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Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10 -8 -10 -6  M) or polyethylene glycol (PEG, molecular weight ∼8,000 Da, 10 -7 -10 -4  M) increase the half-life of a green fluorescent protein expressing adenovirus from ∼48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.

Human papilloma virus (HPV)-16 is the prevalent genotype associated with cervical tumours. Virus-like-particle (VLP)-based vaccines have proven to be effective in limiting new infections of high-risk HPVs, but their high cost has hampered their use, especially in the poor developing countries. Avipox-based recombinants are replication-restricted to avian species and represent efficient and safe vectors also for immunocompromised hosts, as they can elicit a complete immune response. A new fowlpox virus recombinant encoding HPV-L1 (FPL1) was engineered and evaluated side-by-side with a FP recombinant co-expressing L1 and green fluorescent protein (FPL1GFP) for correct expression of L1 in vitro in different cell lines, as confirmed by Western blotting, immunofluorescence, real-time PCR, and electron microscopy. Mice were also immunised to determine its immunogenicity. Here, we demonstrate that the FPL1 recombinant better expresses L1 in the absence of GFP, correctly assembles structured capsomers into VLPs, and elicits an immune response in a preclinical animal model. To our knowledge, this is the first report of HPV VLPs assembled in eukaryotic cells using an avipox recombinant., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A new, simple and green method was developed for the manufacturing of heparin nanoassemblies active against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV. These nanoassemblies were obtained by the auto-association of O-palmitoyl-heparin and α-cyclodextrin in water. The synthesized O-palmitoyl-heparin derivatives mixed with α-cyclodextrin resulted in the formation of crystalline hexagonal nanoassemblies as observed by transmission electron microscopy. The nanoassembly mean hydrodynamic diameters were modulated from 340 to 659 nm depending on the type and the initial concentration of O-palmitoyl-heparin or α-cyclodextrin. The antiviral activity of the nanoassemblies was not affected by the concentration of the components. However, the method of the synthesis of O-palmitoyl-heparin affected the antiviral activity of the formulations. We showed that reduced antiviral activity is correlated with lower sulfation degree and anticoagulant activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1-8 μm, characterised by their core-shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or 'loaded' in purposed-built bubble systems - indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cyclodextrin-based nanosponges (NS) are solid nanoparticles, obtained from the cross-linking of cyclodextrins that have been proposed as delivery systems for many types of drugs. Various NS derivatives are currently under investigation in order that their properties might be tuned for different applications. In this work, new carboxylated cyclodextrin-based nanosponges (Carb-NS) carrying carboxylic groups within their structure were purposely designed as novel Acyclovir carriers. TEM measurements revealed their spherical shape and size of about 400 nm. The behaviour of Carb-NS, with respect to the incorporation and delivery of Acyclovir, was compared to that of NS, previously investigated as a drug carrier. DSC, XRPD and FTIR analyses were used to investigate the two NS formulations. The results confirm the incorporation of the drug into the NS structure and NS-Acyclovir interactions. The Acyclovir loading into Carb-NS was higher than that obtained using NS, reaching about 70% (w/w). In vitro release studies showed the release kinetics of Acyclovir from Carb-NS to be prolonged in comparison with those observed with NS, with no initial burst effect. The NS uptake into cells was evaluated using fluorescent Carb-NS and revealed the nanoparticle internalisation. Enhanced antiviral activity against a clinical isolate of HSV-1 was obtained using Acyclovir loaded in Carb-NS., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Six recently synthesized cyano-substituted heteroaryles, which do not bind to DNA but are highly cytotoxic against the human tumor cell line HeLa, were analyzed for their antitumor mechanisms of action (MOA). They did not interfere with the expression of human papillomavirus oncogenes integrated in the HeLa cell genome, but they did induce strong G1 arrest and result in the activation of caspase-3 and apoptosis. A computational analysis was performed that compared the antiproliferative activities of our compounds in 13 different tumor cell lines with those of compounds listed in the National Cancer Institute database. The results indicate that interference with cytoskeletal function and inhibition of mitosis are the likely antitumor MOA. Furthermore, a second in silico investigation revealed that the tumor cells that are sensitive to the cyano-substituted compounds show differences in their expression of locomotion genes compared with that of insensitive cell lines, thus corroborating the involvement of the cytoskeleton. This MOA was also confirmed experimentally: the cyano-substituted heteroaryles disrupted the actin and the tubulin networks in HeLa cells and inhibited cellular migration. However, further analysis indicated that multiple MOA may exist that depend on the position of the cyano-group; while cyano-substituted naphthiophene reduced the expression of cytoskeletal proteins, cyano-substituted thieno-thiophene-carboxanilide inhibited the formation of cellular reactive oxygen species.

The activity of antivirals can be enhanced by their incorporation in nanoparticulate delivery systems. Peculiar polymeric nanoparticles, based on a β-cyclodextrin-poly(4-acryloylmorpholine) monoconjugate (β-CD-PACM), are proposed as acyclovir carriers. The experimental procedure necessary to obtain the acyclovir-loaded nanoparticles using the solvent displacement preparation method will be described in this chapter. Fluorescent labeled nanoparticles are prepared using the same method for cellular trafficking studies. The biocompatibility assays necessary to obtain safe nanoparticles are reported. Section 4 of this chapter describes the assessment of the antiviral activity of the acyclovir-loaded nanoparticles., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) infection is associated with severe morbidity and mortality in immunocompromised individuals, mainly transplant recipients and AIDS patients, and is the most frequent cause of congenital malformations in newborn children. To date, few drugs are licensed for the treatment of HCMV infections, most of which target the viral DNA polymerase and suffer from many drawbacks, including long-term toxicity, low potency, and poor bioavailability. In addition, the emergence of drug-resistant viral strains is becoming an increasing problem for disease management. Finally, none of the current anti-HCMV drugs have been approved for the treatment of congenital infections. For all these reasons, there is still a strong need for new anti-HCMV drugs with novel mechanisms of action. The first events of the virus replication cycle, including attachment, entry, immediate-early gene expression, and immediate-early functions-in particular that of Immediate-Early 2 protein-represent attractive targets for the development of novel antiviral compounds. Such inhibitors would block not only the expression of viral immediate-early proteins, which play a key role in the pathogenesis of HCMV infection, but also the host immunomodulation and the changes to cell physiology induced by the first events of virus infection. This review describes the current knowledge on the initial phases of HCMV replication, their validation as potential novel antiviral targets, and the development of compounds that block such processes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Prions result in fatal degeneration of the central nervous system (CNS) in the form of diseases known as transmissible spongiform encephalopathies (TSEs). The discovery in 1996 of a new variant of Creutzfeldt-Jakob disease (a human TSE) and experimental confirmation that it is caused by the prion strain responsible for bovine spongiform encephalopathy (BSE) has greatly spurred research in this field. The mechanism underlying prion propagation is now reasonably clear. Prions multiply, in fact, by stimulating their hosts to produce proteins that are initially normal, but acquire an abnormal, prion-like conformation during the coiling stage. A fuller understanding of this mechanism could lead to the employment of molecules capable of making prion proteins revert to the normal conformation in the treatment of both TSEs and other serious CNS disorders.

Ribonucleotide reductase (RNR), a crucial enzyme for nucleotide anabolism, is encoded by all living organisms and by large DNA viruses such as the herpesviruses. Surprisingly, the beta-herpesvirus subfamily RNR R1 subunit homologues are catalytically inactive and their function remained enigmatic for many years. Recent work sheds light on the function of M45, the murine cytomegalovirus R1 homologue; during viral evolution, M45 apparently lost its original RNR activity but gained the ability, via inhibiting RIP1, a cellular adaptor protein, to block cellular signaling pathways involved in innate immunity and inflammation. The discovery of this novel mechanism of viral immune subversion provides further support to the concept of evolutionary tinkering.

TNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates the activation of NF-kappaB, MAPKs, and programmed cell death. It also transduces signals derived from Toll-like receptors and intracellular sensors of DNA damage and double-stranded RNA. Here, we show that the murine CMV M45 protein binds to RIP1 and inhibits TNFalpha-induced activation of NF-kappaB, p38 MAPK, and caspase-independent cell death. M45 also inhibited NF-kappaB activation upon stimulation of Toll-like receptor 3 and ubiquitination of RIP1, which is required for NF-kappaB activation. Hence, M45 functions as a viral inhibitor of RIP1-mediated signaling. The results presented here reveal a mechanism of viral immune subversion and demonstrate how a viral protein can simultaneously block proinflammatory and innate immune signaling pathways by interacting with a central mediator molecule.

Prions result in fatal degeneration of the central nervous system (CNS) in the form of diseases known as transmissible spongiform encephalopathies (TSEs). The discovery in 1996 of a new variant of Creutzfeldt-Jakob disease (a human TSE) and experimental confirmation that it is caused by the prion strain responsible for bovine spongiform encephalopathy (BSE) has greatly spurred research in this field. The mechanism underlying prion propagation is now reasonably clear. Prions multiply, in fact, by stimulating their hosts to produce proteins that are initially normal, but acquire an abnormal, prion-like conformation during the coiling stage. A fuller understanding of this mechanism could lead to the employment of molecules capable of making prion proteins revert to the normal conformation in the treatment of both TSEs and other serious CNS disorders.