Wenhao O. Ouyang

University of Illinois Urbana-Champaign

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Nicholas C. Wu

University of Illinois Urbana-Champaign

Timothy J.C. Tan

University of Illinois Urbana-Champaign

Ruipeng Lei

University of Electronic Science and Technology of China

Ian A. Wilson

Scripps Research Institute

Meng Yuan

University of Edinburgh

Huibin Lv

University of Illinois Urbana-Champaign

Ivana R. Shen

University of Illinois Urbana-Champaign

Ge Song

Jiangsu University

Xinghong Dai

Case Western Reserve University

Natalie So

University of Illinois Urbana-Champaign

Cooperative Institutions

University of Illinois Urbana-Champaign

Scripps Research Institute

University School

University of Hong Kong

Washington University in St. Louis

Scripps Institution of Oceanography

Scripps (United States)

HKU-Pasteur Research Pole

First Affiliated Hospital of Guangzhou Medical University

State Key Laboratory of Respiratory Disease

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A library-on-library screen reveals the breadth expansion landscape of a broadly neutralizing betacoronavirus antibody

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Marya Y. Ornelas Wenhao O. Ouyang Nicholas C. Wu

ABSTRACT Broadly neutralizing antibodies (bnAbs) typically evolve cross-reactivity breadth through acquiring somatic hypermutations. While evolution of breadth requires improvement of binding to multiple antigenic variants, most experimental evolution platforms select against only one antigenic variant at a time. In this study, a yeast display library-on-library approach was applied to delineate the affinity maturation of a betacoronavirus bnAb, S2P6, against 27 spike stem helix peptides in a single experiment. Our results revealed that the binding affinity landscape of S2P6 varies among different stem helix peptides. However, somatic hypermutations that confer general improvement in binding affinity across different stem helix peptides could also be identified. We further showed that a key somatic hypermutation for breadth expansion involves long-range interaction. Overall, our work not only provides a proof-of-concept for using a library-on-library approach to analyze the evolution of antibody breadth, but also has important implications for the development of broadly protective vaccines.

Affinity maturation

10.1101/2024.06.06.597810

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Rapid synthesis and screening of natively paired antibodies against influenza hemagglutinin stem via oPool⁺display

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Wenhao O. Ouyang Huibin Lv W. Liu Zongjun Mou Ruipeng Lei

Antibody discovery is crucial for developing therapeutics and vaccines as well as understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a high-throughput manner represents a major bottleneck in antibody discovery. Here, we presented oPool

10.1101/2024.08.30.610421

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High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike

bioRxiv (Cold Spring Harbor Laboratory) (2022)

Timothy J.C. Tan Zongjun Mou Ruipeng Lei Wenhao O. Ouyang Meng Yuan

Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we established a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrated a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, this method was applied to a region in the S2 domain that includes the first heptad repeat and central helix. Our results revealed that besides K986P and V987P, several mutations simultaneously improved expression and significantly lowered the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.

Heptad repeat

Immunogen

Identification

10.1101/2022.09.24.509341

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Citations (5)

Homeostatic regulation of extracellular signal-regulated kinase 1/2 activity and axonal Kv7.3 expression by prolonged blockade of hippocampal neuronal activity

Frontiers in Cellular Neuroscience (2022)

Brian C. Baculis Harish Kesavan Amanda C. Weiss Edward H. Kim Gregory Tracy

Homeostatic plasticity encompasses the mechanisms by which neurons stabilize their synaptic strength and excitability in response to prolonged and destabilizing changes in their network activity. Prolonged activity blockade leads to homeostatic scaling of action potential (AP) firing rate in hippocampal neurons in part by decreased activity of N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes including KCNQ3 which encodes K v 7.3. Neuronal K v 7 channels are mostly heterotetramers of K v 7.2 and K v 7.3 subunits and are highly enriched at the axon initial segment (AIS) where their current potently inhibits repetitive and burst firing of APs. However, whether a decrease in K v 7.3 expression occurs at the AIS during homeostatic scaling of intrinsic excitability and what signaling pathway reduces KCNQ3 transcript upon prolonged activity blockade remain unknown. Here, we report that prolonged activity blockade in cultured hippocampal neurons reduces the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) followed by a decrease in the activation of brain-derived neurotrophic factor (BDNF) receptor, Tropomyosin receptor kinase B (TrkB). Furthermore, both prolonged activity blockade and prolonged pharmacological inhibition of ERK1/2 decrease KCNQ3 and BDNF transcripts as well as the density of K v 7.3 and ankyrin-G at the AIS. Collectively, our findings suggest that a reduction in the ERK1/2 activity and subsequent transcriptional down-regulation may serve as a potential signaling pathway that links prolonged activity blockade to homeostatic control of BDNF-TrkB signaling and K v 7.3 density at the AIS during homeostatic scaling of AP firing rate.

Homeostatic plasticity

Homeostasis

Premovement neuronal activity

10.3389/fncel.2022.838419

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Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Ruipeng Lei Enya Qing Abby Odle Meng Yuan Timothy J.C. Tan

ABSTRACT The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we performed a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identified mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we showed that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.

Antigenicity

Coronavirus

10.1101/2023.11.28.569051

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Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin

Nature Communications (2024)

Ruipeng Lei Weiwen Liang Wenhao O. Ouyang Andrea Hernandez Garcia Chika Kikuchi

Abstract The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

Epistasis

Antigenicity

10.1038/s41467-024-49487-4

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Engineering an Oxygen‐Binding Protein for Photocatalytic CO₂Reductions in Water

Angewandte Chemie (2023)

Yunling Deng Sudharsan Dwaraknath Wenhao O. Ouyang Cory J. Matsumoto Stephanie Ouchida

Abstract While native CO 2 ‐reducing enzymes display remarkable catalytic efficiency and product selectivity, few artificial biocatalysts have been engineered to allow understanding how the native enzymes work. To address this issue, we report cobalt porphyrin substituted myoglobin (CoMb) as a homogeneous catalyst for photo‐driven CO 2 to CO conversion in water. The activity and product selectivity were optimized by varying pH and concentrations of the enzyme and the photosensitizer. Up to 2000 TON(CO) was attained at low enzyme concentrations with low product selectivity (15 %), while a product selectivity of 74 % was reached by increasing the enzyme loading but with a compromised TON(CO). The efficiency of CO generation and overall TON(CO) were further improved by introducing positively charged residues (Lys or Arg) near the active stie of CoMb, which demonstrates the value of tuning the enzyme secondary coordination sphere to enhance the CO 2 ‐reducing performance of a protein‐based photocatalytic system.

10.1002/ange.202215719

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Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning

Science Advances (2022)

Wenhao O. Ouyang Timothy J.C. Tan Ruipeng Lei Ge Song Collin Kieffer

Increasing the expression level of the SARS-CoV-2 spike (S) protein has been critical for COVID-19 vaccine development. While previous efforts largely focused on engineering the receptor-binding domain (RBD) and the S2 subunit, the amino-terminal domain (NTD) has been long overlooked because of the limited understanding of its biophysical constraints. In this study, the effects of thousands of NTD single mutations on S protein expression were quantified by deep mutational scanning. Our results revealed that in terms of S protein expression, the mutational tolerability of NTD residues was inversely correlated with their proximity to the RBD and S2. We also identified NTD mutations at the interdomain interface that increased S protein expression without altering its antigenicity. Overall, this study not only advances the understanding of the biophysical constraints of the NTD but also provides invaluable insights into S-based immunogen design.

Immunogen

Antigenicity

10.1126/sciadv.add7221

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Citations (26)

Engineering an Oxygen‐Binding Protein for Photocatalytic CO₂Reductions in Water

Angewandte Chemie International Edition (2023)

Yunling Deng Sudharsan Dwaraknath Wenhao O. Ouyang Cory J. Matsumoto Stephanie Ouchida

10.1002/anie.202215719

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Widespread impact of immunoglobulin V gene allelic polymorphisms on antibody reactivity

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Meng Yuan Ziqi Feng Huibin Lv Natalie So Ivana R. Shen

The ability of human immune system to generate antibodies to any given antigen can be strongly influenced by immunoglobulin V gene (IGV) allelic polymorphisms. However, previous studies have provided only a limited number of examples. Therefore, the prevalence of this phenomenon has been unclear. By analyzing >1,000 publicly available antibody-antigen structures, we show that many IGV allelic polymorphisms in antibody paratopes are determinants for antibody binding activity. Biolayer interferometry experiment further demonstrates that paratope allelic mutations on both heavy and light chain often abolish antibody binding. We also illustrate the importance of minor IGV allelic variants with low frequency in several broadly neutralizing antibodies to SARS-CoV-2 and influenza virus. Overall, this study not only highlights the pervasive impact of IGV allelic polymorphisms on antibody binding, but also provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery.

Paratope

Antibody Repertoire

10.1101/2023.06.06.543969

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