Human Diploid Fibroblast Cell Lines - a Model System for Studying Immunodulatory Properties of Modern Immunobiological Drugs and Viruses.

The immunomodulatory properties of immunobiological drugs Glutoxim and Phosprenyl we well as vesicular stomatitis virus and inactivated tick-borne encephalitis vaccine virus were studied using human diploid fibroblast cell line from the collection of M. P. Chumakov Federal Research Center for Research and Development of Immunobiological Products. All tested preparations exhibited immunomodulatory activity in human diploid fibroblast cell line. Glutoxim in doses of 0.1 and 0.25 µg/ml stimulated production of IL-6 and IL-10 during 24-48 h of culturing, but did not stimulate production of IL-1ß. Phosprenyl, on the contrary, increased production of IL-1ß and the levels of IL-6 and IL-10. Vesicular stomatitis virus stimulated the production of IL-1ß, IL-6, and IL-10, while inactivated tick-borne encephalitis vaccine virus stimulated the production of cytokines IL-8 and IL-18. Immunomodulatory activity of inactivated tick-borne encephalitis vaccine virus was first demonstrated in the in vitro system.

The Effect of a Lipopolysaccharide from Rhodobacter capsulatus PG on Inflammation Caused by Various Influenza Strains.

The development of a specific inflammation in mice that had been infected by two influenza virus strains, A/chicken/Kurgan/5/2005 (H5N1) and A/Hamburg/2009 MA (H1N1), was studied. We investigated the effect of a non-toxic lipopolysaccharide from Rhodobacter capsulatus PG on the survival and body weight of the mice, production of IgG antibodies, and the induction of pro- and anti-inflammatory cytokines in blood serum. The administration of the R. capsulatus PG lipopolysaccharide was shown to induce interferon-ß synthesis, both in healthy and influenza A virus-infected mice, and to promote production of antiviral antibodies in the blood of the influenza-infected animals.

Selection of Domestic Cell Lines for the Creation of Diagnostic and Preventive Preparations against Enterovirus 71.

We studied the sensitivity of domestic proprietary human and animal cell lines from the collection of M. P. Chumakov Federal Scientific Center for Research and Development of Immuneand-Biological Products to infection with different enterovirus 71 strains. A cell system based on domestic proprietary permanent cell line 4647 was for the first time used for reproduction of four enterovirus 71 strains (BrCr, 42266, 42934, and 43374). It was shown that strain 4647 is the optimal cell substrate for enterovirus 71 reproduction. The titers of enterovirus 71 for all four strains considerably (by 2 lgTCID50/ml and more) increased during sequential passages in permanent cell line 4647. The prospects of using permanent cell line 4647 for creation of diagnostic and preventive preparations against 71 was demonstrated.

COMPARISON OF RESULTS OBTAINED BY ELISA AND NEUTRALIZATION TEST IN ASSESSING THE PROTECTION OF POPULATION FROM TICK-BORNE ENCEPHALITIS.

The enzyme-linked immunosorbent assay (ELISA) and the neutralization test (NT) are often used to determine the level of seropositive population and to evaluate the immunogenicity of vaccines. ELISA provides information on the total pool of antiviral antibodies, while NT allows the antiviral protection level of a person to be estimated. It is assumed that the 1:100 titer in ELISA and the 1:10 titer in NT are protective. Obviously, the ratio of the total pool and virus neutralizing antibodies can vary as a result of natural immunization or vaccination. In this study, two methods were used to study the blood serum samples taken in a group of inhabitants of the Sverdlovsk region aged from 1 to 60 years. The samples were collected before immunization and 30 days after two immunizations with inactivated vaccines against tick-borne encephalitis of different manufacturers. Immunizations were performed either according to a standard scheme (30-day interval between immunizations), or according to an emergency scheme (14-day interval). It was shown that the data on the presence of antiviral antibodies in protective titers obtained by ELISA and NT were consistent in more than 85% of cases. The discrepancies between the data are due, in the first place, to the difference in the sensitivities of the two methods. The proportion of seropositive people according to NT data is always greater than that according to the results of ELISA. Nevertheless, among 174 children, about 5% of recipients after a double immunization were seropositive according to ELISA, but did not have neutralizing antibodies in protective titers.

Evaluation of immune response and protective effect of four vaccines against the tick-borne encephalitis virus.

Among three main subtypes of the tick-borne encephalitis virus (TBEV), the Siberian subtype is currently dominant in a majority of the endemic regions of Russia. However, inactivated vaccines are based on TBEV strains of the heterologous Far Eastern or the European subtypes isolated 40-77 years ago. To analyze the efficacy of the available vaccines against currently prevailing TBEV isolates of the Siberian subtype, mice were immunized subcutaneously three times (one group per each vaccine). The expression of seven cytokine genes was determined using RT-PCR. Sera were studied using homologous and heterologous ELISA, hemagglutination inhibition (HI) and neutralization tests with TBEV strains of the Far Eastern, Siberian and European subtypes. Cross-protective efficacy of the vaccines was evaluated with the TBEV strain 2689 of Siberian subtype isolated from an ixodid tick from the Novosibirsk, South-Western Siberia, Russia in 2010. The cytokine gene expression profile indicates a predominantly Th2 response due to exogenous antigen presentation. Titers for homologous combinations of vaccine strain and strain in ELISA, HI and neutralization tests exceeded those for heterologous antigen-antibody pairs. Despite antibody detection by means of ELISA, HI and neutralization tests, the mouse protection afforded by the vaccines differed significantly. Complete protection of mice challenged with 100 LD50 virus of the Siberian subtype was induced by the vaccine "Encevir" ("Microgen", Tomsk, Russia). The minimal immunization doze (MID50) of "Encevir" protecting 50% of the mice was less than 0.0016 ml. Partial protective effect of vaccines produced in Moscow, Russia and Austria revealed MID50 within recommended intervals (0.001-0.017 ml). However, the MID50 for the vaccine "Encepur" (Novartis, Germany) 0.04 ml exceeded acceptable limits with total loss of mice immunized with vaccine diluted 32, 100 and 320 fold. These results suggest regular evaluation of TBEV vaccines in regions where heterologous virus subtypes prevail.

[Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect].

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16- to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.

[Synthesis, immunogenicity and antigenic characteristics of the glycoprotein E fragments from the tick-borne encephalitis virus]. / Sintez, immunogennye i antigennye svoistva fragmentov glikoproteina E virusa kleshchevogo éntsefalita.

Six peptide fragments of the envelope protein E of the tick-borne encephalitis virus involving the predicted T-helper epitopes were synthesized. Their ability to induce antibodies without conjugation with any high-molecular-mass carrier was studied in mice of three lines. Five of six synthesized peptides exhibited immunogenic properties, which differed in dependence on the haplotype of immunized mice. The peptide binding to the antiviral antibodies was studied, and two peptides were revealed that demonstrated a high ability to recognize the viral antibodies in the horse and human sera. These peptides are promising for the development of diagnostic agents for the tick-borne encephalitis virus.

[Comparative analysis of tests on in vitro and in vivo quantitative assessment of the immunogenicity of tick-borne encephalitis vaccine]. / Sravnitel'nyi analiz testov in vitro i in vivo kolichestvennoi otsenki immunogennosti vaktsiny kleshchevogo éntsefalita.

Many-year routine use of EIA as an in vitro test demonstrated it as a highly reproducible and technological test for assessing the efficacy of vaccine against tick-borne encephalitis and its semiproducts at the intermediate stages of vaccine production. The reproducibility of mouse protection test is notably inferior to that of EIA.

[Protein E 98-113 sequence is a fusion site of tick-borne encephalitis virus with cellular membrane]. / Posledovatel'nost' 98-113 belka E virusa kleshchevogo éntsefalita iavliaetsia uchastkom sliianiia virusa s kletochnoi membranoi.

The synthetic peptide with the conservative 98-113 sequence of protein E of tick-borne encephalitis virus was studied in order to elucidate its role in the functioning of flaviviruses. The peptide was shown to inhibit the in vitro infection of macrophages with the virus. An antibody that specifically binds this peptide was found among the set of monoclonal antibodies produced against protein E. This antibody was found to prevent penetration of the virus into liposomes. A correlation was found between our results and data on the spatial structure of protein E and its interspecies homology. The protein E 98-113 sequence of the tick-borne encephalitis virus was found to be the fusion site of the viral envelope with a cellular membrane.

[Analysis of the stability of some properties of tick-borne encephalitis virus (strain 205) upon passaging in mice]. / Analiz stabil'nosti nekotorykh svoistv virusa kleshchevogo éntsefalita (shtamm 205) pri passirovanii na myshakh.

Comparative analysis of the characteristics of tick-borne encephalitis (TBE) strain 205 used for the production of vaccine against TBE and of its variants obtained by passages in mouse brain showed the stability of such properties as infective activity, neurovirulence, sensitivity to physical (heating to 50 C) and chemical (sodium deoxycholate treatment) factors. At the same time increased neurovirulence of variants 205/M10 and 205/M20, which undergone through 10 and 20 passages in white mouse brain, for low-sensitive Syrian hamsters was revealed. Use of a panel of 5 monoclonal antibodies to protein E and of 4 monoclonal antibodies to protein E and of 4 monoclonal antibodies to protein NS3 helped differentiate between not only strains 205 and Sofyin, but between variants of strain 205 as well.

[Preparation of recombinant metastazin and characteristics of it]. / Poluchenie rekombinantnogo metastazina i ego kharakteristika.

The recombinant mts-1 protein has been obtained with the use of the pMAL-c expression vector. This protein was shown to bind calcium ions and interact with melittin, a polypeptide from bee venom.

[The protective activity of preparations made from the tick-borne encephalitis virus grown using different cell cultures]. / Izuchenie protektivnoi aktivnosti preparatov iz virusa kleshchevogo éntsefalita, vyrashchennogo s ispol'zovaniem razlichnykh kletochnykh kul'tur.

The preparations of tick-borne encephalitis (TBE) virus grown in swine embryo kidney cell culture have been shown to possess pronounced protective activity per unit of virion protein E in comparison with TBE virus preparations derived from cell culture 4647 and chick embryo cell culture. The antigenic activity of all virus preparations under study has proved to be practically the same. The role of post-translation modifications of TBE virus protein E in the manifestation of some of its biological properties is discussed.

[The process of the pH-dependent fusion of the tick-borne encephalitis virus with artificial membranes]. / Izuchenie protsessa pH-zavisimogo sliianiia virusa kleshchevogo éntsefalita s iskusstvennymi membranami.

Fusion of TBE virus with liposomes was distinctly determined at pH 7.0 or lower, the maximum degree of fusion being observed at pH 6.4. Disorders in the native structure of TBE virus envelope protein E prevented virus-membrane fusion. Pre-incubation of viral preparations at pH 6.0 completely inhibited the fusion process, while rupture of disulfide bonds in protein E reduced the degree of fusion approximately 2-fold. Reduction of TBE virus infectivity upon changes in the native conformation of protein E as a consequence of disorders in the process of fusion of virions with cell membranes is discussed.

[The purification of tick-borne encephalitis virus preparations of cellular DNA]. / Ochistka preparatov virusa kleshchevogo éntsefalita ot kletochnoi DNK.

According to the WHO requirements, the concentration of cellular DNA in vaccine preparations produced by pooling virus from continuous cell lines is limited to 100 ng/dose. In this study, different methods were used for purification of tick-borne encephalitis virus suspensions grown in continuous cultures of cell line 4647 from cellular DNA. Two approaches are proposed based on treatment with DNAse and promamin sulfate which allow one to reduce cellular DNA concentration in the virus preparation to the acceptable level. Prospects of their use in vaccine production are discussed.

[Changes in biological properties of tick-borne encephalitis virus during cleavage of disulfide bonds in protein E]. / Izmeneniia biologicheskikh svoistv virusa kleshchevogo éntsefalita pri razryve disul'fidnykh sviazei v belke E.

The study showed the disruption of disulphide bonds in E protein of tick-borne encephalitis virus (TBE) to lead to the loss of antigenicity, infectivity, hemagglutinating and protective activities. The loss of infectivity under the effect of a thiolic reagent appears to be associated with block of the very first stage of virus-cell interaction, virus adsorption on the target cell. An attempt to reestablish the E protein structure and the above-mentioned virus properties after the removal of the thiolic reagent failed. The role of tertiary structure of E protein in the manifestation of TBE virus main biological properties is discussed.