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Biomedical subjects

V D Smirnov

Publications and source records attributed to V D Smirnov.

At least 19 recordsLinked to original sources
PubMed1998

Detection of hepatitis C virus core protein circulating within different virus particle populations.

Progress in studying pathogenesis and increasing the reliability of hepatitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected persons. Detection of hepatitis C virus (HCV) proteins faces two basic difficulties: low concentration of HCV proteins, and their blocking by antibodies. The aim of this work was to develop a method for the detection of nucleocapsid (core) protein in the plasma of HCV-infected persons using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were negative. The plasmas were centrifuged for 3 hr at 143,000 g and the antigenic activity of core-protein was studied in the pellets by EIA using four MABs able to recognize four nonoverlapping determinants, two at N-terminus and two at C-terminus of recombinant core (1-150 aa). The determinants detected were present in the natural core protein of at least two genotypes (1b and 3a). Maximal efficiency of recombinant protein detection was achieved with 2 MABs, whereas a combination of 4 MABs was necessary for optimal detection of natural core protein. This is indicative of different conformational structures of natural protein and its gene-engineered analog. The sensitivity of core detection by monoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after normalization to the initial plasma volume. To dissociate immune complexes, the pellet was treated with 2.5 M KBr after first treating the pellet with the nonionic detergent Tween 80 to remove the virus lipid envelope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas.

Animals↗
PubMed1998

Specificity of humoral and cellular immune response against recombinant particles of nucleocapsid protein of human hepatitis B virus in rabbits.

Nucleocapsid (core) protein of hepatitis B virus (HBcAg) induces potent cellular and humoral responses that have a clear protective potential. Rabbits were immunized by particles formed by recombinant molecules of HBcAg carrying N-terminally inserted heterologous sequences. Specificity of humoral and cellular immune response against HBcAg and selection of HBcAg epitopes was surveyed. Immunological properties of the recombinant particles were similar to those of the original HBcAg. Recombinant particles were not toxic to the peripheral blood mononuclear cells (PBMC) of non-immune or HBcAg-immunized animals ex vivo. Proliferative response of PBMC (T-lymphocytes) to HBcAg in immunized animals increased in a concentration-dependent manner in the broad interval of HBcAg concentrations (10-104 ng/ml). On the contrary, a narrow bell-shaped HBcAg dose-dependence curve was earlier observed for T-lymphocytes of donors immune to HBV after natural infection that was probably due to the cytotoxic effect of HBcAg on the expressing cells. Specificity of humoral and cellular immune response against HBcAg particles in the immunized animals and in natural infection with hepatitis B virus (HBV) was compared. Immunization with recombinant HBcAg particles induced potent anti-HBcAg antibody responses: high (up to 2.107) titers of anti-HBcAg antibodies were reached. Appearance of anti-HBcAg antibodies was in every case preceded by an increasing T-cell response to the whole protein and HBcAg-derived peptides, thus mimicking immune responses during acute HBV infection in humans. A predominant universal (haplotype-independent) T-helper cell epitope (amino acid residues (aa) 61-85 of HBcAg (p61-85)) was recognized by T-cells of all animals. Transient antibody response against p61-85 was recorded during the early stages of immunization in spite of the fact that a major B-cell epitope localized in this region is supposed to be purely conformational. A sequence representing another cluster of immunodominant T-cell epitopes of mice and HBV infected humans, aa 121-140 (p121-140), was not immunogenic on the T-cell level. However, it appeared to be a potent B-cell immunogen, despite a common assumption that HBcAg and p121-140 are not cross-reactive at the B-cell level. A possibility that anti-p121-140 antibodies were induced by an exposed region of the native particulate HBcAg and not by the denatured protein molecules, was confirmed by recognition of the particulate HBcAg by antibodies specific to synthetic peptides representing aa 120-140 of HBcAg. The data point to the exposition of aa 121-140 on the surface of the particles.

Amino Acid Sequence↗
PubMed1996

HIV-1 epitopes exposed by hybrid hepatitis B core particles affect proliferation of peripheral blood mononuclear cells from HIV-1 positive donors.

Hepatitis B virus core protein (HBc) in particulate form is a potent immunogen for the design of vaccines with a broad T-cell reactivity. Hybrid HBc proteins with N-terminal insertions of human immunodeficiency virus (HIV-1) B- and T-cell epitopes from gp41 and p34 pol, respectively, were constructed HBc hybrids formed particles with HIV-1 epitopes exposed on the surface. The proliferative response of peripheral blood mononuclear cells (PBMC) from HIV-1 infected donors to the hybrids was studied in vitro and compared to that of synthetic peptides representing the same HIV-1 sequences. The epitope from p34 pol induced PBMC proliferation both when inserted into HBc and as a peptide. The epitope from gp41, when inserted into HBc, and to a lesser extent the relevant peptide caused a decreased reactivity. The inhibitory effect of the HBc hybrid carrying the gp41-epitope was pronounced even in the HIV-1 infected donors with proliferative responses to HBc. The similarity between the mode of action of the peptides and hybrid HBcs implies either correct processing of the latter or T-cell recognition of HIV-1 epitopes in the intact hybrid HBc particles.

Amino Acid Sequence↗
PubMed1996

[Expression of HIV-1 epitopes included in particles formed by human hepatitis B virus nucleocapsid protein].

Hybrids of the core protein of hepatitis B virus (HBcAg) have been designed which carry N-terminal insertions of B- and T-cell epitopes of HIV-1 an immunodominant B-epitope from gp41, a T-cell epitope from p34 pol, and a cluster of B- and T-cell epitopes from p17 gag. The hybrids have been synthesized using two expression systems-one based on the thermoinducible PR promoter of bacteriophage lambda and the other one based on phi 10 promoter of bacteriophage T7 with 3-5% and 7-14% yields, respectively. The hybrids have dual HBV and HIV-1 immunospecificity and are assembled into particles similar to those formed by the protein carrier HBcAg. Sandwich ELISA and immune electron microscopy revealed that HIV-1 epitopes are exposed on the surface of the particles.

Amino Acid Sequence↗
PubMed1994

Linear epitopes of HIV-1, presented as hybrids with Escherichia coli beta-galactosidase or synthetic peptides.

HIV-1 B cell epitopes from gp41, the T cell epitope of p34pol, and a cluster of B and T epitopes from p17gag were selected. The epitopes were presented as synthetic peptides and as either N- or C-terminal insertions into beta-galactosidase. Hybrids were efficiently expressed in E. coli and easily purified when epitopes were inserted at the beta-galactosidase C terminus. Sera from HIV-1-infected individuals reacted in peptide- and hybrid protein-based enzyme-linked immunosorbent assays (ELISAs) mostly with the immunodominant site of gp41. The second site of gp41 and also sites from p17 and p34 appeared to be immunorecessive. A few of the HIV-1-positive sera exhibited several immunorecessive reactivities. HIV-1-positive sera from the former Soviet Union and Cuba had reactivities similar to those of American, African, and west European sera. Some sera could not be evaluated as specifically HIV-1 seropositive because of their broad reactivities with a multitude of peptides and proteins, unrelated to HIV-1. Extensive tests were performed to define unspecific reactivities by absorption, blocking, and sandwich ELISAs. The application of the hybrid protein assay substantially improved the specificity of the ELISA tests. Thus, hybrid protein-based ELISAs appeared to be more suitable than peptide-based ELISAs, especially for the evaluation of immunorecessive reactivities.

Amino Acid Sequence↗
PubMed1993

[Localization of an immunodominant site in the hepatitis delta viral antigen using synthetic peptides].

A set of 8 peptides from the immunodominant region (65-80 aa) of delta-antigen was prepared by solid-phase synthesis. Peptide 71-80 was synthesized in two variants--with different amino acid residues in positions 73, 74 and 76. Free peptides and their conjugates with bovine serum albumin were tested for antigenicity in ELISA. The correlation between the peptide chain's length and its antigenic activity was noted. Peptides 65-80 and 69-80 displayed a positive reaction with all individual sera and pools of sera from HDV chronic patients. Both variants of the peptide 71-80 reacted with 100% of sera pools but only with 83% of individual sera. Smaller peptides from the same 65-80 region (73-80, 69-78, 71-78, 71-76) did not bind to any anti-delta positive serum. All synthesized peptides reacted strongly with rabbit antisera raised to the conjugate of peptide 65-80 with bovine albumin. These findings suggest that delta-antigen contains multiple highly immunogenic epitopes associated with the single immunodominant site between 69 and 80 amino acid residues.

Amino Acid Sequence↗
PubMed1992

[Immunobiological properties and therapeutic effectiveness of preparations from Klebsiella bacteriophages].

The purified preparations of Klebsiella bacteriophages, viz. the monovalent preparation of K. pneumoniae bacteriophage and the polyvalent bacteriophage preparation for the treatment of infections caused by K. ozaenae, K. rhinoscleromatis scleromatis and K. pneumoniae sensu lato, have been obtained. The bacteriophage preparations have proved to be nontoxic and safe for laboratory animals after the intraperitoneal injection of these preparations followed by the pathomorphological study of the internal organs of the animals. The clinical study of the newly developed bacteriophage preparations in the course of the treatment of purulent inflammatory diseases in 109 patients has revealed that the preparations are not reactogenic and exhibit sufficient effectiveness in the therapy of ozena, rhinoscleroma and Klebsiella infections with different localization of the infectious process.

Animals↗
PubMed1992

[Synthesis and antigenic activity of peptides of the nucleocapsid protein of the hepatitis delta virus].

Hepatitis delta virus (HDV), a recently discovered infectious agent, participates in severe, often lethal forms of acute and chronic hepatitis and liver cirrhosis. Based on theoretical analysis of secondary structure, hydrophilicity and acrophilicity data, several regions of HDV antigen, presumably containing B-epitopes, have been revealed and the corresponding peptides have been synthesized by the solid phase method. All the peptides obtained reacted with the respective antipeptide rabbit sera. The peptides and their conjugates with BSA or KLH were used for ELISA with individual and pooled anti-HD-positive sera from patients with chronic delta hepatitis. The high antigenicity of the peptide 65-80 shows that one of the antigenically active regions of HDAg is situated between these amino acid residues and that the peptide may be used for detection of anti-HD antibodies in patients blood sera.

Amino Acid Sequence↗
PubMed1991

[Synthesis of peptides of the preS1-region of the viral hepatitis B envelope protein and localization of antigenic determinants].

We synthesized the 24-41, 30-36, 31-36, 24-30 fragments of the preS1-region of the hepatitis B (subtype ayw) envelope. The peptides were prepared by the solid phase synthesis on perfluorpolyethylene polymer grafted with polystyrene. The peptide chains were elongated from C-terminus using activated esters and symmetrical anhydrides of Boc-amino acids, cleaved off the solid phase by HBr or TFMSA in TFA, purified by gel filtration, and, after conjugation with protein carriers, inoculated into test animals. The resultant antibodies were shown to react with peptides. The blood sera from patients with acute hepatitis B reacted with the conjugates of peptides 24-41, 30-36, 31-36 in the immunoenzymic solid phase assay. The monoclonal antibodies for the preS1-polypeptide were shown to react with peptides 24-41, 30-36, 31-36 and with their conjugates. The results obtained were proved by the data of the epitope-mapping with overlapping hexapeptides.

Amino Acid Sequence↗
PubMed1991

The effect of the structure of the terminal regions of the hepatitis B virus gene C polypeptide on the formation of core antigen (HBcAg) particles.

A series of plasmids encoding native and modified sequences of the hepatitis B virus core antigen (HBcAg) was created. Analysis of the products generated by expression of the plasmid genomes in Escherichia coli showed that a polypeptide with primary structure identical to that deduced for native HBcAg forms particles in the bacterial cells which are indistinguishable from the native nucleocapsids in morphological and antigenic properties. Removal of the thirty-nine C-terminal amino acids which form a protamine-like domain caused insignificant impairment of the particle-forming process. Modification of the N-terminal region of the polypeptide showed that at least part of the structural determinant governing particle formation is localised between amino acid residues 3 and 11. When the plasmid genes were expressed in an E. coli cell-free transcription - translation system, polypeptides devoid of ten to twenty N-terminal amino acids were formed in addition to the full-length products. From the results obtained it is proposed that a protease digestion site situated within the region containing amino acid residues 10 - 20 plays a role in the formation of the HBe antigen.

Amino Acid Sequence↗
PubMed1991

[Use of a temporary protective group during the synthesis of oligonucleotides with an unnatural bond].

Methoxygroup has been used for transient P-protection on H-phosphonate oligonucleotide synthesis. Whereas other H-phosphonate linkages can be irreversibly transformed into phosphoramidates by treatment with alkylamines, the protected phosphoralkoxyl groups generate natural phosphodiester linkages after the final deprotection. The method allows for synthesis of oligonucleotides with addressed position of internucleotide phosphoralkylamidate groups.

Base Sequence↗
PubMed1990

[Synthesis and immunological properties of the peptide fragment of the hepatitis B virus envelope preS-protein].

We have synthesized the 24-41 fragment of the preS region of the hepatitis B (subtype ayw) envelope. The peptide was prepared by the solid phase synthesis on perfluoropoly-ethylene polymer grafted with polystyrene. The peptide chain was elongated from C-terminus using pentafluorophenyl- and p-nitrophenyl esters of Boc-amino acids. The peptide was cleaved off the solid phase by HBr in CF3COOH, purified by gel filtration, and, after conjugation with serum albumin (BSA), inoculated into mice. The resultant antibodies were shown to react with the peptide. The blood sera from patients with acute hepatitis B reacted with the peptide-BSA conjugates in the immunoenzymic solid phase assay.

Amino Acid Sequence↗
PubMed1990

[The development of a new generation of pertussis vaccine].

The results of the weight gain test on mice have shown that acellular pertussis vaccine is less toxic than the pertussis component of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine due to a lower content of endotoxin in the acellular vaccine; but the leukocytosis-promoting and histamine-sensitizing activities of JNIH-6 and adsorbed DPT vaccines are indicative of incomplete inactivation of Bordetella pertussis toxin. The content of incompletely inactivated B. pertussis toxin is practically the same in both preparations, constituting 1/100-1/200 of the calculated initial activity. For this reason, the use of the new pertussis vaccine also involves a risk of development of serious postvaccinal reactions and/or complications caused by this toxin. Search for the optimum method of inactivation of B. pertussis main toxin should be continued. As shown by the enzyme immunoassay, acellular pertussis vaccine used in the same immunizing dose as adsorbed DPT vaccine induces a more intensive immune response to hemagglutinin and B. pertussis toxin. This is due to higher residual toxicity of the corpuscular component of adsorbed DPT vaccine. Induction of antibodies to B. pertussis toxin has been shown to decrease in response to injection of acellular pertussis vaccine containing a certain residual amount of incompletely inactivated B. pertussis toxin.

Animals↗
PubMed1989

[Immunobiologic activity of Bordetella pertussis strains defective in various virulence factors].

The immunobiological properties of mutant strains, selectively deprived of certain antigens (hemagglutinin, B. pertussis toxin, dermonecrotic toxin, hemolysin, adenylate cyclase), have been studied with the aim of finding out the relationship between the presence of certain antigens in microbial strains and their protective properties. The results of these studies suggest that the protective potency of pertussis vaccine may be related to the presence of some antigenic substances, including those not pertaining to the known factors of virulence.

Adenylate Cyclase Toxin↗
PubMed1988

Effect of structure of the initiator codon on translation in E. coli.

A set of plasmids carrying different initiator codons--either AUG, or GUG, or UUG, or CUG (as a control) in the hybrid gene lacIZ--was constructed by using synthetic oligonucleotides. GUG and UUG codons were demonstrated to be 2-3 times less effective than AUG in translation initiation. Furthermore, the correlation between the efficiencies of different initiator codons in translation initiation proved to vary, depending on the phase of bacterial growth. The rarely occurring usage in nature of the initiator codons GUG and UUG is supposed to be due to the particular role played by the initiator triplets in regulation of gene expression.

Bacteriophage lambda↗
PubMed1988

[Development of a method for the quantitative determination of Bordetella pertussis toxin and the prospects of its use for diagnosis].

Two variants of the enzyme immunoassay (EIA) systems for the determination of B. pertussis toxin (BPT), the "double sandwich" system and the competitive assay system, have been developed. For the titration of BPT in B. pertussis antigens the use of fetuin as the affinity base is preferable, and not antibodies from different paired animals. Of the two variants, the competitive EIA is more promising for diagnostic purposes.

Animals↗