Antibody to Hepatitis B Core Antigen

Years after infection with hepatitis B virus, chimpanzees may have manifestations of the carrier state as described in man. In addition to serologic evidence for persistent viral infection, percutaneous liver biopsy specimens showed hepatitis B virus surface antigen in the cytoplasm and hepatitis B virus core antigen in the nucleus. Four (3,4). Recently, two distinct types of experimental hepatitis B have been described in chimpanzees, one with rapid resolution and the other with smoldering features characterized by portal inflammatory cell infiltration present for more than 1 year (5). After acute infection, some animals became long-term HBV carriers, maintaining elevated levels of HBsAg, antibody to hepatitis B core antigen (anti-HBc), and Dane particles in the serum. Therefore, it appeared important to study such chimpanzee carriers (a) to establish the histologic features in these chimpanzees, (b) to correlate serologic observations with immunohistochemical findings in the liver, and (c) to characterize the nature of viral DNA in the hepatocytes. This was considered important because human HBV carriers have a significant risk of developing hepatocellular carcinoma (6). In a human hepatocellular carcinoma line (PLC/PRF/5) in tissue culture, HBV DNA has recently been found to be integrated into the host genome (7,8). With development of newer techniques in nucleic acid analysis and availability of recombinant cloned HBV DNA probes (9-14), this question could be addressed in small samples of liver by using very sensitive techniques.We have examined percutaneous liver biopsy specimens and serum from known chimpanzee HBV carriers that were infected experimentally or in the wild. By using a 32P-labeled

contrasting

confidence: 66%

Chronic hepatitis in chimpanzee carriers of hepatitis B virus: morphologic, immunologic, and viral DNA studies.

Shouval¹,

Chakraborty²,

Ruiz-Opazo³

et al. 1980

“…Of this, 40% was found in the medium and 60% was released from the cells by freeze-thawing and sonication. Subtype analysis (22) showed that SVHBV-HBsAg had the same antigenic composition (d+, y -) as the antigen from the original donor of the HBV DNA. SVHBV-infected cells were negative for HBcAg and 8 antigen by immunofluorescence.…”

Section: Methodsmentioning

confidence: 98%

“…HBsAg was detected by the Ausria II radioimmunoassay (Abbott Laboratories) and quantitated by a parallel-line assay with a known standard (BoB HBsAg/adw vaccine, reference lot 1, 40 jig/ml). The d/y subtype of HBsAg was determined by the competition radioimmunoassay method of Hoofnagle (22). All experiments requiring physical containment level P3 were performed at the certified P3 facility of the Georgetown University Division of Molecular Virology and Immunology of ligation assay buffer, and treated with 4 units of T4 DNA ligase for 5 hr at 14°C.…”

Section: Methodsmentioning

confidence: 99%

Expression of the hepatitis B virus surface antigen gene in cell culture by using a simian virus 40 vector

Moriarty

Hoyer

Shih

et al. 1981

139

We have constructed a simian virus 40 recombinant carrying a fragment of DNA from hepatitis B virus. Cultured monkey kidney cells infected with this recombinant produce hepatitis B surface antigen. The antigen is excreted into the culture medium as 22-nm particles with the same physical properties, antigenic composition, and constituent polypeptides as those found in the sera of patients with type B hepatitis.At least half of the world population shows evidence of past or present infection by hepatitis B virus (HBV), and the approximately 200 million carriers in the world are at serious risk of chronic liver disease and, possibly, primary liver cancer. The classic marker for chronic infection by this virus is the surface antigen HBsAg which circulates in the serum of HBV carriers in three forms: 22-nm spherical particles, 22-nm filaments of various lengths, and the 42-nm spherical form known as the Dane particle. The 22-nm particles and filaments are subviral forms containing two predominant polypeptides, with apparent molecular weights of about 23,000 and 29,000, together with several minor polypeptides of larger size (1, 2). The two predominant species, which are probably identical except that the larger is glycosylated, carry both the group (a) and the subtype (d/y) antigenic determinants of HBsAg (3). The Dane particle, which represents the infectious virion, consists of a lipoprotein coat (HBsAg) surrounding an internal core particle which contains a DNA polymerase and the 3200-base pair (bp) circular DNA genome. The 22-nm particle is the predominant form in the sera of chronic carriers and circulates at concentrations as high as 100-200 ,ug/ml.Characterization ofthe life cycle and biology ofHBV has been hampered by its narrow host range, which is restricted to humans and a few other primates, and by its inability to grow in cultured cells. Recently, however, several groups have succeeded in cloning the viral genome in Escherichia coli phage A (4) and plasmid vectors (5, 6) and in determining its primary structure (7)(8)(9). This has allowed the identification of a continuous 892-bp sequence that could encode surface antigen (7), a 549-bp sequence that may specify the core antigen (8), and several additional open sequences of unknown function (9).Although the DNA sequence provides crucial structural information, it clearly is not sufficient to establish all of the HBV gene products or to indicate how these products interact during infection of the target cell. For this purpose it would be useful to develop a system for introducing defined portions ofthe viral genome into cultured cells. Simian virus 40 (SV40), a small DNA tumor virus that can lytically infect cultured monkey cells, provides a useful vector for this purpose. In this paper we describe the construction and propagation of a SV40 recombinant carrying a 1350-bp fragment of HBV DNA that includes the structural sequences for surface antigen. We show that monkey kidney cells infected with this recombinant synthesize surface antigen that is ...

“…Sera were obtained weekly and monitored for anti-HBs by Ausab (A) and anti-P49 by radioimmunoprecipitation as described in Table 2 (B). tibody while the immunoprecipitation assay selects for IgG. The d/y subtype specificity of the anti-HBs response was determined by the method of Hoofnagle et al (21) at wk 2, 3, 4, 6, and 8 for the high-responder chimpanzee 1 and at wk 6 for chimpanzees 2 and 3. The anti-HBs activity of undiluted serum from chimpanzee 1 was totally blocked by HBsAg/ay and not blocked by HBsAg/ad, establishing that the anti-HBs activity throughout the course of response was entirely anti-HBs/y, not anti-HBs/a or d. An anti-HBs/y response was also observed in the wk 6 sera of both chimpanzees 2 and 3, although the low titer prevented the exclusion of anti-HBs/a in one of these.…”

Section: Resultsmentioning

confidence: 99%

“…Sera were obtained weekly and monitored for anti-HBs by a commercial radioimmunoassay (Ausab, Abbott) and antipeptide 49 by radioimmunoprecipitation as described above. The d/y subtype specificity of the anti-HBs response in selected sera was determined by the method of Hoofnagle et al (21).…”

Section: Methodsmentioning

confidence: 99%

Chemically synthesized peptides of hepatitis B surface antigen duplicate the d/y specificities and induce subtype-specific antibodies in chimpanzees.

Gerin¹,

Alexander²,

Shih³

et al. 1983

108

Synthetic peptides, predicted from the nucleotide sequence of the S gene of hepatitis B virus were analyzed in terms of the established specificities of the hepatitis B surface antigen. The analysis indicated that the group-specific a antigen is composed of at least three nonoverlapping sequences and that a relatively hydrophilic region of the surface antigen protein, spanning amino acid residues 110-137, specifies the major d and y subtype system. The d/y subtype appears to depend on changes in one or more variable amino acids at positions 127, 131, and 134 of the hepatitis B surface antigen protein. Peptide 49 (consisting of amino acid sequences of the y subtype for the region 110-137), coupled to a carrier protein and mixed with an adjuvant, stimulated a brisk anti-y response in chimpanzees, the relevant model of human response to hepatitis B virus immunization and infection. Experimental challenge with homologous hepatitis B virus resulted in a pattern of partial protection. The results offer promise for the application of chemically synthesized peptides as vaccines in the prophylaxis of hepatitis B virus disease.Although a number of studies have suggested that the immunogenicity of a protein molecule depends largely on its conformation (1-3), more recent investigations have shown that reasonably short chemically synthesized peptides elicit antibodies reactive with virtually any region of an exposed surface of a protein (4). Coupled with the rapid determination of amino acid sequences via nucleic acid sequence analysis technology, synthetic peptides offer promise for vaccines that, in molecular terms, can be designed with a precision not heretofore possible.Hepatitis B virus (HBV) represents an important test model of the synthetic peptide vaccine approach. Current vaccines for HBV (5) consist of subviral components of the virus surface coat (HBsAg) purified from the plasma of chronically HBV-infected donors and inactivated. HBsAg has been the subject of extensive immunochemical characterization and consists of a groupspecific (a) and two sets of subtype-specific (d/y, w/r) determinants (6, 7); these specificities are associated with a single polypeptide (8, 9), the entire 226-amino acid sequence of which has been determined from the nucleotide sequence of the S gene (10) of It has been shown by radioimmunoprecipitation (15) and commercial solid-phase radioimmunoassays for anti-HBs (16) that antisera to synthetic peptides predicted from the nucleotide sequence of various regions of the S gene of HBV react with native HBsAg. We report here an analysis of the specificities of synthetic peptides in terms of the established HBsAg determinants and the immunogenicity of one such peptide in chimpanzees, the relevant human model of response. The data indicate that the group-specific (a). determinant is composed of at least three nonoverlapping sequences and that a relatively hydrophilic region of the HBsAg protein, residues 110-137 (numbered 1-226 from the amino terminus), specifies the major d/y subtype s...