Publications
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Title & Authors Journal Publication Date

Priming 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, Allen JD, Nogal B, Silva M, Bhiman J, Pauthner M, Irvine DJ, Montefiori D, Crispin M, Burton DR, Silvestri G, Crotty S, Ward AB.
NPJ Vaccines July 12, 2024

Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to prime and elicit antibody responses against the conserved fusion peptide (FP). GC responses and antibody specificities were tracked longitudinally using lymph node fine-needle aspirates and electron microscopy polyclonal epitope mapping (EMPEM), respectively, to show antibody responses to the FP/N611 glycan hole region were primed, although exhibited limited neutralization breadth. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.

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, Burbach S, Truong A, Stubbington MJT, Burton DR, Andrabi R, Ward AB, McDonnell WJ, Briney B.
Cell Reports June 25, 2024

The development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees to isolate over 9,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific monoclonal antibodies (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. Among the recovered antibodies was TXG-0078, an N-terminal domain (NTD)-specific neutralizing mAb that recognizes diverse alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy-chain-dominant binding pattern seen in other NTD-supersite-specific neutralizing Abs with much narrower specificity. We also report CC24.2, a pan-sarbecovirus neutralizing antibody that targets a unique receptor-binding domain (RBD) epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 shows protection in vivo, suggesting their potential use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41.


Schiffner T, Phung I, Ray R, Irimia A, Tian M, Swanson O, Lee JH, Lee CD, Marina-Zárate E, Cho SY, Huang J, Ozorowski G, Skog PD, Serra AM, Rantalainen K, Allen JD, Baboo S, Rodriguez OL, Himansu S, Zhou J, Hurtado J, Flynn CT, McKenney K, Havenar-Daughton C, Saha S, Shields K, Schulze S, Smith ML, Liang CH, Toy L, Pecetta S, Lin YC, Willis JR, Sesterhenn F, Kulp DW, Hu X, Cottrell CA, Zhou X, Ruiz J, Wang X, Nair U, Kirsch KH, Cheng HL, Davis J, Kalyuzhniy O, Liguori A, Diedrich JK, Ngo JT, Lewis V, Phelps N, Tingle RD, Spencer S, Georgeson E, Adachi Y, Kubitz M, Eskandarzadeh S, Elsliger MA, Amara RR, Landais E, Briney B, Burton DR, Carnathan DG, Silvestri G, Watson CT, Yates JR 3rd, Paulson JC, Crispin M, Grigoryan G, Ward AB, Sok D, Alt FW, Wilson IA, Batista FD, Crotty S, Schief WR.
Nature Immunology May 30, 2024

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Use of Transient Transfection for cGMP Manufacturing of eOD-GT8 60mer, a Self-Assembling Nanoparticle Germline-Targeting HIV-1 Vaccine Candidate.


Sharma VK, Menis S, Brower ET, Sayeed E, Ackland J, Lombardo A, Cottrell CA, Torres JL, Hassell T, Ward AB, Tsvetnitsky V, Schief WR.
Pharmaceutics May 30, 2024

. We describe the current Good Manufacturing Practice (cGMP) production and subsequent characterization of eOD-GT8 60mer, a glycosylated self-assembling nanoparticle HIV-1 vaccine candidate and germline targeting priming immunogen. Production was carried out via transient expression in the human embryonic kidney 293 (HEK293) cell line followed by a combination of purification techniques. A large-scale cGMP (200 L) production run yielded 354 mg of the purified eOD-GT8 60mer drug product material, which was formulated at 1 mg/mL in 10% sucrose in phosphate-buffered saline (PBS) at pH 7.2. The clinical trial material was comprehensively characterized for purity, antigenicity, glycan composition, amino acid sequence, and aggregation and by several safety-related tests during cGMP lot release. A comparison of the purified products produced at the 1 L scale and 200 L cGMP scale demonstrated the consistency and robustness of the transient transfection upstream process and the downstream purification strategies. The cGMP clinical trial material was tested in a Phase 1 clinical trial (NCT03547245), is currently being stored at -80 °C, and is on a stability testing program as per regulatory guidelines. The methods described here illustrate the utility of transient transfection for cGMP production of complex products such as glycosylated self-assembling nanoparticles.

Isolation of human antibodies against influenza B neuraminidase and mechanisms of protection at the airway interface.


Wolters RM, Ferguson JA, Nuñez IA, Chen EE, Sornberger T, Myers L, Oeverdieck S, Raghavan SSR, Kona C, Handal LS, Esilu TE, Davidson E, Doranz BJ, Engdahl TB, Kose N, Williamson LE, Creech CB, Gibson-Corley KN, Ward AB, Crowe JE Jr.
Immunity May 29, 2024

Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition-binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo, and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates.

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 24, 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.

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.

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 Reports May 7, 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.

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 . March 27, 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.

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Title & Authors Journal Publication Date

Structural Mapping of Polyclonal IgG Responses to HA After Influenza Virus Vaccination or Infection.


León AN, Rodriguez AJ, Richey ST, Peña AT de la, Wolters RM, Jackson AM, Webb K, Creech CB, Yoder S, Mudd PA, Crowe JE, Han J, Ward AB.

bioRxiv July 8, 2024

A single mutation in dairy cow-associated H5N1 viruses increases receptor binding breadth


Good MR, Ji W, Fernández-Quintero ML, Ward AB, Guthmiller JJ

bioRxiv June 22, 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

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 8, 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, Montiel Garcia D, Brouwer PJM, Sanders RW, Ward AB

bioRxiv March 15, 2024