NIHRDataInsights/HRCSData huggingface dataset:
Title: Statistical inference with mechanistic models on heterogeneous data: improving the control of infectious diseases
Funder: Medical Research Council

Abstract: Understanding what governs the size and severity of seasonal influenza epidemics is a key public health priority. This can be obtained by fitting mechanistic models to the many different and varied surveillance data available in the UK to draw inference on key epidemiological drivers. However, the non-linear and stochastic nature of epidemiological processes pose serious challenges to classical statistical methods. To address these issues we will consider three classes of algorithms that have recently been developed to perform inference on partially observed Markov processes: Approximate Bayesian Computation [Toni et al. 2009 J R Soc Interface], Particle Markov Chain Monte Carlo [Andrieu et al. 2010 J R Stat Soc B] and Maximum Likelihood via Iterated Filtering [Ionides et al. 2006 PNAS]. An attractive feature of these new methodologies is that the only operation applied to the underlying Markov process model is the generation of a draw from the transition density. The accuracy, precision, robustness and practical use of these different methods on simulated data sets will be compared in order to select the most appropriate one to analyse the uniquely rich and detailed influenza data available in the UK. These include serological, virological, immunological, epidemiological and sociological data available at both the individual and population level over many years. These data will be combined and linked to the statistical models through observation processes accounting for noise, overdispersion and biases (e.g. negative-binomial). These heterogeneous data will be exploited in a quantitative and rigorous manner to test alternative hypotheses on the mechanisms underlying disease spread. In the medium term this will facilitate accurate near-to-real-time predictions on the severity and extent of a given epidemic of influenza. In addition, it will help design more efficient and cost-effective vaccination policies.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: INFECTION
Title: Development of a statistical modelling framework to inform real-time immunization strategies for elimination of poliomyelitis and other diseases
Funder: Medical Research Council

Abstract: Objectives and methodology: > Determine spatial and temporal trends in population immunity of children in Africa A cohort model of the immunization status of children aged 0-5 will be developed, and parameterised using surveillance data. Estimates of vaccine associated population immunity will be provided along with measures of uncertainty > Develop a model framework to identify demographic and mobility networks that influence poliomyelitis spread A metapopulation approach (using a susceptible-infected-recovered model and exploring other methods such as transmission trees) will be used to test hypotheses on the factors affecting the spatial and temporal spread of disease at a fine spatial scale (districts within countries within Africa). Further developments of this methodology will be included to account underreporting and asymptomatic infection of poliovirus. > Develop risk maps for poliomyelitis and validate findings using prospective sampling Inferences made from the proposed model will be validated using prospective sampling from historical outbreaks of poliomyelitis in Africa. Methods to assess predictive ability will be reviewed and updated. During the four years of the fellowship, I will also test the findings on emergent outbreaks. > Test different vaccination strategies to limit and prevent poliomyelitis outbreaks From the findings identified above, different vaccination strategies will be trialled using computer simulation. I will identify optimal control strategies that focus on the scale of vaccination that will limit outbreak size and duration. Scientific and medical opportunities: New methodologies will be developed specific to capturing dynamics of infectious diseases that are partially observed. Insights will also be applicable to other infectious diseases in humans and animals. The research will provide valuable insight for policy makers and stakeholders within the GPEI, and will contribute to the eradication of poliomyelitis.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY);3.4 VACCINES
Health categories: INFECTION
Title: Pre-clinical development of a simian adenovirus vectored respiratory syncytial virus (RSV) vaccine
Funder: Medical Research Council

Abstract: RSV is a major cause of respiratory disease in infants worldwide, with a peak incidence of severe disease in infants less than 6 months of age. Repeated infections occur throughout life, and the burden of RSV disease in the elderly is comparable to that of seasonal influenza, while the economic impact of RSV disease in adults is even greater. There is no effective RSV vaccine or anti-viral therapy. Vaccine development has been hampered by the finding that a formalin-inactivated RSV vaccine failed to protect against RSV infection and primed for exacerbated respiratory disease. There is, therefore, a need for a safe and effective RSV vaccine not only to protect infants, but also to boost immunity in adults and the elderly, thereby reducing the circulation of RSV in the community. Ideally, an RSV vaccine should induce rapid, protective T- and B-cell responses, not prime for enhanced disease, and be able to boost immune responses in those whose immunity has declined. To this end, we have generated replication-defective chimpanzee adenovirus (ChAd) vectors, which are not neutralized by pre-existing anti-vector immunity in humans, and an attenuated poxvirus vector, MVA, expressing a string of conserved RSV proteins. Intranasal vaccination with ChAd/RSV, or vaccination by heterologous prime-boost with ChAd/RSV and MVA/RSV, completely protected mice and cotton rats against RSV, and did not prime for exacerbated pulmonary pathology. In this project, we will identify a ChAd/RSV vaccine for clinical application by comparing ChAd/RSV vaccine candidates for genetic stability, productivity, immunogenicity and safety in small animal models. The ability of ChAd/RSV to protect against bovine RSV in calves, a natural host of RSV, the effects of route of vaccination and maternal antibody on vaccine efficacy, and the ability to boost pre-existing immunity will be determined. A vaccine production process in GLP conditions for Phase I clinical trials will also be developed.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Elucidation of a new anti-bacterial killing system
Funder: Medical Research Council

Abstract: Protein secretion systems play a central role in the virulence, host interaction and survival of Gram-negative bacterial pathogens. The recently-identified Type VI secretion system (T6SS) plays a key role in virulence and competitiveness of many bacteria, including important human pathogens, although the mechanisms and roles of T6SSs are not yet well understood. Pathogens can use T6SSs to directly target eukaryotic organisms, as classical virulence factors. Recent work has shown that certain pathogens can also use T6SSs to target other bacterial cells, killing or inhibiting rivals. Such 'anti-bacterial' T6SSs provide a competitive mechanism to allow pathogens to proliferate in polymicrobial infection sites or environmental reservoirs and ultimately cause disease. The mechanisms by which different bacteria use anti-bacterial T6SSs to specifically kill rivals but protect self are currently unknown, something we aim to address using a clinically-relevant and tractable model organism, Serratia marcescens (Sma). Opportunistic enteric bacteria, including Sma, represent an important cause of problematic and antibiotic resistant hospital-acquired infections. We have recently identified a T6SS in Sma Db10 which exhibits potent anti-bacterial killing activity. The primary aim of this project is to elucidate how the model opportunistic pathogen Sma is able to utilise its T6SS to efficiently kill other bacteria. We will identify and characterise anti-bacterial effector proteins secreted by the T6SS, identify self-resistance determinants protecting Sma from killing itself, and determine the cellular target(s) of the Sma T6SS in susceptible bacteria. Additionally we will begin to examine the wider biological relevance and consequences of the process. Our extensive preliminary data, including identification of several candidate T6-secreted effectors, demonstrates the feasibility of the proposal. Our findings may pave the way for future development of new anti-bacterial therapies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: The role of cellular RNA editing in human respiratory syncytial virus and influenza virus biology.
Funder: Medical Research Council

Abstract: Influenza A virus (IAV) and human respiratory syncytial virus (HRSV) are globally important negative stranded RNA virus pathogens responsible for a large disease burden. Whilst there is an effective vaccine to IAV and selective anti-viral chemotherapy, the rapid emergence of IAV pandemics and the build up and spread of drug resistance can lead to current strategies becoming ineffective. No effective therapeutic strategy exists for HRSV. Thus there is an urgent and unmet healthcare need to ensure robust strategies to lower the current and future disease burden caused by these viruses. This proposal focuses on the interaction between IAV and HRSV and the cellular Adenosine Deaminase Acting on RNA (ADAR) pathway, a system whose importance to human disease (both infectious and genetic) is becoming increasingly apparent. Our research will define the interactions of these two important human respiratory pathogens with the ADAR system, furthering our understanding of their replication and potentially leaving us well placed to take advantage of future advances in chemotherapeutic modification of ADAR activity. ADAR activity catalyses adenosine (A) to inosine (I) changes on both viral and cellular mRNAs. For many medically relevant viruses, including IAV, HRSV, HIV-1 and herpesviruses this is thought to be pro-viral, although the mechanisms are not understood. This proposal brings together expertise in both viruses and the necessary technologies to investigate the activity of ADAR on IAV and HRSV to investigate two primary hypotheses. (1). That both IAV and HRSV co-opt the cellular ADAR pathway for pro-viral purposes and (2) that ADAR activity contributes to virus diversity. To achieve this we propose to define which isoforms of ADAR are involved in the IAV and HRSV life cycles, where these activities occur inside the virus infected cells and how this modification process is important for viral fitness and sequence diversity.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: An evolutionary population genomics approach to determine the genetic basis of virulence in the pathogenic fungus Cryptococcus neoformans
Funder: Medical Research Council

Abstract: We will use our cryopreserved collection of >300 clinical and environmental MLST-profiled Cng isolates from SE Asia and Africa to sequence isolates, including the reference H99, to a depth of ~100x. Reads will be aligned to the Broad Institute Cng VNI H99 reference genome to allow assembly, and base-calling. SNPs that are entirely covered in all isolates with high confidence will be concatenated for final alignment against the H99 chromosomes to produce a super-alignment. Aneuploid contigs will be treated as heterozygotes and detected using a depth-dependent binomial technique. In tandem with bioinformatics development, we will continue to expand our collection of environmental isolates from Southern Africa and SE Asia in collaboration with MRC-funded trials. Isolates will be Illumina-sequenced and sequentially added to the population genomics database. These will then be subjected to a genomics analysis aimed at determining the patterns and processes that have led to contemporary distributions of genetic diversity for global Cng. Associations will be sought between genotype and virulence by using clinical vs environmental isolates and, within clinical isolates, using the detailed clinical outcome data available for the source patients. Functional characterisation of outbreak clinical versus arboreal/environmental isolates will be done using the established Cng virulence factors, and a high-throughput analysis of macrophage parasitism using an intracellular proliferation rate assay for representatives of each Cng genotype. Virulence will also be assessed utilising the waxmoth larvae as a surrogate for murine models. Lineage-specific phenotypes will be analysed and panels of isolates that exhibit virulent versus hypovirulent phenotypes will be subjected to analysis of gene-expression using RNAseq. This will set the stage for downstream QTL-mapping of virulence in this sexually-reproducing fungus and the discovery of novel pathogen effectors.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Regulation of HIV-1 Gag expression by SR proteins
Funder: Medical Research Council

Abstract: The HIV-1 genomic RNA (gRNA) has several independent functions in the HIV-1 life cycle and its regulation is critical for virus replication. In the nucleus, the gRNA is the pre-mRNA that is spliced into mRNAs for seven different proteins. In the cytoplasm, the unspliced gRNA is the mRNA for Gag and Gag-Pol, which are polyproteins containing the viral structural proteins and enzymatic activities, and is also packaged into virons as the infectious genome. Therefore, the gRNA must avoid splicing to accumulate in the cytoplasm and be translated. In addition, the gRNA has a highly structured 5' UTR and appears to be a poor substrate for translation. While these properties predict that Gag should be poorly expressed, ~2000 molecules of Gag are required for each virion and a large amount of Gag is expressed in an infected cell. Understanding how Gag expression is regulated will fill a critical knowledge gap in the HIV-1 life cycle. SR proteins are a family of 12 RNA binding proteins in humans that regulate almost all steps of cellular mRNA metabolism. SR proteins also regulate several steps of HIV-1 mRNA metabolism and our preliminary data demonstrate that at least two SR proteins, SRp40 and SRp55, strongly stimulate Gag expression by enhancing gRNA abundance and Gag translation efficiency. In this proposal, we will test the whole SR protein gene family to determine which of these proteins promote Gag expression. To characterise their mechanisms of action, we will perform a structure/function analysis on the active SR proteins to identify the necessary functional domains as well as identify their binding sites in the HIV-1 gRNA and the cellular co-factors they interact with to stimulate Gag expression. Finally, we will determine how SRp40 and SRp55 stimulate Gag translation efficiency. These experiments will increase our knowledge of how HIV-1 Gag expression is controlled and may identify novel antiviral drug targets.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Deciphering mycolic acid transport in Mycobacterium tuberculosis
Funder: Medical Research Council

Abstract: Mycolic acids are vital components of the unique cell wall of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), and are essential for viability and virulence. While the core processes of mycolic acid biosynthesis are now well understood, not much is known about the post biosynthesis processing of newly synthesised mycolic acids inside the cell and their subsequent transport for transfer to cell wall components. The aim of this study is to identify mycolic acid transport mechanisms in M. tuberculosis and this will be achieved by: 1) Generation and characterisation of M. tuberculosis and M. smegmatis conditional mutant strains of mmpL3, a gene encoding putative large transmembrane protein, orthologs of which have been shown to play a role in mycolate transport in the related corynebacteria. A phage based Specialised Transduction method will be used to generate conditional Mycobacterium mutants of mmpL3. 2) Generation and characterisation of mutants/conditional mutants of a group of core mycobaterial genes immediately surrounding mmpL3, including a second mmpL gene, mmpL11 3) Conducting localisation studies with MmpL3 (and MmpL11)-GFP fusions using flourescence microscopy as well as Transmission Electron Microscopy. 4) Isolation of interacting partners of MmpL3 (and MmpL11) with the potential for identifying late processing enzymes that may interact with MmpL3. Bacterial two-hybrid systems, yeast two-hybrid systems and a novel nanodisc method that incorporates co-immunoprecipitation will be used in parallel to achieve this aim.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: NTD Highlight Notice: Melarsoprol-pentamidine cross-resistance in African trypanosomes
Funder: Medical Research Council

Abstract: African trypanosomes are transmitted by the tsetse fly and circulate in the bloodstream of their mammalian hosts. These protozoan parasites cause Human African Trypanosomiasis (HAT) and Nagana in livestock. HAT is typically fatal without chemotherapy but therapies have multiple problems, including toxicity and resistance. Commonly used HAT therapies are pentamidine, only suitable for pre-CNS disease, and melarsoprol for advanced disease. Melarsoprol treatment failure is a major problem and resistant trypanosomes often display pentamidine cross-resistance. Research lead by the applicant recently linked an unusual aquaglyceroporin, AQP2, to this phenotype. Thus, reduced AQP2 expression could explain innate or acquired resistance. Other unpublished data reveal that AQP2 is restricted to the flagellar pocket. The proposal now is to carry out a molecular dissection of the AQP2 sequences required for drug sensitivity. The AQP2 selectivity filter will be genetically dissected using an established set of strains and phenotype assays. Similar assays will be used to characterize a set of naturally occurring AQPs and their role in drug sensitivity. The findings will allow us to predict which AQPs confer drug sensitivity and which mutations lead to resistance. Since mechanisms of flagellar pocket sequestration are of more general interest and may be important for AQP2 function, the molecular determinants of sequestration will be similarly dissected. Identification of the molecular 'zip-codes' involved will allow us to predict subcellular localisation of naturally occurring AQPs. We will also generate a complete aqp null strain to gain insight into natural AQP function in trypanosomes. Our findings will then be translated into a diagnostic test that could rapidly reveal drug-resistant strains; an AQP2-based test that rapidly predicts the melarsoprol and pentamidine sensitivity/resistance phenotype of clinical strains will allow selection of the most appropriate treatment option.


Research activity codes: 5.1 PHARMACEUTICALS;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Virulence gene dynamics in the human malaria parasite
Funder: Medical Research Council

Abstract: The proposed work will investigate a novel mechanism for silencing sub-telomeric var genes and promoting their recombination in Plasmodium falciparum. The var gene family is known to undergo epigenetic silencing and switching, involving sirtuin histone deacetylases. Var genes also recombine readily to generate new variants. However, the mechanisms underlying these virulence processes are not fully understood. G-quadruplex (G4) DNA structures may influence both transcription and recombination of var genes. G4-forming sequences are strikingly rare in the AT-rich P. falciparum genome, and are concentrated around var genes. In model systems, these structures can affect both transcription and recombination by stalling RNA or DNA polymerases. A special class of 'RecQ' helicases unwind G4s and a human RecQ helicase was recently shown to operate in concert with a sirtuin: cells lacking either of these proteins developed genome instability and chromosome fusions at G-rich telomeres. In P. falciparum, I have shown that similar phenotypes are seen in a sirtuin knockout, suggesting that G4s, RecQ helicases and sirtuins may also affect DNA metabolism in the parasite. Several complementary molecular-genetic approaches will address this hypothesis. Parasites will be treated with G4-stablizing drugs and tested for altered var gene transcription, telomere maintenance and karyotypic changes. G4 sequences will be cloned upstream of reporter genes, as integrated transgenes, and their effect on transcription and recombination will be examined. Putative RecQ helicases will be knocked out and the mutant parasites phenotyped, and ChIP studies of the helicases will be carried out in strains with or without a sirtuin. Together, these experiments will provide multiple lines of evidence for the role of G4s, RecQ helicases and sirtuins in controlling transcription and recombination of P. falciparum virulence genes, thus improving our understanding of a fundamental aspect of malaria biology.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Substrate-receptor interactions in the Tat protein transport pathway
Funder: Medical Research Council

Abstract: The Tat system in the model organism Escherichia coli comprises the three integral proteins TatA, TatB, and TatC. Multiple copies of TatB and TatC form a large complex that acts both as the substrate receptor for the Tat system and as the central organising element of the translocation site. Tat substrate proteins possess a signal peptide containing a twin-arginine consensus motif that is recognised by the TatC protein. Although all of the multiple TatC subunits in the TatBC complex are chemically identical TatBC complexes contain two different types of signal peptide binding site: a single high affinity, non-exchangeable site, and multiple low affinity, readily-exchangeable, sites. Building on this work we now aim to: - Probe the molecular nature of the two types of signal peptide binding sites. - Test models for the relationship between these two sites. - Elucidate the molecular events triggered by signal peptide binding to the TatBC complex. Methods to be used include analysis of signal peptide-TatBC interactions in solution using isothermal titration calorimetery and fluorescence labelling, as well as analysis of substrate-transporter interactions in membranes using site-directed photoaffinity crosslinking and in vitro transport assays. These studies will be assisted by the characterization and use of a range of engineered Tat proteins. Further insight into the mechanism of TatBC will be obtained by isolating genetic suppressors of mutations that affect substrate-translocase interactions. These data will provide crucial information on how the substrate protein interacts with the Tat system during the translocation cycle. This fundamental knowledge will support drug discovery activities and underpin commercial efforts to exploit the Tat pathway in the production of proteins of theraputic relevance.


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