Title & Authors | Journal | Publication Date |
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Crystal Structure of a Soluble Cleaved HIV-1 Envelope Trimer |
Science | Dec. 20, 2013 |
HIV-1 entry into CD4+ target cells is mediated by cleaved envelope glycoprotein (Env) trimers that have been challenging to characterize structurally. Here, we describe the crystal structure at 4.7 angstroms of a soluble, cleaved Env trimer that is stabilized and antigenically near-native (termed the BG505 SOSIP.664 gp140 trimer) in complex with a potent broadly neutralizing antibody, PGT122. The structure shows a prefusion state of gp41, the interaction between the component gp120 and gp41 subunits, and how a close association between the gp120 V1/V2/V3 loops stabilizes the trimer apex around the threefold axis. The complete epitope of PGT122 on the trimer involves gp120 V1, V3, and several surrounding glycans. This trimer structure advances our understanding of how Env functions and is presented to the immune system, and provides a blueprint for structure-based vaccine design. |
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Structural Basis for Enhanced HIV-1 Neutralization by a Dimeric Immunoglobulin G Form of the Glycan-Recognizing Antibody 2G12 |
Cell Reports | Dec. 12, 2013 |
The human immunoglobulin G (IgG) 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1) envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two fragment crystalline (Fc) regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab)2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally distinct dimers, demonstrating flexibility of the (Fab)2-Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12. |
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Hepatitis C Virus E2 Envelope Glycoprotein Core Structure |
Science | Nov. 29, 2013 |
Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design. |
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Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site |
Journal of Virology | Nov. 20, 2013 |
ABSTRACT Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines. |
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Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation |
Proceedings of the National Academy of Sciences | Nov. 5, 2013 |
We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120–gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120–gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated. |
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A Next-Generation Cleaved, Soluble HIV-1 Env Trimer, BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies |
PLoS Pathogens | Sept. 20, 2013 |
A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens. |
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Structural Characterization of Cleaved, Soluble HIV-1 Envelope Glycoprotein Trimers |
Journal of Virology | Sept. 20, 2013 |
Human immunodeficiency virus type 1 (HIV-1) infection is a significant global public health problem for which development of an effective prophylactic vaccine remains a high scientific priority. Many concepts for a vaccine are focused on induction of appropriate titers of broadly neutralizing antibodies (bNAbs) against the viral envelope (Env) glycoproteins gp120 and gp41, but no immunogen has yet accomplished this goal in animals or humans. One approach to induction of bNAbs is to design soluble, trimeric mimics of the native viral Env trimer. Here, we describe structural studies by negative-stain electron microscopy of several variants of soluble Env trimers based on the KNH1144 subtype A sequence. These Env trimers are fully cleaved between the gp120 and gp41 components and stabilized by specific amino acid substitutions. We also illustrate the structural consequences of deletion of the V1/V2 and V3 variable loops from gp120 and the membrane-proximal external region (MPER) from gp41. All of these variants adopt a trimeric configuration that appropriately mimics native Env spikes, including the CD4 receptor-binding site and the epitope for the VRC PG04 bNAb. These cleaved, soluble trimer designs can be adapted for use with multiple different env genes for both vaccine and structural studies. |
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Human antibodies that neutralize respiratory droplet transmissible H5N1 influenza viruses |
Journal of Clinical Investigation | Sept. 3, 2013 |
Recent studies described the experimental adaptation of influenza H5 HAs that confers respiratory droplet transmission (rdt) to influenza virus in ferrets. Acquisition of the ability to transmit via aerosol may lead to the development of a highly pathogenic pandemic H5 virus. Vaccines are predicted to play an important role in H5N1 control should the virus become readily transmissible between humans. We obtained PBMCs from patients who received an A/Vietnam/1203/2004 H5N1 subunit vaccine. Human hybridomas were then generated and characterized. We identified antibodies that bound the HA head domain and recognized both WT and rdt H5 HAs. We used a combination of structural techniques to define a mechanism of antibody recognition of an H5 HA receptor–binding site that neutralized H5N1 influenza viruses and pseudoviruses carrying the HA rdt variants that have mutations near the receptor-binding site. Incorporation or retention of this critical antigenic site should be considered in the design of novel H5 HA immunogens to protect against mammalian-adapted H5N1 mutants. |
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Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120 |
Nature Structural & Molecular Biology | July 20, 2013 |
Some broadly neutralizing antibodies to HIV-1 recognize glycan-dependent epitopes on gp120. Now X-ray crystallography and EM approaches, along with functional analyses, reveal how one particular antibody (PGT135) recognizes three glycan groups and can accommodate their conformational and chemical diversity. A substantial proportion of the broadly neutralizing antibodies (bnAbs) identified in certain HIV-infected donors recognize glycan-dependent epitopes on HIV-1 gp120. Here we elucidate how the bnAb PGT 135 binds its Asn332 glycan–dependent epitope from its 3.1-Å crystal structure with gp120, CD4 and Fab 17b. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield and access the gp120 protein surface. EM reveals that PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. Combined structural studies of PGT 135, PGT 128 and 2G12 show that this Asn332-dependent antigenic region is highly accessible and much more extensive than initially appreciated, which allows for multiple binding modes and varied angles of approach; thereby it represents a supersite of vulnerability for antibody neutralization. |
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Influences on Trimerization and Aggregation of Soluble, Cleaved HIV-1 SOSIP Envelope Glycoprotein |
Journal of Virology | July 3, 2013 |
We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies. |
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Rational HIV Immunogen Design to Target Specific Germline B Cell Receptors |
Science | May 10, 2013 |
Vaccine development to induce broadly neutralizing antibodies (bNAbs) against HIV-1 is a global health priority. Potent VRC01-class bNAbs against the CD4 binding site of HIV gp120 have been isolated from HIV-1–infected individuals; however, such bNAbs have not been induced by vaccination. Wild-type gp120 proteins lack detectable affinity for predicted germline precursors of VRC01-class bNAbs, making them poor immunogens to prime a VRC01-class response. We employed computation-guided, in vitro screening to engineer a germline-targeting gp120 outer domain immunogen that binds to multiple VRC01-class bNAbs and germline precursors, and elucidated germline binding crystallographically. When multimerized on nanoparticles, this immunogen (eOD-GT6) activates germline and mature VRC01-class B cells. Thus, eOD-GT6 nanoparticles have promise as a vaccine prime. In principle, germline-targeting strategies could be applied to other epitopes and pathogens. |
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Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9 |
Proceedings of the National Academy of Sciences | March 12, 2013 |
PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. Here, we show that a soluble SOSIP.664 gp140 trimer constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations of the PG9:Env trimer complex. The BG505 SOSIP.664 gp140 trimer is remarkably stable as assessed by electron microscopy (EM) and differential scanning calorimetry. EM, small angle X-ray scattering, size exclusion chromatography with inline multiangle light scattering and isothermal titration calorimetry all indicate that only a single PG9 fragment antigen-binding (Fab) binds to the Env trimer. An ∼18 Å EM reconstruction demonstrates that PG9 recognizes the trimer asymmetrically at its apex via contact with two of the three gp120 protomers, possibly contributing to its reported preference for a quaternary epitope. Molecular modeling and isothermal titration calorimetry binding experiments with an engineered PG9 mutant suggest that, in addition to the N156 and N160 glycan interactions observed in crystal structures of PG9 with a scaffolded V1/V2 domain, PG9 makes secondary interactions with an N160 glycan from an adjacent gp120 protomer in the antibody–trimer complex. Together, these structural and biophysical findings should facilitate the design of HIV-1 immunogens that possess all elements of the quaternary PG9 epitope required to induce broadly neutralizing antibodies against this region. |
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Integrative Structural Biology |
Science | Feb. 22, 2013 |
Integrative approaches using data from a wide variety of methods are yielding model structures of complex biological assemblies. |
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A Blueprint for HIV Vaccine Discovery |
Cell Host & Microbe | Oct. 18, 2012 |
Despite numerous attempts over many years to develop an HIV vaccine based on classical strategies, none has convincingly succeeded to date. A number of approaches are being pursued in the field, including building upon possible efficacy indicated by the recent RV144 clinical trial, which combined two HIV vaccines. Here, we argue for an approach based, in part, on understanding the HIV envelope spike and its interaction with broadly neutralizing antibodies (bnAbs) at the molecular level and using this understanding to design immunogens as possible vaccines. BnAbs can protect against virus challenge in animal models, and many such antibodies have been isolated recently. We further propose that studies focused on how best to provide T cell help to B cells that produce bnAbs are crucial for optimal immunization strategies. The synthesis of rational immunogen design and immunization strategies, together with iterative improvements, offers great promise for advancing toward an HIV vaccine. |
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Cross-neutralization of influenza A viruses mediated by a single antibody loop |
Nature | Sept. 27, 2012 |
Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens. The crystal structure of an influenza antibody that recognizes a small, conserved site in the variable receptor-binding domain of HA is described; this antibody shows broad neutralization across multiple subtypes of influenza A virus through an antibody–antigen interaction dominated by a single heavy-chain complementarity-determining region 3 loop. This manuscript reports the identification and structural characterization of a novel anti-influenza antibody, C05, that recognizes a small conserved site in the variable receptor-binding domain of haemagglutinin. The antibody achieves broad neutralization by the insertion of a single loop of the heavy-chain complementarity-determining region 3 into the small conserved site amplified by the avidity of additional binding interactions. This finding highlights loop insertion into the receptor-binding pocket of haemagglutinin as a possible strategy to achieve broad neutralization of influenza by vaccines and therapeutic antibodies. |
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Highly Conserved Protective Epitopes on Influenza B Viruses |
Science | Sept. 14, 2012 |
Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody–based immunotherapy and “universal” vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody–based treatments and a universal flu vaccine for all influenza A and B viruses. |
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Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers* |
Journal of Biological Chemistry | July 13, 2012 |
The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The glycan-depleted Env trimers should be useful for structural and immunogenicity studies. Background: The heterogeneity and flexibility of HIV-1 envelope glycoprotein N-glycans interfere with structural and vaccine studies. Results: HIV-1 envelope trimers can be partially deglycosylated without affecting trimer integrity. Conclusion: HIV-1 envelope glycoprotein N-glycans do not contribute to trimer integrity once the protein is folded. Significance: Deglycosylated HIV-1 envelope trimers should be useful for structural and vaccine studies. |
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A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield |
Science | Nov. 25, 2011 |
The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man9 at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface. |
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Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9 |
Nature | Nov. 23, 2011 |
Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which—with PG9—involves a site of vulnerability comprising just two glycans and a strand. The crystal structure of V1/V2, the only unresolved portion of the HIV-1 gp120 envelope glycoprotein, is reported in complex with human antibody PG9 and reveals a paradigm of antibody recognition with implications for vaccine development. The V1/V2 variable region of the gp120 envelope glycoprotein of HIV-1, with its extraordinary sequence diversity and N-linked glycosylation, exemplifies the ability of the virus to evade antibody recognition. It has also resisted structural determination. Now Peter Kwong and colleagues have determined the atomic-level structure of gp120 V1/V2 by using an antibody called PG9, which can neutralize most strains of HIV. Instead of being confounded by the N-linked glycan that shields most of gp120 from immune recognition, PG9 uses N-linked glycan for binding through a mechanism shared by a number of antibodies capable of effective HIV neutralization. The structure shows that the antibody recognizes glycopeptide conjugates and avoids diversity in V1/V2 by making sequence-independent interactions, such as hydrogen bonds. |