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Tuberculosis has impacted human health for millennia. The World Health Organization estimated that, in 2014, 9.6 million people developed tuberculosis and 1.5 million people died from the disease. In May 2014, the World Health Assembly endorsed the new "End TB Strategy" that presents a pathway to tuberculosis elimination. The strategy outlines 3 areas of emphasis, one of which is intensified research and innovation. In this article we highlight the essential role for fundamental tuberculosis research in the future of tuberculosis diagnostics, treatment, and prevention. To maximize the impact of fundamental research, we must foster collaboration among all stakeholders engaged in tuberculosis research and control to facilitate open dialogue to assure that critical gaps in outcome-oriented science are identified and addressed. We present here a framework for future discussions among scientists, physicians, research and development specialists, and public health managers for the reinforcement of national and international strategies toward tuberculosis elimination.

Novel tuberculosis (TB) drug regimens are urgently needed, and their development will be enabled by improved preclinical approaches that more effectively inform and ensure safe selection of clinical candidates and drug combination/regimens. An evidence-based approach for the assessment of nonclinical models supporting TB drug development has been proposed by a joint partnership between the National Institute of Allergy and Infectious Diseases (NIAID) and the Critical Path to TB Drug Regimens (CPTR) Consortium.

Christian Lienhardt and colleagues describe the research efforts needed to end the global tuberculosis epidemic by 2035.

Tuberculosis (TB) has been a persistent public health concern for hundreds of years. Despite advances in medicine and science, eliminating this disease has been beyond our reach. Several organizations, including the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), have expressed their commitment to advancing biomedical research in TB in order to increase our understanding of the causative pathogen and the disease. This basic knowledge is a critical first step in the development and implementation of new therapeutics, vaccines and diagnostics. Collaboration between researchers is a key component to accomplishing this goal; product development can no longer be limited to separate programs. Rather, the interconnectedness and possible combination of interventions must be investigated. This review will discuss ongoing TB research including NIAID's role, as well as future research that is needed to improve TB control. Emphasizing the importance of coordination among researchers, funders and advocacy groups, we aim to illustrate the fact that biomedical research, and particularly basic research, is a vital part of a complementary approach to eliminating TB across the globe.

Partially fueled by the human immunodeficiency virus (HIV) pandemic, tuberculosis is the second-leading cause of infectious diseases–associated mortality globally and the leading cause of death among those infected with HIV. In 2010, there were an estimated 8.8 million incident cases of tuberculosis and approximately 1.5 million deaths attributed to tuberculosis. Increased public heath efforts have been effective in decreasing the overall prevalence and mortality associated with this disease, but declines in incidence rates have been outpaced by global population growth, such that absolute incidence rates have not declined as fast as previously expected [1]. Commensurate with increased public recognition of the importance of tuberculosis to global health in the mid-1990s, biomedical research for tuberculosis has increased both in the United States and internationally. Although tools to conduct modern biomedical research involving tuberculosis have only been available for a little over a decade, this research has resulted in several successes, including promising new vaccines to protect against tuberculosis in the highest-risk populations, diagnostic candidates to improve the accuracy and speed of diagnosing all forms of tuberculosis, and new drug candidates with the potential for improved treatment and chemoprevention for drug-sensitive and drug-resistant tuberculosis. Despite these successes, many questions remain in all aspects of tuberculosis research, ranging from fundamental pathogenesis to programmatic implementation, and they are complicated by the complexity of the disease and its chronic nature. Compared with other infections, Mycobacterium tuberculosis infection does not elicit clinically relevant disease in the majority of subjects, and many who are infected carry asymptomatic, paucibacillary disease for decades. This complicates the definition and evaluation of early infection events and initial host responses and the characterization of infecting bacillary populations. The clinical definition of latent infection is limited to positive responses to injection of purified mycobacterial components (ie, the tuberculosis skin test) or to T-cell–recall responses measured through the production of interferon γ; unlike viral infections, latent tuberculous infections cannot be identified on the basis of stable antibody titers. Progression to active disease is not a clear unidirectional process and is prolonged, and many affected persons appear to be able to clear the infection, whereas others progress to active disease. The spectrum of active tuberculosis is complex. Although adult pulmonary tuberculosis is considered of greatest relevance for public health, extrapulmonary disease occurs in a significant portion of subjects, and pediatric manifestations of tuberculosis may be mistaken for other pulmonary infections and are difficult to diagnose. Treatment of active disease is complicated by the fact that the current standard of care involves combinations of drugs whose administration is sequenced on the basis of the clearance of mycobacteria from sputum and that, unlike most other antibacterial treatments, have to be administered for a minimum of 6 months to elicit a stable cure. Hypotheses exist that explain the need for such prolonged therapy, but they are difficult to test experimentally because initial treatment of tuberculosis eliminates bacteria from sputum and thus limits options for studying bacterial populations. Unlike many other infectious diseases, in tuberculosis, patients do not become immune to reinfection at a later time. Thus, a clear definition of the markers of immune protection is dependent on successful trials of new vaccines or has to be derived from data for persons who were infected but did not progress to active disease. However, both criteria are complicated by the limitations listed above. Comprehensive reviews of research priorities that have been defined on the basis of the unique nature of tuberculosis and the associated scientific/technical limitations of studying the disease have been addressed elsewhere [2–5], but several issues merit further exploration.

Tuberculosis (TB) remains a significant cause of morbidity and mortality despite improvements in detection and treatment. Current global control strategies are not sufficient to significantly affect TB rates worldwide and are unlikely to meet global targets without new diagnostic tests, drugs, and vaccines. Increasing rates of drug-resistant TB coupled with co-infection in people with HIV infection, threaten to reverse gains made in controlling both diseases. Control of TB includes many interconnected components. NIH/NIAID supports fundamental, translational, and clinical TB studies and provides resources to facilitate research on candidate products. In an era of limited government funding, clinical use of candidate products, and a changing landscape of organizations participating in TB research and development, it is critical to re-evaluate each partner's contributions. This rethinking will ensure that limited resources focus on medical gaps, and tasks are undertaken by the most suitable organizations. Collaborative efforts are essential to advance in TB research and development.

Background New tools are required for the diagnosis of pre-symptomatic leprosy towards further reduction of disease burden and its associated reactions. To address this need, two new skin test antigens were developed to assess safety and efficacy in human trials. Methods A Phase I safety trial was first conducted in a non-endemic region for leprosy (U.S.A.). Healthy non-exposed subjects (n = 10) received three titrated doses (2.5 µg, 1.0 µg and 0.1 µg) of MLSA-LAM (n = 5) or MLCwA (n = 5) and control antigens [Rees MLSA (1.0 µg) and saline]. A randomized double blind Phase II safety and efficacy trial followed in an endemic region for leprosy (Nepal), but involved only the 1.0 µg (high dose) and 0.1 µg (low dose) of each antigen; Tuberculin PPD served as a control antigen. This Phase II safety and efficacy trial consisted of three Stages: Stage A and B studies were an expansion of Phase I involving 10 and 90 subjects respectively, and Stage C was then conducted in two parts (high dose and low dose), each enrolling 80 participants: 20 borderline lepromatous/lepromatous (BL/LL) leprosy patients, 20 borderline tuberculoid/tuberculoid (BT/TT) leprosy patients, 20 household contacts of leprosy patients (HC), and 20 tuberculosis (TB) patients. The primary outcome measure for the skin test was delayed type hypersensitivity induration. Findings In the small Phase I safety trial, reactions were primarily against the 2.5 µg dose of both antigens and Rees control antigen, which were then excluded from subsequent studies. In the Phase II, Stage A/B ramped-up safety study, 26% of subjects (13 of 50) showed induration against the high dose of each antigen, and 4% (2 of 50) reacted to the low dose of MLSA-LAM. Phase II, Stage C safety and initial efficacy trial showed that both antigens at the low dose exhibited low sensitivity at 20% and 25% in BT/TT leprosy patients, but high specificity at 100% and 95% compared to TB patients. The high dose of both antigens showed lower specificity (70% and 60%) and sensitivity (10% and 15%). BL/LL leprosy patients were anergic to the leprosy antigens. Interpretation MLSA-LAM and MLCwA at both high (1.0 µg) and low (0.1 µg) doses were found to be safe for use in humans without known exposure to leprosy and in target populations. At a sensitivity rate of 20–25% these antigens are not suitable as a skin test for the detection of the early stages of leprosy infection; however, the degree of specificity is impressive given the presence of cross-reactive antigens in these complex native M. leprae preparations. Trial Registration ClinicalTrails.gov NCT01920750 (Phase I), NCT00128193 (Phase II), Author Summary Clinically useful skin test reagents should be safe and sufficiently sensitive to detect infection prior to physical manifestations of leprosy disease. While in these small scale human studies, leprosy reagents were safe for use in humans, they failed in respect of sensitivity at a rate of 20–25% in the key indicator group, BT/TT leprosy patients. Specificity in terms of leprosy vs. tuberculosis at a rate of 95–100% was surprisingly high in light of the extensive presence of cross-reactive antigens in the complex native M. leprae preparations. These results could justify a further trial at lower dosages.

Teicoplanin aglycone 1 has been linked to a solid support via two different double cleavable linkers. The attachment was realized selectively on the 38 COOH function of 1 . Two molecules of 1 were attached on each linker moiety and could be separately released with different mechanisms. The release properties of the resin bound entities were determined by means of analytical and biological characterization.

Xylose-inducible vectors have been constructed for extracellular production of antibody fragments in Staphylococcus carnosus. The pre-pro sequence of S. hyicus lipase was taken as secretional signal sequence, and the S. xylosus Xyl repressor was used to confer xylose inducibility of transcription. Cleavage sites for the IgA protease were engineered between the pre-pro sequence and the antibody fragments to permit removal of the pro sequence. Extracellular expression of the light chain and the Fd fragment of a chimeric Fab fragment containing the variable regions of the anti-lysozyme antibody D1.3 was achieved with these vectors. The pro sequence could be removed from the expression product by IgA protease treatment. When the light chain and the Fd fragment were co-secreted as a protein fusion they accumulated in a structure capable of heterodimerization after IgA cleavage. This fusion contains the pre-pro sequence followed by the light chain, a second IgA site and the Fd fragment.

The development, evaluation, and implementation of new and improved diagnostics have been identified as critical needs by human immunodeficiency virus (HIV) and tuberculosis researchers and clinicians alike. These needs exist in international and domestic settings and in adult and pediatric populations. Experts in tuberculosis and HIV care, researchers, healthcare providers, public health experts, and industry representatives, as well as representatives of pertinent US federal agencies (Centers for Disease Control and Prevention, Food and Drug Administration, National Institutes of Health, United States Agency for International Development) assembled at a workshop proposed by the Diagnostics Working Group of the Federal Tuberculosis Taskforce to review the state of tuberculosis diagnostics development in adult and pediatric populations.

We have investigated the regulation of the operon encoding xylose utilization in Staphylococcus xylosus C2a and Staphylococcus carnosus TM300. For in vivo studies, transcriptional fusions of the xylAB regulatory region to the lipase gene from Staphylococcus hyicus were constructed. Repression of lipase activity depended on a functional xylR gene and an xyl operator palindrome downstream of the promoter, while induction was obtained in the presence of xylose. Inactivation of either xylR or the xyl operator led to constitutive expression in the absence of xylose. Crude protein extracts from xylR+ staphylococci led to gel mobility shifts of the xyl regulatory DNA in the absence but not in the presence of xylose. A copper-phenanthroline footprint of the shifted band revealed protection of 28 phosphodiesters from cleavage in each strand of the xyl operator. Thus, the Xyl repressor covers the DNA over more than 2.5 helical turns. Glucose repression of the xyl operon occurs at the level of transcription and is independent of a functional xylR gene. A potential cis-active sequence element for glucose repression is discussed on the basis of sequence similarities to respective elements from bacilli.

The Staphylococcus xylosus xyl genes were cloned in Staphylococcus carnosus by complementation to xylose utilization. Xylose isomerase assays under inducing (xylose present) and non-inducing (xylose absent) conditions indicated the presence of a regulated xylA gene on the recombinant plasmid. The nucleotide sequence (4520 bases) revealed three open reading frames with the same polarity. They were identified by sequence homologies as xylR, encoding the Xyl repressor, xylA, encoding xylose isomerase and xylB, encoding xylulokinase. Primer extension analyses indicated constitutive transcription of xylR and xylose-inducible transcription of xylA. Promoter consensus sequences were found upstream of both transcriptional start sites. A transcriptional terminator between xylR and xylA separates the different transcriptional units. Potential regulatory elements were identified by sequence analysis and suggest a repressor-operator mechanism for the regulation of xylAB expression.

Globally tuberculosis (TB) is one of the leading causes of death due to an infectious disease second only to HIV/AIDS. Estimates suggest that approximately one-third of the worlds population is infected with Mycobacterium tuberculosis the microbe that causes TB and approximately 10% of infected individuals will develop active TB at some point in their lives. For individuals also infected with HIV the likelihood of developing active TB after infection is much higher. In 2006 approximately 9.2 million people globally developed active TB and it is estimated that 1.7 million people died as a result of TB including 200000 HIV-infected individuals. Although estimates suggest that the rates of new cases and deaths due to TB show signs of slowing throughout the world recent increases in rates of drug-resistant TB have the potential to reverse these gains. (excerpt)

The 2012 Strategic Blueprint for tuberculosis (TB) vaccines presents a cogent vision for developing and introducing safe and effective TB vaccines within a decade. This document offers a valuable reappraisal of how we can best move this critical area of research forward to help achieve our ultimate goal of eliminating tuberculosis worldwide. The new Blueprint builds on more than 10 years of exceptional progress in TB science and product development since the 2000 publication of the first Blueprint, which resulted from a workshop organized by the U.S. National Institutes of Health (NIH). For a research field that began to receive serious attention and commitment of substantial resources only in the mid1990s, remarkable gains have been achieved. The framework laid out in the original Blueprint for creating a sustainable infrastructure for the clinical development of candidate TB vaccines is updated in the current plan to reflect new knowledge gained from studying multiple vaccine candidates in preclinical studies and human clinical trials. This update will enable us to refine our approach and define research gaps that must be filled if we are to make licensure of new TB vaccines a reality. The NIH welcomes the publication of the 2012 Blueprint, and we look forward to continuing to work with the many committed scientists and our public and private partners in the global effort to control and ultimately end the global TB pandemic. TB control programs have led to a recent worldwide decline in the number of people falling ill and dying from TB. However, as noted in the 2011 Global Plan to Stop TB, novel drugs, rapid diagnostics, and, importantly, an effective vaccine are all needed to accelerate the decline of TB cases worldwide and drive TB toward elimination. Multifaceted global efforts will be required to reach this goal. As part of the international team dedicated to TB elimination, NIH will sustain and accelerate our support of the basic and clinical research that plays a critical role in developing medical countermeasures for TB, primarily through research supported by the National Institute of Allergy and Infectious Diseases (NIAID). NIAID’s strategic goals for the developing new TB vaccines are well complemented by and reflected in the new Blueprint. In U.S.-based research and collaborations conducted in more

The luxA,B genes from the Gram-negative marine bacterium Vibrio harveyi MAV were used in Staphylococcus carnosus TM300 as a reporter system for regulated expression of xylose utilization. The luciferase genes were fused to the xyl operon from Staphylococcus xylosus C2a. Expression of bioluminescence was induced through addition of xylose and repressed in the presence of glucose. A method to quantitate bioluminescence directly from the culture is described.

A saturating oligonucleotide-directed mutagenesis of both tet operators in the tet regulatory sequence was performed yielding mutants with four identical base pair exchanges at equivalent positions in the four tet operator half sides. The mutants were cloned between bipolar lacZ and galK indicator genes on a multicopy plasmid allowing the quantitative analysis of their effects in vivo. In the absence of Tet repressor the mutations lead to considerably different expression levels of both genes. They are discussed with respect to the promoter consensus sequences. In particular, the -10 region of the in vivo active tetPR2 promoter is unambiguously defined by these results. In the presence of Tet repressor most of the mutants exhibit a lower affinity for that protein as determined quantitatively by their reduced expression levels. In general, tet operator recognition is most strongly affected by alterations of base pairs near the center of the palindromic sequence. The most important position is the third base pair, followed by base pairs two, four, five and six, the latter showing similar effects as base pair one. At each position, the four possible base pairs show different affinities for Tet repressor. They are discussed according to their exposure of H-bond donors and -acceptors in the major and minor grooves of the B-DNA. The results are in agreement with major groove contacts at positions two, three and five. At position four a low potential correlation of efficiencies with the H-bonding in the minor groove is found, while mutations at position six seem to influence repressor binding by other mechanisms.

Xylose-inducible vectors have been constructed for extracellular production of antibody fragments in Staphylococcus carnosus. The pre-pro sequence of S. hyicus lipase was taken as secretional signal sequence, and the S. xylosus Xyl repressor was used to confer xylose inducibility of transcription. Cleavage sites for the IgA protease were engineered between the pre-pro sequence and the antibody fragments to permit removal of the pro sequence. Extracellular expression of the light chain and the Fd fragment of a chimeric Fab fragment containing the variable regions of the anti-lysozyme antibody D1.3 was achieved with these vectors. The pro sequence could be removed from the expression product by IgA protease treatment. When the light chain and the Fd fragment were co-secreted as a protein fusion they accumulated in a structure capable of heterodimerization after IgA cleavage. This fusion contains the pre-pro sequence followed by the light chain, a second IgA site and the Fd fragment.