Bovis Bacillus Calmette-Guérin Vaccination Research Articles

Evaluation of Immunogenicity and Protective Efficacy Elicited by Mycobacterium bovis BCG Overexpressing Ag85A Protein against Mycobacterium tuberculosis Aerosol Infection.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is currently the only vaccine available for preventing tuberculosis (TB), however, BCG has varying success in preventing pulmonary TB. In this study, a recombinant BCG (rBCG::Ag85A) strain overexpressing the immunodominant Ag85A antigen was constructed, and its immunogenicity and protective efficacy were evaluated. Our results indicated that the Ag85A protein was successfully overexpressed in rBCG::Ag85A, and the Ag85A peptide–MHC complexes on draining lymph node dendritic cells of C57BL/6 mice infected with rBCG::Ag85A were detectable 4 h post-infection. The C57BL/6 mice infected with this strain had stronger antigen-specific interferon-gamma (IFN-γ) responses and higher antibody titers than those immunized with BCG, and the protective experiments showed that rBCG::Ag85A can enhance protection against Mycobacterium tuberculosis (M. tuberculosis) H37Rv infection compared to the BCG vaccine alone. Our results demonstrate the potential of rBCG::Ag85A as a candidate vaccine against TB.

Manipulation of BCG vaccine: a double-edged sword

Mycobacterium bovis Bacillus Calmette-Guérin (BCG), an attenuated vaccine derived from M. bovis, is the only licensed vaccine against tuberculosis (TB). Despite its protection against TB in children, the protective efficacy in pulmonary TB is variable in adolescents and adults. In spite of the current knowledge of molecular biology, immunology and cell biology, infectious diseases such as TB and HIV/AIDS are still challenges for the scientific community. Genetic manipulation facilitates the construction of recombinant BCG (rBCG) vaccine that can be used as a highly immunogenic vaccine against TB with an improved safety profile, but, still, the manipulation of BCG vaccine to improve efficacy should be carefully considered, as it can bring in both favourable and unfavourable effects. The purpose of this review is not to comprehensively review the interaction between microorganisms and host cells in order to use rBCG expressing M. tuberculosis (Mtb) immunodominant antigens that are available in the public domain, but, rather, to also discuss the limitations of rBCG vaccine, expressing heterologous antigens, during manipulation that pave the way for a promising new vaccine approach.

Adverse effects of BCG vaccine 1173 P2 in Iran: A meta-analysis.

Although in the last two decades the World Health Organization (WHO) has introduced tuberculosis as “a threat to global”, the vaccination with the Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is the only way for the prevention of this fatal infectious disease. Despite of the efficacy of BCG vaccine especially against infants’ meningitis, it has still some limitations due to a variety of adverse effects. Many studies have evaluated the side effects of different strains of BCG vaccines in different countries. In Iran, some studies have been done so far to evaluate the adverse effects of 1173 P2 strain which is used for BCG vaccination. Each of these studies have used different standardization and sampling methods. This review will survey all studies that have been published about adverse effects of 1173 P2 strain of BCG vaccine in Iran using data mining methods.

Immunotherapy of Urothelial Carcinoma of the Bladder- from BCG Vaccines to Targeted Therapy

Bladder cancer has high prevalence in men and women. Bladder cancer usually originates from urothelium. More than 75% of cases are classified as nonmuscle invasive bladder cancer. Urothelial bladder carcinoma is usually managed by transurethral resection of the bladder tumor. Role of transurethral resection of the bladder tumor is also essential in bladder cancer staging. Local prophylaxis is used in nonmuscle invasive bladder cancer to reduce risk of recurrence. While local chemoprophylaxis is sufficient in low and middle risk patients, intravesical instillation of Mycobacterium bovis bacillus CalmetteGuerin (BCG) is preferred in high risk bladder cancer. Chemotherapy alone or in combination with locoregional treatment is used in advanced bladder cancer. New immunotherapy modalities have proven their efficacy in several clinical studies in advanced bladder cancer.

Repeated Aerosolized-Boosting with Gamma-Irradiated Mycobacterium bovis BCG Confers Improved Pulmonary Protection against the Hypervirulent Mycobacterium tuberculosis Strain HN878 in Mice.

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only licensed vaccine, shows limited protection efficacy against pulmonary tuberculosis (TB), particularly hypervirulent Mycobacterium tuberculosis (Mtb) strains, suggesting that a logistical and practical vaccination strategy is urgently required. Boosting the BCG-induced immunity may offer a potentially advantageous strategy for advancing TB vaccine development, instead of replacing BCG completely. Despite the improved protection of the airway immunization by using live BCG, the use of live BCG as an airway boosting agent may evoke safety concerns. Here, we analyzed the protective efficacy of γ-irradiated BCG as a BCG-prime boosting agent for airway immunization against a hypervirulent clinical strain challenge with Mycobacterium tuberculosis HN878 in a mouse TB model. After the aerosol challenge with the HN878 strain, the mice vaccinated with BCG via the parenteral route exhibited only mild and transient protection, whereas BCG vaccination followed by multiple aerosolized boosting with γ-irradiated BCG efficiently maintained long-lasting control of Mtb in terms of bacterial reduction and pathological findings. Further immunological investigation revealed that this approach resulted in a significant increase in the cellular responses in terms of a robust expansion of antigen (PPD and Ag85A)-specific CD4+ T cells concomitantly producing IFN-γ, TNF-α, and IL-2, as well as a high level of IFN-γ-producing recall response via both the local and systemic immune systems upon further boosting. Collectively, aerosolized boosting of γ-irradiated BCG is able to elicit strong Th1-biased immune responses and confer enhanced protection against a hypervirulent Mycobacterium tuberculosis HN878 infection in a boosting number-dependent manner.

A live attenuated BCG vaccine overexpressing multistage antigens Ag85B and HspX provides superior protection against Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains one of the most menacing infectious diseases, although attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine has been widely used to protect children against primary TB. There are increasing evidences that rapid growing and dormant Mycobacterium tuberculosis (M. tuberculosis) coexist in vivo after infection. However, BCG vaccine only elicits cell-mediated immune responses to secretory antigens expressed by rapid growing pathogen. BCG vaccine is thus unable to thwart the reactivation of latent tuberculosis infection (LTBI), and its protection wanes over age after neonatal immunization. In order to extend its ability for a durable protection, a novel recombinant BCG (rBCG) strain, named rBCG::XB, was constructed by overexpressing immunodominant multistage antigens of Ag85B and HspX, which are expressed by both rapid replicating and dormant M. tuberculosis. Long-term protective effect and immunogenicity of rBCG::XB were compared with the parental BCG in vaccinated C57BL/6 mice. Our results demonstrated that rBCG::XB provided the stronger and long-lasting protection against M. tuberculosis H37Rv intranasal infection than BCG. The rBCG::XB not only elicited the more durable multistage antigen-specific CD4(+)Th1-biased immune responses and specific polyfunctional CD4(+)T cells but also augmented the CD8(+) CTL effects against Ag85B in vivo. In particular, higher levels of CD4(+) TEM and CD8(+) TCM cells, dominated by IL2(+) CD4(+) and CD8(+) TCM cells, were obtained in the spleen of rBCG::XB vaccinated mice. Therefore, our findings indicate that rBCG::XB is a promising candidate to improve the efficacy of BCG.

Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ(+)) interleukin 2-positive (IL-2(+)) tumor necrosis factor α-positive (TNF-α(+)) polyfunctional CD4(+) T cells concurrent with CD4(+) IL-17A(+) and CD8(+) IFN-γ(+) T cells or, in contrast, virtually absent cytokine responses with induction of CD8(+) regulatory T cells. Significant induction of polyfunctional CD4(+) IFN-γ(+) IL-2(+) TNF-α(+) T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8(+) T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8(+) regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4(+) T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.

Role of Dendritic Cells in Host - Mycobacferium Bovis BCG Interactions

Tuberculosis (TB) remains a global problem that results in as many as 2 million deaths each year. TB infects innate immune cells patrolling the lung. A live-attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) strain is the only strain used in vaccines against tuberculosis. However, its efficacy remains controversial. Dendritic cells (DCs) are the major antigen-presenting cells in the induction of a cellular response to intracellular pathogens, such as mycobacteria. DCs possess the ability to induce both innate and adaptive immune responses. In the context of human infection, little is known about DC−BCG interaction. In this review, we focus on BCG interactions with human DCs with regard to the role of DCs receptors in BCG recognition and the significance of the pro-inflammatory cytokines produced by BCG-infected DCs, especially interferon-γ−producing cells. Insights into human DC responses to BCG are necessary for a better understanding of the lack of a protective response to the current BCG vaccine and for developing new effective anti-TB immunotherapeutics and vaccine strategies.

A new recombinant BCG vaccine induces specific Th17 and Th1 effector cells with higher protective efficacy against tuberculosis.

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.

Genetic diversity and dynamic distribution of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in Thailand.

This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies.

Impact of in vitro evolution on antigenic diversity of Mycobacterium bovis bacillus Calmette-Guerin (BCG)

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only vaccine currently used against tuberculosis, is an attenuated derivative of M. bovis that has been propagated in vitro for more than 40 years. We have previously reported that the experimentally-verified human T cell epitopes of the M. tuberculosis complex (MTBC) are the most conserved elements of the genome; whether immune recognition is the force driving the conservation of epitopes in the MTBC is unknown. Therefore, we sequenced the genomes of 12 BCG strains to determine whether T cell epitopes were under selection pressure during BCG in vitro evolution. We constructed a genome-wide phylogeny and refined the previously-determined BCG phylogeny. Notably, we identified a new cluster between BCG Japan and BCG Russia, and repositioned the relationships of several strains within the lineage. We also compared the sequence diversity of 1530 experimentally verified human T cell epitopes in the BCG vaccines with those in the MTBC. We found 23% of the known T cell epitopes are absent, and that the majority (82%) of the absent epitopes in BCG are contained in 6 proteins encoded in 2 regions of difference (RD) unique to BCG strains. We also found that T cell epitope sequences in BCG are more conserved than non-epitope sequences in the same gene. Finally, we find evidence that epitope sequence variation in BCG potentially affects human T cell recognition. These findings provide new insight into sequence variation in a slow-growing bacterium closely related to the MTBC that has been subjected to prolonged passage outside of a mammalian host, and indicate little difference in the extent of variation in vivo and in vitro.

Inhibition of IFN-γ promotes anti-asthma effect of Mycobacterium bovis Bacillus Calmette-Guerin neonatal vaccination: A murine asthma model

ObjectiveThe Mycobacterium bovis Bacillus Calmette-Guerin (BCG) neonatal vaccination inhibits allergy-induced pathologic changes. However, the mechanisms underlying this process are unclear. This study aimed to investigate the role of interferon (IFN)-γ and interleukin (IL)-17 in the protective effects of the BCG neonatal vaccination on allergic pulmonary inflammation and airway hyperresponsiveness (AHR). MethodsWild type (WT)-neonate and IL-17 knock out (KO) neonate mice were vaccinated with BCG. A murine asthma model was developed by sensitization and then challenging with ovalbumin (OVA). Recombinant IL-17 or recombinant IFN-γ was delivered to the airway to overexpress IL-17 or IFN-γ. An anti-IFN-γ neutralizing antibody was used to block the effects of IFN-γ. ResultsWe found exogenous IL-17 delivered to the airway reversed the anti-asthma effects of the neonatal BCG vaccination. BCG neonatal vaccination further reduced OVA-induced inflammation and AHR in IL-17 KO mice. Inhibition of IFN-γ in BCG neonatal vaccinated OVA-induced asthma model mice led to a further reduction in airway inflammation and AHR. In addition, airway inflammation and AHR were robust following treatment with exogenous IFN-γ. Neutralizing IL-17 was not sufficient to block OVA-induced airway inflammation and AHR. In IL-17 KO mice, airway inflammation and AHR did not occur following treatment with an anti-IFN-γ neutralizing antibody. ConclusionsIn an OVA-induced murine asthma model, inhibition of IFN-γ enhanced the anti-asthma effects of BCG neonatal vaccination.

Mycobacterium bovisBacillus Calmette-Guerin (BCG) and BCG-based Vaccines Against Tuberculosis

Tuberculosis (TB) is the second leading infectious cause of mortality worldwide with about two million deaths per year. The only licensed TB vaccine, Mycobacterium bovis bacillus Calmette-Guerin (BCG) shows limited protection efficacy suggesting an improved vaccination strategy is required. Recently, several TB vaccine candidates have entered clinical trials. These vaccine candidates are live mycobacterial vaccines designed to replace BCG or subunit vaccines designed to boost immunity induced by BCG. Vaccines with different strategy such as therapeutic vaccines, which can also be used in combination with drug therapy, are in the early stages of development to resolve latent TB or reactivation from the latent state. In this review, we discuss about development of BCG and BCG-based vaccines and further studies necessary for novel TB vaccine development to sterilize tuberculosis.

Immunogenicity and Protective Efficacy Conferred by a Novel Recombinant Mycobacterium bovis Bacillus Calmette‐Guérin Strain Expressing Interleukin‐12p70 of Human Cytokine and Ag85A of Mycobacterium tuberculosis Fusion Protein

Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunization provides protection against tuberculosis (TB) in infants, but the antituberculosis protective immunity wanes gradually after initial immunization and lasts less than 15 years. Therefore, more efficacious vaccines are urgently needed. In this study, we constructed a new tuberculosis vaccine of recombinant BCG strain (rBCG-IA), which could express IL-12p70 of human cytokine and Ag85A of M. tuberculosis fusion protein, and investigated its immunogenicity in BALB/c mice by measuring antibody titres, proliferation rate of splenocytes, ratios of CD4(+) T and CD8(+) T cells stimulated by specific antigens and levels of IFN-γ production in antigen-stimulated splenocyte cultures. Meanwhile, we evaluated its protective efficacy against M. tuberculosis H37Rv infection through detecting lung histopathology, organ bacterial loads and lung acid-fast stain. Immunogenicity experiments illustrated that from 2nd to 8th week after immunization, the rBCG-IA vaccine was able to induce the highest level of antibody titres, proliferation rate of splenocytes and IFN-γ production among groups and gained improved ratio of CD4(+) T and CD8(+) T cells from 6th to 8th week after vaccination. And from 2nd to 8th week after M. tuberculosis H37Rv infection, the score of pathology and bacterial loads in the rBCG-IA group were obviously lower than that in rBCG-I group, rBCG-A group or control group (PBST group), but similar to that in BCG group. This study suggested that rBCG-IA was able to elicit stronger humoral and cellular immune responses, but could only confer similar protective efficacy compared with its parental BCG vaccine.

Testing a molasses-based bait for oral vaccination of white-tailed deer (Odocoileus virginianus) against Mycobacterium bovis

White-tailed deer (Odocoileus virginianus) in Michigan, USA, are wildlife reservoirs of bovine tuberculosis (bTB) with documented spread to cattle. In vaccine efficacy trials, Mycobacterium bovis bacillus Calmette–Guerin (BCG) administered orally reduces colonization and bTB-associated lesions in white-tailed deer after experimental challenge with virulent M. bovis. The objective of this study was to develop and evaluate the palatability of a molasses-based bait for oral delivery of BCG to white-tailed deer. Relevant practical properties of the bait such as physical stability under various environmental conditions were evaluated, as well as palatability. Captive deer consumed baits within 3 h of introduction during 48 of 50 trials. Digital game cameras revealed consumption of all placed baits by one deer over 62 % of the time. Addition of BCG vaccine did not negatively impact palatability. Physical stability analysis demonstrated that ice and water significantly reduced bait stability as measured with a compression assay. Storage of BCG-containing baits at 4 °C showed a slight decrease in colony-forming units (CFUs) by day 31. In contrast, storage at −20 or −80 °C over the same 31-day period showed no significant decrease in BCG viability. The results of this study suggest that molasses-based baits, as prepared here, represent a plausible means of oral delivery of BCG to white-tailed deer under most environmental conditions.

Effect of lipopolysaccharide on nitric oxide (NO) production in culture supernatants exposed to Bacillus Calmette-Guerin (BCG) and supplemented with 1, 25(OH) 2 D3

Recently, there has been a striking increase in the number of patients with mycobacterial disease, and this is an important health problem around the world. One essential component of tubercular host defense includes nitric oxide production. The objective of this study was to investigate in vitro the effect of lipopolysaccharide on nitric oxide production in supernatants of mononuclear cell cultures from Mycobacterium bovis BCG-infected goats supplemented with 1, 25 Dihydroxyvitamin D3 [1,25(OH)2 D3]. Twelve animals of goat cattle 20-to 24-month-old sannen selected themselves. Five samplings were made, previous to the inoculation (zero days), 3, 7, 14 and 21 days after applying the treatments. The mononuclear cells by the ficoll-hypaque method were obtained. The nitric oxide concentrations were measured directly by griess method with ELISA reader. The treatment with the 1,25(OH)2D3 stimulated the nitric oxide synthesis indicating, that by itself it is a good modulator of the micobacterial replication and in the treatment with M. bovis-BCG vaccine increased as a result to the treatment with 1,25(OH)2D3. The exhibition to M. bovis-BCG vaccine with 1,25(OH)2D3 was able to increase answer NO2- in exposed animals. The LPS has effect in vitro in the production of nitric oxide in goats exposed to M. bovis BCG vaccine and supplemented with 1,25(OH)2D3. Key words: Nitric oxide, lipopolysaccharide, M. bovis-BCG vaccine, 1, 25 Dihydroxyvitamin D3, goats.

Subcutaneous Administration of a 10-Fold-Lower Dose of a Commercial Human Tuberculosis Vaccine, Mycobacterium bovis Bacillus Calmette-Guérin Danish, Induced Levels of Protection against Bovine Tuberculosis and Responses in the Tuberculin Intradermal Test Similar to Those Induced by a Standard Cattle Dose

Vaccination of cattle with a commercial human tuberculosis (TB) vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG) Danish, at a dose equivalent to 5 human doses of BCG has protected these animals against TB in field and experimental trials. There is interest in determining whether a 10-fold-lower dose could still protect cattle but not induce a tuberculin intradermal test response. Two groups of calves (n = 9/group) were vaccinated subcutaneously with a lyophilized BCG Danish vaccine containing either 0.5 (1 × 10(5) to 4 × 10(5) CFU) or 5 (1 × 10(6) to 4 × 10(6) CFU) human doses of BCG Danish, with an additional group of 10 calves serving as nonvaccinated controls. Fifteen weeks after vaccination, these animals were challenged intratracheally with 5 × 10(3) CFU of virulent M. bovis and another 15 weeks later were slaughtered and examined for the presence of tuberculous lesions. Vaccination of the calves with either 0.5 or 5 equivalent human doses of BCG Danish induced similar levels of protection against challenge with M. bovis, with both groups showing significant reductions in the pathological and microbiological parameters compared to those for the the control group (P < 0.05). Vaccination with either of the two BCG doses induced similar numbers of animals responding to the tuberculin intradermal test at 11 weeks postvaccination. Vaccination with a 0.5 equivalent human dose of a commercial lyophilized BCG vaccine can protect cattle against challenge with M. bovis.

Analysis of the Secretome and Identification of Novel Constituents from Culture Filtrate of Bacillus Calmette-Guérin Using High-resolution Mass Spectrometry

Tuberculosis (TB) is an infectious bacterial disease that causes morbidity and mortality, especially in developing countries. Although its efficacy against TB has displayed a high degree of variability (0%-80%) in different trials, Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been recognized as an important weapon for preventing TB worldwide for over 80 years. Because secreted proteins often play vital roles in the interaction between bacteria and host cells, the secretome of mycobacteria is considered to be an attractive reservoir of potential candidate antigens for the development of novel vaccines and diagnostic reagents. In this study, we performed a proteomic analysis of BCG culture filtrate proteins using SDS-PAGE and high-resolution Fourier transform mass spectrometry. In total, 239 proteins (1555 unique peptides) were identified, including 185 secreted proteins or lipoproteins. Furthermore, 17 novel protein products not annotated in the BCG database were detected and validated by means of RT-PCR at the transcriptional level. Additionally, the translational start sites of 52 proteins were confirmed, and 22 proteins were validated through extension of the translational start sites based on N-terminus-derived peptides. There are 103 secreted proteins that have not been reported in previous studies on BCG [corrected] secretome and are unique to our study. The physicochemical characteristics of the secreted proteins were determined. Major components from the culture supernatant, including low-molecular-weight antigens, lipoproteins, Pro-Glu and Pro-Pro-Glu family proteins, and Mce family proteins, are discussed; some components represent potential predominant antigens in the humoral and cellular immune responses.

Reactivation of immune responses against Mycobacterium tuberculosis by boosting with the CpG oligomer in aged mice primarily vaccinated with Mycobacterium bovis BCG

BackgroundMycobacterium bovis bacillus Calmette Guérin (BCG) vaccine, which has been inoculated to more than one billion people world-wide, has significant effect in preventing tuberculous meningitis and miliary tuberculosis (TB) in neonate and early childhood. However, BCG fails to adequately protect against pulmonary TB and reactivation of latent infections in adults. To overcome this problem, adequate booster is urgently desired in adult who received prior BCG vaccination, and appropriate animal models that substitute human cases would be highly valuable for further experimentation.FindingsThe booster effect of the synthesized CpG oligomer (Oligo-B) on aged mice which had been primarily vaccinated with BCG at the age of 4-week old. The specific Th1 type reaction, production of interferon-γ, in response to TB antigens, purified protein derivatives (PPD) and protection against challenge with Mycobacterium tuberculosis (MTB) H37Rv decreased with increasing age and were not observed in 89-week old mice. In order to rejuvenate the Th1 type response against PPD and protection activity against MTB infection, Oligo-B, which is known to augment Th1 responses, was administered as a booster to 81-90-week old mice (late 50’s in human equivalent) vaccinated with BCG at 4-week old. The boosting with Oligo-B increased the number of CD4+ CD44high CD62Lhigh, central memory type T cell. Furthermore, the Oligo-B boosting rejuvenated the ability of mice to protect against infection with MTB H37Rv.ConclusionsTh1-adjuvant CpG oligo DNA, such as Oligo-B, may be a promising booster when coupled with BCG priming.

Safety and immunogenicity of the recombinant BCG vaccine VPM1002 in a phase 1 open-label randomized clinical trial

BackgroundCurrent vaccination using Mycobacterium bovis bacillus Calmette-Guérin (BCG), fails to prevent pulmonary tuberculosis (TB). New vaccination strategies are essential for reducing the global incidence of TB. We assessed the safety and immunogenicity of VPM1002, a recombinant BCG vaccine candidate. EudraCT (2007-002789-37) and ClinicalTrials.gov (NCT00749034). MethodsHealthy volunteers were enrolled in a phase 1 open-label, dose escalation randomized clinical trial, and received one intradermal dose of VPM1002 (Mycobacterium bovis BCG ΔureC::hly HmR) or BCG. Immunogenicity was assessed by interferon-gamma (IFN-γ) production, cellular immune response markers by flow cytometry and serum antibodies against mycobacterial antigens. ResultsEighty volunteers were randomized into two groups according to previous BCG vaccination and mycobacterial exposure (BCG-naïve, n=40 and BCG-immune, n=40). In each group, 30 individuals were vaccinated with VPM1002 (randomized to three escalating doses) and 10 with BCG. VPM1002 was safe and stimulated IFN-γ-producing and multifunctional T cells, as well as antibody-producing B cells in BCG-naïve and BCG-immune individuals. ConclusionsVPM1002 was safe and immunogenic for B-cell and T-cell responses and hence will be brought forward through the clinical trial pipeline.