E.A. Trifonova

Examination of Biologically Active Nanocomplexes by Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) was first applied to biologically active nanocomplexes to obtain concurrent information on their size, state of aggregation, concentration, and antigenic specificity in liquid. The subject of the NTA was an immunogenic complex (a candidate nanovaccine) comprised of spherical particles (SPs) generated by thermal remodeling of the tobacco mosaic virus and Rubella virus tetraepitopes exposed on the surface of SP.

β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation

Conversion of the rod-like tobacco mosaic virus (TMV) virions into “ball-like particles” by thermal denaturation at 90–98 °C had been described by R.G. Hart in 1956. We have reported recently that spherical particles (SPs) generated by thermal denaturation of TMV at 94–98 °C were highly stable, RNA-free, and water-insoluble. The SPs were uniform in shape but varied widely in size (53–800 nm), which depended on the virus concentration. Here, we describe some structural characteristics of SPs using circular dichroism, fluorescence spectroscopy, and Raman spectroscopy. It was found that the structure of SPs protein differs strongly from that of the native TMV and is characterized by coat protein subunits transition from mainly (about 50%) α-helical structure to a structure with low content of α-helices and a significant fraction of β-sheets. The SPs demonstrate strong reaction with thioflavin T suggesting the formation of amyloid-like structures.

Use of a polycation spacer for noncovalent immobilization of albumin on thermally modified virus particles

The noncovalent immobilization of the protein bovine serum albumin on the surface of spherical nanoparticles 330 ± 60 nm in diameter is described. These nanoparticles are prepared by the thermal treatment of tobacco mosaic virus and are preliminarily covered with a layer of the cationic polymer poly(N-ethyl-4-vinylpyridinium bromide). The electrostatic adsorption of the polycation on the surface of negatively charged spherical nanoparticles (on average 1.2 × 104 macromolecules per particle) is accompanied by recharging of the surface; as a result, the negatively charged protein bovine serum albumin can be adsorbed on it in an amount of 1.7 × 104 molecules per particle. The modification of spherical nanoparticles with the polycation and protein does not cause the aggregation of particles. The spherical-nanoparticle-polycation-protein ternary complex demonstrates increased stability in salt solutions relative to the spherical-nanoparticle-polycation binary complex. Because of the simplicity of the method used to modify the surface of spherical nanoparticles, it shows promise for preparation of functionally active complexes.

Comparative Study of Non-Enveloped Icosahedral Viruses Size

Now, as before, transmission electron microscopy (TEM) is a widely used technique for the determination of virions size. In some studies, dynamic light scattering (DLS) has also been applied for this purpose. Data obtained by different authors and using different methods could vary significantly. The process of TEM sample preparation involves drying on the substrate, which can cause virions to undergo morphology changes. Therefore, other techniques should be used for measurements of virions size in liquid, (i.e. under conditions closer to native). DLS and nanoparticle tracking analysis (NTA) provide supplementary data about the virions hydrodynamic diameter and aggregation state in liquid. In contrast to DLS, NTA data have a higher resolution and also are less sensitive to minor admixtures. In the present work, the size of non-enveloped icosahedral viruses of different nature was analyzed by TEM, DLS and NTA: the viruses used were the encephalomyocarditis virus (animal virus), and cauliflower mosaic virus, brome mosaic virus and bean mild mosaic virus (plant viruses). The same, freshly purified, samples of each virus were used for analysis using the different techniques. The results were compared with earlier published data and description databases. DLS data about the hydrodynamic diameter of bean mild mosaic virus, and NTA data for all examined viruses, were obtained for the first time. For all virus samples, the values of size obtained by TEM were less than virions sizes determined by DLS and NTA. The contribution of the electrical double layer (EDL) in virions hydrodynamic diameter was evaluated. DLS and NTA data adjusted for EDL thickness were in better agreement with TEM results.

Influenza virus aerosols in the air and their infectiousness.

Influenza is one of the most contagious and rapidly spreading infectious diseases and an important global cause of hospital admissions and mortality. There are some amounts of the virus in the air constantly. These amounts is generally not enough to cause disease in people, due to infection prevention by healthy immune systems. However, at a higher concentration of the airborne virus, the risk of human infection increases dramatically. Early detection of the threshold virus concentration is essential for prevention of the spread of influenza infection. This review discusses different approaches for measuring the amount of influenza A virus particles in the air and assessing their infectiousness. Here we also discuss the data describing the relationship between the influenza virus subtypes and virus air transmission, and distribution of viral particles in aerosol drops of different sizes.

Complexes assembled from TMV-derived spherical particles and entire virions of heterogeneous nature

Previously, we described some structural features of spherical particles (SPs) generated by thermal remodelling of the tobacco mosaic virus. The SPs represent a universal platform that could bind various proteins. Here, we report that entire isometric virions of heterogeneous nature bind non-specifically to the SPs. Formaldehyde (FA) was used for covalent binding of a virus to the SPs surface for stabilizing the SP—virus complexes. Transmission and high resolution scanning electron microscopy showed that the SPs surface was covered with virus particles. The architecture of SP–virion complexes was examined by immunologic methods. Mean diameters of SPs and SP–human enterovirus C and SP–cauliflower mosaic virus (CaMV) compositions were determined by nanoparticle tracking analysis (NTA) in liquid. Significantly, neither free SPs nor individual virions were detected by NTA in either FA-crosslinked or FA-untreated compositions. Entirely, all virions were bound to the SPs surface and the SP sites within the SP–CaMV complexes were inaccessible for anti-SP antibodies. Likewise, the SPs immunogenicity within the FA-treated SPs–CaMV compositions was negligible. Apparently, the SP antigenic sites were hidden and masked by virions within the compositions. Previously, we reported that the SPs exhibited adjuvant activity when foreign proteins/epitopes were mixed with or crosslinked to SPs. We found that immunogenicity of entire CaMV crosslinked to SP was rather low which could be due to the above-mentioned masking of the SPs booster. Contrastingly, immunogenicity of the FA-untreated compositions increased significantly, presumably, due to partial release of virions and unmasking of some SPs-buster sites after animals immunization.

Biosafety of plant viruses for human and animals

Currently, virions and virus-like particles (VLPs) of plant viruses are considered as the basis for the development of new biotechnologies for human and veterinary medicine, including production of modern and safe vaccines, targeted delivery systems, and novel diagnostic preparations, as well as for production of therapeutic proteins in plants. Despite the fact that plant viruses cannot replicate in vertebrates, there are data that they are able to reproduce one or another phase of the infectious cycle in mammalian cells. Moreover, it was shown that plant viruses can be permanently present in a human and animal organism and can use it as a vector. In the review, the results of biocompatibility, toxicity, teratogenicity, and distribution of plant viruses are presented. Based on recent data, it can be affirmed that plant viruses are safe for humans and animals. It was shown that the virions are biodegradable and are easily eliminated from an organism of laboratory animals. Furthermore the virions and VLPs of plant viruses are highly immunogenic and presentation of antigenic determinant of human and animal pathogens on their surface allow to simulate a safe viral particle that is able to replace live attenuated vaccines.

Proteins immobilization on the surface of modified plant viral particles coated with hydrophobic polycations

Two hydrophobic cations based on poly-N-ethyl-vinylpyridine were used to produce biologically active complexes. The complexes obtained from tobacco mosaic virus (TMV) spherical particles (SPs), hydrophobic polycation, and a model protein were stable and did not aggregate in solution, particularly at high ionic strengths. The nucleic acid-free SPs were generated by thermal remodeling of the TMV (helical rod-shaped plant virus). The model protein preserved its antigenic activity in the ternary complex (SP–polycation–protein). Immobilization of proteins on the surface of SPs coated with hydrophobic cation is a promising approach to designing biologically active complexes used in bionanotechnologies.

Study of rubella candidate vaccine based on a structurally modified plant virus

Abstract A novel rubella candidate vaccine based on a structurally modified plant virus – spherical particles (SPs) – was developed. SPs generated by the thermal remodelling of the tobacco mosaic virus are promising platforms for the development of vaccines. SPs combine unique properties: biosafety, stability, high immunogenicity and the effective adsorption of antigens. We assembled in vitro and characterised complexes (candidate vaccine) based on SPs and the rubella virus recombinant antigen. The candidate vaccine induced a strong humoral immune response against rubella. The IgG isotypes ratio indicated the predominance of IgG1 which plays a key role in immunity to natural rubella infection. The immune response was generally directed against the rubella antigen within the complexes. We suggest that SPs can act as a platform (depot) for the rubella antigen, enhancing specific immune response. Our results demonstrate that SPs‐antigen complexes can be an effective and safe candidate vaccine against rubella. Graphical abstract Figure. No caption available. HighlightsIn vitro assembly and characterisation of a rubella candidate vaccine based on modified plant virus (spherical particles).Candidate vaccine induces efficient humoral immune response against rubella.Acute and chronic toxicity studies demonstrate biosafety of candidate vaccine.Candidate vaccine demonstrated good results and can be considered as a promising safe tool against the rubella virus.

Obtaining and characterization of spherical particles—new biogenic platforms

The technique of obtaining spherical particles from rod-like plant virus, tobacco mosaic virus, in a preparative scale was developed. The conditions of tobacco mosaic virus isolation for obtaining spherical particles were selected. Spherical particles were examined by methods of electron microscopy, nanoparticle tracking analysis, and dynamic light scattering. Information about inner structure of spherical particles was obtained. High electron density of spherical particles was demonstrated. The analysis of ultrathin sections showed that spherical particles are homogenous within and do not contain any cavities.