Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism

Abstract

The humanized monoclonal antibody HzKR127 recognizes the preS1 domain of the human hepatitis B virus surface proteins with a broadly neutralizing activity in vivo. We present the crystal structures of HzKR127 Fab and its complex with a major epitope peptide. In the complex structure, the bound peptide forms a type IV ␤-turn followed by 310 helical turn, the looped-out conformation of which provides a structural basis for broad neutralization. Upon peptide binding, the antibody undergoes a dramatic complementarity determining region H3 lid opening. To understand the structural implication of the virus neutralization, we carried out comprehensive alanine-scanning mutagenesis of all complementarity determining region residues in HzKR127 Fab. The functional mapping of the antigen-combining site demonstrates the specific roles of major binding determinants in antigen binding, contributing to the rational design for maximal humanization and affinity maturation of the antibody.alanine-scanning mutagenesis ͉ crystal structure ͉ humanized antibody ͉ virus neutralization H uman hepatitis B virus (HBV) is a small enveloped DNA virus that causes acute and chronic hepatitis in humans (1-3). There are approximately 400 million carriers of HBV worldwide, posing a serious health threat. The current HBV vaccines have several limitations, including nonresponsiveness, escape mutants, and low immunogenicity (4). Recently, the potential of immunoprophylaxis for virus prevention has been increased by the discovery of monoclonal antibodies that have broad specificity toward various isolates of infectious viruses (5, 6).The HBV envelope consists of three surface glycoproteins called the large (L), middle (M), and small (S) proteins. All of these proteins are translated from a single ORF comprising preS1, preS2, and S domains (7). Translation of S, preS2ϩS, and preS1ϩpreS2ϩS domains results in S, M, and L proteins, respectively (Fig. 1). The L protein, preferentially localized in infectious HBV particles, plays an essential role in viral infection (7,8), and its preS1 domain is responsible for cell attachment in viral infection (9-12). In particular, the residue 21-47 region of preS1 has been suggested to contain a specific binding site for hepatocyte receptors and also has been shown to carry virusneutralizing epitopes (10-13).In the blood of HBV-infected persons, the noninfectious subviral particles with M and S proteins are present in excess amounts over the infectious virus particles containing L protein (7,8). Thus, neutralizing anti-preS1 antibodies are thought to have advantages over the neutralizing antibodies targeting S protein, which are currently undergoing clinical trials (4). Previously, we generated an anti-preS1 murine antibody KR127 that recognizes adr subtype preS1 (residues 37-45) (15). KR127 and the humanized version of KR127 antibody (HzKR127) bind to the preS1 domain of various clinical HBV isolates, including both adr and ayw subtypes, suggesting their broad neutralizing activity (15, 16). HzKR127 also has been sh...

Free energy perturbation approach for the rational engineering of the antibody for human hepatitis B virus

“…A few years ago, Chi et al reported the X-ray crystal structure of HzKR127 in complex with a major epitope peptide, which provides a structural basis for the neutralization of HBV [16]. The antibody was shown to undergo an opening of CDR to a substantial extent upon binding of the peptide antigen.…”

Broadly neutralizing anti‐HBV antibody binds to non‐epitope regions of preS1

“…In our previous study, HzKR127 Fab displayed one of the largest conformational changes in antigen-antibody recognition [11]. The high conformational flexibility of CDR often contributes to multispecificity of antigen recognition of TCR [24][25][26] and germline antibodies [27,28,31].…”

“…4B). HzKR127 Fab bound to the epitope peptide of preS1 (36- Considering unusually high conformational flexibility observed in the crystal structure [11], the binding site of HzKR127 could change its own structure to accommodate the low-affinity site.…”

“…The antibody exhibits broadly neutralizing activity against various HBV isolates, including adr and ayw subtypes, and protected the chimpanzee from HBV infection [10]. To elucidate the antigen recognition mechanism of the broadly neutralizing antibody of HBV, we determined the crystal structures of free HzKR127 Fab and its complex with HBV preS1 (36-46) peptide epitope [11]. Structural comparison between free and bound forms of the Fab revealed that unusually high conformational change of antibody ( 10 Å in main-chain and 15 Å in side-chain of H3 loop) occurs upon antigen binding.…”

Pre‐structured motifs in the natively unstructured preS1 surface antigen of hepatitis B virus

“…An antiviral fusion inhibitor ''Pentafuside'' was developed against HIV following such a strategy (Chan and Kim 1998). We have been studying structural features of HBV surface antigens to gain insight into interactions between HBV and hepatocyte (Chi et al 2006(Chi et al , 2007. Preliminary CD studies showed that the preS1 exists in an unstructured state without a three-dimensional structure (Lee et al 1994;Maeng et al 2001).…”