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1.  Safety and Immunogenicity of the Recombinant BCG Vaccine AERAS-422 in Healthy BCG-naïve Adults: A Randomized, Active-controlled, First-in-human Phase 1 Trial 
EBioMedicine  2016;7:278-286.
We report a first-in-human trial evaluating safety and immunogenicity of a recombinant BCG, AERAS-422, over-expressing TB antigens Ag85A, Ag85B, and Rv3407 and expressing mutant perfringolysin.
This was a randomized, double-blind, dose-escalation trial in HIV-negative, healthy adult, BCG-naïve volunteers, negative for prior exposure to Mtb, at one US clinical site. Volunteers were randomized 2:1 at each dose level to receive a single intradermal dose of AERAS-422 (> 105–< 106 CFU = low dose, ≥ 106– < 107 CFU = high dose) or non-recombinant Tice BCG (1–8 × 105 CFU). Randomization used an independently prepared randomly generated sequence of treatment assignments. The primary and secondary outcomes were safety and immunogenicity, respectively, assessed in all participants through 182 days post-vaccination. registration number: NCT01340820.
Between Nov 2010 and Aug 2011, 24 volunteers were enrolled (AERAS-422 high dose, n = 8; AERAS-422 low dose, n = 8; Tice BCG, n = 8); all were included in the safety and immunogenicity analyses. All 24 subjects had at least one adverse event, primarily expected local reactions. High dose AERAS-422 vaccination induced Ag85A- and Ag85B-specific lymphoproliferative responses and marked anti-mycobacterial activity in a whole blood bactericidal activity culture assay (WBA), but was associated with varicella zoster virus (VZV) reactivation in two vaccinees. These volunteers displayed high BCG-specific IFN-γ responses pre- and post-vaccination possibly predisposing them to autocrine/paracrine negative regulation of immune control of latent VZV. A systems biology transcriptomal approach identified positive correlations between post-vaccination T cell expression modules and WBA, and negative correlations between post-vaccination monocyte expression modules and WBA. The expression of one key macrophage marker (F4/80) was constitutively elevated in the two volunteers with zoster.
The unexpected development of VZV in two of eight healthy adult vaccine recipients resulted in discontinuation of AERAS-422 vaccine development. Immunological and transcriptomal data identified correlations with the development of TB immunity and VZV that require further investigation.
Aeras, FDA, Bill and Melinda Gates Foundation.
•Novel rBCG first-in-human trial overexpressing 3Mtb Ags induces a more potent human immune response than standard BCG.•Transcriptomal studies demonstrated correlations between mycobacterial growth inhibition growth and potent T cell responses.•rBCG was associated with varicella-zoster reactivation and associated with high interferon gamma and EMR1 expression.A first-in-human trial evaluating safety and immune responses of a recombinant BCG vaccine was reported. The new vaccine expresses additional TB antigens and escapes from seclusion to induce a better immune response in CD8 T cells. The immune response produced in humans could inhibit the growth of the TB pathogen. Unfortunately the vaccine was associated with shingles and studies were done to explore the reasons why that occurred.
PMCID: PMC4909487  PMID: 27322481
Tuberculosis;; Recombinant BCG;; Herpes zoster;; Transcriptomes;; Functional T cell assays
2.  Interleukin 27R regulates CD4+ T cell phenotype and impacts protective immunity during Mycobacterium tuberculosis infection 
The Journal of Experimental Medicine  2015;212(9):1449-1463.
Loss of IL-27R on T cells results in increased protection from Mycobacterium tuberculosis. Torrado et al. demonstrate that IL-27R−/− T cells show improved fitness that is associated with decreased expression of cell death molecules, maintenance of IL-2 production, and preferential accumulation in the lung parenchyma and around infected macrophages.
CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra−/− mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R–deficient T cells is not associated with increased proliferation but with decreased expression of cell death–associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R–deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB.
PMCID: PMC4548054  PMID: 26282876
3.  Antigen expression determines adenoviral vaccine potency independent of IFN and STING signaling 
The Journal of Clinical Investigation  2015;125(3):1129-1146.
Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee-derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines.
PMCID: PMC4362254  PMID: 25642773
4.  Systems-Level Analysis of Innate Immunity 
Annual review of immunology  2014;32:547-577.
Systems-level analysis of biological processes strives to comprehensively and quantitatively evaluate the interactions between the relevant molecular components over time, thereby enabling development of models that can be employed to ultimately predict behavior. Rapid development in measurement technologies (omics), when combined with the accessible nature of the cellular constituents themselves, is allowing the field of innate immunity to take significant strides toward this lofty goal. In this review, we survey exciting results derived from systems biology analyses of the immune system, ranging from gene regulatory networks to influenza pathogenesis and systems vaccinology.
PMCID: PMC4084718  PMID: 24655298
systems biology; transcriptomics; proteomics; lipidomics; influenza; systems vaccinology
5.  Mouse NAIP1 detects the type III secretion system needle protein 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(8):10.4049/jimmunol.1301549.
The NAIP/NLRC4 inflammasomes activate caspase-1 in response to bacterial type III secretion systems (T3SS). Inadvertent injection of the T3SS rod protein and flagellin into the cytosol are detected through murine NAIP2 and NAIP5/6, respectively. Here, we identify the agonist for the orphan murine NAIP1 receptor as the T3SS needle protein. NAIP1 is poorly expressed in resting mouse bone marrow-derived macrophages (BMMs), however, priming with poly(I:C) induces it, and confers needle protein sensitivity. Further, overexpression of NAIP1 in immortalized BMMs by retroviral transduction enabled needle detection. In contrast, peritoneal cavity macrophages basally express NAIP1 and respond to needle protein robustly independent of priming. Human macrophages are known to only express one NAIP gene, which detects the needle protein, but not rod or flagellin. Thus, murine NAIP1 is functionally analogous to human NAIP.
PMCID: PMC3819181  PMID: 24043898
Naip1; NLRC4; inflammasomes; T3SS; needle
6.  Caspase-11 protects against bacteria that escape the vacuole 
Science (New York, N.Y.)  2013;339(6122):975-978.
Caspases are either apoptotic or inflammatory. The inflammatory Caspases-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory disease, only Caspase-1 has an established protective role during infection. Herein, we report that Caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. While Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) that aberrantly enter the cytosol triggered Caspase-11, enhancing clearance of S. typhimurium sifA in vivo. This response did not require NLRP3, NLRC4, or ASC inflammasome pathways. Burkholderia species that naturally invade the cytosol also triggered Caspase-11, protecting mice from lethal challenge with B. thailandensis and B. pseudomallei. Thus, Caspase-11 is critical for surviving exposure to ubiquitous environmental pathogens.
PMCID: PMC3697099  PMID: 23348507
7.  A systems view of host defense 
Nature biotechnology  2009;27(11):999-1001.
Large-scale perturbations unravel the complex networks of activated dendritic cells.
PMCID: PMC3076594  PMID: 19898453
8.  Genome-wide histone acetylation data improve prediction of mammalian transcription factor binding sites 
Bioinformatics  2010;26(17):2071-2075.
Motivation: Histone acetylation (HAc) is associated with open chromatin, and HAc has been shown to facilitate transcription factor (TF) binding in mammalian cells. In the innate immune system context, epigenetic studies strongly implicate HAc in the transcriptional response of activated macrophages. We hypothesized that using data from large-scale sequencing of a HAc chromatin immunoprecipitation assay (ChIP-Seq) would improve the performance of computational prediction of binding locations of TFs mediating the response to a signaling event, namely, macrophage activation.
Results: We tested this hypothesis using a multi-evidence approach for predicting binding sites. As a training/test dataset, we used ChIP-Seq-derived TF binding site locations for five TFs in activated murine macrophages. Our model combined TF binding site motif scanning with evidence from sequence-based sources and from HAc ChIP-Seq data, using a weighted sum of thresholded scores. We find that using HAc data significantly improves the performance of motif-based TF binding site prediction. Furthermore, we find that within regions of high HAc, local minima of the HAc ChIP-Seq signal are particularly strongly correlated with TF binding locations. Our model, using motif scanning and HAc local minima, improves the sensitivity for TF binding site prediction by ∼50% over a model based on motif scanning alone, at a false positive rate cutoff of 0.01.
Availability: The data and software source code for model training and validation are freely available online at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2922897  PMID: 20663846
9.  Role of the transcription factor C/EBPδ in a regulatory circuit that discriminates between transient and persistent Toll-like receptor 4-induced signals 
Nature immunology  2009;10(4):437-443.
The innate immune system is a two-edged sword; it is absolutely required for host defense against infection but, uncontrolled, can trigger a plethora of inflammatory diseases. Here we used systems biology approaches to predict and validate a gene regulatory network involving a dynamic interplay between the transcription factors NF-κB, C/EBPδ, and ATF3 that controls inflammatory responses. We mathematically modeled transcriptional regulation of Il6 and Cebpd genes and experimentally validated the prediction that the combination of an initiator (NF-κB), an amplifier (C/EBPδ) and an attenuator (ATF3) forms a regulatory circuit that discriminates between transient and persistent Toll-like receptor 4-induced signals. Our results suggest a mechanism that enables the innate immune system to detect the duration of infection and to respond appropriately.
PMCID: PMC2780024  PMID: 19270711
10.  Systems biology of innate immunity 
Immunological reviews  2009;227(1):264-282.
Systems biology is the comprehensive and quantitative analysis of the interactions between all of the components of biological systems over time. Systems biology involves an iterative cycle, in which emerging biological problems drive the development of new technologies and computational tools. These technologies and tools then open new frontiers that revolutionize biology. Innate immunity is well suited for systems analysis, because the relevant cells can be isolated in various functional states and their interactions can be reconstituted in a biologically meaningful manner. Application of the tools of systems biology to the innate immune system will enable comprehensive analysis of the complex interactions that maintain the difficult balance between host defense and inflammatory disease. In this review, we discuss innate immunity in the context of the systems biology concepts, emergence, robustness, and modularity, and we describe emerging technologies we are applying in our systems-level analyses. These technologies include genomics, proteomics, computational analysis, forward genetics screens, and analyses that link human genetic polymorphisms to disease resistance.
PMCID: PMC2697920  PMID: 19120490
systems biology; innate immunity; Toll-like receptors; gene regulation; genomics; proteomics
11.  Quantifying gene network connectivity in silico: Scalability and accuracy of a modular approach 
Systems biology  2006;153(4):236-246.
Large, complex datasets that are generated from microarray experiments create a need for systematic analysis techniques to unravel the underlying connectivity of gene regulatory networks. A modular approach, previously proposed by Kholodenko and co-workers, helps to scale down the network complexity into more computationally manageable entities called modules. A functional module includes a gene’s mRNA, promoter and resulting products, thus encompassing a large set of interacting states. The essential elements of this approach are described in detail for a three-gene model network and later extended to a ten-gene model network, demonstrating scalability. The network architecture is identified by analyzing in silico steady-state changes in the activities of only the module outputs -communicating intermediates- that result from specific perturbations applied to the network modules one at a time. These steady-state changes form the system response matrix, which is used to compute the network connectivity or network interaction map. By employing a known biochemical network, we are able to evaluate the accuracy of the modular approach and its sensitivity to key assumptions.
PMCID: PMC2346590  PMID: 16986625
gene networks; reverse engineering; modular approach; connection coefficients
14.  Uncovering a Macrophage Transcriptional Program by Integrating Evidence from Motif Scanning and Expression Dynamics 
PLoS Computational Biology  2008;4(3):e1000021.
Macrophages are versatile immune cells that can detect a variety of pathogen-associated molecular patterns through their Toll-like receptors (TLRs). In response to microbial challenge, the TLR-stimulated macrophage undergoes an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network poses significant challenges and requires the integration of multiple experimental data types. In this work, we inferred a transcriptional network underlying TLR-stimulated murine macrophage activation. Microarray-based expression profiling and transcription factor binding site motif scanning were used to infer a network of associations between transcription factor genes and clusters of co-expressed target genes. The time-lagged correlation was used to analyze temporal expression data in order to identify potential causal influences in the network. A novel statistical test was developed to assess the significance of the time-lagged correlation. Several associations in the resulting inferred network were validated using targeted ChIP-on-chip experiments. The network incorporates known regulators and gives insight into the transcriptional control of macrophage activation. Our analysis identified a novel regulator (TGIF1) that may have a role in macrophage activation.
Author Summary
Macrophages play a vital role in host defense against infection by recognizing pathogens through pattern recognition receptors, such as the Toll-like receptors (TLRs), and mounting an immune response. Stimulation of TLRs initiates a complex transcriptional program in which induced transcription factor genes dynamically regulate downstream genes. Microarray-based transcriptional profiling has proved useful for mapping such transcriptional programs in simpler model organisms; however, mammalian systems present difficulties such as post-translational regulation of transcription factors, combinatorial gene regulation, and a paucity of available gene-knockout expression data. Additional evidence sources, such as DNA sequence-based identification of transcription factor binding sites, are needed. In this work, we computationally inferred a transcriptional network for TLR-stimulated murine macrophages. Our approach combined sequence scanning with time-course expression data in a probabilistic framework. Expression data were analyzed using the time-lagged correlation. A novel, unbiased method was developed to assess the significance of the time-lagged correlation. The inferred network of associations between transcription factor genes and co-expressed gene clusters was validated with targeted ChIP-on-chip experiments, and yielded insights into the macrophage activation program, including a potential novel regulator. Our general approach could be used to analyze other complex mammalian systems for which time-course expression data are available.
PMCID: PMC2265556  PMID: 18369420
15.  Systems analysis of circadian time-dependent neuronal epidermal growth factor receptor signaling 
Genome Biology  2006;7(6):R48.
A systems level analysis of circadian time-dependent signaling via the epidermal growth factor receptor in the suprachiasmatic nucleus suggests several transcription factors that mediate the transcriptional response to epidermal growth factor receptor signaling.
Identifying the gene regulatory networks governing physiological signal integration remains an important challenge in circadian biology. Epidermal growth factor receptor (EGFR) has been implicated in circadian function and is expressed in the suprachiasmatic nuclei (SCN), the core circadian pacemaker. The transcription networks downstream of EGFR in the SCN are unknown but, by analogy to other SCN inputs, we expect the response to EGFR activation to depend on circadian timing.
We have undertaken a systems-level analysis of EGFR circadian time-dependent signaling in the SCN. We collected gene-expression profiles to study how the SCN response to EGFR activation depends on circadian timing. Mixed-model analysis of variance (ANOVA) was employed to identify genes with circadian time-dependent EGFR regulation. The expression data were integrated with transcription-factor binding predictions through gene group enrichment analyses to generate robust hypotheses about transcription-factors responsible for the circadian phase-dependent EGFR responses.
The analysis results suggest that the transcriptional response to EGFR signaling in the SCN may be partly mediated by established transcription-factors regulated via EGFR transription-factors (AP1, Ets1, C/EBP), transcription-factors involved in circadian clock entrainment (CREB), and by core clock transcription-factors (Rorα). Quantitative real-time PCR measurements of several transcription-factor expression levels support a model in which circadian time-dependent EGFR responses are partly achieved by circadian regulation of upstream signaling components. Our study suggests an important role for EGFR signaling in SCN function and provides an example for gaining physiological insights through systems-level analysis.
PMCID: PMC1779538  PMID: 16784547

Results 1-15 (15)