NIHRDataInsights/HRCSData huggingface dataset:
Title: Assembly Cofactors of HIV-1
Funder: Medical Research Council

Abstract: 1) Identifying the RNA(s) that bind HIV-1 Matrix (MA) in living cells. We will use transfected cells to identify RNA(s) and define binding sites at nucleotide-resolution, using a modified iCLIP protocol coupled with next generation sequencing on the MiSeq platform. After demultiplexing and removal of PCR duplicates, sequences will be aligned to the human and HIV-1 genomes. Partitioning of identified RNA(s) to the cell membrane will be investigated using biochemical techniques such as membrane flotation coupled with RT-qPCR; and microscopy techniques, involving tagging RNAs of interest with MS2 loops for visualisation by fluorescent MS2 coat protein. We will use techniques such as si/shRNA and CRISPR to investigate the effect of knockdown of these RNAs on HIV-1 assembly and infectious virion production, as well as transfection of 5'-O-methylated antisense oligonucleotides to block the RNA-MA interaction. 2) Characterising the HIV-1 gRNA-bound proteome in living cells. We will use transduced cells and an MS2-based RNA purification protocol, coupled with protein tagging with TMTsixplex labelling reagents and identification by mass spectrometry (Orbitrap Velos Pro) to identify proteins that interact with HIV-1 gRNA. Peptides will be identified and quantified using MaxQuant/Andromeda software. Proteins that specifically interact with gRNA will be knocked down to assess their effects on virus assembly. The mechanism(s) of action of positively-acting cofactors will be elucidated using a range of well-established molecular virology approaches. We will also perform pairwise comparisons of the gRNA-associated proteome in assembly permissive/non-permissive conditions to further enrich for assembly cofactors. Here, we will take advantage of the fact that murine cells are non-permissive for HIV assembly. Where functional differences between human and murine proteins are found, structure-function analyses will be undertaken to map the motifs responsible.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Investigating the epigenetic regulation of pneumococcal virulence
Funder: Medical Research Council

Abstract: The important human pathogen Streptococcus pneumoniae has a clear in vitro phenotype which correlates with virulence, i.e. opaque colonies correlate to invasive infection and transparent colonies to carriage. We have clear preliminary data of an epigenetic phase variable regulatory system which has a major impact on virulence and upon the colony opacity phenotype. The mechanism of variation is based upon recombination within a restriction modification enzyme subunit gene which allows any pneumococcal population to separate into six coexisting genetically indistinguishable subpopulations. The overall aim of the project is the determination of the mechanisms of epigenetic regulation of S. pneumoniae by the phase variable type I restriction modification system SpnIII. To achieve this aim we will investigate both (i) the molecular mechanisms by which SpnIII modulates gene expression and variations of pneumococcal phenotypes, and (ii) the function of the SpnIII HsdM and HsdR enzymes and the mechanism which allows the re-assortment of the six HsdS variants. Technically we will use a series of mutant strains each expressing just one of the six possible HsdS hybrids, mine databases of target site conservation, exploit SMRT sequencing to define adenine methylation, use RNAseq to monitor gene expression and perform selective substitution of chromosomal methylation targets sites for validation. The functionality of SpnIII will be explored using gene-scan allele quantification, in vitro protein production, and modification and restriction of plasmids containing panels of synthetic SpnIII target sites. These two approaches should help to elucidate SpnIII epigenetic regulation and will allow mathematical modelling of the bistable regulatory circuit which is the basis of the pneumococcal colony opacity phenotype. The integration of biological data and modelling is expected to shed light on the significance of subpopulations in the virulence of this important human pathogen.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: A systems biology approach to understand immunity and pathogenesis of malaria in children
Funder: Medical Research Council

Abstract: We will examine immune response profiles of children developing immunity to Plasmodium falciparum malaria, in order to define a response pattern that distinguishes children who have suffered very frequent clinical malaria episodes from those who have an average number of clinical. We will then sample a cohort prior to surveillance, store the samples, and then test once clinical data are available to examine whether these profiles can prospectively predict multiple clinical episodes and failure to acquire immunity. Using RNA sequencing we will determine the transcriptome profile of the immune response. This will be combined with flow cytometric analyses on leukocytes in whole blood, and measurements of cytokines, chemokines and P. falciparum specific B cell/antibody responses. At each analytical step we will identify potentially important 'predictors', which could be individual up- or down-regulated genes, gene clusters, regulatory pathways etc. These will be collated and iteratively used to build up our predictive model. We will concentrate on the most significant pathways for more in depth analysis by developing mathematical descriptions for pathways and then predict the effects of perturbations in the study population. Detailed cytometric analyses (CyTOF and Flow) and functional B-cell and T-cell assays will be performed, and will inform mechanistic animal model studies. During this study we will develop novel analytical tools and train scientists in systems immunology, build up large databases of childrens' immune responses, and define biomarkers that will predict frequent malaria episodes in children.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Genetic, structural and chemical validation of Aspergillus fumigatus cell wall targets
Funder: Medical Research Council

Abstract: The cell wall of the opportunistic fungal pathogen Aspergillus fumigatus is a dynamic and multi-layered structure consisting of the sugar polymers chitin, glucan and (galacto)mannan. Despite decades of work, there are huge gaps in our knowledge of the enzymes responsible for cell wall biogenesis. This is particularly pressing given the significant rise of fatal A. fumigatus infections in immunocompromised patients, and thus the need for novel, properly genetically and chemically validated, drug targets. This proposal uses a multidisciplinary approach (covering genetics, biochemistry, structural biology, fragment-based inhibitor discovery, synthetic chemistry and cell biology) to genetically, structurally and chemically validate A. fumigatus enzymes involved in fungal cell wall biogenesis as targets. The proposal covers three areas: UDP-GlcNAc biosynthesis - exploiting a significant body of preliminary data to use fragment-based approaches to generate tools for chemical validation of this pathway. Cell wall synthesis - with the aims of generating overexpression systems and crystal structures for enzymes involved in chitin/glucan synthesis and developing novel assays to discover inhibitors that will aid in chemical validation of these targets. Cell wall modification - with the aims of genetically and chemically validating chitinases, chitin deacetylases and glucan transglycosylases. The output will be: 1) A significant increase in our molecular understanding of fungal cell wall biogenesis enzymes, 2) A collection of novel chemical tools for use by the fungal scientific community, and 3) A portfolio of targets (and associated strains, protein structures and chemical starting points) that can serve as a springboard for novel antifungal drug discovery by follow-up translational research and/or the pharmaceutical industry.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Continued follow-up of the United Kingdom Collaborative HIV Cohort (UK CHIC) and the UK HIV Drug Resistance Database (UK HDRD), 2014-2019
Funder: Medical Research Council

Abstract: Whilst current combination antiretroviral (cART) regimens for HIV are extremely effective, there are many questions that remain to be addressed to ensure the optimal outcomes of HIV-positive people. In order to plan the need for health care services and to understand the need for new drugs, it is essential that any changes in the characteristics of the UK epidemic are monitored. These changes may arise from the changing demographics of the HIV-positive population, an increase in viral diversity, the introduction of new drugs, generic forms of existing drugs and changing treatment paradigms (e.g. a move towards earlier initiation of cART), as well as increased use of cART drugs as part of pre- or post-exposure prophylaxis against HIV. Surveillance of clinical and resistance outcomes is therefore required to inform national treatment policies. The primary aim of this application is to continue to expand and maintain the combined UK CHIC and UK HDRD database in order to answer a wide-ranging spectrum of questions relating to the clinical outcomes of those with HIV in the UK. The secondary aim is to continue to ensure that the UK plays a leading role in international HIV cohort research through the provision of high quality data and scientific input to major collaborative studies. The proposed research will be conducted in an environment at University College London that provides expertise in surveillance and audit studies, surveys of sexual behaviour, randomised trials, qualitative methods, mathematical modelling, behavioural and basic science research. UCL provides access to information technology and bioinformatics capability through the Farr Institute, with UCL Platform Technologies providing biobanks and informatic support. This setting therefore provides an ideal environment in which to conduct large-scale HIV cohort studies, with opportunities to translate study findings into interventions for those living with HIV and for wider public health benefit.


Research activity codes: 2.4 SURVEILLANCE AND DISTRIBUTION
Health categories: INFECTION
Title: First Line Antimicrobials in Complicated Severe Acute Malnutrition (FLACSAM)
Funder: Medical Research Council

Abstract: The WHO currently recommends all children with severe acute malnutrition (SAM) receive broad-spectrum antibiotics. At the four hospitals for the proposed trial, 15-18% of admissions aged 2-59 months have SAM, of whom 20-25% have HIV. SAM dramatically increases the case fatality of all common clinical syndromes with overall case fatality up of to 30%. Non-susceptibility to the recommended first-line empiric antimicrobials (penicillin plus gentamicin) is widespread. Our long-term surveillance in Kenya suggests that 38% of bacterial isolates from children with SAM are non-susceptible to this combination, compared to <14% to ceftriaxone. There are no tests that accurately indicate the presence of invasive bacterial infection or non-susceptibility within the vital first few days. Ceftriaxone is a potential alternative first-line antibiotic. Some centres are already changing policy without data on susceptibility or outcomes. Ceftriaxone is given once rather than four times daily, and is now inexpensive. However, a significant concern is the induction of resistance to multiple antibiotic classes, increasing costs and threatening our ability to treat serious infections. Any benefits of reduced mortality must be weighed against potential risks of infections that are difficult and expensive to treat. A second outstanding question in the management of SAM is the use of oral metronidazole. It is active against anaerobic bacteria that cause small bowel overgrowth, and against Giardia. Small cohort studies suggest growth benefits However, metronidazole can cause nausea, anorexia and hepatic toxicity, potentially impairing recovery. Importantly, the one pharmacokinetic study in SAM, reported delayed elimination. We propose a 2x2 factorial trial of ceftriaxone and metronidazole versus standard care, with primary outcomes of mortality and anthropometric recovery. We will examine pharmacokinetics; predictors of infection; antimicrobial resistance; and cost-effectiveness.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Rapid Urine-Based Screening for Tuberculosis to Reduce AIDS-Related Mortality in Hospitalized Patients in Africa (STAMP) Trial
Funder: Medical Research Council

Abstract: TB underlies 30%-67% of HIV/AIDS-related deaths among medical in-patients in hospitals across sub-Saharan Africa and much of this remains undiagnosed. Background studies in South Africa have shown major increases in diagnostic yield of TB are gained from rapid urine-based screening in such patients using a combination of the Determine TB-LAM lateral-flow assay and testing urine (40 ml concentrated by centrifugation) with the Xpert MTB/RIF assay. We hypothesize that urine-based screening will increase the yield and speed of TB diagnosis, accelerating appropriate medical management and reducing mortality risk. However, no studies have yet demonstrated improved clinical outcomes from use of new TB diagnostics with superior sensitivity. Thus, a trial is needed. This pragmatic individually randomised controlled blinded trial will compare between arms the survival of medical in-patients admitted to two regional hospitals in South Africa and Malawi and randomised to one of two diagnostic TB screening strategies on day one of admission. Those in the standard arm will have sputum tested with Xpert whereas those in the intervention arm will, in addition, have urine-based screening using Determine TB-LAM and Xpert. The routine medical teams will be informed of laboratory results as soon as possible to inform treatment decisions but they will remain blinded to study arm allocation. Treatment for TB in the absence of microbiological confirmation will be at the discretion of the medical team. Clinical outcomes will be ascertained during hospital admission and during out-patient follow-up and the primary outcome is all-cause mortality at 56 days after randomization. The findings will be of immediate relevance to public health policy both nationally and internationally and will be underpinned with economic analyses. Both diagnostic assays used are commercially available and Xpert is being widely implemented. Thus, implementation of this strategy is feasible and scalable.


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES;4.3 INFLUENCES AND IMPACT
Health categories: INFECTION
Title: Can improved housing provide additional protection against clinical malaria over current best practice? A household-randomised controlled trial
Funder: Medical Research Council

Abstract: Malaria remains one of the greatest threats to global public health, with 207 million cases of falciparum malaria and 627,000 deaths occurring in 2012, with over 80% of deaths occurring in Africa. More than 80% of malaria is transmitted indoors at night, so the number of infective mosquitoes which enter a house is critical for getting the disease - with people living in 'leaky' houses being at most risk. We have shown that closing the eaves (the gap between the top of the wall and the roof) and screening a house can reduce dramatically the number of malaria vectors entering a house. Here we propose to determine whether we can reduce the incidence of clinical malaria in children living in well-screened houses, which have a (1) a metal roof, (2) closed eaves, (3) screened doors and windows and (4) screened-air bricks which allow the warm air to rise out of the house, but not let any mosquitoes indoors. The control houses will be left with thatched roofs and open eaves. We will also carry out subsidiary studies to find out whether the interventions are cost effective and develop a strategy for scaling-up housing interventions. Across Africa a silent revolution in house design has been happening for several decades. The traditional thatched-roofed houses are being replaced by metal-roofed houses as the continent develops. We hope to ride this wave of cultural change and further improve the design of houses to make them healthier to live in. Improved housing has the potential to improve the lives of millions of people across sub-Saharan Africa.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: INFECTION
Title: A novel vaccine for broad protection against meningococcal disease: progression to phase I clinical trial
Funder: Medical Research Council

Abstract: The aim of this project is to progress a novel vaccine against serogroup B meningococcal disease (MenB) to Phase I clinical trial. The need we are addressing is prevention of MenB disease, the leading infectious cause of death in children in the UK. Two vaccine candidates are under development or recently licensed, based on recombinant proteins and outer membrane vesicles. The recently licensed vaccine may be not cost-effective as it requires up to 4 injections, and the other one was developed for adolescents only. Therefore both have serious limitations in the goal of cost-effective protection of the population at most risk, who are infants under one year of age. Ongoing development of alternative vaccine candidates is therefore required. Our proposed solution is to use a vectored delivery platform expressing a major meningococcus antigen target. A vector which induces both innate and adaptive immune responses in mammalian hosts, including concomitant induction of robust IFN-gamma responses, could provide the ideal conditions for switching B cells to produce complement-fixing bactericidal antibody, unlike conventional unadjuvanted or aluminium-based adjuvanted vaccines. A single dose of such a vectored vaccine has been shown to induce strong and functional antibody responses. A vectored vaccine is also a versatile platform that allows rapid adaptation to changing epidemiology. We have already demonstrated in pre-clinical models that the approach is feasible with a conserved antigen: a single dose induces a strong, functional and long-lived antibody responses, far exceeding those obtained with conventional outer membrane vesicle-based vaccines. Our development plan is now to progress this vaccine candidate to a Phase I clinical trial. We propose taking an optimized vectored vaccine, designed to induce broad protection against heterologous strain and suitable for human use, to GMP manufacture and obtain regulatory and ethical approval to carry out a phase I trial


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Natural Killer (NK) cell regulation of antiviral T cell responses in the pathogenesis of HIV infection
Funder: Medical Research Council

Abstract: Aims objectives: Accumulating evidence indicates the involvement of NK cells in the control of viral infections and shaping of adaptive immunity. Our recent work was the first to identify a novel pathway for direct NK cell regulation of T cells in chronic HBV infection in humans. These emerging data challenge the paradigm of NK cell function during viral infections and may be particularly relevant in HIV infection. We aim to investigate the ability of NK cells to limit the antiviral response in HIV infection and the role of immune activation in driving NK cell mediated T cell lysis. We will also explore the role of Killer Immunoglobulin Receptor (KIR) diversity in controlling NK cell regulation of T cells in HIV, providing a mechanistic model for the striking genetic KIR/HLA associations influencing HIV outcome. Design and methodology: We will utilise PBMC from four diverse and well-characterised cohorts of HIV infected individuals (early, established HIV with different levels of viraemia, well controlled on cART, and elite controllers) to help us evaluate the mechanisms and triggers that could regulate this process during dynamic disease phases. Functional experiments with blocking antibodies and NK cell depletion will allow us to determine the capacity of NK cells to kill autologous virus-specific T cells from patients with different disease profiles, and to investigate whether HIV-specific T cells are more susceptible than T cells of other specificities in the same patients. Subgroup immunogenetic analyses will help determine any independent effects, especially of HLA-C allotypes, ligands for the inhibitory 2 domain KIRs. Scientific and medical opportunities: Uncovering the precise mechanisms in place that regulate NK cell mediated immune suppression of T cells during HIV infection will facilitate the development of novel therapeutic strategies that can supplement ongoing efforts to restore antiviral immunity and reduce disease burden.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: The role of LC3 Interacting Regions in Viral Subversion of Autophagy
Funder: Medical Research Council

Abstract: The molecular mechanisms by which viruses subverts autophagy remain largely unknown. I have discovered that binding of the C-terminus of the Matrix 2 (M2) to ATG8-like molecules occurs via LC3-Interacting Region (LIR), and have shown that this has a role to play in viral stability, filament formation and presumably therefore the critical step in the viral life-cycle of transmission between hosts. The M2 LIR also seems to mediate a process by which the plasma membrane becomes LC3 positive, a phenomenon which we describe for the first time. The utilization of a LIR to subvert autophagy by a virus may be a widespread viral strategy, as an accessory matrix protein of Ebola and a type II membrane protein of HSV also appear to contain a LIR motif. This project will explore these convergently evolved strategies in an effort to understand better the way in which these important human pathogens interact with the cell; exploiting technologies recently developed in the host institutions.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION