Publications
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Title & Authors Journal Publication Date

Influenza chimeric hemagglutinin structures in complex with broadly protective antibodies to the stem and trimer interface


Zhu X, Han J, Sun W, Puente-Massaguer E, Yu W, Palese P, Krammer F, Ward AB, Wilson IA.
Proceedings of the National Academy of Sciences May 20, 2022

Influenza virus hemagglutinin (HA) has been the primary target for influenza vaccine development. Broadly protective antibodies targeting conserved regions of the HA unlock the possibility of generating universal influenza immunity. Two group 2 influenza A chimeric HAs, cH4/3 and cH15/3, were previously designed to elicit antibodies to the conserved HA stem. Here, we show by X-ray crystallography and negative-stain electron microscopy that a broadly protective antistem antibody can stably bind to cH4/3 and cH15/3 HAs, thereby validating their potential as universal vaccine immunogens. Furthermore, flexibility was observed in the head domain of the chimeric HA structures, suggesting that antibodies could also potentially interact with the head interface epitope. Our structural and binding studies demonstrated that a broadly protective antihead trimeric interface antibody could indeed target the more open head domain of the cH15/3 HA trimer. Thus, in addition to inducing broadly protective antibodies against the conserved HA stem, chimeric HAs may also be able to elicit antibodies against the conserved trimer interface in the HA head domain, thereby increasing the vaccine efficacy.

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody


Torres JL, Ozorowski G, Andreano E, Liu H, Copps J, Piccini G, Donnici L, Conti M, Planchais C, Planas D, Manganaro N, Pantano E, Paciello I, Pileri P, Bruel T, Montomoli E, Mouquet H, Schwartz O, Sala C, De Francesco R, Wilson IA, Rappuoli R, Ward AB.
Proceedings of the National Academy of Sciences May 12, 2022

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of the previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryoelectron microscopy (cryo-EM) and X-ray crystallography. We show that mAb J08 has low nanomolar affinity against most VoCs and binds high on the receptor binding domain (RBD) ridge, away from many VoC mutations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy.

Structural mapping of antibody landscapes to human betacoronavirus spike proteins


Bangaru S, Antanasijevic A, Kose N, Sewall LM, Jackson AM, Suryadevara N, Zhan X, Torres JL, Copps J, de la Peña AT, Crowe JE Jr, Ward AB.
Science Advances May 4, 2022

Preexisting immunity against seasonal coronaviruses (CoVs) represents an important variable in predicting antibody responses and disease severity to severe acute respiratory syndrome CoV-2 (SARS-CoV-2) infections. We used electron microscopy–based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against β-CoV spike proteins in prepandemic (PP) sera or SARS-CoV-2 convalescent (SC) sera. We observed that most PP sera had antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the SC sera showed reactivity across all human β-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by preexisting antibodies and SC serum antibodies. Our studies provide an antigenic landscape to β-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-CoV-2–infected individuals.

A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 Delta variant


Hingankar N, Deshpande S, Das P, Rizvi ZA, Wibmer CK, Mashilo P, Ansari MY, Burns A, Barman S, Zhao F, Mukherjee S, Torres JL, Chattopadhyay S, Mehdi F, Sutar J, Rathore DK, Pargai K, Singh J, Sonar S, Jakhar K, Dandotiya J, Bhattacharyya S, Mani S, Samal S, Singh S, Kshetrapal P, Thiruvengadam R, Batra G, Medigeshi G, Ward AB, Bhatnagar S, Awasthi A, Sok D, Bhattacharya J.
PLoS Pathogens April 28, 2022

Although efficacious vaccines have significantly reduced the morbidity and mortality of COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of five neutralizing mAbs from an Indian convalescent donor, out of which two (THSC20.HVTR04 and THSC20.HVTR26) showed potent neutralization of SARS-CoV-2 VOCs at picomolar concentrations, including the Delta variant (B.1.617.2). One of these (THSC20.HVTR26) also retained activity against the Omicron variant. These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein and prevent virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as a cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.

Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit


Hurlburt NK, Homad LJ, Sinha I, Jennewein MF, MacCamy AJ, Wan YH, Boonyaratanakornkit J, Sholukh AM, Jackson AM, Zhou P, Burton DR, Andrabi R, Ozorowski G, Ward AB, Stamatatos L, Pancera M, McGuire AT.
Communications Biology April 11, 2022

Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection. We previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 spike, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and binds to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is maintained against the Alpha, Delta, Gamma and Omicron variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region. Negative stain electron microscopy and a 1.74 Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Structural and functional characterisation of an antibody CV3-25 reveals wide neutralisation spectrum of the antibody against multiple SARS-CoV2 variants.

Structure-guided changes at the V2 apex of HIV-1 clade C trimer enhance elicitation of autologous neutralizing and broad V1V2-scaffold antibodies


Sahoo A, Hodge EA, LaBranche CC, Styles TM, Shen X, Cheedarla N, Shiferaw A, Ozorowski G, Lee WH, Ward AB, Tomaras GD, Montefiori DC, Irvine DJ, Lee KK, Amara RR.
Cell Reports March 1, 2022

HIV-1 clade C envelope immunogens that elicit both neutralizing and non-neutralizing V1V2-scaffold-specific antibodies (protective correlates from RV144 human trial) are urgently needed due to the prevalence of this clade in the most impacted regions worldwide. To achieve this, we introduce structure-guided changes followed by consensus-C-sequence-guided optimizations at the V2 region to generate UFO-v2-RQH173 trimer. This improves the abundance of well-formed trimers. Following the immunization of rabbits, the wild-type protein fails to elicit any autologous neutralizing antibodies, but UFO-v2-RQH173 elicits both autologous neutralizing and broad V1V2-scaffold antibodies. The variant with a 173Y modification in the V2 region, most prevalent among HIV-1 sequences, shows decreased ability in displaying a native-like V1V2 epitope with time in vitro and elicited antibodies with lower neutralizing and higher V1V2-scaffold activities. Our results identify a stabilized clade C trimer capable of eliciting improved neutralizing and V1V2-scaffold antibodies and reveal the importance of the V2 region in tuning this.

High thermostability improves neutralizing antibody responses induced by native-like HIV-1 envelope trimers


Del Moral-Sánchez I, Russell RA, Schermer EE, Cottrell CA, Allen JD, Torrents de la Peña A, LaBranche CC, Kumar S, Crispin M, Ward AB, Montefiori DC, Sattentau QJ, Sliepen K, Sanders RW.
npj Vaccines Feb. 28, 2022

Soluble HIV-1 envelope glycoprotein (Env) immunogens are a prime constituent of candidate vaccines designed to induce broadly neutralizing antibodies. Several lines of evidence suggest that enhancing Env immunogen thermostability can improve neutralizing antibody (NAb) responses. Here, we generated BG505 SOSIP.v9 trimers, which displayed virtually no reactivity with non-neutralizing antibodies and showed increased global and epitope thermostability, compared to previous BG505 SOSIP versions. Chemical crosslinking of BG505 SOSIP.v9 further increased the melting temperature to 91.3 °C, which is almost 25 °C higher than that of the prototype SOSIP.664 trimer. Next, we compared the immunogenicity of a palette of BG505-based SOSIP trimers with a gradient of thermostabilities in rabbits. We also included SOSIP.v9 proteins in which a strain-specific immunodominant epitope was masked by glycans to redirect the NAb response to other subdominant epitopes. We found that increased trimer thermostability correlated with increased potency and consistency of the autologous NAb response. Furthermore, glycan masking steered the NAb response to subdominant epitopes without decreasing the potency of the autologous NAb response. In summary, SOSIP.v9 trimers and their glycan masked versions represent an improved platform for HIV-1 Env based vaccination strategies.

Structural insights into the Venus flytrap mechanosensitive ion channel Flycatcher1


Jojoa-Cruz S, Saotome K, Tsui CCA, Lee WH, Sansom MSP, Murthy SE, Patapoutian A, Ward AB
Nature Communications Feb. 14, 2022

Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is a larger protein and its sequence diverges from previously studied MscS homologs, suggesting it has unique structural features that contribute to its function. Here, we characterize FLYC1 by cryo-electron microscopy, molecular dynamics simulations, and electrophysiology. Akin to bacterial MscS and plant MSL1 channels, we find that FLYC1 central core includes side portals in the cytoplasmic cage that regulate ion preference and conduction, by identifying critical residues that modulate channel conductance. Topologically unique cytoplasmic flanking regions can adopt ‘up’ or ‘down’ conformations, making the channel asymmetric. Disruption of an up conformation-specific interaction severely delays channel deactivation by 40-fold likely due to stabilization of the channel open state. Our results illustrate novel structural features and likely conformational transitions that regulate mechano-gating of FLYC1. Flycatcher1 (FLYC1) is a candidate mechanosensitive channel involved in Venus flytrap touch-induced prey capture. Here, the authors report structural and functional details of FLYC1, with insights into gating conformational transitions.

From structure to sequence: Antibody discovery using cryoEM


Antanasijevic A, Bowman CA, Kirchdoerfer RN, Cottrell CA, Ozorowski G, Upadhyay AA, Cirelli KM, Carnathan DG, Enemuo CA, Sewall LM, Nogal B, Zhao F, Groschel B, Schief WR, Sok D, Silvestri G, Crotty S, Bosinger SE, Ward AB.
Science Advances Jan. 19, 2022

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

Structural Biology Illuminates Molecular Determinants of Broad Ebolavirus Neutralization by Human Antibodies for Pan-Ebolavirus Therapeutic Development


Murin CD, Gilchuk P, Crowe JE Jr, Ward AB.
Frontiers in Immunology Jan. 10, 2022

Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses.

Broadly neutralizing antibodies target a haemagglutinin anchor epitope


Guthmiller JJ, Han J, Utset HA, Li L, Lan LY, Henry C, Stamper CT, McMahon M, O'Dell G, Fernández-Quintero ML, Freyn AW, Amanat F, Stovicek O, Gentles L, Richey ST, de la Peña AT, Rosado V, Dugan HL, Zheng NY, Tepora ME, Bitar DJ, Changrob S, Strohmeier S, Huang M, García-Sastre A, Liedl KR, Bloom JD, Nachbagauer R, Palese P, Krammer F, Coughlan L, Ward AB, Wilson PC.
Nature Dec. 23, 2021

Broadly neutralizing antibodies that target epitopes of haemagglutinin on the influenza virus have the potential to provide near universal protection against influenza virus infection1. However, viral mutants that escape broadly neutralizing antibodies have been reported2,3. The identification of broadly neutralizing antibody classes that can neutralize viral escape mutants is critical for universal influenza virus vaccine design. Here we report a distinct class of broadly neutralizing antibodies that target a discrete membrane-proximal anchor epitope of the haemagglutinin stalk domain. Anchor epitope-targeting antibodies are broadly neutralizing across H1 viruses and can cross-react with H2 and H5 viruses that are a pandemic threat. Antibodies that target this anchor epitope utilize a highly restricted repertoire, which encodes two public binding motifs that make extensive contacts with conserved residues in the fusion peptide. Moreover, anchor epitope-targeting B cells are common in the human memory B cell repertoire and were recalled in humans by an oil-in-water adjuvanted chimeric haemagglutinin vaccine4,5, which is a potential universal influenza virus vaccine. To maximize protection against seasonal and pandemic influenza viruses, vaccines should aim to boost this previously untapped source of broadly neutralizing antibodies that are widespread in the human memory B cell pool. A distinct class of broadly neutralizing antibodies to the influenza virus target a membrane-proximal anchor epitope of the haemagglutinin stalk domain.

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Title & Authors Journal Publication Date

Lassa virus glycoprotein nanoparticles elicit a neutralizing antibody that defines a new site of vulnerability


M Brouwer PJ, Antanasijevic A, Ronk AJ, Müller-Kräuter H, Watanabe Y, Claireaux M, Lloyd NM, L Bijl TP, Perrett HR, Steijaert T, Burger JA, van Haaren MM, Sliepen K, van Gils MJ, Crispin M, Strecker T, Bukreyev A, Ward AB, Sanders RW

Now Published: 10.1016/j.chom.2022.10.018
bioRxiv March 28, 2022

CD4 binding-site antibodies induced by a subtype B HIV-1 envelope trimer


Schorcht A, van den Kerkhof TL, Torres J, Schermer E, LaBranche CC, Bontjer I, Brinkkemper M, Gulzar N, Han AX, Burger J, Ozorowski G, Scott JK, Schuitemaker H, Montefiori D, van Gils MJ, Ward AB, Sanders R

bioRxiv March 23, 2022

Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels


Teng B, Kaplan JP, Liang Z, Kreiger Z, Tu YH, Burendei B, Ward A, Liman ER

Now Published: 10.7554/eLife.77946
bioRxiv March 1, 2022

High-resolution structural analysis of enterovirus-reactive polyclonal antibodies in complex with whole virions


Antanasijevic A, Schulze AJ, Reddy VS, Ward AB

Now Published: 10.1093/pnasnexus/pgac253
bioRxiv Jan. 31, 2022

An infectious virus-like particle built on a programmable icosahedral DNA framework


Yang Y, Xu Y, Yang Y, Shi Q, Ward A, Wang W

Now Published: 10.26434/chemrxiv-2022-20s93
chemRxiv Jan. 17, 2022

Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape


Zhao F, Keating C, Ozorowski G, Shaabani N, Francino-Urdaniz IM, Barman S, Limbo O, Burns A, Zhou P, Ricciardi MJ, Woehl J, Tran Q, Turner HL, Peng L, Huang D, Nemazee D, Andrabi R, Sok D, Teijaro JR, Whitehead TA, Ward AB, Burton DR, Jardine JG

Now Published: 10.1016/j.isci.2022.104914
bioRxiv Jan. 6, 2022

A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 delta variant


Hingankar N, Deshpande S, Das P, Abbas Rizvi Z, Burns A, Barman S, Zhao F, Yousuf Ansari M, Mukherjee S, Torres JL, Chattopadhyay S, Mehdi F, Sutar J, Kumar Rathore D, Pargai K, Singh J, Sonar S, Jakhar K, Bhattacharyya S, Mani S, Singh S, Dandotiya J, Kshetrapal P, Thiruvengadam R, Batra G, Medigeshi G, Ward AB, Bhatnagar S, Awasthi A, Sok D, Bhattacharya J

Now Published: 10.1371/journal.ppat.1010465
bioRxiv Dec. 25, 2021

Molecular insights into antibody-mediated protection against the prototypic simian immunodeficiency virus


Zhao F, Berndsen ZT, Pedreño-Lopez N, Burns A, Allen JD, Barman S, Lee WH, Chakraborty S, Gnanakaran S, Sewall LM, Ozorowski G, Limbo O, Song G, Yong P, Callaghan S, Weisgrau KL, Lifson JD, Nedellec R, Voigt TB, Laurino F, Louw J, Rosen BC, Ricciardi M, Crispin M, Desrosiers RC, Rakasz EG, Watkins DI, Andrabi R, Ward AB, Burton DR, Sok D

Now Published: 10.1038/s41467-022-32783-2
bioRxiv Dec. 22, 2021

Long-lasting germinal center responses to a priming immunization with continuous proliferation and somatic mutation


Lee JH, Sutton H, Cottrell CA, Phung I, Ozorowski G, Sewall LM, Nedellec R, Nakao C, Silva M, Richey ST, Torres JL, Lee WH, Georgeson E, Kubitz M, Hodges S, Mullen TM, Adachi Y, Cirelli KM, Kaur A, Allers-Hernandez C, Fahlberg M, Grasperge BF, Dufour JP, Schiro F, Aye PP, Carnathan DG, Silvestri G, Shen X, Montefiori DC, Veazey RS, Ward AB, Hangartner L, Burton DR, Irvine DJ, Schief WR, Crotty S

Now Published: 10.1038/s41586-022-05216-9
bioRxiv Dec. 20, 2021