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1.  Interleukin-17 Protects against the Francisella tularensis Live Vaccine Strain but Not against a Virulent F. tularensis Type A Strain 
Infection and Immunity  2013;81(9):3099-3105.
Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated strains of Francisella that do not cause disease in humans. However, emerging data indicate that the protective immune response against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-17) confers protection against the live vaccine strain (LVS) of Francisella. While we too have found that IL-17Rα−/− mice are more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that IL-17Rα−/− mice display organ burdens and pulmonary gamma interferon (IFN-γ) responses similar to those of wild-type mice following infection. In addition, oral LVS vaccination conferred equivalent protection against pulmonary challenge with SchuS4 in both IL-17Rα−/− and wild-type mice. While IFN-γ was found to be critically important for survival in a convalescent model of SchuS4 infection, IL-17 neutralization from either wild-type or IFN-γ−/− mice had no effect on morbidity or mortality in this model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL-23p19 mRNA expression was found to be caspase-1 dependent in macrophages infected with LVS but not SchuS4. Collectively, these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and protective immunity differs between attenuated and virulent strains of F. tularensis.
PMCID: PMC3754213  PMID: 23774604
2.  Virulence difference between the prototypic Schu S4 strain (A1a) and Francisella tularensis A1a, A1b, A2 and type B strains in a murine model of infection 
The use of prototypic strains is common among laboratories studying infectious agents as it promotes consistency for data comparability among and between laboratories. Schu S4 is the prototypic virulent strain of Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica (type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S4 belongs to the most virulent subspecies of F. tularensis, subspecies tularensis.
In this study, we investigated the relative virulence of Schu S4 in comparison to A1a, A1b, A2 and type B strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu S4 at drop point.
Survival curve comparisons demonstrate that the Schu S4 strain used in this study resembles the virulence of type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when bacterial burdens were compared between mice infected with Schu S4 or MA00-2987 (A1b) significantly higher burdens were present in the blood and spleen of mice infected with MA00-2987.
The knowledge gained from using Schu S4 as a prototypic virulent strain has unquestionably advanced the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation of results.
PMCID: PMC3923427  PMID: 24502661
Prototypic strains; Murine model; Fever; Francisella tularensis; Tularemia; Schu S4; A1a
3.  A metabolic biosignature of early response to anti-tuberculosis treatment 
The successful treatment of tuberculosis (TB) requires long-term multidrug chemotherapy. Clinical trials to evaluate new drugs and regimens for TB treatment are protracted due to the slow clearance of Mycobacterium tuberculosis (Mtb) infection and the lack of early biomarkers to predict treatment outcome. Advancements in the field of metabolomics make it possible to identify metabolic profiles that correlate with disease states or successful chemotherapy. However, proof-of-concept of this approach has not been provided for a TB-early treatment response biosignature (TB-ETRB).
Urine samples collected at baseline and during treatment from 48 Ugandan and 39 South African HIV-seronegative adults with pulmonary TB were divided into discovery and qualification sets, normalized to creatinine concentration, and analyzed by liquid chromatography-mass spectrometry to identify small molecule molecular features (MFs) in individual patient samples. A biosignature that distinguished baseline and 1 month treatment samples was selected by pairwise t-test using data from two discovery sample sets. Hierarchical clustering and repeated measures analysis were applied to additional sample data to down select molecular features that behaved consistently between the two clinical sites and these were evaluated by logistic regression analysis.
Analysis of discovery samples identified 45 MFs that significantly changed in abundance at one month of treatment. Down selection using an extended set of discovery samples and qualification samples confirmed 23 MFs that consistently changed in abundance between baseline and 1, 2 and 6 months of therapy, with 12 MFs achieving statistical significance (p < 0.05). Six MFs classified the baseline and 1 month samples with an error rate of 11.8%.
These results define a urine based TB-early treatment response biosignature (TB-ETRB) applicable to different parts of Africa, and provide proof-of-concept for further evaluation of this technology in monitoring clinical responses to TB therapy.
PMCID: PMC3918231  PMID: 24484441
Tuberculosis; Metabolomics; Biomarker; Mass spectrometry; Small molecule biosignature; Anti-tuberculosis therapy; Mycobacterium tuberculosis, Urine
4.  Deciphering the proteome of the in vivo diagnostic reagent “purified protein derivative” from Mycobacterium tuberculosis 
Proteomics  2012;12(7):979-991.
Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. FDA standard PPD-S2. Many known M. tuberculosis T cell antigens were detected. Of significance, four heat shock proteins (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.
PMCID: PMC3756804  PMID: 22522804
5.  Isolation of a distinct Mycobacterium tuberculosis mannose-capped lipoarabinomannan isoform responsible for recognition by CD1b-restricted T cells 
Glycobiology  2012;22(8):1118-1127.
Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.
PMCID: PMC3382347  PMID: 22534567
CD1b; lipoarabinomannan; lipoglycans; Mycobacterium tuberculosis; T cells
6.  Post-Exposure Immunization Against Francisella tularensis Membrane Proteins Augments Protective Efficacy of Gentamicin in a Mouse Model of Pneumonic Tularemia 
Vaccine  2012;30(33):4977-4982.
Successful treatment of pneumonic infection with Francisella tularensis, the causative agent of tularemia, requires rapid initiation of antibiotic therapy, yet even then treatment failures may occur. Consequently, new treatments are needed to enhance the effectiveness of antimicrobial therapy for acute pneumonic tularemia. In a prior study, immunization with F. tularensis membrane protein fraction (MPF) antigens 3 days prior to challenge was reported to induce significant protection from inhalational challenge. We therefore hypothesized that MPF immunization might also be effective in enhancing infection control if combined with antibiotic therapy and administered after infection as post-exposure immunotherapy. To address this question, a 24 h post-exposure treatment model of acute pulmonary Schu S4 strain of F. tularensis infection in BALB/c mice was used. Following exposure, mice were immunized with MPF and treated with low-dose gentamicin, alone or in combination and the effects on survival, bacterial burden and dissemination were assessed. We found that immunization with MPF significantly increased the effectiveness of subtherapeutic gentamicin therapy for post-exposure treatment of pneumonic tularemia, with 100% of combination-treated mice surviving long-term. Bacterial burdens in the liver and spleen were significantly reduced in combination MPF-gentamicin treated mice at 7 days after challenge. Passively transferred antibodies against MPF antigens also increased the effectiveness of gentamicin therapy. Thus, we concluded that post-exposure immunization with MPF antigens was an effective means of enhancing conventional antimicrobial therapy for pneumonic tularemia.
PMCID: PMC3576924  PMID: 22652404
Francisella tularensis; membrane proteins; antibodies
7.  Tuberculosis Biomarker and Surrogate Endpoint Research Roadmap 
The Centers for Disease Control and Prevention and National Institutes of Health convened a multidisciplinary meeting to discuss surrogate markers of treatment response in tuberculosis. The goals were to assess recent surrogate marker research and to provide specific recommendations for (1) the qualification and validation of biomarkers of treatment outcome; (2) the standardization of specimen and data collection for future clinical trials, including a minimum set of samples and collection time points; and (3) the creation of a specimen repository to support biomarker testing. This article summarizes these recommendations and provides a roadmap for their implementation.
PMCID: PMC3208659  PMID: 21737585
8.  Use of Temperature for Standardizing the Progression of Francisella tularensis in Mice 
PLoS ONE  2012;7(9):e45310.
The study of infectious agents, their pathogenesis, the host response and the evaluation of newly developed countermeasures often requires the use of a living system. Murine models are frequently used to undertake such investigations with the caveat that non-biased measurements to assess the progression of infection are underutilized. Instead, murine models predominantly rely on symptomology exhibited by the animal to evaluate the state of the animal's health and to determine when euthanasia should be performed. In this study, we used subcutaneous temperature as a non-subjective measurement to follow and compare infection in mice inoculated with Francisella tularensis, a Gram-negative pathogen that produces an acute and fatal illness in mice. A reproducible temperature pattern defined by three temperature phases (normal, febrile and hypothermic) was identified in all mice infected with F. tularensis, regardless of the infecting strain. More importantly and for the first time a non-subjective, ethical, and easily determined surrogate endpoint for death based on a temperature, termed drop point, was identified and validated with statistical models. In comparative survival curve analyses for F. tularensis strains with differing virulence, the drop point temperature yielded the same results as those obtained using observed time to death. Incorporation of temperature measurements to evaluate F. tularensis was standardized based on statistical models to provide a new level of robustness for comparative analyses in mice. These findings should be generally applicable to other pathogens that produce acute febrile disease in animal models and offers an important tool for understanding and following the infection process.
PMCID: PMC3454384  PMID: 23028924
9.  A Novel Metabolite of Antituberculosis Therapy Demonstrates Host Activation of Isoniazid and Formation of the Isoniazid-NAD+ Adduct 
One of the most effective and widely used antituberculosis (anti-TB) drugs is isoniazid (INH), a prodrug activated via oxidation that forms an adduct with NAD+ to inhibit NADH-dependent targets of Mycobacterium tuberculosis, such as enoyl-acyl carrier protein reductase (InhA). The metabolic by-products and potentially toxic intermediates resulting from INH therapy have been identified through a large body of work. However, an INH-NAD adduct or structures related to this adduct have not been identified in specimens from human TB patients or animal models of TB. Analyses by mass spectrometry of urine collected from TB patients in a study conducted by the NIAID-funded Tuberculosis Research Unit identified 4-isonicotinoylnicotinamide (C12H9N3O2) as a novel metabolite of INH therapy. This compound was formed by M. tuberculosis strains in a KatG-dependent manner but could also be produced by mice treated with INH independent of an M. tuberculosis infection. Thus, the 4-isonicotinoylnicotinamide observed in human urine samples is likely derived from the degradation of oxidized INH-NAD adducts and provides direct evidence of host INH activation.
PMCID: PMC3256082  PMID: 22037847
10.  Analysis of Lipids from Crude Lung Tissue Extracts by Desorption Electrospray Ionization-MS and Pattern Recognition 
Analytical biochemistry  2010;408(2):289-296.
A method is described using Desorption Electrospray Ionization (DESI) Mass Spectrometry (MS) to obtain phospholipid mass spectral profiles from crude lung tissue extracts. The measured DESI-mass spectral lipid fingerprints were then analyzed by unsupervised learning principal components analysis (PCA). This combined approach was used to differentiate the effect(s) of two vaccination routes on lipid composition in mice lung. Specifically, the two vaccination routes compared were intra-nasal (i.n.) and intra-dermal (i.d.) inoculation of the Francisella tularensis Live Vaccine Strain (Ft-LVS). Lung samples of control and LVS-inoculated mice were quickly extracted with a methanol/chloroform solution and the crude extract directly analyzed by DESI-MS, with a total turnaround time of less than 10 min per sample. All the measured DESI-mass spectra (in both positive and negative ion mode) were compared via PCA which resulted in clear differentiation of mass spectral profiles of i.n.-inoculated mice lung tissues from those of i.d.-inoculated and control mice lung tissues. Lipid biomarkers responsible for sample differentiation were identified via tandem MS (MS/MS) measurements or by comparison with mass spectra of lipid standards. The DESI-MS approach here described provided a practical and rapid means to analyze tissue samples without extensive extractions and solvent changes.
PMCID: PMC2980589  PMID: 20868645
DESI; mass spectrometry; lipids; lung tissue
11.  Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism 
PLoS ONE  2012;7(1):e29970.
The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.
PMCID: PMC3253812  PMID: 22253841
12.  Differential Chitinase Activity and Production within Francisella Species, Subspecies, and Subpopulations▿‡ 
Journal of Bacteriology  2011;193(13):3265-3275.
Genotyping of Francisella tularensis (A1a, A1b, A2, and type B) and Francisella novicida has identified multiple differences between species and among F. tularensis subspecies and subpopulations. Variations in virulence, geographic distribution, and ecology are also known to exist among this group of bacteria, despite the >95% nucleotide identity in their genomes. This study expands the description of phenotypic differences by evaluating the ability of F. tularensis and F. novicida to degrade chitin analogs and produce active chitinases. Endochitinase activities were observed to vary among F. tularensis and F. novicida strains. The activity observed for F. tularensis strains was predominantly associated with whole-cell lysates, while the chitinase activity of F. novicida localized to the culture supernatant. In addition, the overall level of chitinase activity differed among the subpopulations of F. tularensis and between the species. Bioinformatic analyses identified two new putative chitinase genes (chiC and chiD), as well as the previously described chiA and chiB. However, the presence of these four open reading frames as intact genes or pseudogenes was found to differ between Francisella species and F. tularensis subspecies and subpopulations. Recombinant production of the putative chitinases and enzymatic evaluations revealed ChiA, ChiB, ChiC, and ChiD possessed dissimilar chitinase activities. These biochemical studies coupled with bioinformatic analyses and the evaluation of chiA and chiC knockouts in F. tularensis A1 and A2 strains, respectively, provided a molecular basis to explain the differential chitinase activities observed among the species and subpopulations of Francisella.
PMCID: PMC3133279  PMID: 21531796
13.  Effective, Broad Spectrum Control of Virulent Bacterial Infections Using Cationic DNA Liposome Complexes Combined with Bacterial Antigens 
PLoS Pathogens  2010;6(5):e1000921.
Protection against virulent pathogens that cause acute, fatal disease is often hampered by development of microbial resistance to traditional chemotherapeutics. Further, most successful pathogens possess an array of immune evasion strategies to avoid detection and elimination by the host. Development of novel, immunomodulatory prophylaxes that target the host immune system, rather than the invading microbe, could serve as effective alternatives to traditional chemotherapies. Here we describe the development and mechanism of a novel pan-anti-bacterial prophylaxis. Using cationic liposome non-coding DNA complexes (CLDC) mixed with crude F. tularensis membrane protein fractions (MPF), we demonstrate control of virulent F. tularensis infection in vitro and in vivo. CLDC+MPF inhibited bacterial replication in primary human and murine macrophages in vitro. Control of infection in macrophages was mediated by both reactive nitrogen species (RNS) and reactive oxygen species (ROS) in mouse cells, and ROS in human cells. Importantly, mice treated with CLDC+MPF 3 days prior to challenge survived lethal intranasal infection with virulent F. tularensis. Similarly to in vitro observations, in vivo protection was dependent on the presence of RNS and ROS. Lastly, CLDC+MPF was also effective at controlling infections with Yersinia pestis, Burkholderia pseudomallei and Brucella abortus. Thus, CLDC+MPF represents a novel prophylaxis to protect against multiple, highly virulent pathogens.
Author Summary
Conventional treatment of bacterial infections typically includes administration of antibiotics. However, many pathogens have developed spontaneous resistance to commonly used antibiotics. Development of new compounds that stimulate the host immune system to directly kill bacteria by mechanisms different from those utilized by antibiotics may serve as effective alternatives to antibiotic therapy. In this report, we describe a novel compound capable of controlling infections mediated by different, unrelated bacteria via the induction of host derived reactive oxygen and reactive nitrogen species. This compound is comprised of cationic liposome DNA complexes (CLDC) and crude membrane preparations (MPF) obtained from attenuated Francisella tularensis Live Vaccine Strain (LVS). Pretreatment of primary mouse or human cells limited replication of virulent F. tularensis, Burkholderia pseudomallei, Yersinia pestis and Brucella abortus in vitro. CLDC+MPF was also effective for controlling lethal pulmonary infections with virulent F. tularensis. Thus, CLDC+MPF represents a novel antimicrobial for treatment of lethal, acute, bacterial infections.
PMCID: PMC2877747  PMID: 20523903
14.  Peptides of a Novel Mycobacterium tuberculosis–Specific Cell Wall Protein for Immunodiagnosis of Tuberculosis 
The Journal of infectious diseases  2009;200(4):571-581.
The sequencing of the Mycobacterium tuberculosis genome revealed the existence of several genes encoding novel proteins with unknown functions, one of which is the proline-threonine repetitive protein (PTRP; Rv0538). Genomic studies of various mycobacterial species and M. tuberculosis clinical isolates demonstrate that ptrp is specific to the M. tuberculosis complex and ubiquitous in clinical isolates. Enzyme-linked immunosorbent assay, Western blot analysis, and electron microscopic evaluation of M. tuberculosis subcellular fractions and intact bacteria confirm that PTRP is a cell wall protein. Antibodies to PTRP are present in serum specimens from human immunodeficiency virus (HIV)–negative, tuberculosis (TB)–positive and HIV-positive, TB-positive patients but not purified protein derivative (PPD)–negative or PPD-positive healthy control subjects, demonstrating its diagnostic potential. Epitope mapping of PTRP delineated 4 peptides that can identify >80% of sputum smear–positive and >50% of smear-negative, HIV-negative, TB-positive patients and >80% of HIV-positive, TB-positive patients. These results demonstrate that immunodominant epitopes of carefully selected M. tuberculosis–specific proteins can be used to devise a simple peptide-based serodiagnostic test for TB.
PMCID: PMC2846530  PMID: 19604115
15.  Morphological features and signature gene response elicited by inactivation of FtsI in Mycobacterium tuberculosis 
Universally conserved events in cell division provide the opportunity for the development of novel chemotherapeutics against Mycobacterium tuberculosis. The aim of this study was to use the β-lactam antimicrobials cefalexin and piperacillin to inhibit FtsI and characterize the morphological changes and global transcriptional activities of genes to identify a signature response to FtsI inactivation.
Cefalexin and piperacillin were used to block cell division, and microscopy was used to evaluate the effects on bacterial morphology and ultrastructure. Global transcriptional analysis was performed to determine the impact of FtsI inhibition on cell cycle processes and to identify molecular markers.
Inhibition of FtsI with cefalexin and piperacillin resulted in filamentous cells with multiple concentric rings and occasional branching as visualized by light and electron microscopy. Whole genome microarray-based transcriptional profiling and transcriptional mapping allowed the evaluation of cell cycle processes in response to inhibition of FtsI and characterization of transcriptional response and cell cycle processes.
This study substantiated that FtsZ-ring constriction and septal resolution require the transpeptidase activity of FtsI, making FtsI essential for cell division in M. tuberculosis. Therefore, FtsI is a target for drug discovery, and these studies provided a molecular signature of FtsI inactivation that can be applied to screening strategies for novel FtsI inhibitors.
PMCID: PMC2721702  PMID: 19109339
M. tuberculosis; cell division; microarray; cefalexin; piperacillin
16.  N-Terminal clustering of the O-glycosylation sites in the Mycobacterium tuberculosis lipoprotein SodC 
Glycobiology  2008;19(1):38-51.
SodC is one of two superoxide dismutases produced by Mycobacterium tuberculosis. This protein was previously shown to contribute to virulence and to act as a B-cell antigen. SodC is also a putative lipoprotein, and like other Sec-translocated mycobacterial proteins it was suggested to be modified with glycosyl units. To definitively define the glycosylation of SodC, we applied an approach that combined site-directed mutagenesis, lectin binding, and mass spectrometry. This resulted in identification of six O-glycosylated residues within a 13-amino-acid region near the N-terminus. Each residue was modified with one to three hexose units, and the most dominant SodC glycoform was modified with nine hexose units. In addition to O-glycosylation of threonine residues, this study provides the first evidence of serine O-glycosylation in mycobacteria. When combined with bioinformatic analyses, the clustering of O-glycosylation appeared to occur in a region of SodC with a disordered structure and not in regions important to the enzymatic activity of SodC. The use of recombinant amino acid substitutions to alter glycosylation sites provided further evidence that glycosylation influences proteolytic processing and ultimately positioning of cell wall proteins.
PMCID: PMC2733778  PMID: 18842962
Glycoprotein; lipoprotein; mycobacterium; superoxide dismutase; tuberculosis
17.  IFNγ Response to Mycobacterium tuberculosis, Risk of Infection and Disease in Household Contacts of Tuberculosis Patients in Colombia 
PLoS ONE  2009;4(12):e8257.
Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNγ produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNγ levels in HHCs correlate with tuberculosis development.
A cohort of 2060 HHCs was followed for 2–3 years after exposure to a tuberculosis case. Besides TST, IFNγ responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination.
Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNγ response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNγ responders to CFP-10 (HR 1.82 95% CI 0.79–4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNγ producers was observed (trend Log rank p = 0.007).
CFP-10-induced IFNγ production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.
PMCID: PMC2788133  PMID: 20011589
18.  Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation1 
CD4+ T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an antigen associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell antigen as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4+T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate nor did it inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, since recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by α-mannosidase did not activate T cells. This study demonstrates that the universe of bacterial T cell antigens includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.
PMCID: PMC2710377  PMID: 18424702
Lipoglycoprotein; T cells; mycobacteria; leprosy; human
19.  Genomic Markers for Differentiation of Francisella tularensis subsp. tularensis A.I and A.II Strains▿ †  
Tularemia is caused by two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). F. tularensis subsp. tularensis is further subdivided into two genetically distinct populations (A.I and A.II) that differ with respect to geographical location, anatomical source of recovered isolates, and disease outcome. Using two human clinical isolates, suppression subtractive hybridization was performed to identify 13 genomic regions of difference between A.I and A.II strains. Two PCR assays, one to identify A.I and A.II as well as to discriminate between F. tularensis subsp. holarctica and F. novicida and another specific for A.I, were developed. This is the first report to identify and characterize conserved genomic differences between A.I and A.II.
PMCID: PMC2223193  PMID: 18024683
20.  Biomarkers for Clinical and Incipient Tuberculosis: Performance in a TB-Endemic Country 
PLoS ONE  2008;3(4):e2071.
Simple biomarkers are required to identify TB in both HIV−TB+ and HIV+TB+ patients. Earlier studies have identified the M. tuberculosis Malate Synthase (MS) and MPT51 as immunodominant antigens in TB patients. One goal of these investigations was to evaluate the sensitivity and specificity of anti-MS and –MPT51 antibodies as biomarkers for TB in HIV−TB+ and HIV+TB+ patients from a TB-endemic setting. Earlier studies also demonstrated the presence of these biomarkers during incipient subclinical TB. If these biomarkers correlate with incipient TB, their prevalence should be higher in asymptomatic HIV+ subjects who are at a high-risk for TB. The second goal was to compare the prevalence of these biomarkers in asymptomatic, CD4+ T cell-matched HIV+TB− subjects from India who are at high-risk for TB with similar subjects from US who are at low-risk for TB.
Methods and Results
Anti-MS and -MPT51 antibodies were assessed in sera from 480 subjects including PPD+ or PPD− healthy subjects, healthy community members, and HIV−TB+ and HIV+TB+ patients from India. Results demonstrate high sensitivity (∼80%) of detection of smear-positive HIV−TB+ and HIV+TB+ patients, and high specificity (>97%) with PPD+ subjects and endemic controls. While ∼45% of the asymptomatic HIV+TB− patients at high-risk for TB tested biomarker-positive, >97% of the HIV+TB− subjects at low risk for TB tested negative. Although the current studies are hampered by lack of knowledge of the outcome, these results provide strong support for the potential of these biomarkers to detect incipient, subclinical TB in HIV+ subjects.
These biomarkers provide high sensitivity and specificity for TB diagnosis in a TB endemic setting. Their performance is not compromised by concurrent HIV infection, site of TB and absence of pulmonary manifestations in HIV+TB+ patients. Results also demonstrate the potential of these biomarkers for identifying incipient subclinical TB in HIV+TB− subjects at high-risk for TB.
PMCID: PMC2323108  PMID: 18446210
21.  In Vivo Adaptation of the Wayne Model of Latent Tuberculosis▿  
Infection and Immunity  2007;75(5):2621-2625.
Cultures of Mycobacterium tuberculosis grown under oxygen depletion conditions enter into a state of nonreplicating persistence that may reflect a physiologically latent state. When these cultures were harvested and injected intranasally into mice, no bacteria could be recovered from the lungs for about 3 weeks, but after that evidence of regrowth was observed. Preimmunization of mice with a panel of selected vaccine candidates slowed or prevented this event. This simple model has potential for identifying vaccines targeting latent tuberculosis.
PMCID: PMC1865751  PMID: 17283091
22.  Use of Protein Microarrays To Define the Humoral Immune Response in Leprosy Patients and Identification of Disease-State-Specific Antigenic Profiles▿ †  
Infection and Immunity  2006;74(11):6458-6466.
Although the global prevalence of leprosy has decreased over the last few decades due to an effective multidrug regimen, large numbers of new cases are still being reported, raising questions as to the ability to identify patients likely to spread disease and the effects of chemotherapy on the overall incidence of leprosy. This can partially be attributed to the lack of diagnostic markers for different clinical states of the disease and the consequent implementation of differential, optimal drug therapeutic strategies. Accordingly, comparative bioinformatics and Mycobacterium leprae protein microarrays were applied to investigate whether leprosy patients with different clinical forms of the disease can be categorized based on differential humoral immune response patterns. Evaluation of sera from 20 clinically diagnosed leprosy patients using native protein and recombinant protein microarrays revealed unique disease-specific, humoral reactivity patterns. Statistical analysis of the serological patterns yielded distinct groups that correlated with phenolic glycolipid I reactivity and clinical diagnosis, thus demonstrating that leprosy patients, including those diagnosed with the paucibacillary, tuberculoid form of disease, can be classified based on humoral reactivity to a subset of M. leprae protein antigens produced in recombinant form.
PMCID: PMC1695501  PMID: 16966411
23.  Mycobacterium tuberculosis Malate Synthase- and MPT51-Based Serodiagnostic Assay as an Adjunct to Rapid Identification of Pulmonary Tuberculosis 
Clinical and Vaccine Immunology  2006;13(11):1291-1293.
The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from ∼80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis.
PMCID: PMC1656542  PMID: 17090645
24.  A randomised controlled trial of the effects of albendazole in pregnancy on maternal responses to mycobacterial antigens and infant responses to bacille Calmette-Guérin (BCG) immunisation [ISRCTN32849447] 
Maternal schistosomiasis and filariasis have been shown to influence infant responses to neonatal bacille Calmette-Guérin (BCG) immunisation but the effects of maternal hookworm, and of de-worming in pregnancy, are unknown.
In Entebbe, Uganda, we conducted a randomised, double-blind, placebo-controlled trial of a single dose of 400 mg of albendazole in the second trimester of pregnancy. Neonates received BCG. Interferon-gamma (IFN-γ) and interleukin (IL)-5 responses to a mycobacterial antigen (crude culture filtrate proteins (CFP) of Mycobacterium tuberculosis) were measured in a whole blood assay. We analysed results for binary variables using χ2 tests and logistic regression. We analysed continuous variables using Wilcoxon's tests.
Maternal hookworm was associated with reduced maternal IFN-γ responses to CFP (adjusted odds ratio for IFN-γ > median response: 0.14 (95% confidence interval 0.02–0.83, p = 0.021). Conversely, maternal hookworm was associated with subsequent increased IFN-γ responses in their one-year-old infants (adjusted OR 17.65 (1.20–258.66; p = 0.013)). Maternal albendazole tended to reduce these effects.
Untreated hookworm infection in pregnancy was associated with reduced maternal IFN-γ responses to mycobacterial antigens, but increased responses in their infants one year after BCG immunisation. The mechanisms of these effects, and their implications for protective immunity remain, to be determined.
PMCID: PMC1352364  PMID: 16371154

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