Whole blood RNA signatures in tuberculosis patients receiving H56:IC31 vaccine as adjunctive therapy.

Introduction: Therapeutic vaccination in tuberculosis (TB) represents a Host Directed Therapy strategy which enhances immune responses in order to improve clinical outcomes and shorten TB treatment. Previously, we have shown that the subunit H56:IC31 vaccine induced both humoral and cellular immune responses when administered to TB patients adjunctive to standard TB treatment (TBCOX2 study, NCT02503839). Here we present the longitudinal whole blood gene expression patterns in H56:IC31 vaccinated TB patients compared to controls receiving standard TB treatment only. Methods: The H56:IC31 group (N=11) and Control group (N=7) underwent first-line TB treatment for 182 days. The H56:IC31 group received 5 micrograms of the H56:IC31 vaccine (Statens Serum Institut; SSI, Valneva Austria GmbH) intramuscularly at day 84 and day 140. Total RNA was extracted from whole blood samples collected in PAXgene tubes on days 0, 84, 98, 140, 154, 182 and 238. The expression level of 183 immune-related genes was measured by high-throughput microfluidic qPCR (Biomark HD system, Standard BioTools). Results: The targeted gene expression profiling unveiled the upregulation of modules such as interferon (IFN) signalling genes, pattern recognition receptors and small nucleotide guanosine triphosphate (GTP)-ases in the vaccinated group compared to controls two weeks after administration of the first H56:IC31 vaccine. Additionally, the longitudinal analysis of the Adolescent Cohort Study-Correlation of Risk (ACS-COR) signature showed a progressive downregulation in both study arms towards the end of TB treatment, in congruence with reported treatment responses and clinical improvements. Still, two months after the end of TB treatment, vaccinated patients, and especially those developing both cellular and humoral vaccine responses, showed a lower expression of the ACS-COR genes compared to controls. Discussion: Our data report gene expression patterns following H56:IC31 vaccination which might be interpreted as a lower risk of relapse in therapeutically vaccinated patients. Further studies are needed to conclude if these gene expression patterns could be used as prognostic biosignatures for therapeutic TB vaccine responses.

Baseline gene signatures of reactogenicity to Ebola vaccination: a machine learning approach across multiple cohorts.

Introduction: The rVSVDG-ZEBOV-GP (Ervebo®) vaccine is both immunogenic and protective against Ebola. However, the vaccine can cause a broad range of transient adverse reactions, from headache to arthritis. Identifying baseline reactogenicity signatures can advance personalized vaccinology and increase our understanding of the molecular factors associated with such adverse events. Methods: In this study, we developed a machine learning approach to integrate prevaccination gene expression data with adverse events that occurred within 14 days post-vaccination. Results and Discussion: We analyzed the expression of 144 genes across 343 blood samples collected from participants of 4 phase I clinical trial cohorts: Switzerland, USA, Gabon, and Kenya. Our machine learning approach revealed 22 key genes associated with adverse events such as local reactions, fatigue, headache, myalgia, fever, chills, arthralgia, nausea, and arthritis, providing insights into potential biological mechanisms linked to vaccine reactogenicity.

Impaired resolution of blood transcriptomes through tuberculosis treatment with diabetes comorbidity.

BACKGROUND: People with diabetes are more likely to develop tuberculosis (TB) and to have poor TB-treatment outcomes than those without. We previously showed that blood transcriptomes in people with TB-diabetes (TB-DM) co-morbidity have excessive inflammatory and reduced interferon responses at diagnosis. It is unknown whether this persists through treatment and contributes to the adverse outcomes. METHODS: Pulmonary TB patients recruited in South Africa, Indonesia and Romania were classified as having TB-DM, TB with prediabetes, TB-related hyperglycaemia or TB-only, based on glycated haemoglobin concentration at TB diagnosis and after 6 months of TB treatment. Gene expression in blood at diagnosis and intervals throughout treatment was measured by unbiased RNA-Seq and targeted Multiplex Ligation-dependent Probe Amplification. Transcriptomic data were analysed by longitudinal mixed-model regression to identify whether genes were differentially expressed between clinical groups through time. Predictive models of TB-treatment response across groups were developed and cross-tested. RESULTS: Gene expression differed between TB and TB-DM patients at diagnosis and was modulated by TB treatment in all clinical groups but to different extents, such that differences remained in TB-DM relative to TB-only throughout. Expression of some genes increased through TB treatment, whereas others decreased: some were persistently more highly expressed in TB-DM and others in TB-only patients. Genes involved in innate immune responses, anti-microbial immunity and inflammation were significantly upregulated in people with TB-DM throughout treatment. The overall pattern of change was similar across clinical groups irrespective of diabetes status, permitting models predictive of TB treatment to be developed. CONCLUSIONS: Exacerbated transcriptome changes in TB-DM take longer to resolve during TB treatment, meaning they remain different from those in uncomplicated TB after treatment completion. This may indicate a prolonged inflammatory response in TB-DM, requiring prolonged treatment or host-directed therapy for complete cure. Development of transcriptome-based biomarker signatures of TB-treatment response should include people with diabetes for use across populations.

Differences in Inflammatory Pathways Between Dutch South Asians vs Dutch Europids With Type 2 Diabetes.

CONTEXT: South Asian individuals are more prone to develop type 2 diabetes (T2D) coinciding with earlier complications than Europids. While inflammation plays a central role in the development and progression of T2D, this factor is still underexplored in South Asians. OBJECTIVE: This work aimed to study whether circulating messenger RNA (mRNA) transcripts of immune genes are different between South Asian compared with Europid patients with T2D. METHODS: A secondary analysis was conducted of 2 randomized controlled trials of Dutch South Asian (n = 45; age: 55 ± 10 years, body mass index [BMI]: 29 ± 4 kg/m2) and Dutch Europid (n = 44; age: 60 ± 7 years, BMI: 32 ± 4 kg/m2) patients with T2D. Main outcome measures included mRNA transcripts of 182 immune genes (microfluidic quantitative polymerase chain reaction; Fluidigm Inc) in fasted whole-blood, ingenuity pathway analyses (Qiagen). RESULTS: South Asians, compared to Europids, had higher mRNA levels of B-cell markers (CD19, CD79A, CD79B, CR2, CXCR5, IGHD, MS4A1, PAX5; all fold change > 1.3, false discovery rate [FDR] < 0.008) and interferon (IFN)-signaling genes (CD274, GBP1, GBP2, GBP5, FCGR1A/B/CP, IFI16, IFIT3, IFITM1, IFITM3, TAP1; all FC > 1.2, FDR < 0.05). In South Asians, the IFN signaling pathway was the top canonical pathway (z score 2.6; P < .001) and this was accompanied by higher plasma IFN-γ levels (FC = 1.5, FDR = 0.01). Notably, the ethnic difference in gene expression was larger for women (20/182 [11%]) than men (2/182 [1%]). CONCLUSION: South Asian patients with T2D show a more activated IFN-signaling pathway compared to Europid patients with T2D, which is more pronounced in women than men. We speculate that a more activated IFN-signaling pathway may contribute to the more rapid progression of T2D in South Asian compared with Europid individuals.

Global blood miRNA profiling unravels early signatures of immunogenicity of Ebola vaccine rVSVΔG-ZEBOV-GP.

The vectored Ebola vaccine rVSVΔG-ZEBOV-GP elicits protection against Ebola Virus Disease (EVD). In a study of forty-eight healthy adult volunteers who received either the rVSVΔG-ZEBOV-GP vaccine or placebo, we profiled intracellular microRNAs (miRNAs) from whole blood cells (WB) and circulating miRNAs from serum-derived extracellular vesicles (EV) at baseline and longitudinally following vaccination. Further, we identified early miRNA signatures associated with ZEBOV-specific IgG antibody responses at baseline and up to one year post-vaccination, and pinpointed target mRNA transcripts and pathways correlated to miRNAs whose expression was altered after vaccination by using systems biology approaches. Several miRNAs were differentially expressed (DE) and miRNA signatures predicted high or low IgG ZEBOV-specific antibody levels with high classification performance. The top miRNA discriminators were WB-miR-6810, EV-miR-7151-3p, and EV-miR-4426. An eight-miRNA antibody predictive signature was associated with immune-related target mRNAs and pathways. These findings provide valuable insights into early blood biomarkers associated with rVSVΔG-ZEBOV-GP vaccine-induced IgG antibody responses.

Transcriptional profiles predict treatment outcome in patients with tuberculosis and diabetes at diagnosis and at two weeks after initiation of anti-tuberculosis treatment.

BACKGROUND: Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients. Treatment success is lower among people with diabetes mellitus (DM). Predicting treatment outcome early after diagnosis, especially in TB-DM patients, would allow early treatment adaptation for individuals and may improve global TB control. METHODS: Samples were collected in a longitudinal cohort study of adult TB patients from South Africa (n  =  94) and Indonesia (n  =  81), who had concomitant DM (n  =  59), intermediate hyperglycaemia (n  =  79) or normal glycaemia/no DM (n  =  37). Treatment outcome was monitored, and patients were categorized as having a good (cured) or poor (failed, recurrence, died) outcome during treatment and 12 months follow-up. Whole blood transcriptional profiles before, during and at the end of TB treatment were characterized using unbiased RNA-Seq and targeted gene dcRT-MLPA. FINDINGS: We report differences in whole blood transcriptome profiles, which were observed before initiation of treatment and throughout treatment, between patients with a good versus poor TB treatment outcome. An eight-gene and a 22-gene blood transcriptional signature distinguished patients with a good TB treatment outcome from patients with a poor TB treatment outcome at diagnosis (AUC = 0·815) or two weeks (AUC = 0·834) after initiation of TB treatment, respectively. High accuracy was obtained by cross-validating this signature in an external cohort (AUC = 0·749). INTERPRETATION: These findings suggest that transcriptional profiles can be used as a prognostic biomarker for treatment failure and success, even in patients with concomitant DM. FUNDING: The research leading to these results, as part of the TANDEM Consortium, received funding from the European Community's Seventh Framework Programme (FP7/2007-2013 Grant Agreement No. 305279) and the Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038). The research leading to the results presented in the Indian validation cohort was supported by Research Council of Norway Global Health and Vaccination Research (GLOBVAC) projects: RCN 179342, 192534, and 248042, the University of Bergen (Norway).

Transcriptomic signatures induced by the Ebola virus vaccine rVSVΔG-ZEBOV-GP in adult cohorts in Europe, Africa, and North America: a molecular biomarker study.

BACKGROUND: A recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine has been reported as safe, immunogenic, and highly protective in a ring vaccination trial. We aimed to identify transcriptomic immune response biomarker signatures induced by vaccination and associated signatures with its immunogenicity and reactogenicity to better understand the potential mechanisms of action of the vaccine. METHODS: 354 healthy adult volunteers were vaccinated in randomised, double-blind, placebo-controlled trials in Europe (Geneva, Switzerland [November, 2014, to January, 2015]) and North America (USA [Dec 5, 2014, to June 23, 2015]), and dose-escalation trials in Africa (Lambaréné, Gabon [November, 2014, to January, 2015], and Kilifi, Kenya [December, 2014, to January, 2015]) using different doses of the recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP; 3 × 105 to 1 × 108 plaque-forming units [pfu]). Longitudinal transcriptomic responses (days 0, 1, 2, 3, 7, 14, and 28) were measured in whole blood using a targeted gene expression profiling platform (dual-colour reverse-transcriptase multiplex ligation-dependent probe amplification) focusing on 144 immune-related genes. The effect of time and dose on transcriptomic response was also assessed. Logistic regression with lasso regularisation was applied to identify host signatures with optimal discriminatory capability of vaccination at day 1 or day 7 versus baseline, whereas random-effects models and recursive feature elimination combined with regularised logistic regression were used to associate signatures with immunogenicity and reactogenicity. FINDINGS: Our results indicated that perturbation of gene expression peaked on day 1 and returned to baseline levels between day 7 and day 28. The magnitude of the response was dose-dependent, with vaccinees receiving a high dose (≥9 × 106 pfu) of rVSVΔG-ZEBOV-GP exhibiting the largest amplitude. The most differentially expressed genes that were significantly upregulated following vaccination consisted of type I and II interferon-related genes and myeloid cell-associated markers, whereas T cell, natural killer cell, and cytotoxicity-associated genes were downregulated. A gene signature associated with immunogenicity (common to all four cohorts) was identified correlating gene expression profiles with ZEBOV-GP antibody titres and a gene signatures associated with reactogenicity (Geneva cohort) was identified correlating gene expression profiles with an adverse event (ie, arthritis). INTERPRETATION: Collectively, our results identify and cross-validate immune-related transcriptomic signatures induced by rVSVΔG-ZEBOV-GP vaccination in four cohorts of adult participants from different genetic and geographical backgrounds. These signatures will aid in the rational development, testing, and evaluation of novel vaccines and will allow evaluation of the effect of host factors such as age, co-infection, and comorbidity on responses to vaccines. FUNDING: Innovative Medicines Initiative 2 Joint Undertaking.

Histone acetylation as a new mechanism for bilirubin-induced encephalopathy in the Gunn rat.

Bilirubin neurotoxicity has been studied for decades and has been shown to affect various mechanisms via significant modulation of gene expression. This suggests that vital regulatory mechanisms of gene expression, such as epigenetic mechanisms, could play a role in bilirubin neurotoxicity. Histone acetylation has recently received attention in the CNS due to its role in gene modulation for numerous biological processes, such as synaptic plasticity, learning, memory, development and differentiation. Aberrant epigenetic regulation of gene expression in psychiatric and neurodegenerative disorders has also been described. In this work, we followed the levels of histone 3 lysine 14 acetylation (H3K14Ac) in the cerebellum (Cll) of the developing (2, 9, 17 days after the birth) and adult Gunn rat, the natural model for neonatal hyperbilirubinemia and kernicterus. We observed an age-specific alteration of the H3K14Ac in the hyperbilirubinemic animals. The GeneOntology analysis of the H3K14Ac linked chromatin revealed that almost 45% of H3K14Ac ChiP-Seq TSS-promoter genes were involved in CNS development including maturation and differentiation, morphogenesis, dendritogenesis, and migration. These data suggest that the hallmark Cll hypoplasia in the Gunn rat occurs also via epigenetically controlled mechanisms during the maturation of this brain structure, unraveling a novel aspect of the bilirubin-induced neurotoxicity.

The Biological Effects of Bilirubin Photoisomers.

Although phototherapy was introduced as early as 1950's, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.

Life-long correction of hyperbilirubinemia with a neonatal liver-specific AAV-mediated gene transfer in a lethal mouse model of Crigler-Najjar Syndrome.

Null mutations in the UGT1A1 gene result in Crigler-Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies, we applied AAV liver-specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months postinjection. The therapeutic effect was mediated by the presence of transcriptionally active double-stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver versus skeletal muscle transgene expression. We observed that 5-8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1±1.5 mg/dl). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4±2.0 mg/dl), despite 20-30% of hUgt1a1 expression levels, compared with normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters.

Alterations in the cell cycle in the cerebellum of hyperbilirubinemic Gunn rat: a possible link with apoptosis?

Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.