Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections

Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5 Å resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified delta genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved when compared with the human respiratory coronavirus HCoV-NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S2' activation loop, containing a reduced number of basic amino acids, which participates to rendering the spike largely protease-resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity.IMPORTANCECoronaviruses use transmembrane spike (S) glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic porcine deltacoronavirus (PDCoV), which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is decorated with 78 N-linked glycans obstructing the protein surface to limit accessibility to neutralizing antibodies in a way reminiscent of what has recently been described for a human respiratory coronavirus. PDCoV S is largely protease-resistant which distinguishes it from most other characterized coronavirus S glycoproteins and suggests that enteric coronaviruses have evolved to fine-tune fusion activation in the protease-rich environment of the small intestine of infected hosts.

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Incorporation of apolipoprotein E into HBV–HCV subviral envelope particles to improve the hepatitis vaccine strategy

Scientific Reports ◽

10.1038/s41598-021-01428-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Elsa Gomez-Escobar ◽

Julien Burlaud-Gaillard ◽

Clara Visdeloup ◽

Adeline Ribeiro E. Silva ◽

Pauline Coutant ◽

...

Keyword(s):

Apolipoprotein E ◽

Neutralizing Antibodies ◽

Envelope Proteins ◽

Viral Envelope ◽

Vaccine Strategy ◽

S Protein ◽

Hepatitis Vaccine ◽

Protein Immunization ◽

Neutralizing Potential

AbstractHepatitis C is a major threat to public health for which an effective treatment is available, but a prophylactic vaccine is still needed to control this disease. We designed a vaccine based on chimeric HBV–HCV envelope proteins forming subviral particles (SVPs) that induce neutralizing antibodies against HCV in vitro. Here, we aimed to increase the neutralizing potential of those antibodies, by using HBV–HCV SVPs bearing apolipoprotein E (apoE). These particles were produced by cultured stable mammalian cell clones, purified and characterized. We found that apoE was able to interact with both chimeric HBV–HCV (E1-S and E2-S) proteins, and with the wild-type HBV S protein. ApoE was also detected on the surface of purified SVPs and improved the folding of HCV envelope proteins, but its presence lowered the incorporation of E2-S protein. Immunization of New Zealand rabbits resulted in similar anti-S responses for all rabbits, whereas anti-E1/-E2 antibody titers varied according to the presence or absence of apoE. Regarding the neutralizing potential of these anti-E1/-E2 antibodies, it was higher in rabbits immunized with apoE-bearing particles. In conclusion, the association of apoE with HCV envelope proteins may be a good strategy for improving HCV vaccines based on viral envelope proteins.

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Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

PLoS Pathogens ◽

10.1371/journal.ppat.1008977 ◽

2021 ◽

Vol 17 (4) ◽

pp. e1008977

Author(s):

Rajesh Kumar ◽

Suprit Deshpande ◽

Leigh M. Sewall ◽

Gabriel Ozorowski ◽

Christopher A. Cottrell ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Neutralizing Antibody ◽

Viral Envelope ◽

Negative Stain ◽

Clade C ◽

Negative Stain Electron Microscopy ◽

Env Protein ◽

Hiv 1

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.

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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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Dynamic Interactions of Fully Glycosylated SARS-CoV-2 Spike Protein with Various Antibodies

10.1101/2021.05.10.443519 ◽

2021 ◽

Author(s):

Yiwei Cao ◽

Yeol Kyo Choi ◽

Martin Frank ◽

Hyeonuk Woo ◽

Sang-Jun Park ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Antibody Binding ◽

Viral Envelope ◽

Binding Modes ◽

Dynamic Interactions ◽

Health Crisis ◽

S Protein ◽

Protein Receptor ◽

Dynamics Simulations ◽

Antibody Interactions

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.

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

Nature Communications ◽

10.1038/s41467-020-19013-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Kuan-Ying A. Huang ◽

Daming Zhou ◽

Elizabeth E. Fry ◽

Abhay Kotecha ◽

Peng-Nien Huang ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Enterovirus 71 ◽

Therapeutic Agents ◽

Fold Axis ◽

Effective Protection ◽

Promising Candidate ◽

Cryo Electron Microscopy ◽

Protective Antibodies ◽

Conserved Epitope

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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Structural basis of trehalose recycling by the ABC transporter LpqY-SugABC

Science Advances ◽

10.1126/sciadv.abb9833 ◽

2020 ◽

Vol 6 (44) ◽

pp. eabb9833

Author(s):

Fengjiang Liu ◽

Jingxi Liang ◽

Bing Zhang ◽

Yan Gao ◽

Xiuna Yang ◽

...

Keyword(s):

Electron Microscopy ◽

Abc Transporter ◽

Abc Transporters ◽

Unique Feature ◽

Structural Basis ◽

Human Pathogens ◽

Gram Positive Bacteria ◽

Cryo Electron Microscopy ◽

Uptake Of Nutrients ◽

Initial Mode

In bacteria, adenosine 5′-triphosphate (ATP)–binding cassette (ABC) importers are essential for the uptake of nutrients including the nonreducing disaccharide trehalose, a metabolite that is crucial for the survival and virulence of several human pathogens including Mycobacterium tuberculosis. SugABC is an ABC transporter that translocates trehalose from the periplasmic lipoprotein LpqY into the cytoplasm of mycobacteria. Here, we report four high-resolution cryo–electron microscopy structures of the mycobacterial LpqY-SugABC complex to reveal how it binds and passes trehalose through the membrane to the cytoplasm. A unique feature observed in this system is the initial mode of capture of the trehalose at the LpqY interface. Uptake is achieved by a pivotal rotation of LpqY relative to SugABC, moving from an open and accessible conformation to a clamped conformation upon trehalose binding. These findings enrich our understanding as to how ABC transporters facilitate substrate transport across the membrane in Gram-positive bacteria.

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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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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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