Title & Authors Journal Publication Date

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

Tomris I, van der Woude R, de Paiva Froes Rocha R, Torrents de la Peña A, Ward AB, de Vries RP.
Protein Sci . April 1, 2024

Enveloped viruses carry one or multiple proteins with receptor-binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor-binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here, we report a suite of distinct fluorescent fusions, both N- and C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained three or six fluorescent protein barrels and were applied directly to cells to assess receptor binding properties.

The Role of Force Fields and Water Models in Protein Folding and Unfolding Dynamics

Fischer ALM, Tichy A, Kokot J, Hoerschinger VJ, Wild RF, Riccabona JR, Loeffler JR, Waibl F, Quoika PK, Gschwandtner P, Forli S, Ward AB, Liedl KR, Zacharias M, Fernández-Quintero ML
Journal of Chemical Theory and Computation Feb. 19, 2024

Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes in atomistic detail, including folding and unfolding processes of proteins. However, the accuracy of the conformational ensembles derived from MD simulations inevitably relies on the quality of the underlying force field in combination with the respective water model. Here, we investigate protein folding, unfolding, and misfolding of fast-folding proteins by examining different force fields with their recommended water models, i.e., ff14SB with the TIP3P model and ff19SB with the OPC model. To this end, we generated long conventional MD simulations highlighting the perks and pitfalls of these setups. Using Markov state models, we defined kinetically independent conformational substates and emphasized their distinct characteristics, as well as their corresponding state probabilities. Surprisingly, we found substantial differences in thermodynamics and kinetics of protein folding, depending on the combination of the protein force field and water model, originating primarily from the different water models. These results emphasize the importance of carefully choosing the force field and the respective water model as they determine the accuracy of the observed dynamics of folding events. Thus, the findings support the hypothesis that the water model is at least equally important as the force field and hence needs to be considered in future studies investigating protein dynamics and folding in all areas of biophysics.

Flipping hemagglutinin on its head

Ferguson JA, Han J, Ward AB
Nature Chemical Biology Feb. 6, 2024

Antigen reorientation via oligoD, a label-free, alum-based technique, guides immunofocused, broadly reactive antibody responses.

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

Jojoa-Cruz S, Burendei B, Lee WH, Ward AB.
Structure Dec. 6, 2023

Members of the OSCA/TMEM63 family are mechanically activated ion channels and structures of some OSCA members have revealed the architecture of these channels and structural features that are potentially involved in mechanosensation. However, these structures are all in a similar state and information about the motion of different elements of the structure is limited, preventing a deeper understanding of how these channels work. Here, we used cryoelectron microscopy to determine high-resolution structures of Arabidopsis thaliana OSCA1.2 and OSCA2.3 in peptidiscs. The structure of OSCA1.2 matches previous structures of the same protein in different environments. Yet, in OSCA2.3, the TM6a-TM7 linker adopts a different conformation that constricts the pore on its cytoplasmic side. Furthermore, coevolutionary sequence analysis uncovered a conserved interaction between the TM6a-TM7 linker and the beam-like domain (BLD). Our results reveal conformational heterogeneity and differences in conserved interactions between the TMD and BLD among members of the OSCA family.

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.

Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2/MPER region

Flyak AI, Kuzmina N, Murin CD, Bryan C, Davidson E, Gilchuk P, Gulka CP, Ilinykh PA, Shen X, Huang K, Ramanathan P, Turner H, Fusco ML, Lampley R, Kose N, King H, Sapparapu G, Doranz BJ, Ksiazek TG, Wright DW, Saphire EO, Ward AB, Bukreyev A, Crowe JE Jr
Nature microbiology Oct. 20, 2023

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMappTM that has been shown to be effective in nonhuman primate models of infection1 and has been used under compassionate-treatment protocols in humans2. ZMappTM is a mixture of three chimerized murine monoclonal antibodies (mAbs)3–6 that target EBOV-specific epitopes on the surface glycoprotein (GP)7,8. However, ZMappTM mAbs do not neutralize other species from the Ebolavirus genus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here we describe three naturally-occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane proximal external region (MPER) of GP. The identification of a conserved neutralizing antigenic site in the GP suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2/MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

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.

Title & Authors Journal Publication Date

Anti-Immune Complex Antibodies are Elicited During Repeated Immunization with HIV Env Immunogens

Brown S, Antanasijevic A, Sewall LM, Montiel Garcia D, Brouwer PJM, Sanders RW, Ward AB

bioRxiv March 15, 2024

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire

Altman PX, Ozorowski G, Stanfield RL, Haakenson J, Appel M, Parren M, Lee WH, Sang H, Woehl J, Saye-Francisco K, Joyce C, Song G, Porter K, Landais E, Andrabi R, Wilson IA, Ward AB, Mwangi W, Smider VV, Burton DR, Sok D

bioRxiv Feb. 13, 2024

Broadly inhibitory antibodies against severe malaria virulence proteins

Reyes RA, Sundar Rajan Raghavan S, Hurlburt NK, Introini V, Hussain Kana I, Jensen RW, Martinez-Scholze E, Gestal-Mato M, Bancells Bau C, Lisa Fernández-Quintero M, Loeffler JR, Alexander Ferguson J, Lee WH, Michael Martin G, Theander TG, Ssewanyana I, Feeney ME, Greenhouse B, Bol S, Ward AB, Bernabeu M, Pancera M, Turner L, Bunnik EM, Lavstsen T

bioRxiv Jan. 25, 2024

Defining bottlenecks and opportunities for Lassa virus neutralization by structural profiling of vaccine-induced polyclonal antibody responses

Brouwer PJ, Perrett HR, Beaumont T, Nijhuis H, Kruijer S, Burger JA, Lee WH, Müller-Kraüter H, Sanders RW, Strecker T, van Gils MJ, Ward AB

bioRxiv Dec. 21, 2023

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

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