New TB vaccines are urgently needed because of the apparent lack of effect of the BCG vaccine on rates of adult contagious pulmonary tuberculosis and the risk of disseminated BCG disease in ...immunocompromised individuals. Since BCG appears to protect children, the primary target for vaccine development is a booster vaccine for adults but such vaccines ideally need to be able to efficiently prime mycobacterially naïve individuals as well as boost individuals previously vaccinated with BCG and those latently infected with TB. Protective immunity against Mycobacterium tuberculosis depends mainly on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-γ) production. In the present study, we monitored safety and IFN-γ responses in healthy BCG-vaccinated and prior or latently TB-infected individuals receiving a novel vaccine composed of the fusion protein Ag85B–ESAT-6 combined with the adjuvant IC31®, administered at 0 and 2 months. Vaccination caused few local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against Ag85B–ESAT-6 and both the Ag85B and ESAT-6 components, that could be augmented by second vaccination. The strong responses persisted through 32 weeks of follow-up, indicating the induction of a persistent memory response in the vaccine recipients.
Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins ...are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one Kd-restricted T-cell epitope could be identified. BALB/c and (B6D2)F₁ mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.
Tuberculosis (TB) remains one of the most deadly infectious diseases. One-third to one-fourth of the human population is estimated to be infected with Mycobacterium tuberculosis (Mtb) without showing ...clinical symptoms, a condition called latent TB infection (LTBI). Diagnosis of Mtb infection is based on the immune response to a mixture of mycobacterial antigens (PPD) or to Mtb specific ESAT-6/CFP10 antigens (IGRA), highly expressed during the initial phase of infection. However, the immune response to PPD and IGRA antigens has a low power to discriminate between LTBI and PTB. The T-cell response to a group of so-called latency (DosR-regulon-encoded) and Resuscitation Promoting (Rpf) antigens of Mtb has been proved to be significantly higher in LTBI compared to active TB across many populations, suggesting their potential use as biomarkers to differentiate latent from active TB.
PBMCs from a group LTBI (n = 20) and pulmonary TB patients (PTB, n = 21) from an endemic community for TB of the city of Medellín, Colombia, were in vitro stimulated for 7 days with DosR- (Rv1737c, Rv2029c, and Rv2628), Rpf- (Rv0867c and Rv2389c), the recombinant fusion protein ESAT-6-CFP10 (E6-C10)-, or PPD-antigen. The induced IFNγ levels detectable in the supernatants of the antigen-stimulated cells were then used to calculate specificity and sensitivity in discriminating LTBI from PTB, using different statistical approaches.
IFNγ production in response to DosR and Rpf antigens was significantly higher in LTBI compared to PTB. ROC curve analyses of IFNγ production allowed differentiation of LTBI from PTB with areas under the curve higher than 0.70. Furthermore, Multiple Correspondence Analysis (MCA) revealed that LTBI is associated with higher levels of IFNγ in response to the different antigens compared to PTB. Analysis based on decision trees showed that the IFNγ levels produced in response to Rv2029c was the leading variable that best-classified disease status. Finally, logistic regression analysis predicted that IFNγ produced by PBMCs in response to E6-C10, Rv2029c, Rv0867c (RpfA) and Rv2389c (RpfA) antigens correlates best with the probability of being latently infected.
The Mtb antigens E6-C10, Rv2029c (PfkB), Rv0867c (RpfA) and Rv2389c (RpfA), may be potential candidates to discriminate LTBI from PTB.
Continuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant ...models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection.
Here, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNgamma responses. Antigen 85A-specific IFNgamma secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNgamma levels post-infection as the strongest immunocorrelate for protection (spearman's rho: -0.60).
Both the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates.
Mycobacterium bovis bacille Calmette-Guérin (BCG) prevents extrapulmonary tuberculosis (TB) and death among infants but fails to consistently and sufficiently prevent pulmonary TB in adults. Thus, TB ...remains the leading infectious cause of death worldwide, and new vaccine approaches are urgently needed. T-cells are important for protective immunity to Mycobacterium tuberculosis (Mtb), but the optimal T-cell antigens to be included in new vaccines are not established. T-cells are often thought of as responding mainly to peptide antigens presented by polymorphic major histocompatibility complex (MHC) I and II molecules. Over the past two decades, the number of non-peptidic Mtb derived antigens for αβ and γδ T-cells has expanded rapidly, creating broader perspectives about the types of molecules that could be targeted by T-cell-based vaccines against TB. Many of these non-peptide responsive T-cell subsets in humans are activated in a manner that is unrestricted by classical MHC-dependent antigen-presenting systems, but instead require essentially nonpolymorphic presentation systems. These systems are Cluster of differentiation 1 (CD1), MHC related protein 1 (MR1), butyrophilin 3A1, as well as the nonclassical MHC class Ib family member HLA-E. Thus, the resulting T-cell responses can be shared among a genetically diverse population, creating the concept of donor-unrestricted T-cells (DURTs). Here, we review evidence that DURTs are an abundant component of the human immune system and recognize many antigens expressed by Mtb, including antigens that are expressed in BCG and other candidate whole cell vaccines. Further, DURTs exhibit functional diversity and demonstrate the ability to control microbial infection in small animal models. Finally, we outline specific knowledge gaps and research priorities that must be addressed to realize the full potential of DURTs as part of new TB vaccines approaches.
Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to ...tuberculosis (TB) among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana. We compared 22 polymorphisms of 14 autophagy genes between 1022 Indonesian TB patients and 952 matched controls, and between patients infected with different M. tuberculosis genotypes, as determined by spoligotyping. The same autophagy polymorphisms were studied in correlation with ex-vivo production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 in healthy volunteers. No association was found between TB and polymorphisms in the genes ATG10, ATG16L2, ATG2B, ATG5, ATG9B, IRGM, LAMP1, LAMP3, P2RX7, WIPI1, MTOR and ATG4C. Associations were found between polymorphisms in LAMP1 (p = 0.02) and MTOR (p = 0.02) and infection with the successful M. tuberculosis Beijing genotype. The polymorphisms examined were not associated with M. tuberculosis induced cytokines, except for a polymorphism in ATG10, which was linked with IL-8 production (p = 0.04). All associations found lost statistical significance after correction for multiple testing. This first examination of a broad set of polymorphisms in autophagy genes fails to show a clear association with TB, with M. tuberculosis Beijing genotype infection or with ex-vivo pro-inflammatory cytokine production.
CD8 T-cells contribute to control of Mycobacterium tuberculosis infection, but little is known about the quality of the CD8 T-cell response in subjects with latent infection and in patients with ...active tuberculosis disease. CD8 T-cells recognizing epitopes from 6 different proteins of Mycobacterium tuberculosis were detected by tetramer staining. Intracellular cytokines staining for specific production of IFN-gamma and IL-2 was performed, complemented by phenotyping of memory markers on antigen-specific CD8 T-cells. The ex-vivo frequencies of tetramer-specific CD8 T-cells in tuberculous patients before therapy were lower than in subjects with latent infection, but increased at four months after therapy to comparable percentages detected in subjects with latent infection. The majority of CD8 T-cells from subjects with latent infection expressed a terminally-differentiated phenotype (CD45RA+CCR7(-)). In contrast, tuberculous patients had only 35% of antigen-specific CD8 T-cells expressing this phenotype, while containing higher proportions of cells with an effector memory- and a central memory-like phenotype, and which did not change significantly after therapy. CD8 T-cells from subjects with latent infection showed a codominance of IL-2+/IFN-gamma+ and IL-2(-)/IFN-gamma+ T-cell populations; interestingly, only the IL-2+/IFN-gamma+ population was reduced or absent in tuberculous patients, highly suggestive of a restricted functional profile of Mycobacterium tuberculosis-specific CD8 T-cells during active disease. These results suggest distinct Mycobacterium tuberculosis specific CD8 T-cell phenotypic and functional signatures between subjects which control infection (subjects with latent infection) and those who do not (patients with active disease).
Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is ...determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings.
The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs.
Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas.
Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC.
Tuberculosis (TB) is a prevalent disease causing an estimated 1.6 million deaths and 10.6 million new cases annually. Discriminating TB disease from differential diagnoses can be complex, ...particularly in the field. Increased levels of complement component C1q in serum have been identified as a specific and accessible biomarker for TB disease but the source of C1q in circulation has not been identified. Here, data and samples previously collected from human cohorts, a clinical trial and a non-human primate study were used to identify cells producing C1q in circulation. Cell subset frequencies were correlated with serum C1q levels and combined with single cell RNA sequencing and flow cytometry analyses. This identified monocytes as C1q producers in circulation, with a pronounced expression of C1q in classical and intermediate monocytes and variable expression in non-classical monocytes.
The classical paradigm of tuberculosis (TB) immunity, with a central protective role for Th1 responses and IFN-γ-stimulated cellular responses, has been challenged by unsatisfactory results of ...vaccine strategies aimed at enhancing Th1 immunity. Moreover, preclinical TB models have shown that increasing IFN-γ responses in the lungs is more damaging to the host than to the pathogen. Type 1 interferon signaling and altered Th17 responses have also been associated with active TB, but their functional roles in TB pathogenesis remain to be established. These two host responses have been studied in more detail in autoimmune diseases (AID) and show functional interactions that are of potential interest in TB immunity. In this review, we first identify the role of type 1 interferons and Th17 immunity in TB, followed by an overview of interactions between these responses observed in systemic AID. We discuss (i) the effects of GM-CSF-secreting Th17.1 cells and type 1 interferons on CCR2
monocytes; (ii) convergence of IL-17 and type 1 interferon signaling on stimulating B-cell activating factor production and the central role of neutrophils in this process; and (iii) synergy between IL-17 and type 1 interferons in the generation and function of tertiary lymphoid structures and the associated follicular helper T-cell responses. Evaluation of these autoimmune-related pathways in TB pathogenesis provides a new perspective on recent developments in TB research.
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