Publications
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Title & Authors Journal Publication Date

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.


Yang YR, Han J, Perrett HR, Richey ST, Rodriguez AJ, Jackson AM, Gillespie RA, O'Connell S, Raab JE, Cominsky LY, Chopde A, Kanekiyo M, Houser KV, Chen GL, McDermott AB, Andrews SF, Ward AB.
Cell Rep May 28, 2024

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase 1 clinical trial investigating a ferritin nanoparticle vaccine displaying H2 hemagglutinin (HA) in H2-naive and H2-exposed adults enabled us to perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited. We temporally map the epitopes targeted by serum antibodies after vaccine prime and boost, revealing that previous H2 exposure results in higher responses to the variable HA head domain. In contrast, initial responses in H2-naive participants are dominated by antibodies targeting conserved epitopes. We use cryoelectron microscopy and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the HA head, including the receptor-binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses post-vaccination.

Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.


Steichen JM, Phung I, Salcedo E, Ozorowski G, Willis JR, Baboo S, Liguori A, Cottrell CA, Torres JL, Madden PJ, Ma KM, Sutton HJ, Lee JH, Kalyuzhniy O, Allen JD, Rodriguez OL, Adachi Y, Mullen TM, Georgeson E, Kubitz M, Burns A, Barman S, Mopuri R, Metz A, Altheide TK, Diedrich JK, Saha S, Shields K, Schultze SE, Smith ML, Schiffner T, Burton DR, Watson CT, Bosinger SE, Crispin M, Yates JR 3rd, Paulson JC, Ward AB, Sok D, Crotty S, Schief WR.
Science May 17, 2024

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.


Xie Z, Lin YC, Steichen JM, Ozorowski G, Kratochvil S, Ray R, Torres JL, Liguori A, Kalyuzhniy O, Wang X, Warner JE, Weldon SR, Dale GA, Kirsch KH, Nair U, Baboo S, Georgeson E, Adachi Y, Kubitz M, Jackson AM, Richey ST, Volk RM, Lee JH, Diedrich JK, Prum T, Falcone S, Himansu S, Carfi A, Yates JR 3rd, Paulson JC, Sok D, Ward AB, Schief WR, Batista FD.
Science May 17, 2024

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses.


Ray R, Nait Mohamed FA, Maurer DP, Huang J, Alpay BA, Ronsard L, Xie Z, Han J, Fernandez-Quintero M, Phan QA, Ursin RL, Vu M, Kirsch KH, Prum T, Rosado VC, Bracamonte-Moreno T, Okonkwo V, Bals J, McCarthy C, Nair U, Kanekiyo M, Ward AB, Schmidt AG, Batista FD, Lingwood D.
Immunity May 14, 2024

Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.

Improving antibody language models with native pairing.


Burbach SM, Briney B.
Patterns (N Y) May 10, 2024

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


Jojoa-Cruz S, Dubin AE, Lee WH, Ward AB.
Elife April 9, 2024

The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension (Jojoa-Cruz et al., 2018). Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e. they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). Here, in an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization (Murthy et al., 2018). Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.

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.
NPJ Vaccines April 6, 2024

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

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 AM, 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.
J Chem Theory Comput March 12, 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.

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 Feb. 1, 2024

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.

mRNA lipid nanoparticles expressing cell-surface cleavage independent HIV Env trimers elicit autologous tier-2 neutralizing antibodies.


Guenaga J, Alirezaei M, Feng Y, Alameh MG, Lee WH, Baboo S, Cluff J, Wilson R, Bale S, Ozorowski G, Lin P, Tam Y, Diedrich JK, Yates JR 3rd, Paulson JC, Ward AB, Weissman D, Wyatt RT.
Front Immunol Jan. 1, 2024

The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed ‘native flexibly linked’ (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans.

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Title & Authors Journal Publication Date

HIV BG505 SOSIP.664 trimer with 3M-052-AF/alum induces human autologous tier-2 neutralizing antibodies.


Hahn WO, Parks KR, Shen M, Ozorowski G, Janes H, Ballweber-Fleming L, Woodward Davis AS, Duplessis C, Tomai M, Dey AK, Sagawa ZK, De Rosa SC, Seese A, Siddaramaiah LK, Stamatatos L, Lee WH, Sewall LM, Karlinsey D, Turner HL, Rubin V, Furth S, MacPhee K, Duff M, Corey L, Keefer MC, Edupuganti S, Frank I, Maenza J, Baden LR, Hyrien O, Sanders RW, Moore JP, Ward AB, Tomaras GD, Montefiori DC, Rouphael N, McElrath MJ.

medRxiv May 9, 2024

Broadening sarbecovirus neutralization with bispecific antibodies combining distinct conserved targets on the receptor binding domain


Guerra D, Radić L, Brinkkemper M, Poniman M, Maas L van der, Torres JL, Ward AB, Sliepen K, Schinkel J, Sanders RW, Gils MJ van, Beaumont T.

bioRxiv May 9, 2024

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin.


Berndsen ZT, Akhtar M, Thapa M, Vickers T, Schmitz A, Torres JL, Baboo S, Kumar P, Khatoom N, Sheikh A, Hamrick M, Diedrich JK, Martinez-Bartolome S, Garrett PT, Yates JR 3rd, Turner JS, Laird RM, Poly F, Porter CK, Copps J, Ellebedy AH, Ward AB, Fleckenstein JM.

bioRxiv May 8, 2024

Assessing AF2’s ability to predict structural ensembles of proteins


Riccabona JR, Spoendlin FC, Fischer ALM, Loeffler JR, Quoika PK, Jenkins TP, Ferguson JA, Smorodina E, Laustsen AH, Greiff V, Forli S, Ward AB, Deane CM, Fernández-Quintero ML

bioRxiv April 16, 2024

HIV envelope trimers and gp120 as immunogens to induce broadly neutralizing antibodies in cows.


Altman PX, Parren M, Sang H, Ozorowski G, Lee WH, Smider VV, Wilson IA, Ward AB, Mwangi W, Burton DR, Sok D.

bioRxiv March 20, 2024

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


Brown S, Antanasijevic A, Sewall LM, Garcia DM, Brouwer PJM, Sanders RW, Ward AB.

bioRxiv March 17, 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. 14, 2024

Broadly inhibitory antibodies against severe malaria virulence proteins.


Reyes RA, Raghavan SSR, Hurlburt NK, Introini V, Kana IH, Jensen RW, Martinez-Scholze E, Gestal-Mato M, Bau CB, Fernández-Quintero ML, Loeffler JR, Ferguson JA, Lee WH, Martin GM, 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 PJM, 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. 23, 2023