NIHRDataInsights/HRCSData huggingface dataset:
Title: Utilizing gametocyte immunity to reduce malaria transmission
Funder: Medical Research Council

Abstract: Malaria transmission from man to mosquito requires the uptake of male and female gametocytes from an infected host during a mosquito blood meal. Gametocytes are therefore essential for successful onward infection to new hosts and a major target for interventions. Our recent work demonstrates that gametocytes, together with a subset of asexual parasites, develop in the hematopoietic environment of the bone marrow before being released into circulation as transmission competent mature forms. Our preliminary studies have revealed natural immune responses targeting immature gametocytes that are correlated with reduced gametocyte burden in malaria patients. Using a combination of surface proteomics and immune profiling of immunised mice we have identified a series of putative surface antigens on gametocyte-infected red blood cells. The majority of them is shared with asexual stage parasites suggesting shared function in host cell interactions during development in the bone marrow. Here we propose to systematically investigate the targets and functionality of natural gametocyte immunity as a basis for novel vaccine development. For this purpose, we will combine a series of field studies in malaria endemic areas and experimental human infections, in vitro functional assays to define antigen-specific antibody activity and B-cell epitopes, as well proof-of-principle experiments in the rodent malaria model.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Evaluation of novel TB drug regimens by targeting resuscitation promoting factor-dependent persistent Mycobacterium tuberculosis in the Cornell model
Funder: Medical Research Council

Abstract: Treatment of tuberculosis with novel regimens in a modified Cornell model. The novel drug regimens will be tested using a modified Cornell mouse model in which treatment starts at 6 weeks after a low dose M. tuberculosis H37Rv infection. Treatment will be given for 14 to 16 weeks by daily oral administration. Mouse organs will be harvested at a two-week interval to monitor CFU and broth counts with 7H9 and culture filtrates. After the termination of the chemotherapy, the remaining mice will be administered hydrocortisone for 8 weeks. CFU counts from lungs and spleens will be performed to determine disease relapse. Standardizing resuscitation promoting factor production from M. tuberculosis and preparation of culture filtrates. M. tuberculosis is grown in 7H9 for 15 to 20 days until an optical density of 1 to 1.5 is reached. The cultures are harvested by centrifugation and filtered with 0.2 um filters twice. The culture filtrates will be used immediately and RPF stability testing will be performed before and after storage at -20 or -70-degree centigrade. Resuscitation of M. tuberculosis in mouse organs. Broth counting is performed as serial 10-fold dilutions in triplicate in which 0.5 ml of organ homogenates are added to 4.5 ml of the culture filtrates or 7H9 medium. At 10-day intervals over a 2-month period of incubation, the broth cultures will be examined for visible turbidity and confirmed by colonial morphology on 7H11 agar. The most probable number of viable bacilli is then estimated from the patterns of positive and negative tubes. Resuscitation of RPF-dependent persisters in Mycobacteria Growth Indicator Tube (MGIT). Mouse organ homogenates will be incubated in MGITs, then culture filtrate will be added into MGIT before and after 42-day incubation and continue to incubate for up to 2 months.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Integrating genomic surveillance and ecological modelling to maximise pneumococcal vaccine efficacy
Funder: Medical Research Council

Abstract: Streptococcus pneumoniae (the pneumococcus) is a nasopharyngeal commensal and respiratory pathogen that causes thousands of invasive pneumococcal disease (IPD) cases in the UK annually. Polysaccharide conjugate vaccines (PCVs) have reduced the incidence of infant IPD through eliminating most vaccine-type pneumococcal serotypes. However, their replacement with non-vaccine serotypes has caused the UK IPD incidence in the elderly to rise in recent years. This project will employ a combination of genomics and mathematical modelling to understand these contrasting demographic trends, not commonly observed in other countries. The underlying hypothesis is that carried S. pneumoniae population dynamics will be governed both by PCV-induced herd immunity and multi-locus negative frequency-dependent selection (NFDS), a model of bacterial ecology in which NFDS maintains common accessory genome loci at their pre-vaccination 'equilibrium frequencies'. These will be established by sequencing historic IPD isolates from infants and adults at timepoints matched to carriage surveys, for which genomic data will also be available. The multi-locus NFDS model will be reimplemented to combine carriage and IPD data to enable population dynamic modelling that includes the highly-invasive strains driving post-PCV13 disease trends in UK adults. Recently-developed scalable genomic surveillance methods will be used to integrate these data with global research collections, to understand the contribution of global strain migration patterns to post-PCV population restructuring, and ongoing IPD surveillance, to enable evaluation of models' forecasting accuracy. The model and data will be made flexible and open source, to provide the opportunity for other researchers to test alternative ecological model structures. Ultimately this work aims to aid policy decisions with regard to the introduction of PCV formulations, and guide the design of superior vaccines.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Host factors required for human polyomavirus replication and spread
Funder: Medical Research Council

Abstract: Polyomaviruses are ubiquitous viral pathogens that establish life-long persistent infections in their hosts and can cause serious problems in immune-suppressed patients. Of all the human polyomaviruses BKPyV has the biggest clinical impact due to causing BK polyomavirus-associated nephropathy (BKVAN) in up to 10% of renal transplant patients, and this disease can lead to allograft loss in a high proportion of such patients. Due to a lack of effective antiviral therapies for treating BKVAN, there is a pressing need to understand the basic biology of BKPyV to develop novel therapeutics against this virus. To address this gap in understanding, we have established a CRISPR genome-wide screen for host factors required in early stages of BKPyV infection. This has uncovered specific host factors involved in endocytosis, transcription and splicing. This research will determine the importance and mechanisms of action of the identified host factors during BKPyV infection. Furthermore, these genetic screens will be expanded to encompass the entire replication cycle of this virus within renal epithelial cells to identify and characterise host pathways required for viral late gene expression, virion assembly and virus egress. Additionally, we will characterise the changes to the plasma membrane proteome of BKPyV infected cells, to gain further understanding of how this virus alters cellular secretion pathways to promote its own release, and to define biomarkers of infection. This programme of work will provide the most complete understanding of host factors required for BKPyV to establish infection, replicate and spread in primary renal cells. In addition to providing new therapeutic targets for the treatment of life threatening diseases in the immunocompromised, our work will have wide reaching implications beyond the field of polyomavirus biology as we expect to uncover new insights into cellular biological processes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION
Title: Rapid, whole genome sequencing-based diagnosis of drug-resistant Mycobacterium abscessus complex and new options for treatment
Funder: Medical Research Council

Abstract: The project will include different phases: 1. Selection of resistant laboratory mutants (objective 1) - M. abscessus resistant strains to a single drug will be selected in the laboratory and WGS performed on the resistant isolates. This will allow determining the genetic mutations conferring resistance by applying a process previously described in other mycobacteria, both in M. smegmatis and M. tuberculosis. 2. Next generation sequencing for rapid diagnosis of resistance (objective 2)- Once a set of mutations has been identified in the laboratory mutants, whole genome sequencing will also be performed on resistant clinical isolates to validate the susceptibility profile with the genetic mutations for rapid diagnosis. Susceptibility data to first line antibiotics for all our M. abscessus complex isolates are already available as it was performed as part of the routine clinical service. 3. Susceptibility testing of clinical isolates (objective 3) - Our research collaborators at the National Heart and Lung Institute have an extensive collection of NTM clinical strains (over a thousand) and selected M. abscessus complex isolates from this collection will be tested against various compounds (and combination of drugs) to assess their susceptibility profile to new antibiotics (for example, bedaquiline and delamanid), currently not included in any first line testing. Testing will be performed using a combination of different methods (resazurin and other dilution techniques) to assess the validity and reproducibility of such methods. Additionally, our collaborators from the MRC Centre for Molecular Bacteriology and Infection are already conducting research on new anti-microbial peptides with promising antimycobacterial effect but such compounds have yet to be tested against M. abscessus resistant strains. To complete the picture, bacteriophages from our research partners at the University of Pittsburgh will be tested against such resistant strains.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Ecology or genetics? Adapting machine learning approaches to understand determinants of cross-species transmission and virulence in RNA viruses
Funder: Medical Research Council

Abstract: Emerging infectious diseases remain a prominent threat to global health, e.g., Ebola virus, Zika virus. In 2015, the WHO designated 'Disease X' to indicate the serious potential of previously unknown emerging pathogens to cause public health crises. Though zoonotic RNA viruses are known to present higher risks of emergence, detailed determinants of cross-species transmission remain unclear. Zoonotic viruses also vary widely in their capability to cause severe disease. To predict public health impacts of 'Disease X', a better understanding of which traits drive this variation in infectivity and virulence is urgently needed. Whilst previous approaches have focused on ecological predictors, these traditional frameworks have been unable to capture the information within increasingly available RNA virus sequences. This research aims to capitalise upon the potential power within large genetic data resources and quantify comparative influences of genetic versus ecological traits of RNA viruses and hosts upon cross-species transmission dynamics. To fully integrate novel, high-dimensional genetic data, new analytical approaches are needed. I will apply machine learning as a state-of-the-art statistical methodology, comparing several advanced approaches, e.g. gradient boosting, a method of gradual model learning which outperforms traditional methods. Models will span all known mammal and avian RNA viruses (22 families) using the exceptional breadth of EID2, a large, host-virus infectivity dataset. This project will additionally develop further text-mining tools to capture and integrate virulence data within EID2. The proposed models will allow tests of evolutionary theory across a range of RNA viruses. Quantified model outputs will contribute to public health risk assessments by informing prioritisation for novel viruses and advancing frameworks for emergence predictions, moving towards a 'smarter', empirically-driven strategy to prevent future disease burden.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: How does virus shape relate to infectivity?
Funder: Medical Research Council

Abstract: The influenza A virus (IAV) is responsible for the seasonal flu as well as the recurring global flu pandemics. The WHO has identified the risk of global influenza pandemic as one of the 10 most serious threats to global health in 2019, placing the study of influenza as a high priority for biomedical research. One particularity of IAV is its heterogeneity in shapes and composition suggesting a highly adaptable phenotype in vivo. The importance of this large variability for the virus however remains poorly understood, in part due to the lack of imaging technologies capable of studying the virus at the nanoscale. My research focusses on exploiting and developing novel imaging technologies, by combining super-resolution microscopy (SRM), high-content screening (HCS) and machine learning, to study the virus from the single molecule, single cell to the population level, essential to understand the role of viral heterogeneity. Initially, I will develop quantitative structural and genetic analysis to inform on the architecture of the virus assembly and genome at the single virus level. Then, by correlating this high-content data with infectivity assay at the nanoscale, I will explore how modulation of this heterogeneity affects the infectivity of the virus in primary respiratory epithelial cells, the virus' host in vivo. This combination of SRM and HCS has large potentials for its drug screening capabilities. Finally, by following the virus' replication in real time at the entry and assembly stages, I will be able to understand the effect of shape and composition heterogeneity on its mechanisms. This research is jointly supported by an industrial partner, developing Live Attenuated Influenza Viruses (LAIV) as a vaccine technology, so not only it has the potential to highlight the importance of the virus variability for its replication mechanisms, it will also inform the design of vaccines, joining the global effort to tackle influenza's health and economic burden.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: MRC AMED - The HDAC6/USP7 axis in enveloped RNA viral infection
Funder: Medical Research Council

Abstract: Influenza A virus (IAV) causes annual epidemics and occasional pandemics with continuous threat of emerging and potentially lethal strains derived from reassortment with avian or swine IAV. In addition to development of a universal influenza vaccine, a deeper understanding of cell biology of IAV infections is needed to discover antiviral strategies that target cellular proteins and pathways. Viral uncoating is an early step in infection that is poorly understood but highly druggable. As an example, adamantanes that were once used in human flu patients blocks IAV uncoating by inhibiting the M2 ion channel. IAV entry and uncoating is well-studied compared to many enveloped RNA viruses. Uncoating requires a ubiquitin-mediated, misfolded protein degradation pathway known as aggresome processing. Here, the key host player, histone deacetylase 6 (HDAC6), senses unanchored ubiquitin chains packaged within the virion via the HDAC6 zinc finger ubiquitin-binding domain (ZnF-UBP). In this study we will address the interplay between HDAC6 and a novel host factor, the deubiquitinase USP7, and analyse their role in IAV entry. Furthermore based on knowledge that HDAC6 can be hijacked by multiple viruses, we will test a panel of emerging and re-emerging enveloped RNA viruses on their dependence on HDAC6 and USP7. Our strategy is to use IAV as a prototype enveloped RNA virus. Our findings may contribute to the understanding of general viral uncoating pathways and lead to conceptualisation of a broad spectrum antiviral that can combat emerging and re-emerging viruses of medical importance.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: MRC AMED - Systematic analysis of persistence mechanisms by high-throughput bar-seq and single cell analyses.
Funder: Medical Research Council

Abstract: The research from Japan have utilised the E. coli single-gene deletion library in which each deletion is marked with a unique ~ 20-nucleotide molecular artificial barcode (Otsuka et.al., 2015) to identify 13 persistence mutants. The mutant's competitive growth advantage or disadvantage was determined by pooling the mutants and analyzing the population in a mixed culture using high-throughput DNA sequencers following treatment with various antibiotics (i.e. persisters are often multi-drug tolerant). However, the persistence mechanism was not obvious in the majority of cases, with the average results most likely swamping the persister characteristics. The UK team believe that by averaging results we often miss the exceptional, in this case persisters. The team will analyse the identify mutants at the single cell level. Time-lapse imaging of single cells will be carried out using a bespoke system consisting of a microfluidics platform and a Nikon confocal microscope equipped with an environmental chamber, motorized stage and perfect focus system, with image scans taken every minute. The experiments for persister cell discovery will consist of grow, kill, regrow and rekill stages. This final step is often left out in persister analysis and is a requisite to determining phenotypic resistance, a key characteristic of persistence. Computerised tracking will the be utilised to determine any difference in length at birth, division, elongation rate, division time as described in detail (Hu et al., 2017). Morphology will also be observed. Alongside, both teams will also be constructing fluorescent reporter strains for each of the mutants which will also allow the localisation of gene product at the single cell level. The majority of persistence mechanisms occur at the sub-population level and this research will identify novel persistence and AMR mechanisms, the characterization of which would be integral in combating this global health threat.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: The human T-cell leukaemia virus HTLV-1: transcriptional heterogeneity at the single-cell level
Funder: Medical Research Council

Abstract: The human T-cell leukaemia virus HTLV-1 causes an aggressive leukaemia or inflammatory diseases in ~10% of people it infects. There is no satisfactory treatment available for either condition; the average survival time of the malignancy is less than one year, despite treatment. HTLV-1-infected people typically make a strong immune response to the virus, but the impact on the virus of this response is limited because, as we and others have recently shown, HTLV-1 is transcribed only in intermittent bursts. A central question arises: how does HTLV-1 regulate the balance between transcription and latency, so as to maintain the proviral load while at the same time avoiding destruction by the host's immune response? The aim of this project is twofold: first, to quantify the dynamics and the heterogeneity of HTLV-1 reactivation at the single-cell level, and second, to identify the molecular mechanisms that regulate this reactivation. We will use recent advances in single-cell analytic techniques to elucidate the regulation of transcriptional latency of HTLV-1. In the collaborating laboratories in Japan and UK, there are strong existing research programmes on HTLV-1 molecular virology. The Co-PI in Japan is doing single cell transcriptome analysis, using peripheral blood mononuclear cells. The Co-PI in UK is also investigating HTLV-1 infection at the single-cell level, using real-time fluorescent microscopy, single-molecule RNA-FISH (smFISH), transcriptomics, and mathematical techniques. By combining these techniques and exchanging materials in the two laboratories, we will accelerate the progress of the research and provide training for doctoral students and postdoctoral workers in the two groups. The results of this work will identify both key mechanisms of viral persistence and disease development, molecular markers to predict disease development, and new potential molecular targets for the treatment of leukaemia.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: MRC AMED - Study of the T cell Response During Cryptococcal Meningitis Using New Tools
Funder: Medical Research Council

Abstract: Patients with T-cell deficiencies, such as caused by HIV infection or iatrogenic immune-suppression, are highly susceptible to fungal infections caused by Cryptococcus neoformans. C. neoformans is an environmental fungus that produces airborne spores. CD4 T-cell dysfunction in humans is associated with an inability to contain these spores in the lung, leading to a disease called cryptococcal meningitis whereby the fungus invades the central nervous system (CNS). Cryptococcal meningitis is difficult to diagnose and has few treatment options, leading to high mortality rates. It is therefore critical that we obtain a better understanding of the protective role of CD4 T-cells in defence against C. neoformans so we can develop better immune-based therapeutic strategies and vaccines to help reduce the global burden of this infection on human health. This proposal outlines a collaborative project between experts in antifungal T-cell responses (Dr Kawakami), organ-specific antifungal immunity in the CNS (Dr Drummond), and T-cell signalling (Dr Bending), with the aim of defining pathways controlling the activation and generation of C. neoformans-specific CD4 T-cell responses during infection in the lung and CNS. This research uses clinically-relevant animal models of infection and novel transgenic animal lines available in our laboratories to dissect the mechanisms underlying (1) when fungal-specific T-cells become activated and the influence of innate immunity on this process, (2) how fungal-specific T-cell memory responses are generated and maintained, and (3) how these processes are disrupted by immune-suppression protocols observed in patients. Collectively, our collaboration provides the expertise and tools needed to generate significant new insights into the role and control of functional specialisation of fungal-specific T-cells during cryptococcal meningitis, a key goal of the future treatment and management of this lethal infection.


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