Immunogenicity and protective efficacy of mycobacterial DNA vaccines ...

Immunology and Cell Biology (2010) 88, 651–657 & 2010 Australasian Society for Immunology Inc. All rights reserved 0818-9641/10 $32.00 www.nature.com/icb

ORIGINAL ARTICLE

Immunogenicity and protective efficacy of mycobacterial DNA vaccines incorporating plasmid-encoded cytokines against Mycobacterium bovis Sarah L Young1, Lynn J Slobbe1, Matthew Peacey1, Sarah C Gilbert2, Bryce M Buddle3, Geoffrey W de Lisle4 and Glenn S Buchan1 DNA-based vaccines, alone or in combination with other sub-unit vaccination regimes, represent an alternative to live mycobacterial vaccines for protective immunization against tuberculosis. Here, we have used a murine immunization or Mycobacteriam bovis aerosol challenge model to assess the immunogenicity and protective efficacy of mycobacterial DNA vaccines. Mice that received immunization with DNA constructs encoding M. bovis antigen 85A (Ag85–A) and arget(ESAT-6) produced measurable interferon-gamma (IFN-c) responses to CD4+ T-cell epitope-peptide recall antigens in vitro. The magnitude of these responses was enhanced by co-delivery of a construct encoding murine cytokines (macrophage inhibitory protein (MIP)-1a or interleukin(IL)-7), although they did not the match responses observed in mice that received Bacille CalmetteGuerin(BCG) immunisation. In contrast, DNA priming followed by boosting with modified vaccinia Ankara (MVA) vaccine (expressing M. tuberculosis Ag85–A) invoked higher IFN-c levels, with the most immunogenic regime of Ag85 or ESAT or IL-7 prime followed by MVA boost being of commensurate immunogenicity to BCG. Despite this, neither DNA alone nor DNA-prime or MVA boost regimes conferred measurable protection against aerosol challenge with virulent M. bovis. These data highlight both the promise and the shortcomings of new generation subunit tuberculosis vaccines, with particular emphasis on their potential as vaccines against M. bovis. Immunology and Cell Biology (2010) 88, 651–657; doi:10.1038/icb.2010.25; published online 16 March 2010 Keywords: tuberculosis; Mycobacterium bovis; DNA vaccines; MVA vaccine; prime boost

Tuberculosis (TB) is a re-emerging disease, that is a major human priority as well as an important disease of livestock. TB is due to infection with mycobacteria of the Mycobacterium tuberculosis complex, and is responsible for over two million human deaths annually.1,2 The disease in livestock is important as a zoonosis as well as on account of its causing major economic losses, with an estimated 50 million cattle worldwide infected with Mycobacterium bovis.3 A partly efficacious human TB vaccine has been available for over 80 years, namely M. bovis strain Bacille CalmetteGuerin (BCG); despite its widespread use, the reported protective efficacy of BCG as a clinical vaccine has been highly variable, ranging from 80% to 0.4,5 Partly owing to this variable efficacy, and in part because of the ongoing safety concerns regarding the use of a live attenuated bacillus as an injectable vaccine, recent research objectives have focussed on the development of alternative sub-unit TB vaccines.6 DNA-based vaccines, alone or in combination with recombinant vector or protein

or peptide immunization, have been reported to provide protection against M. tuberculosis or M. bovis challenge in animal models.7–10 However, although reports have shown the induction of strong mycobacterial antigen-inducible interferon-gamma (IFN-g) responses via DNA vaccination,10,11 no sub-unit vaccination regime has yet surpassed the protective efficacy conferred by BCG-based immunization. One strategy to increase the efficacy of DNA-based TB vaccination is to co-deliver the target immunogens with endogenous molecules that can assist in driving the protective response against mycobacterial infection. Triccas et al.12 showed that IFN-g effector responses to DNA vaccination could be enhanced in mice by co-delivery of plasmids expressing the pro-IFN-g cytokines IL-12 or IL-18. Other candidate co-delivery molecules for augmenting cell- mediated immune (CMI) responses include chemokines and molecules associated with the generation of immunological memory. For example, macrophage

1Department of Microbiology & Immunology, University of Otago, Dunedin, New Zealand; 2Jenner Institute, University of Oxford, Oxford, UK; 3AgResearch, Palmerston North, New Zealand and 4AgResearch, Wallaceville Upper Hutt, Upper Hutt, New Zealand Correspondence: Dr SL Young, Department of Microbiology & Immunology, University of Otago, Box 56, Dunedin, New Zealand. E-mail: [email protected] Received 23 November 2009; revised 21 January 2010; accepted 22 January 2010; published online 16 March 2010

DNA-based vaccines against Mycobacterium bovis SL Young et al 652

inflammatory protein (MIP)-1a has been shown to augment CMI responses to mycobacterial DNA vaccination in mice,13 while coimmunization using IL-7 or IL-15 has been reported to improve CMI responses to sub-dominant antigens and promote survival of CD8+ memory T cells.14,15 However, whether these latter co-administered molecules can improve vaccine efficacy against TB is unknown. In our research, we have designed a DNA vaccine that encodes immunodominant antigens from M. bovis, namely antigen85-A (Ag85-A) and ESAT-6. We have also engineered the vaccine to encode either murine MIP-1a or murine IL-7, two cytokines selected for their chemotactic and memory-promoting properties, respectively, as outlined above. In the present study, we have investigated the ability of vaccination regimes based on these DNA constructs to promote effector IFN-g responses in mice, drawing comparisons against responses generated using a standard BCG immunization. As several studies have indicated that DNA vaccines are most effective as immunological priming agents to precede a recombinant vector or proteinboost,7,16,17 we also sought to investigate the performance of the DNA constructs in combination with sub-unit vaccination. For this purpose, we used the modified vaccinia Ankara (MVA) virus expressing Ag85A of M. tuberculosis, as this is currently the most

advanced sub-unit alternative to BCG in TB vaccinology.9 Finally, the ability of different DNA immunization and DNA-prime or MVAboost immunization regimes to confer protection against mycobacterial infection was assessed in mice using an aerosol infection model; for this purpose we used a virulent strain of M. bovis for pulmonary challenge, as previous research has indicated that vaccine-mediated protection in rodents may be more discernible using this pathogen than M. tuberculosis.18 RESULTS IFN-c responses following vaccination Splenocytes prepared from mice following BCG or DNA immunization alone (Experiment 1) produced statistically significant IFN-g responses to recall antigen stimulation in vitro, in comparison with control (non-vaccinated) animals (Figure 1). Mice remained immunoresponsive to vaccination at 3 and 12 weeks post-immunization. The strongest DNA vaccine-induced IFN-g responses were observed among mice that had received the Ag85/ESAT construct in conjunction with interleukin(IL)-7 (Figure 1), following in vitro splenocyte stimulation by addition of a CD4+ T cell-restricted Ag85 epitope plus free ESAT antigen (Figure 1); however, the strongest IFN-g responses

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Figure 1 IFN-g responses in mice following DNA vaccination. Groups of mice (n¼6 per group) were administered two intramuscular. immunizations comprising DNA constructs encoding M. bovis Ag85A and ESAT-6, alone or in conjunction with constructs encoding for murine MIP-1a or IL-7. Control mice received either no immunization or immunization with an empty DNA vector (negative controls), or subcutaneous injection of 106 live BCG. Splenic IFN-g responses were measured 3 (a) and 12 (b) weeks following the final immunization; the in vitro recall antigens used included a CD4+ T-cell-restricted epitope of Ag85A and/or a peptide epitope of ESAT-6 (control cells received no recall antigen in vitro). Data represent pg IFN-g secreted per ml of culture supernatant, each data set representing the mean±s.e.m. for six mice per group. Asterisks refer to group responses following antigen stimulation that were significantly higher than the corresponding responses in control (non-stimulated) cells, *Po0.05, **Po0.001. In addition, antigen-stimulated responses among mice that received DNA constructs co-expressing murine cytokines were significantly higher than the corresponding responses in mice that received DNA constructs expressing M. bovis antigens alone (Po0.05 in each case for responses stimulated by Ag85 epitope, or ESAT 6 or a combination thereof). Immunology and Cell Biology


overall were observed among mice that received a standard BCG immunization (Figure 1). Mice that received BCG immunization alone did not respond to recall stimulation using ESAT-6 antigen (which is expressed only in virulent M. bovis). Splenocytes prepared from mice following BCG immunization alone, or in response to DNA priming followed by MVA boosting (Experiment 2), produced statistically significant IFN-g responses to recall antigen stimulation in vitro, in comparison with control (nonvaccinated) animals (Figure 2). As expected, animals that received BCG immunization alone failed to produce IFN-g in response to recall stimulation with free ESAT-6 antigen (Figure 2) although strong responses were observed in these mice following splenocyte stimulation in the presence of a CD4+ T cell-restricted Ag85 epitope (Figure 2). Among the DNA immunization or MVA-boosting regimes, responses were lowest among animals that received DNA constructs expressing M. bovis antigens alone; these responses were increased in each case by co-delivery of a construct expressing murine cytokines (Figure 2). The strongest DNA-prime or MVA-boost vaccine-induced IFN-g responses were observed among mice that had received the Ag85 or ESAT construct in conjunction with IL-7 (Figure 2); following in vitro splenocyte stimulation by addition of a CD4+ T cell-restricted Ag85 epitope and/or free ESAT antigen, these responses were significantly higher than those among mice that received MVA alone (Figure 2) and commensurate in magnitude to the responses observed in BCG-immunized mice (Figure 2). Protection against M. bovis aerosol challenge In both Experiment 1 (DNA vaccination followed by M. bovis challenge) and Experiment 2 (DNA-prime or MVA boost followed by M. bovis challenge), there was no discernible protection to the lungs by either DNA immunization regime (Tables 1 and 2). In contrast, in both experiments, mice that received BCG vaccination showed protection by 41 log10 reduction in lung M. bovis burdens compared with non-vaccinated control animals. In addition, in Experiment 2, BCG-vaccinated animals showed a significant reduction in M. bovis burden compared with animals that received MVA vaccination only (Table 2). DISCUSSION Previous rodent model studies have investigated the potential for DNA-based vaccines to confer protection against challenge infections of M. tuberculosis, virulent M. bovis or virulent M. avium.6,19–21 In general, however, although DNA vaccines have proved immunogenic, the degree of protection conferred has not surpassed that observed using a standard BCG immunization. In the present study, we first confirmed that DNA constructs expressing two major M. bovis antigens (Ag85A and ESAT-6) were immunogenic in mice. Two intramuscular injections of the construct invoked ex vivo recall IFN-g production in splenocytes, confirming previous reports that intramuscular DNA vaccination can invoke strong systemic-level CMI responsiveness. The fact that these responses were T cell-dependent was shown by the use of CD4-restricted T-cell peptide epitopes. Interestingly, IFN-g production following DNA vaccination was increased significantly by the delivery of a construct co-expressing murine cytokines, confirming previous reports that co-administered cytokines can augment CMI responsiveness.12,22 These previous reports had used expression of pro-interferon monokines (IL-12 or IL-18) as a means of enhancing the IFNg-based effector CMI responses, with the aim of clearing pathogenic mycobacteria more effectively. In contrast, in our study, we utilised a cytokine-expression vector encoding MIP-1a, a chemokine that has been shown to be

important for facilitating lung infiltration by immune effector cells following pulmonary TB challenge of vaccinated mice;23 in addition, we used IL-7 expression, as this cytokine has been shown to be important in the selection of memory T cells and in the stabilization of IFN-g responses during mycobacterial infection.24 However, despite successfully enhancing IFN-g responses, no DNA immunization regime invoked a CMI response that surpassed the response observed using injected BCG as the vaccine, even when cytokine-expressing vectors were used along with DNA vaccination. Recent evidence suggests that systemic-level IFN-g production, although while useful as an index of mycobacterial vaccine immunogenicity sensu stricto, is not a correlative indicator of vaccine-mediated protection,25 and that localized pulmonary immunity, invoked following systemic-level vaccination, may have a more representative role in protection.26 Hence, in addition to measuring systemic-level IFN-g responses in the present study, we also sought to identify any vaccinemediated pulmonary protection induced by the DNA immunization by challenging mice via aerosol exposure to virulent M. bovis. Previous studies have indicated that DNA-based vaccines in rodents have the potential to confer measurable protection against M. bovis.10,11,27 In these studies, one major measure of protection was to observe a postchallenge reduction in pathogen burdens among vaccinees; the same measurement was used here as the means of assessing any vaccinemediated protection against M. bovis. However, in the present study, the only vaccine treatment that conferred significant protection against pulmonary M. bovis challenge was BCG immunization, indicating that the DNA vaccination regime used here is not, on its own, a suitable alternative to the current TB vaccine. Although we have no further evidence here to provide an explanation, recent evidence has indicated that injected DNA-based TB vaccines, although effective in invoking systemic-level CMI, may not invoke pulmonarylevel immune activation sufficiently to control mycobacterial lung infection.23 Recently, the MVA vaccine expressing M. tuberculosis Ag85A has been shown to be immunogenic and protective in animal models of TB and immunogenic in humans in clinical trials.28,29 As the MVA vaccine seems to be most effective when used as a prime or boost in conjunction with other TB vaccination regimes,27 in this study we also investigated whether our DNA constructs could prove immunogenic and protective in mice when used in conjunction with MVA. Interestingly, IFN-g production was strong among mice that received DNA prime followed by MVA boost. The most immunogenic combination proved to be an Ag85 or ESAT DNA construct prime in conjunction with IL-7 followed by an MVA boost; in this case, levels of CD4+ T-cell reactogenicity approached those observed with BCG immunization. Despite this, upon challenge with virulent M. bovis, none of the DNAprime or MVA-boost regimes conferred significant protection, as determined by counting the lung colony forming unitsof M. bovis post-challenge. Similar discrepancies have been recorded in other prime or boost studies that have used DNA vaccines,7,8,16 to the extent that although such strategies undoubtedly generate a strong immunological response, this does not necessarily correlate with pulmonary protection. Previous research has indicated that Ag85 or ESAT DNA prime followed by BCG boost could augment vaccine immunogenicity in mice, although it did not enhance protection against aerosol M. bovis challenge compared with that for BCG alone.20 In the context of the present results, here we conclude that DNA priming offers promise as a component of a prime-boost vaccination regime to provide pulmonary protection against exposure to virulent mycobacteria; however, it is the nature of the boosting agent that determines this Immunology and Cell Biology


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Figure 2 IFN-g responses in mice following DNA priming and MVA boosting. Groups of mice (n¼6 per group) received two intramuscular priming immunizations comprising DNA constructs encoding M. bovis Ag85A and ESAT-6, alone or in conjunction with constructs encoding for murine MIP-1a or IL-7; these mice were then boosted using MVA vaccine injected intradermally. Control mice received either no immunization or immunization with an empty DNA vector (negative controls), or subcutaneous injection of 106 live BCG. Splenic IFN-g responses were measured 4 weeks following the final immunization; the in vitro recall antigens used included a CD4+ T-cell-restricted epitope of Ag85A and/or a peptide epitope of ESAT-6 (a), a mixture of ESAT-6 and Ag85A (b) or Ag85A alone (c) (control cells received no recall antigen in vitro (data not shown)). Data represent pg IFN-g secreted per ml of culture supernatant, each data set representing the mean±s.e.m. for six mice per group. All vaccine groups thatreceived mycobacterial antigens produced significant levels of IFN-g in response to recall stimulation; in addition, asterisks refer to treatment responses that were significantly different from the responses observed in other line-paired groups (*Po0.05).

Immunology and Cell Biology


Table 1 Post challenge lung M. bovis cfu counts in DNA-vaccinated mice Naı¨ve controls

5.2±0.1

BCG

3.4±0.1 *

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Ag85/ESAT

Ag85/ESAT + MIP1a

Ag85/ESAT + IL-7

5.6±0.3

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5.4±0.3

5.2±0.5

Abbreviations: Ag85, Antigen85; BCG, Bacille Calmette-Guerin; cfu, colony forming unit; ESAT, early secretory antigenic target; IL-7, interleukin; MIP, macrophage inflammatory protein. Groups of mice (n¼6 per group) received two i.m. immunisations comprising DNA constructs encoding M. bovis Ag85A and ESAT-6, alone or in conjunction with constructs encoding for murine MIP-1a or IL-7. Control mice received either no immunization or immunization with an empty DNA vector (negative controls), or subcutaneous injection of 106 live BCG. Mice were challenged through pulmonary exposure to virulent M. bovis; lungs were excised and homogenized, and tissue M. bovis burdens were calculated 5 weeks following challenge. Data represent log10 M. bovis cfu’s per lung, each data set representing the mean±s.e.m. for six mice per group. The asterisk refers to a significant reduction in lung M. bovis burden among BCG-vaccinated mice compared with naı¨ve controls.

Table 2 Post-challenge lung M. bovis cfu counts in mice that received DNA priming followed by MVA boost Naı¨ve controls

6.4±0.04*1

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6.0±0.1*2

5.8±0.1

6.0±0.1

5.9±0.1

Abbreviations: Ag85, Antigen85; BCG, Bacille Calmette-Guerin; ESAT, early secretory antigenic target; IL-7, interleukin; MIP, macrophage inflammatory protein; MVA, modified vaccinia Ankara. Groups of mice (n¼6 per group) received two i.m. priming immunizations comprising DNA constructs encoding M. bovis Ag85A and ESAT-6, alone or in conjunction with constructs encoding for murine MIP-1a or IL-7; these mice were then boosted using MVA vaccine injected intradermally. Control mice received either no immunization or immunization with an empty DNA vector (negative controls), or subcutaneous injection of 106 live BCG. Mice were challenged by pulmonary exposure to virulent M. bovis; lungs were excised and homogenized, and tissue M. bovis burdens were calculated 5 weeks following challenge. Data represent log10 M. bovis cfu’s per lung, each data set representing the mean±s.e.m. for six mice per group. The asterisk refers to significant reductions in lung M. bovis burden among BCG-vaccinated mice (Po0.05) compared with naı¨ve controls (*1) and with empty vector controls (*2).

protection. Although a previous report has indicated that Ag85Aencoding DNA prime followed by MVA boost can confer protection to BALB/c mice against M. tuberculosis challenge,30 and that this is commensurate with protection conferred with BCG alone, here we report that prime-boosting regimes based on sub-unit vaccines alone do not offer commensurate protection against virulent M. bovis challenge. METHODS DNA vaccines DNA was isolated from heat-inactivated M. bovis BCG Pasteur to act as a template to amplify the Ag85A gene; heat-inactivated DNA from virulent M. bovis was used as the template to amplify the ESAT-6 gene. Oligonucleotide primers were designed to the N-terminus and C-terminus of both genes and included appropriate sequences for restriction sites to enable directional cloning into the various vectors. First ESAT-6 was cloned into pBluescript KS into which the human immunoglobulin kappa light chain signal sequence had been cloned (IgSS-ESAT-BS; a generous gift from Dr Euan Lockhart, University of Pittsburgh School of Medicine). The Ag85A gene was amplified without its signal sequence or stop codon and inserted between the human immunoglobulin signal sequence and ESAT-6, producing a fusion construct that would be produced as a single contiguous fusions protein (IgSS–Ag85A–ESAT–BS). The expression vector pBudCE4.1 (Invitrogen, Auckland, New Zealand) is designed for the simultaneous expression of two genes, one under the control of human cytomegalovirus (CMV) immediate early promoter and the other under the human elongation factor 1 alpha promoter . The multiple cloning cassette of elongation factor 1 alphaa was altered by inserting the Gateway Conversion cassette pBudgw (Invitrogen, Carlsbad, CA, USA). New primers were designed to amplify the entire IgSS–Ag85–ESAT sequence for cloning in phase into the multiple cloning cassette under the control of the CMV promoter (pBudgw– IgSS–Ag85–ESAT). The DNA profile of each construct was confirmed at each stage by nucleotide sequencing. To clone the macrophage inflammatory protein-1 (MIP-1a) gene, murine alveolar MH-S cells (ATCC, Manassas, VA, USA) were stimulated for 24 h with lipopolysaccharide and total RNA was isolated, converted to complementary DNA and this was

used as the template to amplify the gene. The 5¢ primer included the ATG start codon, while the 3¢ primer removed the gene’s stop codon and inserted a six histidine tag, stop codon and restriction site. The amplified product was cloned into the gateway entry vector pENTR11 (Invitrogen), which had been digested with XmnI and NotI. After sequence confirmation, the LR (L1-genex RI) clonase reaction kit (Invitrogen) was used to transfer the gene by recombination between the entry vector and the destination vector, which in this case was both the pBudgw– IgSS–Ag85–ESAT construct and the pBudgw vector. To clone murine IL-7, mouse thymocytes were stimulated in vitro with 12 mg ml1 Concanavalin A (Sigma, Perth, Australia) for 48 h; total RNA was isolated, converted to complementaryDNA and this was used as the template to amplify the IL-7 gene. Primers were designed as above to clone into pENTR11 and the LR clonase reaction was used to move the gene into both destination vectors.

MVA and BCG vaccines MVA85A vaccine, produced by recombining the shuttle vector pSC11 expressing the Ag85A antigen of M. tuberculosis with wild-type MVA, was prepared as described previously.31,32 The vaccine comprises approximately 106 viral plaque forming units per 50 ml immunizing dose. The BCG vaccine comprised live Mycobacterium bovis BCG (Pasteur strain 1172). Bacilli were cultured in Middlebrook 7H9 broth (Difco Invitrogen life sciences, Carlsbad, CA, USA) until the mid-log phase, before the harvest, washing by centrifugation with phosphate-buffered saline, and subsequent use in experimentation at an immunization dose of 106 colony forming units per animal (by sub-cutaneous injection).

In vivo experiments Specific pathogen-free BALB/c mice (6–10 weeks old) were obtained from the Department of Laboratory Animal Sciences, University of Otago, New Zealand. Two main experiments were conducted: the first to investigate the immunogenicity and protective efficacy of DNA constructs in mice, and the second to further evaluate the immunogenicity and protective efficacy of the constructs in mice that additionally received MVA vaccine as a boost. In Experiment 1, groups of mice (n¼6 per group) were vaccinated intramuscularly in each quadricep with either 50 mg of empty vector or 50 mg Immunology and Cell Biology

DNA-based vaccines against Mycobacterium bovis SL Young et al 656 of constructs encoding Ag85A/ESAT-6, Ag85A/ESAT-6 plus MIP-1a, or Ag85A/ ESAT-6 plus IL-7. One month later, all groups were boosted intramuscularly with the same original DNA vaccine. Mice were left for a further 3 or 12 weeks to assess their immune response to the vaccines. In addition, replicate groups of mice (n¼6 per group) were challenged by aerosolized M. bovis infection to determine vaccine-mediated pulmonary protection, as outlined below, at 20 weeks following the first DNA immunization. In Experiment 2, groups of mice (n¼6 per group) were vaccinated twice intramuscularly in each quadricep with either 50 mg of empty vector or 50 mg of constructs encoding Ag85A/ESAT-6, Ag85A/ESAT-6 plus MIP-1a, or Ag85A/ ESAT-6 plus IL-7, as outlined for Experiment 1. One month after the second DNA immunization, mice were further administered 25 ml of MVA vaccine through an intra-dermal injection into each ear. Immune responses were assessed after 4 weeks following the MVA boost in these mice, while replicate groups of mice (n¼6 per group) were challenged by aerosolized M. bovis infection to determine vaccine-mediated pulmonary protection, as outlined below, at 20 weeks following the first DNA immunization.

Mycobacterium bovis challenge To evaluate the level of protection afforded by DNA vaccines, groups of mice vaccinated as above were given an aerosol challenge of virulent M. bovis 20 weeks after the first DNA vaccination. Single-cell suspensions of the challenge isolate (strain 83 or 6235) were prepared as described previously.33 The mice were infected through the respiratory route by using an aerosol chamber that produces droplet nuclei of size appropriate for entry into alveolar spaces. The concentration of viable M. bovis in the nebulizer fluid was adjusted empirically to result in the inhalation and retention of approximately 10–20 viable organisms per mouse. The mice were killed 5 weeks after challenge and serial dilutions of lung homogenates were plated onto a modified 7H11 agar; each animal was assessed individually. Bacterial colonies were counted 28 days later following incubation at 37 1C.

Assessing cell-mediated immune responses (IFN-c) Spleens were excised from euthanized mice and single-cell suspensions were prepared, washed by centrifugation in phosphate-buffered saline , adjusted to 2106 mononuclear cells per ml and plated in 24-well plates (Nunc, Roskilde, Denmark). Samples were cultured for a period of 72 h along with DMEM (Gibco BRL, Grand Island, NY, USA) as a no-antigen control or 50 mgml1 of a CD4+ T-cell epitope of M. tuberculosis Ag85A,34 or 50 mgml1 of a 20 amino acid peptide sequence from M. tuberculosis ESAT-6,35 both peptides synthesized by AusPep Ltd (Melbourne, Victoria, Australia). Supernatants were removed and analysed for levels of IFN-g using a capture enzyme-linked immunosorbent assay (BD PharMingen, San Diego, CA, USA). The IFN-g response of each animal was assessed individually and expressed as pg IFN-g detected per ml of culture supernatant, in the range 20–5000 pg ml1; data were presented as group mean values for acrossgroup statistical comparisons.

ACKNOWLEDGEMENTS This research was funded by the FRST (NZ) and Otago University. We would like to thank Gary Yates and Maree Joyce for the bacteriology and Frank Cross (Otago University) for his help in the preparation of this paper.

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