Title & Authors Journal Publication Date

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.

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 Pathogens March 22, 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.

Field-Based Affinity Optimization of a Novel Azabicyclohexane Scaffold HIV-1 Entry Inhibitor

Meuser ME, Rashad AA, Ozorowski G, Dick A, Ward AB, Cocklin S
Molecules Feb. 23, 2021

Small-molecule HIV-1 entry inhibitors are an extremely attractive therapeutic modality. We have previously demonstrated that the entry inhibitor class can be optimized by using computational means to identify and extend the chemotypes available. Here we demonstrate unique and differential effects of previously published antiviral compounds on the gross structure of the HIV-1 Env complex, with an azabicyclohexane scaffolded inhibitor having a positive effect on glycoprotein thermostability. We demonstrate that modification of the methyltriazole-azaindole headgroup of these entry inhibitors directly effects the potency of the compounds, and substitution of the methyltriazole with an amine-oxadiazole increases the affinity of the compound 1000-fold over parental by improving the on-rate kinetic parameter. These findings support the continuing exploration of compounds that shift the conformational equilibrium of HIV-1 Env as a novel strategy to improve future inhibitor and vaccine design efforts.

Innate cell markers that predict anti-HIV neutralizing antibody titers in vaccinated macaques

Van Tilbeurgh M, Maisonnasse P, Palgen JL, Tolazzi M, Aldon Y, Dereuddre-Bosquet N, Cavarelli M, Beignon AS, Marcos-Lopez E, Gallouet AS, Gilson E, Ozorowski G, Ward AB, Bontjer I, McKay PF, Shattock RJ, Scarlatti G, Sanders RW, Le Grand R.
Cell Reports Medicine Feb. 23, 2021

Given the time and resources invested in clinical trials, innovative prediction methods are needed to decrease late-stage failure in vaccine development. We identify combinations of early innate responses that predict neutralizing antibody (nAb) responses induced in HIV-Env SOSIP immunized cynomolgus macaques using various routes of vaccine injection and adjuvants. We analyze blood myeloid cells before and 24 h after each immunization by mass cytometry using a three-step clustering, and we discriminate unique vaccine signatures based on HLA-DR, CD39, CD86, CD11b, CD45, CD64, CD14, CD32, CD11c, CD123, CD4, CD16, and CADM1 surface expression. Various combinations of these markers characterize cell families positively associated with nAb production, whereas CADM1-expressing cells are negatively associated (p < 0.05). Our results demonstrate that monitoring immune signatures during early vaccine development could assist in identifying biomarkers that predict vaccine immunogenicity.

Immunogenic Display of Purified Chemically Cross-Linked HIV-1 Spikes

Leaman DP, Lee JH, Ward AB, Zwick MB
Journal of Virology Feb. 9, 2021

ABSTRACT HIV-1 envelope glycoprotein (Env) spikes are prime vaccine candidates, at least in principle, but suffer from instability, molecular heterogeneity and a low copy number on virions. We anticipated that chemical cross-linking of HIV-1 would allow purification and molecular characterization of trimeric Env spikes, as well as high copy number immunization. Broadly neutralizing antibodies bound tightly to all major quaternary epitopes on cross-linked spikes. Covalent cross-linking of the trimer also stabilized broadly neutralizing epitopes, although surprisingly some individual epitopes were still somewhat sensitive to heat or reducing agent. Immunodepletion using non-neutralizing antibodies to gp120 and gp41 was an effective method for removing non-native-like Env. Cross-linked spikes, purified via an engineered C-terminal tag, were shown by negative stain EM to have well-ordered, trilobed structure. An immunization was performed comparing a boost with Env spikes on virions to spikes cross-linked and captured onto nanoparticles, each following a gp160 DNA prime. Although differences in neutralization did not reach statistical significance, cross-linked Env spikes elicited a more diverse and sporadically neutralizing antibody response against Tier 1b and 2 isolates when displayed on nanoparticles, despite attenuated binding titers to gp120 and V3 crown peptides. Our study demonstrates display of cross-linked trimeric Env spikes on nanoparticles, while showing a level of control over antigenicity, purity and density of virion-associated Env, which may have relevance for Env based vaccine strategies for HIV-1. IMPORTANCE The envelope spike (Env) is the target of HIV-1 neutralizing antibodies, which a successful vaccine will need to elicit. However, native Env on virions is innately labile, as well as heterogeneously and sparsely displayed. We therefore stabilized Env spikes using a chemical cross-linker and removed non-native Env by immunodepletion with non-neutralizing antibodies. Fixed native spikes were recognized by all classes of known broadly neutralizing antibodies but not by non-neutralizing antibodies and displayed on nanoparticles in high copy number. An immunization experiment in rabbits revealed that cross-linking Env reduced its overall immunogenicity; however, high-copy display on nanoparticles enabled boosting of antibodies that sporadically neutralized some relatively resistant HIV-1 isolates, albeit at a low titer. This study describes the purification of stable and antigenically correct Env spikes from virions that can be used as immunogens.

Title & Authors Journal Publication Date

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

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, Viswanadh Edara V, Floyd K, Vanderheiden A, Mascola JR, Doria-Rose N, Wang L, Sung Yang E, 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

Now Published:
bioRxiv March 23, 2021

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:
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. 16, 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 CCD, 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. 11, 2021