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1.  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.
PMCID: PMC2632018  PMID: 19064635
2.  Strain-Specific Differences in the Genetic Control of Two Closely Related Mycobacteria 
PLoS Pathogens  2010;6(10):e1001169.
The host response to mycobacterial infection depends on host and pathogen genetic factors. Recent studies in human populations suggest a strain specific genetic control of tuberculosis. To test for mycobacterial-strain specific genetic control of susceptibility to infection under highly controlled experimental conditions, we performed a comparative genetic analysis using the A/J- and C57BL/6J-derived recombinant congenic (RC) mouse panel infected with the Russia and Pasteur strains of Mycobacterium bovis Bacille Calmette Guérin (BCG). Bacillary counts in the lung and spleen at weeks 1 and 6 post infection were used as a measure of susceptibility. By performing genome-wide linkage analyses of loci that impact on tissue-specific bacillary burden, we were able to show the importance of correcting for strain background effects in the RC panel. When linkage analysis was adjusted on strain background, we detected a single locus on chromosome 11 that impacted on pulmonary counts of BCG Russia but not Pasteur. The same locus also controlled the splenic counts of BCG Russia but not Pasteur. By contrast, a locus on chromosome 1 which was indistinguishable from Nramp1 impacted on splenic bacillary counts of both BCG Russia and Pasteur. Additionally, dependent upon BCG strain, tissue and time post infection, we detected 9 distinct loci associated with bacillary counts. Hence, the ensemble of genetic loci impacting on BCG infection revealed a highly dynamic picture of genetic control that reflected both the course of infection and the infecting strain. This high degree of adaptation of host genetics to strain-specific pathogenesis is expected to provide a suitable framework for the selection of specific host-mycobacteria combinations during co-evolution of mycobacteria with humans.
Author Summary
Susceptibility to mycobacterial infection results from a complex interaction between host and bacterial genetic factors. To examine the effect of host and pathogen genetic variability on the control of mycobacterial infection, we infected a panel of genetically related recombinant congenic (RC) mouse strains with two closely related strains of Mycobacterium bovis BCG. Bacterial counts of BCG Russia and BCG Pasteur were determined in the lung and spleen at 1 and 6 weeks following infection and used for genetic analysis. A novel analytical approach was developed to perform genome-wide linkage analyses using the RC strains. Comparative linkage analysis using this model identified a strong genetic effect on chromosome 1 controlling counts of BCG Pasteur at 1 week and of BCG Russia at 1 week and 6 weeks in the spleen. A locus impacting on late BCG Russia counts in the lung and spleen was identified on chromosome 11. Nine additional loci were shown to control bacterial counts in a tissue-, time-, and BCG strain-specific manner. Our findings suggest that the host genetic control of mycobacterial infection is highly dynamic and adapted to the stage of pathogenesis and to the infecting strain. Such a high degree of genetic plasticity in the host-pathogen interplay is expected to favour evolutionary co-adaptation in mycobacterial disease.
PMCID: PMC2965770  PMID: 21060820
3.  BCG-Mediated Protection against Mycobacterium ulcerans Infection in the Mouse 
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.
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.
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.
PMCID: PMC3057947  PMID: 21423646
4.  Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. 
Journal of Bacteriology  1996;178(5):1274-1282.
The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.
PMCID: PMC177799  PMID: 8631702
5.  Immune Response Induced by Three Mycobacterium bovis BCG Substrains with Diverse Regions of Deletion in a C57BL/6 Mouse Model▿  
This study was performed to examine the adaptive immune response generated by three Mycobacterium bovis bacillus Calmette-Guérin (BCG) substrains to determine if the number of genomic regions of deletion played a significant role in determining the magnitude of the immune response or affected their ability to reduce the bacterial burden following low-dose aerosol challenge with a virulent M. tuberculosis strain. BCG Connaught, Pasteur, and Sweden were chosen as representative substrains, as they possessed many, intermediate, and few regions of deletion, respectively, as a result of changes in the genome in various regions. Mice were vaccinated subcutaneously and were then examined at 14, 21, and 42 days postvaccination. BCG was observed in the spleen, lung, and lymph nodes. BCG Connaught induced a greater pulmonary T-cell response than the other two substrains at day 14 postvaccination, although by 42 days postvaccination activated T-cell levels dropped to the levels observed in control mice for all three substrains. Among the three substrains, BCG Connaught induced significantly greater levels of interleukin-12 in bone marrow-derived macrophage cultures. Mice challenged at days 14, 21, and 42 postvaccination displayed an equal capacity to reduce the bacterial burden in the lungs and spleen. The data provide evidence that although the BCG substrains generated qualitatively and quantitatively different immune responses, they induced similar reductions in the bacterial burden against challenge with a virulent M. tuberculosis strain in the mouse model of tuberculosis. The data raise questions about the assessment of vaccine immune responses and the relationship to a vaccine's ability to reduce the bacterial burden.
PMCID: PMC2394844  PMID: 18353924
6.  Rates of Latent Tuberculosis in Health Care Staff in Russia 
PLoS Medicine  2007;4(2):e55.
Russia is one of 22 high burden tuberculosis (TB) countries. Identifying individuals, particularly health care workers (HCWs) with latent tuberculosis infection (LTBI), and determining the rate of infection, can assist TB control through chemoprophylaxis and improving institutional cross-infection strategies. The objective of the study was to estimate the prevalence and determine the relative risks and risk factors for infection, within a vertically organised TB service in a country with universal bacille Calmette-Guérin (BCG) vaccination.
Methods and Findings
We conducted a cross-sectional study to assess the prevalence of and risk factors for LTBI among unexposed students, minimally exposed medical students, primary care health providers, and TB hospital health providers in Samara, Russian Federation. We used a novel in vitro assay (for gamma-interferon [IFN-γ]) release to establish LTBI and a questionnaire to address risk factors. LTBI was seen in 40.8% (107/262) of staff and was significantly higher in doctors and nurses (39.1% [90/230]) than in students (8.7% [32/368]) (relative risk [RR] 4.5; 95% confidence interval [CI] 3.1–6.5) and in TB service versus primary health doctors and nurses: respectively 46.9% (45/96) versus 29.3% (34/116) (RR 1.6; 95% CI 1.1–2.3). There was a gradient of LTBI, proportional to exposure, in medical students, primary health care providers, and TB doctors: respectively, 10.1% (24/238), 25.5% (14/55), and 55% (22/40). LTBI was also high in TB laboratory workers: 11/18 (61.1%).
IFN-γ assays have a useful role in screening HCWs with a high risk of LTBI and who are BCG vaccinated. TB HCWs were at significantly higher risk of having LTBI. Larger cohort studies are needed to evaluate the individual risks of active TB development in positive individuals and the effectiveness of preventive therapy based on IFN-γ test results.
Gamma-interferon assays were used in a cross-sectional study of Russian health care workers and found high rates of latent tuberculosis infection.
Editors' Summary
Tuberculosis (TB) is a very common and life-threatening infection caused by a bacterium, Mycobacterium tuberculosis, which is carried by about a third of the world's population. Many people who are infected do not develop the symptoms of disease; this is called “latent infection.” However, it is important to detect latent infection among people in high-risk communities, in order to prevent infected people from developing active disease, and therefore also reduce the spread of TB within the community. 22 countries account for 80% of the world's active TB, and Russia is one of these. Health care workers are particularly at risk for developing active TB disease in Russia, but the extent of latent infection is not known. In order to design appropriate measures for controlling TB in Russia, it is important to know how common latent infection is among health care workers, as well as other members of the community.
Why Was This Study Done?
The researchers here had been studying the spread of tuberculosis in Samara City in southeastern Russia, where the rate of TB disease among health care workers was very high; in 2004 the number of TB cases among health care workers on TB wards was over ten times that in the general population. There was also no information available on the rates of latent TB infection among health care workers in Samara City. The researchers therefore wanted to work out what proportion of health care workers in Samara City had latent TB infection, and particularly to compare groups whom they thought would be at different levels of risk (students, clinicians outside of TB wards, clinicians on TB wards, etc.). Finally, the researchers also wanted to use a new test for detecting latent TB infection. The traditional test for detecting TB infection (tuberculin skin test) is not very reliable among people who have received the Bacillus Calmette-Guérin (BCG) vaccination against TB earlier in life, as is the case in Russia. In this study a new test was therefore used, based on measuring the immune response to two proteins produced by M. tuberculosis, which are not present in the BCG vaccine strain.
What Did the Researchers Do and Find?
In this study the researchers tested health care workers from all the TB clinics in Samara City, as well as other clinical staff and students, for latent tuberculosis. In total, 630 people had blood samples taken for testing. A questionnaire was also used to collect information on possible risk factors for TB. As expected, the rate of latent TB infection was highest among clinical staff working in the TB clinics, 47% of whom were infected with M. tuberculosis. This compared to a 10% infection rate among medical students and 29% infection rate among primary care health workers. The differences in infection rate between medical students, primary care health workers, and TB clinic staff were statistically significant and reflected progressively increasing exposure to TB. Among primary care health workers, past exposure to TB was a risk factor for having latent TB infection.
What Do These Findings Mean?
This study showed that there was a high rate of latent TB infection among health care workers in Samara City and that infection is increasingly likely among people with either past or present exposure to TB. The results suggest that further research should be carried out to test whether mass screening for latent infection, followed by treatment, will reduce the rate of active TB disease among health care workers and also prevent further spread of TB. There are concerns that widespread treatment of latent infection may not be completely effective due to the relatively high prevalence of drug-resistant TB strains and any new initiatives would therefore need to be carefully evaluated.
Additional Information.
Please access these Web sites via the online version of this summary at
The Stop TB Partnership has been set up to eliminate TB as a public health problem; its site provides data and resources about TB in each of the 22 most-affected countries, including Russia
Tuberculosis minisite from the World Health Organization, providing data on tuberculosis worldwide, details of the Stop TB strategy, as well as fact sheets and current guidelines
The US Centers for Disease Control has a tuberculosis minisite, including a fact sheet on latent TB
Information from the US Centers for Disease Control about the QuantiFERON-TB Gold test, used to test for latent TB infection in this study
PMCID: PMC1796908  PMID: 17298167
7.  Viral Booster Vaccines Improve Mycobacterium bovis BCG-Induced Protection against Bovine Tuberculosis ▿  
Infection and Immunity  2009;77(8):3364-3373.
Previous work with small-animal laboratory models of tuberculosis has shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guérin (BCG) to prime and modified vaccinia virus Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad85A) expressing the mycobacterial antigen Ag85A to boost may increase the protective efficacy of BCG. Here we report the first efficacy data on using these vaccines in cattle, a natural target species of tuberculous infection. Protection was determined by measuring development of disease as an end point after M. bovis challenge. Either Ad85A or MVA85A boosting resulted in protection superior to that given by BCG alone: boosting BCG with MVA85A or Ad85A induced significant reduction in pathology in four/eight parameters assessed, while BCG vaccination alone did so in only one parameter studied. Protection was particularly evident in the lungs of vaccinated animals (median lung scores for naïve and BCG-, BCG/MVA85A-, and BCG/Ad85A-vaccinated animals were 10.5, 5, 2.5, and 0, respectively). The bacterial loads in lymph node tissues were also reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection. Therefore, this study provides evidence of improved protection against tuberculosis by viral booster vaccination in a natural target species and has prioritized potential correlates of vaccine efficacy for further evaluation. These findings also have implications for human tuberculosis vaccine development.
PMCID: PMC2715681  PMID: 19487476
8.  Tuberculosis vaccines: beyond bacille Calmette–Guérin 
Tuberculosis (TB) disease caused by Mycobacterium tuberculosis (M. tb) remains one of the leading infectious causes of death and disease throughout the world. The only licensed vaccine, Mycobacterium bovis bacille Calmette–Guérin (BCG) confers highly variable protection against pulmonary disease. An effective vaccination regimen would be the most efficient way to control the epidemic. However, BCG does confer consistent and reliable protection against disseminated disease in childhood, and most TB vaccine strategies being developed incorporate BCG to retain this protection. Cellular immunity is necessary for protection against TB and all the new vaccines in development are focused on inducing a strong and durable cellular immune response. There are two main strategies being pursued in TB vaccine development. The first is to replace BCG with an improved whole organism mycobacterial priming vaccine, which is either a recombinant BCG or an attenuated strain of M. tb. The second is to develop a subunit boosting vaccine, which is designed to be administered after BCG vaccination, and to enhance the protective efficacy of BCG. This article reviews the leading candidate vaccines in development and considers the current challenges in the field with regard to efficacy testing.
PMCID: PMC3146779  PMID: 21893541
tuberculosis; vaccine; bacille Calmette–Guérin; clinical trials; efficacy; immune correlates
9.  Transcriptional and translational analyses of recA mutant alleles in Pseudomonas aeruginosa. 
Journal of Bacteriology  1988;170(4):1637-1650.
Recombinant plasmids containing the recA gene from Pseudomonas aeruginosa were used in complementation, transcriptional, and translational studies to examine the nature of rec-102 and rec-2, mutations which confer a recA-like mutant phenotype on P. aeruginosa PAO strains. For comparison, recA7::Tn501 mutants of strain PAO were constructed by gene replacement. The rec-2 and rec-102 alleles were shown to be recA alleles; plasmids containing the recA gene complemented the three rec mutant strains for defects associated with recA mutation. Northern blot analyses indicated that the recA gene in P. aeruginosa was transcribed as two distinct mRNAs of approximately 1.2 and 1.4 kilobases (kb). A plasmid encoding both transcripts of recA complemented all defects associated with the three recA mutations rec-2, rec-102, and recA7. However, a 2.4-kb subclone (pJH13) encoding only the smaller transcript of the recA gene was expressed differently in the three recA allele backgrounds and served as a tool to distinguish the nature of the rec-2 and rec-102 mutations in recA. A minicell analysis showed that a plasmid expressing both of the recA gene transcripts or one that expressed only the smaller transcript both produced the same 42-kilodalton recA protein. A chloramphenicol acetyltransferase gene fusion in the 3' end of the recA transcript showed that the recA gene of P. aeruginosa was induced following treatment with a DNA-damaging agent (methyl methanesulfonate). The recA7 mutant constructed here showed no recA-related transcript or protein under inducing conditions, and pJH13 in this host produced only low levels of the smaller recA transcript and low levels of recA protein. The rec-2 mutant produced a detectable transcript but no recA protein following induction. The presence of low levels of activated recA protein encoded by pJH13 in the rec-2 mutant resulted in wild-type transcriptional levels of chromosomally encoded recA, but no recA protein was detectable. Thus, the rec-2 allele of recA was normal with respect to induction of mRNA, but these transcripts were defective in either translation or synthesis of a stable protein. The rec-102 mutant also produced a detectable transcript and no recA protein following induction, but having pJH13 in the cell to produce low levels of activated recA protein resulted in overproduction of chromosomally encoded recA transcripts and active recA protein. Thus, the recA defect in the rec-102 mutant is apparently in the interaction between recA and a lexA-like repressor.
PMCID: PMC211012  PMID: 2450868
10.  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.
PMCID: PMC1187936  PMID: 16110326
11.  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
12.  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.
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.
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.
PMCID: PMC2586085  PMID: 19052643
13.  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.
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.
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.
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.
PMCID: PMC3094199  PMID: 21507239
14.  MVA.85A Boosting of BCG and an Attenuated, phoP Deficient M. tuberculosis Vaccine Both Show Protective Efficacy Against Tuberculosis in Rhesus Macaques 
PLoS ONE  2009;4(4):e5264.
Continuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection.
Methods and Findings
Here, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNγ responses. Antigen 85A-specific IFNγ secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNγ levels post-infection as the strongest immunocorrelate for protection (spearman's rho: −0.60).
Both the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates.
PMCID: PMC2666807  PMID: 19367339
15.  Identification of Surrogates and Correlates of Protection in Protective Immunity against Mycobacterium bovis Infection Induced in Neonatal Calves by Vaccination with M. bovis BCG Pasteur and M. bovis BCG Danish ▿  
Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against infection with virulent M. bovis, the causative agent of bovine tuberculosis (bTB). We compared two strains of BCG, Pasteur and Danish, in order to confirm that the current European human vaccine strain (BCG Danish) induced protective immunity in calves, and we assessed immune responses to determine correlates of protection that could assist future vaccine evaluation in cattle. Both vaccine strains induced antigen (purified protein derivate [PPD])-specific gamma interferon (IFN-γ) in whole-blood cultures. These responses were not significantly different for BCG Pasteur and BCG Danish and peaked at week 2 to 4 postvaccination. Vaccination with either BCG Danish or BCG Pasteur induced significant protection against bTB, with reductions in both lesion score and bacteriological burden evident in both groups of vaccinated calves compared with nonvaccinated control calves. Measurement of IFN-γ-expressing T lymphocytes postvaccination and postchallenge revealed both correlates and surrogates of protective efficacy. The frequency of central memory T lymphocytes present at 12 weeks postvaccination (at the time of M. bovis challenge) correlated significantly with protection. Conversely, the number of IFN-γ-expressing effector T cells present after M. bovis challenge was correlated with disease. These results demonstrate that vaccination of neonatal calves with either BCG Pasteur or BCG Danish induces protective immune responses against TB. In addition, we show that measurement of antigen-specific T lymphocyte populations may provide a reliable means for identifying protective vaccine candidates.
PMCID: PMC3067386  PMID: 21228141
16.  Development and characterization of recA mutants of Campylobacter jejuni for inclusion in attenuated vaccines. 
Infection and Immunity  1994;62(2):426-432.
Isogenic recA mutants of Campylobacter jejuni have been constructed for evaluation of their usefulness in attenuated vaccines against this major worldwide cause of diarrhea. The recA+ gene of C. jejuni 81-176 was cloned by using degenerate primers to conserved regions of other RecA proteins in a PCR. The C. jejuni recA+ gene encodes a predicted protein with an M(r) of 37,012 with high sequence similarity to other RecA proteins. The termination codon of the recA+ gene overlaps with the initiation codon of another open reading frame which encodes a predicted protein which has > 50% identity with the N terminus of the Escherichia coli enolase protein. A kanamycin resistance gene was inserted into the cloned recA+ gene in E. coli and returned to C. jejuni VC83 by natural transformation, resulting in allelic replacement of the wild-type recA gene. The resulting VC83 recA mutant displayed increased sensitivity to UV light and a defect in generalized recombination as determined by natural transformation frequencies. The mutated recA gene was amplified from VC83 recA by PCR, and the product was used to transfer the mutation by natural transformation into C. jejuni 81-176 and 81-116, resulting in isogenic recA mutants with phenotypes similar to VC83 recA. After oral feeding, strain 81-176 recA colonized rabbits at levels comparable to wild-type 81-176 and was capable of eliciting the same degree of protection as wild-type 81-176 against subsequent homologous challenge in the RITARD (removable intestinal tie adult rabbit diarrhea) model.
PMCID: PMC186125  PMID: 8300203
17.  Properties of proteins MPB64, MPB70, and MPB80 of Mycobacterium bovis BCG. 
Infection and Immunity  1986;52(1):293-302.
The immunogenic proteins MPB64 and MPB80 of Mycobacterium bovis BCG were purified to homogeneity and compared with MPB70. MPB70 and MPB80 showed a similar distribution in substrains of BCG, both being present in high concentrations in culture fluids of BCG substrain Tokyo, BCG Moreau, BCG Russia, and BCG Sweden and in only very small amounts in BCG Glaxo, BCG Tice, BCG Copenhagen, and BCG Pasteur. In various physicochemical properties MPB70 and MPB80 were closely similar, but MPB80 had a distinctly lower pI value. The N-terminal amino acid sequence was identical for the first 30 residues. In reactions with anti-MPB70 antibodies and delayed-type hypersensitivity skin reactions, MPB70 and MPB80 also had very similar properties. These results show that MPB70 and MPB80 are two closely similar forms of the same gene product, and postsynthetic changes probably explain the observed differences. By contrast, MPB64 had a higher molecular weight. The N-terminal amino acid sequence showed no homology with MPB70, and these two proteins showed no immunologic similarity. MPB64 and MPB70 showed only very restricted cross-reactivity with other species of mycobacteria but cross-reacted with Nocardia asteroides. The similar occurrence in eight different substrains of BCG indicated that the two proteins are influenced by similar control mechanisms, but in contrast to MPB70, MPB64 occurred in sufficient concentration in two strains of Mycobacterium tuberculosis to give a distinct spot in two-dimensional polyacrylamide gel electrophoresis of their culture fluids.
PMCID: PMC262233  PMID: 3514457
18.  Use of Synthetic Peptides Derived from the Antigens ESAT-6 and CFP-10 for Differential Diagnosis of Bovine Tuberculosis in Cattle 
In Great Britain an independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis infection holds the best long-term prospect for tuberculosis control in British herds. A precondition for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that testing and slaughter-based control strategies can continue alongside vaccination. To date bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only available vaccine for the prevention of tuberculosis. However, tests based on tuberculin purified protein derivative cannot distinguish between M. bovis infection and BCG vaccination. Therefore, specific antigens expressed by M. bovis but absent from BCG constitute prime candidates for differential diagnostic reagents. Recently, two such antigens, ESAT-6 and CFP-10, have been reported to be promising candidates as diagnostic reagents for the detection of M. bovis infection in cattle. Here we report the identification of promiscuous peptides of CFP-10 that were recognized by M. bovis-infected cattle. Five of these peptides were formulated into a peptide cocktail together with five peptides derived from ESAT-6. Using this peptide cocktail in T-cell assays, M. bovis-infected animals were detected, while BCG-vaccinated or Mycobacterium avium-sensitized animals did not respond. The sensitivity of the peptide cocktail as an antigen in a whole-blood gamma interferon assay was determined using naturally infected field reactor cattle, and the specificity was determined using blood from BCG-vaccinated and noninfected, nonvaccinated animals. The sensitivity of the assay in cattle with confirmed tuberculosis was found to be 77.9%, with a specificity of 100% in BCG-vaccinated or nonvaccinated animals. This compares favorably with the specificity of tuberculin when tested in noninfected or vaccinated animals. In summary, our results demonstrate that this peptide cocktail can discriminate between M. bovis infection and BCG vaccination with a high degree of sensitivity and specificity.
PMCID: PMC96103  PMID: 11329460
19.  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.
PMCID: PMC3984997  PMID: 24778634
rBCG; tuberculosis; vaccine; protection; long-term memory; strain differences
20.  CpG Oligodeoxynucleotides and Interleukin-12 Improve the Efficacy of Mycobacterium bovis BCG Vaccination in Mice Challenged with M. tuberculosis 
Infection and Immunity  2000;68(5):2948-2953.
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved for prevention of tuberculosis. It has been postulated that serial passage of BCG over the years may have resulted in attenuation of its effectiveness. Because interleukin-12 (IL-12) and oligodeoxynucleotides (ODN) containing cytidine phosphate guanosine (CpG) motifs have been shown to enhance Th1 responses in vivo, they were chosen as adjuvants to increase the effectiveness of BCG vaccination. In this report, mice were vaccinated with BCG with or without IL-12 or CpG ODN and then challenged 6 weeks later via the aerosol route with the Erdman strain of M. tuberculosis. Mice vaccinated with BCG alone showed a 1- to 2-log reduction in bacterial load compared with control mice that did not receive any vaccination prior to M. tuberculosis challenge. Moreover, the bacterial loads of mice vaccinated with BCG plus IL-12 or CpG ODN were a further two- to fivefold lower than those of mice vaccinated with BCG alone. As an immune correlate, the antigen-specific production IFN-γ and mRNA expression in spleen cells prior to challenge were evaluated. Mice vaccinated with BCG plus IL-12 or CpG ODN showed enhanced production of IFN-γ compared with mice vaccinated with BCG alone. Finally, granulomas in BCG-vaccinated mice were smaller and more lymphocyte rich than those in unvaccinated mice; however, the addition of IL-12 or CpG ODN to BCG vaccination did not alter granuloma formation or result in added pulmonary damage. These observations support a role for immune adjuvants given with BCG vaccination to enhance its biologic efficacy.
PMCID: PMC97508  PMID: 10768993
21.  Mycobacterium bovis BCG recA Deletion Mutant Shows Increased Susceptibility to DNA-Damaging Agents but Wild-Type Survival in a Mouse Infection Model 
Infection and Immunity  2001;69(6):3562-3568.
Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.
PMCID: PMC98336  PMID: 11349014
22.  Genomic and proteomic analyses of Mycobacterium bovis BCG Mexico 1931 reveal a diverse immunogenic repertoire against tuberculosis infection 
BMC Genomics  2011;12:493.
Studies of Mycobacterium bovis BCG strains used in different countries and vaccination programs show clear variations in the genomes and immune protective properties of BCG strains. The aim of this study was to characterise the genomic and immune proteomic profile of the BCG 1931 strain used in Mexico.
BCG Mexico 1931 has a circular chromosome of 4,350,386 bp with a G+C content and numbers of genes and pseudogenes similar to those of BCG Tokyo and BCG Pasteur. BCG Mexico 1931 lacks Region of Difference 1 (RD1), RD2 and N-RD18 and one copy of IS6110, indicating that BCG Mexico 1931 belongs to DU2 group IV within the BCG vaccine genealogy. In addition, this strain contains three new RDs, which are 53 (RDMex01), 655 (RDMex02) and 2,847 bp (REDMex03) long, and 55 single-nucleotide polymorphisms representing non-synonymous mutations compared to BCG Pasteur and BCG Tokyo. In a comparative proteomic analysis, the BCG Mexico 1931, Danish, Phipps and Tokyo strains showed 812, 794, 791 and 701 protein spots, respectively. The same analysis showed that BCG Mexico 1931 shares 62% of its protein spots with the BCG Danish strain, 61% with the BCG Phipps strain and only 48% with the BCG Tokyo strain. Thirty-nine reactive spots were detected in BCG Mexico 1931 using sera from subjects with active tuberculosis infections and positive tuberculin skin tests.
BCG Mexico 1931 has a smaller genome than the BCG Pasteur and BCG Tokyo strains. Two specific deletions in BCG Mexico 1931 are described (RDMex02 and RDMex03). The loss of RDMex02 (fadD23) is associated with enhanced macrophage binding and RDMex03 contains genes that may be involved in regulatory pathways. We also describe new antigenic proteins for the first time.
PMCID: PMC3199284  PMID: 21981907
23.  Attrition of T-Cell Functions and Simultaneous Upregulation of Inhibitory Markers Correspond with the Waning of BCG-Induced Protection against Tuberculosis in Mice 
PLoS ONE  2014;9(11):e113951.
Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live attenuated vaccine. However, the correlates of protection and waning of its immunity against tuberculosis is poorly understood. In this study, we correlated the longitudinal changes in the magnitude and functional quality of CD4+ and CD8+ T-cell response over a period of two years after mucosal or parenteral BCG vaccination with the strength of protection against Mycobacterium tuberculosis in mice. The BCG vaccination-induced CD4+ and CD8+ T cells exhibited comparable response kinetics but distinct functional attributes in-terms of IFN-γ, IL-2 and TNF-α co-production and CD62L memory marker expression. Despite a near life-long BCG persistence and the induction of enduring CD4+ T-cell responses characterized by IFN-γ and/or TNF-α production with comparable protection, the protective efficacy waned regardless of the route of vaccination. The progressive decline in the multifactorial functional abilities of CD4+ and CD8+ T cells in-terms of type-1 cytokine production, proliferation and cytolytic potential corresponded with the waning of protection against M. tuberculosis infection. In addition, simultaneous increase in the dysfunctional and terminally-differentiated T cells expressing CTLA-4, KLRG-1 and IL-10 during the contraction phase of BCG-induced response coincided with the loss of protection. Our results question the empirical development of BCG-booster vaccines and emphasize the pursuit of strategies that maintain superior T-cell functional capacity. Furthermore, our results underscore the importance of understanding the comprehensive functional dynamics of antigen-specific T-cell responses in addition to cytokine polyfunctionality in BCG-vaccinated hosts while optimizing novel vaccination strategies against tuberculosis.
PMCID: PMC4242676  PMID: 25419982
24.  Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product. 
Journal of Bacteriology  1991;173(18):5653-5662.
A fragment of Mycobacterium tuberculosis DNA containing recA-like sequences was identified by hybridization with the Escherichia coli recA gene and cloned. Although no expression was detected from its own promoter in E. coli, expression from a vector promoter partially complemented E. coli recA mutants for recombination, DNA repair, and mutagenesis, but not for induction of phage lambda. This clone produced a protein which cross-reacts with antisera raised against the E. coli RecA protein and was approximately the same size. However, the nucleotide sequence of the cloned fragment revealed the presence of an open reading frame for a protein about twice the size of other RecA proteins and the cloned product detected by Western blotting (immunoblotting). The predicted M. tuberculosis RecA protein sequence was homologous with RecA sequences from other bacteria, but this homology was not dispersed; rather it was localized to the first 254 and the last 96 amino acids, with the intervening 440 amino acids being unrelated. Furthermore, the junctions of homology were in register with the uninterrupted sequence of the E. coli RecA protein. Identical restriction fragments were found in the genomic DNAs of M. tuberculosis H37Rv and H37Ra and of M. bovis BCG. It is concluded that the ancestral recA gene of these species diversified via an insertional mutation of at least 1,320 bp of DNA. Possible processing mechanisms for synthesizing a normal-size RecA protein from this elongated sequence are discussed.
PMCID: PMC208294  PMID: 1909321
25.  A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis 
PLoS ONE  2014;9(11):e112848.
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that is a major public health problem. The vaccine used for TB prevention is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.
PMCID: PMC4232451  PMID: 25398087

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