NIHRDataInsights/HRCSData huggingface dataset:
Title: 'See and Sequence' - Genomic surveillance of antimicrobial resistant and high-risk pathogenic clones within the Philippines
Funder: Medical Research Council

Abstract: The challenges facing public health microbiologists when tackling pathogenic microbial species can be broadly summarised as 1) identification of high-risk lineages, 2) their risk assessment (eg., antimicrobial resistance) and 3) infection control. Whole Genome Sequencing (WGS) of bacterial species provides signatures of resistance and virulence and, uniquely, allows the build up of a 'digital library' for ongoing population comparison, which can be utilised to identify markers for rapid diagnostics. We will facilitate the implementation of WGS within AMR surveillance and clinical diagnostics within the Philippines Antimicrobial resistance surveillance laboratory network (22 sentinel laboratories) and develop bioinformatics to enable local, national and international comparison of data for key indicator species (Salmonella, N. gonorrhoeae, Carbapenem resistant species and S.aureus). The proposal consists of 4 major parts: 1) Establishment of sequencing capacity and expertise in Genomic analysis within the National Reference Laboratory. 2) Retrospective sequencing of susceptible and resistant isolates of key species (to be undertaken at The Sanger Institute). 3) Provision of data throughout the surveillance network using tools developed at Sanger and Imperial College (eg www.microreact.org/project/NJqiECX8 and www.wgsa.net ) 4) Active Prospective sequencing within the Philippines - isolates with novel antibiograms determined by routine susceptibility testing via a 'save for sequencing' flag within the WHONET software. 5) Capacity building in genomics, bioinformatics and sequencing (through staff exchange and training between locations and).


Research activity codes: 2.4 SURVEILLANCE AND DISTRIBUTION
Health categories: INFECTION
Title: Streamlining the detection of latent HIV and investigating the causes of ineffective reactivation
Funder: Medical Research Council

Abstract: I am currently working to confirm that viruses from each latently infected cell are genetically distinguishable from each other. I am also working with my collaborators to define and reduce the errors introduced by virus outgrowth, cDNA and amplicon generation and next generation sequencing. After gathering the pilot data, I will proceed to characterise the repertoire of reactivated viruses in patient blood samples by next generation sequencing. I will look for an increase in the number of distinct viral sequences in a sample after viral reactivation and correlate it with the reactivatable viral load determined by virus outgrowth assay. I will use the cultures generated in virus outgrowth assay to study the causes of ineffective reactivation. I will harvest intracellular RNA from cultures which viruses have not been reactivated, characterise and sequence any viral transcripts detected. I will look for mutations consistently present in viral intracellular RNAs from non-reactivatable cells. I will examine the integration sites of the viruses which have been successfully reactivated. To facilitate this, I will expand the copies of genome available for analysis using low dose IL-7, which causes cellular proliferation without disrupting viral latency. To obtain the integration sites, I will modify a published method that involves DNA digestion, linker ligation, PCR amplification and sequencing. I will optimize bisulphite genomic sequencing using a cell line model of HIV latency. Once optimized, I will perform bisulphite genomic sequencing on 5'LTR and Env in ex vivo cultures from virus outgrowth assay, correlating base modification with reactivability of the virus. I will also correlate DNA base modification with viral transcription activity. I aim to seek further collaboration to study the role of different base modifications such as CpG methylation and hydroxymethylation in HIV gene expression at single cell level.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Can community-wide active case finding for tuberculosis and universal testing and treatment for HIV control the African tuberculosis epidemic?
Funder: Medical Research Council

Abstract: Current strategies are failing to control the HIV-driven African TB epidemic. Two important control measures are active case finding for TB (ACF) and antiretroviral therapy (ART). ACF involves systematic TB screening, to identify and treat infectious cases early and prevent the onward spread of TB. ART decreases the susceptibility to TB among people living with HIV. However, their effect on TB epidemiology is unclear. In HPTN071 a three armed (2 intervention and a control arm) cluster randomised controlled (CRT) HIV-prevention trial in 21 Zambian and South African communities, a community-wide intervention package (including universal testing and treatment for HIV [UTT] and ACF) is being implemented in the intervention arms from 2014-2018. The control arm receives the current standard of care. Intervention effects on HIV incidence will be measured among an adult (18-44 years) cohort (N=52500), followed-up for 36 months from all 21 communities. HPTN071 provides an unprecedented opportunity to determine the combined effect of community-wide ACF and UTT on TB epidemiology (incidence and outcomes). The objectives of this project nested in HPTN071 are to: 1. Determine ACF and UTT effects on population-level TB incidence: effect on TB incidence in the adult cohort (N=52500) and on routine TB case notification rates 2. Determine ACF and UTT effects on TB case characteristics, fatality rates and treatment outcomes 3. Use mathematical modelling to describe the current and projected ACF and UTT effects on TB prevalence and incidence This project involves secondary data analysis using routine and HPTN071 research data. Statistical methods for CRTs will be used. The HPTN071 TB model that has been developed will be refined, parameterised with all study data and used to estimate and project intervention effects on TB epidemiology. This study will provide data to a pressing Global Health question and inform the TB prevention landscape in sub-Saharan Africa.


Research activity codes: 4.4 POPULATION SCREENING
Health categories: INFECTION
Title: The roles of the pre-genomic RNA in Hepatitis B Virus nucleocapsid assembly.
Funder: Medical Research Council

Abstract: This is a two year pump-priming grant to enable us to explore the roles of the HBV pre-genome in assembly of the nucleocapsid. Our objectives are to: define precisely the recognition motif in the RNA packaging signals and their role(s) in binding either the CP or the polymerase, using a combination of bioinformatics, smFCS & cryo-EM, and the consequences of those interactions for efficient assembly of the T=4 HBV nucleocapsid. The role(s) of the polymerase-epsilon complex as well as phosphorylation of the CP in RNA binding will also be examined. In addition we will screen, using mass spectrometry, small numbers of known drug ligands likely to bind nucleic acids for binding to PSs. This work will lay the foundations of a novel anti-viral strategy for treatment of HBV infection.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Establishment of adeno-associated virus latency by the recruitment of KAP1
Funder: Medical Research Council

Abstract: Adeno-associated virus (AAV) is a human dependovirus that appears to have evolved an optimal working relationship with host and helper virus. With the support of viruses such as adenovirus and herpes virus, AAV replicates successfully, causing a cytopathic effect only in those cells which were already destined to suffer from helper virus infection; in the absence of co-infection the virus remains latent without causing any apparent harm. It was generally thought that latency is established through integration into the human genome, however our preliminary data indicate that latency of AAV might already be established prior to integration through interactions with the transcriptional co-repressor KAP1, which render the virus 'invisible' until conditions for replication are met. In addition, we have found that relief of repression is achieved in part through actions of the AAV Rep proteins, which lead to phosphorylation of KAP1. We propose experiments to further investigate the mechanism of KAP1-mediated repression of AAV, to determine a role for helper factors in the release of KAP1-mediated repression of the AAV genome and unravel the mechanism of Rep-mediated phosphorylation of KAP1. Knowledge gained from this proposal does not only establish a new concept in AAV biology but will also inform virus-host interactions of other DNA viruses, including pathogenic viruses. In addition, AAV has become a frontrunner as a vector for human gene therapy based on results from a number of clinical trials. However, the research community around AAV biology has remained very small and thus limited molecular insights into its life cycle is available. As an example of how an understanding of the biology of AAV can further the application of AAV vectors is demonstrated by our finding that KAP1 knockdown leads to increased recombinant AAV vector replication, an observation that could be exploited to help overcome the challenges of production for wide clinical use.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Genomic RNA control of HIV viral assembly and export
Funder: Medical Research Council

Abstract: Production of an infectious HIV viral particle requires the inclusion of a dimeric RNA genome. Capture of the genomic RNA (gRNA) by a small number of molecules of the structural protein Gag appears to be the initiating event. However, Gag interacts with only a small proportion of the gRNA and is joined by cellular proteins to form the trafficking ribonucleoprotein complex that arrives at the viral budding site. At the plasma membrane a series of cellular proteins including those of the ESCRT series and associated factors such as ALIX cooperate to facilitate virus export. There is growing evidence that some of these proteins have RNA binding capability which is critically involved in successful virus production and also that the presence of the viral genome facilitates the correct cleavage rates of the protease on the structural proteins. We have identified RNA interactions with some of these factors and there is published evidence that supports others. We plan to take an RNA focussed view of viral production and to seek the interactions of the RNA genome with these factors by structural and functional analyses. We will determine which interactions are essential for virus export and would thus be possible targets for antiviral therapies. Using techniques based on cross linking and immunoprecipitation (CLIP) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) as well as super resolution microscopy and crystallography we aim to build up a picture of the components and exact structure of the encapsidation complex which allows specific packaging of HIV genomic RNA. Our aim is to understand the interactions of the viral genomic RNA with cellular proteins. Ultimately this would lead to development of small molecule screens targeting those interactions analogous to the work we have performed on HIV packaging which has led to a collaboration with GSK seeking inhibitors of the RNA/protein interaction involved in genomic RNA encapsidation.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Rational Design, Synthesis and Biological Evaluation of Benzimidazoles; Towards a Novel Therapy Selectively Targeting C. neoformans beta-tubulin
Funder: Medical Research Council

Abstract: The aim of this proposal is to carry out the early stage drug discovery activities required to ultimately develop new agents for cryptococcal meningoencephalitis for use in sub-Saharan Africa. The project addresses the need for novel therapies to treat the systemic fungal infection C. neoformans that causes lethal meningoencephalitis. In sub-Saharan Africa C. neoformans causes more deaths annually than tuberculosis and is the most common cause of systemic mycosis in patients with AIDS. An estimated 1 million cases of cryptococcal meningitis occur worldwide each year. It has been reported that selected benzimidazole compounds have good in vitro potency against C. neoformans and this has been confirmed through our own in house MIC screening. Benzimidazoles have been shown to disrupt mitosis in C. neoformans through binding to the beta-tubulin subunit of microtubules. Subsequently the C. neoformans beta-tubulin genes have been characterised. More recently, unpublished studies have shown flubendazole to be active in vivo against C. neoformans in the mouse model. Identification of an active class of compound, against a known target, with established in vitro and in vivo activity provides a solid platform for a targeted medicinal chemistry programme. The benzimidazole template has never been optimised for C. neoformans activity or target selectivity before. Flubendazole and albendazole suffer from extensive drug metabolism and have limiting pharmacokinetic (DMPK) properties. As such new analogues design will be guided by DMPK predictive algorithms, whole cell activity, selectivity for C. neoformans beta-tubulin over human beta-tubulin (measured in vitro and modelled) and measured in vitro DMPK. Compounds satisfying set criteria values will move into an in vivo PK/PD mouse models of C. neoformans infection. Compounds active in the mouse model demonstrating suitable PK will form the basis of funding applications for candidate selection enabling studies and beyond.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Functional dissection of Condensin and Cohesin in atypical mitosis and meiosis in Plasmodium
Funder: Medical Research Council

Abstract: Plasmodium is a unicellular parasite and the causative agent of malaria. These parasites have an atypical cell division that differs from that of the host they invade. They divide within red blood cells (schizogony) in a process resembling endomitosis where the nuclear envelope remains intact and a single cell becomes multinucleated. Moreover, during this stage their chromosomes do not condense. The parasite also shows endo-reduplication during male gametocyte differentiation, as well as a short meiotic stage within the mosquito vector. The molecular mechanisms that regulate these atypical cell divisions in Plasmodium are not well studied. The main aim of this project is to dissect out the molecular mechanisms controlling two important components, Condensin and Cohesin in these types of atypical cell division and establish how they differ from the classical cell division of host cells, ultimately to identify intervention targets. Condensin and cohesin belong to an important family of proteins that have crucial roles in chromosome dynamics and gene regulation, both in mitosis and meiosis in most eukaryotes. This has been widely studied in some cells, yet nothing is known of their localisation, function, regulation or the complexes associated with them during atypical cell division in Plasmodium. Complementary approaches will be used to achieve three main objectives, using Plasmodium berghei as a model system: (1) To identify the subcellular location of condensin and cohesin at various stages of parasite cell division. 2) To study the function and transcriptional regulation of cohesin and condensin during parasite cell division and (3) To characterise the cohesion and condensin complexes using proteomic approaches. We will use reverse genetics, state of the art cell biology methods with fluorescence, confocal, and electron microscopy, and protein biochemistry with proteomics to achieve our goals.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Shifts in the metabolic and virulence profiles of Streptococcus pneumoniae following the introduction of conjugate-polysaccharide vaccine in Malawi
Funder: Medical Research Council

Abstract: Streptococcus pneumoniae is a leading cause of childhood pneumonia, meningitis and bacteraemia. Where PCV13 has been introduced in sub-Saharan Africa, including Malawi, a large direct benefit has been seen early after introduction. However, unlike some countries where there was an extensive catch-up campaign, we have not detected impact on unvaccinated populations and not observed herd immunity nearly four years after PCV13 introduction. Two questions remain: whether direct protection will be long-lasting or be undermined by progressive non-vaccine serotype replacement; and whether as the proportion of the population who have received PCV13 increases, there will be herd immunity. We will exploit ongoing globally rare, carefully phenotyped pneumococcal strain collection, exploring the hypothesis that PCV introduction results in a reduction in vaccine serotype carriage and niche replacement by non-vaccine serotypes which have similar metabolic/ virulence profiles to the vaccine-serotypes, therefore undermining vaccine effectiveness. We speculate that non-vaccine carriage serotypes with a successful metabolic/ virulence profile will be increasingly identified in invasive disease. This hypothesis will be addressed through the following objectives: a) Establish whether there is segregation of pneumococcal metabolic/ virulence profiles in geographically distinct human populations. b) Determine whether there is segregation of metabolic/ virulence profiles in vaccine exposed and non-exposed individuals in Malawi. c) Detect successful metabolic/ virulence pneumococcal profiles (identified above) amongst disease-associated isolates. d) Further develop a theoretical framework to explore the effective use of vaccine. Maintaining PCV impact is critical in resource constrained sub-Saharan African countries. This project will address long term vaccine effectiveness in vulnerable populations with a high prevalence of HIV, malnutrition and malaria.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Innate pathogen sensing by local unconventional T cells during microbial infections
Funder: Medical Research Council

Abstract: This study will 1. Confirm local and systemic pathogen-specific responses of unconventional T cells in PD patients before, during and after acute peritonitis. 2. Demonstrate that local APCs in the peritoneal cavity can present microbial ligands to such unconventional T cells. 3. Demonstrate specific recognition of distinct microorganisms by defined human gamma/delta and MAIT TCR clonotypes. This work will take advantage of unique access to a cohort of individuals receiving PD as life-saving renal replacement therapy. No other team in the UK has similarly convenient, continuous and non-invasive access to local samples from patients to study infections caused by a range of Gram-negative and Gram-positive organisms. Stable individuals will be evaluated 3-6 monthly, to establish pre-infection baselines and determine whether the composition of peritoneal cells remains constant over time, unless disturbed by infection. Patients who develop peritonitis will be analysed on the day of presentation prior to commencing antibiotic treatment, during the treatment period, and after full recovery. We will establish for the first time in vivo whether different unconventional T cell populations sense distinct groups of microbes, by defining gamma/delta, MAIT, NKT cells and GEM T cell responses depending on the nature of the causative pathogen, using polychromatic flow cytometry. We will also characterise the potential of peritoneal macrophages and DCs to present non-peptidic ligands to unconventional T cells via the presenting molecules BTN3, MR1, CD1d and CD1b. To achieve further insight into the underlying mechanisms, we will conduct the first TCR clonotyping analysis of locally activated MAIT and Vd2+ T cells and correlate certain clonotypes with distinct pathogens in support of local recruitment/expansion during infection. In addition, we will establish pathogen-specific T cell clones for further characterisation both functionally and, where feasible, structurally.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: The T6SS toxins are powerful weapons for Pseudomonas' antibacterial strategy
Funder: Medical Research Council

Abstract: The T6SS kills bacteria and manipulates microbial populations. Pseudomonas aeruginosa is a Gram-negative pathogen colonizing the CF lungs where it establishes chronically after eliminating competitors. The P. aeruginosa T6SS delivers toxins of which 2 classes are characterized, peptidoglycan hydrolases, phospholipases. The strain producing the toxin (Tse) also makes immunity (Tsi). The ability to outcompete others in a T6SS-dependent manner relies on the diversity of these toxins. Hypothesis-driven approaches identified 6 toxins (Tse1-Tse6) but a larger reservoir exists. We use TraDIS to systematically identify immunity genes as Tn insertion in such genes is possible in a T6SS inactive but not active strain. We confirmed the identity of Tsi1-6 and pointed 4 new potential toxin/immunity pairs. One of 3 T6SSs has been studied extensively in P. aeruginosa, H1-T6SS, since induced in a retS mutant. We showed that H1-, H2- and H3-T6SS are induced in a rsmA mutant and will use this background in a TraDIS approach to characterize the full toxin repertoire. With this knowledge we can map the T6SS toxins content in P. aeruginosa clinical isolates and assess whether the T6SS landscape is a hallmark of infection strategy/disease severity. The TraDIS approach pointed that in a T6SS positive strain some genes are hit with higher frequency. These genes encode membrane proteins, which suggests they may be T6SS receptors. In their absence the strain is less sensitive to T6SS action. This is ignored in the field and source of novel concepts. Using super-resolution fluorescence microscopy we observed GFP-tagged T6SS inducing blebs in membranes suggesting puncturing. Using distinct fluorescent proteins we aim at visualising all 3 T6SSs simultaneously in one cell, imaging docking onto receptors of preys, following T6SS toxin translocation. Our proposal shall provide a global vision of the P. aeruginosa's antimicrobial strategy which could be mimicked in treatment of resistant bacteria.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION