NIHRDataInsights/HRCSData huggingface dataset:
Title: Fighting AMR of hard-to-reach microbial pathogens by repurposing antibiotics using a targeted liposomal delivery strategy: A Helicobacter pilot study
Funder: Medical Research Council

Abstract: This project has 4 main experimental aims. Aim 1) Antimicrobial susceptibility testing of ~600 available H. pylori (Hp) isolates against standard first and second line drugs (amoxicillin, clarithromycin, levofloxacin, tetracycline, metronidazole and rifampicin) as recommended by the British Society for Antimicrobial Chemotherapy, using Etest strips (bioMérieux SA) on Mueller-Hinton agar with 10% horse blood. The strains will also be tested against antibiotics not traditionally used for Hp, but efficacious in vitro (erythromycin, cefixime and tigecycline). Aim 2) Production of liposomes with extruder technology using established techniques. Once liposomes have been produced, they will functionalized using copper-free 'click' chemistry (conjugation of lipids containing a reactive bicyclo[6.1.0]nonyne (BCN) cyclo-octyne group with an azide). The latter will be added recombinantly to the C-terminal of the H. pylori adhesins LabA and BabA as p-azido-L-phenylalanine, using the pEVOL-pAzF plasmid (Addgene). Aim 3) In vitro binding of functionalized liposomes: The properties of functionalized liposomes will be investigated in vitro using protocols established in our lab. We will use a human gastric epithelial cell line stably transfected with human MUC5AC (a known ligand for BabA), using untransfected AGS cells and BabA binding site mutants as a control. Aim 4) In the final year of the grant, we will test the optimal formulations developed as described above using our C57BL/6 mouse model under an existing project licence. Tc-99m NanoSPECT-CT imaging, which we have previously established, will be used A) to verify increased gastric retention of liposomes functionalized with LabA in the murine stomach, and B) determining the increased efficacy of gastric targeted liposome-formulated antibiotics in vivo, by comparing the effectiveness of our liposomal formulation with the standard triple therapy in eradicating a highly-colonising SS1 strain of Hp in mice.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: The Mycobacterium tuberculosis Cell Envelope: unravelling complex cell wall assembly, degradation and re-cycling pathways
Funder: Medical Research Council

Abstract: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has a unique cell envelope, which accounts for its unusual low permeability and hence, contributes to resistance against common antibiotics. Our integrated studies are aimed at coordinating a continued investigation into the basic biochemistry and physiology, and drug targeting potential of the M. tuberculosis cell envelope. The application will focus on the following key components of this complex; arabinogalactan (AG), lipoarabinomannan (LAM) and peptidoglycan (PG), and in addition we will continue to explore key physical properties of the mycobacterial cell envelope. Firstly, we will build on our previous studies on lipid biosynthesis and investigate the role of echA proteins in lipid degradation and re-cycling pathways. Secondly, we will extend our characterisation of the assembly of the complex AG matrix, with an emphasis on the role of glycosyltransferases and transporters, as well as the related enzymes leading to the key immunogenic cell envelope component, LAM. Thirdly, we will integrate our studies on the cross-linked network of PG, following a multi-pronged approach that will involve the detailed genetic, biochemical and structural characterisation of key M. tuberculosis PG biosynthetic enzymes. Fourthly, we investigate the physicochemical properties of the mycobacterial cell envelope. Addressing these topics will provide fundamental knowledge on M. tuberculosis cell envelope assembly, degradation and recycling pathways. In addition, it is anticipated that the above functional and structural studies, will ultimately prove useful for screening for inhibitors, potentially generating valuable hits and leads, ultimately leading towards new anti-TB drugs. In the longer-term, these studies will offer key translational opportunities for improved therapies and prevention strategies, particularly for populations living in low and middle income countries (LMIC) where the burden of TB is greatest.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Regulation of HIV-1 and Ebola virus replication by CpG dinucleotides, ZAP and TRIM25
Funder: Medical Research Council

Abstract: Synonymous genome recoding can inhibit RNA virus replication by introducing large numbers of synonymous mutations in viral open reading frames. This is a potential new approach to develop novel vaccines. However, whether these mutations inhibit viral mRNA translation efficiency or have a different deleterious mechanism of action has been controversial. Recently, it has become clear that increasing the abundance of CpG dinucleotides using synonymous mutations is sufficient to attenuate picornavirus and influenza A virus replication. Furthermore, we and others have shown that HIV-1 replication is inhibited by increasing the CpG abundance. Therefore, CpG dinucleotides in RNA may be recognised by cellular proteins to inhibit viral replication. The cellular antiviral protein ZAP has been shown to inhibit HIV-1 replication and bind regions of the HIV-1 genome containing CpG dinucleotides. ZAP has also been shown to inhibit Ebola virus (EBOV) replication. Recent progress in EBOV reverse genetics has yielded a safe, non-infectious tetracistronic transcription- and replication-competent virus-like particle (trVLP) system that allows modelling of multiple rounds of the EBOV replication cycle in standard containment level 2 laboratories. We are using this trVLP system to study how cellular proteins regulate EBOV replication. In this proposal, we use HIV-1 and EBOV trVLPs as model systems to characterise how CpGs inhibit RNA virus replication. We will explore how ZAP and its cofactor TRIM25 inhibit HIV-1 and EBOV trVLP replication in a CpG-dependent manner. In addition, other cellular proteins may regulate the response to CpG-containing viral RNA and we will use proteomics approaches to identify and characterise these. Overall, this proposal will increase our understanding of how CpG dinucleotides inhibit HIV-1 and EBOV trVLP replication and the cellular antiviral proteins that restrict these viruses. This may allow the development of novel vaccines and antiviral therapies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Hepatitis C virus genotype 3 NS5A protein: resistance to direct acting antiviral agents and functional analysis
Funder: Medical Research Council

Abstract: Hepatitis C virus (HCV) genotype 3 (GT3) represents both a clinical issue and an unique biological question. GT3 isolates exhibit high baseline resistance to direct acting antivirals (DAAs) such that the sustained virological response rates for GT3 patients are significantly lower than for other GTs, particularly for patients with cirrhosis. GT3 is also associated with more rapid progression of liver disease and a direct correlation with metabolic syndrome, leading to a higher incidence of insulin resistance, steatosis (fatty liver), and hepatocellular carcinoma compared to other GTs. The underlying biological differences between GT3 and other GTs are poorly understood, largely because of a lack of robust in vitro culture systems. However, we and others have recently developed both transiently replicating sub-genomic replicons (SGRs), and infectious viruses that overcome this hurdle. Focussing on the NS5A protein, we will use both SGR and virus to investigate the nature of the baseline resistance to DAAs that target NS5A, using a mutagenic approach but also screening isolates from the STOP-HCV and HCV-Research UK biorepositories which both contain a number of GT3 patients who either failed therapy or relapsed. We will establish viable SGRs and infectious virus constructs containing in-frame tags within NS5A to address how the GT3 NS5A interactome and phosphorylation pattern differs from other GTs. We will follow these studies with phenotypic analysis of interacting proteins and phosphorylation sites and ask how DAA resistance affects the function of the protein. Lastly, we will pursue structural analysis of GT3 NS5A - the protein comprises 3 domains, the structure of the N-terminal domain I has been determined for GT1 isolates so we will attempt to achieve a similar level of structural understanding for GT3. This will help to frame the information obtained from the other aspects of the proposal.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: HPV hijacking of the epidermal growth factor receptor via its oncogenic ion channel, the E5 viroporin: a new mechanism of virus-driven pathogenesis
Funder: Medical Research Council

Abstract: HPV infection causes ~6% of all human cancers. These include cervical cancer in women, which results in an estimated 266,000 deaths annually. Moreover, the past decade has witnessed a new epidemic of HPV-associated head and neck squamous cell carcinomas (HNSCC), primarily in white, middle-aged men. The majority of HPV cancer patients (~85%) reside in low-income countries, with limited health infrastructure and finance. HPV-related health care costs are extreme and are estimated to cost $8 billion per annum in the USA alone. Despite FDA-approved prophylactic vaccines, the burden of HPV disease will remain high for decades due to limited access to vaccines in low-income countries, and also poor coverage in those countries which have access to the vaccines, and the long latency period separating infection from carcinogenesis. Development of HPV-specific therapies to complement the vaccine strategy is clearly warranted. To develop such interventions a detailed understanding of HPV biology is required. Our research has focused on E5, which is the least well understood virus oncoprotein. We previously showed that E5 functioned as a virus-coded ion channel or viroporin. Now we demonstrate a role for the viroporin activity of E5 in hijacking host epidermal growth factor receptor (EGFR) signalling. This serves to maintain keratinocyte proliferation and as a novel means of blocking p53 function, resulting in a delay of Notch-mediated differentiation. Critically, small molecule inhibitors targeting the viroporin activity of E5 prevent EGFR activation. Thus our data greatly expands the role of E5 in the HPV life cycle and provides a clear function for viroporin activity as an activator of EGFR. Using established primary cell cultures that model genital and oropharyngeal infection with oncogenic HPV we will test the hypothesis that E5 activates EGFR function to manipulate keratinocyte proliferation and suppress differentiation during productive infection.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: An investigation of interventions to increase uptake of HIV self-testing and linkage to post-test services among higher education students in Zimbabwe
Funder: Medical Research Council

Abstract: Adolescents and young people in Africa have the worst health outcomes of adolescents and young people globally, in large part because of the high incidence of HIV, particularly among young women. Despite this high incidence, young people are less likely to know their HIV status than older adults and as such are less likely to benefit from prevention and treatment options. Africa's impending demographic transition means that if nothing is done to improve young people's access to HIV prevention and care, the number of new infections in young people will double in the next 30 years. This implementation science proposal targets young people attending higher education institutions with a novel intervention, HIV self- testing, aimed at improving their uptake of HIV testing and subsequent engagement with prevention and care services. HIV self-testing, where people test themselves for HIV infection, has been shown to be safe and accurate and of importance here, highly acceptable to young people who desire privacy and autonomy when testing. I propose to compare the effectiveness and cost effectiveness of two distribution models of HIV self-testing among students in higher education, in a cluster randomised trial. Higher education institutions will be the units of randomisation. Outcomes include: i) uptake of self-testing, ii) linkage to the appropriate post-test services and, iii) cost per person tested, diagnosed positive and started on treatment or preventive service. The two HIV self test distribution models to be compared are a peer-led plus mass-media supported model which will be compared with a mass media only model. I will also develop an mHealth tool to facilitate measurement of self-test kit uptake and support measurement of subsequent engagement to prevention and care. I will then explore how to adapt these self-testing models for young people into other settings and to support prevention and care for other chronic diseases in young people.


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION
Title: Development of a cost-effective protein-based combined glycoconjugate meningococcal-pneumococcal vaccine
Funder: Medical Research Council

Abstract: Despite vaccination, the ongoing threat from bacterial meningitis is unquestionable, especially in low-to-middle income countries where cost is a significant factor, justifying the search for novel approaches to vaccine candidate discovery. In proof-of-principle studies, evidence was provided for the potential utilisation of a sequential approach involving reverse vaccinology 2.0- RV 2.0 - (cloning of human monoclonal antibodies [hmAbs] from convalescing patients) followed by protein-glycan coupling technology (PGCT) to produce a cost-effective combined meningococcal-pneumococcal vaccine with broad coverage. From four ml of patient blood samples, bactericidal antibodies targeting antigens not currently included in licensed meningococcal vaccines were cloned. The functional activity of a pneumococcal antibody with broad serotype-independent specificity is currently being assessed. Furthermore, preliminary data using PGCT (cheaper and time-saving alternative to conventional in vitro chemical coupling systems) shows the successful expression and potential glycosylation of recombinant meningococcal factor-H binding protein in an Escherichia coli mutant expressing the pneumococcal serotype 4 glycan. This study will extend investigations using both RV 2.0 and PGCT to identify functionally-immunogenic protein antigens that will compose a vaccine targeting both meningococcal and pneumococcal strains. Specifically, the study will: (1) Clone and express recombinant hmAbs from patients convalescing from meningococcal and pneumococcal diseases; (2) Assess functional activity of specific hmAbs; (3) Determine antigen targets (and their sequence conservation) of functional hmAbs; (4) Couple antigens of interest to glycan; and (5) Conduct animal efficacy studies (immunogenicity and SBA/OPA) in mice to determine candidate antigens that can progress to Phase 1 human clinical trials.


Research activity codes: 3.4 VACCINES
Health categories: INFECTION
Title: Triggering assembly of the twin-arginine translocase
Funder: Medical Research Council

Abstract: The twin arginine protein transport (Tat) system is a conserved and highly unusual protein transport pathway that exports folded proteins across the cytoplasmic membranes of bacteria. It is required for the virulence of most important human pathogens and is essential for the viability of pathogenic mycobacteria. The Tat system assembles 'on demand' in the presence of a substrate protein and disassembles once transport is completed. The aim of this study is to provide key information about the interaction of Tat substrates with the Tat receptor through a combination of molecular dynamics, modelling, in vivo crosslinking and biochemistry. It has the following specific objectives: 1. To map the signal peptide binding site within the Tat receptor and to determine whether binding of the signal peptide at this site is dependent on the protonmotive force; 2. To determine whether the newly-discovered TatA/B binding site on TatC TM6 is essential for operation of the Tat pathway and; 3. To examine the organisation and activation mechanism of the Tat receptor in Tat translocases that lack the TatB component. 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 therapeutic relevance.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Phenotype to genotype: dissecting meningococcal disease and carriage traits
Funder: Medical Research Council

Abstract: Meningococci cause disease by transitioning from the mucosa into the circulatory system, surviving bactericidal effects of blood, adhering to the meninges and eliciting pathological cytokine responses. Variability in these phenotypes and in transmissibility influences whether meningococci cause disease. We aim to dissect disease potential by linking variability in multiple phenotypes to genotypic variation (allelic, accessory genes or phasotypes). Our hypotheses are:- (1) quantitative differences in specific phenotypes can be predicted from the genotype; (2) disease potential is due to the accumulation of large-effect genetic determinants of multiple phenotypes; (3) virulent phenotypic variants are antagonised by avirulent phenotypic variants. We will develop high throughput assays for twelve disease-related phenotypes by combining multiwell plate formats with bacteriological, microscopic or immunological methods for measuring colony forming units, tissue disruption and cytokine levels. Application of assays to ~330 cc23 MenY and cc11 MenW isolates will generate quantitative data with defined variance levels. Statistical testing of individual and combinatorial phenotypic outputs provides significant power for multicomponent statistical association testing to link patterns of phenotypic variation to disease and carriage traits. Using treeWAS, a genome-wide association testing approach, we will associate phenotypic variation with specific genotypes followed by confirmatory re-testing of isogenic mutant strains in our panel of phenotypic assays. Antagonism will be studied in co-infection studies such as examining inhibition of CaLu3-monolayer disruptive variants by non-disruptive variants. Our long-term goal is to generate predictive algorithms that utilise genotypically-linked phenotypic information to determine disease potential for meningococcal isolates and enable detection of infectivity shifts during epidemiological monitoring of meningococcal populations.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Analysis of the L,D transpeptidation pathway in Clostridium difficile: contribution to peptidoglycan synthesis and antibiotic resistance
Funder: Medical Research Council

Abstract: C. difficile causes gut infections that vary in severity from mild diarrhea to life threatening, inflammatory complications. The bacterium is intrinsically resistant to many antibiotics, including beta-lactams such as cephalosporins, and infection commonly follows antibiotic-induced intestinal dysbiosis that gives C. difficile a competitive advantage. The molecular basis of resistance is not understood but the cell wall peptidoglycan contains an unusually high degree of 3-3 crosslinks, catalysed by a family of L,D transpeptidases (Ldts), the activity of which has been associated with beta-lactam resistance in other species. Most C. difficile strains encode three Ldts; two of these appear to have only a minor contribution to the formation of 3-3 crosslinks, while the third has yet to be characterised in any detail. We aim to fully characterise the L,D transpeptidation pathway in C. difficile: elucidating the role of each Ldt in peptidoglycan synthesis and beta-lactam resistance, and exploring the structural basis of these functions. We will also analyse Ldt diversity in a panel of clinically relevant C. difficile strains and determine how this impacts peptidoglycan structure and beta-lactam resistance. We will adopt a highly multidisciplinary approach, combining expertise in peptidoglycan biochemistry, C. difficile molecular biology, structural biology and clinical microbiology. The insights gained from our analysis of this important biochemical pathway will shed light on C. difficile multidrug resistance and pave the way for the development of Ldt inhibitors to directly kill C. difficile or induce susceptibility to traditional beta-lactams.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Understanding mechanisms of antimicrobial resistance (AMR) in Streptococcus pneumoniae clinical isolates
Funder: Medical Research Council

Abstract: The human pathogen S. pneumoniae is responsible for millions of cases of invasive disease annually with emerging penicillin resistant lineages threatening treatment outcomes for patients. Thus, there is an urgent need to understand the biology underpinning penicillin resistance for this pathogen to safeguard patient outcomes. To achieve this, I have selected clinical isolates with low-level penicillin resistance for further study, reasoning these strains would have a limited number of resistance determinants and would offer a tractable platform for further investigations. I have discovered uncharacterised resistance determinants in these isolates that are essential for beta-lactam resistance in clinically important strain backgrounds. I hypothesise these unknown mechanisms are essential for beta-lactam resistance and support mosaic-pbp acquisition that drive higher levels of resistance. This project will identify mutations in known and previously unknown S. pneumoniae genes linked to beta-lactam resistance in clinical strains with differing resistance profiles (Objective 1). I have made the exciting discovery of a new gene cluster linked to beta-lactam resistance and the function of target PBP enzymes. I will study the function of genes within this cluster alongside those identified in Aim 1 to understand the biological mechanisms underpinning resistance (Objective 2). The ultimate aim of this study is to identify ways to 're-sensitise' strains to antibiotics, to a level where they now respond to treatment. I will use a zebrafish embryo model of infection to test how disruption of identified mechanisms impacts virulence and, importantly, if these disruptions have a favourable impact if infected zebrafish are treated with antibiotics (Objective 3). This unique approach will inform S. pneumoniae lineage surveillance and identify strategic points of weakness in beta-lactam resistance phenotypes that may be exploited for therapeutic gain.


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