preS1 Peptide Research Articles

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody specific for pre-S2 antigen of hepatitis B virus

Binding specificity of a monoclonal antibody (mAb) (κ, γ 2b) H8 which can react with the pre-S2 peptide of hepatitis B virus (HBV) was determined by Western blot analyses. From the hybridoma cell line secreting mAb H8, poly(A) +RNA was prepared and used as a template for cDNA synthesis and cloning. Full-length cDNAs coding for the heavy and κ light chains of the mAb were cloned from the cDNA library and characterized by nucleotide (nt) sequence analyses and N-terminal amino acid sequencing. The sequence analyses revealed that both heavy and light chain-specific cDNAs are functional, and the variable regions of the heavy and light chains are members of mouse heavy chain subgroup III(c) and light chain group I, respectively. Comparison of the nt sequences with mouse immunoglobulin genes listed in the GenBank data base show that the cDNAs have not been previously reported. The cDNAs will be used for the construction of a therapeutic antibody for HBV infection.

Receptor for pre-S1(21-47) component of hepatitis B virus on the liver cell: role in virus cell interaction.

Attachment of hepatitis B virus to a hepatoblastoma cell line (HepG2) was examined using a synthetic peptide corresponding to the pre-S1 (21-47) region of the envelope protein. Scatchard analysis revealed a single class binding site of Kd 104 +/- 27 nM/l and 5.4 +/- 1.2 x 10(5) sites per cell. Competition of HBV with pre-S1 peptides was dose dependent, and demonstrated it as the dominant binding site. In view of the suggested sequence homology between the peptide and IgA, cross-competition studies were carried out. The results indicate no direct role of IgA receptor in HBV binding. The receptor for the pre-S1 peptide was identified as a single major peptide of molecular weight 31 kD using in-situ ligand receptor crosslinking.

Identification of polymerized-albumin receptor domain in the pre-S2 region of hepatitis B virus surface antigen M protein

The pre-S2-coding region in the hepatitis B virus surface antigen M (P31; pre-S2 + S) protein gene was modified to identify a polymerized-albumin receptor (PAR) domain by deleting restriction fragments or performing site-directed mutagenesis. The modified M protein genes (M-P31x; x = d, e, f, h and i) were cloned into the yeast generalized-expression vector pGLD 906-1 and expressed in Saccharomyces cerevisiae under the control of yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. The PAR activities of these gene products suggested that the PAR domain is located in the hydrophilic and highly conserved domain in the pre-S2 region (around Leu 12 ∼ Tyr 21). Antibodies specific for a pre-S2 peptide (Phe 8 ∼ Pro 34, subtype adr), which covers the PAR domain, were purified from sera of rabbits immunized with yeast-derived M protein particles having a natural PAR domain. Immune electron microscopy showed that the purified antibodies could aggregate HBV particles. Therefore, it was speculated that the PAR domain overlapped with the dominant virus-neutralizing and virus-protecting epitopes.

Hepatitis B virus envelope L protein particles. Synthesis and assembly in Saccharomyces cerevisiae, purification and characterization.

The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene. Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus. To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein. Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles. The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated. It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus. Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene. These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form. Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen.

Expression and characterization of the preS1 peptide of hepatitis B surface antigen inEscherichia coli

The infectious particles of hepatitis B virus (HBV) contain 3 related surface antigens, i.e., small, medium, and large, all of which are encoded by one large open reading frame with multiple initiation codons. The large surface antigen (L-Ag) contains preS1, preS2, and S regions while both the middle and small surface antigens lack preS1. Several lines of evidence suggested that the preS1 region is involved in the binding of HBV to human hepatocytes as shown by its binding to HepG2 cells and isolated human hepatocyte membranes. To obtain large quantity of preS1 peptide, an expression vector was constructed containing a lac promoter, the 5' half of the beta-galactosidase gene, the Factor Xa tetrapeptide recognition sequence, and the coding region of preS1 plus preS2. This recombinant plasmid constitutively produced high concentration of a fusion protein in inclusion bodies in Escherichia coli. When the fusion protein was treated with Factor Xa, a peptide consisting of the N-terminal 91 amino acids of the preS1 region was released. This preS1 fragment purified by anion exchange chromatography was able to bind specifically to the isolated plasma membranes from human liver. Hence, this recombinant preS1 peptide can be used to identify and isolate hepatocyte receptors for HBV.

Significance of natural polymerized albumin and its receptor in hepatitis B infection of hepatocytes

Lack of information regarding the presence of native albumin polymer in serum and its structural similarity to the one produced by glutaraldehyde treatment casts doubt on the postulate that hepatitis B virus attachment to hepatocytes is mediated through polymerized albumin. We used a sandwich enzyme-linked immunosorbent assay with murine monoclonal antibodies raised against glutaraldehyde-polymerized albumin to detect native albumin polymer in human serum and its cross-reactivity with other albumin polymers. Presence of polymerized albumin receptor on the HepG2 cell was studied by radioreceptor assay. Purified hepatitis B virus and synthetic peptide analogous to part of pre-S2 sequence (120-145) were used to study polymerized albumin-dependent attachment of the virus to HepG2 cells. Antibodies raised against pre-S2 peptide were used to inhibit the pre-S2 and hepatitis B virus attachment to HepG2 cells. Glutaraldehyde-treated polymerized albumin was found to be immunologically cross-reactive with native albumin polymer. Its levels were found to be significantly raised in sera of patients with liver diseases. Polymerized albumin has specific saturable receptor on HepG2 cells with two classes of binding sites of different equilibrium dissociation constant (Kd1 = (16 +/- 9.6)pmol/L and Kd2 = (1,019 +/- 172)pmol/L. Albumin monomer was unable to compete for the polymerized albumin receptor sites on HepG2 cells. Anti-pre-S2 antibodies inhibit hepatitis B virus and pre-S2 binding to hepatocyte by 40% and 70%, respectively. Added extraneous polymerized albumin and the antibody against it did not interfere with virus attachment to HepG2 cells.

Significance of natural polymerized albumin and its receptor in hepatitis B infection of hepatocytes

Lack of information regarding the presence of native albumin polymer in serum and its structural similarity to the one produced by glutaraldehyde treatment casts doubt on the postulate that hepatitis B virus attachment to hepatocytes is mediated through polymerized albumin. We used a sandwich enzyme-linked immunosorbent assay with murine monoclonal antibodies raised against glutaraldehyde-polymerized albumin to detect native albumin polymer in human serum and its cross-reactivity with other albumin polymers. Presence of polymerized albumin receptor on the HepG2 cell was studied by radioreceptor assay. Purified hepatitis B virus and synthetic peptide analogous to part of pre-S2 sequence (120–145) were used to study polymerized albumin-dependent attachment of the virus to HepG2 cells. Antibodies raised against pre-S2 peptide were used to inhibit the pre-S2 and hepatitis B virus attachment to HepG2 cells. Glutaraldehyde-treated polymerized albumin was found to be immunologically cross-reactive with native albumin polymer. Its levels were found to be significantly raised in sera of patients with liver diseases. Polymerized albumin has specific saturable receptor on HepG2 cells with two classes of binding sites of different equilibrium dissociation constant (Kd1) = (16 ± 9.6)pmol/L and Kd2 = (1,019 ± 172)pmol/L. Albumin monomer was unable to compete for the polymerized albumin receptor sites on HepG2 cells. Antipre-S2 antibodies inhibit hepatitis B virus and pre-S2 binding to hepatocyte by 40% and 70%, respectively. Added extraneous polymerized albumin and the antibody against it did not interfere with virus attachment to HepG2 cells. (HEPATOLOGY 1991;13:134–142).

Binding of the Major and Large Hbsag to Human Hepatocytes and Liver Plasma Membranes: Putative External and Internal Receptors for Infection and Secretion of Hepatitis B Virus

A likely mechanism of the strong hepatotrophism of the hepatitis B virus is the presence of specific receptors for the surface antigen of hepatitis B virus on hepatocyte membranes. To examine this hypothesis, we have performed binding studies using recombinant large (preS1 + preS2 + S) and major (S) proteins with adult human hepatocytes, rat hepatocytes, human fibroblasts, human peripheral blood mononuclear cells and plasma membranes derived from these cell types. We found that major HBsAg was able to bind specifically to human hepatocytes, human fibroblasts and human blood mononuclear cells. This binding could be inhibited by recombinant middle (preS2 + S) protein but not by the recombinant large protein. No binding could be demonstrated between large HBsAg and human hepatocytes. However, large protein bound specifically to plasma membranes derived from human liver tissue, human fibroblasts and HepG2A16 cells. This binding could be partially inhibited by the major protein and by a synthetic preS1 peptide but not by a synthetic preS2 peptide. These results support the assumption that hepatitis B virus absorption and penetration into human hepatocytes is mediated by specific receptors recognizing an amino acid sequence in the S-region. This recognition site is not displayed by the recombinant large protein. However, the large protein is recognized by its preS1 region and by a second binding site in the S-region by a receptor molecule, located on the inner surface of the plasma membranes or intracellular membranes of human hepatocytes and of some other cell types derived from human tissue.

In Vitro Immune Responses to Hepatitis B Surface Antigens Sand preS2 during Acute Infection by Hepatitis B Virus in Humans

This study evaluated the in vitro immune responses to different components of the hepatitis B surface antigen (HBsAg) over the course of acute hepatitis B virus (HBV) infection. Early in the convalescent phase of infection, while HBsAg was present in the serum, peripheral blood mononuclear cells (PBMCs) were stimulated with preS2 peptide or hepatitis B surface protein. Specific IgG directed to different components of HBsAg was produced without a polyclonal increase in total IgG production. Stimulation with preS2 peptide produced IgG to the preS2 peptide (anti-preS2) and to the S antigen (anti-HBs). Hepatitis B surface protein stimulation produced anti-HBs but not anti-preS2. After this initial reactive phase, the PBMCs did not produce specific antibody when stimulated with either component of HBsAg; this effect lasted greater than 1 year. At some time 1-2 years after acute infection, the pattern of in vitro S antigen- and preS2 antigen-stimulated anti-HBs response reemerged in the PBMCs. Reemergence of sustained preS2 peptide-stimulated anti-preS2 response was not observed.

Antibody response to a foreign epitope expressed at the surface of recombinant bacteria: importance of the route of immunization

A genetic procedure has been previously established to expose a foreign epitope at the surface of Escherichia coli by using the outer membrane LamB protein as a carrier. A portion of the pre-S2 region of hepatitis B virus, residues 132-145, has been inserted at amino acid position 153 of the LamB protein, in a cell surface exposed loop. In the present study, we have analysed the antibody responses induced by these recombinant bacteria (live, heat-killed or sonicated) depending upon the route of immunization. The intravenous (i.v.) or intraperitoneal (i.p.) administration of the live recombinant bacteria to mice induced the synthesis of antibodies against both the inserted peptide and the native LamB protein. The antibodies raised recognized HBsAg particles. These mice also had high titres of antibodies against E. coli antigens (as determined using a crude bacterial sonicate). In contrast, mice immunized subcutaneously (s.c.) did not develop antibodies against the pre-S2 peptide nor against the HBsAg particles. Their anti-LamB responses were low compared with the response of mice immunized by the parenteral route. Interestingly, s.c. or i.v. immunizations induced comparable levels of anti-E. coli antibodies. Thus, the antibody response to the inserted peptide generally parallels the response to the LamB protein (and not to the bulk of E. coli antigens). However, this treatment corresponding to a 'pre-processing' of the recombinant bacteria was not sufficient to obtain an anti-peptide response following s.c. immunization.

Hepatitis B virus surface antigen (HBsAg) as carrier for synthetic peptides having an attached hydrophobic tail

B- and T-cell epitopes from three distinct regions of the hepatitis B virus (HBV) envelope (env) protein (preS1, preS2 and S) are involved in eliciting protective immunity. Since preS1 sequences inhibit the secretion of HBV env proteins from eukaryotic cells, it is difficult to prepare immunogens rich in preSl sequences. This problem can be overcome by linking synthetic peptides from the preS1 region to particles containing both S and preS2 sequences. We describe here a novel approach for bincling of synthetic peptides to exposed hydrophobic domains on HBV env proteins. Long chain fatty acids or mercaptans are covalently linked to synthetic peptides. Peptides with the attached hydrophobic tails interact strongly with HBV env proteins (S + preS2), whereby hybrid immunogens are generated. Such immunogens can be used in combination with alum, the only adjuvant approved for human use. The combination of the preS1 peptide [preS(12–47)] with particles containing the S and preS2 regions resulted in an immunogen which: (1) elicits a broad spectrum of protective antibodies; (2) circumvents the nonresponsiveness to: (a) preS1 epitopes in preS1-nonresponder strains of mice; and (b) S-protein in S-protein-nonresponder strains of mice; and (3) augments the immune response to S-protein. The combination of HBV env proteins with a synthetic peptide from the envelope of the human immunodeficiency virus (HIV-1) resulted in an immunogen eliciting anti-HIV-1. Hybrid immunogens consisting of viral proteins and of synthetic peptides represent a feasible approach for the design of future vaccines.

Sequential studies of pre-S2 antigenemia and anti-pre-S2 antibodies in relation to viral replication in acute hepatitis B followed from the early incubation phase.

To determine the time sequence of expression of pre-S2 peptide (120-150) during the presymptomatic phase of acute hepatitis B, we used a monoclonal antibody radioimmunoassay in five subjects followed from 30 to 70 days before the onset of liver damage. Pre-S2 peptide was present in serum at low levels from the early incubation phase and started to increase immediately after the first detection of HBV-DNA in serum, in parallel with the increase in HBsAg levels. During the symptomatic phase, levels of pre-S2 peptide declined rapidly; it was no longer detectable after recovery. Anti-pre-S2 antibodies were detected, in four patients, only in the recovery phase. These results demonstrate that expression of pre-S2 peptide occurs very early in the incubation phase of acute HBV infection and is cleared in parallel with HBsAg. Anti-pre-S2 antibodies seem to play no role in viral clearance in these patients.

A synthetic peptide coded for by the pre-S2 region of hepatitis B virus for adding immunogenicity to small spherical particles made of the product of the S gene

Small spherical particles produced in the non-permissive phase of hepatitis B virus infection, when the viral genome is integrated into the chromosome of hosts, are rich in the product of the S gene, but poor in the product of the pre-S2 region. For the purpose of adding immunogenicity to spherical particles deficient in the pre-S2 region product, they were conjugated with a synthetic peptide of 19 amino acid residues. The peptide reproduced a hydrophilic area of the pre-S2 region product encoded by viral genomes of subtypes adr, ayw and ayr. The spherical particles supplemented with the pre-S2 peptide raised antibody to the pre-S2 region product in mice, in addition to antibody to the product of the S gene. Antibody to pre-S2 region product, prepared from sera of immunized mice by absorption with the S gene product, bound to spherical particles bearing pre-S2 region product, irrespective of adr, adw, ayw or ayr subtype, and agglutinated hepatitis B virions in immune electron microscopy. Based on the results obtained, the synthetic peptide may prove useful in adding protective efficacy to small spherical particles poor in pre-S2 region product.