Publications
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Title & Authors Journal Publication Date

Fluorescent Trimeric Hemagglutinins Reveal Multivalent Receptor Binding Properties.


Nemanichvili N, Tomris I, Turner HL, McBride R, Grant OC, van der Woude R, Aldosari MH, Pieters RJ, Woods RJ, Paulson JC, Boons GJ, Ward AB, Verheije MH, de Vries RP.
J Mol Biol Feb. 15, 2019

Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA–receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA–sfGFP proteins was studied using glycan arrays and tissue staining. The HA–sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA–sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.

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 Jr, Wilson IA, Ward AB.
PLoS Biol Feb. 1, 2019

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The “breathing” of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.

Structure-Based Vaccine Antigen Design.


Graham BS, Gilman MSA, McLellan JS.
Annu Rev Med Jan. 27, 2019

Vaccine-Induced Protection from Homologous Tier 2 SHIV Challenge in Nonhuman Primates Depends on Serum-Neutralizing Antibody Titers.


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.
Immunity Jan. 15, 2019

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ∼1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained.

Deception through Mimicry: A Cellular Antiviral Strategy.


Wu NC, Ward AB.
Cell Dec. 13, 2018

Viruses use mimicry to hijack cellular machinery, but can human cells use mimicry as an antiviral approach? Batra et al. identify a novel antiviral restriction factor, RBBP6, by characterizing the cellular interactome of Ebola virus. RBBP6 targets the Ebola virus transcription factor VP30 by mimicking the binding of Ebola virus nucleoprotein.

Publisher Correction: 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 Dec. 10, 2018

Development of Clinical-Stage Human Monoclonal Antibodies That Treat Advanced Ebola Virus Disease in Nonhuman Primates.


Pascal KE, Dudgeon D, Trefry JC, Anantpadma M, Sakurai Y, Murin CD, Turner HL, Fairhurst J, Torres M, Rafique A, Yan Y, Badithe A, Yu K, Potocky T, Bixler SL, Chance TB, Pratt WD, Rossi FD, Shamblin JD, Wollen SE, Zelko JM, Carrion R Jr, Worwa G, Staples HM, Burakov D, Babb R, Chen G, Martin J, Huang TT, Erlandson K, Willis MS, Armstrong K, Dreier TM, Ward AB, Davey RA, Pitt MLM, Lipsich L, Mason P, Olson W, Stahl N, Kyratsous CA.
J Infect Dis Nov. 22, 2018

For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin.


Laursen NS, Friesen RHE, Zhu X, Jongeneelen M, Blokland S, Vermond J, van Eijgen A, Tang C, van Diepen H, Obmolova G, van der Neut Kolfschoten M, Zuijdgeest D, Straetemans R, Hoffman RMB, Nieusma T, Pallesen J, Turner HL, Bernard SM, Ward AB, Luo J, Poon LLM, Tretiakova AP, Wilson JM, Limberis MP, Vogels R, Brandenburg B, Kolkman JA, Wilson IA.
Science Nov. 2, 2018

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus–mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.

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.

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Title & Authors Journal Publication Date

Structure of the Ebola virus nucleoprotein – RNA complex


Kirchdoerfer RN, Saphire EO, Ward AB

Now Published: 10.1107/S2053230X19004424
bioRxiv Feb. 13, 2019

Structures of the Otopetrin Proton Channels Otop1 and Otop3


Saotome K, Teng B, Chun Tsui CA, Lee WH, Tu YH, P. Sansom MS, Liman ER, Ward AB

Now Published: 10.1038/s41594-019-0235-9
bioRxiv Feb. 6, 2019

Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics


Torrents de la Peña A, Rantalainen K, Cottrell CA, Allen JD, van Gils MJ, Torres JL, Crispin M, Sanders RW, Ward AB

Now Published: 10.1371/journal.ppat.1007920
bioRxiv Dec. 18, 2018

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