NIHRDataInsights/HRCSData huggingface dataset:
Title: Protein Complexes and Human Genetic Disease
Funder: Medical Research Council

Abstract: Many human diseases have a significant genetic component, and so understanding how genetic variation translates into observable phenotypic differences is of tremendous importance for disease prevention, diagnosis and treatment. While consideration of protein structural context has often been useful for explaining and predicting the effects of genetic variants, the fact that most proteins can assemble into complexes is often ignored. The main premise of this proposal, which is strongly supported by preliminary results presented here, is that the quaternary structural context of a mutation will often have a big influence on its phenotype, in ways that have not previously been recognised. Therefore, I propose to use computational approaches to study human genetic variation (both coding and non-coding) from the perspective of protein complexes, taking advantage of the huge amounts of protein structure and gene sequence data now available. Specifically, we will investigate how the position of a mutation within a complex affects its likelihood of having a large phenotypic impact. We will also study how the overall type of quaternary structure formed is related to its dominant or recessive mode of inheritance. Furthermore, we will use simulations to assess how the order of protein complex assembly pathways can influence the effects of genetic variants. Finally, we will build upon the results of these analyses to develop new methods for predicting the phenotypic impacts of mutations, with ultimate the aim of facilitating the prioritisation and identification of causal variants


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: INFECTION
Title: Trial of vitamin D supplementation to prevent acquisition of latent tuberculosis infection in schoolchildren
Funder: Medical Research Council

Abstract: The World Health Organisation aims to eliminate tuberculosis (TB) as a public health problem by 2050. Measures to prevent acquisition of latent TB infection (LTBI) will need to be implemented to achieve this goal. Vitamin D has long been known to enhance innate immunity to Mycobacterium tuberculosis (MTB), and Phase 2 clinical trials show that vitamin D supplementation enhances innate immune resistance to MTB infection. We propose to conduct a Phase 3 double-blind randomised controlled clinical trial to determine whether vitamin D supplementation reduces risk of acquisition of LTBI in 5,400 initially uninfected Cape Town primary schoolchildren aged 6-9 years at enrolment. Study medication will be administered over 3 years as 156 directly-observed weekly oral doses of 14,000 IU (0.35 mg) vitamin D3 or placebo. We will employ a well-validated interferon-gamma release assay (QuantiFERON TB Gold) to determine whether participants have acquired LTBI at 3 years of follow-up (primary outcome). Logistic regression analysis will be performed on the intention-to-treat population to compare rates of LTBI in intervention vs. control arms at 3 years. The effect of vitamin D supplementation on incidence of active TB, absence from school due to acute respiratory infection and body mass index for age will also be determined (secondary outcomes) and an economic evaluation will be performed to estimate the cost-effectiveness of using vitamin D to prevent LTBI. Results of this trial will inform TB control policy in South Africa and beyond. Vitamin D deficiency is highly prevalent globally: supplementation is safe and inexpensive, and intermittent bolus doses of vitamin D are effective in correcting deficiency and easy to administer. Vitamin D supplementation therefore represents a potentially highly cost-effective means of enhancing TB control in low resource settings which bear the highest burden of disease.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: IMPACT OF FUNGAL ADAPTATION UPON HOST RECOGNITION AND PATHOGENESIS
Funder: Medical Research Council

Abstract: An individual's susceptibility to fungal infection is heightened by factors that attenuate immune surveillance. Considerable progress has been made in defining the mechanisms by which innate immune cells recognise and clear Candida albicans, the major fungal pathogen of humans. These depend on the recognition of specific pathogen-associated molecular patterns (PAMPs) by host pathogen recognition receptors (PRRs). To date, C. albicans PAMPs have generally been defined using fungal cells grown under standardised conditions in vitro that do not accurately reflect dynamic host microenvironments. In reality, the fungus is a moving target. It adapts to host signals such as local nutrients, thermal fluctuations and iron limitation by altering its cell surface. We recently showed that the adaptation of C. albicans to physiologically relevant carbon sources triggers dramatic changes in cell wall architecture, immune recognition and virulence. Therefore, in this programme we will test our hypothesis that fungal adaptation within host niches influences the efficacy of local immune surveillance, thereby strongly impacts on disease progression. First, we will establish how fungal adaptation impacts on PAMP exposure at the C. albicans cell surface by combining targeted molecular dissection in C. albicans and the host with genomic-wide screens and biochemical characterisation. Next we will define how these changes influence the dynamics of Candida-phagocyte interactions by combining precise molecular intervention with state-of-the-art 4-D video microscopy. We will also exploit new synthetic reporters to determine the impact of these changes on disease progression in well-established infection models. Furthermore, the molecular characterisation of C. albicans cells from patient samples will establish the relevance of these major findings to human infection. In this way we will establish a key principle in infection biology - that fungal adaptation resists host immunology.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Capturing functional states of type III secretion systems from Chlamydia in situ
Funder: Medical Research Council

Abstract: Chlamydiae are obligate intracellular bacterial pathogens that have worldwide medical impact. Initial chlamydial entry into host cells and subsequent intracellular replication require a type III secretion system (T3SS), a molecular nanosyringe conserved among many medically important pathogens. T3SSs translocate virulence effector proteins into the host that hijack key cellular processes. The conserved T3SS apparatus is an attractive target for the development of molecular inhibitors, which are becoming vital considering ever-increasing bacterial resistance to conventional antibiotics. We have applied cryo-electron tomography to study the structure of infectious chlamydial elementary bodies (EB). Our data revealed important insights into polarised EB architecture, mapped the global arrangement of T3SSs on the EB surface, and revealed early endocytic structures formed during EB interaction with host cells. We propose to study the intact macromolecular structure of the C.trachomatis T3SS in different functional states by cryo-electron microscopy and sub-tomogram averaging. We will determine the intact membrane-embedded structure of the T3SS in situ. Our preliminary data reveal a substantial cytosolic substructure not previously visualised in detergent-solubilise core complexes. We will examine T3SS structure in contact with host membranes, potentially allowing the visualisation of the enigmatic host membrane-embedded translocon complex, and the conformational and compositional changes associated with T3SS activity. We will exploit transformed chlamydial strains to identify T3SS components using fusion proteins and epitope tags. We will study T3SS disassembly following EB internalisation and early vacuole architecture using focussed ion beam milling. The structural data arising from our studies of Chlamydia has the potential to directly contribute to the development of novel therapeutics targeting T3SS-dependent virulence effector secretion in bacterial pathogens.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Characterisation of the essential C. difficile S-layer secretion system
Funder: Medical Research Council

Abstract: C. difficile causes intestinal infections varying in severity from a mild, self-limiting diarrhea to severe, often life threatening, inflammatory complications such as toxic megacolon and pseudomembranous colitis. Infection commonly follows antibiotic-induced intestinal dysbiois that gives C. difficile a competitive advantage. There is an urgent need for novel therapies that treat C. difficile infection without causing further dysbiosis. Our research focuses on the biogenesis of the C. difficile cell surface and, in particular, the translocation of cell wall associated proteins across the membrane. The C. difficile surface is completely covered with a paracrystalline S-layer comprised of the S-layer protein, SlpA, and an additional 28 cell wall proteins which functionalise the surface. SlpA and the dominant cell wall protein, CwpV, are translocated by the accessory SecA2 system, a second parallel Sec machinery found in some Gram positive bacteria. The ATPase at the core of the accessory Sec system in C. difficile, SecA2, is an essential protein, making this an attractive target for therapeutic intervention. SlpA and CwpV are critical virulence factors contributing to colonisation of the host and interactions with the immune system. We aim to fully characterise the C. difficile accessory Sec system, localising protein secretion within the cell, identifying all of the proteins required for efficient secretion and elucidating the molecular basis of substrate recognition. We will adopt a highly multidisciplinary approach, complementing our expertise in molecular microbiology and protein biochemistry with key collaborations adding world-class mass spectrometry and advanced super-resolution microscopy. The insights gained from our analysis of S-layer secretion will contribute to the development of targeted inhibitors that block the accessory SecA2 system and thereby kill C. difficile.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Antibiotic resistance in Gram-negative bacteria: structure and function of TolC-dependent multidrug efflux pumps
Funder: Medical Research Council

Abstract: The increasing prevalence of bacterial multidrug resistance (MDR) presents a growing threat, and it is important to understand antibiotic resistance mechanisms and elicit ways to combat them. We seek a continuation in funding from mid 2015 to extend our analyses of the tripartite efflux pumps that underpin MDR throughout Gram-negative bacteria like E.coli, Salmonella and Pseudomonas. These pumps comprise inner membrane transporters that bind and deliver antibiotics to the cell exit duct TolC that projects across the periplasm and outer membrane, assembly and function aided by the third component, the periplasic adaptor. There are three classes of tripartite MDR efflux pumps, differing in their transporters and the antibiotics they eject. By combining approaches, especially the crystallography and in vivo cross-linking of membrane proteins and complexes, we have described the structure and action of the exit duct and adaptor components common throughout the pumps, and defined their interactions with each other and the transporter in the widespread RND class of drug efflux pumps exemplified by E.coli AcrA-AcrB-TolC. This allowed us to model the first data-based atomic view of a complete assembled multidrug efflux pump, and provide insight into how the TolC periplasmic entrance opens to allow efflux, and how it might be inhibited. We aim to further refine our RND pump model, and shift focus to the component atomic structures and assembly of the two other distinct classes of antibiotic efflux pump, ABC and MFS, exemplified by E.coli MacA-MacB-TolC and EmrA-EmrB-TolC. We will also instigate approaches to establish how pumps bind and translocate antibiotics from live bacteria. In these ways we aim to visualize how all the drug efflux pumps assemble and operate within the cell envelope to offer a better understanding of the resistance mechanisms bacteria use to evade antibiotics, and also identify weak points in the pumps that may open up new ways for us to inhibit them


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Helminth infection and the role of altered macrophage phenotype in tuberculosis immunopathology
Funder: Medical Research Council

Abstract: Tuberculosis is more prevalent in areas with high rates of helminth coinfection. Helminths may alter macrophage phenotype influencing innate immune responses. Preliminary data gained during my Walport Fellowship in Peru shows that helminth infection alters pro-inflammatory phenotype, including gene expression and secretion of matrix metalloproteinases (MMPs). There is limited understanding of the impact of worm infection on TB-specific innate immune responses, particularly in terms of secretion of MMPs by differently polarised macrophages. Elucidating mechanisms behind differently activated macrophages has implications for our understanding of tissue destruction in TB. On the basis of my own and other preliminary data, I shall investigate the hypothesis that helminth infection drives macrophage-dependent immunopathology in human TB. Specific aims are to investigate effect of helminth infection on M1 and M2 macrophage activity in patients with TB. Secondly, I shall dissect mechanisms regulating such M1/M2 macrophage responses. I shall recruit patient/control subjects with/without tuberculosis +/- helminth infection in Lima and the Amazon jungle in Peru. In addition to collecting demographic/clinical data, I shall purify PBMCs, generate M1 and M2 macrophages and stimulate them with PPD from TB or the helminth-antigen chitin. Pro-inflammatory & MMP activity will be analysed at the level of gene expression by qPCR, secretion by luminex and surface expression by FACs. A longitudinal assessment shall also be performed to investigate changes in immune responses when helminths +/- TB disease are treated. Concurrently, I shall investigate regulatory pathways in differentiated M1/M2 human macrophages using techniques including phosphoprotein multiplex array, specific chemical inhibition of paths including PI3K and AMPK and specific siRNA inhibition. Understanding the mechanisms of parasite-TB interactions may identify potential targets to decrease inflammation in TB


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: HIV-1 Reservoir in Gut-Associated Lymphoid Tissue (GALT) in Primary HIV Infection
Funder: Medical Research Council

Abstract: Aims: This project aims to determine if measurement of HIV-1 reservoir in Gut-Associated Lymphoid Tissue (GALT) can predict post-treatment control (PTC). Relevance: Recent cases of PTC of HIV - where virus does not rebound after cessation of ART commenced during primary HIV infection (PHI) - suggest a remission or functional cure is possible. Accurate predictors of PTC are needed to allow safe stopping of ART. Current viral reservoir quantification assays from blood have not predicted PTC, suggesting an alternative source of rebounding viraemia. I propose that the terminal ileum is a key productive reservoir and that quantification of HIV reservoir at this site will predict PTC. Objectives: 1.To establish a GALT HIV-reservoir sub-study from the HEATHER cohort involving ileal and rectal biopsies. 2.To develop and apply virological and immunological assays to characterise the HIV reservoir in GALT and compare with equivalent measures in blood using established assays/platforms. 3.To develop a predictive algorithm of PTC, that will include GALT and blood biomarkers. This will be tested amongst selected individuals with a Treatment Interruption(TI) in the HEATHER study. Methodology: >130 individuals are enrolled and have donated blood samples to the HEATHER study. I will enrol to a GALT sub-study (n=55). Using Ileal and rectal biopsies, I will apply assays of HIV reservoir and immunophenotyping to CD4+ cells from GALT; including Total and integrated DNA, HIV-1 intracellular viral unspliced RNA transcripts, Next Generation Sequencing of Provirus and immunophenotyping using FACS. The measures of reservoir in GALT will be compared with blood and together will inform an algorithm to PTC. Scientific and medical opportunities: The project will provide a a detailed analyses of sustained ART on the GALT reservoir and the role of GALT biomarkers in predicting PTC. Such biomarker data will allow for development of safe TI protocols in HIV cure intervention trials.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Towards an integrated understanding of the CD28/CTLA4 immune checkpoint in the regulation of autoimmunity
Funder: Medical Research Council

Abstract: This programme is focused on understanding how the T cell molecules CD28 and CTLA4 dictate control of tolerance versus immunity, an area of profound importance in immunology. Whilst their involvement is understood in broad principle, the mechanistic details of how the system actually works have not been established and will be crucial for sophisticated manipulation of the immune response. We recently identified a mechanism of action for CTLA4 (transendocytosis) where its inhibitory function is linked to physical destruction of its ligands (CD80 and CD86). Since CD28 binds to the same ligands, CTLA4 controls CD28 engagement. This model generates an integrated view in which the function of each individual molecule must be understood in the context of the other three players. This also leads to an emphasis on the regulation of CD80/CD86 expression and re-expression as the arbiters of immune outcome. Surprisingly, despite the fundamental importance of these ligands in driving immune responses, regulation of their expression is still poorly understood. Furthermore, the rationale for having two biophysically distinct ligands is entirely unclear. It is our contention that a transendocytosis perspective holds the key to understanding why two ligands exist. We will therefore carefully evaluate the differences in the control of their expression, and establish the capacity of each ligand to stimulate responses via CD28 as well as be regulated via CTLA4 transendocytosis. We will use novel mouse models to dissect whether differences between ligand function in vivo reflect inherent functional properties or distinct expression patterns. By utilising a combination of molecular and cellular in vitro and in vivo approaches we will provide new insights into an area of fundamental importance to immunology. This will result in a more complete and predictive understanding of this crucial immune checkpoint enabling us to conceive and test novel therapeutic strategies.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION
Title: Systematic identification of host proteins involved in Human Cytomegalovirus replication, assembly and egress.
Funder: Medical Research Council

Abstract: Disease outcome from virus infection depends on complex host-pathogen interactions. Rational design of therapies, vaccines and disease control depends on understanding these interactions at the molecular level. Identification of host virus interactions provide important potential avenues for novel therapeutics as development of resistance to drugs targeting host genes is less likely than with virus targets. Furthermore, small molecule inhibitors are already available for many host genes. Genome wide siRNA screens have proven a useful tool for deciphering which cellular genes are required for virus replication and which genes have antiviral properties. As well as contributing to our understanding of virus host interactions, siRNA screens are a powerful approach for the identification of antiviral drug targets. The majority of screens performed measure virus replication through the expression of a reporter construct. Although this identifies entry and replication phenotypes, the crucial parameter of virus production is not considered. We have devised a novel siRNA screen that measures both primary replication but crucially, also measures infectious progeny. Using a library of more than 160 siRNA pools against host genes involved in membrane organisation, we have identified multiple genes required for HCMV virus replication, assembly and egress. Crucially we have shown that the correlation between primary replication and infectious progeny is far lower than would be expected, indicating that measurement of primary replication alone may be misleading, especially when attempting to identify host targets for antiviral therapy. Further characterisation reveals the top hit from our screen, VCP/p97, is required for expression of the crucial viral trans-activator IE86. Despite being co-expressed, IE72 protein levels are unaffected. In addition, we show that a small molecule inhibitor of VCP is a potent anti-HCMV drug.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Immunological responses to emerging phlebo- and arenaviruses
Funder: Medical Research Council

Abstract: Emerging New World arena- and phlebo-viruses are responsible for severe hemorrhagic fever in humans with up to 70% fatality rates in as little as 7-10 days. Here, we will study the humoral immune response against these two groups of viruses through the generation of a library of antibodies capable of cross-neutralising several genetically distinct New World arena-viral and phlebo-viral lineages. We will immunise mice with the antigenic glycoprotein-subcomponents of these viruses and apply a prime-boost strategy with glycoproteins from several related virus species to elicit cross-binding monoclonal antibodies. Following elicitation of a robust immune response, we will employ techniques in antigen-specific single B-cell isolation and molecular biology to isolate two sets of monoclonal antibody libraries, one specific to discreet arenviruses and the other to phleboviruses. Following confirmation of neutralisation activity, the conserved viral epitopes targeted by these neutralising antibodies (nAbs) and the mechanism of neutralisation will be studied at a molecular level using, X-ray crystallography. The nAbs produced will be valuable reagents, useful in the serological detection of emerging New World arena- and phlebo-viruses isolated in the field and also as potential antiviral therapeutic reagents. These structural data revealed from this work will reveal sites of vulnerability on New World arena- and phlebovirus surfaces and neutralising epitopes conserved across distant viral species that could be exploited for antiviral and vaccine design.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION