Production of Antiserum against a Specific Herpes Simplex Virus Type 2 Antigen

Antisera were produced in rabbits by inoculation of precipitates cut out of second dimensional agarose gels from crossed immunoelectrophoreses. The precipitating antigen chosen (antigen no. 1) had previously been identified as specific for herpes simplex virus type 2. Crossed immunoelectrophoretic analysis of the produced antisera revealed that the antisera contained a high concentration of precipitating antibodies that reacted exclusively with antigen no. 1 and a minor antigen with the same electrophoretic mobility. The produced antisera were useful in the typing of herpes simplex virus isolates by rocket immunoelectrophoresis.     

Modulation of Mucosal and Systemic Immunity by Enteric Administration of Nonreplicating Herpes Simplex Virus Expressing Cytokines

In this report the ability of enteric immunization with recombinant replication deficient (ICP4−/−) HSV expressing IFNγ to generate protection and modulate mucosal and systemic immunity was evaluated. ICP4−/−HSV, ICP4−/−HSV expressing IL4, live replicating, and uv HSV were used as controls. Following enteric administration of live HSV, a Th₁cytokine response was induced in the spleen, while both Th₁and notable Th₂cytokine production were detected at mucosal sites. Modulation of mucosal and systemic immune response was achieved when nonreplicating recombinant HSV viruses expressing cytokines were used. Compared to the control replication defective viruses, decreased frequency of Th₂cytokine producing cells in Peyer's patches was observed following enteric administration of nonreplicating HSV expressing IFNγ. When IFNγ expressing virus was given enterically, modulation was observed at the systemic level, measured by ELISPOT for cytokine producing cells, ELISA from thein vitrorestimulated splenic cell cultures, and by the increase of the IgG2a/IgG1 ratio in the serum. This report provides evidence that replication defective viruses expressing cytokine genes in contrast to uv HSV, are immunogenic when administered enterically and can generate significant immunomodulatory effects at the mucosal and systemic levels.     

Seroepidemiology of Herpes Simplex Virus and Restriction Endonuclease Cleavage Analysis of Herpes Simplex Virus Type 2 in Okinawa

A seroepidemiologic study of herpes simplex virus (HSV) in Okinawa was performed. A total of 423 serum samples were collected from all over Okinawa, and the positivity rate of antibody against HSV was measured using a passive hemagglutination method. The sero positive rate for HSV in age groups of over 40 years was 100%. Seven HSV type 2 (HSV 2) isolates were obtained in Okinawa, and DNA preparations from Vero cells infected with the isolates were analyzed using five restriction endonucleases: Bam HI, Hind III, Kpn I, Bgl II and Eco Rl. Variations in the genomic region were demonstrated in five of the isolates. Such variations have not been reported previously in HSV 2 in mainland Japan. This is the first report of a seroepidemiologic study of HSV and restriction endonuclease cleavage analysis of HSV 2 in Okinawa, is a subtropical island where HSV is endemic. Acta Pathol Jpn 41: 24–30, 1991.     

Herpes Simplex Virus Type 2 Vaccines: New Ground for Optimism?

The development of effective prophylactic and therapeutic vaccines against genital herpes has proven problematic. Difficulties are associated with the complexity of the virus life cycle (latency) and our relatively poor understanding of the mechanism of immune control of primary and recurrent disease. The types of effector cells and the mechanisms responsible for their activation and regulation are particularly important. Studies from my and other laboratories have shown that recurrent disease is prevented by virus-specific T helper 1 (Th1) cytokines (viz., gamma interferon) and activated innate immunity. Th2 cytokines (viz., interleukin-10 [IL-10]) and regulatory (suppressor) T cells downregulate this immune profile, thereby allowing unimpeded replication of reactivated virus and recurrent disease. Accordingly, an effective therapeutic vaccine must induce Th1 immunity and be defective in Th2 cytokine production, at least IL-10. These concepts are consistent with the findings of the most recent clinical trials, which indicate that (i) a herpes simplex virus type 2 (HSV-2) glycoprotein D (gD-2) vaccine formulated with a Th1-inducing adjuvant has prophylactic activity in HSV-2- and HSV-1-seronegative females, an activity attributed to the adjuvant function, and (ii) a growth-defective HSV-2 mutant (ICP10ΔPK), which is deleted in the Th2-polarizing gene ICP10PK, induces Th1 immunity and has therapeutic activity in both genders. The ICP10ΔPK vaccine prevents recurrent disease in 44% of treated subjects and reduces the frequency and severity of recurrences in the subjects that are not fully protected. Additional studies to evaluate these vaccines are warranted.     

Typing of Clinical Herpes Simplex Virus Type 1 and Type 2 Isolates with Monoclonal Antibodies

The purpose of this study was to evaluate the performance of a herpes simplex virus (HSV) type 1-specific anti-glycoprotein C-1 monoclonal antibody (MAb) and a type 2-specific anti-glycoprotein G-2 MAb for typing of 2,400 clinical HSV-1 isolates and 2,400 clinical HSV-2 isolates, respectively, using an enzyme immunoassay. The anti-HSV-1 MAb showed sensitivity and specificity of 100%, and the anti-HSV-2 MAb showed a sensitivity of 99.46% and 100% specificity, indicating that these MAbs are suitable for typing of clinical HSV isolates.     

Herpes Simplex Virus Type 2 and Cytomegalovirus Perigenital Ulcer in an HIV Infected Woman

We report a case of mucocutaneous Herpes Simplex Virus (HSV)-2 and Cytomegalovirus (CMV) infection in a 39-year-old female with acquired immunodeficiency syndrome, who presented with a perigenital ulcer. The patient was receiving antiretroviral treatment (ART) for 3 months before presentation. Scraping from the perigenital ulcer was positive for HSV-2 and Treponema pallidum using polymerase chain reactions (PCR). The extent and duration of the lesions led us to consider the possibility of coinfection with CMV. The patient also tested positive for CMV by PCR. On subsequent follow-up after 8 weeks, the genital lesions had healed completely. This is possibly ascribable to the ART, which led to significant immune reconstitution.     

Expression Analysis of Recombinant Herpes Simplex Virus Type 1 DNase

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)¹. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503–626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein re aching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear loca lization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association. This revised version was published online in July 2006 with corrections to the Cover Date.     

单克隆抗体作单纯疱疹病毒糖蛋白的特性鉴定以及临床应用

应用本室制备的8株抗单纯疱疹病毒糖蛋白单克隆抗体(抗HSV McAb)进行了系列研究,①鉴定出一种新的HSV糖蛋白g30K、gC的一种新形式,以及gC和gD;②建立了HSV感染的临床标本作抗原抗体分型检测的McAb-ELISA双夹心法,检测961份标本效果良好,并己在全国各地一些医疗单位推广使用;③证明McAb治疗家兔实验性单纯疱疹性角膜炎(HSK)有显著效果,并探讨其治疗机理主要为中和作用和ADCC效应;④对部分志愿者用McAb治疗单纯疱疹性角膜炎、妇女生殖器疱疹和小儿口腔疱疹性糜烂,取得明显疗效。     

Delta-9-Tetrahydrocannabinol Inhibits the Splenocyte Proliferative Response to Herpes Simplex Virus Type 2

The present investigation was undertaken to determine the effect of in vivo Delta-9-tetrahydrocannabinol (Delta-9-THC) treatment on immune responsiveness to secondary exposure to herpes simplex virus type 2 (HSV2) antigens in vitro. A splenocyte proliferative assay, employing HSV2-infected mouse embyro fibroblasts as target cells, was used to measure immune responsiveness. Administration of 50mg/kg or 100mg/kg Delta-9-THC to B6C3F1 mice in concert with HSV2 infection resulted in suppression of the proliferative response to HSV2 cell-surface antigens expressed on virus-infected mouse embryo fibroblasts. Similarly, in vifero treatment of HSV2-infected cells with Delta-9-THC (10 ⁷M to ⁵M) resulted in a dose-dependent suppression of proliferative responsiveness of splenocytes of non-drug-treated HSV2-sensitized mice. These results suggest that Delta-9-THC inhibits immune responsiveness of B6C3F1 mice to homotypic challenge with HSV2. This inhibition may be resultant of drug action on both effector immunocytes and target HSV2 antigen-bearing cells.     

Delta-9-Tetrahydrocannabinol Inhibits the Splenocyte Proliferative Response to Herpes Simplex Virus Type 2

Abstract

The present investigation was undertaken to determine the effect of in vivo Delta-9-tetrahydrocannabinol (Delta-9-THC) treatment on immune responsiveness to secondary exposure to herpes simplex virus type 2 (HSV2) antigens in vitro. A splenocyte proliferative assay, employing HSV2-infected mouse embyro fibroblasts as target cells, was used to measure immune responsiveness. Administration of 50mg/kg or 100mg/kg Delta-9-THC to B6C3F1 mice in concert with HSV2 infection resulted in suppression of the proliferative response to HSV2 cell-surface antigens expressed on virus-infected mouse embryo fibroblasts. Similarly, in vifero treatment of HSV2-infected cells with Delta-9-THC (10 ⁷M to ⁵M) resulted in a dose-dependent suppression of proliferative responsiveness of splenocytes of non-drug-treated HSV2-sensitized mice. These results suggest that Delta-9-THC inhibits immune responsiveness of B6C3F1 mice to homotypic challenge with HSV2. This inhibition may be resultant of drug action on both effector immunocytes and target HSV2 antigen-bearing cells.     

Identification and Characterization of the UL24 Gene Product of Herpes Simplex Virus Type 2

The UL24 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 281 amino acid protein with a molecular mass of 30.5kDa. In this study, the HSV-2 UL24 gene product has been identified by using a rabbit polyclonal antiserum produced against a recombinant protein containing the full-length UL24 gene product of HSV-2 fused to glutathione-S-transferase. The antiserum reacted specifically with a 32kDa protein in HSV-2 186-infected Vero cells and with 31 and 32kDa proteins in UL24-expressing Cos-7 cells. Accumulation of UL24 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. UL24 protein was found to be associated with purified HSV-2 virions and C capsids. Indirect immunofluorescence analysis demonstrated that the UL24-specific fluorescence was detected in perinuclear regions of the cytoplasm and/or in the nucleus as small discrete granules from 9h post infection (hpi). Furthermore, the UL24 protein expressed singly was detected predominantly in the nucleus and slightly in the cytoplasm at 24h after transfection, with branch-like cytoplasmic protruding structures. Strong nucleolus staining was visible in partial cells.     

Identification and Characterization of the UL7 Gene Product of Herpes Simplex Virus Type 2

We have raised a rabbit polyclonal antiserum against a recombinant 6× His-tagged herpes simplex virus type 2 (HSV-2) UL7 fusion protein expressed in Escherichia coli. The antiserum specifically reacted with a 33 kDa protein in HSV-1 and HSV-2-infected cell lysates, and was used to characterize the UL7 gene product of HSV-2. The UL7 protein was produced in the late phase of infection, and its synthesis was highly inhibited, but not abolished by the addition of acyclovir (ACV). The UL7 protein associated with extracellular virions and also with all types of capsids, including A, B, and C capsids, though the association seemed to be weak. Indirect immunofluorescence studies revealed that at 9 h postinfection, UL7 specific fluorescence was detected in part or all of the nucleus, and the specific fluorescence colocalized with the scaffold protein ICP35. However, at later times postinfection, the UL7 protein was mainly detected as a mass in a juxtanuclear cytoplasmic region. In addition, transmission immunoelectron microscopy (TIEM) confirmed the association of the UL7 protein with intracellular capsids and virions in HSV-2-infected cells. The HSV-2 UL7 protein contained a domain highly conserved in all herpesviruses, part of which exhibited a homology with domains in the fission yeast Schizosaccharomyces pombe DNA topoisomerase III. We discuss the possibility that the UL7 protein may play a supplementary role in the viral DNA cleavage/packaging process.     

Optimal Cooling and Warming Rates in the Preservation of Herpes Simplex Virus (Type 2)

Fast rates of cooling and warming were optimal for survival of herpes simplex virus, type 2. Under these conditions cryoprotectants were not necessary for virus preservation.     

Mannan-Binding Protein and Bovine Conglutinin Mediate Enhancement of Herpes Simplex Virus Type 2 Infection in Mice

A broad range of plant lectins have recently been shown to inhibit the infectivity of herpes simplex virus type I (HSV-1) in viiro . We decided to investigate the role of mammalian Icctins in infection witb herpes simplex virus. Two lectins, conglutinin and mannan-binding protein (also called mannose-binding protein. MBP). belonging to the collectin family of lectins, were examined.
Four week-old BALB/c mice were injected subcutaneously with 100 μg bovine conglutinin or 50 μg human MBP 1 day before intravenous infection with 5 × 10⁴ PFU of herpes simplex virus type 2 (HSV-2). A three-fold increase in virus titre of the liver was observed on day 3 of the infection in the mice pretreated with conglutinin or MBP. whereas no effect was seen on days I and 5.
In a standard plaque assay using Vero cells we were not able to demonstrate reproducibly either infection inhibition or infection enhancement, when virus was pre-incubated with differing concentrations ofthe collectins. Tbe concentrations used were similar to tbose used by us in livo , and by others in in vitro experiments showing inhibition of the infectivity of HSV-1 with plant lectins.
In an ELISA with HSV-2 antigens captured on anti-HSV-2 antibodies, calcium-dependent and carbohydrate inhibitabte binding of the collectins was observed. Our results indicate that the effect of endogenous mammalian collectins in vivo may not be neutralization as suggested by the data using plant lectins. Instead, the previously described opsonizing activity of the mammalian collectins may provide the virions witb an alternative port of entry into cells leading to infection enhancement.     

Acute Morphine Administration Reduces Cell-Mediated Immunity and Induces Reactivation of Latent Herpes Simplex Virus Type 1 in BALB/c Mice

Acute morphine administration is known to alter the course of herpes simplex virus infection. In this study, the effect of acute morphine administration on the reactivation of latent herpes was investigated in a mouse model. Because of the important role of cytolytic T lymphocyte （CTL） activity in the inhibition of herpes simplex virus type 1 （HSV-1） reactivation, the effect of acute morphine administration on CTL responses was also evaluated. Furthermore, lymphocyte proliferation and IFN-γ production were evaluated for their roles in the induction of the CTL response. The findings showed that acute morphine administration significantly reduced CTL responses, lymphocyte proliferation, and IFN-γ production. Furthermore, acute morphine administration has been shown to reactivate latent HSV-γ. Previous studies have shown that cellular immune responses have important roles in the inhibition of HSV reactivation. These findings suggest that suppression of a portion of the cellular immune response after acute morphine administration may constitute one part of the mechanism that induces HSV reactivation.     

表达2型单纯疱疹病毒糖蛋白D的重组痘苗病毒对动物的免疫效果

用表达２型单纯疱疹病毒（ＨＳＶ－２）糖蛋白Ｄ的重组痘苗病毒（Ｒ－ｇＤ－Ｖ）免疫ＣＢＡ小鼠，通过间接免疫荧光（ＩＦ）（观察小鼠抗体滴度变化）和四甲基偶氮唑蓝法（ＭＴＴ）动态观察细胞毒性Ｔ细胞活性（ＣＴＬ），均见增高并检测了其产生的抗体对动物的保护作用。同时对Ｒ－ｇＤ－Ｖ和２型单纯疱疹病毒（ＨＳＶ－２）产生的二次ＣＴＬ活性进行了比较，结果表明，Ｒ－ｇＤ－Ｖ产生抗体３周达高峰（１：６４０），７周下降为１：３２０，抗体对病毒攻击动物有保护作用，其中和指数（Ｎ）为６３。Ｒ－ｇＤ－Ｖ在鼠体内产生初次ＣＴＬ８ｄ达高峰，１２ｄ后消失；二次ＣＴＬ持续８周之久，且ＣＴＬ活性明显高于初次。Ｒ－ｇＤ－Ｖ和ＨＳＶ－２相比较产生ＣＴＬ在统计学上有差异（Ｐ＜０．０５）。说明Ｒ－ｇＤ－Ｖ能引起体液免疫和细胞免疫，对动物有保护作用。     

Herpes Simplex Virus Infection Downmodulates NKG2D Ligand Expression

Herpes simplex virus (HSV) type 1 infection may cause orofacial infections in humans. The virus resides in a latent form in neural ganglia and occasionally reactivates and infects epithelial cells. Natural killer (NK) cells have been implicated in immune control of herpes virus infections, possibly by downmodulating major histocompatibility complex (MHC) class I and by other, as yet unidentified, mechanisms. Upon HSV‐1 infection of cell lines, surface levels of NKG2D ligands MHC class I related proteins (MIC) A and UL16 binding protein 2 were downmodulated due to late viral gene product(s). As also MHC class I levels were reduced by HSV‐1, NK cell recognition of HeLa cells was not affected by infection. Total cellular MICA contents remained unchanged, suggesting masking, internalization or intracellular retention of MICA as possible mechanisms of viral downregualtion of MICA surface levels. Furthermore, NK cells from patients with active HSV‐1 infection had a tendency towards increased expression level of the activating receptor NKG2D. These data support a role for NKG2D–MICA interactions in immune responses to HSV‐1 reactivation.     

Detection of Concanavalin A — Binding Herpes Simplex Virus Type 1 and Type 2 Antigens by Crossed Immuno-Affinoelectrophoresis

The presence of four herpes simplex virus (HSV) glycoprotein antigens in cells infected with HSV type 1 and HSV type 2 was demonstrated by the use of crossed immuno-affinoelectrophoresis with con A.     

1型单纯疱疹病毒突变体治疗肿瘤的研究进展

病毒溶瘤治疗的研究由来已久,但治疗多种人类恶性肿瘤的溶瘤病毒的选择构建仍在不断的探索与研究中.目前1型单纯疱疹病毒(HSV-1)被认为是最为有效的溶瘤病毒.作者阐述了溶瘤病毒治疗肿瘤的现状及研究机制、HSV-1的生物学特点、HSV-1突变体构建及其治疗肿瘤的现状,并探讨了放射性核素标记HSV-1突变体的可行性及标记物治疗肿瘤的前瞻性.