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Biomedical subjects

A V Timofeev

Publications and source records attributed to A V Timofeev.

At least 19 recordsLinked to original sources
PubMed2005

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein.

BACKGROUND: Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. RESULTS: The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. CONCLUSION: Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines.

Animals↗
PubMed2005

A synthetic peptide based on the NS1 non-structural protein of tick-borne encephalitis virus induces a protective immune response against fatal encephalitis in an experimental animal model.

Linear immunogenic peptides corresponding to amino acid sequences from the NS1 non-structural protein from tick-borne encephalitis virus (strain Sophyin) were predicted using established algorithms and synthesized. Of the 12 peptides predicted, 11 were able to induce peptide-specific antibodies in BALB/c mice but only 1 of these 11 was able to induce antibodies, which reacted with the native protein in a radio-immune precipitation assay. This peptide corresponds to amino acids 37--55, and forms one of the predicted structurally conserved alpha helices of the virus NS1 protein. It was able to protect 60% of animals against lethal challenge with the homologous highly pathogenic tick-borne encephalitis virus strain, and adoptive transfer experiments indicated the involvement of the antibodies induced by this peptide in its protective activity in mice.

Amino Acid Sequence↗
PubMed2004

Genetic vaccination of mice with plasmids encoding the NS1 non-structural protein from tick-borne encephalitis virus and dengue 2 virus.

Although there is a safe, inexpensive and efficacious vaccine against yellow fever, vaccination against other flavivirus diseases is less successful. There is no licensed vaccine against dengue fever and current vaccines against tick-borne encephalitis (TBE) and Japanese encephalitis are expensive and require several injections. Furthermore novel vaccines containing only virus envelope proteins may raise fears over antibody mediated enhancement (ADE) of disease. Here we report the successful use of genetic vaccination against TBE in an experimental animal model using a plasmid containing the coding sequence of a non-structural protein (NS1). Such vaccines would provide inexpensive protection against disease, without raising concerns over inducing ADE on subsequent exposure to heterotypic infectious virus. Attempts to generate chaemeric plasmids to protect against both TBE and dengue fever were less successful. Although these chaemeric plasmids directed the synthesis and secretion of the virus NS1 protein normally, no protection was observed against either TBE or dengue fever.

Amino Acid Sequence↗
PubMed2004

[Differential diagnosis of diabetes mellitus in adults: assessment of immunogenetic markers of insulin dependence].

To assess immunogenetic markers of insulin dependence (IMID), 117 patients with diabetes mellitus of the adults (DMA) were examined. Of them, 97 patients (77 females and 20 males, 41 and 10 obese, respectively) were insulin dependent and 20 (8 males and 12 females) had classic diabetes mellitus (DM) type 2. All the analysed immunogenetic markers (genotype SS/SS, alleles 04*, 0301 in HLA-DQ*A2, haplotype A1*0301-B1*0302) were associated most often with insulin-dependent DMA. The following conclusions were made: both DMA variants can be definitely differentiated only by clinico-anamnestic screening; there were no differences in distribution of the alleles 0301 in DQ*A1 and 0302 in DQ*B1 in different variants of DMA; alleles 04* and 0301 DQ*A1 significantly more frequently occur in DM type 1 than DM type 2; 0301 allele was more frequent in patients with insulin-dependent DM type 2 vs DM type 2; an expectant genetic marker of insulin-dependence (haplotype 0301/0302) occurs significantly more frequently in insulin-dependent DMA than in healthy subjects and DM type 2.

Autoantibodies↗
PubMed2002

[Connection between severity of the course of tick-borne encephalitis with the concentration of interleukin-2 and interleukin-6 in blood].

AIM: To test IL-6 and IL-2 serum concentrations as indirect prognostic indicators in tick-borne encephalitis (TBE) course. MATERIAL AND METHODS: 13 TBE patients' sera were examined for IL-2 and IL-6. RESULTS: IL-6 was not detected in patients with TBE fever but was detected in meningoencephalytic TBE. CONCLUSION: TBE patients demonstrate a positive correlation between the disease severity and serum IL-6. This criterion is proposed as a prognostic factor of a TBE course.

Adult↗
PubMed1999

A monoclonal antibody that recognizes the predicted tick-borne encephalitis virus E protein fusion sequence blocks fusion.

The fusion motif of tick-borne encephalitis virus E protein has been predicted to be located within its conserved region (98-120). Results are presented to demonstrate that non-neutralizing monoclonal antibody which recognizes a synthetic peptide corresponding to residues 98-113 of the E protein sequence can block the fusion of the virus particles with artificial membranes.

Amino Acid Sequence↗
PubMed1998

Immunological basis for protection in a murine model of tick-borne encephalitis by a recombinant adenovirus carrying the gene encoding the NS1 non-structural protein.

The humoral immune response to flaviviruses is mainly directed to the major envelope protein, E, and a glycosylated non-structural protein, NS1. Cell-mediated immune responses, however, appear to be directed mainly against non-structural proteins. Experiments described here show that a defective recombinant adenovirus (Rad51) containing the gene encoding the NS1 protein of tick-borne encephalitis virus can induce a strong protective immune response against several pathogenic tick-borne flaviviruses in an experimental animal model, and can enhance the efficacy of conventional vaccine preparations. A protective immune response against a lethal virus challenge can also be induced by the passive transfer of antibodies, B cells or T cells from animals vaccinated with Rad51. Raised levels of non-neutralizing antibodies and cytokines associated with a T helper cell-type 1 immune response are also observed. These data demonstrate the importance of non-structural viral proteins in the protective immune response against flaviviruses and support the use of non-structural viral proteins as vaccine components.

Adenoviridae↗
PubMed1998

[Protein E 98-113 sequence is a fusion site of tick-borne encephalitis virus with cellular membrane].

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.

Amino Acid Sequence↗
PubMed1996

Mechanism for dendritic cell dysfunction in retroviral infection of mice.

Dendritic cells (DC) from mice infected with the murine retrovirus Rauscher leukaemia virus (RLV) are poor stimulators of allogeneic and syngeneic T cells and express lower, but still significant, levels of MHC class II. In this paper we further investigated the mechanism of the dysfunction of DC. DC from infected animals did not cause anergy of T cells during coculture for 3 or 6 days. They did not release a substantial amount of soluble factors which could suppress T cell responses. The low T cell responses on stimulation using RLV-infected DC could be overcome by the addition of control DC. Pretreatment of these control DC with monoclonal antibody against MHC class II molecules completely blocked their ability to restore stimulation of T cells in the presence of infected DC. However, antibody against MHC class I or mismatched MHC class II molecules did not prevent restoration of function. The reduced labeling of surface MHC class II molecules previously reported was shown to reflect a loss in total class II molecules within the cells; MHC class I levels were unaltered by exposure to the virus. In DC from RLV-infected mice biosynthesis of MHC class II was decreased by around 50% at the transcriptional level in comparison with beta-actin. Thus, the down-regulation of surface class II molecules observed in DC following RLV infection is a consequence of a specific block in its biosynthesis and the failure of DC to stimulate T cells may be a direct consequence of the reduced class II levels. Since reduced stimulation by DC is also seen in HIV-1 infection in humans we speculate that a similar mechanism might operate in retroviral infection in man.

Animals↗
PubMed1995

Rapid vaccination protocols for commercial vaccines against tick-borne encephalitis.

Although inactivated viral vaccines have been dramatically successful in controlling many of the world's most devastating diseases, they frequently need several injections to ensure high levels of protection, and thus their efficacy is reduced in many situations. We have developed several rapid vaccination protocols for two commercial vaccine preparations against tick-borne encephalitis virus and studied their efficacy in an experimental murine model. Vaccination protocols as brief as two doses given over two days elicit efficient protection against challenge with potentially fatal doses of virus and this protection is afforded as soon as 5 or as long as 100 days after the first vaccination. The very rapid induction of protection and the poor antibody responses observed would suggest that cell-mediated immune responses are the most important mechanisms for the protection elicited by conventional inactivated vaccines against tick-borne encephalitis.

Animals↗
PubMed1994

The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell.

Flaviviruses elicit a humoral immune response to two virus-encoded, membrane-associated glycoproteins. One is the major virion surface envelope protein (E), which is recognized by antibody, whereas the other is a secreted, heavily glycosylated non-structural protein (NS1). Inoculation with either protein can give rise to a protective immune response, as can the passive transfer of E and NS1 monospecific monoclonal antibodies. Experiments reported here demonstrate that the secreted form of NS1, whether from cells infected with tick-borne encephalitis virus (TBEV) or from cells infected with a defective recombinant adenovirus containing the NS1 gene, occurs chiefly as a pentamer or hexamer and occasionally as a decamer or dodecamer. Intracellular forms of this protein however occur only as dimers. The higher M(r) forms secreted from the cell are exquisitely sensitive to detergent, suggesting they are held together by hydrophobic bonds. Both intracellular and extracellular forms of the dimer can be dissociated by heat, but at different temperatures. Unlike similar proteins from mosquito-borne viruses. NS1 from TBEV-infected cells cannot be dissociated at ambient temperatures by extremes of pH. Studies on the antigenic structure of this protein show it to have several highly conserved epitopes, confirming similar earlier conclusions from amino acid sequence analyses.

Adenoviridae↗
PubMed1994

In vivo study of engineered G-domain mutants of Escherichia coli translation initiation factor IF2.

During the IF2-catalysed formation of the 30S initiation complex, the GTP requirement and its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli. In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E. coli IF2, we have introduced seven single amino acid substitutions into its GTP-binding site (Gly for Val-400; Thr for Pro-446; Gly, Glu, Gln for His-448; and Asn, Glu for Asp-501). These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E. coli strain from which the functional chromosomal copy of the infB gene has been deleted. Only one of the mutated proteins (Asp-501 to Glu) was able to sustain cell viability and several displayed a dominant negative effect. These results emphasize that the amino acid residues we substituted are essential for the IF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation. These findings are discussed in relation to a previously proposed theoretical model for the IF2 G-domain.

Amino Acid Sequence↗
PubMed1994

[The effect of transcardiac galvanization on the size of the necrotic mass and on the central hemodynamic indices in patients with an acute myocardial infarct].

The authors examined 110 males and females admitted to hospital within 6 hours since the onset of new macrofocal anterior myocardial infarction running without complications. All the patients underwent transcardiac galvanization (TG) in the initial disease hours. Precardiac mapping, tetrapolar rheography and clinical observation provided evidence on TG course action on the necrosis mass, central hemodynamics and clinical appearance of the disease in the acute and postinfarction periods. TG promoted faster and more marked improvement of cardiac muscle contractility, attaining higher functional level of inotropic myocardial activity up to the disease month 6. This was associated with alleviation of circulatory disorders both at the hospital and postinfarction stage. The response to course TG results from cardioprotective action of constant electric current on ischemic myocardium which limits the zone of the lesion thus reducing early fatal cases number and preventing cardiogenic shock.

Cardiography, Impedance↗
PubMed1992

[The insulin-secreting and proliferative activity of established islet cells in the presence of sulfonylurea].

Exponentially growing rat islet cells (RINr) and hamster islet cells (HIT T-15) were incubated in presence of tolbutamide (10-1000 microM), gliclazide (0.1-10 microM) or glibenclamide (0.01-10 microM) for 15 hrs. Accumulation of insulin in culture medium was estimated by RIA. Effects of sulfonylureas (SU) on cell proliferation were assessed by 3H-thymidine (3H-T) incorporation into cellular DNA. All of SUs used stimulated insulin production in RIN and HIT cell cultures (with an exception of tolbutamide, which markedly suppressed insulin secretion in HIT cells at 1000 microM). 3H-T incorporation into RIN cells was elevated only in presence of gliclazide (10 microM), whereas tolbutamide at 1000 M significantly inhibited RIN cell proliferation. Gliclazide (0.1 microM) and glibenclamide (0.01-10 microM) enhanced 3H-T incorporation into HIT cells. Further detailed investigations of mechanisms of SU effects on islet cell reproduction will be of use for designing optimal strategy of hypoglycemizing therapy of diabetes mellitus.

Animals↗