RESUMO
Epstein-Barr virus (EBV) infects more than 95% of adults worldwide and is closely associated with various malignancies. Considering the complex life cycle of EBV, developing vaccines targeting key entry glycoproteins to elicit robust and durable adaptive immune responses may provide better protection. EBV gHgL-, gB- and gp42-specific antibodies in healthy EBV carriers contributed to sera neutralizing abilities in vitro, indicating that they are potential antigen candidates. To enhance the immunogenicity of these antigens, we formulate three nanovaccines by co-delivering molecular adjuvants (CpG and MPLA) and antigens (gHgL, gB or gp42). These nanovaccines induce robust humoral and cellular responses through efficient activation of dendritic cells and germinal center response. Importantly, these nanovaccines generate high levels of neutralizing antibodies recognizing vulnerable sites of all three antigens. IgGs induced by a cocktail vaccine containing three nanovaccines confer superior protection from lethal EBV challenge in female humanized mice compared to IgG elicited by individual NP-gHgL, NP-gB and NP-gp42. Importantly, serum antibodies elicited by cocktail nanovaccine immunization confer durable protection against EBV-associated lymphoma. Overall, the cocktail nanovaccine shows robust immunogenicity and is a promising candidate for further clinical trials.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Infecções por Vírus Epstein-Barr , Glicoproteínas , Nanovacinas , Animais , Feminino , Humanos , Camundongos , Adjuvantes Imunológicos/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/prevenção & controle , Infecções por Vírus Epstein-Barr/virologia , Glicoproteínas/imunologia , Glicoproteínas/administração & dosagem , Herpesvirus Humano 4/imunologia , Linfoma/imunologia , Linfoma/virologia , Nanovacinas/imunologiaRESUMO
Epstein-Barr Virus (EBV) is closely linked to nasopharyngeal carcinoma (NPC), notably prevalent in southern China. Although type II latency of EBV plays a crucial role in the development of NPC, some lytic genes and intermittent reactivation are also critical for viral propagation and tumor progression. Since T cell-mediated immunity is effective in targeted killing of EBV-positive cells, it is important to identify EBV-derived peptides presented by highly prevalent human leukocyte antigen class I (HLA-I) molecules throughout the EBV life cycle. Here, we constructed an EBV-positive NPC cell model to evaluate the presentation of EBV lytic phase peptides on streptavidin-tagged specific HLA-I molecules. Utilizing a mass spectrometry (LC-MS/MS)-based immunopeptidomic approach, we characterized eleven novel EBV peptides as well as two previously identified peptides. Furthermore, we determined these peptides were immunogenic and could stimulate PBMCs from EBV VCA/NA-IgA positive donors in an NPC endemic southern Chinese population. Overall, this work demonstrates that highly prevalent HLA-I-specific EBV peptides can be captured and functionally presented to elicit immune responses in an in vitro model, which provides insight into the epitopes presented during EBV lytic cycle and reactivation. It expands the range of viral targets for potential NPC early diagnosis and treatment.
Assuntos
Antígeno HLA-A11 , Antígeno HLA-A2 , Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Peptídeos , Humanos , Linhagem Celular Tumoral , China , Epitopos de Linfócito T/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Antígeno HLA-A11/imunologia , Antígeno HLA-A11/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/genética , Carcinoma Nasofaríngeo/imunologia , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/imunologia , Neoplasias Nasofaríngeas/virologia , Peptídeos/imunologia , Peptídeos/química , Proteômica , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Infecções por Vírus Epstein-Barr , Vacinas , Animais , Camundongos , Proteínas do Envelope Viral/química , Glicoproteínas de Membrana , Herpesvirus Humano 4 , Proteínas Virais , Terapia Combinada de Anticorpos , Epitopos , Glicoproteínas , Anticorpos Neutralizantes/uso terapêuticoRESUMO
Cytoskeleton-associated protein 4 (CKAP4) acts as a key transmembrane protein that connects the endoplasmic reticulum (ER) to microtubule dynamics. Researchers have not examined the roles of CKAP4 in nasopharyngeal carcinoma (NPC). The study aimed at evaluating the prognostic value and metastasis-regulating effect of CKAP4 in NPC. CKAP4 protein could be observed in 86.36% of 557 NPC specimens but not in normal nasopharyngeal epithelial tissue. According to immunoblot assays, NPC cell lines presented high CKAP4 expression relative to NP69 immortalized nasopharyngeal epithelial cells. Moreover, CKAP4 was highly expressed at the NPC tumor front and in matched liver, lung, and lymph node metastasis samples. Furthermore, high CKAP4 expression reported poor overall survival (OS) and presented a positive relevance to tumor (T) classification, recurrence, and metastasis. According to multivariate analysis, CKAP4 could independently and negatively predict patients' prognosis. Stable knockdown of CKAP4 expression in NPC cells inhibited cell migration, invasion and metastasis in vitro and in vivo. Moreover, CKAP4 promoted epithelial-mesenchymal transition (EMT) in NPC cells. CKAP4 knockdown was followed by the downregulation of the interstitial marker vimentin, and upregulation of the epithelial marker E-cadherin. In NPC tissues, high CKAP4 expression exhibited a positive relevance to vimentin expression and a negative relevance to E-cadherin expression. In conclusion, CKAP4 is an independent predictor of NPC, and CKAP4 might contribute NPC progression and metastasis, which may be involved in EMT with vimentin and E-cadherin.
Assuntos
Neoplasias Nasofaríngeas , Humanos , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica/genética , VimentinaRESUMO
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.
RESUMO
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.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Anticorpos Antivirais , Imunoglobulina G , Anticorpos MonoclonaisRESUMO
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.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Proteínas Virais , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/uso terapêutico , Microscopia Crioeletrônica , Infecções por Vírus Epstein-Barr/prevenção & controle , Infecções por Vírus Epstein-Barr/terapia , Herpesvirus Humano 4/imunologia , Humanos , Fusão de Membrana , Camundongos , Proteínas Virais/imunologiaRESUMO
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.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Gefitinibe/uso terapêutico , Humanos , Repetição Kelch , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Supressora de Tumor p14ARF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) are two major EBV-associated epithelial malignancies, both of which are characterized by the infiltration of a large number of lymphocytes, including natural killer (NK) cells. Although NK cells can prevent the development of EBV-associated epithelial malignancies, EBV-infected tumor cells often develop resistance to surveillance by NK cells. Elucidating the interactions between NK cells and EBV-infected tumor cells will facilitate the development of more effective NK-mediated therapies for treating EBV-associated malignancies. Here we investigated the cytotoxic function of NK cells in EBV-associated epithelial malignancies and discovered that EBV infection-induced upregulation of F3 expression correlates with NK-cell dysfunction in NPC and EBVaGC. The subsequent inhibitory effect of F3-mediated platelet aggregation on NK-cell function was verified in vitro and in vivo. Mechanistically, EBV latent membrane protein 2A (LMP2A) mediated upregulation of F3 through the PI3K/AKT signaling pathway. In an NPC xenograft mouse model, inhibition of F3 restored the antitumor function of NK cells and showed therapeutic efficacy when administered with NK-cell transfer. On the basis of these findings, EBV infection induces F3-mediated platelet aggregation that inhibits the antitumor function of NK cells, providing a rationale for developing and combining NK-cell-based therapies with F3 inhibitors to treat EBV-associated epithelial malignancies. SIGNIFICANCE: This study reveals a mechanism by which EBV-associated epithelial malignancies escape NK-cell-mediated immune surveillance, providing a new target for improving NK-cell immunotherapy.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Neoplasias Gástricas , Animais , Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4/metabolismo , Humanos , Células Matadoras Naturais , Camundongos , Carcinoma Nasofaríngeo , Fosfatidilinositol 3-Quinases/metabolismo , Agregação PlaquetáriaRESUMO
Background: Although immune checkpoint inhibitors have opened a new mode of treatment for solid tumors, their efficacy in nasopharyngeal carcinoma (NPC) needs to be further investigated. Inhibitors of the PD-1/PD-L1 immune checkpoint are one of the hot topics in tumor immunotherapy. Programmed death ligand-2 (PD-L2) is a less studied ligand of PD-1 and has not yet been fully explored, especially in NPC. Understanding the clinical significance of PD-L2 expression, together with immune cell infiltration, might provide clues for biomarker screening in NPC immunotherapy. This study aimed to evaluate the role of PD-L2 as a prognostic factor for NPC patients as well as its role in immune regulation. Methods: Immunohistochemistry (IHC) was performed on a tissue microarray including 557 NPC specimens using PD-L2 antibody. The immune cell markers CD4, FOXP3 and CD68 were also stained and quantified. The expression of PD-L2 exhibited different spatial patterns among NPC tumor and stromal tissues. Results: A total of 90.8% of the cases showed membranous PD-L2 expression in tumors, and 80.8% showed membranous PD-L2 expression in stromal tissue. High stromal expression of PD-L2 predicted favorable overall and disease-free survival of NPC patients and was negatively correlated with tumor size, recurrence or metastasis and clinical stage. In contrast, high tumor abundance of PD-L2 correlated with poor disease-free survival, but had no obvious correlation with clinicopathological parameters. Multivariate analysis indicated that stromal PD-L2 was an independent and favorable prognostic factor. Furthermore, we found a positive correlation between stromal PD-L2 expression and the infiltration of CD68+ macrophages and CD4+Foxp3+ Treg cells in NPC stromal tissues (Pearson correlation=0.181 and 0.098, respectively). Conclusions: Our results suggest that different PD-L2 expression patterns have distinct predictive values. PD-L2 expressed on stromal cells might play a role in the regulation of NPC progression, and involve in immune activation in the tissue microenvironment and have an independent good prognosis for NPC patients.
RESUMO
Humanized mouse models are used as comprehensive small-animal models of EBV infection. Previously, infectious doses of EBV used in vivo have been determined mainly on the basis of TD50 (50% transforming dose), which is a time-consuming process. Here, we determined infectious doses of Akata-EBV-GFP using green Raji units (GRUs), and characterized dose-dependent effects in humanized mice. We defined two outcomes in vivo, including an infection model and a lymphoma model, following inoculation with low or high doses of Akata-EBV-GFP, respectively. Inoculation with a low dose induced primary B cells to become lymphoblastoid cell lines in vitro, and caused latent infection in humanized mice. In contrast, a high dose of Akata-EBV-GFP resulted in primary B cells death in vitro, and fatal B cell lymphomas in vivo. Following infection with high doses, the frequency of CD19+ B cells decreased, whereas the percentage of CD8+ T cells increased in peripheral blood and the spleen. At such doses, a small part of activated CD8+ T cells was EBV-specific CD8+ T cells. Thus, GRUs quantitation of Akata-EBV-GFP is an effective way to quantify infectious doses to study pathologies, immune response, and to assess (in vivo) the neutralizing activity of antibodies raised by immunization against EBV.
Assuntos
Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/imunologia , Animais , Antígenos CD19/imunologia , Linfócitos B , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/patologia , Humanos , Linfoma , Linfoma de Células B , CamundongosRESUMO
Using Epstein-Barr virus (EBV)-based markers to screen populations at high risk for nasopharyngeal carcinoma (NPC) is an attractive preventive approach. Here, we develop a comprehensive risk score (CRS) that combines risk effects of EBV and human genetics for NPC risk stratification and validate this CRS within an independent, population-based dataset. Comparing the top decile with the bottom quintile of CRSs, the odds ratio of developing NPC is 21 (95% confidence interval: 12-37) in the validation dataset. When combining the top quintile of CRS with EBV serology tests currently used for NPC screening in southern China, the positive prediction value of screening increases from 4.70% (serology test alone) to 43.24% (CRS plus serology test). By identifying individuals at a monogenic level of NPC risk, this CRS approach provides opportunities for personalized risk prediction and population screening in endemic areas for the early diagnosis and secondary prevention of NPC.
Assuntos
Infecções por Vírus Epstein-Barr/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Adulto , Idoso , Anticorpos Antivirais/sangue , China , Detecção Precoce de Câncer , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Feminino , Genótipo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/sangue , Carcinoma Nasofaríngeo/diagnóstico , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/sangue , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/virologia , Polimorfismo de Nucleotídeo Único , Medição de Risco , Fatores de RiscoRESUMO
Glycoprotein B (gB) is an essential fusion protein for the Epstein-Barr virus (EBV) infection of both B cells and epithelial cells and is thus a promising target antigen for a prophylactic vaccine to prevent or reduce EBV-associated disease. T cell responses play key roles in the control of persistent EBV infection and in the efficacy of a vaccine. However, to date, T cell responses to gB have been characterized for only a limited number of human leukocyte antigen (HLA) alleles. Here, we screened gB T cell epitopes in 23 healthy EBV carriers and ten patients with nasopharyngeal cancer (NPC) using a peptide library spanning the entire gB sequence. We identified twelve novel epitopes in the context of seven new HLA restrictions that are common in Asian populations. Two epitopes, gB214-223 and gB840-849, restricted by HLA-B*58:01 and B*38:02, respectively, elicited specific CD8+ T cell responses to inhibit EBV-driven B cell transformation. Interestingly, gB-specific CD8+ T cells were more frequent in healthy viral carriers with EBV reactivation than in those without EBV reactivation, indicating that EBV reactivation in vivo stimulates both humoral (VCA-gp125-IgA) and cellular responses to gB. We further found that most gB epitopes are conserved among different EBV strains. Our study broadens the diversity and HLA restrictions of gB epitopes and suggests that gB is a common target of T cell responses in healthy viral carriers with EBV reactivation. In particular, the precisely mapped and conserved gB epitopes provide valuable information for prophylactic vaccine development.ImportanceT cells are crucial for the control of persistent EBV infection and the development of EBV-associated diseases. The EBV gB protein is essential for virus entry into B cells and epithelial cells and is thus a target antigen for vaccine development. Understanding T cell responses to gB is important for subunit vaccine design. Herein, we comprehensively characterized T cell responses to full-length gB. Our results expand the available gB epitopes and HLA restrictions, particularly those common in Asian populations. Furthermore, we showed that gB-specific CD8+ T cells inhibit B cell transformation ex vivo and that gB-specific CD8+ T cell responses in vivo may be associated with intermittent EBV reactivation in asymptomatic viral carriers. These gB epitopes are highly conserved among geographically separated EBV strains. Precisely mapped and conserved T cell epitopes may contribute to immune monitoring and to the development of a gB subunit vaccine.
RESUMO
Rationale: Epstein-Barr virus (EBV) is the causative pathogen for infectious mononucleosis and many kinds of malignancies including several lymphomas such as Hodgkin's lymphoma, Burkitt's lymphoma and NK/T cell lymphoma as well as carcinomas such as nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBV-GC). However, to date no available prophylactic vaccine was launched to the market for clinical use. Methods: To develop a novel vaccine candidate to prevent EBV infection and diseases, we designed chimeric virus-like particles (VLPs) based on the hepatitis B core antigen (HBc149). Various VLPs were engineered to present combinations of three peptides derived from the receptor binding domain of EBV gp350. All the chimeric virus-like particles were injected into Balb/C mice for immunogenicity evaluation. Neutralizing titer of mice sera were detected using an in vitro cell model. Results: All chimeric HBc149 proteins self-assembled into VLPs with gp350 epitopes displayed on the surface of spherical particles. Interestingly, the different orders of the three epitopes in the chimeric proteins induced different immune responses in mice. Two constructs (149-3A and 149-3B) induced high serum titer against the receptor-binding domain of gp350. Most importantly, these two VLPs elicited neutralizing antibodies in immunized mice, which efficiently blocked EBV infection in cell culture. Competition analysis showed that sera from these mice contained antibodies to a major neutralizing epitope recognized by the strong neutralizing mAb 72A1. Conclusion: Our data demonstrate that HBc149 chimeric VLPs provide a valuable platform to present EBV gp350 antigens and offer a robust basis for the development of peptide-based candidate vaccines against EBV.
Assuntos
Anticorpos Neutralizantes/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Imunização/métodos , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/sangue , Epitopos/genética , Epitopos/imunologia , Infecções por Vírus Epstein-Barr/prevenção & controle , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidade , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinação/métodos , Vacinas/farmacologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
Germline polymorphisms are linked with differential survival outcomes in cancers but are not well studied in nasopharyngeal carcinoma (NPC). Here, a two-phase association study is conducted to discover germline polymorphisms that are associated with the prognosis of NPC. The discovery phase includes two consecutive hospital cohorts of patients with NPC from Southern China. Exome-wide genotypes at 246 173 single nucleotide polymorphisms (SNPs) are determined, followed by survival analysis for each SNP under Cox proportional hazard regression model. Candidate SNP is replicated in another two independent cohorts from Southern China and Singapore. Meta-analysis of all samples (n = 5553) confirms that the presence of rs1131636-T, located in the 3'-UTR of RPA1, confers an inferior overall survival (HR = 1.33, 95% CI = 1.20-1.47, P = 6.31 × 10-8). Bioinformatics and biological assays show that rs1131636 has regulatory effects on upstream RPA1. Functional studies further demonstrate that RPA1 promotes the growth, invasion, migration, and radioresistance of NPC cells. Additionally, miR-1253 is identified as a suppressor for RPA1 expression, likely through regulation of its binding affinity to rs1131636 locus. Collectively, these findings provide a promising biomarker aiding in stratifying patients with poor survival, as well as a potential drug target for NPC.
RESUMO
Epstein-Barr virus (EBV) infection is ubiquitous worldwide and is associated with multiple cancers, including nasopharyngeal carcinoma (NPC). The importance of EBV viral genomic variation in NPC development and its striking epidemic in southern China has been poorly explored. Through large-scale genome sequencing of 270 EBV isolates and two-stage association study of EBV isolates from China, we identify two non-synonymous EBV variants within BALF2 that are strongly associated with the risk of NPC (odds ratio (OR) = 8.69, P = 9.69 × 10-25 for SNP 162476_C; OR = 6.14, P = 2.40 × 10-32 for SNP 163364_T). The cumulative effects of these variants contribute to 83% of the overall risk of NPC in southern China. Phylogenetic analysis of the risk variants reveals a unique origin in Asia, followed by clonal expansion in NPC-endemic regions. Our results provide novel insights into the NPC endemic in southern China and also enable the identification of high-risk individuals for NPC prevention.
Assuntos
Proteínas de Ligação a DNA/genética , Infecções por Vírus Epstein-Barr/complicações , Genoma Viral , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/virologia , Polimorfismo de Nucleotídeo Único , Proteínas Virais/genética , Estudos de Casos e Controles , China/epidemiologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/classificação , Herpesvirus Humano 4/isolamento & purificação , Humanos , Carcinoma Nasofaríngeo/epidemiologia , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/epidemiologia , Neoplasias Nasofaríngeas/genéticaRESUMO
Rationale: Epstein-Barr virus (EBV) is associated with multiple malignancies with expression of viral oncogenic proteins and chronic inflammation as major mechanisms contributing to tumor development. A less well-studied mechanism is the integration of EBV into the human genome possibly at sites which may disrupt gene expression or genome stability. Methods: We sequenced tumor DNA to profile the EBV sequences by hybridization-based enrichment. Bioinformatic analysis was used to detect the breakpoints of EBV integrations in the genome of cancer cells. Results: We identified 197 breakpoints in nasopharyngeal carcinomas and other EBV-associated malignancies. EBV integrations were enriched at vulnerable regions of the human genome and were close to tumor suppressor and inflammation-related genes. We found that EBV integrations into the introns could decrease the expression of the inflammation-related genes, TNFAIP3, PARK2, and CDK15, in NPC tumors. In the EBV genome, the breakpoints were frequently at oriP or terminal repeats. These breakpoints were surrounded by microhomology sequences, consistent with a mechanism for integration involving viral genome replication and microhomology-mediated recombination. Conclusion: Our finding provides insight into the potential of EBV integration as an additional mechanism mediating tumorigenesis in EBV associated malignancies.
Assuntos
DNA Viral/análise , Infecções por Vírus Epstein-Barr/complicações , Genoma Humano , Herpesvirus Humano 4/genética , Neoplasias/virologia , Integração Viral , DNA Viral/genética , Loci Gênicos , Humanos , Análise de Sequência de DNARESUMO
BACKGROUND: The male predominance in the incidence of nasopharyngeal carcinoma (NPC) suggests the contribution of the X chromosome to the susceptibility of NPC. However, no X-linked susceptibility loci have been examined by genome-wide association studies (GWASs) for NPC by far. METHODS: To understand the contribution of the X chromosome in NPC susceptibility, we conducted an X chromosome-wide association analysis on 1615 NPC patients and 1025 healthy controls of Guangdong Chinese, followed by two validation analyses in Taiwan Chinese (n = 562) and Malaysian Chinese (n = 716). RESULTS: Firstly, the proportion of variance of X-linked loci over phenotypic variance was estimated in the discovery samples, which revealed that the phenotypic variance explained by X chromosome polymorphisms was estimated to be 12.63% (non-dosage compensation model) in males, as compared with 0.0001% in females. This suggested that the contribution of X chromosome to the genetic variance of NPC should not be neglected. Secondly, association analysis revealed that rs5927056 in DMD gene achieved X chromosome-wide association significance in the discovery sample (OR = 0.81, 95% CI 0.73-0.89, P = 1.49 × 10-5). Combined analysis revealed rs5927056 for DMD gene with suggestive significance (P = 9.44 × 10-5). Moreover, the female-specific association of rs5933886 in ARHGAP6 gene (OR = 0.62, 95%CI: 0.47-0.81, P = 4.37 × 10-4) was successfully replicated in Taiwan Chinese (P = 1.64 × 10-2). rs5933886 also showed nominally significant gender × SNP interaction in both Guangdong (P = 6.25 × 10-4) and Taiwan datasets (P = 2.99 × 10-2). CONCLUSION: Our finding reveals new susceptibility loci at the X chromosome conferring risk of NPC and supports the value of including the X chromosome in large-scale association studies.
Assuntos
Cromossomos Humanos X , Predisposição Genética para Doença , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Caracteres Sexuais , Adulto , Povo Asiático/genética , China , Feminino , Estudos de Associação Genética , Loci Gênicos , Humanos , Malásia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , TaiwanRESUMO
Background: Oral Epstein-Barr virus (EBV) status reflects host EBV activity and potentially links to EBV-associated diseases, however, factors influencing oral EBV loads or reactivation, such as environmental exposures or host factors, are not fully understood. Methods: A 2-stage, multicenter, cross-sectional study of 6558 subjects from 21 administrative cities of southern China and 3 populations from representative geographical areas in China (referred to as the south, north, and northeastern populations) was performed. The relationships between demographical factors and environmental exposures to EBV loads were analyzed by logistic regression models. Results: Current smoking, with a dose-response effect, was found to be strongly associated with higher oral EBV loads in the pooled data, with an odds ratio of 1.58 (95% confidence interval, 1.39-1.79), as well as in each of the separate populations. The odds ratio increased to 3.06 when current smokers in southern China were compared to never smokers in northern China. Additionally, higher oral EBV loads tended to be detected in older participants, male participants, and participants in southern China. Conclusions: This study provided evidence linking the effect of host-environmental factors, particularly smoking, to oral EBV activity. It could strengthen our understanding of the possible causal roles of EBV-related diseases, which may help to prevent or mitigate EBV-associated diseases.