NIHRDataInsights/HRCSData huggingface dataset:
Title: Bacterial type III secretion systems: from structure to function
Funder: Medical Research Council

Abstract: Aim I -Define entire structure of Shigella T3SS: a) Obtain near-atomic resolution 3D reconstruction of needle and selected mutants by cryoEM; b) Produce 5-9 Å resolution non-symmetrised 3D map of NC by cryoEM (including mapping location & orientation of CIMEA components, integrating views of needle & TC into single high-resolution view of wild-type NC and reconstruction of mutant NCs); c) Obtain tomographic reconstruction of whole T3SS within intact bacteria. Aim II -Define path taken by activation signal from TC until late effector secretion: a) Screen for mutants altered in activation throughout NC (using already identified signal-insensitive mutants in TC components to initiate a colony colour-based suppressor screen); b) Progress towards holistic understanding of regulation of T3SS substrate expression by generating an RNA map of Shigella virulence plasmid; c) Progress towards understanding mechanisms mediating secretion hierarchy by quantifying secretion processes (both of the latter objectives will be executed in different mutants and under conditions representing different stages in T3SS activity); d) Examine location of export substrates and their mRNAs relative to T3S machineries, as well as diffusive properties of the former to understand whether they contribute to secretion hierarchy establishment. Aim III -Build tools to reconstitute export of T3 substrates across the inner membrane in vitro: For technical reasons we will here use the Salmonella flagellar T3SS as a model system to a) Generate & test mutants and genetic constructs for monitoring export activity in a strain overexpressing flagella; b) Generate, affinity purify and characterise inverted inner membrane vesicles (IMVs) derived from above strain; c) Prepare cytosol and substrate (s), mix with IMVs and an energy source and monitor export activity enzymatically or radioactively; d) Perform initial functional characterisation of in vitro assay.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Developmental Clinical Studies - A Phase I study of a novel peptide fusion inhibitor for the treatment of chronic HIV infection
Funder: Medical Research Council

Abstract: Current combination antiretroviral therapy (cART) utilises antiretroviral (ARV) agents from at least 2 different classes and has dramatically decreased HIV-related mortality and morbidity and increased survival. Many classes of ARV are associated with long term toxicities and are drug-drug interactions. With an ageing HIV infected population such factors are increasing challenging to manage making the development of new ARV a critical medical need. The majority of ARV in use and in development targets inhibition of the HIV viral enzymes reverse transcriptase and protease and recently the viral integrase. Only maraviroc and enfuvirtide target the mechanism of viral entry. Maraviroc is a CCR5 inhibitor and therefore is not active against HIV strains with other tropism, which represent a substantial proportion (>40%) of patients with advanced HIV infection and has significant drug-drug interactions. Enfuvirtide, a peptide administered by subcutaneous injection, does not have these limitations. It is safe and well tolerated with a favourable metabolic profile and without propensity for drug-drug interactions. However local injection-site reactions (ISR) are observed in the majority of patients. Its inconvenient administration profile and ISRs have progressively hampered its use. However, 2nd generation fusion inhibitors with improved profiles would fill an important gap in the existing therapeutic armamentarium. We have developed a novel peptide fusion inhibitor, C34-PEG4-Chol, which overcomes the key limitations of enfuvirtide: it has 100-fold more potent antiviral activity in vitro and prolonged duration of action in vivo. Available preclinical data suggest that this new agent should be efficacious in humans at a lower dose and reduced frequency of administration, possibly as a once-weekly subcutaneous injection. We aim to assess the safety, pharmacokinetics, and antiviral efficacy of this peptide in a Phase 1 study in HIV-infected adults.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Analyses of herpes simplex virus latency and reactivation at the single cell level.
Funder: Medical Research Council

Abstract: The major aims of the proposed research are to define the reactivation propensity and molecular characteristics of neurones latently infected with herpes simplex virus type 1 and to explore the mechanisms by which the HSV latency associated transcripts function to maintain latency. This will be achieved at single cell resolution using Ai6 ZsGreen transgenic reporter mice to facilitate the fluorescent detection, purification and subsequent characterization of latently infected neurones. The specific objectives are; 1. To determine the latent virus DNA copy number in individual latently infected neurones that have experienced distinct patterns of lytic cycle promoter activity during latency establishment. 2. To investigate the reactivation potential of isolated populations of latently infected neurones in real time to correlate reactivation propensity to evidence of prior lytic cycle promoter activity and latent DNA loads. 3.To investigate the role of the HSV encoded latency associated transcript (LATs) in latency maintenance. 4. To define the role of latency associated miRNA expression and/or LAT encoded anti apoptotic functions in latency maintenance. 5. To develop a system to restore WT virulence and latency characteristics to an HSV genome cloned as a bacterial artificial chromosome to facilitate in vivo studies.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: A comprehensive survey of protein-protein interactions between Plasmodium falciparum merozoites and human receptors
Funder: Medical Research Council

Abstract: Plasmodium falciparum parasites cause more than a million deaths from malaria each year. All the symptoms and pathology of malaria are caused by the blood stages of P. falciparum development, which are initiated when merozoites invade human erythrocytes. Erythrocyte invasion is potentially an attractive vaccine target because it is essential to parasite survival and the extracellular ligands that catalyse it are exposed on the parasite surface. Although many candidate P. falciparum ligands have been identified, in very few cases are their host receptors known, in part because cell surface protein-protein interactions are often of very low affinity, making them difficult to detect using standard approaches. We will address this important gap in our understanding using AVEXIS, a protein-protein interaction technology developed at the Sanger Institute specifically to detect low affinity interactions. A pilot screen has already identified two novel P. falciparum-erythrocyte interactions, including one that plays a critical role in invasion. To expand these studies, we will: 1) Construct a comprehensive library of recombinant P. falciparum merozoite proteins 2) Screen the library against human erythrocytes to identify erythrocyte binding proteins 3) Identify carbohydrate receptors for merozoite ligands by screening glycan arrays 4) Identify protein receptors by using AVEXIS to screen libraries of human erythrocyte receptors or other soluble blood components 5) Validate the function of identified interactions using in vitro cultured P. falciparum parasites. This approach will generate the first biochemically validated map of interactions between P. falciparum merozoite proteins and human blood cell components, and will identify protein-protein interactions that are of particular interest for vaccine or drug development. All expression constructs will be deposited in not-for-profit reagent resource collections to empower research in other labs.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Prognosis of HIV-positive patients treated with antiretroviral therapy: comparative analyses and treatment strategies
Funder: Medical Research Council

Abstract: Antiretroviral therapy (ART) dramatically reduces rates of AIDS and death in HIV-positive people in Europe, North America and low-income settings, particularly those who start early and attain viral suppression. However mortality rates still exceed those in the general population, and there are increasing numbers of older patients and those treated for many years. Predictors of prognosis are changing to include factors beyond conventional markers of HIV disease progression: long term outcomes remain unclear. This application seeks funding for a collaboration of 19 European and North American HIV cohort studies. Working within regional collaborations, we plan to combine data on >100000 treated patients and compare outcomes with those in Southern and West Africa. Objectives are: A) Examine prognosis for AIDS and deaths from all and specific causes among patients on ART, focusing on non-HIV biomarkers, additional CD4 measures, co-infections, new measures of and interactions with HIV virus burden, and HIV-1 subtype; B) Investigate heterogeneity in outcomes between regions and settings; C) Estimate life expectancy overall and in subgroups; D) Conduct collaborative work on prognosis in low-income settings (comparison of mortality in S. Africa, Europe and N. America allowing for incomplete death ascertainment, and prognosis for HIV-2 in W. Africa). We will use causal inference methods to examine optimal treatment strategies including E) strategies for regimen modification (switching) in patients with virological failure of first ART regimen; F) effect of non-adherence to ART on treatment failure and progression to AIDS and death; G) optimal timing of ART in patients presenting with specific CNS AIDS-defining opportunistic infections such as cryptococcal meningitis and toxoplasmic encephalitis. We expect our research results to be of direct relevance to the care of HIV-positive people and to be published in high impact journals and incorporated in treatment guidelines.


Research activity codes: 8.1 ORGANISATION AND DELIVERY OF SERVICES;6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Calibration and analysis of complex models: methodological development and application to explore the impact of HAART in Africa
Funder: Medical Research Council

Abstract: Complex individual-based stochastic models are increasingly being used across science, engineering, medicine and public health. The utility of models for prediction & planning relies on how well they are calibrated to data and analysed for robustness of predictions. However, development of methods to calibrate and analyse complex stochastic models has greatly lagged behind their application, resulting in unreliable predictions and increasing the probability of incorrect policy and funding decisions Aim Develop and evaluate methods to calibrate and analyse complex individual-based stochastic models, and apply them to explore the impact of HAART on HIV/AIDS in Africa Objectives -Develop novel methods for Bayesian Emulation of stochastic models -Comparative analysis of accuracy/efficiency of existing MCMC, ABC and novel Bayesian Emulation methods for the calibration and analysis of stochastic models -Develop and analyse a novel hybrid model calibration strategy combining the strengths of ABC and Emulation methods -Apply developed and analysed methods to predict the impact of HAART on HIV/AIDS in Africa Methods Development: Bayes linear and fully Bayesian emulation for calibration of stochastic models, and multivariate outputs Hybrid ABC & Emulation methods for efficient generation of probabilistic statements about goodness of fit & joint estimation of posterior probability distributions Experiments: Use new methods to estimate model parameters by calibrating model to simulated data with known 'true' parameter values, varying number and prior bias & variance of model parameters Application: Use new methods to calibrate complex model to uniquely detailed HIV/AIDS data from African cohorts Application/exploitation Application within project to predict impact of HAART on HIV in Africa and engagement with international policy makers/funders to improve policy/funding decisions. Novel methods dissemination to other science areas/public sector/industry


Research activity codes: 8.4 RESEARCH DESIGN AND METHODOLOGIES (HEALTH SERVICES);2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: INFECTION
Title: Structure, Cell Entry and Egress of Lassa Fever Virus
Funder: Medical Research Council

Abstract: Lassa virus (LASV) is a deadly zoonotic pathogen which kills thousands of people each year. Despite the threat that it poses, little is known about the molecular mechanisms it employs for entry into and egression from host cells. Such information is invaluable for the rational development of therapeutics that can prevent and treat LASV infection. Here, we will address this paucity of information using a combined structural approach that incorporates electron cryo-microscopy (cryo-EM) and X-ray crystallography. Cryo-EM will be utilized for medium resolution (10-30 A) imaging of unstained LASV (fully deactivated) and non-pathogenic virus-like-particles (VLP). This technique is highly suited for this work as samples will be kept in a vitreous, near physiological state. As LASV and its VLPs are pleomorphic in shape, we will employ tomographic data acquisition and sub-volume averaging, methods which will allow the structure of the entire virus and subcomponent complexes of structural proteins (e.g. attachment-fusion glycoprotein and nucleocapsid-RNA complexes), to be solved in three-dimensions. Additionally, using cellular cryo-EM we will examine how progeny virus particles assemble and bud from host cells. X-ray crystallography will serve as a complementary method to cryo-EM and will allow high resolution (2-3 A) structural studies of the LASV envelope glycoproteins, GP1 and GP2. These glycoproteins are involved in viral attachment and fusion, respectively, and will be produced using the in-house mammalian expression system designed to control highly heterogeneous N-linked sugars for structural work. Following crystallization of these individual glycoproteins alone and in complex with their cellular receptor, a-dystroglycan, protein crystals will be subjected to synchrotron radiation for structural elucidation at Diamond Light Source. Ultimately, this multidisciplinary work will reveal structural targets for the rational antiviral and vaccine design.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: DEALING WITH THERAPY-RESISTANT CRYPTOCOCCOSIS BY TARGETING INTRACELLULAR PATHOGENS
Funder: Medical Research Council

Abstract: The etiological agents of cryptococcosis, Cryptococcus neoformans and C. gattii , are both facultative intracellular pathogens that can survive and proliferate within macrophages. Intracellular yeast represent a reservoir of disease that is difficult to eradicate with current antifungal regimes. We have recently identified a novel expulsion process, termed vomocytosis, by which intracellular cryptococci exit macrophages. In addition, we have shown that vomocytosis can be enhanced by the application of specific cytokine subsets, gaseous stimuli or chemical agents. Here we propose a programme of work to both investigate the intracellular behaviour of cryptococci and identify agents that manipulate this behaviour. Specifically, we will: a) identify the molecular steps that lead to cryptococcal expulsion b) determine how differing execution of these steps leads to inter-strain variation in these infectious species c) identify compounds that induce vomocytosis, and d) reveal the mode of action of such compounds. In following this line of investigation, our intention is to develop a new therapeutic strategy to accelerate fungal clearance in patients. This strategy is based around combining existing antifungal regimes with new compounds, identified from our screen, which will induce the expulsion of intracellular cryptococci, rendering them susceptible to the antifungal agents. Our approach, focusing on a drug-redeployment screen within current FDA-approved compounds, is likely to identify agents that are safe in patients and can be rapidly deployed into clinical settings. In addition, we are mindful of the fact that analogous pathogen expulsion processes have recently been identified in a range of infectious organisms Thus findings we make as part of this proposal are likely to have far-reaching consequences both in terms of fundamental microbiology and translational approaches for dealing with intracellular infections.


Research activity codes: 5.1 PHARMACEUTICALS;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Identifying regulators of HIV-1 cell-to-cell spread at virological synapses
Funder: Medical Research Council

Abstract: Human Immunodeficiency Virus Type-1 (HIV-1) disseminates rapidly between CD4+ T cells by cell-to-cell transmission at intercellular contacts virological synapses (VS), but how this is regulated is unclear. To address this we propose to couple functional virology and cell biology with proteomics to identify key regulators and signalling pathways implicit in HIV-1 spread. We will use validated cell-to-cell transmission assays to measure HIV-1 transfer between T cells, including quantitative real-time PCR, flow cytometry and reporter assays thereby generating data amenable to robust statistical analysis. HIV-1 infected cells will be treated with chemical inhibitors and putative regulators will be inhibited with RNAi or dominant-negative mutants and we will measure loss of function to identify key proteins and pathways. Signalling pathways will be activated using a reductionist approach of cross-linking specific surface receptors and the effect on T cell polarisation and HIV-1 recruitment will be examined. State-of-the art proteomics analysis will be used to identify novel regulators. We will also perform live-cell imaging of HIV-1 infected cells to follow dynamic processes in real-time. Our working hypothesis is that cell-to-cell transmission of HIV-1 at VS is an energy-dependent, regulated process triggered by cell-cell contact that activates signalling pathways to facilitate efficient viral transmission. The broad aims of this project are to: 1. identify receptors and signalling pathways regulating HIV-1 spread at VS and concomitant T cell polarisation 2. determine how VS are initiated and terminated 3. examine the requirement for T cell polarisation at the VS 4. identify cellular and viral factors implicit in HIV-1 cell-to-cell spread and to investigate strategies of inhibition. The over-arching aim of this research is to identify novel, potential therapeutic targets to interfere this mode of retroviral dissemination to limit progression to AIDS.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: The fungal "septasome": dividing and conquering cells of fungal pathogens
Funder: Medical Research Council

Abstract: Chitin synthesis and septum formation are both essential processes for the viability of fungi. The main objective of this proposal is to examine the molecular mechanisms of chitin synthesis during septation in the dimorphic fungal pathogen of humans Candida albicans, and how this process is regulated. This will inform our understanding of septation in all fungi and create an attractive platform for the identification of novel targets for antifungal therapies. This will be achieved by: (i) Determining how and when the four chitin synthase enzymes are recruited to septation sites in yeast cells. Live cell fluorescence microscopy will be used to observe the position and dynamic recruitment of fluorescently-tagged chitin synthase proteins in relation to known components of the cell division machinery in wild-type cells. Mutant strains and chemical inhibitors will be used to determine the effects of interfering with protein localisation to the mother-bud neck. (ii) Identifying the proteins that the chitin synthases interact with at septation sites, i.e. defining the fungal "septasome". The membrane yeast two-hybrid system will be used to detect direct protein-protein interactions between the chitin synthases themselves and with proteins co-localised at septation sites, and to identify the specific domains of the proteins required for these interactions. In parallel, immuno-precipitation based screens and affinity chromatography will be used to identify novel binding partners of the chitin synthases by pull-down. (iii) Assessing how chitin synthase assembly mechanisms differ in yeast and hyphal cells. Chitin synthase recruitment and assembly at hyphal septa will be defined and will serve as a useful model for filamentous fungi. The differences and, more importantly, points of convergence with septum formation in yeast cells will be identified which might be explored as novel targets for antifungal therapies with future funding.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Identification of a critical receptor mediating fungal recognition, epithelial activation and induction of mucosal innate immunity
Funder: Medical Research Council

Abstract: The ability of mucosal tissues to discriminate between commensal and pathogenic microbes is essential to human health. Recently, we identified an epithelial signalling mechanism instrumental in identifying the commensal-pathogen switch of Candida albicans, a major life-threatening fungal pathogen of humans. This activation mechanism targets the hyphal form of C. albicans, resulting in induction of proinflammatory cytokines and subsequent activation of neutrophils, leading to protection against mucosal fungal infection. We have now identified the hyphal protein (Ece1p) targeted for epithelial recognition of the commensal-pathogen switch and provide strong evidence that Ece1p activates this recognition system via a novel surface epithelial receptor. This proposal will (i) determine the role of Ece1p in C. albicans infection using a novel murine mucosal model, (ii) identify the epithelial receptor that recognises Ece1p and demonstrate functional importance of the Ece1p-receptor interaction in epithelial activation using several proteomic-based approaches, RNA interference, receptor blocking/inhibition and overexpression assays, and (iii) demonstrate the role of the Ece1p-receptor interaction in controlling fungal colonisation and infection in vivo and in mediating protective innate immune responses. This project will provide substantial new insights to our understanding of epithelial-microbial interactions, and will identify new microbial and epithelial targets for the development of new drugs, immune-based treatments, vaccines and adjuvants to prevent mucosal diseases and new diagnostic tests and biomarkers for mucosal infections.


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