Biology subjects

Ouyang, W. O.

Publications and source records attributed to Ouyang, W. O..

7 recordsLinked to original sources

bioRxiv2025

Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity

The conserved stem domain of influenza hemagglutinin (HA), which is classified into group 1 and group 2, is a target of broadly neutralizing antibodies. While many group 1 HA stem antibodies have been described, much less is known about group 2 HA stem antibodies. This study structurally characterizes two group 2 HA stem antibodies, 2F02 and AG2-G02, targeting the central stem epitope and the lower stem epitope, respectively. Unlike prototypic group 2 HA stem antibodies, 2F02 and AG2-G02 do not compete for binding. Both antibodies offer protection in vivo despite having minimal neutralization activity in vitro. We further demonstrate that the natural evolution of HA2 position 32 restricts the binding of AG2-G02 to recent human H3N2 HAs and influences the binding of human plasma samples. Overall, these findings advance our understanding of the antigenicity of HA stem, which has important implications for the development of broadly protective influenza vaccines.

immunology↗

bioRxiv2024

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

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+ display, a high-throughput cell-free platform that combined oligo pool synthesis and mRNA display to rapidly construct and characterize many natively paired antibodies in parallel. As a proof-of-concept, we applied oPool+ display to probe the binding specificity of >300 uncommon influenza hemagglutinin (HA) antibodies against 9 HA variants through 16 different screens. Over 5,000 binding tests were performed in 3-5 days with further scaling potential. Follow-up structural analysis of two HA stem antibodies revealed the previously unknown versatility of IGHD3-3 gene segment in recognizing the HA stem. Overall, this study established an experimental platform that not only accelerate antibody characterization, but also enable unbiased discovery of antibody molecular signatures.

immunology↗

bioRxiv2024

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

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.

biochemistry↗

bioRxiv2023

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

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.

microbiology↗

bioRxiv2023

Widespread impact of immunoglobulin V gene allelic polymorphisms on antibody reactivity

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.

immunology↗

bioRxiv2022

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

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.

biochemistry↗

bioRxiv2022

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

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 N-terminal domain (NTD) has been long overlooked due to 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.

biochemistry↗