NIHRDataInsights/HRCSData huggingface dataset:
Title: Molecular mechanisms of M2-mediated influenza virus budding and scission
Funder: Medical Research Council

Abstract: Recently we have found an essential role for the influenza virus M2 protein amphipathic helix (AH) in the mediation of membrane scission and the release of budding viruses. This research will elucidate the molecular mechanisms of M2-mediated influenza virus budding and has the following objectives: 1. To determine the molecular mechanism by which the M2 AH alters membrane curvature; 2. To correlate M2-mediated alterations in membrane curvature with the process of membrane scission in vivo; 3. To determine the biophysical properties of M2-mediated membrane scission. Each of these objectives will be addressed using a novel collection of state-of-the-art experimental protocols. To elucidate the mechanism by which the M2 AH alters membrane curvature, strategic mutations will be made in M2 peptides and their capacity for budding assessed in Giant Unilamellar Vesicles (GUV) containing multiple different lipid mixtures. The conditions and mutations that significantly affect M2-mediated budding will then be tested in vivo using a novel method for quantifying membrane budding in cellular membranes and by incorporation of the relevant mutations into recombinant viruses. These mutant viruses will then be assessed for function through a variety of assays including: viral assays (growth rate and budding efficiency) and protein assays (cholesterol association, protein incorporation and localization). These functional studies will be supported by biophysical studies of the M2 AH using real-time single GUV imaging and molecular dynamics simulations. The successful completion of this research project will determine the molecular mechanism of M2-mediated budding. This research has significant implications in the understanding of influenza virus budding, establishes in vitro model systems that can be used in the investigation of budding for multiple different viruses, and provides the molecular details necessary for future drug discovery research.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Utilising proteomics to develop anti-HCMV immunotherapy
Funder: Medical Research Council

Abstract: Development of vaccines and therapy requires that research uses viruses that mimic the agent of disease. We have developed the only HCMV BAC containing a complete wildtype genome, and can prevent in-vitro mutation by tet-repressing the unstable regions UL128L and RL13, enabling growth of high titre, genetically wildtype virus. During subsequent infection without repression, the entire virion proteome is expressed for analysis. We will use this virus to define and validate the entire proteome available for therapeutic targeting. For the production of neutralising antibodies and inhibitors of viral entry, the virion proteome of wildtype virus will be defined. These strategies can reduce transmission, but spread in vivo is cell-to-cell, for which antibody dependent cellular cytotoxicity (ADCC) antibodies may be more effective. We will use Stable Isotope Labelling of Amino Acids in Cell Culture (SILAC) of surface proteins to define all potential targets for ADCC antibodies on the infected cell. We will determine which are targets during natural infection, and the functional consequences of viral encoded Fc binding proteins in circumventing ADCC. We will engineer chimeric antibodies against targets, and test their ability to promote ADCC killing of infected cells for therapy. Our viruses permit the study of wildtype HCMV infected cells, but it remains hard to produce high titre cell free wildtype virus for studying virus entry, because RL13 (an Fc binding protein) inhibits virus release. We have defined the binding partners of RL13 using SILAC-IP and will determine, and circumvent, the interactions responsible for this inhibition. This will enable the production of high titre wildtype virus (with reference to our virion proteome). Since RL13 inhibits virus release, we will determine whether clinical virus lacks gpRL13 when secreted at high titres into urine. Finally we will begin determining whether RL13 promotes persistence of Rhesus CMV based vaccine vectors.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Modelling the contribution of relapse infections to the epidemiology and control of Plasmodium vivax malaria
Funder: Medical Research Council

Abstract: Following a bite from a P. vivax infectious mosquito, sporozoites inoculated into the skin migrate to the liver, where they develop to cause blood-stage infection, or transform into hypnozoites, a stage of the parasite that lies dormant in the liver. Hypnozoites re-activate weeks to months later and cause P. vivax relapses. Recent advances in genotyping markers for molecular monitoring have enabled investigation of the genetic diversity and multiplicity of P. vivax infections, allowing relapses to be distinguished from new infections. I will use data on genotyped infections from approximately 4,000 participants in longitudinal studies to estimate the contributions of new and relapse infections. These estimates will be combined with epidemiological data to test hypotheses for the determinants of hypnozoite activation, ranging from febrile illness, exposure to Anopheles proteins, to a seasonally-driven biological clock for activation. I will construct statistical models of P. vivax relapses and fit them to data from treatment-reinfection studies to estimate key parameters describing relapses: (i) the proportion of infections due to relapses; (ii) the frequency of relapse infections; and (iii) the period during which a person is at risk of relapsing after primary infection. Models of relapse infections will be embedded within a transmission model of the P. vivax lifecycle. This will be fitted to data on age and exposure-specific patterns of parasite prevalence and clinical incidence using Bayesian Markov Chain Monte Carlo methods incorporating prior biological knowledge. Using this model, I will estimate the public health benefit of malaria control interventions. I will develop models for the effects of primaquine treatment (the only available drug that can clear hypnozoites), to compare different treatment strategies, from the perspective of an individual receiving treatment, and from a population-level perspective incorporating reductions in transmission.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Developing methods to assess the impact of malaria interventions upon transmission and the progress towards elimination
Funder: Medical Research Council

Abstract: Malaria is estimated to cause around 1 million deaths per year, with the majority of these deaths occurring in sub-Saharan Africa, primarily amongst young children. Over the last decade there has been an increase in funding from high-income countries and global agencies and renewed interest in achieving local elimination in various regions and the prospects of global eradication of the disease. Measuring progress towards elimination requires an estimate of the reduction in transmission that has occurred over time and the proximity to the threshold reproduction number value of one. However, these changes are not linearly related to measures of disease prevalence and burden (the quantities often used to track progress). The first aim of this project is to develop and adapt methods used for reproduction number estimation in epidemic models to estimate the changes in transmission which have occurred in an area of Western Kenya which has served as a key focus for malaria research over the past few decades. This will provide estimates of the additional control necessary to achieve elimination in an area of intense transmission with high LLIN coverage. It will also provide insights into the direct and indirect effects of insecticide treated nets and how this varies during a long-term intervention. The second objective of this fellowship is to estimate the impact of different control strategies involving the mass administration of artemisinin combination therapy on transmission during a forthcoming trial in the same area. The detailed data likely to be collected during this trial will be used to estimate transmission at a high spatial and temporal resolution. Using the results from the first part of the project of the level of transmission and trajectory of changing transmission prior to the trial, I will then assess the extent to which reductions in prevalence are likely to be sustainable and the prospects for elimination, both within the study area and more generally.


Research activity codes: 8.4 RESEARCH DESIGN AND METHODOLOGIES (HEALTH SERVICES);6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Clinical grade cell separation technologies in the Newcastle Cellular Therapies Facility
Funder: Medical Research Council

Abstract: OBJECTIVES Research and development for current and future clinical trials of which Newcastle University is either a sponsor or a partner is our objective. Cell purification by positive or negative selection, or combinations of the two approaches, will be developed in clinical trials of dendritic cell vaccines, and viral-specific T cells for CMV infection, in the first instance. These are technologies that can be extended to multiple indications. For example, viral-specific T cells could be used to treat EBV infection and deployed in solid organ transplant recipients. Partnerships are being developed with trial sponsors, industrial partners, and through academic and applied research projects to exploit the capacity of NCTF to manufacture cellular therapies in a GMP licensed facility. THE CLINIMACS PRODIGY: The instrument permits automated, standardized cell manufacturing processes in a closed system, and is equipped with a cell processing unit for washing and fractionating cells by centrifugation, image recognition to detect macroscopic layers of cell product, and an integral cell separation unit, based on magnetic bead cell-selection techniques. The device also possesses an integrated cell culture chamber. Input ports allow for the sterile removal and replenishment of media, whilst cells can be monitored using an integrated microscope. The device therefore is a tool for the design and optimisation of GMP-Grade processes and provides a means of producing cell-specific products for human use. CLINIMACS PLUS: is an automated cell separation system based on Miltenyi-Biotec magnetic bead separation technology. It enables the operator to perform clinical-scale magnetic enrichment of target cells. Processes using this technology will be validated and made available to other clinical trials. This capability will permit manufacture of cellular therapies for users beyond Newcastle and support the MRC's strategic aims for the UK development of cellular therapies


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT);5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION
Title: Multivalent proteins for the prophylaxis of influenza
Funder: Medical Research Council

Abstract: The influenza virus continues to be a major health threat with the appearance of new assortants (e.g. H1N1 "swine flu" and H5N1 & H7N9 "bird flu"), difficulties in providing fast and effective vaccination and an increasing resistance to licenced antivirals, particularly oseltamivir (Tamiflu). Our approach targets the host rather than the virus, overcoming drug resistance that is a major problem with the rapidly evolving influenza virus with its potential to mutate and genetically reassort. We have engineered a series of multivalent proteins, or biologics, that bind with high affinity to the terminal sialic acids of cell surface glycoconjugates, thereby masking the receptor of the influenza virus and other respiratory pathogens. As this recognition is linkage-independent, the biologics mask sialic acids receptors with (a2,3) and (a2,6) linkages recognized by avian and human viruses, respectively, and these different receptors have been found at varying locations in the human respiratory tract. In our current DPFS grant we have studied a panel of engineered multivalent proteins that differ in both their valency and the origin of their sialic acid binding modules. Our two lead candidates, when administered intranasally as a single dose, protect mice from a subsequent lethal challenge with influenza virus. Significantly, our current candidate is effective even when given as a single 1ug dose 7 days before virus. In the 12 month extension, we are seeking to determine an optimal prophylactic dosage regimen in mice and to initiate studies in ferrets, the preferred animal model for influenza studies, against both H5N1 and H7N9 viruses. Critically, immunogenicity assessment of the lead candidates will be considered through evaluation of the immune response to the biologics upon repeat dosing. Deimmunisation strategies will be addressed. The efficacy of delivery by nebulizer will also be explored.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: The influence of the liver on NK cell education: a role in Hepatitis C?
Funder: Medical Research Council

Abstract: Introduction Hepatitis C virus (HCV) is a chronic infection that leads to liver failure and cancer affecting 216,000 people in the UK and 150 million worldwide. Natural Killer (NK) cells are important components of the innate immune response to HCV. Specific combinations of NK cell receptors, including KIRs and their HLA-I ligands determine both spontaneous and treatment related outcomes, as do other polymorphisms within the innate immune system affecting the expression of IL28B. Why certain diseases are able to escape the NK cell response is unclear, however we suspect that changes occur at the level of NK cell maturation in the liver directing them towards a hypofunctional or tolerant subpopulation. Hypothesis I propose that NK cells enter the liver as an immature subset that is modulated by the intrahepatic cytokine environment in response to a particular HCV genotype. In this way the HCV virus may determine NK cell response. We will investigate how the liver and HCV infection affect NK cell development and function, focusing on STAT4 as a key molecule in these processes. We also aim to establish how Th1 and Th3 cytokines induced by IL28B/IFNL4 polymorphisms may affect this pathway. Methodology Aim 1: Determine the phenotype and function of NK cells in the liver and blood of non-HCV infected patients. Aim 2: Study how STAT4 affects NK cell maturation, which genes it targets and how this relates to NK cell phenotype Aim 3: Investigate whether abnormalities in NK cells in HCV are genotype specific and if this relates to the hepatic microenvironment Medical opportunities Our work will integrate NK cell development and function with clinical outcomes in HCV. Understanding the influence of the liver on NK cell development could be important for therapy for chronic liver disease and developing a vaccine against HCV infection. Insights will also be relevant to other viral infections in which NK cell development could be affected.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: The roles of the Legionella pneumophila effectors PieE and LtpG in formation and maintenance of the Legionella containing vacuole
Funder: Medical Research Council

Abstract: The identification of host cell interaction partners of effectors is essential to understand their function. We developed a novel method that allows discovery of effector signalling complexes under physiological infection conditions. Our method relies on a cell line stably expressing the biotin ligase BirA from E. coli. Following infection with L. pneumophila expressing an effector fused to a Bio-tag consisting of a His6 tag and a BirA specific biotinylation site, the effector-host protein complexes are stabilized by chemical cross-linking and purified under denaturing conditions in two steps using Nickel and Streptavidin columns (Tandem affinity, TA, chromatography). Proteins in the complexes are then identified by mass spectrometry (MS). Unspecific hits are eliminated by cross-comparison with results obtained from infections with L. pneumophila containing the empty pBio plasmid and an unrelated Bio-tagged effector. Specific interactions are confirmed by two hybrid systems, co-IP, pull downs and ELISA. In addition we will: 1. Use live cell imaging to track interaction partners, vesicular trafficking and, employing a new split-GFP system, the effectors during infection. 2. Express host proteins and effectors fused to the miniSOG-tag, a bi-functional tag for correlated, high resolution IF and TEM. 3. Use TR-FRET to characterise protein-protein interactions within the effector complexes. 4. Use chemical probes and click chemistry to determine the effect of effector binding on the post-translational modification of Rabs. 5. Use RNAi to deplete host cell proteins and site directed mutagenesis to delete effector genes from Legionella (and complementation) to study their roles during infection. 6. Purify recombinant proteins for biochemical and cell-free vesicle tethering assays and structural studies. 7. Study the coatomer complex, p62, autophagy, MAP kinase and NF-kB dependent survival signalling and regulation of cytokine expression using IF, WB and ELISA.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Deciphering the Candida albicans Hsp90 Kinase Interactome and Elucidating its Role in Fungal Virulence
Funder: Medical Research Council

Abstract: This proposal aims to understand how the Hsp90/Cdc37 chaperone complex and associated kinases affect virulence in Candida albicans, the leading fungal pathogen of humans. Every year ~700 UK patients die of candidemia with a directly attributable cost of £16 million. Two problems are associated with the treatment of life-threatening candidemia: evolutionary conservation of antifungal drug targets and rapidly emerging drug resistances, potentiated by the essential chaperone Hsp90. Yet, as of now, depleting Hsp90 and treating candidemia with existing drugs causes severe host toxicity. I showed that Hsp90 depletion in combination with specific kinase gene deletions abrogates C. albicans growth. C. albicans Hsp90 interacts with 34 kinases, five of which are fungal-specific and as of yet uncharacterized. This suggests a promising venue for the development of fungal-specific kinases as future drug targets for use in combinatorial therapy with Hsp90 inhibitors already in clinical trial as anti-cancer drugs. To achieve this, three objectives will be pursued in parallel. First, casein kinase 2 mediated regulation of Hsp90 and its kinase-targeting co-chaperone Cdc37 will be characterized by identifying physical interactions, mapping phosphorylation sites using Agilent HPLC-chips and assessing the effects of chaperone phosphorylation in virulence assays, such as biofilm formation, oxidative stress, iron limitation, and killing of non-vertebrate hosts. Second, the virulence potential and regulation by Hsp90/Cdc37of fungal-specific and conserved kinases will be compared. Third, the C. albicans physical and genetic Hsp90 interaction network will be mapped employing a bar-coded mutant library covering 85% of the genome in combination with next-gen sequencing and TAP-pull down followed by mass spectrometry. Novel aspects include CK2-mediated regulation of C. albicans Hsp90, characterization of fungal-specific kinases and genome-scale discovery of novel interactors.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: A Systematic Investigation into Human Cytomegalovirus Gene Function
Funder: Medical Research Council

Abstract: We defined the genetic content of a circulating HCMV strain (Merlin) and generated an infectious BAC clone to provide a secure basis for studies into HCMV pathogenesis. The infectious HCMV Merlin BAC clone provides a reliable source of wild-type genes and genetically intact, defined clonal virus. Detailed transcriptional analyses underpin the definition of HCMV canonical genes. To permit functional screening, 170 HCMV protein-coding genes were inserted into an adenovirus vector and a panel of HCMV 'block' deletion mutants spanning >50% of genome assembled. Ongoing screening of HCMV genes for recognised and novel NK cell modulatory functions will be completed prior to initiation of the project. The literature details 7 HCMV NK evasion functions; to date, we have identified 8 more we wish to characterise. NK cells recognise a combination of activating and inhibitory ligands on the surface of targets cells. We have therefore developed Plasma Membrane Profiling (PMP) to simultaneously track the expression of >700 cell surface viral and cellular proteins in multiple samples. Using bespoke HCMV mutants, PMP will be used to characterise the mechanism-of-action of novel NK evasion functions in the context of virus infection. Where necessary mechanism will be further explored in whole cell proteomic analyses and SILAC-immunoprecipitation. The contribution of host proteins will be elucidated by genetrap retrovirus-mediated mutagenesis in a haploid cell line (KBM7). Remarkably, the HCMV genome contains multiple sets of homologous genes that have been assigned to gene 'families'. Functional screening with HCMV 'block' deletion mutants highlighted roles for multiple members of the RL11 and US12 gene families in immune modulations. Using genetic screening and proteomic technologies pioneered to characterise NK modulatory functions, we shall seek to systematically investigate conservation and divergence of gene function across the two largest HCMV gene families.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Cytoplasmic tail interactions of the influenza M2 protein with lipid and protein.
Funder: Medical Research Council

Abstract: The aim of this grant is to provide structural and functional information on the cytoplasmic tail of the influenza virus protein M2 and its interactions with the membrane, with specific lipids, and with M1. The amino-adamantane drugs, which target the ion channel activity of M2, have been used as antivirals for decades, but currently circulating strains are resistant. However, M2 has recently discovered functions in the budding and scission of nascent viral particles. A membrane proximal amphipathic helix (APH) is implicated in generating the negative membrane curvature required for demarcating the viral budozone. In addition, M2 interacts with the viral protein M1, which facilitates organisation of nascent virion. The cytoplasmic C-terminal domain of M2 is responsible for both membrane and M1 interactions. Whereas the membrane proximal APH has been characterised in some detail by NMR and EPR methods, less is known about the high resolution details of the depth of insertion of the APH in the membrane and the specific interactions with lipids, including cholesterol and PIP2. For the M2/M1 interactions, the M2 residues involved have been mapped to the region around residues ~71-76. However, nothing is known about the corresponding site on M1 and whether M1 or M2 undergoes any conformational changes upon binding. In addition, there are no experimental reports of the site-specific structural elements, if any, in the M2 cytoplasmic tail outside of the APH (residues ~63-97). Therefore, we aim to characterise M2 interactions with the membrane, in particular that of the APH, and correlate these findings across variants and cholesterol concentration with the activity of M2 in GUV membrane budding assays. In addition, we will characterise structurally and functionally the interactions of the M2 APH with both cholesterol and PIP2. Finally, we will determine the molecular basis of M2/M1 interactions and whether complex formation leads to structural rearrangements.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION