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 Leu12 approximately Tyr21). Antibodies specific for a pre-S2 peptide (Phe8 approximately Pro34, 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.     

Antibodies to pre-S and X determinants arise during natural infection with ground squirrel hepatitis virus.

The DNA sequence of the ground squirrel hepatitis virus (GSHV) genome predicts the existence of several proteins in addition to the major surface (S) and core antigens. These include the pre-S1 and pre-S2 proteins, initiated at sites within the open reading frame preceding and continuous with the coding region for the S gene product, and the X protein, the putative product of an independent reading frame. Using an antibody directed against a peptide predicted by codons 130 to 143 of the pre-S1 reading frame, we identified a 43-kilodalton product of the pre-S1 coding region in preparations of GSHV surface antigen purified from the sera of infected animals. In addition, by immunoprecipitation of S- and pre-S-specific in vitro translation products with ground squirrel sera obtained after GSHV infection, we determined that antibodies arise to both S and pre-S determinants. The antibody response to pre-S includes, in some cases, reactivity to pre-S1-specific domains and is not always associated with an anti-S response. Similarly, by production of the viral X gene product in vitro followed by immunoprecipitation with ground squirrel sera, we showed that antibodies to this viral gene product also arise during infection, indicating that X antigenic determinants are synthesized during viral infection and are recognized by the host immune system.     

In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses.

We have developed an adenovirus vector to express foreign proteins under the control of the adenovirus E1a promoter. Two recombinant plasmids, harbouring either the S gene or the pre-S2 region and the S gene of hepatitis B virus under the control of the E1a promoter, were used to construct two recombinant adenoviruses. These two viruses direct the synthesis of hepatitis B virus surface antigen (HBsAg) particles during the time course of an infectious cycle. When the pre-S2 region is present in the constructed virus, the synthesis of particles carrying the receptor for polymerized human serum albumin (pHSA) is observed. Moreover, the inoculation of rabbits with this latter purified recombinant adenovirus elicits the production of antibodies that react with both HBsAg and pHSA receptor.     

The influence of glycosylation on secretion,stability, and immunogenicity of recombinant HBV pre-S antigen synthesized in Saccharomyces cerevisiae

Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1 degrees S2 (Asn15Gln) and pre-S1 degrees S2 degrees (Asn15Gln and Asn123Gln). An N-terminus sequence (Ser5-Ala28) of human interleukin 1 beta (hIL-1 beta) was used as synthetic prosequence of recombinant HBV surface antigen (pre-S), secreted from S. cerevisiae. The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site (-Lys-Arg-), and the surface antigen. The recombinant pre-S1S2 and pre-S1 degrees S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1 degrees S2 degrees was produced without N-glycosylation. It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies. This problem was overcome by eliminating both of the N-glycosylation signals. Despite enhanced immunogenicity, the recombinant pre-S1 degrees S2 degrees showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases. The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1 degrees S2 degrees was somewhat inevitable. Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen.     

Characterization of a 120-Kilodalton pre-S-binding protein as a candidate duck hepatitis B virus receptor.

Infection by human and animal hepadnaviruses displays remarkable host and tissue tropism. The infection cycle probably initiates with binding of the pre-S domain of viral envelope protein to surface receptors present on the hepatocyte. Three types of neutralizing monoclonal antibodies against duck hepatitis B virus (DHBV) have their binding sites clustered within residues 83 to 107 of the pre-S protein, suggesting that this region may constitute a major receptor binding site. A 170- or 180-kDa duck protein (p170 or gp180) which binds DHBV particles through this part of the pre-S sequence has been identified recently. Although the p170 binding protein is host (duck) specific, its distribution is not restricted to DHBV-infectible tissues. Using the pre-S protein fused to glutathione S-transferase and immobilized on Sepharose beads, we have now identified an additional binding protein with a size of 120 kDa (p120). p120 expression is restricted to the liver, kidney, and pancreas, the three major organs of DHBV replication. While optimal p170 binding requires an intact pre-S protein, binding to p120 occurs much more efficiently with a few N- or C-terminally truncated forms. The p120 binding site was mapped to residues 98 to 102 of the pre-S region, which overlaps with a cluster of known virus-neutralizing epitopes. Site-directed mutagenesis revealed residues 100 to 102 (Phe-Arg-Arg) as the critical p120 contact site; nonconservative substitution in any of the three positions abolished p120 binding. Double mutations at positions 100 to 102 markedly reduced DHBV infectivity in cell culture. Short pre-S peptides covering the clustered neutralizing epitopes (also p170 and p120 binding sites) reduced DHBV infectivity in primary duck hepatocyte cultures. Thus, p120 represents a candidate component of the DHBV receptor complex.     

Cloning and expression of the yeast plasma membrane ATPase in Escherichia coli

The yeast plasma membrane ATPase gene PMA1 was cloned into Escherichia coli using the high expression tac and T7 promoters. The gene product is toxic to the bacterial cell leading to very low expression levels and arrested growth of the host cell within minutes of induction. The expressed protein is immunologically cross-reactive with the yeast ATPase, comigrates with the original protein in sodium dodecyl sulfate-polyacrylamide gels, and is isolated in the E. coli membrane fraction. The partially purified protein exhibits ATPase activity.     

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.     

Immune response to the pre-S(1) region of the hepatitis B surface antigen (HBsAg): a pre-S(1)-specific T cell response can bypass nonresponsiveness to the pre-S(2) and S regions of HBsAg

Hepatitis B surface antigen (HBsAg) particles are composed of a major polypeptide, p25, and additional polypeptides of higher m.w., namely p33 and p39, are variably present. All three polypeptides share the 226 amino acid residues of the S region: p33 consists of the p25 sequence plus an NH2-terminal 55 residues (pre-S(2], and p39 consists of the p33 sequence plus an NH2-terminal 108-119 residues (pre-S(1). In previous studies we demonstrated the influence of two Ir genes on the humoral and cellular immune responses to the S region and identified nonresponder phenotypes (H-2f,s). Subsequent studies showed that the immune response to the pre-S(2) region was regulated by H-2-linked genes independently of the S region response, such that immunization of S region nonresponder, pre-(S2) region responder mice (H-2s) with HBsAg/p33 circumvented nonresponse to the S region. In the present study, we have extended this analysis to the pre-S(1) region of HBsAg, with the following results: 1) and pre-S(1) region is immunogenic at the T and B cell levels; 2) anti-pre-S(1) specific antibody production is regulated by H-2-linked genes and can be independent of anti-S and anti-pre-S(2) antibody production; 3) immunization of H-2f strains with HBsAg/p39 particles containing the pre-S(1) region can bypass nonresponsiveness to the S and pre-S(2) regions in terms of antibody production; 4) two synthetic peptides, p32-53 and p94-117, define murine and human antibody binding sites on the pre-S(1) region, and p1-21 and p12-32 define additional human antibody binding sites; 5) pre-S(1)-specific T cells can be elicited in S and pre-S(2) region nonresponder mice (H-2f) and provide functional T cell help for S-pre-S(2)-, and pre-S(1)-specific antibody production; and 6) a T cell recognition site in the pre-S(1) region, p12-32 was identified. These results are relevant to HBV vaccine development, and possibly to viral clearance mechanisms, since the higher m.w. polypeptides are preferentially expressed on intact virions.     

Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae

Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC's surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14 months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines.     

Two distinct but overlapping antibody binding sites in the pre-S(2) region of HBsAg localized within 11 continuous residues

The fine specificity of the humoral immune response to the pre-S(2) region of the hepatitis B surface antigen was studied. It was demonstrated that the murine antibody response to the pre-S(2) region is focused on residues 133 through 143, and two distinct but overlapping epitopes were identified within 11 continuous residues. One epitope, defined by p133-139, is group specific, and the other epitope, defined by p137-143, is influenced by a subtype-dependent amino acid substitution at residue 141. However, the influence of residue 141 was "covert" in that it was only detected when synthetic antigens of 19 amino acids or smaller were used as the solid-phase ligand. The minimum size of both epitopes (p133-139 and p137-143) was seven amino acids. The physical and chemical form of the immunogen (i.e., protein vs peptide; conjugated vs free peptide) influenced antibody fine specificity. In quantitative antibody inhibition studies it was demonstrated that antibodies with nonoverlapping as well as overlapping fine specificities were capable of mutual inhibition. Finally, human HBV-infected, patient sera were shown to possess anti-pre-S(2) region antibodies that recognized sequences in common with the murine antisera. These results have implications relevant to the design of synthetic and recombinant second generation HBV vaccines and diagnostic reagents.     

The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.     

Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.     

Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase.

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.     

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.     

Characterization of a pre-S polypeptide on the surfaces of infectious avian hepadnavirus particles.

DNA from the pre-S region of the duck hepatitis B virus (DHBV) genome was inserted into an open reading frame vector designed to give high-level expression in Escherichia coli. The resulting fusion protein contained the first 8 amino acids of beta-galactosidase, 86 amino acids of the DHBV pre-S region, and 219 amino acids of chloramphenicol acetyltransferase at the C terminus (beta-gal:pre-S:CAT). Rabbit antiserum against purified beta-gal:pre-S:CAT was used to identify pre-S-containing polypeptides in DHBV particles by Western blotting. A dominant species of 36 kilodaltons (kDa) was identified. Antiserum against the major 17-kDa DHBsAg polypeptide also reacted with the 36-kDa protein. This suggests that the DHBV envelope gene polypeptides share the same carboxyl terminus, but differ in the sites from which translation is initiated. N-linked carbohydrate was not detected on either the 17- or 36-kDa envelope proteins. Anti-beta-gal:pre-S:CAT abolished infectivity of the virus in an in vitro assay. Thus, the pre-S region is exposed on the surfaces of infectious virions and may be directly involved in binding of virus to host-cell receptors.     

Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.     

抗乙型肝炎病毒表面抗原preS1（20－47）多抗的制备、性质和应用

ＨＢｓＡｇｐｒｅＳ１（２１－４７）肽参与乙肝病毒与肝细胞受体的结合。我们用固相法合成了ＨＢｓＡｇｐｒｅＳ１（２０－４７）２８肽,并用此肽与ＢＳＡ的偶联物免疫家兔;抗血清经亲和层析纯化后,得到了高纯度、高活力及专一性的抗ＨＢｓＡｇｐｒｅＳ１（２０－４７）多抗,它能成功地用于基因工程表达产物及病人血清中的ＰｒｅＳ１抗原的检测。