NIHRDataInsights/HRCSData huggingface dataset:
Title: A novel immuno-diagnostic strategy for invasive fungal infections in solid organ transplant patients.
Funder: Medical Research Council

Abstract: Key aims and objectives of the project 1) Define the effects of transplant immuno-suppressant drugs on host immunity in the standard hydrocortisone-suppressed (HC) murine model of invasive aspergillosis (IA). 2) Identify the immune deficits that lead to susceptibility to IA in this model. 3) Develop novel diagnostic assays of susceptibility to invasive fungal infections in this model. 4) Validate these diagnostic assays through synergistic studies in renal transplant patients. Design and Methodology 1) Define the additive effects of the transplant immuno-suppressants tacrolimus (FK506) and mycophenolate (MMF) on mortality in the HC-based model of invasive aspergillosis. Doses of FK506 and MMF will be increased until mortality increases from 50% to >80%. The model will be defined through histopathologiocal, pharmocokinetic, fungal burden and peripheral blood profiling. 2) Identify the key defects in immunity that increase susceptibility to IA in this model. Macrophage, T lymphocyte, NKT cell and dendritic cell populations, function and fungal responses in lung, liver, spleen and peripheral blood will be characterized by conidial phagocytosis assays, RT-PCR and Luminex cytokine assay, FACS analysis, NKT cell xenogenic target assays, and dendritic cell na?ve T-cell co-culture. 3) Develop peripheral blood mononuclear cell (PBMC) microarray and anti-Aspergillus T-cell Elispot IFN- assays that identify increased susceptibility to IA in FK506/MMF-immuno-suppressed mice. PBMCs from infected mice will be stimulated with Aspergillus antigens or control mitogens. Cytokine responses will be assayed by Agilent whole genome microarrays and correlated with immuno-suppressive drug levels and transcriptional markers of drug toxicity. Parallel anti-Aspergillus T-cell IFN- Elispot assays will be performed on peripheral blood. The Elispot assay will provide a highly sensitive means of quantifying the effects of FK506/MMF on T-cell mediated antifungal immunity. 4) Define the utility of the PBMC RT-PCR and Elispot assays described in Component 3 through a controlled clinical study of 40 renal transplant patients with invasive fungal infections. For the PBMC assays, select markers of susceptibility to invasive fungal infection will be translated into a rapid RT-PCR assay that can also identify patients who will benefit from IFN- therapy. Scientific and Medical Opportunities 1) Deliver a model in which to define the immuno-pathogenesis of transplant infectious diseases. 2) Identify of the key immunological defects that predispose solid organ transplant patients to IFIs. 3) Deliver peripheral blood assays to identify susceptibility to IFI and guide immuno-therapy. 4) Provide a rational basis for future studies of biopharmaceutical modulators for IFIs.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;6.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Cell mediated immunity against RSV and influenza in a human experimental challenge model
Funder: Medical Research Council

Abstract: Background Influenza and respiratory syncytial virus (RSV) are among the most common causes of viral respiratory infection, causing widespread morbidity and mortality particularly at the extremes of age and exacerbating conditions such as asthma. Vaccine development remains an urgent priority. While cell mediated immunity (CMI) is important in viral clearance and may enhance vaccine efficacy, its relative contribution in humans has not been fully determined. We aim to elucidate its role by comparing the human T cell responses to influenza and RSV. These differ in that re-infection with antigenically identical strains of influenza does not occur, while within 2 months of RSV challenge even subjects with high antibody levels may be re-infected with the same strain. The reasons for this are unclear but may involve immune modulation by viral products or impaired antigen presentation by RSV-infected dendritic cells leading to impaired T cell numbers function. Hypotheses & aims 1. To test the hypothesis that RSV and influenza lead to quantitative and functional differences in T-cell responses. 2. To identify the mechanisms underlying poor antigen-specific CMI to RSV by comparing the transcriptional changes in T-cells following experimental challenge with RSV and live attenuated influenza vaccine (LAIV). Study design Healthy adult volunteers will be challenged with RSV Memphis 37 or LAIV. Samples will be collected periodically from blood, broncho-alveolar lavage (BAL) and nose. After 6 months, a second dose of virus will be administered and further samples obtained to examine the recall response. The number of virus-specific T-cells will be analysed by ELISPOT and by MHC/peptide multimer staining. Differential effector functions will be studied including surface protein expression, cytokine production and proliferative capacity by CFSE staining. Flow cytometry will be used to characterize activation markers, co-stimulatory molecules, cytokines, and cytotoxic molecules. Nasal and bronchial fluid will be analysed for cytokine levels using multiplex bead arrays and Luminex. Using microarrays, we will identify transcriptional changes that occur during T-cell activation and following re-stimulation with RSV and LAIV. These data will recapitulate the predicted functional defects in RSV-infected T cells and will allow us to make inferences regarding underlying mechanisms via differential expression of signalling intermediates in TCR signal transduction. CMI is important against both influenza and RSV infection, but protection against RSV is incomplete. By identifying correlates of immunity absent from the CMI response to RSV we aim to elucidate the basis for previous vaccine failures as well as novel approaches to vaccination.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Structural Studies on Viruses, Viral Proteins and Cell Interactions
Funder: Medical Research Council

Abstract: This application is for the renewal of a programme focused on virus structure analysis which includes technical development, and spans from basic research into fundamental questions of function and taxonomy, through to work to underpin the development of improved vaccines and anti-viral drugs. Technical developments comprise the first aim: novel methods for X-ray structure determination in situ (i.e. avoiding any handling of crystals), the investigation of novel crystal freezing methods and exploration of the limits of X-ray analysis for small arrays of virus particles in cells using diffraction and imaging. These methods underpin three biomedical themes: (i) to tackle some major outstanding questions concerning picornaviruses, (ii) to understand a major vaccine particle, that of hepatitis B, and to determine novel structures to map the landscape of the viral universe, and (iii) to work towards a more comprehensive view of virus fusion. Theme (i) includes the investigation of the atomic structures of hepatitis A, EV71 and a newly assigned nonclassical picornavirus (Ljungan or a parechovirus); investigation of assembly /disassembly pathways including analysis of a representative picornavirus heavy particle; and the design and structure determination for recombinant mutant picornavirus capsids with increased stability, to establish rules for improved vaccines. For theme (ii) we will work towards the high resolution X-ray structure of the hepatitis B surface antigen particle and initiate crystallographic analyses of novel viruses from a virus ecology pipeline. Theme (iii) builds on existing work on viral fusion proteins, with a particular focus on hepatitis C E1 and E2 proteins and E2 of the bovine viral diarrhoea pestivirus. We will also initiate work on a markedly different and complex fusion system from vaccinia virus. The programme includes the analysis of viruses responsible for major global health burdens (including hepatitis A, B and C viruses) and will contribute to the body of structural information freely available in the public domain, which can be used both by academics and commercial organisations for structure-guided drug discovery. In addition we will work in a more translational fashion on vaccine design. As appropriate we will form collaborations to maximise exploitation of the results, a strategy exemplified by our current work on foot-and-mouth disease virus vaccine (with Intervet/Schering-Plough Animal Health).


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: HIV-Host Interactions
Funder: Medical Research Council

Abstract: It is now recognised that animal cells express various factors and pathways that naturally inhibit the replication of viruses. The balance between these innate/intrinsic inhibitors and the many factors that promote replication helps establish the tempo of virus infection and, ultimately, contributes to disease outcome. Here, we outline an ambitious series of discovery-based and mechanistic experiments that will continue to build on our work on the anti-HIV-1 APOBEC3G/F proteins and will explore relationships between HIV-1 infection and other potential anti-viral networks, including the RNA silencing machinery, cellular stress-induced pathways, and the type-1 interferon (IFN) system. First, we will further define the mechanisms by which the APOBEC3 proteins suppress HIV-1 infection. In particular, we will determine how APOBEC3 proteins inhibit HIV-1 reverse transcription in primary CD4 T cells, using new assays to define the cDNAs of inhibited replication intermediates, to map interactions between APOBEC3G/F proteins and viral proteins and RNA, and to define the necessary participating attributes of the APOBEC3G/F proteins. Second, having demonstrated that APOBEC3G/F assemble into large cellular ribonucleoprotein (RNP) complexes, we will define the RNAs to which these proteins bind directly and address the impact of such interactions upon RNA fate and metabolism. Since APOBEC3G/F RNPs accumulate in RNA-rich cellular microdomains called P bodies (PBs) and stress granules (SGs), and some of the factors and complexes that also accumulate there (e.g., the RNA silencing machinery) have been implicated in the suppression of viral and transposon replication, we will investigate the importance of PB-, SG- or RNA silencing components in HIV-1 replication and APOBEC3G/F function. Third, we will use transcriptomic-based methodologies to define the initial (i.e., within hrs) response of CD4 T cells to HIV-1 challenge. Our preliminary results indicate that cells undergo substantial reprogramming, and that many of the induced host genes are also activated by type-1 IFN. Functional analyses will aim to identify cellular sensors of HIV-1 infection, as well as downstream signalling pathways and effector molecules. This work will be interpreted together with parallel studies where the mechanism by which IFN establishes an anti-HIV-1 state in T cells and macrophages is examined, and screens to isolate the contributing genes are undertaken. In sum, we aim to identify novel interactions between HIV-1 and infected host cells, with the goal of better understanding the principles of virus replication, pathogenesis and human cell biology, as well as defining rational targets for future therapeutic exploitation.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Vaccinia virus entry, exit and evasion
Funder: Medical Research Council

Abstract: Vaccinia virus (VACV) is the live vaccine that was used to eradicate smallpox. Thereafter, VACV has continued to be studied due to its potential as a live recombinant vaccine for other diseases, and because it is an excellent model for studying virus-host interactions and immune evasion. This project proposal is for renewal of an existing programme grant and the objectives are to study fundamental aspects of VACV biology including entry, egress, spread and immune evasion. 1. Entry. Studies of VACV entry into cells will focus on the double enveloped extracellular virion. Specifically, we will study how the entry of this virus into cells by a non-fusogenic shedding of the outer envelope is prevented when this virion contacts the A33/A36 complex on the infected cell surface. 2. Intracellular transport. We will investigate how newly formed VACV particles are transported on microtubules to the cell surface and the roles of proteins E2, 12 and A36 in this process. This work will build on our recent demonstration that F12 is a structural mimic of kinesin light chain (part of the kinesin motor) and proteins E2, F12 and A36 all possess tetratricopeptide repeats that mediate protein:protein interactions. 3. Virus spread. Recently we have discovered a novel mechanism that VACV uses to spread from cell to cell at a rate 4 times faster than can be explained by the virus replication kinetics. The mechanism involves repulsion of superinfecting virions by the A33 / A36 protein complex so that virus particles are disseminated to distant uninfected cells without replication being required in the intervening cells. This study has redefined how viruses form a plaque and reveals another target for intervention with anti-viral therapy. We will investigate which parts of these proteins are needed, which proteins on the virus surface are required, and test whether soluble fragments of either can diminish virus spread. 4. Lastly we will investigate immune evasion by VACV, with focus on i) VACV encoded miRNAs that are produced from within the coding region of a VACV mRNA, and ii) an uncharacterised protein, B6, that we hypothesise is a novel chemokine binding protein. The contribution of each to virus virulence and immune evasion will be investigated using appropriate in vitro and in vivo models.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Genetic, structural and functional analyses of flagellar glycosylation in epidemic Clostridium difficile strains
Funder: Medical Research Council

Abstract: The current epidemic of C. difficile infection has been accompanied predominantly by the emergence of two clonal groups of PCR-ribotype 027 and 017 (A-B+) strains that are associated with increased transmissibility and disease severity. Recently, in collaboration with the WTSI, we sequenced the genomes of several 027 and 017 strains and identified a number of genes involved in flagellin glycosylation that are distinct from other C. difficile strains all of which appear to be glycosylated. Further studies have confirmed that flagellin from representative 027 and 017 strains are glycosylated and are structurally different to other C. difficile strains. The modification of flagella by glycosylation has been shown to be increasingly important in the virulence and host adaption of several bacterial pathogens. We hypothesise that flagellar glycosylation is important in C. difficile and that this may be especially relevant to the epidemic 027 and 017 strains that maintain distinct glycoforms To determine the role and importance of flagella and flagellar glycosylation in C. difficile we will construct several 027 and 017 mutants to produce aflagellate mutants, glycosylation deficient mutants and glycosylation modified mutants. These distinct mutants will be rigorously characterised in several physical, in vitro and in vivo assays relevant to the life cycle of the organism. These studies will be augmented by structural analyses of the glycan component of the wild type 027 and 017 strains and the respective mutants. In this way we will dissect the process of flagellar glycosylation by a combination of genetic, bioinformatic and structural analyses enabling us to build up a comprehensive picture of the core and accessory genes involved in flagellar glycan biosynthesis. We will also determine if the same glycosylation pathway also modifies other surface structures. The characterisation of glycosylated surface exposed structures such as flagella should improve our knowledge of how C. difficile causes disease and could also prove useful in the development of diagnostic markers and the identification of therapeutic and vaccine targets.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Immunology and Immunopathology of visceral leishmaniasis
Funder: Medical Research Council

Abstract: Leishmaniasis represents a spectrum of diseases associated with infection by the protozoan parasite, Leishmania. Of global importance, with approximately 2M new cases reported annually from 88 countries, leishmaniasis ranks high amongst parasitic diseases for its impact on public health. Visceral leishmaniasis (VL), caused by L. donovani and L. infantum/chagasi is a leading cause of parasite-associated mortality. Research on VL thus holds potential for major benefits to health. In addition, the study of this intracellular parasite continues to increase our fundamental understanding of the host-parasite interface and immune function. Building on the exciting achievements of our previous MRC supported research, we now seek to develop a fully-integrated programme, using animal models, human liver-slice culture and clinical histopathology, to explore the diverse roles played by mononuclear phagocytes in VL (as host, regulator of inflammation / immunity, and mediator of pathology). To study the biology of tissue-resident macrophages in their natural environment, we will define (at the molecular and cellular level) the in situ response of murine and human Kupffer cells to L. donovani infection and the downstream consequences for the initiation of inflammation. We will perform comparative mRNA and miRNA profiling of human and mouse Kupffer cells, and validate potential mediators of inflammation using dynamic imaging approaches in human and murine liver. New tools will allow us to examine the function of Kupffer cells in established granulomas in mice. We will perform comparative histopathology of spleen / LN from mice and patients with VL, to determine whether the lymphoid tissue remodelling associated with parasite persistence and immunosuppression in mice is also evident in human disease, and using intra-vital imaging in combination with mice that allow cell-specific and temporal depletion and lineage tracing, we will define the involvement of mononuclear phagocytes in this process. Informed by histopathology and transcriptional profiling, we will develop new models of hepatic infection that reflect more closely the diversity of response seen in man. Likewise, we will exploit recently identified sites of cryptic infection in mice to develop a greater understanding of the mechanisms underlying post kala azar dermal leishmaniasis, mucosal disease and secondary infection. Collectively, the data generated in this Programme will test a number of new hypotheses related to mononuclear phagocyte function in VL, help to cross-validate existing and new mouse models and, by providing new comparative data on disease pathogenesis, increase the likelihood of effective translation of our research into new prophylactic or therapeutic interventions.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: An analysis of wild type human cytomegalovirus
Funder: Medical Research Council

Abstract: Genetic changes in the human cytomegalovirus (HCMV) genome during growth in cell culture are associated with loss of virulence and immunomodulatory functions. To address this issue, low passage clinical isolate Merlin was cloned into a bacterial artificial chromosome (BAC). By passages 1-3 in fibroblasts, mutations in RL13 and UL128 had emerged and were then captured in the BAC. These defects were seamlessly repaired to generate (uniquely) an infectious clone of genetically-intact HCMV. Significantly, the repaired virus, and independent clinical isolates, exhibit severe growth impairment until mutants at this locus (re-)emerge. We seek to investigate the molecular mechanisms responsible for RL13 repression of replication, and (ii) whether RL13 expression is compatible with efficient virus replication and secretion in vivo. In order to be able to produce homogeneous RL13+ virus stocks, we engineered HCMV to provide conditional RL13 expression. RL13 is hypervariable and encodes a highly glycosylated virion envelope protein, making gpRL13 a potential target for neutralising antibody. A recent monovalent gB vaccine study demonstrated that humoral immunity can prevent HCMV transmission. In this context, there is an urgent need to identify the full complement of proteins/antigens presented on the surface of the wild-type virus. Having developed the capacity to propagate phenotypically wild-type (RL13+) virus, we now seek to define the proteome of such virions. Our chief objective in accurately defining HCMV gene usage was to provide a reliable basis for studying HCMV immune evasion and modulation. In this context, we seek to systematically investigate the effect of HCMV productive infection on 244 cell surface markers in three cell types. The most significant changes in cellular gene expression will be analysed in detail. Previously whenever HCMV has been demonstrated to modulate a cellular function, there was no obvious means to identify the gene responsible. With MRC support, we have been cloning all 184 predicted HCMV genes into an adenovirus vector as the basis for a high throughput screening system. Interferons and TNF receptor family members together provide extremely effective barriers to virus infection. Remarkably, while HCMV clearly targets multiple member of the TNFR superfamily, the genes responsible have not been identified. While HCMV interferon resistance genes have been mapped, the list may well be incomplete. We therefore seek to screen the complete HCMV genome for novel immunomodulatory functions taking advantage of the luciferase reporter to facilitate high throughput assays.


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