Publications
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Title & Authors Journal Publication Date

A cross-neutralizing antibody between HIV-1 and influenza virus.


Lee CD, Watanabe Y, Wu NC, Han J, Kumar S, Pholcharee T, Seabright GE, Allen JD, Lin CW, Yang JR, Liu MT, Wu CY, Ward AB, Crispin M, Wilson IA.
PLoS Pathog March 1, 2021

Incessant antigenic evolution enables the persistence and spread of influenza virus in the human population. As the principal target of the immune response, the hemagglutinin (HA) surface antigen on influenza viruses continuously acquires and replaces N-linked glycosylation sites to shield immunogenic protein epitopes using host-derived glycans. Anti-glycan antibodies, such as 2G12, target the HIV-1 envelope protein (Env), which is even more extensively glycosylated and contains under-processed oligomannose-type clusters on its dense glycan shield. Here, we illustrate that 2G12 can also neutralize human seasonal influenza A H3N2 viruses that have evolved to present similar oligomannose-type clusters on their HAs from around 20 years after the 1968 pandemic. Using structural biology and mass spectrometric approaches, we find that two N-glycosylation sites close to the receptor binding site (RBS) on influenza hemagglutinin represent the oligomannose cluster recognized by 2G12. One of these glycan sites is highly conserved in all human H3N2 strains and the other emerged during virus evolution. These two N-glycosylation sites have also become crucial for fitness of recent H3N2 strains. These findings shed light on the evolution of the glycan shield on influenza virus and suggest 2G12-like antibodies can potentially act as broad neutralizers to target human enveloped viruses.

Prolonged evolution of the human B cell response to SARS-CoV-2 infection.


Sakharkar M, Rappazzo CG, Wieland-Alter WF, Hsieh CL, Wrapp D, Esterman ES, Kaku CI, Wec AZ, Geoghegan JC, McLellan JS, Connor RI, Wright PF, Walker LM.
Sci Immunol Feb. 23, 2021

Influenza hemagglutinin-specific IgA Fc-effector functionality is restricted to stalk epitopes.


Freyn AW, Han J, Guthmiller JJ, Bailey MJ, Neu K, Turner HL, Rosado VC, Chromikova V, Huang M, Strohmeier S, Liu STH, Simon V, Krammer F, Ward AB, Palese P, Wilson PC, Nachbagauer R.
Proc Natl Acad Sci U S A Feb. 23, 2021

In this study, we utilized a panel of human immunoglobulin (Ig) IgA monoclonal antibodies isolated from the plasmablasts of eight donors after 2014/2015 influenza virus vaccination (Fluarix) to study the binding and functional specificities of this isotype. In this cohort, isolated IgA monoclonal antibodies were primarily elicited against the hemagglutinin protein of the H1N1 component of the vaccine. To compare effector functionalities, an H1-specific subset of antibodies targeting distinct epitopes were expressed as monomeric, dimeric, or secretory IgA, as well as in an IgG1 backbone. When expressed with an IgG Fc domain, all antibodies elicited Fc-effector activity in a primary polymorphonuclear cell-based assay which differs from previous observations that found only stalk-specific antibodies activate the low-affinity FcγRIIIa. However, when expressed with IgA Fc domains, only antibodies targeting the stalk domain showed Fc-effector activity in line with these previous findings. To identify the cause of this discrepancy, we then confirmed that IgG signaling through the high-affinity FcγI receptor was not restricted to stalk epitopes. Since no corresponding high-affinity Fcα receptor exists, the IgA repertoire may therefore be limited to stalk-specific epitopes in the context of Fc receptor signaling.

Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.


Rappazzo CG, Tse LV, Kaku CI, Wrapp D, Sakharkar M, Huang D, Deveau LM, Yockachonis TJ, Herbert AS, Battles MB, O'Brien CM, Brown ME, Geoghegan JC, Belk J, Peng L, Yang L, Hou Y, Scobey TD, Burton DR, Nemazee D, Dye JM, Voss JE, Gunn BM, McLellan JS, Baric RS, Gralinski LE, Walker LM.
Science Feb. 19, 2021

Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles.


Brouwer PJM, Antanasijevic A, de Gast M, Allen JD, Bijl TPL, Yasmeen A, Ravichandran R, Burger JA, Ozorowski G, Torres JL, LaBranche C, Montefiori DC, Ringe RP, van Gils MJ, Moore JP, Klasse PJ, Crispin M, King NP, Ward AB, Sanders RW.
NPJ Vaccines Feb. 9, 2021

The HIV-1 envelope glycoprotein trimer is poorly immunogenic because it is covered by a dense glycan shield. As a result, recombinant Env glycoproteins generally elicit inadequate antibody levels that neutralize clinically relevant, neutralization-resistant (Tier-2) HIV-1 strains. Multivalent antigen presentation on nanoparticles is an established strategy to increase vaccine-driven immune responses. However, due to nanoparticle instability in vivo, the display of non-native Env structures, and the inaccessibility of many neutralizing antibody (NAb) epitopes, the effects of nanoparticle display are generally modest for Env trimers. Here, we generate two-component self-assembling protein nanoparticles presenting twenty SOSIP trimers of the clade C Tier-2 genotype 16055. We show in a rabbit immunization study that these nanoparticles induce 60-fold higher autologous Tier-2 NAb titers than the corresponding SOSIP trimers. Epitope mapping studies reveal that the presentation of 16055 SOSIP trimers on these nanoparticle focuses antibody responses to an immunodominant apical epitope. Thus, these nanoparticles are a promising platform to improve the immunogenicity of Env trimers with apex-proximate NAb epitopes.

The C3/465 glycan hole cluster in BG505 HIV-1 envelope is the major neutralizing target involved in preventing mucosal SHIV infection.


Charles TP, Burton SL, Arunachalam PS, Cottrell CA, Sewall LM, Bollimpelli VS, Gangadhara S, Dey AK, Ward AB, Shaw GM, Hunter E, Amara RR, Pulendran B, van Gils MJ, Derdeyn CA.
PLoS Pathog Feb. 1, 2021

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins.


Bouwman KM, Tomris I, Turner HL, van der Woude R, Shamorkina TM, Bosman GP, Rockx B, Herfst S, Snijder J, Haagmans BL, Ward AB, Boons GJ, de Vries RP.
PLoS Pathog Feb. 1, 2021

The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV). Here we identified the optimal D8 ligand to be chondroitin sulfate E (CS-E). CS-E is characterized by a disaccharide moiety with two sulfated hydroxyl groups at positions 4′ and 6′ of GalNAc. To study the role of antibodies in preventing D8 adhesion to CS-E, we have used a panel of murine monoclonal antibodies, and tested their ability to compete with CS-E for D8 binding. Among four antibody specificity groups, MAbs of one group (group IV) fully abrogated CS-E binding, while MAbs of a second group (group III) displayed widely varying levels of CS-E blocking. Using EM, we identified the binding site for each antibody specificity group on D8. Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E.

Polyclonal epitope mapping reveals temporal dynamics and diversity of human antibody responses to H5N1 vaccination.


Han J, Schmitz AJ, Richey ST, Dai YN, Turner HL, Mohammed BM, Fremont DH, Ellebedy AH, Ward AB.
Cell Rep Jan. 26, 2021

Novel influenza A virus (IAV) strains elicit recall immune responses to conserved epitopes, making them favorable antigenic choices for universal influenza virus vaccines. Evaluating these immunogens requires a thorough understanding of the antigenic sites targeted by the polyclonal antibody (pAb) response, which single-particle electron microscopy (EM) can sensitively detect. In this study, we employ EM polyclonal epitope mapping (EMPEM) to extensively characterize the pAb response to hemagglutinin (HA) after H5N1 immunization in humans. Cross-reactive pAbs originating from memory B cells immediately bound the stem of HA and persisted for more than a year after vaccination. In contrast, de novo pAb responses to multiple sites on the head of HA, targeting previously determined key neutralizing sites on H5 HA, expanded after the second immunization and waned quickly. Thus, EMPEM provides a robust tool for comprehensively tracking the specificity and durability of immune responses elicited by novel universal influenza vaccine candidates.

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


Dingens AS, Pratap P, Malone K, Hilton SK, Ketas T, Cottrell CA, Overbaugh J, Moore JP, Klasse PJ, Ward AB, Bloom JD.
Elife Jan. 13, 2021

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 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.
Front Immunol Jan. 1, 2021

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.

Quantification of the Resilience and Vulnerability of HIV-1 Native Glycan Shield at Atomistic Detail.


Chakraborty S, Berndsen ZT, Hengartner NW, Korber BT, Ward AB, Gnanakaran S.
iScience Dec. 18, 2020

Dense surface glycosylation on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. However, the molecular complexity and flexibility of glycans make experimental studies a challenge. Here we have integrated high-throughput atomistic modeling of fully glycosylated HIV-1 Env with graph theory to capture immunologically important features of the shield topology. This is the first complete all-atom model of HIV-1 Env SOSIP glycan shield that includes both oligomannose and complex glycans, providing physiologically relevant insights of the glycan shield. This integrated approach including quantitative comparison with cryo-electron microscopy data provides hitherto unexplored details of the native shield architecture and its difference from the high-mannose glycoform. We have also derived a measure to quantify the shielding effect over the antigenic protein surface that defines regions of relative vulnerability and resilience of the shield and can be harnessed for rational immunogen design.

Cross-Neutralization of a SARS-CoV-2 Antibody to a Functionally Conserved Site Is Mediated by Avidity.


Liu H, Wu NC, Yuan M, Bangaru S, Torres JL, Caniels TG, van Schooten J, Zhu X, Lee CD, Brouwer PJM, van Gils MJ, Sanders RW, Ward AB, Wilson IA.
Immunity Dec. 15, 2020

Most antibodies isolated from individuals with coronavirus disease 2019 (COVID-19) are specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here, we determined a crystal structure of the COVA1-16 antibody fragment (Fab) with the SARS-CoV-2 receptor-binding domain (RBD) and negative-stain electron microscopy reconstructions with the spike glycoprotein trimer to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long complementarity-determining region (CDR) H3, and competes with the angiotensin-converting enzyme 2 (ACE2) receptor because of steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with the structural and functional rationale for epitope conservation, provide insights for development of more universal SARS-like coronavirus vaccines and therapies.

Polyreactive Broadly Neutralizing B cells Are Selected to Provide Defense against Pandemic Threat Influenza Viruses.


Guthmiller JJ, Lan LY, Fernández-Quintero ML, Han J, Utset HA, Bitar DJ, Hamel NJ, Stovicek O, Li L, Tepora M, Henry C, Neu KE, Dugan HL, Borowska MT, Chen YQ, Liu STH, Stamper CT, Zheng NY, Huang M, Palm AE, García-Sastre A, Nachbagauer R, Palese P, Coughlan L, Krammer F, Ward AB, Liedl KR, Wilson PC.
Immunity Dec. 15, 2020

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.

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Title & Authors Journal Publication Date

A public broadly neutralizing antibody class targets a membrane-proximal anchor epitope of influenza virus hemagglutinin


Guthmiller JJ, Han J, Utset HA, Li L, Yu-Ling Lan L, Henry C, Stamper CT, Stovicek O, Gentles L, Dugan HL, Zheng NY, Richey ST, Tepora ME, Bitar DJ, Changrob S, Strohmeier S, Huang M, García-Sastre A, Nachbagauer R, Palese P, Bloom JD, Krammer F, Coughlan L, Ward AB, Wilson PC

Now Published: 10.1038/s41586-021-04356-8
bioRxiv Feb. 25, 2021

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization.


Turner HL, Andrabi R, Cottrell CA, Richey ST, Song G, Callaghan S, Anzanello F, Moyer TJ, Abraham W, Melo M, Silva M, Scaringi N, Rakasz EG, Sattentau Q, Irvine DJ, Burton DR, Ward AB.

Now Published: 10.1126/sciadv.abh2791
bioRxiv Feb. 17, 2021

Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants


Yuan M, Huang D, Lee CCD, Wu NC, Jackson AM, Zhu X, Liu H, Peng L, van Gils MJ, Sanders RW, Burton DR, Reincke SM, Prüss H, Kreye J, Nemazee D, Ward AB, Wilson IA

Now Published: 10.1126/science.abh1139
bioRxiv Feb. 16, 2021

A combination of cross-neutralizing antibodies synergizes to prevent SARS-CoV-2 and SARS-CoV pseudovirus infection.


Liu H, Yuan M, Huang D, Bangaru S, Lee CD, Peng L, Zhu X, Nemazee D, van Gils MJ, Sanders RW, Kornau HC, Reincke SM, Prüss H, Kreye J, Wu NC, Ward AB, Wilson IA.

Now Published: 10.1016/j.chom.2021.04.005
bioRxiv Feb. 12, 2021

Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM


Antanasijevic A, Sewall LM, Cottrell CA, Carnathan DG, Jimenez LE, Ngo JT, Silverman JB, Groschel B, Georgeson E, Bhiman J, Bastidas R, LaBranche C, Allen JD, Copps J, Perrett HR, Rantalainen K, Cannac F, Yang YR, Torrents de la Peña A, Froes Rocha R, Berndsen ZT, Baker D, King NP, Sanders RW, Moore JP, Crotty S, Crispin M, Montefiori DC, Burton DR, Schief WR, Silvestri G, Ward AB

Now Published: 10.1038/s41467-021-25087-4
bioRxiv Jan. 28, 2021

Canonical features of human antibodies recognizing the influenza hemagglutinin trimer interface


Zost SJ, Dong J, Gilchuk I, Gilchuk P, Thornburg NJ, Bangaru S, Kose N, Finn JA, Bombardi R, Soto C, Nargi R, Irving RP, Suryadevara N, Westover JB, Carnahan RH, Turner HL, Li S, Ward AB, Crowe JE

Now Published: 10.1172/jci146791
bioRxiv Dec. 31, 2020

Neutralizing antibodies induced by first-generation gp41-stabilized HIV-1 envelope trimers and nanoparticles


Kumar S, Lin X, Ngo T, Shapero B, Sou C, Allen JD, Copps J, Zhang L, Ozorowski G, He L, Crispin M, Ward AB, Wilson IA, Zhu J

Now Published: 10.1128/mbio.00429-21
bioRxiv Dec. 2, 2020