Publications
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Title & Authors Journal Publication Date

OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels.


Murthy SE, Dubin AE, Whitwam T, Jojoa-Cruz S, Cahalan SM, Mousavi SAR, Ward AB, Patapoutian A.
Elife Nov. 1, 2018

Cryo-EM structure of the mechanically activated ion channel OSCA1.2.


Jojoa-Cruz S, Saotome K, Murthy SE, Tsui CCA, Sansom MS, Patapoutian A, Ward AB.
Elife Nov. 1, 2018

Mechanically activated ion channels underlie touch, hearing, shear-stress sensing, and response to turgor pressure. OSCA/TMEM63s are a newly-identified family of eukaryotic mechanically activated ion channels opened by membrane tension. The structural underpinnings of OSCA/TMEM63 function are not explored. Here, we elucidate high resolution cryo-electron microscopy structures of OSCA1.2, revealing a dimeric architecture containing eleven transmembrane helices per subunit and surprising topological similarities to TMEM16 proteins. We locate the ion permeation pathway within each subunit by demonstrating that a conserved acidic residue is a determinant of channel conductance. Molecular dynamics simulations reveal membrane interactions, suggesting the role of lipids in OSCA1.2 gating. These results lay a foundation to decipher how the structural organization of OSCA/TMEM63 is suited for their roles as MA ion channels.

HIV-1 vaccine design through minimizing envelope metastability.


He L, Kumar S, Allen JD, Huang D, Lin X, Mann CJ, Saye-Francisco KL, Copps J, Sarkar A, Blizard GS, Ozorowski G, Sok D, Crispin M, Ward AB, Nemazee D, Burton DR, Wilson IA, Zhu J.
Sci Adv Nov. 1, 2018

Overcoming envelope metastability is crucial to trimer-based HIV-1 vaccine design. Here, we present a coherent vaccine strategy by minimizing metastability. For 10 strains across five clades, we demonstrate that the gp41 ectodomain (gp41ECTO) is the main source of envelope metastability by replacing wild-type gp41ECTO with BG505 gp41ECTO of the uncleaved prefusion-optimized (UFO) design. These gp41ECTO-swapped trimers can be produced in CHO cells with high yield and high purity. The crystal structure of a gp41ECTO-swapped trimer elucidates how a neutralization-resistant tier 3 virus evades antibody recognition of the V2 apex. UFO trimers of transmitted/founder viruses and UFO trimers containing a consensus-based ancestral gp41ECTO suggest an evolutionary root of metastability. The gp41ECTO-stabilized trimers can be readily displayed on 24- and 60-meric nanoparticles, with incorporation of additional T cell help illustrated for a hyperstable 60-mer, I3-01. In mice and rabbits, these gp140 nanoparticles induced tier 2 neutralizing antibody responses more effectively than soluble trimers.

Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis.


Kirchdoerfer RN, Wang N, Pallesen J, Wrapp D, Turner HL, Cottrell CA, Corbett KS, Graham BS, McLellan JS, Ward AB.
Sci Rep Oct. 24, 2018

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S is believed to undergo a number of conformational transitions as it is cleaved by host proteases and binds to host receptors. We recently developed stabilizing mutations for coronavirus spikes that prevent the transition from the pre-fusion to post-fusion states. Here, we present cryo-EM analyses of a stabilized trimeric SARS-CoV S, as well as the trypsin-cleaved, stabilized S, and its interactions with ACE2. Neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within stabilized SARS-CoV S or expose the secondary cleavage site, S2′.

Cryo-EM structure of P. falciparum circumsporozoite protein with a vaccine-elicited antibody is stabilized by somatically mutated inter-Fab contacts.


Oyen D, Torres JL, Cottrell CA, Richter King C, Wilson IA, Ward AB.
Sci Adv Oct. 1, 2018

The circumsporozoite protein (CSP) on the surface of Plasmodium falciparum sporozoites is important for parasite development, motility, and host hepatocyte invasion. However, intrinsic disorder of the NANP repeat sequence in the central region of CSP has hindered its structural and functional characterization. Here, the cryo–electron microscopy structure at ~3.4-Å resolution of a recombinant shortened CSP construct with the variable domains (Fabs) of a highly protective monoclonal antibody reveals an extended spiral conformation of the central NANP repeat region surrounded by antibodies. This unusual structure appears to be stabilized and/or induced by interaction with an antibody where contacts between adjacent Fabs are somatically mutated and enhance the interaction. This maturation in non-antigen contact residues may be an effective mechanism for antibodies to target tandem repeat sequences and provide novel insights into malaria vaccine design.

Rational Design of DNA-Expressed Stabilized Native-Like HIV-1 Envelope Trimers.


Aldon Y, McKay PF, Allen J, Ozorowski G, Felfödiné Lévai R, Tolazzi M, Rogers P, He L, de Val N, Fábián K, Scarlatti G, Zhu J, Ward AB, Crispin M, Shattock RJ.
Cell Rep Sept. 18, 2018

The HIV-1-envelope glycoprotein (Env) is the main target of antigen design for antibody-based prophylactic vaccines. The generation of broadly neutralizing antibodies (bNAb) likely requires the appropriate presentation of stabilized trimers preventing exposure of non-neutralizing antibody (nNAb) epitopes. We designed a series of membrane-bound Envs with increased trimer stability through the introduction of key stabilization mutations. We derived a stabilized HIV-1 trimer, ConSOSL.UFO.750, which displays a dramatic reduction in nNAb binding while maintaining high quaternary and MPER-specific bNAb binding. Its soluble counterpart, ConSOSL.UFO.664, displays similar antigenicity, and its native-like Env structure is confirmed by negative stain-EM and glycosylation profiling of the soluble ConSOSL.UFO.664 trimer. A rabbit immunization study demonstrated that the ConSOSL.UFO.664 can induce autologous tier 2 neutralization. We have successfully designed a stabilized native-like Env trimer amenable to nucleic acid or viral vector-based vaccination strategies.

Differential processing of HIV envelope glycans on the virus and soluble recombinant trimer.


Cao L, Pauthner M, Andrabi R, Rantalainen K, Berndsen Z, Diedrich JK, Menis S, Sok D, Bastidas R, Park SR, Delahunty CM, He L, Guenaga J, Wyatt RT, Schief WR, Ward AB, Yates JR 3rd, Burton DR, Paulson JC.
Nat Commun Sept. 12, 2018

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75–90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.

Structural Basis of Pan-Ebolavirus Neutralization by an Antibody Targeting the Glycoprotein Fusion Loop.


Murin CD, Bruhn JF, Bornholdt ZA, Copps J, Stanfield R, Ward AB.
Cell Rep Sept. 4, 2018

Monoclonal antibodies (mAbs) with pan-ebolavirus cross-reactivity are highly desirable, but development of such mAbs is limited by a lack of a molecular understanding of cross-reactive epitopes. The antibody ADI-15878 was previously identified from a human survivor of Ebola virus Makona variant (EBOV/Mak) infection. This mAb demonstrated potent neutralizing activity against all known ebolaviruses and provided protection in rodent and ferret models against three ebolavirus species. Here, we describe the unliganded crystal structure of ADI-15878 as well as the cryo-EM structures of ADI-15878 in complex with the EBOV/Mak and Bundibugyo virus (BDBV) glycoproteins (GPs). ADI-15878 binds through an induced-fit mechanism by targeting highly conserved residues in the internal fusion loop (IFL), bridging across GP protomers via the heptad repeat 1 (HR1) region. Our structures provide a more complete description of the ebolavirus immunogenic landscape, as well as a molecular basis for how rare but potent antibodies target conserved filoviral fusion machinery.

Electron-Microscopy-Based Epitope Mapping Defines Specificities of Polyclonal Antibodies Elicited during HIV-1 BG505 Envelope Trimer Immunization.


Bianchi M, Turner HL, Nogal B, Cottrell CA, Oyen D, Pauthner M, Bastidas R, Nedellec R, McCoy LE, Wilson IA, Burton DR, Ward AB, Hangartner L.
Immunity Aug. 21, 2018

Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.

Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.


Gilchuk P, Kuzmina N, Ilinykh PA, Huang K, Gunn BM, Bryan A, Davidson E, Doranz BJ, Turner HL, Fusco ML, Bramble MS, Hoff NA, Binshtein E, Kose N, Flyak AI, Flinko R, Orlandi C, Carnahan R, Parrish EH, Sevy AM, Bombardi RG, Singh PK, Mukadi P, Muyembe-Tamfum JJ, Ohi MD, Saphire EO, Lewis GK, Alter G, Ward AB, Rimoin AW, Bukreyev A, Crowe JE Jr.
Immunity Aug. 21, 2018

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

Structure of the human volume regulated anion channel.


Kefauver JM, Saotome K, Dubin AE, Pallesen J, Cottrell CA, Cahalan SM, Qiu Z, Hong G, Crowley CS, Whitwam T, Lee WH, Ward AB, Patapoutian A.
Elife Aug. 10, 2018

SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with four other LRRC8 family members, potentially forms a vastly heterogeneous cohort of VRAC channels with different properties; however, SWELL1 alone is also functional. Here, we report a high-resolution cryo-electron microscopy structure of full-length human homo-hexameric SWELL1. The structure reveals a trimer of dimers assembly with symmetry mismatch between the pore-forming domain and the cytosolic leucine-rich repeat (LRR) domains. Importantly, mutational analysis demonstrates that a charged residue at the narrowest constriction of the homomeric channel is an important pore determinant of heteromeric VRAC. Additionally, a mutation in the flexible N-terminal portion of SWELL1 affects pore properties, suggesting a putative link between intracellular structures and channel regulation. This structure provides a scaffold for further dissecting the heterogeneity and mechanism of activation of VRAC.

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.


Saphire EO, Schendel SL, Fusco ML, Gangavarapu K, Gunn BM, Wec AZ, Halfmann PJ, Brannan JM, Herbert AS, Qiu X, Wagh K, He S, Giorgi EE, Theiler J, Pommert KBJ, Krause TB, Turner HL, Murin CD, Pallesen J, Davidson E, Ahmed R, Aman MJ, Bukreyev A, Burton DR, Crowe JE Jr, Davis CW, Georgiou G, Krammer F, Kyratsous CA, Lai JR, Nykiforuk C, Pauly MH, Rijal P, Takada A, Townsend AR, Volchkov V, Walker LM, Wang CI, Zeitlin L, Doranz BJ, Ward AB, Korber B, Kobinger GP, Andersen KG, Kawaoka Y, Alter G, Chandran K, Dye JM.
Cell Aug. 9, 2018

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

A multifunctional human monoclonal neutralizing antibody that targets a unique conserved epitope on influenza HA.


Bangaru S, Zhang H, Gilchuk IM, Voss TG, Irving RP, Gilchuk P, Matta P, Zhu X, Lang S, Nieusma T, Richt JA, Albrecht RA, Vanderven HA, Bombardi R, Kent SJ, Ward AB, Wilson IA, Crowe JE Jr.
Nat Commun July 10, 2018

The high rate of antigenic drift in seasonal influenza viruses necessitates frequent changes in vaccine composition. Recent seasonal H3 vaccines do not protect against swine-origin H3N2 variant (H3N2v) strains that recently have caused severe human infections. Here, we report a human VH1-69 gene-encoded monoclonal antibody (mAb) designated H3v-47 that exhibits potent cross-reactive neutralization activity against human and swine H3N2 viruses that circulated since 1989. The crystal structure and electron microscopy reconstruction of H3v-47 Fab with the H3N2v hemagglutinin (HA) identify a unique epitope spanning the vestigial esterase and receptor-binding subdomains that is distinct from that of any known neutralizing antibody for influenza A H3 viruses. MAb H3v-47 functions largely by blocking viral egress from infected cells. Interestingly, H3v-47 also engages Fcγ receptor and mediates antibody dependent cellular cytotoxicity (ADCC). This newly identified conserved epitope can be used in design of novel immunogens for development of broadly protective H3 vaccines.

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Title & Authors Journal Publication Date

Chimpanzee SIV Envelope trimer: structure and deployment as an HIV vaccine template


Andrabi R, Pallesen J, Allen J, Song G, Zhang J, de Val N, Gegg G, Porter K, Su CY, Pauthner M, Newman A, Bouton-Vervelle H, Garces F, Wilson IA, Crispin M, Hahn BH, Haynes BF, Verkoczy L, Ward AB, Burton DR

Now Published: 10.1016/j.celrep.2019.04.082
bioRxiv Nov. 1, 2018

Vaccine-induced protection from homologous Tier 2 simian-human immunodeficiency virus challenge in nonhuman primates


Pauthner MG, Nkolola JP, Havenar-Daughton C, Murrell B, Reiss SM, Bastidas R, Prévost J, Nedellec R, von Bredow B, Abbink P, Cottrell CA, Kulp DW, Tokatlian T, Nogal B, Bianchi M, Li H, Lee JH, Butera ST, Evans DT, Hangartner L, Finzi A, Wilson IA, Wyatt RT, Irvine DJ, Schief WR, Ward AB, Sanders RW, Crotty S, Shaw GM, Barouch DH, Burton DR

Now Published: 10.1016/j.immuni.2018.11.011
bioRxiv Oct. 29, 2018

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor binding head domains


Turner HL, Pallesen J, Lang S, Bangaru S, Urata S, Li S, Cottrell CA, Bowman CA, Crowe JE, Wilson IA, Ward AB

Now Published: 10.1371/journal.pbio.3000139
bioRxiv Oct. 5, 2018

Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance


Cirelli KM, Carnathan DG, Nogal B, Rodriguez OL, Martin JT, Upadhyay AA, Enemuo CA, Gebru EH, Choe Y, Viviano F, Nakao C, Pauthner M, Reiss S, Cottrell CA, Bastidas R, Gibson W, Wolabaugh AN, Melo MB, Cosette B, Kuman V, Patel N, Tokatlian T, Menis S, Kulp DW, Burton DR, Murrell B, Bosinger SE, Schief WR, Ward AB, Watson CT, Silvestri G, Irvine DJ, Crotty S

Now Published: 10.1016/j.cell.2019.04.012
bioRxiv Oct. 1, 2018

Cryo-EM structure of the mechanically activated ion channel OSCA1.2


Jojoa-Cruz S, Saotome K, Murthy SE, Chun Tsui CA, P. Sansom MS, Patapoutian A, Ward AB

Now Published: 10.7554/elife.41845
bioRxiv Sept. 4, 2018

OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels


Murthy SE, Dubin AE, Whitwam T, Jojoa-Cruz S, Cahalan SM, Ali Reza Mosavi S, Ward AB, Patapoutian A

Now Published: 10.7554/elife.41844
bioRxiv Sept. 4, 2018

HIV-1 vaccine design through minimizing envelope metastability


He L, Kumar S, Allen JD, Huang D, Lin X, Mann CJ, Saye-Francisco KL, Copps J, Sarkar A, Blizard GS, Ozorowski G, Sok D, Crispin M, Ward AB, Nemazee D, Burton DR, Wilson IA, Zhu J

Now Published: 10.1126/sciadv.aau6769
bioRxiv July 3, 2018