Despite triggering strong immune responses, Epstein-Barr virus (EBV) has colonized more than 90% of the adult human population. Successful persistence of EBV depends on the establishment of a balance between host immune responses and viral immune evasion. Here we have extended our studies on the EBV-encoded BILF1 protein, which was recently identified as an immunoevasin that functions by enhancing degradation of major histocompatibility complex class I (MHC-I) antigens via lysosomes. We now demonstrate that disruption of the EKT signaling motif of BILF1 by a K122A mutation impairs the ability of BILF1 to enhance endocytosis of surface MHC-I molecules, while subsequent lysosomal degradation was impaired by deletion of the 21-residue C-terminal tail of BILF1. Furthermore, we identified another mechanism of BILF1 immunomodulation: it targets newly synthesized MHC-I/peptide complexes en route to the cell surface. Importantly, although the diversion of MHC-I on the exocytic pathway caused a relatively modest reduction in cell surface MHC-I, presentation of endogenously processed target peptides to immune CD8 ؉ effector T cells was reduced by around 65%. The immune-modulating functions of BILF1 in the context of the whole virus were confirmed in cells lytically infected with a recombinant EBV in which BILF1 was deleted. This study therefore extends our initial observations on BILF1 to show that this immunoevasin can target MHC-I antigen presentation via both the exocytic and endocytic trafficking pathways. The results also emphasize the merits of including functional T cell recognition assays to gain a more complete picture of immunoevasin effects on the antigen presentation pathway.

Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus, encodes 2 immediate early (IE), >30 early (E) and >30 late (L) phase proteins during its replication (lytic) cycle. Despite this, lytic antigen-induced CD8 responses are strongly skewed towards IE and a few E proteins only, all expressed before HLA I presentation is blocked in lytically-infected cells. For comparison, here we (i) examined CD4+ T cell responses to 8 IE, E or L proteins, screening 14 virus-immune donors to overlapping peptide pools in interferon-gamma Elispot assays, and (ii) established CD4+ T cell clones against 12 defined epitopes for target recognition assays. We find that the lytic antigen-specific CD4+ T cell response differs radically from its CD8 counterpart in that (i) it is widely distributed across IE, E and L antigen targets, often with multiple reactivities detectable per donor and with either IE, E or L epitope responses being numerically dominant, and (ii) all CD4+ T cell clones, whether IE, E or L epitope-specific, show strong recognition of EBV-transformed B cell lines despite the lines containing only a small fraction of lytically-infected cells; efficient recognition occurs because lytic antigens are released into the culture, then acquired and processed by neighbouring latently-infected cells. These findings suggest that (i) lytic antigen-specific CD4 responses are driven by a different route of antigen display than drives CD8 responses, and (ii) such CD4 effectors could be therapeutically useful against EBV-driven lymphoproliferative disease lesions, which contain similarly small fractions of EBV-transformed cells entering lytic cycle.

2020) Preclinical evaluation of an affinity-enhanced MAGE-A4-specific T-cell receptor for adoptive T-cell therapy, OncoImmunology, 9:1, 1682381, ABSTRACT A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line crossreactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768). ARTICLE HISTORY

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE

Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha‐fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer‐specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity‐optimized T‐cell receptor (TCR) with specificity to AFP/HLA‐A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA‐A*02‐restricted AFP158‐166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X‐scan) and testing TCR‐transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR‐transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T‐cell immunotherapy.

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8؉ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8 ؉ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8 ؉ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4 ؉ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8؉ and CD4 ؉ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8؉ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8 ؉ T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8 ؉ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4 ؉ T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. E pstein-Barr virus (EBV) is a gammaherpesvirus found in more than 90% of the human population. Primary infection with EBV is usually followed by establishment of a lifelong latent infection, with occasional reactivation (1). The balance between host immune responses, including CD4 ϩ and CD8 ϩ T cells, and viral immune evasion of these responses is key to the spread and survival of EBV in human populations. Passive evasion through the ability to establish antigenically silent latent infections is an important characteristic of all herpesviruses, ...

Epstein-Barr virus (EBV) elicits primary CD8+ T cell responses that, by T cell cloning from infectious mononucleosis (IM) patients, appear skewed towards immediate early (IE) and some early (E) lytic cycle proteins, with late (L) proteins rarely targeted. However, L antigen-specific responses have been regularly detected in polyclonal T cell cultures from long-term virus carriers. To resolve this apparent difference between responses to primary and persistent infection, 13 long-term carriers were screened in ex vivo IFN-γ ELISPOT assays using peptides spanning the 2 IE, 6 representative E and 7 representative L proteins. This revealed memory CD8 responses to 44 new lytic cycle epitopes that straddle all three protein classes but, in terms of both frequency and size, maintain the IE > E > L hierarchy of immunodominance. Having identified the HLA restriction of 10 (including 7L) new epitopes using memory CD8+ T cell clones, we looked in HLA-matched IM patients and found such reactivities but typically at low levels, explaining why they had gone undetected in the original IM clonal screens. Wherever tested, all CD8+ T cell clones against these novel lytic cycle epitopes recognised lytically-infected cells naturally expressing their target antigen. Surprisingly, however, clones against the most frequently recognised L antigen, the BNRF1 tegument protein, also recognised latently-infected, growth-transformed cells. We infer that BNRF1 is also a latent antigen that could be targeted in T cell therapy of EBV-driven B-lymphoproliferative disease.

Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replication in vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses. IMPORTANCEThis report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor. E pstein-Barr Virus (EBV), one of eight human herpesviruses, is carried by over 90% of the world's adult population. Primary EBV infection occurs in the oropharynx, leading to infection of B lymphocytes (1, 2). These infected B cells can support the lytic cycle, in which more than 80 viral genes are expressed to generate new infectious virus, but they more frequently host nonproductive infections through expression of a limited number of socalled latent EBV genes (latency III genes) that drive lymphoproliferation as an alternative mechanism of expanding the infected cell pool. In vitro, this growth transformation is demonstrated by the ready establishment of lymphoblastoid cell lines (LCLs) following infection of resting B cells. Following initial infection in vivo, EBV downregulates the expression of all viral proteins and enters a true latent phase (latency 0) in the memory B cell population, where it establishes a lifelong infection (1). Periodically the virus reactivates and undergoes fu...