Intravaginal Zinc Oxide Tetrapod Nanoparticles as Novel Immunoprotective Agents against Genital Herpes.

Virtually all efforts to generate an effective protection against the life-long, recurrent genital infections caused by HSV-2 have failed. Apart from sexual transmission, the virus can also be transmitted from mothers to neonates, and it is a key facilitator of HIV coacquisition. In this article, we uncover a nanoimmunotherapy using specially designed zinc oxide tetrapod nanoparticles (ZOTEN) with engineered oxygen vacancies. We demonstrate that ZOTEN, when used intravaginally as a microbicide, is an effective suppressor of HSV-2 genital infection in female BALB/c mice. The strong HSV-2 trapping ability of ZOTEN significantly reduced the clinical signs of vaginal infection and effectively decreased animal mortality. In parallel, ZOTEN promoted the presentation of bound HSV-2 virions to mucosal APCs, enhancing T cell-mediated and Ab-mediated responses to the infection, and thereby suppressing a reinfection. We also found that ZOTEN exhibits strong adjuvant-like properties, which is highly comparable with alum, a commonly used adjuvant. Overall, to our knowledge, our study provides the very first evidence for the protective efficacy of an intravaginal microbicide/vaccine or microbivac platform against primary and secondary female genital herpes infections.

Heparanase is a host enzyme required for herpes simplex virus-1 release from cells.

Herpesviruses exemplified by herpes simplex virus-1 (HSV-1) attach to cell surface heparan sulfate (HS) for entry into host cells. However, during a productive infection, the HS moieties on parent cells can trap newly exiting viral progenies and inhibit their release. Here we demonstrate that a HS-degrading enzyme of the host, heparanase (HPSE), is upregulated through NF-kB and translocated to the cell surface upon HSV-1 infection for the removal of HS to facilitate viral release. We also find a significant increase in HPSE release in vivo during infection of murine corneas and that knockdown of HPSE in vivo inhibits virus shedding. Overall, we propose that HPSE acts as a molecular switch for turning a virus-permissive 'attachment mode' of host cells to a virus-deterring 'detachment mode'. Since many human viruses use HS as an attachment receptor, the HPSE-HS interplay may delineate a common mechanism for virus release.

Zebrafish 3-O-sulfotransferase-4 generated heparan sulfate mediates HSV-1 entry and spread.

Rare modification of heparan sulfate (HS) by glucosaminyl 3-O sulfotransferase (3-OST) isoforms generates an entry receptor for herpes simplex virus type-1 (HSV-1). In the zebrafish (ZF) model multiple 3-OST isoforms are differentially expressed. One such isoform is 3-OST-4 which is widely expressed in the central nervous system of ZF. In this report we characterize the role of ZF encoded 3-OST-4 isoform for HSV-1 entry. Expression of ZF 3-OST-4 into resistant Chinese hamster ovary (CHO-K1) cells promoted susceptibility to HSV-1 infection. This entry was 3-O sulfated HS (3-OS HS) dependent as pre-treatment of ZF 3-OST-4 cells with enzyme HS lyases (heparinase II/III) significantly reduced HSV-1 entry. Interestingly, co-expression of ZF 3-OST-4 along with ZF 3-OST-2 which is also expressed in brain rendered cells more susceptible to HSV-1 than 3-OST-4 alone. The role of ZF-3-OST-4 in the spread of HSV-1 was also evaluated as CHO-K1 cells that expressed HSV-1 glycoproteins fused with ZF 3-OST-4 expressing effector CHO-K1 cells. Finally, adding further evidence ZF 3-OST-4 mediated HSV-1 entry was inhibited by anti-3O HS G2 peptide. Taken together our results demonstrate a role for ZF 3-OST-4 in HSV-1 pathogenesis and support the use of ZF as a model to study it.

Inhibition of myosin light chain kinase can be targeted for the development of new therapies against herpes simplex virus type-1 infection.

BACKGROUND: Herpes simplex virus type-1 (HSV-1) is the leading cause of infectious blindness worldwide. Through a multistep process, HSV-1 enters into naturally susceptible human corneal epithelial (HCE) cells where it establishes an optimal environment for viral replication and spread. HSV-1 employment of cytoskeletal proteins, kinases, and cell signalling pathways is crucial for the entry process. METHODS: Here we demonstrate that non-muscle myosin IIA (NM-IIA) and/or a myosin activating kinase, myosin light chain kinase (MLCK), can be targeted for the development of new and effective therapies against HSV-1. HCE cells were incubated with MLCK inhibitors ML-7 and ML-9 and NM-IIA inhibitor blebbistatin. Following the application of inhibitors, HSV-1 entry and spread to neighbouring HCE cells was evaluated. RESULTS: Upon application of MLCK inhibitors ML-7 and ML-9 and NM-IIA inhibitor blebbistatin, HSV-1 entry into HCE cells was significantly decreased. Furthermore, dramatic impairment of glycoprotein-mediated membrane fusion was seen in cells treated with MLCK inhibitors, thus establishing a role for MLCK activation in cell-to-cell fusion and multinucleated syncytial cell formation. These results also indicate that the activation of motor protein NM-IIA by MLCK is crucial for cytoskeletal changes required for HSV-1 infection of corneal cells. CONCLUSIONS: We provide new evidence that NM-IIA and MLCK can be used as effective antiviral targets against ocular herpes.

An investigative peptide-acyclovir combination to control herpes simplex virus type 1 ocular infection.

PURPOSE: To investigate the efficacy of a combination treatment composed of the cationic, membrane-penetrating peptide G2, and acyclovir (ACV) in both in vitro and ex vivo models of herpes simplex virus 1 (HSV-1) ocular infection. METHODS: The antiviral activity of a combined G2 peptide and ACV therapy (G2-ACV) was assessed in various treatment models. Viral entry, spread, and plaque assays were performed in vitro to assess the prophylactic efficacy of G2, G2-ACV, and ACV treatments. In the ex vivo model of HSV-1 infection, the level of viral inhibition was also compared among the three treatment groups via Western blot analysis and immunohistochemistry. The potential change in expression of the target receptor for G2 was also assessed using immunohistochemistry and RT-PCR. RESULTS: Statistically significant effects against HSV-1 infection were seen in all treatment groups in the viral entry, spread, and plaque assays. The greatest effects against HSV-1 infection in vitro were seen in the G2-ACV group. In the ex vivo model, statistically significant anti-HSV-1 effects were also noted in all control groups. At 24 hours, the greatest inhibitory effect against HSV-1 infection was seen in the ACV group. At 48 hours, however, the G2-ACV-treated group demonstrated the greatest antiviral activity. Syndecan-1, a target of G2, was found to be upregulated at 12-hours postinfection. CONCLUSIONS: This study shows that G2-ACV may be an effective antiviral against HSV-1 (KOS) strain when applied as single prophylactic applications with or without continuous doses postinfection.

Glycoprotein targeted therapeutics: a new era of anti-herpes simplex virus-1 therapeutics.

Herpes simplex virus type-1 (HSV-1) is among the most common human pathogens worldwide. Its entry into host cells is an intricate process that relies heavily on the ability of the viral glycoproteins to bind host cellular proteins and to efficiently mediate fusion of the virus envelope with the cell membrane. Acquisition of HSV-1 results in a lifelong latent infection. Because of the cycles of reactivation from a latent state, much emphasis has been placed on the management of infection through the use of DNA synthesis inhibitors. However, new methods are needed to provide more effective treatment at earlier phases of the viral infection and to prevent the development of drug resistance by the virus. This review outlines the infection process and the common therapeutics currently used against the fundamental stages of HSV-1 replication and fusion. The remainder of this article will focus on a new approach for HSV-1 infection control and management, the concept of glycoprotein-receptor targeting.

Zebrafish encoded 3-O-sulfotransferase-2 generated heparan sulfate serves as a receptor during HSV-1 entry and spread.

Previously we reported the role of zebrafish (ZF) encoded glucosaminyl 3-O-sulfotransferase-3 (3-OST-3) isoform in assisting herpes simplex virus type-1 (HSV-1) entry and spread by generating an entry receptor to HSV-1 envelope glycoprotein D (gD). However, the ability of ZF encoded 3-OST-2 isoform to participate in HSV-1 entry has not been determined although it is predominantly expressed in ZF brain, a prime target for HSV-1 to infect and establish lifelong latency. Here we report the expression cloning of ZF encoded 3-OST-2 isoform and demonstrate HSV-1 entry into resistant Chinese hamster ovary (CHO-K1) cells expressing the clone. Additional significance of ZF encoded 3-OST-2 receptor was demonstrated using medically important isolates of HSV-1. In addition, interference to HSV-1 entry was observed upon co-expression of HSV-1 gD and ZF 3-OST-2. Similarly HSV-1 entry was significantly inhibited by the pre-treatment of cells with enzyme HS lyases (heparinase II/III). Finally, ZF-3-OST-2 expressing CHO-K1 was able to fuse with HSV-1 glycoprotein expressing cells suggesting their role in HSV-1 spread. Taken together our result demonstrates a role for ZF 3-OST-2 in HSV-1 pathogenesis.

Tin oxide nanowires suppress herpes simplex virus-1 entry and cell-to-cell membrane fusion.

The advent of nanotechnology has ushered in the use of modified nanoparticles as potential antiviral agents against diseases such as herpes simplex virus 1 and 2 (HSV-1) (HSV-2), human immunodeficiency virus (HIV), monkeypox virus, and hepatitis B virus. Here we describe the application of tin oxide (SnO(2)) nanowires as an effective treatment against HSV-1 infection. SnO(2) nanowires work as a carrier of negatively charged structures that compete with HSV-1 attachment to cell bound heparan sulfate (HS), therefore inhibiting entry and subsequent cell-to-cell spread. This promising new approach can be developed into a novel form of broad-spectrum antiviral therapy especially since HS has been shown to serve as a cellular co-receptor for a number of other viruses as well, including the respiratory syncytial virus, adeno-associated virus type 2, and human papilloma virus.

Prophylactic, therapeutic and neutralizing effects of zinc oxide tetrapod structures against herpes simplex virus type-2 infection.

The attachment of Herpes simplex virus type-2 (HSV-2) to a target cell requires ionic interactions between negatively charged cell surface co-receptor heparan sulfate (HS) and positively charged residues on viral envelop glycoproteins, gB and gC. Effective blocking of this first step of HSV-2 pathogenesis demonstrates significant prophylactic effects against the viral disease; any in vitro therapeutic effects of blocking this interaction, however, are not clear. Here, we provide new evidence that zinc oxide tetrapod micro-nanostructures synthesized by flame transport approach significantly block HSV-2 entry into target cells and, in addition, demonstrate the potential to stop the spread of the virus among already infected cells. The zinc oxide tetrapods (ZnOTs) also exhibit the ability to neutralize HSV-2 virions. Natural target cells such as human vaginal epithelial and HeLa cells showed highly reduced infectivity when infected with HSV-2 virions that were pre-incubated with the ZnOTs. The mechanism behind the ability of ZnOTs to prevent, neutralize or reduce HSV-2 infection relies on their ability to bind the HSV-2 virions. We used fluorescently labeled ZnOTs and GFP-expressing HSV-2 virions to demonstrate the binding of the ZnOTs with HSV-2. We also show that the binding and hence, the antiviral effects of ZnOTs can be enhanced by illuminating the ZnOTs with UV light. Our results provide new insights into the anti-HSV-2 effects of ZnOT and rationalize their development as a HSV-2 trapping agent for the prevention and/or treatment of infection. The observed results also demonstrate that blocking HSV-2 attachment can have prophylactic as well as therapeutic applications.