NIHRDataInsights/HRCSData huggingface dataset:
Title: Defining early events underpinning Chlamydia trachomatis entry into mammalian host cells
Funder: Medical Research Council

Abstract: Chlamydiae are obligate intracellular bacterial pathogens that have worldwide medical impact. Chlamydiae force their own entry into human host cells where they replicate within a specialised membrane-bound compartment, termed an 'inclusion'. Both bacterial entry and replication require a bacterial type III secretion system (T3SS), a macromolecular machine that deploys virulence effector proteins into the host cytosol, nucleus and the boundary membrane of the inclusion. We have recently examined events that occur during the entry of infectious extracellular C.trachomatis elementary bodies into human cells. We have shown that filopodial capture is a predominant early event during Chlamydia entry. We have described a novel macropinocytosis-like pathway underpinning bacterial entry and demonstrated a pivotal role for the SNX-PX-BAR family protein sorting nexin 9 (SNX9) in filopodial capture. We propose to address important interrelated questions that arise from our findings using established multidisciplinary approaches including emerging genetic techniques, cell biology and bioimaging. We propose to define the major route of actin-dependent entry into target cells involving filopodial capture and macropinocytosis by investigating the T3SS effectors involved in SNX9-dependent signalling, by determining why the actin nucleation promoting factor N-WASP is critical for C.trachomatis entry, and by examining the mechanism of filopodial capture in detail. We will also characterise a novel early trafficking step involving F-actin stress fibres during the biogenesis of early chlamydial-containing vacuoles. Together the data will provide a mechanistic understanding of how this major human pathogen subverts the host actin cytoskeleton to promote its uptake and establish a replicative niche within target cells.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Human live-attenuated rotavirus to assess next-genchallenge with eration rotavirus vaccines in Africa
Funder: Medical Research Council

Abstract: Widespread introduction of oral rotavirus vaccines (ORVs) in Africa has reduced diarrhoeal disease and mortality, but rotavirus remains the leading cause of hospitalised disease as these vaccines are only ~50% effective in low-income countries compared with ~90% in high-income countries. Testing the next-generation of vaccines is challenging because very large trials are needed to establish non-inferiority/superiority and reliable immune correlates of protection (CoPs) have not been identified. Building on pilot work in Zambia, we propose to measure rotavirus shedding after challenge with live-attenuated rotavirus vaccine (Rotarix) to assess mucosal immunity induced by a novel parenteral trivalent VP8 subunit vaccine (SK Chemicals, South Korea) used alone or in combination with ORV. Administration of parenteral VP8 vaccine in a combined schedule promises to maximise protection by boosting immunity in 'mucosally-primed' infants and immunising those that remain naïve. The appropriateness of this human infection model is supported by recent work showing protection against rotavirus disease is mediated by immunity against infection rather than against disease following infection. Also, we will use this novel and safe human infection challenge model to investigate the development of mucosal immunity and measure novel immune CoPs against infection, which can be validated by comparison with stored samples from the ongoing phase 3 efficacy trial of VP8 vaccine against clinical disease. Identification of CoPs as potential alternative clinical trial endpoints and the establishment of a rotavirus infection human challenge model in Africa would greatly facilitate the testing and licensure of RVs under development. More immediately, if we find that combined oral-parenteral immunisation is effective, subsequent programmatic evaluation could support wider introduction of this schedule in Africa.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Development of next generation anti-malarials targeting the essential parasite protein kinase PfCLK3
Funder: Medical Research Council

Abstract: This programme aims to develop an Early Lead and back series of inhibitors against the malaria protein kinase PfCLK3. This protein kinase plays an essential role in RNA splicing and has been validated by us to be a target with the potential to offer prophylactic, curative and transmission blocking activity. To deliver on this aim we have obtained the support of the malaria drug discovery community. In particular, the Medicines for Malaria Venture (MMV), Bill and Melinda Gates Foundation and the Malaria Drug Accelerator (MalDa) are making available in vitro and in vivo assay platforms what will allow this grant to test the properties of optimised PfCLK3 inhibitors against MMV's Target Candidate Profiles (TCPs) for compounds for Lead to Candidate development. The key decision makers at MMV and Gates Foundation (Jeremy Burrows and Gang Lui, respectively) have agreed to sit on our Scientific Advisory Board, which together with Javier Gamo from GSK not only means we have a strong decision making body for GO/NO GO decisions but means that MMV and Gates are kept well informed of the progress of this programme. We will deliver on our aim through three objectives 1. Conduct hit to lead on TCMDC-135051. Our previous studies discovered a potent and selective inhibitor to PfCLK3, namely TCMDC-135051. We plan to further develop this inhibitor in a hit to lead programme. 2. Screen for novel chemical matter and develop in a hit to lead campaign. We have developed a high throughput assay for PfCLK3 that will be used to screen for new chemical matter at the European Lead Factory and in the Dundee Drug Discovery Unit. Hits will be optimised in a hit to lead programme 3. Test front running molecules for properties that meet MMV criteria for subsequent Lead to Candidate optimisation. In collaboration with MMV/Gates Foundation/MalDa we will test our front-running compounds for properties that meet MMV's TCPs for further development.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Using conjugative elements to manipulate bacterial communities
Funder: Medical Research Council

Abstract: Conjugative elements are naturally occurring genetic entities that encode machinery necessary to facilitate their transfer between bacterial cells. They often are responsible for the spread of virulence factors and antibiotic resistance among bacteria, but by replacing these undesired components with genes engineered to modulate gene expression of specific bacterial species, conjugative elements can be exploited as vehicles for a form of microbial gene therapy. In this proposal, we will use conjugative elements to modify Vibrio cholerae bacteria by introducing "anti-virulence" factors that block the expression of genes required for virulence and colonization. We will first develop an efficient conjugative system that can both carry novel genetic functions and be visually tracked using fluorescence microscopy as it spreads among V. cholerae cells. Using time-lapse microscopy, we will determine the impact of environmental conditions, bacterial community organization and multispecies mixing on conjugation. These in vitro experiments will give insight into the in vivo conditions conducive for conjugative element transfer. Finally, conjugative elements will be engineered to carry gene-silencing elements targeting specific V. cholerae virulence and colonization genes. These conjugative elements will then be introduced into V. cholerae infecting infant mice to determine whether such elements can be used to interfere with the ability of V. cholerae to cause disease. Although the scope of this project will focus on V. cholerae, the approaches taken are directly translatable to other enteric pathogens. Furthermore, the range of delivered genes can be expanded to include other beneficial functionalities, such as immunomodulatory factors or biosynthetic enzymes that compensate for metabolic deficiencies. Ultimately, the information gained from our findings will also be applied broadly to study how mobile genetic elements spread in nature.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Medical and biological significance of exhaled M. tuberculosis detected by community level face mask sampling in Pretoria.
Funder: Medical Research Council

Abstract: Through face mask sampling (FMS), we are identifying exhaled M. tuberculosis (Mtb) in a pilot active case finding (ACF) study to detect individuals with pulmonary TB (pTB). Working with the community oriented primary care programme in the Tshwane district of Pretoria (RSA) we have found exceptionally high positivity rates (>15% against a prior estimate of <1%) through unselected household sampling. These findings raise concerns that FMS+ve individuals have active TB and are infectious. Although we have evidence from a pilot ACF study that some FMS+ve individuals progress to overt TB in 1-2 months, no clinically recognised pTB has yet been detected in the Tshwane study. Current follow up is very limited and we now seek to place this important finding into context with recent concerns that much TB transmission may come from asymptomatic infection. Through further sampling on the established Tshwane platform, we will recruit individuals, FMS+ve for Mtb at first contact (cases), into one year follow up allowing us to establish the main patterns of bacillary excretion (e.g. persistent, intermittent and transient) and their clinical status through monthly review. Control individuals, negative on two masks at recruitment, will be matched to cases by HIV status, age and sex, to cases and followed 3 monthly, also for one year. (Target, 75 in each group; expected retention 50) In addition to Xpert assays on masks, we will detect live Mtb in both masks and blood with a novel bacteriophage assay (recently reported positive in blood from symptomless contacts of pTB). We hypothesise that that blood positivity may predict disease progression. In addition to providing participant care supervised by an independent study monitoring group, we aim to assess potential for a future study of FMS+ve asymptomatic individuals for their infectivity to guinea pigs in the Pretoria AIR facility, thereby definitively establishing their likely contribution to TB transmission.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Improving malaria risk assessment in Blantyre district, Malawi by optimizing Anopheles surveillance using open-source and real-time data
Funder: Medical Research Council

Abstract: Malaria is an important public health problem in Malawi. Significant investment in malaria control has reduced malaria burden in the last decade. Unfortunately, it has not been enough to decrease malaria incidence below the annual 4 million cases. To improve malaria control, surveillance of mosquito vectors is essential. Unfortunately, this is costly and time-consuming. Quantitative tools can help maximize the value of data with the least amount of resources required. The aim of this project is to improve mosquito surveillance by developing a targeted surveillance tool. Using transparent and robust statistical methods, this tool will make surveillance more economical, whilst simultaneously improving the quality of data collected. A mosquito database will be established in Malawi by collating data from stakeholders on the species composition, spatial distribution, physiological status and sporozoite rate of malaria vector mosquitoes. This database will be linked to malaria incidence and open access data (e.g. temperature, landcover, human populations), to identify variables associated with Anopheles proliferation and malaria incidence. Next, a real-time spatial predictive model will be developed that identifies areas with a high risk of Anopheles proliferation using real-time data from the daily satellite products (e.g. MODIS, LANDSAT). The model will be implemented, validated and refined in Blantyre district, Malawi by comparing the targeted surveillance to the already established mosquito surveillance. Monthly collections will be done using CDC light traps and proko-pack aspirators. The model will then be adapted into a user-friendly format. I will work with the Ministry of Health to implement this decision supporting tool within the malaria control program. This project will provide an economically feasible sampling strategy which improves the allocation of the limited resources available and provide a new scientific avenue to improve mosquito surveillance.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: MRC Transition Support CSF Nicholas Matheson
Funder: Medical Research Council

Abstract: Approximately 100 years since its transmission to humans, HIV-1 infects almost 40 million people worldwide, and causes around 1 million AIDS-related deaths every year. I previously used TMT-based plasma membrane proteomics to gain a comprehensive, unbiased overview of cell surface proteins regulated by HIV infection. In addition to known targets, I discovered HIV-mediated downregulation of the amino acid transporter SNAT1, and the putative serine carriers SERINC3/5. I found SNAT1 to be dramatically upregulated at the surface of activated T cells, identified alanine as an endogenous SNAT1 substrate, and showed that extracellular alanine is essential for T cell mitogenesis. Along with SNAT1 and SERINC3/5, I also observed regulation of other transmembrane transporters, suggesting a general paradigm for HIV-mediated manipulation of nutrient uptake and cellular metabolism. During the first 2.5 years of my Clinician Scientist Fellowship, I have: developed a reporter virus (HIV-AFMACS) allowing magnetic selection of HIV-infected primary CD4+ T cells; optimised a novel method for metabolite extraction from these cells; and developed a transformative stable isotope-based approach to quantitate amino acid transport. I therefore now wish to leverage these techniques to further elucidate the metabolic processes manipulated by HIV, and define their importance in HIV pathogenesis and T cell immunobiology. In particular, they will enable me to: quantitate amino acid transport by SNAT1 and SERINC3/5 under normal growth conditions in a comprehensive, unbiased fashion; map global metabolic changes in HIV-infected primary CD4+ T cells; and characterise other critical mitogenic amino acid transporters of CD4+ T cells identified by a combined proteomic-genetic screening approach.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: MICA: An evaluation of the antimalarial vaccine candidateR21/matrix M for malaria elimination strategy in the Greater Mekong Subregion
Funder: Medical Research Council

Abstract: We propose the development of the vaccine candidate, R21/matrix M, as an addition to the current malaria elimination tools in the Greater Mekong Subregion (GMS). With prompt case management and the distribution of insecticide treated bed nets, falciparum malaria transmission is now at very low levels in the GMS. We found that the addition of mass anti-malarial drug administration (MDA) can reduce and even interrupt falciparum malaria transmission for short periods. An effective vaccine that can confer protection even for a limited time period combined with MDA may be sufficient to interrupt malaria transmission permanently. R21/matrix M was developed by the Jenner Institute with strong support from the MRC DPFS and is now manufactured by the Serum Institute of India (SII), which produces vaccines in large volumes at affordable prices. To roll out mass R21/matrix-M vaccination, sequential trials are needed. The first is a Phase II trial, for which we apply here, to demonstrate that R21/matrix M is well-tolerated and immunogenic when administered with the antimalarial drug combination dihydroartemisinin-piperaquine and a single low-dose piperaquine. In this trial, three groups of healthy, adult Thai volunteers will be randomised to receive: a) three rounds R21/matrix-M combined with antimalarial drugs (dihydroartemisinin/piperaquine and a single low dose primaquine) b) R21/matrix-M alone and c) antimalarial drugs alone. Study participants will be followed for six months after the first vaccine dose. Adverse events will be recorded initially daily, then weekly and finally monthly. IgG levels against epitopes on the circumsporozoite proteins will be assessed at four time points (M0, M1, M3, M6). This first trial will start at the beginning of 2020 and results should become available shortly after the study completion 6 months later. If R21/matrix-M is found to be safe and immunogenic in this Phase II trial, we will seek funding to proceed to Phase III and IV trials.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: TransLeish: Fitness phenotyping of Leishmania transporter mutants
Funder: Medical Research Council

Abstract: Membrane transporters are fundamental to cellular function, facilitating uptake of nutrients and building blocks, waste disposal, shuttling of molecules between cellular compartments and maintaining physiological pH and osmotic pressure. Leishmania spp. are protists with a parasitic life-style, cycling between an insect vector and mammalian host where they infect macrophages and cause disease through interference with normal immune functions. The specific environments the parasite inhabits determine which transporters are required to meet the basic needs for parasite proliferation and to enable it to adapt and respond to environmental changes. Detailed studies on a few individual transporters underscored the fundamental roles played by Leishmania transporters in meeting basic nutritional needs, intracellular survival and drug resistance. There are however around 300 predicted transporters encoded in the genome, most of which have not been assessed. Here we propose to investigate the L. mexicana transportome to determine which transporters are important on the two major life cycle stages and in the effect of anti-leishmanial drugs. We will exploit a mutant library which we have generated that consists of >200 transporter null mutants and propose to generate additional mutants: around 100 heterozygous mutants for transporters that could not be deleted, and 18 deletions of transporter arrays. Each of these mutants carries a unique barcode that enables "bar-seq" fitness screens, where we will test the relative fitness of each mutant under three types of conditions: (i) growth of promastigote forms under nutrient limitation and pH stress conditions, (ii) growth of amastigotes in macrophages and (iii) survival under drug pressure. These data will identify non-dispensable transporters, and provide the necessary context for targeted screens of novel anti-leishmanial compounds.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Bacterial antibiotic resistance: structure, mechanism and inhibition of ABC transporters responsible for drug efflux and cell wall biogenesis.
Funder: Medical Research Council

Abstract: The emergence of antimicrobial resistant (AMR) bacteria is a well-recognized threat to human health through increased incidence, severity, and persistence of infections. In Gram-negative pathogens like E. coli and P. aeruoginosa, AMR is underpinned in two major ways: the robust outer membrane barrier which prevents many antibiotics reaching their intracellular targets and tripartite efflux pumps which eject drugs from the cell. These pumps consist of an inner membrane (IM) transporter, a periplasmic adaptor protein and the TolC exit duct. Previously, we have determined the structures and interactions of several pump components. More recently, we have revealed the structure of the IM transporter MacB bound to ATP, revealing a novel architecture and mechanism of action where cytoplasmic ATP hydrolysis is coupled to conformational changes in the periplasm, a process we have termed mechanotransmission. Topologically-related to MacB, the LolCDE transporter mediates the extraction of lipoproteins from the inner membrane and their subsequent transfer to the periplasmic chaperone, LolA. Lipoproteins are finally inserted into the outer membrane by LolB. Disruption of the Lol pathway at any stage has been shown to lead to cell death. As such, and with no human ortholog in existence, this system presents an attractive target for the identification, and synthesis of novel antimicrobials. We aim, through our breadth of experience in microbiology, biochemistry, biophysical and structural methods to understand the molecular mechanisms underpinning both antibiotic efflux by MacB tripartite pumps, and lipoprotein trafficking by the Lol system. While the two core transporters bear an overall similarity in fold and likely share the same mechanotransmissive mechanism, they play divergent roles, mediating drug efflux and outer membrane biogenesis. Our studies will further the development of therapeutics capable of interfering with both.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Lung resident memory CD8+ T cells during viral infections - generation and functionality regulated by localisation and type I IFNs
Funder: Medical Research Council

Abstract: Lower respiratory tract infections are a leading cause of death globally and respiratory syncytial virus (RSV) and influenza virus together account for a large number of hospitalisations of especially children and the elderly. Absence of effective vaccines and recurrent infections throughout life demonstrate the difficulties in eliciting protective memory responses in the lung. However, recent evidence indicating the potency of protective mechanisms mediated by tissue resident T cells (Trm) suggest that these cells may be key. In human experimental RSV infection, CD8+ Trm cells correlate with improved outcome and contribute to heterosubtypic immunity against influenza viruses. However, we have limited knowledge of what these cells require in order to become resident, be maintained and function efficiently in the lung. During respiratory viral infections, type I interferons (IFNs) drive early inflammatory responses and modulate T cell effector and memory programming. However, how type I IFNs influence Trm cells has not been investigated. We will address this using established mouse models and a new ex vivo approach involving precision cut lung slices (PCLS). We will use type I IFN receptor deficient (Ifnar1-/-) mice to study the influence of type I IFNs on Trm cell generation and function and will use PCLS to study Trm cell localisation and re-activation after ex vivo infection or antigen re-stimulation. In addition, we will use antibody-mediated blocking and gene silencing tools in PCLS to directly test the impact of type I IFN signalling on Trm cell function. Finally, we will extend PCLS to human lung tissue, providing a unique opportunity to study and manipulate human Trm cells in situ. These studies will help to understand the factors that govern Trm cell residency and function and may ultimately enhance our ability to develop vaccines that better protect against respiratory viral infections.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION