Publications
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Title & Authors Journal Publication Date

LipIDens: simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins


Ansell TB, Song W, Coupland CE, Carrique L, Corey RA, Duncan AL, Cassidy CK, Geurts MMG, Rasmussen T, Ward AB, Siebold C, Stansfeld PJ, Sansom MSP.
Nature Communications Nov. 27, 2023

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities. Interpretation of lipid-like densities in cryo-EM structures of membrane proteins is challenging. Here authors present LipIDens, enabling molecular dynamics analysis of protein-lipid interactions.

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates


Phung I, Rodrigues KA, Marina-Zárate E, Maiorino L, Pahar B, Lee WH, Melo M, Kaur A, Allers C, Fahlberg M, Grasperge BF, Dufour JP, Schiro F, Aye PP, Lopez PG, Torres JL, Ozorowski G, Eskandarzadeh S, Kubitz M, Georgeson E, Groschel B, Nedellec R, Bick M, Kaczmarek Michaels K, Gao H, Shen X, Carnathan DG, Silvestri G, Montefiori DC, Ward AB, Hangartner L, Veazey RS, Burton DR, Schief WR, Irvine DJ, Crotty S.
Nature Communications Nov. 4, 2023

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation. Protein antigens, such as HIV envelope protein, require adjuvants for high immunogenicity. Here the authors show that a combined adjuvant approach with slow antigen delivery and potent ISCOMs adjuvant primes robust germinal center activity and humoral immunity in non-human primates. pSer-modified antigen shifts immunodominance to allow subdominant epitope-targeting of rare B cells.

Glycine Substitution at Helix-to-Coil Transitions Facilitates the Structural Determination of a Stabilized Subtype C HIV Envelope Glycoprotein


Guenaga J, Garces F, de Val N, Stanfield RL, Dubrovskaya V, Higgins B, Carrette B, Ward AB, Wilson IA, Wyatt RT
Immunity Oct. 30, 2023

Advances in HIV-1 envelope glycoprotein (Env) design generate native-like trimers and high-resolution clade A, B, and G structures and elicit neutralizing antibodies. However, a high-resolution clade C structure is critical, as this subtype accounts for the majority of HIV infections worldwide, but well-ordered clade C Env trimers are more challenging to produce due to their instability. Based on targeted glycine substitutions in the Env fusion machinery, we defined a general approach that disfavors helical transitions leading to post-fusion conformations, thereby favoring the pre-fusion state. We generated a stabilized, soluble clade C Env (16055 NFL) and determined its crystal structure at 3.9 Å. Its overall conformation is similar to SOSIP.664 and native Env trimers but includes a covalent linker between gp120 and gp41, an engineered 201-433 disulfide bond, and density corresponding to 22 N-glycans. Env-structure-guided design strategies resulted in multiple homogeneous cross-clade immunogens with the potential to advance HIV vaccine development.

Broad SARS-CoV-2 neutralization by monoclonal and bispecific antibodies derived from a Gamma-infected individual


Guerra D, Beaumont T, Radić L, Kerster G, van der Straten K, Yuan M, Torres JL, Lee WH, Liu H, Poniman M, Bontjer I, Burger JA, Claireaux M, Caniels TG, Snitselaar JL, Bijl TPL, Kruijer S, Ozorowski G, Gideonse D, Sliepen K, Ward AB, Eggink D, de Bree GJ, Wilson IA, Sanders RW, van Gils MJ
iScience Oct. 20, 2023

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has remained a medical threat due to the evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants. A stabilized spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants, with COVA309-35 being the most potent against the autologous virus, as well as Omicron BA.1 and BA.2, and COVA309-22 having binding and neutralization activity against Omicron BA.4/5, BQ.1.1, and XBB.1. When combining the COVA309 mAbs as cocktails or bispecific antibodies, the breadth and potency were improved. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

Structure and Dynamics Guiding Design of Antibody Therapeutics and Vaccines


Fernández-Quintero ML, Pomarici ND, Fischer AM, Hoerschinger VJ, Kroell KB, Riccabona JR, Kamenik AS, Loeffler JR, Ferguson JA, Perrett HR, Liedl KR, Han J, Ward AB.
Antibodies Oct. 18, 2023

Antibodies and other new antibody-like formats have emerged as one of the most rapidly growing classes of biotherapeutic proteins. Understanding the structural features that drive antibody function and, consequently, their molecular recognition is critical for engineering antibodies. Here, we present the structural architecture of conventional IgG antibodies alongside other formats. We emphasize the importance of considering antibodies as conformational ensembles in solution instead of focusing on single-static structures because their functions and properties are strongly governed by their dynamic nature. Thus, in this review, we provide an overview of the unique structural and dynamic characteristics of antibodies with respect to their antigen recognition, biophysical properties, and effector functions. We highlight the numerous technical advances in antibody structure prediction and design, enabled by the vast number of experimentally determined high-quality structures recorded with cryo-EM, NMR, and X-ray crystallography. Lastly, we assess antibody and vaccine design strategies in the context of structure and dynamics.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2


Huang R, Warner Jenkins G, Kim Y, Stanfield RL, Singh A, Martinez-Yamout M, Kroon GJ, Torres JL, Jackson AM, Kelley A, Shaabani N, Zeng B, Bacica M, Chen W, Warner C, Radoicic J, Joh J, Dinali Perera K, Sang H, Kim T, Yao J, Zhao F, Sok D, Burton DR, Allen J, Harriman W, Mwangi W, Chung D, Teijaro JR, Ward AB, Dyson HJ, Wright PE, Wilson IA, Chang KO, McGregor D, Smider VV.
Proceedings of the National Academy of Sciences Sept. 26, 2023

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This “knob” domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

A Lassa virus mRNA vaccine confers protection but does not require neutralizing antibody in a guinea pig model of infection


Ronk AJ, Lloyd NM, Zhang M, Atyeo C, Perrett HR, Mire CE, Hastie KM, Sanders RW, Brouwer PJM, Saphire EO, Ward AB, Ksiazek TG, Alvarez Moreno JC, Thaker HM, Alter G, Himansu S, Carfi A, Bukreyev A
Nature Communications Sept. 12, 2023

Lassa virus is a member of the Arenaviridae family, which causes human infections ranging from asymptomatic to severe hemorrhagic disease with a high case fatality rate. We have designed and generated lipid nanoparticle encapsulated, modified mRNA vaccines that encode for the wild-type Lassa virus strain Josiah glycoprotein complex or the prefusion stabilized conformation of the Lassa virus glycoprotein complex. Hartley guinea pigs were vaccinated with two 10 µg doses, 28 days apart, of either construct. Vaccination induced strong binding antibody responses, specific to the prefusion conformation of glycoprotein complex, which were significantly higher in the prefusion stabilized glycoprotein complex construct group and displayed strong Fc-mediated effects. However, Lassa virus-neutralizing antibody activity was detected in some but not all animals. Following the challenge with a lethal dose of the Lassa virus, all vaccinated animals were protected from death and severe disease. Although the definitive mechanism of protection is still unknown, and assessment of the cell-mediated immune response was not investigated in this study, these data demonstrate the promise of mRNA as a vaccine platform against the Lassa virus and that protection against Lassa virus can be achieved in the absence of virus-neutralizing antibodies. Lassa virus infections in humans can result in severe disease, including hemorrhagic fever. Here the authors describe an mRNA-based Lassa virus vaccine that shows protection without requirement for neutralizing antibody in a guinea pig model of infection.

Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection


Martin GM, Torres JL, Pholcharee T, Oyen D, Flores-Garcia Y, Gibson G, Moskovitz R, Beutler N, Jung DD, Copps J, Lee WH, Gonzalez-Paez G, Emerling D, MacGill RS, Locke E, King CR, Zavala F, Wilson IA, Ward AB.
Nature Communications July 28, 2023

The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3–33 antibodies and highlight key features underlying the potent protection of this antibody family. Here, the authors use cryo-EM to solve the structures of seven potent human antibodies, and demonstrate in vivo protection in a liver burden assay, using chimeric Plasmodium berghei sporozoites expressing Plasmodium falciparum circumsporozoite protein.

Protocol for analyzing antibody responses to glycoprotein antigens using electron-microscopy-based polyclonal epitope mapping


Turner HL, Jackson AM, Richey ST, Sewall LM, Antanasijevic A, Hangartner L, Ward AB.
STAR Protocols July 28, 2023

Electron microscopy-based polyclonal epitope mapping (EMPEM) can delineate epitope specificities of serum antibodies to a given antigen following vaccination or infection. Here, we present a protocol for the EMPEM method for rapid high-throughput assessment of antibody responses to glycoprotein antigens in vaccination and infection studies. We describe steps for antibody isolation and digestion, antigen complex and purification, and electron microscope imaging. We then detail procedures for processing and analysis of EMPEM data. For complete details on the use and execution of this protocol, please refer to Bianchi et al. (2018). 1

Human antibodies that neutralize respiratory droplet transmissible H5N1 influenza viruses


Thornburg NJ, Nannemann DP, Blum DL, Belser JA, Tumpey TM, Deshpande S, Fritz GA, Sapparapu G, Krause JC, Lee JH, Ward AB, Lee DE, Li S, Winarski KL, Spiller BW, Meiler J, Crowe JE Jr
Journal of Clinical Investigation July 25, 2023

Recent studies described the experimental adaptation of influenza H5 HAs that confers respiratory droplet transmission (rdt) to influenza virus in ferrets. Acquisition of the ability to transmit via aerosol may lead to the development of a highly pathogenic pandemic H5 virus. Vaccines are predicted to play an important role in H5N1 control should the virus become readily transmissible between humans. We obtained PBMCs from patients who received an A/Vietnam/1203/2004 H5N1 subunit vaccine. Human hybridomas were then generated and characterized. We identified antibodies that bound the HA head domain and recognized both WT and rdt H5 HAs. We used a combination of structural techniques to define a mechanism of antibody recognition of an H5 HA receptor–binding site that neutralized H5N1 influenza viruses and pseudoviruses carrying the HA rdt variants that have mutations near the receptor-binding site. Incorporation or retention of this critical antigenic site should be considered in the design of novel H5 HA immunogens to protect against mammalian-adapted H5N1 mutants.

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


Martin GM, Russell RA, Mundsperger P, Harris S, Jovanoska L, Trajano LF, Schiffner T, Fabian K, Tolazzi M, Scarlatti G, McFarlane L, Cheeseman H, Aldon Y, Schermer EE, Breemen M, Sliepen K, Katinger D, Kunert R, Sanders RW, Shattock R, Ward AB, Sattentau QJ.
npj Vaccines July 13, 2023

Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4–0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design.

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Title & Authors Journal Publication Date

Germline-targeting SOSIP trimer immunization elicits precursor CD4 binding-site targeting broadly neutralizing antibodies in infant macaques


Nelson AN, Shen X, Vekatayogi S, Zhang S, Ozorowski G, Dennis M, Sewall LM, Milligan E, Davis D, Cross KA, Chen Y, van Schooten J, Eudailey J, Isaac J, Memon S, Weinbaum C, Stanfield-Oakley S, Byrd A, Chutkan S, Berendam S, Cronin K, Yasmeen A, Alam SM, LaBranche CC, Rogers K, Shirreff L, Cupo A, Derking R, Villinger F, Klasse PJ, Ferrari G, Williams WB, Hudgens MG, Ward AB, Montefiori DC, Van Rompay KK, Wiehe K, Moore JP, Sanders RW, De Paris K, Permar SR

bioRxiv Nov. 7, 2023

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding


Tomris I, van der Woude R, de Paiva Droes Rocha R, Torrents de la Peña A, Ward AB, de Vries RP

Now Published: 10.1002/pro.4974
bioRxiv Oct. 23, 2023

Local structural flexibility drives oligomorphism in computationally designed protein assemblies


Khmelinskaia A, Bethel NP, Fatehi F, Antanasijevic A, Borst AJ, Lai SH, Yang Wang JJ, Basu Mallik B, Miranda MC, Watkins AM, Ogohara C, Caldwell S, Wu M, Heck AJ, Veesler D, Ward AB, Baker D, Twarock R, King NP

bioRxiv Oct. 18, 2023

Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli


Jojoa-Cruz S, Dubin AE, Lee WH, Ward A

Now Published: 10.7554/elife.93147
bioRxiv Oct. 3, 2023

Germline-targeting chimpanzee SIV Envelopes induce V2-apex broadly neutralizing-like B cell precursors in a rhesus macaque infection model


Musharrafieh R, Safonova Y, Song G, Roark RS, Lee FH, Zhang S, Hurtado J, Yong P, Wang S, Russell RM, Ding W, Li Y, Rando J, Murphy AI, Lindemuth E, Zhao C, Jesse Connell A, Lee WH, Mishra N, Avillion G, He W, Callaghan S, Dueker K, Vo AL, Li X, Capozzola T, Joyce C, Zhao F, Anzanello F, Liu W, Bibollet-Ruche F, Ramos A, Li H, Lewis MG, Ozorowski G, Landais E, Foley BT, Wagh K, Sok D, Briney B, Ward AB, Hahn BH, Burton DR, Shaw GM, Andrabi R

bioRxiv Sept. 21, 2023

Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines


del Moral-Sánchez I, Wee EG, Xian Y, Lee WH, Allen JD, Torrents de la Peña A, Fróes Rocha R, Ferguson J, León AN, Koekkoek S, Schermer EE, Burger JA, Kumar S, Zwolsman R, Brinkkemper M, Aartse A, Eggink D, Han J, Yuan M, Crispin M, Ozorowski G, Ward AB, Wilson IA, Hanke T, Sliepen K, Sanders RW

Now Published: 10.1038/s41541-024-00862-8
bioRxiv Aug. 27, 2023

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination


Yang YR, Han J, Perrett HR, Richey ST, Jackson AM, Rodriguez AJ, Gillespie RA, O’Connell S, Raab JE, Cominsky LY, Chopde A, Kanekiyo M, Houser KV, Chen GL, McDermott AB, Andrews SF, Ward AB

Now Published: 10.1016/j.celrep.2024.114171
bioRxiv Aug. 23, 2023

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


Ringe RP, Colin P, Ozorowski G, Allen JD, Yasmeen A, Seabright GE, Lee JH, Antanasijevic A, Rantalainen K, Ketas T, Moore JP, Ward AB, Crispin M, Klasse PJ

Now Published: 10.1371/journal.ppat.1011601
bioRxiv Aug. 8, 2023

Focusing antibody responses to the fusion peptide in rhesus macaques


Cottrell CA, Pratap PP, Cirelli KM, Carnathan DG, Enemuo CA, Antanasijevic A, Ozorowski G, Sewall LM, Gao H, Greene KM, Allen JD, Ngo JT, Choe Y, Nogal B, Silva M, Bhiman J, Pauthner M, Irvine DJ, Montefiori D, Crispin M, Burton DR, Silvestri G, Crotty S, Ward AB

bioRxiv June 26, 2023