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

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 (Aβ) 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 Aβ; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aβ1-40 and Aβ1-42. Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aβ 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 β-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

Use of herpes simplex virus type 1-based amplicon vector for delivery of small interfering RNA

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. The use of DNA-based plasmid vectors to achieve transient and stable expression of siRNA has been developed to avoid the problems of double-stranded oligonucleotides transfection. These vectors direct the transcription of small hairpin RNAs (shRNAs) from a polymerase-III (H1 or U6)-RNA gene promoter. However, numerous disadvantages remain, including low transfection efficiency and difficulty in transfecting primary cells. To overcome some of these problems, the use of viral vectors for siRNA delivery has been described. Retroviral, adenoviral, adeno-associated and herpes viral shRNAs delivery systems have been successfully used to silence genes, in vitro and in vivo. The use of a herpes simplex virus type 1 (HSV-1)-based amplicon vector for siRNA delivery into mammalian cells, using human polyomavirus BK (BKV)-transformed cells as a model system is described. The results demonstrate the ability of amplicon vectors to inhibit the expression of BKV T-Ag and tumorigenicity of BKV-transformed cells. We show that the use of the amplicon vector is highly efficient for the delivery of siRNA molecules. The unique ability of these vectors to deliver multiple copies of siRNA may provide a useful tool in the development of novel anticancer therapy

Poster: Herpes simplex vaccine vector co-expressing fused Mycobacterium Tuberculosis immunogens and Tat adjuvant enhances cellular and humoral responses in Balb/c mice

Tuberculosis (TB) caused by the intracellular Mycobacterium Tuberculosis (Mtb), is one of the major health problems in the world and responsible for about 2 million deaths annually. Currently Bacillus Calmette-Guerin (BCG) is the only TB vaccine available for humans. However BCG is the most controversial vaccine in use and its protective efficacy against adult TB is insufficient. To date several types of TB vaccines such as genetic modified BCG, protein-adjuvant combination, plasmid DNA and viral-based vaccines have been made and used in prime and boost strategies. In particular recombinant viral vectors expressing adjuvant molecules and Mtb fusion proteins that can generate a broader cellular and humoral response are highly promising candidates. The main goal of our study was to explore and develop new efficacious and safe Herpes simplex type 1 (HSV-1) vector based vaccines against TB. For this purpose, we have constructed viral vectors (HSVTB5Ag) that contain a gene cassette that express a fusion protein formed by 5 Mycobacterium tuberculosis antigens (TB5Ag: Ag85B, ESAT-6, Mpt 64/63/83) that have been reported to act as efficient activators of the protective immune-responses against Mtb. In order to modulate and increase Th1 and/or Th2 responses against a broader range of the Mtb fusion protein epitopes we construct an HSV vector co-expressing TB5Ag and HIV-1 Tat protein as immune-modulator (HSVTB5Ag/Tat). At this regard we have previously reported the capacity of Tat to reveal new epitopes and to induce protective cellular immune responses against HSV1 lethal infection in a mice model. HSV-TB5Ag and HSV-TB5Ag/Tat vectors have been used to immunize Balb/C mice with different protocols of immunization. The group of mice vaccinated with the vector co-expressing TB5Ag and HIV-1 Tat, compared with mice immunized with the vector expressing only TB5Ag, have shown a broader epitopes specific T cellular and humoral responses. These data strengthen the hypothesis that Tat acts as a potent adjuvant since modulates Th1 and Th2 epitopes hierarchy. The results from the in vivo experiments indicate that HSV-TB5Ag/Tat vector is a potential candidate vaccine against Mtb for further studies on TB protectio

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 (IC50) between 3 ± 0.05 and 48 ± 27 nM, and were not toxic to the cells at concentrations as high as 5 μM. 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