Geoff Connolly

Endogenous Presentation of CD8 T Cell Epitopes from Epstein-Barr Virus-encoded Nuclear Antigen 1

Epstein-Barr virus (EBV)–encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type–dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I–restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8+ T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.

Induction of Therapeutic T-Cell Responses to Subdominant Tumor-associated Viral Oncogene after Immunization with Replication-incompetent Polyepitope Adenovirus Vaccine

The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed in various EBV-associated malignancies have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and potential threat from their oncogenic potential. Here we have developed a replication- incompetent adenoviral vaccine that encodes multiple HLA class I-restricted CTL epitopes from LMP1 and LMP2 as a polyepitope. Immunization with this polyepitope vaccine consistently generated strong LMP-specific CTL responses in HLA A2/Kb mice, which can be readily detected by both ex vivo and in vivo T-cell assays. Furthermore, a human CTL response to LMP antigens can be rapidly expanded after stimulation with this recombinant polyepitope vector. These expanded T cells displayed strong lysis of autologous target cells sensitized with LMP1 and/or LMP2 CTL epitopes. More importantly, this adenoviral vaccine was also successfully used to reverse the outgrowth of LMP1-expressing tumors in HLA A2/Kb mice. These studies demonstrate that a replication-incompetent adenovirus polyepitope vaccine is an excellent tool for the induction of a protective CTL response directed toward multiple LMP CTL epitopes restricted through common HLA class I alleles prevalent in different ethnic groups where EBV-associated malignancies are endemic.

Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition

Many viruses avoid immune surveillance during latent infection through reduction in the synthesis of virally encoded proteins. Although antigen presentation critically depends on the level of viral protein synthesis, the precise mechanism used to regulate the generation of antigenic peptide precursors remains elusive. Here, we demonstrate that a purine overloaded virally encoded mRNA lacking secondary structure significantly impacts the efficiency of protein translation and prevents endogenous antigen presentation. Reducing this purine bias through the generation of constructs expressing codon-modified sequences, while maintaining the encoded protein sequence, increased the stem–loop structure of the corresponding mRNA and dramatically enhanced self-synthesis of the viral protein. As a consequence, a higher number of HLA–peptide complexes were detected on the surface of cells expressing this viral protein. Furthermore, these cells were more efficiently recognized by virus-specific T cells compared with those expressing the same antigen expressed by a purine-biased mRNA. These findings delineate a mechanism by which viruses regulate self-synthesis of proteins and offer an effective strategy to evade CD8+ T cell-mediated immune regulation.

Potent T cell response to a class I‐binding 13‐mer viral epitope and the influence of HLA micropolymorphism in controlling epitope length

The BZLF1 antigen of Epstein‐Barr virus includes three overlapping sequences of different lengths that conform to the binding motif of human leukocyte antigen (HLA) B*3501. These 9‐mer (56LPQGQLTAY64), 11‐mer (54EPLPQGQLTAY64), and 13‐mer (52LPEPLPQGQLTAY64) peptides all bound well to B*3501; however, the CTL response in individuals expressing this HLA allele was directed strongly and exclusively towards the 11‐mer peptide. In contrast, EBV‐exposed donors expressing HLA B*3503 showed no significant CTL response to these peptides because the single amino acid difference between B*3501 and B*3503 within the F pocket inhibited HLA binding by these peptides. The extraordinarily long 13‐mer peptide was the target for the CTL response in individuals expressing B*3508, which differs from B*3501 at a single position within the D pocket (B*3501, 156Leucine; B*3508, 156Arginine). This minor difference was shown to enhance binding of the 13‐mer peptide, presumably through a stabilizing interaction between the negatively charged glutamate at position 3 of the peptide and the positively charged arginine at HLA position 156. The 13‐mer epitope defined in this study represents the longest class I‐binding viral epitope identified to date as a minimal determinant. Furthermore, the potency of the response indicates that peptides of this length do not present a major structural barrier to CTL recognition.

Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes

A significant proportion of endogenously processed CD8+ T cell epitopes are derived from newly synthesized proteins and rapidly degrading polypeptides (RDPs). It has been hypothesized that the generation of rapidly degrading polypeptides and CD8+ T cell epitopes from these RDP precursors may be influenced by the efficiency of protein translation. Here we address this hypothesis by using the Epstein-Barr virus–encoded nuclear antigen 1 protein (EBNA1), with or without its internal glycine-alanine repeat sequence (EBNA1 and EBNA1ΔGA, respectively), which display distinct differences in translation efficiency. We demonstrate that RDPs constitute a significant proportion of newly synthesized EBNA1 and EBNA1ΔGA and that the levels of RDPs produced by each of these proteins directly correlate with the translation efficiency of either EBNA1 or EBNA1ΔGA. As a consequence, a higher number of major histocompatibility complex–peptide complexes can be detected on the surface of cells expressing EBNA1ΔGA, and these cells are more efficiently recognized by virus-specific cytotoxic T lymphocytes compared to the full-length EBNA1. More importantly, we also demonstrate that the endogenous processing of these CD8+ T cell epitopes is predominantly determined by the rate at which the RDPs are generated rather than the intracellular turnover of these proteins.

Selection Pressure-Driven Evolution of the Epstein-Barr Virus-Encoded Oncogene LMP1 in Virus Isolates from Southeast Asia

ABSTRACT The geographically constrained distribution of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) in southeast Asian populations suggests that both viral and host genetics may influence disease risk. Although susceptibility loci have been mapped within the human genome, the role of viral genetics in the focal distribution of NPC remains an enigma. Here we report a molecular phylogenetic analysis of an NPC-associated viral oncogene, LMP1, in a large panel of EBV isolates from southeast Asia and from Papua New Guinea, Africa, and Australia, regions of the world where NPC is and is not endemic, respectively. This analysis revealed that LMP1 sequences show a distinct geographic structure, indicating that the southeast Asian isolates have evolved as a lineage distinct from those of Papua New Guinea, African, and Australian isolates. Furthermore, a likelihood ratio test revealed that the C termini of the LMP1 sequences of the southeast Asian lineage are under significant positive selection pressure, particularly at some sites within the C-terminal activator regions. We also present evidence that although the N terminus and transmembrane region of LMP1 have undergone recombination, the C-terminal region of the gene has evolved without any history of recombination. Based on these observations, we speculate that selection pressure may be driving the LMP1 sequences in virus isolates from southeast Asia towards a more malignant phenotype, thereby influencing the endemic distribution of NPC in this region.

Epstein-Barr Virus Associated Modulation of Wnt Pathway Is Not Dependent on Latent Membrane Protein-1

Previous studies have indicated that Epstein-Barr virus (EBV) can modulate the Wnt pathway in virus-infected cells and this effect is mediated by EBV-encoded oncogene latent membrane protein 1 (LMP1). Here we have reassessed the role of LMP1 in regulating the expression of various mediators of the canonical Wnt cascade. Contradicting the previous finding, we found that the levels of E-cadherin, β-catenin, Glycogen Synthase Kinase 3ß (GSK3β), axin and α-catenin were not affected by the expression of LMP1 sequences from normal B cells or nasopharyngeal carcinoma. Moreover, we also show that LMP1 expression had no detectable effect on the E-cadherin and β-catenin interaction and did not induce transcriptional activation of β-catenin. Taken together these studies demonstrate that EBV-mediated activation of Wnt pathway is not dependent on the expression of LMP1.

Translation efficiency of EBNA1 encoded by lymphocryptoviruses influences endogenous presentation of CD8(+) T cell epitopes

Lymphocryptoviruses (LCV) that infect humans and Old World primates display a significant degree of genetic identity. These viruses use B lymphocytes as primary host cells to establish a long‐term latent infection and express highly homologous latent viral proteins. Of particular interest is the expression of the EBV‐encoded nuclear antigen‐1 (EBNA1), which plays a crucial role in maintaining the viral genome in B cells. Using human and Old World primate homologues of EBNA1, we show that the internal repeat sequences differentially influence their in vitro translation efficiency. Although the glycine‐alanine repeat domain of human LCV (EBV) EBNA1 inhibits its self‐synthesis, the repeat domains within the simian LCV homologues of EBNA1 do not inhibit self‐synthesis. As a consequence, simian LCV EBNA1‐expressing cells are more efficiently recognized by virus‐specific CTL when compared to human EBV EBNA1, even though both proteins are highly stable in B cells. Interestingly, we also show that similar to human EBNA1, CD8+ T cell epitopes from simian LCV EBNA1 are predominantly derived from newly synthesized protein rather than the long‐lived pool of stable protein. These observations provide additional evidence that supports the theory that immune recognition of EBNA1 can occur without compromising the biological maintenance function of this protein.