Animals ; Epstein-Barr Virus Nuclear Antigens ; immunology ; Immunization ; Mice ; Mice, Inbred BALB C ; Viral Matrix Proteins ; immunology ; Viral Vaccines

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.
Epstein-Barr Virus Infections/complications/virology ; Epstein-Barr Virus Nuclear Antigens/genetics/metabolism ; *Genes, Viral ; Herpesvirus 4, Human/*physiology ; Humans ; MicroRNAs/genetics ; Neoplasms/etiology ; Protein Binding ; RNA, Viral/genetics ; Viral Matrix Proteins/genetics/metabolism ; *Virus Latency

OBJECTIVETo evaluate the value of EBNA1-IgA and EA-IgG in serological diagnosis of nasopharyngeal carcinoma (NPC).

METHODSThe serum EBNA1-IgA and EA-IgG of 56 patients with NPC and 58 healthy adults were detected by ELISA. The sensitivity, specificity, positive predictive value, accuracy rate and odds ratio of the two tests used singly or in combination were compared with each other.

RESULTSThe sensitivity of EBNA1-IgA (91.07%) was higher than that of EA-IgG (87.50%), while the specificity of EA-IgG (87.93%) was higher than that of EBNA1-IgA (84.48%). The combination of EBNA1-IgA and EA-IgG could enhance the specificity (94.83%), positive predictive value (0.9375), likelihood ratio (15.5435) and odds ratio (75.0000) for serological diagnosis of NPC. Forty-five patients showed both positive EBNA1-IgA and positive EA-IgG. A positive EA-IgG was detected in 4 out of 5 patients with negative EBNA1-IgA and a positive EBNA1-IgA was founded in 6 out of 7 patients with negative EA-IgG.

CONCLUSIONAlthough relatively high sensitivity and specificity could be obtained by either EBNA1-IgA or EA-IgG test alone, the combination of these two tests with a complementary effect is able to enhance the reliability of serological diagnosis of NPC as most patients have positive ENBA1-IgA and EA-IgG concurrently.

Adult ; Antigens, Viral ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epstein-Barr Virus Nuclear Antigens ; immunology ; Humans ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Nasopharyngeal Neoplasms ; diagnosis ; immunology ; Sensitivity and Specificity

OBJECTIVETo investigate the relationship between changes in high-risk populations and screening detected nasopharyngeal carcinoma (NPC) during the three-year follow-up of high-risk and moderate-risk groups at initial EB virus serology screening.

METHODSWe tested EB virus VCA-IgA and EBNA1-IgA antibody to identify the probability of suffering from NPC of the crowd. The high-risk and moderate-risk groups at initial screening in one county during 2009 to 2010 were followed-up once a year with EB virus serology testing. All the high-risk people during initial screening and follow-up were conducted with nasopharyngeal fiber endoscopy. Through the follow-up of three years, we analyzed changes in the number of high-risk group, detection rate of NPC in high-risk group, and tumor staging. Firstly detected NPC by screening was defined as screening group, and detected by following-up was defined as following-up group.

RESULTSA total of 404 participants were at high-risk and 1 041 participants were at moderate-risk group, 1 445 persons were in the group. All 404 persons were at high-risk at initial screening, the number of high-risk people during follow-up decreased from 371 to 187, 853 people of the all high-risk group were conducted with nasopharyngeal fiber endoscopy, and 38 cases of NPC were detected. NPC detection rate of high-risk group was 6.2% (25/404), 3.2% (12/371), 0.5% (1/188) and 0 (0/187) during the initial screening and three years follow-up respectively. The cumulative incidence of NPC in the high-risk and moderate-risk group were 7.7% (31/404) ,0.8% (8/1 041) . The early diagnosis rate of NPC in screening group and following-up group was 80% (20/25)and 11/13, respectively. With the primary tumor, the rate of T1 in screening group was higher than following-up group (80% to 38%, 20/25 to 5/13; P = 0.028). However, compared with following-up group, the rate of regional lymph node metastasis in screening group was higher (19/25 to 5/13; P = 0.035 ).

CONCLUSIONAlong with the high detection rate of early staging NPC in screening group and following-up group, the detection of NPC in high risk people is mainly at initial screening and the first year following-up and NPC detection rate thereafter is dropping significantly.

Antibodies, Viral ; Antigens, Viral ; Capsid Proteins ; Carcinoma ; Early Detection of Cancer ; Epstein-Barr Virus Nuclear Antigens ; Follow-Up Studies ; Herpesvirus 4, Human ; Humans ; Nasopharyngeal Neoplasms ; Neoplasm Staging ; Risk Factors

We predict in this paper B-cell epitopes of Epstein-Barr virus nuclear antigen-1 (EBNA-1) and analyze the results matched with the related autoantigens sequence of human. We selected EBV-1 standard strain NA-1 amino acid sequence as the basis. We predicted B-cell dominant epitopes of EBNA-1 with the methods of SOPMA, GOR and HNN, combined with the multi-parameter analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenicity index, polarity and average flexibility. The blastp method was adopted to analyze the matched results between the predicted B-cell epitopes of EBNA-1 and the related autoantigens sequence of human. The results have shown that the possible B-cell dominant epitopes of EBNA-1 were located in the N terminal regions of 16-23, 35-78, 332-337, 340-357, 398-404, 419-432 and 620-637, in which different regions gained higher scores when matched with small nuclear ribonucleoprotein SmB, SmD, ribonucleoprotein SSA, heterogeneous nuclear ribonucleoprotein hnRNP A1, hnRNP G, respectively. It was available to predict B-cell dominant epitopes of EBNA-1 with multiparameter methods and to analyze the same or similar autoantigens sequences of human, which laid a theory foundation for the study of pathogenesis, diagnosis and treatment of autoimmune diseases.
Amino Acid Sequence ; Autoantigens ; immunology ; Base Sequence ; Epitopes, B-Lymphocyte ; immunology ; Epstein-Barr Virus Nuclear Antigens ; immunology ; Humans ; Molecular Sequence Data ; Sequence Homology, Amino Acid

This study aimed to construct recombinant adenovirus expressing Epstein-Barr nuclear antigen 1 (EBNA1) against nasopharyngeal carcinoma (NPC). The C-terminal region fragment of the ebna1 gene of Epstein-Barr virus was amplified from the standard strain B95-8 by polymerase chain reaction (PCR). The gene fragment was inserted into the pDC316 shuttle plasmid using the EcoRI and BgIII restriction enzyme sites. The pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells after sequencing. The soluble protein was extracted from HEK293 cells, which caused apparent cytopathic effects. The transcription and expression of the ebna1 gene were confirmed using flow cytometry and Western blotting. rAd-ebna1 titers were measured by the TCID50. rAd-ebna1 was injected into BALB/c mice at a dose of 2 x 10(8) VP per mouse, EBNA1 epitope-specific responses were measured at 1st, 2nd, 4th and 8th weeks post-immunization. The target fragment of ebna1 (939 bp) was obtained by PCR, and was in consensus with the sequence from the standard strain B95-8. Cytopathic effects were observed after the pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells. rAd-ebna1 was successfully recombined in HEK293 cells. EBNA1 protein was detected in HEK293 cells, rAd-ebna1 titers reached 10(8) TCID50/mL. Specific responses to CD4+ epitopes of EBNA1 were detected in the immunized mice. In conclusion, rAd-ebna1 was successfully constructed and induced specific responses to CD4+ epitopes of EBNA1 in immunized mice.
Adenoviridae ; genetics ; immunology ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; virology ; Epstein-Barr Virus Infections ; immunology ; prevention & control ; virology ; Epstein-Barr Virus Nuclear Antigens ; administration & dosage ; genetics ; immunology ; Genetic Vectors ; genetics ; immunology ; Herpesvirus 4, Human ; genetics ; immunology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Viral Proteins ; administration & dosage ; genetics ; immunology

OBJECTIVESTo investigate the immunophenotypes of primary nasopharyngeal non-Hodgkin lymphoma (NPL) and their relationship to Epstein-Barr virus (EBV) infection.

METHODSThe clinical data and biopsies of 73 patients with NPL were collected in Guangzhou. In situ hybridization was performed to detect the EBV-encoded small non-polyadenylated nuclear RNAs (EBERs) on biopsy slides. Immunohistochemistry was used to classify the immunophenotypes of NPL and detect EBV antigen expression.

RESULTSForty-four (60.27%) of the 73 NPLs were of B cell lineage (CD79alpha(+)/CD3(-)/CD56(-)) while the 29 others (39.73%) were of non-B cell lineage. Seventy-three NPLs could be classified into 3 major immunophenotypes: B cell (CD79alpha(+)/CD3(-)/CD56(-), 44 cases), peripheral T cell (CD79alpha(-)/CD3(+)/CD56(-), 22) and NK/T cell (CD79alpha(-)/CD3(+)/CD56(+), 7). The percentages of EBV infection differed among the 3 major immunophenotypes (B cell: 11.36%, 5/44; peripheral T cell: 81.82%, 18/22; NK/T cell: 100%, 7/7). Both CD56(-) positive and CD56(-) negative immunophenotypes could further be divided into 4 subtypes: CD8(-)/CD4(-), CD8(+)/CD4(-), CD8(-)/CD4(+) and CD8(+)/CD4(+). All the CD8(-)/CD4(-) NPLs with CD56(-) positivity (7) or CD56(-) negativity (2) were infected with EBV. The neoplastic cells of a nasopharyngeal Burkitt's lymphoma expressed EBV nuclear antigen 1 (EBNA1) and EBV RNA (EBERs) only. In the other 29 EBV-infected NPLs, most of the lymphoma cells harboring EBV also expressed EBNA1 and EBERs; 21 of the 29 NPLs had a considerable number of neoplastic cells expressing latent membrane protein 1 (LMP1) (21/29, 72.41%) and 23 of 29 NPLs expressed latent membrane protein 2A (LMP2A) (23/29, 79.31%). A few lymphoma cells in 17 (17/29, 58.62%), 23 (23/29, 79.31%) and 22 NPLs (22/29, 75.86%) expressed Zta (Bam HI Z transactivator), viral capsid antigen (VCA) and membrane antigen (MA), respectively.

CONCLUSIONSThe prevalence ratio of the 3 immunophenotypes, namely, B cell, peripheral T cell and NK/T cell lymphoma, is about 6:3:1. However, the EBV infection ratio is reversed, 1:8:10. All the NK/T cell (CD56(+)) and peripheral immature T cell (CD3(+)/CD8(-)/CD4(-)) NPLs were EBV-infected. Except for one Burkitt's lymphoma, the EBV harbored in both B cell and non-B cell NPLs was mainly latent infection, type II, expressing EBNA1, LMP1 and LMP2A. However, the EBV found in a few lymphoma cells could become replicative, expressing lytic proteins.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; CD56 Antigen ; analysis ; Child ; Epstein-Barr Virus Infections ; complications ; Epstein-Barr Virus Nuclear Antigens ; analysis ; Female ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Immunophenotyping ; Lymphoma, Non-Hodgkin ; etiology ; immunology ; virology ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; etiology ; immunology ; virology ; RNA, Viral ; analysis

Different cytokines are needed in the course of culturing cells to do adoptive immunotherapy. This study was aimed to investigate the differentiation directions of lymphocytes and related gene expression characteristics after combined stimulation of lymphocytes by different cytokines or EBV antigen peptide combined with cytokines. The experiment was divided into 4 groups. The levels of total T lymphocytes (CD3(+)), T helper lymphocytes (CD3(+)CD4(+)), cytotoxic T-lymphocyte (CD3(+)CD8(+)), memory T cells (CD3(+)CD8(+)CD45RO(+)), naive T cells (CD3(+)CD8(+)CD45RA(+)), Th2 cells (CD3(+)CD30(+)), B cells (CD19(+)), NK cells (CD56(+)), naive T regulatory cells (CD4(+)CD25(+)), precise T regulatory cells (CD4(+)CD25(+)FOXP3(+)) were detected by flow cytometry. The expression levels of house-keeping gene (mad1, pten), T helper cells transcriptional regulatory gene t-bet (Th1), gata3 (Th2), cytokine IFN-γ(Th1), IL-4(Th2) were detected by using RT-PCR. The results showed that CTL in EBV polypeptide group were dominant cells with certain clinical effects. Comparison of result of EBV polypeptide group with other 3 different cytokine stimulating groups demonstrated that EBV antigen peptide had much more effects on stimulating CTL generation. The expression of IFN-γ gene was significantly increased; the T helper differentiation-related gene t-bet, gata3 also increased evidently, while expression change of house-keeping gene mad1 and pten were not evident. Addition of different cytokines and antigen peptides in culture may be much more effective on stimulating CTL generation. It is concluded that specific CTL can be obtained by using the lymphocytes co-cultured with EBV and cytokines, and the different cytokines play different roles in cell differentiation.
Cells, Cultured ; Cytokines ; immunology ; metabolism ; Epstein-Barr Virus Nuclear Antigens ; genetics ; Flow Cytometry ; Humans ; Immunotherapy, Adoptive ; Lymphocyte Count ; Lymphoma, Extranodal NK-T-Cell ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

The nonviral gene delivery systems are usually not very effective in transferring gene into target cells, and the intensity and duration of the gene expression is very poor. The EBNA1/oriP maintain EBNA1/oriP-based plasmids as episome, contribute to nuclear transport of the plasmid and transcriptional up-regulation of target gene. The EBNA1/oriP based plasmid enhances the transfection rate as well as magnitude and longevity of gene expression. This article reviews recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, the EBNA1/ oriP based plasmid encoding the HSV1-TK suicide gene was combined with a cationic polymer to transfer into HCC cell line. The expression level of TK gene was 100- to 1000-fold higher than the conventional plasmid. The sensitivity of HCC to ganciclovir (GCV) elevated several hundred-fold. The EBNA1/oriP based plasmid equipped with tumor specific promoter, such as CEA promoter, enabled targeted killing of CEA-positive tumor cell. Transfection of EBNA1/oriP based plasmid carrying IL-12 and IL-18 gene either locally, or systemically, induced therapeufic antitumor immune responses including augmentation of the cytotoxic T lymphocyte and natural killer activities and growth retardation of tumors. For gene therapy of congenital diseases and chronic diseases, the EBNA1/oriP based plasmid encoding the adenosine deaminase gene was transfered into human hematopoietic progenitor cells. The ADA activity was elevated 1.5-to 2-fold. Intracardiomuscrlar transfer of the EBNA1/oriP based plasmid encoding the beta-AR gene may be useful for the treatment of severe heart failure. Human tumor necrosis factoralpha (hTNFalpha) is one of the most important inflammatory cytokines. It has been implicated in many autoimmune and inflammatory diseases. sTNFR can efficiently neutralize the bioactivities of hTNFalpha. In primary study we cloned the chimeric protein sTNFR II-IgG Fc and expect to use it in the gene therapy of the inflammatory disease relative to TNF. In summary, The EBNA1/oriP based plasmid shows advantage in gene therapy of cancer, congenital and inflammatory diseases. Moreover, the EBNA1/oriP element may greatly contribute to the engineering of a human artificial chromosome, the ultimate device for controllable gene therapy.
Epstein-Barr Virus Nuclear Antigens ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Herpesvirus 4, Human ; genetics ; metabolism ; Humans ; Muscular Dystrophy, Duchenne ; therapy ; Neoplasms ; therapy ; Plasmids ; genetics ; Replication Origin ; genetics ; Transcription, Genetic ; Transfection ; methods

OBJECTIVETo evaluate the risk of nasopharyngeal carcinoma (NPC) through EB virus antibody profile by enzyme linked immunosorbent assay (ELISA).

METHODSEBNA 1/IgA, EBNA 1/IgG and zta/IgG by ELISA and VCA/IgA by immmunoenzymatic method were detected in 121 NPC patients and 332 healthy subjects (HS) in the Pearl river estuary.

RESULTSThe sensitivity rates were 85%, 83% and 79% for EBNA 1/IgA, EBNA 1/IgG and zta/IgG, all three of which if combined was the highest 92%. The specificity rates were 86%, 86% and 80% for EBNA 1/IgA, EBNA 1/IgG and zta/IgG, all three of which if combined was also the highest 93%. According to the level of odds ratio, nasopharyngeal carcinoma risk could be divided into 3 groups: low, moderate and high-risk groups. 93% of HS had low risk of NPC with the odds ratio 0.0 to 0.3. 0.4% of HS had high risk of NPC with the odds ratio of 137.9%.

CONCLUSIONELISA is more objective than the traditional immunoenzymatic method in the detection and diagnosis of NPC. The combination of EBNA 1/IgA, EBNA 1/IgG and zta/IgG is able to evaluate the risk of NPC.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Viral ; analysis ; Enzyme-Linked Immunosorbent Assay ; Epstein-Barr Virus Nuclear Antigens ; analysis ; Female ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; diagnosis ; virology ; Risk Factors