Influence of Advanced Age on Mycobacterium bovis BCG Vaccination in Guinea Pigs Aerogenically Infected with Mycobacterium tuberculosis

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only tuberculosis (TB) vaccine currently available, but its efficacy against adult pulmonary TB remains controversial. BCG induces specific immune responses to mycobacterial antigens and may elicit protective immunity against TB. TB remains a major public health problem, especially among the elderly, yet the efficacy of BCG in the elderly is unknown. We investigated the ability of BCG vaccination to prevent TB in young (6-week-old), middle-aged (18-month-old), and old (60-month-old) guinea pigs. BCG-Tokyo vaccination reduced the growth of Mycobacterium tuberculosis H37Rv in all three groups. By use of an enzyme-linked immunospot (ELISPOT) assay, antigen-specific gamma interferon (IFN-γ)-producing cells were detected in the 60-month-old guinea pigs after a booster vaccination with BCG-Tokyo. Our findings suggest that BCG-Tokyo has a protective effect against tuberculosis infection regardless of age.Tuberculosis (TB) remains a major public health problem, especially among elderly people. Patients ≥60 years of age account for ≥50% of new cases in Japan (29). The increasing susceptibility of the elderly to Mycobacterium tuberculosis is generally thought to be associated with age-related changes in immune system function, especially losses or delays in antigen-specific CD4⁺ T-cell function (14). Compromised antigen-specific CD4⁺ T-cell responses may contribute to increased susceptibility to M. tuberculosis infection in mice (27).Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only TB vaccine currently available. BCG has been used for more than 80 years (41), and vaccination with BCG is the standard for TB prevention in most countries. BCG induces specific immune responses to mycobacterial antigens and may elicit protective immunity against tuberculosis. BCG provides efficient protection against severe and disseminated TB, such as tuberculosis meningitis and miliary tuberculosis, in children (33, 34, 40). Although the long-term efficacy of BCG has been documented (3, 6), with several reports indicating efficient protection against disseminated TB in newborns and children, it appears to have less efficacy against adult pulmonary TB (2). In fact, its efficacy against pulmonary TB in both adults and the elderly is controversial, as is the efficacy of revaccination (5).In the present study, we examined the efficacy of BCG against TB at different ages in a common guinea pig model (15, 25, 30). We used three age-segregated groups—young (6 weeks old), middle-aged (18 months old), and old (60 months old)—and we measured the number of antigen-specific gamma interferon (IFN-γ)-producing cells as an indicator of the efficacy of the vaccine against TB.     

Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ⁺/TNF⁺) and IFN-γ⁺ CD4⁺ T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.     

Development of a Murine Mycobacterial Growth Inhibition Assay for Evaluating Vaccines against Mycobacterium tuberculosis

The development and characterization of new tuberculosis (TB) vaccines has been impeded by the lack of reproducible and reliable in vitro assays for measuring vaccine activity. In this study, we developed a murine in vitro mycobacterial growth inhibition assay for evaluating TB vaccines that directly assesses the capacity of immune splenocytes to control the growth of Mycobacterium tuberculosis within infected macrophages. Using this in vitro assay, protective immune responses induced by immunization with five different types of TB vaccine preparations (Mycobacterium bovis BCG, an attenuated M. tuberculosis mutant strain, a DNA vaccine, a modified vaccinia virus strain Ankara [MVA] construct expressing four TB antigens, and a TB fusion protein formulated in adjuvant) can be detected. Importantly, the levels of vaccine-induced mycobacterial growth-inhibitory responses seen in vitro after 1 week of coculture correlated with the protective immune responses detected in vivo at 28 days postchallenge in a mouse model of pulmonary tuberculosis. In addition, similar patterns of cytokine expression were evoked at day 7 of the in vitro culture by immune splenocytes taken from animals immunized with the different TB vaccines. Among the consistently upregulated cytokines detected in the immune cocultures are gamma interferon, growth differentiation factor 15, interleukin-21 (IL-21), IL-27, and tumor necrosis factor alpha. Overall, we have developed an in vitro functional assay that may be useful for screening and comparing new TB vaccine preparations, investigating vaccine-induced protective mechanisms, and assessing manufacturing issues, including product potency and stability.The tuberculosis (TB) epidemic is a global public health tragedy that is being fueled by the spread of human immunodeficiency virus/AIDS and the increasing incidence of multiple-drug-resistant Mycobacterium tuberculosis strains. Annually, about 2 million people worldwide die from tuberculosis and 8 to 9 million new cases of this disease are reported (34). Although the current TB vaccine, Mycobacterium bovis BCG, has been widely used for decades, its effectiveness has been shown to be highly variable in well-controlled clinical trials (5). While immunization with BCG is effective against severe childhood disease, BCG does not adequately protect against the most prevalent form of the disease, adult pulmonary tuberculosis (13). Vaccinated individuals who become infected with TB are susceptible to disease progression when the BCG-induced immune responses are suppressed or wane with time (32). Clearly, to curb the global TB epidemic, more effective immunization strategies must be generated and evaluated.The development of new vaccines against TB has been hindered by our limited understanding of the mechanisms of protective immunity against M. tuberculosis. While it is known that acquired cellular immune responses are critical for controlling tuberculosis infections, the cell subsets that confer antituberculosis protective immunity have not been adequately defined (14). In addition, the immune mechanisms that are responsible for inhibiting the intracellular growth of M. tuberculosis have not been fully delineated and the surrogate biomarkers of this growth inhibition remain unknown. Because it is difficult to study the multiple components of the immune system and their numerous interactions in vivo, the development of an in vitro system which models the in vivo immune responses should facilitate the identification of antituberculosis protective immune mechanisms. The availability of a relevant in vitro assay should allow a more direct study of the mediators of protective immunity against M. tuberculosis in a controlled system. Although in vitro mycobacterial growth inhibition assays for human cells have been developed and are being characterized for their capacity to detect vaccine-induced immunogenicity in human clinical trials, the development and assessment of preclinical assays to measure vaccine-induced activity against M. tuberculosis has thus far been limited (4, 6, 16, 18, 30, 35).To accelerate TB vaccine development and investigations of protective immune mechanisms, we initiated studies aimed at developing a murine in vitro functional assay for evaluating the protective activity of TB vaccines. For this assay, antituberculosis protection was evaluated by targeting an important end point, the control of M. tuberculosis growth within its primary host cell, the macrophage. By assessing the immune-mediated inhibition of mycobacterial growth, we hypothesized that our results would correlate more directly with in vivo protection than the measurement of other immune responses, including cytokine expression. In addition to assessing cellular immune mechanisms, a relevant in vivo assay could be useful for screening and comparing new TB vaccine candidates. From a manufacturing viewpoint, a standardized in vitro functional assay could also be adapted to measure vaccine potency, lot-to-lot production consistency, and vaccine stability.Here we describe our initial results from the characterization of a murine in vitro functional assay for assessing the activity of TB vaccines. We show that vaccine-induced protection seen in vitro for five different TB vaccines correlates with the antituberculosis protective immunity detected in a mouse model of pulmonary TB. Also, we establish an in vitro profile of cytokine expression which is associated with the activity of BCG vaccine and demonstrate that similar in vitro cytokine responses were detected for the four other types of TB vaccines that were tested in this study.     

Evaluation of the Immunogenicity of Mycobacterium bovis BCG Delivered by Aerosol to the Lungs of Macaques

Nine million cases of tuberculosis (TB) were reported in 2013, with a further 1.5 million deaths attributed to the disease. When delivered as an intradermal (i.d.) injection, the Mycobacterium bovis BCG vaccine provides limited protection, whereas aerosol delivery has been shown to enhance efficacy in experimental models. In this study, we used the rhesus macaque model to characterize the mucosal and systemic immune response induced by aerosol-delivered BCG vaccine. Aerosol delivery of BCG induced both Th1 and Th17 cytokine responses. Polyfunctional CD4 T cells were detected in bronchoalveolar lavage (BAL) fluid and peripheral blood mononuclear cells (PBMCs) 8 weeks following vaccination in a dose-dependent manner. A similar trend was seen in peripheral gamma interferon (IFN-γ) spot-forming units measured by enzyme-linked immunosorbent spot (ELISpot) assay and serum anti-purified protein derivative (PPD) IgG levels. CD8 T cells predominantly expressed cytokines individually, with pronounced tumor necrosis factor alpha (TNF-α) production by BAL fluid cells. T-cell memory phenotype analysis revealed that CD4 and CD8 populations isolated from BAL fluid samples were polarized toward an effector memory phenotype, whereas the frequencies of peripheral central memory T cells increased significantly and remained elevated following aerosol vaccination. Expression patterns of the α4β1 integrin lung homing markers remained consistently high on CD4 and CD8 T cells isolated from BAL fluid and varied on peripheral T cells. This characterization of aerosol BCG vaccination highlights features of the resulting mycobacterium-specific immune response that may contribute to the enhanced protection previously reported in aerosol BCG vaccination studies and will inform future studies involving vaccines delivered to the mucosal surfaces of the lung.     

Investigating the Induction of Vaccine-Induced Th17 and Regulatory T Cells in Healthy,Mycobacterium bovis BCG-Immunized Adults Vaccinated with a New Tuberculosis Vaccine,MVA85A

Tuberculosis (TB) remains a threat to global health. While advances in diagnostics and treatment are crucial to the containment of the epidemic, it is likely that elimination of the disease can only be achieved through vaccination. Vaccine-induced protection from Mycobacterium tuberculosis is dependent, at least in part, on a robust Th1 response, yet little is known of the ability of TB vaccines to induce other T-cell subsets which may influence vaccine efficacy. Interleukin-17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells which has been associated with both immune pathology and protection against infectious disease. Following vaccination with MVA85A, a viral vector vaccine aimed at enhancing immune responses to M. tuberculosis, antigen-specific IL-17A-producing T cells were induced in the peripheral blood of healthy volunteers. These T cells are detected later than gamma interferon (IFN-γ)-secreting T cells and are of a low magnitude. Preexisting immune responses to mycobacterial antigens were associated with higher CD4⁺ CD25^hi CD39⁺ T-cell levels in the periphery and a reduced capacity to produce IL-17A following immunization. These data highlight the intricate balance of effector and regulatory immune responses induced by vaccination and that preexisting immunity to mycobacterial antigens may affect the composition of vaccine-induced T-cell subsets.Tuberculosis (TB) remains a global health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis, HIV-TB coinfection, and failure of the BCG vaccine to control adult pulmonary TB (9, 13). There is evidence that protection from M. tuberculosis is, at least in part, dependent on a robust Th1 response and the secretion of gamma interferon (IFN-γ) by antigen-specific T cells (1, 15, 22). Although IFN-γ alone is not sufficient for protection, in the absence of a better biomarker, early clinical trials of new TB vaccines use antigen-specific IFN-γ production as the primary gauge of vaccine-induced immune responses (16). In early clinical trials, modified vaccinia virus Ankara expressing antigen 85A from M. tuberculosis (MVA85A), a subunit vaccine designed to increase immune protection conferred by BCG, has been found to induce high levels of antigen-specific IFN-γ-secreting CD4⁺ T cells in individuals previously vaccinated with BCG (28, 29, 31).CD4⁺ T cells can differentiate into diverse effector cell subsets upon antigenic stimulation and the classical Th1/Th2 paradigm has now been expanded to include Th17 and T-regulatory (Treg) cells. Th17 cells are potent inflammatory cells which produce interleukin-17A (IL-17A) as their hallmark cytokine (17, 30). Th17 cells are mainly known for their role in mediating autoimmune pathology (19, 35) but are also thought to be involved in mediating protection against certain extracellular pathogens and fungi which are not effectively cleared by Th1- and Th2-type responses (12, 20). In contrast to Th17 cells, Treg cells comprise a regulatory cell subset of CD4⁺ T cells which act to suppress T-cell responses and are thereby thought to prevent pathology from chronic or excessive immune responses (21, 32, 33).Although a role has been defined for Th17 cells and Treg cells in other diseases, their role in TB remains unclear and even less is known about the effect of vaccination on these T-cell subsets.Clinical trials evaluating the safety and immunogenicity of MVA85A in BCG-vaccinated adults provide an opportunity to further investigate the induction and dynamics of vaccine-induced Th17 cells and Treg cells. Determining the effect of vaccination with MVA85A on these T-cell subsets is important, as protection from TB is likely to be dependent not only upon a Th1 response but also upon the balance between this effector response and the Th17 and Treg responses.     

Intranasal Administration of Mycobacterium bovis BCG Induces Superior Protection against Aerosol Infection with Mycobacterium tuberculosis in Mice

Despite the widespread use of Mycobacterium bovis BCG, the only licensed vaccine against tuberculosis (TB), TB remains a global epidemic. To assess whether more direct targeting of the lung mucosa by respiratory immunization would enhance the potency and longevity of BCG-induced anti-TB protective immunity, the long-term impact of intranasal (i.n.) BCG vaccination was compared to conventional subcutaneous (s.c.) immunization by using a mouse model of pulmonary tuberculosis. Although significantly improved protection in the lung was seen at early time points (2 and 4 months postvaccination) in i.n. BCG-immunized mice, no differences in pulmonary protection were seen 8 and 10 months postvaccination. In contrast, in all of the study periods, i.n. BCG vaccination induced significantly elevated protective splenic responses relative to s.c. immunization. At five of nine time points, we observed a splenic protective response exceeding 1.9 log₁₀ protection relative to the s.c. route. Furthermore, higher frequencies of CD4 T cells expressing gamma interferon (IFN-γ) and IFN-γ/tumor necrosis factor alpha, as well as CD8 T cells expressing IFN-γ, were detected in the spleens of i.n. vaccinated mice. Using PCR arrays, significantly elevated levels of IFN-γ, interleukin-9 (IL-9), IL-11, and IL-21 expression were also seen in the spleen at 8 months after respiratory BCG immunization. Overall, while i.n. BCG vaccination provided short-term enhancement of protection in the lung relative to s.c. immunization, potent and extremely persistent splenic protective responses were seen for at least 10 months following respiratory immunization.     

Differentiation between Mycobacterium bovis BCG-Vaccinated and M. bovis-Infected Cattle by Using Recombinant Mycobacterial Antigens

Tuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection with Mycobacterium tuberculosis or Mycobacterium bovis and vaccination with M. bovis BCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins of M. bovis—MPB59, MPB64, MPB70, and ESAT-6—were tested in a whole-blood gamma interferon (IFN-γ) assay for differentiation between cattle vaccinated with BCG and those experimentally infected with M. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected with M. bovis in BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-γ responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which were M. bovis culture positive following challenge produced IFN-γ responses to PPDB and ESAT-6 which were significantly stronger than those observed in the corresponding M. bovis culture-negative animals. IFN-γ responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-γ assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.     

A Novel Tuberculosis DNA Vaccine in an HIV-1 p24 Protein Backbone Confers Protection against Mycobacterium tuberculosis and Simultaneously Elicits Robust Humoral and Cellular Responses to HIV-1

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major infectious disease worldwide. Moreover, latent M. tuberculosis infection is more likely to progress to active TB and eventually leads to death when HIV infection is involved. Thus, it is urgent to develop a novel TB vaccine with immunogenicity to both M. tuberculosis and HIV. In this study, four uncharacterized T cell epitopes from MPT64, Ag85A, Ag85B, and TB10.4 antigens of M. tuberculosis were predicted, and HIV-1-derived p24, an immunodominant protein that can induce protective responses to HIV-1, was used as an immunogenic backbone. M. tuberculosis epitopes were incorporated separately into the gene backbone of p24, forming a pP24-Mtb DNA vaccine. We demonstrated that pP24-Mtb immunization induced a strong M. tuberculosis-specific cellular response as evidenced by T cell proliferation, cytotoxicity, and elevated frequency of gamma interferon (IFN-γ)-secreting T cells. Interestingly, a p24-specific cellular response and high levels of p24-specific IgG were also induced by pP24-Mtb immunization. When the protective effect was assessed after mycobacterial challenge, pP24-Mtb vaccination significantly reduced tissue bacterial loads and profoundly attenuated the mycobacterial infection-related lung inflammation and injury. Our findings demonstrated that the pP24-Mtb tuberculosis vaccine confers effective protection against mycobacterial challenge with simultaneously elicited robust immune responses to HIV-1, which may provide clues for developing novel vaccines to prevent dual infections.     

Recombinant Mycobacterium bovis BCG Expressing the Chimeric Protein of Antigen 85B and ESAT-6 Enhances the Th1 Cell-Mediated Response

The chimeric protein that relies on the T-cell epitopes of antigen 85B (Ag85B) and the 6-kDa early secreted antigen target (ESAT-6) has been demonstrated to augment the Th1 immune response. In this study, we developed a recombinant Mycobacterium bovis BCG (rBCG) strain that secretes the chimeric protein of Ag85B and ESAT-6 (rBCG-A_N-E-A_C). Immunization with this rBCG strain induced stronger antigen-specific gamma interferon (IFN-γ) activities, as determined by an enzyme-linked immunospot assay, and higher levels of antigen-specific CD4⁺ and CD8⁺ T-cell responses than those in the control groups immunized with either rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) or BCG. Likewise, rBCG-A_N-E-A_C significantly increased the level of production of the major Th1 cytokines IFN-γ and tumor necrosis factor alpha in splenocyte cultures to levels comparable to those elicited by control BCG. Moreover, the antigen-specific immunoglobulin 2c (IgG2c)/IgG1 ratio for mice immunized with rBCG-A_N-E-A_C was also much higher than the ratios for the other immunized groups. Together, these results indicate that this rBCG-A_N-E-A_C strain enhances the Th1 cell-mediated response and may serve as a potential vaccine against M. tuberculosis.Mycobacterium bovis bacillus Calmette Guérin (BCG) is the only vaccine against tuberculosis (TB) currently available and exhibits various levels of efficacy for the prevention of pulmonary TB (range, 0 to 80%) in different trials (9). BCG has a protective effect in children, particularly against tuberculous meningitis; however, it does not satisfactorily prevent the development of pulmonary TB in adults and fails to protect individuals against reinfection (1). Given the rate of mortality from TB worldwide, with more 8 million new cases and 2 million deaths occurring annually (2), newer strategies need to be implemented to improve BCG or vaccines more effective than BCG urgently need to be developed.One approach that might be used to increase the efficacy of BCG could be to construct a recombinant BCG (rBCG) which either overexpresses immunogenic antigens or modulates the ensuing immune response (8). rBCG vaccines are attractive because of the widespread experience with their use, the known immunogenicity associated with protection against the worst forms of the disease in children, and the safety profiles of standard BCG strains (13). Two rBCG vaccines have been entered into clinical trials. This includes rBCG30, which expresses the antigen 85B (Ag85B) protein, and ΔureC hly-positive rBCG, which expresses listeriolysin and which is urease deficient (12, 15). It is hoped that these vaccines will provide a strong and perhaps longer-lasting immune response than that achieved with the conventional BCG vaccine.The most effective defined-antigen TB vaccines will likely require the induction of both cell-mediated and humoral immune responses. Ag85B and the 6-kDa early secreted antigen target (ESAT-6) have been identified as two of the most promising vaccine candidates which are strongly recognized by T lymphocytes (3, 19). In a previous study, we relied on the T-cell epitopes of Ag85B and ESAT-6 to design a chimeric protein by inserting ESAT-6 into Ag85B from amino acids 167 to 182 and demonstrated that this recombination of Ag85B and ESAT-6 could improve the immunogenicity and enhance the T-helper type 1 (Th1) cell-mediated immune response (27). This finding prompted us to explore further the efficacy of rBCG overexpressing this chimeric protein. In this study, we constructed rBCG expressing chimeric protein Ag85B_N-ESAT-6-Ag85B_C (rBCG-A_N-E-A_C) and further compared the immune response to that protein with that to rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) and BCG.     

Diagnosis of Active Tuberculosis in China Using an In-House Gamma Interferon Enzyme-Linked Immunospot Assay

Gamma interferon (IFN-γ) release assays have been proven to be useful in the diagnosis of Mycobacterium tuberculosis infection. Nevertheless, their specificity and sensitivity vary among the different populations studied. Here, we evaluate the value of an in-house IFN-γ enzyme-linked immunospot (ELISPOT) assay in the diagnosis of active tuberculosis (TB) in Shenzhen, China, where the prevalence of tuberculosis is severe and Mycobacterium bovis BCG vaccination is mandatory at birth. A total of 305 patients with active tuberculosis, 18 patients with nontuberculosis lung diseases, and 202 healthy controls were recruited in this study. Among them, 156 individuals were simultaneously tested for IFN-γ responses by the commercial QuantiFERON-TB Gold in-tube (QFT-IT) assay. Tuberculin skin tests (TST) were performed with 202 healthy controls. The overall sensitivities of the ELISPOT and QFT-IT assays for active tuberculosis were 83.60% and 80.85%, respectively; the specificities were 76.6% and 73.26%, respectively. The IFN-γ ELISPOT responses, but not those of the TST, were significantly correlated with TB exposure (r = −0.6040, P < 0.0001). The sensitivities of the ELISPOT assay varied for patients with different forms of tuberculosis, with the highest sensitivity for patients with sputum-positive pulmonary tuberculosis (89.89%) and the lowest for those with tuberculous meningitis (62.5%). In conclusion, the IFN-γ ELISPOT assay is a useful adjunct to current tests for diagnosis of active TB in China. The ELISPOT assay is more accurate than TST in identifying TB infections.Tuberculosis (TB) is a leading cause of morbidity and mortality throughout the world, with 95% of cases and 97% of all deaths occurring in high-prevalence countries, such as China, where the prevalence of active TB is as high as 367/100,000 population (10). For the effective and efficient control of TB in these countries, rapid diagnosis and treatment for active-TB patients are the mainstays of the TB control program. However, the current widely used tests, including acid-fast staining of sputum, mycobacterial culture, and antibody test, are not satisfactory for this purpose (4).Recently, commercial immunodiagnostic tests for TB infection have been introduced. These tests are based on the Mycobacterium tuberculosis-specific antigens early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) and include a whole-blood gamma interferon (IFN-γ) enzyme-linked immunosorbent assay (QuantiFERON-TB Gold in-tube [QFT-IT]; Cellestis Ltd, Victoria, Australia) and an enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB; Oxford Immunotec, Oxfordshire, United Kingdom). Both tests have shown promising results in the detection of latent TB infection (LTBI) (1, 11). In addition, some clinical data also suggest the potential to use these IFN-γ assays for the differential diagnoses of active tuberculosis (3, 8, 9). However, the sensitivities and specificities of these assays varied among the different populations studied, due mostly to the different HLA genetic backgrounds, the prevalence of TB infection, and the coverage of Mycobacterium bovis BCG vaccination (11).In contrast to the wide use of IFN-γ assays for the diagnosis of M. tuberculosis infection in Europe and America, the utilization of IFN-γ assays in China is scarce, and no data are available to evaluate the diagnostic value of the QFT-IT assay mainly because of the high cost of these kits. Since China ranks second on the list of 22 countries with the highest tuberculosis burden in the world (11), the aim of this study was to evaluate the usefulness of IFN-γ assays in the diagnosis of active tuberculosis in the Chinese population in mainland China. Thus, we developed and evaluated an in-house IFN-γ ELISPOT assay by using recombinant ESAT-6 protein and peptide pools derived from ESAT-6 and CFP-10 for diagnosis of active TB. We also compared the performance of our ELISPOT assay with that of the commercial QFT-IT assay and analyzed the influence of clinical manifestation on the accuracy of the ELISPOT assay.     

Bactericidal Activity in Whole Blood as a Potential Surrogate Marker of Immunity after Vaccination against Tuberculosis

The development of new tuberculosis (TB) vaccines will require the identification of correlates of human protection. This study examined the balance between immunity and virulence in a whole blood infection model in which intracellular mycobacterial survival was measured using BACTEC. In the blood of tuberculin-negative donors, counts of Mycobacterium tuberculosis H₃₇Ra organisms fell by 0.14 log₁₀ CFU during 96 h of whole blood culture, whereas counts of Mycobacterium bovis BCG, M. tuberculosis H₃₇Rv, and a clinical TB isolate's organisms increased by 0.13, 0.43, and 1.04 log₁₀ CFU, respectively (P < 0.001), consistent with their relative virulence. Inhibition of tumor necrosis factor alpha by the addition of methylprednisolone or pentoxifylline or removal of CD4⁺ or CD8⁺ T cells by magnetic beads had deleterious effects on immune control of intracellular growth only in the blood of tuberculin-positive donors. Repeated vaccination of eight tuberculin-negative volunteers with M. bovis BCG resulted in a 0.3 log (50%) reduction in BCG CFU counts in the model compared to baseline values (P < 0.05). Three of the volunteers responded only after the second vaccination. These experiments indicate that whole blood culture may be used to measure immunity to M. tuberculosis and that further studies of repeated BCG vaccination are warranted.     

Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ⁺) interleukin 2-positive (IL-2⁺) tumor necrosis factor α-positive (TNF-α⁺) polyfunctional CD4⁺ T cells concurrent with CD4⁺ IL-17A⁺ and CD8⁺ IFN-γ⁺ T cells or, in contrast, virtually absent cytokine responses with induction of CD8⁺ regulatory T cells. Significant induction of polyfunctional CD4⁺ IFN-γ⁺ IL-2⁺ TNF-α⁺ T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8⁺ T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8⁺ regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4⁺ T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.     

Heterologous boost vaccines for bacillus Calmette-Guérin prime immunization against tuberculosis

The current tuberculosis (TB) epidemic continues to call for the development of effective vaccination strategies. The initial TB vaccine research effort mostly focused on the search for a vaccine that might be better than, and thus could replace, the current bacillus Calmette-Guérin (BCG) vaccine. It has increasingly been realized that BCG or an improved BCG will continue to be used as a prime TB vaccine and there is a need to develop effective boost vaccines that could enhance and prolong the protective immunity of BCG prime immunization. Mounting experimental evidence suggests that recombinant vaccines, including both recombinant protein and genetic vector vaccines, are effective in boosting immune activation and protection by BCG vaccination. This review will discuss recent advances and the authors' views in the development of there boost vaccines.     

Tuberculosis vaccines: Time to think about the next generation

Efforts over the last 2 decades have led to a rich research and development pipeline of tuberculosis (TB) vaccines. Although none of the candidates has successfully completed the clinical trial pipeline, many are under advanced clinical assessment. These vaccines aim at prevention of active TB, with most of them being considered for preexposure with recent additions for postexposure or multistage administration. A few therapeutic vaccines are under clinical assessment, as well. Preexposure vaccination with the licensed TB vaccine BCG prevents severe forms of TB in children but not in adolescents and adults. The current vaccine pipeline does not include strategies which prevent or eliminate infection with the causative agent Mycobacterium tuberculosis (Mtb). Rather in a best-case scenario, they are quantitatively superior to BCG in preventing active TB over prolonged periods of time, ideally lifelong in the face of latent Mtb infection. Qualitatively superior vaccines should be capable of preventing or eliminating Mtb infection, in this way eliminating the risk of TB reactivation. The time is now ripe to exploit radically new strategies to achieve this goal.     

Recombinant BCG coexpressing Ag85B, CFP10, and interleukin-12 induces multifunctional Th1 and memory T cells in mice

Mycobacterium tuberculosis (MTB) continues to be a leading cause of human deaths due to an infectious agent. Current efforts are focused on making better TB vaccines. We describe the generation and immunological characterization of recombinant BCG (rBCG). This rBCG was generated by incorporating an expression plasmid encoding two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 into a BCG strain. Immunogenicity studies in mice showed that rBCG coexpressing Ag85B, CFP10, and IL-12 (rBCG::Ag85B-CFP10-IL-12) induces a robust immune response in mice. The rBCG vaccine promotes a T-cell response against MTB that is characterized by a high proportion of polyfunctional and memory T cells in spleen and lung. Our results showed strong immunogenicity and mycobacterial growth inhibition of rBCG::Ag85B-CFP10 plus IL-12 than that of BCG vaccine.     

Protection against Mycobacterium leprae Infection by the ID83/GLA-SE and ID93/GLA-SE Vaccines Developed for Tuberculosis

Despite the dramatic reduction in the number of leprosy cases worldwide in the 1990s, transmission of the causative agent, Mycobacterium leprae, is still occurring, and new cases continue to appear. New strategies are required in the pursuit of leprosy elimination. The cross-application of vaccines in development for tuberculosis may lead to tools applicable to elimination of leprosy. In this report, we demonstrate that the chimeric fusion proteins ID83 and ID93, developed as antigens for tuberculosis (TB) vaccine candidates, elicited gamma interferon (IFN-γ) responses from both TB and paucibacillary (PB) leprosy patients and from healthy household contacts of multibacillary (MB) patients (HHC) but not from nonexposed healthy controls. Immunization of mice with either protein formulated with a Toll-like receptor 4 ligand (TLR4L)-containing adjuvant (glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) stimulated antigen-specific IFN-γ secretion from pluripotent Th1 cells. When immunized mice were experimentally infected with M. leprae, both cellular infiltration into the local lymph node and bacterial growth at the site were reduced relative to those of unimmunized mice. Thus, the use of the Mycobacterium tuberculosis candidate vaccines ID83/GLA-SE and ID93/GLA-SE may confer cross-protection against M. leprae infection. Our data suggest these vaccines could potentially be used as an additional control measure for leprosy.     

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-γ) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-γ-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-γ and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-γ and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.The attenuated Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) is the currently used vaccine against tuberculosis (TB). In spite of its wide use, the BCG vaccine only protects against severe forms of childhood TB and generally does not prevent adult pulmonary TB (11, 30, 47).Considering that one-third of the world population is thought to be infected with Mycobacterium tuberculosis and that only a small proportion of these individuals will develop active disease, new vaccine candidates to prevent the establishment of infection could also boost and improve the cellular immunity of already latently infected individuals. Vaccine candidates currently in clinical trials include improved recombinant BCG vaccines, virus-based recombinant vaccines, and subunit vaccines comprised of dominant secreted antigens (1, 32). Secreted proteins, regularly described as culture filtrate proteins (CFP), are the main targets of the T-cell response in mice, both at the height of infection and in a state of memory immunity, as well as in humans with active TB (1, 4, 5, 7, 23). Immunization with these antigens in the presence of different adjuvants provided protection in mice challenged with M. tuberculosis, and protection was mediated by gamma interferon (IFN-γ)-producing CD4⁺ cells (29, 38).We previously showed that a homologous immunization schedule based on three doses of CFP antigens plus CpG oligodeoxynucleotide adjuvant stimulated significant IFN-γ production by spleen cells and in the lungs of challenged mice. In spite of high IFN-γ concentrations, immunized and challenged mice were not protected and indeed had extensive lung damage (16).Since IFN-γ is the best indicator of protective immunity defined thus far, we changed the schedule of homologous immunization to heterologous immunization, also known as a prime-boost regimen, to induce protection.Several studies have demonstrated the efficacy of prime-boost vaccination strategies in generating cellular immunity to a variety of pathogens (3, 10, 14, 17, 34, 36, 44, 45, 49). Recently, our group also showed that a single dose of a DNA-HSP65 vaccine booster significantly enhanced the protection conferred against TB by a single subcutaneous dose of BCG (18). In addition, secreted antigens such as the 6-kDa early-secretion antigen target (ESAT-6), 85A or 85B antigens, and Mtb72F have proven to be promising candidates for BCG-boosting vaccines in mice, guinea pigs, and nonhuman primates (6, 9, 12, 19, 33, 37, 46, 48). Because a single dominant antigen may not confer the same level of protection to all vaccinated individuals, and based on high CFP antigen-mediated IFN-γ production in the presence of CpG adjuvant, in this study we used CFP plus CpG oligodeoxynucleotides to boost BCG vaccination in order to improve protection and lung preservation following M. tuberculosis challenge.     

Establishment of an Aerosol Challenge Model of Tuberculosis in Rhesus Macaques and an Evaluation of Endpoints for Vaccine Testing

The establishment of an aerosol challenge model in nonhuman primates (NHPs) for the testing of vaccines against Mycobacterium tuberculosis would assist the global effort to optimize novel vaccination strategies. The endpoints used in preclinical challenge studies to identify measures of disease burden need to be accurate and sensitive enough to distinguish subtle differences and benefits afforded by different tuberculosis (TB) vaccine regimens when group sizes are inevitably small. This study sought to assess clinical and nonclinical endpoints as potentially sensitive measures of disease burden in a challenge study with rhesus macaques by using a new protocol of aerosol administration of M. tuberculosis. Immunological and clinical readouts were assessed for utility in vaccine evaluation studies. This is the first example of TB vaccine evaluation with rhesus macaques where long-term survival was one of the primary endpoints. However, we found that in NHP vaccine efficacy studies with maximum group sizes of six animals, survival did not provide a valuable endpoint. Two approaches used in human clinical trials for the evaluation of the gamma interferon (IFN-γ) response to vaccination (enzyme-linked immunospot [ELISpot] assay and enzyme-linked immunosorbent assay [ELISA]) were included in this study. The IFN-γ profiles induced following vaccination were found not to correlate with protection, nor did the level of purified protein derivative (PPD)-specific proliferation. The only readout to reliably distinguish vaccinated and unvaccinated NHPs was the determination of lung lesion burden using magnetic resonance (MR) imaging combined with stereology at the end of the study. Therefore, the currently proposed key markers were not shown to correlate with protection, and only imaging offered a potentially reliable correlate.Tuberculosis (TB) is a reemerging infectious disease and is responsible for nearly 2 million deaths and 9 million new cases each year (36). The global TB pandemic has been exacerbated by the emergence of drug-resistant strains of Mycobacterium tuberculosis, which render treatment less effective, and by the HIV epidemic, where coinfection with HIV greatly increases the risk of reactivation of latent TB and susceptibility to active TB disease.The most effective means of controlling this global epidemic would be by prophylactic immunization. Mycobacterium bovis bacille Calmette-Guérin (BCG), the only licensed TB vaccine, is administered to neonates in high-risk populations as part of the WHO Expanded Programme on Immunization. BCG consistently protects against TB meningitis and disseminated TB in childhood (27, 30), but its efficacy wanes with time, and it affords only variable protection against pulmonary disease (10). A new, more effective TB vaccine is a major global health priority and is an important part of the WHO STOP TB partnership strategy.A large international effort is under way to develop a more effective vaccine. The leading TB vaccine development strategy involves vaccination with BCG followed by a heterologous subunit vaccine boost designed to enhance protective immunity. One such subunit vaccine is the virus-vectored subunit candidate TB vaccine developed at Oxford University, MVA85A (19) (live, replication-deficient, modified vaccinia virus Ankara [MVA] [7], expressing the highly conserved, immunodominant mycobacterial antigen 85A [Ag85A]). The enhancement of BCG with systemically administered (intradermal) MVA85A has been evaluated with several preclinical animal models and is currently the subject of ongoing evaluations in several clinical trials. This BCG-MVA85A vaccination regimen induces a high magnitude of cellular immunity in mice and cattle (19, 32) and can protect against M. tuberculosis in guinea pigs (34), nonhuman primates (NHPs) (31), and cattle (33). It is safe and highly immunogenic in healthy adults, adolescents, children, infants, and HIV- and M. tuberculosis-infected adults (6, 20, 21) and has recently entered a large-scale efficacy trial with South African infants (http://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT00953927","term_id":"NCT00953927"}}NCT00953927).The lack of a defined immunological correlate of protection for TB means that in order to assess efficacy, candidate TB vaccines must enter large clinical trials involving thousands of at-risk individuals in countries where the disease is endemic. Therefore, there is a need for a validated preclinical animal model that can be utilized to accurately predict the effectiveness of a candidate vaccine in humans and to aid in the identification of correlates of protection through challenge studies. Mouse models are generally used as a first screen of vaccine candidates and are very useful for studying detailed immunological responses (24). Guinea pigs are considered a more stringent model than mice to discriminate between vaccines in terms of protective efficacy, since they show a variety of pulmonary and extrapulmonary lesion types that are similar to those observed for humans (4, 17). Although small-animal models are useful, it is widely accepted that larger animals such as cattle and NHPs are potentially the most relevant model species to predict safety, immunogenicity, and protective efficacy of vaccines prior to their large-scale evaluation in humans (8, 18). NHPs are naturally susceptible to infection with M. tuberculosis via the respiratory route and develop a disease that clinically closely mimics human disease. As with the other preclinical species, BCG vaccination of NHPs provides a limited level of protection against M. tuberculosis that can be quantified through a variety of clinical and nonclinical parameters (2, 3, 9, 12, 14).The establishment of an aerosol challenge model in NHPs, in which the M. tuberculosis challenge is delivered by the same route as that which occurs during natural infection, would assist the global effort in optimizing novel vaccination strategies. The endpoints used in preclinical challenge studies to identify measures of disease burden need to be accurate and sensitive enough to distinguish subtle differences and benefits afforded by different TB vaccine regimens, including those that enhance the protective efficacy of BCG, where partial protection is already conferred and the power to detect smaller incremental improvements in small numbers of animals is limited. The aims of this study were to establish an aerosol challenge model of TB in rhesus macaques and to assess clinical and nonclinical endpoints as potentially sensitive measures of disease burden in an NHP challenge study following vaccination with BCG or BCG boosted by MVA85A. The currently most widely used challenge model with macaques uses high-dose, intratracheal administration of M. tuberculosis, and it has been unclear whether low-dose aerosol administration would provide a better model, because although it should replicate the route of natural infection (31), larger group sizes may be necessary to compensate for interindividual heterogeneity to a low-dose challenge (8).     

Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis

Whether the intranasal (i.n.) route of Mycobacterium bovis BCG vaccination provides better protection against pulmonary tuberculosis than subcutaneous (s.c.) vaccination remains an incompletely solved issue. In the present study, we compared both immune responses and protection elicited by single BCG vaccinations via the i.n. or s.c. route in BALB/c mice. While both i.n. and s.c. vaccination triggered comparable levels of primary immune activation in the spleen and draining lymph nodes, i.n. vaccination led to a greater antigen-specific gamma interferon recall response in splenocytes than s.c. vaccination upon secondary respiratory mycobacterial challenge, accompanied by an increased frequency of antigen-specific lymphocytes. There was also a quicker cellular response in the lungs of i.n. vaccinated mice upon mycobacterial challenge. Mice vaccinated i.n. were found to be much better protected, particularly in the lung, than s.c. vaccinated counterparts against pulmonary tuberculosis at both 3 and 6 months postvaccination. These results suggest that the i.n. route of vaccination improves the protective effect of the current BCG vaccine.