NIHRDataInsights/HRCSData huggingface dataset:
Title: Characterisation of a novel AAV vector with enhanced neurotropism
Funder: Medical Research Council

Abstract: Adeno-associated virus (AAV) gene therapy is currently experiencing a renaissance thanks to a number of successful clinical trials. However, vector doses required to achieve therapeutic effect are high and some tissues remain difficult targets for efficient transduction. In general, gene therapy strategies directed at the brain of patients with inherited neurological or neurodegenerative disorders have shown limited efficacy. One of the main challenges for gene therapy for central nervous system (CNS) diseases is the limited distribution of AAV vectors from the injection site to the target cells. Our preliminary studies have shown that mutation of a select number of amino acids (AA) in the AAV2 capsid results in a vector with enhanced transduction and significant spread in the CNS of rodent models. Comparative biodistribution studies demonstrated that our new vector, AAV-TT, has better distribution abilities than benchmark neurotropic serotypes AAV9 and AAVrh10. Importantly, AAV-TT was the only vector able to correct a mouse model of mucopolyssacharidosis IIIC (MPSIIIC), a CNS disease caused by a lysosomal transmembrane enzyme and therefore reliant on efficient transduction throughout the brain. Here we propose a study that is aimed at delineating which mechanisms contribute to the unparalleled transduction and spread of AAV-TT in the CNS. This will be achieved by comparing the abilities of AAV2 and AAV-TT in biological assays that are designed to measure (1) cellular determinants of transduction i.e. viral attachment, cellular uptake, cytoplasmic escape, nuclear entry and uncoating; (2) interstitial transport within the brain parenchyma; (3) retro- and anterograde axonal transport within the CNS. The experiments proposed here will provide insight into the mechanisms that contribute to the increased bioactivity of AAV-TT in the CNS and will further assist the development of treatments for a group of neurological diseases that are defined by global neuropathy.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION
Title: CD43 - a molecular switch for efferocytosis and HIV-1 infection
Funder: Medical Research Council

Abstract: Immune cell interactions are essential for multiple processes including efferocytic removal of dying cells by phagocytes. These interactions are mediated primarily by specific ligand-receptor engagement, but may be regulated by molecules such as CD43. CD43 is a highly expressed leukocyte glycoprotein, unusual because it extends 45 nm from the cell surface and contains ~ 80 sialic acid-terminated O-linked glycans. These features impart steric and electrostatic barriers to immune cell interactions. Genetic removal of CD43 results in increased intercellular interactions, including, as we show in the current application, capture by macrophages. This latter finding demonstrates that CD43 is a previously undescribed 'don't eat-me' leukocyte signal. We also describe a novel pathway by which CD43 is rapidly and almost completely removed from the surface of T cells upon cell death or HIV-1 infection by the plasma membrane sheddase ADAM10. HIV-1 infection of CD4 T cells and macrophages contributes to viral pathogenesis and persistence even in the presence of suppressive immunotherapy. Although CD4 T cells are readily infectable in vitro and in vivo, macrophages are relatively resistant. We recently described an efficient pathway of macrophage infection based upon capture and uptake of HIV-1 infected T cells by macrophages, which mediates macrophage infection even by weakly macrophage-tropic HIV-1. Data provided in the current application reveal that both HIV-1 infection of T cells and their regulated cell death leads to cell surface CD43 loss and macrophage uptake. In the case of HIV-1-infected T cells, this may drive efficient macrophage infection. These findings have implications for both the pathogenesis of inflammatory and autoimmune diseases linked to ADAM10 dysfunction, spread of infection between T cells and from T cells to macrophages, and therapeutic intervention in these conditions via currently available or novel ADAM10 inhibitors.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Deciphering Gram-negative phage-inducible chromosomal island strategies for spreading in nature
Funder: Medical Research Council

Abstract: Bacteria are successful as commensal organisms or pathogens partly because they rapidly adapt to selective pressures imparted by the human host. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information (DNA) among and within bacterial species. Importantly, MGEs encode putative virulence factors and molecules that confer resistance to antibiotics. Inasmuch as bacterial infections are a significant problem worldwide and continue to emerge in epidemic waves, there has been significant effort to understand the agents that effect DNA movement. In recent years, we have extensively characterised a family of pathogenicity islands in Staphylococcus aureus, SaPIs, which contribute substantively to horizontal gene transfer, host adaptation and virulence. We have recently demonstrated that similar elements occur widely in bacteria, that these are not defective prophages, despite their annotations in GenBank, but are rather a unique class of MGEs, the phage-inducible chromosomal islands (PICIs). Although our studies have confirmed the universality of the PICIs, there are two important questions related to the biology of the PICIs present in the Gram-negative bacteria that remain to be answered: i) how these elements sense the presence of their helper phages; and ii) how they hijack the phage machinery for their own specific packaging, blocking phage reproduction. Note that these are the two key processes that define the ability of these elements to spread in nature, generating novel virulent and resistant clones. Answering these questions will be the main goal of the current proposal. Our studies will greatly enhance understanding of the evolution of pathogenic bacteria and apparition of novel virulence clones through the acquisition of MGEs, especially across genera.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Assessing the impact of host and virus genetic variability on Filovirus cell entry and infectivity
Funder: Medical Research Council

Abstract: The West African Ebolavirus outbreak was on an unprecedented scale. There is overwhelming evidence that the risk of future Filovirus outbreaks will increase due to population growth, urbanisation, habitat encroachment and improved transport links. Understanding host and viral factors that influence Ebola and Marburg virus infectivity and outbreak severity will inform future outbreak preparedness, it will also provide important insight into the Filovirus entry process that can guide future therapeutic design; especially interventions that have pan-Filovirus activity. Knowledge of host genetic factors that impact on Filovirus entry efficacy will pave the way for future large-scale genomics studies of Filovirus-exposed populations to determine the impact of host genetic factors, particularly receptor polymorphisms, on human population susceptibility (or resistance) to infection and disease severity. In the long-term this can inform public health policy and also inform patient care. Using the most relevant experimental systems, which will include transgenic mouse models, recombinant infectious VSV-chimeras encoding Filovirus surface glycoproteins, pseudovirus entry, reverse genetics, biochemical assays, cell transduction and live cell imaging, we will determine the impact of virus evolution on host tropism and infectivity. We will also define the impact of inter-species host receptor variation and human allelic variants on Filovirus entry and link these observations to African patient cohorts to determine the possible impact in the context of virus outbreaks. In addition to new understanding of Filovirus infectivity, the project will generate a small animal model to support wider studies of Filovirus entry, a large number of viral clones, genetically modified cell lines and synthetic glycoproteins that will be of wider benefit to the research community, particularly those engaged in treatment and vaccine development.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Profiling of host and parasite factors associated with organ dysfunction in severe malaria
Funder: Medical Research Council

Abstract: In low transmission settings, patients with severe falciparum malaria develop brain, lungs, liver and kidney dysfunction, either alone or in combination. The mechanisms leading to these complications have never been systematically investigated. We propose to implement minimally invasive tissue sampling techniques in well-characterised patients admitted at Ispat General Hospital in India to analyse for the first time multi-omics host and parasite factors leading to specific organ dysfunction in severe malaria. We will collect samples of brain, lung, liver, kidney and blood tissue from fatal cases with or without cerebral malaria, acute respiratory distress syndrome, jaundice and acute kidney injury. Half of the solid tissues will be used for the first extensive immunohistopathology analysis of its kind, and the rest digested to isolate parasitized red blood cells, endothelial cells, and immune cell populations. We will determine the subset of circulating parasite variants that sequestered in different organs of fatal malaria cases and their association with specific clinical complications by cloning, sequencing and comparing DBLa tags between tissues. In parallel, we will perform deep RNA sequencing to characterise global transcription profiles of endothelial cells from the same organs and identify potential PfEMP1/endothelial receptor combinations associated with specific organ dysfunctions. We will also determine the type, amount and transcriptome of immune cell populations present in each organ through single-cell transcriptomics. Lastly, we will carry out global metabolomic and complex lipidomic profiling of the plasma from fatal cases, non-fatal matched cases and uncomplicated malaria controls to identify signatures associated with specific organ failures and with fatality. The primary outcome of this project will be a better understanding of the factors influencing the development of organ dysfunction in severe malaria, with a view to developing new therapies.


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: TConditional ermination of Transcription in Mycobacterium tuberculosis
Funder: Medical Research Council

Abstract: This project will investigate how conditional termination of transcription contributes to gene expression control in M. tuberculosis. More specifically, we will define transcriptional terminator motifs and investigate the genome-wide abundance of conditional terminators in M. tuberculosis by applying Term-seq (3' end enriched RNA-seq) to cultures of M. tuberculosis grown under standard growth conditions. We will also determine to what extent essential metabolites and anti-TB drugs regulate gene expression and in particular conditional termination in M. tuberculosis by applying Term-seq to cultures of M. tuberculosis grown in the presence of S-adenosylmethionine (SAM) and glycine (essential metabolites) or selected anti-TB drugs (Isoniazid, Ethambutol, Bedaquiline and Linezolide). The findings will be further consolidated by using traditional molecular biology methods. To validate termination and antitermination function of selected elements we will employ reporter genes fusions of wildtype and mutated elements expressed under different growth conditions. To identify genes associated with synthesis, modification or turnover, we will express these reporters and in transposon-insertion mutants. To validate predicted structures of selected elements we will perform RNA structure probing with a complement of single-stranded and double-stranded specific RNases. To probe how potential ligands modulate transcription kinetics and termination we will employ single-round in vitro transcription; to screen for putative ligands, we will modify our existing in vitro transcription assay to include the expression of a fluorophore-binding RNA aptamer (Mango), which will enable high-throughput (96-well format).


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: The Role Of Antibody in Enabling Cell-mediated Control of HCMV Infection
Funder: Medical Research Council

Abstract: HCMV treatment relies on antivirals, yet these have significant drawbacks including toxicity and resistance. Many clinical efforts are focussed on the development of neutralising antibodies. However, although antibody titres correlate with control of disease, their neutralising capacity is not critical; in a trial of neutralising human IgG, no reduction in disease was observed. Consistent with this observation, we have shown that dissemination by clinical HCMV is resistant to neutralising antibodies. In contrast to results with neutralising antibodies, in two recent vaccine trials, 50% protection was achieved when the induced antibodies acted through non-neutralising mechanisms. Non-neutralising mechanisms including antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytolysis (CDC) and antibody-dependent cellular phagocytosis (ADCP). Both in vivo and in vitro findings strongly suggest that ADCC is critical for virus control. We have examined the cell surface proteome to define, for the first time, the viral antigens that activate ADCC rapidly after infection. We will determine the relative effectiveness of each viral antigen at activating ADCC in isolation, in the context of the wildtype virus and across a range of clinically-relevant cell types. We will then investigate the relative ability of these viral antigens to activate each non-neutralising effector mechanism (ADCC, CDC, ADCP). On the basis of these findings, humanised monoclonal antibodies will be generated to the strongest candidates, and the relative ability of ADCC, ADCP and CDC to control infection assessed. Finally, we will enable optimisation of therapeutic antibody efficacy by assessing the role of HCMV-encoded Fc-binding proteins in sequestering therapeutic antibodies, before crystallising the viral Fc-binding proteins in complex with Fc. This will guide the development of optimised antibodies that activate human, but not viral, Fc-receptors.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Adaptive variation in sexual and asexual reproduction of endemic malaria parasites
Funder: Medical Research Council

Abstract: This proposal is to undertake a quantitative analysis of sexual and asexual reproduction in malaria parasites from endemic populations, and test a hypothesis of transcriptional control and selection. It addresses the following questions: 1) What are distributions of naturally occurring switch rates to sexual differentiation of Plasmodium falciparum? Rates of commitment to sexual stage differentiation per asexual generation cycle will be measured in parasites isolated from clinical infections in areas with high levels of transmission (in Ghana), moderate seasonal transmission (in The Gambia), and extremely low transmission (in Malaysia). Induction of gametocytogenesis will be performed by transfer to growth medium lacking lysophosphatidylcholine. 2) What are the principal determinants of asexual multiplication rate variation? We will test for correlations between sexual commitment rates and asexual multiplication rates within each of the panels of clinical isolates studied in the previous section. Components of the asexual multiplication cycle will also be quantified by measuring numbers of merozoites per mature schizont, merozoite invasion efficiency, use of alternative receptors for invasion, and cell cycle duration. The overall variance in each of the measured parameters and their associations with asexual multiplication rate will be tested. 3) How do the alternative major haplotypes of the gdv1 3'-intergenic region affect transcription and sexual commitment? The levels of 3'-intergenic and antisense long non-coding RNA transcripts, in comparison with the sense transcripts of gdv1, will be analysed at basal levels and in response to gametocytogenesis induction in these isolates, and tested for differences between the two major gdv1 3'-intergenic allelic types. Complementary to this test association, allelic replacement genetic modifications will be performed to determine the regulatory effects of different parts of the intergenic sequence.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Metabolic Regulation of the Th2 Response
Funder: Medical Research Council

Abstract: Immune responses to infection involve a careful balance. Too weak a response allows pathogen replication and persistence; too strong risks immunopathology. The damage caused by misdirected or inappropriate immune responses is evident in chronic inflammatory conditions such as inflammatory bowel disease and allergic lung disease. In Th2 immunity, the prevalence and chronicity of current helminth infections illustrates the pressing need for novel strategies to reinforce Th2 immunity, while the rising incidence of allergy and atopy also points to an urgent need for targeted interventions that restrict Th2 activity. New therapies that control Th2 immunity have the potential to provide significant health and economic benefit. The activation and differentiation of CD4+ T cells is accompanied by profound changes in cell metabolism, switching from a pattern of catabolic, fatty acid oxidation in naïve T cells, to an anabolic metabolism in activated effector cells that is dominated by aerobic glycolysis. Recent data indicates that the extent of this metabolic shift can influence both the size and the quality of T cell activation. Th2 cells in vitro are reported to be uniquely glycolytic, but Th2 glycolysis has not yet been characterized in vivo or in the context of infection. Our preliminary data suggest that Th2 cells in vivo behave differently to those activated in vitro, using little glycolysis despite being activated, effector cells. Our aim here is to characterise Th2 metabolism in vivo, identifying the factors that control Th2 glycolysis and defining the mechanisms by which metabolism influences Th2 function. We will test the hypothesis that manipulating T cell metabolism can control the strength and impact of the Th2 response. Together, our work will provide new insight into Th2 biology and will evaluate new targets for the therapeutic regulation of Th2 immunity.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Integrating participatory approaches and traditional models to strengthen One Health responses to zoonotic diseases in India's changing environments
Funder: Medical Research Council

Abstract: Diseases transmitted from animals to humans (zoonotic diseases) cause 60% of emerging infectious events worldwide and disproportionately affect people in tropical, resource-poor areas. As well as their impact on human and animal health, zoonotic diseases are detrimental to livelihoods and economies. Although we know that the number and effect of these diseases are increasing globally, we still lack knowledge on how these diseases circulate between wildlife, livestock and people, and the extent to which changes in the landscape and anthropogenic activities are affecting disease emergence and spread. Effective disease control strategies requires cooperation of all relevant policy-makers and managers from across the animal health, human health, agriculture and environment sectors, from national and international decision-makers down to district managers responsible for managing diseases at the local scale. Focussing in India as a key global hotspot for zoonotic diseases this project aims to reduce health, welfare and livelihood impacts of zoonotic diseases by better understanding links between surveillance, knowledge, research and models across sectors and improving current information systems that support intervention. The research underpinning these improvements will include: (1) Mapping of key stakeholders, their priorities and needs for decision-support tools (2) Identifying where relevant knowledge exists to better understand and manage zoonotic diseases (3) Understanding the full range of potential drivers that might cause disease impacts to increase (4) Interpreting patterns in disease impacts to predict outcomes of intervention (5) Building capacity across policy, research and practice. The project platform and approach of developing research and decision support tools together with the relevant stakeholders across sectors will build significant capacity in science, policy and practitioners to respond to these emerging and endemic global threats.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: INFECTION
Title: A multi-stakeholder approach towards operationalising antibiotic stewardship in India's pluralistic rural health system.
Funder: Medical Research Council

Abstract: Antimicrobial Resistance (AMR) is high on India's health policy agenda as the country has one of the highest burdens of bacterial infections in the world. This disease burden is potentially a risk factor for high antibiotic consumption leading to the emergence and spread of AMR. One of the greatest challenges for containing AMR in Low and Middle-Income Countries (LMICs) like India is the complex and pluralistic health system. Over 70% of the population seeks healthcare from private providers; of these, more than 70% do not have a formal medical qualification. These informal providers (IPs) use a variety of antibiotics and in an earlier study we found that the drivers of high antibiotic use included the pharmaceutical industry's aggressive promotion of antibiotics, the weak capacity and ethical dilemmas of the regulatory system to restrict 'excess' without ensuring 'access', the influence of formal doctors' often inappropriate prescribing practices and patchy public perceptions of antibiotics intertwined with providers' perceptions that favoured short term patient outcomes over long term risks. A quarter of IPs also treated animals. Solutions to this situation will necessarily need to engage with multiple stakeholders in building consensus and compromise to move beyond individual gains towards the greater common good. In this study we propose to co-design and pilot a multi-stakeholder antibiotic stewardship intervention to reduce and improve IPs antibiotic use in two rural sites in district South 24 Parganas in West Bengal. We will use a participatory methodology called Deliberative Mapping. The resulting intervention will be evaluated for its feasibility and acceptability, provider level outcomes, and for its effect on the stakeholders, individually and collectively. The evidence generated by this study will inform strategies for improving the performance of informal providers at scale in West Bengal and other Indian states and stimulate international debates.


Research activity codes: 3.1 PRIMARY PREVENTION INTERVENTIONS TO MODIFY BEHAVIOURS OR PROMOTE WELL-BEING
Health categories: INFECTION