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.     

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。     

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.     

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。     

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.     

Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.     

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.     

携带HIV-1抗原的单纯疱疹病毒载体疫苗的构建及鉴定

利用细菌人工染色体技术将串联的HIV-1 gp160、gag和protease基因以及表达元件插入1型单纯疱疹病毒(Herpes simplex virus type 1,HSV-1)内部反向重复序列区,以获得携带HIV-1抗原的单纯疱疹病毒载体疫苗。首先将HIV-1 gp160(B型和C型)、gag和protease基因串联克隆入pc DNA3获得重组质粒pc DNA/g Bgp和pc DNA/g Cgp,重组质粒转染293FT细胞,Western blotting检测HIV抗原表达。继而将pc DNA/g Bgp和pc DNA/g Cgp中包括HIV-1抗原基因和表达元件的表达框克隆入p KO5/BN获得重组穿梭质粒p KO5/BN/g Bgp和p KO5/BN/g Cgp,穿梭质粒电转含BAC-HSV的大肠杆菌,筛选重组菌,提取重组DNA并转染Vero细胞,挑取病毒蚀斑纯化重组病毒,Southern blotting鉴定重组病毒DNA,Western blotting检测重组病毒感染细胞中HIV抗原表达,并分析病毒的增殖特性。结果表明,Western blotting在pc DNA/g Bgp和pc DNA/g Cgp转染的293FT细胞中检测到表达的gp160和gag蛋白。p KO5/BN/g Bgp和p KO5/BN/g Cgp分别电转获得重组菌,并从重组DNA转染的Vero细胞中纯化获得重组HSV,Southern blotting检测表明重组HSV基因组发生特异性重组,重组病毒感染细胞中检测到gp120和gp41,且重组HSV保留了在哺乳动物细胞中的复制能力。本研究获得携载HIV-1 gp160、gag和protease基因的重组HSV,并保留了在哺乳动物细胞中的复制能力,可作为HIV-1复制型病毒载体疫苗。     

Comparison of the host immune response to herpes simplex virus 1 (HSV-1) and HSV-2 at two different mucosal sites

A study was undertaken to compare the host immune responses to herpes simplex virus 1 (HSV-1) and HSV-2 infection by the ocular or genital route in mice. Titers of HSV-2 from tissue samples were elevated regardless of the route of infection. The elevation in titers of HSV-2, including cell infiltration and cytokine/chemokine levels in the central nervous system relative to those found following HSV-1 infection, was correlative with inflammation. These results underscore a dichotomy between the host immune responses to closely related alphaherpesviruses.     

Using HSV-1 Genome Phylogenetics to Track Past Human Migrations

We compared 31 complete and nearly complete globally derived HSV-1 genomic sequences using HSV-2 HG52 as an outgroup to investigate their phylogenetic relationships and look for evidence of recombination. The sequences were retrieved from NCBI and were then aligned using Clustal W. The generation of a maximum likelihood tree resulted in a six clade structure that corresponded with the timing and routes of past human migration. The East African derived viruses contained the greatest amount of genetic diversity and formed four of the six clades. The East Asian and European/North American derived viruses formed separate clades. HSV-1 strains E07, E22 and E03 were highly divergent and may each represent an individual clade. Possible recombination was analyzed by partitioning the alignment into 5 kb segments, performing individual phylogenetic analysis on each partition and generating a.phylogenetic network from the results. However most evidence for recombination spread at the base of the tree suggesting that recombination did not significantly disrupt the clade structure. Examination of previous estimates of HSV-1 mutation rates in conjunction with the phylogenetic data presented here, suggests that the substitution rate for HSV-1 is approximately 1.38×10⁻⁷ subs/site/year. In conclusion, this study expands the previously described HSV-1 three clade phylogenetic structures to a minimum of six and shows that the clade structure also mirrors global human migrations. Given that HSV-1 has co-evolved with its host, sequencing HSV-1 isolated from various populations could serve as a surrogate biomarker to study human population structure and migration patterns.     

NKT cells are not critical for HSV-1 disease resolution

NKT cells are a minor subset of T cells that have important roles in controlling immune responses in disease states including cancer, autoimmunity and pathogenic infections. In contrast to conventional T cells, NKT cells express an invariant TCR and respond to glycolipids presented by CD1d. In this study, we sought to investigate the role of NKT cells in regulating the response to infection with HSV-1, and the mechanism involved, in well-established mouse models. Previous studies of HSV-1 disease in mice have shown clear roles for CD4+ and CD8+ T cells. The role of NKT cells in the resolution of HSV-1 (KOS strain) infection was investigated through flank zosteriform or footpad infection in wild-type versus CD1d-deficient mice, by measurement of viral plaque-forming units at different sites after infection, lesion severity and HSV-1-specific T-cell responses. In contrast to a previous study using a more virulent strain of HSV-1 (SC16 strain), no differences were observed in disease magnitude or resolution, and furthermore, the T-cell response to HSV-1 (KOS strain) was unaltered in the absence of NKT cells. In conclusion, this study shows that NKT cells do not play a general role in controlling the resolution or severity of HSV-1 infection. Instead, the resolution or severity of the infection may depend on the HSV-1 strain under investigation.     

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.     

几种细胞因子对HSV─1感染单核巨噬细胞的影响

以ＨＳＶ─１接种人单核细胞系Ｕ＿（９３７）和小鼠腹腔巨噬细胞,并在接种前后分别以不同的细胞因子处理。经病毒滴定、免疫荧光试验检测病毒抗原及多聚酶链反应技术检测病毒基因,研究了细胞因子对ＨＳＶ─１感染单核巨噬细胞的影响。结果证实,ＴＮＦ─α５０ｕ／ｍＬ　、Ｍ─ＣＳＦ２００ｕ／ｍＬＩＦＮ─γ１０００ｕ／ｍＬ、ＩＦＮ－α１０００ｕ／ｍＬ均能增强单核巨噬细胞对ＨＳＶ─１的抗性,加速细胞对ＨＳＶ─１ＤＮＡ的清除,抑制病毒抗原的表达;并能拮抗ＬＰＳ对ＨＳＶ─１在单核巨噬细胞中表达的促进作用。