OBJECTIVETo express a multi-copy specific epitope recombinant protein of herpes simplex virus type 1 (HSV-1) with immuno-reactivity.

METHODSMulti-copy genes with a specific epitope of HSV-1-glycoprotein G 112-127 were constructed by DNA recombination and cloned in E. coli JM109 pGEM-5Zf. The positive recombinants were determined by SDS-PAGE and Western blotting.

RESULTSThe recombinants with 4, 8, 16 and 32 copies of gG 112-127 were obtained. The 8-copy recombinant was expressed by 17.5%, mainly as inclusion body. And it reacted with antiserum HSV-1, but not with antiserum HSV-2.

CONCLUSIONThe HSV-1-gG112-127 recombinant could be used to distinguish HSV-1 and HSV-2 in ELISA.

Antigen-Antibody Reactions ; DNA, Recombinant ; Epitopes ; biosynthesis ; immunology ; Herpesvirus 1, Human ; immunology ; Herpesvirus 2, Human ; immunology ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Viral Envelope Proteins ; biosynthesis ; immunology

OBJECTIVETo investigate the expression of CD25(+) lymphocytes in nasopharyngeal carcinoma (NPC) tissue and the influence of Epstein-Barr virus (EBV) infection on CD25 expression.

METHODSImmunohistochemistry was used to detect CD25 expression in the NPC tissues and in situ hybridization employed to detect EBV infection with chronic nasopharyngitis tissue as the control sample.

RESULTSSignificant difference was noted in the expression of CD25(+) lymphocytes between NPC and chronic inflammatory tissues. The expression was higher in undifferentiated NPC than in keratinizing squamous cell carcinoma and non-keratinizing carcinomas. The NPC tissue was all EBV-positive except for one sample, which was identified as keratinizing carcinoma, but the control samples were all negative for EBV infection, which was correlated with CD25 expression.

CONCLUSIONThe expression of CD25(+) lymphocytes is higher in NPC tissues and correlated to EBV infection.

Carcinoma, Squamous Cell ; immunology ; virology ; Epstein-Barr Virus Infections ; immunology ; Female ; Herpesvirus 4, Human ; Humans ; Interleukin-2 ; immunology ; Interleukin-2 Receptor alpha Subunit ; biosynthesis ; Lymphocytes ; immunology ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; immunology ; virology

BACKGROUNDTo get early laboratory study of type specific antigenicity of herpes simplex virus II.

METHODSPCR and prokaryotic expression technique.

RESULTSHerpes simplex virus II type specific gene fragment was expressed in E.coli and the products can be used as specific antigen for the detection of anti\HSV in the recovery sera.

CONCLUSIONSCloning and express of HSVII type specific antigen found the basis for developing specific diagnosis methods and vaccine of HSV.

Antigens, Viral ; immunology ; Cloning, Molecular ; Gene Expression ; Herpesvirus 2, Human ; genetics ; Humans ; Immunoglobulin G ; blood ; Polymerase Chain Reaction ; Recombinant Proteins ; immunology

OBJECTIVETo study the improvement of CTL effect by CTL epitopes which are modified by KDEL and recognized by CD8+ T lymphocytes to herpes simplex virus type 2.

METHODSObservations were made on the specific immune response induced by the CD8+ CTL epitopes(SSIEFARL, S1), the CD8+ CTL epitopes modified by KDEL(SSIEFARL-KDEL, S1-KDEL), the tandem four copies CD8+ CTL epitopes[(SSIEFARL)4, S4] and the tandem four copies CD8+ CTL epitopes modified by KDEL[(SSIEFARL)4-KDEL, S4-KDEL], 25 male C57BL/6 mouse were randomly divided into 5 group, 5 mice per group, respectively immunized with istonic Na chloride, S1, S1-KDEL, S4 and S4-KDEL. Lymphocyte proliferation was detected by 3H-TdR and the CTL effect induced by CTL epitopes in vivo was detected by 51Cr.

RESULTSIn the 3H-TdR test, compared with the control, the group S1 and group S1-KDEL, the cpm values of Group S4 and Group S4-KDEL were markedly higher (P < 0.05) and the cpm value of Group S4-KDEL was significantly higher than Group S4 (P < 0.05), but the cpm values of Group S1 and Group S1-KDEL were not significantly different from the control (P > 0.05), nor was that of Group S1 from Group S1-KDEL (P > 0.05). In the experiment in which the EL4 cells sensitized by S1 were attacked as target cells, the CTL activities induced in Group S4 and Group S4-KDEL were markedly higher (P < 0.05) compared with the control, Group S1 and Group S1-KDEL, and that induced in Group S4-KDEL was significantly higher than Group S4 (P < 0.05), but the CTL activity induced in Group S1 and Group S1-KDEL were not significantly different from the control (P > 0.05), nor was that induced in Group S1 from Group S1-KDEL (P > 0.05). In the experiment in which the EL4 cells were attacked as target cells, the kill rate was below 10% in every group, not significantly different from the control.

CONCLUSIONThe tandem four copies CD8+ CTL epitopes, modified by KDEL and recognized by CD8+ T lymphocytes to herpes simplex virus type 2, can improve the CTL effect.

Animals ; CD8-Positive T-Lymphocytes ; immunology ; Cell Line ; Epitopes, T-Lymphocyte ; immunology ; Herpesvirus 2, Human ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Oligopeptides ; immunology ; Protein Sorting Signals ; Random Allocation ; T-Lymphocytes, Cytotoxic ; immunology

To determine the characteristics of seroprevalence of herpes simplex virus type 2 (HSV-2) infection among Korean people, a cross-sectional study was conducted on three groups in 2004. The three groups consisted of the general public who visited public health centers, commercial sex workers (CSWs), and human immunodeficiency virus (HIV)-infected persons. Among the general public, HSV-2 seroprevalence rates for age under the 20s, in the 20s, 30s, 40s and the above 22.6%, 32.7% and 32.3%, respectively, which showed rapid increase of the rate in the 30s (p<0.0001). In case of the above of 19 yr old, women (28.0%) was higher than men (21.7%) (p<0.0001). The rate of CSWs (81.6%) was about 10 times higher than that of general women. In case of HIV-infected men (47.6%), the figure was about 2-3 times higher than that of general men. The low rate in the teens and the 20s proved that it is essential to develop sexually transmitted infections (STIs) prevention programs of education and publicity for them as a precaution measure. This study is the first major study of its kind on HSV-2 and would provide basic data for prevention of STIs including information about target groups subject to vaccination program.
Adult ; Age Factors ; Aged ; Antibodies, Viral/*blood ; Female ; HIV Infections/complications ; Herpes Genitalis ; Herpesvirus 2, Human/*immunology ; Humans ; Korea/epidemiology ; Male ; Middle Aged ; Seroepidemiologic Studies

OBJECTIVETo study the infection of human cytomegalovirus (HCMV) and herpes simplex virus type II (HSV-I) and the morphological characteristics of the infected spermatogenic cells in the semen of infertile men.

METHODSWe washed and concentrated the spermatogenic cells obtained from 83 semen samples of infertile men, extracted DNA and then screened HCMV and HSV-II by polymerase chain reaction (PCR). Immunocytochemistry (ICC) was used to detect the expression of correlative virus antigens of the positive semen cells, and the cytology smear was employed to observe the morphological changes of the spermatogenic cells under the microscope after cytology staining.

RESULTSOf all the semen samples, 8 were HCMV positive, 4 HSV-II positive, but none were both HCMV and HSV-II positive. HCMV late antigens were positively and HCMV early antigens negatively expressed in the spermatogenic cells of the 8 HCMV positive cases. In the 4 HSV-II positive cases, 3 were positively and 1 weakly positively expressed. In the semen of the 12 positive cases were found large numbers of immature spermatogenic cells, with different manifestations of apoptosis, such as chromatin pycnosis, vacuoles, damaged nuclear membrane, and apoptotic bodies, but without virus infection-induced specific morphological alteration. Sperm concentration of the positive group was significantly lower than that of the negative (P < 0. 05).

CONCLUSIONSpermatogenic cells infected by HCMV and HSV-II may cause pathologic lesions and affect spermatogenesis. Morphologically, the infected spermatogenic cells may undergo some pathologic alteration, such as apoptosis. The rate of HCMV infection is higher among infertile males with pathologic cells in the semen.

Adult ; Antigens, Viral ; analysis ; Cytomegalovirus ; genetics ; immunology ; Cytomegalovirus Infections ; pathology ; virology ; DNA, Viral ; genetics ; Herpes Simplex ; pathology ; virology ; Herpesvirus 2, Human ; genetics ; immunology ; Humans ; Immunohistochemistry ; Infertility, Male ; pathology ; virology ; Male ; Polymerase Chain Reaction ; Semen ; cytology ; virology ; Spermatozoa ; cytology ; virology

OBJECTIVETo explore the inhibition effect of RNA interference on the ICP4 expression and DNA replication of herpes simplex virus type 2 (HSV2).

METHODSFour pairs of siRNA targeted to HSV2 ICP4 gene and negative control siRNA were synthetized by chemical method, named as siRNA-1, siRNA-2, siRNA-3, siRNA-4 and siRNA-N respecticely. HSV2 HG52 was used to attack Vero cell after transfection overnight. Vero cell and supernatant were collected at 1d, 2d, 3d, 4d and 5d after virus attacking. Flurogenic quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR)was used to detect the expression of HSV2 ICP4 mRNA, flurogenic quantitative polymerase chain reaction(FG-PCR) was used to detect the expression of HSV2 DNA and Western-Blot was used to detect the expression of HSV2 ICP4 protein.

RESULTSAll the four pairs of siRNA could significantly inhibit the expression of HSV2 ICP4 mRNA and protein, especially siRNA-2. The above siRNAs could significantly decrease HSV2 DNA copy number,too.

CONCLUSIONsiRNAs targeted to HSV2 ICP4 gene could significantly inhibit expression of HSV2 ICP4 mRNA and protein, and decrease HSV2 DNA copy number, suggesting that siRNA can inhibit HSV2 DNA replication through silencing ICP4 gene.

Gene Expression Regulation, Viral ; drug effects ; genetics ; Gene Silencing ; drug effects ; physiology ; Herpesvirus 2, Human ; drug effects ; genetics ; RNA Interference ; drug effects ; immunology ; RNA, Small Interfering ; pharmacology ; RNA, Viral ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Establishing Epstein-Barr virus(EBV)-specific cytolytic T lymphocytes(EBV-CTLs) from peripheral blood mononuclear cells(PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma(NPC). In the current study, we performed ex vivo expansion of tumor-infiltrating lymphocytes(TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody(OKT3), recombinant human interleukin(IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+ T cells, a variable percentage of CD3+CD8+ and CD3+CD4+ T cells, and less than 10% of CD3-CD16+ natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.
Biopsy ; CD3 Complex ; analysis ; CD4 Antigens ; analysis ; CD8 Antigens ; analysis ; Cells, Cultured ; Herpesvirus 4, Human ; immunology ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; pharmacology ; Interleukin-4 ; metabolism ; Lymphocytes, Tumor-Infiltrating ; immunology ; virology ; Monocytes ; pathology ; Muromonab-CD3 ; pharmacology ; Nasopharyngeal Neoplasms ; immunology ; pathology ; therapy ; virology ; Receptors, IgG ; analysis ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the immune responses and protection from virus challenge, induced by the coinjection of IL-2cDNA with herpes simplex virus type 1 (HSV-1) glycoprotein-D (gD) DNA vaccine.

METHODSTwo DNA vaccines (pgD and pIL-2) were constructed by inserting the gD gene and IL-2 cDNA into the eukaryotic expression vector pcDNA3.1, respectively. The BALB/c mice were inoculated intramuscularly three times at 2-week intervals. Two weeks after the final immunization, mice were bled for antibody assay and spleen cells were separated for Th cell proliferation and cytokine assays. Delayed type hypersensitivity (DTH) response was detected by the pinna-swelling test. Corneal protection under HSV-1 virus challenge was continuously observed with slit-lamp microscope.

RESULTSIL-2 cDNA coinjection remarkably enhanced the specific IgG2a level when compared with gD plasmid vaccination alone. Th cell proliferation and secretion of cytokines (IL-2 and IFN-gamma) were significantly increased by IL-2 cDNA coinjection. However, the production of IL-10 was inhibited. The DTH response was also enhanced by IL-2 coinjection. When the mice were challenged with HSV-1, the cornea epithelial lesions were significantly alleviated by IL-2 coinjection as compared with gD vaccination alone.

CONCLUSIONIL-2 cDNA can enhance both the humoral and cellular immune responses, and thus increase the vaccine potency.

Animals ; Antibodies, Viral ; blood ; COS Cells ; Cell Proliferation ; Cercopithecus aethiops ; DNA ; genetics ; Female ; Herpesvirus 1, Human ; pathogenicity ; Hypersensitivity, Delayed ; immunology ; Immunization ; Immunoglobulin G ; blood ; Interferon-gamma ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Th1 Cells ; cytology ; Transfection ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

GITR (glucocorticoid-induced TNF receptor) is a recently identified member of the TNF receptor superfamily. The receptor is preferentially expressed on CD4+CD25+ regulatory T cells and GITR signals break the suppressive activity of the subset. In this study, we wanted to reveal the in vivo function of GITR in herpes simplex virus type 1 (HSV-1) infection. A single injection of anti-GITR mAb (DTA-1) immediately after viral infection significantly increased the number of CD4+ and CD8+ T cells expressing CD25, an activation surface marker, and secreting IFN-gamma. We confirmed these in vivo observations by showing ex vivo that re-stimulation of CD4+ or CD8+ T cells with a CD4+ or CD8+ T-cell-specific HSV-1 peptide, respectively, induced a significant elevation in cell proliferation and in IFN-gamma secretion. Our results indicate that GITR signals play a critical role in the T-cell immunity to HSV-1.
Animals ; Antibodies, Monoclonal/pharmacology ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cell Proliferation ; Female ; Glucocorticoids/*pharmacology ; Herpes Simplex/*immunology ; Herpesvirus 1, Human/pathogenicity ; *Immunity, Cellular ; Interferon Type II/secretion ; *Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/metabolism ; Receptors, Interleukin-2/metabolism ; Receptors, Nerve Growth Factor/genetics/immunology/*metabolism ; Receptors, Tumor Necrosis Factor/genetics/immunology/*metabolism ; Research Support, Non-U.S. Gov't ; T-Lymphocytes/*immunology/metabolism/virology