Title & Authors Journal Publication Date

Fusion of the molecular adjuvant C3d to cleavage-independent native-like HIV-1 Env trimers improves the elicited antibody response

Bale S, Yang L, Alirezaei M, Wilson R, Ota T, Doyle ED, Cottrell CA, Guenaga J, Tran K, Li W, Stamatatos L, Nemazee D, Ward AB, Wyatt RT.
Frontiers in Immunology June 6, 2023

An effective HIV vaccine likely requires the elicitation of neutralizing antibodies (NAbs) against multiple HIV-1 clades. The recently developed cleavage-independent native flexibly linked (NFL) envelope (Env) trimers exhibit well-ordered conformation and elicit autologous tier 2 NAbs in multiple animal models. Here, we investigated whether the fusion of molecular adjuvant C3d to the Env trimers can improve B- cell germinal center (GC) formation and antibody responses. To generate Env-C3d trimers, we performed a glycine-serine- based (G4S) flexible peptide linker screening and identified a linker range that allowed native folding. A 30–60- amino- acid- long linker facilitates Env-to-C3d association and achieves the secretion of well-ordered trimers and the structural integrity and functional integrity of Env and C3d. The fusion of C3d did not dramatically affect the antigenicity of the Env trimers and enhanced the ability of the Env trimers to engage and activate B cells in vitro. In mice, the fusion of C3d enhanced germinal center formation, the magnitude of Env-specific binding antibodies, and the avidity of the antibodies in the presence of an adjuvant. The Sigma Adjuvant System (SAS) did not affect the trimer integrity in vitro but contributed to altered immunogenicity in vivo, resulting in increased tier 1 neutralization, likely by increased exposure of variable region 3 (V3). Taken together, the results indicate that the fusion of the molecular adjuvant, C3d, to the Env trimers improves antibody responses and could be useful for Env-based vaccines against HIV.

Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

Kong R, Xu K, Zhou T, Acharya P, Lemmin T, Liu K, Ozorowski G, Soto C, Taft JD, Bailer RT, Cale EM, Chen L, Choi CW, Chuang GY, Doria-Rose NA, Druz A, Georgiev IS, Gorman J, Huang J, Joyce MG, Louder MK, Ma X, McKee K, O'Dell S, Pancera M, Yang Y, Blanchard SC, Mothes W, Burton DR, Koff WC, Connors M, Ward AB, Kwong PD, Mascola JR
Science May 31, 2023

The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.

Conformational Plasticity in the HIV-1 Fusion Peptide Facilitates Recognition by Broadly Neutralizing Antibodies

Yuan M, Cottrell CA, Ozorowski G, van Gils MJ, Kumar S, Wu NC, Sarkar A, Torres JL, de Val N, Copps J, Moore JP, Sanders RW, Ward AB, Wilson IA.
Cell Host & Microbe May 27, 2023

The fusion peptide (FP) of HIV-1 envelope glycoprotein (Env) is essential for mediating viral entry. Detection of broadly neutralizing antibodies (bnAbs) that interact with the FP has revealed it as a site of vulnerability. We delineate X-ray and cryo-electron microscopy (cryo-EM) structures of bnAb ACS202, from an HIV-infected elite neutralizer, with an FP and with a soluble Env trimer (AMC011 SOSIP.v4.2) derived from the same patient. We show that ACS202 CDRH3 forms a “β strand” interaction with the exposed hydrophobic FP and recognizes a continuous region of gp120, including a conserved N-linked glycan at N88. A cryo-EM structure of another previously identified bnAb VRC34.01 with AMC011 SOSIP.v4.2 shows that it also penetrates through glycans to target the FP. We further demonstrate that the FP can twist and present different conformations for recognition by bnAbs, which enables approach to Env from diverse angles. The variable recognition of FP by bnAbs thus provides insights for vaccine design.

Structural conservation of Lassa virus glycoproteins and recognition by neutralizing antibodies

Perrett HR, Brouwer PJM, Hurtado J, Newby ML, Liu L, Müller-Kräuter H, Müller Aguirre S, Burger JA, Bouhuijs JH, Gibson G, Messmer T, Schieffelin JS, Antanasijevic A, Boons GJ, Strecker T, Crispin M, Sanders RW, Briney B, Ward AB.
Cell Reports May 18, 2023

Lassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences among phylogenetically distinct LASV lineages. Despite the sequence diversity of the GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VII, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of the GPC in complex with GP1-A-specific antibodies suggest their neutralization mechanisms. Finally, we present the isolation and characterization of a trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines.

Structural basis of epitope selectivity and potent protection from malaria by PfCSP antibody L9

Martin GM, Fernández-Quintero ML, Lee WH, Pholcharee T, Eshun-Wilson L, Liedl KR, Pancera M, Seder RA, Wilson IA, Ward AB.
Nature Communications May 17, 2023

A primary objective in malaria vaccine design is the generation of high-quality antibody responses against the circumsporozoite protein of the malaria parasite, Plasmodium falciparum (PfCSP). To enable rational antigen design, we solved a cryo-EM structure of the highly potent anti-PfCSP antibody L9 in complex with recombinant PfCSP. We found that L9 Fab binds multivalently to the minor (NPNV) repeat domain, which is stabilized by a unique set of affinity-matured homotypic, antibody-antibody contacts. Molecular dynamics simulations revealed a critical role of the L9 light chain in integrity of the homotypic interface, which likely impacts PfCSP affinity and protective efficacy. These findings reveal the molecular mechanism of the unique NPNV selectivity of L9 and emphasize the importance of anti-homotypic affinity maturation in protective immunity against P. falciparum. The cryo-EM structure of the highly potent malaria antibody L9 reveals a key role of light-chain derived homotypic interactions in antigen binding and parasite inhibition, enabling antibody engineering and next-generation malaria vaccine design.

Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine

Dzimianski JV, Han J, Sautto GA, O'Rourke SM, Cruz JM, Pierce SR, Ecker JW, Carlock MA, Nagashima KA, Mousa JJ, Ross TM, Ward AB, DuBois RM.
Communications Biology April 25, 2023

Influenza virus poses an ongoing human health threat with pandemic potential. Due to mutations in circulating strains, formulating effective vaccines remains a challenge. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) proteins is a promising vaccine strategy to protect against a wide range of current and future influenza viruses. Though effective in preclinical studies, the mechanistic basis driving the broad reactivity of COBRA proteins remains to be elucidated. Here, we report the crystal structure of the COBRA HA termed P1 and identify antigenic and glycosylation properties that contribute to its immunogenicity. We further report the cryo-EM structure of the P1-elicited broadly neutralizing antibody 1F8 bound to COBRA P1, revealing 1F8 to recognize an atypical receptor binding site epitope via an unexpected mode of binding. Structural studies of a computationally optimized broadly reactive antigen hemagglutinin in complex with a broadly neutralizing antibody reveal its immunogenic properties and provide insights into flu vaccine design.

Bispecific antibodies targeting distinct regions of the spike protein potently neutralize SARS-CoV-2 variants of concern

Cho H, Gonzales-Wartz KK, Huang D, Yuan M, Peterson M, Liang J, Beutler N, Torres JL, Cong Y, Postnikova E, Bangaru S, Talana CA, Shi W, Yang ES, 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.
Science Translational Medicine April 21, 2023

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of existing vaccines and therapeutic antibodies and underscores the need for additional antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells collected from patients with coronavirus disease 2019. The three most potent antibodies targeted distinct regions of the receptor-binding domain (RBD), and all three neutralized the SARS-CoV-2 Alpha and Beta variants. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the angiotensin converting enzyme 2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five bispecific antibodies that inhibit SARS-CoV-2 infection at concentrations of less than 1 ng/mL. Through a distinct mode of action, three bispecific antibodies cross-linked adjacent spike proteins using dual N-terminal domain-RBD specificities. One bispecific antibody was greater than 100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, two bispecific antibodies in our panel comparably neutralized the Alpha, Beta, Gamma and Delta variants and wild-type virus. Furthermore, a bispecific antibody that neutralized the Beta variant protected hamsters against SARS-CoV-2 expressing the E484K mutation. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

Single-component multilayered self-assembling protein nanoparticles presenting glycan-trimmed uncleaved prefusion optimized envelope trimmers as HIV-1 vaccine candidates

Zhang YN, Paynter J, Antanasijevic A, Allen JD, Eldad M, Lee YZ, Copps J, Newby ML, He L, Chavez D, Frost P, Goodroe A, Dutton J, Lanford R, Chen C, Wilson IA, Crispin M, Ward AB, Zhu J.
Nature Communications April 8, 2023

Uncleaved prefusion-optimized (UFO) design can stabilize diverse HIV-1 envelope glycoproteins (Envs). Single-component, self-assembling protein nanoparticles (1c-SApNP) can display 8 or 20 native-like Env trimers as vaccine candidates. We characterize the biophysical, structural, and antigenic properties of 1c-SApNPs that present the BG505 UFO trimer with wildtype and modified glycans. For 1c-SApNPs, glycan trimming improves recognition of the CD4 binding site without affecting broadly neutralizing antibodies (bNAbs) to major glycan epitopes. In mice, rabbits, and nonhuman primates, glycan trimming increases the frequency of vaccine responders (FVR) and steers antibody responses away from immunodominant glycan holes and glycan patches. The mechanism of vaccine-induced immunity is examined in mice. Compared with the UFO trimer, the multilayered E2p and I3-01v9 1c-SApNPs show 420 times longer retention in lymph node follicles, 20-32 times greater presentation on follicular dendritic cell dendrites, and up-to-4 times stronger germinal center reactions. These findings can inform future HIV-1 vaccine development. Here the authors present an HIV-1 vaccine strategy that combines Env stabilization, nanoparticle display, and glycan trimming, which improves neutralizing antibody responses, frequency of vaccine responders, and germinal center reactions in animal models.

Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies

Pritchard LK, Spencer DI, Royle L, Bonomelli C, Seabright GE, Behrens AJ, Kulp DW, Menis S, Krumm SA, Dunlop DC, Crispin DJ, Bowden TA, Scanlan CN, Ward AB, Schief WR, Doores KJ, Crispin M
Nature communications April 3, 2023

The envelope spike of HIV-1 employs a ‘glycan shield’ to protect itself from antibody-mediated neutralization. Paradoxically, however, potent broadly neutralizing antibodies (bnAbs) have been isolated which target this shield. The unusually high glycan density on the gp120 subunit limits processing during biosynthesis, leaving a region of under-processed oligomannose-type structures which is a primary target of these bnAbs. Here we investigate the contribution of individual glycosylation sites to formation of this so-called intrinsic mannose patch. Deletion of individual sites has a limited effect on the overall size of the intrinsic mannose patch but leads to changes in the processing of neighboring glycans. These structural changes are largely tolerated by a panel of glycan-dependent bnAbs targeting these regions, indicating a degree of plasticity in their recognition. These results support the intrinsic mannose patch as a stable target for vaccine design.

Respiratory viruses: New frontiers—a Keystone Symposia report

Cable J, Sun J, Cheon IS, Vaughan AE, Castro IA, Stein SR, López CB, Gostic KM, Openshaw PJM, Ellebedy AH, Wack A, Hutchinson E, Thomas MM, Langlois RA, Lingwood D, Baker SF, Folkins M, Foxman EF, Ward AB, Schwemmle M, Russell AB, Chiu C, Ganti K, Subbarao K, Sheahan TP, Penaloza-MacMaster P, Eddens T.
Annals of the New York Academy of Sciences April 1, 2023

Respiratory viruses are a common cause of morbidity and mortality around the world. Viruses like influenza, RSV, and most recently SARS‐CoV‐2 can rapidly spread through a population, causing acute infection and, in vulnerable populations, severe or chronic disease. Developing effective treatment and prevention strategies often becomes a race against ever‐evolving viruses that develop resistance, leaving therapy efficacy either short‐lived or relevant for specific viral strains. On June 29 to July 2, 2022, researchers met for the Keystone symposium “Respiratory Viruses: New Frontiers.” Researchers presented new insights into viral biology and virus–host interactions to understand the mechanisms of disease and identify novel treatment and prevention approaches that are effective, durable, and broad. Respiratory viruses are a common cause of morbidity and mortality around the world. Viruses like influenza, RSV, and most recently SARS‐CoV‐2 can rapidly spread through a population, causing acute infection and, in vulnerable populations, severe or chronic disease. On June 29 to July 2, 2022, researchers met for the Keystone symposium “Respiratory Viruses: New Frontiers”. The meeting was held jointly with the symposium “Viral Immunity: Basic Mechanisms and Therapeutic Applications.” Researchers presented new insights into viral biology and virus‐host interactions to understand the mechanisms of disease and identify novel treatment and prevention approaches that are effective, durable, and broad.

In vitro evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity

Wu NC, Grande G, Turner HL, Ward AB, Xie J, Lerner RA, Wilson IA
Nature Communications March 8, 2023

The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs. The C05 variants evolve up to 20-fold higher affinity but increase specificity to each HA subtype used in the selection. Structural analysis reveals that the fine specificity is strongly influenced by a highly conserved substitution that regulates receptor binding in different subtypes. Overall, this study suggests that subtle natural variations in the HA RBS between subtypes and species may differentially influence the evolution of high-affinity bnAbs. Broadly neutralizing antibodies (bnAbs) against influenza hemagglutinin (HA) have yielded insights for antiviral development. Here, the authors employ saturated mutagenesis of the paratope region of a bnAb combined with yeast display screening using H1 and H3 HAs, and find that a tradeoff exists between Ab affinity and breadth that influenced by disparate modes of receptor binding.

Site of vulnerability on SARS-CoV-2 spike induces broadly protective antibody to antigenically distinct Omicron subvariants

Changrob S, Halfmann PJ, Liu H, Torres JL, McGrath JJC, Ozorowski G, Li L, Wilbanks GD, Kuroda M, Maemura T, Huang M, Zheng NY, Turner HL, Erickson SA, Fu Y, Yasuhara A, Singh G, Monahan B, Mauldin J, Srivastava K, Simon V, Krammer F, Sather DN, Ward AB, Wilson IA, Kawaoka Y, Wilson PC.
Journal of Clinical Investigation March 2, 2023

The rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identified S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) that was derived from an individual previously infected with wildtype SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrated broad cross-neutralization of all dominant variants including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1/XBB). Furthermore, S728-1157 protected hamsters against in vivo challenges with wildtype, Delta, and BA.1 viruses. Structural analysis showed that this antibody targets a class 1/RBS-A epitope in the receptor binding domain (RBD) via multiple hydrophobic and polar interactions with its heavy chain complementarity determining region region 3 (CDR-H3), in addition to common motifs in CDR-H1/CDR-H2 of class 1/RBS-A antibodies. Importantly, this epitope was more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared to diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential, and may inform target-driven vaccine design against future SARS-CoV-2 variants.

Co-evolution of HIV Envelope and Apex-Targeting Neutralizing Antibody Lineage Provides Benchmarks for Vaccine Design

Rantalainen K, Berndsen ZT, Murrell S, Cao L, Omorodion O, Torres JL, Wu M, Umotoy J, Copps J, Poignard P, Landais E, Paulson JC, Wilson IA, Ward AB
Cell Reports Dec. 22, 2022

Broadly neutralizing antibodies (bnAbs) targeting the HIV envelope glycoprotein (Env) typically take years to develop. Longitudinal analyses of both neutralizing antibody lineages and viruses at serial time points during infection provide a basis for understanding the co-evolutionary contest between HIV and the humoral immune system. Here, we describe the structural characterization of an apex-targeting antibody lineage and autologous clade A viral Env from a donor in the Protocol C cohort. Comparison of Ab-Env complexes at early and late time points reveals that, within the antibody lineage, the CDRH3 loop rigidifies, the bnAb angle of approach steepens, and surface charges are mutated to accommodate glycan changes. Additionally, we observed differences in site-specific glycosylation between soluble and full-length Env constructs, which may be important for tuning optimal immunogenicity in soluble Env trimers. These studies therefore provide important guideposts for design of immunogens that prime and mature nAb responses to the Env V2-apex.

Antibodies targeting the neuraminidase active site inhibit influenza H3N2 viruses with an S245N glycosylation site

Stadlbauer D, McMahon M, Turner HL, Zhu X, Wan H, Carreño JM, O'Dell G, Strohmeier S, Khalil Z, Luksza M, van Bakel H, Simon V, Ellebedy AH, Wilson IA, Ward AB, Krammer F.
Nature Communications Dec. 21, 2022

Contemporary influenza A H3N2 viruses circulating since 2016 have acquired a glycosylation site in the neuraminidase in close proximity to the enzymatic active site. Here, we investigate if this S245N glycosylation site, as a result of antigenic evolution, can impact binding and function of human monoclonal antibodies that target the conserved active site. While we find that a reduction in the inhibitory ability of neuraminidase active site binders is measurable, this class of broadly reactive monoclonal antibodies maintains protective efficacy in vivo. Antibodies that broadly inhibit influenza virus neuraminidase by binding to its active site could be therapeutic candidates, but circulating viruses have acquired a glycosylation site in that region. Here, the authors show that, while the S245N glycosylation site affects binding of tested monoclonal antibodies, protective activity in a mouse model is maintained.

Title & Authors Journal Publication Date

Structure of mechanically activated ion channel OSCA2.3 reveals mobile elements in the transmembrane domain

Jojoa-Cruz S, Burendei B, Lee WH, Ward AB

Now Published: 10.1016/j.str.2023.11.009
bioRxiv June 15, 2023

Evolving spike-protein N -glycosylation in SARS-CoV-2 variants

Baboo S, Diedrich JK, Torres JL, Copps J, Singh B, Garrett PT, Ward AB, Paulson JC, Yates JR

bioRxiv May 8, 2023

Ab initio prediction of specific phospholipid complexes and membrane association of HIV-1 MPER antibodies by multi-scale simulations

Maillie C, Golden J, Wilson IA, Ward AB, Mravic M

bioRxiv May 4, 2023

Broadly neutralizing antibodies targeting a conserved silent face of spike RBD resist extreme SARS-CoV-2 antigenic drift

Song G, Yuan M, Liu H, Capozzola T, Lin RN, Torres JL, He WT, Musharrafieh R, Dueker K, Zhou P, Callaghan S, Mishra N, Yong P, Anzanello F, Avillion G, Lina Vo A, Li X, Makhdoomi M, Feng Z, Zhu X, Peng L, Nemazee D, Safonova Y, Briney B, Ward AB, Burton DR, Wilson IA, Andrabi R

bioRxiv April 26, 2023

Deep repertoire mining uncovers ultra-broad coronavirus neutralizing antibodies targeting multiple spike epitopes

Hurtado J, Rogers TF, Jaffe DB, Adams BA, Bangaru S, Garcia E, Capozzola T, Messmer T, Sharma P, Song G, Beutler N, He W, Dueker K, Musharrafieh R, Stubbington MJ, Burton DR, Andrabi R, Ward AB, McDonnell WJ, Briney B

bioRxiv March 28, 2023

Conformational antigenic heterogeneity as a cause of the persistent fraction in HIV-1 neutralization

Colin P, Ringe RP, Yasmeen A, Ozorowski G, Ketas TJ, Lee WH, Ward AB, Moore JP, Klasse PJ

Research Square Feb. 21, 2023