[Bacterial synthesis of immunogenic epitopes of foot-and-mouth disease virus fused either to human necrosis factor or to hepatitis B core antigen]. / Bakterial'nyi sintez immunogennykh épitopov virusa iashchura v sostave gibridov s faktorom nekroza opukholei i kor-antigenom virusa gepatita B.

Using recombinant DNA technology, construction and bacterial expression of genes was carried out which code for hybrid proteins, human tumor necrosis factor and hepatitis B core protein fused to immunogenic epitopes of foot-and-mouth disease virus, strains A22 and O1-194. Hybrids of tumor necrosis factor with foot-and-mouth disease antigenic determinants protected laboratory animals against the experimental challenge with a homologous strain of foot-and-mouth disease virus. Hybrid protein that contained immunogenic regions of two strains, A22 and O1-194, protected animals against infection with both A and O serotypes. Hybrid proteins based on hepatitis B virus core antigen retained the ability to assemble into core-like particles.

Locations of antibody binding sites within conserved regions of the hepatitis C virus core protein.

The binding sites for human antibodies recognising antigenic domains within the hepatitis C virus (HCV) core protein were analyzed using synthetic peptides. Omission peptide analogues where a pair of residues was sequentially omitted were produced corresponding to the regions 1-18, 11-28, 21-38, 51-68, and 101-118. The N-terminal part of HCV core was found to contain three distinct antibody binding sites, which includes the previously reported one at residues 9-16. The other two were located at residues 19-26 and residues 29-34. Within the region 51-68 two overlapping sites were found, the first at residues 51-60 and the second at residues 59-68. Within the region 101-118, residues 107-114 were identified as the binding site, which contains two residues differing between genotypes I/II and III/VI. Thus the HCV core protein contains at least six distinct linear antibody binding sites, located at regions highly conserved between the genotypes of HCV. The human recognition of these regions show a variation with respect to the amino- and carboxy-terminal extension of each individual binding site. These findings will have implications for the prediction of the structure of the HCV core protein, since these antibody binding sequences are likely to be more or less accessible from the exterior of either, or both, of the native HCV core and its precursor polyprotein.

[PreS-sequence of the hepatitis B virus envelope: molecular biology and immunology]. / PreS-posledovatel'nosti obolochki virusa gepatita B: molekuliarnaia biologiia i immunologiia.

Current data on molecular biology and immunology of the proteins of hepatitis B viral capsid encoded by the preS-region of HBV genome are reviewed. Main attention is paid to study of the immune properties of the capsid proteins, to search of immune dominant epitopes encoded in preS-sequences. The medical and biological significance of the research is shown as exemplified by search of preS-antigens and anti-preS-antibodies in serum from patients suffering hepatitis B. The use of preS-sequences as components of vaccine preparations is discussed.

Immunogenicity of recombinant core particles of hepatitis B virus containing epitopes of human immunodeficiency virus 1 core antigen.

A Gag protein segment of human immunodeficiency virus 1 (HIV-1) has been fused to a C terminally truncated core antigen of hepatitis B virus (HBcAg) using an E. coli expression system. Fusion of 90 amino acids of HIV-1 Gag protein to HBcAg still allowed the formation of capsids presenting on their surface epitopes of HIV-1 core protein, whereas fusion of 317, 189, or 100 amino acids of Gag prevented self-assembly of chimeric particles. Mice immunized with recombinant particles emulsified with Freund's complete adjuvant (CFA) or aluminium hydroxide developed high anti-HBcAg titers. However, anti-HIVp24 antibodies were detected only in mice inoculated with immunogen emulsified with CFA.

Determination of the minimal length of preS1 epitope recognized by a monoclonal antibody which inhibits attachment of hepatitis B virus to hepatocytes.

The minimal amino acid sequence sufficient to be recognized efficiently by virus-attachment inhibiting murine monoclonal anti-preS1 antibody MA18/7 has been determined. We have constructed a recombinant gene library using the cloned coat protein gene of Escherichia coli RNA bacteriophage fr as a carrier. Different fragments of preS1 region from cloned hepatitis B virus (HBV) genomes, subtype ayw and adw, were inserted at position 2 of the 129 amino acid-long fr coat protein gene in the appropriate E. coli expression vectors. Fine mapping of preS1 epitope recognized by MA18/7 was accomplished by bidirectional shortening of the preS1 within original recombinant preS-fr coat protein genes with Bal31 exonuclease. Immunoblot analysis of the obtained recombinant protein library revealed that the tetrapeptide Asp-Pro-Ala-Phe (DPAF), located at the position preS(31-34) and conserved in all known HBV genomes, is sufficient to bind MA18/7 antibody. Recognition of the preS1 region by MA18/7 occurred irrespective of the amino acid context surrounding this DPAF tetrapeptide. Further shortening of this minimal epitope from the left or from the right side completely prevented antibody binding in immunoblots.

[Exposure of the major immunodominant epitope of the gp51 envelope protein of bovine leukosis virus on the surface of the hepatitis B core antigen capsid]. / Eksponirovanie mazhornogo immunodominantnogo épitopa obolochechnogo belka gp51 virusa leikoza krupnogo rogatogo skota na poverkhnosti kapsid kor-antigena virusa gepatita B.

Insertion of 48 amino acid long sequence of envelope protein gp51 of bovine leukemia virus (BLV), located from position 56 till 103 of mature protein, into Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids. These capsids preserve morphology of intact HBcAg but expose on their outer surface BLV epitopes which are localised in the inserted gp51 fragment and responsible for the recognition of chimeras by monoclonal anti-gp51 antibodies MAK14. The anti-genicity of gp51 epitopes within chimeric capsids is not disturbed after shortening of C terminal part of inserted gp51 fragment by deletion of amino acids 73-103. The resulting chimeras show the same capsid-forming ability as well as HBcAg and gp51 antigenic properties.

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

[Study of the antigenic structure of hepatitis B virus proteins. I. Synthesis of pre-S fragments of hepatitis B virus proteins and their immunogenicity]. / Izuchenie antigennoi struktury belkov virusa gepatitaa B. I. Sintez fragmentov pre-S belkov virusa gepatita B i izuchenie ikh immunogennosti.

Fragments of hepatitis B virus envelope proteins corresponding to the parts of the pre-S domain were synthesised and immobilized on the carriers with low own immunogenicity. The highest stimulation of the antibody production was observed for the antigens immobilized on microspherical carriers or gelatin modified by H-Gly-Tyr-OH. Among peptides used for immunization, pre-S fragment 134-144, conjugated with microspherical carrier, proved to be the most active.

Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface.

Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg.

[Genetic transformation of somatic cells. XIV. Expression of gene coding for the human hepatitis B virus surface antigen in mammalian cells]. / Geneticheskaia transformatsiia somaticheskikh kletok. XIV. Ekspressiia gena, kodiruiushchego poverkhnostnyi antigen virusa gepatita B cheloveka, v kletkakh mlekopitaiushchikh.

A set of recombinant plasmids with a gene encoding surface antigen of hepatitis B virus (HBsAg) is constructed. The plasmids were transfected by DEAE-dextran method into different lines of cultured cells and transient expression of the HBsAg gene was studied. The results indicate that: transcriptional enhancer of hepatitis B virus situated downstream from HBsAg gene is active in green monkey kidney cells (CVI), promoter of 5 LTR of bovine leukemia virus is trans-activated in the goat or calf cells infected with BLV. The results are discussed in the light of hypothesis on the role of transcriptional enhancers in determination of tissue-specificity of hepatitis B virus.

[Structure and expression of human hepatitis B virus surface antigen]. / Struktura i ékspressiia gena poverkhnostnogo antigena virusa gepatita B cheloveka.

In view of the growing occurrence rate of the virus-induced hepatitis B and also of the special role played by this particular virus (HBV) in the application of recombinant genetic techniques to the study of complex biological systems, an attempt was made to survey the available evidence concerning the widely investigated and practically the most important part of the viral genome, viz. the gene coding for the surface antigen (HBsAg) and the protein itself. The possible antigenic structure of the protein was investigated using data on the primary structure of 11 cloned HBsAg gene variants and on the synthesis of peptides simulating its immunological properties. Special emphasis was placed on quantitative assessment of antigenicity and immunogenicity. Expression of the gene in homologous systems was studied using cultures of eukaryotic tissues: both as part of HBV nucleotide sequences incorporated into the chromosome and as part of extrachromosomal DNA. The latest findings on HBsAg gene expression in yeast and bacteria are reviewed.

Subtype ayw variant of hepatitis B virus. DNA primary structure analysis.

The entire genome of human hepatitis B virus (HBV) occurring in Latvia was sequenced. This sequence, which is 3182 nucleotides long, was compared with the other previously published HBV genomes and was shown to share maximum homology with HBV subtype ayw DNA. The coordinates of 4 main open reading frames as well as hairpin structures are very well conserved in the two genomes. The distribution of nucleotide substitutions among different HBV genomes suggest that the open reading frames P and X can fulfil a coding function. On the basis of primary structure comparison for hepadnaviral DNAs several evolutionary conclusions can be drawn.

Expression of hepatitis B virus surface antigen gene in Escherichia coli.

Direct expression of hepatitis B virus (HBV) surface antigen (HBsAg) gene under the control of the Escherichia coli tryptophan operon (trp) promoter has been achieved. Synthesis of HBsAg (both complete and lacking its N-terminal segment) as a part of hybrid proteins with the N-terminal portion coded by genes cat, kan or bla is controlled by the appropriate promoters, as well as by the trp promoter. The highest levels of expression, including those for direct synthesis of HBsAg, provide the accumulation of about 10(5) polypeptide molecules per bacterial cell.