Human Monocytes Are Suitable Carriers for the Delivery of Oncolytic Herpes Simplex Virus Type 1 In Vitro and in a Chicken Embryo Chorioallantoic Membrane Model of Cancer.

Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for the treatment of unresectable melanoma. T-VEC, like most OVs, is administered via intratumoral injection, underlining the unresolved problem of the systemic delivery of the oncolytic agent for the treatment of metastases and deep-seated tumors. To address this drawback, cells with a tropism for tumors can be loaded ex vivo with OVs and used as carriers for systemic oncolytic virotherapy. Here, we evaluated human monocytes as carrier cells for a prototype oHSV-1 with a similar genetic backbone as T-VEC. Many tumors specifically recruit monocytes from the bloodstream, and autologous monocytes can be obtained from peripheral blood. We demonstrate here that oHSV-1-loaded primary human monocytes migrated in vitro towards epithelial cancer cells of different origin. Moreover, human monocytic leukemia cells selectively delivered oHSV-1 to human head-and-neck xenograft tumors grown on the chorioallantoic membrane (CAM) of fertilized chicken eggs after intravascular injection. Thus, our work shows that monocytes are promising carriers for the delivery of oHSV-1s in vivo, deserving further investigation in animal models.

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

BACKGROUND: Keratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation. METHODS: We applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease. FINDINGS: We demonstrate that in vivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis in vivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression. INTERPRETATION: Our findings reinforce the critical role played by increased HC activity in the skin pathology associated with KID syndrome. They also underscore the clinical potential of anti-HC mAbs coupled with genetic based delivery systems for treating the underlying mechanistic basis of this disorder. Inhibition of HC activity is an ideal therapeutic target in KID syndrome, and the genetic delivery of mAbs targeted against mutant HCs could form the basis of new therapeutic interventions to treat this incurable disease. FUNDING: Fondazione Telethon grant GGP19148 and University of Padova grant Prot. BIRD187130 to FM; Foundation for Ichthyosis and Related Skin Types (FIRST) and National Institutes of Health grant EY 026911 to TWW.

Giving Oncolytic Viruses a Free Ride: Carrier Cells for Oncolytic Virotherapy.

Oncolytic viruses (OVs) are an emerging class of therapeutics which combine multiple mechanisms of action, including direct cancer cell-killing, immunotherapy and gene therapy. A growing number of clinical trials have indicated that OVs have an excellent safety profile and provide some degree of efficacy, but to date only a single OV drug, HSV-1 talimogene laherparepvec (T-Vec), has achieved marketing approval in the US and Europe. An important issue to consider in order to accelerate the clinical advancement of OV agents is the development of an effective delivery system. Currently, the most commonly employed OV delivery route is intratumoral; however, to target metastatic diseases and tumors that cannot be directly accessed, it is of great interest to develop effective approaches for the systemic delivery of OVs, such as the use of carrier cells. In general, the ideal carrier cell should have a tropism towards the tumor microenvironment (TME), and it must be susceptible to OV infection but remain viable long enough to allow migration and finally release of the OV within the tumor bed. Mesenchymal stem cells (MSCs) have been heavily investigated as carrier cells due to their inherent tumor tropism, in spite of some disadvantages in biodistribution. This review focuses on the other promising candidate carrier cells under development and discusses their interaction with specific OVs and future research lines.

Lentiviral Vectors Expressing Chimeric NEDD4 Ubiquitin Ligases: An Innovative Approach for Interfering with Alpha-Synuclein Accumulation.

One of the main pathological features of Parkinson's disease (PD) is a diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 Ub ligase promotes aS degradation by the endosomal-lysosomal route. Interestingly, NEDD4, as well as being a small molecule able to trigger its functions, is protective against human aS toxicity in evolutionary distant models. While pharmacological activation of E3 enzymes is not easy to achieve, their flexibility and the lack of "consensus" motifs for Ub-conjugation allow the development of engineered Ub-ligases, able to target proteins of interest. We developed lentiviral vectors, encoding well-characterized anti-human aS scFvs fused in frame to the NEDD4 catalytic domain (ubiquibodies), in order to target ubiquitinate aS. We demonstrate that, while all generated ubiquibodies bind to and ubiquitinate aS, the one directed against the non-amyloid component (NAC) of aS (Nac32HECT) affects aS's intracellular levels. Furthermore, Nac32HECT expression partially rescues aS's overexpression or mutation toxicity in neural stem cells. Overall, our data suggest that ubiquibodies, and Nac32HECT in particular, represent a valid platform for interfering with the effects of aS's accumulation and aggregation in neurons.

Targeting the Regulatory Subunit R2Alpha of Protein Kinase A in Human Glioblastoma through shRNA-Expressing Lentiviral Vectors.

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.

Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line.

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

Alix-Mediated Rescue of Feline Immunodeficiency Virus Budding Differs from That Observed with Human Immunodeficiency Virus.

The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains-the matrix (MA), capsid (CA), and nucleocapsid (NC) domains-drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the C377,409S double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding.IMPORTANCE FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.

Antiviral treatment and virological monitoring of oseltamivir-resistant influenza virus A(H1N1)pdm09 in a patient with chronic B lymphocytic leukemia.

We report the virological monitoring and the antiviral therapy adopted for the treatment of a patient affected by chronic B lymphocytic leukemia, who experienced a severe pneumonia with long-term shedding of influenza virus A(H1N1)pdm09, characterized by an early development of oseltamivir resistance.

Lentiviral Vectors as Tools for the Study and Treatment of Glioblastoma.

Glioblastoma (GBM) has the worst prognosis among brain tumors, hence basic biology, preclinical, and clinical studies are necessary to design effective strategies to defeat this disease. Gene transfer vectors derived from the most-studied lentivirus-the Human Immunodeficiency Virus type 1-have wide application in dissecting GBM specific features to identify potential therapeutic targets. Last-generation lentiviruses (LV), highly improved in safety profile and gene transfer capacity, are also largely employed as delivery systems of therapeutic molecules to be employed in gene therapy (GT) approaches. LV were initially used in GT protocols aimed at the expression of suicide factors to induce GBM cell death. Subsequently, LV were adopted to either express small noncoding RNAs to affect different aspects of GBM biology or to overcome the resistance to both chemo- and radiotherapy that easily develop in this tumor after initial therapy. Newer frontiers include adoption of LV for engineering T cells to express chimeric antigen receptors recognizing specific GBM antigens, or for transducing specific cell types that, due to their biological properties, can function as carriers of therapeutic molecules to the cancer mass. Finally, LV allow the setting up of improved animal models crucial for the validation of GBM specific therapies.

Perspectives on immunotherapy via oncolytic viruses.

BACKGROUND: With few exceptions, current chemotherapy and radiotherapy protocols only obtain a slightly prolonged survival with severe adverse effects in patients with advanced solid tumors. In particular, most solid malignancies not amenable to radical surgery still carry a dismal prognosis, which unfortunately is also the case for relapsing disease after surgery. Even though targeted therapies obtained good results, clinical experience showed that tumors eventually develop resistance. On the other hand, earlier attempts of cancer immunotherapy failed to show consistent efficacy. More recently, a deeper knowledge of immunosuppression in the tumor microenvironment (TME) allowed the development of effective drugs: in particular, monoclonal antibodies targeting the so-called immune checkpoint molecules yielded striking and lasting effects in some tumors. Unfortunately, these monoclonal antibodies are not effective in a majority of patients and are ineffective in several solid malignancies. Furthermore, due to their mechanism of action, checkpoint inhibitors often elicit autoimmune-like disease. MAIN BODY: The use of viruses as oncolytic agents (OVs) was considered in the past, while only recently OVs revealed a connection with immunotherapy. However, their antitumoral potential has remained largely unexplored, due to safety concerns and some limitations in the techniques to manipulate viruses. OV research was recently revived by a better knowledge of viral/cancer biology and advances in the methodologies to delete virulence/immune-escape related genes from even complex viral genomes or "to arm" OVs with appropriate transgenes. Recently, the first oncolytic virus, the HSV-1 based Talimogene Laherparepvec (T-VEC), was approved for the treatment of non-resectable melanoma in USA and Europe. CONCLUSION: OVs have the potential to become powerful agents of cancer immune and gene therapy. Indeed, in addition to their selective killing activity, they can act as versatile gene expression platforms for the delivery of therapeutic genes. This is particularly true for viruses with a large DNA genome, that can be manipulated to address the multiple immunosuppressive features of the TME. This review will focus on the open issues, on the most promising lines of research in the OV field and, more in general, on how OVs could be improved to achieve real clinical breakthroughs in cancers that are usually difficult to treat by immunotherapy.

Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment.

Herpes Simplex Virus type 1 (HSV-1), a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS) in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2) to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i) liposomes containing dichloromethylene bisphosphonic acid (clodronate) to deplete tissue macrophages, (ii) CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1 infection and allow recognition of an original pathophysiologic mechanism underlying gastrointestinal diseases as well as identification of novel therapeutic targets.

Antiviral activity of cationic amphiphilic drugs.

INTRODUCTION: Emerging and reemerging viral infections represent a major concern for human and veterinary public health and there is an urgent need for the development of broad-spectrum antivirals. Areas covered: A recent strategy in antiviral research is based on the identification of molecules targeting host functions required for infection of multiple viruses. A number of FDA-approved drugs used to treat several human diseases are cationic amphiphilic drugs (CADs) that have the ability to accumulate inside cells affecting several structures/functions hijacked by viruses during infection. In this review we summarized the CADs' chemical properties and effects on the cells and reported the main FDA-approved CADs that have been identified so far as potential antivirals in drug repurposing studies. Expert commentary: Although there have been concerns regarding the efficacy and the possible side effects of the off-label use of CADs as antivirals, they seem to represent a promising starting point for the development of broad-spectrum antiviral strategies. Further knowledge about their mechanism of action is required to improve their antiviral activity and to reduce the risk of side effects.

Dissecting the Role of K61/K59 Residue in VPS4 Functions.

ESCRTs (Endosomal Sorting Complexes Required for Transport) are required for the formation of the intraluminal vesicles in the multivesicular bodies and are involved in other topologically similar processes such as cytokinesis, nuclear envelope sealing and viral egress. The final complex ESCRT-III is disassembled by the recruitment and activation of the AAA-ATPase VPS4 to the endosomal membranes. This recruitment is due to the binding of VPS4 N-terminal MIT with MIM1 and MIM2 domains present in the CHMPs proteins. By analyzing different cellular membrane remodeling events in which VPS4 is involved, here we provide evidence that the K61 residue, mapping within the MIT domain of VPS4B (K59 in VPS4A), is involved in VPS4 functioning. Posttranslational modifications of this residue might modulate MIT-MIM2 binding affinity and, as a consequence, VPS4 functions.

A novel de novo missense mutation in TP63 underlying germline mosaicism in AEC syndrome: implications for recurrence risk and prenatal diagnosis.

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare autosomal dominant ectodermal dysplasia syndrome. It is caused by heterozygous mutations in TP63, encoding a transcriptional factor of the p53 family. Mutations in TP63, mainly missense in exons 13 and 14 encoding the sterile alpha motif (SAM) and the transactivation inhibitory (TI) domains, account for 99% of mutations in individuals with AEC syndrome. Of these, ≥70% are de novo mutations, present in the affected patient, but not in parents nor in healthy siblings. However, when a mutation appears de novo, it is not possible to differentiate between a sporadic mutation, or germline mosaicism in the parents. In this latter case, there is a risk of having additional affected offspring. We describe two sisters with AEC syndrome, whose parents were unaffected. Both patients carried the heterozygous c.1568T>C substitution in exon 13 of TP63, resulting in a p.L523P change in the SAM domain of the protein. Analyses of DNA from parental blood cells, seminal fluid (from the father) and maternal cells (buccal, vaginal, and cervical) did not reveal the mutation, suggesting that the mosaicism may involve a very low percentage of cells (very low grade somatic mosaicism) or, more likely, maternal gonadal mosaicism. Mosaicism must be considered for the assessment of recurrence risk during genetic counseling in AEC syndrome, and pre-implantation/prenatal genetic diagnosis should be offered to all couples, even when the mutation is apparently de novo.

Feline tetherin is characterized by a short N-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein.

Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2(504) and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2(504) is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2(504) failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2(504) was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2(504) also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction.

Report of the 2011 annual meeting of the Italian Society for Virology.

The 10th annual meeting of the Italian Society for Virology (SIV) comprised seven plenary sessions focused on: General virology and viral genetics; Virus-Host interaction and pathogenesis; Viral oncology; Emerging viruses and zoonotic, foodborne and environmental pathways of transmission; Viral immunology and vaccines; Medical virology and antiviral therapy; Viral biotechnologies and gene therapy. The meeting had an attendance of 143 virologists, about 60% were senior, and the other were young scientists. The submitted abstracts amounted to 88 and the abstracts selected for oral presentation were 41. Complete abstracts of oral and poster presentations are available at the web site www.siv-virologia.it. A summary of the plenary lectures and oral selected presentations is reported.

Herpes simplex virus type 2 infection increases human immunodeficiency virus type 1 entry into human primary macrophages.

Epidemiological and clinical data indicate that genital ulcer disease (GUD) pathogens are associated with an increased risk of human immunodeficiency virus type 1 (HIV-1) acquisition and/or transmission. Among them, genital herpes simplex virus type 2 (HSV-2) seems to play a relevant role. Indeed, the ability of HSV-2 to induce massive infiltration at the genital level of cells which are potential targets for HIV-1 infection may represent one of the mechanisms involved in this process. Here we show that infection of human primary macrophages (MDMs) by HSV-2 results in an increase of CCR5 expression levels on cell surface and allows higher efficiency of MDMs to support entry of R5 HIV-1 strains. This finding could strengthen, at the molecular level, the evidence linking HSV-2 infection to an increased susceptibility to HIV-1 acquisition.

Summary of the 9th annual meeting of the Italian Society for Virology.

The 9th annual meeting of the Italian Society for Virology (SIV) comprised seven plenary sessions focused on: General virology and viral genetics; Virus-Host interaction and pathogenesis; Viral oncology; Emerging viruses and zoonotic, foodborne, and environmental pathways of transmission; Viral immunology and vaccines; Medical virology and antiviral therapy; Viral biotechnologies and gene therapy. Moreover, four hot topics were discussed in special lectures: the Pioneer in human virology lecture regarding the control of viral epidemics with particular emphasis on the human immunodeficiency virus (HIV), the Pioneer in plant virology lecture focused on cell responses to plant virus infection, a Keynote lecture on the epidemiology and genetic diversity of Crimea-Congo Hemorrhagic Fever virus, and the G.B. Rossi lecture on the molecular basis and clinical implications of human cytomegalovirus tropism for endothelial/epithelial cells. The meeting had an attendance of about 160 virologists. A summary of the plenary lectures and oral selected presentations is reported.

View and review on viral oncology research.

To date, almost one and a half million cases of cancer are diagnosed every year in the US and nearly 560,000 Americans are expected to die of cancer in the current year, more than 1,500 people a day (data from the American Cancer Society at http://www.cancer.org/). According to the World Health Organization (WHO), roughly 20% of all cancers worldwide results from chronic infections; in particular, up to 15% of human cancers is characterized by a viral aetiology with higher incidence in Developing Countries. The link between viruses and cancer was one of the pivotal discoveries in cancer research during the past Century. Indeed, the infectious nature of specific tumors has important implications in terms of their prevention, diagnosis, and therapy. In the 21st Century, the research on viral oncology field continues to be vigorous, with new significant and original studies on viral oncogenesis and translational research from basic virology to treatment of cancer. This review will cover different viral oncology aspects, starting from the history of viral oncology and moving to the peculiar features of oncogenic RNA and DNA viruses, with a special focus on human pathogens.