellular mechanisms underlying influenza virus-specific antibody response to acute infection remain unclear. METHODS: We studied the kinetics and magnitude of influenza virus-specific B-cell and serum antibody responses in relation to virus replication during the course of influenza infection in healthy adult volunteers who were previously seronegative and experimentally infected with seasonal influenza H1N1 A/Brisbane/59/07 virus. RESULTS: Our data demonstrated a robust expansion of the virus-specific antibody-secreting cells (ASCs) and memory B cells in the peripheral blood, which correlated with both the throat viral load and the duration of viral shedding. The ASC response was obviously detected on day 7 post-infection when the virus was completely cleared from nasal samples, and serum hemagglutination-inhibition antibodies were still undetectable. On day 28 postinfection, influenza virus-specific B cells were further identified from the circulating compartment of isotype-switched B cells. CONCLUSIONS: Virus-specific ASCs could be the earliest marker of B-cell response to a new flu virus infection, such as H7N9 in humans.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Adulto , Anticorpos Antivirais/sangue , Linfócitos B/metabolismo , Linfócitos B/virologia , Feminino , Humanos , Influenza Humana/virologia , Masculino , Modelos Imunológicos , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Carga Viral/imunologia , Adulto Jovem
7.
Int Immunol ; 24(11): 729-37, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22888216

RESUMO

UNLABELLED: Humans express four MHC-like CD1 molecules CD1a, b, c and d that are capable of presenting a wide variety of self or foreign lipid antigens to T cells. Much progress has been made in elucidating the function of CD1d-restricted NKT cells in both innate and adaptive immune responses. However, knowledge of the other CD1 molecules is less well defined in terms of lipid presentation and immune regulation. We have previously shown that immunoglobulin-like transcript 4 (ILT4) binds to CD1d and inhibits its recognition by NKT cells. In this study, we show that CD1c can also interact specifically with ILT4 with a higher affinity than that of CD1d. Furthermore, changes in CD1c expression seem to modulate CD1d function; up-regulation of CD1c enhances NKT recognition of CD1d and down-regulation reduces CD1d recognition. We propose that CD1c can act as a sink for the inhibitory receptor ILT4: when CD1c is up-regulated, ILT4 is recruited to CD1c, thus reducing the inhibitory effect of ILT4 on CD1d recognition. Consequently, CD1c could be a potential target for modulating NKT activity. KEYWORDS: NKT, CD1d, CD1c, ILT4, antigen presentation.


Assuntos
Antígenos CD1/imunologia , Antígenos CD1d/imunologia , Glicoproteínas/imunologia , Glicoproteínas de Membrana/imunologia , Células T Matadoras Naturais/imunologia , Receptores Imunológicos/imunologia , Antígenos CD1/genética , Antígenos CD1/metabolismo , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Ligação Competitiva/imunologia , Western Blotting , Linhagem Celular , Células Cultivadas , Regulação para Baixo/imunologia , Citometria de Fluxo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Células Jurkat , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Células T Matadoras Naturais/metabolismo , Ligação Proteica/imunologia , Interferência de RNA , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Ressonância de Plasmônio de Superfície , Regulação para Cima/imunologia
8.
ACS Appl Mater Interfaces ; 15(42): 48871-48881, 2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37816068

RESUMO

Virus-like particle (VLP)-based vaccines are required to be associated with a suitable adjuvant to potentiate their immune responses. Herein, we report a novel, biodegradable, and biocompatible polyphosphoester-based amphiphilic cationic polymer, poly(ethylene glycol)-b-poly(aminoethyl ethylene phosphate) (PEG-PAEEP), as a Hepatitis B surface antigen (HBsAg)-VLP vaccine adjuvant. The polymer adjuvant effectively bound with HBsAg-VLP through electrostatic interactions to form a stable vaccine nanoformulation with a net positive surface charge. The nanoformulations exhibited enhanced cellular uptake by macrophages. HBsAg-VLP/PEG-PAEEP induced a significantly higher HBsAg-specific IgG titer in mice than HBsAg-VLP alone after second immunization, indicative of the antigen-dose sparing advantage of PEG-PAEEP. Furthermore, the nanoformulations exhibited a favorable biocompatibility and in vivo tolerability. This work presents the PEG-PAEEP copolymer as a promising vaccine adjuvant and as a potentially effective alternative to aluminum adjuvants.


Assuntos
Antígenos de Superfície da Hepatite B , Vacinas de Partículas Semelhantes a Vírus , Camundongos , Animais , Polímeros , Adjuvantes de Vacinas , Vacinas contra Hepatite B , Adjuvantes Imunológicos/farmacologia , Adjuvantes Farmacêuticos , Imunidade Celular , Camundongos Endogâmicos BALB C
9.
J Biol Chem ; 286(43): 37692-701, 2011 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-21900247

RESUMO

The CD1 family consists of five proteins that are related to the peptide-presenting MHC class I family. T cells can recognize the presentation of both foreign and self-derived lipids on four CD1 family members. The identities of the self-lipids capable of stimulating autoreactive T cell responses remain elusive or controversial. Here, we employed mass spectrometry to analyze the lipid content of highly purified CD1c and CD1d protein samples. We report the identification of 11 novel self-lipids presented by CD1c and nine by CD1d. Rigorous controls provide strong evidence that the identified lipids were specifically loaded into the lipid-binding site of the CD1 molecules. The diverse but distinct population of lipids identified from each CD1 family member implies each present a different subset of self-lipids, and the enrichment of particular motifs indicates that the lipids that are presented by CD1 family members could be predicted. Finally, our results imply the CD1 system surveys the endoplasmic reticulum, Golgi apparatus, and/or secretory compartments, in addition to its well characterized surveillance of the endocytic and lysosomal compartments.


Assuntos
Apresentação de Antígeno/fisiologia , Antígenos CD1/metabolismo , Antígenos CD1d/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Vigilância Imunológica/fisiologia , Lipídeos de Membrana/metabolismo , Antígenos CD1/genética , Antígenos CD1/imunologia , Antígenos CD1d/genética , Antígenos CD1d/imunologia , Sítios de Ligação , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/fisiologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Células HEK293 , Humanos , Espectrometria de Massas , Lipídeos de Membrana/genética , Lipídeos de Membrana/imunologia
10.
Sci Transl Med ; 14(671): eabo5795, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36383686

RESUMO

Interstitial lung disease and associated fibrosis occur in a proportion of individuals who have recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through unknown mechanisms. We studied individuals with severe coronavirus disease 2019 (COVID-19) after recovery from acute illness. Individuals with evidence of interstitial lung changes at 3 to 6 months after recovery had an up-regulated neutrophil-associated immune signature including increased chemokines, proteases, and markers of neutrophil extracellular traps that were detectable in the blood. Similar pathways were enriched in the upper airway with a concomitant increase in antiviral type I interferon signaling. Interaction analysis of the peripheral phosphoproteome identified enriched kinases critical for neutrophil inflammatory pathways. Evaluation of these individuals at 12 months after recovery indicated that a subset of the individuals had not yet achieved full normalization of radiological and functional changes. These data provide insight into mechanisms driving development of pulmonary sequelae during and after COVID-19 and provide a rational basis for development of targeted approaches to prevent long-term complications.


Assuntos
COVID-19 , Armadilhas Extracelulares , Humanos , SARS-CoV-2 , Neutrófilos , Pulmão
11.
J Virol ; 84(2): 1097-109, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19889773

RESUMO

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.


Assuntos
Membrana Celular , Interações Hospedeiro-Patógeno , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Proteínas Estruturais Virais/metabolismo , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Morte Celular , Membrana Celular/patologia , Membrana Celular/virologia , Chlorocebus aethiops , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/virologia , Deleção de Genes , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Humanos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Transfecção , Células Vero/patologia
12.
PLoS Pathog ; 5(5): e1000409, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19412338

RESUMO

The continued spread of highly pathogenic H5N1 influenza viruses among poultry and wild birds, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. Inactivated subvirion or whole-virion H5N1 vaccines have shown promising immunogenicity in clinical trials, but their ability to elicit protective immunity in unprimed human populations remains unknown. A cold-adapted, live attenuated vaccine with the hemagglutinin (HA) and neuraminidase (NA) genes of an H5N1 virus A/VN/1203/2004 (clade 1) was protective against the pulmonary replication of homologous and heterologous wild-type H5N1 viruses in mice and ferrets. In this study, we used reverse genetics to produce a cold-adapted, live attenuated H5N1 vaccine (AH/AAca) that contains HA and NA genes from a recent H5N1 isolate, A/Anhui/2/05 virus (AH/05) (clade 2.3), and the backbone of the cold-adapted influenza H2N2 A/AnnArbor/6/60 virus (AAca). AH/AAca was attenuated in chickens, mice, and monkeys, and it induced robust neutralizing antibody responses as well as HA-specific CD4+ T cell immune responses in rhesus macaques immunized twice intranasally. Importantly, the vaccinated macaques were fully protected from challenge with either the homologous AH/05 virus or a heterologous H5N1 virus, A/bar-headed goose/Qinghai/3/05 (BHG/05; clade 2.2). These results demonstrate for the first time that a cold-adapted H5N1 vaccine can elicit protective immunity against highly pathogenic H5N1 virus infection in a nonhuman primate model and provide a compelling argument for further testing of double immunization with live attenuated H5N1 vaccines in human trials.


Assuntos
Anticorpos Antivirais/sangue , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Administração Intranasal , Animais , Temperatura Corporal , Feminino , Virus da Influenza A Subtipo H5N1/fisiologia , Injeções Intravenosas , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Vírus Reordenados/imunologia , Linfócitos T/imunologia , Vacinação , Vacinas Atenuadas/imunologia , Carga Viral , Replicação Viral , Eliminação de Partículas Virais
13.
J Immunol ; 182(4): 1962-71, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19201849

RESUMO

There is much evidence that T cells may be activated via mechanisms that act independently of direct TCR ligation. Despite this, the question of whether such forms of bystander T cell activation occur during immune responses is hotly debated. To address some outstanding questions, we set up an in vitro system within which to analyze bystander T cell activation in human T cells, in the absence of the possibility for TCR cross-reactivity. In addition, we have investigated the genetic, phenotypic, and functional characteristics of bystander-activated T cells. In this study, we show that bystander T cell activation is, indeed, observed during a specific immune response, and that it occurs preferentially among CD4(+) memory T cells. Furthermore, bystander-activated T cells display a distinct gene expression profile. The mechanism for bystander T cell activation involves soluble factors, and the outcome is an elevated level of apoptosis. This may provide an explanation for the attrition of T cell memory pools of heterologous specificity during immune responses to pathogens such as viruses.


Assuntos
Apoptose/imunologia , Efeito Espectador/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
J Immunol ; 182(2): 1033-40, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19124746

RESUMO

NKT cells recognize lipid Ags presented by CD1d molecules and play an important role in the regulation of innate and adaptive immune responses. In this study, we report the identification of a membrane-associated protein, Ig-like transcript 4 (ILT4), as a novel human CD1d receptor that inhibits CD1d-mediated immune responses. We found that native CD1d tetramer generated by mammalian cells was able to specifically bind human monocytes in the peripheral blood, and this binding was at least partly mediated by monocyte-expressed ILT4. The interaction between ILT4 and CD1d involves the two N-terminal domains of ILT4 and the Ag-binding groove of CD1d (alpha1/alpha2 domain). This interaction has been identified on the cell surface as well as in the endosomal and lysosomal compartments. Functional analysis showed that ILT4 could block the loading of lipid Ags such as alpha-GalCer, and consequently inhibited NKT recognition. The interaction between ILT4 and CD1d may provide new insights into the regulation of NKT-mediated immunity.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Galactosilceramidas/imunologia , Galactosilceramidas/metabolismo , Tolerância Imunológica/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores Imunológicos/fisiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD1d/química , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/imunologia , Citoplasma/metabolismo , Galactosilceramidas/antagonistas & inibidores , Humanos , Imunidade Celular , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Ligação Proteica/imunologia , Ratos , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo
15.
Nat Med ; 9(7): 921-7, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12808447

RESUMO

Dengue virus presents a growing threat to public health in the developing world. Four major serotypes of dengue virus have been characterized, and epidemiological evidence shows that dengue hemorrhagic fever (DHF), the more serious manifestation of the disease, occurs more frequently upon reinfection with a second serotype. We have studied dengue virus-specific T-cell responses in Thai children. During acute infection, few dengue-responsive CD8+ T cells were recovered; most of those present showed an activated phenotype and were undergoing programmed cell death. Many dengue-specific T cells were of low affinity for the infecting virus and showed higher affinity for other, probably previously encountered strains. Profound T-cell activation and death may contribute to the systemic disturbances leading to DHF, and original antigenic sin in the T-cell responses may suppress or delay viral elimination, leading to higher viral loads and increased immunopathology.


Assuntos
Apoptose/fisiologia , Vírus da Dengue/imunologia , Epitopos/imunologia , Antígenos HLA-A/imunologia , Dengue Grave/imunologia , Dengue Grave/virologia , Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Criança , Vírus da Dengue/classificação , Variação Genética , Antígeno HLA-A11 , Humanos , Linfócitos T/imunologia , Linfócitos T/virologia , Tailândia
16.
J Infect Dis ; 201(8): 1173-7, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20210629

RESUMO

The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, suggesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abundant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues.


Assuntos
Colo/virologia , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/virologia , Replicação Viral/fisiologia , Epitélio/virologia , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Mucosa Intestinal/virologia , Técnicas de Cultura de Órgãos , Receptores de Superfície Celular/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Clin Infect Dis ; 51(9): 1028-32, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20887209

RESUMO

BACKGROUND: We followed a cohort of 773 individuals who received a monovalent vaccine against 2009 pandemic influenza A (H1N1). Approximately 6 weeks after vaccination, 12 persons developed the disease. METHODS: Three groups of subjects were studied (12 patients who had or had not received previous monovalent vaccine and 1 group of 49 control subjects who had previously been immunized with the same vaccine). For all patients, clinical features were characterized and the causative viruses sequenced for possible mutations. Nasopharyngeal swabs, serum specimens, and peripheral blood monocyte cells (PBMCs) were collected at different time points up to 11 weeks after symptom onset to measure the virus load and humoral and cellular immune responses. Serum samples and PBMCs were also collected from 49 and 16 vaccinated control subjects, respectively. RESULTS: Both patient groups had similar clinical manifestations. No substantial viral mutations were detected. Compared with unvaccinated patients, viral loads in vaccinated patients were initially higher, but the levels decreased faster to undetectable levels. However, the virus became detectable again for 6 of them. Two weeks after infection, vaccinated and unvaccinated patients had similar neutralizing antibody levels as the vaccinated control subjects. Thereafter, the neutralizing antibody levels decreased markedly in vaccinated patients. During the acute phase, memory T cell counts and tumor necrosis factor-α levels were significantly higher in vaccinated than in unvaccinated patients. CONCLUSIONS: Although the clinical consequences of infection are comparable between vaccinated and unvaccinated patients, humoral and cellular immune responses in vaccinated patients are boosted for some weeks, indicating an additional benefit of vaccination against 2009 pandemic influenza A (H1N1) virus.


Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/patologia , Adolescente , Adulto , Anticorpos Antivirais/sangue , Sangue/imunologia , Sangue/virologia , Estudos de Coortes , Feminino , Humanos , Influenza Humana/virologia , Leucócitos Mononucleares/imunologia , Masculino , Nasofaringe/virologia , Carga Viral , Adulto Jovem
18.
J Virol ; 83(13): 6631-40, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19403678

RESUMO

Viruses such as hepatitis C and the severe acute respiratory syndrome coronavirus (SARS-CoV) encode proteins that are distributed between mitochondria and the nucleus, but little is known about the factors that control partitioning between these sites. SARS-CoV encodes a unique accessory gene called open reading frame (ORF) 3b that, like other unique accessory genes in SARS-CoV, likely contributes to viral pathogenicity. The ORF 3b protein is 154 amino acids and is predicted to express from the second ORF in subgenomic RNA3. In this report, we have characterized the molecular components that regulate intracellular localization of the ORF 3b protein. We demonstrate unique shuttling behavior of ORF 3b, whereby the protein initially accumulates in the nucleus and subsequently translocates to mitochondria. Following nuclear localization, ORF 3b traffics to the outer membrane of mitochondria via a predicted amphipathic alpha-helix. Additionally, ORF 3b contains a consensus nuclear export sequence, and we demonstrate that nuclear export and thus mitochondrial translocation are dependent on a leptomycin B-sensitive nuclear export mechanism. We further show that ORF 3b inhibits induction of type I interferon induced by retinoic acid-induced gene 1 and the mitochondrial antiviral signaling protein. Our observations provide insights into the cellular localization of ORF 3b that may enhance our understanding of the mechanisms by which ORF 3b contributes to SARS-CoV pathogenesis. The findings reported here reveal that for multilocalized proteins, consideration of the spatiotemporal distribution may be crucial for understanding viral protein behavior and function.


Assuntos
Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Fases de Leitura Aberta , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Proteínas Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Humanos , Membranas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Células Vero , Proteínas Estruturais Virais/genética
19.
J Immunol ; 181(9): 5865-74, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18941175

RESUMO

Severe dengue virus (DV) infections can cause the life-threatening condition dengue hemorrhagic fever, which is characterized by a severe plasma leak, thrombocytopenia, hemorrhage, and, in severe cases, circulatory collapse and death. There is now much evidence that pre-existing immunity to DV can enhance disease when an individual becomes infected on a second or sequential occasion. It has been shown that in contrast to infected dendritic cells (DC), noninfected bystander DC underwent maturation in dengue infection. In this study, we show that TNF-alpha and type I IFN contribute to the maturation of bystander DC, whereas the inhibition of DV-infected DC maturation can be overcome by activated T cells. Furthermore, IFN-gamma-inducible chemokines, CXCL9, 10, and 11 produced by infected DC are greatly amplified in the presence of DV-specific T cells. The chemokine secretion is also enhanced in coculture of HUVEC with either DV-infected DC or activated T cells. Finally, we found a close correlation between the serum level of these three chemokines and disease severity.


Assuntos
Citocinas/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/virologia , Linfócitos T/imunologia , Linfócitos T/virologia , Efeito Espectador/imunologia , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Citotoxicidade Imunológica , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Dengue/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Humanos , Ativação Linfocitária/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia
20.
J Immunol ; 181(8): 5490-500, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18832706

RESUMO

Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-gamma ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8(+) T cell responses were more frequent and of a greater magnitude than CD4(+) T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27(+)/CD45RO(+)) with a significantly higher frequency of polyfunctional CD4(+) T cells producing IFN-gamma, TNF-alpha, and IL-2, and CD8(+) T cells producing IFN-gamma, TNF-alpha, and CD107a (degranulation), as compared with the mild-moderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Células Th2/imunologia , Adulto , Estudos de Coortes , Citocinas/imunologia , Feminino , Humanos , Antígenos Comuns de Leucócito/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Masculino , Pessoa de Meia-Idade , Proteoma/imunologia , Síndrome Respiratória Aguda Grave/mortalidade , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Vacinas Virais/imunologia
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