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Two synthetic antibodies that recognize and neutralize distinct proteolytic forms of the ebola virus envelope glycoprotein.

ABSTRACT: Ebola virus (EBOV) is a highly pathogenic member of the Filoviridae family of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann-Pick C1 (NPC1) and may induce conformational changes in GP leading to membrane fusion. However, the precise details of the structural changes in GP associated with proteolysis and the role of these changes in viral entry have not been established. Here, we have employed synthetic antibody technology to identify antibodies targeting EBOV GP prior to and following proteolysis (i.e. in the "uncleaved" [GP(UNCL)] and "cleaved" [GP(CL)] forms). We identified antibodies with distinct recognition profiles: Fab(CL) bound preferentially to GP(CL) (EC(50)=1.7 nM), whereas Fab(UNCL) bound specifically to GP(UNCL) (EC(50)=75 nM). Neutralization assays with GP-containing pseudotyped viruses indicated that these antibodies inhibited GP(CL)- or GP(UNCL)-mediated viral entry with specificity matching their recognition profiles (IC(50): 87 nM for IgG(CL); 1 ?M for Fab(UNCL)). Competition ELISAs indicate that Fab(CL) binds an epitope distinct from that of KZ52, a well-characterized EBOV GP antibody, and from that of the luminal domain of NPC1. The binding epitope of Fab(UNCL) was also distinct from that of KZ52, suggesting that Fab(UNCL) binds a novel neutralization epitope on GP(UNCL). Furthermore, the neutralizing ability of Fab(CL) suggests that there are targets on GP(CL) available for neutralization. This work showcases the applicability of synthetic antibody technology to the study of viral membrane fusion, and provides new tools for dissecting intermediates of EBOV entry.

SUBMITTER: Koellhoffer JF

PROVIDER: S-EPMC3684266 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Two synthetic antibodies that recognize and neutralize distinct proteolytic forms of the ebola virus envelope glycoprotein.

Koellhoffer Jayne F JF Chen Gang G Sandesara Rohini G RG Bale Shridhar S Saphire Erica Ollmann EO Chandran Kartik K Sidhu Sachdev S SS Lai Jonathan R JR

Chembiochem : a European journal of chemical biology 20121030 17

Ebola virus (EBOV) is a highly pathogenic member of the Filoviridae family of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann-Pick C1 (NPC1) and may induce conformationa ...[more]

PMID: 23111988

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Project description:The high viral diversity of HIV-1 is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to efficiently elicit bNAbs. To overcome these obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (MAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive MAbs were isolated using antigen-specific single B-cell sorting. Four of these MAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing MAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles, and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B-neutralizing MAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer-dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic, and 1.7 million people still get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing MAbs, combined with detailed characterization of the neutralization epitope, can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination.

Dataset Information

Two synthetic antibodies that recognize and neutralize distinct proteolytic forms of the ebola virus envelope glycoprotein.

Publications

Two synthetic antibodies that recognize and neutralize distinct proteolytic forms of the ebola virus envelope glycoprotein.

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