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-1 Virions Engineered for Specific Binding to Cell Surface Receptors

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In Vivo Administration of Replication-Deficient Mutant HSV-1 Targets Professional APCs and Induces Efficient CD4+ T Helper Responses

Both neutralizing antibodies and cytotoxic T cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. These findings have critical implications in the design of vaccination strategies aimed at triggering and sustaining antigen specific CD4+ in addition to CD8+ effector immune responses. Here we show that a recombinant replication-deficient HSV-1 vector encoding the HIV-1 matrix protein p17 (T0-p17) is capable to infect professional APCs in vitro and in vivo without interfering with the endogenous MHC class II processing of the transgene encoded antigen. Moreover, we show that injection of T0-p17 in the mouse dermis generates a strong p17specific CD4+ T helper response preceding both cytotoxic and humoral responses. Importantly, T0-p17 infected peritoneal macrophages were capable to trigger a longlasting expansion of p17-specific CD4+ T cells in vitro. Because of their capability to infect professional APCs without interfering with their biological functions, replication-deficient HSV vectors are appealing candidates for the development of vaccines able to trigger strong T helper responses. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 200

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

Herpesvirus/retrovirus chimeric vectors

Over the years, the design of HSV-1 based vectors has developed from different types of replicative-defective and replication-conditioned recombinant viruses to plasmid based amplicon vectors. More recently hybrid or chimeric vectors have incorporated desired elements of different viruses to increase the efficacy of gene delivery in vitro and in vivo. Amongst different systems, herpesvirus/retrovirus chimeras take advantage of the features of the HSV-1 vectors to efficiently transduce large amounts of foreign genetic sequences, remaining episomal, to allow production of recombinant retrovirus vectors able to stably integrate into the cellular genome. This review will focus on three different groups of herpesvirus/retrovirus chimeric vectors aimed to: generate retrovirus particles in cells tranduced with HSV-1 amplicon vectors; express a limited set of retrovirus genes for vaccine purposes; and express herpesvirus/retrovirus chimeric proteins to study cellular targeting signal and improve their biological effect

Capitolo 7. Rapporti microrganismi/ ospite ( A. Rosato, R. Manservigi, P. Grisoli)

L'interesse dell’uomo verso i microrganismi è nato con la scoperta del loro coinvolgimento nelle malattie infettive e si è inizialmente focalizzato su quelli definiti patogeni. Con il tempo, si è reso evidente che la suddivisione in patogeni e non patogeni è inadeguata, non è possibile, cioè, operare una netta separazione in queste due distinte categorie. In realtà, l’incontro con un microrganismo, anche quando esso è definibile come patogeno, non equivale sempre a malattia; la malattia, la sua gravità e l’esito sono, infatti, il risultato del complesso e dinamico rapporto che si stabilisce tra microbo e ospite uomo

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

The C-terminal cytoplasmic tail of herpes simplex virus type 1 gE protein is phosphorylated in vivo and in vitro by cellular enzymes in the absence of other viral proteins

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