6 mice

ARTICLE IN PRESS Tuberculosis (2005) 85, 107–114

Tuberculosis http://intl.elsevierhealth.com/journals/tube

Nasal boost with adjuvanted heat-killed BCG or arabinomannan–protein conjugate improves primary BCG-induced protection in C57BL/6 mice M. Hailea,b, B. Hamasura, T. Jaxmara,b, D. Gavier-Widenc, ¨dere, G. Ka ¨lleniusa,b, M.A. Chambersd, B. Sancheza, U. Schro S.B. Svensona,f, A. Pawlowskia, a

Swedish Institute for Infectious Disease Control, Department of Bacteriology, S-17182 Solna, Sweden Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden c National Veterinary Institute, S-75189 Uppsala, Sweden d TB Research Group, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK e Eurocine AB, Karolinska Science Park, S-17177 Stockholm, Sweden f Department of Bacteriology, Swedish University for Agricultural Sciences, P.O. Box 583, S-75123 Uppsala, Sweden b

Accepted 27 September 2004

KEYWORDS Tuberculosis vaccine; BCG; Booster vaccination

Summary Today it is generally accepted that the Bacillus Calmette–Gue´rin (BCG) vaccine protects against childhood tuberculosis (TB) but this immunity wanes with age, resulting in insufficient protection against adult pulmonary TB. Hence, one possible strategy to improve the protective efficacy of the BCG vaccine would be to boost in adulthood. In this study, using the mouse model, we evaluated the ability of two new TB vaccine candidates, heat-killed BCG (H-kBCG) and arabinomannan–tetanus toxoid conjugate (AM–TT), given intransally in a novel Eurocinet adjuvant, to boost a primary BCG-induced immune response and to improve protection. Young C57BL/6 mice were vaccinated with conventional BCG and, 6 months later, boosted intranasally with adjuvanted H-kBCG or AM–TT, or subcutaneously with BCG. Ten weeks after the booster, mice were challenged intravenously with M. tuberculosis (Mtb) strain H37Rv. In spleens, there was a significant reduction of cfu counts in mice boosted with either H-kBCG or AM–TT vaccines compared to the non-boosted BCGvaccinated mice. None of the boosting regimens significantly reduced bacterial loads in lungs, compared to non-boosted BCG vaccination. However, the extent of

Corresponding author. Tel.: +46 8 457 2441; fax: +46 8 5088 4591.

E-mail address: [email protected] (A. Pawlowski). 1472-9792/$ - see front matter & 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2004.09.013

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M. Haile et al. granulomatous inflammation was significantly reduced in the lungs of mice that received two of the booster vaccines (AM–TT and conventional BCG), as compared with sham-vaccinated mice. All boosted groups, except for mice boosted with the AM–TT vaccine, responded with a proliferation of spleen T cells and gamma interferon production comparable to that induced by a single BCG vaccination. & 2004 Elsevier Ltd. All rights reserved.

Introduction The live attenuated Bacillus Calmette–Gue´rin (BCG) vaccine has been used as the only vaccine against tuberculosis (TB) for over 80 years. Today, more than 100 million children are vaccinated with BCG every year. However, the efficacy of BCG has been inconsistent, with protection varying from zero to 80% in several controlled trials performed in different countries.1,2 Today, there is a general consensus that BCG vaccination significantly protects against childhood TB but that the protection wanes with age. Hence, at an early adult age, when the risk of being exposed to Mtb is very high, the immunity induced by a prior BCG vaccination appears to, more or less, have vanished. The protection afforded by BCG vaccination varies depending on the patient’s immune status, prior exposure to environmental mycobacteria, presence of latent infection, and the time between BCG vaccination and infection.2 The varying efficacy of the BCG vaccine and, in particular, the low efficacy against pulmonary TB in adults has led to an increased interest in the development of new, more efficient TB vaccines. A number of such TB vaccine candidates have been developed but, so far, only very few of them have attained a protective efficacy equivalent to that conferred by BCG in preclinical experiments in laboratory animals.3 Even though BCG may not protect adults against pulmonary TB, it still confers good protection against disseminated TB and tuberculous meningitis in the young child. In addition, BCG provides efficient crossprotection against leprosy.4 These facts, together with the current lack of alternative vaccines, have resulted in the WHO recommending the continued use of the BCG vaccine. This policy is likely to be in practice still for many years to come, in particular in low-income countries with high TB prevalence. One possible explanation for the failure of BCG to protect against adult pulmonary TB is that BCGinduced memory immunity, although rather robust in the child, tends to wane as the individual reaches adulthood.5,6 An early anamnestic vaccination of BCG-primed individuals could possibly boost the waning memory responses and hence restore protective immunity into adulthood.

A second vaccination of adults with BCG is currently not recommended by the WHO7 and there are reports showing no decrease in TB incidence in high prevalence countries where BCG boosting has been used.4,8,9 However, the definitive evidence of the presence or the absence of eventual benefits from boosting BCG is still lacking. It would be of particular interest to explore the ability of new subunit vaccine candidates to boost a primary BCG vaccination. If proven effective in restoring BCG-induced protection in adults, such vaccines could rapidly supplement BCG vaccination in high prevalence countries, in concordance with the current childhood BCG vaccination programmes. Recently, we have reported that two new vaccine candidates, heat-killed BCG(H-kBCG) and mycobacterial arabinomannan–tetanus toxoid conjugate (AM–TT), formulated in a novel adjuvant, confer protection to young mice when administered twice as a subcutaneous priming followed by an intranasal booster.10,11 In the present study, we explore the ability of intranasal booster with H-kBCG and AM–TT, administered at adulthood, to intensify the immune responses induced by primary BCG vaccination and to confer increased protection.

Materials and methods Mice Eight to ten week old female C57BL/6 mice (Anticimex, Sweden) were maintained under BSL3 conditions throughout the study period.

Bacteria Mycobacterium tuberculosis (Mtb) strain H37Rv (American Type Culture Collection) was passaged in mice, propagated in Middlebrook 7H9 medium, and frozen at
70 1C in Middlebrook 7H9 medium containing 10% glycerol at approximately 106 cfu/ ml. Mycobacterium bovis BCG (BCG Vaccine SSI, batch 9773 S/99-01) was obtained from the Statens Serum Institute, Copenhagen, Denmark.

ARTICLE IN PRESS Nasal boost with adjuvanted heat-killed BCG or arabinomannan

Preparation and formulation of antigens in Eurocinet L3 adjuvant (L3) H-kBCG and AM–TT were prepared and formulated in L3 adjuvant emulsion as previously described.10–12 The concentration of antigens in the formulations was 2  108 cfu/ml for H-kBCG and 50 mg=ml for AM–TT (on the basis of carbohydrate content).

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at 37 1C. Only the right lung from each mouse was used for viable counts as described above; the left lung was fixed in 10% saline-buffered formalin, embedded in paraffin, cut into sections, and stained with hematoxylin–eosin for light microscopy. Three sections were prepared from each lung, one from each lobe, cut at the same position in all animals. The variables assessed in the sections were: area occupied by granulomatous lesions (granuloma area, %), area occupied by

Vaccinations and immunogenicity assays Mice were vaccinated subcutaneously (s.c.) in the flank with 100 ml of conventional live BCG (Danish; 5  105 cfu) and 26 weeks later were boosted intranasally (i.n.) with 10 ml of H-kBCG in L3 (2  106 cfu) or AM–TT in L3 ð5 mgÞ; administered in 5 ml portions to each nostril. Control groups included non-boosted mice (single BCG only), mice boosted with live BCG, and naive mice shamvaccinated with adjuvant alone. Proliferation of T cells was determined in spleen cell cultures stimulated with mycobacterial antigens (H37Rv extract or PPD) or concanavalin A as control. Mouse splenocytes were isolated and cultured as described earlier.10 The cultures were carried out for 4 days in the presence of H37Rv extract ð10 mg=mlÞ or PPD (Statens Serum Institute, Copenhagen, Denmark, 10 mg=ml) or for 3 days with concanavalin A ð5 mg=mlÞ: 3H-thymidine incorporation was determined during the last 18 h of culture. For IFNg production assay mouse spleen cells were cultured at a concentration of 2  106 cells/ well in 24-well plates. After 1, 2, and 3 days of culture in the presence of concanavalin A or mycobacterial antigens (H37Rv extract or PPD) culture media were collected and frozen, awaiting assay. IFNg ELISA was performed, as previously described,10 using monoclonal capture-polyclonal detection antibody pairs from R&D Systems (United Kingdom), in accordance with the manufacturer’s recommendations.

Challenge infections and evaluation of bacterial burden and pathology in organs At 10 weeks after booster vaccinations, the mice were challenged intravenously (i.v.) with 3  105 cfu of Mtb H37Rv. Eight weeks following challenge infections, groups of 5–7 mice were sacrificed and their lungs and spleens were harvested, homogenized, and plated at 10-fold dilutions on Middlebrook 7H11 (Difco) agar plates supplemented with 2 mg=ml of TCH (thiophen-2-carboxylic acid hydrazide). Colonies were counted after 3–4 week incubation

Fig. 1 Bacterial loads in spleens (upper panels) and lungs (lower panels) of C57BL/6 mice vaccinated parenterally with conventional BCG and boosted nasally 26 weeks later with H-kBCG or AM–TT conjugate, both in Eurocinet L3 adjuvant. Eight weeks after booster vaccination the mice were challenged intravenously with 3  105 cfu of M. tuberculosis H37Rv strain. Mice were sacrificed 8 weeks post-challenge. Serially diluted organ homogenates were plated onto 7H11 Middlebrook agar and the colonies were enumerated 3 weeks later. Results are expressed as mean log 10(cfu)  SEM of 5–7 animals/ group. Asterisks indicate a significant difference in bacterial load by Student’s t-test between boosted and non-boosted BCG-vaccinated group. po0:01: ‘‘Sham’’, sham-vaccination with adjuvant alone; ‘‘No boost’’, BCG vaccination only; ‘‘BCG’’, BCG vaccination followed by BCG boost; ‘‘H-kBCG’’, BCG vaccination followed by HkBCG boost; ‘‘AM–TT’’, BCG vaccination followed by AM–TT boost.

ARTICLE IN PRESS 110 healthy tissue (%), amounts of lymphocytes in lesions (score 1–5), and amounts of lymphocytes in perivascular cuffs (score 1–5). The analysis was performed by an experienced veterinary pathologist who had no prior knowledge of the identity of the sections.

Statistics T-cell proliferation and cfu data were analyzed using the unpaired two-sided Student’s t-test for comparison of means. In the case of the cfu data, the p value for significance was reduced to po0:01 to take account of the number of pair-wise tests performed. Pathology scores were analyzed using non-parametric ANOVA (Kruskal–Wallis Test) with Dunn’s Multiple Comparison post-test.

Results and discussion Recently, we have shown that two new vaccine candidates, H-kBCG and AM–TT, when given i.n., are protective in the mouse model of Mtb infection and disease.10,11 Here, we have attempted to stimulate the waning immunity induced by BCG in young mice, by boosting the adult mice intranasally with these new vaccine candidates. In spleens 8 weeks post-challenge, the bacterial counts were significantly reduced in mice boosted

M. Haile et al. with H-kBCG (0.4 log unit, p ¼ 0:004) and in mice boosted with AM–TT (0.5 log unit, p ¼ 0:003), but not in mice boosted with live BCG (0.3 log unit, p ¼ 0:17), compared to the non-boosted BCG group (Fig. 1). Analysis of the bacterial load in the lungs revealed that both the non-boosted BCG group and all boosted groups, including conventional live BCG boost, had similar bacterial counts: 1–1.2 log unit lower than in the sham-vaccinated group ðpo0:0004Þ: The mean lung cfu value was lowest in mice boosted with the AM–TT vaccine, compared to all other groups of mice (Fig. 1). Histopathological examination was performed on the lungs of mice euthanized 8 weeks after challenge. In sham-vaccinated mice, the greater part of the pulmonary parenchyma was occupied by granulomatous inflammation (median area ¼ 58%). All test vaccination regimens reduced the extent of granulomatous lesions, compared with that of the sham-vaccination (Fig. 2). The greatest, and highly significant, reduction of the granuloma area was found in mice boosted with AM–TT (44% reduction, po0:01) (Fig. 3), followed by a reduction found in the group boosted with live BCG (40% reduction, po0:01). In all boosted groups, the median percentage of granulomatous inflammation was similar to that found in the non-boosted BCG group. There was significant positive correlation between the percentage of granulomatous inflammation and cfu titre in the lung (r ¼ 0:4068; 95% CI ¼ 0:1082
0:6381; two-tailed p ¼ 0:0075; by Spearman Rank),

Fig. 2 Effect of different booster regimens on the extent of granulomatous inflammation in lungs of C57BL/6 mice 8 weeks after intravenous challenge with 3  105 cfu of M. tuberculosis H37Rv strain. The median percentage area of section occupied by granulomatous inflammation is shown as a line. The box defines the 75th and 25th percentiles and the whiskers define the maximum and minimum values. Groups significantly different from sham-vaccinated control are labeled with asterisk, po0:01: For group designation see Fig. 1.

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Fig. 3 Representative micrographs of mouse lung illustrating the reduction of granulomatous inflammation in a mouse boosted with AM–TT (A, B), compared with a mouse vaccinated once with live BCG (C, D) or sham-vaccinated (E, F). Each low-magnification micrograph exhibits a percentage granulomatous inflammation close to the median value for its group: 12% (A); 25% (C); 65% (E). A detail of the inflammatory reaction in each section is shown in panels B, D, and F at higher magnification. For group designation see Fig. 1.

supporting recent observations with different Mtb genotypes in BALB/c mice.13,14 Evaluation of extension of lymphocytic infiltration in the granulomas and of the number of lymphocytes or size of the perivascular lymphocytic cuffs did not show significant differences among the groups. The apparent lack of effect of booster vaccination on bacterial loads in lungs and only minor effect on lung pathology could be due to the use of the intravenous route of challenge, leading to the distribution and deposition of bacteria in lungs

through mechanisms that differ considerably from those operating during natural airborne infection. This could possibly make the local immune response in the lungs less effective. Another conceivable reason could be too short a time between primary vaccination and subsequent booster and challenge. This time interval was earlier reported to be critically important for boosting the conventional BCG vaccine.15 Accordingly, we have found here that mice challenged 8.5 months post-BCG vaccination still had high level of protection in lungs (over

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Fig. 4 Proliferation of splenocytes from C57BL/6 mice vaccinated parenterally with conventional BCG and boosted nasally 26 weeks later with H-kBCG or AM–TT conjugate, both in Eurocinet L3 adjuvant. Spleen cells were isolated at week 30 and restimulated with PPD (A) or H37Rv extract (B) in 3 day culture. 3[H]Td incorporation was measured and splenocyte proliferation was expressed as proliferation index (incorporation in stimulated sample divided by incorporation in non-stimulated control). Mean values of 4 animals  SEM are shown. For group designation see Fig. 1.

1 log unit cfu lower than sham-vaccinated counterparts). In contrast, mice challenged 20 months post-BCG vaccination had the lung protection considerably reduced (0.47 log unit) as reported by Brooks et al.15 It is possible that in our experimental setting the strong lung immunity induced by the primary BCG vaccination obscured the possible positive effects of the booster vaccinations in the lungs. Mice boosted with live BCG and mice boosted with H-kBCG showed antigen-specific proliferation of spleen T cells similar to that found in nonboosted BCG mice (Fig. 4). Mice boosted with the AM–TT vaccine had significantly lower T-cell proliferation which was equivalent to the level of sham-vaccinated controls (2.6-fold reduction compared to non-boosted BCG, p ¼ 0:025). Despite inhibited proliferation, this group of mice responded to restimulation by producing levels of IFNg that were the highest among all boosted groups (mean value of 3300 pg/ml, H37Rv antigen, day 2), exceeding that induced by live BCG boost (1300 pg/ml) as well as the single parenteral BCG

M. Haile et al. vaccination (950 pg/ml) (Fig. 5). This low proliferative response associated with high IFNg production is reminiscent of findings with the lung T cells of the TB-resistant A/Sn mouse strain16 and of the so-called ‘effector memory’ cell population, which recently has been shown to correlate with resistance of C57BL/6 mice to aerosol challenge with M. tuberculosis.17 Further clarification could be obtained by examining these splenocyte cultures for co-expression of markers such as CD44, CD45, CCR7, and CD62L.18 Robust IFNg responses are considered a prerequisite of an efficient protective immunity against Mtb infections. The predictive value of IFNg levels for the outcome of the disease is, however, controversial. Some authors reported discrepancy between IFNg production and mycobacterial immunity in clinical settings.19 Recently, a number of animal studies have reported no correlation, or even an inverse relationship, between both post-vaccination and post-challenge IFNg levels and the extent of the mycobacteria-induced organ pathology.20–23 We have found here that the protective effect of booster vaccination in spleens generally did not correlate with the IFNg levels produced by restimulated splenocytes in vitro. However, the markedly high levels of IFNg noted in the mice boosted with AM–TT could have been associated with the relatively better protection found in this group. The value of additional BCG vaccination is controversial. It has been reported that revaccination with BCG does not induce enhanced protection against TB in humans,4,8,9 although increased protection against leprosy has been noted.4 It has also been difficult to unequivocally assess the value of revaccination with BCG in laboratory animals. In some studies, parenteral revaccination of mice with BCG was reported to improve protection in terms of reduced bacterial loads.15,24 In contrast, a recent study in cattle revealed that calves revaccinated with BCG had significantly more bacilli in lungs and developed more extensive lung pathology than their counterparts who received BCG only once.21 In the present study, we have not found any protective effect of live BCG booster in terms of bacterial loads in lungs and spleens. However, there was some improvement in lung pathology after revaccination with live BCG, compared to the single vaccination regimen. None of the boosting regimens described here was able to effectively increase lung protection of adult animals against Mtb infection beyond that induced by early BCG vaccination. This observation made in experimental setting reflects difficulties in boosting BCG childhood vaccination in adulthood. Nevertheless, the significant reduction of granulo-

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Fig. 5 IFNg production in splenocytes from C57BL/6 mice vaccinated parenterally with conventional BCG and boosted nasally 26 weeks later with H-kBCG or AM–TT conjugate, both in Eurocinet L3 adjuvant. Spleen cells were isolated at week 30 and restimulated with 5 mg=ml of concanavalin A (upper panel), 10 mg=ml of PPD (middle panel) or 10 mg=ml of H37Rv extract (lower panel) in 3 day culture. Media were collected after 24, 48, and 72 h and IFNg was assayed by sandwich ELISA using mouse IFNg specific antibody pairs from R&D. IFNg concentrations were determined from calibration curve for mouse IFNg (R&D). Individual IFNg production kinetics for each of 4 animals/group is shown. For group designation see Fig. 1.

matous lesions in the lungs (AM–TT and live BCG) and decrease of bacterial load in spleens (H-kBCG and AM–TT) of the boosted mice is encouraging and merits further exploration, especially using aerogenic challenge and a modified mouse model using a prolonged time before boosting and challenge.15 Work along these lines is under way.

CRAFT Project QLK2-CT-2002-71587), the Swedish Medical Research Council (Grant K99-06X), King Oscar II Jubilee Foundation, the Swedish Heart–Lung Association, and the Foundation for Strategic Research (Infection and Vaccinology).

References Acknowledgements This study was supported by grants from the European Community (TS-CT94-0001, BMH4-CT97-2671 and

1. Colditz GA, Brewer TF, Berkey CS, Wilson ME, Burdick E, Fineberg HV, et al. Efficacy of BCG vaccine in the prevention of tuberculosis: meta-analysis of the published literature. JAMA 1994;271:698–702. 2. Fine PEM. Variation in protection by BCG: implications of and for heterologous immunity. Lancet 1995;346:1339–45.

ARTICLE IN PRESS 114 3. Orme IM, McMurray DN, Belisle JT. Tuberculosis vaccine development: recent progress. Trends Microbiol 2001;9: 115–8. 4. Karonga Prevention Trial Group. Randomised controlled trial of single BCG, repeated BCG, or combined BCG and killed Mycobacterium leprae vaccine for the prevention of leprosy and tuberculosis in Malawi. Lancet 1996; 348:17–24. 5. Aydinlioglu H, Caglayan S, Kansoy S, Yaprak I, Seckin E, Bakiler AR, et al. The decline of BCG immunity after neonatal vaccination: what about revaccination at one year? Paediatr Perinat Epidemiol 1993;7:334–8. 6. Sterne JA, Rodrigues LC, Guedes IN. Does the efficacy of BCG decline with time since vaccination? Int J Tuberc Lung Dis 1998;2:200–7. 7. WHO Global Tuberculosis Programme on Vaccines. Statement on BCG revaccination for the prevention of tuberculosis. WHO Weekly Epidem Rec 1995; 70:229–31. 8. Leung CC, Tam CM, Chan SL, Chan-Yeung M, Chan CK, Chang KC. Efficacy of the BCG revaccination programme in a cohort given BCG vaccination at birth in Hong Kong. Int J Tuberc Lung Dis 2001;5:717–23. 9. Sepulveda RL, Parcha C, Sorenson RU. Case-control study of the efficacy of BCG immunization against pulmonary tuberculosis in young adults in Santiago. Chile Tuberc Lung Dis 1992;73:372–7. 10. Haile M, Schro ¨der U, Hamasur B, Pawlowski A, Jaxmar T, Ka ¨llenius G, et al. Immunization with heat killed Mycobacterium bovis Bacille Calmette–Gue´rin (BCG) in Eurocinet L3 adjuvant protects against tuberculosis. Vaccine 2004;22: 1498–508. 11. Hamasur B, Haile M, Pawlowski A, Schro ¨der U, Williams A, Hatch G, et al. Mycobacterium tuberculosis arabinomannan–protein conjugates protect against tuberculosis. Vaccine 2003;21:4081–93. 12. Hamasur B, Kallenius G, Svenson SB. Synthesis and immunologic characterization of Mycobacterium tuberculosis lipoarabinomannan specific oligosaccharide–protein conjugates. Vaccine 1999;17:2853–61. 13. Lopez B, Aguilar D, Orozco H, Burger M, Espitia C, Ritacco V, et al. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 2003;133:30–7. 14. Dormans J, Burger M, Aguilar D, Hernandez-Pando R, Kremer K, Roholl P, et al. Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in a BALB/c mouse model. Clin Exp Immunol 2004;137:460–8. 15. Brooks JV, Framk AA, Keen MA, Bellisle JT, Orme IM. Boosting vaccine for tuberculosis. Infect Immun 2001;69:2714–7.

M. Haile et al. 16. Lyadova IV, Eruslanov EB, Khaidukov SV, Yeremeev VV, Majorov KB, Pichugin AV, et al. Comparative analysis of T lymphocytes recovered from the lungs of mice genetically susceptible, resistant, and hyperresistant to Mycobacterium tuberculosis-triggered disease. J Immunol 2000;165:5921–31. 17. Turner OC, Keefe RG, Sugawara I, Yamada H, Orme IM. SWR mice are highly susceptible to pulmonary infection with Mycobacterium tuberculosis. Infect Immun 2003;71: 5266–72. 18. Bjorkdahl O, Barber KA, Brett SJ, Daly MG, Plumpton C, Elshourbagy NA, et al. Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues. Immunology 2003;110:170–9. 19. Hoft DE, Worku S, Kampmann B, Whalen CC, Ellner JJ, Hirsch CS, et al. Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. J Infect Dis 2002;186: 1448–57. 20. Buddle BM, Wedlock DN, Parlane NA, Corner LAL, de Lisle GW, Skinner MA. Revaccination of neonatal calves with Mycobacterium bovis BCG reduces the level of protection against bovine tuberculosis induced by a single vaccination. Infect Immun 2003;71:6411–9. 21. Skinner MA, Ramsay AJ, Buchan GS, Keen DL, Ranasinghe C, Slobbe L, et al. A DNA prime-live vaccine boost strategy in mice can augment IFN-g responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis. Immunology 2003;108:548–55. 22. Langermans JAM, Andersen P, van Soolingen D, Vervenne RAW, Frost PA, van der Laan T, et al. Divergent effect of Bacillus Calmette–Gue ´rin (BCG) vaccination on Mycobacterium tuberculosis infection in highly related macaque species: implications for primate models in tuberculosis vaccine research. Proc Natl Acad Sci, USA 2001;98: 11497–502. 23. Vordermeier HM, Chambers MA, Cockle PJ, Whelan AO, Simmons J, Hewinson RG. Correlation of ESAT-6-specific gamma interferon production with pathology in cattle following Mycobacterium bovis BCG vaccination against experimental bovine tuberculosis. Infect Immun 2002;70: 3026–32. 24. Goonetilleke NP, McShane H, Hannan CM, Anderson RJ, Brookes RH, Hill AV. Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette–Gue ´rin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara. J Immunol 2003;171:1602–9.