Immunisation Against Hepatitis B in Man

On the basis of theoretical considerations, a peptide (H Discovery of hepatitis virus-specific antigens (1-3) and their association with the outer membrane ofhepatitis B virus (HBV) (4, 5) has led to rapid advances in the prevention of HBV infection. This has been achieved through detection of HBV carrier blood donors by testing for the HBV surface antigen (HBsAg) (6, 7), and by passive (8-12) and active (13-18) immunization.The protective effect of hepatitis B immune globulin, prepared from plasma selected for high anti-HBsAg content, as well as the demonstration that protective immunity can be induced, albeit at high cost, by the purified 23,000-dalton HBsAg polypeptide isolated from HBV-associated subviral particles (19), has suggested that anti-HBsAg is a protective antibody. Other surface specificities on the HBV virion could also play a similar role (18,20).HBsAg also exhibits generally mutually exclusive strain-specific subtype antigenic determinants-e.g., d/y (21) and w/r (22)-in addition to the determinant a common to all strains. Available evidence suggests that the subtype specificities play no role in providing protective immunity (23,24), and thus that HBsAg/a may be the critical determinant.It is thus ofobvious interest to learn the amino acid sequence of the antibody-combining site, or epitope, of HBsAg/a. Progress towards this goal has depended on two key advances: isolation and characterization of HBV DNA (25) and determination of its sequence through recombinant DNA methods (26)(27)(28)(29). This permitted prediction of the amino acid sequence of the product of the HBsAg gene (the s gene) (30,31). Using a theoretical model, Hopp and Woods (32) were then able to predict a putative dominant HBsAg epitope (termed H epitope): the sequence Lys-Pro-Thr-Asp-Gly-Asn corresponding to positions 141-146 on the HBsAg protein. They then synthesized a tetradecapeptide (H peptide) containing residues 138-149 of HBsAg plus two glycine residues at its COOH terminus, using classical solid-phase peptide synthetic methodology. The four cysteinyl residues were replaced by a-aminobutyric acid in order to prevent polymerization and other side reactions common to sulfhydryl-containing peptides. Polystyrene beads covalently coated with H peptide bound more "2I-labeled anti-HBsAg than did uncoated beads (33).We report herein confirmation of the prediction that the H peptide contains a major epitope for HBsAg, and we further demonstrate that this sequence of amino acids contains the HBsAg/a and HBsAg/d epitopes, but not the epitope ofHBsAg/ y or human serum albumin. Furthermore, we demonstrate that when bound to a carrier the peptide induces the synthesis of anti-HBsAg in vivo.MATERIALS AND METHODS H Epitope Polypeptide Preparations. Initial experiments were done with intact polystyrene beads (XE305A resin beads, Rohm and Hass, Philadelphia) on which the H peptide had been synthesized. These beads contained 10 mg of peptide per 25 mg.For immunization it was necessary to remove the peptide from the polystyrene ...

Section: Munizationmentioning

confidence: 99%

Section: Munizationmentioning

confidence: 99%

Hepatitis B virus vaccine: identification of HBsAg/a and HBsAg/d but not HBsAg/y subtype antigenic determinants on a synthetic immunogenic peptide.

Prince¹,

Ikram²,

Hopp³

1982

Section: Introductionmentioning

confidence: 99%

“…HBV DNA is the smallest known mammalian viral genome. These unique characteristics of the genome, the frequency of hepatitis B, and the likely relationship between this virus and primary liver carcinoma (7,8) justify an accurate investigation of the structure of the HBV genome.In this article we present a physical map of the HBV genome. Twenty eight restriction sites are located, and new information concerning the physical structure of HBV DNA is also established.…”

mentioning

confidence: 99%

Cloning in Escherichia coli and physical structure of hepatitis B virion DNA.

Charnay¹,

Pourcel²,

Louise³

et al. 1979

154

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and the 5' end of the short strand was mapped at a fixed position. Hepatitis B is a frequent and sometimes severe disease. In some areas of Asia and tropical Africa, 10% of the population carry the viral surface antigen (1). The Dane particle (2), found in the sera of patients, is considered to be the etiological agent (3). It contains a circular DNA molecule, hepatitis B virion (HBV) DNA, with a nick (or a short gap) and a single-stranded region (4-6). HBV DNA is the smallest known mammalian viral genome. These unique characteristics of the genome, the frequency of hepatitis B, and the likely relationship between this virus and primary liver carcinoma (7,8) justify an accurate investigation of the structure of the HBV genome.In this article we present a physical map of the HBV genome. Twenty eight restriction sites are located, and new information concerning the physical structure of HBV DNA is also established. In particular, the single-stranded region is localized on the restriction map. The position of the nick is discussed. MATERIALS AND METHODSDNA Preparations and Cloning. HBV were purified as described by Summers et al. (4) and their DNA was labeled with the endogenous DNA polymerase (6), with [32P]dTTP (22 Ci/mmol, 1 Ci = 3.7 X 1010 becquerels) as the radioactive precursor. After purification of the HBV DNA (4), it appeared from electron microscope observation that, under the action of the endogenous DNA polymerase, the single-stranded region of about 10% of the DNA molecules was totally repaired.The purified HBV DNA was used for cloning. The circular HBV genome was cleaved by EcoRI endonuclease at its unique restriction site (J. Summers, personal communication) and inserted in vitro into the DNA of bacteriophage Xgt WES. XB as described (9). After transfection of C600 recBC bacteria with the hybrid DNA, recombinant phage clones were purified three times. For each recombinant clone a first phage stock was made and, in order to avoid any genetic drift, all subsequent stocks were made from this initial sample. The cloned EcoRI DNA fragment will be referred to as Eco HBV DNA.After digestion of the DNA of a recombinant clone with EcoRI endonuclease, Eco HBV DNA was purified from the two vector arms by zone centrifugation in a 5-30% sucrose gradient.Enzymatic Digestions and Chemicals. Digestions by the following restriction nucleases (New England BioLabs), EcoRI, BamHI, Sma I,Xho I, Pst I, Xba I, Bgl II, Sst I, Sal I, Kpn I, HincII, and Hae III, were performed in the buffers recommended by New England BioLabs. Alkaline phosphatase (P-L Biochemicals) was used in 10 mM Tris-HCI, pH 8.5/10 mM MgCl2. The 5' ends were labeled by means of [y-32P]ATP (New England Nuclear, 3000 Ci/mmol) and polynucleoti...

“…The global incidence of this disease could be greatly reduced with a safe, effective, and inexpensive vaccine that could be easily administered to all populations at risk. Current immunization against HBV requires intramuscular injection of HBV major surface antigen (HBsAg) purified from plasma of HBV carriers (2)(3)(4) or from recombinant yeast (5,6). Live recombinant viral vaccines represent an attractive alternative to the present HBV vaccines.…”

mentioning

confidence: 99%

Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters.

Morin¹,

Lubeck²,

Barton³

et al. 1987

101

Recombinant adenoviruses carrying the hepatitis B virus surface antigen coding sequence in the adenovirus E3 region were constructed using DNA from either adenovirus type 5 or an adenovirus type 5 E3-region deletion mutant. Both of these recombinant adenoviruses replicated as efficiently as wild-type adenovirus in all human cells tested, including the human diploid cell strain WI-38. This indicates that insertion of the hepatitis B virus surface antigen gene into the E3 region does not significantly affect viral replication. Human cells infected with these recombinant adenoviruses secreted immunoreactive hepatitis B virus surface antigen. Since a practical small animal model for human adenoviruses was lacking, a hamster model was developed to evaluate the immunogenic potential of these recombinant adenoviruses. Upon intranasal inoculation, both wildtype adenovirus and the adenovirus E3-region deletion mutant replicated in the lungs of these animals and induced an antibody response against adenovirus. Hamsters similarly immunized with the live recombinant adenoviruses produced antibody against both adenovirus and hepatitis B virus surface antigen. (7,8). They give rise to asymptomatic intestinal adenovirus infections in humans that induce immunity against adenovirus respiratory disease. These characteristics have prompted us to develop recombinant adenoviruses that direct infected cells to produce HBsAg and thus confer immunity against both adenovirus and HBV. Vaccinia virus recombinants that direct production of HBsAg in animals (9, 10) were designed with a similar strategy in mind; however, live recombinant adenovirus vaccines provide the convenience of oral administration. In this study we describe recombinant adenoviruses that carry the HBsAg-coding sequence in the adenovirus E3 region and that direct the production of immunogenic HBsAg. Hepatitis MATERIALS AND METHODSCells and Viruses. Cell line 293 derived from human embryonic kidney (11) was used for calcium phosphate transfection as described (12). Adenovirus type 5 (Ad5) and the recombinant adenoviruses described below were grown and titrated on 293 cells as well as on A549 cells (13) derived from human lung carcinoma. These viruses were also grown on the human diploid cell strain WI-38 (14).Immunological Reagents. HBsAg was assayed using diagnostic RIA kits from Organon Teknika (Irving, TX) and from Abbott (North Chicago, IL). The levels ofantibodies directed against HBsAg were measured using a diagnostic RIA kit (AUSAB) from Abbott. Antibody levels were converted from RIA units to milliinternational units (mIU) based on an equivalence factor of 3.5 RIA units per 1 mIU. Monoclonal antibody A5C11 against HBsAg was obtained from Centocor (Malvern, PA).Metabolic Radiolabeling and Electrophoretic Analysis. A549 cells were metabolically radiolabeled using L-[35 ]cysteine at 260 p.Ci/ml (1 Ci = 37 GBq) during either the early phase (4.5-9 hr after infection) or the late phase (22.5-27 hr after infection) of infection with either AdS E3HS or AdS AE3HS...