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The tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a pivotal role in the regulation of the human immune system. Studies of the TNF functional topography are a challenging task in bioengineering. We have produced genes encoding the peptides Dl (3-30), D2 (31-85), D3 (86-114), and D4 (115-157), which correspond to isolated fragments of the informational structure of TNF. These genes were expressed in E. coli cells at a high level in a soluble form. We have shown that hybrid proteins SD2 and SD4 tend to form soluble aggregates, which can be destroyed by urea treatment. Purified peptides Dl, D3, and D4 possess a similar secondary structure with dominating beta-structural elements. The analysis of the biological activity of these peptides has shown that they do not exhibit cytotoxic properties on L929 murine fibroblasts. The simultaneous addition of Dl with full-length TNF results in the concentration dependent suppression of TNF activity.

Study of individual hepatitis C (HCV) proteins could help to find a molecular structure and conformation, localization of antigenic and immunogenic determinants, to reveal of protective epitopes. It is necessary for practical medicine - development of diagnostic test-systems, vaccines and therapeutics. Linear and conformation dependent epitopes of HCV proteins was localized in this work and immunogenic properties of phage displayed peptides screened on monoclonal antibodies to HCV proteins have been investigated. Eleven epitopes of four HCV proteins have been studied. Three epitopes was found as linear, two epitopes were dependent on secondary structure of proteins and one epitope was dependent on tertiary structure of NS3 protein. Aminoacid sequences of other determinants have been determined and the distinct localization of these determinants will be continued after discovering of tertiary structure of HCV proteins. It was shown, that phage mimotope 3f4 is immunogenic and could induce specific hu- moral immune response to NS5A HCV protein. The data obtained could be useful for improving of HCV diagnostic test-systems, studying of amino acid substitutions and its influence on antigenic properties of the HCV proteins. The results could help to study an immune response in patients infected with different genotypes of HCV. Phage displayed peptides mimicking the antigenic epitopes of HCV proteins could be applied to development of HCV vaccine.

Adjuvant activities of granulocyte-macrophage colony-stimulating factor (GM-CSF) and synthetic glucosaminyl-muramyl dipeptide (GMDP) were studied in immunization against type 1 herpes simplex virus (HSV1). Gene encoding the gD HSV1 protein (pDNAgD) was used as an immunogen. Gene encoding GM-CSF in pDNAGM-CSF plasmid, which was developed for eukaryotic expression, and GM-DP were used as immune response modulators. GMDP and plasmid DNA with inserted GM-CSF gene enhanced T-cell immune response to HSV1 after a single injection (pDNAGM-CSF) or 24 h before (GMDP) immunization with the gD HSV1 gene. Both adjuvants increased protective effect of DNA-immunization by a virus gene with 63 up to 100% after injection of two genes and up to 96% after the viral gene was inoculated 24 h after GMDP. These high effects indicate that further investigation of anti-HSV1 DNA-based vaccines used with genetic and peptide adjuvant is prospective.

The structure-function relationships in a series of synthetic glucosaminylmuramyl peptides--glycopeptide adjuvants of the bacterial origin--have been investigated. Modification of the N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) molecule affects its adjuvant and pyrogenic activity in vivo. GMDP is shown to readily stimulate the synthesis of the IgG2a and IgG3 of the immunoglobulin subclasses upon the secondary immune response to the protein antigen. The adjuvant effect correlates with the comitogenic test of the reaction of splenocytes blast-transformation in the presence of B-cell mitogen. No direct dependence has been revealed between the level of the glycopeptides adjuvant action and their effect on the IL-1 production by macrophages.

Mild acid hydrolysis of the lipopolysaccharide from Yersinia kristensenii strain 103 (0:12.26) afforded teichoic acid-like polysaccharide. From the results of methylation, dephosphorylation, partial Smyth degradation, and 13C and 31P NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [formula: see text] The structure was confirmed by complete interpretation of polysaccharide 13C NMR spectrum.