Structural and functional analysis of protective antibodies targeting the threefold plateau of enterovirus 71

Abstract Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.

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Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

Journal of Virology ◽

10.1128/jvi.00575-16 ◽

2016 ◽

Vol 90 (19) ◽

pp. 8542-8551 ◽

Cited By ~ 23

Author(s):

Lauren M. Drouin ◽

Bridget Lins ◽

Maria Janssen ◽

Antonette Bennett ◽

Paul Chipman ◽

...

Keyword(s):

Electron Microscopy ◽

Intramolecular Interactions ◽

Single Amino Acid ◽

Fold Axis ◽

Wild Type ◽

Scanning Calorimetry ◽

Genome Packaging ◽

Cryo Electron Microscopy ◽

Empty Capsid ◽

Packaging Efficiency

ABSTRACTThe adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.IMPORTANCEThe mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

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Networks of HIV-1 envelope glycans maintain antibody epitopes in the face of glycan additions and deletions

10.1101/2020.02.21.959981 ◽

2020 ◽

Cited By ~ 2

Author(s):

Gemma E. Seabright ◽

Christopher A. Cottrell ◽

Marit J. van Gils ◽

Alessio D’addabbo ◽

David J. Harvey ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Broadly Neutralizing Antibodies ◽

Site Specific ◽

Glycan Analysis ◽

Cryo Electron Microscopy ◽

Immunogen Design ◽

The Face ◽

Antibody Epitopes ◽

Hiv 1

SUMMARYNumerous broadly neutralizing antibodies (bnAbs) have been identified that target the glycans of the HIV-1 envelope spike. Neutralization breadth is notable given that glycan processing can be substantially influenced by the presence or absence of neighboring glycans. Here, using a stabilized recombinant envelope trimer, we investigate the degree to which mutations in the glycan network surrounding an epitope impact the fine glycan processing of antibody targets. Using cryo-electron microscopy and site-specific glycan analysis, we reveal the hierarchy of importance of glycans in the formation of the 2G12 bnAb epitope, and show that the epitope is only subtly impacted by variations in the glycan network. In contrast, we show that the PG9 and PG16 glycan-based epitopes at the trimer apex are dependent on the presence of the highly conserved surrounding glycans. Glycan networks underpin the conservation of bnAb epitopes and are an important parameter in immunogen design.

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Structures of SARS-CoV-2 B.1.351 neutralizing antibodies provide insights into cocktail design against concerning variants

10.1101/2021.07.30.454402 ◽

2021 ◽

Author(s):

Shuo Du ◽

Pulan Liu ◽

Zhiying Zhang ◽

Tianhe Xiao ◽

Ayijiang Yasimayi ◽

...

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibodies ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Antibody Responses ◽

Spike Protein ◽

Receptor Binding Domain ◽

Humoral Antibody ◽

Cryo Electron Microscopy ◽

Antibody Cocktail

The spread of the SARS-CoV-2 variants could seriously dampen the global effort to tackle the COVID-19 pandemic. Recently, we investigated the humoral antibody responses of SARS-CoV-2 convalescent patients and vaccinees towards circulating variants, and identified a panel of monoclonal antibodies (mAbs) that could efficiently neutralize the B.1.351 (Beta) variant. Here we investigate how these mAbs target the B.1.351 spike protein using cryo-electron microscopy. In particular, we show that two superpotent mAbs, BD-812 and BD-836, have non-overlapping epitopes on the receptor-binding domain (RBD) of spike. Both block the interaction between RBD and the ACE2 receptor; and importantly, both remain fully efficacious towards the B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. The BD-812/BD-836 pair could thus serve as an ideal antibody cocktail against the SARS-CoV-2 VOCs.

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Fast Restoration of a Broad-Spectrum SARS-Cov Therapeutic Antibody for SARS-Cov-2

10.26434/chemrxiv.12891041.v3 ◽

2020 ◽

Author(s):

Sara Zhang ◽

Mike Young ◽

Jason Pan

Keyword(s):

Broad Spectrum ◽

Neutralizing Antibodies ◽

Global Economy ◽

Therapeutic Antibody ◽

Receptor Binding Domain ◽

Promising Candidate ◽

Rapid Spread ◽

Antibody Design ◽

Potential Therapeutics ◽

Conserved Epitope

<p>The rapid spread of SARS-Cov-2 remains a major threat for public health and global economy, both preventative and therapeutic solutions are therefore urgently needed. Through the use of epitope-guided antibody design, we successfully restored a broad-spectrum SARS-Cov therapeutic antibody for SARS-Cov-2. Compared to the precursor antibody CR3022, the newly designed antibody NOVOAB-20 binds to SARS-Cov-2 receptor binding domain (RBD) with a more than 10-fold higher affinity. Because this antibody targets a highly conserved epitope and the mutations on SARS-Cov-2 known so far are all not in this region, it also has the potential to block future SARS-Cov-2 mutants. As a fully humanized antibody, NOVOAB-20 is a promising candidate to be developed as potential therapeutics for SARS-Cov-2, either as monotherapy or in combination with other neutralizing antibodies targeting different epitopes (e.g. the ACE2 binding site).</p>

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Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

10.1101/2020.06.27.174979 ◽

2020 ◽

Cited By ~ 16

Author(s):

Zunlong Ke ◽

Joaquin Oton ◽

Kun Qu ◽

Mirko Cortese ◽

Vojtech Zila ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Structural Rearrangement ◽

Conformational Flexibility ◽

Target Cells ◽

Receptor Binding Site ◽

Cryo Electron Microscopy ◽

High Resolution Structure ◽

Virion Surface

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

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Fast Restoration of a Broad-Spectrum SARS-Cov Therapeutic Antibody for SARS-Cov-2

10.26434/chemrxiv.12891041.v2 ◽

2020 ◽

Author(s):

Sara Zhang ◽

Mike Young ◽

Jason Pan

Keyword(s):

Broad Spectrum ◽

Neutralizing Antibodies ◽

Global Economy ◽

Therapeutic Antibody ◽

Receptor Binding Domain ◽

Promising Candidate ◽

Rapid Spread ◽

Antibody Design ◽

Potential Therapeutics ◽

Conserved Epitope

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Autologous Antibody Responses to an HIV Envelope Glycan Hole Are Not Easily Broadened in Rabbits

Journal of Virology ◽

10.1128/jvi.01861-19 ◽

2020 ◽

Vol 94 (7) ◽

Cited By ~ 7

Author(s):

Yuhe R. Yang ◽

Laura E. McCoy ◽

Marit J. van Gils ◽

Raiees Andrabi ◽

Hannah L. Turner ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Neutralizing Antibodies ◽

Cross Reactivity ◽

High Resolution Data ◽

Subtype B ◽

Cryo Electron Microscopy ◽

Antibody Complex ◽

Subtype A ◽

Hiv Envelope

ABSTRACT Extensive studies with subtype A BG505-derived HIV envelope glycoprotein (Env) immunogens have revealed that the dominant autologous neutralizing epitope in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. To test whether broader neutralization to the common glycan hole can be achieved, we immunized rabbits with B41 SOSIP (gp120-gp41 disulfide [SOS] with an isoleucine-to-proline mutation [IP] in gp41) alone, as well as B41 and BG505 coimmunization. We isolated autologous neutralizing antibodies (nAbs) and described their structure in complex with the B41 Env. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the >97% conserved N241 glycan, which is present in B41. Single-particle cryo-electron microscopy studies confirmed that B41- and BG505-specific nAbs bind to overlapping glycan hole epitopes. We then used our high-resolution data to guide mutations in the BG505 glycan hole epitope in an attempt to broaden the reactivity of a B41-specific nAb, but we recovered only partial binding. Our data demonstrate that the lack of cross-reactivity in glycan hole antibodies is due to amino acid differences within the epitope, and our attempts to rationally design cross-reactive trimers resulted in only limited success. Thus, even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult. IMPORTANCE A glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer-immunized rabbits. Our high-resolution cryo-electron microscopy (cryoEM) studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult.

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Structural basis for antibody resistance to SARS-CoV-2 omicron variant

10.1101/2021.12.21.473620 ◽

2021 ◽

Author(s):

Gabriele Cerutti ◽

Yicheng Guo ◽

Liu Lihong ◽

Zhening Zhang ◽

Liyuan Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Structural Basis ◽

Receptor Binding Domain ◽

Functional Studies ◽

Antibody Recognition ◽

Cryo Electron Microscopy ◽

Antibody Resistance

The recently reported B.1.1.529 Omicron variant of SARS-CoV-2 includes 34 mutations in the spike protein relative to the Wuhan strain that initiated the COVID-19 pandemic, including 15 mutations in the receptor binding domain (RBD). Functional studies have shown omicron to substantially escape the activity of many SARS-CoV-2-neutralizing antibodies. Here we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of the Omicron spike protein ectodomain. The structure depicts a spike that is exclusively in the 1-RBD-up conformation with increased mobility and inter-protomer asymmetry. Many mutations cause steric clashes and/or altered interactions at antibody binding surfaces, whereas others mediate changes of the spike structure in local regions to interfere with antibody recognition. Overall, the structure of the omicron spike reveals how mutations alter its conformation and explains its extraordinary ability to evade neutralizing antibodies.

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Structural basis of Omicron neutralization by affinity-matured public antibodies

10.1101/2022.01.03.474825 ◽

2022 ◽

Author(s):

Daniel J. Sheward ◽

Pradeepa Pushparaj ◽

Hrishikesh Das ◽

Changil Kim ◽

Sungyong Kim ◽

...

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Neutralizing Antibodies ◽

Affinity Maturation ◽

Structural Basis ◽

Binding Motif ◽

Vaccine Protection ◽

Antibody Therapeutics ◽

Cryo Electron Microscopy ◽

Cross Neutralization

The SARS-CoV-2 Omicron Variant of Concern (B.1.1.529) has spread rapidly in many countries. With a spike that is highly diverged from that of the pandemic founder, it escapes most available monoclonal antibody therapeutics and erodes vaccine protection. A public class of IGHV3-53-using SARS-CoV-2 neutralizing antibodies typically fails to neutralize variants carrying mutations in the receptor-binding motif, including Omicron. As antibodies from this class are likely elicited in most people following SARS-CoV-2 infection or vaccination, their subsequent affinity maturation is of particular interest. Here, we isolated IGHV3-53-using antibodies from an individual seven months after infection and identified several antibodies capable of broad and potent SARS-CoV-2 neutralization, extending to Omicron without loss of potency. By introducing select somatic hypermutations into a germline-reverted form of one such antibody, CAB-A17, we demonstrate the potential for commonly elicited antibodies to develop broad cross-neutralization through affinity maturation. Further, we resolved the structure of CAB-A17 Fab in complex with Omicron spike at an overall resolution of 2.6 angstroms by cryo-electron microscopy and defined the structural basis for this breadth. Thus, public SARS-CoV-2 neutralizing antibodies can, without modified spike vaccines, mature to cross-neutralize exceptionally antigenically diverged SARS-CoV-2 variants.

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A broadly neutralizing macaque monoclonal antibody against the HIV-1 V3-Glycan patch

eLife ◽

10.7554/elife.61991 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Zijun Wang ◽

Christopher O Barnes ◽

Rajeev Gautam ◽

Julio C Cetrulo Lorenzi ◽

Christian T Mayer ◽

...

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Neutralizing Antibodies ◽

Geometric Mean ◽

High Dose ◽

Dependent Manner ◽

Peptide Motif ◽

Broadly Neutralizing Antibodies ◽

Cryo Electron Microscopy ◽

Hiv 1

A small fraction of HIV-1- infected humans develop broadly neutralizing antibodies (bNAbs) against HIV-1 that protect macaques from simian immunodeficiency HIV chimeric virus (SHIV). Similarly, a small number of macaques infected with SHIVs develop broadly neutralizing serologic activity, but less is known about the nature of simian antibodies. Here, we report on a monoclonal antibody, Ab1485, isolated from a macaque infected with SHIVAD8 that developed broadly neutralizing serologic activity targeting the V3-glycan region of HIV-1 Env. Ab1485 neutralizes 38.1% of HIV-1 isolates in a 42-pseudovirus panel with a geometric mean IC50 of 0.055 µg/mLl and SHIVAD8 with an IC50 of 0.028 µg/mLl. Ab1485 binds the V3-glycan epitope in a glycan-dependent manner. A 3.5 Å cryo-electron microscopy structure of Ab1485 in complex with a native-like SOSIP Env trimer showed conserved contacts with the N332gp120 glycan and gp120 GDIR peptide motif, but in a distinct Env-binding orientation relative to human V3/N332gp120 glycan-targeting bNAbs. Intravenous infusion of Ab1485 protected macaques from a high dose challenge with SHIVAD8. We conclude that macaques can develop bNAbs against the V3-glycan patch that resemble human V3-glycan bNAbs.

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