Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization.

UNLABELLED: The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 µg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans. IMPORTANCE: To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different antibody binding arms could potentially display neutralization characteristics better than those of any single parental antibody. Here we show that bispecific antibodies contain the binding specificities of the two parental antibodies and that a single bispecific antibody can neutralize 97% of viral strains with a high overall potency. These findings support the use of bispecific antibodies for the prevention or treatment of HIV-1 infection.

Drift of the HIV-1 envelope glycoprotein gp120 toward increased neutralization resistance over the course of the epidemic: a comprehensive study using the most potent and broadly neutralizing monoclonal antibodies.

Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.

Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding.

Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.

A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding.

The entry of primate immunodeficiency viruses into target cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors, CD4 and members of the chemokine receptor family. The gp120 third variable (V3) loop has been implicated in chemokine receptor binding, but the use of the CCR5 chemokine receptor by diverse primate immunodeficiency viruses suggests the involvement of an additional, conserved gp120 element. Through the use of gp120 mutants, a highly conserved gp120 structure was shown to be critical for CCR5 binding. This structure is located adjacent to the V3 loop and contains neutralization epitopes induced by CD4 binding. This conserved element may be a useful target for pharmacologic or prophylactic intervention in human immunodeficiency virus (HIV) infections.

Structure of beta 2-bungarotoxin: potassium channel binding by Kunitz modules and targeted phospholipase action.

BACKGROUND: beta-bungarotoxin is a heterodimeric neurotoxin consisting of a phospholipase subunit linked by a disulfide bond to a K+ channel binding subunit which is a member of the Kunitz protease inhibitor superfamily. Toxicity, characterized by blockage of neural transmission, is achieved by the lipolytic action of the phospholipase targeted to the presynaptic membrane by the Kunitz module. RESULTS: The crystal structure at 2.45 A resolution suggests that the ion channel binding region of the Kunitz subunit is at the opposite end of the module from the loop typically involved in protease binding. Analysis of the phospholipase subunit reveals a partially occluded substrate-binding surface and reduced hydrophobicity. CONCLUSIONS: Molecular recognition by this Kunitz module appears to diverge considerably from more conventional superfamily members. The ion channel binding region identified here may mimic the regulatory interaction of endogenous neuropeptides. Adaptations of the phospholipase subunit make it uniquely suited to targeting and explain the remarkable ability of the toxin to avoid binding to non-target membranes. Insight into the mechanism of beta-bungarotoxin gained here may lead to the development of therapeutic strategies against not only pathological cells, but also enveloped viruses.

Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates.

BACKGROUND: The gp120 exterior envelope glycoprotein of HIV-1 binds sequentially to CD4 and chemokine receptors on cells to initiate virus entry. During natural infection, gp120 is a primary target of the humoral immune response, and it has evolved to resist antibody-mediated neutralization. We previously reported the structure at 2.5 A of a gp120 core from the HXBc2 laboratory-adapted isolate in complex with a 2 domain fragment of CD4 and the antigen binding fragment of a human antibody. This revealed atomic details of gp120-receptor interactions and suggested multiple mechanisms of immune evasion. RESULTS: We have now extended the HXBc2 structure in P222, crystals to 2.2 A. The enhanced resolution enabled a more accurate modeling of less-well-ordered regions and provided conclusive identification of the density in the central cavity at the crux of the gp120-CD4 interaction as isopropanol from the crystallization medium. We have also determined the structure of a gp120 core from the primary clinical HIV-1 isolate, YU2, in the same ternary complex but in a C2 crystal lattice. Comparisons of HXBc2 and YU2 showed that while CD4 binding was rigid, portions of the gp120 core were conformationally flexible; overall differences were minor, with sequence changes concentrated on a surface expected to be exposed on the envelope oligomer. CONCLUSIONS: Despite dramatic antigenic differences between primary and laboratory-adapted HIV-1, the gp120 cores from these isolates are remarkably similar. Taken together with chimeric substitution and sequence analysis, this indicates that neutralization resistance is specified by quaternary interactions involving the major variable loops and thus affords a mechanism for viral adaptation. Conservation of the central cavity suggests the possibility of therapeutic inhibitors. The structures reported here extend in detail and generality our understanding of the biology of the gp120 envelope glycoprotein.

Selective effects of electrostatic changes in the CD4 CDR-3-like loop on infection by different human immunodeficiency virus type 1 isolates.

The role of the CDR-3-like loop of the first domain of the CD4 molecule in infection by the human immunodeficiency virus type 1 (HIV-1) is controversial. In an attempt to determine whether the strong negative charge in the CDR-3-like loop influences HIV-1 infection we have substituted by mutagenesis negative for positively charged residues at position 87/88 and 91/92. These mutations were shown to have no obvious effect on CD4 conformation outside of the CDR-3-like loop. Infection of cells expressing the E87K/D88K substitution mutant resulted in a selective reduction in infectivity for certain HIV-1 viruses compared to cells expressing wile-type CD4. Viruses Hx10, HxB2, and MN were 4- to 13-fold less efficient at infecting the E87K/D88K mutant, whereas SF2, RF, and NDK yielded an efficiency of infection similar to, or slightly greater than, that of the wild type. To investigate the step at which infectivity was selectively reduced, we compared early events in the life cycles of Hx10 and SF2 viruses using PCR entry and gp120-binding assays. Both gp120 binding and virus entry were reduced for Hx10 on the mutant CD4-expressing cells as compared to wild-type CD4-expressing cells, whereas no difference was seen in either assay with SF2. Although relatively small in magnitude, the contribution of the CDR-3-like loop to the overall CD4-gp120 interaction may serve to modify the binding and entry of certain virus isolates.

Analysis of the interaction of antibodies with a conserved enzymatically deglycosylated core of the HIV type 1 envelope glycoprotein 120.

The binding of a panel of monoclonal antibodies to V1, V2, and V3 loop-deleted HIV-1 gp120 was studied by competition analysis. Most of the previously defined relationships between gp120 epitopes were preserved on the variable loop-deleted protein, although interactions between some epitopes were dependent on the presence of the V1, V2, and V3 loops. Enzymatic deglycosylation of the variable loop-deleted protein only minimally altered the binding of most antibodies examined. Thus, a carbohydrate-deficient, conserved HIV-1 gp120 core can be produced that has a structure closely approximating that of the full-length, correctly folded gp120 monomer.

Dimeric association and segmental variability in the structure of human CD4.

CD4 is a co-receptor in the cellular immune response. It increases the avidity of association between a T cell and an antigen-presenting cell by interacting with non-polymorphic portions of the complex between class II major histocompatibility complex (MHC) and T-cell receptor (TCR) molecules, and it contributes directly to signal transduction through its cytoplasmic association with the lymphocyte kinase Lck. CD4 also serves as the high-affinity receptor for cellular attachment and entry of the human immunodeficiency virus (HIV). The extracellular portion of CD4 comprises four immunoglobulin-like domains (D1-D4). This part of human CD4 (residues 1-369) has been characterized as a recombinant soluble protein (sCD4), and crystal structures have been described for the human D1D2 fragment and for the rat D3D4 fragment. We have now determined the structures of intact sCD4 in three crystal lattices. These structures have a hinge-like variability at the D1D2 to D3D4 junction that might be important in immune recognition and HIV fusion, and a common dimeric association through D4 domains. Dynamic light scattering measurements and chemical crosslinking of sCD4 corroborate dimerization at high protein concentration. We suggest that such dimers mayhave relevance as mediators of signal transduction in T cells.

Beta-bungarotoxin. Preparation and characterization of crystals suitable for structural analysis.

Phospholipases in some snake venoms are potent neurotoxins that target their enzymatic action to the synaptic membrane. One of these is the heterodimeric neurotoxin, beta-bungarotoxin, which binds with a protease inhibitor-like subunit to a presynaptic potassium channel and then blocks neurotransmission with a second subunit that has phospholipase A2 activity. We have prepared and characterized well ordered crystals of the most toxic beta-bungarotoxin isoform, beta 1-bungarotoxin. The crystals are monoclinic, space group C2, with unit cell parameters: a = 176.5 A, b = 39.3 A, c = 92.7 A, and beta = 114.8 degrees. Rotation-function analysis of the Patterson function, as calculated from a 2.3-A data set, reveals an asymmetric unit composed of four heterodimers. These heterodimers appear to be associated as two crystallographically distinct (AB)4 tetramers, each having dihedral D2 symmetry. The two are positioned with equivalent molecular 2-fold axes coincident with crystallographic dyads, but rotated by 55 degrees relative to one another. X-ray analysis of these crystals will permit direct visualization of the specific structural motifs and chemical features that underlie phospholipase neurotoxicity.

Considerations on the folding topology and evolutionary origin of cadherin domains.

Cell-cell adhesion in zonula adherens and desmosomal junctions is mediated by cadherins, and recent crystal structures of the first domain from murine N-cadherin provide a plausible molecular basis for this adhesive action. A structure-based sequence analysis of this adhesive domain indicates that its fold is common to all extracellular cadherin domains. The cadherin folding topology is also shown to be similar to immunoglobulin-like domains and to other Greek-key beta-sandwich structures, as diverse as domains from plant cytochromes, bacterial cellulases, and eukaryotic transcription factors. Sequence similarities between cadherins and these other molecules are very low, however, and intron patterns are also different. On balance, independent origins for a favorable folding topology seem more likely than evolutionary divergence from an ancestor common to cadherins and immunoglobulins.

Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus.

The human immunodeficiency virus envelope glycoproteins, gp120 and gp41, function in cell entry by binding to CD4 and a chemokine receptor on the cell surface and orchestrating the direct fusion of the viral and target cell membranes. On the virion surface, three gp120 molecules associate noncovalently with the ectodomain of the gp41 trimer to form the envelope oligomer. Although an atomic-level structure of a monomeric gp120 core has been determined, the structure of the oligomer is unknown. Here, the orientation of gp120 in the oligomer is modeled by using quantifiable criteria of carbohydrate exposure, occlusion of conserved residues, and steric considerations with regard to the binding of the neutralizing antibody 17b. Applying similar modeling techniques to influenza virus hemagglutinin suggests a rotational accuracy for the oriented gp120 of better than 10 degrees. The model shows that CD4 binds obliquely, such that multiple CD4 molecules bound to the same oligomer have their membrane-spanning portions separated by at least 190 A. The chemokine receptor, in contrast, binds to a sterically restricted surface close to the trimer axis. Electrostatic analyses reveal a basic region which faces away from the virus, toward the target cell membrane, and is conserved on core gp120. The electrostatic potentials of this region are strongly influenced by the overall charge, but not the precise structure, of the third variable (V3) loop. This dependence on charge and not structure may make electrostatic interactions between this basic region and the cell difficult to target therapeutically and may also provide a means of viral escape from immune system surveillance.

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.

The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gp120 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 A resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.

The antigenic structure of the HIV gp120 envelope glycoprotein.

The human immunodeficiency virus HIV-1 establishes persistent infections in humans which lead to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope glycoproteins, gp120 and gp41, are assembled into a trimeric complex that mediates virus entry into target cells. HIV-1 entry depends on the sequential interaction of the gp120 exterior envelope glycoprotein with the receptors on the cell, CD4 and members of the chemokine receptor family. The gp120 glycoprotein, which can be shed from the envelope complex, elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gp120 regions that are occluded on the assembled trimer and which are exposed only upon shedding. Neutralizing antibodies, by contrast, must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor-binding regions. Here we describe the spatial organization of conserved neutralization epitopes on gp120, using epitope maps in conjunction with the X-ray crystal structure of a ternary complex that includes a gp120 core, CD4 and a neutralizing antibody. A large fraction of the predicted accessible surface of gp120 in the trimer is composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Understanding the structural basis for the ability of HIV-1 to evade the humoral immune response should assist in the design of a vaccine.

Modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution.

The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.

Molecular characteristics of recombinant human CD4 as deduced from polymorphic crystals.

We have grown crystals of a soluble recombinant form of human CD4, a transmembrane glycoprotein found predominantly on the surface of helper T cells. Crystals composed of the entire extracellular portion of CD4 exhibit extensive polymorphism. Of the five crystal types that have been grown, the best diffracts to Bragg spacings of 4.9 A. Symmetry considerations and characterization of the asymmetric unit by volume-specific amino acid analysis lead to the suggestion that a tetramer is the fundamental unit of crystallization. The characterization also showed that several of the crystal types have unusually high solvent contents. Because high solvent content and weak diffraction are indicative of an extended flexible structure, we examined the molecular shape of the recombinant CD4 with ultracentrifugation and found that it has an axial ratio of roughly 6, when modeled as a prolate ellipsoid. These results, combined with crystal packing constraints, suggest dimensions of approximately 25 x 25 x 125 A for a monomer. The structural features deduced here may be relevant to the biological function of CD4 as a receptor mediating cell-cell and cell-virus interactions.

Probability analysis of variational crystallization and its application to gp120, the exterior envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1).

The extensive glycosylation and conformational mobility of gp120, the envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1), pose formidable barriers for crystallization. To surmount these difficulties, we used probability analysis to determine the most effective crystallization approach and derive equations which show that a strategy, which we term variational crystallization, substantially enhances the overall probability of crystallization for gp120. Variational crystallization focuses on protein modification as opposed to crystallization screening. Multiple variants of gp120 were analyzed with an iterative cycle involving a limited set of crystallization conditions and biochemical feedback on protease sensitivity, glycosylation status, and monoclonal antibody binding. Sources of likely conformational heterogeneity such as N-linked carbohydrates, flexible or mobile N and C termini, and variable internal loops were reduced or eliminated, and ligands such as CD4 and antigen-binding fragments (Fabs) of monoclonal antibodies were used to restrict conformational mobility as well as to alter the crystallization surface. Through successive cycles of manipulation involving 18 different variants, we succeeded in growing six different types of gp120 crystals. One of these, a ternary complex composed of gp120, its receptor CD4, and the Fab of the human neutralizing monoclonal antibody 17b, diffracts to a minimum Bragg spacing of at least 2.2 A and is suitable for structural analysis.

Energetics of the HIV gp120-CD4 binding reaction.

HIV infection is initiated by the selective interaction between the cellular receptor CD4 and gp120, the external envelope glycoprotein of the virus. We used analytical ultracentrifugation, titration calorimetry, and surface plasmon resonance biosensor analysis to characterize the assembly state, thermodynamics, and kinetics of the CD4-gp120 interaction. The binding thermodynamics were of unexpected magnitude; changes in enthalpy, entropy, and heat capacity greatly exceeded those described for typical protein-protein interactions. These unusual thermodynamic properties were observed with both intact gp120 and a deglycosylated and truncated form of gp120 protein that lacked hypervariable loops V1, V2, and V3 and segments of its N and C termini. Together with previous crystallographic studies, the large changes in heat capacity and entropy reveal that extensive structural rearrangements occur within the core of gp120 upon CD4 binding. CD spectral studies and slow kinetics of binding support this conclusion. These results indicate considerable conformational flexibility within gp120, which may relate to viral mechanisms for triggering infection and disguising conserved receptor-binding sites from the immune system.

Selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120.

It is well established that the gp120 V3 loop of T-cell-line-adapted human immunodeficiency virus type 1 (HIV-1) binds both cell-associated and soluble polyanions. Virus infectivity is increased by interactions between HIV-1 and heparan sulfate proteoglycans on some cell types, and soluble polyanions such as heparin and dextran sulfate neutralize HIV-1 in vitro. However, the analysis of gp120-polyanion interactions has been limited to T-cell-line-adapted, CXCR4-using virus and virus-derived gp120, and the polyanion binding ability of gp120 regions other than the V3 loop has not been addressed. Here we demonstrate by monoclonal-antibody inhibition, labeled heparin binding, and surface plasmon resonance studies that a second site, most probably corresponding to the newly defined, highly conserved coreceptor binding region on gp120, forms part of the polyanion binding surface. Consistent with the binding of polyanions to the coreceptor binding surface, dextran sulfate interfered with the gp120-CXCR4 association while having no detectable effect on the gp120-CD4 interaction. The interaction between polyanions and X4 or R5X4 gp120 was readily detectable, whereas weak or undetectable binding was observed with R5 gp120. Analysis of mutated forms of X4 gp120 demonstrated that the V3 loop is the major determinant for polyanion binding whereas other regions, including the V1/V2 loop structure and the NH(2) and COOH termini, exert a more subtle influence. A molecular model of the electrostatic potential of the conserved coreceptor binding region confirmed that it is basic but that the overall charge on this surface is dominated by the V3 loop. These results demonstrate a selective interaction of gp120 with polyanions and suggest that the conserved coreceptor binding surface may present a novel and conserved target for therapeutic intervention.