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1.
Clin Cancer Res ; 28(23): 5221-5230, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36165913

RESUMO

PURPOSE: The favorable prognosis of stage I and II nasopharyngeal carcinoma (NPC) has motivated a search for biomarkers for the early detection and risk assessment of Epstein-Barr virus (EBV)-associated NPC. Although EBV seropositivity is ubiquitous among adults, a spike in antibodies against select EBV proteins is a harbinger of NPC. A serologic survey would likely reveal which EBV antibodies could discriminate those at risk of developing NPC. EXPERIMENTAL DESIGN: Lysates from a new EBV mammalian expression library were used in a denaturing multiplex immunoblot assay to survey antibodies against EBV in sera collected from healthy individuals who later developed NPC (incident cases) in a prospective cohort from Singapore and validated in an independent cohort from Shanghai, P.R. China. RESULTS: We show that IgA against EBV nuclear antigen 1 (EBNA1) discriminated incident NPC cases from matched controls with 100% sensitivity and 100% specificity up to 4 years before diagnosis in both Singapore and Shanghai cohorts. Incident NPC cases had a greater IgG repertoire against lytic-classified EBV proteins, and the assortment of IgA against EBV proteins detected by the immunoblot assay increased closer to diagnosis. CONCLUSIONS: Although NPC tumors consistently harbor latent EBV, the observed heightened systemic and mucosal immunity against lytic-classified antigens years prior to clinical diagnosis is consistent with enhanced lytic transcription. We conclude that an expanding EBV mucosal reservoir (which can be latent and/or lytic) is a risk factor for NPC. This presents an opportunity to identify those at risk of developing NPC using IgA against EBNA1 as a biomarker.


Assuntos
Carcinoma , Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Adulto , Humanos , Antígenos Nucleares do Vírus Epstein-Barr , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/diagnóstico , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/diagnóstico , Estudos Prospectivos , Imunoglobulina A , China , Anticorpos Antivirais , Biomarcadores
2.
Bio Protoc ; 12(6): e4365, 2022 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-35434197

RESUMO

The ubiquitous and cancer-associated Epstein-Barr virus (EBV) is associated with nearly all cases of nasopharyngeal carcinoma (NPC). Nasopharyngeal tissue is comprised of both pseudostratified and stratified epithelium, which are modeled in three-dimensional (3-D) cell culture. The cellular origin of EBV-associated NPC is as yet unknown, but both latent and lytic infections are likely important for preneoplastic mechanisms and replenishing the compartmentalized viral reservoir. Conventional 2-D cultures of nasopharyngeal epithelial cells (as primary cells or immortalized cell lines) are difficult to infect with EBV and cannot mimic the tissue-specific biology of the airway epithelium, which can only be captured in 3-D models. We have shown that EBV can infect the pseudostratified epithelium in air-liquid interface (ALI) culture using primary conditionally reprogrammed cells (CRCs) derived from the nasopharynx. In this protocol, we provide a step-by-step guide for the (i) conditional reprogramming of primary nasopharyngeal cells, (ii) differentiation of CRCs into pseudostratified epithelium in ALI culture (known as pseudo-ALI), and (iii) EBV infection of pseudo-ALI cultures. Additionally, we show that nasopharyngeal CRCs can be grown as organotypic rafts and subjected to EBV infection. These nasopharyngeal-derived 3-D cell cultures can be used to study EBV latent and lytic infection in relation to cell type and donor variation, by immunostaining and single-cell RNA-sequencing methods ( Ziegler et al., 2021 ). These methods are useful for studies of EBV molecular pathogenesis, and can overcome many of the limitations associated with conventional 2-D cell cultures. Graphic abstract: Workflow of nasopharyngeal-derived conditionally reprogrammed cells grown into pseudostratified-ALI and organotypic rafts in 3-D cell culture. Created with Biorender.com.

3.
PLoS Pathog ; 17(4): e1009041, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33914843

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, α-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (~0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC.


Assuntos
Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/virologia , Interações Hospedeiro-Patógeno/imunologia , Neoplasias Nasofaríngeas/virologia , Carcinoma/metabolismo , Carcinoma/virologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Epitélio/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Carcinoma Nasofaríngeo/virologia , RNA Viral/genética
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