[Guest peptide insertions into the surface loops of alkaline phosphatase].

The paper presents the description of the experiments in line with the rational concept for the "safe" insertion of guest polypeptides into the alkaline phosphatase with the minimal influence of the inserts on the enzymatic activity of the protein. Several approaches are described in the paper for the surface loop length estimation and two loops were used as the sites for guest peptides introduction by gene engineering technique. The experiments clearly demonstrate that insertions of several peptides after Ala218 of alkaline phosphatase (the site was selected by loop length analysis) do not block the activity of the enzyme. According the experimental data, the selection of the loops for the guest peptides insertion can be defined by the mobility of backbone dihedral angles during molecular dynamics simulation of alkaline phosphatase. The paper demonstrates the possibility to use in practice the estimation of loop length based on the mobility of backbone dihedral angles during molecular dynamics simulation. Indeed, it looks that the proteins with new features can be constructed by the introduction of new polypeptides into the enzymatically active proteins.

[Experimental study of candidate vaccines against variable or quasi-species pathogenes: multiepitopic synthetic peptide antigenes and new receptor-guiding adjuvants].

The short multiepitopic synthetic peptides from the sequences of hypervariable area of V3-loope of gp120 of HIV don't induce anti-peptides antibodies production in mice themselves. We prepared the potent immunogen by noncovalent conjugations of the multitude peptides with pure peptidoglycans from cell wall of Salmonella typhi. The sera from immunized mice have the anti-peptides antibody titers (3-5) x 10(5) in ELISA, as high as Freund's adjuvant is of use.

[TEpredict: software for T-cell epitope prediction].

A program named TEpredict was developed for T-cell epitope prediction. Original models for T-cell epitope prediction were constructed by means of Partial Least Squares regression method on the basis of data, extracted from the IEDB (Immune Epitope Database)--the most complete resource of experimental peptide-MHC binding data known to date. TEpredict is also able to predict proteasomal processing of protein antigens, and the ability of produced oligopeptides to bind to TAP (Tansporters Associated with Processing). TEpredict could exclude peptides, shearing local similarity with human proteins, from the set of predicted T-cell epitopes. It is also able to estimate expected population coverage by selected peptides, using known HLA allele genotypic frequencies data. The majority of produced models demonstrated high sensitivity of predictions (0.50-0.80) concurrent with high specificity (0.75-0.99). TEpredict was shown to be highly competitive or even superior in comparison with such programs as ProPred1, SVRMHC, SVMHC and SYFPEITHI. TEpredict demonstrated high quality of predictions and we expect that it could become a useful tool in the development ofpolyepitope vaccines against dangerous human pathogens, including HIV, influenza etc. The program and its source code could be freely downloaded from the project web-site: http://tepredict.sourceforge.net.

[Searching for local similarities between HIV-1 and human proteins. Application to vaccines]. / Poisk v belkakh VICh-1 raionov, lokal'no skhodnykh s belkami cheloveka. Primenenie k vaktsinam.

A method for identification of fragments with a high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions, whose fragments are frequent in human proteins, and regions, exhibiting a high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV-infection due either to induction of cross-reacting effectors of immune system or through molecular mimicry of physiologically important human proteins, leading to an alteration of homeostasis of the organism, is discussed. Most of regions, identified in HIV-1 proteins, contain either T-cell (CD8+ CTL or CD4+ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines which enable the exclusion of epitopes, having the (immuno)pathogenic potential, are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions, whose fragments are frequent in human proteins, as well as of regions exhibiting a pronounced local similarity to proteins that provide for important physiological functions.

[Frequency of occurrence of Hepatitis C virus markers and risk factors among hospital personnel in the Novosibirsk region]. / Chastota vstrechaemosti markerov gepatita C i faktory riska u personala bol'nits Novosibirskoi oblasti.

The occurrence of markers, the genotypic variety of isolates and the profile of risk factors with respect to viral hepatitis C among 629 employees of the Regional Clinical Hospital (RCH) in Novosibirsk and 1,020 employees of the Central District Hospital (CDH) in Iskitim were studied in a cross-sectional investigation. The occurrence of hepatitis C virus (HCV) markers was 5.1% in RCH and 2.2% in CDH. Among the risk factors in the population under study were: the medical history of blood transfusions (TF) with 0 TF, anti-HCV = 2.3%; 1 TF, = 5.7% > 1 TF, = 13.5% (p < 0.001); general anesthesia (GA) with < or = 2 GA, anti-HCV = 2.8%; > 2 GA, = 7.8% (p = 0.002); surgical interventions (SU) with 0 SU, = 1.9%; > 0 SU, = 4.3% (p = 0.012); the intravenous use of drugs (OR = 31.8); age (< or = 25 years, anti-HCV IgG = 8.6% > 25 years, = 4.5%); the number of partners of the opposite sex < or = 4 partners, = 2.4%; > 4 partners, = 6.9%; p < 0.001). The probable risk factors at a working place (pricks and cuts, contamination of mucous membranes with blood and other biological fluids, etc.) proved to be faintly related with the status of HBV infection. HBV isolates detected in the examined persons (35 examinees) were distributed by genotypes as follows: 60% of subtype 1b, 28.6% of subtype 2a/2c, 11.4% of subtype 3a. HBV of genotype 1a was not detected in the examined specimens, while the detection rate of genotype 2a/2c was considerably greater than in specimens obtained in the European and Asian parts of Russia (according to the data reported earlier).

[Preparation and selection of monoclonal antibodies for detecting hepatitis B surface antigen in blood sera]. / Poluchenie i otbor monoklonal'nykh antitel dlia vyiavleniia poverkhnostnogo antigena virusa gepatita B v syvorotkakh krovi.

HV monoclonal antibodies (MAb) were produced in order to improve the quality of HBsAg detection and their specific characteristics were compared with those of other MAbs. MAbs were characterized by asymmetric interactions with the antigen when used as first or second antibodies. The reactivity of a panel of HV and X MAb to ad and ay subtypes was studied by enzyme immunoassay. Mutual blocking (epitope mapping) of MAb helped select antibody couples for the creation of highly effective test system for the diagnosis of the major HBsAg subtypes. The sensitivity and specificity of MAbs were evaluated on reference and control panels of HBsAg sera and on serum specimens from a random sampling of 300 blood donors. The sensitivity of the most specific MAb pairs was 0.1 ng/ml for HBsAg subtype ay and 0.25 ng/ml for subtype ad. The specificity of attested MAb was 98.5% in incubation with stirring and 97% in static incubation. The optimal combinations of attested MAbs were used in the manufacture of Recomnathep B test system in the sandwich format.

[Markers of hepatitis C and its various genotypes in patients from Novosibirsk]. / Markery virusa gepatita C i razlichnky ego genotipov u patsientov Novosibirska.

Study of the prevalence of hepatitis C virus (HCV) markers in 153 patients of Municipal Infectious Diseases Clinical Hospital No. 1 in Novosibirsk revealed anti-HCV in 88.2% patients and viral RNA in 69.3% samples. For 93 Siberian HCV isolates 5'-UTR regions were amplified and sequenced. Comparison of these nucleotide sequences with databank showed that 63.4% HCV isolates belonged to subtype 1b, 7.5% to genotype 2, and 18.3% to genotype 3. In the rest HCV isolates the 5'-UTR sequences contained heretofore undescribed nucleotide substitutions, insertions, or deletions.

[Human herpesvirus-6 (HHV-6): current status]. / Virus gerpesa 6-go tipa (HHV-6): sovremennoe sostoianie voprosa.

Molecular, biological, and immunological studies on the recently identified human herpesvirus-6 (HHV-6) suggest that the virus is involved in the etiology of at least several lymphoproliferative diseases. Furthermore, HHV-6 may be an important cofactor in the pathogenesis of several other diseases, including cervical carcinoma, oral carcinoma, multiple sclerosis, and HIV infection. Analyzing the data available in the literature shows it necessary to conduct systematic investigations and to emphasize the importance of the problem for national public health.

[Development of sera reference panels for quality control of ELISA diagnostic kits in Russia]. / Razrabotka standartnykh panelei syvorotok dlia kontrolia kachestva immunofermenthykh test-sistem v Rossii.

In the past 5 years, the investigators of the "VECTOR" SRB VB and the L.A. Tarasevich State Institute of Standardization and Control of Medical Biological Preparations have jointly designed sera reference panels containing anti-HIV-1 IgG, anti-HCV IgG, and anti-HAV IgM which have been approved as national standard panels. The panels are intended for use in controlling the specificity and stability of the most widely used ELISA diagnostic kits and immunoblot test systems during production, control, and application stages. Some problems of development and production of these panels, including the representation of different sera in the panels and the selection of specific IgG concentrations in the different sera in the panel are described. The authors also attract attention to the stabilization of the specific characteristics of panel sera during storage and transportation.

[Design of reference panels of serum for testing the quality of HIV antibody tests in practical laboratories]. / Konstruirovanie otsenochnykh panelei syvorotok dlia testirovaniia kachestva provedeniia analizov na antitela k VICh v prakticheskikh laboratoriiakh.

The authors define the scientific basis for development of panels of reference sera intended for effective control of the quality of enzyme immunoassay of HIV antibody screening. Special attention was paid to developing the technology of preparing standards highly stable serum samples with a preset concentration of anti-HIV antibodies. The resultant panel of reference sera was tried in screening for anti-HIV antibodies by enzyme immunoassay at seven diagnostic laboratories. Mathematical analysis of the results permits the detection of the minimal errors in studies with the use of serum panel of practical laboratories.

[Synthesis of insulin fragments and study of their physicochemical and immunological properties]. / Sintez fragmentov insulina i izuchenie ikh fiziko-khimicheskikh i immunologicheskikh svoistv.

A two-chain peptide was predicted as a receptor binding site of insulin on the basis of theoretical conformational analysis. This dimeric peptide, consisting of the C-terminal A18-A21 tetrapeptide of the insulin A-chain and the C-terminal B17-B30 tetradecapeptide of the insulin B-chain connected with a disulfide bridge, was synthesized along with the C-terminal nonadecapeptide. The analysis of the aggregation state of human insulin and the synthesized linear and dimeric peptides was performed by the small-angle X-ray scattering method. Specific antibodies were produced after rabbit immunization with the dimeric peptide-BSA conjugate. The immunochemical identity of the model dimeric peptide and the corresponding fragment of the insulin molecule were shown by immunoenzyme analysis.

[Constructing the reference panel of sera with standard level of IgG-antibodies]. / Konstruirivanie standartnykh panelei syvorotok c normirovannym urovnem IgG-antitel.

Fundamentals of designing reference panels of sera for effective control of commercial test systems and immunoblotting, intended for detecting antiviral antibodies, have been developed. Reference low-titer panels of anti-IgG antibodies to HIV-a and hepatitis C virus have been designed. A reference panel contains diluted reactive sera with a standard level of IgG antibodies and native sera with undetectable level of antibodies to the major viral antigens from risk group subjects. The reactive sera of a panel contain the whole spectrum of antibodies to all principal viral antigens.

[Ability of a recombinant protein containing fragment 114-122 of myelin basic protein, to cause allergic encephalomyelitis in guinea pigs]. / O sposobnosti rekombinantnogo belka, soderzhashchego fragment 114-122 osnovnogo belka mielina, vyzyvat' allergicheskii éntsefalomielit u morskikh svinok.

Four genetic constructions have been designed, capable of producing in E. coli the hybrid beta-galactosidases containing the encephalitogenic determinant 114-122 of myelin basic protein. The ability of chromatography-purified proteins to cause allergic encephalomyelitis in guinea pigs has been investigated. Only one out of four proteins carrying at least one complete replica of encephalitogenic determinant did induce allergic encephalomyelitis in animals. Effects of the structural context of the encephalitogenic determinant on its functional activity are discussed.

[Antigenic determinants of proteins. The humoral immune response]. / Antigennye determinanty belkov. Gumoral'nyi immunnyi otvet.

There exist two opposing points of view on the organization of antigenic determinants of proteins: 1) the determinants are stringently predetermined regions of the protein molecule; 2) all the surface of the protein globule is potentially antigenic, and the immune response is generated occasionally in every individual, although some sites of the protein surface could be preferable for eliciting the immune response. In this work, taking into account the results of recent investigations, we propose some reconciliation: a correlation between the rigidity of the protein architecture and the antigenicity of the particular sites of the antigen molecule. Some general principles of the antigenic determinants of proteins are formulated.

[Monoclonal antibodies cross-reacting with the tick-borne encephalitis virus and with the Venezuelan equine encephalomyelitis virus]. / Monoklonal'nye antitela, perekrestno reagiruiushchie s virusom kleshchevogo éntsefalita i virusom venesuél'skogo éntsfalomielita loshadei.

Employment of radioimmunoassay led to the demonstration of serological crossing between tick-borne encephalitis (TBE) virus and Venezuelan equine encephalomyelitis (VEE) virus. Using hybridoma technology, three hybridomas were produced secreting monoclonal antibodies (MAb) cross-reacting with these two viruses. With MAb, the epitope of binding of these antibodies was shown to be located on protein E of TBE virus and protein E1 of VEE virus. Despite the low percentage (14%) of homology of amino acid sequences of these proteins, 12 areas with homology from 24% to 63% were demonstrated. Considering conservative replacements, homology of these areas was 53%-75%. The assumed existence of some of these areas in alpha-helical conformation may explain the observed immunological crossing.

[Correlation of antigenic properties of influenza viruses of the H3N2 subtype with changes in the amino acid sequence of hemagglutinins]. / Korreliatsiia antigennykh svoistv virusov grippa podtipa H3N2 s izmeneniiami v aminokislotnoi posledovatel'nosti gemagliutininov.

The effect of amino acid substitutions within the antigenic sites, the residues close to these sites, and the other parts of the hemagglutinin molecule on cross-reactions between influenza subtype H3N2 virus strains in hemagglutination-inhibition tests are considered. Previously we reported a method for calculation of the values of immunochemical cross-reactions between the homologous proteins based on the structural data. On this basis, a method for calculation of the titers of cross-reactions of influenza viruses in hemagglutination-inhibition tests is proposed. The values obtained by this method show a good correlation with the known experimental data. Analysis of the nature of amino acid replacements in region D located in the interface between the hemagglutinin subunits has shown that this region cannot bind with antibodies. The data on the limitation of the resource of the subtype H3N2 variability are presented.

[A method of calculatiing immunochemical cross-reactions between homologous proteins]. / Metod rascheta immunokhimicheskogo perekresta mezhdu gomologichnymi belkami.

The effects of amino acid substitutions within the antigenic sites, within the residues close to these sites and within other parts of the molecule on the cross-reaction with the antisera to homologous protein were considered. The method for calculus the values of cross-reactions, based on using primary structures data, X-ray coordinate of one of the homologues and the locations of the antigenic sites is proposed. The values obtained by this method for the cross-reactions of ten myoglobins from various species with antisperm-whale myoglobin sera have a good correlation with the known experimental data. The possibility of using the method to make the location of protein antigenic sites more precise is discussed.