Construction and Characterization of HIV-1 env-Pseudoviruses of the Recombinant Form CRF63_02A and Subtype A6.

HIV-1 env-pseudoviruses are a useful tool in the search for antiviral drugs (entry inhibitors) and evaluation of the efficacy of HIV-1 vaccines. Given the high genetic variability of HIV-1, it is necessary to regularly update the panels of pseudoviruses in accordance with the emergence of new strains. Based on genetic variants of HIV-1 circulating in the regions of the Siberian Federal District, 13 HIV-1 env-pseudoviruses of recombinant form CRF63_02A and subtype A6 were obtained. Most pseudoviruses have been shown to be sensitive to neutralization by bnAbs VRC01, PGT126, and 10E8, moderately sensitive to bnAbs PG9 and 4E10, and resistant to bnAbs 2G12, PG16, and 2F5. All obtained variants of pseudoviruses are CCR5-tropic.

Concentration of viruses and electron microscopy.

Nearly all lethal viral outbreaks in the past two decades were caused by newly emerging viruses. Viruses are often studied by electron microscopy (EM), which provides new high-resolution data on the structure of viral particles relevant to both fundamental virology and practical pharmaceutical nanobiotechnology. Electron microscopy is also applied to ecological studies to detect viruses in the environment, to analysis of technological processes in the production of vaccines and other biotechnological components, and to diagnostics. Despite the advances in more sensitive methods, electron microscopy is still in active use for diagnostics. The main advantage of EM is the lack of specificity to any group of viruses, which allows working with unknown materials. However, the main limitation of the method is the relatively high detection limit (107 particles/mL), requiring viral material to be concentrated. There is no most effective universal method to concentrate viruses. Various combinations of methods and approaches are used depending on the virus and the goal. A modern virus concentration protocol involves precipitation, centrifugation, filtration, and chromatography. Here we describe the main concentrating techniques exemplified for different viruses. Effective elution techniques are required to disrupt the bonds between filter media and viruses in order to increase recovery. The paper reviews studies on unique traps, magnetic beads, and composite polyaniline and carbon nanotubes, including those of changeable size to concentrate viral particles. It also describes centrifugal concentrators to concentrate viruses on a polyethersulfone membrane. Our review suggests that the method to concentrate viruses and other nanoparticles should be chosen with regard to objectives of the study and the equipment status of the laboratory.

Morphological changes of the red blood cells treated with metal oxide nanoparticles.

The toxic effect of Al2O3, SiО2 and ZrО2 nanoparticles on red blood cells of Wistar rats was studied in vitro using the atomic force microscopy and the fluorescence analysis. Transformation of discocytes into echinocytes and spherocytes caused by the metal oxide nanoparticles was revealed. It was shown that only extremely high concentration of the nanoparticles (2mg/ml) allows correct estimating of their effect on the cell morphology. Besides, it was found out that the microviscosity changes of red blood cell membranes treated with nanoparticles began long before morphological modifications of the cells. On the contrary, the negatively charged ZrO2 and SiO2 nanoparticles did not affect ghost microviscosity up to concentrations of 1µg/ml and 0.1mg/ml, correspondingly. In its turn, the positively charged Al2O3 nanoparticles induced structural changes in the lipid bilayer of the red blood cells already at a concentration of 0.05µg/ml. A decrease in microviscosity of the erythrocyte ghosts treated with Al2O3 and SiO2 nanoparticles was shown. It was detected that the interaction of ZrO2 nanoparticles with the cells led to an increase in the membrane microviscosity and cracking of swollen erythrocytes.

Influence of glycyrrhizin on permeability and elasticity of cell membrane: perspectives for drugs delivery.

Glycyrrhizin or glycyrrhizic acid (GA) - triterpene glycoside extracted from licorice root - has been intensively studied over the past decade and is considered to be a potential drug delivery system. Glycyrrhizin was found to enhance the therapeutic effect of various drugs; however the detailed mechanism of these effects is still unknown and attracts the attention of researchers. In this work, we have made an attempt to clarify the mechanism of Glycyrrhizin activity on molecular and cellular level. The influence of GA on the functional properties of biomembranes was investigated via NMR spectroscopy and atomic force microscopy (AFM) using human erythrocytes as a model system. GA was shown to increase the permeability (about 60%) and to decrease elasticity modulus of cell membranes (by an order of magnitude) even in micromolar concentrations. Changes on the erythrocyte surface were also detected by AFM. These results could provide a new insight on the mechanism of bioavailability enhancement of some drugs in the presence of glycyrrhizin, as well as the mechanism of its own biological activity. The role of cholesterol-glycyrrhizin binding in the observed effects is also discussed.

[An antitumor osteotropic agent based on tumor necrosis factor].

A novel drug for treatment of bone metastases based on human recombinant tumor necrosis factor (TNF-alpha) has been designed. The drug is a molecular structure containing yeast double-stranded ribonucleic acid (dsRNA) covered by the conjugate of polyanion dextran with TNF-alpha and bisphosphonate, alendronic acid. The structure is characterized by the combination of substances possessing antitumor activity (TNF-alpha, dsRNA) and a vector molecule (bisphosphonate) providing tropism to hydroxyapatite, the main mineral component of the bone tissue matrix. The conjugation conditions were optimized and the conjugates of TNF-alpha and alendronic acid with dextran were synthesized. Molecular structures were obtained by self-assembly, and the resulting complexes were separated by gel filtration on Sepharose CL-6B. The electrophoretic analysis method revealed decreased mobility of dsRNA in the complex with the conjugate as compared to the mobility of the original dsRNA. This confirms formation of the designed structures. Transmission electron microscopy confirmed the presence of particles with sizes of 30-40 nm in the drug. Evaluation by the sorption/desorption method showed a higher affinity of TNF-alpha conjugates to hydroxyapatite as compared to the original TNF-alpha molecules (from 1.0 to 1.8 mol/L vs. 0.3 mol/L of potassium phosphate buffer for desorption, respectively).

An atomic force microscopy study in the detection of hepatitis B surface antigen by enzyme-linked immunosorbent assay.

Atomic force microscopy (AFM) was employed to study certain enzyme immunoassay steps for the detection of hepatitis B surface antigen (HBsAg). Physical adsorption of monoclonal antibodies (mAb), blocking of surface active sites free of antibodies by neutral proteins, and capture of HBsAg particles by sensitized surfaces were visualized successively in microplate wells of standard immunological plates from various manufacturers. The previously undescribed details such as "etching holes" up to 20 nm in depth were observed on the surface of plates some companies. The quantitative relationships between the optical density (OD) values obtained by enzyme immunoassay (EIA) and the number of antigen-antibody complexes in AFM were calculated.

Characterization of glycoprotein E C-end of West Nile virus and evaluation of its interaction force with alphaVbeta3 integrin as putative cellular receptor.

Recombinant polypeptide containing the 260-466 amino acid sequence of West Nile virus (WNV) strain LEIV-Vlg99-27889-human glycoprotein E (gpE, E(260-466)) was constructed. Immunochemical similarity between the E(260-466) and gpE of WNV was proven by enzyme immunoassay (EIA), immunoblot, competitive EIA, hemagglutination inhibition, and neutralization tests using polyclonal and monoclonal antibodies against the viral gpE and recombinant E(260-466). Polypeptide E(260-466) induced formation of virus neutralizing and cross-reactive antibodies that were interactive with various epitopes of this recombinant protein. It is shown by evaluation of the interaction of E(260-466) with one of the proposed cell receptors of WNV that average E(260-466)-alphaVbeta3 integrin-specific interaction force measured using atomic force spectroscopy was 80 and 140 pN for single and double interactions, correspondingly. Taken together with previously described interaction between laminin-binding protein (LBP) and WNV gpE domain II, it is proposed that WNV gpE can interact specifically with two cellular proteins (LBP and alphaVbeta3 integrin) during virus entry.

[Validation of the modified algorithm for predicting host susceptibility to viruses taking into account susceptibility parameters of primary target cell cultures and natural immunity factors].

The paper presents results of testing a modified algorithm for predicting virus ID50 values in a host of interest by extrapolation from a model host taking into account immune neutralizing factors and thermal inactivation of the virus. The method was tested for A/Aichi/2/68 influenza virus in SPF Wistar rats, SPF CD-1 mice and conventional ICR mice. Each species was used as a host of interest while the other two served as model hosts. Primary lung and trachea cells and secretory factors of the rats' airway epithelium were used to measure parameters needed for the purpose of prediction. Predicted ID50 values were not significantly different (p = 0.05) from those experimentally measured in vivo. The study was supported by ISTC/DARPA Agreement 450p.

[Quantitative assessment of in vitro neutralizing activity of secretory factors from rat lungs against influenza virus].

Secretory factors were isolated by lung wash followed by centrifugation to remove cells, dialysis of supernatant to remove NaCl salt, lyophilization of the lavage fluid and resuspention of the lyophilization product in an isotonic NaCl solution. It was shown that biological activity of influenza virus /Aichi/2/68 (3N2) significantly decreased (p = 0,01) from 8,17 +/- 0,10 to 7,14 +/- 0,20 IgEID50/ml during its incubation with secretory factors at 37 degrees C for 1 hr and to 7,92 +/- 0,17 IgEID50/ml in isotonic NaCl solution in the absence of these factors. Their concentration in the incubation medium was estimated to be 9.1 +/- 0.7% of their level in the lungs.

[Development of microencapsulating measles live vaccine]. / Razrabotka mikrokapsulirovannoi formy zhivoi korevoi vaktsiny.

Designing of non-injection methods of immunization against measles has recently turned into a topical issue. Development of mucosal vaccines ensuring the "entry gate" immunity, which is highly effective in airborne infection, is in the focus of attention. The authors developed a method of microencapsulating the viral particles into the matrix of pH-dependent polymers. Microencapsulated live measles vaccine shaped as 0.6-2.0 microm particles was obtained. The specific activity of measles virus in the drug was 3.36-4.31 log TCD50/0.5 ml. In subcutaneous immunization of guinea pigs with capsules, the best results were obtained in a single administration of vaccine based on ethylcrylate, sodium alginate/ chitosan and sodium slaginate/HMDA. In the intranasal administration of vaccine based on sodium alginate/spermin and sodium alginate/HMDA, there was a need in 2 and 3 stages of immunization.

[Morphology of the microencapsulated form of measles vaccine based on pH-dependent polymers]. / Morfologiia mikrokapsulirovannoi formy korevoi vaktsiny na osnove pH-zavisimykh polimerov.

Morphology of microparticles of the microencapsulated measles vaccine was studied by cryofractography, transmission electronic microscopy and by atomic force microscopy; co-polymers of polyacrylic acid and of sodium-alginate (spermidine) complexes were used as the matrix. The different-composition microcapsules had clear-cut borders and a certain range of sizes; but they were different in morphology, and their structures and densities varied identically with regard for a medium acidity, which is apparently preconditioned by some conformation-type alterations of matrix molecules. The studied preparations can, probably, protect the viral material in the stomach aggressive medium and release the material to ensure its contact with the intestine lymph tissue; thereof, they can be referred to as promising for further study of mucosal vaccines.

[Directed modification of the early region of T7 bacteriophage DNA by alkylation in the R-loop formed by a modified transcript]. / Napravlennaia modifikatsiia rannei oblasti DNK bakteriofaga T7 putem alkilirovaniia v R-petle, obrazovannoi modifitsirovannym transkriptom.

For addressed modification of T7 phage DNA a polyalkylating derivative of T7 phage early transcript was used. The derivative was obtained by attaching polyfunctional alkylating agent N,N,N,'-tri(beta-chloroethyl)-N'-(p-formylphenyl) propylene diamine-1,3 to the transcript, their molecules being covalently bound by alkylating 4--5% of the RNA adenine and guanine residues. The polyalkylating RNA was used to form R-loops in the complementary site of T7 DNA to alkylate selectively the T7 phage early DNA. Alkylation of DNA by the modified transcript in the region of the R-loop led to the covalent binding of the transcript to the complementary site of DNA. The correct localization of this R-loop was confirmed by electron microscopy. The application of the results for the addressed mutagenesis and inactivation of selected genes is discussed.