Potent human monoclonal antibodies targeting Epstein-Barr virus gp42 reveal vulnerable sites for virus infection.

Epstein-Barr virus (EBV) is linked to various malignancies and autoimmune diseases, posing a significant global health challenge due to the lack of specific treatments or vaccines. Despite its crucial role in EBV infection in B cells, the mechanisms of the glycoprotein gp42 remain elusive. In this study, we construct an antibody phage library from 100 EBV-positive individuals, leading to the identification of two human monoclonal antibodies, 2B7 and 2C1. These antibodies effectively neutralize EBV infection in vitro and in vivo while preserving gp42's interaction with the human leukocyte antigen class II (HLA-II) receptor. Structural analysis unveils their distinct binding epitopes on gp42, different from the HLA-II binding site. Furthermore, both 2B7 and 2C1 demonstrate potent neutralization of EBV infection in HLA-II-positive epithelial cells, expanding our understanding of gp42's role. Overall, this study introduces two human anti-gp42 antibodies with potential implications for developing EBV vaccines targeting gp42 epitopes, addressing a critical gap in EBV research.

Evaluation of serum Epstein-Barr virus envelope glycoproteins antibodies and their association with systemic autoimmune diseases.

Systemic autoimmune diseases (SADs) are a growing spectrum of autoimmune disorders that commonly affect multiple organs. The role of Epstein-Barr virus (EBV) infection or reactivation as a trigger for the initiation and progression of SADs has been established, while the relationship between EBV envelope glycoproteins and SADs remains unclear. Here, we assessed the levels of IgG, IgA, and IgM against EBV glycoproteins (including gp350, gp42, gHgL, and gB) in serum samples obtained from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and found that RA and SLE patients exhibited a statistically significant increase in the levels of 8 and 11 glycoprotein antibodies, respectively, compared to healthy controls (p < 0.05). The LASSO model identified four factors as significant diagnostic markers for RA: gp350 IgG, gp350 IgA, gHgL IgM, and gp42 IgA; whereas for SLE it included gp350 IgG, gp350 IgA, gHgL IgA, and gp42 IgM. Combining these selected biomarkers yielded an area under the curve (AUC) of 0.749 for RA and 0.843 for SLE. We subsequently quantified the levels of autoantibodies associated with SADs in mouse sera following immunization with gp350. Remarkably, none of the tested autoantibody levels exhibited statistically significant alterations. Elevation of glycoprotein antibody concentration suggests that Epstein-Barr virus reactivation and replication occurred in SADs patients, potentially serving as a promising biomarker for diagnosing SADs. Moreover, the absence of cross-reactivity between gp350 antibodies and SADs-associated autoantigens indicates the safety profile of a vaccine based on gp350 antigen.

EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression through recruiting monocytes and regulating their directed differentiation.

Epstein-Barr virus (EBV) is associated with several types of human cancer including nasopharyngeal carcinoma (NPC). The activation of EBV to the lytic cycle has been observed in advanced NPC and is believed to contribute to late-stage NPC development. However, how EBV lytic cycle promotes NPC progression remains elusive. Analysis of clinical NPC samples indicated that EBV reactivation and immunosuppression were found in advanced NPC samples, as well as abnormal angiogenesis and invasiveness. To investigate the role of the EBV lytic cycle in tumor development, we established a system that consists of two NPC cell lines, respectively, in EBV abortive lytic cycle and latency. In a comparative analysis using this system, we found that the NPC cell line in EBV abortive lytic cycle exhibited the superior chemotactic capacity to recruit monocytes and polarized their differentiation toward tumor-associated macrophage (TAM)-like phenotype and away from DCs, compared to EBV-negative or EBV-latency NPC cells. EBV-encoded transcription activator ZTA is responsible for regulating monocyte chemotaxis and TAM phenotype by up-regulating the expression of GM-CSF, IL-8, and GRO-α. As a result, TAM induced by EBV abortive lytic cycle promotes NPC angiogenesis, invasion, and migration. Overall, this study elucidated the role of the EBV lytic life cycle in the late development of NPC and revealed a mechanism underlying the ZTA-mediated establishment of the tumor microenvironment (TME) that promotes NPC late-stage progression.

Super-enhancer driven SOX2 promotes tumor formation by chromatin re-organization in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer with a high incidence in Southern China and Southeast Asia. Patients with remote metastasis and recurrent NPC have poor prognosis. Thus, a better understanding of NPC pathogenesis may identify novel therapies to address the unmet clinical needs. METHODS: H3K27ac ChIP-seq and HiChIP was applied to understand the enhancer landscapes and the chromosome interactions. Whole genome sequencing was conducted to analyze the relationship between genomic variations and epigenetic dysregulation. CRISPRi and JQ1 treatment were used to evaluate the transcriptional regulation of SOX2 SEs. Colony formation assay, survival analysis and in vivo subcutaneous patient-derived xenograft assays were applied to explore the function and clinical relevance of SOX2 in NPC. FINDINGS: We globally mapped the enhancer landscapes and generated NPC enhancer connectomes, linking NPC specific enhancers and SEs. We found five overlapped genes, including SOX2, among super-enhancer regulated genes, survival related genes and NPC essential genes. The mRNA expression of SOX2 was repressed when applying CRISPRi targeting different SOX2 SEs or JQ1 treatment. Next, we identified a genetic variation (Chr3:181422197, G > A) in SOX2 SE which is correlated with higher expression of SOX2 and poor survival. In addition, SOX2 was highly expressed in NPC and is correlated with short survival in patients with NPC. Knock-down of SOX2 suppressed tumor growth in vitro and in vivo. INTERPRETATION: Our study demonstrated the super-enhancer landscape with chromosome interactions and identified super-enhancer driven SOX2 promotes tumorigenesis, suggesting that SOX2 is a potential therapeutic target for patients with NPC. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Non-overlapping epitopes on the gHgL-gp42 complex for the rational design of a triple-antibody cocktail against EBV infection.

Epstein-Barr virus (EBV) is closely associated with cancer, multiple sclerosis, and post-acute coronavirus disease 2019 (COVID-19) sequelae. There are currently no approved therapeutics or vaccines against EBV. It is noteworthy that combining multiple EBV glycoproteins can elicit potent neutralizing antibodies (nAbs) against viral infection, suggesting possible synergistic effects. Here, we characterize three nAbs (anti-gp42 5E3, anti-gHgL 6H2, and anti-gHgL 10E4) targeting different glycoproteins of the gHgL-gp42 complex. Two antibody cocktails synergistically neutralize infection in B cells (5E3+6H2+10E4) and epithelial cells (6H2+10E4) in vitro. Moreover, 5E3 alone and the 5E3+6H2+10E4 cocktail confer potent in vivo protection against lethal EBV challenge in humanized mice. The cryo-EM structure of a heptatomic gHgL-gp42 immune complex reveals non-overlapping epitopes of 5E3, 6H2, and 10E4 on the gHgL-gp42 complex. Structural and functional analyses highlight different neutralization mechanisms for each of the three nAbs. In summary, our results provide insight for the rational design of therapeutics or vaccines against EBV infection.

A gB nanoparticle vaccine elicits a protective neutralizing antibody response against EBV.

Epstein-Barr virus (EBV) is a global public health concern, as it is known to cause multiple diseases while also being etiologically associated with a wide range of epithelial and lymphoid malignancies. Currently, there is no available prophylactic vaccine against EBV. gB is the EBV fusion protein that mediates viral membrane fusion and participates in host recognition, making it critical for EBV infection in both B cells and epithelial cells. Here, we present a gB nanoparticle, gB-I53-50 NP, that displays multiple copies of gB. Compared with the gB trimer, gB-I53-50 NP shows improved structural integrity and stability, as well as enhanced immunogenicity in mice and non-human primate (NHP) preclinical models. Immunization and passive transfer demonstrate a robust and durable protective antibody response that protects humanized mice against lethal EBV challenge. This vaccine candidate demonstrates significant potential in preventing EBV infection, providing a possible platform for developing prophylactic vaccines for EBV.

mRNA-based Vaccines Targeting the T-cell Epitope-rich Domain of Epstein Barr Virus Latent Proteins Elicit Robust Anti-Tumor Immunity in Mice.

Epstein-Barr virus (EBV) is associated with various malignancies and infects >90% of the global population. EBV latent proteins are expressed in numerous EBV-associated cancers and contribute to carcinogenesis, making them critical therapeutic targets for these cancers. Thus, this study aims to develop mRNA-based therapeutic vaccines that express the T-cell-epitope-rich domain of truncated latent proteins of EBV, including truncatedlatent membrane protein 2A (Trunc-LMP2A), truncated EBV nuclear antigen 1 (Trunc-EBNA1), and Trunc-EBNA3A. The vaccines effectively activate both cellular and humoral immunity in mice and show promising results in suppressing tumor progression and improving survival time in tumor-bearing mice. Furthermore, it is observed that the truncated forms of the antigens, Trunc-LMP2A, Trunc-EBNA1, and Trunc-EBNA3A, are more effective than full-length antigens in activating antigen-specific immune responses. In summary, the findings demonstrate the effectiveness of mRNA-based therapeutic vaccines targeting the T-cell-epitope-rich domain of EBV latent proteins and providing new treatment options for EBV-associated cancers.

Neutralizing antibodies against EBV gp42 show potent in vivo protection and define novel epitopes.

Epstein-Barr virus (EBV) is the first reported human oncogenic virus and infects more than 95% of the human population worldwide. EBV latent infection in B lymphocytes is essential for viral persistence. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into B cells. The C-type lectin domain (CTLD) of gp42 plays a key role in receptor binding and is the major target of neutralizing antibodies. Here, we isolated two rabbit antibodies, 1A7 and 6G7, targeting gp42 CTLD with potent neutralizing activity against B cell infection. Antibody 6G7 efficiently protects humanized mice from lethal EBV challenge and EBV-induced lymphoma. Neutralizing epitopes targeted by antibodies 1A7 and 6G7 are distinct and novel. Antibody 6G7 blocks gp42 binding to B cell surface and both 1A7 and 6G7 inhibit membrane fusion with B cells. Furthermore, 1A7- and 6G7-like antibodies in immunized sera are major contributors to B cell neutralization. This study demonstrates that anti-gp42 neutralizing antibodies are effective in inhibiting EBV infection and sheds light on the design of gp42-based vaccines and therapeutics.

Multiple TMA-aided CRISPR/Cas13a platform for highly sensitive detection of IL-15 to predict immunotherapeutic response in nasopharyngeal carcinoma.

BACKGROUND: Immune checkpoint inhibitors (ICIs)-based treatments have been recommended as the first line for refractory recurrent and/or metastatic nasopharyngeal carcinoma (NPC) patients, yet responses vary, and predictive biomarkers are urgently needed. We selected serum interleukin-15 (sIL-15) out of four interleukins as a candidate biomarker, while most patients' sIL-15 levels were too low to be detected by conventional methods, so it was necessary to construct a highly sensitive method to detect sIL-15 in order to select NPC patients who would benefit most or least from ICIs. METHODS: Combining a primer exchange reaction (PER), transcription-mediated amplification (TMA), and a immuno-PER-TMA-CRISPR/Cas13a system, we developed a novel multiple signal amplification platform with a detection limit of 32 fg/mL, making it 153-fold more sensitive than ELISA. RESULTS: This platform demonstrated high specificity, repeatability, and versatility. When applied to two independent cohorts of 130 NPC sera, the predictive value of sIL-15 was accurate in both cohorts (area under the curve: training, 0.882; validation, 0.898). Additionally, lower sIL-15 levels were correlated with poorer progression-free survival (training, HR: 0.080, p<0.0001; validation, HR: 0.053, p<0.0001). CONCLUSION: This work proposes a simple and sensitive approach for sIL-15 detection to provide insights for personalized immunotherapy of NPC patients.

Camrelizumab combined with apatinib in patients with first-line platinum-resistant or PD-1 inhibitor resistant recurrent/metastatic nasopharyngeal carcinoma: a single-arm, phase 2 trial.

Immunotherapy combined with antiangiogenic targeted therapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for refractory recurrent/metastatic nasopharyngeal carcinoma (RM-NPC). We conducted a phase 2 trial to evaluate the safety and activity of camrelizumab plus apatinib in platinum-resistant (cohort 1, NCT04547088) and PD-1 inhibitor resistant NPC (cohort 2, NCT04548271). Here we report on the primary outcome of objective response rate (ORR) and secondary endpoints of safety, duration of response, disease control rate, progression-free survival, and overall survival. The primary endpoint of ORR was met for cohort 1 (65%, 95% CI, 49.6-80.4, n = 40) and cohort 2 (34.3%; 95% CI, 17.0-51.8, n = 32). Grade ≥ 3 treatment-related adverse events (TRAE) were reported in 47 (65.3%) of 72 patients. Results of our predefined exploratory investigation of predictive biomarkers show: B cell markers are the most differentially expressed genes in the tumors of responders versus non-responders in cohort 1 and that tertiary lymphoid structure is associated with higher ORR; Angiogenesis gene expression signatures are strongly associated with ORR in cohort 2. Camrelizumab plus apatinib combination effectiveness is associated with high expression of PD-L1, VEGF Receptor 2 and B-cell-related genes signatures. Camrelizumab plus apatinib shows promising efficacy with a measurable safety profile in RM-NPC patients.

Peptidome-wide association analysis of Epstein-Barr virus identifies epitope repertoires associated with nasopharyngeal carcinoma.

Human leukocyte antigen (HLA) molecules are essential for presenting Epstein-Barr virus (EBV) antigens and are closely related to nasopharyngeal carcinoma (NPC). This study aims to systematically investigate the association between HLA-bound EBV peptides and NPC risk through in silico HLA-peptide binding prediction. A total of 455 NPC patients and 463 healthy individuals in NPC endemic areas were recruited, and HLA-target sequencing was performed. HLA-peptide binding prediction for EBV, followed by peptidome-wide logistic regression and motif analysis, was applied. Binding affinity changes for EBV peptides carrying high-risk mutations were analyzed. We found that NPC-associated EBV peptides were significantly enriched in immunogenic proteins and core linkage disequilibrium (LD) proteins related to evolution, especially those binding HLA-A alleles (p = 3.10 × 10-4 for immunogenic proteins and p = 8.10 × 10-5 for core LD proteins related to evolution). These peptides were clustered and showed binding motifs of HLA supertypes, among which supertype A02 presented an NPC-risk effect (padj = 3.77 × 10-4 ) and supertype A03 presented an NPC-protective effect (padj = 4.89 × 10-4 ). Moreover, a decreased binding affinity toward risk HLA supertype A02 was observed for the peptide carrying the NPC-risk mutation BNRF1 V1222I (p = 0.0078), and an increased binding affinity toward protective HLA supertype A03 was observed for the peptide carrying the NPC-risk mutation BALF2 I613V (p = 0.022). This study revealed the distinct preference of EBV peptides for binding HLA supertypes, which may contribute to shaping EBV population structure and be involved in NPC development.

EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells.

Human cytomegalovirus (HCMV) infection is associated with human glioblastoma, the most common and aggressive primary brain tumor, but the underlying infection mechanism has not been fully demonstrated. Here, we show that EphA2 was upregulated in glioblastoma and correlated with the poor prognosis of the patients. EphA2 silencing inhibits, whereas overexpression promotes HCMV infection, establishing EphA2 as a crucial cell factor for HCMV infection of glioblastoma cells. Mechanistically, EphA2 binds to HCMV gH/gL complex to mediate membrane fusion. Importantly, the HCMV infection was inhibited by the treatment of inhibitor or antibody targeting EphA2 in glioblastoma cells. Furthermore, HCMV infection was also impaired in optimal glioblastoma organoids by EphA2 inhibitor. Taken together, we propose EphA2 as a crucial cell factor for HCMV infection in glioblastoma cells and a potential target for intervention.

Neuropilin 1 is an entry receptor for KSHV infection of mesenchymal stem cell through TGFBR1/2-mediated macropinocytosis.

Kaposi's sarcoma-associated herpesvirus (KSHV) has been implicated in the pathogenesis of Kaposi's sarcoma (KS) and other malignancies. The cellular origin of KS has been suggested to be either mesenchymal stem cells (MSCs) or endothelial cells. However, receptor(s) for KSHV to infect MSCs remains unknown. By combining bioinformatics analysis and shRNA screening, we identify neuropilin 1 (NRP1) as an entry receptor for KSHV infection of MSCs. Functionally, NRP1 knockout and overexpression in MSCs significantly reduce and promote, respectively, KSHV infection. Mechanistically, NRP1 facilitated the binding and internalization of KSHV by interacting with KSHV glycoprotein B (gB), which was blocked by soluble NRP1 protein. Furthermore, NRP1 interacts with TGF-ß receptor type 2 (TGFBR2) through their respective cytoplasmic domains and thus activates the TGFBR1/2 complex, which facilitates the macropinocytosis-mediated KSHV internalization via the small GTPases Cdc42 and Rac1. Together, these findings implicate that KSHV has evolved a strategy to invade MSCs by harnessing NRP1 and TGF-beta receptors to stimulate macropinocytosis.

Anti-EBV antibodies: Roles in diagnosis, pathogenesis, and antiviral therapy.

Epstein-Barr virus (EBV) infection is prevalent in global population and associated with multiple malignancies and autoimmune diseases. During the infection, EBV-harbored or infected cell-expressing antigen could elicit a variety of antibodies with significant role in viral host response and pathogenesis. These antibodies have been extensively evaluated and found to be valuable in predicting disease diagnosis and prognosis, exploring disease mechanisms, and developing antiviral agents. In this review, we discuss the versatile roles of EBV antibodies as important biomarkers for EBV-related diseases, potential driving factors of autoimmunity, and promising therapeutic agents for viral infection and pathogenesis.

Targeting herpesvirus entry complex and fusogen glycoproteins with prophylactic and therapeutic agents.

Herpesviruses are among the most successful viruses found in human populations. They establish lifelong latent infections, which are punctuated by recurrent reactivations. The entry process of herpesviruses into specific target cells requires a well-orchestrated teamwork involving multiple envelope glycoproteins. The conserved glycoprotein B (gB) is the membrane fusogen, of which conformational changes are induced by an entry complex (EC) consisting of at least gH and gL. Despite the high prevalence and heavy disease burdens associated with human herpesviruses (HHVs), vaccines against these pathogens are still lacking, except for varicella zoster virus (VZV). Recent advances in understanding the coordinated mechanisms of action of the key EC glycoproteins and fusogen will help to improve approaches for effective vaccine development and neutralizing antibody (nAb) screening.

A DNA tumor virus globally reprograms host 3D genome architecture to achieve immortal growth.

Epstein-Barr virus (EBV) immortalization of resting B lymphocytes (RBLs) to lymphoblastoid cell lines (LCLs) models human DNA tumor virus oncogenesis. RBL and LCL chromatin interaction maps are compared to identify the spatial and temporal genome architectural changes during EBV B cell transformation. EBV induces global genome reorganization where contact domains frequently merge or subdivide during transformation. Repressed B compartments in RBLs frequently switch to active A compartments in LCLs. LCLs gain 40% new contact domain boundaries. Newly gained LCL boundaries have strong CTCF binding at their borders while in RBLs, the same sites have much less CTCF binding. Some LCL CTCF sites also have EBV nuclear antigen (EBNA) leader protein EBNALP binding. LCLs have more local interactions than RBLs at LCL dependency factors and super-enhancer targets. RNA Pol II HiChIP and FISH of RBL and LCL further validate the Hi-C results. EBNA3A inactivation globally alters LCL genome interactions. EBNA3A inactivation reduces CTCF and RAD21 DNA binding. EBNA3C inactivation rewires the looping at the CDKN2A/B and AICDA loci. Disruption of a CTCF site at AICDA locus increases AICDA expression. These data suggest that EBV controls lymphocyte growth by globally reorganizing host genome architecture to facilitate the expression of key oncogenes.

Retraction notice to "Endogenous production of C-C motif chemokine ligand 2 by nasopharyngeal carcinoma cells drives radioresistance-associated metastasis" [Canc. Lett. 468 (2020) 27-40].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). .

Urgency and necessity of Epstein-Barr virus prophylactic vaccines.

Epstein-Barr virus (EBV), a γ-herpesvirus, is the first identified oncogenic virus, which establishes permanent infection in humans. EBV causes infectious mononucleosis and is also tightly linked to many malignant diseases. Various vaccine formulations underwent testing in different animals or in humans. However, none of them was able to prevent EBV infection and no vaccine has been approved to date. Current efforts focus on antigen selection, combination, and design to improve the efficacy of vaccines. EBV glycoproteins such as gH/gL, gp42, and gB show excellent immunogenicity in preclinical studies compared to the previously favored gp350 antigen. Combinations of multiple EBV proteins in various vaccine designs become more attractive approaches considering the complex life cycle and complicated infection mechanisms of EBV. Besides, rationally designed vaccines such as virus-like particles (VLPs) and protein scaffold-based vaccines elicited more potent immune responses than soluble antigens. In addition, humanized mice, rabbits, as well as nonhuman primates that can be infected by EBV significantly aid vaccine development. Innovative vaccine design approaches, including polymer-based nanoparticles, the development of effective adjuvants, and antibody-guided vaccine design, will further enhance the immunogenicity of vaccine candidates. In this review, we will summarize (i) the disease burden caused by EBV and the necessity of developing an EBV vaccine; (ii) previous EBV vaccine studies and available animal models; (iii) future trends of EBV vaccines, including activation of cellular immune responses, novel immunogen design, heterologous prime-boost approach, induction of mucosal immunity, application of nanoparticle delivery system, and modern adjuvant development.