NIHRDataInsights/HRCSData huggingface dataset:
Title: Single-molecule analysis of influenza virus transcription and replication
Funder: Medical Research Council

Abstract: We will study the molecular mechanisms of the influenza virus transcription and replication. The influenza virus belongs to the family of negative-sense, single-stranded RNA viruses, which include many important animal and humans pathogens that can cause severe disease. The influenza A virus genome comprises 8 segments of viral RNA (vRNA) with highly conserved 5' and 3' termini interacting to form promoter-like structure bound by the viral RNA polymerase (RNAP). The viral RNAP is highly conserved and may serve as an excellent target for developing antivirals. Despite the influenza virus being the best-studied negative-sense ssRNA virus, there are many questions about the molecular mechanisms of its genome transcription and replication, in part due to the lack of single-molecule studies. For example, little is known about the conformational changes in promoter RNA and the polymerase in early transcription and replication. Further, despite the recent discovery that replication of complementary RNPs requires an additional polymerase molecule, the role of that molecule is unclear. We will address these challenges using a combination of powerful single-molecule fluorescence methods and biochemical assays. Aided by recently published structures of the entire RNAP complex and large amounts of influenza proteins, we will prepare labelled polymerases and RNA, and test models of action, revealing rich mechanistic detail about influenza transcription and replication and enabling new ways to combat the disease. Our main tool will be cutting-edge methods, such as single-molecule fluorescence resonance energy transfer and tethered-fluorophore motion. Our studies will provide direct, real-time views of the viral genome duplication and reveal transient intermediates and structural states difficult to observe using other methods. Our work will also help understand polymerase mutations involved in enhanced avian-to-human transmission, with clear implications for human health.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: A genealogical approach to tracking bacterial transmission
Funder: Medical Research Council

Abstract: Whole genome sequencing (WGS) data is increasingly accepted as a powerful approach to track host-to-host transmission of bacterial infections. To correctly analyse WGS data, it is however necessary to account for both within-host evolution and between-host transmission. Here we propose a two-steps pipeline to perform such analysis: first a timed phylogeny is reconstructed based on the time-stamped WGS data, and then this tree is subdivided (i.e. 'coloured') into parts corresponding to the evolution going on in different hosts. For the first step, we propose to exploit the ClonalFrame model that includes the effect of recombination, and to use recently developed maximum likelihood techniques to efficiently reconstruct a timed phylogeny under this model from WGS data. For the second step, we propose to develop a Monte-Carlo Markov Chain (MCMC) algorithm that can colour a phylogeny while accounting for the possibility of missing transmission links, realistic epidemiological models, varying within-host pathogen population dynamics, and the possibility of an incomplete transmission bottleneck. This MCMC algorithm will also be extended to use other epidemiological information, and to allow some hosts to be sampled multiple times. The new pipeline will by applied to both simulated and real datasets, to guarantee its correctness, efficiency, relevance and general applicability to the large genomic datasets that are increasingly becoming available. The approach will be implemented in software that will be distributed freely and open source via the Internet. This software will be developed so that it does not require bioinformatics expertise to be used. This will guarantee that the new approach is available for microbiologists to apply it to a wide range of epidemiological investigations.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: The role of improved housing in the African malaria recession
Funder: Medical Research Council

Abstract: BACKGROUND: Since most malaria transmission occurs indoors in sub-Saharan Africa (SSA), housing quality is an important determinant of malaria risk, through its effect on house entry by mosquitoes. Unprecedented population expansion and rapid socioeconomic development in SSA presents an unrivalled opportunity to advance malaria control and elimination by building healthy homes. AIM: To investigate the role of improved housing in the African malaria recession. METHODS: Two principal data sources will be used: (i) the Demographic and Health Survey (DHS), Malaria Indicator Survey (MIS) and Multiple Indicator Cluster Survey (MICS) programs and (ii) the Repository of Open Access Data of the Malaria Atlas Project (ROAD-MAP II). First, the association between house quality and prevalence of malaria in children will be examined (a) in Upper River Region, The Gambia, using a 2010 parasitological survey of c3800 children and remotely-sensed imagery to classify house type and (b) in 22 countries in SSA, by applying multilevel modelling to MIS and DHS surveys. Second, changes in housing quality at the district level across SSA, 2000-2015, will be estimated by fitting housing data from georeferenced DHS/MIS/MICS surveys within a multilevel model using a Bayesian framework. Third, the relationship between housing improvements and changes in malaria endemicity in SSA, 2000-2015, will be modelled at three spatial scales using data from Objective 2 and ROAD-MAP-II. Fourth, the total number of malaria cases averted in SSA in 2015 due to housing improvements during 2000-2015 will be estimated using a spatiotemporal model and counterfactual framework. SUMMARY: The application of geospatial modelling to large existing datasets will generate novel evidence on the relationship between housing and malaria and the impact of housing improvements on malaria endemicity, yielding critical and timely policy recommendations for sustainable malaria control.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: INFECTION
Title: Bacterial pathogenesis: dissecting the early stages of cellular invasion by the obligate intracellular bacterium Orientia tsutsugamushi
Funder: Medical Research Council

Abstract: Scrub typhus is a leading cause of non-malarial fever in rural parts of Southeast Asia. Characterised in the early stages by fever, rash and myalgia, delayed or inappropriate treatment can lead to disseminated intravascular coagulation, multiple organ failure and mortality. Whilst highly responsive to tetracycline antibiotic therapy, rapid and unambiguous diagnosis remains a major challenge and an obstacle to high quality patient care. Scrub typhus is caused by the obligate intracellular bacteria Orientia tsutsugamushi that is carried and transmitted by Leptotrombidium mites. O. tsutsugamushi is a member of the Rickettsiaceae family, occupying its own genus to reflect significant differences in genome structure, cellular organisation and intracellular lifecycle compared with other Rickettsias. Pathogenicity requires entry into and propagation within mammalian host cells, and we plan to study the early stages of this process. Bacterial attachment to host cells is mediated by specific bacterial surface proteins, and is followed by a clathrin-mediated endocytosis-like mechanism. Bacteria exit from the late endosomal pathway, then induce and escape from host autophagy, and finally employ microtubule-directed motility to move through the cytosol. In this study we will use a combination of systems biology, cell biology and biochemistry approaches to study these early stages of bacterial infection. Specifically, we will use a genome-wide RNAi screen to identify host cell factors required for (i)entry, (ii)escape to the cytosol and (iii)bacterial motility. We will then perform a range of specific experiments to validate and dissect these molecular pathways. Finally, we will use biochemical approaches to identify specific host receptors that bind O. tsutsugamushi and induce bacterial internalisation. The outcome of our research will be a better understanding of the molecular pathways exploited by O. tsutsugamushi in the early stages of host cell internalisation.


Research activity codes: 4.4 POPULATION SCREENING
Health categories: INFECTION
Title: Treatment of multi-drug-resistant Gram-negative bacterial infections using capsule depolymerases
Funder: Medical Research Council

Abstract: The global emergence of multi-drug-resistant clones of Gram-negative bacteria in intensive care units has narrowed treatment options for these life-threatening infections. The burden on healthcare in Thailand is particularly severe and is increasing. For example, in 2010, over 90,000 hospitalized patients acquired such infections and one third died, resulting three million days of hospitalization. In Thailand, as elsewhere, the two most prominent responsible species are Klebsiella pneumoniae and Acinetobacter baumannii; isolates of both species initially colonise the gastrointestinal (GI) tract or lung as a prelude to invasion of the systemic circulation and are usually protected from patients' immune defences by polysaccharide capsules that comprise the outermost layer of the bacterial cell. We have shown that parenteral administration of depolymerase directed against capsules of bacteria causing lethal neonatal sepsis or inhalation anthrax resolve experimental infections; cure rates approach 100%. We wish to apply this technology to emergent Thai strains of K. pneumoniae and A. baumannii. To identify enzymes active against Thai clones, we will sequence the genomes of one hundred recent clinical isolates of each species and compare them with the global phylogeny. Bacteriophage that infect the most frequently encountered clones will be examined for the presence of capsule depolymerase: phage infecting capsulated bacteria usually produce enzymes enabling penetration of the capsular polysaccharide to facilitate viral docking on to the surface. Depolymerases will be purified and characterised, and encoding genes cloned to obtain recombinant enzymes from suitable vectors. We will develop rat models of GI tract and lung colonisation and systemic invasion, infect animals with prominent Thai clones and examine the impact of systemic administration of enzymes, alone and in combination, on the course of infection.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION
Title: The dynamic of anti-dengue antibodies over time.
Funder: Medical Research Council

Abstract: Dengue virus infection is a growing problem in many tropical and subtropical areas such as Thailand. Four dengue serotypes co-circulate. These 4 serotypes have 60-70% amino acid sequence homology. Primary infection with dengue virus is often mild and the more severe form of the infection, dengue haemorrhagic fever tends to occur when individuals are infection on a second or subsequent occasion by another dengue serotype. The antibody dependent enhancement (ADE) theory has been put forward as one explanation for this phenomenon. ADE occurs when pre-existing heterologous antibodies generated during a primary infection, may not be of sufficient avidity or concentration to neutralize a secondarily encountered virus. Studies show that serum from an acute dengue infection frequently show cross-reactivity and neutralize a number of dengue serotypes. However the heterologous immunity wanes over the first year following infection leaving serotype-specific immunity to the previously encountered serotype. Interestingly, surveillance suggests symptomatic cases caused by third or fourth infections are rare, implying that following secondary infection there is some degree of cross protection towards the remaining serotypes. Non structural protein 1(NS1) has been suggested to have effects on complement activation. In this application, we have proposed: 1. Generation of human monoclonal antibodies using 2 techniques, EBV transformation and molecular cloning of immunoglobulin genes as described by Lanzavecchia and Wilson respectively 2. Characterisation of human monoclonal antibodies: antigen recognition, epitope mapping, neutralization, antibody dependent enhancement 3. Comparison of antibody repertoires over a time course and between symptomatic and asymptomatic cases 4. Understanding the role of anti-NS1 antibodies in pathogenesis and protection during secondary infection


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Applying New Tools to Identify Inhibitors of Antimicrobial Resistance Plasmid Transmission or Stability in Gram Negative Bacteria
Funder: Medical Research Council

Abstract: Antimicrobial resistance (AMR), especially by Gram-negative bacteria, poses a threat to human health. Furthermore, developing new antimicrobials has proved increasingly difficult over the past few decades. Therefore, innovative strategies to address the growing problem of AMR are needed. This project uses a new fluorescence based reporter system constructed using three clinically relevant Gram-negative associated global AMR plasmids (pCTblaCTX-M-14, pNDM-HKblaNDM1, and pKpQILblaKPC) which encode genes conferring resistance to some of our 'last line of defence' antibiotics. These tools will be used to screen for novel compounds that inhibit AMR plasmid transmission and/or stability. This will be done in a high throughput fashion at the University of Birmingham Drug Discovery Facility, where we will screen approximately 40,000 compounds, from the Diversity Library and the FDA approved Prestwick Library. Flow cytometry will be used to analyze compound effects on AMR plasmid transmission and stability. Identified target compounds will then be verified, and the specificity of their effect will be determined. Our preliminary work shows that the fluorescent reporters and affiliated flow cytometry assays are an effective way to determine plasmid stability and transmission. The work proposed in this project has significant implications for global levels of AMR. Identified compounds could be used in a number of applications including but not limited to: animals feed, farming, veterinary medicine, waste water treatment and possibly human medicine. Our group has established collaborations that will allow these applications to be examined in future work. Using compounds that reduce the prevalence of AMR genes in people and the environment will sensitize bacteria to already existing antimicrobials.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Neutralization of Apx toxicity as an alternative to antibiotics for control of contagious porcine pleuropneumonia.
Funder: Medical Research Council

Abstract: The aim of this project is to modify the Apx toxins of Actinobacillus pleuropneumoniae to remove the immunologically dominant part of the molecule, which is interfering with stimulation of a neutralizing antibody response that is needed for vaccination to be effective. In this project we will develop a dermal oedema test for Apx toxin and its neutralization. We will also test the effect of those forms of modified Apx found to be able to generate an appropriate response in protection against pleuropneumonia in the target species. Contagious porcine pleuropneumonia (CPP) is a severe disease that kills growing pigs and causes lesions in the pleura which require trimming on the slaughter line. Pig herds can and do become uneconomic and are regularly closed down as a result of losses incurred through morbidity and mortality following infection. Efforts to control CPP by vaccination have been disappointing and vaccines have not yet demonstrated adequate levels of protection. This reults in farmers resorting to use of in-feed prescription antimicrobials, including lincomycin, tylosin, tilmicosin, amoxicillin, chlortetracycline, together with injectables such as florfenicol, the floroquinolone enrofloxacin, and the macrolide tulathromycin. It is essential that to halt this widespread use of prescription antimicrobials in the pig industry, both in Europe and worldwide, and develop alternative strategies for control of this disease. This project is designed to test the hypothesis that neutralizing epitopes of the Apx toxins will be more effective in generating a protective antibody response in pigs compared with the whole toxoid. Recombinant proteins, containing ApxI-III fragments will be generated fused to diphtheria toxoid CRP197. These will be used to immunize pigs, the response measured and the effect of vaccination tested in a novel model of dermal oedema and then in a well-refined challenge model of CPP in the pig.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION
Title: Rapid assessment of phage for combating antimicrobial resistance in Enterobacter cloacae using a novel insect model
Funder: Medical Research Council

Abstract: This proposal will exploit a validated experimental evolution system based on a marked insect adapted strain of the enteric commensal Enterobacter cloacae. It will address how best to deploy phage therapy for the purpose of preserving the efficacy of antibiotics. Experiments will track the spontaneous de-repression of ampC beta-lactamase or the frequency of an IncF plasmid that confers resistance to a wide range of antibiotics. Repeated infection (passage) experiments will measure bacterial clearance and density, transmission, the frequency of antibiotic and phage resistance and the effect of phage on conjugation rates of evolved clones. We will use experimental evolution to assess a range of ways of deploying bacteriophages to slow the evolution of resistance. This will include the use of plasmid-dependent MS2 phage that specifically targets F-plasmid carrying bacteria as well a general lytic phage. We will investigate how to best to deploy antibiotics combination with phage, and will test the prediction that prior treatment with phage is best for limiting spontaneous resistance, while simultaneous or post-antibiotic phage treatment will be more effective at minimizing the transmission of plasmid-encoded resistance. We can rear gnotobiotic insects that are infected with our focal E. cloacae strain and also establish aliquots of a pooled diverse enteric community that can be used to orally infect insects with more diverse enteric microbial communities. We predict that the presence of diverse community might impose between-species competition that could increase the efficacy of phage. We will assess the impact of a diverse microbiota on the efficacy of phage-based resistance management using either a general or a plasmid specific phage. In addition to assessing clearance and evolution of resistance in our focal strain we will confirm differences in microbial diversity between treatments using 16S metagenomics.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION
Title: Improving the management of sepsis through rapid pathogen and antibiotic resistance detection in blood
Funder: Medical Research Council

Abstract: Next generation sequencing (NGS) based pathogen identification in bloodstream infection is very challenging owing to the vast amount of human DNA present compared with pathogen DNA (the ratio can be as high as 10^9:1). Therefore, pathogen DNA enrichment is crucial and we are developing novel strategies to achieve this, removing the vast majority of the human DNA from blood and reducing the ratio of human:pathogen DNA < 10:1. This will enable us to develop sequencing based workflows for the rapid and accurate diagnosis of sepsis which will result in reduced patient morbidity and mortality. Objectives: (1) Further develop and optimise our current pathogen DNA enrichment strategy and to test two new enrichment strategies (2) Test a number of NGS technologies/platforms to determine the most suitable in terms of analysis time, flexibility, complexity of bioinformatics analysis, cost and comprehensiveness of sequencing results (3) Run a clinical diagnostics evaluation study of an optimised NGS based diagnostic method Objective 1: Method 1 (current method): We will continue to develop the current method and determine the limit of detection of this method. Method 2: We have devised a second novel approach to pathogen DNA enrichment involving the removal of human DNA using nucleic acid capture. Method 3: This approach is designed to ensure detection of both intracellular/phagocytosed pathogens and cell free pathogen DNA while readily removing human DNA. Objective 2: Short read (Illumina MiSeq) sequencing technology will be compared to nanopore (MinION and PromethION) sequencing technology to determine which is best suited to infectious diseases diagnostics. Objective 3: The performance of the optimised sepsis diagnostic workflow will be tested on 50 biobanked blood samples from sepsis patients and controls. The arising NGS results will be compared to blood culture, PCR (Septifast) and the clinical gold standard diagnosis.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Mathematical modelling and spatial data analysis to inform TB care and control strategies in high TB incidence settings
Funder: Medical Research Council

Abstract: The overall aim of my Fellowship is to determine how spatial data from TB cases presenting at clinics can best be used to improve the impact and cost-effectiveness of TB care and control interventions in high incidence settings. My objectives and methods are: Theme 1: Using spatial data analysis to understand the distribution of TB, and the effects of TB care and control interventions. 1a: Investigate the distribution of TB disease, and of individual and population level TB risk factors. 1b: Use existing data to understand the spatial and temporal effects of completed community and household based TB case finding interventions. 1c: Identify and cost candidate spatially targeted intervention approaches. Theme 2: Using mathematical modelling to inform the development of spatially targeted case finding strategies. 2a: Develop a spatially structured mathematical model of M. tb. transmission in high incidence settings 2b: Investigate the impact and cost-effectiveness of spatially targeted TB care and control interventions. I will use datasets from four high incidence African settings, all of which have data on the locations of TB cases' households. They are the ZAMSTAR study populations in South Africa and Zambia, and Blantyre and Karonga in Malawi. The work will identify strategies that could improve the impact and cost-effectiveness of TB care and control measures, using spatially targeted interventions. A funding proposal for a pilot study of one or more of the approaches suggested by my work will be developed near the end of the Fellowship.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY);3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: INFECTION