NIH pre-clinical screening program: overview and current status
ABSTRACT 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.
SourceAvailable from: Lisa M. Wolfe[Show abstract] [Hide abstract]
ABSTRACT: The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.PLoS ONE 07/2014; 9(7):e103811. DOI:10.1371/journal.pone.0103811 · 3.53 Impact Factor
[Show abstract] [Hide abstract]
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) . 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/j.tube.2014.10.010 · 3.50 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: The development of adjuvants for vaccines has become an important area of research as the number of protein-based vaccines against infectious pathogens increases. Currently there are a number of adjuvant based Mycobacterium tuberculosis vaccines in clinical trials, which have shown efficacy in animal models. Despite these novel adjuvants there is still a need to design new and more versatile adjuvants that have minimal adverse side effects but produce robust long-lasting adaptive immune responses. To this end, we hypothesized that Bacillus subtilis spores may provide the appropriate innate signals that are required to generate such vaccine mediated responses, which would be sufficient to reduce the mycobacterial burden after infection with M. tuberculosis. In addition we compared the response generated by B. subtilis spores to that generated by monophosphoryl lipid A (MPL), which has been used extensively to test tuberculosis vaccines. The well-characterized, 6-kDa early secretory antigenic target of M. tuberculosis (ESAT-6; Rv3875) was used as a test antigen to determine the T cell activation potential of each adjuvant. Inoculated into mice, B. subtilis spores induced a strong pro-inflammatory response and Th1 immunity, similar to MPL, however unlike MPL formulated with dimethyldioctadecylammonium bromide it failed to induce significant levels of IL-17A and was unable to significantly reduce the mycobacterial burden after pulmonary infection with M. tuberculosis. Further analysis of the activity of MPL/DDA suggested that IL-17A was required for protective immunity. Taken together the data emphasize the requirement for a network of cytokines, working together that are required for protective immunity.Clinical and vaccine Immunology: CVI 01/2014; DOI:10.1128/CVI.00622-13 · 2.37 Impact Factor