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A solid-phase enzyme-linked immunoassay using a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was developed. Antibodies were covalently linked to glutaraldehyde-activated 96-well aminopolystyrene plates. Antigens from test samples were adsorbed to the solid phase and detected using antibodies conjugated with E. coli beta-galactosidase. Glutaraldehyde, N-succinimidyl-3-(2-pyridyldithio)-propionate or N-succinimidyl-6-(4-azido-2-nitrophenylamino)-hexanoate were used as linkers between antibodies and the enzyme. The measurement of fluorescence can be automated for rapid screening of many specimens. The sensitivity limit of the test for HBsAg is about 5-10 pg.

The influence of Murabutide, a muramyl peptide analog, on the response of mice to two synthetic hepatitis B virus antigens was compared with the activity of Al (OH)3 and of FCA. The synthetic peptides represented fragments either of the major component of the hepatitis B surface antigen (HBsAg) or of the pre-S region. They were used either conjugated to toxoid carrier or as a totally synthetic preparation, i.e. copolymerized with a streptococcal peptide. Our results demonstrated that treatment with Murabutide increased the levels of antibodies, modulated their specificity and allowed a better recognition of the natural antigens.

The unavailability of serological tests for detection of several not yet characterized infectious agents transmitted by blood transfusion or by blood products prompted the development of alternative tests based on utilization of labeled nucleic acid probes specific for genomes of each of these agents. The prerequisite for the preparation of such probes is the demonstration in human plasma of nucleic acid sequences distinct from those present in host DNA or in genes of already characterized viruses occurring in plasma of infected individuals. To accomplish this, ultrasensitive tests for nucleic acids not dependent on their base sequence are needed. We describe here a radioimmunoassay (RIA) for picogram quantities of DNA. Plasma (serum) specimens are treated with proteinase K in the presence of sodium dodecyl sulfate and extracted with phenol. Nucleic acids are precipitated with ethanol in the presence of dextran (mol. wt. approximately equal to 5 X 10(5)) as carrier. Subsequently, DNA from the redissolved samples is adsorbed onto polylysine-coated wells of microtiter plates and detected by a double-antibody RIA using anti-DNA autoantibodies from NZB/NZW mice and 125I-labeled antibodies to mouse immunoglobulins. DNA which did not hybridize with human DNA was detected by this method in sera containing hepatitis B virus used as a model system.

A family of antigens, referred to collectively as e antigen (eAg), has been detected in sera of some individuals with liver disease who test positive for hepatitis B surface antigen. Studies on eAg partially purified by affinity chromatography on insolubilized antibodies to eAg revealed the following: (i) eAg has the physicochemical and immunologic properties of an immunoglobulin, predominantly of the IgG4 subclass, and (ii) specific antigenic sites, designated as e determinants, differentiate eAg from other immunoglobulins. We suggest that these determinants represent idiotypic determinants on antibodies that are formed in response to hepatitis B virus infection and that block the host's immune surveillance mechanisms by which virus proliferation is stopped.

Hepatitis B virus (HBV) has not yet been propagated in vitro, and knowledge concerning its reaction with receptors on target cells remained scant. We have located within the HBV envelope proteins a sequence mediating the attachment of HBV to human hepatoma HepG2 cells. A synthetic peptide analog (PLGFFPDHQLDPAFGANSNNPDWDFNP) is recognized by both cell receptors and anti-HBV antibodies and elicits antibodies reacting with native HBV. The synthetic peptide is a promising immunogen expected to elicit protective antibodies based on the concept of the attachment blockade pathway of virus neutralization. The approach described here, based on anti-peptide antisera and the binding of peptide analogs to cell receptors is generally applicable for the delineation of cell receptor binding sites on viruses with known gene sequences.

Thyroxine and triiodothyronine binding sites are present on the 20-nm spherical particles associated with hepatitis B surface antigen (HBsAg). Thyroxine-treated HBsAg has a buoyant density of 1.26 g/cm-3 in CsCl and appears under the electron microscope as a hexagonal particle with a center-to-vertex distance of 10 nm. These results provoke questions concerning the origin of thyroid hormone binding sites on HBsAg and a possible relationship between thyroid status and HBsAg antigenemia in humans.

Studies with synthetic peptides have provided evidence for the presence of preS coded sequences in the envelope (env) proteins of hepatitis B virus (HBV) and indicated that these sequences are involved in the specific attachment of HBV to liver cells. Scanning of the preS sequence for immunodominant continuous epitopes identifies the sequence within residues preS (120-145) as the most immunogenic in eliciting antibodies recognizing HBV and as the most efficiently binding antibodies from sera of rabbits and humans immunized with HBV env proteins. To assess the potential of preS (120-145) as a synthetic vaccine against hepatitis B, in vitro neutralization of the virus by rabbit antiserum to the peptide was assayed in chimpanzees. The animals, subsequently proven to be susceptible to HBV infection, did not develop hepatitis B as judged by negative serological tests for HBV-associated antigens and antibodies and by normal serum alanine aminotransferase levels and normal liver biopsies. These results establish the role of preS domains in the process of virus neutralization and the potential of synthetic preS analogues for hepatitis B vaccination.

A novel minor constituent was isolated from normal human serum by affinity chromatography on columns of insolubilized concanavalin A and antibodies to albumin, followed by rate zonal centrifugation. This component has the following properties: a sedimentation coefficient of approximately 31; a diameter of 14--22 nm, and a buoyant density of 1.302 gm/cu cm. It contains about 1% neutral sugars and is electrophoretically heterogeneous, since two populations of particles with isoelectric points of pH 4.95 and 5.75 were separated by isoelectric focusing. It contains a single major glycopeptide with an apparent molecular weight of 72,000 daltons. Its amino acid composition is distinct from that of albumin. It elicited the formation of antibodies that also reacted with HBsAg.

Immunodominant B and T cell epitopes have been demonstrated recently on the preS1 and PreS2 regions of the hepatitis B virus (HBV) envelope protein. Synthetic peptide analogs corresponding to the preS2 region elicit virus-neutralizing antibodies and protect chimpanzees against HBV infection. Antibodies raised by immunization with peptides derived from the preS1 sequence block the site involved in HBV attachment to cell receptors, and are expected to be virus-neutralizing. Results presented here show that antisera raised against synthetic peptide analogs carrying the immunodominant epitope of the preS1 and preS2 sequence, respectively, and corresponding to two HBV subtypes, adw2 and ayw, each recognized preS1 and preS2 specific epitopes on all serological subtypes of the HBV envelope protein. Thus, the sequence variability within the preS1 and preS2 regions does not represent an impediment to the development of synthetic peptide or genetically engineered hepatitis B preS immunogens for worldwide immunization.

The nucleocapsid of Dane particles (= hepatitis B core antigen; HBcAg) was isolated from human sera either positive or negative for e-antigen (HBeAg)--an apparent marker for the level of infectious hepatitis B virus in serum. HBcAg from the HBeAg-positive serum pool consisted of two distinct populations of particles, one with a buoyant density (d) of 1.358 g/ml and a sedimentation coefficient (S20, w) of approximately 110, and another with d = 1.25 to 1.30 g/ml and S20, w approximately 70. Only the latter type of particles was isolated from an HBeAg-negative serum pool. HBcAg was labelled with 125I-p-hydroxyphenylpropionic acid N-hydroxysuccinimide ester, dissociated and analysed by polyacrylamide gel electrophoresis. One major and one minor polypeptide with apparent mol. wt. of 16000 +/- 500 and 68000, respectively, were detected. Another component have the properties of a glycolipid with a mol. wt. in the order of 10(3) was observed. After isoelectric focusing, HBcAg was recovered in fractions with a pH between 4.0 and 5.8, suggesting heterogeneity in isoelectric points.

A total of 749 persistent carriers of hepatitis B surface antigen (HBsAg) were tested for hepatitis B e antigen/antibody to e antigen (HBeAg/anti-HBe) by a radioimmunoassay method. The prevalence of HBeAg was found to be: 9.1% in carrier-blood donors, 10.3% in mentally retarded patients without Down's syndrome, 23.6% in Chinese-Americans, 35.3% in drug addicts, 39.2% in mentally retarded patients with Down's syndrome, 61.4% in homosexual men, and 71.2% in dialysis patients. The prevalence of HBeAg appears to be independent of sex, race, and HBsAg antigenic subtype. Younger carries tend to be more frequently HBeAg positive than older ones. All individuals acutely infected with hepatitis B virus become at least transiently positive for HBeAg. Appearance or clearance of HBsAg, HBeAg and their corresponding antibodies seem to be interrelated.

Hepatitis B virus (HBV) envelope (env) proteins contain three antigenic domains designated S, preS2 and preS1. Studies with synthetic peptide immunogens demonstrated the role of preS2 epitopes in protection against HBV infection. The preS1 domain is implicated in virus-cell receptor interactions suggesting that anti-preS1-specific antibodies should neutralize the infectivity of HBV by blocking virus attachment to cells. We present here evidence that an antiserum to a peptide from the preS1 sequence, anti-preS(21-47), is virus-neutralizing and that active immunization of chimpanzees with a longer peptide derived from the preS1 sequence, preS(12-47), elicits antibodies protective against HBV infection. These results establish the role of the preS1 domain in the process of virus neutralization and the potential of synthetic preS1 analogues for hepatitis B vaccination.

Immunodominant, disulfide-bond independent epitopes recognized by human antibodies to hepatitis B virus (HBV) are located within the 55-residue amino terminal portion (coded for by the pre-S region of HBV DNA) of minor HBV envelope components larger than the major protein constituents encoded by the S gene. A peptide having the sequence of the first 26 amino acids from the amino terminal methionine was synthesized and elicited antibodies (at dilutions of greater than or equal to 1 to 10(5) ) to the HBV envelope. These antibodies can be utilized for diagnostic tests. The immunogenicity of the peptide was substantially increased by covalent attachment to liposomes. The disulfide bond-independent determinants on sequences coded for by the pre-S gene may be more easily mimicked by peptide analogs than "conformational" determinants on the S-gene product.

Krugman has demonstrated that injection of heated serum containing HBsAg conferred some protection against subsequent challenge with live hepatitis B virus (HBV). It is likely that improved protection will result from injections for larger quantities of HBsAg. This can be readily done with purified antigen. We have carried out preliminary studies in mice to investigate the inactivation kinetics of HBsAg antigenicity and immunogenicity. Rates of inactivation of the two parameters by heat have been found to be parallel. Chimpanzees will be required to determine adequacy of inactivation procedures, and optimal dose and immunization schedule, prior to the initiation of human trials. The use of newly imported chimpanzees for this purpose may be hazardous. Of 27 chimpanzees recently tested in an exporter's compound in Sierra Leone, ten were found to circulate HBsAg and five additional had detectable anti-HBs, suggesting a high risk of hepatitis B transmission within the compound. Thus newly imported animals may be incubating HB infection, and could give rise to false positive results in inactivation trials, unless quarantined for a four to six month period prior to use. We have therefore established a laboratory for chimpanzee trials in West Africa, and plan to utilize only animals captured by trapping and held in strict isolation from the time of capture. It is unlikely that immunization against HBV will ever provide absolute immunity. Three chimpanzees who received a massive transfusion of infective plasma one year after resisting challenge with aliquots of the same plasma inoculated intraperitoneally, all developed severe hepatitis, accompanied in two cases by HBs antigenemia, despite strong anamnestic anti-HBs responses in all three animals.

Hepatitis B surface antigen was adsorbed to insolubilized sialic acid-specific haemagglutinin isolated from the haemolymph of Limulus polyphemus. Treatment of the antigen with Vibrio cholerae neuraminidase (EC 3.2.1.18) resulted in the release of sialic acid and in an increase of the isoelectric point from pH 4-35 (for subtype ad) or 4-9 (for subtype ay) to pH 5-45. Treated, but not untreated, antigen incorporated [14-C]-sialic acid when incubated at 37 degrees C with sialyl transferase (EC 2.4.99.1) and cytidine-5'-monophosphate-[14-C]-sialic acid. The major portion of [14-C]-sialic acid was linked to a glycoprotein with an apparent mol. wt. of 26 x 10-a. De-sialylated antigen had a drastically reduced in vivo life span in rabbit plasma and elicited a higher humoral antibody response than intact antigen (subtype ad). Antigen-stimulated proliferation of lymphocytes, measured 3 months after immunization, was observed only with cells from rabbits injected with neuraminidase-treated antigen.

The region of the hepatitis B virus (HBV) genome coding for the viral envelope proteins contains three inphase ATGs that are conserved among viral subtypes. Each of these ATGs can be used as mRNA initiation codons. The three translated proteins share a carboxy-terminal region (the S protein) and extend amino-terminally to include the pre-S2 region in the middle (M) protein, and the pre-S1 and pre-S2 regions in the large (L) protein. We have inserted the HBV DNA coding for the M protein into a baculovirus expression vector. Infected insect cells transcribe a mRNA that is initiated solely within a baculovirus promoter, and that contains the initiator codons for both M and S proteins. Although these cells primarily secrete the M protein, the major translational product is the S protein, which is not secreted. This preferential translation, the result of the use of an internal initiator codon, demonstrates that the regulation of HBV envelope protein production can occur at the translational level.

A solid-phase radioimmunoassay for antibodies to hepatitis B core antigen (anti-HBc) is described. Polystyrene beads coated with anti-HBc, hepatitis B core antigen prepared from pooled sera of humans infected with hepatitis B virus (HBV) and 125I-labelled anti-HBc were used for the test. Distinct patterns of development and changes of anti-HBc and their immunologic properties are all related to variations of other markers specific for HBV infections. Knowledge concerning the detailed features of the immune response to hepatitis B core antigen may provide deeper insight into the pathogenesis of HBV infections.

A radioimmunoassay for albumin-binding sites associated with hepatitis B surface antigen (HBsAg) is described. Polystyrene beads coated with glutaraldehyde-polymerized human serum albumin were incubated with diluted specimens of HBsAg-positive sera which contained either e-antigen (HBeAg) or anti-HBe or were negative for both HBeAg and anti-HBe as determined by rheophoresis. The quantity of HBsAg attached to the beads, determined by measuring the amount of 125I-labeled anti-HBs adsorbed during a subsequent incubation step, was related to the presence of HBeAg in the specimens.

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bond adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.

Monoclonal antibodies (McAb) specific for the pre-S region of the hepatitis B virus (HBV) envelope protein were prepared using HBV particles of hepatitis B surface antigens (HBsAg) as immunogens. The antibodies reacted in Western blot analyses and in ELISA with pre-S2 sequences of the HBV envelope protein. Pepsin or protease V8 treatment of the antigen abolished reactivity. The fine specificity of one of the McAb (F376) was established by immunoassays using synthetic peptides and a pre-S2-beta-galactosidase fusion protein expressed in E. coli. The shortest peptide recognized by F376 is demarcated by residues pre-S(132) at the N-terminal and pre-S(140)-pre-S(145) at the C-terminal. The corresponding amino acid sequence (for HBV subtype adw2) is: QDPRVRGLY(LPAGG). Additional amino acid residues at the N-terminal, and possibly at the C-terminal ends contribute to the binding of McAb, probably due to conformational influences. The McAb was applied to immunoassays of pre-S2 sequences in purified HBsAg and in human sera containing HBsAg.

A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 delta (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-beta-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to greater than 90% homogeneity by affinity chromatography on p-aminophenyl-beta-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-beta-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-beta-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B.

Hepatitis B antigen-associated particles, isolated from sera of antigen carriers, were submitted to affinity chromatography on columns of insolubilized antibodies to normal human plasma. The particles adsorbed to the immunosorbent at pH 7.2 and were subsequently eluted at pH 2.2. Exposure of the particles to 8 M urea, 5 M KI, pH 2.2, detergents, organic solvents, or proteolytic enzymes failed to prevent their subsequent adsorption to the immunosorbent. This suggests that antigenic determinants related to human plasma proteins are constituent components of hepatitis B antigen-associated particles. These determinants are distinct from the group-specific (a) and subtype-specific (d or y) sites of the hepatitis B antigen and appear to be related to antigenic specificities on prealbumin, albumin, apolipoproteins C and D, and the gamma-chain of immunoglobulin G.

Large-scale purification of hepatitis B surface antigen, applicable to the preparation of potential vaccines for prevention of hepatitis B, is described. The method involves the following steps: precipitation of serum with polyethylene glycol 6000, affinity chromatography on concanavalin A-Sepharose and on omega-aminononyl-Sepharose, and isopycnic centrifugation.

A solid-phase enzyme-linked fluorescence immunoassay (ELFA) for antiviral antibodies using antigen covalently linked to Escherichia coli beta-galactosidase and a fluorogenic substrate (4-methyl-umbelliferyl-beta-D-galactopyranoside) is described. Antibodies to hepatitis B surface antigen (HBsAg) from test samples were adsorbed to wells of 96-well aminopolystyrene plates precoated with HBsAg. The antibodies were subsequently detected using enzyme-conjugated HBsAg. The measurement of fluorescence was automated. The sensitivity of the test was approximately 50 times higher than that of a commercial radioimmunoassay.

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and thus may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA are detected.

We predicted the localization of a major hepatitis B surface antigen (HBsAg) determinant within residues 135-155. A peptide corresponding to this sequence (P135-155) was synthesized, linked to macromolecular carriers and used to immunize rabbits. In accordance with published data on shorter peptides within the same amino acid sequence, antibodies to HBsAg were elicited. A detailed analysis of the immune response to P135-155 revealed the following: A heterogeneous population of IgG and IgM antibodies reacting with P135-155 was detected in the antisera by a variety of radioimmunoassays and enzyme linked fluorescence immunoassays. Only a subpopulation of these antibodies reacted with HBsAg. The equilibrium constant (K) for the reaction of the antibodies with HBsAg (K = 4 X 10(5) X M-1) was approximately two orders of magnitude lower than K for the reaction with P135-155, and was below K for the reaction between HBsAg and antibodies elicited by HBsAg (K greater than 10(7) X M-1). Preimmunization with P135-155 did not result in an enhanced response to subsequent immunization with HBsAg. Peptides more accurately mimicking determinants on HBsAg may have to be synthesized for possible application in antiviral prophylaxis.

The immune response to current hepatitis B vaccines appears to be qualitatively different from the response elicited during recovery from natural infection. Such disparate responses can probably be explained by the absence or under-representation of preS- and the nucleoprotein core-specific determinants in the vaccines. The incorporation of these determinants into future vaccines may improve their efficacy.

Immunoprecipitates obtained by reacting Dane particle cores with human antibodies to hepatitis B core antigen (HBcAg) were chromatographed on columns of Sepharose 4B CL using 3 M-NaSCN as eluant. An antigen having the size and immunological specificity of monomeric e-antigen (HBeAg) was separated from HBcAg by this method. Antisera from animals immunized with HBcAg or HBeAg reacted not only with the antigen used for immunization but also with HBeAg and HBcAg, respectively. This indicates that HBeAg determinants are associated with the core of Dane particles.

Adenovirus type 7 exposed to solutions of LiI was progressively converted into slower sedimenting deoxyribonucleic acid (DNA)-containing particles, and, ultimately, under proper conditions, DNA free or almost free from protein was released from the virus. The degree of viral degradation was dependent on the time of treatment, on the temperature, and on the concentration of the reagent.