Overview of vaccination trials for control of tuberculosis in cattle, wildlife and humans.

Vaccination is a key strategy for control of tuberculosis (TB), and considerable progress has been made in the past 5 years to develop improved vaccines for humans and animals, differentiate vaccinated animals from those infected with Mycobacterium bovis and deliver vaccines to wildlife. Studies have moved from testing vaccines in small animal models to clinical trials in humans and from experimental challenge studies in cattle and wildlife to evaluation of vaccines in the field. Candidate vaccines undergoing testing in humans include live mycobacterial vaccines to replace bacille Calmette Guérin (BCG), subunit vaccines (virus vector or protein) to boost BCG and therapeutic vaccines used as an adjunct to chemotherapy. In cattle, a number of diagnostic tests have been developed and successfully tested for differentiating infected from vaccinated animals, which will facilitate the use of BCG vaccine in cattle. Encouraging results have been obtained from recent field trials in cattle using BCG vaccine to protect against natural exposure to M. bovis. To date, no subunit TB vaccines have induced improved protection compared with that for BCG, but prime-boost combinations of BCG with DNA, protein or virus-vectored vaccines have induced better protection than BCG vaccine alone. Development of an oral bait BCG formulation has demonstrated the practicality of delivering TB vaccines to wildlife. Oral BCG preparations have induced protection against experimental challenge of M. bovis in possums, badgers, wild boar and white-tailed deer and against natural exposure to M. bovis in possums. Recent progress in TB vaccine development has provided much impetus for their future use.

Oral vaccination of brushtail possums with BCG: Investigation into factors that may influence vaccine efficacy and determination of duration of protection.

AIMS: To determine factors that may influence the efficacy of an oral pelleted vaccine containing Mycobacterium bovis bacille Calmette-Guérin (BCG) to induce protection of brushtail possums against tuberculosis. To determine the duration of protective immunity following oral administration of BCG. METHODS: In Study 1, a group of possums (n=7) was immunised by feeding 10 pellets containing dead Pasteur BCG, followed 15 weeks later with a single pellet of live Pasteur BCG. At that time, four other groups of possums (n=7 per group) were given a single pellet of live Pasteur BCG orally, a single pellet of live Danish BCG orally, 10 pellets of live Pasteur BCG orally, or a subcutaneous injection of live Pasteur BCG. For the oral pelleted vaccines, BCG was formulated into a lipid matrix, and each pellet contained approximately 107 colony forming units (cfu) of BCG, while the vaccine injected subcutaneously contained 106 cfu of BCG. A sixth, non-vaccinated, group (n=7) served as a control. All possums were challenged by the aerosol route with a low dose of virulent M. bovis 7 weeks after vaccination, and killed 7-8 weeks after challenge. Protection against challenge with M. bovis was assessed from pathological and bacteriological findings. In Study 2, lipid-formulated live Danish BCG was administered orally to three groups of possums (10-11 per group), and these possums were challenged with virulent M. bovis 8, 29 or 54 weeks later. The possums were killed 7 weeks after challenge, to assess protection in comparison to a non-vaccinated group. RESULTS: The results from Study 1 showed that vaccine efficacy was not adversely affected by feeding dead BCG prior to live BCG. Feeding 10 vaccine pellets induced a level of protection similar to feeding a single pellet. Protection was similar when feeding possums a single pellet containing the Pasteur or Danish strains of BCG. All vaccinated groups had significantly reduced pathological changes or bacterial counts when compared to the non-vaccinated group. In Study 2, oral administration of Danish BCG induced protection against challenge with M. bovis, which persisted for at least 54 weeks after vaccination. Some protection was observed in possums challenged 54 weeks after vaccination, but this protection was significantly less than that observed in groups vaccinated 29 or 8 weeks prior to challenge. There was a strong relationship between the proportion of animals producing positive lymphocyte proliferation responses to M. bovis antigens and protection against challenge with M. bovis. CONCLUSIONS: Factors considered potentially capable of interfering with vaccination, including feeding dead BCG to possums prior to feeding live BCG, feeding multiple doses of BCG at one time, and changing strains of BCG, were shown not to interfere with the acquisition of protective immune responses in possums. Protection against tuberculosis was undiminished up to 29 weeks after vaccination with BCG administered orally. It is concluded that vaccination of possums by feeding pellets containing BCG is a robust and efficient approach to enhance the resistance of these animals to tuberculosis.

Improving protective efficacy of BCG vaccination for wildlife against bovine tuberculosis.

Possums are a wildlife vector of bovine tuberculosis in New Zealand. Vaccination of possums with BCG is being considered as a measure to control the spread of bovine tuberculosis to cattle and deer. Delivery via oral bait is feasible but BCG is degraded in the stomach. The aim was to determine whether ranitidine (Zantac) would reduce gastric acidity and enhance the efficacy of intragastrically administered BCG. A dose of 75 mg reduced gastric acidity for at least 4 h. Thus, possums were vaccinated intragastrically with BCG after receiving 75 mg ranitidine or ranitidine or BCG alone, as controls, before challenge with virulent Mycobacterium bovis. Proliferative responses of blood lymphocytes to M. bovis antigens after vaccination were significantly higher in possums given ranitidine/BCG compared to controls and seven weeks after challenge they had significantly lower lung weights and spleen bacterial counts than ranitidine alone controls. Vaccination with BCG alone only gave a reduction in loss in body weight. Agents that reduce gastric acidity may be useful in formulating BCG for oral bait delivery to wildlife for vaccination against bovine tuberculosis.

Vaccination with DNA vaccines encoding MPB70 or MPB83 or a MPB70 DNA prime-protein boost does not protect cattle against bovine tuberculosis.

SETTING: Bovine tuberculosis is a problem in a number of countries and protection of cattle by vaccination could be an important control strategy. OBJECTIVES: To determine the ability of DNA vaccines, which express the mycobacterial antigens MPB83 and MPB70 and a DNA prime-protein boost strategy to stimulate immune responses in cattle and protect against bovine tuberculosis. DESIGN: Groups of cattle (n=10) were vaccinated with MPB83 DNA, MPB70 DNA, or MPB70 DNA followed by MPB70 protein or injected with BCG or control plasmid DNA. Animals were challenged intratracheally with virulent Mycobacterium bovis at 13 weeks and protection assessed 17 weeks later at postmortem. RESULTS: In contrast to the strong cellular immune responses induced by BCG, the DNA vaccines induced minimal interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) responses. Cattle primed with MPB70 DNA and boosted with MPB70 protein induced a strong antibody response and a weak IFN-gamma response. BCG gave significant reduction in four pathological parameters of disease while the DNA vaccines and MPB70 DNA/protein did not protect animals against challenge with M. bovis. Moreover, cattle vaccinated with MPB70 DNA/protein had a significantly higher proportion of animals with severe lung lesions (>100 lesions) than the MPB70 DNA alone or the control group. Increased bovine PPD-specific IL-4 mRNA expression in cattle, post-challenge, correlated with the presence of tuberculous lung lesions. CONCLUSION: Vaccination of calves with MPB70 or MPB83 DNA vaccines or with a more immunogenic MPB70 DNA prime-protein boost strategy did not induce protection against bovine tuberculosis.

Revaccination of neonatal calves with Mycobacterium bovis BCG reduces the level of protection against bovine tuberculosis induced by a single vaccination.

Cattle may provide a suitable model for testing ways of improving tuberculosis vaccine efficacy in human infants. A vaccination and challenge study was undertaken in calves to determine the optimal time to vaccinate neonatal animals with Mycobacterium bovis bacillus Calmette-Guérin (BCG) for protection against tuberculosis and to determine whether revaccination with BCG was beneficial. Calves (10 per group) were vaccinated with BCG within 8 h of birth or at 6 weeks of age, when immune responses to antigens of environmental mycobacteria were detectable, or vaccinated at birth and revaccinated at 6 weeks. A control group was not vaccinated. BCG vaccination at birth induced strong antigen-specific gamma interferon (IFN-gamma) and interleukin-2 (IL-2) responses and antigen-specific activation in CD4(+), CD8(+), and WC1(+) gammadelta T-cell subsets from blood. The proportions of animals per group with macroscopic tuberculous lesions after challenge were 0/10 for BCG at birth, 1/9 for BCG at 6 weeks, 4/10 for the revaccinated group, and 10/10 for the nonvaccinated group. There was no significant difference in the levels of protection between groups vaccinated at birth or at 6 weeks, while animals vaccinated both at birth and at 6 weeks had significantly less protection than those vaccinated only at birth. The revaccinated calves that subsequently developed tuberculous lesions had significantly stronger IFN-gamma and IL-2 responses to bovine purified protein derivative after the BCG booster than those in the same group that did not develop lesions. The results indicated that BCG vaccination at birth induced a high level of immunity and that the sensitization of very young animals to antigens of environmental mycobacteria by 6 weeks of age did not affect the effectiveness of BCG. However, BCG revaccination of these young animals was contraindicated.

Increased protection against bovine tuberculosis in the brushtail possum (Trichosurus vulpecula) when BCG is administered with killed Mycobacterium vaccae.

SETTING: The Australian brushtail possum is the major wildlife reservoir for Mycobacterium bovis infection in New Zealand. Development of an effective tuberculosis vaccine for possums will reduce the spread of infection to cattle and farmed deer. OBJECTIVES: To determine whether killed M. vaccae can improve the efficacy of vaccination with M. bovis bacillus Calmette Guerin (BCG) against bovine tuberculosis in the possum. DESIGN: Groups of possums (n=6-8) were vaccinated via intranasal and intraconjunctival routes with BCG alone or BCG in combination with heat-killed M. vaccae. Controls were non-vaccinated or vaccinated with heat-killed M. vaccae alone. After challenge with virulent M. bovis, protection was assessed by a reduction in loss of body weight and bacterial counts in lungs and spleens. Blood lymphocyte proliferative responses to M. bovis purified protein derivative were monitored throughout. RESULTS: The earliest lymphocyte responses following vaccination were from animals inoculated with BCG plus 100 microg heat-killed M. vaccae. Loss of body weight was significantly reduced in all BCG-vaccinated groups compared control groups. Spleen bacterial counts were significantly lower in animals vaccinated with M. vaccae plus BCG compared to the non-vaccinated group. Furthermore, vaccination with 100 microg M. vaccae plus BCG significantly reduced spleen bacterial counts compared to vaccination with BCG alone. CONCLUSION: The possum infection model is one of the first to show that novel vaccine strategies may offer better protection against tuberculosis than BCG alone.

Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils.

The physiological effects of intramammary infusions of recombinant bovine cytokines in six lactating dairy cows on the quality and yield of milk and the bactericidal activity of milk neutrophils were investigated. Recombinant bovine interleukin-2 (rboIL-2) and interferon-gamma (rboIFN-gamma) were produced in the yeast Pichia pastoris. Two animals were given rboIL-2 (2 x 10(5) units) in two quarters, two animals were given rboIFN-gamma (6.5 x 10(5) units) in two quarters, and the other two cows received a dose of rboIL-2 in one quarter and rboIFN-gamma in a second quarter. In addition, each animal was given phosphate-buffered saline (PBS) in the other two quarters as a control. Somatic cell counts and conductivity of the fore milk were monitored before and after infusion. Neutrophils were isolated from quarter milk samples 36 h after infusion of cytokine or PBS and their bactericidal activities against Staphylococcus aureus were measured in vitro with a colorimetric assay. Quarters infused with rboIL-2 or rboIFN-gamma showed significant but transitory increases in both milk somatic cell counts and conductivity when compared with preinfusion values and with control quarters. There were minimal effects on daily milk yield. Neutrophils isolated from milk from quarters infused with rboIL-2 showed enhanced bactericidal activity against Staph. aureus. The bacterial killing from rboIL-2 treated quarters was significantly greater, with a mean of 63.5% compared with a mean of 5.4% for neutrophils taken from uninfected quarters to which PBS had been administered. The bactericidal activities for quarters treated with rboIFN-gamma and infected quarters treated with PBS were 15.0 and 30.0% respectively. The results indicate that intramammary infusions of rboIL-2 and rboIFN-gamma to lactating cows are well tolerated, and that rboIL-2 can activate milk neutrophils and augment their bactericidal activity.

Physiological effects and adjuvanticity of recombinant brushtail possum TNF-alpha.

The present paper describes the physiological properties of recombinant possum TNF-alpha and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant possum TNF-alpha was produced in the yeast Pichia pastoris. The recombinant cytokine was secreted into the culture medium and purified by gel filtration. Possum TNF-alpha produced in this expression system was N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, the possum TNF-alpha had lower specific activity compared to human TNF-alpha, while in a possum-specific assay, possum TNF-alpha enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-alpha. The physiological effect of the recombinant possum TNF-alpha was investigated in groups of possums administered doses of 6, 30 or 150 micrograms of cytokine. For each dose, TNF-alpha caused profound effects on the numbers of circulating leucocytes characterized by a three-to-four-fold increase in neutrophil numbers at 6-24 h after injection and an initial sharp decrease in lymphocyte numbers. The efficacy of TNF-alpha as an immunological adjuvant was determined in possums administered KLH (125 micrograms) in an aqueous or Al(OH)3-based formulation with or without added recombinant TNF-alpha (150 micrograms). Serum antibody responses to KLH were monitored by ELISA. The TNF-alpha stimulated two-fold and four-fold increases in antibody levels in aqueous and Al(OH)3-based vaccine formulations, respectively. The strongest antibody responses were observed in the group of possums that received KLH formulated in Al(OH)3 with addition of TNF-alpha.

Molecular cloning and physiological effects of brushtail possum interleukin-1beta.

Interleukin-1beta (IL-1beta) was isolated from LPS-stimulated brushtail possum alveolar macrophages using PCR primers based on conserved regions of mammalian IL-1beta. The complete cDNA was cloned by 5' and 3' rapid amplification of cDNA ends (RACE). The predicted protein of 269 amino acids shared 4346% identity with several mammalian IL-1beta proteins. Constructs were made to express the mature IL-1beta in Escherichia coli and two recombinant IL-1beta proteins, rpIL-1beta1 and rpIL-1beta2, which differed in length by four amino acids at the N-terminus, were produced. Both proteins induced a weak proliferative response in a possum thymocyte assay. Possums injected intravenously with 100 microg of rpIL-1beta1 or rpIL-1beta2 showed profound changes in body temperature and numbers of circulating leukocytes. A sharp decrease in temperature occurred within 2 h of administration followed by an elevation of temperature peaking at 24 h. The smaller rpIL-1beta1 protein had a greater effect on temperature than rpIL-1beta2. Both rpIL-1beta proteins caused a marked decrease in number of neutrophils and lymphocytes at 2-6 h after injection. At 24 h after injection, neutrophil and lymphocyte numbers were elevated 6.0-fold and 2.6-fold, respectively in the possums injected with rpIL-1beta1 and 3.9-fold and 1.5-fold, respectively in the possums injected with rpIL-1beta2. Fibrinogen levels were elevated at 24 and 72 h after injection with both proteins. In comparison, neither recombinant bovine IL-1beta (rbIL-1beta) nor PBS had significant effects on body temperature or blood haematology. The studies have shown that the two recombinant forms of IL-1beta were biologically active in possums and that the IL-1beta with four fewer amino acids at the N-terminus was the more active.

Differentiation between Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle by using recombinant mycobacterial antigens.

Tuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection with Mycobacterium tuberculosis or Mycobacterium bovis and vaccination with M. bovis BCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins of M. bovis-MPB59, MPB64, MPB70, and ESAT-6-were tested in a whole-blood gamma interferon (IFN-gamma) assay for differentiation between cattle vaccinated with BCG and those experimentally infected with M. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected with M. bovis in BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-gamma responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which were M. bovis culture positive following challenge produced IFN-gamma responses to PPDB and ESAT-6 which were significantly stronger than those observed in the corresponding M. bovis culture-negative animals. IFN-gamma responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-gamma assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.

Intraduodenal vaccination of brushtail possums with bacille Calmette-Guérin enhances immune responses and protection against Mycobacterium bovis infection.

SETTING: An effective oral bacille Calmette-Guérin (BCG) vaccine would have advantages for use in humans and as an oral bait vaccine for protecting wild-life against bovine tuberculosis. OBJECTIVE: To compare the level of protection against tuberculosis in intraduodenally BCG-vaccinated possums with those vaccinated intragastrically in order to determine whether degradation of BCG in the stomach lowers vaccine efficacy. DESIGN: Three groups of five possums were vaccinated with BCG by the intraduodenal, intragastric or subcutaneous routes, with a fourth group serving as unvaccinated controls. The animals were later challenged intratracheally with a low dose of virulent Mycobacterium bovis. RESULTS: Possums vaccinated intraduodenally with BCG had significantly greater lymphocyte blastogenic responses to bovine purified protein derivative (PPD) and lower lung bacterial counts in comparison with intragastrically vaccinated animals. In comparison with unvaccinated animals, all of the BCG-vaccinated groups had significant protection against M. bovis infection as assessed by changes in body weight, lung weight and reduction in numbers of mycobacteria and granulomas in the spleen. CONCLUSION: The enhanced immune responses and protection against bovine tuberculosis observed in the intraduodenally BCG-vaccinated possums indicated that if BCG vaccine is protected from degradation in the stomach its efficacy should improve.