Divergent proinflammatory immune responses associated with the differential susceptibility of cattle breeds to tuberculosis.

Tuberculosis (TB) in the bovine is one of the most predominant chronic debilitating infectious diseases primarily caused by Mycobacterium bovis. Besides, the incidence of TB in humans due to M. bovis, and that in bovines (bovine TB, bTB) due to M. tuberculosis- indicates cattle as a major reservoir of zoonotic TB. While India accounts for the highest global burden of both TB and multidrug-resistant TB in humans, systematic evaluation of bTB prevalence in India is largely lacking. Recent reports emphasized markedly greater bTB prevalence in exotic and crossbred cattle compared to indigenous cattle breeds that represent more than one-third of the total cattle population in India, which is the largest globally. This study aimed at elucidating the immune responses underlying the differential bTB incidence in prominent indigenous (Sahiwal), and crossbred (Sahiwal x Holstein Friesian) cattle reared in India. Employing the standard Single Intradermal Tuberculin Test (SITT), and mycobacterial gene-targeting single as well as multiplex-PCR-based screening revealed higher incidences of bovine tuberculin reactors as well as Mycobacterium tuberculosis Complex specific PCR positivity amongst the crossbred cattle. Further, ex vivo mycobacterial infection in cultures of bovine peripheral blood mononuclear cells (PBMC) from SITT, and myco-PCR negative healthy cattle exhibited significantly higher intracellular growth of M. bovis BCG, and M. tuberculosis H37Ra in the crossbred cattle PBMCs compared to native cattle. In addition, native cattle PBMCs induced higher pro-inflammatory cytokines and signaling pathways, such as interferon-gamma (IFN-γ), interleukin-17 (IL-17), tank binding kinase-1 (TBK-1), and nitric oxide (NO) upon exposure to live mycobacterial infection in comparison to PBMCs from crossbred cattle that exhibited higher expression of IL-1ß transcripts. Together, these findings highlight that differences in the innate immune responses of these cattle breeds might be contributing to the differential susceptibility to bTB infection, and the resultant disparity in bTB incidence amongst indigenous, and crossbred cattle.

Augmentation of the Riboflavin-Biosynthetic Pathway Enhances Mucosa-Associated Invariant T (MAIT) Cell Activation and Diminishes Mycobacterium tuberculosis Virulence.

Mucosa-associated invariant T (MAIT) cells play a critical role in antimicrobial defense. Despite increased understanding of their mycobacterial ligands and the clinical association of MAIT cells with tuberculosis (TB), their function in protection against Mycobacterium tuberculosis infection remains unclear. Here, we show that overexpressing key genes of the riboflavin-biosynthetic pathway potentiates MAIT cell activation and results in attenuation of M. tuberculosis virulence in vivo. Further, we observed greater control of M. tuberculosis infection in MAIThi CAST/EiJ mice than in MAITlo C57BL/6J mice, highlighting the protective role of MAIT cells against TB. We also endogenously adjuvanted Mycobacterium bovis BCG with MR1 ligands via overexpression of the lumazine synthase gene ribH and evaluated its protective efficacy in the mouse model of M. tuberculosis infection. Altogether, our findings demonstrate that MAIT cells confer host protection against TB and that overexpression of genes in the riboflavin-biosynthetic pathway attenuates M. tuberculosis virulence. Enhancing MAIT cell-mediated immunity may also offer a novel approach toward improved vaccines against TB. IMPORTANCE Mucosa-associated invariant T (MAIT) cells are an important subset of innate lymphocytes that recognize microbial ligands derived from the riboflavin biosynthesis pathway and mediate antimicrobial immune responses. Modulated MAIT cell responses have been noted in different forms of tuberculosis. However, it has been unclear if increased MAIT cell abundance is protective against TB disease. In this study, we show that augmentation of the mycobacterial MAIT cell ligands leads to higher MAIT cell activation with reduced M. tuberculosis virulence and that elevated MAIT cell abundance confers greater control of M. tuberculosis infection. Our study also highlights the potential of endogenously adjuvanting the traditional BCG vaccine with MR1 ligands to augment MAIT cell activation. This study increases current knowledge on the roles of the riboflavin-biosynthetic pathway and MAIT cell activation in M. tuberculosis virulence and host immunity against TB.

Bacillus Calmette-Guérin Overexpressing an Endogenous Stimulator of Interferon Genes Agonist Provides Enhanced Protection Against Pulmonary Tuberculosis.

BACKGROUND: Stimulator of interferon genes (STING) is a key cytosolic receptor for small nucleotides and plays a key role in anticancer and antiviral immunity. Cyclic dinucleotide STING agonists may comprise a novel class of vaccine adjuvants capable of inducing cellular immune responses and protective efficacy against intracellular pathogens. METHODS: We generated a recombinant Bacillus Calmette-Guérin ([BCG] BCG-disA-OE) that overexpresses the endogenous mycobacterial diadenylate cyclase gene and releases high levels of the STING agonist bis-(3'-5')-cyclic dimeric adenosine monophosphate (c-di-AMP). We used a 24-week guinea pig vaccination-Mycobacterium tuberculosis (M.tb.) challenge model to test the protective efficacy of BCG-disA-OE versus wild-type BCG and measured lung weights, pathology scores, and M.tb. organ colony-forming unit (CFU) counts. RESULTS: BCG-disA-OE elicited significantly stronger tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, interferon (IFN) regulatory factor 3, and IFN-ß levels than BCG-wild type (WT) in vitro in murine macrophages. In vivo in guinea pigs, we found that BCG-disA-OE reduced lung weights, pathology scores, and M.tb. CFU counts in lungs by 28% (P < .05), 34%, and 2.0 log10 CFU units (P < .05) compared with BCG-WT, respectively. CONCLUSIONS: We report a strategy of delivering a STING agonist from within live BCG. Overproduction of the STING agonist c-di-AMP significantly enhanced the protective efficacy of BCG against pulmonary and extrapulmonary tuberculosis. Our findings support the development of BCG-vectored STING agonists as a tuberculosis vaccine strategy.

Inhibition of innate immune cytosolic surveillance by an M. tuberculosis phosphodiesterase.

Mycobacterium tuberculosis infection leads to cytosolic release of the bacterial cyclic dinucleotide (CDN) c-di-AMP and a host-generated CDN, cGAMP, both of which trigger type I interferon (IFN) expression in a STING-dependent manner. Here we report that M. tuberculosis has developed a mechanism to inhibit STING activation and the type I IFN response via the bacterial phosphodiesterase (PDE) CdnP, which mediates hydrolysis of both bacterial-derived c-di-AMP and host-derived cGAMP. Mutation of cdnP attenuates M. tuberculosis virulence, as does loss of a host CDN PDE known as ENPP1. CdnP is inhibited by both US Food and Drug Administration (FDA)-approved PDE inhibitors and nonhydrolyzable dinucleotide mimetics specifically designed to target the enzyme. These findings reveal a crucial role of CDN homeostasis in governing the outcome of M. tuberculosis infection as well as a unique mechanism of subversion of the host's cytosolic surveillance pathway (CSP) by a bacterial PDE that may serve as an attractive antimicrobial target.

Computer-aided pulmonary image analysis in small animal models.

PURPOSE: To develop an automated pulmonary image analysis framework for infectious lung diseases in small animal models. METHODS: The authors describe a novel pathological lung and airway segmentation method for small animals. The proposed framework includes identification of abnormal imaging patterns pertaining to infectious lung diseases. First, the authors' system estimates an expected lung volume by utilizing a regression function between total lung capacity and approximated rib cage volume. A significant difference between the expected lung volume and the initial lung segmentation indicates the presence of severe pathology, and invokes a machine learning based abnormal imaging pattern detection system next. The final stage of the proposed framework is the automatic extraction of airway tree for which new affinity relationships within the fuzzy connectedness image segmentation framework are proposed by combining Hessian and gray-scale morphological reconstruction filters. RESULTS: 133 CT scans were collected from four different studies encompassing a wide spectrum of pulmonary abnormalities pertaining to two commonly used small animal models (ferret and rabbit). Sensitivity and specificity were greater than 90% for pathological lung segmentation (average dice similarity coefficient > 0.9). While qualitative visual assessments of airway tree extraction were performed by the participating expert radiologists, for quantitative evaluation the authors validated the proposed airway extraction method by using publicly available EXACT'09 data set. CONCLUSIONS: The authors developed a comprehensive computer-aided pulmonary image analysis framework for preclinical research applications. The proposed framework consists of automatic pathological lung segmentation and accurate airway tree extraction. The framework has high sensitivity and specificity; therefore, it can contribute advances in preclinical research in pulmonary diseases.

A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis.

Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit type I interferon (IFN) responses, which are crucial to antimicrobial defense. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)-overexpressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-ß, elicits increased macrophage autophagy, and exhibits substantial virulence attenuation in mice. We show that c-di-AMP-mediated IFN-ß induction during M. tuberculosis infection requires stimulator of interferon genes (STING)-signaling. We observed that c-di-AMP induction of IFN-ß is independent of the cytosolic nucleic acid receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), but cGAS nevertheless contributes substantially to the overall IFN-ß response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen-associated molecular pattern (PAMP) driving host type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against tuberculosis.

Crosstalk between Mycobacterium tuberculosis and the host cell.

The successful establishment and maintenance of a bacterial infection depend on the pathogen's ability to subvert the host cell's defense response and successfully survive, proliferate, or persist within the infected cell. To circumvent host defense systems, bacterial pathogens produce a variety of virulence factors that potentiate bacterial adherence and invasion and usurp host cell signaling cascades that regulate intracellular microbial survival and trafficking. Mycobacterium tuberculosis, probably one of the most successful pathogens on earth, has coexisted with humanity for centuries, and this intimate and persistent connection between these two organisms suggests that the pathogen has evolved extensive mechanisms to evade the human immune system at multiple levels. While some of these mechanisms are mediated by factors released by M. tuberculosis, others rely on host components that are hijacked to prevent the generation of an effective immune response thus benefiting the survival of M. tuberculosis within the host cell. Here, we describe several of these mechanisms, with an emphasis on the cyclic nucleotide signaling and subversion of host responses that occur at the intracellular level when tubercle bacilli encounter macrophages, a cell that becomes a safe-house for M. tuberculosis although it is specialized to kill most microbes.

Segmentation of PET images for computer-aided functional quantification of tuberculosis in small animal models.

Pulmonary infections often cause spatially diffuse and multi-focal radiotracer uptake in positron emission tomography (PET) images, which makes accurate quantification of the disease extent challenging. Image segmentation plays a vital role in quantifying uptake due to the distributed nature of immuno-pathology and associated metabolic activities in pulmonary infection, specifically tuberculosis (TB). For this task, thresholding-based segmentation methods may be better suited over other methods; however, performance of the thresholding-based methods depend on the selection of thresholding parameters, which are often suboptimal. Several optimal thresholding techniques have been proposed in the literature, but there is currently no consensus on how to determine the optimal threshold for precise identification of spatially diffuse and multi-focal radiotracer uptake. In this study, we propose a method to select optimal thresholding levels by utilizing a novel intensity affinity metric within the affinity propagation clustering framework. We tested the proposed method against 70 longitudinal PET images of rabbits infected with TB. The overall dice similarity coefficient between the segmentation from the proposed method and two expert segmentations was found to be 91.25 ±8.01% with a sensitivity of 88.80 ±12.59% and a specificity of 96.01 ±9.20%. High accuracy and heightened efficiency of our proposed method, as compared to other PET image segmentation methods, were reported with various quantification metrics.

A computational pipeline for quantification of pulmonary infections in small animal models using serial PET-CT imaging.

BACKGROUND: Infectious diseases are the second leading cause of death worldwide. In order to better understand and treat them, an accurate evaluation using multi-modal imaging techniques for anatomical and functional characterizations is needed. For non-invasive imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), there have been many engineering improvements that have significantly enhanced the resolution and contrast of the images, but there are still insufficient computational algorithms available for researchers to use when accurately quantifying imaging data from anatomical structures and functional biological processes. Since the development of such tools may potentially translate basic research into the clinic, this study focuses on the development of a quantitative and qualitative image analysis platform that provides a computational radiology perspective for pulmonary infections in small animal models. Specifically, we designed (a) a fast and robust automated and semi-automated image analysis platform and a quantification tool that can facilitate accurate diagnostic measurements of pulmonary lesions as well as volumetric measurements of anatomical structures, and incorporated (b) an image registration pipeline to our proposed framework for volumetric comparison of serial scans. This is an important investigational tool for small animal infectious disease models that can help advance researchers' understanding of infectious diseases. METHODS: We tested the utility of our proposed methodology by using sequentially acquired CT and PET images of rabbit, ferret, and mouse models with respiratory infections of Mycobacterium tuberculosis (TB), H1N1 flu virus, and an aerosolized respiratory pathogen (necrotic TB) for a total of 92, 44, and 24 scans for the respective studies with half of the scans from CT and the other half from PET. Institutional Administrative Panel on Laboratory Animal Care approvals were obtained prior to conducting this research. First, the proposed computational framework registered PET and CT images to provide spatial correspondences between images. Second, the lungs from the CT scans were segmented using an interactive region growing (IRG) segmentation algorithm with mathematical morphology operations to avoid false positive (FP) uptake in PET images. Finally, we segmented significant radiotracer uptake from the PET images in lung regions determined from CT and computed metabolic volumes of the significant uptake. All segmentation processes were compared with expert radiologists' delineations (ground truths). Metabolic and gross volume of lesions were automatically computed with the segmentation processes using PET and CT images, and percentage changes in those volumes over time were calculated. (Continued on next page)(Continued from previous page) Standardized uptake value (SUV) analysis from PET images was conducted as a complementary quantitative metric for disease severity assessment. Thus, severity and extent of pulmonary lesions were examined through both PET and CT images using the aforementioned quantification metrics outputted from the proposed framework. RESULTS: Each animal study was evaluated within the same subject class, and all steps of the proposed methodology were evaluated separately. We quantified the accuracy of the proposed algorithm with respect to the state-of-the-art segmentation algorithms. For evaluation of the segmentation results, dice similarity coefficient (DSC) as an overlap measure and Haussdorf distance as a shape dissimilarity measure were used. Significant correlations regarding the estimated lesion volumes were obtained both in CT and PET images with respect to the ground truths (R2=0.8922,p<0.01 and R2=0.8664,p<0.01, respectively). The segmentation accuracy (DSC (%)) was 93.4±4.5% for normal lung CT scans and 86.0±7.1% for pathological lung CT scans. Experiments showed excellent agreements (all above 85%) with expert evaluations for both structural and functional imaging modalities. Apart from quantitative analysis of each animal, we also qualitatively showed how metabolic volumes were changing over time by examining serial PET/CT scans. Evaluation of the registration processes was based on precisely defined anatomical landmark points by expert clinicians. An average of 2.66, 3.93, and 2.52 mm errors was found in rabbit, ferret, and mouse data (all within the resolution limits), respectively. Quantitative results obtained from the proposed methodology were visually related to the progress and severity of the pulmonary infections as verified by the participating radiologists. Moreover, we demonstrated that lesions due to the infections were metabolically active and appeared multi-focal in nature, and we observed similar patterns in the CT images as well. Consolidation and ground glass opacity were the main abnormal imaging patterns and consistently appeared in all CT images. We also found that the gross and metabolic lesion volume percentage follow the same trend as the SUV-based evaluation in the longitudinal analysis. CONCLUSIONS: We explored the feasibility of using PET and CT imaging modalities in three distinct small animal models for two diverse pulmonary infections. We concluded from the clinical findings, derived from the proposed computational pipeline, that PET-CT imaging is an invaluable hybrid modality for tracking pulmonary infections longitudinally in small animals and has great potential to become routinely used in clinics. Our proposed methodology showed that automated computed-aided lesion detection and quantification of pulmonary infections in small animal models are efficient and accurate as compared to the clinical standard of manual and semi-automated approaches. Automated analysis of images in pre-clinical applications can increase the efficiency and quality of pre-clinical findings that ultimately inform downstream experimental design in human clinical studies; this innovation will allow researchers and clinicians to more effectively allocate study resources with respect to research demands without compromising accuracy.

Adjunctive immunotherapy with α-crystallin based DNA vaccination reduces Tuberculosis chemotherapy period in chronically infected mice.

By employing modified Cornell model, we have evaluated the potential of adjunctive immunotherapy with DNA vaccines to shorten the tuberculosis chemotherapy period and reduce disease reactivation. We demonstrate that α-crystallin based DNA vaccine (DNAacr) significantly reduced the chemotherapy period from 12 weeks to 8 weeks when compared with the chemotherapy alone. Immunotherapy with SodA based DNA vaccine (DNAsod) reduced the pulmonary bacilli only as much as DNAvec. Both DNAacr and DNAsod, although significantly delayed the reactivation in comparison to the chemotherapy alone, this delay was associated with the immunostimulatory sequences present in the vector backbone and was not antigen specific. Both DNA vaccines resulted in the production of significantly higher number of TEM cells than the chemotherapy alone, however, only in the case of DNAsod, this enhancement was significant over the DNAvec treatment. Overall, our findings emphasize the immunotherapeutic potential of DNAacr in shortening the duration of TB chemotherapy.

Perspectives for Developing New Tuberculosis Vaccines Derived from the Pathogenesis of Tuberculosis: I. Basic Principles, II. Preclinical Testing, and III. Clinical Testing.

Part I. Basic Principles. TB vaccines cannot prevent establishment of the infection. They can only prevent an early pulmonary tubercle from developing into clinical disease. A more effective new vaccine should optimize both cell-mediated immunity (CMI) and delayed-type hypersensitivity (DTH) better than any existing vaccine. The rabbit is the only laboratory animal in which all aspects of the human disease can be reproduced: namely, the prevention of most primary tubercles, the arrestment of most primary tubercles, the formation of the tubercle's solid caseous center, the liquefaction of this center, the formation of cavities and the bronchial spread of the disease. In liquefied caseum, virulent tubercle bacilli can multiply extracellularly, especially in the liquefied caseum next to the inner wall of a cavity where oxygen is plentiful. The bacilli in liquefied caseum cannot be reached by the increased number of activated macrophages produced by TB vaccines. Therefore, new TB vaccines will have little or no effect on the extracellular bacillary growth within liquefied caseum. TB vaccines can only increase the host's ability to stop the development of new TB lesions that arise from the bronchial spread of tubercle bacilli from the cavity to other parts of the lung. Therefore, effective TB vaccines do not prevent the reactivation of latent TB. Such vaccines only control (or reduce) the number of metastatic lesions that result after the primary TB lesion was reactivated by the liquefaction process. (Note: the large number of tubercle bacilli growing extracellularly in liquefied caseum gives rise to mutations that enable antimicrobial resistance-which is a major reason why TB still exists today). Part II. Preclinical Testing. The counting of grossly visible tubercles in the lungs of rabbits after the inhalation of virulent human-type tubercle bacilli is the most pertinent preclinical method to assess the efficacy of new TB vaccines (because an effective vaccine will stop the growth of developing tubercles before while they are still microscopic in size). Unfortunately, rabbits are rarely used in preclinical vaccine trials, despite their relative ease of handling and human-like response to this infection. Mice do not generate an effective DTH response, and guinea pigs do not generate an effective CMI response. Only the rabbits and most humans can establish the proper amount of DTH and CMI that is necessary to contain this infection. Therefore, rabbits should be included in all pre-clinical testing of new TB vaccines. New drugs (and/or immunological procedures) to reduce liquefaction and cavity formation are urgently needed. A simple intradermal way to select such drugs or procedures is described herein. Part III. Clinical Testing. Vaccine trials would be much more precise if the variations in human populations (listed herein) were taken into consideration. BCG and successful new TB vaccines should always increase host resistance to TB in naive subjects. This is a basic immunological principle. The efficacies of new and old TB vaccines are often not recognized, because these variations were not identified in the populations evaluated.

Development of the first oligonucleotide microarray for global gene expression profiling in guinea pigs: defining the transcription signature of infectious diseases.

BACKGROUND: The Guinea pig (Cavia porcellus) is one of the most extensively used animal models to study infectious diseases. However, despite its tremendous contribution towards understanding the establishment, progression and control of a number of diseases in general and tuberculosis in particular, the lack of fully annotated guinea pig genome sequence as well as appropriate molecular reagents has severely hampered detailed genetic and immunological analysis in this animal model. RESULTS: By employing the cross-species hybridization technique, we have developed an oligonucleotide microarray with 44,000 features assembled from different mammalian species, which to the best of our knowledge is the first attempt to employ microarray to study the global gene expression profile in guinea pigs. To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection. A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection. CONCLUSION: We report the development of first comprehensive microarray for studying the global gene expression profile in guinea pigs and validation of its usefulness with tuberculosis as a case study. An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases.

Over-expression of superoxide dismutase obliterates the protective effect of BCG against tuberculosis by modulating innate and adaptive immune responses.

An efficient global control of tuberculosis requires development of alternative vaccination strategies that can enhance the efficacy of existing BCG vaccine. In this study, we evaluated the protective efficacy of a recombinant BCG (rBCG) vaccine over-expressing iron-cofactored superoxide dismutase (SOD-A), one of the prominent oxidative stress response proteins of Mycobacterium tuberculosis. Contrary to our expectations, over-expression of SOD-A resulted in the abrogation of BCG's ability to confer protection in guinea pig as well as in murine model. Analysis of immune responses revealed that over-expression of SOD-A by rBCG has pleiotropic effects on innate and adaptive immune responses. Macrophages infected in vitro with rBCG exhibited a marked reduction in apoptosis and microbicidal potential. In addition, rBCG vaccination of mice resulted in a reduced IFNγ and increased IL10 production when compared with the BCG vaccination. Further, we show that rBCG vaccination failed to generate an effective multi-functional CD4 T cell response. Altogether, our findings suggest that over-expression of SOD-A in BCG enhances the immuno-suppressive properties of BCG, characterized by skewing of immune responses towards Th2 type, an inefficient multi-functional T cell response and reduced apoptosis and microbicidal potential of macrophages leading to abolishment of BCG's protective efficacy.

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis.

BACKGROUND: In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB. METHODS/PRINCIPAL FINDINGS: In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin--a key latency antigen of M. tuberculosis to boost the BCG induced immunity. 'BCG prime-DNA boost' regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log(10) and 1.96 log(10) fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3(+)) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3(+) Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.

Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines.

BACKGROUND: Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG. METHODS/PRINCIPAL FINDINGS: We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that "rBCG prime-DNA boost" strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)ß and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFß in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.

Boosting with a DNA vaccine expressing ESAT-6 (DNAE6) obliterates the protection imparted by recombinant BCG (rBCGE6) against aerosol Mycobacterium tuberculosis infection in guinea pigs.

Owing to its highly immunodominant nature and ability to induce long-lived memory immunity, ESAT-6, a prominent antigen of Mycobacterium tuberculosis, has been employed in several approaches to develop tuberculosis vaccines. Here, for the first time, we combined ESAT-6 based recombinant BCG (rBCG) and DNA vaccine (DNAE6) in a prime boost approach. Interestingly, in spite of inducing an enhanced antigen specific IFN-gamma response in mice, a DNAE6 booster completely obliterated the protection imparted by rBCG against tuberculosis in guinea pigs. Analysis of immunopathology and cytokine responses suggests involvement of an exaggerated immunity behind the lack of protection imparted by this regimen.

Enhanced and enduring protection against tuberculosis by recombinant BCG-Ag85C and its association with modulation of cytokine profile in lung.

BACKGROUND: The variable efficacy (0-80%) of Mycobacterium bovis Bacille Calmette Guréin (BCG) vaccine against adult tuberculosis (TB) necessitates development of alternative vaccine candidates. Development of recombinant BCG (rBCG) over-expressing promising immunodominant antigens of M. tuberculosis represents one of the potential approaches for the development of vaccines against TB. METHODS/PRINCIPAL FINDINGS: A recombinant strain of BCG - rBCG85C, over expressing the antigen 85C, a secretory immuno-dominant protein of M. tuberculosis, was evaluated for its protective efficacy in guinea pigs against M. tuberculosis challenge by aerosol route. Immunization with rBCG85C resulted in a substantial reduction in the lung (1.87 log(10), p<0.01) and spleen (2.36 log(10), p<0.001) bacillary load with a commensurate reduction in pathological damage, when compared to the animals immunized with the parent BCG strain at 10 weeks post-infection. rBCG85C continued to provide superior protection over BCG even when post-challenge period was prolonged to 16 weeks. The cytokine profile of pulmonary granulomas revealed that the superior protection imparted by rBCG85C was associated with the reduced levels of pro-inflammatory cytokines - interleukin (IL)-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, moderate levels of anti-inflammatory cytokine - transforming growth factor (TGF)-beta along with up-regulation of inducible nitric oxide synthase (iNOS). In addition, the rBCG85C vaccine induced modulation of the cytokine levels was found to be associated with reduced fibrosis and antigen load accompanied by the restoration of normal lung architecture. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of rBCG85C over BCG as a promising prophylactic vaccine against TB. The enduring protection observed in this study gives enough reason to postulate that if an open-ended study is carried out with low dose of infection, rBCG85C vaccine in all likelihood would show enhanced survival of guinea pigs.