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

The Tat Protein of HIV-1 Prevents the Loss of HSV-Specific Memory Adaptive Responses and Favors the Control of Viral Reactivation

The development of therapeutic strategies to control the reactivation of the Herpes Simplex Virus (HSV) is an unaddressed priority. In this study, we evaluated whether Tat, a HIV-1 protein displaying adjuvant functions, could improve previously established HSV-specific memory responses and prevent viral reactivation. To this aim, mice were infected with non-lethal doses of HSV-1 and, 44 days later, injected or not with Tat. Mice were then monitored to check their health status and measure memory HSV-specific cellular and humoral responses. The appearance of symptoms associated with HSV-reactivation was observed at significantly higher frequencies in the control group than in the Tat-treated mice. In addition, the control animals experienced a time-dependent decrease in HSV-specific Immunoglobulin G (IgG), while the Tat-treated mice maintained antibody titers over time. IgG levels were directly correlated with the number of HSV-specific CD8+ T cells, suggesting an effect of Tat on both arms of the adaptive immunity. Consistent with the maintenance of HSV-specific immune memory, Tat-treated mice showed a better control of HSV-1 re-infection. Although further studies are necessary to assess whether similar effects are observed in other models, these results indicate that Tat exerts a therapeutic effect against latent HSV-1 infection and re-infection by favoring the maintenance of adaptive immunity

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

Development and assessment of herpes simplex virus type 1 (HSV-1) amplicon vectors with sensory neuron-selective promoters

Background: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. Methods: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters’ candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. Results: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. Conclusions: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for ND

Magnolia officinalis L. bark extract and respiratory diseases: From traditional Chinese medicine to western medicine via network target

The understanding of the use of Magnolia officinalis L. (Magnoliaceae) as a possible dietary supplement for supporting the treatment of airway pathologies might be of clinical interest. Two commercially available bark extracts (M. officinalis extract [MOE]) were characterized by quantitation in honokiol and magnolol content by means of high-performance liquid chromatography with UV detection. MOE effects, as well as those of the reference compounds per se, on some targets connected to airway pathologies (antibacterial- and lung and trachea relaxing- activities) were investigated. Results showed that MOE possessed interesting antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae. This was accompanied by a spasmolytic and antispasmodic activity, possibly owing to its ability to concurrently modulate different targets such as H-1-, beta(2)- and muscarinic receptors and l-type calcium channels involved in bronchodilation. All these effects were directly related to the MOE content in honokiol and magnolol. In conclusion, the properties of MOE highlighted here strongly encourage its application as dietary supplement in the treatment of airway diseases

Hippocampal FGF-2 and BDNF overexpression attenuates epileptogenesis-associated neuroinflammation and reduces spontaneous recurrent seizures

Under certain experimental conditions, neurotrophic factors may reduce epileptogenesis. We have previously reported that local, intrahippocampal supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) increases neurogenesis, reduces neuronal loss, and reduces the occurrence of spontaneous seizures in a model of damage-associated epilepsy. Here, we asked if these possibly anti-epileptogenic effects might involve anti-inflammatory mechanisms. Thus, we used a Herpes-based vector to supplement FGF-2 and BDNF in rat hippocampus after pilocarpine-induced status epilepticus that established an epileptogenic lesion. This model causes intense neuroinflammation, especially in the phase that precedes the occurrence of spontaneous seizures. The supplementation of FGF-2 and BDNF attenuated various parameters of inflammation, including astrocytosis, microcytosis and IL-1β expression. The effect appeared to be most prominent on IL-1β, whose expression was almost completely prevented. Further studies will be needed to elucidate the molecular mechanism(s) for these effects, and for that on IL-1β in particular. Nonetheless, the concept that neurotrophic factors affect neuroinflammation in vivo may be highly relevant for the understanding of the epileptogenic process

Development of a New Tool for 3D Modeling for Regenerative Medicine

The effectiveness of therapeutic treatment based on regenerative medicine for degenerative diseases (i.e., neurodegenerative or cardiac diseases) requires tools allowing the visualization and analysis of the three-dimensional (3D) distribution of target drugs within the tissue. Here, we present a new computational procedure able to overcome the limitations of visual analysis emerging by the examination of a molecular signal within images of serial tissue/organ sections by using the conventional techniques. Together with the 3D anatomical reconstitution of the tissue/organ, our framework allows the detection of signals of different origins (e.g., marked generic molecules, colorimetric, or fluorimetric substrates for enzymes; microRNA; recombinant protein). Remarkably, the application does not require the employment of specific tracking reagents for the imaging analysis. We report two different representative applications: the first shows the reconstruction of a 3D model of mouse brain with the analysis of the distribution of the β-Galactosidase, the second shows the reconstruction of a 3D mouse heart with the measurement of the cardiac volume

Comunicazione orale, Keynote speaker: Herpes Simplex Vector Expressing Hiv1 Tat: A New Strategy For Vaccine Development

Recent major breakthroughs in the field of HSV-1 technology authorize and support the use of HSV-1 based vectors as vaccine vectors against HSV-1 infection or for the delivery of foreign antigens. One of the major obstacles in developing effective vaccines against intracellular pathogens is the incomplete understanding on the mechanisms that contribute to effective immune responses. The identification of a new class of adjuvants, expressed by HSV vectors, able to direct and broad antigen presentation by the major histocompatibility complexes (MHC) can be used to achieve a stronger effect or a more potent skewing of immune responses. The focus of our laboratory is to develop vaccines able to activate and modulate effective immune responses not only against HSV but also against other intracellular pathogens such as Mycobacterium tuberculosis (Mtb). In this context, we have investigated, whether HIV-1 Tat protein co-expressed with HSV-1 vector antigens, may act as a molecule capable to induce such broad and protective immunity against intracellular pathogens. The concept of Tat as a candidate adjuvant has derived from in vitro and in vivo evidences indicating that the biologically active clade B Tat protein possesses several immunomodulatory features making it an attractive adjuvant for other antigens and suggesting that it can be exploited for vaccination strategies and therapeutic interventions aimed at modulating antigen-specific immune responses in different types of human diseases. In recent studies we have demonstrated that an attenuated replication competent HSV vector expressing Tat, as immunostimulatory molecule, is immunogenic and confers protection in mice challenged with a lethal dose of wild-type HSV1. Moreover, in preliminary experiments using a non-replicative HSV vector expressing Mtb antigens and Tat we observed that Tat can function as a potent adjuvant that modulates the CTL epitope hierarchy and broadens the epitope-specific T cell responses of heterologous antigens. Our results suggest that HSV vector backbone co-expressing an adjuvant molecule is able to broaden epitope responses and that this can be a new concept for a future vaccine against intracellular pathogens

Terapia genica anti HIV-1: effetto di mutanti transdominanti negativi di TAT sulla replicazione e riattivazione del virus dalla latenza

Dottorato di ricerca in biotecnologie. A.a 1993-94. Coordinatore Giuseppe Barbanti Brodano, tutore Roberto ManservigiConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - Piazza Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Herpes simplex virus type 1 and Alzheimer's disease: link and potential impact on treatment

Introduction: Alzheimer's disease (AD), the most common form of dementia worldwide, is a multifactorial disease with a still unknown etiology. Herpes simplex virus 1 (HSV-1) has long been suspected to be one of the factors involved in the pathogenesis of the disease. Areas covered: We review the literature focusing on viral characteristics of HSV-1, the mechanisms this virus uses to infect neural cells, its interaction with the host immune system and genetic background and summarizes results and research that support the hypothesis of an association between AD and HSV-1. The possible usefulness of virus-directed pharmaceutical approaches as potential treatments for AD will be discussed as well. Expert opinion: We highlight crucial aspects that must be addressed to clarify the possible role of HSV-1 in the pathogenesis of the disease, and to allow the design of new therapeutical approaches for AD