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We identified the epitopes on the preS1 which induce antibodies that neutralize both ad and ay subtypes of hepatitis B virus (HBV). Previously we generated murine monoclonal antibodies KR359 and KR127 that bind specifically to the preS1 of HBV. In this study we have performed fine mappings of the epitopes of the antibodies by examining their reactivity with GST fusion proteins, which contain a series of deletion mutants of the preS1. KR359 and KR127 specifically recognize aa 19-26 and 37-45 of the preS1, respectively. The antibodies neutralized both adr and ayw subtypes of the virus in an in vitro neutralization assay using in vitro infection of adult human hepatocyte primary culture by HBV. The epitopes showed little sequence divergence and the antibodies bound to the preS1 of all the HBV subtypes and variants tested., (Copyright 2000 Academic Press.)

We have efficiently generated mouse monoclonal antibodies (MAbs), which bind specifically to amino acids 21-47 of the preS1 domain of hepatitis B virus (HBV) by immunizing mice with the preS1 peptide (amino acids, aa 1-56) conjugated to keyhole limpet hemocyanin. Hybridomas were screened by an indirect enzyme-linked immunosorbent assay (ELISA) using the purified preS1 peptide as a coated antigen. Eighteen positive hybridomas were selected and subjected to isotyping. Of these, 5 clones secreted immunoglobulin G (IgG) and 13 clones secreted IgM. Four (KR1, KR2, KR3, and KR4) of the 5 IgG MAbs bound to preS1 peptide (aa 21-47). Epitope mapping using bacterially expressed GST fusion proteins revealed that three clones (KR2, KR3, KR4) (IgG1, K) recognize aa 21-35, while KR1 (IgG2a, K) recognizes aa 35-47 of the preS1. These MAbs immunoprecipitated HBV particles, demonstrating that they bind to native HBV particles.

We cloned and analyzed the cDNA sequences encoding the variable regions of a human monoclonal antibody (23HN) (gamma1, kappa) that binds to the common a antigenic determinant on hepatitis B surface antigen and its fine epitope is different from those of previously known mAbs with the same specificity. The heavy and light chain variable regions are the members of human heavy chain subgroup III and kappa light chain subgroup III, respectively, and are most homologous to human germline VH segment DP-50 and Vkappa segment DPK-22, respectively., (Copyright 1998 Elsevier Science B.V.)

In vitro HBV infection and neutralization were assayed using an anti-preS1 murine monoclonal antibody (1B3) and anti-preS2 (H69K) and anti-S (CS131A) murine-human chimeric antibodies. The 1B3 (IgG1) and H69K (IgG1) was constructed previously and the CS131A was constructed for this study by expressing stably the chimeric heavy and light chains in Chinese hamster ovary cells and purifying from the culture supernatant. Previous study showed that the H69K and CS131A recognize known virus-neutralizing epitopes, while the 1B3 does not. For the assays, adult human hepatocyte primary culture was infected with the adr or ayw subtype of HBV, and the infectivity and subsequent replication was confirmed both by measuring the kinetics of HB-sAg secretion by the infected cells and detecting the intermediate replicative form of HBV DNA in the cells. Next, the hepatocytes were infected with the adr or ayw subtype of the virus that had been preincubated with various concentrations of each of the antibodies and the neutralization of HBV was analyzed. The results showed that the anti-preS2 and anti-S chimeric antibodies exhibited neutralizing activity against both the adr and ayw subtypes of the virus, with approximately 1,000 and 2,000 times higher specific activity than polyclonal hepatitis B immune globulin, respectively, but the anti-preS1 antibody scarcely neutralized the infection. The neutralizing activities of the antibodies were consistent with their epitope specificity and antigenbinding affinity, suggesting that this neutralization assay is specific. The in vitro neutralization assay will be useful for evaluating the neutralizing activity of anti-HBV antibodies before in vivo testing in chimpanzees.

For the prevention of hepatitis B virus (HBV) infection, monoclonal antibodies (mAbs) against the surface antigens of HBV would offer several advantages over the current human polyclonal antibody. We have developed a humanized antibody with specificity for the preS2 surface antigen of HBV and evaluated the neutralizing activity of the humanized antibody. The complementarity-determining regions of the heavy and light chains of a murine monoclonal antibody (H8) were grafted onto the variable regions of a highly homologous human anti-Sm antibody, which were then combined with the constant regions of human gamma 1 and kappa, respectively. The affinity of the resulting humanized antibody (Z6B) was about one tenth that of the chimeric antibody. The new version (ZP39) of the humanized antibody, which was constructed by substituting the heavy-chain framework residue at position 94 of the Z6B with original mouse residue, showed almost the same affinity as that of the chimeric antibody. The evaluation of the HBV neutralizing activity of ZP39 using in vitro infection of adult human hepatocyte primary culture by HBV showed that it had a specific activity that was approximately 1000 times higher than commercially available polyclonal hepatitis B immune globulin. We expect that the present humanized antibody will be useful in the prevention of HBV infection.

A murine monoclonal antibody H67 was characterized for the binding specificity, which showed that H67 recognizes a disulfide-bond-dependent conformational epitope of common a antigenic determinant on the hepatitis B surface antigen. The result suggested that this antibody may have the potential of replacing hepatitis B immune globulin in the prevention of hepatitis B virus (HBV) infection. Therefore, we have constructed the humanized antibody HuS10 by grafting the complementarity determining regions and some framework amino acid residues of H67 onto the most homologous human antibody variable regions, 21/28 for heavy chain variable region and B1 and J kappa 2 for light chain variable region, followed by combining with human constant regions C gamma 1 and C kappa. The affinity of the HuS10 was the same as that of the H67, 8 x 10(8) x 10(8)M-1, and the HuS10 neutralized the in vitro infection of adult human hepatocyte primary culture by adr or ayw subtype of HBV. The neutralization assay showed that the HuS10 had approximately 2,000-times higher specific activity than commercially available polyclonal HBIG. These results suggest that the humanized antibody will be useful in the prevention or treatment of HBV infection.

The nucleotide (nt) sequences encoding the variable regions of the heavy and light chains of a murine monoclonal antibody (mAb) have been determined. The mAb recognizes a disulfide-bond-dependent conformational epitope on the hepatitis B virus surface antigen. The sequence analyses revealed that the variable regions of the heavy and light chains are members of mouse heavy-chain subgroup II(A) and kappa light-chain subgroup III, respectively.

Binding specificity of a monoclonal antibody (mAb) (kappa, gamma 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 kappa 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.