Publications by authors named "Simon Clare"

117 Publications

Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.

PLoS Pathog 2021 01 22;17(1):e1009224. Epub 2021 Jan 22.

School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.
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http://dx.doi.org/10.1371/journal.ppat.1009224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870057PMC
January 2021

Best practice skin antisepsis for insertion of peripheral catheters.

Br J Nurs 2021 Jan;30(1):8-14

Clinical Director ANTT, The Association for Safe Aseptic Practice.

This article discusses the importance of effective skin antisepsis prior to the insertion of peripheral intravenous catheters (PIVCs) and how best clinical practice is promoted by application of an appropriate method of skin disinfection integrated effectively with a proprietary aseptic non touch technique, or other standard aseptic technique. Historically under-reported, incidence of infection and risk to patients from PIVCs is now increasingly being recognised, with new research and evidence raising concern and helping to drive new clinical guidance and improvement. The risks posed by PIVCs are particularly significant given increasing PIVC dwell times, due to cannula removal now being determined by new guidance for clinical indication, rather than predefined time frames. Clinical 'best practice' is considered in context of the evidence base, importantly including availability and access to appropriate skin antisepsis products. In the UK, and other countries, ChloraPrep is the only skin antisepsis applicator licensed as a drug to disinfect skin and help prevent infections before invasive medical procedures, such as injections, blood sampling, insertion of PIVCs and minor or major surgery.
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http://dx.doi.org/10.12968/bjon.2021.30.1.8DOI Listing
January 2021

Baseline Gut Microbiota Composition Is Associated With Infection Burden in Rodent Models.

Front Immunol 2020 18;11:593838. Epub 2020 Nov 18.

Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.

In spite of growing evidence supporting the occurrence of complex interactions between and gut bacteria in mice and humans, no data is yet available on whether worm-mediated changes in microbiota composition are dependent on the baseline gut microbial profile of the vertebrate host. In addition, the impact of such changes on the susceptibility to, and pathophysiology of, schistosomiasis remains largely unexplored. In this study, mice colonized with gut microbial populations from a human donor (HMA mice), as well as microbiota-wild type (WT) animals, were infected with , and alterations of their gut microbial profiles at 50 days post-infection were compared to those occurring in uninfected HMA and WT rodents, respectively. Significantly higher worm and egg burdens, together with increased specific antibody responses to parasite antigens, were observed in HMA compared to WT mice. These differences were associated to extensive dissimilarities between the gut microbial profiles of each HMA and WT groups of mice at baseline; in particular, the gut microbiota of HMA animals was characterized by low microbial alpha diversity and expanded Proteobacteria, as well as by the absence of putative immunomodulatory bacteria (e.g. ). Furthermore, differences in infection-associated changes in gut microbiota composition were observed between HMA and WT mice. Altogether, our findings support the hypothesis that susceptibility to infection in mice is partially dependent on the composition of the host baseline microbiota. Moreover, this study highlights the applicability of HMA mouse models to address key biological questions on host-parasite-microbiota relationships in human helminthiases.
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http://dx.doi.org/10.3389/fimmu.2020.593838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718013PMC
November 2020

Gut-educated IgA plasma cells defend the meningeal venous sinuses.

Nature 2020 11 4;587(7834):472-476. Epub 2020 Nov 4.

Molecular Immunity Unit, Department of Medicine, University of Cambridge, Cambridge, UK.

The central nervous system has historically been viewed as an immune-privileged site, but recent data have shown that the meninges-the membranes that surround the brain and spinal cord-contain a diverse population of immune cells. So far, studies have focused on macrophages and T cells, but have not included a detailed analysis of meningeal humoral immunity. Here we show that, during homeostasis, the mouse and human meninges contain IgA-secreting plasma cells. These cells are positioned adjacent to dural venous sinuses: regions of slow blood flow with fenestrations that can potentially permit blood-borne pathogens to access the brain. Peri-sinus IgA plasma cells increased with age and following a breach of the intestinal barrier. Conversely, they were scarce in germ-free mice, but their presence was restored by gut re-colonization. B cell receptor sequencing confirmed that meningeal IgA cells originated in the intestine. Specific depletion of meningeal plasma cells or IgA deficiency resulted in reduced fungal entrapment in the peri-sinus region and increased spread into the brain following intravenous challenge, showing that meningeal IgA is essential for defending the central nervous system at this vulnerable venous barrier surface.
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http://dx.doi.org/10.1038/s41586-020-2886-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748383PMC
November 2020

Genomics of the Argentinian cholera epidemic elucidate the contrasting dynamics of epidemic and endemic Vibrio cholerae.

Nat Commun 2020 10 1;11(1):4918. Epub 2020 Oct 1.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA, UK.

In order to control and eradicate epidemic cholera, we need to understand how epidemics begin, how they spread, and how they decline and eventually end. This requires extensive sampling of epidemic disease over time, alongside the background of endemic disease that may exist concurrently with the epidemic. The unique circumstances surrounding the Argentinian cholera epidemic of 1992-1998 presented an opportunity to do this. Here, we use 490 Argentinian V. cholerae genome sequences to characterise the variation within, and between, epidemic and endemic V. cholerae. We show that, during the 1992-1998 cholera epidemic, the invariant epidemic clone co-existed alongside highly diverse members of the Vibrio cholerae species in Argentina, and we contrast the clonality of epidemic V. cholerae with the background diversity of local endemic bacteria. Our findings refine and add nuance to our genomic definitions of epidemic and endemic cholera, and are of direct relevance to controlling current and future cholera epidemics.
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http://dx.doi.org/10.1038/s41467-020-18647-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530988PMC
October 2020

How widely has ANTT been adopted in NHS hospitals and community care organisations in England and Scotland?

Br J Nurs 2020 Sep;29(16):924-932

Research and Practice Development Director, The Association for Safe Aseptic Practice.

Background: To the detriment of patient safety, the important clinical competency of aseptic technique has been notoriously variable in practice, and described ambiguously in the literature, internationally. From a UK perspective, attempts have been made to improve patient safety by reducing variability and improving education and practice through standardisation. The Welsh Government mandated Aseptic Non Touch Technique (ANTT®) as a specific national standard in 2015. All healthcare organisations in England are required by the Health and Social Care Act 2008 to have a single standard aseptic technique, demonstrable by the clinical governance indicators of education, training, competency assessment and compliance audit. In Scotland, an education-based initiative was launched by NHS Education for Scotland in 2012. To review the impact of these and other initiatives on the current status of aseptic technique, all NHS trusts in England and NHS health boards in Scotland were assessed under the Freedom of Information procedure.

Findings: 93% of NHS trusts in England use a single standard for aseptic technique. In 88% of these trusts the single standard was stipulated as being ANTT. In Scotland, 62% of NHS acute and community care hospitals within health boards use a single standard. In 56% of these, the single standard was ANTT. When including those that use ANTT in combination with other techniques ANTT usage is 73%.

Conclusion: These data demonstrate significant progress in standardising aseptic technique education, assessment and governance, and confirms ANTT as the de facto aseptic technique used in NHS trusts in England and health boards in Scotland.
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http://dx.doi.org/10.12968/bjon.2020.29.16.924DOI Listing
September 2020

GM-CSF Calibrates Macrophage Defense and Wound Healing Programs during Intestinal Infection and Inflammation.

Cell Rep 2020 07;32(1):107857

Molecular Immunity Unit, University of Cambridge Department of Medicine, MRC Laboratory of Molecular Biology, Cambridge, UK; Wellcome Sanger Institute, Hinxton, UK; NIHR Cambridge Biomedical Research Centre, Cambridge, UK. Electronic address:

Macrophages play a central role in intestinal immunity, but inappropriate macrophage activation is associated with inflammatory bowel disease (IBD). Here, we identify granulocyte-macrophage colony stimulating factor (GM-CSF) as a critical regulator of intestinal macrophage activation in patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis. We find that GM-CSF drives the maturation and polarization of inflammatory intestinal macrophages, promoting anti-microbial functions while suppressing wound-healing transcriptional programs. Group 3 innate lymphoid cells (ILC3s) are a major source of GM-CSF in intestinal inflammation, with a strong positive correlation observed between ILC or CSF2 transcripts and M1 macrophage signatures in IBD mucosal biopsies. Furthermore, GM-CSF-dependent macrophage polarization results in a positive feedback loop that augmented ILC3 activation and type 17 immunity. Together, our data reveal an important role for GM-CSF-mediated ILC-macrophage crosstalk in calibrating intestinal macrophage phenotype to enhance anti-bacterial responses, while inhibiting pro-repair functions associated with fibrosis and stricturing, with important clinical implications.
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http://dx.doi.org/10.1016/j.celrep.2020.107857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351110PMC
July 2020

Lentiviral gene therapy rescues p47 chronic granulomatous disease and the ability to fight Salmonella infection in mice.

Gene Ther 2020 09 12;27(9):459-469. Epub 2020 Jun 12.

Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, University College London, London, UK.

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder characterised by recurrent and often life-threatening infections and hyperinflammation. It is caused by defects of the phagocytic NADPH oxidase, a multicomponent enzyme system responsible for effective pathogen killing. A phase I/II clinical trial of lentiviral gene therapy is underway for the most common form of CGD, X-linked, caused by mutations in the gp91 subunit of the NADPH oxidase. We propose to use a similar strategy to tackle p47-deficient CGD, caused by mutations in NCF1, which encodes the p47 cytosolic component of the enzymatic complex. We generated a pCCLCHIM-p47 lentiviral vector, containing the chimeric Cathepsin G/FES myeloid promoter and a codon-optimised version of the human NCF1 cDNA. Here we show that transduction with the pCCLCHIM-p47 vector efficiently restores p47 expression and biochemical NADPH oxidase function in p47-deficient human and murine cells. We also tested the ability of our gene therapy approach to control infection by challenging p47-null mice with Salmonella Typhimurium, a leading cause of sepsis in CGD patients, and found that mice reconstituted with lentivirus-transduced hematopoietic stem cells had a reduced bacterial load compared with untreated mice. Overall, our results potentially support the clinical development of a gene therapy approach using the pCCLCHIM-p47 vector.
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http://dx.doi.org/10.1038/s41434-020-0164-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500983PMC
September 2020

Screening of a library of recombinant Schistosoma mansoni proteins with sera from murine and human controlled infections identifies early serological markers.

J Infect Dis 2020 Jun 10. Epub 2020 Jun 10.

Wellcome Sanger Institute, Cambridge, UK.

Schistosomiasis is a major global health problem caused by blood-dwelling parasitic worms, which is currently tackled primarily by mass administration of the drug praziquantel. Appropriate drug treatment strategies are informed by diagnostics that establish the prevalence and intensity of infection, which, in regions of low transmission, should be highly sensitive. To identify sensitive new serological markers of Schistosoma mansoni infections, we have compiled a recombinant protein library of parasite cell-surface and secreted proteins expressed in mammalian cells. Together with a time series of sera samples from volunteers experimentally infected with a defined number of male parasites, we probed this protein library to identify several markers that can detect primary infections with as low as ten parasites and as early as five weeks post infection. These new markers could be further explored as valuable tools to detect ongoing and previous S. mansoni infections, including in endemic regions where transmission is low.
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http://dx.doi.org/10.1093/infdis/jiaa329DOI Listing
June 2020

Leupaxin Expression Is Dispensable for B Cell Immune Responses.

Front Immunol 2020 25;11:466. Epub 2020 Mar 25.

Inflammation Chemokines and Immunopathology, Institut National de la Santé et de la Recherche Medicale (INSERM), Faculté de Médecine, Université Paris-Sud, Université Paris-Saclay, Clamart, France.

The generation of a potent humoral immune response by B cells relies on the integration of signals induced by the B cell receptor, toll-like receptors and both negative and positive co-receptors. Several reports also suggest that integrin signaling plays an important role in this process. How integrin signaling is regulated in B cells is however still partially understood. Integrin activity and function are controlled by several mechanisms including regulation by molecular adaptors of the paxillin family. In B cells, Leupaxin (Lpxn) is the most expressed member of the family and studies suggest that it could dampen BCR signaling. Here, we report that expression is increased in germinal center B cells compared to naïve B cells. Moreover, deficiency leads to decreased B cell differentiation into plasma cells . However, Lpxn seems dispensable for the generation of a potent B cell immune response . Altogether our results suggest that Lpxn is dispensable for T-dependent and T-independent B cell immune responses.
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http://dx.doi.org/10.3389/fimmu.2020.00466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109257PMC
March 2021

Establishment, optimisation and quantitation of a bioluminescent murine infection model of visceral leishmaniasis for systematic vaccine screening.

Sci Rep 2020 03 13;10(1):4689. Epub 2020 Mar 13.

Cell Surface Signalling Laboratory, Wellcome Sanger Institute, Cambridge, UK.

Visceral leishmaniasis is an infectious parasitic disease caused by the protozoan parasites Leishmania donovani and Leishmania infantum. The drugs currently used to treat visceral leishmaniasis suffer from toxicity and the emergence of parasite resistance, and so a better solution would be the development of an effective subunit vaccine; however, no approved vaccine currently exists. The comparative testing of a large number of vaccine candidates requires a quantitative and reproducible experimental murine infection model, but the parameters that influence infection pathology have not been systematically determined. To address this, we have established an infection model using a transgenic luciferase-expressing L. donovani parasite and longitudinally quantified the infections using in vivo bioluminescent imaging within individual mice. We examined the effects of varying the infection route, the site of adjuvant formulation administration, and standardised the parasite preparation and dose. We observed that the increase in parasite load within the liver during the first few weeks of infection was directly proportional to the parasite number in the initial inoculum. Finally, we show that immunity can be induced in pre-exposed animals that have resolved an initial infection. This murine infection model provides a platform for systematic subunit vaccine testing against visceral leishmaniasis.
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http://dx.doi.org/10.1038/s41598-020-61662-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070049PMC
March 2020

IRF5 Promotes Influenza Virus-Induced Inflammatory Responses in Human Induced Pluripotent Stem Cell-Derived Myeloid Cells and Murine Models.

J Virol 2020 04 16;94(9). Epub 2020 Apr 16.

Division of Infection and Immunity/Systems Immunity, University Research Institute, Cardiff, United Kingdom.

Recognition of influenza A virus (IAV) by the innate immune system triggers pathways that restrict viral replication, activate innate immune cells, and regulate adaptive immunity. However, excessive innate immune activation can exaggerate disease. The pathways promoting excessive activation are incompletely understood, with limited experimental models to investigate the mechanisms driving influenza virus-induced inflammation in humans. Interferon regulatory factor 5 (IRF5) is a transcription factor that plays important roles in the induction of cytokines after viral sensing. In an model of IAV infection, IRF5 deficiency reduced IAV-driven immune pathology and associated inflammatory cytokine production, specifically reducing cytokine-producing myeloid cell populations in mice but not impacting type 1 interferon (IFN) production or virus replication. Using cytometry by time of flight (CyTOF), we identified that human lung IRF5 expression was highest in cells of the myeloid lineage. To investigate the role of IRF5 in mediating human inflammatory responses by myeloid cells to IAV, we employed human-induced pluripotent stem cells (hIPSCs) with biallelic mutations in , demonstrating for the first time that induced pluripotent stem cell-derived dendritic cells (iPS-DCs) with biallelic mutations can be used to investigate the regulation of human virus-induced immune responses. Using this technology, we reveal that IRF5 deficiency in human DCs, or macrophages, corresponded with reduced virus-induced inflammatory cytokine production, with IRF5 acting downstream of Toll-like receptor 7 (TLR7) and, possibly, retinoic acid-inducible gene I (RIG-I) after viral sensing. Thus, IRF5 acts as a regulator of myeloid cell inflammatory cytokine production during IAV infection in mice and humans and drives immune-mediated viral pathogenesis independently of type 1 IFN and virus replication. The inflammatory response to influenza A virus (IAV) participates in infection control but contributes to disease severity. After viral detection, intracellular pathways are activated, initiating cytokine production, but these pathways are incompletely understood. We show that interferon regulatory factor 5 (IRF5) mediates IAV-induced inflammation and, in mice, drives pathology. This was independent of antiviral type 1 IFN and virus replication, implying that IRF5 could be specifically targeted to treat influenza virus-induced inflammation. We show for the first time that human iPSC technology can be exploited in genetic studies of virus-induced immune responses. Using this technology, we deleted IRF5 in human myeloid cells. These IRF5-deficient cells exhibited impaired influenza virus-induced cytokine production and revealed that IRF5 acts downstream of Toll-like receptor 7 and possibly retinoic acid-inducible gene I. Our data demonstrate the importance of IRF5 in influenza virus-induced inflammation, suggesting that genetic variation in the IRF5 gene may influence host susceptibility to viral diseases.
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http://dx.doi.org/10.1128/JVI.00121-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163152PMC
April 2020

Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen-presenting cells and promotes resistance to Salmonella infection.

Immunology 2020 04 28;159(4):393-403. Epub 2020 Jan 28.

Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

Signalling lymphocyte activation molecule family member 9 (SLAMF9) is an orphan receptor of the CD2/SLAM family of leucocyte surface proteins. Examination of SLAMF9 expression and function indicates that SLAMF9 promotes inflammation by specialized subsets of antigen-presenting cells. Within healthy liver and circulating mouse peripheral blood mononuclear cells, SLAMF9 is expressed on CD11b , Ly6C , CD11c , F4/80 , MHC-II , CX CR1 mononuclear phagocytes as well as plasmacytoid dendritic cells. In addition, SLAMF9 can be found on peritoneal B1 cells and small (F4/80 ), but not large (F4/80 ), peritoneal macrophages. Upon systemic challenge with Salmonella enterica Typhimurium, Slamf9 mice were impaired in their ability to clear the infection from the liver. In humans, SLAMF9 is up-regulated upon differentiation of monocytes into macrophages, and lipopolysaccharide stimulation of PMA-differentiated, SLAMF9 knockdown THP-1 cells showed an essential role of SLAMF9 in production of granulocyte-macrophage colony-stimulating factor, tumour necrosis factor-α, and interleukin-1β. Taken together, these data implicate SLAMF9 in the initiation of inflammation and clearance of bacterial infection.
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http://dx.doi.org/10.1111/imm.13169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078004PMC
April 2020

High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation.

Nat Immunol 2020 01 16;21(1):86-100. Epub 2019 Dec 16.

Department of Immunobiology, King's College London, London, UK.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.
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http://dx.doi.org/10.1038/s41590-019-0549-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338221PMC
January 2020

Letter to the editor regarding "Survey to explore understanding of the principles of aseptic technique: Qualitative content analysis with descriptive analysis of confidence and training".

Authors:
Simon Clare

Am J Infect Control 2020 02 19;48(2):235-236. Epub 2019 Nov 19.

The Association for Safe Aseptic Practice, Surrey, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.ajic.2019.09.030DOI Listing
February 2020

Systematic screening of 96 cell-surface and secreted antigens does not identify any strongly protective vaccine candidates in a mouse model of infection.

Wellcome Open Res 2019 16;4:159. Epub 2019 Oct 16.

Wellcome Sanger Institute, Cambridge, CB10 1SA, UK.

Schistosomiasis is a major parasitic disease affecting people living in tropical and sup-tropical areas. Transmission of the parasite has been reported in 78 countries, causing significant morbidity and around 200,000 deaths per year in endemic regions. The disease is currently managed by the mass-administration of praziquantel to populations at risk of infection; however, the reliance on a single drug raises the prospect of parasite resistance to the only treatment widely available. The development of an effective vaccine would be a more powerful method of control, but none currently exists and the identification of new immunogens that can elicit protective immune responses therefore remains a priority. Because of the complex nature of the parasite life cycle, identification of new vaccine candidates has mostly relied on the use of animal models and on a limited set of recombinant proteins. In this study, we have established an infrastructure for testing a large number of vaccine candidates in mice and used it to screen 96 cell-surface and secreted recombinant proteins from . This approach, using standardised immunisation and percutaneous infection protocols, allowed us to compare an extensive set of antigens in a systematic manner. Although some vaccine candidates were associated with a statistically significant reduction in the number of eggs in the initial screens, these observations could not be repeated in subsequent challenges and none of the proteins studied were associated with a strongly protective effect against infection. Although no antigens individually induced reproducible and strongly protective effects using our vaccination regime, we have established the experimental infrastructures to facilitate large-scale systematic subunit vaccine testing for schistosomiasis in a murine infection model.
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http://dx.doi.org/10.12688/wellcomeopenres.15487.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833992PMC
October 2019

The flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi-like protein tail fibre.

Mol Microbiol 2019 12 9;112(6):1831-1846. Epub 2019 Oct 9.

Infection and Immunity Program, Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, 3800, Australia.

The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.
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http://dx.doi.org/10.1111/mmi.14396DOI Listing
December 2019

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Nat Commun 2019 09 19;10(1):4280. Epub 2019 Sep 19.

Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000, Antwerp, Belgium.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum β-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.
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http://dx.doi.org/10.1038/s41467-019-11844-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753159PMC
September 2019

Adaptation of host transmission cycle during Clostridium difficile speciation.

Nat Genet 2019 09 12;51(9):1315-1320. Epub 2019 Aug 12.

Host-Microbiota Interactions Laboratory, Wellcome Sanger Institute, Hinxton, UK.

Bacterial speciation is a fundamental evolutionary process characterized by diverging genotypic and phenotypic properties. However, the selective forces that affect genetic adaptations and how they relate to the biological changes that underpin the formation of a new bacterial species remain poorly understood. Here, we show that the spore-forming, healthcare-associated enteropathogen Clostridium difficile is actively undergoing speciation. Through large-scale genomic analysis of 906 strains, we demonstrate that the ongoing speciation process is linked to positive selection on core genes in the newly forming species that are involved in sporulation and the metabolism of simple dietary sugars. Functional validation shows that the new C. difficile produces spores that are more resistant and have increased sporulation and host colonization capacity when glucose or fructose is available for metabolism. Thus, we report the formation of an emerging C. difficile species, selected for metabolizing simple dietary sugars and producing high levels of resistant spores, that is adapted for healthcare-mediated transmission.
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http://dx.doi.org/10.1038/s41588-019-0478-8DOI Listing
September 2019

Using a Systems Biology Approach To Study Host-Pathogen Interactions.

Microbiol Spectr 2019 03;7(2)

Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom.

The rapid development of genomics and other "-omics" approaches has significantly impacted how we have investigated host-pathogen interactions since the turn of the millennium. Technologies such as next-generation sequencing, stem cell biology, and high-throughput proteomics have transformed the scale and sensitivity with which we interrogate biological samples. These approaches are impacting experimental design in the laboratory and transforming clinical management in health care systems. Here, we review this area from the perspective of research on bacterial pathogens.
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http://dx.doi.org/10.1128/microbiolspec.BAI-0021-2019DOI Listing
March 2019

Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis.

Immunity 2019 04 12;50(4):1099-1114.e10. Epub 2019 Mar 12.

Molecular Immunity Unit, University of Cambridge Department of Medicine, Cambridge CB2 0QH, UK; Cellular Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinton CB10 1SA, UK. Electronic address:

Inflammatory bowel disease is a chronic, relapsing condition with two subtypes, Crohn's disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWASs) in UC implicate a FCGR2A variant that alters the binding affinity of the antibody receptor it encodes, FcγRIIA, for immunoglobulin G (IgG). Here, we aimed to understand the mechanisms whereby changes in FcγRIIA affinity would affect inflammation in an IgA-dominated organ. We found a profound induction of anti-commensal IgG and a concomitant increase in activating FcγR signaling in the colonic mucosa of UC patients. Commensal-IgG immune complexes engaged gut-resident FcγR-expressing macrophages, inducing NLRP3- and reactive-oxygen-species-dependent production of interleukin-1β (IL-1β) and neutrophil-recruiting chemokines. These responses were modulated by the FCGR2A genotype. In vivo manipulation of macrophage FcγR signal strength in a mouse model of UC determined the magnitude of intestinal inflammation and IL-1β-dependent type 17 immunity. The identification of an important contribution of IgG-FcγR-dependent inflammation to UC has therapeutic implications.
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http://dx.doi.org/10.1016/j.immuni.2019.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477154PMC
April 2019

FBXO7 sensitivity of phenotypic traits elucidated by a hypomorphic allele.

PLoS One 2019 6;14(3):e0212481. Epub 2019 Mar 6.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

FBXO7 encodes an F box containing protein that interacts with multiple partners to facilitate numerous cellular processes and has a canonical role as part of an SCF E3 ubiquitin ligase complex. Mutation of FBXO7 is responsible for an early onset Parkinsonian pyramidal syndrome and genome-wide association studies have linked variants in FBXO7 to erythroid traits. A putative orthologue in Drosophila, nutcracker, has been shown to regulate the proteasome, and deficiency of nutcracker results in male infertility. Therefore, we reasoned that modulating Fbxo7 levels in a murine model could provide insights into the role of this protein in mammals. We used a targeted gene trap model which retained 4-16% residual gene expression and assessed the sensitivity of phenotypic traits to gene dosage. Fbxo7 hypomorphs showed regenerative anaemia associated with a shorter erythrocyte half-life, and male mice were infertile. Alterations to T cell phenotypes were also observed, which intriguingly were both T cell intrinsic and extrinsic. Hypomorphic mice were also sensitive to infection with Salmonella, succumbing to a normally sublethal challenge. Despite these phenotypes, Fbxo7 hypomorphs were produced at a normal Mendelian ratio with a normal lifespan and no evidence of neurological symptoms. These data suggest that erythrocyte survival, T cell development and spermatogenesis are particularly sensitive to Fbxo7 gene dosage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212481PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402633PMC
November 2019

USP44 is dispensable for normal hematopoietic stem cell function, lymphocyte development, and B-cell-mediated immune response in a mouse model.

Exp Hematol 2019 04 9;72:1-8. Epub 2019 Jan 9.

Department of Physiology, McGill University, Montreal, QC, Canada; McGill University Centre on Complex Traits, McGill University, Montreal, QC, Canada. Electronic address:

Ubiquitin-specific protease 44 (USP44) is a nuclear protein with deubiquitinase (DUB) catalytic activity that has been implicated as an important regulator of cell cycle progression, gene expression, and genomic stability. Dysregulation in the molecular machinery controlling cell proliferation, gene expression, and genomic stability in human or mouse is commonly linked to hematopoietic dysfunction, immunodeficiency, and cancer. We therefore set out to explore the role of USP44 in hematopoietic and immune systems through characterization of a Usp44-deficient mouse model. We report that USP44 is dispensable for the maintenance of hematopoietic stem cell numbers and function under homeostatic conditions, and also after irradiation or serial transplantation. USP44 is also not required for normal lymphocyte development. Usp44-deficient B cells show normal activation, proliferation, and immunoglobulin class switching in response to in vitro stimulation, and Usp44-deficient mice mount normal antibody response to immunization. We also tested the effects of USP44 deficiency on disease progression and survival in the Emu-myc model of mouse B-cell lymphoma and observed a trend toward earlier lethality of Usp44 Emu-myc mice; however, this did not reach statistical significance. Overall, we conclude that USP44 is dispensable for the normal physiology of hematopoietic and immune systems, and its functions in these systems are likely redundant with other USP family proteins.
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http://dx.doi.org/10.1016/j.exphem.2019.01.001DOI Listing
April 2019

Cyclosporine H Overcomes Innate Immune Restrictions to Improve Lentiviral Transduction and Gene Editing In Human Hematopoietic Stem Cells.

Cell Stem Cell 2018 12 8;23(6):820-832.e9. Epub 2018 Nov 8.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, MI 20132, Italy. Electronic address:

Innate immune factors may restrict hematopoietic stem cell (HSC) genetic engineering and contribute to broad individual variability in gene therapy outcomes. Here, we show that HSCs harbor an early, constitutively active innate immune block to lentiviral transduction that can be efficiently overcome by cyclosporine H (CsH). CsH potently enhances gene transfer and editing in human long-term repopulating HSCs by inhibiting interferon-induced transmembrane protein 3 (IFITM3), which potently restricts VSV glycoprotein-mediated vector entry. Importantly, individual variability in endogenous IFITM3 levels correlated with permissiveness of HSCs to lentiviral transduction, suggesting that CsH treatment will be useful for improving ex vivo gene therapy and standardizing HSC transduction across patients. Overall, our work unravels the involvement of innate pathogen recognition molecules in immune blocks to gene correction in primary human HSCs and highlights how these roadblocks can be overcome to develop innovative cell and gene therapies.
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http://dx.doi.org/10.1016/j.stem.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292841PMC
December 2018

Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal .

Proc Natl Acad Sci U S A 2018 10 27;115(41):10428-10433. Epub 2018 Sep 27.

Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom.

Nontyphoidal cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Typhimurium and Enteritidis vaccines. strains were chosen and deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.
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http://dx.doi.org/10.1073/pnas.1807655115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187145PMC
October 2018

Alpha kinase 1 controls intestinal inflammation by suppressing the IL-12/Th1 axis.

Nat Commun 2018 09 18;9(1):3797. Epub 2018 Sep 18.

Kennedy Institute of Rheumatology, University of Oxford, Oxford, OX3 7FY, United Kingdom.

Inflammatory bowel disease (IBD) are heterogenous disorders of the gastrointestinal tract caused by a spectrum of genetic and environmental factors. In mice, overlapping regions of chromosome 3 have been associated with susceptibility to IBD-like pathology, including a locus called Hiccs. However, the specific gene that controls disease susceptibility remains unknown. Here we identify a Hiccs locus gene, Alpk1 (encoding alpha kinase 1), as a potent regulator of intestinal inflammation. In response to infection with the commensal pathobiont Helicobacter hepaticus (Hh), Alpk1-deficient mice display exacerbated interleukin (IL)-12/IL-23 dependent colitis characterized by an enhanced Th1/interferon(IFN)-γ response. Alpk1 controls intestinal immunity via the hematopoietic system and is highly expressed by mononuclear phagocytes. In response to Hh, Alpk1 macrophages produce abnormally high amounts of IL-12, but not IL-23. This study demonstrates that Alpk1 promotes intestinal homoeostasis by regulating the balance of type 1/type 17 immunity following microbial challenge.
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http://dx.doi.org/10.1038/s41467-018-06085-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143560PMC
September 2018

Interleukin-22 promotes phagolysosomal fusion to induce protection against Typhimurium in human epithelial cells.

Proc Natl Acad Sci U S A 2018 10 14;115(40):10118-10123. Epub 2018 Sep 14.

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom.

Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection.
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http://dx.doi.org/10.1073/pnas.1811866115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176607PMC
October 2018