scholarly journals Cryo-EM structure of full-length HIV-1 Env bound with the Fab of antibody PG16

2019 ◽  
Author(s):  
Junhua Pan ◽  
Hanqin Peng ◽  
Bing Chen ◽  
Stephen C. Harrison
Keyword(s):  
Single Molecule ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Good Representation ◽  
Native Conformation ◽  
X Ray Crystallography ◽  
Closed Conformation ◽  
Immunogen Design ◽  
Single Molecule Studies ◽  
Hiv 1

AbstractThe HIV-1 envelope protein (Env) is the target of neutralizing antibodies and the template for vaccine immunogen design. The dynamic conformational equilibrium of trimeric Env influences its antigenicity and potential immunogenicity. Antibodies that bind at the trimer apex stabilize a “closed” conformation characteristic of the most difficult to neutralize isolates. A goal of vaccine development is therefore to mimic the closed conformation in a designed immunogen. A disulfide-stabilized, trimeric Env ectodomain -- the “SOSIP” construct -- has many of the relevant properties; it is also particularly suitable for structure determination. Some single-molecule studies have, however, suggested that the SOSIP trimer is not a good representation of Env on the surface of a virion or an infected cell. We isolated Env (fully cleaved to gp120 and gp41) from the surface of expressing cells using tagged, apex-binding Fab PG16 and determined the structure of the PG16-Env complex by cryo-EM to an overall resolution of 4.6 Å. Placing the only purification tag on the Fab ensured that the isolated Env was continuously stabilized in its closed, native conformation. The Env structure in this complex corresponds closely to the SOSIP structures determined by both x-ray crystallography and cryo-EM. Although the membrane-interacting elements are not resolved in our reconstruction, we can make inferences about the connection between ectodomain and membrane-proximal external region (MPER) by reference to the published cryo-tomography structure of an Env “spike” and the NMR structure of the MPER-transmembrane segment. We discuss these results in view of the conflicting interpretations in the literature.

scholarly journals Reducing V3 Antigenicity and Immunogenicity on Soluble, Native-Like HIV-1 Env SOSIP Trimers

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Rajesh P. Ringe ◽  
Gabriel Ozorowski ◽  
Kimmo Rantalainen ◽  
Weston B. Struwe ◽  
Katie Matthews ◽  
...  
Keyword(s):  
Animal Models ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Tier 2 ◽  
Present Generation ◽  
Coreceptor Binding ◽  
Tier 1 ◽  
Immunogen Design ◽  
V3 Region ◽  
Hiv 1

ABSTRACT Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such “off-target” immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged. IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.

scholarly journals A site of vulnerability at V3 crown defined by HIV-1 bNAb M4008_N1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kun-Wei Chan ◽  
Christina C. Luo ◽  
Hong Lu ◽  
Xueling Wu ◽  
Xiang-Peng Kong
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Design ◽  
Tier 2 ◽  
Broadly Neutralizing Antibodies ◽  
Closed Conformation ◽  
Immunogen Design ◽  
A Site ◽  
Β Sheet ◽  
Hiv 1

AbstractIdentification of vulnerable sites defined by broadly neutralizing antibodies (bNAbs) on HIV-1 envelope (Env) is crucial for vaccine design, and we present here a vulnerable site defined by bNAb M4008_N1, which neutralizes about 40% of a tier-2 virus panel. A 3.2 Å resolution cryo-EM structure of M4008_N1 in complex with BG505 DS-SOSIP reveals a large, shallow protein epitope surface centered at the V3 crown of gp120 and surrounded by key glycans. M4008_N1 interacts with gp120 primarily through its hammerhead CDR H3 to form a β-sheet interaction with the V3 crown hairpin. This makes M4008_N1 compatible with the closed conformation of the prefusion Env trimer, and thus distinct from other known V3 crown mAbs. This mode of bNAb approaching the immunogenic V3 crown in the native Env trimer suggests a strategy for immunogen design targeting this site of vulnerability.

scholarly journals Cooperation between Strain-Specific and Broadly Neutralizing Responses Limited Viral Escape and Prolonged the Exposure of the Broadly Neutralizing Epitope

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Colin Anthony ◽  
Talita York ◽  
Valerie Bekker ◽  
David Matten ◽  
Philippe Selhorst ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Prolonged Exposure ◽  
Vaccine Development ◽  
Viral Evolution ◽  
Neutralizing Antibody ◽  
Viral Escape ◽  
Broadly Neutralizing Antibodies ◽  
Immunogen Design ◽  
Early Strain ◽  
Hiv 1

ABSTRACT V3-glycan-targeting broadly neutralizing antibodies (bNAbs) are a focus of HIV-1 vaccine development. Understanding the viral dynamics that stimulate the development of these antibodies can provide insights for immunogen design. We used a deep-sequencing approach, together with neutralization phenotyping, to investigate the rate and complexity of escape from V3-glycan-directed bNAbs compared to overlapping early strain-specific neutralizing antibody (ssNAb) responses to the V3/C3 region in donor CAP177. Escape from the ssNAb response occurred rapidly via an N334-to-N332 glycan switch, which took just 7.5 weeks to reach >50% frequency. In contrast, escape from the bNAbs was mediated via multiple pathways and took longer, with escape first occurring through an increase in V1 loop length, which took 46 weeks to reach 50% frequency, followed by an N332-to-N334 reversion, which took 66 weeks. Importantly, bNAb escape was incomplete, with contemporaneous neutralization observed up to 3 years postinfection. Both the ssNAb response and the bNAb response were modulated by the presence/absence of the N332 glycan, indicating an overlap between the two epitopes. Thus, selective pressure by ssNAbs to maintain the N332 glycan may have constrained the bNAb escape pathway. This slower and incomplete viral escape resulted in prolonged exposure of the bNAb epitope, which may in turn have aided the maturation of the bNAb lineage. IMPORTANCE The development of an HIV-1 vaccine is of paramount importance, and broadly neutralizing antibodies are likely to be a key component of a protective vaccine. The V3-glycan-targeting bNAb responses are among the most promising vaccine targets, as they are commonly elicited during infection. Understanding the interplay between viral evolution and the development of these antibodies provides insights that may guide immunogen design. Our work contrasted the dynamics of the early strain-specific antibodies and the later broadly neutralizing responses to a common Env target (V3C3), showing slower and more complex escape from bNAbs. Constrained bNAb escape, together with evidence of contemporaneous autologous virus neutralization, supports the proposal that prolonged exposure of the bNAb epitope enabled the maturation of the bNAb lineage.

scholarly journals Structurally related but genetically unrelated antibody lineages converge on an immunodominant HIV-1 Env neutralizing determinant following trimer immunization

2021 ◽  
Vol 17 (9) ◽  
pp. e1009543
Author(s):  
Safia S. Aljedani ◽  
Tyler J. Liban ◽  
Karen Tran ◽  
Ganesh Phad ◽  
Suruchi Singh ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Molecular Mechanisms ◽  
Vaccine Development ◽  
Hypervariable Region ◽  
Variable Region ◽  
Tier 2 ◽  
X Ray Crystallography ◽  
Neutralizing Capacity ◽  
Clade C ◽  
Hiv 1

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184–186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.

scholarly journals Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Andrew J Borst ◽  
Connor E Weidle ◽  
Matthew D Gray ◽  
Brandon Frenz ◽  
Joost Snijder ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
X Ray Crystallography ◽  
Antibody Recognition ◽  
Immunodeficiency Virus ◽  
Gp120 Core ◽  
Clade C ◽  
Immunogen Design ◽  
Cd4 Binding Site ◽  
Variable Loops ◽  
Hiv 1

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1–3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.

scholarly journals Structurally related but genetically unrelated antibody lineages converge on an immunodominant HIV-1 Env neutralizing determinant following trimer immunization

2021 ◽  
Author(s):  
Safia Aljedani ◽  
Tyler J Liban ◽  
Karen Tran ◽  
Ganesh Phad ◽  
Suruchi Singh ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Molecular Mechanisms ◽  
Vaccine Development ◽  
Hypervariable Region ◽  
Variable Region ◽  
Tier 2 ◽  
X Ray Crystallography ◽  
Neutralizing Capacity ◽  
Clade C ◽  
Hiv 1

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 ug/ml (IC 50 ). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184-186) that is unique to the 16055 strain. They also provide an explanation for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.

scholarly journals HIV-1 Envelope Conformation, Allostery, and Dynamics

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 852
Author(s):  
Ashley Lauren Bennett ◽  
Rory Henderson
Keyword(s):  
Host Cell ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Critical Role ◽  
Cell Receptor ◽  
Broadly Neutralizing Antibodies ◽  
Structural Mapping ◽  
Host Cell Receptor ◽  
Immunogen Design ◽  
Hiv 1

The HIV-1 envelope glycoprotein (Env) mediates host cell fusion and is the primary target for HIV-1 vaccine design. The Env undergoes a series of functionally important conformational rearrangements upon engagement of its host cell receptor, CD4. As the sole target for broadly neutralizing antibodies, our understanding of these transitions plays a critical role in vaccine immunogen design. Here, we review available experimental data interrogating the HIV-1 Env conformation and detail computational efforts aimed at delineating the series of conformational changes connecting these rearrangements. These studies have provided a structural mapping of prefusion closed, open, and transition intermediate structures, the allosteric elements controlling rearrangements, and state-to-state transition dynamics. The combination of these investigations and innovations in molecular modeling set the stage for advanced studies examining rearrangements at greater spatial and temporal resolution.

scholarly journals Neutralizing antibodies induced by first-generation gp41-stabilized HIV-1 envelope trimers and nanoparticles

2020 ◽  
Author(s):  
Sonu Kumar ◽  
Xiaohe Lin ◽  
Timothy Ngo ◽  
Benjamin Shapero ◽  
Cindy Sou ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Neutralizing Antibodies ◽  
First Generation ◽  
Heptad Repeat ◽  
X Ray ◽  
X Ray Crystallography ◽  
Clade C ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Hiv 1

ABSTRACTAntigen-specific B-cell sorting and next-generation sequencing (NGS) were combined to isolate HIV-1 neutralizing antibodies (NAbs) from mice and rabbits immunized with BG505 trimers and nanoparticles. Three mouse NAbs potently neutralize BG505.T332N and recognize a glycan epitope centered at the C3/V4 region, as revealed by electron microscopy (EM), x-ray crystallography, and epitope mapping. Three potent NAbs were sorted from rabbit B cells that target glycan holes on the BG505 envelope glycoprotein (Env) and account for a significant portion of autologous NAb response. We then determined a 3.4Å-resolution crystal structure for the clade C transmitted/founder Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend. This clade C Env, as a soluble trimer and attached to a ferritin nanoparticle, along with a clade A Q482-d12 Env trimer, elicited distinct NAb responses in rabbits. Our study demonstrates that nanoparticles presenting gp41-stabilized trimers can induce potent NAb responses in mice and rabbits with Env-dependent breadth.TEASERMouse and rabbit NAbs elicited by gp41-stabilized trimers and nanoparticles neutralize autologous HIV-1 by targeting different epitopes

scholarly journals Conformational Engineering of HIV-1 Env Based on Mutational Tolerance in the CD4 and PG16 Bound States

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Jeremiah D. Heredia ◽  
Jihye Park ◽  
Hannah Choi ◽  
Kevin S. Gill ◽  
Erik Procko
Keyword(s):  
Virus Replication ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Bound States ◽  
Structural Changes ◽  
Electrostatic Repulsion ◽  
Closed Conformation ◽  
Open Conformation ◽  
Outer Domain ◽  
Hiv 1

ABSTRACTHIV-1 infection is initiated by viral Env engaging the host receptor CD4, triggering Env to transition from a “closed” to “open” conformation during the early events of virus-cell membrane fusion. To understand how Env sequence accommodates this conformational change, mutational landscapes decoupled from virus replication were determined for Env from BaL (clade B) and DU422 (clade C) isolates interacting with CD4 or antibody PG16 that preferentially recognizes closed trimers. Sequence features uniquely important to each bound state were identified, including glycosylation and binding sites. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this key observation, mutations were found that increase presentation of quaternary epitopes associated with properly conformed trimers when Env is expressed at the plasma membrane. Many mutations reduce electrostatic repulsion at the Env apex and increase PG16 recognition of Env sequences from clades A and B. Other mutations increase hydrophobic packing at the gp120 inner-outer domain interface and were broadly applicable for engineering Env from diverse strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. Core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. Finally, we demonstrate how our methodology can be adapted to interrogate interactions between membrane-associated Env and the matrix domain of Gag. These findings and methods may assist vaccine design.IMPORTANCEHIV-1 Env is dynamic and undergoes large conformational changes that drive fusion of virus and host cell membranes. Three Env proteins in a trimer contact each other at their apical tips to form a closed conformation that presents epitopes recognized by broadly neutralizing antibodies. The apical tips separate, among other changes, to form an open conformation that binds tightly to host receptors. Understanding how Env sequence facilitates these structural changes can inform the biophysical mechanism and aid immunogen design. Using deep mutational scans decoupled from virus replication, we report mutational landscapes for Env from two strains interacting with conformation-dependent binding proteins. Residues in the Env trimer interface and apical domains are preferentially conserved in the closed conformation, and conformational diversity is facilitated by electrostatic repulsion and an underpacked core between domains. Specific mutations are described that enhance presentation of the trimeric closed conformation across diverse HIV-1 strains.

scholarly journals B-cell–lineage immunogen design in vaccine development with HIV-1 as a case study

2012 ◽  
Vol 30 (5) ◽  
pp. 423-433 ◽  
Author(s):  
Barton F Haynes ◽  
Garnett Kelsoe ◽  
Stephen C Harrison ◽  
Thomas B Kepler
Keyword(s):  
B Cell ◽  
Vaccine Development ◽  
Cell Lineage ◽  
Immunogen Design ◽  
Hiv 1
Sign in / Sign up

Export Citation Format

Share Document