Receptor-mediated transport of oligodeoxynucleotides into hepatic cells.

Receptor-mediated endocytosis was employed for a highly efficient transport of oligodeoxynucleotides into hepatoma cell line PLC/PRF/5. The oligodeoxynucleotides were bound to the asialofetuin-poly-L-lysine conjugate and this complex was internalized by the cells via asialoglycoprotein receptor, an endocytic receptor unique for hepatocytes. Binding of the oligodeoxynucleotides to the complex dramatically increased their cellular uptake more than 20-fold. Chloroquine, a lysosomatropic agent, further increased the transport of the complex but not of the free oligodeoxynucleotides.

Inhibition of hepatitis B virus surface gene expression by antisense oligodeoxynucleotides in a human hepatoma cell line.

We have studied the inhibitory effect of antisense oligodeoxynucleotides on the expression of hepatitis B virus surface antigens. Human hepatoma cell line PLC/PRF/5 harbors several integrated copies of the HBV genome and produces and secretes hepatitis B virus surface antigen (HBsAg) to the medium. Synthetic antisense oligodeoxynucleotides complementary to various regions of the surface antigen gene were synthesized and their ability to block its expression was tested. Oligodeoxynucleotides (17- and 21-mers) complementary to regions covering ATG codons of both preS2 and S genes significantly inhibited preS2 and S protein production. Less efficient inhibition was achieved when the oligonucleotide complementary to the inside S gene region was assayed.

Expression of large hepatitis B envelope protein mutants using a new expression vector.

Aminoterminal deletion mutants of the gene encoding the large hepatitis B surface protein were expressed in COS cells using a new expression vector. The truncated protein showed the same intracellular retention like the wild type protein. The findings show that the secretion block of the protein is not due to its aminoterminal myristylation.

Absence of free core antigen in anti-HBc negative viremic hepatitis B carriers.

Using enzyme immune assay and immune electron microscopy, we have examined the sera of immune-suppressed anti-HBc negative HBV-infected patients for the presence of HBcAg. Our results suggest that free HBV core particles are absent or present only in minute amounts in the blood of chronic carriers and that at the most, only minimal amounts of core antigen are found on the surface of the virus particles.

HBc and HBe specificity of monoclonal antibodies against complete and truncated HBc proteins from E. coli.

We have prepared and used monoclonal antibodies against various populations of full-length and truncated hepatitis B core proteins in order to distinguish between epitopes of HBcAg and HBeAg. Our results show that various epitopes are specific for the different proteins. Certain epitopes, however, are ubiquitous to HBc/e proteins and these are probably exposed on the surface of HBc particles.

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.

Detection of antibodies against hepatitis B core antigen using the avidin-biotin system.

An enzyme avidin-biotin assay for the detection of anti-HBcAg antibody in human sera was developed. The assay uses genetically engineered HBcAg. HBcAg is immobilized on the surface of the wells of microtitre plates and the test serum sample, biotin-labelled HBcAg and streptavidin-labelled horseradish peroxidase are added. The assay was found to be specific and was compared with a commercial radioimmunoassay kit for sensitivity by testing 96 human clinical sera for anti-HBcAg antibody. Both assays gave identical results.

Expression of hepatitis B virus large envelope protein in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for hepatitis B large envelope protein was cloned under the lac promoter in bacterial vector pUC-8 and under the ADH1 promoter in yeast expression shuttle vector pVT103-U, and expression of HBsAg in bacteria and yeast was determined. The strongest expression of large envelope protein was obtained after transformation of the protease-deficient yeast strain BJ1991. The recombinant large envelope protein did not form complex 22-nm particles and was not secreted into medium.

A suppressive mechanism counteracts the production of anti-idiotype antibody.

BALB/c mice were repeatedly, at two-week intervals, immunized with monoclonal antibody against hepatitis B surface antigen and their sera were titrated for anti-idiotype antibody. The highest titres appeared after 1-4 immunizations. Further immunizations led to fast disappearance of the anti-idiotype antibody. These data suggest that anti-idiotype antibody production is a rather complicated process in which some suppressive mechanism is involved.

Monoclonal antibodies against genetically manipulated hepatitis B core antigen.

Four different hybridoma clones secreting anti-HBcAg antibodies were constructed by fusing cells of the mouse myeloma line SP2/0 with lymphocytes from mice immunized with bacterially produced HBcAg. The monoclonal antibodies were immunologically characterized and used for HBcAg detection by ELISA. This monoclonal-antibody-based assay was compared with ELISA based on polyclonal human anti-HBcAg IgG for sensitivity and specificity. The monoclonal antibody reacted specifically both with the bacterially produced HBcAg and HBcAg isolated from human liver, but did not react with HBeAg. The human polyclonal antibody reacted with HBcAg, but also with HBeAg.

Anti-idiotype antibody as a prospective vaccine against hepatitis B.

Rabbit and mouse sera containing anti-idiotype antibodies against a monoclonal and against some polyclonal anti-HBsAg antibodies were prepared. The immunoglobulin fractions of these sera induced the formation of anti-HBsAg antibodies when injected into BALB/c mice. The sera of humans repeatedly immunized with human anti-HBsAg antibodies were shown to contain anti-idiotype antibodies capable of eliciting an anti-HBsAg response in BALB/c mice and Syrian hamsters. Such sera may be a potential source of human anti-idiotype vaccine.

Detection of antibodies to hepatitis B surface antigen (HBsAg) using monoclonal antibody and the avidin-biotin system.

A direct ELISA using biotinylated HBsAg and a competitive ELISA using biotinylated monoclonal antibody were developed for the detection of antibodies to HBsAg. Both tests are capable of detecting 0.1 I.U. of anti-HBsAg antibody/ml. The direct ELISA was compared with a SPRIA test for anti-HBsAg antibody in human sera.

Monoclonal antibodies to hepatitis B surface antigen: production and characterization.

Hybridomas secreting anti-HBsAg antibodies were produced by fusion of the mouse myeloma cell line SP2/0 with lymphocytes from mice immunized with purified HBsAg. All clones produced antibodies of the IgG1 idiotype that react with the subtype a determinant of HBsAg. An enzyme immunoassay for detection of HBsAg in human sera using monoclonal antibodies was developed and compared with commercial Sevatest ELISA HBsAg/micro I kit for detection of HBsAg in clinical serum samples.

Genetic structure of the endogenous proviruses and expression of the gag gene in Brown Leghorn chickens.

Seven loci of endogenous proviruses were detected in the genome of Brown Leghorn chickens. Sets of endogenous proviruses in DNA of the chicken embryos examined were identified by blot hybridization with 32P-labelled DNA of RSV and EcoRI restriction endonuclease digestion. Comparison of the results showed that only one locus (A) of endogeneous provirus was associated with a gs+ phenotype as determined by the immunoperoxidase reaction and antibodies against gag gene products of RSV. Restriction endonuclease analysis with HindIII, BamHI and SacI revealed that proviruses A and F in Brown Leghorn chickens correspond to loci ev-3 and ev-6, respectively, in White Leghorn chickens. Other loci (B, C, D, E, and X) were designated ev-22, ev-23, ev-24, ev-25, ev-26, respectively. None of these loci expressed infectious virions. The structure of most of the endogenous proviruses examined is considerably different from the genome of the endogenous chicken virus RAV-O. The difference in structure may be one possible cause of the absence of endogenous provirus expression.

Amino acid sequence homology between protein products of oncogenes and hormones (v-myc--gastrin and oxytocin; v-sis--secretin).

Computer comparisons of amino acid sequences from 8 retroviral oncogenes and 26 protein hormones were done with respect to structure similarity and so-called uninterrupted structure similarity. Sequence homology was found between v-myc, the transforming protein of MC29 virus, and human gastrin and oxytocin on the one hand, and between v-sis, the transforming protein of simian sarcoma virus, and secretin on the other.

Effect of the expression of an endogenous viral gene on the growth of tumours induced by Rous sarcoma virus in chickens.

The endogenous group-specific antigen of avian retroviruses has been detected in feather pulp of chickens of the inbred CB line (WL breed). Expression of the gs antigen has been demonstrated by ELISA, whereas the classical methods have classified the CB line as gs-. The number of gs+ chickens decreased with increasing age after hatching, and all chickens were gs- already at 20 weeks of age. The longer duration of gs antigen expression in line CB chickens had an adverse effect on their ability to regress Rous sarcomas. Chickens from a closed flock of the BL breed that segregated to gs+ and gs- also significantly differed in the ability to regress Rous sarcomas.

Genetic linkage of endogenous viral loci with the B (MHC) and C histocompatibility loci in chickens.

Genetic linkage of endogenous viral loci and histocompatibility loci B (MHC) and C has been demonstrated serologically in backcross progeny of inbred lines of chickens. The endogenous viral locus linked to the B complex is the first case of the localization of an endogenous viral gene to the microchromosomes in chickens.

A rapid detection of avian oncovirus group-specific antigens in feather pulp by the enzyme-linked immunosorbent assay.

An ELISA procedure for the detection of avian sarcoma-leukosis gs antigen in feather pulp of adult birds is described. The test can be used for identifying gs+ and gs- chickens in leukosis-free populations. The titres of exogenous and endogenous virus-coded gs antigens overlap and the ELISA can be used only for a preliminary screening of unknown chicken populations.

[Analysis of the susceptibility of the Minor inbred chicken strain to Rous sarcoma virus infection]. / Analýza vnímavosti inbrední linie slepic minor k infekci virem Rousova sarkomu.

The sensitivity and resistance of the Minor hen inbred line to two antigenic subgroups of the Rous sarcoma virus were determined by the method of the infection of chorioallantoic membranes. The Minor line is sensitive to infection with the virus of the antigenic B subgroup and, as demonstrated by the testing of the interline hybrids of F1 and B1 generations, the birds of the Minor line are dominant homozygotes of genotype bsbs. The testing with the virus of antigenic A subgroup revealed a heterogeneity of the inbred population as to sensitivity and resistance. A formula was derived for the determination of the frequency of the dominant allele as determining sensitivity.