Role of Herpes Simplex Envelope Glycoprotein B and Toll-Like Receptor 2 in Ocular Inflammation: An ex vivo Organotypic Rabbit Corneal Model.

It has been recently reported, using in vitro studies, that the herpes simplex virus 1 (HSV-1) encoded envelope glycoprotein B (gB1) interacts with cell surface toll-like receptor 2 (TLR2) and induces the secretion of interleukin-8 (IL8), a representative marker of inflammatory cytokine activation. The purpose of this study is to investigate the role of gB1 in activating host inflammatory responses by using a secreted form of gB1 (gB1s) and an ex vivo organotypic rabbit corneal model. Abraded corneas exposed to gB1s alone or to the recombinant protein mixed with anti gB polyclonal antibody were cultured in an air-liquid interface. The corneas exposed to gB1s show the appearance of mydriasis and high levels of TLR2 and IL-8 mRNAs transcripts were detected in the superficial layer of corneal epithelial cells. Histological stain and immunohistochemical analyses revealed morphological changes in the epithelium of the treated corneas and variations in expression and localization of TLR2. Collectively these findings provide new insight into the pathogenesis of HSV-1 ocular infection by demonstrating the leading role of gB in activating an inflammatory response and in the appearance of mydriasis, a sign of HSV-1 anterior uveitis.

Intranasal immunization in mice with non-ionic surfactants vesicles containing HSV immunogens: a preliminary study as possible vaccine against genital herpes.

The purpose of this study was to investigate the potential of intranasal immunization with non-ionic surfactant vesicles (NISV) containing either the secretory recombinant form of glycoprotein B (gBs) of herpes simplex virus type 1 or a related polylysine reach peptides (DTK) for induction of protective immunity against genital herpes infection in mice. NISV were prepared by lipid film hydration method. The mean diameter of vesicles was around 390 nm for DTK-containing NISV (DTK-NISV) and 320 nm for gB1s-containing NISV (gB1s-NISV). The encapsulation efficiency of the molecules was comprised between 57% and 70%. After 7-14 day NISV maintained stable dimensions and a drug encapsulation higher than 48%. We showed that intranasal immunization with gB1s-NISV induces gB-specific IgG antibody and lymphoproliferative responses, whereas vaccination with DTK-NISV was not able to generate a gB-specific immune response. Our results indicate that vaccination of BALB/c mice with gB1s-NISV induced Th1 responses, as characterized by increased titre of IG2a in plasma and IFN-production in CD4+ splenic cells. Intranasal immunization with gB1s-NISV could elicit 90% (almost complete) protection against a heterologous lethal vaginal challenge with herpes simplex virus type 2. These data may have implications for the development of a mucosal vaccine against genital herpes.

Characterization of herpes simplex virus 1 strains as platforms for the development of oncolytic viruses against liver cancer.

BACKGROUND: Diverse oncolytic viruses (OV) are being designed for the treatment of cancer. The characteristics of the parental virus strains may influence the properties of these agents. AIMS: To characterize two herpes simplex virus 1 strains (HSV-1 17syn(+) and HFEM) as platforms for virotherapy against liver cancer. METHODS: The luciferase reporter gene was introduced in the intergenic region 20 locus of both HSV-1 strains, giving rise to the Cgal-Luc and H6-Luc viruses. Their properties were studied in hepatocellular carcinoma (HCC) cells in vitro. Biodistribution was monitored by bioluminescence imaging (BLI) in athymic mice and immune-competent Balb/c mice. Immunogenicity was studied by MHC-tetramer staining, in vivo killing assays and determination of specific antibody production. Intratumoural transgene expression and oncolytic effect were studied in HuH-7 xenografts. RESULTS: The H6-Luc virus displayed a syncytial phenotype and showed higher cytolytic effect on some HCC cells. Upon intravenous or intrahepatic injection in mice, both viruses showed a transient transduction of the liver with rapid relocalization of bioluminescence in adrenal glands, spinal cord, uterus and ovaries. No significant differences were observed in the immunogenicity of these viruses. Local intratumoural administration caused progressive increase in transgene expression during the first 5 days and persisted for at least 2 weeks. H6-Luc achieved faster amplification of transgene expression and stronger inhibition of tumour growth than Cgal-Luc, although toxicity of these non-attenuated viruses should be reduced to obtain a therapeutic effect. CONCLUSIONS: The syncytial H6-Luc virus has a strong oncolytic potential on human HCC xenografts and could be the basis for potent OV.

APP processing induced by herpes simplex virus type 1 (HSV-1) yields several APP fragments in human and rat neuronal cells.

Lifelong latent infections of the trigeminal ganglion by the neurotropic herpes simplex virus type 1 (HSV-1) are characterized by periodic reactivation. During these episodes, newly produced virions may also reach the central nervous system (CNS), causing productive but generally asymptomatic infections. Epidemiological and experimental findings suggest that HSV-1 might contribute to the pathogenesis of Alzheimer's disease (AD). This multifactorial neurodegenerative disorder is related to an overproduction of amyloid beta (Aß) and other neurotoxic peptides, which occurs during amyloidogenic endoproteolytic processing of the transmembrane amyloid precursor protein (APP). The aim of our study was to identify the effects of productive HSV-1 infection on APP processing in neuronal cells. We found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons is followed by multiple cleavages of APP, which result in the intra- and/or extra-cellular accumulation of various neurotoxic species. These include: i) APP fragments (APP-Fs) of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aß; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aß(1-40) and Aß(1-42). Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aß oligomer. The multiple cleavages of APP that occur in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell ß-secretase, γ-secretase, and caspase-3-like enzymes. These findings demonstrate that HSV-1 infection of neuronal cells can generate multiple APP fragments with well-documented neurotoxic potentials. It is tempting to speculate that intra- and extracellular accumulation of these species in the CNS resulting from repeated HSV-1 reactivation could, in the presence of other risk factors, play a co-factorial role in the development of AD.

HSV Recombinant Vectors for Gene Therapy.

The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges.

HSV as a vector in vaccine development and gene therapy.

The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), major human pathogen whose lifestyle is based on a long-term dual interaction with the infected host characterized by the existence of lytic and latent infections, has allowed the development of potential vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous system, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases and targeted infection of specific tissues or organs. Three different classes of vectors can be derived from HSV-1: replication-competent attenuated vectors, replication-incompetent recombinant vectors and defective helper-dependent vectors known as amplicons. This chapter highlights the current knowledge concerning design, construction and recent applications, as well as the potential and current limitations of the three different classes of HSV-1-based vectors.

Inhibition of herpes simplex virus types 1 and 2 in vitro infection by sulfated derivatives of Escherichia coli K5 polysaccharide.

Herpes simplex virus type 1 (HSV-1) and HSV-2 are neurotropic viruses and common human pathogens causing major public health problems such as genital herpes, a sexually transmitted disease also correlated with increased transmission and replication of human immunodeficiency virus type 1 (HIV-1). Therefore, compounds capable of blocking HIV-1, HSV-1, and HSV-2 transmission represent candidate microbicides with a potential added value over that of molecules acting selectively against either infection. We report here that sulfated derivatives of the Escherichia coli K5 polysaccharide, structurally highly similar to heparin and previously shown to inhibit HIV-1 entry and replication in vitro, also exert suppressive activities against both HSV-1 and HSV-2 infections. In particular, the N,O-sulfated [K5-N,OS(H)] and O-sulfated epimerized [Epi-K5-OS(H)] forms inhibited the infection of Vero cells by HSV-1 and -2, with 50% inhibitory concentrations (IC(50)) between 3 +/- 0.05 and 48 +/- 27 nM, and were not toxic to the cells at concentrations as high as 5 muM. These compounds impaired the early steps of HSV-1 and HSV-2 virion attachment and entry into host cells and reduced the cell-to-cell spread of HSV-2. Since K5-N,OS(H) and Epi-K5-OS(H) also inhibit HIV-1 infection, they may represent valid candidates for development as topical microbicides preventing sexual transmission of HIV-1, HSV-1, and HSV-2.

HSV as a vector in vaccine development and gene therapy.

The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), major human pathogen whose lifestyle is based on a long-term dual interaction with the infected host characterized by the existence of lytic and latent infections, has allowed the development of potential vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous system, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases and targeted infection of specific tissues or organs. Three different classes of vectors can be derived from HSV-1: replication-competent attenuated vectors, replication-incompetent recombinant vectors and defective helper-dependent vectors known as amplicons. This chapter highlights the current knowledge concerning design, construction and recent applications, as well as the potential and current limitations of the three different classes of HSV-1-based vectors.

HSV-1-derived recombinant and amplicon vectors for gene transfer and gene therapy.

Herpes simplex virus type 1 (HSV-1) is a major human pathogen whose lifestyle is based on a long-term dual interaction with the infected host characterized by the existence of lytic and latent infections. Although in most cell types infection with HSV-1 will induce toxic effects ending in the death of the infected cells, the very deep knowledge we possess on the genetics and molecular biology of HSV-1 has permitted the deletion of most toxic genes and the development of non-pathogenic HSV-1-based vectors for gene transfer. Several unique features of HSV-1 make vectors derived from this virus very appealing for preventive or therapeutic gene transfer. These include (i) the very high transgenic capacity of the virus particle, authorizing to convey very large pieces of foreign DNA to the nucleus of mammalian cells, (ii) the genetic complexity of the virus genome, allowing to generate many different types of attenuated vectors possessing oncolytic activity, and (iii) the ability of HSV-1 vectors to invade and establish lifelong non-toxic latent infections in neurons from sensory ganglia and probably in other neurons as well, from where transgenes can be strongly and long-term expressed. Three different classes of vectors can be derived from HSV-1: replication-competent attenuated vectors, replication-incompetent recombinant vectors, and defective helper-dependent vectors known as amplicons. Each of these different vectors attempts to exploit one or more of the above-mentioned features of HSV-1. In this review we will update the current know-how concerning design, construction, and recent applications, as well as the potential and current limitations of the three different classes of HSV-1-based vectors.

Immunotherapeutic activity of a recombinant combined gB-gD-gE vaccine against recurrent HSV-2 infections in a guinea pig model.

The guinea pig model of recurrent genital herpes simplex virus type 2 (HSV-2) infection was used to test the immunotherapeutic activity of a glycoprotein subunit vaccine. Vaccine formulation consisted of three recombinant herpes simplex virus (HSV) glycoproteins, namely gB1s, gD2t and gE1t, plus aluminium hydroxide [Al(OH)3)] adjuvant. One month after viral challenge, infected animals were therapeutically immunised by seven subcutaneous injections of a low dose of antigens with a weekly interval for the first five and a fortnightly interval for the last two administrations. Results showed that the treatment was highly effective in ameliorating the recidivist pathology of animals, suggesting that this kind of vaccine formulation and administration may be helpful for therapeutic intervention in humans affected by recurrent herpes infections.

Specific targeted binding of herpes simplex virus type 1 to hepatocytes via the human hepatitis B virus preS1 peptide.

To improve the utility of herpes simplex virus type 1 (HSV-1) vectors for gene therapy, the viral envelope needs to be manipulated to achieve cell-specific gene delivery. In this report, we have engineered an HSV-1 mutant virus, KgBpK(-) gC(-), deleted for the glycoprotein C (gC) and the heparan sulfate-binding domain (pK) of gB, in order to express gC:preS1 and gC:preS1 active peptide (preS1ap) fusion molecules. PreS1, and a 27 amino acid active peptide inside preS1 (preS1ap), are supposed to be the molecules that the human hepatitis B virus (HBV) needs to bind specifically to hepatocytes. Biochemical analysis demonstrated that the gC:preS1ap fusion molecule was expressed and incorporated into the envelope of the recombinant HSV-1 virus KgBpK(-)gC:preS1ap. Moreover, KgBpK(-)gC:preS1ap recombinant virus gained a specific binding activity to an hepatoblastoma cell line (HepG2) with a consequent productive infection. In addition, anti-preS1-specific antibodies were shown to neutralize recombinant virus infectivity, and a synthetic preS1ap peptide was able to elute KgBpK(-)gC:preS1ap virus bound on HpeG2 cells. These data provide further evidence that HSV-1 can productively infect cells through a specific binding to a non-HSV-1 receptor. Furthermore, these data strongly support the hypothesis that the HBV preS1ap molecule is an HBV ligand to hepatocytes.