Biology subjects

Stanton, R. J.

Publications and source records attributed to Stanton, R. J..

10 recordsLinked to original sources

bioRxiv2025

Spike-specific IgG4 generated post BNT162b2 mRNA vaccination is inhibitory when directly competing with functional IgG subclasses

The rapid development of vaccines against SARS-CoV-2 during the COVID-19 pandemic proved vital in controlling viral spread and reducing mortality and morbidity. Both neutralising activity and effector function activity of Spike-specific antibodies have been shown to be important for their protective and therapeutic activity. However, several recent studies have reported that vaccination with mRNA based COVID-19 vaccines can lead to elevated levels of Spike-specific IgG4, an isotype which is often considered anti-inflammatory due to its reduced binding to Fc{gamma} receptors on immune cells. Here we show that the level of Spike-specific IgG4 produced following BNT162b2 vaccination is impacted by the interval between and frequency of vaccines boosts, prior SARS-CoV-2 infection (hybrid immunity), breakthrough infection and bivalent vaccine boosters. Despite the increase in Spike-specific IgG4 between the 2nd and 3rd BNT162b2 vaccine dose, neutralisation, ADCD and ADCP activity all increased. Through expression of SARS-CoV-2 monoclonal antibodies cloned as IgG1, IgG2, IgG3 and IgG4, we demonstrated that whilst Spike-specific IgG4 had reduced effector function activity, including ADCC, ADCD and ADCP, IgG4 was only inhibitory when directly competing with functional IgG subclasses binding to an overlapping epitope. In the context of polyclonal plasma, ADCC and ADCD activity could not be depleted by addition of high concentrations of a Spike-specific IgG4 mAb cocktail suggesting the non-stimulatory effect of Spike-specific IgG4 may be hidden in more complex scenarios, such as polyclonal mixes in serum.

immunology↗

bioRxiv2024

Whole Virion Proteomics of HCMV Reveals that gpUL141 is a Virion Component that Regulates Envelope Glycoprotein Composition to Protect Against Humoral Immunity

Human cytomegalovirus (HCMV) is a clinically important herpesvirus that has co-evolved for millions of years with its human host, and establishes lifelong persistent infection. A substantial proportion of its 235kb genome is dedicated to manipulating host immunity through targeting antiviral host proteins for degradation or relocalisation. Quantitative proteomics of the infected cell has extensively characterised these processes, but the cell-free virion has been less well studied. We therefore carried out proteomic analysis of a clinical HCMV strain (Merlin) virion. This revealed 18 novel components, including the viral protein gpUL141, which is recognised as an NK immune-evasin that targets several host proteins (CD155, CD112, and TRAILR) when expressed within the cell. Co-Immunoprecipitation of gpUL141 from virions identified interactions with viral entry glycoproteins from the trimer (gH/gL/gO), pentamer (gH/gL/UL128/UL130/UL131A), and gH/gpUL116 complexes, as well as gB. Only interactions with gH/gB occurred in the absence of other viral proteins. Analysis supported a model in which gpUL141 homodimers independently interacted with separate gB/gH-containing complexes. gpUL141 encodes an ER retention domain that restricts trafficking through the ER/golgi, and limited the transport of glycoprotein complexes bound by gpUL141. As a result, gpUL141 reduced levels of multiple glycoprotein complexes on the infected cell surface as well as in the virion. This reduced syncytium formation, inhibited antibody-dependent cellular cytotoxicity (ADCC), and reduced susceptibility to neutralising antibodies. Thus, gpUL141 represents an immune-evasin that not only targets host proteins to limit NK-cell attack, but also alters the trafficking of multiple viral glycoprotein complexes in order to evade humoral immunity. SignificanceThe large number of immune-modulators encoded by HCMV has led to it becoming a paradigm for pathogen-mediated immune-modulation. These mechanisms have informed on virus pathogenesis, the evolution and function of host defences, and identified therapeutic targets. Previously discovered immune-evasins functioned by modifying host proteins. gpUL141 represents a novel strategy in which multiple immunological functions are targeted through manipulating eight different viral entry glycoproteins, via interactions with two proteins common to multiple complexes. Entry glycoproteins define virus tropism, mechanisms of infection and spread, and susceptibility to neutralising and non-neutralising antibody activities, yet UL141 is frequently mutated in passaged viruses. This demonstrates the need to work with virus strains encoding the complete repertoire of viral accessory proteins during preclinical therapeutic development.

microbiology↗

bioRxiv2024

Highly Multiplexed Proteomic Analysis of HCMV-Infected Dendritic Cells Reveals Global Manipulation of Adaptive Immunity and Host Restriction of Viral Replication

Human cytomegalovirus (HCMV) is a clinically significant herpesvirus and a paradigm for pathogen-mediated immune-evasion. Its broad tropism includes antigen-presenting cells such as dendritic cells (DCs), which may partly explain a unique, dramatic imprint on host immunity that occurs following lifelong carriage. Despite this breadth of infection, most studies use fibroblasts as a model. We therefore developed systems to isolate pure populations of DCs infected with wild-type HCMV, before applying quantitative temporal proteomic technologies to systematically characterise the virus:DC interaction within cells and at the cell surface. This comprehensive dataset quantifying almost 9,000 proteins throughout the infection timecourse revealed multiple DC-specific viral:host effects, including key impacts on innate, intrinsic, and adaptive immunity. These effects included observations that APOBEC3A is downregulated in infected cells and restricts HCMV infection in ex vivo DCs, delaying the progression of lytic infection, and that cell surface ICOS-Ligand was downregulated by the viral genes US16 and US20, inhibiting the induction of adaptive immunity.

microbiology↗

bioRxiv2023

A virally encoded high resolution screen of cytomegalovirus host dependencies

Genetic screens have transformed our ability to interrogate cellular factor requirements in infection, yet current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information which is often based on infected cell survival. Here, by engineering human cytomegalovirus to express sgRNA libraries directly from the viral genome, we developed a sensitive, versatile, viral centric approach that allows profiling of different stages along viral infection in a pooled format. Using this approach, which we termed VECOS (Virus Encoded CRISPR-based direct readOut Screening system), we identified hundreds of novel host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on viral-host interactions. These high resolution measurements reveal that perturbations that alter late stages in HCMV life cycle mostly regulate HCMV particle quality rather than quantity, defining correct virion assembly as a critical stage that is heavily reliant on viral-host interactions. Overall, VECOS facilitates systematic high resolution dissection of human proteins role along the infection cycle, providing a roadmap for in-depth dissection of host-herpesvirus interactions.

microbiology↗

bioRxiv2023

ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defences. In exploring the finding that HCMV infection upregulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory anti-viral cytokine TNFa, we discovered the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype sheddase, a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with a HCMV double deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (p<0.05) in an ADAM17-dependent fashion. These included known substrates of ADAM17 with established immunological functions such as TNFR2 and Jagged1, but also numerous novel host and viral targets, such as Nectin1, UL8 and UL144. Regulation of TNF-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation. Significance statementHuman cytomegalovirus (HCMV) is an important pathogen, being the commonest infectious cause of brain damage to babies and the primary reason for hospital readmissions in transplant recipients. Even though HCMV induces the strongest immune responses by any human pathogen, it evades host defences and persists for life. This study describes a novel immunoregulatory strategy through which HCMV modulates multiple immune pathways simultaneously, by targeting a single host protein. HCMV UL148 and UL148D impair the maturation of the sheddase, A Disintegrin And Metalloproteinase 17, profoundly altering surface expression of numerous immunoregulatory proteins. This is the first description of viral genes targeting this pathway. Our findings may be relevant for future viral therapies and understanding the impact of HCMV in developmental biology.

microbiology↗

bioRxiv2021

Human cytomegalovirus protein RL1 degrades the antiviral factor SLFN11 via recruitment of the CRL4 E3 ubiquitin ligase complex

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate and adaptive immunity. We have employed two novel, orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins downregulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterised, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion. Significance StatementPrevious proteomic analyses of host factors targeted for downregulation by HCMV have focused on early or intermediate stages of infection. Using multiplexed proteomics, we have systematically identified viral factors that target each host protein downregulated during the latest stage of infection, after the onset of viral DNA replication. Schlafen-11 (SLFN11), an interferon-stimulated gene and restriction factor for retroviruses and certain RNA viruses, potently restricted HCMV infection. Our discovery that the late-expressed HCMV protein RL1 targets SLFN11 for proteasomal degradation provides the first evidence for a viral antagonist of this critical cellular protein. We therefore redefine SLFN11 as an important factor that targets DNA viruses as well as RNA viruses, offering novel therapeutic potential via molecules that inhibit RL1-mediated SLFN11 degradation.

microbiology↗

bioRxiv2021

ADNKA overcomes SARS-CoV2-mediated NK cell inhibition through non-spike antibodies

The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This correlated with a reduction in NK cell activation, identifying a novel mechanism by which SARS-CoV2 antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.

immunology↗

bioRxiv2020

The Virucidal Efficacy of Oral Rinse Components Against SARS-CoV-2 In Vitro

The ability of widely-available mouthwashes to inactivate SARS-CoV-2 in vitro was tested using a protocol capable of detecting a 5-log10 reduction in infectivity, under conditions mimicking the naso/oropharynx. During a 30 second exposure, two rinses containing cetylpyridinium-chloride and a third with ethanol/ethyl lauroyl arginate eliminated live virus to EN14476 standards (>4-log10 reduction), while others with ethanol/essential oils and povidone-iodine (PVP-I) eliminated virus by 2-3-log10. Chlorhexidine or ethanol alone displayed little or no ability to inactivate virus. Studies are warranted to determine whether these formulations can inactivate virus in the human oropharynx in vivo, and whether this might impact transmission risk.

microbiology↗

bioRxiv2020

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the commonly used 68-1 strain of RhCMV has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptation that cause reduced viremia in RhCMV-naive animals and limited shedding compared to low passage isolates. Using sequence information from primary RhCMV isolates we constructed a full-length (FL) RhCMV by repairing all presumed mutations in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naive RM with the reconstituted virus resulted in significant replication in the blood similar to primary isolates of RhCMV and furthermore led to extensive viremia in many tissues at day 14 post infection. In contrast, viral dissemination and viremia was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques. Author SummaryHuman cytomegalovirus (HCMV) infections are generally asymptomatic in healthy immunocompetent individuals, but HCMV can cause serious disease after congenital infection and in individuals with immunocompromised immune systems. Since HCMV is highly species specific and cannot productively infect immunocompetent laboratory animals, experimental infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines against chronic and recurring infections with pathogens such as the human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted with derivatives of the 68-1 strain of RhCMV which has acquired multiple genomic alterations in tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naive RM with FL-RhCMV demonstrated viremia and tissue dissemination that was comparable to that of non-clonal low passage isolates. We further demonstrate that FL-RhCMV is strongly attenuated upon deletion of gene regions absent in 68-1 thus demonstrating the usefulness of FL-RhCMV to study RhCMV pathogenesis.

microbiology↗

bioRxiv2020

Human cytomegalovirus protein pUL36: a dual cell death pathway inhibitor

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterise host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of pro-caspase 8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death. SIGNIFICANCE STATEMENTCell death is a key defence against viral infection, preventing spread from infected to uninfected cells. Correspondingly, certain viruses encode inhibitors of apoptotic and necroptotic cell death pathways in order to facilitate their persistence. Human cytomegalovirus (HCMV) is an important human pathogen that can block apoptosis, but hitherto it has been unclear whether or how the virus blocks necroptosis. Here, we used a proteomic screen to identify human proteins targeted for destruction by HCMV, finding that the key necroptosis mediator MLKL is degraded throughout infection. MLKL is targeted for degradation by HCMV protein pUL36, which is also instrumental in inhibiting apoptosis. Thus, pUL36 is a dual cell death pathway inhibitor, and may represent an important therapeutic target.

microbiology↗