Vol 63, No 3 (2018)

In Russia, about 2000 people get tick-borne encephalitis (TBE) every year. Almost none of them are vaccinated. For the prevention of TBE, inactivated vaccines (IVTBE) are used. IVTBE are safe and protect from TBE not less than 95% of vaccinated. The disadvantages of IVTBE are the need for numerous intramuscular injections by medical personnel, the high cost of vaccination and the vaccination refusals. A new vaccine against TBE should not be inferior to IVTBE in its safety and efficacy, should cause long-term immunity after a single application, and, preferably, be effective after oral administration. Currently, genetic engineering methods for producing replication-defective (single-cycle) flaviviruses that can serve as the basis for creating new types of safe vaccines similar in many characteristics to classic live vaccines based on attenuated strains of viruses have been proposed. The possibility of infecting humans with TBE by the use of milk of naturally infected animals, as well as the experience of using experimental live TBE vaccines, are prerequisites for the creation of a safe oral single-dose TBE vaccine.

Rabies epidemic situation in the Tver Region has been studied. Animals of different species that had confirmed clinical rabies were statistically analyzed. It was established that the features of the course of epizootics in the Tver region correspond to the regularities characteristic of rabies of the natural-focal type. As a result of sequencing of the rabies virus N gene and phylogenetic analysis, the isolates studied were assigned to the central phylogenetic group. With the help of the geoinformatic system, nosological maps of the Tver region were obtained and the spatial- temporal features of the course of the epizootic process of rabies infection were studied.

Virus-like HBc particles formed as a result of the self-assembly of the nuclear antigen of the hepatitis B virus can be used as a highly immunogenic carrier for the presentation of foreign epitopes when creating recombinant vaccines. We use this vehicle to create influenza vaccines based on the conservative antigens of the influenza virus, the extracellular domain of the transmembrane protein M2 (M2e) and the fragment of the second subunit of hemagglutinin (HA2). Presentation on the surface of HBc particles should improve the immunogenicity of these peptides. Using genetic engineering techniques, we obtained a fusion protein in which the HA2 sequence is attached to the N-terminus of the HBc antigen, and the M2e peptide is included in the immunodominant loop region exposed on the surface of HBc particle. The hybrid protein expressed in Escherichia coli and purified under denaturing conditions formed virus-like HBc particles after refolding in vitro. Refolding of this protein in the presence of a previously denatured HBc antigen carrying no inserts resulted in formation of mosaic virus-like particles. The developed method will allow construction of mosaic HBc particles carrying different target epitopes of the influenza virus by combining the corresponding modified HBc proteins, which opens the possibility of creating vaccines with a wider spectrum of protection.

The aim of the study was to investigate immunogenic properties of mosaic recombinant proteins constructed on the data of hepatitis C virus NS4A and NS4B antigens. Four mosaic recombinant proteins, containing the T and B epitopes of the NS4A and NS4B antigens, were created by genetic engineering methods in the E. coli system. To enhance the immune response they were linked in different variations to the nucleotide sequences of murine interleukin-2 (IL-2), the Neisseria meningiditis lipopeptide, and the T helper epitope of the core protein of hepatitis C virus. The immunogenic properties of these recombinant proteins were analyzed by immunoblotting, ELISA and ELISpot using sera from immunized mice and patients infected with hepatitis C virus. Recombinant proteins specifically reacted with the sera of immunized mice and infected patients in immunoblotting. According to the ELISA data, the predominant formation of antibodies to NS4B was observed when mice were immunized with the recombinant proteins containing both antigens. Analysis of gamma-interferon production by T-lymphocytes upon contact with activated dendritic cells showed in ELISpot that the maximum production of this cytokine was detected when adjuvant components were located at the N- and C-ends of the recombinant protein. The highest level of gamma-interferon production during stimulation with this drug was detected in lymphocytes from the bone marrow and lymph nodes. The recombinant protein containing the T and B epitopes of NS4A and NS4B, murine IL-2 and the lipopeptide Neisseria meningiditis had the greatest immunostimulate effect among the four constructions. This recombinant protein formed nanoparticles of 100-120 nm in size.

.