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1.
Sci Rep ; 7(1): 9923, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28855662

RESUMO

Epstein-Barr virus (EBV) is a common gammaherpesvirus associated with various human malignancies. Antibodies with T cell receptor-like specificities (TCR-like mAbs) provide a means to target intracellular tumor- or virus-associated antigens by recognising their processed peptides presented on major histocompatibility complex (MHC) class I (pMHC) complexes. These antibodies are however thought to be relevant only for a single HLA allele. Here, we show that HLA-A*02:01-restricted EBV antigenic peptides EBNA1562-570, LMP1125-133 and LMP2A426-434 display binding degeneracy towards HLA-A*02 allelic microvariants, and that these pMHC complexes are recognised by anti-EBV TCR-like mAbs E1, L1 and L2 raised in the context of HLA-A*02:01. These antibodies bound endogenously derived pMHC targets on EBV-transformed human B lymphoblastoid cell lines expressing A*02:01, A*02:03, A*02:06 and A*02:07 alleles. More importantly, these TCR-like mAbs mediated both complement-dependent and antibody-dependent cellular cytotoxicity of these cell lines in vitro. This finding suggests the utility of TCR-like mAbs against target cells of closely related HLA subtypes, and the potential applicability of similar reagents within populations of diverse HLA-A*02 alleles.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígeno HLA-A2/genética , Herpesvirus Humano 4/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Variação Genética , Antígeno HLA-A2/química , Antígeno HLA-A2/imunologia , Infecções por Herpesviridae/imunologia , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo
2.
Blood ; 128(10): 1396-407, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27338099

RESUMO

Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor-like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg(-/-) mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Antígeno HLA-A2/imunologia , Herpesvirus Humano 4/imunologia , Neoplasias Hepáticas/prevenção & controle , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fagocitose/imunologia
3.
Sci Rep ; 5: 8235, 2015 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-25648885

RESUMO

The Type VI Secretion System cluster 1 (T6SS1) is essential for the pathogenesis of Burkholderia pseudomallei, the causative agent of melioidosis, a disease endemic in the tropics. Inside host cells, B. pseudomallei escapes into the cytosol and through T6SS1, induces multinucleated giant cell (MNGC) formation that is thought to be important for bacterial cell to cell spread. The hemolysin-coregulated protein (Hcp) is both a T6SS substrate, as well as postulated to form part of the T6SS secretion tube. Our structural study reveals that Hcp1 forms hexameric rings similar to the other Hcp homologs but has an extended loop (Asp40-Arg56) that deviates significantly in position compared to other Hcp structures and may act as a key contact point between adjacent hexameric rings. When two residues within the loop were mutated, the mutant proteins were unable to stack as dodecamers, suggesting defective tube assembly. Moreover, infection with a bacterial mutant containing in situ substitution of these hcp1 residues abolishes Hcp1 secretion inside infected cells and MNGC formation. We further show that Hcp has the ability to preferentially bind to the surface of antigen-presenting cells, which may contribute to its immunogenicity in inducing high titers of antibodies seen in melioidosis patients.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Burkholderia pseudomallei/fisiologia , Domínios e Motivos de Interação entre Proteínas , Sistemas de Secreção Tipo VI , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Linhagem Celular , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Melioidose/imunologia , Melioidose/microbiologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/metabolismo
4.
Sci Rep ; 3: 3232, 2013 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-24240815

RESUMO

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1(125-133), LMP2A(426-434) or EBNA1(562-570) in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1(562-570) in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease.


Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Latência Viral/imunologia , Animais , Antígenos Virais/imunologia , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Antígeno HLA-A2/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas da Matriz Viral/imunologia
5.
J Immunol ; 182(6): 3503-9, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19265128

RESUMO

Dendritic cell (DC) immunogenicity correlates with its maturation, which can be induced by toxic microbial products such as LPS. In this study, we report that a nontoxic polysaccharide-protein complex isolated from a Chinese medicinal herb, Lycium barbarum (LBP), induces phenotypic and functional maturation of DCs with strong immunogenicity. LBP up-regulated DC expression of CD40, CD80, CD86, and MHC class II molecules; down-regulated DC uptake of Ag; enhanced DC allostimulatory activity; and induced IL-12p40 and p70 production. All of its five fractions were active. LBP developed enhanced Th1 response, and LBP-treated DCs enhanced Th1 and Th2 responses in vitro and in vivo. Our study provides evidence and rationale on using LBP in various clinical conditions to enhance host immunity and suggests LBP as a potent adjuvant for the design of DC-based vaccines.


Assuntos
Células Dendríticas/imunologia , Medicamentos de Ervas Chinesas/farmacologia , Lycium/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/isolamento & purificação , Feminino , Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Plantas/administração & dosagem , Proteínas de Plantas/farmacologia , Células Th1/imunologia , Células Th2/imunologia
6.
Phytother Res ; 23(8): 1116-22, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19170138

RESUMO

Macrophages play crucial roles in innate immunity. This paper reports that a polysaccharide-protein complex isolated from Lycium barbarum (LBP) is able to activate macrophages. LBP was isolated from Lycium barbarum fruit and separated to five homogenous fractions, designated LBPF1, LBPF2, LBPF3, LBPF4 and LBPF5. It was found that LBP (50 mg/kg, i.p.) markedly upregulated the expressions of CD40, CD80, CD86 and MHC class II molecules on peritoneal macrophages. In vitro studies showed that LBP and LBPF1-5 activated transcription factors NF-kappaB and AP-1 by RAW264.7 macrophage cells, induced TNF-alpha, IL-1beta, IL-12p40 mRNA expression, and enhanced TNF-alpha production in a dose-dependent manner. Furthermore, LBP (50 mg/kg, i.p.) significantly enhanced macrophage endocytic and phagocytic capacities in vivo. These results indicate that LBP enhances innate immunity by activating macrophages. The mechanism may be through activation of transcription factors NF-kappaB and AP-1 to induce TNF-alpha production and upregulation of MHC class II costimulatory molecules.


Assuntos
Lycium/química , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Imunidade Inata , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Fagocitose/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
7.
Hum Immunol ; 66(5): 483-93, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15935885

RESUMO

Nasopharyngeal carcinoma (NPC) is a human epithelial tumor with a high incidence in Southern Chinese population, with contributions from Epstein-Barr virus (EBV), human leukocyte antigen (HLA), and environmental factors to its etiology. It has been shown previously that the recognition of immediate-early transactivator Rta of EBV by CD8+ T cells may have a significant impact on controlling EBV and, indirectly, NPC. The current study used two computer-aided prediction methods and competition-based HLA-peptide binding assays to screen for HLA B2704/B4601/B5801 restricted T-cell epitopes derived from Rta. HLA tetrameric complexes containing these potential T-cell epitopes were synthesized. Rta-specific CD8+ T-cell responses in healthy virus carriers were then defined by these tetramers and IFN-gamma ELISPOT assays. We clearly demonstrated that healthy virus carriers have detectable Rta-specific CD8+ T cells restricted by B2704 in the circulation. However, there were no B4601/B5801 tetramer-reactive T cells specific for Rta in the peripheral blood of matched/mismatched donors. On the other hand, B4601 tetramers containing the computer-predicted B4601 binder EBNA3A (318-326) showed detectable tetramer-reactive T cells in the circulation of healthy virus carriers. topes also elicited IFN-gamma responses as detected by ELISPOT.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-B/imunologia , Proteínas Imediatamente Precoces/imunologia , Transativadores/imunologia , Sequência de Aminoácidos , Linfócitos B/citologia , Linfócitos B/imunologia , Ligação Competitiva/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Simulação por Computador , Epitopos de Linfócito T/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Antígenos HLA-B/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Contagem de Linfócitos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Transativadores/genética , Proteínas Virais
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