NIH pre-clinical screening program: Overview and current status

Department of Microbiology, Colorado State University, Immunology and Pathology, Mycobacterial Research Laboratories, 1682 Campus Delivery, Fort Collins 80523-1682, USA.
Tuberculosis (Impact Factor: 2.71). 01/2005; 85(1-2):25-8. DOI: 10.1016/
Source: PubMed


The increase in the number of cases of Mycobacterium tuberculosis around the world has lead to a greater need for a more efficacious vaccine than the currently used M. bovis BCG. Despite the relative success of this attenuated vaccine there are multiple examples where alternative strategies are desperately needed. In 1996, the National Institutes of Health published a request calling for applications to test newly developed vaccines against tuberculosis. The current screening program at Colorado State University has tested a wide range of novel vaccine candidates.

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    • "). These methods were tested on exosomes (purified using ExoQuick) isolated from a series of guinea pig fluids that were collected 30 days after a low aerosol dose infection [28] and included bronchoalveolar lavage (BAL) fluid, urine and serum. All three fluids were collected from each animal when possible. "
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    ABSTRACT: Exosomes were originally described as small vesicles released from reticulocytes during the maturation process. These 40-200 nm microvesicles were hypothesized to be a mechanism for the removal of membrane proteins in lieu of intracellular degradation by Harding et al. (1984) and Johnstone et al. (1987) [1,2]. Exosomes can be distinguished from other extracellular vesicles (ectosomes, apoptotic blebs) based on their size and the protein indicators intercalated in their membrane (also, linking their derivation from the endocytic pathway) by Simpson (2012) [3]. The exact role which exosomes play in cell-to-cell communication and immune modulation is a topic of intense study. However, the focus of most reports has been directed towards discovering aberrations in exosomal protein and RNA content linked to disease onset and progression, and also primarily related to cancer. Nonetheless, exosomes are now documented to be released from a wide variety of cell types by Mathivanan et al. (2012), Simpson et al. (2012) and Kalra et al. (2012) [4-6] and have been isolated from all bodily fluids; thus, exosomes are an excellent source of biomarkers. Here we describe the discoveries related to the role exosomes play in tuberculosis disease, as well as translational work in vaccine development and how circulation of these dynamic vesicles can be harnessed for diagnostic purposes. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Tuberculosis 11/2014; 95(1). DOI:10.1016/ · 2.71 Impact Factor
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    • "During the last decade, many novel vaccines have been tested in mouse or guinea pig models including DNA, subunit proteins, recombinant BCG and attenuated strains of Mtb [3], [4], [5]. Most DNA or subunit vaccines are based on immuno-dominant Mtb proteins like MPT32, Phos, DnaK, GroES, MPT46, MPT53, MPT63, 19 kDa lipoprotein, Antigen 85 (Ag85) complex (Ag 85A, Ag85B and Ag85C), RD1 encoded proteins like early secretory antigen target-6 (ESAT-6), culture filtrate protein 10 (CFP10), and antigen TB10.4 [5]. "
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    ABSTRACT: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.
    PLoS ONE 05/2012; 7(5):e36198. DOI:10.1371/journal.pone.0036198 · 3.23 Impact Factor
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    • "Guinea pigs were maintained under ABSL-3 barrier conditions in isolator cages (Thoren, Hazleton PA). Bacterial suspensions described above were used to infect 12 guinea pigs with H37Rv by the LDA method (approximately 10 CFU) with a Madison Chamber [66]. In addition, a small aliquot of the suspension is plated on 7H11 media to confirm dose of aerosolized bacilli used in each infection. "
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    ABSTRACT: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo. We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection. Cumulatively, the results of this work may provide the basis for rational drug design - identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection.
    PLoS ONE 11/2010; 5(11):e13938. DOI:10.1371/journal.pone.0013938 · 3.23 Impact Factor
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