Biology subjects

James, W. S.

Publications and source records attributed to James, W. S..

18 recordsLinked to original sources

bioRxiv2026

Dark proteome ensemble superposition gating calibrates biosynthesis of antiviral cocktail

Intrinsically disordered regions (IDRs)-the proteome dark matter-confer dynamic adaptability upon rigid protein scaffolds. How IDRs and their adjacent folded domain (context) modulate enzyme substrate promiscuity and calibrate host-specific immune outputs remains unknown. Addressing this knowledge gap has substantial economic and industrial value in pharmaceutical drug discovery, biotechnological applications, and synthetic biology. Here, we report that the widely studied and conserved radical S-adenosylmethionine enzyme of intrinsic immunity, viperin, has a C-terminal IDR that calibrates synthesis of host-specific antiviral cocktails. We show that a C-terminal tripeptide and its context shape the IDR conformational space (ensemble), keeping active-site entry in a superposition of open and closed states. Consequently, transplanting the rat enzyme C-terminal tripeptide context into its human orthologue alters the antiviral cocktail output. These results revise the prevailing model that vertebrate viperins with identical active-site pockets generate the same antiviral output, establish a new framework in host-specific antiviral responses, and inform IDRs biotechnological applications.

biochemistry↗

bioRxiv2026

Nanobodies against the S2 region of the spike protein potently neutralize SARS-CoV-2 viruses and show resistance to virus escape

Entry of coronaviruses into cells is mediated by the viral spike (S) glycoproteins each consisting of S1 receptor binding and S2 membrane fusion subunits. The sequence of the S2 region is very highly conserved amongst variants of SARS-CoV-2 and compared to the S1 unit shares significant sequence identity amongst different beta-coronavirus lineages. By targeting the S2 of SARS-CoV-2 we have identified two selective and potent neutralizing nanobodies (BA.1-C2 and BA.1-D3) that bind to two different quaternary epitopes in the S2 formed by the Heptad Repeat 2 (HR2) trimer at the base of the spike protein. The HR2 sequence is identical in SARS-CoV and SARS-CoV-2 but differs in other beta-coronaviruses explaining the lack of binding to the spike proteins of MERS-CoV or HuCoV-OC43. No viral escape was observed following serial passaging of SARS-CoV-2 (JN.1) with a combination of BA.1-C2 and BA.1-D3 and the most potent of these nanobodies reduced viral load in the hamster model of COVID-19, following intranasal administration. Overall, the results show the value of nanobody technology for identifying novel neutralising epitopes in the S2 region of beta-coronaviruses with potential for the development of new selective anti-viral agents.

molecular biology↗

bioRxiv2025

Development of a Recombinant Single-Cycle Influenza Viral Vector as an Intranasal Vaccine against SARS-CoV-2 and SARS-like Betacoronaviruses

The COVID-19 pandemic has demonstrated the detrimental potential of zoonotic coronavirus transmission to human populations. Effective vaccines capable of eliciting immunity to SARS-CoV-2 have been pivotal in mitigating the spread of the virus. In this study, we describe the generation of a non-replicating pseudotyped influenza A virus (S-FLU), where the native haemagglutinin (HA) sequence is replaced with the coding sequence of either a membrane-anchored form (TM) or secretory form (Sec) of the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 Wuhan (S-RBD Wuhan). We showed that both S-RBD-TM and S-RBD-Sec viruses can be generated via reverse genetics and grown to high titre. Intranasal immunisation in mice with S-RBD-TM elicits robust serum binding and neutralisation activity against SARS-CoV-2, superior to S-RBD-Sec. Furthermore, we demonstrate that a heterologous prime-boost immunisation regime in mice with S-RBD-TM Wuhan and S-RBD-TM BM48-31 (a distant Clade 3 SARS-like betacoronavirus (sarbecovirus)) increases antibody breadth against mismatched sarbecoviruses compared to homologous prime-boost with S-RBD-TM Wuhan. These results suggest that S-RBD-TM is a promising intranasal vaccine candidate against SARS-CoV-2 and may offer potential as a broadly protective sarbecovirus vaccine.

immunology↗

bioRxiv2025

Tracking tau and cellular responses in human iPSC-microglia from uptake to seedable secretion in extracellular vesicles

The templated spread of tau aggregates in tauopathies has been attributed to neuron-to- neuron spread, but microglia have also been implicated through mouse studies. Here we examine in detail the uptake, processing, release and seeding of tau using human iPS- derived microglia (iMGL). We show that tau is taken up by iMGL via LRP1 and heparan sulfate proteoglycans, with a role for LRRK2 in LRP1 trafficking, and that phagocytosed fibrils can escape into the cytoplasm. Monomeric tau has minimal effects on iMGL, but recombinant or brain-derived tau fibrils induce a shift towards chemokine and interferon response subtypes, alongside downregulation of homeostatic and MHC genes. Endogenous tau protein is undetectable in iMGL, and monomeric internalised tau is digested to completion, but fibrillar tau is more resistant to degradation and becomes phosphorylated on two specific residues. Finally, fibrillar tau is released by iMGL, visualized within extracellular vesicles by cryo-EM, and can seed tau aggregation in downstream neurons.

cell biology↗

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↗

bioRxiv2023

Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology

Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection. One Sentence SummaryA vaccine candidate with polyprotein antigens displayed on nanocages induces neutralizing antibodies to multiple SARS-like coronaviruses.

immunology↗

bioRxiv2022

SAMHD1 controls nucleoside-based cancer therapeutics, deoxyguanosine toxicity, and inflammation

SAMHD1 is a key regulator of dNTP metabolism, being activated by dGTP and converting dNTPs into their respective nucleosides. As a result, it plays a significant role in several distinct diseases. Not only is SAMHD1 a restriction factor for HIV-1 replication, which requires dNTPs for replication, but it is also required for genome stability, is mutated in multiple cancers, and loss of function mutations cause Acardi-Goutieres Syndrome, an early-onset inherited encephalopathy. In this paper we knock out the SAMHD1 gene in pluripotent stem cells, and show that it also affects nucleoside analogue sensitivity of dividing cells and deoxyguanosine toxicity in a cell cycle-independent manner. Comparing wild type and gene-edited macrophages, we also show that deoxyguanosine toxicity is associated with pro-inflammatory cell death, modifications in uric acid production and the formation of monosodium urate crystals within the macrophages. These novel features of the SAMHD1-/- phenotype provide unique insights into the aetiology of Acardi-Goutieres Syndrome, and have implications for certain chemotherapeutic agents.

pharmacology and toxicology↗

bioRxiv2022

Zika virus-induces metabolic alterations in fetal neuronal progenitors that could influence in neurodevelopment during early pregnancy

Neuronal progenitor subtypes have distinct fate restrictions regulated by time-dependent activation of energetic pathways. Thus, the hijacking of cellular metabolism by Zika virus (ZIKV) to support its replication may contribute to damage in the developing fetal brain. Here, we showed that ZIKV replicates differently in two glycolytically distinct hiPSC-derived neuronal progenitors that correspond to early and late progenitors in the forebrain. This differential replication alters the transcription of metabolic genes and upregulates the glycolytic capacity of progenitor subtypes. Analysis using Imagestream(R) revealed that, during early stages of infection, ZIKV replication in early progenitors increases lipid droplet abundance and decreases mitochondrial size and membrane potential. During later stages infection, early progenitors show increased subcellular distribution of lipid droplets, whilst late progenitors show decreased mitochondria size. The finding that there are hi-NPC subtype-specific alterations of cellular metabolism during ZIKV infection may help to explain the differences in brain damage over each trimester.

neuroscience↗

bioRxiv2022

Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSCs to microglia

There is increasing genetic evidence for the role of microglia in neurodegenerative diseases, including Alzheimers, Parkinsons, and motor neuron disease. Therefore, there is a need to generate authentic in vitro models to study human microglial physiology. Various methods have been developed using human induced Pluripotent Stem Cells (iPSC) to generate microglia, however, systematic approaches to identify which media components are actually essential for functional microglia are mostly lacking. Here, we systematically assess medium components, coatings, and growth factors required for iPSC differentiation to microglia. Using single-cell RNA sequencing, qPCR, and functional assays, with validation across two labs, we have identified several medium components from previous protocols that are redundant and do not contribute to microglial identity. We provide an optimised, defined medium which produces both transcriptionally and functionally relevant microglia for modelling microglial physiology in neuroinflammation and for drug discovery.

immunology↗

bioRxiv2022

Type I interferons govern Zika virus resistance of human macrophages and microglia

The role of the human type I interferon (IFN-I) system in restricting Zika virus (ZIKV) is uncertain. Here, genetic and pharmacological ablation of IFN-I signalling enhanced ZIKV replication and cytopathicity in macrophages and microglia, key cells in ZIKV transmission and pathogenesis. Thus, despite the extensive IFN-I countermeasures employed by ZIKV, IFN-I dictates the outcome of infection in macrophages. Therapeutic manipulation of the IFN-I system may bring clinical benefit in ZIKV.

cell biology↗

bioRxiv2021

Naturally-acquired immunity in Syrian Golden Hamsters provides protection from re-exposure to emerging heterosubtypic SARS-CoV-2 variants B.1.1.7 and B.1.351

The ability of acquired immune responses against SARS-CoV-2 to protect after subsequent exposure to emerging variants of concern (VOC) such as B1.1.7 and B1.351 is currently of high significance. Here, we use a hamster model of COVID-19 to show that prior infection with a strain representative of the original circulating lineage B of SARS-CoV-2 induces protection from clinical signs upon subsequent challenge with either B1.1.7 or B1.351 viruses, which recently emerged in the UK and South Africa, respectively. The results indicate that these emergent VOC may be unlikely to cause disease in individuals that are already immune due to prior infection, and this has positive implications for overall levels of infection and COVID-19 disease.

microbiology↗

bioRxiv2020

Differentiation of Human induced Pluripotent Stem Cells to Authentic Macrophages using Fully Defined, Serum Free, Open Source Media

SummaryHuman iPSC and macrophages derived from them are increasingly popular tools for research into both infectious and degenerative diseases. However, as the field strives for greater modelling accuracy, it is becoming ever more challenging to justify the use of undefined and proprietary media for the culture of these cells. We describe here two fully defined, serum-free, open-source media for the culture of iPSC and differentiation of iPSC-derived macrophages. These media are equally capable of maintaining these cells compared to commercial alternatives. The macrophages differentiated in these defined media display improved terminally differentiated cell characteristics, reduced basal expression of induced anti-viral response genes, and improved polarisation capacity. We conclude that cells cultured in these media are an appropriate and malleable model for tissue resident macrophages, on which future differentiation techniques can be built.

immunology↗

bioRxiv2020

Plasmablast-derived antibody response to acute SARS-CoV-2 infection in humans

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 13.0% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-RBD, three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. At last, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

microbiology↗

bioRxiv2020

A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy

In the absence of a proven effective vaccine preventing infection by SARS-CoV-2, or a proven drug to treat COVID-19, the positive results of passive immune therapy using convalescent serum provides a strong lead. We have developed a new class of tetravalent, biparatopic therapy, 89C8-ACE2. It combines the specificity of a monoclonal antibody (89C8) that recognizes the relatively conserved N-terminal domain (NTD) of the viral S glycoprotein, and the ectodomain of ACE2, which binds to the receptor-binding domain (RBD) of S. This molecule shows exceptional performance in vitro, inhibiting the interaction of recombinant S1 to ACE2 and transduction of ACE2-overexpressing cells by S-pseudotyped lentivirus with IC50s substantially below 100 pM, and with potency approximately 100-fold greater than ACE2-Fc itself. Moreover, 89C8-ACE2 was able to neutralize authentic virus infection in a standard assay at low nanomolar concentrations, making this class of molecule a promising lead for therapeutic applications.

immunology↗

bioRxiv2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

microbiology↗

bioRxiv2020

A chemical tool for improved culture of human pluripotent stem cells

The large-scale and cost-effective production of quality-controlled human pluripotent stem cells (hPSC) for use in cell therapy and drug discovery requires chemically-defined xenobiotic-free culture systems that enable easy and homogeneous expansion of pluripotent cells. Through phenotypic screening, we have identified a small molecule, OXS8360 (an optimized derivative of (-)-Indolactam V ((-)-ILV)), that stably disrupts hPSC cell-cell contacts. Proliferation of hPSC in OXS8360 is normal, as are pluripotency signatures, directed differentiation to hallmark lineages and karyotype over extended passaging. In 3D culture, OXS8360-treated hPSC form smaller, more uniform aggregates, that are easier to dissociate, greatly facilitating expansion. The mode of action of OXS8360 involves disruption of the localisation of the cell-cell adhesion molecule E-cadherin, via activation of unconventional Protein Kinase C isoforms. OXS8360 media supplementation is therefore able to yield more uniform, disaggregated 2D and 3D hPSC cultures, providing the hPSC field with an affordable tool to improve hPSC quality and scalability.

cell biology↗

bioRxiv2019

LRRK2 is recruited to phagosomes and co-recruits Rab8 and Rab10 in human pluripotent stem cell-derived macrophages

The Parkinsons disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease, and is expressed in immune subsets. Here, we characterise a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes, using human induced Pluripotent Stem Cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-{gamma} in these cells, including a 187kD cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles, but is recruited to LAMP1(+)/Rab9(+) maturing phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for Rab8a and Rab10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes.

immunology↗

bioRxiv2018

HIV-1 evasion of restriction factors: cyclophilin A and cell fusion provide a helping hand.

Retroviral restriction factors are important regulators of viral infection, targeting vulnerable steps of the virus lifecycle; steps that are also targeted by antiviral drugs. It has become clear that the route of cellular infection can alter the sensitivity of HIV-1 to these agents. Using CRISPR-Cas9 edited pluripotent stem cell-derived macrophages, we have explored the potential of a modified restriction factor (human TRIMCyp) to inhibit HIV-1 replication in both cell free and cell-cell infection models. We show that the expression of TRIMCyp from the endogenous TRIM5 locus potently restricts infection by cell-free HIV-1. Our results also show the importance of the human cyclophilin A-HIV-1 capsid interaction for viral escape from restriction by native human TRIM5, highlighting the evolutionary interplay between virus and this host restriction factor. However, when co-cultured with infected T cells, stem cell-derived macrophages are primarily infected by fusion between the cells. We have termed infected cells that result from these fusions heterocytia, and show that their formation overcomes multiple restriction factors and the reverse transcriptase inhibitor AZT.\n\nImportanceAs sentinels of the immune system, macrophages are relatively resistant to infection by pathogens such as HIV-1. However, infected macrophages are found in infected patients and they play key roles in the pathogenesis of the disease as well as being a component of the viral reservoir that must be targeted before treatment can become cure. In this article, we show that some of the mechanisms by which macrophages restrict HIV-1 can be overcome through a recently described cell-cell interaction leading to cell-cell fusion. We also highlight an evolutionary battle between virus and host and show how the virus has co-opted a host protein to protect it from destruction by an antiviral mechanism. These two key findings suggest potential novel treatment strategies that may reduce the viral reservoir and help our natural defences take back control from the virus.

microbiology↗