Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies

Adrian Chong Nyi Sim,Shu Uin Gan,Michelle Yating Eio,Paul Anthony Macary,Kok Onn Lee,Jianzhu Chen,Chien Tei Too,Diane Ai Lin Tan,Thomas Kwok Seng Loh,Min Zin Oo,Nalini Srinivasan,Soh Ha Chan,Zhenying Song,Shyue Wei Pang,Junyun Lai

doi:10.1038/srep03232

Abstract

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 LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 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.

Highlights

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts
The latent gene products Latent Membrane Protein 1 (LMP1), Latent Membrane Protein 2A (LMP2A) and Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines
The virus can infect cells of both lymphoid and epithelial origin and its latent infection phase is associated with malignancies that arise from these cell types, including Non-Hodgkin’s lymphoma, Hodgkin’s Lymphoma[2] and undifferentiated nasopharyngeal carcinoma (NPC)[3]

Summary

Introduction

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. We describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201 These are employed to map the expression hierarchy of endogenously generated EBV epitopes. Despite the subdominant frequencies of CTLs specific for epitopes derived from these latent gene products (0.05%–1%), they are implicated in the control of EBV infection in vivo[1] The presence of these gene products in the majority of EBV-associated tumors suggests that an analysis of their associated CTL epitopes is essential for the design of immuno-targeting approaches including adoptive T-cell therapy. Antibodies targeting epitopes of EBV latent gene products (LMP1125–133, LMP2A426–434 and EBNA1562–570) were generated and characterized This allowed an analysis of viral epitope expression using a combination of immunological and biochemical methods www.nature.com/scientificreports including flow cytometry, immunohistochemical staining, and confocal microscopy. Our observations of this hierarchy and its differential binding on strain-associated epitope variants have important implications for diagnosis, immuno-targeting and vaccine development

Methods

Results

Conclusion