T. Mark Doherty

The success and failure of BCG — implications for a novel tuberculosis vaccine

Over the past 50 years, the Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccine against tuberculosis (TB) has maintained its position as the worlds most widely used vaccine, despite showing highly variable efficacy (0–80%) in different trials. The efficacy of BCG in adults is particularly poor in tropical and subtropical regions. Studies in animal models of TB, supported by data from clinical BCG trials in humans, indicate that this failure is related to pre-existing immune responses to antigens that are common to environmental mycobacteria and Mycobacterium tuberculosis. Here, we discuss the potential mechanisms behind the variation of BCG efficacy and their implications for an improved TB vaccination strategy.

Biomarkers and diagnostics for tuberculosis: progress, needs, and translation into practice

Human infection with Mycobacterium tuberculosis can progress to active disease, be contained as latent infection, or be eradicated by the host response. Tuberculosis diagnostics classify a patient into one of these categories. These are not fixed distinct states, but rather are continua along which patients can move, and are affected by HIV infection, immunosuppressive therapies, antituberculosis treatments, and other poorly understood factors. Tuberculosis biomarkers-host or pathogen-specific-provide prognostic information, either for individual patients or study cohorts, about these outcomes. Tuberculosis case detection remains difficult, partly because of inaccurate diagnostic methods. Investments have yielded some progress in development of new diagnostics, although the existing pipeline is limited for tests for sputum-smear-negative cases, childhood tuberculosis, and accurate prediction of reactivation of latent tuberculosis. Despite new, sensitive, automated molecular platforms for detection of tuberculosis and drug resistance, a simple, inexpensive point-of-care test is still not available. The effect of any new tests will depend on the method and extent of their introduction, the strength of the laboratories, and the degree to which access to appropriate therapy follows access to diagnosis. Translation of scientific progress in biomarkers and diagnostics into clinical and public health programmes is possible-with political commitment, increased funding, and engagement of all stakeholders.

Immune Responses to the Mycobacterium tuberculosis-Specific Antigen ESAT-6 Signal Subclinical Infection among Contacts of Tuberculosis Patients

ABSTRACT Diagnosis of latent Mycobacterium tuberculosis infection is considered essential for tuberculosis control but is hampered by the lack of specific reagents. We report that strong recognition of tuberculosis complex-specific antigen ESAT-6 by healthy household contacts of tuberculosis patients correlates with the subsequent development of active tuberculosis during a 2-year follow-up period.

Exchanging ESAT6 with TB10.4 in an Ag85B Fusion Molecule-Based Tuberculosis Subunit Vaccine: Efficient Protection and ESAT6-Based Sensitive Monitoring of Vaccine Efficacy

Previously we have shown that Ag85B-ESAT-6 is a highly efficient vaccine against tuberculosis. However, because the ESAT-6 Ag is also an extremely valuable diagnostic reagent, finding a vaccine as effective as Ag85B-ESAT-6 that does not contain ESAT-6 is a high priority. Recently, we identified a novel protein expressed by Mycobacterium tuberculosis designated TB10.4. In most infected humans, TB10.4 is strongly recognized, raising interest in TB10.4 as a potential vaccine candidate and substitute for ESAT-6. We have now examined the vaccine potential of this protein and found that vaccination with TB10.4 induced a significant protection against tuberculosis. Fusing Ag85B to TB10.4 produced an even more effective vaccine, which induced protection against tuberculosis comparable to bacillus Calmette-Guérin vaccination and superior to the individual Ag components. Thus, Ag85B-TB10 represents a new promising vaccine candidate against tuberculosis. Furthermore, having now exchanged ESAT-6 for TB10.4, we show that ESAT-6, apart from being an excellent diagnostic reagent, can also be used as a reagent for monitoring vaccine efficacy. This may open a new way for monitoring vaccine efficacy in clinical trials.

Recognition of Stage-Specific Mycobacterial Antigens Differentiates between Acute and Latent Infections with Mycobacterium tuberculosis

ABSTRACT Mycobacterium tuberculosis is estimated to infect 80 to 100 million people annually, the majority of whom do not develop clinical tuberculosis (TB) but instead maintain the infection in a latent state. These individuals generally become positive in response to a tuberculin skin test and may develop clinical TB at a later date, particularly if their immune systems are compromised. Latently infected individuals are interesting for two reasons. First, they are an important reservoir of M. tuberculosis, which needs to be considered for TB control. Second, if detected prior to recrudescence of the disease, they represent a human population that is making a protective immune response to M. tuberculosis, which is very important for defining correlates of protective immunity. In this study, we show that while responsiveness to early secretory antigenic target 6 is a good marker for M. tuberculosis infection, a strong response to the 16-kDa Rv2031c antigen (HspX or α-crystallin) is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis of, or therapeutic vaccination against, TB.

Ag85B-ESAT-6 adjuvanted with IC31® promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in naïve human volunteers.

Though widely used, the BCG vaccine has had little apparent effect on rates of adult pulmonary tuberculosis. Moreover, the risk of disseminated BCG disease in immunocompromised individuals means that improved TB vaccines ideally need to be able to efficiently prime mycobacterially-naïve individuals as well as boost individuals previously vaccinated with BCG. Protective immunity against Mycobacterium tuberculosis is thought to depend on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-gamma) production. In the present study, we monitored safety and IFN-gamma responses in healthy TB-naïve humans receiving an entirely novel vaccine, composed of the fusion protein Ag85B-ESAT-6, administered at 0 and 2 months either as recombinant protein alone or combined with two concentrations of the novel adjuvant IC31. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against H1 and both the Ag85B and the ESAT-6 components. These strong responses persisted through 2.5 years of follow-up, indicating the induction of a substantial memory response in the vaccine recipients.

Biomarkers for tuberculosis disease activity, cure, and relapse

New drugs, vaccines, and other therapies will be required to realise the goal of global tuberculosis elimination or control. This Review covers the important role biomarkers can have in accelerating drug development by providing validated surrogate endpoints that can bring enhanced statistical power to small short studies. Candidate biomarkers should differentiate people with active tuberculosis from healthy individuals, normalise with therapy, and reproducibly predict clinical outcomes in diverse patient populations. Although a large number of promising candidate biomarkers have been examined to date, few patients in these studies have reached clinically meaningful outcomes, and few of the studies have been conducted to international research standards. These markers must be further studied in tuberculosis treatment trials to evaluate the kinetics of the responses and their relation to long-term clinical outcomes. These studies will benefit from multidisciplinary collaborations including microbiologists, immunologists, clinicians, tuberculosis control personnel, and the pharmaceutical and biotechnology industry.

Healthy Individuals That Control a Latent Infection with Mycobacterium tuberculosis Express High Levels of Th1 Cytokines and the IL-4 Antagonist IL-4δ2

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop disease and identifying what constitutes “protective immunity” is one of the holy grails of M. tuberculosis immunology. It is known that IFN-γ is essential for protection, but it is also apparent that IFN-γ levels alone do not explain the immunity/susceptibility dichotomy. The controversy regarding correlates of immunity persists because identifying infected but healthy individuals (those who are immune) has been problematic. We have therefore used recognition of the M. tuberculosis virulence factor early secretory antigenic target 6 to identify healthy, but infected individuals from tuberculosis (TB)-endemic and nonendemic regions (Ethiopia and Denmark) and have compared signals for cytokines expressed directly ex vivo with the pattern found in TB patients. We find that TB patients are characterized by decreased levels of Th1 cytokines and increased levels of IL-10 compared with the healthy infected and noninfected community controls. Interestingly, the healthy infected subjects exhibited a selective increase of message for the IL-4 antagonist, IL-4δ2, compared with both TB patients or noninfected individuals. These data suggest that long-term control of M. tuberculosis infection is associated not just with elevated Th1 responses but also with inhibition of the Th2 response.

Mucosal Administration of Ag85B-ESAT-6 Protects against Infection with Mycobacterium tuberculosis and Boosts Prior Bacillus Calmette-Guérin Immunity

We have examined the intranasal administration of a vaccine against Mycobacterium tuberculosis (M.tb) consisting of the mucosal adjuvant LTK63 and the Ag Ag85B-ESAT-6. Vaccination with LTK63/Ag85B-ESAT-6 gave a strong and sustained Th1 response mediated by IFN-γ-secreting CD4 cells, which led to long-lasting protection against tuberculosis, equivalent to that observed with bacillus Calmette-Guérin (BCG) or Ag85B-ESAT-6 in dimethyldioctadecylammonium bromide/monophosphoryl lipid A. Because a crucial element of novel vaccine strategies is the ability to boost BCG-derived immunity, we also tested whether LTK63/Ag85B-ESAT-6 could act as a BCG booster vaccine in BCG-vaccinated mice. We found that vaccinating with LTK63/Ag85B-ESAT-6 strongly boosted prior BCG-stimulated immunity. Compared with BCG-vaccinated nonboosted mice, we observed that infection with M.tb led to a significant increase in anti-M.tb-specific CD4 T cells in the lungs of LTK63/Ag85B-ESAT-6-boosted animals. This correlated with a significant increase in the protection against M.tb in LTK63/Ag85B-ESAT-6-boosted mice, compared with BCG-vaccinated animals. Thus, LTK63/Ag85B-ESAT-6 represents an efficient preventive vaccine against tuberculosis with a strong ability to boost prior BCG immunity.

Comparison of different standards for real-time PCR-based absolute quantification

Quantitative real-time PCR (qPCR) is a powerful tool used for both research and diagnostic, which has the advantage, compared to relative quantification, of providing an absolute copy number for a particular target. However, reliable standards are essential for qPCR. In this study, we have compared four types of commonly-used standards--PCR products (with and without purification) and cloned target sequences (circular and linear plasmid) for their stability during storage (using percentage of variance in copy numbers, PCR efficiency and regression curve correlation coefficient (R(2))) using hydrolysis probe (TaqMan) chemistry. Results, expressed as copy numbers/microl, are presented from a sample human system in which absolute levels of HuPO (reference gene) and the cytokine gene IFN-gamma were measured. To ensure the suitability and stability of the four standards, the experiments were performed at 0, 7 and 14 day intervals and repeated 6 times. We have found that the copy numbers vary (due to degradation of standards) over the period of time during storage at 4 degrees C and -20 degrees C, which affected PCR efficiency significantly. The cloned target sequences were noticeably more stable than the PCR product, which could lead to substantial variance in results using standards constructed by different routes. Standard quality and stability should be routinely tested for assays using qPCR.