RESUMO
Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.
Assuntos
Antígenos CD/metabolismo , Hepacivirus/patogenicidade , Receptores Virais/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Sequência Conservada , Hepacivirus/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Tetraspanina 28 , Proteínas do Envelope Viral/genéticaRESUMO
An 83-year-old woman receiving long-term prednisolone treatment presented with a right optic neuropathy and right third, fourth, fifth, and sixth cranial nerve palsies secondary to sino-orbital aspergillosis with cavernous sinus involvement. Because the patient refused conventional treatment, she was given a two-year course of oral itraconazole 200 mg/day, leading to a complete imaging resolution of the lesion. Three years after completing treatment, there is no clinical or imaging evidence of disease recurrence. Visual and ocular motor function did not recover, but ptosis and proptosis improved. We believe this to be the first documented case of successful treatment of such a lesion with oral itraconazole monotherapy.
Assuntos
Antifúngicos/administração & dosagem , Aspergilose/tratamento farmacológico , Aspergillus/isolamento & purificação , Seio Cavernoso/microbiologia , Itraconazol/administração & dosagem , Administração Oral , Idoso de 80 Anos ou mais , Aspergilose/diagnóstico , Aspergilose/microbiologia , Aspergillus/genética , Biópsia , DNA Fúngico/análise , Diagnóstico Diferencial , Feminino , Seguimentos , Humanos , Imageamento por Ressonância MagnéticaRESUMO
The mouse monoclonal antibody (MAb) AP33, recognizing a 12 amino acid linear epitope in the hepatitis C virus (HCV) E2 glycoprotein, potently neutralizes retroviral pseudoparticles (HCVpp) carrying genetically diverse HCV envelope glycoproteins. Consequently, this antibody and its epitope are highly relevant to vaccine design and immunotherapeutic development. The rational design of immunogens capable of inducing antibodies that target the AP33 epitope will benefit from a better understanding of this region. We have used complementary approaches, which include random peptide phage display mapping and alanine scanning mutagenesis, to identify residues in the HCV E2 protein critical for MAb AP33 binding. Four residues crucial for MAb binding were identified, which are highly conserved in HCV E2 sequences. Three residues within E2 were shown to be critical for binding to the rat MAb 3/11, which previously was shown to recognize the same 12 amino acid E2 epitope as MAb AP33 antibody, although only two of these were shared with MAb AP33. MAb AP33 bound to a panel of functional E2 proteins representative of genotypes 1-6 with higher affinity than MAb 3/11. Similarly, MAb AP33 was consistently more efficient at neutralizing infectivity by diverse HCVpp than MAb 3/11. Importantly, MAb AP33 was also able to neutralize the cell culture infectious HCV clone JFH-1. In conclusion, these data identify important protective determinants and will greatly assist the development of vaccine candidates based on the AP33 epitope.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Hepacivirus/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Linhagem Celular , Hepatite C/virologia , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/química , Antígenos da Hepatite C/imunologia , Humanos , Testes de Neutralização , Biblioteca de Peptídeos , Conformação Proteica , Proteínas do Envelope Viral/químicaRESUMO
Patients with infectious mononucleosis (IM) undergoing primary EBV infection show large expansions of EBV-specific CD8+ T cells in the blood. While latent infection of the B cell pool is quickly controlled, virus shedding from lytically infected cells in the oropharynx remains high for several months. We therefore studied how responses localize to the tonsil, a major target site for EBV, during primary infection and persistence. In acute IM, EBV-specific effectors were poorly represented among CD8+ T cells in tonsil compared with blood, coincident with absence of the CCR7 lymphoid homing marker on these highly activated cells. In patients who had recently recovered from IM, latent epitope reactivities were quicker than lytic reactivities both to acquire CCR7 and to accumulate in the tonsil, with some of these cells now expressing the CD103 integrin, which mediates retention at mucosal sites. By contrast, in long-term virus carriers in whom both lytic and latent infections had been controlled, there was 2- to 5-fold enrichment of lytic epitope reactivities and 10- to 20-fold enrichment of latent epitope reactivities in tonsil compared with blood; up to 20% of tonsillar CD8+ T cells were EBV specific, and many now expressed CD103. We suggest that efficient control of EBV infection requires appropriate CD8+ T cell homing to oropharyngeal sites.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Herpesvirus Humano 4/metabolismo , Mononucleose Infecciosa/imunologia , Tonsila Palatina/imunologia , Tonsila Palatina/virologia , Antígenos CD/imunologia , Portador Sadio , Humanos , Mononucleose Infecciosa/virologia , Fenótipo , Receptores CCR7 , Receptores de Quimiocinas/imunologia , Carga ViralRESUMO
BACKGROUND: Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) encompasses a histologically broad range of lesions, arising from the expanded pool of EBV-infected B cells in the immunocompromised host. Identification of the precise cellular origin of these tumours could clarify their pathogenesis. METHODS: Of 13 cases of EBV-positive cases of PTLD characterised by histological analysis, pattern of EBV gene expression, and clinical course, 11 had monoclonal or biclonal lesions in which we determined the progenitor B cell by immunoglobulin heavy chain (IgH) genotyping. RESULTS: Two tumours had a naive B cell genotype and two showed patterns of IgH somatic mutation typical of antigen-selected (post-germinal-centre) memory cells. All four arose early post-transplant and expressed the markers of EBV transformation--Epstein-Barr nuclear antigen (EBNA) 2 and latent membrane protein (LMP) 1. However, seven tumours, either of early or late onset and including some with downregulated EBNA 2 and LMP 1, arose from post-germinal cells with randomly mutated or sterile IgH genotypes usually incompatible with B-cell survival in vivo. INTERPRETATION: PTLD can arise from a broad range of target B cells and not only from the pool of antigen-selected memory cells that EBV generally colonises in immunocompetent individuals. Tumour development seems frequently associated with the EBV-induced rescue and expansion of B cells that have failed the physiological process of germinal centre selection into memory. This finding shows an unexpected connection between pathogenesis of PTLD and that of EBV-positive Hodgkin's lymphoma, another B-cell malignancy of atypical post-germinal-centre cell origin.