Title & Authors Journal Publication Date

Isolation and characterization of cross-neutralizing coronavirus antibodies from COVID-19+ subjects

Jennewein MF, MacCamy AJ, Akins NR, Feng J, Homad LJ, Hurlburt NK, Seydoux E, Wan YH, Stuart AB, Edara VV, Floyd K, Vanderheiden A, Mascola JR, Doria-Rose N, Wang L, Yang ES, Chu HY, Torres JL, Ozorowski G, Ward AB, Whaley RE, Cohen KW, Pancera M, McElrath MJ, Englund JA, Finzi A, Suthar MS, McGuire AT, Stamatatos L.
Cell Reports July 13, 2021

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

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.
mBio June 22, 2021

The immunogenicity of gp41-stabilized HIV-1 BG505 envelope (Env) trimers and nanoparticles (NPs) was recently assessed in mice and rabbits. Here, we combined Env-specific B-cell sorting and repertoire sequencing to identify neutralizing antibodies (NAbs) from immunized animals. A panel of mouse NAbs was isolated from mice immunized with a 60-meric I3-01 NP presenting 20 stabilized trimers. Three mouse NAbs potently neutralized BG505.T332N by recognizing a glycan epitope centered in the C3/V4 region on BG505 Env, as revealed by electron microscopy (EM), X-ray crystallography, and epitope mapping. A set of rabbit NAbs was isolated from rabbits immunized with a soluble trimer and a 24-meric ferritin NP presenting 8 trimers. Neutralization assays against BG505.T332N variants confirmed that potent rabbit NAbs targeted previously described glycan holes on BG505 Env and accounted for a significant portion of the autologous NAb response in both the trimer and ferritin NP groups. Last, we examined NAb responses that were induced by non-BG505 Env immunogens. We determined a 3.4-Å-resolution crystal structure for the clade C transmitted/founder (T/F) Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend in gp41. This clade C Env, in a soluble trimer form and in a multivalent form with 8 trimers attached to ferritin NP, and the gp41-stabilized clade A Q482-d12 Env trimer elicited distinct NAb responses in rabbits, with notable differences in neutralization breadth. Although eliciting a broad NAb response remains a major challenge, our study provides valuable information on an HIV-1 vaccine design strategy that combines gp41 stabilization and NP display.

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.
Journal of Molecular Biology June 2, 2021

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.

Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells

Rossignol ED, Dugast AS, Compere H, Cottrell CA, Copps J, Lin S, Cizmeci D, Seaman MS, Ackerman ME, Ward AB, Alter G, Julg B.
Cell reports May 25, 2021

HIV monoclonal antibodies for viral reservoir eradication strategies will likely need to recognize reactivated infected cells and potently drive Fc-mediated innate effector cell activity. We systematically characterize a library of 185 HIV-envelope-specific antibodies derived from 15 spontaneous HIV controllers (HCs) that selectively exhibit robust serum Fc functionality and compared them to broadly neutralizing antibodies (bNAbs) in clinical development. Within the 10 antibodies with the broadest cell-recognition capability, seven originated from HCs and three were bNAbs. V3-loop-targeting antibodies are enriched among the top cell binders, suggesting the V3-loop may be selectively exposed and accessible on the cell surface. Fc functionality is more variable across antibodies, which is likely influenced by distinct binding topology and corresponding Fc accessibility, highlighting not only the importance of target-cell recognition but also the need to optimize for Fc-mediated elimination. Ultimately, our results demonstrate that this comprehensive selection process can identify monoclonal antibodies poised to eliminate infected cells.

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

Yuan M, Huang D, Lee CD, 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.
Science May 20, 2021

Neutralizing antibodies (nAbs) elicited against the receptor-binding site (RBS) of the spike protein of wild-type SARS-CoV-2 are generally less effective against recent variants of concern. RBS residues E484, K417 and N501 are mutated in variants first described in South Africa (B.1.351) and Brazil (P.1). We analyzed their effects on ACE2 binding and K417N and E484K mutations on nAbs isolated from COVID-19 patients. Binding and neutralization of the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2), which can both bind the RBS in alternate binding modes, are abrogated by K417N, E484K, or both. These effects can be structurally explained by their extensive interactions with RBS nAbs. However, nAbs to the more conserved, cross-neutralizing CR3022 and S309 sites were largely unaffected. The results have implications for next-generation vaccines and antibody therapies.

Cryo-EM Structure of a Fully Glycosylated Soluble Cleaved HIV-1 Envelope Trimer

Lyumkis D, Julien JP, de Val N, Cupo A, Potter CS, Klasse PJ, Burton DR, Sanders RW, Moore JP, Carragher B, Wilson IA, Ward AB
Science May 19, 2021

The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo–electron microscopy reconstruction and structural model of a cleaved, soluble Env trimer (termed BG505 SOSIP.664 gp140) in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 angstrom resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1, and HR2 domains, as well as shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.

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, Zhao F, Lee CD, Peng L, Barman S, Zhu X, Nemazee D, Burton DR, van Gils MJ, Sanders RW, Kornau HC, Reincke SM, Prüss H, Kreye J, Wu NC, Ward AB, Wilson IA.
Cell Host & Microbe May 12, 2021

Coronaviruses have caused several human epidemics and pandemics including the ongoing coronavirus disease 2019 (COVID-19). Prophylactic vaccines and therapeutic antibodies have already shown striking effectiveness against COVID-19. Nevertheless, concerns remain about antigenic drift in SARS-CoV-2 as well as threats from other sarbecoviruses. Cross-neutralizing antibodies to SARS-related viruses provide opportunities to address such concerns. Here, we report on crystal structures of a cross-neutralizing antibody, CV38-142, in complex with the receptor-binding domains from SARS-CoV-2 and SARS-CoV. Recognition of the N343 glycosylation site and water-mediated interactions facilitate cross-reactivity of CV38-142 to SARS-related viruses, allowing the antibody to accommodate antigenic variation in these viruses. CV38-142 synergizes with other cross-neutralizing antibodies, notably COVA1-16, to enhance neutralization of SARS-CoV and SARS-CoV-2, including circulating variants of concern B.1.1.7 and B.1.351. Overall, this study provides valuable information for vaccine and therapeutic design to address current and future antigenic drift in SARS-CoV-2 and to protect against zoonotic SARS-related coronaviruses.

Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines

He L, Chaudhary A, Lin X, Sou C, Alkutkar T, Kumar S, Ngo T, Kosviner E, Ozorowski G, Stanfield RL, Ward AB, Wilson IA, Zhu J.
Nature Communications May 11, 2021

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

Cooperativity Enables Non-neutralizing Antibodies to Neutralize Ebolavirus

Howell KA, Brannan JM, Bryan C, McNeal A, Davidson E, Turner HL, Vu H, Shulenin S, He S, Kuehne A, Herbert AS, Qiu X, Doranz BJ, Holtsberg FW, Ward AB, Dye JM, Aman MJ
Cell Reports April 13, 2021

Drug combinations are synergistic when their combined efficacy exceeds the sum of the individual actions, but they rarely include ineffective drugs that become effective only in combination. We identified several “enabling pairs” of neutralizing and non-neutralizing anti-ebolavirus monoclonal antibodies, whose combination exhibited new functional profiles, including transforming a non-neutralizing antibody to a neutralizer. Sub-neutralizing concentrations of antibodies 2G4 or m8C4 enabled non-neutralizing antibody FVM09 (IC50 >1 μM) to exhibit potent neutralization (IC50 1–10 nM). While FVM09 or m8C4 alone failed to protect Ebola-virus-infected mice, a combination of the two antibodies provided 100% protection. Furthermore, non-neutralizers FVM09 and FVM02 exponentially enhanced the potency of two neutralizing antibodies against both Ebola and Sudan viruses. We identified a hotspot for the binding of these enabling antibody pairs near the interface of the glycan cap and GP2. Enabling cooperativity may be an underappreciated phenomenon for viruses, with implications for the design and development of immunotherapeutics and vaccines.

Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches

Pauthner M, Havenar-Daughton C, Sok D, Nkolola JP, Bastidas R, Boopathy AV, Carnathan DG, Chandrashekar A, Cirelli KM, Cottrell CA, Eroshkin AM, Guenaga J, Kaushik K, Kulp DW, Liu J, McCoy LE, Oom AL, Ozorowski G, Post KW, Sharma SK, Steichen JM, de Taeye SW, Tokatlian T, Torrents de la Pena A, Butera ST, LaBranche CC, Montefiori DC, Silvestri G, Wilson IA, Irvine DJ, Sanders RW, Schief WR, Ward AB, Wyatt RT, Barouch DH, Crotty S, Burton DR
Immunity April 7, 2021

The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.

Envelope proteins of two HIV-1 clades induced different epitope-specific antibody response

Shrivastava T, Samal S, Tyagi AK, Goswami S, Kumar N, Ozorowski G, Ward AB, Chakrabarti BK
Vaccine April 6, 2021

Using HIV-1 envelope protein (Env)-based immunogens that closely mimic the conformation of functional HIV-1 Envs and represent the isolates prevalent in relevant geographical region is considered a rational approach towards developing HIV vaccine. We recently reported that like clade B Env, JRFL, membrane bound Indian clade C Env, 4-2.J41 is also efficiently cleaved and displays desirable antigenic properties for plasmid DNA immunization. Here, we evaluated the immune response in rabbit by injecting the animals with plasmid expressing membrane bound efficiently cleaved 4-2.J41 Env followed by its gp140-foldon (gp140-fd) protein boost. The purified 4-2.J41-gp140-fd protein is recognized by a wide panel of broadly neutralizing antibodies (bNAbs) including the quaternary conformation-dependent antibody, PGT145 with high affinity. We have also evaluated and compared the quality of antibody response elicited in rabbits after immunizing with plasmid DNA expressing the membrane bound efficiently cleaved Env followed by gp140-fd proteins boost with either of clade C Env, 4-2.J41 or clade B Env, JRFL or in combination. In comparison to JRFL group, 4-2.J41 group elicited autologous as well as limited low level cross clade neutralizing antibody response. Preliminary epitope-mapping of sera from animals show that in contrast to JRFL group, no reactivity to either linear peptides or V3-loop is detected in 4-2.J41 group. Furthermore, the presence of conformation-specific antibody in sera from animals immunized with 4-2.J41 Env is observed. However, unlike JRFL group, in 4-2.J41 group of animals, CD4-binding site-directed antibodies cannot be detected. Additionally, we have demonstrated that the quality of antibody response in combination group is guided by JRFL Env-based immunogen suggesting that the selection and the quality of Envs in multicade candidate vaccine are important factors to elicit desirable response.

Title & Authors Journal Publication Date

Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens

Martin G, Russell RA, Mundsperger P, Harris S, Jovanoska L, Farache Trajano L, Schiffner T, Fabian K, Tolazzi M, Scarlatti G, McFarlane L, Cheeseman H, Aldon Y, Breemen M, Sliepen K, Katinger D, Kunert R, Sanders RW, Shattock R, Ward AB, Sattentau QJ

Now Published: 10.1038/s41541-023-00696-w
bioRxiv July 26, 2021

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 W, Yong P, Torres J, 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, Crotty S, Irvine DJ, Ward AB, Wilson IA, Burton DR, Andrabi R

Now Published: 10.1126/scitranslmed.abl9605
bioRxiv July 5, 2021

Structural Insights into the Venus flytrap Mechanosensitive Ion Channel Flycatcher1

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

Now Published: 10.1038/s41467-022-28511-5
bioRxiv June 30, 2021

Changes at V2 apex of HIV-1 Clade C trimer enhance elicitation of autologous neutralizing and broad V1V2-scaffold antibodies

Sahoo A, Hodge EA, LaBranche C, Turner Styles T, Shen X, Cheedarla N, Shiferaw A, Ozorowski G, Lee WH, Ward AB, Tomaras GD, Montefiori DC, Irvine DJ, Lee KK, Rao Amara R

Now Published: 10.1016/j.celrep.2022.110436
bioRxiv June 15, 2021

Protective pan-ebolavirus combination therapy by two multifunctional human antibodies

Gilchuk P, Murin CD, Cross RW, Ilinykh PA, Huang K, Kuzmina N, Borisevich V, Agans KN, Geisbert JB, Carnahan RH, Nargi RS, Sutton RE, Suryadevara N, Zost SJ, Bombardi RG, Bukreyev A, Geisbert TW, Ward AB, Crowe JE

Now Published: 10.1016/j.cell.2021.09.035
bioRxiv May 2, 2021

From Structure to Sequence: Identification of polyclonal antibody families 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

Now Published: 10.1126/sciadv.abk2039
bioRxiv April 13, 2021

Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

Amanat F, Strohmeier S, Lee WH, Bangaru S, Ward AB, Coughlan L, Krammer F

Now Published: 10.1128/mbio.01002-21
bioRxiv April 5, 2021

One dose of COVID-19 nanoparticle vaccine REVC-128 provides protection against SARS-CoV-2 challenge at two weeks post immunization

Gu M, Torres JL, Greenhouse J, Wallace S, Chiang CI, Jackson AM, Porto M, Kar S, Li Y, Ward AB, Wang Y

Now Published: 10.1080/22221751.2021.1994354
bioRxiv April 2, 2021

Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

Cho H, Kay Gonzales-Wartz K, Huang D, Yuan M, Peterson M, Liang J, Beutler N, Torres JL, Cong Y, Postnikova E, Bangaru S, Adrienna Talana C, Shi W, Sung Yang E, Zhang Y, Leung K, Wang L, Peng L, Skinner J, Li S, Wu NC, Liu H, Dacon C, Moyer T, Cohen M, Zhao M, Lee FE, Weinberg RS, Douagi I, Gross R, Schmaljohn C, Pegu A, Mascola JR, Holbrook M, Nemazee D, Rogers TF, Ward AB, Wilson IA, Crompton PD, Tan J

Now Published: 10.1101/2021.04.01.437942
bioRxiv April 1, 2021