NIHRDataInsights/HRCSData huggingface dataset:
Title: Pharmocokinetics of azithromycin in severe malaria bacterial co-infection in African children
Funder: Medical Research Council

Abstract: African children with severe malaria are susceptible to gram-negative bacterial co-infection resulting in a substantially higher rates of in-hospital and post-discharge mortality. Current evidence for treating co-infection is lacking, and there is no consensus on the dosage or length of treatment required. To provide supporting data to inform a future trial we propose to (i) establish the appropriate dose of oral dispersible azithromycin as an antimicrobial treatment for children with severe malaria and (ii) investigate whether antibiotics can be targeted to those at greatest risk of bacterial co-infection using clinical criteria alone or in combination with a rapid diagnostic biomarker tests. We will conduct a Phase I/II trial in 105 Uganda children with severe malaria at highest risk of bacterial co-infection comparing three doses of azithromycin (10, 15 and 20 mg/kg prescribed for feasibility by weight-bands) given over 5 days in order to generate relevant pharmacokinetic (PK) data by sparse sampling during dosing intervals. The goal is to determine, via population PK modelling, the optimal azithromycin dose in severe malaria, and investigate associations between azithromycin exposure and potential mechanisms (PK-pharmacodynamics, PKPD) using change in C-reactive protein, a putative marker of sepsis at 72 hours (continuous) and microbiological cure (7-day) (binary), alone and as a composite with 7-day survival, while providing preliminary data on longer-term survival (to day-90). We also aim to establish whether a mixture of clinical and rapid diagnostic tests can be used to predict which children with severe malaria are at greatest risk of having bacterial co-infections so that the results may be generalizable in future where microbiological services are poorly developed or non-existent both in children enrolled to the trial and in a comparator cohort (n=50) of children hospitalised with severe malaria at low risk of bacterial co-infection.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Molecular mechanisms of sporogonic development in malaria parasites
Funder: Medical Research Council

Abstract: New therapies for prevention and treatment of malaria, and for reducing malaria transmission, are urgently needed. This project aims to increase our understanding of the molecular mechanisms underlying the formation and function of the crystalloid - a malaria parasite organelle found uniquely in the ookinete and young oocyst stages that is essential for sporogonic development and transmission from mosquito to human. A crystalloid-resident palmitoyl-S-acyl transferase (DHHC10) is essential for crystalloid formation, indicating that S-palmitoylation of crystalloid proteins is critical to these processes. This project aims to study the mechanisms of sporogonic development focussing on the role of palmitoylation in crystalloid biogenesis and function. Using the rodent malaria parasite Plasmodium berghei, we will dissect the substrate range, spatiotemporal dynamics and mechanisms of DHHC10-mediated palmitoylation in crystalloids. The objectives are: (1) Using acyl-biotin-exchange (ABE) and label-free quantitative mass spectrometry (LFQMS), the ookinete palmitome will be determined of DHHC10-positive and DHHC10-negative parasite, allowing the identification of DHHC10 substrates and the crystalloid palmitome; (2) Proteins that interact with DHHC10 (either subunits of a DHHC10 protein complex, or its substrates) will be isolated by GFP pull-down using ookinetes expressing GFP-tagged DHHC10, and identified by LFQMS. In a second approach, interactors for DHHC10 will be identified by BioID using ookinetes that express DHHC10 fused to a biotin ligase; (3) New candidate crystalloid proteins identified will be validated using fluorescent protein tagging and gene knockout in transgenic parasite lines. The results will provide important new insights into the molecular mechanisms that facilitate crystalloid genesis and function, and form a platform for the identification of new drug targets specific for sporogonic development, providing new strategies to prevent transmission.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Innate immune restriction of enveloped virus exit
Funder: Medical Research Council

Abstract: Respiratory syncytial virus (RSV) and measles virus (MeV) are significant causes of human mortality, especially among children and the elderly. There is no vaccine for RSV and no licenced antivirals to tackle either virus. My research focuses on the innate immune response to these pathogens, particularly factors that inhibit viral egress. RSV and MeV are both enveloped paramyxoviruses whose exit is coordinated by viral matrix and glyco- proteins. In this project I will apply genetic screens and proteomics to identify inhibitors of these viral proteins and pathways. My first approach combines quantitative assays for viral cell fusion and budding with a human interferon stimulated gene (ISG) library to identify inhibitors. These platforms, assays and approaches have been established in my laboratory and preliminary experiments, presented in my case for support, demonstrate their viability. The second approach is deliberately non-biased - in this system the membrane fraction from infected cells is compared, using quantitative proteomics (SILAC), to uninfected cell fractions to identify enrichment of immune-related proteins. This is relevant because RSV/MeV egress is continually membrane-associated, starting with viral protein trafficking through the ER, Golgi and endosomes and ending with plasma membrane-based budding or fusion events. Again, I have demonstrated the viability of this approach in preliminary experiments which successfully enriched the entire MeV virome. Finally, proteins or pathways that I identify and validate as inhibitors of egress will be broadly characterised to dissect their mechanism of action within the infected cell. This will be performed using an established biochemical and cell-biology based toolbox. Again I provide compelling information in my Case for Support to back-up their application. To summarise this is a cutting-edge application that will improve our understanding of the innate immune response to two globally relevant infections.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: The mechanisms underlying the production of natural mosquito repellents by human beings
Funder: Medical Research Council

Abstract: One of the most effective protections against outdoor-biting mosquitoes is from skin-applied repellents. Whilst repellents have been shown to reduce malaria transmission in some studies, they pose problems for control programmes because people do not use repellents properly and they lack persistence, making them a difficult intervention to exploit. A new generation of personal protection is needed which eliminates the need for application to the skin. Many people report that they are highly prone, or unusually resistant, to being bitten by mosquitoes and we have shown that this is due to differences in chemicals produced in our body odour. Individuals that attract few mosquitoes produce natural, volatile repellent compounds that repel mosquitoes. These chemicals provide complete protection against mosquito bites and are now being developed as products. However, the mechanisms underpinning the production of the repellents by the human body have not yet been elucidated. In a recent pilot study, we demonstrated that attractiveness to mosquitoes is controlled by genetic factors. There is now an exciting opportunity to investigate the genes associated. Our aim is to understand the genetic mechanisms underlying differential attractiveness and determine its biomedical potential to reduce disease transmission. We hypothesise that differential repellency between individuals is mediated by genes that control the production of volatile repellents through the skin. Using cohorts of identical and non-identical twins we will investigate and identify genes that are associated with attractiveness to mosquitoes and those that control the production of natural repellents. In the long term, this could lead to the development of novel small molecules (potentially in pill-form) that enhance the production of natural repellents by the human body, thus eliminating the need for topical application of repellents to the skin, benefitting people at risk of mosquito-borne diseases.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION
Title: Role of the Human Cytomegalovirus Major Immediate-Early 1 Protein in Viral Reactivation from Latency
Funder: Medical Research Council

Abstract: Human cytomegalovirus (hCMV) is the leading cause of birth defects and a major pathogen in immunocompromised patients, yet prevention and treatment options are limited. Following primary infection, hCMV establishes lifelong latent infection in its host. Although much of the disease burden associated with hCMV infection has been linked to reactivation of latent virus, the molecular mechanisms underlying this process have remained unclear. The proposed project will investigate key molecular events required for hCMV reactivation in myeloid cells focussing on the viral major immediate-early 1 protein (IE1). IE1 is among the very first proteins expressed during hCMV reactivation and therefore an obvious candidate for regulating this process. The function of IE1 has been extensively studied in fibroblasts permissive for viral replication. However, how IE1 may contribute to hCMV reactivation from latently infected cells has not been explored. Our previous work in fibroblasts has shown that IE1 targets proteins of the signal transducer and activator of transcription (STAT) family to modulate central cytokine-induced signalling pathways including interferon responses. We have also shown that IE1 docks with the nucleosome surface. We propose that the IE1-STAT and IE1-nucleosome interactions are individually required for efficient hCMV reactivation from myeloid cells. We will elucidate exactly how IE1 may promote hCMV reactivation by rewiring signalling linked to STAT1, STAT2 and STAT3. We will also study how the IE1-nucleosome interaction may impact chromatin composition and function pertinent to viral reactivation including core histone ubiquitylation. Finally, we will explore how the relevant IE1-host interactions may be exploited for novel strategies targeting hCMV reactivation. The results from this study will add considerably to our limited molecular understanding of hCMV reactivation from latency and will identify novel strategies to combat this important virus.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Understanding DNA-binding by type IV pilins: key event during transformation in naturally competent bacteria
Funder: Medical Research Council

Abstract: Natural transformation is the biological process widespread in bacteria during which some species capture free DNA from their surroundings and incorporate it into their genomes. This process can have devastating medical consequences by leading to the emergence of bacterial pathogens with increased virulence and/or resistance to antibiotics. The first step during natural transformation, DNA uptake, is mediated by surface nanomachines composed of type IV pilins, which are known as type IV filaments (Tff). Tff are almost universal in prokaryotes and have been associated with a vast array of properties, many of which are key for the virulence of bacteria causing important human diseases. Our recent work has explained how Tff interact with DNA. We have identified the first type IV pilin with intrinsic DNA-binding ability, ComP from N. meningitidis. We found that ComP specifically binds the DUS motif, which in meningococci promotes selective uptake of homotypic DNA. We found that this important mechanism of protection against indiscriminate transformation by foreign DNA is shared by most species from the Neisseriaceae family that harbour ComP homologs recognizing different DUS. The high-resolution structures we determined for two ComP orthologs identified a novel mode of interaction with DNA. We will address two important questions during this project. How can different ComP orthologs differentiate between closely related DUS motifs? How do other DNA-binding pilins, in competent bacteria where there are no ComP homologs, interact with DNA. This is expected to generate a holistic view on DNA-binding by type IV pilins, which will have important and broad implications for nanomachines that are virtually ubiquitous in prokaryotes. This could ultimately lead to the development of new antivirulence drugs interfering with Tff assembly or Tff-mediated functions, which are needed in this era of rising antibiotic resistance.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Mechanisms underlying enhanced infection prophylaxis for advanced HIV in Africa
Funder: Medical Research Council

Abstract: Mortality is high in HIV-infected patients starting antiretroviral therapy with advanced disease in sub-Saharan Africa. The REALITY trial, which enrolled 1805 adults and children with CD4<100 cells/uL in Kenya, Uganda, Malawi and Zimbabwe, showed that enhanced prophylaxis (cotrimoxazole plus 12 weeks isoniazid and fluconazole, 5 days azithromycin and single-dose albendazole) reduced 24-week mortality by 27% compared to cotrimoxazole. We now need to understand how this bundle works, whether all components are required and whether additional interventions could reduce mortality further. Enhanced prophylaxis reduced TB and cryptococcal disease but not severe bacterial infections. Our first question is therefore whether azithromycin and albendazole are needed in the bundle, or should be refined in a scaled-up intervention. Our second question is to ascertain how enhanced prophylaxis works at the mechanistic level. Understanding this would help to delineate which underlying pathogenic pathways are most tractable to intervention, and inform future approaches. We will use a case-cohort design to evaluate the contribution of subclinical infections, enteropathy and immune activation to mortality in 602 participants and to assess whether the randomised interventions perturb these processes. Using plasma, PBMCs and stool collected at baseline, 4 and 12 weeks, we will evaluate i) subclinical bacteraemia and microbial translocation by qPCR and broad-range 16s rDNA PCR with next-generation sequencing; ii) enteropathy using a range of biomarkers to evaluate bacterial and helminth carriage, enterocyte damage, intestinal inflammation, epithelial regeneration, gut barrier function and microbial translocation; and iii) multiplex soluble inflammatory markers and T-cell immunophenotyping by flow cytometry. We will estimate associations between each marker and mortality using Cox regression models, random effects models and principal components analysis.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: A novel family of type VI-secreted toxins affecting Rho GTPases, actin dynamics and inflammation
Funder: Medical Research Council

Abstract: Our goal is to understand how intramacrophage opportunistic pathogens, in conjunction with an underlying chronic condition, drive actin network defects leading to exaggerated inflammation. In Aim 1 we will functionally characterise TecA by (i) Investigating its mechanism of secretion via the T6SS, and (ii) Identifying novel TecA targets in macrophages and whether TecA plays a role in other T6SS-mediated phenotypes. We will address whether TecA binds to the inner surface of the ring-like Hcp hexamers or to other key proteins of the T6SS apparatus. The studies will involve co-immunoprecipitation (co-IP) with and without crosslinking using Hcp- and TecA-specific antibodies and also combinations of mutant strains lacking one or more T6SS core components. Using purified TecA-FLAG or TecA-6xHis as baits we will isolate complexes and examine their composition by mass spectrometry (MS). A variation of this approach will also be used to address the second part of this aim, where we will perform Co-IPs with macrophage cell lysates to identify TecA-binding eukaryotic partners. We will also use a candidate approach by directly examining the effects of TecA on additional GTPases such a Cdc42, Rab7 and others and also to determine if TecA is responsible for defects in NADPH oxidase assembly and phagocytic capacity. In Aim 2 we will decode how the Pyrin inflammasome senses defective actin polymerization by (i) demonstrating that Pyrin recruited to the sites of ectopic actin nucleation upon infection with Burkholderia; (ii) Identifying Pyrin-interacting proteins recruited in a TecA-dependent fashion to sites of ectopic actin nucleation; and (iii) assessing the status of the Pyrin inflammasome in Burkholderia-infected, CFTR-defective human peripheral monocytes. These experiments involve a combination of cell biology and biochemical approaches probing various potential pathways for Pyrin recruitment and activation.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: MICA: Understanding how bacteria respond to efflux inhibition
Funder: Medical Research Council

Abstract: Antibiotics are a cornerstone of modern medicine and used extensively by healthcare professionals. Therefore, antimicrobial resistance (AMR), especially by Gram-negative bacteria, poses a threat to human health. Furthermore, developing new antimicrobials has proved increasingly difficult over the past few decades. For Gram-negative bacteria this is due to combination of poor drug permeability coupled with active efflux. Therefore, one strategy to address the growing problem of AMR is to develop inhibitors of multidrug resistance (MDR) efflux pumps thereby sensitizing bacteria to already existing antimicrobials. Using efflux inhibitors in combination with antibiotics will increase and prolong their effectiveness. This project will build upon our preliminary data that indicates that bacteria can become resistant to an efflux inhibitor alone and in combination with a fluoroquinolone antibiotic. In this project we will (1) investigate the bacterial response to efflux inhibition, (2) the conditions by which bacteria can evolve resistance, and (3) identify clinically relevant strategies to suppress resistance. We will do this using fluorescence based reporter systems that will detect transcription of RND MDR efflux pump and stress response genes in populations and single cells. We will use methods established in our laboratory including flow cytometry methods to (1) measure efflux activity in bacterial cells, and (2) sort populations of bacterial cells after exposure to drug +/- efflux inhibitor followed by whole genome sequencing. The mechanisms of resistance will be elucidated. We will also use established pharmacokinetic pharmacodynamic models to identify when resistance emerges and how it can be suppressed. The work proposed in this project has significant implications for the treatment of bacterial infections.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Structural biology of immune receptors in disease-carrying mosquitoes
Funder: Medical Research Council

Abstract: This project combines my multifaceted technical skills applied to the characterization of the structural biology of mosquito immune receptors. I will focus on TOLL receptors implicated in resistance to pathogens that have undergone gene duplication in disease-carrying mosquitoes. I will combine structural techniques (X-ray crystallography and electron microscopy) with biophysical tools and molecular modelling to understand the mechanism of activation of TOLL1 and 5 by SPATZLE1 (SPZ1) isoforms; TOLL7 by neurotrophin1 (NT1, also SPZ2) and viral envelope glycoproteins; and orphan TOLL9 and 11. I will use baculovirus expression to produce the extracellular ligand-binding domains of these receptors. Bacterial and insect expression systems are foreseen for the production of SPZ and NT isoforms, the respective TOLL ligands. Ligand fishing experiments with Biacore will help identify the ligands of orphan TOLL9 and 11. I have successfully produced fruit fly TOLL1 and SPZ1 and determined the crystal structure of their 1:1 complex at a resolution of 2.4 Å. Here I will use mosquito TOLL5A and SPZ1C, with established immune functions, to determine their atomic structure. My working hypothesis is that using a different species will facilitate the crystallization of the active dimer in a 2:2 complex, which eluded me using Drosophila material. Comparing monomer and dimer structures will establish the molecular basis of negative cooperativity. I will characterize NT1 and viral glycoprotein recognition by TOLL7 and compare to binding modes of TOLL, mammalian TLRs, and also NT receptors p75, trk and sortilin that have no direct homologue in mosquitoes. Cellular signalling assays will confirm the validity of my structural observations. Fluorescence microscopy will determine the cellular localization of the activated receptors and co-localization of adaptors. Structure-function relationships analysis will give insight into the mechanism of protein network evolution.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: VACCINE - Development of novel picornavirus virus-like particle vaccines
Funder: Medical Research Council

Abstract: Virus-like particles (VLPs) are of interest as novel vaccines. They can present antigenic features of a viral pathogen in a safe and highly immunogenic form. The hepatitis B and human papillomavirus vaccines are well-established examples. Some viruses e.g. the picornavirus, poliovirus, produce empty particles as part of the natural lifecycle. Although these empty particles could be VLP vaccine candidates, they are very unstable and readily undergo an antigenic conversion to a non-native state and therefore would not elicit a protective immune response. Over the past 5 years, we have generated VLPs of poliovirus that are now being expressed in recombinant systems as novel vaccine candidates. By selecting viruses at elevated temperatures and by identifying reversion of temperature-sensitive mutants, we have identified a number of residues in the capsid which renders this stable, even when empty. We now propose to extend our studies to other picornaviruses and thus develop a generic approach to VLP vaccine development. The focus of the current application is the causative agents of human hand, foot and mouth disease (HFMD), enterovirus 71 (EV71) and Coxsackie virus A16 (CVA16). HFMD is a disease of growing concern due to outbreaks affecting children in the SE Asia region, associated with hospitalisation and death. Outbreaks have recently been reported in Europe.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION