NIHRDataInsights/HRCSData huggingface dataset:
Title: Modes of action and resistance mechanisms towards anti-trypanosomal benzoxaboroles
Funder: Medical Research Council

Abstract: Improvements in understanding how new chemical entities can be converted to effective drugs has underpinned development of new drugs for parasitic diseases, including human African trypanosomiasis. An effective series of compounds, the benzoxaborole class, was developed primarily through the implementation of pharmacokinetic and pharmacodynamic (PK/PD) standards. One of the series, SCYX-7158, has entered clinical trials. Less attention, however, was paid to mode of action against trypanosomes. Here we propose a systematic, multifaceted approach to understand the mode of action of the benzoxaborole series. This will be achieved by exploiting a sub-set of distinct members of the class that have demonstrated differential potency, time to kill and PK parameters. We will exploit several orthogonal approaches to answer the question of how these compounds interact with trypanosomes. To determine how the compounds interfere with cellular metabolism, each will be used over a range of concentrations and exposure times against bloodstream form trypanosomes, and metabolites extracted from lysates will be quantified to assess which pathways are impacted. Preliminary work suggests that SCYX-7158 perturbs S-adenosylmethionine metabolism, pointing to a methyltransferase target. The second powerful route to understanding drug mode of action involves selecting parasites for loss of function mutations, using RNA interference to elicit drug resistance. Again our preliminary data with selected compounds indicates that changes to cellular sumoylation and specific dehydrogenase enzymes stimulates resistance to the drugs. Finally we will also use a battery of microscopy based assays to determine alterations to cellular phenotype as cells die. Once data is collected across the series we will then use genetic approaches to validate the putative targets identified though these analyses.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Improving the design and analysis of drug efficacy/effectiveness studies in malaria and in selected neglected tropical diseases (NTDs).
Funder: Medical Research Council

Abstract: We shall investigate how accurately drug efficacy and effectiveness are measured in human tropical infectious diseases. The main methodological challenges are high levels of patient drop-out during follow-up after treatment, and high re-infection rates which mean that drug failures must be distinguished from new infections. We first investigate malaria as an important paradigm disease and then move on to other neglected tropical diseases (NTDs). The basic technique will be to simulate field data appropriate for each disease and local epidemiological setting using our existing expertise in malaria drug treatment modelling and existing epidemiological models of NTDs. drug efficacy/effectiveness can then be assessed using existing methods and compared to novel frequentist and Bayesian methods to be developed in the project. We can then address an equally important second question: are existing data collection protocols optimal, or could they be significantly improved? Specifically, how should the number of patients enrolled be balanced against intensity of drug follow-up, and would enrolling more patients with a less frequent, 'sparse' follow-up schedule provide better data while minimising the onus on individual patients for frequent and inconvenient follow up visits? We then consider how drug effectiveness can be monitored using data generated outside of standard clinical trials by following patients taking drugs either prescribed as standard first-line therapies, or administered during intermittent treatment programmes.


Research activity codes: 8.4 RESEARCH DESIGN AND METHODOLOGIES (HEALTH SERVICES)
Health categories: INFECTION
Title: An essential channel in Clostridium difficile sporulation: structure and function
Funder: Medical Research Council

Abstract: C. difficile infections (CDIs) are a major cause of morbidity and mortality in UK hospitals, with recent outbreaks of more virulent strains posing an increasing challenge to healthcare. Therefore, there is an urgent need for new, effective methods for infection control and treatment. Spores are the transmission agents in CDIs; however, sporulation is poorly understood at the molecular level. A key aspect of spore maturation is the communication between the forespore, isolated from the medium upon engulfment, and the mother cell. We have recently identified a key forespore protein essential for effective sporulation, SpoIIQ. Our current work shows that it forms a complex with a mother cell membrane protein, SpoIIIAH, to create the necessary communication channel. We aim to elucidate 3 key aspects of this channel: - How does the complex form a bi-membrane channel? We will (a) solve the X-ray structures of SpoIIQ and SpoIIIAH and their complex and (b) use NMR and single-channel studies to determine the biochemical and biophysical properties of the complex and identify potential ligands. - What is the role of the complex in communication between forespore and mother cell? We will use the SNAP labelling system to (a) establish the localisation of the complex, (b) identify the channel effects on gene expression control and (c) study the binding/transport of identified potential ligands. - Is SpoIIQ endopeptidase activity required for channel formation? We aim to (a) assess SpoIIQ endopeptidase activity in vitro (turbidity, zymogram essays, MS analysis), (b) describe catalysis at the molecular level using structural analysis and (c) investigate the effect of inactive SpoIIQ in channel localisation and formation. Inhibition of channel function is an attractive target for therapeutic intervention. This work will provide a new understanding into sporulation of C. difficile that would open new avenues for CDI control.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Randomised trial of HPV vaccination for the control of HPV-related diseases in HIV-positive African populations: Preparatory phase (PH01/14-39)
Funder: Medical Research Council

Abstract: Vaccines against HPV hold great promise for the long term prevention of cervical cancer & anogenital warts (AGW). Two vaccines targeting the most frequent genotypes involved in the aetiology of cervical cancer (HPV16/18) & AGW (HPV6/11), in bivalent & quadrivalent forms, have been licensed. A nonavalent vaccine covering 90% of cancer types is being evaluated. HPV vaccination programmes typically target young girls. However, the relevance of extending vaccination to other groups, e.g. HIV+ women, is of public health importance since they are at higher risk of HPV infection, persistence & disease progression. Cervical cancer screening programmes can identify HIV+ women in need of CIN management treatment & prevention through vaccination. Although HPV vaccines have been shown to be safe & immunogenic in HIV+ populations in South Africa (SA), data is lacking on their efficacy against CIN & AGW. Our hypothesis is that HPV vaccination can decrease the incidence of HPV-related disease among HIV+ African women. The goal is to determine the likely effectiveness of adding vaccination to an HPV-based screening programme among HIV+ women in SA to inform the design of a phase III/IV individually randomized trial of the effectiveness of vaccination against CIN2+ & AGW endpoints. We will test HPV serology in stored serum samples taken from SA study cohorts that have also measured cervical HPV DNA & CIN prevalence & incidence to examine current & prior exposure to vaccine preventable HPV genotypes. The data will contribute to estimate & model the fraction of vaccine preventable types & the expected vaccine effectiveness with a bi/quadri/nonavalent vaccine against CIN2+, AGW & persistent HPV, & determine key trial parameters (e.g. sample size, trial duration, frequency of visits, eligibility criteria). We will also extend the analysis to predict the effectiveness on cancers prevented among HIV+ women. Qualitative research will determine the feasibility & acceptability of the trial.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Pilot study: Live real-time imaging of life-threatening invasive fungal infections
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Candida bloodstream infections remain a significant problem in severely ill patients, particularly those in intensive care units and surgical patients. Mortality rates remain high due to difficulties in diagnosis, delays in initiation of antifungal therapy and limited choices of antifungal drugs. Animal models of Candida bloodstream infection remain a vital element in the development of new antifungal agents and diagnostic tests, as well as increasing our understanding of how infection initiates and progresses. However, in temporal studies there is potential for large numbers of animals to be used. Because of inherent biological variation, groups of 3-10 animals are sampled at the each time point, with analyses based upon the mean or median values for each group. In vivo imaging of fungi in a single group of animals over the same time period would significantly (75-90%) reduce the number of animals required for the same study. However, in vivo imaging of invasive fungal infection requires a reporter which can be detected in the internal organs of mice. To date, none of the reporters developed for C. albicans has been able to accurately reflect disease progression. Publication of new near-infrared reporters for mammalian cells has demonstrated that the near-infrared fluorescence can be detected deep within mice. In addition, only an endogenous compound, biliverdin, is required for fluorescence. The aim of this project is to develop a codon-optimized near-infrared reporter (iRFP) for use in in vivo imaging of C. albicans. A range of constructs will be produced, with the Candida optimized reporter (camiRFP) expression controlled by various Candida gene promoters. Strains expressing the various strains will be extensively analysed in laboratory tests to identify the most suitable reporter for in vivo imaging. Finally, the Candida reporter gene will be validated in small scale experimental infection studies.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Developing an in vitro approach to study transmission of respiratory viruses.
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: A key area of current influenza research attempts to understand the genetic basis of transmissibility. We do not know why the particular swine influenza virus that emerged in Mexico in 2008/9 caused a pandemic where as other swine influenza viruses, to which humans are exposed, did not. However the large number of experiments currently performed with ferrets use small animal numbers and crude readouts, and do not adequately measure difference in transmissibility between different viruses that might have large epidemiological impacts. For example, the length of contagiousness is not measured. Instead of using many more sentinel animals, we propose to develop methodology whereby sentinel animals are replaced by cultures of human airway epithelium that recapitulate the innate barriers that respiratory viruses must overcome before they initiate infection. The project will use the H1N1 2009 pandemic strain of influenza A virus as a model because we and others already have a body of results generated in ferrets to compare and validate the in vitro alternative. The idea is to allow infected donor ferrets to breathe into a chamber as if in a small room or confined space. We will ask whether the air breathed out will and initiate infection on a cell culture placed downstream. The days on which infected animals are contagious will be compared for different viruses to measure increases or decreases in transmission. Once methodology is established, we will move to replacing the infected donor animals with infected airway cultures to assess the environmental conditions that affect transmission of different respiratory viruses through the air. Experiments showing influenza virus transmission to be optimal at dry and cold conditions are often used to explain the winter seasonality of influenza. But parainfluenza virus outbreaks occur in summer, so we aim to assess the effect of humidity and temperature on transmission of PIV and other respiratory pathogens through the air.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: A Drosophila model of Candida albicans gastrointestinal infection
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Candida albicans is a commensal fungus that can cause life-threatening bloodstream infections. However, very little is known about the pathogen attributes that allow a mucosal infection to become systemic. As pathogen genomic libraries become available it is financially unrealistic and ethically problematic to individually test them in mammalian models. We have therefore developed the fruit fly Drosophila melanogaster as an alternative host. We propose to screen by gastrointestinal infection a large C. albicans homozygous deletion library to find those strains unable or hyperactive in inducing systemic immunity. This screen will reduce considerably future experiments in mice.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: An in vitro model system to assay kidney-pathogen interactions determining outcome of Candida albicans infection
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Candida albicans is a human commensal organism that can cause life-threatening systemic infections in severely ill patients. The mouse intravenous challenge model is commonly used to model this infection. However, recent research has found that it is early events in the kidney that generate damaging immune responses and determine gross outcome of infection. Therefore, identification of the renal cells involved in these responses will allow development of an in vitro assay to model these events.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Neonatal BCG vaccination: screening for genetic factors that influence host-pathogen interactions and reducing and replacing the requirement for animal infection models in immune mechanism discovery
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Tuberculosis (TB) kills 2 million people each year. Multidrug resistant TB is on the rise globally and BCG, the TB vaccine does not protect against TB in high-prevalence countries. New therapies are urgently required. It is well documented that host genetic factors are important determinants of susceptibility to TB – identification of the specific genes involved could identify critical pathways that control TB infection which could in turn be targets for new treatments and vaccines. Studies aimed at identifying human TB susceptibility genes have been disappointing due to the complexity of the gene– environment interaction that lead to disease. Meanwhile data derived from animals do not extrapolate to humans. Alternative models are required. We have developed a novel human model in which we have identified interferon-gamma (IFN-_) regulatory genes that are significantly linked to immune responses to mycobacterial antigens following BCG vaccination at birth. This project will use a range of laboratory techniques in human samples to further characterise the functional effects of these genes and provide proof of principle that studies on vaccination responses in early life can identify relevant genes and pathways involved in host –pathogen interactions.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Towards an in vitro system of predictive biomarkers of in vivo liposome efficacy
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Adjuvants are essential components in subunit vaccines that are capable of eliciting strong protective responses in vivo. However, whilst Alum-based adjuvants are the most commonly used adjuvants in humans they fail to promote protective immune responses as they induce weak Th1/CTL responses and thus are not applicable to the control of important intracellular infections such as TB, HIV and malaria. Liposomes have moved to the forefront of vaccine design as they can both deliver antigen and exert adjuvant effects. However, the physicochemical composition of liposome formulations can profoundly affect adjuvant activity and consequent in vivo success of the formulation. Thus a major challenge to vaccine development is our current inability to predict which liposome formulations will demonstrate in vivo efficacy. Novel formulations tend to be incremental developments of existing formulations and they rely heavily on animal tests from an early stage. The ultimate aim of our work is to allow liposome formulation library screens and REPLACE animal testing at early formulation testing. We will take a 'systems biology' approach to integrate results from a unique range of in vitro bioassays to develop a predictive model of in vivo efficacy of formulations. We will use a panel of liposome formulations (previously characterized) with varying physicochemical properties and in vivo efficacy. This panel will be screened in a unique series of in vitro bioassays relevant to the generation of immune responses. We will assess how these liposomes attract antigen-presenting cells (APC); interact with APC; stimulate maturation of APC. From the generated data we will correlate the results of these in vitro bioassays to develop a fingerprint of successful in vivo liposome formulations. This will pave the way for developing a library screening approach to speed the development of novel liposome antigen delivery/adjuvant systems


Research activity codes: 3.5 RESOURCES AND INFRASTRUCTURE (PREVENTION);3.4 VACCINES
Health categories: INFECTION
Title: 3Rs of murine models of malaria infection: immunology meets experimental genetics
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Our recent identification of epitopes recognised by CD8+ T cells, which are evoked by malaria liver stages, provides the basis to address fundamental parasitological and immunological questions: Are these proteins essential for the parasite life cycle? Do responses to these targets contribute to protection? How are these targets efficiently presented to CD8+ T cells? We will generate a series of Plasmodium berghei transgenic parasites as tools to answer these questions. We propose a novel strategy that combines methods for positive/negative selection and flow cytometry-mediated sorting of fluorescent transgenic parasites to mutate candidate genes, along with integration of a green fluorescent protein and luciferase expression cassette in the parasite genome. While 'traditional' protocols require up to 30 mice for each cloning step, our strategy will allow the rapid generation of transgenic parasites using as few as three to five mice (Reduction). During the generation of the transgenic parasites, we will administer pyr and 5C (selection drugs) in the drinking water, not by injection (Refinement). The various transgenic parasites will be assessed for their ability to complete the life cycle in both Anopheles stephensi mosquitoes and mice. Since the transgenic parasites express luciferase, their development in the liver can be evaluated by in vivo imaging (Refinement and Reduction). This method permits the non-invasive detection of parasites in intact animals and allows for the observation of the same animal as infection progresses. Gene-deficient (KO) parasites will be characterised based on their interactions with hepatocytes. Parasites with mutated epitopes (EM) will be used to immunise mice to determine the contribution of specific epitopes in protective immunity. Parasites expressing the ovalbumin epitope (OvaEpiSwaps) will be used to develop in vitro assays (Replacement) for antigen processing and presentation.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Investigating tuberculosis by engineering human granulomas
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Tuberculosis (TB) remains a global health pandemic but treatment has remained unchanged for over 30 years. TB research relies extensively on animal studies, including mice, guinea pigs, rabbits and non-human primates, but no model accurately reflects disease in man. We have recently identified matrix metalloproteinases (MMPs) as critical drives of immunopathology in TB and have shown that matrix destruction is a key step leading to TB immunopathology. In this fellowship, I will develop an in vitro granuloma model of TB infection by exploiting bioelectrospray technology to generate spheroids incorporating primary human cells, Mycobacterium tuberculosis (Mtb) and extracellular matrix components. I will determine the effect of different spheroid matrix composition on granuloma formation and MMP and cytokine production. Next, I will investigate regulation of intracellular signalling pathways and mycobacterial growth by the extracellular matrix, and incorporate divergent Mtb lineages and cells from patients with TB. Finally, I will develop the model to screen for new antimycobacterial compounds active within a granuloma and also to assess efficacy of vaccination strategies ex vivo from patients. These studies will dissect TB immunopathology in an entirely novel in vitro system to identify new therapeutic candidates and evaluate vaccine responses, replacing the need for extensive studies in suboptimal animal models. The 3-dimensional cell culture platform will also be applicable to other human diseases where cell-cell and cell-matrix interactions are important, and so has the potential to replace animal modelling in a wide range of diseases.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION