A lentiviral vector-based, herpes simplex virus 1 (HSV-1) glycoprotein B vaccine affords cross-protection against HSV-1 and HSV-2 genital infections

Genital herpes is caused by herpes simplex virus 1 (HSV-1) and HSV-2, and its incidence is constantly increasing in the human population. Regardless of the clinical manifestation, HSV-1 and HSV-2 infections are highly transmissible to sexual partners and enhance susceptibility to other sexually transmitted infections. An effective vaccine is not yet available. Here, HSV-1 glycoprotein B (gB1) was delivered by a feline immunodeficiency virus (FIV) vector and tested against HSV-1 and HSV-2 vaginal challenges in C57BL/6 mice. The gB1 vaccine elicited cross-neutralizing antibodies and cell-mediated responses that protected 100 and 75% animals from HSV-1- and HSV-2-associated severe disease, respectively. Two of the eight fully protected vaccinees underwent subclinical HSV-2 infection, as demonstrated by deep immunosuppression and other analyses. Finally, vaccination prevented death in 83% of the animals challenged with a HSV-2 dose that killed 78 and 100% naive and mock-vaccinated controls, respectively. Since this FIV vector can accommodate two or more HSV immunogens, this vaccine has ample potential for improvement and may become a candidate for the development of a truly effective vaccine against genital herpes.     

Identification of an HSV-1/HSV-2 cross-reactive T cell determinant.

The results from a number of studies have documented that the HSV glycoprotein gD is an important target for neutralizing antibodies. In contrast, little is known about the Th cell determinants present on HSV that are required for anti HSV gD antibody production. In our study we have immunized BALB/c mice with a recombinant source of HSV-1 gD lacking the carboxyl-terminal 93 amino acids. T cell hybridomas produced from the immunized animals recognized a single antigenic peptide (amino acids 246-261) in the context of I-Ad. The determinant expressed by gD peptide 246-261 was generated and presented by both HSV-1 and HSV-2 infected APC. Fine specificity analysis using truncated synthetic gD peptides revealed that the minimal amino acids recognized by the T hybrids were identical between HSV-1 and HSV-2. In addition, the minimal peptide-I-Ad binding analysis demonstrated that the minimal peptide sequence required for the binding to I-Ad and for T cell recognition contained two prolines. Thus, this important HSV antigenic determinant would not be expected to form an amphipathic alpha-helix and could therefore be missed by algorithms currently used to predict which amino acid sequences would be antigenic based on the propensity to form helices.     

Trichosanthin affects HSV-1 replication in Hep-2 cells

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inhibits the replication of both human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1). The mechanism of inhibition is not clear. This investigation explored the effects of TCS on the stages of HSV-1 infection in Hep-2 cells, from attachment to release. We demonstrated that TCS reduced HSV-1 antigen and DNA content and interfered with viral replication as early as 3-15 h after infection. TCS had no effect on HSV-1 attachment, penetration or immediate-early gene expression. However, the expression of early and late genes and virion release were diminished. In summary, this study demonstrates that TCS primarily affects HSV-1 replication in Hep-2 cells during the early to late infection period.     

HSV-1 enhances GvHR-associated parent anti-F1 alloreactivity in vivo and in vitro

The present studies were performed to demonstrate whether concurrent HSV-1 infection could enhance the immune alterations and dysfunction associated with P----F1-induced graft-versus-host reactions. Examination of phenotypic and functional parameters revealed that Gv-HR-related immune abnormalities in the (C3H.SW X H-2bm1)F1 recipient were dependent on the parental donor inoculum. Together with HSV-1 infection, virus was found to exacerbate the phenotypic changes and functional abnormalities induced in this GvHR model. In addition, the presence of concurrent HSV-1 was shown to augment the level of specific in vivo donor anti-host reactivity present in F1 recipient spleen cells. Moreover, in vitro studies demonstrated that HSV-1 also enhanced the levels of parent anti-F1 allospecific cytotoxic activity. In total, these findings support the hypothesis that viral exacerbation of GvHR is mediated by its enhancement of donor anti-host alloreactive responses.     

In vivo role of nectin-1 in entry of herpes simplex virus type 1 (HSV-1) and HSV-2 through the vaginal mucosa

Herpes simplex virus type 2 (HSV-2) is transmitted through the genital mucosa during sexual encounters. In recent years, HSV-1 has also become commonly associated with primary genital herpes. The mechanism of viral entry of HSV-1 and HSV-2 in the female genital tract is unknown. In order to understand the molecular interactions required for HSV entry into the vaginal epithelium, we examined the expression of herpesvirus entry mediator nectin-1 in the vagina of human and mouse at different stages of their hormonal cycle. Nectin-1 was highly expressed in the epithelium of human vagina throughout the menstrual cycle, whereas the mouse vaginal epithelium expressed nectin-1 only during the stages of the estrous cycle in which mice are susceptible to vaginal HSV infection. Furthermore, the ability of nectin-1 to mediate viral entry following intravaginal inoculation was examined in a mouse model of genital herpes. Vaginal infection with either HSV-1 or HSV-2 was blocked by preincubation of the virus with soluble recombinant nectin-1. Viral entry through the vaginal mucosa was also inhibited by preincubation of HSV-2 with antibody against gD. Together, these results suggest the importance of nectin-1 in mediating viral entry for both HSV-1 and HSV-2 in the genital mucosa in female hosts.     

Expression of HSV-1 glycoproteins in tunicamycin-treated monkey kidney cells

The role of glycosylation in transport and expression of HSV-1 glycoproteins on the surface of HSV-1-infected African green monkey kidney cells was investigated by using tunicamycin (TM). A concentration of 0.05 microgram/ml of TM inhibited the replication of HSV-1 by greater than 99%. Immunoblot analysis of TM-treated and virus-infected cells indicated that 0.05 microgram/ml of TM blocked the addition of N-linked oligosaccharides into glycoproteins B, C and D. An immunofluorescence assay of TM-treated (0.05 and 0.1 microgram/ml) and virus-infected cells demonstrated the presence of nonglycosylated gC, gD and a reduced amount of gB on the surface of infected cells. The results suggest that the addition of N-linked oligosaccharides on the studied HSV-1 glycoproteins was not necessary for their transport and expression on the virus-infected cell surface.     

Antiviral activity of recombinant cyanovirin-N against HSV-1

In this study, a standard strain of HSV-1 (strain SM₄₄) was used to investigate the antiviral activity of the recombinant Cyanovirin-N (CV-N) against Herpes simplex virus type 1 (HSV-1) in vitro and in vivo. Cytopathic effect (CPE) and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells. The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR (FQ-PCR). The results showed that CV-N had a low cytotoxicity on Vero cells with a CC₅₀ of 359.03±0.56 μg/mL, and that it could not directly inactivate HSV-1 infectivity. CV-N not only reduced the CPE of HSV-1 when added before or after viral infection, with a 50% inhibitory concentration (IC₅₀) with 2.26 and 30.16μg/mL respectively, but it also decreased the copies of HSV-1 DNA in infected host cells. The encephalitis model for HSV-1 infection was conducted in Kunming mice, and treated with three dosages of CV-N (0.5, 5 &; 10 mg/kg) which was administered intraperitoneally at 2h, 3d, 5d, 7d post infection. The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination (HE staining). Compared with the untreated control group, in the 5mg/kg CV-N and 10mg/kg CV-N treated groups, the mice suffered light symptoms and the number of survival days were more than 9d and 14d respectively. HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups, the brain cells did not show visible changes, except for a slight inflammation. Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo, and it would be a promising new candidate for anti-HSV therapeutics.     

靶向ICP4的小干扰RNA对HSV-1病毒复制能力的影响

HSV-1是一种嗜神经病毒,能引起一系列神经系统严重症状,然而目前抗HSV-1药物易反弹、不能完全清除潜伏的病毒。ICP4对HSV复制、转录起主要调节作用,决定着溶细胞型感染或潜伏状态的平衡点。为了探寻新的抗病毒策略,本课题以HSV-1ICP4基因为靶点,设计合成2对siRNA,并构建重组真核慢病毒表达质粒pL-KO-puror-hU6-siRNA,通过脂质体转染和嘌呤霉素筛选建立靶向ICP4的4个siRNA单克隆细胞系,Real-timePCR法检测细胞系中ICP4的mRNA表达水平,TCID50法检测siRNA对HSV-1病毒复制能力的影响。结果显示靶向siRNA能有效抑制单克隆细胞系中的ICP4表达,并且抑制ICP4的表达后HSV-1病毒复制能力明显减弱,表明靶向ICP4的siRNA对HSV-1复制有明显抑制作用,且多位点siRNA联合干扰对病毒复制有协同抑制效果,有望应用于生物抗病毒药物的制备。     

Ⅰ型单纯疱疹病毒感染的细胞培养系统的建立

目的:建立稳定的HSV-1感染的细胞培养系统,为HSV-1感染性皮肤病的基础及临床研究提供稳定的平台。方法:以猴肾细胞(Vero)为繁殖细胞,选择人鼻咽癌上皮细胞(Hep-2)为靶细胞,观察HSV-1在Hep-2株中的致细胞病变效应(CPE)。结果:HSV-1在Vero细胞中能稳定、大量地繁殖;HSV-1感染Hep-2细胞后可出现明显的细胞病变;结论:以Vero为繁殖细胞,Hep-2为靶细胞的稳定的细胞培养系统可作为HSV-1感染性皮肤病的基础及临床研究的平台。     

Cell type-dependent requirement of autophagy in HSV-1 antiviral defense

Type I interferons (IFNs) are induced during most viral infections and are considered to be the primary and universal means of innate viral control. However, several other innate mechanisms, including autophagy, have recently been shown to play an important role in antiviral defense. In our recent study, we utilized a herpes simplex virus 1 (HSV-1) infection model to investigate the relationship between cell type and innate antiviral immune mechanisms. Our study demonstrates that dorsal root ganglion (DRG) neurons undergo an innate antiviral response to HSV-1 that differs from the antiviral program induced in mitotic cells in three distinct ways. First, DRG neurons produce less type I IFN and undergo a less effective IFN antiviral program vs. mitotic cells in response to HSV-1 infection. Second, the type I IFN program initiated in DRG neurons induces less cell death than in mitotic cells. Third, in the absence of a robust type I IFN response, DRG neurons, but not mitotic cells, repy on autophagy in HSV-1 defense. Our findings reveal a cell type-specific requirement for autophagy in defense against HSV-1, and offer insight into the cell-appropriate antiviral defense mechanism employed by neurons.     

HSV-1溶瘤病毒的研究进展

1型单纯疱疹病毒（Herpes simplex virustype1,HSV-1）感染效率高且易于通过基因工程改造,己广泛应用于肿瘤治疗研究和临床实验。溶瘤HSV-1可通过基因工程改造或从HSV-1自发突变株中筛选获得。研究证实溶瘤HSV-1能够有效抑杀肿瘤细胞,可通过多种机制靶向肿瘤细胞,溶瘤HSV-1与放化疗联合使用治疗肿瘤的研究也取得了理想的结果。目前,已有多个溶瘤HSV-1进入临床试验。     

Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells

Herpes simplex virus type 1 (HSV-1) has the ability to enter many different hosts and cell types by several strategies. This highly prevalent alphaherpesvirus can enter target cells using different receptors and different pathways: fusion at a neutral pH, low-pH-dependent and low-pH-independent endocytosis. Several cell receptors for viral entry have been described, but several observations suggest that more receptors for HSV-1 might exist. In this work, we propose a novel role for the proteolipid protein (PLP) in HSV-1 entry into the human oligodendrocytic cell line HOG. Cells transfected with PLP-EGFP showed an increase in susceptibility to HSV-1. Furthermore, the infection of HOG and HOG-PLP transfected cells with the R120vGF virus–unable to replicate in ICP4-defficient cells- showed an increase in viral signal in HOG-PLP, suggesting a PLP involvement in viral entry. In addition, a mouse monoclonal antibody against PLP drastically inhibited HSV-1 entry into HOG cells. PLP and virions colocalized in confocal immunofluorescence images, and in electron microscopy images, which suggest that PLP acts at the site of entry into HOG cells. Taken together these results suggest that PLP may be involved in HSV-1 entry in human oligodendrocytic cells.     

The dominant-negative herpes simplex virus type 1 (HSV-1) recombinant CJ83193 can serve as an effective vaccine against wild-type HSV-1 infection in mice

By selectively regulating the expression of the trans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral alpha, beta, and gamma1 genes but little or no gamma2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced by d27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced by d27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.