Novel polyprotein antigens designed for improved serodiagnosis of bovine tuberculosis.

Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis-infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis-infected and 57 non-infected cattle showed the best accuracy (∼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis.

Memory B cells and tuberculosis.

Immunological memory is a central feature of adaptive immunity. Memory B cells are generated upon stimulation with antigen presented by follicular dendritic cells in the peripheral lymphoid tissues. This process typically involves class-switch recombination and somatic hypermutation and it can be dependent or independent on germinal centers or T cell help. The mature B cell memory pool is generally characterized by remarkable heterogeneity of functionally and phenotypically distinct sub-populations supporting multi-layer immune plasticity. Memory B cells found in human patients infected with Mycobacterium tuberculosis include IgD+ CD27+ and IgM+ CD27+ subsets. In addition, expansion of atypical memory B cells characterized by the lack of CD27 expression and by inability to respond to antigen-induced re-activation is documented in human tuberculosis. These functionally impaired memory B cells are believed to have adverse effects on host immunity. Human and animal studies demonstrate recruitment of antigen-activated B cells to the infection sites and their presence in lung granulomas where proliferating B cells are organized into discrete clusters resembling germinal centers of secondary lymphoid organs. Cattle studies show development of IgM+, IgG+, and IgA+ memory B cells in M. bovis infection with the ability to rapidly differentiate into antibody-producing plasma cells upon antigen re-exposure. This review discusses recent advances in research on generation, re-activation, heterogeneity, and immunobiological functions of memory B cells in tuberculosis. The role of memory B cells in post-skin test recall antibody responses in bovine tuberculosis and implications for development of improved immunodiagnostics are also reviewed.

A defined antigen skin test for the diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTB) is a major zoonotic disease of cattle that is endemic in much of the world, limiting livestock productivity and representing a global public health threat. Because the standard tuberculin skin test precludes implementation of Bacille Calmette-Guérin (BCG) vaccine-based control programs, we here developed and evaluated a novel peptide-based defined antigen skin test (DST) to diagnose bTB and to differentiate infected from vaccinated animals (DIVA). The results, in laboratory assays and in experimentally or naturally infected animals, demonstrate that the peptide-based DST provides DIVA capability and equal or superior performance over the extant standard tuberculin surveillance test. Together with the ease of chemical synthesis, quality control, and lower burden for regulatory approval compared with recombinant antigens, the results of our studies show that the DST considerably improves a century-old standard and enables the development and implementation of critically needed surveillance and vaccination programs to accelerate bTB control.

The Humoral Immune Response to BCG Vaccination.

Bacillus Calmette Guérin (BCG) is the only currently available vaccine against tuberculosis (TB), but it confers incomplete and variable protection against pulmonary TB in humans and bovine TB (bTB) in cattle. Insights into the immune response induced by BCG offer an underexploited opportunity to gain knowledge that may inform the design of a more efficacious vaccine, which is urgently needed to control these major global epidemics. Humoral immunity in TB and bTB has been neglected, but recent studies supporting a role for antibodies in protection against TB has driven a growing interest in determining their relevance to vaccine development. In this manuscript we review what is known about the humoral immune response to BCG vaccination and re-vaccination across species, including evidence for the induction of specific B cells and antibodies; and how these may relate to protection from TB or bTB. We discuss potential explanations for often conflicting findings and consider how factors such as BCG strain, manufacturing methodology and route of administration influence the humoral response. As novel vaccination strategies include BCG prime-boost regimens, the literature regarding off-target immunomodulatory effects of BCG vaccination on non-specific humoral immunity is also reviewed. Overall, reported outcomes to date are inconsistent, but indicate that humoral responses are heterogeneous and may play different roles in different species, populations, or individual hosts. Further study is warranted to determine whether a new TB vaccine could benefit from the targeting of humoral as well as cell-mediated immunity.

Ag85A-specific CD4+ T cell lines derived after boosting BCG-vaccinated cattle with Ad5-85A possess both mycobacterial growth inhibition and anti-inflammatory properties.

There is a need to improve the efficacy of the BCG vaccine against human and bovine tuberculosis. Previous data showed that boosting bacilli Calmette-Guerin (BCG)-vaccinated cattle with a recombinant attenuated human type 5 adenovirally vectored subunit vaccine (Ad5-85A) increased BCG protection and was associated with increased frequency of Ag85A-specific CD4+ T cells post-boosting. Here, the capacity of Ag85A-specific CD4+ T cell lines - derived before and after viral boosting - to interact with BCG-infected macrophages was evaluated. No difference before and after boosting was found in the capacity of these Ag85A-specific CD4+ T cell lines to restrict mycobacterial growth, but the secretion of IL-10 in vitro post-boost increased significantly. Furthermore, cell lines derived post-boost had no statistically significant difference in the secretion of pro-inflammatory cytokines (IL-1ß, IL-12, IFNγ or TNFα) compared to pre-boost lines. In conclusion, the protection associated with the increased number of Ag85A-specific CD4+ T cells restricting mycobacterial growth may be associated with anti-inflammatory properties to limit immune-pathology.

A multi-antigenic adenoviral-vectored vaccine improves BCG-induced protection of goats against pulmonary tuberculosis infection and prevents disease progression.

The "One world, one health" initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG.

Goats primed with Mycobacterium bovis BCG and boosted with a recombinant adenovirus expressing Ag85A show enhanced protection against tuberculosis.

This is the first efficacy study using the experimental goat model, a natural host of tuberculosis (TB), to evaluate the efficacy of heterologous Mycobacterium bovis bacillus Calmette-Guérin (BCG) prime followed by boosting with a replication-deficient adenovirus expressing the antigen Ag85A (AdAg85A). Three experimental groups of 11 goat kids each were used: BCG vaccinated, BCG vaccinated and AdAg85A boosted, and nonvaccinated. Twenty-two goat kids were vaccinated with ∼5 × 10(5) CFU of BCG (week 0), and 11 of them were boosted at week 8 with 10(9) PFU of AdAg85A. At week 14, all goats were challenged by the endobronchial route with ∼1.5 × 10(3) CFU of Mycobacterium caprae. The animals were euthanized at week 28. Cellular and humoral immunity induced by vaccination and M. caprae infection was measured throughout the study. After challenge BCG-AdAg85A-vaccinated animals exhibited reduced pathology compared to BCG-vaccinated animals in lungs and in pulmonary lymph nodes. There were significant reductions in bacterial load in both groups of vaccinated goats, but the reduction was more pronounced in prime-boosted animals. Antigen-specific gamma interferon (IFN-γ) and humoral responses were identified as prognostic biomarkers of vaccination outcome depending on their correlation with pathological and bacteriological results. As far as we know, this is the first report using multidetector computed tomography (MDCT) to measure vaccine efficacy against pulmonary TB in an animal model. The use in vaccine trials of animals that are natural hosts of TB may improve research into human TB vaccines.

Improved skin test for differential diagnosis of bovine tuberculosis by the addition of Rv3020c-derived peptides.

A peptide cocktail derived from the mycobacterial antigens ESAT-6, CFP-10, and Rv3615c allowed differentiation between Mycobacterium bovis-infected and M. bovis bacillus Calmette-Guérin (BCG)-vaccinated cattle when used as a skin test reagent for a "DIVA" test (i.e., a test capable of differentiating infected and uninfected vaccinated animals). Addition of the antigen Rv3020c improves the diagnostic sensitivity without compromising specificity in the face of BCG or Johne's disease vaccination.

Global gene transcriptome analysis in vaccinated cattle revealed a dominant role of IL-22 for protection against bovine tuberculosis.

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge. RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle. Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-γ. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy.

Immune responses to the enduring hypoxic response antigen Rv0188 are preferentially detected in Mycobacterium bovis infected cattle with low pathology.

The DosR regulon and the Enduring Hypoxic Response (EHR) define a group of M. tuberculosis genes that are specifically induced in bacilli exposed in vitro to conditions thought to mimic the environment encountered by Mycobacteria during latent infection. Although well described in humans, latent mycobacterial infection in cattle remains poorly understood. Thus, the aim of this study was to identify antigens that may potentially disclose cattle with latent M. bovis infection. To this end, we initially screened 57 pools of overlapping peptides representing 4 DosR regulon and 29 EHR antigens for their ability to stimulate an immune response in whole blood from TB-reactor cattle using IFN-γ and IL-2 as readouts. All 4 DosR regulon proteins were poorly recognized (maximum responder frequency of 10%). For the EHR antigens, both IFN-γ and IL-2 revealed similar response hierarchies, with responder frequencies ranging from 54% down to 3% depending on the given EHR antigen. Furthermore, these results demonstrated that responses in the infected cattle were largely IFN-γ biased. To support the concept for their role in latency, we evaluated if EHR antigen responses were associated with lower pathology. The EHR antigen Rv0188 was recognised predominantly in animals presenting with low pathology scores, whereas responses to ESAT-6/CFP-10 or the other EHR antigens tested were prevalent across the pathology spectrum. However, when we determined the production of additional cytokines induced by the M. bovis antigens PPD-B or ESAT-6/CFP-10, we detected significantly greater PPD-B-induced production of the pro-inflammatory cytokine IL-1ß in animals recognizing Rv0188 (i.e. those with limited or no pathology). Thus, these results are consistent with the idea that responses to Rv0188 may identify a subset of animals at early stages of infection or in which disease progression may be limited.

Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM) cells in cattle naturally infected with bovine tuberculosis.

Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+)IL-2(+)TNF-α(+) and IFN-γ(+) TNF-α(+) response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hi)CD45RO(+)CD62L(lo) T-effector memory (T(EM)) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM) cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

Simultaneous measurement of antigen-stimulated interleukin-1 beta and gamma interferon production enhances test sensitivity for the detection of Mycobacterium bovis infection in cattle.

In order to identify cytokines that may be useful as candidates for inclusion in diagnostic tests for Mycobacterium bovis infection in cattle, we compared the levels of gamma interferon (IFN-γ), interleukin 1ß (IL-1ß), IL-4, IL-10, IL-12, macrophage inflammatory protein 1ß (MIP-1ß), and tumor necrosis factor alpha (TNF-α) in whole-blood cultures from tuberculosis (TB) reactor animals or TB-free controls following stimulation with M. bovis-specific antigens (purified protein derivative from M. bovis [PPD-B] or ESAT-6/CFP-10). In addition to IFN-γ responses, the production of IL-1ß and TNF-α was also statistically significantly elevated in TB reactor cattle over that in uninfected controls following stimulation with PPD-B or ESAT-6/CFP-10 peptides. Thus, we evaluated whether the use of these two additional readouts could disclose further animals not detected by measuring IFN-γ alone. To this end, receiver operating characteristic (ROC) analyses were performed to define diagnostic cutoffs for positivity for TNF-α and IL-1ß. These results revealed that for ESAT-6/CFP-10-induced responses, the use of all three readouts (IFN-γ, TNF-α, and IL-1ß) in parallel increased the sensitivity of detection of M. bovis-infected animals by 11% but also resulted in a specificity decrease of 14%. However, applying only IFN-γ and IL-1ß in parallel resulted in a 5% increase in sensitivity without the corresponding loss of specificity. The results for PPD-B-induced responses were similar, although the loss of specificity was more pronounced, even when only IFN-γ and IL-1ß were used as readout systems. In conclusion, we have demonstrated that the use of an additional readout system, such as IL-1ß, can potentially complement IFN-γ by increasing overall test sensitivity for the detection of M. bovis infection in cattle.

Screening of predicted secreted antigens from Mycobacterium bovis identifies potential novel differential diagnostic reagents.

To date, the most promising vaccination strategies for the control of bovine tuberculosis (TB) focus on improving the efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG). However, vaccination with BCG results in sensitization of animals to bovine tuberculin and compromises tests currently used for diagnosis of bovine TB infection. Thus, the development of specific diagnostic reagents capable of discriminating between infected and uninfected vaccinated animals (DIVA) is of high priority. To test the hypothesis that M. bovis-secreted proteins are likely to contain immunogenic antigens that can be used to increase the specificity of diagnostic tests, we screened 379 pools of overlapping peptides representing 119 antigens for their ability to stimulate a gamma inferferon (IFN-gamma) response in vitro using whole blood from both TB reactor and BCG-vaccinated animals. Peptide pools representing antigens Rv3020c and Rv2346c induced responses in 61% and 57% of the TB reactor animals, respectively, without inducing responses in any BCG-vaccinated animal studied. Furthermore, individual peptides contained within pools recognized by BCG vaccinates were identified that were specific and induced IFN-gamma responses in TB reactor animals. From these results, we constructed a cocktail of nine peptides representing multiple antigen targets that was recognized by 54% of TB reactor animals but also failed to induce responses in any BCG-vaccinated animal studied. In summary, we have identified three peptide cocktails for prioritization in larger trials to discriminate between M. bovis infection and BCG vaccination.

Signal regulatory protein alpha (SIRPalpha) cells in the adaptive response to ESAT-6/CFP-10 protein of tuberculous mycobacteria.

BACKGROUND: Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRP)alpha (also referred to as macrophage fusion receptor or CD172a) is essential for multi-nucleate giant cell formation. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, ESAT-6/CFP-10 complex and SIRPalpha interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a(+) (SIRPalpha-expressing) cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail), but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis DeltaRD1). Sorted CD172a(+) cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8alpha). Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c(+), CD172a(+), and CD3(+) cells, including CD172a-expressing multi-nucleated giant cells. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a(+) cells, bind to CD172a(+) cells, and induce multi-nucleated giant cells expressing CD172a.

Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis.

Previous work with small-animal laboratory models of tuberculosis has shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guérin (BCG) to prime and modified vaccinia virus Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad85A) expressing the mycobacterial antigen Ag85A to boost may increase the protective efficacy of BCG. Here we report the first efficacy data on using these vaccines in cattle, a natural target species of tuberculous infection. Protection was determined by measuring development of disease as an end point after M. bovis challenge. Either Ad85A or MVA85A boosting resulted in protection superior to that given by BCG alone: boosting BCG with MVA85A or Ad85A induced significant reduction in pathology in four/eight parameters assessed, while BCG vaccination alone did so in only one parameter studied. Protection was particularly evident in the lungs of vaccinated animals (median lung scores for naïve and BCG-, BCG/MVA85A-, and BCG/Ad85A-vaccinated animals were 10.5, 5, 2.5, and 0, respectively). The bacterial loads in lymph node tissues were also reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection. Therefore, this study provides evidence of improved protection against tuberculosis by viral booster vaccination in a natural target species and has prioritized potential correlates of vaccine efficacy for further evaluation. These findings also have implications for human tuberculosis vaccine development.

Immune responses induced in cattle by vaccination with a recombinant adenovirus expressing Mycobacterial antigen 85A and Mycobacterium bovis BCG.

Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot assay analysis demonstrated that memory responses were induced by all three protocols but were strongest after BCG/rAd85A and rAd85A/BCG vaccination.

Recent advances in the development of adenovirus- and poxvirus-vectored tuberculosis vaccines.

Tuberculosis vaccine research began with the search for a vaccine that might be better than, and thus could replace, the current Bacillus Calmette Guérin (BCG) vaccine. Over the last fifteen years or so, intense research effort has led to the identification of a number of novel tuberculosis (TB) vaccines which can be divided into 4 categories: genetically modified mycobacteria, protein, plasmid DNA and viral. However, it is increasingly believed that the current BCG vaccine will continue to be used as a childhood vaccine and that more effort should be directed to developing appropriate boosting vaccines. Mounting evidence suggests that recombinant genetic vaccines, particularly recombinant viral vaccines, are effective in boosting immune activation and protection by BCG vaccination. Since modified vaccinia virus Ankara (MVA)- and adenovirus-vectored TB vaccines have been most extensively studied, this review will focus on recent advances in the development and applications of these two viral TB vaccines.

Infection biology of a novel alpha-crystallin of Mycobacterium tuberculosis: Acr2.

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the alpha-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Deltaacr2) was unimpaired in log phase growth and persisted in IFN-gamma-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-gamma-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.