Biology subjects

Teo, Q. W.

Publications and source records attributed to Teo, Q. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Mutational fitness landscapes of diverse human influenza H1N1 neuraminidases

While neuraminidase (NA) is emerging as a promising influenza vaccine target, it evolves rapidly and undergoes antigenic drift. However, our understanding of its evolutionary constraints remains limited. Here, we systematically quantified the replication fitness effects of >6,000 mutations for each of three antigenically diverse H1N1 NAs using deep mutational scanning. Moderate correlations among H1N1 NA fitness landscapes revealed mutations with strain-dependent replication fitness effects. Additionally, cross-subtype comparison between N1 and N2 NAs uncovered similar epistatic effects that were enriched in the C-terminal interprotomer interface. We also showed that NA mutational fitness strongly correlated between in vivo and in vitro. Characterization of 40 individually constructed NA mutants further demonstrated that NA mutational fitness depended more on its surface protein expression than on its total protein expression or endoplasmic reticulum stress induction. Overall, the large dataset generated in this work provides critical insights into the evolutionary constraints of NA.

microbiology↗

bioRxiv2026

Somatic evolution of a cross-reactive germline antibody that expands its breadth to neutralize new SARS-CoV-2 variants

Rapid antigenic drift of the SARS-CoV-2 receptor-binding domain (RBD) underlies immune escape and continues to challenge the durability of antibody-mediated protection. Among the major classes of RBD-directed antibodies, germline-encoded IGHV3-53 responses are highly potent against early SARS-CoV-2 variants but are generally compromised by Omicron-associated mutations. Here, we identify an intrinsically cross-reactive IGHV3-53 germline antibody that recognizes multiple pre-Omicron variants, including SARS-CoV-2 wild-type, Alpha, and Delta. Notably, we demonstrate that targeted somatic evolution can further expand this breadth to overcome the immune escape of different Omicron variants. Guided by integrated structural and sequence analyses, we introduce four somatic mutations (G26E, T28I, S53P, and Y58F) into the germline antibody, resulting in markedly enhanced binding and neutralization of Omicron BA.1, BA.2, and BA.4/5. High-resolution crystal structures reveal that these mutations re-establish critical interactions disrupted by substitutions on Omicron RBD and optimize affinity at a remodeled epitope interface. Collectively, our findings delineate a structural and mechanistic pathway through which an inherently cross-reactive germline antibody lineage can be adaptively refined to counter highly divergent SARS-CoV-2 variants. This work highlights the underappreciated breadth encoded within the naive B-cell repertoire and provides a conceptual framework for engineering and eliciting antibody responses resilient to future antigenic drift.

immunology↗

bioRxiv2026

TRIM21 is a molecular rheostat for influenza A virus replication

TRIM21 is a multifunctional E3 ubiquitin ligase and intracellular antibody receptor, yet its role during viral infection remains unclear, with reports describing both antiviral and proviral activities. Here, we show that TRIM21 regulates influenza infection in an expression-dependent manner by functioning as a molecular rheostat rather than a binary restriction factor. This graded activity of TRIM21, which leads to both suppression and promotion of influenza replication, couples linkage-specific ubiquitination of viral nucleoprotein with modulation of innate immune signaling. Additionally, loss of TRIM21 unmasks a compensatory antiviral program centered on PRKDC, which is a ubiquitination target of TRIM21. This positions PRKDC as a latent restriction factor selectively engaged when primary TRIM21 control is lost. Together, these findings reveal a hierarchical and plastic antiviral network in which TRIM21 sets an adjustable threshold for host defense while restraining secondary restriction pathways. This framework highlights the sophisticated layers of regulation of the host ubiquitin-mediated antiviral immunity.

cell biology↗

bioRxiv2026

Structural insights into antibody responses against influenza A virus in its natural reservoir

While influenza A virus undergoes rapid antigenic drift in humans, at least some subtypes, such as H3, have relatively stable antigenicity in natural waterfowl reservoirs, despite the presence of immune pressure. However, the underlying mechanisms remain poorly understood. This study identified and characterized 187 antibodies to H3 hemagglutinin from experimentally infected mallard ducks, 18 of which were further analyzed by cryo-EM. Compared with human H3 antibodies, duck H3 antibodies exhibited higher glycan-binding propensity, more balanced immunodominance hierarchy, and targeted distinct epitopes. Other unique features of duck H3 antibodies included a convergent CDR H3-independent heavy chain-only binding mode and an N-glycosylated CDR H3 as decoy receptor. By annotating duck immunoglobulin germline genes, we also demonstrated the importance of gene conversion in duck H3 antibodies. Overall, our findings provide insights into how millennia of coevolution have shaped the interplay between influenza A virus antigenic drift and antibody responses in the natural reservoir.

immunology↗

bioRxiv2025

Single-cell heterogeneity in interferon induction potential is heritable and governed by variation in cell state

Type I and III interferons (IFNs) are among the first lines of defense against viral infection, yet they are generally only produced by a tiny fraction of infected cells. Here, we show that cellular heterogeneity in IFN induction potential upon treatment with immunostimulatory RNA is not due to variability in sensing of stimuli but instead is shaped by heterogeneity in tonic cell signaling state. Using complementary single-cell approaches, we found that baseline variation in the c-Jun N-terminal kinase (JNK) and activator protein (AP)-1 transcription factor families correlated with IFNL1 expression predisposition. We further show that drug-based inhibition of JNK signaling virtually eliminates the innate antiviral response to immunostimulatory RNA. Finally, we show that single cell heterogeneity in IFN induction potential is heritable and stably maintained over numerous generations. Together, our study emphasizes the influence of intrinsic variability in cell state on innate immune regulation and IFN induction heterogeneity.

systems biology↗

bioRxiv2025

Structure basis for distinct protective mechanisms of IGHV3-23 antibodies targeting influenza hemagglutinin stem

Characterization of antibodies targeting the conserved stem domain of influenza hemagglutinin (HA) is critical for developing broadly protective countermeasures against influenza virus. From a phage display human antibody library, this study discovers three group 1 HA-specific stem antibodies, namely HB31, HB34, and HB315, all of which are encoded by IGHV3-23. While HB31 and HB34 have minimal neutralization activity in vitro, their Fc-mediated effector functions lead to better in vivo protection than the potently neutralizing HB315. Consistently, cryo-EM analysis suggests that HB31 and HB34 have a higher Fc accessibility than HB315, based on their epitopes and approaching angles. HB31 and HB34 engage a pocket in the upper HA stem that is rarely targeted by known HA stem antibodies, whereas the epitope of HB315 involves the lower stem. Overall, our findings provide insights not only into the structure-function relationship of HA stem antibodies, but also into the design of next-generation influenza therapeutics.

immunology↗

bioRxiv2025

Vulnerability in the breadth evolution of an influenza broadly neutralizing antibody

The highly conserved influenza hemagglutinin (HA) stem domain is a major target for broadly neutralizing antibodies (bnAbs). However, despite being discovered more than a decade ago, the IGHV1-69-encoded CR9114 remains the only HA stem bnAb that cross-reacts with both influenza A and B viruses. To investigate the constraints on the breadth evolution of CR9114, this study performs four deep mutational scanning experiments to compare the binding affinity landscapes of the germline and somatic CR9114 against H1 HA, H3 HA, and influenza B HA. Many mutations that minimally affect or even improve the H1 HA binding are detrimental for binding to H3 HA and BHA. We further reveal the prevalence of epistasis in IGHV1-69 HA stem bnAbs. Overall, our findings provide a mechanistic explanation for the scarcity of HA stem bnAbs with cross-reactivity against both influenza A and B viruses, and have important implications for developing broadly protective influenza vaccines.

immunology↗

bioRxiv2025

UCHL3 regulates subgenomic flaviviral RNA condensates to promote virus propagation

In this study we demonstrate a previously uncharacterised post-translational regulatory mechanism governing flavivirus replication through the deubiquitylating enzyme ubiquitin C-terminal hydrolase L3 (UCHL3). Using activity-based protein profiling, we identified UCHL3 as a key cellular factor activated during Zika virus (ZIKV) and dengue virus (DENV) infections. CRISPR-Cas9 knockout experiments demonstrated that UCHL3 deficiency impairs flavivirus replication and viral protein expression across multiple cellular models. The underlying molecular mechanism involves UCHL3-mediated stabilisation of subgenomic flaviviral RNA (sfRNA)-containing biomolecular condensates. Through biotinylated sfRNA-interactome capture assays, we show that UCHL3 physically interacts with sfRNA-containing ribonucleoprotein complexes alongside G3BP1. Importantly, UCHL3 depletion triggers inappropriate RNase L activation, leading to sfRNA relocalisation from protective P-bodies to degradative compartments, such as RNase L-induced bodies (RLBs) as reported previously, resulting in viral RNA decay. Our rescue experiments confirmed that RNase L knockdown restores viral replication in UCHL3-deficient cells. This pro-viral effect of UCHL3 operates through interferon-independent mechanisms, as demonstrated by persistent replication defects even upon exogenous interferon treatment. This work therefore identifies UCHL3 as a molecular switch controlling the balance between pro-viral and antiviral RNA condensates, representing a promising host dependency factor for broad-spectrum flavivirus intervention strategies.

cell biology↗

bioRxiv2025

Evolution of antibody cross-reactivity to influenza H5N1 neuraminidase from an N2-specific germline

The ongoing spread of highly pathogenic avian influenza H5N1 clade 2.3.4.4b virus in animals and its occasional spillover to humans have raised concerns about a potential H5N1 pandemic. Although recent studies have shown that pre-existing human antibodies can recognize H5N1 neuraminidase, the molecular basis of how this cross-reactivity develops remains poorly understood. In this study, we used a phage display antibody library derived from 245 healthy donors to isolate an antibody, HB420, that cross-reacts with neuraminidases of human H3N2 and avian H5N1 clade 2.3.4.4b viruses and confers protection in vivo. Cryo-EM analysis reveals that HB420 targets the neuraminidase active site by mimicking sialic acid binding through a single Asp residue. Furthermore, the inferred germline of HB420 is N2-specific but acquires cross-reactivity to H5N1 neuraminidase through somatic hypermutation. Overall, our findings provide insights into how neuraminidase antibody evolves breadth, which has important implications for the development of broadly protective influenza vaccines.

immunology↗

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↗

bioRxiv2025

Intrinsic OASL expression licenses interferon induction during influenza A virus infection

Effective control of viral infection requires rapid induction of the innate immune response, especially the type I and type III interferon (IFN) systems. Despite the critical role of IFN induction in host defense, numerous studies have established that most cells fail to produce IFNs in response to viral stimuli. The specific factors that govern cellular heterogeneity in IFN induction potential during infection are not understood. To identify specific host factors that license some cells but not others to mount an IFN response to viral infection, we developed an approach for analyzing temporal scRNA-seq data of influenza A virus (IAV)-infected cells. This approach identified the expression of several interferon stimulated genes (ISGs) within pre-infection cells as correlates of IFN induction potential of those cells, post-infection. Validation experiments confirmed that intrinsic expression of the ISG OASL is essential for robust IFNL induction during IAV infection. Altogether, our findings reveal an important role for IFN-independent, intrinsic expression of ISGs in promoting IFN induction and provide new insights into the mechanisms that regulate cell-to-cell heterogeneity in innate immune activation.

immunology↗

bioRxiv2025

Probing the Role of Membrane in Neutralizing Activity of Antibodies Against Influenza Virus

Influenza poses a major health issue globally. Neutralizing antibodies targeting the highly conserved stem region of hemagglutinin (HA) of the influenza virus provide considerable protection against the infection. Using an array of advanced simulation technologies, we developed a high-resolution structural model of full-length, Fab-bound HA in a native viral membrane to characterize direct membrane interactions that govern the efficacy of the antibody. We reveal functionally important residues beyond the antibodys complementary-determining regions that contribute to its membrane binding. Mutagenesis experiments and infectivity assays confirm that deactivating the membrane-binding residues of the antibody decreases its neutralization activity. Therefore, we propose that the association with the viral membrane plays a key role in the neutralization activity of these antibodies. Given the rapid evolution of the influenza virus, the developed model provides a structural framework for the rational design and development of more effective therapeutic antibodies.

biophysics↗

bioRxiv2024

Seasonal influenza A virus lineages exhibit divergent abilities to antagonize interferon induction and signaling

The circulation of seasonal influenza A viruses (IAVs) in humans relies on effective evasion and subversion of the host immune response. While the evolution of seasonal H1N1 and H3N2 viruses to avoid humoral immunity is well characterized, relatively little is known about the evolution of innate immune antagonism phenotypes in these viruses. Numerous studies have established that only a small subset of infected cells is responsible for initiating the type I and type III interferon (IFN) response during IAV infection, emphasizing the importance of single cell studies to accurately characterize the IFN response during infection. We developed a flow cytometry-based method to examine transcriptional changes in IFN and interferon stimulated gene (ISG) expression at the single cell level. We observed that NS segments derived from seasonal H3N2 viruses are more efficient at antagonizing IFN signaling but less effective at suppressing IFN induction, compared to the pdm2009 H1N1 lineage. We compared a collection of NS segments spanning the natural history of the current seasonal IAV lineages and demonstrate long periods of stability in IFN antagonism potential, punctuated by occasional phenotypic shifts. Altogether, our data reveal significant differences in how seasonal and pandemic H1N1 and H3N2 viruses antagonize the human IFN response at the single cell level.

microbiology↗

bioRxiv2024

Deep mutational scanning of influenza A virus NEP reveals pleiotropic mutations in its N-terminal domain

The influenza A virus nuclear export protein (NEP) is a multifunctional protein that is essential for the viral life cycle and has very high sequence conservation. However, since the open reading frame of NEP largely overlaps with that of another influenza viral protein, non-structural protein 1, it is difficult to infer the functional constraints of NEP based on sequence conservation analysis. Besides, the N-terminal of NEP is structurally disordered, which further complicates the understanding of its function. Here, we systematically measured the replication fitness effects of >1,800 mutations of NEP. Our results show that the N-terminal domain has high mutational tolerance. Additional experiments demonstrate that N-terminal domain mutations pleiotropically affect viral transcription and replication dynamics, host cellular responses, and mammalian adaptation of avian influenza virus. Overall, our study not only advances the functional understanding of NEP, but also provides insights into its evolutionary constraints.

cell biology↗

bioRxiv2024

Differential antigenic imprinting effects between influenza H1N1 hemagglutinin and neuraminidase in a mouse model

Understanding how immune history influences influenza immunity is essential for developing effective vaccines and therapeutic strategies. This study investigates the antigenic imprinting of influenza hemagglutinin (HA) and neuraminidase (NA) using a mouse model with sequential infection by four seasonal H1N1 strains. Our findings reveal that, among pre-2009 H1N1 strains, the extent of infection history correlates with the restriction of antibody responses to antigenically drifted HA, but not NA. This suggests the mouse model failed to recapitulate NA imprinting in humans, likely due to the difference in NA immunodominance hierarchy between humans and mice. Nevertheless, pre-existing antibodies induced by infection with pre-2009 influenza virus impeded both functional HA and NA antibody responses against a 2009 pandemic H1N1 strain. Overall, this study provides insights into antigenic imprinting for influenza virus, as well as the limitations of using mouse models for studying antigenic imprinting. ImportanceInfluenza viruses continue to pose a significant threat to human health, with vaccine effectiveness being a persistent concern. One important factor is the individual immune history can influence subsequent antibody responses. While many studies have focused on how pre-existing antibodies influence the induction of anti-HA antibodies after influenza virus infections or vaccinations, the impact on anti-NA antibodies has been less extensively investigated. In this study, using a mouse model, we highlighted within the pre-2009 H1N1 strains, a greater extent of immune history negatively affected anti-HA antibodies but positively influenced anti-NA antibody responses. However, for the 2009 pandemic H1N1 strain, which underwent with antigenic shift, both anti-HA and anti-NA antibody responses have been impeded by the antibodies induced by pre-2009 H1N1 virus infection. These findings have important implications for enhancing our understanding of antigenic imprinting on anti-HA and anti-NA antibody response and for developing more effective vaccination strategies.

immunology↗

bioRxiv2023

An explainable language model for antibody specificity prediction using curated influenza hemagglutinin antibodies

Despite decades of antibody research, it remains challenging to predict the specificity of an antibody solely based on its sequence. Two major obstacles are the lack of appropriate models and inaccessibility of datasets for model training. In this study, we curated a dataset of >5,000 influenza hemagglutinin (HA) antibodies by mining research publications and patents, which revealed many distinct sequence features between antibodies to HA head and stem domains. We then leveraged this dataset to develop a lightweight memory B cell language model (mBLM) for sequence-based antibody specificity prediction. Model explainability analysis showed that mBLM captured key sequence motifs of HA stem antibodies. Additionally, by applying mBLM to HA antibodies with unknown epitopes, we discovered and experimentally validated many HA stem antibodies. Overall, this study not only advances our molecular understanding of antibody response to influenza virus, but also provides an invaluable resource for applying deep learning to antibody research.

immunology↗

bioRxiv2023

Stringent and complex sequence constraints of an IGHV1-69 broadly neutralizing antibody to influenza HA stem

IGHV1-69 is frequently utilized by broadly neutralizing influenza antibodies to the hemagglutinin (HA) stem. These IGHV1-69 HA stem antibodies have diverse complementarity-determining region (CDR) H3 sequences. Besides, their light chains have minimal to no contact with the epitope. Consequently, sequence determinants that confer IGHV1-69 antibodies with HA stem specificity remain largely elusive. Using high-throughput experiments, this study revealed the importance of light chain sequence for the IGHV1-69 HA stem antibody CR9114, which is the broadest influenza antibody known to date. Moreover, we demonstrated that the CDR H3 sequences from many other IGHV1-69 antibodies, including those to HA stem, were incompatible with CR9114. Along with mutagenesis and structural analysis, our results indicate that light chain and CDR H3 sequences coordinately determine the HA stem specificity of IGHV1-69 antibodies. Overall, this work provides molecular insights into broadly neutralizing antibody responses to influenza virus, which have important implications for universal influenza vaccine development.

molecular biology↗

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↗