Host genetic variants, Epstein-Barr virus subtypes, and the risk of nasopharyngeal carcinoma: Assessment of interaction and mediation.

Epstein-Barr virus (EBV) and human leukocyte antigen (HLA) polymorphisms are well-known risk factors for nasopharyngeal carcinoma (NPC). However, the combined effects between HLA and EBV on the risk of NPC are unknown. We applied a causal inference framework to disentangle interaction and mediation effects between two host HLA SNPs, rs2860580 and rs2894207, and EBV variant 163364 with a population-based case-control study in NPC-endemic southern China. We discovered the strong interaction effects between the high-risk EBV subtype and both HLA SNPs on NPC risk (rs2860580, relative excess risk due to interaction [RERI] = 4.08, 95% confidence interval [CI] = 2.03-6.14; rs2894207, RERI = 3.37, 95% CI = 1.59-5.15), accounting for the majority of genetic risk effects. These results indicate that HLA genes and the high-risk EBV have joint effects on NPC risk. Prevention strategies targeting the high-risk EBV subtype would largely reduce NPC risk associated with EBV and host genetic susceptibility.

NOP14-mediated ribosome biogenesis is required for mTORC2 activation and predicts rapamycin sensitivity.

The mechanistic target of rapamycin (mTOR) forms two distinct complexes: rapamycin-sensitive mTOR complex 1 (mTORC1) and rapamycin-insensitive mTORC2. mTORC2 primarily regulates cell survival by phosphorylating Akt, though the upstream regulation of mTORC2 remains less well-defined than that of mTORC1. In this study, we show that NOP14, a 40S ribosome biogenesis factor and a target of the mTORC1-S6K axis, plays an essential role in mTORC2 signaling. Knockdown of NOP14 led to mTORC2 inactivation and Akt destabilization. Conversely, overexpression of NOP14 stimulated mTORC2-Akt activation and enhanced cell proliferation. Fractionation and coimmunoprecipitation assays demonstrated that the mTORC2 complex was recruited to the rough endoplasmic reticulum through association with endoplasmic reticulum-bound ribosomes. In vivo, high levels of NOP14 correlated with poor prognosis in multiple cancer types. Notably, cancer cells with NOP14 high expression exhibit increased sensitivity to mTOR inhibitors, because the feedback activation of the PI3K-PDK1-Akt axis by mTORC1 inhibition was compensated by mTORC2 inhibition partly through NOP14 downregulation. In conclusion, our findings reveal a spatial regulation of mTORC2-Akt signaling and identify ribosome biogenesis as a potential biomarker for assessing rapalog response in cancer therapy.

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.

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.

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.

Are High Levels of Microsatellite Instability and Microsatellite Stability Identical in DNA Mismatch Repair-Deficient Colorectal Cancer Patients?

Purpose: The purpose of the current study was to determine whether there is a difference between high levels of microsatellite instability (MSI-H) and microsatellite stability (MSS) in DNA mismatch repair-deficient (DMMR) colorectal cancer (CRC) patients. Methods: A total of 452 CRC patients with DMMR from December, 2014, to April, 2021, in our hospital were selected retrospectively. However, only 105 patients underwent Sanger or next-generation-sequencing (NGS) to confirm their microsatellite status. Ultimately, 55 MSI-H patients and 20 MSS patients with intact medical record information were included in this study. Results: The MSS group was associated with a higher mutation rate in the KRAS gene (P=0.011). Meanwhile, MSI-H was related to colon cancer (P < 0.01). However, no significant differences in other clinical characteristics were observed between the two groups of patients. There was no significant difference between the MSI-H and MSS groups in terms of overall survival (OS) (P=0.398) and disease-free survival (DFS) (P=0.307). Conclusion: The MSI-H status was associated with colon cancer and a lower mutation rate of the KRAS gene in DMMR patients. In CRC-DMMR patients, the MSS group exhibited better OS and DFS than the MSI-H group, although these differences were not statistically significant. Accordingly, in clinical practice, we should not confuse these two types of patients.

HLA-Bw4 in association with KIR3DL1 favors natural killer cell-mediated protection against severe COVID-19.

Replicating SARS-CoV-2 has been shown to degrade HLA class I on target cells to evade the cytotoxic T-cell (CTL) response. HLA-I downregulation can be sensed by NK cells to unleash killer cell immunoglobulin-like receptor (KIR)-mediated self-inhibition by the cognate HLA-I ligands. Here, we investigated the impact of HLA and KIR genotypes and HLA-KIR combinations on COVID-19 outcome. We found that the peptide affinities of HLA alleles were not correlated with COVID-19 severity. The predicted poor binders for SARS-CoV-2 peptides belong to HLA-B subtypes that encode KIR ligands, including Bw4 and C1 (introduced by B*46:01), which have a small F pocket and cannot accommodate SARS-CoV-2 CTL epitopes. However, HLA-Bw4 weak binders were beneficial for COVID-19 outcome, and individuals lacking the HLA-Bw4 motif were at higher risk for serious illness from COVID-19. The presence of the HLA-Bw4 and KIR3DL1 combination had a 58.8% lower risk of developing severe COVID-19 (OR = 0.412, 95% CI = 0.187-0.904, p = 0.02). This suggests that HLA-Bw4 alleles that impair their ability to load SARS-CoV-2 peptides will become targets for NK-mediated destruction. Thus, we proposed that the synergistic responsiveness of CTLs and NK cells can efficiently control SARS-CoV-2 infection and replication, and NK-cell-mediated anti-SARS-CoV-2 immune responses being mostly involved in severe infection when the level of ORF8 is high enough to degrade HLA-I. The HLA-Bw4/KIR3DL1 genotype may be particularly important for East Asians undergoing COVID-19 who are enriched in HLA-Bw4-inhibitory KIR interactions and carry a high frequency of HLA-Bw4 alleles that bind poorly to coronavirus peptides.

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.

A high-throughput neutralizing assay for antibodies and sera evaluation against Epstein-Barr virus.

BACKGROUND: Epstein-Barr virus (EBV) is a wide-spread human herpesvirus that is highly associated with infectious mononucleosis and several malignancies. Evaluation of EBV neutralizing antibody titers is important for serological studies, vaccine development and monoclonal antibody screening. The traditional method based on antibody inhibition of EBV transformation of B cells is very time-consuming. A more practical flow cytometry-based (FCM) approach to evaluate neutralizing titers is not amenable to achieving high-throughput evaluation of large-scale samples. A high-throughput approach is urgently needed. RESULTS: Here, we present a rapid and high-throughput method based on high content imaging system (HCIS) analysis. EBV titers determined by the HCIS-based assay were similar to those obtained by the FCM-based assay. Neutralizing titers of sera and monoclonal antibodies measured by the HCIS-based assay strongly correlated with titers measured by the FCM-based assay. HCIS assays showed a strong correlation between B cell infection neutralizing titers and the anti-gp350 IgG titers in healthy EBV carriers and monkey sera. Finally, anti-gHgL IgG titers from sera of healthy EBV carriers significantly correlated with epithelial cell infection neutralizing titers. CONCLUSIONS: This HCIS-based assay is a high-throughput assay to determine viral titers and evaluate neutralizing potentials of sera and monoclonal antibodies. This HCIS-based assay will aid the development of vaccines and therapeutic monoclonal antibody against EBV.

Protective anti-gB neutralizing antibodies targeting two vulnerable sites for EBV-cell membrane fusion.

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population and accounts for a significant cancer burden of epithelial and B cell origins. Glycoprotein B (gB) is the primary fusogen essential for EBV entry into host cells. Here, we isolated two EBV gB-specific neutralizing antibodies, 3A3 and 3A5; both effectively neutralized the dual-tropic EBV infection of B and epithelial cells. In humanized mice, both antibodies showed effective protection from EBV-induced lymphoproliferative disorders. Cryoelectron microscopy analyses identified that 3A3 and 3A5 bind to nonoverlapping sites on domains D-II and D-IV, respectively. Structure-based mutagenesis revealed that 3A3 and 3A5 inhibit membrane fusion through different mechanisms involving the interference with gB-cell interaction and gB activation. Importantly, the 3A3 and 3A5 epitopes are major targets of protective gB-specific neutralizing antibodies elicited by natural EBV infection in humans, providing potential targets for antiviral therapies and vaccines.

Itaconate is a lysosomal inducer that promotes antibacterial innate immunity.

Lysosomes are the main organelles in macrophages for killing invading bacteria. However, the precise mechanism underlying lysosomal biogenesis upon bacterial infection remains enigmatic. We demonstrate here that LPS stimulation increases IRG1-dependent itaconate production, which promotes lysosomal biogenesis by activating the transcription factor, TFEB. Mechanistically, itaconate directly alkylates human TFEB at cysteine 212 (Cys270 in mice) to induce its nuclear localization by antagonizing mTOR-mediated phosphorylation and cytosolic retention. Functionally, abrogation of itaconate synthesis by IRG1/Irg1 knockout or expression of an alkylation-deficient TFEB mutant impairs the antibacterial ability of macrophages in vitro. Furthermore, knockin mice harboring an alkylation-deficient TFEB mutant display elevated susceptibility to Salmonella typhimurium infection, whereas in vivo treatment of OI, a cell-permeable itaconate derivative, limits inflammation. Our study identifies itaconate as an endogenous metabolite that functions as a lysosomal inducer in macrophages in response to bacterial infection, implying the potential therapeutic utility of itaconate in treating human bacterial infection.

CRL2KLHDC3 mediates p14ARF N-terminal ubiquitylation degradation to promote non-small cell lung carcinoma progression.

Kelch superfamily involves a variety of proteins containing multiple kelch motif and is well characterized as substrate adaptors for CUL3 E3 ligases, which play critical roles in carcinogenesis. However, the role of kelch proteins in lung cancer remains largely unknown. In this study, the non-small cell lung cancer (NSCLC) patients with higher expression of a kelch protein, kelch domain containing 3 (KLHDC3), showed worse overall survival. KLHDC3 deficiency affected NSCLC cell lines proliferation in vitro and in vivo. Further study indicated that KLHDC3 mediated CUL2 E3 ligase and tumor suppressor p14ARF interaction, facilitating the N-terminal ubiquitylation and subsequent degradation of p14ARF. Interestingly, Gefitinib-resistant NSCLC cell lines displayed higher KLHDC3 protein levels. Gefitinib and Osimertinib medications were capable of upregulating KLHDC3 expression to promote p14ARF degradation in the NSCLC cell lines. KLHDC3 shortage significantly increased the sensitivity of lung cancer cells to epidermal growth factor receptor (EGFR)-targeted drugs, providing an alternative explanation for the development of Gefitinib and Osimertinib resistance in NSCLC therapy. Our works suggest that CRL2KLHDC3 could be a valuable target to regulate the abundance of p14ARF and postpone the occurrence of EGFR-targeted drugs resistance.

Environmental Factors for Epstein-Barr Virus Reactivation in a High-Risk Area of Nasopharyngeal Carcinoma: A Population-Based Study.

Background: Epstein-Barr virus (EBV) reactivation from latent to lytic infection has been considered as a key step in nasopharyngeal carcinoma oncogenesis. However, epidemiological evidence regarding environmental risk factors for EBV reactivation on a population level remains largely lacking. Methods: We enrolled 1916 randomly selected adults from the general population of Guangdong and Guangxi, China, from 2010 to 2014. Information on environmental factors was collected via a structured interview. Serum immunoglobulin A antibodies against EBV viral capsid antigen and nuclear antigen 1 were measured by enzyme-linked immunosorbent assay to evaluate EBV reactivation status. We used logistic regression to calculate odds ratios (ORs) with 95% confidence intervals (CIs) for the associations of EBV reactivation with various environmental factors. Results: No associations were observed between EBV reactivation and extensive environmental factors, including alcohol or tea drinking, a history of chronic ear/nose/throat diseases, use of medications or herbs, consumption of salted fish or preserved foods, oral hygiene, sibship structure, and various residential and occupational exposures. Only cigarette smoking was associated with EBV reactivation (current smokers vs never smokers; OR = 1.37; 95% CI = 1.02-1.83), with positive exposure-response trends with increasing intensity, duration, and pack-years of smoking. Conclusions: Consistent with previous studies, we found an association between cigarette smoking and EBV reactivation. Other examined exposures were not associated with EBV reactivation. These null results could suggest either more complex interactions between exposures and EBV reactivation or a predominant role of host and/or viral genetic variation.

Vesicular Stomatitis Virus-Based Epstein-Barr Virus Vaccines Elicit Strong Protective Immune Responses.

Epstein-Barr virus (EBV), the first identified human tumor virus, is etiologically associated with various kinds of malignant and benign diseases, accounting for 265,000 cancer incident cases and 164,000 cancer deaths in 2017. EBV prophylactic vaccine development has been gp350 centered for several decades. However, clinical studies show that gp350-centered vaccines fail to prevent EBV infection. Advances in the EBV infection mechanisms shed light on gB and gHgL, the two key components of the infection apparatus. In this study, for the first time, we utilized recombinant vesicular stomatitis virus (VSV) to display EBV gB (VSV-ΔG-gB/gB-G) or gHgL (VSV-ΔG-gHgL). In vitro studies confirmed successful virion production and glycoprotein presentation on the virion surface. In mouse models, VSV-ΔG-gB/gB-G or VSV-ΔG-gHgL elicited potent humoral responses. Neutralizing antibodies elicited by VSV-ΔG-gB/gB-G were prone to prevent B cell infection, while those elicited by VSV-ΔG-gHgL were prone to prevent epithelial cell infection. Combinatorial vaccination yields an additive effect. The ratio of endpoint neutralizing antibody titers to the endpoint total IgG titers immunized with VSV-ΔG-gHgL was approximately 1. The ratio of IgG1/IgG2a after VSV-ΔG-gB/gB-G immunization was approximately 1 in a dose-dependent, adjuvant-independent manner. Taken together, VSV-based EBV vaccines can elicit a high ratio of epithelial and B lymphocyte neutralizing antibodies, implying their unique potential as EBV prophylactic vaccine candidates. IMPORTANCE Epstein-Barr virus (EBV), one of the most common human viruses and the first identified human oncogenic virus, accounted for 265,000 cancer incident cases and 164,000 cancer deaths in 2017 as well as millions of nonmalignant disease cases. So far, no prophylactic vaccine is available to prevent EBV infection. In this study, for the first time, we reported the VSV-based EBV vaccines presenting two key components of the EBV infection apparatus, gB and gHgL. We confirmed potent antigen-specific antibody generation; these antibodies prevented EBV from infecting epithelial cells and B cells, and the IgG1/IgG2a ratio indicated balanced humoral-cellular responses. Taken together, we suggest VSV-based EBV vaccines are potent prophylactic candidates for clinical studies and help eradicate numerous EBV-associated malignant and benign diseases.

A Neutralizing Antibody Targeting gH Provides Potent Protection against EBV Challenge In Vivo.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that is associated with 200,000 new cases of cancer and 140,000 deaths annually. To date, there are no available vaccines or therapeutics for clinical usage. Recently, the viral heterodimer glycoprotein gH/gL has become a promising target for the development of prophylactic vaccines against EBV. Here, we developed the anti-gH antibody 6H2 and its chimeric version C6H2, which had full neutralizing activity in epithelial cells and partial neutralizing activity in B cells. C6H2 exhibited potent protection against lethal EBV challenge in a humanized mouse model. The cryo-electron microscopy (cryo-EM) structure further revealed that 6H2 recognized a previously unidentified epitope on gH/gL D-IV that is critical for viral attachment and subsequent membrane fusion with epithelial cells. Our results suggest that C6H2 is a promising candidate in the prevention of EBV-induced lymphoproliferative diseases (LPDs) and may inform the design of an EBV vaccine. IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes lifelong persistence and is related to multiple diseases, including cancers. Neutralizing antibodies (NAbs) have proven to be highly effective in preventing EBV infection and subsequent diseases. Here, we developed an anti-EBV-gH NAb, 6H2, which blocked EBV infection in vitro and in vivo. This 6H2 neutralizing epitope should be helpful to understand EBV infection mechanisms and guide the development of vaccines and therapeutics against EBV infection.