NIHRDataInsights/HRCSData huggingface dataset:
Title: Targeting the RNA helicase, UAP56: understanding KSHV RNA processing mechanisms to novel antiviral approaches
Funder: Medical Research Council

Abstract: The KSHV ORF57 protein orchestrates the formation of vRNPs by recruiting the cellular hTREX complex onto viral mRNAs. We now demonstrate that vRNP formation is driven by an ATP-cycle-dependent remodelling event of hTREX, required for ORF57 binding. Excitingly, this ATP-dependent formation of vRNPs can be prevented by a small molecule, CCT018159, which inhibits the ATPase activity of the hTREX component, UAP56. Further results have demonstrated the effective CCT018159-mediated inhibition of KSHV vRNP formation, lytic replication and infectious virion production. The ability of CCT018159 to specifically disrupt the ORF57-hTREX interaction now presents an ideal opportunity to firstly investigate how KSHV ORF57 manipulates cellular mRNA processing. We present data showing that ORF57 sequestration of hTREX drastically inhibits bulk cellular mRNA nuclear export. However, ORF57 has also been reported to associate with a subset of cellular transcripts, as well as viral mRNAs. We therefore hypothesize that KSHV ORF57 binds this subset of cellular transcripts, enabling hTREX recruitment, to form ORF57-induced cellular RNPs, overriding virus-induced host cell shutoff mechanisms. In turn, we believe these ORF57-processed cellular transcripts probably enhance KSHV replication. As such, our small molecule inhibitor is an ideal tool to test this hypothesis and identify which ORF57-dependent cellular mRNAs are essential for KSHV lytic replication. Secondly, as the ORF57-hTREX interaction is essential for KSHV lytic replication, CCT018159 is a potentially exciting therapeutic reagent for KSHV infection. We have demonstrated the ability of CCT018159 to inhibit KSHV lytic replication and infectious virion production in cell culture-based experiments. We now aim to further develop CCT018159 to enhance its antiviral activity and pharmacokinetic properties prior to assessing its antiviral potential in an appropriate small animal model for human gamma-herpesvirus infection.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Understanding differences in clinical presentations of cutaneous leishmaniasis in an endemic disease focus
Funder: Medical Research Council

Abstract: The incidence of cutaneous leishmaniasis (CL), a neglected vector-borne tropical disease, is increasing with around 1 million new cases every year. We propose to study differences in clinical presentations of CL in an endemic disease focus: infections with Leishmania aethiopica in North West Ethiopia represents a unique opportunity to unravel the biological basis of the differences in clinical phenotypes, as closely related parasites in a single focus can cause three different cutaneous diseases: localised CL (LCL), characterised by single or multiple localised lesions on exposed skin; mucosal CL (MCL), affecting the mucosa of the nose, mouth or pharynx; and diffuse CL (DCL), characterised by numerous non-ulcerating nodules. To elucidate the pathogenesis of this multifactorial disease, we will recruit and follow the clinical history of patients with L. aethiopica infections, including 80 of each presentation. Each case will be reviewed by the clinical team on site. The scientific team will obtain the data for analysis after de-identification of the patients by creating unique patient IDs. The downstream analysis will be performed using the patient IDs. We will isolate parasites from all patients and generate whole-genome data for all isolates to identify parasite genotypes associated with particular manifestations. Detailed phenotyping will use standard cytokine assays and transcriptomics of skin tape strips, lesion biopsies and blood samples. Analysis of these will seek to identify distinct immunological and gene expression signatures of each presentation.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Harnessing the structural flexibility of bovine monoclonal antibodies for the treatment of emerging virus infections
Funder: Medical Research Council

Abstract: Emerging haemorrhagic fever viruses pose a significant threat to global health and prosperity and there is an urgent need for the development of effective vaccines and treatments. In vivo animal studies and anecdotal evidence following use in human outbreaks have shown that monoclonal antibodies are a safe and effective means of treating haemorrhagic fever virus infection, even after symptoms have developed. However, in vivo data have shown that the likely long-term efficacy of existing interventions, where they exist, will be limited by virus escape. This 'weakness' is due to the inherent structure of the antibodies, and the location and nature of epitopes they target. Mouse and human antibodies, which represent the vast majority of available virus-specific antibodies, possess relatively compact antigen binding domains and engage with epitopes that are easily accessible on the surface of the virus. These epitopes are rarely important in terms of function and therefore tolerate genetic drift and resulting antibody escape. Viruses are adept at hiding more mission-critical regions through global conformational masking or glycan occlusion. However, there is increasing evidence that some animals, such as bovines, are able to produce antibodies that have an extended antibody structure (mediated mainly through the presence of an unusually large and structurally complex CDR3 region). These extended antibodies are able to overcome conformational masking and directly bind to functional regions of the virus surface glycoprotein. Such epitopes are intolerant to significant mutational drift and this limits escape. We have developed a bovine monoclonal antibody platform and will use this to generate antibodies to Ebola, Marburg, Lassa Fever and Nipah viruses. We will use immune-assays and pseudovirus neutralisation assays to identify monoclonal antibodies with potent and cross-reactive antiviral activity in readiness for downstream pre-clinical and clinical development.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Molecular Mechanism of IMD (NF-kB) Inhibition by Dengue Virus in the Mosquito Aedes Aegypti, and Implications for Transmission and Emergence
Funder: Medical Research Council

Abstract: Dengue virus (DENV) is the most important mosquito-borne virus of humans, with almost half the world's population at risk in (mostly) low and middle-income countries across the tropics. There are 390 million infections annually, with 96 million cases of dengue fever and 500,000 cases of the potentially fatal dengue haemorrhagic fever. No antivirals are available, and the only licensed vaccine has mixed efficacy. Mosquito control is therefore important for reducing the global disease burden. Vector immunity restricts DENV replication and transmission. However, we showed that DENV-2 inhibits the IMD pathway, an NF-kB response analogous to human TNF receptor signalling, in cell lines derived from Aedes aegypti, the major DENV vector. This grant will (i) characterise the molecular mechanism of DENV-2's IMD antagonism; (ii) evaluate the impact IMD responses have on DENV-2 evolution; (iii) establish the relevance to diverse DENV serotypes and transmission potential in vivo. Aim 1 exploits our unique IMD assays and track record in Ae. aegypti proteomics to identify which DENV-2 protein antagonises IMD activation, along with its cellular targets. In aim 2, which is not dependent on aim 1, we will experimentally evolve DENV-2 in our first-in-field IMD-deficient CRISPR cell lines to assess the impact on the viral quasispecies (the total population of viral sequences) and identify viral sequences required for IMD antagonism. Results will be validated in an established reverse genetics system. Aim 3 will establish real-world relevance by testing transmission potential in mosquitoes infected with wild-type DENV-2 and IMD-sensitive mutants from aim 2. Key experiments will also be repeated with divergent DENV serotypes. We will thus define virus-vector interactions modulating DENV transmission, with implications for ongoing epidemics and future opportunities for vector-targeted control strategies. This will be the first characterisation of mosquito NF-kB evasion for any arbovirus.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Using viruses to study kinesin-1 recruitment, regulation and function
Funder: Medical Research Council

Abstract: The microtubule network and associated kinesin-1 motor mediate the transport of cargo to specific places within cells and this is critical for normal cell function. Defects in kinesin-1 function or regulation can lead to disease, such as Parkinson's, Huntington's and Alzheimer's. How kinesin-1 binds to and moves along microtubules is well known, but how it binds specific cargos and how its activity and target destination are regulated are less well known. Several viruses exploit kinesin-1 to transport newly formed virus particles out of the cell. An example is vaccinia virus (VACV), the vaccine used to eradicate smallpox. Our lab uses the recruitment of kinesin-1 by VACV as a model to study the mechanisms that control the binding of cargo to kinesin-1 and activation of the motor. This is the subject of this application. The kinesin-1 complex contains 2 kinesin heavy chains (KHC) and 2 kinesin light chains (KLC). Its activity is regulated by autoinhibition. Activation involves binding by cargo proteins that induce a structural change in the complex enabling association with microtubules. Mammalian cells express multiple kinesin-1 types (isoforms), each with slightly different biochemical properties that may bind different cargos or move to different destinations. This project will study the mechanisms by which VACV recruits and activates kinesin-1 to transport virus particles out of infected cells, to determine the structure of the virus proteins involved, and to investigate the expression and functions of the different KLC isoforms and splice variants for cell biology and which ones are hijacked by the virus. The information gained will help understanding of the recruitment and regulation of kinesin-1 activity is in normal cells and how dysregulation can lead to disease. Another objective of the project is to study the properties and functions of different kinesin light chain (KLC) isoforms and splice variants to understand their roles in cell biology.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Dissecting the modulation of nucleocytoplasmic signalling, host cell cycle and differentiation by a new family of Legionella protease effectors
Funder: Medical Research Council

Abstract: We identified a new family of Legionella Dot/Icm type IV secretion system effectors with a protease and an Ankyrin-repeat domain, which are widely distributed across the Legionella genus. We hypothesise that these effectors play an important role in the interaction of Legionella spp. with phagocytic cells. Substrate trapping experiments revealed that one of them targets nuclear transport and a receptor involved in Wnt-signalling, while the other effector cleaves itself, localises to the nucleus and seems to interfere with the cell cycle. This project aims to reveal the function of these proteases and their targets during infection. We will: 1. Define the cleavage specificity of the proteases by affinity purification and N-terminal protein sequencing or intact protein by mass spectrometry of the cleavage products. 2. Generate stable, inducible cell lines for the effectors and survey protease substrates at the proteome level using ProtoMAP and Terminal amine isotopic labelling of substrates (TAILS). 3. Validate binding of inactive proteases to known and newly identified substrates by yeast-two hybrid assay, ELISA or co-immunoprecipitation. 4. Investigate the fate of cleaved substrates during infection using Western Blot and follow their localisation by immunofluorescence microscopy (IF) and live cell imaging. 5. Create L. pneumophila protease mutants or deplete the protease substrates by gene-editing or RNA interference and study the effect on intracellular growth and trafficking, NF-kB activation, nuclear and whole cell proteome and cytokine response using microplate assays, IF, qRT-PCR, ELISA and quantitative proteomics. 6. Assess how Legionella modulates Wnt-signal transduction after stimulation of infected cells or cells ectopically-expressing the effectors with Wnt-ligands using qRT-PCR, luciferase reporters and Western Blot. 7. Dissect the effect of Legionella and the protease effector on the host cell cycle using IF, Western Blotting and flow cytometry.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: The T cell immune response to cytomegalovirus across the adult lifecourse
Funder: Medical Research Council

Abstract: Cytomegalovirus (CMV) infection establishes a state of chronic infection in which viral replication is suppressed by the immune system and is thus an outstanding example of how the immune system can control chronic antigenic stimulation without excessive immunopathology. Greater understanding how the immune system controls chronic infection will directly impact upon and translate into improved understanding of immunotherapy therapy and vaccine regimens. CMV is however an important cause of mortality and morbidity both within the UK and across the world. Although infection is well tolerated by immune competent people it is a major problem in patients who are immunosuppressed. Additionally, CMV is the major global infectious cause of congenital abnormalities. In addition, CMV has emerged as an important risk factor for health problems in the elderly and those with inflammatory disease due in part to a marked increased risk of death from cardiovascular disease (CVD). The work will take advantage of several MRC supported, large and richly phenotyped population cohorts Healthy Donors: >100 regular healthy donors aged 18-70 years; 1000+ donors aged 65-90+ years in the 'Thousand Elders' Cohort and a 5 year prospective study of 1000 medical students. '1000 Elders' donors give blood samples prospectively and have participated in a recent trial to assess valaciclovir therapy for suppression of CMV load and immune response (MRC funded DPFS, Moss PI) Patients: 100+/year stem cell transplant patients, 170/year renal transplant patients, >100 patients with chronic lymphocytic leukaemia (CLL) and 100+ patients with vasculitis. This research proposal will - Study the virus-host balance across the human life course. - Analyse the role or checkpoint protein expression on virus-specific T cells in CMV infection. - Investigate CMV-specific T cell responses restricted by HLA-C. - Assess the mechanisms of CMV-mediated immune pathogenesis and potential therapeutic intervent


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Development of a human challenge model of Leishmania major infection as a tool for assessing vaccines against leishmaniasis
Funder: Medical Research Council

Abstract: The leishmaniases are global diseases impacting millions. No vaccine against human leishmaniasis has yet been developed. What is needed is a rapid, cost effective means for early evaluation of the efficacy of candidate vaccines. Our solution to this roadblock is a human challenge model (HCM) of sand fly initiated Leishmania major infection. In stage 1, we will hold focus group discussions to assess acceptability and attitudes towards the study and preferred approaches for conducting it. We will validate/modify a NIH protocol for sand fly bite on healthy volunteers, evaluating sand fly number/feeding time and identifying adverse events. We will develop a clinical parasite bank to the required standards, screening fresh isolates of L. major (through clinical/immune profiling of donors, parasite genome sequencing, drug sensitivity, sand fly infectivity, transmissibility) and selecting one for full scale development. By Milestone 1, we will have assembled the components necessary to initiate human challenge studies. By Milestone 2, we will have received a favourable ethical opinion to proceed with human challenge. In stage 2, we will conduct human challenge infection in healthy adult volunteers, with safety as a primary endpoint. We will minimise risk by careful escalation of infected sand fly number, assessment of take rate and by drug terminating infections at a suitable clinical endpoint. Blood analysis will provide additional safety data, as well as novel information on the host response to infection. Milestone 3 reflects the primary outcome, namely the development of an effective (i.e. high take rate) L. major HCM with 100% lesion clearance after treatment. Secondary outcomes include immune response parameters and analysis of lesion development. Collectively, the project will provide a new tool to assess vaccine efficacy and to allow for evidence-based decisions to be made on down selection/progression of candidate vaccines for leishmaniasis.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Clinical Evaluation of "Prime-Target" Immunisation
Funder: Medical Research Council

Abstract: We propose to assess the feasibility of developing a malaria vaccine using a new "prime-target" immunisation approach that involves CD8+ T cell targeting to the liver by a novel vaccination strategy. Instead of relying on the ability of circulating T cells, induced remotely by an intramuscular immunisation, to find the target tissue and clear an infectious pathogen, the approach here is to use sequential immunisations by different routes to target cellular immunity to an internal organ. We have found that the efficacy of leading liver-stage malaria vaccine candidates in mice can be enhanced with this approach from 0-30% efficacy to 100%. One administers a booster vaccination with a recombinant viral vector, e.g. a recombinant adenovirus, by a route that leads to antigen expression predominantly in the liver. This is best achieved by an intravenous immunization. We have found that this "prime and target" approach, even with repeated use of the same vector, increased substantially the number of tissue resident memory T cells specific for the immunising antigen in the liver. These numbers correlate with the improved efficacy suggesting a mechanism. We propose here to undertake the first clinical trial of the efficacy of this approach in humans using two liver-stage specific antigens. Small-scale safety assessments are underway this year and we here propose to assess efficacy in 2018 using a standard controlled human malaria infection (CHMI) protocol. We have undertaken safely twenty such CHMI trials in human volunteers in over 500 subjects. This work could lead to the identification and development of a cost-effective high efficacy malaria vaccine and may also provide an effective means of targeting cellular immunity to the liver to immunise, either preventatively or therapeutically, against viruses such as hepatitis B and C.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: MRC Centre for Global Infectious Disease Analysis
Funder: Medical Research Council

Abstract: The continued threat posed by emerging infectious diseases has been highlighted in recent years by the emergence of MERS-coronavirus in the Middle East, Ebola in West Africa and Zika in Latin America. Preparedness therefore remains a policy priority for public health agencies and governments worldwide. In addition, as new vaccines, drugs and other interventions are developed for endemic diseases such as malaria, HIV and TB, there is a need to extrapolate from clinical trial results to determine which combination of interventions gives the optimal (or most cost-effective) control strategy for a specific disease in a particular setting. In this context, modern epidemiological modelling and analysis methods have proven themselves increasingly powerful tools for giving insight into the patterns of emergence of novel outbreaks, the transmission dynamics of endemic diseases and the options available for the control of both. The Centre for Global Infectious Disease Analysis is a world leader in research in this area, and in translating our research into improved public-health decision-making. Since it was founded, MRC funding has supported this translation by facilitating the forging of close partnerships with all major global health organisations (e.g. WHO, PHE, CDC, BMGF, The Global Fund, Gavi) and research centres and public health agencies in many low and middle income countries (LMICs). Continued funding for the Centre will allow existing partnerships to be sustained and enhanced, and new collaborations with researchers and health agencies in LMICs to be developed. By supporting PhD training and postdoctoral career development, it will also continue to expand the UK skill base in mathematical modelling and advanced analysis applied to health problems. Last, continued MRC support will pump-prime new priority research areas, including work on improving the development of new vaccines and on tackling the global challenge posed by antimicrobial resistance.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);3.4 VACCINES;3.5 RESOURCES AND INFRASTRUCTURE (PREVENTION)
Health categories: INFECTION
Title: Developing new drugs for pneumonia in the era of antimicrobial resistance
Funder: Medical Research Council

Abstract: Aims The overall aim is to generate new knowledge of antibacterial concentration-time (C-T) profiles in subcompartments of the lung and how these relate to microbiological outcomes (bacterial kill and the suppression of resistance) by: 1. Exploring the consequences of using drug C-T profiles in plasma versus epithelial lining fluid (ELF) for planning regimens in early phase clinical studies for HAP. 2. Characterising the spatial distribution of meropenem in subcompartments of normal and infected lung. 3. Developing a semi-mechanistic physiology-based (PB) pharmacokinetic-pharmacodynamic (PK-PD) model to describe how PK differences in plasma, ELF and target tissue relate to microbiological outcomes. Methodology 1. Dynamic in-vitro experiments will be conducted using a Hollow Fibre Infection Model to quantify how differential C-T profiles in plasma and ELF affect bacterial kill and the emergence of resistance. The clinical effectiveness of a new beta-lactam/beta-lactamase inhibitor will be predicted by benchmarking its in-vitro efficacy at the level of plasma and ELF against meropenem. 2. PK-PD relationships will be evaluated using a murine model of Klebsiella pneumoniae pneumonia. Drug concentrations in plasma, ELF and lung homogenates will be quantified by liquid chromatography-tandem mass spectrometry. Histology-guided high-resolution matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) will characterise spatial distribution and provide a semi-quantitative estimate of meropenem metabolism in lung. 3. A PBPK model will be developed and combined with an experimental PK-PD model, illustrating the potential to predict the time course of bacterial kill and emergence of resistance at clinically relevant sites for HAP. Scientific opportunities This Fellowship will build tools to accelerate the successful development of new antibacterial drugs for patients with pneumonia that are safe, effective and reduce the emergence of resistance


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: INFECTION