Bovis Bacille Calmette-Guérin Vaccination Research Articles

Latency Antigen α-Crystallin Based Vaccination Imparts a Robust Protection against TB by Modulating the Dynamics of Pulmonary Cytokines

BackgroundEfficient 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 FindingsWe 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/SignificanceThese 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.

BCG-mediated protection against Mycobacterium ulcerans infection in the mouse.

BackgroundVaccination 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.MethodsWe 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 FindingsBCG 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.ConclusionsThe efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen.

Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis

Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC-IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.

Lysine Auxotrophy Combined with Deletion of the SecA2 Gene Results in a Safe and Highly Immunogenic Candidate Live Attenuated Vaccine for Tuberculosis

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.

Immunity toMycobacterium tuberculosis

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide. Currently, a third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of TB, and annually there are 10 million new cases of clinical TB and approximately 2 million deaths [1]. TB kills more individuals each year than any other bacterial pathogen, and alarmingly, current control practices have not been able to significantly reduce the incidence of the disease over the past 15 years [1]. The current vaccine in use, Mycobacterium bovis bacille Calmette-Guerin (BCG), has been unable to limit the transmission of the disease, and the problem is compounded by the HIV/AIDS pandemic and the emergence of multidrug resistant strains of M. tuberculosis including extensively drug-resistant strains in multiple countries [2]. There is thus an urgent need to better understand the host response to M. tuberculosis and develop more effective strategies to control TB. This special issue contains original research reports and review articles covering a number of aspects of TB immunity. The first series of articles focuses on immune response to M. tuberculosis in humans. These studies range from the expression of cytolytic mediators following BCG vaccination in children (P. L. Semple et al.), control of antigen presentation function (A. Aquino et al.), changes in cellular makeup during postprimary TB (K. J. Welsh et al.), and treatment of disseminated infection in immuno-compromised individuals (A. A. Alangari et al.). The reviews of S. Meraviglia et al. and H. Saiga et al. describe the role of immune effectors in mycobacterial infection, while articles describing M. tuberculosis strain diversity (E. Nava-Agliluera et al.), TB in myelitis (Y. Feng et al.), and humans T- and B- cell responses to immunodominant mycobacterial antigens (G. C. Macedo et al.) also form part of this special issue. New insights on the interaction of M. tuberculosis with the host are also provided in this special issue. The reviews of M. Abebe et al., S. L. Sampson et al., and S. Ahmad describe pathogenic mechanisms and virulence factors expressed by M. tuberculosis, while the research articles of N. Sanarico et al. and E. Giacomini et al. investigate the transcriptional and cytokine response of host cells to M. tuberculosis infection. Reviews on TB transcriptomics (C. R. Zarate-Blades et al.) and granuloma liquefaction (P.-J. Cardona) provide further insight into the disease process during M. tuberculosis infection. The development of new vaccines is a major goal of TB research programs, and this special issue contains a number of articles investigating vaccine design and testing in animal models. G. G. Guerrero and C. Locht report on the use of recombinant antigens to boost BCG-induced immunity, while C. Wang et al. and M. Okada et al. similarly investigate prime-boost approaches to develop more effective TB vaccine regimens. The immune response following vaccination with M. tuberculosis lipoproteins is described in the article of C. Palma et al., and the use of cattle as a model to study TB immunity is the focus of the review article of W. R. Waters et al. The special issue closes with an overview of biosensing technologies for detection of M. tuberculosis by Z. Zhou et al. In conclusion, the aspects of immunity to M. tuberculosis infection, host-pathogen interaction and vaccine development covered in this special issue may lead to future advances in the treatment and control of TB. James A. Triccas Nathalie Winter Carl G. Feng Nicholas P. West

Non-clinical efficacy and safety of HyVac4:IC31 vaccine administered in a BCG prime–boost regimen

Despite the extensive success with the introduction of M. bovis Bacille Calmette-Guérin (BCG), tuberculosis (TB) remains a major global epidemic infecting between 8 and 9 million people annually with an estimated 1.7 million deaths each year. However, because of its demonstrated effectiveness against some of the most severe forms of childhood TB, it is now realized that BCG vaccination of newborns is unlikely to be replaced. Therefore, BCG or an improved BCG will continue to be used as a prime TB vaccine and there is a need to develop effective boost vaccines that would enhance and prolong the protective immunity induced by BCG prime immunization. We report on a heterologous booster approach using two highly immunogenic TB antigens comprising Ag85B and TB10.4 (HyVac4) delivered as a fusion molecule and formulated in the proprietary adjuvant IC31. This vaccine was found to be immunogenic and demonstrated greater protection in the more stringent guinea pig model of pulmonary tuberculosis than BCG alone when used in a prime/boost regimen. Significant difference in lung involvement was observed for all animals in the HyVac4 boosted group compared to BCG alone regardless of time to death or sacrifice. A vaccine toxicology study of the HyVac4:IC31 regimen was performed and it was judged safe to advance the vaccine into clinical trials. Therefore, all non-clinical data supports the suitability of HyVac4 as a safe, immunogenic, and effective vaccination in a prime–boost regimen with BCG.

TUBERCULOSIS IN EUROPEAN BADGERS (MELES MELES) AND THE CONTROL OF INFECTION WITH BACILLE CALMETTE-GUÉRIN VACCINATION

The eradication of tuberculosis (Mycobacterium bovis infection) from cattle herds may be compromised if infected wildlife species, such as European badgers (Meles meles), share the same environment and contribute to transfer of infection. Options for dealing with tuberculosis in this wild reservoir host are limited by conservation and social concerns, despite a clear implication that infected badgers are involved with the initiation of tuberculosis in cattle herds. Vaccination of badgers against M. bovis, if successfully employed, would directly facilitate the completion of bovine tuberculosis eradication in affected areas. Vaccine trials in captive badgers have established that the M. bovis bacille Calmette-Guérin (BCG) vaccine can induce a protective response that limits the distribution and severity of tuberculosis disease following experimental challenge. The protective effect of the vaccine has been demonstrated when the vaccine was delivered by subcutaneous injection, deposited on mucous membranes, and given orally in a lipid formulation. A large-scale field trial of oral BCG vaccine has been designed to measure the protection generated in wild badgers subjected to natural transmission of infection and to estimate vaccine efficacy. These parameters will be estimated by comparing the prevalence of M. bovis infection in vaccinated and nonvaccinated badgers. The results will provide a framework for the development and implementation of a national strategy to eliminate the disease in badger populations and if successful will remove this major impediment to bovine tuberculosis eradication.

Antigen Load Governs the Differential Priming of CD8 T Cells in Response to the Bacille Calmette Guérin Vaccine or Mycobacterium tuberculosis Infection

One reason proposed for the failure of Mycobacterium bovis bacille Calmette Guérin (BCG) vaccination to adequately control the spread of tuberculosis is a limited ability of the vaccine to induce effective CD8 T cell responses. However, the relative capacity of the BCG vaccine and virulent Mycobacterium tuberculosis to induce activation of CD8 T cells, and the factors that govern the initial priming of these cells after mycobacterial infection, are poorly characterized. Using a TCR transgenic CD8 T cell transfer model, we demonstrate significant activation of Ag-specific CD8 T cells by BCG, but responses were delayed and of reduced magnitude compared with those following infection with M. tuberculosis. The degree of CD8 T cell activation was critically dependent on the level of antigenic stimulation, as modifying the infectious dose to achieve comparable numbers of BCG or M. tuberculosis in draining lymph nodes led to the same pattern of CD8 T cell responses to both strains. Factors specific to M. tuberculosis infection did not influence the priming of CD8 T cells, as codelivery of M. tuberculosis with BCG did not alter the magnitude of BCG-induced T cell activation. Following transfer to RAG-1(-/-) recipients, BCG and M. tuberculosis-induced CD8 T cells conferred equivalent levels of protection against M. tuberculosis infection. These findings demonstrate that BCG is able to prime functional CD8 T cells, and suggest that effective delivery of Ag to sites of T cell activation by vaccines may be a key requirement for optimal CD8 T cell responses to control mycobacterial infection.

Transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with BCG at birth

BackgroundNovel tuberculosis (TB) vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG). Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult.MethodsTo better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD)-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix® HumanRef-8 Expression BeadChip (Illumina) to measure expression of >16,000 genes.ResultsWe found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, including PPAR-γ, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs), including integrin alpha M (ITGAM), which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated.ConclusionOur results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.

Neonatal bacillus Calmette–Guérin vaccination inhibits de novo allergic inflammatory response in mice via alteration of CD4+CD25+ T-regulatory cells

The hygiene hypothesis suggests a lack of bacterial infections would favor the development of allergic diseases. Mycobacterium bovis bacille Calmette-Guérin (BCG) infection can inhibit allergen-induced asthma reactions, but the underlying mechanism of this infection on the immunological responses is unclear. T-regulatory (Treg) cells are thought to play a role as a crucial immunoregulatory cells that are capable of regulating adaptive immune responses. We conducted this study to investigate whether the protective effect of the BCG vaccination on allergic pulmonary inflammation is associated with the alteration of CD4+CD25+ Treg cells in a murine asthma model and the mechanisms of Treg cells. Newborn C57BL/6 mice were vaccinated 3 times with BCG on d 0, 7, and 14 and subsequently sensitized and challenged with ovalbumin. Eosinophil infiltration was investigated. The frequencies of spleen CD4+CD25+ Treg cells and the expression of specific transcriptional factor Foxp3 were assayed. The cytotoxic lymphocyte associated antigen (CTLA)-4 expression and cytokine interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) levels were measured. We showed that treatment of mice with BCG inhibited de novo allergic inflammatory response in a mouse model of asthma. BCG treatments are associated with the increase of CD4+CD25+ Treg cells and Foxp3 expression, accompanied by an increased CTLA-4 expression and cytokine IL-10 and TGF-beta levels (P<0.05). Neonatal BCG vaccinations ameliorate de novo local eosinophilic inflammation induced by allergen and increase the numbers of CD4+CD25+ Treg cells and Foxp3 expression. The cell-cell contact inhibition and regulatory cytokine production may be involved in the regulatory mechanism.

Mycobacterium bovisBacille Calmette-Guérin Infection in the CNS Suppresses Experimental Autoimmune Encephalomyelitis and Th17 Responses in an IFN-γ-Independent Manner

Multiple sclerosis and an animal model resembling multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases of the CNS that are suppressed by systemic mycobacterial infection in mice and BCG vaccination in humans. Host defense responses against Mycobacterium in mice are influenced by T lymphocytes and their cytokine products, particularly IFN-gamma, which plays a protective regulatory role in EAE. To analyze the counter-regulatory role of mycobacterial infection-induced IFN-gamma in the CNS on the function of the pathological Th17 cells and the clinical outcome of EAE, we induced EAE in mice that were intracerebrally infected with Mycobacterium bovis bacille Calmette-Guerin (BCG). In this study, we demonstrate that intracerebral (i.c.) BCG infection prevented inflammatory cell recruitment to the spinal cord and suppressed the development of EAE. Concomitantly, there was a significant decrease in the frequency of myelin oligodendrocyte glycoprotein-specific IFN-gamma-producing CD4(+) T cells in the CNS. IL-17(+)CD4(+) T cell responses were significantly suppressed in i.c. BCG-infected mice following EAE induction regardless of T cell specificity. The frequency of Foxp3(+)CD4(+) T cells in these mice was equivalent to that of control mice. Intracerebral BCG infection-induced protection of EAE and suppression of myelin oligodendrocyte glycoprotein-specific IL-17(+)CD4(+) T cell responses were similar in both wild-type and IFN-gamma-deficient mice. These data show that live BCG infection in the brain suppresses CNS autoimmunity. These findings also reveal that the regulation of Th17-mediated autoimmunity in the CNS can be independent of IFN-gamma-mediated mechanisms.

Assessment of different formulations of oral Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine in rodent models for immunogenicity and protection against aerosol challenge with M. bovis

Bovine tuberculosis (bTB) caused by infection with Mycobacterium bovis is causing considerable economic loss to farmers and Government in the United Kingdom as its incidence is increasing. Efforts to control bTB in the UK are hampered by the infection in Eurasian badgers ( Meles meles) that represent a wildlife reservoir and source of recurrent M. bovis exposure to cattle. Vaccination of badgers with the human TB vaccine, M. bovis Bacille Calmette-Guérin (BCG), in oral bait represents a possible disease control tool and holds the best prospect for reaching badger populations over a wide geographical area. Using mouse and guinea pig models, we evaluated the immunogenicity and protective efficacy, respectively, of candidate badger oral vaccines based on formulation of BCG in lipid matrix, alginate beads, or a novel microcapsular hybrid of both lipid and alginate. Two different oral doses of BCG were evaluated in each formulation for their protective efficacy in guinea pigs, while a single dose was evaluated in mice. In mice, significant immune responses (based on lymphocyte proliferation and expression of IFN-γ) were only seen with the lipid matrix and the lipid in alginate microcapsular formulation, corresponding to the isolation of viable BCG from alimentary tract lymph nodes. In guinea pigs, only BCG formulated in lipid matrix conferred protection to the spleen and lungs following aerosol route challenge with M. bovis. Protection was seen with delivery doses in the range 10 6–10 7 CFU, although this was more consistent in the spleen at the higher dose. No protection in terms of organ CFU was seen with BCG administered in alginate beads or in lipid in alginate microcapsules, although 10 7 in the latter formulation conferred protection in terms of increasing body weight after challenge and a smaller lung to body weight ratio at necropsy. These results highlight the potential for lipid, rather than alginate, -based vaccine formulations as suitable delivery vehicles for an oral BCG vaccine in badgers.

Development of a Mycobacterium bovis intranasal challenge model in mice

Tuberculosis caused by infection with Mycobacterium tuberculosis or Mycobacterium bovis remains one of the most important infectious diseases of man and animals. The current vaccine, M. bovis bacille Calmette-Guérin (BCG) demonstrates variable efficacy in humans and cattle, and so an urgent need exists for a new vaccine to replace or supplement BCG. Novel vaccine development requires the availability of a suitable animal model in which to test potential vaccine candidates. Models for tuberculosis vaccine development include mice, guinea pigs, cattle and non-human primates. Murine models provide an economical and easily manipulated tool, but the natural aerosol infection route requires extensive facilities, equipment and validation. We sought to develop a logistically simpler intranasal M. bovis infection model for use in vaccine development for bovine tuberculosis. Intranasal M. bovis infection model in mice demonstrated distinct airway associated, dose related pathology, and was strikingly more virulent than previously employed intravenous infection with M. bovis. BCG vaccination of intranasal challenged mice induced 2 logs of protection with similar kinetics as those displayed in M. tuberculosis aerosol infection models. In conclusion, we report the development of a virulent, robust, stringent, physiological and inexpensive M. bovis intranasal infection model for the screening of potential vaccine candidates against bovine tuberculosis.

Consequence of prior exposure to environmental mycobacteria on BCG vaccination and diagnosis of tuberculosis infection

The protective efficacy of Mycobacterium bovis-bacille Calmette Guérin (BCG) against tuberculosis (TB) is variable in both humans and cattle. Exposure to environmental mycobacteria is thought to result in inappropriate priming of host immune responses. To investigate the impact of environmental mycobacteria on BCG efficacy, cattle were infected with M. avium, vaccinated with BCG, challenged with M. bovis and skin tested prior to necropsy. Elevated levels of IFNgamma were evident in M. avium-exposed animals before and after BCG vaccination with a bias towards avian purified protein derivative (PPD-A), suggesting that M. avium primed host immune responses. Exposure to M. avium also resulted in a higher frequency of circulatory IFNgamma-producing cells in response to PPD antigens at the time of M. bovis challenge. After M. bovis inoculation, the IFNgamma response to bovine PPD (PPD-B) increased compared to pre-challenge levels, indicating that all animals had been exposed to M. bovis. Skin test responses indicated 2/6 M. avium-BCG-M. bovis animals as reactors and 2/6 as inconclusive compared with 6/6 BCG-M. bovis animals as reactors. M. avium-exposed animals also had fewer lesions and the number of tissues containing viable M. bovis at post-mortem was significantly lower (P<0.02 compared with BCG-M. bovis animals), with two of the animals described as skin test negative with no visible lesions or viable bacteria. Thus, exposure of cattle to environmental mycobacteria such as M. avium prior to BCG vaccination did not dampen BCG-specific immune responses and resulted in lower TB pathology. However, the PPD-A bias associated with M. avium exposure is likely to undermine current TB diagnostic tests and the IFNgamma test in cattle.

Accelerating the secondary immune response by inactivating CD4+CD25+ T regulatory cells prior to BCG vaccination does not enhance protection against tuberculosis

CD4(+)CD25(+) natural T regulatory cells (Tregs) have been shown to suppress protective immune responses in several different vaccination models. Since the effect of Tregs on vaccination against tuberculosis (Tb) was unknown, we used a murine model to investigate whether natural Tregs suppress the development of protective immunity following Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. Using a monoclonal antibody against CD25, natural Tregs were inactivated prior to vaccination with BCG. The primary immune response was evaluated after BCG vaccination and the secondary immune response was assessed after an intranasal BCG challenge 42 days after vaccination. Inactivation of natural Tregs prior to vaccination led to an increased immune response 14 days after vaccination, increased numbers of antigen-responsive lymphocytes immediately prior to secondary challenge and the earlier appearance of IFN-gamma-producing CD4(+) and CD8(+) lymphocytes in the draining lymph nodes and lungs after challenge. Despite this, protection from virulent Mycobacterium tuberculosis or M. bovis aerosol challenge was unaffected by natural Treg inactivation prior to BCG vaccination. This suggests that increasing the primary and accelerating the secondary immune responses by inactivating natural Tregs at the time of vaccination, does not affect the development of protective immunity to Tb.

Adverse reactions to Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccination against tuberculosis in humans, veterinary animals and wildlife species

The Mycobacterium bovis strain, bacille Calmette-Guérin (BCG) is one of the most widely used human vaccines and remains one of the safest vaccines available. It has been used in human populations for over 80 years and 100 million children receive the vaccine annually. It has also been employed extensively for vaccine studies in laboratory animal hosts and is currently being developed for use in a variety of livestock and wild animals. Despite the large number of doses delivered since is first usage in 1921, reports of adverse reactions arising from the use of the BCG vaccine are relatively uncommon and where serious reactions do occur they are often the result of vaccination of immuno-compromised individuals. Factors that may influence the development of adverse reactions to BCG include the potency and dose of the vaccine strain, the route of delivery, the age and immune status of the host, and the skill levels of the operator administering the vaccine. Circumstances affecting the notification of adverse reactions include the lack of clear case definitions of abnormal vaccine reactions, and a scarcity of systematic surveillance and functioning reporting systems. With continued use of the BCG and the development of a new generation of prophylactic and therapeutic vaccines against tuberculosis in different host species, the risk factors associated with adverse reactions may need to be reappraised.

Evidence for enhanced central memory priming by live Mycobacterium bovis BCG vaccine in comparison with killed BCG formulations

Development of cattle vaccines against bovine tuberculosis is a GB research priority. Recently, it has been shown that formalin-killed Bacille Calmette-Guérin (BCG) delivered with the liposomal adjuvant NAX687 imparted significant protection against Mycobacterium bovis infection in the guinea pig aerosol infection model. Extending these studies, we inoculated calves with live BCG, formalin-killed BCG and formalin-killed BCG formulated in NAX687. Live and killed BCG vaccine formulations induced primary effector T-cell populations comparably, both killed BCG formulations also induced potent humoral immune responses. In contrast, live BCG generated enhanced central memory responses against the protective antigen Ag85A whilst killed BCG-induced such responses only poorly. However, the poor capacity of killed BCG to generate central memory could be partially overcome by formulation with NAX687. Measurement of central memory responses induced by TB vaccine candidates in cattle may provide a useful correlate of protection and warrants further investigation in challenge experiments.

Mucosal prime-boost vaccination for tuberculosis based on TLR triggering OprI lipoprotein from Pseudomonas aeruginosa fused to mycolyl-transferase Ag85A

Toll-like receptor (TLR) triggering is an important step in the induction of T helper (Th) type 1 T cells which are key players in protection against the intracellular pathogen Mycobacterium ( M.) tuberculosis. Here we report on the construction of a fusion protein consisting of a tuberculosis vaccine candidate mycolyl-transferase antigen 85A (Ag85A, Rv3804c) coupled to the outer membrane lipoprotein I (OprI) from Pseudomonas ( P.) aeruginosa, a documented TLR2/TLR4 trigger. Subcutaneous boosting with this fusion protein in the absence of adjuvant increased significantly the Ag85A-specific humoral but not cellular immune responses of Ag85A-DNA vaccinated mice. Intranasal priming of C57BL/6 mice with live, attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine, followed by intranasal boosting with OprI-Ag85A increased systemic and local antigen-specific interferon (IFN)-γ and interleukin (IL)-2 responses in spleen, draining cervical and mediastinal lymph nodes and particularly in lung tissue, as compared to responses in mice only vaccinated with BCG vaccine. Despite enhanced immune responses, boosting with OprI-Ag85A did not increase protective efficacy against M. tuberculosis of either plasmid DNA or BCG vaccine in this experimental setting.

Vaccine efficacy of an attenuated but persistent Mycobacterium tuberculosis cysH mutant

The emergence of drug-resistant Mycobacterium tuberculosis strains and the widespread occurrence of AIDS demand newer and more efficient control of tuberculosis. The protective efficacy of the current Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine is highly variable. Therefore, development of an effective new vaccine has gained momentum in recent years. Recently, several M. tuberculosis mutants were tested as potential vaccine candidates in the mouse model of tuberculosis. However, only some of these mutants were able to generate protection equivalent to that of BCG in mice. This study reports the vaccine potential of an attenuated 5'-adenosine phosphosulfate reductase mutant (DeltacysH) of M. tuberculosis. Immunization of mice with either BCG or DeltacysH followed by infection with the virulent M. tuberculosis Erdman strain demonstrated that DeltacysH can generate protection equivalent to that of the BCG vaccine.

Immune Gene Networks of Mycobacterial Vaccine–Elicited Cellular Responses and Immunity

Gene networks of protective lymphocytes after immune activation with live attenuated vaccines remain poorly characterized. Because Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine can confer protection against fatal forms of tuberculosis in humans and monkeys, we made use of macaque models to optimally study immune gene networks after BCG vaccination/infection. We first established and validated a large-scale real-time quantitation system and then used it to measure expression levels of 138 immune genes after BCG vaccination/infection of rhesus macaques. Systemic BCG vaccination induced up to 600-fold increases in expression of 78 immune genes among the 138 genes tested at the time when BCG-elicited T cell responses and immunity were apparent. These up-regulated transcripts constituted multiple gene networks that were linked to various aspects of immune function. Surprisingly, the up-regulation of most of these immune genes in the gene networks occurred at 1 week and was sustained at > or = 6 weeks after BCG vaccination/infection. Although early activation of immune gene networks was an immune correlate of anti-BCG immunity, prolonged up-regulation of these networks coincided with the development of vaccine-elicited T cell responses after BCG vaccination/infection. These findings provide molecular evidence suggesting that the BCG-induced gene networks may represent global transcriptomes and proteomes underlying the development of T cell responses and, ultimately, immunity to mycobacteria.