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1.  Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs 
PLoS ONE  2013;8(10):e77930.
Background
The failure of Mycobacterium bovis Bacille Calmette-Guérin to impart satisfactory protection against adult pulmonary tuberculosis has necessitated the development of more effective TB vaccines. The assumption that the vaccine strain should be antigenically as similar as possible to the disease causing pathogen has led to the evaluation of M.tuberculosis mutants as candidate tuberculosis vaccines.
Methods/Principal Findings
In this study, we have generated a mutant of M.tuberculosis (Mtb∆mms) by disrupting 3 virulence genes encoding a mycobacterial secretory acid phosphatase (sapM) and two phosphotyrosine protein phosphatases (mptpA and mptpB) and have evaluated its protective efficacy in guinea pigs. We observed that Mtb∆mms was highly attenuated in THP-1 macrophages. Moreover, no bacilli were recovered from the lungs and spleens of guinea pigs after 10 weeks of Mtb∆mms inoculation, although, initially, the mutant exhibited some growth in the spleens. Subsequently, when Mtb∆mms was evaluated for its protective efficacy, we observed that similar to BCG vaccination, Mtb∆mms exhibited a significantly reduced CFU in the lungs of guinea pigs when compared with the unvaccinated animals at 4 weeks after challenge. In addition, our observations at 12 weeks post challenge demonstrated that Mtb∆mms exhibited a more sustainable and superior protection in lungs as compared to BCG. However, the mutant failed to control the hematogenous spread as the splenic bacillary load between Mtb∆mms vaccinated and sham immunized animals was not significantly different. The gross pathological observations and histopathological observations corroborated the bacterial findings. Inspite of disruption of phosphatase genes in MtbΔmms, the lipid profiles of M.tuberculosis and MtbΔmms were identical indicating thereby that the phenotype of the mutant was ascribed to the loss of phosphatase genes and the influence was not related to any alteration in the lipid composition.
Conclusions/Significance
This study highlights the importance of M.tuberculosis mutants in imparting protection against pulmonary TB.
doi:10.1371/journal.pone.0077930
PMCID: PMC3799640  PMID: 24205032
2.  The fbpA/sapM Double Knock Out Strain of Mycobacterium tuberculosis Is Highly Attenuated and Immunogenic in Macrophages 
PLoS ONE  2012;7(5):e36198.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.
doi:10.1371/journal.pone.0036198
PMCID: PMC3344844  PMID: 22574140
3.  Enhanced and Enduring Protection against Tuberculosis by Recombinant BCG-Ag85C and Its Association with Modulation of Cytokine Profile in Lung 
PLoS ONE  2008;3(12):e3869.
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 log10, p<0.01) and spleen (2.36 log10, 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)-γ, tumor necrosis factor (TNF)-α, moderate levels of anti-inflammatory cytokine - transforming growth factor (TGF)-β 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.
doi:10.1371/journal.pone.0003869
PMCID: PMC2586085  PMID: 19052643
4.  Immunogenicity and Protective Efficacy of Prime-Boost Regimens with Recombinant ΔureC hly+ Mycobacterium bovis BCG and Modified Vaccinia Virus Ankara Expressing M. tuberculosis Antigen 85A against Murine Tuberculosis▿  
Infection and Immunity  2008;77(2):622-631.
In the light of the recent emergence of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the epidemic of tuberculosis (TB) in populations coinfected with human immunodeficiency virus, and the failure of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to protect against disease, new vaccines against TB are urgently needed. Two promising new vaccine candidates are the recombinant ΔureC hly+ BCG (recBCG), which has been developed to replace the current BCG vaccine strain, and modified vaccinia virus Ankara (MVA) expressing M. tuberculosis antigen 85A (MVA85A), which is a leading candidate vaccine designed to boost the protective efficacy of BCG. In the present study, we examined the effect of MVA85A boosting on the protection afforded at 12 weeks postchallenge by BCG and recBCG by using bacterial CFU as an efficacy readout. recBCG-immunized mice were significantly better protected against aerosol challenge with M. tuberculosis than mice immunized with the parental strain of BCG. Intradermal boosting with MVA85A did not reduce the bacterial burden any further. In order to identify a marker for the development of a protective immune response against M. tuberculosis challenge, we analyzed splenocytes after priming or prime-boosting by using intracytoplasmic cytokine staining and assays for cytokine secretion. Boosting with MVA85A, but not priming with BCG or recBCG, greatly increased the antigen 85A-specific T-cell response, suggesting that the mechanism of protection may differ from that against BCG or recBCG. We show that the numbers of systemic multifunctional cytokine-producing cells did not correlate with protection against aerosol challenge in BALB/c mice. This emphasizes the need for new biomarkers for the evaluation of TB vaccine efficacy.
doi:10.1128/IAI.00685-08
PMCID: PMC2632018  PMID: 19064635
5.  Identification of a Mycobacterium bovis BCG Auxotrophic Mutant That Protects Guinea Pigs against M. bovis and Hematogenous Spread of Mycobacterium tuberculosis without Sensitization to Tuberculin 
Infection and Immunity  2000;68(12):7094-7099.
Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.
PMCID: PMC97820  PMID: 11083835
6.  The Impact of Alcohol on BCG-Induced Immunity Against Mycobacterium tuberculosis 
Background
Alcoholics are at heightened risk for developing active tuberculosis. This study evaluates chronic alcohol consumption in a murine model of vaccination with Mycobacterium bovis Bacille-Calmette Guèrin (BCG) and subsequent pulmonary infection with virulent Mycobacterium tuberculosis.
Methods
BALB/c mice were administered the Lieber-DeCarli liquid ethanol diet (LED) or pair-fed the liquid control diet (LCD) for three weeks either before or after subcutaneous vaccination with M. bovis BCG. At least three weeks after BCG vaccination, groups of mice on the above diets were challenged with intratracheal infection with M. tuberculosis H37Rv. Lung mycobacterial burden, and lung and lung-associated lymph node CD4+ lymphocyte production of TB-specific interferon (IFN)-γ were assayed. Popliteal lymph node lymphocytes from both dietary regimens undergoing BCG vaccination (in the absence of M. tuberculosis infection) were also evaluated for PPD-induced interferon (IFN)-γ production by ELISpot assay.
Results
Mice begun on alcohol prior to vaccination with M. bovis BCG demonstrated impaired control of pulmonary challenge with virulent M. tuberculosis, as well as impaired lung CD4+ and popliteal lymph node T cell IFN-γ responses. If BCG vaccination was delivered prior to initiation of alcohol feeding, the mice remained protected against a subsequent challenge with M. tuberculosis, and BCG-induced immunity was not impaired in either the lung or the popliteal lymph nodes.
Conclusions
Alcohol consumption blunts the development of the adaptive immune response to M. bovis BCG vaccination, which impairs the control of a secondary challenge with M. tuberculosis, but only if the alcohol exposure is begun prior to BCG vaccination. These results provide insight into mechanisms by which alcohol consumption impairs antimycobacterial immunity, including in response to vaccination and subsequent pathogenic challenge.
doi:10.1111/j.1530-0277.2011.01624.x
PMCID: PMC3266991  PMID: 22014229
Alcohol; M. tuberculosis; BCG vaccination; CD4+ lymphocytes; IFN-γ
7.  Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin 
Journal of Clinical Investigation  2005;115(9):2472-2479.
The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly+ rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C–deficient hly+ rBCG (ΔureC hly+ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. ΔureC hly+ rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of ΔureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell–mediated immunity.
doi:10.1172/JCI24617
PMCID: PMC1187936  PMID: 16110326
8.  Secreted Acid Phosphatase (SapM) of Mycobacterium tuberculosis Is Indispensable for Arresting Phagosomal Maturation and Growth of the Pathogen in Guinea Pig Tissues 
PLoS ONE  2013;8(7):e70514.
Tuberculosis (TB) is responsible for nearly 1.4 million deaths globally every year and continues to remain a serious threat to human health. The problem is further complicated by the growing incidence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), emphasizing the need for the development of new drugs against this disease. Phagosomal maturation arrest is an important strategy employed by Mycobacterium tuberculosis to evade the host immune system. Secretory acid phosphatase (SapM) of M.tuberculosis is known to dephosphorylate phosphotidylinositol 3-phosphate (PI3P) present on phagosomes. However, there have been divergent reports on the involvement of SapM in phagosomal maturation arrest in mycobacteria. This study was aimed at reascertaining the involvement of SapM in phagosomal maturation arrest in M.tuberculosis. Further, for the first time, we have also studied whether SapM is essential for the pathogenesis of M.tuberculosis. By deleting the sapM gene of M.tuberculosis, we demonstrate that MtbΔsapM is defective in the arrest of phagosomal maturation as well as for growth in human THP-1 macrophages. We further show that MtbΔsapM is severely attenuated for growth in the lungs and spleen of guinea pigs and has a significantly reduced ability to cause pathological damage in the host when compared with the parental strain. Also, the guinea pigs infected with MtbΔsapM exhibited a significantly enhanced survival when compared with M.tuberculosis infected animals. The importance of SapM in phagosomal maturation arrest as well as in the pathogenesis of M.tuberculosis establishes it as an attractive target for the development of new therapeutic molecules against tuberculosis.
doi:10.1371/journal.pone.0070514
PMCID: PMC3724783  PMID: 23923000
9.  Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur 
BMC Microbiology  2011;11:80.
Background
Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.
Results
The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern.
Conclusions
Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.
doi:10.1186/1471-2180-11-80
PMCID: PMC3094199  PMID: 21507239
10.  Tuberculosis vaccine strain Mycobacterium bovis BCG Russia is a natural recA mutant 
BMC Microbiology  2008;8:120.
Background
The current tuberculosis vaccine is a live vaccine derived from Mycobacterium bovis and attenuated by serial in vitro passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Guérin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy.
Results
As a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains while maintaining their protective efficacy, we aimed to inactivate recA by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia). Homologous gene replacement was achieved successfully in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of recA revealed that a single nucleotide insertion in the 5' part of recA led to a translational frameshift with an early stop codon making BCG Russia a natural recA mutant. At the protein level BCG Russia failed to express RecA.
Conclusion
According to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental M. bovis. We hypothesize that recA inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to M. bovis AF2122/97 wild-type.
doi:10.1186/1471-2180-8-120
PMCID: PMC2483709  PMID: 18637199
11.  BCG-Mediated Protection against Mycobacterium ulcerans Infection in the Mouse 
Background
Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is widely used to reduce the risk of childhood tuberculosis and has been reported to have efficacy against two other mycobacterial diseases, leprosy and Buruli ulcer caused by M. ulcerans (Mu). Studies in experimental models have also shown some efficacy against infection caused by Mu. In mice, most studies use the C57BL/6 strain that is known to develop good cell-mediated protective immunity. We hypothesized that there may be differences in vaccination efficacy between C57BL/6 and the less resistant BALB/c strain.
Methods
We evaluated BCG vaccine efficacy against challenge with ∼3×105 M. ulcerans in the right hind footpad using three strains: initially, the Australian type strain, designated Mu1617, then, a Malaysian strain, Mu1615, and a recent Ghanaian isolate, Mu1059. The latter two strains both produce mycolactone while the Australian strain has lost that capacity. CFU of both BCG and Mu and splenocyte cytokine production were determined at intervals after infection. Time to footpad swelling was assessed weekly.
Principal Findings
BCG injection induced visible scars in 95.5% of BALB/c mice but only 43.4% of C57BL/6 mice. BCG persisted at higher levels in spleens of BALB/c than C57BL/6 mice. Vaccination delayed swelling and reduced Mu CFU in BALB/c mice, regardless of challenge strain. However, vaccination was only protective against Mu1615 and Mu1617 in C57BL/6 mice. Possible correlates of the better protection of BALB/c mice included 1) the near universal development of BCG scars in these mice compared to less frequent and smaller scars observed in C57BL/6 mice and 2) the induction of sustained cytokine, e.g., IL17, production as detected in the spleens of BALB/c mice whereas cytokine production was significantly reduced, e.g., IL17, or transient, e.g., Ifnγ, in the spleens of C57BL/6 mice.
Conclusions
The efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen.
Author Summary
Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is used to reduce the risk of childhood tuberculosis and is reported to have efficacy against two other diseases also caused by mycobacteria, leprosy and Buruli ulcer caused by M. ulcerans. We hypothesized that there may be differences in the effectiveness of BCG vaccination in different mouse strains. We vaccinated two mouse strains with BCG eight weeks before infection with three different strains of M. ulcerans. Two of the bacterial strains make a toxin that is critical for Buruli ulcer disease and the third does not. We observed the progression of disease in vaccinated and mock-vaccinated mice and also evaluated the immune response of the mice. We found that the BALB/c mice respond to BCG vaccination with prominent scars, a vigorous immune response, and delayed or no manifestations of M. ulcerans infection. C57BL/6 mice, on the other hand, usually do not have vaccination scars, make a relatively short-lived and/or weaker immune response, and all show disease at the site of M. ulcerans infection. We conclude that the efficacy of BCG against M. ulcerans, and possibly other diseases, depends on the nature of the host and of the infecting strain of the bacteria.
doi:10.1371/journal.pntd.0000985
PMCID: PMC3057947  PMID: 21423646
12.  Recombinant BCG: Innovations on an Old Vaccine. Scope of BCG Strains and Strategies to Improve Long-Lasting Memory 
Bacille Calmette–Guérin (BCG), an attenuated vaccine derived from Mycobacterium bovis, is the current vaccine of choice against tuberculosis (TB). Despite its protection against active TB in children, BCG has failed to protect adults against TB infection and active disease development, especially in developing countries where the disease is endemic. Currently, there is a significant effort toward the development of a new TB vaccine. This review article aims to address publications on recombinant BCG (rBCG) published in the last 5 years, to highlight the strategies used to develop rBCG, with a focus on the criteria used to improve immunological memory and protection compared with BCG. The literature review was done in April 2013, using the key words TB, rBCG vaccine, and memory. This review discusses the BCG strains and strategies currently used for the modification of BCG, including: overexpression of Mycobacterium tuberculosis (Mtb) immunodominant antigens already present in BCG; gene insertion of immunodominant antigens from Mtb absent in the BCG vaccine; combination of introduction and overexpression of genes that are lost during the attenuation process of BCG; BCG modifications for the induction of CD8+ T-cell immune responses and cytokines expressing rBCG. Among the vaccines discussed, VPM1002, also called rBCGΔureC:hly, is currently in human clinical trials. Much progress has been made in the effort to improve BCG, with some promising candidates, but considerable work is still required to address functional long-lasting memory.
doi:10.3389/fimmu.2014.00152
PMCID: PMC3984997  PMID: 24778634
rBCG; tuberculosis; vaccine; protection; long-term memory; strain differences
13.  Recombinant BCG ΔureC hly+ Induces Superior Protection Over Parental BCG by Stimulating a Balanced Combination of Type 1 and Type 17 Cytokine Responses 
The Journal of Infectious Diseases  2011;204(10):1573-1584.
Background. New vaccines against tuberculosis (TB) are urgently needed because the only available vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), fails to protect against pulmonary TB in adults. The recombinant ΔureC hly+ BCG (rBCG) is more efficient than parental BCG (pBCG) against pulmonary TB in preclinical studies and has proven safe and immunogenic in phase I clinical trials.
Methods. In an attempt to identify the mechanisms underlying the superior protection of rBCG, we compared the immune responses elicited after vaccination and subsequent aerosol infection with Mycobacterium tuberculosis (MTB) in mice.
Results. We demonstrate that both rBCG and pBCG induce marked type 1 cytokine responses, whereas only rBCG elicits a profound type 17 cytokine response in addition. We observed earlier recruitment of antigen-specific T lymphocytes to the lung upon MTB infection of rBCG-vaccinated mice. These T cells produced abundant type 1 cytokines after restimulation, resulting in 10-fold reduced bacterial burden 90 days after infection.
Conclusions. Our findings identify a general immunologic pathway for improved vaccination strategies against TB that can also be harnessed by other vaccine candidates.
doi:10.1093/infdis/jir592
PMCID: PMC3192191  PMID: 21933877
14.  Lysine Auxotrophy Combined with Deletion of the SecA2 Gene Results in a Safe and Highly Immunogenic Candidate Live Attenuated Vaccine for Tuberculosis 
PLoS ONE  2011;6(1):e15857.
Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major global health problem, despite the widespread use of the M. bovis Bacille Calmette-Guerin (BCG) vaccine and the availability of drug therapies. In recent years, the high incidence of coinfection of M. tuberculosis and HIV, as well as escalating problems associated with drug resistance, has raised ominous concerns with regard to TB control. Vaccination with BCG has not proven highly effective in controlling TB, and also has been associated with increasing concerns about the potential for the vaccine to cause disseminated mycobacterial infection in HIV infected hosts. Thus, the development of an efficacious and safe TB vaccine is generally viewed as a critical to achieving control of the ongoing global TB pandemic. In the current study, we have analyzed the vaccine efficacy of an attenuated M. tuberculosis strain that combines a mutation that enhances T cell priming (ΔsecA2) with a strongly attenuating lysine auxotrophy mutation (ΔlysA). The ΔsecA2 mutant was previously shown to be defective in the inhibition of apoptosis and markedly increased priming of antigen-specific CD8+ T cells in vivo. Similarly, the ΔsecA2ΔlysA strain retained enhanced apoptosis and augmented CD8+ T cell stimulatory effects, but with a noticeably improved safety profile in immunosuppressed mice. Thus, the M. tuberculosis ΔsecA2ΔlysA mutant represents a live attenuated TB vaccine strain with the potential to deliver increased protection and safety compared to standard BCG vaccination.
doi:10.1371/journal.pone.0015857
PMCID: PMC3018466  PMID: 21264335
15.  Latency Antigen α-Crystallin Based Vaccination Imparts a Robust Protection against TB by Modulating the Dynamics of Pulmonary Cytokines 
PLoS ONE  2011;6(4):e18773.
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)β 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 TGFβ 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.
doi:10.1371/journal.pone.0018773
PMCID: PMC3078913  PMID: 21533158
16.  IFN-γ Mediates the Rejection of Haematopoietic Stem Cells in IFN-γR1-Deficient Hosts 
PLoS Medicine  2008;5(1):e26.
Background
Interferon-γ receptor 1 (IFN-γR1) deficiency is a life-threatening inherited disorder, conferring predisposition to mycobacterial diseases. Haematopoietic stem cell transplantation (HSCT) is the only curative treatment available, but is hampered by a very high rate of graft rejection, even with intra-familial HLA-identical transplants. This high rejection rate is not seen in any other congenital disorders and remains unexplained. We studied the underlying mechanism in a mouse model of HSCT for IFN-γR1 deficiency.
Methods and Findings
We demonstrated that HSCT with cells from a syngenic C57BL/6 Ifngr1+/+ donor engrafted well and restored anti-mycobacterial immunity in naive, non-infected C57BL/6 Ifngr1−/− recipients. However, Ifngr1−/− mice previously infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) rejected HSCT. Like infected IFN-γR1-deficient humans, infected Ifngr1−/− mice displayed very high serum IFN-γ levels before HSCT. The administration of a recombinant IFN-γ-expressing AAV vector to Ifngr1−/− naive recipients also resulted in HSCT graft rejection. Transplantation was successful in Ifngr1−/− × Ifng−/− double-mutant mice, even after BCG infection. Finally, efficient antibody-mediated IFN-γ depletion in infected Ifngr1−/− mice in vivo allowed subsequent engraftment.
Conclusions
High serum IFN-γ concentration is both necessary and sufficient for graft rejection in IFN-γR1-deficient mice, inhibiting the development of heterologous, IFN-γR1-expressing, haematopoietic cell lineages. These results confirm that IFN-γ is an anti-haematopoietic cytokine in vivo. They also pave the way for HSCT management in IFN-γR1-deficient patients through IFN-γ depletion from the blood. They further raise the possibility that depleting IFN-γ may improve engraftment in other settings, such as HSCT from a haplo-identical or unrelated donor.
Claire Soudais and colleagues investigated the mechanism of rejection of hematopoietic stem cell transplants in patients with interferon-gamma receptor 1 (IFN-γR1) deficiency and show that IFN-γ is an anti-hematopoietic cytokine in vivo.
Editors' Summary
Background.
Normally, the body's immune system efficiently recognizes and kills bacteria and viruses, but in some rare inherited disorders (“primary immunodeficiencies”) part of the immune system works poorly or is missing. This leaves affected individuals susceptible to infections. People with one of these disorders—interferon-gamma receptor 1 (IFN- γR1) deficiency—are very susceptible to infections with mycobacteria. Except for Mycobacterium tuberculosis and M. leprae (which cause tuberculosis and leprosy, respectively), mycobacteria rarely cause human disease. However, most people with IFN-γR1 deficiency die during childhood from multiple, widespread mycobacterial infections, because IFN-γR1 deficiency disables a specific part of their immune system. When most bacteria enter the body, immune system cells called macrophages engulf and kill them, but mycobacteria actually multiply inside macrophages. This infection stimulates lymphocytes and other immune system cells to release IFN-γ, which binds to IFN-γR1 on uninfected macrophages, activates them, and recruits them to the infection site. Here, they form a “granuloma,” a mass of macrophages and activated lymphocytes that “walls off” the infection. Granuloma formation does not occur in patients with IFN-γR1 deficiency, so mycobacteria (including the usually benign tuberculosis vaccination strain M. bovis BCG) spread throughout the body with disastrous consequences.
Why Was This Study Done?
The only effective treatment for patients with IFN-γR1 deficiency is hematopoietic stem cell transplantation (HSCT). HSCs are the source of all the immune system cells, so transplantation of HSCs from a donor with a normal IFNGR1 gene can provide a patient who has IFN-γR1 deficiency with a new immune system that can combat mycobacterial infections. Unfortunately, in this particular immune deficiency, the new HSCs cannot engraft, even when the patient's own immune system is disabled before HSCT by intensive chemotherapy, and when the donor cells come from a close relative and are a good immunological match. In this study, the researchers have investigated why rejection is so common in IFN-γR1 deficiency using a mouse strain called C57BL/6 Ifngr1−/−—C57BL/6 denotes the genetic background of these mice and Ifngr1−/− indicates that, like human patients, these mice make no IFN-γR1.
What Did the Researchers Do and Find?
Ifngr1−/− mice, the researchers report, cannot control M. bovis BCG infections and do not form mature granulomas just like human patients with IFN-γR1 deficiency. Wild-type C57BL/6 (Ifngr1+/+) mice, however, rapidly control M. bovis BCG infections and form mature granulomas. Ifngr1+/+ HSC transplanted into mycobacteria-free Ifngr1−/− mice survived well and protected the mice against later mycobacterial challenge but Ifngr1−/− mice infected with M. bovis BCG before HSCT rejected the transplanted HSCs. Mycobacteria-infected Ifngr1−/− mice and human patients with IFN-γR1 deficiency have blood high levels of IFN-γ. Could this be responsible for HSCT rejection? To find out, the researchers expressed IFN-γ in uninfected Ifngr1−/− mice before HSCT. As in infected mice, these grafts failed. Conversely, transplanted HSCs survived when transplanted into Ifngr1−/− mice that had been genetically altered to express no IFN-γ, even when these mice were infected with M. bovis BCG before transplantation. Finally, when the researchers used antibodies (proteins made by the immune system that recognize specific molecules) to remove circulating IFN-γ from infected Ifngr1−/− mice, HSCT worked well in the animals with the lowest IFN-γ levels.
What Do These Findings Mean?
These findings indicate that in a mouse model of IFN-γR1 deficiency, high circulating IFN-γ concentrations drive the rejection of transplanted HSCs and prevent the development of antimycobacterial immunity, probably by directly killing the transplanted cells and/or stopping them multiplying. They also suggest how HSCT could be improved in patients with IFN-γR1 deficiency although, as with all animal studies, the situation in people might turn out to be very different. Importantly, antibodies that reduce circulating IFN-γ are already being used to treat other human immune diseases, so the clinical use of these antibodies in patients with IFN-γ deficiency before HSCT is feasible. Finally, the researchers speculate that the use of IFN-γ–depleting antibodies might be beneficial in other situations where HSCT often fails such as when a close relative is not available as a donor. However, this possibility will need to be thoroughly tested in mice before human clinical trials can be started.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050026.
General information about primary immunodeficiencies is available from the US National Institute of Child Health and Human Development
Online Mendelian Inheritance in Man (OMIM) provides information about familial predisposition to mycobacterial disease
Wikipedia has pages on hematopoietic stem cell transplantation and on interferon-γ (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Human Genetics of Infectious Diseases Lab focuses on the genetic basis of predicposition or resistance to infectious diseases in humans
doi:10.1371/journal.pmed.0050026
PMCID: PMC2214797  PMID: 18232731
17.  Surface Expression of MPT64 as a Fusion with the PE Domain of PE_PGRS33 Enhances Mycobacterium bovis BCG Protective Activity against Mycobacterium tuberculosis in Mice▿  
Infection and Immunity  2010;78(12):5202-5213.
To improve the current vaccine against tuberculosis, a recombinant strain of Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing a Mycobacterium tuberculosis vaccine candidate antigen (MPT64) in strong association with the mycobacterial cell wall was developed. To deliver the candidate antigen on the surface, we fused the mpt64 gene to the sequence encoding the PE domain of the PE_PGRS33 protein of M. tuberculosis (to create strain HPE-ΔMPT64-BCG), which we have previously shown to transport proteins to the bacterial surface. In a series of protection experiments in the mouse model of tuberculosis, we showed that (i) immunization of mice with HPE-ΔMPT64-BCG provides levels of protection significantly higher than those afforded by the parental BCG strain, as assessed by bacterial colonization in lungs and spleens and by lung involvement (at both 28 and 70 days postchallenge), (ii) rBCG strains expressing MPT64 provide better protection than the parental BCG strain only when this antigen is surface expressed, and (iii) the HPE-ΔMPT64-BCG-induced MPT64-specific T cell repertoire when characterized by β chain variable region-β chain joining region (BV-BJ) spectratyping indicates that protection is correlated with the ability to recruit gamma interferon (IFN-γ)-secreting T cells carrying the BV8.3-BJ1.5 (172 bp) shared rearrangement. These results demonstrate that HPE-ΔMPT64-BCG is one of the most effective new vaccines tested so far in the mouse model of tuberculosis and underscore the impact of antigen cellular localization on the induction of the specific immune response induced by rBCG.
doi:10.1128/IAI.00267-10
PMCID: PMC2981302  PMID: 20921146
18.  The Glycosylated Rv1860 Protein of Mycobacterium tuberculosis Inhibits Dendritic Cell Mediated TH1 and TH17 Polarization of T Cells and Abrogates Protective Immunity Conferred by BCG 
PLoS Pathogens  2014;10(6):e1004176.
We previously reported interferon gamma secretion by human CD4+ and CD8+ T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-α, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-β unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-γ and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-γ and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-α compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.
Author Summary
Tuberculosis (TB), although recognized as an infectious disease for centuries, is still the leading cause of human deaths, claiming a million lives annually. Successful control of TB, either through drugs or effective preventive vaccines has not been achieved despite decades of research. We have studied the role for mannosylated protein Rv1860 of MTB in interfering with the early response of dendritic cells, which belong to the host's innate immune arsenal, to this mycobacterium. We were able to show that incorporating the gene coding for Rv1860 of MTB into the safe vaccine strain BCG resulted in loss of BCG's protective ability in the guinea pig animal model. Using primary mouse bone marrow derived dendritic cells in vitro as well as spleen dendritic cells from infected mice, we show in this study that exposure to mannosylated Rv1860 leads to loss of dendritic cell functions such as cytokine secretion and T cell activation. This leads to defective downstream T cell responses to the mycobacteria. We suggest that altering or extinguishing the expression of such glycoproteins by mycobacteria may be a strategy for developing better vaccines against TB.
doi:10.1371/journal.ppat.1004176
PMCID: PMC4055742  PMID: 24945624
19.  Influence of bovine lactoferrin on expression of presentation molecules on BCG-infected bone marrow derived macrophages 
Biochimie  2008;91(1):76-85.
The current vaccine for tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is an attenuated strain of Mycobacterium bovis bacillus Calmette-Guerin (BCG). BCG has proven to be effective in children, however, efficacy wanes in adulthood. Lactoferrin, a natural protein with immunomodulatory properties, is a potential adjuvant candidate to enhance efficacy of BCG. These studies define bovine lactoferrin as an enhancer of the BCG vaccine, functioning in part by modulating macrophage ability to present antigen and stimulate T-cells. BCG-infected bone marrow derived macrophages (BMMs) cultured with bovine lactoferrin increased the number of MHC II+ expressing cells. Addition of IFN-γ and lactoferrin to BCG-infected BMMs enhanced MHC II expressiona dna increased the ratio of CD86/CD80. Lactoferrin treated BCG-infected BMMs were able to stimulate an increase in IFN-γ production from presensitized CD3+ splenocytes. Together, these results demonstrate that bovine lactoferrin is capable of modulating BCG-infected macrophages to enhance T-cell stimulation through increased surface expression of antigen presentation and co-stimulatory molecules, which potentially explains the observed in vivo bovine lactoferrin enhancement of BCG vaccine efficacy to protect against virulent MTB infection.
doi:10.1016/j.biochi.2008.04.008
PMCID: PMC2626195  PMID: 18486627
Lactoferrin; BCG; Vaccine; Adjuvant; Tuberculosis
20.  Oncogenic activation of Pak1-dependent pathway of macropinocytosis determines BCG entry into bladder cancer cells 
Cancer research  2013;73(3):1156-1167.
Bacille Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that is used widely as a vaccine for tuberculosis and is used as an effective treatment for superficial bladder carcinoma. Despite being the most successful cancer biotherapy, its mechanism of action and response determinants remain obscure. Here we establish a model system to analyze BCG interaction with bladder cancer cells, using it to show that these cells vary dramatically in their susceptibility to BCG infection. Unexpectedly, the uptake of BCG by bladder cancer cells occurs by macropinocytosis rather than phagocytosis. BCG entry into bladder cancer cells relied upon Rac1, Cdc42 and their effector kinase Pak1. The difference in susceptibility between BCG-permissive and BCG-resistant bladder cancer cells was due to oncogenic activation of signaling pathways that activate macropinocytosis, with PI3K activation stimulating BCG uptake independently of Akt. Similarly, activated Ras strongly activated Pak1-dependent uptake of BCG. These results reveal that oncogenic activation of macropinocytosis determines BCG uptake by bladder cancer cells, implying that tumor responsiveness to BCG may be governed by the specific mutations present in the treated cancer cell.
doi:10.1158/0008-5472.CAN-12-1882
PMCID: PMC3756537  PMID: 23378476
21.  Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine 
The current vaccine against tuberculosis, Mycobacterium bovis strain bacille Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for the expression and delivery of protective recombinant antigens (Stover, C.K., V.F. de la Cruz, T.R. Fuerst, J.E. Burlein, L.A. Benson, L.T. Bennett, G.P. Bansal, J.F. Young, M.H. Lee, G.F. Hatfull et al. 1991. Nature [Lond]. 351:456; Jacobs, W.R., Jr., S.B. Snapper, L. Lugosi and B.R. Bloom. 1990. Curr. Top. Microbiol. Immunol. 155:153; Jacobs, W.R., M. Tuckman, and B.R. Bloom. 1987. Nature [Lond.]. 327:532); but as an attenuated intracellular bacterium residing in macrophages, BCG would seem to be best suited for eliciting cellular responses and not humoral responses. Since bacterial lipoproteins are often among the most immunogenic of bacterial antigens, we tested whether BCG expression of a target antigen as a membrane-associated lipoprotein could enhance the potential for a recombinant BCG vaccine to elicit high-titered protective antibody responses to target antigens. Immunization of mice with recombinant BCG vaccines expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi as a membrane-associated lipoprotein resulted in protective antibody responses that were 100- 1,000-fold higher than responses elicited by immunization with recombinant BCG expressing OspA cytoplasmically or as a secreted fusion protein. Furthermore, these improved antibody responses were observed in heterogeneous mouse strains that vary in their immune responsiveness to OspA and sensitivity to BCG growth. Thus, expression of protective antigens as chimeric membrane-associated lipoproteins on recombinant BCG may result in the generation of new candidate vaccines against Lyme borreliosis and other human or veterinary diseases where humoral immunity is the protective response.
PMCID: PMC2191093  PMID: 8315378
22.  Novel Recombinant Mycobacterium bovis BCG, Ovine Atadenovirus, and Modified Vaccinia Virus Ankara Vaccines Combine To Induce Robust Human Immunodeficiency Virus-Specific CD4 and CD8 T-Cell Responses in Rhesus Macaques▿  
Journal of Virology  2010;84(12):5898-5908.
Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA401 as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA401 was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA401 and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration.
doi:10.1128/JVI.02607-09
PMCID: PMC2876636  PMID: 20375158
23.  Elevated ex vivo monocyte chemotactic protein-1 (CCL2) in pulmonary as compared with extra-pulmonary tuberculosis 
BMC Immunology  2005;6:14.
Background
Tuberculosis causes 3 million deaths annually. The most common site of tuberculosis is pulmonary however; extra-pulmonary forms of the disease also remain prevalent. Restriction of Mycobacterium tuberculosis depends on effective recruitment and subsequent activation of T lymphocytes, mononuclear and polymorphonuclear cells to the site of infection. Tumor necrosis factor (TNF)-α is essential for granuloma formation and is a potent activator of monocyte chemotactic protein (MCP-1, CCL2). CCL2 is essential for recruitment of monocytes and T cells and has been shown to play a role in protection against tuberculosis. Interleukin -8 (CXCL8) is a potent activator of neutrophils. Increased levels of CCL2, CXCL8 and TNFα are reported in tuberculosis but their significance in different forms of tuberculosis is as yet unclear. We have used an ex vivo assay to investigate differences in immune parameters in patients with either pulmonary or extra-pulmonary tuberculosis.
Methods
Serum levels of CCL2, CXCL8 and TNFα were measured in patients with pulmonary tuberculosis (N = 12), extra-pulmonary tuberculosis (N = 8) and BCG-vaccinated healthy volunteers (N = 12). Whole blood cells were stimulated with non-pathogenic Mycobacterium bovis bacille-Calmette Guerin (BCG) vaccine strain or bacterial lipopolysaccharide (LPS) and cyto/chemokines were monitored in supernatants.
Results
Circulating serum levels of CXCL8 and TNFα were raised in all tuberculosis patients, while CCL2 levels were not. There was no difference in spontaneous cytokine secretion from whole blood cells between patients and controls. M. bovis BCG-induced ex vivo CCL2 secretion was significantly greater in pulmonary as compared with both extra-pulmonary tuberculosis patients and endemic controls. In response to LPS stimulation, patients with pulmonary tuberculosis showed increased CCL2 and TNFα responses as compared with the extra-pulmonary group. BCG-, and LPS-induced CXCL8 secretion was comparable between patients and controls.
Conclusion
CCL2 is activated by TNFα and is essential for recruitment of monocytes and T cells to the site of mycobacterial infection. Increased CCL2 activation in pulmonary tuberculosis may result in a stronger cellular response as compared with extra-pulmonary tuberculosis patients, and this may contribute to the localization of infection to the pulmonary site.
doi:10.1186/1471-2172-6-14
PMCID: PMC1182368  PMID: 16001981
24.  A Booster Vaccine Expressing a Latency-Associated Antigen Augments BCG Induced Immunity and Confers Enhanced Protection against Tuberculosis 
PLoS ONE  2011;6(8):e23360.
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 log10 and 1.96 log10 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.
doi:10.1371/journal.pone.0023360
PMCID: PMC3157374  PMID: 21858087
25.  Enhanced Protection against Bovine Tuberculosis after Coadministration of Mycobacterium bovis BCG with a Mycobacterial Protein Vaccine-Adjuvant Combination but Not after Coadministration of Adjuvant Alone▿  
Current efforts are aimed at optimizing the protective efficacy of Mycobacterium bovis BCG by the use of vaccine combinations. We have recently demonstrated that the protection afforded by BCG alone is enhanced by vaccinating cattle with a combination of vaccines comprising BCG and a protein tuberculosis vaccine, namely, culture filtrate proteins (CFPs) from M. bovis plus an adjuvant. In the current study, three different adjuvant systems were compared. The CFP was formulated with a depot adjuvant, dimethyldioctadecyl ammonium bromide (DDA), together with one of three different immunostimulants: monophosphoryl lipid A (MPL), a synthetic mycobacterial phosphatidylinositol mannoside-2 (PIM2), and a synthetic lipopeptide (Pam3Cys-SKKKK [Pam3CSK4]). Groups of cattle (n = 10/group) were vaccinated with BCG-CFP-DDA-PIM2, BCG-CFP-DDA-MPL, or BCG-CFP-DDA-Pam3CSK4. Two additional groups (n = 10) were vaccinated with BCG alone or BCG-adjuvant (DDA-MPL), and a control group was left unvaccinated. Protection was assessed by challenging the cattle intratracheally with M. bovis. Groups of cattle vaccinated with BCG-CFP-DDA-PIM2, BCG-CFP-DDA-MPL, BCG-CFP-DDA-Pam3CSK4, and BCG alone showed significant reductions in three, three, five, and three pathological and microbiological disease parameters, respectively, compared to the results for the nonvaccinated group. Vaccination with the combination of BCG and the DDA-MPL adjuvant alone abrogated the protection conferred by BCG alone. The profiling of cytokine gene expression following vaccination, prior to challenge, did not illuminate significant differences which could explain the latter result. Vaccination of cattle with a combination of BCG and protein tuberculosis vaccine enhances protection against tuberculosis.
doi:10.1128/CVI.00034-08
PMCID: PMC2394838  PMID: 18337375

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