The transciptomic landscape of HIV-TB

Includes abstract.Includes bibliographical references.The thesis consists of four main parts. In Part 1 of this thesis I provide a broad overview of HIVTB with an emphasis on systems approaches followed by an overview of a systems-level study aiming at addressing hypotheses relating to transcriptional differences in active tuberculosis and HIV-1 infection, measured in blood and the site of disease in tuberculous pericarditis. The final chapter in this part describes the methods used to generate and analyse the systems-level data, with emphasis on microarray data generation and analysis. Part 2 first presents analysis of transcriptional data generated by RT-PCR at the site of disease compared to blood in study subjects with tuberculous pericarditis, with results showing clear evidence of transcriptional differences between compartments. A Technical Results chapter then provides an overview of the microarray data, and an analytic paradigm based on sample embedding in high-dimensional phenotype space is developed. I then assess the overall quality of the dataset and exclude large-scale systematic bias, while comparison of the IMPI-MA data to exiting TBtranscriptomic data shows a close match. Part 2 concludes with a description of a comprehensive analytic framework developed for the IMPI-MA data. Part 3 presents the results of the analytic pipeline as applied to the transcriptional response in blood to active tuberculosis in an HIV-1 uninfected population. A signature of active tuberculosis is described, and deconvolution analysis finds significant NK cell activation in active tuberculosis. Cell-type specific differential expression identifies CD4 T cells, NK cells and neutrophils as the most likely contributors to the overall “signature” of active tuberculosis. Weighted gene co-expression network analysis reveals multiple modules, whose expression is shown to be differentially regulated based on disease category. Part 4 summarises the results of analysing contrasts in three main contexts: tuberculosis, HIV-1 infection and compartment. Two novel results are presented: Firstly, NK cells are shown to be functionally downregulated at the site of disease, suggesting a possible defence mechanism by M. tuberculosis, and secondly, large-scale metabolic pathway dysregulation at the site of disease, possibly favouring M. tuberculosis, is demonstrated. Part 5 concludes the thesis with a summary and outlines future work

Six host-range restricted poxviruses from three genera induce distinct gene expression profiles in an in vivo mouse model

BACKGROUND: Host-range restricted poxviruses make promising vaccine vectors due to their safety profile and immunogenicity. An understanding of the host innate immune responses produced by different poxvirus vectors would aid in the assessment, selection and rational design of improved vaccines for human and veterinary applications. Novel avipoxviruses are being assessed to determine if they are different from other poxvirus vectors. Analysis of the transcriptome induced in a mouse model would aid in determining if there were significant differences between different poxvirus vectors which may reflect different adjuvant potential as well as establish if they should be further evaluated as vaccine vectors. RESULTS: We compared host transcript abundance in the spleens of BALB/c mice twenty four hours after intravenous infection (10 5 pfu/mouse) with six host-restricted poxvirus species from three genera, namely Lumpy Skin Disease virus (LSDV), Canarypox virus (CNPV), Fowlpox virus (FWPV), modified vaccinia Ankara (MVA) and two novel South African avipoxviruses, Feral Pigeonpox virus (FeP2) and Penguinpox virus (PEPV). These six viruses produced qualitatively and quantitatively distinct host responses with LSDV, followed by MVA, inducing the greatest interferon (IFN) response. FeP2 and PEPV caused very little change to host transcript abundance compared to the other 4 viruses tested. CNPV and FWPV induced the up regulation of two immunoglobulin genes (Ighg and Ighg3 (IgG3)) with CNPV inducing a third, Ighm (IgM). HIV-1-specific IgG3 antibodies have been correlated with decreased risk of HIV-1 infection in the RV144 trial, which included a CNPV-based vector (Yates et al. (Sci Transl Med, 6(228) p228, 2014). Up regulation of IgG3 by CNPV and FWPV but not the other poxviruses tested in vivo, implies that these two avipoxvirus-vector backbones may be involved in stimulation of the clinically important IgG3 antibody subclass. Differential transcript abundance associated with the different poxviruses is further discussed with particular emphasis on responses related to immune responses. CONCLUSION: Six, genetically diverse host-restricted poxviruses produce different responses in a mouse model early after infection. These differences may affect the immune response induced to vaccine antigen in vectors based on these viruses. The two novel avipoxviruses were clearly distinguishable from the other viruses

Prevalence, hemodynamics, and cytokine profile of effusive-constrictive pericarditis in patients with tuberculous pericardial effusion

BACKGROUND: Effusive constrictive pericarditis (ECP) is visceral constriction in conjunction with compressive pericardial effusion. The prevalence of proven tuberculous ECP is unknown. Whilst ECP is distinguished from effusive disease on hemodynamic grounds, it is unknown whether effusive-constrictive physiology has a distinct cytokine profile. We conducted a prospective study of prevalence and cytokine profile of effusive-constrictive disease in patients with tuberculous pericardial effusion. METHODS: From July 2006 through July 2009, the prevalence of ECP and serum and pericardial levels of inflammatory cytokines were determined in adults with tuberculous pericardial effusion. The diagnosis of ECP was made by combined pericardiocentesis and cardiac catheterization. RESULTS: Of 91 patients evaluated, 68 had tuberculous pericarditis. The 36/68 patients (52.9%; 95% confidence interval [CI]: 41.2-65.4) with ECP were younger (29 versus 37 years, P=0.02), had a higher pre-pericardiocentesis right atrial pressure (17.0 versus 10.0 mmHg, P 15 mmHg (odds ratio [OR] = 48, 95%CI: 8.7-265; P 200 pg/ml (OR=10, 95%CI: 1.1, 93; P=0.04) were independently associated with ECP. CONCLUSION: Effusive-constrictive disease occurs in half of cases of tuberculous pericardial effusion, and is characterized by greater elevation in the pre-pericardiocentesis right atrial pressure and pericardial and serum IL-10 levels compared to patients with effusive non-constrictive tuberculous pericarditis

HIV-1 infection alters CD4 1 memory T-cell phenotype at the site of disease in extrapulmonary tuberculosis

HIV-1-infected people have an increased risk of developing extrapulmonary tuberculosis (TB), the immunopathogenesis of which is poorly understood. Here, we conducted a detailed immunological analysis of human pericardial TB, to determine the effect of HIV-1 co-infection on the phenotype of Mycobacterium tuberculosis (MTB)-specific memory T cells and the role of polyfunctional T cells at the disease site, using cells from pericardial fluid and blood of 74 patients with (n 5 50) and without (n 5 24) HIV-1 co-infection. The MTB antigen-induced IFN-c response was elevated at the disease site, irrespective of HIV-1 status or antigenic stimulant. However, the IFN-c ELISpot showed no clear evidence of increased numbers of antigen-specific cells at the disease site except for ESAT-6 in HIV-1 uninfected individuals (p 5 0.009). Flow cytometric analysis showed that CD4 1 memory T cells in the pericardial fluid of HIV-1-infected patients were of a less differentiated phenotype, with the presence of polyfunctional CD4 1 T cells expressing TNF, IL-2 and IFN-c. These results indicate that HIV-1 infection results in altered phenotype and function of MTB-specific CD4 1 T cells at the disease site, which may contribute to the increased risk of developing TB at all stages of HIV-1 infection

Poor Penetration of Antibiotics Into Pericardium in Pericardial Tuberculosis

Pericardial tuberculosis (TB) is associated with high therapy failure and high mortality rates. Antibiotics have to penetrate to site of infection at sufficient non-protein bound concentrations, and then enter bacteria to inhibit intracellular biochemical processes. The antibiotic concentrations achieved in pericardial fluid in TB pericarditis have never been measured before. We recruited two cohorts of patients with TB pericarditis, and left a pigtail catheter in-situ for serial drug concentration measurements over 24 h. Altogether, 704 drug concentrations were comodeled for pharmacokinetic analyses. The drug concentrations achieved in pericardial fluid were compared to the minimum inhibitory concentrations (MICs) of clinical Mycobacterium tuberculosis isolates. The total rifampicin concentration pericardial-to-serum ratios in 16 paired samples were 0.19 ± 0.33. The protein concentrations of the pericardial fluid in TB pericarditis were observed to be as high as in plasma. The non-protein bound rifampicin concentrations in pericardial fluid were 4-fold lower than rifampicin MICs in the pilot study, and the peak concentration was 0.125 versus 0.208 mg/L in the second (p = 0.001). The rifampicin clearance from pericardial fluid was 9.45 L/h versus 7.82 L/h in plasma (p = 0.002). Ethambutol peak concentrations had a pericardial-to-plasma ratio of 0.55 ± 0.22; free ethambutol peak concentrations were 2.30-lower than MICs (p < 0·001). The pericardial fluid pH was 7.34. The median pyrazinamide peak concentrations were 42.93 mg/L versus a median MIC of 800 mg/L at pH 7.34 (p < 0.0001). There was no significant difference between isoniazid pericardial fluid and plasma concentrations, and isoniazid peak concentrations were above MIC. This is the first study to measure anti-TB drug concentrations, pH and protein in the pericardial TB fluid. Pericardial concentrations of the key sterilizing drugs for TB were below MIC, which could contribute to poor outcomes. A new regimen that overcomes these limitations might need to be crafted

Tuberculous meningitis in children is characterized by compartmentalized immune responses and neural excitotoxicity

CITATION: Rohlwink, U. K., et al. 2019. Tuberculous meningitis in children is characterized by compartmentalized immune responses and neural excitotoxicity. Nature Communications, 10:3767, doi:10.1038/s41467-019-11783-9.The original publication is available at https://www.nature.comENGLISH ABSTRACT: Tuberculous meningitis (TBM) is the most severe form of TB with high rates of mortality and morbidity. Here we conduct RNA-sequencing on whole blood as well as on ventricular and lumbar cerebrospinal fluid (CSF) of pediatric patients treated for TBM. Differential transcript expression of TBM cases are compared with healthy controls in whole blood and with non-TB cerebral infection controls in CSF. Whole blood RNA-Seq analysis demonstrates a distinct immune response pattern in TBM, with significant increase in both canonical and non-canonical inflammasome activation and decrease in T-cell activation. In ventricular CSF, a significant enrichment associated with neuronal excitotoxicity and cerebral damage is detected in TBM. Finally, compartmental comparison in TBM indicates that the ventricular profile represents brain injury whereas the lumbar profile represents protein translation and cytokine signaling. Together, transcriptomic analysis shows that disease processes differ between the periphery and the central nervous system, and within brain compartments.https://www.nature.com/articles/s41467-019-11783-9Publisher's versio