NIHRDataInsights/HRCSData huggingface dataset:
Title: SENBIOTAR: Sensiting Pseudomonas aeruginosa biofilms to antibiotics and reducing virulence through novel target inhibition
Funder: Medical Research Council

Abstract: The traditional approach to combating bacterial infections has been based on the use of antibiotics which kill bacteria or inhibit their growth. There has also been a strong emphasis on the identification of essential gene targets for drug intervention. A major problem with therapeutic approaches targeting viability is that they induce strong selective pressures resulting in the rapid emergence of antimicrobial resistance. An alternate approach is to inhibit virulence rather than bacterial viability and this will be explored in the SENBIOTAR project. In the opportunistic human pathogen Pseudomonas aeruginosa, virulence is co-ordinately controlled at the bacterial population level through quorum sensing (QS), a global cell-to-cell communication system employing diffusible signal molecules. P. aeruginosa strains with mutations in the Pseudomonas Quinolone Signal (PQS) QS pathway are avirulent in experimental animal infections. The partners of this consortium will exploit this information by optimising hit compounds and peptide nucleic acids (PNAs) they identified previously which target PQS biosynthesis and/or PQS signal transduction. These hits have been shown to not only render P. aeruginosa avirulent but also to sensitize biofilms to the action of antibiotics. One of the limitations of using inhibitors of virulence is the fact that immunocompromised patients may not be able to clear the targeted pathogen efficiently. However, if the pathogen can, at the same time, be sensitised to antibiotics then there is great scope for dual therapy with PQS inhibitors and antibiotics. SENBIOTAR will bring together world experts in QS, medicinal chemistry, PNAs, drug delivery and preclinical studies to optimise the activity of the hit compounds and PNAs (hit-to-lead optimization) with the intention of identifying lead compounds which strongly inhibit QS, attenuate virulence and sensitise biofilms to conventional antibiotics at sub-micromolar concentrations. This will be achieved by improving (a) their physicochemical properties without the emergence of cytotoxicity, and (b) delivery to the site of infection. These studies will be performed using a combination of in vitro virulence and biofilm bioassays alongside experimental animal lung infection models. The lead compounds developed by SENBIOTAR will also have significant potential for the treatment of wound, bloodstream and medical-device associated infections caused by P. aeruginosa.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Cambridge-Chennai Centre Partnership on Antimicrobial Resistant Tuberculosis
Funder: Medical Research Council

Abstract: The need for a joint India-UK Centre Partnership focused on drug-resistant tuberculosis (TB) is founded on the scale of the problem both in India and globally, combined with the pressing need to develop new tools and therapeutics to combat it. There were 1,467,585 cases of TB notified in India during 2012 (the largest number of cases in any country worldwide), with a sharp rise in the number of people diagnosed with multidrug resistant (MDR) TB (from 308 to 16,588 laboratory confirmed cases between 2008 and 2012). Our partnership between the University of Cambridge and the National Institute for Research in Tuberculosis (NIRT) in Chennai, India, will bring together a multidisciplinary team to focus on novel diagnostics and therapeutics for TB. This includes the use of emerging sequence-based diagnostics to improve the accuracy of individual patient treatment for MDR and extensively drug resistant (XDR) TB; new drug targets for TB and prediction/investigation of the impact of resistance mutations based on modelling of bacterial genome data; the development of an in-depth understanding of the bacterial genes in diverse populations of Mycobacterium tuberculosis associated with so-called 'drug tolerance'; and novel approaches to treatment of TB based on immunomodulation (enhancement of autophagy and novel enhancers of T cell responsiveness). The Centre Partnership will generate a rich and lasting clinical and genomic dataset. Our objectives require detailed clinical and biological phenotyping and genotyping of patient cohorts, which can be mined to address future questions and will further enhance collaborative research to tackle the burden of drug-resistant TB. We will also innovate in training and knowledge transfer. Leveraging of novel technologies will increase relevant and collaborative research experience for UK and Indian investigators. Transfer of scientific training and technology to India enhances independent research capacity and fosters future international collaborative projects. This will be achieved through mobility and exchange of junior (training) and senior (discipline-hopping) researchers.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: UK-India Centre for Advanced Technology for Minimising the indiscriminate use of
Funder: Medical Research Council

Abstract: Resistance to antibiotics is becoming one of the most important challenges of our age. A number of challenges exist if we are to avoid decent into a post-antibiotic age, including development of: new antibiotics; new diagnostics and improved delivery systems. The UK-India Centre for Advanced Technology for Minimising the indiscriminate use of Antibiotics (UKICAT-MA) will establish materials for both detection, at the point-of-care, of ocular infections and the targeted delivery of antibiotics to infected sites in the eye. UKICAT-MA will also promote the use of these technologies in other infective diseases. The Centre involves a multidisciplinary team from the University of Sheffield (Polymer Chemistry, Clinical and Mechanistic Microbiology and LV Prasad Ophthalmology). Much of the work is based around the idea that certain materials respond to bacteria by changing state on binding to bacteria. These conformational changes are then either monitored to provide a signal of the presence of the bacteria or they provide a basis for targeting antibiotics to bacteria whilst reducing effects on surrounding tissues. UKICAT-MA will develop these smart materials to produce contact lenses that give colourimetric indications of bacterial infections and systems for local delivery of therapeutics to infected ocular tissues. The Centre involves basic science activities, which are aimed at understanding the binding interactions that are key aspects of these materials, materials development and development of manufacturing strategies through to clinical trials. Close collaborative working between the UK and India will facilitate this ambitious programme and each partner brings important expertise to progress these technologies efficiently and quickly to the clinic. UKICAT-MA will also look to collaborate with new partners: industrial, clinical and scientific with expertise of infections other than in eyes. To facilitate these further researches we will run a series of workshops in both India and the UK.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES;5.3 MEDICAL DEVICES;6.3 MEDICAL DEVICES
Health categories: INFECTION
Title: Spatially-resolved PKPD modelling for optimised treatment of central nervous system infection due to Mycobacterium tuberculosis
Funder: Medical Research Council

Abstract: Aims This work aims is to define safe and effective therapeutic regimens to improve clinical outcomes in central nervous system (CNS) infection due to M. tuberculosis (Mtb). Methods An in vivo model that recapitulates the pathology of CNS infection will be used. Concentration-time profiles of anti-Mtb drugs will be quantified in plasma, cerebrospinal fluid (CSF), brain and meninges using standard LC-MS/MS. The spatial distribution of anti-Mtb drugs within CNS subcompartments will be visualised using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Adjacent tissue sections will be stained for acid fast bacilli and histopathological architecture. Clinically relevant biochemical markers (CSF total protein, glucose, and white cell count) and novel genetic PD endpoints (bacterial chromosome equivalents and Mtb 16s rRNA) will be investigated and compared with quantitative culture. Experimental data will be used to construct and fit structural PKPD models. Innovative spatially-resolved PKPD models, which describe drug kinetics as a function of both time and space, will be developed. The clinical implications of experimental data will be examined by simulating humanised dosing regimens in silico. Scientific and Medical Opportunities This work will benefit the agencies working to improve clinical outcomes for the millions of patients infected with Mtb worldwide, for which effective and affordable treatment regimens are urgently needed. PKPD modelling is an important tool to identify therapeutic targets and predict outcomes of novel regimens. Current models describe drug kinetics in homogeneous non-physiological compartments, an assumption which is known to have major limitations for anti-Mtb drugs. The development of spatially-resolved models to describe the PKPD of drugs within infected tissue would represent a significant advancement in the field, with broad application to drug development programs and optimised clinical use.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: The relationship between malarial anaemia, neutrophil function and susceptibility to invasive bacterial disease.
Funder: Medical Research Council

Abstract: A recent episode of clinical malaria is a known risk for subsequent life-threatening invasive bacterial disease, due primarily (but not exclusively) to non-Typhoidal Salmonella (NTS) infection. We have shown that malaria-induced haemolysis increases plasma concentrations of heme and heme-oxygenase-1 (HO-1) which in turn leads to profound neutrophil dysfunction and failure to control NTS. Observations in a mouse model (Cunnington et al, 2012, Nature Medicine) were subsequently verified in children with acute malaria infection (Cunnington et al, 2012, J. Immunol). We have also shown that neutrophil dysfunction is compounded by reduced resistance to bacterial colonisation (Mooney et al, 2015, Sci Rep.) and impaired mucosal immunity (Mooney et al, 2014, Mucosal Immunol.) leading to dissemination of bacteria from the gut to the periphery (Lokken, Mooney et al, 2014, PLoS Pathog.). However, in most malaria endemic settings, the major burden of malarial anaemia is due to chronic, low grade (often, erroneously, described as "asymptomatic") infections. "Asymptomatically" infected individuals thus represent a large, hitherto unrecognized, population that may be at high risk of invasive bacterial disease due to persistent neutrophil dysfunction. If so, treatment of "asymptomatic" infections might restore neutrophil function and reduce the burden of bacteraemia in malaria-endemic communities. The aim of this research is to determine the extent to which persistent, low density, "asymptomatic" malaria infection impairs immunity to secondary bacterial infections; to further characterise neutrophil dysfunction; and to test whether anti-malarial treatment reverses these defects and restores functional immunity to NTS. Preliminary experiments in mouse models of anaemia will be followed by cross-sectional studies of Gambian children with persistent "asymptomatic" malaria and a proof-of-principle study of the impact of antimalarial therapy on neutrophil function.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Modulation of immune recognition by P. aeruginosa through engagement of lectin receptors.
Funder: Medical Research Council

Abstract: Chronic P. aeruginosa (PA) infections are underpinned by the formation of biofilms. Biofilms consist of an extracellular matrix containing two main carbohydrate polymers, Psl and Pel. Our recent results show that two major lectin receptors expressed by immune cells, DC-SIGN (CD209) and the mannose receptor (MR, CD206), interact with PA biofilms. These findings suggest that biofilms do not just provide an effective barrier against immune attack but could also modulate immune cell activation through engagement of lectin receptors. For MR, biofilm binding is largely mediated through recognition of Psl. DC-SIGN also recognises Psl but in addition binds directly to PA cells independently of the presence of Psl or Pel. Our preliminary results strongly suggest that the common polysaccharide antigen (CPA) LPS, shared by all PA serotypes, is the DC-SIGN ligand in planktonic PA. We hypothesise that engagement of lectin receptors by PA (MR by biofilms and DC-SIGN by planktonic cells and biofilms) provides an advantage to PA by modulating immune cells towards a regulatory phenotype, hence facilitating the establishment of chronic infections. The overall aim of this proposal is to determine the contribution of MR and DC-SIGN to immunity against PA. In objectives (1) and (2) we will dissect how MR and DC-SIGN engagement by PA biofilms and planktonic cells contributes to human myeloid cell activation. For this we will use (i) PA biofilms deficient in Psl or Pel, (ii) DC-SIGN-binding and non-binding planktonic PA and (iii) human monocyte-derived and dermis-derived MR and DC-SIGN expressing cells. In objective (3) we will employ cystic fibrosis samples (3.1) and huDC-SIGN transgenic mice (3.2) to assess the specific contribution of DC-SIGN to PA pathogenesis. This study will unveil novel mechanisms exploited by PA to modulate immune responses which may ultimately lead to the design of therapies aimed at enhancing the effectiveness of anti-PA immunity.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Enabling rapid conversion of antigen to vaccine, applied to multi-stage malaria vaccination
Funder: Medical Research Council

Abstract: Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, ideal for multimerising foreign antigens to create safe and effective vaccines. However, decorating VLPs with target-antigens is time-consuming and often leads to capsid misassembly or antigen misfolding; this is a major limiting factor for vaccine development. We have recently established a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein we designed to form a spontaneous irreversible isopeptide bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria and cancer antigens. This gave potent antibody responses in mice after only a single immunisation. Here we will greatly enhance the stability, purity and scale of production of this "Plug-and-display" vaccination platform. We will make major advances to the platform by establishing super-high VLP valency, twin conjugation using a second unique covalent labelling technology, and Plug-and-display on the clinically validated Hepatitis B VLP platform. Using recently established blood-stage antigens from Plasmodium falciparum, we will test the activity of the platform for generating high level protection from red blood cell invasion. By optimising a synergistic immune response from co-display of two different antigens at high density on VLPs, we will help the progress towards a broadly protective malaria vaccine. Beyond malaria, establishing the simple, efficient and modular decoration of antigen onto VLPs should accelerate and enhance vaccine development to a wide range of human and animal diseases.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: The molecular mechanism for trypanosome cell death induced by ApoLI and its inactivation in human infective T. b. rhodesiense.
Funder: Medical Research Council

Abstract: This project combines the technical expertise of the Higgins, Carrington and Gilbert laboratories, allowing an integrated structural, biophysical and cellular approach. We will combine structural techniques (x-ray crystallography, NMR and electron microscopy) with biophysical tools. Bacterial expression systems allow us to produce ApoLI and SRA, while hybridoma culture is used to generate monoclonal antibodies. We have obtained a crystal structure of SRA and will also use crystallography (and possibly NMR) to obtain structures of ApoLI and the SRA:ApoLI complex. Electron microscopy will be used to study ApoLI integrated into nanodiscs and liposomes. Liposome flotation and dye-release will be used to study membrane binding and pore formation. TIRF microscopy with photobleaching will reveal pore stoichiometries. Hydrogen-deuterium exchange mass spectrometry will reveal binding interfaces and biophysical tools, such as SPR, will characterize interactions. This combined set of structural and biophysical tools will reveal how ApoLI forms pores and how SRA inhibits these pores. Cellular studies will allow us to integrate structural information into the context of trypanosome cell biology. We will use the latest microscopy techniques to study fluorescently labeled single molecules in live cells that have specifically labeled organelles. We will use these to study the dynamics of ApoLI and SRA movement and to correlate these changes with the effects of cell killing. We will use TIRF microscopy to study ApoLI at the cell surface and FRET-FLIM to determine if it forms clusters. We will design novel fluorescently labeled ApoLI variants that fluoresce when they integrate into membranes. We will use these tools, together with the integration of structural and biophysical information, to assess the molecular basis for ApoLI pore formation and its inactivation by SRA.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Staphylococcus aureus subversion of phagosomal maturation in macrophages
Funder: Medical Research Council

Abstract: Staphylococcus aureus is a major human pathogen with several adaptations to aid immune evasion. Macrophages that phagocytose S. aureus struggle to kill internalised bacteria. I have shown S. aureus inhibits phagosomal maturation. I hypothesise that S. aureus gene expression impairs critical steps in phagosomal maturation and inhibits phagosome fusion with lysosomes, leading to bacterial persistence. My aims are to: 1) Identify microbial determinants of impaired phagosomal maturation; 2) Characterise the host responses critical for phagosomal maturation and establish the steps inhibited by S. aureus; 3) Confirm that phagosomal maturation plays a critical role in S. aureus killing in vivo. I will establish high-content automated microscopy screens of phagosomal maturation in differentiated THP-1 cells. These screens will involve challenging macrophages with S. aureus labelled with pHrodo, to measure bacteria in a phagosome of pH 6 or less and an automated fluorometric assay of activation of the lysosomal protease cathepsin D as markers of phagosomal maturation. These screens will be used with the Nebraska transposon mutant library of a USA300 S. aureus strain and with a targeted siRNA library of host factors regulating membrane trafficking to identify factors influencing the maturation of S. aureus containing phagosomes. A candidate approach using a panel of antibodies against phagosomal markers of lysosomal tethering and fusion will also be studied to characterise the features of S. aureus containing phagosomes. Key findings will be confirmed with clinical strains of S. aureus, in primary human macrophages using CRISPR gene-editing and in a zebrafish model of S. aureus infection, to establish the role of regulators of phagosomal maturation in bacterial killing. This will define the S. aureus induced phagosomal maturation block and it's role in intracellular bacterial persistence in vivo.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Systematic characterisation of the HCMV pathogenicity domain spontaneously lost from commonly used passaged strains
Funder: Medical Research Council

Abstract: Prior to this application, the genome and transcriptome of intact human cytomegalovirus (HCMV) strain Merlin was first defined (with Dr. Davison, Glasgow). Bacterial artificial chromosome (BAC)-cloned Merlin DNA provides a stable source of infectious virus. For functional screens, 10 HCMV 'block' mutants with deletions of 2-9 genes and adenovirus (Ad) vector library expressing all HCMV ORFs were constructed. Whilst the Ad library is routinely used to screen individual HCMV functions, single HCMV gene deletion mutants are used as gold-standard reagents. All 22 single deletion HCMV UL/b' mutants (UL131A-UL150A) have been generated, validated by whole genome sequencing (Dr. Davison), and functionally, leading to discovery of UL148, as described in this application. These reagents allow for effective loss of function screens of the entire UL/b' region. Mass spectrometry (MS) technologies were developed specifically for HCMV proteomics (with Dr. Weekes and Prof. Lehner, Cambridge), for analysis of plasma membrane (PM) and whole cell lysate (WCL) proteomes over time, deletion mutant analysis is a rational next step. Proteomics employs 10-plex Tandem Mass Tag (TMT) labels for higher throughput analysis. Proteomics is now established in the lab and used to analyse 10 HCMV US12 family deletion mutants confirming its feasibility. Dr. Fielding (named RA) is trained in proteomic techniques and analysis. Access to HpRP-HPLC, Orbitrap Fusion MS etc. is via collaboration with Dr. Weekes and Prof. Lehner (letter of support attached). NK cell techniques are well established, including NK expansion/cloning, multicolour phenotyping, matched effector/target cells. Delineating NK function is challenging, due to multiple activating and inhibiting receptor inputs. For the first time, we are able to examine NK functional effect against changes in PM profile of 1,200 cell surface markers and connect functional screens and mechanistic studies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION