NIHRDataInsights/HRCSData huggingface dataset:
Title: Understanding how viral innate immune evasion strategies affect adaptive immunity, and the application to vaccine development
Funder: Medical Research Council

Abstract: Although many successful vaccines exist, effective vaccines against important diseases such as HIV, malaria and dengue are still lacking. Vaccine development requires a better understanding of how to elicit strong memory responses. The contribution of innate immunity to memory immunity is currently not well understood, but work in our laboratory has demonstrated that mutants of vaccinia virus (VACV) that lack innate immune inhibitory genes are more immunogenic and efficacious vaccines. These data highlight the importance of innate immunity in shaping immune memory and can be used not only to design more effective vaccines based on VACV vectors, but also to enhance current understanding of how stimulating innate immunity positively impacts on vaccine design in general. We have demonstrated already that removal of specific immunomodulators can improve either the CD8+ T-cell or the NK-cell memory responses to immunisation. The question we will address in this study is how does the removal of these innate immune inhibitors from VACV improve memory immunity? Our hypothesis is that in the absence of innate immunomodulators the cytokine, and hence cellular, environment following vaccination is altered in a way that positively impacts on development of memory immune responses. We will characterise the immune response in detail shortly after VACV vaccination. First, we will measure which cells are infected and what cytokines are produced. Then we will determine which cells are recruited to the site of vaccination, what is their activation state and how they present antigen to activate T and B-cells. By comparing these parameters following infection with wild-type versus mutant VACVs lacking inhibitors of innate immunity we will correlate differences with enhanced immunological protection. Finally, we will use this information to improve vaccine design.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Using MHC class I peptides to modulate NK cell activity, as a basis for immunotherapy
Funder: Medical Research Council

Abstract: We will use transporter associated with antigen processing (TAP) deficient cell lines for peptide loading experiments to identify specific peptides that bind HLA and also either bind the activating receptor KIR2DS2 or function as peptide antagonists for KIR2DL2/3. Peptides will be designed based on our on-going experiments and the current literature. We have identified an HCV derived peptide involved in the direct recognition of virus-expressing cells by KIR2DS2. We have used this to synthesize related peptides that induce KIR2DS2-binding and these experiments will inform the design of additional peptides that will engage KIR2DS2 to stimulate NK cells in vitro. KIR3DS1 is a receptor associated with protective responses to HIV and hepatocellular carcinoma. We have identified that KIR3DS1 is also protective against chronic HCV infection. We will use HLA-B*2705 as an allele with which to perform a screen for peptides that may bind KIR3DS1, taking a motif-based approach and going on to perform a large scale screen if the initial approach is unsuccessful. To investigate inhibitory receptors, we will make use of our published and pilot data to identify antagonist peptides for KIR2DL2/3. This will inform the design and testing of peptides that may antagonize inhibition by other HLA-C alleles. To translate these into a potential therapeutic reagent we will set up assays of endogenous peptide presentation using minigenes and vector constructs to express the peptide with or without its cognate MHC class I ligands on the cell surface. Cell lines expressing these constructs, and exosomes derived from these cell lines, will be tested for their potential to activate and proliferate NK cells expressing specific KIR. We will also express our peptides transiently in monocyte derived dendritic cells in cultures with autologous NK cells so that HLA class I is completely matched as would be the case for in vivo therapy.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION
Title: Genetic Admixture and Host-Pathogen Interactions in Helicobacter pylori Infection
Funder: Medical Research Council

Abstract: The main goal of this research is to assess whether individual genomic ancestry and genomic variation of both humans and associated H. pylori affect susceptibility to infection in the admixed population of Cape Verde. To this end, we propose to screen volunteers, of both sexes and aged >35 years, and to collect blood and gastric mucus samples from 700 infected individuals in the two main hospitals of Cape Verde. The blood samples will be used for human DNA extraction, determination of H. pylori infection status through detection of Immunoglobulin G against H. pylori, and for indirect evaluation of the severity of gastric mucosa damage through measurement of the plasma biomarkers pepsinogen I and pepsinogen II. The gastric mucus samples will be used for H. pylori culture, and will be collected on an adhesive nylon string that the participants swallow and that is retrieved an hour later (gastric string). We will then perform high-throughput genotyping of human genomes and next-generation sequencing of complete bacterial genomes. This will allow us to characterise the genomic variation, the admixture structure and the correlation between the individual genomic ancestries (IA) of hosts and H. pylori in Cape Verde. We will also be able to examine the correlation between IA's and the degree of gastric mucosa damage. A significant correlation will indicate the presence of human and bacterial genetic factors that are responsible, at least in part, for the phenotypic variation in disease risk to H. pylori infection observed within and between the ancestral populations to Cape Verde. The relative contribution of these genetic factors can then be quantitated. In addition, we will be able to perform association scans between human and bacterial genotypes or between human and bacterial local ancestry (admixture mapping) and human phenotypes to gain a preliminary characterisation of the genetic architecture underlying susceptibility to H. pylori infection in Cape Verde.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Identifying new members of virulence signalling networks: three new sensor kinases in the GacS network of Pseudomonas aeruginosa
Funder: Medical Research Council

Abstract: Pathogens rely on sensory networks to make decisions that are critical for survival during infection. With antibiotic resistance levels rising, interfering with these networks is a promising new way of treating infection. Many of these networks employ multiple sensor kinases that work together to detect and integrate different signals. However, determining which kinases collaborate to form these multikinase-networks (MKNs) is challenging and is a major rate limiting step for devising ways of targeting these networks to reduce virulence. In this proposal, we will trial an approach for predicting which kinases work together in MKNs, based on the premise that these kinases should share their specificity determining residues to enable them to interact with the same receiver domains. Using this approach, we have predicted three new kinases in the GacS network controlling virulence in Pseudomonas aeruginosa. Our aim is to validate this predictive approach by showing that these new kinases signal via the GacS network and play an important role in infection. We will determine the role of the kinases by investigating the behaviour of deletion mutants in virulence assays. Next, the regulon controlled by each kinase will be determined using RNA-seq, and compared with the regulon of the GacS network to determine the extent to which the new kinases signal via the GacS network. Finally, we will test for interactions between the new kinases and the other proteins in the GacS network to find how the new kinases fit into this network. In addition to determining the role of the new kinases in the GacS network, this study will, more widely, serve as proof-of-principle for using shared specificity residues to predict new members of virulence signalling networks in other pathogens. Indeed, similar to P. aeruginosa, many other pathogens have groups of kinases that share their specificity residues and therefore this work will help to reveal these potential drug targets.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Characterisation of novel virus-host cell interactions essential for herpes simplex virus envelopment
Funder: Medical Research Council

Abstract: Many clinically important human and animal viruses, ranging from human immunodeficiency virus to ebola virus, are enclosed in envelopes derived from cellular membranes. These viruses utilize both the secretory and endocytic transport pathways to direct their envelope proteins to specific sites of virus envelopment. Hence, enveloped viruses subvert vesicle transport in their morphogenesis, and interact with specific cellular factors in the process. These transport factors represent tangible, and potentially broad-spectrum, antiviral targets. This proposal aims to take one such enveloped virus - the human herpesvirus herpes simplex virus (HSV1) - and dissect the essential virus-host interactions involved in its envelopment process. HSV1 is an often-disregarded human infection that nonetheless causes significant disease, infecting 24 million annually, and resulting in considerable morbidity and mortality. Our recent studies showing specific roles for three Rab GTPases in HSV1 trafficking means that we already have a broad understanding of HSV1 envelopment that can act as a model for many other enveloped viruses. We will take three approaches to discover novel factors essential for envelope protein trafficking: (1) a large-scale but rational siRNA screening of cellular transport factors; (2) a directed analysis of Rab6-specific trafficking to the plasma membrane; and (3) a focused proteomic study to find cellular binding partners of gK and UL20, the only two glycoproteins that are essential for envelopment. This work will be underpinned by confocal imaging studies and ultrastructural analyses of virus trafficking. Taken together, the results will provide vital new information on general virus exploitation of cellular trafficking pathways, and will contribute to future rational treatment design, potentially in a broad-spectrum fashion.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: A genome-scale census of virulence factors in the major mould pathogen of human lungs
Funder: Medical Research Council

Abstract: Spores of the common Aspergillus moulds are agents of widespread, often fatal, human and avian diseases, however, amongst a repertoire of more than 400 predicted transcription factors, fewer than ten have been characterised for their role in disease. We have developed a 'first-in-field' collection of 401 transcription factor knock out (TFKO) mutants for Aspergillus fumigatus, the major mould pathogen of humans, and now propose to identify those which govern pathogenicity. We have also developed and optimised a high throughput parallel fitness screen with which to functionally annotate transcription factor activities. We will use this method to assess the fitness of each mutant under infection-relevant laboratory-imposed stress, and in two murine models of aspergillosis. This will enable the functional annotation of the entire genomic cohort of transcription factors and identification of those which support pathogenicity (P-TFs). We will construct isolates expressing epitope-tagged P-TFs to identify the regulatory network of gene products they govern. We will use chromatin immunoprecipitation to capture DNA bound by P-TFs and massively parallel DNA sequencing to identify them. We will then use RNA-Seq to verify the functional significance of promoter binding by P-TFs. This work will allow us to construct a map of the functional regulatory network which governs pathogenicity and elevate our understanding of this important pathogen.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Investigating the trypanosomatid lysosome and its role in lipid catabolism.
Funder: Medical Research Council

Abstract: This proposal follows up our purification of intact lysosomes from procyclic T.brucei. This study will advance our understanding of the parasites lysosome and the functions within it that are required for its survival. This multidisciplinary proposal will address the following: 1)Identify novel lysosomal proteins; initially focussing on the procyclic proteome, by undertaking a large-scale preparation. This will be followed up byin vitro preparations with bloodstream form, prior to using rodents to obtain larger amounts of material. Cellular localisation of some hits will be undertaken for validation purposes. 2)Study the enzyme activities associated with the lysosome; this will be done directly and indirectly. Directly, using tagged (fluorescently, radiolabelled, stable isotope labelled) probes (proteins or lipids) to investigate their catabolism, utilizing appropriate methodologies. 3)Validate novel drug targets associated with lysosomal lipid catabolism; we have identified a putative lysosomal proteins acidic sphingomyelinase, acid phosphatase and phosphodiesterase, that will be genetically validated. Localisation will be verified prior to recombinant expression, assays and screening efforts. 4)Undertake lipidomic / metabolomic studies of the lysosome; fractionation of the lysosomes obtained from under varying conditions, will allow lipidomic and metabolomics analyses 5)Identify new chemical entities that can interfere with lysosomal lipid catabolism; knowledge from this study, will highlight chemical entities (non-natural lipids) that cannot be processed by the lysosomal, thus poisoning the lysosomal activities. Collectively this proposal will allow us to open a novel avenue of chemotherapeutic approaches against T. brucei and potentially other pathogens that rely heavily upon scavenging lipids and other essential metabolites from its mammalian hosts. We believe this work has real potential to impact on anti-parasitic drug discovery in the medium term.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Metabolic regulation of hepatic immunopathology by myeloid-derived suppressor cells
Funder: Medical Research Council

Abstract: The liver has evolved a highly tolerising environment which preserves the functional integrity of this vital organ in the face of the high antigenic load it receives through the portal venous system. Hepatotropic infections and tumours take advantage of this niche to subvert immune responses. In this proposal we explore the metabolic regulation of hepatic T cell immunity by granulocytic myeloid-derived suppressor cells (gMDSC), based on our previous work implicating amino acid starvation in chronic hepatitis B pathogenesis (CHB) (Das, JEM 2008). Our unpublished data suggest that the disparate degrees of immune-mediated tissue injury manifested in this highly prevalent infection may be determined by the differential expansion of gMDSC. Arginase-expressing gMDSC expand transiently in acute resolving HBV, their decrease coinciding with the peak of hepatic injury. In CHB, gMDSC are likewise most increased in those patients replicating HBV without evidence of immunopathology, their expansion correlating with increases in circulating arginase and a depletion of L-arginine. gMDSC accumulate in the liver and our preliminary data indicate a role for activated hepatic stellate cells in sustaining them. We now propose to characterise which pathological insults drive their expansion in the liver and test the hypothesis that stellate cells, HBV-infected hepatocytes and hypoxia make mechanistic contributions. We find that gMDSC can potently inhibit T cell responses by depriving them of L-arginine; we will further probe the impact of this starvation on T cell signaling, function and amino acid transporters. We will test the postulate that T cells can undergo compensatory changes in amino acid transporters promoting metabolic reprogramming. To investigate the therapeutic potential of gMDSC, we will deplete them (and their metabolic mediators) in mouse models of HBV infection. Our findings will have broad relevance for the understanding and manipulation of hepatic tolerance.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION
Title: A dynamic insight into the interaction of Chlamydia trachomatis with host cells
Funder: Medical Research Council

Abstract: Chlamydia trachomatis (CT) infection imparts a large burden on the human population, particularly amongst young adults, contributing significantly to the prevalence of sexually transmitted diseases, infertility and preventable blindness. CT is an obligate intracellular bacterium that is exquisitely adapted to manipulate the environment of the host cell. In contrast to gene expression studies, there is no information on the global protein changes that occur during the infectious process. Thus, we will utilise a powerful multiplexed proteomic technique that allows the relative abundance of chlamydia and host proteins to be determined accurately over the course of the infectious life cycle in both human epithelial and dendritic cells. This approach will provide the first opportunity to interrogate the chlamydia and host protein responses in tandem and provide a dynamic insight into the chlamydia-host interaction. A quantitative proteomics approach provides the opportunity to interrogate the biology of CT which has not been readily amenable to genetic manipulation approaches. CT infection is also characterised by silent infection and inappropriate or inadequate cellular immunity to prevent reinfection. The plasma membrane provides a critical nexus with immune system cells and we hypothesize that changes in host cell plasma membrane proteins induced by CT infection may be critical in the modulation of immune responses. To address this, we will compare proteomic changes in the surface exposed membrane of human epithelial and dendritic cells infected CT. Both these approaches will identify pathways in the host cell response that that may be critical for either CT replication, induction of inflammatory responses and deviation of immune system responses. Most importantly, this study will inform potential approaches to prevent successful Chlamydia infection and its inflammatory sequelae in humans.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: MICA: Does phase variation contribute to virulence and immune evasion of Neisseria meningitidis?
Funder: Medical Research Council

Abstract: Asymptomatic carriage of Neisseria meningitidis (Nm) in the upper respiratory tract is frequent and is associated with the induction of antibodies against surface antigens. Multiple genes of meningococci undergo phase variation due to mutations in simple sequence repeat (SSR) tracts. Recently, we have shown that persistent carriage of Nm is associated with reductions in expression of outer membrane proteins (OMP) due to changes in SSR tracts. Serum samples from these carriers were found to contain antibodies specific for PorA, a major OMP, and bactericidal antibodies against the capsule. We hypothesize that antigen-specific antibodies select for variants with reduced expression of phase-variable determinants and that these variants have a reduced capacity to cause disease. Expression states of multiple phase-variable genes will be analysed in comparable sets of Nm disease and carriage isolates. Data sets will include isolates from a longitudinal Novartis-sponsored carriage study and the phase-variable genes required for synthesis of O-linked glycans. Repeat numbers of phase-variable genes will be determined by dideoxy Sanger sequencing and PCR-based GeneScan assays of DNA extracts from isolates. Statistical tests will be performed to determine whether Nm disease requires a specific pattern of phase-variable gene expression and if persistent isolates have an expression pattern indicative of a reduced capacity to cause disease. Antibody levels for allelic variants of OMPs and for different glycan structures will be measured using bioplex assays, which detect binding of antibodies to purified, recombinant proteins linked to fluorescently-labelled microspheres. Bactericidal antibody levels will be measured using strains expressing target antigens but having mismatches in other antigens. Statistical testing will determine whether reductions in antigen expression are due to selection by specific antibodies.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Dissecting the Red Blood Cell Invasion Pathways of the Malaria Parasite Plasmodium knowlesi
Funder: Medical Research Council

Abstract: Clinical symptoms of malaria are associated with the cycles of parasite invasion of, and multiplication within, host red blood cells (RBCs). The ligands used by the parasite to target host cell receptors and invade RBCs are key determinants of virulence. Two parasite protein families are implicated in this process, the reticulocyte binding-like (RBL) and Duffy binding-like (DBL) proteins, but little is known about their role or their host cell receptors. I will define the role of these protein families in Plasmodium knowlesi, a parasite causing severe and fatal disease in South East Asia. Taking advantage of the high transfection efficiency and unique capability to grow this parasite in two different host cells, I will generate gene knock-out lines for each of the 5 P. knowlesi DBL/RBL genes - to define which proteins are essential for invasion of the two different host RBC. Transgenic parasite lines will be generated in which each of the RBL/DBL proteins has been modified by the addition of a fluorescent/epitope tag, which will be used to determine subcellular localisation, and through immunoprecipitation, identify host cell receptors and interacting parasite proteins. I will use electron tomography and super resolution microscopy for the phenotypic analysis of knock-out and tagged lines to determine the precise role of each protein in the invasion process. To identify proteins that are essential for invasion of both host RBC, I will adapt the Cas9 and DiCre conditional recombinase system for use in P. knowlesi. Finally, I will develop a transgenic P. knowlesi model to assess both naturally acquired and vaccine induced antibodies targeting P. vivax, an important malaria parasite that currently lacks an in vitro model. The unique advantages of P. knowlesi will provide an unprecedented insight into the mechanistic role of RBL/DBL proteins during invasion, and novel tools to support development of vaccines to target them.


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