Title & Authors Journal Publication Date

Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses

He WT, Musharrafieh R, Song G, Dueker K, Tse LV, Martinez DR, Schäfer A, Callaghan S, Yong P, Beutler N, Torres JL, Volk RM, Zhou P, Yuan M, Liu H, Anzanello F, Capozzola T, Parren M, Garcia E, Rawlings SA, Smith DM, Wilson IA, Safonova Y, Ward AB, Rogers TF, Baric RS, Gralinski LE, Burton DR, Andrabi R.
Nature Immunology June 2, 2022

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines. A broadly neutralizing antibody (bnAb) response is required to combat SARS-CoV-2 variants of concern (VOCs). The authors isolated and characterized a large panel of sarbecovirus bnAbs from vaccinated individuals who had recovered from COVID-19, finding that many of these antibodies were able to neutralize all VOCs, including Omicron, and demonstrate prophylaxis in mice infected with diverse sarbecoviruses.

Convergence of a common solution for broad ebolavirus neutralization by glycan cap-directed human antibodies

Murin CD, Gilchuk P, Ilinykh PA, Huang K, Kuzmina N, Shen X, Bruhn JF, Bryan AL, Davidson E, Doranz BJ, Williamson LE, Copps J, Alkutkar T, Flyak AI, Bukreyev A, Crowe JE Jr, Ward AB.
Cell reports June 1, 2022

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1–69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies.

Influenza H7N9 Virus Neuraminidase-Specific Human Monoclonal Antibodies Inhibit Viral Egress and Protect from Lethal Influenza Infection in Mice

Gilchuk IM, Bangaru S, Gilchuk P, Irving RP, Kose N, Bombardi RG, Thornburg NJ, Creech CB, Edwards KM, Li S, Turner HL, Yu W, Zhu X, Wilson IA, Ward AB, Crowe JE Jr.
Cell Host & Microbe May 20, 2022

H7N9 avian influenza virus causes severe infections and might have the potential to trigger a major pandemic. Molecular determinants of human humoral immune response to N9 neuraminidase (NA) proteins, which exhibit unusual features compared with seasonal influenza virus NA proteins, are ill-defined. We isolated 35 human monoclonal antibodies (mAbs) from two H7N9 survivors and two vaccinees. These mAbs react to NA in a subtype-specific manner and recognize diverse antigenic sites on the surface of N9 NA, including epitopes overlapping with, or distinct from, the enzyme active site. Despite recognizing multiple antigenic sites, the mAbs use a common mechanism of action by blocking egress of nascent virions from infected cells, thereby providing an antiviral prophylactic and therapeutic protection in vivo in mice. Studies of breadth, potency, and diversity of antigenic recognition from four subjects suggest that vaccination with inactivated adjuvanted vaccine induce NA-reactive responses comparable to that of H7N9 natural infection.

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-resolution mapping of the neutralizing and binding specificities of polyclonal sera post-HIV Env trimer vaccination

Dingens AS, Pratap P, Malone KD, Hilton SK, Ketas T, Cottrell CA, Overbaugh JM, Moore JP, Klasse PJ, Ward AB, Bloom JD.
eLife Feb. 28, 2022

Mapping polyclonal serum responses is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual antibodies, which incompletely defines the polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. We used mutational antigenic profiling to determine how all mutations in Env affected viral neutralization and electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize serum Fabs bound to Env trimers. The dominant neutralizing specificities were generally only a subset of the more diverse binding specificities. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Furthermore, we refined residue-level epitope specificity directly from sera, revealing subtle differences across sera. Together, mutational antigenic profiling and EMPEM yield a holistic view of the binding and neutralizing specificity of polyclonal sera.

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.

Fully synthetic platform to rapidly generate tetravalent bispecific nanobody–based immunoglobulins

Misson Mindrebo L, Liu H, Ozorowski G, Tran Q, Woehl J, Khalek I, Smith JM, Barman S, Zhao F, Keating C, Limbo O, Verma M, Liu J, Stanfield RL, Zhu X, Turner HL, Sok D, Huang PS, Burton DR, Ward AB, Wilson IA, Jardine JG.
Proceedings of the National Academy of Sciences Jan. 19, 2022

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor–binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

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 to SARS-related viruses can be readily induced in rhesus macaques

He WT, Yuan M, Callaghan S, Musharrafieh R, Song G, Silva M, Beutler N, Lee WH, Yong P, Torres JL, Melo M, Zhou P, Zhao F, Zhu X, Peng L, Huang D, Anzanello F, Ricketts J, Parren M, Garcia E, Ferguson M, Rinaldi W, Rawlings SA, Nemazee D, Smith DM, Briney B, Safonova Y, Rogers TF, Dan JM, Zhang Z, Weiskopf D, Sette A, Crotty S, Irvine DJ, Ward AB, Wilson IA, Burton DR, Andrabi R.
Science Translational Medicine Dec. 23, 2021

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting broadly neutralizing antibody responses to CoVs. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein with a two-shot protocol generated potent serum receptor binding domain cross-neutralizing antibody responses to both SARS-CoV-2 and SARS-CoV-1. Furthermore, responses were equally effective against most SARS-CoV-2 variants of concern (VOCs) and some were highly effective against Omicron. This result contrasts with human infection or many two-shot vaccination protocols where responses were typically more SARS-CoV-2 specific and where VOCs were less well neutralized. Structural studies showed that cloned macaque neutralizing antibodies, particularly using a given heavy chain germline gene, recognized a relatively conserved region proximal to the angiotensin converting enzyme 2 receptor binding site (RBS), whereas many frequently elicited human neutralizing antibodies targeted more variable epitopes overlapping the RBS. B cell repertoire differences between humans and macaques appeared to influence the vaccine response. The macaque neutralizing antibodies identified a pan-SARS–related virus epitope region less well targeted by human antibodies that could be exploited in rational vaccine design.

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