Influence of sensitisation to environmental mycobacteria on subsequent vaccination against bovine tuberculosis.

Bacille Calmette-Guerin (BCG) is the world's most widely used vaccine, but there are concerns that it provides little protection against pulmonary tuberculosis of humans in countries that have a high prevalence of environmental mycobacteria. Experiments in cattle provide a model to investigate this situation and to develop an improved tuberculosis vaccine. In the third of a series of BCG vaccination trials, calves had high interferon-gamma (IFN-gamma) responses to purified protein derivative (PPD) from Mycobacterium avium prior to vaccination, indicating that infection with environmental mycobacteria had occurred. The calves vaccinated with BCG had minimal protection against an experimental intratracheal challenge with virulent Mycobacterium bovis. In comparison, calves vaccinated with either of two newly-derived attenuated M. bovis strains had significantly better but not complete protection against the development of tuberculous lesions compared to both BCG-vaccinated and non-vaccinated animals. Vaccination with the newly-derived attenuated M. bovis strains induced strong IFN-gamma and interleukin-2 (IL-2) responses to PPD from M. bovis at 2 weeks after vaccination, while BCG vaccination induced only a weak response at this time. In association with the previous two trials, the results suggest that sensitisation of the calves to environmental mycobacteria adversely affected subsequent protective efficacy of BCG. However, the results of vaccination with the other two attenuated M. bovis strains indicated that improved tuberculosis vaccines could be developed for such sensitised animals.     

Oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in brushtail possums

A method was developed for formulating Mycobacterium bovis bacille Calmette-Guerin (BCG) for oral vaccination against tuberculosis. Selected lipid-based formulations of BCG were tested in the brushtail possum for their ability to elicit immune responses and protection against bovine tuberculosis. Formulation of BCG in lipid matrices maintained bacteria in a dormant but viable state. Oral delivery of 2 x 10(8) colony forming units of formulated BCG to possums induced strong lymphocyte proliferation responses to bovine purified protein derivative (PPD) in peripheral blood lymphocytes. Oral vaccination of possums also reduced the severity of disease following aerosol challenge with virulent M. bovis compared with animals vaccinated with non-formulated BCG. In a second experiment, levels of protection with lipid-formulated oral BCG were similar to those seen with subcutaneous BCG vaccination. Our data shows that formulated oral BCG is an efficient means of inducing protection against bovine tuberculosis in possums and should be a practical means of vaccinating wildlife against tuberculosis.     

Lack of correlation between BCG-induced tuberculin skin test sensitisation and protective immunity in cattle

Vaccination of cattle with Mycobacterium bovis Bacille Calmette-Guérin (BCG) can provide significant protection against bovine tuberculosis (TB). However, BCG vaccination sensitises animals to respond to the tuberculin skin-test. This provides a potential operational impediment to the use of BCG as a cattle vaccine since the tuberculin skin-test is the primary surveillance tool used by many countries with ‘test and slaughter’ control strategies. Currently, it is also unclear what BCG-induced skin-test conversion means in respects to BCG's protective immunity. In the current study we first investigated the duration of tuberculin skin-test sensitisation in calves neonatally vaccinated with BCG. BCG vaccination induced strong skin-test responses in calves during their first 6 months. However, a rapid decay in skin-test sensitivity was observed after this time. Between 6 and 9 months this represented a reduction from 80% to 8% of calves providing a positive response in the single intradermal comparative cervical tuberculin test at standard interpretation. We next investigated the relationship between BCG induced skin-test sensitivity and retention of protective immunity. Calves were neonatally vaccinated with BCG and subsequently divided into 2 groups based on retention or loss of tuberculin skin-test responses after 6 months. In contrast to their skin-test responsiveness, these vaccinates maintained their tuberculin specific IFN-γ blood responses. Moreover, irrespective of their pre-challenge skin-test responses, following M. bovis challenge both groups of BCG vaccinated calves demonstrated comparable levels of protection, as evidenced by reduced TB-associated pathology. Therefore, we have demonstrated that following neonatal BCG vaccination of cattle, tuberculin skin-test responder frequencies waned rapidly after 6 months but importantly, loss of skin-test sensitivity did not correlate with loss of protective immunity. These findings could have implications for the practical application of BCG based cattle vaccines.     

An ESAT-6:CFP10 DNA vaccine administered in conjunction with Mycobacterium bovis BCG confers protection to cattle challenged with virulent M. bovis

The potency of genetic immunization observed in the mouse has demonstrated the utility of DNA vaccines to induce cell-mediated and humoral immune responses. However, it has been relatively difficult to generate comparable responses in non-rodent species. The use of molecular adjuvants may increase the magnitude of these suboptimal responses. In this study, we demonstrate that the co-administration of plasmid-encoded GM-CSF and CD80/CD86 with a novel ESAT-6:CFP10 DNA vaccine against bovine tuberculosis enhances antigen-specific cell-mediated immune responses. ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinated animals exhibited significant (p<0.01) antigen-specific proliferative responses compared to other DNA vaccinates. Increased expression (p< or =0.05) of CD25 on PBMC from ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinates was associated with increased proliferation, as compared to control DNA vaccinates. Significant (p<0.05) numbers of ESAT-6:CFP10-specific IFN-gamma producing cells were evident from all ESAT-6:CFP10 DNA vaccinated animals compared to control DNA vaccinates. However, the greatest increase in IFN-gamma producing cells was from animals vaccinated with ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA. In a low-dose aerosol challenge trial, calves vaccinated as neonates with Mycobacterium bovis BCG and ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA exhibited decreased lesion severity in the lung and lung-associated lymph nodes following viruluent M. bovis challenge compared to other vaccinated animals or non-vaccinated controls. These data suggest that a combined vaccine regimen of M. bovis BCG and a candidate ESAT-6:CFP10 DNA vaccine may offer greater protection against tuberculosis in cattle than vaccination with BCG alone.     

Vaccination of white-tailed deer (Odocoileus virginianus) with Mycobacterium bovis bacillus Calmette Guerín

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer in order to interrupt the cycle of deer to deer and deer to cattle transmission. Thirty-one white-tailed deer were assigned to one of three groups; 2 SC doses of 10(7)CFU of M. bovis BCG (n=11); 1 SC dose of 10(7)CFU of M. bovis BCG (n=10); or unvaccinated deer (n=10). After vaccination, deer were inoculated intratonsilarly with 300 CFU of virulent M. bovis. Gross lesion severity scores of the medial retropharyngeal lymph node were significantly reduced in deer receiving 2 doses of BCG compared to unvaccinated deer. Vaccinated deer had fewer lymph node granulomas than unvaccinated deer, and most notably, fewer late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli. BCG was isolated from 7/21 vaccinated deer as long as 249 days after vaccination. In one case BCG was transmitted from a vaccinated deer to an unvaccinated deer. In white-tailed deer BCG provides measurable protection against challenge with virulent M. bovis. However, persistence of vaccine within tissues as well as shedding of BCG from vaccinates remain areas for further investigation.     

Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the specificity of the comparative intradermal tuberculin skin test

The current tuberculin test and slaughter strategy for the control of bovine tuberculosis in cattle has failed to prevent a sharp rise in cases over recent years, especially in the south-west of England. A recent scientific review has concluded that the development of a cattle vaccine holds the best prospect for tuberculosis control in British herds. In order to continue with test and slaughter-based control strategies, the development of TB vaccines that do not compromise the specificity of the tuberculin skin test are required. This report describes results of cattle vaccination experiments with TB DNA vaccines expressing the mycobacterial antigens MPB70, MPB83, and Ag85A and constitutes the first published vaccination study with DNA vaccines undertaken in a target host species. All calves vaccinated with the MPB83 expressing plasmid demonstrated potent cellular immune responses, characterised by CD4(+) T cells producing interferon-gamma as well as humoral immunity characterised by IgG1 biased specific antibodies. Vaccination with MPB70 was less effective with immune responses only observed in half of the vaccinated animals, while vaccination with Ag85A did not result in vaccine-induced immune responses. Intramuscular vaccination was found to stimulate stronger cellular responses than intradermal immunisation. Significantly, the specificity of tuberculin skin testing was not compromised by DNA vaccination since none of the vaccinated calves showed positive skin test reactivity.     

Indications for,and the significance of,the tuberculin test in the Netherlands

The almost 100-year-old tuberculin skin test still is the gold standard for diagnosing Mycobacterium tuberculosis infection. The sensitivity of this test with the usual cut-off values is high, but may be decreased with impaired cellular immunity and at older age. The specificity is primarily determined by cross-reactivity to atypical mycobacterial infections and vaccination with Bacillus Calmette-Guérin (BCG). Positivity of the skin test after BCG vaccination decreases with time after vaccination and depends on the age when vaccinated. The tuberculin reaction can be boosted by repeated tuberculin skin tests over a short time period, whereby the anamnestic immune response is stimulated. This boosting phenomenon occurs mostly with atypical mycobacterial infections, after BCG vaccination and at older age. Interpretation of the tuberculin skin test depends on the indication for the test, the expected risk of latent tuberculosis infection, higher prevalence of 'old' tuberculosis in elderly Dutch people and immigrants, BCG vaccination status and, if a baseline value is available, the boosting phenomenon. Its role in the diagnosis of tuberculosis is limited.     

The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs

The Mycobacterium tuberculosis phoP mutant strain SO2 has previously been shown to have reduced multiplication in mouse macrophages and in vivo using the mouse intravenous-infection model. In this study we demonstrate that the M. tuberculosis SO2 is highly attenuated when compared with the parental M. tuberculosis MT103 strain and also more attenuated than BCG in severe combined immunodeficiency disease (SCID) mice. Complementation of the M. tuberculosis SO2 with the wild-type phoP gene restored the virulence of the strain in the SCID mice, confirming that the attenuated phenotype is due to the phoP mutation. In Balb/c mice subcutaneously vaccinated with either M. tuberculosis SO2 or BCG, the proportions of CD4+ and CD8+ populations measured in the spleen were significantly higher in the M. tuberculosis SO2 vaccinated group. In addition, the proportion of antigen-stimulated CD4+/CD8+ cells expressing IFN-gamma was significantly higher in the M. tuberculosis SO2 vaccinated group when compared with the BCG group. Balb/c mice subcutaneously vaccinated with the M. tuberculosis SO2 strain were also protected against intra-venous challenge with M. tuberculosis H37Rv at levels comparable to mice vaccinated with BCG, as measured by reduced bacterial counts in lung and spleens. Guinea pigs subcutaneously vaccinated with the M. tuberculosis SO2 strain were protected against aerosol challenge with M. tuberculosis H37Rv delivered at different doses. A high dose aerosol challenge of M. tuberculosis SO2 vaccinated guinea pigs resulted in superior levels of protection when compared with BCG vaccination, as measured by guinea pig survival and reduction in disease severity in the lung.     

Development of a simple, sensitive, rapid test which discriminates BCG-vaccinated from Mycobacterium bovis-infected cattle

Bovine tuberculosis (bTB) is increasing in incidence in the UK. Effective control strategies could involve vaccination; BCG, either alone or in prime-boost strategies, remains the most effective vaccine against bovine tuberculosis. However, BCG vaccination of cattle would require development of diagnostic tests able to accurately discriminate Mycobacterium bovis-infected from BCG-vaccinated animals. Herein, we demonstrate that the detection of secreted IFN-gamma following short term culture (4h) of whole blood with purified protein derived from M. bovis (PPD-B) allows such discrimination. This reflects, in part, the differential kinetics of IFN-gamma secretion in infected compared to vaccinated cattle. This is the first study to demonstrate that accurate, rapid distinction of BCG-vaccinated from M. bovis-infected cattle can be achieved in a short time period without the need for production of M. bovis-specific antigens, complex antigen mixtures or extensive laboratory procedures. We were also able to detect PPD-specific IFN-gamma release during short term culture of blood from a number of humans with active TB indicating that this test may have wider application and is potentially useful for the rapid diagnosis of disease in humans.     

CD80 and CD86, but not CD154, augment DNA vaccine-induced protection in experimental bovine tuberculosis

DNA vaccination is known to elicit robust cellular and humoral responses to encoded antigen. The co-administration of costimulatory molecules CD80 (B7-1), CD86 (B7-2) and CD154 (CD40L) has been shown to enhance immune responses in several murine models. The role of specific costimulatory molecules in non-rodent species remains incompletely characterized. In these studies, we demonstrate that the co-administration of CD80 and CD86, but not CD154, to an existing candidate subunit DNA vaccine (ESAT-6) against bovine tuberculosis, enhances protection after aerosol challenge with virulent Mycobacterium bovis. Additionally, we have shown that vaccination with M. bovis BCG is protective against tuberculosis following aerosol challenge in cattle. Two independent trials were conducted in cattle to determine the adjuvant effect of encoded antigen + CD80/CD86 and directly compare the adjuvant activities of CD80/CD86 to those of CD154. Co-administration of either CD80/CD86 or CD154 enhanced ESAT-6-specific IFN-gamma responses as compared to animals vaccinated with ESAT-6 DNA alone. However, following aerosol challenge, only animals vaccinated with CD80/CD86 possessed decreased pathology of the lungs and associated lymph nodes, as measured by gross examination, radiographic lesion morphometry and bacterial recovery. Collectively, these results demonstrate that the co-administration of costimulatory molecules with a protective antigen target enhances bovine immune responses to DNA vaccination, and that CD80/CD86 is superior to CD154 in augmenting DNA vaccine-induced protection in experimental bovine tuberculosis.     

Mycobacterium bovis DeltaleuD auxotroph-induced protective immunity against tissue colonization, burden and distribution in cattle intranasally challenged with Mycobacterium bovis Ravenel S

Bovine tuberculosis is a chronic granulomatous disease caused by Mycobacterium bovis. Lack of definitive diagnostics and effective vaccines for domestic animals are major obstacles to the control and eradication of bovine tuberculosis. Auxotrophic mutants of Mycobacterium tuberculosis have shown promise as vaccine candidates for preventing human tuberculosis. Similarly, we constructed a leucine auxotroph of M. bovis, by using allelic exchange to delete leuD (encoding isopropyl malate isomerase), creating a strain requiring exogenous leucine for growth in vitro. We vaccinated 10 cattle subcutaneously with 10(9)CFU of M. bovis DeltaleuD and 10 age-matched, gender-matched controls were injected with phosphate-buffered saline. Vaccinated cattle had significantly increased in vitro antigen-specific T-cell-mediated responses. All cattle were challenged intranasally on day 160 post-immunization with 10(6)CFU of virulent M. bovis Ravenel S. On day 160 post-challenge vaccinated cattle had significantly reduced tissue mycobacterial burdens and 6 of 10 had complete clearance of the challenge strain and histopathological lesions were dramatically less severe in the vaccinated group. Thus, a single subcutaneous immunization of the M. bovis DeltaleuD mutant produced highly significantly protective immunity as measured by a reduction in tissue colonization, burden, bacilli dissemination, and histopathology caused by virulent M. bovis Ravenel S challenge.     

Immunogenicity and protection induced by Mycobacterium tuberculosis mce-2 and mce-3 mutants in a Balb/c mouse model of progressive pulmonary tuberculosis

Mycobacterial proteins coded by the mammalian cell entry (mce) genes allow for cell invasion into the host. The Mycobacterium tuberculosismce-2 and mce-3 mutants have impaired synthesis of mce proteins and are attenuated in BALB/c mice. Intra-tracheal infection of Balb/c mice with either mce mutant induced lower but progressive production of IFN-gamma and TNF-alpha, as well as larger delayed type hypersensitivity (DTH) reactions, than their parental H37Rv strain. When used as a subcutaneous vaccine and, before challenge, both mutants were more attenuated than BCG in Balb/c and immunodeficient nude mice. Cell suspensions from lymph nodes and spleen from mce mutant vaccinated mice stimulated with mycobacterial culture filtrate antigens (CFA) or immunodominant antigens (ESAT-6, Ag85) produced more INF-gamma than BCG-vaccinated animals. Used as subcutaneous vaccines, 60 days before intra-tracheal challenge with the hypervirulent strain of M. tuberculosis (Beijing code 9501000), both mutants induced a higher level of protection than BCG; 72% and 63% of the mice vaccinated with the mce-2 and mce-3 mutants, respectively, survived for 16 weeks after the challenge as compared to 30% of those vaccinated with BCG. Likewise, there was less tissue damage (pneumonia) and lower colony forming units (CFU) in the mice vaccinated with either of the two mutants as compared to the findings in mice vaccinated with BCG. These data suggest that lack of mce-2 and -3 gene expression decreases virulence and increases immunogenicity of live vaccines, favouring their ability to protect against tuberculosis, which was better than the protection conferred by BCG.     

Neonatal revaccination with Bacillus Calmette-Guérin elicits improved, early protection against Mycobacterium tuberculosis in mice

The protective effect of revaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) against Mycobacterium tuberculosis in animals is controversial. To investigate whether revaccination of neonates with BCG could improve the protection against M. tuberculosis, C57BL/6 neonates were vaccinated with BCG on day 1, or on days 1, 7, and 14, and the mice at eight weeks of age were challenged with M. tuberculosis and monitored for survival. The M. tuberculosis burden in their livers and lungs, the pathological changes in the lungs, their splenic T cell responses and serum cytokines were examined longitudinally post-challenge. BCG vaccination significantly prevented the M. tuberculosis-related mouse death and reduced the burden of M. tuberculosis in the liver and lungs, and lung damage in the mice, particularly at early stage of the pathogenic process in the BCG-revaccinated mice. However, the BCG revaccination-induced protection waned over time. BCG vaccination did not significantly modulate the levels of serum IFN-γ and the frequency of splenic PPD-reactive IFN-γ-secreting T cells, but significantly decreased the levels of serum TNF-α and PPD-specific IL-4 responses at 3 weeks post challenge. Taken together, these data suggest that revaccination of neonates with BCG elicits improved, early protection against M. tuberculosis by modulating cytokine responses in adult mice.     

BCG scar and positive tuberculin reaction associated with reduced child mortality in West Africa. A non-specific beneficial effect of BCG?

Previous studies have suggested that the bacille Calmette-Guérin (BCG) vaccine may have a non-specific beneficial effect on childhood survival in areas with high mortality. We examined whether BCG-vaccinated children with a BCG scar or a positive tuberculin reaction had better survival than children without such reactions. As part of an ongoing two-dose measles vaccine trial for which children were recruited at 6 months of age, we examined 1813 children for BCG scar at 6 months of age and 813 BCG-vaccinated children were skin-tested for delayed hypersensitivity to tuberculin, tetanus and diphtheria. We found that BCG-vaccinated children with a BCG scar had significantly lower mortality compared with BCG scar-negative children, the mortality ratio in the first 12 months of follow-up being 0.41 (0.25-0.67). BCG-vaccinated children with a positive tuberculin test had a mortality ratio of 0.45 (0.24-0.85) compared with tuberculin negative children. These results were unchanged by control for potential confounders or using different cut-off points for a tuberculin-positive response. Exclusion of dead children who had HIV antibodies did not modify the estimate (mortality rate (MR)=0.46 (0.23-0.94)). After censoring for tuberculosis (TB) exposure at home, the mortality ratios for having a scar and being tuberculin-positive were 0.46 (0.27-0.79) or 0.42 (0.21-0.84), respectively. Children positive to tetanus or diphtheria in the skin test had the same mortality as children not responding to these vaccine-related antigens. Thus, BCG scar and a positive tuberculin reaction were associated with better survival in early childhood in an area with high mortality. Since nothing similar was found for responders to diphtheria-tetanus-pertussis (DTP) vaccine, and the effect could not be explained by protection against tuberculosis, the effect of BCG vaccination could be due to non-specific immune-stimulation protecting against other infections.     

PPD induced in vitro interferon gamma production is not a reliable correlate of protection against Mycobacterium tuberculosis

Correlates of protection against tuberculosis are crucial for the evaluation of new vaccine candidates and for the demonstration of their potential efficacy. Such correlates can be proposed on the basis of animal models. In this study, we hypothesized that protection against tuberculosis (TB) induced by bacillus Calmette-Guerin (BCG) correlates with in vitro TB antigen-specific IFN-gamma production. BCG vaccination, known to provide effective protection against TB in animals, was used to investigate the use of in vitro IFN-gamma production as a marker of BCG-induced protection against TB. Our results show that BCG vaccination does provide substantial protection against challenge with Mycobacterium tuberculosis. However, despite previous compelling evidence that Th1 type immune responses are essential for TB immunity, the magnitude of in vitro purified protein derivative (PPD)-specific IFN-gamma production assessed during the course of TB infection did not correlate with protection. This emphasizes the need to identify further correlates of protection, in addition to IFN-gamma, to be used as markers of protective immunity against M. tuberculosis and/or to identify M. tuberculosis antigens inducing IFN-gamma that correlate with protective immunity.     

Tuberculin reactivity in a population of schoolchildren with high BCG vaccination coverage.

OBJECTIVE: To investigate the influence of BCG vaccination or revaccination on tuberculin skin test reactivity, in order to guide the correct interpretation of this test in a setting of high neonatal BCG vaccination coverage and an increasing BCG revaccination coverage at school age. METHODS: We conducted tuberculin skin testing and BCG scar reading in 1 148 children aged 7-14 years old in the city of Salvador, Bahia, Brazil. We measured the positive effect of the presence of one or two BCG scars on the proportion of tuberculin skin test results above different cut-off levels (induration sizes of > or = 5 mm, > or = 10 mm, and > or = 15 mm) and also using several ranges of induration size (0, 1-4, 5-9, 10-14, and > or = 15 mm). We also measured the effects that age, gender, and the school where the child was enrolled had on these proportions. RESULTS: The proportion of tuberculin results > or = 10 mm was 14.2% (95% confidence interval (CI) = 8.0%-20.3%) for children with no BCG scar, 21.3% (95% CI = 18.5%-24.1%) for children with one BCG scar, and 45.0% (95% CI = 32.0%-58.0%) for children with two BCG scars. There was evidence for an increasing positive effect of the presence of one and two BCG scars on the proportion of results > or = 5 mm and > or = 10 mm. Similarly, there was evidence for an increasing positive effect of the presence of one and two scars on the proportion of tuberculin skin test results in the ranges of 5-9 mm and of 10-14 mm. The BCG scar effect on the proportion of results > or = 5 mm and > or = 10 mm did not vary with age. There was no evidence for BCG effect on the results > or = 15 mm. CONCLUSIONS: In Brazilian schoolchildren, BCG-induced tuberculin reactivity is indistinguishable, for results under 15 mm, from reactivity induced by Mycobacterium tuberculosis infection. BCG revaccination at school age increases the degree of BCG-induced tuberculin reactivity found among schoolchildren. This information should be taken into account in tuberculin skin test surveys intended to estimate M. tuberculosis prevalence or to assess transmission patterns as well as in tuberculin skin testing of individuals used as an auxiliary tool in diagnosing tuberculosis. Taking this information into consideration is especially important when there is increasing BCG revaccination coverage.     

Assessment of safety and interferon gamma responses of Mycobacterium bovis BCG vaccine in goat kids and milking goats

Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed.BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination.BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible.     

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: Effect of reducing or eliminating BCG load on cell-mediated immunity

Oral delivery of lipid-encapsulated BCG represents an effective method for vaccination against tuberculosis (Tb). This method establishes live, replicating BCG in the lymphatic tissues of the alimentary tract, and promotes systemic-level cell-mediated immunity (CMI) and consequent protection against virulent mycobacterial challenge. Here, we investigated the effects of reducing or eliminating the BCG load on CMI responses in mice. Mice receiving a standard immunising dose of approximately 10(7) BCG (range, 1-5 x 10(7)) developed mycobacterial antigen-specific lymphocyte transformation (LT) responses, as well as interleukin-2 (IL-2) and gamma-interferon (IFN-gamma) secretion, at 8 and 18 weeks post-oral vaccination. These responses were concurrent with establishment of viable, replicating BCG in the alimentary tract lymphatics in over 90% of cases. Reducing the immunising dose by 10-fold reduced the magnitude of CMI, concurrent with abridged establishment of BCG in the lymphatics; reducing the dose 100-fold ablated BCG establishment, and diminished the production of IFN-gamma by antigen-stimulated lymphocytes of these mice. In mice immunised using the standard dose, replicating BCG were eliminated from the alimentary tract lymphatics using selective antibiotics. Interestingly, while lymphocyte transformation and interleukin-2 responses remained largely unaltered in these mice, levels of IFN-gamma produced by antigen-stimulated lymphocytes were shown to be reduced significantly. This study identifies a dosage threshold for effective oral vaccination using lipid-encapsulated BCG, and furthermore highlights the requirement of on-going intra-lymphatic BCG replication for the maintenance of strong IFN-gamma production, above other indicator CMI responses.     

Immune reactions of CD4- and CD8-positive T cell subpopulations in spleen and lymph nodes of guinea pigs after vaccination with Bacillus Calmette Guerin

Vaccination of guinea pigs with Mycobacterium bovis BCG confers partial resistance against infection with Mycobacterium tuberculosis. Induction of immunity is associated with a strong T cell response. The reactions of the cytotoxic and helper T lymphocyte subsets after BCG vaccination were analyzed by cytofluorometry and in functional tests. The relative number of CD8(+) T cells in the spleen increased substantially after injection of BCG. In vitro restimulation after immunization induced a strong proliferative response but no cytotoxic reactions of CD8(+) T cells against BCG-infected macrophages. A specific induction of IFN-gamma and RANTES mRNA was observed after vaccination particularly in CD8(+) but not in CD4(+) T cells of the lymph nodes.     

A novel live recombinant mycobacterial vaccine against bovine tuberculosis more potent than BCG

Mycobacterium bovis infection of cattle and other domesticated animals exacts a significant economic toll in both economically developing and industrialized countries. Vaccination of herds and/or wild animals that share their grazing land and serve as reservoirs of infection has been proposed as a strategy to combat bovine tuberculosis. However, the only currently available vaccine, M. bovis Bacille Calmette-Guerin (BCG), is not highly efficacious. Here we show that a live recombinant vaccine, rBCG30, which expresses large amounts of the Mycobacterium tuberculosis 30 kDa major secretory protein, is more efficacious against bovine tuberculosis than BCG in the highly demanding guinea pig model of pulmonary tuberculosis. Compared with the parental wild-type BCG strain, rBCG30 administered intradermally induced significantly greater cell-mediated and humoral immune responses against the 30 kDa protein, as determined by measuring cutaneous delayed-type hypersensitivity and antibody titers. As for potency, in three independent experiments, rBCG30 induced greater protective immunity than BCG against aerosol challenge with a highly virulent strain of M. bovis, reducing the burden of M. bovis by 0.4 +/- 0.2 log colony-forming units (CFU) in the lung (P < 0.05) and by 1.1 +/- 0.4 log CFU in the spleen (P = 0.0005) below the level in BCG-immunized animals. A recombinant BCG vaccine overexpressing the identical M. bovis 30 kDa protein, rBCG30Mb, also induced greater cell-mediated and humoral immunity against the 30 kDa protein than BCG and greater protective immunity against M. bovis challenge; however, its potency was not significantly different from rBCG30. As rBCG30 is significantly more potent than BCG against M. bovis challenge, it has potential as a vaccine against bovine tuberculosis in domesticated animals and in wild animal reservoirs.