Structural basis for enhanced HIV-1 neutralization by a dimeric immunoglobulin G form of the glycan-recognizing antibody 2G12.
Ontology highlight
ABSTRACT:
SUBMITTER: Wu Y
PROVIDER: S-EPMC3919625 | biostudies-literature | 2013 Dec
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.
| S-EPMC2612297 | biostudies-literature
Project description:OBJECTIVE:Increasing data support a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV-1 infection. We recently isolated a naturally occurring dimeric form of the anti-HIV-1 antibody 2G12 and found it to be significantly more potent than 2G12 monomer in neutralizing primary virus strains. However, given the unusual structure of dimeric 2G12 with two Fc regions, it was not clear whether 2G12 dimer could bind to the CD16 Fc receptor on ADCC effector cells or trigger ADCC. Here we compared the in-vitro ADCC activities of 2G12 monomer and dimer and investigated the effects of including ADCC-enhancing mutations in both forms of 2G12. METHODS:An in-vitro ADCC assay using target cells stably expressing gp160 was developed to evaluate the activities of 2G12 monomer and dimer with and without ADCC-enhancing mutations that increase the CD16-binding affinity of the 2G12 Fc region. RESULTS:Both 2G12 monomer and 2G12 dimer elicited ADCC, although the dimer showed increased potency [lower half-maximal concentration (EC(50))] in triggering ADCC, thus confirming its ability to bind CD16 and trigger ADCC. The ADCC-enhancing mutations improved the ADCC activity of 2G12 monomer more than 2G12 dimer such that their EC(50) values were nearly equal. However, no increase in nonspecific ADCC activity was observed using 2G12 IgGs with these mutations. CONCLUSION:Given the likelihood that ADCC plays a role in protecting against initial infection and/or controlling chronic infection, these data suggest 2G12 dimers and/or addition of ADCC-enhancing mutations could augment the prophylactic and/or therapeutic potential of 2G12.
| S-EPMC2898891 | biostudies-literature
Project description:Despite structural knowledge of broadly neutralizing monoclonal antibodies (NMAbs) complexed to HIV-1 gp120 and gp41 envelope glycoproteins, virus inactivation mechanisms have been difficult to prove, in part because neutralization assays are complex and were previously not understood. Concordant with recent evidence that HIV-1 titers are determined by a race between entry of cell-attached virions and competing inactivation processes, we show that NMAb 2G12, which binds to gp120 N-glycans with α (1, 2)-linked mannose termini and inhibits replication after passive transfer into patients, neutralizes by slowing entry of adsorbed virions. Accordingly, apparent neutralization is attenuated when a kinetically competing virus inactivation pathway is blocked. Moreover, removing 2G12 from media causes its dissociation from virions coupled to accelerated entry and restored infectivity, demonstrating the reversibility of neutralization. A difference between 2G12 dissociation and infectivity recovery rates implies that the inhibited complexes at virus-cell junctions contain several 2G12's that must dissociate before entry commences. Quantitative microscopy of 2G12 binding and dissociation from single virions and studies using a split CCR5 coreceptor suggest that 2G12 competitively inhibits interactions between gp120's V3 loop and the tyrosine sulfate-containing CCR5 amino terminus, thereby reducing assembly of complexes that catalyze entry. These results reveal a unique reversible kinetic mechanism for neutralization by an antibody that binds near a critical V3 region in the glycan shield of gp120.
| S-EPMC3356642 | biostudies-literature
Project description:The monoclonal antibody (MAb) 2G12 recognizes a cluster of high-mannose oligosaccharides on the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 and is one of a select group of MAbs with broad neutralizing activity. However, subtype C viruses are generally resistant to 2G12 neutralization. This has been attributed to the absence of a glycosylation site at position 295 in most subtype C gp120s, which instead is typically occupied by a Val residue. Here we show that N-linked glycans in addition to the one at position 295 are important in the formation of the 2G12 epitope in subtype C gp120. Introduction of the glycosylation site at position 295 into three subtype C molecular clones, Du151.2, COT9.6, and COT6.15, did increase 2G12 binding to all three mutagenized gp120s, but at various levels. The COT9-V295N mutant showed the strongest 2G12 binding and was the only mutant to become sensitive to 2G12 neutralization, although very high antibody concentrations were required. Introduction of a glycosylation site at position 448 into mutant COT6-V295N, which occurs naturally in COT9, resulted in a virus that was partially sensitive to 2G12. Interestingly, a glycosylation site at position 442, which is common among subtype C viruses, also contributed to the 2G12 epitope. The addition of this glycan increased virus neutralization sensitivity to 2G12, whereas its deletion conferred resistance. Collectively, our results indicate that the 2G12 binding site cannot readily be reconstituted on the envelopes of subtype C viruses, suggesting structural differences from other HIV subtypes in which the 2G12 epitope is naturally expressed.
| S-EPMC2045459 | biostudies-literature
Project description:We previously showed that broadly neutralizing anti-HIV-1 antibody 2G12 (human IgG1) naturally forms dimers that are more potent than monomeric 2G12 in in vitro neutralization of various strains of HIV-1. In this study, we have investigated the protective effects of monomeric versus dimeric 2G12 against HIV-1 infection in vivo using a humanized mouse model. Our results showed that passively transferred, purified 2G12 dimer is more potent than 2G12 monomer at preventing CD4 T cell loss and suppressing the increase of viral load following HIV-1 infection of humanized mice. Using humanized mice bearing IgG "backpack" tumors that provided 2G12 antibodies continuously, we found that a sustained dimer concentration of 5-25 µg/ml during the course of infection provides effective protection against HIV-1. Importantly, 2G12 dimer at this concentration does not favor mutations of the HIV-1 envelope that would cause the virus to completely escape 2G12 neutralization. We have therefore identified dimeric 2G12 as a potent prophylactic reagent against HIV-1 in vivo, which could be used as part of an antibody cocktail to prevent HIV-1 infection.
| S-EPMC3002980 | biostudies-literature
Project description:Carbohydrate-binding antibodies play a critical role in basic and clinical research. Monoclonal antibodies that bind glycans are used to measure carbohydrate expression, and serum antibodies to glycans can be important elements of the immune response to pathogens and vaccines. Carbohydrate antigen arrays, or glycan arrays, have emerged as powerful tools for the high-throughput analysis of carbohydrate-protein interactions. Our group has focused on the development and application of neoglycoprotein arrays, a unique array format wherein carbohydrates are covalently attached to a carrier protein prior to immobilization on the surface. The neoglycoprotein format permits variations of glycan structure, glycan density, and neoglycoprotein density on a single array. The focus of this study was on the effects of neoglycoprotein density on antibody binding. First, we evaluated binding of five monoclonal antibodies (81FR2.2, HE-195, HE-193, B480, and Z2A) to the blood group A antigen and found that neoglycoprotein density had a substantial effect on recognition. Next, we profiled serum antibodies in 15 healthy individuals and showed that inclusion of multiple neoglycoprotein densities helps distinguish different subpopulations of antibodies. Finally, we evaluated immune responses induced by a prostate cancer vaccine and showed that variations in neoglycoprotein density enable one to detect antibody responses that could not be detected otherwise. Neoglycoprotein density is a useful element of diversity for evaluating antibody recognition and, when combined with variations in glycan structure and glycan density, provides multidimensional glycan arrays with enhanced performance for monoclonal antibody development, biomarker discovery, and vaccine optimization.
| S-EPMC3462433 | biostudies-literature
Project description:Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti-HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation.
| S-EPMC3271928 | biostudies-literature
Project description:The arenavirus Lassa causes severe hemorrhagic fever and a significant disease burden in West Africa every year. The glycoprotein, GPC, is the sole antigen expressed on the viral surface and the critical target for antibody-mediated neutralization. Here we present the crystal structure of the trimeric, prefusion ectodomain of Lassa GP bound to a neutralizing antibody from a human survivor at 3.2-angstrom resolution. The antibody extensively anchors two monomers together at the base of the trimer, and biochemical analysis suggests that it neutralizes by inhibiting conformational changes required for entry. This work illuminates pH-driven conformational changes in both receptor-binding and fusion subunits of Lassa virus, illustrates the unique assembly of the arenavirus glycoprotein spike, and provides a much-needed template for vaccine design against these threats to global health.
| S-EPMC6007842 | biostudies-literature
Project description:Glycoprotein D (gD) of herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD-nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.
| S-EPMC3792640 | biostudies-literature
Project description:Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.
| S-EPMC8916737 | biostudies-literature