PurposeTo develop a general method for NP fabrication from various proteins with maintenance of biological activity.MethodsA novel general approach for producing protein nanoparticles (NP) by nanoprecipitation of the protein solutions in 1,1,1,3,3,3-hexafluoroisopropanol is described. Protein NP sizes and shapes were analyzed by dynamic light scattering, scanning electron and atomic force microscopy (SEM and AFM). Chemical composition of the NP was confirmed using ultraviolet (UV) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and circular dichroism (CD). Biological properties of the NP were analyzed in ELISA, immunofluorescent analysis and lysozyme activity assay.ResultsWater-insoluble NP were constructed from globular (bovine serum albumin (BSA), lysozyme, immunoglobulins), fibrillar (fibrinogen) proteins and linear polylysines by means of nanoprecipitation of protein solutions in fluoroalcohols. AFM and SEM revealed NP sizes of 20–250 nm. The NP chemical structure was confirmed by UV spectroscopy, protease digestion and EDX spectroscopy. CD spectra revealed a stable secondary structure of proteins in NP. The UV spectra, microscopy and SDS-PAA gel electrophoresis (PAGE) proved the NP stability at +4°C for 7 months. Co-precipitation of proteins with fluorophores or nanoprecipitation of pre-labeled BSA resulted in fluorescent NP that retained antigenic structures as shown by their binding with specific antibodies. Moreover, NP from monoclonal antibodies could bind with the hepatitis B virus antigen S. Besides that, lysozyme NP could digest bacterial cellular walls.ConclusionThus, the water-insoluble, stable protein NP were produced by nanoprecipitation without cross-linking and retained ligand-binding and enzymatic activities.

Background Experimental data accumulated over more than a decade indicate that cross-strain protection against influenza may be achieved by immunization with conserved influenza proteins. At the same time, the efficacy of immunization schemes designed along these lines and involving internal influenza proteins, mostly NP and M1, has not been sufficient.Objective To test the immunogenicity and protective efficacy of DNA vaccination with a combination of NP, M1 and NS1 genes of influenza virus.Methods The immunogenicity and protective efficacy of DNA vaccination with NP, M1 and NS1 was tested in mice and chickens. Mice were challenged with mouse-adapted viral strains H3N2 and H5N2 and chicken challenged with avian H5N3 virus.Results In these settings, wild-type NS1 did not impede the cellular and humoral response to NP ⁄ M1 immunization in vivo. Moreover, addition of NS1-encoding plasmid to the NP ⁄ M1 immunization protocol resulted in a significantly increased protective efficacy in vivo.Conclusions The addition of NS1 to an influenza immunization regimen based on conserved proteins bears promise. It is feasible that upon further genetic modification of these and additional conserved influenza proteins, providing for their higher safety, expression and immunogenicity, a recombinant vaccine based on several structural and non-structural proteins or their epitopes will offer broad anti-influenza protection in a wide range of species.

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The prevalence of HCV genotypes in four republics of the former Soviet Union (Russia, Belarus, Moldova, and Uzbekistan) was investigated. Overall, 197 HCV isolates from 66 blood donors and 131 patients with chronic hepatitis were typed. Viral sequences from sera of infected subjects were amplified by nested RT-PCR using primers from the core region and typed by one or two techniques: (1) DNA enzyme immunoassay (DEIA) and (2) PCR with a set of type-specific primers. Only three major HCV genotypes were identified in this study population. HCV 1b was found to be the predominant virus type both among blood donors and chronic hepatitis patients, followed by 3a, 2a, and 1a (chronic hepatitis patients: 1b-82%; 3a-10%; 2a-4%, 1a-5% and 2c-1%; blood donors: 1b-77%; 3a-17%; and 2a-6%). No significant difference in genotype distribution was observed between different countries or between blood donors and chronic hepatitis patients within the same country. Results of the genotyping procedures were confirmed by direct sequencing of 216 nt PCR fragments corresponding to part of HCV core gene. Phylogenetic analysis of HCV 1b sequences from this study and from the Genbank demonstrated that the sequences from the former Soviet Union do not form evolutionary lineage(s) different from those of strains of the same subtype circulating in other geographical regions.

There was studied the therapeutic efficacy ofinhalations ofaerosolform ofaprotinin delivered by a propellant in the metered-dose inhaler (MDI). This clinical trial was performed during the winter-spring outbreak caused with pandemic Influenza H1N1pdmO9. Aprotinin (a natural antiprotease low molecular weight polypeptide from bovine lungs) is known to be an antiviral drug, which inhibits influenza virus proteolytic activation accomplished by host respiratory proteases. Patients inhaled 2 aerosol doses of aprotinin (160 Kallikrein-inhibiting Units (KIU)) each 2 hours for 5 days. In comparison group, patients were treated with Ingavirin (a synthetic peptidoamine with unknown antiviral target), 90 mg per dayfor 5 days. On day 2 after treatment viral loads in naso-pharyngeal washes were determined. In aprotinin patients about the tenfold decrease of viral load was determined in comparison to Ingavirin patients. Duration of clinical symptoms, such as rhinorrhea, weakness, headache, sore throat, cough, chest pains, fever, in aprotinin group was 1 -2 days shorter then in Ingavirin group. No side effects neither patient discomfort were revealed in aprotinin group of patients. MDI, containing aprotinin as an active substance, can be recommended as a drug against Influenza caused by different viruses, including seasonal H1 N1, H3N2, swine-like H1 N1 pdmO9, and avian-like H7N9 viruses.