UV-C irradiation as an effective tool for sterilization of porcine chimeric VP1-PCV2bCap recombinant vaccine.

Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV-C light necessary for baculovirus inactivation by measurement of fluorescent GFP protein produced by baculovirus expression system after the irradiation of baculovirus culture in doses ranging from 3.5 to 42 J/m2 was determined. At a dose of 36.8 J/m2, only 0.5% of GFP-expressing cells were detected by flow cytometry and confocal microscopy. The stability of purified VP1-PCV2bCap protein produced by baculovirus expression system was analyzed after the irradiation at doses ranging from 3.5 to 19.3 J/m2. Up to the dose of 11 J/m2, no significant effect of UV-C light on the stability of VP1-PCV2bCap was detected. We observed a dose-dependent increase in VP1-PCV2bCap-specific immune response in BALB/c mice immunized by recombinant protein sterilized by irradiation in dose 11 J/m2 with no significant difference between vaccines sterilized by UV-C light and filtration. A substantial difference in the production of VP1-PCV2bCap specific IgG was observed in piglets immunized with VP1-PCV2bCap sterilized by UV-C in comparison with protein sterilized by filtration in combination with the inactivation of baculovirus by binary ethylenimine. UV-C irradiation represents an effective method for vaccine sterilization, where commonly used methods of sterilization are not possible.

Development of modern immunization agent against bovine papillomavirus type 1 infection based on BPV1 L1 recombinant protein.

Bovine papillomavirus type 1 L1 protein was produced in a baculovirus expression system and purified as virus-like particles (VLPs) by affinity chromatography using lectins. The morphological integrity of VLPs was confirmed by electron microscopy. Differences between the two detected variants were deciphered by mass spectrometry of peptides (MALDI-TOF). Mice were immunized with purified VLPs in doses of 10, 25, or 50 µg in combination with 1% saponin and 15% alhydrogel per dose as adjuvants. Analysis of the humoral immune response revealed increased levels of specific antibodies detected 3 weeks after the first immunization in all groups of animals. This was further significantly increased by the booster applied 3 weeks after the first dose, with the best immune response in a group of mice immunized by the largest dose of antigen. BPV1 L1 VLPs purified by affinity chromatography using lectins could be used for prophylactic immunization in veterinary medicine.

Diphyllin Shows a Broad-Spectrum Antiviral Activity against Multiple Medically Important Enveloped RNA and DNA Viruses.

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.

FDA-Approved Drugs Efavirenz, Tipranavir, and Dasabuvir Inhibit Replication of Multiple Flaviviruses in Vero Cells.

Vector-borne flaviviruses (VBFs) affect human health worldwide, but no approved drugs are available specifically to treat VBF-associated infections. Here, we performed in silico screening of a library of U.S. Food and Drug Administration-approved antiviral drugs for their interaction with Zika virus proteins. Twelve hit drugs were identified by the docking experiments and tested in cell-based antiviral assay systems. Efavirenz, tipranavir, and dasabuvir at micromolar concentrations were identified to inhibit all VBFs tested; i.e., two representatives of mosquito-borne flaviviruses (Zika and West Nile viruses) and one representative of flaviviruses transmitted by ticks (tick-borne encephalitis virus). The results warrant further research into these drugs, either individually or in combination, as possible pan-flavivirus inhibitors.

Preparation of nanoliposomes by microfluidic mixing in herring-bone channel and the role of membrane fluidity in liposomes formation.

Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with "herring-bone" geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.

Viral RNA-Dependent RNA Polymerase Inhibitor 7-Deaza-2'-C-Methyladenosine Prevents Death in a Mouse Model of West Nile Virus Infection.

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2'-C-methyl- or 4'-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2'-methylated nucleosides, 7-deaza-2'-C-methyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2'-C-methyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4'-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro However, the strong anti-WNV effect of 4'-azidocytidine and 4'-azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2'-C-methylated or 4'-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.

N-Oxy lipid-based click chemistry for orthogonal coupling of mannan onto nanoliposomes prepared by microfluidic mixing: Synthesis of lipids, characterisation of mannan-coated nanoliposomes and in vitro stimulation of dendritic cells.

New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.

Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α-Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44.

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

Arbidol (Umifenovir): A Broad-Spectrum Antiviral Drug That Inhibits Medically Important Arthropod-Borne Flaviviruses.

Arthropod-borne flaviviruses are human pathogens of global medical importance, against which no effective small molecule-based antiviral therapy has currently been reported. Arbidol (umifenovir) is a broad-spectrum antiviral compound approved in Russia and China for prophylaxis and treatment of influenza. This compound shows activities against numerous DNA and RNA viruses. The mode of action is based predominantly on impairment of critical steps in virus-cell interactions. Here we demonstrate that arbidol possesses micromolar-level anti-viral effects (EC50 values ranging from 10.57 ± 0.74 to 19.16 ± 0.29 µM) in Vero cells infected with Zika virus, West Nile virus, and tick-borne encephalitis virus, three medically important representatives of the arthropod-borne flaviviruses. Interestingly, no antiviral effects of arbidol are observed in virus infected porcine stable kidney cells (PS), human neuroblastoma cells (UKF-NB-4), and human hepatoma cells (Huh-7 cells) indicating that the antiviral effect of arbidol is strongly cell-type dependent. Arbidol shows increasing cytotoxicity when tested in various cell lines, in the order: Huh-7 < HBCA < PS < UKF-NB-4 < Vero with CC50 values ranging from 18.69 ± 0.1 to 89.72 ± 0.19 µM. Antiviral activities and acceptable cytotoxicity profiles suggest that arbidol could be a promising candidate for further investigation as a potential therapeutic agent in selective treatment of flaviviral infections.

Antiviral activity of the adenosine analogue BCX4430 against West Nile virus and tick-borne flaviviruses.

There are currently no approved antiviral therapies against medically important human flaviviruses. The imino-C-nucleoside BCX4430 shows broad-spectrum antiviral activity against a wide range of RNA viruses. Here, we demonstrate that BCX4430 inhibits tick-borne species of the genus Flavivirus; however, the antiviral effect varies against individual species. Micro-molar BCX4430 levels inhibited tick-borne encephalitis virus (TBEV); while, approximately 3-8-fold higher concentrations were needed to inhibit louping ill virus and Kyasanur Forest disease virus. Moreover, the compound strongly inhibited in vitro replication of West Nile virus, a typical mosquito-transmitted flavivirus. Two chemical forms of the compound, i.e. BCX4430 and BCX4430 hydrochloride, were compared and both exerted similar inhibitory profiles in our in vitro antiviral assay systems and no or negligible cytotoxicity in porcine kidney stable and Vero cells. The obtained data indicate that, in addition to mosquito-borne flaviviruses, the compound has strong antiviral activity against members of the TBEV serocomplex.

Antiviral activities of 2,6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: In vitro study results with pseudorabies virus (PrV, SuHV-1).

Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100µM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models.

Preparation and biological activity of 6-benzylaminopurine derivatives in plants and human cancer cells.

To study the structure-activity relationships of aromatic cytokinins, the cytokinin activity at both the receptor and cellular levels, as well as CDK inhibitory and anticancer properties of 38 6-benzylaminopurine (BAP) derivatives were compared in various in vitro assays. The compounds were prepared by the condensation of 6-chloropurine with corresponding substituted benzylamines. The majority of synthesised derivatives exhibited high activity in all three of the cytokinin bioassays employed (tobacco callus, wheat senescence and Amaranthus bioassay). The highest activities were obtained in the senescence bioassay. For some compounds tested, significant differences of activity were found in the bioassays used, indicating that diverse recognition systems may operate and suggesting that it may be possible to modulate particular cytokinin-dependent processes with specific compounds. Position-specific steric and hydrophobic effects of different phenyl ring substituents on the variation of biological activity were confirmed. In contrast to their high activity in bioassays, the BAP derivatives were recognised with much lower sensitivity than trans-zeatin in both Arabidopsis thaliana AHK3 and AHK4 receptor assays. The compounds were also investigated for their effects on cyclin-dependent kinase 2 (CDK2) and for antiproliferative properties on cancer and normal cell lines. Several of the tested compounds showed stronger inhibitory activity and cytotoxicity than BAP. There was also a significant positive correlation of the inhibitory effects on human and plant CDKs with cell proliferation of cancer and cytokinin-dependent tobacco cells, respectively. This suggests that at least a part of the antiproliferative effect of the new cytokinins was due to the inhibition of CDK activity.