NIHRDataInsights/HRCSData huggingface dataset:
Title: Molecular arrows: DNA markers for electron cryotomography
Funder: Medical Research Council

Abstract: Thanks to new detectors, electron cryotomography (cryoET) has unprecedented potential to reveal molecular detail inside cells and viruses. Unlike heavy metal stain-based biological electron microscopy, cryoET uses frozen, hydrated samples that retain a near native state. Data interpretation for cryoET remains challenging however, as biological specimens are crowded and composed mainly of light atoms, lowering the contrast. Although strategies have been proposed to overcome these challenges, they have limited applicability and potential toxicity. Here, we propose an alternative labelling toolkit for cryoET, with high contrast and low toxicity, based on DNA nanostructures conjugated to DNA aptamers. DNA nanostructures are self-assembled DNA structures incorporating stretches of complementary oligonucleotide sequences. They can be designed into many different shapes and sizes, and the phosphate in the DNA backbone provides higher contrast. Nanostructures can be engineered to contain DNA aptamers, short (20-40 nucleotide) single stranded DNA sequences that recognise a biomolecule with high specificity and affinity. We propose to use DNA aptamers to target nanostructures to the entry machinery of herpes simplex virus type 1 (HSV-1). Herpesviruses are a family of large enveloped viruses, 8 of which cause lifelong infection in humans. These viruses are a major health concern due to their worldwide prevalence, ease of spread, and severity. An important target for therapies is entry of the virus into host cells, which despite much research, is poorly understood. We will use our DNA toolkit to determine the organization of glycoproteins responsible for fusion of the viral and host membranes, and to identify aptamers that block entry. We will then apply our "DNA arrows" intracellularly to follow the packaging of these glycoproteins during HSV-1 infection. This work will develop both a valuable resource for cryoET and provide important information about HSV-1 infection.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: A pilot assessment of miltefosine's efficacy and tolerability for treating cutaneous Leishmania tropica in Afghanistan
Funder: Medical Research Council

Abstract: Background. Cutaneous leishmaniasis (CL) is a neglected tropical disease par excellence and attracts scant attention from the global research community, pharma, and governments. It is an unsightly disease that scars and causes misery, psychological distress, and poor quality of life. Old World (OW) CL, caused by Leishmania tropica and L. major, is a significant public health challenge in the Middle East and west Asia, notably in Afghanistan, Palestine, Syria, and Yemen (Karimkhani 2016). The WHO estimates a case burden of ~500,000/year and war related epidemics are well documented in Afghanistan and in Syrian refuges (Al-Salem 2016). Treatment options for OWCL are limited. Most countries rely on toxic pentavalent antimonials that must be given by injection (intralesion or parenterally). Better tolerated oral alternatives are needed. Miltefosine, a 28-day, effective oral treatment for visceral leishmaniasis, has variable efficacy in New World CL, and appears promising in OWCL case reports. There has never been a study to assess simultaneously clinical cure and the miltefosine parasite clearance relationship. Methods. Our randomised pilot trial will assess efficacy and tolerability of miltefosine (n=50) and SSB (n=50) in Afghan patients aged 1 year or more with L. tropica CL needing chemotherapy. Miltefosine will be dosed allometrically (Dorlo 2012). Follow up is 6 months. The primary end point is cure (complete re-epithelisation of ulcers/return to normal skin). Key secondary endpoints include quality of life and the modelled relationship between miltefosine pharmacokinetics and parasite clearance. Results & future direction. A miltefosine cure rate similar to that of SSB or at least 70% defines success for designing a large randomised noninferiority trial of 14 and 28-day miltefosine vs. SSB, including an economic analysis. We will work with WHO, and the Afghan MoPH to promote positive results and advocate for more research on OWCL.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: The Clean Study
Funder: Medical Research Council

Abstract: Infection Prevention & Control (IPC) is critical to minimizing Healthcare-associated Infections (HAI), currently affecting around 15% of hospital patients in Tanzania & similar to other low-income settings. Cleanliness of surfaces prevents direct & indirect (via hands air or equipment) contamination of patients & also contributes to a respectful & welcoming maternity environment. Poor cleanliness of the maternity & paediatric wards has been reported in Tanzania & in other parts of sub-Saharan African. The "Clean Study" will be conducted in three Tanzanian hospitals over 18 months from March 2018, & represents the development phase for a cluster-randomized trial to reduce HAIs in mothers & newborns by improving environmental hygiene in maternity & newborn units. The main aim of the development phase is to adapt the intervention & to assess its potential for improving environmental hygiene in maternity & newborn units (including the antenatal, labour & postnatal wards; obstetric operating theatre; & newborn intensive care units). The composite intervention comprises the training & mentorship of local "cleaning champions" (e.g. IPC leaders, or cleaning managers), who in turn educate & supervise existing cleaners of maternity & newborn units employing standard Quality Improvement tools. We will also test & refine a) environmental microbiology procedures to measure cleanliness, & b) assessment methods to capture clinical HAI. This information collected during the development phase will inform key design features of the main trial, in particular, sample size & primary outcome measurement. With an increasing number of women delivering in Tanzanian facilities, no formal in-country cleaners' training, & a recent governmental commitment to improve hygiene, our intervention could provide the Ministry of Health with the means to improve women's & newborn health, & potentially patient outcomes in other wards too, with wide significance across LMICs.


Research activity codes: 5.6 PSYCHOLOGICAL AND BEHAVIOURAL;5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: INFECTION
Title: Male partner-assisted contact tracing for HIV and tuberculosis in Malawi: an adaptive multi-arm multi-stage randomised trial (mPATCH-TB)
Funder: Medical Research Council

Abstract: Men are currently a neglected group in responses to the HIV and TB co-epidemics in sub-Saharan Africa, despite having high prevalence of undiagnosed disease, and facing considerable barriers to testing, treatment and prevention services. Through epidemiological and meta-analysis research, we have demonstrated that men in Africa have 2-fold greater prevalence of tuberculosis than women, and can spend up to a year longer than women infectious in the community before achieving a diagnosis. Efforts to engage men in TB and HIV care earlier are likely to have substantial health benefits for men, women and children. In this development grant, we will use qualitative methods and an innovative multi-arm adaptive study design to identify the most promising candidate interventions to increase TB/HIV screening among male partners of women attending primary health care in Malawi with symptoms of TB. Eligible women will be randomly allocated by day of clinic attendance to either standard of care (including access to a Fast Track TB screening clinic), or one of 4 interventions. Interventions will be based on previous research we have done to increase partners' HIV self-testing study, and will refined by qualitative interviews. Interviews will likely include: phone reminders, partner delivery of sputum cups; HIV self-testing; and financial incentives. The primary outcome will compare the proportion of male partner household contacts that complete TB screening (defined as receipt of result of a sputum Xpert or smear result) within four weeks of recruitment in standard of care with each intervention group. By the completion of this development grant we will have strong evidence to indicate which interventions have potential to achieve public health effectiveness in Africa. Selected interventions will then subsequently be evaluated in a Phase 3 clinic cluster-randomised trial.


Research activity codes: 4.3 INFLUENCES AND IMPACT
Health categories: INFECTION
Title: Analysis of flavivirus infection on the cellular lipidome - implications for virus particle production and replication.
Funder: Medical Research Council

Abstract: The overall aim of the UK-Thai collaboration is to increase our understanding of how flavivirus infection impacts cellular lipid metabolism and identify cellular lipids that are key for flavivirus replication and particle assembly. Initially human hepatic Huh-7 cells will be infected with the four serotypes of DENV, JEV and ZIKV, which are flaviviruses of most relevance to Thailand. The infected and mock infected cells will be used to prepare total cell lysates and a heavy membrane fraction containing the virus replication/assembly complex. We will also collect the culture supernatants from the virus infected cells and use them to isolate virus particles, a total supernatant fraction and exosomes. Lipids extracted from the samples will be analysed by ultraperformance liquid chromatography - mass spectrometry (UPLC-MS). The large datasets resulting from the analyses will be subject to bioinformatic analysis to identify classes of lipids modulated during virus infection. More detailed targeted analysis will be undertaken to identify lipid species that are either associated with flavivirus replication and/or particle morphogenesis or modulated in response to infection with multiple flaviviruses. The role of the identified lipids in the flavivirus lifecycle will be assessed by i) localisation of the lipids by immunological techniques and ii) siRNA knockdown or compound inhibition of key proteins involved in lipid metabolism, in combination with flavivirus replicon and infection assays. The findings will be validated using a monocyte-derived dendritic cell infection model. The lipid composition of recombinant virus-like-particles (VLP) will be analysed to determine if it differs from virus particles and whether the composition depends on remodelling of host membranes during virus replication. The effects of differences in VLP lipid composition will be analysed by examining the effects on VLP yield, immunogenicity and host cell signalling.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Dissecting global protective immune response to dengue virus at a single-cell resolution
Funder: Medical Research Council

Abstract: Dengue virus (DENV) infects 390 million people each year, mostly in the tropics, with endemic regions expanding rapidly. As yet no effective vaccine or antiviral therapy exists. Here we propose a comprehensive study of the immune responses in asymptomatic vs symptomatic DENV-infected individuals at the level of single cells, as the basis for improvements in vaccine design and better treatment strategies. Recently published data using a bulk microarray approach showed differences in the immune response in DENV-infected individuals with and without symptoms. However, bulk gene expression data has limited resolution. In contrast, single cell analysis generates information on the specific subtypes of immune cells that are activated and that can clear the virus. Utilizing a Dengue patient cohort at Mahidol University, Thailand and cutting-edge single-cell technology at WTSI, we propose to dissect the global protective immune response to DENV infection. Single cell transcriptomes will be obtained from pre-collected peripheral blood mononuclear cells (PBMCs) from asymptomatic and symptomatic DENV infected individuals to identify factors associated with protection versus pathogenesis. Innate immune responses will be assessed by single-cell analysis of T and B cell-depleted PBMCs using an emulsion-based single-cell technology (10x sequencing). In addition, we will perform an in-depth analysis of activated and DENV-specific T cells and activated B cells using the SMART-seq2 protocol, which allows the analysis of TCR and BCR gene segment usage. In cell sub-populations linked to viral clearance, we will identify differentially regulated markers and perform validation experiments to confirm the identified severity-associated factors at the protein level by means of surface marker expression analysis by FACS and cytokine array studies on patient plasma. This will provide crucial information on desirable type immune responses against DENV, paving a way for future vaccine design


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: INFECTION
Title: Deep Mutational Scanning of Dengue Viruses for Vaccine Development
Funder: Medical Research Council

Abstract: Dengue poses a major threat to public health in most tropical and subtropical countries. There are up to four-hundred-million infections per year and three-billion people are at risk of infection. Despite intensive work, the only available licensed vaccine cannot effectively protect against all four serotypes of dengue viruses (DENV). Host antiviral responses by type I interferon (type I IFN) plays a critical role in determining the outcomes of dengue infection and the efficacy of vaccines for many viruses. DENV antagonizes the thuman type I IFN response using its NS5 protein to target the key regulator STAT2 for degradation. However, deleting NS5 or a large segment of it to eliminate STAT2-targeting activity is not possible as NS5 is the essential viral RNA polymerase and proved to be intolerant to such drastic changes. A new approach that can extensively explore the viral sequence-function relationship is needed. The current project aims to implement an approach that combines deep mutational scanning and a virus genetic engineering tool developed in our lab to comprehensively test DENV mutations for their effects on STAT2-antagonizing activity. The proposed exercise will identify a collection of DENV mutants with varying degrees of resistance to STAT2, enabling us to test different level of viral attenuation which is critical for finding optimal immune responses for developing effective vaccines. Characterizations of these viruses by a battery of immunological and virological assays will give us an idea of a relationship between viral attenuation and the ensuing immune response, key information for fine tuning the immunogenicity of a dengue vaccine. Further application of this exercise to investigate multiple interactions between the virus and host immunity could accelerate our path to identify the elusive correlates of protection for dengue in humans.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Using whole genome sequencing to characterise drug resistant Mycobacterium tuberculosis in Thailand
Funder: Medical Research Council

Abstract: Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is an important global public health issue, with increasing drug resistance. Understanding the factors underlying Mtb transmission is essential for disease control, but surprisingly little is known about the role of pathogen genomic variation, where most infections occur, the importance of host factors, or who transmits to whom. Mtb can be grouped into 7 lineages or phylogenetic clades, and further into sub-lineages, which may vary in propensity to transmit and cause disease. By sequencing Mtb in a population-based setting it is possible to construct transmission chains using phylogenetic-based algorithms: strains with near identical genomes are most likely to be due to a transmission event, and will appear on the same branch of the phylogenetic tree. However, there are few large population-based studies in high TB prevalence areas with known drug resistance outbreaks that can apply long-term large-scale whole genome sequencing. Our work will focus on the Kanchanaburi province of Thailand, where there have been outbreaks of multi-drug resistant TB bacterial strains. Mtb strains from 2,000 patients attending the largest hospital (Makarak Hospital), during the period 2003 to 2019, will be whole genome sequenced and important meta data (e.g. HIV status, diabetes) curated. By including all genomic variants, it will be possible to build a probabilistic model of transmissions, and hence assess effects of pathogen variation and host factors (age, sex, HIV status, diabetes, proximity) on transmissibility using regression-based methods. Further, it will be possible to adopt a genome-wide approach to identify loci in Mtb that are associated with or under evolutionary selective pressure from drug resistance and transmissibility phenotypes. Loci identified as being associated with drug resistance and transmissibility will be validated, including by considering sequence data from other Thai or global populations.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: INFECTION
Title: Population differences in vaccine response: the role, reversibility and mediators of immunomodulation by chronic parasitic infections in the tropics
Funder: Medical Research Council

Abstract: We propose that parasite infections contribute substantially to population differences in vaccine response; and that their effects are mediated partly by "transkingdom" pathways (activation of herpesviruses; intestinal translocation of microbial products), and ultimately by pre-immunisation immune characteristics of the host. We will test this hypothesis through four linked objectives among Ugandan adolescents. An immunisation programme comprising relevant live and inert vaccines will be given over one school year, with primary endpoints one month post-immunisation. A secondary endpoint at one year will assess response waning. 1. We will compare vaccine response profiles in urban adolescents (low parasite burden) with two rural cohorts, chosen for high schistosomiasis and high malaria prevalence. 2. We will establish whether current parasite infections have causal effects using individually-randomised, placebo-controlled interventions targeting the dominant infection in each rural cohort. Sample sizes will be robust, powered to detect vaccine response differences of 0.14log10 between study arms (modest effects compared to preliminary data). 3. We will assess herpesvirus-specific antibodies (Luminex), viral loads (droplet digital PCR) and cellular responses (ELISpot); and microbial translocation (MT; PCR for bacterial 16s ribosomal DNA, ELISA for lipopolysaccharide and other biomarkers). Markers of viral activation and MT will be related to parasite exposures and vaccine outcomes. 4. We will investigate pre-immunisation immunological characteristics using simple biomarkers and cell phenotyping, and with in-depth studies (including by mass cytometry) in smaller, representative groups; and link findings to both parasite exposures and vaccine outcomes. Our data will be integrated using causal mediation analyses to determine how urban-rural environment, parasites, "transkingdom" effects and immune responses relate to determine vaccine responses.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Defining the role of src family kinases in cytomegalovirus reactivation
Funder: Medical Research Council

Abstract: Human cytomegalovirus (HCMV) reactivation is dependent on the activation of cellular signalling pathways. We have demonstrated that interleukin-6 (IL6) activation of ERK promotes reactivation in dendritic cells (DCs) but not progenitors. Thus why IL-6 activation of ERK only promotes reactivation in DCs is unclear. We hypothesised that the concomitant activation of additional pathways underpinned this DC-specific phenotype. A phospho-proteomic approach revealed that a src family kinase, haematopoietic cell kinase (HCK), was activated more efficiently by IL6 in a DC subtype that supported reactivation. Further evidence for a role was the observation that pharmacological inhibitors of src family kinase signalling inhibited reactivation. Preliminary data suggest that the role of SFK activity is to act in concert with ERK signalling to promote the post-translational modification of histones bound to a key viral promoter to ensure efficient viral gene expression. In this study we will: 1) Use genetic approaches to delete HCK from DCs to demonstrate a role for HCK in HCMV reactivation focusing on the impact of this deletion of the induction of the viral gene expression 2) Test whether we can rescue HCMV reactivation in cells that do not normally support reactivation through introduction of a constitutively active version of HCK 3) Using Chromatin IP approaches, demonstrate that the role of HCK and SFK activity is to potentiate the chromatin remodelling initiated by ERK-MAPK signalling 4) Demonstrate that a peptide mimic of gp130 (the signalling arm of the IL-6 receptor) inhibits HCMV reactivation through the targeted blockade of IL-6 induced HCK activation The study will improve our understanding of HCMV reactivation and, furthermore, enhance our understanding how crosstalk between pathways regulates eukaryotic gene expression using HCMV as a paradigm. Ultimately, it may lead to a strategy to target a host response to inhibit HCMV reactivation therapeutically.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Mechanisms of Infection Chronicity and Pathogenesis in Gastrointestinal Chagas disease
Funder: Medical Research Council

Abstract: Chronic infections cause a massive global burden of morbidity and mortality. They are caused by bacteria, viruses, protozoa, helminths and fungi against which host immune responses are insufficient to clear infection. Understanding the mechanisms that explain pathogen persistence and how this leads to disease is crucial to developing new treatments. This project aims to address these questions in an animal model of digestive Chagas disease (DCD), which is caused by the parasite Trypanosoma cruzi. At least 6 million people are infected with T. cruzi and approximately 15% will develop DCD. This disease involves damage to gastrointestinal neurons and smooth muscle leading to massive enlargement of the colon and/or oesophagus. Very little is understood about the mechanism of pathogenesis. The principal applicant recently uncovered specific regions of the gut that serve as major reservoirs of T. cruzi infection. This discovery was made possible by an infection imaging system based on parasites expressing a codon-optimized, red-shifted luciferase gene. This project will combine this technology with advanced techniques to study the GI nervous, immune and smooth muscle systems to investigate how T. cruzi causes DCD. A comprehensive series of experiments will be used to optimise methods to investigate these systems and establish a framework for the first in vivo trial of a front-line drug (benznidazole) to prevent DCD. This is a hypothesis-driven project with the potential to reveal novel insights into the mechanisms of T. cruzi persistence and the immuno-pathogenesis of DCD. The models and the underpinning data will support trials of new anti-T. cruzi compounds, immunotherapies and regenerative approaches, such as enteric neuron stem cell therapy. The project will generate fundamental insights relevant to other chronic diseases and will help to define the complex interactions at the interface of gastrointestinal, immune and neurological systems.


Research activity codes: 6.1 PHARMACEUTICALS;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION