Use of resuscitation promoting factors to screen for tuberculosis infection in household-exposed children in The Gambia.

BACKGROUND: Interferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children. Children often develop paucibacillary TB and their immune response differs from that of adults, which together affect TB disease diagnostics and immunodiagnostics. We assessed the ability of Rpf to identify infection among household TB-exposed children in The Gambia and investigated their ability to discriminate Mycobacterium tuberculosis complex (MTBC) infection from active TB disease in children. METHODS: Detailed clinical investigations were done on 93 household TB-exposed Gambian children and a tuberculin skin test (TST) was administered to asymptomatic children. Venous blood was collected for overnight stimulation with ESAT-6/CFP-10-fusion protein (EC), purified protein derivative and RpfA, B, C, D and E. Interferon gamma (IFN-γ) production was measured by ELISA in supernatants and corrected for the background level. Infection status was defined by IGRA with EC and TB disease by mycobacterial confirmation and/or clinical diagnosis. We compared IFN-γ levels between infected and uninfected children and between infected and TB diseased children using a binomial logistic regression model while correcting for age and sex. A Receiver Operating Characteristics analysis was done to find the best cut-off for IFN-γ level and calculate sensitivity and specificity. RESULTS: Interferon gamma production was significantly higher in infected (IGRA+, n = 45) than in uninfected (IGRA-, n = 20) children after stimulation with RpfA, B, C, and D (P = 0.03; 0.007; 0.03 and 0.003, respectively). Using RpfB and D-specific IFN-γ cut-offs (33.9 pg/mL and 67.0 pg/mL), infection was classified with a sensitivity-specificity combination of 73-92% and 77-72% respectively, which was similar to and better than 65-75% for TST. Moreover, IFN-γ production was higher in infected than in TB diseased children (n = 28, 5 bacteriologically confirmed, 23 clinically diagnosed), following RpfB and D stimulation (P = 0.02 and 0.03, respectively). CONCLUSION: RpfB and RpfD show promising results for childhood MTBC infection screening, and both performed similar to and better than the TST in our study population. Additionally, both antigens appear to discriminate between infection and disease in children and thus warrant further investigation as screening and diagnostic antigens for childhood TB.

Differential transcriptomic and metabolic profiles of M. africanum- and M. tuberculosis-infected patients after, but not before, drug treatment.

The epidemiology of Mycobacterium tuberculosis (Mtb) and M. africanum (Maf) suggests differences in their virulence, but the host immune profile to better understand the pathogenesis of tuberculosis (TB) have not been studied. We compared the transcriptomic and metabolic profiles between Mtb- and Maf-infected TB cases to identify host biomarkers associated with lineages-specific pathogenesis and response to anti-TB chemotherapy. Venous blood samples from Mtb- and Maf-infected patients obtained before and after anti-TB treatment were analyzed for cell composition, gene expression and metabolic profiles. Prior to treatment, similar transcriptomic profiles were seen in Maf- and Mtb-infected patients. In contrast, post treatment, over 1600 genes related to immune responses and metabolic diseases were differentially expressed between the groups. Notably, the upstream regulator hepatocyte nuclear factor 4-alpha (HNF4α), which regulated 15% of these genes, was markedly enriched. Serum metabolic profiles were similar in both group pre-treatment, but the decline in pro-inflammatory metabolites post treatment were most pronounced in Mtb-infected patients. Together, the differences in both peripheral blood transcriptomic and serum metabolic profiles between Maf- and Mtb-infected patients observed over the treatment period, might be indicative of intrinsic host factors related to susceptibility to TB and/or differential efficacy of the standard anti-TB treatment on the two lineages.

Differential gene expression of activating Fcγ receptor classifies active tuberculosis regardless of human immunodeficiency virus status or ethnicity.

New diagnostics and vaccines for tuberculosis (TB) are urgently needed, but require an understanding of the requirements for protection from/susceptibility to TB. Previous studies have used unbiased approaches to determine gene signatures in single-site populations. The present study utilized a targeted approach, reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), to validate these genes in a multisite study. We analysed ex vivo whole blood RNA from a total of 523 participants across four sub-Saharan countries (Ethiopia, Malawi, South Africa, and The Gambia) with differences in TB and human immunodeficiency virus (HIV) status. We found a number of genes that were expressed at significantly lower levels in participants with active disease than in those with latent TB infection (LTBI), with restoration following successful TB treatment. The most consistent classifier of active disease was FCGR1A (high-affinity IgG Fc receptor 1 (CD64)), which was the only marker expressed at significantly higher levels in participants with active TB than in those with LTBI before treatment regardless of HIV status or genetic background. This is the first study to identify a biomarker for TB that is not affected by HIV status or geo-genetic differences. These data provide valuable clues for understanding TB pathogenesis, and also provide a proof-of-concept for the use of RT-MLPA in rapid and inexpensive validation of unbiased gene expression findings.

Identification of biomarkers for tuberculosis disease using a novel dual-color RT-MLPA assay.

Owing to our lack of understanding of the factors that constitute protective immunity during natural infection with Mycobacterium tuberculosis (Mtb), there is an urgent need to identify host biomarkers that predict long-term outcome of infection in the absence of therapy. Moreover, the identification of host biomarkers that predict (in)adequate response to tuberculosis (TB) treatment would similarly be a major step forward. To identify/monitor multi-component host biomarker signatures at the transcriptomic level in large human cohort studies, we have developed and validated a dual-color reverse-transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) method, permitting rapid and accurate expression profiling of as many as 60-80 transcripts in a single reaction. dcRT-MLPA is sensitive, highly reproducible, high-throughput, has an extensive dynamic range and is as quantitative as QPCR. We have used dcRT-MLPA to characterize the human immune response to Mtb in several cohort studies in two genetically and geographically diverse populations. A biomarker signature was identified that is strongly associated with active TB disease, and was profoundly distinct from that associated with treated TB disease, latent infection or uninfected controls, demonstrating the discriminating power of our biomarker signature. Identified biomarkers included apoptosis-related genes and T-cell/B-cell markers, suggesting important contributions of adaptive immunity to TB pathogenesis.

Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells.

BACKGROUND: Serpins form a widely distributed protein superfamily, but no integral membrane serpins have been described. OBJECTIVES: To anchor three serpins -α(1) -proteinase inhibitor (α(1) PI) (M358R), antithrombin (AT), and heparin cofactor II (HCII) - in the plasma membranes of transfected mammalian cells and assess their ability to inhibit thrombin. METHODS: Serpin cDNAs were altered to include N-terminal, non-cleavable plasma membrane-targeting sequences from the human transferrin receptor (TR) (TR-serpin) or the human asialoglycoprotein receptor (AR) (AR-serpin), and used to transfect COS-1 or HEK 293 cells. Cells were analyzed for serpin expression by immunoblotting of subcellular fractions, by immunofluorescence microscopy, or by flow cytometry, with or without exposure to exogenous thrombin; AR-serpins and TR-serpins were also compared with their soluble recombinant counterparts. RESULTS: Both TR-α(1) PI (M358R) and AR-α(1) PI (M358R) were enriched in the integral membrane fraction of transfected COS-1 or HEK 293 cells, and formed inhibitory complexes with thrombin, although less rapidly than soluble α(1) PI (M358R). Thrombin inhibition was abrogated by an additional T345R mutation in AR-α(1) PI (M358R). Surface-displayed AR-AT also formed serpin-enzyme complexes with thrombin, but to a lesser extent than AR-α(1) PI (M358R); AR-HCII inhibitory function was not detected. Immunofluorescence detection and flow cytometric quantification of bound thrombin also supported the status of AR-α(1) PI (M358R) and AR-AT as thrombin inhibitors. CONCLUSIONS: Two of three thrombin-inhibitory serpins retained functionality when expressed as integral membrane proteins. Our findings could be applied to create and screen hypervariable serpin libraries expressed in mammalian cells, or to confer protease resistance on engineered cells in vivo.

Antibody and T-cell responses during acute and convalescent stages of invasive pneumococcal disease.

OBJECTIVE: To understand the pattern of immune responses to pneumococcal proteins during invasive disease as a guide to their development as vaccine candidates. METHODS: The antibody concentration and avidity, as well as frequency of interferon-gamma (IFN-γ)-, interleukin-10 (IL-10)-, and tumor necrosis factor-alpha (TNF-α)-containing CD4+ T-lymphocytes in response to pneumolysin, pneumococcal surface protein A (PspA), and choline-binding protein A (CbpA), during and after invasive pneumococcal disease (IPD) in 20 children were compared to those of 20 healthy matched controls. RESULTS: During the acute phase of IPD, the concentrations of antibodies against these three pneumococcal proteins were lower, whereas the frequencies of IL-10- and TNF-α-producing CD4+ T-cells were higher, compared to values obtained during convalescence and in healthy controls (p < 0.01). In addition, the concentrations of antibodies against the capsular polysaccharides for the serotypes isolated from these patients, were all below the detection level of the assay during both the acute and convalescent phases of IPD. CONCLUSION: These data indicate that the recognition of these antigens by the immune system occurs in variable proportions according to the stage of infection, implying the important role of these in the pathogenesis of IPD, and support their usefulness in vaccine development.