Biology subjects

Dejnirattisai, W.

Publications and source records attributed to Dejnirattisai, W..

13 recordsLinked to original sources

bioRxiv2026

Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

Diverse human viruses deploy a common immune-evasion strategy: viral 2-O-methylation coupled with STAT2 antagonism. Yet the importance of this coupling and whether targeting the coupled functions offers benefits in vaccine development, remains unknown. Using deep mutational scanning of the dengue NS5 protein, we discovered that the two immune-evasion functions work synergistically to cripple interferon signaling. Disrupting this synergy exposes a reciprocal regulatory relationship between the host proteins IFIT1 and STAT2 that controls both viral restriction and immune cell activation. Dengue viruses with mutations that disrupt the synergy are potently attenuated yet trigger antigen-presenting cell activation that exceeds a licensed dengue vaccine benchmark. Our results establish a generalizable principle: targeting synergies between viral immune evasion mechanisms can simultaneously achieve maximal viral attenuation and innate immune priming. This work provides a rational design framework for developing high-performance vaccines against many human viruses that exploit the IFIT1-STAT2 axis. TeaserBreaking synergistic viral immune evasion unlocks potent innate immunity for next-generation live-attenuated vaccines.

microbiology↗

bioRxiv2024

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 4,471,579 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of June 2024, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

bioinformatics↗

bioRxiv2023

Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.

bioinformatics↗

bioRxiv2023

Cooperativity and induced oligomerisation control the interaction of SARS- CoV-2 with its cellular receptor and patient-derived antibodies

Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV-2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant-dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets. One-Sentence SummaryMultivalent interactions between viral proteins, cell-surface receptors, and anti-viral antibodies regulate infection and inhibition.

biophysics↗

bioRxiv2023

The molecular reach of antibodies determines their SARS-CoV-2 neutralisation potency

Key functions of antibodies, such as viral neutralisation, depend on bivalent binding but the factors that influence it remain poorly characterised. Here, we develop and employ a new bivalent model to mechanistically analyse binding between >45 patient-isolated IgG1 antibodies interacting with SARS-CoV-2 RBD surfaces. Our method reproduces the monovalent on/off-rates and enables measurements of the bivalent on-rate and the molecular reach: the maximum antigen separation that supports bivalent binding. We find large variations in these parameters across antibodies, including variations in reach (22-46 nm) that exceed the physical antibody size ([~]15 nm) due to the antigen size. The bivalent model integrates all parameters, including reach and antigen density, to predict an emergent binding potency for each antibody that matches their neutralisation potency. Indeed, antibodies with similar monovalent affinities to the same RBD-epitope but with different reaches display differences in emergent bivalent binding that match differences in their neutralisation potency. Together, our work highlights that antibodies within an isotype class binding the same antigen can display differences in molecular reach that can substantially modulate their emergent binding and functional properties. Lay SummaryAntibodies are soluble proteins that can neutralise pathogens by sticking to them. They contain two identical arms that allow them to simultaneously bind two identical antigen molecules on pathogen surfaces. Although we know that bivalent binding is important for neutralisation, we dont know how different antibodies achieve it. We developed a new model to analyse the mechanism of bivalent binding and used it to study over 45 antibodies from COVID-19 patients that bind the RBD antigen of SARS-CoV-2. Unexpectedly, we found that the molecular reach of an antibody, which is the maximum antigen separation that supports bivalent binding, varied widely between antibodies and exceeded their physical size. We show how antibody binding emerges from the interplay of multiple factors, including reach, and that this emergent binding predicts their neutralisation function. The ability to analyse and predict bivalent binding should improve our understanding and exploitation of antibodies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/556503v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1674563org.highwire.dtl.DTLVardef@fc7feborg.highwire.dtl.DTLVardef@1325847org.highwire.dtl.DTLVardef@41ad51_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Inhibition of Phosphodiesterase 12 results in Antiviral Activity against several RNA Viruses including SARS-CoV-2.

Phosphodiesterase 12 (PDE12) is a negative regulator of the type 1 interferon (IFN) response and here we show that PDE12 inhibitors (lead compounds 63 and 17) are associated with increased RNAseL activity, are well tolerated at the therapeutic range and inhibit, both in vitro and in vivo, the replication of several RNA viruses including hepatitis C virus (HCV), dengue virus (DENV), West Nile Virus (WNV) and SARS-CoV-2.

microbiology↗

bioRxiv2022

CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever

Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional: the intracellular dimeric NS1 (iNS1) forms part of the viral replication complex, the extracellular multi-oligomeric secreted NS1 (sNS1) is a major factor contributing to DHF. The structure of the iNS1 is well studied but not sNS1. Here we show the tetrameric (stable and loose conformation) and hexameric structures of sNS1. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain - elongated {beta}-sheet or {beta}-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics.

molecular biology↗

bioRxiv2021

Receptor binding and escape from Beta antibody responses drive Omicron-B.1.1.529 evolution

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.

microbiology↗

bioRxiv2021

The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 B.1.351 and other variants of concern in preclinical studies.

There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), high titre binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) are induced. In addition, a strong and polyfunctional T cell response was measured in these booster regimens. These data support the ongoing clinical development and testing of this new variant vaccine.

immunology↗

bioRxiv2021

Antibody evasion by the Brazilian P.1 strain of SARS-CoV-2

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.

microbiology↗

bioRxiv2021

Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

microbiology↗

bioRxiv2020

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFN{gamma} based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4+ and/or CD8+ epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8+ T cells than spike-specific CD8+ T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8+ to CD4+ T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.

immunology↗

bioRxiv2020

A high resolution view of an adolescent flavivirus

Mosquito-transmitted flaviviruses, such as Dengue virus (DENV) or Zika virus (ZIKV), are responsible for significant economic damage and human misery. In infected cells, flaviviruses first assemble into an immature form within the endoplasmatic reticulum (ER), and then undergo further processing by furin protease in the trans-Golgi. Despite substantial efforts, previous cryogenic electron microscopy (cryo-EM) studies of immature flaviviruses were restricted to low to medium resolutions, limiting our understanding of maturation. To better grasp the process of maturation, we have carried out cryo-EM reconstructions of immature Spondweni virus (SPOV), an emerging human flavivirus belonging to the same serogroup as ZIKV (~75% amino acid identity). By combining localized reconstruction and focused refinement, we were able to improve the resolution to 3.8 [A], yielding unprecedented insight into the immature form. The structure elucidates how, at neutral pH, polar interactions conceal the furin recognition site within trimeric envelope (E) protein spikes. Furthermore, we identify how a strictly conserved pH sensor anchors the precursor membrane (prM) protein to immature E. We reconstructed mature forms of SPONV and DENV to 2.6[A] and 3.1[A], respectively. Comparison with immature virus shows a conserved binding pocket for a lipid headgroup, which forms as a consequence of the rearrangement of amphipathic stem-helices of E. We propose a structural role for the pocket and suggest it stabilizes mature E. Taken together, our data suggest a compelling rationale for low-pH triggered conformational rearrangement in the Golgi, which occurs during flavivirus maturation.

microbiology↗